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Poult Sci 2007. 86:59-62
© 2007 Poultry Science Association
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IMMUNOLOGY, HEALTH, AND DISEASE: Research Note

Histopathology of Two Serotypes of Infectious Bronchitis Virus in Laying Hens Vaccinated in the Rearing Phase

K. K. Chousalkar*,1, J. R. Roberts* and R. Reece{dagger}

* Animal Science, School of Rural Science and Agriculture, University of New England, Armidale, New South Wales, 2351, Australia; and {dagger} Elizabeth MacArthur Agricultural Institute, Menangle, New South Wales, 2568, Australia

1 Corresponding author: kchousal{at}une.edu.au


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The comparative histopathology of 2 different strains of infectious bronchitis virus (T and N1/ 88) in vaccinated hens was studied at 110 wk of age. The Harderian gland showed similar histopathology in T- and N1/88-infected hens. The trachea and kidney of challenged vaccinated hens were protected to a moderate extent, but the oviduct was protected to only a small extent. The severity and persistence of lesions were greater in tubular shell gland, shell gland pouch, and kidney of the T-infected hens, whereas, for the magnum, N1/88 had a greater effect.

Key Words: histopathology • infectious bronchitis virus • vaccinated hen


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infectious bronchitis is an acute, highly contagious infection in chickens, occurring at all ages. Infectious bronchitis virus (IBV) has a great economic effect on the layer industry, because it affects egg production and quality. Besides being pathogenic for the respiratory system, this virus has an ability to replicate in epithelial layers of various organs (Cavanagh, 2003). In Australia, Ignjatovic et al. (2002) categorized major IBV strains as respiratory and nephropathogenic. However, major strains are undergoing variation in their pathogenicity (Ignjatovic et al., 1997). The 2 IBV strains T and N1/88 were isolated by Cumming (1962) and Ignjatovic and McWaters (1991), respectively, and were placed in 2 different groups by Sapats et al. (1996). The N1/88 strain was isolated from a vaccinated broiler flock, whereas the T strain was isolated from cases of uremia from 3-wk-old pullets and adult laying hens.

At present all over the world, a range of vaccines is available commercially, with various administration protocols recommended. According to Cavanagh and Naqi (1997), the vaccine protocol for laying pullets includes 1 or 2 revaccinations during rearing and boosters during lay at 8- to 10-wk intervals. Sulaiman et al. (2002) reported that regular revaccination during lay has a deleterious effect on egg production and quality. However, when hens vaccinated during rearing were challenged in mid and late lay, there were negative effects on production and quality of eggs (Jolly, 2005). Relatively little information is available from previous studies regarding histopathological changes occurring in challenged birds. The present study was designed to investigate the extent of protection offered by vaccination on various organs in hens vaccinated during rearing and challenged at 110 wk of age with 2 phylogenetically different Australian strains of IBV.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
In total, 36 Hy-Line Gray hens (Hy-Line Australia Pty Ltd., Maitland, New South Wales) were used in this experiment. All birds were vaccinated with commercial vaccine at the age of 1 d, 4 wk, and 12 wk. Twelve Hy-Line birds from 2 groups at the age of 110 wk were challenged with 2 different Australian strains of virus, T and N1/88 (obtained from Jagoda Ignjatovic, Commonwealth Scientific and Industrial Research Organisation, Geelong, Australia). Six birds were kept as a control and maintained throughout the experiments, whereas another 6 control birds were killed. Two birds from each treatment and 1 bird from the control group were killed on d 3, 6, 10, 13, 16, and 21 postinfection (p.i.). The Harderian gland, trachea, kidney, and different regions of oviduct, magnum, tubular shell gland, and shell gland pouch (Solomon, 1991) were fixed in 10% neutral buffered formalin. The tissues were processed by standard histological procedures, embedded in paraffin, and cut in 5-micron sections. All sections were stained with hematoxylin and eosin. Additionally, some of the kidney and magnum sections were stained also with Alcian blue, which stains endothelial cells producing acid mucopolysaccharides. All stained slides were viewed by light microscopy.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Harderian Gland
In control birds, the main features were some plasma cells in the subepithelium, an intact collecting duct epithelium and acinar epithelium, and migratory infiltration of lymphocytes in the interstitium.

On the third day p.i., in both T- and N1/88-infected birds, the collecting duct epithelium displayed moderate damage. On 6, 10, and 13 d p.i., lymphoid cells around blood vessels and globular leukocytes in the subepithelium were numerous. On 16 and 21 d p.i., most of the duct epithelium had regenerated and the number of globular leukocytes was reduced, but the lymphocyte infiltration in the interstitium was at a peak. Migration of lymphocytes into the subepithelium was mild on 3, 13, 16, and 21 d p.i., but moderate at 6 and 10 d p.i. In N1/88-infected hens, most of the lesions were similar to birds infected with T-strain IBV.

Exfoliative epithelium, along with a few inflammatory cells, was a constant finding in all infected birds, as well as control birds, throughout the experiment.

Trachea
The pathology in the trachea was mild to moderate. In both T- and N1/88-infected groups, there was occasional loss of cilia on d 6 p.i., with the change of some epithelial cells from columnar to squamous. Cilia in most parts of the trachea appeared normal for the remainder of the experiment. The hypertrophy of glandular cells was noted on d 6 p.i. and continued up to d 13 p.i. However, the severity of glandular hypertrophy was greater in N1/88-infected birds than for T-infected birds. At 21 d p.i., most of the tissue appeared normal, but there was extensive thickening of the mucosa due to heavy infiltration of lymphocytes in both the infected groups.

In vaccinated control birds, there were no changes except persistent heavy lymphocytic infiltration in the mucosal layer.

Kidney
In control birds, occasional focal infiltration of lymphocytes and granulocytic casts were observed.

In both the infected groups, granulocytic and urate casts in the collecting duct and occasional necrotic foci were persistent throughout the experiment, beginning at d 6 p.i. In the T-infected group, there was lymphocytic infiltration in the interstitial space at 16 and 21 d p.i., and heavy lymphocytic infiltration was also recorded in 1 bird from the N1/88-infected group killed 16 d p.i. Moderate edema in Bowman’s capsule was observed in the T-infected group, but edema was mild in N1/88-infected birds on the 10 and 13 d p.i. The remainder of the tissue appeared normal.

Oviduct
As compared with challenged vaccinated birds, in control vaccinated birds, there was no prominent pathology in any of the oviduct, magnum, tubular shell gland, or shell gland pouch, but there was mild infiltration of lymphocytes around blood vessels in the muscularis area. A small number of plasma cells were also recorded in the interglandular space.

In the magnum of T-infected birds, there was moderate loss of cilia on d 10 p.i., which continued up to d 16 p.i. However, occasional cilia loss was also observed in 1 bird killed on d 21 p.i. In the magnum of N1/88-infected birds, most of the epithelial cells showed patchy loss of mucopolysaccharides (which stain bright blue with Alcian blue stain) on d 10 p.i. (Figure 1Go). In contrast, the epithelium of the control birds showed continuous staining (Figure 2Go). There was occasional loss of cilia until 16 d p.i. Lymphocytic and plasma cell infiltration in the subepithelial space was persistent in the magnum of both the infected groups.


Figure 1
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  		Figure 1. Magnum of N1/88-infected bird d 10 postinfection. Alcian blue stain showing blue-stained endothelial cells with patches of non-staining (arrows); x400. Scale bar represents 25 µm. Soft-shelled egg in shell gland pouch of oviduct.

 

Figure 2
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  		Figure 2. Magnum of control-vaccinated bird d 10 postinfection. Alcian blue stain showing blue stained endothelial cells (arrow); x400. Scale bar represents 25 µm. Soft-shelled egg in shell gland pouch of oviduct.

 
In T- and N1/88-infected birds, the findings and time frame of the effects of IBV infection in both the tubular shell gland and shell gland pouch were similar. There was occasional loss of cilia and lymphocyte and plasma cell infiltration in both the tubular shell gland and shell gland pouch on d 10 p.i., which continued up to the d 21 p.i. in the shell gland of T-infected birds (Figure 3Go). Most of the blood vessels in the muscularis area were surrounded by lymphocytes, although infiltration was moderate in the subepithelium. Moderate dilatation of glands was noted in the shell gland of T-infected birds on d 10 p.i. and continued up to d 16 p.i.


Figure 3
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  		Figure 3. Shell gland pouch of T-infected bird d 13 postinfection. Lymphocyte infiltration (arrow A) and cilia loss from epithelial lining (arrow B). Hematoxylin and eosin x200. Scale bar represents 50 µm. Egg in lower magnum.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The exfoliative epithelium in the Harderian gland, observed in all 3 treatment groups, may result from exposure to a naturally occurring irritant such as dust. In both challenged groups (T and N1/88) of vaccinated birds, the severity and time frame of lesions in the Harderian gland were almost the same. This indicates that both strains are equally pathogenic for the Harderian gland, although regeneration occurred more quickly in the N1/88-infected birds. Our findings agree with those of Davelaar and Kouwenhoven, (1976) and Toro et al. (1996). The presence of globular leukocytes originating from mast cells and eosinophilic granulocytes could be the result of active immunostimulation episodes.

For the trachea, lesions were similar for both IBV strains, indicating a similar predilection of both strains for the trachea. However, most parts of the trachea appeared normal by the end of the experiment. This indicates that the trachea was protected to a moderate extent by the vaccination protocol used. The cause of the extensive thickening of the mucosal layer due to infiltration of lymphocytes in both control and treated birds needs further investigation.

The histopathological changes observed in kidney of the T-infected matches previous findings (Ratanasethakul and Cumming, 1983). Severity of kidney lesions was greater in T-infected as compared with N1/88-infected hens. Jolly (2005) also reported lymphocytic infiltration in the kidneys of T-infected birds. Granulocytic and urate casts in the collecting duct are indicators of urolithiasis, which is a possible manifestation during IBV infection in layers (Cavanagh and Naqi, 1997).

Most of the changes in the oviduct of the challenged groups were noticeable on d 10 p.i., as was recorded also by Sevoian and Levine (1957). However, the moderate pathology of T strain in the tubular shell gland and shell gland pouch and the severe pathology of N1/88, particularly in the magnum, indicates the disparity between the strains of virus and their ability to induce pathology in different parts of the oviduct. Our finding regarding loss of mucopolysaccharides from epithelial cells in the magnum is in accordance with Davidson (1986), who reported a similar observation in the magnum of hens producing watery whites.

The histopathological findings in the trachea and kidney of both T- and N1/88-infected vaccinated birds suggest that the vaccination protocol offered moderate protection in both of these organs. On the other hand, the extent of the histopathology in the oviduct indicates that the vaccination protocol offered only limited protection for the oviduct against infection by both the strains of infectious bronchitis at a very late stage of lay. The significant pathology and affinity of both IBV strains for the oviduct of laying hens suggests that, despite vaccination, the fully functional oviduct can be affected, which could influence egg production and egg quality. However, intrinsic factors such as age could also influence the pathogenesis of IBV effects on the oviduct (Crinion and Hofstad, 1972) as well as egg production and quality (Roberts, 2004). It is difficult to compare our results of the effects of IBV on the oviduct of vaccinated hens, owing to a scarcity of literature regarding the response of the normal and fully functional oviduct during IBV infection. The current research is focused on a detailed study of the pathogenesis of IBV in birds in full lay, to form a basis for further studies. Despite the fact that IB revaccination could be disadvantageous (Roberts et al., 2004), the extent to which regular IBV revaccination or intercurrent IBV infection can induce microscopic pathology in the oviduct of the mature laying hen needs further investigation.

Received for publication April 20, 2006. Accepted for publication July 20, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cavanagh D. 2003. Severe acute respiratory syndrome vaccine development: Experiences of vaccination against avian infectious bronchitis coronavirus. Avian Pathol. 32:567–582.[ISI][Medline]

Cavanagh, D., and S. A. Naqi. 1997. Infectious bronchitis. Pages 511–526 in Diseases of Poultry. 10th ed. B. W. Calnek, H. J. Barnes, C. W. Beard, L. R. McDougald, and M. Y. Saif, ed. Iowa State Univ. Press, Ames.

Crinion, R. A. P., and M. S. Hofstad. 1972. Pathogenicity of four serotypes of avian infectious bronchitis virus for the oviduct of young chickens of various ages. Avian Dis. 16:351–363.[ISI][Medline]

Cumming R. B. 1962. The etiology of "uraemia" of chickens. Aust. Vet. J. 38:554.[Medline]

Davelaar, F. G., and B. Kouwenhoven. 1976. Changes in Harderian gland of the chicken following conjunctival and intranasal infection with infectious bronchitis virus in one and 20 day old chickens. Avian Pathol. 5:39–50.

Davidson M. F. 1986. Histological studies of changes in the magnum of domestic hen associated with the production of "watery white" eggs. Br. Poult. Sci. 27:353–354.[ISI][Medline]

Di Matteo, A. M., M. C. Sonez, C. M. Plano, and I. von Lawzewitsch. 2000. Morphologic observation on respiratory tracts of chickens after hatchery infections bronchitis vaccination and formaldehyde fumigation. Avian Dis. 44:507–518.[ISI][Medline]

Ignjatovic, J., D. F. Ashton, R. Reece, P. Scott, and P. Hooper. 2002. Pathogenicity of Australian strains of avian infectious bronchitis virus. J. Comp. Pathol. 126:115–123.[ISI][Medline]

Ignjatovic, J., and P. G. McWaters. 1991. Monoclonal antibodies to three structural proteins of avian infectious bronchitis virus: Characterization of epitopes and antigenic differentiation of Australian strains. J. Gen. Virol. 72:2915–2922.[Abstract/Free Full Text]

Ignjatovic, J., S. Saptas, and F. Ashton. 1997. A long-term study of Australian infectious bronchitis viruses indicates a major antigenic change in recently isolated strains. Avian Pathol. 26:535–552.[Medline]

Jolly, M. 2005. Protection of egg quality in laying hens by rearing phase infectious bronchitis vaccination. PhD Diss. Univ. N. Engl., Armidale, Australia.

Ratnasethakul, C., and R. B. Cumming. 1983. The effect of route of infection and strain of virus on the pathology of Australian infectious bronchitis. Aust. Vet. J. 60:209–213.[ISI][Medline]

Roberts J. R. 2004. Factors affecting egg internal quality and egg shell quality in laying hens. J. Poult. Sci. 41:161–177.

Sapats, S., P. J. Wright, and J. Ignjatovic. 1996. Sequence analysis of S1 glycoprotein of Australian strains of infectious bronchitis virus. Pages 33–38 in Proc. Aust. Poult. Sci. Symp., Poult. Res. Found., Univ. Sydney, Australia.

Sevoian, M., and P. P. Levine. 1957. Effects of infectious bronchitis on reproductive tracts egg production and egg quality of laying chickens. Avian Dis. 1:136–164.

Solomon, S. E. 1991. Egg and Egg Shell Quality. Wolfe Publ. Ltd., London, UK.

Sulaiman, A., J. R. Roberts, and W. Ball. 2002. Effects of vaccine strain, route of administration of IB vaccine and revaccination on egg quality in laying hens. Pages 117–120 in Proc. Aust. Poult. Sci. Symp. Poult. Res. Found., Univ. Sydney, Australia.

Toro, H., V. Godoy, J. Larenas, E. Reyes, and E. F. Kaleta. 1996. Avian infectious bronchitis: Viral persistence in the Harderian gland and histological changes after eye drop vaccination. Avian Dis. 40:114–120.[ISI][Medline]




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K. K. Chousalkar and J. R. Roberts
Ultrastructural Observations on Effects of Infectious Bronchitis Virus in Eggshell-Forming Regions of the Oviduct of the Commercial Laying Hen
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