Source: UNIVERSITY OF ILLINOIS submitted to
AVIAN INFECTIOUS BRONCHITIS VIRUS: POSSIBLE CAUSE OF REPRODUCTIVE IMPAIRMENT IN ROOSTERS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0200605
Grant No.
2004-35203-14770
Project No.
ILLU-538-542
Proposal No.
2004-01451
Multistate No.
(N/A)
Program Code
41.0
Project Start Date
Sep 1, 2004
Project End Date
Aug 31, 2008
Grant Year
2004
Project Director
Bahr, J. M.
Recipient Organization
UNIVERSITY OF ILLINOIS
2001 S. Lincoln Ave.
URBANA,IL 61801
Performing Department
ANIMAL SCIENCES
Non Technical Summary
Several years ago, our lab made a serendipitous discovery while preparing the reproductive tract of roosters for histology. Abundant small stones, consisting mainly of calcium carbonate, were found in the epididymal roosters. Further tests indicated that the number of sperm produced per day and fertility of the semen produced were greatly reduced to the point that these factors may have been a major reason for low hatchability in broiler breeders. A link between immunization with the avian infectious bronchitis virus (AIBV) and these epididymal stones and reduced fertility was suggested. Our first experiment in which we used specific pathogen free (SPF) chicks which had never been exposed to AIBV and either immunized these chicks against the live attenuated AIBV or did not vaccine the chicks gave solid data documenting the role of AIBV immunization and subsequent formation of stones and reduction in fertility. The proposed studies planned for the next three years will clearly delineate the role of AIBV in the formation of epididymal stones and reduced fertility. Important outcomes of this research will be a better understanding of the reproductive physiology of the rooster and will indicate if new types of vaccines need to be developed that will protect chicken from getting avian bronchitis but will not have severe negative effects on reproduction efficiency.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
30132201020100%
Goals / Objectives
The major objective of this research is to determine if immunization with the live attenuated avian infectious bronchitis virus (AIBV), as done in the poultry industry, causes the formation of stones in the reproductive tract, specifically the epididymal region, and causes a 40% reduction in fertility in roosters. AIBV exhibits a tropism for ciliated cells such as found in the trachea, kidney, and oviduct. The epididymal epithelium also has many ciliated cells. The hypothesis to be tested is that AIBV is the causative agent responsible for the development of epididymal stones and reduced fertility in rosters. To test this hypothesis, answers to four questions will be sought. 1) Is AIBV the direct cause of epididymal stone formation and reduced fertility? 2) Does AIBV have the ability to enter and replicate in epididymal epithelial cells? 3) Does immunization with AIBV cause a change in gene expression of ion transporters and regulators of pH balance of the epididymal epithelial cells or epididymal lumen? 4) Does immunization with AIBV decrease fertility by altering composition of seminal plasma, sperm concentration, motility and morphology and/or survivability in the hen and ability of sperm to fertilize the egg?
Project Methods
Four experiments will be performed to accomplish the objective. First it is necessary to determine that AIBV, a RNA virus, is the cause of epididymal stones by using specific pathogen free (SPF) roosters that are either not immunized (control) or immunized with either the live attenuated AIBV, the killed AIBV (not able to replicate within ciliated cells) or the Newcastle disease virus (NDV-a RNA virus). Second, it will be determined that the AIBV can enter and replicate in the epididymal cells which would be a basis for the virus usurping the cellular activities of these cells. Third, expression of ion transporters and regulators of pH balance will be evaluated because these molecules are responsible for the maintenance of the proper ionic environment in the cell and also the epididymal fluid. A change in these molecules could be involved in the formation of epididymal stones and also the reduced fertility. Fourth, specific reproductive markers will be evaluated to determine the reason(s) for the reduced sperm production and fertility in roosters with epididymal stones. The endpoints that will evaluated are composition of seminal plasma, sperm concentration, motility, morphology and ability of sperm to survive in the female tract and to penetrate the perivitelline membrane.

Progress 09/01/04 to 08/31/08

Outputs
OUTPUTS: Several years ago, while processing reproductive tracts from roosters, we found many small stones in the reproductive tract, especially in the tubules carrying sperm. We also found that fertility and serum testosterone concentrations were low in roosters with stones. Our initial hypothesis was that the stones were the result of a high calcium diet because the roosters ate the same diet as laying hens. Extensive testing of diets with different amounts of calcium and vitamin D had no effect on the number of stones in the tract. Next, we tested the hypothesis that the stones were the result of roosters being immunized prepubertally against the avian infectious bronchitis virus (AIBV). This virus has a tropism for ciliated cells such as those found in the trachea, the reproductive tract, and kidney. Specific pathogen free (SPF) eggs were hatched and roosters were immunized at 2, 6, 10, and 14 weeks of age with the live attenuated AIBV according to the commercial schedule or not immunized (controls). At 26 weeks of age, which is about 10 weeks after roosters start producing sperm, 80% of roosters immunized with AIBV had stones, decreased daily sperm production, and lower serum testosterone concentrations. In a second study, we tested if immunization with a killed AIBV would also cause epididymal stones in adult roosters. We found that the killed AIBV triggered a rapid and robust antibody response and also epididymal stones. Next we did an in vitro study to test the ability of AIBV to enter the ciliated cells of the epididymis and undergo replication. Using 3"RACE we showed that AIBV entered and replicated in the epididymal region. To understand the etiology of the stones, we examined the roosters at 12, 16, 20, and 24 weeks of age to determine when stones first appeared following immunization with AIBV. Numerous stones were being formed in the epididymis as early as week 20. We did find that the formation of stones following AIBV immunization did not occur in roosters immunized with the Newcastle virus, another common avian pathogen. Early studies indicated that fertility was significantly reduced, following either natural mating or artificial insemination, as indicated by the number of eggs hatched when sperm were obtained from roosters with stones. Extensive studies were done to determine the cause for this reduced fertility. Sperm concentrations, sperm viability, sperm motility, sperm quality and fertilizing ability, acrosome integrity, in vivo sperm fertility, and serum testosterone concentrations were examined among roosters immunized against AIBV compared with non-immunized roosters. We found that pre-pubertal vaccination against AIBV had a limited effect on these different endpoints. Using a commercial kit to measure antibody titers against AIBV, we found AIBV antibodies in the blood serum as expected but also in the seminal fluid. Western blot analysis indicated that these AIBV present in blood serum and seminal plasma reacted with sperm membranes. Further analysis using 2-D gel analyses and sequencing of the proteins bands of interest showed that AIBV antibodies reacted with several proteins unique to the sperm membrane. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The formation of stones in the rooster reproductive tract is an international problem. The presence of stones has a significant negative impact on fertility. In the broiler industry, hatchability is 83%. A 1% increase in hatchability/year would increase profits $36-38 M. Therefore any management change that could increase fertility even slightly will have a significant impact on the financial gains of the poultry industry. For this reason, the findings from our research on the role of AIBV in causing reproductive impairment in the rooster are of significant value to the poultry industry and to the production of other animals that are immunized. Our findings do not only apply to poultry but even have implications for humans. There are a number of outcomes from our research. 1) Immunization with either the live attenuated or killed AIBV causes stones in the reproductive tract of the rooster and has a negative impact on fertility. 2) Stones are formed in young roosters, even within four weeks after roosters have started to produce sperm and cause erosion of the epithelial cells which are responsible for the transfer of ions, fluids, and proteins in and out of the reproductive tract. The correct ion concentration and osmolarity of the epididymal fluid are necessary for proper sperm maturation. The AIBV can enter and replicate in the ciliated cells of the reproductive tract; however, this replication is not the primary cause for the formation of epididymal stones. Sperm produced by roosters with epididymal sperm are impaired so that fertility is reduced whether under natural mating circumstances or the use of artificial insemination. In response to immunization with the AIBV, anti-AIBV antibodies are found in the blood serum but also in the seminal plasma. These anti-AIBV antibodies bind to sperm membranes and cause clumping of sperm. The binding of anti-AIBV antibodies to sperm membranes indicates that AIBV shares one or more common epitopes with proteins in sperm membranes. Clumps of sperm, as indicated by the staining for actin, were visible as calcification of the clumps was occurring. To eliminate the formation of epididymal stones, it is necessary to identify the common epitopes shared by AIBV and sperm membrane proteins and then synthesize fragments of AIBV which will still induce immunity against this virus but will not result in the production of epididymal stones and significant reduction of fertility.

Publications

  • Zimmerman, C.R. 2005. Vaccination of roosters against avian infectious bronchitis virus (AIBV): Elucidating the problem of reduced fertility by sperm quality analysis and detection of sperm autoimmunity. M.S. thesis, University of Illinois.
  • Janssen, S.J., Kirby, J.D., Hess, R.A., Rhoads. M., Bailey, K., Bunick, D., Wang, H. and Bahr, J.M. 2000. Characterization of a new poultry disease: Epididymal calcium stones in diverse rooster populations. Poult Sci 79:568-574.
  • Boltz, D.A., Nakai, M. and Bahr, J.M. 2004. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. Avian Diseases 48:909-915.
  • Jackson, U., Boltz, D.A., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Prepubertal exposure to the avian infectious bronchitis virus induces epididymal stones in the rooster after puberty. Journal of Poultry Science 43:280-285.
  • Bahr, J.M., Dalponte, M., Janssen, S.J. and Nakai, M. 2006. Ion transporters for fluid resorption in the rooster (Gallus domesticus) epididymal region. Animal Reproduction Science 95 331-337.
  • Boltz, C.R., Belton, R.A., Boltz, D.A., Dirks, A., Bunick, D., Scherba, G. and Bahr, J.M. 2007. Vaccination of roosters against avian infectious bronchitis virus results in autoimmunity to sperm (Submitted).
  • Jackson, U.H. 2004. Avian infectious bronchitis virus (AIBV) vaccine: Possible cause of epididymal region stone formation. M.S. thesis, University of Illinois.
  • Boltz, D.A. 2005. Avian infectious bronchitis virus: A tropism for the rooster reproductive tract and effects on reproductive performance. Ph.D. dissertation, University of Illinois.


Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: We have previously reported that roosters vaccinated with the live attenuated avian infectious bronchitis virus (AIBV) have decreased serum testosterone concentrations, epididymal stones, and reduced fertility. The objectives of our research were to determine: 1) if the epididymal stones were caused by anti-AIBV antibodies binding to sperm membranes, and 2) if the formation of epididymal stones was due specifically to immunization against AIBV or could also be caused by immunization against another common viral disease, Newcastle disease. To accomplish the first objective, specific pathogen free Leghorn roosters were divided into 3 groups that received one of the following treatments: no vaccination (NONVAC), vaccination with the killed virus (KVAC), or vaccination with the live attenuated virus (LVAC). Semen was collected from NONVAC, sperm obtained and sperm membrane proteins were isolated. Sperm membrane proteins were run on a western blot. Blood serum and epididymal fluid obtained from the three groups of roosters as well as blood serum obtained from NONVAC and LVAC hens were used as the source of the primary antibody. Also testis and the reproductive tracts from NONVAC roosters were removed, fixed and histological sections prepared. Immunohistochemistry was performed by reacting the sections with blood serum from NONVAC and LVAC. To accomplish the second objective, 16 specific free roosters were divided into two groups: NONVAC and Newcastle virus vaccinated. Blood was taken every other week starting at week 16 and semen was collected weekly starting at week 18. Roosters were euthanized at 30 weeks of age to check for presence of epididymal stones. PARTICIPANTS: David Boltz, graduate student, Claire Zimmerman (Boltz), graduate student, Joseph Esch, undergraduate student, and Jessica Sweet, undergraduate student. TARGET AUDIENCES: Poultry scientists, industry, and vaccine manufacturers.

Impacts
The performance of western blots using sperm membrane proteins from NONVAC reacted with blood serum or epididymal fluid from the NONVAC, LVAC, and KVAC roosters and blood serum from NONVAC and LVAC hens indicated that there were antibodies in the blood serum and epididymal fluid of LVAC, KVAC and LVAC hens that bound to sperm membrane proteins. These results were confirmed with immunohistochemistry. Vaccination with the Newcastle virus did induce a robust antibody response in the roosters as indicated by the measurement of the Newcastle viral antibodies in the blood. However, vaccination with this virus, in contrast to immunization with AIBV, did not cause epididymal stones nor any change in number of sperm ejaculated. These results clearly show that immunization with AIBV causes the production of antibodies which are present in the blood serum and the epididymal fluid and which bind to sperm membranes. Previous results showed the clumping of sperm in the reproductive tract as early as 20 weeks of age. The clumping of sperm is the precursor to stone formation which also induces an inflammation of the region, as indicated by the infiltration of immune cells. The binding of AIBV antibodies to sperm membranes appear to be specific for AIBV because immunization with the Newcastle virus did not cause stone formation. Apparently AIBV antibodies and sperm membranes share several common epitopes, also referred to as molecular mimicry. The importance of these findings is that new vaccines need to be developed which will provide protection against AIBV but will not cause the formation of antibodies which will bind to sperm membranes.

Publications

  • Boltz, D.A., Zimmerman, C.R., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus. Avian Diseases 50:594-598.
  • Boltz, C.R., Boltz, D.A., Bunick, D., Scherba, G. and Bahr, J.M. 2007. Vaccination against the avian infectious bronchitis virus affects sperm concentration, sperm quality, and blood testosterone concentrations in cockerels. British Poultry Science 48; 617-624.


Progress 01/01/06 to 12/31/06

Outputs
Avian infectious bronchitis virus (AIBV), a respiratory virus, has a tropism for the rooster reproductive tract and is capable of infecting the epididymal region of the rooster. Vaccination of roosters with live attenuated AIBV causes stones and reduces fertility. Our objective was to determine if vaccinating with killed AIBV would cause stones and reduced fertility. Killed AIBV, unlike live attenuated AIBV, is inactivated and unable to cause infection. Specific Pathogen Free (SPF) roosters were divided into three groups: a non-vaccinated group (SPF), a live attenuated AIBV vaccinated group (LVAC), and a killed AIBV vaccinated group (KVAC). Roosters were vaccinated at 2, 6, 10, and 14 wks. At 28 wks the reproductive tract was removed and examined for stones and prepared for histology. Testis weight and daily sperm production were determined. Stones were present in one (1/8) SPF bird, whereas 50% (5/10) of the KVAC and 64% (7/11) of LVAC roosters had stones. Testes weights were not different among the three groups. Daily sperm production (sperm/testis/day) was reduced in KVAC (4.4 +/- 0.7 x 108) and in LVAC (3.9 +/- 0.3 x 108, p<0.05) when compared to SPF roosters (4.9 +/-0.4 x 108). When comparing roosters within groups: LVAC with stones had an increased testis weight (11.3+/-1.0 g) when compared to LVAC without stones (10.9+/-1.9 g). Daily sperm production was reduced in LVAC roosters with stones (3.8+/-0.4 x 108) when compared to LVAC without stones (4.2 +/- 0.7 x 108). Testis weight was reduced in KVAC with stones (9.9 +/- 0.9 g, p<0.05) when compared to KVAC without stones (13.3 +/- 1.8 g). Daily sperm production was reduced in KVAC with stones (3.4 +/- 0.4 x 108, p<0.05) when compared to KVAC without stones (6.3 +/- 1.0 x 108). Histologically, seminiferous tubules were normal in KVAC and LVAC when compared to SPF controls. Efferent ductules (ED) from roosters without stones were normal. ED from roosters with stones were infiltrated with immune cells surrounding the area where a stone was present. We conclude that the use of a killed vaccine does not reduce the incidence of epididymal stones. It is possible AIBV proteins resemble those of the rooster resulting in an autoimmune response. Current studies are determining if the formation of epididymal stones is unique to exposure to the AIBV. Two groups of roosters, one control and one vaccinated with the Newcastle virus, are being examined to determine if exposure to this virus causes stones, reduces serum testosterone concentrations and alters daily sperm production.

Impacts
Our previous research indicated that vaccination of roosters with a live, attenuated avian infectious bronchitis virus caused epididymal stones, reduced daily sperm production and blood testosterone concentrations. We had hypothesized that the changes were caused by the virus entering and replicating in the cells of the reproductive tract. However, this study in which roosters were immunized with a killed avian infectious bronchitis virus indicates that entrance of the virus into the cells of the reproductive tract followed by replication is not necessary for this virus to have extensive negative effects on the reproductive performance of roosters.

Publications

  • Boltz, D.A., Nakai, M. and Bahr, J.M. 2004. Avian infectious bronchitis virus: A possible cause of reduced fertility in the rooster. Avian Diseases 48:909-915.
  • Jackson, U., Boltz, D.A., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006 Prepubertal exposure to the avian infectious bronchitis virus induces epididymal stones in the rooster after puberty. Journal of Poultry Science 43:280-285.
  • Boltz, D.A., Zimmerman, C.R., Nakai, M., Bunick, D., Scherba, G. and Bahr, J.M. 2006. Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus. Avian Diseases (In Press).
  • Bahr, J.M., Dalponte, M., Janssen, S.J. and Nakai, M. 2006. Ion transporters for fluid resorption in the rooster (Gallus domesticus) epididymal region. Animal Reproduction Science 95 331-337.


Progress 01/01/05 to 12/31/05

Outputs
Avian infectious bronchitis virus (AIBV), a respiratory virus, has a tropism for the rooster reproductive tract and is capable of infecting the epididymal region of the rooster. Vaccination of roosters with live attenuated AIBV causes stones and reduces fertility. Our objective was to determine if vaccinating with killed AIBV would cause stones and reduced fertility. Killed AIBV, unlike live attenuated AIBV, is inactivated and unable to cause infection. Specific Pathogen Free (SPF) roosters were divided into three groups: a non-vaccinated group (SPF), a live attenuated AIBV vaccinated group (LVAC), and a killed AIBV vaccinated group (KVAC). Roosters were vaccinated at 2, 6, 10, and 14 wks. At 28 wks the reproductive tract was removed and examined for stones and prepared for histology. Testis weight and daily sperm production were determined. Stones were present in one (1/8) SPF bird, whereas 50% (5/10) of the KVAC and 64% (7/11) of LVAC roosters had stones. Testes weights were not different among the three groups. Daily sperm production (sperm/testis/day) was reduced in KVAC (4.4 +/- 0.7 x 108) and in LVAC (3.9 +/- 0.3 x 108, p <0.05) when compared to SPF roosters (4.9 +/- 0.4 x 108). When comparing roosters within groups: LVAC with stones had an increased testis weight (11.3 +/- 1.0 g) when compared to LVAC without stones (10.9 +/- 1.9 g). Daily sperm production was reduced in LVAC roosters with stones (3.8 +/- 0.4 x 108) when compared to LVAC without stones (4.2 +/- 0.7 x 108). Testis weight was reduced in KVAC with stones (9.9 +/- 0.9 g, p <0.05) when compared to KVAC without stones (13.3 +/- 1.8 g). Daily sperm production was reduced in KVAC with stones (3.4 +/- 0.4 x 108, p < 0.05) when compared to KVAC without stones (6.3 +/- 1.0 x 108). Histologically, seminiferous tubules were normal in KVAC and LVAC when compared to SPF controls. Efferent ductules (ED) from roosters without stones were normal. ED from roosters with stones were infiltrated with immune cells surrounding the area where a stone was present. We conclude that the use of a killed vaccine does not reduce the incidence of epididymal stones. It is possible AIBV proteins resemble those of the rooster resulting in an autoimmune response.

Impacts
Our previous research indicated that vaccination of roosters with a live, attenuated avian infectious bronchitis virus caused epididymal stones, reduced daily sperm production and blood testosterone concentrations. We had hypothesized that the changes were caused by the virus entering and replicating in the cells of the reproductive tract. However, this study in which roosters were immunized with a killed avian infectious bronchitis virus indicates that entrance of the virus into the cells of the reproductive tract followed by replication is not necessary for this virus to have extensive negative effects on the reproductive performance of roosters.

Publications

  • Boltz, D.A., Zimmernman, C.R. and Bahr, J.M. 2005. Epididymal stone formation and decreased sperm production in roosters vaccinated with a killed strain of avian infectious bronchitis virus. Biology of Reproduction, Abst. # 214, p. 129.