Comparison of PCR and culture methods for detecting mastitis causing mycoplasma in bulk tank milk from commercial dairy herds

Abstract Text:

In this study we compared the mycoplasma detection of direct culture, broth enhanced culture and PCR from 1299 bulk tank and composite pen milk samples collected from 62 commercial dairies in 11 states. Culture of bulk tank milk directly onto specialized mycoplasma culture media (such as modified Hayflick agar) has proven effective in detection of mycoplasma mastitis outbreaks. Many laboratory methods also incorporate a mycoplasma broth enhancement protocol to increase sensitivity. This routine, direct and broth enhanced mycoplasma culture protocol, of bulk tank milk samples and pooled pen samples is widely practiced as a primary screening method for detection of mycoplasma infection status of a herd. However disease detection can be delayed up to 10 days due to slow colony formation with culture. Direct mycoplasma detection in enriched broth by the use of PCR may have the advantage of detecting the pathogen much sooner, if it is shown to detect the pathogen in bulk tank milk at least as well as culture. All samples in the study were cultured by plating 10 µL of the milk sample directly onto commercial mycoplasma isolation agar. All samples also were enriched by inoculating a 100 µL aliquot into 3 mL of mycoplasma broth and then incubated for 48 h, prior to plating a 10 µL aliquot of this enriched broth onto mycoplasma agar. Any sample where one or more colonies were detected by either direct or broth enrichment were deemed culture positive. A 2 µL aliquot of the same enriched broth from the culture method was used for the PCR detection method. The PCR method used was a four-way multiplex real time assay including primers for general Mycoplasma spp., Mycoplasma bovis, Mycoplasma bovigenitalium, and an internal positive control. When an organism was detected by any of the mycoplasma primers, the sample was deemed to be PCR positive. Of the 58 PCR detection events 35 (60.3%) were classified as Mycoplasma bovis, 16 (27.6%) as Mycoplasma bovigenitalium, and 7 (12.1%) were only detected by the Mycoplasma spp. primers. The comparison summary presented below shows a high level (98.6%) of agreement between the two methods. Direct PCR amplification of a broth enriched milk sample can rapidly detect Mycoplasma spp. in bulk tank and pen samples, and can give results comparable to conventional culture methods.  PCR also offers the possibility of providing species differentiation of positive samples.

Keywords: mastitis, mycoplasma, PCR