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This study evaluated the isolation, molecular typing and celular proliferation of C. perfringens in small intestine of cattle and sheep distributed in case and control groups. In the case group, out of the 61 bovine samples analyzed, C. perfringens was isolated in 29 (47.54%) and in 4 (33.34%) from 12 sheep samples. In the control group, we did not isolate the microorganism in the bovine samples (73 samples), however it was possible in 5 (20.83%) out of the 24 sheep samples. All isolates (100%) were classified as type A, and cell quantification showed that in control group, all cattle samples resulted in 10 CFU/g of intestinal content while in the study group presented a median value of 104 CFU/g, varying from 10 CFU/g through 108 CFU/g. In ovine group there was a clear divison of values between the two groups. Molecular typing corroborates with other studies regardin the epidemiological importance of type A in enterotoxaemia in ruminants, thus cellular quantification data allow us to conclude that healthy animals present a basal level of C. perfringens lower than 10 CFU/g that does not allow its isolation.

We herein describe a typical episode of bacillary hemoglobinuria in buffalloes, triggered by liver fluke invasion proved by pathognomonic macro and micro lesions of fasciolosis in the biliary ducts and the nephrosis and hepatic focal necrosis characterizing the toxic-infectious process caused by Clostridium haemolyticum. This report is quite similar to another one described by AHOURAI et al. (1990) in cattle following liver fluke invasion and C. haemolyticum isolation and we both agree with the fact that foci of ischemic of toxic necrosis serve as a focus in which clostridial spores might vegetate and cause the disease by producing toxins. Vaccination with bacterin or toxoids is the most effective measure for preventing these histotoxic clostridial diseases since the endospores of the microorganism persist in the environment. Also, as the process is triggered by fluke liver migration, the prevention should cover the control of this trematode as well. Although the pathogenicity of bacillary hemoglobinuria is well known, there are only a few reports currently available, and to our knowledge this is the first case of the disease described in buffaloes.

RESUMO A pleuropneumonia suína, causada pelo Actinobacillus pleuropneumoniae, é uma importante doença respiratória, responsável por prejuízos e queda de produtividade nas criações. Este trabalho teve como objetivo determinar a ocorrência de A. pleuropneumoniae em amostras de campo, mediante a adaptação e emprego de uma técnica de nested-PCR dirigida ao gene Apx IV. Definiu-se a sensibilidade analítica das técnicas de PCR e nested-PCR utilizando a amostra padrão A. pleuropneumoniae sorotipo III, em concentrações de DNA variando entre 30 µg/mL a 0,01 ng/ mL. Um total de trinta e sete amostras de campo encaminhadas ao Instituto Biológico entre 1995 a 2007 foram analisadas pelas técnicas de PCR e nested-PCR. A avaliação da sensibilidade analítica revelou que a PCR possui capacidade de gerar sinal a partir de 2 ng/mL de DNA extraído e a nested-PCR a partir de 0,4 ng/mL. Uma vez que a nested-PCR apresentou sensibilidade analítica cinco vezes maior se comparada à PCR para detecção de A. pleuropneumoniae em amostra padrão, o seu emprego pode minimizar a ocorrência de resultados tipo “falso-negativo”. Dentre as amostras testadas, dez foram positivas à nested-PCR, sendo observada a ocorrência de A. pleuropneumoniae em nove diferentes animais, um deles javali. A presente técnica de nested-PCR pode ser utilizada para detecção direta de A. pleuropneumoniae em amostras de campo, mesmo após congelamento da amostra por longos períodos e sem necessidade de isolamento bacteriano prévio. ABSTRACT Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an important respiratory disease, responsible for economic losses and reduced productivity. The aim of this study was to determine occurrence of A. pleuropneumoniae in field samples, using an adapted nested-PCR reaction targeting the Apx IV gene. Different DNA concentrations (from 30 µg/mL to 0.01 ng/mL) of A. pleuropneumoniae serotype III reference strain were used to determine the level of sensitivity of first generation and nested-PCR reactions. Thirty-seven field samples sent to Instituto Biológico from 1995 to 2007 were tested by PCR and nested-PCR. Determination of the level of sensitivity showed that PCR could amplify to 2 ng/mL of extracted DNA and nested-PCR to 0.4 ng/mL. Since the nested reaction exhibited a level of sensitivity 5 times greater than the PCR reaction to detect a reference strain, using nested-PCR could minimize the occurrence of false-negative results. Among tested samples, 10 of them were nested-PCR positive, showing occurrence of A. pleuropneumoniae in 9 different animals (including one wild boar). This nested-PCR reaction can be used for direct detection of A. pleuropneumoniae in field samples, even after frozen storage for long periods, without the need for previous bacterial isolation.

This study aimed to describe an outbreak of abomasal bloat in kid goats and its clinical, pathological, microbiological, molecular, and epidemiological characteristics. In the kidding season, increased mortality of kid goats with a history of abdominal bloating, dullness, and death was reported. Clinical examinations were carried out, and biological samples from necropsied kids (n = 11) were collected for pathological, microbiological, and molecular diagnosis. Likewise, an epidemiological survey was carried out in order to verify possible associated factors related to the disorder. A therapeutic protocol was also implemented. The main necropsy findings were dehydration, pale mucosa, ascites, abomasal and intestinal meteorism and congestion, emphysematous abomasitis, and cranial areas of lung consolidation. Through staining techniques for cytological evaluations of the abomasum, it was possible to identify Gram positive bacteria, coccoid, with a cuboid shape suggestive of Clostridium ventriculi, Gram positive bacilli suggestive of Clostridium perfringens and ovoid basophilic yeasts compatible with Saccharomyces cerevisiae. By anaerobic culture and molecular tests, C. ventriculi and C. perfringens type A were confirmed. The main histopathological findings were cholangiohepatitis, nephrosis, emphysematous abomasitis, hyalinization of the gastric and intestinal walls, gastroenteritis, intestinal thromboembolism, pulmonary edema, and non-purulent pneumonia, overall suggesting a systemic enterotoxemia picture. The early detection of sick kids and quick initiation of treatment were the primary determinants of the prognosis of each case. There was a final mortality rate of 24.4% (20/82), and the agents C. perfringens type A and C. ventriculi were identified as the main ones involved, with the possible participation of S. cerevisiae. Among the possible associated factors, the erroneous use of the milk replacer associated with inadequate kid management was verified. Among the prophylactic measures, hygiene care, proper use of milk replacer, vaccination plan containing C. perfringens alpha toxoid associated with a good colostrum management were suggested.

Doença bacteriana zoonótica, a campilobacteriose é responsável mundialmente por frequentes casos de gastroenterite humana. Campylobacter spp. apresenta fator de virulência associado à diarreia, denominado toxina citoletal distensiva (CDT), sendo codificado pelos genes do complexo cdt. Os objetivos do presente estudo foram: 1) isolar e identificar estirpes de Campylobacter spp. de 102 suabes de carcaças e 102 suabes retais de ovinos (Ovis aries) e de sete amostras de água dos efluentes, antes e depois do tratamento de desinfecção de abatedouro localizado no estado de São Paulo; e 2) detectar, pela técnica de multiplex-PCR, a presença do complexo de genes cdt. Foram isoladas e identificadas, por métodos fenotípicos e genotípicos, sete estirpes de Campylobacter coli provenientes de 4/102 (3,92%) das amostras de suabes retais, 1/102 (0,98%) de suabes de carcaças e 2/7 (28,5%) das águas dos efluentes. Dos isolados de suabes retais, em 2/7 (28,6%) estirpes foi detectada a presença dos genes cdt. Trata-se do primeiro relato de isolamento de estirpes de Campylobacter coli provenientes de abatedouro de ovinos e das estirpes portadoras do complexo de genes cdt nessa espécie animal no Brasil.

The goal of this work was to verify the effect of the fructooligossaccharide prebiotic (FOS) addition in the diet containing varying levels of alfalfa hay, on the microbiology and intestinal morphology of weaned piglets. It was used 72 piglets, crossed, both sexes, with initial weight of 5.95 ± 0.73 Kg, with approximate age of 21 days. It was evaluated the treatment: T1 – Basal feed + 0% FOS; T2 – Basal feed + 0.3% FOS; T3 – Feed with 5% of alfalfa + 0% FOS; T4 – Feed with 5% of alfalfa + 0.3% FOS; T5 – Feed with 10% of alfalfa + 0% FOS; Feed with 10% of alfalfa + 0.3% FOS. In the end of the experimental period one piglet of each replication was slaughtered for the collection of sample aiming to analyze Salmonela sp. and coliformes and intestinal morphology (height of villus and depht of crypt). The analysis for coliformes didn’t show statistic difference among the levels of FOS and alfalfa hay. It wasn’t find Salmonella sp. colonies in the samples. No difference was observe in the crypt depht (P>0.05) among the treatments. The height of villus and the relation of height of villus per depht of crypt, the group without the addition of FOS didn’t show difference (P>0.05), however, the group supplemented with 0.3% of FOS showed a significant improvement (P

Background Diarrhea in piglets directly affects commercial swine production. The disease results from the interaction of pathogens with the host immune system and is also affected by management procedures. Several pathogenic agents such as Campylobacter spp., Clostridium perfringens, Escherichia coli, Salmonella spp., group A rotavirus (RV-A), coronaviruses (transmissible gastroenteritis virus; porcine epidemic diarrhea virus), as well as nematode and protozoan parasites, can be associated with disease cases. Results All bacterial, viral, protozoan, and parasitic agents here investigated, with the exception of Salmonella spp. as well as both coronaviruses, were detected in varying proportions in piglet fecal samples, and positive animals were equally distributed between case and control groups. A statistically significant difference between case and control groups was found only for Cystoisospora suis (p = 0.034) and Eimeria spp. (p = 0.047). When co-infections were evaluated, a statistically significant difference was found only for C. perfringens β2 and C. suis (p = 0.014). Conclusions The presence of pathogens in piglets alone does not determine the occurrence of diarrhea episodes. Thus, the indiscriminate use of antibiotic and anthelminthic medication should be re-evaluated. This study also reinforces the importance of laboratory diagnosis and correct interpretation of results as well as the relevance of control and prophylactic measures.

C. perfringens é uma bactéria anaeróbia presente no intestino delgado do homem e animais em equilíbrio e, sob a ação de alguns fatores predisponentes como mudança brusca de alimentação ou super alimentação, stress no manejo ou alto parasitismo intestinal, há a proliferação do microrganismo com a consequente produção de potentes toxinas que provocam a morte do animal. Dentre as toxinas principais destaca-se a toxina alfa, importante fator de virulência, produzida por todos os tipos de C. perfringens, sendo os pertencentes ao tipo A os maiores produtores. A fim de caracterizar o microrganismo em suspeitas de enterotoxemia em ruminantes, trabalhamos com 61 amostras de intestino delgado de bovinos e 12 de ovinos como grupo estudo e no grupo controle composto de animais hígidos levados ao abate, 73 amostras de intestino delgado de bovinos e 24 de ovinos. Foram realizados procedimentos de isolamento e tipagem molecular de C. perfringens e quantificação celular, detecção molecular da toxina β2, além de avaliações moleculares qualitativa (PCR convencional) e quantitativa (PCR em tempo real) do gene da toxina alfa dos diferentes isolados. Em 29 amostras do grupo estudo bovino (47,54%) e em 4 (33,33%) do grupo estudo ovino isolou-se o microrganismo, em contrapartida no grupo controle bovino não houve isolamento do bacilo e 5 amostras do grupo controle ovino (20,83%) foram positivas. Houve diferença estatisticamente significante somente entre os grupos de bovinos (p

Toxigenic types of Clostridium perfringens are significant causative agents of enteric disease in domestic animals, although type E is presumably rare, appearing as an uncommon cause of enterotoxemia of lambs, calves and rabbits. We report herein the typing of 23 C. perfringens strains, by the polymerase chain reaction (PCR) technique, isolated from small intestine samples of bovines that have died suddenly, after manifesting or not enteric or neurological disorders. Two strains (8.7%) were identified as type E, two (8.7%) as type D and the remainder as type A (82.6%). Commercial toxoids available in Brazil have no label claims for efficacy against type E-associated enteritis; however, the present study shows the occurrence of this infection. Furthermore, there are no recent reports on Clostridium perfringens typing in the country.

Mycobacterium was verified in animals from a Brazilian dairy herd, a total of 42 samples from 30 cows were submitted to culture and the isolated strains were analyzed by two polymerase chain reaction (PCR), the first specific for species belonging to the Mycobacterium complex (MTBC) and the other for differentiating M. tuberculosis from M. bovis. Twenty seven samples (64.3%) from 18 animals (60%) were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5%), 9 prescapular lymphnodes (33.3%), 2 lungs (7.4%), and 1 liver (3.7%). All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.

A total of 192 samples of illegal cheese from different regions of the states of São Paulo and Minas Gerais, Brazil, were analyzed for the isolation and detection of Brucella spp. DNA by means of microbiological culture and polymerase chain reaction (PCR), respectively. Samples that yielded positive results were submitted to the analysis of the occurrence of Brucella abortus (biovars 1, 2 e 4), as well as to the differentiation of DNA in B19 vaccinal strain or Brucella abortus field strain using PCR. Although the microorganism was not isolated from any sample, PCR detected 37 positive samples (19.27%) using genus-specific primers. From these, all (100%) were Brucella abortus. Differentiation of the strain showed that 30/37 samples (81.08%) were vaccinal strain B19 and seven (18.92%) were Brucella abortus field strains. Results showed that diagnostic sensitivity of PCR was greater than that of microbiological culture. The standardization of the reaction for the differentiation of vaccinal and field strains enabled the analysis of all samples positive for Brucella abortus. It is, therefore, a reliable method, also applicable to natural infections caused by the microrganism.Foram analisadas 192 amostras de queijo clandestinas provenientes de várias regiões do Estado de São Paulo e Minas Gerais, Brasil, para isolamento e detecção de DNA de Brucella spp. através das técnicas de cultivo microbiológico e reação da polimerase em cadeia (PCR), respectivamente. Para as amostras positivas foi pesquisada a ocorrência da espécie Brucella abortus (biovares 1, 2 e 4), além da diferenciação do DNA em cepa vacinal B19 ou de campo por PCR. Não foi possível isolar o microrganismo de nenhuma amostra, porém, na PCR, 37 amostras (19,27%) foram positivas na reação com primers gênero específicos e destas, todas (100%) foram comprovadas como sendo Brucella abortus. A diferenciação da cepa revelou que 30/37 amostras (81,08%) eram cepa vacinal B19 e sete (18,92%) eram cepas de Brucella abortus de campo. Os resultados mostraram uma maior sensibilidade diagnóstica da PCR em relação ao cultivo microbiológico, e a padronização da reação de diferenciação da cepa em vacinal ou campo permitiu que todas as amostras positivas para Brucella abortus fossem analisadas, sendo uma metodologia confiável e aplicável a infecções naturais pelo microrganismo.

A total of 192 samples of illegal cheese from different regions of the states of São Paulo and Minas Gerais, Brazil, were analyzed for the isolation and detection of Brucella spp. DNA by means of microbiological culture and polymerase chain reaction (PCR), respectively. Samples that yielded positive results were submitted to the analysis of the occurrence of Brucella abortus (biovars 1, 2 e 4), as well as to the differentiation of DNA in B19 vaccinal strain or Brucella abortus field strain using PCR. Although the microorganism was not isolated from any sample, PCR detected 37 positive samples (19.27%) using genus-specific primers. From these, all (100%) were Brucella abortus. Differentiation of the strain showed that 30/37 samples (81.08%) were vaccinal strain B19 and seven (18.92%) were Brucella abortus field strains. Results showed that diagnostic sensitivity of PCR was greater than that of microbiological culture. The standardization of the reaction for the differentiation of vaccinal and field strains enabled the analysis of all samples positive for Brucella abortus. It is, therefore, a reliable method, also applicable to natural infections caused by the microrganism.

The objective of the present trial was to characterize genetically strains of Campylobacter jejuni subsp. jejuni isolated from humans and several animal sources (bovines, swine, dogs, primates, wild boars and poultry). A total of 828 different animal samples (feces, carcass, aborted fetus and hysterectomized uterus) were analysed by means of routine bacteriological methods, and 36 C. jejuni strains were isolated. Thirty strains of human fecal origin were obtained in clinical analysis laboratories in the city of São Paulo. The 66 C. jejuni strains isolated were submitted to genetic characterization. Primers based on fla A gene were used in a polymerase chain reaction (PCR) and amplified a fragment of the 702 bp. PCR products were evaluated by means of sequencing and genealogic analysis. Genetic variability analysis of 66 strains showed 44 different subtypes of C. jejuni. One subtype was identical to a C. jejuni strain of human origin with the sequence in the GenBank (GENBANK accession number AF050186). Subtyping analysis of C. jejuni strains based on sequencing of the fla A gene variable region and analysis of sequence alignment by the Maximum Parsimony method showed to be highly discriminatory, providing the best conditions to differentiate strains involved in outbreaks from those sporadically isolated. This is the first study of molecular subtyping analysis of human and animal C. jejuni strains using sequencing technique and genealogic analysis in the state of São Paulo, Brazil.O objetivo do presente trabalho foi caracterizar geneticamente estirpes de Campylobacter jejuni subsp. jejuni isoladas de humanos e de diferentes origens animais (bovinas, suínas, cães, primatas, javalis, suínos e aves de corte). Um total de 828 amostras (fezes, carcaças, fetos abortados e útero histerectomizado) foram analisadas por métodos de rotina bacteriológica e 36 estirpes de C. jejuni foram isoladas. Trinta estirpes de origem fecal humana foram obtidas de laboratórios de análises clínicas da cidade de São Paulo. As 66 estirpes de C. jejuni isoladas foram submetidas à caracterização genética. Oligonucleotídeos baseados no gene fla A foram usados na reação de polimerase em cadeia (PCR) e amplificou um fragmento de 702 pb. Os produtos obtidos pela PCR foram avaliados pelas técnicas de seq��enciamento e análise genealógica. Análise da variabilidade genética das 66 estirpes revelou 44 diferentes subtipos de C. jejuni. Um subtipo de origem humana apresentou seqüência idêntica à de C. jejuni depositada no GenBank (GENBANK acesso número AF050186). A subtipagem das estirpes de C. jejuni baseadas no seqüenciamento da região variável do gene fla A e na análise do alinhamento das seqüências pelo método da Máxima Parcimônia, mostraram-se altamente discriminatórios fornecendo melhores condições para a correta diferenciação entre estirpes originárias de surto e as isoladas esporadicamente. Este foi o primeiro estudo de subtipagem molecular de estirpes de C. jejuni de origem humana e animal utilizando a técnica do seqüenciamento com análise genealógica realizado no Estado de São Paulo, Brasil.

Leptospirosis is a widely distributed zoonosis that affects domestic and wild animals, and that has the man as the end point of its epidemiological chain. Leptospirosis diagnosis in primates is more difficult than in other animal species, as clinical signs and lesions are less evident and antibody response is detected only for short periods. The aim of this article was to describe the detection of Leptospira spp using polymerase chain reaction (PCR), in clinical samples from one captive black-capped Capuchin monkey (Cebus apella), which presented characteristics compatible with leptospirosis (jaundice and haemorrhagic kdney) in the macroscopic post-mortem examination. A friable kidney fragment and urine sample were cultured and submitted to experimental inoculation in guinea pigs and PCR using genus specific primer pair targeting the 16S rRNA region from Leptospira interrogans serovar canicola. Isolation of the agent was negative both in culture and experimental inoculation. The PCR amplification of the clinical samples showed a 330 pb amplified fragment that corresponds to the Leptospira genus. Based on these results PCR was considered an important tool for leptospira detection in nonhumam primates, more sensitive and specific than other techniques, especially considering that the viability of the pathogen was not possible. These advantages enable the detection of the leptospiras in urine and kidney, even when autolysed, frozen or badly conserved, which prevented the isolation and experimental inoculation from positive results.A leptospirose é uma zoonose de ampla distribuição geográfica que acomete os animais domésticos e silvestres, tendo o homem como ponto final da cadeia epidemiológica. O diagnóstico da leptospirose em primatas é dificultado em relação a outras espécies animais, porque os sinais clínicos e lesões são menos evidentes e a resposta de anticorpos é detectada apenas em curtos períodos. O presente trabalho tem por objetivo descrever a detecção de Leptospira spp empregando-se a Reação em Cadeia pela Polimerase (PCR) em amostras clínicas provenientes de um macaco prego (Cebus apella) criado em cativeiro, que apresentou ao exame macroscópico realizado durante a necrópsia, características sugestivas de leptospirose (icterícia e rins hemorrágicos). Amostras de urina e fragmento de rim friável foram submetidas às técnicas cultivo e inoculação experimental em cobaias e PCR empregando-se primers gênero específicos da seqüência do gene 16S rRNA de Leptospira interrogans sorovar canicola. Na PCR as amostras clínicas revelaram um fragmento amplificado de 330 pb correspondente ao gênero Leptospira. O isolamento do agente foi negativo nas demais técnicas empregadas. Pelos resultados obtidos, verifica-se que a técnica de PCR representou uma importante ferramenta de detecção de leptospiras em primatas não-humanos, principalmente considerando-se que não foi necessária a viabilidade do patógeno, permitindo a detecção do agente em amostras autolisadas, congeladas ou amostras mal conservadas, que prejudicaram o isolamento e a inoculação experimental.

Caseous lymphadenitis (CLA) is a contagious disease caused by Corynebacterium pseudotuberculosis. It occurs worldwide and is responsible for economic losses in sheep raising. The aim of this study was to standardize an indirect ELISA to detect antibodies anti-C. pseudotuberculosis in sheep. Serum samples from animals previously tested serially using microbiological culture and PCR from the puncture of lymph nodes and lungs, made up an initial panel with the aim of standardizing the test. When it was applied to field samples, the ELISA reaction showed sensitivity of 100% and specificity of 100% based on microbiological culture and PCR results. Although microbiological culture is the gold standard in C. pseudotuberculosis diagnosis, the ELISA standardized in this study seems to be advantageous because it is faster to process the samples, and may be used as a screening test, or even for animals trade, once the disease is often unapparent, making clinical and microbiological diagnosis difficult to be performed.

Isolaram-se estirpes de Campylobacter spp. em amostras de carcaças (n=65), fezes (n=65) e linfonodos mesentéricos (n=65) de suínos abatidos em frigoríficos do estado de São Paulo e detectaram, pela técnica da Multiplex-PCR, a presença do complexo de genes cdt, responsáveis pela expressão do fator de virulência da toxina CDT. Do total de 195 amostras de origem suína, Campylobacter spp. foi isolado de 31 (15,9%), sendo 29 (93,6%) de amostras de suabe retal, 1/65 (3,2%) de suabe de carcaça e um (3,2%) de linfonodo. Vinte e oito estirpes de C. coli foram positivas para a detecção dos genes cdt, e três estirpes de C. jejuni foram negativas para a detecção desses genes. Foi detectada, pela primeira vez no estado de São Paulo, a presença dos genes cdt em 100% das estirpes de Campylobacter coli provenientes de suínos abatidos em frigoríficos. The purposes of this study were to isolate and identify Campylobacter spp. strains from the carcasses (n=65), feces (n=65) and mesenteric lymph nodes (n=65) of swine slaughtered in abattoirs in the State of Sao Paulo and to detect the presence of the cdt gene complex - responsible for the expression of the virulence factor cytolethal distensive toxin - in these Campylobacter spp. strains through Multiplex-PCR. From 195 samples analyzed, Campylobacter spp. was isolated in 31 (15.9%): 29 (93,6%) samples of rectal swab, 1 (3.2%) carcass swab and 1 (3.2%) lymph node sample. The 28 strains of isolated C. coli were positive for CDT toxin genes and the three strains of isolated C. jejuni were negative for these genes. It was also the first time that the cdt gene cluster was detected in strains isolated from swine in the state of São Paulo. These findings indicate swine as a potential spreading source of virulent strains of Campylobacter coli, either for slaughterhouse staff or consumers of carcasses and sub products.

Nos últimos anos, laboratórios de instituições públicas que atuam em sanidade vêm padronizando seus ensaios com a finalidade de atender à demanda internacional dos agronegócios. Esse fenômeno vem provocando mudança da cultura organizacional com vistas à melhoria da rotina, rastreabilidade e credibilidade dos resultados das análises. Contudo, o processo de implantação e implementação pode ser comprometido pelos custos relacionados a insumos e calibração dos equipamentos, bem como à falta de tempo e dedicação do pessoal especializado. Este trabalho tem como objetivo relatar o processo de transição da norma ISO 9001 para a ISO/ IEC 17025 em laboratórios com o mesmo perfil de serviço. Três laboratórios foram avaliados no processo de transição. Os avanços da implantação da ISO/IEC 17025 foram comprovados pela análise documentária, contextualização, auditorias e questionário. Verificou-se que laboratórios com sistema de gestão de qualidade implantado em normas mais genéricas apresentaram mais facilidade de implantar e implementar normas técnicas.

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.

This paper aimed to determine the excretion period of B19 vaccine strain during a complete reproductive cycle (from estrus synchronization, artificial insemination, pregnancy and until 30 days after parturition) of dairy cows from 3 to 9 years old that were previously vaccinated from 3 to 8 months. Three groups were monitored with monthly milk and urine collection during 12 months: G1 with seven cows from 3 to 4 years old; G2 with three cows from 5 to 6 years old; and G3 with four cows from 7 to 9 years old. Urine and milk samples were submitted to bacteriological culture and urine and PCR reactions for detection of Brucella spp. and PCR-multiplex for B19 strain identification. Ring test (RT) was also performed in the milk samples, and serum samples were tested by buffered acidified plate antigen test (BAPA). All animals were serologically negative at BAPA and Brucella spp. was not isolated from both urine and milk samples. RT revealed 13/210 (6.2%) positive milk samples. PCR reactions detected DNA of Brucella spp. in 86/420 (20.5%) samples. In urine it was found a significantly higher frequency (35.2%; 74/210) than in milk (5.7%; 12/210), more frequently from the estrus to 150 days of pregnancy and after parturition (6.7%; 10/150), and from 150 days of pregnancy to parturition (3.4%; 2/60), and they were all identified as B19 strain. In three groups, intermittent excretion of B19 strain was detected mainly in urine samples, which confirmed its multiplication and persistence in cows for until 9 years.

Torque teno sus virus (TTSuV) is emergent in swine herds. Recent studies have shown an increased frequency of TTSuV2 in Porcine circovirus type 2 (PCV2)-associated diseases (PCVAD), which are endemic in many swine-producing countries, including Brazil. Coinfection with several other viral and bacterial agents results in an increased incidence of more severe PCVAD. Given the limited information on TTSuV and PCV2 coinfection, especially in Brazilian swine herds, this study made a preliminary estimation of the occurrence of coinfection in swine herds by testing samples from different categories. Between 2008 and 2009, 111 samples of feces and 23 serum samples from 5 swine herds were tested for PCV2 and TTSuVs and the results analyzed for associations between these agents. No significant differences in coinfection frequency were observed for PCV2 + TTSuV1 or for PCV2 + TTSuV2 between nursery piglets (P = 0.730), growing pigs (P = 0.331), or sows (P = 0.472). However, a significant difference was observed for PCV2 + TTSuV1 + TTSuV2 between nursery piglets and growing pigs (P = 0.004; Fisher’s exact test). Phylogenetic studies agreed with the grouping of TTSuV1 and TTSuV2 into 2 different clades, with no distinct pattern of clustering of these isolates with the animal categories.

The objectives of the present study were the subtyping of Campylobacter jejuni subsp. jejuni strains obtained from humans and different animal species using PCR-RFLP, and the detection, by means of the same technique, of strains related to serotype PEN O19:LIO 7, the main C. jejuni serotype linked to Guillain-Barré Syndrome (GBS). Seventy C. jejuni strains isolated from human feces (n=33), primates (n=15), dogs (n=5), swine (n=2), bovines (n=1), abortion material from goats (n=2) and poultry carcasses (n=12), all collected in the state of São Paulo, were subtyped by means of PCR-RFLP of fla A gene, using restriction endonucleases Hae III, Afa I and Mbo I. Seven subtypes were observed when using the enzyme Hae III; eight when using Mbo I; and seven when using Afa I. The combination of the three endonucleases led to 16 fla-RFLP subtypes, from which ten subtypes shared strains of human and animal origin. From these, seven subtypes were observed in human and broiler strains. In eight subtypes, the other animal species shared patterns with human strains. It was inferred that, besides broilers, swine, goats, dogs and primates may be sources of infection for human in São Paulo. PCR-RFLP is a highly discriminatory technique that may be applied to molecular epidemiology studies of samples from different origins. Besides, the study also enabled the detection of two human strains and two primate strains related to serotype PEN O19: LIO 7.

The extent of knowledge regarding the diversity of globally distributedEhrlichia canisstrains has been limited to information gained from a few evolutionarily conserved genes. In this study,E. canisstrains from the United States (strain Jake [US]), Brazil (strain São Paulo [BR]), and Israel (strain 611 [IS] and Ranana [IS-R]) were used to examine the antigenic and genetic diversities of four well-characterized major immunoreactive protein genes/proteins. gp36 and gp200 were the most divergent genes, and nucleotide substitutions in the gp36 tandem repeat region of the IS strain, but not the IS-R strain, resulted in two amino acid differences (S→P and P→T) in each nine-amino-acid repeat (epitope-containing region). DNA sequences of gp19 and gp140 were completely conserved in the US and BR strains, but differences were found in the Israeli strains, including two fewer tandem repeats in gp140 and a single amino acid substitution in gp19 from the IS strain.E. caniswhole-cell lysates from each isolate were examined by Western immunoblotting using sera from naturally infected dogs from each country, and four major immunoreactive proteins (gp19, gp36, gp140, and gp200) were identified in each strain using protein-specific antisera. The US and BR strains exhibited highly conserved immunoreactive protein profiles, while some differences were identified in the IS strain. Sera from naturally infected Israeli dogs confirmed gene sequencing information, which demonstrated two distinctE. canisstrains, defined by the gp36 gene. Conversely, gp19 was strongly reactive and present in allE. canisisolates. gp140 and gp200 were also present in all strains, although gp140 in the IS strain had two fewer tandem repeats and exhibited a smaller mass.

C. chauvoei presence was detected by means of polymerase chain reaction (PCR) from supernatant of culture in cooked meat medium of liver, muscle and metatarsian bone marrow samples of seven calves with blackleg symptoms. The isolation under anaerobic conditions of one muscle sample revealed Clostridium perfringens in pure culture.

The objective of the present trial was to characterize genetically strains of Campylobacter jejuni subsp. jejuni isolated from humans and several animal sources (bovines, swine, dogs, primates, wild boars and poultry). A total of 828 different animal samples (feces, carcass, aborted fetus and hysterectomized uterus) were analysed by means of routine bacteriological methods, and 36 C. jejuni strains were isolated. Thirty strains of human fecal origin were obtained in clinical analysis laboratories in the city of São Paulo. The 66 C. jejuni strains isolated were submitted to genetic characterization. Primers based on fla A gene were used in a polymerase chain reaction (PCR) and amplified a fragment of the 702 bp. PCR products were evaluated by means of sequencing and genealogic analysis. Genetic variability analysis of 66 strains showed 44 different subtypes of C. jejuni. One subtype was identical to a C. jejuni strain of human origin with the sequence in the GenBank (GENBANK accession number AF050186). Subtyping analysis of C. jejuni strains based on sequencing of the fla A gene variable region and analysis of sequence alignment by the Maximum Parsimony method showed to be highly discriminatory, providing the best conditions to differentiate strains involved in outbreaks from those sporadically isolated. This is the first study of molecular subtyping analysis of human and animal C. jejuni strains using sequencing technique and genealogic analysis in the state of São Paulo, Brazil. O objetivo do presente trabalho foi caracterizar geneticamente estirpes de Campylobacter jejuni subsp. jejuni isoladas de humanos e de diferentes origens animais (bovinas, suínas, cães, primatas, javalis, suínos e aves de corte). Um total de 828 amostras (fezes, carcaças, fetos abortados e útero histerectomizado) foram analisadas por métodos de rotina bacteriológica e 36 estirpes de C. jejuni foram isoladas. Trinta estirpes de origem fecal humana foram obtidas de laboratórios de análises clínicas da cidade de São Paulo. As 66 estirpes de C. jejuni isoladas foram submetidas à caracterização genética. Oligonucleotídeos baseados no gene fla A foram usados na reação de polimerase em cadeia (PCR) e amplificou um fragmento de 702 pb. Os produtos obtidos pela PCR foram avaliados pelas técnicas de seqüenciamento e análise genealógica. Análise da variabilidade genética das 66 estirpes revelou 44 diferentes subtipos de C. jejuni. Um subtipo de origem humana apresentou seqüência idêntica à de C. jejuni depositada no GenBank (GENBANK acesso número AF050186). A subtipagem das estirpes de C. jejuni baseadas no seqüenciamento da região variável do gene fla A e na análise do alinhamento das seqüências pelo método da Máxima Parcimônia, mostraram-se altamente discriminatórios fornecendo melhores condições para a correta diferenciação entre estirpes originárias de surto e as isoladas esporadicamente. Este foi o primeiro estudo de subtipagem molecular de estirpes de C. jejuni de origem humana e animal utilizando a técnica do seqüenciamento com análise genealógica realizado no Estado de São Paulo, Brasil.

Submitted by Guilherme Lemeszenski (guilherme@nead.unesp.br) on 2013-08-22T19:05:10Z No. of bitstreams: 1 S0036-46652006000600001.pdf: 111171 bytes, checksum: 6a61c02469fcfb243fe994861a44c6ed (MD5) Made available in DSpace on 2013-08-22T19:05:10Z (GMT). No. of bitstreams: 1 S0036-46652006000600001.pdf: 111171 bytes, checksum: 6a61c02469fcfb243fe994861a44c6ed (MD5) Previous issue date: 2006-12-01 Made available in DSpace on 2013-09-30T17:55:11Z (GMT). No. of bitstreams: 2 S0036-46652006000600001.pdf: 111171 bytes, checksum: 6a61c02469fcfb243fe994861a44c6ed (MD5) S0036-46652006000600001.pdf.txt: 22884 bytes, checksum: 3834f0c254088d96f42a50066277c70a (MD5) Previous issue date: 2006-12-01 Submitted by Vitor Silverio Rodrigues (vitorsrodrigues@reitoria.unesp.br) on 2014-05-20T13:15:27Z No. of bitstreams: 2 S0036-46652006000600001.pdf: 111171 bytes, checksum: 6a61c02469fcfb243fe994861a44c6ed (MD5) S0036-46652006000600001.pdf.txt: 22884 bytes, checksum: 3834f0c254088d96f42a50066277c70a (MD5) Made available in DSpace on 2014-05-20T13:15:27Z (GMT). No. of bitstreams: 2 S0036-46652006000600001.pdf: 111171 bytes, checksum: 6a61c02469fcfb243fe994861a44c6ed (MD5) S0036-46652006000600001.pdf.txt: 22884 bytes, checksum: 3834f0c254088d96f42a50066277c70a (MD5) Previous issue date: 2006-12-01 Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) O gênero Campylobacter tem grande destaque em saúde pública, principalmente por pertencerem a este gênero várias espécies que podem causar diarréia. Estas espécies podem ser encontradas em amostras de água, alimentos e no trato intestinal das aves. Este estudo investigou a presença de Campylobacter jejuni e Campylobacter coli em abatedouros de aves no Estado de São Paulo. As 288 amostras foram coletadas em seis estabelecimentos e incluíram: fezes; penas; água de escaldamento, de evisceração e de resfriamento; e água de enxaguadura de carcaça não eviscerada, eviscerada e resfriada. Após o isolamento microbiológico das amostras positivas de Campylobacter spp. foi realizada uma Reação em Cadeia da Polimerase (PCR) utilizando o gene HIP, da hipuricase, específico para Campylobacter jejuni e o gene da enzima aspartoquinase, específico para Campylobacter coli. A porcentagem de amostras positivas para Campylobacter spp. foi de 4,9% (14/288), sendo que o isolamento foi maior em amostras de fezes (22%, 8/36). Foi isolada uma amostra positiva para C. coli. em conclusão, os resultados indicam que há uma necessidade de melhorar a qualidade higiênico-sanitária do controle de Campylobacter em abatedouros de aves. The genus Campylobacter is of great importance to public health because it includes several species that may cause diarrhea. These species may be found in water, food and in the intestinal tract of chickens. This study investigated the presence of Campylobacter jejuni and Campylobacter coli in chicken abattoirs in São Paulo State, Brazil. A total of 288 samples of feces, feathers, scald water, evisceration water, chiller water, and the rinse water of eviscerated, not eviscerated and chilled carcasses were collected in six chicken abattoirs. Polymerase Chain Reaction (PCR) was performed in Campylobacter spp.-positive isolates using the gene HIP, specific for hippuricase enzyme from Campylobacter jejuni and aspartokinase gene, specific to detect Campylobacter coli. The percentage of positive isolates of Campylobacter jejuni was 4.9% (14/288). Isolation was greater in feces samples (22%, 8/36). One sample was positive for the species C. coli. In conclusion, the results indicate that it is necessary to improve quality control for Campylobacter spp. in chicken abattoirs. Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Medicina Veterinária Preventiva e Reprodução Animal Instituto Biológico Universidade Estadual Paulista Faculdade de Ciências Agrárias e Veterinárias de Jaboticabal Departamento de Medicina Veterinária Preventiva e Reprodução Animal

A técnica de PCR, utilizando-se \"primers\" desenhados a partir do gene que codifica uma proteína imunogênica de 31 KDa de Brucella abortus, foi empregada na detecção de Brucella spp. em 300 amostras de produtos lácteos clandestinos apreendidas em municípios dos Estados de São Paulo (SP) e de Minas Gerais (MG). Foram analisadas 49 amostras de queijo minas frescal e 18 de queijo meia-cura provenientes de MG, e 92 amostras de queijo minas frescal, 33 amostras de queijo meia-cura e 108 amostras de leite cru provenientes de SP. O isolamento bacteriano foi tomado como referência, porém não se isolou Brucella spp. de nenhuma das amostras cujos cultivos mostraram-se contaminados com outros gêneros bacterianos. Pela PCR foi observado que 37 amostras de queijo foram positivas: 29/141 (20,56 %) das amostras de queijo minas frescal e 8/51 (15,68%) das amostras de queijo meia-cura. Todas as amostras positivas na PCR para Brucella spp. foram confirmadas como sendo da espécie Brucella abortus pela técnica de PCR utilizando-se \"primers\" espécie-específicos. Foi instituída a reação de hemi-nested PCR para a diferenciação da cepa em B19 ou campo tomando-se como base a deleção genética de 702 pb existente na cepa vacinal B19. A padronização da técnica permitiu que todas as cepas de Brucella spp. fossem diferenciadas, revelando que 30 (81,08%) amostras eram B19 e 7 (18,92%) eram cepas de Brucella abortus de campo. A concordância estimada entre as técnicas de PCR e do cultivo microbiológico através do indicador Kappa foi considerada baixa (K = 0) devido ao não isolamento do microrganismo. A PCR assay using primers designed based on the gene that encodes a 31 KDa immunogenic protein from Brucella abortus was used in the detection of Brucella spp. in 300 samples of illegal dairy products obtained in cities of the states of São Paulo (SP) and Minas Gerais (MG), Brazil. A total of 49 samples of minas frescal cheese and 18 samples of meia-cura cheese from MG were analyzed, besides 92 samples of minas frescal cheese, 33 samples of meia-cura cheese and 108 samples of raw milk from SP. Although bacterial isolation was used as reference, Brucella spp. was not isolated from any samples in which culture was contaminated by other genera of bacteria. PCR showed that 37 samples were positive for Brucella spp.: 29/141 (20,56 %) of the minas frescal cheese samples and 8/51 (15,68%) of the meia-cura cheese samples. All positive samples for Brucella spp. were confirmed as Brucella abortus by PCR using species-specific primers. Hemi-nested PCR was used for the differentiation between B19 and field strains, based on the genetic deletion of 702 pb in the vaccinal strain. Standardization of the technique enabled the differentiation of all Brucella spp. strains, showing that 30 of them (81,08%) were B19 and 7 (18,92%) of them were Brucella abortus field strains. Estimated agreement between PCR and microbiological culture as determined by Kappa indicator was considered to be poor (K = 0) due to the absence of isolation of the microorganism.

Leptospirosis is a widely distributed zoonosis that affects domestic and wild animals, and that has the man as the end point of its epidemiological chain. Leptospirosis diagnosis in primates is more difficult than in other animal species, as clinical signs and lesions are less evident and antibody response is detected only for short periods. The aim of this article was to describe the detection of Leptospira spp using polymerase chain reaction (PCR), in clinical samples from one captive black-capped Capuchin monkey (Cebus apella), which presented characteristics compatible with leptospirosis (jaundice and haemorrhagic kdney) in the macroscopic post-mortem examination. A friable kidney fragment and urine sample were cultured and submitted to experimental inoculation in guinea pigs and PCR using genus specific primer pair targeting the 16S rRNA region from Leptospira interrogans serovar canicola. Isolation of the agent was negative both in culture and experimental inoculation. The PCR amplification of the clinical samples showed a 330 pb amplified fragment that corresponds to the Leptospira genus. Based on these results PCR was considered an important tool for leptospira detection in nonhumam primates, more sensitive and specific than other techniques, especially considering that the viability of the pathogen was not possible. These advantages enable the detection of the leptospiras in urine and kidney, even when autolysed, frozen or badly conserved, which prevented the isolation and experimental inoculation from positive results. A leptospirose é uma zoonose de ampla distribuição geográfica que acomete os animais domésticos e silvestres, tendo o homem como ponto final da cadeia epidemiológica. O diagnóstico da leptospirose em primatas é dificultado em relação a outras espécies animais, porque os sinais clínicos e lesões são menos evidentes e a resposta de anticorpos é detectada apenas em curtos períodos. O presente trabalho tem por objetivo descrever a detecção de Leptospira spp empregando-se a Reação em Cadeia pela Polimerase (PCR) em amostras clínicas provenientes de um macaco prego (Cebus apella) criado em cativeiro, que apresentou ao exame macroscópico realizado durante a necrópsia, características sugestivas de leptospirose (icterícia e rins hemorrágicos). Amostras de urina e fragmento de rim friável foram submetidas às técnicas cultivo e inoculação experimental em cobaias e PCR empregando-se primers gênero específicos da seqüência do gene 16S rRNA de Leptospira interrogans sorovar canicola. Na PCR as amostras clínicas revelaram um fragmento amplificado de 330 pb correspondente ao gênero Leptospira. O isolamento do agente foi negativo nas demais técnicas empregadas. Pelos resultados obtidos, verifica-se que a técnica de PCR representou uma importante ferramenta de detecção de leptospiras em primatas não-humanos, principalmente considerando-se que não foi necessária a viabilidade do patógeno, permitindo a detecção do agente em amostras autolisadas, congeladas ou amostras mal conservadas, que prejudicaram o isolamento e a inoculação experimental.

Background Human monocytotropic ehrlichiosis is an emerging life-threatening zoonosis caused by obligately intracellular bacterium, Ehrlichia chaffeensis. E. chaffeensis is transmitted by the lone star tick, Amblyomma americanum, and replicates in mononuclear phagocytes in mammalian hosts. Differences in the E. chaffeensis transcriptome in mammalian and arthropod hosts are unknown. Thus, we determined host-specific E. chaffeensis gene expression in human monocyte (THP-1) and in Amblyomma and Ixodes tick cell lines (AAE2 and ISE6) using a whole genome microarray. Methodology/Principal Findings The majority (∼80%) of E. chaffeensis genes were expressed during infection in human and tick cells. There were few differences observed in E. chaffeensis gene expression between the vector Amblyomma and non-vector Ixodes tick cells, but extensive host-specific and differential gene expression profiles were detected between human and tick cells, including higher transcriptional activity in tick cells and identification of gene subsets that were differentially expressed in the two hosts. Differentially and host-specifically expressed ehrlichial genes encoded major immunoreactive tandem repeat proteins (TRP), the outer membrane protein (OMP-1) family, and hypothetical proteins that were 30–80 amino acids in length. Consistent with previous observations, high expression of p28 and OMP-1B genes was detected in human and tick cells, respectively. Notably, E. chaffeensis genes encoding TRP32 and TRP47 were highly upregulated in the human monocytes and expressed as proteins; however, although TRP transcripts were expressed in tick cells, the proteins were not detected in whole cell lysates demonstrating that TRP expression was post transcriptionally regulated. Conclusions/Significance Ehrlichia gene expression is highly active in tick cells, and differential gene expression among a wide variety of host-pathogen associated genes occurs. Furthermore, we demonstrate that genes associated with host-pathogen interactions are differentially expressed and regulated by post transcriptional mechanisms.