广西山羊传染性胸膜肺炎病原的二重PCR快速诊断方法的建立

根据绵羊肺炎支原体标准株Y98和丝状支原体山羊亚种标准株PG3的16S rRNA两端的保守区序列设计了2对引物,建立了广西山羊传染性胸膜肺炎病原的二重PCR快速检测方法.试验结果显示,所建立的二重PCR能特异地扩增绵羊肺炎支原体和丝状支原体山羊亚种的基因片段,其敏感性可达lPg,,用建立的二重PCR检测了20份临床病例肺组织,检出率为70%(14/20),其中6份扩增出丝状支原体山羊亚种的基因片段,10份扩增出绵羊肺炎支原体的基因片段,有2份同时扩增出两种支原体的基因片段.培养法检出率为40%(8/20),而这8份病料均为PCR阳性.     

山羊传染性胸膜肺炎PG3组织灭活苗的研制

山羊传染性胸膜肺炎是山羊发展的大敌,其病原为支原体（亦称霉形体Mycopiasma）,是一种接触性传染病。该病的发生给养羊业造成极大的损失和威胁,对山羊传染性胸膜肺炎的免疫苗,目前国内只有中国农科院兰州兽科所定点生产,而该苗主要是预防绵羊肺炎支原体Y98引起的山羊传染性胸膜肺炎,而对丝状支原体山羊亚种PG3引起的山羊传染性胸膜肺炎无预防作用,为保障我省养羊业的健康发展,2000年,我们利用在我省罗甸县分离培养鉴定的丝状支原体山羊亚种PG3贵州地方株进行了山羊传染性胸膜肺炎组织灭活苗的研制,报告于下。     

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。     

山羊支原体山羊肺炎亚种甘肃株的分离及鉴定

2007年甘肃某地羊场山羊发生以呼吸困难、咳嗽和胸膜肺炎为特征的呼吸道传染病,经临床诊断为羊支原体性肺炎.用改良的Thiaucourt's培养基对病山羊的肺组织进行病原分离,成功分离到1株支原体M1601,经菌落形态观察、生化试验、PCR鉴定、以16S rRNA基因序列构建的分子进化树分析和动物致病性试验,证明该分离株属于山羊支原体山羊肺炎亚种.     

贵州山羊传染性胸膜肺炎病原的分离鉴定

经临床症状，病理剖检，病原分离培养，生化试验，生长抑制试验，代谢抑制试验，人工接种发病，间接血凝诊断，对贵州省习水县和罗甸县山羊群以呼吸道疾患为主要特征的传染病进行诊断，确诊为山羊传染性胸膜肺炎，分离出的两株支原体均为丝状支原体山羊亚种PG3，分别定名为贵习PG3／99和贵罗PG3／99。     

一株致病性丝状支原体山羊亚种支原体的分离鉴定

通过培养纯化、形态学观察、生化试验、生长抑制试验、代谢抑制试验、人工感染试验、间接血凝试验,对疑似山羊传染性胸膜肺炎病原分离株SY2进行初步鉴定,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1对引物,提取人工感染分离株SY2培养物的菌体DNA进行PCR扩增并克隆、测序。将该序列与NCBI中已知支原体序列比较,结果证明SY2株与丝状支原体山羊亚种标准株PG3同源性为99%,故确定疑似山羊传染性胸膜肺炎病原分离株SY2为丝状支原体山羊亚种。     

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。     

山羊支原体山羊肺炎亚种培养基的筛选及生长曲线测定

为筛选出山羊支原体山羊肺炎亚种的适宜培养基,将山羊支原体山羊肺炎亚种分离株M1601分别接种Thiaucourt肉汤、MEM-KM2和TSA 3种不同的培养基,测定其在3种不同培养基中生长滴度、生长速度,并测定了M1601在最适培养基中在不同培养阶段的生长滴度。结果表明,添加2g/L丙酮酸钠和150mL/L马血清的Thiaucourt肉汤培养基最适宜山羊支原体山羊肺炎亚种的生长,生长滴度可达109 CCU/mL,在培养6h后开始进入对数生长期,培养60h后进入稳定期,培养72h时后进入衰亡期。此试验结果为山羊支原体山羊肺炎亚种培养特性研究提供了参考数据。     

山羊支原体的分离与鉴定

1997年对怀化市山羊疫病血清学调查时发现山羊传染性胸膜肺炎阳性率为7.25%。为证实本病的存在，从1998年以来共采集山羊肺脏病理材料60份，分离出二株支原体，将分离株接种山羊气管后复制出本病。根据国际兽疫局的标准诊断方法以及分离物培养特性、显微镜观察、生长抑制试验、生化试验等，初步鉴定为丝状支原体山羊亚种和绵羊肺炎支原体。     

间接ELISA法对山羊传染性胸膜肺炎的快速诊断

为了完善和提高山羊传染性胸膜肺炎的快速诊断技术,试验应用贵州省畜牧兽医研究所1998年分离的山羊传染性胸膜肺炎丝状支原体山羊亚种贵罗PG3作为全细胞包被抗原建立的ELISA方法,对贵州省4个不同养羊场进行了血清学调查。结果表明:4个羊场的丝状支原体山羊亚种PG3血清阳性率分别为30.2%、15.9%、46.5%和47.7%;间接ELISA法与间接血凝试验、鼻腔拭子培养法相比具有良好的敏感性和特异性,其阳性检出率更高。     

绵羊肺炎支原体GH3-3株在改良KM2培养基中的增殖情况

绵羊肺炎支原体GH3-3 MoGH3-3株是我国绵羊支原体肺炎灭活疫苗制苗菌株.为研究MoGH3-3株在改良KM2培养基中的生长及繁殖特性,利用活菌计数方法对其在不同培养阶段的生长滴度(颜色变化单位)进行测定,并绘制生长曲线.结果表明,传代稳定的MoGH3-3株在改良KM2培养基中培养12 h开始进入对数生长期,培养7...     

Immunoblot assays using recombinant antigens for the detection of Mycoplasma hyopneumoniae antibodies

The 36kDa L-lactate dehydrogenase (LDH) and a 29kDa partial fragment of an ABC transporter ATP-binding protein analogue/multidrug resistance protein homologue (PR2) of Mycoplasma hyopneumoniae were tested for their potential as diagnostic antigens. Recombinant LDH was genetically engineered to contain six histidine residues at its C-terminal end, expressed in Escherichia coli and purified to a high degree using Ni(2+)-chelate affinity chromatography. A partial 262 amino acid segment representing the C-terminal end of the PR2 protein was cloned as a glutathione S-transferase (GST) fusion protein, expressed in E. coli and purified by urea extraction. Purified recombinant LDH-6xHis and PR2-GST were then reacted with pig sera in immunoblot assays. Our immunoblots showed that both proteins detected anti-M. hyopneumoniae antibodies in field and experimentally infected pig sera but not in any of the SPF control sera. The two proteins were specific for M. hyopneumoniae as they did not react with sera of pigs infected with the closely related Mycoplasma flocculare and Mycoplasma hyorhinis which are frequently isolated in pigs but are not of particular concern.     

Detection of specific Mycoplasma conjunctivae antibodies in the sera of sheep with infectious keratoconjunctivitis

The serological cross reactions between Mycoplasma conjunctivae, the etiological agent of infectious keratoconjunctivitis (IKC), and the antigenetically and phylogenetically closely related Mycoplasma ovipneumoniae, which is often found in sheep, were analysed. Cross reacting antigens were identified using sera from sheep with IKC and from sheep of herds known to be free of IKC, as well as rabbit hyperimmune serum specific to the two Mycoplasma species. Cross reactions were predominantly due to the strongly antigenic proteins of 42 kDa and 83 kDa. Serospecific antigens of M. conjunctivae could be separated from cross-reacting antigens by the extraction of Tween 20-soluble membrane proteins. The Tween 20-extracted proteins of the M. conjunctivae strain HRC/581T were used for the development of an indirect ELISA test. This ELISA test was shown to be a useful serological method for the diagnosis of M. conjunctivae infections and to identify infected sheep herds.     

Analysis of heat shock protein 60 encoding genes of mycoplasmas and investigations concerning their role in immunity and infection

Only little is known about the heat shock proteins (Hsp) and Hsp-encoding genes of mycoplasmas. The aim of this study was to identify and sequence the hsp60 gene of Mycoplasma agalactiae, Mycoplasma arthritidis, Mycoplasma bovis, and Mycoplasma hyopneumoniae, and to investigate the immune response to Hsp60.Fragments of the hsp60 genes of M. agalactiae, M. arthritidis, M. bovis and M. hyopneumoniae representing almost the entire coding region were amplified by PCR. Two fragments of a hsp60 gene were cloned in Escherichia coli and the antibody response of pigs infected with M. hyopneumoniae against the recombinant Hsp60 fusion proteins was analysed. Within the mycoplasmas, the hsp60 genes showed sequence identities of nearly 100%, with the exception of the hsp60 gene of Mycoplasma genitalium, which was determined to be only 76.5-77.7% identical. Identities to Clostridium perfringens, Bacillus subtilis and E. coli were determined between approximately 50 and 60%. The predicted amino acid sequences of Hsp60 showed an identity of 90 to nearly 100% among mycoplasmas and 50-60% to the other bacteria indicated above. Two Hsp60 derived glutathione-S-transferase fusion proteins containing mycoplasma peptides of 28 and 35kDa were isolated. M. hyopneumoniae-ELISA positive porcine convalescent sera reacted strongly with the recombinant Hsp60 fusion proteins in Western immunoblotting indicating for the first time that mycoplasmal Hsp60 is immunogenic in natural infection.     

猪肺炎支原体膜蛋白的电泳分析及免疫印迹检测

以猪肺炎支原体Z株为试验材料,采用SDS-PAGE和Western印迹对该菌膜蛋白进行了分析。研究表明:猪肺炎支原体经A_(26)液体培养基培养,15000 r/min离心收集菌体细胞,低渗超声法破膜获得膜制剂后,应用12.5％的凝胶对膜蛋白进行SDS-PAGE分离,在电泳图谱上呈现出36条蛋白带,相对分子质量范围为1.18×10~4～9.12×10~4。同时以膜制剂作为抗原,分4次免疫试验兔,心脏采血,提取抗血清,并分离纯化得到IgG,然后进行Western印迹法检测。其结果发现在SDS-PAGE分离得到的36种膜蛋白(或多肽)中,有6种蛋白具有免疫原性。     

牛支原体单克隆抗体的制备与鉴定

以牛支原体(Mycoplasma bovis)湖北分离株HB0801作为抗原免疫8周龄BALB/c小鼠,利用杂交瘤技术筛选出了6株能稳定分泌抗牛支原体的单克隆抗体细胞株,分别生产腹水并对单抗进行了纯化和特性鉴定。经亚型测定,这些单抗都属IgG类。腹水ELISA效价在1×10⁵~1.6×10⁶。ELISA特异性分析结果表明,6株单抗与临床分离的牛支原体菌株以及ATCC标准株PG45都显阳性反应,但与牛的其他常见病原菌如多杀性巴氏杆菌、化脓隐秘杆菌等都显阴性反应。所有制备的单抗都与无乳支原体有交叉反应,其中两株单抗1A5和1C11只与无乳支原体有交叉反应,与其他支原体无交叉反应。经Western blotting验证,6株单抗分别识别牛支原体全菌蛋白中的不同条带,说明分别针对不同的蛋白抗原。这些牛支原体单克隆抗体为后期建立牛支原体检测方法及致病机理研究奠定了良好基础。     

Demonstration of neuraminidase activity in Mycoplasma neurolyticum and of neuraminidase proteins in three canine Mycoplasma species

Neuraminidases are virulence factors in many pathogenic microorganisms. They are present also in some Mycoplasma species that cause disease in birds, dogs and alligators. Thirty-seven Mycoplasma species have been examined previously for neuraminidase (sialidase) activity, whereas many of the species causing disease in man, ruminants, pigs, rodents and other animals have not. In this study neuraminidase enzymatic activity (NEAC) was examined in 45 previously untested Mycoplasma species, including those causing diseases in man, farm animals and laboratory animals. The only species in which NEAC was found was Mycoplasma neurolyticum, specifically, its type strain (Type A(T)) which is capable of inducing neurologic signs in inoculated young mice and rats. The NEAC of washed cells was relatively weak, but it differed even more than 10-fold among cells of cultures derived from individual colonies of M. neurolyticum. A weak NEAC was also detected in the supernatant of the M. neurolyticum broth culture. Canine Mycoplasma spp. with high sialidase activity reported previously, Mycoplasma canis, Mycoplasma cynos and Mycoplasma molare had 100-fold more NEAC than M. neurolyticum, but apparent differences in NEAC levels existed among strains of M. canis and of M. cynos. Zymograms using neuraminidase-specific chromogenic substrate were used to show proteins having NEAC. In M. canis (a field isolate Larissa and the type strain PG14(T)), M. cynos (isolate 896) and M. molare (type strain H542(T)) proteins with NEAC had molecular masses of ～130kDa, 105kDa and 110kDa, respectively. Identification of these neuraminidases could provide the basis for their molecular characterization.     

Immunological and pathological reactions in piglets experimentally infected with Mycoplasma hyopneumoniae and/or Mycoplasma flocculare.

The aim of this work was to examine in vivo whether infection with Mycoplasma hyopneumoniae (M. hyop) and/or Mycoplasma flocculare (M. floc) would interact and influence the severity of enzootic pneumonia in piglets. Specific pathogen-free, hysterectomy-derived piglets were allocated to six groups and experimentally inoculated with M. hyop. and/or M. floc at the age of 2 or 8 weeks. Clinical symptoms, frequency of coughing and temperature measurement were noted daily. Lung lesions were recorded by post-mortem examination and histological observations. The cross-inoculation with both mycoplasmas did not influence the clinical or the pathological picture of the disease. Evolution of specific and crossreacting antibodies was analyzed by ELISA and immunoblotting. Animals inoculated with M. floc did not develop any lesions but showed a weak antibody response 6-8 weeks post-infection (p.i.). No cross-reacting antibodies against M. hyop proteins were detected. In animals inoculated with M. hyop, the first antibody response was detectable 4-5 weeks p.i. and was stronger in piglets infected at the age of 2 weeks than at the age of 8 weeks. Three cross-reacting antibodies against M. floc proteins with molecular weights of 110, 47 and 33 kDa were detected by antibodies to M. hyop. Experimental infections with both mycoplasmas did not show differences in the pattern of species-specific proteins.     

猪肺炎支原体黏附蛋白的研究进展

猪肺炎支原体是导致猪慢性肺炎的致病因子,其致病过程主要由表面黏附蛋白介导完成。综述了肺炎支原体表面黏附蛋白（P36、P46、P65、P97、P102、P110、P159和P216）的研究进展,为猪慢性肺炎的治疗和诊断研究奠定了基础。