Resumo Staphylococcus coagulase-negativos (SCN) são os principais micro-organismos responsáveis pela mastite ovina e o tratamento ao final da lactação pode ser usado como método de controle contra a doença. Porém, o longo período seco apresentado pelas ovelhas em alguns sistemas de criação pode prejudicar os efeitos positivos do tratamento antimicrobiano. Os objetivos deste trabalho foram identificar as principais espécies de SCN na etiologia da mastite ovina antes e após o tratamento ao final da lactação das ovelhas, bem como investigar a persistência das espécies mais prevalentes na glândula mamária na lactação seguinte. Sessenta ovelhas foram divididas em dois grupos experimentais, um deles formado por animais sem tratamento antimicrobiano, enquanto o outro era composto por ovelhas cujas metades mamárias foram tratadas com cloxacilina-benzatina por via intramamária. As amostras de leite foram obtidas antes da secagem e aos 15 e 30 dias após o parto da lactação seguinte. As espécies prevalentes foram S. warneri, S. simulans e S. epidermidis. Clones das três espécies de maior ocorrência foram identificados antes e depois do tratamento, ou seja, mesmo com o extenso período seco entre as duas lactações consecutivas, os micro-organismos ainda foram identificados no interior da glândula mamária.

Abstract Coagulase-negative Staphylococci (CNS) are the main microorganisms responsible for mastitis in sheep and the treatment in the end of lactation can be used as a control method against disease. However, the long dry period presented by ewes in some breeding systems may impair the positive effects of antimicrobial treatment. The aims of this study were to identify the main CNS species in the etiology of ovine mastitis before and after treatment at dry-off of the ewes, as well as investigate the persistence of the most prevalent species in the mammary gland at the next lactation. Sixty sheep were divided into two experimental groups, one consisting of animals without antimicrobial treatment, while the other was composed by sheep whose mammary halves were treated with cloxacillin-benzathine intramammary. The milk samples were obtained before drying-off and at 15 and 30 days after calving the next lactation. The prevalent species were S. warneri, S. simulans and S. epidermidis. Clones of the three species more prevalent were identified before and after treatment, i.e., even with the extensive dry period between the two consecutive lactations, the microorganisms were still identified in the mammary gland.

RESUMO: A mastite influencia negativamente na sobrevivência e no ganho de peso em ovinos com aptidão para produção de carne. O objetivo do presente estudo foi investigar, em ovelhas para produção de carne, a ocorrência de mastite ovina subclínica, no final de lactação e no início da lactação consecutiva, bem como avaliar as características composicionais e celulares do leite em função de diferentes índices pluviométricos. Metades mamárias (821) das raças 'Santa Inês' (479) e 'Morada Nova' (342) foram analisadas. As amostras de leite foram colhidas em dois estágios diferentes de lactação: no desmame e após o parto da lactação consecutiva. Os períodos de colheita de amostras foram denominados "seco" ou "chuvoso", conforme o índice de chuva no mês imediatamente antes ao mês da colheita. A ocorrência de mastite subclínica na desmama nas raças 'Santa Inês' e 'Morada Nova' foi de 16,4 e 12,6% no período seco, e 17,7 e 23,5% no período chuvoso, respectivamente. Na lactação consecutiva, as ocorrências foram 26,7 e 27,7% no período seco e 41,8 e 39,1% no período chuvoso, nas ovelhas 'Santa Inês' e 'Morada Nova', respectivamente. O período pós-parto foi crítico para a ocorrência de mastite subclínica, comparado àquele no final da lactação anterior. A ocorrência da doença teve uma influência negativa na CCS do leite no início da lactação e alterou sua composição, principalmente nos períodos mais chuvosos, provavelmente por dificuldade em manter a higiene no ambiente onde os animais permaneceram.

ABSTRACT: Mastitis negatively influences the survival and weight gain of ovines for meat production. The purpose of this study was to investigate, in sheep for meat production, the occurrence of subclinical mastitis in ewes at the end of lactation and beginning of the consecutive lactation and to assess the composition and cellular characteristics of milk as a function of different rainfall indices. Mammary halves (821) of Santa Ines (479) and Morada Nova (342) ewes were examined. Milk samples were collected in two different moments of lactation: at weaning and postpartum of the consecutive lactation. Sample collection periods were called "dry" or "rainy" according to the rainfall index in the month immediately before the month of collection. The occurrence of subclinical mastitis at weaning in the Santa Ines and Morada Nova ewes were 16.4 and 12.6% in the dry period, and 17.7 and 23.5% in the rainy period, respectively. In the consecutive lactation period, the occurrences were 26.7 and 27.7% in the dry period and 41.8 and 39.1% in the rainy period, for the Santa Ines and Morada Nova ewes, respectively. Postpartum stage was critical for the occurrence of subclinical mastitis, as compared to that at the end of the previous lactation. Occurrence of the disease negatively influenced the SCC in the milk at the beginning of lactation and changed its composition, mainly in the rainiest periods, probably due to a difficulty in maintaining hygiene in the environment where the animals remained.

Resumo: A inflamação da glândula mamária é uma das principais causas de prejuízo na ovinocultura. Este estudo teve como objetivo investigar as taxas de cura do tratamento da mastite subclínica após infusão intramamária de princípio ativo antimicrobiano no momento da secagem, em formulações convencional e nanoparticulada. Os rebanhos estavam localizados em São Carlos, São Paulo, Brasil. Analisou-se um total de 584 glândulas mamárias de 307 ovelhas de aptidão para produção de carne. Triagem prévia dos casos subclínicos de mastite foi efetuada por meio do California Mastitis Test (CMT) e/ou da contagem de células somáticas (CCS). Análises microbiológicas foram realizadas para confirmação da etiologia infecciosa. As glândulas mamárias com mastite subclínica foram distribuídas em três grupos: G1 (Controle; glândulas mamárias que não receberam tratamento antimicrobiano); G2 (glândulas mamárias em que foi administrado 100 mg de cloxacilina benzatina em estrutura convencional) e G3 (glândulas mamárias em que foi administrado 50 mg de cloxacilina benzatina em estrutura nanoencapsulada). O tratamento aplicado ao G3 mostrou-se mais eficiente (P=0,047) na cura de glândulas mamárias com mastite subclínica. O uso da cloxacilina nanoencapsulada no momento da secagem de ovelhas de corte auxilia no controle da mastite subclínica infecciosa e reduz os prejuízos consequentes.

Abstract: Inflammation of the mammary gland is one of the main causes of losses in sheep-rearing. This study aimed to investigate the cure rates from treating subclinical mastitis after intramammary infusion of active antimicrobial agents as conventional formulations or as nanoparticles, at the time when the ewes are being dried off. A total of 584 mammary glands in 307 ewes in meat-producing herds located in São Carlos, São Paulo, Brazil, were analyzed. Prescreening of subclinical mastitis cases was done using the California mastitis test (CMT) and/or the somatic cell count (SCC). Microbiological analyses were performed to confirm the infectious etiology. The mammary glands with subclinical mastitis were distributed into three groups: G1 (control; mammary glands that did not receive any antimicrobial treatment); G2 (mammary glands to which 100mg of benzathine cloxacillin in conventional form were administered); and G3 (mammary glands to which 50mg of benzathine cloxacillin in nanoparticulate form were administered). The treatment applied to G3 was more efficient (P=0.047) in curing mammary glands with subclinical mastitis. Use of cloxacillin nanoparticles at the time when the ewes are being dried off helps to control infectious subclinical mastitis and reduces consequential losses among meat-producing herds.

The objective of this study was to characterize the clonal profile, virulence factors and antimicrobial resistance, particularly oxacillin resistance, of Staphylococcus aureus isolated from sheep milk. Milk samples were collected from all teats for the California Mastitis Test (CMT), somatic cell count, identification of S. aureus, investigation in these strains of genes encoding toxins (sea, seb, sec, sed, tst), biofilm (icaA, icaC, icaD, bap), leukocidin (luk-PV) oxacillin resistance by mecA gene detection and susceptibility testing (12 antibiotics). Messenger RNA expression was evaluated by RT-PCR in isolates carrying toxin and biofilm genes. Biofilm formation was also evaluated phenotypically by adherence to polystyrene plates. The clonal profile of S. aureus was investigated by pulsed-field gel electrophoresis. A total of 473 milk samples were collected from 242 animals on three farms and 20 S. aureus strains were isolated and none carried the mecA gene. The two sec gene-positive isolates and the isolates carrying the tst and luk-PV genes were positive by RT-PCR. Staphylococcus aureus isolated from the three flocks studied showed high susceptibility to the drugs tested and none was biofilm producer, indicating that biofilm formation was not a virulence factor causing infection by these strains. The typing of 17 S. aureus isolates revealed the presence of a common clone on the three farms studied, and the presence and expression of the sec and tst genes in one strain of this clone suggest the possible acquisition of virulence genes by this clone, a fact that is important for animal health and food hygiene.

O objetivo desse estudo foi avaliar a ocorrência da mastite subclínica ovina em duas lactações e a evolução da doença em duas lactações consecutivas. As amostras de leite foram originadas de um rebanho com 160 ovelhas da raça Santa Inês. Na primeira lactação, colheram-se amostras em ovelhas aos 14 dias pós-parto e no período próximo ao desmame. Na segunda lactação, as amostras de leite foram colhidas dos mesmos animais aos 14 dias pós-parto, 52 dias pós-parto e ao desmame. A triagem dos casos de mastite subclínica foi feita preliminarmente por meio do California Mastitis Test (CMT). As amostras de leite para a investigação microbiológica foram obtidas de metades mamárias positivas e negativas ao CMT. Os estafilococos coagulase-negativos foram os micro-organismos de maior ocorrência nas duas lactações. Houve a predominância de ovelhas que permaneceram com mastite subclínica durante a Lactação 1. As curas espontâneas foram significativamente inferiores no período entre 52 dias de lactação e o desmame dos cordeiros. Nenhuma diferença foi observada quanto à evolução dos casos de mastite subclínica infecciosa quando comparadas duas lactações (P>0,05).

The aim of this study was to evaluate the occurrence of ovine subclinical mastitis in two lactations, and the disease evolution in two consecutive lactations. Milk samples originated from a herd with 160 Santa Inês breed sheep. In the first lactation, milk samples were collected from ewes at 14 days post-partum and during the period near weaning. In the second lactation, milk samples were collected from the same animals at 14 days postpartum, at 52 days postpartum and at weaning. Screening of subclinical mastitis was carried out preliminarily by means of California Mastitis Test (CMT). Milk samples for microbiological culture were obtained from CMT-positive and CMT-negative mammary halves. Coagulase-negative Staphylococci were the most prevalent microorganisms isolated in two lactations. Ewes that remained with subclinical mastitis in lactation 1 were predominant. Spontaneous recoveries were significantly lower in the period between 52 days of lactation and weaning of lambs. No difference was observed regarding the progression of subclinical mastitis cases compared the two lactations (P>0.05).

Biofilm formation is considered to be a selective advantage for Staphylococcus aureus mastitis isolates by facilitating bacterial persistence in the udder. It requires attachment to mammary epithelium, proliferation and accumulation of cells in multilayers. The objective of this study was to determine the sensitivity and specificity of three techniques for the detection of S. aureus biofilm-positive strains. Two phenotypic tests, including growth on microtitre plates and Congo red agar, were compared with a PCR technique using 94 S. aureus strains obtained from cows with subclinical mastitis from two farms in the state of São Paulo. These strains were characterised by in vitro slime production on Congo red agar, biofilm formation on microtitre plates and the presence of the icaA and icaD genes. The results revealed that 85% of the isolates tested produced slime on the Congo red agar, 98.9% of the isolates produced biofilms in vitro by adhering to sterile 96-well "U" bottom polystyrene tissue culture plates, and 95.7% of the isolates carried the icaA and icaD genes. The results of the phenotypic tests for biofilm formation were compared with those of the molecular analysis, and the sensitivity and specificity of the Congo red agar test were 88.9% and 100%, respectively, while those of the microtitre plate test were 100% and 25%, respectively. When the phenotypic methods for the detection of biofilm producers, namely growth on microtitre plates and Congo red agar, were compared, the sensitivity and specificity were 86% and 100%, respectively. Therefore, growth on Congo red agar and the microtitre plate test are methods that could be used to determine whether an isolate has the potential for biofilm production.

Foram submetidas a PCR-Ribotipagem e aos testes de sensibilidade in vitro frente a 12 antimicrobianos 77 estirpes de Staphylococcus aureus isoladas em amostras de leite procedentes de 40 vacas da raça holandesa que apresentaram mastite subclínica, em uma propriedade rural localizada no Estado de São Paulo, Brasil. Os resultados obtidos revelaram quatro diferentes padrões de resistência a antimicrobianos, sendo observada a predominância de resistência à lincomicina entre 19 (24,7%) estirpes de S.aureus. As 58 (75,3%) estirpes restantes foram sensíveis aos 12 antimicrobianos testados. A PCR-ribotipagem revelou a ocorrência de nove padrões genotípicos distintos, além de apresentar uma capacidade discriminatória maior (D = 0,82) que a obtida nos antibiogramas (D = 0,42). Entre as 19 estirpes resistentes aos antimicrobianos, 14 (73,7%) foram agrupadas em três padrões de ribotipagem e, destas, 13 (92,9%) apresentaram resistência à eritromicina e à lincomicina, isoladamente ou em associação. O grande número de ribotipos e de padrões de resistência a antimicrobianos observados nesta propriedade demonstrou que há grande heterogeneidade genética em populações naturais de S. aureus, fato este que deve ser levado em consideração em programas de controle da mastite bovina.

Seventy-seven S. aureus strains, isolated in milk samples obtained from 40 Holstein cows suffering from subclinical mastitis in a dairy herd in the state of São Paulo - Brazil, were undergone to PCR-ribotyping and in vitro susceptibility testing to 12 antimicrobial drugs. PCR-ribotyping revealed nine different patterns and presented a higher discriminatory power (D = 0.82) in comparison with the in vitro antibiotic susceptibility test (D = 0.42). Moreover,, the results showed four antibiotic resistance patterns, with the resistance to lincomycin being the most predominant and comprising 19 (24.7%) S. aureus strains. The other 58 (75.3%) isolates were sensitive to all antibiotics tested. Of the 19 isolates which presented resistance to antibiotics, 14 (73.7%) were grouped in three ribotypes, and 13 (92.9%) of them were resistant to eritromycin and/or lincomycin. The large number of ribotypes and antibiotic resistance patterns found in this single dairy herd showed that one must be careful when considering the results of in vitro susceptibility tests obtained from a few S. aureus strains, because there is a high heterogeneity among naturally occurring populations of S. aureus, which must be taken into consideration in control programs against bovine mastitis.

Foram estudadas as provas de condutividade elétrica, utilizando-se um medidor manual, e do conteúdo de cloretos do leite como métodos auxiliares para o diagnóstico da mastite subclínica bovina na identificação de quartos mamários doentes em que Staphylococcus aureus e microrganismos do grupo Corynebacterium foram isolados posteriormente. Os exames foram realizados durante o período de dois anos, em animais da raça Holandesa, em propriedade rural produtora de leite do tipo C, onde a ordenha era realizada uma vez ao dia. A sensibilidade das provas de condutividade elétrica e do conteúdo de cloretos do leite originado dos quartos mamários em que foram isolados o Corynebacterium sp (65,3% e 78,3%, respectivamente) foi superior à encontrada para os quartos mamários em que o Staphylococcus aureus foi identificado (55,4% e 68,2%, respectivamente). As eficiências das duas provas diagnósticas foram semelhantes. Foi demonstrada significância estatística nas análises de regressão das duas provas acompanhadas para os quartos mamários sadios e quartos com mastite subclínica por Staphylococcus aureus.

Electrical conductivity measured by a hand-held meter and chloride concentration of milk were studied as auxiliary methods for diagnosis of bovine subclinical mastitis in the identification of affected mammary quarters where Staphylococcus aureus and Corynebacterium sp were later isolated. Tests were made during 2 years in Holstein cows of a dairy farm producing type C milk, where milking was performed once a day. Sensitivities of electrical conductivity and chloride concentration tests from mammary quarters, where Corynebacterium sp was isolated (65.3% and 78.3%, respectively), were superior to the found in mammary quarters where S. aureus was identified (55.4% and 68.2%, respectively). The efficacies of the two diagnostic tests were similar. Statistical significance was demonstrated with regression analysis of both tests of healthy mammary quarters and subclinical mastitis quarters infected with Staphylococcus aureus.