F型肉毒毒素的制备及类毒素免疫原性初探

将F型肉毒梭菌经适宜条件产毒培养后,以硫酸铵盐析和酸沉两种不同工艺制备的F型肉毒毒素,用分段脱毒法脱毒制备类毒素,分别免疫豚鼠、马匹后测定免疫血清抗体效价。结果显示,两种工艺制备的毒素其类毒素都具有较好的免疫原性。     

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。     

肉毒毒素研究进展

肉毒毒素是肉毒梭菌产生的一种神经毒素,能够通过抑制神经肌肉接头处的乙酰胆碱释放而引起肌肉麻痹.肉毒毒素在培养液中以复合物形式存在,其中的毒性组分由3个非同源性结构域组成,是一种新型的金属蛋白酶.不同血清型的肉毒毒素能够特异性地作用于不同底物,这些底物在神经细胞的胞外分泌过程中发挥重要作用.肉毒毒素在胞吞胞吐机制的研究以及临床医学应用方面具有宝贵的价值.     

一株D型肉毒梭菌的分离和鉴定

从我国东海地区的海泥培养物中,分离出一株厌氧性芽孢杆菌。经细菌学、毒素血清学．细菌代谢产物的气相色谱分析及DNA中G+ct001％测定,鉴定为D型肉毒梭菌,编号为D85501。与国际参考菌株D359对照,D85 501菌株具有D型肉毒梭菌的总特性,还有自身的特点,是D型肉毒梭菌中的一新株,是我国分离的首株D型肉毒梭菌。     

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。     

E型内毒毒素的分子生物学研究进展

Ｅ型肉毒毒素不同于Ａ型肉毒毒素及具有蛋白分解性的Ｂ,Ｆ型肉毒毒素,具有自身的特点。本文就Ｅ型肉毒毒素的结构,激活及作用机制,Ｅ型肉毒毒素的精制特点,基因结构及酪酸梭菌产生Ｅ型肉毒毒素的机制探讨等四个方面予以介绍。     

E型肉毒毒素的分子生物学研究进展

Ｅ型肉毒毒素不同于Ａ型肉毒毒素及具有蛋白分解性的Ｂ、Ｆ型肉毒毒素,具有自身的特点。本文就Ｅ型肉毒毒素的结构、激活及作用机制、Ｅ型肉毒毒素的精制特点、基因结构及酪酸梭菌产生Ｅ型肉毒毒素的机制探讨等四个方面予以了介绍。     

肉毒梭菌培养条件的初步比较试验小结

为了配合肉毒梭菌检验工作的开展,我们用实验室保存的各型(A～F)肉毒梭菌菌种对培养基的类型、培养温度及培养时间等条件进行了生长与产毒的初步比较试验。权衡各个因素,我们认为采用明胶琼脂半固体培养基和30℃的培养温度这个统一条件较好;培养3至5天,对于所有各型的生长或产毒,均可获得符合检验要求的结果。     

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。     

婴儿肉毒中毒

<正> 一、肉毒中毒一般概念[1-3]肉毒毒素是由肉毒梭菌产生的一种高分子蛋白的神经毒素,它能引起死亡率很高的人和易感动物的肉毒中毒,尽管根据抗原的不同已将肉毒毒素分为A、B、C_1、C_2、D、E、F和G8个型,但它们都有抑制周围胆硷能运动神经末梢释放乙酰胆硷,妨碍神经传导,引起肌肉松弛性麻痹的药理作用。它们的结构和分子量也是相似的。     

An unusually heavy contamination of honey products by Clostridium botulinum type F and Bacillus alvei

Many spores (1-60/g) of Clostridium botulinum type F were detected in different containers of honey products of the same brand. Microbiological and physicochemical properties of the contaminated honey were compared with those of the negative one. No difference in pH, hydroxymethyl furfural contents or diastase activity was found between them. The total counts of anaerobes other than C. botulinum and of yeast were also similar, whereas the aerobe counts, which were proportionally related with the C. botulinum counts, were higher in the positive honey than in the negative one. Motile colony-forming Bacillus alvei was predominant among the aerobes. B. alvei stimulated the toxin production by C. botulinum type F in culture medium incubated under aerobic conditions. The high count of C. botulinum in the honey might have been due to the possible stimulation of growth by B. alvei or some other microorganisms at some stage of honey ripening.     

Response of type B and E Botulinum toxins to purified sulfhydryl-dependent protease produced by Clostridium botulinum type F.

A sulfhydryl-dependent protease (SHP) was purified from a culture of Clostridium botulinum type F. The enzyme can activate type E progenitor toxin completely but type B progenitor toxin only partially. This may suggest that SHP by itself could completely activate the toxin of proteolytic C. botulinum types A and F in culture. The toxicity of type E progenitor toxin potentiated by the treatment with SHP persisted, whereas that of derivative toxin decreased rapidly by further incubation with SHP. This may indicate that only the progenitor toxin, the complex of the toxic and nontoxic components, activated by SHP withstands the subsequent exposure to the enzyme in cultures of proteolytic C. botulinum.     

Monoclonal antibody to type F Clostridium botulinum toxin

Hybridomas synthesizing monoclonal antibodies (MAbs) against type F Clostridium botulinum toxin were developed. MAb from one stable hybridoma, hybridoma 223, consisted of kappa light chains and an immunoglobulin G subclass 2a heavy chain. This MAb was used in a double-sandwich enzyme-linked immunosorbent assay to detect type F toxin in foods, culture fluids, and purified toxin preparations. The sensitivity of the double-sandwich enzyme-linked immunosorbent assay was approximately 10 mouse lethal doses of toxin per ml of toxic fluid.     

Detection of Clostridium botulinum type G toxin by enzyme-linked immunosorbent assay.

Clostridium botulinum type G toxin was detected and quantified readily with the enzyme-linked immunosorbent assay. With the double-sandwich technique and alkaline phosphatase as the enzyme indicator, C. botulinum toxin type G was detected in quantities equaling those required for one mouse intraperitoneal median lethal dose. The time required for the procedure was approximately 6.5 h, but this requirement could have been reduced to 5.5 h or less with the use of precoated plates stored at -70 degrees C. Cross-reactions did not occur with culture extracts of C. sporogenes of C. botulinum types B, C, D, E, and F. Acidic preparations of C. botulinum type A exhibited nonspecific reactivity. Likewise, 50% of the C. subterminale isolates tested were cross-reactive in the assay. These latter isolates express similar metabolic and physiological characteristics with C. botulinum type G.     

The Inhibition of Clostridium Botulinum Type B and E in Salami Sausage

Vegetative cells and spores of Clostridium botulinum type B and E were inoculated into salami sausages with and without the preservatives sodium nitrite and sodium benzoate. The growth and toxin production of Clostridium botulinum type B and E were inhibited in this type of salami sausages, even without any addition of preservatives. The use of a starter culture with pH-lowering components has both technological and hygienic advantages.     

Sequence of the gene encoding type F neurotoxin of Clostridium botulinum

Primers designed to conserved regions of botulinum and tetanus clostridial toxins were used to amplify DNA fragments from non-proteolytic Clostridium botulinum type F (202F) DNA using polymerase chain reaction technology. The fragments were cloned and the complete nucleotide sequence of the gene encoding type F toxin determined. Analysis of the nucleotide sequence demonstrated the presence of an open frame encoding a protein of 1274 amino acids, similar to other botulinum neurotoxins. Upstream of the toxin gene is the end of an open reading frame which encodes the C-terminus of a protein with homology to non-toxic-non-hemagglutinin component of type C progenitor toxin.     

Real-time PCR detection of the nontoxic nonhemagglutinin gene as a rapid screening method for bacterial isolates harboring the botulinum neurotoxin (A-G) gene complex

Botulinum neurotoxin (BoNT) producing clostridia contain genes encoding a specific neurotoxin serotype (A-G) and nontoxic associated proteins that form the toxin complex. The nontoxic nonhemagglutinin (NTNH) is a conserved component of the toxin complex in all seven toxin types. A real-time PCR assay that utilizes a locked nucleic acid hydrolysis probe to target the NTNH gene was developed to detect bacterial strains harboring the botulinum neurotoxin gene cluster. The specificity of the assay for Clostridium botulinum types A-G, Clostridium butyricum type E and Clostridium baratii type F was demonstrated using a panel of 73 BoNT producing clostridia representing all seven toxin serotypes. In addition, exclusivity of the assay was demonstrated using non-botulinum toxin producing clostridia (7 strains) and various enteric bacterial strains (n=27). Using purified DNA, the assay had a sensitivity of 4-95 genome equivalents. C. botulinum type A was detected directly in spiked stool samples at 10(2)-10(3) CFU/ml. Stool spiked with 1 CFU/ml was detected when the sample was inoculated into enrichment broth and incubated for 24 h. These results indicate that the NTNH real-time PCR assay can be used to screen enrichment cultures of primary specimens at earlier time points (24 h) than by toxin detection of unknown culture supernatants (up to 5 days).     

Enzyme-linked immunosorbent assay for detection of Clostridium botulinum type E toxin.

The enzyme-linked immunosorbent assay using the "double-sandwich" technique was utilized to determine Clostridium botulinum type E toxin. With this technique, about 80 mouse intraperitoneal 50% lethal doses of toxin could be detected. Cross-reaction was hardly observed with C. botulinum type A and B toxins. No cross-reaction was observed with culture supernatants of C. botulinum type C or other Clostridium strains. In all probability this was due to the high specificity of the antiserum prepared aginst the toxic component of type E toxin.