Differential Proteomic Analysis of Ewe’s Milk During Staphylococcal Experimental Intra-Mammary Infections to Evaluate the Impact of Selection on Milk Somatic Cell Count

Mastitis, defined as inflammation of the mammary gland, usually in response to infection, can lead to irreversible damage to the secretory tissue. It results in decreased milk production, abnormalities in milk inducing altered technological quality, and possible diffusion of pathogenic bacteria into the food chain. Despite considerable efforts in selection, prevention and early diagnosis, mastitis remains one of the most important diseases in dairy cattle and small ruminants worldwide. With its huge impact both at the economical level and on animal welfare, methods for early stage detection of clinical or even subclinical mastitis are still to be found. In dairy small ruminants the main ethiological agents are Staphylococci.

An extensive differential proteomic analysis of ewe’s milk was undertaken during the course of mastitis to assess the possible effects of milk somatic cell count (SCC) on mastitis resistance and to detect any useful markers of udder infection. To this purpose, two groups of dairy Lacaune sheep having high (SCC+) and low (SCC-) milk somatic cell counts were selected and subjected to experimental infection protocols at one half udder whereas the other one served as control. In trial A, 4 ewes from each group were injected with Staphylococcus aureus. Milk samples were collected immediately before (d0) and at days 1 (d1) and 7 (d7) after infection. In trial B, 7 ewes from each group were injected with S. epidermidis. Milk samples were collected immediately before and at days 1, 3, 7, 10, 15, 22 and 32 after infection. The proteomic analysis was performed as follows: major milk proteins (both in trial A and B) and 4% TCA soluble peptides (trial A) were separated by RP-HPLC, minor whey proteins (trial A) were analysed by two-dimensional LC (Beckman, PF2D). Relevant fractions were collected and their protein contents identified by mass spectrometry.

The percentage of major milk proteins was almost unaffected in control half udders whereas a marked caseinolysis and increased amounts of serum albumin were observed in the infected quarters. These phenomena were more apparent in the SCC+ group, suggesting a reduced resistance to mastitis. In trial A, 2 casein fragments were detected at days 1 and 7 after infection.They were not observed at day 0 and in control udders. In trial B, it was possible to identify an acute phase protein (Serum Amyloid A3), early after infection. Among the selected peptides fractions that were analysed, 10 SAA fragments were also identified. When analysing minor whey proteins, several proteins (including lactoferrin, calpastatin, and SAA from Ovis aries and Q6GE43 hypothetical protein from S. aureus), showing differential expression levels between SCC+ and SCC- ewes, were identified at day 7 after infection. For each one, a possible role in intra-mammary infection and inflammation response is proposed.
 
It was concluded that SCC- ewes might have a better ability to react against bacterial invasion, as indicated by major protein patterns, possibly because of different expression of proteins regulating cell metabolism and inflammation. Moreover, possible markers of mammary infections were identified amongst proteins and peptide fractions.