魏氏梭菌类毒素疫苗研制及其免疫家兔抗体消长规律

本研究分别从山东省的莱芜、泰安、潍坊的三个养兔场发生疑似梭菌性下痢的病兔体分离到三株魏氏梭菌，鉴定为A型。利用该分离菌株和A型魏氏梭菌标准株（CVCC37）所产外毒素经甲醛灭活并加入氢氧化铝胶佐剂吸附浓缩后制备了A型魏氏梭菌类毒素疫苗。对该疫苗分别进行了安全性检验、有效免疫剂量试验和接种家兔1～23周血清中抗毒素（Ab）消长规律研究。结果表明，魏氏梭菌外毒素用03％甲醛32h能够彻底灭活，并最大可能地保持其抗原性；制备的魏氏梭菌类毒素疫苗无毒副作用，安全可靠；用野毒株和标准株制备的类毒素疫苗对家兔的有效免疫剂量为2mL／只，免疫保护效果可靠；采用野毒株和标准株制备的类毒素疫苗接种家兔后第二周血清抗毒素效价迅速升高，到第四周达最高峰。分别为6．6一和7．125log2，较高抗体滴度维持约17周后缓慢下降，至23周时血清平均抗毒素滴度仍维持在4．0-和3．4log2以上，所以，该类毒素疫苗的免疫保护期可以设定为6个月。     

魏氏梭菌毒素疫苗研制及其免疫家兔抗体消长规律

从山东省莱芜、泰安、潍坊等3个养兔场发生疑似梭菌性下痢的病兔体内分离到3株魏氏梭菌，鉴定为A型。利用该分离菌株和A型魏氏梭菌标准株(CVCC37)所产外毒素经甲醛灭活并加入氢氧化铝胶佐荆吸附浓缩后制备了A型魏氏梭菌类毒素疫苗。对该疫苗分别进行了安全性检验、有效免疫剂量试验和接种家兔1～23周血清中抗毒素(Ab)消长规律研究。结果表明，魏氏梭菌外毒素用0．3％甲醛32h能够彻底灭活，并最大可能地保持其抗原性；制备的魏氏梭菌类毒素疫苗无毒副作用，安全可靠；用野毒株和标准株制备的类毒素疫苗对家兔的有效免疫剂量为2mL／只，免疫保护效果可靠；采用野毒株和标准株制备的类毒素疫苗免疫接种家兔后第2周血清抗毒素效价迅速升高，到第4周达最高峰，分别为6．6 log2和7．125 log2，较高抗体滴度维持约17周后缓慢下降，至23周时血清平均抗毒素滴度仍维持在4．0 log2和3．4 log2以上，所以，该类毒素疫苗的免疫保护期可以设定为6个月。     

山东疫区魏氏梭菌血清型的多重PCR检测

采用多重PCR对从流行过魏氏梭菌病的牧场粪便样品中分离魏氏梭菌的α、β、ε、ι毒素基因进行了检测,确定了血清型。电泳成像显示,仅魏氏梭菌扩增出了特异性条带,其他对照菌株无,说明该方法具有很高的特异性。通过对山东德州、枣庄、泰安、临沂、青岛等地养殖场的418个样品分离的62个菌株的检测,检出率为14．8％,均为A型。研究确认,多重PCR是魏氏梭菌血清型鉴别的一种快速、简便的研究方法,山东省流行型别与国外报道不完全一致。     

不同方法对牛舍气载魏氏梭菌型别的研究

分别采用ELISA、多重PCR方法对从一个犊牛舍分离到的魏氏梭菌进行型别研究，结果显示：ELISA检测结果为88％的菌种属于A型，3．4％为C型，8．6％是D型；而多重PCR结果均为A型。     

山东疫区魏氏梭菌血清型的多重PCR检测

采用多重PCR对从流行过魏氏梭菌病的牧场粪便样品中分离魏氏梭菌的α、β、ε、ι 毒素基因进行了检测,确定了血清型。电泳成像显示,仅魏氏梭菌扩增出了特异性条带,其他对 照菌株无,说明该方法具有很高的特异性。通过对山东德州、枣庄、泰安、临沂、青岛等地养殖场 的418个样品分离的62个菌株的检测,检出率为14．8％,均为A型。研究确认,多重PCR是 魏氏梭菌血清型鉴别的一种快速、简便的研究方法,山东省流行型别与国外报道不完全一致。     

鲜猪肉中A型产气荚膜梭菌污染情况调查

对贵阳市部分市场销售的鲜猪肉A型产气荚膜梭菌的污染情况进行了调查，从被检肉样中分离获得3株A型产气荚膜梭菌，分离率为6．25％（3／48），表明贵阳市部分市场销售的鲜猪肉存在A型产气荚膜梭菌的污染，对消费者的健康构成潜在的威胁。     

毛皮动物魏氏梭菌病的诊治

<正>魏氏梭菌病又称产气荚膜杆菌病,该菌广泛存在于土壤、饲料、蔬菜、污水、粪便中,在某些应激因素诱导下引起动物发病并导致其死亡。该菌分A,B,C,D,E,F六个型,闫新华教授等分离于狐狸、貉、水貂、兔、犬的菌株鉴定后证明均为A型,该菌对经济动物威胁性较大,近几年,随着我国特种毛皮动物养殖业的发展,有关魏氏梭菌侵害特种毛皮动物的报道逐年增多,本单位研究所今年6~9月份接到9个病例,其中狐狸4例,貉子3例,水貂2例,通过对发病情况、临床症状及尸体     

绵羊肠毒血症的病原诊断

青海省海北州祁连县默勒镇2006年至2008年绵羊发生持续死亡,死亡数达3358只,从4只病死羊回肠中分离到疑似魏氏梭菌,经中和试验及毒素测定,证实分离到的菌株为D型魏氏梭菌,静注小白鼠最小致死量为200—1600MLD／mL,从而诊断为D型魏氏梭菌引起的肠毒血症。     

两种兔源肠致病性菌原生质体融合的耐药性遗传标记选择

在兔源肠致病性大肠杆菌WF-03-B3菌株和兔源A型魏氏梭菌XT-04-X5菌株原生质体融合的研究中，为了对融合子进行选择鉴定，在呼吸缺陷选择的基础上WF—03-B3菌株O2^r（氧气抗性）；XT-04-X5菌株O2^r（氧气敏感性），再应用定性和定量药物敏感性试验，对两种菌原生质体融合的耐药性遗传标记进行了筛选．结果表明，WF-03-B3菌株对丁胺卡那霉素AKN^s（丁胺卡那霉素敏感）；XT-04-X5菌株对丁胺卡那霉素AKN^r（丁胺卡那霉素抗性），在此基础上，测定了丁胺卡那霉素在选择培养基中的适用浓度。筛选出兔源肠致病性大肠杆菌WF-03-B3菌株的遗传标记为（AKN^s，O2^r）和兔源A型魏氏梭菌XT-04-X5菌株的遗传标记为（AKNr，O2^s）。     

黑鹿魏氏梭菌的分离与鉴定及部分生物学特性观察

黑鹿是哺乳纲偶蹄目鹿科大型食草动物,学名为Cervus unicolor,是我国的特产动物,有较高的研究、经济价值,属国家二级保护动物,性喜水,雨后活动频繁,常到溪涧喝水或沐浴.即使冬季,也常流连于水塘,故名之"水鹿".目前黑鹿人工饲养数量较大,但该动物饲养技术和疫病防控尚缺乏经验.本实验室从某动物园发生"猝死症"的观赏黑鹿肠道中分离出1株梭菌,通过对该株梭菌进行培养特性观察、生化试验和动物接种等试验,确诊此次黑鹿致死原因为魏氏梭菌感染.应用魏氏梭菌分型血清确定魏氏梭菌分离株的血清型为A型.由于该病没有有效的疫苗用于预防,本文同时探索了魏氏梭菌灭活苗的制备工艺,提出了防治黑鹿魏氏梭菌病的措施.     

鸡传染性支气管炎病毒血凝谱的研究

用家兔A型魏氏梭菌培养液处理的6株鸡传染性支气管炎病毒,以方阵试验分别与人O型红细胞及羊、猪、兔、鸡、鸭、鹌鹑、麻雀、小鼠等8种动物的红细胞作血凝试验,结果证明,鸡传染性支气管炎病毒H_(120)株和M_(41)株均能凝集人O型以及羊、猪、兔、鸡、鸭、鹌鹑、小鼠的红细胞,而不能凝集麻雀的红细胞;GIBV株和Connecticut株能凝集人O型以及免、鸡、鹌鹑、麻雀的红细胞,而不能凝集猪、羊、鸭、小鼠的红细胞;Gray株能凝集兔、鸡、鹤鹑的红细胞,不能凝集人O型以及猪、羊、鸭、麻雀、小鼠的红细胞;而经家兔A型魏氏梭菌培养液处理的T株和未经处理的6株病毒对人O型以及8种动物的红细胞都没有凝集性。试验还证明,M_(41)株和H_(120)株对人O型及8种动物的血凝活性水平有差异。     

Initial studies on "six months disease" in sheep.

Enterotoxaemia in sheep due to Clostridium welchii type D was indicated by field and laboratory investigations in Nepal. Morphological, cultural, biochemical, biological and toxin-producing characteristics observed were used to type the isolates. In anaerobic meat medium, all isolates produced pinkish discoloration of meat. All the strains fermented lactose, maltose, dextrose and sucrose whereas, salicine was fermented only by 17 strains. All but five strains were MR negative. Out of 200 isolated, 166 produced both alpha and epsilon toxins and the remaining 34 non-toxogenic strains are likely to be variants which have lost their toxogenicity. Epidemiologically the local name "Six months disease" and enterotoxaemia are considered to be identical diseases.     

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。     

A型魏氏梭菌α毒素Asp-56基因定点突变与结构分析

为探索A型魏氏梭菌α毒素第56位天冬氨酸(Asp-56)对其生物学活性的影响,将α毒素Asp-56密码子(GAT)定点突变成甘氨酸(Gly-56)密码子(GGT),构建了含α毒素D56G突变基因表达质粒的重组菌株BL21(DE3)(pM-D56G),该突变体蛋白表达量约为22.48%。α毒素和α毒素D56G突变体蛋白的二级结构主要由α螺旋和无规则卷曲组成,两者的圆二色(CD)光谱检测有微小变化。生物学活性检测结果表明,α毒素D56G突变体蛋白失去了α毒素的磷脂酶C活性,且α毒素D56G突变体蛋白免疫的小鼠能够保护1MLD的A型魏氏梭菌标准株C57-1毒素攻击。此研究为进一步研究α毒素分子结构与生物学功能的关系奠定了基础。     

Double intussusception fatally complicated by clostridial infection in a dog (a case report)

A 10-month-old Alsation dog with history of anorexia, diarrhoea, dehydration and vomition developed a double intussusception which affected the distal jejunum and proximal ileum. Necropsy revealed the intussusception to be swollen and congested with fibrinous adhesions between the intussusceptum and intussuscepiens. Two pieces of bone believed to be the inciting cause were found within the intussusceptum. Clostridium welchii and Clostridium bifermentans were isolated from the lesions.     

Gas gangrene in the dog

Three cases of gas gangrene in the dog are described. Symptoms followed fracture of a femur in two cases and amputation of a hindlimb in the third. Clostridium welchii was isolated in each case. Two animals recovered following drainage of the lesion together with local and parenteral administration of the appropriate antibiotics.
Résumé. On décrit trois cas de gangrène gazeuse chez le chien. Des symptômes ont suivi une fracture du fémur dans deux cas et une amputation dans le troisième. On a isolé un Clostridium welchii dans chaque cas. Deux animaux se sont rétablis après drainage de la lésion avec à la fois une administration locale et parentérale des antibiotiques appropriés.
Zusammenfassung. Drei Fälle von Gasgangrän in Hunden sind beschrieben. In zwei Fällen folgten Symptome einer Oberschenkelfraktur und in einem dritten Fall einer Amputation. Clostridium welchii wurde in jedem Fall isoliert. Zwei Tiere erholten sich nach Dränage der Läsion kombiniert mit örtlicher und parenteraler Verabreichung der entsprechenden Antibiotika.     

Isolation and characterization of Clostridium perfringens from apparently healthy animals of the Shandong province of China

In a pilot study the presence and frequency of Clostridium (C.) perfringens was investigated among apparently healthy farm animals in the Shandong province of China. 748 faecal samples were collected from 9 pig-, 4 sheep-, 7 cattle- and 5 rabbit farms. C. perfringens was isolated from 124 samples (16.6%). The isolates were classified into major toxin types by using PCR analysis detecting the genes encoding these toxins. All isolates were identified as C perfringens toxin type A. There are also some reports from different regions in China linking C. perfringens toxin type A strains to gastrointestinal diseases. Therefore further investigations about the epidemiologic role of C perfringens toxin type A strains in the Shandong region are necessary. Currently, cases of enterotoxemia from this region are investigated for the presence of C perfringens.     

牛出血性肠炎A型产气荚膜梭菌的分离鉴定

从新疆维吾尔自治区某牛场采集的3份疑似出血性肠炎病料中分离到8株产气荚膜梭菌,用PCR扩增保守基因16SrRNA,并进行序列测定和同源性分析,再通过多重PCR方法扩增型特异性基因进行分离菌株的分型鉴定。结果显示,所分离的8株产气荚膜梭菌之间16S rRNA基因同源型为100%,与GenBank参比序列同源性在99.8%以上,确定为产气荚膜梭菌。遗传进化分析表明,本次分离的8株产气荚膜梭菌之间拥有共同起源,但与所用的参考菌株分属不同来源。多重PCR扩增结果显示,8株菌株均为产气荚膜梭菌A型。     

Identification of virulence factors by multiplex PCR in Escherichia coli isolated from calves in Minas Gerais, Brazil

In this study, multiplex PCR was employed to investigate the virulence factors of Escherichia coli strains isolated from 60-day-old calves. Faecal samples were collected from 54 calves at 12 dairy farms in the state of Minas Gerais, Brazil. A total of 156 isolates were obtained after culture and microbiological isolation and were tested by multiplex PCR for the presence of genes encoding toxins (Stx1, Stx2 and STa) and adherence factors (intimin, F41 and F5). Seventy of 156 isolates were positive for at least one virulence factor: ten (14.3?%) from diarrhoeic animals and 60 (85.7?%) from healthy calves. The virulence markers identified were: Stx1 (82.8?%), eae (24.3?%), F41 (11.4?%), F5 (10?%), STa (4.28?%) and Stx2 (4?%). In diarrhoeic animals, Stx1 (70?%) and F41 (30?%) were identified, while Stx1 (83.3?%), eae (28.3?%), F41 (8.3?%), F5 (11.6?%), STa (5?%) and Stx2 (1.6?%) were detected in isolates from healthy calves. Mixed infections with pathotypes Shiga toxin-producing E. coli (STEC)/enteropathogenic E. coli, STEC/enterohaemorrhagic E. coli and STEC/other (eae/F5, Stx1/STa) were detected in five healthy calves. Pathogenic E. coli were identified in 59.26?% of all calves and on 75?% of the dairy farms studied, not only in diarrhoeic (five of six) but also in healthy calves (27 of 48), which demonstrates the importance of this agent in the aetiology of diarrhoea in calves in the state of Minas Gerais.     

Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals.

Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animals may be susceptible to all subtypes of C. perfringens type C. A simple method of using blood agar plates coated with type A antiserum for demonstration of hemolytic patterns was found advantageous in differentiation of C. perfringens strains.