An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon components of multivalent sheep vaccines

Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins. The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test.     

Quantitative detection of Clostridium perfringens in the broiler fowl gastrointestinal tract by real-time PCR

Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 10(2) CFU/g of ileal material, but only about 10(4) CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract.     

Diagnosis of spontaneous Clostridium spiroforme iota enterotoxemia in a barrier rabbit breeding colony

Five New Zealand White rabbits (Oryctolagus cuniculus) in a rigid barrier rabbit breeding colony developed acute diarrhea 1 week after weaning. Both Clostridium spiroforme and an iota-toxin were isolated from cecal and colon contents of all five rabbits. When pure isolates of C. spiroforme were administered to two normal healthy rabbits, the rabbits developed identical disease and shed both the organism and the iota-toxin. Results of this study suggested that C. spiroforme is an important enteric pathogen of weanling rabbits and the etiology of this diarrhea complex can be rapidly confirmed using four diagnostic criteria.     

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.     

Chicken intestine microbiota following the administration of lupulone, a hop-based antimicrobial

The use of antibiotic growth promotants in poultry rearing is a public health concern due to antibiotic resistance in bacteria and the harborage of resistance genes. Lupulone, a hop β-acid from Humulus lupulus, has been considered as a potential feed additive growth promotant. Here, the effect of lupulone was evaluated for its effect on the microbiota of the chicken intestine. The intestinal microbiota of broilers was quantified after the addition of 125 mg L(-1) lupulone to water and challenge with Clostridium perfringens. Microbial DNA was extracted from the broiler midgut and cecal sections and bacterial groups were quantified using real-time PCR. The predominant cecal bacterial groups were Clostridium leptum subgroup 16S rRNA gene Cluster IV, Clostridium coccoides subgroup 16S rRNA gene Clusters XIVa and XIVb and Bacteroides, whereas Lactobacillus, the Enterobacteriaceae family and Enterococcus dominated the midgut. Lupulone at 125 mg L(-1) significantly decreased the C. perfringens subgroup 16S rRNA gene Cluster I, which contains several pathogenic species, in both the midgut and the cecum and Lactobacillus in the midgut. No significant changes were noted in the overall microbiota for the cecum or the midgut. Lupulone warrants further evaluation as a botanical agent to mitigate C. perfringens overgrowth in antibiotic-free reared poultry.     

Characterization of cecal microbiota and response to an orally administered lactobacillus probiotic strain in the broiler chicken

A probiotic Lactobacillus strain was given in drinking water to young broiler chickens from 1 to 19 days of age. Cecal contents were collected from 4- and 19-day-old chickens in treated and control groups. Enumeration of bacteria by culture on selective media showed a decrease in Clostridium perfringens carriage in the 4-day-old treated chickens, whereas coliforms and Lactobacillus populations were not significantly affected by the treatment. Fluorescent in situ hybridization analysis with 7 phylogenetic probes targeting the major groups of intestinal bacteria revealed that the Clostridium coccoides group accounted for more than 50% of the total bacteria in the cecum of 4-day-old chickens, whereas the bacterial community of 19-day-old chickens evolved towards a more diverse microbiota with Faecalibacterium prausnitzii (36%) and C. coccoides (22%) groups representing the predominant bacteria. No effect of the Lactobacillus strain supplementation was observed in the composition of the cecal microbiota assessed by fluorescent in situ hybridization with the 7 probes. Nevertheless, profiling of the cecal microbiota using temporal temperature gradient gel electrophoresis in combination with principal component analysis demonstrated an impact of the probiotic treatment on the overall bacterial community as well as on the Lactobacillus population.     

Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature

Toxigenic types of Clostridium perfringens are important causes of enteric disease in domestic animals, although type E is putatively rare, appearing as an uncommon cause of enterotoxemia of lambs, calves, and rabbits. We report here two geographically distinct cases of type E enterotoxemia in calves, and diagnostic findings which suggest that type E may play a significant role in enteritis of neonatal calves. The cases had many similarities, including a history of diarrhea and sudden death, abomasitis, and hemorrhagic enteritis. In both cases, anaerobic cultures of abomasum yielded heavy growth of C. perfringens genotype E. Four percent of > 1000 strains of C. perfringens from cases of enteritis in domestic animals were type E, and all (n=45) were from neonatal calves with hemorrhagic enteritis. Furthermore, type E isolates represented nearly 50% of all isolates submitted from similar clinical cases in calves. Commercial toxoids available in North America have no label claims for efficacy against type E infections. Consideration should be given to type E-associated enteritis when planning for the health care of calves.     

Pregnancy interrupting effects of some bacterial toxins.

Embryotoxic properties of Shigella dysenteriae and Clostridium perfringens toxins, of E. coli endotoxin, V. cholerase and E. coli enterotoxins were compared in mice. E. coli endotoxin has embryotoxic effects at all stages of pregnancy. E. coli enterotoxin V. cholerae enterotoxin and Shigella dysenteriae toxin are most effective mainly at earlier stages of pregnancy. Clostridium perfringens toxin has no embryotoxic effect.     

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.     

Clostridium perfringens in the Environment

Clostridium perfringens was isolated from samples collected in Puget Sound in the state of Washington and areas considered as possible sources of these organisms to Puget Sound. The distribution of C. perfringens in the total Clostridium population was determined for fish gut contents and sediments collected in highly polluted and less polluted areas, sewage samples, freshwater sediments, and soils. The greatest numbers of C. perfringens were obtained from marine sediments collected near the sewage outfall at West Point. Fewer isolates were made from fish collected from less polluted stations, although the number of C. perfringens remained high in sediments from other Puget Sound stations. The proportion of C. perfringens in the total Clostridium populations varied between 56 and 71% for sewage samples and only 0.4 to 4.1% for freshwater sediments and soil samples. Only 25 C. perfringens isolates out of 137 from fish guts, or 18%, were identifiable serologically and these fell into 12 groups. C. perfringens were fed to fish and the fish were sacrificed after varying lengths of time. The number of C. perfringens increased slightly in the gut during the first 24 h and then the numbers decreased rapidly for the next 120 h.     

Sporulation of Clostridium perfringens Type A in Vacuum-Sealed Meats

Type A strains of Clostridium perfringens inoculated into vacuum-sealed packages of hamburger, roast beef, ground beef plus cream sauce, and turkey roll produced high levels of spores.     

Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

ABSTRACT: BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown whether faecal samples are representative of proximal compartments. The objectives were to investigate the prevalence of C. difficile and C. perfringens in different intestinal compartments of healthy adult horses and to determine whether faecal samples are representative of colonization in proximal sites and overall carrier status. RESULTS: Toxigenic C. difficile was isolated from 14/135 (10.3%) samples from 8/15 (53.3%) horses. Between zero and three sites were positive per horse, and multiple sites were positive in four horses. Isolates were recovered from duodenum, jejunum, ileum, right dorsal colon, small colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent with previous reports. Faecal samples were representative for presence of C. difficile in proximal compartments in 5/8 horses (63%) but were not representative for the specific strain.     

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.     

Absence of cecal secondary bile acids in gnotobiotic mice associated with two human intestinal bacteria with the ability to dehydroxylate bile acids in vitro.

Germ-free mice were orally inoculated with human intestinal 7alpha-dehydroxylating bacterial strains to evaluate their ability to transform bile acids in vivo. Three weeks after inoculation of the bacteria, cecal bile acids were examined. Among free-form bile acids, only beta-muricholic acid was detected in the cecal contents of gnotobiotic mice associated with Bacteroides distasonis strain K-5. No secondary bile acid was observed in the cecal contents of any of the gnotobiotic mice associated with 7alpha-dehydroxylating bacteria, Clostridium species strain TO-931 or Eubacterium species strain 36S.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens.

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E. coli and C. perfringens. Following shuttle vector construction in E. coli, plasmid pAK201 was transformed into E. coli HB101 and C. perfringens ATCC 3624A, using intact cell electroporation. The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E. coli and C. perfringens, respectively. Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical. Reciprocal transformation experiments in E. coli and C. perfringens indicated no difference in transformation frequency. Plasmid pAK201 was stable in C. perfringens following repeated transfer in the absence of chloramphenicol pressure. The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C. perfringens gene library construction.     

Shuttle plasmids for Escherichia coli and Clostridium perfringens.

Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E. coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C. perfringens. Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C. perfringens plasmids were cloned into them. Both tet systems made E. coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids. The shuttle vectors have been used to transform L-phase variants and autoplasts of C. perfringens. In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration. Reciprocal transformation experiments in which plasmid DNAs derived from E. coli or C. perfringens were used suggest that restriction barriers exist between these two organisms. The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.     

Microbial dysbiosis in rabbit mucoid enteropathy

The cecal contents of normal rabbits and rabbits with mucoid enteropathy (ME) were examined microscopically. Rabbits with ME consistently had dramatic cecal dysbiosis characterized by a loss of protozoa, large metachromatic bacilli and other gram-positive organisms and an increase in the size and stain retention of gram-negative organisms. Experimentally induced cecal hyperacidity in fistulated rabbits produced dysbiosis similar to that observed in natural cases of ME. Cecal pH measurements in normal young and adult rabbits revealed that pH values consistently were lower in young rabbits and frequently were low enough to induce microbial changes. Spontaneous cecal hyperacidity in young rabbits, therefore, appears to account for the cecal distention and diarrhea seen initially in ME. Late manifestations such as mucus hypersecretion and impaction, on the other hand, appear to be the result of specific microbial factors which develop when dysbiosis persists. The gradual change in the dysbiotic flora to one which closely resembles that of the normal rabbit colon appears to be the stimulus that permits these late manifestations to develop.     

Cloning and characterization of a conjugated bile acid hydrolase gene from Clostridium perfringens.

The gene encoding a conjugated bile acid hydrolase (CBAH) from Clostridium perfringens 13 has been cloned and expressed in Escherichia coli, and its nucleotide sequence has been determined. Nucleotide and predicted amino acid sequence analyses indicated that the gene product is related to two previously characterized amidases, a CBAH from Lactobacillus plantarum (40% identity) and a penicillin V amidase from Bacillus sphaericus (34% identity). The product is apparently unrelated to a CBAH from C. perfringens for which N-terminal sequence information was determined. The gene product was purified from recombinant E. coli and used to raise antibody in rabbits. The presence of the protein in C. perfringens was then confirmed by immunoblot analysis. The protein was shown to have a native molecular weight of 147,000 and a subunit molecular weight of 36,100, indicating its probable existence as a tetramer. Disruption of the chromosomal C. perfringens CBAH gene with a chloramphenicol resistance cartridge resulted in a mutant strain which retained partial CBAH activity. Polyacrylamide gel electrophoresis followed by enzymatic activity staining and immunoblotting indicated that the mutant strain no longer expressed the cloned CBAH (CBAH-1) but did express at least one additional CBAH (CBAH-2). CBAH-2 was immunologically distinct from CBAH-1, and its mobility on native polyacrylamide gels was different from that of CBAH-1. Furthermore, comparisons of pH optima and substrate specificities of CBAH activities from recombinant E. coli and wild-type and mutant C. perfringens provided further evidence for the presence of multiple CBAH activities in C. perfringens.     

Deoxyribonuclease Inhibitory Activity in Pig Intestinal Contents

The inhibitory effect of pig intestinal contents on some microbial DNases and bovine pancreas DNase was examined employing an agar diffusion test. The molecular weight of 1 inhibitor as well as the electrophoretic patterns of the inhibitors were determined. DNases from Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pyogenes, Clostridium perfringens and bovine pancreas were inhibited by intestinal contents. DNases from Clostridium septicum, Serratia marcescens, Proteus mirabilis, Aeromonas hydropnila and Pseudomonas aeruginosa were not inhibited. The concentrations of the inhibitory substances were considerably higher in contents from the small intestine than from the large intestine. Using electrophoretic procedures on intestinal contents, 3 different fractions showing DNase inhibition were demonstrated. The molecular weight of one of the inhibitors was estimated to be about 30000.