Sandwich enzyme-linked immunosorbent assay by using monoclonal antibody for detection of Clostridium perfringens enterotoxin.
(1/1215)
Sandwich enzyme-linked immunosorbent assay (ELISA) was developed for the quantitative estimation of Clostridium perfringens enterotoxin (CPE) with monoclonal and polyclonal antibodies as capturing and detecting antibodies, respectively. The dose-dependent relationship between absorbance at 405 nm and concentration of purified CPE was obtained over the range of 0.64-400 ng/ml. The sandwich ELISA was fond to detect crude CPE in culture and CPE in 10% fecal extracts. This method is convenient, rapid and sensitive for specific detection of CPE. (+info)
Hemorrhagic enteritis associated with Clostridium perfringens type A in a dog.
(2/1215)
A female Shetland sheep dog died suddenly with hemorrhagic diarrhea and vomitting, and was examined pathologically and microbiologically. Gross pathological change was restricted to the intestinal tract. The intestine contained watery, blood-stained fluid. Histopathologically, the principal intestinal lesion was superficial mucosal hemorrhagic necrosis at the jejunoileum. Many Gram-positive bacilli were found adhering to the necrotic mucosal surface in parts of the intestinal tract. Clostridium perfringens in pure culture were isolated from jejunal contents by anaerobic culture. These results suggested that the typical lesion of this case coincided with canine hemorrhagic enteritis and enterotoxemia due to C. perfringens infection could be the cause of sudden death. (+info)
Cationic currents induced by Clostridium perfringens type A enterotoxin in human intestinal CaCO-2 cells.
(3/1215)
Clostridium perfringens type A produces an enterotoxin that induces diarrhoea experimentally in man and animals. The enterotoxin causes increased membrane permeability in susceptible cells which is thought to be due to pore formation in the host cell membrane. The effect of purified C. perfringens enterotoxin on intact intestinal CaCO-2 monolayers was examined in Ussing chambers and on single cells by whole-cell patch clamp. Mucosal application of C. perfringens enterotoxin resulted in prompt increases in short-circuit current coupled with a reduction in transepithelial resistance consistent with movement of sodium and other cations smaller than diethanolamine from mucosa to serosa. These changes were independent of extracellular calcium. Increases in short-circuit current were also observed in the apical membranes of CaCO-2 monolayers permeabilised across the basolateral membrane with nystatin. Currents were blocked by subsequent exposure to mucosal barium and zinc. Zinc also prevented the development of the current increases in apical membranes. Cationic currents were also observed following exposure of single CaCO-2 cells in whole-cell patch clamp recordings. These data indicate that C. perfringens enterotoxin is able to form cation permeant pores in the apical membrane of human intestinal CaCO-2 epithelia and the increases in short-circuit current can be prevented by pre-exposure to zinc ions. (+info)
Production of phospholipase C (alpha-toxin), haemolysins and lethal toxins by Clostridium perfringens types A to D.
(4/1215)
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. (+info)
Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity.
(5/1215)
The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli. The purified GST-beta-toxin fusion protein from the E. coli transformant cells was not lethal. The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T. Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C. perfringens type C. Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity. Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity. The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity. In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not. These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin. Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity. Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin. These observations suggest that the site essential for the activity is close to the cysteine residue. (+info)
Molecular subtyping of Clostridium perfringens by pulsed-field gel electrophoresis to facilitate food-borne-disease outbreak investigations.
(6/1215)
Clostridium perfringens is a common cause of food-borne illness. The illness is characterized by profuse diarrhea and acute abdominal pain. Since the illness is usually self-limiting, many cases are undiagnosed and/or not reported. Investigations are often pursued after an outbreak involving large numbers of people in institutions, at restaurants, or at catered meals. Serotyping has been used in the past to assist epidemiologic investigations of C. perfringens outbreaks. However, serotyping reagents are not widely available, and many isolates are often untypeable with existing reagents. We developed a pulsed-field gel electrophoresis (PFGE) method for molecular subtyping of C. perfringens isolates to aid in epidemiologic investigations of food-borne outbreaks. Six restriction endonucleases (SmaI, ApaI, FspI, MluI, KspI, and XbaI) were evaluated with a select panel of C. perfringens strains. SmaI was chosen for further studies because it produced 11 to 13 well-distributed bands of 40 to approximately 1,100 kb which provided good discrimination between isolates. Seventeen distinct patterns were obtained with 62 isolates from seven outbreak investigations or control strains. In general, multiple isolates from a single individual had indistinguishable PFGE patterns. Epidemiologically unrelated isolates (outbreak or control strains) had unique patterns; isolates from different individuals within an outbreak had similar, if not identical, patterns. PFGE identifies clonal relationships of isolates which will assist epidemiologic investigations of food-borne-disease outbreaks caused by C. perfringens. (+info)
Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner.
(7/1215)
The phospholipase C gene (plc) of Clostridium perfringens possesses three phased A-tracts forming bent DNA upstream of the promoter. An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A-tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A-tracts, and more prominent at lower temperature. A gel retardation assay and hydroxyl radical footprinting revealed that the phased A-tracts facilitate the formation of the RNAP-plc promoter complex through extension of the contact region. The upstream (UP) element of the Escherichia coli rrnB P1 promoter stimulated the downstream promoter activity temperature independently, differing from the phased A-tracts. When the UP element was placed upstream of the plc promoter, low temperature-dependent stimulation was observed, although this effect was less prominent than that of the phased A-tracts. These results suggest that both the phased A-tracts and UP element cause low temperature-dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature-dependent activation by the phased A-tracts may be due to an increase in the bending angle at a lower temperature. (+info)
Differences in the carboxy-terminal (Putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes.
(8/1215)
The phospholipases C of C. perfringens (alpha-toxin) and C. bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties. We report here the purification and characterisation of alpha-toxin and Cbp. The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes. However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp. To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin. The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic. These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins. (+info)