2 VIRAL

DISEASES
W. L. Mengeling, Editor
 Adenovirus
4 J. B. Derbyshire
The first isolation of a porcine adenovirus was made
from a rectal swab from a piglet with diarrhea by Haig et
al. (1964). A second isolation was made by Kasza (1966)
from the brain of a pig with encephalitis. Subsequently,
porcine adenoviruses were isolated from a variety of
sources, including stocks of hog cholera virus, swine fe-
ces, and kidney tissue used for routine cell culture pro-
duction. Serologic surveys have indicated that aden-
ovirus infections are widespread and probably
ubiquitous in swine (Bibrack 1974). Strains of serotype 4
appear to be the most widely distributed, both in Europe
and North America. While the majority of infections are
asymptomatic, these viruses have been associated with
encephalitis, pneumonia, kidney lesions, and diarrhea.
Porcine adenoviruses are not known to be infectious for
other species, although pigs can be infected with certain
human adenoviruses (Betts et al. 1962), and some DNA
homology has been demonstrated between porcine and
bovine subgroup 1 adenoviruses (Benko et al. 1990).
ETIOLOGY
Physicochemical Characteristics
The basic characteristics of porcine adenoviruses resem-
ble those of other members of the Adenoviridae. The
roughly spherical, nonenveloped virions of about 75 nm
diameter contain a genome of double-stranded deoxyri-
bonucleic acid (DNA) surrounded by an icosahedral cap-
sid of 252 capsomeres. Strains of porcine adenovirus
type 4 hemagglutinate various species of red blood cells.
The porcine adenoviruses are stable at pH 4 or when
treated with chloroform or ether. They are relatively
heat-resistant viruses, surviving for more than 10 days at
room temperature (Kasza 1966). Effective chemical dis-
infectants for porcine adenoviruses include sodium
hypochlorite, formaldehyde, phenolic compounds, ethyl
alcohol, and sodium hydroxide (Derbyshire and Arkell
1971). In liquid manure, porcine adenoviruses are inacti-
vated by aeration or treatment with calcium hydroxide
(Derbyshire and Brown 1979).
Laboratory Cultivation
Porcine adenoviruses can be cultivated fairly readily in
primary pig kidney (PK) cell cultures and in certain es-
tablished porcine cell lines. Relatively high yields were
reported from secondary pig thyroid cell cultures by Dea
and Elazhary (1984a). In unstained cultures the cyto-
pathic effects (CPE) are characterized by enlargement
and rounding of the cells, followed by detachment; in
stained monolayers characteristic nuclear inclusion bod-
ies are seen. Ultrastructural studies of infected cells
showed that the virus was intranuclear, sometimes in
crystalline arrays (Chandler 1965).
Serologic Classification
The porcine adenoviruses contain the mammalian aden-
ovirus group-specific antigen, detectable by immunodif-
fusion or complement fixation. Virus-neutralization
(VN) tests have failed to reveal relationships between
porcine adenoviruses and those of other species. Four
serotypes of porcine adenovirus are currently recognized
(Table 4.1) on the basis of VN tests, and there is evidence
that additional serotypes may exist (Derbyshire et al.
1975).
EPIDEMIOLOGY
Strains of porcine adenovirus type 4 appear to be the
most widely distributed infection; this virus has been
identified either serologically or by virus isolation in Aus-
tralia, Belgium, Bulgaria, Canada, Denmark, Germany,
Hungary, Japan, the Netherlands, the United Kingdom,
and the United States. Transmission of porcine aden-
oviruses is probably by the fecal-oral route. Inhalation of
infectious aerosols may also occur. Virus is excreted in
the feces, most frequently by pigs in the postweaning pe-
riod (Derbyshire et al. 1966). Adults rarely excrete virus,
but they frequently have high serum antibody levels.
Nursing piglets are probably protected by antibodies in
the milk of the sow.
Table 4.1. Serologic classification of porcine
adenoviruses
Serotype No. WHO Reference Strain
1 25R
2 A47
3 6618
4 F618
89
90 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
CLINICAL SIGNS
The reference strain of porcine adenovirus type 4 was
isolated from the brain of a pig that showed anorexia, en-
teritis, incoordination, muscle twitching, and frequent
recumbency (Kasza 1966); other strains of the same
serotype have been isolated from pigs with respiratory or
gastrointestinal disease (Genov and Bodon 1976). Other
serotypes have been isolated from piglets with diarrhea
(Coussement et al. 1981), but adenoviruses have also
been isolated fairly frequently from the feces of clinically
normal piglets. The only consistent clinical sign in
piglets infected experimentally with porcine adenovirus
type 4 was diarrhea (Shadduck et al. 1967), and infec-
tions with other strains have also produced diarrhea ex-
perimentally (Coussement et al. 1981). Although experi-
mentally infected piglets may develop lesions in the
brain, lungs, and kidneys, clinical signs associated with
these lesions have not been described. In a study of two
herds with respiratory disease, Watt (1978) concluded
that porcine adenovirus type 4 may have been directly as-
sociated with the disease, and Kirkbride and McAdaragh
(1978) reported the isolation of adenovirus from a small
proportion of abortions in swine. Narita et al. (1985)
found skin cyanosis and subcutaneous edema in new-
born piglets with natural transplacental adenovirus in-
fections.
PATHOGENESIS
Natural infection with porcine adenoviruses is acquired
by ingestion or inhalation. Experimentally, intranasal in-
oculation of virus was the most effective route of infec-
tion in terms of the widespread distribution of virus and
production of lesions (Shadduck et al. 1967). Experi-
mental pathogenesis studies with various strains of ade-
novirus (Sharpe and Jessett 1967; Shadduck et al. 1968;
Ducatelle et al. 1982) suggest that the primary sites of vi-
ral replication are the tonsil and lower small intestine.
Spread of the infection to a variety of tissues, including
the central nervous system, lung, heart, liver, kidney, and
spleen, was recorded. Viremia was detected in one piglet.
Viral excretion in the feces continued for several weeks
after infection, and there was also evidence of persis-
tence of the virus in the kidney and other tissues. Some
limited findings suggest that adenoviruses may play a
role in the pathogenesis of bacterial infections of the res-
piratory tract. Thus Kasza et al. (1969) found that
porcine adenovirus type 4 and Mycoplasma hyopneumo-
niae produced a more severe experimental pneumonia
when inoculated together than either agent did alone.
However, Smith et al. (1973) found that the experimental
pneumonia produced by porcine adenovirus type 4 was
not enhanced by infection with Pasteurella septica.
LESIONS
Although most of the descriptions of lesions associated
with porcine adenoviruses are based on experimental in-
fections, there are several reports of adenovirus-associat-
ed lesions in field material. Porcine adenovirus type 4
was isolated from the brain of a pig that showed lesions
of encephalitis, including perivascular cellular infiltra-
tion and microglial nodule formation (Kasza 1966).
Rondhuis (1972) isolated a strain of the same serotype
from pneumonic lung. Pavlov (1977) described kidney
lesions in pigs naturally infected with adenovirus. The le-
sions involved dystrophy of the kidney tubules and cap-
illary dilatation, which gave the gross appearance of pe-
techiation. Nuclear inclusion bodies have been found in
the enterocytes of the terminal jejunum and ileum in
natural infections (Ducatelle et al. 1982; Sanford and
Hoover 1983). Transplacentally infected piglets showed
vascular lesions, with nuclear inclusions in the endothe-
lial cells (Narita et al. 1985).
In experimentally infected piglets, gross lesions have
been described only in the lungs. These consisted of ar-
eas of atelectasis of variable extent (Shadduck et al.
1967). Microscopically, the lungs showed interstitial
pneumonia characterized by thickened alveolar septa
due to proliferated septal cells, some of which contained
inclusion bodies, infiltrating lymphocytes, plasma cells,
and histiocytes. The histologic lesion produced most
consistently with porcine adenovirus type 4 was a severe
peritubular infiltration in the kidney, although menin-
goencephalitis, characterized by focal accumulations of
microglia and oligodendrocytes within the brain sub-
stance and perivascular lymphocytic cuffing, was also
produced by this virus (Edington et al. 1972). Brack et al.
(1969) produced meningoencephalitis experimentally
with porcine adenovirus types 1 and 3 and also described
chronic inflammatory changes in the liver, heart, pan-
creas, and adrenal. In a sequential study of the enteric le-
sions produced by porcine adenovirus type 3, Ducatelle
et al. (1982) described stunting of the villi in the lower je-
junum and ileum and demonstrated infected enterocytes
by histology, immunoperoxidase staining, and electron
microscopy.
DIAGNOSIS
Clinical signs and gross lesions are minimal or absent in
porcine adenovirus infections. Histologically, nuclear in-
clusion bodies in the lung, kidney, or intestinal epitheli-
um may be suggestive of adenovirus infection, but they
are not diagnostic unless viral antigen is demonstrated
by immunofluorescence or immunoperoxidase staining.
A virologic diagnosis can best be made by isolation of
the virus from infected tissue, which can be fairly readily
 CHAPTER 4 ADENOVIRUS Derbyshire
accomplished in porcine cell cultures. Some strains of
the virus require several blind passages in cell culture be-
fore CPE are evident. Stained coverslip preparations of
the infected cells show nuclear inclusion bodies, and the
virions may be demonstrated by negative staining of
lysates of the infected cells. The virus can also be identi-
fied by specific immunofluorescence of the infected cells
(Dea and Elazhary 1984b). Serologic typing of the isolat-
ed virus may be attempted by VN if reference antisera
are available. Serologic diagnosis of suspected aden-
ovirus infections in swine may be attempted by the
demonstration of rising titers of antibody in VN or im-
munodiffusion tests or by an indirect fluorescent anti-
body test (Dea and Elazhary 1984b).
TREATMENT AND PREVENTION
No specific antiviral treatment is available. Porcine aden-
ovirus infections have not been shown to be of sufficient
economic importance to justify the development of vac-
cines, although adenovirus vaccines have been used suc-
cessfully in other species. Repopulation of herds with
specific-pathogen-free stock does not appear to be a reli-
able means of excluding infection with porcine aden-
oviruses (Derbyshire and Collins 1971).
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Coussement, W.; Ducatelle, R.; Charlier, G.; and
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———. 1984b. Prevalence of antibodies to porcine ade-
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Meningoencephalitis in gnotobiotic pigs inoculated
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Genov, I., and Bodon, L. 1976. Vurkhu tipiziraneto i
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Sharpe, H. B. A., and Jessett, D. M. 1967. Experimental
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H. S. 1973. Experimental infections with Pasteurella
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gnotobiotic piglets. J Comp Pathol 83:1–12.
Watt, R. G. 1978. Virological study of two commercial
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24:147–153.
 African Swine Fever
5 J. M. Sanchez-Vizcaino
African swine fever (ASF) was first described by Mont-
gomery in Kenya in 1921. It was subsequently shown to
be caused by an unusual virus, ASF virus (ASFV), that
under natural conditions infects only porcine species
and whose properties differ markedly from those of any
other virus of veterinary importance (Murphy et al.
1995). Depending on the circumstances, the clinical re-
sponse to infection with ASFV ranges from peracute to
inapparent. The acute form is most dramatic in regard to
signs and lesions and is characterized by high fever, high
mortality, extensive hemorrhages, pulmonary edema,
and extensive necrosis of lymphoid tissue (Mebus 1988;
Gomez-Villamandos et al. 1995). Clinically and patho-
logically, ASF can resemble several other diseases of pigs
that sometimes present similar signs and lesions, espe-
cially hog cholera and erysipelas. Therefore, laboratory
tests are required to establish a definitive diagnosis
(Sanchez-Vizcaino 1986). ASFV replicates primarily in
mononuclear phagocytic cells (Malmquist and Hay
1960; Minguez et al. 1988). However, it also replicates in
endothelial cells (Wilkinson and Wardley 1978), hepato-
cytes (Sierra et al. 1987), renal tubular epithelial cells
(Gomez-Villamandos et al. 1995), and neutrophils
(Casals et al. 1984; Carrasco et al. 1996).
Immunologically speaking, ASF is characterized by
lymphopenia due to apoptosis of lymphocytes mainly in
the T area of the lymphoid organs (Carrasco et al. 1996),
but there is no evidence of virus replication in T or B cells
(Minguez et al. 1988; Gomez-Villamandos et al. 1995).
Humoral and cell-mediated immunity do not seem to be
affected (De Boer 1967). However, the mechanism of
protection is poorly understood.
Inapparent infections with ASFV are common in two
species of wild boars: warthogs (Phacochoerus aethiopi-
cus) and bushpigs (Potamochoerus porcus). Both act as
reservoir hosts in Africa (De Tray 1957; Heuschele and
Coggins 1965). Soft ticks have been shown to be both
reservoirs and vectors of ASFV, especially Ornithodoros
moubata (Plowright et al. 1970) and O. erraticus (Sanchez
Botija 1963a).
From the epidemiological point of view it is believed
that the entrance of ASFV into a free area is related main-
ly to feeding swine uncooked, infected pork products
from international airports and ports. However, once
ASF is established in domesticated swine, infected and
carrier pigs are the most important source for virus dis-
semination thereafter.
ASF is currently endemic in Sardinia and in parts
of Africa, particularly in many sub-Saharan coun-
tries. Although ASF has appeared elsewhere in the
world at one time or another, these areas are now ASF-
free.
There is no treatment or effective vaccine available
for ASF, and therefore its control is based on a rapid lab-
oratory diagnosis and the enforcement of strict sanitary
measures.
ETIOLOGY
ASFV is a complex, icosahedral, DNA virus classified as
the only member of an unnamed floating genus (Mur-
phy et al. 1995).
Size and Structure
Virus particles have an average diameter of 200 nm
(Breese and De Boer 1966). The architecture of individ-
ual particles (virions) includes several concentric struc-
tures with an external hexagonal membrane (Fig. 5.1)
that is acquired during budding through the cell mem-
brane (Carrascosa et al. 1984). The ASFV genome is a
double-stranded, linear DNA molecule that, depending
on the particular strain (Blasco et al. 1989), comprises
between 170 and 190 kilobases (kb). It is further charac-
terized by terminal inverted repeats (Sogo et al. 1984), a
conserved central region of about 125 kb pairs (kbp),
and variable ends (Blasco et al. 1989). Recently, the com-
plete DNA sequence of the BA71v strain of ASFV was
analyzed and found to comprise 170,101 nucleotides and
151 open reading frames and to encode five multigene
families (Yañez et al. 1995).
ASFV is a very complex virus. At least 28 structural
proteins have been identified in intracellular virus parti-
cles (Tabares et al. 1980), and more than 100 virus-in-
duced proteins have been identified in infected porcine
macrophages. At least 50 of the latter react with sera
from infected or recovered pigs (Alcaraz et al. 1992), and
40 are incorporated into the viral particle (Carrascosa et
al. 1985). Some of these proteins, such as p73, p54, p30,
and p12, are very antigenic. Although their role in in-
ducing protective immunity is still unresolved, they are
93
94 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
5.1. Electron micrograph of an
ASF virus particle.
very good antigens and are used in ASF serodiagnosis
(Arias and Sanchez-Vizcaino 1992).
Virus Replication and Persistence
In an infected pig, ASFV replicates primarily in mono-
cytes and macrophages (Malmquist and Hay 1960;
Minguez et al. 1988; Mebus 1988). However, ASFV also
replicates in endothelial cells (Wilkinson and Wardley
1978), hepatocytes (Sierra et al. 1987), renal tubular ep-
ithelial cells (Gomez-Villamandos et al. 1995) and neu-
trophils (Casals et al. 1984; Carrasco et al. 1996). No in-
fection has been observed in lymphocytes (Minguez et
al. 1988; Gomez-Villamandos et al. 1995). In nature, AS-
FV also replicates in some soft ticks, principally O.
moubata (Plowright et al. 1970) and O. erraticus (Sanchez
Botija 1963a). ASFV has been adapted to grow in a large
number of stable cell lines as well, such as VERO, MS,
and CV (Hess et al. 1965). ASFV is very resistant to inac-
tivation in environmental conditions such as tempera-
ture changes and acid pH. It can be isolated from serum
or blood kept at room temperature for 18 months. How-
ever, it is inactivated by heat treatment at 60˚C for 30
minutes (Plowright and Parker 1967) and by many lipid
solvents and commercial disinfectants.
ASFV may persist for several weeks or months in
frozen or uncooked meat. In meat products such as Par-
ma ham, viral infectivity was not demonstrated after 300
days of processing and curing (McKercher et al. 1987).
In typical Spanish dry-cured meat products, such as Ser-
rano ham and Iberian ham and shoulder, the virus sur-
vives for 140 days in Iberian and Serrano hams and
for 112 days in loin (Mebus et al. 1993). No infectious
ASFV was found in cooked or canned hams heated at
70˚C.
EPIDEMIOLOGY
History and Distribution
ASF was described for the first time in Kenya by Mont-
gomery in 1921. In this case the virus had spread from in-
fected warthogs (Phacochoerus aethiopicus) to domesti-
cated pigs (Sus scrofa), causing 100% mortality. Since
then, ASF has been recognized, and is currently endem-
ic, in many African countries, namely, Angola, Zimbab-
we, Sudan, Republic of South Africa, Mozambique, São
Tomé, and Príncipe. In 1957, ASF was detected for the
first time outside the African continent. It appeared in
Lisbon, Portugal, as a peracute form with a mortality of
almost 100% (Manso Ribeiro et al. 1963). In 1960 it reap-
peared near Lisbon, apparently as a new outbreak, and it
spread through the rest of Portugal and to Spain the
same year (Polo Jover and Sanchez Botija 1961). ASF re-
mained endemic in Portugal and Spain until 1995, when,
as a result of an intensive eradication program, both
countries were declared ASF-free. In 1978, ASF again ap-
peared outside Africa, namely, in Malta, Sardinia, Brazil,
and the Dominican Republic. In 1979 it appeared in
Haiti and 1980 it appeared in Cuba. Today, ASF is pres-
ent only in Africa, mainly in sub-Saharan countries, and
in Sardinia. Elsewhere it has been successfully eradicat-
ed.
Reservoirs and Susceptible Animals
Several different species of soft ticks have been shown to
be ASFV reservoirs and vectors, including O. moubata in
Africa (Plowright et al. 1969) and O. erraticus in the Iber-
ian Peninsula (Sanchez Botija 1963a). Transovarial and
transstadial transmission of ASFV has been described
for O. moubata (Plowright et al. 1970). More recently, a
number of other tick species widely distributed in North
and South America have been identified as harboring
and transmitting ASFV (Groocock et al. 1980), and a new
species of soft ticks, O. Savignyi, which is present in
Africa, can experimentally transmit ASFV to domesticat-
ed pigs (Mellor and Wilkinson 1985).
Pigs are the only domesticated animals that are natu-
rally infected by ASFV. Wild boars also are susceptible to
ASFV infection, with clinical signs and mortality rates
similar to those observed in naturally infected domesti-
cated pigs in Spain and Portugal (Sanchez Botija 1982)
and in Sardinia (Contini et al. 1981) and in experimen-
tally infected feral pigs in Florida (McVicar et al. 1981).
Wild boars can transmit the disease directly to domesti-
cated pigs, as well as to one another. In Africa, it has been
observed that ASFV induces an inapparent infection in
two species of wild boars, warthogs (Phacochoerus
aethiopicus) and bushpigs (Potamochoerus porcus).
ASFV Transmission
ASFV is maintained in Africa by a cycle of infection be-
 CHAPTER 5 AFRICAN SWINE FEVER Sanchez-Vizcaino
tween wild boars and soft ticks. In some of these wild
boars infection is characterized by low levels of virus in
the tissues and low or undetectable levels of viremia
(Plowright 1981); however, even these low levels of ASFV
are enough for its transmission to domesticated pigs via
tick vectors. This transmission cycle makes it very diffi-
cult to eradicate ASF in Africa.
In Sardinia, where ASFV is still present, neither inap-
parent infection of wild or domesticated pigs nor soft
tick populations have been observed. Wild pigs are as
susceptible as domesticated pigs, and their role in the
epidemiology of ASF is similar to that of domesticated
pigs. However, in Sardinia, recovered, ASFV carrier, do-
mesticated pigs have been recognized, and their role in
the epidemiology of ASF is a major consideration in de-
signing a strategy for ASF eradication. Notably, the sero-
logical recognition of carrier pigs was an important facet
of the successful ASF eradication program in Spain
(Arias and Sanchez-Vizcaino 1992). Due to the lack of
neutralizing antibodies in ASF, the correlation between a
new outbreak and the possible source of virus strain has
been very difficult to establish. Even the use of the
hemadsorption inhibition reaction (Malmquist 1963) is
not conclusive. New studies on molecular epidemiology
comparing the patterns of DNA from different ASFV iso-
lates appear most promising, especially for differentiat-
ing African and European ASFV strains (Blasco et al.
1989). However, it is epidemiologically well established
that the entrance of ASFV into an ASFV-free country is
related to the introduction of uncooked infected pork
through international ports or airports where garbage
containing uncooked pork can be found and used for pig
feeding. Once ASF is established in domesticated pigs,
ASFV carrier pigs are the most important source for sub-
sequent virus dissemination.
IMMUNOLOGY
The immune mechanisms involved in protection against
ASF are still poorly understood, and all attempts to de-
velop an effective vaccine have been unsuccessful. The
difficulty in inducing an effective immunity may be relat-
ed to the great variability observed among different AS-
FV isolates. It may also be related to the fact that ASFV
replicates in some cells typically involved in the immune
response. Although there is no evidence for replication of
ASFV in either T or B cells (Minguez et al. 1988; Gomez-
Villamandos et al. 1995), it does replicate in monocytes
and macrophages. If immune suppression has a role,
however, it is not obvious. ASFV is highly antigenic, and
high titers of antibody are detected by in vitro testing of
sera collected from pigs after exposure to the virus. IgM
and IgG were detected in sera collected at 4 days and at
6–8 days after exposure, respectively (Hortiguela and
Sanchez-Vizcaino 1984), and high titers of antibody
were detected for a long period of time. Antibodies
95
against ASFV have been shown to delay the onset of ASF
clinical signs, to reduce the levels of viremia, and to pro-
tect pigs against the potentially fatal consequence of in-
fection (Schalafer et al. 1984; Onisk et al. 1994). Early ex-
periments demonstrated the absence of neutralizing
antibodies against ASFV in sera from naturally or exper-
imentally infected pigs. Yet, recovered pigs produce a
normal level of neutralizing antibodies in response to
foot-and-mouth disease virus vaccine, an event suggest-
ing that humoral responses are not adversely affected by
ASFV (De Boer 1967). Other authors (Ruiz Gonzalvo et
al. 1986) have demonstrated that different ASFV isolates
are largely neutralized by convalescent porcine sera;
however, a persistent 10% fraction of nonneutralized
virus remained. More recently, Gomez-Puertas et al.
(1996) reported that ASFV-induced antibodies in serum
collected from a convalescent pig effectively neutralized
ASFV before and after it was bound to susceptible cells.
However, ASFV-specific antibodies have never been
demonstrated to entirely fulfill the classic definition of
virus neutralization. On the other hand, T cytotoxic lym-
phocytes from recovered pigs can destroy infected
macrophages (Martins and Leitao 1994)—suggesting
that cell-mediated immunity may be an important com-
ponent of the protective response. However, the relative
roles of antibodies and cell-mediated immunity in pro-
tection against ASF are still not well understood.
CLINICAL SIGNS
Clinically, ASF can resemble several other diseases of
pigs that often have similar signs, especially hog cholera
and erysipelas. Therefore, laboratory tests are required
to establish a definitive diagnosis (Sanchez-Vizcaino
1986). Moreover, ASF can present different clinical signs,
depending mostly on virus virulence, infectious dose,
and route of exposure. The clinical forms of ASF range
from peracute (i.e., sudden death with few, if any, previ-
ous clinical signs) to subclinical or inapparent. In Africa
ASF appears mostly as an acute disease characterized by
loss of appetite, high temperature (40–41˚C), leukope-
nia, hemorrhages in internal organs, hemorrhages in the
skin (especially in the skin of the ears and flanks), and
high mortality (Moulton and Coggins 1968; Mebus et al.
1983).
Outside Africa it is also possible to see an acute ASF
outbreak; however, subacute or chronic forms of the dis-
ease are more common. The subacute form is character-
ized by transitory thrombocytopenia and leukopenia
and numerous hemorrhagic lesions (Gomez-Villaman-
dos et al. 1997). The chronic form is characterized by res-
piratory alteration, abortion, and low mortality (Arias et
al. 1986).
The incubation periods in natural infection vary from
as short as 4 days to as long as 19 days. In experimental
infections, the incubation period is generally shorter and
96 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
varies from 2 to 5 days depending on the virus dose and
the route of inoculation (Mebus et al. 1983).
PATHOGENESIS
Generally ASFV is spread in domesticated pigs via oral
and nasal routes of dissemination and exposure (Col-
grove et al. 1969; Plowright et al. 1968). However, pigs al-
so can be infected by a number of other routes, including
tick bites (Plowright et al. 1969), cutaneous scarification,
and intramuscular, intravenous, subcutaneous, and in-
traperitoneal injection (McVicar 1984).
Infection usually starts in monocytes and
macrophages of tonsils and mandibular lymph nodes
and then spreads through the draining lymph nodes and
blood to the target organs (lymph nodes, bone marrow,
spleen, lung, liver, and kidney), which are the main sites
of secondary replication. The viremia in ASF starts be-
tween 6 and 8 days after infection and lasts for a long
time thereafter.
ASFV is associated with red blood cell membranes
(Quintero et al. 1986) and with platelets (Gomez-Villa-
mandos et al. 1996) and causes hemadsorption (HA) in
affected pigs (Sierra et al. 1991). The pathogenesis of he-
morrhages observed in the acute form is believed to be
phagocytic activation of endothelial cells aggravated by
virus replication in the same cells in the final stages of
the disease. In the subacute form hemorrhages are due
mainly to an increase of vascular permeability (Gomez-
Villamandos et al. 1995). The pathogenesis of the lym-
phopenia in the acute form has been related to apoptosis
of lymphocytes mainly on the T area of lymphoid organs
(Carrasco et al. 1996), but there is no evidence of virus
replications in T or B cells (Minguez et al. 1988; Gomez-
Villamandos et al. 1995).
The subacute form is characterized by a transitory
5.2. Enlarged and darkened
spleen from acute ASF.
thrombocytopenia (Gomez-Villamandos et al. 1996).
The alveolar edema observed in the last stages of the
acute and subacute forms of ASF (and the main cause of
death) is a consequence of activation of pulmonary in-
travascular macrophages (Sierra et al. 1990; Carrasco et
al. 1996).
LESIONS
A wide variety of lesions have been observed in ASF, de-
pending on the virus virulence. The acute and subacute
forms are characterized by extensive hemorrhages and
lymphoid tissue destruction. Conversely, lesions may be
minimal or absent in the subclinical and chronic forms
(Mebus et al. 1983; Gomez-Villamandos et al. 1995).
Gross Lesions
The main gross lesions are in spleen, lymph nodes, kid-
neys, and heart (Sanchez Botija 1982). The spleen may
be darkened, enlarged, infarcted, and friable (Fig. 5.2).
Sometimes lesions are large infarcts with subcapsular he-
morrhages. Lymph nodes are hemorrhagic, edematous,
and friable (Fig. 5.3). They often look like dark-red
hematomas. Because of congestion and subcapsular he-
morrhage, cut sections of affected lymph nodes some-
times have a marmoreal appearance. Kidneys usually
have petechial hemorrhages on the cortical (Fig. 5.4) and
cut surfaces, as well as in the renal pelvis. An intense hy-
dropericardium with serohemorrhagic fluid is present in
some cases. Petechial and ecchymotic hemorrhages can
be observed in epicardium and endocardium. Other le-
sions can also be observed in acute ASF, such as serohe-
morrhagic fluid in the abdominal cavity, with edema and
hemorrhages throughout the alimentary tract. Conges-
tion of the liver and the gall bladder can be observed, as
well as petechial hemorrhages in the mucosa of the uri-
 CHAPTER 5 AFRICAN SWINE FEVER Sanchez-Vizcaino
nary bladder. Hydrothorax and petechial hemorrhages
of the pleura are frequently found in the thoracic cavity,
and lungs are usually edematous. Intense congestion is
observed in the meninges, chorioid plexus, and en-
cephalon (Arias et al. 1986).
For the last fifteen years, the most predominant form
of ASF outside Africa has been the subacute form, which
is similar to the acute form except for milder lesions. The
subacute form is characterized by large hemorrhages in
lymph nodes and kidneys. The spleen is enlarged and he-
morrhagic. Congestion and edema can be observed in
lungs and in some cases an interstitial pneumonia has
been found (Arias et al. 1986).
Microscopic Lesions
In the acute form of ASF histopathological lesions are
present in blood vessels and in lymphoid organs. These
lesions are characterized by hemorrhages, microthrom-
bosis, and damage of the endothelial cells with accumu-
lations of dead cells in the subendothelium (Gomez-Vil-
lamandos et al. 1995). Hemorrhagic splenomegaly,
characteristic of the acute and subacute forms, is a con-
sequence of a loss of splenic architecture caused by the
viral replication and resulting necrosis of splenic fixed
97
5.3. Lymph nodes from a nor-
mal pig (left), a pig with subacute
ASF (center), and a pig with acute
ASF (right).
5.4. Kidney from a pig with
acute ASF showing numerous
petechiae on the cortical surface.
macrophages (Carrasco et al. 1997). The lymphoid tissue
destruction in the acute form is mainly observed on the
T area of the lymphoid organs, but no evidence of virus
replications has been observed (Minguez et al. 1988 and
Carrasco et al. 1996). The chronic form of ASF is charac-
terized mainly by alterations in the respiratory tract.
These include fibrinous pleuritis, pleural adhesions,
caseous pneumonia, and hyperplasia of the lymphoretic-
ular tissues. Fibrinous pericarditis and necrotic skin le-
sions are also common (Moulton and Coggins 1968;
Arias et al. 1986).
DIAGNOSIS
Laboratory studies are essential to establish a definitive
diagnosis of ASF due to the great similarity of ASF clini-
cal signs and lesions with those of other hemorrhagic pig
diseases (e.g., hog cholera, erysipelas, and septicemic sal-
monellosis). As in other virus diseases, the laboratory di-
agnosis of ASF can be based on the demonstration of in-
fectious virus, viral antigens, viral DNA, or specific
antibodies. A wide variety of laboratory tests are avail-
able for detecting either ASFV or homologous antibodies
(Sanchez-Vizcaino 1986).
98 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
ASFV Detection
Several techniques have been adapted for the identifica-
tion of ASFV. Some of them, such as the complement fix-
ation test (Cowan 1961), immunoperoxidase staining
(Pan et al. 1978), enzyme-linked immunosorbent assay
(ELISA) sandwich (Wardley and Wilkinson 1980), elec-
tron microscopy, and, more recently, DNA-hybridization
methods (Pastor and Escribano 1990), are, for one rea-
son or another, not practical for routine diagnosis
(Sanchez-Vizcaino 1986).
At present, the most convenient, safe, and frequently
used techniques are direct immunofluorescence (DIF)
(Bool et al. 1969) and HA (Malmquist and Hay 1960).
DIF is the test recommended to be used first when ASF is
suspected. It is based on the demonstration of viral anti-
gens in impression smears or frozen tissue sections from
spleen, lung, lymph node, or kidney, reacted with a con-
jugated immunoglobulin against ASFV. It is a fast and
economical test with high sensitivity for the acute form
of ASF. However, for subacute or chronic forms, DIF has
a sensitivity of only 40%. This decrease in sensitivity
seems to be related to the formation of antigen-antibody
complexes in tissues of an infected pig, which, in turn,
block the reaction between ASFV antigens and the ASF
conjugate when such tissues are tested in the laboratory
(Sanchez-Vizcaino 1986).
HA, because of its sensitivity and specificity, is useful
under the widest range of circumstances. It should be
performed to confirm any new outbreak, and especially
when other tests are negative. In the laboratory the HA
test is based on the attachment of erythrocytes to the ex-
ternal (cytoplasmic) membrane of ASFV-infected
porcine macrophages cultured in vitro. Typically the
erythrocytes form a rosette around the infected
macrophage before the appearance of ASFV-induced cy-
topathic effects (Fig. 5.5) (Malmquist and Hay 1960).
5.5. Hemadsorption phenome-
non: the attachment of erythro-
cytes to macrophages infected with
ASFV.
However, even though HA is the most sensitive test
for ASFV identification, it is important to point out that
a few field strains of ASFV have been isolated that induce
cytopathic effects in macrophages but do not induce HA
(Sanchez Botija 1982). These strains were identified us-
ing DIF sediments of these cell cultures.
The exposure of susceptible pigs to tissues from sus-
pected cases of ASF may be used as an additional testing
procedure when the recognition of ASF is especially crit-
ical (e.g., the possible introduction of ASFV into an AS-
FV-free area). Two groups of pigs are necessary: one vac-
cinated against hog cholera and one not vaccinated and
susceptible to hog cholera (because ASF and hog cholera
are easily confused on the basis of clinical signs and le-
sions). After the inoculation of both groups with suspect
samples, they should be observed daily for clinical signs,
and blood samples should be collected and tested for AS-
FV by HA and for homologous antibody.
ASFV-DNA Detection
The detection of a wide range of ASFV isolates by poly-
merase chain reaction (PCR) has been made possible by
using primers from a highly conserved region of the viral
genome. PCR has been used to identify ASFV isolates
from all of the known virus genotypes, including those of
low virulence and those that fail to induce HA. It has
been particularly useful for identifying viral DNA in tis-
sues unsuitable for other diagnostic tests (e.g., tissues in
which the virus had been inactivated or further degraded
by tissue changes such as putrefaction). It is an excellent
and rapid technique to be included in ASF diagnostic
testing.
Antibody Detection
The study of antibodies to ASFV is especially important
for two main reasons. First, a better understanding of
 CHAPTER 5 AFRICAN SWINE FEVER Sanchez-Vizcaino
the humoral immune response may contribute to the de-
velopment of an efficacious vaccine. Second, detection of
antibodies is a useful tool in the diagnosis of ASF. Spe-
cific ASFV IgG is detectable in blood from the 6th to 8th
day after inoculation and for a long time, even years,
thereafter. The early appearance and subsequent persis-
tence of antibodies is the reason they are so useful in
studying subacute and chronic forms of the disease. For
the same reason they play an important role in testing
strategies that are part of eradication programs (Arias
and Sanchez-Vizcaino 1992).
Several techniques have been adapted to ASF anti-
body detection. These include complement fixation
(Cowan 1961), indirect immunofluorescence (IIF) (Bool
et al. 1969), immunoelectroosmophoresis (IEOP) (Pan et
al. 1972), ELISA (Sanchez-Vizcaino et al. 1979, 1982), ra-
dioimmunoassay (RIA) (Wardley and Wilkinson 1980),
and immunoblotting assay (IB) (Pastor et al. 1987). IIF,
ELISA, and IB are the most frequently used.
IIF is a fast technique with high sensitivity and speci-
ficity for the detection of ASF antibodies from either sera
or tissue exudates (Sanchez Botija et al. 1970). It is based
on the detection of ASF antibodies which bind to a
monolayer of cell lines infected with a cell culture adapt-
ed ASF virus. The antibody-antigen reaction is detected
by a labeled fluorescein A-protein (Hortiguela and
Sanchez-Vizcaino 1984). Using both IIF and DIF, it is
possible to detect from 85% to 95% of ASF cases (acute,
subacute, and chronic) in less than 3 hours (Sanchez-Viz-
caino 1986).
At present, ELISA is the most useful method for large-
scale ASF serological studies. It is based on the detection
of ASF antibodies bound to the viral proteins by addi-
tion of protein A, which is conjugated with an enzyme
that produces a visible color reaction when it reacts with
the appropriate substrate.
The IB test is a highly specific, sensitive, and easy-to-
interpret technique that has been used successfully as an
alternative method to IIF for confirmation of question-
able ELISA results (Arias and Sanchez-Vizcaino 1992).
Samples that should be collected for ASF laboratory
diagnosis are lymph nodes, kidneys, spleen, lungs,
blood, and serum. Tissues are used for virus isolation
(HA) and viral antigen detection (DIF), blood is used for
virus isolation (HA), and serum and tissue exudates are
used for antibody detection (IIF, ELISA, IB).
TREATMENT
At present, no treatment or effective vaccine against AS-
FV is available. Since 1963, when the first live-attenuated
vaccine was used in Portugal (Manso Ribeiro et al. 1963),
many efforts have been made to develop a satisfactory
vaccine. Inactivated vaccine does not produce any pro-
tection. Live-attenuated vaccine protects some pigs
against challenge with the homologous strain of virus,
99
but some of these pigs become carriers and develop
chronic lesions, the likelihood of which increases when
large numbers of pigs are vaccinated (Manso Ribeiro et
al. 1963; Sanchez Botija 1963b). Other studies have
shown that serum from pigs resistant to homologous
and some heterologous strains of ASFV inhibits (in vit-
ro) infection of cells with different but related heterolo-
gous strains (Ruiz Gonzalvo et al. 1986). However, ASFV-
specific antibodies have never been demonstrated to
neutralize virus in the classic concept of neutralization.
The recent analysis of the complete nucleotide sequence
of ASFV (Yañez et al. 1995) offers new opportunities to
discover the role of various ASFV genes in protective im-
munity. However, the successful eradication of ASF from
Portugal and Spain proved that vaccine is not essential
for an eradication program even where the disease is en-
demic.
PREVENTION
Because ASF is a costly disease and because there are no
effective vaccines for its control, it is especially important
that ASF-free areas be maintained free by preventing the
introduction of ASFV. Retrospective epidemiological
studies have shown that the most frequent source of AS-
FV has been contaminated garbage from international
airports or ports. Therefore, all food leftovers from
planes and ships should be incinerated. In infected Euro-
pean areas such as Sardinia, where the disease is endem-
ic and where mild or inapparent clinical signs have been
recognized, the most important aspects of prevention
are controlling pig movements and implementing exten-
sive serological surveys to detect carrier pigs. In areas of
Africa where ASF is endemic, the most important aspects
of prevention are controlling natural reservoirs, namely,
soft ticks (O. moubata) and warthogs, and preventing
their contact with domesticated pigs. When ASF is sus-
pected for any reason, pig movement should be restrict-
ed and diagnostic tests should be performed immediate-
ly. Moreover, it is important to remember that
low-virulence ASFV strains do not cause signs or lesions
that signal their presence.
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 Blue Eye Disease
6 A. H. Stephano
Blue eye (BE) is a disease of swine associated with infec-
tion by a member of the Paramyxoviridae family. It is
characterized clinically by central nervous system disor-
ders, corneal opacity, and reproductive failure.
In 1980 the disease was reported in central Mexico,
with numerous outbreaks of encephalitis and corneal
opacity in piglets from which a hemagglutinating virus
was isolated (Stephano et al. 1981). This virus was iden-
tified as a serologically distinct (Stephano et al. 1986b)
member of the family Paramyxoviridae (Stephano and
Gay 1983, 1984, 1985a) and was subsequently named BE
paramyxovirus (BEP).
Members of the Paramyxoviridae family also have
been isolated from pigs in other countries. In Japan,
Sasahara et al. (1954) isolated a hemagglutinating virus
from pigs with porcine influenza and encephalitis; this
virus, named hemagglutinating virus of Japan or Sendi
virus, proved to be pathogenic for pigs, producing cen-
tral nervous system, respiratory system, and reproduc-
tive system disorders. In Canada, Greig et al. (1971) iso-
lated a paramyxovirus from the brain of a pig during an
investigation of pigs with nervous signs. This virus was
not able to produce signs and lesions in experimentally
inoculated piglets. In Israel, Lipkind et al. (1986) isolated
a paramyxovirus from nasal swabs of apparently healthy
pigs before slaughter. In the United States, a paramyxo-
like virus was isolated from finishing pigs with respirato-
ry system and central nervous system disease. This virus
was shown to be antigenically related to paramyxovirus
1, 3, and 4b but not to the BEP (Paul et al. 1994).
The first reported outbreak of BE was observed on a
commercial farm with 2500 sows located in La Piedad,
Michoacán, Mexico (Stephano et al. 1982). That year
similar outbreaks were observed on other farms in the
same area, as well as in the states of Jalisco and Guanaju-
ato. From an economic perspective, BE became especial-
ly important in central Mexico, where it was diagnosed
in the states of Nuevo León, Hidalgo, Tlaxcala, Estado de
México, Federal District, Querétaro, Tamaulipas, Puebla,
and Campeche (Stephano et al. 1988b). Serological evi-
dence indicated that BEP was present in at least 16 states
(Fuentes et al. 1992). The disease is still recognized occa-
sionally in central Mexico in the states of Michoacán,
Jalisco, and Guanajuato as cases of nervous system dis-
orders, corneal opacity, reproductive failure in sows, and
orchitis with epididymitis in boars. However, its eco-
nomic impact has been considerably reduced. BE has not
been reported outside Mexico.
Differences in clinical signs became evident during
the first years of the disease. In 1980 mainly piglets were
affected. Mortality and nervous system disorders in pigs
older than 30 days were uncommon. In 1983, severe out-
breaks of encephalitis with high mortality in pigs weigh-
ing 15–45 kg were observed on badly managed farms, al-
ways with concomitant viral and bacterial diseases
(Stephano and Gay 1985b, 1986a). These and subse-
quent observations have indicated that BE is frequently
associated with other infectious diseases. It was not until
1983 that reproductive failure in sows and transient in-
fertility in boars were identified (Stephano and Gay
1984, 1985a). In 1988 severe problems of orchitis, epi-
didymitis, and testiclar atrophy in boars became evident
(Campos and Carbajal 1989; Stephano et al. 1990).
ETIOLOGY
Recently the family Paramyxoviridae was reorganized and
BEP was classified as a member of the subfamily
Paramyxovirinae, genus Rubulavirus (mumps virus)
(Fields et al. 1996). Extensive molecular work has been
carried out in Sweden (Sundqvist et al. 1990; Berg et al.
1991; Sundqvist et al. 1992; Berg et al. 1992). BEP repli-
cates and produces cytopathic effects in a wide variety of
cell cultures and agglutinates erythrocytes of both mam-
malian and avian origin (Stephano and Gay 1985a;
Moreno-Lopez et al. 1986; Stephano et al. 1986b).
Size and Morphology
Electron microscopic examination of BEP revealed parti-
cles similar to other paramyxoviruses, measuring from
135–148 nm to 257–360 nm (Fig. 6.1). The virion is pleo-
morphic but usually more or less spherical; no filamen-
tous forms have been observed. The envelope is covered
with a layer of closely spaced surface projections or
spikes. Nucleocapsids from disrupted virus particles are
frequently seen as single entities with a diameter of 20
nm and a length of 1000–1630 nm (Fig. 6.2) or more
(Stephano and Gay 1985a).
Physicochemical Properties
The infectivity of BEP is abolished by treatment with
ether, chloroform, formalin, and beta propiolactone, but
103
104 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

6.1. Blue eye paramyxovirus particles

showing surface projections by negative-
stain electron microscopy (×108,200).

6.2. Fraction of a nucleocapsid from a dis-

rupted BEP (negative-stain electron mi-
croscopy; ×203,000).
 CHAPTER 6 BLUE EYE DISEASE Stephano
BEP is resistant to actinomycin D. Formalin treatment
abolishes both infectivity and hemagglutinating activity.
BEP is inactivated at 56˚C after 4 hours. Intact virions
have a buoyant density of 1.21 g/mL in sucrose gradients
(Stephano and Gay 1985a).
Biological Properties and Replication
BEP has been grown with cytopathic effect in monolayer
cultures of pig kidney (PK), bovine thyroid, bovine em-
bryo, equine dermis, swine testicle, cat kidney, and baby
hamster kidney–21 and Vero cells. In monolayer cultures
of primary PK cells and cells of an established PK cell
line (PK-15), the cytopathic effects start between 24 and
48 hours after inoculation and are complete between 5
and 7 days. They consist of individual rounded cells, cy-
toplasmic vacuoles, and syncytium formation (Fig. 6.3);
dead cells detach from the culture vessel surface, leaving
small plaques. In PK-15 cells the virus was seen free in cy-
toplasm and within vesicles. Some cells also contained
viral inclusion bodies (Stephano and Gay 1985a;
Stephano et al. 1986a). Cytopathic effects have also been
observed in other types of monolayer cell cultures of
porcine origin (turbinate, choroid plexus), as well as in
105
monolayer cell cultures of bovine (turbinate, kidney, tes-
ticle, skin, palatine, choroid plexus), monkey (GMK),
mink (lung), and human (fetal) origin. Syncytia were ob-
served in BEP-infected monolayer cultures of porcine
turbinate cells and bovine turbinate and kidney cells
(Moreno-Lopez et al. 1986). The chick embryo also sup-
ports BEP replication.
Hemagglutination has been tested in erythrocytes
from chickens, guinea pigs, mice, rats, rabbits, hamsters,
horses, pigs, goats, cats, and dogs, as well as in the four
groups (A, B, AB, and O) of human erythrocytes. Spon-
taneous elution at 37˚C occurred after 30–60 minutes.
The infected PK-15 cells are also positive to hemadsorp-
tion with chicken erythrocytes (Stephano and Gay
1985a; Stephano et al. 1986b).
Serology
Antiserum prepared against paramyxoviruses 1, 2, 3, 4,
6, and 7 and parainfluenza viruses 1, 2, 3, 4a, 4b, and 5
do not affect BEP infectivity (Stephano et al. 1986b).
No morphological, physicochemical, or serological
differences have been observed among different strains
(Gay and Stephano 1987).
6.3. Syncytium formation
in pig kidney-15 monolayer culture
72 hours after infection (Giemsa;
×500).
106 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
EPIDEMIOLOGY
Pigs are the only animals that are known to be affected
clinically by BEP in nature. Experimentally BEP affects
mice, rats, and chick embryos; rabbits, dogs, cats, and
peccaries do not show clinical signs, but rabbits, cats,
and peccaries produce antibodies (Stephano and Gay
1985a; Stephano et al. 1988a; Flores 1991; Arellanes et al.
1994; Cuetero et al. 1995).
Subclinically infected pigs from affected farms are the
main source of BEP infection. The virus seems to be dis-
seminated by nose-to-nose contact between infected and
susceptible pigs.
Transmission through semen has not been estab-
lished, but BEP can be recovered from testis, epididymis,
prostate, seminal vesicles, and bulbo-urethral glands.
The virus may be disseminated by people and vehi-
cles, and possibly by birds and wind; other sources of in-
fection have not been demonstrated.
The disease is self-limiting in closed herds. Sentinel
pigs introduced to the farm 6–12 months after an out-
break remain asymptomatic and do not produce anti-
bodies against BEP (Stephano and Gay 1986b; Stephano
et al. 1986a).
Naturally infected pigs develop antibodies that usual-
ly persist throughout their lives. However, the disease
can recur in susceptible progeny and when susceptible
pigs are added to the herd. Farms with a continuous sys-
tem of production may have cases periodically.
CLINICAL SIGNS
BE may start in any area of commercial breeding units,
but it is usually observed first in the farrowing house,
with central nervous system signs and high piglet mor-
tality. At about the same time, the farmer may observe
corneal opacity in some weaned or fattening pigs
(Stephano and Gay 1985a, 1986a; Stephano et al. 1988a).
The mortality rate increases rapidly and then decreases
within a short time. Once the initial outbreak is over, no
new clinical cases appear unless susceptible pigs are in-
troduced, as has been observed on farms that operate on
a continuous-flow pattern.
The clinical signs are variable and depend mainly on
the age of the pig. Piglets 2–15 days old are most suscep-
tible, and the clinical signs are sudden in onset. Healthy
piglets may suddenly become prostrate, generally in lat-
eral recumbency, or show nervous system signs. But the
disease usually runs a course that starts with fever, a
rough hair coat, and an arched back, sometimes accom-
panied by constipation or diarrhea. These signs are fol-
lowed by progressive nervous signs of ataxia, weakness,
rigidity (mainly of the hindlegs), muscle tremor, and ab-
normal posture, such as a sitting position. Anorexia does
not occur while the piglets can still walk. Some piglets
are hyperexcitable, squealing and showing paddling
movements when handled. Other signs include lethargy
with some involuntary movements, dilated pupils, ap-
parent blindness, and sometimes nystagmus. Some
piglets suffer from conjunctivitis, with swollen eyelids
and lacrimation. Often the eyelids are closed and adher-
ent with exudate. In 1–10% of the affected piglets, either
unilateral or bilateral corneal opacity is present. Fre-
quently, corneal opacity occurs without other signs and
resolves spontaneously. In the first cases observed,
piglets usually died within 48 hours of the appearance of
clinical signs, but in later cases, death occurred after 4–6
days.
Of the litters farrowed during an outbreak, 20–65%
are affected. In these litters the piglet morbidity is be-
tween 20% and 50%, and mortality of affected piglets is
between 87% and 90%; deaths occur for 2–9 weeks fol-
lowing an initial outbreak depending mainly on the sys-
tem of management.
Most of the sows of affected litters are clinically nor-
mal. Some of them show moderate anorexia 1 or 2 days
before the appearance of clinical signs in the piglets, and
corneal opacity has also been observed in the farrowing
house during outbreaks.
In pregnant sows a reproductive failure which lasts
2–11 months (usually 4 months) is observed. During
outbreaks there is an increase in the number of animals
returning to estrus, a reduction in farrowing rate, and an
increase in the weaning-to-service interval and nonpro-
ductive sow days. Also, an increase in stillbirths and
mummified fetuses and, consequently, a reduction in the
number of pigs born alive and later the total pigs born
by farrow are observed (Table 6.1). Abortion is not a fea-
ture, but it is observed in some dams during an acute
outbreak. Gilts and other adult pigs also occasionally de-
velop corneal opacity.
Pigs more than 30 days old show moderate and tran-
sient clinical signs such as anorexia, fever, sneezing, and
coughing. Nervous system signs are less common and
less obvious, but when present they consist of listless-
ness, ataxia, circling, and rarely swaying of the head. As
in piglets, unilateral or bilateral corneal opacity and con-
junctivitis continue to appear on the farm for another
month without other signs. Only 1–4% of pigs older than
30 days are affected and the mortality is generally low.
Outbreaks with 20% mortality and severe central ner-
vous system manifestations have been observed in 15–45
kg pigs; corneal opacity was present in up to 30% of these
pigs (Stephano and Gay 1985b).
Boars, like other adult animals, generally do not show
clinical signs, but mild anorexia and corneal opacity have
been observed. Semen evaluation demonstrated that in
herds infected by the BEP, 29–73% of the boars show
temporary or permanent infertility, with a decrease in
concentration, an increase in abnormalities, and a de-
crease in motility and viability of spermatozoa. In some
boars there is azospermia; the ejaculate becomes clear
 CHAPTER 6 BLUE EYE DISEASE Stephano
Table 6.1. Reproductive parameters affected during BEP infection outbreaks
Parameter
Repeat service (%) Increase
Farrowing rate (%) Decrease
Weaning to first service interval Increase
Bred by 7 days (%) Decrease
Abortions (%) Increase
Sow deaths (%) Increase
Total born/litter Decrease
Pigs born alive/litter Decrease
Stillborn pigs (%) Increase
Mummies (%) Increase
Preweaning mortality (%) Increase
Note: Most parameters reach the figures shown above, but some persist with lower numbers. Nine farms were evaluated.
and resembles coconut water. Some boars develop
swollen testicles. The testis and epididymis become
turgid with marked edema; later some develop a granu-
lar texture and most atrophy (generally unilateral) or be-
come soft and flabby with or without granular epi-
didymitis. Boars with severe lesions lose libido (Campos
and Carbajal 1989; Stephano et al. 1990).
PATHOGENESIS
It has been presumed that the natural infection with BEP
is acquired by inhalation. Experimental intratracheal or
intranasal exposures by either instillation or aerosol are
effective routes of infection and result in clinical signs
and lesions similar to those observed in natural cases. In
previous experiments 1-day-old piglets developed a ner-
vous syndrome at 20–66 hours postinoculation, some
weaned pigs (21–50 days old) developed a nervous syn-
drome at 11 days postinoculation, and pregnant sows de-
veloped reproductive failure when inoculated during
pregnancy. The corneal opacity is also occasionally re-
produced in these cases. The disease was also reproduced
in susceptible pigs placed in contact with experimentally
infected pigs for up to 19 days after experimental infec-
tion (Stephano and Gay 1983; Perez et al. 1988;
Stephano et al. 1988b).
The initial site of BEP replication has not been estab-
lished; however, the nasal mucosa and tonsils have been
suggested based on the fact that infectious virus has been
recovered from nasal and tonsillar swabs. Moreover, vi-
ral antigens are easily detected by immunofluorescence
in these tissues when they are collected from either natu-
rally or experimentally infected pigs. The virus also has
been observed in the axon of neurons. From the initial
site of replication BEP spreads early in the infection to
the brain and lung, and histological lesions and central
nervous system manifestations occur early in the disease.
The brain is the best tissue for isolation and immunoflu-
orescence.
107
Duration
Range (months)
5.8–22.1 2–6
6.0–30.2 1–4
1.0–2.9 2–8
10.0–26.1 2–8
0–4.7 0–2
0.1–0.8 0–2
0–2.1 1–4
0.8–4.1 4–8
4.5–19.6 2–11
6.8–36.2 3–12
32.0–51.8 1–7
The interstitial pneumonia observed suggests dis-
semination through the blood. In experimentally inocu-
lated piglets, the virus could be isolated from the brain,
lung, tonsil, liver, turbinate, spleen, kidney, mesenteric
lymph node, heart, and blood. Brain, lung, and tonsil are
the most common sites for isolation (Stephano et al.
1988a).
The cause of the corneal opacity that is sometimes as-
sociated with BE is unknown. It is not always reproduced
experimentally, but histological lesions such as anterior
uveitis are commonly observed in the cornea (Stephano
and Gay 1986b; Perez et al. 1988). Usually the opacity oc-
curs late in the course of the disease. The histological le-
sions and signs suggest that the opacity is due to an im-
munologic reaction similar to that produced by canine
adenovirus hepatitis. Recent results indicate that the
virus replicates in the cornea since intracytoplasmic in-
clusion body formation in the epithelial cells close to the
corneo-scleral angle was observed in acutely affected
pigs. The corneal opacity has been observed in pigs oth-
erwise clinically normal and resistant to the infection,
and it usually disappears after some period.
It has been suggested that BEP reaches the uterus
through the blood. In pregnant sows this produces re-
productive failure, with embryonic mortality and return
to estrus when infection is in the first one-third of gesta-
tion, and stillbirths and mummified fetuses when infec-
tion is later in gestation (Stephano and Gay 1984).
Experimental inoculation of young hybrid boars
showed that nasal instillation of BEP resulted in inflam-
mation and edema of the testis and epididymis by 15
days after inoculation. By 30 days after inoculation there
was necrosis of the seminiferous tubules and rupture of
the epithelial wall of the epididymis with leakage of sper-
matozoa out from the lumen leading to abscess forma-
tion. Boars sacrificed 80 days after infection showed fi-
brosis and granuloma formation in the epididymis as
well as testicular atrophy (Ramirez et al. 1995).
BEP was recovered from testis, epididymis, prostate,
108 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
seminal vesicles, and bulbo-urethral glands in mature
Mexican hairless boars from 10 to 45 days after inocula-
tion (Vizuet et al. 1996).
The BEP infection has commonly been associated
with pneumonia, especially that due to Actinobacillus
pleuropneumoniae, but experimental inoculation of BEP
prior to infection with Pasteurella multocida types A and
D did not result in colonization of the bacteria in the
lung (Garcia et al. 1988).
LESIONS
Gross Lesions
There are no specific gross changes. In piglets, a mild
pneumonia is frequently observed at the ventral tips of
the cranial lung lobes; also mild gastric distension with
milk, distension of the urinary bladder with urine, and a
slight accumulation of fluid with fibrin in the peritoneal
cavity may be observed. Brain congestion and an in-
crease in the cerebrospinal fluid occur. Conjunctivitis,
chemosis (Fig. 6.4), and varied degrees of corneal opaci-
ty (Fig. 6.5), usually unilateral, are observed. Vesicle for-
mation, ulcers, and queratocono (keratoconus) have been
observed in the cornea, as well as exudate in the anterior
chamber. Pericardial and kidney hemorrhages are occa-
sionally observed (Stephano and Gay 1985a, 1986b).
Boars develop swollen testicles and epididymis, with
increase in diameter and weight due to edema. These
changes are frequently unilateral. Orchitis, epididymitis,
and, later, atrophy of the testicle with or without granu-
lomatous formation in the epididymis occur. Hemor-
rhages are occasionally observed in the tunica albuginia,
6.4. Thickening of the cornea due to edema and
inflammatory cell infiltration.
6.5. Corneal opacity in a 7-day-old piglet.
epididymis, or testis (Campos and Carbajal 1989;
Stephano et al. 1990; Ramirez et al. 1995; Vizuet et al.
1996).
Microscopic Lesions
The main histological changes are located in the brain
and spinal cord. These reflect a nonsuppurative en-
cephalomyelitis affecting mainly the gray matter of the
thalamus, midbrain, and cerebral cortex and include a
multifocal and diffuse gliosis; perivascular cuffing with
lymphocytes (Fig. 6.6), plasma cells, and reticular cells;
neuronal necrosis; neuronophagia; meningitis; and
choroiditis (Ramirez and Stephano 1982). Intracytoplas-
mic inclusion bodies are found in neurons. There are
variations in the severity and extent of these lesions
(Stephano and Gay 1986b; Perez et al. 1988; Stephano et
al. 1988a).
The lungs have scattered localized areas of interstitial
pneumonia characterized by thickened septa with
mononuclear cell infiltration.
Changes in the eye are mainly corneal opacity, char-
acterized by corneal edema, and anterior uveitis. Neu-
trophils, macrophages, or mononuclear cells infiltrate
the iridocorneal endothelium, corneo-scleral angle, and
cornea. The external sheet of the cornea often has cyto-
plasmic vesicles (Fig. 6.7) (Stephano and Gay 1986b;
Perez et al. 1988; Stephano et al. 1988a).
Many affected pigs have a mild tonsillitis with
desquamated epithelium and inflammatory cells in the
crypts.
In boars the affected testes show degeneration and
necrosis of the germinal epithelium. The interstitial tis-
sue shows Leydig cell hyperplasia, mononuclear cell in-
 CHAPTER 6 BLUE EYE DISEASE Stephano 109

6.6. Perivascular cuffing and diffuse gliosis in the cerebellum (H&E; ×225).

6.7. Mononuclear cell

infiltration and edema of the
lamina propria of the cornea,
with cytoplasmic vesicles
in the external epithelium
(H&E; ×225).
110 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
filtration, vascular wall hialinization, and fibrosis. The
epididymis shows vesicle formation, loss of cilia of the
epithelial cells, rupture of the epithelial wall, presence of
spermatozoa in the intertubular space, and severe infil-
tration of inflammatory mononuclear cells with
macrophage phagocytosis of fragmented sperm. Fibrosis
and spermatic granuloma are organized.
DIAGNOSIS
Clinical signs such as encephalitis, corneal opacity, and
reproductive failure in the sow and orchitis and epi-
didymitis in the boar are consistent with a diagnosis of
BE. Additional diagnostic evidence is provided by histo-
logical lesions such as nonsuppurative encephalitis, ante-
rior uveitis, keratitis, orchitis, and epididymitis. The
presence of intracytoplasmic inclusion bodies in neu-
rons and corneal epithelium confirms the diagnosis.
Serological tests such as hemagglutination inhibition
(HI), virus neutralization, microtitration serum-virus
neutralization, and enzyme-linked immunosorbent as-
say (ELISA) have been used diagnostically to identify an-
tibody-positive pigs. HI is the most used test, but false-
positive titers of 1:16 have been detected when chicken
erythrocytes are used or when the BEP antigen is grown
in chicken embryos; therefore, bovine erythrocytes are
recommended. Direct immunofluorescence has been
performed with tissue sections and monolayers, using a
conjugate prepared with rabbit or pig serum (Stephano
and Gay 1985a; Stephano et al. 1988a; Ramirez et al.
1996). Paired serum samples, 15 days apart, are recom-
mended for the diagnosis of the disease.
BEP is easily isolated by adding a homogenated or
triturated suspension of brain or tonsil from infected
pigs to the medium of monolayer cultures of PK-15 cells
or primary porcine kidney cells. The virus-induced cyto-
pathic effect is characterized by syncytium formation.
A differential diagnosis must include other causes of
encephalitis and reproductive disease, especially porcine
reproductive and respiratory syndrome virus and Au-
jeszky’s disease virus. Only BEP produces corneal opaci-
ty (in up to 30% of infected pigs) as well as orchitis and
epididymitis in boars (Stephano and Gay 1985b;
Stephano 1986; Stephano et al. 1988a; Campos and Car-
bajal 1989; Stephano et al. 1990).
TREATMENT
As with most viral diseases of swine, there are no specif-
ic treatments for BEP. Once the clinical signs are evident,
nothing can be done to modify the course of the disease.
Pigs with corneal opacity frequently spontaneously re-
cover whereas pigs with central nervous disturbances
generally die.
Serum from sows who previously had affected litters
has been administered orally to piglets, apparently with-
out effective results. Antimicrobial therapy is commonly
used to treat and prevent secondary infections. Medica-
tion is commonly used for controlling associated respira-
tory problems. Good management, such as maintaining
a healthy environment and providing proper housing
and nutrition, reduces the detrimental effect of the dis-
ease in the herd.
PREVENTION AND CONTROL
Health control programs are the most reliable method of
preventing the introduction of BEP to a farm. Swine pop-
ulations must be established or replaced from a healthy
pig herd. Perimeter fencing; separate load-out areas,
changing rooms, and showers; control of personnel, vis-
itors, and vehicles; control of birds, rats, and mice; waste
removal and dead pig disposal; and a quarantine—all
provide insurance against infection. Serological analysis
should be performed in the replacement animals.
Elimination of BEP from infected herds has been ac-
complished by management practices such as closing the
herd, cleaning and disinfecting, all-in/all-out, elimina-
tion of clinically affected animals (pigs with nervous
signs or infertile boars), and dead pig disposal. These
procedures followed by serological testing, herd perfor-
mance analyses, and introduction of sentinel BEP
seronegative pigs confirm the elimination of BEP
(Stephano et al. 1986b).
To reduce the economic impact of BEP, different
measures have to be taken. Infertile boars, with or with-
out orchitis, must be eliminated. Artificial insemination
should be performed if necessary. Sows and gilts that are
presumed to be pregnant should be carefully observed
for signs of estrus and, if possible, examined by ultra-
sound to confirm pregnancy. It is unknown whether pur-
poseful, and thus quicker, dissemination of the virus in
an infected herd has a tendency to limit the total eco-
nomic impact of the disease.
Various experimental killed-virus vaccines have been
produced in cell monolayer cultures and chicken em-
bryos and suspended in oil or OH2Al3 adjuvant
(Stephano et al. 1992; Martinez et al. 1996).
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112 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
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and Cordoba, D. J. 1992. Eficacia de una vacuna inacti-
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L. 1996. Experimental inoculation in Mexican Hairless
boars with the paramyxovirus of the blue eye disease.
Proc Int Congr Pig Vet Soc 14:127.
 Bovine Viral Diarrhea and
7 Border Disease
P. Vannier and E. Albina
One of the best known and most widely studied viral
pathogens of the pig is hog cholera (HC) virus (HCV), a
pestivirus of the family Flaviviridae. There are, however,
other pestiviruses to which pigs may be exposed, includ-
ing bovine viral diarrhea (BVD) virus (BVDV) and bor-
der disease (BD) virus (BDV).
The discovery by Darbyshire (1961) of an antigenic
relationship between the pestiviruses stimulated further
investigations into natural and experimental BVDV and
BDV infections of swine. The first report of natural in-
fection of swine with BVDV came from Australia in
1964, but BVDV was not isolated from a naturally infect-
ed pig until 1973 (Fernelius et al. 1973). The presence of
BVD-BD antibodies in pig sera interferes with HC eradi-
cation programs. Therefore, it is essential to identify the
origin of any pestivirus antibodies encountered in such
programs. In addition, the teratogenic property of pes-
tiviruses has been well established (Terpstra and
Wensvoort 1988; Vannier et al. 1988; Wensvoort and
Terpstra 1988), and the infection of pregnant sows by
BVDVs and BDVs induces a pathology partially resem-
bling that of congenital HC.
ETIOLOGY
BVDVs and BDVs have been classified together with the
HCVs in the Pestivirus genus, Flaviviridae family (Wen-
gler et al. 1995). Morphological and structural character-
istics of the three pestiviruses are similar (Laude 1979;
Collett et al. 1989). Their genome is single-stranded RNA
of positive polarity. Virions are enveloped and range in
diameter from 25 to 120 nm. This wide range in size is
the result of either the entire particle or only the core be-
ing measured (Hsien-Jue Chu and Yuan Chung Zee
1984). Their density ranges from 1.09 to 1.16 g/cm3 and
is dependent on the type of cell host used for their prop-
agation (Horzinek and Van Berlo 1987; Laude 1979).
BVD was first described in 1946 as an infectious and
contagious bovine disease (Olafson et al. 1946), and
“mucosal disease” was reported in 1953 (Ramsey and
Chivers 1953). Further studies showed that the viruses
responsible for these two conditions were similar. They
have been referred to by most authors as BVDV
(Gillespie et al. 1961). Some isolates induced cytopathic
changes in cell culture, while others were noncytopathic
(Gillespie et al. 1960). The relationship between the cy-
topathic effect and the pathogenesis of the disease has
been investigated. It is now well established that mucos-
al disease occurs only in immunotolerant calves persis-
tently infected with a noncytopathic strain of BVDV and
subsequently superinfected with a cytopathic strain of
BVDV with the similar antigenic properties (Brownlie et
al. 1984, 1987). Infection with the cytopathic strain may
be of endogenous origin (i.e., a cytopathic mutant of the
noncytopathic strain) or it may be the result of exoge-
nous exposure to a strain that, by chance, is antigenical-
ly similar. The third member of the Pestivirus genus is the
causative agent of BD in sheep. This disease, character-
ized by congenital disorders in lambs, was first recog-
nized in 1959 in Great Britain near the border between
England and Wales (Hughes et al. 1959), but the im-
munological relationship of BDV with BVDV was dis-
covered later (Hamilton and Timoney 1972).
The terms BVDV and BDV are used to indicate that
the virus was isolated from either cattle or sheep, al-
though these two viruses cannot be differentiated mor-
phologically or structurally (Laude 1979). There is also
evidence that BVDV can infect sheep and BDV can infect
cattle (Barlow et al. 1980; Terlecki et al. 1980). In pigs,
pestivirus isolates are usually HCV. However, BVDV and
BDV can be isolated from naturally infected pigs (Car-
brey et al. 1976; Terpstra and Wensvoort 1988). More-
over, it has been demonstrated through cross-neutraliza-
tion tests and tests using monoclonal antibodies
(Wensvoort et al. 1989b; Leforban 1990) that, in the
past, BVDV may have been isolated from pigs but misla-
beled as HCV on the basis of tests using polyclonal anti-
bodies only.
Up to now, pestiviruses have been named according
to the species in which they cause disease. As previously
indicated, cross-species transmission within the Artio-
dactyla has been reported for BVDV as well as BDV. The
molecular analysis and the use of a panel of 76 mono-
clonal antibodies revealed a fourth group within the
genus Pestivirus which consists of ovine and bovine iso-
lates (Becher et al. 1995; Paton et al. 1995). The authors
proposed that the taxonomy of pestiviruses be reconsid-
ered and suggested the following: pestivirus type 1 (clas-
113
114 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
sical BVDV strains), pestivirus type 2 (HCV strains), pes-
tivirus type 3 (true BDV strains), and pestivirus type 4
(BVDV-like strains of bovine and ovine origins).
More recently, Harasawa (1996) constructed a phylo-
genetic tree of pestiviruses by analyzing their 5′-untrans-
lated region. His results indicated that the genetic rela-
tionship between BDV and HCV is much closer than that
between genotypes of various BVDVs. Therefore, Hara-
sawa proposed five groups of pestiviruses. Genetic dis-
criminations between pestiviruses actually rely on se-
quence comparison of short fragments of the pestivirus
genome. It would not be surprising that in the future, ad-
ditional subgroups will be identified through sequencing
and analysis of other parts of the genome. Studies on
pestiviruses isolated from wild ruminants or wild boars
could also give interesting new pestivirus genotypes.
EPIDEMIOLOGY
From HC-free countries like Australia, Ireland, Great
Britain, and Denmark, prevalences of BVD antibodies
within the pig population have been described as varying
from 1.6% to 43.5% depending on the age of the pigs and
possibly to some extent on the degree of their contact
with cattle (Holm Jensen 1985). In countries where HC is
present, the situation with regard to BVD antibodies
seems to be about the same. In the Netherlands, even
though up to 15% of slaughtered sows had antibodies
against BVDV, only 13 naturally occurring infections
have been encountered over a 10-year period (Terpstra
and Wensvoort 1988). Cattle are usually regarded as the
source of BVDV infection in pigs. One source of infec-
tion is the feeding of whey or milk to sows, a common
practice in units with dairy farming (Terpstra and
Wensvoort 1988). In some cases, pigs had contact with
cattle recently vaccinated with BVDV (Stewart et al.
1971). In other cases the pigs and cattle were kept in sep-
arate lots and buildings, but personnel and equipment
moved freely between the different farm units (Carbrey
et al. 1976).
Nevertheless, pigs can be infected without any con-
tact with cattle and without being fed with bovine milk
or offal (Terpstra and Wensvoort 1988). The use of con-
taminated modified-live-virus vaccines (HC or Aujeszky’s
disease) or contaminated biological products can be the
source of infection in pigs (Vannier et al. 1988;
Wensvoort and Terpstra 1988). In these cases, ovine or
bovine contaminants are involved in the disorders.
In all probability, persistent infection of congenitally
infected pigs is responsible for the dissemination of
BVDV or BDV within a herd and virus transmission to
other susceptible pregnant sows (Terpstra and
Wensvoort 1988; Vannier et al. 1988). For example, it has
been shown that persistent BDV infection of litters oc-
curs when sows are infected during early pregnancy;
then the fetuses are transplacentally infected and most
piglets show persistent infection and immunotolerance
(Vannier et al. 1988). When the sow is experimentally in-
fected, the litter may comprise a mixture of virus-posi-
tive and antibody-positive pigs. This suggests some vari-
ability in the time at which individual fetuses became
infected (Edwards et al. 1995). The course of the infec-
tion is quite similar to that described after a BVDV infec-
tion in pregnant cows (Baker 1987). Such congenitally
BDV-infected piglets appear to excrete large amounts of
virus, since other young animals kept in contact have a
rapid seroconversion with high antibody titers. Con-
versely, when the pigs are infected after birth, spread of
infection to in-contact pigs under the same experimental
conditions does not occur, which suggests a low or nil ex-
cretion of virus (Vannier et al. 1988).
BVDVs and BDVs can be isolated or detected by im-
munofluorescence from blood, tonsils, spleen, kidneys,
ileum, and lymph nodes. Isolation of virus from ileum
indicates that fecal excretion of the virus is a possibility.
CLINICAL SIGNS
Under Natural Conditions
Natural infection of pigs with BVDV usually occurs with-
out clinical signs. However, in some cases, natural infec-
tion of pig herds with pestiviruses other than HC is asso-
ciated with breeding problems like poor conception,
small litters, and a few abortions. Hyperthermia and
clonic spasms have also been described (Carbrey et al.
1976; Stewart et al. 1977). More recently, in the Nether-
lands and France, signs resembling those of congenital
HC infections were described, namely, an increased
death rate in piglets of up to 2–5 weeks of age which
were born to sows vaccinated 4 months earlier against
HC or Aujeszky’s disease with batches of vaccines acci-
dentally contaminated with a ruminant pestivirus (Terp-
stra and Wensvoort 1988; Vannier et al. 1988). The clini-
cal signs in the piglets included anemia, rough hair coat,
growth retardation, wasting, and congenital tremors.
Conjunctivitis, diarrhea, polyarthritis, petechiae in the
skin, and blue eartips were also observed (Terpstra and
Wensvoort 1988).
Natural infection of sows with BDV often resulted in
repeat-breeding and many litters containing dead and
mummified fetuses. Clinical signs of eyelid edema and
locomotor disorders, sometimes associated with diar-
rhea and arthritis, occurred in a high proportion of
piglets from these sows. The mortality rate up to 2 days
of age varied between 30% and 70% (Vannier et al. 1988).
Under Experimental Conditions
Several trials of BVDV and BDV inoculation of pigs,
mainly pregnant sows, have been done using oral, in-
tranasal, intramuscular, or intrauterine routes with in-
consistent results (Fernelius et al. 1973; Wrathall et al.
1978; Stewart et al. 1980; Mengeling 1988; Leforban et
 CHAPTER 7 BOVINE VIRAL DIARRHEA AND BORDER DISEASE
al. 1990b). The result is mainly dependent on the strain
used and on the stage of pregnancy. The inoculation of
pregnant sows with the NADL strain of BVDV (Gutekun-
st and Malquist 1963) between the 28th and 54th day of
gestation did not result in subsequent transplacental in-
fection of the fetuses (Stewart et al. 1980), but eyelid ede-
ma was seen in a few piglets (Leforban et al. 1990b). The
inoculation of 9–18 kg piglets with the Singer strain of
BVDV (Coria and McClurkin 1978) did not produce dis-
ease, but the virus was recovered from blood and tissues
of inoculated pigs, and antibodies were detected in their
sera after 3 weeks. When later challenged with a virulent
HCV strain, these inoculated piglets developed a severe
disease, but 6 of 7 survived (Stewart et al. 1971). The
same strain used to inoculate fetuses between 41 and 65
days by transuterine injection caused either death or
small fetuses (Mengeling 1988). In another study, groups
of weaner pigs were intranasally inoculated with BVDV
strain OSLOSS/2482. Four weeks later, the animals were
challenged with decreasing doses of HCV. After HCV
challenge, fever occurred in only one animal, while none
of the others reacted clinically. However, most animals
developed viremia (Dahle et al. 1993).
Field BDV strains inoculated into pregnant sows be-
tween the 30th and 34th day of gestation infected the
corresponding fetuses and caused disease characterized
by low body weights and shorter lengths of newborn
piglets (Wrathall et al. 1978). In another experiment
(Leforban et al. 1990b) perinatal death rate was in-
creased; and eyelid edema, increased rectal temperature,
and anemia were observed in survivors during their sec-
ond week of life. Subsequently, growth retardation asso-
ciated with respiratory signs and diarrhea developed in
some pigs which eventually died within 2 months. Pigs
without respiratory and enteritic signs survived and had
normal growth despite a marked snout deformation with
prognathism observed in one of them. Virus was isolat-
ed from blood and organs of all dead piglets but not
from those that survived. When 40-day-old new specific-
pathogen-free pigs were placed in contact with these
transplacentally infected piglets, they did not show any
disease, but they developed a high level of antibody to
BDV which protected them completely against a chal-
lenge with a virulent strain of HCV.
PATHOGENESIS
The ability of BVDV and BDV to establish intrauterine
infection in swine has been well demonstrated by several
authors (Wrathall et al. 1978; Stewart et al. 1980; Van-
nier et al. 1988). These two pestiviruses are embryotoxic,
but they have little or no pathogenicity for pigs infected
after birth. The intensity of the signs is associated with
the stage of gestation: clinical signs are more serious if
the sows are infected during the first third of pregnancy.
Indeed, the most spectacular clinical signs or lesions in
Vannier, Albina 115
fetuses or piglets are observed when the sows are infect-
ed at 25–41 days of gestation (Mengeling 1988; Leforban
et al. 1990b).
Under experimental conditions, most of the congen-
itally infected piglets showed persistent infection and im-
munotolerance. After the disappearance of the maternal
passively acquired antibodies, no active immunity was
detected in the serum of a majority of piglets. The virus
could be isolated from slaughtered piglets and was ex-
creted by some of them, as evidenced by the contamina-
tion of young pigs placed in contact with them (Vannier
et al. 1988). In some experimental infections of pregnant
sows with BDV, for unknown reasons, the onset of the
clinical signs in the piglets was delayed until 13–14 days
after birth. It can be assumed that colostral antibodies
ingested by the piglets blocked the multiplication of the
pestiviruses or delayed disease in transplacentally infect-
ed piglets (Vannier et al. 1988; Leforban et al. 1990b).
However, the delayed postnatal clinical expression of the
infection in piglets when sows are infected during early
pregnancy remains unexplained.
The pathogenicity of BVDVs and BDVs seems vari-
able depending on strains used and on experimental con-
ditions. BDV seems to be more consistently pathogenic
for fetuses, whereas variable results are obtained with
BVDV strains. The Singer strain, adapted to replicate in
porcine cells, and strain 87/6 can infect and cause death
of porcine fetuses, whereas the NADL strain does not in-
duce a real disease in the piglets (Mengeling 1988; Lefor-
ban et al. 1990b; Edwards et al. 1995).
LESIONS
Pigs infected after birth with BVDVs or BDVs have very
mild or no lesions. Stewart et al. (1971) saw hyperemia of
the small intestine of a pig euthanized and necropsied 11
days after it had been placed in contact with NADL-
strain-infected calves. Carbrey et al. (1976) identified a
transient leucopenia during the first week following ex-
perimental infection of pigs with a pig isolate of BVDV.
The prenatal infection of fetuses by diaplacental
transmission of the viruses from the infected sow is fol-
lowed by consistent pathological disorders in fetuses or
piglets. In the 13 naturally occurring BVD outbreaks in
the Netherlands, chronic gastroenteritis and septicemia
with hemorrhages in lymph nodes, epicardium, and kid-
neys were the most consistent lesions reported. The in-
flammation of the digestive tract was frequently charac-
terized by catarrh, hypertrophy, or ulceration of the
mucosa. Necrotic tonsillitis, icterus, polyserositis, pol-
yarthritis, and atrophy of the thymus were also noted
(Terpstra and Wensvoort 1988).
When a pig isolate of BVDV was given to gilts be-
tween 42 and 46 days of pregnancy, significant micro-
scopic lesions were observed in the leptomeninges and
the choroid plexus of the fetus as collections of lympho-
116 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
cytes and histiocytes and cellular accumulation in the
vascular adventitia and perivascular spaces (Stewart et
al. 1980).
After experimental inoculation of 34-day-pregnant
sows with BDV, postmortem dissection of newborn
piglets revealed cerebellar hypoplasia in 9 out of 19 live-
born piglets, associated with a small meningocoele in 1
of the 9 (Wrathall et al. 1978). The French-isolated Avey-
ron strain of BDV (Chappuis et al. 1984) inoculated into
30-day-pregnant sows was responsible for lesions in lym-
phoid tissues in some piglets: marked hemorrhages in
lymph nodes and other lymphoid tissues were found in
stillborn fetuses or in piglets that died shortly after birth.
The histological examination of lymph nodes, spleen,
and tonsil revealed marked subacute inflammatory le-
sions characterized by accumulations of lymphocytes,
plasmocytes, and eosinophilic polymorphonuclear
leukocytes, numerous secondary follicles, increased pop-
ulations of reticulocytes, and lymphoid hypoplasia with
pyknosis and karyorrhexis. Thymus, liver, and nervous
tissues were normal (Leforban et al. 1990b).
DIAGNOSIS
Isolation of the virus may be achieved from tissues sub-
mitted for HC diagnosis (i.e., tonsil, lymph node, spleen,
and heparinized blood). In HC-free countries BVD and
BD must be considered as differential diagnoses of HC
and all suspect cases of HC should be tested for BVDVs
and BDVs.
The diagnosis can be readily achieved using immuno-
fluorescence or immunoenzymatic tests on cryostat sec-
tions of tissues. Isolation of the virus in pestivirus-free
cell culture can also be used. Cells can be of either
porcine or ruminant origin. If BVDV or BDV is isolated
from pigs, it is reported to grow better with a higher titer
in cells from ruminant origin than in porcine cells
(Wensvoort et al. 1989a). However, the definitive identi-
fication of an isolate can only be done by using a panel of
monoclonal antibodies able to link specifically to rumi-
nant pestiviruses or to HCV. Three types of monoclonal
antibodies are used for this purpose: those that detect all
strains of pestivirus, including HCV; those that detect
HCV only; and those that detect ruminant pestivirus
strains only (Peters et al. 1986; Bolin et al. 1988; Edwards
et al. 1988; Hess et al. 1988; Wensvoort et al. 1989b; Pa-
ton et al. 1995).
Molecular genetic probes for the detection of pes-
tiviruses were generated from genomic RNA of HCV af-
ter synthesis of cDNA and cloning. A panel of probes
was selected according to their place on the viral
genome—as determined by sequencing and comparison
with the BVDV sequence. This panel seems to be able to
differentiate between pestiviruses (Cruciere et al. 1991;
Schelp et al. 1991). A reverse transcriptase-polymerase
chain reaction (RT-PCR) allowed differentiation between
pestiviruses by the expected size of the amplified frag-
ments. The threshold of sensitivity was 100 TCID50
(median tissue culture infectious doses) for HCV and 1
TCID50 for BVDV. The RT-PCR provides a rapid and sen-
sitive diagnostic tool for the detection and differentia-
tion of HCV from ruminant pestiviruses (Wageck Canal
et al. 1996).
Since pestiviruses share common antigenic struc-
tures (or patterns), the serological tests for the detection
of antibodies to HCV also detect antibodies to ruminant
pestiviruses. The practical importance of this is that the
presence of ruminant pestivirus antibodies in pig sera
very often causes a false-positive result in serological sur-
veys for HC (Holm Jensen 1985).
These cross-reactivities are causing difficulties in
eradication and epidemiosurveillance programs for HC.
However, the differentiation between antibodies to HCV
and antibodies to ruminant pestivirus can be done by the
neutralization test (Holm Jensen 1981) or the ELISA test
(Leforban et al. 1990a) by comparing the antibody titers
to the two viruses (i.e., HCV and ruminant pestivirus) or
through a monoclonal-antibody-based HC ELISA that is
able to specifically detect antibodies to HCV while ignor-
ing antibodies to other pestiviruses (Wensvoort et al.
1988).
PREVENTION
To prevent the infection of pigs by BVDVs or BDVs, it is
necessary to prevent direct or indirect contact with cattle
or sheep. Also, because natural infection with BVDV of-
ten occurs when pigs are fed cow’s milk or bovine offal,
such additions to pig feed should be avoided.
The biological risk is also important when live-virus
vaccines are used. Indeed, the cells used for multiplica-
tion of master seed virus to prepare vaccine batches can
be contamined by BVDVs or BDVs. HC and Aujeszky’s
disease vaccines were contaminated by a pestivirus
(probably BDV) because secondary lamb kidney cells
were used to multiply the vaccinal strains (Wensvoort
and Terpstra 1988; Vannier et al. 1988).
Thirty-five of 158 tested bovine kidney or testis pri-
mary cells were found to be contaminated by BVDV
(Wellemans and Van Opdenbosch 1987). For this reason,
the use of primary or secondary cells to prepare modi-
fied-live-virus vaccines has to be prohibited. Moreover,
both bovine and nonbovine cell lines might be infected,
and all cell lines have to be controlled very carefully in re-
gard to pestiviruses (Wellemans and Van Opdenbosch
1987; Potts et al. 1989). The main source of contamina-
tion for cells is the bovine serum that is added to the nu-
trient medium. The high prevalence of BVDV in the
world and the existence of persistently infected calves
and bovine fetuses increase the risk of contamination of
bovine serum. Rossi et al. (1980) reported that up to 62%
of examined batches of nonirradiated bovine fetal sera
might be found positive for BVDV. Therefore, the sys-
tematic test and treatment of bovine serum and of bio-
 CHAPTER 7 BOVINE VIRAL DIARRHEA AND BORDER DISEASE
logical products used for the preparation of vaccines are
firmly recommended.
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8 Porcine Circovirus
P. D. Lukert and G. M. Allan
In 1982 a novel virus, subsequently named porcine cir-
covirus (PCV), was identified by Tischer et al. (1982) as a
contaminant of a porcine kidney cell line (PK-15, ATCC-
CCLL33). It was found to contain a single-stranded, cir-
cular, DNA genome and to be smaller than other DNA
viruses. Largely on the basis of these properties it has re-
cently been placed in a new family of DNA viruses called
Circoviridae by the International Committee on Taxono-
my of Viruses (Lukert et al. 1995). Serological surveys in
Germany (Tischer et al. 1982), Canada (Dulac and Af-
shar 1989), New Zealand (Horner 1991), Great Britain
(Edwards and Sands 1994), Northern Ireland (Allan et al.
1994b), and the United States (Hines and Lukert 1995)
indicated widespread infection with PCV in adult pigs in
all of the countries. It is likely that the virus is ubiquitous
throughout the world.
Hines and Lukert (1994) reported PCV as a cause of
congenital tremors in newborn pigs, and more recently,
Canadian and Northern Ireland workers (Clark 1997) as-
sociated PCV with a new disease called postweaning mul-
tisystemic wasting syndrome (PMWS) in weanling pigs.
The infectious nature of congenital tremors has been
known for many years, but PMWS appears to be an
emerging disease in swine. If the association of PCV to
these clinical diseases is confirmed, this virus would rep-
resent a very significant pathogen of swine.
8.1. Electronmicrograph of
negatively stained 17 nm virions of
PCV from a PK-15 cell culture. Bar
represents 100 nm.
ETIOLOGY
PCV is a member of the Circoviridae family. Its mean di-
ameter is 17 nm (Fig. 8.1) and its buoyant density in CsCl
is 1.33–1.34. It resists inactivation when exposed to an
acidic environment (pH 3), chloroform, or high temper-
atures (56˚C and 70˚C) (Allan et al. 1994b). Its entire sin-
gle-stranded, circular, DNA genome comprising 1759
bases has been cloned and sequenced (Buhk et al. 1985;
Meehan et al. 1997).
PCV replicates in some porcine cell lines and is de-
pendent upon cellular proteins expressed during the S-
phase of the cell cycle (Tischer et al. 1982, 1987). Tisch-
er et al. found that viral replication was enhanced
following treatment of cell cultures with d-glucosamine,
resulting in an increase of up to 30% in the number of
cells containing PCV antigens. The virus does not cause
detectable cytopathic effects (CPE) in cell culture, and vi-
ral-infected cells are detected using immunostaining
techniques (Fig. 8.2). Persistent infections of cell cul-
tures with PCV, notably PK-15 cell lines, have been
recorded. Allan et al. (1994a) have demonstrated replica-
tion of PCV in monocyte/macrophage cultures derived
from porcine bone marrow, peripheral blood, lung wash-
ings, and lymph nodes (Fig. 8.3). Mononuclear cell cul-
tures derived from the peripheral blood of a bovine were
119
120 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
8.2. PCV-infected cells in PK-15 cell
cultures detected using a monoclonal
antibody to PCV in an IIF assay.
8.3. Porcine macrophage cultures,
inoculated with PCV and immuno-
stained 48 hours after infection with
a monoclonal antibody to PCV using
an IIF test. Note PCV replication in
some cells.
also susceptible to infection with PCV, but not similar
cultures from sheep or humans. McNeilly et al. (1996)
studied the effect of PCV infection on porcine alveolar
macrophage immune function in vitro. They found no ef-
fect on the expression of Fc and complement receptors
or phagocytosis. They did observe an up-regulation of
MHC class I antigen at 4 days after infection and a re-
duction in the number of cells expressing MHC class II
antigen at 8 days after infection. A significant reduction
in macrophage-mediated, mitogen-induced lymphocyte
proliferation was also observed following PCV infection.
Such results could indicate that PCV infections may in-
terfere with normal immune function. Two other cir-
coviruses, chicken anemia virus (CAV) and psittacine
beak-and-feather disease virus (PBFDV), have been asso-
ciated with immune dysfunction (Adair et al. 1991; La-
timer et al. 1992). The genome of PCV has either six
(Bukh et al. 1988) or seven (Meehan et al. 1997) overlap-
ping open reading frames (ORFs) and an absence of in-
tergenic regions. The largest predicted ORF encodes a 36
kDa protein which exhibits high similarity with the pu-
tative replication-associated protein of plant circoviruses
and geminiviruses. A similar ORF region has been iden-
tified in the genome of PBFDV. Another animal cir-
covirus, CAV, has no significant nucleic acid or protein
similarities (Meehan et al. 1997).
EPIDEMIOLOGY
As previously stated, serological surveys in several coun-
tries indicated that PCV infections are widespread and
the seropositive incidence of PCV in most herds ranges
from 20% to 80%. It is quite likely that the virus is present
worldwide. Allan et al. (1994b) performed a longitudinal
survey of PCV antibody profiles in sera from two litters
of piglets in a PCV-infected breeder-finisher herd in
Northern Ireland. They found that maternally derived
antibody to PCV disappeared at 8–9 weeks after birth
and serum antibody reappeared at 13–15 weeks, indicat-
ing exposure of the animals to PCV challenge around
11–13 weeks of age. This corresponded to the movement
of pigs to the fattening unit within the farm.
 CHAPTER 8 PORCINE CIRCOVIRUS Lukert
Tischer et al. (1982) and Allan et al. (1994b) were un-
able to detect PCV antibodies in sera from cattle, sheep,
chickens, turkeys, goats, mice, rabbits, or humans using
indirect immunofluorescence microscopy (IIF). In a later
study by Tischer et al. (1995) using enzyme-linked im-
munosorbent assay (ELISA) and IIF, a low level of PCV
antibody was reported in 30% of human sera, 12–69% of
mouse sera, and 35% of cattle sera. They suggest that this
reactivity reflects infection of other species with a cir-
covirus related to PCV. These findings have yet to be con-
firmed.
CLINICAL SIGNS
The discussion of clinical signs will relate to two diseases
that have recently been associated with PCV infections:
infectious congenital tremors and PMWS. Earlier studies
with experimental infections of seronegative swine of
several ages with PCV failed to induce clinical disease
(Tischer et al. 1986; and Allan et al. 1995). Tischer et al.
(1986) were able to reisolate virus from infected pigs,
and Allan et al. (1995) demonstrated the organs and tis-
sues where the virus replicated using immunostaining.
Both of these studies used PCV that originated in the PK-
15 (ATCC-CCLL33) cell line, and the possibility exists
that this is an attenuated strain of PCV since it may have
resided in this cell line for a large number of cell and
virus generations.
Infectious Congenital Tremors
The clinical signs of congenital tremors are highly vari-
able and the tremors vary from mild to severe. The num-
ber of pigs affected within a litter may also vary consid-
erably. The severe tremors can cause death during the
first week of life because of the inability to nurse; pigs
die of starvation. Pigs that live for a week usually survive,
and most will recover by 3 weeks. The tremors are bilat-
eral, affecting the skeletal muscle, and the tremors sub-
side when the pigs are recumbent or asleep. Tremors may
be initiated or accentuated by external stimuli such as
sudden noises or chilling. Some producers report that
some pigs never fully recover and continue to shake
throughout the growing and fattening period. The affect-
ed litters usually are from young breeding stock recently
introduced to a farm, which would indicate exposure of
seronegative breeding stock to the virus at a critical stage
of gestation.
PMWS
PMWS primarily affects pigs between 6 and 8 weeks of
age and rarely affects suckling pigs. It has been diag-
nosed in porcine reproductive and respiratory syndrome
(PRRS) positive and negative herds but the diagnosis of
PMWS is complicated in PRRS-positive herds by sec-
ondary infections. The clinical signs include weight loss,
emaciation, tachypnea, dyspnea, and jaundice. Less
121
common signs are diarrhea, coughing, and central ner-
vous system disturbances. Morbidity is usually low, with
mortality high in clinical cases. The morbidity and mor-
tality rates in postweaning pigs have been reported to be
as high as 50% in some swine populations (Harding
1997).
PATHOGENESIS
Tischer et al. (1986) reported the first experimental
pathogenesis study with PCV. They infected 6 nine-
month-old seronegative pigs with virus purified from PK-
15 infected cells. They recovered virus from nasal swabs
from 3–6 days postinfection and from feces 13–15 days
postinfection. A noninfected contact pig became infect-
ed, and virus was recovered from the same anatomical lo-
cations, indicating lateral transmission of the virus. No
clinical or histologic changes were observed. Experimen-
tal infections of 4 one-day-old pigs also failed to produce
signs of disease. Antibody to PCV first appeared 1 week
after exposure and increased up to 5 weeks after infec-
tion. The IIF antibody titers ranged from 1:40 to 1:160 at
the peak of the response.
Allan et al. (1995) infected colostrum-deprived, one-
day-old pigs and the infection was followed by a combi-
nation of virus isolation and immunofluorescent (IF)
staining of cryostat sections of organs and tissues. The
virus was found to replicate in several lymphoid tissues
(thymus, spleen, and the mesenteric, bronchial, and
retropharyngeal lymph nodes), nasal mucosa, lung, and
small intestine, and it was associated with
macrophage/monocytes, histocytes, and thymic
macrophages (Figs. 8.4 and 8.5). The virus was not de-
tected in kidney, brain, or liver.
Allan et al. (1995) also reported a survey of a large
number of sera and tissues from fetuses involved in out-
breaks of reproductive disorders in Northern Ireland.
No antibody to PCV was detected in any sera from fetus-
es; however, two isolations of PCV were made from still-
born pigs. They concluded that the absence of antibody
to PCV in fetal sera suggests that transplacental infection
would be either extremely rare or occurs prior to fetal im-
munocompetence.
Hines (1994) isolated PCV from primary kidney cell
cultures derived from a pig with congenital tremors. The
isolate was used to infect four seronegative sows in the
third trimester of gestation. They were infected with
5000 TCID50 (median tissue culture infectious doses) of
PCV by intranasal/oral and subcutaneous routes. The
majority of pigs that were delivered from these sows ex-
hibited tremors which persisted for 2–3 weeks. These
tremors were milder than those of the pig from which
the virus was isolated. The virus was reisolated in cell cul-
tures from the kidney, small intestine, cecum, and colon
of five pigs from the experiment with congenital
tremors. The virus was visualized by immunostaining of
122 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
8.4. IIF staining of cryostat section,
using monoclonal antibodies of spleen
from a pig inoculated with PCV and
sacrificed 4 days after inoculation.
Note foci of PCV antigen in spleen
cells.
8.5. IIP staining of tissue section of
thymus from a pig inoculated with PCV
and sacrificed 8 days after infection.
Note immunostaining of PCV antigen
throughout the section.
all of the same tissues plus the mesenteric lymph nodes
and liver.
Gustafson and Kanitz (1974) had previously de-
scribed transmission of a virus causing congenital
tremors. The electron micrograph of the virus that they
isolated (Gustafson 1981) was similar to PCV, and like
PCV it did not cause a CPE in cell culture. They reported
that the virus could induce tremors when sows were ex-
posed at several different stages of gestation.
Detailed studies on the pathogenesis of PMWS await
a definitive identification of its cause and a better under-
standing of what is needed to consistently produce the
associated signs and lesions.
LESIONS
All of the pathogenesis studies with PCV have failed to
report either macroscopic or microscopic changes in or-
gans or tissues from experimentally infected swine. The
lesions normally found with congenital tremors and with
PMWS will be discussed separately.
Congenital Tremors
The only reported lesion associated with severe cases of
congenital tremors is retardation of myelin deposition in
the spinal cord. Lamar (1971) reported lack of myelina-
tion on nerves in the spinal cord from 1 to 13 days of age
in affected pigs. There are no gross lesions in pigs with
congenital tremors. The PCV antigens have not been
demonstrated in the cells of the spinal cord nor has the
virus been isolated from these tissues.
PMWS
The gross and histopathologic lesions of PMWS have
been described by Clark (1997) and Daft et al. (1996).
Carcasses of pigs with PMWS are in poor condition and
display varying degrees of muscle wasting. The skin
shows moderate pallor and is icteric in about 20% of the
cases. All lymph nodes are enlarged three to four times
and are homogeneously white on cut surfaces.
Lungs are diffusely noncollapsed and are heavy and
firm or rubbery. Lung surfaces are mottled with gray-tan
lobules interspersed with normal yellow to pink lobules.
 CHAPTER 8 PORCINE CIRCOVIRUS Lukert
In severe cases alveolar hemorrhages are indicated by
large patches of dark red or brown lobules. Gray-red at-
electatic or consolidated areas are common in cranial
and middle lung lobes.
The liver is grossly normal in half the cases, and the
rest show varying degrees of mottling with mild to mod-
erate liver atrophy. The interlobular connective tissue is
prominent, and in severe cases, all lobules are smaller
and more prominent than normal. Spleens are usually
enlarged and meaty and noncongested on cut surfaces.
In half the cases the kidneys are spotted with white
foci visible on the subcapsular surfaces. All kidneys with
visible lesions are enlarged and pale and may be up to
five times normal size due to edema. There may be mot-
tled areas in the cecum, and spiral colon mucosa may be
hyperemic and petechiated.
Histopathologic changes are found in the lungs, kid-
neys, liver, pancreas, and all lymphoid tissues. The com-
mon finding in all of the tissues is lymphocytic-histiocyt-
ic infiltrations. Lungs exhibit partial to complete
sloughing of epithelium with fibroplasia. Lymphoid or-
gans and tissues show loss of B-cell follicles and expan-
sion of T-cell areas. Kidneys may show cortical and
medullary atrophy and edema of the intertubular con-
nective tissues. Glomeruli are not involved. The livers
show mild to severe hepatocyte necrosis, and existing he-
patocytes are swollen.
Electron microscopy and immunoperoxidase stain-
ing indicate that PCV is involved in lesions found in all
organs.
DIAGNOSIS
The diagnosis of PCV infection can be made by isolation
of the virus in primary pig kidney cell cultures or in per-
manent cell lines of pig kidney. The presence of virus is
detected using immunostaining. Both IIF and indirect
immunoperoxidase staining have been used. Detection
of a rising titer of PCV antibodies would also indicate a
recent infection. Congenital tremors is recognized by the
clinical signs and ruling out either genetic or chemical
causes of the tremors. As the association of PCV with
congenital tremors and PMWS becomes more substanti-
ated, the use of immunofluorescence and in situ hy-
bridization to detect virus antigen or nucleic acid in tis-
sue and organs will be available for diagnosis.
TREATMENT
There are no known treatments for PCV infections. Pigs
severely affected with congenital tremors may have a
greater survival rate if help in nursing is provided by the
caretaker.
123
PREVENTION
There are no vaccines available and none have been ex-
perimentally tried. When PCV was shown to cause
tremors, many in the swine industry believed that the
condition was not economically severe enough to war-
rant the development of a vaccine. The emergence of
PMWS may change this attitude in the future, especially
in those areas where the syndrome is apparent. Artificial
exposure of new breeding stock to the environment of a
new farm might prevent the occurrence of congenital
tremors. However, until we understand the epidemiolo-
gy and pathogenesis of both congenital tremors and
PMWS, it is difficult to make recommendations for con-
trol of PCV infections.
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 Cytomegalovirus
9 N. Edington
Large basophilic intranuclear inclusions in cytomegalic
cells were first described in the mucous glands of porcine
turbinate mucosa by Done (1955). Their occurrence in
pigs with rhinitis led to the designation “inclusion body
rhinitis.” Experimental transmission aligned with ultra-
structural examination (Duncan et al. 1965; Valiček et al.
1970) indicated that herpeslike virions were the causal
agent and that infection frequently also involved the
lachrymal and salivary glands, as well as renal tubular
epithelium. Subsequently, the characteristic histologic
lesions have been described in the turbinates of pigs
from countries throughout the world. The nature and
distribution of the lesions suggested that the agent be-
longed to that group of herpesviruses affecting humans
and animals described as “salivary gland viruses” (Smith
1959) and then as cytomegaloviruses (Weller et al. 1960;
Plummer 1973) and finally as Betaherpesvirinae
(Matthews 1982). Inasmuch as they form a subgroup of
the herpesviruses, they are slow growing and produce cy-
tomegaly with distinctive intranuclear inclusions; they
tend to be species-specific; they usually induce a clinical-
ly silent infection in the adult but often a fatal, general-
ized infection in the young animal; and they have the
ability to cross the placenta and infect the fetus. Since the
virus of “inclusion body rhinitis” shows all these fea-
tures, it is justifiable to call it porcine cytomegalovirus
(PCMV). PCMV also shares with the other herpesviruses
the capacity to induce latent infection and to be excreted
in the presence of circulating antibody. In susceptible
herds the virus may cause fetal and piglet deaths, runt-
ing, rhinitis, and pneumonia, as well as poor liveweight
gain. However, in herds under good management the
virus may be endemic without any apparent economic
loss.
ETIOLOGY
Size and Structure
The morphology of PCMV is typical of a herpes virion
with an electron-dense core 45–70 nm in diameter, a sur-
rounding icosahedral capsid of 80–100 nm, and an out-
er single or, more rarely, double membrane 120–150 nm
across (Duncan et al. 1965; Valiček et al. 1970). The core
has a usually elongated, variable outline that is oval, rec-
tangular, or dumbbell in shape. The nuclear nucleocap-
sids appear to acquire an electron-dense coat, which is
separated by a translucent halo from the envelope; the
envelope of extracellular and cytoplasmic virions has ex-
ternal projections and a clear unit-membrane structure
(Valiček et al. 1973). As with other herpesviruses, parti-
cles without cores or with translucent cores, as well as cy-
toplasmic or extracellular nucleocapsids without en-
velopes, are frequently seen in vivo and in vitro.
Physicochemical Properties
PCMV is sensitive to chloroform and to ether (Booth et
al. 1967), but other physicochemical properties have not
been investigated.
Cultivation
Propagation of PCMV in vitro has been problematic. L’E-
cuyer and Corner (1966) passaged an isolate five times in
primary pig lung cells, while Watt et al. (1973), investi-
gating the susceptibility of a wide range of tissues, found
that only lung macrophages from 3- to 5-week-old pigs
were highly sensitive for both primary isolation and the
serial propagation of virus. Cytomegaly and the forma-
tion of intranuclear and, occasionally, small intracyto-
plasmic inclusions (Fig. 9.1) were seen 3–14 days
postinoculation (DPI), with the time of their appearance
dependent on the titer of PCMV used as inoculum. Prog-
eny virus reaches a maximum titer of up to 5.5 median
cell culture infectious doses of virus/ml at 11–14 DPI.
However, the limitations of lung macrophages are that
they do not replicate, and therefore, primary cultures
must be screened for contaminant viruses, including
PCMV and porcine reproductive and respiratory syn-
drome (PRRS) virus (PRRSV). Also, the absence of an
identifiable cytopathic effect (CPE) in unstained prepara-
tions necessitates the reading of the macrophages either
with Giemsa or labeled antibody (Watt et al. 1973;
Plowright et al. 1976) (Fig. 9.2). Bouillant et al. (1975) re-
ported that a cell line derived from porcine fallopian
tube will support viral replication, while Kawamura and
Matsuzaki (1996) reported that 12-O-tetrade-
canoylphorbol 13-acetate accelerated viral replication in
this system.
Replication
Systematic studies of in vitro replication have not been
reported. Infected cells are about six times as large as
normal cells, with swelling of the mitochondria, endo-
125
9.1. Cultured pig lung macrophages
11 days after infection with PCMV. The
basophilic intranuclear inclusions stand
out clearly in the enlarged cells (May-
Grünwald-Giemsa; ×720). (Courtesy
R. G. Watt.)
plasmic reticulum, and Golgi apparatus (Duncan et al.
1965). Much smaller acidophilic inclusions are seen,
sometimes in the nucleus but more often in the cyto-
plasm. Ultrathin sections show that the large intranu-
clear inclusions correlate with the formation of nucleo-
capsids, often in crystalloid arrays. The capsids acquire
an electron-dense coat in the nucleus, and the envelope is
taken from the inner nuclear membrane, the virions
coming to lie free or within membranous sacs in the cy-
toplasm. In the late stages of replication, as the cell is dis-
integrating, crystalloid arrays of viruses may also be seen
in the cytoplasm (Valiček et al. 1973).
Serology
An indirect immunofluorescence (IIF) test on infected
lung macrophages fixed in acetone has been described
9.2. Lung macrophage cultures showing fluorescence
after IIF, using specific PCMV antiserum. The nuclear
staining (N) is most intense at the membrane. Cytoplas-
mic and discrete paranuclear fluorescence (P) can also be
seen (×480).
(Plowright et al. 1976) (Fig. 9.2). IIF titers up to 1:64 to
1:128 were frequent in commercial pig sera, the test be-
ing at least eightfold more sensitive than virus-neutraliz-
ing assays (VN). There was no evidence of serologic vari-
ations among the limited number of U.K. isolates. Assaf
et al. (1982) and Tajima et al. (1993) describe ELISAs that
are more sensitive than VN and have been adapted to
distinguish IgG and IgM responses (Tajima et al. 1994).
Host Range
PCMV appears to be host-specific both in vivo and in vit-
ro. The virus has failed to replicate in rabbits, mice, ham-
sters, chick embryos, and cattle.
EPIDEMIOLOGY
The virus has worldwide distribution. Serologic evidence
in the United Kingdom and Japan indicates that over 90%
of herds have been exposed to infection, with more than
98% of pigs being seropositive by ELISA or IIF. The pos-
sibility of transplacental infection demands that hystero-
tomy-derived litters be carefully monitored.
The virus may be recovered from nasal and ocular se-
cretions, urine, and cervical fluids of pregnant sows ex-
posed to PCMV for the first time; isolation of virus from
the testis and epididymis (Booth et al. 1967; Shirai et al.
1985) indicates that boars should be carefully examined.
Dissemination of PCMV and infection by PCMV proba-
bly most commonly occurs via the nasal route, with the
environment also being contaminated by urine.
In a longitudinal survey of winter and summer co-
horts of pigs, the majority shed virus nasally between 3
and 8 weeks of age, correlating with the mixing of stock
in the early postweaning period. Antibody levels de-
 CHAPTER 9 CYTOMEGALOVIRUS Edington
creased during the period of virus excretion but rose
again at 8–11 weeks, continuing up to slaughter at 23
weeks (Plowright et al. 1976). The pattern suggested that
maternal antibody was replaced by active immunity,
which has been confirmed by Tajima et al. (1994).
In a smaller number of pigs, virus excretion was pre-
dominant at 3 weeks and had terminated at 5 weeks, sug-
gesting that early postnatal or congenital infection had
occurred. This has been associated with mummification
and stillbirths, neonatal deaths, and runt pigs with rhini-
tis and/or pneumonia in field situations (Cameron-
Stephen 1961; Rac 1961; Corner et al. 1964) and has been
confirmed experimentally (L’Ecuyer et al. 1972; Edington
et al. 1977). The reactivation of PCMV excretion after
the administration of corticosteroids (Edington et al.
1976c; Narita et al. 1985) points to the possible reactiva-
tion of replication when new breeding stock is intro-
duced or when routine is otherwise disturbed.
The recovery of virus from the lung macrophages of
quiescent pigs indicates that the macrophage is a reser-
voir of infection; this finding has serious implications in
relation to the restricted in vitro tropism of European
isolates of PRRSV (see Chapter 18). PCMV may also
modify the outcome of concurrent infections, as does
murine cytomegalovirus, which modifies the host defen-
sive mechanism, particularly inhibiting T-cell function
(Booss and Wheelock 1977; Kelsey et al. 1977). This ef-
fect has been more elegantly demonstrated for human
cytomegalovirus (Bonifacino 1996). Nowhere has the
“opportunistic” component of cytomegaloviruses been
more devastating than in humans infected with HIV.
CLINICAL SIGNS
The susceptible pregnant sow is frequently listless and
anorexic while viremic but shows no pyrexia or any oth-
er clinical abnormality throughout pregnancy. Piglets
may be born dead or die soon after birth without clinical
symptoms. Others are stunted, are pale due to anemia,
and show a variable edema which is often most notice-
able around the jaw and tarsal joints. Field outbreaks
have included symptoms of shivering, sneezing, and res-
piratory distress. Up to 25% of the litter may be lost, with
the remainder showing poor liveweight gains. The possi-
bility of surviving pigs being persistent excreters must
also be considered.
Uncomplicated infection with PCMV is usually a clin-
ically silent event in pigs older than 3 weeks but can be
fatal for the fetus or newborn pig. Such a statement is
qualified by the observations that some adult animals ex-
posed to PCMV for the first time may develop the lesions
of a generalized infection and be anorexic and lethargic
without pyrexia during the period of viremia (i.e., 14–21
DPI), while some piglets less than 3 weeks old develop
only the lesions of disseminated epithelial infection and
survive.
PCMV does not induce atrophic rhinitis but it will
127
produce a mild rhinitis in young pigs. In short-term trials
no synergistic effect was observed between PCMV and
Bordetella bronchiseptica (Edington et al. 1976b), but pro-
longed exposures warrant investigation, as these results
contradict field observations (Cameron-Stephen 1961;
Corner et al. 1964).
PATHOGENESIS
Pregnant sows inoculated with virus were lethargic and
anorexic 14–21 DPI, coinciding with the period of
viremia, and this was immediately followed by the recov-
ery of virus from nasal swabs. PCMV was isolated from
cervical fluids later, at 30–35 DPI, and radiographic as-
sessment of growth arrest indicated that the majority of
fetal deaths occurred in this period. This suggests that
the virus takes a further 14–20 days to replicate in the fe-
tus and that cervical shedding is of fetal, rather than ma-
ternal, origin. Neither virus nor inclusions were detected
in the cervix or endometrium at this stage. In a small
number of fetuses, the virus was detected as late as
60–80 DPI, but it was not clear whether this represented
in utero spread by contact infection, delayed replication
in these fetuses, or virus crossing the placenta on more
than one occasion. The small number of congenitally in-
fected animals that were examined excreted virus persis-
tently and died suddenly with the histologic lesions of
generalized infection within 7 days of birth (Edington et
al. 1977). Late-term fetuses and congenitally infected
neonates were all seronegative.
Superinfection of sows with low levels of circulating
antibody has been associated with transplacental infec-
tion (Edington et al. 1988a). Infection of the embryo
shortly after implantation certainly can occur and may
result in embryonic deaths. The virus was most com-
monly found in the meninges, Kupffer cells, and peri-
toneal macrophages of these early fetuses (Edington et
al. 1988b).
In experimental intranasal infections of young pigs
the occasional isolation of virus from nasal and conjunc-
tival swabs prior to viremia suggests that the primary site
of replication is in the nasal mucous glands or the lachry-
mal or harderian glands. Viremia was detected 14–16
DPI in 3-week-old pigs inoculated intranasally, and over
an extended period (5–19 DPI) in neonatal pigs (Eding-
ton et al. 1976c). Infectivity in blood was leukocyte asso-
ciated. Subsequently, the virus may be recovered from
nasal secretions for up to 32 days, pharyngeal and con-
junctival shedding being of shorter duration, while the
persistence of viruria has been difficult to determine.
The site of tertiary viral replication varies with age.
At an age of 3 weeks or more, when most animals ex-
perience infection, the virus disseminates to epithelial
sites—particularly the glands of the nasal mucosa, hard-
erian and lachrymal glands, kidney tubules, and more
rarely the epididymis and mucous glands of the esopha-
gus—to hepatocytes, and to duodenal epithelium.
128 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

In the fetal or neonatal pig there is a predilection for

reticuloendothelial (RE) cells, particularly capillary en-
dothelium and the sinusoids of lymphoid tissues, thus
giving rise to generalized lesions (Edington et al. 1988b).
While there is a tendency for replication to predominate
in either RE or epithelial cells, they are not mutually ex-
clusive, some individuals developing inclusions in both
groups of cells. Infectivity persists at least in lung
macrophages, since the virus is recoverable from cul-
tured lung washings from pigs that were not excreting
virus. Viral shedding has been restimulated in quiescent
animals following administration of corticosteroids (Ed-
ington et al. 1976c; Narita et al. 1985). Experimentally,
IIF antibody is detected 3 weeks after exposure to PCMV,
rising to a peak at 6 weeks and persisting until slaughter.
However, in rearing units in which PCMV is endemic, the
virus appears to be released in the presence of circulating
maternal antibody (Plowright et al. 1976).
Although inclusion bodies have occasionally been
seen in the epididymis, attempts to isolate virus from se-
men have been unsuccessful. Nor has it been possible to
establish infection by preputial inoculation. Neverthe-
less, boars can excrete virus in urine and nasal secretions.

LESIONS

It is useful to distinguish between disseminated infection

of epithelial tissues in the older pig and generalized in-
fection of RE tissues in the fetus or neonate (Edington et
al. 1976a).
Epithelial Lesions
No macroscopic changes are seen. Histologically, the
characteristic basophilic intranuclear inclusion bodies
and cytomegaly are seen in the nasal mucous glands (Fig.
9.3), acinar and duct epithelium of harderian and lachry-
mal glands, and renal tubular epithelium. In these tis-
sues the number of affected cells may be extensive. Iso-
lated inclusions are more rarely seen in the mucous
glands of the esophagus, the epithelial lining of the duc-
tus epididymis, and the seminiferous epithelium, as well
as the epithelial lining of the duodenum and jejunum. In
these minor sites the desquamation of infected cells
leaves no trace of infection; but in the major sites lym-
phocytes, plasma cells, and macrophages accumulate
around the affected epithelial tissue and invade the acini
as the cells are shed. Where complete acini are lost, there
may be some attempted replacement by a more squa-
mous type of epithelium from the duct; but frequently in
the nasal mucosa the remnants of the acinus have the ap-
pearance of focal lymphoid hyperplasia (Fig. 9.4). In nat-
ural cases the simultaneous infection with B. bronchisep-
tica or possibly Pasteurella spp. will produce changes in
the ciliated epithelium lining the nasal mucosa and in the
lesion of atrophic rhinitis, but these ostoid changes are
not seen in pigs that are infected with PCMV only. The
9.3. The basophilic intranuclear inclusion, translucent
halo, and defined nuclear membrane are prominent in
the enlarged superficial mucous gland epithelium of an
animal 18 days after experimental intranasal inoculation
(H&E; ×480).
reparative lesions in the kidney are those of an intersti-
tial nephritis (Kelly 1967). Sparsely distributed focal
gliosis is seen in the central nervous system (CNS), with
inclusions occasionally seen in the glial cells.
Generalized Lesions
The salient macroscopic lesions in young pigs are wide-
spread petechiae and edema. The edema most common-
ly involves the thoracic cavity and subcutaneous tissues.
In the thorax, pericardial and pleural effusions are seen,
while the pulmonary edema occurs throughout the
lungs, the interlobular septae being most distended and
the ventral tips of the lobes appearing purple and con-
solidated (Fig. 9.5). The subcutaneous edema was most
marked around the throat and tarsal joints. The lymph
nodes were all enlarged and edematous, with petechiae.
Although petechiae were also distinct at the other sites of
edema, they were most extensive in the kidneys, particu-
larly subcapsular, so that the appearance varied from
speckling to completely purple or black. In the small in-
 CHAPTER 9 CYTOMEGALOVIRUS Edington
9.4. The lamina propria is heavily infiltrated with
lymphocytes and plasma cells 24 days after inoculation
of PCMV. Many of the acini of the mucous glands still
show cytomegaly and prominent inclusion bodies (H&E;
×120).
testine, hemorrhages were seen, but rarely, varying from
complete involvement to focal areas less than 1 cm long.
Fetal infection did not result in pathognomonic
macroscopic lesions. The pattern was one that is typical-
ly seen in cases of reproductive failure characterized by
stillbirth, mummified fetuses, embryonic death, and in-
fertility (SMEDI). The mummified fetuses were random-
ly distributed and sometimes of variable age. In the
acute fatal syndrome most inclusions are seen in capil-
lary endothelial and sinusoidal cells and thus occur in all
lymphoid and parenchymatous tissues. The fact that
these cells are smaller than the epithelial cells of the dis-
seminated disease means that the cytomegaly and inclu-
sion bodies are not so prominent and may be overlooked
(Fig. 9.6). The damaged endothelium is associated with
the local edema and/or hemorrhage and with
macrophages and erythrocytes in the distended extracel-
lular space. Mononuclear cells with inclusions are found
in blood vessels and also in the spleen; infected
macrophages are prevalent in alveolar tissues. Replica-
tion in hepatocytes results in focal necrosis. In the kidney
the inclusions are most common in areas of differentiat-
129
9.5. This pig was inoculated with PCMV when 1 day
old. It died at 16 days of age with widespread petechiae
and subcutaneous edema. The interlobular septae of the
lungs are distended with transudate, and the tips of the
apical, cardiac, and diaphragmatic lobes are also consoli-
dated.
ing renal tissue and in glomerular capillary endothelium
(Fig. 9.6). Hemorrhage and gliosis occur throughout the
CNS, with a predilection for the choroid plexus, cerebel-
lum, and olfactory lobes (Stephano-Hornedo and Eding-
ton 1987).
In field outbreaks these lesions may be complicated
by concurrent infections—mucopurulent rhinitis, pneu-
monia, and enteritis being reported (Cameron-Stephen
1961; Corner et al. 1964).
DIAGNOSIS
The presence of PCMV in a herd of pigs is most easily
confirmed by detecting antibody by IIF or ELISA on ran-
dom samples of sera from fattening pigs; this is also the
most practical way of monitoring hysterotomy-derived
herds.
In sows showing SMEDI-like reproductive disorders,
differentiation must be made from parvovirus and possi-
bly from Aujeszky’s disease. The virus may be isolated
from neonates or fetuses by sampling nasal mucosa,
lung, and kidney and by making direct cultures of lung
macrophages if possible. If the carcass is kept at 4˚C, vi-
ral antigen can be detected by immunostaining on frozen
sections of these tissues at least 24 hours after infectivity
has been lost. Alternatively, the pathognomonic inclu-
sions and cytomegaly may be detected in histologic sec-
tions.
In determining the presence of PCMV as a compo-
130 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
9.6. Section of kidney from a
piglet with viral inclusions (arrows)
in capillary endothelium in both the
glomerulus and interstitial tissue.
Infected cells show enlargement but
only reach the size of normal tubu-
lar epithelium (H&E; ×240).
nent of infectious rhinitis in a herd, virus isolation must
be made from nasal swabs or confirmed by immuno-
staining from nasal scrapings.
The relatively recent emergence of PRRS as a wide-
spread and economically important disease of swine
leads to the speculation that PCMV may be recognized
more often in the future. Diagnostic testing for PRRSV is
now common, and porcine lung macrophages, which are
often used for primary isolation of PRRSV, may fortu-
itously reveal the presence of PCMV in some cases.
Detecting PCMV will likely become an even more im-
portant consideration in the future because of the ex-
panding field of xenotransplantation and the potential
for transfer of porcine tissues and organs to people.
TREATMENT
No specific treatment can be given, although medication
against concurrent bacterial infection is used in out-
breaks of infectious rhinitis. Where rhinitis and/or
SMEDI have been associated with the detection of
PCMV, the outbreaks have usually been self-limiting
(Cameron-Stephen 1961; Corner et al. 1964).
PREVENTION
In populations where PCMV is endemic, it does not ap-
pear to be a problem under good management systems.
However, the introduction of new stock must always be
regarded as hazardous since it may stimulate latent in-
fection in the presence of circulating antibody or give
rise to the problems of primary infection in susceptible
herds. Virus-free herds can be established by hysteroto-
my, but the ability of PCMV to cross the placenta dic-
tates that offspring must be carefully monitored serolog-
ically for at least 70 days.
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 CHAPTER 9 CYTOMEGALOVIRUS Edington
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 Eastern Equine Encephalomyelitis
10 F. Elvinger and C. A. Baldwin
Eastern equine encephalomyelitis (EEE) is endemic in the
eastern United States. Naturally occurring EEE in swine
was first reported in 1972 (Pursell et al. 1972) when 160
of 200 pigs in one herd died in an outbreak in the state of
Georgia. Exposure of domestic and feral swine to the
agent had been described earlier in serologic surveys in
the states of Georgia, Massachusetts, and Wisconsin
(Karstad and Hanson 1958, 1959; Feemster et al. 1958).
The disease is diagnosed sporadically in porcine case ma-
terial at veterinary diagnostic laboratories but is likely
underrecognized in the field.
EEE first was recognized in horses on the eastern
coast of the United States (Giltner and Shahan 1933;
TenBroeck and Merrill 1933). Its etiologic agent, EEE
virus (EEEV), soon was reported to be a human pathogen
as well (Fothergill et al. 1938). A median annual number
of three human cases has been reported in the United
States since 1955 (Anonymous 1990). In addition, EEE
has been diagnosed and reported in cattle, dogs, goats,
swine, and a variety of wild mammalian species (Pursell
et al. 1972, 1976, 1983; Bigler et al. 1976; McGee et al.
1992). Wild and domesticated birds are susceptible to
disease caused by EEEV (Scott and Weaver 1989). Clini-
cal signs range from inapparent to severe with high death
losses. Histologic lesions in birds include inflammation
and hemorrhage or necrosis of visceral organs, which
differs from the central nervous system disease seen in
mammals (Dein et al. 1986; Tully et al. 1992; Guy et al.
1994).
The virus is transmitted by mosquito vectors in two
distinct cycles. The endemic cycle, which involves or-
nithophilic mosquito species, maintains the virus in
birds considered to be reservoirs and amplifiers of the
virus. The epidemic cycle, which involves a variety of
mosquito species feeding on various avian and mam-
malian hosts, leads to the occasional transmission of
virus to mammalian hosts (Scott and Weaver 1989).
ETIOLOGY
The virus of EEE belongs to the genus Alphavirus in the
family Togaviridae. Presently there are 26 confirmed
members in the genus Alphavirus, including western
equine encephalomyelitis (WEE) virus (WEEV) and
Venezuelan equine encephalomyelitis virus (Hahn et al.
1988). EEEV was described and serologically differentiat-
ed from WEEV in 1933 (TenBroeck and Merrill 1933).
Nucleic acid analysis has demonstrated that EEEV has
evolved independently into North and South American
antigenic varieties (Casals 1964; Weaver et al. 1991), each
with distinct virulence characteristics (Walder et al.
1980). Isolates appear to be genetically stable over a dis-
crete time period within a geographic region of trans-
mission (Roehrig et al. 1990).
EPIDEMIOLOGY
EEEV circulates endemically among many bird species
considered to be reservoirs and amplifiers of virus. Virus
is transmitted from bird to bird mainly by the
ornithophilic mosquito vector Culiseta melanuris. Other
mosquito species from genera such as Aedes and
Anopheles also transmit the EEEV. Mosquito species with
opportunistic feeding habits on both birds and mam-
mals are responsible for the epidemic virus transmission
to mammals. The appearance of EEE in mammals is cor-
related to climatic conditions that influence population
dynamics of vectors (Letson et al. 1993; Francy and Wag-
ner 1992). In Florida, virus circulates among birds and
infects mammals year-round, but farther north, virus ac-
tivity is limited to summer months.
Exposure of swine to EEEV was first noted in the late
1950s in serosurveys in Georgia, Massachusetts, and
Wisconsin (Karstad and Hanson 1958, 1959; Feemster et
al. 1958), and clinical outbreaks have been reported from
Georgia and Florida (Pursell et al. 1972; Elvinger et al.
1994, 1996b). Swine tested on 9 (20%) of 45 farms in
southern Georgia had antibodies to EEEV, and 60 (16%)
of 376 feral swine on a Georgia barrier island had serum-
virus neutralization test titers that ranged from 4 to 128
(Elvinger et al. 1996b). In general, mammals have been
considered to be dead-end hosts due to low virus titers
that are insufficient to infect vectors. However, nursing
pigs that were experimentally infected developed high-
titer viremia that lasted up to 168 hours. Virus could be
recovered from oropharyngeal and rectal swabs up to 96
hours after virus administration, and virus could be iso-
lated from tonsils of pigs up to 20 days after experimen-
tal infection. Contact control pigs, in addition, have se-
roconverted in several experimental infection studies
(Karstad and Hanson 1958; Baldwin et al. 1997a). Thus,
it is conceivable that infected nursing pigs are a source of
133
134 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
virus both for vectors and, by direct transmission, for
mammals in close proximity.
CLINICAL SIGNS
EEE predominantly affects nursing pigs. Clinical signs
include depression, anorexia, ataxia, prostration, lateral
recumbency, convulsions, and ultimately death. Howev-
er, clinical disease is not commonly observed following
experimental or natural exposure to EEEV. Most nursing
pigs exhibit only a transient temperature increase, even
when high doses of EEEV are experimentally given by
oral, intradermal, or intravenous routes (Elvinger et al.
1996a; Baldwin et al. 1997a). Predisposing factors, in-
cluding adverse environmental conditions or concurrent
disease, may contribute to the high mortality observed in
natural outbreaks of EEE in swine (Pursell et al. 1972;
Elvinger et al. 1994). Experimental infection of older an-
imals did not lead to clinical disease (Karstad and Han-
son 1959), and EEE has not been described in adult
swine. The oldest pig recorded with clinical EEE was a 2-
month-old female (Pursell et al. 1983).
PATHOGENESIS
Natural infection of pigs likely occurs through transmis-
sion of virus by hematophagous vectors; experimental
infection has been achieved by intracranial, intradermal,
intravenous, and oral routes of virus administration
(Karstad and Hanson 1959; Pursell et al. 1972; Baldwin
et al. 1997a). Rectal temperature increases within 24
hours of virus administration and is elevated for less
than 12 hours in all experimentally infected pigs; howev-
er, clinical signs of central nervous system disease are ob-
served in only a few pigs between 18 and 72 hours after
virus administration (Baldwin et al. 1997a). Circulating
virus was isolated 6 hours after experimental infection,
regardless of route of infection, and was present in
blood for up to 168 hours. Neutralizing antibodies are
detectable approximately 120 hours after experimental
infection. The virus also was recovered from oropharyn-
geal and rectal swabs 6–96 hours after infection. After
the acute viremic phase, EEEV was isolated only from
tonsils and central nervous tissues. The virus was isolat-
ed or demonstrated by oligonucleotide probe in tonsils
up to 20 days after experimental infection. Consequent-
ly, tonsillar tissues of infected pigs could be a significant
source of virus dispersion, especially since contact pigs
become infected (Karstad and Hanson 1958; Baldwin et
al. 1997a). Oligonucleotide probing revealed EEEV in
transient liver lesions that appeared early during the
viremic phase of the infection, prior to the appearance of
lesions in myocardium and the central nervous system
(Baldwin et al. 1997a, b). The presence of the virus in liv-
er lesions may indicate virus tropism for hepatic tissue
and viral replication in the liver.
LESIONS
No gross lesions indicative of EEE have been observed in
naturally or experimentally infected pigs (Karstad and
Hanson 1959; Pursell et al. 1972; Elvinger et al. 1994,
1996a; Baldwin et al. 1997a). Microscopically, necrotiz-
ing hepatitis appears within 12 hours of experimental in-
fection. Lesions increase in size over the next 24 hours
and resolve partially by 48 hours and completely by 72
hours after infection (Baldwin et al. 1997a). Central ner-
vous system lesions may not be present in pigs that die in
the acute phase of disease. Earliest lesions appear in
brain approximately 48 hours after infection. The ap-
pearance of these lesions is concurrent with the appear-
ance of mild lesions in heart and resolution of lesions in
liver. Encephalitis is characterized by inflammatory cell
infiltration, disseminated perivascular cuffing, neuronal
necrosis and neuronophagia, nodules of glial prolifera-
tion, and malacia (Pursell et al. 1972; Elvinger et al. 1994,
1996a; Baldwin et al. 1997a). Viral inclusions have not
been observed. Initially, neutrophils predominate in in-
filtrates and perivascular cuffs, whereas lymphocytes are
more common at later stages. In addition, histiocytes,
adventitial cells, eosinophils, and cellular debris are ob-
served. Hyaline or granular thrombi are found in blood
vessels. Lesions occur primarily in gray matter of brain
and spinal cord, but white matter may also be affected.
Patches of inflammatory cells are noted in the meninges.
Multifocal myocardial necrosis has been reported in nat-
urally infected pigs and reproduced in experimental in-
fection (Elvinger et al. 1994, 1996a; Baldwin et al.
1997a). Swine have not developed hemorrhagic intesti-
nal lesions such as those described in whooping cranes
and emus (Dein et al. 1986; Tully et al. 1992). Pigs that
survived experimental infection with no apparent clini-
cal signs occasionally had lesions of mild encephalitis
with lymphocytic perivascular cuffs, focal areas of glio-
sis, and foci of myocardial necrosis that were partially
mineralized and surrounded by macrophages (Baldwin
et al. 1997a).
DIAGNOSIS
Death losses in nursing pigs following clinical signs of
central nervous system disease in EEE endemic areas are
indicators of virus activity, particularly when climatic
conditions are conducive to the development of vector
populations. Histopathologic lesions may first appear in
liver and myocardium, but lesions in brain consistent
with EEE may not have developed yet in pigs that died
during the early viremic phase. A definitive diagnosis can
be supported by virus isolation or detection of virus
RNA or antigen. Serologic evidence of exposure in sur-
viving pigs is a good indicator of EEEV activity, although
antibody titers may be due to earlier exposure of sows
and transmission of antibodies through colostrum.
 CHAPTER 10 EASTERN EQUINE ENCEPHALOMYELITIS
A wide spectrum of tests to isolate virus, demon-
strate and identify EEEV RNA or antigen, or detect anti-
bodies to EEEV is used to diagnose natural and experi-
mental infections with EEEV. The virus has been isolated
in cell cultures using the Vero (continuous African green
monkey) cell line, BHK-21 (baby hamster kidney) cells,
or other vertebrate cell lines susceptible to EEEV (Fergu-
son et al. 1979; Monath et al. 1981; Scott et al. 1988;
Elvinger et al. 1996a). Virus antigen has been demon-
strated in tissues by immunohistochemistry and en-
zyme-linked immunosorbent assay (Monath et al. 1981;
Scott et al. 1988; Patterson et al. 1996), and EEEV RNA
was detected with DNA in situ hybridization or after am-
plification with polymerase chain reaction (Vodkin et al.
1993; Armstrong et al. 1995; Gregory et al. 1996). Meth-
ods used to detect antibodies to EEEV include virus-
serum neutralization test, enzyme-linked immunosor-
bent assay, complement fixation test, and
hemagglutination inhibition test (Ferguson et al. 1979;
Tesh and McCammon 1979; Sahu et al. 1994).
Fresh and formalin-fixed specimens of brain, spinal
cord, liver, heart, and tonsil should be submitted for
histopathologic examination and virus isolation when
EEE is suspected in a swine herd. Virus isolation can be
attempted on oropharyngeal swabs from herd mates,
since EEEV can persist in tonsillar tissue. Precautions
should be taken to avoid accidental human infection
when collecting the specimens. Serum from affected and
nonaffected pigs should be submitted for serologic ex-
amination, although disease and death likely will pre-
cede the onset of a detectable immune response.
TREATMENT AND PREVENTION
Treatment of swine clinically affected with EEE has not
been attempted. No treatment of affected horses has
been described, and therapeutic efforts in human cases
are directed toward management of symptoms (Craven
1991).
Two routes for prevention of EEE can be taken: (1)
vaccination of animals at risk and (2) vector control.
In endemic areas, most horses are routinely vaccinat-
ed at least once, but usually twice, per year to prevent
EEEV infection (Wilson et al. 1986; Gibbs et al. 1988).
Human experimental vaccines are available for research
and for laboratory personnel who may become exposed
to the virus. Vaccination of sows is an option for protect-
ing swine producers from catastrophic losses due to
EEEV infection in pigs. Sows vaccinated experimentally
with commercial equine vaccines have developed mater-
nal antibodies that were transmitted to pigs via
colostrum (Elvinger et al. 1996a). Maternally derived an-
tibodies are protective for experimentally infected, early-
weaned pigs. Pigs from vaccinated sows did not develop
the fever, clinical signs, or histopathologic lesions related
to EEEV infection that the pigs from nonvaccinated sows
Elvinger, Baldwin 135
developed. Although two of six experimentally infected
pigs from vaccinated sows were viremic, virus titers were
lower and duration of viremia was shorter than in pigs
from nonvaccinated sows. Pigs from vaccinated sows are
not likely to be a source of virus for infection of mosqui-
to vectors. Naturally infected sows should transfer pro-
tective antibodies against EEEV to their litters. Dams
transferred maternal antibodies 5 months after vaccina-
tion even though antibody levels in serum from dams
were below detection level by virus-serum neutralization
test. Maternally derived antibodies were measured in
pigs for up to 11 weeks after colostrum intake and should
be protective until natural susceptibility to clinical dis-
ease is outgrown.
The second approach to prevention of EEE is control
of exposure to EEE vectors. Incident cases of EEE in any
mammalian or avian species can serve as sentinel cases
signaling the need to initiate application of insecticides
by local and state governments. Some communities in
endemic areas have established surveillance programs
for testing sera of sentinel birds for the appearance of
antibodies to EEE and other arboviral diseases and/or
for trapping mosquitoes to determine the presence of
EEEV or other viruses. Pheasants are more sensitive for
detection of EEEV activity than chickens; however, chick-
ens are more commonly used as sentinel birds (Morris et
al. 1994; Day and Stark 1996). Detection of virus activity
in an area prompts the release of warnings to the public
through news media and implementation or expansion
of vector control measures, including a general, area-
wide application of pesticides like malathion or fenthion
(Carter et al. 1981). One southern Georgia county initiat-
ed chemical mosquito control following the diagnosis of
EEE in swine (Elvinger et al. 1994). Insecticidal interven-
tions are costly and are designed to prevent human cas-
es. Costs for treatment of a human case have been esti-
mated to range from $20,000, when there are no residual
sequelae, up to a lifetime cost of $3 million for patients
that suffer permanent sequelae (Villari et al. 1995). Aeri-
al application of insecticides benefits swine producers in
endemic areas directly by preventing outbreaks and eco-
nomic losses and indirectly by preventing amplification
of EEEV in pigs that would put the producer and farm la-
bor at risk of contracting infection.
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 Encephalomyocarditis Virus
11 H. S. Joo
Encephalomyocarditis virus (EMCV) has been recog-
nized as a swine pathogen since it was first isolated dur-
ing an outbreak of acute disease in Panama (Murnane et
al. 1960). Subsequently, outbreaks have been reported in
many different countries. Major epizootics with high
mortality in baby pigs have been reported in Florida
from 1960 to 1966 (Gainer 1967), New South Wales,
Australia, in 1970 and 1984 (Acland and Littlejohns
1975; Seaman et al. 1986), and Cuba (Ramos et al. 1983).
Clinical disease has also been reported in New Zealand
(Sutherland et al. 1977), South Africa (Williams 1981),
Brazil (Roehe et al. 1985), Italy (Sidori et al. 1988),
Switzerland (Hani et al. 1992), Greece (Paschaleri-Pa-
padopoulou et al. 1992), and Canada (Dea et al. 1991),
while detection of EMCV antibodies without clinical dis-
ease has been reported in the United Kingdom (Sangar et
al. 1977) and Japan (Kudo et al. 1995).
Besides baby pig mortality, EMCV has been implicat-
ed as a cause of reproductive failure. Reproductive prob-
lems associated with EMCV infections have been report-
ed in Cuba (Gomez et al. 1982), Australia (Littlejohns
1984; Links et al. 1986), Canada (Dea et al. 1991), South
Korea (Park et al. 1990), Belgium (Koenen et al. 1992),
and the United States (Joo et al. 1988; Kim et al. 1989a;
Christianson et al. 1992).
ETIOLOGY
The EMCV group is classified as genus Cardiovirus of the
family Picornaviridae. The first EMCV isolation was
made from a chimpanzee with myocarditis in Florida
(Helwig and Schmidt 1945), and subsequently, the virus
was isolated from a variety of animal species over a wide
geographic area (Tesh and Wallace 1977). During the
1940s several virus strains, including Columbia-SK,
Mengo, and M.M., were isolated. These viruses were
found to be antigenically similar and are considered to
be in the same group as EMCV. EMCV has been reported
to be antigenically related to the cricket paralysis virus of
insects (Tinsley et al. 1984).
The author deeply acknowledges Dr. H. M. Acland and Dr. I. R. Little-
johns, the authors of the EMCV chapter in the 6th edition of Diseases of
Swine. The present chapter is based on their chapter in the 6th edition.
Many properties of EMCV are shared by other picor-
naviruses. It is ether-resistant and stable over a wide
range of pH. It is inactivated after 30 minutes at 60˚C,
but some strains have shown a marked thermal stability.
EMCV replicates well in cell cultures originating from
several animal species, including rodents, swine, and hu-
mans. The virus also replicates in mice and chicken em-
bryos and is pathogenic to many laboratory animal
hosts.
Acute fatal disease is most often produced in mice
and hamsters following inoculation by various routes.
Neurologic disease due to encephalitis is observed, but
myocarditis may also be seen at necropsy. Pathogenicity
in rats, guinea pigs, rabbits, and monkeys appears to
vary depending on the age of the animals and the virus
strains used.
The virus has hemagglutinating ability with guinea
pig, rat, horse, and sheep erythrocytes, and most EMCV
strains require KCl-borate (0.12M KCl; 0.05M H3BO3)
buffered solution for an optimal hemagglutination reac-
tion. Some differences in hemagglutinating activity be-
tween EMCV strains have been reported (Sangar et al.
1977; Kim et al. 1991b). The EMCV virions contain a sin-
gle strand of RNA of molecular weight 2.6 × 106 daltons,
which makes up 31% of the virion mass and is enclosed
in a protein capsid shell. The viral proteins show four
nonidentical polypeptide bands in SDS-polyacrylamide
gel electrophoresis. Other molecular characteristics, in-
cluding the nucleotide sequence of the RNA, are rela-
tively well understood.
EPIDEMIOLOGY
The EMCV group is generally regarded as a rodent virus,
although the virus naturally infects a wide range of ver-
tebrate species. The host range includes chimpanzees,
monkeys, elephants, lions, squirrels, mongooses,
racoons, and swine. Pigs are the domestic animals most
susceptible to clinical disease by EMCV infection.
Rats and mice are believed to be the principal reser-
voir of the virus (Tesh and Wallace 1977). EMCV neu-
tralizing antibodies have been demonstrated in sera
from wild rats, Rattus spp., trapped in several areas of
the United States and Canada. Many rodents are suscep-
tible to experimental infection, showing high levels of
the virus in their tissues, and infected rodents excrete the
139
140 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
virus in their feces and urine. EMCV has been isolated
from dried feces and from intestines of rats and mice
captured on farms where swine disease had previously
occurred (Gainer 1967; Acland and Littlejohns 1975). Al-
though the virus has been isolated from mosquitoes
caught in Africa, Brazil, and the United States, and from
ticks in India (Tesh and Wallace 1977), there is no evi-
dence that natural EMCV infection in swine is vector-
borne.
The most important sources for swine infection ap-
pear to be feed or water contaminated with the virus by
rats, other rodents, or infected carcasses. Feed contami-
nated with infected carcasses may contain high doses of
the virus, since a large amount of the virus is found in the
carcasses. An episode of lion deaths at a zoo was found
to be due to feeding carcasses of African elephants that
had died of EMCV infection (Simpson et al. 1977).
The mode of virus transmission is not clear, but ro-
dent-to-pig transmission is probably common. Several
outbreaks in Australia were found to be closely associat-
ed with rat and mouse plagues (Acland and Littlejohns
1975; Seaman et al. 1986). Pig-to-pig transmission has
been questioned because sentinel pigs failed to become
infected after contact with experimentally infected and
sick pigs (Littlejohns and Acland 1975; Horner and
Hunter 1979). However, the role of the infected pigs in
natural transmission, either directly or indirectly, cannot
be excluded, since infected pigs have been shown to ex-
crete the virus at least for a short period.
The virulence of EMCV isolates in pigs may vary de-
pending on the virus strains maintained in reservoir
hosts within geographical areas. For example, Australian
strains were shown to be more virulent in experimental
infection of pigs than New Zealand strains (Littlejohns
and Acland 1975; Horner and Hunter 1979), and certain
isolates in Florida were found to cause only myocarditis,
without death (Gainer et al. 1968).
To date there is no clear evidence to support the role
of EMCV as a pathogen in livestock other than swine. Ev-
idence for human infection with EMCV has been demon-
strated by antibody detection in human populations
(Tesh 1978), but there are no reports that the virus caus-
es human heart disease. However, EMCV has been uti-
lized in different research models for human diseases, in-
cluding the investigation of the pathogenesis of
myocarditis, vasculitis, and viral-induced diabetes melli-
tus.
CLINICAL SIGNS
EMCV infection in young pigs is characterized most
commonly by acute disease with sudden deaths due to
myocardial failure. Other clinical signs, such as anorexia,
listlessness, trembling, staggering, paralysis, or dyspnea,
are also observed. Experimentally infected swine (Craig-
head et al. 1963; Littlejohns and Acland 1975) have
shown temperatures up to 41˚C and death between days
2 and 11, usually 3–5 days, postinoculation or occasional
recovery with chronic myocarditis. The severity of the
disease in pigs appears to vary depending on the virus
strain and the age of the pigs at the time of infection.
Mortality approaching 100% is usually confined to pigs
of preweaning age. Infections in pigs from postweaning
age to adulthood are usually subclinical, although some
mortality may be observed even in adult pigs.
In breeding females, clinical signs vary from no obvi-
ous illness to severe reproductive problems. The first
clinical signs of anorexia and fever may be observed in
infected sows. Such sows may show various forms of re-
productive failure, including abortions and increased
numbers of mummified and stillborn fetuses (Dea et al.
1991). Increased neonatal deaths and preweaning mor-
tality will also be observed.
PATHOGENESIS
The natural infection of swine occurs most likely by the
oral route. Following experimental oral infection in
piglets, viremia was demonstrated as early as 2 days
postinoculation and persisted for 2–4 days (Craighead et
al. 1963). The virus was present in the feces for as long as
9 days following oral administration. The persistence of
the virus beyond the viremic period suggests some viral
replication in the intestine. Large amounts of the virus
were found in the spleen and mesenteric lymph nodes,
indicating that lymphatic tissue was a site of virus repli-
cation. The highest virus titers were recovered from heart
muscle in both experimental and natural infections, and
myocardial lesions were predominant at necropsy. Liver,
pancreas, and kidney also contained virus, usually at a
greater concentration than in blood. Animals that sur-
vived the acute disease produced EMCV antibodies. Af-
ter antibody formation, the virus was no longer recover-
able. The course of infection in swine appears to be
influenced by virus strain, viral dose, history, and level of
viral passage and susceptibility of the individual animal.
The pathogenesis of transplacental infection with
EMCV in pregnant sows is not well understood. Follow-
ing intramuscular infection of pregnant sows with EM-
CV, a transplacental infection with fetal deaths was ob-
served in one of the three sows infected in late gestation,
while fetal infection in sows during early pregnancy was
not conclusive (Love and Grewal 1986). Infected and
dead fetuses showed myocardial lesions varying from
multiple small foci to large diffuse patches. Some diffi-
culty in producing experimental reproductive disease in
pregnant sows was also observed with a U.S. isolate.
However, transplacental infection was successful when
the virus was passaged in young pigs rather than cell cul-
ture before inoculation (Christianson et al. 1992). Fetal
deaths following infection in sows appear to occur as ear-
ly as 2 weeks postinfection. At this time it is not known
 CHAPTER 11 ENCEPHALOMYOCARDITIS VIRUS Joo 141

whether all EMCV strains can cause both the typical my-
ocarditis observed in young pigs and reproductive fail-
ure.
Pathogenic variability in swine fetuses by different
EMCV isolates has been reported (Kim et al. 1989b). Lit-
tle pathogenicity was observed following infection of
swine fetuses in utero with laboratory-passaged strains.
Pathogenic effects of field isolates were obvious in fetus-
es of both mid- and late gestational ages. It has been sug-
gested that virulent strains may have been attenuated
and lost pathogenicity by prolonged laboratory pas-
sages.
Among the laboratory animals, clinical manifesta-
tions and the pathogenesis of EMCV infection were vari-
able. Experimental infection has resulted in fatal my-
ocarditis in adult guinea pigs and in some strains of
white rats. Owl monkeys (night monkeys) and mar-
mosets were reported to be highly susceptible to infec-
tion. The virus has seldom been pathogenic to rabbits
and rhesus monkeys, causing only inapparent infections
despite high viremic levels. It is interesting to note that
the pathogenicity of EMCV can be modified by laborato- 11.1. Heart of pig with EMCV infection showing
ry manipulations, and EMCV variants that differ in or- multiple white foci in myocardium.
gan tropism and in pathogenicity have been developed.
Certain strains cause predominantly fatal en-
cephalomyelitis (encephalotropism) or widespread my- tion of mononuclear cells (Fig. 11.3), vascular conges-
ocardial damage (cardiotropism) or even specific de- tion, edema, and degeneration of the myocardial fibers
struction of pancreatic beta cells (pancreotropism) with necrosis. Mineralization of necrotic heart muscle is
(Craighead 1966; Cerutis et al. 1989). common but not always present. Congestion with
meningitis, perivascular infiltration with mononuclear
LESIONS cells, and some neuronal degeneration may be observed
in the brain (Murnane et al. 1960; Gainer et al. 1968;
Pigs dying in the acute phase of cardiac failure may show Acland and Littlejohns 1975). Nonsuppurative en-
only epicardial hemorrhage or no gross lesions. At cephalitis and myocarditis have also been observed in
necropsy of experimentally infected young pigs, hy- swine fetuses with natural EMCV infection (Kim et al.
dropericardium, hydrothorax, pulmonary edema, and 1989a).
ascites are frequently observed, and gross myocardial le-
sions are prominent. The heart is usually enlarged, soft, DIAGNOSIS
and pale, with visible yellowish or white necrotic foci
(2–15 mm in diameter) or large ill-defined pale areas The clinical history of reproductive failure, along with
(Fig. 11.1). The lesions are most commonly observed on high preweaning mortality, is a useful tip in the diagno-
the epicardium of the right ventricle and may extend to sis of EMCV infection. EMCV-induced reproductive
varying depths within the myocardium. The virus is problems should be differentiated from those of infec-
present in heart muscle in most cases, even when my- tion with other pathogens. EMCV causes reproductive
ocardial lesions are minimal or occasionally absent problems in sows of all parities, along with high neona-
(Gainer et al. 1968; Littlejohns and Acland 1975). tal mortality, while porcine parvovirus infection is mani-
Infected fetuses become mummified in various sizes fested in an increase in mummified fetuses mainly in gilt
depending on the stage of infection and may be hemor- litters, without neonatal mortality. Other infections,
rhagic, edematous, or apparently normal (Fig. 11.2). The such as porcine reproductive and respiratory syndrome
myocardial lesions may be seen in some infected fetuses virus, pseudorabies virus, and Leptospira spp., should al-
but are difficult to observe under field conditions. The so be considered.
gross abnormalities of the fetuses are not easy to distin- Dyspnea manifested as rapid abdominal breathing
guish from those caused by other viral infections, unless due to heart failure may be observed among EMCV-in-
the myocardial lesions are observed. fected newborn pigs. Gross lesions of white necrotic ar-
Histopathologically, the most significant finding in eas in the heart muscle are characteristic of EMCV infec-
young pigs is myocarditis with focal or diffuse accumula- tion, although such lesions resemble those of vitamin E
142 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

11.2. Normal-appearing,
edematous, hemorrhagic, and
mummified swine fetuses (left to
right) from a sow infected intra-
muscularly with EMCV at 50
days of gestation and examined
28 days after infection.

11.3. Focal infiltration of

mononuclear cells in myocardium
of a stillborn swine fetus with
natural EMCV infection.

and selenium deficiency and heart infarcts by septic em-
bolism.
A definitive diagnosis should be based on virus isola-
tion and identification. Heart is the best tissue for virus
isolation from swine fetuses and young pigs. Tissue sam-
ples can be frozen without any detectable loss of virus in-
fectivity. The virus can be isolated by inoculating sam-
ples into mice, chicken embryos, or cell cultures. Infected
mice or chicken embryos will die 3–6 days after inocula-
tion. For virus isolation in cell culture, baby hamster kid-
ney (BHK-21), HeLa, or Vero cell line cultures are com-
monly used. For primary cell culture, fetal mouse
fibroblasts are suitable. Infected cell monolayers will
show a rapid and complete cytolysis, and viral identifica-
tion can then be made by staining the infected monolay-
ers with EMCV fluorescent antibody conjugate or by the
inhibition of the cytopathic effects by specific immune
serum. Virus isolation is usually successful from pigs
during the acute phase but is difficult from pigs in the
stage after development of circulating antibody.
Histopathologic lesions consistent with EMCV infec-
tion may play an important role in making a diagnosis.
As described previously, myocarditis of varying stages
with infiltration of mononuclear cells, along with non-
suppurative encephalitis, is indicative of EMCV infec-
tion. Serologic methods may also help in the diagnosis of
 CHAPTER 11 ENCEPHALOMYOCARDITIS VIRUS
EMCV infection. Detection of antibody specific to EM-
CV from stillborn or large mummified fetuses is particu-
larly significant for fetal infection (Joo et al. 1988; Kim et
al. 1991a, b) because there is no transmission of mater-
nal immunoglobulins across the placenta in swine. Inter-
pretation of sow serology is often confusing, since posi-
tive antibody titers have not always been associated with
clinical disease. Both serum neutralization and hemag-
glutination inhibition tests (Joo et al. 1988) can be used
to detect EMCV antibody in serum samples. Although
the specificity of each test is not well defined, antibody
titers of ≥1:16 appear to be significant. An enzyme-
linked immunosorbent assay can also be used to detect
EMCV antibody (Shanley 1980).
TREATMENT AND PREVENTION
There is no treatment, but mortality may be minimized
by avoiding stress or excitement of the pigs at risk. It is
important to control rodents on pig farms or minimize
their contact with pigs either directly or indirectly via
contamination of feed or water. Although pig-to-pig
transmission is not clear, introduction of pigs from pre-
viously infected farms should be avoided. Basic rules of
sanitation and hygiene should be applied. Animals dying
of the disease should be promptly and sanitarily dis-
posed. The virus can be inactivated in water containing
0.5 ppm residual chlorine. For disinfectants, iodine-
based preparations or mercuric chloride can be used. An
inactivated vaccine for EMCV infection in swine is com-
mercially available in the United States. The vaccine ap-
pears to be effective, since high humoral immunity is de-
tected in vaccinated pigs.
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 Enterovirus
12 J. B. Derbyshire
Porcine enteroviruses are ubiquitous, and no conven-
tional herd of pigs has been shown to be free of infec-
tion. While the majority of infections are asymptomatic,
porcine enteroviruses have been associated with a variety
of clinical conditions, including polioencephalomyelitis,
female reproductive disorders, enteric disease, and pneu-
monia. They have also been isolated from the male geni-
tal tract (Phillips et al. 1972), although the insemination
of gilts with contaminated semen did not influence their
fertility (De Meurichy and Pensaert 1977). A possible as-
sociation between porcine enteroviruses and cutaneous
lesions resembling swine vesicular disease has been pro-
posed (Knowles 1988).
The first evidence of porcine enterovirus infection to
be reported was the occurrence of Teschen disease, a po-
lioencephalomyelitis with high mortality, in Czechoslo-
vakia over 60 years ago. This severe disease has contin-
ued to occur sporadically, mainly in Central Europe but
also in Africa; milder forms of polioencephalomyelitis
(Talfan disease, benign enzootic paresis), caused by sero-
logically related but less virulent strains of porcine en-
terovirus, have been reported in the last 35 years in West-
ern Europe, North America, and Australia. In France, the
encephalomyelitis is intermediate in severity between
Teschen and Talfan diseases (Métianu 1986). The associ-
ation of porcine enteroviruses with reproductive, en-
teric, and respiratory disorders in swine is less well es-
tablished than their role in polioencephalomyelitis.
Strains that have not been shown to be pathogenic have
been referred to as enteric cytopathogenic swine orphan
(ECSO) or enteric cytopathogenic porcine orphan
(ECPO) viruses. The only known natural host for porcine
enteroviruses is the pig, although experimentally infect-
ed pregnant guinea pigs developed placental lesions
(Lieu 1976).
ETIOLOGY
Physicochemical Characteristics
Porcine enteroviruses resemble the enteroviruses of oth-
er species in their basic properties and are classified
within the family Picornaviridae. The virions are spheri-
cal, 25–31 nm in diameter, and nonenveloped, with a
buoyant density in cesium chloride of 1.34. They contain
a core of single-stranded ribonucleic acid (RNA), which
is surrounded by a cubic capsid. Lipoprotein is lacking,
and the viruses are stable when treated with lipid sol-
vents. Porcine enteroviruses are also relatively stable to
heat and pH values between 2 and 9. Hemagglutination
has not been demonstrated for porcine enteroviruses.
They are relatively resistant to many disinfectants; of 10
commonly used disinfectants tested by Derbyshire and
Arkell (1971) against Talfan virus, only sodium
hypochlorite or 70% ethanol completely inactivated it.
Porcine enteroviruses are also highly resistant to the en-
vironment, with Teschen disease virus surviving for
more than 168 days at 15˚C (Ottis 1976). They survive
for long periods in liquid manure, in which they are in-
activated more rapidly if the manure is aerated (Lund
and Nissen 1983); they are also inactivated in liquid ma-
nure by ionizing radiation (Simon et al. 1983), and by
anaerobic digestion (Derbyshire et al. 1986).
Laboratory Cultivation
Porcine enteroviruses are readily cultivated in the labora-
tory in cell cultures of porcine origin. They are most fre-
quently grown in primary or secondary pig kidney (PK)
cell cultures or in established kidney cell lines such as
IBRS-2, but they may also be cultivated in other cells of
porcine origin such as the SST cell line. Some strains can
be grown in HeLa, monkey kidney, or baby hamster kid-
ney (BHK) cell lines. Different strains produce one of
three kinds of cytopathic effect (CPE) in PK cultures, and
this characteristic, together with the cytopathogenicity
of the viruses for BHK-21, HeLa, and Vero cells, enables
strains to be placed in one of four groups (Knowles et al.
1979).
Serologic Classification
The serologic classification of porcine enteroviruses is
based upon the virus-neutralization (VN) test; numerous
attempts to achieve such a classification have been
recorded in the literature. The early work in this area was
by Dunne et al. (1971) and extended by Knowles et al.
(1979); Table 12.1 is based mainly upon these authors’
findings. A complement-fixation test, suitable for the
rapid screening and typing of porcine enteroviruses, has
also been described (Knowles and Buckley 1980). Subse-
quent findings (Knowles 1983) suggest that additional
serogroups may exist. Some limited cross-reactivity
145
146 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

among the existing serogroups is evident, and Hazlett CLINICAL SIGNS

and Derbyshire (1978) showed that gastrointestinal anti-
bodies were more broadly specific than serum antibod-
ies.
EPIDEMIOLOGY
Although porcine enterovirus infection is most frequent-
ly asymptomatic, various clinical syndromes have been
associated with certain serogroups, as indicated in Table
12.2 and outlined below.

The virulent serogroup 1 strains associated with classical Polioencephalomyelitis

Teschen disease appear to be restricted to those areas in
which the disease occurs, and they have not been isolat-
ed in North America. Less virulent serogroup 1 viruses
and representatives of the other serogroups appear to be
ubiquitous (Odend’hal 1983). Transmission of porcine
enterovirus infection is most frequently by the fecal-oral
route, and indirect transmission by fomites is extremely
likely to occur since the viruses are relatively resistant.
Endemic infection with several serogroups of porcine en-
teroviruses can always be demonstrated in conventional
herds and is probably maintained in groups of weaned
piglets. Singh and Bohl (1972) demonstrated waves of
infection with six different serotypes over a period of 26
months in a long-term study of enterovirus infection in a
single herd. Infection is normally acquired by piglets
shortly after weaning when maternally derived antibod-
ies are withdrawn and pigs from several litters are mixed
together, and it persists for at least several weeks. Adults
rarely excrete virus but have high antibody levels. Pigs of
any age are, however, fully susceptible to infection with a
porcine enterovirus belonging to a serogroup to which
they have not previously been exposed.
The most severe form of polioencephalomyelitis is that
produced by the highly virulent serogroup 1 strains that
cause Teschen disease. This is a disease of high morbidi-
ty and high mortality, affecting all ages of swine and as-
sociated with major economic losses. The early signs of
Teschen disease include fever, anorexia, and listlessness,
rapidly followed by locomotor ataxia. In severe cases
there may be nystagmus, convulsions, opisthotonus, and
coma. Paralysis ensues, and the animal may assume a
dog-sitting posture or remain in lateral recumbency.
Stimulation by sound or touch may elicit uncoordinated
limb movements or opisthotonus. Death is common
within 3 or 4 days of the onset of clinical signs. Since the
appetite returns after the acute phase, some animals may
be kept alive by careful nursing, but these cases show
muscle wasting and residual paralysis. The less virulent
type 1 strains (Talfan disease, benign enzootic paresis)
and the other serogroups associated with polioen-
cephalomyelitis produce a milder disease with relatively
low morbidity and mortality. Mainly young piglets are
affected, and the disease rarely progresses to complete
paralysis.

Table 12.1. Serologic classification of porcine en- Reproductive Disorders

teroviruses The term SMEDI was introduced initially (Dunne et al.
Sero- 1965) to designate a group of viruses, subsequently
group Reference shown to be porcine enteroviruses, that had been isolat-
No. CPE Strain Other Strains ed in association with stillbirth (S), mummified fetuses
1 I Teschen Talfan, PS34 (SMEDIC), PS35, (M), embryonic death (ED), and infertility (I). Subse-
F65, J1, WR1, T1, ECPO-3, E1, quent studies by the same group of workers and by oth-
PE6 ers (De Meurichy et al. 1976) indicated that the syn-
2 I T80 T52A, O3b, F17, F59, E4, J2, T3 drome could be reproduced experimentally. However, it
3 I O2b PS2–PS13, PS14 (SMEDI B),
PS15–PS20, F34, PE1, PE3, is now well established that parvovirus infection may al-
PE10 so lead to embryonic death and fetal mummification,
4 I PS36 PS38, F78, DE8 and this virus may be more frequently associated with
5 I F26 F12, J3 these disorders of early and midgestation. Other find-
6 I PS37 (SMEDI E) T4, WR4, F7, ECPO-2, J5
7 I F43 WR2
8 II PS27 (SMEDI A) PS23, PS25, PS26, PS28–PS30,
PS32 (SMEDI D), V13, A1, Table 12.2. Natural or experimental clinical syn-
WR3, WR5, WR6, ECPO-1, dromes associated with porcine enterovirus infection
ECPO-5, CHICO, PE4, PE5, Syndrome Serogroups
PE7
9 III UKG410/73 UKG139/3, UKG194/73, Polioencephalomyelitis 1, 2, 3, 5
UKG298/73(c), UKG380/73, Reproductive disorders 1, 3, 6, 8
Pd2 Diarrhea 1,2,3,5,8
10 III LP54 GF50 Pneumonia 1,2,3,8
11 I UKG173/74 MV1-76 Pericarditis and myocarditis 2,3
 CHAPTER 12 ENTEROVIRUS Derbyshire
ings (Cropper et al. 1976) substantiate a role for en-
teroviruses as well as parvoviruses in these disorders,
and experimental (Bielanski and Raeside 1977) and field
(Kirkbride and McAdaragh 1978) data confirm an asso-
ciation between enterovirus infection and abortion in
swine. These reproductive disorders are not usually ac-
companied by clinical signs in the sow or gilt.
Diarrhea
The role of porcine enteroviruses as enteric pathogens is
uncertain. They have frequently been isolated from the
feces of piglets with diarrhea; but since they can be read-
ily isolated from normal piglets, particularly postwean-
ing, and since diarrhea can be caused by a variety of oth-
er viral and bacterial agents, their presence may be
coincidental. However, diarrhea has been produced ex-
perimentally by enteroviruses in piglets believed to be
free of other pathogens. The diarrhea is mild and rela-
tively transient, and it seems clear that porcine en-
teroviruses are considerably less important enteric
pathogens than rotaviruses or coronaviruses. When
piglets were infected with porcine enteroviruses together
with rotaviruses, the disease was less severe than in
piglets infected only with the rotavirus (Janke et al.
1988).
Pneumonia, Pericarditis, and Myocarditis
The role of enteroviruses as respiratory pathogens is also
uncertain. It is probable that alone they rarely cause clin-
ical signs of respiratory disease, although Pospisil et al.
(1971) noted increased respiration, coughing, snorting,
reduced appetite, and depression in piglets exposed to
an aerosol of porcine enterovirus. While pathogenic
studies indicate some degree of tropism of these viruses
for the lung, the pneumonia produced is usually subclin-
ical. Two serotypes of porcine enterovirus have been
shown experimentally to be capable of producing peri-
carditis, and in one experiment myocardial involvement
occurred (Long and Koestner 1969). These findings
might lead to a suspicion of enterovirus infection in the
case of sudden death in piglets, although encephalomy-
ocarditis virus might be a more likely candidate.
PATHOGENESIS
Natural infection occurs by ingestion of the virus, and it
is well established (Long 1985) that initial replication oc-
curs in the tonsil and intestinal tract. The large intestine
and ileum are infected more frequently than the upper
small intestine, and the former tissues contain higher
titers of virus. It has not been clearly established which
cells in the intestine support viral replication, but by
analogy with experiments on poliovirus (Kanamitsu et
al. 1967) it is probable that the reticuloendothelial tissue
147
of the lamina propria is involved. Epithelial cell destruc-
tion is not a feature of enterovirus infections. Viremia
follows regularly in infections with the virulent
serogroup 1 viruses but occurs less regularly with the less
virulent strains, leading to infection of the central ner-
vous system (CNS) (Holman et al. 1966). It may be as-
sumed that the pregnant uterus is also infected by
viremic spread of the virus, since embryonic or fetal in-
fections were demonstrated in gilts following nasal or
oral inoculation of enterovirus (Huang et al. 1980). In-
tranasal inoculation of the virus may lead experimental-
ly to lung infection (Meyer et al. 1966), but the signifi-
cance of the natural inhalation of viral aerosols is not
known. It has also been clearly demonstrated that when
piglets are inoculated parenterally with swine en-
teroviruses, the virus rapidly infects the intestine. Ex-
traintestinal infections are relatively transient, whereas
the virus persists in the large intestine for several weeks.
LESIONS
No specific changes have been associated with intestinal
enterovirus infections. They do not appear to cause vil-
lous atrophy, which is characteristic of primary intesti-
nal pathogens such as coronaviruses and rotaviruses.
Other than muscle atrophy in chronic cases, no gross le-
sions are found in polioencephalomyelitis. The histolog-
ic lesions associated with the latter are widely distributed
in the CNS but are especially numerous in the ventral
columns of the spinal cord, the cerebellar cortex, and the
brain stem. The changes are more marked and extensive
in Teschen disease than in milder encephalomyelitides
such as Talfan disease. The neurons show progressive,
diffuse chromatolysis (Koestner et al. 1966), and there
are focal areas of gliosis and perivascular lymphocytic
cuffing. Some meningeal infiltration with lymphocytes,
particularly over the cerebellum, may also occur.
The SMEDI syndrome is remarkable for the lack of
specific lesions in stillborn or neonatal piglets, although
mild focal gliosis and perivascular cuffing in the brain
stem have been found occasionally. Placental changes are
restricted to nonspecific degeneration.
Pneumonic lesions have been produced by several in-
vestigators. Smith et al. (1973) described areas of grayish
red consolidation in the ventral anterior lobes of lungs
infected with a serogroup 2 strain. There were exudates
in the alveoli and bronchi, slight perivascular and peri-
bronchiolar cuffing, and some hyperplasia of the bron-
chiolar epithelium.
A serogroup 3 strain consistently produced serofibri-
nous pericarditis experimentally, and the more severely
affected piglets showed focal myocardial necrosis (Long
and Koestner 1969).
148 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
DIAGNOSIS
The occurrence of clinical signs associated with polioen-
cephalomyelitis is suggestive of viral infection, but the
differentiation of enteroviral infection from other neu-
rotropic viruses requires isolation of the virus from the
CNS or the demonstration of viral antigen by specific im-
munofluorescence. Virus isolation from the CNS re-
quires the collection of tissues from a piglet showing ear-
ly nervous signs; animals that have been paralyzed for
several days may no longer contain infectious virus in the
CNS (Lynch et al. 1984). The virus may be isolated in PK
cell cultures from suspensions of spinal cord, brain stem,
or cerebellum; it may subsequently be identified on the
basis of its physicochemical characteristics, by immuno-
fluorescence (Watanabe 1971), or immunoperoxidase
staining (Sulochana and Derbyshire 1978). Serologic
identification of the isolate is desirable. The isolation of
an enterovirus from the gastrointestinal tract of a piglet
with nervous signs does not establish the etiology of the
disease, since the enteric infection may be coincidental.
An enzyme-linked immunosorbent assay, suitable for
mass screening, has been described for the detection of
antibodies against Teschen disease virus (Hubschle et al.
1983). In the SMEDI syndrome, mummified fetuses car-
ried to term rarely contain live virus but may contain vi-
ral antigen detectable by immunofluorescence. En-
terovirus isolation in PK cell culture may be attempted
from tissues of aborted or stillborn fetuses. Lung tissue
appears to be the most reliable source for the isolation of
porcine enteroviruses from fetuses (Huang et al. 1980).
VN tests on the body fluids of such fetuses can be carried
out against the SMEDI-associated serogroups. Serology
on the sow is not of value. In the investigation of pneu-
monia or diarrhea, virus isolation from the respiratory or
intestinal tract may be attempted, but the virologic find-
ings should be cautiously interpreted, especially in rela-
tion to diarrhea, since enteric infections with en-
teroviruses are common in healthy piglets.
TREATMENT
As in most viral infections, control measures for porcine
enteroviruses depend on prevention rather than treat-
ment. Potential antiviral chemotherapeutics for porcine
enteroviruses have received little attention. Piglets with
mild polioencephalomyelitis may recover if nursing care
is provided during the period of transient paresis.
PREVENTION
Vaccination has been practiced in the field only for the
control of Teschen disease. The earlier Teschen disease
vaccines, containing inactivated virus of pig tissue ori-
gin, have been superseded by attenuated or inactivated
cell culture vaccines. Mayr and Correns (1959) attenuat-
ed Teschen disease virus by cell culture passage and
showed that live or formalin-inactivated vaccines pre-
pared from this virus induced similar levels of protection
in piglets. Success has been claimed for a Teschen disease
eradication program involving ring vaccination and
slaughter (Schaupp 1968). Restrictions on the import of
swine and pork products from areas in which Teschen
disease is endemic seem to be effective in limiting the
spread of virulent serogroup 1 viruses. If such strains
were introduced into North America, they would be con-
trolled by a policy of quarantine and slaughter.
Vaccination has not been practiced against the milder
forms of polioencephalomyelitis or against the other
clinical manifestations of enterovirus infection in swine.
Of the latter, only the SMEDI syndrome is of sufficient
economic importance to justify specific control measures
in the field, but the multiplicity of serogroups that may
be involved complicates the development of an effective
vaccine. The best current approach to the prevention of
reproductive disorders associated with enteroviruses
would appear to be the application of management prac-
tices that ensure that gilts are exposed to infection with
endemic enteroviruses at least 1 month before breeding.
This can be achieved naturally if the animals remain in a
single building from birth to breeding, with thorough
mixing of piglets from different litters at weaning; but if
breeding stock is segregated at an early age, they should
be contaminated with fecal material from recently
weaned piglets. This can be readily accomplished by
adding fresh feces to the feed of gilts or by dosing gilts
with capsules of feces, which should be a pooled sample
collected from weaned piglets in several pens to ensure
exposure to as wide a range as possible of the virus pres-
ent in the herd. The operation of a closed-herd system re-
duces the risk of introducing extraneous viruses, but it is
not possible to eliminate this risk, since the relatively re-
sistant enteroviruses can be transmitted by a variety of
fomites. If the introduction of fresh stock is essential for
breeding purposes, before the gilts or sows are bred they
should be exposed (by fecal contamination as described
above) to any virus that may be present or introduced.
Exclusion of porcine enteroviruses by repopulation
of herds with specific-pathogen-free (SPF) stock seems to
be difficult or impossible to achieve over a prolonged pe-
riod, since enteroviruses have been isolated from a com-
mercial SPF herd (Derbyshire et al. 1966) and the acci-
dental introduction of Talfan virus into SPF gilts
maintained under strict isolation has been described
(Parker et al. 1981).
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tured cells infected with porcine enteroviruses. Jpn J Vet
Res 19:1–6.
 Hemagglutinating
13 Encephalomyelitis Virus
M. B. Pensaert
In Canada in 1962, Greig et al. isolated a previously un-
recognized viral pathogen of swine from the brain of
suckling pigs with encephalomyelitis. The virus respon-
sible for this disease was called hemagglutinating en-
cephalomyelitis virus (HEV) because of its hemaggluti-
nating properties; it was later classified as a coronavirus
(Greig et al. 1971; Phillip et al. 1971).
In 1969, an antigenically similar, if not identical,
virus was isolated in England from suckling pigs showing
anorexia, depression, and vomiting, but without signs
clearly associated with encephalomyelitis (Cartwright et
al. 1969). Animals that did not die remained stunted in
growth; the condition was therefore called vomiting and
wasting disease (VWD). Mengeling and Cutlip (1976)
were later able to experimentally reproduce both of the
major forms of the disease (i.e., the clinically apparent
encephalomyelitis and VWD) using the same field iso-
lates. Although epizootiologic studies have revealed that
infection of swine with HEV is prevalent, naturally oc-
curring disease is uncommon. Neonatal pigs are usually
protected by passively acquired colostral antibody, and
they subsequently develop an age-related resistance to
the potential clinical effects of the virus. Therefore, stud-
ies on interactions between the virus and swine have
been scarce in recent years.
ETIOLOGY
Virus Structure
HEV has an electron microscopic appearance similar to
other coronaviruses. Negatively stained particles have a
spherical shape, with an overall diameter of 120 nm
(Greig et al. 1971). Club-shaped surface projections
arranged as a “solar corona” protrude from the envelope.
Lamontagne et al. (1981) showed that the viral particle
contains two concentric membranes (an external enve-
lope and an inner membranous bag) encircling a central
core.
Studies on the chemical composition revealed that
the virus contains five polypeptides, four of which are
glycosylated, with molecular weights from 31,000 to
180,000 daltons (Pocock and Garwes 1977; Callebaut
and Pensaert 1980). The viral nucleic acid is considered
to be of the RNA type since the growth of HEV is not af-
fected by inhibitors of DNA metabolism (Greig and Gi-
rard 1969). The buoyant density is 1.21 g/cm3 in cesium
chloride (Mengeling and Coria 1972) and 1.18 g/cm3 in
potassium tartrate (Greig and Bouillant 1972).
Physicochemical Properties
The virus is stable between pH 4 and 10 and moderately
sensitive to heat (Chappuis et al. 1975). All viral infectiv-
ity is lost after 30 minutes at 56°C, but the infectivity titer
is reduced by only 0.8 log10 after 7 days at 4°C. HEV was
shown by Greig and Girard (1969) to be sensitive to lipid
solvents, including sodium desoxycholate. Lipid solvent
agents are thus suitable for disinfection. Ultraviolet irra-
diation also results in a significant reduction of viral in-
fectivity (Pensaert and Callebaut 1974).
Biologic Properties
The natural host of HEV is the pig. The virus also has
been adapted experimentally to replicate in mice (Kaye
et al. 1977; Yagami et al. 1986) and Wistar rats (Hirano
et al. 1993). In mice, the virus is neurotropic, and mouse
susceptibility was found to be influenced by age and in-
oculation routes (Yagami et al. 1993). Four-week-old
Wistar rats died of encephalitis after inoculation of HEV
via different routes (Hirano et al. 1993).
HEV was first isolated in primary cultures of pig kid-
ney (PK) cells by Greig et al. (1962), who described a cy-
topathic effect (CPE) characterized by the appearance of
syncytia. Many of these syncytia degenerated soon after
their formation, detached from the cell sheet, and float-
ed in the medium as semiopaque gelatinous masses. The
incorporation of a noncytotoxic amount of diethylami-
noethyl-dextran may enhance the CPE (Sato et al. 1983).
Using the immunofluorescence (IF) test, HEV was al-
so shown to propagate in several other porcine cell cul-
tures, such as adult thyroid gland, embryonic lung, testi-
cle cell line, PK-15 cell line (Pirtle 1974), IBRS2 cell line
(Chappuis et al. 1975), SK cell line (Lucas and Napthine
1971), SK-K cell line (Hirano et al. 1990), and swine em-
bryo kidney cell line KSEK6 (Kadoi et al. 1994). Non-
porcine cell cultures were shown to have little suscepti-
bility for growth of HEV.
Virus particles can be seen by electron microscopy in
cytoplasmic vesicles of infected cells. Assembly occurs
by budding through membranes of the endoplasmic
reticulum (Ducatelle et al. 1981).
HEV was demonstrated to possess a virion-associated
hemagglutinin. The virus spontaneously agglutinates
151
152 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
erythrocytes of mice, rats, chickens, and several other
animals (Girard et al. 1964). Elution of HEV from red
blood cells has not been observed. The S protein of HEV
is shown to be a hemagglutinin requiring N-acetyl-9-O-
acetylneuraminic acid as a receptor determinant on the
surface of erythrocytes (Schultze and Herrler 1993). Us-
ing the same kinds of erythrocytes, a hemadsorption test
can be used to demonstrate viral growth in inoculated
cell cultures.
Antigenic and Genomic Characteristics
Although HEV is known to be the cause of different clin-
ical syndromes, only one serotype of the virus is known
to exist. The susceptibility of the infected pigs and strain
differences in virulence are probably responsible for the
fact that usually only one of the syndromes is seen in a
particular outbreak. An antigenic relationship exists be-
tween HEV and the bovine coronavirus (BCV), as shown
by seroneutralization (SN), hemagglutination inhibition
(HI) (Sato et al. 1980), IF, and immunoelectron mi-
croscopy (Pensaert et al. 1981). HEV is also related to hu-
man respiratory coronavirus OC43 (Kaye et al. 1977) and
mouse hepatitis virus (Pedersen et al. 1978). Nucleotide
sequence analysis of the region covering the S2 probe re-
vealed 92.6% nucleotide sequence homology to BCV and
91.9% to HCV-OC43 (Vieler et al. 1995). Moderate cross-
reactivity was observed between HEV and turkey enteric
coronavirus (Dea and Tijssen 1989).
EPIDEMIOLOGY
Pigs are the only species known to be naturally suscepti-
ble to HEV infection. Most of the infections in this
species are subclinical, and the economic importance of
the disease is low. The spread of HEV in the pig popula-
tion has been studied in several countries. The HI and SN
tests proved to be almost equally sensitive in the demon-
stration of specific antibodies to HEV in swine sera
(Mengeling 1975). Serologic surveys revealed that infec-
tion of swine with HEV is very common and probably
worldwide. In fattening pigs, 31% of the sera were posi-
tive in Canada (Girard et al. 1964), 46% in Northern Ire-
land (McFerran et al. 1971), 49% in England (Cartwright
and Lucas 1970), 52% in Japan (Hirai et al. 1974), 75% in
Germany (Hess and Bachmann 1978), and 0–89% in the
United States, depending on the region surveyed (Men-
geling 1975). The percentage of sows with antibodies at
slaughter varied from 43% in Northern Ireland to 98% in
the United States. A high number of seropositive ani-
mals was also found in Denmark (Sorensen 1975),
France (Vannier et al. 1981), Australia (Forman et al.
1979), Belgium (Pensaert et al. 1980), and Austria (Möstl
1990). Conversely, Neuvonen et al. (1982) found 40
Finnish elite breeding pig herds to be free of seropositive
animals.
HEV was isolated from the respiratory tracts of pigs
with respiratory illness in Japan in 1984 (Hirahara et al.
1987). The first isolation of this virus in Taiwan was ob-
tained from 30- to 50-day-old pigs with central nervous
disease (Chang et al. 1993).
Under experimental conditions, disease has been
produced in most instances when nonimmune pigs have
been exposed oronasally to HEV sometime during the
first few weeks of life (Alexander 1962; Appel et al.
1965). Clinical signs may vary, however, and in a study in
which several field isolates of HEV were compared as to
virulence, the severity of such signs was related to a dif-
ference in host susceptibility (even among littermates) as
well as to the apparent virulence of each isolate (Men-
geling and Cutlip 1976). In contrast, older pigs and
neonatal pigs that had received antibody in colostrum
were usually clinically unaffected when exposed to HEV
under otherwise similar conditions (Appel et al. 1965).
These observations are believed to explain why naturally
occurring disease is relatively uncommon even though
HEV is ubiquitous among swine. In herds where HEV in-
fection is enzootic, most pigs receive protective antibody
in colostrum, and circulating maternal antibodies persist
for about 4–18 (mean 10.5) weeks (Paul and Mengeling
1984). By the time such antibody wanes, the pigs have al-
ready developed an age-related resistance to the disease.
Additional support for this idea is provided by a serolog-
ic study on two Belgian breeding farms, which showed
that passively acquired colostral immunity was replaced
by active immunity as a consequence of subclinical in-
fection of pigs between 8 and 16 weeks of age (Pensaert
et al. 1980).
CLINICAL SIGNS
Upon infection with HEV, two clinical syndromes are
possible: an acute, clinically apparent encephalomyelitis
and VWD. Both syndromes are mainly confined to pigs
less than 3 weeks of age, although older swine may occa-
sionally vomit and have a brief period of inappetence,
listlessness, and nervous signs. Encephalomyelitis
caused by HEV was indeed diagnosed in 30- to 50-day-
old pigs in Taiwan (Chang et al. 1993). Prior to the Tai-
wanese report, a typical encephalomyelitic form had
been described only in Canada (Alexander et al. 1959)
and the United States (Werdin et al. 1976). Both syn-
dromes show many signs in common, and between the
acute encephalomyelitis and the chronic VWD, all de-
grees of severity may occur.
At the start of a VWD outbreak, sneezing or cough-
ing may occur. The primary sign seen after an incubation
period of 4–7 days is repeated retching and vomiting.
Pigs under 4 weeks of age start suckling but soon stop,
withdraw from the sow, and vomit the milk they have
taken in. They huddle together, look pale and listless,
and often have an arched back. Body temperature can be
elevated at the beginning of the disease but returns to
 CHAPTER 13 HEMAGGLUTINATING ENCEPHALOMYELITIS VIRUS
the normal range within 1–2 days. Affected pigs are often
observed to grind their teeth. They dip their mouths into
water bowls but drink little, if at all, indicating a possible
pharyngeal paralysis. The persistent vomiting and de-
creased food intake result in constipation and a rapid de-
cline of condition. The youngest pigs become severely
dehydrated after a few days, exhibit dyspnea and
cyanosis, fall into a coma, and die. Older pigs lose their
appetite and rapidly become emaciated. They continue
to vomit, although less frequently than in the early stage
of the disease. Some pigs develop a large distension of
the cranial abdomen. This “wasting” state may persist for
several weeks until the pigs die of starvation. Mortality
approaches 100% within the same litter, and survivors
remain permanently stunted.
An outbreak of the encephalomyelitic form may start
as a VWD outbreak. Some pigs begin to vomit 4–7 days
after birth. The vomiting is continued intermittently for
1–2 days but is rarely severe and does not result in dehy-
dration. In other outbreaks, the first sign is acute depres-
sion and a tendency to huddle. Pigs may become sick as
soon as 2 days after birth. Occasionally, sneezing, cough-
ing, or upper respiratory embarassment is observed. The
pigs lose weight rapidly and their hair loses its luster and
becomes rough.
After 1–3 days, symptoms of a severe en-
cephalomyelitis arise. Younger pigs are most severely af-
fected and exhibit various combinations of nervous
signs. Generalized muscle tremors and hyperesthesia are
common findings. Pigs that are able to stand usually
have a jerky gait, and they tend to walk backward, often
ending in a dog-sitting position. They soon become very
weak, are unable to rise, and paddle their limbs. Their
noses and feet become cyanotic. Blindness, opistho-
tonus, and nystagmus can also occur. Finally, the pigs be-
come prostrate, lying on their sides, and have dyspnea.
In most cases, coma precedes death. Mortality in
younger pigs is usually 100%.
Older pigs usually suffer a mild transient illness in
which posterior paralysis may be the most common sign.
The paresis in a few cases is accompanied by blindness.
The outbreak described in Taiwan (Chang et al. 1993)
in 30- to 50-day-old pigs was characterized by fever, con-
stipation, hyperesthesia, muscular tremor, progressive
anterior paresis, posterior paresis, prostration, recum-
bency, and paddling movements with a morbidity rate of
4% and a mortality rate of 100%. The pigs died within
4–5 days after the onset of clinical signs.
The interval for onset of the disease in the first litter
to cessation of the disease or its failure to appear in a lit-
ter is usually 2–3 weeks (Werdin et al. 1976). Disappear-
ance of the disease coincides with the time it takes sows
to develop immunity and to pass this protection on to
their offspring. It has been shown that pigs exposed to
HEV develop seroneutralizing and hemagglutination-in-
hibiting antibodies (Pensaert and Callebaut 1974).
Pensaert 153
PATHOGENESIS
Several observations indicate that HEV is able to repli-
cate in the upper respiratory tract with or without pro-
ducing clinical signs. Mengeling et al. (1972), Pensaert et
al. (1980), and Hirahara et al. (1989) recorded the isola-
tion of HEV from the nasal cavity, trachea, and lungs of
diseased and healthy pigs. Respiratory signs such as
sneezing and nasal secretion were produced after in-
tranasal or oral inoculation of conventional pigs at the
age of 50 days but not after subcutaneous inoculation
and not in intranasally inoculated 70-day-old animals
(Hirahara et al. 1989). Hirahara et al. (1989) obtained
only sneezing and nasal secretion in 10-day-old,
colostrum-deprived piglets upon intranasal inoculation;
intracerebral inoculation resulted in nervous signs in on-
ly two of seven animals. The virus is excreted for 8–10
days in oronasal secretions (Pensaert and Callebaut
1974; Hirahara et al. 1989). The mode of transmission
occurs through nasal secretions. Most infections under
field circumstances have a subclinical course.
Typical clinical disease was reproduced by oronasal
inoculation of colostrum-deprived piglets. In a series of
studies on the pathogenesis of the disease, Andries and
Pensaert (1980c) inoculated newborn colostrum-de-
prived pigs oronasally with an HEV strain from pigs
showing the VWD syndrome. Anorexia and vomiting
were seen after an incubation period of 4 days. Pigs were
killed at different times after inoculation; results of the
examination by the immunofluorescent antibody tech-
nique revealed that the epithelial cells of nasal mucosa,
tonsils, lungs, and small intestine served as sites of pri-
mary viral replication.
After local replication near the sites of entry, the
virus spread via the peripheral nervous system to the
central nervous system (CNS). At least three pathways
appeared to be involved. A first pathway led from the
nasal mucosa and tonsils to the trigeminal ganglion and
the trigeminal sensory nucleus in the brain stem. A sec-
ond pathway occurred along the vagal nerves via the va-
gal sensory ganglion to the vagal sensory nucleus in the
brain stem. A third pathway led from the intestinal
plexuses to the spinal cord, also after replication in local
sensory ganglia. Earlier studies had already shown that
viremia is probably of little or no importance in the
pathogenesis (Andries and Pensaert 1980b).
In the CNS, the infection started in well-defined nu-
clei of the medulla oblongata but progressed later into
the entire brain stem, the spinal cord, and sometimes al-
so the cerebrum and cerebellum. Fluorescence in the
brain was always restricted to the perikaryon and
processes of neurons (Fig. 13.1). Vomiting was induced
by viral replication in the vagal sensory ganglion (gan-
glion distale vagi) or by impulses to the vomiting center
produced by infected neurons at different sites (Andries
1982).
154 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
To elucidate the pathogenesis of the wasting, radio-
logic studies were performed on chronically infected
pigs, vagotomized pigs, and controls (Andries 1982).
The stomach of control pigs was always empty within 10
hours, whereas barium was retained in the stomach for
2–7 days in pigs with HEV. In pigs with a bilateral ab-
dominal vagotomy, the stomach emptying was less dis-
turbed. This indicated not only that the delayed empty-
ing in pigs with HEV was due to earlier viral replication
in the vagal ganglion and vagal nuclei in the brain but
that the virus-induced lesions in the intramural plexuses
of the stomach were probably also responsible for the
gastric stasis. The disturbance of the stomach emptying
was considered to play an important role in the patho-
genesis of the wasting.
LESIONS
The only significant gross lesions reported in natural
HEV infections are cachexia and a distension of the ab-
domen, which develops in some chronically affected pigs
(Schlenstedt et al. 1969; Hoorens et al. 1977). The stom-
achs of such pigs are dilated and filled with gas.
Microscopic lesions are found in the tonsils, the ner-
vous system, respiratory system, and stomach of acutely
diseased pigs. The lesions tend to disappear in animals
surviving acute stages of the disease. Tonsillar changes
are characterized by epithelial degeneration and lym-
phatic cell infiltration in the crypts (Narita et al. 1989a).
Degeneration of the epithelial cells in the turbinates,
bronchi, and alveoli and interstitial peribronchiolar
pneumonia with infiltration of neutrophils and
macrophages were observed in 20% of naturally infected
pigs (Hoorens et al. 1977) and to a much larger extent in
experimentally infected pigs (Cutlip and Mengeling
1972; Hirahara et al. 1989).
13.1. Viral multiplication in the
brain stem of a pig inoculated with
HEV. Fluorescence is seen in the
axon and the perikaryon of a neuron
(×500).
A nonsuppurative encephalomyelitis was reported in
70–100% of pigs with nervous signs and in 20–60% of
pigs showing the VWD syndrome. The lesions are char-
acterized by perivascular cuffing, gliosis, and neuronal
degeneration (Richards and Savan 1960; Alexander
1962; Hoorens et al. 1977; Narita et al. 1989b; Chang et
al. 1993). They are most pronounced in the gray matter
of the pons Varolii, the medulla oblongata, and the dor-
sal horns of the upper spinal cord. It was suggested that
encephalitic lesions are a specific immune response to
HEV following its replication in the CNS (Narita et al.
1989b). Neuritis of peripheral sensory ganglia, particu-
larly the trigeminal ganglia, also occurs.
Microscopic changes in the stomach wall and the
lungs were found only in pigs showing the VWD syn-
drome. Degeneration of the ganglia of the stomach wall
and perivascular cuffing were present in 15–85% of dis-
eased animals. The lesions were most pronounced in the
pyloric gland area (Steinicke and Nielsen 1959; Schlen-
stedt et al. 1969; Hoorens et al. 1977).
DIAGNOSIS
To effect diagnosis by virus isolation, the tonsils, brain,
and lungs are dissected aseptically from young diseased
piglets slaughtered as soon as possible after the first
signs of infection. It is very difficult to isolate the virus
from pigs that have been sick for more than 2 days.
Crude suspensions of selected tissues are inoculated into
primary PK cells or secondary pig thyroid cells. The pres-
ence of HEV is shown by the developing of syncytia,
hemadsorption, and hemagglutination (Andries and
Pensaert 1980a). A single blind passage with cells and
culture fluid is recommended, since clinical specimens
from pigs infected with HEV often contain very small
amounts of infectious virus.
 CHAPTER 13 HEMAGGLUTINATING ENCEPHALOMYELITIS VIRUS
Antibodies to the virus can be detected by the SN,
plaque reduction, or HI test (Mengeling 1975; Sato et al.
1983). Since subclinical infections with the virus are very
common, antibody titers must be evaluated very careful-
ly. Moreover, a significant rise in antibody titer can be
obtained only if acute sera are taken as soon as possible
after the appearance of clinical signs. Pigs that become ill
after an incubation period of 6–7 days may indeed have
already built up a high antibody titer at that moment,
making an interpretation of paired sera very difficult.
Differential diagnosis must be made between HEV in-
fection, Teschen-Talfan disease, and Aujeszky’s disease.
Clinical signs of encephalomyelitis associated with the
latter two diseases are usually more severe than those as-
sociated with HEV infection and may appear among old-
er pigs as well as piglets. Respiratory signs in older pigs
and abortions in sows are typical for Aujeszky’s disease.
The viruses can be grown in PK and pig thyroid cells; in
PK cells they are distinguishable by their CPE. They can
be further differentiated by virus identification tests and
the production of hemagglutinin by HEV.
PREVENTION
On most breeding farms, HEV infection persists enzoot-
ically and is maintained through a subclinical infection
of the respiratory pathways. Sows usually will have come
into contact with the virus before the time of first far-
rowing. They will protect their offspring against clinical
signs by colostral antibodies. When infection occurs in
such pigs, it will remain subclinical. Only when sows are
not immune at the time of farrowing (on newly populat-
ed farms or on small farms in which the virus is not
maintained through lack of sufficient numbers of litters)
will an infection of pigs within the first weeks after birth
result in clinical signs. Maintaining the virus on farms to
obtain immune sows at the time of their first farrowing
creates a favorable situation in preventing break-
throughs of disease in piglets.
Once clinical signs are evident, the disease will run its
course; spontaneous recoveries are rare. Litters born 2–3
weeks after the onset of disease are usually protected by
maternal immunity. Before that time, piglets born from
nonimmune sows can be protected by specific hyperim-
mune serum injected at birth. However, the time lapse
between diagnosis and cessation of the disease is usually
too short to gain much profit from this procedure.
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 Classical Swine Fever (Hog Cholera)
14 J. T. Van Oirschot
To avoid confusion with the acronym HCV for hepatitis
C virus of humans, the term “classical swine fever virus”
instead of “hog cholera virus” is increasingly used. Con-
sequently, this designation will be adopted in this chap-
ter.
Classical swine fever (CSF) (hog cholera) is a highly
contagious viral disease of swine. The infection can run
an acute, subacute, chronic, atypical, or inapparent
course. Acute CSF is caused by virulent virus and gener-
ally results in high morbidity and mortality, whereas in-
fections with low-virulence virus can go unnoticed.
The question of whether CSF originated in the Unit-
ed States or elsewhere will probably remain conjectural.
According to Hanson (1957), the first description of a
cholera-like disease of swine was from Tennessee, in
about 1810. Later outbreaks were reported from Ohio in
the early 1830s. CSF may possibly have occurred in
France in 1822 and in Germany in 1833, but other re-
ports suggest that the disease first appeared outside the
United States in England in 1862 and subsequently
spread to the European continent (Fuchs 1968). CSF was
reported from South America in 1899 and from South
Africa in 1900.
Classical swine fever virus (CSFV) still has a world-
wide distribution, but gradually more countries succeed
in eradicating the virus. Countries free of the disease
are Australia, Belgium, Canada, France, Great Britain,
Iceland, Ireland, New Zealand, Portugal, the Scandina-
vian countries, Spain, Switzerland, and the United
States.
A state-federal CSF eradication program was begun
in the United States in 1962. The last U.S. outbreak oc-
curred in 1976. The total cost of the effort to eradicate
CSF amounted to about $140 million.
The European Community (EC) supports a program
to eliminate CSFV from its member countries. It is based
on the killing of infected herds and is supported by oth-
er veterinary legislative and zoo sanitation measures. In
spite of these combined efforts, outbreaks of CSF are
still reported from the EC countries, causing severe eco-
nomic losses; for example, in the Netherlands the direct
cost of the control program amounted to $93 million
in the period 1983–85. Severe epizootics plagued Bel-
gium and Germany in 1994, and the Netherlands in
1997.
ETIOLOGY
CSFV belongs to the genus Pestivirus. Bovine viral diar-
rhea virus (BVDV) and border disease virus (BDV),
which can infect cattle, sheep, and swine, are the two oth-
er members of this genus. In 1991, the genus Pestivirus
was allocated to the family Flaviviridae.
CSFV measures 40–50 nm in diameter, with a nucle-
ocapsid of about 29 nm. It is an enveloped RNA virus,
the membrane surrounding an isometric core. Fringelike
projections of 6–8 nm have been demonstrated on the
surface of the virion. The buoyant density, depending on
the gradient material and on the cells used to propagate
the virus, has been reported to be between 1.12 and 1.17
g/mL. Sedimentation coefficient values of 140–180 S
have been found (Horzinek 1981).
The virion has a single-stranded RNA of positive po-
larity and is about 12.3 kb long. The genome, which has
been completely sequenced, contains one large open
reading frame, coding for one large polyprotein of ap-
proximately 3900 amino acids (Meyers et al. 1989;
Moormann et al. 1990) that is cleaved co- and posttrans-
lationally by host-cell- and virus-encoded proteases to
yield mature viral proteins. The open reading frame is
flanked by a 5′ noncoding region of almost 400 nu-
cleotides and a 3′ noncoding region of approximately
200 nucleotides. The order of the gene products along
the open reading frame is as follows:
NH 2 -(N pro -C-E rns -E1-E2-p7-NS2.3-NS4A-NS4B -
NS5A-NS5B)-COOH
The (putative) function of individual proteins has
been described (Meyers and Thiel 1996). The nucleocap-
sid protein precedes the Erns protein (formerly called
gp44/48); the latter is located at the surface of the virion
as a homodimer but is also secreted from cells. The Erns
protein has ribonuclease activity, which is unique among
viruses (Schneider et al. 1993; Hulst et al. 1994). The
function of this enzymatic activity is as yet unclear. The
E1 protein (gp33) is present in the viral envelope as an
E1-E2 heterodimer, and the E2 (gp55), which is the most
immunogenic protein of CSFV, is present as a homo-
dimer and as a heterodimer with E1. The p7 protein is
probably not incorporated in the virion, and the remain-
159
160 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
ing C-terminal part of the open reading frame is coding
solely for nonstructural proteins.
The analysis of nucleotide sequences of the 5′ non-
coding region, the N-terminal part of E2, and a region of
NS5B yielded indications to divide CSFV strains into two
major groups (Hofmann et al. 1994; Lowings et al. 1996).
A marked antigenic variation also exists among strains of
CSFV (Wensvoort 1989; Edwards and Sands 1990) and
probably resides on the nonconserved antigenic unit on
the N-terminal half of E2 (Van Rijn et al. 1994) and on
E1 as well (Weiland et al. 1992). Even within some strains
of CSFV, antigenic heterogeneity is present (Wensvoort
et al. 1989). The use of monoclonal antibodies (MABs)
to study the diversity of CSFV strains supports the ge-
netic division of CSFV into two major groups (Lowings
et al. 1996). No relation was observed between antigenic
variants and virulence (Wensvoort et al. 1989). On the
other hand, field isolates that were neutralized more
readily by BVDV antibody than by CSFV antibody were
of reduced virulence (Kamijyo et al. 1977).
Although CSFV differs from BVDV and BDV on the
genetic and antigenic level, it also shows many similari-
ties with these other pestiviruses. The differences mostly
occur on the E2 protein, because most of the MABs that
differentiate CSFV from BVDV and BDV are directed
against E2 (Wensvoort 1989; Edwards et al. 1991; Paton
1995).
The close antigenic relationship between CSFV and
other pestiviruses is evidenced by cross-reactions in im-
munodiffusion, immunofluorescence, and, to a lesser de-
gree, neutralization tests. Certain strains of CSFV also in-
duce neutralizing antibody to BVDV, and pigs can be
partially immunized against CSF with BVDV. The com-
mon antigens among pestiviruses reside largely on the
nonstructural protein NS2.3. There is an overall homol-
ogy of about 70% on the amino acid level between CSFV
and BVDV strains (Paton 1995).
Field strains of CSFV vary widely in virulence. Strains
of high virulence induce acute disease and high mortali-
ty, whereas moderately virulent strains generally give rise
to subacute or chronic infections. Postnatal infection
with low-virulence CSFV results in mild disease or sub-
clinical infection. However, such low-virulence strains
can produce mortality in porcine fetuses and newborn
piglets. The outcome of CSFV infections with strains of
moderate virulence is partially determined by host fac-
tors such as breed, age, immune competence, and nutri-
tional condition, whereas in infections with highly viru-
lent or avirulent CSFV, host factors seem to play a minor
role. It has been reported that the virulence of CSFV may
be an unstable property, since enhancement of virulence
has been observed after one or more passages in pigs
(Dunne 1975).
Although CSFV can replicate in nonporcine cells,
porcine kidney cells are used most frequently for virus
growth. Virus replication is restricted to the cytoplasm of
the cell and does not result in a cytopathic effect. The
first-progeny virus is released from the cells at 5–6 hours
postinfection. Under single growth cycle conditions,
there is an exponential increase in virus titer until 15
hours postinfection, after which virus production con-
tinues at a high level for several days. In cell cultures, CS-
FV spreads by means of the medium, by cytoplasmic
bridges to neighboring cells, and from mother to daugh-
ter cells. CSFV can persist in cell culture. The virus seems
to mature at intracytoplasmic membranes, which is in
accordance with the inability to detect CSFV antigens at
the surface of the infected cell (Van Oirschot 1980).
Thermal and pH stability may vary per strain of CS-
FV. The higher the temperature, the quicker CSFV is in-
activated. Inactivation is partly dependent on the medi-
um containing the virus. Thus cell culture fluid
infectivity is lost after 10 minutes at 60˚C, whereas in de-
fibrinated blood the virus is not inactivated after 30 min-
utes at 68˚C. The virus is quite stable at pH 5–10. Above
and below these pH values, infectivity is relatively rapid-
ly destroyed. The inactivation rate below pH 5 is depen-
dent on the temperature: at pH 4 a half-life of 260 hours
was found at 4˚C and of 11 hours at 21˚C (Depner et al.
1992). Lipid solvents such as ether, chloroform, and de-
oxycholate quickly inactivate the virus. For disinfection,
2% sodium hydroxide is still considered most suitable. In
pens the virus appears to be inactivated in a few days; in
liquid manure of pigs, CSFV (initial titer of 105.5 TCID50
[median tissue culture infectious doses] per mL) may
survive for 2 weeks at 20˚C and more than 6 weeks at 4˚C
(Haas et al. 1995). In pork and pork products it can re-
main infective for months, which is of great epizootio-
logic importance.
EPIDEMIOLOGY
The pig is the only natural host and is the significant
source of spread of CSFV. Direct contact between infect-
ed and susceptible pigs is the principal means of viral
transmission. Infected pigs may shed the virus before the
onset of disease and continue to do so during the entire
disease period. CSFV is notably excreted with oronasal
and lacrimal secretions, urine, and feces. Pigs that recov-
er from CSF generally shed virus until specific antibodies
have developed. Thus pigs infected with virulent CSFV
may shed large amounts of virus for 10–20 days, where-
as postnatal infections with low-virulence strains are
characterized by short virus excretion periods. Conse-
quently, virulent CSFV will usually spread faster in a
herd and induce higher morbidity than low-virulence
strains. Chronically infected pigs shed the virus continu-
ously or intermittently till death.
When a pregnant sow is exposed to CSFV strains of
low virulence, the infection initially will often go unno-
 CHAPTER 14 CLASSICAL SWINE FEVER (HOG CHOLERA)
ticed, but the virus can be transmitted to the fetuses in
utero. Such a congenital infection usually results in still-
birth and/or birth of weak pigs, which die shortly after
birth. Because CSFV persists in fetuses, large quantities
of virus may then be disseminated at farrowing. Howev-
er, if congenitally infected piglets are born healthy and
remain so for months, they act as a hardly recognizable
and continuous source of viral spread (Van Oirschot and
Terpstra 1977). Thus CSFV infection may smolder in a
herd for months before the eventual diagnosis is made.
These persistent congenital infections are therefore of
utmost importance in the epidemiology of CSF, particu-
larly in regions where viruses of reduced virulence pre-
dominate.
The introduction of newly purchased, healthy-looking
infected pigs into a herd is the most common cause of CSF
outbreaks. The infection may originate from breeding
farms, or pigs may be exposed to the virus at places where
many pigs are assembled or during transport in contami-
nated vehicles. An important source of infection is virus-
containing garbage that has not been properly sterilized.
This mode of virus spread was responsible for 22% of the
outbreaks of CSF in the United States in 1973 (Dunne
1975). The virus can be carried over long distances in pork
and (frozen) pork products, which may lead to the in-
troduction of CSF into countries free of the disease.
Mechanical vectors contribute to the spread of CSFV
between premises. In this respect, trucks, farmers, vet-
erinarians, and other personnel with their equipment
constitute a great risk, but virus may also be mechanical-
ly transmitted by pets, birds, and arthropods (Stewart
1981). Airborne transmission has been demonstrated ex-
perimentally (Hughes and Gustafson 1960; Terpstra
1987) and thus may be a (probably infrequent) mode of
spread of CSFV between herds at close proximity.
CSFV can circulate in wild pig populations, in some
countries posing a threat to domestic pig husbandry.
Several countries, including those of the EC, have
eradication programs in force, based on rapid diagnosis
and killing of infected herds, supplemented by other
control measures. Despite these efforts, CSF has still not
been eliminated in many countries. This may be ac-
counted for by the high density of pigs in certain areas,
the movement over long distances of pigs and pork and
pork products, and the frequent inability to trace the
source of outbreaks. During the 1982–85 epizootic in
the Netherlands, the source of infection was recorded as
“unknown” in 54% of breeding and mixed herds and in
29% of fattening herds (Terpstra 1987). In Europe, the
origin of 13 out of 32 outbreaks (42%) in 1988 was un-
known (Picard 1989); in Germany 50 of the 117 cases
(43%) in 1994 had an unknown origin (Pittler et al.
1995). The emergence of CSFV strains of reduced viru-
lence is also a major factor in explaining hitherto unsuc-
cessful eradication programs. Such strains give rise to
Van Oirschot 161
persistent and inapparent infections that are clinically
hard to recognize and therefore may be diagnosed only
after the virus has spread to other herds.
CLINICAL SIGNS
When virulent CSFV first appears in a herd, only a few
pigs will show clinical signs of disease. Initially, the pigs
may only appear drowsy or less active; if they are dis-
turbed and made to stand, some will have arched backs
and others may appear chilled. Still others may stand
with drooping heads and straight tails. At this time, a re-
duced appetite is noticed. Later, it progresses to a
marked anorexia exemplified by the pig nosing around
in the feed for a short while before returning to its resting
place.
At the first sign of inactivity, fever can be recorded.
Within 6 days of exposure to CSFV, the temperature of
an affected animal may become higher than 42˚C, al-
though values between 41˚C and 42˚C are more common
during the course of the disease. Concurrent with the
temperature rise is a corresponding drop in the leuko-
cyte count. Total leukocyte counts of 9000 to as low as
3000/mm3 of blood may be found.
Early in the course of the disease, the eyes show a
marked discharge associated with conjunctivitis, which
may progress until the eyelids are completely adhered.
Constipation commonly develops during the period of
initial high temperature, followed by a severe, watery,
yellowish gray diarrhea. Sick pigs become chilled and
will huddle or pile on each other seeking warmth. It is
not uncommon for pigs to vomit a yellowish fluid con-
taining much bile. Convulsions may occur in a few swine,
which usually die within hours or at most a few days af-
ter the convulsions begin. Hyperemia of the skin may oc-
cur concurrently with the initial temperature rise.
As the disease progresses, more pigs become affected,
and those that were sick first become gaunt and tucked
up in the flank and have a characteristic weaving, stag-
gering gait that appears to be directly related to a weak-
ness in the hindquarters. This is usually followed by a
posterior paresis. A purplish discoloration extending
over the abdomen, snout, ears, and medial sides of the
legs may occur near the terminal stage of the disease
(Stewart 1981). Most pigs that suffer from acute CSF die
between 10 and 20 days postinfection. In subacute CSF,
pigs show less severe signs of disease and succumb with-
in 30 days (Dunne 1975).
Persistence of CSFV in the host can result in different
clinical syndromes. Mengeling and Packer (1969) de-
scribed three phases of chronic CSF based on the clinical
signs observed. In the first, or acute, phase of illness,
anorexia, depression, elevated temperature, and
leukopenia were present. After several weeks, the ap-
petite and general appearance of the pigs improved
162 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

markedly, and their temperatures decreased to normal
or slightly above normal values. Leukopenia usually per-
sisted. This general clinical improvement characterized
the second phase of illness. In the third phase, pigs again
became anorectic and depressed, with temperatures of-
ten elevated until shortly before death. Runt pigs can de-
velop during the course of chronic CSF. Such pigs are se-
verely retarded in growth, have skin lesions, and
frequently stand with arched backs. Pigs with chronic
CSF may survive for more than 100 days.
Van Oirschot and Terpstra (1977) reported a “late-on-
set” disease as a sequel of congenital CSFV infection. The
syndrome was characterized by an initial, relatively long
period during which pigs remained free of disease. Not
until a few months after primary exposure did the pigs
develop mild anorexia and depression, conjunctivitis,
dermatitis, diarrhea, and locomotive disturbances lead-
ing to posterior paresis. Body temperatures were normal.
Most pigs survived for more than 6 months, but all even-
tually died (Table 14.1).
Persistent, virtually inapparent infections can also
develop after postnatal inoculation with CSFV strains of
reduced virulence (Carbrey et al. 1977). Leukopenia is a
consistent manifestation of persistent CSF, but in the
terminal stages of disease, leukocytosis may develop.
Low-virulence strains of CSFV generally induce mild dis-
ease or subclinical infections, which can be accompanied
by leukopenia.
A congenital CSFV infection can result in abortion,
fetal mummification, malformations, stillbirth, and the
birth of weak pigs with tremors or of healthy-looking yet
infected piglets. Among piglets infected in utero, skin he-
morrhages are common and neonatal mortality is high.
However, piglets can recover from an in utero–acquired
CSFV infection.
PATHOGENESIS
Under natural conditions, the mode of entry of CSFV in
the pig is the oronasal route. Occasionally, the virus gains
access to the host through conjunctival or genital mu-
cous membranes or skin abrasions. The tonsil is the pri-
mary site of virus replication after oral and parenteral in-
fection.
The virus initially infects epithelial cells of the tonsil-
lar crypts and subsequently spreads to the surrounding
lymphoreticular tissue. From the tonsil, CSFV is trans-
ferred via lymphatic vessels to the lymph nodes draining
the tonsillar region. The virus replicates in the regional
lymph nodes, then reaches the peripheral blood, and
from that time grows to high titers in spleen, bone mar-
row, visceral lymph nodes, and lymphoid structures lin-
ing the small intestine. As a result of the growth in lym-
phoid tissue and in circulating leukocytes and
mononuclear cells, the level of viremia is high. The virus
probably does not invade parenchymatous organs until
late in the viremic phase. Generally, CSFV titers in lym-
phoid tissues are higher than in parenchymatous organs.
The spread of virulent virus throughout the pig is usual-
ly completed in 5–6 days (Ressang 1973).
The multiple hemorrhages seen in acute CSF are
caused by degeneration of endothelial cells in conjunc-

Table 14.1. General features of acute, chronic, and late-onset CSF

Feature Acute Chronic Late-Onset
Virulence High Moderate Low
of virus
Time of Postnatal Postnatal Prenatal
infection
Course of Short incubation period, severe Short incubation period, three Late onset of disease, gradually
illness depression, high fever, anorexia, phases of illness: (1) depression, aggravating depression and
conjunctivitis, constipation, fever, anorexia; (2) clinical anorexia, normal to slightly
diarrhea, convulsions, incoor- improvement; (3) terminal, elevated body temperatures,
dination, hemorrhages of skin exacerbation of disease conjunctivitis, dermatitis,
locomotion disturbances
Viremia High level Temporary reduction or disappearance Persistent high level
Leukopenia Develops quickly Develops quickly, followed by Develops late during infection
leukocytosis
Immune Absent Present Absent
response to
CSF
Death 10–20 days 1–3 months 2–11 months
Gross lesions Multiple hemorrhages (especially Ulcers of cecum and colon, Lymph node swelling, thymic
in lymph nodes and kidney), infarction of spleen, rib atrophy
infarction of spleen lesions
Microscopic Degeneration of endothelial cells, Degeneration of endothelial cells, Degeneration of endothelial
proliferation of reticular cells, severe lymphocyte depletion, cells, severe lymphocyte
encephalitis histiocytic hyperplasia, depletion, histocytic hyper-
glomerulonephritis plasia
 CHAPTER 14 CLASSICAL SWINE FEVER (HOG CHOLERA)
tion with severe thrombocytopenia and disturbance in fi-
brogen synthesis. Virtually all pigs die from acute CSF.
The mechanism actually responsible for death is not
clear, but severe disturbance of the circulatory system
appears to be the most likely cause (Fuchs 1968).
During acute CSF, the pig’s immune reactivity
changes. A depressed secondary antibody response to
lysozyme (Charley et al. 1980), highly abnormal respons-
es of peripheral blood and organ lymphocytes to T- and
B-cell mitogens (Van Oirschot et al. 1981, 1983), and B-
lymphocyte deficiency (Susa et al. 1992) have been re-
ported.
Persistent CSFV infections are generally caused by
strains of reduced virulence. Arbitrarily, two forms of
persistence may be distinguished: chronic and late-onset
CSF (Table 14.1).
The first phase of chronic CSF resembles acute CSF,
but the virus spreads more slowly and virus titers in
serum and organs tend to be lower. In the period of clin-
ical improvement, virus titers in serum are low or absent
and viral antigen is usually limited to epithelial cells of
tonsil, salivary gland, ileum, and kidney. A specific anti-
body response and/or decrease in the number of cells
producing virus probably account for the temporary dis-
appearance of CSFV from the serum. The simultaneous
circulation of viral antigens and antibody may result in
deposition of antigen-antibody complexes in the kid-
neys, which eventually can lead to glomerulonephritis.
During the exacerbation of acute disease, virus is again
distributed throughout the body. Its spread may be pro-
moted by the immune exhaustion that appears to devel-
op in pigs with chronic CSF (Cheville and Mengeling
1969).
Late-onset CSF initially runs an inapparent course. It
may not be until several months after primary contact
with the virus that pigs develop signs of disease. These
infections are the sequel of exposure to CSFV strains of
low virulence during fetal life (Van Oirschot and Terpstra
1977). The pigs have a lifelong high level of viremia,
which can be transiently decreased after the ingestion of
colostral antibody. CSFV antigen is widespread through-
out epithelial, lymphoidal, and reticuloendothelial tis-
sues. Pigs with congenital persistent CSF do not mount a
neutralizing antibody response to the virus. However,
the antibody response to antigens unrelated to CSFV is
generally normal, indicating a specific immune tolerance
to the virus. The response of peripheral blood lympho-
cytes to mitogens is only slightly depressed in these per-
sistently infected pigs (Van Oirschot 1979).
The pathogenesis of postnatal infections with low-
virulence CSFV is not well known. The growth of virus
seems to be mainly restricted to tonsil and regional
lymph nodes, but the virus can be disseminated via the
bloodstream, as evidenced by the frequently occurring
transplacental transmission in pregnant sows. CSFV ap-
pears to grow across the placental barrier at one or more
sites, and it subsequently spreads from fetus to fetus. The
Van Oirschot 163
developmental age of the fetus and the virulence of the
virus strain largely determine the outcome of a congeni-
tal infection. Generally, the risk for fetal damage is high-
er the earlier infection occurs during pregnancy.
Pigs that have recovered from CSF possess antibodies
to CSFV, but neutralizing antibodies can also be pro-
duced in subacute fatal cases. Whereas pigs with chronic
CSF are able to elicit a specific antibody response, pigs
with congenital persistent CSF appear to be immunotol-
erant to the virus. Although porcine fetuses gain im-
munocompetence around midgestation, only some of
them may produce antibodies to CSFV in the last phase
of fetal development. Occasionally, after postnatal infec-
tion with certain strains of CSFV, neutralizing antibodies
are only transiently detectable or completely absent.
This may be the consequence of a poor immunogenicity
of the virus. Such strains are generally of reduced viru-
lence.
In pigs that do not mount a normal antibody re-
sponse after a primary contact with CSFV, two phenom-
ena have repeatedly been observed. Reexposure of such
pigs to CSFV can result in a kind of hyperreactivity char-
acterized by a shorter incubation period and a more se-
vere illness than in primarily infected pigs. In contrast
with this sensitization phenomenon, an enhanced resis-
tance to virulent CSFV has been described. Pigs persis-
tently infected with CSFV of reduced virulence have a
markedly prolonged survival when inoculated with viru-
lent CSFV. It is conceivable that these phenomena play a
role under field conditions.
The role of cell-mediated immunity in CSF has not
yet been elucidated. Corthier (1978) and Remond et al.
(1981) detected a transient blastogenic response to CSFV
antigens in subclinically affected or vaccinated pigs. A
proliferative response of T lymphocytes against CSFV
has been induced by infective virus, which appeared not
to be directed to the immunodominant E2 protein. In ad-
dition, a considerable nonspecific lymphoproliferation
has been observed (Kimman et al. 1993). An epitope rec-
ognized by CSFV-specific cytotoxic T lymphocytes has
been identified near the cleavage site between the pro-
teins NS2.3 and NS4A (Pauly et al. 1995).
The precise pathogenic mechanisms of CSFV infec-
tions are not well understood. Some hypotheses are de-
scribed in extensive reviews by Fuchs (1968) and Mahnel
and Mayr (1974).
LESIONS
In peracute cases of CSF, pathologic lesions are often
absent. In acute to subacute CSF, the pathologic picture
is that of a septicemic disease characterized by multiple
hemorrhages of various sizes (caused by hydropic degen-
eration and necrosis of endothelial cells lining the vascu-
lar system) in conjunction with defects in the blood
coagulation mechanism. In addition, catarrhal, fibri-
nous, and hemorrhagic inflammatory reactions are
164 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
present in digestive, respiratory, and urogenital tracts.
Pathologic changes are most frequently observed in
lymph nodes and kidney. The lymph nodes become
swollen, edematous, and then hemorrhagic. They com-
monly have peripheral or more diffuse hemorrhages, giv-
ing the nodes a marbled or red to near black appearance
respectively (Fig. 14.1). Virtually all lymph nodes may be
affected. Microscopically, depletion of lymphocytes and
reticular hyperplasia are seen.
Hemorrhages of the kidney may vary in size from
hardly visible petechiae to ecchymotic hemorrhages.
They frequently occur on the surface of the cortex (Fig.
14.2) and are less common in the medullary pyramids
and hilus. Petechial to ecchymotic hemorrhages can also
be observed in urinary bladder, larynx and epiglottis,
14.1. Peripheral hemorrhage
of the mandibular lymph node.
(Courtesy W. C. Stewart.)
14.2. Kidney showing numerous
petechial hemorrhages. (Courtesy
W. C. Stewart.)
heart, intestinal mucosa, serous and mucous mem-
branes, and skin. The skin may also become cyanotic.
Infarction is the result of a disrupted blood flow into
a certain area resulting from the occlusion of blood cap-
illaries by thrombi. The thrombi in turn may be caused
by the hydropic degeneration of endothelial cells cou-
pled with the tendency of erythrocytes to clump in a
manner termed “sludging.” Infarction of the spleen (Fig.
14.3) is considered to be almost pathognomonic for CSF.
Infarctions occur as dark blebs of various sizes, raised
slightly above the surrounding surfaces. They may ap-
pear singly or as a series of lesions coalescing to form a
continuous border of infarcts along the edge of the
spleen. Infarction of the gallbladder and tonsil may oc-
cur. In the latter organ it causes necrosis, which upon
 CHAPTER 14 CLASSICAL SWINE FEVER (HOG CHOLERA)
bacterial invasion leads to suppurative tonsillitis.
Pigs with acute to subacute CSF can show infarctions
and hemorrhages in the lung. Presumably as a conse-
quence of secondary bacterial infections, catarrhal to
fibrinous bronchopneumonia and pleuritis may develop.
The heart is usually flabby and shows some myocardial
congestion. In an animal dying of CSF, the stomach is
usually empty except for a yellow, bilious fluid and a
small amount of feed. The fundus is often markedly con-
gested and hemorrhagic. The mucosal surface may con-
tain petechiae and mild to severe erosions. A mild to
moderate catarrhal or necrotic enteritis is found in the
intestines. Mesenteric blood vessels are usually marked-
ly engorged. Occasionally, subserous ecchymotic and
suffusive hemorrhages occur in the small and large in-
testines (Stewart 1981).
Most CSFV-infected swine show an encephalitis at
necropsy, of which the principal lesion is perivascular
cuffing. Proliferation of endothelial cells, microgliosis,
Van Oirschot 165
14.3. Infarction of the spleen.
(Courtesy L. D. Miller.)
and focal necrosis can also be observed in the brain.
In persistent CSF, hemorrhages and infarctions are
less pronounced or are completely absent. Although de-
generation of endothelial cells is a consistent finding in
persistently infected pigs, it does not result in severe cir-
culatory disturbances. The most outstanding lesions in
pigs with persistent CSF are atrophy of the thymus and
severe depletion of lymphocytes and germinal follicles in
peripheral lymphoid organs. Histiocytic hyperplasia
with phagocytosis of lymphocytic debris is also fre-
quently seen. The plasmacytosis and glomerulonephritis
that occur in chronic CSF were not present in pigs with
late-onset CSF. Persistent CSFV infections induce adren-
al cortical hyperplasia characterized by an increased
width of the zona fasciculata and atrophy of the zona
glomerulosa and zona reticulata (Cheville and Men-
geling 1969; Van der Molen and Van Oirschot 1981).
Necrosis and ulcerations, sometimes in the form of but-
ton ulcers (Fig. 14.4), in the cecum and colon and rib le-
14.4. Button ulcers in the cecum
and colon. (Courtesy L. D. Miller.)
166 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
sions are common in persistent CSF. Chronic rib lesions
are caused by a sudden calcification of large numbers of
mature cartilage cells and appear as a marked transverse
line of semisolid bone structure across the rib proximal
from the costochondral junction (Dunne 1975) (Table
14.1).
Congenital CSFV infections can result in mummifica-
tion, stillbirth, and malformations (Fig. 14.5). General-
ized subcutaneous edema, hydrops ascites, and hy-
drothorax are the most pronounced lesions in stillborn
pigs. Malformations consist of deformities of head and
limbs, hypoplasia of cerebellum and lungs, and hy-
pomyelogenesis. In utero–infected piglets that die short-
ly after birth often show petechial hemorrhages of skin
and internal organs.
DIAGNOSIS
Outbreaks of typical acute CSF can be diagnosed in the
field with reasonable certainty on the basis of an accu-
rate anamnesis and a thorough clinical and pathologic
investigation. An anamnesis of a CSF outbreak often in-
cludes one or more of the following: recent purchase of
pigs, cases of CSF on adjacent or nearby farms, garbage
feeding, or a recent visit by persons having close contact
14.5. Mummified fetuses and stillborn pigs of a sow
exposed to low-virulence CSFV at day 43 of gestation.
(Courtesy W. C. Stewart.)
with swine. In acute CSF there is a rapid spread of the
disease among pigs of all ages and high mortality at 1–2
weeks after the onset of clinical signs. Leukopenia is a
consistent finding in pigs with CSF. At necropsy, lesions
of diagnostic significance are hemorrhages in lymph
nodes, kidney, and other organs and infarction of the
spleen.
Acute CSF may be confused on clinical and patholog-
ic grounds with African swine fever, porcine reproduc-
tive and respiratory syndrome, septicemic salmonellosis,
pasteurellosis, streptococcosis, erysipelas, or
Haemophilus suis infections; therefore, it is recommend-
ed that clinical diagnosis be confirmed in the laboratory.
In many countries that have eradication programs that
include destruction of positive herds, any tentative field
diagnosis has to be confirmed by laboratory investiga-
tions.
Unlike in acute CSF, it is virtually impossible to make
a clinical diagnosis in cases of subacute, chronic, or late-
onset CSF because of the wide variability of clinical signs
and pathologic lesions. Clinical signs are milder than in
acute CSF; they can occur intermittently, or the infection
may go unnoticed for months. In addition, persistent in-
fections may involve only a few pigs in a herd. In such
cases, counting of blood leukocytes may prove useful. Fi-
nally, infected swine may not have signs indicative of
CSF because of concurrent infections with other infec-
tious agents. Persistent CSFV infections are difficult to
recognize, so the diagnosis is often delayed. Consequent-
ly, such an infected herd may act as a source for further
dissemination of the virus. Therefore, in countries where
CSF is present, samples should be submitted to the labo-
ratory when the clinical signs, combined with the anam-
nesis, raise even the slightest suspicion of CSF. CSFV in-
fections by strains of reduced virulence seem to increase
in importance, particularly in areas where CSF has been
endemic for a long period and in final stages of eradica-
tion programs. In the United States 55% of field isolates
in the period 1965–75 were characterized as being of re-
duced virulence (Carbrey et al. 1977).
Laboratory diagnosis of CSF is based on detection of
viral antigen, isolation of virus, or demonstration of vi-
ral antibody.
The direct fluorescent antibody (FA) test on frozen
tissue sections is the method of choice for detecting viral
antigen. Samples should be collected from a number of
dead or sick pigs and submitted fresh (preferably on ice)
and without the addition of any preservatives to the lab-
oratory. Tissues to submit for laboratory diagnosis are
tonsil, spleen, kidney, and distal part of the ileum. The
tonsil, which is the first tissue to become positive after
exposure to CSFV, is by far the most important organ for
detecting viral antigen (Fig. 14.6). The ileum is frequent-
ly found to be positive in more prolonged cases of CSF.
An absence of viral antigen in several tissues does not ex-
 CHAPTER 14 CLASSICAL SWINE FEVER (HOG CHOLERA)
14.6. CSF viral antigen in epithelial cells of tonsillar crypts detected by FA test. (Courtesy C. Terpstra.)
clude CSF as the cause of the outbreak; where suspicion
remains, more pigs should be examined. The FA test on
frozen tissue sections is a simple, rapid (can be complet-
ed in 2 hours), and reliable technique and has become
the official diagnostic test in many CSF eradication pro-
grams.
CSFV shares common antigens with BVDV and BDV.
Therefore, the direct FA test fails to discriminate be-
tween CSFV and BVDV/BDV antigens. Because pigs can
become infected with BVDV or BDV, the FA test may
yield false-positive results. It is crucial to distinguish CS-
FV from BVDV/BDV, because a BVDV or BDV infection
erroneously diagnosed as CSF leads, in most countries,
to killing of the herd and other control measures and
may result in loss of the CSF-free status. To differentiate
between CSFV and BVDV/BVD it no longer is necessary
to perform time-consuming cross-neutralization tests
with sera of in-contact pigs or pigs inoculated with the
isolate. A rapid differentiation can be made by staining
frozen tissue sections or infected cell cultures with conju-
gates of MABs directed against conserved epitopes of
CSFV. When the selected MABs react with the isolate, it
is CSFV, and when antigen is not detected, BVDV/BVD is
involved (Fig. 14.7) (Wensvoort et al. 1986, 1989). Such
nonreactive isolates need to be confirmed as BVDV or
BDV.
Van Oirschot 167
Molecular biology has provided genetic assays that
rapidly differentiate between CSFV and other pestivirus-
es. These are based on reverse transcription followed by
polymerase chain reaction (RT-PCR) and direct DNA se-
quencing of the amplified cDNA from the 5′ noncoding
region (Hofmann et al. 1994), on discriminatory RT-PCR
of sequences of E2 (Katz et al. 1993) or of a nonstruc-
tural protein (Wirz et al. 1993), or on RT-PCR followed
by restriction enzyme mapping of the 5′ noncoding re-
gion (Vilcek et al. 1994).
In pigs vaccinated with the attenuated Chinese (C)
strain of CSF, viral antigen can be demonstrated by the
direct FA test until about 2 weeks postvaccination (Terp-
stra 1978). Thus in areas where vaccination is practiced,
it may sometimes be necessary to differentiate between
field virus and vaccine virus. For this purpose, a pair of
conjugated MABs can be used, one of which reacts with
a conserved epitope of CSFV and the other with most
CSFV strains but not with vaccine strains. A positive re-
action by the first MAB and a negative by the second in-
dicate that vaccine virus may be present (Fig. 14.7) and
warrant further investigation. Lapinized vaccine strains
can be distinguished from field strains by their ability to
induce fever and antibodies to CSFV after intravenous in-
oculation in rabbits.
CSFV may be isolated by inoculation of a porcine kid-
14.7. Frozen sections of tonsils of pigs infected with the virulent Brescia strain (1, 2, 3) or the
attenuated vaccine strain C (4, 5, 6) of CSVF or a strain of BVDV (7, 8, 9). Sections were reacted
with peroxidase conjugates prepared from a polyclonal anti-CSFV serum (1, 4, 7) or from MABs that
recognize different epitopes of CSFV (MAB 1 [2, 5, 6], MAB 2 [3, 6, 9]). (Courtesy G. Wensvoort.)
 CHAPTER 14 CLASSICAL SWINE FEVER (HOG CHOLERA)
ney (PK-15) cell line with a 2–10% mixed homogenate of
tonsil and spleen of a suspected pig. After 24–72 hours,
the cultures are examined for viral antigen by the FA test
(Fig. 14.7). The virus isolation procedure is more sensi-
tive than the FA on frozen tissue sections.
A promising method to support a rapid diagnosis of
CSF in large numbers of live pigs is antigen detection by
enzyme-linked immunosorbent assay (ELISA) in blood
and tissue samples. However, this method is not as sensi-
tive as virus isolation, in that virus isolation can detect
infection some days earlier. In addition, the tests are not
yet specific for CSFV (Shannon et al. 1993; Depner et al.
1995).
Antibody detection can be a useful diagnostic tool for
suspected farms where the usual virus detection proce-
dures have failed and in the last phase of an eradication
program to detect subclinically infected herds. Serologic
tests are imperative if a country desires to become inter-
nationally recognized as free from CSF.
Several tests are available for the detection of anti-
bodies to CSFV. The virus neutralization test in its vari-
ous modifications (Holm Jensen 1981; Terpstra et al.
1984) is often used. However, it may measure antibodies
to BVDV because low neutralizing antibody titers to CS-
FV can be induced by infections with BVDV (Liess et al.
1977); therefore, such sera need to be tested in a BVDV-
neutralization test. Higher antibody titers to BVDV than
to CSFV indicate infection with BVDV, but a concurrent
infection with CSFV may not be completely excluded. In
such cases, more pigs from the herd should be tested.
ELISAs, which are rapid and simple to perform, have
been developed for large-scale serologic investigations.
An ELISA that differentiates between antibodies to CSFV
and BVDV employs two MABs directed against different
epitopes on glycoprotein E2. Because neither MAB rec-
ognizes BVDV, the chance that BVDV antibodies are de-
tected is virtually negligible (Wensvoort et al. 1988). The
E2 antigen is produced by recombinant baculovirus in
insect cells. This ELISA is used for seroepizootiologic sur-
veys in various countries (Wensvoort et al. 1988).
Because antibodies to field strains of CSFV cannot
yet be distinguished from antibodies to vaccine virus,
vaccination limits the use of serologic tests for diagnostic
purposes.
PREVENTION
To prevent the reintroduction of CSFV, countries free of
CSF ban the import of live pigs, pork, and insufficiently
heated pork products from countries where CSF is pres-
ent. In addition, swill is generally destroyed or effective-
ly sterilized before being fed to pigs. Cleansing and dis-
infecting of mechanical vectors, such as trucks, are too
often neglected as preventive measures against reintro-
duction of CSFV into an area. Should a case of CSF arise
in countries free of CSF, veterinary “police” may be
Van Oirschot 169
called upon and zoo sanitation measures may be insti-
tuted to help eradicate the virus. All pigs in infected
herds are destroyed, the source of infection and possible
contacts are traced, “standstill” restrictions become op-
erative, and infected farms are intensively disinfected.
Liquid manure can best be disinfected by chemical treat-
ment. Countries with sporadic outbreaks of the disease
usually apply similar control and eradication proce-
dures.
In countries where CSF is enzootic, vaccination is of-
ten practiced, and in some countries vaccination is sup-
plementary to the killing of infected herds. Vaccination
usually ceases when no more outbreaks occur or when a
stage is reached where destruction of infected herds
alone may eliminate the residual virus.
In the Netherlands, a strict vaccination regime pur-
sued for 1 year and supported by the usual veterinary po-
lice and zoo sanitation measures succeeded in eradicat-
ing CSF from three enzootic areas. The program
consisted of mass vaccination of all pigs over 2 weeks of
age and supplementary vaccination of previously unvac-
cinated 6- to 8-week-old pigs and newly introduced stock
at monthly intervals. Vaccination was compulsory, and
all vaccinated pigs were identified by ear tagging. The
number of outbreaks in the vaccinated areas declined in
2 weeks, and the areas were free of CSF from the fifth
month after the start of the program (Terpstra and
Robijns 1977). In an emergency vaccination campaign,
supplementary vaccination was conducted in piglets
born from vaccinated sows at the age of 7–9 weeks, and
breeding gilts born from vaccinated sows were revacci-
nated when 6–7 months old to boost herd immunity
(Terpstra and Wensvoort 1987). The European Union
has adopted a nonvaccination policy for CSF, which im-
plies that emergency vaccinations are also prohibited.
Vaccines originally attenuated by serial passages in
rabbits (C strain) or attenuated by passages in cell cul-
tures (Japanese GPE− strain, French Thiverval strain) are
used as an aid in the control of CSF. The C strain has an
insertion of 13 nucleotides in the 3′ noncoding region
compared with that of virulent CSFV strains (Moor-
mann et al. 1996). The GPE− strain differs in 225 nu-
cleotides from its parental strain (Ishikawa et al. 1995).
These vaccines are highly efficacious and safe (Aynaud
1988). The simultaneous vaccination method and use of
inactivated vaccines have been abandoned in most coun-
tries. In addition, the early attenuated CSFV vaccines are
no longer used because they induced a variety of fetal ab-
normalities after vaccination of pregnant sows.
The C strain is the most extensively used vaccine. The
growth of the C strain appeared to be mainly restricted
to lymphoid tissues, especially the tonsil, although C vi-
ral antigen has occasionally been detected in kidneys
(Terpstra 1978). The C strain can pass the placental bar-
rier of pregnant sows but does not seem to produce any
abnormality in infected fetuses (Tesmer et al. 1973). It is
170 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
innocuous for pregnant sows and newborn piglets. The C
strain can be transmitted from vaccinated pigs to non-
vaccinated contact pigs (Van Bekkum 1977). However,
the chance of reversion to virulence appears to be mini-
mal, because it retained its attenuated characteristics af-
ter 20–30 serial passages in pigs (Aynaud 1988) and be-
haved similarly in pigs treated with corticosteroids as in
nontreated pigs (Florent et al. 1969; Kamijyo et al. 1976).
The attenuated vaccines usually confer immunity
within 1 week after inoculation, and the immunity per-
sists for at least 2–3 years, and probably lifelong. Vacci-
nation not only protects against disease but also greatly
reduces the replication of virulent CSFV upon challenge
(Biront et al. 1987) and prevents transmission of chal-
lenge virus from pigs with moderate neutralizing anti-
body titers to in-contact pigs (Terpstra and Wensvoort
1988). Vaccinated sows transmit antibodies to their off-
spring via colostrum. Because the intestine of the new-
born piglet is permeable for immunoglobulins during
the first 36–48 hours of life, antibodies to CSFV enter di-
rectly into the circulation. These maternally derived anti-
bodies have a half-life of approximately 14 days. Piglets
born to vaccinated sows are protected for 5–8 weeks
against mortality from CSF but not against replication
and shedding of virulent virus (Terpstra 1977).
Maternally derived antibodies inhibit the develop-
ment of active immunity after vaccination. Although a
high percentage of piglets born to vaccinated sows can
effectively be vaccinated around 6 weeks of age, revacci-
nation of such pigs around 5 months will probably in-
crease herd immunity. The suppression by maternally
derived antibodies can be reduced by substantially in-
creasing the amount of vaccine virus per dose (Lin et al.
1982). Aerosol immunization of pigs has yielded varying
results with regard to efficacy.
The development of marker vaccines that allow dif-
ferentiation between infected and vaccinated pigs is in
progress. Such a marker vaccine can be composed of the
E2 protein that is excreted from insect cells infected with
recombinant E2-baculovirus and that is subsequently
formulated in an adjuvant (Hulst et al. 1993). In this
case, serologic differentiation can be based on detection
of antibody against Erns. A CSFV marker vaccine may al-
so be based on a genetically engineered live deletion mu-
tant virus. The engineering of full-length cDNA of the
genomes of the C strain (Moormann et al. 1996) and of
a virulent strain (Meyers et al. 1996) from which infec-
tious RNA can be transcribed provides the basis for the
development of live marker vaccines against CSF.
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 Japanese B Encephalitis
15 H. S. Joo and R. M. Chu

Japanese B encephalitis (JE) is a mosquito-borne viral

disease in animals and humans. Most domestic animals
are vulnerable to the virus, including horses, cattle,
sheep, goats, and pigs. Other animals, such as rabbits,
rats, pigeons, dogs, ducks, chickens, wild birds, and rep-
tiles, are also susceptible. Experimental infection has
been reported in mice and in a few species of lizards.
The pig is considered the most important natural am-
plifying animal for JE virus (JEV). The disease is impor-
tant in pregnant sows because infection causes stillbirth
and other reproductive disturbances; affected boars may
have an acute inflammatory reaction in the testes. Hors-
es infected by the virus develop lesions in the central ner-
vous system (CNS), and similar lesions have been found
in donkeys and monkeys. In other animals, infection is
usually subclinical.
JE is of primary significance in humans, epidemics
having been recorded in Japan (1935), Korea (1949), and
India and Nepal (1978). The disease is one of the most
common mosquito-borne diseases of the human CNS in 15.1. Geographic distribution of JE outbreaks in Asia.
Japan, China, and other western Pacific countries. Isola-
tion and identification of the virus were first described
by Fujita (1933) and Taniguchi et al. (1936). Episodes of There are three structural and several nonstructural pro-
infection in humans occur annually during mosquito teins. The structural proteins are envelope glycoprotein
(Culex tritaeniorhynchus) season. In most people disease E (54 kDa), nonglycosylated envelope protein M (8 kDa),
is subclinical or mild, but fatal encephalitis develops in and capsid protein C (14 kDa). Envelope protein E carries
some children, and abortion is reported in pregnant epitopes that elicit neutralization antibody. At least eight
women (Chaturvedi et al. 1980). epitopes are found on protein E, and one of the critical N
The geographic distribution of the disease is restrict- sites on the protein shows JEV specificity (Kimura-Kuro-
ed to eastern Asia (Fig. 15.1); infection has been recog- da and Yasui 1986). The oligonucleotide fingerprinting
nized in Japan, eastern Soviet Union, Korea, China, Tai- technique (Hori et al. 1986) reveals that mutations of
wan, the Philippines, Indonesia, Singapore, Malaysia, JEV are present in strains that were isolated years apart
Hong Kong, Vietnam, Laos, Bangladesh, Nepal, Thai- in Japan and Thailand.
land, Burma, Sri Lanka, India, and the Pacific islands. Use of monoclonal antibodies in hemagglutination
inhibition, neutralization, enzyme-linked immunosor-
ETIOLOGY bent assay (ELISA), and competitive binding assay has
shown that the closest relative of the JEV is the Murray
Physicochemical Properties Valley encephalitis virus; the next closest relative is the
JE is caused by a filterable virus, of the genus Flavivirus, West Nile virus; the St. Louis encephalitis virus is the
the only genus in the family Flaviviridae, formerly least closely related (Kimura-Kuroda and Yasui 1986).
grouped in the family Togaviridae. The virus is spherical, These relationships are confirmed by comparing the ho-
about 40 nm in diameter, and enveloped, and it contains mology of the viruses’ nucleotides and amino acids
single-stranded ribonucleic acid (RNA) with icosahedral (McAda et al. 1987).
capsids. The complete nucleotide sequence of the JEV The JEV is unstable in the environment and easily in-
genome RNA contains 10,976 nucleotides correspond- activated by disinfectants. The virus is sensitive to ether,
ing to 3432 amino acid residues (Sumiyoshi et al. 1987). chloroform, and sodium desoxycholate and also to pro-

173
174 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
teolytic or lipolytic enzymes. It is readily inactivated at
56˚C after 30 minutes and has an optimal pH stability of
8.5.
Replication
The JEV replicates well and produces a cytopathic effect
in a wide range of host cell culture systems, such as pri-
mary cells and cell lines from mammals, including Vero
cells, baby hamster kidney (BHK)-21 cells, and L-M
mouse fibroblast cells. Cells originating from mosquitoes
are also frequently used. Examples include C6/36 cell
lines from larval cells of Aedes albopictus and the A. ae-
gypti cell line from embryonic tissue. Leukocyte cultures
from various animals are good media for growing the
virus (Kadarnath et al. 1987). Hormones such as insulin,
adrenocorticotropic hormone (ACTH), hydrocortisone,
and concanavalin A have been shown to enhance JEV
replication in cell culture systems (Kelkar 1985; Kokorev
and Kolotvinova 1986).
Like the other flaviviruses, JEV proteins are encoded
by a single open reading frame that continues uninter-
rupted throughout the region that is sequenced (McAda
et al. 1987). A short pulse of actinomycin D has no effect
on early replication of the virus, but continuous expo-
sure partially inhibits replication. Mitomycin, a DNA-
synthesis inhibitor, does not exert any influence on repli-
cation of the virus. In addition, nuclear involvement
apparently does not occur (Leary and Blair 1983).
EPIDEMIOLOGY
The epidemiologic features of JE have been described in
detail by Konno et al. (1966) and Kono and Kim (1969).
In nature, infection with JEV is maintained cyclically, in-
volving vector mosquitoes (Self et al. 1973), birds (arde-
ids), and mammals.
JE is of public health concern. A correlation between
infection in pigs and infection in humans is apparent,
with evidence indicating that swine play an important
role in the buildup of the virus within a population. In
enzootic zones, pigs are invariably found in high concen-
tration and are favored feeding sources for mosquitoes.
Consistent development of viremia in susceptible pigs
ensures a continued supply of infected mosquitoes. In
Japan and Korea, the mosquito season starts in late June,
varying somewhat according to latitude, and the pig-
mosquito cycle becomes evident shortly afterward. At
that time, the pig population contains a high proportion
of young, susceptible breeding stock in which passive
immunity to JEV has waned during the winter (Konno et
al. 1966). This buildup of susceptible stock is not as
prevalent in tropical areas of Southeast Asia, where the
pig-mosquito cycle may continue throughout the year, al-
lowing most young stock to develop an early active im-
munity. Epidemiologic patterns may differ among areas
of Southeast Asia, as vector activity is modified by dif-
fering climatic conditions.
Other animals and different mosquito species could
alter infection cycles and are factors involved in the
transmission of JE. The disease is a growing threat in In-
dia, where pigs are much less numerous compared with
other endemic areas. In southern Thailand, 70% of the
pigs are infected with JEV, and the JEV vector mosquito is
prevalent; however, the encephalitis in humans is rare
(Burke et al. 1985).
In temperate zones, JEV survives the winter, but in-
fection of pigs in winter is not an important problem
(Takashima et al. 1988). Chickens and wild birds, espe-
cially herons and egrets, are able to maintain positive
sera year-round (Hammon et al. 1958; Bhattacharya et al.
1986). The JEV may also be carried by cold-blooded ver-
tebrates throughout the winter; experimentally, the virus
survives in two species of lizard in hibernation (Doi et al.
1983). The virus can also be isolated naturally from
snakes, and bats are possible reservoirs. Multiplication
of JEV has been demonstrated in a variety of mosqui-
toes, including the species in Culex, Aedes, and Anopheles.
The role of ticks has been described in animals with over-
winter infection. Transovarial vertical infection is ob-
served in some mosquitoes, such as Aedes, which may be
another mechanism for the infection.
CLINICAL SIGNS
Although young susceptible piglets may occasionally
show clinical signs, clinical illness is not a feature of JEV
infection in adults or pregnant pigs. However, various de-
grees of abnormality in fetuses may be evident when in-
fected pregnant pigs come to term. Litters commonly
contain varying numbers of stillborn and mummified fe-
tuses (Fig. 15.2), weak piglets with nervous system signs,
and apparently normal piglets. Experimental induction
of reproductive failure by infection with JEV causes sim-
ilarly damaged litters, an additional observation being
the presence of subcutaneous edema and hydrocephalus
in some stillborn piglets (Shimizu et al. 1954). Experi-
mental infection of susceptible pregnant sows causes no
clinical signs in dams but results in infection of fetuses in
utero and subsequent abnormal farrowings: varying
numbers of mummified fetuses of different sizes and
stillborn and weak piglets with subcutaneous edema and
hydrocephalus (Shimizu et al. 1954). Abortion has not
been a feature of intrauterine infection in experimental
studies.
Infertility in boars in summer appears to be associat-
ed with JEV infection. Hashimura et al. (1976) isolated
JEV from the testicles of boars with orchitis, and Ogasa
et al. (1977) demonstrated that JEV infection of suscepti-
ble boars resulted in invasion of the sexual organs and
disturbance of spermatogenesis. Such boars developed
 CHAPTER 15 JAPANESE B ENCEPHALITIS
edematous, congested testicles, hardening of the epi-
didymis, and reduced libido, and virus was excreted in
the semen, which had significantly depressed total and
motile sperm counts, with numerous abnormalities of
spermatozoa. In most boars, damage was temporary,
with subsequent complete recovery, but occasionally,
boars with severe infection became permanently infer-
tile.
PATHOGENESIS
Pigs become infected with JEV via the bites of mosqui-
toes carrying the virus. Infected pigs develop viremia,
which persists for approximately 12 hours to a few days.
The multiplication model of the JEV in pigs has not been
thoroughly studied; however, some data are available for
humans, monkeys, and mice.
After the initial viremia, the virus disseminates to
vascular tissues such as the liver, spleen, and muscle,
where further replication augments the viremia. The
virus enters the CNS via cerebral spinal fluid; by en-
dothelial cell, macrophage, or lymphocyte infection; or
by a hematogenous route. In humans and mice, JEV in-
fects and destroys neurons selectively, mostly in the areas
of the brain stem, thalamus, basal ganglion, and the
lower layer of the cortex (Johnson 1987).
In mosquitoes, JEV infection is noncytopathic; the
virus is multiplied in phagocytic or hemolymph cells of
the fat body in 2 days (Johnson 1987). After infection,
the viral antigens spread widely in many organs, but se-
lective infection in the CNS (Leake and Johnson 1987) is
always present 4 days postinfection; 1 or 2 days later the
virus can be found in salivary glands.
Macrophages play an important role in the patho-
genesis of the disease. After intraperitoneal inoculation
Joo, Chu 175
15.2. Mummified and stillborn
fetuses from a gilt following a
natural infection with JEV.
in mice, the virus replicates first in the peritoneal
macrophages and later, on day 3, in the splenic
macrophages of the prefollicular region. Viral produc-
tive infection is observed in macrophages and T cells.
Macrophages are also claimed to be involved in the de-
velopment of latent infection.
Experimentally, JEV infection induces the generation
of suppressor T cells through the production of a soluble
suppressor factor and suppresses the humoral- and cell-
mediated immune responses of the infected animal. This
is manifested by depressed activities of plaque-forming
cells and delayed hypersensitivities. These suppressions
involve at least two generations of suppressor T cells. In-
fection with JEV initially stimulates the production of
first-generation phenotypic Ly 1–2 suppressor cells,
which release the suppressor factor, which has a molecu-
lar weight of 12,000 Da. This factor is nondialyzable and
sensitive to hydrocortisone. Suppressor factor adheres to
the peritoneal or splenic macrophages and further stim-
ulates the generation of another subpopulation of sup-
pressor T cells, which then exert their suppressor activi-
ties on plaque-forming cells and delay hypersensitivities
(Mathur et al. 1986; Rawat et al. 1986).
Transplacental infection of JEV has been reported in
pigs and mice. In pregnant pigs, fetuses may become in-
fected during the viremic period. Experimentally, after
intravenous infection of pregnant pigs with JEV, the
virus is recoverable from fetuses as early as 7 days postin-
fection. In some animals, the virus fails to cross the pla-
centa. Shimizu et al. (1954) have suggested that success-
ful transplacental infection may depend on the time of
gestation at which the dam was infected or on the strain
of virus. When infection of pregnant dams takes place in
the mid-third of gestation, transplacental infection and
pathogenic effects are more obvious (Shimizu et al.
176 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
1954). Field observations show that fetal death and
mummification are associated with JEV infection of
dams between 40 and 60 days of gestation; fetuses from
gilts infected after 85 days of gestation were little affect-
ed (Sugimori et al. 1974).
Fetal deaths are assumed to be associated with un-
controlled multiplication of the virus and subsequent
destruction of vital stem cells in fetuses that have not
reached the stage of immunocompetence. The stage of
gestation at which fetuses become immunocompetent
for JEV has not yet been determined. The average time of
immunocompetence for other viruses, such as porcine
parvovirus, is 70 days of gestation (Joo et al. 1976), and
the findings of Sugimori et al. (1974) suggest that there
are no pathogenic effects of JEV on the fetus when the
virus crosses the placenta after this stage of gestation.
Thus, the pathogenesis of JEV for the porcine conceptus
seems comparable to that described for porcine par-
vovirus (Joo et al. 1976); that is, for pathogenic effects to
occur, the virus must reach the conceptus before the
stage of immunocompetence.
In mice (Fujisaki et al. 1982), transplacental infection
was demonstrated when pregnant females were exposed
to the virus any time after the third day of gestation—
with the incidence of infection highest when exposure
was between 7 and 10 days of gestation. It was conclud-
ed that transplacental infection of JEV depends on the
degree of development between placenta and fetal tis-
sues, not on the intensity of viremia.
LESIONS
Major pathologic lesions have not been found in pigs
postnatally infected with JEV. However, various abnor-
malities have been observed in litters from dams infected
with the virus during pregnancy. Gross pathologic le-
sions noted in stillborn or weak neonatal piglets include
hydrocephalus, subcutaneous edema, hydrothorax, as-
cites, petechial hemorrhages on serous membranes, con-
gestion of lymph nodes, necrotic foci in liver and spleen,
and congested meninges or spinal cord (Burns 1950). It
was further observed that the CNS appeared hypoplastic
in areas, the cerebral cortex particularly being extremely
thin in some hydrocephalic piglets (Shimizu et al. 1954).
Cerebellar hypoplasia and spinal hypomyelinogenesis
have also been described (Morimoto 1969).
Histopathologically, significant lesions in affected
piglets or stillborn pigs are restricted to the CNS; few da-
ta on changes in other tissues have been recorded. Most
CNS lesions, mainly involving the cortex, basal ganglion,
brain stem, and spinal cord, occur in pigs up to 6 months
of age. Diffuse nonsuppurative encephalitis and
spondylitis are also reported. The highly selective dam-
age to neurons found in humans is not well described in
pigs; however, scattered neuronal degeneration and
necrosis are found in the cerebrum and cerebellum in
pigs. Neuronal degeneration is prominent in the gray
matter and Purkinje layers. Most cells in perivascular
cuffs in humans are T-helper/inducer cells; however,
one-fourth of the cells are suppressor/cytotoxic T cells.
The data are similar for mice, in which T-suppressor cells
proliferate during JEV infection. Involvement of visceral
organs, including lung, liver, kidney, and spleen, is docu-
mented in humans and experimentally in monkeys and
horses. Pathologic changes are seen mostly in the reticu-
loendothelial system, such as focal hyperplasia of germi-
nal centers in the spleen; interstitial pneumonia is also
seen (Rawat et al. 1986; Johnson 1987; R. M. Chu and M.
Y. Liao, unpublished data—1990).
Pathologic changes in the testes associated with JEV
infection are not well described. In naturally affected
pigs, a large amount of mucous fluid is observed in the
cavity tunica vaginalis, as well as fibrous thickening
along the edge of the epididymis and the visceral layer of
the tunica vaginalis. Microscopically, such testicles show
edema and inflammatory changes, with cellular infiltra-
tions in the interstitial tissue of the epididymis and tuni-
ca vaginalis. Cell infiltration and hemorrhage are also ev-
ident in the interstitial tissue of the testes. Degenerative
changes are often seen in the seminiferous epithelium
(Hashimura et al. 1976; Ogasa et al. 1977).
DIAGNOSIS
The definitive diagnosis of JEV infection is based upon
isolation of the virus from fetuses and infected pigs. A
differential diagnosis must include porcine reproductive
and respiratory syndrome, parvovirus infection, Au-
jeszky’s disease, toxoplasmosis, and hog cholera. Other
infections associated with reproductive failure such as
cytomegalovirus, leptospirosis, and enterovirus infec-
tions are also considered. Lack of clinical signs in sows
and piglets with JEV infection is useful in excluding many
diseases. Seasonal distribution also indicates JEV infec-
tion.
Several serologic tests are available that detect anti-
body titers of JEV infection in pig serum, such as the
hemagglutination inhibition test, ELISA, antigen biotin-
labeled ELISA, single radial hemolysin, and serum neu-
tralization technique. Epitope-blocking immunoassay
(Burke et al. 1987) and detection of cerebral spinal fluid
IgM, using ELISA diagnostic kits, are used in humans.
However, in an area where vaccination is routine, the di-
agnostic value of the serologic tests is challenged, and
paired serum samples should be considered. Presence of
the antibody in fetuses is also of diagnostic value.
Although isolation of the virus from suitable speci-
mens is essential to make a definitive diagnosis, detect-
ing viral antigens in infected tissues such as brains, pla-
centa, and mummified fetuses is also diagnostically
important. Methods such as avidin-biotin staining and
fluorescent antibody staining have been used to detect JE
 CHAPTER 15 JAPANESE B ENCEPHALITIS
viral antigen in formalin-fixed tissues following treat-
ment of such tissues with trypsin or another proteolytic
enzyme (Kurata et al. 1983; Iwasaki et al. 1986).
Isolation of the virus can be performed by intracere-
bral inoculation of brain extracts into suckling mice be-
tween 1 and 5 days of age. Signs of CNS disturbance or
death follow between 4 and 14 days postinoculation, and
virus in mouse brain tissue can readily be identified by in
vivo neutralization tests in suckling mice or in cell cul-
ture. The commonly used cell cultures are derived from
hamster, pig kidney, and mosquito, in all of which the
virus causes a cytopathic effect; the mosquito cell line
from A. albopictus clone C6/36 is the most efficient. It
should be recognized that tissue suspected of being in-
fected with JEV must be handled with care, because the
virus is not only heat labile but also pathogenic to hu-
mans.
PREVENTION
The treatment of JE in humans with human recombinant
interferon-αA gives clinically satisfactory results. This
treatment of JE in swine is not available. Breaking the in-
fectious cycle in this arthropod-borne disease is usually a
good step toward management; however, JEV multiplies
in a variety of species of mosquitoes, and the infectious
patterns vary according to local ecology. It is impractical
to control the insect; therefore, immunizing the breeders
with JEV vaccine is widely applied as an applicable con-
trol and preventive measure.
A variety of mostly live-attenuated vaccines have
been developed for the prevention of JE in pigs and are
being used successfully in the field (Hsu et al. 1972; Fu-
jisaki et al. 1975; Kwon et al. 1976). Inactivated vaccines
have proved to be less efficient and have become less
popular. It is recommended that attenuated vaccines be
given to young gilts and boars twice at an interval of 2–3
weeks before the start of the mosquito season. As an ex-
tra precaution, it is advisable that during the mosquito
season, young gilts and boars selected for breeding be
vaccinated before mating. The vaccine may be used si-
multaneously with other viral vaccines such as hog
cholera vaccine.
Attempts have also been made to develop a genetical-
ly engineered vaccine, but no commercial product is
available. Some studies have demonstrated that mono-
clonal antibodies to epitopes on the E envelope of JEV
prevent mice from being infected by JEV (Kimura-Kuro-
da and Yasui 1988). However, application of this to pigs
needs further investigation.
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 Porcine Epidemic Diarrhea
16 M. B. Pensaert
In 1971, previously unrecognized acute outbreaks of di-
arrhea were observed in feeder pigs and fattening swine
in England (Oldham 1972). The clinical appearance was
similar to that of an infection with transmissible gas-
troenteritis (TGE) virus except for the important differ-
ence that suckling pigs (under 4–5 weeks of age) did not
become sick. TGE virus (TGEV) and other known en-
teropathogenic infectious agents were ruled out, but a vi-
ral etiology of unknown identity was suspected. The dis-
ease spread to other European countries and the name
“epidemic viral diarrhea” (EVD) was adopted.
In 1976, TGE-like outbreaks of acute diarrhea were
observed in swine of all ages, including suckling pigs
(Wood 1977), but again TGEV and other known en-
teropathogenic agents were ruled out as the cause. The
name “EVD type 2” was used to differentiate them from
the outbreaks described in the early 1970s (designated
type 1). The difference between types 1 and 2 was that in
type 2 outbreaks baby piglets were involved.
In 1978, a coronavirus-like agent was found to be as-
sociated with the type 2 outbreaks (Chasey and
Cartwright 1978; Pensaert and DeBouck 1978). Experi-
mental inoculations with an isolate designated CV777 re-
vealed its enteropathogenic character both for piglets
(DeBouck and Pensaert 1980) and for fattening swine. It
appeared that this coronavirus could be involved in out-
breaks of type 1 as well as of type 2, and the name
“porcine epidemic diarrhea” (PED) was proposed (Pen-
saert et al. 1982) and is still used. An explanation for the
clinical difference between type 1 and type 2 has not
been provided until now.
ETIOLOGY
On a morphologic basis, PED virus (PEDV) particles
show characteristics of the family Coronaviridae (Chasey
and Cartwright 1978; Pensaert and DeBouck 1978). The
particles detected in fecal material are pleomorphic, with
a tendency to a spherical shape. Their mean diameter,
projections included, is 130 nm, with a range of 95–190
nm. Many particles have an electron-opaque central
area. The club-shaped projections measure 18–23 nm
and are radially spaced from the core. An internal struc-
ture cannot be recognized. The morphogenesis of PEDV
in intestinal epithelial cells was found to be identical to
that of other coronaviruses. Assembly of the virus oc-
curs by budding through intracytoplasmic membranes
(Ducatelle et al. 1981b; Sueyoshi et al. 1995).
Physicochemical and biological characterization
studies have confirmed the classification of PEDV as a
member of the Coronaviridae family. The virus is ether
and chloroform sensitive. Its density in sucrose is 1.18
g/mL. Concentrated and purified virus containing in-
testinal perfusate from infected piglets failed to aggluti-
nate erythrocytes of 12 different animal species (Calle-
baut and DeBouck 1981; Witte et al. 1981). Cell
culture–adapted PEDV loses its infectivity when heated
to ≥60˚C for 30 minutes but is moderately stable at 50˚C.
The virus is stable between pH 5.0 and 9.0 at 4˚C and be-
tween pH 6.5 and 7.5 at 37˚C. Viral infectivity is not im-
paired by ultrasonication or by multiple freezing and
thawing. Replication is not inhibited by 5-iodo-
2′-deoxyuridine, indicating that the viral nucleic acid is
RNA (Hofmann and Wyler 1989).
The pattern of the structural proteins of PEDV is
similar to that of other coronaviruses. The virus possess-
es a glycosylated peplomer protein with a molecular
weight of 85,000–135,000 daltons, a glycosylated enve-
lope protein of 20,000–32,000 daltons, and an unglyco-
sylated RNA-binding nucleocapsid protein of 58,000 dal-
tons (Egberink et al. 1988).
Using direct immunofluorescence (IF) and immuno-
electron microscopy, PEDV was found to be antigenical-
ly distinct from the two known porcine coronaviruses
(TGEV and hemagglutinating encephalomyelitis virus)
and canine coronavirus, neonatal calf diarrhea virus, in-
fectious bronchitis virus, and feline infectious peritonitis
virus (Pensaert et al. 1981; Witte et al. 1981). However,
examination with the aid of more sensitive techniques
such as immunoblotting and immunoprecipitation
showed that PEDV has antigenic determinants in
common with feline infectious peritonitis virus. These
determinants are located on the nucleocapsid protein
(Yaling et al. 1988). Identification of the membrane gly-
coprotein supports classification with group I coron-
aviruses (Utiger et al. 1995). On the basis of genomic
analysis, PEDV holds an intermediate position between
human coronavirus 229E and TGEV (Bridgen et al.
1993). All these data definitely confirm that PEDV is a
coronavirus.
179
180 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
Cultivation of the virus was originally accomplished
by orally inoculating piglets and subsequently collecting
the small intestine and its contents during the early
stages of diarrhea. A virus stock containing 105 pig infec-
tious doses per milliliter was easily obtained (DeBouck
and Pensaert 1980).
The adaptation of PEDV to grow in an artificial host
under laboratory conditions has been fastidious. At-
tempts with different virus isolates were unsuccessful in
intestinal and tracheal explants from swine fetuses and
newborn pigs. Also, numerous cell types were employed
with or without treatments with trypsin and pancreatin
and were nonpermissive for PEDV replication (Hess et al.
1980; Callebaut and DeBouck 1981; Witte et al. 1981).
However, Vero (African green monkey kidney) cells were
found to support the serial propagation of PEDV.
Growth was successful in some Vero cell lines and not in
others, so that cells of different origin should be at-
tempted. Viral growth depends on the presence of
trypsin in the cell culture medium. Cytopathic effects
consist of vacuolation and formation of syncytia. The
syncytia may contain over 100 nuclei. Growth kinetics
show peak titers of 105.5 plaque-forming units per milli-
liter 15 hours after inoculation (Hofmann and Wyler
1988, 1989). Vero cell–adapted PEDV has been success-
fully propagated in other cell types such as MA104 (Ku-
sanagi et al. 1992).
PEDV or viral antigens have until now been detected
in fecal material by electron microscopy (EM) and en-
zyme-linked immunosorbent assay (ELISA). The sensi-
tivity of the ELISA test is much higher than that of EM
(Callebaut et al. 1982). With ELISA, PED viral antigens
were demonstrated in rectal swabs of pigs until 11 days
postinoculation (Carvajal et al. 1995b).
Specific antibodies have been detected in sera from
swine after natural or experimental infection with PEDV.
They can be demonstrated by immunoelectron mi-
croscopy, ELISA, ELISA blocking, indirect IF, IF block-
ing, and seroneutralization performed in pigs or in Vero
cell cultures (Pensaert et al. 1981; Prager and Witte
1981a, b; Callebaut et al. 1982; Witte and Prager 1987;
Hofmann and Wyler 1989, 1990). Using ELISA, antibod-
ies are easier to detect against the viral peplomer protein
than against the nucleocapsid protein (Knuchel et al.
1990).
There are no indications that different serotypes of
PEDV exist. Isolates from Europe, Korea, and China are
serologically identical to the prototype CV777 strain
(Hess et al. 1980; Witte et al. 1981; Vannier and DeBouck
1983; Q. Yongqing, pers. comm.—1997). Additionally,
polypeptide bands detected by immunoblotting with the
Korean isolate showed a molecular weight similar to that
of CV777 (Kweon et al. 1993). Also, the nucleotide se-
quence of the N gene of PEDV isolated in Korea showed
97% homology with that of the CV777 strain isolated in
Belgium (Park et al. 1995).
EPIDEMIOLOGY
Serologic examinations to determine the presence of
PEDV were carried out between 1982 and 1990, and spe-
cific antibodies were detected in Belgium, England, Ger-
many, France, the Netherlands, Switzerland, Bulgaria,
and Taiwan but not in Austria and North America (De-
Bouck et al. 1982; Hofmann and Wyler 1987; Möstl et al.
1990). The virus itself has been isolated in most swine-
raising countries in Europe and also in China (Qinghua
et al. 1992), Korea (Kweon et al. 1993), and Japan (Taka-
hashi et al. 1983; Kuwahara et al. 1988). No confirmation
of the occurrence of PEDV in North or South America
has been given until now.
In recent years, severe outbreaks of PED with high
mortality figures have been reported from Asia. In the
winter of 1996, a PED epizootic occurred in Japan on 108
mostly farrow-to-finish swine farms in 9 prefectures.
Diarrhea and high mortality were encountered in
baby piglets: 39,509 suckling pigs died out of a total
of 56,256 (T. Ogawa, pers. comm.—1997). Also in
Japan, outbreaks between September 1993 and June
1994 have been described, with 14,000 deaths and mor-
tality between 30% and 80% in suckling pigs. During
these epi- zootics, adult pigs showed only a transient in-
appetence and decreased milk secretion (Sueyoshi et al.
1995).
In Korea, PED causes outbreaks of diarrhea in swine
of all ages. Of 71 viral enteric cases requested for diag-
nosis at the Veterinary Research Institute between Janu-
ary 1992 and December 1993, 56.3% were identified as
PED. PED occurred all year round but was more preva-
lent during the cold season. The disease occurred nation-
wide, and 90% of the outbreaks were detected in piglets
less than 10 days old (Hwang et al. 1994).
Recent serologic surveys or diagnostic studies have
not been published in Europe, where epizootics of PED
in suckling pigs have become rare. Outbreaks of diarrhea
due to PEDV are mainly confined to feeder and fattening
pigs and to young breeding animals. Very likely, this is
due to the enzootic character of the virus and the pres-
ence of lactogenic immunity, which protects suckling
pigs. In a longitudinal study in 10 groups of multisource
feeder pigs entering a fattening unit in September 1993,
none seroconverted, whereas the pigs in another 7
groups, entering in February 1994, had seroconverted 4
weeks after arrival (Van Reeth and Pensaert 1994). These
7 groups showed diarrhea during the first week after en-
tering and PED was demonstrated in feces.
In Spain, PED was identified as the cause of an epi-
zootic of acute watery diarrhea in 7 of 15 farms, and the
 CHAPTER 16 PORCINE EPIDEMIC DIARRHEA
diarrhea became persistent in a small number of sows on
one of the farms (Carvajal et al. 1995a). In a Spanish
serosurvey carried out in 1992–93, PEDV specific anti-
bodies were detected in 1513 of 5098 sows, and positive
animals were found in 55.9% of 798 breeding farms.
Prevalence was highest on farms with 20 or more sows
(Carvajal et al. 1995c).
In the Netherlands, a clinical and virological study of
an acute outbreak of PEDV diarrhea in a combined
breeding and finishing pig herd was described (Pijpers et
al. 1993). The first signs of disease were observed in fat-
tening pigs, and the infection spread rapidly to sows and
their suckling pigs, gilts, and weaners housed in separate
barns. Diarrhea was most striking in fattening pigs and
pregnant sows. Diarrhea in suckling pigs and young
weaners was mild or not at all present. The virus became
enzootic and persisted in 6- to 10-week-old pigs and in
gilts newly introduced to the farm for at least 1.5 years af-
ter the original outbreak. This outbreak was clinically
similar to the type 1 outbreaks observed in the early sev-
enties.
The situation in Asia, where diarrhea with high pig
mortality occurs, differs from the current situation in Eu-
rope. These Asian epizootics appear to be so severe that
they cannot be differentiated clinically from typical acute
TGEV outbreaks, and they incur heavy economic losses.
The virus in this continent, as well as in Japan, appears to
have found nonimmune populations. Presumably an
evolution to enzootic prevalence and to population im-
munity will follow, as it did in Europe. A serosurvey in
Korea in 1994 involving 469 sera from slaughtered pigs
in 7 provinces showed 17.6–79% of pigs with PEDV anti-
bodies, with an overall average of 45%, clearly suggesting
that the virus was endemic in some areas of that country
(Kweon et al. 1994).
Presently in Europe, the virus appears to persist
rather easily in densely swine populated areas but studies
on the mechanism or modalities of this persistence have
not been carried out.
PEDV is transmitted by feces from infected animals.
The natural infection starts after oral uptake. The feces-
oral route of transmission is probably the main, if not
the only, one. Acute outbreaks of PED on susceptible
farms often occur within 4-5 days after sale or purchase
of pigs. Virus probably enters by infected pigs, by way of
contaminated trucks, boots, or other virus-carrying
fomites. After an outbreak has occurred on a breeding
farm, the virus may either disappear or become enzootic.
The enzootic status can be established on a farm if suffi-
cient litters of pigs are produced and weaned so that the
virus is maintained through infection of consecutive lit-
ters of pigs that have lost their lactogenic immunity at
weaning. PEDV may be a cause of persistent weaning di-
arrhea in 5- to 8-week-old pigs on such breeding farms.
Pensaert 181
PEDV does not differ markedly from TGEV with re-
gard to modes of transmission, but it appears to persist
more easily on a farm once the acute infection has
passed.
CLINICAL SIGNS
The main and often the only obvious clinical sign of PED
is watery diarrhea. Acute outbreaks on breeding farms
involving neonatal pigs have become rare in regions or
countries where the virus has become widespread and
where the sow population is largely immune.
Outbreaks in susceptible breeding herds may show
much variation in morbidity and mortality. On some
farms, pigs of all ages become sick, with morbidity ap-
proaching 100%. The disease is then very similar to TGE,
except for a slower spread and a somewhat lower mortal-
ity in baby piglets. Piglets up to 1 week of age may die
from dehydration after the diarrhea has lasted 3–4 days.
Mortality averages 50% but may be as high as 80%. Older
pigs recover after about 1 week. After the acute outbreak
has passed, diarrhea may persist in pigs on the farm
around 2–3 weeks after weaning, and newly introduced
pigs may systematically become sick. Acute outbreaks
with high mortality figures in neonatal pigs have been
described in recent years in Japan and Korea (Ogawa,
pers. comm.—1997; Y. S. Lyoo, pers. comm.—1996).
On some farms in Europe, however, weaned pigs and
even adult pigs have been severely affected while suck-
ling pigs have had no or only mild diarrhea even in the
absence of immunity. Morbidity in baby pigs is then low.
No explanation can be given at this time for the high vari-
ation in clinical signs observed on breeding farms. Sever-
al isolates have been tested for variation in virulence for
piglets, but no variation has been found.
Much less variation is observed when an acute PED
outbreak occurs in multisource feeder pigs or during the
fattening period. All the pigs in the unit will show diar-
rhea within a week. The animals are somewhat anorectic,
depressed, and their feces are watery (no blood). A PEDV
infection toward the end of the fattening period results
in more severe disease than with TGEV. The animals ap-
pear to have more abdominal pain. Recovery occurs as a
rule after 7–10 days. Mortality of 1–3% may be seen in
fattening pigs infected toward the end of the fattening
period. They die acutely, usually in the early stages of di-
arrhea or even prior to the appearance of diarrhea. A
common necropsy finding in these animals is acute back
muscle necrosis. The highest mortality is found on farms
with stress-sensitive pig breeds. In general, PEDV repli-
cation starts more easily in the intestine of feeder and
fattening pigs than in baby pigs. Consequently, fattening
pigs are more susceptible to the virus, and 100% morbid-
ity during an outbreak is common.
182 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
Compared to TGEV, PEDV spreads more slowly
throughout the premises of closed-breeding farms and
also within and between fattening units. On breeding
farms that have several separate units and on fattening
farms with separate buildings, it may take 4–6 weeks be-
fore the virus has infected different groups. Some units
may even remain free of infection.
PATHOGENESIS
The pathogenesis of PED has been studied in hysterecto-
my-derived, colostrum-deprived piglets. Piglets were
orally inoculated with the CV777 isolate at the age of 3
days (DeBouck et al. 1981a). Upon inoculation, the pigs
became sick after 22–36 hours. Viral replication, as
demonstrated by IF and transmission EM, occurred in
the cytoplasm of villous epithelial cells throughout the
small intestine and also in the colon.
Infected epithelial cells were observed as early as
12–18 hours postinoculation, and a maximum was
reached between 24 and 36 hours. Viral replication in
the small intestine resulted in cell degeneration leading
to villous shortening. A reduction of villous height:crypt
depth ratio from the normal 7:1 value to 3:1 was ob-
served. No cell degeneration was seen in the colonic ep-
ithelial cells. Fluorescent cells were detectable until 5
days postinoculation.
The pathogenetic features of PEDV in the small in-
testine of piglets are very similar to those of TGEV. How-
ever, the events with TGEV are of a more rapid and dras-
tic nature, leading to a villous shortening that is more
extensive and occurs within 18–24 hours postinocula-
tion. Since viral replication and progress of the infection
in the small intestine with PEDV occurs at a slower rate,
a longer incubation period is observed. PEDV replication
in piglets has not been detected in cells outside the in-
testinal tract.
The pathogenesis of PEDV in older swine has not
been studied in much detail, but fluorescence was found
in the epithelial cells of the small intestinal and colonic
villi of conventional fattening swine after experimental,
as well as natural, infection. Witte et al. (1981), however,
did not observe this but found the colon positive only in
1-day-old piglets and not in older specific-pathogen-free
swine.
It is not clear how much the colonic infection adds to
the severity of clinical signs. Also, no pathogenetic ex-
planation can be given for the sudden death accompa-
nied by acute back muscle necrosis often observed in fin-
ishing pigs and adult pigs. A recent study proved that
halothane-positive fattening swine of 40–70 kg showed
markedly increased concentrations of the skeletal mus-
cle enzymes creatine phosphokinase and lactate dehy-
drogenase in their blood, starting 3 days after inocula-
tion with PEDV.
Using the ELISA tests, PEDV antigens can be detected
in rectal swabs of experimentally inoculated pigs until 11
days (Carvajal et al. 1995b).
Pathogenetic features described in Korea and Japan
are identical to those observed in Europe (Hwang et al.
1994; Sueyoshi et al. 1995).
LESIONS
Lesions have been described in experimentally and natu-
rally infected piglets (Pospischil et al. 1981; Ducatelle et
al. 1982a, b; Hwang et al. 1994; Sueyoshi et al. 1995).
They are confined to the small intestine, which is dis-
tended with yellow fluid. Microscopically, vacuolation
and exfoliation of enterocytes on the small-intestinal vil-
li were observed starting at 24 hours postinoculation,
which coincided with the onset of diarrhea. From that
time on, shortening of the villi occurred. These findings
were confirmed in scanning EM studies. Histochemical-
ly, the enzymatic activity in the small intestine was found
to be markedly reduced (Ducatelle et al. 1981a). This
pathology is very similar to that described for TGE
(Pospischil et al. 1981; Sueyoshi et al. 1995). No
histopathologic changes have been observed in the
colon.
Ultrastructural changes occurred mainly in the cyto-
plasm of enterocytes (Horvath and Moscari 1981;
Pospischil et al. 1981), in which cell organelles had de-
creased, leaving electron translucent areas. Later, the mi-
crovilli and terminal web disappeared, and parts of the
cytoplasm protruded into the intestinal lumen. The cells
became flattened, the tight junction was lost, and cell re-
lease occurred into the gut lumen. Intracellular virus for-
mation was seen by budding through membranes of the
endoplasmic reticulum (Ducatelle et al. 1981b). In the
colon, some cellular changes were observed in entero-
cytes containing virus particles, but no exfoliation was
seen.
DIAGNOSIS
A diagnosis of PED cannot be made on a clinical basis
only. Acute PED outbreaks in which diarrhea is observed
in pigs of all ages, baby pigs included, cannot be clinical-
ly differentiated from TGE. In Europe, outbreaks with a
rapidly spreading watery diarrhea in weaned pigs and
older animals on a breeding farm, in which no baby
piglets are involved, point toward PED.
An etiologic diagnosis can be made in the laboratory
by direct demonstration of PEDV and/or its antigens or
by detection of antibodies.
A direct IF test and an immunohistochemical tech-
nique applied on sections of the small intestine of baby
pigs with diarrhea are the most sensitive, rapid, and reli-
able methods. They can only be used on the intestines of
pigs killed during the acute phase of diarrhea, preferably
within 3 days of onset. The results of these techniques
 CHAPTER 16 PORCINE EPIDEMIC DIARRHEA
are often not reliable on pigs that die naturally, because
of the severe villous atrophy (DeBouck et al. 1981b;
Sueyoshi et al. 1995; Bernasconi et al. 1995).
PEDV particles can be demonstrated in the feces of
pigs with diarrhea by direct EM. Examination of feces for
coronaviruses is often difficult because the virus particles
are not easy to detect if the spikes are lost or not clearly
visible. The highest percentage of positive fecal samples
obtained in experimentally inoculated piglets was 73% in
feces collected the first day after the onset of diarrhea.
Furthermore, immunoelectron microscopy has to be ap-
plied to differentiate PEDV from TGEV, since both virus-
es have the same morphology.
So far, field strains of PEDV from diarrheic feces need
to be adapted to cell culture before they can be routinely
cultivated. Therefore, cell cultures cannot be used rou-
tinely for diagnosis of PED (Hofmann and Wyler 1988).
A number of ELISA techniques have been developed
for detection of PEDV antigens in feces as well as for
demonstration of specific antibodies in serum. They are
sensitive and reliable for diagnosis, particularly on a
group basis. For the antigen ELISAs, polyclonal and
monoclonal antibodies were used with pig-cultivated
virus (Callebaut et al. 1982; van Nieuwstadt and Zetstra
1991; Carvajal et al. 1995a). For the antibody ELISA, anti-
gen consists of semipurified virus, either pig cell culti-
vated (Callebaut et al. 1982; Kweon et al. 1994; Carvajal
et al. 1995b; De Arriba et al. 1995) or S and N viral pro-
tein extracted from infected Vero cells (Knuchel et al.
1992). The antibody test has also been used for detection
of immunoglobulins in sow’s milk.
PEDV antigens can be demonstrated in fecal swabs
from 3 until 11 days after experimental inoculation, with
peak excretion being at 4 and 5 days. The time periods of
antigen excretion are shorter in naturally infected pigs.
Fecal material should be collected from several pigs,
preferably during the acute phase of diarrhea. The
ELISA antigen test is of sufficient sensitivity to detect the
virus in situations where it is endemic and is a cause of
persistent diarrhea in breeding farms and where the
amount of virus is too low to be detected by any other
methods.
A serologic diagnosis can be made by demonstration
of PEDV antibodies. Several methods have been report-
ed: the indirect IF test and the IF-blocking test on PEDV-
positive cryostat sections of pig intestine, the ELISA, and
the ELISA-blocking test. Antibodies are detectable by
blocking ELISA at 7 days postinoculation and by indirect
IF between 10 and 13 days postinoculation (Carvajal et
al. 1995b). With all tests, paired serum samples should
be examined. The second (convalescent) serum sample
should be collected no sooner than 2–3 weeks after the
onset of diarrhea.
The antibodies demonstrated with the indirect IF
technique are reported to be temperature labile, which
appears to make the use of this method difficult under
Pensaert 183
practical diagnostic circumstances (Prager and Witte
1981a, b). The indirect IF test has also been found to be
less sensitive than the IF-blocking test and the ELISA
(Prager and Witte 1981a, b; Witte and Prager 1987; Hof-
mann and Wyler 1990; Carvajal et al. 1995b).
PEDV antibodies demonstrated in the serum with the
ELISA-blocking and IF-blocking tests have been shown to
persist for at least 1 year. Reinfection of the intestine,
with development of diarrhea, may occur in seropositive
pigs that have recovered from a first infection 5 months
previously. However, such pigs will show a rapid booster
reaction (DeBouck and Pensaert 1984; Witte and Prager
1987).
TREATMENT AND PREVENTION
Definite recommendations with regard to measures to be
taken during a PED outbreak cannot be given at this
time. Antibiotic treatments are of no help. Pigs with di-
arrhea should have free access to water to diminish de-
hydration. It is advisable to withhold feed, particularly in
fattening swine. Since PEDV does not spread very quick-
ly, preventive measures to temporarily prevent virus en-
trance into farrowing units with newly born piglets may
be of help to postpone the infection of these piglets un-
til a later age, resulting in fewer deaths. In the meantime,
artificial spread of the virus to pregnant sows will stimu-
late a rapid lactogenic immunity and thus shorten the
outbreak on the farm. This artificial exposure can be ac-
complished by use of fecal material from pigs with wa-
tery diarrhea or of intestinal contents from pigs that
have died. This approach is similar to that used with
TGEV. If persistence of the virus is diagnosed in consec-
utive litters of weaned piglets after an outbreak has oc-
curred on a breeding farm, virus elimination can be at-
tempted by interrupting consecutive infections through
removing pigs immediately after weaning to another site
for at least 4 weeks. Also, introduction of new pigs
should be stopped temporarily.
Sanitary measures should prevent introduction of
the virus to the farm. Present epizootiologic knowledge
indicates that virus introduction occurs mainly (if not
only) by animal and human traffic (Bollwahn 1983).
In Europe, the disease caused by PEDV in 1997 was
not of sufficient economic importance to initiate re-
search to develop a vaccine. Outbreaks in Asia have been
so severe that attempts have been made to develop an at-
tenuated virus. Bernasconi et al. (1995) reported that cell
culture adaptation of the CV777 virus made it strikingly
different with regard to genomic sequences. Also, the vir-
ulence of this cell-culture-adapted virus was much lower
for newborn cesarean-derived piglets, since only 5 of 21
had a mild diarrhea at 40 hours postinoculation and
histopathological changes were decreased in severity. A
Korean strain, the KPEDV strain, when passaged 93
times in Vero cells, also showed a reduced pathogenicity
184 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
for neonatal pigs and was found to be safe for pregnant
sows. Therefore, the possible use of such a cell-culture-
adapted virus as a candidate for a vaccine has been pro-
posed (C. H. Kweon, pers. comm.—1996).
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 Porcine Parvovirus
17 W. L. Mengeling
Porcine parvovirus (PPV) causes reproductive failure of
swine characterized by embryonic and fetal infection
and death, usually in the absence of outward maternal
clinical signs. The disease develops mainly when
seronegative dams are exposed oronasally to the virus
anytime during about the first half of gestation, and con-
ceptuses are subsequently infected transplacentally be-
fore they become immunocompetent. There is no defini-
tive evidence that infection of swine other than during
gestation is of any clinical or economic significance. The
virus is ubiquitous among swine throughout the world
and is enzootic in most herds that have been tested. Di-
agnostic surveys have indicated that PPV is the major in-
fectious cause of embryonic and fetal death (Cartwright
and Huck 1967; Mengeling 1978b; Thacker and Leman
1978; Vannier and Tillon 1979; Mengeling et al. 1991).
ETIOLOGY
PPV is classified in the genus Parvovirus (Latin parvus =
small) of the family Parvoviridae (Siegl 1976; Bachmann
et al. 1979). All isolates of PPV that have been compared
have been found antigenically similar if not identical
(Cartwright et al. 1969; Johnson and Collings 1969; Mo-
rimoto et al. 1972a; Johnson et al. 1976; Ruckerbauer et
al. 1978). PPV is also antigenically related to several oth-
er members of the genus (Cotmore et al. 1983; Men-
geling et al. 1986, 1988). However, its identity can be es-
tablished by relatively stringent serologic tests such as
serum neutralization (SN) and hemagglutination inhibi-
tion (HI).
Biophysical and Biochemical Properties
The biophysical and biochemical properties of PPV have
been extensively studied (Siegl 1976; Molitor et al. 1983;
Berns 1984) and are summarized as follows. A mature
virion has cubic symmetry, two or three capsid proteins,
a diameter of approximately 20 nm, 32 capsomeres, no
envelope or essential lipids, and a weight of 5.3 × 106 dal-
tons. The viral genome is single-stranded deoxyribonu-
cleic acid (DNA) with a molecular weight of 1.4 × 106
(i.e., about 26.5% of the weight of the complete virion).
Buoyant densities (g/mL in cesium chloride) of com-
plete infectious virions, incomplete “empty” virions, and
extracted virion DNA are 1.38–1.395, 1.30–1.315, and
1.724 respectively. Viral infectivity, hemagglutinating ac-
tivity, and antigenicity are remarkably resistant to heat, a
wide range of hydrogen ion concentrations, and en-
zymes.
Replication
Replication of PPV in vitro is cytocidal and characterized
by “rounding up,” pyknosis, and lysis of cells (Fig.
17.1A). Many of the cell fragments often remain at-
tached, eventually giving the affected culture a ragged
appearance. Intranuclear inclusions develop (Cartwright
et al. 1969) but they are often sparsely distributed (Rond-
huis and Straver 1972). Infected cultures may hemad-
sorb slightly (Cartwright et al. 1969) (Fig. 17.1B). Cyto-
pathic changes are extensive when cell culture–adapted
virus is propagated under appropriate conditions. How-
ever, on initial isolation several serial passages of the
virus (Cartwright et al. 1969) or, better, the infected cul-
ture may be necessary before the effects are recognized.
The use of immunofluorescence (IF) microscopy greatly
increases the likelihood of detecting minimally infected
cultures (Lucas and Napthine 1971; Mengeling 1975).
Primary and secondary cultures of fetal or neonatal
porcine kidney cells are most often used for propagation
and titration of PPV, although other kinds of cultures
are also susceptible (Pirtle 1974). Replication is en-
hanced by infection of mitotically active cultures (Mayr
et al. 1968; Cartwright et al. 1969; Bachmann 1972; Hal-
lauer et al. 1972). Many cells in such cultures are in the S
phase (i.e., the DNA synthesis phase) of their cell cycle,
wherein the DNA polymerases of cell origin needed for
viral replication are available (Tennant 1971; Siegl and
Gautschi 1973a, b).
If either fetal or adult bovine serum is incorporated
in the nutrient medium of cell cultures used to propagate
PPV, it should be pretested for viral inhibitors (Coackley
and Smith 1972; Johnson 1973; Pini 1975). The same
may apply to sera of several other species (Joo et al.
1976d). Because replication of PPV is affected by mitotic
activity, the effect of the serum on the cells is also espe-
cially important. In addition, cultures should be pretest-
ed for PPV contamination (Lucas and Napthine 1971;
Mengeling 1975). Cultures are sometimes unknowingly
prepared from infected tissues of fetal (Mengeling 1975)
and postnatal (Huygelen and Peetermans 1967; Bach-
mann 1969; Cartwright et al. 1969; Hafez and Liess
1979) pigs. Moreover, PPV can be accidentally intro-
187
188 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
17.1. Cell cultures infected with
PPV. (A) Cytopathic effect, sec-
ondary fetal porcine kidney cells,
120 hours after infection (×250)
(Mengeling 1972). (B) Hemadsorp-
tion, secondary adult porcine thy-
roid cells, guinea pig erythrocytes,
22 hours after thyroid cells were
infected and then subcultured A B
(May-Grünwald-Giemsa; ×100).
duced into cultures in several ways (Hallauer et al. 1971),
including the use of contaminated trypsin (Croghan and
Matchett 1973; Croghan et al. 1973). If contamination is
detected before all cells are infected, the virus can be
eliminated by repeatedly subculturing the cells in the
presence of nutrient medium containing PPV antiserum
(Mengeling 1978a).
Several investigators have used IF microscopy to fol-
low the development of PPV in cell culture (Cartwright
et al. 1969; Lucas and Napthine 1971; Mengeling 1972;
Siegl et al. 1972; Bachmann and Danner 1976). In gener-
al, the sequence of events is as follows. Viral antigen is
detected in the cytoplasm of cells soon after infection if
the inoculum contains a high titer of virus and viral anti-
gen. Most, if not all, of this early cytoplasmic fluores-
cence is the result of antigen phagocytized from the in-
oculum (Mengeling 1972; Mengeling and Cutlip 1975).
By sequential examinations, such antigen can be demon-
strated first on the external surface of the cytoplasmic
membrane and later within the cytoplasm, often rela-
tively concentrated in a juxtanuclear location. The first
unequivocal evidence of viral replication is the appear-
ance of nascent viral antigen in the nucleus (Fig. 17.2A).
In at least some infected cells, nascent antigen next ap-
pears in the cytoplasm in sufficient quantity that both cy-
toplasm and nucleus are brightly fluorescent. Infected
cells commonly seen in the lung of fetuses that develop a
high titer of antibody for PPV probably represent this
stage of replication (see Fig. 17.8C). Affected cells subse-
quently round up, become pyknotic, and disintegrate
with release of virus and viral antigen (Fig. 17.2B). Other
cells in the culture that are not at the appropriate stage to
support viral replication continue to phagocytize and ac-
cumulate viral antigen in their cytoplasm (Fig. 17.2C). A
second wave of viral replication can be induced if these
cells are stimulated to enter the S phase of the cell cycle
as, for example, by the addition of fresh culture medium.
Hemagglutination
PPV agglutinates human, monkey, guinea pig, cat, chick-
en, rat, and mouse erythrocytes. Erythrocytes of other
kinds of animals that have been tested are relatively or
completely insensitive, or the results have been equivocal
(Darbyshire and Roberts 1968; Mayr et al. 1968;
Cartwright et al. 1969; Hallauer et al. 1972; Mengeling
1972; Morimoto et al. 1972a). Several parameters of the
hemagglutination (HA) test—such as the temperature of
incubation (Mayr et al. 1968; Mengeling 1972), the
species of erythrocyte used, and in the case of chicken
erythrocytes the genetic composition (Cartwright et al.
1969; Pini 1975; Ruckerbauer et al. 1978) and age (Mori-
moto et al. 1972a) of the donor—may quantitatively af-
fect results. The HA test is most commonly conducted at
room temperature, at approximately neutral pH, and
with guinea pig erythrocytes. Higher HA titers have been
recorded when the diluent used in the test was veronal
buffer rather than phosphate-buffered saline (Rucker-
bauer et al. 1978). Elution of virus (the hemagglutinin is
part of the virion) can be induced by suspending eryth-
rocytes in alkaline buffer, pH 9 (Hallauer et al. 1972).
Infectivity Titrations
Infectivity titrations are conducted in a standard manner
except that, because cytopathic changes at terminal dilu-
tions are often vague, endpoints of infectivity are often
determined either by examining cell cultures for intranu-
 CHAPTER 17 PORCINE PARVOVIRUS Mengeling 189

A B C
17.2. Secondary cultures of fetal porcine kidney cells infected with PPV and examined by IF
microscopy (×500). (A) 14 hours after infection, culture fixed and then reacted with fluorescent
antibodies (FA). (B) 24 hours after infection, culture reacted with FA and then fixed; only
extracellular antigen and antigen in cells with disrupted cytoplasmic and nuclear membranes are
identified. (C) 48 hours after infection, culture fixed and then reacted with FA.

clear inclusions after appropriate staining or by examin-
ing cell culture medium for viral hemagglutinin
(Cartwright et al. 1969). A titration procedure wherein
infected cells are made evident by IF microscopy (Men-
geling 1972) and a plaque assay (Kawamura et al. 1988)
also have been described.
Serologic Tests
The HI test is frequently used for detection and quantita-
tion of humoral antibody for PPV. Antibody sometimes
can be detected as early as 5 days after swine are exposed
to live virus, and it may persist for years (Johnson et al.
1976). Sera examined by the HI test are usually pretreat-
ed by heat inactivation (56˚C, 30 minutes) and by ad-
sorption with erythrocytes (to remove naturally occur-
ring hemagglutinins) and kaolin (to remove or reduce
nonantibody inhibitors of HA) (Mengeling 1972; Mori-
moto et al. 1972a). Trypsin also has been used to remove
nonantibody inhibitors of HA (Cartwright et al. 1969).
Parameters of the HI test have been studied in detail
(Kim 1974; Joo et al. 1976c).
The SN test is occasionally used for detection and
quantitation of humoral antibody for PPV. Neutraliza-
tion of infectivity is usually confirmed by the absence or
reduction either of intranuclear inclusions or fluorescent
cells in cultures or of viral hemagglutinin in the culture
medium (Mengeling 1972; Johnson 1973; Joo et al.
1975). The SN test has been reported to be more sensi-
tive than the HI test (Johnson and Collings 1971; Joo et
al. 1975). A microtechnique for application of the SN
test has been described (Joo et al. 1975).
Immunodiffusion (Joo et al. 1978), a modified direct
complement-fixation test (Ruckerbauer et al. 1978), and
enzyme-linked immunosorbent assay (Hohdatsu et al.
1988; Westenbrink et al. 1989) also have been used suc-
cessfully to detect antibody for PPV.
EPIDEMIOLOGY
Porcine parvovirus is ubiquitous among swine through-
out the world. In major swine-producing areas such as
the midwestern United States, infection is enzootic in
most herds, and with few exceptions sows are immune.
In addition, a large proportion of gilts are naturally in-
fected with PPV before they conceive, and as a result they
develop an active immunity that probably persists
throughout life. Collectively, the seroepidemiological da-
ta indicate that exposure to PPV is common. They also
emphasize the high risk of infection and reproductive
disease among gilts that have not developed immunity
before conception. The most common routes of infec-
tion for postnatal and prenatal pigs are oronasal and
transplacental respectively.
Pigs nursing immune dams absorb a high titer of an-
tibody for PPV from colostrum. These titers decrease
progressively with time by dilution as pigs grow as well
190 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
as by biological degradation. They usually reach subde-
tectable levels in 3–6 months if sera are examined by the
HI test (Etoh et al. 1979; Paul et al. 1982). Sometimes
passively acquired antibody persists for a longer interval.
Moreover, levels of antibody too low to be detected by
the HI test may be detected by the SN test (Johnson et al.
1976). The primary significance of passively acquired
antibody is that it interferes with the development of ac-
tive immunity. High levels of such antibody can prevent
infection, and lower levels can minimize dissemination
from infected pigs (Suzuki and Fujisaki 1976; Paul et al.
1980). Consequently, some groups of gilts are not fully
susceptible to infection and dissemination of virus until
either shortly before conception or during early gesta-
tion.
Contaminated premises are probably major reser-
voirs of PPV. The virus is thermostable, is resistant to
many common disinfectants (Brown 1981), and may re-
main infectious for months in secretions and excretions
from acutely infected pigs. It was shown experimentally
that although pigs transmitted PPV for only about 2
weeks after exposure, the pens in which they were initial-
ly kept remained infectious for at least 4 months (Men-
geling and Paul 1986). The ubiquity of PPV also raises
the possibility that some pigs are persistently infected
and at least periodically shed virus. However, shedding
beyond the interval of acute infection has not been
demonstrated (Johnson et al. 1976). The possibility of
immunotolerant carriers of PPV as a result of early in
utero infection has been suggested (Johnson 1973).
When gilts were infected with PPV before day 55 of ges-
tation, their pigs were born infected but without anti-
body. Virus was isolated from kidneys, testicles, and
seminal fluid of such pigs killed at various times after
birth up to the time they were 8 months of age; at which
time the experiment was terminated (Johnson and
Collings 1971). Results of another study, wherein dams
were infected early in gestation and their pigs were born
infected but without antibody, also suggest an acquired
immunotolerance (Cartwright et al. 1971). A possible ex-
ample of an infected, immunotolerant, sexually active
boar was reported (Johnson et al. 1976).
Boars may play a significant role in dissemination of
PPV at a critical time. During acute infection the virus is
shed by various routes, including semen, and the isola-
tion of PPV from semen of naturally infected boars has
been reported (Cartwright and Huck 1967; Cartwright et
al. 1969; McAdaragh and Anderson 1975). Semen may
be contaminated externally, as for example with virus-
containing feces, or within the male reproductive tract.
The virus was isolated from a testicle of a boar 5 days af-
ter it was injected into the boar’s prepuce (Lucas et al.
1974) and from testicles of boars killed 5 and 8 days af-
ter they were infected oronasally (Mengeling, unpub-
lished data—1976). Virus was also isolated from scrotal
lymph nodes of boars killed 5, 8, 15, 21, and 35 days af-
ter oronasal exposure. After day 8, isolation was accom-
plished by cocultivating lymph node fragments with fetal
porcine kidney cells (Mengeling, unpublished data—
1976). Irrespective of their immune status, boars can al-
so function as a vehicle for mechanical dissemination of
PPV among susceptible females.
CLINICAL SIGNS
Acute infection of postnatal pigs, including pregnant
dams that subsequently develop reproductive failure, is
usually subclinical (Johnson and Collings 1969; Cutlip
and Mengeling 1975a; Fujisaki et al. 1975; Johnson et al.
1976; Joo et al. 1976a; Mengeling and Cutlip 1976). How-
ever, in young pigs and probably in older breeding stock
as well, the virus replicates extensively and is found in
many tissues and organs with a high mitotic index. Viral
antigen is especially concentrated in lymphoid tissues
(Cutlip and Mengeling 1975a; Fujisaki et al. 1975) (Fig.
17.3A, B). Many pigs, irrespective of age or sex, have a
transient, usually mild, leukopenia sometime within 10
days after initial exposure to the virus (Johnson and
Collings 1969, 1971; Joo et al. 1976a; Mengeling and Cut-
lip 1976). PPV and other structurally similar viruses have
been identified in the feces of pigs with diarrhea (Dea et
al. 1985; Yasuhara et al. 1989). However, there is no ex-
perimental evidence to suggest that PPV either replicates
extensively in the intestinal crypt epithelium or causes
enteric disease as do parvoviruses of several other
species (Cutlip and Mengeling 1975a; Brown et al. 1980).
PPV also has been isolated from pigs with lesions de-
scribed as vesiclelike. The etiologic role of PPV in such
lesions has not been clearly defined (Kresse et al. 1985).
The major and usually only clinical response to infec-
tion with PPV is maternal reproductive failure. Patholog-
ic sequelae depend mainly on when exposure occurs dur-
ing gestation. Dams may return to estrus, fail to farrow
despite being anestrus, farrow few pigs per litter, or far-
row a large proportion of mummified fetuses. All can re-
flect embryonic or fetal death or both. The only outward
sign may be a decrease in maternal abdominal girth
when fetuses die at midgestation or later and their asso-
ciated fluids are resorbed. Other manifestations of ma-
ternal reproductive failure, namely, infertility, abortion,
stillbirth, neonatal death, and reduced neonatal vitality,
also have been ascribed to infection with PPV
(Cartwright and Huck 1967; Johnson 1969; Morimoto et
al. 1972b; Narita et al. 1975; Forman et al. 1977). These
are normally only a minor component of the disease.
The presence of mummified fetuses in a litter can pro-
long both gestation (Narita et al. 1975) and the farrowing
interval (Mengeling et al. 1975). Either may result in still-
birth of apparently normal littermates, whether or not
they are infected.
 CHAPTER 17 PORCINE PARVOVIRUS Mengeling 191

17.3. Cryostat-micro-
tome sections of tissues
from PPV-infected 8-
week-old pigs, examined
by IF microscopy
(×312.5). (A) Viral anti-
gen in germinal center,
tonsil. (B) Viral antigen
in osteogenic layer of
periosteum, rib: a =
connective tissue, b =
A B cortical bone, c =
marrow cavity.

There is no evidence that either fertility or libido of al consequences of infection at different stages of gesta-
boars is altered by infection with PPV (Biront and Bonte tion are summarized in Table 17.1.
1983; Thacker et al. 1987). When only part of a litter is infected transplacental-
ly, as is often the case, one or more littermates are fre-
PATHOGENESIS quently infected by subsequent intrauterine spread of
virus. The same would apply if initial infection were
Dams are susceptible to PPV-induced reproductive fail- through contaminated semen. As a result, any combina-
ure if infected anytime during about the first half of ges- tion or all of the sequelae indicated in Table 17.1 can de-
tation. This interval of maternal susceptibility is indicat- velop in the same litter. Intrauterine dissemination is
ed by the collective results of several experimental probably less common when early embryos are infected
studies (Joo et al. 1976a; Mengeling and Cutlip 1976; because they are quickly resorbed after death, effectively
Mengeling 1979; Mengeling et al. 1980a), by in-depth removing the intrauterine reservoir of virus (Mengeling
epidemiological investigations (Donaldson-Wood et al. et al. 1980a). In such cases there is no evidence at far-
1977; Gillick 1977), and by estimates of the time of rowing for the cause of fewer pigs per litter.
death of fetuses collected during epidemiological sur-
veys (Mengeling 1978b; Mengeling et al. 1991). Conse-
quences of maternal infection during this interval are Table 17.1. Consequences of infection with PPV at
embryonic and fetal death followed by resorption and different intervals of gestation
mummification respectively. Transplacental infection al- Interval of Gestation
so follows maternal exposure after midgestation, but fe- (days)a
tuses usually survive without obvious clinical effects in Infection Infection Description Consequences
utero. The likely reason is that transplacental infection of of of of
often requires 10–14 days (Mengeling et al. 1978, 1980a) Dam Conceptusb Conceptus Infection
or longer (Joo et al. 1976a), and by 70 days of gestation ≤56 10–30 Embryo Death and
most fetuses are able to develop a protective immuno- resorption
logic response to the virus. In general, fetuses experi- 30–70 Fetus Death and
mentally infected by transuterine inoculation of the mummification
>56 70–term Fetus Immune response
virus have died when infected before day 70 of gestation, and usually
but they have survived and produced antibody when in- survival in utero
fected later in gestation (Redman et al. 1974; Bachmann
et al. 1975; Cutlip and Mengeling 1975b; Mengeling and a
Intervals are approximations.
Cutlip 1975). A strain of PPV of slightly greater viru- b
Assuming transplacental infections 10–14 days after mater-
lence also has been reported (Choi et al. 1987). The usu- nal exposure.
192 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
The effect, if any, of PPV on the ovum before ovula-
tion is unknown. The virus adheres tenaciously to the ex-
ternal surface of the zona pellucida of the fertilized
porcine ovum (Wrathall and Mengeling 1979a, b), and
although it apparently cannot penetrate this layer, spec-
ulation is that it could pose a threat to the embryo after
hatching (Wrathall and Mengeling 1979a).
Despite strong circumstantial evidence (Cartwright
et al. 1971), a direct causal role of PPV-contaminated se-
men in reproductive failure has not been established un-
equivocally (Lucas et al. 1974). The zona pellucida could
protect the early embryo while local immunity is devel-
oping. Conversely, the virus may cause uterine changes
incompatible with gestation (Wrathall and Mengeling
1979c). In any event, a female infected through semen
provides a focus of infection for others.
With the possible exception of the uterine changes al-
luded to in the preceding paragraph, PPV-induced repro-
ductive failure is caused by the direct effect of the virus
on the conceptus. In the absence of an immune re-
sponse, the virus replicates extensively throughout these
tissues. By the time the conceptus dies, most of its cells
contain large quantities of intracytoplasmic viral antigen
that can be demonstrated by IF microscopy. The relative
lack of nuclear fluorescence at the time of death, com-
pared to earlier stages of the disease, indicates that when
the conceptus is severely affected, mitotic activity and
the associated conditions necessary for viral replication
are suppressed more than phagocytic activity.
Death of the conceptus probably results from the col-
lective damage by the virus to a variety of tissues and or-
gans, including the placenta (Cutlip and Mengeling
1975b). However, in the absence of an immune re-
sponse, changes in almost any vital organ are probably
sufficient to eventually cause death. One of the most
striking features of viral distribution is the extensive in-
volvement of endothelium. This seems to preclude fur-
ther development of the vascular network of the concep-
tus. Preparation for cellular mitosis (i.e., the S phase)
results in concomitant viral replication and cell death.
Damage to the fetal circulatory system is indicated by
edema, hemorrhage, and the accumulation of large
amounts of serosanguineous fluids in body cavities.
Necrosis of the endothelium is microscopically evident
(Lenghaus et al. 1978).
The mechanism of transplacental infection has been
investigated by using IF microscopy to identify infected
cells in maternal and fetal tissues at progressively longer
intervals after maternal oronasal exposure (Mengeling et
al. 1978). Examination of tissues contiguous with the
maternal-fetal junction revealed viral antigen in en-
dothelial and mesenchymal cells of the chorion, with in-
creasing involvement of these tissues at progressively lat-
er stages of gestation. Viral antigen was never detected
unequivocally in either uterine epithelium or trophecto-
derm. Consequently, there was no evidence for maternal-
fetal transfer of the virus by replicating through these tis-
sues. However, this route cannot be excluded, since only
a small part of the total area of contact was examined.
Transfer of the virus within macrophages has been con-
sidered (Paul et al. 1979). Whatever the route, maternal
viremia seems a likely prerequisite for transplacental in-
fection (Joo et al. 1976a; Mengeling and Cutlip 1976).
LESIONS
Neither macroscopic nor microscopic lesions have been
reported for nonpregnant pigs (Cutlip and Mengeling
1975a; Brown et al. 1980). It is conceivable that cellular
infiltrations subsequently described for fetuses could be
induced by infection during the perinatal interval.
Macroscopic lesions have not been reported in preg-
nant dams; however, microscopic lesions have been seen
in tissues of gilts killed after their fetuses were infected
by transuterine inoculation of virus. Gilts that were
seronegative when their fetuses were infected at 70 days
of gestation had focal accumulations of mononuclear
cells adjacent to the endometrium and in deeper layers of
the lamina propria when they were killed 12 and 21 days
later. In addition, there were perivascular cuffs of plasma
cells and lymphocytes in the brain, spinal cord, and
choroid of the eye (Hogg et al. 1977). When fetuses were
infected earlier in gestation (35, 50, and 60 days) and
their dams were killed 7 and 11 days later, the lesions
were similar. However, uterine lesions were more severe
and also included extensive cuffing of myometrial and
endometrial vessels with mononuclear cells (Lenghaus et
al. 1978). Only focal accumulations of lymphocytes were
seen in uteruses of gilts that were seropositive when their
fetuses were infected (Cutlip and Mengeling 1975b).
Macroscopic changes of embryos are death followed
by resorption of fluids (Fig. 17.4) and then soft tissues
(Fig. 17.5). Virus and viral antigen are widely distributed
in tissues of infected embryos and their placentas (Men-
geling et al. 1980a), and it is probable that microscopic
lesions of necrosis and vascular damage, subsequently
described for fetuses, also develop in advanced embryos.
There are numerous macroscopic changes in fetuses
infected before they become immunocompetent (Fig.
17.6). These include a variable degree of stunting and
sometimes an obvious loss of condition before other ex-
ternal changes are apparent; occasionally, an increased
prominence of blood vessels over the surface of the fetus
due to congestion and leakage of blood into contiguous
tissues; congestion, edema, and hemorrhage with accu-
mulation of serosanguineous fluids in body cavities;
hemorrhagic discoloration becoming progressively dark-
er after death; and dehydration (mummification). Many
of these changes also apply to the placenta. Microscopic
lesions consist primarily of extensive cellular necrosis in
a wide variety of tissues and organs (Joo et al. 1977;
Lenghaus et al. 1978) (Fig. 17.7A). Inflammation (Joo et
 CHAPTER 17 PORCINE PARVOVIRUS Mengeling 193

17.4. Embryos from a gilt experimentally infected oronasally immediately after breeding and
killed 22 days later. Bar = 1 cm. (Top) Noninfected, clinically normal embryo (arrow) and
associated extraembryonic membranes; (bottom) PPV-infected, dead littermate embryo (arrow)
and associated extraembryonic membranes, recent death, no obvious resorption of soft tissues.
(Mengeling et al. 1980a.)

cephalitis characterized by perivascular cuffing with pro-

liferating adventitial cells, histiocytes, and a few plasma
cells was seen in the gray and white matter of the cere-
brum and in the leptomeninges of PPV-infected stillborn
pigs. These lesions were believed to be pathognomonic
for PPV infection (Narita et al. 1975). Similar lesions
have been observed in PPV-infected, live fetuses collected
late in gestation (Hogg et al. 1977; Joo et al. 1977) (Fig.
17.7B).
Both general types of microscopic lesions (i.e., necro-
sis and mononuclear cell infiltration) may develop in fe-
tuses infected near midgestation (Lenghaus et al. 1978)
when the immune response is insufficient to provide pro-
tection.

DIAGNOSIS

PPV should be considered in a differential diagnosis of

17.5. Segment of uterus opened to show necrotic
remnants of a partially resorbed PPV-infected embryo
(arrows) and associated extraembryonic membranes of a
gilt experimentally infected oronasally immediately after
breeding and killed 22 days later; remnants are laden
with virus and viral antigen. Bar = 1 cm. (Mengeling et
al. 1980a.)
al. 1977) and intranuclear inclusions (Lenghaus et al.
1978) also have been described.
In contrast, macroscopic changes have not been re-
ported for fetuses infected after they become immuno-
competent for PPV. Microscopic lesions are primarily
endothelial hypertrophy (Hogg et al. 1977) and mononu-
clear cell infiltrations consistent with an immune re-
sponse (Hogg et al. 1977; Joo et al. 1977). Meningoen-
reproductive failure of swine whenever there is evidence
of embryonic or fetal death or both. The pathologic se-
quelae of maternal infection during gestation have been
described (see the section on clinical signs). If gilts but
not sows are affected, maternal illness is not seen during
gestation, there are few or no abortions or fetal develop-
mental anomalies, and other evidence suggests an infec-
tious disease, then a tentative diagnosis of PPV-induced
reproductive failure can be made. The relative lack of
maternal illness, abortions, and fetal developmental
anomalies differentiates PPV from most other infectious
causes of reproductive failure. However, a definitive di-
agnosis requires laboratory support.
Several mummified fetuses (<16 cm in length) or
lungs from such fetuses, if sufficiently developed, should
be submitted to the diagnostic laboratory. Larger mum-
mified fetuses (i.e., more than about 70 days of gesta-
tional age) (Marrable and Ashdown 1967), stillborn pigs,
and neonatal pigs are not recommended for submission
194 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

A B

17.6. PPV-infected fetuses. Bars = 5 cm. (A) Litter of a gilt experimentally infected oronasally
˜
on day 47 of gestation and killed 34 days later; fetuses from left (L) and right (R) horn of uterus,
numbered 1–4 from cervix toward ovary; fetuses L1 and L4 stunted but alive at necropsy, fetus L3
recently dead, others later stages. (B) Fetuses from litter of a naturally infected gilt, collected at
about 114 days of gestation, advanced stage of dehydration (mummification). (Mengeling et al.
1975.)

unless they are the only samples available. If infected,
their tissues will usually contain antibody that interferes
with laboratory tests for either virus or viral antigen.
If females fail to farrow despite being anestrus and
are sent to an abattoir, their uteruses should be collected
and examined for affected fetuses. Sometimes only rem-
nants of fetal tissues remain when fetuses die early in the
middle third of gestation. Nevertheless, these are ade-
quate samples if tested for viral antigen by IF microscopy
(Mengeling and Cutlip 1975; Mengeling 1978b). The ab-
sence of affected fetuses or fetal remnants does not ex-
clude PPV-induced reproductive failure. When all em-
bryos of a litter die and are completely resorbed after the
first few weeks of gestation, the dam may remain en-
docrinologically pregnant and not return to estrus until
after the expected time of farrowing (Rodeffer et al.
1975).
Identification of viral antigen by IF microscopy is a
reliable and sensitive diagnostic procedure. Sections of
fetal tissues are prepared with a cryostat microtome and
are then reacted with standardized reagents (Mengeling
et al. 1975; Mengeling 1978b). The test can be complet-
ed within a few hours. In the absence of a fetal antibody
response, antigen is seen throughout fetal tissues (Fig.
17.8A, B); even when antibody is present, infected cells
usually can be detected in fetal lung (Fig. 17.8C).
Detection of viral hemagglutinin also has been rec-
ommended as a diagnostic technique (Joo et al. 1976b;
Joo and Johnson 1977a). Tissues are triturated in diluent
and then sedimented by centrifugation. The supernatant
fluid is tested for agglutinating activity for guinea pig
erythrocytes. This test requires a minimum of laborato-
ry equipment and is effective in the absence of antibody.
Virus isolation is less suitable as a routine diagnostic
procedure than either of the aforementioned tests. Infec-
tivity is slowly but progressively lost after fetal death
 CHAPTER 17 PORCINE PARVOVIRUS Mengeling 195

A B
17.7. Tissues of PPV-infected fetuses of gilts experimentally infected oronasally. (A) Necrotic
focus in liver of live fetus of a gilt infected on day 40 of gestation and killed 42 days later; fetus
had numerous macroscopic lesions (H&E; ×400). (B) Perivascular cuffing with mononuclear cells
in cerebrum of live fetus, littermate of A; fetus had no macroscopic lesions (H&E; ×320). (Insert)
Viral antigen associated with endothelium of cerebral vessel of fetus of a gilt infected on day 46 of
gestation and killed 25 days later (IF microscopy; ×312.5). All fetuses were probably infected by
intrauterine spread of PPV from transplacentally infected littermates. (Photographs A and B
courtesy of T. T. Brown, Jr., National Animal Disease Center.)

(Mengeling and Cutlip 1975); as a result, isolation of
virus from mummified fetuses that have died as a result
of infection is sometimes unsuccessful (Mengeling
1978b). Moreover, the procedure is time-consuming,
and contamination is a constant threat because of the
stability of PPV in the laboratory (Cartwright et al. 1969)
and because cell cultures are sometimes unknowingly
prepared from infected tissues (Huygelen and Peeter-
mans 1967; Bachmann 1969; Cartwright et al. 1969;
Mengeling 1975; Hafez and Liess 1979). IF microscopy is
often used to determine whether PPV has been isolated
in cell culture (Cartwright 1970; Johnson 1973; Men-
geling 1978b).
In general, serologic procedures are recommended
for diagnosis only when tissues from mummified fetuses
are not available for testing as previously described. Re-
sults with maternal sera are of value if antibody is not
detected, thus excluding PPV as a cause, and if samples
collected at intervals reveal seroconversion for PPV coin-
cident with reproductive failure (Morimoto et al. 1972b;
Mengeling et al. 1975; Rodeffer et al. 1975). Because PPV
is ubiquitous, the presence of antibody in a single sam-
ple is otherwise meaningless. However, a determination
of relative amounts of antibody present as immunoglob-
ulin M and G can indicate the recency of infection (Kim
1974; Joo et al. 1978). Detection of antibody in sera of fe-
tuses and stillborn pigs and in sera collected from neona-
tal pigs before they nurse is evidence of in utero infec-
tion, since maternal antibody does not cross the
maternal-fetal junction (Johnson and Collings 1969,
1971; Cartwright et al. 1971; Mengeling 1972; Chaniago
et al. 1978). When serum is not available, body fluids col-
lected from fetuses or their viscera that have been kept in
a plastic bag overnight at 4˚C have been used successful-
ly to demonstrate antibody (Cropper et al. 1976; Joo et al.
1976b).
196 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

A B C
17.8. Cryostat-microtome sections of lungs of PPV-infected fetuses examined by IF microscopy.
(A) Lung of mummified fetus reacted with FA plus nonimmune serum (×312.5). (B) Replicate
section reacted with FA plus PPV-immune serum (i.e., blocking control) (×312.5). (C) Lung of live
fetus with HI antibody titer of 640 reacted with fluorescent antibodies (FA) plus nonimmune
serum, two infected cells (arrow) (× 162.5). (Insert) Two similar infected cells in the same section
as C (×500). (Mengeling 1978b.)

TREATMENT AND PREVENTION
There is no treatment for PPV-induced reproductive fail-
ure.
Gilts should be either naturally infected with PPV or
vaccinated for PPV before they are bred. To promote nat-
ural infection, a common practice is to arrange contact
between seronegative gilts and seropositive sows, with
the expectation that one or more of the sows will be
shedding virus. Moving gilts to a potentially contaminat-
ed area, either currently or recently inhabited by
seropositive swine, also can be recommended. Once in-
fection is started, the virus spreads rapidly among fully
susceptible swine. Just how effective these procedures are
in increasing the incidence of natural infection is un-
known. For whatever reasons, infection is common, and
probably well over one-half of all gilts in areas where
PPV is enzootic are infected before they are bred for the
first time (Mengeling 1972).
The use of vaccine is the only way to ensure that gilts
develop active immunity before conception. Both inacti-
vated (Suzuki and Fujisaki 1976; Ide et al. 1977; Joo and
Johnson 1977b; Mengeling 1977; Fujisaki 1978; Fujisaki
et al. 1978b; Mengeling et al. 1979, 1980b) and modified
live-virus (MLV) vaccines (Paul and Mengeling 1980; Fu-
jisaki and Murikami 1982) have been developed. An in-
activated vaccine has been tested under field conditions
(Fujisaki 1978; Fujisaki et al. 1978a), and both types of
vaccines were effective when tested under controlled lab-
oratory conditions (Mengeling et al. 1979, 1980b; Paul
and Mengeling 1980).
Vaccines should be administered several weeks be-
fore conception to provide immunity throughout the
susceptible period of gestation but after the disappear-
ance of passively acquired colostral antibody, which
could interfere with the development of active immunity
(Paul and Mengeling 1986). These limits may define a
very brief interval for effective vaccination of gilts that
are bred before 7 months of age. Although inactivated
vaccine provides maximum safety, there is experimental
evidence that PPV can be sufficiently attenuated so that
it is unlikely to cause reproductive failure even if inad-
vertently administered during gestation (Paul and Men-
geling 1980). The apparent safety of MLV vaccine may be
due to its reduced ability to replicate in tissues of the in-
tact host and cause the level of viremia needed for
transplacental infection (Paul and Mengeling 1984).
Moreover, it has been shown by transuterine inoculation
of both virulent and attenuated virus that a much larger
dose of attenuated virus is required to establish infection
of fetuses (Mengeling et al. 1984). Duration of immuni-
ty following vaccination is unknown; however, in one
study antibody titers were maintained for at least 4
months after administration of an inactivated vaccine
(Joo and Johnson 1977b). Low levels of antibody found
to be protective allow speculation that, once the immune
system has been primed with PPV, subsequent exposure
to virulent virus during gestation is unlikely to result in
transplacental infection even if antibody from vaccina-
tion is no longer detected (Mengeling et al. 1979).
Vaccination is recommended also for seronegative
sows and boars. Seronegative sows are usually found on-
ly in PPV-free herds; in such cases, inactivated vaccine is
indicated. Experience has shown that few herds can be
expected to remain free of PPV even if access is carefully
controlled. Introduction of PPV into a totally susceptible
 CHAPTER 17 PORCINE PARVOVIRUS Mengeling
herd can be disastrous (Donaldson-Wood et al. 1977).
Vaccination of boars should reduce their involvement in
dissemination of the virus.
Vaccines are used extensively in the United States and
in several other countries where PPV has been recog-
nized as an economically important cause of reproduc-
tive failure. All federally licensed vaccines marketed in
the United States are inactivated.
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 Porcine Reproductive and
18 Respiratory Syndrome
D. A. Benfield, J. E. Collins, S. A. Dee, P. G. Halbur,
H. S. Joo, K. M. Lager, W. L. Mengeling, M. P. Murtaugh,
K. D. Rossow, G. W. Stevenson, and J. J. Zimmerman
Previously unrecognized epidemics of an acute porcine
reproductive disease characterized by a marked increase
of late-term abortions, stillborn and weak pigs, lowered
farrowing rates, high death rates among weaned pigs,
and delayed returns to estrus were first reported in the
United States in 1987. In many of the epidemics severe
respiratory disease in suckling and weaned pigs was also
a prominent feature (Keffaber 1989; Loula 1991). Clini-
cally similar epidemics were recognized in Canada in the
autumn of 1987 (Dea et al. 1990), in Japan in 1989
(Shimizu et al. 1994), in Germany in 1990 (Lindhaus and
Lindhaus 1991), in the Netherlands, Spain, France, and
the United Kingdom in 1991 (Wensvoort et al. 1991;
White 1991; Meredith 1992), in Denmark in 1992
(Bøtner et al. 1994), and since 1992 in much of the rest
of the world wherever large numbers of swine are raised
(Meredith 1995).
In 1991 the name “porcine reproductive and respira-
tory syndrome” (PRRS) was proposed as the internation-
al designation for such epidemics (European Commis-
sion 1991), with the intent of supplanting an array of
different and somewhat confusing names that, until that
time, were being used in various parts of the world to de-
scribe the same syndrome. Among these were “mystery
swine disease” (often used in the United States before the
cause of PRRS was identified), “blue ear disease” (based
on a transient, bluish discoloration of the ears of some
affected sows and gilts), “swine infertility and abortion
This chapter was compiled by the section editor from invited contribu-
tions of individuals recognized as being among the international experts in
specific subject areas dealing with PRRS and PRRS virus. It was believed that
this approach would provide the most comprehensive and incisive treatment
of what is currently known about PRRS. Contributors and their correspond-
ing subject areas are as follows: D. A. Benfield (Introduction, Etiology); J. E.
Collins and K. M. Lager (Diagnosis); S. A. Dee and H. S. Joo (Prevention and
Control); P. G. Halbur and K. M. Lager (Lesions); W. L. Mengeling (Vaccines);
M. P. Murtaugh (Immunity); K. D. Rossow (Pathogenesis); G. W. Stevenson
(Clinical Signs); and J. J. Zimmerman (Epidemiology). In addition, numerous
other individuals knowledgeable about PRRS or PRRS virus or both were
asked for their input. Every effort was made to blend the individual subject
areas into a single coherent, highly informative, error-free document.
syndrome,” and “porcine epidemic abortion and respira-
tory syndrome.” Today PRRS is the name used almost ex-
clusively, and it is the name used hereafter in this chap-
ter.
Although epidemiological data from even the very
early cases of PRRS suggested that it was an infectious
disease, it was not until several years later that the cause,
PRRS virus (PRRSV), was identified with certainty
(Wensvoort et al. 1991; Collins et al. 1992). The origin of
PRRSV is still unknown, but it is intriguing that North
American and European isolates of the virus have
marked genotypic and phenotypic differences, suggest-
ing that if they originated from a common ancestor, they
developed along different evolutionary lines.
PRRS continues to plague the swine industry both as
an endemic respiratory disease, often as part of what has
been referred to as the “porcine respiratory disease com-
plex,” and as a sporadic, acute reproductive disease most
strikingly manifested as an unusually high incidence of
abortions. Recent severe epidemics of reproductive dis-
ease described in the United States as “acute” or “atypi-
cal” PRRS (Halbur and Bush 1997; Epperson and Holler
1997; Mengeling et al. 1997; Zimmerman et al. 1997a)
and caused by what may be new, more virulent strains of
PRRSV, suggest that PRRSV is continuously changing—
a concept that is supported in part by base sequence (Ka-
pur et al. 1996; Murtaugh et al. 1997) and restriction en-
zyme (Wesley et al. 1996; Mengeling et al. 1997) analysis.
If so, the potential for genotypic and related phenotypic
change, and the ability of PRRSV to persist in herds for
extended periods of time, pose serious challenges for the
control and possible eradication of PRRS.
ETIOLOGY
The first major step in establishing the etiology of PRRS
was made by Collins et al. (1990), who reproduced the
respiratory facet of the syndrome by exposing gnotobi-
otic pigs to filtered homogenates of tissues from field
cases. About a year later Koch’s postulates were fulfilled
when investigators at the Central Veterinary Research In-
201
202 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
stitute in Lelystad, Netherlands, caused the reproductive
facet of the syndrome with a virus they had isolated in
porcine alveolar macrophages (PAMs) and identified as
Lelystad virus (LV) (Terpstra et al. 1991; Wensvoort et al.
1991). Soon thereafter, similar results were obtained by
American investigators with a virus they had isolated in
an established cell line and identified as VR-2332 (Ben-
field et al. 1992; Collins et al. 1992). Additional studies
indicated that while LV and VR-2332 were sufficiently re-
lated to be considered strains of PRRSV, they were also
genetically (Meng et al. 1995a; Murtaugh et al. 1995) and
antigenically (Wensvoort et al. 1992a; Nelson et al. 1993)
distinguishable—with LV the prototype of most strains
of PRRSV subsequently isolated in Europe, and VR-2332
the prototype of most strains of PRRSV subsequently
isolated in North America and Asia.
In addition to the substantial differences that have
been identified between LV and VR-2332 there are also
differences among North American isolates of PRRSV
(i.e., VR-2332-like isolates), including those isolated at
the same time from the same herd (Mengeling et al.
1997). Usually these are no more than what appear to be
minor sequence (base) changes that may have little or no
effect on phenotypic expression. However, some North
American isolates have been shown to differ serological-
ly (E. A. Nelson, unpublished data—1998) and in viru-
lence (Halbur et al. 1995; Halbur et al. 1996b; Mengeling
et al. 1996c). Despite the fact that most isolates can be
differentiated on the basis of minor to marked sequence
changes, and some on the basis of phenotypic expres-
sion, there is still no consensus as to exactly what prop-
erties define a “strain” versus an “isolate” of PRRSV. As a
consequence, these two terms are often used inter-
changeably in various subject areas of this chapter (e.g.,
18.1. A complete PRRSV particle
and (indicated by the open arrow) an
incomplete (coreless) PRRSV particle.
The insert shows a complete PRRSV
particle with the 35 nm nucleocapsid
core (black arrow) surrounded by an
envelope. Bars = 50 nm. (Courtesy of
D. Robison, South Dakota State
University.)
etiology, epidemiology) depending largely on the per-
sonal preference of the author, or authors, who con-
tributed that information rather than on any generally
accepted criteria.
A number of studies (Benfield et al. 1992; Conzelman
et al. 1993; Dea et al. 1992; Wensvoort et al. 1992b; Meu-
lenberg et al. 1993a, b; Saito et al. 1996) have indicated
that PRRSV is closely related biologically, structurally,
and genetically to equine arteritis virus (EAV), lactate de-
hydrogenase-elevating virus (LDV) of mice, and simian
hemorrhagic fever virus (SHFV). On the basis of these
common properties, PRRSV, EAV, LDV, and SHFV
(Plagemann and Moenning 1992; Plagemann 1996) have
been grouped together in a newly created genus Ar-
terivirus, of the family Arterividae, order Nidovirales (Ca-
vanagh 1997). Among the properties of arteriviruses
that are especially important from a clinical perspective
are their ability to (1) cause asymptomatic, persistent in-
fections as well as severe and often fatal diseases; (2)
replicate in macrophages; and (3) exhibit considerable
genome plasticity (Plagemann 1996).
Size and Morphology
The causative agent of PRRS is an enveloped virus with a
diameter of 50–65 nm, a relatively smooth surface, and
a cubical, nucleocapsid core with a diameter of 25–35
nm (Benfield et al. 1992; Dea et al. 1995; Wensvoort et al.
1992b) (Fig. 18.1). Occasionally, short (8–12 nm) granu-
lar projections are observed on the surface of negatively
stained extracellular viral particles (Dea et al. 1995). In
ultrathin sections of infected PAMs, PRRSV appears as a
45–65 nm spherical particle with a 30–35 nm nucleocap-
sid core and a smooth external lipid bilayer membrane
(Dea et al. 1995; Wensvoort et al. 1992b).
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
Density, Temperature, and pH Stability
PRRSV has a buoyant density of 1.19 in cesium chloride,
and glycerol-tartrate gradients; and 1.14–1.15 g/mL in
sucrose gradients (Benfield et al. 1992; Wensvoort et al.
1992b; Bautista et al. 1996).
The temperature stability of PRRSV is similar to that
of the porcine coronavirus (transmissible gastroenteritis
virus). PRRSV is stable for a long time (months to years)
at temperatures of −70˚C and −20˚C. Approximately
90% of PRRSV infectivity is lost within 1 week at 4˚C, but
low titers of infectious virus can still be detected for at
least 30 days. PRRSV infectivity persists for 1–6 days at
20–21˚C, 3–24 hours at 37˚C, and 6–20 minutes at 56˚C.
The thermal stability of PRRSV in serum and tissues is
similar to that described for virus stored in media.
PRRSV was isolated from 47%, 14%, and 7% of porcine
serum samples stored at 25˚C for 24, 48, and 72 hours,
respectively. When serum was stored at 4˚C or −20˚C,
PRRSV was isolated from 85% of the samples after 72
hours (Van Alstine et al. 1993). In another study, serum
and muscle collected from 14 pigs 5 days after they had
been exposed to PRRSV were tested for infectious virus
after subsequent storage at 4˚C. Infectious PRRSV was
isolated from all of the sera after 48 hours in storage and
from muscle of 2 of the 14 pigs after 5 days in storage
(Bloemraad et al. 1994).
PRRSV is stable at pH 6.5–7.5, but infectivity is rapid-
ly lost at pH below 6 and above 7.5 (Benfield et al. 1992;
Bloemraad et al. 1994).
Collectively, these studies indicate that PRRSV is
thermal and pH labile and is not likely to survive in the
environment for extended periods of time. It can be as-
sumed that the labile nature of PRRSV is part of the rea-
son why it is not consistently isolated from aborted fe-
tuses and stillborn pigs during epidemics of PRRS.
Serum and tissues collected for virus isolation (VI)
should be stored at − 20˚C or 4˚C to preserve the infec-
tivity of PRRSV in the sample.
Sensitivity to Lipid Solvents and
Hemagglutinating Activity
PRRSV is inactivated by treatment with the lipid sol-
vents, namely, chloroform and ether (Benfield et al.
1992; Dea et al. 1992; Wensvoort et al. 1992b). Most ar-
teriviruses, including PRRSV, are also highly unstable in
solutions containing low concentrations of detergents,
which disrupt the envelope with concomitant release of
the noninfectious core particles and loss of infectivity
(Plagemann 1996).
North American and European PRRSV isolates (Ben-
field et al. 1992; Dea et al. 1992; Wensvoort et al. 1991)
and other arteriviruses (Plagemann and Moenning 1992;
Plagemann 1996) failed to agglutinate erythrocytes of
any of the species tested. However, recently, two Japan-
Benfield et al. 203
ese PRRSV isolates, antigenically related to VR-2332,
were shown to agglutinate mouse erythrocytes. Pretreat-
ment of these isolates with detergent increased the
hemagglutinating activity, indicating possible release of
an envelope glycoprotein responsible for hemagglutina-
tion (Jusa et al. 1996).
Genomic Organization
The genome of PRRSV is similar in organization to those
of other arteriviruses and the coronaviruses (Plagemann
and Moenning 1992; Conzelman et al. 1993; Meulenberg
et al. 1993a, b; Plagemann 1996). Virions contain a 15.1
kilobase (kb), linear, single-stranded, polyadenylated
RNA of positive polarity (Conzelman et al. 1993; Meu-
lenberg et al. 1993a, b; Murtaugh et al. 1995). The base
sequence has been determined for the entire genome of
LV (Meulenberg et al. 1993a, b) and for the 3′ terminal
3.5 kb portion of the genome of other European and
many North American isolates of PRRSV (Conzelman et
al. 1993; Mardassi et al. 1994a; Meng et al. 1995a, b;
Murtaugh et al. 1995; Saito et al. 1996; Suarez et al.
1996). The genome contains eight open reading frames
(ORFs) which encode specific viral proteins. ORF 1a and
1b, which span the 5′ terminal 12 kb part of the genome,
encode proteins involved in RNA replication and tran-
scription, including the RNA-dependent RNA poly-
merase. The remaining 3.5 kb located on the 3′ terminal
of the genome comprise ORFs 2 through 7, which encode
the putative viral structural proteins. The order of the
genes is 5′-ORF 1a and 1b (genes encoding the RNA
polymerase)-ORFs 2 through 6 (genes encoding the
membrane-associated proteins)-ORF 7 (gene encoding
the nucleocapsid protein)-3′ end. This sequence of gene
transcription and expression is the same in EAV, LDV,
toroviruses, and coronaviruses.
Genomic transcription involves the formation of a 3′-
coterminal nested set of six subgenomic messenger
RNAs for each of the ORFs 2 through 7. Some PRRSV
isolates produce seven subgenomic mRNAs. The extra
mRNA has a molecular mass between those of the mR-
NAs transcribed from ORFs 2 and 3 (Meng et al. 1996).
The significance of this extra subgenomic mRNA is un-
known, but the number of subgenomic mRNAs pro-
duced by PRRSV does not correlate with either pneu-
movirulence or with RNA from defective-interfering
particles (Meng et al. 1996). All isolates of LDV synthe-
size seven subgenomic mRNAs, none of which are pack-
aged into virions (Plagemann and Moenning 1992;
Plagemann 1996). Each subgenomic mRNA carries a
nonencoding leader at its 5′ end that is derived from the
5′ end of the viral genome. The ORFs of LDV and
PRRSV are read in alternate frames, with ORFs 2, 5, and
7 read in one frame, ORF 4 in another, and ORFs 3 and 6
in the third frame (Plagemann 1996).
204 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
Viral Proteins
Experiments in which the mRNAs have been cloned into
Escherichia coli, baculovirus, and Semliki Forest virus in-
dicate that each of the subgenomic mRNAs codes for one
protein. In cells infected with LV or VR-2332, at least
three viral proteins of 15, 19, and 26 kDa can be detect-
ed by radioimmunoprecipitation and western blotting
using polyclonal antisera from pigs previously infected
with VR-2332 (Nelson et al. 1993; Nelson et al. 1995;
Bautista et al. 1996). Similar proteins have been detected
in lysates of cells infected with either LV or the Canadian
IAF-exp91 isolate of PRRSV (Meulenberg et al. 1995;
Meulenberg and Den Besten 1996; Mardassi et al. 1995).
These three proteins have also been observed in purified
preparations of virions, suggesting that the 15, 19, and
26 kDa proteins are structural. The molecular weights of
these proteins correspond well with the reported molec-
ular weights of the major structural proteins of LDV and
EAV, which contain a nucleocapsid protein of 12–15
kDa, a nonglycosylated membrane-spanning protein of
16–19 kDa, and at least one glycosylated envelope pro-
tein of 24–44 kDa (Plagemann and Moenning 1992).
Recent experiments using proteins expressed from
ORFs 2 through 7 of LV indicate that the products of
ORFs 2 through 5 are glycosylated, whereas the products
of ORFs 6 and 7 are not (Meulenberg et al. 1995; Meu-
lenberg and Den Besten 1996; Van Nieuwstadt et al.
1996). The products of ORFs 2 through 4 were identified
in preparations of purified virions using monoclonal an-
tibodies or polyclonal antisera that had been prepared by
using either PRRSV proteins expressed in vitro or syn-
thetic peptides prepared from the sequence of each viral
protein. These antibodies were then used to identify the
corresponding ORF product in lysates of either PRRSV-
infected cell cultures or purified virions by radioim-
munoprecipitation and western blotting. Results showed
that ORFs 2, 3, and 4 encode for N-glycosylated proteins
with molecular weights of 29–30, 45–50, and 31–35
kDa, respectively (Meulenberg et al. 1995; Meulenberg
and Den Besten 1996; Van Nieuwstadt et al. 1996). Mon-
oclonal antibodies to the ORF 4 protein of LV neutralize
viral infectivity, indicating that at least part of this pro-
tein is exposed at the surface of the virion (Van Nieuw-
stadt et al. 1996).
LV is the first arterivirus shown to contain six struc-
tural proteins, consisting of four membrane glycopro-
teins, a membrane-spanning protein (M), and a nucleo-
capsid protein (N). Only three to four structural proteins
have been identified on North American PRRSV isolates
(Nelson et al. 1993; Nelson et al. 1995; Mardassi et al.
1995; Mardassi et al. 1996; Bautista et al. 1996) and the
other arteriviruses EAV (De Vries et al. 1992) and LDV
(Harty and Plagemann 1988; Plagemann and Moenning
1992; Faaberg and Plagemann 1995; Plagemann 1996).
Currently, there is little information on the biologic
function of each of these viral proteins in terms of viral
assembly, attachment, virulence, pathogenicity, and
antigenicity.
Biologic and Genetic Variability
of PRRSV Isolates
There is ample biologic and genetic evidence to suggest
the presence of different PRRSV strains or isolates. RNA
synthesis is inherently error prone, and RNA genomes
have high mutation frequencies as a result of point mu-
tations, deletions, additions, and substitutions (Holland
1995). The biologic and genetic differences of PRRSV are
determined on the basis of (1) different clinical presen-
tations of the disease; (2) experimental evidence to indi-
cate differences in pneumovirulence and reproductive
virulence; (3) antigenic differences defined by reactivity
with polyclonal and monoclonal antibodies in serologic
assays; and (4) differences in the RNA sequences.
LV and VR-2332 produce remarkably similar repro-
ductive and respiratory clinical syndromes. However, the
first biologic difference noted between the North Ameri-
can and European PRRSV isolates was the frequent ob-
servation of blue discoloration of ears, teats, snout, ven-
tral cervical skin, vulva, and abdomen in outbreaks of
PRRS in Europe, but not in North America (Goyal 1993).
Comparative pathogenicity studies in colostrum-de-
prived, cesarean-derived pigs indicated that PRRSV iso-
lates could be classified in high- and low-virulence
groups based on the severity of gross and microscopic
lung lesions (Halbur et al. 1995; Halbur et al. 1996b). In
addition, some PRRSV isolates were more likely to in-
duce rhinitis, encephalitis, and myocarditis (Halbur et al.
1996b), and some U.S. isolates were more or less virulent
than LV (Halbur et al. 1995). While the least virulent iso-
lates had greater sequence variation in ORFs 2 through 4
than did other U.S. isolates to which they were com-
pared, virulence could not be correlated with any specif-
ic changes in the sequence of the viral genome (Meng et
al. 1995b). Similar studies done with high- and low-
pneumovirulent PRRSV isolates in pregnant gilts sug-
gested that PRRSV isolates differ in their effect on the re-
productive tract. However, there was no clear indication
that the virulence of a particular PRRSV isolate for the
respiratory tract was a predictor of its virulence for the
reproductive tract (Mengeling et al. 1996c).
Assessment of antigenic differences between PRRSV
isolates has been difficult due to technical problems in
producing monoclonal antibodies to the PRRSV struc-
tural proteins and the unreliability of monospecific poly-
clonal serum for use in neutralization assays for the dif-
ferentiation of serotypes. A comparison of 24 field sera
and 7 PRRSV isolates from Europe and North America
failed to reveal two-way cross-reactions between Euro-
pean and North American PRRSV isolates using the im-
munoperoxidase macrophage assay (Wensvoort et al.
1992b). While the North American PRRSV isolates re-
acted with antisera against the European strains, the Eu-
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
ropean strains did not react with antisera from the U.S.
field cases (Wensvoort et al. 1992a, b). In other studies,
pigs seropositive for LV were found to be seronegative for
VR-2332, and vice versa (Bautista et al. 1993). Mono-
clonal antibodies specific for epitopes on the 15 kDa nu-
cleocapsid protein indicate that there are conserved and
variable epitopes between European and North Ameri-
can isolates (Nelson et al. 1993; Drew et al. 1995; Magar
et al. 1995). Nelson et al. (1993) suggested that the North
American and European isolates be classified into two
antigenic subgroups on the basis of their reactivity with
nucleocapsid monoclonal antibodies. The monoclonal
antibodies SDOW 12 and 17, which are specific for con-
served epitopes on the nucleocapsid protein of North
American and European isolates, are used as universal
diagnostic reagents. Monoclonal antibodies to the N
protein and a 45 kDa protein encoded by ORF 3 of the
Humberside isolate of PRRSV react with European, but
not U.S., isolates (Drew et al. 1995). Antigenic differ-
ences on the M protein of North American and Euro-
pean isolates of PRRSV were also demonstrated using
monoclonal antibodies to the M protein of a Canadian
isolate of PRRSV, which reacts only with North Ameri-
can PRRSV isolates (Magar et al. 1997).
The antigenic variation observed between the Euro-
pean and North American PRRSV isolates has been con-
firmed by analysis of the nucleotide and amino acid se-
quences of LV and VR-2332. Nucleotide sequence
analysis of VR-2332 and LV indicated that the character-
istics of ORFs 2 through 7 are very similar, but the pre-
dicted protein structures indicate numerous differences
in the molecular weights, isoelectric points, and predict-
ed glycosylation sites of the corresponding proteins.
Amino acid sequences for VR-2332 and their similarities
to those of LV are 76% ORF 2, 72% ORF 3, 80% ORF 4,
80% ORF 5, 91% ORF 6, and 74% ORF 7 (Murtaugh et al.
1995). Despite significant sequence differences, the hy-
dropathicity profiles of VR-2332 and LV indicated that
PRRSV tends to conserve the structure of the proteins,
and that the extensive amino acid differences in the pro-
teins account for the differences in serologic cross-reac-
tivity (Murtaugh et al. 1995). The amino acid homolo-
gies among North American isolates are 90% or greater
for ORFs 2 through 7 (Kapur et al. 1996; Meng et al.
1995a, b; Mardassi et al. 1994a). Among U.S. isolates,
ORF 5 is the most variable and ORF 6 the most con-
served (Kapur et al. 1996). Sequence analysis of PRRSV
isolates from midwestern swine herds indicates the
viruses are evolving by random mutation and by intra-
genic recombinations between different PRRSV isolates
(Kapur et al. 1996).
Differences between Field Isolates
and Vaccine Viruses
There are currently two commercially available modi-
fied-live-virus vaccines: RespPRRS® (recently renamed
Benfield et al. 205
RespPRRS/Repro®) and Prime Pac® PRRS. Both of these
vaccines are modified-live viruses that have been attenu-
ated in virulence by serial passage in monkey kidney
cells. Genetic and antigenic differences between the vac-
cine strains and virulent field strains of PRRSV have
been identified and used diagnostically. For example, the
ORF 5 sequence of RespPRRS® contains several uncom-
mon restriction sites. As a consequence, digestion of
RespPRRS® with selected restriction endonucleases gives
a gel pattern distinct from that of at least most field iso-
lates of PRRSV (Wesley et al. 1996). Likewise, Prime
Pac® PRRS does not react with the SDOW 12 or 17 mon-
oclonal antibodies, indicating that the nucleocapsid pro-
tein of Prime Pac® PRRS is missing an epitope present
on a majority of field isolates of PRRS—including the
virulent field isolate from which it originated (R. A.
Hesse, unpublished data—1998; E. A. Nelson, unpub-
lished data—1998). The antigenic difference between
Prime Pac® PRRS and its parent strain suggests a change
associated with serial passage in vitro.
Cultivation and Morphogenesis
Most isolates of PRRSV replicate to titers of 105–107
median tissue culture infectious doses (TCID50) in PAMs,
African green monkey kidney cells (MA-104), derivatives
of MA-104 (CL-2621 and MARC-145), and CRL-11171
cells (Wensvoort et al. 1991; Benfield et al. 1992; Dea et
al. 1992; Kim et al. 1993; Goyal 1993; Halbur et al. 1995).
The virus replicates cytocidally in each cell type, causing
cells to become rounded, clumped, and pyknotic. Affect-
ed cells eventually detach from the culture vessel. The cy-
topathic effect usually develops within 1–4 days in PAM
cultures and within 2–6 days in CL-2621 cell cultures
(Benfield et al. 1992; Pol et al. 1992). Virus replication is
restricted to the cytoplasm, as demonstrated by fluores-
cent antibody or immunoperoxidase staining (Benfield
et al. 1992; Dea et al. 1992; Wensvoort et al. 1991).
A few North American isolates of PRRSV have been
found to grow exclusively in either PAMs or CL-2621
cells, but most grow in both cell types (Bautista et al.
1993; Goyal 1993). Conversely, most, if not all, European
isolates of PRRSV replicate better or exclusively in
PAMs. Vaccine viruses replicate at levels 100–1000 times
lower on PAMs than on derivatives of a monkey kidney
cell line (D. A. Benfield, unpublished data—1998). Al-
though data are lacking, it is probable that the use of CL-
2621 or MARC-145 cells versus PAMs selects for the iso-
lation of vaccine viruses rather than field isolates of
PRRSV.
There is limited information on the morphogenesis
of PRRS virions. Pol et al. (1992) have described the mor-
phogenesis of LV in PAMs. These investigators observed
viral nucleocapsids budding through the smooth endo-
plasmic reticulum (ER) by 6 hours after inoculation. By 9
hours after inoculation they noted enveloped viral parti-
cles in the lumen of the smooth ER and in the Golgi re-
206 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
gion. Viral replication was restricted to the cytoplasm.
Intact viral particles were released from infected cells by
exocytosis as early as 9–12 hours after inoculation.
Pulse-chase experiments with a Canadian isolate of
PRRSV propagated in MARC-145 cells indicated that vi-
ral assembly starts in the ER, where the matrix and enve-
lope proteins are linked by disulfide bonds and then in-
teract with the nucleocapsid protein that has previously
accumulated in the cytoplasm of the perinuclear region
of the cell. Thereafter, vesicles containing enveloped nu-
cleocapsids are derived from the ER and transported to
the Golgi region, where budding takes place and some
envelope proteins undergo terminal glycosylation. At the
end of the transit from the ER to the Golgi region, the en-
velope protein has acquired its mature structure, which
appears to influence the onset of virus release by exocy-
tosis (Mardassi et al. 1996).
EPIDEMIOLOGY
Currently available information suggests that PRRSV en-
tered the domestic swine population relatively recently
and spread rapidly thereafter. The earliest evidence of
PRRSV infection in domestic swine comes from a retro-
spective serologic study of herds in Ontario, Canada.
Carman et al. (1995) found that none of 50 herds sam-
pled in 1978 were serologically positive for antibodies
against PRRSV by enzyme-linked immunosorbent assay
(ELISA) or indirect fluorescent antibody (IFA), but anti-
bodies were detected in sera of 2 of 51 (3.9%) herds sam-
pled in 1979 and in sera of 8 of 51 (15.7%) herds sampled
in 1980.
Apparently, PRRSV did not enter the United States
for several years after the first infections in Canada. None
of 1425 serum samples collected from 118 Iowa swine
herds in 1980 were antibody positive by IFA (Zimmer-
man et al. 1997c). One of 26 herds (3.8%) sampled in
1985 was PRRSV-infected. Each successive year showed a
marked increase in prevalence. By 1988, 17 of 27 herds
(63.0%) and 313 of 658 (47.6%) pigs were seropositive.
The low prevalence in 1985 suggests that entry of the
virus into Iowa occurred during or shortly prior to 1985.
Similar to the Iowa data, the earliest evidence of infec-
tion in the state of Minnesota comes from serum sam-
ples collected in 1986 (Yoon et al. 1992).
The infection did not remain limited to North Amer-
ica. In Asia, antibodies against PRRSV were retrospec-
tively documented in South Korea in serum from pigs
imported in October 1985 (Shin et al. 1993), and in
Japan in samples collected in June 1988 (Hirose et al.
1995). In Europe, clinical outbreaks of PRRS were first
reported in Germany in November 1990 (European
Commission 1991), but there is serologic evidence sug-
gestive of the presence of PRRSV in Germany as early as
1988.
Thus, in a period of approximately 10 years, PRRSV
entered and became endemic in a large proportion of the
world’s domestic swine population. The original source
of the virus and the circumstances under which it came
into the domestic swine population are not known. A
wildlife reservoir is the most logical explanation, with
feral swine the most likely candidate species, but in
North America, preliminary data suggest that the virus
has moved into feral swine from domestic swine, rather
than the reverse (J. J. Zimmerman, unpublished data—
1998). Further complicating the puzzle, the prototypic
European and North American PRRSV isolates are suffi-
ciently different from each other, despite the temporal
proximity of their emergence, to suggest that either they
originated from two independent sources, or they devel-
oped along different evolutionary lines over a period of
at least several years. Although PRRSV has become ubiq-
uitous in domestic swine, the question of its origin re-
mains relevant because, until we know otherwise, we
cannot discount the possibility that new strains of
PRRSV may emerge from the original reservoir.
Geographic Distribution and Prevalence
Although survey information is not available from all
countries, PRRSV appears to have become endemic in
nearly all swine-producing areas of the world. Although
some countries claim to be free of PRRSV infection, only
Australia and Sweden have documented their virus-free
status. In Australia, a survey of 875 serum samples from
163 herds selected across all states revealed no evidence
of antibodies against PRRSV (Garner et al. 1996). In
Sweden, a serosurvey of 11,003 samples collected be-
tween 1993 and 1996 also revealed no evidence of infec-
tion (Elvander et al. 1997). However, given the demon-
strated transmissibility of PRRSV, it will not be easy to
maintain these areas free of infection.
Reliable estimates of the prevalence of infection in
endemically infected areas are not generally available for
several reasons. Most published studies are not based on
statistically valid population sampling procedures and,
therefore, may lack accuracy. More recently, prevalence
studies have become confounded by the use of modified-
live-PRRSV vaccines, the use of which results in serum
antibodies that are indistinguishable from those pro-
duced against field isolates. Epidemiologic investigations
are further complicated by the fact that vaccine virus is
disseminated from vaccinated to naive pigs.
In the United States the best prevalence estimate is
based on a 1995 National Animal Health Monitoring
System (NAHMS) study (U.S. Department of Agricul-
ture 1997). In the NAHMS study, 8038 serum samples
were collected from 286 herds in 16 swine-producing
states. Among herds not using modified-live-PRRSV vac-
cine, 129 of 217 (59.4%) herds were infected. Among
6376 unvaccinated animals, 41.3% had antibody titers
detectable by IFA. When animals were identified by sta-
tus, 23.5% of unvaccinated breeding animals and 51.7%
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
of unvaccinated finishers were IFA positive. As in the
United States, reports from other parts of the world in-
dicate that the prevalence of infection is high in endemi-
cally infected regions.
Hirose et al. (1995) reported that 46.4% of pigs sam-
pled in 1993 in Chiba Prefecture, Japan, were serological-
ly positive for PRRSV. In Belgium, Maes (1997) found
that 50 of 50 herds and 96% of market hogs tested were
seropositive. And although PRRS was first reported in
Germany in November 1990, Geue (1995) found that
490 of 689 herds (71.1%) in the Nordfriesland district of
Germany were already infected by October 1992. One ex-
ception to the trend of rapid spread and high prevalence
is the “Pays de la Loire” region of France. By implement-
ing a rigorous program of serologic monitoring and
strict control measures, prevalence was held below 2%
nearly 2.5 years after PRRSV had entered the region (Le
Potier et al. 1997).
Transmission
Efficient transmission has been a hallmark of PRRS since
it was first identified clinically. In the absence of vaccines
or treatments effective in stopping shedding of virus or
eliminating infection in chronic carrier animals, strate-
gies for the prevention, control, and elimination of
PRRSV must be grounded upon a thorough understand-
ing of the means by which transmission occurs. Swine
are susceptible to PRRSV by a number of routes of ex-
posure, including oral, intranasal, intramuscular, in-
traperitoneal, and vaginal. The virus is highly infectious
and swine are readily infected by inoculation of 10 or
fewer PRRSV particles by either intranasal or intramus-
cular routes (K. J. Yoon, unpublished data—1998). The
infectious dose by other routes has not been determined,
but studies with LDV, a closely related virus of mice, are
suggestive of what we might expect. For LDV, the mini-
mum infectious dose has been shown to vary consider-
ably depending on the route of exposure. Cafruny and
Hovinen (1988) found the minimum infectious dose by
intraperitoneal or tail cartilage injections approached 1
virus particle; that is, similar to PRRSV. In contrast, ex-
posure via mucosal surfaces such as ocular, vaginal, or
oral routes required a minimum infectious dose of
103.3–105.3 virions, depending on the specific anatomical
location (Cafruny and Hovinen 1988). Although we
know PRRSV is highly infectious, additional work is
needed to further define the effect of route of exposure
on minimum infectious dose.
Infection results in the shedding of virus in saliva,
urine, semen, and mammary secretions. Wills et al.
(1997b) detected PRRSV in saliva for up to 42 days and
in urine for up to 14 days. Swenson et al. (1994b) found
infectious virus in the semen of experimentally infected
boars for as long as 43 days following exposure. Using a
reverse transcription–polymerase chain reaction, viral
RNA was detected in the semen of experimentally infect-
Benfield et al. 207
ed boars through day 92 postexposure (Christopher-
Hennings et al. 1995a). Transmission of PRRSV to fe-
males artificially inseminated with undiluted semen
from experimentally infected boars has been demon-
strated (Yaeger et al. 1993), and transmission of PRRSV
to females using extended semen from experimentally
infected boars has also been proven (Gradil et al. 1996).
Virus has also been recovered from mammary secretions
collected from gilts and sows challenged in the third
trimester of gestation (J. J. Zimmerman, unpublished
data—1998). The prevalence of fecal shedding of
PRRSV remains an open question. Yoon et al. (1993) re-
ported extensive fecal shedding by young pigs over a 35-
day observation period. In contrast, Rossow et al. (1994)
found only intermittent shedding, and Wills et al.
(1997b) found no infectious virus in fecal samples col-
lected from pigs over a 42-day postinoculation observa-
tion period.
Shedding of virus in saliva, urine, and perhaps feces
results in environmental contamination. In general,
PRRSV is a fragile virus that is quickly inactivated in the
environment; however, it can remain infectious for an ex-
tended period of time under specific conditions of tem-
perature and moisture. Bloemraad et al. (1994) found
both temperature and pH to influence the stability of in-
fectious virus. At 37˚C and a pH of 6.0, the half-life (in-
activation of one-half of the virus population) of LV was
estimated at 6.5 hours, but half-life declined to 0.65
hours at pH 5.0 and 1.28 hours at pH 8.5. At 4˚C and a
pH of 7.5, the half-life of LV was estimated to be 140
hours. The virus is quickly inactivated in the absence of
moisture or in hostile solutions. Pirtle and Beran (1996)
examined the persistence of PRRSV in or on 16 fomites,
including plastic, stainless steel, rubber, alfalfa, wood
shavings, straw, corn, swine starter feed, denim cloth,
phosphate-buffered saline, saline G, well water, city wa-
ter, swine saliva, urine, and fecal slurry. At 25–27˚C virus
did not persist on fomites beyond day zero, except for
phosphate-buffered saline through day 3, saline G
through day 6, well water through day 8, and city water
through day 11. From a practical perspective, standard
cleaning and disinfection procedures should be effective
for inactivation of PRRSV in facilities and on equipment.
Although the data indicate that virus cannot persist in fe-
cal slurry, the potential for persistence of virus in la-
goons has not been examined.
Infection produces a prolonged carrier state. Zim-
merman et al. (1992) reported transmission by direct
contact from sows infected 99 days earlier to commin-
gled susceptible animals. Albina et al. (1994a, b) subse-
quently demonstrated transmission of PRRSV by pigs
infected 15 weeks earlier. More recently, isolation of
virus from oropharyngeal samples for up to 157 days af-
ter experimental inoculation was reported (Wills et al.
1997c). Overall, these data have provided definitive evi-
dence that PRRSV persists in swine for an extended peri-
208 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
od of time. Exactly how long animals remain potentially
infectious is not known.
Although PRRSV is highly infectious (i.e., has a low
minimum infectious dose), it is not highly contagious.
Transmission usually occurs by close contact between
animals. This finding is in sharp contrast to earlier
thought. It was once suggested that aerosolized virus was
the primary route of transmission of PRRSV, with
aerosol transmission postulated to occur routinely and
over distances of up to 20 kilometers. Since PRRSV is
present in the upper respiratory tract and oropharyngeal
area of infected pigs for an extended period of time, this
was not an unreasonable hypothesis. However, Wills et
al. (1997a) found that transmission among penmates oc-
curred much more readily than transmission across a
space of 40 inches or less. In further testimony to the
limited contagiousness of the virus, Torremorell et al.
(1997) documented the transmission of PRRSV from
acutely infected pigs to only 2 of 8 contact controls un-
der experimental conditions. These studies highlight our
limited understanding of the process of transmission it-
self.
Given that the exact mechanism of transmission be-
tween pigs is uncertain, studies of LDV in mice provide
clues to the importance of certain types of interactions
between animals. For LDV, it was observed that certain
laboratory strains of young male mice rarely became in-
fected when placed in the same cage as infected mice,
compared to approximately 50% infection rates when
General Purpose (GP) Swiss male mice were placed in the
same conditions (Notkins and Shochat 1963; Notkins et
al. 1964). However, it was noted that GP Swiss mice were
more prone to fighting than were the other strains of
mice, and if the incisors of both inoculated and exposed
6-month-old GP Swiss male mice were removed, trans-
mission rarely occurred. In contrast, transmission always
occurred if the incisors of the inoculated mice were
present, regardless of whether or not the incisors of the
exposed mice were removed. If the incisors of only the
inoculated mice were removed, transmission still oc-
curred a majority of the time. Overall, the results sug-
gested that, during the course of fighting, infectious
mice could transmit LDV by the injection of saliva or
susceptible mice could contract the infection by the in-
gestion of blood or tissue from infectious mice.
As in the case of LDV, the social dominance behavior
which occurs among pigs in direct contact is ideal for the
transmission of PRRSV. The fighting which invariably
occurs when pigs are mixed, and the low minimum in-
fectious dose and the persistence of virus in the oropha-
ryngeal region for up to several months after infection,
make the movement of pigs both within and between
farms the ideal mode of transmission.
Nonporcine hosts exist, but their role in the epidemi-
ology of PRRSV is uncertain. Although rodents are not
susceptible to PRRSV (Hooper et al. 1994), some avian
species, mallard ducks in particular, are susceptible to
PRRSV (Zimmerman et al. 1997b). Mallards exposed to
PRRSV in drinking water shed virus in feces for an ex-
tended period of time. At the termination of one experi-
ment, virus was recovered from fecal samples collected
from 8 of 20 birds 39 days after exposure. In a second ex-
periment, mallard-to-mallard transmission was demon-
strated by infecting ducks with feces from ducks
shedding PRRSV. And finally, pigs were shown to be sus-
ceptible to mallard-derived virus. That is, pigs
intranasally exposed to PRRSV isolated from mallard
feces became viremic, seroconverted by ELISA, and
transmitted the virus to sentinel pigs. Although it is
unlikely that mallards are active participants in the
transmission of PRRSV to and among swine and swine
herds, they could theoretically serve as a source of new
virus types, as in the case of the influenza viruses. At this
juncture it would seem to be more important to further
investigate the susceptibility of other birds and animals
known to frequent swine facilities and potentially serve
as carriers.
Transmission within Herds
Once infected, PRRSV tends to circulate within a herd in-
definitely. Rarely, spontaneous elimination of PRRSV
from commercial herds has been reported by producers
or veterinary practitioners, but the circumstances under
which this occurs are not well defined. At this point, the
mechanisms that make endemicity possible are not en-
tirely clear. However, the key components appear to be
persistent PRRSV infection in clinically normal carrier
animals and the continual introduction of susceptible
animals, either through birth or purchase (Wills et al.
1997c).
Typically, the virus is perpetuated by a cycle of trans-
mission from dams to pigs either in utero or postpartum,
or by commingling naive animals with infected animals
in later stages of production. In neonatal pigs, maternal
antibodies may provide some immunologic resistance to
infection, but the protection is not absolute and is of
short duration. Under conditions in which susceptible
and infected pigs are mixed (e.g., at weaning), a large
proportion of the population may quickly become in-
fected. Dee and Joo (1994a), for example, reported
80–100% of the pigs in 3 swine herds were infected by
8–9 weeks of age, and Maes (1997) found 96% of market
hogs sampled from 50 herds to be seropositive.
However, it has become increasingly clear that the
pattern of infection in PRRSV-endemic herds often devi-
ates from this description of rapid, uniform spread. And
even within infected herds, marked differences in infec-
tion rates between groups, pens, or rooms of pigs are of-
ten observed in the field. Houben et al. (1995b) found
transmission to vary even within litters, with some litter-
mates seroconverting as early as 6–8 weeks and other in-
dividuals as late as 10–12 weeks of age, and described a
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
slow spreading pattern. In some cases, litters of pigs
reached 12 weeks of age, the end of the monitoring peri-
od, still free of PRRSV. Thus, it is possible for animals in
endemically infected herds to escape infection for an ex-
tended period of time, as when Le Potier et al. (1997) ob-
served seroconversion in young sows on farms using in-
herd gilt replacements.
Transmission between Herds
Herd-to-herd transmission occurs but is generally recog-
nized too long after the fact to accurately determine the
origin of the virus. Known sources of herd-to-herd trans-
mission include infected carrier animals and the use of
PRRSV-contaminated semen. Potential but unproved
sources include contaminated biologics or needles, wa-
terborne virus, airborne virus, nonporcine hosts, arthro-
pods, and fomites. In the course of the PRRSV control
program in France, Le Potier et al. (1997) reported that
the infection was introduced to 56% of herds through in-
fected pigs, 20% through infected semen, 21% through
fomites, and 3% through unidentified sources.
Area spread, that is, herd-to-herd transmission in the
absence of any apparent animal or human contact, is al-
so known to occur but is generally more restricted in dis-
tance than once believed. This may be attributed to the
fact that, until the existence of subclinical infections was
recognized, the distance between clinical outbreaks was
used to measure virus travel. More recently, Le Potier et
al. (1997) found that 45% of herds suspected to have be-
come infected through area spread were located within
500 meters (0.3 miles) of the postulated source herd and
only 2% were 1 kilometer from the initial outbreak.
Area spread is most commonly attributed to aerosols
of infectious PRRSV traveling downwind from infected
herds. Of course, aerosol transmission is known to occur
in the case of many viral infections. Foot-and-mouth dis-
ease virus (FMDV), in particular, is understood in such
detail that the behavior of aerosolized FMDV can be pre-
dicted with a high degree of accuracy using mathemati-
cal models. Transmission of PRRSV in aerosols, howev-
er, has been difficult to achieve experimentally.
Torremorell et al. (1997) were able to transmit PRRSV
from a group of acutely infected pigs over a distance of
just 1 meter in only one of two attempts. Wills et al.
(1997a) described similar results across a comparable
distance, and Robertson (1992) reported no success in
isolating virus from the airspace in which acutely infect-
ed pigs were housed. Specific information is needed at
present. In particular, we need to know the quantity of
virus shed by acutely infected animals in respired air and
the stability of aerosolized PRRSV under various condi-
tions of humidity and temperature. Until such data are
available, it will not be possible to fully evaluate the im-
portance of aerosols in transmission. Even so, the avail-
able information makes it clear that aerosol transmission
is much more limited than once thought.
Benfield et al. 209
CLINICAL SIGNS
Descriptions of the clinical signs of PRRS in swine herds
are similar in North America (Keffaber 1989; Moore
1990; Bilodeau et al. 1991; Loula 1991; Sanford 1992)
and Europe (de Jong et al. 1991; Leyk 1991; Wensvoort et
al. 1991; Anon. 1992; Busse et al. 1992; Gordon 1992;
Hopper et al. 1992; White 1992a, b). Clinical signs of
PRRS are extremely variable and influenced by strain of
virus (Halbur et al. 1996b), immune status of the herd
(Keffaber 1989; Wensvoort 1993), and management fac-
tors (Blaha 1992; White 1992a). Clinical disease in a herd
is primarily the consequence of acute viremia in individ-
uals (Pol et al. 1991; Terpstra et al. 1991; Collins et al.
1992) and transplacental transmission of virus from
viremic dams to their fetuses (Terpstra et al. 1991), which
occurs most efficiently in the third trimester of pregnan-
cy (Christianson et al. 1993; Mengeling et al. 1994).
Strains of PRRSV vary remarkably in virulence (Hal-
bur et al. 1996b). Low-virulence strains can cause com-
pletely subclinical epidemic or endemic infections of
herds (Morrison et al. 1992), whereas highly virulent
strains can cause severe clinical illness. In clinical epi-
demics occurring in immunologically naive herds, all
ages are susceptible, and acute infection is most consis-
tently evidenced by inappetence, fever, and dyspnea. For
the next 1–4 months, many sows farrow prematurely,
usually after 100 days of gestation. Fewer farrow after a
prolonged gestation of 115–118 days. Affected litters
that are born early, full term, or late are composed of any
or all of the following: stillborn pigs, mummified fetuses,
late-term dead fetuses, variably sized weak-born pigs,
and variably sized, apparently normal pigs. Preweaning
mortality is high. In endemically infected herds, pre-
dominant signs are in nursery-grower pigs and include
reduced thrift, dyspnea, exacerbation of other endemic
diseases, and increased mortality. Less commonly, there
is sporadic reproductive failure in subpopulations of sus-
ceptible gilts.
Epidemic Infection of Herds
In clinically evident epidemic infections in immunologi-
cally naive herds, the first phase of PRRS lasts approxi-
mately 2 weeks and is an acute systemic illness in 5–75%
of animals that is caused by initial viremia and that is
most often characterized by anorexia and lethargy. It be-
gins in one or more stages of production and quickly
spreads within 3–7 days to all stages of production. Indi-
viduals are anorectic for 1–5 days, and the spread of the
disease through a segregated group of pigs usually re-
quires 7–10 days, giving rise to the descriptive term
“rolling inappetence.”
In addition to anorexia and lethargy in acutely ill an-
imals, there are also less consistent clinical signs that
may be seen in animals of all ages. Frequently, ill animals
are lymphopenic and/or pyretic with rectal tempera-
210 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
tures from 39 to 41˚C (102 to 106˚F) and/or hyperpneic
and dyspneic (“thumping”). A few (1–2%) exhibit tran-
sient “blotchy” cutaneous hyperemia or cyanosis of ex-
tremities, which is most visible on the ears, snouts, mam-
mary glands, and vulvas. In addition to these clinical
features seen in all stages of production, there are fea-
tures that are unique to the different stages of produc-
tion.
SOWS. During the phase of acute illness, abortions
may occur in 1–3% of sows that are from 21 to 109 days
of gestation (Hopper et al. 1992; White 1992a). Some re-
ports emphasize abortions in the last one-half to one-
third of gestation; however, in these reports early loss of
pregnancy was alternatively reported as an immediate
increase in the number of sows with irregular returns to
heat as well as a subsequent reduction in farrowing rate
associated with an increase in the number of sows that
were not pregnant (Keffaber 1989; Loula 1991). Also, in
some herds, there is a 1–4% mortality in acutely ill sows
(Loula 1991) that is sometimes associated with lesions of
pulmonary edema or cystitis/nephritis (Hopper et al.
1992). Recently in the United States, a very virulent form
of PRRS was described (sometimes referred to as “sow
abortion and mortality syndrome”) with abortion rates
of 10–50% and mortality rates of up to 10% in sows. Cen-
tral nervous system clinical signs such as ataxia, circling,
and falling to one side were sometimes seen as complica-
tions of abortion (Epperson and Holler 1997; Halbur
and Bush 1997). Other inconsistently reported signs in
acutely ill sows include agalactia (Hopper et al. 1992), in-
coordination (de Jong et al. 1991), and a dramatic exac-
erbation of endemic diseases such as sarcoptic mange,
atrophic rhinitis, or cystitis/pyelonephritis (White
1992a).
Beginning approximately 1 week after the onset of
acute illness, a second phase of the disease begins. It is a
consequence of transplacental transmission of the virus
and is characterized by late-term reproductive failure. It
occurs in sows without previous clinical signs as well as
in those clinically affected during the first phase of the
disease. The second phase initially overlaps the first, but
it typically lasts much longer, usually for 1–4 months.
During the second phase, 5–80% of sows may have re-
productive failure at any time between 100 and 118 days
of gestation. Most affected sows farrow prematurely, but
they may also farrow full term or late term, or they may
abort. For example, Gordon (1992) described a herd dur-
ing an outbreak of PRRS in which the proportion of
sows farrowing prematurely from days 110 to 114 of ges-
tation rose from a normal of 20% to a peak of 76% by
week 4 of the outbreak. Affected litters contain variable
numbers of normal pigs, weak small pigs, and dead pigs
that are fresh stillborn (intrapartum deaths), autolytic
stillborn (prepartum deaths), and partially mummified
or completely mummified fetuses. Typically, pigs born
dead compose 0–100% of each affected litter and 7–35%
of the total pigs born in a farrowing group. As this 1- to
4-month phase progresses, the majority of abnormal
pigs in each litter typically shifts from stillborn pigs and
large partially mummified pigs to smaller more com-
pletely mummified pigs to small weak-born pigs to pigs
of normal size and vigor (Keffaber 1989; Loula 1991;
White 1992a). In some herds, the majority of abnormal
pigs are born alive, premature, weak, and small, but few
are born dead (Gordon 1992).
Among sows farrowing abnormal litters, periparturi-
ent mortality may be 1–2% (Keffaber 1989; de Jong et al.
1991). Typically, when these sows are bred again, they ex-
hibit delayed returns to estrus and suffer low conception
rates. These low conception rates, combined with the
previously mentioned increases in overt abortions, irreg-
ular returns to estrus, and nonpregnant sows, result in a
depression in farrowing rate for a complete reproductive
cycle.
BOARS. During the first phase of acute illness, in ad-
dition to anorexia, lethargy, and respiratory clinical
signs, boars may lack libido and have variable reduction
in semen quality (de Jong et al. 1991; Feitsma et al. 1992;
Prieto et al. 1994). Changes in sperm occur 2–10 weeks
after infection with virus and include reduced motility
and acrosomal defects. Although transmission of
PRRSV through semen occurs and may be a significant
portal of PRRSV entry into susceptible herds (Yaeger et
al. 1993; Swenson et al. 1994a), the impact of PRRS
viremia in boars on conception is not clear (Yaeger et al.
1993; Swenson et al. 1994a; Lager et al. 1996; Prieto et al.
1996a, c).
SUCKLING PIGS. During the 1- to 4-month phase of
late-term reproductive failure, high preweaning mortali-
ty (up to 60%) occurs in both pigs born weak and those
born with normal vigor. Nearly all of the premature
weak pigs die within hours of birth. In the rest, mortali-
ty is greatest within the first week after birth but contin-
ues to weaning and beyond. A variety of clinical signs are
reported in suckling pigs with PRRS. Most consistently
reported are listlessness, emaciation/starvation, splay-
legged posture, hyperpnea, dyspnea (“thumping”), and
chemosis. The chemosis that occurs in some pigs is se-
vere, causing a characteristic swelling of the eyelids and
ocular conjunctiva that some consider a nearly “diagnos-
tic” lesion of PRRS when observed in pigs under 3 weeks
of age (White 1992a). Watery diarrhea that is nonre-
sponsive to antibiotic therapy is consistent in the United
Kingdom (Gordon 1992; Hopper et al. 1992; White
1992a) and less consistent elsewhere (Keffaber 1989;
Leyk 1991). Also reported in some herds in piglets are
tremors or paddling (Keffaber 1989; Loula 1991), slight
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
doming of foreheads (Gordon 1992), and thrombocy-
topenia with consequent hemorrhage from navels, sites
of injections, and tails following docking, as well as ane-
mia (Hopper et al. 1992; White 1992a). An increased
number of secondary bacterial infections, such as pol-
yarthritis, also contributes to morbidity and mortality.
WEANLING AND GROWER PIGS. Acute PRRSV in-
fection in weaned pigs in the nursery or grower pigs in
finishing barns is characterized most consistently by
anorexia, lethargy, hyperpnea, dyspnea (“thumping”),
and cutaneous hyperemia as described above. Coughing
is not a consistent clinical feature of uncomplicated
PRRS in weaned pigs. Consistently reported in PRRSV-
infected weaned pigs is an “ill-thrift” that is characterized
by rough hair coats and a variable reduction in average
daily gain and feed efficiency which causes a large varia-
tion in size of pigs in age-matched groups (Moore 1990;
White 1992b). Frequently associated with PRRS in nurs-
ery-grower pigs is a higher than usual incidence of a
number of endemic diseases, which results in elevated
mortality of up to 12–20% (Moore 1990; Loula 1991; Bla-
ha 1992; Keffaber et al. 1992; White 1992a; Stevenson et
al. 1993). These endemic diseases include septicemic sal-
monellosis, Glasser’s disease, streptococcal meningitis,
septicemia and polyarthritis, exudative dermatitis, acti-
nobacillary pleuropneumonia, mycoplasmal pneumo-
nia, bacterial bronchopneumonia, atrophic rhinitis,
postweaning colibacillosis, proliferative enteritis, swine
dysentery, spirochetal colitis, and sarcoptic mange. This
PRRSV-associated enhancement of endemic disease is
most significant in herds with lower health status and/or
poor management (Blaha 1992). However, most at-
tempts to experimentally reproduce PRRSV enhance-
ment of bacterial disease have been unsuccessful (Galina
et al. 1994; Carvalho et al. 1995; Pol et al. 1995; Solano et
al. 1995; Van Alstine et al. 1996).
Endemic Infection of Herds
Swine herds often remain infected with PRRSV for years
(Stevenson et al. 1992; Stevenson et al. 1993; Terpstra et
al. 1992) through various mechanisms, including pro-
longed or persistent viremia and viral shedding in indi-
vidual pigs (Albina et al. 1994a, b; Wills et al. 1995b;
Benfield et al. 1997), continuous introduction of suscep-
tible breeding stock (Dee and Joo 1994b), and replication
of virus in susceptible weaned pigs (Stevenson et al.
1994). The clinical signs of endemic PRRS infection in
herds are most often obvious in weaned pigs in the nurs-
eries or early in the finishing barns when a limited out-
break occurs in a susceptible subpopulation of pigs (Kef-
faber et al. 1992; Stevenson et al. 1993). This can occur in
two general nonexclusive ways. Infected pigs in continu-
ous-flow facilities can shed virus to negative younger sus-
ceptible pigs. Alternatively, pigs born persistently infect-
Benfield et al. 211
ed with virus can shed virus to other pigs within their
age-segregated cohort as these pigs become increasingly
susceptible due to decline of maternal immunity. The
clinical disease in these subpopulations of pigs is the
same as that seen in weaned pigs in a generalized whole-
herd epidemic as described above.
Clinical signs in the breeding herd in endemically in-
fected herds are usually limited to susceptible gilts or re-
placement boars that are exposed to PRRSV after intro-
duction into the herd (Grosse-Beilage and
Grosse-Beilage 1992; Dee and Joo 1994b; Dee et al.
1996). Acute clinical disease in gilts or boars is as de-
scribed above. The reproductive manifestation of en-
demic PRRS depends on the number of gilts infected and
the stage of their reproductive cycle when infected, both
of which may vary widely (Torrison et al. 1994). If scat-
tered gilts are infected in a somewhat continuous and
random manner, then there may be scattered abortions,
irregular returns to estrus, nonpregnant gilts, and late-
term reproductive failure with abnormal litters that may
only be recognized if records are evaluated on a parity-
specific basis (White 1992b). Alternatively, gilts may es-
cape exposure to PRRSV until there is a significant sub-
population of susceptible gilts in various stages of
gestation. In this situation, endemic PRRS in the breed-
ing herd manifests as periodic mini-outbreaks of PRRS
in gilts and possibly first-parity sows. The clinical signs
in affected gilts/sows in these parity-specific mini-out-
breaks are identical to those in a whole-herd outbreak
but on a smaller scale (Dee and Joo 1994b).
Conclusion and Caveat
This description of the clinical signs of PRRS is the dis-
tillation of observations reported by swine clinicians
from around the world and presents the most consistent
clinical features of a “classical” PRRS outbreak or en-
demic herd infection and attempts to highlight signifi-
cant or unique clinical features of the disease that may be
useful diagnostically. However, probably the only com-
pletely consistent clinical feature of PRRSV infection in
pigs is that there is no single consistent feature of PRRSV
infection in pigs. For nearly every clinical sign described,
there are exceptions. On a broader scope, there are ex-
ceptions involving entire phases of production. For ex-
ample, there are reports of outbreaks in single-site farms
where there was severe clinical disease in the grower pigs
and no clinical signs in the breeding herd (Kuwahara et
al. 1994). Probably more than for any other disease in
swine, clinicians need a thorough understanding of the
pathogenesis and epidemiology of PRRSV in order to ac-
curately recognize the various clinical presentations of
PRRS in individuals and populations. Otherwise, one
could erroneously “see” PRRS in nearly every set of clin-
ical signs or fail to recognize PRRS in its many clinical
variations.
212 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
PATHOGENESIS
The pathogenesis of PRRSV infection is based on PRRSV
infection and replication within cells of the mono-
cyte/macrophage lineage (Wensvoort et al. 1991; Voicu
et al. 1994; Rossow et al. 1995; Rossow et al. 1996a; Moli-
tor et al. 1996). Exposure of a mucosal surface to PRRSV
(Fig. 18.2) results in viremia within 12 hours after infec-
tion (Rossow et al. 1995), and PRRSV antigen is identi-
fied in macrophages of the nasal mucosa, lung, and ton-
sil (Rossow 1996). Sows can be infected with PRRSV by
exposure of the nasal mucosa or uterine endometrium
(Christianson et al. 1992; Yaeger et al. 1993; Swenson et
al. 1994c; Lager et al. 1996). PRRSV is also transmitted
by inoculation (Mengeling et al. 1994) and presumably
by bite wounds. Macrophages within a mucosal surface
are the site of primary PRRSV replication, with distribu-
tion to regional lymphoid tissue and subsequent sys-
temic distribution to macrophages in multiple tissues
and/or to monocytes (Rossow et al. 1995; Rossow et al.
1996a).
18.2. Pathogenesis of PRRSV infection. (Courtesy of K. D. Rossow, South Dakota State University.)
PRRSV was first isolated in PAMs (Wensvoort et al.
1991) and has also been reported to replicate in mono-
cytes (Voicu et al. 1994), in microglial cells (Molitor et al.
1996), in two MA-104 (embryonic monkey kidney cell)
clones known as CL-2621 (Benfield et al. 1992) and
MARC-145 (Kim et al. 1993), and in CRL-11171 cells (Hal-
bur et al. 1995). PRRSV replication can be selective be-
tween these cell types (Bautista et al. 1993). PAMs from a
pig less than 6 weeks old are most susceptible to PRRSV
infection, and within a PAM population, PRRSV has a
preference for replication within immature macrophages
(Choi et al. 1994; Mengeling et al. 1995). The predilec-
tion of PRRSV for PAMs from immature swine corre-
sponds with the severe disease most commonly de-
scribed in young pigs (Goyal 1993). Activation or
maturation of monocytes or PAMs treated with
macrophage-colony stimulating factor may modulate
surface marker expression and susceptibility to PRRSV
replication (Choi et al. 1994). PRRSV antigen has been
identified in macrophages of multiple tissues, mono-
cytes, endothelial cells, smooth muscle cells, and fibro-
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
blasts by in situ hybridization and immunohistochem-
istry (Magar et al. 1993; Halbur et al. 1995; Rossow et al.
1996a; Sur et al. 1996). Replication of PRRSV in endoge-
nous porcine cells has only been demonstrated in PAMs,
monocytes, and microglial cells (Wensvoort et al. 1991;
Voicu et al. 1994; Molitor et al. 1996). Resident
macrophages within a variety of tissues should be con-
sidered susceptible to PRRSV infection (Rossow et al.
1995; Rossow et al. 1996a). Because macrophages are
found in all tissues, there is no apparent limitation to the
types of tissue that can be infected by PRRSV. However,
macrophages residing in different tissues are constitu-
tively different because of their varied local environ-
ment, and thus their permissiveness and the productivi-
ty of PRRSV replication vary (Rossow et al. 1995; Rossow
et al. 1996a).
Microscopic lesions develop subsequent to PRRSV
infection of macrophages in multiple tissues, and the
type and degree of inflammation depend on the virus
strain, pig age, infection with other bacterial or viral
agents, host genetic composition, and environmental
stressors (Halbur et al. 1995; Rossow 1996). Interstitial
pneumonia, encephalitis, myocarditis, lymphadenopa-
thy, and arteritis (Rossow 1996) characterize microscop-
ic lesions resulting from PRRSV infection. Lesions result-
ing from PRRSV infection occur in other tissues less
frequently (Rossow 1996). Placental lesions have been
observed only by electron microscopy and were charac-
terized by placental-uterus microseparation with epithe-
lial cell necrosis and desquamation (Stockhofe-Zur-
wieden et al. 1993). The severity of systemic disease will
determine the clinical presentation of PRRSV infection.
The influence of the immune response on the devel-
opment and expression of PRRSV infection has not been
determined; however, anti-PRRSV antibody enhances
the phagocytosis of PRRSV by macrophages (antibody-
dependent enhancement [ADE]), resulting in increased
virus replication (Christianson et al. 1993; Yoon et al.
1996). The significance of ADE in natural PRRSV infec-
tion has not been determined.
PRRSV infection results in clinical or subclinical dis-
ease with resolution or persistent infection (Rossow et al.
1995; Benfield et al. 1996). The clinical presentation of
disease is age dependent. PRRSV infection in neonatal
pigs is characterized by dyspnea, central nervous signs,
and mortality rates up to 100% (Collins et al. 1992; Goy-
al 1993).
PRRSV infection in weaned pigs is characterized by
fever, pneumonia, lethargy, failure to thrive, and a
marked increase in mortality from single to multiple con-
current bacterial infections (Rossow 1996). Concurrent
bacterial infections drive the marked increases in mor-
tality associated with PRRSV infections in immature
swine, and the increased mortality subsequent to PRRSV
infection is clinically obvious from observations of in-
creases in postweaning mortality from 1–2% to 10–15%
when all other factors apparently remain unchanged
Benfield et al. 213
(Hopper et al. 1992; Goyal 1993; Zeman et al. 1993;
Stevenson et al. 1994). Although coinfection with single
or multiple bacteria (Goyal 1993; Rossow 1996), and less
frequently with other viruses (Larochelle et al. 1994), is a
severe complicating factor of PRRSV infection, an in-
crease in susceptibility to bacterial superinfections has
only been experimentally demonstrated once (Galina et
al. 1994). Since the PRRSV-bacterial interaction is not
easily reproduced experimentally, there may be multiple
factors involved in susceptibility to secondary bacterial
infections, including the PRRSV strain, swine genetic
composition, recruitment of susceptible macrophages
by bacterial coinfections, and environmental factors.
PRRSV infection in finishing pigs, unbred gilts or
sows, and boars is commonly characterized by a tran-
sient fever and inappetence (Goyal 1993; Christopher-
Hennings et al. 1995a; Christopher-Hennings et al. 1996;
Rossow 1996). Mortality associated with PRRSV infec-
tion of adult swine has been reported and may reach 5%.
Some PRRSV-infected boars demonstrate a loss of li-
bido, while most appear clinically unaffected (Feitsma et
al. 1992; Yaeger et al. 1993; Christopher-Hennings et al.
1995a; Prieto et al. 1996b). PRRSV is identified in semen
of intact and vasectomized boars, indicating a similar
route of shedding independent of testicular involvement
(Christopher-Hennings et al. 1996). PRRSV field and
vaccine strains can be shed in semen of infected or vacci-
nated boars (Christopher-Hennings et al. 1995a; Molitor
and Shin 1995; Christopher-Hennings et al. 1996;
Christopher-Hennings et al. 1997). The source of PRRSV
in the semen has not been identified. Changes in sperm
morphology and function have been described subse-
quent to PRRSV infection (Christopher-Hennings et al.
1997): a decrease in sperm motility, a decrease in num-
ber of sperm with normal acrosomes, and an increase in
sperm with distal cytoplasmic droplets. However, there
also are reports describing no changes in sperm after
PRRSV infection (Prieto et al. 1996b). PRRSV can be
identified in semen prior to seroconversion and after the
end of viremia (Christopher-Hennings et al. 1995a).
Infection of sows in their third trimester results in
the typical clinical presentation of abortion or prema-
ture farrowing (Christianson et al. 1992; Mengeling et al.
1994). Fetuses in all stages of gestation can support repli-
cation of PRRSV when infected intraamniotically (Chris-
tianson et al. 1993; Lager and Mengeling 1995). Howev-
er, fetal PRRSV infection is less likely to occur after
intranasal infection of midgestation sows (Christianson
et al. 1993; Mengeling et al. 1994). Postcoital intrauter-
ine inoculation of PRRSV at or near the time of concep-
tion has little or no effect on reproductive performance,
and 4- to 16-cell-stage porcine embryos infected with
PRRSV develop normally and do not support PRRSV
replication (Lager et al. 1996; Prieto et al. 1996c). There
are anecdotal reports of PRRSV-induced abortion in
first- and second-trimester sows. The pathogenesis of fe-
tal PRRSV infection has not been determined. A conse-
214 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
quence of late-gestation PRRSV infection is persistent
virus infection of surviving fetuses (Benfield et al. 1996).
Persistence resulting from RNA-virus infection is de-
fined as the continued presence of virus within a host for
extended periods of time after acute infection (Ahmed et
al. 1996). Evidence for persistence of PRRSV infection is
limited but is based on four different reports drawing
similar conclusions: (1) PRRSV RNA has been demon-
strated by the polymerase chain reaction (PCR) in se-
men, but not serum, collected from boars 92 days after
they were exposed experimentally to PRRSV when a con-
current serum sample was negative for PRRSV RNA
(Christopher-Hennings et al. 1995a). (2) PRRSV naive,
specific-pathogen-free pigs were commingled with
PRRSV seronegative pigs that had been exposed to
PRRSV 22 weeks previously (Albina et al. 1994a, b). The
PRRSV-exposed pigs were treated with exogenous gluco-
corticoids prior to commingling (Albina et al. 1994a, b).
The PRRSV-naive pigs developed anti-PRRSV antibod-
ies, had clinical signs of PRRSV infection, and became
viremic within 1–2 weeks of exposure to the PRRSV car-
rier pigs (Albina et al. 1994a, b). (3) PRRSV has been iso-
lated from oropharyngeal scrapings up to 157 days
postinoculation, 134 days after the last isolation of
PRRSV from serum (Wills et al. 1995b). (4) Sera of pigs
exposed to PRRSV at 85–90 days of gestation contained
PRRSV RNA (PCR technique) at 110 days postfarrowing
(Benfield et al. 1996). Sentinel pigs cohoused with the
PRRSV persistently infected pigs (98 days postfarrow-
ing) developed anti-PRRSV antibodies 14 days later
(Benfield et al. 1996).
These experiments indicate that PRRSV persists in
infected swine in a viable state that may not stimulate an-
tibody production and that PRRSV can be recovered
long after initial exposure from serum, the oropharyn-
geal cavity, and semen. Persistently infected pigs may be
an important factor for PRRSV survival and transmis-
sion within a herd. Differentiation of PRRSV strains by
their ability to cause persistent infections or acute, re-
solved infections has not been demonstrated, although
this difference has been verified for another arterivirus
(Gravell et al. 1986).
LESIONS
PRRSV induces a multisystemic infection in pigs result-
ing in the potential for all tissues to become infected with
virus. However, gross lesions are usually observed in on-
ly a few organ systems (e.g., respiratory and lymphoid).
Gross and microscopic lesions characteristic of PRRSV
infection are most remarkable in neonatal and nursery
pigs. In older pigs, lesions attributed to PRRSV infection
are similar but much less remarkable. Under field condi-
tions, most PRRSV-infected pigs are coinfected with one
or more pathogens, which complicates the diagnosis of
PRRS based on lesions. PRRSV-induced lesions in the re-
productive tracts of dams and in fetuses are not as well
characterized and are less consistently observed.
Neonatal Pigs
Gross and microscopic lesions in neonatal pigs often are
quite remarkable. PRRSV-infected lungs are mottled tan
and red and fail to collapse. It is often difficult to clearly
demarcate affected and unaffected tissue. Lesions are
most commonly observed in the cranioventral portion,
and the extent of lung affected macroscopically may vary
from 0% to 100% depending in part on the virulence of
the strain and the time postinfection (Halbur et al. 1995;
Halbur et al. 1996a, b). Lymph nodes are moderately to
severely enlarged and tan in color (Halbur et al. 1995;
Rossow et al. 1994; Rossow et al. 1995). Lymph nodes in
the cervical, cranial thoracic, and inguinal regions are the
most obvious at necropsy. Fluid-filled spaces in the
lymph nodes are inconsistently observed.
Microscopic examination reveals moderate-to-severe
multifocal interstitial pneumonia characterized by three
hallmark changes: (1) alveolar septal infiltration by a
mixed population of mononuclear cells; (2) type 2 pneu-
mocyte hypertrophy and hyperplasia; and (3) marked ac-
cumulation of mixed inflammatory and necrotic alveolar
exudate. Lymph nodes are reactive, with marked follicu-
lar hyperplasia, foci of follicular necrosis, increased
numbers of tingible-body macrophages, and karyorrhec-
tic debris within follicles (Halbur et al. 1995; Halbur et
al. 1996a; Rossow et al. 1994; Rossow et al. 1995). The
follicular mitotic index is increased, and the paracortex is
expanded with mixed inflammatory cells. Other lesions
such as lymphoplasmacytic rhinitis, encephalitis, and
myocarditis are also observed in neonatal pigs.
Nursery Pigs
Lungs from affected pigs fail to collapse and have a vari-
able amount of tan and red mottling (Fig. 18.3). Practi-
tioners sometimes describe the gross lung lesions as
“thymus-like” or “liver-like.” Even if gross lung lesions
are not apparent, microscopic examination usually re-
veals interstitial pneumonia similar to that seen in
neonatal pigs but often less severe and more multifocal.
The most consistent gross lesion, which has become the
hallmark of PRRSV infection in nursery pigs, is the
markedly enlarged tan lymph nodes (Fig. 18.4). Other
less consistent gross lesions include chemosis and in-
creased amounts of clear fluid in the abdomen, thoracic
cavity, and pericardial space.
Microscopic examination of brain, tonsil, lymph
nodes, and heart is of diagnostic value. The majority of
PRRSV-infected pigs have some degree of lymphohistio-
cytic meningoencephalitis and choroiditis characterized
by perivascular cuffing, vasculitis, gliosis, and glial nod-
ule formation (Halbur et al. 1995; Rossow et al. 1994;
Rossow et al. 1995). Severity varies from very mild to se-
vere enough to resemble pseudorabies virus infection.
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
Lymphoid tissues exhibit lymphoid hyperplasia and fo-
cal follicular necrosis. Lymphoplasmacytic and histiocyt-
ic myocarditis is often detected beyond 7 days postinfec-
tion. Myocardial inflammation is often most severe in
the subendocardial and perivascular areas of the my-
ocardium. Mild-to-severe nonsuppurative rhinitis with
epithelial metaplasia has also been attributed to PRRSV
infection (Halbur et al. 1995).
Grower-Finisher Pigs
Gross lesions in grower-finisher pigs attributed to
PRRSV are similar to, but less remarkable than, those in
nursery age pigs. Lymphadenopathy is consistently ob-
served. Lung lesions are often complicated by mixed in-
fections (Zeman et al. 1993). “Porcine respiratory disease
Benfield et al. 215
18.3. Lateral view of a lung
from a pig infected 10 days
previously with a virulent strain of
PRRSV. The lung has failed to
collapse, and nearly the entire
lung is firm and mottled tan in
color. (Courtesy of P. G. Halbur,
Iowa State University.)
18.4. Ventral view of enlarged
lymph nodes (arrows) located at
the thoracic inlet in a pig infected
28 days previously with a virulent
strain of PRRSV. T = trachea,
H = heart. (Courtesy of W. L.
Mengeling and K. M. Lager,
National Animal Disease Center.)
complex” is commonly used to describe a syndrome that
has become more problematic since the mid-1990s.
PRRSV is thought to be a major primary pathogen in the
complex. Mycoplasma hyopneumoniae, Pasteurella multo-
cida, and swine influenza virus are common coinfectors,
resulting in dark red and tan lungs and cranioventral
consolidation of 30–70% of the lung in grower-finisher
pigs. Coinfection with mycoplasma and bacteria results
in the presence of exudate in airways and clearer lines of
demarcation between affected and unaffected lung tis-
sue.
Microscopic examination reveals typical PRRSV-in-
duced interstitial pneumonia underlying the endemic
pneumonia or bacterial bronchopneumonia. Encephali-
tis and myocarditis lesions are similar to, but usually less
216 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
common and less severe than, those observed in younger
pigs.
Boars
The presence of PRRSV in boar semen and the transmis-
sion of PRRSV by boar semen have been demonstrated
(Swenson et al. 1994b; Christopher-Hennings et al.
1995b). Consistent or remarkable lesions in the adult
boar attributable to PRRSV infection have not been ob-
served following experimental infection (Swenson et al.
1994b). PRRSV has been isolated from the bulbourethral
gland of a boar euthanized at 104 days after inoculation,
suggesting that infectious PRRSV may be shed from ac-
cessory sex glands (Christopher-Hennings 1995a). De-
tailed chronological and morphological studies to char-
acterize PRRSV-induced lesions and antigen distribution
in the adult boar have not been reported to date.
Sows
Natural epidemics and experimental inoculation studies
of PRRSV-induced reproductive failure are characterized
primarily by late-term abortions, premature farrowings,
and increased numbers of autolyzed or mummified fe-
tuses, stillborn pigs, and weak-born pigs. There general-
ly are no pathognomonic gross or microscopic lesions in
the pregnant animal; however, only a limited number of
naturally or experimentally infected sows have been ex-
amined histologically in detail. Microscopic examination
of the uterus of uncomplicated PRRSV-induced abor-
tion may reveal mild-to-moderate lymphoplasmacytic
endometritis and myometritis (Christianson et al. 1993;
Lager and Halbur 1996). Endometrial edema is also com-
mon. Lymphoplasmacytic placentitis is less consistently
observed (Stockhofe-Zurwieden et al. 1993). Mild lym-
phoplasmacytic encephalitis, mild multifocal histiocytic
interstitial pneumonia, and lymphoplasmacytic my-
ocarditis are inconsistently observed in sows and gilts.
Fetuses
A spectrum of fetal lesions following transplacental in-
fection have been reported. Fetal lesions are useful when
present; however, they are inconsistently observed and
are not pathognomonic for PRRS. A typical PRRSV-in-
fected litter may contain some normal fetuses, stillborn
pigs, fetuses that have been dead for several days in
utero, resulting in brown discoloration and autolysis,
and fetuses that are covered with a sticky mixture of
meconium, blood, and amniotic fluids. The most consis-
tent gross lesion in fetuses is segmental to full-length he-
morrhages in the umbilical cord. Perirenal and colonic
mesenteric edema is also commonly observed in PRRSV-
infected fetuses (Lager and Halbur 1996).
Microscopic examination may reveal segmental
necrotizing umbilical arteritis characterized by fibri-
nosuppurative inflammation and necrosis of the tunica
intima and tunica media with intramural and perivascu-
lar hemorrhage (Lager and Halbur 1996). Interstitial
pneumonia characterized by mild septal infiltration by
mononuclear cells, type 2 pneumocyte hypertrophy and
hyperplasia, and increased amounts of mixed inflamma-
tory and necrotic alveolar exudate is observed in some
cases (Lager and Halbur 1996; Rossow et al. 1996b).
Necrotizing and lymphoplasmacytic pulmonary arteritis
is also of diagnostic value when observed (Lager and Hal-
bur 1996; Rossow et al. 1996b). Bronchiolar bud necrosis
and pulmonary hemorrhage have also been reported in
fetuses experimentally infected between 45 and 49 days
of gestation (Lager and Ackermann 1994). Lymphoplas-
macytic myocarditis, myocardial fibrosis, and lympho-
plasmacytic perivascular encephalitis are less frequently
observed (Rossow et al. 1996b).
DIAGNOSIS
A diagnosis of PRRS is based on subjective (history, clin-
ical signs, gross and microscopic lesions) and objective
(production record analysis, serology, virus detection)
factors. PRRS should be considered when there are clini-
cal signs of respiratory disease occurring at any stage of
production, when reproductive failure occurs, and when
herd performance is suboptimal. Mild or subclinical dis-
ease is common, so absence of clinical signs does not en-
sure a PRRSV-free herd. The herd history, clinical signs,
and lesions associated with PRRS have been previously
discussed and will not be repeated here.
Record Analysis
Herd production records are becoming a useful adjunct
to PRRS investigations. A review of production records
in clinically active PRRS herds often reveals evidence of
abortions, early farrowings, reduced numbers of live-
born pigs, increased preweaning mortality, reduced fer-
tility, and increased nonproductive sow days. The rate
and duration of farrowing rate reduction, if it occurs,
vary with the speed at which the infection is transmitted
within the herd. While PRRSV is generally highly infec-
tious, farm design, pig flow through the facilities, and
production system type influence herd transmission and
the duration and severity of clinical signs. Thus, farrow-
ing rates may be depressed for only 3–4 consecutive
weeks on some farms, whereas on others they may de-
cline for 6–8 weeks or more.
The relative proportion of irregular and delayed re-
turns to estrus sometimes increase dramatically at the
expense of normal returns to estrus, but the explanation
for this is unclear since PRRSV seems to have minimal ef-
fects on conception (Lager et al. 1996; Prieto et al.
1996a). All parities of sows are affected during epi-
demics of PRRS, whereas in endemically infected herds
lower-parity sows are typically more at risk of PRRS re-
productive failure. Endemically infected PRRS herds may
therefore show either no change in fertility or a paradox-
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
ical improvement in fertility with service number (i.e.,
fertility would normally fall with number of services that
a sow has after weaning) (Collins and Dial 1996; G. D.
Dial, unpublished data—1998).
Serology
PRRSV antibodies are detected using a variety of sero-
logic tests, including IFA (Yoon et al. 1992), immunoper-
oxidase monolayer assay (IPMA) (Wensvoort et al. 1991;
Drew 1995), ELISA (Albina et al. 1992; Cho et al. 1996),
and serum virus neutralization assay (SVN) (Yoon et al.
1994). Advantages of the automated ELISA test are that
large numbers of sera can be tested in a short time, and
results are potentially more consistent among diagnostic
laboratories. An additional advantage is that ELISA can
detect North American and European PRRSV strains,
whereas IFA and IPMA detect only those PRRSV isolates
that are antigenically close to the PRRSV isolate used in
the test. The PRRS vaccination status and age of the pigs
and the regional prevalence of PRRS must be considered
when interpreting serologic test results because serolog-
ic tests do not distinguish vaccinated from naturally in-
fected pigs and because young pigs may possess passive-
ly acquired antibodies for 5–10 weeks after infection
(Houben et al. 1995a).
The PRRSV antibody kinetics of the IFA, IPMA, and
ELISA tests are similar. PRRSV antibodies are detected
7–14 days after infection, reach maximal titers by 30–50
days, and then gradually decline to low or undetectable
levels by 4–6 months after infection. The SVN test is less
sensitive than other serologic tests for detecting recent
infection with PRRSV because neutralizing antibodies
develop slowly. However, it provides better long-term ev-
idence for past infection because PRRSV neutralizing an-
tibodies persist for at least 1 year (Yoon et al. 1994). Use
of an IFA test that detects anti-PRRSV IgM antibodies
has been advocated because of its ability to detect recent
infection (Park et al. 1995). Acute and convalescent
serum samples should be provided to a diagnostic labo-
ratory when seeking evidence of recent infection.
Virus Detection
As with all diagnostic investigations, the first and most
important step is to select the samples most likely to
yield useful laboratory information. In general, speci-
mens collected from younger, rather than older, pigs are
preferred because PRRSV is found in greater amounts
and for longer periods of time in young pigs. Virus can
be isolated from serum for 4–6 weeks after infection of
suckling, weanling, and grower pigs (Mengeling et al.
1996d) and for 1–2 weeks after infection in boars
(Christopher-Hennings et al. 1995a) and sows (Men-
geling et al. 1996c). Virus can typically be isolated from
bronchioalveolar lavage samples for at least several
weeks following the cessation of detectable viremia
(Mengeling et al. 1995; Mengeling et al. 1996b, c, d). The
Benfield et al. 217
influence of passively acquired maternal antibodies on
the success of VI is unknown, so obtaining presuckle
serum in addition to other samples should be attempted
when investigating abortion outbreaks.
Specimens for VI must be refrigerated or frozen soon
after collection. PRRSV is rapidly degraded by heat and
is preserved by refrigeration and freezing (Benfield et al.
1992; Van Alstine et al. 1993; Bloemraad et al. 1994).
Care should be used to prevent inadvertent heating be-
fore and during shipment. For example, transport of
samples to the veterinary clinic on warm summer days or
excessive warming of blood samples during centrifuga-
tion may subject PRRSV to heat inactivation and thereby
preclude VI. The virus has a narrow range of pH stabili-
ty so sterility must be maintained to avoid pH alterations
caused by bacterial contamination. Specimens should be
packaged in an insulated, leak-proof container with am-
ple amounts of refrigerant and shipped by overnight
courier.
Several laboratory techniques have been used to
identify PRRSV antigen. Immunohistochemistry (IHC)
(Halbur et al. 1994; Larochelle and Magar 1995) and IFA
(Yoon et al. 1992; Lager and Ackermann 1994) tests de-
tect PRRSV antigen in many tissues, although lung and
tonsil are preferred. Tissues should be submitted refrig-
erated for IFA and fixed in 10% neutral buffered formalin
for IHC. Unfortunately, the IHC and IFA test results are
influenced by technician skill, so results should be con-
firmed by VI or PCR. In situ hybridization is a sensitive
method to identify viral nucleic acid (Larochelle et al.
1996; Sur et al. 1996; Haynes et al. 1997); however, it
finds greater application in research than in routine di-
agnosis.
PCR is a highly sensitive test for detecting viral RNA.
It is useful when VI is troublesome, such as when testing
semen (Christopher-Hennings et al. 1995b) and when
virus has been partially degraded by autolysis (as in
necrotic fetal tissues). Nested PCR tests have been devel-
oped which appear as sensitive as conventional VI tech-
niques for detecting an acute virus infection in pigs
(Mardassi et al. 1994b; Van Woensel et al. 1994; Kono et
al. 1996; Gilbert et al. 1997). Moreover, these tests may
be more sensitive than VI during the convalescent stages
of infection. Automated PCR tests, which are less prone
to contamination, have enabled diagnostic laboratories
to offer sensitive and specific PCR testing at much lower
cost (Molitor et al. 1997). PCR samples should be col-
lected and handled like VI samples.
To date, PRRSV has only been propagated in vitro in
a limited number of cell types. These are PAMs
(Wensvoort et al. 1991), the MA-104 cell line and its de-
rivatives (the MARC-145 and CL-2621 cell lines; Kim et
al. 1993; Benfield et al. 1992), the CRL-11171 cell line
(Halbur et al. 1995), blood-derived monocytes (Voicu et
al. 1994), and microglia (Molitor et al. 1996). Different
strains or isolates of PRRSV vary in their ability to repli-
218 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
cate in different cell types (Bautista et al. 1993); there-
fore, the use of PAMs in conjunction with a cell line such
as MA-104 maximizes VI success. Heparin prevents
PRRSV from infecting MA-104 cells, so heparin should
not be added to cell cultures (Jusa et al. 1997). Blood
samples for VI should be collected in serum or EDTA
tubes but not in tubes containing heparin.
Serum, buffy coat, lung, alveolar washings, lymph
node, and tonsil are the specimens of choice for VI. For
isolation, the sample is inoculated onto cell cultures and
cultures are observed for cytopathic effect. The presence
of PRRSV can then be confirmed by IFA. Monoclonal an-
tibodies directed against the highly conserved nucleo-
capsid protein are useful for immunofluorescence testing
because they detect antigenically diverse PRRSV strains
(Nelson et al. 1993). The cytopathic effect of PRRSV is
usually obvious by 2–7 days after initial exposure of a
susceptible cell culture to a diagnostic sample containing
a field strain of the virus. However, it may escape notice
if the sample contains little of the virus or if the particu-
lar isolate replicates poorly in cell culture. Consequently,
at least one additional cell culture passage is recom-
mended to increase the reliability of the isolation proce-
dure.
Diagnosis by VI is complicated by the use of PRRS
modified-live-virus vaccines because vaccinated pigs may
be viremic for up to several weeks and may transmit vac-
cine virus to penmates (Torrison et al. 1996). Therefore,
attempts have been made to distinguish PRRS field virus
from vaccine virus by restriction fragment length poly-
morphism (Wesley et al. 1996), genetic analysis, and
monoclonal antibody profiling (Nelson et al. 1997). Re-
sults of these tests should be interpreted cautiously be-
cause vaccine and field isolates may in some instances
provide similar results. Also, only small regions of the
PRRSV genome are sequenced for routine diagnostic
testing, so important differences between isolates might
go undetected (K. S. Faaberg, unpublished data—1998).
Diagnosis Summary
The clinical manifestations of PRRS are variable and are
affected by many factors that confound the diagnosis of
this disease. A definitive diagnosis is dependent on the
collection and submission of correct samples.
RESPIRATORY DISEASE. Pigs should be evaluated
for gross and microscopic lesions of PRRS. Portions of
lung, heart, brain, lymph node, kidney, and tonsil should
be submitted refrigerated and fixed in 10% neutral
buffered formalin. IFA, IHC, PCR, and VI on lung, tonsil,
and lymph nodes can be attempted. Collecting alveolar
washings enhances VI in late stages of PRRSV infection
and from older animals (Mengeling et al. 1995; Men-
geling et al. 1996b, c, d). A variety of sampling strategies
have been proposed for serologic detection of PRRSV
(Collins et al. 1996).
REPRODUCTIVE DISEASE. Diagnosis of PRRSV in-
fection is difficult to achieve by examining fetal tissues.
Fetus and placenta seldom have microscopic lesions
(Rossow et al. 1996b), and if present the lesions are non-
diagnostic (Lager and Ackermann 1994). VI is trouble-
some because typically only some of the fetuses and/or
piglets of an affected sow are infected at the time of de-
livery and because virus is degraded by in utero fetal au-
tolysis. Moreover, PRRSV-infected sows may abort in the
absence of in utero infection. For the same reasons, at-
tempts to identify PRRSV antigen in fetal tissues by IHC
and IFA are often unsuccessful. PRRS viral detection by
PCR has provided promising results presumably because
this technique does not depend on the presence of live
virus (K. D. Rossow, unpublished data—1998), but it
does not circumvent the problem of identifying which fe-
tuses and pigs are infected at the time of sample collec-
tion.
Collecting pre- and postsuckle serum from several
weak-born and live-born pigs per litter improves the
chances of successful virus detection and provides an op-
portunity to measure PRRSV antibodies. The presence of
PRRSV antibodies in presuckle sera indicates in utero in-
fection. Unfortunately, PRRSV antibodies are present in
only a minority of PRRSV-infected pigs at delivery (Men-
geling et al. 1996c). Using PCR to identify PRRSV in sera
of aborting sows often yields positive results, whereas VI
from the same samples is seldom successful (J. E. Collins,
unpublished data—1998). Serologic examination of
acute and convalescent sow sera may provide evidence of
seroconversion.
PCR has proven to be a highly sensitive test for de-
tecting PRRSV in semen (Christopher-Hennings et al.
1995b; Shin et al. 1997). In most cases a positive PCR test
correlates with a positive swine bioassay (Christopher-
Hennings et al. 1995b), indicating that viral RNA detect-
ed in semen represents infectious virus. Collecting at
least 3–5 successive semen samples at weekly intervals
has been recommended because boars may shed PRRSV
intermittently over prolonged time periods (Christo-
pher-Hennings et al. 1995a). The cost of PCR testing se-
men samples can be reduced by pooling in groups of
3–5, although decreased test sensitivity may occur when
semen contains low amounts of the virus.
IMMUNOLOGY
Pigs infected with PRRSV produce an immune response
that is easily detected by the presence of serum antibod-
ies to the virus. The appearance of antibodies correlates
with decreases in disease signs, viremia, and viral shed-
ding. Animals that recover from infection are immune
and are protected from subsequent challenge. The anti-
body response is detected by the appearance of specific
IgM by 7 days and of specific IgG by 14 days after infec-
tion (Vezina et al. 1996).
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
Humoral immune responses are monitored by a vari-
ety of assays, including IFA, IPMA, ELISA, and SVN.
Most serologic tests indicate that antibodies reach a
maximum titer by 5–6 weeks postinfection and persist
for the life of a commercial feeder pig. However, virus-
neutralizing antibodies appear more slowly, usually be-
ing detected first at 4–5 weeks postinfection, and reach-
ing a maximal titer about 10 weeks postinfection (Yoon
et al. 1995b). The appearance of neutralizing antibodies
seems to be delayed in boars, occurring at about 10
weeks and reaching a maximum at about 18 weeks (Nel-
son et al. 1994).
The duration of antibody responses in sows and
boars is not well described since most experiments are of
relatively short duration. Sows that are seropositive se-
crete antibodies in the colostrum (Eichhorn and Frost
1997). Noninfected piglets born to seropositive sows are
seronegative at birth and acquire antibodies passively
from the colostrum.
Antibodies are presumed to play a role in protection
against reinfection. Antibody responses to the major
proteins of the virion, including the nucleocapsid prod-
uct of ORF 7, the envelope glycoprotein product of ORF
5, and the integral membrane protein encoded by ORF 6,
as well as to the protein products of the ORF 2, ORF 3,
and ORF 4 genes, are readily detected. Antibodies to the
envelope glycoprotein neutralize viral infectivity (S. Dea,
unpublished data—1998) and immunization with re-
combinant proteins from ORF 5 or ORF 3 appears to
provide partial protection from infection (Plana Duran
et al. 1997). Antinucleocapsid antibodies are not neu-
tralizing, since the protein is not on the surface of the
virion. Interestingly, antibodies to the ORF 4 proteins are
reported to neutralize infectivity of LV (Van Nieuwstadt
et al. 1996). Whether the ORF 4 protein is on the virion
surface and subject to neutralization in North American
strains of PRRSV is not known. Antigenic variation is
well documented in the amino acid sequences of PRRSV
proteins based on DNA sequencing and is also reflected
in the specificities of monoclonal antibodies to PRRSV
isolates. Thus, serologic diagnostics that rely on detec-
tion of whole virus or virus-infected cells with swine
serum are more reliable than antibody tests that rely on
monoclonal antibodies or purified recombinant viral
proteins (Yoon et al. 1995a). However, some monoclonal
antibodies have a range of cross-reactivity that exceeds
that of many polyvalent sera (Nelson et al. 1993).
Little is known about the cellular immune responses
to PRRSV infection. Lymphocytopenia appearing tran-
siently at 3–9 days after infection is a hallmark of PRRSV
infection. However, by 14 days after infection total pe-
ripheral blood lymphocyte numbers are normal. T cells
positive for CD8 increase, consistent with the hypothesis
that PRRS-specific cytotoxic T cells are produced
(Shimizu et al. 1996). Nevertheless, the role of cellular
immunity in resistance to PRRS is unknown, due in part
Benfield et al. 219
to the difficulty in developing antigen-specific, major-
histocompatibility-complex-restricted T-cell prolifera-
tion and cytotoxicity assays in swine.
Immunosuppression, perhaps due to destruction of
PAMs during viral replication, has been suggested to ac-
count for reports of increased incidences of respiratory
disease and loss of productivity on farms affected with
PRRS and for episodes of apparent vaccine failure. How-
ever, efforts to demonstrate immunosuppressive effects
of PRRSV infection, or exacerbation of other diseases by
concurrent infection with PRRS, have generally been un-
successful (Grosse-Beilage 1995; Van Alstine et al. 1996).
Likewise, it has been proposed that the presence of anti-
PRRSV antibodies in animals might exacerbate the
severity of infection due to antibody-mediated entry of
virus into macrophages via the Fc receptor. However,
there are few, if any, convincing data in support of this
hypothesis.
The development of licensed vaccines for prevention
of PRRS has led to widespread application and serologic
testing in North America. The resulting “experiments of
nature” have revealed cases in which animals fail to
seroconvert following exposure to vaccine, potential lack
of cross-protection, and potential examples of immuno-
logic nonresponsiveness following multiple exposures to
an attenuated vaccine virus. Some of these examples are
predictable since genetic and environmental differences
in host animals influence the magnitude of an immune
response. Ongoing genetic and antigenic variation
in the wild-type virus and immunologic selection due to
vaccination make it likely that variants will arise which
escape the protection afforded by currently available vac-
cines.
Our knowledge of PRRS immunology is at present
rather rudimentary and descriptive. There are many
more questions than answers regarding the essential fea-
tures of a protective immune response. These include
the effect of antibodies on progression of viral infection
in macrophages, the existence and consequences of
PRRS-mediated immunosuppression, and the immuno-
logic responses in piglets which are born viremic. An-
swers to these questions will be important in the contin-
uing effort to control and prevent PRRS.
PREVENTION AND CONTROL
Reducing the clinical and economic effects of PRRSV
within infected swine farms is a challenge to veterinari-
ans throughout the world. Despite the high degree of
frustration experienced with PRRS since the disease was
first reported, progress has been made in the under-
standing of its epidemiology and in the accuracy of the
diagnostic testing procedures. Although numerous ques-
tions still remain unanswered, this section provides an
overview of existing strategies known to be effective for
the control of PRRS, as well as a review of specific steps
220 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
necessary to prevent the introduction of the virus into a
naive farm.
Prevention
Despite the fact that PRRSV infection is widespread
throughout the world, noninfected herds still exist.
While all routes of virus introduction into a naive herd
are not completely understood at this time, the primary
source is the infected pig. Therefore, it is critical to rou-
tinely isolate and test breeding stock before introducing
them to a PRRS-negative herd. Replacement stock to be
added to naive herds should be obtained from known
negative sources that carry out a regular schedule of herd
monitoring. In addition, there should be clear and open
communication between veterinarians responsible for
the health of the source and recipient herds, respective-
ly, prior to purchase of replacement stock.
Isolation facilities should be located on another farm
site and visited at the end of the working day. Following
the arrival of new stock, all animals should be blood-test-
ed 14 days following entry into the isolation building.
Isolation periods should be at least 30 days to allow suf-
ficient time to obtain laboratory results prior to animal
introduction. If diagnostic results indicate that incoming
stock are infected with PRRSV, all animals of the isolat-
ed group should be removed from the building and mar-
keted. The isolation facility should then be cleaned using
95˚C water, disinfected using phenolic- or formaldehyde-
based products, and allowed to remain empty for a min-
imum of 7 days. Finally, in accordance with strict isola-
tion and testing policies, sound principles of biosecurity
previously described for other swine pathogens (e.g.,
transmissible gastroenteritis virus) should be practiced
at all times (Moore 1992).
Control
Following the identification of PRRSV and the develop-
ment of diagnostic tests, practitioners throughout the
world have attempted to control the effects of PRRS. The
initial attempts were based on the use of strategies
known to be effective for controlling other diseases of
swine (e.g., segregated early weaning); however, results
were inconsistent (Dee et al. 1993). The central compo-
nent of PRRS control is the reduction of the spread of
the virus within the breeding herd, thereby preventing
the infection of offspring prior to weaning. Recently, a
report describing the presence of seronegative subpopu-
lations in chronically infected breeding herds was pub-
lished (Dee et al. 1996). This report concluded that in-
consistent levels of virus exposure exist within
endemically infected herds, and the formation of sub-
populations assisted in the maintenance of virus trans-
mission in the breeding herd over time.
Recently, a model for the control of PRRS which fo-
cuses on the elimination of subpopulations was devel-
oped. The components of the model are as follows:
1. Understanding of the pattern of viral spread
throughout the system through the application of popu-
lation-based diagnostic strategies
2. Proper development of replacement stock prior to in-
troduction into PRRSV-infected herds
3. Prevention of the transmission of the virus from
sow to piglet through breeding-herd stabilization
4. Control of the spread of virus in the nursery or
finisher populations through weaned-pig management
The purpose of such a model is to provide a generally
applicable systems approach for the control of PRRS-re-
lated disease problems. The individual components of
the model are described in greater detail as follows.
POPULATION-BASED DIAGNOSTIC STRATEGIES.
In today’s industry, swine production systems are a series
of interdependent populations, consisting of the breed-
ing, gestating, and lactating populations, the nursery
and finisher populations, and the replacement gilt pool.
Previously published reports have indicated that PRRSV
may be limited to an individual population (e.g., the
nursery) of a particular farm (Dee and Joo 1994a).
Therefore, it is critical to accurately determine the pat-
tern of viral transmission within a farm on a population
basis.
Serologic profiling using IFA or ELISA is the preferred
method of data collection. Randomly selected swine are
tested using cross-sectional sampling in order to assess
the prevalence of infection and the pattern of viral trans-
mission within each population. Increased sample sizes
may be required in larger herds, and statistical tables
should be referenced to ensure that the correct number
of samples are collected. An example of a testing proto-
col is provided in Table 18.1. Tables 18.2 and 18.3 pro-
vide examples of virus infection localized to the nursery
or widespread throughout all populations, respectively.
The data presented in Table 18.2 are indicative of a
chronically infected farm experiencing postweaning,
PRRS-related, respiratory disease, whereas the data pre-
sented in Table 18.3 are typical of an acute epidemic.
In addition to the technique of cross-sectional profil-
ing, serial testing of weaned pigs is helpful not only for
detecting the point of seroconversion to PRRSV but also
for determining the role of other pathogens in the dis-
ease process (e.g., Mycoplasma hyopneumoniae or swine
influenza virus). Using this strategy, a minimum of 10
pigs are identified and sampled throughout the nursery
and/or finishing periods at 4-week intervals. Although
this method requires a longer period of time to obtain re-
sults, it can be used in conjunction with cross-sectional
profiling to enhance the accuracy of the sampling
process.
Ideally, the diagnostic evaluation should take place
before vaccination. Serologic assessment is much more
difficult following vaccination, since tests for differenti-
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME Benfield et al. 221

Table 18.1. Serologic profiling protocol for evaluation of a PRRSV-infected herd

Key Points concerning Profiling:
1. Ideally, profiling should take place before vaccination.
2. Statistical tables should be consulted to ensure adequate sample sizes are collected.
3. Results are indicative of infection, not immunity.
4. Tissue samples from infected pigs should be submitted simultaneously for verification of viral antigen and microscopic evidence of an
infectious disease process.
Step 1: Diagnostic Program
Randomly select the following number of samples from the designated populations:
1. Breeding herd: n = 30 sows (5 samples/parity)
2. Recently weaned pigs (3–4 weeks old): n = 10
3. Pigs at the end of the nursery stage (8–10 weeks old): n = 10
4. Premarket finishers (5–6 months old): n = 10
5. Replacement gilts prior to entry into breeding herd: n = 10
Step 2: Monitoring Program
1. Breeding herd
Quarter 1 Month 1: From the initial sampling of 30 animals, select 10 monitor sows for serial testing.
Months 2 and 3: Retest monitor sows, along with 20 randomly selected animals monthly for a total of 30 samples each
testing period.
Quarters 2 through 4: Repeat quarter 1 protocol, selecting 10 new monitor sows.
2. Gilt pool: Randomly test 30 replacement gilts prior to entry into the breeding herd on a monthly basis.
3. Weaned-pig populations: Repeat sampling of 10 recently weaned pigs, 10 (8- to 10-week-old) nursery pigs, and 10 premarket finishers on
a quarterly basis.

Table 18.2. Cross-sectional serologic (ELISA) Table 18.3. Cross-sectional serologic (ELISA)
profile indicating infection in the nursery within a profile indicating active infection in all stages of
nonvaccinated herd production within a nonvaccinated herd
Sample Sample
No. Breeding 4 Weeks 8 Weeks 6 Months No. Breeding 4 Weeks 8 Weeks 6 Months
1 0.35* 0.18 1.95 0.50 1 2.53* 1.95 2.50 0.85
2 1.00 0.33 2.01 0.65 2 2.20 3.25 3.15 3.05
3 0.44 0.21 1.00 1.20 3 2.54 2.70 0.81 3.20
4 0.61 0.19 1.59 1.20 4 3.44 0.31 0.05 1.79
5 0.59 0.46 1.49 0.60 5 0.32 2.95 1.75 0.50
6 0.37 0.20 2.24 1.00 6 0.81 3.10 2.30 0.06
7 0.36 0.12 1.28 1.00 7 2.95 0.65 1.65 1.79
8 0.63 0.55 0.83 0.82 8 1.27 1.85 0.50 2.55
9 0.51 0.63 1.95 0.95 9 0.18 0.20 0.31 3.10
10 0.20 0.01 3.00 0.50 10 0.25 0.55 0.26 0.75
*ELISA S/P ratio (≥ 0.4 = positive). *ELISA S/P ratio (≥0.4 = positive).
Additional diagnostics: PRRSV isolated from tissues and sera Additional diagnostics: PRRSV isolated from tissues and
of nursery pigs. sera.
Clinical picture: Pigs healthy at weaning, developing evi- Clinical picture: Reproductive failure in late gestation, respi-
dence of respiratory disease 2–4 weeks postweaning. ratory disease in weaned pigs.
Production data: Nursery average daily gain = 0.25 kg; nurs- Production data: Stillborns = 35%; abortions = 10%; post-
ery mortality = 8.5%. weaning mortality = 10%.

ating antibodies produced against vaccine virus versus
those produced against field virus are not available. Fi-
nally, in conjunction with serology, tissue submissions
from affected pigs will assist in the verification of a
PRRSV-related disease process. Microscopic evidence of
PRRSV infection and demonstration of the presence of
viral antigen in tissue frequently support the serologic in-
terpretation.
DEVELOPMENT OF REPLACEMENT STOCK.
Proper management of replacement stock is essential for
controlling subpopulation formation and the spread of
the virus in the breeding herd. Episodes of recurrent re-
productive failure following direct introduction of naive
replacement gilts into an actively infected herd have
been described (Dee and Joo 1994b). Therefore, it is im-
portant to understand whether there are differences be-
tween the PRRS status of incoming replacement stock
and the recipient breeding herd, and to establish pro-
grams for animal introduction.
Isolation and acclimatization of replacement stock
have been used to reduce the risk of introducing new
222 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
pathogens to an existing herd, as well as to properly pre-
pare animals for entry into a PRRS-positive system. In
the past, isolation periods typically ranged from 21 to 30
days; however, due to the ability of PRRSV to induce pro-
longed periods of viremia following infection, isolation
and acclimatization periods need to be extended. There-
fore, development may begin early in the life of the ani-
mal and take place in facilities specifically designed for
this purpose (Dee et al. 1994). Animal introduction may
take place during the suckling period (i.e., 2 days of age),
at the time of weaning, or at 25 or 100 kg of body weight.
In order to accommodate the need for extended develop-
ment periods, it is recommended that larger groups of
younger animals be introduced less frequently through-
out the year.
During the development period, a standard program
of immunization against PRRSV and exposure to previ-
ously infected swine is practiced to enhance the develop-
ment of protective immunity. Serologic profiling of ani-
mals following the completion of the development
program can assess the PRRS status of the replacement
population prior to their addition to the breeding herd.
Previously published summaries provide additional in-
formation on specific development programs (Dee
1997a, b).
In conclusion, improved understanding of the rela-
tionship between replacement stock and the control of
PRRS-related reproductive disease has emphasized the
importance of this population. The challenge to the in-
dustry will be not only to improve these existing systems
but to apply their principles to the control of other in-
fectious disease problems of swine.
BREEDING-HERD STABILIZATION. While replace-
ment stock programs are critical for controlling the for-
mation of subpopulations, commercially available mod-
ified-live-virus vaccines have been used to induce a
consistent immune response throughout the adult popu-
lation. Currently, such products are only approved for
use in nonpregnant females 3–4 weeks prior to breeding;
however, they have been used extensively in pregnant
sows and in boars in the field (Dee 1996). Use of vaccines
in this manner is at the discretion of the veterinarian,
and it must be realized that vaccination of naive preg-
nant sows during the third trimester of gestation may re-
sult in fetal infection (Mengeling et al. 1996c). Only lim-
ited published data generated following implementation
of breeding-herd vaccination strategies exist at this time;
however, one study has indicated the ability of vaccina-
tion to reduce the spread of virus among members of the
adult population and prevent the infection of piglets pri-
or to weaning (Dee et al. 1997a).
Finally, although efficacious killed-virus vaccines are
not yet commercially available, such a product may be
beneficial to control subpopulation formation, maintain
protective immunity within the breeding herd, and pro-
tect noninfected herds from production and economic
losses if they become exposed to the virus.
WEANED-PIG MANAGEMENT. For the control of
chronic PRRS in the weaned-pig population, two prima-
ry strategies exist: partial depopulation and pig vaccina-
tion. Again, the success of either strategy depends on
preventing the introduction of actively infected animals
into the nursery.
Partial depopulation consists of a strategic adjust-
ment in pig flow to prevent the lateral spread of
PRRSV within chronically infected populations. Previ-
ous studies have indicated that the spread of the virus
within the nursery or finishing stage is maintained
through the transmission of the virus to recently
weaned, naive piglets from older, previously infected
pigs (Dee and Joo 1994a). Partial depopulation has
been applied extensively to the nursery, and published
data have indicated highly significant improvements
(p < .0001) in average daily gain and mortality (Dee et al.
1997c).
The economic benefit of nursery depopulation has
been demonstrated as well. In a recently published study,
improved profitability was demonstrated in 94% of the
study farms, despite the fact that PRRSV was not elimi-
nated in all cases (Dee et al. 1997b). Limitations of this
strategy include the logistics involved in depopulating
large nurseries and the fact that depopulation may need
to be repeated periodically to maintain improvements in
performance over extended periods of time.
Commercially available vaccines for the control of
PRRS in pigs of 3–18 weeks of age became available in
the United States in June 1994. Prior to implementing a
vaccination program, it is important to determine the
timing of virus exposure in order to immunize the ani-
mal prior to infection. Because transmission of modi-
fied-live-vaccine virus to negative swine can take place,
the use of this product is not recommended in cases
where a seronegative status is desired (Torrison et al.
1996).
Finally, if the suckling piglet population is acutely in-
fected, a series of management strategies have been out-
lined (Henry 1994). Emphasized within this report is
limiting the movement of piglets between litters to the
initial 24 hours of life, humanely destroying chronically
infected offspring prior to weaning, and maintaining
strict all-in/all-out animal flow in the nursery.
In conclusion, although the application of this model
has resulted in successful control of PRRS-related dis-
ease problems over a wide range of production systems,
elimination of the virus is not easily attained at this time.
The ability of the virus to establish persistent infections
and the lack of regulatory control over the sale of infect-
ed breeding stock are key issues that must be resolved
prior to the initiation of large-scale eradication pro-
grams.
 CHAPTER 18 PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME
VACCINES
Several studies have established that vaccination against
PRRS can result in protective immunity (Gorcyca et al.
1995; Gorcyca et al. 1996b; Hesse et al. 1996a, b; Men-
geling et al. 1996a; Plana Duran et al. 1995). Such stud-
ies have led to the extensive use, especially in the United
States, of commercial vaccines as an aid in reducing the
clinical consequences of infection with virulent PRRSV.
Although both attenuated and inactivated vaccines have
been described, the former are generally believed to be
more efficacious. As a result, virus inactivation has been
used mostly as a method of preparing autogenous vac-
cines.
Currently, two attenuated vaccines are commercially
available for use as an aid in the prevention and control
of PRRS. RespPRRS/Repro® has been marketed in
North America since 1994 for administration to pigs be-
tween 3 and 18 weeks of age for the prevention of the
respiratory component of PRRS, and since 1996 for ad-
ministration to nonpregnant gilts and sows for the pre-
vention of the reproductive component of PRRS. (The
same vaccine is marketed in Europe under the name In-
gelvac® PRRS MLV.) Prime Pac® PRRS has been market-
ed in the United States since 1996 for administration to
nonpregnant gilts and sows for the prevention of the re-
productive component of PRRS.
Much of what is now known about vaccination
against PRRS is based on controlled laboratory studies
with RespPRRS/Repro® and with three experimental
vaccines developed at the National Animal Disease Cen-
ter (NADC) (Mengeling et al. 1996a). All of these vac-
cines, as well as Prime Pac® PRRS, were derived (i.e., at-
tenuated) by repeated passage of virulent, North
American field strains of PRRS virus in cell culture. They
differ genetically from one another to some extent, but
they are more closely related to each other than to LV.
Information from field use of PRRS vaccine is limited
mostly to RespPRRS/Repro®, which has been used ex-
tensively since it was first licensed and marketed in 1994
under the name RespPRRS®. Although RespPRRS® was
not approved for use in gilts and sows for the prevention
of the reproductive component of PRRS until 1996
(when the name was changed to RespPRRS/Repro®), it
was often used “off label” for this purpose.
The following discussion relative to the safety, effica-
cy, and limitations of PRRS vaccines is based almost en-
tirely on information derived from laboratory studies
with RespPRRS/Repro® and NADC vaccines and from
field use of RespPRRS/Repro®. The relative merits of the
more recently introduced Prime Pac® PRRS remain to be
determined through independent laboratory and field
comparisons.
It is generally believed that, when used judiciously, at-
tenuated vaccines can be of value in preventing and con-
trolling PRRS (Dee 1996). However, to achieve greatest
Benfield et al. 223
success a number of factors need to be considered when
designing a vaccination program. (1) Vaccine virus can
persist for weeks and perhaps months (Mengeling et al.
1996c), and its persistence may be similar to that of vir-
ulent virus (Mengeling et al. 1996a, c; Wills et al. 1995a,
b). (2) Vaccine virus can be transmitted from vaccinated
pigs to naive pigs (Torrison et al. 1996; Bøtner et al.
1997; Mengeling et al. 1998). (3) Vaccine virus can cross
the placenta to cause congenital infection (Mengeling et
al. 1996c). (4) Vaccine virus can persist in boars and be
disseminated through semen (Christopher-Hennings et
al. 1997). (5) Vaccine-induced protective immunity is
slow to develop (Mengeling et al. 1996a; W. L. Men-
geling, K. M. Lager, and A. C. Vorwald, unpublished da-
ta—1998). (6) The time required to develop protective
immunity is likely to be strain related; that is, immunity
may develop more quickly and more forcefully (W. L.
Mengeling, K. M. Lager, and A. C. Vorwald, unpublished
data—1998) and last longer (Lager and Mengeling, un-
published data—1998) against the homologous and
antigenically similar strains than against more distantly
related strains of PRRSV. (7) Some vaccinated pigs, gilts,
and sows may fail to seroconvert for PRRSV following
vaccination (W. L. Mengeling, K. M. Lager, and A. C.
Vorwald, unpublished data—1998). (8) Successful vacci-
nation of adult females (as judged by seroconversion)
may not ensure the complete absence of transplacental
infection and vertical transmission of virulent virus
(Mengeling et al. 1996a). In addition, evidence has been
presented for the transmission of vaccine virus from vac-
cinated sows to nonvaccinated sows, and from vaccinat-
ed herds to nonvaccinated herds, with the subsequent
development of vaccine-virus-induced reproductive fail-
ure (Bøtner et al. 1997).
With these issues in mind, a vaccination program
that emphasizes safety as well as efficacy might be de-
fined by the following. For at least several weeks after
vaccination (the longer the better), vaccinated pigs, gilts,
and sows would be isolated to minimize transmission of
vaccine virus. Neither mature boars (used for natural ser-
vice or semen collection) nor pregnant gilts and sows
would be vaccinated. Gilts would be vaccinated twice be-
fore breeding to increase the likelihood of seroconver-
sion. The age of gilts at the time of the first vaccination
would depend on the limitations of the particular opera-
tion, but in general, the earlier the better so that the sec-
ond dose of vaccine, administered 1–2 months after the
first (preferably the latter), could be administered at least
1 month before breeding. Moreover, PRRSV replicates
more extensively in young pigs and, as a consequence,
may induce a better immune response. Assuming that
sows are immune and their suckling pigs are protected
by passively acquired antibodies, a booster vaccination
could probably be administered safely during the nurs-
ing interval. The objective of this schedule is to provide
the maximum time for the development of protective
224 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
immunity against the widest range of virus strains, to
minimize the chance that vaccination interferes with
conception or gestation, and to maintain a high level of
maternal immunity.
The temporal parameters for vaccinating young pigs
are less well defined than they are for vaccinating gilts
and sows. What is known is that passively acquired anti-
bodies can be protective and interfere with replication of
at least the homologous strain of PRRSV (Gorcyca et al.
1996a; Morrison et al. 1996). Consequently, vaccine is
likely to be less effective when it is administered too soon
to pigs that suckle immune dams. However, a decision to
delay vaccination until the disappearance of passively ac-
quired antibodies is complicated by the fact that protec-
tive immunity is slow to develop—which, in turn, em-
phasizes the importance of early vaccination. Therefore,
to help ensure active and protective immunity as soon as
possible in young pigs, it may be necessary to administer
two doses of vaccine, the first before (assuming at least
some stimulation of the immune system), and the sec-
ond after, the disappearance of passively acquired anti-
bodies.
Once a vaccination program is selected, the next deci-
sion may be whether to use attenuated virus in herds that
appear free of virulent field virus. The fact that Prime
Pac® PRRS is (with the advice of a veterinarian), and
RespPRRS/Repro® is not, recommended by the manu-
facturers for use in virus-free herds may be more a mat-
ter of judgment than a reflection on the particular prop-
erties of the two vaccines. A possible compromise might
be the use of attenuated virus in a PRRSV-free herd only
when there is reason to suspect imminent introduction
of virulent virus with the potential for clinical disease
and financial loss.
Although vaccines, especially attenuated vaccines,
have played a role in the prevention and control of PRRS
and are likely to continue to do so in the foreseeable fu-
ture, there is still much to be learned about their efficacy
and safety and about the minimal interval between vac-
cination and exposure to virulent virus necessary for pro-
tective immunity. And because of strain differences
there may be a need for multistrain vaccines to provide
the earliest protection against the widest range of het-
erologous strains. Also, inactivated vaccines (Plana Du-
ran et al. 1995; Swenson et al. 1995), because of their
added level of safety, need to be further evaluated for
their potential for the control of PRRS, either when used
alone or as an adjunct to attenuated vaccines.
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Wills, R. W.; Zimmerman, J. J.; Swenson, S. L.; Yoon, K.–J.;
Hill, H. T.; Bundy, D. S.; and McGinley, M. J. 1997a.
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Hoffman, L. J.; McGinley, M. J.; Hill, H. T.; and Platt, K.
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 Pseudorabies
19 (Aujeszky’s Disease)
J. P. Kluge, G. W. Beran, H. T. Hill, and K. B. Platt
The history of pseudorabies (PR) has been summarized
by Baskerville et al. (1973) and more recently by
Wittmann and Rziha (1989). The disease was first de-
scribed in the United States in 1813 in cattle. The cattle
suffered from extreme pruritus and eventually died; con-
sequently the disease was termed “mad itch.” The term
“pseudorabies” was first used in Switzerland in 1849 be-
cause the clinical signs in cattle were similar to those of
rabies. In 1902 Aujeszky established the etiologic agent
as nonbacterial, and in 1910 Schmiedhofer confirmed
that the agent was viral by filtration experiments. In
1934 Sabin and Wright identified the virus as a her-
pesvirus that was immunologically related to herpes sim-
plex and herpes B viruses.
Prior to the 1960s the disease was important in East-
ern Europe but not considered of economic importance
in the United States. Subsequently, PR has emerged as an
important disease in the United States and most areas of
the world where swine are raised. Several reasons have
been postulated for the apparent increase in disease
severity, prevalence, and distribution. First, new and
more virulent viral strains may have emerged. A second
reason involves the decline of hog cholera and the devel-
opment of effective hog cholera vaccines and specific di-
agnostic tests. Prior to fluorescent antibody techniques,
cases of PR may have been misdiagnosed as hog cholera.
Porcine hyperimmune serum was often used in conjunc-
tion with virulent hog cholera virus as part of the vacci-
nation regimen. Shope (1935) demonstrated that most
of the serum contained both PR and hog cholera viral an-
tibodies, which provided some passive immunity to both
diseases. The third reason commonly cited is the dra-
matic change in swine management systems during the
past 30 years. The advent of total confinement systems
with large numbers of animals and the incorporation of
continuous farrowing have created an environment that
facilitates the maintenance and spread of a virus within
herds.
ETIOLOGY
PR virus belongs to the alphavirus subfamily of the fam-
ily Herpesviridae. Alpha herpesviruses include viruses
with both broad (as in the case of PR virus) and narrow
host ranges. Other biological characteristics of the alpha
herpesviruses include lytic replication cycles of less than
24 hours and the ability to establish latent infections in
sensory ganglia of the nervous system and lymphoid tis-
sue of the tonsils (Wheeler and Osorio 1991).
The pig is the only natural host of PR virus, which ac-
counts for its ability to be subclinically and latently in-
fected. Other common farm animals that the virus in-
fects are cattle, sheep, goats, cats, and on rare occasions
horses. Infection of these animals is lethal, although
there is one report documenting the survival of an in-
fected cow. Several wild animal species are also suscepti-
ble to lethal PR virus infection. The most common of
these species are the raccoon, opossum, rat, and mouse,
all of which are frequently found around farms. Experi-
mental studies in nonhuman primates have shown that
chimpanzees and Barbary apes are refractory to PR virus
infection but rhesus monkeys and marmosets are sus-
ceptible. Reports of human infection have been limited
and are inadequately documented. None of these reports
have been based on virus isolation. A report in 1987 de-
scribed three patients in Denmark and France who de-
veloped low-level transient seroconversion to PR virus
following an illness that was possibly transmitted by
cats. The possibility that the transient seroconversions
were due to a serologic cross-reaction induced by anoth-
er herpesvirus was not considered. The most recent re-
port of possible human infection by PR virus was de-
scribed in 1992 (Anusz et al.). Six of seven workers who
were in direct contact with PR virus–infected cattle in
Poland developed a transient pruritus which occurred
first on the hands and subsequently spread to the shoul-
ders and back.
PR virus consists of an enveloped nucleocapsid that
surrounds a linear genome of about 145 kb of DNA. The
PR virus genome is about 30 times the size of the small-
est known DNA-containing viral pathogen of swine (i.e.,
porcine parvovirus) and is large enough to code for
about 100 proteins. The overall dimension of the virus
ranges from 150 to 180 nm in diameter. The nucleocap-
sid is reported to be 105–110 nm in diameter. The nucle-
ocapsid is composed of at least eight proteins ranging in
size from 22.5 to 142 kDa. Proteins with molecular
weights of 142, 34, and 32 kDa are the most prominent
233
234 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
(Stevely 1975). The viral envelope contains at least nine
structural proteins with molecular weights ranging from
50 to 130 kDa (Hampl et al. 1984; Wittmann and Rziha
1989; Klupp et al. 1992). Eight of these proteins have
sugar residues attached to them and are referred to as
glycoproteins.
Originally these glycoproteins were designated by Ro-
man numerals or by their molecular mass (i.e., kilodal-
tons). In recent years this system of protein nomencla-
ture has been increasingly replaced by the letter system
used to designate the proteins of human herpesvirus
(i.e., herpes simplex virus 1 and 2). The old and new
nomenclature of PR virus envelope glycoproteins is sum-
marized in Table 19.1. The reason for the change is to fa-
cilitate comparison of the biological properties of ho-
mologous proteins of different herpesviruses. For
example, gE of PR virus (formerly referred to as gI) is the
counterpart of gE of herpes simplex virus 1. Other im-
portant proteins that are encoded by the viral genome
include the nonstructural glycoprotein gG (formerly
known as gX) and thymidine kinase (TK). Genes that
code for many of the above proteins are not essential for
viral replication. For example, vaccine strains of PR virus
have been genetically engineered to be deficient in one or
more of the following proteins: gE, gC, gG, and TK.
The role that individual PR viral proteins play in vir-
ulence and in the induction of immunity has been par-
tially characterized. Virulence of PR virus is controlled
synergistically by several genes, most notably the genes
encoding glycoproteins gE, gD, gI, and TK (Wittmann
and Rziha 1989; Kritas et al. 1994; Mulder et al. 1996).
Glycoproteins gB, gC, and gD appear to be most impor-
tant with respect to the induction of immunity. This con-
clusion is based on observations that monoclonal anti-
bodies that represent multiple epitopes of gB neutralize
PR virus in vitro and are also active in antibody-depen-
dent cell-mediated cytotoxicity. Specific gB monoclonal
antibodies also confer protective immunity to mice that
are experimentally infected with PR virus. Monoclonal
antibodies to gC also neutralize PR virus in vitro and
confer protective immunity to both mice and pigs. Pro-
tective immunity has also been conferred to both mice
and swine injected with gD-specific monoclonal anti-
body (Marchioli et al. 1988) or immunized with recom-
Table 19.1. Comparison of the old and new sys-
tem of nomenclature for the envelope proteins of PR
virus
Old New
Nomenclature Nomenclature
gI gE
gIIa, b, and c gB
gIII gC
gp50 gD
gp63 gI
gX gG
binant-derived gD and gI (Marchioli et al. 1987; Kost et
al. 1989).
Only one serotype of PR virus is recognized, although
distinct differences between some strains can be demon-
strated by panels of monoclonal antibodies. PR virus
strains can also be differentiated with a high degree of
certainty by biological and physical markers. Vaccine
and field strains of PR virus have been reliably differen-
tiated by using the heat and trypsin inactivation markers
in combination with the mouse and rabbit virulence
markers. Genomic differences, as revealed by restriction
fragment length polymorphism (RFLP), can also be used
to reliably differentiate PR virus strains. All of these
characteristics have been shown to be stable in several
PR virus strains after serial passage in pigs (Platt 1981;
Mengeling et al. 1983), which makes them useful in epi-
zootiological studies. Differentiation of virus strains may
also help resolve questions of liability. However, in this
respect it must be remembered that although it is possi-
ble to say with a high degree of certainty that strains are
different, it is not possible to say with the same certainty
that virus strains with genomic similarities and identical
biological markers are one and the same.
EPIDEMIOLOGY
A primary outbreak of PR in a naive immunologically
unprotected herd can be a devastating event, with spread
through the entire herd within 1 week and ending with
more than 90% of suckling pigs dead, nursery pigs stunt-
ed in their growth, and, depending on virus strains and
severity of exposure, with febrile respiratory disease in
older hogs and abortion in those in gestation. PR virus is
shed in nasal and oral secretions and is aerosolized in
droplets, which are moved rapidly by airflow to suscepti-
ble swine within and adjacent to shared airspaces; the
virus is also transmitted transplacentally and via vaginal
mucosa, semen, and milk. The outbreak ends with es-
sentially every convalescent animal permanently latently
infected (Davies and Beran 1980). If other respiratory in-
fectious agents, including Actinobacillus pleuropneumoni-
ae or porcine influenza virus, are present in subclinical or
low-grade form, they will exacerbate the outbreak fur-
ther.
A wide variety of factors may prevent this devastating
course in a primary-exposure episode. Vaccination of the
breeding herd with the genetically engineered gene-
deleted vaccines available today will protect the sows and
gilts from acute clinical signs. Depending upon severity
of exposure to the field virus, these swine will still be in-
fected and develop latency as they did following in-
tranasal or intramuscular exposure to the vaccine virus
(Henderson et al. 1991). In the naive unvaccinated herd,
the course of clinical events will depend on the strain of
introduced virus; the stages of gestation in the breeding
herd; the air, water, and feed management system; the
 CHAPTER 19 PSEUDORABIES (AUJESZKY’S DISEASE)
size and age segregation of the herd; the overall health
maintenance of the herd; and the sanitary practices in
operation. Pigs nursing immunized sows or gilts will be
protected from all but overwhelming exposures.
Characteristically, the swine herd returns to normal
productivity following the primary episode, with the la-
tently infected pigs being marketed while the breeding
herd remains latently infected. Reactivation of latent in-
fections can occur at any time of stress (Davies and Be-
ran 1980), but intensive vaccination, usually recom-
mended to be performed 3–5 times each year, reduces
occurrence, levels, and duration of shedding. Only ex-
ceptionally—and usually only in small herds with
turnover of the breeding stock and their replacement
with vaccinated gilts within 2 years or less—will the en-
demicity be eliminated. Usually, a rigorous combination
of herd cleanups and protective interventions are need-
ed, including intensive vaccination of the breeding herd
(De Smet et al. 1992) and, if infected, of finisher pigs
with one or two doses of vaccine each between 10 and 16
weeks of age (De Smet et al. 1994). Intensive culling of
seropositive breeding stock and replacement by infec-
tion-free gilts, strong biosecurity and sanitation mea-
sures, all-in/all-out movement and segregation by age,
and personnel access and movement restrictions (Thaw-
ley et al. 1982) are important. Progress in herd cleanups
must be monitored every 6–12 months and continued as
long as risk of reexposure is present.
Exposure and reexposure risks are by far the greatest
from proximate, infected, shedding, introduced swine.
Epizootiological investigations of over 3000 newly rec-
ognized herd infections in the United States during the
period 1994–March 1997 reported that 20% were trans-
mitted by exposure to introduced, infected breeding
swine and feeder pigs and that 2.0% were transmitted by
contact with feral swine. Surveillance studies in the Unit-
ed States have identified PR-seropositive feral swine in 11
of 13 sites investigated, with 34.8% of 1662 sera from the
11 infected sites in Florida being positive by at least one
test (Vander der Leek et al. 1993), and studies in Ger-
many have yielded 13/640 ELISA test positive findings
(Oslage et al. 1994).
So far as has been recognized, all livestock mammals
are susceptible to PR, with transmission from swine to
other livestock occurring either directly onto mucous
membranes or broken skin, leading to neurological dis-
ease, usually with intense pruritus emanating from the
site of inoculation, or indirectly by aerosol inhalation,
leading to rapidly progressing encephalitic disease.
These infected animals almost without exception die by
the second clinical day without having shed PR virus
through routes which would endanger other swine; that
is, they are true dead-end hosts. Sheep are highly suscep-
tible, and flocks in contact with swine often inadvertent-
ly serve as sentinels of active or reactivated latent infec-
tions in the herds. Cats are highly susceptible; dogs,
Kluge, Beran, Hill, Platt 235
raccoons, and skunks are moderately susceptible; and
rats and mice are less susceptible to direct exposure by
ingestion of dead swine carcasses or indirect exposure by
inhalation of aerosolized virus or ingestion of contami-
nated feed or water. The incubation periods in these ani-
mals are commonly short, within 3 days; the clinical pe-
riods are characterized by rapidly progressing
encephalitis with variable pruritus; and death is certain,
usually within 2–3 days. The short incubation and clini-
cal periods usually restrict transmission to a single farm,
with swine exposed by ingestion of contaminated feed,
water, or animal carcasses. Occasionally dogs drag car-
casses of pigs that died of PR from one area to another,
or rodents that died of PR are unknowingly milled in
swine feed or are present in bedding from infected farms.
There is little field or experimental evidence that do-
mestic or synanthropic birds play any role in PR virus
transmission. Experimental oral exposures have been un-
successful. Externally contaminated feed rapidly loses in-
fectivity (Beran 1991). Insect transmission has not been
demonstrated as playing an epizootiological role, al-
though PR virus experimentally fed to houseflies re-
mained viable in the gut with a half-life of 13 hours at
10˚C and 3 hours at 30˚C. Results of experimental expo-
sure via virus-contaminated flies onto corneas or abrad-
ed skin of susceptible swine have been variable, usually
negative (Zimmerman et al. 1989).
Interherd and intraherd PR virus transmission by air
or water or by contaminated fomites (especially move-
ment of feed or bedding from farms of undetermined PR
status) is hypothesized with some frequency. PR virus
has a relatively low survivability compared to nonen-
veloped RNA viruses in the environment. It is most sta-
ble between pH 6 and 8 in moist environments at cool,
nonfluctuating temperatures. It is inactivated in 1–7
days at pH 4.3 or 9.7 at temperatures between 37˚C and
4˚C (Davies and Beran 1981). It is highly susceptible to
drying, especially in the presence of direct sunlight.
Times required for infectious levels of PR virus to drop
to noninfectious levels when experimentally suspended
in porcine saliva and held in contact with moist fomites
at 25˚C are summarized in Table 19.2 (Beran 1993;
Schoenbaum et al. 1993). Other studies at colder tem-
peratures, usually below 4˚C, have shown long survival
times, but PR virus is unstable at fluctuating tempera-
tures, even below freezing (Beran 1993; Davies and Be-
ran 1981). Full advantage needs to be taken of these sur-
vival factors outside the living host in waste
management, in cleaning and disinfecting contaminated
premises, and in biosecurity of swine units.
Epizootiological evidence of airborne transmission
even over distances up to many kilometers is occasional-
ly reported both in the United States and in Europe,
sometimes during major atmospheric events, such as fol-
lowing the path of a tornado (Christensen et al. 1993;
Scheidt et al. 1991). Experimentally, infective levels of
236 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

Table 19.2. Survival times of PR in contact with Genetically engineered modified-live-virus vaccines
fomites with compatible serologic tests to differentiate vaccine-
Survival in Saliva at and field-infection-induced antibodies have proven es-
Fomites (at 25˚C) Infectious Levels (Days) sential in area control efforts and in infection cleanups in
Nonfomite control test <4 all but the smallest herds of swine. Although commonly
Steel <4 used only in swine, inactivated vaccines and some of the
Concrete <4 TK gene-deleted live-virus vaccines have been shown to
Plastic <3 be safe for cattle and sheep. While the genetically engi-
Rubber <2
Denim cloth <1 neered vaccines afford relatively long protection of swine
Loam soil <7 from clinical PR, they effectively reduce transmission for
Green grass <2 only a few months, decreasing occurrence level and du-
Shelled corn <4 ration of shedding by reactivation of latent infections.
Pelleted hog feed <3
For a period of weeks to a few months, swine vaccinated
Meat and bone meal <2
Alfalfa hay <1 with these vaccines have enhanced resistance to infection
Straw bedding <4 by field viruses, especially following intranasal vaccina-
Sawdust bedding <2 tion, but infections with reduced shedding and develop-
Swine feed <2 ment of latency must be expected in vaccinates exposed
Well water <7
Chlorinated water <2 to field viruses (Mengeling et al. 1992). Intensive vacci-
Anaerobic lagoon effluent <2 nation programs must be accompanied by rigorous mon-
Swine unit pit effluent <1 itoring with culling and turnover of infected herds, by
preventing exposure of uninfected sectors of the herd to
infected swine, usually in the breeding herd, and by ef-
virus have persisted in aerosols up to 7 hours at 55% or fective biosecurity (Van Oirschot et al. 1996).
higher relative humidity. Droplets of infective salivary or
nasal mucus can carry infective doses over short dis- CONTROL AND ERADICATION
tances and when coalesced into droplet nuclei can be
moved over long distances within the experimental sur- Area control and elimination of infection are being pro-
vival time (Schoenbaum et al. 1990). gressively undertaken by major swine-producing coun-
All of these factors of survival and transmission of tries worldwide. The United Kingdom (Taylor 1989) and
PR virus are classified together into area spread (US- Denmark (Anderson et al. 1989) have achieved major
DA/APHIS/VS 1994–1997). During the period success. The U.S. eradication program was initiated in
1994–March 1997, it was concluded that movement of 1989 with a 10-year projected target for achievement.
contaminated feed or bedding was the source in 1% of Working on a state-by-state basis, this program progress-
the outbreaks investigated. In 38% of the outbreaks in- es through five stages according to a program standards
vestigated, the source could not be differentiated as to in- document that is revised annually by a broad-based com-
terherd movements of companion and wild animals, mittee representing national and state agencies, swine
trucks and other vehicles, people, insects, or air and wa- producers, and researchers (USDA/APHIS/VS 1997).
ter movement. Finally, 39% of these epizootiological in- Stage I is a time of preparation and formation of state
vestigations failed to identify exposure sources. plans and movement regulations. Stage II is a time of
In a cost-benefit study of the U.S. PR eradication pro- surveillance testing for prevalence and location of infect-
gram, productivity and economic impacts were calculat- ed herds and of implementing movement controls and
ed for PR-infected and noninfected herds, including cleanup of infected herds. It is the active period of iden-
preweaning, nursing, grower, and finisher pig mortality, tifying the infected herds statewide and of eliminating
breeding-herd mortality, feed conversion, labor, and vet- infection and preventing transmission to uninfected
erinary and biologic/pharmaceutic expenses. The study herds. Stage III is a time of intense surveillance and
calculated that there was a $6.00/hundredweight reduc- mandatory cleanup of all identified infected herds in the
tion of profitability due to PR herd infection (Anderson state. Area prevalence must be below 1% of herds. Stage
et al. 1989; Miller et al. 1996). IV is a time of continuing surveillance and is reached on-
Recombination has been demonstrated between field ly when no newly infected herds have been identified in
strains of PR virus and between field strains and live-vac- the past 12 months. Vaccination is prohibited except by
cine virus strains, in both experimental and field studies. special permission. Stage V is the certified free state, with
Only in a small number of field studies in Europe have surveillance at a maintenance level and entry of swine in-
prevailing field strains been identified as having recom- to the state only from other Stage IV or V states. Vacci-
binant vaccine virus components (Henderson et al. 1991; nation is prohibited.
Mayfield 1990). The PR eradication program in the United States is
 CHAPTER 19 PSEUDORABIES (AUJESZKY’S DISEASE) Kluge, Beran, Hill, Platt 237

based on active partnership of swine producers, state of infected vaccinated breeding swine in the herd with
governments, and the federal government in organiza- the active surveillance based on differential serologic
tion and funding. All three are active in education, both testing until culling and turnover of the breeding herd
for the swine industry and the public. Practicing veteri- has been completed.
narians are active participants in case finding and As of March 1, 1997, the U.S. National Pseudorabies
cleanup of infected herds. Case finding is based on re- Control Board has granted Stage V status to 23 states
porting by veterinarians, by on-farm area testing, by cir- plus Puerto Rico, Stage IV status to 7 states plus U.S. Vir-
cle testing of herds within 1–2 km of identified infected gin Islands, split Stage III/IV status to 1 state, Stage III
herds, and by tracing back from slaughter surveillance status to 13 states, split Stage II/III status to 5 states, and
findings. On-farm area testing and slaughter surveillance Stage II status to 1 state (USDA/APHIS/VS 1994-1997).
are performed by statistical sampling, commonly based
on testing breeding swine at 95% probability of detecting CLINICAL SIGNS
infection in herds in which at least 10%, 20%, or 30% of
swine are seropositive (Table 19.3). The clinical signs associated with PR are primarily de-
A system of qualified negative herds, of qualified pendent on the strain of virus, the infectious dose, and,
negative vaccinated herds, and of monitored feeder pigs most important, the age of the swine affected. Like her-
is based on statistical testing at intervals varying from pesviruses of other animal species, younger swine are the
monthly to yearly. Vaccination is controlled on a state- most severely affected by PR virus. The virus has a
by-state basis; only genetically engineered vaccines with predilection for respiratory and nervous tissue; thus the
deletion of at least the gene encoding glycoprotein gE, majority of clinical signs are associated with dysfunction
with companion differential serologic tests, are permit- of these two organ systems. Generally, nervous signs are
ted, except in special conditions. more commonly observed in suckling and weaned pigs,
Herd cleanup plans are individually developed and while respiratory signs are observed in finisher pigs and
implemented in infected herds, adapting four basic adult swine.
plans. Cleaning and disinfection of individual swine The response to a PR viral infection can differ
units or entire premises are essential components. In de- markedly between herds. The disease may manifest itself
population-repopulation, the entire herd is removed, the as a rapidly spreading disease affecting all ages of swine
premises are cleaned and disinfected, and the farm is re- on the farm, or it may be a completely inapparent infec-
populated with clean stock. In herds with low prevalence tion detected only when a serologic evaluation of the
of infection (usually with fewer than 20% seropositive herd is made. PR is more often inapparent when the in-
breeding stock and all immature swine negative), testing fection occurs while neonatal pigs are not present, as in a
of the entire breeding herd, removal of the positive pigs, period between farrowing groups in a group-type farrow-
and active continuing surveillance testing may be effec- ing operation. Inapparent herd infections rarely occur
tive. Offspring segregation involves segregated rearing of when PR virus is introduced into a herd for the first time
progeny of infected breeding herds with rapid removal where neonatal pigs are present, because pigs of this age
of the breeding stock following weaning and monitoring are so highly susceptible. Inapparent infections in breed-
of all gilts raised as replacements. Active surveillance in- ing herds or separate finishing barns may be mild PR res-
cludes two negative tests 90 days apart and again at 12 piratory infections that are ignored or misdiagnosed as
months to declare the herd free of infection. Offspring some other disease, such as swine influenza.
segregation with intensive vaccination of all breeding The first clinical signs observed in a farrow-to-finish
stock is permitted in some programs, allowing retention herd will vary depending on the age group first infected.
Typically, the first clinical signs will be a few gilts or sows
Table 19.3. Size of the sample required to detect aborting; or finisher pigs coughing and becoming listless
reactor animals by population size for 95% and 99% and anorectic; or rough-haired suckling pigs that become
certainty of detection listless, anorectic, and within 24 hours ataxic and con-
Number of Reactor Animals in the Population vulsive. If any of these scenarios occurs, immediate di-
agnosis is imperative, because early vaccination in the
≥10% ≥20% ≥30%
face of an outbreak can limit losses.
Population Size 95% 99% 95% 99% 95% 99%
≤100 25% 36% 13% 19% 9% 13% Neonatal Pigs
101–200 27% 40% 13% 20% 9% 13% The incubation period in suckling pigs is usually quite
201–300 28% 41% 14% 20% 9% 13% short, ranging from 2 to 4 days. Before severe clinical
301–500 28% 42% 14% 21% 9% 13% signs are noted, suckling pigs will become listless,
501–1000 29% 43% 14% 21% 9% 13%
anorectic, and febrile (41˚C). Some pigs will develop cen-
>1000 29% 44% 14% 21% 9% 13%
tral nervous system (CNS) signs within 24 hours of the
238 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
onset of clinical signs, which progress from trembling,
hypersalivation, incoordination, ataxia, and nystagmus
to opisthotonos, to severe epileptiform-like seizures. Af-
fected pigs may sit like dogs because of posterior paraly-
sis, while others will circle or become recumbent and
paddle. Vomiting and diarrhea may also occur, but nei-
ther of these clinical signs is consistent. Pigs with CNS
signs usually die within 24–36 hours of onset. Mortality
in suckling pigs is quite high, often approaching 100%. In
herds with dams of varying immune status to PR virus,
clinical signs may be seen in some litters, or pigs within a
litter, while neighboring litters or littermates remain nor-
mal. If susceptible sows or gilts are infected close to
term, pigs may be born weak and show clinical signs im-
mediately and die within the first day or two of life.
Weaned Pigs (3–9 Weeks)
Younger pigs in this age group tend to have clinical signs
similar to those described for suckling pigs. However, the
clinical signs are less severe and fewer pigs develop the
severe CNS involvement, which invariably leads to coma
and death. Mortality in 3- to 4-week-old pigs may ap-
proach 50% in severe outbreaks. Older pigs in this age
group become listless, anorectic, and febrile (41–42˚C)
within 3–6 days of exposure. Often, respiratory signs are
present, characterized by sneezing, nasal discharge, and
dyspnea, and progress to a severe cough. Pigs displaying
these clinical signs will lose significant body condition
and weight. Duration of clinical signs is usually only
5–10 days; most pigs make a rapid full recovery once the
fever and anorexia disappear. Pigs that develop CNS
signs often die, as do pigs with a PR viral respiratory in-
fection that develop a secondary or concurrent bacterial
infection such as Pasteurella multocida or Actinobacillus
pleuropneumoniae. However, the mortality in pigs 5–9
weeks of age, if cared for properly and treated for sec-
ondary infections, will usually not exceed 10% and is of-
ten lower. The more severely affected pigs that survive,
especially those developing CNS signs, will often be
stunted and sometimes show lasting signs, such as head
tilt. These pigs reach market weight 1–2 months after the
rest of the group.
Grower-Finisher Swine
Respiratory signs have become the hallmark of PR in
grower-finisher pigs. Morbidity is usually very high, ap-
proaching 100%, but in uncomplicated cases mortality is
low, 1–2%. CNS signs do occur, but only sporadically,
and they may vary from mild muscle tremors to violent
convulsions. Typically, clinical signs appear in 3–6 days
and are characterized by a febrile response (41–42˚C),
depression, anorexia, and mild to severe respiratory
signs. A rhinitis develops causing sneezing and a nasal
discharge that progress to pneumonia, resulting in a
harsh cough and labored breathing, especially when the
pigs are forced to move about. These pigs become gaunt
and lose considerable body weight. The duration of clin-
ical signs is usually 6–10 days, and recovery is rapid once
the fever disappears and the appetite returns. Although
compensatory gain negates some of the lost body
weight, grower-finisher pigs will lose at least a week in
the production cycle. Losses may be dramatically in-
creased if a PR virus infection is superimposed on an A.
pleuropneumoniae infection. It has been reported that PR
virus inhibits the function of alveolar macrophages (Igle-
sias et al. 1989), thus reducing the capacity of these de-
fense cells to process and destroy bacteria.
Adult Swine
Sows and boars develop clinical signs, primarily respira-
tory in nature, very similar to those described above for
grower-finisher pigs. Pregnant gilts often abort, and in a
farrow-to-finish operation this may be the first clinical
sign observed. Pregnant females infected with PR virus
in the first trimester may resorb the fetuses and return to
estrus. Reproductive failure due to PR in the second or
third trimester is usually manifested by abortion or still-
born or weak pigs if the time of infection is close to term.
If the gilt or sow is very close to farrowing when infected,
pigs may be born with PR, in which case they die within
a day or two. The PR virus can cross the placenta and in-
fect and kill fetuses in utero, resulting in abortion. Fortu-
nately, reproductive failure usually has a low incidence,
occurring in 20% or fewer of the pregnant females on a
farm. Mortality in gilts, sows, and boars infected with PR
virus rarely exceeds 2%.
PATHOGENESIS
The pathogenesis is variable depending on viral strain,
age of the pig, size of inoculum, and route of infection.
There is an increased resistance to development of
clinical signs with age; strains of low virulence may not
produce clinical signs in adult animals, and the viral
replication may be limited locally to the site of introduc-
tion. To produce clinical disease experimentally, a mini-
mum dose of virus is required; however, under field con-
ditions very low quantities of virus may cause pigs to
seroconvert and even become latent carriers although no
clinical signs can be detected in the herd. Experimental
infection by intranasal inoculation requires about 10
TCID50 in pigs under 2 weeks of age, about 103 TCID50 in
6-week-old pigs, and about 104 TCID50 in swine 4 months
of age or older (McCullough 1989).
Experimentally, pigs develop infection after inocula-
tion via the following routes: intramuscular, -venous,
-cerebral, -gastric, -nasal, -tracheal, -conjunctival, -uter-
ine, -testicular, and oral. Intranasal inoculation results in
clinical signs and lesions similar to the natural disease,
and under field conditions the oral-nasal routes are the
most common.
Under natural conditions the primary site of replica-
 CHAPTER 19 PSEUDORABIES (AUJESZKY’S DISEASE)
tion is epithelium in the nasopharynx and tonsil (Masic
et al. 1965). From these sites there is lymphatic spread to
regional lymph nodes and replication in the nodes. The
virus also spreads via nerves from the primary site of in-
fection to the CNS. Examples include spread within axo-
plasm of the trigeminal nerve to the medulla and pons
and spread along olfactory nerves and the glossopharyn-
geal nerve to the medulla. After replication in neurons in
the medulla and pons, there may be spread to other por-
tions of the brain. As noted earlier, with low-virulence
strains and moderate-virulence strains in older animals,
viral spread may be limited to those locations.
Strains of higher virulence will follow a course simi-
lar to the above, but in addition there is widespread virus
distribution in the body, and older swine will have clini-
cal signs and may have gross lesions (McFerran and Dow
1965; Sabó et al. 1968, 1969; Wittmann et al. 1980).
Probably all strains have a tropism for the upper respira-
tory tract and the CNS. Strains of higher virulence pro-
duce a brief viremia and virus can be demonstrated in
the serum and also associated with buffy-coat cells. High-
virulence strains can be isolated from alveolar
macrophages (Iglesias et al. 1992), epithelium of termi-
nal bronchioles, hepatocytes, lymphoid cells of the
spleen and lymph nodes, adrenal cortical cells, tro-
phoblasts and embryos from the gravid uterus, and
luteal cells of the ovary. The virus has been isolated from
semen but probably originated from preputial lesions,
since attempts to isolate virus from the testes and acces-
sory sex glands have failed (Gueguen and Aynaud 1980;
Medveczky and Szabó 1981). The observed decrease in
sperm quality is probably the result of fever and systemic
disease. Embryos are resistant to infection as long as the
zona pellucida is intact; thus, associated virus can be
washed from embryos, and embryo transplantation can
be used as a method of salvaging genetic material from
an infected herd (Bolin et al. 1983; Bolin and Bolin
1984).
Virus excretion precedes or coincides with the onset
of clinical disease; in clinically inapparent infection, ex-
cretion begins after an incubation period of 2–5 days. PR
virus may be recovered from nasal exudate or secretions
at levels of approximately 102 TCID50 per swab for 1 to
more than 2 weeks and for similar periods but in lesser
quantities from tonsillar scrapings, vaginal secretions,
prepuce, milk, and occasionally urine (Wittmann and
Rziha 1989). Active immunity from vaccination or prior
infection decreases the period of viral shedding. As with
other herpesviruses, infection with PR virus results in a
high percentage of latent infections in host pigs (Rziha et
al. 1982). The virus persists in ganglia such as the trigem-
inal ganglia and in tonsil (Cheung 1995). Latently infect-
ed pigs often have detectable viral recrudescence during
times of stress, such as farrowing, crowding, and trans-
port. Experimentally, corticosteroid injection results in
subsequent viral recrudescence and nasal shed of virus.
Kluge, Beran, Hill, Platt 239
This fact should be considered when administering
steroids to animals in an infected herd that is in the
midst of an eradication program. Vaccination with live
or killed vaccines cannot be relied upon to prevent the
development of latency in postvaccination exposed
swine. Some live vaccines have the potential to induce la-
tent CNS infection.
LESIONS
Gross Lesions
Gross lesions are often absent or are minimal and unde-
tected. When they are present, they aid in arriving at a
tentative diagnosis when combined with herd history
and clinical signs. Serous to fibrinonecrotic rhinitis is
common but may be missed unless the head is split to ex-
pose the entire nasal cavity (Csontos and Széky 1966;
Olander et al. 1966). The lesions may extend to the lar-
ynx and even down the trachea. Necrotic tonsillitis is fre-
quently seen along with swollen and hemorrhagic lymph
nodes of the oral cavity and upper respiratory tract
(Narita et al. 1984a). When present, lesions in the lower
respiratory tract range from pulmonary edema to scat-
tered small foci of necrosis, hemorrhage, and/or pneu-
monia (Becker 1964; Corner 1965).
Keratoconjunctivitis is common and often more obvi-
ous in white breeds because of the discoloration caused
by excessive lacrimation and periocular deposits of exu-
date (Schneider and Howarth 1973).
Typical herpetic yellow-white foci (2–3 mm) of necro-
sis may be scattered through the liver and spleen (Fig.
19.1) and may be seen just beneath the serosal surface.
These lesions are most frequently seen in very young pigs
that lack passive immunity.
Sows that have recently aborted may have a mild en-
dometritis, and the wall of the uterus is often thickened
and edematous (Kluge and Maré 1978; Hsu et al. 1980).
If the placenta is available for examination, a necrotic
placentitis is often observed. Aborted fetuses may appear
fresh, macerated, or occasionally mummified. Some pigs
in an affected litter may be normal and others weak or
dead at birth. Infected fetuses or neonatal pigs frequent-
ly have the previously described necrotic foci in liver and
spleen as well as hemorrhagic necrotic foci in lungs and
tonsils (Csontos et al. 1962; Kluge and Maré 1976;
Wohlgemuth et al. 1978). A history of abortion along
with the presence of necrotic foci is strongly suggestive
of a tentative diagnosis of PR. The only gross lesion re-
ported in the male reproductive tract is scrotal edema.
Necrotic enteritis in the lower jejunum and ileum has
been reported in young pigs (Narita et al. 1984c).
Microscopic Lesions
Microscopic lesions are most frequently reported in the
CNS and persist for many weeks (12–24 weeks postin-
fection) (Olander et al. 1976). They may also be present
240 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
19.1. Spleen and liver with
multiple small focal areas of
necrosis.
in pigs that failed to develop clinical signs, but they are
often absent in aborted fetuses. Nonsuppurative menin-
goencephalitis and ganglioneuritis are the characteristic
lesions (Dow and McFerran 1962; Sabó et al. 1969;
Baskerville 1972). Lesions are present in both the gray
and the white matter, and the distribution is dependent
on the route of entry into the CNS. Affected areas are
characterized by predominantly mononuclear cell
perivascular cuffing (Fig. 19.2) and glial nodules. A few
granulocytes may be mixed with the mononuclear cells,
and pyknosis and karyorrhexis of the latter cells are of-
ten prominent. The endothelium of affected vessels ap-
pears normal. Neuronal necrosis may be focal and the
neurons surrounded by mononuclear cells, or affected
neurons may be diffusely scattered. Similar lesions may
be present in the spinal cord, especially in the cervical
19.2. Brain with perivascular
cuffing.
and thoracic regions. Meninges over affected areas of
brain and cord may be thickened because of infiltrates of
mononuclear cells. Similar lesions have been reported in
ganglia and ganglia cells (cerebrospinal, cardiac, and
celiac ganglia, Meissner’s and Auerbach’s plexuses, and
the ganglion cell layer of the retina). Intranuclear inclu-
sion bodies may be present in neurons, astrocytes, and
oligodendroglia, but in our experience they are much
more common in lesions outside the nervous system.
In tonsils, necrosis begins in the subepithelial area
and then spreads to the epithelium and deeper into the
lymphoid tissue (Narita et al. 1984c). Intranuclear inclu-
sion bodies are common in crypt epithelial cells adjacent
to necrotic foci. The upper respiratory tract lesions con-
sist of mucosal epithelial necrosis and submucosal infil-
trations of mononuclear cells (Baskerville 1971, 1973).
 CHAPTER 19 PSEUDORABIES (AUJESZKY’S DISEASE)
Lung lesions consist of necrotic bronchitis, bronchioli-
tis, and alveolitis. Peribronchial mucous gland epitheli-
um may be necrotic. There is often hemorrhage and fib-
rin exudation because of involvement of connective
tissue and endothelium. Lesions often are patchy in ma-
jor airways, and healing by fibrosis is often observed in
areas adjacent to acute lesions. Intranuclear inclusion
bodies are frequently present in the epithelial lining of
the airways, connective tissue cells, and cells sloughed in-
to alveolar spaces.
Focal necrotic lesions are similar regardless of the tis-
sue involved. They are most frequently found in spleen,
liver (Fig. 19.3), lymph nodes, and adrenal glands.
Necrotic foci are randomly distributed and surrounded
by a few inflammatory cells, or the latter may be absent.
Parenchymal cells at the margins of necrosis commonly
contain intranuclear inclusion bodies (Fig. 19.4). In mac-
erated fetuses microscopic necrotic foci often are visible
even though cellular detail cannot be discerned since the
pyknotic nuclei stain with hematoxylin.
Uterine infection is characterized by multifocal to dif-
fuse lymphohistocytic endometritis and vaginitis and
necrotic placentitis with coagulative necrosis of chorion-
ic fossae (Bolin et al. 1985). Intranuclear inclusion bodies
are present in degenerate trophoblasts associated with
the necrotic lesions (Kluge and Maré 1978; Hsu et al.
1980). Depending upon the stage of infection, corpora
lutea may be necrotic and contain neutrophils, lympho-
cytes, plasma cells, and macrophages (Bolin et al. 1985).
In the male reproductive tract there may be degener-
ation of seminiferous tubules and necrotic foci in the tu-
nica albuginea of the testicles (Corner 1965; Larsen et al.
1980; Hall et al. 1984). Boars with exudative periorchitis
have necrotic and inflammatory lesions in the serosa cov-
ering the genital organs. Spermatozoa abnormalities in-
clude tail abnormalities, retained distal cytoplasmic
Kluge, Beran, Hill, Platt 241
droplets, knobbed cystic acrosomes, double heads, and
detached heads. These changes may be the result of
pyrexia and not the result of viral infection of spermato-
genic epithelium.
Enteric lesions consist of focal necrosis of the mucos-
al epithelium and may involve the underlying muscularis
mucosa and tunica muscularis (Narita et al. 1984b). In-
tranuclear inclusion bodies may be present in degenerate
crypt epithelial cells.
There may be necrotizing vasculitis of arterioles,
venules, and lymphatic vessels around tonsils and sub-
maxillary lymph nodes (Narita et al. 1984a). Endothelial
nuclei are pyknotic and karyorrhectic, and the vessel
walls are infiltrated by neutrophils. Intranuclear inclu-
sion bodies often are present in affected endothelial cells.
Two types of intranuclear inclusion bodies may be
observed (Corner 1965): a homogenous basophilic body
that fills the entire nucleus and an eosinophilic body that
has a definite halo between it and the marginated chro-
matin. In either case, specificity of the inclusion must be
determined by demonstrating viral particles or antigen
by electron microscopy or immunohistochemistry.
DIAGNOSIS
The diagnosis of PR is usually made on a herd basis us-
ing a combination of herd history, clinical signs, gross le-
sions, microscopic lesions, serology, and virus detection
by either the fluorescent antibody tissue section (FATS)
test or virus isolation (VI). Classic clinical signs will often
lead to a presumptive diagnosis that is often supported if
gross lesions (focal hepatic and splenic necrosis and
necrotic tonsillitis) are observed in neonatal pigs. PR is
more difficult to diagnose if only grower-finisher pigs or
adult swine are involved. A PR outbreak in these age
groups can easily be misdiagnosed as swine influenza if
19.3. Liver with focal area of
necrosis.
242 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
19.4. Liver with intranuclear
inclusion bodies in hepatocytes at
margins of necrotic focus.
the disease is manifested only by respiratory signs. If,
however, a few individuals develop CNS signs, it is easier
to make a presumptive diagnosis of PR. PR virus is rela-
tively easy to demonstrate in infected swine if samples
are taken at the right time and handled properly. Serolo-
gy is not the test of choice when trying to diagnose an
acute PR virus infection because of the delay required for
humoral antibodies to develop. Microscopic lesions can
confirm a viral etiology and are helpful in finalizing the
diagnosis (see the section on microscopic lesions).
Virus Isolation
Brain, spleen, and lung are the organs of choice for VI.
The tissues should be collected from two or three acutely
affected pigs. Tissues should be refrigerated, preferably
not frozen unless dry ice is used, and transported to the
laboratory as soon as possible. If only grower-finisher
and/or adult swine are affected and none have died,
nasal swabs collected in cold phosphate-buffered cell cul-
ture media with antibiotics may be utilized. If cell cul-
ture fluid or a balanced salt solution is not available, a
cold sterile saline solution may be used. The sample is in-
oculated onto cell cultures, and the cultures are observed
for the presence of characteristic cytopathic effects. The
identity of the suspected isolate can be confirmed by uti-
lizing a fluorescent antibody test. Usually, PR virus will
be isolated within 2–5 days. However, most laboratories
will conduct two blind passages 1 week apart before re-
porting the specimen as negative. Tissues from aborted
fetuses can also be submitted for VI; however, if the
abortion was due to a febrile response in the dam rather
than a direct effect of the virus on the fetuses, VI results
will be negative. Caution needs to be taken when inter-
preting these results, and additional diagnostic tests may
be required to confirm a diagnosis of a PR virus–induced
abortion.
In developing countries or in remote areas where di-
agnostic laboratories are not available, the supernatant
fluid of a 10% brain suspension can be injected intra-
muscularly into the hindleg of a rabbit. Typical signs of
intense pruritus and self-mutilation at the site of injec-
tion within 48–96 hours will confirm the diagnosis of
PR. This method should only be used as a last resort due
to concern for the experimental animal.
Fluorescent Antibody Tissue Section Test
The FATS test is a rapid, reliable method of detecting PR
virus in tissue. The tissue of choice is tonsil, but brain or
pharyngeal smears may also be used. In the pig, the ton-
sil is easily identified, for it is in the dorsal area of the an-
terior pharynx and it has visible crypts. The advantages
of the FATS test are that it can be completed in 1 hour
and the results in neonatal pigs with typical clinical signs
are comparable to VI results. In grower-finisher pigs and
in adults, the FATS test is not as sensitive as VI. If clinical
signs and history are suggestive of PR, a negative FATS
test result in older animals must be confirmed by VI.
Serodiagnosis
Numerous serologic tests have been developed to assay
serum for PR antibodies. The most widely utilized sero-
logic assays are serum virus neutralization (SVN), latex
agglutination (LA), automated latex agglutination,
screening enzyme-linked immunosorbent assay (ELISA),
particle concentration fluorescence immunoassay, and
the differential ELISA for glycoproteins gE, gC, and gG.
Other serologic tests have been developed, such as com-
plement-fixation, immunodiffusion, countercurrent im-
munoelectrophoresis, and indirect immunofluores-
cence; all have limitations of sensitivity, require
considerable time to conduct, or are difficult to perform.
Consequently, they have received limited acceptance.
 CHAPTER 19 PSEUDORABIES (AUJESZKY’S DISEASE)
The SVN test is a reliable yet time-consuming test
and requires 48 hours to perform. The SVN test is con-
sidered the standard and is the test used to compare new
test procedures. It can be used to titrate PR viral anti-
body in serum, and it is still the most widely used test for
this purpose.
The ELISA has replaced the SVN test in laboratories
that test large numbers of sera for PR antibody, primari-
ly because of the short time required to conduct the test
and its superior sensitivity. Several commercial ELISA
kits are available worldwide that allow large numbers of
sera to be tested in 3–4 hours.
The LA test is the simplest of these three tests to per-
form, and it is commercially available worldwide. The LA
test can be performed on a single serum sample in 10
minutes. Specificity problems can be minimized by di-
luting the test serum 1:40 instead of 1:4 without signifi-
cantly affecting sensitivity. The high sensitivity of the LA
test makes it an excellent test for quick screening of sera
for PR antibodies. Generally, the LA test will detect sero-
conversion in 6–7 days, whereas the ELISA requires 7–8
days and the SVN requires 8–10 days. The automated
format of the LA test has replaced the screening ELISA in
some high-volume laboratories.
Interpretation of serologic results can be difficult, es-
pecially in young pigs. Maternal antibodies can be pres-
ent for up to 4 months of age. The half-life of the mater-
nal antibodies that pigs receive in the colostrum of
PR-immune sows is approximately 18 days. It takes 18
days for the antibody titer to be divided in half (i.e., be
reduced from 1:16 to 1:8). If pigs from an immune sow
are tested too early, they could be identified as being in-
fected when the antibody is of maternal origin and not
the result of exposure and active infection.
The most important technological breakthroughs in
PR control and eradication programs have been the de-
velopment of the gene-deleted PR vaccines and their ac-
companying differential ELISA. These tests detect a spe-
cific antibody produced against one of the viral
glycoproteins. There are vaccines and companion diag-
nostic serology tests for gE, gC, and gG. Animals vacci-
nated with a gene-deleted vaccine will lack antibody
against the specific glycoprotein encoded for by the
deleted gene, allowing a vaccinated pig to be differentiat-
ed from an infected pig. Once the pig is infected, it will
develop antibody against all of the PR viral proteins
(antigens) and be positive on all differential serology
tests. Some countries have restricted commercial compa-
nies as to which gene can be deleted from the vaccine,
but in other countries different vaccines have different
deletions. These vaccines would not be compatible in a
single herd, because a pig vaccinated with two different
gene-deleted vaccines would have antibodies specific for
all the glycoproteins and be falsely identified as a PR
virus–infected pig. Therefore, it is important to know
which vaccine is used and to avoid vaccinating pigs with
Kluge, Beran, Hill, Platt 243
vaccines with different deletions. Countries with PR
eradication programs will eventually need to conduct
slaughter-plant surveillance once the prevalence of PR is
low. This surveillance will be possible only if a universal
gene deletion is used in all vaccines.
TREATMENT: VACCINES
Modified-live-virus (MLV), inactivated, and gene-deleted
vaccines have been developed for the control of PR and
are available in most countries in which PR is endemic.
These vaccines have proven to be quite effective in reduc-
ing or preventing clinical signs of PR and thus have re-
duced the economic impact of the disease.
Vaccinated swine that subsequently become infected
have less invasion of tissues, usually limited to the upper
respiratory system, and do not transmit virus to the fetus
in utero. Vaccinated swine shed lesser amounts of virus,
in most studies at least 1000-fold less; and for a shorter
period of time, in most studies a 4- to 7-fold reduction.
Latent infections may not be prevented in swine infected
subsequent to vaccination. However, recent studies have
demonstrated that certain strains of MLV vaccines will
colonize the latency target tissue, the trigeminal ganglia,
of vaccinated animals and block the establishment of la-
tency by a superinfecting challenge of wild-type PR virus
(Schang 1994). The efficiency of a particular vaccine in
blocking a wild-type virus is dependent on the strain,
dose, and route of inoculation. MLV vaccines that have
extensive deletions of not only glycoproteins gE and gG
but also other gene deletions for attenuation such as TK
were less efficient at colonizing the trigeminal ganglia.
Prevention of latency by wild-type virus was also en-
hanced by MLV vaccines that were of higher titer and ad-
ministered intranasally. Blocking or reducing the state of
latency by a wild-type virus is of paramount importance
in herds undergoing PR cleanup.
The MLV vaccines replicate principally at the site of
injection and regional lymph nodes. They have been
shown to be shed in nasal secretions and tonsillar mucus,
but at levels too low for there to be any practical risk of
transmission to swine or any other animals. These vac-
cines vary in virulence in animal species other than
swine. Appropriate precautions must be taken when us-
ing MLV vaccines in species other than swine, and ex-
treme care must be taken that syringes and needles used
with PR MLV vaccines are not then used when inoculat-
ing other animals with other vaccines. This hazard and
the concern for the potential for recombination of MLV
with wild-type virus have led to the improvement of
killed PR vaccines. The employment of new adjuvants
has made the efficacy of some of these killed vaccines
quite competitive with MLV vaccines, especially when
multiple doses are used. However, killed vaccines do not
colonize latency target tissues and thus do not prevent or
reduce latency.
244 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
Killed vaccines have been developed from wild-type
strains and from the Bucharest and Bartha MLV vaccine
strains. Genetically engineered PR vaccines and some of
the conventionally attenuated vaccines have deletions in
nonessential glycoprotein genes such as gE, gC, gG, and
gI. The absence of these glycoproteins makes them use-
ful as negative immunological markers and has been uti-
lized in serologic tests designed to identify virus-infected
vaccinated swine.
The PR vaccines which have a gE deletion have be-
come the vaccines of choice throughout the world be-
cause of the superiority of the gE differential serologic
tests (White et al. 1996). The gG gene-deleted vaccines
are very immunogenic but the companion gG differen-
tial ELISA lacks both sensitivity and specificity. Many an-
imal health regulatory bodies have mandated that all PR
vaccines must have a gE gene deletion.
The TK gene has also been deleted from genetically
engineered PR vaccines. TK facilitates PR infections of
neurons, permitting viral replication. Since the natural
endogenous level of TK in differentiated neural tissue is
low, the virulence of a PR vaccine can be reduced by re-
moving the TK gene so that the virus can no longer en-
code for the production of this enzyme.
The recombination of gene-deleted vaccines that
might lead to a virulent virus that still contains a negative
immunological marker is a concern (Henderson et al.
1991). Studies demonstrated that when pigs were inocu-
lated with two different gene-deleted vaccines, recombi-
nation did occur. It is therefore imperative that only one
type of gene-deleted vaccine be used in a single herd an-
imal to avoid potential recombination between vaccine
viruses. Recombination potentially could also occur
when gene-deleted MLV vaccines are used in the face of
an outbreak with field PR virus, resulting in a gene-delet-
ed virulent virus as a result of recombination between
the field strain and the gene-deleted vaccine strain. This
apparently does not occur or occurs very infrequently,
for gene-deleted MLV vaccines have been widely used in
PR outbreaks, and as yet, there are no reports of a viru-
lent PR virus with a deletion of a gene for one of the gly-
coproteins.
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Anderson, J. B.; Bitsch, V.; Christensen, L. S.; Hoff-Jor-
gensen, R. and Petersen, K. 1989. The control and eradi-
cation of Aujeszky’s disease in Denmark: Epidemiologi-
cal aspects. In Vaccination and Control of Aujeszky’s
Disease. Ed. J. T. Van Oirschot. Brussels and Luxem-
bourg: ECSC, EEC, EAEC; Boston: Kluwer Academic
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 Rabies
20 L. G. Morehouse
Rabies is a highly infectious viral disease of humans and
animals that dates to antiquity. The dramatic clinical
signs of this disease were described in the writings of
Plutarch and Celsus in A.D. 100 (Kelser 1947). The earli-
est reference to the disease in Asia is in the pre-Mosaic
Eshnunna Code (ca. 2000 B.C.), which predates the eigh-
teenth-century B.C. code of Hammurabi of ancient Baby-
lon.
Despite the ancient history of the disease, many as-
pects of rabies are not clearly understood. Although al-
most all warm-blooded animals are susceptible, the long-
held belief in the fatal termination of the disease may
not be applicable to all species. In fact, recovery from hu-
man rabies (U.S. Dept. HEW 1970) and recovery of dogs
from experimental rabies infection (Nillson and Cortes
1975; D. C. Blenden, pers. comm.—1979; Fekadu et al.
1983) have been reported. Evidence also indicates that
some animal species are more resistant to the infection
than others (Bisseru 1972; Botros et al. 1979); the discus-
sion in this chapter suggests that swine fall in this cate-
gory. Nonetheless, the overwhelming mortality associat-
ed with rabies infection makes it one of the most feared
diseases of humans and animals.
ETIOLOGY
Rabies is caused by a filterable virus of the rhabdovirus
group (Melnick and McCombs 1966). The external di-
mensions of the rabies particle are 750 × 180 nm. Its
surface has a honeycomb appearance with projections
6–7 nm long (Witkor 1971). The particle is cylindrical,
with one round or conical end and one planar or concave
end (Murphy 1975). The virus is classified as a ribonu-
cleic acid (RNA)–containing virus. The RNA is a single-
stranded molecule with a molecular weight of 4.6 × 106
daltons per nucleocapsid (Witkor 1971). Sokol et al.
(1969) have separated the different components of ra-
Some of the material presented in this chapter is based on experience en-
countered in an outbreak of rabies in a herd of Missouri swine. The author
acknowledges contributions of his colleagues Dr. L. D. Kintner, Dr. S. L. Nel-
son, and Dr. B. L. Moseley of the University of Missouri; the Veterinary Pub-
lic Health Unit, Jefferson City, Mo.; and Veterinary Services Laboratories,
APHIS, USDA, Ames, Iowa.
bies virions from the purified virus. By treatment with
sodium deoxycholate and combined velocity and equi-
librium gradient centrifugation, the viral coat and the
nucleocapsid are separated. The latter contains all the
RNA and is made up of 96% protein and 4% RNA. The
five structural proteins and conclusions regarding their
function and arrangement within the virion have been
reviewed (Cox 1982).
EPIDEMIOLOGY
Traditionally, the disease has been said to exist in two
epidemiologic forms: the urban type, spread mainly by
dogs; and the wildlife type, seen principally in skunks,
foxes, wolves, jackals, coyotes, weasels, and bats (Bisseru
1972). A marked shift to wildlife rabies occurred in the
United States during the 1960s and 1970s. By 1964, 75%
of the rabies in the United States was in wildlife, and less
than 10% was reported from dogs (U.S. Dept. HEW
1964). A more recent report confirms this trend, with
88.4% of recorded cases in wildlife and only 2.7% in dogs
(U.S. Dept. HHS 1989).
With this increased percentage of rabies occurring in
wild animals, farm animals have been placed at greater
risk of exposure. The true incidence of rabies in swine is
difficult to assess. In the United States 854 cases were re-
ported over the 17-year period from 1938 to 1955
(Schoening 1956), representing 6.6% of cases reported in
all farm animals. Cases of rabies on record in swine in
the United States dropped to 3–8 annually, and in Cana-
da and Mexico to 10–21 annually (U.S. Dept. HEW
1969–83). This trend has continued through 1988 (U.S.
Dept. HHS 1989). In a review of the global rabies situa-
tion, however, specific reference has been made to con-
firmed cases of porcine rabies in 25 different countries
on five continents (Bisseru 1972). Yates et al. (1983) de-
scribe rabies outbreaks in 15 swine herds in western
Canada over a 14-year period.
Epidemiologic studies are now aided by monoclonal
antibody analysis of various strains of rabies virus from
different animals and geographic regions (Smith et al.
1984). Major antigenic groups of rabies virus in Canada
have been determined by antinucleocapsid monoclonal
antibodies, allowing classification of antigenic groups by
geographic area (Webster et al. 1986).
247
248 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
CLINICAL SIGNS
The typical course of the disease is one of sudden onset,
loss of coordination, dullness, and later prostration.
Death will likely ensue within 72 hours from the appear-
ance of clinical signs (Merrimann 1966; Morehouse et al.
1968). Peculiar twitching and preoccupation with the
nose, similar to that observed in swine with rings recent-
ly placed in their noses, has been noted. This may be fol-
lowed by prostration, rapid chewing movements, exces-
sive salivation, and generalized clonic muscular spasms,
which become less marked as the disease progresses un-
til muscular activity is manifested only by fine tremors.
Affected swine may be unable to squeal, and elevation of
body temperature may be absent (Morehouse et al.
1968).
Oldenberger (1963) reported a paralytic form of ra-
bies in swine in the USSR, with a duration of illness of
5–6 days. Gigstad (1971) reported clinical signs of rabies
in experimentally infected swine that included an inter-
mittent weakness in the rear legs, weakness in the shoul-
ders, incoordination, posterior paralysis, prostration,
paddling, and death. Merrimann (1966) documented
one case of the furious form of rabies in a pig, but this
pattern of behavior was observed in only 1 of 17 affected
pigs. Hazlett and Keller (1986) reported an outbreak of
porcine rabies in a closed feeder barn where clinical signs
were not observed.
Although most reports suggest that swine die within
72–96 hours following development of clinical signs,
there is indication of a wide variation in the incubation
period. Merrimann’s report (1966) suggests an incuba-
tion period of approximately 70 days for one group of
swine. Morehouse et al. (1968) reported that since there
was a progression of rabies cases in a drove of swine over
a 2-month period, the incubation period would have
been extremely variable if the disease resulted from a si-
multaneous exposure of a number of swine. Reichel and
Möckelmann (1963) reported an outbreak of rabies in
swine where the exposure time from rabid foxes was doc-
umented. Incubation periods of 9, 56, and 123 days were
recorded for the affected swine. Gigstad (1971) reported
incubation periods in experimentally infected swine that
varied from 12 days when exposed intracerebrally to 98
days when exposed intramuscularly.
PATHOGENESIS
The spread of rabies virus along peripheral or cranial
nerves appears to be the major avenue of access to the
central nervous system (CNS) following intramuscular
or subcutaneous exposure to the virus. Dean et al. (1963)
published convincing evidence that fixed (attenuated)
and street (virulent) rabies viruses are ordinarily trans-
mitted from the site of inoculation to the CNS by way of
the peripheral nerves. Johnson (1971) emphasized that
integrity of the nerve is necessary for CNS invasion, thus
confirming the case for a neural pathway for the virus.
The rabies virus may enter the host via many routes,
but the importance of oral and respiratory transmission
is uncertain (Johnson 1971). Kucera et al. (1985) report
three routes of entry from the eye to the brain: the ocu-
lomotor parasympathetic nerve, the preopticoretinal
pathway, and the ophthalmic nerve. The mechanism by
which the virus gains access to the CNS has been studied
by a number of workers. A progression of rabies infec-
tions in hamsters has been demonstrated, starting in
striated muscle cells near the site of inoculation, with in-
fection of myocytes and shedding of the virus into extra-
cellular spaces. A working hypothesis was proposed that
the route of passive centripetal passage of the rabies
virus to the CNS is via peripheral nerve axoplasm from
invasion at unmyelinated distal end organs to progeny
release at synaptic junctions in the spinal cord (Murphy
et al. 1973a). An infection of myocytes at the site of in-
oculation with the rabies virus in skunks has been re-
ported (Charlton and Casey 1979). Immunofluorescence
in muscle fibers remote from the inoculation site oc-
curred only after infection of lumbar cord and dorsal
root ganglia, suggesting infection as a result of centrifu-
gal neural migration of virus.
In the development of infection within the CNS, neu-
rons of spinal cord, brain stem, hippocampus, septal nu-
clei, and limbic cortex appear especially vulnerable.
Meningeal, ependymal, vascular endothelial, and glial
cells show no antigen (Johnson 1971). However, Jackson
and Reimer (1989) have reported infection of ependymal
cells lining lateral ventricles in mice following intracere-
bral exposure and postulate that virus entry occurs at
least in part by a cerebrospinal fluid pathway. Murphy et
al. (1973b) proposed a mechanism of viral spread to in-
clude ascending infection within the CNS and centrifugal
peripheral neural spread, with passive intraneuronal
movement of subviral entities interspersed with active
viral replication at cell surfaces, such that progeny virus
might invade contiguous neurons or move within inter-
cellular spaces to involve neural elements elsewhere.
Charlton and Casey (1979) suggested direct transneu-
ronal transfer of virus from perikarya and dendrites to
adjacent axon terminals as a mechanism in dissemina-
tion of rabies in the CNS of striped skunks.
Whether or not factors such as the mechanism of
virus spread via the nerves, significance of oral and res-
piratory transmission, selective cellular vulnerability,
and host cell–virus relationships that might allow for la-
tent infection can result in a variability in species suscep-
tibility remains an unanswered question in the patho-
genesis of rabies. This is particularly true in the case of
swine, where the lack of detailed case reports or experi-
mental studies and the relatively low reported number of
cases in this species compound the problem.
 CHAPTER 20 RABIES Morehouse 249

LESIONS cord was characterized by perivascular cuffing that in-

volved a high percentage of the larger vessels in both
Necropsy examination of rabid swine will likely fail to re- gray and white matter. Figure 20.2 shows marked
veal gross changes (Merrimann 1966; Morehouse et al. perivascular cuffing of a vessel in the medulla.
1968; Hazlett and Koller 1986). Histopathologic changes
may be observed in the brain and spinal cord but may be
variable. This is true for many species, where changes
may range from indiscernible, except for early necrosis of
neurons and specific cytoplasmic inclusions in affected
nerve cells, to a diffuse encephalitis (Smith et al. 1972).
The variability in neuropathologic response in other
species infected with rabies seems true also for swine. In
one text the authors indicate that neuronal degeneration
may be very slight in pigs (Jubb et al. 1985). Merrimann
(1966) reported the presence of Negri bodies in only one
of three brains from rabid swine, although they were
positive to the fluorescent rabies antibody test and had
shown clinical signs of the disease. Gigstad (1971) re-
ported an extensive nonsuppurative encephalitis in four
pigs that had died following experimental inoculation
with rabies virus. One animal had been inoculated intra-
muscularly and died 42 days postinoculation, while the
other three had been inoculated intracerebrally and died
12–18 days postinoculation. Negri bodies were not ob-
served. Hazlett and Koller (1986) reported only one Ne-
gri body in 10 sections of brain from a rabid pig.
Neuropathologic changes were observed in pigs dy-
ing from rabies, varying from a mild reaction in the brain
consisting primarily of a mild vasculitis and focal gliosis
to marked changes throughout the brain and spinal
cord. Negri bodies were not observed (Morehouse et al.
1968). The changes included a diffuse meningitis of the
brain and spinal cord, which was most severe in the lep-
tomeninges covering the folia of the cerebellum (Fig. 20.1. Diffuse leptomeningitis of the cerebellar folia
20.1). The inflammatory response in the brain and spinal (H&E; ×135).

20.2. Marked perivascular cuffing and neuronal degeneration in the medulla (H&E; ×100).
250 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

Neuronal destruction observed throughout the brain

and spinal cord was manifested by degeneration of neu-
rons in the Purkinje cell layer of the cerebellum. Phago-
cytosis of pyramidal cells was apparent (Fig. 20.3). The
inflammatory change associated with the vessels in Fig-
ure 20.3 was primarily intramural in nature, with the Vir-
chow-Robin space appearing clear. Degenerating neu-
rons in other areas of the hippocampus were shrunken
and stained heavily with eosin (Fig. 20.4). There was
necrosis of neurons in the pons with accompanying
satellitosis (Fig. 20.5) and neuronophagia (Fig. 20.6).
Large motor neurons in the ventral horn of some seg-
ments of the spinal cord were undergoing degeneration
(Fig. 20.7).

DIAGNOSIS

Animal Inoculation Test

The intracerebral inoculation of laboratory animals with
brain material from animals suspected of being rabid is
the oldest method of diagnosing rabies. Symptoms in
weanling mice may be observed within 6–14 days, and
the diagnosis of rabies is confirmed by the fluorescent
antibody (FA) test (or the demonstration of Negri bodies
in the mouse brains). For a negative result it is common
to wait at least 21 days, although some workers have re-
ported at least a 27-day incubation period in mice receiv- 20.3. Neuronal degeneration in the pyramidal
ing brain material from rabid swine (Morehouse et al. cell layer of the hippocampus (H&E; ×100).

20.4. Necrosis of neurons in the hippocampus (H&E; ×100).

 CHAPTER 20 RABIES Morehouse 251

20.5. Satellitosis of a neuron in the pons 20.7. Degenerating neuron in the ventral horn of the
(H&E; ×100). spinal cord (H&E).

20.6. Phagocytosis of a dying neuron in the pons (H&E; ×400).

252 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
1968; R. Dillman, pers. comm.—1973; M. Keiffer, pers.
comm.—1973). Additional confirmation includes a neu-
tralization test in mice, using specific rabies immune
serum.
Histopathologic Examination
Negri (1903) established the first definitive pathologic
diagnosis of rabies when he described the eosinophilic
cytoplasmic inclusions in nerve cells and dendrites. A
number of stains are available for demonstrating these
inclusion bodies (Gradwohl 1943). Bosch (1966) de-
scribed the use of acidified phenolphloxine for staining
Negri bodies in paraffin sections. Negri bodies, however,
are not found in all rabid animals. Runnells et al. (1965)
stated that Negri bodies are not present in about 25% of
rabid animals, and in many of the remaining 75% they
are so few in number as to be overlooked. Lennette et al.
(1965) reported that in a series of 365 rabies-positive
specimens, only 239 (65.8%) were detected by the pres-
ence of Negri bodies; the variability of their presence in
swine rabies has been reported by Merrimann (1966),
Morehouse et al. (1968), Gigstad (1971), Dhillon and
Dhingra (1973), and Hazlett and Koller (1986).
Fluorescent Antibody Test
Goldwasser and Kissling (1958) were the first to apply
the FA technique to the diagnosis of rabies. It has proved
to be a rapid and accurate test. McQueen et al. (1960)
demonstrated its dependability for diagnosing rabies in
brain tissue that was Negri-body negative. Lennette and
Emmons (1971) have emphasized that fluorescent anti-
gen will be present and detectable in the brain at any
stage of the disease in which the animal could possibly
have transmitted rabies. Blenden (1978) described FA
identification of rabies virus antigen in the skin of mice
before clinical signs developed; he later reported its val-
ue for early detection of rabies virus antigen (Blenden et
al. 1983). Several authors, however, have found swine
specimens negative on FA in cases later diagnosed posi-
tive by mouse inoculation (Beauregard et al. 1965; More-
house et al. 1968; Dhillon and Dhingra 1973).
Other Methods
Bourgon and Charlton (1987) demonstrated rabies anti-
gen in paraffin-embedded tissue by the peroxidase-an-
tiperoxidase method and found it superior to the FA
technique for detection of antigen and preservation of
morphologic detail. Perrin and Sureau (1987) described
an experimental kit for rapid rabies enzyme immunodi-
agnosis (RREID). They found it to be specific, conve-
nient, and a useful tool for epidemiologic studies and for
laboratories without ultraviolet microscopes; it was not
as sensitive as the FA technique.
Comment
Gigstad (1971) concluded from experimental studies
that swine may have a relatively high natural resistance
to the rabies virus and this might be responsible for the
unpredictable behavior of the disease when it occurs as a
herd problem. Blenden (pers. comm.—1979) inoculated
two weanling pigs by the intramuscular route with street
rabies virus. No clinical signs were observed, although
high titers of antibody were found in cerebrospinal fluid
and blood. Whether natural resistance of swine to rabies
results in aberrant clinical signs, thus confusing the diag-
nosis, remains open to question.
These observations do emphasize problems that may
be encountered in the clinical and laboratory diagnosis
of rabies in swine, and they point to the value of careful
neuropathologic examination and other laboratory pro-
cedures in differentiating rabies from other encephali-
tides of swine described in this volume.
TREATMENT AND PREVENTION
The extremely limited literature and experience with ra-
bies in swine give no indication of effective treatment for
this disease, nor is there any indication of vaccine devel-
opment for this species. However, producers and veteri-
narians should be aware of the unpredictable behavior of
swine affected with this disease when it occurs as a herd
problem. Preventive measures consist principally of lim-
iting exposure, particularly where rabies is endemic in
wildlife populations adjacent to areas where swine are
maintained.
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 Rotavirus and Reovirus
21 P. S. Paul and G. W. Stevenson
PORCINE ROTAVIRUS
Rotaviruses are important enteric pathogens in neonates
of many species, including pigs. The enteropathogenici-
ty of porcine rotaviruses has been demonstrated by their
ability to produce severe gastroenteritis and villous atro-
phy under experimental conditions in gnotobiotic and
colostrum-deprived pigs in the absence of other
pathogens. They are also frequently detected in pigs with
diarrhea. Subclinical infections with porcine rotaviruses
are equally common and suggest that other factors such
as host, agent, and environment may be important in the
pathogenesis of disease by porcine rotaviruses.
Rotaviruses were first detected in calves (Mebus et al.
1969) and were subsequently identified in humans and
other animals (Estes et al. 1983). Serologic evidence for
rotavirus infection in pigs was first shown using bovine
rotavirus as an antigen (Woode and Bridger 1975). Sub-
sequently, porcine rotaviruses were detected by other re-
searchers (Rodger et al. 1975; Lecce et al. 1976; McNulty
et al. 1976; Woode et al. 1976; Chasey and Lucas 1977;
Bohl et al. 1978; Tzipori and Williams 1978). Porcine ro-
taviruses have now been detected in many of the swine-
producing countries (Lecce et al. 1976; McNulty et al.
1976; Woode et al. 1976; Bohl et al. 1978; Lecce and King
1978; Bridger 1980; Corthier et al. 1980; Saif et al. 1980;
Bohl et al. 1982; Askaa et al. 1983; Nilsson et al. 1984;
Utrera et al. 1984; Theil et al. 1985; Dea et al. 1986; Fer-
rari et al. 1986; Fu and Hampson 1987; Kim et al. 1987;
Nagesha and Holmes 1988; Paul et al. 1988a; Atii et al.
1990; Bellinzoni et al. 1990; Magar and Larochelle 1992;
Akopian et al. 1992; He et al. 1992; Gouvea et al. 1994;
Ciarlet and Liprandi 1994; Ciarlet et al. 1995, 1996;
Pongsuwanna et al. 1996). Rotaviruses, regardless of
species of origin, were initially shown to share a com-
mon group antigen (Woode et al. 1976; Thouless et al.
1977; Estes et al. 1983). It was later shown that ro-
taviruses are quite diverse serologically (Bridger 1980;
Saif et al. 1980; Askaa and Bloch 1981; McNulty et al.
1981). To date seven distinct serogroups of rotaviruses
have been described (Pedley et al. 1986; Bridger 1987).
Group A rotaviruses appear to be the most common and
best studied. Therefore, the majority of the information
presented here pertains to porcine group A rotaviruses.
In recent years, however, information on other groups of
rotaviruses has begun to emerge and will be included
here where appropriate.
ETIOLOGY
Virus Morphology
Rotavirus particles are nonenveloped and have a distinc-
tive wheel-like appearance when examined by electron
microscopy. Rota is “wheel” in Latin—hence, they were
classified as rotavirus. Complete rotavirus particles have
a well-defined outer rim, a feature that distinguishes
them from the other two genus members, reovirus and
orbivirus, of family Reoviridae. Three forms of virus par-
ticles are visible by negative-staining electron mi-
croscopy. The double-capsid virus particles have an out-
er capsid layer, an inner capsid layer, and an icosahedral
core and measure approximately 75 nm in diameter (Fig.
21.1A). The single-capsid virus particles are approxi-
mately 60 nm in diameter and lack the outer capsid layer
(Fig. 21.1B). Cores are approximately 52 nm in diameter
and contain viral genome and RNA-dependent RNA
polymerase. Only double-capsid virus particles are infec-
tious.
Classification
Rotaviruses are classified in the family Reoviridae under
the genus Rotavirus. Early studies demonstrated that all
rotaviruses, regardless of species of origin are antigeni-
cally related (Woode et al. 1976; Thouless et al. 1977;
Estes et al. 1983). The common group antigen is con-
tained in viral structural protein VP6 on the inner capsid
(Estes and Cohen 1989). It became clear, however, that
rotaviruses are quite diverse antigenically, for rotaviruses
that resembled classical rotaviruses but lacked common
group antigen were detected (Bridger 1980; Saif et al.
1980; Askaa and Bloch 1981; McNulty et al. 1981;
Bridger et al. 1982). Such rotaviruses have been termed
non–group A rotaviruses, novel rotaviruses, rotavirus-
like viruses, pararotaviruses, atypical rotaviruses, and
antigenically distinct rotaviruses (Bridger 1980; Saif et
al. 1980; Bridger et al. 1982; Pedley et al. 1983; Snodgrass
et al. 1984; Theil et al. 1985; Pedley et al. 1986; Bridger
1987). Recent studies show that some epitopes on VP6
are conserved and are shared among different rotavirus
groups (Tsunemitsu et al. 1992c).
Serogroups with common group antigen on the inner
capsid were identified by serologic tests such as im-
255
256 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

21.1. Rotavirus particles in feces viewed by negative-staining electron microscopy (×130,000).

(A) Double-shelled virus particles with intact outer capsids have characteristic smooth outlines.
(B) Single-shelled particles lack outer capsids and have spiked outlines.

munofluorescence, enzyme-linked immunosorbent as-
say (ELISA), or immune electron microscopy (Bridger
1980; Saif et al. 1980; Pedley et al. 1983; Theil and Saif
1985; Chasey et al. 1986; Pedley et al. 1986). Seven anti-
genically distinct groups (A–G) of rotaviruses have now
been reported, four (A, B, C, and E) of which affect pigs
(Bridger 1980; Saif et al. 1980; Bohl et al. 1982; Pedley et
al. 1983; Chasey et al. 1986; Pedley et al. 1986). Group A
rotaviruses represent the earliest recognized rotaviruses,
which appear to be most commonly associated with gas-
troenteritis and are best characterized. Group B ro-
taviruses, also referred to as atypical rotaviruses or ro-
tavirus-like viruses, have been detected in pigs, cattle,
and humans (Bridger 1980; Bridger et al. 1982; Pedley et
al. 1983; Theil et al. 1985). Group C rotaviruses, also des-
ignated as pararotaviruses, have been detected in pigs,
cattle, and humans (Saif et al. 1980; Bohl et al. 1982;
Pedley et al. 1983; Espejo et al. 1984; Bridger et al. 1986;
Tsunemitsu et al. 1991). Group E rotaviruses have been
detected only in pigs in the United Kingdom (Chasey et
al. 1986), whereas rotaviruses of groups D, F, and G have
been detected in chickens and turkeys (Pedley et al. 1986;
Bridger 1987).
Genomes of rotaviruses, when subjected to elec-
trophoresis in a polyacrylamide gel, yield a characteristic
electrophoretic migration commonly referred to as an
electropherotype. Rotaviruses of serogroups identified
thus far have unique electropherotypes, which have been
used to differentiate rotaviruses into groups (Pedley et al.
1986). RNA segments cluster in four regions, I–IV (Fig.
21.2). Group A rotaviruses have 4:2:3:2 segments in re-
gions I, II, III, and IV respectively. This pattern is 4:2:2:3
for group B rotaviruses, and 4:3:2:2 for group C ro-
taviruses. In group E rotaviruses the pattern is similar to
that seen with group B rotaviruses except segments 7–11
migrate equidistant from each other (Pedley et al. 1986).
There appears to be a correlation between electro-
pherotype and serogroup; however, it is emphasized that
serologic and nucleic acid–based assays are more reli-
able.
Rotaviruses within group A have been further divided
into subgroups and serotypes. Two subgroups, SI and SII,
within group A rotaviruses have been reported (Green-
berg et al. 1983a). Evidence indicates that viruses of ad-
ditional subgroups may be prevalent in nature (Svensson
et al. 1988). Serotypes within a serogroup are defined by
plaque reduction or fluorescent focus reduction assays
using hyperimmune serum (Bohl et al. 1984; Hoshino et
al. 1984; Paul et al. 1988a). A minimum of a 20-fold dif-
ference in the neutralization titer between the homolo-
gous and the heterologous rotavirus is required for a
virus to be considered a different serotype.
Serotyping is complex and sometimes gives ambigu-
ous results, due to involvement of two surface proteins,
VP4 and VP7, which induce neutralizing antibodies
(Greenberg et al. 1983b). The genes coding for these sur-
 CHAPTER 21 ROTAVIRUS AND REOVIRUS Paul, Stevenson 257

face proteins segregate independently (Hoshino et al.

21.2. Electrophoretic patterns (electropherotypes) of
A/OSU, B/IA1146, and C/Cowden strains of groups
A, B, and C rotaviruses in polyacrylamide gel stained
with silver. Each rotavirus group has a distinct
electropherotype. Double-stranded RNA segments cluster
in four regions, I–IV. The numbers of bands in the four
regions are 4:2:3:2, 4:2:2:3, and 4:3:2:2 for rotavirus
groups A, B, and C respectively.
1985). A majority of hyperimmune antisera prepared
against the purified virions contain antibodies to VP7;
therefore, classic neutralization assays using hyperim-
mune antisera recognize serotypes based on VP7. A new
classification scheme has been proposed that takes into
account typing of both VP4 and VP7 surface proteins
(Estes and Cohen 1989). VP7 types are designated as G
types (for glycoprotein), and VP4 types are designated as
P types (for protease-sensitive protein).
A virus is designated by group/strain/subgroup/G
type/P type. At least 10 G (VP7) serotypes of porcine ro-
taviruses have been detected (Bohl et al. 1984; Hoshino
et al. 1984; Nagesha and Holmes 1988; Paul et al. 1988a;
Ruiz et al. 1988; Paul et al. 1990a, b, 1991; Gouvea et al.
1994; Bellinzoni et al. 1990; Ciarlet and Liprandi 1994;
Rosen et al. 1994): G1, G2, G3, G4, G5, G6, G8, G9, G10,
and G11. Information on G type and other serotype des-
ignations of selected rotavirus strains is presented in
Table 21.1. At least four P (VP4) types among porcine
group A rotaviruses have been detected (Estes 1996);
however, information on P type on most porcine ro-
tavirus strains is not available at this time. Monoclonal
antibodies (Kang et al. 1989; Nagesha et al. 1989; Kang et
al. 1993), nucleic acid probes (Johnson et al. 1990; Paul
et al. 1990a, c; Rosen et al. 1992), and polymerase chain
reaction (PCR) methods (Winiarzyk and Paul 1997) have
become available, which are allowing typing of porcine
rotaviruses by G and P types in selected laboratories. Re-
cent studies suggest that serotypic diversity also exists
among group C rotaviruses, as evidence has been pre-

Table 21.1. Serogroup and serotype designations of selected porcine rotavirus isolates
Serotypea
Serogroup Strain(s) G (VP7) P (VP4)b Reference
A C60, C80, and C95 1 ? Bellinzoni et al. 1990; Ciarlet and Liprandi 1994
C134 2 Bellinzoni et al. 1990
CRW-8 3 7 Nagesha and Holmes 1988
ISU-65 3 ? Paul et al. 1988a, 1991
Gottfried 4 Bohl et al. 1984; Hoshino et al. 1984
SB-IAc 4 7 Hoshino et al. 1984, 1988
OSU 5 7 Bohl et al. 1984; Hoshino et al. 1984
6 6 Gouvea et al. 1994
8 Gouvea et al. 1994
ISU-64 9 7 Paul et al. 1988a, 1991
10 Gouvea et al. 1994
YM 11 Ruiz et al. 1988
YM-like 11 Rosen et al. 1994
B Ohio Theil et al. 1985
NIAD-1 Pedley et al. 1983
IA1146 Paul, unpublished data—1990; Lyoo et al. 1993
C Cowden Bohl et al. 1982
IA850 Proescholdt 1991
E DC-9 Pedley et al. 1986
a
Based on classification scheme proposed by Estes and Cohen (1989).
b
Although four P (VP4) types of porcine rotavirus have been identified by nucleic methods (Estes 1966), only two P (VP4) types
(listed here) have been conclusively identified by serologic methods.
c
Natural reassortant.
258 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
sented for the existence of at least two serotypes of
group C porcine rotaviruses (Tsunemitsu et al. 1992b).
Physicochemical and Biological Properties
Rotaviruses are extremely stable and are resistant to en-
vironmental conditions such as temperature, pH, chemi-
cals, and disinfectants. Rotaviruses are stable at pH 3–9.
In fecal preparations rotaviruses are resistant to temper-
atures of 60˚C for 30 minutes and are resistant to tem-
peratures of 18–20˚C for at least 7–9 months (Woode
1986). Cell culture–adapted rotaviruses vary in suscepti-
bility to heat. Strains A/OSU and C/Cowden were inacti-
vated by heat at 56˚C after 30 minutes, whereas the
A/Gottfried strain was resistant to heat at 56˚C (Terrett
et al. 1987). In the presence of organic material ro-
taviruses are inactivated by 2% acid glutaraldehyde, 70%
ethanol, 3.7% formaldehyde, 10% povidone-iodine, 67%
chloramine T, and 0.5% triclosan (Sattar et al. 1983;
Woode 1986). Rotaviruses are resistant to treatments by
ether, chloroform, and genetron. Calcium chloride, but
not magnesium chloride, stabilizes infectivity. Calcium
ion chelators such as ethylenediaminetetraacetic acid
(EDTA) at 5 mM concentration remove the outer layer
and destroy infectivity.
The rotavirus genome is double-stranded RNA and
has 11 segments. The total genomic size of rotavirus is
estimated at approximately 18,522 base pairs by nucleic
acid sequence and electron microscopy (Rixon et al.
1984; Estes and Cohen 1989). The inner capsid is com-
posed of two major structural proteins: VP2 with a mol-
ecular mass (Mr) of 94 kDa and VP6 with a Mr of 41 kDa
(Estes and Cohen 1989). The VP6 protein contains the
major group and subgroup antigens. Minor subgroup-
specific epitopes are also located on VP2 (Greenberg et
al. 1983a; Svensson et al. 1990). Other capsid proteins in-
clude VP1 with a Mr of 125 kDa and VP3 with a Mr of 88
kDa. The outer capsid has two major surface proteins—
VP4 with a Mr of 88 kDa and VP7 with a Mr of 37 kDa—
and several other proteins, including VP5 with a Mr of
60 kDa and VP8 with a Mr of 28 kDa (Estes and Cohen
1989). The VP4 protein is nonglycosylated, contains
hemagglutinin, and is important for infectivity and viru-
lence (Greenberg et al. 1983a; Kalica et al. 1983; Offit et
al. 1986a). Proteolytic cleavage of VP4 into VP5 and VP8
is important for viral infectivity. It is coded in gene seg-
ment 4. The VP7 is the second most abundant viral
structural protein, is glycosylated, and is coded in either
gene segment 8 or 9 depending upon the strain of virus.
Proteins analogous to VP6 and VP7 have been described
for group C rotaviruses (Jiang et al. 1990). Both VP4 and
VP7 induce neutralizing antibodies (Greenberg et al.
1983b; Offit and Blavat 1986), are important in induc-
tion of immunity (Offit et al. 1986b), and segregate in-
dependently (Hoshino et al. 1985). Thus, theoretically,
reassortant rotavirus strains containing any combina-
tion of P and G types are possible. One such strain, SB-
IA, with a G4 and P7 type, has been described (Hoshino
et al. 1984; Midthun et al. 1987).
The buoyant density of the double-shelled, single-
shelled, and core particles in cesium chloride is 1.36
g/mL, 1.38 g/mL, and 1.44 g/mL respectively (Bridger
and Woode 1976; Bican et al. 1982). Some strains of
porcine rotavirus possess a hemagglutinin (Eiguchi et al.
1987; P. S. Paul, unpublished data—1990) and aggluti-
nate human type O, guinea pig, and rat erythrocytes.
The cellular receptor for group A rotaviruses has been
identified as monosialoganglioside or a family of mono-
sialogangliosides with sialic acid as an important epitope
for virus binding (Rolsma et al. 1994).
Cultivation
Rotaviruses have been difficult to cultivate in cell cul-
tures. Porcine rotaviruses were first adapted to grow in
porcine primary kidney cell cultures by pretreatment of
virions with trypsin or pancreatin (Theil et al. 1977).
Viruses were later successfully propagated in African
monkey kidney cell line MA-104 (Bohl et al. 1984). Use of
roller cultures of MA-104 and addition of proteolytic en-
zymes, trypsin or pancreatin, were essential in isolation
of rotaviruses. Trypsin enhances the growth of bovine
rotaviruses as much as 1000-fold (Almeida and Hall
1978). Many strains of group A porcine rotaviruses have
now been cultivated in roller cultures of the MA-104 cell
line by pretreatment of virions with trypsin (10 µg/mL
for 30 minutes) or pancreatin before infection. Alterna-
tively, trypsin or pancreatin (0.5–1.0 µg/mL) is added to
serum-free culture medium after virus adsorption. Cell
culture–adapted rotavirus strains produce a cytopathic
effect characterized by rounding of cells followed by re-
moval of cells from the surface. Viral antigen can be
demonstrated in cytoplasm of virus-infected cells by im-
munofluorescence (Fig. 21.3) or immunochemical meth-
ods. Rotaviruses form plaques under agarose in the pres-
ence of neutral red.
One strain of group C rotaviruses has been propagat-
ed in porcine primary kidney cell cultures (Terrett and
Saif 1987). Roller cultures and incorporation of high
concentrations of pancreatin in culture medium were es-
sential for virus propagation (Terrett and Saif 1987). Af-
ter nine passages in primary kidney cells, the group C ro-
tavirus was adapted to grow in the MA-104 cell line (Saif
et al. 1988). A porcine intestinal cell line has been suc-
cessfully used to propagate C/Cowden and an Iowa
strain, C/IA850, of group C rotaviruses (Proescholdt
1991). Roller cultures and addition of high concentra-
tions of pancreatin were required for virus growth.
Groups B and E rotaviruses and some strains of group A
rotaviruses still cannot be serially propagated in cell cul-
tures. Group B porcine rotaviruses produce syncytia in
monolayers of MA-104 following centrifugation of virus
(Theil and Saif 1985). Viral antigen is present in inocu-
lated cells without evidence for productive infection.
 CHAPTER 21 ROTAVIRUS AND REOVIRUS
21.3. Immunofluorescent staining of MA-104 cell
line infected with group A porcine rotavirus showing
cytoplasmic accumulation of rotaviral antigens (× 400).
Such cultures with cytoplasmic viral antigen have proven
useful in a cell culture immunofluorescent assay (CCIF)
for the detection of antibodies (Theil and Saif 1985).
EPIDEMIOLOGY
Porcine rotavirus is ubiquitous in swine herds wherever
swine are raised. Serologic surveys show that a high
percentage (77–100%) of the adult swine are seropositive
for porcine groups A, B, and C rotaviruses (Bridger and
Brown 1985; Terrett et al. 1987). Antibodies to group E
rotaviruses are also common in the United Kingdom
(Chasey et al. 1986). It is difficult to raise pigs free of ro-
taviruses under normal husbandry conditions.
There appears to be an age-related variation in sero-
logic prevalence, for antibodies to group B and C were
lowest in pigs 3–8 weeks of age, whereas all pigs of this
age had antibodies to the group A rotaviruses (Bridger
and Brown 1985). Similarly, antibodies to group C ro-
taviruses were detected in 100% of adults, 59% of wean-
lings, and 86% of nursing pigs tested from Ohio (Terrett
et al. 1987).
Infection with porcine rotaviruses is enzootic in
swine herds. The virus spreads among pigs via the fecal-
oral route. Following natural infection with a rotavirus,
pigs develop circulating and secretory antibodies and are
immune to infection with homologous rotaviruses. Anti-
bodies are transferred to newborns through colostrum
and milk; the duration of persistence of these passively
acquired antibodies varies with the initial antibody level
Paul, Stevenson 259
acquired and appears to last 3–5 weeks (Tzipori et al.
1980a). Piglets born to gilts are more susceptible to ro-
tavirus infection than those born to sows (Askaa et al.
1983; Svensmark et al. 1989a). Pigs usually become in-
fected between 7 and 41 days of age and are infrequent-
ly infected at less than 7 days of age (Bohl et al. 1978). In
one cohort study (Fu and Hampson 1987), pigs became
infected at an average age of 19 days, with a range of
13–39 days. The virus is shed in feces and this is the ma-
jor source of infection; the greatest amount of the virus
is shed during the acute stages of infection. The duration
of group A porcine rotavirus shedding in feces is about
7.4 days, with a range of 1–14 days. Virus shedding is af-
fected by the level of passive immunity and serogroup of
porcine rotavirus. In group B rotavirus infections, lower
amounts of rotavirus are shed for a shorter duration
(Bridger 1980; Theil et al. 1985).
Recurrent infections with porcine rotaviruses with in-
termittent virus shedding have been detected (Debouck
and Pensaert 1983). These possibly represent infections
of different serogroups or serotypes. Adult animals do
not generally shed rotavirus (Debouck and Pensaert
1983; Fu and Hampson 1987); however, occasionally
they may shed rotavirus near farrowing (Benfield et al.
1982) and may serve as sources of infection for suscepti-
ble pigs.
Limited data are available on the relative prevalence
of rotaviruses, especially of different serogroups and
serotypes. Rotavirus infections account for about 14% of
diarrheas in pigs (Janke et al. 1989, 1990; Paul et al.
1990c). In one study (Atii et al. 1990) porcine rotavirus
was detected in 43 of 96 (44.8%) samples from diarrheic
pigs but not in any of 41 samples from nondiarrheic
pigs. There was also a higher infection rate (30.2%) in
piglets aged 1–3 weeks compared to pigs aged 4–6 weeks
(14.6%) (Atti et al. 1990). Group A rotavirus is by far the
predominant porcine rotavirus detected in diarrheic pigs
(Janke et al. 1989; Paul et al. 1990c). Janke et al. (1989)
examined 90 rotavirus-positive samples and found that
60 contained group A, 9 contained group B, 11 contained
group C, and 10 contained a mixture of these groups. In
nursing pigs the majority were group A; whereas in
weaned pigs 38% were group A, 19% were group B, and
24% were group C. In another study, 89% of the ro-
tavirus-positive samples were positive for group A ro-
tavirus (Paul et al. 1990c). In a study in Thailand, 26 of
the 557 fecal samples were positive for rotavirus by poly-
acrylamide gel electrophoresis of RNA: 23 were group A,
1 was group B, and 2 were group C (Pongsuwanna et al.
1996). Group A rotaviruses with P types 6 and 7 are most
abundant (Zaberezhny et al. 1994). In one study, out of
26 rotavirus-positive diarrheic field samples, 17 samples
(65.4%) had P type 7; 5 (15.4%) had P type 6; and 4
(15.4%) were negative for both P6 and P7 (Zaberezhny et
al. 1994). Rotaviruses with G types 1, 2, 3, 4, 5, 6, 8, 9, 10,
and 11 have been detected, with G types 3, 4, 5, 8, and 11
260 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
being predominant (Rosen et al. 1994; Gouvea et al.
1994; Winiarzyk and Paul 1997). Additional studies are
needed to fully determine the relative prevalence of ro-
tavirus serogroups and serotypes.
Contaminated environment plays an important role
in maintenance of rotaviruses in swine herds. Porcine ro-
taviruses are stable at extreme environmental conditions
and are resistant to commonly used disinfectants.
Porcine rotaviruses can be detected in dry feces, dust,
and sewage of farrowing and weaner barns (Fu et al.
1989). Porcine rotaviruses have survived in swine barns
kept empty for 3 months (Fu et al. 1989).
CLINICAL SIGNS
Numerous rotavirus inoculation studies in nonimmune
gnotobiotic or colostrum-deprived pigs fed a liquid diet
have resulted in consistent production of clinical dis-
ease. Disease is most severe in pigs inoculated when 1–5
days of age (Woode et al. 1976; Pearson and McNulty
1977; Tzipori and Williams 1978; Debouck and Pensaert
1979; McAdaragh et al. 1980; Narita et al. 1982a). After
an incubation period of 12–24 hours, pigs become
anorexic and listless and occasionally vomit. Severe, pro-
fuse diarrhea begins 1–4 hours later. Feces are watery
and yellow to white and contain variable amounts of
flocculent material. Diarrhea continues for 3–5 days,
then feces progressively return to normal in 7–14 days.
Pigs become dehydrated and may die 2–5 days following
the onset of diarrhea; mortality may reach 50–100%. Di-
arrhea and dehydration are less severe in pigs innoculat-
ed when 7–21 days old (Crouch and Woode 1978; Theil
et al. 1978; Shaw et al. 1989a). Disease is limited to tran-
sient diarrhea of 1–1.5 days’ duration in pigs inoculated
when 28 days old (Tzipori et al. 1980b; Lecce et al. 1982).
Mortality decreases as the age at inoculation increases;
death is unusual in pigs older than 14 days. No clinical
disease results when 21- to 28-day-old pigs that have con-
sumed a dry diet for more than 3 days are inoculated
with rotavirus (Tzipori et al. 1980b; Stevenson 1990;
Stevenson et al. 1990).
Rotavirus inoculation studies conducted in nonim-
mune pigs likely represent the more severe limits of ro-
tavirus-induced clinical disease. Naturally occurring ro-
tavirus infection in pigs often results in less severe
clinical disease or is subclinical. Rotavirus is endemic in
most swine herds; that is, nearly all gilts and sows are to
some degree immune. A commensurate degree of immu-
nity is passed to suckling pigs via colostrum and milk
(Askaa et al. 1983). Levels of specific rotavirus antibod-
ies in colostrum and milk decline dramatically in the first
few days of lactation, and rotavirus replication occurs in
pigs when the oral challenge level of virus exceeds the
consumed-milk-borne passive immunity. Management
practices that impact either the level of passive immuni-
ty or the level of oral virus challenge differ among swine
herds and may result in consistent yet differing patterns
of rotavirus-associated disease.
Naturally occurring rotavirus-associated diarrheal
disease is most often reported in 7- to 41-day-old suckling
pigs (Bohl et al. 1978; Roberts et al. 1980; Askaa et al.
1983; Debouck and Pensaert 1983; Svensmark et al.
1989b) or within 7 days following weaning (Woode et al.
1976; Bohl et al. 1978; Lecce and King 1978; Tzipori et al.
1980a). The age of onset is often consistent in a given
herd. Where rotavirus is not complicated by concurrent
infection with other enteric pathogens, diarrhea in suck-
ling pigs is usually mild and limited to 2–3 days’ dura-
tion. Feces are yellow or white, watery to creamy, and
variably flocculant. Dehydration is usually mild and
mortality is less than 15% of clinically ill pigs. Morbidity
is variable but is often 10–20%. Diarrhea sometimes pro-
gresses to younger pigs in a farrowing room as more pigs
are affected. Dehydration is more severe in the younger
pigs. Gilt litters may be affected at a younger age with
more severe disease than are sow litters. Diarrheic suck-
ling pigs frequently shed rotavirus along with other en-
teric pathogens such as transmissible gastroenteritis
(TGE) virus (Bohl et al. 1978), coccidia (Roberts et al.
1980), or enterotoxigenic Escherichia coli (Bohl et al.
1978). Diarrhea is generally more severe and morbidity
and mortality are greater in combined infections.
The role of rotaviruses in diarrheic syndromes in re-
cently weaned pigs is unclear. Rotaviruses have been im-
plicated as a primary cause of severe diarrhea in recently
weaned pigs resulting in 10–50% mortality (Woode
1986); however, rotavirus inoculation studies in weaned
pigs do not support such conclusions. Where rotavirus
shedding has been associated with severe diarrhea in
weaned pigs, it has been in combination with TGE virus
(Bohl et al. 1978) or with hemolytic colony types of en-
terotoxigenic E. coli (Bohl et al. 1978; Tzipori et al. 1980a;
Lecce et al. 1982). Inoculation studies in weaned pigs
with rotavirus and hemolytic colony types of enterotoxi-
genic E. coli suggest a subservient yet potentially impor-
tant role for rotaviruses in severe diarrheic postweaning
syndromes in pigs (Tzipori et al. 1980b; Lecce et al.
1982). Inoculation of weaned pigs with rotavirus or he-
molytic E. coli alone results in mild transient diarrhea or
no clinical disease; whereas inoculation of weaned pigs
with rotavirus followed by hemolytic E. coli results in ef-
ficient hemolytic E. coli colonization and severe protract-
ed diarrhea typical of that reported in natural outbreaks.
Differences in virulence among group A rotavirus
strains have been reported in humans (Kapikian and
Chanock 1990) and calves (Bridger and Pocock 1986).
Differences in the outer capsid protein VP4 have been
implicated as the cause of the differing virulence among
human group A rotavirus strains (Kapikian and Chanock
1990). It is not clear whether there are differences in vir-
ulence among serotypes or strains of swine group A ro-
taviruses. Inoculation studies in pigs with serotype 4
 CHAPTER 21 ROTAVIRUS AND REOVIRUS Paul, Stevenson 261

(Bohl et al. 1984; Benfield et al. 1988), serotype 5 (Theil
et al. 1978; Janke et al. 1988), and a new serotype repre-
sented by strain ISU-64 (Stevenson 1990; Stevenson et al.
1990) have resulted in similar clinical disease and similar
lesions. One inoculation study suggested a difference in
virulence between two strains of group A rotavirus in
pigs (Woode et al. 1976); whereas other studies have
shown no difference in virulence among group A ro-
tavirus strains (Debouck and Pensaert 1979; Collins et al.
1989). Genes encoding VP3, VP4, VP7, and NS28 pro-
teins are believed to play an important role in viral viru-
lence (Hoshino et al. 1985). Little is known regarding
natural disease in swine caused by groups B, C, and E ro-
taviruses. Inoculation studies in gnotobiotic pigs with
group C rotaviruses resulted in clinical disease typical of
group A rotaviruses (Bohl et al. 1982; Snodgrass et al.
1984). In contrast, inoculation studies in gnotobiotic
pigs with group B rotaviruses (Bridger et al. 1982; Theil
et al. 1985) and group E (Chasey et al. 1986) resulted in
less severe diarrhea of shorter duration than that ob-
served with groups A or C.
PATHOGENESIS
Rotaviruses replicate predominately in the cytoplasm of
differentiated small-intestinal villous epithelial cells and
to a lesser extent in the cytoplasm of M cells overlying
Peyer’s patches (Buller and Moxley 1988) and in cecal or
colonic epithelial cells (Theil et al. 1978; Collins et al.
1989). Rotavirus replication results in small-intestinal
villous epithelial cell dysfunction and death; lysis or
desquamation of infected cells leads to villous atrophy.
The degree of villous atrophy and the distribution of at-
rophic villi in the small intestine vary relative to the age
of the pig and are primary factors determining the sever-
ity of clinical disease. In general, villous atrophy is more
severe and extensive in younger pigs.
Numerous rotavirus inoculation studies in 1- to 7-
day-old pigs have demonstrated that virus replication is
usually most extensive in the distal one-half to two-
thirds of the small intestine (Pearson and McNulty 1977;
Theil et al. 1978; Narita et al. 1982a; Collins et al. 1989;
Stevenson 1990). Rotaviral antigen may be demonstrat-
ed in the cytoplasm in a few epithelial cells on villous tips
in the duodenum and in nearly all villous epithelial cells
in the jejunum and ileum by 12–48 hours postinocula-
tion (Fig. 21.4). Villous atrophy is most severe by 24–72
hours postinoculation, when villi may be as short as one-
tenth normal length in the jejunum and ileum. In con-
trast, rotavirus inoculation studies in 21- to 24-day-old
pigs revealed the most extensive virus replication in the
proximal one-half to two-thirds of the small intestine
(Shaw et al. 1989a; Stevenson 1990). Virus-infected ep-
ithelial cells were most often on the distal half of villi
(Fig. 21.4), resulting in moderate villous atrophy. The
most atrophic villi were one-third normal length.
Diarrhea begins slightly before or concurrent with
villous atrophy and has been attributed to multiple path-
ogenic mechanisms. Most proven mechanisms are relat-
ed to decreased small-intestinal digestive and absorptive
functions caused by the loss of differentiated entero-
cytes. Decreased small-intestinal disaccharidase activi-
ties (primarily lactase) result in the retention of disac-
charides (primarily lactose) in the lumen of the small

A B
21.4. Rotavirus antigen in the cytoplasm of villous epithelial cells as viewed by the indirect
fluorescent antibody method (× 90). (A) Ileum from 1-day-old gnotobiotic pig 16 hours post-
inoculation with porcine rotavirus. Nearly all villous epithelial cells contain viral antigen.
(B) Midjejunum from a 27-day-old weaned conventional pig 3 days postinoculation.
Viral antigen is in epithelial cells on villous tips.
262 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
intestine. The excess disaccharides result in hyperosmo-
larity of the intestinal contents, which causes an osmot-
ic diarrhea (Davidson et al. 1977; Graham et al. 1984).
Decreased small-intestinal Na+K+-ATPase activity and
glucose-coupled Na+ absorption result in decreased Na+
absorption at villous tips. The normal secretion of Na+
and water from crypts into the small-intestinal lumen is
not balanced by adequate absorption at villous tips and
may result in net Na+ and water flux toward the small-in-
testinal lumen (Davidson et al. 1977; Graham et al.
1984). Recent studies in children have also suggested
that prostaglandins, which are produced as a result of ro-
tavirus-induced enteric inflammation, may enhance the
severity of rotavirus-associated diarrhea (Yamashiro et
al. 1989). Children with rotavirus-associated diarrhea
had higher serum and fecal levels of two prostaglandins
than did clinically normal children. Furthermore, oral as-
pirin therapy, which decreased serum and fecal
prostaglandin levels, also shortened the duration of di-
arrhea. Similar studies in pigs have not been reported.
Several interrelated factors may be involved in caus-
ing the death of rotavirus-inoculated pigs. Diarrhea
causes dehydration and electrolyte imbalances and may
lead to exhaustion of extracellular fluid reserves. Malab-
sorption results in malnutrition and may lead to energy
deficiency and hypothermia. Observed higher mortality
in neonatal pigs is most likely related to more severe and
extensive villous atrophy, coupled with decreased extra-
cellular fluid and energy reserves, compared to slightly
older pigs. Suboptimal ambient temperatures have been
shown to increase mortality in rotavirus-infected pigs.
Inoculation of newborn pigs with a human strain of
group A rotavirus resulted in no mortality when pigs
were maintained at 35˚C and resulted in 70–90% mortal-
ity when pigs were maintained at 26˚C (Steel and Torres-
Medina 1984). Differences in mortality were attributed
to increased energy demands at lower ambient tempera-
tures combined with rotavirus-induced malnutrition.
Mortality may also be affected by virus dose. The mini-
mum infective dose of two strains of porcine group A ro-
tavirus has been shown to be as low as 1 plaque-forming
unit of virus (Graham et al. 1987; Shaw et al. 1989b). In
another study, as few as 90 viral particles were infectious
and caused diarrhea in 4-day-old pigs (Payment and
Morin 1990). Virus titration studies using a porcine
group A rotavirus strain and done in a limited number of
neonatal colostrum-deprived pigs suggested higher mor-
tality in pigs inoculated with lower dilutions (higher
virus dose) of inoculum (Shaw et al. 1989b).
The duration of rotavirus-induced diarrhea is deter-
mined by the time needed for the restoration of an ade-
quate number of differentiated villous epithelial cells in
the small intestine. Complete restoration of morpholog-
ically normal villi in 3-day-old pigs requires 6–10 days;
whereas only 2–4 days are required in 21-day-old pigs.
Recovery is slower in younger pigs owing to more severe
villous atrophy and to sluggish crypt epithelial cell
turnover. Complete replacement of villous epithelial
cells requires 8–10 days in newborn pigs and 2–3 days in
21-day-old pigs (Moon 1971).
The pathogenesis of groups B, C, and E rotaviruses in
pigs has been studied much less extensively than that of
group A rotaviruses. The limited information gained
through inoculation studies in pigs with group B ro-
taviruses (Pedley et al. 1983; Theil et al. 1985), group C
rotaviruses (Bohl et al. 1982; Snodgrass et al. 1984), and
group E rotaviruses (Chasey et al. 1986) suggests a patho-
genesis similar to that described for group A rotaviruses.
Virus replication was most extensive in the distal small
intestine for all rotavirus groups; however, villous atro-
phy was more severe with groups A and C than with
groups B and E.
The pathogenesis of combined infections of ro-
tavirus and other commonly recognized en-
teropathogens has been examined in a limited number of
studies. Inoculation of 3-day-old germ-free pigs with
group A rotavirus followed 24 hours later by K99 pilus-
producing enterotoxigenic E. coli caused more severe di-
arrheal disease than did inoculation of pigs with either
agent separately. Villous atrophy was no more severe in
pigs infected with both agents than in pigs inoculated
with rotavirus alone (Benfield et al. 1988). Inoculation of
28-day-old pigs with rotavirus followed by hemolytic
strains of enterotoxigenic E. coli resulted in more severe
diarrheal disease than was seen in pigs inoculated with
either agent separately (Tzipori and Williams 1978; Lec-
ce et al. 1982). It was concluded that rotavirus infection
enhances colonization of the small intestine by hemolyt-
ic strains of enterotoxigenic E. coli. Inoculation of 7-day-
old germ-free pigs with rotavirus and TGE virus resulted
in more severe diarrheal disease than in pigs inoculated
with rotavirus alone (Woode and Crouch 1978). In con-
trast, simultaneous inoculation of 1-day-old pigs with ro-
tavirus and enterovirus resulted in less severe diarrheal
disease and less severe villous atrophy than was seen in
pigs inoculated with rotavirus alone (Janke et al. 1988). It
was concluded that nonlethal enterovirus infection of
villous epithelial cells interfered with subsequent ro-
tavirus infection.
LESIONS
The lesions caused by rotaviruses in pigs are limited to
the small intestines and are the sum of the degenerative
and functional consequences of rotavirus-induced vil-
lous epithelial cell destruction and the adaptive and re-
generative responses of the small intestine.
Gross lesions are visible slightly before or concurrent
with the onset of diarrhea and are most severe in 1- to 14-
day-old pigs (Pearson and McNulty 1977; Theil et al.
1978; Benfield et al. 1988; Janke et al. 1988; Collins et al.
1989; Stevenson 1990). The stomachs usually contain
 CHAPTER 21 ROTAVIRUS AND REOVIRUS
food and the distal one-half to two-thirds of the small in-
testine is thin walled and flaccid and dilated with a large
volume of watery, flocculant, yellow or gray fluid. Fol-
lowing 24 hours of diarrhea, increased small-intestinal
motility may result in less distension and a less thin-
walled, more nearly normal appearance. The lacteals in
the distal two-thirds of the intestine contain no chyle,
and the associated mesenteric lymph nodes are small
and tan. The cecum and colon are likewise dilated with
similar contents. Gross lesions are less severe or are ab-
sent in pigs that are 21 days of age or older (Shaw et al.
1989a; Stevenson 1990).
Light microscopic lesions have been described in nu-
merous rotavirus inoculation studies in suckling pigs
(Woode et al. 1976; Pearson and McNulty 1977; Crouch
and Woode 1978; Theil et al. 1978; McAdaragh et al.
1980; Benfield et al. 1988; Janke et al. 1988; Collins et al.
1989; Stevenson 1990). Villous epithelial cell degenera-
tion begins in cells on villous tips and in groups of cells
on lateral villi by 16–18 hours postinoculation. Degener-
ative cells are swollen and have rarified cytoplasm,
swollen nuclei, and irregular brush borders. Degenera-
tive cells are frequently partially detached from adjacent
cells or from the basement membrane. By 16–24 hours
postinoculation, sloughing of cells results in significant
villous atrophy, which is most severe by 24–72 hours
postinoculation (Fig. 21.5). The tips of atrophic villi may
be eroded or may be covered by attenuated, nearly squa-
mous epithelial cells, and there is a variable amount of
cellular debris in the lamina propria. Contact of the ex-
posed lamina propria of adjacent villi results in villous
fusion, which may be observed for 24–168 hours
postinoculation. Crypt epithelial hyperplasia results in
significantly deeper crypts beginning 48–72 hours
A B
21.5. Ileum from 3-day-old gnotobiotic pigs (H&E; × 35). (A) Normal villi in an uninoculated
control pig. (B) Severe villous atrophy present 18 hours postinoculation.
Paul, Stevenson 263
postinoculation. The time required for complete regen-
eration of normal villi depends on the age of the pig.
Scanning electron microscopy has allowed detailed
study of the surface topography of affected small-intesti-
nal mucosa in numerous rotavirus inoculation studies
(McAdaragh et al. 1980; Torres-Medina and Underdahl
1980; Collins et al. 1989; Stevenson 1990). Degenerate
villous epithelial cells lyse and/or detach, resulting in vil-
lous erosion and shortening (Fig. 21.6A, B). Within a few
hours, eroded villous tips are covered with a continuous
layer of epithelial cells and there are few remain-
ing swollen and degenerate epithelial cells (Fig.
21.6C).
Ultrastructural studies in rotavirus-inoculated or nat-
urally infected pigs have revealed lesions in the cyto-
plasm of virus-infected villous epithelial cells that are
typical of those described for rotaviruses in many other
mammalian and avian species (Chasey 1977; Saif et al.
1978; Pearson and McNulty 1979; Narita et al. 1982b).
Virus-infected cells contain within their cytoplasm mul-
tiple, variably sized, electron-dense granular viroplasms
that often have dense subviral cores or single-shelled par-
ticles on the periphery. Single-shelled viral particles ob-
tain the outer capsid by budding through the mem-
branes of the rough endoplasmic reticulum (Fig. 21.7).
Mature, 75–78 nm double-shelled virus particles accu-
mulate in the cysternae of the endoplasmic reticulum
and are released by cell lysis. Other degenerative changes
in virus-infected cells include cell swelling, mitochondri-
al swelling, nuclear swelling, dilatation of the cytocavity
network, and fragmentation of microvilli. Macrophages
in the lamina propria contain cellular membrane pro-
files, virus particles, viroplasm, and other cellular debris
in phagosomes.
264 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

A B C
21.6. Scanning electron micrograph of the ileum of 3-day-old gnotobiotic pigs. (A) Normal villi
in an uninoculated control pig (× 70). (B) Sloughing, degenerate villous epithelial cells and exposed
lamina propria on severely atrophic villi, 18 hours postinoculation (×165). (C) The tips of atrophic
villi are covered by a continuous layer of sometimes swollen and degenerate epithelial cells, 24
hours postinoculation (× 230).

21.7. Ultrastructure of a swollen

and degenerate rotavirus-infected
villous epithelial cell containing
dense granular viroplasm (arrow-
head). Virus particles form and
obtain outer capsids through
budding into the rough
endoplasmic reticulum (small
arrow). Many double-shelled virus
particles are within the cysternae
of the endoplasmic reticulum
(large arrows) (× 23,800).

DIAGNOSIS hours after the start of diarrhea. This is especially critical

Rotavirus should be considered as a cause of diarrhea in
neonatal pigs 1–8 weeks of age. Fecal samples or intesti-
nal contents should be collected in acute phases of dis-
ease and submitted for diagnosis. Highest concentra-
tions of porcine rotavirus are shed during the first 24
for the detection of certain groups of rotaviruses, for the
amount of virus shed is lower and the duration of virus
shedding is shorter in pigs infected with group B ro-
taviruses (Bridger 1980; Theil et al. 1985; P. S. Paul, un-
published data—1990).
 CHAPTER 21 ROTAVIRUS AND REOVIRUS
A number of methods have been employed for the
detection of rotaviruses, including electron microscopy,
immune electron microscopy, immunohistochemistry,
immunofluorescence on frozen sections or impression
smears of small intestines, ELISA, virus isolation, latex
agglutination, dot blot hybridization, RNA electro-
pherotyping, and PCR (Paul et al. 1988b; Johnson et al.
1990; Rosen et al. 1990a, b; Koromyslov et al. 1990;
Sanekata et al. 1991; Ojeh et al. 1992; Magar and
Larochelle 1992; Jiang et al. 1992; Ojeh et al. 1993; Rosen
et al. 1994; Zaberezhny et al. 1994). Electron microscopy
has been extensively used for the detection of rotavirus-
es and remains a reference method for their detection.
This method allows detection of different serogroups of
rotaviruses. The morphology of the virus particles de-
tected by negative-staining electron microscopy is affect-
ed by the staining methodology utilized and the
serogroup of rotavirus. Negative staining with phospho-
tungstic acid (PTA) of neutral pH results in predomi-
nantly single-shelled capsids; whereas PTA of pH 4.5 or
uranyl acetate gives double-shelled particles (Nakata et
al. 1987; Suzuki et al. 1987). Core particles have been de-
tected in group B rotaviruses (Bridger et al. 1982; Theil et
al. 1985). Immune electron microscopy allows their dif-
ferentiation into serogroups.
Immunohistochemistry employing immunogold
staining with protein A gold and specific antisera has
been used to detect group A rotaviruses in formalin-fixed
paraffin-embedded sections of small intestines (Magar
and Larochelle 1992).
ELISA is frequently used for the detection of rotaviral
antigens in fecal samples or intestinal contents. It is
more sensitive than the latex agglutination test (Goyal et
al. 1987) but less sensitive than electron microscopy
(Benfield et al. 1984; Benfield 1990). Human-based diag-
nostic kits may be used for the detection of porcine
group A rotaviruses. Monoclonal antibody capture
ELISA has been developed for the detection of group C
rotaviruses (Ojeh et al. 1992; Tsunemitsu et al. 1992a).
Electropherotyping of viral RNA is often used for the
detection and differentiation of rotavirus groups. Ro-
taviruses of different serogroups have distinct electro-
pherotypes (Fig. 21.2), which only provide a tentative di-
agnosis of a serogroup and should be confirmed by
serologic or nucleic acid–based methods. For electro-
pherotyping, viral RNA can be isolated in a crude form
directly from feces and analyzed by polyacrylamide gel
electrophoresis (Paul et al. 1988b) or purified by CF-11
cellulose chromatography (Theil et al. 1981). RNA bands
in gels are visualized by ethidium bromide or silver-stain-
ing methods.
Terminal oligonucleotide fingerprinting has been
used in differentiating porcine rotaviruses and has been
useful in molecular epidemiological studies (Clarke and
McCrae 1981). Nucleic acid probes and the PCR method
offer attractive alternatives for the detection and differ-
Paul, Stevenson 265
entiation of rotavirus groups and serotypes (Dimitrov et
al. 1985; Johnson et al. 1990; Paul et al. 1990a, c; Rosen
et al. 1990a, b; Koromyslov et al. 1990; Ojeh et al. 1993;
Gouvea et al. 1994; Rosen et al. 1994; Zaberezhny et al.
1994).
Serologic tests are of little value in the diagnosis of
rotavirus infection because antibodies are common in
most swine herds; however, antibody titers and isotypes
shed light on the immune status of animals. Antibodies
to rotavirus can be detected by a number of serologic
tests. The indirect immunofluorescence test has often
been used to detect antibodies to porcine rotavirus
(Theil and Saif 1985; Chasey et al. 1986). Monolayers of
MA-104 cells are inoculated with group A, B, or C ro-
tavirus. The virus is allowed to adsorb onto cells in the
case of group A rotaviruses or is centrifuged onto cells in
the case of group B or C rotaviruses. Cells are fixed with
an acetone-methanol mixture and used as antigen in an
indirect immunofluorescence test. Alternatively, cryostat
intestinal sections from pigs infected with noncultivable
rotaviruses may be used as the source of antigens. Anti-
bodies to serogroups B, C, and E have been detected by
this method. Indirect ELISA has also been used to detect
antibodies to rotaviruses. Antibodies detected by the
above methods are usually directed against the common
group antigen. Antibodies to group C have also been de-
tected by blocking ELISA using monoclonal antibodies
(Tsunemitsu et al. 1992a). ELISA combined with isotype-
specific monoclonal antibodies (Paul et al. 1985, 1989)
has been used to detect classes of antibodies to rotavirus
(Paul et al. 1986). Plaque reduction and fluorescent focus
reduction neutralization assays have been used to detect
neutralizing antibodies (Hoshino et al. 1984; Paul et al.
1988a). The hemagglutination inhibition test has also
been developed for the detection of antibodies to
porcine rotaviruses (Eiguchi et al. 1987).
TREATMENT
No known therapeutic agents are available for the specif-
ic treatment of porcine rotavirus infections. General sup-
portive therapy, management procedures, and antibi-
otics are recommended to minimize mortality due to
rotaviruses and secondary bacterial infections. Elec-
trolyte solutions containing glucose-glycine fed ad libi-
tum minimize dehydration and weight loss induced by
rotavirus infection (Bywater and Woode 1980; Bywater
1983). Ambient environment should be optimized, min-
imizing draft and temperature fluctuations. Antibiotic
therapy may be used to reduce losses from secondary
bacterial infections. In herds with a persistent problem
of postweaning diarrhea with high mortality, a change in
weaner diet and weaning procedure should be consid-
ered. Scheduled feeding of a high-energy weaner diet has
been successfully used to reduce morbidity and mortali-
ty (Tzipori et al. 1980a; Hampson and Beban 1985).
266 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
PREVENTION
Management
Rotavirus infection persists endemically in most swine
herds. Rotaviruses survive in dust and organic matter
and may persist as subclinical infections in adult swine
(Benfield et al. 1982). They are therefore difficult, if not
impossible, to eliminate. Studies have shown that in-
creasing concentrations of rotavirus in the environment
cause more severe disease at a younger age (Lecce et al.
1978). Thus, management practices should be directed
at reducing the virus load for susceptible pigs and boost-
ing passive immunity. Virus load may be reduced by san-
itation and limiting contact between young susceptible
pigs and older pigs more likely to be shedding virus. The
floors in farrowing and nursing houses should be con-
structed to minimize feces buildup during use and to fa-
cilitate cleaning. All-in/all-out management practices
should be followed and rooms should be thoroughly
cleaned and disinfected between groups. Recommended
disinfectants include formaldehyde and chlorine-based
disinfectants such as chlorox or chloramine T (Multi-
chlor). The farrowing interval should be minimized to
prevent virus buildup and infection of later-farrowed lit-
ters. Mixing animals of different ages should be discour-
aged because it promotes virus transmission from older
to younger pigs. To enhance passive immunity, replace-
ment gilts should be exposed to the feces of older sows.
Attention to lactation diet, feed intake, sow comfort, and
farrowing-crate design is important to ensure adequate
milk supply and effective suckling necessary for success-
ful immunity.
Immunoprophylaxis
Pigs recovered from a natural infection with rotavirus are
immune to infection with homologous rotavirus for at
least a short time; the duration of immunity is unknown.
The mechanisms of immunity have not been completely
elucidated. Protection is generally correlated with the
presence of antibodies in secretions. Antibodies in the
intestinal lumen produced by local lymphoid cells bathe
susceptible villous epithelial cells and protect them from
infection (Chen et al. 1995; Saif and Fernandez 1996).
Secretory IgA (sIgA) is more effective than IgG and IgM
because sIgA is more resistant than IgG and IgM to pro-
teolytic degradation in the intestinal tract and IgA is the
predominant immunoglobulin (Ig) in secretions. Circu-
lating serum antibodies do not provide protection
against rotavirus infections (Offit and Clark 1985). Stud-
ies in mice indicate that cellular immunity may also be
important in protection against rotavirus infections (Of-
fit and Dudzik 1988, 1989).
Piglets suckling immune sows are protected from ro-
tavirus gastroenteritis during the nursing period. This
passive immunity is related to the continuous presence
of specific virus-neutralizing antibodies in the gastroin-
testinal tract of suckling pigs. Secretory IgA is the pre-
dominant Ig in milk of lactating sows and is the primary
Ig providing passive protection.
The route of immunization of sows influences the
classes of Igs produced. Primary immunization by the
oral route is the best method known to date for induction
of sIgA in milk. Parenteral routes, however, may be used
to boost sIgA in animals previously primed by oral im-
munization. The level of attenuation of a rotavirus also
influences its ability to induce lactogenic immunity. In-
activated vaccines are not immunogenic but modified-
live-virus vaccines are immunogenic. The best immunity
is induced by virulent virus because it induces optimal
intestinal immune responses (Saif and Fernandez 1996).
Induction of active immunity against postweaning diar-
rhea in the presence of passive milk antibodies is prob-
lematic. Oral immunization is required for optimal prim-
ing of the sIgA response, yet passively acquired milk
antibodies will likely interfere in replication of immuniz-
ing rotavirus. Immunization by the parenteral route is
not as likely to result in protective immunity.
Immunity to rotaviruses, in general, is serotype spe-
cific (Bohl et al. 1982; Losonsky et al. 1986). Pigs orally
immunized with the A/OSU strain were resistant to in-
fection and were protected from gastroenteritis follow-
ing challenge with the A/OSU strain, but not from chal-
lenge with the A/Gottfried strain (Bohl et al. 1982).
Similarly, pigs immunized with the A/Gottfried strain
were protected from challenge with A/Gottfried, but not
from the A/OSU strain. This study demonstrated feasi-
bility for the development of a modified-live-rotavirus
vaccine. Two surface proteins, VP4 and VP7, are impor-
tant in induction of neutralizing antibodies and in pro-
tection (Greenberg et al. 1983b; Offit et al. 1986b; Hoshi-
no et al. 1988). In addition to serotype-specific epitopes,
these proteins also possess heterotypic epitopes, which
are conserved among different serotypes. Heterotypic
immunity is not as effective as that induced by homotyp-
ic strains. Reassortant rotaviruses containing the VP4
and VP7 genes from two different serotypes may be used
to induce immunity to both serotypes (Offit et al. 1986b;
Hoshino et al. 1988). The reassortant rotavirus SB-1A,
containing the VP4 gene from the A/OSU strain and the
VP7 gene from the A/Gottfried strain, induced neutral-
izing antibodies for both the OSU and Gottfried strains
and protected orally immunized pigs from challenge
with both the OSU and Gottfried strains (Hoshino et al.
1988). Protection, however, was greater against the
Gottfried strain than against the OSU strain.
Modified-live-virus and inactivated-virus vaccines are
commercially available for immunization of sows as well
as nursing pigs. Modified-live-virus vaccines are admin-
istered orally, orally and intramuscularly, or intramuscu-
larly. Inactivated-virus vaccines are administered intra-
muscularly in sows and intraperitoneally in nursing pigs.
Not all of the commercially available vaccines have been
 CHAPTER 21 ROTAVIRUS AND REOVIRUS
evaluated by independent investigators. Two separate
studies of a modified-live-rotavirus vaccine for post-
weaning diarrhea resulted in conflicting data. In one
study, immunization of nursing pigs improved the
weight gain and reduced the severity of disease (Wester-
camp 1986). In the second study, neither improvement
in weight gain nor curtailment of virus shedding was ob-
served (Hoblet et al. 1986). Bovine rotavirus vaccine is
not effective in preventing rotaviral diarrhea in pigs (Lec-
ce and King 1979). Recent identification of new porcine
PORCINE REOVIRUS
Reoviruses have been detected from healthy pigs as well
as from pigs with respiratory and reproductive diseases
(Kasza 1970; Kirkbride and McAdaragh 1978; McFerran
1986). Their role in these swine diseases is unclear. Re-
oviruses are associated with tenosynovitis in chickens
(Van der Heide 1977; Robertson and Wilcox 1986) and
systemic illness affecting the majority of organs in ro-
dents (Tyler and Fields 1990) and thus have the potential
to be significant pathogens in swine.
ETIOLOGY
Virus Morphology
Reovirus was the first genus identified in the family Re-
oviridae. The other two genuses of importance to ani-
mals are Rotavirus and Orbivirus. “Reo” is an acronym for
“respiratory and enteric orphan.” Reovirus particles are
nonenveloped and icosahedral in shape with a fuzzy out-
er rim; they measure 75 nm in diameter (Fig. 21.8). The
21.8. Electron micrograph of reovirus particles viewed
by negative-staining electron microscopy (×115,000).
Paul, Stevenson 267
rotavirus serotypes (Table 21.1) may require their incor-
poration into vaccines if they are found to be a signifi-
cant cause of gastroenteritis in swine. The VP4 and VP7
genes of porcine rotavirus have been cloned and se-
quenced (Gorziglia et al. 1986, 1988, 1990; Nishikawa
and Gorziglia 1988), and VP4 of one strain has been ex-
pressed in baculovirus vector. VP4 protein and viruslike
particles are immunogenic. Recombinant proteins and
reassortant rotaviruses offer potential alternatives as im-
munogens for rotavirus gastroenteritis.
inner capsid is about 45–50 nm in diameter. Reovirus
replicates in the cytoplasm.
Physicochemical and Biological Properties
Reoviruses have a segmented (10 segments), double-
stranded, RNA genome. The density of a complete (ma-
ture) virion in cesium chloride is 1.36 g/cm2. Porcine re-
oviruses are resistant to ether, chloroform, and trypsin.
They are stable at acidic pH 3 and are sensitive to 0.1%
sodium deoxycholate (Hirahara et al. 1988). Porcine re-
oviruses are susceptible to heat at 50˚C for 1 hour but are
stabilized by the presence of 1 molar MgCl2. They pos-
sess a hemagglutinin that agglutinates human group O
and porcine erythrocytes at 4˚C, 22˚C, and 37˚C, but not
erythrocytes from guinea pig, rat, hamster, cattle, dog,
cat, or chicken. Mammalian reoviruses share a group
antigen that can be detected by complement fixation, im-
munofluorescence, and immunodiffusion (Sabin 1959).
Avian reoviruses are not antigenically related to mam-
malian reoviruses but possess a group antigen that is
shared by avian reoviruses. All mammalian isolates can
be divided serologically into three types: types 1, 2, and
3. Reoviruses of different types can be distinguished by
serum neutralization and hemagglutination inhibition
tests.
Cultivation
Reoviruses can be cultivated in a wide variety of cell cul-
tures from many species: cell cultures of porcine, bovine,
feline, simian (African monkey kidney cell lines Vero and
MA-104), human (HeLa cell line), and canine (canine
thyroid adenocarcinoma cell line) origin (Kasza 1970;
Hirahara et al. 1988; P. S. Paul, unpublished data—
1990). Reovirus replication is slow and the majority
(80%) of the nascent rotavirus population remains cell
associated (McFerran 1986).
The cytopathic effect of reoviruses varies somewhat,
dependent on the cell line used. In general, cells round
up, become granular, and slough off the surface.
Eosinophilic intracytoplasmic inclusion bodies can easi-
268 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
ly be seen in cultures stained with May-Greenwald-
Giemsa stain.
EPIDEMIOLOGY
Porcine reovirus infections appear to be widespread in
swine herds, and antibodies to all three types have been
detected in pigs (Harkness et al. 1971; Zindel et al. 1980).
In a study by Zindel et al. (1980), pigs in 54% of the swine
herds surveyed were seropositive for all three reovirus
types. Passively acquired antibodies to reoviruses persist
in neonatal pigs for about 11 weeks, at which time pigs
become susceptible to infection (Watt 1978). Reoviruses
are spread via fecal-oral and respiratory routes. Follow-
ing experimental infection, virus is shed in nasal secre-
tions and feces for 6 and 14 days, respectively. Virus is
spread by contact exposure (McFerran et al. 1971).
CLINICAL SIGNS
Reoviruses have been isolated from clinically healthy
pigs as well as pigs with respiratory or enteric disease
(Kasza 1970; McFerran and Connor 1970; Robl et al.
1971; Elazhary et al. 1978; McFerran 1986) and from
aborted fetuses (Kirkbride and McAdaragh 1978).
Experimental studies have not consistently resulted
in reproduction of disease. In a majority of studies, in-
tranasal, intraperitoneal, or intracerebral inoculation of
conventional and gnotobiotic pigs of 1–6 weeks of age
with porcine reovirus type 1 or human reovirus type 1
did not result in clinical disease except for a transient
febrile reaction (Kasza 1970; McFerran and Connor
1970; Baskerville et al. 1971; McFerran et al. 1971; Watt
1978). Virus could be isolated from respiratory and ali-
mentary tracts for up to 2 weeks postinfection. In one
study, a rise in body temperature and diarrhea were de-
tected in cesarean-derived colostrum-deprived 1-week-
old pigs inoculated orally with type 1 reovirus; however,
no clinical disease was detected in gnotobiotic pigs
(Elazhary et al. 1978). Mild respiratory disease charac-
terized by pyrexia, sneezing, inappetance, and listless-
ness was reproduced in cesarean-derived colostrum-de-
prived pigs and conventional pigs inoculated
intranasally with or exposed via aerosol to reovirus type
1 (Hirahara et al. 1988). Intravenous or intramuscular in-
oculation of seronegative sows between 40 and 85 days
of gestation with reovirus resulted in term litters con-
taining a mixture of mummified, stillborn, weak, and
normal pigs.
PATHOGENESIS
Reoviruses replicate mainly in intestinal and respiratory
tracts and can be isolated from nasal and fecal swabs.
The virus is excreted in nasal secretions and feces as ear-
ly as 24 hours postinfection, and excretion may continue
for 14 days. The virus can also be isolated from boar se-
men (McAdaragh and Anderson 1975). The virus was
isolated from fecal tissues and placenta of sows inoculat-
ed intravenously or intramuscularly with type 3 reovirus.
The virus was recovered only from fetal tissues of sows
inoculated at 30–60 days of gestation, from both placen-
tal and fetal tissues of sows inoculated at 50–70 days of
gestation, and from only placenta of sows inoculated af-
ter 70 days of gestation (McAdaragh and Anderson
1975). Hemagglutination-inhibiting antibodies may be
detected at 7 days postinfection and reach peak titers be-
tween 11 and 21 days postinfection (McFerran 1986).
LESIONS
A limited number of reovirus inoculation studies in
swine have revealed few gross lesions and only mild mi-
croscopic lesions. Although intravenous or intramuscu-
lar inoculation of sows between 40 and 85 days of gesta-
tion with reovirus resulted in litters containing a mixture
of mummified fetuses, stillborn pigs, small weak pigs,
and normal pigs, no specific gross or histopathologic le-
sions were reported (McAdargh and Robl 1976). Oral in-
oculation of 1-week-old cesarean-derived colostrum-de-
prived pigs with enteric-origin reovirus resulted in focal
villous atrophy in the jejunum and ileum (Elazhary et al.
1978). Aerosol exposure of 4-week-old specific-
pathogen-free (SPF) pigs to porcine-origin type 1 re-
ovirus resulted in no gross lesions; however, there were
consistent microscopic lesions in the lungs consisting of
multifocal aggregates of lymphocytes and macrophages
in alveoli and alveolar septae and mild peribronchiolar
nodular lymphocytic hyperplasia (Baskerville et al.
1971). Intranasal inoculation of 70 kg SPF pigs with a
porcine respiratory isolate of type 3 reovirus resulted in
lobular atalectasis and vesicular emphysema and peri-
bronchiolar nodular lymphocytic hyperplasia, which
varied in intensity between lobules. Additional studies
are needed to clearly characterize the clinical disease and
lesions in swine caused by porcine reoviruses.
DIAGNOSIS
A number of methods may be employed for the detec-
tion of reoviruses, including electron microscopy, im-
mune electron microscopy, immunofluorescence, virus
isolation, and RNA electropherotyping. Virus isolation
has commonly been used for diagnosis. A wide variety of
cells may be used; primary swine kidney cells and
African monkey kidney cell lines such as MA-104 are ex-
tremely susceptible and are commonly available. Typing
of reovirus is achieved by virus neutralization and
hemagglutination inhibition tests with reference antisera
to three reovirus types.
 CHAPTER 21 ROTAVIRUS AND REOVIRUS
TREATMENT AND PREVENTION
No methods are available for treatment or prevention of
porcine reovirus infections, and possibly none are war-
ranted until the clinical significance of reovirus infec-
tions in swine is documented.
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 Swine Influenza
22 B. C. Easterday and K. Van Reeth
Swine Influenza (SI) is an acute, infectious, respiratory
disease of swine caused by type A influenza viruses. The
disease is characterized by a sudden onset, coughing,
dyspnea, fever, and prostration, followed by a rapid re-
covery. Lesions generally develop rapidly in the respira-
tory tract and regress quickly, but in a few cases severe vi-
ral pneumonia may be followed by death. The course,
nature, and severity of the disease are likely to vary with
the strain of the virus, the age and immune status of the
pig, and the presence of intercurrent infections.
SI has received considerable attention since the dis-
ease was first described in 1918. In the late summer of
that year an epizootic disease, having many clinical and
pathologic similarities to influenza in humans, appeared
among swine in the north-central United States. The ap-
pearance of the disease in swine coincided with the 1918
influenza pandemic that was responsible for the death of
an estimated 20 million people throughout the world.
The exact date and locality of the initial occurrence of SI
are unknown, but observers stated that cases were seen
in August 1918 on farms in western Illinois (Shope
1964). Although the infection and disease may have ex-
isted among swine populations before that time, it is un-
likely that a disease with such distinctive characteristics
would have gone unnoticed and unreported.
According to Dorset et al. (1922), Dr. J. S. Koen, an
inspector in the division of hog cholera control of the
U.S. Department of Agriculture, Bureau of Animal In-
dustry, was the first to recognize the disease as being dif-
ferent from any previously encountered. Koen was im-
pressed by the coincidental prevalence of epidemic
human influenza and the similarity of signs and symp-
toms seen in humans and in swine. He was convinced
that the two diseases were the same and he was the first
to apply the name “flu” to this new disease of hogs. His
opinion that the condition represented a new epizootic
disease of swine and that swine had been infected from
humans was shared by veterinarians and farmers in the
area.
Various aspects of the disease, such as signs, lesions,
and course, were described by Dorset et al. (1922),
McBryde (1927), and McBryde et al. (1928) during the
decade following the first report. However,it was not un-
til 1930 that the SI virus (SIV) was isolated and identified
by Shope (1931b). For the next 25 years Shope expanded
his work to include studies of immunity, transmission,
adaptation of the virus to laboratory hosts, antigenic re-
lationships with other influenza viruses, and mainte-
nance of the disease in nature, including his controver-
sial lungworm/earthworm hypothesis for the
interepizootic survival of the virus. From all accounts,
the infection and disease in swine have not changed sig-
nificantly in the north-central United States since SI was
first observed in 1918. However, as will be described lat-
er, the infection and disease may be caused by type A in-
fluenza viruses with different antigenic and/or biologic
characteristics.
It is clear, based on clinical observations and serolog-
ic and virologic surveillance studies, that the infection
and disease are found throughout the United States.
About 25–33% of all pigs 6–7 months old slaughtered in
the United States have antibody against SIV. The rate is
higher (about 45%) among older swine (2 or more years)
that are slaughtered (i.e., those animals that have been
used for breeding purposes) (Woods 1975; Pirtle et al.
1976; Hinshaw et al. 1978; Chambers et al. 1991). Those
studies indicated no significant seasonal variation in the
rate of infection.
Prior to 1975, there were few reports of SI in places
other than the United States. However, since that time,
clinical SI and various type A viruses and their antibod-
ies have been observed and reported in most parts of the
world where swine are found. In Europe, SI viruses of the
H1N1 subtype were reported occasionally in Czechoslo-
vakia (Harnach et al. 1950), the United Kingdom (Blake-
more and Gledhill 1941), and West Germany (Kaplan
and Payne 1959) between 1940 and 1950. Thereafter, the
virus/disease was not reported until 1976, when clinical
SI reemerged on farms in northern Italy. The viruses re-
sponsible for these first outbreaks were closely related to
classic H1N1 viruses and probably had been introduced
to Italy with a shipment of pigs from the United States
(Nardelli et al. 1978).
From 1979, H1N1 influenza virus epizootics occurred
in France, Belgium, the Netherlands, Germany, and oth-
er countries of continental Europe (Vandeputte et al.
1980; Ottis et al. 1981; Soerensen et al. 1981; Gourreau et
al. 1983; Masurel et al. 1983; Sinnecker et al. 1983; Mar-
tinsson et al. 1983). The viruses isolated in Europe after
1979 were related to, but clearly distinguishable from,
the classic H1N1 viruses. Their hemagglutinins were
more closely related to the avian H1 and they were most
277
278 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
likely transmitted to swine from ducks (Pensaert et al.
1981). These avian viruses generally are more invasive
and pathogenic than the SI viruses in the United States,
resulting in economically devastating outbreaks and
widespread occurrence. Classic H1N1 viruses have been
reported in only a few European countries and have
largely been replaced by the avianlike variants (Abusug-
ra et al. 1987; Barigazzi et al. 1996).
In Great Britain, however, the situation has not been
the same as on the European continent. Beginning in
1986, classic SIV similar to the North American SIV cir-
culated at a moderately high prevalence, but it was of mi-
nor clinical importance (Roberts et al. 1987). In 1992, a
large increase in outbreaks of respiratory disease was as-
sociated with the isolation of an H1N1 virus antigenical-
ly distinguishable from the previous Great Britain strains
(Brown et al. 1993b). That virus represented the first
avianlike H1N1 virus transmitted from pigs in continen-
tal Europe, where it had been circulating for more than
10 years. Currently, classic and avian H1N1 viruses are
cocirculating in the British swine population, along with
H3N2 viruses (Brown et al. 1995). The latter represent
human viruses that emerged from Asia in 1968 and be-
came established in pigs throughout Europe during the
1970s (Kundin 1970; Tumova et al. 1976; Ottis et al.
1982). Evidence of clinical disease in swine caused by
H3N2 viruses, however, was not reported until 1984 in
continental Europe (Haesebrouck et al. 1985) and a few
years later in Great Britain (Wibberley et al. 1988).
In recent years, H1N2 and H3N2 viruses have been
associated with severe disease of swine in Canada. Cana-
dian investigators have isolated several different influen-
za viruses from pigs with influenza-like disease. These
viruses have included H1N1 similar to the early (1930)
H1N1 and an H1N1 more similar to the more recent
North American H1N1 viruses (Bikour et al. 1995a) and
H3N2 similar to the early human H3N2 viruses (Bikour
et al. 1995b). Interestingly, the prevalence of infection
with the H3N2 subtype viruses in the United States has
been very low.
Since the late 1970s, there have been several reports
of SI in various places in Asia. Most of those outbreaks
have been due to swine H1N1 or H3N2 viruses. Out-
breaks due to H1N1 viruses similar to the classic H1N1
were reported in Taiwan in 1975 (Hsu et al. 1976), on a
large farm in Sarawak after breeding stock was intro-
duced from the United States (Teik and Lan 1985), and in
pigs with influenza-like disease in Thailand (Kupradinun
et al. 1991). Swine H1N1 virus was isolated. In China,
there are reports of the isolation of H3N2 viruses from
pigs in which the H3 is similar to the early human H3
antigen (Kida et al. 1988), and other H3N2 viruses from
pigs are similar to the more recent human H3 viruses. Be-
tween October 1991 and January 1992, 20 H1N1 viruses
isolated from asymptomatic pigs at a Beijing abattoir
were shown to be of swine, not avian or human, origin
(Guo et al. 1992). Another 72 H1N1 viruses collected at
random in southern China from 1976 to 1982 were all
shown to be characteristic of the classic H1N1 viruses. In
Japan, influenza viruses isolated during outbreaks of in-
fluenza-like disease in swine, or recognized indirectly by
serologic studies, include swine H1N1, human H1N1,
human H3N2 (Goto et al. 1992), and an H1N2 which is a
reassortant with the H1 from a recent-lineage swine
H1N1 and the N2 from an early H3N2 (Ouchi et al.
1996). Antibodies against H3N2 virus were demonstrat-
ed in 10% of 140 swine tested on Andaman and Nicobar
Islands (Joshi et al. 1993). Yamaoka et al. (1991) report-
ed that 19% of 240 swine sera tested had antibody
against type C influenza virus.
ETIOLOGY
SI is caused by influenza A viruses, in the family
Orthomyxoviridae. An enormous amount of information
is available on antigenic, genetic, structural, and biologic
characteristics of influenza A viruses (for review, see
Murphy and Webster 1990 and Lamb and Krug 1996). SI
viruses are pleomorphic, medium-sized, enveloped viri-
ons with glycoproteins, commonly called “spikes,” ex-
tending from their surface. These glycoproteins are the
major surface antigens and are of two distinct kinds:
hemagglutinin (H) and neuraminidase (N). Hemagglu-
tinin is responsible for attachment of viruses to cells and
causes agglutination of erythrocytes. Neuraminidase is
responsible for the enzymatic elution of virus from ery-
throcytes, and it may play a role in the release of virus
from infected cells. Antibodies to the hemagglutinin are
of the most importance for preventing infection with an
influenza virus containing the same hemagglutinin,
whereas antibodies against the neuraminidase restrict
the spread of virus from infected cells. The H and N gly-
coprotein “spikes” are embedded in a lipid envelope that
surrounds the core of the virus particle. Matrix (M) pro-
tein molecules line the underside of the envelope and
surround the core, inside which is a helical complex of
molecules consisting of ribonucleic acid (RNA) in associ-
ation with viral nucleoprotein (NP) and polymerases (en-
zymes that initiate replication). Influenza viruses are
classified as type A, B, or C, based on the antigenic relat-
edness of the NP and M proteins. The viral genome con-
sists of 8 single-stranded RNA segments that encode 10
viral proteins.
The antigenic characteristics of the two types of sur-
face glycoproteins serve as the basis for dividing the
viruses into subtypes. So far, 15 hemagglutinins and 9
neuraminidases have been identified among all influenza
A viruses. The current system of nomenclature of in-
fluenza viruses, introduced in 1980, designates the type,
host, place, strain number (if any), year of isolation, and
antigenic subtype (WHO 1980). For example, a SIV iso-
lated in Wisconsin in 1984 would be designated
 CHAPTER 22 SWINE INFLUENZA Easterday, Van Reeth
A/Swine/Wis/1/84(H1N1). The first character, A, iden-
tifies the type and is followed by the host of origin (ex-
cept human), geographic origin, strain number, the year
of isolation, and the antigenic subtype of the H and N in
parentheses.
The three most common subtypes of influenza that
circulate among swine worldwide are the “classic” H1N1,
the avianlike H1N1, and the humanlike H3N2. Immuni-
ty against an H1N1 influenza virus does not provide pro-
tection against the viruses of the H3N2 subtype. Anti-
genic characterizations of classic H1N1 viruses (Sherrar
et al. 1989) and avian H1N1 variants (Scholtissek et al.
1983) indicate that these viruses have remained remark-
ably conserved since their introduction into swine popu-
lations. An H1N1 virus was isolated in the United States
with slight antigenic variations in its hemagglutinin
(Olsen et al. 1993), but such events are uncommon.
H3N2 viruses are somewhat less stable, and recent iso-
lates may show minor antigenic variations when com-
pared to the older prototype viruses (Haesebrouck and
Pensaert 1988; Kaiser et al. 1991). However, Bikour et
al.(1995a) showed that one of viruses responsible for SI
in Quebec was similar to early human H3N2 viruses. In
contrast to the influenza viruses that circulate in swine,
the human H1N1 and H3N2 viruses continue to have
marked antigenic changes. The reason for this is not en-
tirely clear, but it seems likely that there is little immune
pressure on swine viruses because pigs are short-lived
and their viruses are continually transmitted to nonim-
mune, susceptible pigs.
Because the viral RNA is segmented, genetic ex-
change, or reassortment, between different influenza A
viruses can (and does) occur during mixed infections.
Genetic reassortment between influenza viruses of hu-
man and nonhuman origin is considered a likely mecha-
nism for the origin of new human pandemic strains. In
support of this, human and avian virus reassortants
(H1N1) have been isolated from pigs reared and kept un-
der commercial conditions and from children associated
with infected pigs (see below). In addition, other reassor-
tant viruses have also been isolated from swine. Viruses
of the H1N2 subtype were isolated from swine in Japan
in 1978 (Sugimura et al. 1980), in France in 1987 and
1988 (Gourreau et al. 1994), and in Great Britain in 1994
(Brown et al. 1995). H3N1 (Brown et al. 1993b) and
H1N7 (Brown et al. 1994) virus subtypes were isolated in
Great Britain in the early 1990s. Except for the H1N2
virus in Great Britain, these reassortants did not spread
in the swine populations. Antibodies against type B and
type C influenza viurses have been reported in swine in
Great Britain (Brown et al. 1995).
Laboratory Cultivation
SIV grows readily when inoculated into embryonated
chicken eggs. It may be inoculated by either the intraal-
lantoic or the intraamniotic routes. The temperature of
279
incubation ranges from 33˚C to 37˚C. The infected em-
bryos usually do not die, and the presence of the virus is
demonstrated after 48–72 hours of incubation by
hemagglutination tests on the allantoic and/or amniotic
fluids. With the diversity of influenza viruses that have
been recovered from swine, it is reasonable to expect cul-
tural differences, for example, temperature of incuba-
tion for optimal growth, time of incubation, and embryo
death. Although the embryonated chicken egg is the cul-
ture system most commonly used, various cell culture
systems have been used for the growth and assay of SIV.
These include calf kidney cells, fetal pig lung cells, canine
kidney cells, pig kidney cells, chicken embryo fibro-
blasts, human diploid cells, and Chang conjunctival cells
(Easterday 1975). Other cell culture systems include a pig
oviduct cell line (Bouillant et al. 1975) and a swine testi-
cle cell line (Potgeiter et al. 1977). Fetal pig trachea, lung,
and nasal epithelial organ cultures and tracheal organ
cultures from chickens, horses, and ferrets also support
the growth of SIV (Nakamura and Easterday 1970;
Schmidt et al. 1974).
EPIDEMIOLOGY
Major considerations in any discussion of the epidemi-
ology of SI include the epizootic characteristics in swine
and interspecies transmission and public health aspects.
Acute Swine Influenza
The first appearance of SI in a swine population is com-
monly associated with the movement of animals, for ex-
ample, the introduction of breeding stock, introduction
of feeder pigs, or return of show stock to the farm. Many
outbreaks are clearly related to the movement of animals
from infected to susceptible herds. It is commonly re-
ported that outbreaks are explosive, with all of the pigs
in the herd becoming ill at the same time. However, own-
ers who observe their herds closely are usually aware of
one to a few pigs with signs of the disease 2–5 days be-
fore the whole herd is involved.
The primary route of transmission is presumed to be
direct pig-to-pig transmission by the nasopharyngeal
route. Nasal secretions are laden with virus during the
acute febrile stages of infection, providing an abundant
source of infectious materials to infect susceptible ani-
mals. Swine are readily infected under experimental pro-
cedures by instillation of virus suspensions into the nos-
trils or by exposure to small-particle aerosols. Contact
transmission is easily demonstrated under experimental
conditions. In densely swine-populated regions, air-
borne spread may contribute to explosive epidemics over
large geographic areas, particularly in an immunologi-
cally naive population. This was the case in the late 1970s
and early 1980s in Belgium and France (Vandeputte et al.
1980; Gourreau et al. 1980). In Brittany (France), the first
outbreaks occurred in December 1981, and 70% of the
280 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
herds (4 million swine) were infected by September 1982
(Gourreau et al. 1980).
Once the infection has appeared in a swine breeding
operation or any situation where there is no complete de-
population, the possibility of continuous virus circula-
tion exists (Madec et al. 1985). Under these conditions,
pigs may become infected at a very young age in the pres-
ence of waning maternal immunity. In most instances,
however, influenza viruses disappear from a herd after an
outbreak. Depending on their prevalence in a particular
region, the viruses may be introduced at some later time
(months or years), causing infections of the seronegative
breeding and fattening stock. The prevalence and distri-
bution of influenza viruses and the prevailing subtypes
are different in different parts of the world, and there
may be regional differences within a country or conti-
nent.
Episodes of acute SI in the north-central United
States have historically been reported from the late sum-
mer into the winter, although it is clear that the viruses
are circulating throughout the year. These outbreaks of-
ten coincide with the onset of marked fluctuations in
outdoor temperatures from moderate daytime tempera-
tures to below freezing at night and with cold autumn
rains. As more swine are held in confinement housing
and not subjected to those environmental fluctuations,
the disease may become less seasonal in nature. During
one period of 14 months, Hinshaw et al. (1978) conduct-
ed extensive virologic and serologic surveillance on
slaughter swine in the north-central and south-central
United States. They recovered 478 influenza viruses from
approximately 9400 swine tested (about 5% infectivity
rate). Antibodies against SIV (H1N1) were found in 21%
of those swine. Studies in both areas revealed the pres-
ence of viruses in the pigs throughout the year, but the
frequency of virus isolation was much higher in the
northern states. The north-central states showed peak
virus activity from October to December, whereas the
southern states did not have a marked peak other than a
slight increase in the spring months. These results are in
agreement with those of Nakamura et al. (1972) and Pir-
tle et al. (1976), who reported a similar prevalence and
presence throughout the year. A serologic survey (Cham-
bers et al. 1991) on U.S. pigs in 1988–89 indicated that
51% of pigs in the north-central United States had anti-
bodies to H1N1 viruses, emphasizing the continued cir-
culation of this virus at high frequency.
Over the years there has been speculation about a car-
rier state that would provide for the interepizootic sur-
vival of SIV. The widespread occurrence of SI in the Unit-
ed States and other parts of the world at all times of the
year supports the probability that the virus is constantly
circulating. There are no clear data to support or reject
the existence of a long-term true carrier state of influen-
za viruses in swine.
In Europe, avianlike H1N1 and H3N2 influenza virus
strains cocirculate continuously at a high frequency and
have become enzootic in many regions. Serologic exami-
nation of finishing pigs has shown the prevalence of the
H1N1 and H3N2 strains to be 92% and 57%, respective-
ly, in Belgium (Maes et al. 1996), 60% and 30% in the
Netherlands (Elbers et al. 1990), 73% and 62% in Spain
(Yus et al. 1992), and 55% and 51% in Germany
(Groschup et al. 1993). In some countries the distribu-
tion is not uniform throughout the country, a situation
which is most likely a function of the differences in swine
density and management-related risk factors.
In the enzootic regions, it is unlikely that many farms
escape repeated exposure to the viruses. Consequently,
the majority of the litters on a farm are born from im-
mune sows and acquire serum antibodies through
colostrum. The colostral antibody may provide protec-
tion against disease but may not provide protection
against infection. In herds with continuous virus circula-
tion, young pigs may become infected in the presence of
maternal antibody. A typical infection pattern has been
observed in commercial finishing herds operating on an
all-in/all-out basis. In this situation, pigs usually enter
such units at 9–12 weeks of age, and they are likely to
originate from multiple breeding herds. The chance is
high that some of those animals will be infected when
they arrive in the finishing unit. A serologic study in
commercial fattening herds in Belgium showed that 17 of
17 groups became infected with H1N1 virus within the
first few weeks after entry and that 9 of 17 groups had
been infected with the H3N2 virus during the same peri-
od (Van Reeth and Pensaert 1994a). Some of the groups
that were negative for H3N2 became infected later dur-
ing the fattening period. Serologic data from this and
other studies (Madec et al. 1987; Elbers et al. 1990; Laval
et al. 1991; Houben et al. 1995) document that intercur-
rent infections with H1N1 and H3N2 influenza viruses,
the porcine respiratory coronavirus (PRCV), and the
porcine reproductive and respiratory syndrome virus
(PRRSV) are common in intensive production systems in
Europe.
INTERSPECIES TRANSMISSION
Influenza A viruses infect many different species in na-
ture, including humans, lower mammals (including ma-
rine mammals), and birds. It is clear that swine are in-
volved in the natural exchange of these viruses (Hinshaw
et al. 1984). Swine H1N1 viruses can be introduced into
avian populations and have been responsible for disease
in turkeys (Mohan et al. 1981; Hinshaw et al. 1983; An-
dral et al. 1985; Ludwig et al. 1994).
Infection of swine with human H3N2 viruses has
been conclusively demonstrated. An H3N2 virus, like the
A/Hong Kong/68 virus, was isolated from pigs in Taiwan
soon after it appeared in the human population (Kundin
1970). Subsequently, several of the later variants of the
human H3N2 viruses were found to have been transmit-
 CHAPTER 22 SWINE INFLUENZA Easterday, Van Reeth
ted to pigs (Romvary and Tanyi 1975; Tumova et al.
1976). These viruses continue to be isolated from pigs
many years after their disappearance as epidemic strains
from the human population (Haesebrouck et al. 1985;
Mancini et al. 1985; Pritchard et al. 1987; Castro et al.
1988; Bikour et al. 1995b; W. Loeffen, pers. comm.—
1996) and cause significant disease outside the United
States.
Public Health Aspects
A major milestone in the chronology of zoonotic in-
fluenza came in January 1976 with the isolation of an in-
fluenza virus, A/NJ/8/76(H1N1), closely related to SI
viruses, from sick U.S. military recruits at Fort Dix, N.J.
(Goldfield et al. 1977; Kendal et al. 1977). There were
many cases of acute respiratory disease, and serologic in-
vestigations revealed that several hundred recruits had
been infected. Extensive epidemiologic investigations
could not identify infected or sick pigs that might have
been the source of the virus for that outbreak. There was
speculation and concern that the virus would be the new
epidemic strain of influenza for humans—perhaps a re-
turn of the virus that was responsible for the 1918 pan-
demic. Consequently, a national program was initiated
to vaccinate humans against A/NJ/8/76(H1N1) virus,
and a surveillance program was also initiated to investi-
gate pigs and their human contacts.
Speculation about the zoonotic nature of SIV came to
an end in November 1976 when SIV (H1N1) was isolat-
ed from pigs and their caretaker on a farm in southern
Wisconsin (Easterday et al. 1976; Hinshaw et al. 1978).
Pigs had been sick for 2 or 3 days with classic SI clinical
signs when one of the caretakers also became ill. He had
moderate to severe symptoms of influenza requiring bed
rest for 2 days. Nasal swabs from the pigs and throat
washings from the man were collected on the same day.
The virus was recovered from six of eight pigs sampled
and from three throat washings collected from the young
man over a period of approximately 18 hours. There was
no evidence that the virus spread from the caretaker to
any of his family or other human contacts. The charac-
terization of the viruses from the man and the pigs indi-
cated they were identical (Palese and Ritchey 1977; Hin-
shaw et al. 1978). Two weeks later, a similar case
occurred in a 14-year-old boy on a farm approximately
100 km away. In that case the virus was isolated from one
of five pigs and from one throat washing taken from the
boy. Further testing indicated that the virus had spread
from that boy to at least one and probably three of his
close schoolmates.
Dasco et al. (1980) reported the isolation of an H1N1
virus from a young man who had worked at a large swine
show. Patriarca et al. (1984) recovered an H1N1 virus
similar to contemporary strains isolated from swine in
the United States following a fatal infection in an im-
munocompromised child. Extensive epidemiologic in-
281
vestigation did not reveal a probable animal source of
the virus. Other cases include the recovery of a swine
H1N1 virus from a 32-year-old pregnant woman who
died of primary viral pneumonia after she attended a
livestock show where sick pigs were present (Kaufman et
al. 1988; Wells et al. 1991); the recovery of a swine H1N1
virus from a 27-year-old male animal caretaker who died
with acute respiratory distress syndrome (Wentworth et
al. 1994); and the recovery of H1N1 viruses from two
people in Switzerland and one person in the Nether-
lands, two of whom had been in contact with diseased
pigs. The Swiss patients had mild symptoms and the
Dutch patient had severe pneumonia (de Jong et al.
1988). Infection and mild disease occurred in two indi-
viduals who became infected with a swine H1N1 virus
while doing experimental infections in pigs (Wentworth
et al. 1997). In 1993, two H3N2 viruses, probably of pig
origin, were isolated from young children in the Nether-
lands (Claas et al. 1994).
These documented cases of transmission of SIV re-
sulting in acute, fatal respiratory disease in humans in
contact with swine constitute a clear signal of the
zoonotic potential of influenza viruses that infect swine
and the potential role of pigs in the transmission of new
pandemic strains to humans. Since there are thousands
of human-swine interactions daily in occupational and
casual situations, the zoonotic potential of SI viruses
must be recognized and respected.
CLINICAL SIGNS
Classic SI is a herd disease. The signs of the disease ob-
served now are essentially as they were described in the
1920s (Dorset et al. 1922; McBryde 1927; Shope 1931a).
The onset is sudden, after an incubation period of 1–3
days. Most of the animals in the herd show signs at the
same time. There is anorexia, inactivity, prostration,
huddling, and piling. One can walk among the inactive
animals and they will not move, even with vigorous prod-
ding. There is also open-mouthed, labored, jerking, and
abdominal breathing, especially when the animals are
forced to move. Furthermore, moving may be accompa-
nied by severe paroxysms of coughing that may sound
like a herd of barking dogs. Fever is usually in the range
of 40.5–41.7˚C. One may also observe conjunctivitis,
rhinitis, nasal discharge, and sneezing. There is obvious
loss of weight and weakness related to the anorexia and
inactivity. Morbidity is high (near 100%) but mortality is
low (usually less than 1%) unless there are intercurrent
infections and/or the pigs are very young. Generally, re-
covery begins 5–7 days after onset and is as sudden and
remarkable as the onset. Acute outbreaks of clinically
typical SI, as described here, are generally limited to ful-
ly susceptible, seronegative pigs.
On the European continent, the sudden reintroduc-
tion of influenza viruses into temporarily negative
282 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
breeding-fattening farms is frequently associated with
acute disease in fattening swine of 50 kg and more. A
1996 longitudinal study in breeding-fattening herds in
the Netherlands demonstrated that the infection and
characteristic disease predominantly occur around the
age of 18 weeks (W. Loeffen, pers. comm.—1996). Most
breeding animals that have acquired active immunity as
a result of previous infections and their maternally im-
mune offspring remain unaffected. Infections with
H1N1 and H3N2 subtype viruses are clinically alike, and
both viruses have been correlated with acute respiratory
episodes in Belgium, France (Laval et al. 1991; Madec et
al. 1987), Spain (Castro et al. 1988), Italy (Barigazzi et al.
1996), and the Netherlands (W. Loeffen, pers. comm.—
1996).
Typical outbreaks of SI can be reproduced experi-
mentally only under very specific conditions. Inocula-
tion of fattening swine (±100 kg) with high virus doses
(107–107.5 egg infective doses 50 [EID50] per pig) of either
of the two influenza viruses H1N1 or H3N2 directly into
the trachea results in high fever, complete anorexia, and
dyspnea within 24 hours after inoculation (Maes et al.
1984). Mean growth arrest periods of 5–8 days and
mean weight losses of 5–6 kg have been recorded. Nev-
ertheless, recovery was very rapid, with clinical signs
lasting only 2 days. Conversely, inoculation of the same
amount of virus by the less invasive oronasal route leads
to mild clinical signs or a subclinical infection. So far,
there are no indications of differences in the site of repli-
cation in the lungs with different influenza virus strains.
In addition to the clinically apparent disease, subclin-
ical infections frequently occur, as indicated by the high
seroprevalence of both subtypes in finishing pigs in the
absence of significant respiratory disease during the fat-
tening period. Multiple factors, including immune sta-
tus, age, infection pressure, intercurrent infections, cli-
matic conditions, and housing, may determine the
clinical outcome of an infection with influenza virus. Al-
though infection occurs throughout the year, the clinical
disease is observed mainly in the colder seasons.
Intercurrent infections are among the most impor-
tant complicating factors of an influenza virus infection.
It has been known for years that secondary infections by
respiratory bacteria such as Actinobacillus pleuropneumo-
niae, Pasteurella multocida, Haemophilus parasuis, and
Streptococcus suis type 2 complicate the severity and
course of an infection with influenza viruses. More re-
cent observations under natural conditions suggest that
respiratory viruses, as well, act as complicating factors. It
has been shown that the prevalence of dual or triple in-
fections with influenza virus(es) and either PRCV or
PRRSV is extremely high in intensive fattening units in
Europe (Madec et al. 1987; Van Reeth and Pensaert
1994a; Houben et al. 1995). The associated clinical pic-
ture is less characteristic than that of acute SI outbreaks,
and usually only 20–50% of pigs in the herd show respi-
ratory disease problems, fever, and decreased feed in-
take. Nevertheless, financial losses result from high costs
for medication, increased mortality, and generally poor
performance.
The hypothesis that influenza viruses may precipitate
a more severe disease in combination with other respira-
tory viruses has been supported by experimental dual-in-
fection studies (Van Reeth and Pensaert 1994a; Van
Reeth et al. 1996a; Van Reeth et al. 1996b). In these stud-
ies, fever, respiratory disease, and growth retardation
were significantly more severe and lasting in dual infec-
tions than in single-virus-infected pigs. Nevertheless,
several unknown factors seem to be critical to the devel-
opment of clinical effects following dual virus infections.
To illustrate, the effect of experimental PRRSV-SIV in-
fections is not reproducible to the same level in different
experimental groups. Furthermore, other researchers
did not observe an enhancement of clinical signs follow-
ing simultaneous inoculation of pigs with PRCV and ei-
ther H1N1 or H3N2 viruses (Lanza et al. 1992).
A growing body of evidence suggests that the “avian
variant,” or avianlike, H1N1 strains circulating in Europe
have acquired a more pathogenic character than the
“classic” H1N1 strains that circulate in the United States
as well as in other countries. Observations and reports
suggest that SI now occurs more frequently and has a
greater economic significance in Europe than in the Unit-
ed States.
Producers and veterinarians occasionally report re-
productive, farrowing, and neonatal problems such as
abortions, stillbirths, infertility, and small weak litters
associated with outbreaks of SI. There are insufficient
data to conclude that SI viruses have a specific and sig-
nificant adverse association with reproductive, farrow-
ing, and perinatal problems.
PATHOGENESIS
Infection with influenza virus is generally limited to the
respiratory tract, and viremia has only rarely been de-
tected. Brown et al. (1993a) reported virus isolation from
the serum of experimentally infected pigs 1 and 3 days
postinoculation. However, the virus could be isolated
during only one day, and virus titers in the serum sam-
ples were low. Attempts to demonstrate virus replication
in extrarespiratory sites have been largely unsuccessful.
Virus replication has been demonstrated in nasal mu-
cosa, tonsils, trachea, tracheobronchial lymph nodes,
and lungs (Lanza et al. 1992; Nayak et al. 1965). The
lungs seem to be the major target organ.
Following intratracheal inoculation of pigs, virus
titers in the lungs may be greater than 108 EID50 /gram of
tissue (Haesebrouck and Pensaert 1986). The amount of
virus that reaches the deeper airways and the resulting
production of infectious virus in the lungs seem to de-
termine the severity of illness. Experimental inoculation
 CHAPTER 22 SWINE INFLUENZA Easterday, Van Reeth
of high virus quantities (107–107.5 EID50) via the nasal
route in fattening swine (±100 kg) resulted in subclinical
infections, whereas intratracheal inoculation at the same
virus dose produced typical clinical signs within 24
hours (Maes et al. 1984). Immunofluorescence studies of
lung tissue demonstrate the rapidity of virus replication
and the highly specific tropism for bronchiolar epitheli-
um (Nayak et al. 1965; Haesebrouck and Pensaert 1986;
Brown et al. 1993a). So far, there are no indications of
differences in the site of replication in the lungs with dif-
ferent influenza virus strains.
In the studies by Nayak et al. (1965), bronchial ep-
ithelial cells stained positive within 2 hours postinfec-
tion. By 16 hours, there were large fluorescent areas of
bronchial epithelium. Staining was intense through 72
hours postinfection and then diminished. Antigen was
also detected in the alveolar septa within 4 hours after in-
fection, and at 24 hours there were numerous fluorescent
cells in the alveoli and alveolar ducts. Fluorescent stain-
ing in both bronchioli and alveoli disappeared by day 9.
Immunofluorescence of bronchi may involve nearly
100% of the epithelial cells (Haesebrouck and Pensaert
1986). Typically, bronchi and bronchioli contain exudate
with degenerated and detached fluorescent mucosal cells
and neutrophils.
Little information is available about influenza virus
pathogenesis at the cellular level. Recent studies in
colostrum-deprived pigs suggest that bronchoalveolar
production of the cytokines tumor necrosis factor and
interleukin-1 contributes to the typical constitutional ef-
fects and lung inflammatory changes following virus in-
fection (Van Reeth and Pensaert 1997). In most experi-
mental studies, virus clearance was extremely rapid. SIV
could not be isolated from lungs or other respiratory
tract tissues on or after day 7 (Brown et al. 1993a; Van
Reeth and Pensaert 1994b). Using highly sensitive ELISA
techniques, influenza-specific antibodies could be de-
tected in serum on day 3 and nasal swabs on day 4 (Lee
et al. 1995).
LESIONS
Gross Lesions
The gross lesions found in uncomplicated SI are mainly
those of a viral pneumonia. The changes are most often
limited to the apical and cardiac lobes of the lungs, al-
though in severe cases more than half of the lung may be
affected. Generally, there is a sharp line of demarcation
between the affected and normal lung tissue. The in-
volved areas will be purple and firm. Some interlobular
edema may be evident. The airways may be filled with
blood-tinged, fibrinous exudate, and the associated
bronchial and mediastinal lymph nodes are usually en-
larged. In severe cases there may be fibrinous pleuritis
(Shope 1931a; Nayak et al. 1965; Nakamura 1967). The
lesions in naturally occurring SI are often complicated or
283
masked by intercurrent, especially bacterial, infections.
Morin et al. (1990) described a severe proliferative
and necrotizing pneumonia (PNP) in pigs as a newly rec-
ognized disease in the province of Quebec, Canada. They
reported confluent consolidation of the cranial and mid-
dle lobes and the lower half of the caudal lobes. The
lobes were not collapsed; they were red or gray, “moist
and meaty,” and interlobular edema was common. Type
A influenza virus was isolated from the lungs of affected
animals. Subsequently, Girard et al. (1992) reproduced
the disease with the virus isolated by Morin et al. (1990).
Dea et al. (1992) reported similar lesions, especially the
consolidated lungs. They concluded that the virus was a
swine H1N1 distantly related to the contemporary H1N1
viruses circulating in North America. Rekik et al. (1994)
concurred that a new antigenic variant of H1N1 swine
influenza virus was associated with the PNP. However,
Bikour et al. (1994a) described a virus isolated from cas-
es of PNP in Quebec as a “new strain isolated for the first
time in the porcine population of Canada [that] is relat-
ed to A/Sw/Hong Kong/76 H3N2 swine influenza
virus.” Thus, it is not clear what factor(s) are responsible
for this unique pathological lesion. Winkler and Cheville
(1986) reported that following inoculation of virus di-
rectly into the trachea of anesthetized pigs, they ob-
served dark areas of consolidation over all lung lobes.
These areas were small and covered a small portion of
the lung 24 hours after infection, but by 48–96 hours,
confluent areas of consolidation completely covered the
lung lobes, and the lung tissue was consistently firm.
Differences in lesion severity have been observed
with the different influenza virus variants circulating in
Great Britain. Classical H1N1 and H3N2 strains circulat-
ing since 1986 produced minimal gross lesions and a
very mild interstitial pneumonitis, both in natural cases
and experimentally (Done et al. 1994). The more recent
H1N1 variant, on the other hand, produced marked
gross lesions and more severe histopathological changes
involving severe necrosis of bronchial epithelium, alveo-
lar exudation, and neutrophil infiltration (Brown et al.
1993a). These findings have been confirmed experimen-
tally. The pathological data provide further evidence of
an increased pathogenicity of the avian H1N1 strains in
Europe compared to the classic H1N1 strains circulating
in the United States.
Microscopic Lesions
As with the gross lesions, the microscopic lesions in un-
complicated cases of SI are consistent with viral pneu-
monia. Bachmann (1989) provided a concise summary
of the microscopic lesions of SI: “Histologically, a wide-
spread degeneration and necrosis of the epithelium in
bronchi and bronchioli can be observed. The lumen of
bronchi, bronchioli, and alveoli are filled with exudate
containing desquamated cells and neutrophils, later
mostly monocytes. Furthermore, variable hyperemia
284 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
with dilatation of the capillaries and infiltration of the
alveolar septae with lymphocytes, histiocytes, and plas-
ma cells occurs. Widespread alveolar atelectasis, intersti-
tial pneumonia, and emphysema accompany these le-
sions. There is also peribronchial and perivascular
cellular infiltration (Shope 1931a; Urman et al. 1958;
Witte et al. 1981).” The PNP histopathologic lesions are
described extensively by Morin et al. (1990), Girard et al.
(1992), and Dea et al. (1992). Lesions described include
exudative lesions characterized by alveoli filled with pro-
tein-rich edema and large macrophages; necrotizing
bronchiolitis affecting mainly terminal bronchioles; co-
agulates of necrotic cells in alveolar ducts and alveoli;
and proliferative lesions marked by proliferation of type
II pneumocytes responsible for alveolar epithelialization
and hyaline membrane formation in the lumen of the
terminal alveolar ducts.
DIAGNOSIS
Virus isolation and/or the detection of specific antibody
are necessary for a specific diagnosis of SI. SI may be sus-
pected when there is an outbreak of acute respiratory
disease involving most or all of the pigs in a herd, espe-
cially in the colder seasons of fall and early winter. SI
must be differentiated from a variety of respiratory dis-
eases of swine.
The best sample for virus isolation from a live animal
is nasal mucus obtained by swabbing the nasal passages,
or in the case of very small pigs where it is difficult to
swab the nasal passages, pharyngeal mucus may be ob-
tained by swabbing. The virus is more likely to be found
in nasal and pharyngeal secretions during the febrile pe-
riod than after the fever has subsided. The swab is sus-
pended in a suitable transport medium (such as glycerol
saline). It is very important to keep the sample moist and
cold (refrigerator temperature, 4˚C) during transport to
the laboratory. If the samples for virus isolation can be
tested within 48 hours after collection, they should be
kept at 4˚C. If the samples must be held longer, storage
at −70˚C is recommended. Storage in sealed containers
in dry ice or liquid nitrogen is appropriate, but the virus-
es are not stable at −20˚C. Filtration of the samples
should be avoided to conserve small amounts of virus
that might be in the transport medium. Adventitious
bacterial and fungal agents may be controlled with the
addition of appropriate antimicrobial agents to the
transport medium. The virus may also be isolated from
lung tissue of pigs that die or are killed during the acute
stage of the disease. The lung tissue should be held un-
der the same conditions as swab material until ready for
culturing. At that time the tissue is ground (minced) and
suspended in saline.
Chicken eggs, embryonated for 10 days, remain a re-
liable and economical culture medium for isolating in-
fluenza viruses. The inoculated eggs are generally incu-
bated at 35˚C. Since SIV usually does not kill the chick
embryo, allantoic fluid is collected after 72 hours incuba-
tion and tested for its ability to agglutinate chicken red
blood cells, which is presumptive evidence for the pres-
ence of an influenza virus. The hemagglutinin subtype is
determined by the hemagglutination-inhibition (HI)
test, and the neuraminidase subtype is determined with
the neuraminidase-inhibition (NI) test (Centers for Dis-
ease Control 1982). Cell culture systems are more com-
monly used for the isolation of influenza viruses from
human specimens.
Serologic diagnosis of SI requires the use of paired
serum samples, one obtained during the acute phase of
the disease and the second 3–4 weeks later, to demon-
strate an increase in the amount of antibody when tested
against appropriate antigens. The most common test for
antibody is the HI test. The diagnostician must be aware
of the possibility of the presence of nonspecific in-
hibitors and nonspecific agglutinins found in swine
serum that may interfere with the test.
Other methods for detecting virus, viral antigen, or
specific antibody are direct immunofluorescence tech-
niques applied to lung tissue (Barigazzi et al. 1993), indi-
rect immunofluorescence applied to nasal epithelial cells
(Onno et al. 1990), immunofluorescence microscopy ap-
plied to bronchioalveolar lavage contents (Runge et al.
1996), immunohistochemical detection in fixed tissue
(Haines et al. 1993), enzyme-linked immunosorbent as-
say (ELISA) (Lee et al. 1993a, b, 1995), polymerase chain
reaction (PCR) (Schorr et al. 1994), rapid cell culture as-
say using immunoperoxidase staining for typing and
subtyping (Ziegler et al. 1995), and an enzyme im-
munoassay membrane test (Directigen FLU-A) which
rapidly detects influenza A antigen in clinical specimens
(Ryan-Poirier et al. 1992). Because of the large number of
reagents necessary for the specific identification of in-
fluenza viruses, the definitive determination of the anti-
genic character of the virus(es) involved is generally
done by a designated reference center.
Positive diagnosis of the disease by serologic and vi-
rologic means in suckling or weanling pigs from dams
with antibody to the virus may be complicated. Maternal
antibody persists for 2–4 months, depending on the ini-
tial level. It has been shown that weanling pigs with ma-
ternal antibody may be infected and may shed virus. The
rate of virus recovery and severity of signs of disease are
inversely related to level of maternal antibody (Easter-
day 1971, 1972; Renshaw 1975). Depending on the level
of antibody, it may be very difficult to isolate virus. In ad-
dition, the level of antibody in the convalescent-phase
serum will be lower than in the acute-phase serum be-
cause of the inhibition of active antibody production by
maternal antibody (Mensik 1960, 1963, 1966; Easterday
1971; Renshaw 1975). After maternal antibody is deplet-
ed, pigs may be infected again, shed virus, have signs of
disease, and have a typical primary antibody response.
 CHAPTER 22 SWINE INFLUENZA Easterday, Van Reeth
TREATMENT
There is no specific therapy for SI. Careful nursing is im-
portant, with the provision of comfortable, draft-free
shelter and clean, dry, dust-free bedding. To avoid addi-
tional stress, pigs should not be moved or transported
during the acute stages of the disease. Fresh, clean water
should be accessible at all times, because most animals
will be febrile. There is a marked loss of appetite during
the course of the disease, but it returns quickly with clin-
ical improvement. Expectorants are commonly used as a
herd treatment and are administered in the drinking wa-
ter. Antibiotics and other antimicrobial treatments have
been used on a herd basis to control concurrent or sec-
ondary bacterial infections. Individual animals may re-
quire additional supportive and antibacterial treatment
in severe cases.
Amantadine (1-adamantanamine) has been shown to
be effective in reducing the febrile response and shed-
ding of virus in experimentally infected pigs (McGregor
and Easterday 1979). However, there are no antiviral
treatments licensed for use against influenza in swine.
PREVENTION AND CONTROL
Standard biosecurity measures to prevent susceptible
animals from contacting infected animals are appropri-
ate to prevent the introduction of SI into a herd. Because
of the known occurrences of interspecies transfer of in-
fluenza viruses, biosecurity measures should include
measures to prevent contact with other species, especial-
ly avian species. Also important, humans suspected of
having influenza virus infection should be excluded from
contact with swine. Animals recovered from SI develop
hemagglutination-inhibiting, virus-neutralizing, com-
plement-fixing, and neuraminidase-inhibiting antibody.
The degree and duration of resistance to subsequent in-
fection in swine recovered from influenza have not been
precisely determined. Substantial levels of antibody may
be found for at least 6 months after infection. As with
many other infections, the relationship of the amount of
antibody in the serum or respiratory secretions to the de-
gree of resistance has not been defined, and there is con-
siderable variation in the antibody response of individ-
ual pigs following exposure.
Inactivated-virus vaccines have been developed
and/or tested by several investigators (Woods and
Mansfield 1976, 1978, 1980; Pirtle 1977). There is con-
siderable variation in antibody response and protection
from infection following vaccination. In a study using a
temperature-sensitive strain of H1N1, it was shown that
nonvaccinated, experimentally infected pigs required 2
weeks longer to reach market weight than their vaccinat-
ed exposed cohorts (Easterday et al. 1977). There is one
licensed vaccine, MaxiVac®-FLU, available in the United
States for the prevention of SI (Brown and McMillen
285
1994). It is an oil-in-water vaccine containing an unspec-
ified H1N1 virus that was developed for the protection of
weaned pigs and breeding animals against SI. The au-
thors conclude that it “is a safe and efficacious vaccine for
the prevention of clinical disease caused by SIV.” Bikour
et al. (1994b) have shown that a combination of Quil A
and Alhydrogel is a valuable immunopotentiating adju-
vant for immunizing swine against influenza virus.
In Europe, vaccination against SIV is a common prac-
tice. Inactivated whole-virus vaccines and split-product
vaccines prepared from detergent-treated virus are com-
mercially available. These vaccines contain H1N1 and
H3N2 influenza virus strains of human origin. The
H1N1 component, the prototype A/New Jersey/1/76
strain, confers a high degree of protection against the
“avian” H1N1 strains currently circulating in the Euro-
pean swine populations. As for the H3N2 component,
there should be careful consideration of the strain to be
included in vaccine to provide the best protection, be-
cause several H3N2 strains circulate in the swine popula-
tions (Haesebrouck et al. 1985).
The SIV vaccines are administered intramuscularly,
and protection is based on the presence of hemaggluti-
nation-inhibiting antibodies in serum. Two injections of
vaccine 3 weeks apart are preferred. Under experimental
conditions, one vaccination of fattening swine with
monovalent vaccine based on the A/New Jersey/1/76
(H1N1) strain or the A/Port Chalmers/1/73(H3N2)
strain resulted in clinical protection and reduced virus
replication in the lungs at challenge (Vandeputte et al.
1980; Haesebrouck and Pensaert 1986). Two doses of the
H1N1 vaccine a month apart induced complete protec-
tion against infection (Haesebrouck and Pensaert 1986).
There are contradictory reports about the economic
benefit of SI vaccination in the field. In commercial fat-
tening herds, SIV infections occur very soon after entry
of feeder pigs, so that immunity induced by vaccination
with inactivated vaccines comes too late. On closed
breeding-fattening farms in Belgium, vaccination of
feeder pigs is considered cost-efficient, particularly dur-
ing winter when infections are more harmful. Care
should be taken not to vaccinate before the age of 10
weeks to avoid interference by maternal immunity. One
vaccine for intradermal vaccination (by PIGJET) of feed-
er pigs has recently been licensed in Europe. Though vac-
cination by this method is highly laborsaving, the effica-
cy in the field remains to be proven. In view of the
extensive circulation of H1N1 virus in pigs and the anti-
genic conservation of these viruses, vaccination should
be a valuable component of SI control programs.
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 Swine Pox
23 J. A. House and C. A. House
Swine pox (SwP) was first reported in 1842 by Spinola in
Europe, and in 1929 by McNutt et al. in North America.
Worldwide in distribution, SwP is usually associated
with swine operations that have poor sanitation and/or
intensive breeding with open-herd management. SwP
causes little economic loss and can usually be differenti-
ated from other diseases by its clinical signs and epi-
demiology.
The clinical signs of infection may be caused by ei-
ther vaccinia virus (VV) or SwP virus (SwPV) (Man-
ninger et al. 1940; Shope 1940). Since the eradication of
smallpox (variola) and the subsequent cessation of vac-
cinia vaccination and VV infection of swine, the occur-
rence of SwP has decreased (Meyer and Conroy 1972).
ETIOLOGY
SwPV is classified in the family Poxviridae and is the only
member of the genus Suipoxvirus (Francki et al. 1991).
The large virion (300–450 × 176–260 nm) contains dou-
ble-stranded DNA in a characteristic poxvirus core or
nucleoid bordered by lateral bodies and surrounded by
an outer coat of numerous proteins. Because SwPV is en-
veloped, it is ether sensitive (Cheville 1966a). Typical of
poxviruses, it shares antigenic epitopes with members of
other poxvirus genera (Ouchi et al. 1992). The large
genome has room for many insertions, and the SwPV has
been considered a vector for the delivery of immunogens
to swine (Foley et al. 1991).
The virus, once adapted to grow in cell culture, read-
ily reaches high titers and maintains its pathogenicity for
swine (Kasza et al. 1960; Kasza and Griesemer 1962).
EPIDEMIOLOGY
SwP is a disease of pigs only; SwPV does not infect cattle,
horses, sheep, dogs, cats, domestic fowl, rabbits, guinea
pigs, rats, mice, or humans (Shope 1940; Schwarte and
Biester 1941; Datt 1964).
The pig louse (Haematopinus suis) serves as a me-
chanical vector and is considered the primary means of
transmission of SwPV (Shope 1940). The ventral distrib-
ution of pox lesions can be correlated to the habitat of H.
suis. Flies and mosquitoes have been implicated as me-
chanical vectors, and in this situation, pox lesions are
distributed on the backs and sides of affected pigs
(Schwarte and Biester 1941).
Occasionally, horizontal transmission may occur in
the absence of insects, since SwPV is shed in nasal and
oral secretions and from lesions. The SwPV is present in
infected epithelium and in dry scabs produced in the lat-
er stages of infection. Abraded skin can serve as the
route of entry for SwPV. The virus may persist for up to
a year in the desiccated form, likely accounting for the
persistence of SwP in affected herds.
The morbidity of SwP may approach 100% in young
stock up to 4 months of age where poor hygiene occurs.
The disease may have a seasonal incidence related to the
prevalence of insects. Mortality is usually less than 5%.
CLINICAL SIGNS
SwP is a typical pox disease, with the development of the
lesions progressing through the classic stages of macule
(reddening), papule (reddening with edema), vesicle (flu-
id exuding from the pox lesion), and pustule or crust for-
mation (drying of the lesion with scab formation) (Fig.
23.1). The vesicular stage is not readily observed in SwP.
The time from macule formation to scabbing and resolu-
tion of lesions is 3–4 weeks. A lymphadenitis may occur.
Complication with bacterial infection prolongs the reso-
lution of the lesion (Miller and Olson 1978, 1980).
Young animals are more severely affected than adults.
Suckling piglets may have lesions in the perioral epitheli-
um or a generalized disease with lesions all over the
body. Lesions are more prominent on the hairless parts
of the skin. Mechanical transmission by pig lice results
in lesions on the lower parts of the body, including the
udder and vulva. Transmission by flies and mosquitoes is
less common and may result in lesions on the dorsal
parts of the body, including the snout and ears. Sporadic
congenital infection has been reported (Borst et al. 1990;
Paton et al. 1990).
The incubation period following experimental intra-
dermal inoculation is 2–5 days. The incubation period
following intravenous inoculation is 10–14 days (Shope
1940) but can be as short as 5 days (Kasza and Griesemer
1962), the range likely being related to the inoculation
dosage. Incubation under field conditions may be up to
14 days.
291
292 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
PATHOGENESIS
SwP is usually initiated by SwPV entering a break in the
skin such as a louse bite or an abrasion. The SwPV repli-
cates in the cytoplasm of the cells of the stratum spi-
nosum. Lesions in local lymph nodes are not extensive,
and the virus is not readily isolated from them. Sec-
ondary lesions are believed to result exclusively from the
spread of the virus from an existing lesion, not by a cell-
associated viremia as in most other poxvirus infections.
The virus has not been recovered from the blood (Kasza
and Griesemer 1962; Shope 1940), but this may be due
to the fact that the SwPV is difficult to isolate, especially
when in low concentrations. Viremia must occur, as evi-
denced by congenital infection in piglets.
Following intravenous inoculation, gnotobiotic
piglets developed skin lesions uniformly over their entire
bodies but not in internal organs (Meyer and Conroy
1972), confirming the propensity of SwPV to infect the
integument. In gnotobiotic pigs, the macular stage was
not observed; therefore, the early reddening may be the
combined effect of reaction to louse bites, opportunistic
bacteria, and SwPV.
LESIONS
The gross lesions of SwP are described in the section on
clinical signs.
The histopathological changes caused by SwPV are
similar to those of other poxviruses (Kasza and Griese-
mer 1962; Cheville 1966a, b; Conroy and Meyer 1971;
Meyer and Conroy 1972; De Boer 1975). The virus repli-
cates in the stratum spinosum, causing hydropic degen-
eration. Sections stained with hematoxylin and eosin re-
veal one to three eosinophilic-staining intracytoplasmic
inclusion bodies (ICIBs) in some cells during all stages of
the infection. Characteristic central nuclear clearing,
23.1. Swine pox lesions on
the belly of a pig. Pa = papule;
Pu = pustule; N = nipple.
which is also observed with capripoxvirus infection of
sheep, goats, and cattle, occurs in infected epithelial cells
containing ICIBs and is a result of margination of chro-
matin with nuclear filamentous matrix formation. There
may be intercellular edema, which could account for the
transient vesicular stage. Acantholysis, the separation of
individual cells of the stratum spinosum with loss of
desmosomes and detachment into the vesicular fluid,
was not found with SwPV infection (Meyer and Conroy
1972). Necrosis of epithelial cells and the presence of
neutrophils and macrophages in the dermis and epider-
mis are common in later stages of the pox lesion (Fig.
23.2). The pustules involve the full thickness of epitheli-
um and may heal, leaving a light scar. Poxvirus particles
can be observed in the cytoplasm of infected cells by
electron microscopy (Fig. 23.3).
DIAGNOSIS
SwP is typified by the presence of pox lesions on the skin
of the belly, ears, snout, vulva, and back. Secondary bac-
terial infections may cause more extensive lesions and lo-
cal abscess formation. Diseases to consider in the differ-
ential diagnosis of SwP include classic vesicular diseases,
bites from needle teeth, pityriasis rosea, parakeratosis,
parasitic skin disorders, allergic skin reactions, early
stages of ringworm, staphylococcal or streptococcal epi-
dermitis, and cutaneous erysipelas.
The SwPV infection may be confirmed by detection
of viral antigens using immunofluorescence and electron
microscopy. The presence of ICIBs along with central nu-
clear clearing in affected epithelial cells is pathognomon-
ic for SwPV, as VV infection does not cause the central
nuclear clearing (Teppema and De Boer 1975). The virus
may be isolated by multiple passages (up to five) in swine
cell cultures and definitively identified by neutralization
with specific-reference antiserum.
 23.2. Swine pox lesions in the epidermis: ballooning
degeneration of cytoplasm (B), intracytoplasmic inclusion
bodies (I), and central nuclear clearing (N).

23.3. Electron micrograph of an epidermal cell with cytoplasmic degeneration and numerous
poxvirus particles (V); a second cell has marginated chromatin (MC) and a filamentous matrix
(F) in the nucleus, which corresponds to the central nuclear clearing seen histologically. Insert
shows typical poxvirus particles. (Courtesy of D. A. Gregg.)
294 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
Serologic diagnosis utilizes the agar gel immunodif-
fusion test or the more sensitive counterelectrophoresis
test (De Boer 1975). Swine do not develop high levels of
neutralizing antibody (Shope 1940; Kasza et al. 1960),
and negative results on the virus neutralization test
should not be interpreted as the absence of SwPV infec-
tion.
TREATMENT
There is no known treatment for SwP, although antibi-
otics may diminish the complications caused by infec-
tion with secondary bacteria. Affected animals should be
isolated. Ectoparasites should be controlled by the use of
licensed insecticides, and the premises should be aggres-
sively cleaned and disinfected.
PREVENTION
Vaccines for SwP have not been developed due to the rel-
atively low economic importance of this disease. Recov-
ered animals are specifically immune to SwP (Shope
1940) even in the absence of demonstrable neutralizing
antibody, implying the importance of cell-mediated im-
munity.
Animals introduced into herds should be carefully in-
spected for skin lesions and ectoparasites. Good hygien-
ic practices are essential for the prevention and control of
SwP.
REFERENCES
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skin of swine. Am J Pathol 49:339–352.
———. 1966b. Immunofluorescent and morphologic stud-
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Conroy, J. D. And Meyer, R. C. 1971. Electron microscopy of
swinepox virus in germfree pigs and in cell culture. Am J
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Datt, N. S. 1964. Comparative studies of pigpox and vac-
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De Boer, G. F. 1975. Swinepox: Virus isolation, experimental
infections and the differentiation from vaccinia virus in-
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Foley, P. L.; Paul, P. S.; Levings, R. L.; Hanson, S. K.; and
Middle, L. A. 1991. Swinepox virus as a vector for the de-
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Francki, R. B.; Faquot, C. M.; Knudsen, D. L.; and Brown, F.
1991. Classification and Nomenclature of Viruses. Fifth
Report of the International Committee on Taxonomy of
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Kasza, L.; Bohl, E. H.; and Jones, D. O. 1960. Isolation and
cultivation of swinepox virus in primary cell cultures of
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Manninger, R.; Csontos, J.; and Salyi, J. 1940. Über die Ati-
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Am Vet Med Assoc 74:752.
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cutaneous streptococcal abscesses and swinepox in a
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———. 1980. Experimental induction of cutaneous strep-
tococcal abscesses as a sequela to swinepox. Am J Vet Res
41:341–347.
Ouchi, M.; Fujiwara, M.; Hatano, Y.; Yamada, M.; and Shi-
ro, N. H. 1992. Analysis of swinepox virus antigens us-
ing monoclonal antibodies. J Vet Med Sci 54:731–737.
Paton, D. K.; Brown, I. H.; Fitton, J.; and Wrathall, A. E.
1990. Congenital pig pox: A case report. Vet Rec
127:204.
Schwarte, L. H., and Biester, H. E. 1941. Pox in swine. Am J
Vet Res 2:136–140.
Shope, R. E. 1940. Swine pox. Arch Gesamte Virusforsch
1:457–467.
Spinola, M. 1842. Krankheiten der Schweine. Ed. A.
Hieschwald. Berlin, p. 204.
Teppema, J. S., and De Boer, G. F. 1975. Ultrastructural as-
pects of experimental swinepox with special reference to
inclusion bodies. Arch Virol 49:151–163.
24 Transmissible Gastroenteritis and
Porcine Respiratory Coronavirus
L. J. Saif and R. D. Wesley

Transmissible gastroenteritis (TGE) is a highly conta- early spring; and (4) the commercial vaccines available
gious, enteric viral disease of swine characterized by are of limited effectiveness (Bohl et al. 1975; Moxley and
vomiting, severe diarrhea, and a high mortality (often Olson 1989a; Saif and Theil 1990).
100%) in piglets under 2 weeks of age. Although swine of
all ages are susceptible to this viral infection, the mortal- ETIOLOGY
ity in swine over 5 weeks of age is low.
The disease was first reported by Doyle and Hutch- The viral etiology of TGE was suggested by the initial re-
ings (1946) as occurring in the United States in 1945, al- port of Doyle and Hutchings (1946) when they described
though it undoubtedly had existed prior to this time. the filterable nature of the infectious agent. TGEV is a
Subsequent to its recognition in the United States, TGE species belonging to the genus Coronavirus of the family
was reported in Japan in 1956 (Sasahara et al. 1958) and Coronaviridae (Cavanagh et al. 1995).
England in 1957 (Goodwin and Jennings 1958). Since
then it has been reported in most countries worldwide. Size, Morphology, and Morphogenesis
The disease is most frequently diagnosed and causes TGEV is enveloped and pleomorphic, with an overall di-
the most loss when it occurs in herds at farrowing time. ameter of 60–160 nm as viewed by negative-staining
In contrast, it often goes undiagnosed when it occurs in electron microscopy (EM) (Fig. 24.1) (Okaniwa et al.
growing, finishing, or adult swine because of the mild 1968; Phillip et al. 1971; Saif et al. 1977; Granzow et al.
clinical signs, which usually consist only of inappetence 1981). It has a single layer of club-shaped surface projec-
and diarrhea of a few days’ duration. Because of these tions that are 12–25 nm in length and widely spaced.
undiagnosed infections, serologic surveys are a more ac- The morphogenesis of coronaviruses has been re-
curate indication of the prevalence of TGE virus (TGEV). viewed (Siddell et al. 1983). TGEV antigen can be
Such surveys indicated that 19–54% of sampled swine demonstrated in the cytoplasm by immunofluorescence
herds in Europe and North America were seropositive for (IF) as early as 4–5 hours postinfection (Pensaert et al.
TGEV antibodies (Gagnon et al. 1974; Toma et al. 1978; 1970a). Maturation of virus occurs in the cytoplasm by
Egan et al. 1982), and a more recent survey confirmed a
similar TGEV seroprevalence (36%) in the United States
(USDA APHIS VS 1992). Currently in many European
countries, nearly 100% of swine herds are seropositive
for TGEV antibodies. This situation is due to a TGEV res-
piratory variant, porcine respiratory coronavirus
(PRCV), that appeared in 1984 and rapidly spread in Eu-
ropean swine (Brown and Cartwright 1986; Pensaert et
al. 1986; Pensaert 1989; Pensaert and Cox 1989; Laude et
al. 1993; Pensaert et al. 1993). Economic losses from TGE
can be severe, as reviewed in earlier studies (Pritchard
1982, 1987; Mousing et al. 1988). In North America, the
economic impact is still serious, but in Europe it has de-
creased following the establishment of enzootic PRCV
(Pensaert and Cox 1989; Laude et al. 1993).
In the densely swine-populated areas of North Amer-
ica, TGE remains a major cause of sickness and death in
piglets. Swine producers are especially apprehensive
about this disease because (1) mortality is high in new-
born pigs; (2) there is no effective, practical treatment; 24.1. Electron micrograph of a TGEV particle show-
(3) strict biosecurity measures are needed to prevent en- ing typical coronavirus morphology. Arrow points to the
try of the virus into a herd, especially in the winter and virus peplomers, or spikes. Bar = 100 nm.

295
296 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

budding through endoplasmic reticulum, and viral parti-

cles (65–90 nm in diameter) are often observed within
cytoplasmic vacuoles (Fig. 24.2A) (Thake 1968; Pensaert
et al. 1970b; Wagner et al. 1973). The virus is frequently
seen lining the host cell membranes after exit from in-
fected cells (Fig. 24.2B). TGEV glycoproteins have been
identified on the surface of infected swine testicular (ST)
cells (Laviada et al. 1990).

Biological Properties
TGEV is very stable when stored frozen but somewhat la-
bile at room temperature or above. No detectable drop in
titer occurred when virus of pig intestine origin was
stored at −20˚C for 6-18 months (Young et al. 1955; Hael-
terman and Hutchings 1956). In contrast, virus of pig in-
testine origin, when allowed to dry and putrefy at 21˚C,
was rather labile; after 3 days only two of four inoculated
pigs became sick; and after 10 days no viable virus was
detected by pig inoculation (Bay et al. 1952). When held
at 37˚C, there was a log 10 reduction in infectivity titer
every 24 hours (Young et al. 1955). Storage of cell culture
virus at −20˚C, −40˚C, or −80˚C for 365 days did not re-
sult in any significant drop in titer, whereas storage at
37˚C for 4 days resulted in total loss of infectivity (Hara-
da et al. 1968). Infectious virus persisted in a liquid ma-
nure slurry for more than 8 weeks at 5˚C, 2 weeks at
20˚C, and 24 hours at 35˚C (Haas et al. 1995).
The virus is highly photosensitive. Haelterman
(1963) reported that fecal material containing 105 pig-in-
fectious doses (PID) was inactivated within 6 hours when
exposed to sunlight. Cartwright et al. (1965) reported the
photosensitivity of a cytopathic strain when it was ex-
posed to ultraviolet light on a laboratory bench.
In terms of chemical stability, TGEV is inactivated by
exposure to 0.03% formalin, 1% Lysovet (phenol and
aldehyde), 0.01% beta propiolactone, 1 mM binary ethyl-
enamine, sodium hypochlorite, NaOH, iodines, quater-
nary ammonium compounds, ether, and chloroform
(Harada et al. 1968; Nakao et al. 1978; Brown 1981; Van
Cott et al. 1993).
As reported for other enteric viruses, TGEV field
strains are trypsin resistant, relatively stable in pig bile,
and stable at pH 3 (Harada et al. 1968; Moscari 1980a;
Laude et al. 1981). These properties allow the virus to
survive in the stomach and small intestine. However, at-
tenuated strains, as well as field strains, of TGEV vary in
these properties, and most studies have failed to show a
correlation between susceptibility to these various treat-
ments and cell culture passage level or degree of viru-
lence (Furuuchi et al. 1975; Hess and Bachmann 1976; 24.2. (A) TGEV in vesicles of the endoplasmic
Moscari 1980a; Laude et al. 1981). reticulum of a pig kidney cell (36 hours postinfection).
Bar = 100 nm. (B) TGEV lining the cell membrane of a
pig kidney cell (36 hours postinfection). N = nucleus;
Molecular Properties bar = 200 nm.
TGEV and the deletion mutant, PRCV, are enveloped
viruses containing one large, polyadenylated, single-
stranded, genomic RNA of positive-sense polarity. The
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
genome organization, replication strategy, and expres-
sion of viral proteins are similar to those of other coron-
aviruses (Laude et al. 1990; 1993; Enjuanes and Van der
Zeijst 1995). The genomic RNA of TGEV is infectious
(Brian et al. 1980), and the complete sequence of the
Purdue-115 strain of TGEV is 28,579 nucleotides long
(Kapke and Brian 1986; Rasschaert and Laude 1987;
Rasschaert et al. 1987; Eleouet et al. 1995).
TGEV has a buoyant density in sucrose of 1.18–1.20
g/mL (Brian et al. 1980; Jimenez et al. 1986). The phos-
pholipids and glycolipids incorporated into the virus en-
velope are derived from the host cell, and thus, the enve-
lope composition is host cell dependent (Pike and
Garwes 1977).
Intact TGEVs contain four structural proteins: a
large surface glycoprotein (spike or S); a small mem-
brane protein (sM); an integral membrane glycoprotein
(M); and a nucleocapsid protein (N) (Garwes and Pocock
1975; Spaan et al. 1988; Godet et al. 1992). The N protein
(47 kDa) interacts with viral RNA to form a helical ri-
bonucleoprotein complex. This structure in association
with M protein forms a spherical subviral internal core
(Risco et al. 1996). The 29–36 kDa M glycoprotein, firm-
ly embedded in the viral envelope by three or four mem-
brane-spanning regions, is present in two topological
forms (Risco et al. 1995). In one conformation, the N ter-
minus of the M protein is external to the virus envelope
and the C terminus is internal, whereas, in the second
conformation, both ends of the molecule are on the out-
side of the virion. In both conformations, the hy-
drophilic N terminus with a single accessible glycosyla-
tion site is responsible for interferon induction (Charley
and Laude 1988). Epitopes on protuding N-terminal and
C-terminal ends of M protein molecules bind comple-
ment-dependent neutralizing monoclonal antibodies
(MABs) (Woods et al. 1988; Laude et al. 1992; Risco et al.
1995). S glycoproteins, probably as trimer complexes
(Delmas and Laude 1990), are visible in electron micro-
graphs as the virus “corona” (Fig. 24.1). Functions attrib-
uted to the S glycoprotein (170–220 kDa) include virus
neutralization (complement-independent), virus-cell at-
tachment, membrane fusion, as well as hemagglutina-
tion. Garwes et al. (1978/79) have shown that purified S
glycoprotein elicited the production of TGEV-neutraliz-
ing antibodies, which neutralize at multiple steps in the
virus replication cycle (Nguyen et al. 1986; Sune et al.
1990).
Aminopeptidase N and a second 200 kDa surface
protein on ST cells and on porcine intestinal enterocytes
have been identified as virus receptors (Delmas et al.
1992; Weingartl and Derbyshire 1994). The receptor
binding site for aminopeptidase N and the major neu-
tralizing site (site A) on the S protein are located within
the same domain (Godet et al. 1994). Treatment of
TGEV with sialidase enhanced hemagglutinating activity
(Noda et al. 1987; Schultze et al. 1996). This hemaggluti-
Saif, Wesley 297
nating activity is located at the N-terminal region of the
TGEV S protein, a region that is missing from the PRCV
S protein; thus, determining the presence or absence of
hemagglutinating activity (Schultze et al. 1996) could be
a method to differentiate PRCV and TGEV strains.
MABs have been produced against attenuated
(Jimenez et al. 1986; Laude et al. 1986; Correa et al. 1990;
Delmas et al. 1990) and virulent (Welch and Saif 1988;
Zhu et al. 1990; Simkins et al. 1992, 1993) strains of
TGEV and used to characterize TGEV proteins and map
epitopes which elicit antibodies. The major neutraliza-
tion determinants on the S protein were highly con-
served for TGEV and PRCV strains (Jimenez et al. 1986;
Laude et al. 1986; Garwes et al. 1987; Welch and Saif
1988; Sanchez et al. 1990; Simkins et al. 1992, 1993). Use
of neutralizing MABs to map epitopes on the S glyco-
protein revealed four distinct antigenic sites (A, B, C, D),
with site A-B as the highly conserved immunodominant
epitope recognized by strongly neutralizing MABs (Gar-
wes et al. 1987; Simkins et al. 1992, 1993; Correa et al.
1990; Delmas et al. 1990; Gebauer et al. 1991), although
other sites (D, C) also can induce neutralizing antibodies
(Laude et al. 1986; Posthumus et al. 1990). The locations
of each of the four major antigenic sites were mapped on
the primary structure of the S glycoprotein. Starting
from the amino terminal end, these sites were designat-
ed C, B, D, and A by the Madrid group (Correa et al.
1990) and D, C, and A-B by the Paris group (Delmas et al.
1990). Paris sites D, C, and A-B (the designation used in
this chapter unless specified otherwise) overlap Madrid
sites B, D, and A, respectively. Three subsites (Aa, Ab,
Ac) for site A were recognized using TGEV-MAB-resis-
tant mutants (Correa et al. 1990).
Antigenic and Genomic Relationships
Only one serotype of TGEV is known (Kemeny 1976).
PRCV strains are not distinguished from enteropatho-
genic strains of TGEV by the virus neutralization (VN)
test (Pensaert 1989; Pensaert and Cox 1989) but can be
distinguished by a blocking enzyme-linked immunosor-
bent assay (ELISA) test (see the section on diagnosis be-
low) or by a large deletion in the S gene of PRCV result-
ing in a smaller S protein (170–190 kDa) than the S
protein of TGEV (220 kDa).
Seven coronaviruses are related antigenically or by
their genomic sequence (Enjuanes and Van der Zeijst
1995; Siddell 1995). These are TGEV, PRCV, canine cor-
navirus (CCV), feline infectious peritonitis virus (FIPV),
feline enteric coronavirus (FECV), porcine epidemic di-
arrhea virus (PEDV), and human coronavirus (HCV
229E). Within this group, only TGEV, PRCV, CCV, FIPV,
and FECV form an antigenically related subset based on
cross-reactivity in VN and IF tests and with MABs to the
S, N, or M protein (Pedersen et al. 1978; Reynolds et al.
1980; Woods 1982; Enjuanes and Van der Zeijst 1995).
Moreover, all members of this antigenic subset share the
298 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
antigenic subsite Ac on the S protein (Enjuanes and Van
de Zeijst 1995). Additional cross-reactivity between the
S, M, and N proteins of TGEV, CCV, and FIPV was
shown by radioimmunoprecipitation, immunoblotting,
and ELISA, and it was suggested that these viruses may
actually represent host range mutants of an ancestral
virus strain (Horzinek et al. 1982). Similar cross-reactivi-
ty among the S, M, and N structural proteins of TGEV
and PRCV was observed using polyclonal antisera in im-
munoblotting assays (Callebaut et al. 1988).
PEDV is another coronavirus of pigs that causes a
disease similar to TGEV; this disease has been docu-
mented only in swine in Europe and Asia. Antibodies to
PEDV have not been detected in a limited survey of adult
swine sera from the United States (DeBouck et al. 1982;
L. J. Saif and M. B. Pensaert—unpublished data, 1990).
Some one-way, immunoblotting cross-reactivity with the
N protein has been reported for PEDV, FIPV, CCV, TGEV,
and a putative mink coronavirus (Yaling et al. 1988; Have
et al. 1992). However, no antigenic cross-reactivity be-
tween PEDV and TGEV-related coronaviruses has been
detected using polyclonal antisera or MABs in other
serologic assays (Pensaert and DeBouck 1978; Callebaut
et al. 1988; Enjuanes and Van der Zeijst 1995). Another
coronavirus of pigs, hemagglutinating en-
cephalomyelitis virus, is antigenically unrelated to the
coronaviruses in group I (Enjuanes and Van der Zeijst
1995; Siddell 1995).
Although TGEV, CCV, and FIPV are antigenically
closely related, Reynolds et al. (1980) suggested that
TGEV and CCV could be distinguished serologically
(with sera from naturally infected animals) by employing
two-way cross-neutralization tests. Additional in vitro bi-
ological differences also have been detected; whereas
both TGEV and CCV will grow in either canine kidney
cells (Welter 1965; Reynolds et al. 1980) or an estab-
lished feline cell line (Woods 1982), neither CCV nor
FIPV will grow in ST or porcine thyroid cells, both of
which support the growth of TGEV isolates (Reynolds et
al. 1980).
In vivo biological differences also exist among TGEV,
CCV, and FIPV in their pathogenicity for neonatal pigs
(Binn et al. 1974; Woods and Pedersen 1979). Whereas
virulent FIPV caused diarrhea and intestinal lesions sim-
ilar to those of virulent TGEV, CCV caused no clinical
signs and only slight villous atrophy. IF, using a porcine
anti-TGEV serum conjugate, occurred in villous entero-
cytes of TGEV- and FIPV-infected pigs but predominated
in crypt enterocytes of CCV-infected pigs. CCV shed by
acutely infected dogs was shown to infect baby pigs and
induced serum neutralizing antibodies for both CCV and
TGEV (Woods and Wesley 1992). However, baby pigs
and pregnant gilts experimentally infected with FIPV did
not produce TGEV-neutralizing antibodies but did devel-
op some immunity to a TGEV challenge (Woods and
Pedersen 1979).
Molecular probes and MABs are also used to detect
and differentiate among these antigenically related coro-
naviruses. In terms of the S glycoprotein gene that con-
fers host range specificity, the 300 amino acid residues at
the N terminus are the most variable. In this domain
CCV and FIPV are more similar to each other than to
TGEV (Jacobs et al. 1987; Wesseling et al. 1994). Thus,
cDNA probes developed from this domain of the TGEV
S gene reacted with TGEV RNA but failed to recognize
CCV or FIPV RNA under conditions of high stringency.
Either cDNA probes or RT-PCR (reverse transcriptase-
polymerase chain reaction) techniques are used to differ-
entiate U.S. strains of PRCV from prototype strains of
TGEV (Bae et al. 1990; Vaughn et al. 1994; Jackwood et
al. 1995). Similar differentiation of the TGEV-related
coronaviruses was possible using specific MABs to the S
glycoprotein of TGEV that recognized TGEV strains but
failed to react with PRCV, FIPV, or CCV (Laude et al.
1988; Callebaut et al. 1989; Sanchez et al. 1990; Simkins
et al. 1992).
PRCV strains, isolated during the mid-1980s and
1990s, have been characterized and partially sequenced
(Rasschaert et al. 1990; Wesley et al. 1991b; Britton et al.
1991; Vaughn et al. 1995). An overall nucleotide and
amino acid sequence homology of 96% between TGEV
and PRCV suggests that PRCV evolved from TGEV and
that this occurred on a number of independent occa-
sions. Two striking features characterize the PRCV
genome: (1) a large deletion (621–681 nucleotides) at a
consistent site near the N terminus of the S gene that
gives rise to a smaller S protein; and (2) a variable region
with deletions that often eliminate the open reading
frame (ORF) immediately downstream of the S gene.
These genetic changes may account for the altered tissue
tropism of PRCV. From sequence data, significant ho-
mologies occur in the three genes encoding the three
main structural proteins within TGEV-related viruses
(TGEV, PRCV, CCV, FECV, FIPV) and between TGE-re-
lated viruses and HCV 229E and PEDV (Enjuanes and
Van der Zeijst 1995). Thus, sequencing data show a ge-
nomic relationship between these latter two viruses and
TGEV-related viruses that is not demonstrated by sero-
logic analysis (Enjuanes and Van der Zeijst 1995; Siddell
1995).
An altered phenotype was reported for a small plaque
(SP) variant of TGEV (Woods 1978; Woods et al. 1981).
Although the S gene of the SP variant and the S gene of
wild-type TGEV are similar, a large deletion (462 nu-
cleotides) is present in the SP viral genome just down-
stream from the S gene (Wesley et al. 1990a). This dele-
tion eliminated the ORF of one potential viral encoded
protein and eliminated the N-terminal portion of a sec-
ond potential viral protein. These two ORFs are also
missing from the high cell culture–passaged Nouzilly
strain, a second attenuated TGEV with an SP phenotype
(Britton et al. 1994). Presumably, the altered pathogenic-
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
ity of SP virus, the Nouzilly virus, and the PRCVs results
from these genetic deletions.
EPIDEMIOLOGY
The epizootiology of TGE has been reviewed (Haelter-
man 1962; Ferris 1973; Toma et al. 1978; Wood 1979;
Pritchard 1982, 1987). On a herd basis, two epizootio-
logic forms of TGE can be described: epizootic and en-
zootic. In addition, infections with the TGEV variant
PRCV present a different pattern and greatly complicate
seroprevalence studies of the epizootiology of TGEV
(Pensaert 1989; Pensaert and Cox 1989).
Epizootic TGE
Epizootic TGE refers to the occurrence of TGE in a herd
where most, if not all, of the animals are susceptible.
When TGEV is introduced in such a herd, the disease
usually spreads rapidly to swine of all ages, especially
during the winter. Some degree of inappetence, vomi-
tion, or diarrhea will occur in most animals.
Suckling pigs become very sick, dehydrating rapidly;
mortality is very high in pigs under 2–3 weeks of age but
gradually decreases in older pigs. Lactating sows often
become sick, developing anorexia and agalactia, which
further contribute to piglet mortality. The history and se-
vere clinical signs aid in diagnosis of epizootic TGE in
the United States, since other similar diseases have not
been reported. However, in Europe, PED has similar clin-
ical signs (Pensaert and DeBouck 1978), and the pres-
ence of PRCV appears to have greatly reduced the inci-
dence and severity of epizootic TGE (Pensaert and Cox
1989; Brown and Paton 1991; Laude et al. 1993; Pensaert
et al. 1993).
Enzootic TGE
Enzootic TGE refers to a persistence of the virus and dis-
ease in a herd, occurring as a result of a continual or fre-
quent influx of susceptible swine that, when infected,
tend to perpetuate the disease. Enzootic TGE is limited
to seropositive herds that have frequent farrowings
(Stepanek et al. 1979) or have frequent additions or com-
mingling of susceptible swine and represents a common
sequel to a primary outbreak in large breeding herds. In
these situations, TGEV spreads slowly among adult
swine, particularly herd replacements (Morin et al. 1978,
1983; Pritchard 1987). Females saved for breeding will be
immune and will transfer via their colostrum and milk a
variable degree of passive immunity to their progeny
during the suckling period. Sows usually are not sick. In
these herds, rather mild TGE viral diarrhea will be seen
primarily in pigs from the age of about 6 days until about
2 weeks after weaning. The pig is clinically affected when
viral exposure exceeds the pig’s passive immunity, and
the age when this occurs is related to, or reflects, the
management system used in the herd and the degree of
Saif, Wesley 299
immunity of the sow. Mortality is usually less than
10–20%, being determined by the age when infected and
by the variable degree of immunity obtained from im-
mune sows. Enzootic TGE in suckling or recently weaned
pigs can be difficult to diagnose and must be differenti-
ated from other types of enzootic diarrheal problems
commonly occurring in young pigs, such as rotaviral di-
arrhea and colibacillosis. Enzootic TGE will persist in the
herd as long as susceptible or partially immune swine are
exposed to TGEV.
The recrudescence or reintroduction of TGEV may
occur in herds that contain immune sows, resulting in
discrete episodes of disease (Pritchard 1987). This situa-
tion commonly occurs in herds in the concentrated
swine-rearing areas of the United States or other coun-
tries. Each winter, herds often become reinfected, and
the disease is especially seen in growing and finishing
swine. Such animals are susceptible, since the herd infec-
tion from the previous winter has usually terminated
during the intervening summer and autumn. If the dis-
ease enters the farrowing house, the disease in suckling
or weaned pigs will resemble that described above since
the sows will usually be immune. It is unclear whether
the source of virus in these circumstances comes from re-
activation of virus shedding in carrier swine or reintro-
duction of virus into the herd from an external source.
Porcine Respiratory Coronavirus
Evidence that led to the discovery of PRCV was obtained
from serologic surveys of slaughterhouse sows or swine
tested for international trade. In 1984, this led to the first
isolation of PRCV, a nonenteropathogenic coronavirus,
in Belgium (Pensaert et al. 1986). In 1989, two herds in
the United States unexpectedly were seropositive for
TGEV antibodies although these herds had not been vac-
cinated for TGE nor had they experienced clinical disease
(Hill et al. 1990; Wesley et al. 1990b). PRCV is a TGEV
variant that infects epithelial cells of the respiratory tract
and alveolar macrophages (Pensaert 1989; Pensaert and
Cox 1989; Paul et al. 1994). After experimental challenge,
PRCV infects only a few unidentified cells in the small in-
testine, and thus, virus shedding in feces is limited or not
detected (O’Toole et al. 1989; Cox et al. 1990a, b; Van
Cott et al. 1993, 1994; Brim et al. 1994, 1995). PRCV-in-
fected pigs produce antibodies that neutralize TGEV.
Swine density, distances between farms, and season
of the year influence the epizootiology of PRCV. PRCV
infects pigs of all ages by airborne transmission or by
contact, and in areas with high swine density, the virus
can spread to pigs on neighboring farms that are several
kilometers away (Pensaert and Cox 1989). In addition,
the risk of a farm becoming infected increases as the size
of a neighboring herd increases (Henningsen et al.
1989). PRCV infections are subclinical and the virus has
spread rapidly and extensively in pigs of Western Euro-
pean countries (Brown and Cartwright 1986; Jestin et al.
300 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
1987; Lange et al. 1988; Henningsen et al. 1989; Pensaert
1989; Van Nieuwstadt and Pol 1989; Laude et al. 1993;
Martin et al. 1994) and even into European countries
that were previously free of TGEV (Pensaert 1989). A
similar rapid and extensive spread of PRCV has not been
detected in the United States (USDA APHIS VS 1992);
however, a limited serologic survey in 1995 suggested
that many asymptomatic swine herds in Iowa were
seropositive for PRCV (Wesley et al. 1997).
PRCV has become enzootic in many European swine
herds (Pensaert 1989; Lanza et al. 1993; Laude et al.
1993; Pensaert et al. 1993). The virus circulates in the
herd infecting pigs before the age of 20–26 weeks, after
passively acquired maternal antibodies have declined
(Pensaert et al. 1993). Introduction of pigs into fattening
units and commingling of PRCV-negative and PRCV-
positive pigs from diverse sources resulted in serocon-
version to PRCV in pigs shortly after introduction into
most units (Van Reeth and Pensaert 1994a). Susceptible
pigs experimentally infected with PRCV were shown to
shed virus from nasal secretions for a period of less than
2 weeks (Onno et al. 1989; Wesley et al. 1990b; Bourgueil
et al. 1992; Van Cott et al. 1993; Brim et al. 1994). There
is no evidence for the fecal-oral transmission of PRCV.
Pensaert et al. (1993) have shown that pigs on closed
breeding farms become infected shortly after weaning
even in the presence of maternal antibodies, and thus,
PRCV persists in the herd by regularly infecting newly
weaned pigs. PRCV can persist in the herd throughout
the entire year or it can disappear temporarily from a
herd mainly in the spring and summer months and reap-
pear in the nursery and fattening units during the colder
months of the year (Pensaert et al. 1993). Thus, waves of
infection, without clinical disease, coincide with the fog-
gy and rainy season in Europe (Laude et al. 1993).
The seroprevalence of TGEV infections among Euro-
pean swine was examined by blocking ELISA following
the widespread dissemination of PRCV infections
(Brown and Paton 1991; Lanza et al. 1993; Pensaert et al.
1993). The investigators reported no or low (0.6%)
TGEV-positive serum samples from breeding sows in
Spain (Lanza et al. 1993) and from pigs in Great Britain
(Brown and Paton 1991), respectively. By comparison,
7.6% of sera (of 160 tested) from slaughterhouse sows in
Belgium were TGEV positive (Pensaert et al. 1993). Thus,
the seroprevalence of TGEV infections in Europe has de-
creased coincident with the spread of PRCV.
Transmission and Reservoirs
One of the significant epizootiologic features of TGE is
its seasonal appearance, that is, during the winter
months, usually from the middle of November to about
the middle of April. Several explanations have been giv-
en for this seasonal occurrence. Haelterman (1962) has
suggested that this is probably due to the characteristics
of the virus, since the virus is very stable when frozen but
rather labile when exposed to warm temperature or to
sunlight. This would allow a greater opportunity for the
virus to be transmitted in a viable state between herds in
winter, particularly on inanimate objects, such as during
transport of feed or animals. The observation has been
made that reduced or fluctuating ambient temperatures
markedly predispose feeder pigs to clinical manifesta-
tions of TGE (Shimizu et al. 1978; Shimizu and Shimizu
1979a), thus contributing to transmission in winter be-
cause of the diarrheic state.
What constitutes the reservoir of TGEV between sea-
sonal epizootics? Haelterman (1962) proposed at least
three reservoirs: (1) associated pig farms in which the
virus spreads subclinically; (2) hosts other than swine;
and (3) carrier pigs. The most probable explanation for
maintenance of the disease is that it exists in the enzoot-
ic form in feeder-pig operations (Morin et al. 1978) or in
herds that are on a continuous farrowing program. These
situations could constitute foci for maintenance of the
disease during the warmer months and for dissemina-
tion during the winter months. This hypothesis is sup-
ported by the finding that TGEV infection can spread
rather slowly through a group of growing swine under
certain conditions, such as during the summer months
(Maes and Haelterman 1979).
There is also evidence for the existence of TGEV in
nonporcine hosts. Cats, dogs, and foxes have been sug-
gested as possible carriers of TGEV from one herd to an-
other since they can shed virus in their feces for variable
periods (Haelterman 1962; McClurkin et al. 1970). Al-
though cats and dogs fed TGEV showed no clinical signs
(except after repeated passage in dogs; Klemm and Ristic
1976) and seroconverted to TGEV, virus excreted by dogs
was shown to be infectious for pigs (Haelterman 1962;
Reynolds and Garwes 1979).
Massive concentrations of starlings (Sturnus vulgaris)
in winter in feeding areas of swine may provide a method
by which TGEV can be mechanically carried from one
farm to another. Pilchard (1965) reported that TGEV was
detected in the droppings of starlings for as long as 32
hours after they were fed TGEV. Houseflies (Musca do-
mestica) have also been proposed as possible mechanical
vectors for TGEV. TGEV antigen was detected in flies
within a swine herd in which TGE was enzootic, and ex-
perimentally inoculated flies excreted TGEV for 3 days
(Gough and Jorgenson 1983).
A third possibility relating to the transmission of
TGE is the length of time infected swine eliminate viable
TGEV and the role of the carrier pig. Only one report
(Lee et al. 1954) has indicated that fecal shedding under
natural conditions lasted more than the commonly re-
ported 2-week period (Pensaert et al. 1970a). As for res-
piratory shedding of TGEV, the virus has been detected
in nasal swabs for postexposure periods up to 11 days
(Kemeny et al. 1975). However, from lung homogenates,
the virus was detected for postexposure periods up to
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
104 days (Underdahl et al. 1975). TGEV has also been re-
covered from milk of infected sows during the acute
phase of the disease (Kemeny et al. 1975; Kemeny and
Woods 1977) and following intramammary infusion or
injection of lactating sows with live TGEV (Saif and Bohl
1983). In the latter study, demonstration of TGEV anti-
gen in mammary gland tissue indicates the virus may
replicate in the mammary glands of lactating sows,
which may contribute to agalactia in sows. Transmission
of the virus via milk to suckling piglets, as suggested in
the same study, may account, in part, for the rapid
spread of the infection among a litter.
Although TGEV has been detected in the intestinal
and respiratory tracts for periods of up to 104 days
postinfection, it is unknown whether such virus can be
eliminated from the body in a viable state that will result
in new infections. Addition of sentinel pigs to a herd at
3, 4, and 5 months after a previous TGE outbreak result-
ed in no infections in the introduced pigs, as determined
by serologic tests (Derbyshire et al. 1969).
Nasal shedding of PRCV in experimentally infected
pigs occurs through postexposure day 10 (Onno et al.
1989; Wesley et al. 1990b). However, it is unknown for
how long pigs recovered from PRCV infections in the
field remain infectious. The possible role of the long-
term carrier hog in transmitting TGEV or PRCV is un-
known.
CLINICAL SIGNS
Epizootic TGE
The typical clinical signs of TGE in piglets are transient
vomiting, accompanied or rapidly followed by a watery
and usually yellowish diarrhea, rapid loss of weight, de-
hydration, and high morbidity and mortality in pigs un-
der 2 weeks of age. Diarrhea in young pigs is usually pro-
fuse, and feces will often contain small curds of
undigested milk. The odor of the feces is very offensive.
Severity of the clinical signs, duration of the disease, and
mortality are inversely related to the age of the pig. Most
pigs under 7 days of age will die in 2–7 days after first
showing clinical signs. Most suckling pigs over 3 weeks
of age will survive but are likely to remain unthrifty for a
time.
Clinical signs of TGE in growing and finishing swine
and in sows are usually limited to inappetence and diar-
rhea for 1 or a few days, with vomiting observed in an oc-
casional animal. The very few deaths observed are prob-
ably due to complicating factors such as stress or
concurrent infections, which frequently occur after
weaning. However, Bachmann et al. (1972) reported a
mortality between 25% and 30% in a group of 2- to 6-
month-old swine in Germany. Some lactating sows be-
come very sick, with an elevated temperature, agalactia,
vomiting, inappetence, and diarrhea. These severe signs
may be due to a high degree of exposure to the virus from
Saif, Wesley 301
close contact with their affected piglets or to hormonal
factors that may influence susceptibility. In contrast,
sows in the field having no contact with young infected
pigs usually have rather mild clinical signs or none.
The incubation period is short, usually 18 hours to 3
days. Infection generally spreads rapidly through the en-
tire group of swine so that in 2–3 days most animals are
affected, but this is more likely to occur in winter than
summer (Maes and Haelterman 1979).
Enzootic TGE
Enzootic TGE is most likely to occur in large herds that
have frequent farrowings, as discussed in the section on
epidemiology. The clinical signs shown by infected pigs
will usually be similar but less severe than those seen in
susceptible pigs of the same age. Death loss is usually
low, especially if pigs can be kept warm. The clinical
signs in suckling pigs can resemble those of “white
scours,” which is most commonly caused by rotavirus
(Bohl et al. 1978). In some herds, depending on manage-
ment, enzootic TGE is manifested mainly in weaned pigs
and may be confused with Escherichia coli, coccidia, or ro-
tavirus infections (Morin et al. 1983; Pritchard 1987).
Porcine Respiratory Coronavirus
The severity of clinical signs and the degree of patho-
genicity appear to be PRCV strain dependent, and it is
speculated that these differences might correlate with
slightly different genetic deletions. In addition, infection
of swine with PRCV and other respiratory viruses, espe-
cially in association with porcine reproductive and respi-
ratory syndrome virus (PRRSV), can alter the severity of
disease and clinical signs. Ultimately, the manifestation
of respiratory disease is dependent on a number of vari-
ables, including environmental and seasonal factors,
management practices, virus load or dose, and presence
of other bacterial or viral pathogens in the herd (Van
Reeth and Pensaert 1994a, b).
Many European and American PRCV strains cause
subclinical infections, and by histological examination, a
mild interstitial pneumonia may be present (Van Reeth
and Pensaert 1992; Laude et al. 1993; Enjuanes and Van
der Zeijst 1995). Experimentally infected feeder pigs had
only a transient weight loss, whereas 4- and 6-day-old
pigs infected with PRCV showed only a reduced rate of
weight gain in comparison with control pigs (Vannier
1990; Lanza et al. 1992; Van Reeth et al. 1996; Wesley
and Woods 1996). In one study, when American PRCV
strains were compared in pigs under the same condi-
tions, differences were observed in pathogenicity among
strains (Halbur et al. 1994). Two PRCV isolates, AR310
and LEPP with the same S gene deletion, induced signs of
moderate respiratory disease in pigs from 4 to 10 days
postinfection, while a third American strain (1894) pro-
duced only mild respiratory disease in pigs. However, at
a lower dose, even the AR310 strain was asymptomatic in
302 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
gnotobiotic pigs (Halbur et al. 1993). A Canadian PRCV
strain (1Q90) isolated in Quebec caused severe pneumo-
nia and 60% mortality in 1-week-old pigs given 108.5
TCID50 (median tissue culture infectious doses) of virus.
It is noteworthy, however, that littermates of these pigs
exposed to PRCV by contact, presumbly at a lower dose,
exhibited only rapid breathing and fever (Jabrane et al.
1994). Thus, clinical signs seen after experimental PRCV
exposure may be highly dose related and also influenced
by the age of the pigs and inoculation techniques. The re-
sults of experimental studies were also influenced by the
health status of the pigs and their subsequent treatment
(Vannier 1990).
Besides a single-virus infection with PRCV, pigs can
be exposed concurrently to other respiratory viruses and
agents. Often this occurs when pigs from multiple
sources are grouped together in nurseries or grower-fin-
isher units (Van Reeth and Pensaert 1994a). The effects
of dual-virus inoculations have been investigated and,
generally, found to be more severe than for a single-virus
infection. In particular, respiratory signs were enhanced
in PRCV-infected pigs when the pigs were inoculated 2
days later with either swine influenza virus or pseudora-
bies virus (Van Reeth and Pensaert 1994b, 1996). Com-
bined infections with PRRSV followed by PRCV also re-
sulted in a prolonged fever with respiratory disease and a
reduced rate of weight gain (Van Reeth et al. 1996). Be-
cause many respiratory viruses and bacteria are common
in swine populations, these combined infections with
PRCV may increase the occurrence and severity of respi-
ratory disease. This subject requires additional research.
PATHOGENESIS
The pathogenesis of TGE has been reviewed (Hooper
and Haelterman 1966; Moon 1978; Shepherd et al.
1979). The early events have been described as follows:
TGEV is ingested, infects the mucosa of the small intes-
tine, and causes villous atrophy because of a rapid and
extensive loss of functional epithelial cells.
Intestinal Replication
Whether by the oral or by the nasal route, the virus is
swallowed and, being able to resist the effects of a low
pH and proteolytic enzymes, remains viable until it
comes in contact with the highly susceptible villous ep-
ithelial cells of the small intestine. Infection and rapid
destruction or alteration in function of a high propor-
tion of these cells result in a marked reduction in enzy-
matic activity in the small intestine, which disrupts di-
gestion and cellular transport of nutrients and
electrolytes, causing an acute malabsorption syndrome
(Moon 1978). Hooper and Haelterman (1966) suggested
that the inability of infected pigs to hydrolyze lactose,
and possibly other nutrients, results in the marked dep-
rivation of nutrients that can be so critical to the young
pig. Furthermore, they suggested that the presence of
undigested lactose exerts an osmotic force which causes
a retention of fluid in the lumen of the intestine and
even a withdrawal of fluid from the tissues of the body
and thus contributes to diarrhea and dehydration.
Additional mechanisms contributing to diarrhea in
TGEV-infected pigs include altered sodium transport in
the jejunum, resulting in accumulation of electrolytes
and water in the intestinal lumen (Butler et al. 1974), and
loss of extravascular protein (Prochazka et al. 1975). The
ultimate cause of death, as suggested by Cornelius et al.
(1968), is probably dehydration and metabolic acidosis
coupled with abnormal cardiac function resulting from
hyperkalemia.
A marked shortening or atrophy of the villi occurs in
the jejunum (Fig. 24.3) and to a lesser extent in the ileum,
but it is often absent in the proximal portion of the duo-
denum (Hooper and Haelterman 1966). Both virus pro-
duction and villous atrophy were greater in newborn
pigs than in 3-week-old pigs (Moon et al. 1973; Norman
et al. 1973), suggesting higher susceptibility of neonates
to TGEV infection. Several mechanisms have been pro-
posed to account for this age-dependent resistance to
clinical disease. First, the rapidity with which infected
villous epithelial cells can be replaced by migration of ep-
ithelial cells from crypts of Lieberkühn may partially ac-
count for the lower fatality rate in older than in newborn
pigs. Moon (1978) reported that 3-week-old pigs nor-
mally replace villous enterocytes in the small intestine
about three times more rapidly than newborn pigs.
These newly replaced villous enterocytes are reportedly
resistant to TGEV infection (Pensaert et al. 1970b; Shep-
herd et al. 1979), possibly due to onset of the immune re-
sponse, presence of intestinal interferon (LaBonnardiere
and Laude 1981), or inability of these regenerating cells
to support virus growth. Second, TGEV accumulates and
replicates in the apical tubulovascular system of villous
absorptive cells in newborn pigs; this system is lacking in
pigs older than 3 weeks (Wagner et al. 1973). Third, virus
dose may play a major role in infections. Witte and
Walther (1976) demonstrated that the infectious dose of
TGEV needed to infect a market hog (about 6 months
old) was 104 times greater than that needed to infect a 2-
day-old piglet.
However, the severity of clinical signs due to TGE is
also increased when pigs are (1) fed a zinc-deficient ra-
tion (Whitenack et al. 1978); (2) anemic (Ackerman et al.
1972); (3) exposed to a low temperature or fluctuation in
temperature (Shimizu et al. 1978); or (4) injected with a
synthetic corticosteroid, dexamethazone (Shimizu and
Shimizu 1979a). In regard to the last two items, the
mechanism is thought to be due to an interference with
the early initiation of a local cell-mediated immune re-
sponse (Shimizu and Shimizu 1979a).
The failure of cell-cultured attenuated strains of
TGEV to infect the epithelial cells located in the cranial
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
A B
24.3. Villi of the jejunum from a normal pig (A) and from a TGEV-infected pig (B), as viewed
through a dissecting microscope (approximately ×10).
portion of the small intestines of pigs probably explains
why such strains do not cause diarrhea as severe as that
observed with virulent strains (Frederick et al. 1976;
Hess et al. 1977; Furuuchi et al. 1979; Pensaert 1979).
Furthermore, there was a reverse correlation between the
level of cell culture attenuation of TGEV and the extent
of intestinal infection (Hess et al. 1977).
Cell-cultured strains of TGEV of reduced virulence in
combination with a mildly virulent E. coli have been
shown to cause a more severe disease in germfree pigs
than when either organism was given alone (Underdahl
et al. 1972). Concurrent infections with TGEV and E. coli
or porcine rotavirus have been reported (Hornich et al.
1977; Theil et al. 1979).
Extraintestinal Replication Sites
TGEV. Although ingestion is undoubtedly the most
common portal of entry for the virus, the nasal route, or
airborne infection, may be important, but the efficiency
of this route of transmission for the spread of TGEV ap-
pears much less significant than for PRCV. Macroscopic
lung lesions were observed in gnotobiotic pigs inoculat-
ed oronasally with TGEV, but no clinical pneumonia re-
sulted (Underdahl et al. 1975). A preliminary report
also indicated that TGEV was present in alveolar
macrophages of infected neonatal pigs, suggesting a pos-
sible role for these cells in lung infection. However, only
cell culture–adapted, but not virulent, TGEV replicated
in cultures of alveolar macrophages in vitro (Laude et al.
1984). Highly attenuated strains of TGEV have also been
reported that replicate in the upper respiratory tract and
lung but not in the intestine of newborn pigs, resem-
bling PRCV infections (Furuuchi et al. 1979). Moreover,
nasal shedding of TGEV was detected in infected piglets
(Van Cott et al. 1993) and in lactating sows exposed to in-
Saif, Wesley 303
fected piglets (Kemeny et al. 1975).
In addition, studies have shown that TGEV is capable
of replicating in mammary tissue of lactating sows (Saif
and Bohl 1983) and that infected sows shed virus in milk
(Kemeny and Woods 1977). The significance of possible
mammary gland infection with TGEV under field condi-
tions is unclear. Whether it plays a role in the agalactia,
often seen in TGEV-infected sows, or rapid spread of in-
fection among piglets warrants investigation.
Although natural infection of the porcine fetus with
TGEV has not been reported, intrafetal inoculation re-
sults in the production of villous atrophy and serocon-
version to TGEV (Redman et al. 1978).
PRCV. PRCV has a tropism for cells of the respiratory
tract. It replicates to high titer in porcine lungs and also
infects epithelial cells of the nares, trachea, bronchi,
bronchioles, and alveoli and alveolar macrophages (Pen-
saert et al. 1986; O’Toole et al. 1989). Viremia occurs fol-
lowing primary infection, and virus spreads to parenchy-
mal organs and lymph nodes.
Only a few scattered cells containing PRCV antigen
are found in the small intestine even when the virus is di-
rectly inoculated into the intestinal lumen. These infect-
ed cells are located in or underneath the epithelial layer
of the intestinal villi and crypts, and the virus does not
spread to adjacent cells (Cox et al. 1990a, b). This limited
intestinal replication of PRCV explains why infectious
virus is seldom detected in feces of PRCV-infected swine
(Van Cott et al. 1993, 1994).
Researchers have attempted to use MAB neutraliza-
tion–resistant mutants and recombinant TGEV strains
to explore the molecular basis for the difference in path-
ogenicity and tissue tropism between TGEV and PRCV
strains (Bernard and Laude 1995; Ballesteros et al. 1997).
304 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
Bernard and Laude (1995) reported that most TGEV
(Purdue-115 strain) MAB-resistant mutants selected
with MABs to S protein site D exhibited a reduced en-
teropathogenicity in pigs, which was correlated with a
point mutation or small deletion in the S protein gene
encoding the N-terminal subregion of the S protein (sim-
ilar region deleted in PRCV strains). Using recombinants
generated between enteric/respiratory (PUR46) and res-
piratory (PTV) strains of TGEV, Ballesteros et al. (1997)
concluded that a substitution in amino acid 219 of the S
protein of the PUR46 recombinants was responsible for
the loss of enteric tropism observed after inoculation of
pigs with the PUR46 recombinants. The authors specu-
lated that two different domains of the S protein (around
amino acid 219) are required to infect intestinal epithe-
lial cells: one domain binds to the cellular receptor,
aminopeptidase N, and the other domain may be a bind-
ing site for an undefined intestinal coreceptor.
LESIONS
Gross Lesions
Gross lesions with TGE are usually confined to the gas-
trointestinal tract, with the exception of dehydration.
The stomach is often distended with curdled milk. Con-
gestion of the mucosa is a variable sign. Hooper and
Haelterman (1969) reported that about 50% of the pigs
euthanized during the first 3 days of infection had a
small area of hemorrhage on the diaphragmatic side of
the stomach at the border of the diverticulum ventricu-
lus.
The small intestine is distended with yellow and fre-
quently foamy fluid and usually contains flecks of cur-
dled undigested milk. The wall is thin and almost trans-
parent, probably due to atrophy of the villi.
Although lung lesions have been observed in experi-
mentally infected gnotobiotic pigs (Underdahl et al.
1975), they have not been reported in pigs naturally in-
fected with TGEV.
Subgross Lesions
A highly significant lesion of TGE is the markedly short-
ened villi of the jejunum and ileum, which Hooper and
Haelterman (1969) referred to as villous atrophy (Fig.
24.3). However, this is also seen in rotavirus diarrhea but
is not usually as severe or extensive as in TGE (Bohl et al.
1978). Infections with some strains of E. coli and coccidia
have also been reported to produce this lesion (Hornich
et al. 1977). However, the pathologic findings and extent
of villous atrophy were highly variable in pigs from en-
zootically infected herds (Pritchard 1987).
Microscopic Lesions
The degree of villous atrophy can be judged in histologic
sections by comparing the length of the jejunal villi with
the depth of the crypts of Lieberkühn. In normal piglets
these dimensions average about 795 µm and 110 µm, re-
spectively, giving a villi-crypt ratio of about 7:1; in in-
fected piglets the corresponding figures are about 180
µm and 157 µm, giving a ratio of about 1:1 (Hooper and
Haelterman 1969). Other lesions reported in experimen-
tally challenged 8-week-old pigs include microulceration
of the dome epithelium over Peyer’s patches, especially
in the cranial portion of the small intestine (Chu et al.
1982a).
Scanning EM has been used to reveal the develop-
ment of intestinal lesions of TGE and correlates well
with lesions observed by light microscopy (Waxler 1972;
Moxley and Olson 1989b). Using scanning EM, Moxley
and Olson (1989b) showed that the level of passive im-
munity in TGEV-infected pigs influenced not only the de-
gree of villous atrophy but also its segmental distribu-
tion. Villous atrophy was minimal in pigs nursing sows
previously infected with virulent TGEV, compared to
pigs nursing seronegative sows or sows given live attenu-
ated vaccines. In partially protected pigs, villous atrophy
was seen primarily in the ileum instead of the jejunum.
Similar observations were noted in pigs from herds with
enzootic TGE.
Transmission EM of TGEV-infected epithelial cells of
the small intestine has revealed alterations in the mi-
crovilli, mitochondria, endoplasmic reticulum, and oth-
er cytoplasmic components. Virus particles, primarily in
cytoplasmic vacuoles, were observed in villous entero-
cytes and in M cells, lymphocytes, and macrophages in
the dome regions of Peyer’s patches (Thake 1968; Wagn-
er et al. 1973; Chu et al. 1982a).
PRCV Lesions
In the case of PRCV, as noted earlier, villous atrophy is
not observed. However, microscopic examination of
lungs from asymptomatic pigs reveals that PRCV causes
a diffuse interstitial pneumonia in a high percentage of
inoculated animals (O’Toole et al. 1989; Van Nieuwstadt
and Pol 1989; Cox et al. 1990a). Others reported more se-
vere respiratory lesions dependent upon the strain and
dose of PRCV used to experimentally infect pigs (Halbur
et al. 1993, 1994; Jabrane et al. 1994; Paul et al. 1994).
DIAGNOSIS
This subject has been reviewed by Bohl (1981). Collec-
tion and preservation of appropriate clinical specimens
are necessary for reliable diagnosis. Although villous at-
rophy is a consistent lesion in severely affected pigs, it
frequently occurs in other enteric infections as well (ro-
tavirus, PED, coccidia, and sometimes E. coli). Laborato-
ry diagnosis of TGE may be accomplished by one or
more of the following procedures: detection of viral anti-
gen, detection of viral nucleic acids, microscopic detec-
tion of virus, isolation and identification of virus, or de-
tection of a significant antibody response.
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
Unfortunately, the serologic assays are complicated
by the failure of polyclonal antibodies to differentiate be-
tween PRCV and TGEV (discussed under differential
blocking ELISA). However, evaluation of clinical signs,
histologic lesions, and tissue distribution of viral antigen
may provide a presumptive diagnosis, because PRCV
does not cause diarrhea or villous atrophy and replicates
almost exclusively in respiratory tissues (Pensaert 1989;
Pensaert and Cox 1989). Thus PRCV is suspected if there
is antigen in lung tissues, seroconversion to TGEV/PRCV
by VN test, and no signs of enteric disease.
Detection of Viral Antigen
The detection of TGE viral antigen in epithelial cells of
the small intestine is probably the most common
method for diagnosing TGE in young pigs. Either IF
(Pensaert et al. 1970a) or immunoperoxidase (Becker et
al. 1974; Chu et al. 1982b; Shoup et al. 1996) techniques
may be used, but the former is more common. For best
results, pigs in the early stages of diarrhea are killed. Mu-
cosal scrapings (Black 1971) or frozen sections from the
jejunum and ileum are prepared and stained by either
the direct (Fig. 24.4) or indirect IF method. Mucosal
scrapings generally yield a greater sampling of the in-
testinal mucosa. Problems that may be encountered in IF
tests include (1) lack of sensitivity or specificity of
reagents (primary or secondary reagents used must be
free of antibodies to other enteric organisms, particular-
ly rotavirus); (2) failure to obtain specimens early after
onset of diarrhea before the loss of infected cells (piglets
must be euthanized to obtain specimens); and (3) cross-
reactions with FIPV, CCV, and PRCV. However, replica-
tion of PRCV in villous enterocytes is uncommon, and IF
or immunoperoxidase staining of villous enterocytes in
conjunction with diarrhea almost certainly indicates
TGEV.
24.4. Immunofluorescing cells from a TGEV-infected
pig. A compression smear was made from a mucosal
scraping of the jejunum and stained by the direct
fluorescent antibody test (×350).
Saif, Wesley 305
Recently an immunoperoxidase technique using
MAB to the highly conserved N protein of TGEV has
been applied to the detection of TGEV (intestinal tis-
sues) or PRCV (lungs) using formalin-fixed paraffin-em-
bedded tissues (Shoup et al. 1996). This permits the di-
agnosis of TGEV/PRCV on the same tissues as used for
histopathology and allows retrospective screening of
fixed tissues for TGEV/PRCV. Although polyclonal anti-
bodies will not differentiate between TGEV and PRCV,
certain MABs have been produced that react with TGEV
but fail to recognize PRCV. These differentiating MABs
have been used in IF and immunoperoxidase tests (Gar-
wes et al. 1988; Van Nieuwstadt and Pol 1989). PRCV has
been detected in respiratory tissues and nasal epithelial
cells by IF and immunoperoxidase tests, but use of dif-
ferentiating MABs is necessary for confirmation, since
enteric strains of TGEV may also replicate in these tis-
sues.
A double antibody sandwich ELISA using monoclon-
al or polyclonal antibodies to TGEV as capture or sec-
ondary antibodies is becoming more widely used to de-
tect TGEV antigens in cell culture, feces, and intestinal
contents (Bernard et al. 1986; Van Nieustadt et al. 1988;
Lu et al. 1991; Cornaglia et al. 1994; Lanza et al. 1995) or
PRCV antigen in nasal secretions or lung homogenates
(Cornaglia et al. 1994). The ELISA tests are rapid, applic-
able for use on samples from live pigs, and suitable for
testing large numbers of samples.
Detection of Viral Nucleic Acids
Recently, nucleic acid hybridization probes have been de-
veloped to detect TGEV genome sequences in fecal sam-
ples, infected tissues, or infected cell cultures (Shockley
et al. 1987; Benfield et al. 1991). Moreover, nucleic acid
probes derived from the 5′ end of the TGEV peplomer
gene can distinguish between PRCV and TGEV. In a hy-
bridization assay, these probes selectively differentiated
enteric TGEV isolates from the United States, Japan, and
England, including live attenuated TGEV vaccine strains
from U.S. isolates of PRCV, FIPV, and CCV (Bae et al.
1990; Wesley et al. 1991a). RT-PCR and nonradioactive
cDNA probes have also been used to differentiate TGEV
and PRCV isolates (Vaughn et al. 1994, 1996).
Electron Microscopy
TGEV has been demonstrated in the intestinal contents
and feces of infected pigs by negative-contrast transmis-
sion EM (Fig. 24.5) (Saif et al. 1977). In laboratories hav-
ing access to an electron microscope and personnel
trained in virus recognition by EM, this procedure has
had increasing use. Furthermore, immune EM (IEM) has
advantages over conventional EM techniques in that it is
more sensitive for detecting TGEV and can provide sero-
logic identification of the virus from either clinical spec-
imens or cell culture harvests. In addition, use of IEM en-
ables one to more readily differentiate TGEV from
306 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
common enveloped membranous debris and to concur-
rently detect the presence of other enteric viruses (Saif et
al. 1977). IEM is at least as sensitive as IF for detection of
TGEV. IEM is also applicable for detection of PRCV
shedding in nasal secretions (L. J. Saif, unpublished—
1990). However, this method cannot distinguish be-
tween TGEV and PRCV unless MABs are used, although
shedding of large numbers of PRCV in feces would not
be expected (Van Cott et al. 1993, 1994).
Isolation and Identification of Virus
Oral exposure of young pigs is probably the most sensi-
tive method for isolating or detecting TGEV (Dulac et al.
1977). However, this procedure is very expensive; conse-
quently, cell cultures are more frequently used.
Primary and secondary pig kidney cells (Bohl and Ku-
magai 1965) or pig kidney cell lines (Laude et al. 1981),
primary porcine salivary gland cells (Stepanek et al.
1971), porcine thyroid cells (Witte 1971), and the Mc-
Clurkin swine testicle (ST) cell line (McClurkin and Nor-
man 1966) have been successfully used for isolating
TGEV from feces or gut contents of infected pigs. The
virus also replicates in organ cultures from pig esopha-
gus, ileum, cecum, and colon (Rubenstein et al. 1970).
However, parvovirus contamination of some batches of
cells prepared from porcine thyroid glands may be a dis-
advantage to the use of these cells (Dulac et al. 1977).
Distinct cytopathogenic effect (CPE) may be negligible
24.5. Typical virus-antibody aggregates observed by
IEM of TGEV and gnotobiotic pig anti-TGEV serum.
Bar = 100 nm.
upon primary isolation of field strains; additional pas-
sages may be required before CPE is evident. A charac-
teristic type of CPE usually seen in ST and porcine thy-
roid cells consists of greatly enlarged, rounded, or
elongated cells that have a balloonlike appearance (Ke-
meny 1978). The ST cell line has been used for detecting
field strains of TGEV by CPE, plaques, or IF (Kemeny
1978; Bohl 1979). For detecting viral CPE or plaques, the
sensitivity of ST cells can be further enhanced by adding
pancreatin or trypsin to cell culture media (Bohl 1979;
Woods 1982) and using older cells (Stark et al. 1975).
Pocock and Garwes (1975) reported that TGEV replicat-
ed at highest titer in a slightly acidic medium in sec-
ondary pig thyroid cells.
Pig kidney and particularly ST cells have been the
cells of choice for isolation of PRCV, but PRCV also
grows in a continuous cat fetus cell line (Laude et al.
1993). Nasal swab fluids or lung tissue homogenates
from affected swine are used for isolation of PRCV. The
CPE resembles that produced by TGEV strains, with syn-
cytia formation frequently observed for PRCV (Pensaert
1989; Pensaert and Cox 1989).
Identification of cell culture virus can be done by VN,
IF, or IEM using specific TGEV antiserum. However,
MABs specific for TGEV are required to confirm TGEV
and exclude PRCV (Garwes et al. 1988; Laude et al.
1988), or isolates can be differentiated using RT-PCR or
specific cDNA probes (Laude et al. 1993; Enjuanes and
Van der Zeijst 1995). Confusion with cross-reacting CCV
and FIPV should not occur, since these viruses do not
replicate in ST or secondary pig thyroid cells (Reynolds
et al. 1980).
Serologic Diagnosis
The detection of TGEV antibodies can assist in diagnosis
and control in several different ways. However, the sero-
logic diagnosis of TGEV is complicated by the finding
that both TGEV and PRCV induce neutralizing antibod-
ies that are qualitatively and quantitatively similar (Pen-
saert 1989; Pensaert and Cox 1989). A blocking ELISA
test (described later) is necessary to differentiate these
antibodies. A rise in antibody titer between acute and
convalescent serum samples provides retrospective evi-
dence for epizootic TGE or infection with PRCV. Howev-
er, the history of the herd with respect to disease and
serologic status is needed to help interpret serologic find-
ings. To determine if enzootic TGE or PRCV is a problem
in a herd, serum samples from 2- to 6-month-old swine
can be tested for antibodies. At this age, passively ac-
quired antibodies should be absent (Derbyshire et al.
1969); thus, positive results suggest enzootic TGEV or
PRCV. Serologic tests can also be used to monitor the
TGEV or PRCV infection status of a herd. The entrance
of only serologically negative swine will also help main-
tain a herd free of TGEV and PRCV. Neutralizing anti-
bodies to TGEV can be detected in serum as early as 7–8
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
days after infection and may persist for at least 18
months: little is known about the persistence of neutral-
izing antibodies to PRCV within a herd (Cartwright
1968; Vannier et al. 1982).
TGEV antibodies have been detected by several dif-
ferent serologic tests. The VN test has been the most
widely used, using cell culture–adapted viruses in cell
culture systems by a variety of procedures: inhibition of
CPE in microtiter plates (Toma and Benet 1976) and
plaque reduction assays (Bohl and Kumagai 1965;
Thomas and Dulac 1976) are the most common. An IF
test was developed (Benfield et al. 1978) but was less sen-
sitive and reliable than the VN test (Hortig et al. 1980).
Very sensitive passive hemagglutination tests (Labadie et
al. 1977; Shimizu and Shimizu 1977) and ELISA tests
(Nelson and Kelling 1984; Paul et al. 1986; Hohdatsu et
al. 1987; Garwes et al. 1988; Bernard et al. 1989; Calle-
baut et al. 1989; Van Nieuwstadt et al. 1989; Berthon et
al. 1990) have been described, but both require concen-
trated purified virus for sensitizing red blood cells or
coating ELISA plates, respectively. Other serologic tests
include an indirect immunoperoxidase test adapted to
detect immunoglobulin (Ig) class-specific antibodies
(Kodama et al. 1981) and radioimmunoprecipitation
tests (Kodama et al. 1980). Complement-fixing antibod-
ies could not be demonstrated in convalescing swine
(Dulac et al. 1977).
Blocking ELISA Test for Serologic
Differentiation of PRCV and TGEV
Studies using MABs to TGEV have shown that certain
antigenic sites on TGEV are not present on the spike pro-
tein of PRCV (Laude et al. 1988; Callebaut et al. 1989;
Sanchez et al. 1990; Simkins et al. 1992, 1993). Thus,
some antigenic determinants on the spike protein of
TGEV are absent because of the deletion in the S protein
of PRCV. Differences in the capacity of TGEV MABs to
bind to PRCV serve as the basis of serologic tests to de-
termine if a swine herd is infected with TGEV or PRCV
(Garwes et al. 1988; Bernard et al. 1989; Callebaut et al.
1989; Van Nieuwstadt and Boonstra 1992; Simkins et al.
1993). In the blocking ELISA, TGEV antigen is reacted
with either TGEV or PRCV antiserum followed by the
distinguishing MAB. TGEV antiserum contains compet-
ing antibody that blocks the binding of the MAB, where-
as the PRCV antiserum does not block and allows the
MAB to bind. Thus, a negative (no blocking) reaction in
ELISA and a positive result in the VN test are evidence of
a PRCV infection. The test should only be evaluated on a
herd basis because some pigs with low TGEV or PRCV
antibody titers, as occurs early in the infection process
(7–14 days) or after infection with some TGEV strains,
may go undiagnosed (Callebaut et al. 1989; Van Nieuw-
stadt and Boonstra 1992; Simkins et al. 1993). Presently,
to export TGE-free swine from countries where PRCV in-
fections occur, only this test provides the differential in-
Saif, Wesley 307
formation required for TGEV seropositive animals. Cur-
rently, the reliability of commercial ELISAs for differen-
tiating U.S. strains of PRCV and TGEV is unclear.
TREATMENT
Antiviral agents have not yet been developed for the spe-
cific treatment of TGE. Some inhibition of TGEV repli-
cation in cell culture has been reported for the antiviral
compounds amantadine (Dimitrov 1982) and isathia-
zone (Potopalsky et al. 1983). Although high levels of
type 1 interferon were detected in the intestine in pigs in
the early phase of TGEV infection, the role of this inter-
feron in the recovery or pathogenesis of TGE was unde-
termined (LaBonnardiere and Laude 1981). Recent stud-
ies suggest that interferon may activate natural killer
cells in newborn pigs, thereby contributing a degree of
resistance to challenge with TGEV (Lesnick and Der-
byshire 1988; Loewen and Derbyshire 1988). In addi-
tion, during a field outbreak of TGE, 1- to 12-day-old
piglets treated orally for 4 days with 1–20 IU of human
interferon-α had significantly greater survival rates than
placebo-treated piglets (Cummins et al. 1995). However,
no increased survival was seen in piglets given human in-
terferon-α shortly after birth. Whether such therapy
could be cost-effective for treatment of TGEV-infected
piglets was not assessed.
The only treatment for TGE presently available is to
alleviate starvation, dehydration, and acidosis. Parenter-
al treatment with fluids, electrolytes, and nutrients
would be effective in treating young pigs, but not practi-
cal under farm conditions. Oral therapy with balanced
electrolyte and glucose solutions is contraindicated in
young pigs (Moon 1978). The following measures are
suggested: provide a warm (preferably above 32˚C),
draft-free, and dry environment and make water or nu-
trient solution freely accessible to the thirsty TGEV-in-
fected pigs. Such measures will tend to reduce mortality
in pigs that are infected at more than 3–4 days of age.
Antibacterial therapy might be beneficial in 2- to 5-week-
old pigs, especially if there is a concurrent infection with
pathogenic strains of E. coli. Cross-suckling or putting in-
fected or susceptible litters onto TGE-immune sows was
found useful in various field outbreaks (Stepanek et al.
1979; Pritchard 1982).
PREVENTION
Management
PREVENTING ENTRANCE OF TGEV INTO A HERD.
Swine in the incubative stage of the disease or those in
the viral shedding or carrier state can provide a source of
entrance of TGEV into a herd. Some precautions to help
prevent these possibilities are to introduce swine that
originate from herds known to be free of TGE, are sero-
logically negative, and/or have been placed in isolation
308 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
on the farm for 2–4 weeks before being added to the herd
proper. A frequent question is, How soon after a TGE
outbreak can pigs be moved to another herd without
spreading the disease? A practical answer to this ques-
tion is that a period of 4 weeks should elapse from the
last sign of disease before introducing such animals into
a “clean” herd.
Starlings have been incriminated as a means by
which the disease is spread between herds in winter
months, owing largely to their tendencies to gather in
large flocks and feed around swine. Cats, dogs, or foxes
might play a role in spreading TGE between herds under
certain situations (see the section on epidemiology).
Feces from TGEV-infected swine can be carried on
boots, shoes, clothing, truck beds, feeds, etc., and can be
a source of infection to other herds. Especially in winter
this is probably an important means by which TGEV is
transmitted during the transport of livestock and feed.
Consequently, precautions should be taken to minimize
such occurrences. If visitors, especially animal and feed
truckers, have direct contact with swine during the win-
ter months, it is well to provide them with clean
footwear.
AFTER ONSET OF TGE. When TGE has occurred on
a farm and pregnant animals have not yet been exposed,
two procedures may minimize losses of newborn pigs.
(1) If the animals are due to farrow more than 2 weeks
hence, purposely expose them to virulent virus, such as
the minced intestines of infected pigs, so they will be im-
mune at farrowing time. (2) If the animals will farrow in
less than 2 weeks, attempt to provide facilities and man-
agement procedures so they will not be exposed to TGEV
until at least 3 weeks postfarrowing. To minimize deaths,
provide young pigs with a warm, dry, draft-free environ-
ment and access to water, nutrient solution, or milk re-
placer (see the section on treatment).
Some success has been achieved in elimination of
TGEV from epizootically infected closed breeder herds
without depopulation by the following procedures (Har-
ris et al. 1987): (1) Bring in all breeding stock replace-
ments for the next 4–6 months. (2) In the face of an out-
break, feed TGEV-infected minced intestines
simultaneously to all pigs in the herd (including replace-
ment stock) to eliminate susceptible hosts, thereby
shortening the time the disease progresses through the
herd and ensuring more uniform exposure levels in all
pigs. (3) Maintain strict all-in/all-out production in far-
rowing and nursery units. (4) Add sentinel seronegative
pigs about 2 months after clinical signs of TGE disappear
and monitor these pigs for seroconversion to TGEV. Po-
tential hazards associated with feed-back control of TGE
include possible spread of other pathogens to pregnant
sows and throughout the herd.
ENZOOTIC TGE. Two approaches can be considered
in attempting to control or terminate an enzootic TGE
herd problem. First, pregnant seropositive sows can be
vaccinated intramuscularly (IM) or intramammarily late
in gestation or shortly after farrowing with live attenuat-
ed TGEV to boost immunity. Although only limited in-
formation is available, this procedure should boost milk
antibody levels (Saif and Bohl 1983), providing longer
passive immunity to suckling pigs (Leopoldt and Meyer
1978; Stepanek et al. 1979; Lutter et al. 1982). Although
this procedure may only delay the onset of TGE in ex-
posed pigs, it can be beneficial in reducing mortality.
Second, alterations in management can be made to
break the cycle of infection by eliminating reservoirs of
susceptible pigs in a unit: prevent the continual influx of
susceptible animals into the herd (e.g., by temporarily al-
tering the farrowing schedule); temporarily utilize other
facilities; and have smaller farrowing and nursing units,
to more nearly achieve an all-in/all-out management sys-
tem.
Immunoprophylaxis
ACTIVE IMMUNITY. The duration of active immuni-
ty in swine after oral infection with virulent TGEV has
not been well characterized. Intestinal infection of
breeding-age swine results in detectable serum antibod-
ies that persist for at least 6 months to possibly several
years (Stepanek et al. 1979). However, the serum anti-
body titer, although providing serologic evidence of
TGE, provides little indication of the degree of active im-
munity. Swine that have recovered from TGE are im-
mune to subsequent short-term challenge, presumably
due to local immunity within the intestinal mucosa (Van
Cott et al. 1994; Saif et al. 1994; Brim et al. 1995). The
age and immune status of the animal at initial infection
and the severity of the challenge may greatly influence
the completeness and duration of this active immunity.
The mechanism of active immunity in the gut proba-
bly relates to stimulation of the secretory IgA (sIgA) im-
mune system with production of intestinal sIgA anti-
bodies by lymphoid cells within the lamina propria (Van
Cott et al. 1993, 1994; Saif et al. 1994). IgA TGEV anti-
bodies and antibody-secreting cells (ASCs) have been de-
tected in the intestine and serum of pigs after oral, but
not parenteral, inoculation with TGEV (Kodama et al.
1980; Sprino and Ristic 1982; Van Cott et al. 1993, 1994;
Saif et al. 1994). Kodama et al. (1980) proposed that de-
tection of IgA antibody in the serum, presumably in-
testinally derived, might serve as an indicator of active
immunity to TGE. In another study, oral inoculation of
gnotobiotic pigs with TGEV resulted in development of
both serum and intestinal TGEV-neutralizing antibodies
detectable from 5 to at least 35 days postexposure (DPE)
(Saif 1976). IgM (5–15 DPE) and IgA immunocytes
(which remained predominant from 7 to 35 DPE) were
detected in the intestinal lamina propria of the TGEV-in-
fected gnotobiotic pigs. More recently, an enzyme-linked
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
immunospot (ELISPOT) technique was used to investi-
gate the kinetics of IgA and IgG TGEV antibody produc-
tion by the pig’s systemic and local gut associated lym-
phoid tissues (GALT). High numbers of IgA ASCs were
induced in GALT only by virulent TGEV: live attenuated
(vaccine) TGEV or PRCV strains induced significantly
fewer IgA ASCs (Van Cott et al. 1993, 1994; Saif et al.
1994; Berthon et al. 1990). These and other studies
(Stone et al. 1982) indicate that the pig is immunocom-
petent at birth in regard to humoral and mucosal anti-
body production, but in the intestine, additional matu-
rational time may be required for antibody responses to
reach adult levels.
Besides local antibody-mediated immunity, cell-me-
diated immune responses may also be important in ac-
tive immunity against TGEV infections. A number of
tests have been used to demonstrate cell-mediated im-
munity (CMI) to TGEV, including macrophage migra-
tion inhibition (Frederick and Bohl 1976), leukocyte mi-
gration inhibition (Woods 1977; Liou 1982), direct
lymphocyte cytotoxicity (Shimizu and Shimizu 1979b),
lymphocyte proliferation (Shimizu and Shimizu 1979c;
Welch et al. 1988; Brim et al. 1994, 1995; Anton et al.
1995, 1996), spontaneous cell-mediated cytotoxicity
(SCMC), and antibody-dependent cell-mediated cytotox-
icity (ADCMC) (Cepica and Derbyshire 1983). Only indi-
rect evidence exists concerning the role of CMI in resis-
tance to TGEV infection. CMI was demonstrated with
lymphocytes obtained from GALT of swine orally infect-
ed with virulent TGEV (Frederick and Bohl 1976;
Shimizu and Shimizu 1979c; Welch et al. 1988; Brim et
al. 1994, 1995); swine parenterally or oronasally inocu-
lated with attenuated TGEV or PRCV developed CMI
mainly in systemic sites (spleen or peripheral blood lym-
phocytes). Lymphoproliferative responses to TGEV per-
sisted within GALT, but not systemic lymphocytes, for at
least 110 days after oral infection of 6-month-old swine
(Shimizu and Shimizu 1979c) but for only about 14–21
days after infection of younger (7- to 11-day-old) pigs
(Welch et al. 1988; Brim et al. 1994). Recently, it was con-
firmed that CD4 T-helper cells are involved in lympho-
proliferative responses to TGEV (Anton et al. 1996).
Although T-cell epitopes were identified by lymphopro-
liferation studies for each of the three major proteins of
TGEV, a dominant functional T-helper epitope was de-
fined on the N protein (N321) (Anton et al. 1995). The N321
peptide-induced T cells collaborated in the in vitro syn-
thesis of TGEV-neutralizing antibodies specific for the S
protein. These investigators further reported that pro-
duction of high levels of TGEV-specific antibodies in vit-
ro (from in vivo TGEV-primed mesenteric lymph node
cells) required stimulation by at least two TGEV struc-
tural proteins, with maximal responses induced by na-
tive S protein rosettes in combination with recombinant
N protein. Such findings have important implications for
the optimal design of TGEV subunit or other recombi-
Saif, Wesley 309
nant TGEV vaccines.
Because lymphocyte cytotoxicity was absent in new-
born piglets and decreased in parturient sows, it was pro-
posed that a lack of K and NK cell activity against TGEV-
infected cells may correlate with the increased
susceptibility of newborn piglets and parturient sows to
TGEV infection (Cepica and Derbyshire 1984). Thus,
CMI may play a role in either recovery from TGEV infec-
tion or resistance to reinfection via the rapid elimination
of TGEV-infected epithelial cells by any one or all of a
combination of SCMC, ADCMC, or sensitized T lym-
phocyte-mediated cytotoxicity.
VACCINATION OF NEONATAL OR WEANED PIGS.
TGEV Vaccines. Neonatal pigs have been orally vac-
cinated with attenuated TGEV in an attempt to induce
rapid protection via either interference or local immuni-
ty. No early interference has been demonstrated, and
generally more than 5 days were required before protec-
tion due to active immunity could be induced (Pensaert
1979). One study reported a slightly earlier onset of pro-
tection, by 3–4 DPI, after maintaining vaccinated pigs at
a lowered temperature (18–20˚C) to enhance replication
of the attenuated virus (Furuuchi et al. 1976). Failure to
induce an early interference phenomenon and the delay
required for development of active immunity make
neonatal vaccination an unlikely method of providing
immediate protection against TGEV within the critical
first few days of life.
Active immunization of suckling or feeder pigs could
be important for control of enzootic infections, especial-
ly in newly weaned pigs, in which TGEV infections may
result in increased mortality. Live attenuated and inacti-
vated TGEV vaccines have been federally licensed for oral
or intraperitoneal administration, respectively, shortly
after birth. One limited preliminary study reported
greater protection in vaccinated, compared to control,
seropositive suckling pigs even though serum antibody
levels were not enhanced but were comparable in the two
groups (Graham 1980). However, challenge in older
piglets usually is much more difficult to standardize, due
to age resistance to infection. Further studies reported
that the presence of maternal antibodies in vaccinated
pigs decreased (Hess et al. 1982; Lanza et al. 1995; Sestak
et al. 1996) or completely suppressed (Furuuchi et al.
1978) active antibody production following oral admin-
istration of live TGEV. In the latter study, conducted in
suckling piglets nursing naturally infected sows (Furu-
uchi et al. 1978), high levels of both passive circulating
and intestinal antibodies in the suckling piglets probably
accounted for the complete interference with active im-
munization by an attenuated strain of TGEV.
Other approaches have been used in attempts to ac-
tively immunize young pigs against TGEV. Woods and
Pedersen (1979) noted 33% mortality in challenged pigs
(3 of 9) vaccinated orally/intraperitoneally with two dos-
310 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
es of the antigenically related live virulent FIPV. In com-
parison, 100% of the challenged pigs (3 of 3) died that
were orally vaccinated once with an attenuated SP vari-
ant of TGEV. Gough et al. (1983a) reported that 10
young weaned pigs inoculated IM with two or three dos-
es of an adjuvanted, soluble, undefined TGEV subunit
(23 kDa) vaccine were protected against virus challenge.
However, parenteral administration of three doses of a
baculovirus-expressed recombinant S protein (contain-
ing the four major antigenic sites, A–D) in adjuvant to
suckling piglets elicited neutralizing antibodies to TGEV
in serum but failed to protect pigs against TGEV chal-
lenge (Shoup et al. 1997).
PRCV. The observation that epizootic outbreaks of
TGE in Europe have declined dramatically following the
widespread dissemination of PRCV among European
swine prompted researchers to examine if a respiratory
PRCV infection could induce active intestinal immunity
and protection against TGEV. The consensus from sever-
al studies was that prior infection of nursing or weaned
pigs with PRCV provides partial immunity against TGEV
challenge, evident by a reduced duration and level of
virus shedding and diarrhea in most (Cox et al. 1993; Van
Cott et al. 1994; Brim et al. 1995; Wesley and Woods
1996), but not all (Van Nieuwstadt et al. 1989), pigs stud-
ied. The mechanism of this partial immunity presum-
ably is related to the rapid increase in TGEV-neutralizing
antibody titers (Cox et al. 1993; Wesley and Woods 1996)
and numbers of IgG and IgA ASCs observed in the in-
testines of PRCV-exposed pigs after TGEV challenge
(Van Cott et al. 1994; Saif et al. 1994). The latter re-
searchers speculated that the migration of PRCV-specific
IgG and IgA ASCs from the bronchus-associated lym-
phoid tissues (BALT) to the gut of the PRCV-exposed
pigs after TGEV challenge might explain the rapid
anamnestic response and the partial protection induced.
However, after PRCV exposure of neonatal pigs, at least
6–8 days were required to develop partial immunity to
TGEV challenge (Wesley and Woods 1996), suggesting
that induction of active immunity might be too late to
protect seronegative newborn piglets from epizootic
TGE.
PASSIVE IMMUNITY. Passive immunity is of prima-
ry importance in providing newborn piglets with imme-
diate protection against TGEV infection. Swine are born
devoid of immunoglobulins (Igs), which they acquire af-
ter birth via colostrum. Colostral Igs, which consist pri-
marily of IgG, represent a serum transudate that is trans-
ferred from the dam across the piglet’s intestinal
epithelium to its circulation, thus providing the neonate
with the same complement of serum antibodies as in the
dam (Porter and Allen 1972; Bourne 1973). These pas-
sively acquired humoral antibodies function mainly in
protection against systemic infection but do not protect
against intestinal infection (Hooper and Haelterman
1966). The concentration of IgG decreases about 30-fold
during the first week of lactation, whereas sIgA concen-
trations decline only about 3-fold, becoming the pre-
dominant Ig in milk (Porter and Allen 1972). Cells seed-
ed from the intestine produce sIgA locally in the
mammary tissue (Roux et al. 1977). The piglet does not
absorb sIgA milk antibodies, but they play an important
role in passive intestinal immunity.
Mechanisms of passive immunity to TGEV infections
have been reviewed (Pensaert 1979; Saif and Bohl 1979a,
1981a; Saif 1985; Saif and Theil 1990). Swine that have
recovered from TGE can transmit passive immunity to
their suckling pigs (Bay et al. 1953). Suckling pigs are
protected as a result of the frequent ingestion of
colostrum or milk that contains TGEV-neutralizing anti-
bodies. Such antibodies in the lumen of the intestine
neutralize any ingested TGEV and thus protect the sus-
ceptible epithelial cells of the small intestine. Haelter-
man (1963, 1965) referred to this immunogenic mecha-
nism as lactogenic immunity. This is accomplished
naturally when immune sows allow their piglets to suck-
le about every 2 hours. Passive protection can also be ac-
complished artificially by continuous feeding of anti-
serum to piglets (Haelterman 1963; Noble 1964).
Although presently impractical because of the expense
of the antiserum and the necessity of frequent adminis-
tration, these problems might eventually be overcome by
use of MABs in some type of slow-release delivery sys-
tem. A question of major importance in possible appli-
cation of MABs is whether they could be used in the face
of an outbreak to reduce morbidity and mortality.
TGEV antibodies in colostrum and milk of sows are
primarily associated with IgA or IgG (Abou-Youssef and
Ristic 1972; Bohl et al. 1972; Saif et al. 1972). TGEV milk
antibodies of the IgA class provide the most effective
protection, but IgG antibodies were also protective if
high titers could be maintained in milk (Bohl and Saif
1975) or by artificial feeding of colostral IgG (Stone et al.
1977). The greater efficacy of IgA TGEV antibodies is
probably because (1) they occur in higher levels in milk
(Porter and Allen 1972), (2) they are more resistant to
proteolytic enzymes (Underdown and Dorrington 1974),
and (3) they selectively bind to gut enterocytes (Nagura
et al. 1978). TGEV antibodies in milk of the IgG class are
produced as a result of parenteral or systemic immu-
nization. IgA TGEV antibodies occur in the milk follow-
ing intestinal infection. To explain their occurrence it
was proposed that after antigenic sensitization in the
gut, IgA immunocytes migrate to the mammary gland,
where they localize and secrete IgA antibodies into the
colostrum and milk (Saif and Bohl 1979a, 1981a; Saif
1985; Saif and Theil 1990; Saif et al. 1994). This “gut-
mammary” immunologic axis, first proposed in refer-
ence to TGEV infections in swine (Bohl et al. 1972; Saif et
al. 1972), is an important concept in designing optimal
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
vaccination procedures to provide effective lactogenic
immunity.
TGEV VACCINATION OF THE SERONEGATIVE
PREGNANT DAM. A variety of viral vaccines (viru-
lent, attenuated, inactivated, and subunit) and routes of
administration (oral, intranasal, intramuscular, subcuta-
neous, and intramammary) (Bohl et al. 1975; Kaji and
Shimizu 1978; Pensaert 1979; Saif and Bohl 1979a; Voets
et al. 1980; Moxley and Olson 1989a; Saif and Theil
1990; Saif et al. 1994) have been tested for induction of
lactogenic immunity. Oral administration of live viru-
lent virus to pregnant sows generally gave the highest lev-
el of immunity, resulting in protective immunity for the
sow and consistently producing high titers of persisting
IgA TGEV antibodies in milk associated with protective
lactogenic immunity for suckling pigs.
There are presently several federally licensed TGEV
vaccines. All contain inactivated or live attenuated TGEV
and are approved for use in pregnant or neonatal swine.
These vaccines and their efficacy will be considered in
the following sections according to their respective
routes of administration. Many variables complicate the
evaluation of both experimental and commercial TGEV
vaccines, often resulting in conflicting data. These in-
clude the challenge dose and strain of TGEV; the age of
the pig at challenge; environmental conditions, especial-
ly temperature; the milking efficiency of the vaccinated
sow; and the immune status of the dam at the time of
vaccination.
Oral and/or Intranasal Vaccination. Based on the
observation that sows infected with TGEV during gesta-
tion could transmit immunity to their piglets, “planned”
infection of pregnant swine with virulent TGEV has been
used to mimic this natural immunity. This procedure is
usually accomplished by feeding virulent autogenous
virus to pregnant swine at least 2 weeks before farrowing.
The virus may consist of minced guts from young pigs
acutely infected with TGEV and is administered to sows
with food or in frozen gelatin capsules given orally by
means of a balling gun.
Oral vaccination of pregnant swine with attenuated
TGEV would appear to be the logical route of vaccina-
tion for stimulating milk IgA TGEV antibodies so as to
duplicate the natural route of infection and induction of
immunity. The intranasal (IN) route has been used alone
or together with the oral route, because attenuated
strains of TGEV are known to replicate in the respirato-
ry tract (Furuuchi et al. 1979) and upon being swallowed
might seed additional virus to the gut. However, results
using attenuated strains orally and/or IN have generally
been disappointing (Saif and Bohl 1979a, 1981a; Voets et
al. 1980; Henning and Thomas 1981; Moxley and Olson
1989a). In previous studies using the high-passaged Pur-
due strain of TGEV orally, or orally and IM, few IgA
Saif, Wesley 311
TGEV antibodies were evident, and mortality among
challenged pigs from vaccinated dams ranged from 25%
to 100% (Saif and Bohl 1979a, b; Voets et al. 1980; Mox-
ley and Olson 1989a; Saif and Theil 1990).
Concerns that attenuated strains of TGEV might not
survive passage through the acidic environment of the
stomach prompted studies using lyophilized attenuated
virus in enteric-coated gelatin capsules (Hess et al. 1978;
Voets et al. 1980). Hess et al. (1978), using the high-
titered B1 strain of TGEV (300 cell culture passages), re-
ported high levels of IgA TGEV antibodies in milk and
only 10% piglet mortality. Voets et al. (1980) used the
high-passaged Purdue strain and found that six of nine
sows failed to seroconvert after oral vaccination; even the
three sows that seroconverted had a 44% piglet mortality
rate. Fichtner et al. (1982) noted 30% mortality in chal-
lenged piglets after feeding attenuated Riems virus to
their dams for 10 days during gestation. In further efforts
to ensure that vaccine virus reached the small intestine,
two studies used direct inoculation of attenuated viruses
into the intestinal lumen. Again, protection was poor
(62% mortality) (Voets et al. 1980) in challenged piglets
from dams given a single intralumenal inoculation of at-
tenuated Purdue TGEV during gestation, whereas
greater protection (10% mortality) was noted in chal-
lenged piglets after their dams received intralumenal in-
oculation of attenuated Riems TGEV for several days
during gestation (Fichtner et al. 1982).
Other researchers selected variants of high- and low-
passaged TGEV strains resistant to low pH and prote-
olytic enzymes in vitro and used these strains as vaccines
for passive protection studies (Aynaud et al. 1985; Chen
1985; Shirai et al. 1988; Bernard et al. 1989). They re-
ported inconsistent results, with mortality varying from
0% to 73% among litters challenged with virulent TGEV.
In the latter two studies, data interpretation was con-
founded somewhat by variations in the ages of the pigs
at challenge, a factor shown in other studies to dramati-
cally influence piglet survivability (Moxley and Olson
1989a).
A live attenuated SP variant TGEV grown in a persis-
tently infected porcine leukocyte cell line has been used
to vaccinate pregnant swine by the oral/IN and/or intra-
mammary routes (Woods 1978, 1984). Challenge of the
suckling pigs resulted in mortality of 14–34%. In the lat-
ter study the author reported generally high TGEV anti-
body titers in both IgA and IgG fractions of 3–4 days
postfarrowing (DPF) milk. However, three of eight sows
vaccinated with SP TGEV became mildly sick after chal-
lenge exposure of their nursing pigs. Although diarrhea
was observed in pigs nursing SP-TGEV vaccinates (48%
morbidity), it was reportedly mild and delayed in onset
(3 DPF). The SP TGEV has been reported to be avirulent
for newborn pigs, replicating within the intestinal lami-
na propria but not epithelial cells (Woods et al. 1981).
Molecular analysis of several attenuated TGEV strains
312 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
revealed changes in mRNA 2 and 3, affecting the S pro-
tein and nonstructural protein 3 (Register and Wesley
1994).
Moscari (1980b) reported induction of IgA TGEV an-
tibodies in milk after oral administration of the CKp at-
tenuated strain of TGEV; however, protection studies
were not described. Inconsistent results were noted in
vaccination studies using a commercial vaccine (Ambico,
Inc.) administered twice orally (in feed) and once IM.
Whereas Welter (1980) reported 8% mortality among
challenged pigs, others reported higher mortality, simi-
lar to that in piglets suckling unvaccinated sows (Saif
and Bohl 1981b; Bohl et al. 1982; Moxley and Olson
1989a).
The generally poor results obtained with oral or IN
vaccination of sows using attenuated TGEV strains may
be attributed to the superficial or limited replication of
most attenuated strains in the sow’s intestine (Frederick
et al. 1976; Hess et al. 1977). Consequently, this results in
little antigenic stimulation of underlying intestinal IgA
plasma cells and correspondingly little IgA antibody se-
cretion in milk. Attempts to use a low cell culture–pas-
saged TGEV to induce passive immunity led to erratic re-
sults in terms of both seroconversion in orally/IN
exposed sows and protection in piglets (Saif and Bohl
1979b). The dilemma remains of how to commercially
develop a TGEV vaccine capable of stimulating an IgA
response in the gut of sows but being sufficiently attenu-
ated or noninfectious so as not to produce disease in
newborn pigs.
Parenteral Vaccination. Various experimental and
two commercial vaccines were comprised of live attenu-
ated virus administered IM about 6 and 2 weeks prefar-
rowing. Experimental evaluations of this vaccination
regime have generally indicated reduced piglet mortality
(38–56% in vaccinates compared with 71–92% in con-
trols) but not morbidity (Bohl et al. 1975; Voets et al.
1980; Moxley and Olson 1989a). However, vaccination
results were poor when compared with almost complete
protection (0–9% mortality) in litters of naturally infect-
ed sows. Henning and Thomas (1981) and Matisheck et
al. (1982) reported more favorable vaccination results
with mortality of 10% and 18% using two commercial
vaccines.
The IM vaccination procedure has two major disad-
vantages. (1) Vaccinated swine develop little or no gut
immunity; they usually get sick when exposed to TGEV.
If this occurs during lactation, their suckling pigs will be
deprived of adequate milk. (2) The TGEV antibodies
found in the milk of these vaccinated sows are of the IgG
class and of low titer and fail to provide optimal passive
protection to suckling pigs.
Intramammary injection of seronegative pregnant
swine with TGEV resulted in high titers of primarily IgG
TGEV antibodies in milk, whereas similar injections in
lactating sows resulted in IgA and IgM TGEV antibodies.
Specific antibody activity was found not only in milk
from injected glands but also in milk from noninjected
glands (Bohl and Saif 1975; Saif and Bohl 1983). Protec-
tion was good (14–26% mortality) in litters of intramam-
marily vaccinated pregnant swine, presumably because
exceptionally high levels of IgG antibodies persisted in
the milk at the time of challenge, 3 DPF (Shibley et al.
1973; Bohl and Saif 1975). A similar, greatly enhanced,
predominantly IgG milk antibody titer was noted in two
sows vaccinated IM/IN with the high-titered (108–109.3
TCID50) attenuated TO163 strain of TGEV. No mortality
occurred in either of these litters, confirming the protec-
tive ability of IgG TGEV antibodies when present in high
titers in milk (Kaji and Shimizu 1978).
Heterologous Vaccines. The antigenic relationship
between TGEV and FIPV was the basis for studies of the
possible efficacy of FIPV as a heterologous coronavirus
vaccine in swine. Preliminary studies indicated that
some immunity (25% mortality) against TGE was con-
ferred in pigs nursing two sows vaccinated during gesta-
tion orally/IN and intramammarily with live virulent
FIPV. However, this FIPV was also pathogenic in new-
born pigs (Woods and Pedersen 1979). Subsequent stud-
ies using cell culture–adapted attenuated FIPV in sows
by the same routes of inoculation resulted in higher litter
mortality (52%) and low TGEV antibody titers of the IgG
class in milk (Woods 1984).
VACCINATION OF TGEV-INFECTED SWINE. Vac-
cines have been used on two populations of pregnant
swine: those that have and those that have not previous-
ly been naturally infected with TGEV. There are signifi-
cant differences in the immune responses and, conse-
quently, piglet protection in these two groups of animals
following vaccination. These differences may account for
some of the discrepant results seen in vaccine challenge
studies if previously infected swine were unknowingly
used. This possibility can only be eliminated by using a
very sensitive test (such as plaque reduction VN) to mea-
sure TGEV antibodies and by knowing the herd history
of test animals in terms of previous TGE outbreaks. Oc-
currence of PRCV in herds may further complicate fu-
ture TGEV vaccine studies.
Limited laboratory research has indicated that par-
enteral inoculation of previously infected swine during
gestation using attenuated TGEV resulted in a boost in
TGEV milk antibodies in both the IgA and IgG classes
(Saif and Bohl 1981a, b, 1983; Saif 1985). Others have al-
so reported greatly increased milk TGEV antibody titers
after intramammary inoculation of previously infected
swine with inactivated TGEV (Thorsen and Djurickovic
1971). These titers were about four- to sevenfold greater
than in seronegative intramammarily vaccinated gilts or
nonvaccinated infected sows. Currently available par-
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
enterally administered TGEV vaccines may be more ef-
fective in boosting immunity in previously infected preg-
nant swine than in initiating immunity in previously un-
infected seronegative pregnant swine. These vaccines
may be especially useful in herds in which enzootic TGE
is a problem (Leopoldt and Meyer 1978; Stepanek et al.
1979; Lutter et al. 1982).
NEW EXPERIMENTAL VACCINE APPROACHES
Native Subunit Vaccines. In two studies, virus sub-
units purified from virulent TGEV and administered
with adjuvants by parenteral routes were evaluated as
vaccines (Garwes et al. 1978/79; Gough et al. 1983b).
Garwes et al. (1978/79) injected pregnant sows intra-
mammarily with virus surface projections (S glycopro-
tein) or subunit particles: neutralizing antibodies were
present only in serum and colostrum of sows injected
with S glycoproteins. Colostral antibodies were associat-
ed with IgG, except in one sow that had both IgG and IgA
antibodies. However, protection of challenged piglets
was poor (100% mortality in four of six litters).
Gough et al. (1983b) used a low molecular weight un-
defined soluble subunit (about 23 kDa) purified from
TGEV by sonication, isopycnic centrifugation, and gel fil-
tration to IM inject pregnant gilts. These investigators
detected neutralizing antibodies in serum and milk and
observed only 4% mortality among challenged piglets.
The Ig isotype of TGEV antibodies in the milk of vacci-
nates was not determined. These results have not been
reproduced further or confirmed.
rDNA Subunit Vaccines. Limited data exist con-
cerning the immunogenicity of rDNA-produced viral
subunits or peptides of TGEV. The S protein fragments
expressed in E. coli were not glycosylated, were difficult
to isolate due to aggregation and insolubility, and did not
induce neutralizing antibodies to TGEV in mice (Hu et
al. 1985). Posthumus et al. (1990) noted that a peptide
from the S protein (residues 379–386) raised neutraliz-
ing antibodies to TGEV in rabbits. Higher titers were in-
duced when this peptide was coupled to a second S pro-
tein peptide (residues 1160–1180), but the protective
efficacy of these peptides was not tested in swine.
Eukaryotic vectors have been used to express TGEV S
protein genes encoding determinants dependent on gly-
cosylation (site A-B). The S glycoprotein of TGEV ex-
pressed in vaccinia induced low titers of neutralizing an-
tibodies but no protection (Hu et al. 1985). The S, M, and
N proteins of TGEV were also expressed in insect cells
using a baculovirus expression system (Pulford et al.
1990; Godet et al. 1991; Tuboly et al. 1994, 1995; Shoup
et al. 1997). The baculovirus-expressed S glycoproteins
elicited neutralizing antibodies to TGEV in the serum of
rats (Godet et al. 1991) and pigs (Tuboly et al. 1994,
1995; Shoup et al. 1997). Using TGEV S gene constructs
of various lengths, Tuboly et al. (1994, 1995) showed
Saif, Wesley 313
that only the recombinant S protein fragments contain-
ing antigenic site A induced neutralizing antibodies in
serum which delayed the onset of clinical signs and virus
shedding when artificially fed to TGEV-challenged
piglets. Similarly, Shoup et al. (1997) reported that only
full or partial (789 residues) length baculovirus recombi-
nant S glycoprotein constructs containing the four major
antigenic sites (A–D) induced moderate to high neutral-
izing antibody titers to TGEV in serum and primed pigs
for secondary antibody responses after challenge. A re-
combinant S protein containing only antigenic sites C
and D (397 residues) induced low neutralizing antibody
titers in serum but also primed pigs for a secondary anti-
body response after TGEV challenge. Parenteral inocula-
tion of pigs or sows with the recombinant glycoproteins
failed to induce a high level of active or passive immuni-
ty, respectively, against TGEV challenge. The parenteral-
ly inoculated sows had IgG, but not IgA, antibodies to
TGEV in colostrum and low or no TGEV antibody titers
in milk, confirming previous reports that the presence of
serum antibodies and low titers of colostral antibodies
(of the IgG isotype) are not associated with passive pro-
tection against TGEV (Haelterman 1965; Saif and Bohl
1979a, b, 1981a, 1983).
Live Recombinant Vector Vaccines. Because stim-
ulation of intestinal immunity is important to induce
protection against TGEV, investigators have attempted
to develop genetically engineered vaccines using live re-
combinant vectors that replicate in the intestinal tract.
Studies of a live-vector vaccine for TGEV initially focused
on the use of a prokaryotic expression system such as at-
tenuated strains of Salmonella typhimurium, which pos-
sess a tropism for intestinal Peyer’s patches (Smerdou et
al. 1996). The TGEV S protein site D (similar to Paris site
C) was expressed as a fusion protein with E. coli labile
toxin B in a recombinant S. typhimurium. Oral inocula-
tion of rabbits with the recombinant bacteria induced
TGEV antibodies in serum and intestinal secretions as
measured by a radioimmunoassay but only low titers of
neutralizing antibodies in the intestinal secretions. How-
ever, no pig protection studies have been reported.
Studies are in progress using a live eukaryotic vector
with a respiratory and enteric tropism (human adeno-
virus 5 and porcine adenovirus) to express the TGEV S
glycoprotein (Torres et al. 1996). A human adenovirus 5
recombinant expressing the TGEV S protein (containing
sites A–D) or the amino terminal 26% of the S protein
(Madrid sites C and B; Paris site D) induced neutralizing
antibodies to TGEV in the serum of swine inoculated
oronasally and intraperitoneally. Newborn piglets were
orally challenged with a TGEV-immune serum mixture
and then artificially fed immune serum (3 times per day)
from the immunized swine. Only in the group fed serum
from swine inoculated with the adenovirus–TGEV S pro-
tein recombinant (containing sites A–D) were newborn
314 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
piglets passively protected against diarrheal mortality,
but they were not protected against TGEV infection.
However, no results were reported for direct TGEV chal-
lenge of recombinant TGEV-adenovirus-inoculated pigs.
Others (Callebaut et al. 1996) developed a recombi-
nant human adenovirus 5 expressing the S protein of
PRCV. Piglets inoculated oronasally with the recombi-
nant adenovirus developed low PRCV-neutralizing anti-
bodies in serum and shed virus nasally after PRCV chal-
lenge at similar levels as control pigs. However, the
recombinant-inoculated pigs had a shorter duration of
virus shedding coinciding with a rapid anamnestic neu-
tralizing antibody response after PRCV challenge.
A recent novel strategy for generation of recombi-
nant vaccines is based on the use of synthetic
minigenomes from TGEV defective interfering RNAs
and helper rescue virus to express heterologous proteins.
For example, a recombinant minigenome expressing the
S protein gene of TGEV used in combination with a res-
piratory TGEV strain as a helper virus has been used to
create chimeric viruses expressing the S protein from
TGEV and the respiratory strain (Izeta et al. 1997). If suf-
ficiently attenuated, such chimeric viruses might prove
useful as new candidate vaccines for TGEV.
PRCV Vaccines to Prevent TGEV. Since PRCV be-
came widespread in the European swine population, the
incidence and severity of TGE in countries with PRCV
have declined (Pensaert and Cox 1989; Laude et al.
1993). This suggests that previous exposure of swine to
PRCV imparts a degree of immunity to a subsequent
TGEV infection (Pensaert 1989; Pensaert and Cox 1989).
A number of researchers have examined the relation-
ship between PRCV infection of sows and passive immu-
nity to TGEV in piglets. Prior natural exposure of sows to
PRCV induced a variable degree (44–53% mortality) of
passive protection against experimental TGEV challenge
of suckling pigs (Bernard et al. 1989; Paton and Brown
1990). Variable protection in the field during TGE out-
breaks was also noted among litters of PRCV-exposed
sows (Pensaert and Cox 1989; Callebaut et al. 1990).
Similar variable levels of protection (average of
30–67% mortality) were reported after TGEV challenge
of piglets suckling sows that had been experimentally in-
fected or reinfected with PRCV during pregnancy (Calle-
baut et al. 1990; DeDiego et al. 1992; Wesley and Woods
1993; Lanza et al. 1995; Sestak et al. 1996). Noteworthy
in the latter two studies, litter mortality was lowest
(range = 0–27%, average = 14%) and IgA and IgG milk an-
tibody titers were highest in sows multiply exposed to
PRCV during two subsequent pregnancies. These experi-
mental findings agree with other reports (Callebaut et al.
1990) that naturally PRCV-exposed sows that are rein-
fected with PRCV during pregnancy secreted IgA TGEV
antibodies in milk and provided a high degree of protec-
tion (0–12.5% mortality in three of six litters) to their off-
spring after TGEV challenge. Besides levels of IgA anti-
bodies in milk, a hallmark of protection in these and an-
other study (Wesley and Woods 1993) of passive immu-
nity to TGEV induced by PRCV appeared to be induction
of active immunity to TGEV in the sow, preventing ill-
ness and/or agalactia. Diarrhea mortality was consis-
tently lower in litters of PRCV-exposed sows that did not
become ill or develop agalactia after TGEV challenge of
their litters.
The mechanism for the induction of IgA antibodies
observed inconsistently in the milk of PRCV-exposed
sows is unclear, as is the correlation of such antibodies
with passive protection to TGEV challenge (Pensaert and
Cox 1989; Callebaut et al. 1990). After a primary expo-
sure to PRCV, IgA antibodies occurred in the milk of on-
ly 30% of sows; this percentage increased to 84% in sows
reinfected with PRCV. These results concur with a report
by Sestak et al. (1996) that the titers of IgA (and IgG) an-
tibodies to TGEV were significantly increased in the milk
of sows multiply exposed to PRCV during two sequential
pregnancies. The variable IgA antibody responses ob-
served in the milk of sows after primary exposure to
PRCV could relate to the low numbers of virus-specific
IgA ASCs observed in BALT, GALT, and mesenteric
lymph nodes of seronegative pigs exposed to PRCV (Van
Cott et al. 1993, 1994). Reinfections with PRCV may be
necessary to boost numbers of IgA ASCs in BALT and to
increase the efficiency of a possible sIgA immunologic
link between BALT and the mammary gland. Mainly
virus-specific IgG ASCs were observed in BALT after
oronasal inoculation of seronegative pigs with PRCV
(Van Cott et al. 1993). Following TGEV challenge, in-
creased numbers of IgG ASCs (and IgA ASCs) appeared
in GALT of the PRCV-exposed pigs (Van Cott et al. 1994).
Because IgG antibodies are also prevalent in the milk of
sows exposed to viruses that replicate in the respiratory
tract, for example, attenuated TGEV strains and
pseudorabies virus (Saif and Bohl 1977), it is conceivable
that an IgG immunologic link may also exist between
BALT and the mammary gland in sows, or alternatively,
BALT stimulation contributes serum IgG antibodies that
are subsequently transudated into the milk.
Besides quantitative differences in the levels of IgA
antibodies induced in milk after exposure to TGEV or
PRCV, researchers have investigated potential differ-
ences in virus epitopes recognized by the IgA milk anti-
bodies induced after TGEV versus PRCV infection of
sows (DeDiego et al. 1992, 1994). In TGEV-infected sows,
antigenic subsite A (Aa, Ab, Ac), followed by antigenic
subsite D (Madrid), was the best inducer of IgA antibod-
ies, whereas after PRCV infection, antigenic site D and
subsite Ab were immunodominant. These authors con-
cluded that only IgA recognizing at least antigenic sites A
and D conferred good protection in vivo, whereas any Ig
class recognizing only one antigenic site neutralized
virus in cell culture.
 CHAPTER 24 TRANSMISSIBLE GASTROENTERITIS
Clearly, additional studies are necessary to clarify the
levels and mechanisms of active and passive immunity
to TGEV established in swine by previous exposure to
PRCV. In particular, it is important to elucidate the
mechanism by which IgA antibodies are induced after in-
fection with PRCV, if a BALT-mammary gland immuno-
logic link exists but is less efficient than the gut-mamma-
ry link for induction of IgA in milk, why IgA antibodies
occur only in some PRCV-exposed sows, and the effec-
tiveness of these IgA antibodies in protecting suckling
pigs against intestinal TGEV infections.
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 Vesicular Diseases
25 J. A. House and C. A. House

Vesicular diseases in swine are identical clinically. They
can be caused by infection with the viruses of foot-and-
mouth disease (FMD), vesicular stomatitis (VS), vesicu-
lar exanthema of swine (VES), and swine vesicular dis-
ease (SVD). FMD, VES, and SVD are exotic to the United
States, while VS is enzootic. The importance of vesicular
diseases in today’s world-trade environment remains
high. Because FMD is extremely contagious and has such
a dramatic economic impact, vesicular diseases must be
properly diagnosed, reported, and controlled.
In Italy in 1546, Fracastorius made what is probably
the first report of a vesicular disease (Bulloch 1927).
FMD was the first animal disease shown to be caused by
a filterable agent (Loeffler and Frosch 1898). VS was rec-
ognized in horses and cattle in the United States during
World War I (Cotton 1927), but infection in swine was
not reported until 1943 (Schoening 1943). The first re-
port of VES was in 1932 in California (Traum 1934), al-
though it was initially thought to be FMD. A new vesicu-
lar disease, SVD, emerged in Italy in 1966 (Nardelli et al.
1968). San Miguel sea lion viruses (SMSVs), isolated
from marine mammals, were shown to cause vesicular
disease in inoculated swine (Smith et al. 1973; Berry et al.
1990). The history and distribution of these viruses are
given in Table 25.1.
ETIOLOGY
The characteristics of the viruses that cause vesicular dis-
eases are shown in Table 25.2.

Table 25.1. Chronological and geographical distribution of vesicular diseases

Disease First Agent
Virus Reported Identified Distribution Areas Free
Foot-and-mouth disease 1546 1898 Sporadic: Europe North and Central America,
Enzootic: South America, Australia, New Zealand,
Asia, Africa Japan
Swine vesicular disease 1966 1968 Europe, Japan, Hong Kong, Taiwan North, Central, and South
America; Africa; United
Kingdom
Vesicular exanthema of swine 1932 1934 Last North American case in 1956 World
San Miguel sea lion None 1973 Pacific coast of North America Remainder of world
Vesicular stomatitis 1916 1927 North, Central, and South America Remainder of world

Table 25.2. Classification of the etiologic agents of vesicular diseases

Disease Agents Classification Nucleic Acid Size and Stability Proteins/Antigens Serotypes
a
Foot-and-mouth Picornaviridae SS RNA 22–30 nm; labile 4 structural 7: numerous subtypes
disease virus Aphthovirus (positive below pH 7.0; proteins, plus a show varied degrees of
sense) ether-resistant group of reactive cross-protection within
nonstructural each serotype
proteins
Swine vesicular Picornaviridae SS RNA 22–30 nm; acid- 4 structural 1: related to Coxsackie
disease virus Enterovirus (positive stable; ether- proteins B-5 virus
sense) resistant
Vesicular Caliciviridae SS RNA 35–39 nm; labile 1 polypeptide in 26 or more (13 known for
exanthema of Calicivirus (positive below pH 3; ether- coat; plus group VESVs; at least 13 for
swine/San Miguel sense) resistant reactive antigens SMSVs)
sea lion viruses
Vesicular stomatitis Rhabdoviridae SS RNA 70 × 170 nm; bullet- 1 immunogenic 2 of importance to swine:
virus Vesiculovirus (negative shaped; ether-labile; glycoprotein New Jersey and
sense) stable at pH 5–10 Indiana 1
a
SS RNA = single-stranded RNA.
327
328 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

Foot-and-Mouth Disease closest relative is the human enterovirus Coxsackie B-5

FMD viruses (FMDVs) are in the family Picornaviridae
and the genus Aphthovirus (Francki et al. 1991c). Aphtha
is the Greek word for “vesicles in the mouth.” These
viruses are small (22–30 nm), nonenveloped, and labile
below pH 7.0, and they have single-stranded positive-
sense RNA with approximately 8450 nucleotides that
serve as messenger RNA. The high rate of nucleotide
substitutions in the single-stranded RNA viruses ac-
counts for their wide variation (Domingo et al. 1993;
Vosloo et al. 1996). Perhaps one of the most important
sites for substitutions is the GH loop (codons 133–158)
of viral protein 1 (VP1) (Acharya et al. 1989), which is
adjacent to the common aphthovirus receptor site RGD
(Fox et al. 1989). There are four structural viral proteins,
of which VP1 has been shown to elicit protective immu-
nity in cattle (Bachrach 1977). Viral RNA polymerase, a
nonstructural protein, has been termed “virus infection
associated antigen” (VIAA) for diagnostic purposes but
is more accurately termed “FMDV nonstructural protein
3D.” One copy of 3D occurs in each viral particle (New-
man et al. 1994). The FMDV nonstructural protein 2C is
present in clarified FMD vaccines at levels insufficient to
elicit an immune response (Lubroth et al. 1996).
There are seven serotypes of FMDV: A, O, C, Asia 1,
and South African Territories (SAT) 1, 2, and 3. There is
no cross-protection between the serotypes (Brooksby
1982). All serotypes have numerous subtypes, which
may arise during acute or persistent infection (Gebauer
et al. 1988; Vosloo et al. 1996). The serologic relatedness
of subtypes is determined by “r-values,” the ratio of an-
tibody titers between new and reference viruses (Pereira
1977).
Swine Vesicular Disease
SVD virus (SVDV) is in the family Picornaviridae, genus
Enterovirus (Francki et al. 1991c). The virions are small
(22–30 nm), nonenveloped, and acid-stable. Each has a
single-stranded positive-sense RNA and four structural
viral proteins. There is one serotype of SVDV, whose
(Graves 1973).
Vesicular Exanthema of Swine and San
Miguel Sea Lion Viruses
The VESVs and SMSVs, members of the Caliciviridae
family, are closely related caliciviruses (Smith et al.
1973), named from the Latin calices (cups) for the cup-
shaped depressions on the virion. The viruses are 35–39
nm in diameter, nonenveloped, moderately acid-stable,
and contain single-stranded positive-sense RNA (Francki
et al. 1991b). The capsomers are composed of one major
polypeptide.
There are 13 known serotypes of VESVs, which are
sequentially designated (except for strains 1934B and
101) by a letter followed by the year of isolation (e.g.,
VESV A48). There are at least 13 SMSV serotypes, which
are numbered consecutively (with no SMSV 3, which
does not exist) in order of discovery.
Vesicular Stomatitis
The bullet-shaped VSVs belong to the family Rhabdoviri-
dae, genus Vesiculovirus. The viral particles are 70 × 170
nm, enveloped, ether-labile, and stable at pH 5–10, and
they contain a single-stranded, negative-sense RNA.
There are five structural viral proteins: L, G, N, NS, and
M. The glycosylated G protein confers the type specifici-
ty and elicits neutralizing and hemagglutinating anti-
bodies (Francki et al. 1991a). The viral nucleoprotein N
and the matrix protein M account for serologic-group
cross-reactions. There are at least 11 vesiculovirus
serotypes (Tesh et al. 1983; Travassos da Rosa et al.
1984), of which serotypes New Jersey (NJ), Indiana 1, In-
diana 2, and Indiana 3 are of importance to livestock.
EPIDEMIOLOGY
The epidemiological features of the vesicular diseases are
summarized in Table 25.3.

Table 25.3. Epidemiological features of vesicular diseases

Main Species Contamination of
Disease Agent Affected Transmission Morbidity Carrier State Meat and By-Products
Foot-and-mouth Swine, cattle, sheep, Aerosol, contact, High Cattle, sheep, goats, Frozen meat, lymph
disease virus goats, African fomites African buffaloes nodes, bone marrow,
buffaloes; some milk products
strains are host
restricted
Swine vesicular Swine, humans Contact, fomites Moderate No Virus persists in cured meat
disease virus and gland products
Vesicular Swine, various Contact, fomites Moderate No Meat
exanthema of marine animals
swine/San Miguel
sea lion viruses
Vesicular stomatitis Horses, cattle, Biting insects; some Moderate to No Not documented
virus swine, humans by contact low
 CHAPTER 25 VESICULAR DISEASES House, House
Foot-and-Mouth Disease
FMD is considered the most contagious disease of live-
stock. Essentially all cloven-footed species are suscepti-
ble to FMDV. In some texts, the possibility of human in-
fection is stated, but this has yet to be documented.
Infected animals shed large quantities of virus into their
surroundings for at least 7 days. Aerosol exposure is the
major means of spread, particularly in temperate cli-
mates, where high humidity and moderate temperatures
can favor long-distance airborne spread (Sellers and
Parker 1969; Thomson 1994). Tremendous aerosols are
generated by swine, which are often called an “amplifier
host” (Sellers and Parker 1969; Donaldson and Ferris
1980; Donaldson et al. 1987). Swine may produce up to
108 infectious doses contained in aerosols per day during
peak viremia (Sellers et al. 1971), which is up to 1500
times greater than that produced by cattle (Donaldson
and Ferris 1980). In hot, arid regions, the disease is main-
ly spread by direct contact, as aerosols cannot travel long
distances due to low humidity and high temperatures.
Other means of spread include contaminated semen,
meat and milk products, and fomites. Semen from in-
fected cattle contained and transmitted FMDV by artifi-
cial insemination (AI) (Cottral et al. 1968), whereas se-
men from infected swine contained FMDV but did not
transmit the disease by AI (McVicar et al. 1978).
The most dramatic proof of carrier buffaloes infect-
ing contact animals was shown by Vosloo et al. (1996).
Sporadically over two years, both contact cattle and buf-
faloes became infected by a cloned SAT-2 virus. The nu-
cleotide sequence varied significantly from the original
clone (1.649% nucleotide substitution/year). An inadver-
tent introduction of a SAT-1 strain infected some buf-
faloes, and ultimately these carrier animals infected an-
other buffalo. The nucleotide substitution rate for the
SAT-1 strain was 1.549% per year. African buffaloes have
clearly transmitted FMDV to cattle (Dawe et al. 1994;
Hedger and Condy 1985), as well as under experimental
conditions (Dawe et al. 1994). The major transmission of
FMDV from African buffaloes to cattle occurs when year-
ling buffalo calves, whose maternal antibody has waned,
become infected with virus from carrier buffaloes. It is
hypothesized that buffalo calves then shed large quanti-
ties of virus and pass the virus to susceptible cattle dur-
ing close contact or by sharing water holes (Thomson
1995).
Some strains of FMDV may be host restricted under
field conditions, as exemplified by the porcinophilic na-
ture of the O1 strain that infected virtually the entire
swine population of Taiwan in the spring of 1997. No in-
fections were reported in the 300,000 Taiwanese cattle.
Experimentally, O1 Taiwan did not readily infect cattle
even when administered in massive doses (Dunn and
Donaldson 1997).
On the North American continent, FMD last oc-
curred in Mexico in 1946–54 and in Canada in 1952. It
329
has occurred nine times in the United States, with the
last outbreak in 1929. Serotypes A, O, and C occur in
South America, and six serotypes (A, O, and C, SAT-1,
SAT-2, and SAT-3) occur in Africa. The SAT serotypes are
generally restricted to sub-Saharan Africa. Serotypes A,
O, C, and Asia 1 occur in Asia and the Middle East.
Japan, Australia, New Zealand, and certain Pacific is-
lands are free of FMD. A consistent vaccination program
in Western Europe and most of Eastern Europe greatly
diminished the prevalence of FMD. The European Union
stopped vaccinating for FMD in January 1992. Sporadic
local outbreaks occurred in Italy in 1993 (Maragon et al.
1994), in the Balkans in 1995, and in sheep in Greece in
1996. These outbreaks were controlled either by stamp-
ing out or by stamping out and vaccination.
Stringent importation regulations (Section 306A of
the Tariff Act of 1930 and acts of 1890 and 1903) on an-
imals and animal products have effectively protected the
FMD-free status of the United States. The U.S. Depart-
ment of Agriculture (USDA) conducts research and diag-
nostic studies with FMDV at its high-containment facili-
ties (greater than biosafety level 3) at the Plum Island
Animal Disease Center (PIADC).
FMD results in direct economic losses through the
loss of meat, milk, draft power, and genetic stocks, and
indirect losses through embargoes and loss of trade. The
direct loss from FMD during the first year of an outbreak
in the United States was estimated at $4 billion, with in-
direct costs up to 10 times more (Blackwell 1984).
Swine Vesicular Disease
SVD is a moderately contagious disease of swine. Mice
may be infected experimentally. Persons working with or
around infected pigs can harbor SVDV in their nasal pas-
sages (Sellers and Herniman 1974), and human infection
has been observed (Brown et al. 1976). Swine vesicular
disease virus is spread primarily by contact with infected
swine or by garbage feeding of SVDV-contaminated
meat products. Fecal transmission is not common (Chu
et al. 1979; Mann and Hutchings 1980), which is unusu-
al for an enterovirus. The SVDV can survive up to 38
days in a pH range of 3.9–9.1 at refrigerator tempera-
tures (Herniman et al. 1973). Earthworms from soil
above pits used to bury infected pigs yielded SVDV when
their gut tracts and surfaces were sampled, emphasizing
the ability of SVDV to persist in the environment
(Coombs 1973). SVDV persisted for 560 days in the
lymph nodes of hams prepared from infected swine
(Mebus et al. 1993b). The feeding of infected meat rep-
resents a disease threat even though the oral route of in-
fection may require at least 300,000 infectious units
(Mann et al. 1975; Mann and Hutchings 1980). Abraded
mucosa is more readily infected, requiring only about
100 infectious particles.
There is no known carrier state for SVDV. Outbreaks
of SVD in Hong Kong (1970) and in England (1972) once
330 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
defined its eastern and western limits. Recently, SVD has
been reported in Italy (1995 and 1996) and in Portugal
(1995).
Vesicular Exanthema of Swine and San
Miguel Sea Lion Viruses
VES is a moderately contagious disease. Under certain
management situations—namely, the feeding of infect-
ed seal meat or contaminated garbage—swine were the
only livestock to show clinical VES. Marine animals (fish,
sea lions, fur seals, elephant seals, etc.) along the Pacific
coast from California to Alaska and likely along the coast
of northeastern Russia are apparently the natural hosts
of the numerous serotypes of VESVs and SMSVs. A cali-
civirus was isolated from a California sea lion (Zalophus
californianus) from San Miguel Island and designated
San Miguel sea lion virus (Smith et al. 1973). Numerous
serotypes of SMSVs were isolated from fish and marine
mammals (Smith et al. 1977, 1979, 1980, 1983b) and
from frozen meat of fur seals fed to mink (Sawyer et al.
1978). Bovine calicivirus (BCV, Tillamook virus), a cali-
civirus serotype isolated from three dairy calves in Ore-
gon (Smith et al. 1983a), may be of marine origin, as an-
tibodies to BCV were found in various marine mammals
(Barlough et al. 1987). Many SMSVs have been shown to
produce vesicular disease in experimentally inoculated
swine (Smith et al. 1973; Berry et al. 1990). Low levels of
antibodies to VESVs and SMSVs have been found in ter-
restrial mammals (feral swine, foxes, buffaloes, donkeys,
and cattle) on the west coast of the United States and on
Santa Barbara Channel Islands (Smith and Latham
1978). The relationship of antibodies to natural disease
is not clear. The infection of humans with SMSVs is in-
ferred but not corroborated.
The VESVs were shown to be present in the meat of
infected swine (Mott et al. 1953) and are spread mainly
by feeding of infected meats, contact, or fomites
(Bankowski et al. 1955). Long-term carriers have not
been shown for VESV.
VES was confined to California from its first descrip-
tion in 1932 until 1951, when pork trimmings from an
interstate California passenger train were fed to swine in
Wyoming. The ensuing epizootic involved 42 states plus
the District of Columbia. The last foci of VES were three
large garbage-feeding operations in Secaucus, New Jer-
sey. The epizootic was controlled in 1956, costing $33
million in direct costs and indemnities (Mulhern 1953).
Two cases of VES occurred outside the United States.
Butcher hogs aboard ships en route to Hawaii in 1946
and 1947 were intercepted and were slaughtered. The
second occurrence was in swine fed uncooked garbage
from a U.S. military base in Iceland in 1955, which was
also promptly controlled (Bankowski 1965). After enact-
ment of laws prohibiting feeding of raw garbage to
swine and requiring the slaughter of infected animals,
VES was eradicated in the United States. VES was de-
clared an exotic disease in 1959 in the United States, and
all countries worldwide remain free of VES.
Vesicular Stomatitis
VS is considered to be a disease of moderate to low con-
tagious potential, as it is primarily an insect-borne dis-
ease. The VSVs can cause clinical disease or subclinical
infections in a wide range of animals, including swine,
cattle, horses, and humans.
The origin of outbreaks and means of dissemination
of VSV is not thoroughly understood. The ecological dis-
tribution of VS is river basins and wooded areas, point-
ing to insect transmission. Serotypes Indiana 1 and NJ
have been isolated from sand flies (Lutzomyia shannoni),
and transovarial transmission was demonstrated (Tesh
et al. 1971; Comer et al. 1990). Sand flies infected with
VSV NJ transmitted the virus to suckling mice (Comer et
al. 1990). Studies on Ossabaw Island, Georgia, have
shown that feral swine become seropositive to VSV NJ
during late spring months when insect activity resumes
(Stallknecht et al. 1985). Black flies were shown to bio-
logically transmit VSV NJ in their saliva for up to 10 days
after feeding on viral fluids (Cupp et al. 1992). Fomites
may play a role in the spread of VS in swine. Contact
transmission may occur (Schoening 1943) and apparent-
ly requires abrasions in the epithelium (Patterson et al.
1955). A carrier state for VSV was not detected by virus
isolation or nucleic acid hybridization studies (Redelman
et al. 1989).
VS has been recognized in the United States since at
least 1916 (Cotton 1927). Vesicular disease outbreaks in
cattle and horses in 1925 and 1926 were caused by infec-
tion with two distinct serotypes of VSV (Cotton 1927),
later called Indiana 1 and New Jersey (NJ). VS was first
described in U.S. swine that were used for the production
of hog cholera hyperimmune serum in Missouri in 1943
(Schoening 1943). In the United States, epizootics, most-
ly serotype NJ, occur about every 10–13 years, starting in
early summer and ending with the onset of freezing
weather. These epizootics generally start in the south-
western states and then spread northward into the Rocky
Mountain states. The disease primarily affects horses
and cattle, with an occasional spillover to swine. The
1982–83 VS NJ epizootic persisted into winter, causing
speculation that VS may be altering its epidemiological
pattern. Movement of infected animals extended the
scope of this outbreak to Idaho and California. The 1995
outbreak of VS NJ was typical, being confined to western
states and disappearing with the onset of cold weather.
VS NJ is endemic in the coastal regions of Georgia (Stall-
knecht et al. 1985).
In Central and South America and in Mexico,
serotypes NJ and Indiana 1 have been associated with VS
in cattle, horses, and swine annually. Subtypes Indiana 2
and Indiana 3 were isolated from mites from rice rats in
Trinidad (Jonkers et al. 1964) and in outbreaks of VS in
 CHAPTER 25 VESICULAR DISEASES House, House
Argentina and Brazil (Garcia Perazzi et al. 1963), respec-
tively. Indiana 2 and Indiana 3 (Federer et al. 1967) occur
in Central and South America, respectively, but have not
been reported to produce clinical disease in swine under
field conditions. There are numerous other serotypes of
vesiculoviruses (House and Wilks 1983; Tesh et al. 1983;
Travassos da Rosa et al. 1984) but none has been report-
ed to cause vesicular disease in swine under natural con-
ditions.
CLINICAL SIGNS
The clinical signs of FMD, VS, VES, SVD, and SMSV
cannot be distinguished from each other in the field.
Generally, within 1–5 days postexposure, the body tem-
perature rises sharply to 40.5˚C or higher. Areas of the
331
epithelium become blanched, followed by the formation
of vesicles and erosions after loss of the epithelium. Vesi-
cles up to 3 cm in diameter may be found on the snout,
with vesicular lesions extending into the nares, on the
lips, tongue, hard and soft palate, coronary band, on the
soft tissues of the feet (interdigital clefts and bulbs), and
soft tissues around the dewclaws (see Figs. 25.1–25.4).
Vesicular lesions may occur in other areas of the skin, es-
pecially where there is mechanical pressure or abrasion.
Nursing sows may have vesicles on the teats. Slobbering
and chomping are common. Pregnant sows may abort
due to fever. The viruses of FMD, SVD, VES, and VS are
not reported to infect the fetus.
Vesicles yield a serous fluid if they rupture (usually
6–24 hours after formation), and the epithelial layers
above the stratum germinativum may detach. Eroded,
25.1. Lesions on a pig’s foot, the
second day of clinical signs of
FMD. Note blanching (arrow A),
cracking of the epithelium (arrow
B), and an erosion left where
epithelium has been lost (arrow C).
25.2. Lesions on a pig’s foot,
the third day of clinical signs of
FMD. Note the blanched coronary
band of the dewclaw (arrow A)
and debris adhering to vesicular
tissue on the coronary band of the
foot (arrow B).
332 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

25.3. Lesions on a pig’s snout,

the first day of clinical signs of
FMD. Note the vesicles (arrows).

25.4. Lesions on a pig’s snout,

the third day of clinical signs of
FMD. Note the crusty areas (arrow
A) and darker areas where vesicles
have ruptured and fluid has leaked
through the epithelium (arrow B).

hyperemic, hemorrhagic lesions are present. The hoof
may become detached if the vesicles have coalesced
around the coronary band. Severe laminitis may be fol-
lowed by a chronically deformed foot. After the initial
stage of infection, the animal’s temperature may drop to
40˚C. The lesions heal within 7–14 days if no secondary
bacterial infection occurs.
FMD infects many or all of the exposed animals with
no immunity in temperate climates or confined quarters.
In partially immune populations or in tropical climates,
morbidity may be decreased. VES is moderately conta-
gious, with highly pathogenic strains affecting greater
numbers of animals. With SVD, the entire herd may not
be affected, and subclinical cases may occur. VS may
spread horizontally in swine in confined quarters.
Low mortality (<5%) is usually seen with vesicular
diseases. In young animals infected with FMDV or
SVDV, myocardial necrosis may occur and mortality may
reach 50–100%. There have been occasional reports of
specific FMD isolates causing high mortality in mature
animals. Cattle affected with a strain of C4 in Argentina,
sheep in northern Africa affected with a strain of O1, and
mountain gazelles in Israel affected with another strain
of O1 (Shimshony et al. 1986) represent some examples
of 50% or greater mortality in adult animals.
PATHOGENESIS
Foot-and-Mouth Disease
The major route of transmission for FMDV is aerosol
(Sellers and Parker 1969; Donaldson et al. 1970; Thom-
son 1995). Relatively large aerosol particles (3–6 microns
 CHAPTER 25 VESICULAR DISEASES House, House
or greater) adhere to the upper respiratory tract, while
smaller particles (3 microns or less) reach the lower res-
piratory tract. Viral nucleic acid, as detected by in situ
hybridization, was widespread in epithelial tissues of
cattle only 6 hours after aerosol exposures (Brown et al.
1992). In swine following intradermal inoculation in the
snout, widespread FMDV nucleic acid was detected by in
situ hybridization on the first sampling 24 hours after in-
fection (Brown et al. 1995). Langerhans cells may be the
primary means of this rapid distribution of virus. The
pattern of distribution is “segmentally diffuse.” Follow-
ing seeding of secondary targets (epithelium, mucosa,
and myocardium), the virus replicates, and viremia gen-
erally persists for 3–5 days (Sutmoller and McVicar
1976). Within 2–3 days, vesicles develop where stress
and mechanical abrasions occur. In swine this includes
the snout, feet, dewclaws, skin over the joints and pres-
sure points of the extremities, mouth, tongue, and teats
of nursing sows.
In addition, FMDV replication in the epithelial cells
of the mammary gland is well documented. The FMDV
is generally shed in the milk of cattle for up to 10 days
postexposure, corresponding to the development of
virus-neutralizing antibodies (Blackwell et al. 1982,
1983). In some cattle, however, the virus may be shed in
the milk for up to 7 weeks, even in the presence of neu-
tralizing antibodies in the serum (Burrows et al. 1971).
Similar patterns of virus shedding in milk probably oc-
cur in swine. The virus is found in large quantities in oral
and respiratory secretions 2–7 days postexposure. In
young animals severe myocardial necrosis may occur.
Swine Vesicular Disease
Lesions are evident 3–11 days after eating contaminated
food (McKercher and Graves 1981) and as early as 2–4
days after experimental inoculation (Burrows et al. 1973;
Lai et al. 1979). The first lesions generally appear on the
coronary band. A fever, which persists for up to 5 days,
essentially coincides with the development of a cell-free
viremia and vesicular lesions. The peak viremia occurs
2–4 days postexposure and persists for about 7 days. The
virus persists for at least 7 days in the tissues of the
snout, tongue, coronary band, tonsil, cardiac muscle,
and central nervous system. The stratum spinosum is the
primary site of viral replication in the epithelium. A non-
suppurative meningoencephalitis most frequently oc-
curs in the cerebrum, thalamus, brain stem, and olfacto-
ry lobes. Necrosis and an inflammatory reaction occur in
the endocardium and myocardium (Lai et al. 1979).
Vesicular Exanthema of Swine
VES is primarily introduced by feeding contaminated
raw garbage. However, oral infection with VESV is esti-
mated to require 100–1000 times the amount of virus
needed to produce a lesion by intradermal inoculation
into the snout (Mott et al. 1953). Following entry into the
333
epithelium via an abrasion, the virus multiplies in the
stratum germinativum of the epidermis. Hydropic de-
generation occurs, followed by necrosis of infected cells.
Intracellular edema (hydropic degeneration) and inter-
cellular edema account for vesicle formation (Madin and
Traum 1955). Virus spreads from cell to cell as the infec-
tion progresses. Local lymph nodes may become in-
volved, with extensive lymphocyte destruction and con-
gestion; a low-level viremia occurs and may account for
some secondary lesions.
Vesicular Stomatitis
VSV apparently requires introduction into the epitheli-
um through insect bites or via fomites into minor abra-
sions of the oral or nasal mucosa, teats, or feet (Patterson
et al. 1955). Virus replicates in the stratum germina-
tivum. A vesicle and an exudate form, usually 2–3 days
postinoculation. Without secondary infection, which
may involve the subcutaneous tissue, the lesion heals in
1–2 weeks.
The presence of a viremia is controversial in VSV in-
fection. Early literature indicates that there may be a
viremia. However, this has not been confirmed by more
recent reports. In pigs, virus was isolated from local
lymph nodes but not blood following intradermal inocu-
lation in the snout to approximate the natural route of
infection (Redelman et al. 1989).
LESIONS
The principal lesion, the vesicle, of FMD, SVD, VES, and
VS is identical by gross pathology (Jubb et al. 1985). Vesi-
cles occur in the epithelium of the oral and stratified
squamous nasal mucosa, feet, teats, pressure points on
the extremities, interdigital spaces, eyelids, and around
the coronary bands.
A vesicular lesion begins as a small blanched area and
progresses to a blanched, slightly raised area that ex-
pands as vesiculation progresses. The epithelium may
separate from the stratum basale, leaving a red lesion
with shreds of torn epithelium. Vesicular fluid may leak
through the stratum corneum without the formation of a
true vesicle (Seibold and Sharp 1960). Since vesicles are
most often seen at the site of mechanical stress, they usu-
ally rupture quickly, leaving a red erosion. Often, lesions
are contaminated with dirt and debris (Fig. 25.2), result-
ing in infection with opportunistic bacteria. The foot le-
sions, especially of VES, may be associated with a celluli-
tis with persistent swelling, resulting in lameness and
local lymph node involvement. In young animals infect-
ed with FMDV, myocardial necrosis may produce pale
necrotic areas referred to as “tiger heart.”
Histopathology
The histopathology of FMD, SVD, and VES is considered
quite similar. The lesions begin in the stratum spinosum,
334 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor

where vesicles form because of hydropic degeneration DIAGNOSIS

and intercellular edema. Keratinocytes die, become
spherical, and float as single or clustered cells in the
vesicular fluid (spongiosa) (Fig. 25.5A). The stratum
basale essentially remains intact but may be somewhat
disrupted by VESV infection (Mebus 1977).
The histopathology of VS in cattle has been carefully
studied by Seibold and Sharp (1960) and described by
Jubb et al. (1985) and is probably equivalent for swine.
The lesions develop in the stratum spinosum with inter-
cellular edema that stretches the intercellular bridges
(desmosomes). Cells remain attached at the ends parallel
to the stratum basale but are separated lengthwise by in-
tercellular edema, giving a reticular or “Japanese
lantern” appearance (Fig. 25.5B). Cells become necrotic
in the later stages of the lesion. The epithelial layers
above the stratum basale separate in about 30% of the
cases. Loss of vesicular fluid through the stratum
corneum occurs frequently, and vesicles may not be ob-
served.
The diagnosis of FMD, SVD, VES, and VS must be con-
ducted in the laboratory, for the diseases are clinically in-
distinguishable from each other. In countries where
vesicular diseases are endemic, national animal disease
diagnostic laboratories are generally available. Samples
may be shipped to the World Reference Laboratory for
Foot-and-Mouth Disease at Pirbright, Great Britain, or to
regional vesicular disease diagnostic laboratories includ-
ing the Foreign Animal Disease Diagnostic Laboratory
(FADDL) at the PIADC in Plum Island, New York, Unit-
ed States, or the Pan American Foot-and-Mouth Disease
Laboratory in Rio de Janeiro, Brazil. The director of the
laboratory should be contacted before shipment. Per-
mits may be needed for shipment to some laboratories,
and ever-changing international shipping regulations for
diagnostic samples must be followed.
Samples from acutely ill swine should include vesicu-
lar tissue and, if possible, vesicular fluid from gently

A B
25.5. (A) A multilocular microvesicle of FMD with necrosis of the stratum spinosum (acanthol-
ysis) and keratinocytes floating in the vesicular fluid (spongiosa). (B) Severe intercellular edema
with separation of the cells of the entire stratum spinosum, giving a reticulated pattern typical of
VS; the vesicle forms when the full thickness of epithelium separates from the basal layer (arrow).
(Courtesy of D. A. Gregg.)
 CHAPTER 25 VESICULAR DISEASES House, House
washed, active lesions on the tongue, lips, gums, nares,
feet, dewclaws, extremities, or teats. Heparinized and
clotted blood should be collected for virus isolation and
serology, respectively. Clotted blood for serum should be
collected from recovered animals. Necropsy samples of
tissues for virus isolation should include vesicular lesions
and, particularly from newborn or young animals, skele-
tal muscle and myocardium. These samples should be
shipped on wet ice if they will arrive at a diagnostic lab-
oratory within 24 hours. If a longer shipping time is an-
ticipated, freezing the tissue in airtight vials and ship-
ping on dry ice is recommended. For histopathology, a
complete set of tissues representing all organ systems,
but with emphasis on organs with lesions, should be col-
lected and preserved in buffered 10% formalin.
In swine, the differential diagnosis of FMD, SVD,
VES, and VS should include swine pox, hog cholera,
pseudorabies, porcine parvovirus infection (Kresse et al.
1985), parsnip or celery contact dermatitis (Mont-
gomery et al. 1987), chemical burns, trauma, and my-
cotic stomatitis.
Epithelial and vesicular fluid samples are tested by
complement fixation (CF) or an antigen capture ELISA
for the presence of viral antigens of FMD, SVD, and VS.
ELISA can detect and identify serotypes of FMD viral
antigens at lower concentrations than the CF test under
experimental (Roeder and LeBlanc Smith 1987) and field
conditions (Westbury et al. 1988). The results of these
tests can be available within 4 hours of receipt of the
sample. Virus isolation in cell cultures (Snowdon 1966;
House and House 1989) of suckling mice, electron mi-
croscopy (EM), and animal host inoculation studies may
be done. In nonenzootic areas, virus isolation should be
performed. A positive FMDV isolation with serotype
identification may be completed as early as 24–72 hours
after receipt of the sample. A negative virus isolation
study may take up to 2 weeks, as multiple passages may
be required.
Serum samples may be tested for precipitating anti-
bodies to the FMD VIAA, for neutralizing antibodies to
FMDVs, SVDV, VESVs, and VSVs, and for CF antibodies
to VSVs, with results available in 1–3 days. Virus-neu-
tralizing antibodies are serotype-specific for FMDV, and
animals infected for the first time characteristically de-
velop the highest titer against the homologous subtype.
Antibodies to FMD VIAA were used historically to dif-
ferentiate the immune response to active infection from
that of vaccination. However, repeated vaccination with
killed FMD vaccine may elicit a transient antibody re-
sponse to VIAA (Pinto and Garland 1979). An ELISA em-
ploying baculovirus-expressed FMD nonstructural pro-
teins 3D and 2C can be used to differentiate vaccinated
from convalescent animals (Meyer et al. 1997).
RNA fingerprinting, which uses ribonuclease T1 to
specifically cleave the RNA at unique sites and produce a
335
spectrum of different oligonucleotides, has been used to
study the relationship of FMDVs (Frisby et al. 1976).
However, nucleotide sequencing, which provides the or-
der of the nucleotides in the native RNA, is a more pre-
cise tool to study the variable areas of the genome of
FMDV (Beck and Strohmaier 1987; Westbury et al. 1988;
Marquardt and Adam 1990). The polymerase chain reac-
tion (Meyer et al. 1991; Donn et al. 1994; Marquardt et
al. 1995), combined with nucleotide sequencing, pro-
vides definitive epidemiological information on the ori-
gin of viral strains.
TREATMENT
No specific treatments are known for the vesicular dis-
eases. Palliative measures include feeding soft feeds, re-
moving animals from hard-surfaced or concrete floors,
and providing access to clean water. Providing hygienic
conditions, using antiseptic solutions on affected epithe-
lium, and administering antibiotics may help prevent
and control secondary infections.
PREVENTION
Exotic Vesicular Diseases
It is imperative to prevent the infection of swine with
FMDV since they can produce tremendous infectious
aerosols. Prevention of FMD and SVD is accomplished
by strictly regulating movement of animals and animal
products. Swine from FMD-endemic areas were first im-
ported into the United States from China in 1989 using
the methods developed for cattle importation (House et
al. 1983). Swine semen has also been imported using
testing and quarantine procedures. The importation of
swine embryos may be complicated by the depth of
sperm tracts and other undefined characteristics of
porcine embryos that may cause adherence of microbial
agents to the zona (Callis 1996).
Regulations for the international movement of ani-
mal products other than germ plasm are based on virus
inactivation studies. Inactivation of FMDV and SVDV in
meat products has been reviewed (Blackwell 1984). Pork
products from swine infected with FMDV and SVDV
(McKercher et al. 1985, 1987) and from swine infected
with FMDV, SVDV, African swine fever virus, and hog
cholera virus (classic swine fever virus) (Mebus et al.
1993a, b) were free of virus when subjected to prolonged
curing processes. Virus in the milk of FMDV-infected
cattle was inactivated by pasteurization at ultrahigh tem-
peratures for a short time (148˚C for 2 seconds) (Cunliffe
et al. 1979).
Regulations for cooking of garbage fed to swine re-
sulted in the control and eradication of VES (Madin and
Traum 1953). Swine fed seal meat or other marine-origin
feed supplements could become infected with cali-
336 SECTION 2 VIRAL DISEASES W. L. Mengeling, Editor
civiruses, but such practices are no longer in use and VES
has not recurred.
Domestic Vesicular Diseases
Vaccination has not been used to prevent VS in swine in
the United States (Buisch 1983). The segregation of
swine from infected cattle, horses, and wildlife, including
feral swine, in epizootic regions is a practical suggestion.
The role of insects in the spread of VS justifies an insect
control program during an epizootic.
CONTROL
In case of an outbreak of exotic vesicular disease in
swine, the USDA emergency disease guidelines call for
stamping out of the disease upon identification of the
exotic agent. The plan calls for immediate quarantine of
the affected premises, restriction of animal movement,
public-alert statements, eradication by slaughter, and
close communication and cooperation with industry.
Today, the loss of genetic material and animal pro-
tein, environmental regulations, and animal welfare con-
cerns warrant the consideration of vaccination as a tool
for controlling and eradicating FMD.
The North American Foot-and-Mouth Disease Vac-
cine Bank, maintained by USDA in cooperation with
Canada and Mexico and other international FMD vac-
cine banks, has reserves of inactivated, safety- and po-
tency-tested FMD vaccine antigens available to be for-
mulated into FMD vaccines. Vaccinated animals could be
permanently identified and their movement restricted.
Vaccines for FMD, used throughout the world in
FMD-endemic areas, are inactivated, adjuvanted vac-
cines. These vaccines are produced in BHK (baby ham-
ster kidney) suspension cell cultures and inactivated with
ethylenamine derivatives. Vaccination with the prevalent
serotypes 2–4 times a year is recommended. The tradi-
tional aluminum hydroxide–saponin adjuvanted FMD
vaccines used for cattle are less efficacious in swine than
are oil-adjuvanted FMD vaccines (McKercher and Gior-
dano 1967).
No genetically engineered vaccines for FMD are com-
mercially available, and their practicality is uncertain.
Experimentally, genetic sequences for the key immuno-
genic FMD VP1 were cloned into Escherichia coli, and
VP1 was successfully expressed (Kleid et al. 1981). The
cloned protein protected vaccinated cattle and swine
against a virulent challenge inoculation. The system was
successful with selected subtypes of serotypes A and C,
but for serotype O, the cloned proteins did not provide
consistent protection. The amino acid sequence of criti-
cal polypeptides, which could induce neutralizing anti-
bodies to serotype O, was deduced from the nucleotide
sequence. Synthetic polypeptides protected guinea pigs
(Bittle et al. 1982) and cattle (DiMarchi et al. 1986)
against a virulent challenge inoculation. Roosien et al.
(1990) have synthesized empty capsids of FMDV in in-
sect cells using baculovirus-expression vectors, but there
are no reports of their efficacy.
Inactivated VS vaccines were produced and provi-
sionally licensed for use in cattle during the 1982–83 epi-
zootic, and for horses and cattle in 1995. Efficacy data on
these products are not available. There is no vaccine
available for SVD, since eradication is the preferred
method of control.
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