TL;DR: In this article, the usefulness of cytopathic indicators for the titration of Cl perfringens beta and epsilon toxins has been investigated and it was concluded that cell culture titration offers a valid in vitro alternative to the use of mouse lethal and guinea-pig dermonecrotic indicators.
Abstract: 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.
TL;DR: In this paper, an alternative indicator system based on the use of cell cultures is described, and evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity.
Abstract: The assay of Clostridium septicum antitoxin currently requires the inoculation of test mixtures intravenously into mice or intradermally into guinea-pig skin. An alternative indicator system based on the use of cell cultures is described. Evidence is presented to show that the toxins detected by the in vivo and in vitro indicators are indistinguishable in terms of molecular weight, charge and hydrophobicity and that there is a close agreement between the two methods of titration. Cell culture indicators are more sensitive than their in vivo counterparts, permitting detection of substantially lower titres than is possible using in vivo indicators. It is suggested that cell culture indicators may prove useful for the titration of Cl septicum antitoxin in sera from vaccine field trials and potency tests. Cell culture methods could also be used for the potency testing of antitoxin preparations.
TL;DR: The diphtheria antitoxin titres determined in human sera by the intradermal titration method have been compared with the titres obtained by a range of ELISA assays and an assay based on capture of toxoid by a non-neutralizing monoclonal antibody produced results that correlated very well.
Abstract: The diphtheria antitoxin titres determined in human sera by the intradermal titration method have been compared with the titres obtained by a range of ELISA assays. A direct ELISA based on highly purified diphtheria toxoid correlated poorly with the in vivo method but an assay based on capture of toxoid by a non-neutralizing monoclonal antibody produced results that correlated very well. The performance of assays involving partial blockade of antigen and non-neutralizing monoclonal antibodies was intermediate.
TL;DR: The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells, and the crystal structure of ε‐toxin reveals similarity to aerolysin from Aeromonas’hydrophila, parasporin‐2 from Bacillus’thuringiensis and a lectin from Laetiporus’sulphureus.
Abstract: Clostridium perfringens e-toxin is produced by toxinotypes B and D strains. The toxin is the aetiological agent of dysentery in newborn lambs but is also associated with enteritis and enterotoxaemia in goats, calves and foals. It is considered to be a potential biowarfare or bioterrorism agent by the US Government Centers for Disease Control and Prevention. The relatively inactive 32.9 kDa prototoxin is converted to active mature toxin by proteolytic cleavage, either by digestive proteases of the host, such as trypsin and chymotrypsin, or by C. perfringens λ-protease. In vivo, the toxin appears to target the brain and kidneys, but relatively few cell lines are susceptible to the toxin, and most work has been carried out using Madin-Darby canine kidney (MDCK) cells. The binding of e-toxin to MDCK cells and rat synaptosomal membranes is associated with the formation of a stable, high molecular weight complex. The crystal structure of e-toxin reveals similarity to aerolysin from Aeromonas hydrophila, parasporin-2 from Bacillus thuringiensis and a lectin from Laetiporus sulphureus. Like these toxins, e-toxin appears to form heptameric pores in target cell membranes. The exquisite specificity of the toxin for specific cell types suggests that it binds to a receptor found only on these cells.
TL;DR: The results highlight the importance of beta toxin for type C-induced toxemia and surveyed a large collection of type C isolates to determine their toxin-producing abilities.
Abstract: The gram-positive anaerobe Clostridium perfringens produces a large arsenal of toxins that are responsible for histotoxic and enteric infections, including enterotoxemias, in humans and domestic animals. C. perfringens type C isolates, which cause rapidly fatal diseases in domestic animals and enteritis necroticans in humans, contain the genes for alpha toxin (plc), perfringolysin O (pfoA), beta toxin (cpb), and sometimes beta2 toxin (cpb2) and/or enterotoxin (cpe). Due to the economic impact of type C-induced diseases, domestic animals are commonly vaccinated with crude type C toxoid (prepared from inactivated culture supernatants) or bacterin/toxoid vaccines, and it is not clear which toxin(s) present in these vaccines actually elicits the protective immune response. To improve type C vaccines, it would be helpful to assess the contribution of each toxin present in type C supernatants to lethality. To address this issue, we surveyed a large collection of type C isolates to determine their toxin-producing abilities. When late-log-phase vegetative culture supernatants were analyzed by quantitative Western blotting or activity assays, most type C isolates produced at least three lethal toxins, alpha toxin, beta toxin, and perfringolysin O, and several isolates also produced beta2 toxin. In the mouse intravenous injection model, beta toxin was identified as the main lethal factor present in type C late-log-phase culture supernatants. This conclusion was based on monoclonal antibody neutralization studies and regression analyses in which the levels of alpha toxin, beta toxin, perfringolysin O, and beta2 toxin production were compared with lethality. Collectively, our results highlight the importance of beta toxin for type C-induced toxemia.
TL;DR: Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4- sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate.
Abstract: A new cytotoxicity assay for determining the activity of epsilon toxin produced by Clostridium perfringens type D has been developed. Viability of cultured cells was determined by the ability of only live cells to convert 5-(3-carboxymethoxyphenyl)-2-(4,5-dimethylthiazolyl)-3-(4-sulfophenyl)tetrazolium to the coloured product formazan in the presence of phenazine methosulfate. Of the 12 cell lines tested, only the MDCK cell line was susceptible to epsilon toxin. Specificity was confirmed by the ability of only specific monoclonal antibodies to inhibit cytotoxicity. Good correlation was obtained with the mouse lethality assay (r = 0.991) and over a wide range of viability (15–75%) as determined by ethidium bromide/acridine orange staining (r = 0.995).
TL;DR: Recombinant beta-toxin from Clostridium perfringenstype C was found to increase the conductance of bilayer lipid membranes by inducing channel activity, and the hypothesis that the lethal action of beta-Toxin is based on the formation of cation-selective pores in susceptible cells is supported.
Abstract: Beta-toxin is produced by Clostridium perfringens type B and C strains and is the primary lethal factor in the type C strains. No molecular mechanism has been elucidated for beta-toxin which could be used as a basis for investigating its role in the pathogenesis of these clostridial pathogens. It has been suggested that beta-toxin may be a pore-forming toxin on the basis of weak similarities (10% identity) between the primary structure of beta-toxin and those of the pore-forming alpha-hemolysin and gamma-hemolysin and the leukocidin from Staphylococcus aureus (9). Whether or not beta-toxin is cytotoxic remains unclear; only a single report has suggested that beta-toxin is weakly cytotoxic on intestinal 407 cells (6). However, a previous study suggested that the cytotoxicity associated with beta-toxin preparations was not linked to the beta-toxin itself, but to minor contaminants in the toxin preparation from C. perfringens (11). Recently, Steinthorsdottir et al. demonstrated that beta-toxin could induce the release of arachidonic acid and inositol from human umbilical vein endothelial cells (HUVECs) (32). No cytolytic effects were reported, suggesting that beta-toxin may not be necessarily lethal to these cells. Several other cell types were also tested by these investigators, but they were unresponsive to beta-toxin. C. perfringens type C strains cause necrotic enteritis primarily in pigs, chickens, cattle, sheep, and goats. Although adult animals can contract this disease, it most frequently occurs in the young of these species (34). Piglets are particularly susceptible to type C infections (5, 10, 18, 33), although a similar infection occurs in neonatal calves (7), lambs (8), and goats. During a type C infection, necrosis of the intestine can be extensive; death appears to be the result of toxemia with beta-toxin (reviewed in reference 29). Acute and peracute deaths frequently occur in these animals, suggesting that systemic effects of the toxin are important. In a C. perfringens type C disease of adult sheep, termed “struck,” the animals succumb to the infection so rapidly that they appear to have been struck by lightning. Prior to death, nervous signs such as tetani and opisthotonus have been observed in these animals (reviewed in reference 29), suggesting neurological involvement. Infection of humans by type C strains appears to be largely restricted to certain tribal populations in Papua New Guinea, although infrequent cases of type C infection have occurred in humans throughout the world. Type C infections result in necrotizing enterocolitis (“pigbel”) in these individuals after consumption of undercooked pork during certain ritualistic practices (13). Typically, type C necrotizing enterocolitis in humans resembles the disease in animals. The importance of beta-toxin in both animal and human disease has been demonstrated by immunization studies using a toxoid of beta-toxin. When immunized with the toxoid of beta-toxin, the Papua New Guinea tribespeople experienced a fivefold reduction in the incidence of necrotic enteritis (13), whereas a beta-toxin toxoid administered to infant pigs during an outbreak of necrotizing enterocolitis reduced mortality by approximately 30% (30). In the case of agriculturally important animals, vaccination against type C infections is universally advocated in order to avoid devastating losses. Therefore, beta-toxin plays a key role in the lethal outcome of type C infections, yet we know very little about its mechanism or the cell types it affects. The results presented below demonstrate that beta-toxin is an efficient pore-forming toxin which generates potential-dependent, cation-selective channels in membranes. The channels formed by beta-toxin exhibit characteristics that may provide some insight into the lethal activity of this toxin.
TL;DR: A "best-fit" combined exponential regression model was used to predict the optimal timing for booster vaccinations against diphtheria, and data support a 3, 5, 12 month schedule followed by a fourth dose 4-5 years after the third dose, depending upon the vaccine used.
Abstract: Data from two Swedish pertussis vaccine trials with various combination vaccines were used to compare anti-diphtheria antitoxin concentrations over time between different vaccines, vaccine lots and vaccine schedules. The immune responses were measured with a validated ELISA method. Results are given for 1326 children, born 1992, that were recruited to the placebo (DT)-controlled Trial I which used a 2, 4, 6 month schedule. Two DTP acellular and one DTP whole cell vaccine were used. No DT boosters were given until 5 years of age. Trial II recruited children born 1993–94 and compared three DTP acellular vaccines with one DTP whole cell vaccine. Results are given for 306 children in a 2, 4, 6 month schedule and for 531 children in a 3, 5, 12 month schedule. The latter schedule gave significantly higher diphtheria antitoxin concentrations post third dose. The various DTP acellular vaccines and an inefficacious DTP whole cell vaccine gave lower antitoxin concentrations than both an efficacious DTP whole cell vaccine and the DT vaccine. The larger differences in antigen response between vaccines was reduced in the course of time. Generally, an initial rapid decline of antitoxin concentration was followed by a slower decline; the change typically occurring when the antitoxin concentration reached 0.13–0.16 EU/ml. The time needed to reach this level was between 6 and 10 months based on the initial vaccine response. A “best-fit” combined exponential regression model was used to predict the optimal timing for booster vaccinations against diphtheria. Our data support a 3, 5, 12 month schedule followed by a fourth dose 4–5 years after the third dose, depending upon the vaccine used.