Mastitis is the most economically important disease affecting dairy cattle worldwide. Staphylococcus aureus is a highly prevalent cause of mastitis, causing infections ranging from sub-clinical to gangrenous. However, the interaction between the genotype of the infecting strain of S . aureus and the host response remains largely uncharacterised. To better understand the variation in presentation and outcomes of S . aureus -mediated bovine mastitis, we studied the interaction of a panel of mastitis isolates from several prominent bovine-associated lineages with bovine mammary epithelial cells (bMEC) and neutrophils. Significant differences in immune gene expression by infected primary or immortalised bMEC, or their elaboration of neutrophil chemoattractants, were observed and were dependent on the lineage of the infecting strain. Differences were also apparent in the invasiveness of S . aureus strains and their ability to survive killing by neutrophils. Our results demonstrate that a range of immune responses occur, suggesting the importance of S . aureus strain in dictating mastitis disease course. S . aureus lineages may therefore have adopted differing strategies for exploitation of the intramammary niche. Consequently, improved diagnosis of infecting lineage may enable better prognosis for S . aureus mastitis and reduce morbidity and economic loss.

Staphylococcus aureus is an important cause of bovine mastitis, and intramammary infections caused by this pathogen are often characterized as mild, chronic, or persistent. The strains of Staph. aureus associated with mastitis belong to several distinct bovine-adapted bacterial lineages. Studies of host-pathogen interactions have demonstrated that significant differences exist between Staph. aureus strains and lineages in their ability to internalize and to elicit expression of chemokines and pro-inflammatory mediators in bovine cells in vitro. To determine the effect of bacterial strain on the response to intramammary infection in vivo, 14 disease-free, first-lactation cows were randomly allocated to 2 groups and challenged with Staph. aureus strain MOK023 (belonging to CC97) or MOK124 (belonging to CC151). Clinical signs of infection, as well as somatic cell count (SCC), bacterial load, IL-8 and IL-1β in milk, anti-Staph. aureus IgG in milk and serum, anti-Staph. aureus IgA in milk, and white blood cell populations in milk and blood were monitored for 30 d after the challenge. Cows infected with MOK023 generally developed subclinical mastitis, whereas cows infected with MOK124 generally developed clinical mastitis. Milk yield was reduced to a greater extent in response to infection with MOK124 compared with MOK023 in the first week of the study. Significantly higher SCC, IL-8, and IL-1β in milk as well as higher anti-Staph. aureus IgG and IgA in milk and anti-Staph. aureus IgG in serum were also observed in response to MOK124 compared with the response to MOK023. Higher proportions of neutrophils were observed in milk of animals infected with MOK124 than in animals infected with MOK023. Higher neutrophil concentra-tion in blood was also observed in the MOK124 group compared with the MOK023 group. Overall, the results indicate that the outcome of mastitis mediated by Staph. aureus is strain dependent.

Most new pathogens of humans and animals arise via switching events from distinct host species. However, our understanding of the evolutionary and ecological drivers of successful host adaptation, expansion, and dissemination are limited. Staphylococcus aureus is a major bacterial pathogen of humans and a leading cause of mastitis in dairy cows worldwide. Here we trace the evolutionary history of bovine S. aureus using a global dataset of 10,254 S. aureus genomes including 1,896 bovine isolates from 32 countries in 6 continents. We identified 7 major contemporary endemic clones of S. aureus causing bovine mastitis around the world and traced them back to 4 independent host-jump events from humans that occurred up to 2,500 y ago. Individual clones emerged and underwent clonal expansion from the mid-19th to late 20th century coinciding with the commercialization and industrialization of dairy farming, and older lineages have become globally distributed via established cattle trade links. Importantly, we identified lineage-dependent differences in the frequency of host transmission events between humans and cows in both directions revealing high risk clones threatening veterinary and human health. Finally, pangenome network analysis revealed that some bovine S. aureus lineages contained distinct sets of bovine-associated genes, consistent with multiple trajectories to host adaptation via gene acquisition. Taken together, we have dissected the evolutionary history of a major endemic pathogen of livestock providing a comprehensive temporal, geographic, and gene-level perspective of its remarkable success.