Ultrastructural Observations on Effects of Infectious Bronchitis Virus in Eggshell-Forming Regions of the Oviduct of the Commercial Laying Hen K. K. Chousalkar1 and J. R. Roberts Animal Science, School of Rural Science and Agriculture, University of New England, Armidale, New South Wales, 2351, Australia ABSTRACT The pathogenesis of 2 strains of infectious bronchitis virus (T and N1/88 strains) was studied in the eggshell-forming regions of the oviduct of commercial laying hens. There were no external shell deformities, except for paler shells. There was no decline in egg production in either of the infected groups. One hen from the N1/88-infected group and 2 hens from the T-infected group were out of lay. The light, scanning, and transmission electron microscopic changes in infected shell-forming regions of the oviduct were compared with the control oviducts at different egg positions. The ultrastructural finding revealed that the extent of cytopathology in the isthmus was greater than in the tubular shell gland and
shell gland pouch. The T strain of infectious bronchitis virus was more pathogenic compared with the N1/88 strain. Severe cytopathology was recorded in the shellforming region of hens that were out of production, and virus particles were observed in hens that had stopped laying. Virus particles were recorded in the dilated rough endoplasmic reticulum and Golgi complex of the isthmus, tubular shell gland, and shell gland pouch of all 3 hens that had stopped laying. Although a decrease in egg production and deterioration in eggshell quality were not observed in this trial, cessation of egg production in a small number of hens could be due to severe cytopathology in the eggshell-forming regions of the oviduct.
Key words: transmission electron microscope, scanning electron microscope, infectious bronchitis virus, oviduct, hen 2007 Poultry Science 86:1915–1919
INTRODUCTION Infectious bronchitis is a common disease of poultry worldwide. The egg industry all around the world loses millions of dollars a year as the result of egg quality problems, and a disease such as infectious bronchitis virus (IBV) can contribute to these losses. It is reported in the literature that IBV is responsible for deterioration of eggshell quality (Sevoian and Levine, 1957; Cook, 1971; Munner et al., 1986), although the effects of the Australian strains of IBV on egg production and quality of laying hens have not been studied or documented previously. Also, the causes of pale eggshells and thin or soft eggshells during IBV infection are not yet clear. Information regarding cytopathology in the shell-forming regions of the oviduct of hens that had stopped laying during IBV infection, under experimental as well as field conditions, has not been reported previously. Australian strains can affect the fully functional oviduct in unvaccinated Leghorn hens (Chousalkar et al., 2007a) as well as vaccinated Hy-Line hens (Chousalkar et al., 2007b). This study ex-
©2007 Poultry Science Association Inc. Received March 25, 2007. Accepted May 25, 2007. 1 Corresponding author:
[email protected]
tends our earlier report regarding ultrastructural cytopathology in the infundibulum and magnum of the fully functional oviduct (Chousalkar and Roberts, 2007). In the present study, the cellular pathogenesis of 2 strains of IBV was studied in the shell-forming regions, isthmus, tubular shell gland (TSG), and shell gland pouch (SGP) of the fully functional oviduct of ISA Brown hens.
MATERIALS AND METHODS One hundred fifty 1-d-old ISA Brown layers were obtained from a commercial hatchery. At 1 d old, all the chickens received Rispens vaccine against Marek’s disease but no other vaccinations. Birds were raised in isolation sheds at the University of New England campus, Armidale, New South Wales, Australia. All equipment, sheds, and clothing were fumigated or washed with antiseptic or ethanol before use. Hens were transferred from floor pens to individual cages in different isolation sheds at 28 wk of age and were divided into 3 groups, 1 control with 48 hens and 2 IBV treatment groups each with 51 hens. The hens were kept in 3 different sheds that were physically separated from one another. The IBV-free status of the hens was maintained by isolation and strict biosecurity. All hens were found free from IBV antibodies before virus challenge (Flock Check IBV ELISA test kit, IDEXX Laboratories Inc., Westbrook, ME).
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At 30 wk of age, each bird was challenged with T or N1/88 strains of IBV (virus strains obtained from Jagoda Ignatovic, CSIRO, Geelong, Australia) at the dose rate of 2 × 105 embryo infective dose intraocularly, and the control birds were sham inoculated with normal saline. Two hens from each challenge group and 1 hen from the control group were euthanized at 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, and 24 d postinfection (p.i.). The isthmus and SGP undergoes ultrastructural variation depending on the position of the egg in the oviduct (Wyburn et al., 1973; Draper et al., 1972). Therefore, hens from the control group were deliberately killed with eggs at different positions in the oviduct to provide information about any variation due to position of the egg. Two hens that were out of lay in the T-infected group were killed on d 30 p.i. and 1 hen from the N1/88 group that was out of lay was killed on d 24 p.i. The shell-forming regions of the oviduct, isthmus, TSG, and SGP were placed in fixative (2% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 5.5). The tissues were processed by standard electron microscopy procedures and embedded in epoxy resin, which was allowed to polymerize at 62°C overnight. Semithin sections from 5 resin-embedded blocks of each tissue from each hen were collected and stained with toluidine blue. Ultrathin sections (80 to 90 nm) were then collected on grids and stained with a saturated solution of uranyl acetate followed by lead citrate. Sections were washed in CO2-free water, allowed to dry, and examined under a transmission electron microscope [Jeol, JEM-1200 EX, Jeol (Australasia) Pty. Ltd., Brookvale, Australia]. For scanning electron microscopy, the above samples, after fixation, were critical point dried, mounted on aluminum stubs, gold coated, and observed under a scanning electron microscope (Joel, JSM-5800LV). Tissue samples were also fixed in neutral buffered formalin and processed for histology as described earlier (Chousalkar et al., 2007a).
Figure 1. A) Isthmus of a control hen. Egg was in mid magnum. ×900. Scale bar represents 20 m. B) Isthmus at 12 d postinfection. Hen infected intraocularly with T strain of IBV at 30 wk of age. Note the loss of cilia from the individual cell. Egg was in mid-magnum. ×900. Scale bar represents 20 m.
RESULTS AND DISCUSSION Out of 50 challenged hens from the N1/88 group, only 1 hen went out of lay and was found to have an atrophied oviduct. Two hens from the T strain IBV-infected group were out of lay. Hens from the control group remained negative for IBV antibodies throughout the experiment (Chousalkar and Roberts, 2007). There were no external defects in shell quality, except for the visual appearance of paler shells during 4 to 8 d p.i. and change in egg shape index among the infected groups. There was no drop in egg production. The surface epithelium of the isthmus is lined by a pseudostratified columnar epithelium of ciliated and nonciliated cells. Ciliated and nonciliated cells are interspersed with mitochondrial cells that are characterized by a large number of mitochondria and no secretory granules. The lamina propria is lined by type 1 or type 2 tubular gland cells, which are in different phases of secretory activity in the same cell type (Draper et al., 1972;
Solomon, 1975). Scanning micrographs of the isthmus were similar to those published by Bakst and Howarth (1975; Figure 1, panel A). All parts of the isthmus of control hens appeared normal. No prominent microscopic lesions were recorded in the isthmus of the N1/88-infected group except for moderate infiltration of inflammatory cells in the submucosa and muscularis area in the hen with an atrophied oviduct on d 24 p.i. In T-infected hens, there was loss of cilia in 1 hen killed on each of d 10 and 12 p.i (Figure 1, panel B). One hen killed on d 12 p.i. showed occasional tubular gland dilatation. Virus particles were observed in the isthmus of this hen only. Occasional lymphocyte infiltration was observed in the interglandular spaces in the isthmus of T strain-infected hens at 16, 20, and 24 d p.i. Severe pathology was recorded in the isthmus of both the hens from the T-infected group that were out of lay when killed on d 30 p.i. These hens showed extensive
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Figure 2. Isthmus at 30 d postinfection. Hen infected intraocularly with T strain of IBV at 30 wk of age. Note the virus particles in the cisternae of rough endoplasmic reticulum (A). No egg in the oviduct. ×25,000. Scale bar represents 200 nm.
infiltration of lymphocytes into the interglandular space and large lymphoid nodules in the muscularis area in the isthmus. There was loss of cilia from ciliated cells, phagocytic vacuoles, dilated cisternae, and degeneration of mitochondria in surface as well as glandular epithelium. Virus particles were observed mostly in dilated cisternae. The intracisternal granules in virus-bearing and dilated rough endoplasmic reticulum (RER) were mostly absent (Figure 2). Virus particles were occasionally recorded in ciliated cells. Few mitochondria in mitochondrial cells appeared degenerated. Type 2 gland cells were most common in all parts of the isthmus, and some of the glands in the isthmus of these hens appeared dilated. Necrotic cells, along with an elongated mass of secretion, were observed in the lumen of some dilated gland cells. Pathology was not recorded in the isthmus of any of the hens from the control group. The TSG, also known as the red region, is the distal part of the isthmus where the process of shell formation is initiated (Solomon, 1975). The SGP (uterus) is lined by a pseudostratified columnar epithelium with apical and basal cells. Apical cells are ciliated, whereas basal cells are nonciliated (Wyburn et al., 1973).
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Most of the changes seen in the TSG and SGP were similar. Pathological changes were not recorded in the TSG and SGP of hens from the N1/88 group. However, in one hen that had an atrophied oviduct and was killed on d 24 p.i., large lymphoid nodules were recorded in the interglandular space of the TSG and SGP. Virus particles were observed in the gland cell epithelium of the shell gland pouch of this hen. In T-strain-infected hens, at 20 and 24 d p.i., large lymphoid nodules were recorded in the muscularis area. Severe pathology was observed in the TSG and SGP of hens that were out of production. In hens that had stopped laying, patchy cilia loss and increased deposits of RER with virus particles were recorded in ciliated cells. Vacuoloids were not recorded in most of the granular cells of the SGP, although vacuoloids were observed in the SGP of a control hen with no egg in the oviduct. The glands in the TSG and SGP were occasionally dilated. Mitochondria in nonciliated cells, as well as glandular epithelium, appeared swollen. In some nonciliated cells, the cristae of the mitochondria were disrupted. A few lipid droplets were also recorded in gland cells of the SGP. The apical border of most of the ciliated cells in the SGP and TSG appeared disrupted, and all mitochondria had degenerated (Figure 3). Budding of virus particles was observed in dilated RER in both ciliated and nonciliated cells. 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. In the present study, there was no deterioration of eggshell quality (except for eggshell color) or production unlike the findings of Sevoian and Levine (1957), Cook (1971), and Munner et al. (1986). All these studies were also conducted using limited sample numbers and reported variation in the individual response to IBV infection. However, it is important to note that, in the above studies, the Massachusetts or Arkansas strain of IBV was used. This indicates that a severe decline in eggshell quality is not necessarily a common feature of Australian strains of IBV. The small number of hens used in the present study may have made production changes difficult to detect. In our preliminary study we observed pathology in the TSG and SGP of T- and N1/88-infected unvaccinated Leghorn hens at 65 wk of age (Chousalkar et al., 2007a). The differences between the findings of our previous and present studies could be attributed to age and breed of the hens. However, cytopathology in the albumen-forming (Chousalkar and Roberts, 2007) and shell-forming regions of the oviduct of hens that were out of production suggests that IBV has the potential to infect all parts of the oviduct. Virus particles were observed in shell-forming regions of the T-strain-infected hens up to 30 d p.i., and such hens are a potential source of virus shedding in the flock. During eggshell formation, the isthmus plays an important role in the formation of eggshell membranes. Cytopathology in the isthmus could cause abnormal shell
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Figure 3. Shell gland pouch at 30 d postinfection. Hen infected intraocularly with T strain of infectious bronchitis virus at 30 wk of age. Note the loss of cilia from ciliated cell (B). Also note the degenerated mitochondria (A). No egg in the oviduct. ×5,000. Scale bar represents 1 m.
membrane formation. The intracisternal granules in the RER deposits of the isthmus are a means of regulating protein synthesis (Solomon, 1983), and their absence could be responsible for disturbance in protein synthesis. Mammillary cores in the eggshell are the initial templates for the calcified shell and are secreted by epithelial cells of the TSG (Solomon, 1975). Pathology in the epithelial cells (both surface and glandular) of the tubular shell gland could be responsible for formation of abnormal mammillary cores resulting in formation of a poor quality mammillary layer. The mammillary layer is the crystalline foundation of the eggshell (Brackpool, 1995), hence any alteration in this layer could affect the quality of whole eggshell. However cytochemical studies are essential to clarify this possibility. Moreover, the TSG and SGP play a vital role in the process of eggshell calcification. Solomon (1983) suggested that the columnar epithelium contributes calcium during egg formation. Mitochondria also store calcium ions (Simkiss and Taylor, 1971). The degeneration of surface epithelial cells and changes in mitochondria may alter the rate of calcification and all this could affect the process of eggshell formation. It is inter-
esting to note the paler shells during d 4 to 8 p.i., without any pathology in the shell gland on those days, but further investigation is essential to explain this finding. Eggshell color is an important aspect of egg quality for the markets in Europe and Australia where there is a preference for dark brown shell eggs. Hence, paler eggshells may not be regarded well by consumers. In this trial, severe pathology was observed in the shell gland mucosa of hens that were out of production. Breen and De Bruyn (1969) suggested that vacuoloids in the shell gland resynthesize the disintegrated granules, which are utilized for the secretory product. The absence of vacuoloids in the shell gland of T-infected hens that have stopped laying indicates the absence of the secretory cycle and hence one of the possible causes of reduced production of secretory product. It is also possible that degenerative changes in the shell gland of IBV-infected hens are responsible for insufficient sex steroid stimulation resulting in cessation of egg production. This fact was suggested earlier by Nevalainen (1969) who found degenerative changes in the shell gland mucosa of calcium-deficient hens that were out of lay. However, studies regarding changes in hormonal levels during infection are necessary to prove this. The degenerative changes in the shell-forming regions could be reversible or irreversible in some hens, and hens may stop laying temporarily or permanently. Such changes may not be very common during infection with T and N1/88 strains of IBV in adult ISA Brown hens because only 3 hens that 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. The eggshell-forming region of the avian oviduct is highly complex and secretes a range of eggshell matrix proteins; hence, it would still be difficult to confirm the cause of thin or soft shells during infection. We did not observe soft or thin-shelled eggs mentioned by earlier researchers as a typical finding of IBV infection in hens (Sevoian and Levine, 1957). However, it would be interesting to conduct ultrastructural studies with the Massachusetts strain of IBV, the strain known to cause decline in egg production and deterioration of shell quality on a larger scale. Such studies cannot be conducted in Australia due to quarantine restrictions. The Massachusetts strain of IBV has been reported to be more pathogenic for the oviduct of baby chickens as compared with T strain (Crinion and Hofstad, 1972). Because there are no published records of direct effects of Australian IBV strains on the mature oviduct and eggshell quality, the present study addresses earlier speculations about Australian strains of IBV. It would be interesting to study effects of IBV infection on eggshell matrix proteins.
ACKNOWLEDGMENTS The technical assistance from Patrick Littlefield, electron microscope unit manager, is gratefully acknowledged. The Physiology Teaching Unit, University of New
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England, provided financial support to K. Chousalkar for this study.
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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. Draper, M. H., M. F. Davidson, G. M. Wyburn, and H. S. Johnston. 1972. The fine structure of the fibrous membrane forming region of the isthmus of the oviduct of Gallus domesticus. Q. J. Exp. Physiol. 57:297–309. McMartin, D. A. 1968. The pathogenicity of an infectious bronchitis virus for laying hens, with observations on pathogenesis. Br. Vet. J. 124:576–581. Munner, M. A., D. A. Halvorson, V. Sivnandan, J. A. Newman, and C. N. Coon. 1986. Effects of Infectious bronchitis virus (Arkansas strain) on laying chickens. Avian Dis. 30:644–647. Nevalainen, T. J. 1969. Electron microscope observations on the shell gland mucosa of calcium-deficient hens (Gallus domesticus). Anat. Rec. 164:127–140. Sevoian, M., and P. P. Levine. 1957. Effects of infectious bronchitis on the reproductive tracts, egg production and egg quality of laying chickens. Avian Dis. 1:136–164. Simkiss, K., and T. G. Taylor. 1971. Shell formation. Pages 1331– 1342 in Physiology and Biochemistry of Domestic Fowl. D. J. Bell and B. M. Freeman, ed. Vol. 3. Acad. Press, London, UK. Solomon, S. E. 1975. Studies on the isthmus region of the domestic fowl. Br. Poult. Sci. 16:255–258. Solomon, S. E. 1983. Oviduct. Pages 379–419 in Physiology and Biochemistry of Domestic Fowl. D. J. Bell and B. M. Freeman, ed. Vol. 4. Acad. Press, London, UK. Wyburn, G. M., H. S. Johnston, M. H. Draper, and M. F. Davidson. 1973. Ultra structure of shell forming region of the oviduct and development of shell of Gallus domesticus. Q. J. Exp. Physiol. 58:143–151.
