Flagella are an important virulence factor in the subclinical persistence of Escherichia coli in bovine mammary gland.

We compared the virulence profile and REP-PCR genotypes of Escherichia coli strains isolated from subclinical and clinical mastitis cases and dairy farm environments in Minas Gerais State, Brazil, to determine virulence factors and genotypes potentially associated with subclinical persistence in the udder. The virulence profile was obtained by the search for three virulence genes: lpfA (long polar fimbriae), fliC (flagella), and escN (type III secretion system). Subclinical isolates exhibited mainly the fliC gene (33.33%) and fliC + escN genes (30.30%). Clinical isolates exhibited mainly fliC + escN genes (50%) and environmental isolates the lpfA + escN genes (58.04%). Strains isolated from subclinical mastitis showed 6.75 times more positivity to fliC than environmental isolates. Thirty-four genotypes were observed in the REP-PCR analysis, and clinical mastitis isolates indicated more genetic proximity to dairy farm environment isolates than subclinical mastitis isolates. In conclusion, the results suggested that flagella may be an important virulence factor for mammary persistent E. coli infection in cattle, however, none of the E. coli REP-PCR genotypes were associated with subclinical infection.

Estimate of the economic impact of mastitis: A case study in a Holstein dairy herd under tropical conditions.

The aim of this study was to estimate the economic impact of mastitis at the herd level and the weight (percent) of the components of this impact in a Holstein dairy herd under tropical conditions. Three estimates of the economic impact of mastitis were performed. In estimates 1 and 2 the real production and economic indices from February 2011 to January 2012 were considered. In the estimate 1, indices for mastitis classified as ideal were considered, whereas in the estimate 2, the mastitis indices used were those recorded at the farm and at Holstein Cattle Association of Minas Gerais State database (real indices). Ideal mastitis indices were bulk milk somatic cell counts less than 250,000 cells/mL, incidence of clinical mastitis less than 25 cases/100 cows/year, number of culls due to udder health problems less than 5% and the percentage of cows with somatic cell counts greater than 200,000 cells/mL less than 20%. Considering the ideal indices of mastitis, the economic impact was US$19,132.35. The three main components of the economic impact were culling cows (39.4%) and the reduction in milk production due to subclinical and clinical mastitis (32.3% and 18.2%, respectively). Estimate 2 using real mastitis indices showed an economic impact of US$61,623.13 and the reduction in milk production due to mastitis (77.7%) and milk disposal (14.0%) were the most relevant components. The real impact of culling cows was approximately 16 times less than the weight that was considered ideal, indicating that this procedure could have been more frequently adopted. The reduction in milk production was 27.2% higher than the reduction in Estimate 1, indicating a need to control and prevent mastitis. The estimate 3 considered the same indices as estimate 2, but for the period from February 2012 to January 2013. Its economic impact was US$91,552.69. During this period, 161 treatments of cows with an intramammary antibiotic were performed to eliminate Streptococcus agalactiae, and eight cows chronically infected with Staphylococcus aureus were culled. The reduction in milk production due to mastitis was the main component of the economic impact (54.9%). The culling of cows with chronic infection was associated with an increase in the economic impact of mastitis and a reduction in the average productivity per cow. At the herd level reduction in milk production was the component that presented the largest weight in the economic impact of the disease.

Characterisation of penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine milk samples in Minas Gerais, Brazil.

This Regional Research Communication describes the characterisation of ampicillin, penicillin and tetracycline resistance in Staphylococcus aureus isolated from bovine subclinical mastitis in Minas Gerais State, Brazil. Ninety S. aureus isolates from bovine mastitis exhibiting phenotypic resistance to ampicillin, penicillin and/or tetracycline were selected for this study. The minimum inhibitory concentration (MIC) of each antibiotic was determined using the E-Test® and the production of beta-lactamase was determined by cefinase disks. The resistance genes blaZ, tet(K), tet(L), tet(M), and tet(O) were investigated by PCR in all of the isolates. The MIC results classified 77, 83 and 71% of the isolates as resistant to ampicillin, penicillin and tetracycline, respectively. The MIC50 and MIC90 were, respectively, 1 and 2 µg/ml for ampicillin, 0·5 and 1 µg/ml for penicillin and 32 and 64 µg/ml for tetracycline. Eighty-six per cent of beta-lactamase producing isolates were detected. Of the 90 isolates investigated, 97% amplified blaZ, 84% amplified tet(K), 9% amplified tet(L), 2% amplified tet(M) and 1% amplified tet(O). Seventy-nine isolates (88%) showed blaZ together with at least one tet gene. S. aureus isolates showed high MIC50 and MIC90 values for the three antimicrobials. The blaZ and tet(K) genes were widespread in the herds studied, and most of the isolates harboured blaZ and tet(K) concomitantly.

Genotypic and phenotypic detection of capsular polysaccharide and biofilm formation in Staphylococcus aureus isolated from bovine milk collected from Brazilian dairy farms.

Staphylococcus aureus is a pathogen that frequently causes mastitis in bovine herds worldwide. This pathogen produces several virulence factors, including cell-associated adhesins, toxic and cytolytic exoproteins, and capsular polysaccharides. The aim of the present study was to test for the presence of genes involved in capsular polysaccharide production and biofilm formation in S. aureus isolated from bovine mastitis samples collected from 119 dairy herds located in three different Brazilian regions, as well as to assay the production of capsular polysaccharides and biofilm, in vitro. The detection of the cap, icaAD, and bap genes was performed using PCR. The detection and quantification of capsular polysaccharide production was performed using ELISA assays. The ability of the isolates to form a biofilm was examined using the polystyrene surface of microtiter plates. All 159 S. aureus isolates investigated harboured the cap gene: 80 % carried the cap5 gene and 20 % carried the cap8 gene. Sixty-nine percent of the isolates expressed capsular polysaccharide (CP) in vitro, 58 % expressed CP5 and 11 % expressed CP8. All of the isolates harboured the icaA and icaD genes, and 95.6 % of the isolates carried the bap gene. Of the 159 isolates analysed, 97.5 % were biofilm producers. A significant association between the capsular genotype and phenotype and the amount of biofilm formation was detected: cap5/CP5 isolates tended to form more biofilm and to produce a thinner CP layer than cap8/CP8 isolates. The results indicate a high potential for pathogenicity among S. aureus isolated from bovine milk collected from three different regions in Brazil.