First Molecular Evidence and Phylogenetic Analysis of Bovine Herpesvirus-4 in Bovine Milk of Mastitic and Healthy Cows in Pakistan

Bovine herpesvirus-4 (BoHV-4), a member of the Herpesviridae family, has been increasingly associated with reproductive and mammary gland pathologies in cattle. Despite its widespread global detection, limited data is available on its presence in Pakistan, particularly in relation to bovine mastitis. This study aimed to detect the presence and estimate the prevalence of BoHV-4 in cattle milk samples collected from Pakistan. A total of 174 milk samples were obtained from clinically mastitic (n=87) and non-mastitic (n=87) cattle and were subjected to DNA extraction followed by nested polymerase chain reaction (PCR) targeting the thymidine kinase (TK) gene of BoHV-4. Positive samples were confirmed via gel electrophoresis, and selected amplicons were sequenced for phylogenetic analysis. Out of 174 samples, 7 tested positive for BoHV-4, with a slightly higher prevalence in mastitic animals (5.74%) compared to non-mastitic ones (2.29%). However, statistical analysis using chi-square tests indicated no significant association between BoHV-4 presence and mastitis (p > 0.05). Phylogenetic analysis of local isolates revealed close genetic similarity (98–99%) to strains previously reported from Turkey, Japan, and Belgium. Risk factor analysis did not show significant associations between BoHV-4 detection and herd size, parity, vaccination status or respiratory illness (p > 0.05). This study confirms the first molecular detection of BoHV-4 in milk of both healthy and mastitic cows in Narowal, Pakistan. This study provides baseline molecular evidence for BoHV-4 circulation in Pakistan. Although detected at a low rate, BoHV-4 may contribute to subclinical infections and complicate mastitis pathogenesis, warranting further epidemiological investigations and routine surveillance.