Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.     

Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.     

Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.     

Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts.     

Haemagglutinin production by enterotoxigenic strains of Clostridium perfringens type A

Thirty-nine enterotoxigenic cultures of Clostridium perfringens type A were studied for enterotoxin and haemagglutinin production. Enterotoxin was quantitated by sandwich ELISA and DOT-ELISA techniques and haemagglutinin titres were determined using sheep and human erythrocytes. Haemagglutinins from only six cultures reacted against both sheep and human erythrocytes; a further 13 reacted only against human erythrocytes, and another five only against sheep cells.The authors are with the Department of Veterinary Public Health and Epidemiology, Ranchi Veterinary College, Birsa Agricultural University, Ranchi-834007 (Bihar), India.     

Raffinose increases sporulation and enterotoxin production by Clostridium perfringens type A.

Replacement of starch with raffinose in Duncan and Strong sporulation medium improved percent sporulation in six of eight strains tested. Enterotoxin concentration in cell extracts was increased in the case of four of five known enterotoxin-positive strains. With strain NCTC 10240, levels of 0.3, 0.4, and 0.5% raffinose produced the highest enterotoxin concentration 300 to 320 micrograms of enterotoxin per mg of cell extract protein. At a level of 0.4% raffinose the highest enterotoxin concentration in cell extracts of NCTC 10240 occurred after 8 h of growth in Duncan and Strong medium. Enterotoxin produced in the presence of starch or raffinose by three separate strains all migrated at similar Rm by polyacrylamide gel electrophoresis.     

Raffinose increases sporulation and enterotoxin production by Clostridium perfringens type A.

Replacement of starch with raffinose in Duncan and Strong sporulation medium improved percent sporulation in six of eight strains tested. Enterotoxin concentration in cell extracts was increased in the case of four of five known enterotoxin-positive strains. With strain NCTC 10240, levels of 0.3, 0.4, and 0.5% raffinose produced the highest enterotoxin concentration 300 to 320 micrograms of enterotoxin per mg of cell extract protein. At a level of 0.4% raffinose the highest enterotoxin concentration in cell extracts of NCTC 10240 occurred after 8 h of growth in Duncan and Strong medium. Enterotoxin produced in the presence of starch or raffinose by three separate strains all migrated at similar Rm by polyacrylamide gel electrophoresis.     

TpeL-producing strains of Clostridium perfringens type A are highly virulent for broiler chicks

Clostridium perfringens type A and type C are causative agents of necrotic enteritis (NE) in poultry. TpeL, a recently-described novel member of the family of large clostridial cytotoxins, was found in C. perfringens type C. Others have since reported TpeL in type A isolates from NE outbreaks, suggesting that it may contribute to the pathogenesis of NE. The virulence of TpeL-positive and -negative C. perfringens strains from cases of NE was examined by challenge of broiler chicks. Gross lesions typical of NE were observed in all challenged birds, and those inoculated with TpeL(pos) strains had higher average macroscopic lesion scores than those inoculated with a TpeL(neg) strain. Infection with TpeL(pos) strains may yield disease with a more rapid course and higher case fatality rate. Thus, TpeL may potentiate the effect of other virulence attributes of NE strains of C. perfringens. However, TpeL(pos) and Tpel(neg) strains compared here were not isogenic, and definitive results await the production and testing of specific TpeL mutants.     

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.     

Thermal inactivation of ileal loop-reactive Clostridium perfringens type A strains in phosphate buffer and beef gravy.

The thermal resistance of spore crops produced from each of two ileal loop-reactive strains of Clostridium perfringens type A was determined in two suspending vehicles consisting of 0.067 M (pH 7.0) phosphate buffer and a commercial beef gravy. D115.6 values obtained in buffer and enumerated after pretreatment with sodium ethylenediaminetetraacetate and recovery in plating medium containing lysozyme were two- to threefold greater than those obtained without this treatment. D115.6 values obtained with beef gravy were less than those obtained in buffer with or without lysozyme; however, the D98.9 and D104.4 values were 1.3 to 2 times greater than those obtained in buffer with lysozyme. The z values were within the ranges reported by previous investigators.     

Effect of diolein on hydrolysis of phosphatidylcholine by phospholipase C from Clostridium perfringens.

The activity of phospholipase C from Clostridium perfringens on 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) as a monolayer at an air/water interface was examined. With a pure POPC monolayer, sharp cut-off of the enzyme activity was observed on increase in surface pressure. However, this cut-off disappeared on addition of a 0.3 molar fraction of 1,2-dioleoylglycerol (1,2-DO) to the monolayer. An abrupt change in the enzyme activity was observed with molar fractions of between 0.2 and 0.3 1,2-DO in the POPC monolayer at an initial surface pressure of 35 mN/m. For examination of the effect of 1,2-DO on the phospholipase C activity, the quantity of [125I]phospholipase C adsorbed to the surface was determined. The enzyme was found to be adsorbed nonspecifically to all lipid films except that of POPC only. The adsorption of enzyme was not affected by the presence or absence of Ca2+ and Zn2+. The rate constant for enzyme adsorption to a 1,2-DO film was 4.5 times that for its adsorption to a POPC film. The adsorption decreased linearly with increase in the surface concentration of POPC, and increased with increase in the surface concentration of 1,2-DO. These data suggest that 1,2-DO (a reaction product) regulates the interaction of phospholipase C with films containing substrate and may also regulate the enzyme activity.     

Enterotoxic strains of Clostridium perfringens and sporulation

Clostridium perfringens strains of A type capable of enterotoxin (CPE) synthesis may be a potential source of food-poisoning. Suitability of methods for CPE detection on the protein level is limited by difficulties in inducing sporulation in vitro. A number of unknown facts concerning coregulation the sporulation processes and CPE synthesis are recognised. The goal of the work was to determine the level of correlation between CPE synthesis and spores formation. Enterotoxin and cpe gen were detected by RPLA after sporulation induction test and by methods based on amplification on the DNA and mRNA levels. Sixty-four C. perfringens strains of A type isolated from patients with food poisoning symptoms and from food samples were analysed. Collection of isolates was differentiated as not enterotoxic, enterotoxic, and potentially enterotoxic strains based on appropriate strain profile: plc(+), cpe(-), CPE(-); plc(+), cpe(+), CPE(+); and plc(+), cpe(-), CPE(-), respectively. No significant difference between expression of cpe mRNA in vegetative and sporulation phase was found. The obtained results indicate that sporulation is not an essential factor for cpe gene expression.     

Enterotoxin formation by Clostridium perfringens type A in a defined medium.

Enterotoxin was produced by 9 of 10 strains of Clostridium perfringens type A when grown in a defined medium. Additional dextrin increased the amount of enterotoxin in extracts of sporulating cells of strain NCTC 10239.     

Properties of four temperate bacteriophages active on Clostridium perfringens type A.

Four temperature bacteriophages (designated as PF1, PF2, PF3 and PF4) were isolated from lysogenic strains of Clostridium perfringens type A. On the basis of plaque morphology, pH stability, DNase and RNase resistance, buoyant density, one-step growth parameters and electron microscope phage dimensions, it seems that these phages are different and unrelated. Calcium was required for better phage replication. Bacterial strain S107 appears to be the only UV-inducible strain as compared with the other three lysogenic strains. PF2 has a unique pattern of pH stability showing two optima values: one at pH 5 and the second at pH 8-9. Generally, all four phages have a better resistance in acid than in alkaline pH values. The CsC1 equilibrium centrifugation patterns reveal low figures for phage PF1, PF2 and PF3 and show off the fact that PF4 lysates contain two viral particules different with respect to their densities, a property which other determinations failed to demonstrate. Each phage, except PF4, is well characterized by the parameters of the one-step growth cycle.