The expression of Staphylococcus aureus virulence proteins is under the control of RNA III, a central pleiotropic regulator transcribed from the agr locus. RNA III is activated by at least two two-component systems, one encoded by the agr locus (AgrC-AgrA) and another encoded outside of this locus (TRAP-RAP). In this work, we developed new typing methods based on genes encoding these two systems, which we used to characterize a nonclonal population of S. aureus bovine mastitis isolates. Twelve agr restriction types were identified in this population, but the majority of strains (56.3%) were grouped in the R III-A1 type. No strain isolated from humans, whose agr sequence is available from GenBank, was found to belong to this major type. Restriction maps constructed for all of those agr variants allowed the linking of all types in an evolution scheme and their grouping in one of the four agr interference groups. This analysis indicates that groups 2, 3, and 4 probably evolved from the more frequently encountered type, which belongs to group 1. agr group 1 was also found to be the most prevalent (69.0% of the strains) and the most polymorphic interference group. By developing an agr group-specific multiplex PCR, we confirmed the above classification of strains in the agr interference groups. Four allelic variants of trap were also identified, indicating that this two-component system is also polymorphic. The majority of strains was grouped in the trap 1 type (71.8%). Whereas no relationships between agr group and trap types were found, strains of similar agr restriction type were also of similar trap type (with the exception of strains belonging to the agr R IV-A5 and R VI-A8 types). Our analysis indicates that S. aureus isolated from cows has predominantly a clonal structure and that the highly prevalent agr R III-A1, trap 1 type (56.3% of the strains) probably possesses a genetic background which endows it with superior ability to infect the bovine mammary gland.

To study the dynamics of bovine tuberculosis (bTB) in France, 4,654 M. bovis strains isolated mainly from livestock and wildlife since 1978 were characterized by spoligotyping and MLVA based on MIRU-VNTR. In our study spoligotyping allowed the discrimination of 176 types although 3 spoligotypes are predominant and account for more than half of the total strain population: SB0120 (26%), SB0134 (11%) and SB0121 (6%). In addition, 11% of the isolates, principally from Southern France, showing close spoligotypes and MIRU-VNTR types have been gathered in a family designated as the “F4-family”. MLVA typing allowed extensive discrimination, particularly for strains with predominant spoligotypes, with a total of 498 genotypes, several of which were highly regionalized. The similarity of the strains’ genetic relationships based on spoligotyping and MIRU-VNTR markers supports the co-existence of different clonal populations within the French M. bovis population. A genetic evolution of the strains was observed both geographically and in time. Indeed, as a result of the reduction of bTB due to the national control campaigns, a large reduction of the strains’ genetic variability took place in the last ten years. However, in the regions were bTB is highly prevalent at present, cases in both livestock and in wildlife are due to the spread of unique local genotype profiles. Our results show that the highly discriminating genotyping tools used in this study for molecular studies of bTB are useful for addressing pending questions, which would lead to a better insight into the epidemiology of the disease, and for finding proper solutions for its sustainable control in France.

Among the toxins that Staphylococcus aureus is able to secrete, bi-component toxins named leukotoxins target specifically leukocytes, mainly phagocytic cells. Isolates from cows, goats and ewes with mastitis were selected on the basis of the presence or not of the genes encoding the recently described LukM/LukF-PV leukotoxin. Of the 128 isolates tested, 126 had moderate to high leukotoxic activity to bovine polymorphonuclear cells (PMN). The supernatants of lukM-positive isolates were much more leukotoxic than the supernatants of lukM-negative isolates: mean leukotoxic titers were 122 versus 20 and 581 versus 26 for isolates of bovine and caprine origin, respectively. Among lukM/lukF-PV positive isolates, those of caprine and ovine origins were more leukotoxic than were isolates of bovine origin (P < 0.01). The two most abundant proteins in the culture supernatant of a highly toxic isolate were purified and identified as the two components of LukM (LukM and LukF-PV) on the basis of their molecular mass, N-terminal amino acid sequence, and high synergistic activity. LukM/LukF-PV induced the flattening of bovine PMN at a concentration as low as 3.6 ng/ml (0.1 nM). A higher concentration (18 ng/ml) was necessary to produce the same effect on caprine or ovine PMN. Affinitypurified antibodies to LukM or to LukF-PV neutralized the leukotoxic effect of all the culture supernatants. They neutralized with the same efficiency the toxic activity of supernatants from lukM/lukF-PV positive or negative isolates. These results establish that LukM/LukF-PV is very active on PMN of ruminants and suggest that this leukotoxin could be the most active leukotoxin produced by mastitis isolates. They prompt further studies to delineate the contribution of LukM/LukF-PV to the pathogenesis of mastitis in ruminants and the protective effect of antibodies to this leukotoxin.