PESQUISA VETERINÁRIA BRASILEIRA

Abstract in English:

ABSTRACT.- Costa M.M., Silva M.S., Spricigo D.A., Witt N.M., Marchioro S.B., Kolling L. & Vargas A.P.C. 2006. [Epidemiology, molecular characterization and resistance to antimicrobials of Escherichia coli isolated from South-Brazilian pig herds.] Caracterização epidemiológica, molecular e perfil de resistência aos antimicrobianos de Escherichia coli isoladas de criatórios suínos do Sul do Brasil. Pesquisa Veterinária Brasileira 26(1):5-8. Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: agueda@ccr.ufsm.br Colibacillosis is an enteric disease with a major impact to the swine industry and is caused by enterotoxigenic strains of Escherichia coli. Forty clinical isolates from pigs with diarrhea and 13 environmental isolates were analysed regarding their genotypic profile, genetic relationship and antibiotic resistance. The most prevalent gene was Stb, identified in 50% of the isolates from clinical cases, and Sta and Lt were detected in 35% of them. Among the adesine factors investigated, F18 was found in 27.5% of the E. coli strains. The ERIC-PCR technique used for epidemiological characterization of the isolates did not show the expected discriminatory power. However, the test allowed separation of the isolates in groups, but did not evidence groups related to virulence factors. In the susceptibility test, the highest values for resistance were to tetracycline, in 88.6%. The index of multiple resistance to antimicrobials varied from 0 to 0.69.

• Escherichia coli genotypic profile resistance.

Abstract in Portuguese:

ABSTRACT.- Costa M.M., Silva M.S., Spricigo D.A., Witt N.M., Marchioro S.B., Kolling L. & Vargas A.P.C. 2006. [Epidemiology, molecular characterization and resistance to antimicrobials of Escherichia coli isolated from South-Brazilian pig herds.] Caracterização epidemiológica, molecular e perfil de resistência aos antimicrobianos de Escherichia coli isoladas de criatórios suínos do Sul do Brasil. Pesquisa Veterinária Brasileira 26(1):5-8. Departamento de Medicina Veterinária Preventiva, Centro de Ciências Rurais, Universidade Federal de Santa Maria, 97105-900 Santa Maria, RS, Brazil. E-mail: agueda@ccr.ufsm.br Colibacillosis is an enteric disease with a major impact to the swine industry and is caused by enterotoxigenic strains of Escherichia coli. Forty clinical isolates from pigs with diarrhea and 13 environmental isolates were analysed regarding their genotypic profile, genetic relationship and antibiotic resistance. The most prevalent gene was Stb, identified in 50% of the isolates from clinical cases, and Sta and Lt were detected in 35% of them. Among the adesine factors investigated, F18 was found in 27.5% of the E. coli strains. The ERIC-PCR technique used for epidemiological characterization of the isolates did not show the expected discriminatory power. However, the test allowed separation of the isolates in groups, but did not evidence groups related to virulence factors. In the susceptibility test, the highest values for resistance were to tetracycline, in 88.6%. The index of multiple resistance to antimicrobials varied from 0 to 0.69.

Abstract in English:

Simionatto S., Vaz E.K., Michelon A., Seixas F.K., Dellagostin O.A. 2005. [Development and evaluation of new strategies for immunization against swine colibacillosis.] Desenvolvimento e avaliação de novas estratégias de imunização contra colibacilose suína. Pes-quisa Veterinária Brasileira 25(2):84-90. Laboratório de Biologia Molecular, Centro de Bio-tecnologia, UFPel, Campus Capão do Leão, Cx. Postal 354, Pelotas, RS 96010-900, Brazil. E-mail: ssimionatto@bol.com.br Swine colibacillosis caused by enterotoxigenic Escherichia coli remains one of the main sanitary problems in pig farms. The recombinant DNA technology offers the possibility of developing new immunization strategies. This paper describes the development of a subunit vaccine through the expression and purification of the E. coli K88 FaeC fimbrial protein. The gene that codes for this antigen was amplified by PCR and cloned into an E. coli expression vector fused to a 6X histidine tag. The recombinant protein was purified by affinity chromatography and used for mice immunization. In parallel, the same gene was cloned into an eucariotic expression vector with the addition of the Kozak sequence for improving translation of this gene in muscle cells. The resulting plasmid named pUP310 was purified in large scale and used to immunize mice. The immune response afforded by both forms of immunization was monitored by ELISA. There was an immune response in mice inoculated with pUP310 and purified FaeC. It was possible to detect anti-FaeC antibodies 42 days after the first inoculation. The antibody titer increased with time, being still detectable 7 months after the first inoculation. It is concluded that recombinant FaeC and pUP310 are potential tools for immunization of swine against E. coli K88.

• Swine colibacillosis FaeC Escherichia coli DNA vaccine recombinant vaccine.

Abstract in Portuguese:

Simionatto S., Vaz E.K., Michelon A., Seixas F.K., Dellagostin O.A. 2005. [Development and evaluation of new strategies for immunization against swine colibacillosis.] Desenvolvimento e avaliação de novas estratégias de imunização contra colibacilose suína. Pes-quisa Veterinária Brasileira 25(2):84-90. Laboratório de Biologia Molecular, Centro de Bio-tecnologia, UFPel, Campus Capão do Leão, Cx. Postal 354, Pelotas, RS 96010-900, Brazil. E-mail: ssimionatto@bol.com.br Swine colibacillosis caused by enterotoxigenic Escherichia coli remains one of the main sanitary problems in pig farms. The recombinant DNA technology offers the possibility of developing new immunization strategies. This paper describes the development of a subunit vaccine through the expression and purification of the E. coli K88 FaeC fimbrial protein. The gene that codes for this antigen was amplified by PCR and cloned into an E. coli expression vector fused to a 6X histidine tag. The recombinant protein was purified by affinity chromatography and used for mice immunization. In parallel, the same gene was cloned into an eucariotic expression vector with the addition of the Kozak sequence for improving translation of this gene in muscle cells. The resulting plasmid named pUP310 was purified in large scale and used to immunize mice. The immune response afforded by both forms of immunization was monitored by ELISA. There was an immune response in mice inoculated with pUP310 and purified FaeC. It was possible to detect anti-FaeC antibodies 42 days after the first inoculation. The antibody titer increased with time, being still detectable 7 months after the first inoculation. It is concluded that recombinant FaeC and pUP310 are potential tools for immunization of swine against E. coli K88.

Abstract in English:

Penatti M.P.A., Silva A.S., Valadares G.F. & Leite D.S. 2005. Occurrence of F42 colonization factor in Escherichia coli strains isolated from piglets with diarrhea. Pesquisa Veterinária Brasileira 25(1):31-33. Depto Microbiologia e Imunologia, Instituto de Biologia, Unicamp, Campinas, SP 13081-970, Brazil. E-mail: domingos@unicamp.br The objective of this study was to determine the presence of the colonization factor F42 in 168 strains of Escherichia coli isolated from diarrheic stools of newborn piglets. The presence of F42 in 12 (7.1%) strains was detected with the agglutination test. Through the Polymerase Chain Reaction (PCR) of F42 positive strains, gene encoding enterotoxins (ST-I, ST-II, LT-I and LT-II) were detected. The finding of ST-I/ST-II genes in 50% of the strains, ST-I (16%) and ST-II (25%) indicates a strong association of FC F42 with heat-stable enterotoxins (91%). In contrast, the thermolabile enterotoxin (LT-I and LT-II) genes were not detected. Serogroups of F42 positive strains were determined, serogroup O8 being the most prevalent (41,7%). Other serogroups, as there are O9, O11, O18, O32, O35, O98 and O101, were also identified. Thus, FC F42 was confirmed as an additional factor of virulence in the pathogenesis of porcine colibacillosis.

• Escherichia coli F42 piglets fimbriae diarrhea PCR.

Abstract in Portuguese:

Penatti M.P.A., Silva A.S., Valadares G.F. & Leite D.S. 2005. Occurrence of F42 colonization factor in Escherichia coli strains isolated from piglets with diarrhea. Pesquisa Veterinária Brasileira 25(1):31-33. Depto Microbiologia e Imunologia, Instituto de Biologia, Unicamp, Campinas, SP 13081-970, Brazil. E-mail: domingos@unicamp.br The objective of this study was to determine the presence of the colonization factor F42 in 168 strains of Escherichia coli isolated from diarrheic stools of newborn piglets. The presence of F42 in 12 (7.1%) strains was detected with the agglutination test. Through the Polymerase Chain Reaction (PCR) of F42 positive strains, gene encoding enterotoxins (ST-I, ST-II, LT-I and LT-II) were detected. The finding of ST-I/ST-II genes in 50% of the strains, ST-I (16%) and ST-II (25%) indicates a strong association of FC F42 with heat-stable enterotoxins (91%). In contrast, the thermolabile enterotoxin (LT-I and LT-II) genes were not detected. Serogroups of F42 positive strains were determined, serogroup O8 being the most prevalent (41,7%). Other serogroups, as there are O9, O11, O18, O32, O35, O98 and O101, were also identified. Thus, FC F42 was confirmed as an additional factor of virulence in the pathogenesis of porcine colibacillosis.

Abstract in English:

The diarrheic processes caused by pathogenic enterobacteria are of great economicsanitary importance, with limiting effects on cattle raising. In the present study 106 fecal samples from 52 diarrheic and 54 non-diarrheic calves aged 1 to 90 days were analyzed, in arder to determine the occurrence of enterotoxigenic Escherichia coli (ETEC) and Salmonella sp among calves reared in the agreste region of the state of Pernambuco and to determine the behavior of the isolates in response to 16 antimicrobial agents. The strains were cultured in selective media and submitted to biochemical characterization, serologic identification and detection of heat-stable enterotoxin. E. coli was isolated from all 106 calves studied and five of these strains (4,7%) were proclucers of heat-stable enterotoxin. Salmonellasp was isolated from six (5,7%) samples belonging to serovars Dublin, Muenchen and Infantis. The Salmonella strains were highly sensitive to the drngs tested, whereas the E. co/istrains were more resistent to tetracycline (50,9%), sulfonamide (40,6%), streptomycin (35,9%) and ampicillin (22,6%). Particularly outstancling in the general analysis was the multiresistance of E. coli (52,0%), with a predominance of the tetracycline-sulfonamide association. The present results serve as a warning regarding the incliscriminated use of antimicrobial agents. Low percentages of ETEC and Salmonella in calves show the necessity of other diagnostic procedures to clarify the etiology.

• Diarrheia calves Escherichia coli Salmonella sp

Abstract in Portuguese:

Os processos diarréicos acarretados por enterobactérias patogênicas têm sido incriminados como de grande importância econômica-sanitária, desempenhando um papel limitante na criação de bezerros. Com o objetivo de estudar a ocorrência de Escherichia coli enterotoxigênica e Salmonella sp em bezerros no agreste meridional de Pernambuco e o compottamento dos isolados frente aos antimicrobianos, foram analisadas 106 amostras fecais provenientes de 52 bezerros diarréicos e 54 não diarréicos de 1 a 90 dias de idade. O cultivo das amostras foi realizado em meios seletivos com posterior caracterização bioquímica, identificação sorológica e detecção de enterotoxina termoestável. Para verificação do comportamento dos isolados, foram selecionados 16 antimicrobianos. E. coli foi isolada dos 106 bezerros estudados, sendo cinco (4,7%) produtoras de enterotoxina termoestável e, Salmonella isolada de seis (5,7%) amostras pertencentes aos sorovares Dublin, Muenchen e Infantis. As amostras de Salmonella demonstraram uma elevada sensibilidade frente as drogas testadas, enquanto que as de E. coli revelaram maior resistência à tetraciclina (50,9%); sulfonamida (40,6%); estreptomicina (35,9%) e ampicilina (22,6%). No cômputo geral destaca-se a multirresistência de E. coli (52%), predominando a associação tetraciclina-sulfonamida. Face aos resultados auferidos adverte-se quanto ao uso indiscriminado de antimicrobianos. Os baixos percentuais de ETEC e Salinonella salientam a necessidade de outros procedimentos de diagnóstico visando esclarecer a etiologia das diarréias.

• Diarréia bezerros Eschericbia coli Salmonella sp

Abstract in English:

An experimental study was conducted to evaluate the possibility of preventing Escherichia coli post-weaning diarrhea reducing the protein level and/or acidifying the diet. Sixteen SPF (Specific Pathogen Free) Landrace piglets, 36 days old (weaning day) were used in four treatments (T) as follow: TA) control ration with 20% crude protein (CP); TB) ration similar to TA with 2% citric acid; TC) ration with 16% CP plus lisine up to 0.9%; and TD) ration similar to TC plus 2% citric acid. Rations were formulated with corn, soybean meal, minerals and vitamins. The pigs received the experimental diets from Do (weaning day) to D10 (slaughter day). All piglets were inoculated with 20 ml of E. coli culture 0141 K85 with 109 CFU/ml on the day D3 and D4. The parameters analysed were, occurrence of diarrhea, faeces elimination and intestinal colonization with the E. coli sample inoculated, gastrointestinal content pH and small intestine histology. The addition of citric acid (TB) as well as CP reduction (TC) in the diet caused a benefic effect on the E. coli diarrhea. This effect was more evident with the CP reduction and addition of the acid simultaneously (TD). The diarrhea was prevented, probably, by the reduction of stomach and jejunum pH and by avoiding an exaggerated E. coli multiplication in the intestinal content.

• Pigs post-weaning diarrhea diet protein reduction diet acidification

Abstract in Portuguese:

Estudou-se experimentalmente a possibilidade de se prevenir a diarréia pós-desmama por Escherichia coli através da redução da proteína e/ou acidificação da dieta. Utilizaram-se 16 leitões Landrace SPF (Specific Pathogen Free) com 35 dias de idade (dia da desmama), distribuídos em quatro tratamentos (T): TA - ração controle com 20% de PB; T B - ração do TA com 2% de ácido cítrico; TC - ração com 16% PB mais lisina até nível de 0,9%; TD - ração do TC mais·2% do ácido cítrico. As rações foram formuladas com milho, farelo de soja, minerais e vitaminas. Os leitões receberam as dietas experimentais do D0 (dia da desmama) até D10 (dia do sacrifício). Todos os leitões foram inoculados com 20 ml de um cultivo de E. coli 0141 K85 contendo 109 UFC/ml, nos dias D3 e D4. Os parâmetros analisados foram: ocorrência de diarréia, eliminação nas fezes e colonização intestinal da amostra de E. coli inoculada, pH do conteúdo gastrointestinal e histologia do intestino delgado. Tanto a adição do ácido cítrico (TB) como a redução da PB (TC) na dieta tiveram efeito benéfico na ocorrência da diarréia por E. coli. Este efeito mostrou-se mais evidente com a redução da PB e adição do ácido simultaneamente (T D). A diarréia foi prevenida, possivelmente pela redução do pH do estômago e jejuno e por evitar a multiplicação exagerada de E. coli no conteúdo intestinal.

• Leitões diarréia pós-desmama redução de proteína acidificação

Abstract in English:

Four hundred and seventy-seven cultures of Escherichia coli isolated in the region of Concórdia, Santa Catarina, Brazil, were examined for the presence of ''virulence factors", such as: thermolabile (LT) enterotoxin; thermostable (STa and STb) enterotoxins and the colonization factors K88, K99 and 987P. Paired samples of sera from some sick piglets· were also collected for the search of anti-LT antibodies. Eighty-six (18,02%) E. coli cultures produced ·STa enterotoxin whereas LT enterotoxin was detected in 8 (1,67%) only. Among 391 STa- cultures 49 (12,53%) were STb+. Among 28 K88+ cultures, 1 produced LT; 9 STa and the remaining were non-enterotoxigenic. None of the L T+ cultures codified for K99 antigen, which, on the other hand, was found in 5 STa+ and in 17 non-entérotoxigenic cultures. 987P antigen was found in 3 non-enterotoxigenic cultures. Four out of 10 samples of the examined paired sera showed serological conversion in relation to titres found in ·the passive immunehemolysis (PIH) test. The results of serogrouping revealed that all LT+ cultures belonged to serogroup 01.49. Among 86 STa+ cultures, 1 was grouped as serogroup 09, 2 as 010, 4 as 035; 2 as 064; 3 as 0108; 2 as 0138; 1 as 0149 and 1 as 0157. Seventy STa+ cultures were not classified.· Among 49 STb+ cultures 1 belonged to serogroup 09; 4 to 010; 1 to 035; 1 to 0139; 2 to 0149; 1 to 0157 and 39 cultures could not be serogrouped. These results suggest that in porcine colibacillosis of the region of Concórdia, SC, Brasil the thermostable enterotoxins (STa and STb) may play an important role. It could be possible that among these cultures other colonization factors, different from K88, K99 and 987P may occur. Also, most cultures examined could not be grouped among the serogroups generally accepted as enteropathogenic for swine.

• Virulence factors porcine colibacillosis Concórdia Brazil

Abstract in Portuguese:

Quatrocentos e setenta e sete amostras de Escherichia coli, isoladas na região de Concórdia, SC, Brazil, foram examinadas quanto a presença de fatores de virulência a saber: enterotoxína termolábil (LT), enterotoxinas termoestáveis (STa e STb) e os fatores de colonização K88, K99 e 987P. Amostras pareadas de soro de alguns leitões doentes foram também coletadas para a pesquisa de anticorpos anti-LT. Oitenta e seis (18,02%) amostras de E. coli produziram a enterotoxina STa enquanto a enterotoxina LT-foi detectada em apenas 8 (1.67%). Entre as-381 amostras STa-, 49 (12.53%) foram STb+. Entre 28 amostras K88+, uma produziu LT, 9 STa e as restantes eram não enterotoxigenicas. Nenhuma das amostras ·LT+ codificou para o antígeno K99, porém, este foi encontrado em 5 amostras STa+ e em 17 não entérotoxigênicas. Quatro entre os 10 soros examinados apresentaram conversão sorológica nos níveis de anticorpos anti-LT quando os soros foram examinados pelo teste da imunohemólise passiva (PIH). Os resultados dos exames sorológicos revelaram que todas as amostras LT+ pertenciam ao sorogrupo 0149. Entre 86 amostras STa+, 1 foi classificada como pertencendo ao sorogrupo 09, 2 ao 010; 4 ao 035; 2 ao 064; 3 ao 0108; 2 ao 0138; 1 ao 0149; 1 ao 0157 e 70 amostras não foram Classificadas. Entre as 49 amostras STb+ 1 pertencia ao sorogrupo 09; 4 ao 010; 1 ao 035; 1 ao 0139; 2 ao 0149; 1 ao 0157 e 39 não foram classificadas. Estes resultados sugerem que na colibacilose suína da região· de Concórdia as enterotoxinas termoestáveis (STa e STb) possam desempenhar um importante papel, existindo a possibilidade de que nelas possam ocorrer outros fatores de colonização que não K88, K99 e 987P. As amostras estudadas também, em sua maioria, não se enquadraram entre· aqueles sorogrupos geralmente aceitos como enteropatogênicos para suínos.

• Fatores de virulência colibacilose suína Concórdia Brazil