牦牛肺炎病原多杀性巴氏杆菌和殊异支原体的分子鉴定与分析     

张学勇  郭志宏  简莹娜  朵红  沈秀英  马怡隽  付永  宋永武  才让周在 《青海畜牧兽医杂志》2019,(2):39-42,55

青海省刚察县某牦牛产业基地牦牛发生咳嗽,鼻孔有分泌物流出,呼吸困难等症状的疾病。为快速确诊发病牛的致病原因,及时防控和治疗,从发病和病死的牦牛中,采集鼻腔拭子和肺脏、胸腔积液,利用PCR方法进行分子鉴定与分析。结果显示:此牛场中引发肺炎的病原是A型多杀性巴氏杆菌和殊异支原体两种病原体混合感染,测序结果显示5个有效样品中鉴定的为完全相同的病原,P.multocida-GC1的Kmt基因与参考序列中国株CP031554/3/2/1同源性在99%以上,M.dispar-GC1与参考序列的16S rRNA基因同源性在97%以上。同时在遗传进化角度再次分析确认了鉴定的P.multocida与中国和俄罗斯的A型多杀性巴氏杆菌聚成一大支;鉴定的M.dispar与殊异支原体聚为一支。结果表明,青海刚察地区牦牛存在感染多杀性巴氏杆菌和殊异支原体的潜在风险,该研究为牦牛呼吸道疾病与肺炎的病原学调查和病原的流行病学调查提供了参考数据。  相似文献   

牛源荚膜A型多杀性巴氏杆菌疫苗候选株的筛选及鉴定     

朱玲  柳旭伟  剡文亮  张锐  韩小丽  剡根强 《中国畜牧兽医》2018,45(2):471-478

为了筛选生长快、毒力强、免疫原性好、副反应小的牛源荚膜A型多杀性巴氏杆菌（Pasteurella multocida，Pm）灭活疫苗菌株，本试验选取6株来自不同地区致犊牛肺炎死亡的牛源荚膜A型多杀性巴氏杆菌分离株，测定了培养基生长曲线、小鼠毒力、菌体脂多糖（LPS）含量及各菌株灭活菌苗免疫小鼠和家兔后的抗体效价，并进行了攻毒保护试验。结果显示，分离株Pm2、Pm3、Pm5生长速度较快、毒力较强、LPS含量较多，均含有与毒力和免疫相关的ptfA和fimA基因；免疫小鼠及家兔未发现明显不良反应，在二免后14 d血清抗体达1：64~1：128，强毒攻毒后全部存活，而PBS对照组全部死亡。本试验结果表明，Pm2、Pm3、Pm5均可作为多杀性巴氏杆菌灭活菌苗的候选菌株，其中Pm3作为首选株。  相似文献   

多杀性巴氏杆菌中recN基因的克隆、原核表达及生物信息学分析     

章泸尹  郑义盈  王成强  安琪  张萌萌  黄海峰  张振兴  李宝宝  朱姝  杨小健  曹瑞勇  聂鑫  杜丽  王凤阳 《中国畜牧兽医》2019,46(3):661-668

试验旨在对多杀性巴氏杆菌recN基因进行克隆和原核表达，并对其表达蛋白进行生物信息学分析。参照GenBank中recN基因序列（登录号：CP003313.1）设计1对引物，通过PCR扩增获得目的基因片段，构建pET-28a（+）-recN重组质粒并转化E.coliDH5α感受态细胞，提取质粒进行酶切鉴定，将鉴定正确的重组质粒转化E.coliBL21（DE3）感受态细胞，经IPTG诱导表达，对融合蛋白进行SDS-PAGE及Western blotting鉴定。结果表明，试验成功克隆了大小约为1 677 bp的recN基因序列，通过诱导表达的His-Tag融合蛋白大小约为66.94 ku，主要以包涵体形式存在。经生物信息学分析，recN蛋白的分子式为C₂₇₃₅H₄₄₂₈N₇₈₆O₈₅₅S₁₆，消光系数为24 785，不稳定系数为43.99，属于不稳定蛋白；理论等电点（pI）为5.62，为酸性蛋白；总平均亲水性为-0.316，与试验表达的包涵体蛋白性质相同，即同为疏水性蛋白；recN蛋白在哺乳动物网织红细胞的半衰期为30 h，在酵母（体内）中的半衰期>20 h，在大肠杆菌（体内）中的半衰期>10 h；二级结构主要以α-螺旋（64.87%）及无规则卷曲（21.00%）为主；经疏水性分析，预测该蛋白有3个高疏水性区域和9个高亲水性区域。本试验结果为进一步探究多杀性巴氏杆菌recN基因的功能提供了参考依据。  相似文献   

血清1和3型多杀性巴氏杆菌铁调控外膜蛋白共同抗原的初步分析     

余旭平  邬栋栋  刘晓宁  胡东建  杜鑫  陈玉银 《畜牧兽医学报》2008,39(2):245-250

用普通BHI和缺铁BHI培养基培养禽多杀性巴氏杆菌C48-19（血清1型）、P1059株（血清3型）,提取外膜蛋白（OMPs）,SDS-PAGE电泳比较不同培养条件下两株细菌的外膜蛋白,发现缺铁培养时2株细菌均增加了87、81、79、64、60．5ku铁调控外膜蛋白（IROMPs）。以C48-19株攻毒后耐过的鸡血清作为抗体进行免疫印迹,检测到其中的87、79和60．5ku蛋白是2株细菌共同的、具有交叉免疫反应性的IROMPs,推测这3种蛋白可能是这2个菌株的共同抗原,并可能在C48-19感染的鸡体内特异表达;对鸡胚尿囊液中培养的C48-19株OMPs的SDS-PAGE电泳和免疫印迹试验,也观察到类似IROMPs的表达。这些IROMPs的发现为亚单位疫苗的研制提供了候选蛋白。  相似文献   

Neurological bacterial diseases in possums and gliders: a review of eight cases     

Holz PH  Graham KJ 《Australian veterinary journal》2008,86(8):301-302

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Proliferation and transmission patterns of <Emphasis Type="Italic">Pasteurella multocida</Emphasis> B:2 in goats     

Shafarin MS  Zamri-Saad M  Khairani BS  Saharee AA 《Tropical animal health and production》2008,40(5):335-340

This report describes the proliferation and transmission patterns of Pasteurella multocida B:2 among stressful goats, created through dexamethasone injections. Thirty seven clinically healthy adult goats were divided into three groups consisted of 15 goats in group A, 11 goats in group B and the remaining 11 in group C. At the start of the study, all goats of group A were exposed intranasally to 1.97 × 10¹⁰ CFU/ml of live P. multocida B:2. Dexamethasone was immediately administered intramuscularly for 3 consecutive days at a dosage rate of 1 mg/kg. The exposed goats were observed for signs of HS for a period of 1 month. At the end of the 1-month period, 11 goats from group B were introduced into and commingled with the surviving goats of group A before all goats from both groups were immediately injected intramuscularly with dexamethasone for 3 consecutive days. The treatment with dexamethasone was then carried out at monthly interval throughout the 3-month study period. Goats of group C were kept separately as negative control. Three surviving goats from each group were killed at 2-week interval for a complete post-mortem examination. Two (13%) goats of group A were killed within 24 hours after intranasal exposure to P. multocida B:2 while another two (13%) goats from the same group were killed on day 40, approximately 10 days after the second dexamethasone injection. All four goats showed signs and lesions typical of haemorrhagic septicaemia. Bacteraemia was detected in 3 goats of group A that were having rectal temperature higher than 41°C. The P. multocida B:2 isolation pattern was closely associated with dexamethasone injections when significantly (p < 0.05) higher rate of isolations from both groups were observed after each dexamethasone injection. Transmission of P. multocida B:2 from goats of group A to group B was successful when P. multocida B:2 was isolated from goats of group B for a period of 28 days. There was a strong correlation between dexamethasone injections, rate of bacterial isolation and serum cortisol level. The IgG level showed an increasing trend 2 weeks after exposure to P. multocida B:2 and remained high throughout the study period.  相似文献   

多杀性巴氏杆菌中recN基因的克隆、原核表达及生物信息学分析     

章泸尹  郑义盈  王成强  安琪  张萌萌  黄海峰  张振兴  李宝宝  朱姝  杨小健  曹瑞勇  聂鑫  杜丽  王凤阳 《中国畜牧兽医》2019,(3):661-668

试验旨在对多杀性巴氏杆菌recN基因进行克隆和原核表达,并对其表达蛋白进行生物信息学分析。参照GenBank中recN基因序列(登录号:CP003313.1)设计1对引物,通过PCR扩增获得目的基因片段,构建pET-28a(+)-recN重组质粒并转化E.coli DH5α感受态细胞,提取质粒进行酶切鉴定,将鉴定正确的重组质粒转化E.coli BL21(DE3)感受态细胞,经IPTG诱导表达,对融合蛋白进行SDS-PAGE及Western blotting鉴定。结果表明,试验成功克隆了大小约为1 677 bp的recN基因序列,通过诱导表达的His-Tag融合蛋白大小约为66.94 ku,主要以包涵体形式存在。经生物信息学分析,recN蛋白的分子式为C₂₇₃₅H₄₄₂₈N₇₈₆O₈₅₅S₁₆,消光系数为24 785,不稳定系数为43.99,属于不稳定蛋白;理论等电点(pI)为5.62,为酸性蛋白;总平均亲水性为-0.316,与试验表达的包涵体蛋白性质相同,即同为疏水性蛋白;recN蛋白在哺乳动物网织红细胞的半衰期为30 h,在酵母(体内)中的半衰期>20 h,在大肠杆菌(体内)中的半衰期>10 h;二级结构主要以α-螺旋(64.87%)及无规则卷曲(21.00%)为主;经疏水性分析,预测该蛋白有3个高疏水性区域和9个高亲水性区域。本试验结果为进一步探究多杀性巴氏杆菌recN基因的功能提供了参考依据。  相似文献   

Differential susceptibility to tetracycline,oxytetracycline and doxycycline of the calf pathogens Mannheimia haemolytica and Pasteurella multocida in three growth media     

Andrew Mead  Peter Lees  John Mitchell  Andrew Rycroft  Joseph F. Standing  Pierre‐Louis Toutain  Ludovic Pelligand 《Journal of veterinary pharmacology and therapeutics》2019,42(1):52-59

For clinical isolates of bovine Mannheimia haemolytica and Pasteurella multocida, this study reports minimum inhibitory concentration (MIC) differences for tetracycline, oxytetracycline and doxycycline between cation‐adjusted Mueller‐Hinton broth (CAMHB), foetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) medium. MICs were determined according to CLSI standards and additionally using five overlapping sets of twofold dilutions. Matrix effect: (a) free drug MICs and minimum bactericidal concentrations (MBC) for all drugs were significantly higher in FBS than in CAMHB for both pathogens (p < 0.001); (b) MICs and MBCs were higher for CAMHB and FBS compared to RPMI for P. multocida only. Net growth rate for P. multocida in CAMHB was significantly slower than in FBS and higher than in RPMI, correlating to MIC and MBC ranking. Drug effect: doxycycline MICs and MBCs were significantly lower (p < 0.001) in both CAMHB and FBS than tetracycline and oxytetracycline for both pathogens. Only for M. haemolytica were oxytetracycline MIC and MBC significantly lower than tetracycline, precluding the use of tetracycline to predict oxytetracycline susceptibility in this species. Determining potencies of tetracyclines in a physiological medium, such as FBS, is proposed, when the objective is correlation with pharmacokinetic data for dosage determination.  相似文献   

猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌复合PCR检测方法的建立   总被引：2，自引：0，他引：2  

朱吕昌  陈义平  李明义  郭玉广  马爽  周洁 《中国动物检疫》2008,25(11):22-25

目的建立可以同时检测猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速而可靠的PCR检测方法。方法和结果根据胸膜肺炎放线杆菌的Apx-VIA基因序列、多杀性巴氏杆菌和副猪嗜血杆菌的16SrRNA基因序列设计5条引物。猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌模板的PCR扩增产物大小分别为342bp,485bp和1258bp。复合PCR对1～12型猪胸膜肺炎放线杆菌标准株,6株多杀性巴氏杆菌标准株,1～15型副猪嗜血杆菌以及25株经生化鉴定确认为上述三种细菌的分离株的基因组DNA作为模板进行检测,均获得预期大小的扩增产物。以猪放线杆菌、吲哚放线杆菌等14种常见细菌作为阴性对照进行PCR检测,结果仅有支气管败血波氏杆菌产生了可以和上述三个特异性条带明显区分的PCR产物。复合PCR针对胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的敏感性分别为14pg、34pg和37pg。结论本研究建立的复合PCR特异性好,敏感性高,可以用于猪胸膜肺炎放线杆菌、多杀性巴氏杆菌和副猪嗜血杆菌的快速检测。  相似文献   

猪巴氏杆菌、大肠杆菌二联苗的制备及免疫效果观察     

马索南才让 《中国畜牧兽医》2008,35(2):91-94

以分离某猪场的多杀性巴氏杆菌、致病性大肠杆菌制备猪巴氏杆菌和大肠杆菌二联灭活苗,同时选择病变明显的病料制备组织灭活苗,探讨它们的免疫效果。结果发现它们的免疫保护率分别为：二联灭活苗为80%以上,组织灭活苗为60%。因此二联苗可试用于控制该猪场巴氏杆菌病的流行。  相似文献