CASE REPORT
Equine Herpesvirus I Infection in Mares Vaccinated with a Live-virus Rhinopneumonitis Vaccine Attenuated in Cell Culture M.D. EAGLESOME, J.N.R. HENRY AND J.D. McKNIGHT*
Summarv Vaccination, in July and again in either November or December 1976, of 55 pregnant Standardbred mares with a live-virus rhinopneumonitis vaccine attenuated in cell culture failed to protect some mares from infection with equine herpesvirus 1. From 1976-12-08 to 1977-03-08, 33 mares foaled healthy foals, 16 mares foaled dead foals or live foals which died usually within 48 hours and six mares aborted. Gross and histological examinations and virus isolation studies confirmed that equine herpesvirus I caused 18 of the 22 neonatal deaths, stillbirths or abortions. Resume Infection par l'herpesvirus equin du type 1 chez des juments vaccinees contre la rhino-pneumonite avec un vaccin attenue sur culture cellulaire La vaccination de 55 juments gravides Standard bred, contre la rhino-pneumonite equine, avec un vaccin atftnue sur culture cellulaire, en juillet et de nouveau en novembre ou en decembre 1976, ne reussit pas A proteger certaines d'entre elles contre cette maladie. Du 8 decembre 1976 au 8 mars 1977, 33 juments donnerent naissance A des poulains en sante, 16 autres donnerent des poulains mort-nes ou des poulains vivants qui moururent en 1'espace de 48 heures, tandis que six autres avorterent. Les lesions macroscopiques et microscopiques, ainsi que les epreuves d'isolement du virus, demontrerent que l'herpesvirus equin du type I etait responsable de 18 des 22 mortinatalites, mortalites neonatales et avortements. Introduction Equine herpesvirus I (EHVI) is one of three antigenically distinct species of herpesvirus re-
coverable from horses (7). It causes equine rhinopneumonitis characterized by respiratory disease (4), neurological disorders (6) and both fetal and foal mortality (4). Abortions most often occur after eight months gestation. Infected term foals may be born alive, but usually die within a few hours. For prophylaxis, three live-virus vaccines have been licensed for use in Canada; one contains a hamster-adapted virus,' the other two cell culture-attenuated strains of EHVI.2'3 Vaccination is claimed to have reduced the incidence of virus abortion but it has not abolished it. This paper records the clinical and laboratory findings, during the 1976-1977 foaling season, in a group of 55 pregnant Standardbred mares vaccinated with a live-virus rhinopneumonitis vaccine attenuated in cell culture.2
Clinical Historv The farm is a large Standardbred stud where home-owned and visiting mares are stabled for breeding and/or foaling. A "planned infection" program with the hamster adapted vaccine' was practiced until January 1974, when an abortion storm occurred among vaccinated mares. Subsequently, a program of vaccinating all horses with a cell culture-attenuated vaccine' was introduced. During the first week of November 1976, 55 home-owned mares were assembled in preparation for foaling. They had been vaccinated twice in 1975 and revaccinated in July and in either November or early December 1976. The mares were stabled in three barns: a foaling barn (Barn 1), 32 mares, barn G (Barn 2), 13 mares and barn S (Barn 3), 10 mares. They were managed as a unit with no additions and all 55 mares were to foal at Barn 1. Both Barns I and 2 have stalls and loafing yards. Pregnant mares at Barn I were held in the loafing area and moved into the stalls to foal; after foaling, mares were then moved to Barn 2. Barn I and 3 were isolated, but the yard at Barn 2 was separated only by a six metre wide roadway from the loafing yard of an adjacent barn where, in the fall of 1976, barren and maiden mares were stabled. At Barn I from December 8, 1976 to January 1, 1977, 14 mares foaled and one aborted; 12 foals were born healthy, one foal died at six days and one within 24 hours after birth. No cause for the abortion or the death of the two foals was established. On January 3, 1977, two mares, moved from Barn 3 to Barn 2 on December 14, aborted; one at Barn 2 and the other, following a further move on December 30, at Barn 1. The following day, another mare, stabled at Barn 2 since before
*Animal Pathology Division, Health of Animals Branchi, Agriculture Canada, Animal Diseases Research Institute (E), Box 11300, Station H,Ottawa, Ontario K2H 8P9(Eaglesome), Veterinary Services Laboratory, Ontario Ministry of Agriculture and Food, Guelph, Ontario(Henry) and Armstrong Bros., Box 1000, Brampton,Ontario(McKnight). 'Pneumabort, Fort Dodge Laboratories, Fort Dodge, lowa. -Rhinomune, Norden Laboratories, Lincoln, Nebraska. RIRhinoquin, Plhilips Roxane, St. Joseplh, Missouri.
Can. vet. J. 20: 145-147 (May 1979)
145
d:>W* .i';SN4V w
w
FIGURE 1. Necrosis of thymic medulla. X15.75.
FIGURE 2. Focus of necrosis in the liver. XIOO0
December 10, aborted; no furtlher losses occurred at this barn. Following a tentative diagnosis of EHVI abortion based on gross patlhological lesions found in these three fetuses, 36 pregnant mares were revaccinated on January 4 or 5. At Barn I from January 5 to March 8, 21 healtlhy foals were born, four weak foals died soon after birth, ten foals were stillborn and two fetuses were aborted. Of the 22 mares which lost their foals, ten were originally stabled at Barn 1, seven at Barn 2 and five at Barn 3. Clinical signs of respiratory disease were not observed in the pregnant mares or in the in-contact barren and maiden mares. The gestation periods in days were: 322-358 for healthy foals, 320-347 for dead foals and 301-319 for aborted fetuses. The duration of the outbreak was 63 days.
Histological Findings Tissue specimens were fixed in 10% formalin solution, processed routinely and stained witl hematoxylin and eosin. Necrosis of the thymic medulla was a frequent finding. The alveolar septa of the lungs were thickened. There was a moderate bronchiolitis in some cases, while in others a necrotizing bronchiolitis was present. Foci of necrosis were present in the liver. In a few cases, dystrophic mineralization was present in the necrotic foci. The splenic lymphoid follicles were hyperplastic and the centre of the follicles was, in many cases, necrotic. Focal necrosis was seen in the zona glomerulosa of the adrenal gland in a few cases. Intranuclear inclusion bodies were observed in the reticuloendothelial cells of the thymus and spleen adjacent to areas of necrosis. They were also seen in hepatocytes bordering necrotic foci and in cells near the necrotic foci in the adrenal gland. Inclusion bodies were also numerous in the bronchiolar epithelial cells and alveolar septal cells of the lung.
Necropsj Findings
All carcasses were submitted to the laboratory immediately after death or abortion and were in an excellent state of preservation. The lungs were firm and rubbery, and a tenacious frothy exudate was commonly found in the bronchi. In most cases, the liver was swollen with yellow-white foci, varying in size from pin head to 1-2 mm in diameter, scattered througlhout the parenchyma. Varying degrees of icterus reflected the liver damage. The spleen was often swollen and the splenic lymphoid follicles were more prominent than normal.
146
Electron Microscopic Finclings Sections of paraffin-embedded thymic tissue, in wliclh inclusion bodies had been seen, were removed from the paraffin blocks, and deparaffinized in graded xylol and acetone solution. After a brief washing in distilled water, the tissues were fixed in 1% osmium tetroxide, embedded in
gross and histopathological lesions for EHVI infection and virus isolation studies were not attempted. The gross and histological changes observed in the remaining 18 were typical of EHV I infection (2). Seventeen of these were used for virus isolation studies and were positive.
44
AX~~
I'w
t 9
if'
,
Nucleus of thymic reticuloendothelial cell lilled with viral particles. X48.575.
FIGURE 3.
plastic,4 stained with uranyl acetate and lead citrate and examined with an electron microscope.5 Ultrastructural examination revealed cells in whicil the nuclei were filled with viral particles. Virus Isolation Portions of lung, liver and spleen were routinely submitted for virus isolation. Tissues were minced witlh scissors and ground in a Ten Broeck grinder with ten volumes of Hanks' balanced salt solution containing 20,000 IU of penicillin and 250 mg of streptomycin per ml. Following centrifugation at 400 g for ten minutes, the supernatant was harvested and filtered through a 0.45 j,m Millipore' filter. This suspension was then inoculated into first subculture cell structures of either embryonic bovine spleen or equine kidney from which the medium had been removed. After 90 minutes adsorption at 370C, tubes were washed three times with tissue culture medium and I ml of Earle's minimal essential medium with 2% fetal bovine serum. The tubes were then incubated at 370 C. Most isolates caused cytopathic ettects on cell cultures within 24 hiours. When about 50% of the cell sheet was affected, cultures were examined by electron microscopy to detect virus particles. Isolates were subsequently identified by a neutralization test using EHVI antiserum.
Diagnosis Twenty-one of the 22 aborted fetuses and dead foals were examined. The first tlhree were free ot 4Epon-Shell Chemical Company. Plastic & Resins
Division. Clicago.
Illinois.
iHitachi HS-9 Electron Microscope. M illipore Corporation. Bedftord. Massaichiusetts.
Discussion The mechanisms involved in protecting the equine fetus from EHVI infection are not clearly understood (7). Both humoral and cellular immunity are thought to be involved (5), but viraemia and transfer of virus across the placenta can occur in the presence of circulating antibodies (1). While the immunity against abortion, which may be cell mediated, is more durable it is incomplete (3). Despite these deficiencies in our knowledge, modified live-virus rhinopneumonitis vaccines have been widely used to prevent EHVI abortion. In some cases they have failed to provide satisfactory protection as was the experience on the farm in this report where the combined clinical and laboratory findings indicated that infection with EHVI, despite vaccination, was the major causeof the serious fetal and foal mortalities observed.
Acknowledgments We wish to thank Drs. A.N. Gagnon and G. Papp-Vid for conducting the virus isolation studies.
Note Claims on behalf of Rhinomune for the prevention of abortion have been deleted (September 1977) and Rhinoquin has been voluntarily withdrawn from the market because of adverse reactions (August 1977). Referenc es 1. BRYANS. J.T. On immunity to disease caused by equine herpesvirus 1. J. Am. vet. med. Ass. 155: 294-300. 1969. 2. CORNER. A.H.. D. MITCHEIlt. and E.B. MEADS. Equine virus abortion in Canada. 1. Pathological studies on aborted fetuses. Cornell Vet. 53: 78-88. 1963. 3. DOLl.. E.R. Immunization against viral rliinopneumonitis of hiorses with live virus propagated in hamsters. J. Am. vet. med. Ass. 139: 1324-1330. 1961. 4. DOL.L. E.R. and I.T. BRYANS. Epizootiology of equine viral rhinopneumonitis. J. Am. vet. med. Ass. 142: 31-37. 1963. 5. DL'TTA. S.K. and Dl.. CAMPBEL.I Cell mediated immunitv in equine herpesvirus type I infection 1. In vitro lymphlocyte blastogenesis and serum neutralization antibody in normal parturient and aborting mares. Can. J. comp. Med. 41: 404-408. 1977. 6. JACKSON. T. and w.w KENDRICK. Paralysis ot hlorses associated with equine herpesvirus I inlection. J. Am. vet. med. Ass. 158: 135 1-1357. 1971. 7. STUD)DERT. MJ. Comparative aspects of equine lierpesviruses. Cornell Vet. 64: 94-122. 1974.
147
Equine Herpesvirus I Infection in Mares Vaccinated with a Live-virus Rhinopneumonitis Vaccine Attenuated in Cell Culture M.D. EAGLESOME, J.N.R. HENRY AND J.D. McKNIGHT*
Summarv Vaccination, in July and again in either November or December 1976, of 55 pregnant Standardbred mares with a live-virus rhinopneumonitis vaccine attenuated in cell culture failed to protect some mares from infection with equine herpesvirus 1. From 1976-12-08 to 1977-03-08, 33 mares foaled healthy foals, 16 mares foaled dead foals or live foals which died usually within 48 hours and six mares aborted. Gross and histological examinations and virus isolation studies confirmed that equine herpesvirus I caused 18 of the 22 neonatal deaths, stillbirths or abortions. Resume Infection par l'herpesvirus equin du type 1 chez des juments vaccinees contre la rhino-pneumonite avec un vaccin attenue sur culture cellulaire La vaccination de 55 juments gravides Standard bred, contre la rhino-pneumonite equine, avec un vaccin atftnue sur culture cellulaire, en juillet et de nouveau en novembre ou en decembre 1976, ne reussit pas A proteger certaines d'entre elles contre cette maladie. Du 8 decembre 1976 au 8 mars 1977, 33 juments donnerent naissance A des poulains en sante, 16 autres donnerent des poulains mort-nes ou des poulains vivants qui moururent en 1'espace de 48 heures, tandis que six autres avorterent. Les lesions macroscopiques et microscopiques, ainsi que les epreuves d'isolement du virus, demontrerent que l'herpesvirus equin du type I etait responsable de 18 des 22 mortinatalites, mortalites neonatales et avortements. Introduction Equine herpesvirus I (EHVI) is one of three antigenically distinct species of herpesvirus re-
coverable from horses (7). It causes equine rhinopneumonitis characterized by respiratory disease (4), neurological disorders (6) and both fetal and foal mortality (4). Abortions most often occur after eight months gestation. Infected term foals may be born alive, but usually die within a few hours. For prophylaxis, three live-virus vaccines have been licensed for use in Canada; one contains a hamster-adapted virus,' the other two cell culture-attenuated strains of EHVI.2'3 Vaccination is claimed to have reduced the incidence of virus abortion but it has not abolished it. This paper records the clinical and laboratory findings, during the 1976-1977 foaling season, in a group of 55 pregnant Standardbred mares vaccinated with a live-virus rhinopneumonitis vaccine attenuated in cell culture.2
Clinical Historv The farm is a large Standardbred stud where home-owned and visiting mares are stabled for breeding and/or foaling. A "planned infection" program with the hamster adapted vaccine' was practiced until January 1974, when an abortion storm occurred among vaccinated mares. Subsequently, a program of vaccinating all horses with a cell culture-attenuated vaccine' was introduced. During the first week of November 1976, 55 home-owned mares were assembled in preparation for foaling. They had been vaccinated twice in 1975 and revaccinated in July and in either November or early December 1976. The mares were stabled in three barns: a foaling barn (Barn 1), 32 mares, barn G (Barn 2), 13 mares and barn S (Barn 3), 10 mares. They were managed as a unit with no additions and all 55 mares were to foal at Barn 1. Both Barns I and 2 have stalls and loafing yards. Pregnant mares at Barn I were held in the loafing area and moved into the stalls to foal; after foaling, mares were then moved to Barn 2. Barn I and 3 were isolated, but the yard at Barn 2 was separated only by a six metre wide roadway from the loafing yard of an adjacent barn where, in the fall of 1976, barren and maiden mares were stabled. At Barn I from December 8, 1976 to January 1, 1977, 14 mares foaled and one aborted; 12 foals were born healthy, one foal died at six days and one within 24 hours after birth. No cause for the abortion or the death of the two foals was established. On January 3, 1977, two mares, moved from Barn 3 to Barn 2 on December 14, aborted; one at Barn 2 and the other, following a further move on December 30, at Barn 1. The following day, another mare, stabled at Barn 2 since before
*Animal Pathology Division, Health of Animals Branchi, Agriculture Canada, Animal Diseases Research Institute (E), Box 11300, Station H,Ottawa, Ontario K2H 8P9(Eaglesome), Veterinary Services Laboratory, Ontario Ministry of Agriculture and Food, Guelph, Ontario(Henry) and Armstrong Bros., Box 1000, Brampton,Ontario(McKnight). 'Pneumabort, Fort Dodge Laboratories, Fort Dodge, lowa. -Rhinomune, Norden Laboratories, Lincoln, Nebraska. RIRhinoquin, Plhilips Roxane, St. Joseplh, Missouri.
Can. vet. J. 20: 145-147 (May 1979)
145
d:>W* .i';SN4V w
w
FIGURE 1. Necrosis of thymic medulla. X15.75.
FIGURE 2. Focus of necrosis in the liver. XIOO0
December 10, aborted; no furtlher losses occurred at this barn. Following a tentative diagnosis of EHVI abortion based on gross patlhological lesions found in these three fetuses, 36 pregnant mares were revaccinated on January 4 or 5. At Barn I from January 5 to March 8, 21 healtlhy foals were born, four weak foals died soon after birth, ten foals were stillborn and two fetuses were aborted. Of the 22 mares which lost their foals, ten were originally stabled at Barn 1, seven at Barn 2 and five at Barn 3. Clinical signs of respiratory disease were not observed in the pregnant mares or in the in-contact barren and maiden mares. The gestation periods in days were: 322-358 for healthy foals, 320-347 for dead foals and 301-319 for aborted fetuses. The duration of the outbreak was 63 days.
Histological Findings Tissue specimens were fixed in 10% formalin solution, processed routinely and stained witl hematoxylin and eosin. Necrosis of the thymic medulla was a frequent finding. The alveolar septa of the lungs were thickened. There was a moderate bronchiolitis in some cases, while in others a necrotizing bronchiolitis was present. Foci of necrosis were present in the liver. In a few cases, dystrophic mineralization was present in the necrotic foci. The splenic lymphoid follicles were hyperplastic and the centre of the follicles was, in many cases, necrotic. Focal necrosis was seen in the zona glomerulosa of the adrenal gland in a few cases. Intranuclear inclusion bodies were observed in the reticuloendothelial cells of the thymus and spleen adjacent to areas of necrosis. They were also seen in hepatocytes bordering necrotic foci and in cells near the necrotic foci in the adrenal gland. Inclusion bodies were also numerous in the bronchiolar epithelial cells and alveolar septal cells of the lung.
Necropsj Findings
All carcasses were submitted to the laboratory immediately after death or abortion and were in an excellent state of preservation. The lungs were firm and rubbery, and a tenacious frothy exudate was commonly found in the bronchi. In most cases, the liver was swollen with yellow-white foci, varying in size from pin head to 1-2 mm in diameter, scattered througlhout the parenchyma. Varying degrees of icterus reflected the liver damage. The spleen was often swollen and the splenic lymphoid follicles were more prominent than normal.
146
Electron Microscopic Finclings Sections of paraffin-embedded thymic tissue, in wliclh inclusion bodies had been seen, were removed from the paraffin blocks, and deparaffinized in graded xylol and acetone solution. After a brief washing in distilled water, the tissues were fixed in 1% osmium tetroxide, embedded in
gross and histopathological lesions for EHVI infection and virus isolation studies were not attempted. The gross and histological changes observed in the remaining 18 were typical of EHV I infection (2). Seventeen of these were used for virus isolation studies and were positive.
44
AX~~
I'w
t 9
if'
,
Nucleus of thymic reticuloendothelial cell lilled with viral particles. X48.575.
FIGURE 3.
plastic,4 stained with uranyl acetate and lead citrate and examined with an electron microscope.5 Ultrastructural examination revealed cells in whicil the nuclei were filled with viral particles. Virus Isolation Portions of lung, liver and spleen were routinely submitted for virus isolation. Tissues were minced witlh scissors and ground in a Ten Broeck grinder with ten volumes of Hanks' balanced salt solution containing 20,000 IU of penicillin and 250 mg of streptomycin per ml. Following centrifugation at 400 g for ten minutes, the supernatant was harvested and filtered through a 0.45 j,m Millipore' filter. This suspension was then inoculated into first subculture cell structures of either embryonic bovine spleen or equine kidney from which the medium had been removed. After 90 minutes adsorption at 370C, tubes were washed three times with tissue culture medium and I ml of Earle's minimal essential medium with 2% fetal bovine serum. The tubes were then incubated at 370 C. Most isolates caused cytopathic ettects on cell cultures within 24 hiours. When about 50% of the cell sheet was affected, cultures were examined by electron microscopy to detect virus particles. Isolates were subsequently identified by a neutralization test using EHVI antiserum.
Diagnosis Twenty-one of the 22 aborted fetuses and dead foals were examined. The first tlhree were free ot 4Epon-Shell Chemical Company. Plastic & Resins
Division. Clicago.
Illinois.
iHitachi HS-9 Electron Microscope. M illipore Corporation. Bedftord. Massaichiusetts.
Discussion The mechanisms involved in protecting the equine fetus from EHVI infection are not clearly understood (7). Both humoral and cellular immunity are thought to be involved (5), but viraemia and transfer of virus across the placenta can occur in the presence of circulating antibodies (1). While the immunity against abortion, which may be cell mediated, is more durable it is incomplete (3). Despite these deficiencies in our knowledge, modified live-virus rhinopneumonitis vaccines have been widely used to prevent EHVI abortion. In some cases they have failed to provide satisfactory protection as was the experience on the farm in this report where the combined clinical and laboratory findings indicated that infection with EHVI, despite vaccination, was the major causeof the serious fetal and foal mortalities observed.
Acknowledgments We wish to thank Drs. A.N. Gagnon and G. Papp-Vid for conducting the virus isolation studies.
Note Claims on behalf of Rhinomune for the prevention of abortion have been deleted (September 1977) and Rhinoquin has been voluntarily withdrawn from the market because of adverse reactions (August 1977). Referenc es 1. BRYANS. J.T. On immunity to disease caused by equine herpesvirus 1. J. Am. vet. med. Ass. 155: 294-300. 1969. 2. CORNER. A.H.. D. MITCHEIlt. and E.B. MEADS. Equine virus abortion in Canada. 1. Pathological studies on aborted fetuses. Cornell Vet. 53: 78-88. 1963. 3. DOLl.. E.R. Immunization against viral rliinopneumonitis of hiorses with live virus propagated in hamsters. J. Am. vet. med. Ass. 139: 1324-1330. 1961. 4. DOL.L. E.R. and I.T. BRYANS. Epizootiology of equine viral rhinopneumonitis. J. Am. vet. med. Ass. 142: 31-37. 1963. 5. DL'TTA. S.K. and Dl.. CAMPBEL.I Cell mediated immunitv in equine herpesvirus type I infection 1. In vitro lymphlocyte blastogenesis and serum neutralization antibody in normal parturient and aborting mares. Can. J. comp. Med. 41: 404-408. 1977. 6. JACKSON. T. and w.w KENDRICK. Paralysis ot hlorses associated with equine herpesvirus I inlection. J. Am. vet. med. Ass. 158: 135 1-1357. 1971. 7. STUD)DERT. MJ. Comparative aspects of equine lierpesviruses. Cornell Vet. 64: 94-122. 1974.
147
