Proteases of Clostridium botulinum. V. Studies on the serological relationship between proteases from Clostridium botulinum and other spore-forming bacteria

By the use of the electrophoretic casein precipitating inhibition test (CPI-test) the serological relationship between proteolytic enzymes produced by different species within the genera Clostridium and Bacillus has been tested. The proteases produced by Clostridium botulinum types A, B, C, D and F cross-reacted with each other. Clostridium botulinum strain 84 was inhibited by antiproteases produced against Clostridium sporogenes, Clostridium botulinum types C and F (protease F I and F II), but not by antiproteases against Clostridium botulinum types B and F (protease II), Clostridium bifermentans and Clostridium perfringens. The protease of the newly described Clostridium botulinum strain 89 (type G) was inhibited by Clostridium sporogenes antiprotease, but not by any of the other antiproteases. It is not possible to differentiate between Clostridium botulinum, Clostridium sporogenes and Clostridium perfringens by use of serological differentiation of their proteolytic enzymes. The protease of Clostridium bifermentans is not serologically related to any of the species tested in this investigation. Proteases produced by different Bacilli were not inhibited by antiproteases from Clostridium botulinum types B, C and F, Clostridium sporogenes, Clostridium bifermentans, and the two strains of Clostridium perfringens tested. This investigation indicates a serological relationship between proteases from different Clostridium species, but not a serological relationship between proteases produced by the Clostridium species and Bacillus species tested.     

Proteases of Clostridium botulinum. VI. The role of trypsin, Clostridium botulinum proteases and protease inhibitors in the formation and activation of toxin in growing cultures of Clostridium botulinum

In this study the influence of bovine serum protease inhibitors, trypsin and proteases produced by different types of Clostridium botulinum has been investigated. Trypsin and botulinum proteases had the capability of increasing the toxicity in growing cultures in Clostridium botulinum types A, B and E. Trypsin increased the toxin level to a greater extent than proteases from Clostridium botulinum types A, B, C and F. Protease inhibitors did not influence the toxin formation to any extent compared with the controls. The combined effects of proteases and protease inhibitors on the development of toxin in Clostridium botulinum type B were also investigated by adding proteases and protease inhibitors to the same culture at different time intervals. Protease inhibitors did not reduce the toxicity of the cultures as compared to the controls. Altogether a complex relationship seems to exist between protoxin, toxin, proteases and inhibitors in the culture, and the order and time sequence of addition seem to be of importance. The results obtained in this investigation indicate that proteases of Clostridium botulinum play a part in the formation and/or activation of toxin in growing cultures of proteolytic strains such as Clostridium botulinum types A and B. As to the activation of protoxin and progenitor toxin produced by non-proteolytic Clostridium botulinum types B and E, botulinum proteases showed a marked capability of increasing the toxicity in these cultures. Trypsinization may be valuable for the detection of Clostridium botulinum types A and B in foods, as well as for type E, where it is commonly used.     

Proteases of Clostridium Botulinum: II. The Relationship Between Growth Medium and the Production of Proteases by Clostridium Botulinum Types A,B, C,D, E and F

The present investigation was carried out in order to find a suitable medium for the production of proteolytic enzymes from different types of Clostridium botulinum. Proteolytic activity was found in Clostridium botulinum types A, B, C, D and F, while supernatants of Clostridium botulinum type E did not possess any proteolytic activity at all.Skim milk medium possessed the greatest ability for the production of proteolytic enzymes from the different cultures of Clostridium botulinum tested, while Robertson’s meat broth produced lowest amounts. Highest titres were usually found after 4–5 days of incubation and, after this period, the level of proteolytic activity decreased.     

肉毒梭菌毒素灭鼠研究进展

就肉毒梭菌毒素用于灭鼠的研究和应用情况进行了综述，充分肯定了C型肉毒梭菌毒素灭鼠制剂毒力强，适口性好，对非靶动物毒性低，作用缓慢，无2次中毒，易降解和适合于规模灭鼠的特点，为鼠害防治提供了新的思路。     

荧光定量聚合酶链反应检测乳品中肉毒梭菌

通过实时荧光定量聚合酶链反应方法检测乳粉与乳清粉中的A、B、E、F四型肉毒梭菌,对肉毒梭菌荧光定量聚合酶链反应试剂盒中的阳性对照品及10、102、103、104倍梯度稀释的阳性对照品做出AB混合型和EF混合型的标准曲线,循环域值具有极好的特异性、重复性和准确性.对通过国标方法与中华人民共和国出入境检验检疫行业标准方法检测长出疑似菌落的82份乳粉与乳清粉采用实时荧光定量聚合酶链反应法检测,均检测不到样品中含A、B、E、F四型肉毒梭菌.     

重组C型肉毒梭菌毒素的表达与免疫保护性分析

本研究旨在获得重组C型肉毒梭菌毒素蛋白,并评价其免疫保护性。将麦芽糖蛋白（MBP）和C型肉毒梭菌毒素重链C末端（CH_C）的编码基因序列进行优化和串联,获得基因片段GMBPCH_C。将GMBPCH_C克隆至pET28a-（＋）后转化大肠杆菌BL21（DE3）感受态细胞,分别在15和37℃两种温度条件下诱导表达。利用Ni-IDA亲和层析方法对可溶性表达的目的蛋白进行纯化,从而获得重组蛋白rMBPCH_C。将rMBPCH_C与Montanide ISA 201佐剂混合制备成疫苗,免疫4只家兔,剂量为100 μg/只。根据《中华人民共和国兽药典》（2015年版）规定的方法检测一免后21 d及二免后14 d家兔血清的中和抗体效价。同时,在二免后14 d对家兔进行攻毒。结果表明,rMBPCH_C在37℃的诱导温度下,主要以包涵体的形式表达;在15℃的诱导温度下,可溶性表达的比例可达50%。一次免疫后,免疫组4只家兔血清对C型肉毒梭菌天然毒素（简称天然毒素）的中和效价均可达到1（0.1 mL血清中和1个小鼠最小致死量（MLD）的天然毒素）。二免后,家兔血清的中和抗体效价可达到4~8。用10个家兔MLD的天然毒素攻毒后,免疫组家兔得到了100%（4/4）的保护,而用1个家兔MLD的天然毒素攻毒后,对照组家兔100%（2/2）死亡。以上结果说明,rMBPCH_C具有良好的免疫原性,从而为C型肉毒梭菌病基因工程亚单位疫苗的研制提供了重要的试验数据。     

D型肉毒梭菌菌苗及诊断用血清的研究

利用在国内首次从动物体内分出的D型肉毒梭菌,研制了D型肉毒梭菌菌苗及诊断血清。结果表明:采用透析培养技术制备的D型肉毒梭菌磷酸铝苗,免疫性良好,对家兔和豚鼠的免疫保护力均为100%;制备的诊断血清每毫升可中和10000 MLD毒素,可用于肉毒梭菌菌型鉴定。     

3种诱饵材料对C型肉毒梭菌外毒素灭鼠效果的研究

为了提高C型肉毒梭菌外毒素燕麦毒饵的采食率和灭鼠效果,对韭菜、葱和白菜3种诱饵材料的添加效果进行研究。结果表明:3种诱饵材料均在一定程度上起到了提高毒饵采食率的效果,其中韭菜的诱导效果最佳,采食率和灭洞率较对照分别提高了34.7%和12.5%,较白菜分别提高了29.4%和5.4%,较葱分别提高了17.0%和5.5%。     

Invasive slug populations (Arion vulgaris) as potential vectors for Clostridium botulinum

Background

Norwegian meadows, including those for silage production, are recently found heavily invaded by the slug Arion vulgaris in exposed areas. As a consequence, large numbers of slugs might contaminate grass silage and cause a possible threat to animal feed quality and safety. It is well known that silage contaminated by mammalian or avian carcasses can lead to severe outbreaks of botulism among livestock. Invertebrates, especially fly-larvae (Diptera), are considered important in the transfer of Clostridium botulinum type C and its toxins among birds in wetlands. C. botulinum form highly resistant spores that could easily be consumed by the slugs during feeding. This study aimed to determine whether Arion vulgaris could hold viable C. botulinum and enrich them, which is essential knowledge for assessing the risk of botulism from slug-contaminated silage. Slug carcasses, slug feces and live slugs were tested by a quantitative real-time PCR (qPCR) method after being fed ≅ 5.8 × 10⁴ CFU C. botulinum type C spores/slug.

Results

Low amounts of C. botulinum were detected by qPCR in six of 21 slug carcasses with an even spread throughout the 17 day long experiment. Declining amounts of C. botulinum were excreted in slug feces up to day four after the inoculated feed was given. C. botulinum was only quantified the first two days in the sampling of live slugs. The viability of C. botulinum was confirmed for all three sample types (slug carcasses, slug feces and live slugs) by visible growth in enrichment media combined with obtaining a higher quantification cycle (Cq) value than from the non-enriched samples.

Conclusions

Neither dead nor live invasive Arion vulgaris slugs were shown to enrich Clostridium botulinum containing the neurotoxin type C gene in this study. Slugs excreted viable C. botulinum in their feces up to day four, but in rapidly decreasing numbers. Arion vulgaris appear not to support enrichment of C. botulinum type C.     

Comparative immunogenicity of two bivalent botulinum vaccines

OBJECTIVE: To compare the ability of a new single-dose botulinum vaccine containing a non-mineral oil adjuvant with a single dose of a conventional botulinum vaccine product to produce antibody to Clostridium botulinum types C and D in cattle in Northern Australia. DESIGN AND PROCEDURE: One hundred and fifty Brahman steer weaners were randomly divided into two groups receiving either a single dose of CSL Bivalent Botulinum vaccine or Websters Singvac. Blood samples were collected at 0, 8 and 24 weeks and tested by antibody ELISA. The final samples were also tested by the toxin neutralisation test, to test titres of neutralising antibody. RESULTS: Six months after inoculation, cattle vaccinated with Websters Singvac had ELISA antibody response twice that of CSL conventional product. However, this difference was only evident for neutralising antibody to type C botulinum toxin. Both products produced similar titres of type D neutralising antibody after a single dose. CONCLUSION: Websters' Singvac produces a greater neutralising antibody response to type C botulism upon single inoculation than a conventional vaccine. The product produces an equivalent neutralising antibody response to type D.     

一种致命的隐形杀手:肉毒杆菌与肉毒毒素

2013年8月,因肉毒杆菌污染,问题奶粉再次进入公众视野。肉毒杆菌(Clostridium botulinum)是一种革兰阳性粗短杆菌,严格厌氧,有A～G 7个亚型,每个亚型都可产生一种剧毒的大分子外毒素,即肉毒毒素(botulinum toxin)。这种毒素可引起人和动物发生以松弛性麻痹为主症的肉毒中毒(botulism),虽然并不常见,但却是一种致命的中毒性疾病。本文综述了肉毒杆菌生物学特征、肉毒毒素结构与毒性、肉毒中毒临床表现以及检验鉴定等,并介绍了食品安全生产中肉毒杆菌与肉毒毒素的安全风险与控制措施。     

奶牛和乳制品中的肉毒杆菌

本文回顾了全球范围内牛的肉毒中毒事件和因食用乳及乳制品引发的人的肉毒中毒事件,分析了肉毒污染的可能原因.重点阐述了乳及乳制品中的肉毒毒素或肉毒杆菌引起人肉毒中毒或感染肉毒杆菌及引发乳品加工链中肉毒污染的风险,并对如何防控乳及乳制品发生肉毒污染提出了建议举措.     

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.     

Optimization of polymerase chain reaction for detection of Clostridium botulinum type C and D in bovine samples

Botulism is a rare but serious paralytic illness caused by a nerve toxin that is produced by the bacterium Clostridium botulinum. The economic, medical and alimentary consequences can be catastrophic in case of an epizooty. A polymerase chain reaction (PCR)-based assay was developed for the detection of C. botulinum toxigenic strains type C and D in bovine samples. This assay has proved to be less expensive, faster and simpler to use than the mouse bioassay, the current reference method for diagnosis of C. botulinum toxigenic strains. Three pairs of primers were designed, one for global detection of C. botulinum types C and D (primer pair Y), and two strain-specific pairs specifically designed for types C (primer pair VC) and D (primer pair VD). The PCR amplification conditions were optimized and evaluated on 13 bovine and two duck samples that had been previously tested by the mouse bioassay. In order to assess the impact of sample treatment, both DNA extracted from crude samples and three different enrichment broths (TYG, CMM, CMM followed by TYG) were tested. A 100% sensitivity was observed when samples were enriched for 5 days in CMM followed by 1 day in TYG broth. False-negative results were encountered when C. botulinum was screened for in crude samples. These findings indicate that the current PCR is a reliable method for the detection of C. botulinum toxigenic strains type C and D in bovine samples but only after proper enrichment in CMM and TYG broth.     

Effects of vaccination against brucellosis and clostridia on the intake,performance, feeding behavior,blood parameters,and immune responses of dairy heifers calves

The aim of this study was to identify possible effects of different vaccination strategies (concomitantly or not) against brucellosis and clostridia on intake, performance, feeding behavior, blood parameters, and immune responses of dairy heifers calves. Fifty heifers calves were enrolled [38 Gyr (Zebu, Bos taurus indicus) and 12 5/8 Holstein Gyr]. At 120 d of age, animals were randomly distributed among 3 groups: B (n = 18), vaccinated against brucellosis; C (n = 14), vaccinated against clostridia and CB (n = 18), vaccinated concomitantly for both. Rectal and thermographic temperatures were evaluated on days 1, 0, 1, 2, 3, 5, 7,10, 14, and 28 relatives to the vaccination day. Feed and water intake, body weight (BW), and feeding behavior were monitored daily by an electronic feeding system. Blood was sampled on days 0, 3, 7, 14, and 28, relative to the vaccination day for determination of glucose and -hydroxybutyrate (BHBA) concentrations. Blood sampled on day 0 (prevaccination) and on days 28 and 42 were used to evaluate the immune response against Brucella abortus and clostridia. There was an increase in rectal temperature between the first and the third day postvaccination in the 3 groups. The thermography revealed an increase of local temperature for 7 d on groups B and CB. Group C had increased local temperature for a longer period, lasting for up to 14 d. Dry mater intake was reduced for groups B and CB, but no alteration was observed for group C. No alterations regarding initial BW, final BW, average daily weight gain, and feed efficiency were observed. No differences were observed for the 3 vaccination groups for blood parameters throughout the evaluation period. The concomitant vaccination against brucellosis and clostridia led to lower neutralizing antibody titers against epsilon toxin of Clostridium perfringens and botulinum toxin type C of C. botulinum (C > CB > B). When cellular proliferation assay and serological tests to B. abortus were evaluated, no differences were observed between groups B and CB. The present results indicate that the concomitant vaccination against brucellosis and clostridia has no relevant impact on the intake, performance, and feeding behavior of dairy calves. However, the concomitant vaccination of vaccines against these 2 pathogens impacts animal immunity against clostridial infections.     

鸡源大肠杆菌胞外蛋白酶的检测

用脱脂奶琼脂平板法检测了 1 0 0株大肠杆菌的胞外蛋白酶。结果 ,在 88株鸡源株中 ,阳性者为 2 2株( 2 5%) ,1 2株参考株中有 3株为阳性 ( 2 5%) ,显示较高比例的大肠杆菌产生胞外蛋白酶这一致病因子。另外 ,用嗜水气单胞菌 J-1株的胞外蛋白酶 ( ECPase54)抗血清作 Dot-ELISA,检测上述 1 0 0株大肠杆菌 ,3株鸡源株表现阳性 ,其脱脂奶琼脂平板法检测结果亦为阳性。     

牛魏氏梭菌病概述

魏氏梭菌(Clostridium welchii),又称产气荚膜梭菌(Clostridium perfringens),是引起牛的猝死病的主要病原,对养牛业的危害极大。本文简要介绍了牛魏氏梭菌病的病原、流行病学、临床症状、病理变化、诊断和防制。