Differential quantitative proteomics study of experimental Mannheimia haemolytica mastitis in sheep

Abstract

Objective was the differential quantitative proteomics study of ovine mastitis induced by Mannheimia haemolytica; clinical, microbiological, cytological and histopathological methods were employed for confirmation and monitoring. Proteins were separated by two-dimensional gel electrophoresis (2-DE) for all samples and differentially abundant proteins were identified by mass spectrometry; comparisons were performed with pre- (blood, milk) and post- (milk of contralateral gland) inoculation findings. Animals developed mastitis, confirmed by isolation of challenge strain and increase of neutrophils in milk and by histopathological evidence. In blood plasma, 33 differentially abundant proteins (compared to findings before challenge) were identified: 6 with decrease, 13 with new appearance and 14 with varying abundance. In a post-challenge milk whey protein reference map, 65 proteins were identified; actin cytoplasmic-1, beta-lactoglobulin-1/B, cathelicidin-1 predominated. Further, 89 differentially abundant proteins (compared to findings before challenge) were identified: 18 with decrease, 53 with new appearance, 3 with increase and 15 with varying abundance; 15 proteins showed status changes in blood plasma and milk whey. Differential abundance from inoculated and contralateral glands revealed 74 proteins only from the inoculated gland. Most differentially abundant proteins in milk whey were involved in cell organisation and biogenesis (n = 17) or in inflammatory and defence response (n = 13). SignificanceThe proteomes of blood and milk from ewes with experimental mastitis caused by Mannheimia haemolytica and the differential proteomics in sequential samples after challenge are presented for the first time. This is the first detailed proteomics study in M. haemolytica-associated mastitis in ewes. An experimental model fully simulating natural mastitis has been used. Use of experimentally induced mastitis minimised potential variations and allowed consistency of results. The study included evaluation of changes in blood plasma and milk whey. Protein patterns have been studied, indicating with great accuracy changes that had occurred as part of the disease process and development, during the acute phase of infection. Relevant protein-protein interactions were studied. The entirety of proteomics findings has suggested that affected ewes had mounted a defence response that had been regulated by many proteins (e.g., cathelicidins, haptoglobin, serum amyloid A) and through various pathways (e.g., acute phase response, binding and transporting significant ions and molecules); these were interdependent at various points. Potential biomarkers have been indicated for use in diagnostic assays of mastitis.