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IMMUNOLOGY, HEALTH, AND DISEASE |
* Animal Science, School of Rural Science and Agriculture, University of New England, Armidale, New South Wales 2351, Australia; and Elizabeth Macarthur Agricultural Institute, Menangle, New South Wales 2568, Australia
1 Corresponding author: kchousal{at}une.edu.au
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: histopathology • infectious bronchitis virus • laying hen • cockerel
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Infectious bronchitis virus strains vary greatly in their tissue tropism. Infectious bronchitis virus may occur as a respiratory syndrome, with clinical signs being difficulty in breathing, rales, coughing, or sneezing with or without nasal discharge (Parsons et al., 1992; McMartin, 1993). Maximum IBV antibody titers were recorded in the trachea from 5 to 10 d postinfection (p.i.; Ambali and Jones, 1990; Otsuki et al., 1990). However, the T strain of IBV may persist longer than 20 wk p.i. (Alexander and Gough, 1977). Infectious bronchitis virus infection of the trachea is mainly restricted to ciliated and mucus-secreting cells. Different IBV strains can grow at many epithelial surfaces in addition to the respiratory tract, including the kidney, oviduct, and parts of the gastrointestinal tract (Dhinakar Raj and Jones, 1997). Besides a primary predilection toward the respiratory tract, certain strains of IBV have been found to be severely nephropathogenic. The nephropathogenicity of IBV was first reported in Australia (Cumming, 1962), followed by the United States and many parts of Europe (Picault et al., 1988; Zanella, 1988; Butcher et al., 1990), Japan (Shimakura and Hirai, 1971), India (Bayry et al., 2005), and China (Liu et al., 2005). In layers, viral infection at an early age causes permanent damage to the oviduct (Crinion et al., 1971), along with some respiratory signs. In adult laying chickens, respiratory signs may be in milder form and can remain unnoticed. Infectious bronchitis virus usually causes reproductive disorders, with a decline in egg production accompanied by soft-shelled and misshapen eggs, inferior shell quality, and thin, watery albumen (Ignjatovic and Sapats, 2000). A range of histopathological studies in IBV infection have been carried out in the past in either broilers or pullets. In addition, these studies were mainly restricted to individual organ systems. However, very little information is available on the histopathological changes occurring in a range of tissues over time, in hens and cockerels challenged with IBV. Hence, the present study was designed to investigate the details of pathological changes occurring in various tissues in hens and cockerels challenged with the nephropathogenic T strain, as compared with the more respiratory N1/88 strain, which was isolated from a vaccinated broiler flock (Ignjatovic and McWaters, 1991).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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All chickens were kept unvaccinated against IBV and reared under strict isolation conditions with ad libitum feed and water. The IBV antibody-free status of hens and cockerels was maintained for 65 wk for hens (51 chickens) and 10 wk for cockerels (50 chickens), as confirmed by IBV antibody ELISA (IDEXX) and serum neutralization tests (Fontaine et al., 1963).
IBV
The 2 Australian strains of IBV used, T and N1/88, were obtained from Jagoda Ignjatovic, Commonwealth Scientific and Industrial Research Organization, Geelong, Australia. The dose of each challenge virus was adjusted to ensure a final estimated dose of 2 x 105 50% egg infective dose per bird and was administered intraocularly.
Experiment 1
White Leghorn hens at 65 wk of age were divided into 2 groups of 22 hens, each of which was challenged with either T or N1/88 strains of IBV. Seven hens were kept unchallenged as a control. Three hens from each challenge treatment group and 1 hen from the control group were killed and examined on d 3, 6, 10, 13, 16, and 21 p.i. Harderian gland, trachea, cecum, kidney, magnum, tubular shell gland (TSG; Johnston et al., 1963; Solomon, 1975; Stemberger et al., 1977), and shell gland pouch (SGP) were collected for histopathological examination.
Experiment 2
White Leghorn cockerels were divided into 2 groups each of 20 chickens that were challenged with the same strains of IBV mentioned above. Eight chickens were kept as a control. Four chickens from both treatment groups and 2 chickens from the control group were killed and examined on d 2, 4, 6, 8, and 10 p.i. Harderian gland, trachea, cecum, and kidney were collected for examination.
Histopathology
All tissues collected were fixed in 10% neutral buffered formalin. The tissues were processed by standard histological procedures, embedded in paraffin, and cut in 5-µm sections. All the sections were stained with hematoxylin and eosin (Stevens, 1990), whereas some of the kidney and magnum sections were stained also with alcian blue (Humason, 1962).
All stained sections of Harderian gland, trachea, kidney, cecum, magnum, tubular shell gland, and SGP were examined by light microscopy, and the most prominent lesions were scored as no change (–, 0), mild (+, 1), moderate (++, 2), or severe (+++, 3), after the methods of Nakamura et al. (1991) and Chen et al. (1996; Table 1
). The average lesion score was obtained by counting affected cells in 5 randomly distributed microscopic areas at 200x magnification (field diameter of 920 µm). The mean lesion score from 3 hens or 4 cockerels was then calculated. The incidence of globular leukocytes in the Harderían gland and goblet cells in the trachea, mentioned elsewhere in the paper, was not scored.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In control hens, the main features were some plasma cells (mature B lymphocytes that are specialized for antibody production) in the subepithelium, intact collecting duct epithelium, and occasional lymphocyte infiltration around the blood vessels in the glandular interstitium. Similar findings were recorded in the control cockerels.
In hens, there were no statistically significant main effects of IBV strain on the histopathological lesions in the Harderian gland. However, there was a significant main effect of days p.i. on all but 1 of the lesions investigated (Table 2). In T-strain-infected hens, on d 3 p.i., there was infiltration of plasma cells and globular leukocytes in the subepithelium (Figure 1), and the collecting duct epithelium was severely damaged. On 6, 10, and 13 d p.i., plasma cells and lymphocytes around blood vessels in the septa (interlobular space) were frequently observed. On 16 and 21 d p.i., most of the collecting duct epithelium had regenerated, but the lymphocyte infiltration in the interstitium and globular leukocytes in the subepithelium were still extensive. Exfoliative epithelium, along with inflammatory cells, was seen occasionally in the collecting duct lumen from d 3 to 16 p.i. Migration of heterophils into the subepithelium was mild at 6 and 10 d p.i. In N1/ 88-infected hens, most of the lesions were similar to those of T-strain infection, but the lesions were less severe.
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). In addition, there was a significant main effect of sex of chicken only for the incidence of heterophils in the subepithelium. In cockerels, degeneration of collecting duct epithelium, heterophilic infiltration in the subepithelium, and debris of exfoliated epithelium in the collecting duct lumen were mild at 2 d p.i. These changes were mild to moderate at 4 and 6 d p.i., and, on d 8 and 10 p.i., most of the collecting duct epithelium had regenerated. However, mild to moderate infiltration of heterophils in the subepithelial space continued. There was an increase in the number of plasma cells from 2 d p.i., which was constant throughout the experiment. Lymphocyte infiltration in the septal area was extensive throughout the experiment. The T strain of IBV was more pathogenic as compared with N1/88.
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Normal tracheal epithelia, with healthy cilia and mucus glands, were seen in the control hens and cockerels (Randall and Reece, 1996).
In hens, there were no statistically significant main effects of IBV strain on any of the histopathological lesions investigated, but there was a significant main effect of days p.i. on all the lesions scored (Table 4). There were no microscopic changes at 3 d p.i., in either T- or N1/88-infected hens. Severe pathology occurred mainly from d 6 p.i. in the form of loss of cilia and hypertrophy of alveolar mucus glands, changes in the mucosal epithelium from columnar to squamous, edema in the subepithelium, and occasional heterophilic exudate in the tracheal lumen. Most of the above lesions persisted in moderate to mild form in both infected groups on d 10 p.i. On d 13 p.i., most of the cilia and the epithelium had regenerated in the N1/88 group. The hypertrophied alveolar mucus glands were normal, with occasional exudate in the lumen. Goblet cells appeared to be less frequent in both the T- and N1/88-infected groups on d 3 to 10 p.i., but, after d 10, this appeared to be the case only in the N1/88-infected group. On d 16 and 21 p.i., most features of the tracheas appeared normal. However, thickening of the mucosa with infiltration of lymphocytic nodules persisted from d 13 to 21 p.i.
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). In addition, there was a significant main effect of sex of chicken only for the incidence of hypertrophy of alveolar mucus glands and edema in the mucosa. In cockerels, cilia loss, along with degeneration of the epithelium, was severe to moderate in T-infected chickens and moderate to mild in N1/ 88-infected chickens on 4 and 6 d p.i. Lesions were moderate in both IBV groups on the 8 d p.i. Moderate to severe hypertrophy of alveolar mucus glands, with more lymphocytes and mucosal edema, was persistent from 4 to 10 d p.i. The severity of lesions in both challenge groups, in hens as well as cockerels, was similar. However, IBV seemed to be more pathogenic in the trachea of males as compared with females.
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The kidneys of the chickens from the control groups were normal.
In hens, there was no statistically significant main effect of IBV strain on any of the lesions investigated, but there was a significant main effect of days p.i. on 3 out of the 7 lesions scored (Table 6). In hens, the main kidney lesions consisted of necrosis of proximal convoluted tubules, distension of distal convoluted tubules (not scored), necrotic foci, infiltration of lymphocytes in the interstitial space, edema of Bowman’s capsule, urates, and granulocytic casts in collecting ducts. The lesions were more apparent on 10 d p.i. in both T- and N1/88-infected hens (Figure 2). The pathology continued up to d 13 p.i. in the infected groups. Granulocytic casts and edema of Bowman’s capsule in the T-infected group persisted until the 16 d p.i. Infected tissue was cleared out by inflammatory cells, with diffuse lymphocyte infiltration in the cortex as well as the medulla from d 13 p.i. until the end of the experiment in the T-infected group.
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). There were no significant main effects between hens and cockerels. In both groups of infected cockerels, pathology was observed mainly from d 4 and 6 p.i. On 8 d p.i., most of the changes in the N1/88 group had subsided, but granulocytic and urate casts, along with necrotic foci, were evident in the T-infected group. On 10 d p.i., no changes were recorded except for lymphocyte infiltration, which was a consistent finding from d 4 p.i.
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No changes were observed in the cecum in any of the groups of either hens or cockerels.
Oviduct
Different parts of oviduct, magnum, TSG, and SGP were examined separately. However, as the findings for TSG and SGP were similar, they are presented together in Table 9.
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). In the magnum, on d 10 p.i., severe tubular gland dilatation and complete absence of mucopolysaccharides from epithelial cells in the N1/88-infected group were the prominent findings (Figure 3). These findings were moderate in T-infected hens. Lymphocyte cell infiltration in both T- and N1/88-infected groups was mild on d 10 p.i. and was mild to moderate at d 13 to 21 p.i. In both infected groups, edema in the submucosa was moderate on d 10 p.i. and moderate to mild on d 13 p.i.
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). In the TSG and SGP of both infected groups, on d 10 p.i., cilia loss, changes in the epithelium, tubular gland dilatation, edema in the submucosa, and lymphocyte infiltration were the main findings. Tubular gland dilatation was severe in the T group and moderate in the N1/88 group on d 10 p.i. (Figure 4). However, tubular gland dilatation was mild in the T-infected group and moderate in the N1/88-infected group on d 13 p.i. The dilated glands were filled with homogenous pink-stained material, which was also observed on d 13 p.i. in T-infected hens. Oedema in the submucosa was moderate on d 10 p.i. but mild on d 13 p.i. The intensity of infiltration of inflammatory cells was greater in the TSG and SGP of T-infected hens on d 16 and 21 p.i.. From 13 to 21 d p.i., lymphocyte infiltration in the lamina propria and muscularis layers was moderate in the T-infected group but mild in the N1/88-infected group. Most of the tissues had regenerated with the appearance of mitotic figures at 21 d p.i. All the parts of oviduct in the control hens appeared normal throughout the experiment (Figure 5).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In the past, the histopathology of IBV in the trachea has been extensively described, but most of the studies were limited to young chickens. In the present study, histopathological lesions and the time course of infection observed in the trachea after IBV challenge can be compared with the findings of Nakamura et al. (1991), who compared the histopathology of IBV in the trachea of 2 chicken lines using the M41 IBV strain. However, these authors reported time frames for the various lesions that differed between the 2 lines of chickens studied and also differed from those found in the present study. Fulton et al. (1993), using respiratory tract lavage in 2-wk-old chickens, found increasing numbers of inflammatory cells at different time intervals up to 4 d following M41 and T-strain IBV infection. In addition, more total cells and more inflammatory cells were recovered for the M41-infected chickens than for the T-strain-infected or the control chickens. However, Fulton studied the response at 2, 8, 24, 48, 72, and 96 h p.i. in 2-wk-old chickens, whereas our findings are from adult hens and cockerels at 2- to 3-d intervals. A similar trend of increasing numbers of inflammatory cells was found in the present study up to 13 to 21 d p.i. In addition, lesions were similar for both IBV strains, indicating a similar predilection of both strains for the trachea. Lesions were more severe and persistent in both the groups of cockerels as compared with the hens, which suggests that the virus is highly pathogenic for the trachea of young cockerels, as evidenced by a higher incidence of hypertrophy of alveolar mucus glands and edema in the mucosa. It is likely that the severity and time course of lesions is influenced by strain of IBV, strain of chickens, and age of chickens.
The histopathological changes observed in the kidney match previous findings (Purcell et al., 1976; Chen et al., 1996), except for the microscopical lesion of interstitial edema, which was not observed in the present study. The T-strain IBV was more nephropathogenic (Chong and Apostolov, 1982) as compared with N1/88 in hens, and similar observations were recorded in the younger cockerels. Granulocytic casts and urate casts could be a manifestation of urolithiasis, and Cavanagh and Naqi (1997) also reported an increased incidence of casts following IBV infection.
Most of the changes in the oviduct were noticeable on d 10 p.i., a finding that is in accordance with Sevoian and Levine (1957). Glandular dilatation and loss of mucopolysaccharides in the epithelial layer of the magnum may be the contributory factor in albumen thinning (Butler et al., 1972). The moderate inflammatory cell debris in the lumen of the oviduct may lead to the presence of meat spots in egg albumen, as reported by McDougall (1968). The duration and severity of effects suggest that T strain has more affinity and pathogenicity for the TSG and shell gland pouch, but N1/88 is more pathogenic for the magnum of the oviduct. This finding suggests that the fully functional oviduct is susceptible to IBV infection. The significant difference in lesion score in different parts of the oviduct with respect to time could be compared with studies in infected oviduct cell culture by Pradhan et al. (1984), who studied ciliary movement in oviduct cells. However, there is a dearth of literature regarding in vivo quantitative assessment of other lesions in the oviduct, which precludes comparison of our findings with those of other workers. Further investigation is required to fully describe the effects of IBV on the shell-forming regions of the oviduct, tubular shell gland, and shell gland pouch, including the mechanisms causing misshapen and soft-shelled eggs and cessation of (or reduced) egg production.
In both the IBV-infected hen and cockerels, the intensity of lesions in the kidney was not as severe and persistent as reported in earlier literature. However, the reverse was observed for lesions in the trachea, which were both persistent and severe in both T- and N1/88-infected chickens. The T strain (N1/62) has been regarded as highly nephropathogenic (Cumming, 1962; Klieve and Cumming, 1988) and has been reported as replicating in the trachea with mild lesions (Ignjatovic et al., 2002). Our findings suggest that, besides being nephropathogenic, the T strain of IBV has an ability to produce severe pathology in the trachea. Similarly, Sapats et al. (1996) reported that N1/88 did not replicate in kidney. However, our findings were that N1/88 can produce kidney lesions in hens as well as in cockerels. In addition, the N1/88 strain has been isolated from the kidney by Roberts (2005).
At the same time, the possibility of an intrinsic factor such as age influencing the pathogenesis for kidney (Albassam et al., 1986) and oviduct (Crinion et al., 1971) cannot be ignored. After experimental challenge with IBV, the sequential observations by histopathology suggest that the IBV replicates first in the Harderian gland, then the tracheal mucosa, and then simultaneously replicates in the kidney and oviduct. On the other hand, in cockerels, the IBV first replicates in the Harderian gland and then simultaneously in the trachea and kidney.
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ACKNOWLEDGMENTS |
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Received for publication June 21, 2006. Accepted for publication August 23, 2006.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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