Clonagem, expressão, caracterização molecular da proteína de superfície MSP5 da amostra PR1 de Anaplasma marginale e sua aplicação em um teste de ELISA por competição / Cloning, expression, molecular characterization of the MSP5 protein from PR1 strain of Anaplasma marginale and its application in a competitive enzyme-linked immunosorbent test

No presente trabalho, um teste imunoenzimático por competição (cELISA) foi padronizado com a proteína recombinante de superfície rMSP5, clonada a partir do gene msp5 do isolado PR1 de A. marginale. O sequenciamento mostrou que o gene que codifica a rMSP5/PR1 tem 98 por cento de identidade com os isolados Flórida e Santa Maria, 97 por cento com isolados de Pernambuco, Brasil e Havana, Cuba e 91 por cento com A. centrale. O teste de cELISA-PR1 foi comparado com os testes de IFI e cELISA-USA. Para a padronização e validação do cELISA-PR1, foram utilizados 380 soros bovinos comprovadamente positivos ou negativos pelo cELISA-USA. Desse total, 245 soros positivos eram de animais de área endêmica e 135 soros eram negativos, de área livre de anaplasmose. Na padronização, foram utilizados 283 soros de bovinos, dos quais 135 eram negativos e 148 positivos. Os testes de cELISA-PR1 e IFI apresentaram especificidade de 100 e 99,3 por cento, sensibilidade de 100 e 98 por cento, com coeficiente kappa de 0,993 e 0,978, respectivamente. Na validação do teste, foram utilizados 245 soros de bovinos de áreas endêmicas para anaplasmose, testados pelo cELISA-PR1 e IFI e apresentaram especificidade de 96,7 e 69,1 por cento, sensibilidade de 98,9 e 96,3 por cento e coeficiente kappa de 0,956 e 0,699, respectivamente. Esses resultados permitem afirmar que o teste de cELISA-PR1 apresentou performance equivalente ao cELISA-USA, podendo também ser utilizado em estudos epidemiológicos, programas de controle e movimentação internacional de animais, enquanto a IFI, com os resultados menos precisos apresentados, não deve ser utilizada em situações que requerem maior rigor no diagnóstico.

A competitive enzyme-linked immunosorbent test using the PR1 recombinant major surface protein 5 (rMSP5-PR1-ELISA) of Anaplasma marginale was standardized and validated using sera from anaplasmosis free and endemic regions. The sequencing of the msp5 gene of PR1 isolate showed 98 percent of identity with the Florida and Saint Maries isolates, 97 percent with Brazil (Pernambuco) and Havana isolates; and 91 percent with A. centrale. The cELISA-PR1 test was compared to IFI and cELISA-USA. For the standardization and validation of the cELISA-PR1, 380 bovine sera were used, whereas 245 truly positives and 135 truly negatives sera tested by the cELISA-USA. In the standardization of the cELISA-PR1 135 negative and 148 positive bovine sera were used. The cELISA-PR1 and IFI tests showed 100 and 99.3 percent specificity, 100 and 98 percent, sensibility, and a kappa coefficient of 0.993 and 0.978, respectively. For test validation, 245 bovine sera from an anaplasmosis endemic area were analyzed by the cELISA-PR1 and IFI, which showed 96.7 and 69.1 percent specificity, 98.9 e 96.3 percent sensibility and kappa coefficient of 0.956 and 0.699, respectively. These results indicate that the cELISA-PR1, likewise the cELISA-USA, could sensitively and specifically detect cattle naturally infected with A. marginale and would be recommended for epidemiological studies, eradications program, and regulation of international cattle movement, while IFI, which presented lower specificity should not be used in situations that demand more specific diagnosis.

Frequency of virulence genes in Escherichia coli strains isolated from piglets with diarrhea in the North Parana State, Brazil / Frequência de genes de virulência em Escherichia coli isolada de suínos com diarréia no norte do Estado do Paraná, Brasil

Identification of Escherichia coli causing porcine postweaning diarrhea requires knowledge regarding the prevalent pathotypes within a given region. A total of 100 Escherichia coli isolates from piglets with diarrhea in Londrina city, Parana State, South Brazil, were screened for the presence of genes for F4, F5, F6, F18, F41 fimbrial antigens by specific probes and for enterotoxins (STa, STb, LT and STx2e) by polymerase chain reaction (PCR). The results showed that 60% of the isolates were positive for one or more of the fimbrial antigens and 92% were positive at least for one of the virulence factors examined. Virulence factor genesdetected were F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa (40%), STb (47%) andSTx2e (3%). Twenty four patterns of virulence factor according to the different virulence genes form werefound and the most frequent virulence gene pattern was F4, F18, F41, STa, STb and LT. Most of the isolates that carried genes for adhesins also harboured genes for toxins.

A identificação de amostras de Escherichia coli responsáveis por diarréia pós-desmame em suínos requerconhecimento dos patotipos prevalentes dentro de uma dada região. Cem amostras de Escherichia coli isoladas de leitões com diarréia no Estado do Paraná, Brasil, foram testadas para apresença dos genes que codificam antígenos fimbriais F4, F5, F6, F18, F41 e para a produção de enterotoxinas (STa, STb, LT and STx2e), através de sondas e da técnica da PCR (polymerasechain reaction). Os resultados mostraram que 60% dos isolados foram positivos para um ou mais antígenos fimbriais e 92% foram positivos para pelo menos um dos fatores de virulência examinados. Os genes de virulência detectados foram F4 (44%), F18 (38%), F5 (30%), F41 (32%), F6 (25%), LTp-I (71%), STa(40%), STb (47%) e STx2e (3%). Vinte e quatro padrões de virulência, de acordo com as diferentes combinações dos genes de virulência, foram encontrados e o mais prevalente foi F4,F18, F41, STa, STb e LT. A maioria das amostras que carreiam genes para adesinas também transportam genes para produção de toxinas.

Clonagem, sequenciamento, expressão e caracterização do gene tsh de uma cepa de Escherichia coli patogênica de aves / Cloning, sequencing, expression, and characterization of the tsh gene from an avian pathogenic Escherichia coli strain

A hemaglutinina temperatura sensível (Tsh) pertence à família das serino-proteases autotransporte de Enterobacteriacea (SPATE), as quais são capazes de clivar diferentes substratos. Nós isolamos e caracterizamos o gene de Escherichia coli patogênica aviária (APEC) amostra APEC 13, sorotipo O2:H9, clonado em pET 101. A região de 4.2 kb do DNA clonado codificou uma proteína de aproximadamente 140 kDa (r-Tsh). O plasmídio recombinante pET 101-tsh conferiu um fenótipo de hemaglutinação positivo para a linhagem BL21 (tsh) para eritrócitos de galinha. A proteína r-Tsh foi purificada em coluna de níquel e utilizada na produção de anticorpos anti-Tsh. Um fragmento de 1.6 kb foi amplificado e subclonado em pCR4, e a seqüência parcial mostrou alta homologia com outras seqüências analisadas. O anti-Tsh reagiu com as proteínas r-Tsh e Tsh nativa da amostra APEC13, como demonstrado pela técnica de Western blot, mostrando que a r-Tsh tem epitopos conservados e que sua antigenicidade foi preservada. O anti-Tsh também inibiu a atividade hemaglutinante das amostras APEC 13 e BL12/pET 101-tsh

The temperature-sensitive hemagglutinin (Tsh) belongs to a family of high-molecular-weight serineprotease autotransporters of Enterobacteriaceae (SPATEs), which can cleave different substrates. Weisolated and characterised the tsh gene from an avian pathogenic Escherichia coli (APEC) strain, APEC13serotype O2:H9, which was cloned in pET101. The 4.2 kb region of cloned DNA coded one protein ofapproximately 140 kDa (r-Tsh). The recombinant plasmid pET101-tsh conferred to E. coli BL21 strain(tsh) the hemagglutination-positive phenotype against chicken erythrocytes. The r-Tsh was purified byNi-NTA column and used to produce antibody anti-Tsh. A 1.6 kb fragment of the tsh sequence was alsoamplified and cloned in pCR4, and a partial sequence showed high homology with other sequenceanalysed. The anti-Tsh reacted with the protein r-Tsh and native Tsh of APEC13, as demonstrated byWestern blot, showing that r-Tsh has conserved epitopes and that its antigenicity was preserved. Theanti-Tsh also inhibited the hemagglutinating activity of strains APEC13 and BL21/pET101-tsh

Molecular identification of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea in Londrina, Parana, Brazil

In this work, the prevalence of enteropathogenic Escherichia coli (EPEC) in children in Londrina-PR, Brazil, was evaluated by means of digoxigenin-labelled DNA probes which identify the plasmid responsible for EPEC adherence factor (EAF), and virulence genes for EPEC as bundle-forming pilus (bfp) and E. coli attaching-effacing factor (eae). In addition, the isolated strains were serotyped and tested for adherence to HEp-2 cells. From 102 children with diarrhoea, 19 strains hybridized with at least one probe, and eleven of them were identified as typical EPEC because they hybridized with the three probes used, showed a localized adherence (LA) pattern, and presented no genes for enterotoxins (ST and LT) or invasion as detected by PCR. Six of the typical EPEC strains belonged to the classical serotype O119:H6 43(per cent); in four strains O antigens could not be determined using antisera against O1 to O173, they were all ONT:H7 29(per cent); one strain belonged to O111:H6. Three strains were classified as atypical EPEC: O26H-, O111:H9 and O119:HNT. Strains O26H- and O111:H9 hybridized with the eae probe only and showed localized adherence like (LAL) pattern; strain O119:HNT hybridized with the bfp and eae probes, and showed a localized adherence/diffuse adherence (LA/DA) pattern after 6 h. A DA pattern was observed in two strains isolated from children with diarrhoea (ONT:H11 and O142:H34), which hybridized with the eae probe. From 46 controls, five strains hybridized with one or two probes, but none hybridized with all probes or presented the LA pattern. Three strains with the DA pattern hybridized with the eae probe. No EPEC strain belonging to classical EPEC serotypes was isolated from faeces of control children.