Experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs

The enteric diseases of hares, European and cottontail rabbits, which are caused by members of the genus Clostridium are reviewed. Disease caused by C. perfringens Types A and E, C. spiroforme, C. difficile, C. sordellii, C. tympany cuniculi and clostridial enterotoxins are included. Tyzzer's disease also is discussed.     

Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin

We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C. botulinum C2 toxin component I or the ia chain of C. perfringens E iota toxin. C. spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells. When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C. spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded. The Sa cross-reacted immunologically with either the light chain of C. perfringens E iota toxin or the ADP-ribosyl transferase from C. difficile 196 strain. No immunological relatedness was observed between Sa and C2 toxin component I. C. spiroforme toxin is thus another binary toxin close to iota.     

De-ADP-ribosylation actin by Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin

The reverse reaction of the ADP-ribosylation of actin by Clostridium botulinum C2 toxin and Clostridium perfringens iota-toxin was studied. In the presence of nicotinamide (30-50 mM) C2 toxin and iota-toxin decreased the radioactive labeling of [32P]ADP-ribosylated actin and catalyzed the formation of [32P]NAD. The pH optima for both reactions were 5.5-6.0. Concomitant with the removal of ADP-ribose, the ability of actin to polymerize was restored and actin ATPase activity increased. Neither ADP-ribosylation nor removal of ADP-ribose was observed after treatment of actin with EDTA, indicating that the native structure of actin is required for both reactions. ADP-ribosylation of platelet actin by C2 toxin was reversed by iota-toxin, confirming recent reports that both toxins modify the same amino acid in actin. However, C. botulinum C2 toxin was not able to cleave ADP-ribose from skeletal muscle actin which had been incorporated by iota-toxin, corroborating the different substrate specificities of both toxins.     

Isolation of Clostridium spiroforme from rabbits

The isolation of Clostridium spiroforme from intestinal contents of rabbits was achieved by sampling the supernatant-pellet interphase of centrifuged specimens processed for routine toxin analysis. High-speed centrifugation at 20,000x for 15 minutes provided a rapid and effective means of separating this anaerobic pathogen from the majority of both indigenous and non-indigenous intestinal microbial flora. The unusual helically-coiled, semicircular shape of the microorganism is considered, at least in part, responsible for this phenomenon.     

Effects of human intravenous immune globulin on diarrhea caused by Shiga-like toxin I and Shiga-like toxin II in infant rabbits.

Shiga toxin and the related Shiga-like toxins (SLT), produced by Escherichia coli, can cause hemorrhagic colitis and hemolytic uremic syndrome (HUS). Human intravenous immune globulin (HIVIg) blocks the cytotoxicity of some SLTs in vitro. To examine the ability of HIVIg to modify disease caused by Shiga-like toxin I or Shiga-like toxin II (SLT-I or SLT-II), we injected 3-day-old rabbits intraperitoneally with SLT-containing cell-free supernatants from Escherichia coli O157: H7. A subset of rabbits was treated with subcutaneous HIVIg. All rabbits given 10(4) CD50 of SLT-I developed severe diarrhea, and 5 died. When HIVIg 500 mg/kg was given in addition to SLT-I, only 6 of 18 rabbits (33.3%) developed diarrhea (P < 0.0001), and 1 died. HIVIg 500 mg/kg or 1,000 mg/kg protected against diarrhea when given one hour prior to toxin. HIVIg 1,000 mg/kg was protective when administered one hour after toxin, but not at 6 or 24 hr. Seventeen of 18 rabbits given 10(6) CD50 of SLT-II developed severe diarrhea, and 4 died. In contrast to SLT-I-associated disease, HIVIg had no effect on diarrhea in rabbits given SLT-II. We conclude that HIVIg protects infant rabbits from diarrhea and death caused by intraperitoneally administered SLT-I, but does not affect the course of SLT-II-associated illness.     

Coprobacillus catenaformis gen. nov., sp. nov., a new genus and species isolated from human feces

Three strains of Eubacterium-like isolates from human feces were characterized by biochemical tests and 16S rDNA analysis. The phenotypic characteristics of the three strains resembled those of the genus Collinsella transferred from the genus Eubacterium recently. However, Eubacterium-like strains were phylogenetically members of the Clostridium subphylum of gram-positive bacteria, and these showed a specific phylogenetic association with Clostridium ramosum and C. spiroforme. C. ramosum and C. spiroforme are gram-positive, anaerobic, spore-forming bacteria that belong to the genus Clostridium, and the G + C contents are 26.0 and 27.4 mol%, respectively. However, the three Eubacterium-like strains had G + C contents of 32.1 to 33.1 mol% and were non-spore-forming rods. Based on phenotypic characteristics, we can differentiate these species, and furthermore, a 16S rDNA sequence divergence of greater than 9% with a new related genus, Coprobacillus, is proposed for the three strains, with one species, Coprobacillus catenaformis. The type strain of C. catenaformis is JCM 10604T.     

Intravenous Shiga toxin 2 promotes enteritis and renal injury characterized by polymorphonuclear leukocyte infiltration and thrombosis in Dutch Belted rabbits

Enterohemorrhagic Escherichia coli (EHEC) infection causes hemolytic uremic syndrome, a leading cause of acute renal failure in children. Dutch Belted (DB) rabbits are susceptible to EHEC-induced disease. Using real-time quantitative RT-PCR we measured the renal mRNA expression of cytokines and fibrinolytic factors in DB rabbits challenged with intravenous Shiga toxin 2 (Stx2) (1200 ng/kg). Group 1 rabbits received an incremental dose during an 8-day period whereas Group 2 rabbits received a single dose. Group 1 rabbits developed mild disease. In contrast, Group 2 rabbits developed severe diarrhea, higher levels of circulating polymorphonuclear leukocytes, increased mean platelet volume, and increased fibrinogen levels. Group 2 rabbits developed polymorphonuclear leukocyte infiltration in the intestine and kidney as well as glomerular congestion, luminal constriction, and mesangial glomerulonephropathy. These renal lesions were associated with up-regulation of interleukin-8 (P<0.006), plasminogen activator inhibitor-1 (P<0.04), and tissue plasminogen activator (P<0.05). Circulating Stx2 promoted dose-dependent enteritis and renal injury characterized by inflammation and impaired fibrinolysis leading to thrombosis.     

Effects of dietary carbohydrate on cholesterol metabolism in rats and rabbits.

Semipurified diets produce a marked hypercholesterolemia in rabbits and tend to elevate the level of plasma cholesterol in rats. They also decrease rates of oxidation of [26-14C]cholesterol to respiratory 14CO2 and excretion of label in fecal lipid, compared with commercial feed. In both species, the hypercholesterolemia was prevented and the rate of oxidation of [26-14C]cholesterol increased by using unmodified potato starch as the carbohydrate component of the semipurified diets. Potato starch was poorly digested by rats but appeared to be well utilized by rabbits. A semipurifed diet containing cooked potato starch gave results in rats comparable to those obtained with diets containing other types of carbohydrate. Cooked potato starch produced diarrhea in rabbits, thus complicating interpretation of the results. When the diarrhea was treated with antibiotics, the results were similar to those obtained with other carbohydrates. Rats fed commercial diet which had been heated in an oven or autoclaved had higher plasma levels than those fed untreated commercial diet but no significant differences in rates of oxidation or excretion of cholesterol were observed.     

Mechanisms of the cytopathic action of actin-ADP-ribosylating toxins

Clostridium botulinum C2 toxin, Clostridium perfringens iota toxin, and Clostridium spiroforme toxin ADP-ribosylate actin monomers. Toxin-induced ADP-ribosylation disturbs the cellular equilibrium between monomeric and polymeric actin and traps monomeric actin in its unpolymerized form, thereby depolymerizing actin filaments and destroying the microfilament network. Furthermore, the toxins ADP-ribosylate gelsolin actin complexes. These modifications may contribute to the cytopathic action of the toxins.     

Rabbit enterotoxaemia: Purification and preliminary characterisation of a toxin produced by Clostridium spiroforme

Abstract A culture filtrate of Clostridium spiroforme grown in a brain-heart-infusion peptone medium at 37°C for 24 h was concentrated by ultrafiltration, and the toxic fraction separated by ion exchange chromatography. This material was dermonecrotic in depilated guinea pigs, lethal in mice, enterotoxic in rabbit ileal loops and infant mice and cytophathic for HeLa cells. All activity was neutralised by Clostridium perfringens Type E antitoxin. Analysis by size exclusion high-performance liquid chromatography produced a single symmetrical peak corresponding to an M _r of 41 000 to 42 000.     

Ileal Loop Fluid Accumulation and Production of Diarrhea in Rabbits by Cell-free Products of Clostridium perfringens

The ability of cell extracts and culture filtrates of various strains of C. perfringens to produce ileal loop fluid accumulation and overt diarrhea in rabbits was tested. Good correlation was obtained in the ability of whole cells and a toxic factor (present in cell extracts and concentrated culture filtrates) to produce both fluid accumulation in ileal loops and diarrhea when injected into the normal ileum of the rabbit. The toxic factor was present in cell-free preparations when cells were grown in a sporulation medium, but not when they were grown in an asporogenic medium. The factor was shown to be heat labile, nondialyzable, and was inactivated by Pronase but not by trypsin, lipase, or amylase. Loss of activity occurred at pH 1.0, 3.0, 5.0, and 12.0.     

Attempts to establish experimental Cyclospora cayetanensis infection in laboratory animals

Attempts were made to develop an animal model for Cyclospora cayetanensis to identify a practical laboratory host for studying human cyclosporiasis. Oocysts collected from stool of infected humans in the United States, Haiti, Guatemala, Peru, and Nepal were held in potassium dichromate solution to allow development of sporozoites. The following animal types were inoculated: 9 strains of mice, including adult and neonatal immunocompetent and immune-deficient inbred and outbred strains, rats, sandrats, chickens, ducks, rabbits, jirds, hamsters, ferrets, pigs, dogs, owl monkeys, rhesus monkeys, and cynomolgus monkeys. Most animals were inoculated by gavage, although some of the primates were fed oocysts on food items. The animals were examined for signs of infection, particularly diarrhea, and stool samples were examined for 4-6 wk after inoculation. None of the animals developed patent infections or signs of infection. We conclude that none of the animals tested is susceptible to infection with C. cayetanensis.     

Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes. Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia.

The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail. Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers. The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl. Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner. The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6. The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific. Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride. The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore. Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane. Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them. In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin. The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.     

Use of 16S-23S rRNA spacer-region (SR)-PCR for identification of intestinal clostridia

The suitability of a species identification technique based on PCR analysis of 16S-23S rRNA spacer region (SR) polymorphism for human intestinal Clostridium species was evaluated. This SR-PCR based technique is highly reproducible and successfully differentiated the strains tested, which included 17 ATCC type strains of Clostridium and 152 human stool Clostridium isolates, at the species or intraspecies level. Ninety-eight of 152 stool isolates, including C. bifermentans, C. butyricum, C. cadaveris, C. orbiscindens, C. paraputrificum, C. pefringens, C. ramosum, C. scindens, C. spiroforme, C. symbiosum and C. tertium, were identified to species level by SR-PCR patterns that were identical to those of their corresponding ATCC type strains. The other 54 stool isolates distributed among ten SR-PCR patterns that are unique and possibly represent ten novel Clostridium species or subspecies. The species identification obtained by SR-PCR pattern analysis completely agreed with that obtained by 16S rRNA sequencing, and led to identification that clearly differed from that obtained by cellular fatty acid analysis for 23/152 strains (15%). These results indicate that SR-PCR provides an accurate and rapid molecular method for the identification of human intestinal Clostridium species.     

Rotavirus infection impairs intestinal brush-border membrane Na(+)-solute cotransport activities in young rabbits

The mechanism of rotavirus diarrhea was investigated by infecting young, specific pathogen-free, New Zealand rabbits with a lapine rotavirus, strain La/RR510. With 4-wk-old animals, virus shedding into the intestinal lumen peaked at 72 h postinfection (hpi), and a mild, watery diarrhea appeared at 124 hpi. No intestinal lesions were seen up to 144 hpi, indicating that diarrhea does not follow mucosal damage but can precede it, as if cell dysfunction were the cause, not the consequence, of the histological lesions. Kinetic analyses with brush-border membrane vesicles isolated from infected rabbits revealed strong inhibition of both Na(+)-D-glucose (SGLT1) and Na(+)-L-leucine symport activities. For both symporters, only maximum velocity decreased with time. The density of phlorizin-binding sites and SGLT1 protein antigen in the membrane remained unaffected, indicating that the virus effect on this symporter is direct. Because SGLT1 supports water reabsorption under physiological conditions, the mechanism of rotavirus diarrhea may involve a generalized inhibition of Na(+)-solute symport systems, hence, of water reabsorption. Massive water loss through the intestine may eventually overwhelm the capacity of the organ for water reabsorption, thereby helping the diarrhea to get established.     

Development of a porcine epidemic diarrhea virus M protein-based ELISA for virus detection

A membrane (M), protein-based ELISA was developed to detect porcine epidemic diarrhea virus (PEDV). The M gene of PEDV was expressed in Escherichia coli. The purified recombinant M protein was used to immunize rabbits to generate a polyclonal antibody. Immunofluorescence analysis indicated that the anti-PEDV-M antibody reacted with PEDV-infected cells. The antibody was utilized to develop an indirect ELISA to detect PEDV. Other viruses, porcine transmissible gastroenteritis coronavirus, avian infectious bronchitis coronavirus, porcine reproductive and respiratory syndrome virus, classic swine fever virus and porcine pseudorabies virus, were unreactive.     

Cytotoxicity and antimicrobial susceptibility of Clostridium difficile isolated from hospitalized children with acute diarrhea

Clostridium difficile is an important pathogen associated with outbreaks of pseudomembranous colitis and other intestinal disorders such as diarrhea. In this study, 181 stool samples from children with and without acute diarrhea were analysed. Eighteen children with acute diarrhea were positive to C. ramosum, C. difficile, C. limosum, C. clostridioforme, C. septicum, C. butyricum, C. innocuum and Clostridium sp. Nineteen children without diarrhea harbored C. ramosum, C. septicum, C. barattii, C. butyricum, C. innocuum, C. sphenoides, C. bifermentans, C. clostridioforme and C. paraputrificum. No patient with diarrhea harbored C. barattii, C. bifermentans, C. paraputrificum and C. sphenoides. In addition, ten C. difficile strains were detected in 5 (5.5%) of the children with diarrhea. Also, no children from control group harbored C. difficile, C. limosum and Clostridium sp. Most of the tested strains were resistant to all the used antimicrobial. Nine C. difficile were toxigenic on VERO cells and by multiplex PCR, six strains showed both toxin A and B genes and three strains showed only toxin B gene. In this study, the presence of C. difficile was not significant, and it is suggested the need of more studies to evaluate the role of clostridia or C. difficile play in the childhood diarrhea and these organisms must be looked for routinely and a periodic evaluation of antimicrobial susceptibility should be performed.     

The APUD-system function of the intestines and the morphological changes in the internal organs of adult rabbits infected with toxigenic Vibrio cholerae

V. cholerae infection with clearly pronounced diarrhea was reproduced in adult rabbits with the use of the RITARD system. The state of the APUD system of the intestine of the animals and morphological changes in internal organs in experimental cholera were described. As noted in this study, the manifestation of changes in the intestine and other organs, as well as the reaction of apudocytes to V. cholerae infection, were linked with the intensity of diarrhea and the time of the death of the animals.     

Antimicrobial Agent-Associated Colitis and Diarrhea

Although antimicrobial agent-associated colitis has been recognized as a clinicopathologic entity for years, the cause of this disease has been determined only recently. Virtually all cases of pseudomembranous colitis and some cases of antimicrobial agent-associated nonspecific colitis or diarrhea have been shown to be caused by a toxin of Clostridium difficile. Methods for cultivating C difficile from feces and for detecting the toxin have been developed. Oral administration of vancomycin has proved to be effective for the treatment of C difficile-induced colitis, although isolated instances of relapse after treatment have been documented.The discovery of C difficile as a human intestinal pathogen has provided an explanation for some, but not all cases of antimicrobial agent-associated diarrhea. The epidemiology, pathogenesis and means of prevention of C difficile toxin-induced diarrhea remain to be determined.     

Campylobacteriosis--a "new" disease in laboratory animals

Campylobacter fetus ss jejuni is now recognized as a leading cause of diarrhea in humans. With the development of proper culture techniques, C jejuni has been isolated from humans with diarrhea as frequently as Salmonella or Shigella species. Laboratory animal models are being developed to study the pathogenesis of campylobacter-induced diarrhea; also, identification of C jejuni is occurring more frequently in a variety of laboratory animals, whether asymptomatic carriers or those with clinical diarrhea. Animals shedding C jejuni also have been incriminated as a source of campylobacter diarrhea in humans.