title: Evaluating a commercial PCR assay against bacterial culture for diagnosing Streptococcus uberis and Staphylococcus aureus throughout lactation.
abstract: The performance of a commercial, real-time PCR assay was compared with traditional bacterial culture for the identification of Streptococcus uberis and Staphylococcus aureus in bovine milk collected at different stages of lactation. Initial validation tests using fresh and frozen quarter milk samples identified factors that affected the success of the PCR. Therefore, the standard protocol was adjusted for samples collected at the first milking postpartum (colostrum) and from clinical mastitis cases. The adjustment involved PCR testing both undiluted and diluted (1 in 10 with sterile water) DNA extracts. The performance comparison between culture and the PCR assay used milk samples collected aseptically from individual quarters of mixed-age spring-calving dairy cows, during early, mid, and late lactation. Bacterial culture results were used to select a subset of samples for PCR testing (n = 315) that represented quarters with a current or prior Strep. uberis or Staph. aureus infection. Compared with culture, PCR had a sensitivity of 86.8% and specificity of 87.7% for detecting Strep. uberis (kappa = 0.74) and 96.4% and 99.7%, respectively, for detecting Staph. aureus (kappa = 0.96). The dilution of DNA extracts for colostrum and clinical samples increased the relative sensitivity from 79.2% to 86.8% for Strep. uberis detection and from 92.9% to 96.4% for Staph. aureus, presumably through diluting unidentified PCR inhibitors. The sensitivity for detecting Strep. uberis using PCR, relative to culture, was similar throughout lactation (85-89%), whereas relative specificity was lowest immediately postcalving (64%) but improved in mid and late lactation (98%). Specificity estimates for samples collected in early lactation can be optimized by reducing the cutoff cycle threshold (Ct) value from the recommended value of 37 to 34. Although using this value improved specificity (77%), it reduced test sensitivity (77%). The PCR assay lacked agreement with culture in early lactation, specifically for diagnosing Strep. uberis. Thus, PCR should not be used as the only tool for diagnosing mastitis in early lactation.
authors.affiliation: DairyNZ, Private Bag 3221, Hamilton, 3240, New Zealand; Department of Dairy Sciences, Virginia Polytechnic Institute and State University, Blacksburg 24061. Electronic address: Nicole.Steele@dairynz.co.nz.
authors.forename: N M
authors.initials: NM
authors.lastname: Steele
authors.affiliation: DairyNZ, Private Bag 3221, Hamilton, 3240, New Zealand.
authors.forename: J H
authors.initials: JH
authors.lastname: Williamson
authors.affiliation: Livestock Improvement Corporation, Riverlea, Hamilton, 3216, New Zealand.
authors.forename: R
authors.initials: R
authors.lastname: Thresher
authors.affiliation: Institute of Veterinary, Animal and Biomedical Sciences, Massey University, Palmerston North, 4471, New Zealand.
authors.forename: R A
authors.initials: RA
authors.lastname: Laven
authors.affiliation: Drumlanrig, Cambridge, New Zealand 3496.
authors.forename: J E
authors.initials: JE
authors.lastname: Hillerton
mesh_terms: D000818:Animals; D002417:Cattle; D005260:Female; D007774:Lactation; D008414:Mastitis, Bovine / Q000382:microbiology*; D008892:Milk / Q000382:microbiology; D013203:Staphylococcal Infections / Q000662:veterinary; D013211:Staphylococcus aureus / Q000235:genetics*; D013290:Streptococcal Infections / Q000662:veterinary; D013291:Streptococcus
keywords: colostrum; mastitis pathogen; polymerase chain reaction