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Journal of Clinical Microbiology, July 2001, p. 2584-2589, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2584-2589.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development of a Rapid and Sensitive Test for Identification of Major Pathogens in Bovine Mastitis by PCR

Renée Riffon,1 Khampoune Sayasith,1 Hayssam Khalil,1 Pascal Dubreuil,2 Marc Drolet,1 and Jacqueline Lagacé1,*

Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Succursale Centre-Ville, Montreal, Quebec H3C 3J7,1 and Department of Clinical Science, Faculty of Veterinary Medicine, University of Montreal, 3200, Saint-Hyacinthe, Montreal, Quebec J2S 2M2,2 Canada

Received 4 December 2000/Returned for modification 8 April 2001/Accepted 29 April 2001

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 bovine mastitis is not actually available. Molecular probes reacting in PCR with bacterial DNA from bovine milk, providing direct and rapid detection of Escherichia coli, Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae, Streptococcus parauberis, and Streptococcus uberis, have been developed. Two sets of specific primers were designed for each of these microorganisms and appeared to discriminate close phylogenic bacterial species (e.g., S. agalactiae and S. dysgalactiae). In addition, two sets of universal primers were designed to react as positive controls with all major pathogens of bovine mastitis. The sensitivities of the test using S. aureus 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 3.125 × 102 CFU/ml of milk when S. aureus DNA was extracted with the pre-PCR enzymatic step compared to 5 × 103 CFU/ml of milk in the absence of the pre-PCR enzymatic step. This latter threshold of sensitivity is still compatible with its use as an efficient tool of diagnosis in bovine mastitis, allowing the elimination of expensive reagents. The two PCR tests avoid cumbersome and lengthy cultivation steps, can be performed within hours, and are sensitive, specific, and reliable for the direct detection in milk of the six most prevalent bacteria causing bovine mastitis.

* Corresponding author. Mailing address: Department of Microbiology and Immunology, Faculty of Medicine, University of Montreal, Case Postal 6128, Succursale Centre-Ville, Montreal, Quebec H3C 3J7, Canada. Phone: (514) 343-2180. Fax: (514) 343-6358. E-mail: jacqueline.lagace{at}umontreal.ca.

Journal of Clinical Microbiology, July 2001, p. 2584-2589, Vol. 39, No. 7
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.7.2584-2589.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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