Identification of bovine mastitis pathogens using MALDI-TOF mass spectrometry in Brazil.

In this Research Communication we evaluate the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify 380 bacteria isolated from cases of bovine mastitis in Brazil. MALDI-TOF MS identifications were compared to previous identifications by biochemical tests and 16S rRNA sequencing. MALDI-TOF MS achieved a typeability of 95.5%. The accuracy of MALDI-TOF MS for the identification of Staphylococcus isolates was 93.2%. The agreement between MALDI-TOF MS and biochemical identification of Streptococcus agalactiae was 96%, however, the agreement between these techniques for identifying other catalase-negative, Gram-positive cocci was lower. Agreement in identifying Gram-negative bacteria at the genus level was 90.5%. Our findings corroborate that MALDI-TOF MS is an accurate, rapid and simple technique for identifying bovine mastitis pathogens. The availability of this methodology in some research institutions would represent a significant step toward increasing the diagnosis and epidemiological studies of bovine mastitis and other animal infectious diseases in Brazil.

Characterization of biofilms and antimicrobial resistance of coagulase-negative Staphylococcus species involved with subclinical mastitis.

Biofilm formation is a central feature to guarantee staphylococcal persistence in hosts and is associated with several diseases that are difficult to treat. In this research paper, biofilm formation and antimicrobial susceptibility were investigated in staphylococcal strains belonging to several species. These strains were isolated from the milk of cows with subclinical mastitis and most of them were coagulase-negative, with the prevalence of Staphylococcus chromogenes. High genetic diversity was observed among the strains by pulsed field gel electrophoresis. Antimicrobial resistance was assessed by disk diffusion and more than 50% of the strains were resistant to ampicillin and penicillin G, with multi-resistance profiles (13.6%) also being observed. Most strains (65.9%) formed biofilms when cultivated in BHI supplemented with 1% glucose. Most strains (72.7%) carried the intercellular adhesion gene (icaA), while less than half (36.3%) carried the biofilm-associated protein gene (bap). Concentrations of up to 10xMIC of erythromycin and tetracycline were not sufficient to suppress cell viability in preformed biofilms. Our results revealed that a genetically diverse group of biofilm-forming Staphylococcus species can be involved in subclinical mastitis. Since high antimicrobial concentrations cannot eradicate biofilm cells in vitro, their use in dairy animals may be ineffective in controlling infections, while supporting selection of resistant microorganisms. These data reinforce the need for alternative therapies aiming at disrupting biofilms for effective disease control.

Staphylococcus spp. Isolated from Bovine Subclinical Mastitis in Different Regions of Brazil: Molecular Typing and Biofilm Gene Expression Analysis by RT-qPCR.

Bovine mastitis is mainly caused by bacteria of the genus Staphylococcus spp., which possess different virulence factors, including the capacity for biofilm formation that provides enhanced protection against the action of immune system components and serves as a barrier against the penetration of antimicrobial agents. This study aimed to characterize 181 Staphylococcus spp. Strains-including Staphylococcusaureus and coagulase-negative staphylococci (CoNS) isolated from bovine subclinical mastitis in six Brazilian states-by molecular methods. RT-qPCR was used to verify the expression of genes of the ica operon-mainly responsible for biofilm formation-as well as bap and bhp. Chromosome similarity among the isolates was investigated by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). The icaA gene was detected in 79 (43.6%) isolates, icaB in 24 (13.2%), icaC in 57 (31.4%), and icaD in 127 (70.1%). The bap gene was identified in 66 (36.4%) isolates, while the bhp gene was found in nine (4.9%). RT-qPCR confirmed the expression of the icaA gene in 60 (75.9%) isolates, of icaB in six (25%), of icaC in 26 (45.6%), and of icaD in 80 (63%). Clonal typing of the isolates by PFGE permitted the identification of eight Staphylococcusaureus clusters that simultaneously included ≥3 strains, with a similarity of ≥80%. Regarding the other species studied, three clusters were observed for Staphylococcuschromogenes and four clusters for Staphylococcusepidermidis. Only one cluster each was identified for Staphylococcussaprophyticus and Staphylococcussimulans, while the other species did not form any cluster. With respect to MLST, ST126 and ST1 were the prevalent sequence types in S. aureus, while in S.epidermidis all sequence types were different. These results reveal strains with the same evolutionary origin as other isolates, which might cause infections in humans and animals, suggesting their ability to spread between these species.

Cloxacillin benzathine-loaded polymeric nanocapsules: Physicochemical characterization, cell uptake, and intramammary antimicrobial effect.

The present work shows the development and evaluation of the veterinary antibiotic cloxacillin benzathine (CLOXB) loaded into poly-ε-caprolactone (PCL) nanocapsules (NC), as a potential new treatment strategy to manage bovine intramammary infections, such as mastitis. Staphylococcus aureus-induced mastitis is often a recurrent disease due to the persistence of bacteria within infected cells. CLOXB-PCL NC were prepared by interfacial deposition of preformed biodegradable polymer followed by solvent displacement method. The mean diameter of NC varied from 241 to 428 nm and from 326 to 375 nm, when determined by dynamic light scattering and by atomic force microscopy, respectively. The zeta potential of NC was negative and varied from -28 to -51 mV. In vitro release studies from the NC were performed in two media under sink conditions: PBS with 1% polyethylene glycol or milk. A reversed-phase HPLC method was developed to determine the NC entrapment efficiency and kinetics of CLOXB release from the NC. Free CLOXB dissolution occurred very fast in both media, while drug release from the NC was slower and incomplete (below 50%) after 9 h. CLOXB release kinetics from polymeric NC was fitted with the Korsmeyer-Peppas model indicating that CLOXB release is governed by diffusion following Fick's law. The fluorescence confocal microscopy images of macrophage-like J774A.1 cells reveal NC uptake and internalization in vitro. In addition, antimicrobial effect of the intramammary administration of CLOXB-PCL NC in cows with mastitis resulted in no clinical signs of toxicity and allowed complete pathogen elimination after treatment. The in vivo results obtained in this work suggest that CLOXB-PCL NC could be a promising formulation for the treatment of intramammary infections in cattle, considering their physicochemical properties, release profiles and effects on bovine mastitis control.

Accurate identification of atypical Staphylococcus chromogenes plasma-clotting strains causing bovine mastitis / Identificação acurada de cepas atípicas de Staphylococcus chromogenes que coagulam o plasma envolvidas com mastite bovina

ABSTRACT: We compared the potential of routine techniques used for the identification of Staphylococcus species, aiming to evaluate their accuracy in the detection of 43 Staphylococcus chromogenes strains isolated from bovine mastitis that, despite being a coagulase-negative species, are able to clot plasma. These strains could be mistakenly suspected to be S. aureus and lead to an unappropriated treatment of the disease. MALDI-TOF, PCR-RFLP of the chaperonine gene groEL, and sequencing of the 16S rRNA and elongation factor Tu gene tuf were employed. Results from the four methods were coincident for only half of the strains because of the low accuracy of the groEL PCR-RFLP (51.2% accuracy). Even though all the sequencing results were identical, the high accuracy of the MALDI-TOF results (97.7% accuracy, with only one strain misidentified) encourage the use of this technique, since it does not require laborious sample preparation, being fast and simple to perform.

RESUMO: Nós comparamos o potencial de técnicas rotineiras utilizadas para a identificação de espécies de Staphylococcus, com o objetivo de avaliar a acurácia delas na detecção de 43 isolados de Staphylococcus chromogenes envolvidos com mastite bovina que, apesar de ser uma espécie coagulase-negativa, são capazes de coagular o plasma. Essas cepas poderiam ser erroneamente suspeitas de serem S. aureus e levarem a um tratamento não adequado da doença. MALDI-TOF, PCR-RFLP do gene da chaperonina groEL e sequenciamento do gene do rRNA 16S e do gene do fator de elongação Tu, tuf, foram avaliados. Os resultados dos quatro métodos foram coincidentes para apenas metade das cepas, devido à baixa precisão da PCR-RFLP com groEL (51,2% de acurácia). Apesar de todos os resultados do sequenciamento serem idênticos, a alta precisão dos resultados do MALDI-TOF (97,7% de acurácia, com apenas uma cepa identificada incorretamente) encoraja o uso dessa técnica, pois, não requer preparação laboriosa de amostras, sendo rápida e simples de executar.

Short communication: ß-Lactam resistance and vancomycin heteroresistance in Staphylococcus spp. isolated from bovine subclinical mastitis.

The use of antimicrobial agents has led to the emergence of resistant bacterial strains over a relatively short period. Furthermore, Staphylococcus spp. can produce ß-lactamase, which explains the survival of these strains in a focus of infection despite the use of a ß-lactam antibiotic. The aim of this study was to evaluate the resistance of Staphylococcus spp. isolated from bovine subclinical mastitis to oxacillin and vancomycin (by minimum inhibitory concentration) and to detect vancomycin heteroresistance by a screening method. We also evaluated ß-lactamase production and resistance due to hyperproduction of this enzyme and investigated the mecA and mecC genes and performed staphylococcal cassette chromosome mec typing. For this purpose, 181 Staphylococcus spp. isolated from mastitis subclinical bovine were analyzed. Using the phenotypic method, 33 (18.2%) of Staphylococcus spp. were resistant to oxacillin. In contrast, all isolates were susceptible to vancomycin, and heteroresistance was detected by the screening method in 13 isolates. Production of ß-lactamase was observed in 174 (96%) of the Staphylococcus spp. isolates. The mecA gene was detected in 8 isolates, all of them belonging to the species Staphylococcus epidermidis, and staphylococcal cassette chromosome mec typing revealed the presence of type I and type IV isolates.

Staphylococcus chromogenes, a Coagulase-Negative Staphylococcus Species That Can Clot Plasma.

Staphylococcus chromogenes is one of the main coagulase-negative staphylococci isolated from mastitis of dairy cows. We describe S. chromogenes isolates that can clot plasma. Since the main pathogen causing mastitis is coagulase-positive Staphylococcus aureus, the coagulase-positive phenotype of S. chromogenes described here can easily lead to misidentification.

Presence of mecA-positive multidrug-resistant Staphylococcus epidermidis in bovine milk samples in Brazil.

Bacteria of the genus Staphylococcus are one of the major pathogens causing bovine mastitis. In recent decades, resistance of this genus to oxacillin (methicillin) has been a matter of concern due to the possibility of reducing the effectiveness of mastitis treatments and the transfer of resistance determinants to other bacteria. Oxacillin resistance was studied in 170 staphylococci from bovine milk samples, including 79 Staphylococcus aureus and 91 coagulase-negative staphylococci (CNS). The susceptibility profile of 10 antimicrobial agents used in veterinary practice was determined by the Etest method. In addition to the Etest, the phenotypic characterization of oxacillin resistance was tested using the cefoxitin disk diffusion test. All isolates were screened by PCR to detect the mecA gene in 2 different regions of the gene. The isolates with an oxacillin minimum inhibitory concentration ≥0.5 µg/mL or resistant to cefoxitin were identified by sequencing a 536-bp fragment of the 16S rRNA gene. This group of isolates was also evaluated for the presence of blaZ and mecC genes. Molecular analysis of the mecA gene was carried out by typing of the staphylococcal cassette chromosome mec (SCCmec). The relatedness of the mecA-positive isolates was evaluated by macrorestriction of chromosomal DNA followed by pulsed-field gel electrophoresis. With the exception of penicillin and oxacillin, 86% of the isolates showed susceptibility to cephalothin, gentamicin, erythromycin, sulfonamide, trimethoprim-sulfamethoxazole, and tetracycline. All S. aureus isolates were susceptible to oxacillin, whereas 47% (n=43) of the CNS isolates were resistant. The CNS isolates showed a higher resistance to cephalothin, erythromycin, tetracycline, and gentamicin in comparison with S. aureus. The mecA gene was only detected in 10 CNS isolates, identified as Staphylococcus epidermidis, and classified into 3 pulsotypes (A, B, and C) and 4 subtypes (A1, B1, B2, and B3). Among the isolates with an oxacillin resistance phenotype, 12 were positive for the blaZ gene, and 9 of them were mecA-positive. Two of the oxacillin-resistant isolates amplified the mecA homolog gene of Staphylococcus sciuri and none amplified mecC. Three SCCmec types, I, IV, and V, were found. Our results suggest that Staphylococcus epidermidis can be a reservoir for mecA for other Staphylococcus species. Studies investigating the molecular and phenotypic profile of antimicrobial resistance in staphylococcal species should be performed for controlling the spread of resistance and the selection of appropriate therapeutic measures.

Microbiological safety of Minas Frescal Cheese (MFC) and tracking the contamination of Escherichia coli and Staphylococcus aureus in MFC processing.

Minas Frescal cheese (MFC) is a traditional food produced and consumed in Brazil, characterized by its soft texture, low sodium, and high moisture content. This study characterized the microbiological contamination by coliforms, Escherichia coli and Staphylococcus aureus, in 99 MFC samples obtained in retail sale and produced by three distinct industrial procedures. Dairy processors were selected to investigate the key points of E. coli and S. aureus contamination during cheese processing. MFC samples produced by the addition of lactic culture presented higher counts of coliforms and E. coli, when compared to other samples (p<0.05). MFC samples produced by the addition of rennet alone presented higher counts of S. aureus when compared to other samples (p<0.05). Fourteen of 19 MFC samples produced by the addition of lactic culture presented E. coli counts higher than 5 × 10(2) colon-forming units/g. The processing steps after pasteurization were identified as the main sources of E. coli and S. aureus contamination of MFC. Based on the results, MFC was characterized as a potential hazard for consumers due to the high frequency of samples contaminated with E. coli and S. aureus counts in noncompliance with Brazilian standards for sanitary quality and safety.

Relationship between total bacteria counts and somatic cell counts from mammary quarters infected by mastitis pathogens / Relação entre contagem total de bactérias e contagem de células somáticas de quartos mamários infectados por patógenos da mastite

This study was conducted to establish the relationship between somatic cell count (SCC) and bacterial shedding from mammary quarters according to mastitis pathogens. Milk samples from 638 mammary quarters were examined for mastitis pathogens, SCC and total bacterial count (TBC). The raw data of SCC and TBC were used to perform descriptive statistics. The significance of the arithmetic mean differences between SCC and TBC according to bacteriological examination results was determined by a two-tailed unpaired t-test. Pearson and Spearman´s correlations were done with logarithmic data and linear regression analyses. The geometric means of the bacteriological examination results were (cells mL-1; CFU mL-1): no growth (52,000; 12,000), coagulase-negative staphylococci (85,000; 17,000), Staphylococcus aureus (587,000; 77,000); other streptococci (432,000; 108,000) and Streptococcus agalactiae (1,572,000; 333,000). The Pearson and Spearman's correlations between SCC and TBC were higher than 0.60 for all mastitis pathogens. The regression analyses slopes showed different increase in TBC with the same increase in SCC according to mastitis pathogens. The slope for S. agalactiae (0.542) was higher than that for other mastitis pathogens. The results suggest that the intensity of inflammatory process was associated with number of mastitis pathogens shedding from the mammary gland.

Este estudo foi realizado com objetivo de estabelecer a relação entre contagem de células somáticas (CCS) e a liberação de bactérias de quartos mamários de acordo com os patógenos da mastite. Amostras de leite de 638 quartos mamários foram examinadas para identificação dos patógenos da mastite, CCS e contagem total de bactérias (CTB). Estatísticas descritivas foram utilizadas para avaliar os dados brutos de CCS e CTB. A diferença entre médias para CCS e CTB de acordo com os resultados dos exames bacteriológicos foi avaliada pelo teste T para amostras independentes. Foram realizadas a correlação de Pearson, de Spearman e regressão linear com os dados transformados. As médias geométricas de acordo com os resultados dos exames bacteriológicos foram (células mL-1; UFC mL-1): sem crescimento (52.000; 12.000), estafilococos coagulase negativo (85.000; 17.000), Staphylococcus aureus (587.000; 77000); outros estreptococus (432.000; 108.000) e Streptococcus agalactiae (1.572.000; 333.000). A correlação de Pearson e Spearman entre CCS e CTB foi maior que 0,60 para todos os patógenos da mastite. O coeficiente angular das regressões lineares mostrou diferentes aumentos da CTB como o mesmo aumento da CCS de acordo com os patógenos da mastite. O coeficiente angular para o S. agalactiae (0.542) foi maior em relação aos outros patógenos da mastite. Os resultados sugerem que a intensidade do processo inflamatório foi associada com a quantidade de bactérias da mastite liberada pela glândula mamária.

Antimicrobial activity of marine sponges against coagulase-negative staphylococci isolated from bovine mastitis.

Bovine mastitis remains worldwide a major challenge for the dairy industry despite the widespread implementation of control strategies. The increasing number of coagulase-negative staphylococci (CNS) causing mastitis and of bacteria resistant to conventional antibiotics has become a serious problem in recent years. Marine sponges are a rich source of bioactive compounds, and many species can be useful for the development of new antimicrobial drugs. In the present study, 49 CNS strains were isolated from bovine mastitis cases from 21 different dairy herds kept at farms in Southeast Brazil. Strains were analyzed for antimicrobial susceptibility and mecA gene detection. Fifty-nine percent of the CNS strains were resistant to at least one of the drugs tested and 12.2% were classified as multiresistant. Three strains carried the mecA gene, confering resistance to the beta-lactamic antibiotics. In addition, the CNS strains were submitted to in vitro screening for antimicrobial activities of extracts from marine sponges. Extracts from the sponge species Cinachyrella sp., Haliclona sp. and Petromica citrina showed antibacterial activity against 61% of the CNS strains, including strains resistant to conventional antibiotics. Extracts from P. citrina showed the largest spectrum of inhibitory activity. The aqueous extract inhibited 51% of the CNS strains and presented a bactericidal effect over susceptible and multiresistant-bacteria at a minimal inhibitory concentration of 1.024µg/ml. This study shows the potential of marine sponges as new sources of antibiotics and disinfectants for the control of CNS involved in bovine mastitis.

Composition and bulk tank somatic cell counts of milk from dairy goat herds in Southeastern Brazil / Composição e contagem de células somáticas no leite de rebanhos caprinos do Sudeste do Brasil

The milk composition and somatic cell count (SCC) are requirements for assessment milk quality and mastitis in goat herds. Studies conducted with dairy goat herds indicated that the milk composition differed among them to due to factors such as genetic, feeding, system of production, stage of lactation, year and year-season. The objective of this study was to assess SCC and other milk quality indicators (fat, protein, lactose, and total solids) for goat milk bulk tank. The influence of the herd and year-season on the milk composition as well as herd, milking system and year-season on SCC was also evaluated. Thirteen Brazilian dairy goat herds with about 1,400 dairy goats were included in the study. Six herds were milked by hand and in the remaining seven machine milking was used. Herds were sampled at weekly intervals during two lactations. A total of 913 bulk milk samples were analysed using a automated equipment. The general average percent values for fat, protein, lactose and total solids were, respectively,3.44, 2.95, 4.45 and 11.69. The effect of herd and season was significant for all milk components and bulk milk goat somatic cell count (SCC).The SCC average of all 13 herds was 779,000 cells/ml. The average SCC values of herds milked by hand and by machine were 1,121,000and 848,000 cells/ml respectively. In both groups, the SCC was lowerin the winter and higher in the autumn. Herd characteristics were responsible for higher variability on components and SCC in

A composição do leite bem como a contagem de células somáticas (CCS) são requisitos para avaliar a qualidade do leite e mastite em rebanhos caprinos. Estudos conduzidos indicaram que a composição do leite varia entre os rebanhos devido a fatores genéticos, alimentação, sistema de produção, estágio de lactação, ano e estação do ano. O objetivo do estudo foi avaliar a CCS e outros indicadores de qualidade(gordura, proteína, lactose e sólidos totais) no leite de rebanhos caprinos. A influência do rebanho e estação do ano sobre a composição bem como a influência de rebanho, tipo de ordenha e estação do ano sobre a CCS também foi avaliado. Treze rebanhos caprinos localizados no Brasil, com aproximadamente 1.400 matrizes foram incluídos no estudo, Sendo ordenhados manualmente e os outros sete com equipamento de ordenha. As amostras de leite dos rebanhos foram coletadas semanalmente durante duas lactações. O total de 913 amostras foi analisado no equipamento automatizado. A média dos valores percentuais para gordura, proteína, lactose e sólidos totais foram, respectivamente; 3,44; 2,95; 4,45 e 11,69. O efeito de rebanho e estação do ano foi significante para todos os componentes do leite e CCS. A média para CCS de todos 13 rebanhos foi 779.000 células/ml. As médias para CCS dos rebanhos ordenhados manualmente e mecanicamente foram, respectivamente, 1.121.000 e 848.000 células/ml. Em ambos grupos, a CCS foi menor no inverno e maior no outono. As características de rebanho foram responsáveis pela maior variação dos componentes e CCS no leite de rebanhos caprinos

Contagem, isolamento e caracterização de bactérias psicrotróficas contaminantes de leite cru refrigerado / Counting, isolation and characterization of psychrotrophic bacteria from refrigerated raw milk

Com os objetivos de quantificar, isolar e caracterizar bactérias psicrotróficas contaminantes de leite cru refrigerado, produzido na região da Zona da Mata de Minas Gerais e Sudeste do Rio de Janeiro, foram analisadas amostras de leite coletadas de 20 tanques coletivos e 23 tanques individuais. As contagens de bactérias psicrotróficas nas amostras de leite para os dois tipos de tanques de refrigeração variaram entre 10² e 10(7) Unidades Formadoras de Colônias (UFC) ml-1, porém, um maior número de tanques coletivos apresentou contagens acima de 1 x 10(5) UFC ml-1. Foi verificada a predominância de bactérias psicrotróficas gram-negativas (81,2 por cento), que foram identificadas pelos sistemas API 20E e API 20NE nos gêneros: Aeromonas, Alcaligenes, Acinetobacter, Burkholderia,Chryseomonas, Enterobacter, Ewingella, Klebsiella, Hafnia, Methylobacterium, Moraxella, Pantoea, Pseudomonas, Serratia, Sphingomonas e Yersinia. As bactérias gram-positivas (18,8 por cento) foram identificadas com API 50 CH, API Coryne e API Staph, nos gêneros: Bacillus, Brevibacterium, Cellum/Microbacterium, Kurthia e Staphylococcus. Os sistemas API utilizados não identificaram todos os isolados bacterianos. Pseudomonas foi o gênero mais isolado e P. fluorescens foi a espécie predominante. A maioria dos isolados bacterianos apresentou atividade proteolítica e/ou lipolítica a temperaturas de refrigeração de 4ºC, 7ºC e 10ºC, evidenciando seu alto potencial de deterioração do leite e dos produtos lácteos. Os resultados ressaltam que maior atenção deve ser dada aos procedimentos que impeçam a contaminação do leite por esses microrganismos.

This study aimed to quantify, isolate and characterize psychrotrophic bacteria from refrigerated raw milk produced at the 'Mata' Region of Minas Gerais State and Southeast of Rio de Janeiro State, Brazil. Raw milk samples, were collected at the farms, from 20 collective refrigerated tanks and 23 individual refrigerated tanks. The psychrotrophic bacteria counting ranged from 10² to 10(7) Colony Forming Units (CFU) ml-1 for both types of refrigerated tanks, but most of the collective tanks showed counts higher than 1 x 10(5) CFU ml-1. Predominance of psychrotrophic gram-negative bacteria (81.2 percent), that were identified by API 20E and API 20NE as belonging to genera: Aeromonas, Alcaligenes, Acinetobacter, Burkholderia, Chryseomonas, Enterobacter, Ewingella, Klebsiella, Hafnia, Methylobacterium, Moraxella, Pantoea, Pseudomonas, Serratia, Sphingomonas e Yersinia were oserved. The gram-positive bacteria (18.8 percent), were identified by API 50 CH, API Coryne and API Staph, to genera: Bacillus, Brevibacterium, Cellum/Microbacterium, Kurthia e Staphylococcus. The API systems utilized could not identify all the bacterial isolates. Pseudomonas was the genus most isolated with P. fluorescens as the predominant species. Most of the isolates presented proteolytic and/or lipolytic activity at 4ºC, 7ºC and 10ºC showing high potential for milk and milk products spoilage. The results indicated that more attention must be taken to the procedures necessaries to reduce milk contamination with psychrotrophic bacteria.

Identification of coagulase-negative staphylococci from bovine mastitis using RFLP-PCR of the groEL gene.

Coagulase-negative staphylococci (CNS) have become the predominant pathogens causing bovine mastitis in many countries. CNS infections are associated with damage to milk secretory tissue of the mammary gland by increased connective tissue stroma, moderate increases of somatic cells count in milk and significant production decreases. These consequences impose serious economic losses for the farmers and the dairy industry. Routine veterinary laboratories do not usually identify CNS at the species level. Thereby, the aims of this study were to identify the most common staphylococcal pathogens involved in bovine mastitis using PCR-restriction fragment length polymorphism (RFLP) analysis of a partial groEL gene sequence and to compare our results with the identification carried out by the conventional method. A total of 54 isolates of Staphylococcus, involved in bovine mastitis, were analyzed by this method. The size and number of the fragments obtained by either AluI or HindIII/PvuII digestions made possible to form clear patterns differentiating, among the isolates, 11 of the most common species of animal staphylococcal pathogens. Most of the isolates clustered together with the reference strain of Staphylococcus chromogenes (28) and the type strain of Staphylococcus epidermidis (8). Besides, some isolates clustered together with the type strain of Staphylococcus aureus (5). All patterns were confirmed by the conventional biochemical method, showing concordant results. Thus, the PCR-RFLP of the groEL gene constitutes a reliable and reproducible molecular method for identification of CNS species responsible for bovine mastitis.

Esquema simplificado para identificaçäo de estafilococos coagulase-positivos isolados de mastite bovina / Simplifieed scheme for identification of coagulase-positive staphylococci isolated from bovine mastitis

Os testes de produçäo de acetoína, determinaçäo da atividade da enzima ß-galactosidase e utilizaçäo anaeróbica do manitol em conjunto com a susceptibilidade à acriflavina foram avaliados para diferenciaçäo de amostras de Staphylococcus coagulase-positivas (SCP) isoladas de mastite bovina. As amostras foram classificadas no gênero Staphylococcus por meio da sensibilidade a furazolidona, resistência à bacitracina, produçäo de ácido em aerobiose a partir de glicerol na presença de 0,4µg ml(-1) de eritromicina e catalase, e foram positivas no teste de coagulase do plasma de coelho em tubos. A susceptibilidade à acriflavina foi testada em placas de ágar P com 7,0µg ml(-1) de acriflavina. Como controle dos testes, foram incluídas cinco amostras coagulase-negativas de S. hyicus isoladas de leite bovino e identificadas pelo sistema API Staph e a amostra de S. aureus ATCC 29213. Trinta e oito das 49 amostras de SCP foram identificadas como S. aureus e 11 como S. hyicus, näo sendo identificada nenhuma como S. intermedius. O sistema API Staph foi empregado para confirmar a identificaçäo das amostras coagulase-positivas de S. hyicus, sete amostras de S. aureus negativas no teste de produçäo de acetoína e quatro negativas na fermentaçäo anaeróbica do manitol. Todas as amostras de S. aureus foram resistentes a acriflavina, enquanto as de S. hyicus foram sensíveis. Concluiu-se que a sensibilidade a acriflavina pode ser empregada juntamente com os testes de coagulase e produçäo de acetoína na diferenciaçäo de SCP isolados de mastite bovina.

Contagem bacteriana da superfície de tetas de vacas submetidas a diferentes processos de higienizaçäo, incluindo a ordenha manual com participaçäo do bezerro para estimular a descida do leite / Bacterial counts on the surface of the teats of cows milked and different methods of udder preparation, including cows milked by hand and stimulated by suckling a calf

A pele da teta é uma das principais fontes de contaminaçäo microbiana do leite cru, além de ser uma fonte de infecçäo da glândula mamária. Este estudo comparou diferentes métodos de preparaçäo do úbere, incluindo a participaçäo do bezerro para estimular a descida do leite, em relaçäo à contaminaçäo microbiana da pele da teta. Um experimento com 36 vacas ordenhadas mecanicamente foi conduzido para comparar três procedimentos (A, B, C) de preparaçäo do úbere. Duas tetas de cada vaca foram amostradas para contagem total de bactérias (CTB) e de coliformes, antes e após lavagem com água e secagem com papel toalha descartável (A); procedimento A, mais imersäo das tetas em soluçäo de iodo (4.000ppm) e secagem com papel toalha descartável (B); higienizaçäo com toalha de papel embebida em soluçäo desinfetante contendo clorexidina (C). O segundo experimento incluiu 16 vacas ordenhadas manualmente, na presença do bezerro. Permitiu-se a cada bezerro mamar as quatro tetas por alguns segundos, de acordo com o manejo do rebanho. Amostras para bacteriologia de duas tetas de cada vaca foram coletadas, antes, após a mamada do bezerro, e após higienizaçäo semelhante ao procedimento C, citado acima. Os tratamentos foram analisados usando-se o teste näo-paramétrico de Kruskal-Wallis, considerando-se os dados de tCTB após o uso de um dos métodos de preparaçäo do úbere ou da mamada do bezerro. Usou-se a transformaçäo das contagens bacterianas para a escala logarítmica em base 10, com a fórmula tCTB=log10 (CTB+0,5). Para os grupos A,B e C (primeiro experimento), Os valores médios de diminuiçäo de tCTB foram 1,8, 2,2 e 2,4, respectivamente. No segundo experimento, as CTB's aumentaram significativamente (p<0,05) de 3,2 para 4,3 após a mamada do bezerro, mas houve uma reduçäo (P<0,01) de mais de dez vezes após a aplicaçäo do desinfetante (2,0). Os resultados indicaram que: (a) lavagem das tetas com água seguida de secagem com papel toalha reduz o número de microrganismos da superfície das tetas, mas a reduçäo é muito maior quando se emprega um desinfetante; (b) a mamada do bezerro antes da ordenha aumenta em mais de dez vezes o número de bactérias na superfície das tetas; (c) o número de bactérias na pele das tetas pode ser reduzido em mais de 90 por cento após a mamada do bezerro, se as tetas forem cuidadosamente desinfetadas. Nas condiçöes dos experimentos realizados, a presença de coliformes foi insignificante.

Mastite bovina causada por Prototheca zopfii: relato de um caso / Clinical bovine mastitis caused by Prototheca zopfii: a case study

Um caso de mastite clínica causada por Prototheca zopfii em uma vaca de um rebanho leiteiro localizado na zona da Mata do Estado de Minas Gerais foi estudado. O animal apresentou sinais clínicos nos quartos mamários anteriores e queda acentuada na produçäo de leite. Após o diagnóstico inicial, o animal foi observado durante onze meses, do início da manifestaçäo da doença até 12 dias após o parto. Exames microbiológicos do leite foram realizados aos 7, 30, 39, 49, 65, 326 e 331 dias após o isolamento inicial, sendo os dois últimos exames realizados 7 e 12 dias após o parto. As amostras de P. zopfii isoladas apresentaram resistência in vitro a: ampicilina, canamicina, cefaloridina, enrofloxacina, estreptomicina, gentamicina, neomicina, oxacilina, penicilina, sulfonamidas e trimetoprim x sulfametoxazol. Foi avaliada a susceptibilidade in vitro das amostras de P. zopfii a um produto natural constituído de extrato de sementes de frutas cítricas, obtido comercialmente. A menor concentraçäo que inibiu totalmente a alga foi 1:500. Recomendou-se um tratamento com a diluiçäo a 1:200 do extrato em soluçäo fisiológica estéril contendo timerosal a 1:30.000 como conservante, em doses diárias de 20ml, por via intramamária. O tratamento foi realizado inicialmente durante sete dias. Após este período houve reduçäo, mas näo a completa eliminaçäo dos organismos do leite. Foi, entäo, recomendado mais um período de 15 dias de tratamento, de modo semelhante ao primeiro. Trinta e nove dias após o diagnóstico inicial näo foi mais isolado P. zofii do leite dos quartos afetados e a produçäo retornou a níveis semelhantes de antes da infecçäo. Amostras de leite naturalmente infectadas foram congeladas a 20§C. Células viáveis de P. zofii foram recuperadas de amostras mantidas até 38 dias nestas condiçöes.

Sensibilidade e especificidade do "California Mastitis Test" como recurso diagnóstico da mastite subclínica em relação à contagem de células somáticas / Sensitivity and specificity of the California Mastitis Test as a diagnostic tool for subclinical mastitis in quarter somatic cell count estimation

O "California Mastitis Test" (CMT) estima o conteúdo de células somáticas no leite e é interpretado subjetivamente, estabelecendo-se escores que, na maioria dos casos, variam de 1 a 5. O escore 1 indica uma reação completamente negativa e os de 2-5 indicam graus crescentes de resposta inflamatória do úbere, sendo normalmente considerados como indicativos de mastite subclínica. Dependendo da interpretação dos escores, o CMT pode produzir resultados falso-positivos ou falso-negativos. Esse trabalho teve o objetivo de avaliar a sensibilidade e a especificidade do CMT em relação à contagem de células somáticas (CCS). Foram utilizadas 3.012 amostras de leite provenientes de 760 vacas Holandesas ou mestiças Holandês-Zebu, pertencentes a 15 rebanhos. Todas as amostras foram submetidas ao CMT e processadas para CCS em equipamento Fossomatic 90. Os valores médios de CCS (x 1.000 células/ml) obtidos para os escores de CMT foram 1 (79,9), 2 (333,5), 3 (670,3), 4 (1.354,0) e 5 (4.455,6). Três opções de interpretação (doente/não-doente) para o CMT foram testadas, em relação aos valores de CCS, iniciando com 100.000 células/ml: (a) 1 versus 2, 3, 4, e 5; (b) 1 e 2 versus 3, 4 e 5; (c) 1, 2, 3 versus 4 e 5. As sensibilidades do CMT em identificar corretamente quartos mamários acima de 200.000 células/ml foram 79%, 61% e 34%, para as opções a, b e c, respectivamente. Para identificar corretamente contagens acima de 500.000 células/ml, as sensibilidades do CMT, para as opções a, b e c, foram, respectivamente: 93%, 82% e 54%. A sensibilidade do CMT em identificar quartos mamários com mastite subclínica foi adequada (acima de 80%) quando a interpretação do teste foi mais rigorosa (opções a e b). A interpretação da reação 3 como negativa (opção c) só alcançou sensibilidade de 80% para contagens entre 1.200.000 e 1.400.000 células/ml. As especificidades do CMT, para CCS de 200.000 e 500.000 foram, respectivamente, 90% e 80% (opção a), 97% e 90% (opção b) e 99% e 97% (opção c)

Detecção de soroalbuminas e imunoglobulinas no leite bovino como indicadores de mastite subclínica / Evaluation of serum albumin and immunoglobulins in bovine milk as an indicator of subclinical mastitis

Com o objetivo de verificar a utilidade da detecção de soroalbumina (BSA) e imunoglobulinas (Ig) no leite como diagnóstico da mastite subclínica, avaliou-se a relação entre a concentração dessas proteínas, o exame microbiológico e o teste "California Mastitis Test" (CMT). Amostras de leite de 172 quartos mamários previamente examinadas pelo CMT foram colhidas e levadas imediatamente ao laboratório para exames bacteriológicos. Uma alíquota de cada amostra foi congelada a -20°C e posteriomente testada para a presença de BSA e Ig, por meio da técnica de imunodifusão radial simples. Das amostras testadas, 111 apresentaram níveis fisiológicos de BSA (< 0,2mg/ml) e de Ig (< 0,5 mg/ml), 22 apresentaram níveis elevados de ambas proteínas e 39 apresentaram níveis elevados de Ig, mas níveis fisiológicos de BSA. Concentrações de BSA e Ig acima dos limites fisiológicos foram observadas mais frequentemente em amostras onde o CMT apresentou reação de maior severidade (+++) e naquelas onde foram isoladas Streptococcus spp. e Staphylococcus aureus. Entre as 88 amostras positivas no exame bacteriológico, níveis de BSA e Ig acima do limite fisiológico foram observados em 10 e 35, respectivamente. Em amostras onde não se isolou bactérias (84 das 172 examinadas), níveis fisiológicos de BSA e Ig foram observados em 72 e 58 delas, respectivamente. A detecção de BSA e Ig nas amostras de leite forneceu informações adicionais sobre o grau de lesão tecidual, mas não pode ser considerada como um indicador sensível para detecção precoce da reação inflamatória.

This work aimed to evaluate the feasibility of milk serum albumin (BSA) and immunoglobulin (Ig) detection for the diagnosis of subclinical mastitis. Bacteriological examinations and concentrations of BSA and Ig were determined in 172 milk samples. The samples were also examined by the California Mastitis Test (CMT). Immediately after collection, the samples were taken to the laboratory and bacteriologically examined. An aliquot of each sample was kept at -20°C and later, concentrations of BSA and Ig were determined by radial immunodiffusion. A total of 111 milk samples had physiological levels of BSA (< 0.2 mg/ml) and Ig (< 0.5mg/ml). Twenty-two samples had higher levels of both proteins and 39 samples had higher levels of Ig and physiological levels of BSA. Concentrations of BSA and Ig above the physiological levels were more frequently observed in quarters samples with severo (+++) CMT reaction and udder infection caused by Streptococcus spp and Staphylococcus aureus. In bacteriologically positive quarters (88 out of 172), increased levels of BSA and Ig were found in 10 and 35 samples, respectively. In the bacteriologically negativo quartors (84 out of 172), physiological levels of BSA and Ig were found in 72 and 58 samples, respectively. Although BSA and Ig measurements in milk samples gave further information about udder tissue damage, they can not be used as a sensitive parameter to detect early inflammatory reaction.