gp53 a major envelope glycoprotein of bvdv xue w et al vet microbiol 57 105 118 1997 and 20 10 6 specific for ns3 p80 a conserved nonstructural protein w v corapi et al am j vet res 51 1388 1394 1990 after washing with pbs and tween 20 to remove unbound antibodies peroxidase conjugated rabbit anti mouse igg jackson immuno research lab west grove pa was added after a short incubation period unbound peroxidase conjugated antibody was removed by washing with pbs and tween 20 finally the enzyme substrate aminoethyl carbazole zymed laboratories inc south san francisco calif which produces a reddish brown color when oxidized by horseradish peroxidase was added color change was visualized under light microscopy and compared to known positive and negative controls on each plate all samples other than stock virus aliquots were also passaged in tissue culture to optimize isolation of bvdv upon initial thawing 200 u03bcl of each sample was inoculated onto a 2 cm 2 well seeded 24 h previously with mdbk cells passages were incubated 5 days d at 38 5 u00b0 c in an atmosphere of 5 co 2 and humidified air passages were frozen at u2212 80 u00b0 c for storage tissue culture passage samples were thawed and assayed by virus isolation if isolation of bvdv was unsuccessful from the original.