Ligated Rabbit Ileal Loops Research Articles

Alterations in the pathogenicity of Escherichia coli K-12 after transfer of plasmid and chromosomal genes from Shigella flexneri.

A 140-megadalton plasmid (pWR110), which has previously been associated with virulence in Shigella flexneri, was transferred to Escherichia coli K-12. Segments of S. flexneri chromosomal material were then transferred to the plamid-bearing K-12 strains. The virulence of these transconjugant hybrids was assessed in the HeLa cell model, in ligated rabbit ileal loops, or in the Sereny test. A K-12 strain which harbored only pWR110 invaded HeLa cells, produced minimal lesions in the rabbit ileal mucosa, and was negative in the Sereny test. Plasmid-containing K-12 hybrids which had incorporated various shigella chromosomal regions gave differential reactions in the rabbit ileal loops and in the Sereny test. Analysis of these transconjugants indicated that three regions were linked with virulent phenotypes. These included the his region (when the genes responsible for O-antigen synthesis were cotransferred) and the kcp locus (linked to the lac-gal region). Either of these chromosomal regions was sufficient to allow invasion of the rabbit ileal mucosa. In addition to both of these regions, another shigella chromosomal segment linked to the arg and mtl loci was necessary for fluid production in the rabbit ileal loop and for a positive Sereny reaction. Thus, derivatives of an E. coli K-12 strain, constructed by the stepwise conjugal transfer of a large plasmid and three chromosomal segments from S. flexneri, appeared to contain the necessary determinants for full pathogenicity in a variety of laboratory models.

Characterization of Deoxyribonuclease-Positive Enterococci Isolated from Milk and Milk Products

Five hundred and ten isolates of enterococci recovered from milk and milk products were screened for deoxyribonuclease (DNase) production. Of the 166 (32.5%) DNase-positive cultures, 29 (5.7%) produced thermonuclease that resisted boiling for 15 min. Although the incidence of thermolabile DNase-positive enterococci was 50.5% in Cheddar cheese, the majority of thermonuclease-producing enterococci was recovered from dried milks and infant foods. On the basis of biochemical, physiological and serological tests, all DNase-producing enterococci were characterized as S. faecalis var. faecalis (21), S. faecalis var. zymogenes (9), S. faecalis var. liquefaciens (90), S. faecium (23) and S. durans (16). The predominant thermonuclease-positive type was S. faecalis var. faecalis followed by S. faecalis var. zymogenes. Six strains of enterococci were found to be toxigenic when tested in ligated rabbit ileal loops, infant mice and rabbit skin.

Lincomycin increases synthetic rate and periplasmic pool size for cholera toxin.

Increased enterotoxigenicity of Vibrio cholerae 569B grown with low concentrations of lincomycin, previously described in terms of increased extracellular biological activity (capillary permeability factor and fluid accumulation in ligated rabbit ileal loops), was further characterized. Polyacrylamide gel electrophoresis and single radial immunodiffusion showed that lincomycin-stimulated cells produced increased molar quantities of cholera toxin (CT) both extra- and intracellularly. The intracellular CT was released in comparable amounts by sonication, deoxycholate extraction, and polymyxin B treatment. Polymyxin B release of CT was nearly complete under conditions wherein only 6% of total cellular beta-galactosidase was released, implying a periplasmic pool of CT in stimulated cells. No intracellular choleragenoid (CT subunit B) was found in stimulated cells by polymyxin B release. No proteolysis of 14C-labeled CT was detected after prolonged incubation with sonicated nonstimulated cultures or sonicated concentrated cells. These data support the conclusion that the stimulatory effect of lincomycin involves an increase in the rate of synthesis of the CT molecule, and argue against alternative models involving inhibition of putative normal degradation of CT, increased release of otherwise cell-bound CT, or activation of inactive, or less active, forms of CT.

Mechanism of action of Yersinia enterocolitica enterotoxin.

Enterotoxin derived from three clinical isolates of Yersinia enterocolitica was compared with the heat-stable enterotoxin of Escherichia coli. Both toxins were biologically active in infant mice examined at 2 h and in ligated rabbit ileal loops at 6 h. Neither substance, however, produced changes in ligated ileal loops at 18 h or in Chinese hamster ovary or Y1 adrenal tissue cultures. In addition, both Y. enterocolitica enterotoxin concentrated approximately 20 times by ammonium sulfate precipitation and ultrafiltration and a similarly prepared sample of E. coli heat-stable enterotoxin stimulated the activity of guanylate cyclase but not that of adenylate cyclase in infant mouse intestine. These findings suggest that the role of enterotoxin in the pathogenesis of intestinal Y.enterocolitica infection may be similar to that of heat-stable enterotoxin in E. coli diarrhea.

Partial Purification of a Toxin Found in Hamsters with Antibiotic-Associated Colitis

A toxin with cytotoxic and enterotoxic activities was isolated from cecal contents of hamsters receiving lincomycin. The toxin was partially purified by ultracentrifugation, ultrafiltration, (NH4)2SO4 precipitation, and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa cells, was restricted to material that eluted in the molecular weight range of 107,000 +/- 6,000 daltons. Cytotoxicity of crude AAC toxin could be demonstrated at concentrations as low as 0.04 microgram/ml. The toxin was heat labile (55 degrees-60 degrees C for 0.5 hr) and sensitive to trypsinization, acidification at pH 3, or alkalinization at pH 9. Cytotoxic activity was inhibited by Clostridium sordellii antitoxin. Enterotoxic activity of the crude toxin and the cytotoxic fraction from gel filtration was demonstrated by fluid secretion in ligated rabbit ileal loops. Studies were done in vitro with cholestyramine resin, vancomycin, or gentamicin to determine if the toxin was bound or denatured by these drugs. It was demonstrated that cholestyramine bound the toxin, significantly reducing its cytotoxicity. Reversible binding of the cytotoxic material was demonstrated by salt gradient elution. Neither vancomycin nor gentamicin had any effect on the in vitro cytotoxic activity of the toxin.

Isolation of a Cytotoxin-Enterotoxin from Entamoeba histolytica

A heat-labile material with cytotoxic and enterotoxic activities was isolated from axenically cultivated Entamoeba histolytica. The cytotoxin-enterotoxin was partially purified from the particulate-free supernatant of sonicated trophozoites by ammonium sulfate precipitation and gel filtration. Cytotoxic activity, assayed on monolayers of HeLa or BHK-21 cells, was restricted to proteins that eluted in the molecular weight range of 25,000--35,000 daltons. Cytotoxicity was demonstrated at protein concentrations as low as 2--4 microgram/ml, was heat-labile (75 C, 30 min), and was inhibited by specific immune IgG and by an undetermined factor in nonimmune serum. Enterotoxic activity of the partially purified toxin was demonstrated by induction of fluid secretion in ligated rabbit ileal loops. The cytotoxin-enterotoxin of E. histolytica may play an important role in the production of diarrhea and mucosal injury in amoebic colitis.

Identification of a novel enterotoxigenic activity associated with Bacillus cereus.

A strain of Bacillus cereus isolated from a food poisoning outbreak characterized by vomiting has been shown to be capable of causing vomiting when cultures grown on rice, but not other media, were fed to Rhesus monkeys. In contrast, a strain isolated from a diarrhoeal outbreak produced diarrhoea, but not vomiting, when grown on various media in similar feeding trials. Furthermore, culture filtrates from the diarrhoeal strain caused fluid accumulation in ligated rabbit ileal loops whereas those from the vomiting strain did not. It is proposed that at least two enterotoxins are involved, one responsible for the vomiting and one for the diarrhoeal symptoms.

An outbreak of Bacillus cereus food poisoning resulting from contaminated vegetable sprouts.

In an outbreak of gastrointestinal illness caused by consumption of home-grown raw vegetable sprouts contaminated by Bacillus cereus, victims developed symptoms after an incubation period of 6-15 hours. Four persons initially experienced nausea and vomiting, and this was followed in 3 cases by abdominal cramps and diarrhea. Bacteriologic investigation indicated that B. cereus on unsprouted seeds proliferated during germination in a commercially sold seed sprouting kit and reached levels in excess of 10(7) per gram. B. cereus isolated from the incriminated sprouts exhibited enterotoxigenic activity when tested by the ligated rabbit ileal loop technique, the dermal reaction in guinea pigs, and the rabbit skin capillary permeability test. The diversity of symptoms and incubation periods attributed to B. cereus requires analysis for this often overlooked organism whenever food-borne gastroenteritis is suspected.

Genetic basis of toxin production and pathogenesis in Vibrio cholerae: evidence against phage conversion.

The pathogenicity of Vibrio cholerae strains "cured" of "Kappa-type" phage was not significantly altered relative to that of their "Kappa" lysogenic parental strains. Unlike Corynebacterium diphtheriae, the capacity of V. cholerae to produce exotoxin was not stimulated as a consequence of active phage multiplication. Toxin production in cultures in which Kappa-type phage multiplication was initiated either by inducing Kappa lysogens or by infecting naturally occurring or "cured" Kappa-sensitive strains was greatly reduced compared to normally growing control cultures. Kappa-sensitive El Tor strain Mak 757 and a Kappa lysogen derived from it did not differ in their capacity to colonize ligated rabbit ileal loops nor in their sensitivites to ultraviolet radiation, acidic pH, or osmotic shock. We conclude that Kappa-type phages do not directly affect the pathogenicity of these V. cholerae strains.

Pathogenesis of salmonellosis. Studies of fluid secretion, mucosal invasion, and morphologic reaction in the rabbit ileum.

Strains of Salmonella typhimurium were studied in the ligated rabbit ileal loop model to gain insight into the mechanisms whereby bacteria which invade the gastrointestinal mucosa evoke fluid exsorption. The organisms employed differed in various biologic attributes including the ability to invade the ileal epithelium, multiply within the mucosa, elicit an acute inflammatory reaction, and disseminate across the intestinal wall. Some strains provoked small intestinal fluid exsorption although these did not elaborate enterotoxin. Only those strains which invaded the mucosa were accompanied by either mucosal inflammation or fluid exsorption. Noninvasive strains produced neither histologic abnormalities nor fluid secretion. While strains which invaded the mucosa caused an acute inflammatory reaction, not all such strains evoked fluid secretion. Furthermore, there was no correlation in ability of invasive organisms to evoke fluid secretion or in the intensity of mucosal inflammation, number of intramucosal salmonellae, or in ability to disseminate from the rabbit ileum. These observations suggest that, as is the case in shigellosis, mucosal invasion may be a necessary factor for the intestinal fluid loss in salmonellosis. A bacterial property or factor, in addition to invasion of the gastrointestinal mucosa, seems to be responsible for fluid exsorptin. However, it is unlikely that a salmonella enterotoxin comparable to that elaborated by Vibrio cholerae, toxigenic Escherichia coli, or Shigella dysenteriae 1 is related to fluid secretion in salmonellosis.

Bacillus cereus-induced fluid accumulation in rabbit ileal loops.

The usefulness of the ligated rabbit ileal loop as an experimental model of Bacillus cereus food poisoning was investigated. Positive responses, as measured by fluid accumulation in the loop, were obtained from 19 of 22 strains of B. cereus. Four of six strains of B. thuringiensis also elicited fluid accumulation, but eight strains of other Bacillus spp. failed to evoke a response. The growth medium employed markedly affected the ability of a given strain of B. cereus to provoke a response. Brain heart infusion broth (BHI) (Difco) proved to be best for this purpose. Loop fluid-inducing activity was produced by exponentially growing cells and was present in cell-free culture filtrates and associated with washed vegetative cells. Intraluminal growth of B. cereus did not elicit fluid accumulation. Cultures grown at temperatures in the range of 18 C to 43 C were loop active. When BHI cultures of selected loop positive strains were injected intraluminally into the normal ileum of rabbits, they failed to elicit diarrhea.

Bacillus cereus-Induced Fluid Accumulation in Rabbit Ileal Loops

The usefulness of the ligated rabbit ileal loop as an experimental model of Bacillus cereus food poisoning was investigated. Positive responses, as measured by fluid accumulation in the loop, were obtained from 19 of 22 strains of B. cereus. Four of six strains of B. thuringiensis also elicited fluid accumulation, but eight strains of other Bacillus spp. failed to evoke a response. The growth medium employed markedly affected the ability of a given strain of B. cereus to provoke a response. Brain heart infusion broth (BHI) (Difco) proved to be best for this purpose. Loop fluid-inducing activity was produced by exponentially growing cells and was present in cell-free culture filtrates and associated with washed vegetative cells. Intraluminal growth of B. cereus did not elicit fluid accumulation. Cultures grown at temperatures in the range of 18 C to 43 C were loop active. When BHI cultures of selected loop positive strains were injected intraluminally into the normal ileum of rabbits, they failed to elicit diarrhea.