A new verotoxin (VT) variant, designated vt2g, was identified from a bovine strain of verocytotoxigenic Escherichia coli (VTEC) serotype O2:H25. When vt2g was aligned with published sequences of vt2 and vt variants, it exhibited the highest DNA sequence homology with vt2 and vt2c. However, vt2g was not detected by vt2-specific primers and probes, although it was partially neutralized by an antiserum to the VT2A subunit. VT2g was cytotoxic for Vero and HeLa cells and was not activated by mouse intestinal mucus. The vt2g gene was detected in 3 of 409 (0.7%) bovine VTEC strains, including serotypes O2:H25, O2:H45 and Ont:H ؊

SUMMARYThe aim of the study was to define the prevalence of verotoxin-producing Escherichia coli ( VTEC) in cattle and pigs in a Hong Kong abattoir. Faecal and carcass samples collected from 986 cattle and 487 pigs from an abattoir were tested for verotoxin ( VT) by PCR and cytotoxicity assays. VTEC was isolated from 41n5 and 1n8 % of cattle faecal and carcass samples and from 2n1 and 0n2 % of porcine faecal and carcass samples, respectively. Amongst 409 VTEC isolates from cattle, 9 were serotype O157 : H7 and eaeA + . The most prevalent t genotype among bovine VTEC was t1 + t2 + (73n8 %) and in porcine VTEC was t2e + (30 %). None of the porcine VTEC isolates and 9n3 % of the bovine VTEC isolates was eaeA + . The non-O157 serogroup VTEC isolates carrying eaeA and EHEC-hlyA belonged to serogroups O172, O15, O84, O91, O110 and O121. The local dietary preference for pork or chicken (rather than beef ), the low VTEC carriage in pigs, the rarity of additional virulence factors (eaeA) in VTEC isolated from cattle may explain the apparently low incidence of human diarrhoeal disease associated with VTEC in Hong Kong hitherto. However, the presence of non-O157 VTEC strains carrying the eaeA virulence marker in cattle highlights the fact that sole reliance on sorbitol-MacConkey agar for screening human VTEC isolates may underestimate the human disease burden. The changing dietary habits of the population in Hong Kong reinforce the need for continued vigilance.

The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione S-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing Escherichia coli (VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% ؎ 7.9% and 3.6% ؎ 2.3%, respectively, and those of VT2 were 1.7% ؎ 2.3% and 82.5% ؎ 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.Verotoxin (VT) is the principal virulence factor of verotoxigenic Escherichia coli (VTEC), an emerging food-borne pathogen associated with diseases ranging from uncomplicated diarrhea to the hemolytic-uremic syndrome (1, 24). There are two types of VT, VT1 and VT2; the latter type has variants, including VT2c and VT2e. All VTs belong to the Shiga toxin family, in which the C terminus of the A subunit is encircled by a pentameric ring formed by five identical B subunits (7). This A-B bipartite molecule first binds to a eukaryotic glycolipid receptor via the pentamer of the VT B subunits. Then the catalytic VT A subunit dissociates from the VT B subunit pentamer and translocates into the cytoplasm through a retrograde secretory pathway. Subsequently, inhibition of the 28S rRNA in 60S ribosomal subunits induces programmed cell death (12,14,19). The N terminus of the VT A subunit is the A1 fragment, which is a catalytic domain essential for the cytotoxicity of VT (8). The C terminus A2 fragment facilitates the noncovalent interaction between subunits A and B (2). The A1 and A2 fragments are separated by a trypsin-sensitive region. Finally, there is a transmembrane region at the C terminus of fragment A1. This region is involved in toxin translocation across the endoplasmic reticulum membrane (23). The minimal sequence of VT1A required for activity includes the transmembrane region, and the deletion of this region retarded the functional activity of VT1A (11). Structural and biological properties of the VTs have been studied extensively (15); subunits of VTs have been prepared in large quantities as fusion proteins and used in seroepidemiology (10).Antibodies against VTs have been produced for diagnostic or therapeutic purposes. These antibodies were produced by immuniz...