Assessment of methods to minimize transmission of bovine herpesvirus associated with embryos

Abstract

Bovine herpesvirus 1 (BHV-1) and bovine viral diarrhea virus (BVDV) cause both acute and persistent or latent infections in cattle. With the exception of a few countries and territories from which these agents have been eradicated, the viruses are distributed worldwide, and each is capable of causing economically significant reproductive and respiratory diseases. Because of their presence in serum and reproductive tissues, BHV-1 and BVDV have been the focus of risk assessments associated with pathogen transmission via embryos produced by in vitro production or somatic cell nuclear transfer. Further, it is well known that neither washing nor trypsin treatment will completely remove these viruses from in vitro-produced embryos. Therefore, the goals of this study were to identify an effective and non-cytotoxic anti-herpesvirus agent that could be used in fertilization and culture media, determine if a recombinant trypsin product would be effective for disinfection of embryos, and develop a real-time quantitative polymerase chain reaction (qPCR) that would simultaneously detect BHV-1 and BVDV and facilitate quality assurance testing. The first objective of this study determined if foscarnet or lactoferrin would inhibit BHV-1 in cell culture and if in vitro-derived embryos could develop normally when fertilized or cultured in these antiviral agents. While foscarnet effectively inhibited replication of BHV-1 in cell culture, it also inhibited viability of in vitro-produced bovine embryos when present in fertilization and culture media. Lactoferrin also significantly inhibited BHV-1 in cell culture, and supplementation of in vitro culture medium with lactoferrin inhibited blastocyst development of in vitro-produced embryos. The second objective of this study determined if TrypLE™ Express (1X) or TrypLE™ Select (10X) would be effective at inactivating BHV-1 associated with Day 7 in vivo- and in vitro-derived embryos after they had been exposed to the virus. TrypLE™ Select (10X) when used for 5 and 10 minutes effectively inactivated BHV-1 associated with Day 7, zona pellucida-intact, in vivo-derived embryos. Treatment of in vitro-derived embryos with TrypLE™ Select (10X) for 7 and 10 minutes and with TrypLE™ Select (10X) diluted 1:2 for 10 minutes appeared to be effective at removing BHV-1. However, when these treated embryos were cultured on uterine tubal cells for 48 hours, the cells became infected. While the amount of embryo-associated virus required to infect a recipient is not known, ideally the embryo should be completely free of virus. Therefore, TrypLE™ is not a feasible product for treatment of in vitro-derived embryos. The third objective of this study validated a duplex qPCR for simultaneous detection of BHV-1 and BVDV type I and II. Pooled follicular fluid was diluted 1:5 to 1:107 with both BHV-1 and BVDV contaminated follicular fluid. The duplex qPCR was able to simultaneously detect diluted BHV-1 (1:100) and BVDV I (1:1,000). Therefore, the duplex qPCR might be used for quality assurance testing to identify these two viruses in cells, fluids and tissues collected from donor animals and used in reproductive technologies.