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。     

Occurrence ofClostridium botulinum in the soil of the vicinity of Rome

After the isolation of two strains ofClostridium butyricum that produced type E botulinal toxin from the feces of two babies in Rome affected by infant botulism (P. Aureli, L. Fenicia, M. Gianfranceschi, L. McCroskey, and C. Hatheway, J Infect Dis 154:207–211, 1986), extensive research was carried out to detect botulinal toxin-producing clostridia in soil samples from the vicinity of Rome. A total of 520 specimens from cultivated and pasture lands, obtained from 52 collection stations, were examined.Enrichment cultures were tested for botulinal toxin, and the isolated neurotoxigenic micro-organisms were characterized both enzymatically and gas chromatographically.Clostridium botulinum type A and B proteolytic, the former being the most common, were found in 7 (1.3%) out of 520 samples from 5 (9.6%) of the 52 locations in close association with pasture lands. Possible relationships between the presence of botulinal organisms and the properties of the soil in which they were found have also been investigated.     

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.     

DETECTION OF TYPE A BOTULINAL TOXIN-PRODUCING ORGANISMS SUBCULTURED FROM CHEESE USING AN AMPLIFIED ELISA SYSTEM

Mascarpone cheese implicated in a botulism outbreak was examined for preformed and cultural botulinal toxins using the mouse bioassay. The cheese was also assayed for cultural toxins and for the most probable number (MPN) of toxin-producing organisms/g using an amplified ELISA. No preformed botulinal toxins were discovered in the cheese samples (pH range 5.84-5.86) using the mouse bioassay. However, after cheese subculture in tryptone-peptone-glucose-yeast extract broth, type A botulinal toxin-producing organisms that formed more than 10,000 MLD (mouse lethal dose)/mL in culture were detected. The ELISA results also revealed that type A toxin was present in the culture with a sensitivity of ∼ 10 MLD/mL. The MPN of type A toxin-producing organisms/g in 12 cheese samples examined ranged from < 0.3-9.33. No ELISA cross-reactivity was noted between the type A toxic cultures and other types (B, E, or F). The ELISA sensitivity was ∼5 MLD/mL casein buffer using purified type A neurotoxin. The advantages of the ELISA test are that the toxin type and approximate lethal dose can be determined within one day compared to the mouse bioassay which takes 3–5 days.     

DETECTION OF BOTULINAL TOXIN GENES: TYPES A AND E OR B AND F USING THE MULTIPLEX POLYMERASE CHAIN REACTION

Multiple primer sets were combined with the polymerase chain reaction (PCR) and examined for use in the amplification of toxin gene fragments from four Clostridium botulinum types (A, B, E, and F). Vegetative cells obtained from overnight cultures were used directly in the PCR analyses without purification of chromosomal DNA. Gene fragments were amplified from the different botulinal toxin genes that code for types A, B, E and F toxins using a single PCR protocol. Toxin gene fragments were amplified from types B and F toxigenic organisms using the PCR and specific primer sets for these types in a single PCR tube. Type A and E toxin genes were also examined using type A and E specific primers in a separate PCR tube. The PCR-amplified products were separated by electrophoresis on agarose gels containing ethidium bromide. The identity of the PCR products were confirmed by DNA hybridization using type specific probes. We conclude that this method is useful for the rapid and direct identification of toxigenic botulinal organisms that code for the toxin types A, B, E, or F.     

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.     

An outbreak of type E botulism among common loons (Gavia immer) in Michigan's upper peninsula

An epizootic of type E botulism (Clostridium botulinum) occurred among common loons (Gavia immer) along the Lake Michigan shore of Michigan's Upper Peninsula (USA) during October and November 1983. An estimated 592 dead loons washed ashore along the Garden Peninsula. Type E botulinal toxin was demonstrated in blood samples and stomach contents of dead loons, and in samples of three species of dead fish found on the Lake Michigan shore. We suspect that loons acquired botulism by ingesting sick or dead fish containing type E toxin.     

Dependence of Clostridium botulinum gas and protease production on culture conditions.

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.     

Dependence of Clostridium botulinum gas and protease production on culture conditions

Reports that Clostridium botulinum toxin can sometimes be detected in the absence of indicators of overt spoilage led to a systematic study of this phenomenon in a model system. Media with various combinations of pH (5.0 to 7.0) and glucose (0.0 to 1.0%) were inoculated with vegetative cells of C. botulinum 62A and incubated anaerobically at 35 degrees C. Although growth and toxin production occurred at all pH and glucose combinations, accumulation of gas was delayed or absent in media with low pH, low glucose levels, or both. Other proteolytic C. botulinum strains gave similar results. Trypsin activation was required to detect toxin in some low pH cultures. The trypsinization requirement correlated with low proteolytic activity in the cultures. Proteolytic activity of the strains examined was 5- to 500-fold lower in botulinal assay medium than in cooked meat medium. The results indicate that the absence of gas accumulation does not preclude the presence of botulinal toxin and that proteolytic cultures grown under adverse conditions may require trypsinization for the detection of toxin.     

Cloning of a Clostridium botulinum type B toxin gene fragment encoding the N-terminus of the heavy chain

Two lambda gt11 clones of the toxin gene of Clostridium botulinum type B were identified by the monoclonal antibody specific to the heavy chain of type B toxin. Neither of the expressed fusion proteins from the lysates of lysogenic E. coli Y1089 showed any botulinal toxic activity. One of the clones hybridized to the oligonucleotide probe which was synthesized according to the amino acid sequence of N-terminus of heavy chain. The sequence analysis revealed that highly homologous regions in N-terminus of heavy chain exist among botulinum neurotoxins (type A, B) and tetanus toxin on the amino acid sequence level.     

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.     

首次自肉毒中毒食品中分离的一株产生E型肉毒毒素的酪酸梭菌

对某卫生防疫站委托本研究室分离病原菌的一份引起肉毒中毒的食品一＂黄豆冬瓜酱＂进行检测和病原菌的分离,从中再次检出了Ｅ型肉毒毒素并分离到一产毒菌种,对该菌种的生物学及生化学特性进行检查,并检测其毒素基因（ＰＣＲ试验）。结果：该分离菌能产生Ｅ型肉毒毒素,ＰＣＲ检测结果也证明其具有Ｅ型肉毒神经毒素基因,但其多项生化特性与Ｅ型肉毒梭菌有明显差异,而与酪酸梭菌完全一致。结果说明该分离菌系产生Ｅ型肉毒毒素的酪酸梭菌,而非Ｅ型肉毒梭菌。由酪酸梭菌引起的食物中毒型肉毒中毒并从中毒食品中分离到该病原菌,这在国际上尚属首次报告。     

Evaluation of Type A Botulinal Toxin Assays that Use Antitoxin to Crystalline Toxin

The type A botulinal toxin assay by the reverse passive hemagglutination procedure which uses antitoxin to crystalline toxin was examined for specificity. The analysis was based on the fact that crystalline type A toxin is a complex of neurotoxic protein (Aalpha) and a nontoxic protein (Abeta). By using these components, obtained in essentially pure forms, it was shown that the antitoxin to crystalline toxin has a significantly higher titer to Abeta than to Aalpha. When Formalin-treated red blood cells were sensitized with this antitoxin, the antibodies coupled to the cells were, for practical results, only anti-Abeta. When the suspension is reacted with dilutions of type A toxic solutions, the limiting dilutions are determined by Abeta and not by the neurotoxin, which should be the determinant if the assay is to measure toxicity. These observations may be pertinent to the development of serological assays for other botulinal toxin types.     

Identification of Type A, B, E, and F Botulinum Neurotoxin Genes and of Botulinum Neurotoxigenic Clostridia by Denaturing High-Performance Liquid Chromatography

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products. We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes. PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C. butyricum type E, and C. baratii type F strains were subjected to both DHPLC analysis and sequencing. Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences. We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level. A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis. Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.     

Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish

A rapid, quantitative PCR assay (TaqMan assay) which quantifies Clostridium botulinum type E by amplifying a 280-bp sequence from the botulinum neurotoxin type E (BoNT/E) gene is described. With this method, which uses the hydrolysis of an internal fluoregenic probe and monitors in real time the increase in the intensity of fluorescence during PCR by using the ABI Prism 7700 sequence detection system, it was possible to perform accurate and reproducible quantification of the C. botulinum type E toxin gene. The sensitivity and specificity of the assay were verified by using 6 strains of C. botulinum type E and 18 genera of 42 non-C. botulinum type E strains, including strains of C. botulinum types A, B, C, D, F, and G. In both pure cultures and modified-atmosphere-packaged fish samples (jack mackerel), the increase in amounts of C. botulinum DNA could be monitored (the quantifiable range was 10² to 10⁸ CFU/ml or g) much earlier than toxin could be detected by mouse assay. The method was applied to a variety of seafood samples with a DNA extraction protocol using guanidine isothiocyanate. Overall, an efficient recovery of C. botulinum cells was obtained from all of the samples tested. These results suggested that quantification of BoNT/E DNA by the rapid, quantitative PCR method was a good method for the sensitive assessment of botulinal risk in the seafood samples tested.     

A case report on human type B botulism

A case of type B human botulism was found in Tochigi Prefecture in November, 1984. Botulinum type B toxin was detected in the serum and feces of the patient. The serum toxin was activated by trypsinization. Type B toxin was demonstrated in cooked meat medium cultures of the fecal specimens. The patient recovered after administration of type A, B, E and F quadrivalent antitoxin.     

Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.

The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.     

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.     

A Comparison of Toxin Production by Clostridium botulinum Type E in Irradiated and Unirradiated Vacuum Packed Fish

S ummary . The rates of toxin production at 10° by inocula of 10⁵, 10⁴, 10³ and 10² spores of Clostridium botulinum type E in vacuum packed herring, cod and haddock were compared with that of equivalent numbers of spores surviving γ-irradiation at 0.3 Mrad. There was little difference between the rates of toxin production in unirradiated and irradiated fish. More toxin was produced in irradiated herring than in unirradiated, but in haddock the reverse was obtained. In cod about equal amounts of toxin were found without trypsinization; after trypsinization toxin levels were generally higher in irradiated samples.