Removable Intestinal Tie-adult Rabbit Diarrhea Research Articles

Studies on formulation of a combination heat killed immunogen from diarrheagenic Escherichia coli and Vibrio cholerae in RITARD model

Multiple diarrheagenic enteric bacterial infections cause global morbidity and mortality. A combination vaccine is needed to combat different diarrhea-causing organisms. In our present work, we formulated a combination of antigens from three different diarrheagenic Escherichia coli strains and three different Vibrio cholerae strains. We demonstrated that our newly formulated combination immunogen was able to raise species-specific immunogenicity. This formulation also gave protection against different diarrheagenic E. coli strains in the removable intestinal tie-adult rabbit diarrhea model. However, protective efficacy was not found against the V. cholerae El Tor Ogawa Haitian variant, but challenged with V. cholerae El Tor Inaba or O139 showed protection in rabbits.This is the first report of a single formulated nonliving heat-killed combination immunogen from different diarrheagenic E. coli and V. cholerae that could bestow protection against different bacteria in an animal model.

Development and characterization of recA mutants of Campylobacter jejuni for inclusion in attenuated vaccines.

Isogenic recA mutants of Campylobacter jejuni have been constructed for evaluation of their usefulness in attenuated vaccines against this major worldwide cause of diarrhea. The recA+ gene of C. jejuni 81-176 was cloned by using degenerate primers to conserved regions of other RecA proteins in a PCR. The C. jejuni recA+ gene encodes a predicted protein with an M(r) of 37,012 with high sequence similarity to other RecA proteins. The termination codon of the recA+ gene overlaps with the initiation codon of another open reading frame which encodes a predicted protein which has > 50% identity with the N terminus of the Escherichia coli enolase protein. A kanamycin resistance gene was inserted into the cloned recA+ gene in E. coli and returned to C. jejuni VC83 by natural transformation, resulting in allelic replacement of the wild-type recA gene. The resulting VC83 recA mutant displayed increased sensitivity to UV light and a defect in generalized recombination as determined by natural transformation frequencies. The mutated recA gene was amplified from VC83 recA by PCR, and the product was used to transfer the mutation by natural transformation into C. jejuni 81-176 and 81-116, resulting in isogenic recA mutants with phenotypes similar to VC83 recA. After oral feeding, strain 81-176 recA colonized rabbits at levels comparable to wild-type 81-176 and was capable of eliciting the same degree of protection as wild-type 81-176 against subsequent homologous challenge in the RITARD (removable intestinal tie adult rabbit diarrhea) model.

Non-O1 Vibrio cholerae intestinal pathology and invasion in the removable intestinal tie adult rabbit diarrhea model

A modified removable intestinal tie adult rabbit diarrhea (RITARD) model was used to investigate the intestinal pathology, intestinal bacterial colonization, intestinal fluid volume, and onset of diarrhea caused by non-O1 Vibrio cholerae. Three strains of non-O1 V. cholerae were studied. RITARD rabbits challenged with 10(3) CFU of strain NRT36S (a strain previously shown to cause diarrhea in volunteers) developed grade 3 diarrhea at 48 to 72 h. The mean counts of non-O1 V. cholerae isolated were 9.3 +/- 0.07 and 8.7 +/- 0.7 CFU/g from the small and large intestines, respectively. Histologic examination showed necrosis of the luminal epithelium in the colon and mild inflammatory cell infiltration in the adjacent lamina propria. The severity and extent of intestinal damage by strain NRT36S was dose dependent. Higher doses of strain NRT36S caused severe necrotizing colitis and enteritis, with bacteremia and mortality at less than 24 h in RITARD rabbits challenged with 10(9) CFU and at less than 48 h in RITARD rabbits challenged with 10(4) CFU. Electron and light microscopy demonstrated invasion of NRT36S into the luminal epithelial cells of the intestine. Challenge of RITARD rabbits with non-O1 V. cholerae A-5 and 2076-79 (strains which did not cause diarrhea in volunteers) did not cause diarrhea or intestinal pathology. Intestinal colonization was transient: at 72 h postchallenge, animals inoculated with strain A-5 were culture negative, while only low numbers of strain 2076-79 were detectable (approximately 0.4 to 0.8 CFU/g). Our data highlight the utility of the RITARD model, when combined with appropriate pathologic and bacteriologic studies, for obtaining insights into pathophysiologic mechanisms of enteric disease by non-O1 V. cholerae. In agreement with volunteer studies, non-O1 V. cholerae NRT36S is clearly pathogenic in this model; direct cell invasion may play a role in its ability to cause illness.

Efficacy of enteric-coated protease in preventing attachment of enterotoxigenic Escherichia coli and diarrheal disease in the RITARD model

In this study, we report on a novel approach based on modification of the intestinal surface to prevent diarrhea caused by enterotoxigenic Escherichia coli (ETEC). The removable intestinal tie adult rabbit diarrhea (RITARD) model was used to test the efficacy of an enteric-coated protease preparation (Detach; Enzacor Technology Pty. Ltd.) in the prevention of bacterial attachment and diarrheal disease caused by colonization factor antigen I-positive (CFA/I+) E. coli H10407. Protease was administered orally to rabbits 18 h prior to challenge with 10(11) bacteria. Four groups of rabbits were inoculated with different ETEC strains which produced different combinations of adhesin and enterotoxin or with sterile phosphate-buffered saline. Occurrence of diarrhea during the subsequent 24-h incubation period was recorded. Oral administration of protease was successful in reducing diarrhea and diarrhea-induced death in six of seven (86%) rabbits infected with CFA/I+, heat-stable and heat-labile toxin-positive E. coli (H10407). Seven of eight (87%) rabbits not protected by protease treatment died or developed severe diarrhea. Quantitative analysis of bacterial cultures obtained from the small intestine of rabbits showed a significant (P less than 0.001) 2,000-fold reduction in CFU per centimeter of intestine following treatment with protease. The efficacy of protease treatment was 99.5%, with very wide confidence limits (greater than 0 to 99.9%). The data indicate that the use of protease to prevent ETEC diarrheal disease has considerable potential.

Roles of motility and flagellar structure in pathogenicity of Vibrio cholerae: analysis of motility mutants in three animal models

Wild-type Vibrio cholerae of both El Tor and classical biotypes (strains N16961 and 395, respectively) and nonmotile mutant derivatives with and without flagellar structures were characterized in three different animal models: (i) the rabbit ileal loop, (ii) the removable intestinal tie adult rabbit diarrhea (RITARD) model, and (iii) the suckling mouse model. Both the wild-type strains and nonmotile mutants were toxinogenic in the rabbit ileal loop and the suckling mouse models. However, all of the nonmotile mutants produced significantly less fluid accumulation than did the wild-type parental strains. The two nonmotile mutants of strain N16961 did not adhere to rabbit ileal mucosa, but both nonmotile mutants derived from strain 395 exhibited adherence. In the RITARD model, the motile El Tor strains were more virulent than both the flagellate and aflagellate nonmotile mutants (all infected rabbits died within 18 h), while the nonmotile mutants, when fatalities occurred, required 78 to 105 h to produce a fatal outcome. Likewise, the motile classical parent 395 produced a fatal outcome within ca. 25 h, while nonmotile mutants required 69 to 96 h. The nonmotile flagellate strain KR31 was not significantly more virulent than the nonmotile aflagellate strain KR26. Of the two classical nonmotile mutants, KR1, which produces a coreless sheathlike structure, was clearly more virulent (5 of 10 rabbits died within 96 h), while KR3 (nonmotile, aflagellate) did not produce fatalities in any of the 10 rabbits tested. Similarly, no significant difference in diarrheagenicity or colonizing ability was detected between the two nonmotile mutants derived from the El Tor strain, but the classical nonmotile mutant with the coreless sheath caused significantly greater diarrhea and colonized for a longer time than did the isogenic nonmotile aflagellate strain, KR3. No significant differences between the nonmotile mutants were detected in competition studies done with suckling mice. Analysis of the wild-type and mutant strains in these three animal models clearly demonstrated a role for motility in V. cholerae pathogenicity, while analysis of only the nonmotile mutants derived from the classical parent suggested a role for flagellar structures.

Significance of flagella in colonization resistance of rabbits immunized with Campylobacter spp

Cross-protection among different Lior and Penner serogroups of Campylobacter spp. was studied. Rabbits were orally immunized by gastric feeding with Campylobacter spp., and 27 to 30 days later, they were challenged with matched or unmatched serogroups by the removable intestinal tie adult rabbit diarrhea (RITARD) procedure. When immunized animals were challenged with different Lior serotypes, no protection against colonization was seen; however, when challenged with homologous Lior serogroups, protection was demonstrated. Immune animals were colonized for an average of 1 day or less versus at least 6 days for nonimmune animals. Rabbits challenged with matched Penner-unmatched Lior strains showed only marginal protection. Our study also demonstrated that flagella are important in initiating colonization and eliciting protective immunity. Campylobacter coli VC167B3, an isogenic, nonflagellated mutant, did not colonize rabbits regardless of the route of administration. Single feeding of the mutant strain did not protect the host, whereas three feedings, 48 h apart, resulted in complete protection against the flagellated parent strain. When mutant strain immunized rabbits were challenged with other strains of the same Lior serotype, marginal protection was obtained. Immunogold labeling indicated that there is one or more antigens on the cell surface of the nonflagellated mutant which reacts with a polyclonal antiserum from organisms of the same Lior serogroup. These data implicated the flagellum as the cross-strain protective component of the Lior antigen complex.

Hafnia alvei, a probable cause of diarrhea in humans

Hafnia alvei, a member of the family Enterobacteriaceae, was the only species of bacteria cultured from the stool of a 9-month-old child who was admitted with a 3-day history of watery diarrhea. The isolated strain of H. alvei failed to produce heat-labile or heat-stable enterotoxins or Shiga-like toxin I or II and did not invade HeLa cells, nor did it cause keratoconjunctivitis (determined by the Sereny test) in a guinea pig's eye. The strain, however, induced diarrhea in 8 of 12 adult rabbits with removable intestinal ties (removable intestinal tie-adult rabbit diarrhea [RITARD] assay) and in 1 of 2 orally fed animals. No diarrhea could be induced with Escherichia coli K-12 in eight RITARD assay rabbits and three orally fed rabbits, respectively. Microscopic examination of affected animals revealed moderate inflammatory cellular infiltration of the intestinal mucosa, in which bacterial attachment to the surface epithelium and loss of the microvillus border were evident in the ileum and colon. Electron microscopy demonstrated cellular modifications of the apical surface, with cupping or pedestal formation and increased terminal web density at sites of bacterial "attachment-effacement," a well-known characteristic and mechanism of diarrhea of enteropathogenic E. coli. Identical lesions were also induced by H. alvei in rabbit ileal loops, which ruled out naturally occurring rabbit enteropathogenic E. coli strains, which are known to produce similar lesions. It is concluded that at least some strains of H. alvei have the potential to cause diarrhea and that attachment-effacement is a virulence characteristic shared by bacteria other than E. coli.

Modification to the RITARD Surgical Model

The Removable Intestinal Tie-Adult Rabbit Diarrhea (RITARD) procedure is the surgical model of choice for studying pathogenesis of diarrheas of Campylobacter, Escherichia coli, and Aeromonas origin. Other than nonhuman primates, the RITARD rabbit is the only model that closely resembles human infection. Classically, the RITARD model is created by isolating the terminal ileum using umbilical tape placed in a slipknot fashion, and injecting the occluded segment with the organism of choice. The slipknot is removed at 4 h postinoculation and patency of the gut reestablished. We found removal of the umbilical tape to be extremely difficult, usually requiring a second surgery to gently untie the material and prevent destruction of the fragile intestinal tissue. This complication of the described procedure appeared to be related to the absorptive and abrasive nature of the tape material. The modification described uses the same idea, but a different material and a different knot pattern. The tubing was easily removed at the prescribed 4 h postsurgery, without an additional surgery. The tubing did not adhere to or damage the gut, yet did provide the occlusive seal required for a 4-h static gut. Histologically, no evidence of pressure could be found at the tie site.

In vivo antigenic variation of Campylobacter flagellin.

Campylobacter coli VC167 cells producing either antigenic phase 1 (P1) or phase 2 (P2) flagellins (as determined by characteristic protein and DNA patterns) were used to infect rabbits by the removable intestinal tie-adult rabbit diarrhea (RITARD) procedure. Rabbits infected with P2 cells shed predominantly P2 cells throughout the infection; in rabbits infected with P1 cells, a transition of fecal isolates from P1 to P2 was observed.

Determinants of immunogenicity and mechanisms of protection by virulent and mutant Vibrio cholerae O1 in rabbits

The colonizing capacities of 16 Vibrio cholerae strains, including nine genetically and/or phenotypically defined parent-mutant pairs, were determined in unobstructed adult rabbit small bowel. There were marked interstrain differences in colonizing capacity, which was enhanced by bacterial motility and the production of cholera holotoxin but was unrelated to production of cholera toxin B-subunit or hemolysin or to bacterial serotype or biotype. The role of colonizing capacity and other bacterial features in determining the immunizing efficiency of live V. cholerae was studied by determining the efficiency with which graded inocula of each strain immunized against attempted recolonization of the ileum or induction of choleralike diarrhea by the RITARD (removable intestinal tie-adult rabbit diarrhea) challenge technique using standard inocula of virulent V. cholerae. Mucosal colonizing capacity was the only quantitative predictor of bacterial immunizing capacity; none of the other bacterial features cited above influenced bacterial immunogenicity against either type of challenge, except as they affected colonizing capacity. Live V. cholerae immunized much more efficiently than Formalin-killed bacteria; the former caused marked protection after a single inoculum of 10(2) CFU, whereas the latter gave only partial protection after three inoculations of 10(11) killed organisms. Protection induced by live bacteria was due largely to resistance to colonization and included marked inhibition of bacterial growth within the bowel lumen. These findings strongly suggest that an optimally efficient oral cholera vaccine would be composed of avirulent live V. cholerae selected for their capacity to colonize the small-bowel mucosa.

Mucosal and systemic immunity to Campylobacter jejuni in rabbits after gastric inoculation.

The mucosal and systemic immune responses to Campylobacter jejuni were studied in rabbits receiving gastric inoculation with live organisms. A lavage procedure was used to facilitate repeated monitoring of the intestinal immune response to C. jejuni. Immunity to C. jejuni was determined by secondary challenge by using the removable intestinal tie adult rabbit diarrhea (RITARD) model and monitoring for resistance to colonization and bacteremia. Oral-gastric inoculation of normal rabbits produced a transient intestinal colonization without diarrhea. C. jejuni serotypes differed in their ability to colonize the intestines of rabbits and to stimulate primary intestinal and serum antibody responses. Animals previously colonized were resistant to recolonization and the development of bacteremia after homologous challenge by the RITARD procedure but were not resistant to heterologous challenges. Anticampylobacter intestinal and serum IgA titers before this secondary infection were the most reliable predictors of resistance to colonization and bacteremia.

Vibrio cholerae infection and acquired immunity in an adult rabbit model

We modified the rabbit model for enteric infection by Vibrio cholerae developed by Spira et al. and designated the RITARD (for removable intestinal tie-adult rabbit diarrhea) model (20). Our modification DISC comprises a permanent ligation of the cecum (C) to prevent resorption of the fluid secreted by the small intestine, a temporary ligation of the small intestine (S) to enable the bacteria to colonize, and duodenal inoculation (DI) of the challenge material. The main difference between RITARD and DISC is that in the latter model the challenge material is injected into the duodenum approximately 10 cm distal to the stomach instead of into the jejunum. Four out of 5 V. cholerae strains tested, including 2 serotypes and 2 biotypes, were able to elicit a massive and usually fatal cholera-like diarrhea. The virulence depended strongly on the culturing conditions. One strain, C5, caused fatal diarrhea in a dose of about 1000 organisms, even if the temporary ligation was omitted (DIC model). Other modifications were the DIS and the DI model in which the permanent ligature of the cecum or both ligatures were omitted. Duodenal inoculation of organisms in a dose of 100 × the minimum infective dose (MID) in the DIS or DI model did not cause any disease symptom. However, such inoculations were found to cause protection against subsequent challenges with 100 × MID of homologous and heterologous organisms up to 52 weeks after duodenal inoculation. Subcutaneous injection with classical, whole cell cholera vaccine gave only partial protection of short duration. This model might contribute to the understanding of the pathogenesis of cholera as well as to the improvement of efficacy testing of cholera vaccines.

Successful colonization and immunization of adult rabbits by oral inoculation with Vibrio cholerae O1.

Adult rabbits were inoculated orally (or duodenally) with virulent Vibrio cholerae O1. Jejunal colonization occurred only when hypoperistalsis was induced at the time of inoculation by tincture of opium given intraperitoneally (or by temporary ileal obstruction). For oral inoculation, prior neutralization of gastric acid was also required. Inoculation with 10(9) V. cholerae caused jejunal colonization for 1 to 2 days and ileal colonization for 5 to 6 days. The extent of small bowel colonization 18 h after oral inoculation was related to inoculum size but also reflected limited multiplication of small inoculum sizes and net death, clearance of large inoculum sizes, or both. Serious diarrhea occurred only in rabbits fed large inoculum sizes, i.e., 10(10) V. cholerae, and then rarely. Rabbits colonized once with 10(10) V. cholerae became highly resistant to recolonization with either the same or opposite serotype. After 18 weeks, these rabbits were still partially protected, whereas twice-colonized rabbits were highly protected. Protection against recolonization appeared to be due, at least partly, to interference with the adherence of V. cholerae to the bowel mucosa, thus allowing rapid removal of V. cholerae when peristalsis resumed. Prior colonization also protected against cholera-like diarrhea in rabbits challenged by the removable intestinal tie-adult rabbit diarrhea technique, the 50% effective dose for severe or lethal diarrhea being increased more than 100-fold, and probably more than 10,000-fold, for challenge with either the homologous or heterologous serotype of V. cholerae. The described rabbit model appears well suited for the study of immunity evoked by enteric colonization with V. cholerae O1.

Kinetics of early cholera infection in the removable intestinal tie-adult rabbit diarrhea model.

The colonization of the small intestine of adult rabbits challenged with 5 X 10(7) cells of Vibrio cholerae strain Ogawa 395 has been examined in the removable intestinal tie-adult rabbit diarrhea (RITARD) model. During the first 6 h of infection, numbers of both free and adherent vibrios increased at a rate representing a generation time of about 71 min. Detectable fluid output in response to infection began at about 4 to 5 h postchallenge, and overt diarrhea was observed as early as 11 h. By 8 h after challenge, adherent V. cholerae reached a saturation concentration on the intestinal epithelium of approximately 5 X 10(8) cells per g of intestine, whereas numbers of free cells continued to increase at an exponential rate for at least 12 to 14 h. The concentration of adherent cells remained relatively constant at the saturation level during this period. This saturation level was similar in all parts of the small intestine. The concentration of adherent organisms increased significantly in moribund animals, suggesting that factors responsible for the earlier saturation equilibrium began changing as animals neared death.

Simple adult rabbit model for Vibrio cholerae and enterotoxigenic Escherichia coli diarrhea.

We developed an adult rabbit model for enteric infection by Vibrio cholerae and enterotoxigenic Escherichia coli. The cecum of each animal was first ligated to prevent it from retaining fluid secreted by the small intestine. A temporary reversible obstruction (a slip knot tie) of the small bowel was introduced at the time of challenge and removed 2 h later. With this modification, we were able to elicit a massive and usually fatal cholera-like diarrhea in adult (3.5- to 6-lb [1.6- to 2.7-kb]) animals challenged with V. cholerae. Animals challenged with enterotoxigenic E. coli also developed diarrhea which was severe and watery but less explosive and less rapidly fatal than that produced by V. cholerae. The susceptibility of animals in this model to infection by V. cholerae was similar to the susceptibility of infant rabbits challenged intraintestinally. The death rate was almost 25% when 10(3) Vibrio cells were given and 90% or more when the dose was greater than or equal to 10(6) cells per animal. We designated this procedure the RITARD (for removable intestinal tie-adult rabbit diarrhea) model.