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PubMed1984

An affinity technique for the isolation of Clostridium perfringens type C from man and pigs in Papua New Guinea.

Necrotising enteritis caused by Clostridium perfringens type C (CwC) is an important cause of morbidity and death in highland regions of Papua New Guinea (PNG) and the organism is readily isolated from clinical cases. Although detected in fluorescent antibody stains of faecal cultures, CwC has previously only once been isolated from normal people or from pigs in PNG. The difficulty results from the greater numbers of the much more common Cl. perfringens type A (CwA) which is morphologically and culturally indistinguishable from CwC. Separation of CwC from the more numerous CwA has been achieved by utilising their antigenic dissimilarity. Silicate beads covered with specific antibody to CwC added to a mixed culture bound CwC cells while most of the unbound cells were washed away. When the beads were plated out type C organisms were recovered. In laboratory tests CwC was isolated from mixtures with CwA where the type A organisms predominated by one million to one. With this system type C organisms have been isolated from human and pig faeces from PNG.

Animals↗
PubMed1989

Analysis of plasmid profiling as a method for rapid differentiation of food-associated Clostridium perfringens strains.

Plasmid analysis of over 120 strains of Clostridium perfringens, isolated during food-poisoning incidents and from animal carcasses and food constituents with no association with food poisoning, showed the potential of plasmid profiling as a means of differentiating epidemiologically related strains. On average 65% of freshly isolated strains contained one or more plasmids which could be used in the analysis. Comparison of profiles of strains from unrelated sources or unrelated strains from the same source showed a particularly wide variety of plasmid profiles. Thus the possibility that epidemiologically-unrelated strains might possess similar profiles appears to be very low in this organism. Analysis of serologically-related strains from the same source revealed similar plasmid profiles in all the plasmid-bearing strains examined. A high proportion (71%) of fresh and well-characterized food-poisoning strains possessed plasmids of 6.2 kb in size (compared with 19% of non-food-poisoning strains). The possible role of these plasmids is discussed, since the structural gene encoding the enterotoxin type A was not present on any of the plasmids in the food-poisoning strains tested.

Animals↗
PubMed1992

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak.

Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype.

Bacterial Typing Techniques↗
PubMed2005

Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene.

Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in <15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, >75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that >97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is approximately 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD.

Amino Acid Sequence↗
PubMed2005

Antibiotic-induced expression of a cryptic cpb2 gene in equine beta2-toxigenic Clostridium perfringens.

The cpb2 gene of beta2-toxigenic Clostridium perfringens isolated from horses, cattle, sheep, human and pigs was sequenced. The cpb2 gene of equine and other non-porcine isolates differed from porcine isolates by the absence of an adenine in a poly A tract immediately downstream of the start codon in all non-porcine C. perfringens strains. This deletion involved formation of a cryptic gene harbouring a premature stop codon after only nine amino acid codons, while the full beta2-toxin protein consists of 265 amino acids. Immunoblots carried out with antibodies directed against a recombinant beta2-toxin showed the absence of expression of the beta2-toxin in equine and the other non-porcine strains under standard culture conditions. However, treatment of C. perfringens with the aminoglycosides gentamicin or streptomycin was able to induce expression of the cpb2 gene in a representative equine strain of this group, presumably by frameshifting. The presence of the beta2-toxin was revealed by immunohistology in tissue samples of small and large intestine from horses with severe typhlocolitis that had been treated before with gentamicin. This result may explain the finding that antibiotic treatment of horses affected by beta2-toxigenic C. perfringens leads to a more accentuated and fatal progression of equine typhlocolitis. Clinical observations show a reduced appearance of strong typhlocolitis in horses with intestinal complications admitted to hospital care since the standard use of gentamicin has been abandoned. This is the first report on expression of a bacterial toxin gene by antibiotic-induced ribosomal frameshifting.

Amino Acid Sequence↗
PubMed2006

Haemorrhagic bowel syndrome in dairy cattle: possible role of Clostridium perfringens type A in the disease complex.

A survey based on clinical, pathological and microbiological investigations was performed on 11 Brown Swiss cattle affected with depression, anorexia, agalaxia, ruminal hypomotility, abdominal pain and melaena. In eight animals, macroscopical lesions consisted in haemorrhagic enteritis in the small intestine. Seven of eight isolates from tissue samples were identified as Clostridum perfringens type A, and four were identified as C. perfringens type A with the beta2 toxin gene. Based on these observations, animals were considered affected with haemorrhagic bowel syndrome.

Animals↗
PubMed1990

Leukotoxic activity of Clostridium perfringens of equine origin.

Leukotoxic activity of equine isolates of Clostridium perfringens type A was examined. Thirty-seven isolates (94.9%) of 39 isolates demonstrated leukotoxic effects on mouse peritoneal macrophages. Phase contrast microscopy revealed that the toxic preparations induced rounded protoplasmic extrusion and sometimes destruction of the cells, leaving some membrane fragments. These findings suggest that the leukotoxic activity could be considered to be a virulence factor of C. perfringens.

Animals↗
PubMed1996

Plasmid profiling for strain differentiation and characterization of Clostridium perfringens isolates.

Plasmid profiling was used for the characterization of 13 Clostridium perfringens collection strains as well as 85 clinical and food isolates. Methodological details and limitations of the plasmid isolation procedure are outlined and discussed. For 19 clinical isolates obtained during seven group outbreaks, a close connection between at least some strains from each individual outbreak was established for six of them; only results from one outbreak were completely inconclusive, due to missing plasmids in one of the two isolates. The presence of multiple plasmid types within seven out of 12 given food samples, from which at least two C. perfringens isolates were obtained, indicates the importance of multiple isolates for meaningful typing results in epidemiological investigations. By including results from a previous report from this laboratory, baseline data on plasmid profiles for a total of 133 isolates are provided. The results of this study revealed that 36% of the food isolates unrelated to disease outbreaks carried no plasmids, as compared with 19-25% among disease-related isolates. A high prevalence (24.8%) of a 8.9 +/- 0.5 MDa plasmid was found among the 133 isolates, which contributed to one of four occurrences of identical plasmid profiles among strains that were initially considered unrelated. Two of these identical plasmid profiles were found among strains from the same culture collection, indicating the possibility of a common ancestor strain or cross-contamination.

Animals↗
PubMed1998

Strain differentiation of Clostridium perfringens by bacteriocin typing, plasmid profiling and ribotyping.

Bacteriocin typing, plasmid profiling and ribotyping were used to type 34 food and patient Clostridium perfringens isolates from 10 food poisoning cases, respectively, outbreaks. In nine cases/outbreaks bacteriocin patterns showed identical main groups. Subgroups differed within all cases/outbreaks. Plasmid profiles were identical for all isolates within each of three outbreaks. In eight food poisoning cases and outbreaks, all the ribotypes of each food and stool isolate were found to be identical. All three typing methods give valuable results for the characterization of C. perfringens beyond the species level. Bacteriocin typing represents a suitable addition to plasmid typing, particularly since the results do not show any correlation between losses of plasmids and changes in bacteriocin sensitivity patterns. Ribotyping was found to be a suitable tool to determine the genetic relationship of C. perfringens isolates in the context of food-borne poisoning.

Animals↗
PubMed1992

A highly sensitive enzyme-linked immunosorbent assay for Clostridium perfringens enterotoxin.

A highly sensitive enzyme-linked immunosorbent assay (ELISA) for quantitation of Clostridium perfringens enterotoxin (CPE) by a sandwich method with polystyrene beads was elaborated. The ELISA was very sensitive with a detection limit of 1 pg/ml of CPE. Clostridium perfringens culture fluid did not interfere with the assay. This ELISA may be useful for the mass screening for Cl. perfringens producing small amounts of CPE.

Biotechnology↗
PubMed2005

Multiplex PCR assay for toxinotyping Clostridium perfringens isolates obtained from Finnish broiler chickens.

AIMS: The aim of the study was to determine the presence of genes coding for alpha (cpa), beta (cpb), epsilon (etx), iota (iA) and enterotoxin (cpe) from Clostridium perfringens broiler chicken isolates, using multiplex PCR assay established in the study. METHODS AND RESULTS: The multiplex PCR assay was shown to be specific when tested with 10 C. perfringens strains representing different toxin types, and 15 strains of other bacterial species. All 118 broiler chicken C. perfringens isolates were shown to carry the cpa gene but not cpb, etx, iap or cpe genes, signifying that all isolates represented type A and were cpe-negative. CONCLUSIONS: The assay established in the study enables the simultaneous detection of the major toxin genes and the cpe gene from C. perfringens isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study offers a new primer pair for detecting cpa, combined with a multiplex PCR assay. In addition, the study provides data of the presence of different toxin genes in C. perfringens isolates obtained from broiler chickens.

ADP Ribose Transferases↗
PubMed2006

In vitro inhibitory effect of hen egg white lysozyme on Clostridium perfringens type A associated with broiler necrotic enteritis and its alpha-toxin production.

AIMS: Clostridium perfringens type A causes both clinical and subclinical forms of necrotic enteritis in domestic avian species. In this study the inhibitory effect of hen egg white lysozyme on the vegetative form of Cl. perfringens type A and the production of alpha-toxin in vitro was investigated. METHODS AND RESULTS: A micro-broth dilution assay was used to evaluate the minimal inhibitory concentrations (MIC) of lysozyme against three clinical isolates of Cl. perfringens type A in 96-well microtitre plates. The MIC of lysozyme against Cl. perfringens isolates was found to be 156 microg ml(-1). Scanning electron micrographs of the cells treated with 100 microg ml(-1) of lysozyme revealed extensive cell wall damage. A quantitative sandwich ELISA for alpha-toxin produced by Cl. perfringens was developed based on a commercial ELISA kit allowing only qualitative detection. Addition of 50 microg ml(-1) of lysozyme did not inhibit the growth of Cl. perfringens but significantly inhibited the toxin production. CONCLUSIONS: Lysozyme inhibited the growth of Cl. perfringens type A at 156 microg ml(-1). At sublethal levels, lysozyme was able to inhibit the alpha-toxin production. SIGNIFICANCE AND IMPACT OF STUDY: Inhibition of Cl. perfringens type A and its alpha-toxin production by hen egg white lysozyme had never previously been reported. By inhibiting this avian pathogen and its toxin production, lysozyme showed potential for use in the treatment and prevention of necrotic enteritis and other Cl. perfringens type A related animal diseases.

Animals↗