Polymerase chain reaction based screening of bovine mastitis milk to detect leptospira and mycoplasma

Abstract

In the present study an attempt has been made to detect the Leptospira and Mycoplasma from bovine mastitis milk by PCR. Simultaneously trials were also made to isolate these fastidious organisms from these milk samples.

Direct screening of milk sample by PCR could not successfully detect the DNA of either of these organisms. All the previous studies had revealed that inhibitory factors present in the milk might interfere with the amplification of target DNA present in the milk. To overcome this difficulty initially all the milk samples were preenriched in the broth media, subsequently the target DNA was extracted.

The genus specific primer A and B were used to detect the leptospires in milk samples. Out of fifty haemagalactic milk samples screened by PCR only one sample was positive for Leptospira.

Fletcher’s semisolid media that could support the growth of reference strain of Leptospira pomona was used for the isolation study. But all the attempts to recover the organism from milk sample were unsuccessful.

For the detection of Mycoplasma, genus specific primers MGSO and GPO3 were used. Out of the fifty samples screened only one sample was positive for Mycoplasma. But the M.bovis specific primers PpMB 920-1 and PpMB 920-2 failed to amplify the DNA that was found positive in the genus specific PCR. Since several species of Mycoplasma was found to be associated with the mastitis, organisms detected in this study might not be M.bovis.

The media used for the isolation of Mycoplasma were BHI broth and BHI agar. Media were standardized with the reference strain and were found to be capable of supporting the growth of organisms. But all the attempts to isolate the Mycoplasma from milk samples were unsuccessful.

It was concluded that PCR is a rapid and sensitive technique to detect the fastidious organisms that are difficult to isolate in the ordinary laboratory conditions. But one difficulty encountered with the PCR from milk samples was the extraction of good quality template DNA that contained fewer inhibitors. Since the rapidity and ease of PCR is largely dependent upon the extraction procedure adopted in each bio materials, the protocol followed in this study needs some modifications.