鸽源支原体的分离鉴定

对来自武汉市郊某养鸽专业户及市场食用鸽共７０只采集口腔咽部粘膜拭子及其中１５只鸽子的气管、气囊和肺等材料进行了支原体的分离鉴定，其中口腔咽部拭子几乎１００％能分离到支原体，但均与细菌共存而不易纯化，其他部位均未分离到支原体。对已纯化的３４个分离物中的１９个进行了鉴定，用鸽支原体ＭＭＰ１株、鸽口支原体ＭＭＰ４株及鸽鼻支原体６９４株作为标准抗原制备抗血清，以代谢抑制试验进行了血清学鉴定。结果表明，所有     

新疆石河子地区牛支原体的分离鉴定及药物敏感性分析

【目的】确定引起新疆石河子地区集约化牛场常发性肺炎的主要病原同时进行病原的体外药物敏感性分析。【方法】采集有典型咳嗽、流涕症状的牛鼻拭子10份和病死牛肺脏组织1份,用牛支原体特异性引物进行PCR检测,将检测为阳性的样本进行病原培养纯化,对纯化后的分离株菌落进行形态学观察、Dienes染色、生化试验及16S rRNA测序和进化分析,通过测定颜色变化单位(CCU)测定分离株生长曲线,并对分离株进行药物敏感性试验。【结果】PCR结果显示,10份鼻拭子中检测出7份牛支原体阳性样本,1份病死牛肺脏组织也检测为阳性;在涂有肺脏组织研磨液培养液的PPLO固体培养基上长出针尖状的菌落,纯化后分离株菌落形态为典型的煎蛋状;Dienes染色可见明显的深蓝色中心脐;生化试验结果显示,分离株不水解明胶、精氨酸、七叶苷,不发酵乳糖、葡萄糖和甘露醇,不分解尿素,可还原氯化三苯基四氮唑;16S rRNA测序结果显示,分离株与牛支原体国际标准株PG45相似性为99.7%,与国内牛支原体地方流行株XBY01、Ningxia-1、NM2012、Tibet-10的相似性最高,均为99.9%;生长曲线测定结果显示,分离株在培...     

鸡滑液囊支原体的分离与鉴定

某鸡场鸡群表现关节、爪垫肿胀、行走困难等症状，经血清平板凝集试验检测，鸡毒支原体呈阴性，鸡滑液囊支原体呈阳性。在此基础上，对关节囊内容物进行了病原体的分离和鉴定。分离菌落呈典型支原体菌落，能通过４５０ｎｍ滤膜，发酵葡萄糖，还原四氮唑，不利用精氨酸和尿素，菌落能吸附鸡红细胞，在固体培养基上的生长被鸡滑液囊支原体阳性血清所抑制。分离物在抑菌剂培养基上连续传代１０次，未见返祖Ｌ型细菌。用分离物对鸡做人工感染试验，出现与自然病例相同的症状，故认为分离物为鸡滑液囊支原体。     

山羊莱氏无胆甾原体FJ-NP株的分离鉴定

为明确福建省某羊场发生的疑似羊支原体性肺炎病例的病原,利用支原体培养基对发病羊肺脏组织的病原进行分离培养和纯化,通过生化试验和特异性PCR方法进行鉴定,并对分离株16S rRNA进行测序分析。结果显示,分离株菌落呈油煎蛋状,有棕黄色中心突起;能发酵葡萄糖,不能水解精氨酸,不分解尿素,氯化四氮唑还原反应、胆固醇需要试验、血细胞吸附试验及溶血试验的结果均为阴性,美兰还原反应阳性。经PCR扩增出莱氏无胆甾原体支原体（Acholeplasma laidlawii,AL）大小为505 bp的特异性目的片段,分离株16S rRNA序列与莱氏无胆甾原体标准株PG8同源性为99.8%。鉴定结果表明,本次从山羊肺脏组织分离到的支原体为莱氏无胆甾原体,命名为FJ-NP株,但该分离株与山羊发病性的关系有待进一步研究。     

犬肾（DK）传代细胞中霉形体的分离与鉴定

取经电镜观察证实有霉形体污染的ＤＫ传代细胞，分别接种于霉形体检验用液体、半流体和固体培养基，证明为霉形体混合感染，即该培养物在液体培养基和半流体培养基上，同时显示出水解精氨酸和发酵葡萄糖２种特性，前者使培养基ｐＨ上升，后者使ｐＨ下降。通过反复挑选单个菌落进行克隆传代和有目的地加入单一种属的霉形体抗血清的方法，将混合的２株霉形体纯化分开。经鉴定，一株为精氨酸霉形体，一株为莱氏无（需）胆甾醇霉形体。     

鸡毒支原体C株的分离鉴定及与其它北京株结构蛋白的比较分析

本文对北京某鸡场气囊炎病进行了支原体的分离和鉴定,并将此分离株与北京另两个分离株ＢＧ４４Ｔ、ＮＢ７２的结构蛋白通过ＳＤＳ－ＰＡＧＥ进行了比较分析。结果表明此分离株（命名为Ｃ株）可发酵葡萄糖,不水解尿素和利用精氨酸,是一具有毒力较强的鸡毒支原体株。ＳＤＳ－ＰＡＧＥ结果表明,此分离株与ＮＢ７２株相当一致,与ＢＧ４４Ｇ株有相对较大的区别。我们认为,Ｃ株和ＮＢ７２株具有同源性,鸡胚液制造的活疫苗中支原体的污染可能源于鸡胚培养时采用了ＭＧ感染病鸡所产蛋,另外,还可看出北京地区的支原体感染存在着一定的多样性。     

鸡滑液囊支原体的分离和鉴定研究

从河南某肉种鸡场疑似鸡滑液囊支原体感染的病鸡跗关节样品进行病原的分离与鉴定.分离菌经瑞氏染色,在油镜下呈球形或椭圆形.在固体培养基上表现为细小、光滑、致密的小菌落,菌落形态为“煎蛋状”.L型细菌鉴定发现菌体仍保持原有形态.菌体能够吸附红细胞,分解葡萄糖,但不能分解精氨酸和尿素,还可致SPF鸡胚死亡,说明该分离菌为支原体.进一步的血清学试验结果表明,该分离菌为鸡滑液囊支原体,而不是鸡毒支原体.根据已发表的鸡滑液囊支原体血凝素基因序列vlhA设计合成一对引物,经PCR扩增,该菌体能够扩增出鸡滑液囊支原体(773 bp)的特异性片段.人工感染试验结果显示,SPF鸡能够复制出自然病例.     

一株牛支原体的分离鉴定及致病性初步研究

对采集到的疑似牛支原体肺炎肺组织病料进行病原的分离,并对分离株进行形态学、生化和分子生物学鉴定,结果显示成功分离获得1株牛支原体,命名为NM001。该分离株的菌落形态呈典型的“荷包蛋状”,不能发酵葡萄糖,不能水解精氨酸,不分解尿素。PCR能够扩增出牛支原体特异的P48基因条带,16S rRNA基因序列与Ningxia-1序列同源性为99.03%。将该分离株接种2头6月龄犊牛均出现明显的临床症状,剖检后胸腔中少量淡黄色渗出液,肺脏出现肉样实变。试验结果表明,分离到的牛支原体NM001株对牛具有较强的致病性,为牛支原体攻毒模型的建立奠定了基础。     

不同地区鸡毒支原体的分离鉴定及药物敏感性研究

【目的】了解中国不同地区鸡毒支原体（Mycoplasma gallisepticum，MG）的流行和耐药情况，为禽支原体病的监测与防控提供科学依据。【方法】在广东、福建和安徽3个省份疑似MG感染的养殖场采集的43份气囊样品中分离培养和纯化MG流行菌株，并对其进行培养特性观察、生化及染色鉴定、血清学特异性鉴定、PCR测序分析、致病性试验和对常见抗菌药物敏感性试验。【结果】分离到3株疑似MG，均可使培养基颜色变黄，在固体培养基上呈典型"煎蛋"样菌落，可发酵葡萄糖，不水解精氨酸，不能利用尿素，符合MG培养特性。姬姆萨染色观察菌体形态、血清学特异性鉴定结果进一步证实分离株为MG。mgc2基因测序结果显示，3株分离株与强毒代表株亲缘关系更近。致病性试验结果显示，3株分离株与MG感染临床症状一致，发病率为70%~90%，致病力较强。药物敏感性试验结果显示，3株分离株均对恩诺沙星、替米考星、土霉素、氟苯尼考耐药，对泰万菌素和沃尼妙林均敏感，对大观霉素、金霉素、泰乐菌素和泰妙菌素的敏感性具有地域差异：福建株对大观霉素表现明显耐药，安徽株对泰妙菌素表现耐药，而广东株对大观霉素、金霉素和泰妙菌素均较敏感。【结论】本试验成功分离到3株MG，致病力较强，且均存在一定程度耐药，对药物的敏感程度有地区性差异，今后仍需加强各地区MG的药物敏感性监测，减少耐药性的产生与扩散。     

塔城地区某奶牛场牛支原体的分离鉴定及药敏试验

为确定新疆塔城地区某奶牛场部分断奶犊牛出现咳嗽、流脓性鼻涕、腹泻等症状的发病病因,本试验通过对采集到的发病犊牛鼻拭子样品进行分离培养、形态学观察、生化试验、16S rRNA基因和oppD/F基因PCR扩增与测序、药敏试验等。分离培养显示3株分离株在改良Thiaucourt’s琼脂培养基平板上生长出典型的“煎蛋样”菌落,狄氏染色菌落中心呈深蓝色。生化试验中分离株不发酵葡萄糖与甘露醇,不水解精氨酸,不分解尿素,明胶液化试验为阴性。PCR扩增分离株16S rRNA基因测序结果显示,分离株与GenBank数据库中牛支原体16S rRNA基因序列的相似性在98.73%以上;牛支原体特异性基因oppD/F阳性且相似性达99.74%以上。通过药敏试验从9种药物里选择出替加环素、红霉素2种最为敏感的药物。研究结果表明,造成此次犊牛肺炎的病原菌为牛支原体,为临床防治牛支原体病提供了参考。     

鸡毒支原体与鸡滑液囊支原体双重PCR检测方法的建立

为建立能同时检测鸡毒支原体(Mycoplasma gallisepticum, MG)和鸡滑液囊支原体(Mycoplasma synoviae,MS)的双重PCR诊断方法,该研究根据GenBank中登录的MG gapA基因序列和MS heat shock ATP-dependent protease基因序列,设计2对特异性引物,通过对PCR扩增条件的优化,建立了能够同时检测MG和MS的双重PCR诊断方法。特异性检测结果显示,该方法能够扩增出729 bp的MG和309 bp的MS特异性片段,对禽巴氏杆菌、大肠杆菌、鸡白痢沙门菌、副鸡禽杆菌核酸扩增均为阴性;敏感性检测结果显示,对MG和MS DNA的最低检出量均为5×10^-2 ng/μL;临床样品的检测结果显示,所建立的双重PCR方法可同时有效地检测出MG、MS混合感染和单独感染。该研究建立的鸡毒支原体与鸡滑液囊支原体双重PCR方法具有良好的特异性、敏感性、重复性,为快速、高效检测MG和MS提供了技术支持。     

An abattoir survey of pneumonia and pleuritis in slaughter weight swine from 9 selected herds. II. Enzootic pneumonia of pigs: microbiological findings and their relationship to pathomorphology

Lungs from 191 slaughter pigs with gross lesions indicative of enzootic pneumonia of pigs (EPP) and 80 grossly normal lungs, all originating from 9 different herds, were subjected to microbiological and pathological examinations. The microbiological studies included both bacterial and mycoplasmal culture and also testing for Mycoplasma hyopneumoniae antigen in tissue by indirect immunofluorescent technique. M. hyopneumoniae, Pasteurella multocida and Mycoplasma hyorhinis were detected in 83%, 43% and 37% of the pneumonic lungs, respectively. Mycoplasma flocculare was the most frequently isolated organism in the non-pneumonic lungs. The greatest amounts of macroscopic pneumonia (25.2%) were recorded in lungs with all the three agents M. hyopneumoniae, P. multocida and M. hyorhinis present. The amounts of pneumonia in lungs with M. hyopneumoniae alone and in concurrence with P. multocida, were 9.3% and 15.6%, respectively. M. hyorhinis was also, in this study, associated with higher frequency of diffuse pleuritis. These findings indicate that M. hyorhinis might be involved in the pathogenesis of pneumonia in slaughter pigs. Ninety-six per cent of the isolates of P. multocida from pneumonic lungs could be characterized as type A. In the herds which had the most severe pneumonia problems, toxin production was detected in 83% of the P. multocida strains while only 28% were toxigenic in herds with subclinical to moderate pneumonia problems.     

Development and Application of a Duplex PCR Assay for Detecting Mycoplasma ovipneumiae and Mycoplasma arginini

In order to establish a duplex PCR method for simultaneous detection of Mycoplasma ovipneumiae and Mycoplasma arginini, specific primers of Mycoplasma ovipneumiae and Mycoplasma arginini were designed, and evaluated its sensitivity and specificity after optimizing the reaction conditions of PCR.Then, a total of 40 nasal swabs were tested by duplex PCR.The assay could specifically amplify PCR fragments of 545 and 806 bp from Mycoplasma ovipneumiae and Mycoplasma arginini, respectively.While no PCR products were detected for other pathogens.The detection limits of the assay were determined to be 100 pg/μL for Mycoplasma ovipneumiae and 10 pg/μL for Mycoplasma arginini.The duplex PCR could detect Mycoplasma ovipneumiae and Mycoplasma arginini, and the coincidence rate could reach as high as 92.5% with enrichment culture about the 40 nasal swabs.The results suggested that the duplex PCR could be useful for clinical detection of Mycoplasma ovipneumiae and Mycoplasma arginini.     

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。     

猪肺炎支原体和猪鼻支原体双重PCR检测方法的建立与应用

根据猪肺炎支原体(Mhp)和猪鼻支原体(Mhr)的16S rRNA基因设计3条引物, 建立Mhp和Mhr的双重PCR检测方法,并对该方法进行了特异性和敏感性试验,并使用建立的方法检测了临床样品和疫苗样品。结果显示该方法具有良好的特异性,最低可检测到0.66ng 的Mhp基因组DNA和0.58 ng Mhr基因组DNA,临床样品和疫苗样品检测结果与普通PCR检测结果一致。该双重PCR方法,可用于Mhp与Mhr的鉴别、诊断以及疫苗纯粹性检查,快速而准确。     

鹅败血支原体及滑液囊支原体感染的血清学调查

本研究对珠江三角洲部分地区鹅群的185例血清样品，进行败血支原体、滑液囊支原体感染的平板凝集试验，检测结果表明：鹅败血支原体的感染率为29.7%，滑液囊支原体的感染率为19.5%，两者的混合感染率为14.6%。     

Canine mycoplasmas

This review aims to summarise our current understanding of the role of mycoplasmas in domestic dogs. Canine mycoplasmology is a small field, with less than 50 publications in the past 40 years. In this time we have gained knowledge about the number of species and have made associations with infections in dogs. However much evidence is still lacking. The importance of all canine mycoplasmas remains unknown, yet certain species are associated with canine anaemia (Mycoplasma haemocanis), respiratory disease (Mycoplasma cynos) and urogenital tract infections (Mycoplasma canis). Mycoplasmas can be isolated in pure culture from canine clinical specimens and it is hoped that this review will stimulate veterinarians to consider mycoplasmas as a potential cause of disease in dogs, especially when antibiotic therapy is failing.     

Growth and interaction of Mycoplasma bovirhinis and Mycoplasma dispar in vitro

Two mycoplasmas were grown in pure and mixed cultures in glucose calf-serum broth with initial pH 7.8. Sensitivity to pH was also tested. The main data for Mycoplasma bovirhinis and Mycoplasma dispar, respectively, in pure cultures were as follows: lag phase, days: <1, 1–2; log phase: 1–2, 1–4; relatively stationary phase: 0–1, 2–4; decline phase (to the extent roughly logarithmic): 2–6, 5–10; maximal titers: 5 × 10⁷ to 10⁹, 5 × 10⁶ to 10⁸ color changing units per 0.2 ml; highest pH, approximately, at which decline started: 7.7 and 7.0; definitely toxic initial pH: 5.6, 6.8; relative production of acidity; less, more. Decline either shortly ended in loss of viability or, correlated with higher pH levels, led to a prolonged maintenance in lower numbers. The decline of M. bovirhinis was postulated to be essentially caused by an autotoxic product/products other than H⁺.

In mixed cultures an antagonistic effect due to the faster growing M. bovirhinis against M. dispar was recorded. The effect varied according to the initial numbers of organisms and their ratio. Two mechanisms seemed to be active: 1. decrease of pH somewhat below neutrality led to the death of the sensitive M. dispar; 2. M. dispar, when present in relatively high initial numbers, was inhibited by M. bovirhinis during the latter's logarithmic growth at pH-levels above 7.0, the inhibition ending shortly afterwards. A rapidly inactivated product/products of M. bovirhinis metabolism, inhibitory to M. dispar was posited. The results offer an improved insight into diagnostic practices for M. dispar.     

18种抗菌药物对绵羊肺炎支原体和丝状支原体分离株的抗菌活性

应用微量法测定了18种常用抗菌药物对绵羊肺炎支原体分离株HD1-goat和丝状支原体分离株XT1-goat的抗菌活性.结果表明,喹诺酮类药物及盐酸多西环素、酒石酸泰乐菌素对两个分离株均有很高的抗菌活性;硫氰酸红霉素对分离株HD1-goat无抑制生长作用,但对分离株XT1-goat有很高的抑制和杀灭活性;氨基糖苷类药物和...     

Utilisation of glucose by Mycoplasma suipneumoniae and Mycoplasma flocculare.

The utilisation of glucose by Mycoplasma suipneumoniae and Mycoplasma flocculare was examined by chemical determination of glucose disappearance during growth, and by examination for hexokinase activity in cell preparations. Both species degraded glucose during growth and possessed hexokinase activity as evidence of the presence of a glycolytic pathway. The glucose utilisation capacity was found to be greater for M. flocculare than for M. suipneumoniae.