Immunogenic properties of Clostridium perfringens type A anatheta-hemolysin

Guinea-pigs were immunized with anatheta-hemolysin preparations adsorbed on aluminum hydroxide, as well as with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated. Anatheta hemolysin preparations were obtained with the use of homogeneous theta hemolysin, as well theta hemolysin of various purification degrees. As a result, antatheta hemolytic guinea-pig sera capable of neutralizing 2,000-8,000 HU of theta-hemolysin were obtained. Tests made to establish the degree of protection in the immunized guinea-pigs did not show that the animals immunized with the mixture of anatheta-hemolysin and type A Cl. perfringens toxoid, purified and concentrated, had any advantages in the degree of protection over the animals immunized with the toxoid alone. But there is no doubt that this component plays a positive role under the conditions of natural gas gangrene when the hemolytic action of Cl. perfringens toxin becomes considerably pronounced.     

Clostridium perfringens invasiveness is enhanced by effects of theta toxin upon PMNL structure and function: The role of leukocytotoxicity and expression of CD11/CD18 adherence glycoprotein

Abstract Clostridium perfringens infections are characterized by the lack of an inflammatory response at the site of infection and rapidly progressive margins of tissue necrosis. Studies presented here investigated the role of theta toxin from C. perfringens in the pathophysiology of these events. Mice passively immunized with neutralizing monoclonal antibody against theta toxin and challenged with an LD₁₀₀ of log phase C. perfringens had significantly less mortality than untreated controls. Intramuscular injection of killed, washed C. perfringens in mice induced a massive time-dependent influx of polymorphonuclear leukocytes (PMNL) into tissue; injection of either viable, washed C. perfringens or killed organisms plus theta toxin dramatically attenuated PMNL influx although PMNL accumulated in adjacent vessels. The anti-inflammatory effects could not be attributed to an absence of chemoattractants since C. perfringens proteins had chemotactic factor activity, and killed bacilli generated serum-derived chemotactic factors. Scanning and transmission electron microscopy demonstrated the dramatic leukocidal effects of high doses of theta toxin on PMNL. In contrast, sublethal concentrations of theta toxin primed PMNL chemiluminescence, disrupted PMNL cytoskeletal actin polymerization/disassembly, and stimulated functional upregulation of CD11b/CD18 adherence glycoprotein. In summary, these results demonstrate that theta toxin is an important virulence factor in C. perfringens infection. In a concentration-dependent fashion, theta toxin contributes to the pathogenesis of clostridial gangrene by direct destruction of host inflammatory cells and tissues, and by promoting dysregulated PMNL/endothelial cell adhesive interactions.     

Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis

Clostridium perfringens (C. perfringens) has been developed as a potential oral delivery vehicle to deliver antigens or therapeutic compounds to Gut-Associated Lymphoid Tissue (GALT). However, this recombinant C. perfringens carries a plasmid-encoded expression system, which raises several safety concerns regarding possible horizontal plasmid transfer and spread of plasmid-associated antibiotic resistant genes. Furthermore, this bacterium produces the extracellular theta toxin, which poses a potential safety issue for general administration. Using a Clostridium-specific-targetron donor plasmid, we inserted the Simian Immunodefiency Virus (SIV) p27 gene into the theta toxin gene (pfoA) on the C. perfringens chromosome, which simultaneously inactivated the theta gene and introduced SIV p27 gene onto the bacterial chromosome. Such mutant C. perfringens without an input plasmid or antibiotic resistant gene stably produced a large amount of SIV p27 protein during sporulation and did not produce theta toxin. Upon oral feeding of the mutant bacteria to mice, intact p27 protein was detected in the lower GI tract. The re-engineered C. perfringens provides a biosafe efficient oral vehicle to deliver antigen to the gastrointestinal tract.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

On the in vitro neutralization test of Clostridium perfringens type A toxin

Data are presented on the detection in crude animal and human sera of Cl. perfringens phospholipase C (PLC) inhibitor. When the level of Cl. perfringens type A antitoxin is determined in the in vitro toxin neutralization test the inhibitor is found to decrease PLC activity in the test dose of experimental homologous toxin. The extent of decrease accounts for the variation of results obtained in the in vitro and in vivo toxin neutralization tests. The variation may be cancelled out by introducing a corresponding coefficient to calculate the level of alpha-antitoxin. It is suggested that the isolation and investigation of the PLC inhibitor will contribute to the development of preparations for treatment of gas gangrene due to Cl. perfringens type A.     

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.     

Vero cell assay for rapid detection of Clostridium perfringens enterotoxin.

A rapid assay which measured the biological activity of Clostridium perfringens enterotoxin was developed. The method involved the rapid killing of Vero cells by enterotoxin produced by C. perfringens grown in Duncan and Strong sporulation medium. Serial dilutions of toxin were added to Vero cells either in suspension or grown as monolayers in wells of a 96-well cell tissue culture cluster plate. Vital staining of Vero cells with neutral red, followed by extraction of the dye, allowed toxin levels to be determined either visually or by optical density measurements with a micro-ELISA M580 computer program. The toxin produced was confirmed as different from the Vero toxin of Escherichia coli and the alpha and theta toxins of C. perfringens.     

Changes in the electrical activity of the cerebral cortex under the combined influence of Cl. perfringens type A toxin and products of Cl. butyricum activity]

A study of the changes of the electrocorticogram (ECoG) and the electrocardiogram (ECG) in combined action of Cl. perfringens, type A, and of the Cl. butyricum broth culture filtrate showed that desynchronization of the cortical electrical activity and its subsequent depression occurred at earlier periods than in the case of isolated administration of Cl. perfringens toxin. The general character of the changes in the cortical rhythmic activity remained the same as in intoxication caused by Cl. butyricum toxin alone. The ECoG and ECG changes occurred at shorter intervals. Cl. butyricum filtrates induced no ECoG and ECG changes. It is supposed that the effect of the products of the Cl. butyricum vital activity consisted in increase in the tissue barrier permeability and, in this connection, in a greater penetration of Cl. perfringens toxin into the tissues, including the central nervous system.     

In vitro tests for the measurement of clostridial toxins, toxoids and antisera. II. Titration of Clostridium perfringens toxins and antitoxins in cell culture

The usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated. Neutralization experiments with monoclonal antibodies have shown that the entities responsible for the lethal and dermonecrotic effects of Cl perfringens beta toxin preparations are identical. However, the cytopathic effects of the same preparations are caused by other entities. Therefore, titrations based upon lethal and dermonecrotic indicators of beta toxin are equally valid but those based on cytopathic effects are not. Similar experiments with Cl perfringens epsilon preparations have shown that their lethal, dermonecrotic and cytopathic activities are all caused by the same entity. It follows that all three activities can be valid indicators for toxin neutralization tests. Cell culture titrations of Cl perfringens epsilon antitoxin performed on rabbit sera at the levels of test prescribed by the European Pharmacopoeia have produced consistent results which agree closely with the dermonecrotic test. This test has, in turn, been shown to reflect the results of the mouse lethal test accurately. Titrations of cattle and sheep sera at lower levels of test have also produced results in close agreement with the in vivo test. It is concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators for the titration of sera generated in the course of potency tests and field trials of Cl perfringens epsilon vaccines.     

Method for Estimating the Presence of Clostridium perfringens in Food

The methods currently used for the enumeration of Clostridium perfringens in food are often inadequate because of the rapid loss of viability of this organism when the sample is frozen or refrigerated. A method for estimating the presence of C. perfringens in food which utilizes the hemolytic and lecithinase activities of alpha toxin was developed. The hemolytic activity was measured in hemolysin indicator plates. Lecithinase activity of the extract was determined by the lecithovitellin test. Of 34 strains of C. perfringens associated with foodborne disease outbreaks, 32 produced sufficient alpha toxin in roast beef with gravy and in chicken broth to permit a reliable estimate of growth in these foods. Alpha toxin was extracted from food with 0.4 m saline buffered (at pH 8.0) with 0.05 mN-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid and concentrated by dialysis against 30% polyethylene glycol. A detectable quantity of alpha toxin was produced by approximately 10(6)C. perfringens cells per g of substrate, and the amount increased in proportion to the cell population. Results obtained with food samples responsible for gastroenteritis in humans indicate that a correlation can be made between the amount of alpha toxin present and previous growth of C. perfringens in food regardless of whether the organisms are viable when the examination is performed.     

Theta-toxin of Clostridium perfringens. I. Purification and some properties.

Theta-Toxin, an oxygen-labile hemolysin produced by Clostridium perfringens, was purified 3300 fold from culture filtrate by successive chromatography on DEAE-Sephadex A-50 and Sephadex G-150. The purified toxin gave two distinct bands in disc electrophoresis, while the same material, after mild reduction with dithiothreitol, yielded a single band, indicating that the purified theta-toxin contained, as well as a reduced, active form, an oxidized, inactive form of toxin. These two forms of the toxin had a similar, if not identical molecular size. The purified preparation gave a single band in a sodium dodecyl sulfate polyacrylamide gel electrophoresis and formed a single precipitin line with National Standard gas gangrene (C. perfringens) antitoxin. By sodium dodecyl sulfate polyacrylamide gel electrophoresis, the molecular weight of theta-toxin was estimated to be 51 000, the value being in exact accordance with that obtained by amino acid analysis. The amino acid composition of theta-toxin was very close to that of cereolysin, an oxygen-labile hemolysin produced by Bacillus cereus. The amino-terminal residue of theta-toxin was lysine as determined by the Dansyl method.     

Molecular typing of Clostridium perfringens isolated from turkey meat by multiplex PCR

Aims:  To determine the presence of toxin genes in 22 Clostridium perfringens isolated from turkey meat samples by molecular typing.
Methods and Results:  For this purpose, alpha ( cpa ), beta ( cpb ), beta 2 ( cpb2 ), epsilon ( etx ), iota ( iA ) and enterotoxin ( cpe ) toxin genes were analysed by multiplex PCR. All 22 turkey meat Cl. perfringens isolates were found to carry the cpa , gene but in none of the isolates cpb , etx, iap or cpe genes were detected. Results showed that all isolates represented type A and were cpe negative.
Conclusions:  Our results indicate that Cl. perfringens type A is the most common type in turkey meat. Also multiplex PCR is effective and rapid method for typing of Cl. perfringens .
Significance and Impact of the Study:  It is the first study about molecular typing of Cl. perfringens using multiplex PCR in turkey meat samples in Turkey.     

Medium for Toxin Production by Clostridium perfringens in Continuous Culture

A tryptone-salts medium for continuous growth and alpha toxin production of Clostridium perfringens type A in an adapted chemostat is described. In such steady-state cultures, fermentative and biochemical activity of C. perfringens remained unchanged. Toxigenic ability to produce alpha, theta, and nu toxins was preserved.     

COMPARATIVE KINETICS OF HEMOLYSIS INDUCED BY BACTERIAL AND OTHER HEMOLYSINS

A study has been made of the kinetics of lysis induced by various hemolytic agents. The course of bemolysis was followed by mixing lysin with washed human erythrocytes, removing samples from the mixture, and estimating colorimetrically the hemoglobin in the supernatant fluid of the centrifuged samples. Most of the curves (but not all of them, e.g. tyrocidine) obtained by plotting degree of hemolysis against time, were S-shaped. The initiation of lysis by streptolysin S'' was delayed, and in this property, streptolysin S'' was similar to Cl. septicum hemolysin. None of the other lysins studied exhibited a long latent period preceding lysis. The maximum rate of hemoglobin liberation was found, in the range of lysin concentrations studied, to be a linear function of concentration when theta toxin of Cl. welchii, pneumolysin, tetanolysin, or streptolysin S'' was the lytic agent. With comparable concentrations of saponin, sodium taurocholate, cetyl pyridinium chloride, tyrocidine, duponol C, lecithin-atrox venom mixture, or streptolysin O, the relation between rate and concentration was non-linear. The critical thermal increment associated with hemolysis was determined for systems containing pneumolysin, theta toxin, streptolysin S'', streptolysin O, tetanolysin, and saponin. The findings concerning the effect of concentration and temperature on the rate of hemolysis provide a basis for classifying hemolytic agents (Tables I and II). Hemolysis induced by Cl. septicum hemolysin was found to be preceded by two phases: a phase of alteration of the erythrocytes and a phase involving swelling. Antihemolytic serum inhibited the first but not the second phase while sucrose inhibited the second but not the first phase.     

Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid

The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl. perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations). When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations. In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl. perfringens antitoxin. The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl. perfringens type A toxoid preparations.     

Detection of Clostridial Hemolysin Formed In Vivo

Experimental clostridial myonecrosis closely resembling gas gangrene was produced in goats by the intramuscular injection of washed clostridia suspended in adrenalin. The preparation provides a model for the study of clostridial myonecrosis that is relatively isolated from preceding trauma. A new test for the demonstration of clostridial exotoxins, the hemolysin indicator plate test, was described. This test gave reliable results within 6 hr, and only minute quantities of test material were required. Application of this test to would exudates of goats with experimental clostridial myonecrosis demonstrated the presence of the gamma toxin of Clostridium novyi, alpha toxin of C. septicum, and the alpha toxin of C. perfringens in exudates from animals injected with washed Clostridium bacilli suspended in adrenalin. Exudates from goats injected with C. perfringens were lethal for mice and produced necrosis of guinea pig skin typical of C. perfringens. The convincing demonstration of clostridial exotoxins formed in vivo in this experimental model could be the basis for definition of clostridial exotoxin actions which have evaded study in the past.     

Study of leukotoxic activity of Cl. perfringens type A toxin and its fractions obtained by gel filtration and ion exchange chromatography]

In gel-filtration of Cl. perfringens type A toxin on Sephadex F-100 and F-50 there was revealed relationship between the leukotoxic activity and a high-molecular component. A method of ion-exchange chromatography on a column with DEAE-Sephadex A-25 from the Cl. perfringens toxin there were obtained 8 fractions 3 of which possessed a marked leukotoxic activity: the capacity to destroy neutrophils in the Svejcar and Vancurik test and to-depress the phagocytic activity of leukocytes. Lecithinase and lethal activity was revealed in one of these fractions only. Leukotoxic fractions differed by the capacity to destroy neutrophils and to decrease their phagocytic activity.     

Selection of herbal therapeutics against deltatoxin mediated Clostridial infections

Clostridium perfringens (a versatile pathogenic bacterium) secretes enterotoxins (the deltatoxin, virulent factor) and causes food borne gastroenteritis and gasgangrene. The organism was isolated and characterized from improperly cooked meat and poultry samples. The isolated organism showed multiple drug resistance indicating that the treatment is challenging. Hence, there is need for improved therapeutic agents. The rational design of improved therapeutics requires the crystal structure for the toxin. However, the structure for the toxin is not yet available in its native form. Thus, we modeled the toxin structure using α- hemolysin of Staphylococcus aureus (PDB: 3M4D chain A) as template. The docking of the toxin with the herbal extract curcumin (1,7-bis(4-hydroxy-3- methoxyphenyl)hepta-1,6-diene-3,5-dione) showed a binding energy of -8.6 Kcal/mol, in comparison to the known antibiotic Linezolid with binding energy of -6.1 Kcal/mol. This data finds application in the design and development of novel compounds against the deltatoxin from Clostridium perfringens.     

Detection of α- and ε-toxigenic Clostridium perfringens Type D in sheep and goats using a DNA amplification technique (PCR)

Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the α-, β- and ε-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the α- and ε-toxin genes but were devoid of the β-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the α-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the α- and ε-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the ε-toxin gene, whereas the majority of the colonies were of type A with the α-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens . The β-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation.     

Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts

Davie, Joseph M. (Indiana University, Bloomington), and Thomas D. Brock. Action of streptolysin S, the group D hemolysin, and phospholipase C on whole cells and spheroplasts. J. Bacteriol. 91:595-600. 1966.-The effect of streptolysin S, the group D hemolysin, and phospholipase C (the alpha toxin of Clostridium perfringens) on whole cells and spheroplasts or protoplasts of three strains of streptococci and Micrococcus lysodeikticus was tested. Viability, C(14)-glycine uptake, and lysis were measured. The group D hemolysin and phospholipase C were active against whole bacteria; streptolysin S was not. All three substances were active on spheroplasts. A partially resistant mutant derived from a strain sensitive to the group D hemolysin was also partially resistant to streptolysin S and phospholipase C. Antimycin A protected spheroplasts from streptolysin S but not from the group D hemolysin.