Development of a rapid and sensitive test for identification of major pathogens in bovine mastitis by PCR

Bovine mastitis is the most important source of loss for the dairy industry , A rapid and specific test for the detection of the main pathogens of bovi ne mastitis is not actually available. Molecular probes reacting in PCR wit h bacterial DNA from bovine milk, providing direct and rapid detection of E scherichia coil, Staphylococcus aureus, Streptococcus agalactiae, Streptoco ccus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed, Two sets of specific primers were designed for each of the se microorganisms and appeared to discriminate close phylogenic bacterial s pecies (e,g,, S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all majo r pathogens of bovine mastitis, The sensitivities of the test using S. aure us DNA extracted from milk with and without a pre-PCR enzymatic lysis step of bacterial cells were compared. The detection limit of the assay was 3.12 5 x 10(2) CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 x 10(3) CFU/ml of milk in the absence of the p re-PCR enzymatic step. This latter threshold of sensitivity is still compat ible with its use as an efficient tool of diagnosis in bovine mastitis, all owing the elimination of expensive reagents. The two PCR tests avoid cumber some and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the s ix most prevalent bacteria causing bovine mastitis.