Bluetonge virus infection in cattle after transfer of bovine in vivo-derived embryos.

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 is depending on virus strains (Waldrop et al., 2004). Also BTV-8 has unique characteristics in comparison with other strains of BTV (De Clercq et al., 2008). 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 800µL of MEM, containing 104.9 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). 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 two consecutive trypsin (Invitrogen, 25050-014) washes of 45 sec each at 39°C in 5% CO2 in air and finally, another five 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 7µL of medium and a new tip was used after every wash step. Washes 1 to 5 and washes 6 to 10 were pooled and analyzed for BTV-8 (RT-qPCR). After these washes, 3 pairs of embryos (n=6) were loaded in straws and transferred to three BTV-8 negative recipients. Two sentinel cows served as control. Cows were bled twice weekly and blood and serum samples were analyzed for BTV-8 (RT-qPCR) and BTV-8 antibodies.
Viral BTV-RNA was detected in all three 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.
In conclusion, washing and trypsin treatment did not succeed in removing BTV-8 from in vitro spiked in vivo derived bovine embryos. This partial in vitro study stresses 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 reacts differently with the zona compared to other strains? Are alternative washing procedures needed for in vivo embryos?
De Clercq et al., Transboundary and Emerging Diseases 2008;55:352-359
Vandaele et al., Veterinary Research 2011;42:14-21
Waldrop et al., Theriogenology 2004;62:45-55