Multiplex PCR for the detection of Brucella ovis , Actinobacillus seminis and Histophilus somni in ram semen

Objective  To develop a multiplex polymerase chain reaction (PCR) assay for the rapid detection of Brucella ovis , Actinobacillus seminis , Histophilus somni in fresh ram semen samples. Design  The multiplex assay was based on the single PCR assays published for the detection of A seminis and B ovis , and the forward primer published for the detection of H somni ; an alternative reverse primer for H somni was designed in this study. Procedure  Culture and PCR of 295 fresh semen samples were carried out. Results  The multiplex PCR was far more successful in the detection of H somni (45/295) than culture (23/295). A seminis was also detected in more semen samples by multiplex PCR (29/295) than culture (13/295) and B ovis was detected in three samples using both PCR and culture. No amplifications were detected with DNA from a range of bacterial isolates including species associated with epididymitis in rams. Conclusion  This PCR could be used as a complementary test, or alternative to culture of ram semen and other biological samples for the detection B ovis , H somni and A seminis .