Genotypic evaluation of antimicrobial resistance in Staphylococcus spp. isolated from bovine clinical mastitis / [Avaliação genotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite clínica bovina]

Bovine clinical mastitis caused by Staphylococcus spp. is a serious and widespread disease in the world of dairy farming. Antimicrobial therapy is of fundamental importance in the prevention and treatment of infectious mastitis, but the indiscriminate use of antimicrobials acts as a determining factor for the spread of the disease. The present study evaluated the resistance profiles of 57 Staphylococcus spp. isolated from bovine clinical mastitis to beta-lactams and gentamicin, relating characteristics of phenotype (in vitro susceptibility tests) and genotype (detection and expression of genes encoding resistance - mecA, mecALGA251, blaZ, femA, femB, and aacA-aphD - using PCR and RT-PCR, respectively). One or more genes coding for resistance to different antimicrobials were detected in 50 Staphylococcus spp. isolates. The femA and femB genes were the most frequent (75.4% for both). The observed expression of the genes was as follows: blaZ (60%), femA (39.5%), aacA-aphD (50%), femB (32.6%), mecA (8.3%), and mecALGA251 (0%). Considering the relevance of the genus Staphylococcus to bovine mastitis, this study aimed to elucidate aspects regarding the genotypic and phenotypic profiles of these microorganisms so as to contribute to the development of effective strategies for mastitis control.(AU)

A mastite clínica bovina causada por Staphylococcus spp. é uma doença grave e generalizada no mundo da pecuária leiteira. A terapia antimicrobiana é de fundamental importância na prevenção e no tratamento da mastite infecciosa, mas o uso indiscriminado de antimicrobianos atua como fator determinante para a disseminação da doença. O presente estudo avaliou os perfis de resistência de 57 Staphylococcus spp. isolados de mastite clínica bovina em relação ao uso de betalactâmicos e gentamicina, relacionando características do fenótipo (testes de suscetibilidade in vitro) e genótipo (detecção e expressão de genes que codificam resistência - mecA, mecALGA251, blaZ, femA, femB, e aacA-aphD - usando PCR e RT-PCR, respectivamente). Um ou mais genes que codificam resistência a diferentes antimicrobianos foram detectados em 50 Staphylococcus spp. isolados. Os genes femA e femB foram os mais frequentes (75,4% para ambos). A expressão observada dos genes foi a seguinte: blaZ (60%), femA (39,5%), aacA-aphD (50%), femB (32,6%), mecA (8,3%) e mecALGA251 (0%). Considerando-se a relevância do gênero Staphylococcus para a mastite bovina, este estudo teve como objetivo elucidar aspectos referentes aos perfis genotípico e fenotípico desses microrganismos, a fim de contribuir para o desenvolvimento de estratégias eficazes para o controle da mastite.(AU)

Chlamydia felis: Lack of association between clinical signs and the presence of the cryptic plasmid.

Chlamydia felis is an obligate intracellular bacterial pathogen that infects cats, causing severe conjunctivitis associated with upper respiratory tract disease (URTD). In the present study, 186 cats from three non-commercial catteries in São Paulo, SP, Brazil were evaluated. The detection of Chlamydia felis was performed by PCR. The clinical severity was scored from 1 to 4, with a score of 4 as the most severe manifestation. The total occurrence of C. felis was of 18.82% (35/186) of cats overall, but notably, 58.06% (18/31) of infected cats originated from a single cattery. All animals harboring C. felis had URTD clinical signs and higher scores (3 and 4). In addition, C. felis occurrence was associated with the presence of cryptic plasmid. However, the virulence and clinical severity were not correlated.

Microbiological analysis and sensitivity profile of Staphylococcus spp. in subclinical mastitis of dairy goats / Análise microbiológica e perfil de sensibilidade do Staphylococcus spp. em mastite subclínica de caprinos leiteiros

Subclinical mastitis in goats causes economic losses and risks to public health. Given the need for research that shows the most isolated staphylococci species and sensibility tests comparing the resistance between coagulase-negative (CNS) and positive Staphylococcus (CPS) goats with subclinical mastitis, the aim of this study was to identify the microorganisms isolated from milk samples of goats with subclinical mastitis, as well as define the staphylococci species and determine the sensitivity profile of Staphylococcus spp. to antimicrobials. To collect samples, tests were performed for mug of black background and California mastitis test (CMT), collecting milk from CMT positive animals. A total of 226 samples from seven herds of dairy goats was collected and forwarded to the laboratory, where they were seeded for the isolation of the microorganism and implementing the antibiotic sensibility test. Of these, 122 samples had bacterial growth and the most isolated staphylococci species were: S. epidermidis (24.55%), S. lugdunensis (15.40%) and S. intermedius (13.64%). Samples showed increased resistance to antimicrobials: penicillin (81.8%), oxacillin (60.0%) and ampicillin (55.5%). Greater sensitivity to: enrofloxacin (99.1%), erythromycin (98.2%), gentamicin (98.2%) and vancomycin (98.2%) were observed. The S. epidermidis showed higher antimicrobial resistance to amoxicillin and penicillin than S. lugdunensis and S. intermedius. Similar resistance in vitro between CNS and CPS was observed to most antimicrobials. It is important to control the overuse of antibiotics to prevent the emergence of resistant strains.

A mastite subclínica em caprinos acarreta prejuízos econômicos e riscos à saúde pública. Tendo em vista a necessidade de pesquisas que demonstrem as espécies de estafilococos mais isoladas e os testes de sensibilidade que comparem a resistência entre Staphylococcus coagulase negativa (SCN) e positiva (SCP) de cabras com mastite subclínica, os objetivos do presente estudo foram identificar os microrganismos isolados de amostras de leite de cabras com mastite subclínica, bem como definir as espécies de estafilococos e determinar o perfil de sensibilidade de Staphylococcus spp. aos antimicrobianos. Para realizar a coleta das amostras, foram executados os testes da caneca de fundo preto e California mastitis test (CMT) com o leite dos animais reagentes ao CMT. Coletaram-se 226 amostras provenientes de sete rebanhos de caprinos leiteiros, as quais foram encaminhadas para o laboratório, onde foram semeadas para o isolamento do microrganismo e a realização do teste de antibiograma. Dessas amostras, 122 tiveram crescimento bacteriano e as espécies mais isoladas de estafilococos foram: S. epidermidis (24,55%), S. lugdunensis (15,40%) e S. intermedius (13,64%). As amostras apresentaram maior resistência aos antimicrobianos penicilina (81,8%), oxacilina (60,0%) e ampicilina (55,5%). Observou-se maior sensibilidade para enrofloxacina (99,1%), eritromicina (98,2%), gentamicina (98,2%) e vancomicina (98,2%). O S. epidermidis apresentou maior resistência antimicrobiana para a amoxicilina e a penicilina do que o S. lugdunensis e o S. intermedius. Foi verificada uma resistência in vitro semelhante entre os estafilococos coagulase negativa e positiva para a maioria dos antimicrobianos testados. É importante o controle do uso abusivo de antimicrobianos para evitar o surgimento de cepas resistentes.

Function of milk polymorphonuclear neutrophil leukocytes in bovine mammary glands infected with Corynebacterium bovis.

Corynebacterium bovis is one of the most commonly isolated bacteria from aseptically collected bovine milk samples. The objective of the current study was to characterize the bovine innate immune response by evaluating milk polymorphonuclear neutrophilic leukocytes (PMNL) in mammary glands infected with C. bovis. Twenty quarters infected with C. bovis and 28 culture-negative quarters (with milk somatic cell count <1×10(5) cells/mL) were used. The percentages of milk PMNL and the PMNL expression of L-selectin (CD62L), ß2-integrin (CD11b), and one of the endothelial-selectin ligands (CD44), as well as the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus, were evaluated by flow cytometry. The apoptosis and necrosis rates of the PMNL were quantified using dual-color flow cytometry with fluorescein-labeled annexin and propidium iodide. The present study revealed a higher percentage of PMNL in the milk from C. bovis-infected quarters, although no significant differences were found in levels of CD44, CD62L, or CD11b expression among the PMNL. A lower percentage of apoptotic PMNL was observed in C. bovis-infected quarters, as well as higher percentages of viable PMNL and of PMNL that produced intracellular ROS. However, no alterations were observed in phagocytosis of Staph. aureus by the PMNL or in intensity of intracellular ROS production by PMNL. Thus, results from this investigation of the PMNL function support, at least in part, the fact that intramammary infections by C. bovis may offer protection against intramammary infections by other bacteria.

Lactation stage and udder health status of Santa Ines ewes / Estágio da lactação e saúde da glândula mamária em ovelhas da raça Santa Inês

Avaliou-se o risco de infecção em diferentes fases da lactação em 33 ovelhas da raça Santa Inês. Inicialmente a glândula foi submetida ao exame físico e à prova de fundo escuro. Posteriormente, amostras de leite foram coletadas assepticamente para a realização de exame bacteriológico - California Mastitis teste -, e contagens microscópica e automática de células somáticas. Nenhuma diferença foi observada entre as distintas fases de lactação. Observou-se alta persistência de infecções intramamárias, e tendência a maior contagem de células somáticas no último período de lactação, que pode ser oriunda da maior resistência a infecções neste período.

Antimicrobial susceptibility of coagulase-negative staphylococci isolated from meat-producing ewes with mastitis / Susceptibilidade antimicrobiana de cepas de estafilococcos coagulase-negativa isolados de ovinos de corte com mastite

Avaliou-se a sensibilidade antimicrobiana in vitro de 121 cepas de estafilococos coagulase-negativa isolada de leite de ovelhas Santa Inês, aos fármacos: penicilina, amoxicilina, ampicilina, estreptomicina, oxaciclina, neomicina, cefalotina, gentamicina e sulfonamida. A resistência à sulfonamida foi a mais frequente (27,3 por cento), seguida pela estreptomicina (14,0 por cento) e pela oxaciclina (14,0 por cento), enquanto da gentamicina (1,6 por cento) foi a menos frequente. Todas as cepas foram sensíveis a pelo menos um antimicrobiano, e 20,3 por cento das cepas apresentaram resistência múltipla. Os resultados mostram a importância de Staphylococci coagulase-negativas como agentes causadores de mastite em ovinos, e o perfil de resistência múltipla indica a importância da determinação da resistência à oxaciclina como indicador da presença de ilhas de patogenicidade que contêm fatores de virulência e resistência a outros antimicrobianos que contribuem para a sobrevivência da bactéria ao tratamento.

Evaluation of the microbiological status of milk and various structures in mammary glands from naturally infected dairy cows.

A knowledge of the microbiological status of milk and of the different structures in the mammary glands has great importance in elucidating the pathogenesis of mammary gland infections. The objective of this study was to evaluate the microbiological status of various structures in the mammary glands from naturally infected dairy cows following slaughter. A total of 94 samples of milk, 184 samples of mammary parenchyma, 168 samples of gland cisterns, and 168 samples of teat cisterns were collected for microbiological examination. Microorganisms were detected in 59.9% of all samples, 67.0% of the milk samples, 70.1% of the mammary parenchymas, 55.9% of the gland cisterns and 48.8% of the teat cistern samples. When all samples were considered, coagulase-negative Staphylococcus were the most prevalent (35.7%) followed by coagulase-positive Staphylococcus (12.2%), Corynebacterium bovis (2.4%), Prototheca sp. (1.9%), and Streptococcus dysgalactiae (1.5%). There was a significantly higher occurrence of microorganisms in the milk and mammary parenchyma compared to the gland cisterns and teat cisterns.

Aetiology and histopathology of bovine mastitis of espontaneous occurrence.

Infectious bovine mastitis is responsible for serious economic losses in dairy cattle breeding. Information about the damages caused by micro-organisms is of great interest and importance. The purpose of the present study was to report on the microbiological and histopathological aspects of the mammary parenchymas of slaughtered dairy cows. A total of 184 mammary glands were examined and samples of mammary parenchyma were collected for microbiological and histopathological examinations. Micro-organisms were isolated from 69.6% samples; 23 (12.5%) of the 184 samples did not show histological changes; inflammatory response was observed in 56 (30.4%) samples; inflammatory response and repair were present in 82 (44.6%) samples; repair process was verified in 23 (12.5%) of the mammary glands. Coagulase-negative Staphylococci were observed in 53.8% of the samples followed by coagulase-positive Staphylococci (7.6%), Prototheca sp. (2.2%) and Streptococcus dysgalactiae (1.6%). Coagulase-negative Staphylococci and coagulase-positive Staphylococci were associated mainly to chronic inflammatory response and chronic inflammatory response and repair. Samples from which no micro-organisms were isolated (n = 56) had no histological changes in 82.6% of the cases. These results were higher (P < 0.05) when compared to the samples with micro-organisms and without histological changes (17.4%).

Susceptibility and features of the ultrastructure of Prototheca zopfii following exposure to copper sulphate, silver nitrate and chlorexidine.

One of the most important forms of the occurrence of protothecosis is bovine mastitis. Studies on the "in vivo" and "in vitro" susceptibility to antimicrobials have shown that the microorganism is resistant to most of them. Looking for alternative treatments this study aimed to study the susceptibility to copper sulphate (which has an important algicide effect) and silver nitrate (used in dairy cattle breeding for the cauterization of mammary glands) and also to chlorexidine (an important post-dipping anti-septic used in dairy practice), and the effect of these antimicrobials in the ultrastructure of Prototheca zopfii before and after the exposure to these drugs. The "in vitro" susceptibility tests to chlorexidine, silver nitrate and copper sulphate of the strains of Prototheca zopfii for the determination of their minimal microbicidal concentrations (MMC), were performed using the tube dilution method in Sabouraud dextrose broth and evaluation of colony growth after plating in Sabouraud dextrose agar. The MMCs of chlorexidine, copper sulphate and silver nitrate of the 50 strains tested were 0.01%, 0.1% and 0.3%, respectively. The tubes containing the material used in the antimicrobial susceptibility tests were prepared for the examination in an electron microscope. The untreated controls of P. zopfii showed a similar ultrastructural appearance with the typical characteristics of the microorganism. Cells exposed to silver nitrate showed changes suggesting thickness of the cell wall. Cells exposed to chlorexidine showed changes suggesting degradation of intra-cellular organelles present in the cytoplasm. P. zopfii treated with copper sulphate showed changes suggesting fibrilation of inner layer of cell wall.

Antimicrobial susceptibility of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows.

The high prevalence of the great resistance to antimicrobials shown by the aetiological agents of infectious bovine mastitis, often leads to chronic recurrent mastitis, one of the main causes of early culling of dairy cows. The purpose of this study was to determine the in vitro susceptibility pattern of Staphylococcus spp. isolated from mammary parenchymas of slaughtered dairy cows, to different anti-microbials. A total of 45 Staphylococcus spp. strains [33 coagulase-negative Staphylococcus (CNS) and 12 Staphylococcus aureus (SA)] were used. These strains were tested with 12 different antimicrobials by the Kirby and Bauer standardized disc diffusion method; 84.44% and 86.66% of the 45 strains were resistant to ampicillin and penicillin, respectively. The highest sensitivity was to cephalothin (84.44%), gentamicin (80%) and to sulphazotrin (77.77%). CNS showed higher resistance (P < 0.05) than SA. The in vitro susceptibility pattern of Staphylococcus spp. strains isolated from mammary parenchymas studied in the present investigation was similar to that observed in recent studies of in vitro and in vivo susceptibility patterns of Staphylococcus spp. isolated from cases of bovine mastitis.

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization.

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72-75 degrees C/15 seconds, 72-75 degrees C/20 seconds and 62-65 degrees C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. as representing a public health risk.

Infectious bovine mastitis caused by environmental organisms.

The purpose of this study was to determine the prevalence of environmental mastitis in dairy herds and identify the main environmental pathogens, and to evaluate the influence of season, housing and management. A total of 20,310 quarters of 5216 animals from 52 dairy herds in 32 counties was examined. Milk samples were aseptically collected for laboratory examination from mammary glands testing positive to any of the field tests. From these, 736 environmental infections were identified. The most frequently isolated environmental pathogens were algae of the genus Prototheca sp. (41.2%), Streptococcus uberis (21.1%), fungi (19.5%), enterobacteriacea (8.3%) and Nocardia sp. (6.6%). The occurrence of mastitis was not influenced by the herd size, use of dry cow therapy, or post milking teat dipping. A tendency for increased occurrence of environmental mastitis during the months of September to February (hot and wet weather) was observed, suggesting a seasonal influence.

Métodos para diagnóstico microbiológico de mastite clínica bovina / Microbiological methods for diagnosis of clinical bovine mastitis

O objetivo deste trabalho foi comparar três procedimentos de semeadura do leite para diagnóstico microbiológico da mastite bovina: "in natura", pós-congelamento de 12 horas a -20ºC e pós-incubaçäo a 37§C por 12 horas. Foram examinados 256 amostras de leite procedentes de 200 vacas com mastite clínica de cinco propriedades leiteiras. Os resultados bacteriológicos após os tratamentos descritos mostraram aumento dos microrganismos isolados. Observou-se aumento significativo do Staphylococcus sp nas amostras congeladas e incubadas, aumento da Klebsiella sp e do Pseudomonas sp apenas no pós-incubaçäo, e reduçäo do Corynebacterium bovis, nos isolamentos pós-incubaçäo. Näo se obervou diferença entre os tratamentos "in natura" e pós-congelamento. Pode-se concluir que a melhor conduta para diagnóstico da mastite seria a associaçäo destes procedimentos para se obter maior taxa de isolamento, permitindo estabelecer o diagnóstico e instituir a conduta terapêutica adequada

Epidemiologic study of environmental sources in a Prototheca zopfii outbreak of bovine mastitis.

Bovine mastitis represents the main form of occurrence of protothecosis in animals. The detection of mastitis caused by Prototheca sp. indicates a serious problem which can affect an entire herd. The purpose of this study is to explain some aspects of the epidemiology of mastitis due to Prototheca zopfii with the evaluation of the presence of these microorganisms in samples collected from potential sources in the dairy herd. This study was performed during a Prototheca zopfii outbreak of clinical bovine mastitis in the State of São Paulo, Brazil. The following samples were aseptically collected for microbiological examination: milk (n = 211); rectal swabs (from 15 calves and 2 lactating cows); swabs from tea cup rubbers during milking (n = 2); water (n = 6). Prototheca zopfii was isolated from 77 (36.49%) of the 211 milk samples; 11 calves and 2 cows showed Prototheca zopfii in faecal samples; both swabs collected from the tea cup rubbers showed viable forms of Prototheca zopfii; this microorganism was also isolated from 2 water samples, and 1 soil sample collected from the dry cow pasture. Prototheca zopfii seemed to be widespread throughout the dairy herd environment where this outbreak of bovine mastitis occurred.

Prototheca sp. outbreak of bovine mastitis.

Prototheca sp., a colourless algae, is quite common in dairy environments, particularly in wet areas contaminated with manure. The main purpose of this paper is to describe an outbreak of clinical bovine mastitis in an 86-cow dairy herd in the State of Säo Paulo, Brazil. Prototheca sp., an achlorophyllous algae, were isolated on blood agar (incubated for 24 h at 37 degrees C) from 11 quarters of seven lactating Holstein cows, and from one quarter of a cow at the end of the dry period. Treatments were applied, but there was only a microbiological cure, not a functional one. Diagnosis of Prototheca sp. in any of the cows in the herd indicates a herd problem. Infected animals usually have markedly reduced milk production and granulomatous changes often occur in the mammary gland. All sources of contact between the teat ends and drainage water or damp areas should be corrected. An all-out effort for strict sanitation, including during milking, should be made so that the teat ends will not become contaminated.

Bovine mastitis due to algae of the genus Prototheca.

Protothecosis was described in many animals, with bovine mastitis being the main form. The increasing number of isolations of Prototheca spp. from bovine mastitis cases indicates the need of a detailed evaluation of this problem. Besides this, these algae do not respond to treatment with the antimicrobians most frequently applied, leading to elimination of the affected animals, as the best method to control the disease. In two dairy farms in the State of São Paulo, Brazil, a total of 155 lactating cows and 52 dry cows were examined. Milk samples were aseptically collected from lactating cows with clinical or subclinical mastitis. From dry cows, secretion samples from all quarters were collected. All samples were then taken to microbiological exams. At dairy farm 1, Prototheca spp. was isolated from 14.95% milk samples from lactating cows, (all of them cases of subclinical mastitis), and from 8.06% samples from dry cows. At dairy farm 2, Prototheca spp. was isolated from 5.1% milk samples and there were ten cases of clinical mastitis due to this agent. Although clinical mastitis has been considered the main form of occurrence of this pathology until now, it is important to consider Prototheca spp. as subclinical mastitis pathogen. Prototheca zopfii was the main specie isolated. In this study a high rate of intramammarian infections, as clinical or subclinical mastitis during lactation as well as in dry period, was observed, high lighting the importance of suitable diagnosis, so that control and preventive measures can be implemented to avoid dissemination of the agent.