Avian Pathology

ISSN: 0307-9457 (Print) 1465-3338 (Online) Journal homepage: https://www.tandfonline.com/loi/cavp20

Histopathology and immunohistochemistry of

renal lesions due to avian infectious bronchitis
virus in chicks uninoculated and previously
inoculated with highly virulent infectious bursal
disease virus

B. Y. Chen & C. Itakura

To cite this article: B. Y. Chen & C. Itakura (1997) Histopathology and immunohistochemistry
of renal lesions due to avian infectious bronchitis virus in chicks uninoculated and previously
inoculated with highly virulent infectious bursal disease virus, Avian Pathology, 26:3, 607-624, DOI:
10.1080/03079459708419238

To link to this article: https://doi.org/10.1080/03079459708419238

Published online: 12 Nov 2007.

Submit your article to this journal

Article views: 890

View related articles

Citing articles: 9 View citing articles

Full Terms & Conditions of access and use can be found at

https://www.tandfonline.com/action/journalInformation?journalCode=cavp20
Avian Pathology (1997) 26, 607-624

Histopathology and immunohistochemistry of

renal lesions due to avian infectious bronchitis
virus in chicks uninoculated and previously
inoculated with highly virulent infectious bursal
disease virus

B. Y. CHEN & C. ITAKURA

Laboratory of Comparative Pathology, Graduate School of Veterinary Medicine,

Hokkaido University, Sapporo 060, Japan

SUMMARY
A nephropathogenic strain of infectious bronchitis virus (IBV) was inoculated
intra-tracheally into 14-day-old specific-pathogen-free chicks or ones previously
inoculated with highly virulent infectious bursal disease virus (IBDV) at 7 days of
age. The renal lesions were examined histopathologically and immunohistochemi-
cally at intervals up to 30 days post-inoculation. The mortality was 20% in the
IBDV + IBV-inoculated group, but not in the IBV-inoculated one. Swollen and pale
kidneys due to IBV infection were more severe and of longer duration in dually
infected chicks. At the early stage of infection, the histopathological changes in the
kidneys were similar in both groups, but the ducto-tubular damage was more severe
in the dually infected chicks. At the late stage of infection, the renal lesions were
characterized by chronic interstitial nephritis with formation of lymphoplasmacytic
nodules in IBV-inoculated chicks and by chronic active nephritis which consisted of
tubular degeneration, lymphoid cell reaction and interstitial fibrosis in IBDV + IBV-
inoculated ones. More IBV antigen-positive cells persisted longer in the kidneys of
dually infected chicks than in those of IBV-inoculated ones.

INTRODUCTION
The pathogenicity of infectious bronchitis virus (IBV) for the kidneys as well as
the respiratory tract of chickens is well established (King & Cavanagh, 1991).
Humoral immune incompetence can markedly affect the nephropathogenicity of
IBV (Albassam et al.} 1986; Chandra, 1988; Kinde et at, 1991). Infectious bursal
disease virus (IBDV) is widespread in young chickens and can cause prolonged
immunodeficiency and induce or exacerbate other infectious diseases (Lukert &
Saif, 1991).
Mixed infection of IBDV and IBV has been reported in field chickens and the
IBV-induced renal lesions were very severe in such cases (Butcher et al., 1989;

Received 20 August 1996; Accepted 23 December 1996.

0307-9457/97/030607-18 © 1997 Houghton Trust Ltd

608 B. Y. CHEN & C. ITAKURA

Goryo et al, 1984). The renal lesions were characterized by chronic active
nephritis at the late stage of experimental IBV infection in chemically-bursec-
tomized chickens, which was regarded as a feature of persistent IBV infection,
evidenced by virus isolation (Albassam et al, 1986; Chandra, 1988). However,
virus isolation from tissues only indicated that viable virus was present and no
attempt was made to clarify the pathogenesis of the infection.
Immunohistochemical methods have been used successfully to detect IBV
antigen in affected tissues (Chen et al, 1996; Janse et al, 1994; Jonsson &
Engstrom, 1986; Naqi, 1990; Owen et al, 1991; Tsukamoto et al, 1996; Yagyu
& Ohta, 1990). To the best of our knowledge, however, no sequential immuno-
histochemical study of the IBV-induced renal lesions has been performed using
immunodeficient chickens.
This paper investigates the pathogenesis of chronic active renal lesions in
IBV-infected immunodeficient chicks through sequential histopathological and
immunohistochemical studies in chicks uninoculated or previously inoculated
with highly virulent IBDV.

MATERIALS AND METHODS

Infectious bronchitis virus
The nephropathogenic strain of IBV (MA-87) (Iritani & Takigami, 1995) was
inoculated into the allantoic cavity of 10-day-old specific-pathogen-free (SPF)
chicken embryos and at 3 days post-inoculation (PI) the allantoic fluid was
collected and frozen. A 1:4 dilution of the stored fluid in 0.01 M phosphate-
buffered saline (PBS, pH 7.4) was used as the inoculum at a virus titre of 10 42
median embryo infection dose (EID50)/0.1 ml.

Infectious bursal disease virus

The highly virulent IBDV strain Ehime/91 was isolated from a field outbreak of
IBD in Japan (Tsukamoto et al, 1992). The virus was passaged once in 4-week-
old SPF chicks by oral inoculation. Infected bursae were harvested at 3 days PI,
homogenized as a 10% weight/volume suspension in 0.01M PBS (pH 7.4), and
clarified by centrifugation at 2500 g for 10 min. The supernatant was used as the
inoculum and the virus titre was 1 0 ° EID50/0.1 ml.

Experimental design
One-hundred-and-twenty 7-day-old SPF white leghorn chicks were divided into
an IBDV inoculated group {n = 65) and a control group (w = 55). Chicks in the
former group were inoculated orally with 10 47 EID 50 of IBDV, while the late
group was inoculated orally with 0.1 ml of 0.01 M PBS. At 14 days of age, 60 of
the surviving chicks in the IBDV inoculated group were subdivided into
IBDV + IBV-inoculated group (w = 40) and IBDV-inoculated group (« = 20).
 RENAL LESIONS IN IBV-INFECTED IMMUNODEFICIENT BIRDS 609

Fifty-five chicks of control group were subdivided into an IBV-inoculated group

(n = 35) and a control group (n = 20). Chicks of the IBDV + IBV-inoculated and
IBV-inoculated groups were inoculated intratracheally with 0.3 ml of IBV, and
those of IBDV-inoculated and control groups with 0.3 ml of 0.01 M PBS via the
same route. The four groups were housed separately in isolators under positive
pressure, and feed and water were provided ad libitum.
Clinical signs and mortalities in each group were recorded daily throughout the
experimental period. Three chicks in both IBV infected groups and two birds in
the other two groups were killed at 2, 4, 6, 8, 10, 13, 16, 20 and 25 days PI. All
surviving chicks were killed at 30 days PI.

Histopathology
All killed or dead chicks were necropsied and examined grossly. Tracheas,
kidneys and bursae were removed and fixed in 10% neutral buffered formalin.
Tissues were routinely processed, embedded in paraffin, sectioned at 4 to 5
and stained with haematoxylin and eosin (HE).

Immunohistochemistry
Sections of the tracheas and kidneys were deparaffinized and treated with 0.1%
trypsin (pH 7.4) at 37°C for 30 min. The endogenous peroxidase was blocked by
dipping in methanol containing 0.3% H 2 O 2 at room temperature for 20 min.
Sections were then washed in 0.01 M PBS and immunostained for IBV antigen
by the streptavidin-biotin peroxidase (SAB-PO) method using a Histofine SAB-
PO kit (Nichirei, Tokyo). Sections were treated with a mouse monoclonal
antibody (1:300) to the nucleoprotein of IBV strain M41 (IBV-MoAb, Nippon
Institute for Biological Science, Tokyo) at 4°C overnight. After washing in PBS
they were treated with peroxidase-labelled streptavidin and washed again in PBS.
Sections were then treated with 0.02% diaminobenzidine (DAB)-HCl in buffered
solution at room temperature for 3 min and counter-stained with Mayer's
haematoxylin. Normal mouse serum was used as control substitute for the
primary antibody.

RESULTS
Clinical and gross findings
The incidence of clinical and gross observations is summarized in Table 1.
Respiratory signs such as tracheal rales, coughing and gasping were noticed from
2 to 9 days PI in the IBV-inoculated chicks and from 2 to 20 days PI in
IBDV + IBV-inoculated ones. In the IBV-inoculated group, some chicks had
whitish-green watery diarrhoea from 5 to 10 days PI, but no chicks died. More
severe diarrhoea was observed in IBDV + IBV-inoculated chicks from 4 to 30
days PI. Out of 40 chicks, eight died, one each at 6, 7, 17, 21, 25 and 28 days,
610 B. Y. CHEN & C. ITAKURA

Table 1. Clinical and gross findings in chicks inoculated with IBDVand/or IBV

Group inoculated with

IBDV + IBV IBV IBDV Control

Respiratory signs 40/40 (100)a 35/35 (100) 0/20 (0) 0/20 (0)
Diarrhoea 34/40 (85) 14/35 (40) 0/20 (0) 0/20 (0)
Mortality 8/40 (20) 0/35 (0) 0/20 (0) 0/20 (0)
Tracheal catarrhal exudation 13/40 (33) 6/35 (17) 0/20 (0) 0/20 (0)
Renal swelling 25/40 (63) 9/35 (26) 0/20 (0) 0/20 (0)
Atrophy of bursa 40/40 (100) 0/35 (0) 20/20 (100) 0/20 (0)

"Number of positive chicks/number examined (%).

and two at 8 days PI. Neither clinical signs nor mortalities were observed in the
IBDV-inoculated and control groups throughout the experimental period.
Excess mucus on the tracheal or bronchial mucosae was seen in IBV-inoculated
chicks from 2 to 6 days PI and in IBDV + IBV-inoculated ones from 2 to 13 days
PI. Swollen and pale kidneys were observed in IBV-inoculated chicks from 6 to
16 days PI and in IBDV + IBV-inoculated ones from 4 to 30 days PI. Gross renal
lesions were more severe in dead chicks. Urate deposits were noticed on the
serous capsules of the liver and heart in three chicks that died at 6, 17 and 28 days
PI in the dually infected group. Severely atrophic bursae were detected in all
chicks of both IBDV + IBV-inoculated and IBDV-inoculated groups. No compar-
able gross lesions were observed in control chicks.

Microscopical findings
Histopathology of trachea
Table 2 summarizes the microscopic tracheal lesions in IBDV + IBV-inoculated
and IBV-inoculated chicks. At 2 days PI, compared with the tracheal mucosa of
control chicks (Figure 1A), deciliation, vacuolar degeneration and necrosis of
mucosal epithelial cells were prominent in birds of the two groups inoculated with
IBV (Figure IB). The goblet cells and epithelial cells of the alveolar mucous
glands were markedly decreased in number. Occasionally, heterophilic infiltration
in the lamina propria was more severe in IBDV + IBV-inoculated chicks than in
IBV-inoculated ones. Desquamted epithelial cells with heterophils were fre-
quently seen in the tracheal lumen.
At 4 days PI, hyperplasia of epithelial cells with scattered mucosal epithelial
necrosis and heterophilic infiltration in the lamina propria were more severe in
IBDV + IBV-inoculated chicks. Epithelial hyperplasia was also observed in the
alveolar mucous glands (Figure 1C).
From 6 to 8 days PI, in IBV-inoculated chicks, occasional groups of ciliated
epithelial cells were recognized in the mucosal lining which was slightly thickened
due to epithelial hyperplasia and lymphocytic infiltration. The tracheal mucosa in
 Table 2. Histopathology and immunohistochemistry oftracheas ofIBDV+ IBV-inoculated and IBV-inoculated chicks

Group inoculated with

IBDV + IBV IBV

Tracheal lesion 8 10 13 16 20 25 30 6 8 10 13 16 20 25 30
a
Epithelial deciliation _L

Epithelial
degeneration
Epithelial necrosis
Decrease of mucus
cells
Heterophil infiltration
Epithelial hyperplasia
Lymphoid infiltration O
IBV antigen in
epithelium a
"Days post-inoculation.
b
Mean severity index from three chicks: —, no change; + , mild; + + , moderate; + + + , severe.
c
Mean no. of antigen-positive epithelial cells per 3.15 mm2 tracheal tissue from three chicks: —, no antigen; + , 1 to 4; + + , 5 to 9; + + + , over 10. a
612 B. Y. CHEN & C. ITAKURA

11: **.

£P££3& ilfeijftlli

:»S

Hife
Figure 1. Tracheal lesions in IBDV + IBV-inoculated chicks. (A) Tracheal mucosa of control
chick showing ciliated epithelial cells and goblet cells (arrow); 16 days old. (B) At 2 days PL Note
the diffuse deciliation and vacuolated cytoplasm in degenerated epithelial cells and heterophil
infiltration (arrow) in the epithelial layer with purulent exudate in the tracheal lumen. (C)At4days
PI. Note the epithelial hyperplasia and mucus depletion of the alveolar mucous glands (arrow) and
mitoticfigure (arrowhead). (D) At 8 days PI. Marked thickening oftracheal mucosa with epithelial
necrosis and lymphocytic infiltration into the lamina propria. (E) At 13 days PI. Hyperplastic
mucosa accompanied by necrosis of epithelial cells and mitotic figures in the epithelial cells of
regenerated alveolar mucous glands (arrows). HE stain, bar = 30 (im.
 RENAL LESIONS IN IBV-INFECTED IMMUNODEFICIENT BIRDS 613

• *

Figure 2. JBF an8#»i »n trachea ofIBDV+ IBV-inoculated chicks. (A) At 4 days PL IBV
antigen in cytoplasm ofhyperplastic mucosalepithelial cells. (B) At 8 days PL IBVantigen in some
surface squamous mucosal epithelial cells. (C) At 13 days PL IBV antigen in a few surface mucosal
epithelial cells. Immunostain, bar = 30 fim.

IBDV + IBV-inoculated birds was irregularly thickened and covered with

squamous or cuboidal epithelial cells on the surface, accompanied with severe
deciliation and epithelial degeneration and necrosis (Figure ID).
Epithelial recovery was observed in all IBV-inoculated chicks by at least 13 days
PI. However, IBDV + IBV-inoculated chicks were still showing irregular epithe-
lial hyperplasia with mucosal epithelial deciliation, degeneration or necrosis and
lymphocytic infiltration in the lamina propria until 20 days PI (Figure IE). The
tracheal epithelium in IBDV + IBV-inoculated chicks had recovered by at least 25
days PI.

Immunohistochemistry of trachea
IBV antigen was detected in the tracheal epithelium from 2 to 6 days PI in
IBV-inoculated chicks and from 2 to 13 days PI in IBDV + IBV-inoculated ones
(Table 2). At 2 days PI, in both groups, IBV antigen appeared in a scattered
pattern in the mucosal epithelium, and in a few epithelial cells of alveolar mucous
glands.
At 4 days PI, IBV antigen was also demonstrated in the hyperplastic epithelial
cells in both groups (Figure 2A). The number of IBV antigen-positive cells
increased in IBDV + IBV-inoculated chicks, but decreased in IBV-inoculated
ones. A strong antigen-positive reaction was also seen in some degenerated and
desquamated epithelial cells in IBDV + IBV-inoculated birds.
At 6 days PI, in IBV-inoculated chicks, a few antigen-positive cells were
detected in the hyperplasic epithelium but had disappeared by 8 days PI. In
IBDV + IBV-inoculated chicks, some surface squamous tracheal epithelial cells
614 B. Y. CHEN & C. ITAKURA

Figure 3. Kidney from IBDV+ IBV-inoculated chick at 6 days PI showing degenerate and
necrotic epithelial cells (arrows) mainly in the dilated collecting tubules (CT), distal convoluted
tubules (DCT) and Henle's loops (HL), with cellular debris in some dilated lumina of tubules. PCT:
proximal convoluted tubule. HE stain, bar = 30 \im.

were antigen-positive from 6 to 8 days PI (Figure 2B). Also in the dually infected
birds, scattered antigen-positive cells were identified in the epithelium up to 13
days PI (Figure 2C), but no viral antigen was detectable from 16 days PI.

Histopathology of kidney
Table 3 summarizes the histopathological changes in the kidneys in both IBV
inoculated groups. At 4 days PI, no significant lesions were observed in the
IBV-inoculated chicks. In the IBDV + IBV-inoculated group, some epithelial cells
with swollen nuclei and vacuolated cytoplasm were observed mainly in the dilated
collecting ducts (CD), collecting tubules (CT) and distal convoluted tubules
(DCT) of the medulla, while these in the proximal convoluted tubules (PCT)
were spared. A few heterophils infiltrated in the interstitium or presented within
tubules and ducts.
From 6 to 8 days PI, the renal lesions in IBV-inoculated chicks were less severe
than those in the dually infected ones. Cell degeneration and necrosis were
detected in all segments of tubules and ducts of the medulla and cortex, particu-
larly in CD, CT and DCT in both groups. Desquamated epithelial cells, het-
erophils and macrophages were frequently seen in the dilated lumina of tubules
and ducts in the dually infected chicks (Figure 3). Interstitial oedema with
infiltration of heterophils, lymphocytes and a few macrophages were frequently
observed in the vicinity of degenerating tubules and ducts in both groups.
From 10 to 13 days PI, in IBV-inoculated birds, some affected tubules and
ducts had epithelial hyperplasia. Multifocal infiltrations of lymphocytes and
plasma cells were more pronounced. In IBDV + IBV-inoculated chicks, degener-
ated and necrotic epithelial cells were seen in some tubules or ducts and
 Table 3. Histopathology of kidneys ofIBDV+ IBV-inoculated and IBV-inoculated chicks gj

Group inoculated with E

IBDV + IBV IBV §

Renal lesion 2a 4 6 8 10 13 16 20 25 30 2 4 6 8 10 13 16 20 25 30 a

Ductotubular 52
b
dilation — + + + ++ + + + + + + + + _ — + + + + _ _ — — _ a
Epithelial ^5
degeneration — + + + ++ + + + + + + + + + + + — — + + + + + + — — — O
Heterophil 3
infiltration — + + + + + + — — + — — — + + — — — — — — O
Lymphoid cell 2
infiltration — — + + + + + + + + + + — — + + + + + + + + + + + g
Epithelial cell §
regeneration — — — + + + + + + + — — — + + + — — — — O
Lymphoplasmacytic §
nodule — — — — — — — — + — — — — — — — + + + + + + + 3
Fibroblastic a
proliferation — — — + + + + + + + + + + — — — — + + + — — — Z

"Days post-inoculation. a
b
Mean severity index from three chicks: —, no change; + , mild; + + , moderate; + + + , severe. o
616 B. Y. CHEN & C. ITAKURA

Figure 4. Kidney from IBDV+ IBV-inoculated chick at 10 days PI having marked interstitial
oedema accompanied byfibrocyticproliferation, lymphocytic infiltration and tubular degeneration
(arrow). HE stain, bar = 30 fim.

regeneration of them occurred in others. Occasionally, severe oedema with

fibroblastic proliferation was recognized in association with lymphocytic
infiltration in the interstitium (Figure 4).
From 16 to 30 days PI, in IBV-inoculated chicks, most of the damaged tubules
and ducts recovered and the epithelial damage in ducts and tubules was minimal.
Lymphocytes and plasma cells frequently formed lymphoplasmacytic nodules in
the interstitium. In IBDV + IBV-inoculated chicks, however, degeneration, ne-
crosis, desquamation and regeneration of tubular epithelial cells were still ob-
served, frequently accompanied by interstitial oedema and periducto-tubular
proliferation of fibrocytes (Figure 5). Sometimes, glomerular fibrosis was ob-

Figure 5. Kidney from IBD V+IB V-inoculated chick at 20 days PI having tubular dilation with
epithelial degeneration (arrows), lymphocytic infiltration (*) and fibrocytic reaction (arrowheads)
in the interstitium. HE stain, bar = 30 fim.
 RENAL LESIONS IN IBV-INFECTED IMMUNODEFICIENT BIRDS 617

Figure 6. Kidneyfrom IBDV + IBV-inoculated chick at 30 days PI with necrotic tubules (arrow)
associated with interstitial lymphocytic infiltration andfibrosis involving a glomerulus (arrowhead).
HE stain, bar = 30 fim.

served (Figure 6). Interstitial infiltration of lymphocytes with occasional het-

erophils was observed, but lymphoplasmacytic nodules were rare findings.

Immunohistochemistry of kidney
Distribution of IBV antigen in the kidneys of IBDV + IBV-inoculated and IBV-
inoculated chicks is summarized in Table 4. At 4 days PI, IBV antigen was first
detected in some epithelial cells of CD, CT and DCT of the medullary region in
dually infected birds (Figure 7). At that time, no antigen-positive cells were
detected in the IBV-inoculated chicks.

Figure 7. Kidney from IBDV+ IBV-inoculated chick at 4 days PI showing IBV antigen in
cytoplasm of epithelial cells of collecting ducts (CD) and collecting tubules (CT). Immunostain,
bar = 30 \im.
 oo

Table 4. Immunohistochemistry of kidneys ofIBDV+ IBV-inoculated and IBV-inoculated chicks

Group inoculated with

IBDV + IBV IBV

w
Segment of tubule 2a 4 6 8 10 13 16 20 25 30 2 4 6 8 10 13 16 20 25 30 •<
O
Proximal f§
convoluted ^<
tubule —b — + + — + — _ _ _ _ _ _ _ _ — _ _ — _ *
Henle's loop — — + + + — — + — — — — — + — + — — — —
Distal 3
convoluted 75
tubule — + + + ++ + + + + — + + + — — — + + — — — — — S
Collecting tubule — + + + + + + + + + + + + — — + + + + — — — — >
Collecting d u c t _ — — + + + + — + — + — — — — + + — — — — —

"Days post-inoculation.
b
M e a n n o . of IBV antigen-positive epithelial cells p e r 3.15 m m 2 renal tissue from three chicks: —, n o antigen; + , 1 t o 4 ; + + , 5 t o 9; + + + , over 10.
 RENAL LESIONS IN IBV-INFECTED IMMUNODEFICIENT BIRDS 619

Figure 8. Kidney from IBDV+ IBV-inoculated chick at 8 days PI showing IBV antigen in the
degenerated tubules and with lymphocytic infiltration and slight interstitialfibrosis.Immunostain,
bar = 30 fim.

From 6 to 8 days PI, the antigen-positive cells were mainly detected in CD,
CT, DCT and Henle's loops (HL), particularly in the dilated tubules and ducts
in chicks of both IBV-inoculated groups. In IBV-only inoculated chicks, however,
the IBV antigen was less detectable than that in the dually inoculated ones. It was
also detected in the macula densa of DCT, but rarely in PCT. Some desqua-
mated epithelial cells in the tubular lumina were antigen-positive. Inflammatory
cells frequently accumulated in the vicinity of the degenerate, antigen-positive
tubules or ducts (Figure 8).
From 10 to 13 days PI, a few antigen-positive cells were scattered mainly in the
lower nephrons in IBV-inoculated chicks. In dually infected birds, some antigen-
positive cells were degenerate or necrotic and accompanied by peritubular
lymphocytic infiltration and fibrosis. The other slightly degenerated tubules with
a few antigen-positive cells lacked this peritubular inflammatory reaction (Figure
9).
From 16 to 30 days PI, IBV antigen was occasionally seen in the epithelial cells
of lower nephrons and ducts in IBDV + IBV-inoculated chicks, but not detected
in IBV-inoculated ones. Variable interstitial lymphocytic infiltration and fibrosis
were frequently observed around some degenerated antigen-positive tubules or
ducts (Figure 10).
Abnormalities were not seen in tracheas and kidneys of IBDV-inoculated or
control chicks.

Histopathology of the bursa of Fabricius

There was a marked decrease of follicular lymphoid cells with fibroplasia and
infolding of the surface epithelium in all the bursae of IBDV + IBV-inoculated
and IBDV-inoculated chicks. Slight lymphocytic depletion was also noted in the
620 B. Y. CHEN & C. ITAKURA

Figure 9. Kidney from IBDV+ IBV-inoculated chick at 13 days PI having a severely degenerated
antigen-positive tubule (arrow), near which there arepentubularfibrosis andlymphocytic infiltration
(*). Slightly degenerated antigen-positive tubules show no inflammatory response in their vicinity.
DCT: distal convoluted tubules. Immunostain, bar = 30 jxm.

bursae of some IBV-inoculated chicks having marked renal lesions. No abnor-

malities were detected in the bursae of control chicks.

DISCUSSION
In the present study, mortality was observed in the IBDV + IBV-inoculated
group, with none in the IBV-inoculated one. The clinical signs and gross and
histological lesions in the trachea and kidneys due to IBV infection were more
severe and of longer duration in dually infected chicks than in ones inoculated
with IBV alone. The detection of IBV antigen in the renal epithelial cells
correlated with the development of chronic active renal lesions in IBDV + IBV-

Figure 10. Kidney from IBDV+ IBV-inoculated chick at 25 days PI having IBV antigen-
positive tubules with peritubularfibrosis.Immunostain, bar= 30 fxm.
 RENAL LESIONS IN IBV-INFECTED IMMUNODEFICIENT BIRDS 621

inoculated chicks. These findings suggest that the pathogenicity of nephropatho-

genic strains of IBV is increased in chicks previously infected with IBDV.
Renal lesions consisting of tubular degeneration and an inflammatory cell
reaction in the interstitium and tubules have been described in IBV-infected
chickens (Chong & Apostolov, 1982; Fulton et al, 1993; Pohl, 1974; Purcell et
al, 1976; Siller & Cumming, 1974). It was suggested that the renal lesions might
be initiated as nephrosis that progresses to nephrosis-nephritis and lastly to
interstitial nephritis (Chen et al, 1996). In the present study, the early renal
lesions due to IBV infection were similar in both IBV-inoculated groups. In the
late stage of infection, however, the response was chronic and active in the dually
inoculated group, while it was chronic in the IBV only one. These findings are
similar to those of Chandra (1988) who found IBV-induced chronic and active
lesions in kidneys of chemically-bursectomized chickens. He regarded these
lesions as a feature of IBV persistent infection as evidenced by virus isolation. The
present study provides morphological evidence, by using immunostaining, that
the active renal lesions in IBDV + IBV-inoculated chicks frequently accompany
persistent IBV antigen mainly in epithelial cells of the lower nephrons and ducts.
In the present investigation, the lesions and IBV antigen in tracheas of dually
infected chicks also persisted for longer than in those of IBV-only inoculated
ones. Compared with the renal lesions, the trachéal lesions recovered faster in
both IBV inoculated groups. The nephropathogenic property of the present IBV
might be longer lasting than the respiratory involvement.
Highly virulent IBDV causes severe clinical symptoms and high mortality
(Okoye & Uzoukwu, 1990; Tanimura et al., 1995). In 3-week-old SPF chicks
infected with strain Ehime/91 IBDV, the mortality was over 60% (Tsukamoto et
al., 1992). In the 7-day-old chicks infected here with the same IBDV strain, the
mortality was about 8%. This variation may be attributed to a difference in the
age of infection. The highest sensitivity to IBDV infection was recorded during
the first 3 to 6 weeks of age (Lukert & Saif, 1991). From the present observations,
IBV nephrotropism, especially in the persistent infection, may be related to a state
of immunodeficiency in the affected chicks. This study is a useful model to study
active renal lesions induced by IBV infection in immunosuppressed chicks.

ACKNOWLEDGEMENTS
The authors thank Dr Y. Iritani & S. Y. Aoyama, Aburahi Laboratories, Shionogi
and Company Limited, Koka, Shiga, Japan, for supplying the MA-87 strain of
IBV and Dr S. Hosi, Nippon Institute for Biological Science, Tokyo, for supply-
ing the IBV monoclonal antibody.

REFERENCES
Albassam, M.A., Winterfield, R.W. & Thacker, H.L. (1986). Comparison of the nephropathogenicity of
four strains of infectious bronchitis virus. Avian Diseases, 30, 468-476.
Butcher, G.D., Winterfield, R.W. & Shapiro, D.P. (1989). An outbreak of nephropathogenic H13
infectious bronchitis in commercial broilers. Avian Diseases, 33, 823-826.
Chandra, M. (1988). Comparative nephropathogenicity of infectious bronchitis virus in bursectomized and
nonbursectomized chickens. American Journal of Veterinary Research, 49, 831-834.
622 B. Y. CHEN & C. ITAKURA

Chen, B.Y., Hosi, S., Nunoya, T. & Itakura, C. (1996). Histopathology and immunohistochemistry of
renal lesions due to infectious bronchitis virus in chicks. Avian Pathology, 25, 269-283.
Chong, K.T. & Apostolov, K. (1982). The pathogenesis of nephritis in chickens induced by infectious
bronchitis virus. Journal of Comparative Pathology, 92, 199-211.
Fulton, R.M., Reed, W.M. & Thacker, H.L. (1993). Cellular response of the respiratory tract of chickens
to infection with Massachusetts 41 and Australian T infectious bronchitis viruses. Avian Diseases, 37,
951-960.
Goryo, M., Umemura, T. & Itakura, C. (1984). Concurrence of nephrosis-nephritis due to infectious
bronchitis virus and infectious bursal disease in broiler chickens. Avian Pathology, 13, 191-200.
Iritani, Y. & Takigami, S. (1995). Comparative characteristics among infectious bronchitis viruses isolated
from broiler chickens with nephritis. Journal of the Japan Veterinary Medical Association, 48, 75-78. (In
Japanese with English summary.)
Janse, E.M., Roozelaar, D.V. & Koch, G. (1994). Leukocyte subpopulations in kidney and trachea of
chickens infected with infectious bronchitis virus. Avian Pathology, 23, 513-523.
Jönsson, L.G.O. & Engström, B.E. (1986). Immunohistochemical detection of infectious bursal disease
and infectious bronchitis viral antigens in fixed, paraffin-embedded chicken tissues. Avian Pathology,
15, 385-393.
Kinde, H., Daft, B.M., Castro, A.E., Bickford, A.A., Gelb, Jr, J. & Reynolds, B. (1991). Viral pathogenesis
of a nephrotropic infectious bronchitis virus isolated from commercial pullets. Avian Diseases, 35,
415-421.
King, D.J. & Cavanagh, D. (1991). Infectious bronchitis. In B.W. Calnek, H.J. Barnes, C.W. Beard, W.M.
Reid & H.W. Yoder Jr (Eds) Diseases of poultry (9th edn, pp. 471-484). Ames: Iowa State University
Press.
Lukert, P.D. & Saif, Y.M. (1991). Infectious bursal disease. In B.W. Calnek, H.J. Barnes, C.W. Beard,
W.M. Reid & H.W. Yoder Jr (Eds) Diseases of poultry (9th edn, pp. 690-699). Ames: Iowa State
University Press.
Naqi, S.A. (1990). A monoclonal antibody-based immunoperoxidase procedure for rapid detection of
infectious bronchitis virus in infected tissues. Avian Diseases, 34, 893-898.
Okoye, J.O.A. & Uzoukwu, M. (1990). Pathogenesis of infectious bursal disease in embryonally bursec-
tomised chickens. Avian Pathology, 19, 555-569.
Owen, R.L., Cowen, B.S., Hattel, A.L., Naqi, S.A. & Wilson, R.A. (1991). Detection of viral antigen
following exposure of one-day-old chickens to the Holland 52 strain of infectious bronchitis virus.
Avian Pathology, 20, 663-673.
Pohl, R. (1974). The histopathogenesis of the nephrosis-nephritis syndrome. Avian Pathology, 3, 1-13.
Purcell, D.A., Tham, V.L. & Surman, P.G. (1976). The histopathology of infectious bronchitis in fowls
infected with a nephrotropic T ' strain of virus. Australian Veterinary Journal, 52, 85-91.
Siller, W.G. & Cumming, R.B. (1974). The histopathology of an interstitial nephritis in the fowl produced
experimentally with infectious bronchitis virus. Journal of Pathology, 114, 163-173.
Tanimura, N., Tsukamoto, K., Nakamura, K., Narita, M. & Maeda, M. (1995). Association between
pathogenicity of infectious bursal disease virus and viral antigen distribution detected by immunohis-
tochemistry. Avian Diseases, 39, 9-20.
Tsukamoto, K., Tanimura, N., Hihara, H., Shirai, J., Imai, K., Nakamura, K., Narita, M. & Maeda, M.
(1992). Isolation of virulent infectious bursal disease virus from field outbreaks with high mortality in
Japan. Journal of Veterinary Medical Science, 54, 153-155.
Tsukamoto, Y., Kotani, T., Shiraishi, Y., Kawamura, H. & Sakuma, S. (1996). Epithelial cell proliferation
of collecting ducts and ureters in the regenerating process of interstitial nephritis caused by infectious
bronchitis virus. Avian Pathology, 25, 95-102.
Yagyu, K. & Ohta, S. (1990). Detection of infectious bronchitis virus antigen from experimentally infected
chickens by indirect immunofluorescent assay with monoclonal antibody. Avian Diseases, 34, 246-252.

RÉSUMÉ
Histologie et immunohistochimie des lésions rénales dues à un virus de la
bronchite infectieuse chez des poulets préalablement inoculés avec un
virus très pathogène de la maladie de Gumboro
Une souche néphropathogène du virus de la bronchite infectieuse (IBV) a été inoculée, par
voie intratrachéale, à des poussins exempts de microorganismes pathogènes spécifiés âgés de 14
 RENAL LESIONS IN IBV-INFECTED IMMUNODEFICIENT BIRDS 623.

jours. Ces poussins ont été préalablement inoculés ou non avec un virus très pathogène de la
maladie de Gumboro (IBDV) à l'âge de 7 jours. Les lésions rénales ont été examinées en
histologie et en immunohistochimie à intervalles réguliers jusqu'à 30 jours suivant
l'inoculation. La mortalité a été de 0 et 20% respectivement dans les groupes inoculés IBV et
IBDV + IBV. Un aspect pâle et gonflé des reins, dû à l'infection par le virus IBV, a été observé
de façon plus intense et plus durable chez les poussins infectés par les deux virus. Tout au
début de l'infection, les lésions au niveau des reins ont été similaires dans les deux groupes,
mais celles des tubes et canaux collecteurs ont été plus sévères chez les poussins infectés par
les deux virus. Lors de la dernière phase de l'infection, les lésions rénales ont été caractérisées
par une néphrite interstitielle chronique avec la formation de nodules lymphoplasmocytaires
chez les poussins inoculés IBV et par une néphrite active chronique qui consiste en une
dégénérescence tubulaire, une réaction de cellules lymphoïdes et une fibrose interstitielle chez
les poussins inoculés IBDV + IBV. Les cellules positives en antigène IBV ont été plus
nombreuses et ont persisté plus longtemps au niveau des reins des poussins inoculés avec les
deux virus que chez ceux inoculés avec le virus IBV.

ZUSAMMENFASSUNG
Histopathologie und Immunohistochemie von Nierenveränderungen
durch Bronchitisvirus bei unvorbehandelten und bei vorher mit stark
virulentem Bursitisvirus infizierten Hühnerküken
Ein nephropathogener Stamm des Bronchitisvirus (IBV) wurde 14 Tage alten spezifisch
pathogenfreien Küken oder zuvor im Alter von 7 Tagen mit stark virulentem Bursitisvirus
(IBDV) infizierten Küken intratracheal verabreicht. Die Nierenveränderungen wurden peri-
odisch bis zum 30. Tag nach der Infektion histologisch und immunhistochemisch untersucht.
Zu einer Mortalität von 20% kam es in der IBDV + IBV-infizierten Gruppe, aber nicht in der
nur mit IBV infizierten Gruppe. Die durch die IBV-Infektion verursachte Nierenschwellung
und -blässe waren bei doppelt infizierten Küken stärker ausgeprägt und hielten länger an. In
den frühen Stadien der Infektion waren die histopathologischen Veränderungen in den Nieren
bei beiden Gruppen ungefähr gleich, aber die ductotubuläre Schädigung war bei den doppelt
infizierten Küken schlimmer. Im späten Infektionsstadium waren die Nierenveränderungen bei
IBV-infizierten Küken durch eine chronische interstitielle Nephritis mit der Bildung von
lymphoplasmazytären Knötchen charakterisiert und bei IBDV + IBV-infizierten Küken durch
eine chronisch aktive Nephritis, die aus Tubulus-Degeneration, lympoider Zellreaktion und
Interstitiumfibrose bestand. IBV-Antigen-positive Zellen persistierten in den Nieren doppelt
infizierter Küken länger als in denen von alleine mit IBV infizierten Küken.

RESUMEN
Histopatología e inmunohistoquímica de las lesiones renales producidas
por el virus de la bronquitis infecciosa aviar en gallinas inoculadas con
virus altamente virulento de la bursitis infecciosa
Se inoculó una cepa nefropática del virus de la bronquitis infecciosa (IBV) a pollitos de 14 días
de edad libres de patógenos específicos así como a otro grupo de pollitos que habían sido
inoculados previamente con una cepa virulenta del virus de la bursitis infecciosa (IBDV) a los
7 días de edad. Se examinaron las lesiones renales microscópicamente y mediante inmunohis-
toquímica durante los 30 días siguientes a la inoculación. La mortalidad fue del 20% en el
grupo IBDV + IBV pero no en el grupo inoculado sólamente con IBV. La tumefacción y
palidez de los ríñones debidas a la infección por IBV fue más intensa y de una duración más
624 B. Y. CHEN & C. ITAKURA

prolongada en los pollitos infectados con ambos virus. Los cambios microscópicos en las etapas
iniciales de la infección fueron similares en ambos grupos pero la lesión tubular fue más intensa
en los pollitos infectados con ambos virus. En las etapas finales de la infección las lesiones
renales consistieron en una nefritis intersticial crónica con formación de nódulos linfoplasmoc-
itarios en los pollos inoculados con IBV y en una nefritis crónica activa caraterizada por
degeneración tubular, infiltrados linfoides y fibrosis intersticial en las aves infectadas por
partida doble. Se observó más antígeno y éste persistió más tiempo en los ríñones de los
pollitos infectados con ambos virus que en los inoculados únicamente con IBV.