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）。     

一例兔大肠杆菌与魏氏梭菌混合感染病例诊断与药敏试验

为诊断患病死亡兔,对死亡兔进行临床剖检、细菌分离培养,获得PL01、PL02、PL03、PL04、PL05、PL06、PL07、PL08、PL09、PL10、PL11等11株细菌。对以上菌株使用生化鉴定试剂盒(GB4789.38-2012)进行生化鉴定,结果:PL01、PL02、PL03、PL04靛基质+、MR+、VP-、西蒙氏柠檬酸盐-,与大肠杆菌一致;利用多重PCR方法测定PL05、PL06、PL07、PL08、PL09、PL10为A型魏氏梭菌,PL11为C型魏氏梭菌。按照琼脂扩散法做药敏试验,显示大肠杆菌对替米考星、丁胺卡那敏感,魏氏梭菌对头孢唑林、强力霉素、氧氟沙星敏感。     

兔肠致病性大肠杆菌和魏氏梭菌毒力基因的快速检测

以一中型肉兔场为研究对象,采集病、死兔肠内容物,粪便样品,饮水,污水,饲料和以兔舍为中心不同距离处的尘土样品,共得到样品135份,运用双重PCR法同时检测样品中的肠致病性大肠杆菌(eaeA基因 )和魏氏梭菌(α-毒素基因 )。结果表明在病、死兔肠内容物中、粪便、生活污水中这两种菌同时存在和仅一种菌单独存在的几率都较大;兔舍周围尘土由近到远肠致病性大肠杆菌(eaeA基因 )存在的几率呈递减趋势,魏氏梭菌(α-毒素基因 )在兔舍周围尘土中存在的几率较少,且变化不大。     

利用多重PCR检测兔肠致病性大肠杆菌和魏氏梭菌的毒力基因

我们建立了一种同时检测兔肠致病性大肠杆菌和魏氏梭菌毒力基因的二重PCR方法。在一次PCR反应中同时扩增兔肠致病性大肠杆菌（REPEC）的eae毒力基因和魏氏梭菌的α-toxin毒力基因，扩增产物经电泳，出现与阳性对照细菌条带大小一致的条带即判断为该标本阳性，本试验还对79份腹泻兔粪便或肠内容物样品进行了检测，结果35.4％（28／79）检出eae＋的REPEC，而魏氏梭茵的检出率为。6．3％。敏感性试验结果表明，该二重PCR能检出10 CFU的REPEC，10 CFU的魏氏梭菌，具有简便、快速、特异、经济等特点，宜于临床应用。     

断奶幼兔腹泻肠致病性大肠杆菌LEE毒力岛的分子检测

为了了解 L EE毒力岛在山东省兔场的流行情况 ,自 2 0 0 0年 2月到 2 0 0 3年 6月从潍坊、曲阜、恒台、烟台、莱芜、泰安等地兔场的断奶前后腹泻和健康兔肠内容物或粪便中分离的大肠埃希氏菌 ,用 PCR法检测 L EE毒力岛上的 eae A基因。结果发现 ,在 2 0～ 30日龄、30～ 5 5日龄、5 5～ 80日龄的腹泻幼兔中 ,携带 L EE毒力岛的大肠埃希氏菌检出率分别为 0 (0 / 5 )、72 .7%(2 4 / 33)、2 3.5 % (4 / 17) ,而在相同日龄的健康幼兔中检出率为 0 (0 / 2 4 )。从其他肠道致病菌的检出情况来看 ,沙门氏菌和魏氏梭菌检出率极低 ,沙门氏菌在腹泻兔为 9% ,健康兔为 4 .2 % ,魏氏梭菌在健康兔和腹泻兔均为 0。生化试验表明 eae A阳性的大肠杆菌为乳糖发酵试验弱阳性的菌株。结论 eae A阳性的肠致病性大肠杆菌 (EPEC)应是导致上述兔场断奶后幼兔发生腹泻症的主要细菌性病原 ;L EE毒力岛的检测能够作为兔肠致病性大肠杆菌 (REPEC)的诊断和流行分子病学调查的主要手段     

肠致病性大肠杆菌和魏氏梭菌PCR诊断方法的建立

本研究建立了一种同时检测大肠杆菌和魏氏梭菌的二重PCR方法,根据兔肠致病性大肠杆菌(REPEC)的eae毒力基因和魏氏梭菌的α-toxin毒力基因的基因文库,设计了两对分别与eae基因和α-toxin基因互补的引物,用这两对引物对同一样品中的REPEC和魏氏梭菌模板进行二重PCR扩增。结果均得到了两条特异性大小与试验设计相符的833bp(REPEC)和324bp(魏氏梭菌)的扩增条带,而对其它3种病原菌的PCR扩增结果均为阴性;敏感性试验结果表明,该二重PCR技术能检出10cfu的REPEC,10cfu的魏氏梭菌。     

肠致病性大肠杆菌和魏氏梭菌PCR诊断方法的建立

本研究建立了一种同时检测大肠杆菌和魏氏梭菌的二重PCR方法,根据兔肠致病性大肠杆菌（REPEC）的eae毒力基因和魏氏梭菌的α—toxin毒力基因的基因文库,设计了两对分别与eae基因和α—toxin基因互补的引物,用这两对引物对同一样品中的REPEC和魏氏梭菌模板进行二重PCR扩增。结果均得到了两条特异性大小与试验设计相符的833bp（REPEC）和324bp（魏氏梭菌）的扩增条带,而对其它3种病原菌的PCR扩增结果均为阴性;敏感性试验结果表明,该二重PCR技术能检出10cfu的REPEC,10cfu的魏氏梭菌。     

兔肠致病性大肠杆菌和魏氏梭菌毒力基因的快速检测

以一中型肉兔场为研究对象,采集病、死兔肠内容物,粪便样品,饮水,污水,饲料和以兔舍为中心不同距离处的尘土样品,共得到样品135份,运用双重PCR法同时检测样品中的肠致病性大肠杆菌(eaeA基因 )和魏氏梭菌(α-毒素基因 ).结果表明在病、死兔肠内容物中、粪便、生活污水中这两种菌同时存在和仅一种菌单独存在的几率都较大;兔舍周围尘土由近到远肠致病性大肠杆菌(eaeA基因 )存在的几率呈递减趋势,魏氏梭菌(α-毒素基因 )在兔舍周围尘土中存在的几率较少,且变化不大.     

鸭疫里默氏杆菌和大肠杆菌双型融合菌株的构建

以耐药标记的鸭疫里默氏杆菌TXRA1(Chlr,Erys)和大肠杆菌ZBO78(Eryr,Chls)作为亲本菌株,在DnaseⅠ始终存在的条件下,采用Lysozyme-EDTA法制备2菌株的原生质体,原生质体制备率分别达到了92.00%和91.94%,再生率分别达到了38.77%和37.29%。通过PEG法进行2菌株原生质体融合,成功获得31株具有双亲本耐药性(Chlr,Eryr)的融合菌株,并应用双重PCR方法对融合株的部分外膜蛋白A(ompA)基因进行扩增,其中有11株融合菌株能够表达2亲本的ompA基因,有20株仅表达亲本大肠杆菌的ompA基因。融合菌株的形态、染色特性和生化特性等介于2亲本菌株之间,连续传15代后融合菌株的上述性状仍能够稳定遗传。     

兔致病性大肠杆菌生物学特性的研究

分离自不同地区的 １５ 株兔源致病性大肠杆菌分属于 ７ 个血清型,均能利用乳糖、葡萄糖、甘露醇、山梨醇、海藻糖、麦芽糖、阿拉伯糖产酸产气,触酶、靛基质呈阳性反应。对氟哌酸、庆大霉素、卡那霉素、丁胺卡那霉素、妥布霉素敏感;所有菌株均能引起试验兔和试验鼠发病和死亡。     

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。     

产气荚膜梭菌α毒素C末端的三拷贝串联表达与免疫原性分析

本研究旨在获得产气荚膜梭菌α毒素（CPA） C末端（第247－370位氨基酸,CPA_C）三拷贝串联融合蛋白,并评价其免疫原性。对已知的A型产气荚膜梭菌CPA_C编码基因（GenBank登录号：AY823400.1）进行优化设计,以同向串连的方式串联成三拷贝基因（GCPA_C3）,片段之间用柔性氨基酸linker （GGGS）连接,经人工合成后克隆至原核表达载体pET-30a （＋）中进行表达与纯化,获得CPA_C的三拷贝重组蛋白（rCPA_C3）。利用Western blotting方法检测rCPA_C3与A型产气荚膜梭菌毒素抗血清的反应性。将rCPA_C3与Montanide ISA 201佐剂混合乳化制备疫苗,免疫4只健康家兔,检测一免及二免后兔血清的中和抗体效价。在二免21 d后,对家兔经耳缘静脉注射1个家兔最低致死剂量（MLD）的A型产气荚膜梭菌毒素,检测rCPA_C3对家兔的免疫保护效果。结果显示,rCPA_C3主要以包涵体的形式表达,且能与A型产气荚膜梭菌毒素抗血清反应;每毫升的一免抗血清可中和30~50个、二免抗血清可中和70~100个小鼠MLD的A型产气荚膜梭菌毒素。采用1个家兔MLD的A型产气荚膜梭菌毒素攻毒后,对照组家兔100%（4/4）死亡,免疫组得到了100%（4/4）的保护。以上结果说明,rCPA_C3具有良好的免疫原性,为A型产气荚膜梭菌病基因工程疫苗的研制提供了重要的试验数据。     

产气荚膜梭菌多重PCR定型方法的建立及其应用

建立了多重PCR检测产气荚膜梭菌α、β、ε和ι毒素基因的方法。该方法具有良好的特异性和敏感性,只有产气荚膜梭菌呈现阳性,被检验的其他梭菌以及大肠杆菌、葡萄球菌均为阴性;将肉汤菌液样品10倍系列稀释后进行检测,检测灵敏度达到1.2×104CFU/mL。收集40份牛粪便样品,进行PCR检测,32份样品中成功扩增出589 bp的α毒素基因片段,阳性率为80%。结果显示,建立的多重PCR检测方法可取代血清中和试验,用于产气荚膜梭菌分型,同时表明A型产气荚膜梭菌在当地奶牛场中较为普遍。     

Population-based study of fecal shedding of Clostridium perfringens in broodmares and foals

OBJECTIVE: To determine the percentage of broodmares and foals that shed Clostridium perfringens in their feces and classify the genotypes of those isolates. DESIGN: Prospective cross-sectional study. ANIMALS: 128 broodmares and their foals on 6 equine premises. PROCEDURES: Anaerobic and aerobic bacteriologic cultures were performed on feces collected 3 times from broodmares and foals. All isolates of C. perfringens were genotyped. RESULTS: Clostridium perfringens was isolated from the feces of 90% of 3-day-old foals and 64% of foals at 8 to 12 hours of age. A lower percentage of broodmares and 1- to 2-month-old foals shed C. perfringens in their feces, compared with neonatal foals. Among samples with positive results, C. perfringens type A was the most common genotype identified (85%); C. perfringens type A with the beta2 toxin gene was identified in 12% of samples, C. perfringens type A with the enterotoxin gene was identified in 2.1% of samples, and C. perfringens type C was identified in < 1% of samples. CONCLUSIONS AND CLINICAL RELEVANCE: Clostridium perfringens was identified from the feces of all but 6 foals by 3 days of age and is likely part of the normal microflora of neonatal foals. Most isolates from broodmares and foals are C. perfringens type A; thus, the clinical relevance of culture results alone is questionable. Clostridium perfringens type C, which has been associated with neonatal enterocolitis, is rarely found in the feces of horses.     

山东省部分地区兔大肠杆菌病发病特点与药敏试验分析

于2011年1月至2012年7月,自山东省潍坊、临沂、淄博、滨州等部分地区送检114份病死兔病例中,经细菌分离鉴定,共分离到45株大肠杆菌。对上述大肠杆菌发病病例及发病日龄分析发现,1~3月龄发病数量占到64.4%;通过对其他病原的分离鉴定发现,不同病例存在球虫、魏氏梭菌、巴氏杆菌、波氏杆菌、链球菌、兔瘟等混合感染问题,混合感染病例占总病例近50%;对分离菌进行了药敏试验,发现其存在不同程度的耐药情况,其中对复方新诺明耐药性高达95.6%,对环丙沙星、阿莫西林、庆大霉素、新霉素的耐药性比率超过60%,而且二重耐药、多重耐药的情况较为普遍。     

东北地区鸡源大肠埃希菌耐药性试验

为了选择敏感药物以避免抗药性的产生,选用新霉素、头孢噻肟钠、甲磺酸培氟沙星等8种抗菌药对东北地区主要养鸡场分离出的优势血清型致病性大肠埃希菌进行药敏试验.结果8种抗菌药的抑菌圈直径分别是氟苯尼考30 mm,头孢噻污钠28 mm,新霉素27 mm,甲磺酸培氟沙星21 mm,丁胺卡那25 mm ,磷霉素钙19 mm,庆大霉素6 mm,阿莫西林15 mm.根据药物敏感试验结果表明,抑菌作用较强的为氟苯尼考、头孢噻肟钠和新霉素.     

母猪子宫内膜炎的病原分离及药敏试验

为了解和掌握吉林农业科技学院猪场猪子宫内膜炎的情况,取母猪的子宫内容物分别采用显微镜观察法、鉴别培养法和生化试验方法,鉴定出有葡萄球菌、沙门氏菌、大肠杆菌、链球菌这四种细菌,并将鉴定出来的细菌做药敏试验。结果显示葡萄球菌和链球菌对左氧氟沙星、米诺环素、复方新诺明敏感,对链霉素中度敏感,对青霉素、苯唑青霉素、阿米卡星、头孢噻肟钠耐药;沙门氏菌对左氧氟沙星、米诺环素、复方新诺明、头孢噻肟钠敏感,对链霉素、阿米卡星中度敏感,对青霉素、苯唑青霉素产生耐药性;大肠杆菌对左氧氟沙星、米诺环素、复方新诺明、阿米卡星、头孢塞肟钠敏感,对链霉素中度敏感,对青霉素、苯唑青霉素耐药;中药复方制剂对体外抑菌试验有效果。     

A型产气荚膜梭菌灭活疫苗类毒素含量与疫苗攻毒保护率之间的平行关系研究

测定了制苗菌液中外毒素对小白鼠的最小致死量,并将该制苗菌液按《兽用生物制品规程》（以下简称《规程》）[1]方法制成A型产气荚膜梭菌灭活疫苗,按《规程》方法进行安全和效力检验。结果表明：6批疫苗安全检验全部合格;疫苗保护率与疫苗中类毒素含量呈正相关。     

Comparison of the odds of isolation, genotypes, and in vivo production of major toxins by Clostridium perfringens obtained from the gastrointestinal tract of dairy cows with hemorrhagic bowel syndrome or left-displaced abomasum

OBJECTIVE: To compare the frequency of isolation, genotypes, and in vivo production of major lethal toxins of Clostridium perfringens in adult dairy cows affected with hemorrhagic bowel syndrome (HBS) versus left-displaced abomasum (LDA). DESIGN: Case-control study. ANIMALS: 10 adult dairy cattle with HBS (cases) and 10 adult dairy cattle with LDA matched with cases by herd of origin (controls). PROCEDURE: Samples of gastrointestinal contents were obtained from multiple sites during surgery or necropsy examination. Each sample underwent testing for anaerobic bacteria by use of 3 culture methods. The genotype of isolates of C. perfringens was determined via multiplex polymerase chain reaction assay. Major lethal toxins were detected by use of an ELISA. Data were analyzed with multivariable logistic regression and chi2 analysis. RESULTS: C. perfringens type A and type A with the beta2 gene (A + beta2) were the only genotypes isolated. Isolation of C. perfringens type A and type A + beta2 was 6.56 and 3.3 times as likely, respectively, to occur in samples from cattle with HBS than in cattle with LDA. Alpha toxin was detected in 7 of 36 samples from cases and in 0 of 32 samples from controls. Beta2 toxin was detected in 9 of 36 samples from cases and 0 of 36 samples from controls. CONCLUSIONS AND CLINICAL RELEVANCE: C. perfringens type A and type A + beta2 can be isolated from the gastrointestinal tract with significantly greater odds in cattle with HBS than in herdmates with LDA. Alpha and beta2 toxins were detected in samples from cows with HBS but not from cows with LDA.     

Clostridial enteric infections in pigs.

Clostridium perfringens types A and C and Clostridium difficile are the principal enteric clostridial pathogens of swine. History, clinical signs of disease, and gross and microscopic findings form the basis for a presumptive diagnosis of C. perfringens type-C enteritis. Confirmation is based on isolation of large numbers of type-C C. perfringens and/or detection of beta toxin in intestinal contents. Diagnosis of C. perfringens type-A infection, however, remains controversial, mostly because the condition has not been well defined and because type-A organisms and their most important major (alpha) toxin can be found in intestinal contents of healthy and diseased pigs. Isolation of large numbers of C. perfringens type A from intestinal contents, in the absence of other enteric pathogens, is the most reliable criterion on which to base a diagnosis. Recently, beta2 (CPB2) toxin-producing C. perfringens type A has been linked to disease in piglets and other animals. However, implication of CPB2 in pathogenesis of porcine infections is based principally on isolation of C. perfringens carrying cpb2, the gene encoding CPB2, and the specific role of CPB2 in enteric disease of pigs remains to be fully defined. Clostridium difficile can also be a normal inhabitant of the intestine of healthy pigs, and diagnosis of enteric infection with this microorganism is based on detection of its toxins in feces or intestinal contents.