Rapid identification of bacterial pathogens causing subclinical bovine mastitis with special reference to Staphylococcus aureus Escherichia coli and predominant Streptococcal species by molecular methods

Abstract

The present study was carried out with objectives to screen milk samples for newlinesubclinical mastitis (SCM) by employing somatic cell count and electrical conductivity tests, to isolate and identify major bacterial pathogens Staphylococcus aureus, Escherichia coli and predominant Streptococcal species from SCM cases and to newlinestandardize simplex and multiplex PCR for rapid detection of these pathogens in milk newlinesamples. Out of 246 milk samples screened for SCM, 186 milk samples were subjected for isolation and 85 Streptococci, 95 S. aureus, 95 CoNS and 48 E. coli isolates were newlineobtained. Polymerase chain reaction was standardized targeting tuf gene to identify newlineStreptococci and Staphylococci at genus level, 16S rRNA gene to identify S. agalactiae, S. dysgalactiae, S. uberis and further, sip and pauA gene to identify S. agalactiae and S. uberis respectively. The screening of 85 Streptococcal isolates revealed seven isolates as S. agalactiae. Staphylococcus aureus was identified by nuc and sodA gene based PCR. Screening of 95 S. aureus isolates revealed the presence of nuc gene in all isolates and sodA gene in 87 isolates. Forty eight isolates of E. coli were screened and confirmed by alr gene based simplex and by a multiplex PCR (m-PCR). A two tube m-PCR was standardized to simultaneously detect the five major mastitis pathogens from milk samples. Screening of 147 bulk milk samples detected major pathogens in 81 bulk milk samples. Staphylococcus aureus was the predominant pathogen detected, followed by E. newlinecoli, S. dysgalactiae and S. uberis. The m-PCR assay developed in the present study was newlinean easy and rapid method to simultaneously detect the five major mastitis pathogens in newlinebulk milk. The regular analysis of bulk milk by m-PCR developed in the study may newlinebecome an useful tool for determining the herd status in the detection of contagious and environmental mastitis pathogens.