羊梭菌病快速鉴别PCR方法的建立及初步应用

根据GenBank已公布的产气荚膜梭菌和腐败梭菌的α毒素基因序列,分别设计并合成针对2种α毒素基因的特异性引物,通过扩增条件的优化,建立快速鉴别产气荚膜梭菌和腐败梭菌的双重PCR方法。特异性试验显示,产气荚膜梭菌和腐败梭菌参考菌株均扩增出了相应的预期目的条带,而大肠埃希菌、沙门菌、金黄色葡萄球菌和链球菌则均未能扩增出相应条带。灵敏度试验结果显示,产气荚膜梭菌和腐败梭菌基因组DNA最低检测量分别为17.95pg/μL和1.261pg/μL。应用该方法对样品进行检测,其中5份为产气荚膜梭菌阳性。成功建立了特异性强、敏感性高的双重PCR方法,可以有效进行产气荚膜梭菌和腐败梭菌的快速鉴别,对羊梭菌病病原快速鉴定及流行病学调查具有重要意义。     

二重PCR方法鉴别气肿疽梭菌和腐败梭菌

用包含16S～23S rDNA间隔区和23S rDNA的部分序列作为气肿疽梭菌特异性标志,以α毒素部分序列作为腐败梭菌特异性标志,建立了鉴别气肿疽梭菌和腐败梭菌的二重PCR方法。结果显示：气肿疽梭菌C54-1株扩增出大小为509 bp的条带,腐败梭菌C55-1株、C55-16株均扩增出大小为148 bp的条带,均与预期吻合;而产气荚膜梭菌C57-1株、C59-37株,肉毒梭菌C62-4株,诺维梭菌C61-4株均未扩增出任何条带。扩增产物的测序结果进一步证实了本方法的特异性。菌株的生物学特性试验结果也符合相应气肿疽梭菌和腐败梭菌的特点。本研究所建立的二重PCR方法可用于气肿疽梭菌和腐败梭菌的快速鉴定。     

仔猪去势创口感染引发恶性水肿病的诊治

<正>猪恶性水肿病是由腐败梭菌为主的多种梭菌引起的猪的急性传染病,发病迅速,病情严重,具有很高的致死率。特征为创伤局部发生急剧性炎性水肿,并伴有发热和全身毒血症。病原为梭菌属中的腐败梭菌、魏氏梭菌及诺氏梭菌、溶组织梭菌等。其中主要是腐败梭菌,其次是魏氏梭菌,而诺氏梭菌、溶组织梭菌引起的发病情况较少。该病病原菌广泛存在于自然界中,猪的肠道中和土壤中较多。病猪不能直接接触传染健康猪,但可污染外界环境。病猪的水肿部位     

重组腐败梭菌α毒素替代羊三联四防苗中腐败梭菌组分的免疫效果评价

为了评价重组腐败梭菌α毒素在联苗中的免疫效果,试验用不同含量的重组腐败梭菌α毒素替代羊三联四防苗中腐败梭菌组分制备联苗,分别免疫兔和绵羊,在免疫后的第10,14,21天分别测定血清中和效价,免疫后第25天用1个最小致死量(MLD)的腐败梭菌毒素对免疫兔和绵羊分别进行攻毒,以期得到能使动物获得足够保护的抗原含量。结果表明:对于兔和绵羊联苗中重组腐败梭菌α毒素蛋白最小免疫剂量分别为0. 3 mg、0. 25 mg时,在免疫后第14天腐败梭菌毒素血清中和效价均能达到1;免疫后第25天进行攻毒保护试验,免疫兔和绵羊攻毒保护率分别为100%、75%。说明重组腐败梭菌α毒素可以替代羊三联四防苗中腐败梭菌组分用来进行免疫。     

羊恶性水肿病的病因、诊断与防控

羊恶性水肿病主要是由腐败梭菌引起的急性、创伤性传染病,经口感染腐败梭菌时称快疫,是常见病,经创伤感染腐败梭菌时称恶性水肿,较少见。1病因分析引起本病的主要病原为腐败梭菌,其次A型魏氏梭菌也可感染致病。病菌由伤口进入受伤组织中,在厌氧条件下进行繁殖产生毒素,损害血管引起局     

不同源性腐败梭菌产毒特性的试验

用自制的6种腐败梭菌培养基(Ⅰ、Ⅱ、Ⅲ、Ⅳ、Ⅴ、Ⅵ),对分离到的3株不同源性的腐败梭菌LF2010、LF9601、YF9801进行培养基和产毒能力的筛选试验。结果,此3株菌在Ⅱ号培养基上的产毒能力分别达到3 000,1 000和500MLD/mL,这为今后研制腐败梭菌疫苗的研制提供了一定的技术依据。     

腐败梭菌α毒素和产气荚膜梭菌ε毒素的 共表达及其免疫效力评价

为获得腐败梭菌α毒素和D型产气荚膜梭菌ε毒素的共表达产物,并鉴定其致死活性和反应原性,评价其免疫保护效力,利用PCR扩增腐败梭菌α毒素和产气荚膜梭菌ε毒素基因Gcsa和Gcpe并克隆至pETDuet-1质粒,转化E.coli BL21(DE3),自诱导培养基诱导这2段基因共表达;用SDS-PAGE和Western-blot以及小鼠毒性试验、抗毒素血清中和试验对表达产物进行鉴定;将诱导后的大肠杆菌灭活,制成铝佐剂疫苗1 m L/只皮下注射免疫家兔2次,测定免疫血清中和抗体效价,用1 MLD的腐败梭菌毒素和D型产气荚膜梭菌毒素分别攻击。结果显示,所构建的E.coli BL21(pETDuet-Gcsa-Gcpe)可以同时表达Gcsa和Gcpe,表达产物能够与腐败梭菌和D型产气荚膜抗毒素血清反应;且具有小鼠致死毒性,只有用2种抗毒素一同作用,才能将其毒性中和;制成灭活疫苗免疫家兔,免疫血清对腐败梭菌毒素的中和抗体效价为2 MLD/0.1 m L,对D型产气荚膜梭菌毒素的中和抗体效价≥50 MLD/0.1 m L;用2种毒素攻击,免疫组家兔全部保护,对照组全部死亡,达到《中华人民共和国兽药典》(2015版)效力检验标准。结果表明,所构建的表达载体可以实现α毒素和ε毒素的活性共表达,本研究为腐败梭菌和产气荚膜梭菌的多联基因工程灭活疫苗的研究提供了实验基础。     

绵羊恶性水肿病的治疗

恶性水肿病是一种急性、创伤性传染病,它的特征是局部的炎性水肿和全身毒血症,病原为腐败梭菌。羊经口感染腐败梭菌时称快疫,是较普遍存在的疾     

水貂腐败梭菌感染的诊疗试验

<正>腐败梭菌主要经消化道感染水貂,引起水貂急性致死性传染性疾病,主要症状为胃肠道出血、水肿以及败血症[1]。腐败梭菌病的发生无明显季节性,春、秋季多发,常呈散发或地方性流行。我国腐败梭菌病的发病率较低,但一经暴发会给水貂养殖业带来巨大的经济损失。腐败梭菌广泛存在于自然界中,各种动物的肠道及土壤中均有分布并可随灰尘飞扬,可通过     

羊快疫发病机制及防治

羊快疫(Braxy)是由腐败梭菌(Clostridium septicum)引起的一种急性、高度致死性传染病,腐败梭菌α毒素是其主要的致死毒素。因腐败梭菌会引起家畜恶性水肿,故曾被称为恶性水肿杆菌,对人类健康和畜牧业生产造成严重危害。目前,国内外对于腐败梭菌及其毒素的研究尚有空白,疾病的诊断和防控技术还存在不足。文章从羊快疫的发病机制及其防治措施等方面进行了综述,介绍了目前国内外对于此种疾病的研究进展并进行了展望。     

Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema

The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined. The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema. Five (22%) of 23 C. septicum strains, five (71%) of seven C. perfringens strains and all strains of C. sordellii showed resistance to oxytetracycline. These oxytetracycline-resistant strains carried tetracycline-resistance genes [tetA(P), tetA408(P), tetB(P) and tetM]. The sequences of the tetracycline-resistance genes of some C. septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species. This is the first report of resistance of C. septicum to tetracycline.     

Amplification of the 16S-23S r spacer region for rapid detection of Clostridium chauvoei and Clostridium septicum

Amplification of the 16S-23S rDNA spacer region by polymerase chain reaction (PCR) was used for the rapid detection of Clostridium chauvoei and C septicum. To assess its specificity, PCR was performed with total DNA from 42 strains of clostridia and three strains of other genera. PCR products specific to C chauvoei or to C septicum were generated from homologous cultures only. Clostridium chauvoer-specific or C septicum-specific amplicons were also generated from tissues of cows experimentally infected with C chauvoei or C septicum and in DNA samples from cows clinically diagnosed as having blackleg or malignant oedema. These results suggest that a species-specific PCR may be useful for the rapid and direct detection of C chauvoei and C septicum in clinical specimens.     

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.     

Protective effect of Clostridium septicum alpha-toxoid vaccine against challenge with spores in guinea pigs

The protective effect of an alpha-toxoid vaccine of Clostridium septicum purified alpha-toxin was investigated in guinea pigs. Purified alpha-toxin was treated with formalin to make toxoid, and alpha-toxoid vaccine was prepared by mixing alpha-toxoid (4 to 64 microg/dose) with an aluminum phosphate gel as adjuvant. Guinea pigs were immunized twice with different doses of alpha-toxoid vaccine, and challenged with spores of C. septicum. The guinea pigs surviving after challenge had been immunized with 8 microg/dose or more of alpha-toxoid. All these animals produced titers of 20 units or higher of antitoxin at the challenge. The results suggest that C. septicum alpha-toxin plays an important role in protection against challenge with spores in guinea pigs.     

PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed,paraffin-embedded tissues

The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.     

Genetic variation and cross-reactivity of Clostridium septicum alpha-toxin

Clostridium septicum alpha-toxin genes were sequenced with the polymerase chain reaction (PCR) products amplified from DNAs of 25 C. septicum strains, and were classified into 10 patterns. Alpha-toxins were purified from the culture supernatant of four C. septicum strains (strains No. 44, Kagoshima 8, Mie and Tokachi) which were specially chosen from patterns of the deduced amino acid sequences. The molecular weights of the alpha-toxins were not different according to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. However, the isoelectric points between the alpha-toxins of No. 44 and Tokachi strains differed markedly. Cross-neutralization tests were performed with purified alpha-toxins and antitoxins in mice and in Vero cells. Each antitoxin showed roughly the same titers against the four alpha-toxins in mice and completely identical titers against these in Vero cells. Calves immunized with toxoid prepared from the culture supernatant of No.44 strain were challenged by exposure to spores of Mie strain. The toxoid conferred protection against the challenge in calves. From these results, although genetic variation has been observed within the C. septicum alpha-toxin gene, C. septicum strains toxoid of strain No.44 induces protective immunity against exposure to C. septicum that produce other subtypes of alpha-toxin containing several different amino acid residues.     

Malignant oedema associated with blood-sampling in sheep

Malignant oedema is a fatal disease of several animal species, produced by one or more members of the Clostridium genus. We report here a case of malignant oedema in a 1-year-old Friesian sheep after a blood sample was collected from the jugular vein. Clostridium septicum and Clostridium sordellii were isolated from the lesions and also demonstrated by a fluorescent antibody test. This report stresses the need for maintaining a clean environment for animals and for strict hygienic measures during procedures that generate wounds, together with immunity acquired by proper vaccination, for prevention of malignant oedema.     

Immunogenicity of Clostridium septicum in guinea pigs

Five strains of Clostridium septicum were used to prepare bacterins, bacterin-toxoids, toxoid, and combinations of bacterins or bacterin-toxoids. These preparations were tested for immunogenicity in guinea pigs vaccinated subcutaneously with 1.0 ml of product. Usually, a second vaccination was given 21 to 24 days later. The immunity of groups of vaccinated guinea pigs was challenged with as many as 22 strains of C septicum. When challenge exposed with homologous strains at 21 to 24 days after one vaccination or 10 t0 18 days after a second vaccination, 60% to 100% of the guinea pigs in each group survived. Demonstrable cross-protection among strains of C septicum varied from none to 100% protection in vaccinated guinea pigs. A combination of bacterin-toxoid prepared from four selected strains protected 70% to 100% of the vaccinated guinea pigs challenge exposed with 21 strains. Duration-of-immunity studies demonstrated a twofold to fourfold decrease in protection when the vaccination-to-challenge interval was extended an additional 3 weeks. Strains of C septicum do not have an effective common immunogen and the stimulated immunity appears to be of short duration. Antitoxin was demonstrated to be less important than other factors in protecting against C septicum infection.     

Differences in the pathogenicity of various bacterial isolates used in an induction model for gangrenous dermatitis in broiler chickens

A gangrenous dermatitis model was developed in broiler chickens, in which birds previously vaccinated at 14 days of age with a bursal disease virus vaccine were challenged at 4 wk of age with various bacterial combinations with the combination of subcutaneous and intramuscular injection. Gangrenous dermatitis lesions were not produced in birds injected with one of the Staphylococcus aureus isolates, either alone or in combination with various Clostridium septicum isolates. Other S. aureus isolates produced significant levels of gangrenous dermatitis either alone or in combination with the same C. septicum isolates. These same C. septicum isolates when given alone did not produce gangrenous lesions. Data from this experiment show the highest level of mortality occurred in birds challenged with a mixture of C. septicum and S. aureus isolates, whereas lower or no mortality was associated with the same isolates given separately. The data clearly demonstrate that the pathogenicity of isolates responsible for gangrenous dermatitis varies widely, indicating that the frequency and severity of lesion production, as well as the occurrence of mortality, are largely dependent upon the specific isolate or isolates with which the birds are challenged.     

湖羊黑疫快疫的研究：Ⅰ.病原诊断

自１９６４年起，浙江杭嘉湖地区湖羊发生了一种高发病率高死亡率的急性传染病，仅３个县的不完全统计在一年内即死去一万余头，经病原学研究证明此病主要由水肿梭菌、黑疫的病原所引起，较少的病例是由水肿梭菌和腐败梭菌混合感染所致，后者是快疫的病原，研究工作早在３０年前结束，但迟至今日始发表。