A. Kaeckenbeeck

A role for the Clostridium perfringens β2 toxin in bovine enterotoxaemia

Abstract Non-enterotoxigenic type A Clostridium perfringens are associated with bovine enterotoxaemia, but the α toxin is not regarded as responsible for the production of typical lesions of necrotic and haemorrhagic enteritis. The purpose of this study was to investigate the putative role of the more recently described β2 toxin. Seven hundred and fourteen non-enterotoxigenic type A C. perfringens isolated from 133 calves with lesions of enterotoxaemia and high clostridial cell counts (study population) and 386 isolated from a control population of 87 calves were tested by a colony hybridisation assay for the β2 toxin. Two hundred and eighteen (31%) C. perfringens isolated from 83 calves (62%) of the study population and 113 (29%) C. perfringens isolated from 51 calves (59%) of the control population tested positive with the β2 probe. Pure and mixed cultures of four C. perfringens (one α+β2+, one α+enterotoxin+ and two α+) were tested in the ligated loop assay in one calf. Macroscopic haemorrhages of the intestinal wall, necrosis and haemorrhages of the intestinal content, and microscopic lesions of necrosis and polymorphonuclear and mononuclear cell infiltration of the intestinal villi were more pronounced in loops inoculated with the α and β2-toxigenic C. perfringens isolate. These results suggest in vivo synergistic role of the α and β2 toxins in the production of necrotic and haemorrhagic lesions of the small intestine in cases of bovine enterotoxaemia. However, isolation of β2-toxigenic C. perfringens does not confirm the clinical diagnosis of bovine enterotoxaemia and a clostridial cell counts must still be performed.

DNA sequences coding for the F18 fimbriae and AIDA adhesin are localised on the same plasmid in Escherichia coli isolates from piglets

The adhesin-involved-in-diffuse-adherence (AIDA) afimbrial adhesin is produced by human, but not by animal, Escherichia coli, with the exception of German porcine verotoxigenic Escherichia coli (VTEC) [Clin. Diagn. Lab. Immunol. 8 (2001) 143]. Presence and localisation of DNA sequences (aidA) coding for and production of an AIDA adhesin were investigated in a collection of Belgian VTEC and non-VTEC E. coli isolated from piglets at weaning time. The 174 isolates were also studied by colony hybridisation for the presence of DNA sequences coding for the Stx2e verocytotoxin and the F18 fimbrial adhesin (fed): 71 were Stx2+F18+AIDA+, 26 were F18+AIDA+, 12 were AIDA+, two were Stx2+AIDA+, and one was Stx2+ only. Fifty-four of the 58 (F18+)AIDA+ isolates tested positive in a western blotting assay with an immune serum raised against the AIDA protein. Hybridisation with the AIDA gene probe on plasmid DNA profiles identified a probe-positive plasmid band in the 10 AIDA+ and in 24 of the 25 F18+AIDA+ isolates studied. Moreover in F18+AIDA+ isolates, only one plasmid band hybridised with both F18 and AIDA probes. These results confirm the presence of aidA-related genes in not only VTEC, but also non-VTEC, isolates from piglets and the production of an antigenically AIDA-related protein by the majority of probe-positive E. coli. Moreover the plasmid DNA hybridisation results suggest a localisation on the same plasmid of the aidA- and fed-related DNA sequences.

Virulence plasmids of enterotoxigenic Escherichia coli isolates from piglets

Virulence plasmids of 68 ETEC isolates from piglets belonging to different pathotypes and six ETEC isolates from calves with pathotypes typical of porcine ETEC were identified with seven virulence probes for the heat-stable (STa and STb) and heat-labile (LT) enterotoxins, for the F4, F5, F6, and F41 fimbrial adhesion subunit, and also with five Rep probes for the RepFIA and RepFIB basic replicons, and the RepFIC family of basic replicons. With the exception of the F41 probe, the other virulence probes hybridized with at least one plasmid band of a size range from 65 to more than 100 Mda. Common associations of virulence factor-encoding genes on plasmid bands were: STb/LT, STa/F5, STa/F6, STa/STb. Other associations, STa/F4, STa/F4/F6, and STa/STb/LT/F6, were rarer. On the other hand the F4 adhesin-encoding genes were isolated on one plasmid band in all but three F4+ isolates. All but one of the 92 virulence plasmids which were studied have Rep probe hybridization profiles and replicon types typical of the uni- or multireplicon plasmids belonging to the various incompatibility groups of the F incompatibility complex.