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.     

Response of ligated rabbit ileal loop to Clostridium perfringens type C strains and their toxic filtrates

The vegetative cells and toxic filtrates of Clostridium perfringens type C strains were injected into ligated rabbit ileal loops and the responses were observed. Out of 12 strains examined, 2 strains showed positive reaction in this test, when the vegetative cells were injected. One of these 2 strains was an enterotoxigenic and beta-toxigenic and the other was beta- and delta-toxigenic but not enterotoxigenic. Culture filtrates containing beta or delta toxin also showed fluid accumulation in the rabbit ileal loop. Histological findings of loops injected with culture filtrates containing beta toxin showed separated and effaced villi, hemorrhage in the mucosa, engorged vessels, inflammatory cell infiltration, and hyperplasia of the lymphoid tissue.     

Inactivation of the gene (cpe ) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C. perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops

Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C. perfringens type A isolates, including C. perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea. To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C. perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C. perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene). Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene. When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects. However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression. Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C. perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C. perfringens type A isolates.     

Scaled-up production and purification of Clostridium perfringens type A enterotoxin

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38.8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119.3 micrograms/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty micrograms of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.     

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.     

Scaled-up production and purification of Clostridium perfringens type A enterotoxin

Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism. Rapid, efficient harvesting of 40 1 batch culture of Cl. perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system. Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell. The overall yield of purified enterotoxin was 38·8%. The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate. These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100. Purified monomer enterotoxin had biological activities of 119·3 μ g/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty μg of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.     

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.     

Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D.

To obtain high yields of extracellular enzymes and toxins for immunological analysis, type culture collection strains of Clostridium perfringens types A to D and 28 fresh isolates of C. perfringens type A from humans were grown in fermenters under controlled conditions in a pre-reduced proteose peptone medium. The type culture collection strains all showed different characteristics with respect to growth rates and pH optima for growth. Production of phospholipase C (alpha-toxin), haemolysin and lethal activity varied considerably between the different types. Growth and extracellular protein production in fermenters with pH control and static or stirred cultures were compared. Production of all extracellular proteins measured was markedly improved by cultivation in fermenters with pH control. Strain ATCC13124 produced five times more phospholipase C than any of 28 freshly isolated strains of C. perfringens type A, grown under identical conditions. Haemolytic and lethal activities of the ATCC strain were equal or superior to the activities of any of the freshly isolated strains. There were no differences in the bacterial yields and in the production of extracellular toxins between type A strains isolated from clinical cases of gas gangrene and abdominal wounds, and those isolated from faecal samples from healthy persons.     

Isolation and characterization of a cytolysin produced by Vibrio cholerae serogroup non-O1

A thermolabile toxin (molecular weight, 52 711; isoelectric point, 8.65) produced by a clinical isolate of Vibrio cholerae serogroup non-O1 was cytotoxic for Y-1 mouse adrenal cells and Chinese hamster ovary cells. The toxin lysed rabbit red blood cells and produced a hemorrhagic zone in rabbit skin. When injected intravenously into adult mice, the cytolysin was rapidly lethal and caused fluid accumulation in both 5- and 18-h rabbit ileal loops. Strains of V. cholerae that produced cytolysin but no cholerae enterotoxin were able to cause fluid accumulation in rabbit intestinal loops.     

A型产气荚膜梭菌经小鼠传代的毒力变化

目的:探讨A型产气荚膜梭菌经小鼠腹腔传代后的毒力变化。方法:选择10只小鼠,腹腔注射浓度为5.0×108 cfu/mL的A型产气荚膜梭菌菌液1 mL,记录第一代小鼠存活时间;收集第一代死亡最早的小鼠腹腔内的细菌,经血平板厌氧培养24 h后配制5.0×108 cfu/mL浓度的菌液,并腹腔注射10只小鼠,每只1 mL,记录第二代小鼠存活时间;重复实验并记录第三代、第四代小鼠的存活时间;死亡小鼠腹腔收集的细菌以革兰染色涂片镜检,血平板培养,用API 20A试剂条进行生化鉴定;用SPSS 13.0软件分析每代小鼠的存活时间组间差异。结果:小鼠腹腔收集的标本经革兰染色后均可见两端钝圆棒状的革兰阳性杆菌,血平板厌氧培养均为边缘呈锯齿状有双溶血环的较大菌落,API 20A的结果为产气荚膜梭菌;小鼠存活时间随小鼠腹腔传代数不断延长,第一代与第二代、第三代、第四代之间的存活时间差异有统计学意义,第二代、第三代与第四代两两之间的存活时间差异无统计学差异。结论:A型产气荚膜梭菌经小鼠腹腔第一次传代后毒力显著减弱,随后传代中毒力基本不变,与传统的细菌经动物腹腔培养后毒力增强的观点不一致。     

Rabbit Ileal Loop Response to Strains of Clostridium perfringens

The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops.     

Cooperativity of peptidoglycan synthases active in bacterial cell elongation

Clostridium perfringens possesses at least two functional quorum sensing (QS) systems, i.e. an Agr-like system and a LuxS-dependent AI-2 system. Both of those QS systems can reportedly control in vitro toxin production by C. perfringens but their importance for virulence has not been evaluated. Therefore, the current study assessed whether these QS systems might regulate the pathogenicity of CN3685, a C. perfringens type C strain. Since type C isolates cause both haemorrhagic necrotic enteritis and fatal enterotoxemias (where toxins produced in the intestines are absorbed into the circulation to target other internal organs), the ability of isogenic agrB or luxS mutants to cause necrotizing enteritis in rabbit small intestinal loops or enterotoxemic lethality in mice was evaluated. Results obtained strongly suggest that the Agr-like QS system, but not the LuxS-dependent AI-2 QS system, is required for CN3685 to cause haemorrhagic necrotizing enteritis, apparently because the Agr-like system regulates the production of beta toxin, which is essential for causing this pathology. The Agr-like system, but not the LuxS-mediated AI-2 system, was also important for CN3685 to cause fatal enterotoxemia. These results provide the first direct evidence supporting a role for any QS system in clostridial infections.     

An investigation of the prevalence of the toxigenic types of Clostridium perfringens in horses with anterior enteritis: preliminary results

Equine anterior enteritis is an acute syndrome with unknown aetiology, although salmonellosis and infection with Clostridium perfringens have both been suggested as potential causes. The main aim of this preliminary study was to compare the prevalence of toxigenic types of C. perfringens in clinically healthy horses and in horses with anterior enteritis. From horses admitted with colic at Phillip Leverhulme Large Animal Hospital in 1995-1996, samples of gastric reflux, small intestinal contents and faeces were taken for isolation of C. perfringens. Five of those horses were admitted as anterior enteritis cases, of which C. perfringens was isolated in pure culture in all five horses. Two of the anterior enteritis cases from which viable bacterial counts had been performed revealed 10(6) CFU/g faeces C. perfringens. Samples of gastric reflux and small intestinal contents submitted from one of these horses revealed 10(4) CFU/mL and 10(5) CFU/mL respectively. The number of C. perfringens observed in the gastric reflux was considered significant as the total volume removed was 12 L. The counts observed in faeces taken from horses admitted with anterior enteritis, were significantly higher than the <10(2) CFU/g faeces observed in faeces from healthy horses and horse presenting with colic and with other diagnoses. The major toxigenic types of C. perfringens in both healthy and diseased horses are being investigated using the polymerase chain reaction (PCR) to amplify target DNA sequences of the toxin genes. Primers have been designed from the published DNA sequences of the enterotoxin, alpha, beta, epsilon and iota toxin genes. PCR products obtained from NCTC strains of C. perfringens have been cloned and the sequenced, to verify that the amplicon sequence is correct. Initial typing suggests that C. perfringens type A is the predominant toxin type isolated from healthy horses and horses with colic with other diagnoses.C. perfringens strains isolated from horses with anterior enteritis are of type D.     

Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model

Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.     

Assay Methods for Clostridium perfringens Type A Enterotoxin

Enterotoxin produced by a sporulating culture of Clostridium perfringens type A NCTC 8798 was purified to a level of 3,500 mouse mean lethal doses per mg of nitrogen. High-titer sera were obtained from rabbits injected with enterotoxin and used to compare the sensitivity of serological tests and bioassays for C. perfringens enterotoxin. Reversed passive hemagglutination was by far the most sensitive test, followed by microslide diffusion, single gel diffusion and electroimmunodiffusion, guinea pig skin test, mouse test, and rabbit ileal loop test.     

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.     

Diagnosis of Clostridium perfringens intestinal infections in sheep and goats

Clostridium perfringens produces disease in sheep, goats and other animal species, most of which are generically called enterotoxemias. This micro-organism can be a normal inhabitant of the intestine of most animal species including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates in large numbers and produces several potent toxins that are absorbed into the general circulation or act locally with usually devastating effects on the host. History, clinical signs and gross post-mortem findings are useful tools for establishing a presumptive diagnosis of enterotoxaemia by C. perfringens in sheep and goats, although no definitive diagnosis of these diseases can be made without laboratory confirmation. Because all types of C. perfringens can be normal inhabitants of the intestine of most animals, culture of this micro-organism from intestinal contents of animals has no diagnostic value unless a colony count is performed and large numbers (usually more than 10(4)-10(7)CFU/g) of C. perfringens are found. The most accepted criterion in establishing a definitive diagnosis of enterotoxaemia by C. perfringens is the detection of its toxins in intestinal contents. However, some of the major toxins of C. perfringens (i.e. epsilon toxin) can also be found, albeit in small amounts, in the small intestine of clinically normal sheep, and this poses a diagnostic challenge. In such cases the histopathology of the brain must be used as an alternative diagnostic tool, since the lesions produced by epsilon toxin in the brains of sheep and goats are unique and pathognomonic for C. perfringens type D enterotoxaemia. Ancillary tests, such as measurement of urine glucose or observation of Gram stained smears of intestinal mucosa can be used and, although they have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxaemia if they are negative. In conclusion, the diagnosis of C. perfringens infections in animals is complex and it is appropriate to rely on a combination of diagnostic techniques rather than one singe test.     

Serologic properties of strains of Cl. perfringens isolated from the intestinal contents of healthy subjects]

A study was made of the quantitative content in the intestine of C1. perfringens strains in 6 healthy persons who stayed in a hermetically sealed space for 1 month and for 1 year. C1. perfingens strains were isolated from the fecal samples of each of the volunteers at various periods of the trial. A total of 570 strains of C1. perfringens of type A with anticellular sera obtained to the strains of various serological groups were studied. Serological properties of C1. perfringens strains of type A present in the intestinal contents of man were nonhomogeneous. This pointed to the simultaneous presence in the intestine of strains belonging to several serological types. A partial or complete replacement of one strain by another (differing by serological properties) occurred in the course of not over one month. C1. perfringens strains of type A present in the intestine of each volunteer were subdivided into serological types individual for each of the persons under observation. This pointed to the fact than no interexchange of strains of the mentioned bacteria occurred between different persons in the hermetically sealed space.     

Trypsin-dependent production of an antibacterial substance by a human Peptostreptococcus strain in gnotobiotic rats and in vitro.

An antibacterial substance appeared within 1 day in feces of gnotobiotic rats harboring a human intestinal Peptostreptococcus strain. It disappeared when the rat bile-pancreatic duct was ligatured or when the rats ingested a trypsin inhibitor. Anaerobic cultures of the Peptostreptococcus strain in a medium supplemented with trypsin also exhibited an antibacterial activity, which was also inhibited by the trypsin inhibitor. In vitro the antibacterial substance from both feces and culture medium was active against several gram-positive bacteria, including other Peptostreptococcus spp., potentially pathogenic Clostridium spp. such as C. perfringens, C. difficile, C. butyricum, C. septicum, and C. sordellii, Eubacterium spp., Bifidobacterium spp., and Bacillus spp. Whatever the order of inoculation of the strains, a sensitive strain of C. perfringens was eliminated within 1 day from the intestine of rats monoassociated with the Peptostreptococcus strain. These findings demonstrate for the first time that very potent antibacterial substances can be produced through a mechanism involving intestinal bacteria and exocrine pancreatic secretions.     

New medium for rapid screening and enumeration of Clostridium perfringens in foods.

A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry. The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods. The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate. Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h. Tubes were scored as positive if a stormy fermentation was observed. All tubes demonstrating stormy fermentation were confirmed as containing C. perfringens. Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C. perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar. C. perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar. Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM.