Detection of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk of dairy cattle with history of BoHV-4 infection

Abstract

We have demonstrated, by PCR and restriction enzyme analysis of the PCR product, the presence of bovine herpesvirus 4 (BoHV-4) DNA in the cell fraction of milk from dairy cattle with a history of BoHV-4 infection. We next evaluated the infectious nature of BoHV-4 DNA in those cells. Cocultivation of a BoHV-4-sensitive cell line with BoHV-4 DNA-positive milk cell samples produced cytopathic effects. The same result was obtained from frozen and thawed milk cell fraction coming from the cell milk fraction PCR-positive cows, ensuring that cells were killed and only infectious virus could be recovered after cocultivation with sensitive cells. This report shows that infectious BoHV-4 can be present in milk cells and that therefore nursing may be one of the transmission routes of BoHV-4.

FIG. 1

(A) Specific amplification of BoHV-4 DNA fragment from PBMC samples from BoHV-4 IFAT-positive cows. PCR amplified a 2,538-bp fragment of the BoHV-4 genome containing ORF1 overlapping ORF2 gene, ORF3 corresponding to the thymidine kinase gene, and ORF4 homologous to the herpes simplex virus type 1 glycoprotein H gene. Lanes 1 to 7 correspond to PBMC samples; lanes − and + correspond to negative (DNA from PBMC of BoHV-4 seronegative cow) and positive controls, respectively. 1 kb, molecular size marker. (B) Predicted location of HindIII restriction sites and respective expected restriction fragment sizes (not shown to scale). (C) Ethidium bromide-stained gel of HindIII-digested 2,538-bp BoHV-4 amplified sequence from PBMC DNA samples (lanes 1 to 7). 1 kb, molecular size marker.

FIG. 2

(A) Specific amplification, performed with primers α and β, of DNA from milk cell fractions of milk coming from PBMC PCR-positive cows and producing a 2,538-bp product. Lanes 1 to 7 correspond to milk cell fraction samples; lanes − and + correspond to negative and positive controls respectively. 1 kb, molecular size marker. (B) Ethidium bromide-stained gel of HindIII-digested 2,538-bp BoHV-4 amplified sequence from PBMC DNA samples (2, 3, and 6). 1 kb, molecular size marker.