Invited Review

Veterinary Pathology
2014, Vol. 51(6) 1076-1089
Advancement of Knowledge of Brucella ª The Author(s) 2014
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Over the Past 50 Years DOI: 10.1177/0300985814540545
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S. C. Olsen1 and M. V. Palmer1

Abstract
Fifty years ago, bacteria in the genus Brucella were known to cause infertility and reproductive losses. At that time, the genus was
considered to contain only 3 species: Brucella abortus, Brucella melitensis, and Brucella suis. Since the early 1960s, at least 7 new species
have been identified as belonging to the Brucella genus (Brucella canis, Brucella ceti, Brucella inopinata, Brucella microti, Brucella neotomae,
Brucella ovis, and Brucella pinnipedialis) with several additional new species under consideration for inclusion. Although molecular studies
have found such high homology that some authors have proposed that all Brucella are actually 1 species, the epidemiologic and
diagnostic benefits for separating the genus based on phenotypic characteristics are more compelling. Although pathogenic Brucella spp
have preferred reservoir hosts, their ability to infect numerous mammalian hosts has been increasingly documented. The maintenance
of infection in new reservoir hosts, such as wildlife, has become an issue for both public health and animal health regulatory personnel.
Since the 1960s, new information on how Brucella enters host cells and modifies their intracellular environment has been gained.
Although the pathogenesis and histologic lesions of B. abortus, B. melitensis, and B. suis in their preferred hosts have not changed,
additional knowledge on the pathology of these brucellae in new hosts, or of new species of Brucella in their preferred hosts, has been
obtained. To this day, brucellosis remains a significant human zoonosis that is emerging or reemerging in many parts of the world.

Keywords
Brucella, pathogenesis, zoonosis, reservoir hosts, laboratory models

In 1966, the first issue of Pathologia Veterinaria—now known
as Veterinary Pathology—published an article on the charac-
teristics of Brucella granulomas by K. L. Jacob, and the third
issue included a manuscript by B. I. Osburn and P. C. Kennedy
describing pathologic and immunologic responses of fetal
lambs after infection with Brucella ovis. As the journal
embarks on its second half-century of publication, it seems
appropriate to assess the advancement of knowledge on this
topic since these articles were published in the first issues of the
journal.
Fifty years ago, bacteria in the genus Brucella were known
to cause infertility and reproductive losses with predilection
for causing placentitis, fetal pneumonia, and mastitis. At that
time, the genus contained only Brucella abortus, Brucella
melitensis, and Brucella suis, the 3 classic species of brucel-
lae, and these were generally thought to be somewhat host-
specific pathogens of cattle, sheep and goats, and swine,
respectively. They were also known to be zoonotic, with the
capability for causing clinical disease in humans. At that time,
taxonomy placed the 3 classic species in the family Brucella-
ceae, with other bacterial genera such as Bordetella, Pasteur-
ella and so on. Although scientific and practical knowledge of
brucellosis has increased dramatically, in many countries,
brucellosis continues to be a continuing or reemerging zoono-
sis, causing significant economic losses, not only from costs
associated with clinical treatment and lost productivity in
human infections, but also by economic costs associated with
reproductive losses in livestock.
Although a number of studies demonstrated that Brucella
could be recovered for a period of time from environments
associated with infected animals, knowledge at that time indi-
cated that maintenance in a susceptible host was critical for dis-
ease transmission. The literature of 50 years ago would suggest
that environmental persistence was of little epidemiologic
importance. Data gathered since that time have supported the
requirement for maintenance in susceptible hosts.
Addition of New Species to the
Brucella Genus
Since the early 1960s, at least 7 new species have been identi-
fied as belonging to the Brucella genus. The first new Brucella
1
Bacterial Diseases of Livestock Research Unit, National Animal Disease
Center, Agricultural Research Service, United States Department of
Agriculture, Ames, IA, USA
Corresponding Author:
S. C. Olsen, Bacterial Diseases of Livestock Research Unit, National Animal
Disease Center, Agricultural Research Service, United States Department of
Agriculture, 1920 Dayton Ave, Ames, IA 50010, USA.
Email: Steven.olsen@ars.usda.gov
Olsen and Palmer
species added in the late 1960s was Brucella ovis. Isolated in
1952 from a ram in New Zealand, B. ovis was believed to be
the cause of epididymitis, ewe abortion, and lamb neonatal
mortality.80,115 It took 18 years after the first report before
B. ovis was recognized as a member of the genus.81 The con-
troversy regarding its inclusion was partly due to the fact that
it differed from the existing criteria for recognition of brucellae
and because the manifestations of infection in sheep did not fit
the classic disease pattern associated with brucellosis.37 Also,
B. ovis was a rough strain, lacking expression of the O side
chain on its lipopolysaccharide (LPS). At that time, it was
believed that only smooth strains, such as the classic species,
which expressed the LPS O side chain, were virulent and could
maintain themselves within populations of reservoir hosts.37
Beginning in 1966, an increased incidence of abortion, epi-
didymitis, and reproductive failure was reported in dogs in the
United States primarily from large commercial breeding ken-
nels.26 A Gram-negative bacterium, similar to members of the
genus Brucella, was isolated from placental and fetal tissues of
aborted pups. Although first thought to be a biotype of B. suis
because of its biochemical properties, the bacterium was later
named Brucella canis.25,84 Like B. ovis, B. canis is a rough
strain that lacks expression of the O side chain on its LPS.
One of the least controversial of the new strains was Brucella
neotomae. This strain was first isolated in 1957 from the desert
wood rat (Neotoma lepida),116 a rodent that inhabits the western
regions of the United States. It was readily accepted as a member
of the genus due to its smooth colony morphology and because it
fit criteria used to identify Brucella organisms.66 Only about 25
cultures of B. neotomae have been isolated, of which none have
been recovered from domestic livestock or humans.5
In 1994, it was reported that a Gram-negative bacterium iso-
lated from an aborted bottlenose dolphin fetus had metabolic,
biochemical, and bacteriologic characteristics that placed it in
the Brucella genus.43 In following years, numerous isolates
of marine mammal brucellae have been recovered from the
Mysticeti and Odontoceti suborders of cetaceans. Further stud-
ies have led to taxonomic classification of these isolates into
Brucella ceti (predominantly from porpoises and dolphins) and
Brucella pinnipedialis (predominantly from seals) strains, each
of which has several subgroups.46 Bacteriologic studies have
indicated that these strains are phenotypically smooth, like the
classic Brucella strains. Seropositive responses to marine bru-
cellae have been observed in 53 cetaceans, with isolation or
identification of the bacteria having occurred in samples from
18 marine mammal species.59 It is suspected that cetacean bru-
cellosis may be distributed worldwide in the oceans. The ST27
genotype in Pacific B. ceti and B. pinnipedialis strains, charac-
terized by a specific genetic location for the mobile genetic ele-
ment IS711, has been associated with zoonotic infections in
Peru and New Zealand.31
Common voles (Microtus arvalis) were found to be a host
for another new species, Brucella microti, which has also been
recovered from wild red foxes (Vulpes vulpes).60,111 Data sug-
gest potential long-time survival of B. microti in soil, as this
species was isolated from soil samples after storage for 6
1077
months at 4 C.112 Prolonged survivability in soil suggests
B. microti may have a reservoir outside mammalian hosts.
Experimental studies show close similarity of this Brucella
sp to B. suis. To date, there is no evidence of domestic animal
or human infection with B. microti.93
Another new strain, Brucella inopinata, was isolated from
an infected human breast implant.113 Phylogenetic studies most
closely link this strain to B. ovis.93 Additional isolates recov-
ered from wild Australian rodents and from a human patient
with chronic destructive pneumonia have been proposed to
be novel lineages of B. inopinata.120,121
Several other bacterial isolates have been identified that
may eventually be classified in the Brucella genus. These
include 2 atypical Brucella strains isolated in 2008 from 2
foxes from eastern Austria that had oxidative, bacteriologic,
and molecular characteristics that placed them in the Brucella
genus, but data indicated they represented a novel species sep-
arate from other Brucella, including B. microti.61 Other poten-
tial new strains, with characteristics typical of Brucella, have
been recently recovered from 2 stillborn baboons (Papio
spp)109 and African bullfrogs (Pyxicephalus edulis).40
Molecular Characterization of the
Brucella Genus
Molecular studies have found that most Brucella strains have 2
circular chromosomes encoding approximately 3.2 kb with 2
replicons. Bacteriophages have been isolated from Brucella
strains,32 but plasmids have not. The extent to which Brucella
are able to undergo exchange among themselves or with other
bacteria is unknown. It has been proposed that intracellular
niches inhabited by Brucella during residence in the host limit
their opportunities for genetic exchange with other bacteria. It
should be emphasized that Brucella infections are polyclonal
rather than clonal infections, and clinical infection is generally
associated with multiple bacteria crossing mucosal barriers and
colonizing tissues under in vivo conditions. Cumulative data
suggest dosages of virulent Brucella species required for
50% infection across mucosal surfaces of hosts other than
guinea pigs or mice are most likely in the range of 103
to 104 colony-forming units (CFU).78,119
Development of new molecular techniques, such as DNA-
DNA hybridization, GþC base ratio determinations, and
DNA–ribosomal RNA reciprocal hybridizations, demonstrated
that the genus Brucella was more closely related to plant patho-
gens in the Agrobacterium-Rhizobium complex of organ-
isms.24 Studies have found such high homology within the
genus that some authors have proposed that all Brucella were
actually 1 species.62,123 On average, the genome sequences of
6 species of Brucella (B. abortus, B. suis, B. melitensis, B. ovis,
B. canis, and B. neotomae) display an average of >94% identity
at the nucleotide level,29,124 with B. abortus and B. melitensis
being the most closely related. A close relationship has also
been detected between B. canis and B. suis, whereas B. neoto-
mae and B. ovis demonstrate greater divergence levels from
1078
other Brucella species. With the exception of biovar 5, B. suis
isolates cluster together.126
Although genomic techniques have suggested that Brucella
strains may comprise a single genetic ‘‘species,’’ the epidemio-
logic and diagnostic benefits obtained by assigning Brucella
strains to separate ‘‘nomenspecies‘‘ based on their distinctive
phenotypic characteristics are more compelling than data
gained through genomic analysis. Information gathered over
the years has demonstrated that most pathogenic species are
capable of infecting numerous mammalian hosts even though
most species of Brucella have preferred reservoir hosts. How-
ever, over the past 50 years, the epidemiologic importance of
virulent Brucella species in hosts other than the ‘‘traditional’’
preferred host has increased. For example, cattle or camels in
Central Asia or the Middle East have become established as
reservoir hosts for B. melitensis with zoonotic implications, and
increasing isolations of B. suis in cattle in the southeastern
United States have public health significance due to the shed-
ding of high numbers of bacteria within milk of infected cattle.
Impact of Brucellosis in Humans
Brucellosis remains one of the most important zoonotic dis-
eases worldwide and is reemerging in some countries. The
highest prevalence of human disease is currently found in areas
of Africa, Asia, Latin America, and the Middle East. Mortality
is uncommon; symptoms include fever, night sweats, anorexia,
polyarthritis, meningitis, and pneumonia. Endocarditis, primar-
ily associated with B. melitensis infection, is the principal cause
of human mortality. The most common manifestations of loca-
lized disease are osteoarticular (ie, peripheral arthritis, sacroi-
liitis, spondylitis).10 Incubation periods can be long (up to 6
months), and symptoms may persist for years in the absence
of treatment. Brucellosis in humans almost always originates
from an animal reservoir.55 Infection most commonly occurs
through consumption of nonpasteurized dairy products from
Brucella-infected animals but can also occur by direct contact
with infected animals or tissues or fluids associated with abor-
tion. Human infection occurs across mucosal surfaces: by aero-
solization into respiratory tissues, by oral consumption, or by
penetration through breaks in the epidermis. Inadvertent expo-
sure to live vaccine strains, most commonly via needle sticks,
has also been a frequent source of human infections, especially
in the veterinary profession.15 While rare, human-to-human
dissemination of brucellosis through breast milk or venereal
transmission has been reported.27,81
More than half a million cases are reported worldwide,
although due to the wide-ranging and nonspecific nature of
clinical signs, the true number may be much higher.55 In ende-
mic countries, prevalence rates exceed 10 per 100 000. Multi-
ple studies have demonstrated that addressing brucellosis in
animal reservoirs is the most cost-efficient mechanism for
controlling human brucellosis.16,71,100 As such, most control
strategies involve control of livestock brucellosis through
vaccination and use of test and removal strategies. Livestock
vaccination has proved to successfully decrease human
Veterinary Pathology 51(6)
brucellosis and can be especially helpful in developing nations.
One example is illustrated in the vaccination of more than 33
million sheep and goats over an 11-year period in Mongolia.73
The incidence rate of human brucellosis declined from 4.8 per
10 000 to 0.23 per 100 000. Concurrently, abortions in small
ruminants dropped to 20% of prevaccination levels.
Although still considered uncommon, increasing numbers
of reports over the past decade have documented human infec-
tion with B. canis causing endocarditis, peritonitis, and other
clinical symptoms.52,128 The most common routes of transmis-
sion to humans are through contact with infected dogs or their
secretions, fetal membranes, or aborted puppies. Diagnosis of
B. canis infection in humans is likely underdiagnosed due to
a low index of suspicion, nonspecific symptoms (eg, fever and
fatigue), and lack of cross-reaction on common serologic tests
using smooth Brucella antigens.88 Human zoonosis with B.
canis appears to be an emerging problem primarily in people
of low socioeconomic status living in urban slums where there
are high numbers of free-roaming dogs.79 Epidemiologic
investigations of a human B. canis infection in a slum envi-
ronment in Argentina revealed 29 of 97 (29.9%) neighbor-
hood dogs and 13 of 69 (18.6%) neighbors of the index case
were serologically positive for B. canis.79 Likewise, a serolo-
gic survey in Turkey documented the highest prevalence
(7.8%) in patients from an urban area where stray dogs out-
numbered pet dogs.107 Some reports suggest there may be a
link between B. canis infection and patients with immunosup-
pressive, inflammatory, or metabolic disorders, such as endo-
carditis, Gaucher disease, human immunodeficiency virus
(HIV), and Guillain-Barré syndrome.76,79,128
Currently in the United States, brucellosis is predominantly
a disease associated with international travel or as a food-borne
disease caused by ingestion of nonpasteurized dairy products
originating from Mexico.39,122
Role of Wildlife Reservoirs
Fifty years ago, only domestic livestock were considered of
significant epidemiologic importance with regard to public
health or regulatory programs. In the United States, although
the spillover of B. abortus into elk (Cervus elaphus) and plains
bison (Bison bison) in the Greater Yellowstone Area (Yellow-
stone National Park and surrounding areas) had been identified
decades previously,106 brucellosis in these hosts was not con-
sidered of epidemiologic importance partly due to the high pre-
valence of disease in domestic livestock. In subarctic areas, the
prevalence of B. suis biovar 4 in reindeer and caribou (Rangifer
tarandus) was known and suspected to be of public health
importance.64 Serologic surveys had identified brucellosis in
African wildlife,38,104,108 but the epidemiologic significance
of disease in these reservoir hosts and the significance to public
health and livestock industries were unknown. In Europe, B.
abortus had been detected by culture in Swiss chamois (Rupi-
capra rupicapra)18 and B. suis by culture or serology in red
foxes in Russia, Bulgaria, and Wales.101 In general, it may be
concluded that 50 years ago, the long-term health impacts of
Olsen and Palmer 1079

brucellosis on these wildlife populations were thought to be Cellular Entry

limited, and their role in transmission of disease to humans
Over time, a great deal of knowledge has been gained on the
or domestic livestock was considered minimal.
interactions of Brucella with host phagocytic cells. Entry of
Since that time, numerous wildlife reservoirs of both clas-
nonopsonized smooth Brucella relies on the cytoskeleton of
sic and new species of Brucella have been identified. As bru-
the host cell for internalization. Smooth strains of Brucella
cellosis has been eradicated from domestic livestock in the
interact with cholesterol-rich microdomains (lipid rafts)
United States and other countries, persistence in wildlife
within the plasma membrane that facilitate contact with the
reservoirs has become of greater concern. This is demon-
host cell and mediate internalization into phagocytic cells.
strated by the controversy regarding B. abortus in elk and
Lipid rafts contain glycosphingolipids, cholesterol, and
bison in the Greater Yellowstone Area and the capability of
glycosyl-phosphatidylinositol anchored proteins34 and facili-
these species to transmit brucellosis to cattle under field
tate membrane-associated sorting events, such as the forma-
and/or experimental conditions. B. melitensis has been iso-
tion of multi-subunit membrane complexes and signaling
lated from wild bovids such as ibex (Capra ibex) and chamois
across membranes and membrane fusion. Besides the plasma
in the European Union51 and from Nile catfish (Clarias gar-
membrane, lipid rafts are also found in intracellular orga-
iepinus).41 The infection of B. suis in wild swine populations
nelles and vesicles. The Brucella LPS O-polysaccharide
in the United States, Europe, Australia, and other countries
appears to be a key molecule for interaction with lipid rafts
not only has led to zoonotic infections but has caused regula-
on host cells but also prevents complement-mediated bacter-
tory issues by inducing positive responses on serologic tests
ial lysis and host cell apoptosis.9 Opsonization of smooth
when B. suis is transmitted to domestic livestock. The infec-
strains of Brucella increases entry 10-fold and occurs through
tion of wild boar (Sus scrofa) and European brown hares
IgG (Fc) and complement (C3b and 4b) receptors on the sur-
(Lepus europaeus) in Europe with B. suis biovar 2 has also
face of phagocytes, which diverts smooth bacteria from lipid
confounded diagnostics due to positive serology when domes-
rafts and targets entry to the phagolysosomal compartment.
tic livestock, particularly swine, are infected.
Receptor-mediated phagocytosis leads to greater killing of
Although Brucella infections in wildlife reservoirs may
internalized Brucella by monocytes.
have minimal impact on the viability of free-ranging popu-
Entry of rough strains of Brucella differs from smooth
lations, data demonstrating disease transmission to domestic
strains as rough strains are unable to sustain interactions with
livestock and humans under field conditions convey their
lipid rafts and are readily phagocytosed following either Toll-
epidemiologic importance. The difficulties in controlling
like receptor 4 (TLR4) or mannose receptor recognition of the
disease in free-ranging wildlife make the problem very com-
LPS-deficient bacterial surface. As a result, rough strains
plex. In addition, spillover of Brucella species into other
demonstrate elevated invasion possibly due to exposure of
hosts has occurred under field and experimental conditions.
ligands that are normally hidden by the O side chain and may
For example, B. abortus infection led to high mortality in
have increased capability to adhere to macrophages.62 Conse-
bighorn sheep (Ovis canadensis) after inadvertent transmis-
quently, nonopsonized rough Brucella are internalized as effi-
sion from aborting elk.74 Wild red foxes have been found to
ciently as opsonized Brucella, are rapidly targeted to the
be reservoirs for both B. suis biovar 2 and B. microti most
phagolysosomal compartment, and are generally unable to
likely due to eating diseased hares and rodents, respectively.
replicate.103 Entry of smooth and rough Brucella strains into
Historical experiences suggest that spillover of disease to
the cells through different pathways may also involve recep-
other hosts could become important and should not be dis-
tors with a distinct ability to regulate the level of phagocyto-
missed as insignificant.
sis. In contrast to rough strains that are defective at
intracellular replication, the intracellular smooth Brucella
that survive after opsonin-mediated phagocytosis are capable
Knowledge on Cellular Entry and of significant intracellular replication.14 Genes necessary for
Intracellular Activities of Brucella O side chain synthesis (ie, manB, wboA) play a significant role
in establishing the intracellular replicative compartment for
Fifty years ago, it was known that Brucella penetrate the muco-
smooth strains of Brucella.
sal epithelium and are transported as free bacteria, or within
phagocytic cells, to regional lymph nodes. Localization within
regional lymph nodes results in lymph node hypertrophy, lym-
phatic and reticuloendothelial hyperplasia, and inflammation.
Intracellular Trafficking and Survival
If bacteria are not localized and killed within regional lymph Smooth Brucella that enter via lipid rafts quickly traffic
nodes draining the site of infection, they replicate and spread through the early endosomal compartment and depart the pha-
via blood or lymph to other lymphoreticular tissues and organs gosome to form the modified phagosome (termed brucello-
such as the spleen, reproductive tract, and/or mammary gland. some) by acquiring components of endoplasmic reticulum in
It was also known that the bacteremia associated with most a manner similar to autophagosome biogenesis. Brucella ini-
Brucella species is short, and live bacteria are not readily iso- tially localize within acidified phagosomes,102 where they are
lated from blood samples. exposed to free oxygen radicals generated by the respiratory
1080
burst. Brucella require acidification of the phagosomal com-
partment to a pH <4.5 before they display wild-type intracellu-
lar replication. The requirement for low pH is transient and
extends only through the initial stages of intracellular infection.
Localization in an acidified environment induces expression of
the VirB operon (virB 1–10), which controls expression of
genes associated with a type IV secretion system. The VirB
operon interacts with the endoplasmic reticulum to neutralize
the pH of the phagosome.28 The Brucella-induced modifica-
tions of the phagosome prevent fusion with the lysosome. The
brucellosome environment provides Brucella with conditions
of nutrient depletion and limited oxygen availability. It should
be noted that under in vitro conditions, up to 90% of virulent
Brucella and 99% of nonvirulent Brucella may be killed fol-
lowing intracellular entry.21,98
Another mechanism used by Brucella for intracellular sur-
vival involves modification of the lipid content of the phago-
some limiting membrane. Virulent Brucella strains express a
cyclic glucan synthase (cgs) that produces and secretes low-
molecular-weight cyclic glucans. These molecules disrupt the
lipid raft microdomain structures within intracellular mem-
branes surrounding the bacteria. This modification of lipid raft
distribution in phagosomal membranes inhibits phagosome
maturation, prevents fusion with lysosomes, and is independent
from VirB and type IV secretion systems.8
Greater understanding of the mechanisms that Brucella use
to survive under intracellular conditions continues to occur.
Since oxidative killing is the primary mechanism employed
by host phagocytes to control replication of intracellular patho-
gens, it has been found that Brucella have multiple mechanisms
to detoxify free radicals. Brucella expresses 2 superoxide dis-
mutases, SodA and SodC, with SodC believed to be most
important for detoxification of superoxide anions generated
by the respiratory burst of phagocytes. The Brucella gene ahpC
(alkyl hydroperoxide reductase C) may also protect against oxi-
dative killing; in other bacteria, it protects against low levels of
hydrogen peroxide and can detoxify the oxidizing compound
peroxynitrite (ONOO–). The dps gene, which plays a critical
role in stationary-phase resistance to oxidative killing and
acidic pH in other bacteria, has also been identified in Brucella.
The O side chain also appears to protect against cellular catio-
nic peptides and oxygen metabolites.
Brucella probably use both stationary and exponential
stages for intracellular survival, with stationary-phase physiol-
ogy providing Brucella with benefits for adapting to the harsh
conditions encountered in the phagosome and exponential
stages associated with replication under favorable conditions.
Molecular mechanisms may regulate adaptation. For example,
VirB has been found to be maximally expressed during expo-
nential growth, but expression is repressed upon entry into the
stationary phase.105 Like other intracellular pathogens, Bru-
cella have adapted to their intracellular lifestyle and no longer
require the accumulation of energy-storing molecules.29 The
presence of cytochromes (cytochrome bc1 complex or quinol
oxidase) with a high oxygen affinity may represent an impor-
tant adaptation of Brucella to their intracellular survival.
Veterinary Pathology 51(6)
Several Brucella have been shown to use heme as an iron
source in vitro and have a critical need for heme during resi-
dence in the phagosomal compartment and during replication
in trophoblasts.34 Macrophages, the preferred host cell for Bru-
cella, are important for heme recycling in mammals. Brucella
scavenge iron through siderophores such as 2,3 dihydoroxy-
benzoic acid or brucebactin,14 which are regulated by iron
response regulators and AraC-like transcriptional activators.7
Mutation of iron regulatory genes has been demonstrated to
attenuate virulent Brucella strains in mammalian hosts and
make Brucella more sensitive to oxidative killing.
Avoidance of Innate Immunity
In accordance with its stealthy nature, Brucella has developed
mechanisms to minimize stimulation of pattern recognition
receptors (PRRs) by the innate immune system of the host. The
Brucella cell envelope has high hydrophobicity and its LPS has
a noncanonical structure that elicits a reduced and delayed
inflammatory response compared with other Gram-negative
bacteria102 and has lower stimulatory activity on TLR4 recep-
tors.103 The O side chain on the LPS can form complexes with
the major histocompatibility complex class II molecules that
interfere with the ability of macrophages to present exogenous
proteins. Brucella ornithine-containing lipids and lipoproteins
in the outer membrane are poor activators of innate immunity.
Brucella bacteria are also devoid of many classic structures
involved in virulence such as pili, fimbriae, capsules, and plas-
mids that stimulate PRRs. The ability of Brucella to prevent
phagosome maturation and fusion with lysosomes may inter-
fere with other innate and adaptive immune processes. As pro-
teins have been identified in Brucella that demonstrate
significant homology with TLR adaptor molecules, these pep-
tides may be a mechanism to interfere with or subvert TLR sig-
naling.90 In addition, the ability of smooth Brucella strains, but
not rough strains, to inhibit macrophage apoptosis is believed
to enhance bacterial survival in the host by maintaining the
favorable environment of the phagocyte, preventing release
of the bacteria into the extracellular environment, where it
exhibits reduced replication, and contributing to avoidance of
antibody and complement inactivation.95 Compared with other
Gram-negative bacteria, Brucella induces a reduced innate
immune response and a lower rate of maturation and activation
of dendritic cells, which may impair development of adaptive
immune responses.
Although it was known that latent infections of Brucella
could occur, the mechanisms for long-term in vivo survival
without immune recognition remain unknown. It is known
that Brucella may reside in a dormant, nonreplicative state
in phagocytic cells in calves infected with B. abortus at a
young age, and recrudescence of clinical brucellosis is also
a problem in human patients. The epidemiologic significance
of latency with Brucella spp in other reservoir hosts has not
been well characterized. Currently, the molecular mechan-
isms controlling recrudescence or host physiology influencing
in vitro replication of Brucella remain unidentified in human
Olsen and Palmer
and animal hosts. Although the significance of erythriol meta-
bolism remains controversial, it has been hypothesized that
the preference for Brucella to metabolize erythritol as an
energy source, as well as the presence of erythritol within pla-
cental tissues, plays a role in the tropism of B. abortus for the
pregnant reproductive tract.33 However, this postulated rela-
tionship between erythritol metabolism and Brucella viru-
lence in ruminants has not been directly tested under
experimental conditions.
Pathogenesis of Brucella Species in
Preferred Hosts
Brucella abortus
First described by veterinarian Bernard Bang11 as early as
1906, B. abortus remains an important zoonotic disease in
ruminants, causing economic losses due to fetal wastage and
infertility.
Regardless of the route of infection, adherence to mucosal
epithelium is a requisite. With the exception of intestinal
mucosa, it is not entirely clear how Brucella, once adhered to
mucosal epithelium, translocate across the epithelial barrier.
Oral exposure to aborted fetuses or placental membranes and
ingestion of contaminated milk by calves are considered pri-
mary routes of infection. Using ligated ileal loops, B. abortus
S19 was seen inside ileal lymphoepithelial cells and inside sub-
mucosal lymphatics, suggesting mucosal translocation via lym-
phoepithelial cells or M cells.1 As Brucella pass to draining
lymph nodes, acute lymphadenitis results. In experimental
infection studies, acute lymph node changes have been charac-
terized as hyperplastic with either neutrophilic or eosinophilic
lymphadenitis.2 Chronic lymph node changes are characterized
by granulomatous lymphadenitis with deep cortical and para-
cortical histiocytosis and germinal center expansion.30,110,127
Other lymph node lesions described include multifocal hemor-
rhage and extramedullary hematopoiesis.42
Brucella can be found in parotid lymph nodes and placenta
of cattle as early as 2 days and 14 days, respectively, after con-
junctival exposure.42,94 Extension of disease from the regional
lymph nodes is chiefly hematogenous. Bacteremia may last
several months and, with time, ceases or becomes intermittent,
recurring in 5% to 10% of animals.127 Bacteremia is followed
by seeding of infection in preferred sites such as gravid uterus,
placenta, spleen, mammary gland, and lymph nodes. In males,
testes and accessory sex glands (ie, epididymis, ampulla, semi-
nal vesicles) are preferentially colonized.19,42,110 Arthritis,
tenosynovitis, and bursitis have also been reported.110 Once
infection is established in sexually mature animals, it often per-
sists indefinitely.110
Cattle, goats, and sheep have cotyledonary, villous, epithe-
liochorial, nondeciduate placentas, and there is similarity in
the pathogenesis and morphology of placentitis in cattle
infected with B. abortus and goats and sheep infected with
B. melitensis.6,26,82,83,85 That is, all are characterized by maternal
bacteremia, massive intracellular replication of Brucella in
1081
chorioallantoic trophoblastic cells, peri- and interplacentomal
exudate, necrosis, high numbers of Brucella in tissues and exu-
dates, and abortion.
In cattle, the disease course is long, being several months
from the time of placental infection to abortion,108 and the
development of disease does not progress at the same rate for
all animals.92 Late-term (ie, 7–9 months of gestation) abortion
and birth of a nonviable calf are the most obvious and often the
only clinical signs. Gross lesions of the placenta vary in sever-
ity and are similar to those of placentitis of other bacterial or
fungal origin108 but generally focus on periplacentomal and
intercotyledonary placenta. Most commonly, there is a necro-
tizing placentitis with viscous, brown exudate that may contain
blood, fibrin, and flocculent detritus.85,108,125 Not all placen-
tomes are equally affected, and some placentomes appear
grossly normal. The post-abortion uterus may be characterized
by diffuse suppurative metritis with greenish-hemorrhagic exu-
date covering the endometrium and caruncles.6 Cows may
present with hemorrhagic and suppurative vaginal discharge
for several days after abortion.6
Placental entry, localization, and proliferation of Brucella
have been described in experimental infection studies of goats
inoculated with B. abortus.6 Using this model, it was shown
that Brucella were first present in hematomas present at the
junction of the maternal septal tip and chorionic villi base, a
region containing erythrophagocytic trophoblastic cells. Infec-
tion extends to adjacent chorioallantoic trophoblastic cells.6
Massive intracellular replication of Brucella in chorioallantoic
trophoblastic cells precedes fetal infection. This occurs as Bru-
cella-laden trophoblastic cells die and are shed into the uterine
lumen. Trophoblastic cell death results in erosion and ulcera-
tion of the chorioallantoic membrane, allowing for widespread
hematogenous dissemination of Brucella to other parts of the
placenta and fetus.6
Microscopically, there is superficial to deep caruncular
necrosis and hemorrhage with neutrophilic and mononuclear
exudates; however, a characteristic finding is the presence
of numerous coccobacilli within both intact and desquamated
trophoblastic epithelial cells and macrophages (Figs. 1, 2).
Organisms are also found free in the exudates that fill the uter-
ochorionic space. Maternal portions of the placenta are variably
affected, ranging from superficial necrosis and multifocal neu-
trophilic and lymphohistiocytic endometritis to severe and
extensive ulcerative endometritis (Fig. 3).92 Once placentitis has
progressed to the point of inevitable abortion, endometritis is dif-
fuse, severe, and necrotizing, with endometrial mucosa replaced
by inflammatory granulation tissue.94,110 There may also be
severe neutrophilic vasculitis within endometrium and caruncu-
lar peduncles.125 Mastitis is characterized as multifocal, intersti-
tial, and lymphosuppurative or lymphohistiocytic.127
It is believed that abortion is due to compromised fetal-
maternal metabolic exchange.94 Aborted fetuses may be
autolytic. The most characteristic fetal lesions are those of
bronchitis and bronchopneumonia (Fig. 4); however, experi-
mental infection studies show fibrinous pleuritis, peritonitis,
and pericarditis may also be seen.127 The most commonly
1082 Veterinary Pathology 51(6)

Figures 1 and 2. Brucellosis; placenta, cow. Figure 1. Intracellular Brucella abortus are labeled red within trophoblastic epithelial cells, des-
quamated cells, and cells of inflammatory exudate. Immunohistochemistry (IHC) for Brucella. Figure 2. Interplacentomal placenta. Note
marked exudate between maternal and fetal membranes. Several trophoblastic epithelial cells contain intracellular brucellae (black arrows),
while epithelial cells of the uterine mucosa do not (white arrow). Hematoxylin and eosin (HE). Figure 3. Brucellosis; uterus, bison. Submu-
cosal gland is characterized by focally extensive erosion with intraluminal neutrophils and necrotic cell debris. HE. Figure 4. Brucellosis; lung,
aborted fetal bison. Bronchioles and alveoli are variably filled with neutrophils, free erythrocytes, and fibrin. There is multifocal interstitial
thickening due to infiltrates of neutrophils and vascular congestion (inset). HE. Figure 5. Brucellosis; epididymis, Bighorn sheep. Hydropic
Olsen and Palmer
infected fetal organ is the spleen, thought to be infected as a
result of bacteremia.94
Swelling of the testis, within the confines of the tunica albu-
ginea, results in changes ranging from testicular atrophy to vas-
cular compromise and infarction. Gross lesions are generally
multifocal, containing liquefied necrotic material. Lesions are
often chronic, with fibrosis and dense adhesions between parietal
and visceral tunics. Lesions may resemble abscesses surrounded
by a fibrous capsule. Microscopically, the granulomatous to pyo-
granulomatous infiltrate is centered on tubules. Intracellular Bru-
cella may be seen within macrophages. More chronic lesions
exhibit degeneration and necrosis of the tubular epithelium,
decreased mitotic activity, and decreased spermatogenesis.
Lesions may extend along the epididymis and involve the semi-
nal vesicles and ampullae.
Brucella melitensis
Widely considered the most virulent, this species has proved
difficult to eliminate from domestic livestock. After more than
a century, no major country has been able to eradicate the dis-
ease following its widespread establishment.87 Pathogenesis
and lesion development in goats infected with B. melitensis are
similar to that of cattle infected with B. abortus. Goats are very
susceptible to infection with B. melitensis and are the primary
source of B. melitensis infection for humans.97 Although sheep
are also susceptible to B. melitensis, the disease is more vari-
able and often self-limiting.3
In goats, early infection is subclinical; however, bacteremia
may produce severe clinical signs and even death in some ani-
mals. Abortion occurs in late gestation, with or without reten-
tion of fetal membranes, and these may be the only clinical
signs.3 The gross lesions of placentitis in the goat are similar
to those seen in cattle aborting due to B. abortus infection.
Entry, localization, and proliferation of Brucella in the goat
placenta have been described in experimental infections of
goats with B. abortus.6
A common site of infection in goats and sheep is the mam-
mary gland. Mastitis is a more common feature of caprine bru-
cellosis compared with bovine brucellosis. The affected
mammary gland may be characterized by multinodular firm-
ness with watery, clotted milk.34 Excretion of organisms in
milk may be protracted in goats but less so in sheep.3,97
Although late-gestation abortion is generally the rule, it can
occur at other stages. Aborted fetuses often appear grossly nor-
mal; however, bronchopneumonia, hemorrhagic pleural fluid,
and enlargement of lymph nodes, liver, and spleen are com-
mon.3,97 In male goats, infection of the testis, epididymis, semi-
nal vesicle, and deferent ducts can occur. Infection of male
genitalia often results in decreased fertility.
Figure 5. (continued) degeneration (long arrows) and intraepithelial cysts (short arrows) are present in the lining of the epididymal duct.
Small numbers of lymphocytes are present in the epithelium. HE. (Courtesy of Jack Rhyan, Animal and Plant Health Inspection Service, United
States Department of Agriculture.) Figure 6. Brucellosis; lung, harbor seal. Cross sections of nematodes contain reproductive organs filled
with larvae and myriad brown-staining Brucella. IHC for Brucella. (Courtesy of Jack Rhyan, Animal and Plant Health Inspection Service, United
States Department of Agriculture.)
1083
Brucella suis
First described in 1914 from an aborted pig fetus in Indiana,
this species has multiple biovars that vary in host specificity
and phenotype.4 Biovars 1, 2, and 3 infect primarily domestic
and feral swine and wild boar. Biovar 1 is most often found
in South America and Asia. Both biovars 1 and 3 have been
reported in the United States, Australia, and China.35 Biovar
2 is the most common strain in Europe.50,118 Biovars 1, 2, and
4 can be transmitted from swine to cattle, inducing transient
seroconversion, which can confound B. abortus diagnostic
assays.85 Biovar 2 differs from the other biovars. It was isolated
from European hares (Lepus capensis) in Germany, Switzer-
land, France, and Denmark.34 It was first thought to be a
mutated B. abortus or B. melitensis since at that time, swine bru-
cellosis did not exist in France and B. suis had not been isolated
from domestic animals. We now know that biovar 2 can estab-
lish a reservoir of infection in hares and can be horizontally
transmitted to swine and cattle. Although pathogenic in cattle
and swine, biovar 2 is not zoonotic.
Abortion in swine infected with B. suis is much less com-
mon than abortion in cattle infected with B. abortus. However,
abortion may be the only clinical sign observed in female swine
infected with B. suis. Early fetal loss may go unnoticed and
appear as infertility.87,97 Abortion generally occurs during the
second or third month of gestation, and stillborn or weak
full-term piglets are common.65,110 Placentas from aborting
sows have been described as hyperemic and edematous, with
yellowish purulent cheese-like miliary nodules.35
Orchitis in boars is generally characterized by unilateral or
bilateral enlargement that may be uneven and bulging.65,72 In
chronic cases, the affected testes may be small and atrophic.
On cut surface, the interlobular septa are thickened, and the
tubules may appear as sharply rounded, granular areas.65
Abscesses of the head of the epididymis have also been
described.65 Arthritis with lameness and occasional posterior
paralysis can be seen in both males and females.35
Histologic lesions of orchitis vary in severity but are gener-
ally characterized by multifocal to coalescent degeneration and
necrosis of seminiferous tubules.65,72 In some cases, there may
be extensive liquefactive necrosis with abscess formation.
Intertubular tissues may be thickened with infiltrates of vari-
able numbers of lymphocytes and macrophages. Langhans-
type multinucleated giant cells may also be present.65 Within
the epididymis, there may be infiltrates of lymphocytes and
macrophages with abscess formation. Azoospermia may be
present.
In reindeer, B. suis biovar 4 causes abortions, stillbirths,
weak calves, retained placenta, orchitis, epididymitis, metritis,
mastitis, arthritis, hygromas, lymphadenitis, and nephritis.44
1084
However, experimental infection of cattle with biovar 4 results
in no clinical signs of disease or lesion formation but can cause
seropositive responses on standard brucellosis assays.45
Brucella canis
First recognized in 1966, B. canis caused widespread abortions
in Beagles.22,61,121 Although B. abortus and B. melitensis can
transiently infect dogs, only B. canis infection results in clinical
disease. The host range of B. canis is limited as experimental
infections of pregnant and nonpregnant cattle, sheep, and swine
have been unable to induce clinical signs.96 However, there is a
high zoonotic risk for persons who handle breeding dogs in
kennels or are exposed to infected animals.77,84
Transmitted both venereally and orally, lesions of B. canis
infection in males include epididymitis, prostatitis, and orchi-
tis. In females, lesions include endometritis, placentitis, and
late-term abortion. Infection can result in infertility in either
males or females. Ocular and skeletal lesions (diskospondyli-
tis) may occur but are less common. Clinical infection is usu-
ally afebrile, and signs are generally limited to spontaneous
abortion or failure to conceive.26 Some dogs remain asympto-
matic despite active infection.125 Puppies may be born dead or
weak, with some litters containing both live and dead puppies.
After abortion, there may be prolonged vaginal discharge.
Infectious materials include fetuses, placenta, lochia,
semen, and urine. Lochia may contain as many as 1010 bac-
teria/ml. Male urine generally contains more Brucella than
does female urine, reaching 103 to 106 bacteria/ml.23 Bactere-
mia is an important feature of the disease and can be prolonged
(ie, 2 years or more) and intermittent. Transmission usually
occurs at the time of mating or abortion.
Gross lesions of lymphadenopathy and splenomegaly can be
seen in adults and puppies of both sexes. Males may have acute
epididymal swelling as fluid accumulates between tunics. Tes-
ticular atrophy can result from chronic infection. Females often
have few gross lesions Aborted fetuses are often partially auto-
lyzed, but fetal lesions may include bronchopneumonia, myo-
carditis, multifocal renal hemorrhage, lymphadenitis, and
hepatitis.
Lymphadenopathy is the result of diffuse lymphoid hyperpla-
sia with increased perifollicular lymphoblasts and infiltration of
numerous plasma cells into subcapsular and medullary
sinuses. Macrophages with intracellular bacteria may be seen
in lymph nodes and spleen if bacteremia is present. In males,
necrotizing vasculitis with infiltrates of lymphocytes, plasma
cells, and neutrophils may be seen in the prostate gland, epi-
didymis, and testes. Spermatogenesis is decreased and even-
tually ceases as the disease progresses. In females, there
may be subacute to chronic endometritis. Endometrial and
myometrial lesions have been described as lymphoplasmacy-
tic with endometrial gland hyperplasia. Hepatic necrosis,
myocarditis, meningoencephalitis, iridocyclitis, and retinitis
have also been described.57
As epididymitis, orchitis, and infertility are common fea-
tures of the disease, seminal fluid cytology may be an
Veterinary Pathology 51(6)
important part of the diagnostic workup. Sperm abnormalities
are common and include immature sperm, deformed acro-
somes, bent tails, swollen midpieces, double tails, heads lack-
ing tails, retained protoplasmic droplets, head-to-head
agglutination, and decreased motility.23,57 As early as 2 weeks
after experimental infection, abnormal sperm are noted; by 20
weeks, 90% of sperm may be abnormal. Neutrophils and
macrophages with phagocytized sperm are often increased in
number. When chronic infection has progressed to bilateral tes-
ticular atrophy, azoospermia is seen.
Brucella ovis
B. ovis has a predilection for the genital tract of sheep. Most
important, it is a cause of epididymitis. It is contagious and pro-
gressive, resulting in testicular atrophy and infertility. Lesions
may be unilateral or bilateral, and bilaterally infected rams are
usually sterile. Clinical signs are not obvious and may go
unrecognized until fertility problems are noted;117 moreover,
Brucella can be found in the semen of infected rams with and
without epididymitis.58 In general, transmission occurs
between rams mating the same ewe during the same estrous
cycle. Clinically normal rams at the beginning of breeding sea-
son have been shown to have palpable epididymitis and Bru-
cella-containing semen by the end of breeding season.20,58
Ram-to-ram transmission may also occur from rams mounting
other rams.58
Disease progression is slow. The organism can persist on
mucous membranes (eg, prepuce) for 1 to 2 months. Acute
infection lasting 2 to 5 weeks may go unnoticed, especially
when occurring during breeding season. In the acute phase, the
testis, particularly the epididymis, is hot, swollen, and
doughy.70 Lesions may be unilateral or bilateral.17 Grossly, ser-
ofibrinous exudate and edema are present in the loose scrotal
fascia and between the parietal and visceral tunics. Chronic
infection focuses on the epididymis, specifically the tail, which
becomes enlarged and firm.
Although relatively avirulent for the nongravid uterus, B.
ovis induces placentitis and abortion in pregnant ewes.110 A
yellowish purulent exudate may be seen, particularly in inter-
cotyledonary areas.47,92 The placenta is edematous, gelatinous,
and thickened with adherence of amnion to chorioallantois.
Intercotyledonary placenta is thickened with multifocal to coa-
lescent areas of plaque-like thickening resembling yellow-
white chamois leather.92,110 Cotyledons may show various
stages of necrosis and detachment.
As with macroscopic lesions, microscopic lesions focus on
the tail of the epididymis; however, lesions can also be seen
in the ampulla ductus deferens, seminal vesicle, and testis.
Lesions in these glands are usually secondary to epididymitis.47
As such, in contrast to bovine brucellosis, there is no primary
orchitis in B. ovis infection.48 The epididymal epithelium is
hyperplastic with hydropic degeneration and intraepithelial
lumina or cyst formation (Fig. 5).48 Epithelial hyperplasia com-
bined with fibrosis results in obstruction and spermiostasis.
Exacerbation of lesions is dependent on spermatozoa
Olsen and Palmer
extravasation and the formation of sperm granulomas.48,70
Much of the resulting pathology during the chronic disease
phase is not the result of the virulence of B. ovis per se but
rather the host reaction to extravasated spermatozoa. Free sper-
matozoa entering the cavity of the tunica vaginalis incite gran-
uloma formation that contributes to testicular atrophy.48
In the uterus, periarteritis and arteritis, with or without fibri-
nous thrombi, are distinctive features,92,110 as is necrosis of the
intercotyledonary and chorionic epithelium. As with Brucella-
induced placentitis in other species, trophoblastic epithelial cells
can be seen to contain numerous intracellular Brucella. Lymph
nodes and spleen show signs of activation and hyperplasia.
Acute interstitial nephritis focused on the corticomedullary junc-
tion and hepatitis of the portal triads are also described.92
Marine Brucella spp: B. ceti and B. pinnipedialis
Marine Brucella spp have been isolated from numerous ceta-
ceans with no evidence of Brucella-induced pathology.43 Those
lesions that have been associated with Brucella infection include
blubber and sub-blubber abscesses; meningoencephalomyelitis;
hepatic, splenic, or lymph node necrosis; pneumonia; myocardi-
tis; osteoarthritis; orchitis; mastitis; endometritis; placentitis; and
abortion.13,49,53,56,89
Cetaceans from which B. ceti has been isolated are often
stranded and in poor condition.49,56 Many emaciated dolphins
are heavily parasitized by a lung nematode. Within reproduc-
tive and intestinal organs of these nematodes are found numer-
ous coccobacilli, identified by immunohistochemistry and
polymerase chain reaction (PCR) as Brucella sp.36,75 Bru-
cella-laden nematodes have been seen in the lungs of both ceta-
ceans and pinnipeds (Fig. 6). Nematode-associated lesions are
characterized as multifocal pulmonary granulomas containing
degenerate nematodes and nematode larvae, macrophages,
lymphocytes, multinucleated giant cells, and abundant immu-
nohistochemically positive Brucella. Ultrastructural studies
show Brucella organisms in close association with developing
larvae. The species identity of the pulmonary nematodes is
unclear. Nematode morphology suggests Parafilaroides sp36
or Otostrongylus circumlitus.75 Parafilaroides decorum is a
common parasite of sea lions; however, Brucella has also been
isolated from another lungworm of harbor porpoises with sim-
ilar morphology, Pseudalius inflexus.99 Although the role of
nematodes in the pathogenesis of disease in cetaceans is
unclear, the primary localization of B. ceti in parasites raises
the intriguing possibility that they may serve as a vector for
transmission of this Brucella spp.
In contrast to B. ceti, most isolations of B. pinnipedialis have
been from clinically normal animals, with no Brucella-associ-
ated pathology.56 However, there are cases in which B. pinni-
pedialis was isolated from emaciated seals, particularly from
pups in rehabilitation centers.49 The relative paucity of gross
lesions in pinnipeds infected with B. pinnipedialis suggests
either a difference in virulence between the 2 Brucella spp or
a difference in susceptibility to brucellosis between pinnipeds
and cetaceans.
1085
Brucella neotomae
First isolated in 1957 from a desert wood rat (N. lepida), B.
neotomae is not pathogenic for domestic animals or
humans.54,117 Infection was rapidly cleared with minimal
lesion development in experimentally inoculated, guinea pigs,
wood rats, and mice, despite dosages as high as 8  109 bac-
teria/mouse.13,54 Swine inoculated intravenously with 1 to 50
 109 bacteria developed bacteremia of up to 5 weeks duration
and agglutinating antibodies but did not show lesions or persis-
tent colonization of tissues.13
Brucella microti
First identified in 1999, B. microti was implicated in an epizoo-
tic infection in common voles in the Czech Republic.63
Affected voles displayed edematous extremities, lymphadeno-
megaly, draining subcutaneous abscesses, orchitis, splenome-
galy, hepatomegaly, and hepatic granulomas.63 It has also
been isolated from mandibular lymph nodes of red foxes in
lower Austria; however, it is not clear if lesions were present
in affected lymph nodes. Although most closely related to B.
suis, B. microti replicates more rapidly in human-derived
monocytes and is more virulent in experimentally inoculated
mice compared with B. suis.
Vaccines
Vaccination is a valuable tool for helping to control brucellosis.
Current vaccines with widespread use under field conditions are
attenuated live strains that are highly effective in preventing the
clinical effects of brucellosis (abortion and/or infected off-
spring), which lead to transmission of disease. Currently avail-
able vaccines are less effective at preventing transient
infection or seroconversion after exposure to virulent field
strains. In the early 1960s, live vaccines containing B. abortus
strain 19 and B. melitensis strain Rev1, as well as the killed B.
abortus strain 45/20 vaccine, were being used under field condi-
tions around the world.91 A the present time, use of the killed 45/
20 vaccine has ceased. Except for the introduction of B. abortus
strain RB51 in the United States in 199691 and the implementa-
tion of the use of B. abortus strain 82 in the Russian Federation
in 1974,69 there has been a paucity of vaccines developed over
the past 50 years that have progressed to significant use under
field conditions in natural hosts. Similarly, a brucellosis vaccine
with acceptable safety and efficacy in humans remains elusive.
Although data from new vaccine candidates continue to be
published, most work has been conducted in murine models.
At the present time, none of the new vaccine candidates have
progressed to significant field use. Due to safety issues associ-
ated with current live vaccines, technologic advances in in vivo
antigen expression, adjuvants, and delivery methods are attrac-
tive for development of nonliving Brucella vaccines with
equivalent or greater efficacy. Although some genes have been
identified as playing a role in protection, progress in the devel-
opment of subunit vaccines is impaired by the difficulties in
1086
fully characterizing which Brucella antigens mediate protec-
tive immunity. The possibility cannot be excluded that protec-
tive antigens may differ by Brucella spp and reservoir host.
Although it is anticipated that developmental work will con-
tinue, it is impossible to predict when a new vaccine will be
developed that demonstrates properties that are an improve-
ment over currently available vaccines.
Laboratory Models for Studying Brucella
Pathogenesis and Vaccine Efficacy
Inbred mice, particularly BALB/c and C57BL strains, have been
used most frequently as laboratory models for studying the
pathogenesis of brucellosis.114 Most studies have used intraper-
itoneal infection, but aerosol, oral, and intranasal routes have
also been used. Some strains such as the C57BL/10 have been
described as being more resistant to Brucella colonization due
to T-helper 1 polarization of the immune response. Bacterial
loads in the spleen and/or liver are most commonly used to
assess pathogenesis. Murine models may more closely relate
to human infection where splenomegaly is a common clinical
observation. However, in preferred hosts, Brucella spp localize
more frequently in lymphatic tissues, mammary glands, and
reproductive organs, and replication of clinical signs in preferred
hosts (ie, abortion) in murine models is rare. The fact that natural
hosts are genetically outbred compared with inbred strains of
mice may also be responsible for differences in pathogenesis and
immunologic responses between laboratory models and reser-
voir hosts. Although murine models are popular due to lower
cost and reduced requirements for animal facilities, the authors’
opinion is that murine models have significant limitations with
regard to evaluating vaccine efficacy and understanding the
pathogenesis of brucellosis in natural hosts. It should be noted
that with the exception of abortion, natural hosts generally do not
demonstrate clinical disease. In comparison, humans appear to
be aberrant hosts for Brucella and often have significant symp-
toms of clinical disease that are frequently chronic.
Other models that have been used include guinea pigs, rab-
bits, rats, and nonhuman primates. The guinea pig is probably
the laboratory model with the greatest susceptibility to Brucella
infection.114 It has been used to facilitate isolation of classic spe-
cies of Brucella, evaluate effectiveness of antibiotic treatments,
and also assess the efficacy of vaccines and vaccine-adjuvant
combinations. Lesions of brucellosis are observed in the liver,
spleen, lungs, and lymph nodes.67 Rats have also seen limited
use as laboratory models of brucellosis. Brucella-induced abor-
tion is rare, but rats can venereally transmit B. abortus, and off-
spring can be latently infected.67,68 Lesions of necrosis in
periplacentomal chorionic epithelium and metritis have been
observed in the uterus of pregnant rats infected with B. abortus.
Nevertheless, the rat model appears to have similar limita-
tions to those of murine models. Rabbits are partially suscep-
tible to Brucella, with susceptibility increasing with pregnancy.
However, rabbits are not considered a model of choice for study-
ing the pathogenesis of brucellosis. Nonhuman primate models
(Macaca arctoides and Macaca mulatta) have been infected
Veterinary Pathology 51(6)
through oral, subcutaneous, and aerosol routes with virulent
strains of Brucella. Bacteremia was demonstrated for up to
8 weeks, and lesions of focal granulomatous hepatitis, spleni-
tis, lymphadenitis, and occasionally orchitis, epididymitis,
and endometritis were reported, similar to lesions in humans
infected with Brucella.114 Current knowledge would suggest
that primate models most closely mimic pathogenesis of dis-
ease in humans but are probably of limited value for under-
standing the disease in preferred hosts.
The validity of murine models of brucellosis has been ques-
tioned due to differences between natural transmission across
mucosal surfaces and intraperitoneal experimental infection, as
well as differences between natural reservoirs and murine hosts
in tissue localization and clinical signs. Key differences between
human and mice immune systems have been noted.12 These con-
cerns are also supported by observations that efficacy of brucel-
losis vaccines in the murine models does not always correlate
with protection observed after experimental vaccination and
infection of natural hosts. These observations support the value
of studying the pathogen in its preferred hosts, particularly when
the goal is resolution of disease issues in preferred hosts. By
studying the pathogen in its natural host, we may be able to
devise efficient control measures in that host, thereby disrupting
transmission of the pathogen to humans.12 Although laboratory
models continue to have value for basic research, they cannot
circumvent the need for studies in natural hosts of Brucella.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
The author(s) received no financial support for the research, authorship,
and/or publication of this article.
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