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Resistance of castrated male horses to attempted establishment of the carrier state with equine arteritis virus
J. Comp. Path. 1994 Vol. 111, 383 388
R e s i s t a n c e o f C a s t r a t e d M a l e H o r s e s to A t t e m p t e d E s t a b l i s h m e n t o f the Carrier State w i t h E q u i n e Arteritis V i r u s W. H. M c C o l l u m , T. V. Little, P.J. T i m o n e y and T. W. Swerezek Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099, USA
Summary Twelve geldings all became infected when inoculated intranasally with the KY-84 strain of equine arteritis virus (EAV), a strain previously shown to be capable of establishing the carrier state in the stallion. With the exception of one animal that showed no effects other than pyrexia, all of the geldings developed clinical signs characteristic of equine viral arteritis (EVA). The geldings were febrile for varying periods within the range of 2 10 days after inoculation. Viraemia occurred from day 2 onwards, for periods varying from 9 to at least 19 days. Nasal shedding of virus began 2 4 days after inoculation and persisted for periods ranging from 7-14 days. All geldings "seroconverted" to EAV by day 11, with serum neutralization titres ranging from 8 to 64. The titres ranged from 8 to 32 after 4 weeks. Low concentrations of EAV were detected in the kidney and blood of one gelding killed 30 days after inoculation and in the blood of another killed after 57 days. Virus was not isolated from any tissue or fluid sample collected from the remaining I0 geldings, all of which were killed between days 30 and 148. The findings confirm that persistent EAV infection is unlikely to occur in geldings and support the results of previous studies, which demonstrated that testosterone plays an essential role in the establishment and maintenance of the carrier state,
Introduction T h e existence o f a c a r r i e r state in stallions infected with equine arteritis virus (EAV), a l t h o u g h s u s p e c t e d since the late n i n e t e e n t h a n d early twentieth c e n t u r y (Pottie, 1888; Clark, 1892; B e r g m a n , 1913), was u n e q u i v o c a l l y c o n f i r m e d for the first t i m e in T h o r o u g h b r e d stallions e x p o s e d d u r i n g the 1984 equine viral arteritis (EVA) e p i d e m i c in central K e n t u c k y (Timoney, 1984). T h e r e is no evidence t h a t m a r e s can b e c o m e persistently infected with the virus. T h e e s t a b l i s h m e n t a n d m a i n t e n a n c e o f the c a r r i e r state in n a t u r a l l y infected stallions was p r o v e d to be t e s t o s t e r o n e - d e p e n d e n t b y Little et al. (1992). Earlier studies (Neu et al., 1988) h a d c o n f i r m e d t h a t persistent infection could be established b y the e x p e r i m e n t a l infection o f m a t u r e intact stallions, EAV b e c o m i n g localized in the accessory sex glands. H o l y o a k et al. (1993) failed to establish l o n g - t e r m infection in p r e - p u b e r t a l colts a n d in the m a j o r i t y o f perip u b e r t a l colts e x p o s e d to a similar dose o f virus. T h i s study extends these 0021-99751941080383 +06 $08.0010
9 1994 AcademicPress Limited
384
W.H. McCollum et al.
observations a n d summarizes the results o f attempts to establish the carrier state in m a t u r e geldings. Materials and Methods
Geldings Twelve geldings aged 2 to 20 years were used. They comprised five Thoroughbreds, six horses of mixed breeding and one Appaloosa, and none had neutralizing antibodies to EAV before inoculation. A direct iodine-125 testosterone assay kit (Pantex, Santa Monica, CA, USA), validated for equine sera (Little, 1993), was used to assay serum testosterone concentrations in accordance with the manufacturer's instructions. Preinfection serum testosterone concentrations were < 100 pcg/ml, values characteristic of geldings. The animals were kept at pasture throughout the study.
Experimental Design After intranasal inoculation with the KY-84 strain of EAV (McCollum and Timoney, 1984) by means of a fenestrated catheter, the 12 geldings were examined for evidence of persistent infection. They were observed for the appearance of clinical signs of EVA and rectal temperatures were recorded twice daily for the first 21 days after infection. Nasopharyngeal swabs, buffy coat and serum samples were collected daily during this period for attempted virus isolation and for serological testing. Two geldings were killed on each of days 30, 57, 88, 122, 136 and 148, and necropsied. The following samples were collected and cultured for EAV in RK-13 cells (ATCC No. CCL37): lung, liver, kidney, spleen; inguinal, colonic, mesenteric, splenic and bronchial lymph nodes; bilateral mid and distal vas deferens; bilateral proximal, mid and distal ampullae; bulbourethral, vesicular and prostate glands; thoracic, pericardial and abdominal fluids; urine and unclotted blood.
Viral Inoculum The KY-84 strain of EAV was used, its source being a 10% homogenate of mesenteric lymph node collected from a horse killed 6 days after being infected with the second horse passage of the strain. The EAV inoculum (dose volume 5 ml) contained 2"5 x 103 plaque-forming units/ml. The mode of preparation and administration of the inoculum has been described previously (McCollum and Timoney, 1984; Neu et al., 1988).
Collection and Examination of Specimens The methods described by McCollum (1970) and McCollum et al. (1971) were used for the collection of nasopharyngeal swabs, buffy coat and serum samples, necropsy tissues and body fluids; for the propagation and maintenance of the RK-13 cell cultures; and for the use of these cells in isolating, assaying and identifying isolates of EAV. Serum neutralizing antibody titres to EAV were determined by the microtitre procedure of Senne et al. (1985), with RK-13 cells. Results
Clinical, Virological and Serological Responses All 12 geldings b e c a m e febrile for 5 to 8 days after inoculation, with m a x i m u m t e m p e r a t u r e s r a n g i n g f r o m 39"6 to 40"6~ (Table 1). T w o animals d e v e l o p e d severe signs o f EVA, nine mild signs, and one h a d no signs o t h e r t h a n pyrexia. Severe signs consisted o f extensive leg o e d e m a , conjunctivitis, nasal discharge
Equine Viral Arteritis in Geldings
385
and depression, and mild signs consisted of a slight ocular and nasal discharge. EAV was recovered from the nasopharynx for 7 to 14 days and from the blood for 9 to 19 days. All geldings had "seroconverted" to EAV by day 11. The serum neutralization titres ranged from 8 to 32 at day 28. Attempted b~rus Isolation from 77ssues and Body Fluids Tissues and body fluids, representing 30 sites from each gelding, were cultured from animals killed in pairs between days 30 and 148. Virus was recovered from the buff,/ coat and kidney of one gelding (no. 12) killed 30 days after challenge and from the buffy coat of one (no. 2) of the two horses killed on day 57. EAV was not isolated from the tissues or body fluids of any gelding killed subsequently. Discussion
The occurrence of the EAV carrier state has been recorded in numerous stallions (Timoney et al., 1987; Little et al., 1992) since it was first reported by Timoney et al. (1986a and b). Its existence had been postulated a century earlier in relation to a clinically indistinguishable disease of horses (Pottie, 1888; Clark, 1892), but soon after confirmation in the stallion it was determined experimentally that persistent infection was confined to the urogenital tract, the preferential site being the ampulla of the vas deferens (Neu et al., 1988). Little et al. (1992) confirmed that persistence of EAV infection in stallions was testis,dependent and that this dependence was mediated by testosterone. Efforts to establish long-term persistence in colts before the onset of pubertal development and the appearance of detectable concentrations of circulating testosterone were unsuccessful (Holyoak et al., 1993). Chronic EAV infection has never been confirmed in the mare. Although persistent infection with EAV has not been reported in naturally infected geldings, it was considered necessary to investigate the possibility of its occurrence by means of a controlled experimental study. In the light of what is known about virus clearance and persistent infection with EAV in castrated stallions (Little et al., 1992), it was not surprising that the carrier state was not established in any of the 12 geldings in this study. Although EAV is completely cleared from carrier stallions 4 to 26 days after castration (Little et al., 1992), viral output can be maintained by the administration of replacement testosterone (Little et al., 1992). The mechanism by which castration effects viral clearance is not clear. There may be a decrease in viral replication, an increase in viral elimination, or a combination of both. Androgen ablation or castration is known to cause rapid regression of the rat prostate (Kerr and Searle, 1973; Sanford et al., 1984; Evans and Chandler, 1987). This regression results in a decrease in secretory activity, a reduction in cell proliferation and a loss of androgen-dependent cells (Lamb et al., 1992). Castration of mature stallions also causes rapid involution of the accessory sex glands (Thompson et al., 1980; Little, 1993), presumably by similar mechanisms. If persistent virus is restricted to androgen-dependent epithelial
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cells or relies on the secretory apparatus of those cells for viral release, castration might be expected to result in viral clearance (Little, 1993). By definition, a persistent viral infection must avoid immune surveillance or immune system effectors. The reproductive tract of the mature male is an "immunologically privileged" site, allowing for the expression of sperm antigens in the testes and efferent ducts without the development of sperm antibodies. It has been postulated that EAV establishes persistence in the ampullae as a result of this site being immunologically privileged (Little et al., 1992). Androgens are known to play an essential role in the production and maintenance of physical (Setchell, 1986) and T-suppressor cell barriers (Ritchie et al., 1984) and of immunosuppressive secretions (Anderson and Tarter, 1982; Shivaji et al., 1990), which are believed responsible for the immunological unresponsiveness of the male reproductive tract. Castration may therefore cause changes in these barriers which allow the host's immune system to attack and eliminate persistent virus. Acknowledgment The technical assistance of Mr Y. Liu and the care and handling of the geldings throughout this study by Mr P. Stotler are gratefully acknowledged. This study was supported by the Frederick Van Lennep Professorship Endowment Fund and is published as paper 94-4-64 by permission of the Dean and Director, College of Agriculture and Kentucky Agricultural Experiment Station. References Anderson, D.J. and Tarter, T. H. (1982). Immunosuppressive effects of mouse seminal plasma components in vivo and in vitro. Journal of Immunology, 128, 535-539. Bergman, A. M. (1913). Beitrage zur Kenntnis der Virustrager bei Kotlaufseuche, Influenza erysipelatosa, des Pferdes. 7,/itschriftfiir Infektionskrankheiten, 13, 161-174. Clark, J. (1892). Transmission of pink-eye from apparently healthy stallions to mares. journal of Comparative Pathology and Therapeutics, 5, 261-264. Evans, G. E and Chandler, J. A. (1987). Cell proliferation studies in the rat prostate: II. The effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation. The Prostate, 11, 339 351. Holyoak, G. R., Little, T. V., McCollum, W. H. and Timoney, E J. (1993). The relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt. oTournal of Comparative Pathology, 109, 29-46. Kerr, J. E R. and Searle, J. (1973). Deletion of cells by apoptosis during castrationinduced involution of the rat prostate. FirchowsArchiv. B. CellPathology, 13, 87 102. Lamb, J. C., English, H., Levandoski, E L., Rhodes, G. R., Johnson, R. K. and Isaacs, J. T. (1992). Prostatic involution in rats induced by a novel 5-alphareductase inhibitor, SK-and-F-105657--Role for testosterone in the androgenic response. Endocrinology, 130, 685-694. Little, T. V. (1993). An endocrinological basis for the persistence of equine arteritis virus in the reproductive tract of the stallion. PhD Dissertation, University of Kentucky. Little, T. V., Holyoak, G. R., Timoney, E J. and McCollum, W. H. (1992). Output of equine arteritis virus from persistently infected carrier stallions is testosterone dependent. Proceedings of the 6th International Conference of Equine Infectious Diseases, Cambridge, 1991, pp. 225-229. McCollum, W. H. (1970). Vaccination for equine viral arteritis. Proceedings of the 2nd
388
W. H. McGollum et al.
International Conference of Equine Infectious Diseases, Paris 1969. Karger, Basel and New York, pp. 143 151. McCollum, W. H., Prickett, M. E. and Bryans, J. T. (1971). Temporal distribution of equine arteritis virus in respiratory mucosa, tissues and body fluids of horses infected by inhalation. Research in Veterinary Science, 12, 459-464. McCollum, W. H. and Timoney, R J. (1984). The pathogenic qualities of the 1984 strain of equine arteritis virus. In: Proceedings of the Grayson Foundation International Conference of Thoroughbred Breeders Organizations on Equine Viral Arteritis, Grayson Foundation Inc., Lexington, KY, pp. 34 37. Neu, S. M., Timoney, R J. and McCollum, W. H. (1988). Persistent infection of the reproductive tract in stallions experimentally infected with equine arteritis virus. In: Proceedings of the 5th International Conference of Equine Infectious Diseases, Lexington, pp. 149 154. Pottie, A. (1888). The propagation of influenza from stallions to mares. Journal of Comparative Pathology and Therapeutics, 1, 37 38. Ritchie, A. W. S., Hargreave, T. B., James, K. and Chisholm, G. D. (1984). Intraepithelial lymphocytes in the normal epididymis: a mechanism for tolerance to sperm auto-antigens. British Journal of Urology, 56, 79 83. Sanford, N. L., Searle, J. W. and Kerr, J. F. R. (1984). Successive waves of apoptosis in the rat prostate after repeated withdrawal of testosterone stimulation. Pathology, 16, 406-410. Senne, D. A., Pearson, J. E. and Carbrey, E. A. (1985). Equine viral arteritis: a standard procedure for the virus neutralization test and comparison of results of a proficiency test performed at five laboratories. Proceedings of the United States Animal Health Association Meeting, Louisville, KY, pp. 29-33. Setchell, B. R (1986). The movement of fluids and substances in the testis. Australian Journal of Biological Sciences, 39, 193 207, Shivaji, S., Scheit, K.-H. and Bhargava, R M. (1990). Immunosuppressive factors of seminal plasma. In: Proteins of Seminal Plasma and Secretions of the Male Reproductive Tract. John Wiley and Sons, New York, pp. 375-389. Thompson, D. L., Pickett, B. W., Squires, E. L. and Nett, T. M. (1980). Sexual behavior, seminal pH, and accessory sex gland weights in geldings administered testosterone and(or) estradiol- 17B. Journal of Animal Science, 51, 1358 1366. Timoney, R J. (1984). Clinical, virological and epidemiological features of the 1984 outbreak of equine viral arteritis in the Thoroughbred population in Kentucky, USA. In: Proceedings of the Grayson Foundation International Conference of Thoroughbred Breeders Organizations on Equine Viral Arteritis, Grayson Foundation Inc., Lexington, KY, pp. 24 33. Timoney, RJ., McCollum, W. H. and Roberts, A. W. (1986a). Detection of the carrier state in stallions persistently infected with equine arteritis virus. In: Proceedings of the 32nd Annual Meeting of the American Association of Equine Practitioners, Nashville, pp. 57 65. Timoney, R J., McCollum, W. H., Roberts, A. W. and Murphy, T. W. (1986b). Demonstration of the carrier state in naturally acquired equine arteritis virus infection in the stallion. Research in Veterinary Science, 41, 279 280. Timoney, RJ., McCollum, W. H., Murphy, T. W., Roberts, A. W., Willard, J. G. and Carswell, G. D. (1987). The carrier state in equine arteritis virus infection in the stallion with specific emphasis on the venereal mode of virus transmission, journal of Reproduction and Fertility, Suppl., 35, 95-102. Received, May 3rd, 1994 ] Accepted, July 29th, 1994J
R e s i s t a n c e o f C a s t r a t e d M a l e H o r s e s to A t t e m p t e d E s t a b l i s h m e n t o f the Carrier State w i t h E q u i n e Arteritis V i r u s W. H. M c C o l l u m , T. V. Little, P.J. T i m o n e y and T. W. Swerezek Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099, USA
Summary Twelve geldings all became infected when inoculated intranasally with the KY-84 strain of equine arteritis virus (EAV), a strain previously shown to be capable of establishing the carrier state in the stallion. With the exception of one animal that showed no effects other than pyrexia, all of the geldings developed clinical signs characteristic of equine viral arteritis (EVA). The geldings were febrile for varying periods within the range of 2 10 days after inoculation. Viraemia occurred from day 2 onwards, for periods varying from 9 to at least 19 days. Nasal shedding of virus began 2 4 days after inoculation and persisted for periods ranging from 7-14 days. All geldings "seroconverted" to EAV by day 11, with serum neutralization titres ranging from 8 to 64. The titres ranged from 8 to 32 after 4 weeks. Low concentrations of EAV were detected in the kidney and blood of one gelding killed 30 days after inoculation and in the blood of another killed after 57 days. Virus was not isolated from any tissue or fluid sample collected from the remaining I0 geldings, all of which were killed between days 30 and 148. The findings confirm that persistent EAV infection is unlikely to occur in geldings and support the results of previous studies, which demonstrated that testosterone plays an essential role in the establishment and maintenance of the carrier state,
Introduction T h e existence o f a c a r r i e r state in stallions infected with equine arteritis virus (EAV), a l t h o u g h s u s p e c t e d since the late n i n e t e e n t h a n d early twentieth c e n t u r y (Pottie, 1888; Clark, 1892; B e r g m a n , 1913), was u n e q u i v o c a l l y c o n f i r m e d for the first t i m e in T h o r o u g h b r e d stallions e x p o s e d d u r i n g the 1984 equine viral arteritis (EVA) e p i d e m i c in central K e n t u c k y (Timoney, 1984). T h e r e is no evidence t h a t m a r e s can b e c o m e persistently infected with the virus. T h e e s t a b l i s h m e n t a n d m a i n t e n a n c e o f the c a r r i e r state in n a t u r a l l y infected stallions was p r o v e d to be t e s t o s t e r o n e - d e p e n d e n t b y Little et al. (1992). Earlier studies (Neu et al., 1988) h a d c o n f i r m e d t h a t persistent infection could be established b y the e x p e r i m e n t a l infection o f m a t u r e intact stallions, EAV b e c o m i n g localized in the accessory sex glands. H o l y o a k et al. (1993) failed to establish l o n g - t e r m infection in p r e - p u b e r t a l colts a n d in the m a j o r i t y o f perip u b e r t a l colts e x p o s e d to a similar dose o f virus. T h i s study extends these 0021-99751941080383 +06 $08.0010
9 1994 AcademicPress Limited
384
W.H. McCollum et al.
observations a n d summarizes the results o f attempts to establish the carrier state in m a t u r e geldings. Materials and Methods
Geldings Twelve geldings aged 2 to 20 years were used. They comprised five Thoroughbreds, six horses of mixed breeding and one Appaloosa, and none had neutralizing antibodies to EAV before inoculation. A direct iodine-125 testosterone assay kit (Pantex, Santa Monica, CA, USA), validated for equine sera (Little, 1993), was used to assay serum testosterone concentrations in accordance with the manufacturer's instructions. Preinfection serum testosterone concentrations were < 100 pcg/ml, values characteristic of geldings. The animals were kept at pasture throughout the study.
Experimental Design After intranasal inoculation with the KY-84 strain of EAV (McCollum and Timoney, 1984) by means of a fenestrated catheter, the 12 geldings were examined for evidence of persistent infection. They were observed for the appearance of clinical signs of EVA and rectal temperatures were recorded twice daily for the first 21 days after infection. Nasopharyngeal swabs, buffy coat and serum samples were collected daily during this period for attempted virus isolation and for serological testing. Two geldings were killed on each of days 30, 57, 88, 122, 136 and 148, and necropsied. The following samples were collected and cultured for EAV in RK-13 cells (ATCC No. CCL37): lung, liver, kidney, spleen; inguinal, colonic, mesenteric, splenic and bronchial lymph nodes; bilateral mid and distal vas deferens; bilateral proximal, mid and distal ampullae; bulbourethral, vesicular and prostate glands; thoracic, pericardial and abdominal fluids; urine and unclotted blood.
Viral Inoculum The KY-84 strain of EAV was used, its source being a 10% homogenate of mesenteric lymph node collected from a horse killed 6 days after being infected with the second horse passage of the strain. The EAV inoculum (dose volume 5 ml) contained 2"5 x 103 plaque-forming units/ml. The mode of preparation and administration of the inoculum has been described previously (McCollum and Timoney, 1984; Neu et al., 1988).
Collection and Examination of Specimens The methods described by McCollum (1970) and McCollum et al. (1971) were used for the collection of nasopharyngeal swabs, buffy coat and serum samples, necropsy tissues and body fluids; for the propagation and maintenance of the RK-13 cell cultures; and for the use of these cells in isolating, assaying and identifying isolates of EAV. Serum neutralizing antibody titres to EAV were determined by the microtitre procedure of Senne et al. (1985), with RK-13 cells. Results
Clinical, Virological and Serological Responses All 12 geldings b e c a m e febrile for 5 to 8 days after inoculation, with m a x i m u m t e m p e r a t u r e s r a n g i n g f r o m 39"6 to 40"6~ (Table 1). T w o animals d e v e l o p e d severe signs o f EVA, nine mild signs, and one h a d no signs o t h e r t h a n pyrexia. Severe signs consisted o f extensive leg o e d e m a , conjunctivitis, nasal discharge
Equine Viral Arteritis in Geldings
385
and depression, and mild signs consisted of a slight ocular and nasal discharge. EAV was recovered from the nasopharynx for 7 to 14 days and from the blood for 9 to 19 days. All geldings had "seroconverted" to EAV by day 11. The serum neutralization titres ranged from 8 to 32 at day 28. Attempted b~rus Isolation from 77ssues and Body Fluids Tissues and body fluids, representing 30 sites from each gelding, were cultured from animals killed in pairs between days 30 and 148. Virus was recovered from the buff,/ coat and kidney of one gelding (no. 12) killed 30 days after challenge and from the buffy coat of one (no. 2) of the two horses killed on day 57. EAV was not isolated from the tissues or body fluids of any gelding killed subsequently. Discussion
The occurrence of the EAV carrier state has been recorded in numerous stallions (Timoney et al., 1987; Little et al., 1992) since it was first reported by Timoney et al. (1986a and b). Its existence had been postulated a century earlier in relation to a clinically indistinguishable disease of horses (Pottie, 1888; Clark, 1892), but soon after confirmation in the stallion it was determined experimentally that persistent infection was confined to the urogenital tract, the preferential site being the ampulla of the vas deferens (Neu et al., 1988). Little et al. (1992) confirmed that persistence of EAV infection in stallions was testis,dependent and that this dependence was mediated by testosterone. Efforts to establish long-term persistence in colts before the onset of pubertal development and the appearance of detectable concentrations of circulating testosterone were unsuccessful (Holyoak et al., 1993). Chronic EAV infection has never been confirmed in the mare. Although persistent infection with EAV has not been reported in naturally infected geldings, it was considered necessary to investigate the possibility of its occurrence by means of a controlled experimental study. In the light of what is known about virus clearance and persistent infection with EAV in castrated stallions (Little et al., 1992), it was not surprising that the carrier state was not established in any of the 12 geldings in this study. Although EAV is completely cleared from carrier stallions 4 to 26 days after castration (Little et al., 1992), viral output can be maintained by the administration of replacement testosterone (Little et al., 1992). The mechanism by which castration effects viral clearance is not clear. There may be a decrease in viral replication, an increase in viral elimination, or a combination of both. Androgen ablation or castration is known to cause rapid regression of the rat prostate (Kerr and Searle, 1973; Sanford et al., 1984; Evans and Chandler, 1987). This regression results in a decrease in secretory activity, a reduction in cell proliferation and a loss of androgen-dependent cells (Lamb et al., 1992). Castration of mature stallions also causes rapid involution of the accessory sex glands (Thompson et al., 1980; Little, 1993), presumably by similar mechanisms. If persistent virus is restricted to androgen-dependent epithelial
386
W. H. M c C o l l u m et al. ?2
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cells or relies on the secretory apparatus of those cells for viral release, castration might be expected to result in viral clearance (Little, 1993). By definition, a persistent viral infection must avoid immune surveillance or immune system effectors. The reproductive tract of the mature male is an "immunologically privileged" site, allowing for the expression of sperm antigens in the testes and efferent ducts without the development of sperm antibodies. It has been postulated that EAV establishes persistence in the ampullae as a result of this site being immunologically privileged (Little et al., 1992). Androgens are known to play an essential role in the production and maintenance of physical (Setchell, 1986) and T-suppressor cell barriers (Ritchie et al., 1984) and of immunosuppressive secretions (Anderson and Tarter, 1982; Shivaji et al., 1990), which are believed responsible for the immunological unresponsiveness of the male reproductive tract. Castration may therefore cause changes in these barriers which allow the host's immune system to attack and eliminate persistent virus. Acknowledgment The technical assistance of Mr Y. Liu and the care and handling of the geldings throughout this study by Mr P. Stotler are gratefully acknowledged. This study was supported by the Frederick Van Lennep Professorship Endowment Fund and is published as paper 94-4-64 by permission of the Dean and Director, College of Agriculture and Kentucky Agricultural Experiment Station. References Anderson, D.J. and Tarter, T. H. (1982). Immunosuppressive effects of mouse seminal plasma components in vivo and in vitro. Journal of Immunology, 128, 535-539. Bergman, A. M. (1913). Beitrage zur Kenntnis der Virustrager bei Kotlaufseuche, Influenza erysipelatosa, des Pferdes. 7,/itschriftfiir Infektionskrankheiten, 13, 161-174. Clark, J. (1892). Transmission of pink-eye from apparently healthy stallions to mares. journal of Comparative Pathology and Therapeutics, 5, 261-264. Evans, G. E and Chandler, J. A. (1987). Cell proliferation studies in the rat prostate: II. The effects of castration and androgen-induced regeneration upon basal and secretory cell proliferation. The Prostate, 11, 339 351. Holyoak, G. R., Little, T. V., McCollum, W. H. and Timoney, E J. (1993). The relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt. oTournal of Comparative Pathology, 109, 29-46. Kerr, J. E R. and Searle, J. (1973). Deletion of cells by apoptosis during castrationinduced involution of the rat prostate. FirchowsArchiv. B. CellPathology, 13, 87 102. Lamb, J. C., English, H., Levandoski, E L., Rhodes, G. R., Johnson, R. K. and Isaacs, J. T. (1992). Prostatic involution in rats induced by a novel 5-alphareductase inhibitor, SK-and-F-105657--Role for testosterone in the androgenic response. Endocrinology, 130, 685-694. Little, T. V. (1993). An endocrinological basis for the persistence of equine arteritis virus in the reproductive tract of the stallion. PhD Dissertation, University of Kentucky. Little, T. V., Holyoak, G. R., Timoney, E J. and McCollum, W. H. (1992). Output of equine arteritis virus from persistently infected carrier stallions is testosterone dependent. Proceedings of the 6th International Conference of Equine Infectious Diseases, Cambridge, 1991, pp. 225-229. McCollum, W. H. (1970). Vaccination for equine viral arteritis. Proceedings of the 2nd
388
W. H. McGollum et al.
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