Mastitis pathogen identification using polymerase chain reaction in New Zealand milk samples : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Animal Science at Massey University, Palmerston North, New Zealand

Abstract

Rapid identification of the pathogen responsible for an intramammary infection in a dairy cow can support mastitis management decisions. Polymerase chain reaction (PCR) has become available to identify mastitis pathogens in milk, offering a rapid and sensitive test. The performance of a commercial, real-time PCR assay (PathoProof Complete-12 Mastitis PCR assay; Thermo Fisher Scientific Ltd., Vantaa, Finland) was compared with traditional bacterial culture for the identification of the most frequent pathogens in New Zealand, 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘶𝘣𝘦𝘳𝘪𝘴 and 𝘚𝘵𝘢𝘱𝘩𝘺𝘭𝘰𝘤𝘰𝘤𝘤𝘶𝘴 𝘢𝘶𝘳𝘦𝘶𝘴, during three stages of lactation. Aseptically collected quarter milk samples were analysed by culture and a subset (n=343) selected for PCR analysis based on infection status in culture. Using culture as the reference test, PCR had a relative sensitivity and specificity of 86.8%, and 87.7% (kappa=0.74) for detecting 𝘚. 𝘶𝘣𝘦𝘳𝘪𝘴 and 96.4% and 99.7% (kappa = 0.96) for detecting 𝘚. 𝘢𝘶𝘳𝘦𝘶𝘴. Relative sensitivity for detecting 𝘚. 𝘶𝘣𝘦𝘳𝘪𝘴 was similar throughout lactation whereas relative specificity was lower at the first milking post-calving (64%) and higher in mid-late lactation (97.7%). Initial validation of the PCR assay identified issues in 𝘚. 𝘶𝘣𝘦𝘳𝘪𝘴 detection, particularly when milk samples were from freshly calved cows or from cows whose milk contained clots indicating clinical mastitis. Dilution of some colostrum and some clinical samples was required for detection of bacteria by PCR, due to the presence of PCR inhibitors in the milk. The PCR assay used in this study is not recommended for mastitis pathogen identification in early lactation as the majority of infections caused by 𝘚. 𝘶𝘣𝘦𝘳𝘪𝘴 occur in the first month of lactation. PCR testing offers a number of opportunities and advantages to improve udder health and milk quality but for uptake in New Zealand, development is required to better suit colostrum samples. Greater clarity is required regarding the interpretation of PCR results and the use of information from such tests for decision-making.