Application of a Dot Blot Hybridization Platform to Assess Streptococcus uberis Population Structure in Dairy Herds.

Streptococcus uberis is considered one of the most important pathogens associated with bovine mastitis. While traditionally acknowledged as an environmental pathogen, S. uberis has been shown to adopt a contagious epidemiological pattern in several dairy herds. Since different control strategies are employed depending on the mode of transmission, in-depth studies of S. uberis populations are essential to determine the best practices to control this pathogen. In this work, we optimized and validated a dot blot platform, combined with automatic image analysis, to rapidly assess the population structure of infective S. uberis, and evaluated its efficiency when compared to multilocus sequence analysis (MLSA) genotyping. Two dairy herds with prevalent S. uberis infections were followed in a 6 month period, in order to collect and characterize isolates from cows with persistent infections. These herds, located in Portugal (Barcelos and Maia regions), had similar management practices, with the herd from Barcelos being smaller and having a better milking parlor management, since infected cow segregation was immediate. A total of 54 S. uberis isolates were obtained from 24 different cows from the two herds. To overcome operator-dependent analysis of the dot blots and increase the technique's consistency and reliability, the hybridization signals were converted into probability values, with average probabilities higher than 0.5 being considered positive results. These data allowed to confirm the isolates' identity as S. uberis using taxa-specific markers and to determine the presence of virulence- and antibiotic resistance-related genes. In addition, MLSA allowed to disclose the most prevalent S. uberis clonal lineages in both herds. Seven different clusters were identified, with Barcelos showing a high clonal diversity and Maia a dominant lineage infecting most cows, suggesting distinct epidemiological patterns, with S. uberis displaying an environmental or contagious transmission pattern depending on the herd. Overall, this work showed the utility of dot blot and MLSA to characterize population structure and epidemiological patterns of mastitis-causing S. uberis. This approach allowed to disclose prevalent virulence patterns and clonal lineages of S. uberis in two distinct herds, and gain insights on the impact of herd management practices on pathogen population structure.

Detection and discrimination of common bovine mastitis-causing streptococci.

Detection and typing of bovine mastitis pathogens are currently limited by time-consuming and culture-based techniques. In this work, a novel genus-specific DNA marker for Streptococcus and species-specific DNA markers for the prevalent mastitis pathogens Streptococcus agalactiae and Streptococcus uberis were designed and assessed. In order to enable further discrimination of these mastitis-causing streptococci, metabolic and pathogenicity-related genes were used to infer additional functional markers. A total of 12 DNA markers were validated with a set of 50 reference strains and isolates, representative of the Streptococcus genus, of closely related species and of microorganisms with matching habitats. The experimental validation, using dot blot hybridization under high stringency conditions, confirmed the specificity of the selected markers. The broad-spectrum taxonomic marker (ST1) was specific to the Streptococcus genus and the markers selected for S. agalactiae (A1 and A2) and S. uberis (U1 and U2) were shown to be species-specific. The functional markers revealed strain-specific patterns of S. agalactiae and S. uberis. Markers derived from the fructose operon (FO1 and FO3) were specific to bovine isolates of S. agalactiae, and the nisin operon markers (NU1 and NU3) were able to discriminate isolates belonging to S. agalactiae and S. uberis. The virulence-associated markers (V1, V2 and V3) allowed the detection of S. uberis and of closely related species. This work suggests that the combined use of these novel taxa-specific markers coupled with discriminatory functional markers presents a promising approach for the rapid and cost-effective detection and discrimination of common bovine mastitis-causing pathogens, which will contribute to an improved treatment and control of this disease.

Identification of immunoreactive extracellular proteins of Streptococcus agalactiae in bovine mastitis.

Streptococcus agalactiae is a common pathogen that causes bovine mastitis. The aims of this study were to evaluate the antibody response against S. agalactiae extracellular proteins in the whey and serum of naturally infected bovines and to identify possible immunodominant extracellular antigens. IgG1 antibodies against S. agalactiae extracellular proteins were elevated in the whey and serum of naturally infected bovines. In the whey, the levels of IgG1 specific for S. agalactiae extracellular proteins were similar in infected and noninfected milk quarters from the same cow, and the production of antibodies specific for S. agalactiae extracellular proteins was induced only by infection with this bacterium. The immunoreactivity of extracellular proteins with bovine whey was clearly different in infected versus control animals. Group B protective surface protein and 5'-nucleotidase family protein were 2 major immunoreactive proteins that were detected only in the whey of infected cows, suggesting that these proteins may be important in the pathogenesis of S. agalactiae-induced mastitis. This information could be used to diagnose S. agalactiae infection. In addition, these antigens may be useful as carrier proteins for serotype-specific polysaccharides in conjugate vaccines.