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Standard sanitary protocol for embryo washing does not decontaminate equine embryos previously exposed to equine herpes virus-1
Animal Reproduction Science 94 (2006) 387–390
Abstract
Standard sanitary protocol for embryo washing does not decontaminate equine embryos previously exposed to equine herpes virus-1夽 I. Hebia a , G. Duchamp b , S. Destrumelle a , S. Zientara d , J.-F. Vautherot c , F. Fieni a , J.-F. Bruyas a,∗ a
UPSP Sanitary Risks and Biotechnology of Reproduction, National Veterinary School, Nantes, France b UMR PRC INRA-CNRS-University Tours-Haras Nationaux, 37380 Nouzilly, France c UR 86 BASE INRA-Tours, 37380 Nouzilly, France d UMR 1161 AFSSA, INRA-ENVA, Agence Fran¸ caise de S´ecurit´e Sanitaire des Aliments, 94703 Maisons-Alfort, France Available online 25 April 2006
1. Introduction European and French sanitary regulations for the inter- and intra-European trade of equine embryos recommend that embryos be washed 10 times in accordance with the standardized procedure described by Seidel (1987). Detection of equine herpesvirus-1 (EHV-1) by PCR in an equine embryo immediately after recovery from a donor mare has been reported by Carvalho et al. (2000) and clearly indicates that natural infection of equine embryos with herpes virus can occur. EHV-1 is a major cause of abortion, perinatal mortality, respiratory disease, and neurological disorders in horses worldwide (O’Callaghan et al., 1983), causing severe economic losses in the horse-breeding industry in many countries. Most horses are serologically positive against EHV-1. Several mammalian herpesviruses, including bovine herpesvirus type 1 (BHV-1) and pseudorabies virus (PRV) have been implicated in the infection of embryos. When bovine embryos were exposed to BHV-1 in vitro and then washed, approximately 65% of the embryos retained 夽
This paper is part of the special issue entitled Proceedings of the Ninth International Symposium on Equine Reproduction, Guest Edited by Margaret J. Evans. ∗ Correspondence to: Department of Pathology and Biotechnology of Reproduction, National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France. Tel.: +33 2 40687711; fax: +33 2 40687748. E-mail address: [email protected] (J.-F. Bruyas). 0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.03.064
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the virus. In porcine embryos exposed to PRV in vitro, the washing process recommended by the International Embryo Transfer Society (IETS; Seidel, 1987) was unable to eliminate the PRV that adhered to the zona pellucida (ZP) (Bolin et al., 1983). The risk of EHV-1 transmission through equine embryo transfer has not yet been evaluated. The objective of this study was to determine whether the standard embryo washing protocol, used following collection and prior to transfer, effectively decontaminates equine embryos exposed to EHV-1 in vitro. 2. Materials and methods Twenty embryos were collected transcervically on day 7 after ovulation (blastocyst stage) from 40 pony mares previously inseminated with fresh semen from three stallions. The mares and stallions were regularly vaccinated against EHV-1. The ZP-intact embryos were randomly split into two groups: 10 embryos were contaminated with the virus (Group I) and 10 embryos were used as negative controls (Group II). The strain of EHV-1 used was Kentucky D (KyD; Doll and Wallace, 1954). Embryos in Group I were individually transferred to separate wells of a cell culture plate containing 1 ml of viral suspension (106 TCID50 /ml) mixed with 1 ml of the culture medium Menezo B2 (upgraded B2 INRA Medium CCD lab., France), supplemented with 15% fetal calf serum (FCS), 50 g penicillin, and 50 g streptomycin per ml. They were then incubated in a humid atmosphere of 5% CO2 in air at 37 ◦ C for 24 h. After viral exposure, they were individually washed in accordance with IETS guidelines (Seidel, 1987). Immediately after the 10 wash cycles, embryos were incubated individually for 24 h on rabbit kidney cell (RK13) cultures to detect any cytopathic effects. Embryos from Group II were not exposed to the virus and not washed. They were immediately incubated for 24 h on rabbit kidney cell cultures, in the same way as the embryos from Group I, to detect any cytopathic effects. The embryos (Groups I and II), samples of wash media, semen, uterine flushes, and leucocytes from the mares and stallions, were then frozen and stored until DNA extraction. DNA was extracted using NucleoSpin® RNA virus (Macherey-Nagel EURL, France). The PCR used in this experiment was a type-specific PCR (Kirisawa et al., 1993). The sequences of two primers were selected from the nucleotide sequence of EHV-1 and EHV-4 glycoprotein B (gB) genes. FC3 (5 -GCGTTATAGCTATCACGTCC-3 ) was a forward primer common to EHV-1 and EHV-4 located at positions: EHV-1: 2699–2718 and EHV-4: 2003–2022. R1 (5 ATACGATCACATCCAATCCC-3 ) was a specific EHV1 primer located at position 2886–2867. 3. Results Results of serological testing, using virus neutralization against EHV-1, of donor mares and stallions were positive due to anti-rhinopneumonitis vaccination against EHV-1 and EHV-4. EHV-1 DNA was not detected by PCR in any of the leucocyte samples from the mares and stallions. No EHV-1 DNA was found in the semen, uterine flushing medium, or in embryos of Group II (negative controls). EHV-1 DNA was detected by PCR in 7/10 embryos exposed to EHV-1 for 24 h and washed (Fig. 1). Viral DNA was detected by PCR in the first three washes from all embryos in Group I, in the fourth wash of five embryos (5/10), and in the fifth wash of one embryo (1/10); however no viral DNA was detected in the sixth to tenth washes from any of the embryos. A cytopathic effect was observed in the RK13 culture incubated with the five first washes of those embryos.
I. Hebia et al. / Animal Reproduction Science 94 (2006) 387–390
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Fig. 1. Electrophoresis of EHV-1 DNA (190 bp) gB gene amplified by PCR with primers FC3/R1 from contaminated embryo DNA extracts. M: DNA-size marker; C−: negative control; C+: positive EHV-1 DNA control; E: contaminated embryos. Lanes 2, 3, and 5 are negative; lanes 1, 4, 6, 7, 8, 9, and 10 are positive.
No virus was detected in RK13 cells co-cultured with any embryo of either group. 4. Discussion This study clearly demonstrates that the standard embryo washing protocol as recommended by the IETS did not completely eliminate EHV1 after in vitro contamination of equine embryos. Previous studies have reported that BHV-1 and PRV virus can be associated with embryos contaminated in vitro (Bolin et al., 1982; Singh et al., 1982). Thus, the potential for BHV-1 and PRV transmission with washed embryos has been established (Bolin et al., 1983). In the present study, viral DNA testing on circulating leucocytes from all the animals used in this study, on the stallions’ semen, on uterine flushing media, and on the control embryos (Group II) was negative. These results indicate that in the present experiment embryos were not naturally contaminated by EHV-1; the detection of viral DNA in 7/10 of the embryos from Group I after the standard washing procedure was due to in vitro contamination and not to natural contamination in the genital tracts of the donor mares. The elimination of EHV-1 with the 10 successive washes and the absence of detection of viral DNA in the final washes of all embryos from Group I demonstrates that the virus detected in the seven embryos was either adherent to the surface of the embryos, or had penetrated inside. Viruses could be associated with the ZP by adsorbing to the glycoprotein fibres, or by entering the pores and becoming trapped. The virus persisted after the washing process, making the embryo a means of transmitting the disease to recipient mares. In our study, the embryos were experimentally contaminated with EHV-1 in vitro, but Carvalho et al. (2000) collected an embryo naturally contaminated by this virus from a clinically healthy donor mare. This study also proves the relative inadequacy of the standarized washing procedure in removing EHV-1 from equine embryos. These results concur with those observed for bovine embryos contaminated by BHV-1 and porcine embryos by PRV. In conclusion, equine embryos appear to be susceptible to association with EHV-1 following experimental exposure. Further studies are needed to explore whether the enzymatic treatment of
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I. Hebia et al. / Animal Reproduction Science 94 (2006) 387–390
equine embryos that have been contaminated with herpesvirus could effectively decontaminate them, as for bovine embryos infected by BHV-1. References Bolin, S.R., Runnels, L.J., Sawyer, C.A., Gustafson, D.P., 1982. Experimental transmission of pseudorabies virus in swine by embryo transfer. Am. J. Res. 43, 278–280. Bolin, S.R., Turek, J.J., Runnels, L.J., Gustafson, M.P., 1983. Pseudorabies virus, porcine parvovirus and porcine enterovirus interactions with the zona pellucida of the porcine embryo. Am. J. Vet. Res. 44, 1036–1039. Carvalho, R., Passos, L.M.F., Oliveira, A.M., Henry, M., Martins, A.S., 2000. Detection of equine herpesvirus 1 DNA in a single embryo and in horse semen by polymerase chain reaction. Arq. Bras. Med. Vet. Zootec., 52–54. Doll, E.R., Wallace, E., 1954. Cultivation of equine abortion and equine influenza viruses on the chorioallantoic membrane of chick embryos. Cornell Vet. 44, 453–461. Kirisawa, R., Endo, A., Iwai, H., Kawakami, Y., 1993. Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction. Vet. Microbiol. 36, 57–67. O’Callaghan, D.J., Gentry, G.A., Randall, C.C., 1983. The equine herpesviruses. In: Roizman, B. (Ed.), The Herpesviruses. Plenum Press, New York, pp. 215–318. Seidel, S.M., 1987. In: Carmichael, R.A., et al. (Eds.), Recommendations for the sanitary handling of embryos, Manual of the International Embryo Transfer Society, IETS, Champaign, IL, pp. 31–38. Singh, E.L., Thomas, F.C., Papp-Vid, G., Eaglesome, M.D., Hare, W.C.D., 1982. The in vitro exposure of preimplantation bovine embryos to infectious bovine rhinotracheitis virus. Theriogenology 18, 133–140.
Abstract
Standard sanitary protocol for embryo washing does not decontaminate equine embryos previously exposed to equine herpes virus-1夽 I. Hebia a , G. Duchamp b , S. Destrumelle a , S. Zientara d , J.-F. Vautherot c , F. Fieni a , J.-F. Bruyas a,∗ a
UPSP Sanitary Risks and Biotechnology of Reproduction, National Veterinary School, Nantes, France b UMR PRC INRA-CNRS-University Tours-Haras Nationaux, 37380 Nouzilly, France c UR 86 BASE INRA-Tours, 37380 Nouzilly, France d UMR 1161 AFSSA, INRA-ENVA, Agence Fran¸ caise de S´ecurit´e Sanitaire des Aliments, 94703 Maisons-Alfort, France Available online 25 April 2006
1. Introduction European and French sanitary regulations for the inter- and intra-European trade of equine embryos recommend that embryos be washed 10 times in accordance with the standardized procedure described by Seidel (1987). Detection of equine herpesvirus-1 (EHV-1) by PCR in an equine embryo immediately after recovery from a donor mare has been reported by Carvalho et al. (2000) and clearly indicates that natural infection of equine embryos with herpes virus can occur. EHV-1 is a major cause of abortion, perinatal mortality, respiratory disease, and neurological disorders in horses worldwide (O’Callaghan et al., 1983), causing severe economic losses in the horse-breeding industry in many countries. Most horses are serologically positive against EHV-1. Several mammalian herpesviruses, including bovine herpesvirus type 1 (BHV-1) and pseudorabies virus (PRV) have been implicated in the infection of embryos. When bovine embryos were exposed to BHV-1 in vitro and then washed, approximately 65% of the embryos retained 夽
This paper is part of the special issue entitled Proceedings of the Ninth International Symposium on Equine Reproduction, Guest Edited by Margaret J. Evans. ∗ Correspondence to: Department of Pathology and Biotechnology of Reproduction, National Veterinary School, BP 40706, 44307 Nantes Cedex 03, France. Tel.: +33 2 40687711; fax: +33 2 40687748. E-mail address: [email protected] (J.-F. Bruyas). 0378-4320/$ – see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.anireprosci.2006.03.064
388
I. Hebia et al. / Animal Reproduction Science 94 (2006) 387–390
the virus. In porcine embryos exposed to PRV in vitro, the washing process recommended by the International Embryo Transfer Society (IETS; Seidel, 1987) was unable to eliminate the PRV that adhered to the zona pellucida (ZP) (Bolin et al., 1983). The risk of EHV-1 transmission through equine embryo transfer has not yet been evaluated. The objective of this study was to determine whether the standard embryo washing protocol, used following collection and prior to transfer, effectively decontaminates equine embryos exposed to EHV-1 in vitro. 2. Materials and methods Twenty embryos were collected transcervically on day 7 after ovulation (blastocyst stage) from 40 pony mares previously inseminated with fresh semen from three stallions. The mares and stallions were regularly vaccinated against EHV-1. The ZP-intact embryos were randomly split into two groups: 10 embryos were contaminated with the virus (Group I) and 10 embryos were used as negative controls (Group II). The strain of EHV-1 used was Kentucky D (KyD; Doll and Wallace, 1954). Embryos in Group I were individually transferred to separate wells of a cell culture plate containing 1 ml of viral suspension (106 TCID50 /ml) mixed with 1 ml of the culture medium Menezo B2 (upgraded B2 INRA Medium CCD lab., France), supplemented with 15% fetal calf serum (FCS), 50 g penicillin, and 50 g streptomycin per ml. They were then incubated in a humid atmosphere of 5% CO2 in air at 37 ◦ C for 24 h. After viral exposure, they were individually washed in accordance with IETS guidelines (Seidel, 1987). Immediately after the 10 wash cycles, embryos were incubated individually for 24 h on rabbit kidney cell (RK13) cultures to detect any cytopathic effects. Embryos from Group II were not exposed to the virus and not washed. They were immediately incubated for 24 h on rabbit kidney cell cultures, in the same way as the embryos from Group I, to detect any cytopathic effects. The embryos (Groups I and II), samples of wash media, semen, uterine flushes, and leucocytes from the mares and stallions, were then frozen and stored until DNA extraction. DNA was extracted using NucleoSpin® RNA virus (Macherey-Nagel EURL, France). The PCR used in this experiment was a type-specific PCR (Kirisawa et al., 1993). The sequences of two primers were selected from the nucleotide sequence of EHV-1 and EHV-4 glycoprotein B (gB) genes. FC3 (5 -GCGTTATAGCTATCACGTCC-3 ) was a forward primer common to EHV-1 and EHV-4 located at positions: EHV-1: 2699–2718 and EHV-4: 2003–2022. R1 (5 ATACGATCACATCCAATCCC-3 ) was a specific EHV1 primer located at position 2886–2867. 3. Results Results of serological testing, using virus neutralization against EHV-1, of donor mares and stallions were positive due to anti-rhinopneumonitis vaccination against EHV-1 and EHV-4. EHV-1 DNA was not detected by PCR in any of the leucocyte samples from the mares and stallions. No EHV-1 DNA was found in the semen, uterine flushing medium, or in embryos of Group II (negative controls). EHV-1 DNA was detected by PCR in 7/10 embryos exposed to EHV-1 for 24 h and washed (Fig. 1). Viral DNA was detected by PCR in the first three washes from all embryos in Group I, in the fourth wash of five embryos (5/10), and in the fifth wash of one embryo (1/10); however no viral DNA was detected in the sixth to tenth washes from any of the embryos. A cytopathic effect was observed in the RK13 culture incubated with the five first washes of those embryos.
I. Hebia et al. / Animal Reproduction Science 94 (2006) 387–390
389
Fig. 1. Electrophoresis of EHV-1 DNA (190 bp) gB gene amplified by PCR with primers FC3/R1 from contaminated embryo DNA extracts. M: DNA-size marker; C−: negative control; C+: positive EHV-1 DNA control; E: contaminated embryos. Lanes 2, 3, and 5 are negative; lanes 1, 4, 6, 7, 8, 9, and 10 are positive.
No virus was detected in RK13 cells co-cultured with any embryo of either group. 4. Discussion This study clearly demonstrates that the standard embryo washing protocol as recommended by the IETS did not completely eliminate EHV1 after in vitro contamination of equine embryos. Previous studies have reported that BHV-1 and PRV virus can be associated with embryos contaminated in vitro (Bolin et al., 1982; Singh et al., 1982). Thus, the potential for BHV-1 and PRV transmission with washed embryos has been established (Bolin et al., 1983). In the present study, viral DNA testing on circulating leucocytes from all the animals used in this study, on the stallions’ semen, on uterine flushing media, and on the control embryos (Group II) was negative. These results indicate that in the present experiment embryos were not naturally contaminated by EHV-1; the detection of viral DNA in 7/10 of the embryos from Group I after the standard washing procedure was due to in vitro contamination and not to natural contamination in the genital tracts of the donor mares. The elimination of EHV-1 with the 10 successive washes and the absence of detection of viral DNA in the final washes of all embryos from Group I demonstrates that the virus detected in the seven embryos was either adherent to the surface of the embryos, or had penetrated inside. Viruses could be associated with the ZP by adsorbing to the glycoprotein fibres, or by entering the pores and becoming trapped. The virus persisted after the washing process, making the embryo a means of transmitting the disease to recipient mares. In our study, the embryos were experimentally contaminated with EHV-1 in vitro, but Carvalho et al. (2000) collected an embryo naturally contaminated by this virus from a clinically healthy donor mare. This study also proves the relative inadequacy of the standarized washing procedure in removing EHV-1 from equine embryos. These results concur with those observed for bovine embryos contaminated by BHV-1 and porcine embryos by PRV. In conclusion, equine embryos appear to be susceptible to association with EHV-1 following experimental exposure. Further studies are needed to explore whether the enzymatic treatment of
390
I. Hebia et al. / Animal Reproduction Science 94 (2006) 387–390
equine embryos that have been contaminated with herpesvirus could effectively decontaminate them, as for bovine embryos infected by BHV-1. References Bolin, S.R., Runnels, L.J., Sawyer, C.A., Gustafson, D.P., 1982. Experimental transmission of pseudorabies virus in swine by embryo transfer. Am. J. Res. 43, 278–280. Bolin, S.R., Turek, J.J., Runnels, L.J., Gustafson, M.P., 1983. Pseudorabies virus, porcine parvovirus and porcine enterovirus interactions with the zona pellucida of the porcine embryo. Am. J. Vet. Res. 44, 1036–1039. Carvalho, R., Passos, L.M.F., Oliveira, A.M., Henry, M., Martins, A.S., 2000. Detection of equine herpesvirus 1 DNA in a single embryo and in horse semen by polymerase chain reaction. Arq. Bras. Med. Vet. Zootec., 52–54. Doll, E.R., Wallace, E., 1954. Cultivation of equine abortion and equine influenza viruses on the chorioallantoic membrane of chick embryos. Cornell Vet. 44, 453–461. Kirisawa, R., Endo, A., Iwai, H., Kawakami, Y., 1993. Detection and identification of equine herpesvirus-1 and -4 by polymerase chain reaction. Vet. Microbiol. 36, 57–67. O’Callaghan, D.J., Gentry, G.A., Randall, C.C., 1983. The equine herpesviruses. In: Roizman, B. (Ed.), The Herpesviruses. Plenum Press, New York, pp. 215–318. Seidel, S.M., 1987. In: Carmichael, R.A., et al. (Eds.), Recommendations for the sanitary handling of embryos, Manual of the International Embryo Transfer Society, IETS, Champaign, IL, pp. 31–38. Singh, E.L., Thomas, F.C., Papp-Vid, G., Eaglesome, M.D., Hare, W.C.D., 1982. The in vitro exposure of preimplantation bovine embryos to infectious bovine rhinotracheitis virus. Theriogenology 18, 133–140.