Detection of the enteroaggregative Escherichia coli heat-stable enterotoxin 1 (EAST1) gene and its relationship with fimbrial and enterotoxin markers in E. coli isolates from pigs with diarrhoea
Introduction
Escherichia coli strains are important causes of diarrhoea in humans and animals (Holland, 1990, Nataro and Kaper, 1998). E. coli isolates associated with enteric diseases have been classified into major categories, on the basis of their distinct virulence properties: enterotoxigenic (ETEC), enteropathogenic (EPEC), enterohemorrhagic (EHEC), and enteroinvasive (EIEC) and enteroaggregative (EAEC) (Nataro and Kaper, 1998). Among them, ETEC is a dominant bacterial agent of neonatal and postweaning diarrhoeal diseases in piglets (Nagy and Fekete, 1999). Bacteria of this group are defined as E. coli that elaborate at least one member of two defined categories of enterotoxins: heat-labile LT and heat-stable ST. Each of these toxin groups comprises at least two toxin variants: LTI and LTII, and STI and STII (Sears and Kaper, 1996, Nair and Takeda, 1998). LTI, STI, and STII have been found in both human and pig ETEC, whereas LTII is only sometimes reported from E. coli strains of pig origin (Seriwatana et al., 1988, Celemin et al., 1994). Most porcine ETEC possess one of the pilus adherence factors: F4, F5, F6, F18, or F41 (Gaastra and De Graaf, 1982); of those, F4 fimbriae seem to play a main role in the adherence of toxigenic E. coli bacteria to porcine intestinal epithelial cells.
Strains of enteroaggregative E. coli (EAEC), the most recently recognised category of diarrhoeagenic E. coli, adhere to tissue culture cells in an aggregative adherence pattern and are associated with watery diarrhoea in young children in the developing world (Scaletsky et al., 1984, Nataro and Kaper, 1998). The pathogenesis of EAEC infection is not fully understood; however, a characteristic histopathological lesion and several candidate virulence factors have been described (Scaletsky et al., 1984, Nataro and Kaper, 1998). One of them is a 38-amino-acid protein named enteroaggregative E. coli heat-stable enterotoxin 1 (EAST1) (Savarino et al., 1993). EAST1 of EAEC and STI of ETEC are genetically and immunologically distinct enterotoxins but both activate guanylate cyclase in the intestinal epithelial cells (Savarino et al., 1991). The gene (astA) encoding the production of EAST1 has been found either on plasmids, on the chromosome, or on both (Yamamoto et al., 1997). The role of EAST1 in intestinal secretion has not been clearly determined, however, a strong association of the toxin with several human colonisation factors of ETEC strains has been demonstrated (Yamamoto et al., 1997).
Recently, Yamamoto and Nakazawa (1997) and Choi et al. (2001) reported that EAST1 enterotoxin was present not only in EAEC strains but also in other human and animal (especially pig) diarrhoeagenic E. coli, including ETEC, EPEC, and EHEC. Although enteric colibacillosis is common in weaned piglets, there is very little information concerning the prevalence of EAST1-positive E. coli among bacteria isolated from diarrhoeal cases in these animals. In this study, the presence of the astA gene of EAST1 enterotoxin in ETEC strains isolated from piglets with diarrhoea was investigated by polymerase chain reaction (PCR) and its relationship with fimbrial and enterotoxin markers was determined.
Section snippets
Bacterial strains
E. coli strains (n=207) were isolated from weaned pigs (4–6 weeks old) with diarrhoea and were characterised previously (Osek, 1999a, Osek, 2000). The strains were obtained from six geographically separated pig farms in the western part of Poland. Rectal swabs were taken from 207 piglets, plated on MacConkey’s agar (Oxoid) and from each sample five colonies were picked. Those identified as E. coli using the API-20E biochemical system (bioMerieux) were tested by the PCR test with primers
EAST1 toxin genes
PCR identification of the astA gene revealed that among 207 E. coli strains analysed, 96 (46.4%) isolates were positive as determined by the presence of the 111 bp amplified product. The remaining 111 (53.6%) strains tested with the PCR method for the astA marker with primers EAST1-1 and EAST1-2 did not generate a PCR amplicon of 111 bp or any other size.
EAST1 toxin, fimbriae and serotypes of E. coli
Enterotoxin profiles and the presence of F4 fimbrial antigen in ETEC strains used in the study are shown in Table 1. Among the astA-positive
Discussion
In the present study, the prevalence of the EAST1 gene and its relationship with serotype, fimbriae, and enterotoxin genes in E. coli isolated from piglets with postweaning diarrhoea, was examined. It was clearly demonstrated that E. coli strains possessing genes for at least one enterotoxin type (classified as ETEC) harboured the additional marker encoding the production of EAST1 toxin. Moreover, a close association of the astA gene with the presence of porcine fimbrial colonisation factor F4
Acknowledgements
The author thanks Ms. Ewa Kufel for excellent technical assistance and Dr. Yolande Bertin for the E. coli EAST1 reference strain.
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