A Comparative Study of Bovine Herpesvirus 1247 and Equine Herpesvirus 1 in Ponies R. A. Crandell, Sandra Drysdale and Tina L. Stein*
ABSTRACT
INTRODUCTION
The clinical and immunological response of ponies exposed to a bovine herpesvirus isolate and equine herpesvirus 1 were compared. Each virus was inoculated into two ponies by the intranasal route. One uninoculated pony was used with each group as a contact control. The four inoculated ponies developed a mild rhinitis with an increase in rectal temperature. Virus was recovered from nasal secretions collected from the four inoculated and one contact pony. All ponies developed a serum neutralizing antibody to each virus. The data show that the two viruses are similar.
In a previous report (1) a bovine herpesvirus (BHV 1247) isolated from the lung of an aborted Hereford fetus was described as being unrelated to the known bovine herpesviruses. Later studies have shown that a serological relationship exists between that isolate and equine herpesvirus 1 (EHV) (2,3). This report describes experimental transmission studies with BHV 1247 and EHV-1 viruses in ponies. The serological relatedness of the two viruses is discussed.
RASUMA Cette experience visait a comparer les reactions cliniques et immunologiques, chez deux groupes de deux poneys auxquels on avait respectivement administre, par la voie intranasale, une souche d'herpesvirus bovin et l'herpesvirus equin 1. On plaga un poney t6moin dans chacun des deux groupes. Les quatre poneys expeimentaux manifesterent une legere rhinite et une elevation de la temperature rectale. On isola du virus dans les secretions nasales des quatre poneys experimentaux et d'un tdmoin. Tous les poneys developperent des anticorps seriques neutralisants a l'endroit de chacun des deux virus, indice de la parente de ces deux virus. *Laboratories of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801. Supported in part by the Illinois Department of Agriculture Cattle Disease Research Fund. Submitted May 25, 1978.
94
MATERIALS AND METHODS ANIMALS One stallion, two castrated males and three female Shetland ponies were used (Table I). The ponies were divided into two groups (No. 1, 2, 4), (No. 5, 6, 7) and placed into separate isolation rooms and observed for one week before inoculation. ANIMAL INOCULATION Ponies No. 1 and 2 were each inoculated intranasally with 5 ml of a recent field isolate of EHV-1 containing 1O52' TCID50/ 0.1 ml. Ponies No. 5 and 6 were each inoculated intranasally with 5 ml of BHV 1247 containing 103 -TCID5o/O.1 ml. Twentyone days later ponies 1 and 5 were each inoculated intramuscularly with 2.0 ml of EHV-1 and ponies 2 and 6 each received
Can. J. comp. Med.
TABLE I. Experimental Animals, Viruses, Doses and Routes of Exposure
Initial Pony Number
Virus
Group I
4
Sex F M M
Age
1 2
1 yr 14 yr 12 yr
EHV-1 EHV-1
Group II
5 6 7
F M F
2.5 yr 2.5 yr
1
yr
1247 1247
2.0 ml of BHV 1247 intramuscularly. Ponies 4 and 7 served as contact controls (Table I).
COLLECTION
OF
SPECIMENS
Serum samples for serological tests were obtained before inoculation, 21 days after inoculation and 14 days after challenge. Nasal swabs for virus isolation were collected before inoculation and periodically after inoculation. Swabs were obtained by streaking the nasal septum with a cottontipped applicator. The swabs were stored in cell culture medium at -70°C until tested.
CELL CULTURES Pig kidney (PK-15) cells were grown in tubes and flasks with Eagle's minimal essential medium (MEM) with nonessential amino acids, glutamine, 0.5% lactalbumin hydrolysate plus 10% fetal bovine serum, and antibiotics. Equine embryonic kidney (EEK) cells in third and sixth subculture were grown in MEM. Primary bovine embryonic kidney (BEK) were obtained from a commercial source. MEM with 2% fetal bovine serum was used as maintenance medium. All cell culture media contained 200 units of penicillin, 200 ug of streptomycin and 2.5 ,ug of amphotericin B per ml. VIRUS ISOLATIONS Bovine embryonic kidney (BEK) and EEK cell cultures were inoculated with 0.2 ml of each nasal specimen. The cultures were observed daily for cytopathic changes (CPC). Specimens were considered negative for virus if CPC was absent after one blind passage.
Volume 43
-
January, 1979
Virus Inoculation Challenge (21 days later)
Amount Route IN 5 ml 5 ml IN
Virus
Amount Route
EHV-1 2 ml 1247 2 ml Contact Exposure to Animals 1 and 2 5 ml IN EHV-1 2 ml. 5 ml IN 1247 2 ml Contact Exposure to Animals 5 and 6
IM IM
IM IM
NEUTRALIZATION TESTS
Reciprocal serum-virus neutralization (SVN) tests were performed with BHV 1247 and EHV-1 viruses by the microtiter method using PK-15 cells (4). Serum and virus mixtures containing 200-300 TCID5o doses of virus were incubated at 37°C for one hour before adding the cells. Serum titers are expressed as the reciprocal of the final serum dilution in which CPC was absent.
RESULTS Four ponies developed a mild rhinitis without a cough. On the second postinoculation (PI) day, ponies 1, 2, 5 and 6 had a slight serous bilateral nasal discharge. The discharge changed rapidly to mucopurulent in nature and persisted for four to five days. Ponies 2 and 6 exhibited an ocular discharge between two to five PI days. The febrile response is shown in Table II. Ponies 1, 5 and 6 had rectal temperature increases of 39.9°C, 39.4°C and 40.80 C on the second PI day respectively. Ponies 2 and 4 had a slight temperature increase (38.3°C, 38.5°C) on PI day 5 respectively. The temperature of pony 7 remained normal during the seven day observation period at which time all ponies were considered normal. No complications developed in any pony during the eight week PI period. Virus isolation results are given in Table III. EHV-1 was recovered from ponies 1 and 2 on PI day 1 and from pony 1 on the PI day 2. No further virus recoveries were made from these two animals. Virus was not isolated from contact pony 4. The 1247 virus was isolated from No. 5 on PI day 1
TABLE II. Febrile Response of Ponies after Intranasal Inoculations of Virus
Pony No. 1 2 4 5 6 7
2 39.9 37.6 37.8 39.4 40.8 37.9
1 38.4 37.2 37.5 38.0 37.2 38.0
0 37.9 37.2 37.9 38.3 37.2 38.0
Temperature (C) Postinoculation Days 4 3 37.9 37.8 37.7 37.6 38.5 37.6 38.3 38.5 38.8 38.0 37.8 37.8
5 37.9 38.3 38.4 38.5 39.2 37.9
6 38.0 38.0 37.8 38.0 38.1 37.5
7
38.1 37.8 38.3 38.2 37.8 37.9
TABLE III. Isolation of BHV 1247 and EHV-1 from Nasal Secretions from Experimentally Infected Ponies
Pony No. 1 2 4
5 6 7
Days Postinoculation 5 3 4
1
2
-
+
-
-
+ -
+
+
+ +
-
-
-
-
0
6
7
21-25
a+ -
_
_
_
_
_
-
_
_
_
_
_
+
+
+ +
+ _
+
_
+ _
+ _
+
-
-
aNegative bIsolation but not from pony 6. Pony 5 shed virus between one to seven PI days whereas pony 6 shed virus between two to seven PI days. Virus was recovered from contact pony 7 only on PI day 6. No virus was recovered between 21-25 PI days. The isolates were identified by SVN tests using known immune serum. With the exception of the isolate from pony 7, all isolates were made in both BEK and EEK cells. That isolate was recovered in EEK cells only. The preinoculation, postinoculation and postchallenge serum neutralizing antibody titers are given in Table IV. Ponies 5 and 6 did not have a demonstrable preinoculation antibody titer to either virus. In general, the postinoculation antibody titers were one dilution higher with the EHV-1 antigen except for ponies 2 and 5. In pony 2 the titers were identical and the titer of pony 5 against the EHV-1 antigen was fourfold higher than with the homologous virus. None of the ponies exhibited a rise in titer following the intramuscular challenge.
DISCUSSION The clinical and serological response of 96
both groups of animals to the two viruses were similar. The transient elevation in temperature and rhinitis observed in this study are characteristic signs in horses of infection with EHV-1. Four inoculated and two contact ponies seroconverted following exposure to the respective viruses. The abortifacient property and neurotropism of the two viruses were not investigated. However, no neurological disturbances were observed during the eight weeks following primary exposure to the viruses. The finding that the 1247 virus was shed longer than the EHV-1 virus may be explained by the fact that ponies 1 and 2 had a slight SN antibody against EHV-1 before inoculation whereas no antibody was demonstrated in ponies 5 and 6. Also, the ponies in the group receiving the 1247 isolate were younger than those receiving EHV-1. The recovery of virus for at least seven PI days in ponies 5 and 6 is evidence that the 1247 virus was established and replicated in the upper respiratory tract of these two animals. Since these animals were shedding virus for at least one week PI, the contact animal (No. 6) had a greater exposure to virus than did the contact animal (No. 4) in group 1. The second isolation of a virus serologically related to BHV 1247 isolate from
Can. J. comp. Med.
TABLE IV. Serum Neutralizing Antibody Titers in Experimental Ponies
Virus Preinoculation 2a EHV-1 -b 1247 4 EHV-1 2.. 1247 4 4. 4 EHV-1 1247 4 5. EHV-1 1247 EHV-1 6................... 1247 4 EHV-1 7................... 4 1247 aTiters are reported as the reciprocal of the endpoint dilution bIndicates negative at a final dilution of 1:2 Pony Number 1...................
.................
.................
.................
tissues of an aborted Hereford fetus in Massachusetts (2) emphasized the potential importance of this agent to the bovine population. Although the exact identity and source of the agent in nature are unknown, the data in this study clearly shows that the BHV 1247 virus produces a clinical illness in ponies and is serologically related to EHV-1. The abortifacient property of EHV-1 in horses is well documented (5). An EHV-1 vaccine strain serially passaged in hamsters was shown to produce abortions in hamsters (6). To speculate on the origin of the BHV 1247 virus, at least two possibilities must be considered. Perhaps the virus is a wild aberrant strain of EHV-1 or modified-live vaccine strain which has become adapted to cattle by close association with horses. Calves have been reported to be refractory to infection with EHV-1 (7). In that study the virus was serially passed intranasally twice in two calves. Virus was not established in the animals but a moderate febrile response was reported. An attempt to infect colostrum-deprived calves with BHV 1247 by the intranasal route was unsuccessful (Crandell, unpublished data, 1975). Virus was not recovered from the calves and a neutralizing antibody was not
Volume 43
-
January, 1979
21 Days 14 Days Postinoculation Postchallenge 128 128 64 64 128 64 128 64 256 256 128 64 128 64 32 16 64 32 32 32 128 128 64 64
demonstrated by the standard tube method. Additional studies are necessary to fully identify the BHV 1247 virus and to determine its abortifacient potential in cattle and horses under experimental conditions. In the meantime, it is suggested that the BHIV 1247 isolate be referred to only as herpesvirus 1247.
REFERENCES
1. CRANDELL, R. A., D. M. SELLS and A. M. GALLINA. The isolation and characterization of a new bovine herpesvirus associated with abortion. Theriogenology 6: 1-19. 1976. 2. SMITH, P. C. The bovine herpesviruses: An overview. Proc. 80 a. Meet. U. S. Anim. Hlth Ass. pp. 149-158. 1976. 3. KANITZ, C. L. and M. E. WOODRUFF. A herpesvirus isolated from a skunk serologic relationship to other viruses of the herpes group. Proc. 19 a. Meet. Am. Ass. Vet. Laboratory Diagnosticians. pp. 1-12. 1976. 4. CRANDELL, R. A., G. G. JELLY, D. C. HOEFLING and A. M. GALLINA. Problems encountered in the laboratory diagnosis of porcine pseudorabies (Aujeszky's disease). Proc. 19 a. Meet. Am. Ass. Vet. Laboratory Diagnosticians. pp. 13-20. 1976. 5. BAGUST, T. J. The equine herpesviruses. Vet. Bull. 41: 79-92. 1971. 6. BUREK, J. D., R. P. ROOS and D. NARAYAN. Virus-induced abortion. Studies of equine herpesvirus I (abortion virus) in hamsters. Lab. Invest. 33: 400-406. 1975. 7. JONES, T. C., C. A. GLEISER, F. D. MAURER, M. W. HALE and T. 0. ROBY. Transmission and immunization studies on equine influenza. Am. J. vet. Res. 9: 243-253. 1948.
97
ABSTRACT
INTRODUCTION
The clinical and immunological response of ponies exposed to a bovine herpesvirus isolate and equine herpesvirus 1 were compared. Each virus was inoculated into two ponies by the intranasal route. One uninoculated pony was used with each group as a contact control. The four inoculated ponies developed a mild rhinitis with an increase in rectal temperature. Virus was recovered from nasal secretions collected from the four inoculated and one contact pony. All ponies developed a serum neutralizing antibody to each virus. The data show that the two viruses are similar.
In a previous report (1) a bovine herpesvirus (BHV 1247) isolated from the lung of an aborted Hereford fetus was described as being unrelated to the known bovine herpesviruses. Later studies have shown that a serological relationship exists between that isolate and equine herpesvirus 1 (EHV) (2,3). This report describes experimental transmission studies with BHV 1247 and EHV-1 viruses in ponies. The serological relatedness of the two viruses is discussed.
RASUMA Cette experience visait a comparer les reactions cliniques et immunologiques, chez deux groupes de deux poneys auxquels on avait respectivement administre, par la voie intranasale, une souche d'herpesvirus bovin et l'herpesvirus equin 1. On plaga un poney t6moin dans chacun des deux groupes. Les quatre poneys expeimentaux manifesterent une legere rhinite et une elevation de la temperature rectale. On isola du virus dans les secretions nasales des quatre poneys experimentaux et d'un tdmoin. Tous les poneys developperent des anticorps seriques neutralisants a l'endroit de chacun des deux virus, indice de la parente de ces deux virus. *Laboratories of Veterinary Diagnostic Medicine, College of Veterinary Medicine, University of Illinois, Urbana, Illinois 61801. Supported in part by the Illinois Department of Agriculture Cattle Disease Research Fund. Submitted May 25, 1978.
94
MATERIALS AND METHODS ANIMALS One stallion, two castrated males and three female Shetland ponies were used (Table I). The ponies were divided into two groups (No. 1, 2, 4), (No. 5, 6, 7) and placed into separate isolation rooms and observed for one week before inoculation. ANIMAL INOCULATION Ponies No. 1 and 2 were each inoculated intranasally with 5 ml of a recent field isolate of EHV-1 containing 1O52' TCID50/ 0.1 ml. Ponies No. 5 and 6 were each inoculated intranasally with 5 ml of BHV 1247 containing 103 -TCID5o/O.1 ml. Twentyone days later ponies 1 and 5 were each inoculated intramuscularly with 2.0 ml of EHV-1 and ponies 2 and 6 each received
Can. J. comp. Med.
TABLE I. Experimental Animals, Viruses, Doses and Routes of Exposure
Initial Pony Number
Virus
Group I
4
Sex F M M
Age
1 2
1 yr 14 yr 12 yr
EHV-1 EHV-1
Group II
5 6 7
F M F
2.5 yr 2.5 yr
1
yr
1247 1247
2.0 ml of BHV 1247 intramuscularly. Ponies 4 and 7 served as contact controls (Table I).
COLLECTION
OF
SPECIMENS
Serum samples for serological tests were obtained before inoculation, 21 days after inoculation and 14 days after challenge. Nasal swabs for virus isolation were collected before inoculation and periodically after inoculation. Swabs were obtained by streaking the nasal septum with a cottontipped applicator. The swabs were stored in cell culture medium at -70°C until tested.
CELL CULTURES Pig kidney (PK-15) cells were grown in tubes and flasks with Eagle's minimal essential medium (MEM) with nonessential amino acids, glutamine, 0.5% lactalbumin hydrolysate plus 10% fetal bovine serum, and antibiotics. Equine embryonic kidney (EEK) cells in third and sixth subculture were grown in MEM. Primary bovine embryonic kidney (BEK) were obtained from a commercial source. MEM with 2% fetal bovine serum was used as maintenance medium. All cell culture media contained 200 units of penicillin, 200 ug of streptomycin and 2.5 ,ug of amphotericin B per ml. VIRUS ISOLATIONS Bovine embryonic kidney (BEK) and EEK cell cultures were inoculated with 0.2 ml of each nasal specimen. The cultures were observed daily for cytopathic changes (CPC). Specimens were considered negative for virus if CPC was absent after one blind passage.
Volume 43
-
January, 1979
Virus Inoculation Challenge (21 days later)
Amount Route IN 5 ml 5 ml IN
Virus
Amount Route
EHV-1 2 ml 1247 2 ml Contact Exposure to Animals 1 and 2 5 ml IN EHV-1 2 ml. 5 ml IN 1247 2 ml Contact Exposure to Animals 5 and 6
IM IM
IM IM
NEUTRALIZATION TESTS
Reciprocal serum-virus neutralization (SVN) tests were performed with BHV 1247 and EHV-1 viruses by the microtiter method using PK-15 cells (4). Serum and virus mixtures containing 200-300 TCID5o doses of virus were incubated at 37°C for one hour before adding the cells. Serum titers are expressed as the reciprocal of the final serum dilution in which CPC was absent.
RESULTS Four ponies developed a mild rhinitis without a cough. On the second postinoculation (PI) day, ponies 1, 2, 5 and 6 had a slight serous bilateral nasal discharge. The discharge changed rapidly to mucopurulent in nature and persisted for four to five days. Ponies 2 and 6 exhibited an ocular discharge between two to five PI days. The febrile response is shown in Table II. Ponies 1, 5 and 6 had rectal temperature increases of 39.9°C, 39.4°C and 40.80 C on the second PI day respectively. Ponies 2 and 4 had a slight temperature increase (38.3°C, 38.5°C) on PI day 5 respectively. The temperature of pony 7 remained normal during the seven day observation period at which time all ponies were considered normal. No complications developed in any pony during the eight week PI period. Virus isolation results are given in Table III. EHV-1 was recovered from ponies 1 and 2 on PI day 1 and from pony 1 on the PI day 2. No further virus recoveries were made from these two animals. Virus was not isolated from contact pony 4. The 1247 virus was isolated from No. 5 on PI day 1
TABLE II. Febrile Response of Ponies after Intranasal Inoculations of Virus
Pony No. 1 2 4 5 6 7
2 39.9 37.6 37.8 39.4 40.8 37.9
1 38.4 37.2 37.5 38.0 37.2 38.0
0 37.9 37.2 37.9 38.3 37.2 38.0
Temperature (C) Postinoculation Days 4 3 37.9 37.8 37.7 37.6 38.5 37.6 38.3 38.5 38.8 38.0 37.8 37.8
5 37.9 38.3 38.4 38.5 39.2 37.9
6 38.0 38.0 37.8 38.0 38.1 37.5
7
38.1 37.8 38.3 38.2 37.8 37.9
TABLE III. Isolation of BHV 1247 and EHV-1 from Nasal Secretions from Experimentally Infected Ponies
Pony No. 1 2 4
5 6 7
Days Postinoculation 5 3 4
1
2
-
+
-
-
+ -
+
+
+ +
-
-
-
-
0
6
7
21-25
a+ -
_
_
_
_
_
-
_
_
_
_
_
+
+
+ +
+ _
+
_
+ _
+ _
+
-
-
aNegative bIsolation but not from pony 6. Pony 5 shed virus between one to seven PI days whereas pony 6 shed virus between two to seven PI days. Virus was recovered from contact pony 7 only on PI day 6. No virus was recovered between 21-25 PI days. The isolates were identified by SVN tests using known immune serum. With the exception of the isolate from pony 7, all isolates were made in both BEK and EEK cells. That isolate was recovered in EEK cells only. The preinoculation, postinoculation and postchallenge serum neutralizing antibody titers are given in Table IV. Ponies 5 and 6 did not have a demonstrable preinoculation antibody titer to either virus. In general, the postinoculation antibody titers were one dilution higher with the EHV-1 antigen except for ponies 2 and 5. In pony 2 the titers were identical and the titer of pony 5 against the EHV-1 antigen was fourfold higher than with the homologous virus. None of the ponies exhibited a rise in titer following the intramuscular challenge.
DISCUSSION The clinical and serological response of 96
both groups of animals to the two viruses were similar. The transient elevation in temperature and rhinitis observed in this study are characteristic signs in horses of infection with EHV-1. Four inoculated and two contact ponies seroconverted following exposure to the respective viruses. The abortifacient property and neurotropism of the two viruses were not investigated. However, no neurological disturbances were observed during the eight weeks following primary exposure to the viruses. The finding that the 1247 virus was shed longer than the EHV-1 virus may be explained by the fact that ponies 1 and 2 had a slight SN antibody against EHV-1 before inoculation whereas no antibody was demonstrated in ponies 5 and 6. Also, the ponies in the group receiving the 1247 isolate were younger than those receiving EHV-1. The recovery of virus for at least seven PI days in ponies 5 and 6 is evidence that the 1247 virus was established and replicated in the upper respiratory tract of these two animals. Since these animals were shedding virus for at least one week PI, the contact animal (No. 6) had a greater exposure to virus than did the contact animal (No. 4) in group 1. The second isolation of a virus serologically related to BHV 1247 isolate from
Can. J. comp. Med.
TABLE IV. Serum Neutralizing Antibody Titers in Experimental Ponies
Virus Preinoculation 2a EHV-1 -b 1247 4 EHV-1 2.. 1247 4 4. 4 EHV-1 1247 4 5. EHV-1 1247 EHV-1 6................... 1247 4 EHV-1 7................... 4 1247 aTiters are reported as the reciprocal of the endpoint dilution bIndicates negative at a final dilution of 1:2 Pony Number 1...................
.................
.................
.................
tissues of an aborted Hereford fetus in Massachusetts (2) emphasized the potential importance of this agent to the bovine population. Although the exact identity and source of the agent in nature are unknown, the data in this study clearly shows that the BHV 1247 virus produces a clinical illness in ponies and is serologically related to EHV-1. The abortifacient property of EHV-1 in horses is well documented (5). An EHV-1 vaccine strain serially passaged in hamsters was shown to produce abortions in hamsters (6). To speculate on the origin of the BHV 1247 virus, at least two possibilities must be considered. Perhaps the virus is a wild aberrant strain of EHV-1 or modified-live vaccine strain which has become adapted to cattle by close association with horses. Calves have been reported to be refractory to infection with EHV-1 (7). In that study the virus was serially passed intranasally twice in two calves. Virus was not established in the animals but a moderate febrile response was reported. An attempt to infect colostrum-deprived calves with BHV 1247 by the intranasal route was unsuccessful (Crandell, unpublished data, 1975). Virus was not recovered from the calves and a neutralizing antibody was not
Volume 43
-
January, 1979
21 Days 14 Days Postinoculation Postchallenge 128 128 64 64 128 64 128 64 256 256 128 64 128 64 32 16 64 32 32 32 128 128 64 64
demonstrated by the standard tube method. Additional studies are necessary to fully identify the BHV 1247 virus and to determine its abortifacient potential in cattle and horses under experimental conditions. In the meantime, it is suggested that the BHIV 1247 isolate be referred to only as herpesvirus 1247.
REFERENCES
1. CRANDELL, R. A., D. M. SELLS and A. M. GALLINA. The isolation and characterization of a new bovine herpesvirus associated with abortion. Theriogenology 6: 1-19. 1976. 2. SMITH, P. C. The bovine herpesviruses: An overview. Proc. 80 a. Meet. U. S. Anim. Hlth Ass. pp. 149-158. 1976. 3. KANITZ, C. L. and M. E. WOODRUFF. A herpesvirus isolated from a skunk serologic relationship to other viruses of the herpes group. Proc. 19 a. Meet. Am. Ass. Vet. Laboratory Diagnosticians. pp. 1-12. 1976. 4. CRANDELL, R. A., G. G. JELLY, D. C. HOEFLING and A. M. GALLINA. Problems encountered in the laboratory diagnosis of porcine pseudorabies (Aujeszky's disease). Proc. 19 a. Meet. Am. Ass. Vet. Laboratory Diagnosticians. pp. 13-20. 1976. 5. BAGUST, T. J. The equine herpesviruses. Vet. Bull. 41: 79-92. 1971. 6. BUREK, J. D., R. P. ROOS and D. NARAYAN. Virus-induced abortion. Studies of equine herpesvirus I (abortion virus) in hamsters. Lab. Invest. 33: 400-406. 1975. 7. JONES, T. C., C. A. GLEISER, F. D. MAURER, M. W. HALE and T. 0. ROBY. Transmission and immunization studies on equine influenza. Am. J. vet. Res. 9: 243-253. 1948.
97
