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猪肺炎支原体DJ-166株的分离鉴定 总被引：1，自引：0，他引：1
从山西某猪场采集疑似猪支原体肺炎肺组织病料,接种CH培养基培养,克隆纯化获得一株菌株。该菌株经PCR、培养特性、生化特性和血清学特性试验鉴定为猪肺炎支原体,命名为DJ-166株。该分离菌株在人工合成培养基上传8代稳定后,活菌计数达到108CCU/m L;菌液培养物气管注射猪后,致病性较弱;免疫原性试验证明该分离菌株具有良好的免疫原性,此结论为猪支原体肺炎疫苗的研究提供了必要条件。 相似文献
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我们从外地引进几株猪肺炎支原体标准株和流行株,并从慈溪分离出一株猪肺炎支原体,对其继续培养方法和菌株保存方法进行了一些工作,现小结如下。材料和方法1.猪肺炎支原体液体培养基我们参照中监所的培养基配方,配制了适合于支原体分离和培养的液体培养基,其组成成分为: 0.2%水解乳蛋白Hank’s液400ml 牛心消化液240ml 相似文献
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1976年从猪喘气病的病肺中分离到两株支原体培养物。在液体培养基中生长迅速,菌体以姬姆萨染色呈紫色小点状多形态。固体培养基上生长小圆球状菌落,中间有一小突起。鸡胚卵黄囊内接种,7至10天后,出现心包炎。实验感染三周龄仔猪,腹腔内接种能诱发典型的多发性浆膜炎。作者曾暂定为猪鼻支原体。后经上海畜牧兽医研究所进行生长抑制试验鉴定,N_3和 N_5株与猪鼻支原体国际标准首株 BTS_7栋为同种(10)。所以 N_3和 N_5株应为我国首次分离的猪鼻支原体菌株,并证实我国猪喘气病病猪的病肺中也存在着猪鼻支原体。 相似文献
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从典型猪肺炎支原体病变肺组织传代分离到一株支原体,经培养特性、血清学鉴定、生化鉴定、PCR检测及测序分析证明其为猪肺炎支原体,纯化后命名为S株。将S株P46基因和P97基因R1区的氨基酸序列与其他菌种的相应序列进行同源性分析,该菌株P46基因氨基酸序列与其他菌种同源高达99%以上,P97基因R1区的氨基酸重复数为11个,不同于其他菌株;免疫原性试验结果表明该菌株具有良好的免疫原性。 相似文献
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本研究旨在对山东泰安地区鸡毒支原体的流行菌株进行分离培养及鉴定和纯化,并针对该分离株筛选体外敏感的中药,为后续进一步研究提供材料。采用液体培养法与固体培养法从病鸡组织样品中分离、培养和纯化鸡毒支原体,并通过瑞士染色和姬姆萨染色、血清学方法及分子生物学方法对分离株进行初步鉴定,在此基础上,采用微量稀释法测定8种中药对分离菌株的最小抑菌浓度（MIC）,从而筛选到对分离菌株敏感的中药。结果显示,分离菌株在鸡毒支原体液体培养基中增殖后,培养基颜色由红变黄且呈半透明状态,在固体培养基中培养后呈典型的"煎蛋"样儿菌落,均符合鸡毒支原体培养特性;瑞士染色和姬姆萨染色结果显示,菌体形态符合鸡毒支原体的特征;血清学鉴定结果显示,分离株与鸡毒支原体阳性血清发生凝集;分子生物学鉴定结果显示,所扩增的核酸序列与鸡毒支原体匹配度高达99%;MIC测定结果显示,中药黄连和黄柏对分离的鸡毒支原体的抑菌活性较强,属敏感范畴,其MIC分别为≤0.98和0.39 mg/mL,而中药金荞麦和鱼腥草对鸡毒支原体中度敏感,MIC均≥125 mg/mL,板蓝根、白鲜皮、当归、艾叶对鸡毒支原体均不敏感。综上所述,本研究成功分离到1株鸡毒支原体,并且筛选出4种对该菌株敏感的中药,分别为黄连、黄柏、金荞麦和鱼腥草,为后期进一步研究防治鸡毒支原体病的中药组方时提供参考依据。 相似文献
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通过猪肺炎支原体CJ株接种3种猪肺炎支原体培养基后的生长活性研究发现,与其他2种培养基相比,接种改良Friis培养基培养2~3 d含菌量可达到1.0×109~10CCU/m L,具有生长迅速、含菌量高的特点,可用于猪肺炎支原体CJ株的培养。 相似文献
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为了对从广东地区若干个屠宰场采集的164份疑似猪肺炎支原体感染的猪肺脏组织进行猪肺炎支原体(Mycoplasma hyopneumoniae,MHp)的分离鉴定,并了解其主要抗原蛋白基因的遗传变异特点,试验先对采集的肺脏组织进行16S rRNA基因PCR检测,阳性样本进行病原的分离培养、形态学观察、双重PCR鉴定、分离株主要抗原蛋白膜蛋白P46全基因与黏附蛋白P97基因R1R2区序列分析及效价的测定。结果表明：164份临床样本经16S rRNA鉴定后102份为MHp阳性,阳性率为62.2%;阳性样本接种江苏二号(KM2)液体培养基生长良好,颜色呈规律性变黄,在固体培养基上均能呈现特异性菌落形态;各代次分离菌株经16S rRNA双重PCR鉴定均能获得3个大小约1 000 bp的目的条带,测序结果与国内外MHp各参考株核苷酸相似性为96.42%～98.21%,说明3株分离株均属于MHp,且可稳定遗传,分别命名为JM-24、JM-39、JM-44,分离株生长效价可达1×105.67 CCU/mL;3株分离株主要抗原蛋白P46全基因和P97基因R1R... 相似文献
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Six field strains of Mycoplasma hyopneumoniae isolated from pneumonic lungs of pigs, reference strains 11 and J of M hyopneumoniae, Ms 42 strain of Mycoplasma flocculare, and BTS 7 strain of Mycoplasma hyorhinis were compared serologically, using hyperimmune antisera produced in rabbits. All strains of M hyopneumoniae were closely related as determined with the disk growth-inhibition test; however, differences in zone sizes indicated that some antigenic heterogeneity existed. Cross-reactions were not detected between M hyopneumoniae, M flocculare, and M hyorhinis with the growth-inhibition test. The metabolic-inhibition test was more useful for detection of intraspecies antigenic difference than was the growth-inhibition test, since antigenic diversity was clearly detected among M hyopneumoniae strains. Slight cross-reactions were observed between M hyopneumoniae and M flocculare. Using 2-dimensional immunoelectrophoresis, antigenic differences were observed among M hyopneumoniae strains, although many common components also were detected in electropherograms. Mycoplasma flocculare possessed a close antigenic relationship to M hyopneumoniae, as determined by two-dimensional immunoelectrophoresis, whereas both organisms were less related to M hyorhinis. Evidence obtained in this study indicated that strains of mycoplasmas tentatively identified as M hyopneumoniae were similar antigenically, but evidence was obtained also of some diversity in antigenic structure among these strains. 相似文献
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An indirect immunoperoxidase staining technique was evaluated for detection of Mycoplasma hyopneumoniae in formalin-fixed, paraffin-embedded porcine lung. Lungs from swine with induced (n = 4) or naturally occurring M hyopneumoniae infection (n = 31) were examined grossly, by light and immunofluorescent microscopy, and by an indirect immunoperoxidase test, using antibody raised in swine against M hyopneumoniae as the primary antibody. Organisms stained by the indirect immunoperoxidase method were identified in tissue sections as pleomorphic brown-staining structures corresponding to those observed with immunofluorescence. Mycoplasma hyosynoviae, M hyorhinis, and Acholeplasma laidlawii did not stain with the indirect immunoperoxidase method, using antibody raised against M hyopneumoniae. 相似文献
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Protective effects of oral microencapsulated Mycoplasma hyopneumoniae vaccine prepared by co-spray drying method 总被引：2，自引：0，他引：2
Lin JH Weng CN Liao CW Yeh KS Pan MJ 《The Journal of veterinary medical science / the Japanese Society of Veterinary Science》2003,65(1):69-74
The efficacy of Mycoplasma hyopneumoniae oral vaccine was investigated in microsphere dosage form. A co-spray drying process was used to apply an encapsulating material, Eudragit L30 D-55, to microspheres containing Mycoplasma hyopneumoniae antigens. The microspheres were generally effective (>93%) with protein release at pH 7.4, but almost none were released at pH 1.2, for 3 hr in an in vitro dissolution test. An SPF-swine model was used to evaluate the effectiveness of the microspheres as an oral vaccine, and the related immune responses. The serum's systemic IgG against M. hyopneumoniae was evoked by ELISA analysis, after a 2nd immunization of all pigs. The vaccinated groups' mean lesion score was significantly lower after the Mycoplasma hyopneumoniae challenge than that of the nonvaccinated/challenged groups (P<0.05). This study strongly suggests that the oral microspheres vaccine prepared by a co-spray drying method can provide effective protection against M. hyopneumoniae infection in pigs. 相似文献
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The effect of dextran sulfate (DS), known to be cytotoxic to macrophages, on the cell-mediated and humoral immune response to nonviable Mycoplasma hyopneumoniae in pigs was investigated. The cell-mediated immune response was determined by means of lymphocyte transformation a test, using uptake of [3H]thymidine in a microculture system and the humoral immune response by means of a microplate complement-fixation test. Peripheral blood lymphocytes from pigs vaccinated with nonviable M hyopneumoniae and DS incorporated substantially more [3H]thymidine than did those from pigs given Mycoplasma or DS alone. The transformation of lymphocytes from M hyopneumoniae-DS vaccinated pigs was enhanced when M hyopneumoniae cells used in the assay system were heated at 60 C for 30 minutes. Similarly prepared M flocculare and M hyorhinis cells also stimulated lymphocytes from M hyopneumoniae-DS vaccinated pigs, but not nearly as great as when M hyopneumoniae cells were used. The humoral antibody response and the cell-mediated immune response to nonviable M hyopneumoniae was markedly enhanced by DS. Pigs were vaccinated with nonviable M hyopneumoniae and/or DS 4 times and challenge exposed intratracheally with viable M hyopneumoniae. Pigs vaccinated with M hyopneumoniae and DS had less severe pneumonia than did nonvaccinated pigs. 相似文献
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Application and field validation of a PCR assay for the detection of Mycoplasma hyopneumoniae from swine lung tissue samples. 总被引：1，自引：0，他引：1
Hugh Y Cai Tony van Dreumel Beverly McEwen Geoff Hornby Patricia Bell-Rogers Pat McRaild Gaylan Josephson Grant Maxie 《Journal of veterinary diagnostic investigation》2007,19(1):91-95
A PCR assay was validated for the detection of Mycoplasma hyopneumoniae in porcine lung tissue. The detection limit of the assay was 0.18 colony-forming units/g of lung sample spiked with M. hyopneumoniae. In field validation, 426 pigs from 220 cases were examined for M. hyopneumoniae infection by M. hyopneumoniae PCR and a fluorescent antibody (FA) test. In total, 103 pig lungs (24.2%) were positive in the PCR test, and 69 pig lungs (16.2%) were positive in the FA test, among which, 62 pigs were positive for both PCR and FA test. Most of the PCR-positive but FA test-negative cases had lesions compatible with M. hyopneumoniae infection. With Bayesian modeling, the diagnostic sensitivity and specificity of the PCR were determined to be 97.3% and 93.0%, respectively. 相似文献
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The effect of nonviable Mycoplasma hyopneumoniae on transformation of swine peripheral blood lymphocytes by mitogen was investigated. Lymphocyte transformation was evaluated as incorporation of [3H]-thymidine, using a microculture system. Mycoplasma hyopneumoniae was grown in Friis medium, inactivated with sodium azide, and washed with phosphate-buffered saline solution. Four strains of M hyopneumoniae, strain J, strain 11, and 2 low-passage isolates (1361A, 1375C), were found to suppress phytohemagglutinin-induced lymphocyte transformation. Mycoplasma hyopneumoniae strains J, 11, and 1361A reduced lymphocyte transformation by about 50%, whereas strain 1375C reduced lymphocyte transformation by 98.7%. The suppressive effect was abrogated by heating M hyopneumoniae at 60 C or at higher temperatures for 30 minutes. Sonication of the heated M hyopneumoniae cells partially restored the suppressive effect. 相似文献
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Hemagglutination and hemagglutination inhibition of turkey red blood cells with Mycoplasma hyopneumoniae 总被引：2，自引：0，他引：2
T F Young B Z Erickson R F Ross Y Wannemuehler 《American journal of veterinary research》1989,50(7):1052-1055
The ability of Mycoplasma hyopneumoniae to agglutinate RBC was evaluated to develop an in vitro cytadsorption assay. Using swine RBC in a microtitration hemagglutination test, no agglutination or partial agglutination was detected. Comparison of RBC from various other species indicated that improved hemagglutination was obtained with RBC from turkeys. This hemagglutination was detected only when mycoplasma cells used in the assay had been frozen and thawed, heated at 50 C for 30 minutes, or treated with trypsin. Treatment of RBC with trypsin or neuraminidase enhanced hemagglutination. Possible surface lectin activity in M hyopneumoniae was evaluated by use of carbohydrates in a blocking assay; hemagglutination was not inhibited by any of 13 carbohydrates evaluated. Mycoplasma hyopneumoniae convalescent porcine serum and monoclonal antibodies against 2 M hyopneumoniae immunogens of molecular weights of 64,000 and 41,000 inhibited hemagglutination. 相似文献