牛舍空气微生物及向环境传播的研究

采用４ 台 Ａ Ｎ Ｄ Ｅ Ｒ Ｓ Ｅ Ｎ微生物空气样品收集器对一个犊牛舍内及环境空气细菌含量进行了测量, 需氧菌总数分别为８５３６ ～４６６９１ Ｃ Ｆ Ｕ／ｍ ３ 和２６４９ ～２４７７５ Ｃ Ｆ Ｕ／ｍ ３ , 厌氧菌浓度计３０１７ ～２４７７５ Ｃ Ｆ Ｕ／ｍ ３ 和６６１ ～４１２２ Ｃ Ｆ Ｕ／ ｍ３ 。根据 Ｈ Ｅ Ｓ Ｋ Ｅ Ｔ Ｈ （１９７４） 计算公式和 Ｋ Ｌ Ｕ Ｇ （１９６９） 气溶胶颗粒扩散模式, 得出犊牛舍菌源强度, 即向环境排放需氧菌为１１００００ ～１４００００ Ｃ Ｆ Ｕ／ｓ , 厌氧菌在２７０００ ～１７５０００ Ｃ Ｆ Ｕ／ｓ 之间。细菌浓度实际测量值与估算一致, 该公式和扩散模式适合于动物舍菌源强度和舍外环境不同距离空气微生物浓度的估算。距离犊牛舍１００ ｍ 下风处, 需氧菌高达６５０ ～８３９ Ｃ Ｆ Ｕ／ｍ ３ , 厌氧菌１６０ ～１０３４ 细菌／ｍ ３ , 各项均超过正常值１０ ～１５ 倍。     

ELISA对动物舍气载魏氏梭菌型别的研究

采用ELISA方法对从一个犊牛舍空气中分离到的魏氏梭菌进行型别的鉴定。结果显示：88％的菌种属于A型，3.4％为C型，8.6％是D型，B型未能确认。     

仔猪产气荚膜梭菌肠毒血症病原诊断及疫苗研究IV．肠毒血症C型疫苗生产用菌种的确定

试验证明用单一菌株C59-2生产C型产气荚膜梭菌肠毒血症干粉灭活疫苗,其效力可以达到《规程》要求,并且简化了生产工艺,降低了生产成本,提高了生产效率。疫苗免疫的兔血清可分别中和原来3个制苗用菌株所产生的外毒素。     

Antimicrobial associated diarrhoea in the horse. Part 1: Overview,pathogenesis and risk factors

Antimicrobial associated diarrhoea (AAD) is the most commonly recognised adverse effect of antimicrobial treatment in horses, although its incidence is probably low given the frequency of antimicrobial administration. Clinical signs vary from transient self‐limiting diarrhoea to rapidly fatal toxic enterocolitis. AAD prolongs the duration of hospitalisation, increases diagnostic and therapeutic costs, and was associated with a lower case survival rate than other types of acute diarrhoea in one study. Virtually all antimicrobials have been implicated in AAD, but some pose a greater risk than others.     

猝死羊产气荚膜杆菌某些特性测定

应用溶血试验和动物试验方法，检测来源于正常羊肠内容物和猝死羊肝、脾及肠内容物中产气荚膜杆菌及其毒素的溶血性和致病性。结果显示，在1％葡萄糖血平板上，所有14株猝死羊分离株均表现出典型的双溶血环(100％)，而在22株正常羊分离株中5株为双溶血环(22．7％)、15株为单溶血环(68．2％)和2株不溶血(9．1％)；对1％绵羊红细胞的溶血作用上，猝死羊分离株毒素的溶血OD50值多在0．40～0．90，而正常羊分离株毒素的OD50值多在0．40以下：所有分离株及其毒素对小白鼠的致死性无显著差异。     

Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing,China

Clostridium perfringens is an important zoonotic pathogen. This study was designed to explore the prevalence and toxin types of C. perfringens in retail beef collected from Beijing, China. Among 221 beef samples collected, 53 samples were positive for C. perfringens, resulting in the average prevalence as 23.98%. By toxin gene-based typing, the most C. perfringens strains belong to type A (96.23%, 51/53), only 2 strains were identified as type D. By a multi-locus sequence typing (MLST)-based analysis, a total of 36 sequence types (STs) were detected, and the most STs (n=30) represented just a single strain. These finding suggested that the prevalence of C. perfringens in retail beef in Beijing was considerably high and these bacteria displayed extreme diversity in genetics.     

D型产气荚膜梭菌ε毒素基因表达及其免疫保护作用的初步研究

利用PCR技术,从D型产气荚膜梭菌标准株(C60-2)染色体DNA中扩增出ε毒素基因。该基因产物大小为906 bp,序列分析结果表明,与GenBank报道的产气荚膜梭菌参考菌株ε毒素基因序列同源性大于99%。将扩增的ε毒素基因定向克隆到原核表达载体pET32a中,得到重组质粒pET32a-ETX,将重组质粒转化大肠杆菌BL21(DE3)plys中,经IPTG诱导后,SDS-PAGE分析可见大小为54 ku的特异条带;经Western blotting和ELISA检测结果表明,表达的ε毒素能与抗天然ε毒素抗体发生特异性反应,说明ε毒素蛋白具有较好的反应原性。将表达的ε毒素蛋白用0.4%的甲醛溶液制成类毒素疫苗,免疫小鼠后,具有一定的保护力,这表明该重组菌株有望成为产气荚膜梭菌基因工程类毒素疫苗的候选菌株。     

麋鹿产气荚膜梭菌分离鉴定方法的建立与初步应用

探索了产气荚膜梭菌在营养琼脂、甘露醇卵黄琼脂和血琼脂平板上的菌落形态和培养特点,优化了产气荚膜梭菌的分离方法,并对分离菌株进行生化鉴定;在此基础上对北京南海子麋鹿苑和江苏大丰麋鹿国家级自然保护区的麋鹿自然感染情况进行了调查。结果发现,粪样中的产气荚膜梭菌在营养琼脂上生长呈半透明边缘不整的白色菌落,接种甘露醇卵黄琼脂和血琼脂平板,分别出现伴有卵磷脂酶乳光浑浊带的粉红色火山口状菌落和伴有双溶血环的灰绿色勋章样菌落。生化试验结果确认这些分离株均为产气荚膜梭菌。对北京南海子麋鹿苑和江苏大丰麋鹿国家级自然保护区麋鹿粪样检测发现,阳性率分别为13.04%(3/23)和19.51%(8/41)。说明本研究建立的麋鹿产气荚膜梭菌分离鉴定方法简便快速,确实可行;麋鹿产气荚膜梭菌自然感染率较高,应加强麋鹿产气荚膜梭菌病的防控。     

Cloning,Expression of Clostridium perfringens α-toxin Gene and Preparation of its Antisera

One pair of primers had been designed and synthesized based on the α-toxin gene of Clostridium perfringens.The complete α-toxin gene fragment was amplified by polymerase chain reaction (PCR), and then was cloned into pGEM-T Easy vector to construct pGEM-T-α.Digested with EcoRⅠ and Hind Ⅲ, a fragment of 1125 bp was cloned into the expression plasmid vector pET-28a(+).The recombinant plasmid was transformed into the BL21(DE3)plys and induced by 1.0 mmol/L IPTG at 37 ℃.The expression product was found to be 46.1 ku as expected one identified by SDS-PAGE, and confirmed by Western blotting with Clostridium perfringens type A antisera, indicating similar reactivity with native α-toxin.Recombinant α-toxin protein was simultaneously found in culture supernatant, postsonic supertanant and inclusion bodies, most protein was expressed in inclusion bodies, which indicated recombinant α-toxin protein was expressed in the extracellular, periplasm and cytoplasm.Recombinant α-toxin protein in postsonic supertanant could not make mice die, indicating its non-toxicity.Toxin-antitoxin neutralization test showed that antisera of recombinant α-toxin protein were specific to α-toxin.Upon immunization of rabbit with the recombinant α-toxin protein, antisera with high antibody titer neutralizing 100 MLD toxin per 1 mL were prepared.     

Bacillus subtilis and yeast cell wall improve the intestinal health of broilers challenged by Clostridium perfringens

1. The objective was to investigate the effects of Bacillus subtilis, yeast cell wall (YCW) and their combination on intestinal health of broilers challenged by Clostridium perfringens over a 21-d period.

2. Using a 5 × 2 factorial arrangement of treatments, 800 1-d-old male Cobb 500 broilers were used to study the effects of feed additives (without additive or with zinc bacitracin, B. subtilis, YCW, and the combination of B. subtilis and YCW), pathogen challenge (without or with Clostridium perfringens challenge), and their interactive effects.

3. C. perfringens infection increased intestinal lesions scores, damaged intestinal histomorphology, increased serum endotoxin concentration, cytokine mRNA expression and intestinal population of C. perfringens and Escherichia coli and decreased ileal bifidobacteria numbers. The 4 additives decreased serum endotoxin. Zinc bacitracin tended to decrease cytokine mRNA expression and the intestinal number of C. perfringens and E. coli. B. subtilis, YCW and their combination increased cytokine mRNA expression. B. subtilis and YCW decreased the number of C. perfringens and E. coli in the ileum, and their combination decreased pathogens numbers in the ileum and caecum.

4. In conclusion, B. subtilis, YCW and their combination improved the intestinal health of NE-infected broilers, and could be potential alternatives to antibiotics.