Proteases of Clostridium botulinum. I. Classification of proteases and literature survey

Proteolytic activity has long been regarded as an important characteristic for distinguishing between different types of Clostridium botulinum. While all strains of Clostridium botulinum type A examined so far possess proteolytic activity, the types B and F have both proteolytic and non-proteolytic varieties. Clostridium botulinum types C, D and E were generally regarded as non-proteolytic, but different investigators have shown proteolytic activity in certain strains of these types. A summary of the classification of proteolytic enzymes in general is given and further, investigations are reviewed on the proteolytic activity in Clostridium botulinum.     

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.     

Susceptibility of Foxes to Clostridium Botulinum Type C and E Toxins

Investigations were performed to determine the exact susceptibility of foxes to Clostridium botulinum type C and E toxins.Doses of 5 mill. MLD type C toxin mixed with the feed did not cause symptoms of botulism in either cubs or adult foxes. Subcutaneous injections of 300,100(0 MLD or more were fatal to cubs, while 750,000 MLD caused the death of all adults.Regarding type E toxin, doses of 1 mill. MLD affected neither cubs nor adults on oral administration. Subcutaneously injected doses of 5,000 MLD or more killed all cubs, while 10,000 MLD was required to produce lethal effect on adult animals.The conclusion made is that foxes are highly resistant to both type C and E Clostridium botulinum toxins following oral application. It is further revealed that foxes are 60–70 times more susceptible to type E than to type C toxin when injected subcutaneously.     

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.     

The occurrence of Clostridium botulinum at aquatic sites in and around Auckland and other urban areas of the North Island

Samples of bottom muds from lakes and waterways in the Auckland area were inoculated into bottles of cooked meat medium. After incubation, the media were tested for production of Clostridium botulinum toxin which was detected with samples from 11 of the 20 sites examined. Although toxins produced from all positive samples were neutralized by specific antisera of both type C and D toxins, it is likely that C. botulinum type Calpha is the only type present in the Auckland area. All samples from other urban areas of the North Island gave negative results.     

Two Cases of Equine Grass Sickness with Evidence for Soil‐borne Origin Involving Botulinum Neurotoxin

Botulism is caused by different types of Clostridium botulinum, a soil bacterium. Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease. On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C. botulinum. Different types and type mixtures (A–E) of C. botulinum and botulinum neurotoxin were found. For the first time, it has been shown that green grass blades contain botulinum toxin. The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil‐borne disease.     

The detection of Clostridium botulinum in fecal samples of cattle and swine and in the raw material and animal meal of different animal body rendering plants

Fecal samples of cattle and swine and samples of raw material and pulverized dehydrated meat taken from three rendering plants were investigated with special enrichment methods on the presence of Clostridium botulinum to get a view about the hygienic risk by the incidence of C. botulinum in rendering plants. Eight-six specimens were examined: 25 fecal specimens each of swine and cattle, 11 of raw material and 25 of pulverized dehydrated meat of three rendering plants. Twelve specimens contained C. botulinum: 7 fecal specimens, 6 of swine and one of cattle, 4 raw material specimens and one of pulverized dehydrated meat. C. botulinum was detected by its toxin production in culture medium. Six times C. botulinum type E, twice C. botulinum type B and one time C. botulinum type C was identified. C. botulinum could not be typed in other cases because the toxin quantities were too small. C. botulinum type E was detected in raw material and pulverized dehydrated meat in one of the three examined rendering plants.     

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.     

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。     

Susceptibility of mink to Clostridium botulinum type E toxin

Experiments aiming at elucidation of the toxicity of Clostridium botulinum type E for mink are described. The observations indicate that amounts in the order of 2 x10^s intraperitoneal MLD (mice) or approximately 200 MLD per g of type E toxin will kill a mink after oral administration. The symptoms observed in the animals were atypical as there was an unusually short period between administration of the toxin and the onset of symptoms and deaths of the animals. Similar results were obtained when Clostridium botulinum type E toxin was fed to Swiss mice. When mice were protected by subcutaneous injections of type E antitoxin prior to feeding the animals survived without showing any symptoms.Subcutaneous injection of type E toxin in amounts of the order of 2 x10^s intraperitoneal MLD (mice) killed mink, and typical symptoms of botulism were observed. This quantity corresponds to ap-proximately 2 intraperitoneal MLD (mice) per g.Comparison is made with previous observations obtained in similar experiments made with Clostridium botulinum type C toxin. It is shown that mink arc substantially less susceptible to type E than to type C toxin when the toxins are given by mouth. On this basis previous results in reports on outbreaks of botulism in mink caused by Clostridium botulinum type E may be regarded as questionable.     

A型肉毒毒素基因的克隆及序列分析

目的:克隆肉毒梭菌(Clostridium botulinum)A型肉毒毒素(BoNTa)编码基因。方法:提取肉毒梭菌国际标准株(62A)基因组DNA,根据肉毒梭菌BoNTa基因(GenBank登录号M30196)序列设计引物,采用LA-PCR方法,扩增出目的基因片段,与pMD18-T载体连接,通过酶切鉴定、测序分析克隆到的A型肉毒毒素基因序列。结果:该基因片段与Genbank中的BoNTa基因序列(GenBank登录号M30196)一致性为100%,预测氨基酸序列一致性为100%。结论:成功克隆肉毒梭菌的A型肉毒毒素基因序列,为肉毒梭菌的快速检测,以及进一步用基因工程方法生产A型肉毒毒素奠定了基础。     

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。     

Clostridium botulinum type D intoxication in a dairy herd in Ontario

Thirty-four Holstein cows died after exposure to Clostridium botulinum type D toxin, presumably from contaminated haylage. The presence of type D toxin in ruminal contents was confirmed by mouse inoculation. This is the first confirmation by direct toxin isolation of C. botulinum type D toxin in cattle in North America.     

Susceptibility of mink to Clostridium botulinum type C toxin

Investigations to determine the exact susceptibility of mink to Clostridium botulinum type C toxin clearly showed that mink were considerably less resistant to this toxin than has previously been described. Mink weighing approximately 900 g were killed by 360 MLD when toxin was mixed into the feed. By subcutaneous injection, the lethal dose was determined to be in the range of 18 to 36 MLD.When comparing the susceptibility per g of body weight after parenteral application of the toxin, mink proved to be less resistant than mice to this type of toxin. Continued feeding tests in mink with suspected material is pointed out as a preferable method for practical demonstrations of Clostridium botulinum type C toxin in cases where the toxin content in the suspected material is very low (1 MLD per g or less).     

Presumptive diagnosis of Clostridium botulinum type D intoxication in a herd of feedlot cattle

Fifty-two feedlot cattle exhibited clinical signs suggestive of botulism. Clostridium botulinum type D organisms were recovered from ruminal fluid of 4 of the 5 affected animals tested and were isolated from bakery waste fed to the cattle. Clostridium botulinum type D has not been reported previously in Canadian cattle.     

Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene

The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia.     

Observations on the possible invasiveness of Clostridium botulinum for waterfowl.

Twenty-four ducklings given multiple doses of Clostridium botulinum type C spores and toxin per os and 27 affected waterfowl from four natural outbreaks of the disease were examined bacteriologically. No evidence of invasion of the blood or liver was found in any bird and it is suggested that invasiveness and toxigenesis in internal organs are probably of little, if any, importance in the causation of botulism in waterfowl.     

Quantitative real-time PCR for detection of neurotoxin genes of Clostridium botulinum types A,B and C in equine samples

Botulism in horses in the USA is attributed to Clostridium botulinum types A, B or C. In this study, a duplex quantitative real-time PCR (qPCR) for detection of the neurotoxin genes of C. botulinum types A and B, and a singleplex qPCR for detection of the neurotoxin gene of C. botulinum type C, were optimized and validated for equine gastrointestinal, faecal and feed samples. The performance of these assays was evaluated and compared to the standard mouse bioassay (MBA) using 148 well-characterized samples, most of which were acquired from a repository of veterinary diagnostic samples from cases of botulism: 106 samples positive for C. botulinum (25 type A, 27 type B, 28 type C, 1 type D and 25 type E) and 42 negative samples. The sensitivities of the qPCR assays were 89%, 86% and 96% for C. botulinum types A, B and C, respectively. The overall sensitivity of the mouse bioassay for types A, B and C was 81%. The specificities of the qPCR assays were 99–100% and the specificity of the mouse bioassay was 95%.