Pathogenesis and inflammatory response in experimental caprine mastitis due to Staphylococcus chromogenes.

Coagulase-negative staphylococci (CNS) are the most frequently isolated bacteria in cases of subclinical mastitis in dairy cows. CNS species may differ in their pathogenicity, but very little is known about their virulence factors or their immune response in intramammary infections. To our knowledge, no experimental studies into the mastitis pathogenesis caused by CNS have been described in lactating goats. The aim of this study was to induce an experimentally Staphylococcus chromogenes mastitis in lactating goats aimed at verifying if the model can be used to evaluate the inflammatory response, the dynamics of infection and the pathological findings within the first hours of intramammary inoculation. Six Saanen goats in mid-lactation were inoculated with 1 × 107 colony forming units of S. chromogenes. Bacterial growth peaked in milk from the challenged right halves of the mammary glands (RMG) at 4 h post inoculation (PI). Shedding of viable bacteria showed a marked decrease at 12 h PI. An increase in mean somatic cell counts was observed in the milk samples from 8 h PI onwards. Mild clinical signs were evoked by intramammary inoculation. Staphylococcus chromogenes could be isolated in tissue from all RMG. Histological examination of specimens of the RMG and lymph nodes of the goats showed an increased inflammatory response throughout the experiment with respect to control halves. In conclusion, the experimental inoculation of S. chromogenes in lactating goats is capable of eliciting an inflammatory response and capable of causing pathological changes. This research represents a preliminary study for a better knowledge of the mastitis pathogenesis caused by S. chromogenes.

Conventional identification of Streptococcus uberis isolated from bovine mastitis in Argentinean dairy herds.

The objective of this study was to evaluate a conventional scheme for identifying Streptococcus uberis strains isolated from bovine mastitis. Seventy-five gram-positive, catalase-negative cocci were collected from cows with mastitis from 19 dairy herds located in the east-central region of Argentina. Five American Type Culture Collection strains and bovine isolates were identified by the API 20 Strep system and by restriction fragment length polymorphism analysis of 16S rDNA. A conventional scheme based on 11 biochemical tests was selected for identification of Strep. uberis strains: the Christie-Atkins-Munch-Petersen reaction; hydrolysis of Arg, esculin, and sodium hippurate; growth in inulin, mannitol, raffinose, salicin, and sorbitol; and growth at 45 degrees C and in 6.5% NaCl. Reference strains and 25 bovine isolates were classified accurately to the species level by the conventional scheme in a blind assay. Each reference strain and each bovine isolate were identified as belonging to the same species following these 3 methods. The remaining 50 isolates identified as Strep. uberis by the API 20 Strep system and 16S rDNA RFLP were assayed by the conventional scheme. This scheme correctly identified 47 (94%) of 50 isolates as Strep. uberis by comparing their biochemical profile with that of the reference strain. Three (6%) of the 50 isolates were classified as Strep. uberis by the API 20 Strep system and by 16S rDNA RFLP and were identified as Enterococcus faecalis by the conventional scheme. Thirty percent of the Strep. uberis strains showed biochemical profiles identical to the Strep. uberis American Type Culture Collection 27958 strain. Seventy percent of the Strep. uberis strains demonstrated variability compared with the reference strain, resulting in 19 different biochemical profiles. The conventional scheme proposed in this study resulted in a relatively low number of misidentifications and could biochemically identify not only typical, but also atypical Strep. uberis strains. This conventional scheme can be considered an adequate method for identifying Strep. uberis strains isolated from bovine mastitis because of its affordable cost in developing countries, and it may contribute to determining the frequency of isolation of Strep. uberis strains in Argentinean dairy herds.

Field trials of a vaccine against bovine mastitis. 2. Evaluation in two commercial dairy herds.

A vaccine against bovine mastitis was developed. The vaccine was based on inactivated, highly encapsulated Staphylococcus aureus cells; a crude extract of Staph. aureus exopolysaccharides; and inactivated unencapsulated Staph. aureus and Streptococcus spp. cells. In this study, the vaccine was evaluated in 164 cows from two commercial dairies (A and B) during a 4-mo period. Two doses of the vaccine were administered subcutaneously to 82 cows in the brachiocephalicus muscle of the neck within a 4-wk interval. The results of this trial revealed significantly fewer intramammary infections caused by Staph. aureus at various levels of severity (clinical, subclinical, and latent) in cows that were vaccinated. The odds ratios of all types of intrammammary infections caused by Staph. aureus for dairies A and B, which were determined by a logistic model, were 1.84 and 1.89, respectively, for quarters of vaccinated cows and quarters of control cows. The colony counts for Staph. aureus in milk from infected quarters of vaccinated cows were significantly lower than those in milk from infected quarters of control cows. Also, the somatic cell counts per milliliter in milk from vaccinated cows were significantly decreased when the initial somatic cell count was < 500,000 cells/ml at the start of the trial. The vaccine had no observable effect on fat production in milk or on streptococcal infections.

[Comparison of detection methods for the Staphylococcus aureus capsule]. / Comparación de métodos de detección de cápsula de Staphylococcus aureus.

Eleven methods for capsule detection of Staphylococcus aureus were compared. The most suitable of them were transmission electron microscopy, determination of the presence of clumping factor, determination of colonial morphology in serum-soft agar, estimation of cell volume and staining with safranine. The determination of clumping factor is a fast and effective method for presumptive diagnosis of capsulated strains, but need to be confirmed by another method. The cell volume estimation is useful for determination of capsule production in liquid cultures, while staining with safranine is suitable for genetic studies of capsule production. The other methods analyzed in this work (Indian ink staining, use of anticapsular antisera, determination of virulence for mice, lisostaphin susceptibility, resistance to phages and resistance to phagocytosis) were laborious, too slow, or need components and/or equipment not available in all laboratories. In addition, two methods of induction of capsule production were assayed, one in vitro by several passages in broth with 10% bovine serum and the other in vitro by intraperitoneal inoculation in mice. Both methods induced capsule production.

Pathogenicity in mice of Staphylococcus aureus mutants deficient in exoprotein synthesis.

Twelve mutants were isolated from a Staphylococcus aureus strain derived from bovine mastitis after mutagenesis by ultraviolet light. These mutants were found to be deficient for several characteristics such as production of most exoproteins and had altered phage type and/or colonial morphology in serum-soft agar medium. They also differed in virulence when assayed in mice by intraperitoneal administration; the ratio of the LD50 of the mutants vs. that of the parental strain ranged from 1 to 123. The different virulence of the mutants could not be associated with lack of production of exoproteins or altered colonial morphology. On the other hand, a clear correlation was evidenced between lowered virulence and slower growth rate at 37 degrees C. Three mutants were assayed in the mouse mastitis model. One of them, which was about 40 times less virulent when assayed by intraperitoneal administration, induced a histopathological lesion similar to that produced by the parent strain; the other two mutants, which were about 70 to 120 times less virulent by intraperitoneal administration, induced only a very slight lesion. Mice were vaccinated by the intraperitoneal route with two of the less virulent mutants; the LD50 in the vaccinated mice that were challenged with the parental strain increased 11 to 14 times compared with that for the unvaccinated mice.