Acute phase proteins, proteomics and metabolomics in the diagnosis of bovine mastitis

Bovine mastitis continues to pose a major economic challenge to the dairy industry worldwide. Critical to the management and control of this condition, is the need for prompt and accurate diagnosis in field conditions, therefore a search for more sensitive and reliable biomarkers is required. In this thesis, studies focused on assessing milk samples from cows with various forms of mastitis were undertaken with a view to identifying new biomarkers for bovine mastitis. Three acute phase proteins (APP); haptoglobin (Hp), mammary associated serum amyloid A3 (M-SAA3) and C-reactive protein (CRP) were measured in milk samples from composite milk samples of all lactating cows in a commercial dairy herd, mastitis cases, submitted to a diagnostic laboratory and following an experimental mastitis challenge of cows with Streptococcus uberis. A new enzyme linked immunosorbent assay (ELISA) was developed for measuring Hp, while commercial ELISA assay kits were used to assay M-SAA3 and CRP. Other mastitis related parameters evaluated in the samples included the somatic cell counts (SCC) and the presence of pathogens. A reliable and sensitive ELISA was developed and optimized for measuring milk Hp. A cut off value for Hp of 7.9 μg/ml was established for milk with SCC less than 200,000 cells/ml. Pathogen-specific variations were observed in the concentration of each APP in mastitic milk. It was observed that the environmental pathogens showed higher concentrations of APP compared to other pathogens, from the study of mastitis milk samples submitted to the diagnostic laboratory. Also, it was possible to distinguish between samples from subclinical and clinical mastitis and between samples from subclinical and healthy udders using each of the APP (P<0.05). Haptoglobin, M-SAA3 and CRP showed corresponding variation with stage of infection during the course of experimental mastitis, and specifically CRP was observed to rise earlier than other two APP. Furthermore, characterization of the profile of these APP in the immediate post-calving milk samples was carried out to determine how valuable they would be in recognizing new mastitis infections arising at the post-partum period. It was observed that there is a general moderately-high level of APP in milk immediately following parturition which drops a few days later in healthy milk. The immunohistochemical localization of Hp in the bovine mammary gland was also assessed. It could be concluded from that study that neutrophils and the mammary epithelial cells secrete Hp into milk during mastitis. Gel and non-gel based proteomics approaches were employed to study the protein profiles and variation in mastitic milk from normal samples. Several proteins were identified that confirmed previous findings and project new mastitis markers, for example, serotransferrin, serpins, alpha-macroglobulin and neutrophil gelatinase associated lipocalins. A capillary electrophoresis mass spectrometry system (CE-MS) was also employed to elucidate the changing peptidome in milk during the course of an experimental mastitis, which lead to the generation of a panel of 77 polypeptides, which were able to significantly differentiate critical stages of mastitis. Three of these polypeptides were found in mastitic milk samples from previous peptidomic analyses thereby indicating strong biomarker value. Finally, a liquid chromatography mass spectrometry based metabolomics approach was used to study the changing profile of small metabolites in milk during the course of an experimental infection. Several pathway-based changes that highlighted metabolites of potential significance in mastitis diagnosis were recognized including lactose synthesis, nitrogen containing compounds such as betaine, L-carnitine and lipid metabolites pathways namely sn-glycerophosphocholine and choline among others. Overall, this study has shown the value of APP, milk proteomics and metabolomics in bovine mastitis diagnosis; the changing proteins and metabolites or their patterns need to be further experimentally and clinically validated as specific and sensitive markers of mastitis. Ultimately, the applicability of APP, proteins, peptides and metabolites and/or their changing patterns as mastitis biomarkers would require their adaptation to rapid (on farm) and robust measurement formats.