Caracterização de estirpes de Campylobacter coli isoladas de carcaças de ovinos e de efluentes de abatedouro do estado de São Paulo / Characterization of Campylobacter coli strains isolated from the carcasses of sheep and of wastewater of abattoir in the state of São Paulo

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.

A zoonosis and bacterial disease, campylobacteriosis is responsible for frequent cases of human gastroenteritis worldwide. Campylobacter spp. presents the virulence factor called cytolethal distensive toxine (CDT), responsible for diarrhea and codified by the cdt gene. The aims of this study were: 1) to isolate and identify Campylobacter spp. strains from 102 carcass swabs and 102 rectal swabs of sheep (Ovis aries) and seven samples of wastewater, before and after the disinfection treatment, collected from the abattoir of the state of São Paulo; and 2) to detect the presence of cdt gene complex by Multiplex-PCR in strains of Campylobacter spp. Seven strains of Campylobacter coli were isolated and identified by phenotypic and genotypic methods: 4/102 (3.92%) from rectal swabs, 1/102 (0.98%) from carcass swabs and 2/7 (28.5%) from wastewater. From the rectal swab samples 2/7 (28.6%) strains were detected with the cdt gene. This is the first report on the isolation of Campylobacter coli from sheep abattoir, and of strains carrying the cdt gene complex in this animal species in Brazil.

Detecção de fatores de virulência em estirpes de Campylobacter spp. isoladas de carcaças de suínos abatidos em frigoríficos / Virulence factors in strains of Campylobacter spp. isolated from the carcasses of swine slaughtered in abattoirs

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.

Caracterização molecular e fenotípica de estirpes de Escherichia coli produtoras de shiga-toxina (STEC) não-O157 de fezes e carcaças bovinas / Molecular and phenotypic characterization of shiga toxin producing Escherichia coli (STEC) non-O157 strains from bovine feces and carcass

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.

Excretion of Brucella abortus vaccine B19 strain during a reproductive cycle in dairy cows

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.

Detecção direta de Actinobacillus pleuropneumoniae em órgãos de suídeos do estado de São Paulo pela técnica de reação em cadeia pela polimerase (nested-PCR) / Direct detection of Actinobacillus pleuropneumoniae in suidae organs in São Paulo state, Brazil, by polymerase chain reaction (nested-PCR)

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.

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.

Use of pcr-rflp of thefla a gene for detection and subtyping of Campylobacter jejuni strains Potentially related to Guillain-barré syndrome, isolated from humans and animals

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.

Genotyping of Clostridium perfringens associated with sudden death in cattle

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 percent) were identified as type E, two (8.7 percent) as type D and the remainder as type A (82.6 percent). 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.(AU)

Identification of Clostridium chauvoei in clinical samples cultures from blackleg cases by means of PCR

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.

Foi detectada presença de Clostridium chauvoei pela reação de PCR a partir de cultivo em cooked meat medium de amostras de fígado, músculo e medula óssea metatarsiana de sete bezerros acometidos de carbúnculo sintomático. O isolamento de uma amostra de músculo sob condições anaeróbias revelou Clostridium perfringens em cultura pura.

Isolation and identification of bovine tuberculosis in a Brazilian herd (São Paulo)

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 percent) from 18 animals (60 percent) were positive for mycobacteria by culture, including samples from 15 retrofaryngeal lymphnodes (55.5 percent), 9 prescapular lymphnodes (33.3 percent), 2 lungs (7.4 percent), and 1 liver (3.7 percent). All isolated colonies were confirmed by PCR to contain MTBC organisms, and were identified as M. bovis by the same methodology.

Clostridium perfringens types A and D associated with enterotoxemia in an 18-month-old goat

Postmortem examination of a Boer buck that died peracutely revealed bowel and liver diffusely congested and edematous. Kidney was apparently edematous. Clostridium perfringens type A was isolated from bowel and type D from kidney. Microscopic examination revealed large areas of necrosis in the renal cortex and medulla (pulpy kidney disease), hyperemia and centrilobular necrosis of the liver, necrosis of the small-intestine wall, pulmonary edema and congestion, intense hyperemia of the cerebellum, hyperemia and edema of the brain.