Vibrio cholerae causes the potentiafly lethal disase cholera through the elaboration of the intestinal secretogen cholera toxin. A second toxin of V. cholerae, Zot, decreases intestinal tissue resistance by modifying interceflular tight junctions. In this report, a third toxin of V. cholerae, Ace (accessory cholera enterotoxin), is described. Ace increases short-circuit current in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. The predicted protein sequence of Ace shows striking similarit to eukaryotic iontransporting ATPases, including the product of the cystic fibrosis gene. The gene encoding Ace is located immediately upstream of the genes encoding Zot and cholera toxin. The ctx, zot, and ace genes, which are located on a dynamic sector of the chromosome, comprise a V. cholerae "virulence cassette." Development oflive attenuated Vibrio cholerae oral vaccines by recombinant technology initially targeted the genes encoding cholera toxin (ctx) for deletion. Although these initial vaccine candidates, such as JBK70 and CVD101, elicited high antibody responses and did not cause severe diarrhea in volunteers, more than half ofthe vaccinees developed mild to moderate diarrhea and many experienced abdominal cramps, anorexia, and low-grade fever (1). In search of an additional toxin which could explain this reactogenicity, we reported a second toxin, Zot (zonula occludens toxin), which decreases ileal tissue resistance by affecting intercellular tightjunctions (2). The diarrhea observed in volunteers fed Actx V. cholerae vaccine strains was hypothesized to be caused by alteration of tight junctions, with a subsequent increase in intestinal permeability.The gene encoding Zot is located immediately upstream of ctx (3) on a 4.5-kb region termed the "core region" (4). In many strains of V. cholerae, this 4.5-kb region is flanked by one or more copies of a 2.7-kb sequence called RS1; homologous recombination between RS1 elements can lead to tandem amplification of the 4.5-kb region (4). Since the zot and ctx genes comprise only 55% of the 4.5-kb core region, we investigated the potential pathogenic role ofthe remaining portion of this region. Our results show that the gene for a third toxin is included in this regionll and that this third toxin increases the short-circuit current (IS) in Ussing chambers and causes fluid secretion in ligated rabbit ileal loops. MATERIALS AND METHODSBacterial Strains. V. cholerae E7946 is an El Tor Ogawa strain previously demonstrated to produce cholera in volunteers (5). Strain CVD110 was constructed from E7946 by homologous recombination of the RS1 elements, resulting in deletion of the core region (J.M., J.E.G., A.F., and J.B.K.,The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. unpublished work). In brief, cloned ctx and zot sequences were inserted into the suicide vector pGP704 (7) and conjugated ...

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BackgroundIn sub-Saharan Africa, non-typhoidal Salmonella (NTS) are emerging as a prominent cause of invasive disease (bacteremia and focal infections such as meningitis) in infants and young children. Importantly, including data from Mali, three serovars, Salmonella enterica serovar Typhimurium, Salmonella Enteritidis and Salmonella Dublin, account for the majority of non-typhoidal Salmonella isolated from these patients.MethodsWe have extended a previously developed series of polymerase chain reactions (PCRs) based on O serogrouping and H typing to identify Salmonella Typhimurium and variants (mostly I 4,[5],12:i:-), Salmonella Enteritidis and Salmonella Dublin. We also designed primers to detect Salmonella Stanleyville, a serovar found in West Africa. Another PCR was used to differentiate diphasic Salmonella Typhimurium and monophasic Salmonella Typhimurium from other O serogroup B, H:i serovars. We used these PCRs to blind-test 327 Salmonella serogroup B and D isolates that were obtained from the blood cultures of febrile patients in Bamako, Mali.Principal FindingsWe have shown that when used in conjunction with our previously described O-serogrouping PCR, our PCRs are 100% sensitive and specific in identifying Salmonella Typhimurium and variants, Salmonella Enteritidis, Salmonella Dublin and Salmonella Stanleyville. When we attempted to differentiate 171 Salmonella Typhimurium (I 4,[ 5],12:i:1,2) strains from 52 monophasic Salmonella Typhimurium (I 4,[5],12:i:-) strains, we were able to correctly identify 170 of the Salmonella Typhimurium and 51 of the Salmonella I 4,[5],12:i:- strains.ConclusionWe have described a simple yet effective PCR method to support surveillance of the incidence of invasive disease caused by NTS in developing countries.

The genes of the bacterial sialidases from Clostridium sordellii G12, C. perfringens A99, Salmonella typhimurium LT-2 and Vibrio cholerae 395 sequenced so far were examined for homologies and were compared with sequences of viral sialidases. Each of the bacterial sialidases contains a short sequence of twelve amino-acids, which is repeated at four positions in the protein. All these sequences exhibit significant similarities. Comparing the repeated sequences of the four sialidases, five amino-acids were found to be highly conserved at defined positions: Ser-X-Asp-X-Gly-X-Thr-Trp. Additionally, most of the distances between the four repeated regions are also conserved among the different sialidases. The conserved bacterial sequences show similarity with sialidases of influenza A H7N1 and H13N9.