内蒙古地区绵羊肺炎支原体的分离鉴定及序列分析

本试验无菌采取内蒙古地区某发病羊场的绵羊病变肺脏组织,接种于支原体液体培养基进行分离培养后获得1株支原体,根据分离株的培养特性、形态学观察及生化试验等,初步鉴定为绵羊肺炎支原体。然后提取分离株的基因组,用通用引物体外扩增出分离株16S rRNA序列,将该序列与GenBank中已知33种支原体序列进行比较,结果表明该序列与绵羊肺炎支原体标准株Y-98的16S rRNA序列的同源性为99%,鉴定该分离株为绵羊肺炎支原体。     

绵羊肺炎支原体标准株Y98与丝状支原体丝标准株PG3、绵羊肺炎支原体分离株HD-1、标准株FH抗原的差异性研究

试验采用SDS—PAGE和免疫印迹分析技术研究了绵羊肺炎支原体标准株Y98与绵羊肺炎支原体分离株HD-1、丝状支原体丝状亚种PG3以及肺炎支原体标准株FH间细胞膜蛋白免疫原性的异同。结果表明：绵羊肺炎支原体标准株Y98同绵羊肺炎支原体分离株HD-1间细胞膜蛋白免疫印迹结果基本一致,而绵羊肺炎支原体标准株Y98与丝状支原体丝状亚种PG3和肺炎支原体标准株FH抗原差异较大,缺乏共同抗原成分。     

绵羊肺炎支原体人工感染SPF鸡胚的研究

为探索SPF鸡胚作为绵羊肺炎支原体实验室感染模型的可行性,本试验用不同浓度绵羊肺炎支原体（10⁸、10⁹、10¹⁰ ccu/mL）经由卵黄囊和尿囊腔两个部位接种7日龄SPF鸡胚,通过统计鸡胚死亡情况和不同鸡胚组织样品中绵羊肺炎支原体检测阳性率（PCR检测和支原体分离鉴定）,确定绵羊肺炎支原体鸡胚感染方式、感染剂量和最佳分离部位,再用3株不同来源的绵羊肺炎支原体分离株感染鸡胚,观察其对鸡胚的致病力。结果表明,绵羊肺炎支原体感染鸡胚最佳接种途径为卵黄囊接种,感染剂量为10⁹ ccu/mL、0.2 mL/只,最佳分离部位为卵黄液。3株支原体均能感染和致死鸡胚,并均能从卵黄液中分离到绵羊肺炎支原体,但对鸡胚的致病性存在一定差异：FL3株致鸡胚死亡率为45%,略高于MoGH3-3株（40%）,二者均高于A3株（25%）,但差异均不显著（P>0.05）;FL3株鸡胚检测阳率为100%,高于MoGH3-3株（85%）及A3株（90%）,但差异也不显著（P>0.05）。本试验初步确定了绵羊肺炎支原体可感染和致死SPF鸡胚,不同分离株对SPF鸡胚致病力有差异,表明SPF鸡胚可作为下一步建立绵羊肺炎支原体实验室感染模型的候选,为绵羊肺炎支原体致病性研究和疫苗研制奠定基础。     

山羊支原体性肺炎流行病学调查

山羊支原体性肺炎是威胁山羊养殖的重要传染病,为了解其流行情况,对四川省主要山羊养殖地区的山羊支原体性肺炎进行了流行病学调查。从四川省7个地区山羊养殖场采集肺脏和鼻腔棉拭子样本共135份,经过分离鉴定得到42株支原体,其中绵羊肺炎支原体36株,丝状支原体6株;其中6个羊场仅分离到绵羊肺炎支原体,1个羊场同时分离到绵羊肺炎支原体和丝状支原体。本试验结果表明,绵羊肺炎支原体是引起四川省山羊支原体性肺炎的主要病原,个别地方存在绵羊肺炎支原体和丝状支原体混合感染。     

山羊传染性胸膜肺炎的实验室诊断

为明确贵州省开阳县1例疑似山羊传染性胸膜肺炎(CCPP)病原种类,实验通过病原分离培养、形态学观察、生化试验和PCR方法对病原进行了分离与鉴定。结果显示,所分离病原具有支原体的生长特性,菌落形态与支原体相似;支原体分离株与Mo模式株生化试验结果相同,与Mmc模式株存在差异;PCR试验进一步确定分离株为Mo模式株。试验结果表明,本次山羊传染性胸膜肺炎疫情应为绵羊肺炎支原体感染所致。     

绵羊肺炎支原体内蒙古毒株的分离与鉴定

对疑似为患有绵羊肺炎支原体的病羊无菌采取病料,经临床症状、病理剖检、病原分离培养、形态学观察、生化试验、生长抑制试验,代谢抑制试验,人工接种发病,间接血凝试验,对内蒙古克什克腾旗羊群以呼吸道疾病为主要特征的传染病进行初步诊断,参考国际已知支原体16S r RNA序列,选取共同保守性强的片段设计出一对引物,提取病原分离株(MY1)和人工感染分离株(SY1)培养物的菌体DNA进行PCR扩增并克隆、测序。将该序列与Gen Bank中支原体序列比较,结果证明,分离株MY1和SY1与Y-98序列与绵羊肺炎支原体标准株Y-98同源性为99.7%,与丝状支原体山羊亚种标准株PG3同源性为78.7%,故确定分离株为绵羊肺炎支原体。     

山羊传染性胸膜肺炎病原分离与鉴定

本试验从传染性胸膜肺炎的山羊体内分离获得两株支原体Y1和Y2，通过病原分离、形态学观察、生化试验、HA、生长抑制试验和代谢抑制试验等试验，证实Y1和Y2的形态与培养特性、生化反应特性和血清学特性分别与模式株丝状支原体山羊亚种PG3和绵羊支原体Y98相接近；动物回归试验成功复制出山羊传染性胸膜肺炎的典型临床症状和病理剖解变化。结果表明Y1和Y2分别为丝状支原体山羊亚种和绵羊支原体。     

绵羊肺炎支原体和精氨酸支原体双重PCR检测方法的建立及应用

为建立绵羊肺炎支原体和精氨酸支原体的双重PCR检测方法,本试验分别设计了绵羊肺炎支原体和精氨酸支原体的特异性引物,优化反应条件后对其特异性和敏感性进行评价,并对40份鼻拭子进行了检测。结果显示,该方法能同时扩增出绵羊肺炎支原体545 bp和精氨酸支原体806 bp的特异性片段,而对其他病原的DNA扩增均为阴性。该双重PCR方法对绵羊肺炎支原体和精氨酸支原体的最低检测限分别为100和10 pg/μL。40份鼻拭子检测结果显示,双重PCR检测方法与分离培养法符合率高达92.5%,均能鉴定出绵羊肺炎支原体和精氨酸支原体。结果表明,本研究建立的双重PCR方法可用于绵羊肺炎支原体和精氨酸支原体的临床快速诊断。     

绵羊支原体肺炎诊断及病原的分离与鉴定

通过对送检病羊的临床症状、剖检变化、病科染色镜检,初步诊断为绵羊支原体肺炎。又经无茵采取病料接种改良Frey培养氏液,再取培养液接种于固体培养液后分离到可疑绵羊肺炎支原体。然后将培养生长的支原体分别进行革兰氏染色、姬姆萨染色、瑞氏染色和碱性复红染色;进行了各种生化试验,进行动物和鸡胚接种试验和平扳凝集等血清学试验;最后确诊为绵羊支原体肺炎,并分离获得了绵羊肺炎支原体。该结果为绵羊支原体肺炎的防制奠定了基础。     

山羊源绵羊肺炎支原体的耐药谱型分析

采用微量稀释法对从四川自贡、简阳、乐至等地分离的33株山羊源性绵羊肺炎支原体进行19种抗菌药物MIC值测定,试验数据利用WHONET5.4软件处理并分析其耐药谱型。试验结果表明,33株绵羊肺炎支原体对19种抗菌药物表现出多重耐药性,其耐药谱型以耐氯霉素、链霉素、红霉素、头孢噻呋、阿米卡星、利福平为主,约占78.79%。     

D型多杀性巴氏杆菌和绵羊肺炎支原体引起山羊的混合感染

2018年9月广西某羊场部分山羊发生流涕、咳嗽、呼吸困难和体温升高等临床症状的疾病,为确诊发病原因并提供治疗方案,采用病原分离培养、PCR扩增鉴定的方法进行诊断,并对分离菌进行生化鉴定、致病性试验和药敏试验。结果显示,病料在血平板有圆形的小菌落生长,革兰阴性小球短杆菌,而在PPLO培养基上不生长;病料的PCR扩增结果显示绵羊肺炎支原体和多杀性巴氏杆菌均为阳性;所分离到的病原菌经生化鉴定,该菌符合多杀性巴氏杆菌的特性;用多杀性巴氏杆菌种属和D型多杀性巴氏杆菌特异性引物扩增为阳性;致病性试验显示该菌对小鼠有很强的致病性;药敏试验显示该菌对头孢他啶、头孢噻肟、氧氟沙星高度敏感,对复方新诺明、强力霉素、红霉素、青霉素为耐药。结果表明该病是由绵羊肺炎支原体和D型多杀性巴氏杆菌混合感染引起。     

山羊中绵羊肺炎支原体的分离及鉴定

从四川省乐至县发生胸膜肺炎性传染病的山羊群中采集12个鼻拭子及4个肺组织病料,进行病原分离培养和特异性PCR检测。结果从9个鼻拭子和4个肺组织中分离到支原体,经鉴定均为绵羊肺炎支原体,未发现丝状支原体簇成员及多杀性巴氏杆菌和溶血性曼氏杆菌。结果表明,绵羊肺炎支原体是引起该山羊群发生胸膜肺炎的病原,同时说明绵羊肺炎支原体也是山羊支原体性肺炎的重要病原之一。     

丝状支原体簇和多杀性巴氏杆菌双重PCR方法的建立及应用

本研究旨在建立丝状支原体簇和多杀性巴氏杆菌的双重PCR检测方法,从而为临床上同时检测这2类病原的感染提供一种更方便、快捷、准确的工具。本研究采用2对特异性检测丝状支原体簇和多杀性巴氏杆菌的引物,对PCR反应体系和反应条件进行了优化,并对双重PCR的特异性及敏感性进行了评价,随后采用该方法对52份临床样本进行了检测。结果显示,所建立的双重PCR方法能同时扩增丝状支原体簇成员和多杀性巴氏杆菌的DNA,而对来源于其他常见病原的DNA均无扩增;对丝状支原体簇和多杀性巴氏杆菌的最低检测限分别为24.8和28.9 pg;能成功地从临床样本中检测丝状支原体簇成员和多杀性巴氏杆菌。结果表明,本研究所建立的双重PCR方法具有很好的特异性和敏感性,为临床丝状支原体簇和多杀性巴氏杆菌感染的快速诊断、病原鉴定及流行病学调查提供了有效的方法。     

The humoral immune response of lambs experimentally infected with Mycoplasma ovipneumoniae

Using sera from lambs experimentally infected with Mycoplasma ovipneumoniae and Pasteurella haemolytica, the development of a good humoral immune response to M. ovipneumoniae was detected by ELISA. The antibody titres peaked 41 days post-infection and good antibody titres were maintained over the 16-week experimental period. Immunoblotting revealed that antibodies to specific antigens appeared in the sera in a sequential manner, some being seen shortly after infection and others developing only after a substantial time lag. Antibodies were raised against almost all the major antigens detected in one laboratory strain (956/2) and against all antigens previously shown to be conserved in 22 Scottish field isolates of M. ovipneumoniae.     

Serological comparison of type strains of porcine, bovine, and ovine mycoplasmas with atypical colony morphology

The type strains of Mycoplasma hyopneumoniae, M. flocculare, M. dispar, and M. ovipneumoniae, all characterized by nipple-less colonies on solid media, were compared serologically. By indirect hemagglutination and by complement fixation tests they were found to constitute a related group. By crossed immunoelectrophoresis a sharing of common antigens was demonstrated, whereas no cross reactivity was noted by the metabolism inhibition test.The type strains of Mycoplasma hyorhinis and Mycoplasma bovirhinis were included in the study for comparison. Although some cross reaction was noted, they appeared just moderately related to the nipple-less group as well as to each other.     

Comparison of virulence of ovine respiratory mycoplasmas in the mouse mammary gland

The virulence of isolates of Mycoplasma ovipneumoniae and M. arginini from pneumonic and unaffected ovine lungs was compared in a mouse mammary gland model. The isolates varied in their ability to induce a neutrophilic response in the mammary gland. A moderate to severe form of mastitis was induced by 3 M. ovipneumoniae isolates recovered from pneumonic lungs, while the remaining M. ovipneumoniae isolates from pneumonic lungs and those from unaffected lungs induced a very mild histopathological response. The severity of the mastitis could not be increased by the simultaneous inoculation of a mixture of 5 mycoplasma isolates. Mycoplasma arginini isolates induced only a very mild histopathological response despite having been isolated from pneumonic lungs. The finding that the 3 most virulent M. ovipneumoniae isolates were initially recovered from pneumonic ovine lungs suggested that these virulent isolates may contribute to ovine pneumonia. However, the isolation of M. ovipneumoniae from pneumonic ovine lungs does not necessarily imply that these organisms are the causal agents, since M. ovipneumoniae isolates may vary in virulence.     

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

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。     

Mycoplasmas and acholeplasmas isolated from ducks and their possible association with pasteurellas

Two hundred and sixty-three cases of clinically diseased ducks of all ages were examined for the presence of mycoplasmas. Mycoplasmas and acholeplasmas belonging to more than eight serogroups were cultured from 68 of them, and comprised 12 M anatis, one M columbinasale, two M gallinaceum, two M gallinarum, nine M synoviae, three unidentified Mycoplasma species, 37 Acholeplasma laidlawii and one unclassified acholeplasma belonging to each of serogroups 7 and 8. They were identified by biochemical characterisation, disc growth inhibition and agar gel diffusion tests. Fifty-three (78 per cent) of the isolates occurred with species of Pasteurella: 33.8 per cent with Pasteurella anatipestifer, 32.4 per cent with P multocida and 11.8 per cent with both P anatipestifer and P multocida. Nine of the isolates (13.2 per cent) were in pure culture and six (8.8 per cent) with other agents. Of the ducks negative for mycoplasmas 33.3 per cent were infected with P anatipestifer, 25.1 per cent with P multocida and 14.4 per cent with both P anatipestifer and P multocida. There was no correlation between the infections with mycoplasmas and P anatipestifer but there was a weak association between the infections with mycoplasmas, especially M anatis and P multocida.     

In vitro antimicrobial susceptibility of Pasteurella haemolytica and Pasteurella multocida recovered from cattle with bovine respiratory disease complex

The antimicrobial susceptibilities of 421 Pasteurella haemolytica and 158 P. multocida isolates recovered from cattle with respiratory disease were determined with a microdilution minimal inhibitory concentration test system. Isolates were analyzed for patterns of resistance to ampicillin, ceftiofur, erythromycin, gentamicin, penicillin, spectinomycin, sulfachlorpyridazine, sulfadimethoxine, tetracycline, and tylosin. All isolates tested were found susceptible to ceftiofur and sulfachlorpyridazine. Pasteurella haemolytica isolates were resistant to ampicillin, penicillin, sulfadimethoxine, tetracycline, and tylosin. Pasteurella multocida isolates were resistant to sulfadimethoxine, tetracycline, and tylosin.     

宁夏固原部分地区肉犊牛呼吸道6种病原流行病学调查

[目的]为了掌握宁夏固原地区示范村杨坪村和马沟村的肉犊牛呼吸道疾病的流行情况,[方法]2019年-2021年对2村犊牛呼吸道疾病发病情况进行统计并采用分子生物学技术对采集的犊牛呼吸道鼻拭子样本进行6种病原的鉴定。[结果] 2019年-2021年杨坪村犊牛呼吸系统疾病发病率呈下降趋势,但不同年份间差异不显著(p>0.05);马沟村犊牛呼吸系统疾病发病率呈下降趋势,2021年与2019年相比差异显著(p<0.05)。2019年-2021年,病毒性呼吸道疾病呈下降趋势,而牛支原体和多杀性巴氏杆菌由于没有有效的疫苗预防而呈现较高的阳性率。[结论]本研究揭示了牛支原体和多杀性巴氏杆菌为目前犊牛主要的呼吸道致病病原,做好牛支原体病和多杀性巴氏杆菌病的预防、早期诊断和治疗可有效降低犊牛呼吸道疾病的发病率。