112 BLUETONGUE VIRUS INFECTION IN CATTLE AFTER TRANSFER OF BOVINE IN VIVO-DERIVED EMBRYOS

Abstract

Bluetongue virus (BTV) has been categorized by the OIE as a category 1 disease agent, for which proper handling between collection and transfer is thought to be sufficient to prevent transmission through embryo transfer. For bovine viral diarrhoea virus, it was shown that effectiveness of washing procedures depends on virus strains (Waldrop et al. 2004 Theriogenology 62, 45–55). Also BTV-8 has unique characteristics in comparison with other strains (De Clercq et al. 2008 Transbound. Emerg. Dis. 55, 352–359). The aim here was to investigate whether embryo transfer of in vivo-derived bovine embryos after in vitro exposure to BTV-8 can be performed without risk for infection of the recipients if IETS washing and trypsin treatment procedures are followed. Donor cows (n = 2) were synchronized and superovulated using Stimufol^® (Ulg, Liége, Belgium) and subsequently inseminated. At 6.5 days post-insemination (dpi), flushed embryos (n = 14 and n = 3) were placed in 800 μL of minimal essential medium (MEM), containing 10^4.9 50% tissue culture infectious doses (TCID50) of BTV-8 (Bel 2006/2 P5, VAR, Brussels, Belgium) and incubated for 1 h at 39°C in 5% CO₂ in air (Vandaele et al. 2011 Vet. Res. 42, 14–21). Next, embryos were washed in pairs in 5 consecutive Petri dishes containing PBS with antibiotics and 0.4% BSA, w/o Ca and Mg. Then, embryos were exposed to 2 consecutive trypsin (Invitrogen, Carlsbad, CA, 25050-014) washes of 45 s each at 39°C in 5% CO₂ in air and finally, another 5 consecutive washes in PBS with 2% FCS. Each Petri dish contained at least 2 mL of medium and was gently agitated between washes. Embryos were transferred in a maximum of 7 μL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analysed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n = 6) were loaded in straws and transferred to 3 BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analysed for BTV-8 (RT-qPCR) and BTV-8 antibodies. Viral BTV-RNA was detected in all 3 recipient cows at 7 days after transfer and viraemia was confirmed by the establishment of high antibody titers at 14 days after transfer. Viral BTV-RNA was detected in washes 1 to 5 for each pair of embryos (Cp-value around 29), whereas washes 6 to 10 had Cp-values around the cut-off value (40), indicating that probably the last wash was BTV-8 negative. None of the recipients was pregnant at 28 days post-transfer. In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro-spiked in vivo-derived bovine embryos. These unexpected results stress the need for further in vivo research, e.g. what is the virus load in vivo embryos may be exposed to in utero during viraemia? Does BTV-8 react differently with the zona compared with other strains? Are alternative washing procedures needed to remove BTV-8 from the zona?