Problems encountered with the capillary tube immunodiffusion method for detection of botulinal toxin.

By using antitoxin specific for the neurotoxin molecule, the capillary tube immunodiffusion method did not detect low levels of crystalline toxin. Reactions described earlier with crude toxin and less specific antitoxin were probably due to nontoxigenic botulinal antigens.     

Problems encountered with the capillary tube immunodiffusion method for detection of botulinal toxin

By using antitoxin specific for the neurotoxin molecule, the capillary tube immunodiffusion method did not detect low levels of crystalline toxin. Reactions described earlier with crude toxin and less specific antitoxin were probably due to nontoxigenic botulinal antigens.     

In vitro acetylcholinesterase inhibition by type A botulinum toxin

Type A botulinum toxin was studied for its ability to inhibit the action of acetyl-cholinesterase. The chromogenic substrate, indophenyl acetate, was used for assay of enzyme activity. Inhibition of enzyme function was detected through use of both 6.6 x 10(-6) mg (20 ld(50)) and 6.6 x 10(-10) mg (2 x 10(-3)ld(50)) of type A botulinal toxin. Control assays were performed by use of both homologous antitoxin and heterologous antitoxins (types B and E). Enzyme inhibition was effectively prevented by use of homologous antitoxin only. The inhibition noted was specific and reproducible for given substrate, enzyme, and toxin concentrations.     

Correlation of Clostridium botulinum type C antitoxin titers in mink and guinea pigs to protection against type C intoxication in mink

In USA, the potency of commercial vaccines containing Clostridium botulinum type C toxoid is determined by a mink vaccination-challenge assay outlined in the Code of Federal Regulations, Title 9, Part 113.110. A more humane potency test is desired, and this study provides preliminary data in support of a serological assay that correlates post-vaccination antitoxin titers of guinea pigs to vaccine efficacy in mink. Mink and guinea pigs were injected with varying dilutions of a vaccine containing C. botulinum type C toxoid. Blood samples were collected from each animal prior to challenging the mink with type C toxin. Serum antitoxin titers of mink and guinea pigs were measured by a mouse protection test, and the results were compared to the outcome of the toxin challenge in mink. A dose-dependent antitoxin response was observed in guinea pigs vaccinated with the critical dilutions of vaccine bracketing the minimum protective dose in mink. These preliminary data suggest that it may be possible to correlate post-vaccination antitoxin titers in guinea pigs to vaccine efficacy in mink. This correlation could be used as the basis for a more humane potency test for C. botulinum type C toxoids.     

Toxoid of Clostridium botulinum type F: purification and immunogenicity studies.

Toxin from Clostridium botulinum type F was recovered from dialysis cultures and partially purifed by: (i) ammonium sulfate and ethanol precipitation; (ii) O-(diethylaminoethyl)-cellulose chromatography; or (iii) diethylaminoethyl-cellulose chromatography followed by O-(carboxymethyl)-cellulose chromatography. Toxin purities as reflected by specific activity were 1.83 X 10(6), 9.8 X 10(6), and 2.0 X 10(7) mouse 50% lethal doses (LD50)/mg of N, respectively, for toxins purified by the three methods. The toxins were converted to toxoids by incubation at 35 C in the presence of 0.3 to 0.45% formalin for 21 to 35 days. Toxoids were immunogenic in guinea pigs, as demonstrated by serum antitoxin response and the immunized animals' resistance to challenge by type F botulinal toxin. The immune response to type F toxoids was lower when toxoids of serotypes A, B, C, D, and E were combined with the type F toxoid than when the type F toxoid only was administered. The toxoid prepared from the most highly purified toxin (method [iii]) conferred the highest immunity in guinea pigs at a given dose level. A relation between serum antitoxin level and resistance to challenge was observed. At least 50% of the groups of guinea pigs with 0.015 antitoxin units or more per ml survived challenge by 10(5) mouse LD50 of type F botulinal toxin. A dose of 3.75 mug of N of the most highly purified type F toxoid in combination with the other five serotypes of botulinal toxoid invoked an immune response in guinea pigs comparable to that considered adequate for the other toxoids.     

Serological studies of types A, B, and E botulinal toxins by passive hemagglutination and bentonite flocculation

Johnson, H. M. (Robert A. Taft Sanitary Engineering Center, Cincinnati, Ohio), K. Brenner, R. Angelotti, and H. E. Hall. Serological studies of types A, B, and E botulinal toxins by passive hemagglutination and bentonite flocculation. J. Bacteriol. 91:967-974. 1966.-Formalinized sheep red blood cells (SRBC), sensitized with types A, B, and E botulinal toxoids and toxins by bis-diazotized benzidine (BDB), were tested against A, B, and E antitoxins prepared in horses and rabbits. Type B antitoxin cross-reacted with A toxoid SRBC, but the reciprocal cross-reaction was not observed. E toxin SRBC were specifically agglutinated by E antitoxin. Flocculation of antigen-sensitized bentonite particles was less sensitive in titration of antitoxin than hemagglutination. Also, reciprocal cross-reactions were observed between types A and B antitoxins. Cross-reactions in both serological tests were eliminated by titration of antitoxins in the presence of the heterologous antigens, with no inhibitory effect on the homologous antitoxins. Generally, equine antitoxins were less suitable for agglutinations, especially of antigen-sensitized bentonite particles. Types A, B, and E antitoxins were specifically inhibited by 43, 39, and 245 mouse ld(50) of their respective homologous toxins in the hemagglutination-inhibition test. A, B, and E antitoxins were specifically inhibited by 500, 950, and 1,500 mouse ld(50) of their respective homologous toxins in bentonite flocculation inhibitions. Formalinized SRBC sensitized with rabbit types A and B antitoxins by BDB were respectively clumped by as little as 0.75 to 1.3 mouse ld(50) of A toxin and 2.3 ld(50) of B toxin, whereas bentonite particles sensitized by the same antitoxins were specifically clumped by 150 ld(50) of A toxin and 630 ld(50) of B toxin. E antitoxin sensitization of SRBC or bentonite particles was not successful. Evidence is presented that indicates that the serological procedures are applicable to the detection of botulinal toxins in food.     

A Sandwich Enzyme-Linked Immunosorbent Assay for the Bacillus sphaericus Binary Toxin

Bacillus sphaericus (Bs) binary toxin was purified from recombinant E. coli DH5α harboring the recombinant plasmid pAR5, which carries a 3.6-kb DNA fragment of Bs 1593M encoding mosquito larvicidal activity. The binary toxin preparation, designated BsEcAg, contained mainly 51- and 42-kDa toxin proteins and was toxic to 50% of Culex quinquefasciatus larvae at a concentration of 9.22 ng toxin protein/ml. This preparation was used to raise antibodies in sheep and mice. The sandwich ELISA used sheep antitoxin antibody as primary antibody (coating antibody), mouse antitoxin antibody as second antibody, and goat antimouse antibody as an alkaline phosphatase-conjugated detecting antibody. The assay sensitivity was 200 ng/ml for both BsEcAg and binary toxin antigen (BsAg) from Bs 2362 cells. There is a significant correlation between toxin level determined by ELISA and bioassay. This procedure has also been used to monitor toxin levels in batch fermentations of Bs 2362. Received: 2 July 1997 / Accepted: 12 August 1997     

Rapid Detection of Clostridium botulinum Toxin by Capillary Tube Diffusion

A micro capillary agar-gel diffusion system for the detection of botulinal toxin in foods and cultures was developed and evaluated. Toxins types A, B, and E, produced in culture broth with and without added trypsin, and type E toxin, produced in inoculated canned clams, were tested with this system and with the mouse bioassay procedure. With nontrypsinized toxin, the capillary diffusion system detected as little as 100 minimal lethal doses (MLD) per ml but was effective only at higher levels, 10(6) to 1.5 x 10(7) MLD/ml, when used with trypsinized toxin. The inability to detect lower levels of trypsinized toxin was due to thioglycolate present in the medium used to produce toxin. Evidently, trypsinization of toxin produces polypeptides still held together by disulfide bonds. Cleavage of these bonds by reduction with thioglycolate reduces the sensitivity of the capillary method. Trypsinized toxin produced in broth without thioglycolate was detected as readily as nontrypsinized toxin. Toxin was detected in canned clams containing as low as 100 MLD/ml. No cross-reactions were observed with type E toxin and types A and B antitoxins. Extensive studies using the capillary method for detecting types A and B toxins were not performed; however, a suspected sample of commercially canned mushrooms gave a positive type B reaction but not a type A reaction. This typing was confirmed later by the mouse bioassay. Toxin was present at a level of 100 MLD/ml. The procedure developed may prove useful as a rapid screening method for the detection of botulinal toxin in foods, with final identification made by using the mouse bioassay.     

Assembly Dynamics and Stability of the Pneumococcal Epsilon Zeta Antitoxin Toxin (PezAT) System from Streptococcus pneumoniae

The pneumococcal epsilon zeta antitoxin toxin (PezAT) system is a chromosomally encoded, class II toxin antitoxin system from the human pathogen Streptococcus pneumnoniae. Neutralization of the bacteriotoxic protein PezT is carried out by complex formation with its cognate antitoxin PezA. Here we study the stability of the inhibitory complex in vivo and in vitro. We found that toxin release is impeded in Escherichia coli and Bacillus subtilis due to the proteolytic resistance of PezA once bound to PezT. These findings are supported by in vitro experiments demonstrating a strong thermodynamic stabilization of both proteins upon binding. A detailed kinetic analysis of PezAT assembly revealed that these particular features of PezAT are based on a strong, electrostatically guided binding mechanism leading to a stable toxin antitoxin complex with femtomolar affinity. Our data show that PezAT complex formation is distinct to all other conventional toxin antitoxin modules and a controlled mode of toxin release is required for activation.     

Functional annotation of a novel toxin–antitoxin system Xn-RelT of <Emphasis Type="Italic">Xenorhabdus nematophila</Emphasis>; a combined in silico and in vitro approach

Toxin–antitoxin (TA) complexes play an important role in stress responses and programmed cell death in bacteria. The RelB-RelE toxin antitoxin system is well studied in Escherichia coli. In this study, we used combined in silico and in vitro approaches to study a novel Xn-RelT toxin from Xenorhabdus nematophila bearing its own antitoxin Xn-RelAT—a RelB homolog of E. coli. The structure for this toxin–antitoxin pair is yet unknown. We generated homology-based models of X. nematophila RelT toxin and antitoxin. The deduced models were further characterized for protein–nucleic acid, protein–protein interactions and gene ontology. A detrimental effect of recombinant Xn-RelT on host E. coli was determined through endogenous toxicity assay. When expressed from a isopropyl β-d-1-thiogalactopyranoside-regulated LacZ promoter, Xn-RelT toxin showed a toxic effect on E. coli cells. These observations imply that the conditional cooperativity governing the Xn-RelT TA operon in X. nematophila plays an important role in stress management and programmed cell death.     

Detection of Type E Botulinal Toxin in Cultures by Fluorescent-Antibody Microscopy

Vegetative cells of toxigenic Clostridium botulinum type E cultures were stained with fluorescent antitoxin prepared against purified toxin. The staining seems to be specific.     

沿微山湖地区神经毒素原性酪酸梭菌的土壤分布调查

为了解神经毒素原性酪酸梭菌在微山湖地区土壤的分布情况,我们采集了该地区的土壤样品进行调查。５０份土样培养上清中,有１８份（３６％）检出了肉毒毒素,经中和试验证实均为Ｅ型。从其中的７份阳性样品中分离到产毒菌株,对分离菌株进行毒性测定、生化特性检查及其神经毒素基因的ＰＣＲ检测,所有菌株均具有Ｅ型肉毒神经毒素基因并产生相应毒素,但其生化特性与Ｅ型肉毒梭菌不同,而与酪酸梭菌一致。结果说明沿微山湖地区土壤中确实存在神经毒素原性酪酸梭菌,而且阳性率很高。对神经毒素原性酪酸梭菌的分布进行流行病学调查并从土壤中分离到该病原菌,这在国际上尚属首次。     

A continuous fluorometric assay for the assessment of MazF ribonuclease activity

Plasmids maintain themselves in their bacterial host through several different mechanisms, one of which involves the synthesis of plasmid-encoded toxin and antitoxin proteins. When the plasmid is present, the antitoxin binds to and neutralizes the toxin. If a plasmid-free daughter cell arises, however, the labile antitoxin is degraded (and not replenished) and the toxin kills the cell from within. These toxin-antitoxin (TA) systems thereby function as postsegregational killing systems, and the disruption of the TA interaction represents an intriguing antibacterial strategy. It was recently discovered that the genes for one particular TA system, MazEF, are ubiquitous on plasmids isolated from clinical vancomycin-resistant enterococci (VRE) strains. Thus, it appears that small molecule disruptors of the MazEF interaction have potential as antibacterial agents. The MazF toxin protein is known to be a ribonuclease. Unfortunately, traditional methods for the assessment of MazF activity rely on the use of radiolabeled substrates followed by analysis with polyacrylamide gel electrophoresis. This article describes a simple and convenient continuous assay for the assessment of MazF activity. The assay uses an oligonucleotide with a fluorophore on the 5' end and a quencher on the 3' end, and processing of this substrate by MazF results in a large increase in the fluorescence signal. Through this assay, we have for the first time determined K(M) and V(max) values for this enzyme and have also found that MazF is not inhibited by standard ribonuclease inhibitors. This assay will be useful to those interested in the biochemistry of the MazF family of toxins and the disruption of MazE/MazF.     

Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay

In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451. In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs. The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin. A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin. The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin. No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C. novyi, C. perfringens, or C. sordellii. The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin. Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs. The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.     

Novel Cell-Based Method To Detect Shiga Toxin 2 from Escherichia coli O157:H7 and Inhibitors of Toxin Activity

Escherichia coli O157:H7 is a leading cause of food-borne illness. This human pathogen produces Shiga toxins (Stx1 and Stx2) which inhibit protein synthesis by inactivating ribosome function. The present study describes a novel cell-based assay to detect Stx2 and inhibitors of toxin activity. A Vero cell line harboring a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) was used to monitor the toxin-induced inhibition of protein synthesis. This Vero-d2EGFP cell line produced a fluorescent signal which could be detected by microscopy or with a plate reader. However, a greatly attenuated fluorescent signal was detected in Vero-d2EGFP cells that had been incubated overnight with either purified Stx2 or a cell-free culture supernatant from Stx1- and Stx2-producing E. coli O157:H7. Dose-response curves demonstrated that the Stx2-induced inhibition of enhanced green fluorescent protein fluorescence mirrored the Stx2-induced inhibition of overall protein synthesis and identified a picogram-per-milliliter threshold for toxin detection. To establish our Vero-d2EGFP assay as a useful tool for the identification of toxin inhibitors, we screened a panel of plant compounds for antitoxin activities. Fluorescent signals were maintained when Vero-d2EGFP cells were exposed to Stx1- and Stx2-containing medium in the presence of either grape seed or grape pomace extract. The antitoxin properties of the grape extracts were confirmed with an independent toxicity assay that monitored the overall level of protein synthesis in cells treated with purified Stx2. These results indicate that the Vero-d2EGFP fluorescence assay is an accurate and sensitive method to detect Stx2 activity and can be utilized to identify toxin inhibitors.Shiga toxin-producing Escherichia coli, with E. coli O157:H7 as the most common serotype, is an enteric pathogen known to cause human gastrointestinal illnesses ranging from bloody diarrhea and hemorrhagic colitis to life-threatening hemolytic-uremic syndrome (HUS) (1, 20). It has been estimated that E. coli O157 causes approximately 73,000 cases of illness per year in the United States from food- and waterborne sources. Shiga toxins (Stx1 and Stx2) are major virulence factors in E. coli O157 pathogenicity. These toxins inhibit protein synthesis by inactivating the ribosome and are thought to contribute to the development of HUS, a potentially fatal disease for which treatment is currently limited to supportive care (13, 14, 26). Toxin inactivation would prevent the development of HUS, but antitoxin therapeutics are not currently available (26). Detection methods to prevent the distribution of E. coli O157 in foods are thus an important component of food safety programs.The rise in food-related outbreaks of E. coli O157 infection has heightened the importance of developing better methods to rapidly detect and characterize Stxs from E. coli O157 strains (26). Several methods have been developed to examine Stx activity against mammalian cells. Current assays that measure the viability of intoxicated Vero cells require several days of incubation and often produce poor quantitative data (5, 9, 19). Other methods that are more quantitative and sensitive measure the incorporation of radioactive amino acids into newly synthesized proteins (6, 15). However, these radioactivity assays are complex and laborious and allow only a limited number of conditions to be examined. A quantitative luciferase-based assay was recently developed to measure Stx toxicity in a high-throughput format (31), but this system requires several preparatory and processing steps to detect luciferase expression.In the present study, we describe a simple cell-based assay for the detection of Stx2 and inhibitors of toxin activity by using a Vero cell line that expresses a destabilized variant (half-life, 2 h) of the enhanced green fluorescent protein (d2EGFP) to monitor the Stx2-induced inhibition of protein synthesis. This cell-based Vero-d2EGFP assay was used to screen a panel of natural compounds for anti-Stx activities, and we found that grape seed and grape pomace extracts both provided strong cellular protection against Stx2.     

Radioimmunoassay for Type A Toxin of Clostridium botulinum

A preparation of pure type A toxin of Clostridium botulinum was labeled with (131)I in the presence of chloramine-T and carrier-free isotope. The radioactive toxin ((131)I Tox) was used in a radioimmunoassay procedure similar to that of Berson and Yalow. Dilutions of antibody to the toxin, capable of binding 50% of the added (131)I Tox, were mixed with a sample of the labeled toxin and various concentrations of unlabeled toxin ('Cold' Tox). When concentration of Cold Tox were plotted against the ratio (131)I bound/(131)I not bound, a standard curve was established that could be used to estimate the concentration of Cold Tox in a test mixture. This assay was sensitive to as little as 100 mouse minimum lethal dose and was highly specific for the serological type of the toxin used.     

Detection of Clostridium botulinum Toxin by Local Paralysis Elicited with Intramuscular Challenge

Clostridium botulinum toxin can be identified by a characteristic, acute local paralysis that follows its injection into the gastrocnemius ("calf" muscle) of mice. The local botulism can be elicited with slightly less than one-tenth the toxin amount that is needed to kill mice by the intraperitoneal (i.p.) challenge route. The practical sensitivities of the intramuscular (i.m.) versus i.p. tests are about equal, however, because maximum sample volume injectable i.m. is 0.1 ml as compared to the 0.5-ml range that can be given i.p. i.m. injection of 10 or more mouse i.p. mean lethal doses causes paralysis in about 1 h, and an i.m. injection of about 0.5 i.p. mean lethal doses causes paralysis in 3 to 4 h. Toxin neutralization by homologous type of antitoxin only can be demonstrated with an incubated mixture of toxin and antitoxin. Although not as convenient as the i.p. method for routine use to detect botulinum toxin, the i.m. method has characteristics which could make it a useful supplement to the presently accepted i.p. procedure.     

重组蓖麻毒素A链与几种天然单链核糖体失活蛋白的活性研究

采用ｐＥｘＳｅｃⅠ载体系统进行了蓖麻毒素Ａ链的原核表达,经ＣＭ－Ｓｅｐｈａｒｏｓｅ一步纯化后,获得了纯度约８０％的重组蓖麻毒素Ａ链．将其与几种天然单链核糖体失活蛋白进行了超螺旋ＤＮＡ裂解研究和无细胞体系中蛋白合成抑制试验,结果表明,重组蓖麻毒素Ａ链具有类似于天然单链核糖体失活蛋白的活性,但两种测活方法之间没有明显的相关性     

Incidence of Clostridium botulinum Type E in Salmon and Other Marine Fish in the Pacific Northwest

Salmon, sole, cod, oysters, clams, and crabs from ocean waters along the coast of Oregon and Washington were examined for the presence of Clostridium botulinum type E. The organism was detected by identification of the type E toxin in enrichment cultures of the viscera of individual fish. Of 369 salmon specimens, 48 yielded cultures containing toxin lethal to mice, and almost half of the toxic cultures were shown to contain botulinal toxin, chiefly type E. Eighteen of 113 sole and cod specimens, 4 of 22 Dungeness crab specimens, 5 of 16 oyster specimens, and 27 of 115 clam specimens gave rise to cultures containing botulinal toxin which was usually type E, although types A and B were occasionally encountered.