Based on our findings, the presence of a NAS IMI was associated with changes in the peptide composition of water buffalo milk. The differential peptides identified were derived from proteins with very different functions and localizations. As milk quality and technological properties may be affected, this deserves consideration27.
We detected four differential peptides from serum amyloid A (SAA) in NAS-positive animals. SAA is associated with high SCC and mastitis in bovine cows25,28, being an acute phase protein25 that is overexpressed in milk during mastitis29,30. The mammary gland produces a specific form of SAA, the M-SAA331,32, which can be upregulated by S. aureus lipoteichoic acid33. One differential peptide originating from SAA A-3 (VISNARETIQGITDPLLKGMTRDQVREDSKADQ FANEWGR) was found uniquely in NAS-positive milk, in line with our previous finding of the SAA protein only in the milk of water buffaloes with staphylococcal IMI by shotgun proteomics8. Interestingly, in another shotgun peptidomics study, we detected SAA peptides only in cows with NAS IMI20. Thus, our detection of SAA only in the milk of animals with NAS IMI further supports its diagnostic potential in the dairy buffalo34. Nevertheless, the influence of other physiological variables including parity and stage of lactation on M-SAA levels will have to be assessed35.
Three unique and significantly differential peptides originating from osteopontin were found in NAS-positive milk. This is also in line with our previous peptidomic study on bovine cows20, although we did not identify the intact protein by proteomics in the water buffalo8. Among other biological roles, osteopontin upregulates interferon-gamma and interleukin-12 and downregulates interleukin-10 and plays a role in inducing type I immunity36. In cows, osteopontin peptides have been reported in subclinical mastitis37 and experimental Escherichia coli IMI22,23. As also indicated in a recent review on NAS affecting cows, this further indicates that NAS indeed elicit an inflammatory response in the mammary gland, as confirmed by the increased milk SCC. The present finding may support the hypothesis that NAS provide cross-protection against other mastitis pathogens38 as components of the mammary gland microbiota39,40.
On the other hand, most of the unique peptides found in healthy milk belonged to proteins of the milk fat globule membrane (perilipin 2, butyrophilin, GLYCAM-1, sodium-dependent phosphate cotransporter, annexins, glycoprotein-2)26,41, in line with the observations made by shotgun proteomics8. The predominance of MFG membrane proteins in healthy milk might be related to the high fat content of buffalo milk, and therefore to the higher abundance of these lipid secretion vesicles compared to cow milk. MFG are an important source of nutraceutical components, including membrane proteins, and the possible influence of NAS IMI on their integrity may deserve further consideration concerning nutritional value, product quality, and technological properties27. When looking at the differential distribution of peptides in terms of abundance, we observed that healthy milk was characterized by a higher abundance of casein proteolytic peptides, and NAS-positive milk by a higher abundance of peptides derived from MFG membrane proteins and immune defence proteins. While the first finding might be influenced by the higher abundance of caseins in healthy vs infected milk, the second finding further highlights the impact of NAS IMI on integrity and abundance of MFG membrane proteins and immune defence proteins, respectively, reinforcing the above considerations42.
The distribution of unique and differential peptides based on their C-terminal aminoacid showed a higher frequency of peptides ending with R in healthy milk as opposite to peptides ending especially with K, V, and F, in NAS-positive milk, in line with the observations made by our previous peptidomic work in bovine cows20. According to the MEROPS database, plasmin generates peptides ending with R and K at the C-term, while elastase, cathepsin D and cathepsin G generate peptides ending with V and F at their C-term43. Our results suggest a more intense proteolytic activity by plasmin and endogenous proteases released by inflammatory cells in NAS-positive milk.
The impact of NAS IMI on the buffalo milk peptidome was less intense than observed in cows in our recent work20. However, as mentioned above, many findings were consistent including the presence in NAS-positive milk of peptides derived from osteopontin and SAA, and the different frequency of C-terminal aminoacids in the proteolytic peptides of the two sample groups20.
Concerning the etiologic agent, the identification of S. microti as the predominant species in the milk of water buffaloes with subclinical mastitis is noteworthy as only one study reported its association with mastitis in bovine cows44. S. microti is closely associated with S. rostri and S. muscae, and it has been first isolated from Microtus arvalis, the common vole. Since its description, it has been isolated from rodents/insectivores and a female sandfly44. Therefore, the role of animal vectors might be relevant in this case. Adding to anatomical and physiological characteristics, important differences characterize bubaline cows and bovine cows in terms of animal management, farming practices (housing, feeding, bedding, milking routine), environmental temperature and humidity, and presence of water ponds, and consequently contact with different microbial reservoirs including wild and domestic animals. This may lead to mammary gland exposure and colonization by other NAS species than the bovine dairy cows, as well as to different bacterial loads in the farm environment, and should be carefully considered.