Date of Award
Master of Science
Veterinary Diagnostic and Production Animal Medicine
Bovine mastitis, prevalent in dairy cattle, is often caused by a bacterial infection in the mammary gland. Bovine mastitis is costly to the dairy industry for loss of milk production.
Causative bacteria are determined by microbiological culture. Culture remains popular for its low cost, simple procedure and interpretation however, has limitations. Culture based assays are subjective, timely and may not support fastidious organisms. Nearly 30% of clinical mastitis cases are culture-negative especially in cows treated with antibiotics. Treated cows with negative milk cultures may indeed still be infected, shedding low numbers of bacteria that do not appear in culture causing false negative results.
One potential alternative to culture is a commercial assay, PathoProof™ Mastitis PCR Assay (ThermoFisher Scientific, Waltham, MA). PathoProof is capable of detecting 11 mastitis-causing bacteria from milk. To evaluate PathoProof, the analytical specificity and limit of detection (LOD) was determined using 20 culture isolates of target and non-target bacteria. The LOD was determined by inoculating phosphate-buffered saline and milk with three different bacteria. Serial dilutions, standard plate count and PCR were performed. Further evaluation used cows that were treated with antibiotics for mastitis. Milk samples were collected from cows on days 0, 3, 7, 14 and 30 post-treatment. Samples were evaluated using culture and PathoProof PCR.
PathoProof PCR only detected target bacteria from a group of 20 target and non-target isolates resulting in an analytical specificity of 100%. Average LOD ranged from 103 to 104 CFU/mL and 101 to 103 CFU per PCR reaction, relatively high values compared to previous investigations of other mastitis PCR assays. The high LOD suggests concerns about false negative results from cows shedding bacteria at low levels.
Data from 25 cows treated for mastitis were used to compare culture to PathoProof. More cows were PCR-positive on days 3, 7, 14 and 30 post-treatment demonstrating that PathoProof may be helpful in detecting bacteria in milk from treated cows. Information from PathoProof may be useful in evaluating efficacy of treatment and assist veterinarians and producers in making decisions. Further investigation into the assay’s sensitivity and quantitative abilities is needed to better determine its value.
Lacey Marshall Lund
Marshall Lund, Lacey, "Evaluation of a commercial real-time multiplex PCR assay for the detection of bovine mastitis pathogens directly from milk" (2016). Graduate Theses and Dissertations. 15765.