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Pathological changes associated with equine arteritis virus infection of the reproductive tract in prepubertal and peripubertal colts
j. Comp. Path. 1993 Vol. 109, 281-293
Pathological Changes Associated with Equine Arteritis Virus Infection of the Reproductive Tract in Prepubertal and Peripubertal Colts G. R . H o l y o a k * ,
R . C. G i l e s , W. H . M c G o l l u m , a n d P. J. T i m o n e y
T. V. L i t t l e
Department of Veterinary Science, Cluck Equine Research Center, Universityof Kentucky, Lexington, KY, 40546-0099, U.S.A. Summary The nature and extent of changes associated with equine arteritis virus (EAV) infection of the reproductive tract was documented in 21 .prepubertal and 15 peripubertal colts. This study was part of an investigation mto the relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with EAV. After intranasal challenge with a field isolate of EAV, all colts developed clinical signs of equine viral arteritis (EVA) from which they recovered rapidly. Clinical signs during the acute phase consisted of fever, serous to mucopurulent ocular and nasal discharge, oedema of the limbs, scrotum or prepuce, scleral injection, conjunctivitis, icterus, cough, diarrhoea, stiff gait, lethargy, inappetence and depression. At necropsy, the most significant macroscopic lesions included excessive accumulation of fluid within the thoracic and abdominal cavities, lymph node enlargement and oedema of the reproductive tract. Colts killed 7 to 14 days after challenge had acute necrotizing vasculitis involving the testes, epididymides, vasa deferentia, ampullae, prostatic lobes, vesicular glands and bulbourethral glands. Vasculitis was characterized by striking fibrinoid necrosis of small muscular arteries with extravasation of erythroeytes and proteinaceous material into the media, adventitia and perivascular tissues. Colts examined on days 28-180 had lymphocytic and plasmacytic inflammatory cell infiltrates in the lamina propria and muscularis of the epididymides and accessory sex glands. The vascular lesions found during the acute phase of EAV infection contrasted with the multifocal lympho-plasmacytic infiltrates found within the parenchyma of the reproductive tract during the chronic phase. One peripubertal colt was found to be persistently infected with EAV 15 months after challenge. This colt had marked lympho-plasmacytic infiltrates in the ampullae at necropsy.
Introduction Equine viral arteritis (EVA) is a contagious disease first aetiologically defined in the early 1950s, when a virus was isolated during an outbreak of abortion and respiratory illness on a horse farm near Bucyrus, Ohio (Doll eL aL, 1957a,b). The virus was named equine arteritis virus (EAV) and the disease was called EVA because of the distinctive vascular lesions (Doll et al., 1957a). * Present address: Department of"Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-5600, U.S.A. 0021-9975/93/070281 + 13 $08.00/0
© 1993 Academic Press Limited
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Jones et aL (1957) were the first to describe the lesions associated with experimental EAV infection in horses. Medial necrosis of small muscular arterioles followed by adventitial oedema and infiltration of lymphocytes was evident in many organs. Thrombosis occurred in the most severely affected vessels, and infarction was observed in the intestine and lung. In a later study, Prickett et al. (1973) detected oedema and congestion of lymph nodes and accumulation of pleural and peritoneal fluids early in the course of the experimentally induced disease. This accompanied the development of an acute panvasculitis, consisting of vessel wall necrosis and mild infiltration of polymorphonuclear cells (PMNs) and lymphocytes. Crawford and Henson (1973) noted a correlation between endothelial cell damage and the appearance of viral antigen within endothelial cells. Endothelial cell injury was characterized by cellular hyperplasia, cytoplasmic rounding, nuclear hyperchromatism and pyknosis. Infiltration of neutrophils in and around capillaries and thin-walled vessels occurred simultaneously with endothelial damage. The pathological changes in the reproductive tract of the mare following experimentally induced EAV abortion consist primarily of endometrial vascular damage (Coignoul and Cheville, 1984). Lesions in the reproductive tract of the acutely and persistently infected stallion have also been described (Neu, 1988). In the latter study, stallions were experimentally infected by intranasal inoculation with a field isolate of EAV obtained during the 1984 epidemic in Kentucky. Histopathological changes seen in the reproductive tract during the acute phase of infection consisted of a mild vasculitis characterized by multifocal infiltrates of inflammatory cells. Stallions that remained persistently infected longer than 90 days were reported to have focal accumulations of lymphocytes in the testes, epididymides and deferent ducts for at least 134 to 148 days after challenge. The ampullae of the vasa deferentia had extensive inflammatory infiltrates in the lamina propria. Prdiminary observations on a limited number of young horses indicated that the incidence of persistent EAV infection in sexually immature colts with antibodies to the virus was either very low or nil (McCollum and Timoney, unpublished data). Arising from these observations, an investigation of the relationship of reproductive tract maturity and susceptibility to persistent infection with EAV has already been reported (Holyoak et al. 1993). The present paper describes pathological changes observed in the prepubertal and peripubertal colts used in the previous study. Materials and M e t h o d s Horses
Colts (n=36) of mixed breeding, aged 5 to 12 months, were purchased from a commercial source. An assessment of the maturity of the reproductive tract of each animal was based on age, serum testosterone concentration and ultrasonographie findings. The colts were assigned to either a prepubertal (nos 48-61, 64--70) or a peripubertal (nos 17-31) group. Ten age-matched colts were used as controls (nos 111-118, 255, 257). All of the animals were confirmed as seronegative to EAV and equine infectious anaemia virus before inoculation with EAV.
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Virus Inoculum
T h e challenge inoculum was prepared from a strain of EAV similar to that used by Neu et al. (1988), isolated from Thoroughbred stallions during the 1984 epidemic in Kentucky (McCollum and Timoney, 1985). Animal Inoculation
Colts in each experimental group were challenged intranasopharyngeaUy on day 0 with 5 ml of a 10 per cent splenic tissue suspension administered by fenestrated nasal catheter. The inoculum contained ca 104 plaque forming units (PFU) per ml, both before challenge and after inoculation. Experimental Design
The study consisted of two experiments. The first, on peripubertal colts, extended from March 1990 to J u n e 1991. The second experiment, on prepubertal colts, extended from October 1990 to May 1991. Monitoring of body temperature and accessory sex gland'dimensions, and collection of serum and nasopharyngeal swabs from each group of animals began one day before challenge and continued through the acute phase of infection to day 28 after challenge. All colts were examined daily at ca 8.00 am and 4.00 pm for clinical signs of infection. The amount of peripubertal development of the colts was monitored monthly in both experimental groups by ultrasonographic examination (Little and Woods, 1987) and determination of serum testosterone concentrations (Holyoak, Little, Vernon, McCollum and Timoney, unpublished data). In the first experiment (peripubertal colts) two animals were killed on days 7, 60, and 120 after challenge and tissues from the reproductive tract and non-reproductive sites, as well as various body fluids, were harvested for attempted virus isolation and, in the case of tissues, for histopathoiogical examination. The remaining nine colts were held until it was feasible to collect semen samples for virus isolation; they were subsequently killed ca 15 months after challenge. In the second experiment (prepubertal colts) two animals were killed on days 7, 14, 28, 56, 93, I 19, 150 and 180 after challenge. The remaining five colts were killed on ca day 210. All colts were killed by the intravenous injection of sodium pentobarbital solution (Beuthanasia, Schering) at a dosage of 1 ml per 10 lb body weight. After this overdose, the major cervical vessels were transected. Tissues and body fluids were collected as soon after death as possible. Collection and Processing of Specimens for Histological Examination A range of tissues was collected from each prepubertal and peripubertal colt at necropsy, Reproductive tract tissues comprised right and left testes, epididymides, vasa deferentia, ampullae, vesicular glands, prostatic lobes, bulbourethral glands and proximal and distal urethra. Non-reproductive tissues included lung, liver, spleen, kidneys, ileum, and inguinal, mesenteric, colonic, splenic and bronchial lymph nodes. Specimens were collected from the reproductive tract in the following order: bulbourethral glands, prostate, vesicular glands (carefully avoiding the peMe urethra), and then the ampullae, deferent ducts, epididymides and testes. Specific sites sampled in the reproductive tract included the following: anterior, middle and posterior testis; head, body and tail of the epididymis; proximal, middle and distal vas deferens; proximal, middle and distal ampulla; vesicular glands; prostate; bulbourethral glands; and the pelvic and penile urethra, as well as urethral glands and uterus masculinus where discernible. Both right and left sides were sampled in the case of paired organs.
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Tissue specimens for light microscopy were fixed in 10 volumes of 10 per cent neutral buffered formalin. Fixed specimens were dehydrated through a graded series of alcohols, embedded in paraffin wax, and sectioned at 5 gm and stained with haematoxylin and eosin (HE). Results
All of the challenged prepubertal and peripubertal colts became infected with EAV and developed clinical signs of EVA consisting primarily of fever, varying degrees of limb and scrotal oedema, oculonasal discharge and conjunctivitis.
Gross Pathological Findings External examination of the four colts killed on day 7 after challenge revealed subcutaneous oedema of the hind limbs and scrotum, oculonasal discharge, and conjunctivitis. One of the two colts killed on day 14 had hind limb and scrotal oedema. Increased straw-coloured fluid was present in the thoracic (300-500 ml) and abdominal (1000 ml) cavities of the four colts killed on day 7. Engorgement of visceral lymphatics with concomitant lymph node enlargement involving principally the inguinal, colonic, splenic, bronchial a n d mesenteric lymph nodes was evident. Two of these colts had congestion of the vessels of the small intestine, primarily the duodenum and jejunum. O f the six colts killed within the first 14 days after challenge, all had an accumulation of clear yellow to sero-sanguinous fluid between the visceral and parietal vaginal tunics of the testes. Clear yellow fluid was also found in the scrotum, external to the tunics. In three of these colts, the testicular parenchyma was oedematous. There was marked oedema of the head of each epididymis in both peripubertal colts on day 7. All four of the prepubertal colts, two from d a y 7 and two from day 14, had mild to moderate oedema of the epididymides. Gross changes were not detected in prepubertal or peripubertal colts killed after d a y 14.
Histopathological Findings in the Reproductive Tract Testis. Colts killed on day 7 had a necrotizing vasculitis characterized by endothelial cell necrosis and disruption of the intima with adherence of P M N s and mononuclear ceils to the intimal surface. Inflammatory ceils infiltrated the media, adventitia and perivascular regions of small muscular arteries (Fig. 1). There was necrosis of vascular smooth muscle with karyorrhexis of cell nuclei. Vasculitis was most pronounced in the tunica albuginea; however, vessels in the deeper parenchyma were also affected. Perivascula~ and interstitial o e d e m a with lymphatic engorgement and focal haemorrhage were present between seminiferous tubules. Mild to moderate perivasculitis was seen in colts beginning on day 28. Multifocal accumulations of mononuclear cells, primarily lymphocytes and plasma cells, infiltrated the perivascular region around blood vessels in the tunica albuginea and deeper parenchyma in colts killed on days
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Fig. 1.
Fig, 2.
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'Testis of peript~bertal colt 17 on day 7. Necrotizing vasculitis present in small muscular artery located in h~terstitium between seminiferous tubules. Inflammatory cells infiltrate blood vessel wall and perivasculm" stroma. Note dilated lymphatic with interstitial oedema and inflammatory ceils distributed in stroma. HE. x 150. Testis of prepubertal colt 60 on day 28. Lymphocytes infiltrate perivaseular regions of testicular interstitium, Note inactive semlniferous tubules (cords). HE, x 100.
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28 to 210 (Fig. 2). Small focal accumulations of mononuclear cells were present in the interstitial regions of age-matched control animals.
Epididymis.
Vasculitis observed in the epididymis was more severe than that observed in the body of the testis or the other accessory sex glands. In addition to the vascular changes described in the testes, blood vessels in the epididymis exhibited striking fibrinoid necrosis of the vessel walls in the colts examined on day 7 (Fig. 3). Necrotic blood vessels often showed intensely eosinophilic staining due to extravasation of erythrocytes into the vessel wall (Fig. 3). Changes were most pronounced in the epididymal head and body. By day 28, lesions were those of a subacute to chronic vasculitis characterized by lymphoplasmacytic infiltrates around affected blood vessels, Colts examined on days 60 to 210 had focal accumulations of lymphocytes and plasma cells in the connective tissue between tubules.
Vas deferens and ampulla. Vessels running parallel to the Vasa deferentia and ampullae (proximal and distal sites) exhibited necrotizing vasculitis similar to that described in the testes and epididymis of colts examined on days 7 and 14 (Fig. 4). By day 28, multifocal perivascular infiltrates of lymphocytes and plasma cells were seen around parallel subadventitial blood vessels and vessels within the muscularis and lamina propria (Fig. 5). Large lympho-plasmacytic loci were present in the lamina propria of the ampulla on day 60 (Fig. 6). Similar inflammatory cell infiltrates were observed in colts examined between days 60 and 210 (Fig. 7). Control colts had focal lympho-plasmacytic infiltrates in the lamina propria of the vas deferens and the ampulla, but increased numbers of inflammatory cells were present in the lamina propria of infected colts. Of the nine peripubertal colts killed 15 months after challenge, only colt 29 had significantly more mononuclear cell infiltrates than were observed in the age-matched controls. This colt had no observable lesions in the vasa deferentia, but there was dense plasma cell infiltration immediately beneath the luminal epithelium in the ampullae (Fig. 8). Additionally, there were large focal [ympho-plasmacytic infiltrates deeper within the secretory parenchyma. This was the only colt found to be persistently infected with EAV when killed (Holyoak et al., 1993). Vesicular glands. The vesicular glands were less affected than testes, epididymides, vasa deferentia or ampullae. Lesions observed in colts killed on days 7 and 14 consisted of mild vascular degeneration with mononuclear cell infiltration around a few small muscular arteries. One of the prepubertal colts killed •on day 7 had more intense vasculitis with fibrinoid necrosis similar to that seen in the proximal reproductive tract. There were no significant changes in the vesicular glands from colts examined between days 28 and 210. Prostate.
The findings were similar to those observed in the vesicular glands of colts examined on days 7 and 14. Increased lympho-plasmacytic perivascular
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Fig. 3. Fig. 4.
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Epididymis (head); peripubertal colt 19 on post-challenge day 7. Marked fibrlnoid necrosis present in blood vessel walls. Leucocytes and erythroeytes are also present in vessel wall. Note dilated lymphatics and oedema in stroma. HE. x 200. Ampulla ofprepubertal colt 54 on clay 7. Blood vessel exhibits vascular necrosis with infihration of leucocytes into the media. HE. x 200.
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Fig. 5. Fig+ 6.
G . R . H o l y o a k e t al,
Vas det~rens ofprepubertaI colt 69 on day 28, Inflammatory celts ;,ufiltrate outer edge o[" the vasal muscu[aris. HE. × 100. Ampulla o|'peripubertal colt 28 on clay 60. Large numbers ofinflamnlatory cells infiltrate between glandular structures. Focal aggregates of cells infiltrate the nauseularis. HE. × 15.
Pathology of Equine Arteritis Infection
l"ig.
7.
Fig. 8.
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Ampulla of prepubertal colt 51 on day 150. Lympho-plasmacytic infiltrate located in lamina propria just above the muscularly. HE. x 150. Ampulla of persistently infected colt 29 at 15 months after challenge (A) and non-persistently inl~cted colt 27 also at 15 momhs after challenge (B). In (A), inflammatory cells infiltrate lamina propria beneath the luminal epithelium. FIE. x 200.
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Fig, 9.
G . R . H o l y o a k e t al.
Bulbouretl~ral gland ofperipubertal colt 19 on day 7, Ocdema separates skeletal muscle fibres oftbe bulbourethralis muscle, Note fibrinoid necrosis of small muscular artery with e×travasation of erythrocytes into the media. HE. x 250.
infiltrates were present in colts at day 14. There were no significant changes after day 28, except in prostate glands from colts examined at days 120 to 210; these contained increased numbers of lymphocytes and plasma ceils in the subnmcosa when compared with age-matched controls.
Bulbourethralgfands. Colts killed on day 7 exhibited fibrinoid necrosis of blood vessels with extravasation of erythrocytes into the media and adventitia of small muscular arteries of the bulbourethralis muscle (Fig. 9). Skeletal muscle fibres were separated by oedematous fluid. Colts examined on day 14 had less severe lesions and changes were similar to those found in the prostate and vesicular glands on day 14. Bulbourethral glands from colts examined after day 14 did not differ significantly from age-matched controls except for a single colt on day 210, which had several large lympho-plasmacytic infiltrates within the parenchyma of the bulbourethral glands.
Urethra. In colts killed on day 7, small vessels in the lamina propria just beneath the transitional epithelium of the pelvic and penile urethra had swollen endothelial cells and mild inflammatory changes. Significant changes were not observed after day 7.
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Histological Findings in Non-Reproductive Tracl Tissues" Spleen. Splenic lymphoid follicles were enlarged and exhibited lymphoid hyperplasia on days 7 to 28. Lymph Nodes. The inguinal, mesenteric, colonic, splenic and bronchial lymph nodes exhibited lymphoid follicular hyperplasia on day 7. A single colt killed on day 14 had mild vasculitis characterized by lymphocytic infiltration into the media and periadventitia of several small blood vessels adjacent to the mesenteric lymph node. Lung. Colts killed on day 7 had mild vasculitis characterized by swollen endothelial cells with focal areas of necrosis within the media of small muscular arteries. Mononuclear cells lightly infiltrated the media and adventitia. By day 14, there were increased numbers of lymphocytes and plasma cells in the adventitia of small blood vessels. Bronchiole-associated lymphoid tissue was increased compared with that observed in the controls. Liver. Colts killed on days 7 and 14 had mild vasculitis and perivascular oedema in the portal regions of the liver. Small aggregates of PMNs were distributed within the parenchyma. Kidney. Increased numbers of PMNs were lightly dispersed in the glomerular capillaries in colts killed on days 7 and 14. Discussion
All of the challenged prepubertal and peripubertal colts became infected with EAV and developed clinical signs of EVA. The clinical manifestations of EVA observed in this study included: fever, serous to mucopurulent ocular and nasal discharge, dependant oedema of the limbs and scrotum or prepuce, scleral injection, conjunctivitis, icterus, cough, diarrhoea, stiff gait, lethargy, inappetence and depression. The clinical signs recorded in this study were very similar to those reported by others (Doll et al., 1957a; McCollurn and Timoney, 1985; Neu, 1988). The lesions found in both the prepubertal and peripubertal cotts were similar to those reported in previous studies (Jones el al., 1957; Estes and Cheville, 1970; Prickett et al., 1973) in which lesions were not described in the reproductive tract. The acute microscopic lesions ranged fi'om mild inflammatory cell infiltration immediately adjacent to small blood vessels, to inflammatory interstitial oedema and necrotizing vasculitis in both groups of colts. Lesions observed in the reproductive tract during the acute phase of infection are a result of viral replication and lysis of endothelial cells. This was the conclusion reached in two earlier studies (Crawford and Henson, 1973; Henson and Crawford, 1974), which demonstrated the presence of viral antigen and the lack of immune complexes associated with lesions during the acute phase of infection. The vascular lesions found in the reproductive tract of the colts in the present study differed from those described in a study of
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experimental infection in stallions with the same strain of EAV (Neu, 1988). T h e lesions found in the reproductive tract of the stallion during the acute phase of infection were less severe than those found in the colts, but closely similar to those observed in the colts during the chronic phase of infection. During the chronic phase the most prominent microscopic lesions in the reproductive tract were multifocal lympho-plasmacytic infiltrates in the stroma adjacent to blood vessels or glandular structures in both prepubertal and peripubertal colts. There seemed to be a trend for the focal inflammatory cell infiltrates to persist over time in the parenchyma and adluminally in the vas deferens and ampulla. The infiltrates consisted primarily of plasma cells, with smaller numbers of lymphocytes representing the overall cell population. All five prepubertal colts killed by day 210 had cleared the virus from the reproductive tract (Holyoak et al., 1993), and four of five had few residual lesions in that site. The other prepubertal colt, however, had a relatively high number of focal infiltrates in every reproductive tract tissue sampled. The exact time of viral clearance is not known and this colt was the least sexually mature animal of the prepubertal group. It is not known whether the lack of tract maturity modulated the inflammatory response or whether the lesions reflected recent viral clearance, Of the peripubertal colts killed 15 months after challenge only one had distinct lesions in the reproductive tract, and this animal was also persistently infected with EAV (Holyoak et al., 1993). While only a single animal continued to harbour EAV for the duration of the experiment, the presence of distinct histological lesions appeared to be related to viral persistence. The peripubertal colt that was persistently infected had multiple subluminal plasmacytic infiltrates, as well as focal infiltrates, mostly of plasma ceils, deeper in the secretory parenchyma of the ampullae. These Iesions were much more apparent than those observed in sections from the other eight colts challenged at the same time. The lesions seen in this colt were similar to those observed in tissue sections of ampulla from naturally occurring, long-term carrier stallions (Timoney, McCollum and Little, unpublished), in which the virus also appears to have been localized primarily in the ampulla (Little et al., 1992). The large numbers of focal plasmacytic infiltrates noted in the reproductive tract of the persistently infected colt indicated that antigenic stimulation continues to occur in the affected tissues. Such plasma cells may assist in boosting the systemic anti-EAV titre found in these animals, or perhaps even play a role in viral persistence (Mims, 1989; Oldstone, 1989). Additional studies are needed to determine whether or not the changes in distribution and extent of the histopathological lesions over the course of infection are associated with the localization and establishment of persistent EAV infection in the reproductive tract of the colt.
Acknowledgments The assistance of Dr T. W. Swerczek and Mr K. Boll in conducting the many neeropsies is gratefully acknowledged. Thanks are also due to the:histology staff at the University of Kentucky Livestock Disease Diagrtosis Center for assisting in slide preparation, Support for this study was provided by the Frederick Van Lennep
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Professorship Endowment Fund. This paper (93-4-63) is punished by permission of the Dean and Director, College of Agriculture and Kentucky Experiment Station. References
Coignoul, F. L. and Cheville, N. F. (1984). Pathology of maternal genital tract, placenta, and fetus in equine viral arteritis. Veterinary Pathology, 21, 333-340. Crawford, T. B. and Henson, J. B. (1973). Immunofluorescent, light microscopic and immunologic studies of equine viral arteritis. In Proceedings of the 3rd International Conference on Equine Infectious Diseases, J. T. Bryans and H. Gerber, Eds, S. Karger, Basel, pp. 282-302. Doll, E. R., Bryans, J. T., McCollum, W. H. and Crowe, M. E. W. (1957a). Isolation of a filterable agent causing arteritis of horses and abortion by mares. Its differentiation from equine abortion (influenza) virus. Cornell Veterinarian, 47, 3-41. Doll, E. R., Knappenberger, S. and Bryans, J. T. (1957b). An outbreak of abortion caused by equine arteritis virus. Cornell Veterinarian, 47, 69-75. Estes, P. C. and Cheville, N. F. (1970). The ultrastructure of vascular lesions in equine viral arteritis. American Journal of Pathology, 58, 235-253. Henson, J. B. and Crawford, T. B. (1974). The pathogenesis of virus-induced arterial disease. Aleutian disease and equine arteritis. In: Advanced Cardiology, Vol. 13, F. Homberger and I. Lueas, Eds, S. Karger, Basel, pp. 183-191. Holyoak, G. R., Little, T. V., McCollum, W. H. and Timoney, P.J. (1993). The relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt. Journal of Comparative Pathology, 109, 29-46. Jones, T. C., Doll, E. R. and Bryans, J. T. (1957). The lesions of equine viral arteritis. CornelI Veterinarian, 47, 52-68. Little, T. V., Holyoak, G. R., Timoney, P. J. and McCollum, W. H. (1992). Equine arteritis virus output in the semen of chronically infected carrier stallions is testosterone dependent. Proceedings of the 6th International Conference on Equine Infectious Diseases, Cambridge, UK, 1991, pp. 225-229. Little, T. V. and Woods, G. L. (1987). Ultrasonography of the accessory sex glands in the stallion, journal of Reproduction and Fertility, Supplement, 35, 87-92. McCollum, W. H. and Timoney, P.J. (1985). The pathogenic qualities of the 1984 strain of equine arteritis virus. In: Proceedings of the Grayson Foundation International Conference of Thoroughbred Breeders ONankation on Equine Viral Arterilis, Grayson Found. Inc., Lexington, KY, pp. 34-37. Mires, C. A. (1989). The pathogenetic basis of viral tropism. American Journal of Pat/7ology, 135, 44.7-455. Neu, S. M. (1988). A study of the pathogenesis and pathologic ett?cts of persistent infection with equine arteritis virus in experimentally infected stallions. Ph.D. Thesis. University of Kentucky, pp. 150-200. Neu, S. M., Timoney, P.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 Disease.s; Lexington, KY, pp. 149-154. Otdstone, M. B. A. (1989). Viral alteration of cell function. Scientific American, August, pp. 42-48. Prickett, M. E., McCollum, W. H. and Bryans, J. T. (1973). The gross and microscopic pathology observed in horses experimentally infected with equine arteritis virus. In: Proceedings of the 3rd International Conference on Equine IT!/'eclious Diseases, J. T. Bryans and H. Gerber, Eds, S. Kargcr, Basel, pp. 265-272.
April 28lh, ~Received, Accepted, July 15th,
I993-] 1993J
Pathological Changes Associated with Equine Arteritis Virus Infection of the Reproductive Tract in Prepubertal and Peripubertal Colts G. R . H o l y o a k * ,
R . C. G i l e s , W. H . M c G o l l u m , a n d P. J. T i m o n e y
T. V. L i t t l e
Department of Veterinary Science, Cluck Equine Research Center, Universityof Kentucky, Lexington, KY, 40546-0099, U.S.A. Summary The nature and extent of changes associated with equine arteritis virus (EAV) infection of the reproductive tract was documented in 21 .prepubertal and 15 peripubertal colts. This study was part of an investigation mto the relationship between stage of reproductive tract maturity and susceptibility to the experimental establishment of persistent infection with EAV. After intranasal challenge with a field isolate of EAV, all colts developed clinical signs of equine viral arteritis (EVA) from which they recovered rapidly. Clinical signs during the acute phase consisted of fever, serous to mucopurulent ocular and nasal discharge, oedema of the limbs, scrotum or prepuce, scleral injection, conjunctivitis, icterus, cough, diarrhoea, stiff gait, lethargy, inappetence and depression. At necropsy, the most significant macroscopic lesions included excessive accumulation of fluid within the thoracic and abdominal cavities, lymph node enlargement and oedema of the reproductive tract. Colts killed 7 to 14 days after challenge had acute necrotizing vasculitis involving the testes, epididymides, vasa deferentia, ampullae, prostatic lobes, vesicular glands and bulbourethral glands. Vasculitis was characterized by striking fibrinoid necrosis of small muscular arteries with extravasation of erythroeytes and proteinaceous material into the media, adventitia and perivascular tissues. Colts examined on days 28-180 had lymphocytic and plasmacytic inflammatory cell infiltrates in the lamina propria and muscularis of the epididymides and accessory sex glands. The vascular lesions found during the acute phase of EAV infection contrasted with the multifocal lympho-plasmacytic infiltrates found within the parenchyma of the reproductive tract during the chronic phase. One peripubertal colt was found to be persistently infected with EAV 15 months after challenge. This colt had marked lympho-plasmacytic infiltrates in the ampullae at necropsy.
Introduction Equine viral arteritis (EVA) is a contagious disease first aetiologically defined in the early 1950s, when a virus was isolated during an outbreak of abortion and respiratory illness on a horse farm near Bucyrus, Ohio (Doll eL aL, 1957a,b). The virus was named equine arteritis virus (EAV) and the disease was called EVA because of the distinctive vascular lesions (Doll et al., 1957a). * Present address: Department of"Animal, Dairy and Veterinary Sciences, Utah State University, Logan, UT 84322-5600, U.S.A. 0021-9975/93/070281 + 13 $08.00/0
© 1993 Academic Press Limited
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ctl.
Jones et aL (1957) were the first to describe the lesions associated with experimental EAV infection in horses. Medial necrosis of small muscular arterioles followed by adventitial oedema and infiltration of lymphocytes was evident in many organs. Thrombosis occurred in the most severely affected vessels, and infarction was observed in the intestine and lung. In a later study, Prickett et al. (1973) detected oedema and congestion of lymph nodes and accumulation of pleural and peritoneal fluids early in the course of the experimentally induced disease. This accompanied the development of an acute panvasculitis, consisting of vessel wall necrosis and mild infiltration of polymorphonuclear cells (PMNs) and lymphocytes. Crawford and Henson (1973) noted a correlation between endothelial cell damage and the appearance of viral antigen within endothelial cells. Endothelial cell injury was characterized by cellular hyperplasia, cytoplasmic rounding, nuclear hyperchromatism and pyknosis. Infiltration of neutrophils in and around capillaries and thin-walled vessels occurred simultaneously with endothelial damage. The pathological changes in the reproductive tract of the mare following experimentally induced EAV abortion consist primarily of endometrial vascular damage (Coignoul and Cheville, 1984). Lesions in the reproductive tract of the acutely and persistently infected stallion have also been described (Neu, 1988). In the latter study, stallions were experimentally infected by intranasal inoculation with a field isolate of EAV obtained during the 1984 epidemic in Kentucky. Histopathological changes seen in the reproductive tract during the acute phase of infection consisted of a mild vasculitis characterized by multifocal infiltrates of inflammatory cells. Stallions that remained persistently infected longer than 90 days were reported to have focal accumulations of lymphocytes in the testes, epididymides and deferent ducts for at least 134 to 148 days after challenge. The ampullae of the vasa deferentia had extensive inflammatory infiltrates in the lamina propria. Prdiminary observations on a limited number of young horses indicated that the incidence of persistent EAV infection in sexually immature colts with antibodies to the virus was either very low or nil (McCollum and Timoney, unpublished data). Arising from these observations, an investigation of the relationship of reproductive tract maturity and susceptibility to persistent infection with EAV has already been reported (Holyoak et al. 1993). The present paper describes pathological changes observed in the prepubertal and peripubertal colts used in the previous study. Materials and M e t h o d s Horses
Colts (n=36) of mixed breeding, aged 5 to 12 months, were purchased from a commercial source. An assessment of the maturity of the reproductive tract of each animal was based on age, serum testosterone concentration and ultrasonographie findings. The colts were assigned to either a prepubertal (nos 48-61, 64--70) or a peripubertal (nos 17-31) group. Ten age-matched colts were used as controls (nos 111-118, 255, 257). All of the animals were confirmed as seronegative to EAV and equine infectious anaemia virus before inoculation with EAV.
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Virus Inoculum
T h e challenge inoculum was prepared from a strain of EAV similar to that used by Neu et al. (1988), isolated from Thoroughbred stallions during the 1984 epidemic in Kentucky (McCollum and Timoney, 1985). Animal Inoculation
Colts in each experimental group were challenged intranasopharyngeaUy on day 0 with 5 ml of a 10 per cent splenic tissue suspension administered by fenestrated nasal catheter. The inoculum contained ca 104 plaque forming units (PFU) per ml, both before challenge and after inoculation. Experimental Design
The study consisted of two experiments. The first, on peripubertal colts, extended from March 1990 to J u n e 1991. The second experiment, on prepubertal colts, extended from October 1990 to May 1991. Monitoring of body temperature and accessory sex gland'dimensions, and collection of serum and nasopharyngeal swabs from each group of animals began one day before challenge and continued through the acute phase of infection to day 28 after challenge. All colts were examined daily at ca 8.00 am and 4.00 pm for clinical signs of infection. The amount of peripubertal development of the colts was monitored monthly in both experimental groups by ultrasonographic examination (Little and Woods, 1987) and determination of serum testosterone concentrations (Holyoak, Little, Vernon, McCollum and Timoney, unpublished data). In the first experiment (peripubertal colts) two animals were killed on days 7, 60, and 120 after challenge and tissues from the reproductive tract and non-reproductive sites, as well as various body fluids, were harvested for attempted virus isolation and, in the case of tissues, for histopathoiogical examination. The remaining nine colts were held until it was feasible to collect semen samples for virus isolation; they were subsequently killed ca 15 months after challenge. In the second experiment (prepubertal colts) two animals were killed on days 7, 14, 28, 56, 93, I 19, 150 and 180 after challenge. The remaining five colts were killed on ca day 210. All colts were killed by the intravenous injection of sodium pentobarbital solution (Beuthanasia, Schering) at a dosage of 1 ml per 10 lb body weight. After this overdose, the major cervical vessels were transected. Tissues and body fluids were collected as soon after death as possible. Collection and Processing of Specimens for Histological Examination A range of tissues was collected from each prepubertal and peripubertal colt at necropsy, Reproductive tract tissues comprised right and left testes, epididymides, vasa deferentia, ampullae, vesicular glands, prostatic lobes, bulbourethral glands and proximal and distal urethra. Non-reproductive tissues included lung, liver, spleen, kidneys, ileum, and inguinal, mesenteric, colonic, splenic and bronchial lymph nodes. Specimens were collected from the reproductive tract in the following order: bulbourethral glands, prostate, vesicular glands (carefully avoiding the peMe urethra), and then the ampullae, deferent ducts, epididymides and testes. Specific sites sampled in the reproductive tract included the following: anterior, middle and posterior testis; head, body and tail of the epididymis; proximal, middle and distal vas deferens; proximal, middle and distal ampulla; vesicular glands; prostate; bulbourethral glands; and the pelvic and penile urethra, as well as urethral glands and uterus masculinus where discernible. Both right and left sides were sampled in the case of paired organs.
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Tissue specimens for light microscopy were fixed in 10 volumes of 10 per cent neutral buffered formalin. Fixed specimens were dehydrated through a graded series of alcohols, embedded in paraffin wax, and sectioned at 5 gm and stained with haematoxylin and eosin (HE). Results
All of the challenged prepubertal and peripubertal colts became infected with EAV and developed clinical signs of EVA consisting primarily of fever, varying degrees of limb and scrotal oedema, oculonasal discharge and conjunctivitis.
Gross Pathological Findings External examination of the four colts killed on day 7 after challenge revealed subcutaneous oedema of the hind limbs and scrotum, oculonasal discharge, and conjunctivitis. One of the two colts killed on day 14 had hind limb and scrotal oedema. Increased straw-coloured fluid was present in the thoracic (300-500 ml) and abdominal (1000 ml) cavities of the four colts killed on day 7. Engorgement of visceral lymphatics with concomitant lymph node enlargement involving principally the inguinal, colonic, splenic, bronchial a n d mesenteric lymph nodes was evident. Two of these colts had congestion of the vessels of the small intestine, primarily the duodenum and jejunum. O f the six colts killed within the first 14 days after challenge, all had an accumulation of clear yellow to sero-sanguinous fluid between the visceral and parietal vaginal tunics of the testes. Clear yellow fluid was also found in the scrotum, external to the tunics. In three of these colts, the testicular parenchyma was oedematous. There was marked oedema of the head of each epididymis in both peripubertal colts on day 7. All four of the prepubertal colts, two from d a y 7 and two from day 14, had mild to moderate oedema of the epididymides. Gross changes were not detected in prepubertal or peripubertal colts killed after d a y 14.
Histopathological Findings in the Reproductive Tract Testis. Colts killed on day 7 had a necrotizing vasculitis characterized by endothelial cell necrosis and disruption of the intima with adherence of P M N s and mononuclear ceils to the intimal surface. Inflammatory ceils infiltrated the media, adventitia and perivascular regions of small muscular arteries (Fig. 1). There was necrosis of vascular smooth muscle with karyorrhexis of cell nuclei. Vasculitis was most pronounced in the tunica albuginea; however, vessels in the deeper parenchyma were also affected. Perivascula~ and interstitial o e d e m a with lymphatic engorgement and focal haemorrhage were present between seminiferous tubules. Mild to moderate perivasculitis was seen in colts beginning on day 28. Multifocal accumulations of mononuclear cells, primarily lymphocytes and plasma cells, infiltrated the perivascular region around blood vessels in the tunica albuginea and deeper parenchyma in colts killed on days
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Fig. 1.
Fig, 2.
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'Testis of peript~bertal colt 17 on day 7. Necrotizing vasculitis present in small muscular artery located in h~terstitium between seminiferous tubules. Inflammatory cells infiltrate blood vessel wall and perivasculm" stroma. Note dilated lymphatic with interstitial oedema and inflammatory ceils distributed in stroma. HE. x 150. Testis of prepubertal colt 60 on day 28. Lymphocytes infiltrate perivaseular regions of testicular interstitium, Note inactive semlniferous tubules (cords). HE, x 100.
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28 to 210 (Fig. 2). Small focal accumulations of mononuclear cells were present in the interstitial regions of age-matched control animals.
Epididymis.
Vasculitis observed in the epididymis was more severe than that observed in the body of the testis or the other accessory sex glands. In addition to the vascular changes described in the testes, blood vessels in the epididymis exhibited striking fibrinoid necrosis of the vessel walls in the colts examined on day 7 (Fig. 3). Necrotic blood vessels often showed intensely eosinophilic staining due to extravasation of erythrocytes into the vessel wall (Fig. 3). Changes were most pronounced in the epididymal head and body. By day 28, lesions were those of a subacute to chronic vasculitis characterized by lymphoplasmacytic infiltrates around affected blood vessels, Colts examined on days 60 to 210 had focal accumulations of lymphocytes and plasma cells in the connective tissue between tubules.
Vas deferens and ampulla. Vessels running parallel to the Vasa deferentia and ampullae (proximal and distal sites) exhibited necrotizing vasculitis similar to that described in the testes and epididymis of colts examined on days 7 and 14 (Fig. 4). By day 28, multifocal perivascular infiltrates of lymphocytes and plasma cells were seen around parallel subadventitial blood vessels and vessels within the muscularis and lamina propria (Fig. 5). Large lympho-plasmacytic loci were present in the lamina propria of the ampulla on day 60 (Fig. 6). Similar inflammatory cell infiltrates were observed in colts examined between days 60 and 210 (Fig. 7). Control colts had focal lympho-plasmacytic infiltrates in the lamina propria of the vas deferens and the ampulla, but increased numbers of inflammatory cells were present in the lamina propria of infected colts. Of the nine peripubertal colts killed 15 months after challenge, only colt 29 had significantly more mononuclear cell infiltrates than were observed in the age-matched controls. This colt had no observable lesions in the vasa deferentia, but there was dense plasma cell infiltration immediately beneath the luminal epithelium in the ampullae (Fig. 8). Additionally, there were large focal [ympho-plasmacytic infiltrates deeper within the secretory parenchyma. This was the only colt found to be persistently infected with EAV when killed (Holyoak et al., 1993). Vesicular glands. The vesicular glands were less affected than testes, epididymides, vasa deferentia or ampullae. Lesions observed in colts killed on days 7 and 14 consisted of mild vascular degeneration with mononuclear cell infiltration around a few small muscular arteries. One of the prepubertal colts killed •on day 7 had more intense vasculitis with fibrinoid necrosis similar to that seen in the proximal reproductive tract. There were no significant changes in the vesicular glands from colts examined between days 28 and 210. Prostate.
The findings were similar to those observed in the vesicular glands of colts examined on days 7 and 14. Increased lympho-plasmacytic perivascular
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Fig. 3. Fig. 4.
287
Epididymis (head); peripubertal colt 19 on post-challenge day 7. Marked fibrlnoid necrosis present in blood vessel walls. Leucocytes and erythroeytes are also present in vessel wall. Note dilated lymphatics and oedema in stroma. HE. x 200. Ampulla ofprepubertal colt 54 on clay 7. Blood vessel exhibits vascular necrosis with infihration of leucocytes into the media. HE. x 200.
288
Fig. 5. Fig+ 6.
G . R . H o l y o a k e t al,
Vas det~rens ofprepubertaI colt 69 on day 28, Inflammatory celts ;,ufiltrate outer edge o[" the vasal muscu[aris. HE. × 100. Ampulla o|'peripubertal colt 28 on clay 60. Large numbers ofinflamnlatory cells infiltrate between glandular structures. Focal aggregates of cells infiltrate the nauseularis. HE. × 15.
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l"ig.
7.
Fig. 8.
289
Ampulla of prepubertal colt 51 on day 150. Lympho-plasmacytic infiltrate located in lamina propria just above the muscularly. HE. x 150. Ampulla of persistently infected colt 29 at 15 months after challenge (A) and non-persistently inl~cted colt 27 also at 15 momhs after challenge (B). In (A), inflammatory cells infiltrate lamina propria beneath the luminal epithelium. FIE. x 200.
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Fig, 9.
G . R . H o l y o a k e t al.
Bulbouretl~ral gland ofperipubertal colt 19 on day 7, Ocdema separates skeletal muscle fibres oftbe bulbourethralis muscle, Note fibrinoid necrosis of small muscular artery with e×travasation of erythrocytes into the media. HE. x 250.
infiltrates were present in colts at day 14. There were no significant changes after day 28, except in prostate glands from colts examined at days 120 to 210; these contained increased numbers of lymphocytes and plasma ceils in the subnmcosa when compared with age-matched controls.
Bulbourethralgfands. Colts killed on day 7 exhibited fibrinoid necrosis of blood vessels with extravasation of erythrocytes into the media and adventitia of small muscular arteries of the bulbourethralis muscle (Fig. 9). Skeletal muscle fibres were separated by oedematous fluid. Colts examined on day 14 had less severe lesions and changes were similar to those found in the prostate and vesicular glands on day 14. Bulbourethral glands from colts examined after day 14 did not differ significantly from age-matched controls except for a single colt on day 210, which had several large lympho-plasmacytic infiltrates within the parenchyma of the bulbourethral glands.
Urethra. In colts killed on day 7, small vessels in the lamina propria just beneath the transitional epithelium of the pelvic and penile urethra had swollen endothelial cells and mild inflammatory changes. Significant changes were not observed after day 7.
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Histological Findings in Non-Reproductive Tracl Tissues" Spleen. Splenic lymphoid follicles were enlarged and exhibited lymphoid hyperplasia on days 7 to 28. Lymph Nodes. The inguinal, mesenteric, colonic, splenic and bronchial lymph nodes exhibited lymphoid follicular hyperplasia on day 7. A single colt killed on day 14 had mild vasculitis characterized by lymphocytic infiltration into the media and periadventitia of several small blood vessels adjacent to the mesenteric lymph node. Lung. Colts killed on day 7 had mild vasculitis characterized by swollen endothelial cells with focal areas of necrosis within the media of small muscular arteries. Mononuclear cells lightly infiltrated the media and adventitia. By day 14, there were increased numbers of lymphocytes and plasma cells in the adventitia of small blood vessels. Bronchiole-associated lymphoid tissue was increased compared with that observed in the controls. Liver. Colts killed on days 7 and 14 had mild vasculitis and perivascular oedema in the portal regions of the liver. Small aggregates of PMNs were distributed within the parenchyma. Kidney. Increased numbers of PMNs were lightly dispersed in the glomerular capillaries in colts killed on days 7 and 14. Discussion
All of the challenged prepubertal and peripubertal colts became infected with EAV and developed clinical signs of EVA. The clinical manifestations of EVA observed in this study included: fever, serous to mucopurulent ocular and nasal discharge, dependant oedema of the limbs and scrotum or prepuce, scleral injection, conjunctivitis, icterus, cough, diarrhoea, stiff gait, lethargy, inappetence and depression. The clinical signs recorded in this study were very similar to those reported by others (Doll et al., 1957a; McCollurn and Timoney, 1985; Neu, 1988). The lesions found in both the prepubertal and peripubertal cotts were similar to those reported in previous studies (Jones el al., 1957; Estes and Cheville, 1970; Prickett et al., 1973) in which lesions were not described in the reproductive tract. The acute microscopic lesions ranged fi'om mild inflammatory cell infiltration immediately adjacent to small blood vessels, to inflammatory interstitial oedema and necrotizing vasculitis in both groups of colts. Lesions observed in the reproductive tract during the acute phase of infection are a result of viral replication and lysis of endothelial cells. This was the conclusion reached in two earlier studies (Crawford and Henson, 1973; Henson and Crawford, 1974), which demonstrated the presence of viral antigen and the lack of immune complexes associated with lesions during the acute phase of infection. The vascular lesions found in the reproductive tract of the colts in the present study differed from those described in a study of
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experimental infection in stallions with the same strain of EAV (Neu, 1988). T h e lesions found in the reproductive tract of the stallion during the acute phase of infection were less severe than those found in the colts, but closely similar to those observed in the colts during the chronic phase of infection. During the chronic phase the most prominent microscopic lesions in the reproductive tract were multifocal lympho-plasmacytic infiltrates in the stroma adjacent to blood vessels or glandular structures in both prepubertal and peripubertal colts. There seemed to be a trend for the focal inflammatory cell infiltrates to persist over time in the parenchyma and adluminally in the vas deferens and ampulla. The infiltrates consisted primarily of plasma cells, with smaller numbers of lymphocytes representing the overall cell population. All five prepubertal colts killed by day 210 had cleared the virus from the reproductive tract (Holyoak et al., 1993), and four of five had few residual lesions in that site. The other prepubertal colt, however, had a relatively high number of focal infiltrates in every reproductive tract tissue sampled. The exact time of viral clearance is not known and this colt was the least sexually mature animal of the prepubertal group. It is not known whether the lack of tract maturity modulated the inflammatory response or whether the lesions reflected recent viral clearance, Of the peripubertal colts killed 15 months after challenge only one had distinct lesions in the reproductive tract, and this animal was also persistently infected with EAV (Holyoak et al., 1993). While only a single animal continued to harbour EAV for the duration of the experiment, the presence of distinct histological lesions appeared to be related to viral persistence. The peripubertal colt that was persistently infected had multiple subluminal plasmacytic infiltrates, as well as focal infiltrates, mostly of plasma ceils, deeper in the secretory parenchyma of the ampullae. These Iesions were much more apparent than those observed in sections from the other eight colts challenged at the same time. The lesions seen in this colt were similar to those observed in tissue sections of ampulla from naturally occurring, long-term carrier stallions (Timoney, McCollum and Little, unpublished), in which the virus also appears to have been localized primarily in the ampulla (Little et al., 1992). The large numbers of focal plasmacytic infiltrates noted in the reproductive tract of the persistently infected colt indicated that antigenic stimulation continues to occur in the affected tissues. Such plasma cells may assist in boosting the systemic anti-EAV titre found in these animals, or perhaps even play a role in viral persistence (Mims, 1989; Oldstone, 1989). Additional studies are needed to determine whether or not the changes in distribution and extent of the histopathological lesions over the course of infection are associated with the localization and establishment of persistent EAV infection in the reproductive tract of the colt.
Acknowledgments The assistance of Dr T. W. Swerczek and Mr K. Boll in conducting the many neeropsies is gratefully acknowledged. Thanks are also due to the:histology staff at the University of Kentucky Livestock Disease Diagrtosis Center for assisting in slide preparation, Support for this study was provided by the Frederick Van Lennep
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Professorship Endowment Fund. This paper (93-4-63) is punished by permission of the Dean and Director, College of Agriculture and Kentucky Experiment Station. References
Coignoul, F. L. and Cheville, N. F. (1984). Pathology of maternal genital tract, placenta, and fetus in equine viral arteritis. Veterinary Pathology, 21, 333-340. Crawford, T. B. and Henson, J. B. (1973). Immunofluorescent, light microscopic and immunologic studies of equine viral arteritis. In Proceedings of the 3rd International Conference on Equine Infectious Diseases, J. T. Bryans and H. Gerber, Eds, S. Karger, Basel, pp. 282-302. Doll, E. R., Bryans, J. T., McCollum, W. H. and Crowe, M. E. W. (1957a). Isolation of a filterable agent causing arteritis of horses and abortion by mares. Its differentiation from equine abortion (influenza) virus. Cornell Veterinarian, 47, 3-41. Doll, E. R., Knappenberger, S. and Bryans, J. T. (1957b). An outbreak of abortion caused by equine arteritis virus. Cornell Veterinarian, 47, 69-75. Estes, P. C. and Cheville, N. F. (1970). The ultrastructure of vascular lesions in equine viral arteritis. American Journal of Pathology, 58, 235-253. Henson, J. B. and Crawford, T. B. (1974). The pathogenesis of virus-induced arterial disease. Aleutian disease and equine arteritis. In: Advanced Cardiology, Vol. 13, F. Homberger and I. Lueas, Eds, S. Karger, Basel, pp. 183-191. Holyoak, G. R., Little, T. V., McCollum, W. H. and Timoney, P.J. (1993). The relationship between onset of puberty and establishment of persistent infection with equine arteritis virus in the experimentally infected colt. Journal of Comparative Pathology, 109, 29-46. Jones, T. C., Doll, E. R. and Bryans, J. T. (1957). The lesions of equine viral arteritis. CornelI Veterinarian, 47, 52-68. Little, T. V., Holyoak, G. R., Timoney, P. J. and McCollum, W. H. (1992). Equine arteritis virus output in the semen of chronically infected carrier stallions is testosterone dependent. Proceedings of the 6th International Conference on Equine Infectious Diseases, Cambridge, UK, 1991, pp. 225-229. Little, T. V. and Woods, G. L. (1987). Ultrasonography of the accessory sex glands in the stallion, journal of Reproduction and Fertility, Supplement, 35, 87-92. McCollum, W. H. and Timoney, P.J. (1985). The pathogenic qualities of the 1984 strain of equine arteritis virus. In: Proceedings of the Grayson Foundation International Conference of Thoroughbred Breeders ONankation on Equine Viral Arterilis, Grayson Found. Inc., Lexington, KY, pp. 34-37. Mires, C. A. (1989). The pathogenetic basis of viral tropism. American Journal of Pat/7ology, 135, 44.7-455. Neu, S. M. (1988). A study of the pathogenesis and pathologic ett?cts of persistent infection with equine arteritis virus in experimentally infected stallions. Ph.D. Thesis. University of Kentucky, pp. 150-200. Neu, S. M., Timoney, P.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 Disease.s; Lexington, KY, pp. 149-154. Otdstone, M. B. A. (1989). Viral alteration of cell function. Scientific American, August, pp. 42-48. Prickett, M. E., McCollum, W. H. and Bryans, J. T. (1973). The gross and microscopic pathology observed in horses experimentally infected with equine arteritis virus. In: Proceedings of the 3rd International Conference on Equine IT!/'eclious Diseases, J. T. Bryans and H. Gerber, Eds, S. Kargcr, Basel, pp. 265-272.
April 28lh, ~Received, Accepted, July 15th,
I993-] 1993J