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. 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.     

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。     

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。     

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%。     

丁酸梭菌对致病菌和有益菌的体外作用效果研究

试验研究了丁酸梭菌在体外对致病菌的抑制作用和对有益菌的增殖作用。将丁酸梭菌分别与大肠杆菌、沙门氏菌、产气荚膜梭菌、乳酸杆菌、双歧杆菌按不同的比例混合后进行培养,并与菌种单独培养进行比较。结果表明:培养36～48 h,联合培养组大肠杆菌、沙门氏菌、产气荚膜梭菌菌数均显著低于单独培养组;菌数比例为(1～100)∶1至100∶1时,丁酸梭菌对沙门氏菌均有较强的抑菌作用;菌数比例为100∶1时,丁酸梭菌对大肠杆菌和产气荚膜梭菌抑菌作用较强;菌数比例为10∶1时,丁酸梭菌对乳酸杆菌和双歧杆菌的增殖效果最好。联合培养8 h后,菌数比例为10∶1时,丁酸梭菌对双歧杆菌有显著的增殖效果。     

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.     

仔猪产气荚膜梭菌肠毒血症病原诊断及疫苗研究Ⅲ.7株A型产气荚膜梭菌菌株的产毒素试验

本试验对7株A型产气荚膜梭菌菌株在不同培养基、不同培养温度和不同培养时间的产毒力进行了试验。结果表明，培养基的种类和培养时间对菌株的产毒素能力影响较大，而培养温度则影响较小。所试菌株中C57-1菌株的产毒素能力最强，是制备疫苗的最佳菌株。     

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.     

Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanased and surviving cases of chronic grass sickness

Serum from 12 horses suffering from chronic grass sickness (CGS) were assayed for IgG antibodies against botulinum neurotoxins C and D (BoNT/C and BoNT/D) and to a surface antigen extract of a neurotoxin negative strain of Clostridium botulinum type C. Collectively, the six surviving CGS cases demonstrated significantly higher initial IgG levels (P=0.05) against surface antigens than the six that were subsequently euthanased. The surviving animals also demonstrated higher initial IgG levels against the BoNT/C but not reaching significance (P=0.06). The two groups demonstrated no difference between IgG levels against BoNT/D. This study supports existing evidence of the involvement of C. botulinum type C in the aetiology of grass sickness.     

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.     

A negative serological relationship between cases of grass sickness in Scotland and Clostridium perfringens type A enterotoxin

In an attempt to compare the equine grass sickness as reported in Europe with that described in the Republic of Colombia, sera from horses experiencing grass sickness in Scotland were used in neutralisation tests with Clostridium perfringens type A enterotoxin. The sera, from acute and chronic cases of the disease, failed to neutralise either crude or partially-purified enterotoxin. Neither were precipitin lines formed when the sera were treated against the toxin in immunoelectrophoresis. These results suggest that grass sickness in Europe and the equine disease in Colombia have a different aetiology.     

牛源破伤风梭菌的分离与鉴定

1头进行了瘤胃瘘管手术的黄牛发病死亡,临床症状表现为肌肉强直性痉挛。本试验取其手术伤口部位的炎性肌肉进行了细菌分离,分离到1株破伤风梭菌CT-JX-1。该分离菌株为具有顶端芽孢的革兰氏阳性杆菌;在普通琼脂培养基形成出灰白色、扁平、粗糙的菌落,菌落中心部分致密,外缘呈蜘蛛网状;该菌株能液化明胶,对多种糖均不发酵。