Improved diagnostics and further investigations of bovine mastitis caused by mollicutes

The ultimate aims of this thesis were to improve the diagnostics of mollicute-associated mastitis and confirm their pathogenic role in bovine mastitis. Three types of experiments were carried out on improving diagnostics of mollicute associated mastitis (PCR, real time PCR-high resolution melting curve analysis (HRM), and Enzyme linked immunosorbent assay (ELISA)) on 368 milk samples from a single commercial dairy farm in South Australia (Farm 1), and were compared to conventional culture. Samples were collected purposively only from cows with high somatic cell count and/or mastitis treatment failure. For some tests (e.g. ELISA) samples from a second farm (n=40) in South Australia were also used (Farm 2). Novel primers of 16S ribosomal RNA were used in the PCR and HRM methodologies. A phylogenetic relationship among field isolates of mycoplasmas and acholeplasmas was created based on 16S rRNA sequences. An indirect ELISA, based on a recombinant fragment of the Mycoplasma immunogenic lipase A (MilA) protein was tested in milk for M. bovis antibodies, and compared to PCR and culture. For estimation of the pathogenic role of mollicutes associated with bovine mastitis, milk herd test data was analysed from 7,560 cow-tests. Four types of media (glycerol (GLY) + dimethyl sulphoxide (DMSO), gelatine + DMSO, foetal bovine serum (FBS) + DMSO, and original milk (CON)) were used to test the survivability of Mycoplasma bovis over time at 1, 2, 4, 8 and 16 weeks freezing in domestic and -80º C freezers. Very high prevalence of mollicutes was detected in the 288 purposively sampled cows (76.7%) using species-specific PCR. Culture was inferior in detecting infected milk samples (25.1%). The novel universal PCR demonstrated best concordance with species-specific PCR (Cohen’s Kappa= 0.747 ± 0.031). The novel HRM analysis was able to discriminate between four of the field isolates of Mycoplasma spp. and Acholelasma laidlawii. Mycoplasma bovis antibodies were detected only in 68/291 samples (23.4%). The co-infection with two or more mollicutes had a similar effect on milk composition to other major mastitis pathogens. Long-term stored milk samples should be enriched with some of the cryopreservatives used in this thesis. All cryopreservatives improved the survivability of M. bovis in milk samples stored under freezing conditions. The combination of GLY and DMSO resulted in significantly higher recovery rates at week 16, compared to CON with 57.1% (95% CI = 21.43–133.34) and 19.1% (95% CI = 11.73–60.27), respectively. The use of GLY and DMSO should therefore be encouraged for use as a cryoprotectant for M. bovis at − 20 and − 80 °C. Microbiological and molecular techniques used in this thesis should result in improved diagnostics of mollicute-associated mastitis providing rapid and accurate screening techniques. This should become a cornerstone in control strategies of mollicute-associated bovine mastitis.Thesis (Ph.D.) -- University of Adelaide, School of Animal and Veterinary Sciences, 201