Bovine Sub-Clinical Mastitis Detection by Targeting Major Pathogens & Bovine (Gapdh Gene, Internal Control) by M-PCR

Mastitis is an inflammatory disease of mammary gland, caused by bacterial intramammary infection. It accounts for huge economic
loss to the dairy industry. Detection of major pathogens in the udder during inflammation would help in saving the animal from
clinical mastitis by avoiding permanent damage to the udder. Hence there is a requirement for rapid, sensitive and specific detection of the pathogens causing mastitis at an early stage of infection. Keeping this in mind we have standardized genus specific multiplex PCR (m-PCR) to detect major pathogens i.e. Staphylococcus aureus, Streptococcus spp. and Escherichia coli directly from milk samples. Method of DNA extraction directly from milk was standardized and milk from suspected cases and from the animals apparently looked healthy was subjected to the test. Genus specific primers were designed for these three genera involved in mastitis and tested in m-PCR using total DNA isolated from milk. To account for the efficiency of the DNA isolation method, we have included primers in one of the exon of the Glyceraldehyde phosphate dehydrogenase (gapdh gene) as internal control. The sensitivity and specificity of the test were evaluated using standard cultures. The test was compared with the conventional method of isolation and found to have a positive correlation. Of the total 147 samples tested 111 found to be positive for the presence of these pathogens. In our studies the test could detect the presence of a pathogen up to 1x105 - 1x107 CFU depends on the genus. Therefore the test may be considered as a rapid and efficient for diagnosis of bovine mastitis at subclinical level. The m-PCR test has several advantages like, avoids cumbersome and lengthy culturing steps, rapid, sensitive and specific and hence reliable for the detection of major pathogens causing bovine mastitis directly from milk. The results suggest that the assay could be used as an alternative method for routine detection of S. aureus, Streptococcus spp. and E. coli in milk samples in suspected cases of mastitis at subclinical levels before taking up antibiotic
therapy.