Intestinal Loop Test Research Articles

Virulence of Cholera Toxin Gene-Positive Vibrio cholerae Non-O1/non-O139 Strains Isolated From Environmental Water in Kolkata, India.

Cholera toxin (CT)-producing Vibrio cholerae O1 and O139 cause acute diarrheal disease and are proven etiological agents of cholera epidemics and pandemics. On the other hand, V. cholerae non-O1/non-O139 are designated as non-agglutinable (NAG) vibrios and are not associated with epidemic cholera. The majority of NAG vibrios do not possess the gene for CT (ctx). In this study, we isolated three NAG strains (strains No. 1, 2, and 3) with ctx from pond water in Kolkata, India, and examined their pathogenic properties. The enterotoxicity of the three NAG strains in vivo was examined using the rabbit ileal intestinal loop test. Strain No. 1 induced the accumulation of fluid in the loop, and the volume of fluid was reduced by simultaneous administration of anti-CT antiserum into the loop. The volume of fluid in the loop caused by strains No. 2 and 3 was small and undetectable, respectively. Then, we cultured these three strains in liquid medium in vitro at two temperatures, 25°C and 37°C, and examined the amount of CT accumulated in the culture supernatant. CT was accumulated in the culture supernatant of strain No.1 when the strain was cultured at 25°C, but that was low when cultured at 37°C. The CT amount accumulated in the culture supernatants of the No. 2 and No. 3 strains was extremely low at both temperature under culture conditions examined. In order to clarify the virulence properties of these strains, genome sequences of the three strains were analyzed. The analysis showed that there was no noticeable difference among three isolates both in the genes for virulence factors and regulatory genes of ctx. However, vibrio seventh pandemic island-II (VSP-II) was retained in strain No. 1, but not in strains No. 2 or 3. Furthermore, it was revealed that the genotype of the B subunit of CT in strain No. 1 was type 1 and those of strains No. 2 and 3 were type 8. Histopathological examination showed the disappearance of villi in intestinal tissue exposed to strain No. 1. In addition, fluid accumulated in the loop due to the action of strain No. 1 had hemolytic activity. This indicated that strain No. 1 may possesses virulence factors to induce severe syndrome when the strain infects humans, and that some strains of NAG vibrio inhabiting pond water in Kolkata have already acquired virulence, which can cause illness in humans. There is a possibility that these virulent NAG vibrios, which have acquired genes encoding factors involved in virulence of V. cholerae O1, may emerge in various parts of the world and cause epidemics in the future.

Clostridium perfringens Epsilon Toxin Increases the Small Intestinal Permeability in Mice and Rats

Epsilon toxin is a potent neurotoxin produced by Clostridium perfringens types B and D, an anaerobic bacterium that causes enterotoxaemia in ruminants. In the affected animal, it causes oedema of the lungs and brain by damaging the endothelial cells, inducing physiological and morphological changes. Although it is believed to compromise the intestinal barrier, thus entering the gut vasculature, little is known about the mechanism underlying this process. This study characterizes the effects of epsilon toxin on fluid transport and bioelectrical parameters in the small intestine of mice and rats. The enteropooling and the intestinal loop tests, together with the single-pass perfusion assay and in vitro and ex vivo analysis in Ussing's chamber, were all used in combination with histological and ultrastructural analysis of mice and rat small intestine, challenged with or without C. perfringens epsilon toxin. Luminal epsilon toxin induced a time and concentration dependent intestinal fluid accumulation and fall of the transepithelial resistance. Although no evident histological changes were observed, opening of the mucosa tight junction in combination with apoptotic changes in the lamina propria were seen with transmission electron microscopy. These results indicate that C. perfringens epsilon toxin alters the intestinal permeability, predominantly by opening the mucosa tight junction, increasing its permeability to macromolecules, and inducing further degenerative changes in the lamina propria of the bowel.

Purification and characterization of a novel metalloprotease isolated from Aeromonas caviae

A novel protease produced by Aeromonas caviae was purified and characterized. The molecular weight of the protease (AP19) was estimated as 19 kDa on SDS–polyacrylamide gel electrophoresis. The protease activity was not inhibited completely by heating at 100 °C for 15 min. The proteolytic activities were inhibited by metalloprotease inhibitor. The N-terminal amino acid sequence of AP19 was VTASVSFSGRCTN. AP19 did not activate Aeromonas proaerolysin, and did not show fluid accumulation in the rabbit intestinal loop test. A high concentration of the protease showed cytotoxic activity against Vero cells.

Characterization of Vibrio cgolerae non-O1 serogroups obtained from an outbreak of diarrhea in Lima, Peru.

In February 1994, an outbreak of diarrhea caused by non-O1 Vibrio cholerae occurred among volunteers in a vaccine trial study area in Lima, Peru. Clinically, 95% of the patients presented with liquid diarrhea with either no or mild dehydration. Serogrouping of 58 isolates recovered from diarrheal patients affected in the outbreak revealed seven different serogroups, with serogroups O10 (21%) and O12 (65%) being predominant. Most of these isolates were susceptible to a variety of antimicrobial agents. None of the 58 isolates hybridized with a DNA probe previously used to detect the gene encoding the heat-stable enterotoxin NAG-ST or produced cholera toxin as assessed by GM1 ganglioside enzyme-linked immunosorbent assay. Ribotyping exhibited 10 different BglI ribotype patterns among the 58 V. cholera non-O1 strains studied. However, ribotyping showed that all isolates belonging to serogroup O12 exhibited identical ribotypes and that 83% of the serogroup O10 isolates belonged to another identical ribotype, thus showing excellent correlation between ribotypes and serogroups. Among a group of O10 and O12 isolates selected for virulence studies, none produced enterotoxin whereas the majority produced a cytotoxin, as assessed in Y1 and HeLa cells. These isolates were also negative for the gene encoding zonula occludens toxin (Zot) as assessed by a PCR assay. The isolates tested showed strong adherence and some degree of invasion in the HEp-2 cell assay, whereas none of the isolates was positive in the PCR assay for the gene encoding the toxin coregulated pilus subunit A antigen (tcpA). In the removable intestinal tie adult rabbit diarrhea model, O10 and O12 serogroup isolates produced severe diarrhea and occasionally death when rabbits were challenged with 10(10) bacterial cells. Fluid accumulation was shown in the rabbit intestinal loop test when whole cultures were injected. No significant difference in virulence was shown between serogroup O10 and O12 isolates. This study provides further evidence that V. chlorae non-O1 non-O139 strains have diarrhegenic potential for humans through a yet-undefined mechanism(s) and that such strains can cause outbreaks.

Production of heat-stable enterotoxin II by chicken clinical isolates of Escherichia coli.

Enterotoxigenic Escherichia coli isolated from diarrhea stools of chickens were examined for production of heat-stable enterotoxin II which is considered to be implicated only in diarrhea of pigs. Seven out of 38 strains examined were found to contain heat-stable enterotoxin II gene, determined by colony hybridization and the polymerase chain reaction. The culture supernatants of these strains caused fluid accumulation in the mouse intestinal loop test. This fluid accumulation activity was not lost by heating at 100 degrees C and was neutralized by anti-heat-stable enterotoxin II antiserum. Purified heat-stable enterotoxin II caused fluid accumulation in the chicken intestinal loop assay. These results indicate that STII-producing E. coli is implicated in chicken diarrhea.

Infektionen mit enteropathogenen Escherichia coli (EPEC) beim Rind

In several animals species, enteropathogenic Escherichia coli (EPEC) were described as agents causing diarrhea. The histopathogenic pattern of EPEC is due to a typical adherence to enterocytes, called "attaching and effacing" (AE). This lesions are characterized by the formation of pedestals, cups and a marked loss of microvilli on enterocytes. In view of using an "in vitro system" (HeLa-cell culture) to test the adherence of EPEC, we first tested bovine EPEC in several laboratory animals. Various strains of mice, one day chicks (peroral) and a three day old calf (ligated intestinal loops) were inoculated with a bovine pathogenic EPEC (S 102-9). The adherence of EPEC "in vivo" was histologically, electron microscopically and bacteriologically investigated and compared to adherence to HeLa cell cultures. AE-lesions were found on calf enterocytes as well as on HeLa-cells, no lesions were seen in mice and chicks. The ligated intestinal loop test seems to be a useful model to compare "in vivo" to "in vitro" adherence.

Enzyme-linked immunosorbent assay for Escherichia coli heat-stable enterotoxin type II.

The gene for Escherichia coli heat-stable enterotoxin type II (STII) was fused to the genes for protein A from Staphylococcus aureus and beta-galactosidase in two different expression systems. Antibodies raised in rabbits against the protein A-STII fusion protein recognized the beta-galactosidase-STII fusion protein. The latter fusion protein was used as the immobilized antigen in an enzyme-linked immunosorbent assay (ELISA) for detection of STII. The correlation between the results of the ELISA and the intestinal loop test in piglets was 95%, suggesting that the ELISA can be used to reliably detect STII.

Toxins, putative cell adhesins and fibronectin binding properties of Salmonella dublin.

Fifty Salmonella dublin strains isolated from cattle and human diarrhoeal cases were assayed for toxin production, haemagglutination, cell-surface hydrophobicity and fibronectin-binding properties. Most strains (65% of tested) produced cytotonic toxins and cytotoxic factors when tested on Chinese hamster ovary (CHO) cells and rabbit skin test. However, only three strains produced a skin-permeability factor as determined in pig skin intra-dermal tests. None of the strains were positive in pig intestinal loop tests. Six of the 32 strains tested for 125I-fibronectin and its 125I-29 kDa N-terminal domain binding showed 10-17% and 6-10% binding, respectively. Most of the strains expressed mannose-sensitive haemagglutination (MSHA) (76%) and high cell-surface hydrophobicity (74%) when grown at 37 degrees C. At 20 degrees C the expression of MSHA and especially the expression of high cell-surface hydrophobicity were reduced. Twelve strains grown at 37 degrees C did not haemagglutinate erythrocytes from five animal species used in this study, while six of these strains expressed high cell-surface hydrophobicity. Salmonella dublin strains isolated in Denmark appeared to express a higher frequency of fimbriae type 1 (MSHA) and a lower frequency of high cell-surface hydrophobicity than the strains from external sources.

Purification and characterization of Clostridium sordellii lethal toxin and cross-reactivity with Clostridium difficile cytotoxin.

Lethal toxin (LT) was purified from Clostridium sordellii IP82 by DEAE-Trisacryl, Ultrogel AcA3-4 gel filtration, and hydroxyapatite column chromatography. The molecular weight of purified LT was estimated to be 240,000 to 250,000, and the pI was at pH 4.55. LT was lethal for mice by intraperitoneal injection (3.4 X 10(5) mouse lethal doses per mg of protein), cytotoxic for Vero cells (6.1 X 10(4) cytotoxic units per mg of protein), erythematous and edematous by intradermal injection in guinea pigs, and induced a moderate fluid accumulation in the guinea pig intestinal loop test. The lethal activity was inactivated by N-bromosuccinimide, N-chlorosuccinimide, chloramine-T, and sodium dodecyl sulfate. The data suggest that tryptophan and methionine residues present in the toxin are important for lethal activity. Furthermore, LT was inactivated by oxidized glutathione and activated by dithiothreitol. Inactivation by sulfhydryl-group reagents 5,5'-dithiobis(2-nitrobenzoic acid) and iodoacetamide was only obtained with dithiothreitol-treated LT. Thiol groups which are protected as a disulfide bond(s) seem to be essential for the LT activity. A specific antiserum against LT neutralized the biological activities of LT and also cytotoxic activity and lethal activity of Clostridium difficile toxin B but not of C. difficile toxin A. However, this serum did not recognize antigen from C. difficile culture supernatant by immunoblotting. It was concluded that antibodies prepared from C. sordellii LT that neutralized C. difficile cytotoxic activity recognized a low number of epitopes or tertiary structures of C. difficile cytotoxin.

Comparison between enterotoxic activity and methanol solubility in heat-stable enterotoxins (STa and STb) from Escherichia coli of human, porcine and bovine origins

We have investigated the enterotoxic activity of culture filtrates and their methanol extracted fractions from 10 ST (STa or STb) producing Escherichia coli strains from human, porcine and bovine origin, in the infant mouse test (IMT) as well as in the rabbit intestinal loop test (RILT). Unconcentrated culture filtrates and methanol-soluble fractions from the eight STa-producing strains were positive in the IMT while methanol-insoluble fractions obtained from these STa-positive strains, like methanol-soluble and -insoluble fractions from the two strains producing only STb, lacked activity in the IMT. Unconcentrated culture filtrates from all ST-producing strains were unable to cause fluid accumulation in the rabbit ligated intestinal loops after 6 h incubation. When this material, concentrated 5-fold, was tested again, the culture filtrates and methanol-soluble fractions from all STa-producing strains yielded strongly positive fluid accumulation in the RILT, whereas culture filtrates and their methanol extracted fractions from the strains producing only STb, like methanolinsoluble fractions from four STa-producing strains, caused slight fluid secretion in the rabbit intestinal loops.

Intestinal and serum antibody response in gnotobiotic piglets to oral immunization with Escherichia coli

The local and systemic immune response to a formolized E. coli oral vaccine was investigated in 13 gnotobiotic piglets. Beginning at ten days of age animals received a daily dose of 10 10 or 10 11 bacteria, on ten consecutive days. Intestinal loop tests with one animal of each group on day 26 showed protection which was more pronounced in the animal dosed 10 10 bacteria compared with the other immunized piglet. Immunoglobulin class-specific antibodies to O and K antigens were determined by ELISA technique. In serum no IgG or IgA antibodies were found, whereas IgM-anti O149 antibodies in both immunized groups reached their highest level at day 4 of dosing and decreased thereafter. IgM-anti K88 antibodies were first detected at day 10 of dosing. Both immunized groups had comparable serum levels at days 20 and 30. Also in gut secretion the IgM antibody response was predominant, and higher levels were found in the 10 10 group than in the 10 11 group. IgG and IgA antibody response were also detected in secretion.

Bacteroides fragilis: a possible cause of acute diarrheal disease in newborn lambs.

Bacteroides fragilis with enterotoxin-like activity (BFEL) was isolated from the feces of 24- to 48-h-old lambs with acute diarrheal disease on three different sheep ranches in the Northern Rocky Mountain region of the United States. The lamb intestinal loop test was used to enrich for the bacterium before its initial isolation. Pure cultures of B. fragilis from the feces of diarrheic lambs caused fluid accumulation in five of five lamb intestinal loop tests and in three of three calf intestinal loop tests. Two different serogroups of BFEL were found in diarrheic lambs, and a third serogroups was found in the feces of a diarrheic ewe. Enteric disease characterized by diarrhea, depression, and inappetence occurred in three of eight newborn, colostrum-fed lambs after oral challenge inoculation with viable BFEL. One of the three lambs died 32 h after challenge. Clinical signs of disease were similar to those observed in the naturally occurring disease. B. fragilis is an obligately anaerobic bacterium found in the intestinal tract of most animals and humans. It is a serious cause of extraintestinal infection in humans. The bacterium has not been reported to cause fluid accumulation in the intestine or to cause diarrhea in any species of animal.

Cytotonic enterotoxin from Aeromonas hydrophila

Aeromonas hydrophila produces two hemolysins and an enterotoxin during growth. Enterotoxin, separated from the hemolysins, gave positive reactions in the rabbit intestinal loop test, the rabbit skin test and the adrenal Y1 cell test. Neutralization experiments in the rabbit loop, rabbit skin and Y1 cell tests failed to demonstrate any immunological relationship between Aeromonas enterotoxin and cholera toxin or Escherichia coli heat-labile enterotoxin. Prior incubation of Aeromonas enterotoxin with gangliosides did not inhibit the positive test results in these systems. A co-agglutination test with antiserum to purified cholera toxin was negative for Aeromonas enterotoxin, which therefore seems to be immunologically distinct from cholera toxin. The Aeromonas enterotoxin induced steroid secretion in adrenal Y1 cells and increased the intracellular cyclic adenosine monophosphate (cAMP) content of Y1 cells as well as of rabbit intestinal epithelial cells. It thus seems to act via the adenylate cyclase-cAMP pathway and should be classified as a cytotonic enterotoxin according to the classification of Keusch and Donta (1975).

Separation and characterization of enterotoxin and two haemolysins from Aeromonas hydrophila.

Aeromonas hydrophila produces two haemolysins, now designated alpha- and beta-haemolysin (formerly aerolysin or cytotoxic protein), and one enterotoxin. These toxins were separated and purified by isoelectric focusing and gel chromatography. Alpha- and beta-haemolysin differ with regard to prerequisites for their elaboration, such as selection of A. hydrophila strains and temperature and time for cultivation. The nature of their lytic effects on erythrocytes and toxic effects on tissue culture cells differs significantly. Alpha- but not beta-haemolysin was inactivated by reducing agents and activated by oxygen, while beta-haemolysin was more resistant than alpha-haemolysin to proteolytic enzymes but was inactivated by crude gangliosides. When separated from the two haemolysins, the enterotoxin gave positive reactions in the rabbit intestinal loop test, the rabbit skin and the adrenal Y1 cell test, though the sensitivity of the Y1 cell was low as compared with cholera toxin and E. coli LT toxin. No cytotoxic effects were obtained in HeLa cells.

Purification and partial characterization of heat-stable enterotoxin of enterotoxigenic Escherichia coli

Heat-stable enterotoxin was purified from a strain of enterotoxigenic Escherichia coli 53402 A-1 from human intestine. The cells were cultured in Casamino Acids-yeast extract-salts medium, and the purification procedure consisted of protamine sulfate treatment of the culture supernatant, ultrafiltration with an Amicon PM-10 membrane, diethylaminoethyl-cellulose column chromatography, hydroxyapatite column chromatography, Bio-Gel P-10 gel filtration, 90% ethanol extraction, and preparative polyacrylamide gel disc electrophoresis. About 300-fold purification was achieved, with a yield of about 12%. However, the homogeneity of the purified heat-stable enterotoxin was not rigorously demonstrated. The purified heat-stable enterotoxin had an absorption maximum at about 275 nm, and its isoelectric point was about 3.90. The molecular weight of the purified heat-stable enterotoxin was ca. 4,000 by Sephadex G-100 gel filtration. The minimum effective dose of purified heat-stable enterotoxin was about 2.5 ng in the suckling mouse assay. The purified heat-stable enterotoxin gave a positive reaction in not only the suckling mouse assay but also the mouse intestinal loop test and the guinea pig skin permeability test.

Fluid accumulation in mouse ligated intestine inoculated with Clostridium perfringens enterotoxin

Clostridium perfringens enterotoxin, when inoculated into the ligated intestinal loop of mice, caused marked distension due to fluid accumulation. The increase in weight of the intestinal loop was proportional to the log dose of enterotoxin within a range from 1 to 16 micrograms. The fluid accumulation was arrested by washing the loop with saline or by injection of the specific anti-enterotoxin serum into the loop 5 or even 30 min after inoculation of the enterotoxin. A significant increase in weight of the loop was found as early as 10 min after inoculation of the toxin. These results may suggest that entergotoxin is neither bound firmly to the mucosal membrane nor permeates into the cells of the intestinal wall. The mouse intestinal loop test is economical, simple to perform, and applicable for quantitative determination of the enteropathogenic activity of C. perfringens enterotoxin.

Increased Escherichia coli enterotoxin detection after concentrating culture supernatants: possible new enterotoxin detectable in dogs but not in infant mice

The heat-stable enterotoxin (ST) of Escherichia coli can be detected by infant mouse or dog intestinal loop tests. These tests differ in that the dog assay uses concentrated culture supernatants and is based on measurements of net intestinal absorption, whereas the mouse test uses unconcentrated supernatants and depends on gross fluid accumulation. To compare the relative sensitivities of these assays, culture supernatants of randomly selected E. coli isolates from 34 Bangalee diarrhea patients were tested for ST in dog loops and infant mice. Supernatants were also tested for heat-labile enterotoxin (LT) in dog loops, Y-1 adrenal cells, and Chinese hamster ovary cells. E. coli supernatants that produced positive responses for both ST and LT in the dog loop assay (ST+/LT+) also produced positive responses when tested for ST in infant mice and for LT in cell lines. Supernatants of strains negative for ST and LT in dog loop (ST-/LT) were also negative in other assays. Of 10 strains positive for just ST in the dog loop test (ST+/LT-), only 5 were ST positive in the standard infant mouse test. Supernatants of the other five strains (dog loop positive, mouse test negative) were then concentrated 100-fold and retested in mice. Three of these five gave consistently positive results after concentration, and two were only intermittently positive. Concentrated supernatants of negative control strains (ST-/LT-) were all negative in mice. The dog assay detects more strains producing ST than the infant mouse test. The infant mouse test, which detects only gross fluid accumulation, failed to detect approximately half of the 10 strains which produced ST alone (ST+/LT-; P = 0.025). Concentrating supernatants for the mouse assay increases sensitivity for detection of ST, but certain E. coli strains produce a variety of ST to which infant mice do not respond.

Heat-labile enterotoxins of Escherichia coli

Heat-labile high molecular weight enterotoxin from porcine and human strains of Escherichia coli has been purified in several laboratories in recent years. Data reported concerning enterotoxin purified from cell lysates, cell envelope-associated toxin and toxin in the culture fluids are different and do not seem reproducible from one laboratory to another, due to at least two facts: (1) aggregation of enterotoxin with high molecular weight material such as endotoxin (ii) activation of protoxin by bacterial protease. Conventional methods such as ion exchange and molecular sieve chromatography did not yield toxin of high purity while preparative isotachophoresis and isoelectric focusing in density gradiants and granulated gels were used with increased recovery of purified enterotoxin. The toxin was found to be heterogeneous concerning charge properties and focused in pH ranges 4·8–5·5 and 7·2–7·9. Sepharose 2B chromatography resolved two enterotoxins with different specific activities in the intestinal loop test and the adrenal cell test. The relationship of these preparations of partially purified toxin to enterotoxins with different relative activities in the rabbit intestinal loop and skin test, different cell tests and the adenylate cyclase test is discussed in relation to the subunit composition of cholera toxin which has a similar mode of action, but different chemical composition despite immunological cross-reaction between the cholera and E. coli enterotoxins.

Further studies on the development of a live oral cholera vaccine.

SUMMARYThe possibility of developing a living oral cholera vaccine with naturally avirulent El Tor cultures isolated in a Middle East country or from water sources in Calcutta in the absence of cholera El Tor in these areas has been further studied.In an extended series of experiments the markedly low pathogenicity of the proposed vaccine strains in laboratory animals has been confirmed. The vaccine strains have been shown to get established and multiply regularly in the ligated intestinal loops of adult rabbits and in the intestine of infant rabbits without producing pathogenic reactions.The apathogenic character of the vaccine strains has been found to be stable. When propagated serially in these two laboratory models of experimental cholera the vaccine strains show no enhancement of pathogenicity.Intra-intestinal administration of the live vaccine has been shown to protect adult rabbits fully in the intestinal loop test. Immunized animals were also protected against challenge with V. cholerae strains, though to a somewhat lesser extent. Protective immunity could not, however, be demonstrated in infant rabbits probably because of the immunity-forming mechanism being still rudimentary.Immunization with live vaccine was found to inhibit the growth of homologous strains in the ligated intestinal loop of the adult rabbit. The growth rate of pathogenic V. eltor and V. cholerae strains was also seen to be markedly reduced throughout the 24 hr. period of observation, but more markedly during the first 4–8 hr. However, the total count of pathogenic V. eltor and V. cholerae strains at 24 hr. after inoculation in ligated ileal loops of immunized rabbits reached levels that invariably caused inflammation and accumulation of fluid in normal rabbits' loops. The absence of gut-inflammatory reaction in immunized rabbits under these conditions has been discussed in relation to the nature of the immunity produced by administration of live vaccine.