Characterization of a putative pathogenicity island from bovine Staphylococcus aureus encoding multiple superantigens.

Previous studies have demonstrated that a proportion of Staphylococcus aureus isolates from bovine mastitis coproduce toxic shock syndrome toxin (TSST) and staphylococcal enterotoxin C (SEC). In this study, molecular genetic analysis of one such strain, RF122, revealed the presence of a 15,891-bp putative pathogenicity island (SaPIbov) encoding the genes for TSST (tst), the SEC bovine variant (sec-bovine), and a gene (sel) which encodes an enterotoxin-like protein. The island contains 21 open reading frames specifying hypothetical proteins longer than 60 amino acids including an integrase-like gene. The element is bordered by 74-bp direct repeats at the left and right junctions, and the integration site lies adjacent to the 3' end of the GMP synthase gene (gmps) in the S. aureus chromosome. SaPIbov contains a central region of sequence identity with the previously characterized tst pathogenicity island SaPI1 (J. A. Lindsay et al., Mol. Microbiol. 29:527-543, 1998). A closely related strain, RF120, of the same multilocus enzyme electrophoretic type, random amplified polymorphic DNA type, and ribotype, does not contain the island, implying that the element is mobile and that a recent insertion/deletion event has taken place. TSST and TSST/SEC-deficient mutants of S. aureus strain RF122 were constructed by allele replacement. In vitro bovine Vbeta-specific lymphocyte expansion analysis by culture supernatants of wild-type strains and of tst and sec-bovine allele replacement mutants revealed that TSST stimulates BTB13-specific T cells whereas SEC-bovine stimulates BTB93-specific T cells. This suggests that the presence of SaPIbov may contribute to modulation of the bovine immune response.

Molecular population and virulence factor analysis of Staphylococcus aureus from bovine intramammary infection.

Staphylococcus aureus isolates from cows in Ireland (n = 102) and the USA (n = 42) were characterized by RAPD-PCR and analysed for the production of a number of putative virulence factors. Of these strains 63 representative isolates were screened for the corresponding virulence factor genes by PCR or Southern hybridization or both. The isolates were divided into 12 distinct clonal types on the basis of their RAPD fingerprint profiles. Of the isolates, 107 (74.3%) tested positive for clumping factor in a slide agglutination test, all 24 RAPD type 7 isolates being negative for clumping factor. PCR analysis of region R, a repeat region of the clfA gene, revealed eight region-R sizes. There was a strong association between RAPD type and the clfA region-R genotype among Irish isolates. Of the RAPD type 7 isolates, 21 (87.5%) coproduced toxic shock syndrome toxin-1 (TSST-1) and staphylococcal enterotoxin C (SEC). Over 90% of isolates demonstrated haemolytic activity on sheep or rabbit red blood cells and all isolates harboured the gamma-haemolysin (hlg) locus. Of the Irish isolates, all those of RAPD type 7 were sensitive to penicillin G, whereas 86% of RAPD types 4 and 5 strains were resistant. Furthermore, RAPD types 5 and 7 were more likely to be associated with clinical mastitis whereas RAPD type 4 isolates were more often associated with a latent infection. The current study identifies some of the putative virulence factors produced by the predominant clonal types of bovine Staph. aureus that may be considered as components of a vaccine.

Fine-structure molecular epidemiological analysis of Staphylococcus aureus recovered from cows.

Sixty-three Staphylococcus aureus isolates recovered from bovine sources in the USA and the Republic of Ireland were characterized by multilocus enzyme electrophoresis (MLEE), ribotyping, and random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) typing at two separate laboratories. The S. aureus isolates were assigned by MLEE to 10 electrophoretic types (ETs) (Index of Discrimination, D = 0.779). In contrast, the same isolates were assigned to 13 ribotypes (D = 0.888), and to 12 RAPD types (D = 0.898). A common clone, ET3, of worldwide distribution, was represented by six distinct combinations of ribotypes and RAPD types. S. aureus clones recovered from cows in Ireland were also associated with mastitis in dairy cows in the USA. These findings are consistent with the hypothesis that only a few specialized clones of S. aureus are responsible for the majority of cases of bovine mastitis, and that these clones have a broad geographic distribution.

A toxic shock syndrome toxin mutant of Staphylococcus aureus isolated by allelic replacement lacks virulence in a rabbit uterine model.

The gene coding for toxic shock syndrome toxin-1 in S. aureus was inactivated by allelic replacement in two TSS-associated strains. One mutant derived from FRI1169 (a non-enterotoxigenic strain) lacked virulence in the rabbit uterine chamber infection model. This suggests that TSST-1 is the only determinant produced by this strain that can induce the symptoms of shock in rabbits. A novel method for allelic replacement involving transduction of plasmid integrants is described.

Expression of the cloned toxic shock syndrome toxin 1 gene (tst) in vivo with a rabbit uterine model.

Toxic shock syndrome (TSS) toxin 1 (TSST1) is produced by strains of Staphylococcus aureus associated with TSS. Purified TSST1 induces in rabbits a shock-like illness with many features similar to TSS in humans. These symptoms were also induced by TSST1-producing bacteria in diffusion chambers implanted in the rabbit uterus. Naturally occurring TSST1+ strains and a TSST1- strain harboring a pE194-derived plasmid carrying the cloned TSST1 determinant tst gave the same symptoms. TSST1- strains and a TSST1- strain carrying a pE194-tst plasmid with a deletion of the tst gene had no effect in rabbits. The results with the plasmid-carrying TSST1+ and TSST1- strains, which were isogenic apart from tst, show that the toxin is responsible for the illness in rabbits and suggest that it is a major factor in the pathogenesis of TSS.

Gram-negative endotoxins and staphylococcal toxic shock syndrome.

Strains of Staphylococcus aureus isolated from toxic shock syndrome (TSS) produce toxic shock syndrome toxin 1 (TSST1) which causes a shock-like illness in rabbits with many features similar to TSS in humans. TSST1 is lethal per se in rabbits and also acts synergistically with endotoxin to potentiate lethality. The mode of action of TSST1 is as yet unknown; it has been suggested that it may act by inhibiting the reticuloendothelial system thus allowing endotoxic shock to occur. Rabbits pretreated with polymyxin-B, which prevents the effect of endotoxin, were found to be protected from death by TSST1 indicating that endotoxin is indeed implicated in the pathogenesis of TSS. Specific pathogen-free rabbits which presumably have negligible levels of circulating LPS were susceptible to TSST1 suggesting that very small amounts of endotoxin are sufficient to potentiate lethality. The ways in which TSST1 may allow shock to occur is discussed.

Fertilisation and embryonic survival rates in dairy cows culled as repeat breeders.

Seventy-three Friesian dairy cows culled as repeat breeders were slaughtered three to six, 16 to 19 or 40 to 49 days after insemination to establish fertilisation and embryo survival rates. Fertilisation rate following a single insemination was 72 per cent. The estimated embryo survival rates for days 16 to ¿19 and 40 to 49 were 67 per cent and 76 per cent, respectively. The previous breeding history of the cows showed that only eight (11 per cent) had consistently regular returns (18 to 24 days) to service. Fifty-three (75 per cent) had a combination of both long and short returns to service. Before service on experiment, 64 (89 per cent) cows had regular cycles and the six cows which repeated from the 40 to 49 days group, five (83 per cent) had regular return intervals. It is suggested that errors in oestrus detection contribute substantially to the problem of repeated returns to service of dairy cows in Ireland.