K.K. CHOUSALKAR
J.R. ROBERTS
University of New England, NSW, Australia
The pathogenesis of Infectious Bronchitis Virus (IBV) for different parts of the oviduct was studied in unvaccinated Isa Brown laying hens exposed to T and N1/88 strains of IBV. Two hens from T and one hen from N1/88 infected groups went out of lay. Ultrastructual findings revealed that the infundibulum and magnum were more negatively affected than the isthmus, tubular shell gland and shell gland pouch. However, all parts of the oviduct of hens out of lay showed severe cytopathology. Overall, T strain was more pathogenic for the fully functional oviduct than N1/88. Virus particles were recorded on the 10th day post infection (p.i.) and severe cytopathology was observed between 10 and 12 days p.i.
I. Introduction
Infectious bronchitis, which is caused by a coronavirus, can be a devastating disease to the layer industry. Despite the fact that IBV infection can cause serious problems with egg production and quality, very little research has been undertaken to understand and define the pathogenesis of IBV in the functional oviduct. Earlier studies have been conducted on laying hens using the Massachusetts strain of IBV (Sevoian and Levine, 1957) but still the basic causes of watery whites, thin-shelled eggs and cessation of egg production are unresolved. Also, the extent of pathogenesis of Australian strains of IBV for the reproductive tract is still not clear. Our preliminary studies have shown that Australian strains of IBV can cause severe pathology in the oviduct of unvaccinated (Chousalkar et al, 2006a) and vaccinated hens (Chousalkar et al, 2006b). The present study was undertaken to compare the degree of cellular pathogenesis in the fully functional oviduct of hens challenged with either T or N1/88-strains of IBV.
II. Materials and methods
Isa Brown day-old laying birds (150) were obtained from a commercial hatchery. At day-old, birds received Rispens vaccine against Marek's disease. The birds were divided equally into three groups, one control and two treatment groups. IBV-free status of the birds was maintained by isolation and strict biosecurity. The antibody free status of hens for IBV prior to the challenge experiment was confirmed by antibody ELISA (IDEXX). Birds were fed commercial broiler starter to 3 weeks of age, chick starter to 5 weeks followed by pullet grower up to 16 weeks and layer mash thereafter. All the hens were in full lay at 30 weeks of age when they were challenged with one of two different strains of IBV, T and N1/88 (obtained from Dr. Jagoda Ignatovic, CSIRO, Geelong). Two hens from each challenge group and one hen from the control group were euthanased at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 days post infection (p.i.). At the time that the birds were sacrificed, the position of the egg in the oviduct was noted. Two hens from the T infected group, which stopped laying on the 13th day p.i., were euthanased on the 30th day p.i. Different parts of the oviduct, infundibulum, magnum, isthmus, tubular shell gland (TSG) and shell gland pouch (SGP) were fixed in fixative (2 % paraformaldehyde and 2.5 % glutaraldehyde in 0.1M phosphate buffer, pH 5.5). The tissues were processed by standard electron microscopy procedures and embedded in epoxy resin. Semi-thin sections from 5 blocks of each tissue of individual birds were collected and stained with toluidine blue. Ultra-thin sections (80 to 90 nm) were then collected on grids and stained with a saturated solution of uranyl acetate followed by lead citrate in a carbon dioxide free environment. Sections were washed in CO2-free water, allowed to dry and examined under a transmission electron microscope (Joel, JEM- 1200 EX). Some of the above samples, after fixation, were critical point dried, mounted on aluminium stubs, gold coated and observed under a scanning electron microscope (Joel, JSM -5800LV). Samples were also processed for histology as described earlier (Chousalkar et al, 2006).
III. Results
a) Autopsy findings of the oviduct
Out of 24 hens killed from the N1/88 infected group, the oviduct and ovary were atrophied in one hen that stopped laying at the 12th day p.i. On the 18th day p.i., a fully formed egg was recorded in the infundibulum of another hen. The remaining 22 hens from this group had visibly normal and fully functional oviducts and ovaries. In the T-strain infected group, out of 24 hens, two hens, which had stopped laying from the 13th day p.i., showed thin and inspissated yolk material in the oviduct. However, their ovaries were functioning normally. No visible abnormality was recorded in the oviduct of the remaining hens except for the occurrence of meat spots in the magnum of 12 hens. None of the hens from the control group showed any abnormality.
b) Infundibulum
Pathology was not observed in the infundibulum of hens from the N1/88 group except for one with an atrophied oviduct. In T-strain infected hens, the histopathological findings in the infundibulum included infiltration of plasma cells and lymphocytes into the lamina propria. There was patchy loss of cilia from mucosal folds in both the anterior and posterior parts of the infundibulum from the 10th day p.i. onwards. The cilia loss was observed until the 14th day p.i. Most of the cilia appeared normal from day 16 until the end of the experiment. The posterior surface of the infundibulum of the hen with an atrophied oviduct was devoid of areas of granular cells. However, cilia loss was not observed in that hen. Patchy loss of cilia in the anterior and posterior infundibulum was observed in the hens which were still not laying even after the 30th day p.i. When tissues were examined under the electron microscope, there were increased deposits of endoplasmic reticulum, irrespective of egg position in the oviduct, in the surface epithelial and gland cell. Endocytosis of virus particles was observed on day 10 p.i. in the granular cells. Virus particles were also noticed in the golgi complex and rough endoplasmic reticulum (RER).
c) Magnum
The histopathological findings were similar to our earlier findings in unvaccinated hens except for the dilatation of tubular glands in N1/88-infected hens observed in the earlier study but not in the present study (Chousalkar et al., 2006). Scanning electron micrographs revealed loss of cilia which was extensive in the T group and patchy in the N1/88 group between 10 and 14 days p.i. Cilia in the magnum from both the challenge groups appeared normal for the remainder of the experiment. The loss of cilia was extensive in hens which had stopped laying. Changes observed by electron microscopy were increased deposits of rough endoplasmic reticulum, free ribosomes, and endocytic vesicles. Virus particles were recorded in the Golgi complex and dilated endoplasmic reticulum. Granular cells were devoid of secretory granules. Both A and B type tubular glands were dilated and type C glands were inconspicuous. The microvilli of dilated glands were disrupted. The secretory granules in the dilated glands were gathered near cell surface and the Golgi complex was non functional. Virus particles were also recorded occasionally in cytoplasmic vesicles. After the 14th day p.i., the only findings were lymphoid nodules in the muscularis area and scanty secretory granules in granular cells in T-infected hens. There was degeneration of mitochondria in surface as well as glandular epithelial cells. Phagocytic vacuoles were frequent during 12 to 14 days p.i. The above changes were also recorded in the magnum of hens killed on the 30th day p.i. The tissues from all parts of the Infundibulum and magnum in control hens appeared normal throughout the experiment.
d) Isthmus
All parts of the isthmus of control hens appeared normal. No prominent microscopic lesions were recorded in the isthmus of the N1/88 group except for mild infiltration of inflammatory cells in the submucosa and muscularis area in one hen on the 16th day p.i.. In T-infected hens, there was patchy loss of cilia in two hens on the 10th and 12th days p.i., respectively. There was no glandular dilatation. Lymphoid nodules were observed in the submucosa in the isthmus of hens at 16, 20 and 24 days p.i. Severe pathology was recorded in the isthmus of hens from the T-infected group which had stopped laying. There was extensive infiltration of lymphocytes into the submucosa and large lymphoid nodules in the muscularis area. In granular cells, RER deposits were increased and some secretory granules were observed at the apex of individual cells. Virus particles were recorded in dilated cisternae of RER, dilated golgi vesicles and cytoplasmic vesicles in surface and glandular epithelium. There was patchy cilia loss and virus particles were occasionally recorded in these cells. Mitochondria appeared enlarged in both ciliated and mitochondrial cells. Type 2 gland cells were evident in all parts of the isthmus. The glands appeared dilated. Necrotic cells, along with an elongated mass of secretion, were observed in the lumen of some dilated gland cells. Phagocytic vacuoles were also observed in glandular cells.
e) Tubular shell gland and shell gland pouch
Most of the changes seen in the TSG and SGP were similar. Pathological changes were not recorded in TSG and SGP of hens from the N1/88 group. In T-strain infected hens, at 20 and 24 days p.i., large lymphoid nodules were recorded in the muscularis area. Severe pathology was observed in the TSG and SGP of hens which were out of production. Cilia loss and increased deposits of RER with virus particles were recorded in ciliated cells. Vacuoloids were not recorded in any granular cells of the SGP. The glands in both TSG and SGP were occasionally dilated. Mitochondria in non-ciliated cells, as well as glandular epithelium, appeared swollen. A few lipid droplets were also recorded in gland cells of the shell gland pouch. The apical border of most of the ciliated cells in the SGP and TSG appeared disrupted and all mitochondria had degenerated. The infiltration of plasma cells and lymphocytes into the submucosa was intense. The microvilli of the surface epithelium and glandular epithelium were not affected. All parts of TSG and SGP in control hens appeared normal.
IV. Discussion
In the present study, the autopsy findings of the oviduct of hens which were out of lay are in accordance with the findings of Jones and Jordan (1972). The present study found that most of the hens recovered from infection and resumed laying but some hens stopped laying and this was associated with presence of virus particles in the oviduct. Such hens, despite appearing healthy, can act as persistent virus shedders. Histopathological findings regarding the magnum are similar to our preliminary study except that gland dilatation in the magnum of N1/88 infected group and severe pathology was not recorded in the TSG and SGP of T-strain infected hens in the present study, other than in hens which were out of production. This could be attributed to a difference in age or breed of the hens. Thinning of albumen also observed in this study could be due to dilatation of the tubular glands of the magnum and disturbance in physiology of the Golgi complex and RER. Absence of severe pathology in the lower part of the oviduct indicates that the upper oviduct is more susceptible. However, pathology in the oviduct of hens which were out of production suggests that IBV has the potential to infect all parts of the oviduct in some hens. Pathology in the tubular shell gland could be responsible for formation of abnormal mammillary cores which are the initial templates for the calcified shell, resulting in formation of a poor quality mammillary layer. Moreover, pathology in the shell gland pouch could result in alteration in mineral deposition during egg shell formation. All this could result in formation of wrinkled or thin shelled eggs. However, cytochemical studies are essential to clarify this. Such changes may not be common during infection with T and N1/88 strains of IBV in adult Isa Brown hens, as only two hens which were out of lay showed changes in the isthmus, TSG and SGP. All the above findings support the view of McMartin (1968) that the response of individual hens during IBV infection varies greatly. Loss of cilia in most parts of oviduct could pose the potential risk of secondary bacterial infection and may affect fertility in breeder hens. It is still difficult to conclude the exact reason for cessation of egg production in some hens, but non-functional Golgi bodies, dilated RER and hence the possible alteration in protein synthesis of affected cells could be a contributory factor. Overall, T-strain was more pathogenic for the fully functional oviduct than N1/88 strain. In the present study, out of 50 infected hens in each challenge group, only three hens, one from N1/88 with a non-patent oviduct and two from the T-infected group with patent oviducts, were out of lay. By way of contrast, Crinion et al. (1971), found 26 % non layers with 83 % non-patent and 17 % patent oviducts following infection with IBV as young chicks, which supports the view of Bradfoot et al., (1956) that the oviduct of younger chickens is more likely to be affected by IBV than older birds.
Acknowledgements
The excellent technical assistance from Mr. Patrick Littlefield, Electron Microscope Unit Manager, is gratefully acknowledged. The Physiology Teaching Unit, University of New England, provided financial support to K. Chousalkar for this study.
References
Bradfoot, D.I., Pomeroy, B.S. and Smith, W.M. (1956). Poultry science. 35: 757
Chousalkar, K .K., Roberts, J.R. and Reece, R.L. (2006a). Poultry science. 86 (In press)
Chousalkar, K .K., Roberts, J.R. and Reece, R.L. (2006b). Poultry science (In press)
Crinion, R. A. P., Ball, R.A. and Hofstad, M. S. (1971). Avian Diseases 15: 42-48.
Jones R.C. and Jordan, F.T.V. (1971). Veterinary Record. 89: 317-318
McMartin, D.A. (1968). The British Veterinary Journal, 124:576-581
Sevoian, M. and Levine, P. P. (1957). Avian Diseases, 1: 136-164.
From Proceedings of the "19th Australian Poultry Science Symposium", New South Wales, Australia.
