Bluetongue virus serotype 8 (BTV-8) causes some unique characteristics compared with other BTV strains, such as transplacentary transmission, infertility, and diminished health of the offspring (De Clercq et al. 2008 Transboundary and Emerging Diseases 55, 352–359), and concerns exist about the risk of the transmission of the disease via embryo transfer (Vandaele et al. 2012). It is known that most pathogenic agents can be eliminated by washing and trypsin treatment of intact embryos according to the IETS guidelines, but some viruses adhere strongly to the zona pellucida and are not removed by this process (Ali al Ahmad et al. 2011 Theriogenology 76, 126–132). The aim of this study was to investigate decontaminating methods for bovine in vitro embryos that had been infected in vitro with BTV-8, which were earlier shown to be effective in goat embryos (REF). In vitro bovine blastocysts (n = 105) were placed in 800 μL of minimal essential medium (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 Vet. Res. 42, 14–21). The embryos were exposed to trypsin either at 37°C [Group 1 (G1)] or at room temperature [Group 2 (G2)], with 3 treatments per group (5 embryos/treatment), consisting of 5 washes in PBS without BSA; 2 washes in 0.25% trypsin for 45 s each [treatment 1 (T1)], 2 washes in 0.25% trypsin-EDTA for 60 s each [treatment 2 (T2)], or 2 washes in 0.25% trypsin for 90 s each [treatment 3 (T3)]; and 10 washes in PBS + 0.4% BSA. All the treatments were done in triplicate. The efficiency of the different washing techniques and trypsin temperature for virus removal was evaluated by RT-quantitative PCR (qPCR) on embryos and washes. Virus isolation was performed on embryonated chicken eggs as described by Vandaele et al. (2011 Vet. Res. 42, 14–21) for the first and last washing fluids and for the embryos. Room temperature was 24.9°C. Viral BTV RNA was detected by RT-PCR in the first 5 washes in all groups and treatments. After the trypsin wash, all samples remained negative until the last wash procedure. Viral isolation was positive in the first 3 washes and negative in the 10th wash. The embryos were positive on RT-PCR in at least 2 replicates of each treatment, but all samples remained negative on virus isolation. The results show that the wash procedure is efficient to remove the virus from the wash media, but it failed to remove the virus from bovine embryos produced in vitro. The temperature (37°C or room temperature) did not influence the efficiency of the trypsin treatment.
Development and evaluation of an in vitro virus isolation procedure as a replacement for the mouse inoculation test in rabies diagnosis.
