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.     

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.     

Use of the passive hemagglutination test for the purpose of determining antibody levels following animal immunization with Cl. perfringens toxoid]

Data are presented on the study of possibilities of the application of the passive hemagglutination test for titration of the blood sera of mice, guinea pigs and rabbits immunized with Cl. perfringens toxoid. A diagnostic agent obtained by the sensitization of formalin- and tannin-treated sheep erythrocytes with the serologically pure toxoid, and homologous sera (as standard) were used in this test. A high immune response of BALB/c and C3H mice to the Cl. perfringens toxoid permits to suggest inbred mice as a model for the immunological and immunogenetic studies connected with this toxoid.     

Immunization with an alphatoxin variant 121A/91-R212H protects mice against Clostridium perfringens alphatoxin

As shown previously, a recombinant alphatoxin variant (rAT121A/91) constructed from the naturally occurring Clostridium perfringens mutant strain 121A/91, was devoid of enzymatic (PLC), hemolytic and lethal activity (18). In the present study, the recombinant variant was altered by an oligonucleotide-directed reversion of an arginine in position 212 for a histidine residue, corresponding to the sequence of the wild-type alphatoxin. The new variant rAT121A/91R212H proved to be negative in enzymatic, hemolytic and lethal activity as well. RAT121A/91 as well as rAT121A/91R212H was used for i.p. immunization of balb/c mice. The immune response was studied in ELISA as well as in the mouse neutralization test. Furthermore, immunized mice were challenged by i.p. application of active C. perfringens alphatoxin. In all immunized groups, mice developed high anti-alphatoxin titers (up to 1:128000). Antisera of both groups were able to reduce the hemolytic effect of native alphatoxin with predominance of anti-rAT121A/91R212H sera. During neutralization experiments, mice receiving a mixture of anti-rAT121A/91R212H and wild-type toxin were protected completely, whereas an anti-rAT121A/91/toxin mixture prolonged time until death but failed in protection. I.p immunization with rAT121A/91R212H yielded a significant protection rate (76%) when mice were challenged intraperitoneal with wild-type toxin. Our cumulative data indicates that the reversion of arginine in position 212 to histidine for rAT121A/91R212H was necessary to induce production of protective antibodies against wild-type alphatoxin of C. perfringens.     

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.     

C型肉毒毒素的制备及类毒素保护力初探

将C型肉毒梭菌经适宜条件的产毒培养后纯化,并进行相关鉴定。制备的C型肉毒毒素用分段脱毒法脱毒,并进行类毒素保护力的初步研究。以不同蛋白含量C型肉毒类毒素免疫小鼠后攻毒,结果显示,蛋白含量为0.625μg的类毒素免疫2针或蛋白含量为1.25μg的类毒素免疫1针均可保护50LD50的C型肉毒毒素攻击。蛋白含量为5μg的C型肉毒类毒素与福氏不完全佐剂配制的抗原免疫小鼠3次所得抗血清的保护力(Anti LD50/ml)为4.3×104。说明用该纯化工艺制备的C型肉毒类毒素具有很好的免疫原性,作为抗原成分用于C型肉毒疫苗和C型肉毒抗毒素的研究和生产具有较好的应用潜力。     

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.     

Purification and properties of staphylococcal beta hemolysin. II. Purification of beta hemolysin

Staphylococcal beta hemolysin from the 681 strain of Staphylococcus aureus grown in a Heart Infusion dialysate semisolid medium under 10% carbon dioxide was obtained in an immunoelectrophoretically pure form by a combination of procedures of precipitation with 2 volumes of acetone followed by chromatography on diethylaminoethyl cellulose at pH 6.0. The acetone precipitation procedure did not show any deleterious effect on the hemolytic activity of the beta hemolysin unless the precipitate was left in contact with the acetone for at least 4 hr. The crude preparations contained two types of beta hemolysin. One of these represented the major portion of the total activity of beta hemolysin and behaved as a cation. The other represented a minor (1/5,000) portion of the total beta hemolysin activity and behaved as an anion. These active principles were designated as cationic and anionic beta hemolysins, respectively. An unexpected increase in the total beta hemolysin activity of the crude preparations was noted when these were concentrated by dialysis against polyethylene glycol (20 m). This effect was probably due to polyethylene glycol. A further unexpected increase in the titer of the acetone-precipitated preparations occurred when these were lyophilized. The reason for this incremental increase is not known. It may be due to fragmentation of the beta hemolysin.     

Protective properties of anatoxin obtained from a homogeneous preparation of Pseudomonas aeruginosa exotoxin A

The protective properties of formulated toxoid obtained from the highly purified preparation of P. aeruginosa exotoxin A have been studied in the test of the active immunization of mice. The study has revealed that the preparation when introduced in 1 or 2 injections in a dose of 15 micrograms, shows faint protective potency with respect to P. aeruginosa strains differing in virulence. Immunization with this toxoid in 3 and 4 injections has been found to ensure 60-100% and 50-60% protection of mice infected with P. aeruginosa toxigenic and proteolytic strains respectively. Immunization with toxoid has been found to induce the appearance of short-term antibacterial immunity which loses its capacity to protect the immunized animals, challenged with both toxigenic and proteolytic P. aeruginosa strains, as early as on day 28. The immunization of mice with toxoid in 4 injections has been shown to induce the development of antitoxic immunity capable of neutralizing up to 150 LD50 of purified exotoxin A.     

Conjugation of tetanus toxoid with Salmonella typhimurium PTCC 1735 O-specific polysaccharide and its effects on production of opsonizing antibodies in a mouse model

Serious enteric and extra-intestinal infections with Salmonella typhimurium are very common in many human populations. Phagocytosis is the main defense mechanism against this bacterium; however, the unique structure of S. typhimurium lipopolysaccharide (LPS) makes it resistant to opsonization by complement components. In the present study, the S. typhimurium LPS O-chain was purified and conjugated with tetanus toxoid and the effects of the conjugated vaccine (O-specific polysaccharide tetanus toxoid (O-SP-TT)) on induction of specific antibodies were investigated in a mouse model. In vitro assays measuring phagocytosis in the presence of opsonizing antibodies were performed. Three subcutaneous injections of the O-SP-TT conjugate conferred protection against the intraperitoneal challenge with S. typhimurium and the LD50 was greater for immunized animals than for controls. The mean number of ingested S. typhimirium / mouse peritoneal cell in the presence of sera obtained from immunized mice with purified O-chain, O-SP-TT conjugate, heat-killed bacteria, and negative control were 6.96, 14.24, 15.96, and 6.67, respectively. In summary, we developed an O-SP-TT conjugate that induced opsonizing antibodies that increased phagocytosis, as determined by in vitro assays. In addition, chemiluminescence results, an indicator of oxidative burst, indicated that peritoneal cells respond better to live S. typhimurium cells in the presence of sera obtained from O-SP-TT conjugate immunized mice.     

Outcome of simian-human immunodeficiency virus strain 89.6p challenge following vaccination of rhesus macaques with human immunodeficiency virus Tat protein

The regulatory proteins Nef, Rev, and Tat of human immunodeficiency virus type 1 (HIV-1) are attractive targets for vaccine development, since induction of effective immune responses targeting these early proteins may best control virus replication. Here we investigated whether vaccination with biologically active Tat or inactive Tat toxoid derived from HIV-1(IIIB) and simian-human immunodeficiency virus (SHIV) strain 89.6p would induce protective immunity in rhesus macaques. Vaccination induced high titers of anti-Tat immunoglobulin G in all immunized animals by week 7, but titers were somewhat lower in the 89.6p Tat group. Dominant B-cell epitopes mapped to the amino terminus, the basic domain, and the carboxy-terminal region. Tat-specific T-helper responses were detected in 50% of immunized animals. T-cell epitopes appeared to map within amino acids (aa) 1 to 24 and aa 37 to 66. In addition, Tat-specific gamma interferon responses were detected in CD4+ and/or CD8+ T lymphocytes in 11 of 16 immunized animals on the day of challenge. However, all animals became infected upon intravenous challenge with 30 50% minimal infective doses of SHIV 89.6p, and there were no significant differences in viral loads or CD4+ T-cell counts between immunized and control animals. Thus, vaccination with HIV-1(IIIB) or SHIV 89.6p Tat or with Tat toxoid preparations failed to confer protection against SHIV 89.6p infection despite robust Tat-specific humoral and cellular immune responses in some animals. Given its apparent immunogenicity, Tat may be more effective as a component of a cocktail vaccine in combination with other regulatory and/or structural proteins of HIV-1.     

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.     

The isolation of the ribosomal fraction of Clostridium perfringens type A and an evaluation of its protective activity

A method for isolation of the ribosomal fraction (RF) from the cytoplasm of type-A C. perfringens strain BP6K was developed and its chemical and antigenic properties characterized. RF has been found to possess protective properties: two subcutaneous immunizations of mice with RF preparations adsorbed on Al(OH)3 in doses of 250 and 500 micrograms (dry weight) has ensured, on the average, the protection of 41.9% of the immunized animals from 1 DCL of type-A C. perfringens strain BP6K culture.     

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.     

Pseudomonas aeruginosa toxoid

P. aeruginosa adsorbed toxoid has been obtained. The stabilization of exotoxins and the content of proteases, hemolysin, lecithinase in their structure have been found to enhance the immunogenic potency of preparations which protect test animals from death caused by the experimental injections of toxins, homologous and heterologous to bacterial strains of different O-serogroups, into these animals. Antibodies neutralizing the lethal action of P. aeruginosa exotoxin have been detected in the blood sera of immunized animals.     

Purification of the toxoids of Cl. Perfringens and Cl. pedematiens using silicagel]

A possibility was shown of purification of the concentrated toxoids of the Cl. perfringens and Cl. oedematiens species in examination of KSK-2, KSK-2,5; MCA-2 silicagels. The specific activity of the preparations obtained and the yield approached the analogous indices obtained in stratification of Sephadex G-75. The effect of preliminary silicagel treatment and also of the nature of buffer solutions on the efficacy of the process of purification of the gangrenous toxoids was established.     

Purification of Clostridium toxoids.

A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids. The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration. Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid.     

Characteristics of the immunologic response of animals to Clostridium perfringens anatoxin]

Experiments were conducted on guinea pigs, rabbits and mice (mongrel and inbred); immunogenic properties of Cl. perfringens toxoids of different purity were studied. Toxin neutralization and passive hemagglutination tests were used to determine the antitoxic immunity level. It appeared that in the immunization of guinea pigs and rabbits the degree of immunogenicity of the preparations increased with the elevation of their specific activity. Under the same conditions both the mongrel and the inbred mice displayed the maximum immune response in the immunization with the least purified preparations, and the minimum after the injection of a highly purified antigen.     

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 μ g/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin. Thirty μg of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops. Aggregated enterotoxin had no demonstrable biological or immunological activity.     

Effect of carrier molecules on production and properties of extracellular hemolysin produced byStreptococcus agalactiae

Streptococcus agalactiae type la strain 090 produced a cell-associated hemolysin during exponential growth in medium lacking proteins. Growth of the organism in medium containing proteins or medium supplemented with Tween 40 resulted in the appearance of extracellular hemolytic activity that was filterable. Maximum extracellular hemolytic activity was obtained in the late exponential phase of growth corresponding to the maximum number of cells. Extracellular hemolysin released in medium containing proteins could be precipitated by ammonium sulfate. Cell-associated hemolysin could be extracted in the cold by purified lipoteichoic acid from the organism. Purification and characterization of the extracellular hemolysin by column chromatography showed that the hemolysin was associated with molecules eliciting its release. Hemolysin associated with lipoteichoic acid or Tween 40 had an apparent molecular weight of 1,800,000 or 60,000 daltons, respectively.