Foram coletados 100 suabes retais e 100 suabes de carcaças bovinas em matadouros do estado de São Paulo, e um total de 326 estirpes de E. coli foram identificadas, sendo 163 de amostras retais e 163 de amostras de carcaça. Todos os isolados submetidos à PCR para detecção dos genes das toxinas Stx1 e Stx2 foram identificados como não-O157 e fenotipados pelo teste da citotoxicidade em células Vero. Das 26 estirpes que apresentaram apenas o gene stx1, das 56 que apresentaram apenas o gene stx2 e das 30 estirpes que apresentaram ambos os genes, 17 (65,4%), 42 (75%) e 22 (73,3%), respectivamente, foram positivas ao teste de citotoxicidade. Não houve diferença estatística entre os três perfis genéticos e na positividade ao teste de citotoxicidade. Os resultados mostram a alta frequência de expressão dos fatores de virulência das STEC de bovinos.

In the present study 100 rectal and 100 carcass swabs were collected from bovines at slaughterhouses in São Paulo state, and the total of 326 E. coli strains were identified (163 from rectal samples and 163 from carcass samples). All the isolates were submitted to PCR for Stx1 and Stx2 toxin gene detection and all strains were identified as non-O157 and phenotyped by the citotoxicity test in Vero cells. Out of 26 strains that presented only the stx1 gene, 56 that presented only the stx2 gene and 30 that presented both genes, 17 (65.4%), 42 (75%) and 22 (73.3%), respectively, were positive for the citotoxicity test. There was no statistically significant difference among these three toxinotyping profiles and positivity in the citotoxicity test, but the results show high frequency of virulence factor expression of bovine.

RESUMO As formigas têm uma distribuição mundial e representam importante papel no ecossistema. Algumas espécies são consideradas pragas para a agricultura e um risco potencial à vida humana e à saúde pública veiculando mecanicamente agentes patogênicos. Formigas como Paratrechina longicornis e Camponotus spp. têm sido encontradas em ambientes hospitalares. O foco do presente estudo foi a identificação de micobactérias em 138 amostras de formigas (137 Paratrechina longicornis e apenas uma Camponotus sp.), que tiveram acesso a áreas de laboratórios de diagnóstico de tuberculose. Essas formigas foram suspensas em solução salina estéril que foi semeada em meios de Petragnani e Stonebrink, incubadas a 37º C por até 90 dias e as estirpes de micobactérias isoladas foram identificadas pelas técnicas clássicas como micobactérias ambientais (sendo 1 Mycobacterium smegmatis, 2 Mycobacterium fortuitum peregrinum e 1 Mycobacterium tuberculosis). Esses resultados mostram que as formigas podem também se constituir vetores de dispersão de micobactérias em ambientes de risco, reforçando sua importância em saúde pública.

ABSTRACT Ants are found worldwide playing an important environmental role. Some species are considered as agricultural pests and potential risk to human life and public health acting as pathogens carriers. Ants as Paratrechina longicornis and Camponotus spp. have been found inside hospitals. The aim of this study was the research of mycobacteria in 138 samples of ants (137 Paratrechina longicornis and only one Camponotus spp.) which got into the laboratories of tuberculosis diagnosis. These ants were suspended in sterile saline solution and inoculated into Petragnani and Stonebrink media, incubated at 37° C until 90 days and the isolates were identified as environmental mycobacteria (1 Mycobacterium fortuitum peregrinum, 1 Mycobacterium smegmatis) and 1 Mycobacterium tuberculosis complex. These results showed that ants should also act as mechanical vectors of mycobacteria dissemination in risk environments, reinforcing their significance in public health.

RESUMO A tuberculose bovina é uma doença crônica causada por Mycobacterium bovis, caracterizada por desordens respiratórias, emaciação e linfadenomegalia. Em raros casos, sinais neurológicos associados com lesões granulomatosas no sistema nervoso central são observados. O Brasil iniciou o Programa Nacional para Controle e Erradicação dessa doença em 2001, tornando a notificação compulsória. Um caso de meningomieloencefalite tuberculosa é relatado em uma bezerra leiteira com um ano de idade, que apresentou sinais neurológicos (irritabilidade e agressividade, fraqueza e tontura, decúbito) que progrediram para morte em três dias. A medula espinhal apresentou meningomielite granulomatosa, com células gigantes tipo Langhans nas meninges e manguito perivascular mononuclear na substância branca e cinzenta. Havia alguns granulomas no córtex cerebral, com área central de necrose circundada por infiltrado mononuclear. No neurópilo observou-se manguito perivascular mononuclear, satelitose, hiperemia e severa meningite granulomatosa com células gigantes tipo Langhans. Amostras de cérebro foram inoculadas nos meios de cultivo Petragnani e Stonebrink e M. bovis foi isolado e identificado por PCR com primers JB21-JB22. Considerando as 2.888 amostras de SNC bovino enviadas para a vigilância epidemiológica de síndrome neurológica e negativas para raiva, este foi o único caso (0,035%) de meningomieloencefalite tuberculosa diagnosticado pelo Laboratório de Anatomia Patológica do Instituto Biológico no período de 2004 a 2008. Embora a meningomieloencefalite tuberculosa não seja tão freqüente, é importante incluir a infecção por M. bovis no diagnóstico diferencial de lesões do sistema nervoso central de bovinos. De acordo com a legislação brasileira, medidas sanitárias no rebanho infectado (teste e abate) devem ser adotadas.

ABSTRACT Bovine tuberculosis is a chronic disease caused by Mycobacterium bovis, characterized by respiratory disorders, emaciation and lymphadenomegaly. Neurological signs associated with granulomatous lesions in the central nervous system are rarely seen. In Brazil, the National Program for Control and Eradication of bovine tuberculosis has begun in 2001, and notification of the disease is compulsory. A case of tuberculous meningomyeloencephalitis is reported in a dairy calf less than one year old presenting neurological signs (irritability, aggressiveness, weakness, dizziness and recumbency) which progressed to death in three days. Brain, spinal cord, and lung tissue samples were examined histopathologically. The spinal cord showed granulomatous meningomyelitis with Langhans giant cells in the meninges and perivascular mononuclear cuffing in the white and gray matter. There were a few granulomas in the cerebral cortex with a central area of necrosis surrounded by mononuclear infiltrate. Mononuclear perivascular cuffing, satellitosis, hyperemia, and severe granulomatous meningitis with Langhans giant cells were observed in the neuropile. Samples of brain were inoculated into Petragnani and Stonebrink media. M. bovis was isolated and identified by PCR with JB21-JB22 primers. Considering the 2,888 samples of bovine CNS sent for surveillance of neurological syndrome and negative for Rabies, this was the only case of tuberculous meningomyeloencephalitis (0,035%) diagnosed from 2004 to 2008 by the Laboratory of Anatomopathology of Instituto Biológico. Although tuberculous meningomyeloencephalitis is not so frequent, it’s important to diagnose M. bovis as a causative agent of bovine neurological disorders. In accordance with Brazilian regulations, sanitary measures in the infected herd (test-and-slaughter) should be adopted.

RESUMO A Escherichia coli é uma bactéria pertencente ao grupo das enterobactérias, considerada um dos integrantes da flora intestinal de bovinos e aves. As E. coli patogênicas para aves causam, com freqüência, infecções extraintestinais agrupadas de forma generalizada nas denominadas colibaciloses das aves. No presente estudo, 77 cepas de E.coli foram isoladas e identificadas bioquimicamente. Destas,seis apresentaram reação positiva quando submetidas à soroaglutinação frente ao antisoro O78:K80. Foi verificada uma prevalência de E.coli O78:K80 de, aproximadamente, 1% em órgãos de origem aviária, da região oeste do Estado de São Paulo.

ABSTRACT Escherichia coli bacterium belongs to the enterobacteria group and is considered part of the bovine and avian intestine microbiota. The pathogenic poultry E. coli frequently causes extraintestinal infections named colibacillosis. In the present study 77 E. coli strains were isolated and biochemically identified. Of this total, 6 showed positive reaction to agglutination against the O78:K80 antiserum. The results demonstrated a prevalence of almost 1% of E.coli O78:K80 on the samples from aviar origin in the west region of São Paulo State.

RESUMO Clostridium chauvoei causa uma infecção conhecida no Brasil como “manqueira” e geralmente desenvolve-se em bovinos entre os 6 meses e 2 anos de idade, raramente acomete animais com menos de 6 meses de vida, sendo que a mortalidade é de quase 100%. Um animal de um ano de idade e sem raça definida, vacinado contra as clostridioses entre 10 a 15 dias antes de ser atendido na Clínica de Bovinos do Centro de Pesquisa e Diagnóstico de Enfermidades de Ruminantes (CPDER), FMVZ-USP, apresentava dificuldade de locomoção do membro anterior direito, sendo que na região acometida observou-se mionecrose com crepitação ao toque, e alterações na temperatura do membro acometido. O animal foi tratado com antibioticoterapia sistêmica e limpeza do local da ferida obtendo cura das lesões após 6 meses do início do tratamento, sendo confirmado o diagnóstico de clostridiose pelo isolamento bacteriano do Clostridium chauvoei.

ABSTRACT Clostridium chauvoei infection typically develops in young cattle between 6 months and 2 years of age, and is rarely found affecting calves younger than 6 months of age. Mortality is nearly 100%. In the Clinic of Bovines and Small Ruminants of the FMVZ-USP, assistance was given to a one-year old beef calf with no definite race, vaccinated against Clostridium sp. The animal showed movement troubles of the right anterior member, gas presence with volume and temperature increase on the affected area. The animal was submitted to aseptical cleanliness on the affected region and treated with penicillin against C. chauvoei. Six months later the lesion was reduced and the animal showed only a moderate limp.

Clostridium is an anaerobic bacterial genus. The clostridia produce more protein toxins than any other bacterial genus and are a rich reservoir of toxins for research and medicinal uses. Clostridia are widely spread in the environment: soil, dust and water, presenting more than 120 described species, although few can cause diseases. Diseases can grossly be divided into neurotropic disorders (nervous system is primarily affected), enterotoxemias (affecting intestinal tract and parenchymatous organs), and gas gangrene (myonecrosis with toxemia). Undoubtedly the most widely recognized infection due to anaerobes was clostridial myonecrosis, but recently interest has arisen for the role of clostridia in intestinal diseases. This report describes the most important species, the diseases caused by them, and their occurrence in Brazil, focusing on cattle raising.

RESUMO O Clostridium perfringens, microrganismo anaeróbio, está presente no solo e no trato intestinal dos mamíferos. Provoca gangrena gasosa e intoxicação alimentar nos seres humanos e doenças enterotoxêmicas nos animais domésticos. O C. perfringens é classificado em 5 tipos (A, B, C, D e E) mediante a produção de quatro toxinas principais (alfa-α, beta-β, gama-γ, delta-δ e épsilon-ε). A identificação destas toxinas é realizada através da reação de soroneutralização em animais utilizando anti-soros específicos, os quais, além do alto custo, são de difícil obtenção em laboratórios de referência internacionais. Visando contribuir para a tipificação de amostras de C. perfringens, em nosso meio, o presente trabalho teve como objetivo avaliar a reação em cadeia da polimerase (PCR) na detecção dos genes que codificam as toxinas alfa (cpa), beta (cpb) e épsilon (etx) em isolamentos provenientes de bovinos. A sensibilidade analítica da técnica de PCR padronizada a partir do DNA total bacteriano foi de 2,27 ng/?L para o gene cpa e 227 pg/?L para os genes cpb e etx. Das 35 amostras de C. perfringens isoladas e tipificadas por PCR, 16 (45,7%) foram do tipo A, 18 (51,4%) foram do tipo C e 1 (2,9%) foi do tipo B.

ABSTRACT The Clostridium perfringens, an aerobic microorganism, is present in soil and intestinal tracts of mammals. It causes gaseous gangrene and food intoxication in human beings and enterotoxemic diseases in domestic animals.C. perfringens is classified into 5 types (A,B,C,D and E) according to the production of four main toxins: (alfa-α, beta-β, gama-γ, delta-δ e épsilon-ε). The identification of these toxins is made through serum neutralization test in animals, using specific anti-sera, however these reagents are of high cost and difficult to be obtained from international reference laboratories. To aid in the typing of the C. perfringens strains in our environment, the present work aimed to assess the use of the polymerase chain reaction (PCR) for the detection of the genes coding the alpha (cpa), beta (cpb) and epsilon (etx) toxins in bovines isolates.The analytical sensitivity of the PCR technique, standardized from the total bacterial DNA, was 2.27 ng/?L for the cpa gene and 227 pg/?L for the cpb and etx genes. Among the 35 C. perfringens isolates typed by PCR, 16 (45.7%) were of type A, 18 (51.4%) of type C, and 1 (2.9%) belonged to type B.

A polymerase chain reaction (PCR) assay to detect Clostridium perfringens alpha toxin gene (cpa) was used to identify eighty-nine C. perfringens strains obtained from bovine clinical material. The strains were biochemically characterized as C. perfringens. The isolated strains were cultured on plates containing brain heart infusion agar with 5% sheep blood under anaerobic conditions. DNA extraction was performed by boiling. The 324 bp amplification product of cpa was observed in all isolates. C. sordellii, C. botulinum, C. novyi, and C. septicum were also tested but did not produce any alpha toxin gene amplification. These findings suggest that PCR is a useful assay in identifying C. perfringens toxin types.

Ten out of eighty-nine strains biochemically identified as Clostridium perfringens, isolated from bovine organs, were selected by their different results showed in toxigenicity test on mice. Those and the standard strains, ATCC types A, B, C, and D, had their virulence exalted through serial intramuscular inoculation into guinea pigs. Results showed that, for toxigenic strains (6), one or two passages were enough to cause exaltation, while for the atoxigenic (4), five or six inoculations were needed. Esterase electrophoresis of standard and isolated strains, with and without exaltation, was performed. Electrophoresis analysis permits the following conclusions: strains that do not show any clinical symptoms in mice, when exalted demonstrate decreased esterase activity; on the contrary, it is increased when correlated with animal symptoms.

Eighty-nine of 144 isolates of Clostridium perfringens obtained from 187 samples of 71 bovine in several Brazilian states were submitted to esterase electrophoresis for typing. Mobilites electrophoresis, as parameter, were settled down by isolates from ATCC pattern of types A, B, C, and D. Of the 89 isolates, 43 (48.3%) were characterized as electrophoretic type A, 20 (22.5%) as D, 18 (20.2%) as C, and 3 (3.4%) as B. Five (5.6%) isolates did not identity with any type. Similarly, esterase electrophoresis enabled the typing of 94.4 % of the isolates, demonstrating to be an appropriate method for animal sample analyses.

Clinical samples from 71 bovine from different Brazilian states were processed for the analysis of anaerobe organisms with emphasis on the isolation and characterization of Clostridium spp. From these, eighty-nine Clostridium perfringens strains were recovered: 32 from liver, 19 from intestinal contents, 14 from kidney, 6 from rumen, 5 from nervous system, 4 from bone marrow, 2 from udder tract, blood, spleen and lung, and one from muscle. Four reference Clostridium perfringens types A, B, C, and D were used as controls in this study. All isolates were cultivated in appropriate media, and after centrifugation the supernatant and sediment were separated. From pure supernatant post exopolysaccharide (EPS) extraction, mouse toxigenicity tests were performed, determining protein and protein plus carbohydrate, respectively. ELISA was performed from sediments. The results showed that 51 (57.3%) of the isolates were toxigenic to mice when inoculated by intraperitoneal route; bacteria from different organs had variable patterns of toxigenicity. Toxigenicity of EPS extracts was only expressed when protein concentration was 0.04 mg/mL and between 0.31 and 0.5 mg/mL for carbohydrate. Isolates were characterized as toxigenic when showing optimum protein and carbohydrate concentrations.