Vet. Pathol.

21: 564-569 (1984)

Lesions in Broiler and Layer Chickens in an Outbreak of Highly Pathogenic

Avian Influenza Virus Infection

BACHIN,and R. J. ECKROADE
H. M. ACLAND,L. A. SILVERMAN

Laboratory of Large Animal Pathology and Laboratory of Poultry Pathology, School of Veterinary Medicine,
University of Pennsylvania, New Bolton Center, Kennett Square, PA

Abstract. Fifteen chickens, five broilers and ten layers, from the Pennsylvania 1983 outbreak of highly pathogenic avian
influenza virus infection, were examined. Gross lesions in the broilers were limited to serosal petechiae and dehydration. In
the layers there was comb edema, vesiculation, and necrosis. Microscopic lesions were mild to severe diffuse nonsuppurative
encephalitis, very mild to severe diffuse necrotizing pancreatitis, and very mild to severe subacute necrotizing myositis
involving numerous skeletal muscles and most severe in the external ocular muscles and limbs. While many of these lesions
have been seen in experimental infections of chickens with influenza viruses, the pattern of organs involved in this group of
chickens is distinctive.

Outbreaks of fowl plague, an often fatal disease Until now, a major outbreak of fowl plague-like
caused by influenza A viruses, caused severe losses of disease had not occurred spontaneouslyin North Amer-
chickens in Pennsylvania, New York, Indiana, Michi- ican chickens since 1929. We describe the lesions in 15
gan, and New Jersey in 1924-25 and 1929.12*I4 Limited chickens, five from a broiler flock and ten from two
outbreaks of avian influenza viruses infection occurred layer flocks, involved in an outbreak of highly patho-
in chickens on three farms in Alabama in 19757and genic avian influenza virus infection that started in
one farm in Minnesota in 1978.6Lesions of fowl plague Pennsylvania in 1983.’
included cyanosis and edema of the head, vesicles on
the comb, edema of the feet and tibiometatarsal joint, Materials and Methods
and petechiae of the abdominal fat and mucosa of the Chickens were obtained from flocks which were in the
proventriculus. It has been recommended that the term process of being depopulated because they had been declared
“fowl plague” be discarded, except for historical pur- to be infected with highly pathogenic avian influenza virus by
poses. ’ the United States Department of Agriculture, on the basis of
Commencing in 1964, infection with influenza virus clinico-pathological findings and history of mortality. Chick-
ens 1 to 5 came from a flock of45,000 seven-week-oldbroilers
caused respiratory disease and increased mortality rates which had experienced 35% mortality over the past six days.
in turkeys in limited regions of North A r n e r i ~ a . ~ . ~Depression
*’~ and ataxia were prominent signs in the flock.
Unlike the situation in chickens, influenza virus infec- Birds selected for examination were moribund. Chickens 6 to
tion in the turkey flocks recurs, causing low or negligible 10 came from a flock of 21,300 layers aged 90 weeks which
mortality. The only recent detailed description of le- had experienced an 8% mortality. Egg production had
dropped from 58% to 19% over the past seven days. Severely
sions in chickens caused by an American influenza depressed chickens, with obvious comb lesions, were selected.
virus isolate is from experimental infection with virus Chickens 11 to 15 came from a flock of 75,000 layers aged
derived from turkeys. Lesions were multiple foci of 35 weeks which had experienced a 2% mortality and a drop
necrosis in various organs, together with edema of the in egg production from 90 to 61% over the past five days.
head.’ I Other experimental infections of chickens in Birds with the most distinctive gross external lesion, which
which lesions have been described in detail have been was swelling of the combs and wattles, were chosen for
examination.
produced with influenza virus isolates from South Af- Virology:The presence of influenza virus was determined
rican terns and Scottish chickens13and with the Dutch using a standard technique involving the inoculation of chick
East Indies strain of fowl plague virus.8 embryos, testing of allantoic cavity fluid for hemagglutinating
 Lesions of Avian Influenza
activity, and the examination of chorioallantoic membranes
for the presence of type A internal antigen of influenza virus
by an agar gel precipitin test.3 A range of tissues from each
chicken was examined, but for purposes of this study, the
result on only the first positive tissue will be reported. A
sample of each allantoic fluid with hemagglutinatingactivity
was sent to the National Veterinary Services Laboratory,
Ames, Iowa, for determinationof the influenza virus subtype.
Bacteriology: Liver and heart blood from chickens 11 to
15 and a hock joint of chickens 12, 13,and 14 were cultured
on blood agar and MacConkey agar aerobically at 37°C for
48 hours.
Serology: Sera from chickens 1 to 15 were tested in a plate
agglutinationtest for antibodiesto Mycoplasmagallisepticum.
Histology: Tissues collected from each of the 15 chickens
were, with minor exceptions: pancreas, duodenum, heart,
brain, spleen, liver, kidney, trachea, lung, crop, jejunum,
colon, cecum, gizzard, comb, hard palate, breast muscle,
medial thigh muscle, esophagus, turbinates, air sac, gonad,
eye, eyelids, femur, and sacral nerve plexus. In addition, the
bursa of Fabriciuswas collected from the broilers, and oviduct
(posterior magnum) from the layers. Tissues were fixed in
10% buffered formalin, embedded in parafin, sectioned at 6
pm, and stained with hematoxylin and eosin.
Results
Influenza virus was identified in the brain of each
bird, except for chickens 5 and 9 in which it was found
in the comb and conjunctiva, respectively. Except for
isolates from chickens 3, 11, and 15, for which there
were technical problems, each isolate was subtyped as
H5N2 by the National Veterinary Services Laboratory.
No pathogenic bacteria were recovered from the livers
and hearts of chickens 1 I to 15 or the hock joints of
chickens 12, 13, and 14. All birds were negative in the
plate agglutination test for antibodies to Mycoplasma
gallisepticum.
Broilers (chickens1 to 5):The two major gross lesions
were dehydration with shrinkage of the breast muscle,
and diffuse mild petechiae of the serosal surface of the
sternum and abdominal fat, especially that associated
with the gizzard, proventriculus, and ribs. The thymus
had undergone severe atrophy. The bursa of Fabricius
was about half normal size.
Layers (chickens 6 to 10): Edema of the comb was
severe in three birds and mild in the others. Four of the
combs were pale, while one was dark purple with a
scaly surface. One of the severely edematous combs
had multiple round, rough brown depressed areas 0.5
to 1.0 cm in diameter (fig. 1). One bird had purple-red
discoloration of several scales of the lateral part of the
shank. All eyelids were mildly edematous. In one tra-
chea there were mucosal hyperemia, and a small
565
amount of mucus and fibrin in the lumen. Petechiae
were present in the esophageal mucosa of one bird. Egg
yolk peritonitis was present in three chickens.
Layers (chickensII to 15):,Moderate to severe edema
of the comb and wattles was present in four birds,
hyperemic hocks in one bird, and hyperemic and swol-
len hocks in two birds. Swelling was caused by localized
subcutaneous edema; the joints were normal. Egg yolk
peritonitis was present in four chickens.
Histology
The main lesions were encephalitis, myocarditis, pan-
creatitis, myositis, and edema and inflammation of the
comb. Except for the comb, the lesions varied between
broilers and layers in severity but not in type. Findings
are summarized in table I.
There was very mild to severe diffuse nonsuppurative
encephalitis, and very mild diffuse nonsuppurative
meningitis. In all regions there was perivascular cuffing
with lymphocytes and macrophages and occasional
pyknotic cells. Multifocally, especially in the medulla,
midbrain and cerebellar molecular layer, there were
microgliosis and vascular endothelial swelling (fig. 2).
If these foci involved a nucleus, there was necrosis of a
few neurones. Spongiosis of the white matter in these
foci, especially in the brainstem, was apparent in the
more severely affected brains. Mixed mononuclear cells
infiltrated the meninges to a minimal or very mild
extent.
In the ventricular myocardium, there was mild mul-
tifocal to severe multifocal coalescing interstitial infil-
tration by a variety of mononuclear cells, occasional
heterophils and a few pyknotic unidentifiable cells (fig.
3). A few muscle fibers passing through the inflamma-
tory foci on some hearts had undergone necrosis, with
hyperchromic cytoplasm and pyknotic nuclei. Most
atria were not examined.
Endocrine areas and a surrounding zone of normal
exocrine tissue remained as islands in the otherwise
diffusely inflamed pancreas. Acinar cells were replaced
by vacuoles containing an eosinophilic globule or irreg-
ular eosinophilic granules and a little chromatin debris
(fig. 4). Gomori trichrome stains served to emphasize
acinar collapse while demonstrating little or no increase
in collagen. Small numbers of heterophils and mild
edema sometimes were present in the affected paren-
chyma and the interlobular connective tissue. In milder
cases, affected foci were in the areas where acini have
lower epithelium, most distant from endocrine areas.
Associated with margination of chromatin, intranuclear
inclusions with a distinct, pale eosinophilic border and
566 Acland, Silverman Bachin, and Eckroade

Fig. 1: Head of layer chicken with avian influenza (chicken 9). Edema, necrosis, and ruptured vesicles and bullae.
Fig. 2: Cerebellum of broiler (chicken 3) with avian influenza. Multifocal microgliosis and vascular endothelial swelling in
molecular layer.
Fig. 3 Heart of broiler (chicken 1) with avian influenza. Subacute myocarditis and necrosis of a few myocardial fibers.
Fig. 4: Pancreas of layer (chicken 7) with avian influenza. Severe necrotizing pancreatitis with acinar collapse.

a very pale homogeneous eosinophilic interior were
present in one to 12 of the cells of most islets of
Langerhans’ of chicken 11. They were single or less
often multiple, small, round, ranging to large round,
oval or irregular bodies that almost filled the nucleus.
Chickens 7, 12, 14, and 15 had few (approximately ten)
inclusions in their endocrine pancreas, and even fewer
were present in chickens 3,4, and 5.
Mild to moderately severe multifocal subacute nec-
rotizing myositis was present in breast and medial thigh
muscles, as well as external ocular muscles, ciliary
muscle, and skeletal muscles external to the trachea,
dorsal to the palate, and within the eyelid (fig. 5).
External ocular muscle was most severely affected, fol-
lowed by leg muscle. Short segments of muscle were
swollen, fragmented, and infiltrated by heterophils and
macrophages, while there was an associated mild inter-
stitial infiltrate of mononuclear cells and heterophils.
Lesions occasionally were more acute or more chronic.
The more acute changes were swollen fragmented mus-
cle segments without cellular infiltration and the more
chronic lesions had a dense interstitial cellular infiltrate
through which thin basophilic multinucleated bands of
regenerating muscle traversed (fig. 6).
Chicken combs are composed of epidermis, collage-
nous dermis, in the intermediate zone of which mucoid
material accumulates as the bird matures, and a fatty
subcutis. Mild heterophil infiltration and prominent
lymphoid tissue were present in the subcutis of the
comb of one broiler and several small discrete dermal
granulomas with central caseation and peripheral giant
cells were present in another broiler. The dermis of the
comb was severely edematous in five layer chickens.
Inflammation of the superficial zone of the dermis was
mild, acute, multifocal to severe, diffuse, and necrotiz-
ing. Overlying epithelium developed multiple subepi-
dermal vesicles and bullae (fig. 7) and occasional small
intraepidermal vesicles. It underwent necrosis in con-
cert with the dermis, and the vesicles and bullae rup-
tured. A lymphocytic perivasculitis was present around
the vessels of the dermis-subcutis junction, and the
subcutis of the comb was mildly edematous and infil-
 Lesions of Avian Influenza 567
Table I. Histologic lesions in broiler and layer chickens with avian influenza Virus infection
Class Myositis Comb S&
Pancrea- Sinus- Tra- in-
pin-
Chicken Encephalitis Myocarditis itis cheitis flamma-
Of
chicken number titis Breast Limb External
ocular gitis
tion
Broiler 1 ++ +++ +++ + ++ +++ + + - M
2 +++ ++ ++ + ++ ++ + +- +
-
M
3 +++ + + + ++ +++ - -
M
4 +++ + + + ++ +++ - - ND
5 ++ + + + ++ +++ ND + + ND
Layer 6 - + - - - + + ++ - ++
7 ++ ++ +++ + + + +++ + - +
8 + + - - - + + - +++ -
9 + + - - + +++ + + +++ -
10 + + - - - + - + + ND
Layer 11 + + + + ++ ++ - - - -
12 + ++ + + + ++ + + ++ ++-
13 + + - - + ++ + +- ++
14 + + + - + ++ - - +
15 ++ + + + + +++ - + - +
+++ = Severe; ++ = moderately severe; + = mild, very mild; M = male; ND = not done.
trated by a few heterophils, lymphocytes, and macro- colitis; another had moderately severe multifocal acute
phages. The skin of the shank was the only other area necrotizing hepatitis, and a third bird had moderately
of integument examined. There was mild edema. severe acute diffuse bronchiolitis. Single cases of mild
Thickening of the tracheal mucosa, caused by hyper- multifocal acute ventriculitis and mild multifocal sub-
plasia of surface and glandular epithelium and elonga- acute interstitial nephritis were observed in layers. In
tion of the glands and supporting pillars of lamina the spleen of two layers there was mild diffuse necrosis
propria, occurred in two broilers and seven layers (fig. in the periarteriolar lymphoid sheaths, and one of these
8). Capillariesin the lamina propria were distended and birds also had moderately severe diffuse acute enteritis.
there was mild to moderate edema. A few heterophils One layer had moderately severe acute multifocal ven-
infiltrated the lamina propria and the epithelium. Small triculitis, plus fibrinoid necrosis of the small vessels of
numbers of vacuoles containing cellular debris were the eyelid. Thrombi were present in vessels of the eyelid
present in the epithelium. In the glands there was a of another bird.
smaller than normal proportion of goblet cells. Cilia
were less numerous than normal on the surface epithe- Discussion
lium. This outbreak of avian influenza started in Pennsyl-
In the nasal cavity, there was very mild diffuse infil- vania in April, 1983 as a respiratory disease with low
tration of the respiratory mucosa with heterophils in mortality, and spread slowly to involve 23 flocks. Then
two broilers and five layers while in one layer chicken suddenly the character of the disease changed in Octo-
there was severe multifocally extensive diphtheritic in- ber, with the advent of high mortality, dramatic drop
flammation. Mild infiltration of the conjunctival mu- in egg production, milder respiratory signs, and rapid
cosa by heterophils was a common finding in the eye- spread to a large number of flocks.' The H5N2 subtype
lids, while in one broiler moderate numbers of hetero- of influenza virus was the only one isolated in the
phils were present; another broiler had severe acute outbreak, and it was isolated repeatedly in both the
focal ulceration. When the oviduct of laying hens was early, mild, and later, severe forms of the disease. The
affected there was minimal to moderately severe diffuse ten layer chickens had gross and microscopic lesions
heterophil infiltration beneath or within the surface that were representative of those seen in infected layers
epithelium, or moderate numbers of heterophils in the received after October as routine diagnostic accessions
lumen. Broilers had very mild to mild lymphoid necro- by the Laboratory of Poultry Pathology, University of
sis in follicles of the bursa of Fabricius. Pennsylvania. Some additional lesions seen in this gen-
All other tissues were normal except for minor lesions eral population included poorly defined areas of myo-
in individual birds. One broiler had mild diffuse acute cardial pallor and yellow subcutaneous edema on the
 568 Acland, Silverman Bachin, and Eckroade

7 8
Fig. 5 Ciliary muscle and ciliary body (pars plana), eye of layer (chicken 13) with avian influenza. Moderately severe
subacute necrotizing myositis.
Fig. 6 Skeletal muscle dorsal to palate of broiler with avian influenza (chicken 4). Necrosis and fragmentation of muscle
fibers. Interstitial subacuteinflammation and thin regenerating bands.
Fig. 7: Comb of layer (chicken 8) with avian influenza. Subepidermal vesicle, and acute inflammation of dermis.
Fig. 8: Trachea of broiler (chicken 5 ) with avian influenza. Hyperplastic surface and glandular epithelium. Vacuoles
containing cellular debris in epithelium.

shank, feet, and neck. Small irregular coalescing white
opaque areas were sometimes present throughout the
pancreas. Cloudy mucus exuded from the glands of
some proventriculi when slight pressure was applied.
Histologically, there was moderately severe diffuse
acute catarrhal proventriculitis, affecting glands. The
most notable lesion missing from the ten selected layers
were petechiae that were often visible on the intestinal
serosa, mucosa of the proventriculus, and in the epicar-
dial fat and the fat around the gizzard and proventric-
ulus. Masking by egg yolk peritonitis in seven chickens
may be partly responsible for failure to observe pete-
chiae. Overall, the gross lesions caused by this highly
pathogenic avian influenza virus were very similar to
those seen in fowl plague in 1924-25 and 1929.'2*'4No
description of the microscopic lesions in the earlier
outbreaks is available.
Gross lesions of the five broilers were also represent-
ative of those of broilers among the diagnostic acces-
sions, in that changes in both groups were nonspecific.
Petechiae and dehydration can be the only gross lesions
in several bacterial infections and viscerotropic velo-
genic Newcastle disease.
In the frequently affected organs (brain, heart, pan-
creas, and skeletal muscle), microscopic lesions were
more severe in the five broilers than they were in the
ten layer chickens. The difference may be due to age or
strain of bird, to different virulence or tissue tropisms
of the viral isolates, or to our having selected birds in
different stages of the disease. The broilers were mori-
bund, whereas the layers were not.
There are no detailed descriptions of the gross and
microscopic lesions in chickens with naturally acquired
influenza virus infection. Therefore, lesions in the 15
chickens are compared with those found in four exper-
imental infections. These involved two- to three-
month-old chickens given influenza viruses from Ca-
'
nadian turkeys,' six-week-old chickens given influenza
viruses from Scottish chickens and South African
terns,I3and chickens of unspecified age given the Dutch
 Lesions of Avian Influenza 569

East Indies strain of fowl plague virus.8 The outstanding References

difference is that skeletal muscle lesions, present in all 1 BANKOWSKI, R.A..: Introduction and objectives of the
15 of our chickens, were mentioned only in infections symposium. In: Proceedings of the First International
with the tern virus. Experimental chickens had florid Symposium on Avian Influenza, pp. vii-xiv. Beltsville,
MD, 1981
lesions of the external ocular muscles. Late in the course 2 BANKOWSKI, R.A.; MIKAMI, T.: An apparently new res-
of the disease, pharyngeal and intercostal muscles were piratory disease of turkeys. 68th Annu Proc US Livestock
affected, but the lesions were much less intense. Pectoral Sanit Assoc 68:495-5 17, 1964
muscle had minimal lesions. Additionally, in the four 3 BEARD,C.W.: Avian influenza. In: Isolation and Identi-
experimental infections, no lesions of the trachea, tur- fication of Avian Pathogens, ed. Hitchner, Domermuth,
binates, or oviducts were noted. Lesions seen in the Purchase and Williams, pp. 67-69,2nd ed. The American
Association of Avian Pathologists, Texas, 1980
other organs of our 15 chickens were found in one or 4 BEAUDETTE, F.R.; HUDSON, C.B.; SAXE,A.H.: An out-
more of the four experimental infections of chickens, break of fowl plague in New Jersey in 1929. J Agric Res
but no one set corresponds exactly. The closest combi- 49: 83-92, 1934
nation was produced by a virus from turkeys." En- 5 ECKROADE, R.J.; SILVERMAN, L.A.; ACLAND,H.M.: Avian
cephalitis was similar, myocarditis was more necrotiz- influenza in Pennsylvania. Proc 33rd Western Poultry Dis
Conf, pp. 1-2, 1984
ing and a wider range of lesions occurred in the pan- 6 HALVORSON, D.A.; KARUNAKARAN, D.; NEWMAN, J.A.:
creas-from acinar necrosis to fibrosis and fibrinoid Avian influenza in caged laying chickens. Avian Dis
necrosis of arteriolar walls. These chickens, however, 24:288-294, 1980
also had renal tubular necrosis and conspicuous lym- 7 JOHNSON, D.C.; MAXFIELD, B.G.: An occurrence of avian
phoid depletion in the spleen-changes not seen in our influenza virus infection in laying chickens. Avian Dis
15 chickens. Multifocal liver necrosis, seen in only one 20:422-424, 1976
8 JUNGHERR, E.L.; TYZZER, E.E.; BRANDLY, C.A.; MOSES,
of our broilers, occurred frequently. Chickens expen- H.E.: The comparative pathology of fowl plague and
mentally infected with influenza virus from terns and Newcastle disease. Am J Vet Res 1250-288, 1946
chickens had similar brain and comb lesions but lacked 9 LANG,G.; NARAYAN, 0.; ROUSE,B.T.; FERGUSON, A.E.;
pancreatic lesions. With the tern virus, but not the CONNELL, M.C.: A new influenza A virus infection in
chicken virus, there was similar myocarditis. The Dutch turkeys. 11. A highly pathogenic variant, A/turkey/On-
tario 7732/66. Can Vet J 9:151-160, 1968
East Indies strain of fowl plague virus caused similar 10 LANG,G.; ROUSE,B.T.; NARAYAN, 0.;FERGUSON, A.E.;
ulceration of the combs, but also had as major effects CONNELL, M.C.: A new influenza virus infection in tur-
foci of necrosis in the spleen, liver, lung, kidney, intes- keys. I. Isolation and characterization of virus 62 13. Can
tine, and pancreas. The pattern of lesions in the 15 field Vet J 9:22-29, 1968
cases suggests that the effects of the viruses involved in 1 1 NARAYAN 0.;LANG,G.; ROUSE, B.T.: A new influenza A
the Pennsylvania outbreak are distinctive. virus infection in turkeys. V. Pathology of the experimen-
tal disease by strain Turkey Ontario 7732/66. Arch Ges-
The significance of the intranuclear inclusions in the amte Virusforschung 26: 166-182, 1969
endocrine pancreas of most of the chickens with nec- 12 STUBBS, E.L.: Fowl pest. J Am Vet Med Assoc 21:3-12,
rotizing inflammation of the exocrine pancreas is un- 1926
certain. Inclusion bodies are not a characteristic of 3 UYS,C.J.; BECKER, W.B.: Experimentalinfection of chick-
influenza virus infection in any species. Immunospe- ens with Influenza A/Tern/South Afnca/l961 and
cific staining, however, occurred consistently in the Chicken/Scotland/l959 viruses. J Comp Pathol77: 167-
173, 1967
nucleus of cells of chick embryos experimentally in- 4 WATSON, B.K.; COONS,A.H.: Studies of influenza virus
fected with influenza virus,I4 so it is possible that these infection in the chick embryo using fluorescent antibody.
inclusions are related to the presence of the virus. J Exp Med 99:4 19-428, 1954

Request reprints from Dr. Helen Acland, Univ. of Pennsylvania, School of Veterinary Medicine, New Bolton Center, 382 W.
Street Rd., Kennett Square, PA 19348 (USA).