A radioimmunoassay for parathymosin

A slide latex agglutination (SLA) assay was developed for rapid screening for Clostridium perfringens type A enterotoxin (CPE). SLA specifically detected CPE added to buffer or normal feces (sensitivity limit of 1 μg CPE/g feces). Using clinical fecal samples from C. perfringens food poisoning cases, a strong correlation was shown between (1) SLA results and results from other CPE assays and (2) between SLA results and illness status.     

Clostridium perfringens Type A Enterotoxin (CPE): More Than Just Explosive Diarrhea

Abstract

The bacterial pathogen Clostridium perfringens is the most prolific toxin-producing species within the clostridial group. The toxins are responsible for a wide variety of human and veterinary diseases, many of which are lethal. C. perfringens type A strains are also associated with one of the most common forms of food-borne illness (FBI). The toxicosis results from the production and gastrointestinal absorption of a protein-enterotoxin known as CPE. The regulation, expression, and mechanism of action of CPE has been of considerable interest as the protein is unique. CPE expression is sporulation associated, although the mechanism of cpe-gsne regulation is not fully elucidated. Cloning studies suggest the involvement of global regulators, but these have not been identified. Although very few type A strains are naturally enterotoxigenic, the cpe gene appears highly conserved. In FBI strains, cpe is chromosomally encoded; whereas in veterinary strains, cpe may be plasmid-encoded. Variation in cpe location suggests the involvement of transposable genetic element(s). CPE-like proteins are produced by some C. perfringens types C and D; and silent remnants of the cpe gene can be found in C. perfringens type E strains associated with the iota toxin gene. CPE has received attention for its biomedical importance. The toxin has been implicated in sudden infant death syndrome (SIDS) because of its superantigenic nature. CPE can destroy a wide variety of cell types both in vitro and in vivo, suggesting that it could have potential in the construction of immunotoxins to neoplastic cells. It is obvious that CPE is an interesting protein that deserves continued attention.     

Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin

Purpose: Clostridium perfringens is a significant cause of nosocomial AAD. The prevalence of C. perfringens enterotoxin (CPE)-positive stool specimens in hospitalised patients is very low in the Indian setting making the diagnostics very expensive. Therefore, a cost-effective diagnostic approach to screen faecal specimens for CPE was devised. Materials and Methods: Faecal specimens from 540 hospitalised patients with various ailments and from 340 healthy subjects were investigated for CPE. An aliquot of pooled faecal supernatants was made by mixing 100 μl each of 10 specimens to be tested. Each aliquot was investigated for the presence of CPE by an enzyme immunoassay. A repetition of the assay was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Results: Of the 540 patient specimens tested, 405 (75%) patients were on antibiotics, the predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides, etc. During the time of sampling, diarrhoea was present in 481 (89%), abdomen pain in 203 (37.6%) and fever in 242 (44.8%) patients. C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. Conclusion: Only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described herein will help to save institutional resources.     

Clostridium perfringens Type A Enterotoxin Damages the Rabbit Colon

Clostridium perfringens enterotoxin causes the gastrointestinal (GI) symptoms of C. perfringens type A food poisoning and CPE-associated non-food-borne human GI diseases. It is well established that CPE induces fluid accumulation and severe tissue damage in ligated small intestinal loops of rabbits and other animals. However, a previous study had also reported that CPE binds to rabbit colonic cells yet does not significantly affect rabbit colonic loops. To the contrary, the current study determined that treatment with 50 or 100 μg/ml of CPE causes significant histologic lesions and luminal fluid accumulation in rabbit colonic loops. Interestingly, a CPE-neutralizing monoclonal antibody blocked the development of CPE-induced histologic damage but not luminal fluid accumulation in these loops. Similar luminal fluid accumulation, without significant histologic damage, also occurred after treatment of colonic loops with heat-inactivated CPE, antibody alone, or bovine serum albumin (BSA), indicating that increased osmolarity was causing or contributing to fluid accumulation in CPE-treated colonic loops. Comparative studies revealed the similar development of histologic damage and luminal fluid accumulation in both small intestinal loops and colonic loops after as little as a 1-h treatment with 50 μg/ml of CPE. Consistent with the CPE sensitivity of the small intestine and colon, Western blotting detected CPE binding and large-complex formation in both organs. In addition, Western blotting demonstrated the presence of the high-affinity CPE receptors claudin-3 and -4 in both organs of rabbits, consistent with the observed toxin binding. Collectively, these results offer support for the possible involvement of the colon in CPE-mediated GI disease.     

Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases.     

Assessing the quality of the anaerobic environment — a method developed to support EUCAST disk diffusion of anaerobic bacteria

The purpose of this study is to establish a method for assessing the anaerobic environment for EUCAST disk diffusion antimicrobial susceptibility testing (AST) of anaerobic bacteria on fastidious anaerobe agar with 5% mechanically defibrinated horse blood (FAA-HB). The method utilizes the association between a decrease in the metronidazole disk zone diameter and increasing oxygen levels with an aerotolerant Clostridium perfringens strain DSM 25589 (CCUG 75076 and NCTC 14679). The C. perfringens strain was tested on FAA-HB with a McFarland 1 inoculum and a metronidazole 5 μg disk. FAA-HB was incubated for 16–20 h at 35–37°C. The association between oxygen levels (0, 0.16, 1, 2, and 4% oxygen) and the metronidazole zone diameter was determined. Reproducibility at 0% oxygen was investigated as part of a European multi-centre study of disk diffusion of anaerobic bacteria. The median zone diameters (n=12) at each oxygen level were 29 mm (0%), 21 mm (0.16%), 16 mm (1%), 15 mm (2%), and 15 mm (4%). The metronidazole zone diameters at 0% oxygen from the multi-centre reproducibility-study had a median of 29 mm and a 95%-percentile range of 25–33 mm (n=236). Only one reading was below 25 mm. Based on our results, a zone diameter of ≥25 mm using a metronidazole 5 μg disk and the C. perfringens strain, tested with EUCAST recommendations, can be used to indicate that the anaerobic environment is of sufficient quality for culture and disk diffusion AST. EUCAST has included the method as part of the quality control for AST of anaerobic bacteria.

     

Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens

The chorioallantoic membrane (CAM) of chicken embryo eggs is a suitable model for viral and bacterial infections. In the present study, a new approach for testing the pathogenesis and virulence of Clostridium perfringens and Eimeria tenella dual infections as a model using the CAM of embryonated chicken eggs was developed. For this purpose, 24 specific pathogen-free (SPF) embryonated chicken eggs were divided into four groups (n?=?6) and designated group E, group CP, group CPE, and NC. Sporozoites of E. tenella (20,000 sporozoites) were inoculated into 10-day-old embryonated SPF chicken eggs (groups E and CPE) via allantoic sac route. At 15-day-old, eggs of groups CP and CPE were infected with 10 ⁴  cfu C. perfringens via the same route. Assessment of pathogenicity was assessed using gross and histopathological lesions. Embryo mortality reached 17 % after mono-infection with C. perfringens and/or E. tenella and 50 % in the mixed-infected group. Lesions in the CAMs were most numerous and most severe in co-infected eggs (group CPE), reaching the maximum score of 3 in 50 % of the inoculated eggs (P?<?0.01). In Eimeria spp.-infected eggs (group E), lesions of score were between 1 and 2. Mono-infection with C. perfringens did not lead to a significant occurrence of lesions. Histopathological investigations of the CAM revealed clusters of Gram-positive bacteria, infiltration with leukocytes, lymphocytes, and developmental stages of E. tenella in the co-infected group. These data suggest that embryonated eggs could be an in ovo model for studying the pathogenesis of mixed infection with Eimeria and C. perfringens.     

Pseudorabies virus infectivity for swine skin characterized in vitro

Summary The infectivity of pseudorabies virus (PrV) was demonstrated in a cell substrate derived from swine skin explant cultures designated primary porcine skin cells (^c/cSLA PPSC).^c/cSLA PPSC infected with either wild type or TK^– PrV strain Kaplan (Ka) developed typical cytopathologic changes (CPE) as early as 4 h post inoculation (p.i.). The CPE caused by PrV on^c/cSLA PPSC was specifically neutralized by covalescent swine sera. Synthesis of late viral proteins was demonstrated in PrV-infected^c/cSLA PPSC by indirect fluorescent antibody staining using monoclonal antibodies (mAbs) specific for PrV gIII. PrV induced protein synthesis was further confirmed by specific immunoprecipitation of³⁵S-methionine labeled viral polypeptides from PrV-infected^c/cSLA PPSC with PrV convalescent swine serum, PrV immune mouse serum or mAb to PrV gIII. Moreover, the virus progeny derived from^c/cSLA PPSC was shown to be infectious for MDBK cells and this infection was specifically neutralized by PrV convalescent swine serum. The capacity^c/cSLA PPSC to support a complete growth cycle of PrV and the relative ease of deriving these cells from pigs can be applied in an autologous fashion in studies of cellular immunity where the MHC needs to be matched.     

Elimination of Angiostrongylus costaricensis larvae in feces from experimentally infected Swiss mice: circadian rhythm and correlation with survival

Angiostrongylus costaricensis is a nematode which harbors mesentery arteries of rodents. In these animals, a circadian rhythm of elimination of first-stage larvae (L1) and a relation between the amount of L1 in feces and survival are unknown. We assessed fecal elimination of A. costaricensis L1 from experimentally infected Swiss mice and tried to correlate L1 elimination with survival. Thirteen Swiss mice were infected by gavage with ten A. costaricensis L3 larvae obtained from Phyllocaulis slugs. Feces were weighed at 7 a.m. and 7 p.m. starting from the 24th day post-infection until animal death. Feces sediment was examined in microscope for L1 counting. The mice were dead after a period ranging 19–61 days post-infection. Compared to diurnal samples, both feces’ weight (2.3 ± 0.7 vs. 1.8 ± 0.5 g; P < 0.0001) and L1 total count [median 1,950 vs. 1,250; P = 0.015] were higher in feces eliminated at night. No difference was observed between diurnal and nocturnal elimination when counting L1 by gram of feces (725 vs. 650 L1/g; P = 0.821). A significant correlation was observed between survival and total number of L1 in feces (r = 0.84; P = 0.0007). This study suggests that mice experimentally infected with A. costaricensis eliminate more L1 at night due to higher fecal volume at this period. The correlation between number of L1 in feces and survival suggests a phenomenon of tolerance to A. costaricensis infection in mice with longer survival.     

Evidence That the Enterotoxin Gene Can Be Episomal in Clostridium perfringens Isolates Associated with Non-Food-Borne Human Gastrointestinal Diseases

Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C. perfringens type A food poisoning. CPE-producing C. perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genotypes of 43 cpe-positive C. perfringens isolates obtained from diverse sources. All North American and European food-poisoning isolates examined in this study were found to carry a chromosomal cpe, while all non-food-borne human GI disease isolates characterized in this study were determined to carry their cpe on an episome. Collectively, these results provide the first evidence that distinct subpopulations of cpe-positive C. perfringens isolates may be responsible for C. perfringens type A food poisoning versus CPE-associated non-food-borne human GI diseases. If these putative associations are confirmed in additional surveys, cpe RFLP and PFGE genotyping assays may facilitate the differential diagnosis of food-borne versus non-food-borne CPE-associated human GI illnesses and may also be useful epidemiologic tools for identifying reservoirs or transmission mechanisms for the subpopulations of cpe-positive isolates specifically responsible for CPE-associated food-borne versus non-food-borne human GI diseases.     

BEC,a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.     

A comparison of methods for the detection of candida antigens. Evaluation of a new latex reagent

The sensitivity of various serological techniques for the detection of C citalbicans cytoplasmic antigen (Ag) in buffer and serum diluents was compared, with special reference to variations of the ELISA method and a new microtitre latex particle agglutination (MLA) test. Of the assays evaluated, immunodiffusion, counterimmunoelectrophoresis and slide latex particle agglutination (SLA) were the least sensitive. ELISA tests were more sensitive (50–150 ng/ml Ag in buffer) although sensitivity decreased in serum or heat-inactivated serum (HIS) (250 ng/ml-4 μg/ml). The new MLA test had better sensitivity (16 ng/ml Ag in buffer) than any of the ELISA tests and was unaffected by the presence of serum or HIS (2.5 and 20 ng/ml Ag respectively). MLA seems worthy of further evaluation as an alternative to ELISA for use in antigen detection systems in general and for the serodiagnosis of systemic candidosis in particular.     

Xylanase supplementation to a wheat-based diet alleviated the intestinal mucosal barrier impairment of broiler chickens challenged by Clostridium perfringens

The present study was carried out to evaluate the protective effects of xylanase on the intestinal mucosal barrier in broiler chickens challenged with Clostridium perfringens in a 21-day experiment. A total of 336 1-day-old male broiler chicks (Ross 308) were assigned to four treatment groups. A 2×2 factorial arrangement of treatments was used in a randomized complete block design to study the effects of enzyme addition (with or without xylanase 5500 U/kg wheat-based diet), pathogen challenge (with or without C. perfringens challenge), and their interactions. Most C. perfringens-challenged birds had a congested mucosa and focal haemorrhagic lesions in the jejunum. Xylanase addition tended to reduce (P=0.09) the intestinal lesion score in the challenged birds. C. perfringens challenge resulted in decreased villus height/crypt depth ratio in the jejunum and ileum (P<0.05). Xylanase supplementation significantly increased this ratio in the jejunum (P<0.05) and also had the tendency to decrease crypt depth (P=0.065) and increase this ratio in the ileum (P=0.087). Xylanase addition significantly decreased the plasma endotoxin levels of the birds challenged with C. perfringens (P<0.05). Occludin mRNA expression in the jejunum and ileum was significantly decreased by C. perfringens challenge (P<0.05), but xylanase addition significantly increased its expression in the ileum. Xylanase supplementation also significantly increased MUC2 mRNA expression in the ileum (P<0.05). C. perfringens challenge resulted in a significant increase in apoptotic index in all three intestinal segments (P<0.05), but xylanase supplementation obviously decreased apoptotic index in the ileum (P<0.05). In conclusion, xylanase supplementation could alleviate the impairment of intestinal mucosal barrier induced by C. perfringens challenge.     

Inflammatory responses to a Clostridium perfringens type A strain and α-toxin in primary intestinal epithelial cells of chicken embryos

The causative pathogen of necrotic enteritis is the Gram-positive bacterium Clostridium perfringens. Its main cell wall component, peptidoglycan (PGN), can be recognized by Toll-like receptor 2 and nucleotide-binding oligomerization domain (NOD). Consequently, the immune response is initiated via activation of nuclear factor kappa B (NF-κB) signalling pathway. An in vitro study was conducted to investigate chicken intestinal inflammatory responses to C. perfringens type A and one of its virulence factors, α-toxin. In primary intestinal epithelial cells, C. perfringens as well as commercially available PGN and α-toxin challenge upregulated mRNA expression of interleukin (IL)-6, IL-8 and inducible nitric oxide synthase (iNOS) with a dosage-dependent manner at 3 h post infection (p.i.; P ≤ 0.001). Time-course effects of three stimulators at high concentration were further examined. C. perfringens infection elevated IL-6, IL-8 and iNOS levels from 1 h to 9 h p.i., while PGN treatment increased IL-6 and IL-8 expression at 1 h and 3 h p.i. (P < 0.05). Bacterial and PGN treatments induced NOD1 expression at 6 h p.i. and only bacterial infection boosted NF-κB p65 expression at 6 h and 9 h p.i. (P < 0.05). α-Toxin treatment upregulated IL-6 and IL-8 expression throughout infection, as well as iNOS, TNF-α and NF-κB p65 expression at later hours p.i. (P < 0.05). In conclusion, both C. perfringens and α-toxin challenge induced intense cytokine expression associated with NF-κB activation in chicken intestinal epithelial cells. The receptors for the recognition of PGN component of C. perfringens need further investigation.     

Seroprevalence ofFasciola hepatica infection in sheep in northwestern Spain

To estimate the prevalence ofFasciola hepatica infection in sheep in the León province (northwestern Spain), we conducted a survey between October 1992 and May 1993. A total of 767 samples of feces and serum were collected from sheep over 1 year of age belonging to 152 flocks randomly selected from the 4 natural regions of León province. Samples were analyzed by a standard coprological sedimentation method and an indirect enzyme-linked immunosorbent assay (ELISA) using excretory-secretory products fromF. hepatica as the antigen. The results showed the feasibility of using the indirect ELISA to facilitate the serodiagnosis of ovine fasciolosis in seroepidemiology studies (95% sensitivity and >99% specificity). No serological cross-reaction with infection by the trematodeDicrocoelium dendriticum was found. Furthermore, a statistically significant association was demonstrated between the mean flock prevalence results as determined by ELISA (77.6%) and by coprological examination (23.7%;P<0.001). Differences in the results obtained by the two diagnostic methods could have been due to fluctuations in the numbers of fluke eggs detected in feces and to the persistence of specific antibodies in serum after and efficacious fasciolicide treatment. The median number ofF. hepatica eggs detected per gram of feces was 10 (range, 5–450 eggs/g feces). The geographical distribution ofF. hepatica infection in León province was similar in all natural regions, probably due to the observation that meteorological conditions are not limiting for the maintenance of the parasite life cycle in any area of the province and to the abundance of irrigated areas together with the lack of planned control strategies. No significant association between trematode infection and sheep breed, flock size, or number of treatments was found, but the results showed a significant association between infection and untreated sheep (P<0.05).     

A Synthetic Peptide Corresponding to the Extracellular Loop 2 Region of Claudin-4 Protects against Clostridium perfringens Enterotoxin In Vitro and In Vivo

Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (_rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as _rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either _rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since _rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.     

Detection of antibodies to opioid and glutamate receptors by latex agglutination and enzyme immunoassay

We studied adsorption capacity of 5 latexes to synthetic peptide fragments of μ- and δ-opioid receptors and to GluR1 and NR2A subunits of glutamate receptor. Levels of autoantibodies to opioid receptors in the latex agglutination test and enzyme immunoassay were in good correlation. The level of autoantibodies to opioid receptors measured by these methods was increased in patients with opium narcomania, while the content of autoantibodies to the glutamate receptor subunits was increased in epileptics.__________This revised version was published online in July 2005 with the addition of the issue titleTranslated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 139, No. 1, pp. 94–97, January, 2005     

Clostridium perfringens Type E Animal Enteritis Isolates with Highly Conserved, Silent Enterotoxin Gene Sequences

Several Clostridium perfringens genotype E isolates, all associated with hemorrhagic enteritis of neonatal calves, were identified by multiplex PCR. These genotype E isolates were demonstrated to express α and ι toxins, but, despite carrying sequences for the gene (cpe) encoding C. perfringens enterotoxin (CPE), were unable to express CPE. These silent cpe sequences were shown to be highly conserved among type E isolates. However, relative to the functional cpe gene of type A isolates, these silent type E cpe sequences were found to contain nine nonsense and two frameshift mutations and to lack the initiation codon, promoters, and ribosome binding site. The type E animal enteritis isolates carrying these silent cpe sequences do not appear to be clonally related, and their silent type E cpe sequences are always located, near the ι toxin genes, on episomal DNA. These findings suggest that the highly conserved, silent cpe sequences present in most or all type E isolates may have resulted from the recent horizontal transfer of an episome, which also carries ι toxin genes, to several different type A C. perfringens isolates.     

Noncytotoxic Clostridium perfringens enterotoxin (CPE) variants localize CPE intestinal binding and demonstrate a relationship between CPE-induced cytotoxicity and enterotoxicity

Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE_168-319. Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE_168-319 binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE_168-319 was able to partially inhibit CPE-induced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.