PHENOTYPIC AND MOLECULAR ASPECTS OF SUBCLINICAL MASTITIS IN DAIRY CATTLE

The focus of this PhD thesis was to better evaluate the phenotypic and molecular aspects associated with udder inflammation and subclinical IMI in dairy cows, with the specific focus on the effects of subclinical mastitis on some milk important traits (i.e. protein fractions) and also for better explaining the biological complexity behind the development of mastitis. Therefore specific aims of the thesis were: i) to study the association between DSCC, firstly combined with SCC and then expressed in a quantitative way as combined count of PMN-LYM and MAC, and the detailed milk protein profile expressed both quantitatively (g/L) and qualitatively (% of milk nitrogen) in 1,482 Holstein cows (Chapter I); ii) to deeper investigate the association between naturally occurring subclinical intramammary infection (IMI) caused by different pathogens (Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis and Prototheca spp) in combination with SCC, as well as their interaction, on the detailed milk protein profile assessed at the quarter level in Holstein cows (Chapter II); iii) to better assess the molecular mechanisms behind the pathogenesis of naturally occurring subclinical mastitis and the potential difference in the host immune response by: i) evaluating the milk SCs transcriptome in cows with natural and subclinical infection from S. agalactiae and Prototheca spp., and ii) integrate the transcriptomic and phenotypic information (i.e. milk immune cells population, milk composition traits, SCC and DSCC) to better explain the complexity behind the development of mastitis and the identification of putative informative variables for its detection and prediction (Chapter III). Results from the first contribution firstly confirmed the detrimental effects of the increase in SCC on the milk protein profile, and especially on the casein fractions. Then, when we focused on the contribution of the leucocyte’s populations extracted from the DSCC parameter, which were expressed as combined count of polymorphonuclear cells and lymphocytes (PMN-LYM) and macrophages (MAC) count, we observed that the most detrimental effects towards the protein profile, and once again, especially on the casein fractions, was carried out by the MAC proportion. In the second study we observed that both subclinical IMI, and especially increased SCC significantly reduced the proportion of two of the most abundant casein fractions (i.e., β-CN and αS1-CN), which is ascribed to the increased activity of both milk endogenous and microbial proteases. In terms of whey proteins, both IMI and SCC significantly increased lactoferrin, being a glycoprotein with direct and undirect antimicrobial activity. Overall, we observed that the inflammation status driven by the increase in SCC, was associated with the most significant alteration in the protein profile, suggesting that the increase in endogenous proteolytic enzymes might be the pivotal aspect driving the alteration of the milk protein profile. The third study allowed us to shed some light on the molecular mechanisms behind subclinical IMI and on the immune reaction of the animal in response to the infection. Results from the RNA-Seq allowed us to observe the changes in the gene expression from healthy animals to the ones infected by S. agalactiae or Prototheca. From the pathway analyses even though we observed some differences in terms of immune-related pathways and gene expression between the two infections, there was a strong “core” immune response that was commonly shared by the two pathogens. Then, integrating the transcriptomic and phenotypic information highlighted a strong correlation between the transcriptome and the immune cell populations, and udder health traits (i.e., SCC, DSCC, lactose and conductivity). Also, we identified some putative hub genes that could be helpful for the animal’s response to subclinical mastitis, once validated in a larger population.