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.

Species-level identification of staphylococci isolated from bovine mastitis in Brazil using partial 16S rRNA sequencing.

Staphylococci isolated from bovine milk and not classified as Staphylococcus aureus represent a heterogeneous group of microorganisms that are frequently associated with bovine mastitis. The identification of these microorganisms is important, although it is difficult and relatively costly. Genotypic methods add precision in the identification of Staphylococcus species. In the present study, partial 16S rRNA sequencing was used for the species identification of coagulase-positive and coagulase-negative staphylococci isolated from bovine mastitis. Two hundred and two (95%) of the 213 isolates were successfully identified at the species level. The assigning of an isolate to a particular species was based on ≥99% identity with 16S rRNA sequences deposited in GenBank. The identified isolates belonged to 13 different Staphylococcus species; Staphylococcus chromogenes, S. aureus and Staphylococcus epidermidis were the most frequently identified species. Eight isolates could not be assigned to a single species, as the obtained sequences showed 99% or 100% similarity to sequences from two or three different Staphylococcus species. The relatedness of these isolates with the other isolates and reference strains was visualized using a cladogram. In conclusion, 16S rRNA sequencing was an objective and accurate method for the proper identification of Staphylococcus species isolated from bovine mastitis. Additional target genes could be used in non-conclusive cases for the species-level identification of these microorganisms.

Uso de PCR e sequenciamento do rDNA 16S para identificação de bactérias do gênero staphylococcus isoladas de mastite bovina / Identification of staphylococcus strains isolated from bovine mastitis by PCR and 16S rDNA sequencing

O objetivo deste trabalho foi identificar espécies de Staphylococcus (n=100) isoladas de mastite em rebanhos bovinos do Estado de Minas Gerais. Para esta finalidade foram utilizadas reações de PCR empregando oligonucleotídeos iniciadores descritos anteriormente para amplificar genes específicos de S. aureus (femA), S. intermedius (rDNA 16S) e S. hyicus (rDNA 16S-23S) e o sequenciamento do rDNA 16S. De acordo com as reações de PCR, 83 isolados foram identificados como S. aureus, 13 isolados como S. intermedius, dois como S. hyicus e dois isolados não foram identificados. Foram submetidos ao sequenciamento do rDNA 16S seis isolados identificados como S. aureus e os 17 restantes. Os seis isolados identificados como S. aureus confirmaram essa identificação. Dos outros 17 isolados, 13 foram identificados como S. chromogenes e quatro como S. hyicus, com similaridade igual ou superior a 99%. Baseando-se nos resultados da reação de PCR do gene femA e do sequenciamento do rDNA 16S, foram identificados 83 S. aureus, 13 S. chromogenes e quatro S. hyicus. Neste estudo os oligonucleotídeos iniciadores empregados na reação de PCR para S. intermedius não foram específicos, pois amplificaram também S. chromogenes; e os empregados na reação de PCR para S. hyicus não foram sensíveis, pois falharam na identificação de dois isolados de S. hyicus. A identificação definitiva das duas últimas espécies somente foi possível pelo sequenciamento do rDNA 16S.

The objective of this study was to identify the species of 100 isolates of Staphylococcus from mastitis in dairy cows from herds located in the state of Minas Gerais, Brazil. PCR reactions were carried out using specific primers described previously for S. aureus (femA gene), S. intermedius (16S rDNA) and S. hyicus (16S-23S rDNA spacer region). In addition, products of amplification of variable regions of the 16S rDNA gene of the strains were sequenced. According to the results of the PCR, 83 strains were identified as S. aureus, 13 as S. intermedius, two as S. hyicus and two isolates were not identified. The sequencing of 16S rDNA was applied to 23 strains identified by PCR amplifications: six S. aureus and the strains identified as S. intermedius (n=13), S. hyicus (n=2) or not identified (n=2). The sequencing of 16S rDNA confirmed the six strains as S. aureus. The others 17 strains were identified as S. chromogenes (13 isolates) and S. hyicus (four isolates). Each sample was related to a specie according to the smallest E-value and highest similarity (≥ 99%). The identification of S. hyicus and S. chromogenes was accomplished only by 16S rDNA sequencing.

Retail survey of Brazilian milk and Minas frescal cheese and a contaminated dairy plant to establish prevalence, relatedness, and sources of Listeria monocytogenes isolates.

A study was designed to recover Listeria monocytogenes from pasteurized milk and Minas frescal cheese (MFC) sampled at retail establishments (REs) and to identify the contamination source(s) of these products in the corresponding dairy processing plant. Fifty milk samples (9 brands) and 55 MFC samples (10 brands) were tested from REs located in Juiz de Fora, Minas Gerais, Brazil. All milk samples and 45 samples from 9 of 10 MFC brands tested negative for L. monocytogenes; however, "brand F" of MFC obtained from REs 119 and 159 tested positive. Thus, the farm/plant that produced brand F MFC was sampled; all samples from the milking parlor tested negative for L. monocytogenes, whereas several sites within the processing plant and the MFC samples tested positive. All 344 isolates recovered from retail MFC, plant F MFC, and plant F environmental samples were serotype 1/2a and displayed the same AscI or ApaI fingerprints. Since these results established that the storage coolers served as the contamination source of the MFC, plant F was closed so that corrective renovations could be made. Following renovation, samples from sites that previously tested positive for the pathogen were collected from the processing environment and from MFC on multiple visits; all tested negative for L. monocytogenes. In addition, on subsequent visits to REs 159 and 119, all MFC samples tested negative for the pathogen. Studies are ongoing to quantify the prevalence, levels, and types of L. monocytogenes in MFC and associated processing plants to lessen the likelihood of listeriosis in Brazil.

Molecular analysis of the plasmid-borne aacA/aphD resistance gene region of coagulase-negative staphylococci from chickens.

OBJECTIVES: The aim of this study was to analyse selected coagulase-negative staphylococci from chickens for the genetic basis of plasmid-borne resistance to gentamicin, tobramycin and kanamycin, and also for the presence of Tn4001-like elements. METHODS: Three staphylococcal strains, two Staphylococcus warneri, and one Staphylococcus sciuri, were included in this study. The gene aacA/aphD coding for a bifunctional enzyme that mediates resistance to gentamicin, tobramycin and kanamycin was detected by hybridization with a specific probe. Plasmid location of this gene was also confirmed by hybridization and conjugation. The resistance gene and its adjacent regions were cloned and sequenced. RESULTS: Three different types of Tn4001-like elements in which the IS256 elements were largely truncated and replaced by IS257 elements were identified on large conjugative and non-conjugative plasmids of 33-43 kb in the staphylococcal strains from chickens. Seven different types of IS257-analogous insertion sequences were identified. CONCLUSIONS: Resistance to gentamicin, tobramycin and kanamycin in three staphylococcal strains from chickens was mediated by plasmid-borne aacA/aphD genes located on structurally modified Tn4001-like elements. In one of the three plasmids studied, the arrangement of the elements in the aacA/aphD resistance gene area closely resembled that on plasmids pSH6, pSK41 and pUW3626 from Staphylococcus aureus of human origin.