Trans R Soc Trop Med Hyg, 1995 Sep-Oct, 89(5), 542 - 5 Immunological response to Vibrio cholerae O1 infection and an oral cholera vaccine among Peruvians; Sanchez JL et al.; A 'double-blind', randomized, placebo controlled study of an oral inactivated whole cell plus recombinant B subunit (WC/rBS) cholera vaccine was conducted during February-March 1992 in Peru in 346 military recruits, 307 (89%) of whom received 2 oral doses of vaccine or Escherichia coli K12 placebo, 2 weeks apart . Paired serum samples were obtained from 155 (50%) of the recipients of 2 doses . An epidemic of cholera took place between doses . No difference in cholera attack rates was detected between vaccine and placebo recipients after one dose (8% versus 14%) . Seroconversion (4-fold or higher increase in vibriocidal antibody titres) was detected in 90% and 80% of vaccine and placebo recipients, respectively, with low pre-existing vibriocidal titres (< 0.01) . The anti-cholera toxin seroconversion rate among those with low pre-existing titres was higher in vaccinated subjects (97%) than in placebo recipients (68%) (P < 0.01) . Administration of 2 doses of WC/rBS vaccine concomitantly with natural V . cholerae O1 infection enhanced the serum anti-cholera toxin response . The immune response to the whole cell component of the vaccine was reduced by high pre-existing vibriocidal antibody titres. Protein Sci, 1995 Sep, 4(9), 1931 - 3 Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Vibrio mimicus; Musayev FN et al.; Single crystals of arylesterase (EC 3.1.1.2) from Vibrio mimicus have been obtained from ammonium sulfate as a precipitant at room temperature for 2 months . The present crystals diffract up to 2.2 A resolution and belong to monoclinic space group P2(1) . The cell dimensions are a = 55.65(1) A, b = 53.46(1) A, c = 65.79(1) A, and beta = 106.54(1) degrees . There are two molecules of molecular weight 22 kDa in an asymmetric unit with a solvent content of 43%. Arch Microbiol, 1995 Sep, 164(3), 223 - 30 Cell-surface charge and cell-surface hydrophobicity of collagen-binding Aeromonas and Vibrio strains; Ascencio F et al.; Partitioning in aqueous polymer two-phase systems of polyethylene glycol and dextran was used to detect and compare cell-surface charge and cell-surface hydrophobicity of Aeromonas hydrophila, A . caviae, A . sobria, Vibrio cholerae, and V . anguillarum strains . These strains have cell-surface components that bound either native or thermally denatured type I collagen (i.e., a mixture of the alpha1+alpha2 chains) and gelatin immobilized on latex beads . Our goals were: (1) to compare the possible relationship between the cell-surface charge/hydrophobicity and binding to collagen and (2) to evaluate the influence of the culture media on the expression of surface properties . There was no apparent relationship between cell-surface charge, cell-surface hydrophobicity, and binding to collagen . The expression of surface properties was dependent on the culture media . There was no relationship between binding to immobilized collagen and binding to soluble 125I-labeled collagen . Particle-agglutination reactivity differed when using various collagen-coated microbead preparations . There were general differences in the particle-agglutination reactivity when collagen-coated latex beads were prepared using different coating procedures . The negative charge and hydrophobicity of the various collagen-coated microbead preparations were also studied by partitioning in the two-phase system of polyethylene glycol and dextran . Under these conditions, the alpha1+alpha2 collagen-chain mixture covalently immobilized on carboxy-modified latex beads was less hydrophobic and negatively charged than gelatin and native collagen immobilized on the same kind of latex beads . For latex beads passively coated with collagen preparations, the alpha1+alpha2 collagen-chain mixture was more hydrophobic than gelatin and native collagen . We suggest that for screening collagen-binding among Vibrio and Aeromonas strains, a reliable and sensitive particle-agglutination assay should consider the collagen preparation and the coating procedure for the immobilization of collagen onto the latex beads . In this regard, carboxy-modified latex beads coated with an alpha1+alpha2 collagen-chain mixture gave the best results. J Bacteriol, 1995 Sep, 177(17), 5158 - 60 Isolation of the polar and lateral flagellum-defective mutants in Vibrio alginolyticus and identification of their flagellar driving energy sources; Kawagishi I et al.; Vibrio alginolyticus has two types of flagella (polar and lateral) in one cell . We isolated mutants with only a polar flagellum (Pof+ Laf-) or only lateral flagella (Pof- Laf+) . Using these mutants, we demonstrated that the energy sources of the lateral and polar flagellar motors in V . alginolyticus are H+ and Na+ motive forces, respectively, as in the related species V . parahaemolyticus. J Infect Dis, 1995 Sep, 172(3), 883 - 6 Initial clinical studies of CVD 112 Vibrio cholerae O139 live oral vaccine: safety and efficacy against experimental challenge; Tacket CO et al.; Since October 1992, epidemics of cholera associated with Vibrio cholerae O group 139 have occurred in India, Bangladesh, and much of the rest of Asia . A volunteer model was used to determine the safety, immunogenicity, and efficacy of an attenuated delta ctxA delta zot delta ace delta cep V . cholerae O139 vaccine strain, designated CVD 112 . Six volunteers received 10(6) cfu and 6 received 10(8) cfu of CVD 112 . No subject who received the 10(6) dose had diarrhea or other severe symptoms after vaccination; 3 vaccinees developed mild diarrhea (mean stool volume, 648 mL) after receiving the higher dose . Five weeks after vaccination, 8 vaccinees and 15 unvaccinated control subjects underwent challenge with 10(6) cfu of wild type V . cholerae O139 AI1837 . One vaccinee (13%) and 12 control subjects (80%) developed diarrhea after challenge (P = .003) . The short-term protective efficacy conferred by vaccine strain CVD 112 was 84% and was remarkably similar to that conferred by primary wild type clinical infection (80%). J Med Microbiol, 1995 Sep, 43(3), 216 - 20 Prevalence of Vibrio cholerae with heat-stable enterotoxin (NAG-ST) and cholera toxin genes; restriction fragment length polymorphisms of NAG-ST genes among V . cholerae O serogroups from a major shrimp production area in Thailand; Dalsgaard A et al.; A total of 148 Vibrio cholerae isolates from a major shrimp production area in Southern Thailand were examined by colony hybridisation for genes encoding heat-stable enterotoxin (NAG-ST) and cholera toxin (CT) . Only non-O1 V . cholerae strains were found to harbour NAG-ST (14 of 146) whereas no strains hybridised with the CT probe . NAG-ST-positive V . cholerae non-O1 strains were isolated from shrimp farms situated close to urban areas . Five different O serogroups were found among NAG-ST positive non-O1 strains . Southern blot and restriction endonuclease analysis of NAG-ST-positive strains revealed a high degree of genetic divergence . A total of seven classes of enterotoxin gene patterns were found with HindIII and EcoRI restriction endonucleases . Enterotoxin gene patterns correlated with O-antigen expression in 84% of isolates tested . In combination with other molecular techniques Southern blot analysis with an NAG-ST oligonucleotide probe could be useful for studying the molecular epidemiology of V . cholerae non-O1 strains. Infect Immun, 1995 Sep, 63(9), 3726 - 8 Immunogenicity in Peruvian volunteers of a booster dose of oral cholera vaccine consisting of whole cells plus recombinant B subunit; Begue RE et al.; Forty-nine subjects received two doses of oral cholera vaccine consisting of whole cells plus recombinant B subunit; this was followed by a booster dose one year later . After the primary series, a significant (greater than twofold) increase in the levels of vibriocidal, anti-cholera toxin immunoglobulin G and anti-cholera toxin immunoglobulin A antibodies occurred in 54, 88, and 81% of the subjects, respectively . Within one year, titers decreased to levels close to baseline . A booster dose then induced rises to those which occurred after the initial vaccination . The results suggest that 1-year booster doses may be necessary to maintain immunity against cholera in Latin America. J Appl Bacteriol, 1995 Sep, 79(3), 264 - 73 Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae and to biotype Vibrio cholerae O1; Shangkuan YH et al.; A multiplex polymerase chain reaction (PCR) was developed to identify cholera toxin-producing Vibrio cholerae and to biotype V . cholerae O1 . Enterotoxin-producing V . cholerae strains were identified with a primer pair that amplified a fragment of the ctxA2-B gene . Vibrio cholerae O1 strains were simultaneously differentiated into biotypes with three primers specified for the hylA gene in the same reaction . The hlyA amplicon in the multiplex PCR serves as an internal control when testing toxin-producing strains, as hlyA gene sequences exist in all tested V . cholerae strains . Enrichment of V . cholerae present on oysters for 6 h in alkaline peptone water before detection by a nested PCR with internal primers for ctxA2-B gene yielded a detection limit lower than 3 colony-forming units (cfu) per gram of food. Appl Environ Microbiol, 1995 Sep, 61(9), 3476 - 8 Nested PCR method for rapid and sensitive detection of Vibrio vulnificus in fish, sediments, and water; Arias CR et al.; A nested PCR for the detection of Vibrio vulnificus in fish farms was developed as an alternative to cultural methods by using universal primers flanking the V . vulnificus-specific sequences directed against 23S rRNA genes . This specific assay detected 10 fg of DNA or 12 to 120 cells in artificially inoculated samples without enrichment and within 24 h. Appl Environ Microbiol, 1995 Sep, 61(9), 3468 - 70 Reexamination of tetrodotoxin production by bacteria; Matsumura K; Vibrio alginolyticus has been reported as a good producer of tetrodotoxin (TTX), but the toxin extracted from this bacterium did not react to the monoclonal antibody against TTX . Surprisingly, chromatographic analyses detected high TTX peaks for polypeptone and yeast extracts used as medium materials, which were, as expected, all negative by the mouse bioassay . These results may require us to revise the bacterial production of TTX. Appl Environ Microbiol, 1995 Sep, 61(9), 3436 - 42 Application of antisera raised against sulfate-reducing bacteria for indirect immunofluorescent detection of immunoreactive bacteria in sediment from the German Baltic Sea; Lillebaek R; Polyclonal rabbit antisera raised against sulfate-reducing bacteria (SRB) could detect several distinct populations of bacteria in sediment from the German Baltic Sea . The depth distribution of immunoreactive bacteria was determined by an indirect immunofluorescence filter method . Anti-Desulfovibrio desulfuricans DSM 1926 serum showed maximum bacterial numbers at a depth of 18 cm, with a concentration of 60 x 10(6) cells cm-3 . With anti-Desulfovibrio baculatus DSM 2555 serum, counts were highest at the same depth, approaching 0.7 x 10(6) cells cm-3 . Other significantly smaller populations were observed . Anti-SRBStrain 1 (lactate,vibrio) maxima were at 0 to 4 cm and at 17 to 18 cm . Anti-SRBStrain 2 (lactate,vibrio) serum showed several local maxima . Anti-SRBStrain 3 (lactate,oval) serum detected one single peak at a depth of 10 to 12 cm . Also determined were rates of sulfate reduction, total bacterial counts by acridine orange staining, and the viable counts by dilution series on anaerobic lactate medium . The total bacterial counts were highest (180 x 10(6) cells cm-3) at 3 to 4 cm and dropped to 24 x 10(6) cells cm-3 at 10 to 11 cm but showed additional local maxima reaching 140 x 10(6) cells cm-3 at a depth of 17 to 18 cm . Viable counts probable number) were above 10(5) CFU cm-3 at 0 to 3.6 cm but remained below 10(3) CFU at 7.2 to 18 cm . The sulfate reduction rate was maximal (107 nmol cm-3 day-1) at a depth of 1 to 2 cm, dropped to 10 nmol cm-3 day-1 at 12 to 13 cm, and reached 38 nmol cm-3 day-1 at 17 to 18 cm. Appl Environ Microbiol, 1995 Sep, 61(9), 3379 - 84 Oxidative metabolism in nonculturable Helicobacter pylori and Vibrio vulnificus cells studied by substrate-enhanced tetrazolium reduction and digital image processing; Gribbon LT et al.; Growing and nonculturable cells of Helicobacter pylori and Vibrio vulnificus were studied for the capacity to reduce tetrazolium salts in order to elucidate the possible physiological basis for the proposed "viable but nonculturable" (VNC) state . Initial difficulties in obtaining consistent reduction of rho-iodonitrotetrazolium violet (INT) by H . pylori led us to develop a method for studying the effect of adding exogenous substrates on these reactions . The established procedure provided a profile of substrate enhancement of oxidative activity revealed by INT reduction which was related to both the identity and physiological state of the organism studied . Representation and interpretation of these enhancement profiles were facilitated by digital image processing . Nonculturable cells of H . pylori produced by carbon and nitrogen starvation in air lost all INT-reducing capacity in 24 h when stored at 37 degrees C, while 99% of those produced at 4 degrees C retained oxidative activity for at least 250 days when tested in the presence but not in the absence of succinate, alpha-ketoglutarate, or aspartate . Activity was detected at similar levels in cells with coccoid and spiral shapes . In contrast, only 1% of nonculturable cells of V . vulnificus, produced under conditions previously reported to induce the VNC state in this organism, retained intrinsic INT-reducing capacity; no substrate-enhanced activity occurred in the remainder of the population . Thus, there was no common pattern of oxidative activity indicative of a VNC state in both test organisms . Nonculturable cells of H . pylori can retain several different oxidative enzyme activities; whether these indicate viability or the persistence of cells as "bags of enzymes" remains to be established. Eur J Biochem, 1995 Sep 1, 232(2), 391 - 6 Structure of the capsular polysaccharide of Vibrio cholerae O139 synonym Bengal containing D-galactose 4,6-cyclophosphate; Knirel YA et al.; The capsular polysaccharide (CPS) of Vibrio cholerae O139 synonym Bengal, which is thought to carry determinants of O-specificity, was isolated by phenol/water extraction followed by delipidation of the contaminating lipopolysaccharide at pH 4.2 and gel-permeation chromatography . The CPS contained D-galactose, 3,6-dideoxy-L-xylo-hexose (colitose, Col), 2-acetamido-2-deoxy-D-glucose, 2-acetamido-2,6-dideoxy-D-glucose (N-acetyl-D-quinovosamine, D-QuiNAc), D-galacturonic acid (D-GalA), and phosphate . The CPS was studied by NMR spectroscopy, methylation analysis, and selective degradations, including partial acid hydrolysis at pH 3.1 and dephosphorylation with aqueous 48% hydrofluoric acid, which both resulted in complete cleavage of Col . It was concluded that the CPS is built up of hexasaccharide repeating units containing inter alia D-galactose 4,6-cyclophosphate and having the following structure {structure: see text} These data basically confirm the structure of the V . cholerae CPS proposed on the basis of an NMR study {L . M . Preston et al . (1995) J . Bacteriol . 177, 835-838} and specify exactly the absolute configurations of the constituent monosaccharides and the position of the cyclic phosphate. Infect Immun, 1995 Sep, 63(9), 3537 - 42 Cloning and expression of rfb genes from Vibrio anguillarum serotype O2 in Escherichia coli: evidence for cross-reactive epitopes; Amor PA et al.; Vibrio ordalii and Vibrio anguillarum O2 express lipopolysaccharide (LPS) O antigens containing both specific and cross-reactive epitopes . The localization of these epitopes on the O antigen is not known . We have cloned and expressed the rfb gene cluster for O-antigen synthesis from V . anguillarum O2 (rfbVaO2) in Escherichia coli . E . coli DH5 alpha containing the recombinant plasmid pAM86 expressed O antigens which reacted with polyclonal antisera to V . ordalii and to V . anguillarum O2 LPS and with monoclonal antibody (MAb) 7B4, which is specific for V . anguillarum O2 O antigens . The recombinant strains were also protected from bactericidal killing by normal fish serum . Surprisingly, the LPS expressed from the cloned rfbVaO2 genes also reacted with MAb A16, which is specific for V . ordalii O antigens . Western immunoblot analysis revealed that MAb 7B4 reacted with recombinant LPS bearing shorter O-antigen repeat units, while MAb A16 reacted with the longer O antigens . Similar results were obtained when pAM86 was transformed into E . coli CLM4, which has a deletion spanning the sbcB-rfb region, indicating that the changes in antigenic profiles of O antigens from the recombinant strains were not due to genes within the E . coli rfb cluster . These data suggest that the epitope recognized by the MAb A16 is expressed by V . anguillarum O2 strains but it is apparently not accessible to the antibody in the native O polysaccharide . Cloning of the rfbVaO2 gene cluster resulted in expression of a novel O antigen . The modification(s) which leads to the alterations in antigenic profile of these recombinant LPS remains to be determined. Microbiology, 1995 Sep, 141 ( Pt 9), 2101 - 9 Induction of heat shock response in Vibrio cholerae; JeevanJyot et al.; General properties of the heat shock response in Vibrio cholerae were examined . Enhanced or de novo synthesis of 24 proteins was observed upon heat shock from 30 degrees C to 42 degrees C in cells labelled with {35S}methionine . A similar response could also be induced by a rise in temperature from 30 degrees C to 37 degrees C . Of these heat shock proteins, two were determined to be homologues of GroEL and DnaK, based upon their immunological cross-reactivity with antibodies raised against the Escherichia coli proteins . Three proteins, of molecular sizes 38, 44 and 48 kDa, which were undetectable in the 30 degrees C grown culture, appeared de novo after the heat shock . As in other prokaryotic systems thermal induction of many of the proteins was transient, but both DnaK and GroEL remained induced for at least 28 min after heat shock . DNA hybridization studies revealed that genes analogous not only to dnaK and groEL but also to dnaJ of E . coli exist in V . cholerae . Heat shock induced thermotolerance in V . cholerae but made the cells more sensitive to UV radiation . Unlike in E . coli, however, heat shock had no effect on the progeny virus yield in V . cholerae. Public Health, 1995 Sep, 109(5), 389 - 95 Outbreak of Vibrio cholerae 01 in Hong Kong related to contaminated fish tank water; Kam KM et al.; An outbreak of 12 cholera cases, caused by Vibrio cholerae eltor inaba, occurred in Hong Kong during a three week period in June-July 1994 . Only adults of both sexes were affected . Epidemiological investigations showed linkage in all cases with consumption of seafood, including shellfish, mantis shrimps and crabs . Microbiological findings demonstrated that contaminated seawater in fish tanks used for keeping alive these seafoods is the most likely vehicle of transmission . Aggressive control measures, promptly instituted, included prohibition of use of contaminated typhoon shelter water in fish tanks, use of seawater with E . coli counts below 610 organisms/100 ml, and the banning of unlicensed food sampans in typhoon shelters . These measures, coupled with public announcements and an active health education campaign on food safety and personal hygiene, abruptly terminated the outbreak . Places which practise the use of seawater, from probable contaminated sources, to keep alive their seafood for human consumption should be alerted to the possibility of transmission of Vibrio cholerae through this route. Nippon Rinsho, 1995 Sep, 53(9), 2296 - 300 {Detection of bacterial protein toxins by a bead-ELISA}; Kurazono H et al.; A highly sensitive bead-enzyme-linked immunosorbent assay to detect bacterial protein toxins was developed . Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as a substrate . As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG . The sensitivities of the bead-ELISA for various bacterial protein toxins were as follows: less than 40 pg/ml for cholera enterotoxin (CT), less than 20 pg/ml for VT1 and less than 6 pg/ml for VT2 of enterohemorrhagic Escherichia coli . The bead ELISA was evaluated for direct detection of CT from stool specimens of patients with acute secretory diarrhea . Of the 75 stool samples examined, 59 yielded biochemically and serologically confirmed strains of Vibrio cholerae O1 . The bead ELISA was positive for CT in stool supernatants in 50 (84.7%) of the 59 samples from which V . cholerae O1 was isolated . In addition, the bead ELISA was positive for three stool specimens which were negative by culture . These data indicate that the bead ELISA is a sensitive and simple method for direct detection of CT in nonsterile stool samples. J Mol Biol, 1995 Aug 25, 251(4), 550 - 62 The 2.4 A crystal structure of cholera toxin B subunit pentamer: choleragenoid; Zhang RG et al.; Cholera toxin, a heterohexameric AB5 enterotoxin released by Vibrio cholera, induces a profuse secretory diarrhea in susceptible hosts . Choleragenoid, the B subunit pentamer of cholera toxin, directs the enzymatic A subunit to its target by binding the GM1 gangliosides exposed on the luminal surface of intestinal epithelial cells . The crystal structure of choleragenoid has been independently solved and refined at 2.4 A resolution by combining single isomorphous replacement with non-crystallographic symmetry averaging . The structure of the B subunits, and their pentameric arrangement, closely resembles that reported for the intact holotoxin, choleragen, the heat-labile enterotoxin from Escherichia coli, and for a choleragenoid-GM1 pentasaccharide complex . In the absence of the A subunit the central cavity of the B pentamer is a highly solvated channel . The binding of choleragenoid to the A subunit or to its receptor pentasaccharide modestly affects the local stereochemistry without perceptibly altering the subunit interface. J Mol Biol, 1995 Aug 25, 251(4), 471 - 6 Immunoglobulin mutant library genetically screened for folding stability exploiting bacterial signal transduction; Kolmar H et al.; A model repertoire of variants of immunoglobulin kappa variable domain REIv with different folding stabilities was generated by oligonucleotide-directed randomization of position 29, a key conserved residue of hypervariable loop 1 . Fused to ToxR', the membrane-anchored cytoplasmic domain of the Vibrio cholerae ToxR transcription activator, different members of the library induce different levels of transcription from the ctx promoter in Escherichia coli . Differences in transcription activation correlate positively with folding stabilities of the corresponding REIv domains . Since conformationally stabilized REIv derivatives elicit a dark red colony phenotype on EMB-lactose indicator plates, this procedure constitutes a genetic screen for immunoglobulin folding stability. EMBO J, 1995 Aug 15, 14(16), 3895 - 904 Membrane insertion of the bacterial signal transduction protein ToxR and requirements of transcription activation studied by modular replacement of different protein substructures; Kolmar H et al.; The Vibrio cholerae protein ToxR is an integral membrane protein that acts as a transcription activator in response to environmental signals; it controls expression of toxin genes ctxA and ctxB, along with a variety of other genes related to pathogenicity . Here it is shown that: (i) ToxR has a modular architecture and that activation of transcription starting at the ctx promoter depends strictly on dimerization of the periplasmic ToxR domain; (ii) the transmembrane (TM) region of ToxR is sufficient as a topogenic signal but not for stable membrane anchoring of the protein; (iii) the TM region has no special function in signal transduction and (iv) a proline residue located within the TM region minimizes background transcription activation, most plausibly by reducing TM-TM interaction . Possible applications of ToxR as a technical tool for analysing protein-protein interactions between pairs of arbitrary TM domains are discussed. Biochem Biophys Res Commun, 1995 Aug 15, 213(2), 475 - 83 Vibrio mimicus arylesterase has thioesterase and chymotrypsin-like activity; Chang RC et al.; A Vibrio mimicus serine arylesterase and an Escherichia coli thioesterase/serine protease share 49.4% amino acid identity . The arylesterase has thioesterase activity for benzoyl-CoA and chymotrypsin-like activity for N-carbobenzoxy-L-phenylalanine p-nitrophenyl ester (NBPNPE) . The gene encoding the V . mimicus enzyme is designated etpA . Substituting Ser31 of the V . mimicus enzyme with a glycine or an alanine altered its activity . In comparison with wild type enzyme, the S31A enzyme showed a 5-fold increase and 57% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively, and the S31G enzyme showed a 3.6-fold increase and 43% decrease in the catalytic efficiency for benzoyl-CoA and NBPNPE, respectively . For the two mutant enzymes an 8-fold decrease and a 6- to 7-fold increase in Km were seen for benzoyl-CoA and NBPNPE, respectively . The mutagenesis results prove that residue 31 plays an important role in the substrate-specificity. FEMS Microbiol Lett, 1995 Aug 15, 131(1), 69 - 74 Characterization of phage phi O139, a Vibrio cholerae O139 temperate bacteriophage with cohesive DNA termini; Pajni S et al.; A temperate bacteriophage isolated from Vibrio cholerae O139, the new epidemic strain of cholera, was found to have a polyhedral head 65 nm in diameter and a rigid contractile tail 120 nm in length . The phage chromosome was a double-stranded DNA of 35 kb, with unique cohesive ends and had a G + C content of 58.8% . A restriction map of the phage DNA was constructed using the restriction endonucleases AvaI and BstEII . The phage, whose presence could be detected in nine out of 13 V . cholerae O139 isolates tested, was found to have identical chromosomal integration sites in all the strains . The phage attachment site (attP) was found to be located very close to one end of the genome. FEBS Lett, 1995 Aug 7, 369(2-3), 173 - 6 The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN; Pfenninger-Li XD et al.; The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures . The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g . menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1 . Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex . Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme . FAD but no FMN were also present in V . alginolyticus membranes . FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme . The FAD was copurified with the NADH dehydrogenase . The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein . Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD. J Mol Biol, 1995 Aug 4, 251(1), 50 - 8 High-speed rotation and speed stability of the sodium-driven flagellar motor in Vibrio alginolyticus; Muramoto K et al.; The Na(+)-driven flagellar motor in Vibrio alginolyticus rotates very fast . Rotation of a single flagellum on a stuck cell was measured by laser darkfield microscopy with submillisecond temporal resolution . The rotation rate increased with increasing external concentration of NaCl, and reached 1000 r.p.s . at 300 mM NaCl . The Na+ influx through the motor should determine the rotation period (tau) and affect the speed stability . Fluctuation of the rotation period was analyzed at various rotation rates (from approximately 50 r.p.s . to approximately 1000 r.p.s.), which were changed by changing the external concentration of NaCl and the addition of a protonophore or a specific inhibitor . At high rotation rates (over 400 r.p.s.), the observed rotation was stable, and the standard deviation of tau (sigma tau) ranged from 7% to 16% of the average rotation period (< tau >) . At low rotation rates (under 100 r.p.s), the rotation period tended to fluctuate, and the distributions of tau were non-Gaussian . The value of sigma tau ranged from 10 to 30% of < tau > . However, the observed minimum value of sigma tau at various rotation rates was approximately equal to the calculated standard deviation due to the rotational diffusion of the flagellar filament . These results suggest that the torque was stably generated at various Na+ influxes through the motor . We observed large fluctuations that cannot be explained by rotational diffusion . We discuss the factors that induce the large fluctuation. Mol Microbiol, 1995 Aug, 17(4), 747 - 56 Antisense RNA regulation of the fatB iron transport protein gene in Vibrio anguillarum; Waldbeser LS et al.; We have recently identified an antisense RNA (RNA alpha) that regulates the expression of the fatA iron transport gene encoding the outer membrane receptor for the iron-anguibactin complex . In this work, we demonstrate that RNA alpha also inhibits the expression of fatB, which encodes a 35 kDa iron transport protein and has domains homologous to other periplasmic transport proteins . The expression of fatA and fatB is repressed under iron-rich conditions, in which RNA alpha is induced . RNA alpha is homologous to two-thirds of the coding region of fatB . By cloning RNA alpha coding sequences immediately downstream of a tet promoter, we were able to obtain constitutive expression of the antisense RNA . The cloned region contains approximately 83% of the 650 nucleotide RNA alpha and is complementary to only 51% of the fatB mRNA but is still capable of causing a repression of the expression of the fatB gene . Our results in this work demonstrate that RNA alpha probably affects the stability of the fatB-specific mRNA. Microb Pathog, 1995 Aug, 19(2), 65 - 72 Construction and characterization of recombinant Vibrio cholerae strains carrying heterologous genes encoding non-01 antigen or cholera enterotoxin; Smirnova NI et al.; In an attempt to study the effect of heterologous genes on the virulence of Vibrio cholerae 01 and non-01, rfb genes encoding biosynthesis of non-01 antigens were introduced by homologous recombination into the chromosome of V . cholerae 01 strain 569B (serotype Inaba, biotype classical) . Recombinant strains were obtained which were not agglutinated with the diagnostic cholera 01 antiserum and were not sensitive to the cholera diagnostic bacteriophage, but produced as much cholera toxin as 569B and were highly virulent in the infant rabbit intraintestinal injection model . These data indicate that the rfb genes from the studied V . cholerae non-01 did not alter the virulence phenotype of V . cholerae 01 . In contrast, cloned ctxAB genes from V . cholerae 01 encoding cholera toxin introduced into a non-pathogenic strain lead to efficient secretion of cholera toxin but to only low virulence in the infant rabbit model. Infect Immun, 1995 Aug, 63(8), 3137 - 42 A conserved Aeromonas salmonicida porin provides protective immunity to rainbow trout; Lutwyche P et al.; A protein with an apparent M(r) of 28,000 was isolated from outer membrane preparations of Aeromonas salmonicida A440 . The protein was tested for the ability to form pores, using a planar lipid bilayer model membrane system . The protein appeared to be a monomer with a single-channel conductance in 1.0 M KCl of 1.96 nS and a cation/anion permeability ratio of 2.91 +/- 0.68 . These data show that the porin channel is comparable in size to OmpC and OmpF of Escherichia coli and is relatively nonselective, having some preference for cations over anions . The porin was purified by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a polyclonal antibody was raised . Immunoblot analysis showed that an immunologically cross-reactive protein was present in other Aeromonas strains but not in strains of Vibrio or Yersinia . The N-terminal amino acid sequence of the porin was determined and was found to show some homology to an Aeromonas hydrophila outer membrane protein . This is the second porin species of A . salmonicida to be described, and it differs from the other in subunit molecular weight, aggregation properties, peptidoglycan association, pore size, and antigenicity . Rainbow trout (Oncorhynchus mykiss) immunized intraperitoneally with the purified porin protein were significantly protected from experimental A . salmonicida challenge . This is the first report of successful vaccination against A . salmonicida with a purified outer membrane component. Infect Immun, 1995 Aug, 63(8), 2906 - 11 Capsular polysaccharide-protein conjugate vaccines of carbotype 1 Vibrio vulnificus: construction, immunogenicity, and protective efficacy in a murine model; Devi SJ et al.; Vibrio vulnificus causes septicemia and wound infections in immunocompromised humans . The capsular polysaccharide of Vibrio vulnificus (VvPS) is critical for virulence . We synthesized conjugate vaccines of carbotype 1 VvPS under conditions and in formulations suitable for human use . Purified VvPS was conjugated to tetanus toxoid (TT) or to inactivated V . vulnificus cytolysin or elastase by two different schemes . All conjugates elicited elevated anticapsular immunoglobulin G (IgG) and IgM and antiprotein IgG responses in mice compared with saline placebo . The conjugates prepared through caboxyl activation of VvPS (VvPS-TTa, VvPS-cytolysin, and VvPS-elastase) were more immunogenic than the one prepared through hydroxyl activation (VvPS-TTb) . The protective efficacy of conjugated and unconjugated formulations of VvPS and that of protein carriers were evaluated in a mouse septicemia model . Eighty percent of mice actively immunized with VvPS-TTa vaccine survived challenge with carbotype 1 V . vulnificus, while VvPS-cytolysin and VvPS-elastase conjugates conferred 44 and 40% protection, respectively . Control mice immunized with VvPS, cytolysin, or elastase alone, or saline only, showed 70 to 100% mortality . VvPS-TTa vaccine is nontoxic, immunogenic, and protective in mice. Gastroenterology, 1995 Aug, 109(2), 381 - 6 Calcium-dependent intestinal chloride secretion by Vibrio parahaemolyticus thermostable direct hemolysin in a rabbit model; Raimondi F et al.; BACKGROUND & AIMS: Vibrio parahaemolyticus is a major agent of seafood gastroenteritis that induces intestinal secretion in the rabbit through its thermostable direct hemolysin . The aim of this study was to characterize the enterotoxicity of purified hemolysin in vitro . METHODS: Rabbit ileum was mounted in Ussing chambers, and changes in potential difference and short-circuit current were monitored after addition of hemolysin . Intracellular calcium concentrations in the nontumoral rat crypt-derived cell line IEC-6 were measured using microspectrofluorometry . RESULTS: In Ussing chamber experiments, mucosal toxin addition up to 50 hemolytic units per milliliter induced a proportional increase of the electrical parameters in normal but not Cl(-)-free Ringer's solution . The response to the toxin was not additive to that of calcium ionophore A23187 and was eliminated by preloading the tissue with 1-2-bis(o-aminophenoxy)ethane N,N,N',N'-tetraacetic acid (BAPTA), a calcium buffer . In IEC-6 cells, a 10-fold increase in intracellular calcium level was found after addition of hemolysin . Such an increase was totally quenched by BAPTA . Finally, preincubation with trisialoganglioside GT1b, but not monosialoganglioside GM1, eliminated toxin-induced increases in potential difference and short-circuit current . CONCLUSIONS: These data support the hypothesis that the thermostable direct hemolysin induces intestinal chloride secretion using GT1b as a putative receptor and Ca2+ as a second messenger. J Appl Bacteriol, 1995 Aug, 79(2), 135 - 40 The interaction of trimethoprim and quinolones against gram-negative fish pathogens; Inglis V et al.; The effect of combination of trimethoprim with other non-sulphonamide antibacterial agents, in particular oxolinic acid and nalidixic acid, was evaluated against Gram-negative fish pathogens . The species included Aeromonas salmonicida, Yersinia ruckeri, some Vibrio spp . and Escherichia coli as a reference . The extent of synergy found by other workers with these substances against human Gram-negative bacteria was not apparent here . Some positive interaction between trimethoprim and oxolinic acid was found with Aer . salmonicida, Y . ruckeri and E . coli and between trimethoprim and nalidixic acid with Y . ruckeri in double disc diffusion tests but was not supported by fractional inhibitory concentration indices . The combinations were not effective in preventing emergence of resistance in passage on a drug gradient . Trimethoprim-resistant isolates of Aer . salmonicida were inhibited by low levels of oxolinic acid but the converse did not apply. Chem Phys Lipids, 1995 Aug 1, 77(1), 41 - 9 Aggregation properties of semisynthetic GD1a ganglioside (IV3Neu5AcII3Neu5AcGgOse4Cer) containing an acetyl group as acyl moiety; Brocca P et al.; GD1a ganglioside containing an acetyl group as acyl moiety, GD1a(acetyl), was synthesized from natural GD1a . The aggregative properties in aqueous solution of GD1a(acetyl) have been studied by static and dynamic laser light-scattering measurements . GD1a(acetyl) spontaneously aggregates as small micelles showing a hydrodynamic radius and molecular mass of 33 A and 96 kDa, respectively . Vibrio cholerae sialidase showed a very high activity on the micelles of GD1a(acetyl), compared to GD1a . This has been explained as a consequence of the high surface curvature of the the small micelles . High resolution proton NMR spectra were recorded from micelles of GD1a(acetyl) in deuterated water . The low overall correlation time of the GD1a(acetyl) micelles was calculated to be about 2 x 10(-8)s, a value one order of magnitude lower than that determined for natural GD1a. J Clin Microbiol, 1995 Aug, 33(8), 2186 - 7 Evaluation of DNA probes for specific detection of Vibrio cholerae O139 Bengal; Nair GB et al.; Two DNA probes, 2R1 and 2R3, prepared from a region in the chromosome specific for the lipopolysaccharide O side chains of Vibrio cholerae O139 (M.K . Waldor and J.J . Mekalanos, Lancet 343:1366, 1994) were examined for their specificity and sensitivity . Both probes did not hybridize with any strain of V . cholerae belonging to serogroups other than O139 and to any of the other species examined belonging to the family Vibrionaceae . Among the 126 strains of V . cholerae O139 examined, probe 2R1 hybridized with 125 strains while probe 2R3 hybridized with all 126 strains . Both probes were found to be highly specific and sensitive and can be used for the specific identification of V . cholerae O139. Microbiology, 1995 Aug, 141 ( Pt 8), 1977 - 83 Cholera toxin (CTX) genetic element in Vibrio cholerae O139; Bhadra RK et al.; PFGE analysis of the NotI- and SfiI-digested genome of Vibrio cholerae O139 strains isolated from different epidemic regions of India showed that all the strains are of clonal origin and the genome size is about 2.2 Mb . An analysis of the electrophoretic profiles of the genome of O139 strains, the RFLP of the cholera toxin (ctx) gene and Southern blot hybridization of NotI-digested genomes of classical, El Tor and O139 with a NotI-linking clone of classical strain 569B, suggest that these strains closely resemble V . cholerae O1 biotype El Tor, but are widely different from the classical O1 vibrios . Using restriction enzymes which cleave a single site in either the core region or in the direct repeat sequence (RS) of the CTX genetic element, it has been shown that the genome of most of the O139 strains has two copies of the ctx gene in tandem connected by two RSs . The chromosomal location of the CTX genetic element in the O139 strain is the same as that reported for El Tor vibrios . The organization of the virulence gene cassettes in different O139 strains shows genetic heterogeneity in the population . Whilst most of the epidemic strains have two copies of the CTX genetic element, in some strains the number of elements has been amplified and in at least one strain a single copy of the element has been deleted. J Biol Chem, 1995 Jul 21, 270(29), 17627 - 32 Purification and properties of periplasmic 3':5'-cyclic nucleotide phosphodiesterase . A novel zinc-containing enzyme from the marine symbiotic bacterium Vibrio fischeri; Callahan SM et al.; The 3':5'-cyclic nucleotide phosphodiesterase (CNP) of Vibrio fischeri, due to its unusual location in the periplasm, allows this symbiotic bacterium to utilize extracellular 3':5'-cyclic nucleotides (e.g . cAMP) as sole sources of carbon and energy, nitrogen, and phosphorus for growth . The enzyme was purified to apparent homogeneity by a four-step procedure: chloroform shock, ammonium sulfate precipitation, and chromotography on DEAE-Sephacel and Cibacron Blue 3GA-agarose . The active enzyme consists of a single polypeptide with a mass of 34 kDa . At 25 degrees C, it has a pH optimum of 8.25, a Km for cAMP of 73 microns, and a Vmax of 3700 mumol of cAMP hydrolyzed/min/mg protein (turnover number of 1.24 x 10(5)/min) . The specific activity of the V . fischeri enzyme is approximately 20-fold greater than that of any previously characterized CNP when comparisons of activity are made at the same assay temperature . Activity increases with temperature up to 60 degrees C . The CNP contains 2 atoms of zinc/monomer, and zinc, copper, magnesium, and calcium can restore activity of the apoenzyme to varying degrees . The exceptional specific activity of the enzyme and its unusual location in the periplasm support proposals that the enzyme enables the bacterium to scavenge 3':5'-cyclic nucleotides in seawater and that the enzyme plays a role in cAMP-mediated host-symbiont interactions. J Biol Chem, 1995 Jul 14, 270(28), 16813 - 9 Catalytically active forms of the individual subunits of Vibrio harveyi luciferase and their kinetic and binding properties; Choi H et al.; Contradictory findings have recently been reported regarding the (in)abilities of individual subunits of the Vibrio harveyi alpha beta dimeric luciferase to catalyze bioluminescence . We have produced individual alpha and beta subunits separately in Escherichia coli JM109 cells by recombinant DNA techniques . Both subunits were purified to more than 90% homogeneity and found to be catalytically active, with their general catalytic properties and the specific activities similar to those reported earlier (Sinclair, J . F., Waddle, J . J., Waddill, E . F., and Baldwin, T . O . (1993) Biochemistry 32, 5036-5044) . Individual subunits were significantly distinct from the native luciferase with respect to inactivations by trypsin and N-ethylmaleimide, and the stability of the flavin 4a-hydroperoxide intermediate . The active species in isolated alpha and beta samples were each the predominant protein species, corresponding to a 42,000 M(r) alpha monomer and a 67,000 M(r) beta dimer, respectively . These findings clearly indicate that the activities of the individual subunits are not due to trace contaminations of the respective counter subunits . The much reduced specific activities of the individual subunits are, in part, a consequence of diminished abilities to oxidize the aldehyde substrate . Kinetic and equilibrium measurements indicate that alpha and beta 2 each contained a reduced flavin site, an aldehyde substrate site, and an aldehyde inhibitor site . The on and off rates of the decanal inhibitor binding were substantially slower than the bindings of decanal and reduced riboflavin 5'-phosphate substrates . These findings are consistent with a scheme that the aldehyde inhibitor blocks the binding of the reduced flavin substrate. Wkly Epidemiol Rec, 1995 Jul 14, 70(28), 201 - 8 Cholera in 1994 . Part I; Regulation of angR et al.; Department of Molecular Microbiology and Immunology, Oregon Health Sciences University, Portland 97201, USAAngR and the product(s) encoded in the trans-acting factor (TAF) region are necessary for the full expression of the pJM1 plasmid-mediated anguibactin iron-uptake system in Vibrio anguillarum (Va) . In this report, we analyzed the factors that affect the expression of the angR gene . Northern blot analysis showed that angR encodes a 3.1-kb transcript which is expressed only under iron-limiting conditions . Measurement of steady-state RNA levels show that, under iron-limiting conditions, angR is positively regulated at the transcriptional level by product(s) of the Va TAF region . However, this enhancement of angR expression by TAF does not occur at high levels of the AngR protein, as assessed by using an angR::lacZ fusion in the presence of a construct containing angR under the control of ptac . We also report that repression of angR by iron could possibly be mediated by an endogenous Va antisense RNA beta, which contains a stem-loop structure complementary to the stem-loop structure located at the 5' end of angR. Int J Syst Bacteriol, 1995 Jul, 45(3), 421 - 8 Classification of fish-pathogenic vibrios based on comparative 16S rRNA analysis; Wiik R et al.; No systematic classification of fish-pathogenic vibrios has been accomplished previously despite the use of serological, physiological, and genetical classification systems . In this study, a comparative 16S rRNA analysis of 34 strains (representing seven species) of fish-pathogenic vibrios was performed . The 16S rRNA sequences were obtained by using reverse transcriptase . Nearly complete sequences were obtained for nine strains . On the basis of the results of this analysis, the remaining strains were investigated by analyzing selected stretches containing a total of 560 nucleotides . With the exception of a few strains, including ATCC 43313 (serovar O9), our comparative 16S rRNA analysis confirmed that strains preliminarily identified as Vibrio anguillarum were phylogenetically closely related . Strains of V . anguillarum could be divided into groups, with the main group containing serotype O1 and O2 strains isolated from Atlantic salmon, rainbow trout, turbot, cod, and saithe . The other distinctive group was represented by type strain NCMB 6 . This strain was nearly indistinguishable from the type strains of Vibrio ordalii and Vibrio damsela on the basis of the 16S rRNA stretches compared . The results of a comparative 16S rRNA analysis justified the status of Vibrio salmonicida as a distinct species . Originally, this species was characterized biochemically as a very homogeneous species . However, two strains, which were isolated from diseased halibut and from the intestines of healthy cod, could not be distinguished from V . salmonicida strains phylogenetically, although they differed from the original species description in several phenotypic traits . Our results indicate that V . salmonicida and Vibrio fischeri form a cluster that is clearly separated from the cluster that includes V . anguillarum. Microb Pathog, 1995 Jul, 19(1), 39 - 48 Pathogenesis studies on Vibrio alginolyticus in the grouper, Epinephelus malabaricus, Bloch et Schneider; Lee KK; A pathogenic vibrio, strain S3y was isolated from an outbreak of vibriosis in grouper (Epinephelus malabaricus) fry in Kaohsiung, Southern Taiwan in November 1991 . It was identified as Vibrio alginolyticus on the basis of a number of biochemical tests . The bacterium was therefore identified as Vibrio, a Gram-negative, straight rod, motile, oxidase and catalase positive, swarming on TSA plate, fermentative and produced yellow colonies on TCBS agar . It did not utilise sodium citrate, but it was gelatinase positive and sensitive to the vibriostatic agent, 0/129 . It could grow on TSA with up to 11% NaCl . Its G+C ratio was 47% . The minimum lethal dose of strain S3y was 500 cfu/g fish body weight in the grouper (Epinephelus malabaricus) . The same bacterial species could be reisolated from the kidney of moribund fish following bacterial challenge . The extracellular products (ECP) of S3y were lethal to fish with a minimum lethal dose of 0.52 microgram/g fish body weight . A 34 kDa protease was purified from the ECP of S3y and demonstrated to be a toxin by intraperitoneal injection in the grouper . The minimum lethal dose of the purified protease was 0.17 microgram/g fish body weight . Intraperitoneal injection of the bacteria, ECP or the 34 kDa protease all caused slight exophthalmia with corneal opaqueness in moribund and dead fish . The results suggested that the 34 kDa protease might play an important role in the pathology of vibriosis caused by this bacterium. Microb Pathog, 1995 Jul, 19(1), 11 - 8 Thymine auxotrophy as an attenuating marker in Vibrio cholerae; Attridge SR; Vibrio cholerae CVD102 is a thymine-dependent auxotroph of CVD101, a cholera toxin A-B+ candidate live oral cholera vaccine . Previous clinical experience with these strains suggested that, by restricting intestinal growth, thymine auxotrophy is attenuating for V . cholerae . Studies in the infant mouse cholera model cast doubt upon this conclusion however . Stable thyA mutants selected from each of three pathogenic strains showed unimpaired gut colonization in mixed-infection competition experiments . Similar results were obtained using thyA mutants selected from two atoxigenic strains, including CVD101 . Further studies with CVD102 showed that the reduced colonization potential of this strain could not be compensated by the provision of a functional thyA+ gene in trans . CVD102 shows reduced synthesis of toxin-coregulated pili (TCP) during in vitro growth, suggesting the presence of a second, undefined mutation in this strain . Given the critical role of TCP in intestinal colonization, it seems probable that this previously unrecognized mutation is responsible for the poor in vivo performance of CVD102. J Infect, 1995 Jul, 31(1), 45 - 7 Epidemiology of Vibrio cholerae O139 with special reference to intrafamilial transmission in Calcutta; Sengupta PG et al.; A total of 27 families of hospitalised patients (index case families) suffering from acute watery diarrhoea caused by Vibrio cholerae O139, and 14 neighbourhood families were bacteriologically screened for 4 consecutive days to determine the extent of V . cholerae O139 infection amongst healthy contacts and other suspected vehicles of transmission at the intrafamilial level . V . cholerae O139 was isolated from faeces of 14.6% of healthy contacts in index case families as compared to none in neighbourhood families (P = 0.002) . The organism could be recovered from 3.7% of handwashings of contacts of index cases and also from stored drinking water (8.0%), open well water (28.6%), flies (3.8%) and pond water (25.0%) used by the index case families and none from neighbourhood families . The large number of asymptomatic infected persons indicate an epidemiological similarity to that of eltor cholera . The organisms may be carried on hands and may act as a potential source of infection to other inmates through contamination of stored drinking water, open wells etc . The results will be useful in formulating strategies for intervention of transmission of V . cholerae O139 at the community level. Ecotoxicol Environ Saf, 1995 Jul, 31(2), 149 - 52 Toxicity of diphenylamine and some of its nitrated and aminated derivatives to the luminescent bacterium Vibrio fischeri; Drzyzga O et al.; Aqueous samples containing various nitrated and aminated diphenylamine derivatives were subjected to the luminescent bacterium Vibrio fischeri NRRL-B-11177 to determine their ecotoxicological potential . As the most important toxicological parameter, EC50, the concentration needed to reduce bacterial luminescence by 50%, was calculated . All compounds tested must be classified to the category "very toxic to aquatic organisms" using the widely accepted classification scheme of D . Strupp, H.P . Luhr, H . T . Grunder, J . Gerdesmann, and J . Ahlers (1990, UWSF--Z . Umweltchem . Okotox . 2, 151-156) . Only 2, 4-diaminodiphenylamine can be classified as "less toxic to aquatic organisms" . EC50 values after 30, 60, and 90 min of incubation of the test compounds are presented . For many of the compounds tested in this study there are no toxicological data in the literature. J Infect Dis, 1995 Jul, 172(1), 173 - 9 The emerging diversity of the electrophoretic types of Vibrio cholerae in the Western Hemisphere; Evins GM et al.; Since the Latin American cholera epidemic began in 1991, 447 isolates of Vibrio cholerae O1 from the Western Hemisphere have been assayed by multilocus enzyme electrophoresis (MEE) to determine allelic variation among 16 enzyme-encoding genes . Two electrophoretic types (ETs) were identified among toxigenic isolates from Latin America: 323 were ET 4, the ET associated with the Latin American epidemic, and 29 were ET 3 . Twenty-three of these ET 3 isolates had a distinctive antimicrobial resistance pattern also seen in isolates imported into the United States from Latin America and Southeast Asia . These resistant isolates had an identical ribotype and nearly identical pulsed-field gel electrophoresis (PFGE) patterns . Most nontoxigenic isolates analyzed were not precursors or descendants of toxigenic epidemic strains . MEE provided a population genetic frame-work for the interpretation of PFGE and ribotype data from the isolates in this study . All three methods identified 2 distinct strains of toxigenic V . cholerae O1 currently epidemic in Latin America. Infect Immun, 1995 Jul, 63(7), 2689 - 96 Heterologous antigen expression in Vibrio cholerae vector strains; Butterton JR et al.; Live attenuated vector strains of Vibrio cholerae were derived from Peru-2, a Peruvian El Tor Inaba strain deleted for the cholera toxin genetic element and attRS1 sequences, which was developed as a live, oral vaccine strain . A promoterless gene encoding the Shiga-like toxin I B subunit (slt-IB) was inserted in the V . cholerae virulence gene irgA by in vivo marker exchange, such that slt-IB was under transcriptional control of the iron-regulated irgA promoter . slt-IB was also placed under transcriptional control of the V . cholerae heat shock promoter, htpGp, and introduced into either the irgA or lacZ locus, or both loci, on the chromosome of Peru-2, generating JRB10, JRB11, or JRB12, respectively . A new technique was used to perform allelic exchange with lacZ . This method uses plasmid p6891MCS, a pBR327 derivative containing cloned V . cholerae lacZ, to insert markers of interest into the V . cholerae chromosome . Recombinants can be detected by simple color screening and antibiotic selection . In vitro measurements of Slt-IB produced by the vector strains suggested that expression of Slt-IB from the irgA and htpG promoters was synergistic and that two copies of the gene for Slt-IB increased expression over a single copy . The V . cholerae vectors colonized the gastrointestinal mucosa of rabbits after oral immunization, as demonstrated by very high serum antibody responses to V . cholerae antigens . Comparison of the serologic responses to the B subunit of cholera toxin (CtxB) following orogastric inoculation either with the wild-type C6709 or with Peru-10, a strain containing ctxB regulated by htpGp, suggested that both the cholera toxin and heat shock promoters were active in vivo, provoking comparable immunologic responses . Orogastric inoculation of rabbits with vector strains evoked serum immunoglobulin G (IgG) responses to Slt-IB in two of the four strains tested; all four strains produced biliary IgA responses . No correlation was observed between the type of promoter expressing slt-IB and the level of serum IgG or biliary IgA response, but the vector strain containing two copies of the gene for slt-IB evoked greater serum IgG responses than strains containing a single copy, consistent with the increased expression of Slt-IB from this strain observed in vitro . A comparison of the serum and biliary antibody responses to Slt-IB expressed from htpGp versus CtxB expressed from the same promoter suggested that CtxB is a more effective orally delivered immunogen. Klin Lab Diagn, 1995 Jul-Aug, (4), 50 - 3 {Choice of commercial elective nutrient media for the isolation of pathogenic vibrios of different species}; Danilkina EB et al.; Commercial nutrient media and their modifications are assessed, including elective differentiation medium for V . cholerae developed at the Rostov Research Anti-Plague Institute for the isolation of pathogenic vibrios . V . cholerae cholerae P-1 (145), V . cholerae el tor M-878, V . cholerae non 01 P-9741, E . coli 18, P . vulgaris 19, and 48 strains of vibrios belonging to different species were used in the study . All the strains used were characterized as to their nutritive requirements . Alkaline agar manufactured by the I . I . Mechnikov Research Institute of Vaccines and Sera was found to be the optimal medium for the isolation of pathogenic vibrios, for it provided the growth of all the examined strains from the inoculation dose of 100 bacterial cells . Addition of potassium tellurite to this medium in a dose of 30 to 50 mg/ml improved its selective properties . Elective differential medium for V . cholerae, though somewhat inferior to alkaline agar in sensitivity, provided the growth of colonies of the overwhelming majority of the strains and inhibited the growth of associated bacteria . Hence, these two media are recommended as elective media for the diagnosis and selection of pathogenic vibrio . Endo and Ploskirev's media with alkaline agar in 1 to 1 ratio may be recommended as additional ones for the isolation of vibrios from patients and from environmental objects. Appl Environ Microbiol, 1995 Jul, 61(7), 2624 - 30 Entry into, and resuscitation from, the viable but nonculturable state by Vibrio vulnificus in an estuarine environment; Oliver JD et al.; Using plate counts, total cell counts, and direct viable counts, we examined the fate of cells of Vibrio vulnificus placed into natural estuarine waters during both winter and summer months . Cells inoculated into membrane diffusion chambers and placed into estuarine waters entered into a viable but nonculturable (VBNC) state in January and February, when the water temperatures were low (average, < 15 degrees C) . In contrast, when cells in the VBNC state were placed into the same waters in the warmer months of August through November (average water temperature of ca . 21 degrees C), the cells appeared to undergo a rapid (typically, within 24 h) resuscitation to the fully culturable state . These results were independent of whether the cells were in the logarithmic or stationary phase and whether they were encapsulated or not . This study indicates that the inability to isolate V . vulnificus from cold estuarine sites may be accounted for by entrance of the cells into a VBNC state and that recovery from this state in natural environments may result from a temperature upshift. Appl Environ Microbiol, 1995 Jul, 61(7), 2620 - 3 In vivo resuscitation, and virulence towards mice, of viable but nonculturable cells of Vibrio vulnificus; Oliver JD et al.; Vibrio vulnificus is an estuarine bacterium responsible for 95% of all seafood-related deaths in the United States . The bacterium occurs naturally in molluscan shellfish, and ingestion of raw oysters is typically the source of human infection . V . vulnificus is also known to enter a viable but nonculturable (VBNC) state, wherein the cells are no longer culturable on routine plating media but can be shown to remain viable . Whether or not this human pathogen remains virulent when entering the VBNC state has not been definitively demonstrated . In this study, the VBNC state was induced through a temperature downshift to 5 degrees C, with cells becoming nonculturable (< 0.1 CFU/ml) within 7 days . As they became nonculturable, virulence was determined by employing an iron overload mouse model . At the point of nonculturability (7 days), injections of the diluted microcosm population resulted in death when < 0.04 CFU was inoculated, although > 10(5) cells in the VBNC state were present in the inoculum . Culturable cells of V . vulnificus, with identification confirmed through PCR, were recovered from the blood and peritoneal cavities of mice which had died from injections of cells present in the VBNC state for at least 3 days . Thus, our data suggest that cells of V . vulnificus remain virulent, at least for some time, when present in the VBNC state and are capable of causing fatal infections following in vivo resuscitation . Our studies also indicate, however, that virulence decreases significantly as cells enter the VBNC state, which may account, at least to some extent, for the decrease in infections caused by this bacterium during winter months. Appl Environ Microbiol, 1995 Jul, 61(7), 2493 - 8 Analysis of antigens present in the extracellular products and cell surface of Vibrio anguillarum serotypes O1, O2, and O3; Santos Y et al.; Antigens present in the extracellular products (ECP) and cell walls of strains of Vibrio anguillarum of serotypes O1, O2, and O3 isolated from different fish species in distinct geographic areas were characterized . The usefulness of slide agglutination, dot blot assay, and quantitative agglutination for subtyping V . anguillarum serovars was also evaluated . The three serological assays used to establish the serogroups within V . anguillarum isolates demonstrated that serotype O1 constitutes a homogeneous group, whereas within serotypes O2 and O3, two different patterns of serological reactions were detected . Among the three serological methods used, only dot blot and quantitative agglutination assays differentiated subgroups within serotypes O2 and O3 with unabsorbed sera . Electrophoretic analysis and immunoblot assays of cell envelope and ECP components showed that strains belonging to serotype O1 possessed immunologically related lipopolysaccharide (LPS) and proteins, while V . anguillarum isolates grouped in serotypes O2 and O3 exhibited internal heterogeneity in their LPS and protein banding patterns . On the other hand, although the LPS present in the ECP and those obtained from cell envelopes of V . anguillarum strains showed apparently different gel patterns, a strong relationship between both types of LPS was seen by immunoblot assay . From these results, it can be concluded that V . anguillarum strains representative of each of the antigenic groups (O1, O2 alpha, O2 beta, O3A, and O3B) and their ECPs should be included in the formulation of vaccines against vibriosis in areas where the three serotypes coexist. J Bacteriol, 1995 Jul, 177(14), 4131 - 3 Induction of cold-responsive proteins in Vibrio vulnificus; McGovern VP et al.; We have studied the response of Vibrio vulnificus to temperature shifts (23 to 13 degrees C) within the organism's permissive growth range . Cold shift induced a diminution in protein synthesis . Following a short lag, cells began growth at a new rate . Forty proteins were induced by this downshift. Lipids, 1995 Jul, 30(7), 677 - 9 Comparison of the fatty acids of the tunicate Botryllus schlosseri from the Black Sea with two associated bacterial strains; Carballeira NM et al.; The fatty acid composition of the tunicate Botryllus schlosseri and of two bacterial strains found within the tunicate, namely Vibrio parahaemolyticus and of an associated but previously unreported gram positive cocci were studied . The polyunsaturated fatty acids 6,9,12-octadecatrienoic acid, 5,8,11,14,17-eicosapentaenoic acid, and 4,7,10,13,16,19-docosahexaenoic acid were particularly abundant in B . schlosseri and were not detected in the two bacterial strains found in the tunicate . The iso/anteiso pair, 13-methyltetradecanoic acid and 12-methyltetradecanoic acid, were the principal fatty acids in the gram positive cocci, and the 9- and 11-hexadecenoic acids were particularly abundant in V . parahaemolyticus . The diunsaturated fatty acid 9,12-octadecadienoic acid was also shown to be present in V . parahaemolyticus . The fatty acid composition of a third bacterial strain, characterized as either a Pseudomonas or an Alteromonas species, and shown to be present only in the sea water from the Black Sea and not in B . schlosseri, is also reported . This is the first investigation on fatty acids from Black Sea bacteria. Kansenshogaku Zasshi, 1995 Jul, 69(7), 826 - 34 Rapid detection of Vibrio cholerae with a new selective enrichment medium and polymerase chain reaction; Kida N et al.; The inhibitory effect of metallic EDTA compounds on growth of Vibrio cholerae and Escherichia coli was studied . Only Fe-EDTA among the compounds tested showed pH-dependent growth inhibition on E . coli at pH 9.0, but no inhibition of V . cholerae at the same pH . By addition of Fe-EDTA as a selective inhibitor, a novel enrichment broth (tentatively designated as VCF broth) for the selective isolation and cultivation of V . cholerae from other Gram-negative bacilli has been developed, and the selective enrichment capacity of VCF broth for V . cholerae and selective inhibiting activity against E . coli were significantly higher than those of alkaline peptone water . A simple procedure for rapid detection of V . cholerae by selective enrichment for 6 hr with VCF broth and then amplification of the cholera toxin target DNA fragment by the polymerase chain reaction was presented . VCF broth may be a useful tool for the selective enrichment of V . cholerae in bacterial examinations. New Microbiol, 1995 Jul, 18(3), 289 - 97 First data on the distribution and ecology of Vibrio spp . of the Straits of Magellan (South America); Monticelli LS et al.; During the austral summer of 1991 a study was carried out on the presence and distribution of the genus Vibrio in the Straits of Magellan . Vibrios strains were isolated using membrane filters and Marine Agar 2216 in anaerobiosis . Variations of the populations of total heterotrophic bacteria and vibrios were observed both on the surface and along the column of water . All vibrios are psychrotrophic and were grouped in 4 cluster among which cluster 1, identified as presumed V . anguillarum, seems the most important including 73% of strains . A certain habitat segregation of clusters was noted . Cluster 4 was found only in a deep and permanently colder water mass . The relations between 20 environmental parameters and the bacterial population were also studied . Significant positive correlations were observed between the vibrios population and various fractions of suspended particulate matter. J Foot Ankle Surg, 1995 Jul-Aug, 34(4), 354 - 7 Lower extremity manifestations of Vibrio vulnificus infection; Laughlin TJ et al.; Vibrio vulnificus is a potentially lethal marine bacterium that has not been previously described in podiatric literature . A review of the microorganism's characteristics, susceptible patient population, and lower extremity manifestations of infection is presented . V . vulnificus is found as part of the normal flora of the Gulf of Mexico, Atlantic, and Pacific coastal waters and is often isolated from the filter feeding shellfish of these regions . Its pathogenicity is generally reserved for the immunocompromised host, and is specifically related to disease states which exhibit high serum iron levels . V . vulnificus infections present in two distinct clinical syndromes: primary sepsis secondary to raw oyster ingestion, or localized infection from wound exposure to V . vulnificus-inhabited salt water . Both syndromes demonstrate characteristic skin lesions of the trunk and extremities that present as hemorrhagic bullae and progress to necrotic ulcerations . Although V . vulnificus infection is rare, its extreme virulence in patients suffering from a chronic disease process and its manifestation of characteristic lower-extremity lesions require the podiatric physician to be able to recognize and treat such a condition. Mol Gen Mikrobiol Virusol, 1995 Jul-Sep, (3), 30 - 4 {Phenotypic analysis of two morphologically different types of Vibrio cholerae 0139 colonies}; Smirnova NI et al.; A clinical isolate Vibrio cholerae P16064 serogroup 0139 was shown to produce two different kinds of colonies: opaque encapsulated and translucent devoid of capsule . Low virulence of translucent colonies seems to be due to not only loss of capsule which determines serum resistance, but also to decreased expression of genes controlling the motility, and production of protease and mannose-sensitive adhesion pili . Analysis of the lysogenic properties of the two types of colonies permitted us to propose that simultaneous spontaneous alteration of capsule production and the above-mentioned virulence factors in translucent colonies may be caused by a temperate phage in turbid clones of strain P16064. Mol Microbiol, 1995 Jul, 17(1), 197 - 204 Characterization and regulation of the expression of FatB, an iron transport protein encoded by the pJM1 virulence plasmid; Actis LA et al.; The pJM1-encoded genes fatDCBA are essential for iron acquisition via the siderophore anguibactin . Sequence analysis indicated that the open reading frame corresponding to the fatB gene possesses domains that are characteristic of periplasmic proteins that bind the ferric siderophore . In this work, a monospecific antiserum against an oligopeptide containing the last 27 amino acids of the carboxy-terminal region from this open reading frame was used to demonstrate that fatB encodes a 35 kDa protein that is essential for iron transport . By using this antibody we were able to demonstrate that expression of the fatB gene is negatively regulated by the Fur protein at high iron concentrations . Conversely, its expression was positively regulated by the combined action of the AngR protein and products of the TAF region . FatB, the product of the fatB gene, is isolated with the membrane fraction . In accordance with these findings is the fact that the first 23 amino acid residues of this protein have the properties of a lipoprotein signal sequence . The lipoprotein nature of FatB is supported by the fact that treatment of Vibrio anguillarum cells with globomycin, an inhibitor of the lipoprotein signal peptidase, results in the accumulation of a 38 kDa proFatB precursor protein. Nippon Saikingaku Zasshi, 1995 Jul, 50(3), 863 - 70 {Causative agent of the so-called "light disease of shrimps" is luminescent Vibrio cholerae non-O1}; Shimada T et al.; A number of luminous fresh-water shrimps were found in a fish preserve in Lake Biwa, Shiga Prefecture, in the middle of July, 1994, and most of them died within several hours after collection (the so-called "light disease of shrimp") . Four luminous organisms were isolated from a dead shrimp . Although the phenotypic properties of these strains were similar to those of V . cholerae or V . mimicus, a representative strain, 838-94, was shown to have a high level (79%) of DNA homology with V . cholerae type strain, ATCC 14035 and a low level (45%) of relatedness to V . mimicus type strain, ATCC 33653 . Therefore, these isolates were identified as V . cholerae . The four strains fell into serogroup O28 of V . cholerae . On the other hand, none of the isolates had CT nor NAG-ST genes . The results obtained herein clearly demonstrate that these organisms isolated from luminous shrimps are luminescent V . cholerae serogroup O28. Biochim Biophys Acta, 1995 Jun 30, 1230(3), 170 - 6 Three aspartic residues in membrane-spanning regions of Na+/H+ antiporter from Vibrio alginolyticus play a role in the activity of the carrier; Nakamura T et al.; The Na+/H+ antiporter gene from Vibrio alginolyticus restores the growth of an nhaA-defective strain of Escherichia coli, NM81, in a high NaCl medium (Nakamura, T., Komano, Y., Itaya, E., Tsukamoto, K., Tsuchiya, T . and Unemoto, T . (1994) Biochim . Biophys . Acta 1190, 465-468) . This gene, named nhaAv, allowed the nhaA-defective E . coli strains, NM81(delta nhaA) and RS1 (delta nhaA, chaA-), to extrude Na+ at alkaline pH . The extrusion of Na+ occurred against its chemical gradient in the presence of membrane-permeable amine . Thus, the nhaAv gene product is functional as an electrogenic Na+/H+ antiporter in E . coli cells . The NhaAv protein has only four acidic amino acid residues in the putative membrane-spanning regions, that is, Asp-57, Asp-125, Asp-155 and Asp-156, and these Asp residues are conserved in NhaA from E . coli . Asp-111, which is predicted to be in a loop region between the transmembrane segments is also conserved in NhaA . Thus, each conserved Asp residue was replaced with asparagine by a site-directed mutagenesis . E . coli NM81 cells containing a plasmid harboring the nhaAv gene mutated at Asp-125, -155, or -156 could neither grow in a high NaCl medium nor extrude Na+ at alkaline pH against its chemical gradient . These results show that Asp-125, -155, and -156, but not Asp-57 and -111, play a role in the activity of the Na+/H+ antiporter, NhaAv. J Biol Chem, 1995 Jun 9, 270(23), 13956 - 60 A mutant at position 87 of the GroEL chaperonin is affected in protein binding and ATP hydrolysis; Weiss C et al.; The highly conserved aspartic acid residue at position 87 of the Escherichia coli chaperonin GroEL was mutated to glutamic acid . When expressed in an E . coli groEL mutant strain deficient for phage morphogenesis, plasmid-encoded GroEL mutant D87E restored T4 phage morphogenesis . It did not, however, reactivate the transcription of a recombinant luciferase operon from Vibrio fischeri . In vitro, GroEL mutant D87E was found to be impaired in the ability to bind nonnative proteins and to hydrolyze ATP, resulting in less efficient refolding of urea-denatured ribulose-1,5-bisphosphate carboxylase/oxygenase . Mutant oligomer D87E GroEL14 was able to bind GroES7 as efficiently as wild-type GroEL14 . The conserved aspartic acid residue at position 87 located in the equatorial domain of GroEL (Braig, K., Otwinowski, Z., Hegde, R., Boisvert, D.C., Joachimiak, A., Horwich, A.L., and Sigler, P.B . (1994) Nature 371, 578-586) is thus inferred to have a dual effect on the binding of nonnative proteins to the GroEL14 core chaperonin and on ATP hydrolysis. Southeast Asian J Trop Med Public Health, 1995 Jun, 26(2), 342 - 6 Widespread emergence of Vibrio cholerae 0139 in India; Sachdeva V et al.; The National Institute of Communicable Diseases (NICD) has been monitoring the incidence of laboratory confirmed cases of cholera in Delhi in collaboration with Infectious Diseases Hospital (IDH) since 1965 . Cholera and cholera-like cases from all hospitals in Delhi are admitted in IDH and the rectal swabs of all such cases are processed for isolation of Vibrio cholerae at NICD laboratory . Since April 1993, there has been isolation of Vibrio cholerae serotype 0139, in increasing numbers (831 out of 2,830, 29.2%) The isolates have been characterized and enterotoxin studies carried out . As a referral laboratory NICD has also confirmed the causative role of Vibrio cholerae 0139 in diarrhea outbreaks from various parts of the country . The implications of establishment of this newer serotype of Vibrio cholerae, as a potential epidemic strain are discussed. Appl Environ Microbiol, 1995 Jun, 61(6), 2292 - 6 Evidence for the existence of distinct populations of Vibrio anguillarum serogroup O1 based on plasmid contents and ribotypes; Pedersen K et al.; A total of 103 Vibrio anguillarum serogroup O1 strains displaying 15 different plasmid profiles were characterized with respect to biochemical properties and ribotypes . The results confirmed that V . anguillarum O1 is a biochemically homogeneous group . The 103 strains could be allocated to three main clusters with high similarity coefficients . None of the biochemical properties were connected with the presence of plasmids . In total, 12 different ribotypes were demonstrated, with HindIII being used as the restriction enzyme . Forty of the strains were isolated from the same Danish fish farm, some from the kidneys of diseased fish and some from the environment, and some strains were isolated from the mucus, gills, and feces of healthy fish . Nineteen of these isolates possessed the 67-kb virulence plasmid alone or in combination with other plasmids, while 21 had no plasmids . All strains isolated from the kidneys of diseased fish on this farm had plasmids . Irrespective of their origin (kidneys, gills, or mucus), all 19 strains carrying the 67-kb virulence plasmid had the same ribotype, profile 1, while isolates without plasmids belonged to five different profiles, all different from profile 1 . These results suggest that pathogenic V . anguillarum O1 strains possessing a virulence plasmid and nonpathogenic strains without plasmids from a small geographical area and even from the same fish may constitute two essentially distinct populations . Thus, it may be suggested that an exchange of virulence plasmids among strains is unlikely to occur in vivo. J Med Microbiol, 1995 Jun, 42(6), 399 - 403 Presence of lysogenic phage in the outbreak strains of Vibrio cholerae O139; Mitra SN et al.; Four outbreak strains of Vibrio cholerae O139 from endemic areas of India and Bangladesh were found to carry lysogenic phage(s) . All of these phage(s) produced turbid plaques characteristic of lysogeny on V . cholerae MAK 757 (El Tor, Ogawa) cells as well as on their VcA-1 lysogens but were unable to infect V . cholerae 154 (classical) cells, the universal host for all classical phages . Colonies in the turbid plaques were O139 lysogens and these developed an auxotrophic requirement, mainly for purines suggesting the integration of the prophage into the host chromosome . The immunity profile of the O139 phage(s) was similar to that of phage alpha but differed in the sensitivity of the phage lysogen of V . cholerae MAK 757 to subsequent infection by phage beta. FEMS Microbiol Lett, 1995 Jun 1, 129(1), 97 - 101 Intragenic suppression of a luxR mutation: characterization of an autoinducer-independent LuxR; Poellinger KA et al.; The Vibrio fischeri luminescence genes are activated by the LuxR protein and a diffusible signal termed the autoinducer . LuxR consists of two domains, a C-terminal transcriptional activator domain, and an N-terminal autoinducer-binding domain, which serves to regulate the function of the C-terminal domain . We have isolated and characterized an intragenic suppressor of a mutation that maps to the N-terminal domain and blocks autoinducer binding . The suppressor changes an alanine residue at position-221 in the C-terminal domain to a valine . In Escherichia coli, the suppressor allows partial activation of the V . fischeri luminescence genes although E . coli containing this protein remains unable to bind autoinducer . To further analyze the influence of the second-site mutation on luxR function, we constructed a luxR gene that coded for a protein with a wild-type N-terminal domain and with the ala-221 to val substitution in the C-terminal domain . This protein activated the luminescence genes in the presence or absence of autoinducer, and it bound autoinducer at levels comparable to the wild-type LuxR protein . Apparently, the alanine to valine substitution at position-221 allows activity of the C-terminal domain in a fashion independent of whether autoinducer is bound to the N-terminal domain. Dig Dis Sci, 1995 Jun, 40(6), 1257 - 60 Fatal Vibrio parahemolyticus septicemia in a patient with cirrhosis . A case report and review of the literature; Hally RJ et al.; Vibrio parahemolyticus has been well documented to cause outbreaks of infectious diarrhea, usually related to poor food handling; only rarely has it been reported to cause fetal septicemia . In contrast, Vibrio vulnificus is a well-known cause of septicemia, especially in patients with cirrhosis . A 31-year-old woman with cirrhosis who developed fatal V . parahemolyticus sepsis after ingesting raw seafood is described . We review the clinical syndromes associated with sepsis caused by these two organisms . Leg pain and bullous skin lesions may be a clue to the diagnosis . Febrile patients with cirrhosis should be questioned regarding recent seafood ingestion, and appropriate antibiotics chosen if this history is obtained . Physicians should inform patients at risk to avoid raw seafood in an attempt to prevent this potentially lethal syndrome. J Pediatr, 1995 Jun, 126(6), 882 - 6 Clinical characteristics and risk factors for Vibrio cholerae infection in children; Fukuda JM et al.; Surveillance was conducted during February and March 1991 in the pediatric emergency department of Cayetano Heredia Hospital, Lima, Peru, to contrast the characteristics of children with epidemic cholera with those of children with noncholera-associated diarrhea . Among 626 patients 14 years of age or younger, Vibrio cholerae O1 was isolated from stool specimens of 310 patients (49%), more commonly from children older than 24 months of age (66%; p < 0.0001) than from younger children . Cholera was clinically characterized by a more sudden onset; watery diarrhea; and associated abdominal pain, muscle cramps, and vomiting, which led to more severe dehydration and hospitalization more often than in noncholera cases . Only one patient with cholera died, for a case-fatality rate of 3.2 deaths per 1000 persons . Nonpotable water and uncooked foods were identified as probable vehicles for V . cholerae . The frequency of diarrhea among relatives of patients with cholera suggested intrafamily transmission . This study of epidemic cholera describes the clinical features and the risk factors for acquisition of the infection, and points out the low case-fatality rate with prompt and appropriate treatment. Curr Microbiol, 1995 Jun, 30(6), 331 - 6 Isolation of bacteriophage infectious for Vibrio vulnificus; Pelon W et al.; Nine phage isolates infectious for Vibrio vulnificus and falling into four morphological groups were isolated from estuarine waters collected in Louisiana . Of the 60 V . vulnificus strains tested, 87% were susceptible to one or more of the isolates . With the exception of V . fluvialis, Vibrio species other than vulnificus were resistant to infection . A spectrum of enteric bacterial strains were similarly resistant . Susceptibility differences were seen between opaque (virulent) V . vulnificus strains and those with translucent (nonvirulent) colony types, with the former being more susceptible . Susceptibility patterns to infection by the nine phage isolates among the V . vulnificus test strains suggest that the latter may fall into several groups . Other aspects relating to the phage isolates are presented. J Infect Dis, 1995 Jun, 171(6), 1653 - 6 Impact of infection by Helicobacter pylori on the risk and severity of endemic cholera; Clemens J et al.; To evaluate the relationship between Helicobacter pylori infection and the subsequent risk and severity of endemic Vibrio cholerae O1 diarrhea among rural Bangladeshis, 285 children and adults with cholera (cases) and 881 contemporaneously selected community controls were studied . Cases and controls were contrasted for H . pylori infection, as manifested by serum IgG anti-H . pylori antibodies . Although the overall risk of cholera was not significantly increased among H . pylori-infected subjects, the risk of cholera of life-threatening severity was significantly elevated (relative risk {RR} = 1.61; 95% confidence interval {CI} = 1.07-2.42) . A significant increase in the risk of severe cholera was seen in subjects who lacked natural serum vibriocidal antibodies (RR = 2.88; 95% CI = 1.28-6.48) but not in those with such antibodies . Thus, H . pylori infection was associated with a significant increase in the risk of life-threatening cholera, but only among persons lacking natural vibriocidal immunity. Infect Immun, 1995 Jun, 63(6), 2356 - 60 Construction of nontoxic derivatives of cholera toxin and characterization of the immunological response against the A subunit; Fontana MR et al.; Using computer modelling, we have identified some of the residues of the A subunit of cholera toxin (CT) and heat-labile toxin that are involved in NAD binding, catalysis, and toxicity . Here we describe the site-directed mutagenesis of the CT gene and the construction of CT mutants . Nine mutations of the A subunit gene were generated . Six of them encoded proteins that were fully assembled in the AB5 structure and were nontoxic; these proteins were CT-D53 (Val-53-->Asp), CT-K63 (Ser-63-->Lys), CT-K97 (Val-97-->Lys), CT-K104 (Tyr-104-->Lys), CT-S106 (Pro-106-->Ser), and the double mutant CT-D53/K63 (Val-53-->Asp, Ser-63-->Lys) . Two of the mutations encoded proteins that were assembled into the AB5 structure but were still toxic; these proteins were CT-H54 (Arg-54-->His) and CT-N107 (His-107-->Asn) . Finally, one of the mutant proteins, CT-E114 (Ser-114-->Glu), was unable to assemble the A and the B subunits and produced only the B oligomer . The six nontoxic mutants were purified from the culture supernatants of recombinant Vibrio cholerae strains and further characterized . The CT-K63 mutant, which was the most efficient in assembly of the AB5 structure, was used to immunize rabbits and was shown to be able to induce neutralizing antibodies against both the A and B subunits . This molecule may be useful for the construction of improved vaccines against cholera. Int J Food Microbiol, 1995 Jun, 26(1), 77 - 91 Culture media for the isolation and enumeration of pathogenic Vibrio species in foods and environmental samples; Donovan TJ et al.; The genus Vibrio now includes a large number of species . Clear evidence is only available for the aetiological role of V . cholerae, V . vulnificus and V . parahaemolyticus in foodborne diseases . Until recently, V . cholerae serogroup 0:1 was accepted as the cause of epidemic cholera . However, the designation of outbreaks of diarrhoeal diseases caused by V . cholerae 0:139 as clinical cholera has lead to renewed interest in Non 0:1 serogroups of V . cholerae . A wide range of enrichment and selective media for the isolation of vibrios has been developed . These media are reviewed with respect to their ability to recover and differentiate the target vibrios . Alkaline peptone water (APW) remains the recommended enrichment medium for vibrios in parallel with either salt polymyxin broth (SPB) or glucose teepol (or sodium dodecylsulphate) salt broth (GTSB) when tests for V . parahaemolyticus are required . Thiosulphate citrate bile salt agar (TCBS) in parallel with polymyxin mannose tellurite (PMT) or sodium dodecylsulphate polymyxin sucrose agar (SPS) are the recommended selective plating media. J Trop Pediatr, 1995 Jun, 41(3), 139 - 42 Epidemiology of cholera in Delhi--1992; Singh J et al.; Cholera is endemic in Delhi and is a highly seasonal disease . Suspected cholera cases are referred to Infectious Diseases Hospital, Delhi . Rectal swabs from 2783 cases were bacteriologically examined during 1992, out of which 1075 were found to be positive for Vibrio cholerae O1 biotype El Tor . First isolation was made on 3 April and the last on 14 December . About 87 per cent isolations were made between May and September, which are summer and monsoon months in Delhi . Detailed epidemiological information was collected for about 198 cases of diarrhoea out of which 103 were confirmed cases of cholera . Half of these cases occurred in children below 10 years of age . The other major group affected was adult females, especially housewives . All the cholera cases occurred in those who were illiterate or educated up to primary level . Important risk factors were: contact with person having similar illness, storage of water in wide-mouthed containers, use of glass or mug to draw water from containers, absence of sanitary latrines and habit of washing hands with water alone after defecation, before cooking and eating food . About 30 percent cases had access to piped water supply which was found safe in Delhi during 1992 . The findings suggest that the hygienic practices were more important than contaminated water sources for transmission of cholera in Delhi during the year 1992. Kansenshogaku Zasshi, 1995 Jun, 69(6), 637 - 41 {Studies on Vibrio cholerae non-O1 isolated from diarrheal patients arrived from overseas}; Tsutsumi H et al.; Between the period January 1991 to June 1993, there were 23,976,238 travellers who arrived from overseas to Narita Airport, of which 20,501 stool specimens were collected from diarrheal patients for bacteriological examination, and infectious agents were detected from 2,751 cases (13.4%) including 250 cases (1.2%) of Vibrio cholerae non-O1 . Countries suspected of infection of these patients were Thailand, the most in number, and followed by Indonesia, India and so on these mostly distributed in South-east or South Asia . About fifty percent of the patients were associated with abdominal pain and some with vomiting or fever . Diarrhea was mostly mild except in 16 patients who had severe diarrhea of more than ten times a day . 237 of the 250 isolated V . cholerae non-O1 strains were classified into 48 serogroups . There were 2 rough strains and 11 other strains which were out of the confirmed serogroups . Positive rate of these strains to haemolysin, cholera toxin (CT) and NAG-ST tests were 87.2, 0.4 and 0.8% respectively. J Appl Bacteriol, 1995 Jun, 78(6), 621 - 9 The effect of incubation temperature and sodium chloride concentration on the growth kinetics of Vibrio anguillarum and Vibrio anguillarum-related organisms; Guerin-Faublee V et al.; The effect of temperature and NaCl concentration on the growth kinetics of Vibrio anguillarum and V . anguillarum-related (VAR) strains was studied . For one wild VAR strain, NaCl concentration interfered with growth temperature parameters, in particular, with the maximum growth temperature but also with the optimum temperature (defined as the temperature at which mumax equals its maximal value muopt), and with muopt itself . For the same strain, optimal growth required the adding of NaCl to the medium to a final concentration of 1.5% . These results were not confirmed by tests on a V . anguillarum collection strain . When the NaCl concentration in the culture media was 1.5%, the optimum temperature for the nine strains studied ranged from 29.7 degrees C to 34 degrees C whereas the maximum temperature ranged between 35.3 degrees C and 38.5 degrees C . Hence, antibiotic susceptibility testing as well as biochemical identification might be carried out at 30 degrees C in the presence of 1.5% NaCl, which corresponded to a suboptimal growth. J Diarrhoeal Dis Res, 1995 Jun, 13(2), 127 - 9 Vibriocidal activities of some local herbs; Akinsinde KA et al.; Four of the seven tested medicinal plants exhibited antimicrobial activity against Vibrio cholerae . These 7 plants are: Ficus capensis, Mitragyna stipulosa, Entada africana, Piliostigma reticulatum, Terminalia avicennoides, Mimosa pudica, and Lannea acida . Of them Terminalia avicennoides showed higher antimocrobial activity than others . Potentials of these herbs in the control of cholera need to be determined. J Diarrhoeal Dis Res, 1995 Jun, 13(2), 118 - 21 Plasmid profiles and antimicrobial susceptibility patterns of Vibrio cholerae O1 strain isolated during a recent outbreak in Nigeria; Olukoya DK et al.; In a study on the outbreak of cholera in Nigeria in 1992, 86 strains of Vibrio cholerae O1 (79 Ogawa serotype and 7 Inaba serotype) were isolated . Antimicrobial susceptibility testing and plasmid profile analysis of the strains were done . Most isolates were highly sensitive to ciprofloxacin, cefotaxime, chloramphenicol, gentamicin, erythromycin, nalidixic acid, and nitrofurantoin, and less sensitive to ampicillin, penicillin, cloxacillin, cotrimoxazole, streptomycin, and tetracycline . The strains showed 13 resistant patterns; the commonest resistant patterns were Apr, Smr, and ApTcr . A total of 41 (47.6%) strains contained one or more plasmid(s) with sizes ranging from 4.5 kilobase to 150 kilobase . Ten isolates were able to transfer resistant plasmids to Escherichia coli K-12 by conjugation . Antibiogram patterns distinguished more isolates than in plasmid profile analysis . Plasmids specifying resistance to ampicillin, tetracycline, and trimethoprim were found . The differing patterns of antibiogram and plasmid profiles indicated that many circulating strains were responsible for the last outbreak in the country. J Diarrhoeal Dis Res, 1995 Jun, 13(2), 113 - 7 Molecular epidemiology of Vibrio cholerae O1 isolated in Nepal by southern hybridization with a cholera toxin gene probe; Yamamoto K et al.; A cholera epidemic broke out in 1992 due to Vibrio cholerae O1 biotype El Tor in the eastern and southern belt of Nepal mainly among the Bhutanese refugees . Restriction fragment profiles (RFP) of DNA fragments of V . cholerae O1 isolates hybridized with an enzyme-labelled oligonucleotide probe for cholera toxin gene (ctx) by Southern Hybridization were compared . The probe hybridized with the 13- and 8-kb fragments of PstI-digested total DNA in all isolates observed in the epidemic . This RFP in the Nepalese strain was not observed in the strains isolated during other epidemics but was observed in the strains isolated from the exported marine products from Taiwan and Thailand. Hybridoma, 1995 Jun, 14(3), 271 - 8 Development of highly specific monoclonal antibodies for the diagnosis of Vibrio cholerae 01; Castillo L et al.; We report here the development of two monoclonal antibodies, termed 5G8 and 5C12, belonging to the IgM and IgG1 class, respectively, suitable for the identification of Vibrio cholerae 01 in clinical and environmental samples . The specificities of the monoclonals were evaluated by ELISA and indirect immunofluorescent microscopy of microorganisms normally present in stool samples and with two bacterial panels . One panel included 72 potentially antigenically related bacterial strains and the second panel included 20 pathogenic bacterial strains involved in diarrhea cases . The results of these extensive analyses indicate that monoclonal antibodies 5G8 and 5C12 are highly specific and suitable for the clinical diagnosis of Vibrio cholerae 01 in human stool samples by indirect immunofluorescent microscopy . Although the antigenic sites recognized by these antibodies were not identified in this study, the observation of Western blot patterns suggested that 5G8 and 5C12 monoclonal antibodies bind to LPS epitopes, a good structural marker for the detection of V . cholerae 01 because it is present in all bacterial cell walls. Pigment Cell Res, 1995 Jun, 8(3), 147 - 52 Characterization of the melanogenic system in Vibrio cholerae, ATCC 14035; Ruzafa C et al.; The nature of the pigment formed by Vibrio cholerae and the characterization of its biosynthetic pathway is shown . This microorganism is able to synthesize melanin-like pigment when cultured in the presence of L-tyrosine . Other phenolic chemicals related to L-tyrosine do not lead to pigment production . The microorganism has no tyrosine hydroxylase activity, and the levels of dopa oxidase activity are very low, making the existence of a tyrosinase very unlikely . However, Vibrio cholerae contained transaminases that transforms L-tyrosine into p-hydroxyphenylpyruvate . Moreover, Vibrio cholerae is able to go further in the catabolic pathway, releasing a great amount of homogentisic acid . This acid can spontaneously be oxidized to its p-quinone form, which subsequently polymerizes leading to pigment formation . It is concluded that the pigment formed by Vibrio cholerae is not synthesized by the Raper-Mason pathway, but by a L-tyrosine catabolism pathway leading to homogentisic acid . Some simple properties of that melanin are compared to model eu- and pheomelanin, but no clear distinction could be stated, indicating the similarity between all these pigments. Clin Infect Dis, 1995 Jun, 20(6), 1485 - 90 Ciprofloxacin for the treatment of cholera: a randomized, double-blind, controlled clinical trial of a single daily dose in Peruvian adults; Gotuzzo E et al.; We conducted a randomized, double-blind clinical trial to compare ciprofloxacin (250 mg once a day for 3 days) with tetracycline (500 mg four times a day for 3 days) in terms of efficacy and safety in the treatment of moderate-to-severe cholera in Peruvian adults . The baseline characteristics of the groups were similar . A total of 202 patients (102 in the tetracycline group and 100 in the ciprofloxacin group) were included in the efficacy analysis . The clinical and bacteriologic efficacies of the two regimens were similar . The study drugs were well tolerated . We conclude that ciprofloxacin given once a day is as effective as the standard tetracycline regimen for the treatment of cholera in adults . The ciprofloxacin regimen may represent an alternative to the standard treatment in areas where Vibrio cholerae O1 strains that are resistant to commonly used antimicrobials are prevalent. Clin Infect Dis, 1995 Jun, 20(6), 1480 - 4 Efficacy and tolerability of ciprofloxacin prophylaxis in adult household contacts of patients with cholera; Echevarria J et al.; We conducted a randomized double-blinded study in Lima, Peru, to assess the tolerability and efficacy of a single 250-mg dose of ciprofloxacin in preventing diarrhea and Vibrio cholerae O1 infection among household contacts of bacteriologically confirmed index cases . Adult household contacts with negative baseline stool cultures were included . A total of 213 household contacts were evaluable . The study drugs were well tolerated in both groups . Ciprofloxacin did not prevent the acquisition of V . cholerae O1 infection nor the development of diarrhea . However, in a subgroup of 30 household contacts with positive baseline stool cultures a reduction in the bacterial load and a trend toward prevention of diarrhea were observed among ciprofloxacin recipients . When all household contacts were evaluated, a trend toward prevention of diarrhea was observed with the prophylactic regimen . Ciprofloxacin failed to prevent V . cholerae O1 infections during a period of low transmissibility. Surg Endosc, 1995 Jun, 9(6), 730 - 2 Acute acalculous cholecystitis due to Vibrio cholerae; Gomez NA et al.; The case of a 57-year-old woman admitted with symptoms and signs suggesting an intestinal infection caused by Vibrio cholerae, and who also developed a clinical picture compatible with acute cholecystitis, is presented . Cholera was diagnosed by examining a fresh sample of stools and cultures . An abdominal sonogram disclosed signs of acute acalculous cholecystitis . She underwent cholecystectomy, and cultures of a clear fluid and a "milky" sediment found within the gallbladder were also positive for V . cholerae . This microorganism was seen at the gallbladder mucosa microscopically . The strain was serotyped V . cholerae 01 (El Tor) Ogawa and was the etiology of the acute acalculous cholecystitis in this patient. Ugeskr Laeger, 1995 May 29, 157(22), 3202 - 4 {Severe systemic infection with Vibrio vulnificus}; Hansen LN et al.; Infections with Vibrio vulnificus are not common in Denmark, but in 1994 several cases were identified, probably due to the very hot weather conditions, with seawater temperatures above 20 degrees C . Two cases of infection with V . vulnificus are presented. Gene, 1995 May 26, 158(1), 1 - 7 Putative O-antigen transport genes within the rfb region of Vibrio cholerae O1 are homologous to those for capsule transport; Manning PA et al.; The nucleotide sequence of that part of the Vibrio cholerae (Vc) O1 rfb region encompassing rfbG, rfbH and rfbI is presented . Expression of these genes has enabled the products for rfbG and rfbI to be confirmed, but the rfbH product has not been detected . Comparisons with the sequences of known proteins reveals that RfbH and RfbI are likely to be involved in the export of lipopolysaccharide (LPS) . RfbH shows considerable homology to a number of integral membrane proteins, some of which have been identified as possibly having a role as an export channel for capsular polysaccharides . RfbI corresponds to an ATP-binding protein usually found linked to the membrane protein and is thought to be required for energizing this export process . Thus, we propose that RfbH and RfbI form a complex for the export of Vc O1 LPS . The function of RfbG is unknown, but it would appear to be a relatively hydrophilic protein and we can only speculate that it may be either a specific transferase or possibly the O-antigen polymerase. Biochemistry, 1995 May 23, 34(20), 6581 - 6 Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution; Fisher AJ et al.; Luciferases are a class of enzymes that generate light in the visible spectrum . Luciferase from luminous marine bacteria is an alpha-beta heterodimer monooxygenase that catalyzes the oxidation of FMNH2 and a long-chain aliphatic aldehyde . The X-ray crystal structure of bacterial luciferase from Vibrio harveyi has been determined to 2.4 A resolution . The structure was solved by a combination of multiple isomorphous replacement and molecular averaging between the two heterodimers in the asymmetric unit . Each subunit folds into a (beta/alpha)8 barrel motif, and dimerization is mediated through a parallel four-helix bundle centered on a pseudo 2-fold axis that relates the structurally similar subunits . The vicinity of the active site has been identified on the alpha subunit by correlations with similar protein motifs and previous biochemical studies . The structure presented here represents the first molecular model of a bioluminescent enzyme. FEMS Microbiol Lett, 1995 May 15, 128(3), 265 - 9 Cloning and sequencing of a novel hemolysis gene of Vibrio cholerae; Nagamune K et al.; A hemolysis gene (hlx) which lyses sheep erythrocytes on blood agar plates when expressed in Escherichia coli was cloned from Vibrio cholerae . The cloned gene is predicted to encode a polypeptide of 92 amino acid residues with a deduced molecular mass of 10,451 . E . coli transformed with this gene lysed sheep, goose, horse and chicken erythrocytes but not those of guinea pig and human . The hlx gene was observed in classical- and El Tor-biotype V . cholerae O1, V . cholerae non-O1, and V . mimicus, but not in V . parahaemolyticus. Klin Lab Diagn, 1995 May-Jun, (3), 8 - 11 {Vibrios pathogenic to humans and laboratory diagnosis of diseases caused by them}; Smolikova LM et al.; Present-day taxonomic status of vibrios pathogenic for humans is discussed, as are the phenotypical characteristics of some species, factors of virulence, and clinical manifestations of diseases caused by these agents . A scheme of isolation and identification of vibrios pathogenic for humans is offered. Med Microbiol Immunol (Berl), 1995 May, 184(1), 37 - 44 Characterization of Vibrio cholerae El Tor cytolysin as an oligomerizing pore-forming toxin; Zitzer A et al.; V . cholerae El Tor cytolysin is a secreted, water-soluble protein of M(r) 60,000 that may be relevant to the pathogenesis of acute diarrhea . In this communication, we demonstrate that the toxin binds to and oligomerizes in target membranes to form SDS-stable aggregates of M(r) 200,000-250,000 that generate small transmembrane pores . Pores formed in erythrocytes were approximately 0.7 nm in size, as demonstrated by osmotic protection experiments . Binding was shown to occur in a temperature-independent manner preceding the temperature-dependent oligomerization step . Pores were also shown to be formed in L929 and HEp-2 cells, human fibroblasts and keratinocytes, albeit with highly varying efficacy . At neutral pH and in the presence of serum, human fibroblasts were able to repair a limited number of lesions . The collective data identify V . cholerae El Tor cytolysin as an oligomerizing toxin that damages cells by creating small transmembrane pores. FEMS Microbiol Lett, 1995 May 1, 128(2), 195 - 200 Utilization of hemin and hemoglobin as iron sources by Vibrio parahaemolyticus and identification of an iron-repressible hemin-binding protein; Yamamoto S et al.; Several clinical isolates of Vibrio parahaemolyticus were examined for their ability to utilize either hemin or hemoglobin as a sole source of iron . Both compounds appeared to be equally good iron sources . Maximum growth was obtained at 5 microM hemin or 1.25 microM hemoglobin under the conditions tested . Using a hemin-agarose batch affinity method, the hemin-binding protein was isolated from crude total membranes of a hemin-utilizing strain, WP1, grown under iron-deficient but not under iron-sufficient conditions . This protein was identical to the 83 kDa outer membrane protein which was expressed in response to iron limitation . The protein was susceptible to proteinase K cleavage in whole cells, indicating its exposure at the cell surface . Hemin and hemoglobin, but not protoporphyrin IX, inhibited binding of the protein to hemin-agarose. South Med J, 1995 May, 88(5), 531 - 3 Vibrio vulnificus wound infections from the Mississippi Gulf coastal waters: June to August 1993; Penman AD et al.; Vibrio vulnificus, part of the normal marine flora of the Gulf of Mexico, is being increasingly recognized as an important human pathogen . V vulnificus contamination of superficial wounds can cause a severe, rapidly progressive, necrotizing cellulitis with bullous skin lesions that may require surgical debridement and is occasionally fatal . We summarize information about six cases of V vulnificus wound infection reported to the Mississippi State Department of Health from June to August 1993 . Five of the six patients required hospitalization for intravenous antibiotic treatment and, in two cases, surgery . Two patients died from septicemia, despite aggressive antibiotic treatment; both had preexisting medical conditions that could have contributed to immune compromise and fulminant infection . This report underscores the virulence of this organism and the need for awareness by both the clinician and diagnostic laboratory personnel when dealing with superficial wounds occupationally or recreationally exposed to seawater. Infect Immun, 1995 May, 63(5), 1987 - 92 Purification and characterization of a cell-associated hemagglutinin of Vibrio parahaemolyticus; Nagayama K et al.; We found a positive correlation between cell-associated mannose-sensitive hemagglutination and adherence of Vibrio parahaemolyticus to rabbit enterocytes by investigating 35 strains of V . parahaemolyticus for cell-associated hemagglutinin (cHA) and for the ability to adhere to the enterocytes . We purified a mannose-sensitive cHA from a Kanagawa phenomenon-positive clinical strain of V . parahaemolyticus that exhibited a high level of mannose-sensitive hemagglutination and strongly adhered to the enterocytes . The purified cHA is a heat-labile, tetrameric protein consisting of four identical subunits of approximately 26 kDa each . The adherence to rabbit enterocytes was inhibited in a dose-dependent manner by pretreatment of the bacterial cells with D-mannose and with the Fab fraction of immunoglobulin G against the purified cHA . Furthermore, pretreatment of the enterocytes with the purified cHA inhibited the adherence of V . parahaemolyticus . Immunogold electron microscopy revealed that the cHA is located on the bacterial cell surface and is not associated with pili . These results suggest that cHA is involved in the adherence mechanisms of V . parahaemolyticus to the enterocytes and that the receptors for cHA on the enterocyte appear to be a D-mannose-containing compound. J Exp Med, 1995 May 1, 181(5), 1693 - 703 Trypanosoma cruzi trans-sialidase: enhancement of virulence in a murine model of Chagas' disease; Chuenkova M et al.; Trypanosoma cruzi, the etiological agent of Chagas' disease, expresses a trans-sialidase at highest levels in infective trypomastigotes, where it attaches to the plasma membrane by a glycophosphoinositol linkage . Bound enzyme sheds into the extracellular milieu in a soluble form . Experiments performed in vitro suggest that the trans-sialidase participates in several parameters of T . cruzi-host interactions, like cell adhesion and complement resistance . However, the role that membrane-bound and soluble trans-sialidase plays in the infection of mammals is not understood . To begin to study the role the enzyme may play in vivo, T . cruzi trypomastigotes were inoculated subcutaneously into mice that had been sensitized for various times with the purified protein . A single dose of either endogenous or recombinant trans-sialidase injected into the connective tissues of BALB/c mice greatly enhanced parasitemia and mortality . Maximum enhancement was achieved with 1-2-h priming . Injection of the enzyme after the parasites had been established in the inoculation site had little, if any, consequence in modifying virulence . The enhancement did not seem to be through a direct effect of the enzyme on trypomastigote-host cell interactions because it occur