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.     

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.     

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.     

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.     

The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine

Necrotic enteritis in sucking piglets constitutes a serious problem in piglet rearing units because of the high morbidity and mortality associated with the disease. The primary causal agent is Clostridium perfringens type C. The beta-toxin plays a decisive role in the pathogenesis of this disease. A toxoid vaccine for use in sows has been developed and studied in field trials. The European Pharmacopoeia Monograph on vaccines for use in animals lays down a method of the efficacy testing based on the immunization of rabbits, the collection of pooled sera and the subsequent assay of anti-toxin antibodies in mice using an appropriate test toxin. The vaccine is regarded as effective if it induces a minimum of 10 IU of beta-anti-toxin per ml of rabbit serum. We have established a range of 17.14-98.23 IU beta-anti-toxin per ml rabbit serum induced by a sample of C. perfringens toxoid vaccine. The vaccine has been used under field conditions in different rearing units at the same time, mostly in the form of emergency vaccinations following the outbreak of disease. The outcome of vaccination was evaluated by recording the total numbers of piglets born alive and the piglet losses. Use of the vaccine, coupled with other measures, resulted in an approximately 30% reduction in the number of losses.     

Protective properties of theta-hemolysin obtained by affinity chromatography

The data on the study of the protective activity of theta hemolysin and Cl. perfringens lecithinase preparations and the corresponding antitoxic sera obtained by indirect immune affinity chromatography are presented. Experiments in mice and guinea pigs indicate that the injection of antihemolytic serum and immunization with anatheta hemolysin ensures the protection of the animals from theta toxin. The enrichment of analecithinase preparation with anatheta hemolysin has been found to increase its protective properties against Cl. perfringens culture and toxin.     

The use of the in vitro toxin binding inhibition (ToBI) test for the estimation of the potency of tetanus toxoid

The in vitro toxin binding inhibition (ToBI) test was used to determine antitoxin responses in mice immunized with tetanus toxoid. The ToBI test showed good correlation with the in vivo toxin neutralization (TN) test in titration of sera of mice immunized with various doses of DPT-Polio, DT-Polio and a tetanus reference preparation. Estimates of potency of tetanus toxoid obtained in mice by ToBI test correlated significantly with those obtained in mice by the lethal challenge test. In addition, potency values of the European reference preparation, succeedingly estimated by ToBI test and lethal challenge test in a single group of guinea-pigs, showed good correlation. From the study it is concluded that the ToBI test is a promising alternative to the toxic challenge procedure in the potency assay of tetanus toxoid vaccines. A substantial refinement and reduction in the use of animals can be achieved. Additional savings can be made by combining diphtheria and tetanus potency testing.     

A Study of Rapid and Simplified Confirmatory Tests for Clostridium perfringens

Strains of Clostridium perfringens and culturally similar species which also may grow on selective isolation media for this organism were examined by conventional confirmatory tests, the API ZYM system and by individual tests for phosphatase and glutamic acid decarboxylase activity.
API ZYM tests, involving 19 different enzymes, confirmed the known similarity between Cl. perfringens, Cl. absonum, Cl. paraperfringens and Cl. sardiniensis but effectively distinguished this group from Cl. bifermentans, Cl. celatum, Cl. perenne and Cl. sordellii. A similar separation was achieved by a single test for acid phosphatase which could be applied to individual colonies on a plating medium.
Because the acid phosphatase test was found to be of greater value than nitrate reduction in distinguishing Cl. perfringens , it could replace the latter in the usual series of confirmatory tests. It is suggested that strains from Cl. perfringens isolation media should be screened for acid phosphatase activity at the purification stage and only positive strains subjected to further tests.
It was found that Cl. perfringens could not be distinguished from the other species on the basis of glutamate decarboxylase activity.     

Homogeneous Cl. perfringens alpha-anatoxin: its physicochemical and immunogenic properties]

The authors present a method of obtaining relatively homogeneous preparations of alpha-toxoid of Cl. perfringens, type A, including the primary conception of the alpha-toxin proteins, their chromatography on DEAE-cellulose, fractionation with (NH4)2SO4, detoxication, with the subsequent gel-filtration through sephadex and isoelectric focussing. Sedimentation coefficient of the preparation proved to be 3.8 S, isoelectric point-4.83 +/- 0.07. In studying the immunogenic properties of alpha-toxoid in experiments on guinea pigs and rabbits their high immunogenicity, exceeding that of the industrial toxoid 8- and 6-fold, respectively, was established. Homogeneous preparations of alpha-toxoid provided intense anti-microbial immunity. Interlinear differences in the levels of the immune response of inbred mice, highly-reactive (BALB/c) and low-reactive (C57BL/6) to alpha-toxoid, reached 20-fold; in combination with the high immunogenicity of this antigen for mice this permits to recommend it for immunogenic studies.     

An investigation of a mouse model to estimate the potency of the diphtheria component in vaccines

A mouse model to estimate the potency of the diphtheria toxoid components in vaccines using Vero cells to detect the neutralizing antibodies in the sera from immunized mice is described. The results obtained with this mouse model correlated significantly with those obtained in the lethal challenge test in guinea-pigs. For this reason it is suggested that the potency test in guinea-pigs be replaced by this mouse model because a considerable reduction in the number of animals used and costs can be achieved by the introduction of this mouse test for the routine control of the potency of the diphtheria component of vaccines.     

Detection by in vitro amplification of the alpha-toxin (phospholipase C) gene from Clostridium perfringens

A polymerase chain reaction (PCR) with thermostable DNA polymerase from Thermus aquaticus is described for the specific amplification of the phospholipase C (alpha-toxin) gene of Clostridium perfringens. A set of primers selected for their high specificity could detect Cl. perfringens in stools with a detection limit of approximately 5 x 10² bacteria, after bi-amplification. A modified PCR without thermal steps was performed to rapidly amplify, with a yield of 60%, the DNA template. With this PCR method Cl. perfringens alpha-toxin gene could be detected within 2 h. The PCR method detected alpha-toxin positive Cl. perfringens but did not react with phospholipase C-producing Bacillus cereus, Pseudomonas aeruginosa, Cl. sordellii and Cl. bifermentans.
The amplified PCR products were screened through ethidium bromide agarose gel electrophoresis or, in only 1 h, with the PhastSystem (Pharmacia). This PCR satisfies the criteria of specificity, sensitivity and rapidity required for a useful tool in epidemiology and for the diagnosis of the pathogen Cl. perfringens as it may be used directly on stool samples.     

PCR detection of Clostridium perfringens producing different toxins in faeces of goats

A polymerase chain reaction (PCR) was used to identify the genes encoding the major toxins of Clostridium perfringens in faeces of goats. When pure cultures of Cl. perfringens types A, B, C, D and E were used as templates in the PCR, amplicons were observed on the agarose gel as bands at approximately the 247 (alpha primers), 1025 (beta primers), 403 (epsilon primers) and 298 (iota primers) bp level of the DNA marker. When used to identify different types of Cl. perfringens in samples artificially spiked with these micro-organisms, the PCR detected as few as 1–1·5×10² cfu g⁻¹ of the five types of Cl. perfringens tested. The PCR technique allowed the identification and typing of Cl. perfringens strains in faeces of goats, without recourse to other techniques such as the mouse neutralization test.     

Changes in the serum acid phosphatase activity of guinea pigs poisoned with C1 perfringens type A toxin and a mixture of toxin and a broth culture filtrate

It was shown in experiments on guinea pigs that the injection of C1. perfringens type A toxoid induced an increase in acid phosphatase activity of animal blood serum. The action of the toxoid increased under the effect of C1. butyricum cultural filtrate, which gave rise to an earlier enhancement of the specific activity of the enzyme as compared to the injection of the toxoid alone. Increased activity of acid phosphatase may play a pathogenetic role in cases of anaerobic infection caused by association of C1. perfringens and C1. butyricum.     

Characteristics of a sporulation stimulating factor from Clostridium perfringens type A

A product in the culture supernatant fluid of Clostridium perfringens NCTC 8239 stimulated the sporulation of a test strain, NCTC 8679, of the same organism. The responsible factor, termed sporulation factor (SF), was present in seven cultures of Cl. perfringens grown in either a defined or complex medium. The SF reversed glucose-mediated catabolite repression of sporulation by this organism. Preliminary characterization of the SF demonstrated a resistance to elevated temperatures and proteases and a molecular weight of less than 500 Da. The known association of Cl. perfringens enterotoxin with sporulation highlights the importance of interactions between strains of this organism as may occur in the human intestine during foodborne illness.     

An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon components of multivalent sheep vaccines

Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice. ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins. The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test.     

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.     

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.     

Further research on the detoxifying effect of RNA

Type A Cl. perfringens toxin caused necrosis in guniea-pigs and killed mice. Sodium nucleinate, administered orally to these animals simultaneously with the injection of the toxin and 2 hours later significantly prevented dermonecrosis in guinea-pigs and death in mice, as well as the development of pathomorphological changes in the liver, the kidneys and the spleen, the fatty degeneration of hepatocytes and disturbances in nucleic acid metabolism in these cells.     

Production and characterization of Clostridium perfringens recombinant β toxoid

In this work, we produced and evaluated a vaccine based on a β toxoid of Clostridium perfringens type C produced in Escherichia coli (rBT). The non-toxic rBT was innocuous for mice and induced 14 IU mL(-1) of β antitoxin in rabbits, complying with the European Pharmacopeia and CFR9 - USDA guidelines.     

Comparison of latex agglutination and ELIS A for the detection of Clostridium perfringens type A enterotoxin in faeces

One hundred and thirty one faecal specimens from cases of suspected Clostridium perfringens food poisoning were examined by both a reverse passive latex agglutination test and a standard ELISA test for the presence of Cl. perfringens enterotoxin. The latex agglutination test proved as sensitive and specific as the ELISA, and required less time at the bench without the need for specialized equipment.