Epizootic Outbreaks of Gizzard Erosion Associated with Adenovirus Infection in Chickens

Author(s): Masaaki Ono, Yo Okuda, Shigeto Yazawa, Isao Shibata, Nobuhiko Tanimura, Kumiko
Kimura, Makoto Haritani, Masaji Mase and Shizuo Sato
Source: Avian Diseases, Vol. 45, No. 1 (Jan. - Mar., 2001), pp. 268-275
Published by: American Association of Avian Pathologists
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AVIAN DISEASES 45:268-275, 2001

CaseReport-

Epizootic Outbreaks of Gizzard Erosion Associated with

Adenovirus Infection in Chickens
Masaaki Ono,A Yo Okuda,A Shigeto Yazawa,AIsao Shibata,A Nobuhiko Tanimura,B
Kumiko Kimura,BMakoto Haritani,BMasaji Mase,Band Shizuo SatoA

AZen-nohInstitute of Animal Health, 7 Ohja-machi,Sakura,Chiba 285-0043, Japan

BNationalInstitute of Animal Health, 3-1-1 Kannondai,Tsukuba,Ibaraki305-0856, Japan
Received 12 June 2000

SUMMARY. Two outbreaks of gizzard erosion in slaughtered broiler chickens in Japan

were examined pathologically and microbiologically. The prevalencesof such lesions were
9%-11% and 4%-50% in the affected flocks. Affected chickens had no clinical signs.
Group I fowl adenovirus (FAV) serotype 1 was isolated from gizzard lesions. Histologi-
cally, gizzard mucosa were necrotic. Intranuclear inclusion bodies were seen in the en-
larged nuclei of degenerating epithelial cells of the gizzard. The keratinoid layer in the
erosion was edematous and desquamated and contained degenerative cells. Moderate to
marked inflammatory cell infiltration was observed in the lamina propriaand perivascular
connective tissue in the submucosa and muscle layer. Immunohistochemical staining
showed evidence of FAV antigens in the intranuclearinclusion bodies within degenerating
epithelial cells. Ultrastructurally,numerous viral particles were demonstrated in the in-
clusions.

RESUMEN. Reportede Caso-Brotes epizo6ticos de erosions en la molleja en pollos de

engorde asociadoscon infecciones por adenovirus.
Se realizaaronexamenes patologicos y microbiologicos durante dos brotes de erosiones
en la molleja en pollos de engorde al sacrificio en Jap6n. La prevalencia de estas lesiones
fue de 9% a 11% y de 4% a 50% en las parvadas afectadas. Los pollos afectados no
mostraron signos clinicos. Se aislo el adenovirus aviar del serotipo I de las lesiones de la
molleja. Histologicamente, se observ6 necrosis en la mucosa de la molleja, corpusculos
de inclusi6n intranuclear en los nucleos agrandadosde las celulas epiteliales degeneradas.
En el sitio de erosi6n se observ6 la capa de queratina edematosa y descamada con con-
tenido de celulas degeneradas. En la lamina propia se observ6 infiltracion celular infla-
matoria de moderada a severa y en la submucosa y en la capa muscular se observo tejido
conectivo perivascular. Con la tincion inmunohistoquimica se observaron antigenos de
adenovirus aviar y cuerpos de inclusi6n intranuclear dentro de las celulas epiteliales de-
generadas. Ultraestructuralmente, se observaron numerosas particulas de virus en las in-
clusiones.
Key words: adenovirus,broilerchicken, erosion, gizzard
Abbreviations:CK = chicken kidney; CPE = cytopathic effect; FAV = fowl adenovirus;
H&E = hematoxylin and eosin; PCR = polymerase chain reaction; RFLP = restriction
fragmentlength polymorphism

Adenovirus infections are widespread in birds
and may affect various organs (11). Fowl ade-
noviruses (FAVs) have been associated with
spontaneous gizzard erosions in broiler chick-
ens, leghorn chickens, and quail (7,17). In
these previous studies, basophilic intranuclear
inclusion bodies contained adenoviruslike par-
tides that were observed in the epithelial cells
of the gizzard. However, such cases were spo-
radic. This present report describes epizootic
outbreaks of gizzard erosion associated with
FAV infection in broiler chickens.

268

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 Adenovirusgizzarderosionin chickens 269

Table 1. Prevalenceof gizzarderosions in slaugh- chickens from the same broiler houses were
tered broiler chickens.
slaughtered; however, no gizzard lesion was ob-
sereved in those flocks. After this case, out-
% Birds
No. with breaks of gizzard erosion have not been ob-
Out- Date of Broiler chickens gizzard served in the slaughterhouse nor on the farm.
break slaughter house in flockA esions Five gizzards were selected from the pool col-
1 9 Sep. 1998 A 4300 0 lected from the two broiler houses for patho-
B 4100 11 logic examination.
C 4100 9 Outbreak 2. In June-July 1999, numerous
10 Sep. 1998 D 8500 0 cases of gizzard erosion were confirmed by vet-
O
16 Sep. 1998 AB 4200 0 erinary inspection at a slaughterhouse in east
BB 4300 0
Japan. The lesions were observed in chickens
CB 4300 0
processed from a commercial broiler farm dur-
17 Sep. 1998 DB 8300 0
ing an 8-day period. Prevalence of lesions was
2 28 Jun. 1999 E 12,400 NEC dependent on the flock (Table 1). Age at the
F 12,400 0 time of slaughter was 55 days. The total mor-
30 Jun. 1999 G 12,400 0
tality rate in each of the flocks from hatching
H 12,400 0 to slaughter was below 3%, and there were no
1 Jul. 1999 I 12,400 50
J 12,400 0 apparent clinical signs. On day 5 of the out-
2 Jul. 1999 K 12,400 13 break, 100 53-day-old chickens were euthana-
L 12,400 0 tized by cervical dislocation for necropsy; of
3 Jul. 1999 M 12,400 32 these, five chickens were found to have gizzard
N 12,400 0 erosion. Affected chickens exhibited no clinical
5 Jul. 1999 O 12,400 6 signs and no macroscopic lesions in other or-
P 12,400 0 gans and tissues. These 51 affected chickens
6 Jul. 1999 Q 12,400 4 and 20 gizzards that were condemned by vet-
R 12,400 0
erinary inspection were examined pathological-
7 Jul. 1999 S 12,400 0
T 12,400 0O ly and virologically.

AApproximation. MATERIALSAND METHODS

BFemalechickens.
CNumerousgizzard erosions were observed;how-
ever, prevalencewas not estimated.
CASE REPORT
Outbreak 1. In September 1998, 12,500
male chickens from three broiler houses in a
broiler production farm were slaughtered at a
slaughterhouse in west Japan. Approximately
34 kg of gizzard from two flocks was con-
demned as eroded and/or ulcerated upon vet-
erinary inspection. The prevalences of gizzard
lesions in the two flocks were approximately
9% and 11%. No gizzard erosion was observed
in slaughtered chickens from the third broiler
house from the same farm on the same day
(Table 1). Age of all chickens at the time of
slaughter was 51 days. Total mortality rate from
hatching to slaughter was approximately 2%.
No apparent clinical signs were observed be-
forehand. These affected flocks originated from
different hatcheries. One week later, female
Pathology. Tissues for pathology were fixed in
20% neutral buffered formalin. Paraffin-embedded
tissues were sectioned at 2 Jim and stained with he-
matoxylin and eosin (H&E).
Tissue sections of a gizzardfrom outbreak 1 and
five gizzardsfrom euthanatizedchickens involved in
outbreak2 were immunostainedwith a commercially
availableavidin-biotin-peroxidasecomplexkit (Vector
Laboratories,Burlingame,CA.) in orderto detect FAV
antigens.The stainingwas performedon serialsections
of tissue blocks from which H&E-stained sections
were cut. The primaryantibodywas rabbitantiserum
againstthe mixed antigens containingserotypes1, 4,
and 8 of group I FAV (preparedin the National In-
stitute of Animal Health, Japan).The serum was di-
luted to 1:2048 in 0.01 M phosphate-buffered saline,
pH 7.6. Tissue sections on which antiserumto FAV
was omitted or substitutedwith nonimmune rabbit
serum during stainingservedas controls.
For the electron microscopy,a 20% neutral-buff-
ered-formalin-fixedsampleof erosivemucosa of a giz-
zard from outbreak 1 was transferredto 2.5% glu-
taraldehyde,postfixed in 1% osmium tetroxide, de-
hydratedthrough an alcohol series,and embeddedin

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270 M. Ono et al.

Fig. 1. Multifocal gizzard erosion (arrows)in outbreak 2. The keratinoid layer in the lesions is rough,
thick, swelled, desquamated,cloudy, and occasionallyhemorrhagic.

Quetol 812 resin (Nissin EM Inc., Tokyo, Japan).
Ultrathin sections were cut and stained with uranyl
acetate and lead citrate and examined with a JEOL
1200EX transmissionelectron microscope.
Virus isolation. Ten ground suspensions of
pooled gizzardmucosa were made from 20 affected
gizzards and five euthanatized chickens from out-
break 2. Five pancreas homogenates were also col-
lected from euthanatizedchickens from outbreak2.
Sampleswere diluted 1:10 (w/v) in Eagle'sminimum
essentialmedium containing 10% tryptosephosphate
broth (Difco, Sparks,MD), 5% fetal calf serum, and
antibiotics;samples were separatelyclarifiedby cen-
trifugation at 3000 rpm for 30 min. The harvests
were inoculated on a chicken kidney (CK) cell cul-
ture and observed for 7 days for cytopathic effect
(CPE). If a CPE was observed,viruseswere identified
by a cross-neutralizingtest (12). Tissue culturefluids
were then collected, and viruses were classified by
polymerase chain reaction (PCR)-restriction frag-
ment length polymorphism (RFLP) (15). If a CPE
was not observed after 7 days, two blind passages
were performed. Cell cultures showing a CPE were
fixed in 20% neutral buffered formalin and stained
with H&E for observationof inclusion bodies.
RESULTS
Pathology. Macroscopically, erosions were
single or multiple in number. As regards shape,
erosionswere irregularor arrangedas small foci.
Erosions varied in size. The keratinoidlayer in
the lesions was degenerating(Fig. 1). The sur-
face of gizzard mucosa under the degenerative
keratinoid layer was opaque and frequentlyul-
cerative.
The main microscopic lesions are summa-
rized in Table 2. The keratinoid layer in the
erosion was edematous and desquamated and
contained degenerative epithelial cells and/or
inflammatorycells. The lumen of the gland was
expanded, and the column of keratinoid ma-
terial in the lumen of the gland was no longer
there. The keratinoid layer of other areas was
normal. Focal necrosiswas accompaniedby de-
generation, desquamation, and the disappear-
ance of epithelial cells in the affected gland
(Fig. 2). In advanced cases, lesions were ulcer-
ated and fibrous tissue at the base of the ulcers
was observed. Largebasophilic intranuclearin-
clusion bodies were present in the enlargednu-
clei of degenerating epithelial cells of affected
glands (Fig. 3). Rarely,there were also eosino-
philic inclusions with a halo. The edematous
lamina propria mucosae contained inflamma-
tory cell infiltrationsconsisting of lymphocytes,

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 Adenovirus gizzard erosion in chickens 271

Table 2. Histologic and immunohistochemicalfindings in the affected chickens.

Gizzard Spleen
and cecal
Inclusion Adenovirus and
bodies in Inflammatory antigens,
No. Necrosis epithelial cells in immunohis- Lymphoid
Tissue from samples and erosion cells muscle layer tochemically depletion
Outbreak 1 5 5/5A 5/5 5/5 1/1 NTB
Outbreak2 20 20/20 9/20 20/20 NT NT
Euthanatizedchicken 5 5/5 3/5 5/5 4/5 4/5
ANo. pos./no. examined.
BNT = not tested.

macrophages,and heterophils. Marked hetero-
philic infiltrationwas observedin gizzardsfrom
outbreak 2; such infiltration was mild in out-
break 1. Slight hemorrhagesin the necroticarea
were rarely observed. There was mononuclear
cell infiltration of the perivascularconnective
tissue in the submucosa and muscle layer, es-
pecially under the erosivelesion (Fig. 4). In five
euthanatizedchickens from outbreak 2, intra-
nuclear inclusion bodies were seen only in the
gizzard lesion. Slight lymphoid depletion and
macrophage infiltration were observed in the
spleen and cecal tonsil. No significant lesions
were found in other organsor tissues. However,
the bursa of Fabricius,bone marrow, thymus,
and brain were not examined.
The intranuclearinclusion bodies and debris
in the lumen of the affected glands were found
positive for group I FAV antigen, as demon-
strated by immunohistochemicalstaining (Fig.
5). Some of the eosinophilic inclusions tested
negative for the virus antigen.
Ultrastructuralanalysisof the gizzardmucosa
was performedby electron microscopy.The in-
tranuclear inclusion bodies were identified as
adenovirusreplicationsites. The basophilic in-

Fig. 2. Photomicrographof erosion of gizzardin outbreak2. The lesion consistsof a degenerativekeratinoid

layer (arrow)and necrotic mucosa (arrowhead).H&E. Bar = 2 mm.

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272 M. Ono et al.

c :
d-Anz;'.tIt-

iJ: S3 lt7

Fig. 3. Gizzardmucosa in outbreak 1. The epithelial cells of affected glands with basophilic intranuclear
inclusion bodies (arrow).Atrophy and degenerationalso are observed in other epithelial cells (arrowhead).
The lumen is filled with desquamatedcells. The lamina propriais edematous. H&E. Bar = 20 !xm.

tranuclearinclusion bodies consisted of numer- PCR-RFLP methods. No viruses were isolated

ous round or hexagonalviral particlesthat were
approximately 70 nm in diameter (Fig. 6).
These particles existed either throughout the
nucleus or in paracrystallinearrangements.
Virus isolation. As shown in Table 3, after
one blind passage, a CPE was observed in all
CK cell culturesinto which gizzardmucosa had
been inoculated. The harvestedcells frequently
had intranuclearinclusion bodies. The isolates
were neutralizedat 1:1600 by antiserumagainst
the Ote strain, which was the representativese-
rotype 1 strain of group I adenovirus(11). The
isolates were not neutralizedby 1:20 dilution
of the antisera to other serotypes of group I
FAV. The isolates were identified again by
from the pancreas. However, viral genomic
RNA of FAV serotype 1 was detected by PCR-
RFLP in all of the pancreassamples.
DISCUSSION
Two epizootic outbreaks of gizzard erosion
are describedin this report.The resultsof path-
ologic and virologic study suggest that FAVwas
responsible for the gizzard erosion. Basophilic
intranuclearinclusions, immunohistochemical-
ly positive for FAV group I-specific antigens,
were ultrastructurallyconsistent with numerous
viral particles; such inclusions were observed
only in or around the lesional areas and were

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 Adenovirusgizzarderosionin chickens 273

'. "_"C' " .-;-.?

C C.; r?- ?h`. ?i
.C
r--?-- cl
?1Cu 'Lt, 'C ?L
2 rs? =--r. r'
L?,
.?
':b rrJ ,C..II E'?
*?r? ???
iCli?; ..,- ??I- hr

P* ati'r.r? tt t

s? I --
??' Irn 4 1 ol?'ni; I;rW
tj? r

Fig. 4. The muscle layer just below the submucosa of a gizzard from outbreak 1. Mononuclear cell
infiltrationis observedin the perivasculararea (arrow).SM = submucosa. H&E. Bar = 200 Irm.

1.=.

Fig. 5. Immunohistochemistryof intranuclearinclusion bodies in the epithelialcells of an affectedgizzard

from outbreak 1. Inclusion bodies tested positive when reacted with rabbit antiserum against FAV.Bar =
200 ,Im.

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 I -

274 M. Ono et al.

Vi w
q
-F
.%f
-**.!

Jrt
?hk ,

Fig. 6. Electron micrographof an intranuclearinclusion body in the gland epithelial cells of a gizzard
from outbreak 1. Viral particles(arrow)(about 70 nm in diameter) fill the nucleus. Bar = 500 nm.

not found in unaffectedportions of the gizzard. Group I FAVs have been classified by their
FAV serotype 1 was isolated from chickens in- serologic relationships, by growth in cell cul-
volved in outbreak2. tures, and by their nucleic acid characteristics.
Spontaneousor experimentalgizzarderosion Twelve fowl serotypes have already been rec-
associated with FAV has been reported ognized (11). Many different serotypes have
(8,10,17). In previous cases, necrotizing pan- been associated with naturally occurring out-
creatitis or hepatitis with inclusion bodies also breaks of inclusion body hepatitis (11). How-
was observed. However, no inclusion bodies ever, no relationship has been found between
were observed in the liver, pancreas, or other serotype and virulence with particularisolates
examined organs or tissues in five euthanatized associated with outbreaks of inclusion body
chickens in the present study. These findings hepatitis (3). Some serotypes may also cause
suggest that the isolated virus has tropism for gizzarderosion. Isolated FAV from the gizzards
gizzardtissue. of outbreak 2 in this report was identified as
belonging to the FAV serotype 1. However,
FAV serotype 8 was isolated from erosive giz-
Table3. Microbiologicalidentification
of the iso- zards of
latesfromchickensin outbreak2. layer chickens (17). Some FAVs (sero-
types not described here) that had originally
Cross- been isolated from either the proventriculusor
Inclusionneutraliz- PCR- a gastrointestinal pool of tissues of broiler
CPEin bodiesin ing test RFLP chickenscaused erosion by experimental
CK cell CK cell for FAV for FAV infection (10). gizzard
Tissue culture culture serotype1 serotypeI
We have shown by experimental infection
Gizzard 10/10A 10/10 10/10 10/10 that isolated FAV alone can cause gizzard ero-
Pancreas 0/5 0/5 NTB 5/5 sion in layer and broiler chickens (14). How-
ANo.positive/no.examined. ever,infection by other pathogenswas not stud-
BNT= not tested. ied in the present cases. The majorityof group

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 Adenovirus gizzard erosion in chickens
I FAVs do appear to play a role as secondary
pathogens in association with other pathogens.
Chicken infectious anemia virus (1) and infec-
tious bursal disease virus (4) enhance the path-
ogenicity of FAV. In the previous case of gizzard
erosion with adenoviral intranuclear inclusion
bodies (17), the affected layer chickens were
positive for both FAV serotype 8 and chicken
infections anemia virus. Bone marrow and bur-
sa of Fabricius, which are important in the di-
agnosis of chicken infectious anemia or infec-
tious bursal disease, were not examined in the
present cases. It is unclear what pathogen
caused slight lymphoid depletion in the spleen
and cecal tonsil. Nevertheless, a virulent strain
of FAV has been found to induce lymphoid de-
pletion in the lymphoid organs (16).
Spontaneous gizzard erosions are common in
broiler chickens. Gross lesions similar to those
resulting from FAV infection can be caused by
other factors. The many potential causes in-
clude histamine (9), gizzerosine (13), mycotox-
ins (6), vitamin deficiencies (2), and bacterial
complications (5). Final diagnosis must depend
on microscopic examination and isolation of
FAV. Observation of intranuclear inclusion
bodies and immunohistochemical detection of
FAV antigen were involved in the present dif-
ferential diagnoses. Mononuclear cell infiltra-
tion of the perivascular connective tissue in the
muscle layer of the gizzard was also a common
finding in this study; nevertheless, these chang-
es appear not to be specific to FAV infection.
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