Quantitation of Clostridium perfringens Type A Enterotoxin by Electroimmunodiffusion

Conditions for quantitation of Clostridium perfringens type A enterotoxin by electroimmunodiffusion are described. As little as 0.01 mug of enterotoxin could be detected. Electroimmunodiffusion was more sensitive than either single gel diffusion or quantitation based on erythemal activity of the toxin in guinea pig skin.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

Relationship of sporulation, enterotoxin formation, and spoilage during growth of Clostridium perfringens type A in cooked chicken.

Sporulation and enterotoxin formation were determined for 17 strains of Clostridium perfringens type A in autoclaved chicken dark meat and in Duncan-Strong sporulation medium. The mean numbers of heat-resistant spores detected after 24 h at 37 degrees C were log10 1.13 to log10 7.64/ml in Duncan-Strong medium and log10 4.93 to log10 6.59/g in chicken. Of 17 strains, 7 formed enterotoxin in Duncan-Strong culture supernatant (1.0 to 60 microgram/ml) and 8 produced enterotoxin in chicken (0.21 to 24 microgram/g). Additional studies with chicken were conducted with C. perfringens NCTC 8239. With an inoculum of 10(6) cells per g, greater than log10 7.99 vegetative cells per g were detected by 4 h in chicken at 37 degrees C. Heat-resistant spores occurred by 4 and 6 h and enterotoxin occurred by 8 and 6 h in autoclaved chicken dark meat and barbecued chicken drumsticks, respectively. Enterotoxin was detected in autoclaved dark meat after incubation at 45 degrees C for 1.5 h followed by 37 degrees C for 4.5 h, but not after incubation at 45 degrees C for 1.5 to 8 h. With an inoculum of 10(2) cells per g in oven-cooked or autoclaved chicken, greater than log10 8.00 vegetative cells per g were detected by 6 to 8 h at 37 degrees C, heat-resistant spores were detected by 8 h, and enterotoxin was detected by 12 h. A statistical analysis of odor determinants of chicken after growth of C. perfringens indicated that, at the 95% confidence level, the product was considered spoiled (off or unwholesome odor) by the time spores or enterotoxin were formed.     

A 6 h microslide immunodiffusion assay for confirmed detection of staphylococcal enterotoxins

Use of appropriately balanced immunological reagents and incubation of microslides at 45 degrees C resulted in confirmed assay results in 6 h for staphylococcal enterotoxins. The sensitivity of the 45 degrees C-6 h assay was comparable to the 37 degrees C-24 h assay: 0.1 micrograms of staphylococcal enterotoxin A/ml.     

Identification of enterotoxigenic staphylococci from sheep and sheep cheese

The total of 127 Staphylococcus aureus strains obtained from sheep and sheep cheese were examined for their biochemical activities, biotypes, phage patterns, and ability to produce enterotoxins. Of the 83 staphylococcal strains isolated from animals 77 (93%) were classified as the C biotype. Of this group of sheep-adapted strains, 61 (79%) were sensitive to phage 78, and 46 (60%) produced enterotoxin C exclusively. The three isolated belonging to the A biotype produced enterotoxin D, and two of the three unclassifiable strains produced enterotoxin A. Of the 44 staphylococcal strains isolated from sheep cheese, there were 37 (84%) identified as the C biotype. From this series, 31 (84%) strains were lysed with phage 78, 6 (16%) strains produced enterotoxin C, and 1 strain produced enterotoxin A. One of the six strains determined as the A biotype produced enterotoxin D. C biotype strains, especially of ovine origin, are an exception among animal staphylococci, because a large number of them are enterotoixgenic. The C antigenic type is the most usual of the known enterotoxins in staphylococci of animal provenance.     

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.     

Generation of scFv phages specific to Staphylococcus enterotoxin C1 by panning on related antigens

A method for generation of highly specific miniantibodies within the phage particle has been developed, and used to produce antibodies against Staphylococcus enterotoxin type C1. Under successive panning of the non-immune phage miniantibody (scFv) library with enterotoxins SE (types A, B, C1, D, E, G, and I) adsorbed on the plate surface, we generated 11 individual phage clones to Staphylococcus enterotoxin type C1. Five of them interacted specifically only with SEC1 and had no cross-reactions with the other enterotoxins.     

DETECTION of ENTEROTOXIGENIC CLOSTRIDIUM PERFRINGENS IN GROUND BEEF

Clostridium perfringens is widely distributed in foods. This experiment was performed to assess occurrence of C. perfringens cultures and toxigenic strains isolated from ground beef Samples (118) were collected from 20 locations in Northeast Kansas and the number of C. perfringens was enumerated in these samples by Fung's Double-tube method with tryptose sulfite cycloserine agar medium. Out of 118 samples, 54 (46%) were found positive for C. perfringens. Pure isolates of C. perfringens were further grown in cooked meat medium for 24 h at 42C then heat shocked at 75C for 20 min and inoculated into modified Duncan and Strong medium for production of C. perfringens enterotoxin. Presence of enterotoxin was tested by the reverse passive latex agglutination test (Oxoid), which can detect enterotoxin up to a minimum level of 2 ng/mL. the data indicate that 46% of the beef samples harbored C. perfringens , but only 32 (6%) of 525 isolates were found to produce enterotoxin. This study emphasized the importance of continued surveillance of C. perfringens in meats and meat products and assessment of the toxigenesis of isolates.     

On the cross-reactivity of staphylococcal enterotoxins A, B, and C.

Strong cross-reactions were demonstrated for staphylococcal enterotoxins B (SEB) and C1 (SEC1) by antigen-binding capacity and by competitive binding ability. Both SEB and SEC1 combined completely with the heterologous antibody although requiring four times as much antiserum as the homologous enterotoxin and both displaced about one-third of the other enterotoxin from a heterologous antigen-antibody system. It is proposed that one of the three major antigenic determinants of these enterotoxins possesses a significant similarity but probably not an identity of structure. SEB and SEC1 did not combine with antiserum to enterotoxin A nor inhibit the reaction of SEA with anti-SEA. SEA had no intrinsic binding capacity for anti-SEB or anti SEC1 nor did it inhibit the binding of either enterotoxin to its own antibody. Affinity chromatography was employed to demonstrate that a small apparent binding of SEA to anti-SEB was due to antibody to SEA in the anti-SEB serum and that an almost complete displacement of SEC1 binding to anti-SEC1 was caused by contaminating SEC (about 0.01%) in preparations of enterotoxin A.     

Complete amino acid sequence of staphylococcal enterotoxin A

The amino acid sequence of staphylococcal enterotoxin A is presented. Staphylococcal enterotoxin A is a single-chain polypeptide which consists of 233 amino acid residues with a molecular weight of 27,078 and has the amino acid composition Cys2, Asp17, Asn19, Thr16, Ser13, Glu15, Gln12, Pro4, Gly15, Ala7, Val13, Met2, Ile10, Leu23, Tyr18, Phe8, His6, Lys24, Arg7, Trp2, with serine as both amino- and carboxyl-terminal amino acids. Automated sequence analysis of intact enterotoxin A, as well as characterization of the peptides obtained from cyanogen bromide treatment and trypsin and chymotrypsin digestion, led to the elucidation of the complete primary structure of this protein. Less structural homology is observed among staphylococcal enterotoxins A, B (Huang, I-Y., and Bergdoll, M. S. (1970) J. Biol. Chem. 245, 3518-3525), and C1 (Schmidt, J. J., and Spero, L. (1983) J. Biol. Chem. 258, 6300-6306) than that seen between enterotoxins B and C1.     

The effect of a Clostridium perfringens 8-6 enterotoxin on viability and macromolecular synthesis in Vero cells

Clostridium perfringens type A 8-6 enterotoxin causes gross morphological damage to Vero cells grown in tissue culture. Damage was observed to occur after only 30 minutes exposure at concentrations as low as 20 ng/ml, and within 60 minutes 95% of the cells had detached. Concentrations as low as 0.01 ng were able to cause detectable inhibition of plating efficiency. The enterotoxin inhibited DNA, RNA and protein synthesis and caused the reversal of glucose transport. Heat inactivated enterotoxin had no effect on cell function or morphology.     

A scanning electron microscope study of the effect of an enterotoxin from Clostridium perfringens 8-6 on mice of different ages

Intestinal damage to mice caused by an enterotoxin from a coatless spore mutant of Clostridium perfringens type A (8-6) was examined by scanning electron microscopy. Two distinct types of damage were observed, both of which could be correlated with animal age. Damage appeared to occur in a specific sequence similar to that found in previous studies in rabbits. We conclude that the type of ileal tissue damage reflects the mode of toxin incorporation from the gut, which is a function of animal age.     

Experimental diarrhea in cynomolgus monkeys by oral administration with Clostridium perfringens type A viable cells or enterotoxin.

Purified C. perfringens type A enterotoxin fed orally in an amount of 5 mg caused both vomiting and diarrhea in the monkey only when the gastric juice had been neutralized. Exposure of enterotoxin to pH 4.0 or below rapidly destroyed the activity. All three monkeys receiving sodium bicarbonate and 2.4 X 10(10) viable cells grown in DS medium developed diarrhea, and only one of them vomited once. The diarrhea lasted for 13, 18 and 19 hr. The symptoms were similar to those reported in human cases of C. perfringens food poisoning. These results have verified the general notion that C. perfringens food poisoning should be categorized as a true "intravital intoxication". The reversed passive hemagglutination test detected enterotoxin directly in most fecal samples. This method may be applicable for diagnosis of human cases of C. perfringens food poisoning. Neither enterotoxin nor anti-enterotoxin was detected in serum samples taken from any monkey up to 21 days after the challenge. We are tempted to conclude, therefore, that no significant amount of C. perfringens enterotoxin is absorbed from the intestine.     

Identification of a Fourth Staphylococcal Enterotoxin, Enterotoxin D

A fourth staphylococcal enterotoxin was identified serologically with antiserum to the very crude enterotoxic products of growth of a strain which also produces enterotoxin C, and then with antiserum to the considerably purified enterotoxic antigen of a strain which produces only the new enterotoxin. The identification of this antigen as enterotoxin D was based on the following observations. It was produced by strains which do not produce enterotoxins A, B, or C; it was absent in the growth products of nonenterotoxigenic strains; when appreciably purified, it was associated with emetic activity in the cat, and its biological activity was neutralized only by antisera containing its specific antibody and not by antibodies to enterotoxins A, B, and C. Staphylococcal strain 494 (ATCC 23235) was selected as the prototype strain. The production of this enterotoxin alone and together with enterotoxin A by strains of food-poisoning origin indicates that its role in food poisoning is second in frequency only to that of enterotoxin A. The incidence of production of enterotoxins A, B, C, and D, and of unidentified cat emetic substances by strains from several source categories, is presented.     

Generation of scFv phages specific to Staphylococcus enterotoxin C1 by panning on related antigens

A method for generation of highly specific miniantibodies within the phage particle has been developed and used to produce antibodies against Staphylococcus enterotoxin type C1. Under successive panning of the non-immune phage miniantibody (scFv) library with enterotoxins SE (types A, B, C1, D, E, G and I) adsorbed on the plate surface, we generated 11 individual phage clones to Staphylococcus enterotoxin type C1. Five of them interacted specifically only with SEC1 and had no cross-reactions with the other enterotoxins.Key words: scFv, phage display, antibody specificity, Staphylococcus aureus enterotoxins     

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.     

Electroporation-mediated transformation of lysostaphin-treated Clostridium perfringens

A reliable and efficient method has been developed for the electroporation-mediated transformation of Clostridium perfringens with plasmid DNA. Transformation of vegetative cells of C. perfringens strain 13 with the 7.9-kb Escherichia coli-C. perfringens shuttle plasmid pHR 106 required pretreatment with lysostaphin (2 to 20 micrograms/ml) for 1 h at 37 degrees C. Cells harvested early in the logarithmic stage of growth were transformed more efficiently than cells at other growth phases. The transformation frequency increased with the DNA concentration, to a saturating level at 5 to 10 micrograms DNA/ml. The transformation frequency was proportional to the field strength and time constant of the electroporation pulse; however, the field strength was a far more important parameter. A cell density between 1 x 10(8) and 5 x 10(8) cells/ml proved to be optimal for transformation. The procedure was capable of generating up to 3.0 x 10(5) transformants per micrograms DNA. The potential value of the method for the cloning of C. perfringens genes was demonstrated by the cloning of the clostridial tetracycline-resistance determinant, tetP, from the E. coli recombinant plasmid pJIR71, into C. perfringens strain 13.     

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.