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Trop Anim Health Prod (2009) 41:1643–1651 DOI 10.1007/s11250-009-9360-5

Evaluation of PCR test for detecting major pathogens of bubaline mastitis directly from mastitic milk samples of buffaloes
P. Anand Kumar

Accepted: 27 April 2009 / Published online: 7 May 2009 # Springer Science + Business Media B.V. 2009

Abstract Present study is aimed to evaluate the PCR test for detecting themajor pathogens of bubaline mastitis (BM) directly from the mastitic milk samples of the buffaloes. Cell lysates of the enriched mastitic milk samples were directly used as template DNA in PCR and the reactivity of genus and/or species specific primers against the selected pathogens of BM was tested in individual simplex PCR assays. Out of the 60 mastitic milk samples tested 30 were positive for E.coli, 21 were positive for S. aureus, 4 were positive for S. agalactiae, 9 were positive for S. dysgalactiae and 7 were positive for S. uberis. Certain samples were positive for more than one pathogen thus indicating the mixed infection of the udder. Although certain mastitic milk samples reacted with Staphylococcus genus specific primers, they didn’t react with S. aureus specific primers in PCR,which indicates the implication of other species of Staphylococcus in BM. The processing of mastitic milk samples was also simplified by eliminating the use of expensive reagents. The detection limits of PCR with the cell lysates are sensitive enough for PCR to be used as diagnostic tool for identifying the pathogens of BM.

Keywords Bubaline mastitis . PCR . Oligonucleotide primers . Mastiticmilk samples . Cell lysates . Detection limits

Introduction Mastitis is the most important cause of economic losses to Indian dairy industry due to its adverse impact on milk production of cows and buffaloes (Sasidhar et al. 2002). The mastitis in buffaloes is usually due to microbial infection of udder (Moroni et al. 2006) and the pathogens responsible for the mastitis are to be identifiedrapidly and accurately in order to monitor and control the infections in the dairy herd. Although the microbial pathogens responsible for mastitis may be identified by conventional in vitro culture and biochemical tests, they are time consuming (3 to 7 days), laborious and not highly specific. Moreover, sub-clinically infected animals intermittently shed the bacteria that may yield no bacteria withmilk culture (Phuektes et al. 2001). Therefore Riffon et al. (2001) developed a polymerase chain reaction (PCR) test to identify the major pathogens of mastitis in spiked milk samples of cows. This PCR test was found to be rapid, highly specific and sensitive. Later on a multiplex PCR test was developed to detect certain selected pathogens of mastitis simultaneously in mastitic milk samples of cowsat herd level (Phuektes et al. 2001). The PCR

P. Anand Kumar (*) Department of Veterinary Microbiology, NTR College of Veterinary Science, Sri Venkateswara Veterinary University, Gannavaram 521 102 A.P., India e-mail:


Trop Anim Health Prod (2009) 41:1643–1651

test is widely employed for detecting the mastitis pathogens in bovine milk samples (Graber et al.2007; Chiang et al. 2008). However, the processing of mastitic milk samples for PCR was not simplified and it involves use of either expensive commercial kits or hazardous chemicals like phenol-chloroform. Therefore keeping in view the advantages of PCR test, this study was designed to evaluate the PCR test for detecting major pathogens of bubaline mastitis (BM) in mastitic milk samples ofbuffaloes, directly. This study is also aimed to simplify processing of the mastitic milk samples for PCR test so that the test could be completed rapidly. Among the prevalent mastitis pathogens Staphylococcus aureus, Escherichia coli, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis were selected in this study for their detection in mastitic milk samples of buffaloes, by...
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