山羊传染性胸膜肺炎病原的分离鉴定
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摘要
本研究从四川地区某羊场具有传染性胸膜肺炎临床症状的患病山羊的肺脏、气管等材料中成功地分离和鉴定了两株霉形体(分别命名为Y_1和Y_2)。主要通过病原分离培养特性、形态学观察、生化试验、间接血凝试验、生长抑制试验和代谢抑制试验、动物回归试验等方法进行鉴定。
鉴定结果如下：分离株Y_1和Y_2都能通过0.45μm的微孔滤器，进行过滤传代培养。在形态学观察中，都具有革兰氏染色不易着色，瑞氏染色良好呈淡紫色；在透射电镜下观察，Y_1和Y_2的菌体呈球状、丝状、杆状、螺旋状等不规则形态，大小为0.2～0.4μm。在含1％营养琼脂的固体培养基上均形成圆形、露滴状的小菌落；在40倍和100倍光学显微镜下观察时，可看见典型的煎蛋样菌落，但其形态不完全相同。Y_1的菌落形态较小，菌落生长比较密集，融合菌落较多，中心脐向上突起；Y_2的菌落形态较大，菌落较分散，无中心脐。在生化试验中，Y_1和Y_2均能够发酵葡萄糖、还原美蓝、液化血清；均不水解尿素、不水解精氨酸、膜斑形成试验呈阴性、磷酸酯酶活性呈阴性。在间接血凝试验中，提纯的Y_1和Y_2抗原均能与PG_3和Y_(98)标准阳性血清发生凝集反应，均不与PG_3和Y_(98)的标准阴性血清发生凝集反应。在生长抑制试验中，PG_3和Y_(98)标准阳性血清均能抑制Y_1和Y_2生长，并形成大于2mm的抑制圈；PG_3和Y_(98)的标准阴性血清均不能抑制Y_1和Y_2生长，不形成明显的抑制圈。在代谢抑制试验中，加入的标准阳性血清浓度较高时(如1：2~3、1：2~4)，均能抑制Y_1和Y_2的生长；当加入的标准阳性血清浓度适中时(如1：2~5、1：2~6、1：2~7)，PG_3的标准阳性血清能在一定程度上抑制Y_1的生长，Y_(98)的标准阳性血清能在一定程度上抑制Y_2的生长；当加入的标准阳性血清浓度较低时(如1：2~8、1：2~9)，均不抑制Y_1和Y_2的生长。在动物回归试验中，均能成功复制出山羊传染性胸膜肺炎的典型临床症状和病理解剖变化。
试验表明，Y_1和Y_2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霉形体山羊亚种PG_3和绵羊肺炎霉形体Y_(98)相接近；结果证实Y_1和Y_2分别为丝状霉形体山羊亚种和绵羊肺炎霉形体。
Two kind of pleomorphism microorganisms (named Y_1 and Y_2) were isolated from the lung tissues and trachea of infected cattle from a farm in SiChuan. They were indentified as Mycoplasma capri by the following methods, culture characterization, morphological tests, biochemical reactions, growth inhibition tests, metabolism inhibition tests, serological identification and animal experiments in goats.
Results of above experiments indicated that both microorganisms could be lautered by micr-opore filter of 0.45μrn aperture. And the lautered products could be serially subculti-vated. In morphological tests, both microorganisms were hard to be dye by Gelan Dye. In contrast, they showed amethyst by M-Wright's stain. Under the eyesight of transmission electron microscope, they both showed polymorphism, such as globe, thread, pole, spire and so on. The size of them was between 0.2-0.4μm. During the course of cultivation, conglobate and petite colonies like dew point on solid medium with 1% nutrient agar could be obwerved. If observed with 40 times magnification, though they had tiny difference, the shape of them were all like fried egg. The colonies of Y_1 with compact protuberance in the center of which were small and compressive to mix together. In contrast, the colonies of Y_2 were bigger, dispressive and with no gibbous center hylum.The results of biochemical tests indicated that they were positive with Glucose fermentation( + ), Equine serum liquifaction ( + ), Methylene Blue disoxidation ( + ), and negative with Urea hydrolysis (-) ,Arginine hydrolysis (-) , Film and pot form (-) , PhosphataseActivity (-) . The indirect hemagglutination tests showed that the antigen of Y_1 and Y_2 could agglutinate with positive serum of PG_3 and Y_(98); not agglutinate with negative serum of PG_3 and Y_(98).G rowth inhibition tests demonstrated that the positive serum of PG_3 and Y_(98) could inhibit growth of Y_1 and Y_2 to form inhibiting collar of diameter exceeding 2mm.In the contrary, the negative serum of PG_3 and Y_(98) could not inhibit the growth of Y_1 and Y_2.In the metabolism
鉴定结果如下：分离株Y_1和Y_2都能通过0.45μm的微孔滤器，进行过滤传代培养。在形态学观察中，都具有革兰氏染色不易着色，瑞氏染色良好呈淡紫色；在透射电镜下观察，Y_1和Y_2的菌体呈球状、丝状、杆状、螺旋状等不规则形态，大小为0.2～0.4μm。在含1％营养琼脂的固体培养基上均形成圆形、露滴状的小菌落；在40倍和100倍光学显微镜下观察时，可看见典型的煎蛋样菌落，但其形态不完全相同。Y_1的菌落形态较小，菌落生长比较密集，融合菌落较多，中心脐向上突起；Y_2的菌落形态较大，菌落较分散，无中心脐。在生化试验中，Y_1和Y_2均能够发酵葡萄糖、还原美蓝、液化血清；均不水解尿素、不水解精氨酸、膜斑形成试验呈阴性、磷酸酯酶活性呈阴性。在间接血凝试验中，提纯的Y_1和Y_2抗原均能与PG_3和Y_(98)标准阳性血清发生凝集反应，均不与PG_3和Y_(98)的标准阴性血清发生凝集反应。在生长抑制试验中，PG_3和Y_(98)标准阳性血清均能抑制Y_1和Y_2生长，并形成大于2mm的抑制圈；PG_3和Y_(98)的标准阴性血清均不能抑制Y_1和Y_2生长，不形成明显的抑制圈。在代谢抑制试验中，加入的标准阳性血清浓度较高时(如1：2~3、1：2~4)，均能抑制Y_1和Y_2的生长；当加入的标准阳性血清浓度适中时(如1：2~5、1：2~6、1：2~7)，PG_3的标准阳性血清能在一定程度上抑制Y_1的生长，Y_(98)的标准阳性血清能在一定程度上抑制Y_2的生长；当加入的标准阳性血清浓度较低时(如1：2~8、1：2~9)，均不抑制Y_1和Y_2的生长。在动物回归试验中，均能成功复制出山羊传染性胸膜肺炎的典型临床症状和病理解剖变化。
试验表明，Y_1和Y_2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霉形体山羊亚种PG_3和绵羊肺炎霉形体Y_(98)相接近；结果证实Y_1和Y_2分别为丝状霉形体山羊亚种和绵羊肺炎霉形体。
Two kind of pleomorphism microorganisms (named Y_1 and Y_2) were isolated from the lung tissues and trachea of infected cattle from a farm in SiChuan. They were indentified as Mycoplasma capri by the following methods, culture characterization, morphological tests, biochemical reactions, growth inhibition tests, metabolism inhibition tests, serological identification and animal experiments in goats.
Results of above experiments indicated that both microorganisms could be lautered by micr-opore filter of 0.45μrn aperture. And the lautered products could be serially subculti-vated. In morphological tests, both microorganisms were hard to be dye by Gelan Dye. In contrast, they showed amethyst by M-Wright's stain. Under the eyesight of transmission electron microscope, they both showed polymorphism, such as globe, thread, pole, spire and so on. The size of them was between 0.2-0.4μm. During the course of cultivation, conglobate and petite colonies like dew point on solid medium with 1% nutrient agar could be obwerved. If observed with 40 times magnification, though they had tiny difference, the shape of them were all like fried egg. The colonies of Y_1 with compact protuberance in the center of which were small and compressive to mix together. In contrast, the colonies of Y_2 were bigger, dispressive and with no gibbous center hylum.The results of biochemical tests indicated that they were positive with Glucose fermentation( + ), Equine serum liquifaction ( + ), Methylene Blue disoxidation ( + ), and negative with Urea hydrolysis (-) ,Arginine hydrolysis (-) , Film and pot form (-) , PhosphataseActivity (-) . The indirect hemagglutination tests showed that the antigen of Y_1 and Y_2 could agglutinate with positive serum of PG_3 and Y_(98); not agglutinate with negative serum of PG_3 and Y_(98).G rowth inhibition tests demonstrated that the positive serum of PG_3 and Y_(98) could inhibit growth of Y_1 and Y_2 to form inhibiting collar of diameter exceeding 2mm.In the contrary, the negative serum of PG_3 and Y_(98) could not inhibit the growth of Y_1 and Y_2.In the metabolism
引文
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2 Gillespie J H, Timon, ey J F. [Hagan and Bruner's infectious of diseases of domestic animals][J]. Seventh Edition. Comell University Press, U. S. A, 1981: 124-132.
3 Jones, G. E. Contagious Caprinwe Pleuropneumonia in: Manual of Standaards for diagnostic tests and vaccines. 2nd Ed. OIE Standands Commission Paris[J]: OIE Press, 1992. 376-392.
4 Kusiluka L J, Ojeniyi B, Friis NF, et al. Mycoplasmas isolated from the respiratory tract of cattle and goats in Tanzania[J]. Acta Vet Scand JT-Acta veterinaria Scandinavica. 41(3). Denmark, 2000. 299-309.
5 Alley MR, Clarke JK. The experimental transmission of ovine chronic non-progressive pneumonia[J]. N Z Vet J JT-New Zealand veterinary journal. 27(10). NEW ZEALAND, 1979. 217-20.
6 Carmichael LE, St George TD, Sullivan ND, et al. Isolation, propagation, and characterization studies of an ovine Mycoplasma responsible for proliferative interstitial pneumonia[J]. Comell Vet JT-The Comell veterinarian. 62(4). UNITED STATES, 1972. 654-79.
7 中国农业科学院哈尔滨兽医研究所．家畜传染病学[J]：农业出版社，1989．383-390．
8 Thiaucourt F, Bolske G. Contagious caprine pleuropneumonia and other pulmonary mycoplasmoses of sheep and goats[J]. Rev Sci Tech JT-Revue scientifique et technique (International Office of Epizootics), 1996, 15(4): 1397-414.
9 Harbi MS, El Tahir MS, Macowan KJ, et al. Mycoplasma strain F38 and contagious caprine pleuropneumonia in the Sudan[J]. Vet Rec JT-The Veterinary record, 1981, 108(12): 261.
10 MacOwan, K. J. A mycoplasma from chronic contagious pleuropneumonia in Kenya[J]. Trop. Anim. Health Prod, 1976, 8: 26-38.
11 Cottew, G. S, Yeats, F. R. Occurrence of mycoplasmas in clinically normal goats[J]. Aust. Vet. J, 1981, 57: 52-53.
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