Demonstration of invasiveness of Vibrio parahaemolyticus in adult rabbits by immunofluorescence.

To determine possible pathogenesis of Vibrio parahaemolyticus-host-organ system interactions, studies of invasiveness were made by a direct fluorescent-antibody method. Broth cultures of live cells isolated from seafish or symptomatic humans were inoculated separately into ligated ileal loops of young New Zealand white rabbits. After suitable incubation, rabbits were sacrificed, and ileal loops and tissue specimens were aseptically removed. Ileal loops were prepared and stained with specific fluorescein-tagged antibody, and organ specimens were cultured for isolation of the inoculated Vibrio strain. All strains tested penetrated into the lamina propria of the ileum and were isolated from the cultured tissue specimens, indicating that the organism is capable of more than a superficial colonization of the gut. The presence of Vibrio in cultured tissue specimens suggests invasion of deeper tissue by either the lymphatic or the circulatory system.     

The effect on enterotoxicity of protease purified from Vibrio cholerae O1

Abstract The effect on enterotoxicity of protease purified from Vibrio cholerae O1 was investigated by the inoculation of live vibrio cells into the protease-treated loops of the ileal loop model. Fluid accumulation ratios in the protease-treated loops were elevated in a dose-dependent manner by challenge with live vibrio cells byt not that with toxin. An enhancement effect of protease on enterotoxicity was observed in both serotypes of V. cholerae O1 and V. cholerae non-O1. It is suggested, therefore, that the enterotoxicity was enhanced by treatment with protease when live vibrio cells were inoculated into the ileal loops of rabbits.     

Toxin Isolation from a Kanagawa-Phenomenon Negative Strain of Vibrio parahaemolyticus

Production of a toxin by Vibrio parahaemolyticus Kanagawa-phenomenon negative strains was examined. Ammonium sulfate fractions of broth culture filtrates were dialyzed, concentrated by lyophilization, and tested for toxic effects by mouse intraperitoneal injection. One fraction, which we think is a toxin, was isolated from a broth culture filtrate of V. parahaemolyticus FC 1011 (a Kanagawa-phenomenon negative strain) and consistently produced lethal effects in mice at high concentrations and diarrhea in lower concentrations. The toxin was assayed for mouse LD50 and ability to produce diarrhea via forced feeding in mice. V. parahaemolyticus FC 1011 toxin was found to be protein, to be inactivated by heat or trypsin hydrolysis, and to produce positive skin permeability reactions in rabbits. However, it failed to induce fluid accumulation in ligated ileal loops in rabbits.     

Histopathological changes in experimental cholera with a non toxigenic non- O1 non-O139 Vibrio cholerae strain isolated from Kolkata, India

This study was conducted to understand the pathophysiological changes in experimental rabbit ileal loop model using the Vibrio cholerae strain non-O1non-O139, isolated as sole pathogen from clinically diagnosed cholera patients in Kolkata. Significant amount of haemorrhagic fluid accumulation was observed in all the test loops of rabbit model where the strain of V.cholerae was inoculated as compared to control loops. Microscopic examination of the accumulated fluid showed the presence of erythrocytes and pus cells. Histology revealed structural alteration of the villous epithelium with inflammatory cells infiltration in all the layers of the gut mucosa including the nerve plexus region. Preliminary observation with a haemagglutinin protease extracted from the non-O1 non-O139 strain, was also studied in different concentrations in the same animal model which showed similar type of macroscopic and microscopic response in the ileal loops as seen with the original strain. The results highlight that along with other pathways, inflammatory cells and the enteric neurons have an important role in the pathophysiology of diarrhoea and the isolated protease may be the probable virulence factor in initiating the disease process in this non-O1non-O139 strain induced cholera.     

Effects of crude and pure cholera toxin on prostaglandin.

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE2 equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE2/g tissue when incubated with crude toxin for 1-4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions. Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Selective association and transport of Campylobacter jejuni through M cells of rabbit Peyer's patches

M cells in the Peyer's patches may facilitate transport of pathogens such as Campylobacter jejuni from the intestine. We evaluated this hypothesis by using electron microscopy to examine Peyer's patches in ligated adult rabbit ileal loops inoculated with 5-mL suspensions of 10(9) cfu/mL of Campylobacter jejuni. Peyer's patches taken at intervals from 15 min to 2 h after inoculation of loops in anaesthetized rabbits provided evidence that Campylobacter jejuni selectively adhered to M cells as opposed to absorptive epithelial cells and was transported, apparently intact, into the M cell follicle. Although intercellular organisms were seen within the follicle, many others were phagocytosed by lymphoid cells. The proximity of the lymphatic and blood circulatory systems to the M cell follicle makes this a probable route for systemic spread of Campylobacter jejuni.     

Rabbit Ileal Loop Response to Strains of Clostridium perfringens

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.     

Effects of crude and pure cholera toxin on prostaglandin release from the rabbit ileum

Investigations were made into the effects of crude and pure preparations of cholera toxin on the release of prostaglandin-like substances (PLS) from rabbit ileum. Perfusion of ileal loops in vivo with buffer containing crude toxin was followed by a release of PLS into the perfusate, in amounts up to 37.5 ng/30 min (PGE₂ equivalents). In contrast, no detectable PLS was released when ileal loops were perfused with pure toxin. Similarly, pieces of ileum opened longitudinally released PLS in amounts up to 107 ng PGE₂/g tissue when incubated with crude toxin for 1–4 hr, but no release of PLS was detected in the presence of pure toxin under comparable conditions.Treatment of rabbits with indomethacin, 1.6 mg/kg p.o., had no effect on the accumulation of fluid in ileal sacs injected with crude or pure cholera toxin. These results support the view that prostaglandins do not play an essential role in the action of cholera toxin.     

Effective ileal loop dose of Kanagawa-positive Vibrio parahaemolyticus.

Groups of 16 rabbits per strain were injected with broth culture dilutions of three Kanagawa-positive Vibrio parahaemolyticus strains. The effective dose required to produce ileal loop dilatation in 50% of rabbits for pure cultures of strains 10136-76 and 553-72 from patients stools and NY 477 from incriminated food was 1.1 x 10(6), 2.6 x 10(5), and 7.7 x 10(6) organisms, respectively. When each of these cultures was admixed with greater than or equal to 10(9) Vibrio alginolyticus cells, the 50% effective dose was 1.2 x 10(6), 1.1 x 10(7), and 1.3 x 10(8) cells, respectively. Although concomitant injection of large numbers of competitive nonvirulent cells did not affect the 50% effective dose for strain 10136-76, that for the remaining two was increased 20- to 40-fold. The initiation of ileal loop response as estimated from sigmoidal plots of proportion of positive loops versus cell concentrations was given by as few as 10(2) cells of strain 553-72. Strains NY 477 and 10136-76 required approximately 10(5) cells. Half of the maximal response from these plots corresponded well with the 50% effective dose for the strains. These results suggest that pathogenicity of Kanagawa-positive V. parahaemolyticus strains may involve the participation of some virulence mechanisms in addition to the Kanagawa hemolysin.     

Properties of a hemolysin related to the thermostable direct hemolysin produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus

A hemolytic toxin (Vp-TRH) produced by a Kanagawa phenomenon negative, clinical isolate of Vibrio parahaemolyticus was further characterized. The purified Vp-TRH showed various biological activities, such as fluid accumulation in rabbit ileal loops, increase of rabbit skin vascular permeability, and cardiotoxicity on cultured myocardial cells, all of which are essentially similar to the activities found with thermostable direct hemolysin (Vp-TDH), a pathogenic toxin produced by Kanagawa phenomenon positive V. parahaemolyticus. Immunological similarities of Vp-TRH not only to Vp-TDH but also to hemolytic toxins produced by Vibrio hollisae and Vibrio cholerae non-O1, both of which are also enteropathogens closely related to V. parahaemolyticus, were demonstrated. The amino acid composition and sequence of N-terminal amino acids of Vp-TRH were determined. These results suggest that Vp-TRH has biological and immunological characters similar to Vp-TDH, although they are distinct molecules.     

Isolation of Vibrio alginolyticus and Vibrio splendidus from aquacultured carpet shell clam (Ruditapes decussatus) larvae associated with mass mortalities

Two episodes of mortality of cultured carpet shell clams (Ruditapes decussatus) associated with bacterial infections were recorded during 2001 and 2002 in a commercial hatchery located in Spain. Vibrio alginolyticus was isolated as the primary organism from moribund clam larvae that were obtained during the two separate events. Vibrio splendidus biovar II, in addition to V. alginolyticus, was isolated as a result of a mixed Vibrio infection from moribund clam larvae obtained from the second mortality event. The larval mortality rates for these events were 62 and 73%, respectively. Mortality was also detected in spat. To our knowledge, this is the fist time that these bacterial species have been associated with larval and juvenile carpet shell clam mortality. The bacterial strains were identified by morphological and biochemical techniques and also by PCR and sequencing of a conserved region of the 16S rRNA gene. In both cases bacteria isolated in pure culture were inoculated into spat of carpet shell clams by intravalvar injection and by immersion. The mortality was attributed to the inoculated strains, since the bacteria were obtained in pure culture from the soft tissues of experimentally infected clams. V. alginolyticus TA15 and V. splendidus biovar II strain TA2 caused similar histological lesions that affected mainly the mantle, the velum, and the connective tissue of infected organisms. The general enzymatic activity of both live cells and extracellular products (ECPs), as evaluated by the API ZYM system, revealed that whole bacterial cells showed greater enzymatic activity than ECPs and that the activity of most enzymes ceased after heat treatment (100 degrees C for 10 min). Both strain TA15 and strain TA2 produced hydroxamate siderophores, although the activity was greater in strain TA15. ECPs from both bacterial species at high concentrations, as well as viable bacteria, caused significant reductions in hemocyte survival after 4 h of incubation, whereas no significant differences in viability were observed during incubation with heat-killed bacteria.     

Assessment of in vivo revival, growth, and pathogenicity of Escherichia coli strains after copper- and chlorine-induced injury.

Cells of one enteroinvasive and three enterotoxigenic strains of Escherichia coli were exposed to sublethal concentrations of copper and chlorine to produce 85 to 94% injury. Injured cells were intraluminally inoculated into ligated ileal loops of anesthetized mice, and injury was assessed at timed intervals. Substantial recovery (72-84%) of copper- and chlorine-injured cells was observed in the inoculated loops at 4 and 3 h, respectively. No appreciable increase in total numbers was observed during these time intervals. In vitro revival of copper-injured cells in phosphate-buffered saline alone after incubation at 35 degrees C for 4 h was not observed. However, a 60 to 70% revival occurred when 200 micrograms of protein per ml of mouse intestinal mucosal homogenate was incorporated into saline cell suspensions. The enterotoxigenic activity of copper-injured cells in rabbit ileal loops was somewhat reduced compared with that of chlorine-injured or uninjured cells. These results show that injured pathogenic E. coli cells can revive in the small intestine and appear to retain their enterotoxigenic activity.     

Assessment of in vivo revival, growth, and pathogenicity of Escherichia coli strains after copper- and chlorine-induced injury

Cells of one enteroinvasive and three enterotoxigenic strains of Escherichia coli were exposed to sublethal concentrations of copper and chlorine to produce 85 to 94% injury. Injured cells were intraluminally inoculated into ligated ileal loops of anesthetized mice, and injury was assessed at timed intervals. Substantial recovery (72-84%) of copper- and chlorine-injured cells was observed in the inoculated loops at 4 and 3 h, respectively. No appreciable increase in total numbers was observed during these time intervals. In vitro revival of copper-injured cells in phosphate-buffered saline alone after incubation at 35 degrees C for 4 h was not observed. However, a 60 to 70% revival occurred when 200 micrograms of protein per ml of mouse intestinal mucosal homogenate was incorporated into saline cell suspensions. The enterotoxigenic activity of copper-injured cells in rabbit ileal loops was somewhat reduced compared with that of chlorine-injured or uninjured cells. These results show that injured pathogenic E. coli cells can revive in the small intestine and appear to retain their enterotoxigenic activity.     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia.

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization and distribution of Vibrio alginolyticus and Vibrio parahaemolyticus isolated in Indonesia

Previous studies have shown that Vibrio alginolyticus and Vibrio parahaemolyticus can be isolated from similar types of marine samples. In this report, the results of an examination of 567 V. alginolyticus and V. parahaemolyticus strains, isolated from seawater in Jakarta Bay and from more than 30 types of seafood from markets in Jakarta, Indonesia, are presented. Most isolates were from mackerel, shrimp, or squid. Numerical taxonomic analyses clustered 337 isolates and three V. alginolyticus reference strains at S greater than or equal to 80%. These strains produced acid from sucrose, but only approximately 80% produced acetoin or grew in the presence of 10% NaCl. The frequency of occurrence of V. alginolyticus in seawater samples ranged from 0% (in February and March 1972) to 100% (in September and December 1972) and was highest in seafood samples from August to December 1972. A second cluster of 230 isolates and seven V. parahaemolyticus reference strains was observed at S greater than or equal to 82%. These strains did not produce acetoin or acid from sucrose, and approximately 20% grew in the presence of 10% NaCl. V. parahaemolyticus was detected in seawater samples each month, with the highest frequency of occurrence (83.3%) in May 1972. Twenty-nine K antigen serotypes were demonstrated in V. parahaemolyticus isolates, and another 40% were untypable. The modal antibiotic resistance pattern for each species included five drugs. Only 12% of the V. parahaemolyticus strains were Kanagawa positive, and 10% elicited fluid accumulation in ligated rabbit ileal loops. All of the 7 V. alginolyticus strains and 94 (70%) of the V. parahaemolyticus strains tested killed mice when inoculated intraperitoneally.(ABSTRACT TRUNCATED AT 250 WORDS)     

Virulence genes in environmental strains of Vibrio cholerae

The virulence of a pathogen is dependent on a discrete set of genetic determinants and their well-regulated expression. The ctxAB and tcpA genes are known to play a cardinal role in maintaining virulence in Vibrio cholerae, and these genes are believed to be exclusively associated with clinical strains of O1 and O139 serogroups. In this study, we examined the presence of five virulence genes, including ctxAB and tcpA, as well as toxR and toxT, which are involved in the regulation of virulence, in environmental strains of V. cholerae cultured from three different freshwater lakes and ponds in the eastern part of Calcutta, India. PCR analysis revealed the presence of these virulence genes or their homologues among diverse serotypes and ribotypes of environmental V. cholerae strains. Sequencing of a part of the tcpA gene carried by an environmental strain showed 97.7% homology to the tcpA gene of the classical biotype of V. cholerae O1. Strains carrying the tcpA gene expressed the toxin-coregulated pilus (TCP), demonstrated by both autoagglutination analysis and electron microscopy of the TCP pili. Strains carrying ctxAB genes also produced cholera toxin, determined by monosialoganglioside enzyme-linked immunosorbent assay and by passage in the ileal loops of rabbits. Thus, this study demonstrates the presence and expression of critical virulence genes or their homologues in diverse environmental strains of V. cholerae, which appear to constitute an environmental reservoir of virulence genes, thereby providing new insights into the ecology of V. cholerae.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

Production of ex-germfree rabbits for establishment of specific pathogen-free (SPF) colonies.

Nine groups of ex-germfree (GF) rabbits were produced by inoculation of hysterectomy-derived GF rabbits with various combinations of cecal bacteria isolated from conventional (CV) rabbits in order to establish a barrier-sustained colony. Six strains of Bacteroides and two strains of Streptococcus isolated from CV rabbits (2 to 3 weeks old) were used for pretreatment. The mortality of ex-GF rabbits inoculated with the anaerobic growth (CF) on EG or SM10 plates inoculated with a 10(-5) dilution of cecal contents was 71.4 to 94.4% when given without pretreatment. All ex-GF rabbits pretreated with Bacteroides alone survived, but the mortality of ex-GF rabbits inoculated with Bacteroides plus Streptococcus strains as pretreatment was 20 and 45.4%. The mortality of ex-GF rabbits inoculated with only Bacteroides was 43%. All ex-GF rabbits inoculated with Bacteroides plus anaerobic growth (CF), cecal suspension of ex-GF mice which had been inoculated with cecal suspensions of CV rabbits (MF) or chloroform-treated cecal suspension (CHF) survived, but CHF inoculated ex-GF rabbits were in an unhealthy condition with slight diarrhoea. These data indicate that inoculation with Bacteroides strains as pretreatment plus CF or MF was required to convert GF rabbits to the normal state.     

Serotyping of Vibrio anguillarum

A serotyping scheme based on the detection of O antigens by slide agglutination in fish-pathogenic strains of Vibrio anguillarum is presented. Over a period of 5 years 270 Vibrio strains from feral and cultured fish, 189 strains from the environment, and 36 strains from invertebrates were collected. The strains were divided into 10 distinct serotypes (O1 through O10). More than 90% of the fish-pathogenic strains, but only 40% of the environmental strains, were typable; 71% of the strains isolated from cultured rainbow trout were serotype O1, whereas 78% of the strains isolated from feral fish were serotype O2. No dominating environmental serotype was found. A serotyping system for V. anguillarum is proposed. A total of 90 strains received from culture collections and laboratories in different countries were typed according to the present system.