支原体和肺支原体双通道荧光定量PCR法检测鼠支原体感染试验

采用TaqMan探针法支原体通用型和肺支原体特异型双通道荧光定量PCR法,对清洁级SD大鼠150只I、CR小鼠120只,C57小鼠100只,普通级SD大鼠104只I、CR小鼠97只,C57小鼠76只,检测支原体和肺支原体感染情况。清洁级SD大鼠中检测到1只支原体阳性;清洁级ICR小鼠和C57小鼠中支原体和肺支原体未见阳性;普通级SD大鼠中检测到2只支原体阳性,1只肺支原体阳性;普通级ICR小鼠中检测到1只支原体阳性,2只肺支原体阳性;普通级C57小鼠检测到1只支原体阳性。表明清洁级实验动物大鼠中仍然存在支原体感染;普通级实验动物大鼠和小鼠支原体感染明显高于清洁级动物。     

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。     

Biochemical Characterization of Mycoplasma bovirhinis,Mycoplasma dispar and Recent Bovine Isolates of Mycoplasma canis

The pattern and kinetics of substrate utilization by the type strains of Mycoplasma canis, M. bovirhinis and M. dispar and ten recent M. canis isolates from cattle were determined. Metabolism of a range of sugars and organic acids by M. dispar was detectable by measurement of oxygen uptake. Organic acids were not utilized by M. bovirhinis or M. canis, and there was no oxygen uptake during metabolism of glucose or other sugars, as monitored by a pH-change method. The M. canis strains varied in their ability to metabolize sugars; seven of the isolates from cattle had the distinctive ability to metabolize sucrose, and one isolate, plus the type strain (from a dog), metabolized N-acetylglucosamine. The M. bovirhinis strain metabolized maltose. However, all the test strains oxidized glycerol at high rates and with a high affinity. Oxidation of glycerol has been reported for other mycoplasmas from the bovine respiratory tract and leads to the production of hydrogen peroxide, a potential virulence factor.     

监测支原体

肥育期后期的健康问题可以反映出母猪场内青年母猪的健康状况,肥育猪后期控制支原体非常重要。     

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.     

Species-specific polymerase chain reactions for the detection of Mycoplasma buteonis, Mycoplasma falconis, Mycoplasma gypis, and Mycoplasma corogypsi in captive birds of prey

Mycoplasmas are pathogens of different avian species, but the role of Mycoplasma in raptors is not yet completely determined. As Mycoplasma isolation and identification present several difficulties, species-specific polymerase chain reactions (PCRs) for the detection of mycoplasmas found in birds of prey (Mycoplasma buteonis, Mycoplasma corogypsi, Mycoplasma falconis, and Mycoplasma gypis) were established. The specificity of the PCR methods were investigated using known avian Mycoplasma reference strains and isolates as well as related bacteria and was found to be specific. Amplificons obtained with these PCRs from field samples showed no false-positive results in restriction enzyme analysis and sequencing. The sensitivities of the different PCR assays varied between 50 fg and 1 pg DNA. Twenty-five tracheal swabs from healthy captive birds of prey were investigated by culture and immunobinding assay as comparison to the PCRs. Mycoplasmal DNA was detected in 88% of the samples, with negative results only from vultures. Mycoplasma falconis and M. buteonis were regularly found in falcons, and M. gypis was found in a common buzzard. Mycoplasma corogypsi was not demonstrated. Several isolates could not be differentiated using an immunobinding assay as well as the described PCR methods.     

应用多重套式PCR检测鸡毒支原体和鸡滑液囊支原体

根据已发表的鸡毒和鸡滑液支原体血凝素基因序列pMGA和vlhA各设计两对引物,建立鉴别诊断两种支原体的多重套式PCR方法,对其进行温度条件、Ⅱ步模板浓度优化及特异性、敏感性实验。该方法在两步PCR后能特异性地扩增出MG(408 bp)和MS(688 bp)两个目的片段。应用于临床样品检测,与支原体分离、SPA检测比较结果PCR灵敏度高于病原分离。     

用探针杂交法检测鸡毒支原体和滑液囊支原体

根据鸡毒支原体(MG)和滑液囊支原体(MS)的16SrRNA基因序列，设计并合成了探针MG和MS共同目的基因，跨幅为580bP的一对引物。用这对引物对2个标准的MG和1个标准的Ms菌株DNA模板进行聚合酶锭反应(PcR)，结果得到了预期大小约580bP的PcR产物。回收纯化琼脂糖电泳凝肢中的MG扩增产物克隆至T载体中，DIG随机引物法合成核酸探针，斑点杂交试验对MG和MS均呈阳性，检测灵敏度分别为2ng和3ng，其它对照菌(毒)株为阴性。     

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.     

Identification of species-specific and interspecies-specific polypeptides of Mycoplasma gallisepticum and Mycoplasma synoviae

Temporal antisera (TA) prepared in susceptible Leg-horn-type chickens against Mycoplasma gallisepticum and M synoviae were evaluated to determine the extent of cross-reactivity in ELISA and hemagglutination inhibition tests. Species-specific and interspecies-specific polypeptides were identified after electrophoretic separation and protein immunoblotting with reference antisera, TA, and a monoclonal antibody specific for M gallisepticum. Mycoplasma gallisepticum antiserum cross-reacted with M synoviae polypeptides in ELISA and TA immunoblots. Two major M synoviae polypeptides (88 and 53 kilodaltons [kD]) cross-reacted with M gallisepticum antisera in TA immunoblots. An M gallisepticum polypeptide of 70 kD cross-reacted with M synoviae in TA immunoblots. In contrast, M gallisepticum and M synoviae reference antisera cross-reacted when immunoblotted with heterologous antigens. A monoclonal antibody specific for M gallisepticum bound to a 69-kD polypeptide in lectin-purified and whole-cell M gallisepticum protein fractions in immunoblot assays. The lectin-purified fraction hemagglutinated chicken RBC. Seemingly, the 69-kD polypeptide may constitute all or part of the M gallisepticum hemagglutinin.     

Immunogenicity of Mycoplasma gallisepticum

The serological response and protective immunity elicited in the chicken by the pathogenic Ap3AS strain and the moderately pathogenic 80083 strain of Mycoplasma gallisepticum and variants of strain 80083 attenuated by repeated passage in mycoplasma broth were investigated. Strain 80083 elicited a substantial serum antibody response after administration either in drinking water or by conjunctival sac instillation to 7-week-old SPF chickens. No vaccinated chickens developed air sac lesions when challenged by intra-abdominal (IA) injection with the virulent Ap3AS strain. Chickens vaccinated with strain 80083M (50 broth passages) showed only a weak serological response but were substantially protected when challenged 4 weeks after vaccination. Chickens vaccinated with 80083H (100 broth passages) were serologically negative 4 weeks after vaccination and developed severe air sac lesions after challenge. Thirty-seven-week-old hens vaccinated 6 months previously with strain 80083 had high serum antibody levels and were completely protected against IA challenge with the homologous strain. However, 4/6 showed mild air sac lesions when challenged intra-abdominally with strain Ap3AS. Another group showed high M. gallisepticum serum antibody levels 6 months after vaccination with strain Ap3AS but 4/6 and 2/6 showed mild lesions after IA challenge with strains Ap3AS or 80083, respectively. Strains 80083 or 80083M were administered by conjunctival sac instillation to susceptible 11-week-old commercial pullets at the time of fowl pox vaccination. The concurrent use of both vaccines had no apparent adverse effect on the health of the chickens. Similar protection against IA challenge with strain Ap3AS was produced with the M. gallisepticum vaccines whether used alone or in combination with fowl pox.     

“猪地方性肺炎及其防治控制”专栏三：猪肺炎支原体的诊断

猪肺炎支原体可引发猪地方性肺炎,仍然是导致养猪业疫病的重要病原体之一。这种复杂的小型微生物可定殖在猪呼吸道的纤毛细胞中,使其几乎不暴露于免疫系统下。证实猪肺炎支原体的存在、并确定其在呼吸道疾病和肺炎中的作用,对兽医行业仍然具有挑战性。虽然猪肺炎支原体的培养仍然是对其作出鉴定的黄金标准,但血清学方法、聚合酶链反应和检测组织中猪肺炎支原体的各种不同方法仍是实验室常用的检测技术。由于猪肺炎支原体在由并发的细菌和病毒感染引发的严重肺炎中所起的作用日益增加,了解猪肺炎支原体的发病机制和现有的诊断方法对开发有效的干预措施以及在猪群水平上控制猪群呼吸道疾病非常关键。