BOVINE MILK PROTEOMICS IN HEALTH AND MASTITIS

Abstract

Bovine milk contains different components with nutritional and immunological benefits. It is easily accessible and a rich source of potential markers reflective of pathophysiological conditions. However, there is little information on the changes in protein abundance associated across breeds and seasons. To this end, a study was designed to investigate the change in whey proteome abundance in two conventional milch breeds, Holstein Friesian (HF) cows and Murrah (Mu) buffaloes. Collectively, 490 proteins were identified with extensive breed and season specific changes between the two animal groups. For example, antimicrobial, acute phase proteins and proteins involved in lipid homeostasis were differentially expressed across seasons and breeds. Selected representative mass spectrometric proteins (e.g. gelsolin and osteopontin) were confirmed and validated by Western blot analysis. This study indicates proteins associated with diseases may be breed or season specific. Bovine mastitis, caused by Staphylococcus aureus, is a major impediment to milk production causing huge economic loss to the dairy industry worldwide. Of the different stages of mastitis, subclinical mastitis (SCM) is the asymptomatic form causing major devastation economically as symptoms typically precede by many days and weeks before a diagnosis could be established. In contrast, clinical mastitis (CM) is easily detected through clinical symptoms. Considering the paucity of markers to detect SCM as well as the progression to CM, our subsequent study was designed to investigate changes in proteomic abundance indicating the transition of disease from H to SCM to CM. To this end, milk samples were collected from animals with somatic cell counts (SCC) from healthy (H) (< 105), SCM (5-10 x 105 cells/ml) and CM (> 106) confirmed by California Mastitis Test kit. Furthermore, microbiological and biochemical assays like coagulase and catalase tests were performed to confirm S. aureus infection in milk samples. Prior to milk sample collection, surgically excised biopsy samples from blocked teats of infected mammary glands were histopathologically confirmed for infiltration of polymorphonuclear cells, fibrous tissue proliferation and degeneration by necrosis of epithelial cells. Subsequently, whey proteins extracted from milk samples were analyzed by mass spectrometry in four replicates each from H, SCM and CM of HF and Mu resulting in 24 proteome datasets (12 datasets of each HF and Mu). Statistical analysis was performed based on a combination of fold change (. 0.5 or . 2) and q value < 0.05 to identify significant proteins indicative of disease progression. Collectively, 1479 proteins were identified of which 128 and ii 163 proteins were differentially expressed in cow and buffalo, respectively. Furthermore, these proteins were categorized in 12 groups to identify the signature pattern of the protein expression from H to SCM to CM. For instance, haptoglobin and fibronectin from HF while spermadhesin and osteopontin from Mu showed a linear increase in expression pattern during disease progression. Similarly, angiogenin and cofilin-1 were upregulated in SCM as compared to H and CM exclusively in HF while ubiquitin family members showed a linear decrease in expression pattern during disease transition exclusively in Mu. Protein interaction analysis of differentially expressed proteins in CM/SCM revealed crosstalk among potential candidate proteins such as fibronectin, tetraspanins, haptoglobin and butyrophilin. Subsequently, Western blot validated proteins such as osteopontin showed low expression pattern in both SCM and CM possibly offering resistance to wound healing. On the contrary, CAP-1 was found to be overexpressed in SCM of both HF and Mu suggesting a potential role in the early stage of mastitis. The results of the present study provide an attempt to understand the complexity of milk during the progression of mastitis for early therapeutic intervention and improve animal health. Research on the bovine milk proteome has been carried out over the past three decades with no consolidation of data to better understand milk as a biological fluid. Thus, we developed a manually curated, open online database of bovine milk proteome, BoMiProt (http://bomiprot.org), constituted with over 3100 proteins from whey, fat globule membranes and exosomes. BoMiProt has a user-friendly interface with a broad coverage on a number of useful features including protein functions, biochemical properties and post translational modifications specific to bovine milk. The database will provide an insight into the existing literature and possible future directions to further investigate milk proteins to improve their beneficial effects on human health.