Development of a specific immunomagnetic capture-PCR for rapid detection of viable Mycoplasma agalactiae in sheep milk samples

Aims:To develop an immunomagnetic capture (IMC) to detect viableMycoplasma agalactiaein routine ovine milk samples.Methods and Results:Polyclonal antibodies against twoM. agalactiaemembrane surface proteins (P80 and P55) were covalently conjugated to magnetic beads (MBs) to form MB-Ab80 and MB-Ab55.Mycoplasma agalactiaecells were captured by a specific antigen–antibody reaction and magnetic separation. Immunomagnetic capture (IMC) was used to isolate and concentrateM. agalactiaein serial decimal dilutions and in artificially contaminated milk to facilitate subsequent detection by PCR. A 375-bp fragment ofM. agalactiaewas amplified using a pair ofM. agalactiae-specific primers in PCR. The limit of detection of IMC-PCR method ranged from 10 to 102CCU ml-1when mycoplasmas were resuspended in PBS and from 102to 103CCU ml-1when mycoplasmas were resuspended in uncontaminated ovine milk. This study also describes the application of IMC-PCR method to test forM. agalactiaein 516 milk samples collected from sheep with suspected contagious agalactia. Its performance was evaluated relative to culture.Conclusions:This report has demonstrated for the first time, the effective use of rapid and reliable IMC combined with PCR assay for the detection of viableM. agalactiae.Significance and Impact of the Study:The method IMC-PCR provides an alternative to conventional microbiological detection, method and it could be applied to quick detection ofM. agalactiaein routine sheep milk samples.