Rabbit Ileal Loop Research Articles

Sunlight-induced propagation of the lysogenic phage encoding cholera toxin.

In toxigenic Vibrio cholerae, the cholera enterotoxin (CT) is encoded by CTXPhi, a lysogenic bacteriophage. The propagation of this filamentous phage can result in the origination of new toxigenic strains. To understand the nature of possible environmental factors associated with the propagation of CTXPhi, we examined the effects of temperature, pH, salinity, and exposure to direct sunlight on the induction of the CTX prophage and studied the transmission of the phage to potential recipient strains. Exposure of cultures of CTXPhi lysogens to direct sunlight resulted in approximately 10,000-fold increases in phage titers. Variation in temperature, pH, or salinity of the culture did not have a substantial effect on the induction of the prophage, but these factors influenced the stability of CTXPhi particles. Exposure of mixed cultures of CTXPhi lysogens and potential recipient strains to sunlight significantly increased both the in vitro and in vivo (in rabbit ileal loops) transduction of the recipient strains by CTXPhi. Included in these transduction experiments were two environmental nontoxigenic (CTXPhi(-)) strains of V. cholerae O139. These two O139 strains were transduced at high efficiency by CTXPhi, and the phage genome integrated into the O139 host chromosome. The resulting CTXPhi lysogens produced biologically active CT both in vitro and in rabbit ileal loops. This finding suggests a possible mechanism explaining the origination of toxigenic V. cholerae O139 strains from nontoxigenic progenitors. This study indicates that sunlight is a significant inducer of the CTX prophage and suggests that sunlight-induced transmission of CTXPhi may constitute part of a natural mechanism for the origination of new toxigenic strains of V. cholerae.

A putative heat-labile enterotoxin expressed by strains of Aeromonas media.

Fifteen isolates of Aeromonas media (seven from diarrhoeal stools, four from water and four from superficial skin ulcers of catfish) were examined for enterotoxin production. Ten of these isolates (six diarrhoeal, one from water and three from fish) caused accumulation of fluid in the initial rabbit ileal loop (RIL) tests. Isolates from diarrhoeal stools and fish caused relatively more fluid accumulation than those from water. Those strains that caused little or no fluid accumulation in the initial experiments became enterotoxin producers after one passage through RILs, regardless of source, and also showed gradual enhancement of fluid outpouring after each subsequent passage. Inocula of c. 1 x 10(4) viable cells and 0.25 ml of culture filtrate (CF) caused fluid accumulation similar to that of toxigenic Vibrio cholerae 569B. The enterotoxic factor(s) were inactivated when held at 56 degrees C for 20 min or 65 degrees C for 10 min and showed biological activity over a wide range of pH values. These results suggest that strains of A. media, whether from diarrhoeal stools, water or infected fish, are potentially enterotoxigenic and may have the potential to produce a heat-labile and pH-stable diarrhoeagenic factor in the same way as other known heat-labile and pH-stable enterotoxins.

IEM101, a naturally attenuated Vibrio cholerae strain as carrier for genetically detoxified derivatives of cholera toxin

Two mutants of cholera toxin (CTS106 containing a Pro106→Ser substitution and CTK63 containing a Ser63→Lys substitution) with greatly reduced or no toxicity respectively, were expressed in the naturally attenuated IEM101 Vibrio cholerae strain (El Tor, Ogawa) which does not express cholera toxin (CT). Expression was driven by the natural promoter of CT, or by a promoter known to induce strong in vivo expression such as nirB. In the rabbit ileal loop assay, where 10 4 wild type bacteria were sufficient to induce fluid accumulation, 10 9 IEM101 expressing CTS106 bacteria were needed to induce some fluid accumulation, while IEM101 expressing CTK63 was inactive, even when 10 10 cells were used. When used to immunize mice intranasally, all bacteria induced vibriocidal antibodies; however, anti-CT antibodies were not induced by bacteria expressing low levels of CTK63 under the control of the ct promoter. Anti-CT antibodies were successfully induced by bacteria expressing high levels of CTK63 under the control of the nirB promoter, or by bacteria expressing low levels of CTS106. These data show that antibodies against cholera toxin can be induced in vivo by high level expression of a non toxic mutant, or by using a mutant with residual ADP-ribosyltransferase activity. In conclusion, we have shown that IEM101, a naturally attenuated Vibrio strain known to be safe and immunogenic in humans, can be engineered to express immunogenic levels of CTK63, and may represent a good candidate for vaccination against cholera.

Production of heat-labile enterotoxin by strains of Aeromonas veronii bv veronii.

Three isolates of Aeromonas veronii bv veronii (2 environmental, one blood infection) were examined to see if they produce any enterotoxin and, if so, to determine its characteristics. Two isolates caused fluid accumulation in the initial rabbit ileal loop tests. The other strain did so after a single passage through the rabbit ileal loop. All the isolates showed gradual enhancement of fluid secretion after each subsequent passage. Inocula of 1 x 10(4) viable cells and 0.25 mL of culture filtrate caused fluid accumulation comparable to those of toxigenic Vibrio cholerae 569B. The enterotoxic activity was inactivated at higher temperature, and showed biological activity over a wide range of pH. The only histopathological change observed was depletion of mucous from goblet cells. The findings of the study indicate that strains of A. veronii bv veronii produce a heat-labile, pH-stable diarrhoeagenic factor without causing any damage to the intestinal mucosa.

Identification of a Gene within a Pathogenicity Island of Enterotoxigenic Escherichia coli H10407 Required for Maximal Secretion of the Heat-Labile Enterotoxin

Studies of the pathogenesis of enterotoxigenic Escherichia coli (ETEC) have largely centered on extrachromosomal determinants of virulence, in particular the plasmid-encoded heat-labile (LT) and heat-stable enterotoxins and the colonization factor antigens. ETEC causes illnesses that range from mild diarrhea to severe cholera-like disease. These differences in disease severity are not readily accounted for by our current understanding of ETEC pathogenesis. Here we demonstrate that Tia, a putative adhesin of ETEC H10407, is encoded on a large chromosomal element of approximately 46 kb that shares multiple features with previously described E. coli pathogenicity islands. Further analysis of the region downstream from tia revealed the presence of several candidate open reading frames (ORFs) in the same transcriptional orientation as tia. The putative proteins encoded by these ORFs bear multiple motifs associated with bacterial secretion apparatuses. An in-frame deletion in one candidate gene identified here as leoA (labile enterotoxin output) resulted in marked diminution of secretion of the LT enterotoxin and lack of fluid accumulation in a rabbit ileal loop model of infection. Although previous studies have suggested that E. coli lacks the capacity to secrete LT, our studies show that maximal release of LT from the periplasm of H10407 is dependent on one or more elements encoded on a pathogenicity island.

The sigA gene which is borne on the she pathogenicity island of Shigella flexneri 2a encodes an exported cytopathic protease involved in intestinal fluid accumulation.

In this study, the sigA gene situated on the she pathogenicity island of Shigella flexneri 2a was cloned and characterized. Sequence analysis showed that sigA encodes a 139.6-kDa protein which belongs to the SPATE (serine protease autotransporters of Enterobacteriaceae) subfamily of autotransporter proteins. The demonstration that SigA is autonomously secreted from the cell to yield a 103-kDa processed form and possesses a conserved C-terminal domain for export from the cell were consistent with the autotransporter pathway of secretion. Functional analysis showed that SigA is a secreted temperature-regulated serine protease capable of degrading casein. SigA was cytopathic for HEp-2 cells, suggesting that it may be a cell-altering toxin with a role in the pathogenesis of Shigella infections. SigA was at least partly responsible for the ability of S. flexneri to stimulate fluid accumulation in ligated rabbit ileal loops.

Role of anaerobiosis in virulence of Salmonella typhimurium.

Intestinal pathogens are exposed to various stress conditions during their infectious cycle. Anaerobiosis, one of such hostile condition, is offered by the host within gut and intestinal lumen, where survival, multiplication and entry into intestinal epithelial cells is priority for the invading pathogen. In the present study, a virulent strain of S. typhimurium (1402/84) was grown under anaerobic conditions and its virulence characteristics such as host cell binding, penetration and intracellular survival were compared with aerobic S. typhimurium. Anaerobically grown S. typhimurium showed significantly higher binding to immobilized mice enterocytes and intestinal mucus as compared to bacteria grown aerobically. Anaerobic bacteria also showed an early penetration of mucus and subsequent binding to underlying immobilized enterocytes, in vitro. Anaerobic S. typhimurium exhibited increased intracellular survival within spleen macrophages of mice and caused significantly higher fluid accumulation in ligated rabbit ileal loops as compared to aerobic bacteria. LD50 of anaerobic S. typhimurium was also observed to be 2 fold lower when compared to aerobic bacteria. Cell surface hydrophobicity of anaerobic S. typhimurium was also found to be significantly higher than aerobic bacteria. Thus, it appears that exposure of S. typhimurium to anaerobiosis results in its enhanced virulence, adhesion and penetration of host cells.

Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe‐positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Inactivation of the gene (<i>cpe</i>) encoding <i>Clostridium perfringens</i> enterotoxin eliminates the ability of two <i>cpe</i>‐positive <i>C. perfringens</i> type A human gastrointestinal disease isolates to affect rabbit ileal loops

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on Int 407 and HeLa cells

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.

Morphological and cytoskeletal changes caused by non-membrane damaging cytotoxin of Vibrio cholerae on int 407 and HeLa cells.

Vibrio cholerae produces a non-membrane damaging cytotoxin (NMDCY), also known as cell rounding factor, which causes rapid rounding of cultured cells like HeLa, CHO and Vero and reportedly elicits enterotoxic activity in the rabbit ileal loop assay. Pursuing the concept that NMDCY might be an accessory factor contributing to the diarrhea caused by V. cholerae, we investigated the effect of NMDCY on Int 407 (intestinal cell line) and HeLa (non-intestinal cell line) cells using light, fluorescent and electron microscopy to gain insight into the cellular response evoked by NMDCY. Binding assays showed that NMDCY has affinity for both Int 407 and HeLa cells. Changes in the internal organelles and cytoskeletal structures of the cell lines were documented indicating changes in the secretory and metabolic function of the toxin-treated cells. Toxin-treated cells visualized under the electron microscope revealed retraction of cell body, formation of blebs on cell surface, changes in mitochondria having dilated and rarefied matrix and an extensively developed Golgi apparatus, endoplasmic reticulum and lysosomes compared to those in normal cells. Immunofluorescence study showed restructuring of microfilament network represented by actin, filamin and vinculin, as also of the microtubular component, tubulin and the intermediate filament, vimentin. Immunogold study further revealed that the toxin is internalized even within the nucleus. Moreover, a rise in the intracellular calcium level of the NMDCY-treated cells leads us to hypothesize that a cascade of events results in the final impairment of the cell machinery.

Purification and characterization of novel toxin produced by Vibrio cholerae O1.

Vibrio cholerae WO7 (serogroup O1) isolated from patients with diarrhea produces an extracellular toxin despite the absence of ctx, zot, and ace genes from its genome. The toxin elongates Chinese hamster ovary cells, produces fluid accumulation in ligated rabbit ileal loops, and agglutinates freshly isolated rabbit erythrocytes. Maximal production of this toxin (WO7 toxin) was seen in AKI medium with the pH adjusted to 8.5 at 37 degrees C under shaking conditions. We purified this toxin to homogeneity by sequential ammonium sulfate precipitation, affinity chromatography using a fetuin-Sepharose CL-4B column, and gel filtration chromatography, which increased the specific activity of the toxin by 1.6 x 10(6)-fold. The toxin is heat labile and sensitive to proteases and has a subunit structure consisting of two subunits with molecular masses of about 58 and 40 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Agglutination of GM1-coated sheep erythrocytes by toxin suggests that GM1 might be the physiologic receptor for WO7 toxin on the enterocytes. An immunodiffusion test between the antiserum raised against the purified WO7 toxin and the purified toxin gave a well-defined precipitation band. In the immunoblot assay, two bands were observed in the 58- and 40-kDa region. At the same time, antiserum against WO7 toxin failed to show any cross-reactivity with cholera toxin or Escherichia coli heat-labile toxin (LT1) in an immunodiffusion test or immunoblot assay. The enterotoxic activity of WO7 toxin could be inhibited by antiserum against purified WO7 toxin. Our results indicate that WO7 toxin is structurally and functionally distinct from other cholera toxins and that the enterotoxic activities expressed by WO7 toxin appear to contribute to the pathogenesis of disease associated with V. cholerae O1 strains.

Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops.

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.

Evaluation of Salmonella typhimurium mutants in a model of experimental gastroenteritis.

Salmonella typhimurium strains harboring independent, defined mutations in aroA, invA, ssrA, or msbB were assessed for their ability to induce fluid accumulation, tissue damage, and local inflammation in rabbit ileal loops. Three wild-type strains of S. typhimurium, TML, HWSH, and SL1344, and two mutant strains, S. typhimurium SL1344 ssrA and S. typhimurium SL1344 msbB, consistently induced fluid accumulation in the lumen of loops and inflammation of loop-associated tissues. In contrast, three different S. typhimurium aroA strains and an invA mutant of SL1344 did not induce significant fluid accumulation in the rabbit ileal loops. However, the S. typhimurium aroA strains did induce an inflammatory infiltrate and some local villus-associated damage, but the invA mutant did not. Histologically, wild-type S. typhimurium, S. typhimurium SL1344 ssrA, and S. typhimurium SL1344 msbB demonstrated more severe effects on villus architecture than S. typhimurium aroA strains, whereas S. typhimurium invA-infected loops showed no detectable damage. This suggests that villus damage most likely contributes to fluid accumulation within the loop.

Construction of a recombinant live oral vaccine from a non-toxigenic strain of Vibrio cholerae O1 serotype Inaba biotype El Tor and assessment of its reactogenicity and immunogenicity in the rabbit model

The disease cholera is an important cause of mortality in many developing countries. Though it can be controlled through improved sanitation, this goal is not easily attainable in many countries. Development of an efficacious vaccine offers the best immediate solution. A new oral candidate vaccine has been constructed from a non-toxigenic strain of Vibrio cholerae El Tor, Inaba, which is not only devoid of the cholera toxin (CT) virulence cassette but also is completely non-reactogenic in rabbit ileal loop assay. The strain, however, had toxR and tcpA genes. Through a series of manipulations, the ctxB gene of V. cholerae, responsible for the production of the ‘B’ subunit of the cholera toxin (CTB) was introduced into the cryptic hemolysin locus of the strain. The resulting strain, named vaccine attempt 1.3 (VA1.3), was found to be able to produce copious amounts of CTB. In the RITARD model this strain was found to be non-reactogenic and provided full protection against the challenge doses of both V. cholerae O1, classical and El Tor. In the immunized rabbit it invoked significant levels of anti-bacterial and anti-toxin immunity.

Differential Induction of Cytokine mRNA by Gram Negative and Gram Positive Bacterial Infections in a Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Differential Induction of Cytokine mRNA by Gram Negative and Gram Positive Bacterial Infections in a Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Role of Bacteria and Immunosuppressive Agent in a Weanling Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Role of Bacteria and Immunosuppressive Agent in a Weanling Rabbit Ileal Loop Model of Necrotizing Enterocolitis

Ferrioxamine-mediated Iron(III) utilization by Salmonella enterica.

Utilization of ferrioxamines as sole sources of iron distinguishes Salmonella enterica serotypes Typhimurium and Enteritidis from a number of related species, including Escherichia coli. Ferrioxamine supplements have therefore been used in preenrichment and selection media to increase the bacterial growth rate while selectivity is maintained. We characterized the determinants involved in utilization of ferrioxamines B, E, and G by S. enterica serotype Typhimurium by performing siderophore cross-feeding bioassays. Transport of all three ferric siderophores across the outer membrane was dependent on the FoxA receptor encoded by the Fur-repressible foxA gene. However, only the transport of ferrioxamine G was dependent on the energy-transducing protein TonB, since growth stimulation of a tonB strain by ferrioxamines B and E was observed, albeit at lower efficiencies than in the parental strain. Transport across the inner membrane was dependent on the periplasmic binding protein-dependent ABC transporter complex comprising FhuBCD, as has been reported for other hydroxamate siderophores of enteric bacteria. The distribution of the foxA gene in the genus Salmonella, as indicated by DNA hybridization studies and correlated with the ability to utilize ferrioxamine E, was restricted to subspecies I, II, and IIIb, and this gene was absent from subspecies IIIa, IV, VI, and VII (formerly subspecies IV) and Salmonella bongori (formerly subspecies V). S. enterica serotype Typhimurium mutants with either a transposon insertion or a defined nonpolar frameshift (+2) mutation in the foxA gene were not able to utilize any of the three ferrioxamines tested. A strain carrying the nonpolar foxA mutation exhibited a significantly reduced ability to colonize rabbit ileal loops compared to the foxA+ parent. In addition, a foxA mutant was markedly attenuated in mice inoculated by either the intragastric or intravenous route. Mice inoculated with the foxA mutant were protected against subsequent challenge by the foxA+ parent strain.

Enterotoxigenicity of &lt;i&gt;Klebsiella pneumoniae &lt;/i&gt;Associated with Childhood Gastroenteritis in Madras, India

Klebsiella pneumoniae was isolated as the predominant growth from 12 of 100 children under 3 years of age suffering from acute diarrhea. Of the 12 isolates, SA1, SA2, SA4, SA5, SA6 produced a secretogenic response in the ligated rabbit ileal loop, and one isolate, SA3, induced a diarrheagenic response in suckling mice. Two isolates, SA7 and SA8, were diarrheagenic in both assays. Strains SA9, SA10, SA11, and SA12 were found to be non-enterotoxigenic. These isolates belonged to serotypes K6, 16, 25, 30, 39, 46, 49, 53, 66, and 81. All eight enterotoxigenic strains were resistant to ampicillin, streptomycin, ceftazidime, cefuroxime, and cotrimoxazole. Only quinolones such as ciprofloxacin and norfloxacin appear to be effective against enterotoxigenic K. pneumoniae.

Phenotypic and genetic features of Escherichia coli strains showing simultaneous expression of localized and diffuse adherence.

We have previously shown that some Escherichia coli strains isolated from children with diarrhea present the so-called 'localized and diffused adherence (LA/DA) pattern' in which both localized adherence (LA) and diffused adherence (DA) are expressed simultaneously. In the present study, we show that the LA adherence of these strains is genetically and phenotypically similar to that so far described for enteropathogenic E. coli (EPEC) as determined by DNA hybridization and electron microscopy. On the other hand, the DA is encoded by genes not homologous to the DAEC or AIDA-I DNA probes. In addition, the LA/DA strains are able to invade eukaryotic cells both in vitro and in vivo. In the rabbit ileal loop assay their invasion capacity goes beyond the enterocyte and reaches the muscularis mucosae as determined by transmission electron microscopy. These findings suggest that the LA/DA adherence pattern may be linked to a new E. coli virulence category which in the case of the strains studied may be associated to other virulence traits that enable them to more deeply invade the intestinal mucosa.

Phenotypic and genetic features of Escherichia coli strains showing simultaneous expression of localized and diffuse adherence

We have previously shown that some Escherichia coli strains isolated from children with diarrhea present the so-called `localized and diffused adherence (LA/DA) pattern' in which both localized adherence (LA) and diffused adherence (DA) are expressed simultaneously. In the present study, we show that the LA adherence of these strains is genetically and phenotypically similar to that so far described for enteropathogenic E. coli (EPEC) as determined by DNA hybridization and electron microscopy. On the other hand, the DA is encoded by genes not homologous to the DAEC or AIDA-I DNA probes. In addition, the LA/DA strains are able to invade eukaryotic cells both in vitro and in vivo. In the rabbit ileal loop assay their invasion capacity goes beyond the enterocyte and reaches the muscularis mucosae as determined by transmission electron microscopy. These findings suggest that the LA/DA adherence pattern may be linked to a new E. coli virulence category which in the case of the strains studied may be associated to other virulence traits that enable them to more deeply invade the intestinal mucosa.