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Detection of bovine viral diarrhoea virus antigen and RNA in oviduct and granulosa cells of persistently infected cattle

Large-scale in vitro bovine embryo production systems commonly use genital tracts obtained from an abattoir as a source of both cumulus–oocyte complexes and co-culture feeder cells. Tissues derived from this source may be contaminated with non-cytopathogenic bovine viral diarrhoea virus (BVDV) since, in several countries surveyed, approximately 1% of animals tested are persistently infected with this pathogen. Therefore, the use of such material in in vitro fertilization systems presents a potential risk for the transmission of BVDV to bovine embryos and via embryo transfer. This potential was investigated by obtaining oviduct epithelial cells and granulosa cells, which are commonly used as feeder cells, from cattle persistently infected with BVDV and examining them for the presence of BVD viral antigen (p80 non-structural protein and gp53 envelope glycoprotein) by indirect immunofluorescent histochemistry, and also viral RNA (encoding the p80 region) by in situ hybridization. In addition, titres of virus present in oviduct, ovary and blood were assayed by immunodetection on calf testis cell cultures. Luminal epithelial cells from the oviduct and primary cultures of granulosa cells and oviduct epithelial cells from such cattle were shown to contain both viral antigen and RNA. The susceptibility of both cell types to BVDV infection was further established by inoculating primary cell cultures of cells derived from cattle not infected with BVDV with a cloned isolate of non-cytopathogenic BVDV (Pe515). RNA encoding BVDV and the antigen were detected 12 h after inoculation. Viral titres present in oviduct, ovary and blood were between 10^2.2 and 10⁷; 10^2.2 and 10^6.75; and 10^3.5 and 10^4.25 tissue culture infective doses, (TCID)₅₀ g^–1, respectively. Control tissues from cattle not infected with BVDV, tested in each of the preceding techniques, were negative. These data establish that ovary and oviduct of persistently infected animals harbour non-cytopathogenic BVDV and that granulosa cells and oviduct epithelial cells, which are used as co-culture cells during bovine embryonic development in vitro and which, in the case of granulosa cells, constitute the cumulus investment surrounding the oocyte, are a vehicle for the potential transmission of BVDV to developing embryos.

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