Virulence profiles of enterotoxigenic Escherichia coli isolated from piglets with post-weaning diarrhea and classification according to fecal consistency

Pesquisa Veterinária Brasileira

Cardoso, Marisa R.I.¹  Tagliari, Vinícius²  Bernardi, Mari L.¹  Andrade, Mariana R.⁴  Koerich, Priscilla K.V.³  Sato, José Paulo H.⁴  Barcellos, David E.S.N.⁴  Takeuti, Karine L.⁴ 

[1] UFRGS, Porto Alegre, Brazil;BRF - Brasil Foods, Videira, Brazil;BRF - Brasil Foods, Curitiba, Brazil;Universidade Federal do Rio Grande do Sul, Porto Alegre, Brazil

关键词:  ETEC;    swine;    diarrhea;    enterotoxigenic;    Escherichia coli;    piglets;    virulence factors;    fecal consistency;   

DOI  :  10.1590/S0100-736X2016000400001

来源: Colegio Brasileiro de Patologia Animal-CBPA

【 摘 要 】

The aim of this study was to assess the frequency and association of virulence factors of Escherichia (E.) coli isolated from weaned piglets with diarrhea and to correlate it with fecal consistency. A total of 152 rectal swabs were collected from 25-40 day-old piglets with diarrhea, in farms of Southern Brazil. Phenotypical and molecular techniques were used for bacterial isolation, characterization and classification of enterotoxigenic E. coli (ETEC) pathotypes. Statistical analysis was carried out to determine the frequency of virulence factors and virotypes, of fimbriae F4, F5, F6, F18, F41 and toxins LT, STa, STb and STx2e. Out of 456 E. coli isolates, 287 (62.9%) samples showed significant growth of E. coli. Among them, 194 (67.6%) samples showed at least one virulence factor, indicating that ETEC is an important etiological agent of diarrhea in weaned piglets. Higher frequencies were found of fimbria F4 and F18 and enterotoxins LT, STa and STb. Significant association was found to F4, LT, STa and STb; between F18 and STa and STx2e; between F5 and LT, STa and STb. The most frequent virotypes were F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb and F18-STa-STx2e. Beta-hemolysis was observed in 47.4% of samples and there was significant association between hemolytic samples and virulence factors F4, F18, STa and STx2e. Regarding fecal consistency, there was significant association of liquid feces and F4 fimbria, STa toxin and virotypes F4-STa and F4-F5-LT-STa-STb. Since there was significant association of ETEC and liquid feces in nursery piglets, it is important to prioritize the sampling of liquid feces for the diagnosis etiologic cause of diarrhea.Index Terms: ETEC; swine; diarrhea; enterotoxigenic; Escherichia coli; piglets; virulence factors; fecal consistencyResumoO objetivo deste estudo foi avaliar a frequência e associação de fatores de virulência de Escherichia (E.) coli isoladas de leitões desmamados com diarreia e correlacioná-la com consistência fecal. Suabes retais foram coletados em leitões com 25-40 dias de idade com sinal clínico de diarreia, em granjas do Sul do Brasil, totalizando 456 amostras. Foram utilizadas técnicas fenotípicas e moleculares para isolamento bacteriano, caracterização e classificação de patotipos de E. coli enterotoxigênica (ETEC). A análise estatística foi realizada para determinar a frequência de fatores de virulência e virotipos, de fímbrias F4, F5, F6, F18, F41 e toxinas LT, STa, STB e STx2e. Duzentas e oitenta e sete (62,9%) amostras apresentaram crescimento significativo de E. coli. Entre os quais, 194 (67,6%) amostras apresentaram pelo menos um fator de virulência, indicando que ETEC é um importante agente etiológico de diarreia em leitões desmamados. As frequências mais elevadas foram encontradas para as fímbrias F4 e F18 e enterotoxinas LT, STa e STb. Associação significativa foi encontrada para F4, LT, STa e STb; entre F18 e STa e STx2e; entre F5 e LT, STa e STb. Os virotipos mais frequentes foram F18-STa, F4-LT-STa-STb, F4-STa, F4-LT-STb e F18-STa-STx2e. Beta-hemólise foi observada em 47,4% das amostras e houve associação significativa entre amostras hemolíticas e fatores de virulência F4, F18, STa e STx2e. Quanto consistência fecal, houve associação significativa de fezes líquidas e fímbria F4, toxina STa e virotipos F4-STa e F4-F5-LT-STa-STb. A associação significativa da ETEC e fezes líquidas em leitões de creche, é importante para priorizar a amostragem de fezes com essa consistência para no diagnóstico etiológico da diarreia.Termos de Indexação: ETEC; suíno; diarreia; Escherichia coli enterotoxigênica; fatores de virulência; consistência fecalIntroductionEnterotoxigenic Escherichia coli (ETEC) is one of the main causes of diarrhea in pigs and is associated with neonatal colibacillosis and post-weaning colibacillosis (Fairbrother & Gyles 2006), resulting in significant economic losses to pig production (Ewing & Cole 1994, Barcellos et al. 2011). In order to cause diarrhea, E. coli must be able to adhere to enterocytes, avoiding elimination through peristalsis (Menin et al. 2008) and to produce enterotoxins (Fairbrother & Gyles 2006). Adhesion is mediated by fimbriae, being F4 (K88), F5 (K99), F6 (P987), F7 (F41) and F18 the most commonly found in pathogenic E. coli (Francis, 2004). Among the enterotoxins associated with ETEC strains, the thermolabile (LT) and thermostable (STa, STb) are the most relevant ones (Dubreuil 2008).In Brazil, the routine diagnostic of colibacillosis in piglets is often accomplished only by E. coli isolation, without determining the virulence factors in the isolates. However, to confirm that the isolate is pathogenic, it is essential to identify the set of virulence factors (fimbriae and toxins), and, consequently, classification in virotypes (Nataro & Kaper 1998). The association between hemolysis in blood agar cultures and certain E. coli virotypes has been previously described (Frydendahl 2002, Chapman et al. 2006, Do et al. 2005). Therefore, hemolytic E.coli strains isolated from diarrheic fecal samples have often been identified as pathogenic. However, this criterion is not enough to characterize enteropathogenicity (Frydendahl 2002), being the virotype determination the best approach.The detection of genes codifying fimbriae and enterotoxins in E. coli isolates by polymerase chain reaction (PCR) has contributed to make a more accurate and straightforward diagnosis of virotypes (Fairbrother 2006). Multiplex PCR improved the diagnosis, allowing the investigation of different virulence factors in the same reaction (Francis 2004), speeding results and reducing genotyping costs (Ruiz-Rueda et al. 2011). Real-time polymerase chain reaction (qPCR) generate results in higher speed, sensitivity and specificity when compared to traditional PCR methods (Elizaquivel et al. 2010). The objective of this study was to investigate the ETEC virotypes associated with post-weaning diarrhea in pig farms from Southern Brazil, using a multiplex qPCR protocol.Materials and MethodsSampling was performed in thirteen pig farms located from seven municipalities of the following states of southern Brazil: Rio Grande do Sul (Lajeado and Casca), Santa Catarina (Concordia and Videira) and Parana (Cascavel, Marechal Candido Rondon and Toledo). These farms adopted a similar housing, feed formulation and medication managements. Pigs were weaned around the 21 days of age, and housed in cages, with fully slatted metal floor. Pigs between 25 and 40 days of age that presented diarrhea and had not been previously medicated were included in the study. Animals were selected for sampling based on diarrhea staining on the hindquarters; reddened and/or humid perineal area; apathy and dehydration. The presence of liquid feces on the floor, walls or below crates was also assessed. Immediately before sampling, feces consistency was evaluated with the following visual criteria: normal; pasty; creamy and liquid.Rectal swabs were collected from 12 to 15 piglets in each farm. Immediately after collection, swabs were individually stored in transport medium (Stuart - Larbor Import©), shipped under refrigeration to the laboratory, and processed within 48 hours. Swabs were streaked on Blood agar base (Difco®) enriched with 5% defibirinated sheep blood and MacCon

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