广西山羊传染性胸膜肺炎病原的分离鉴定
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摘要
山羊传染性胸膜肺炎是山羊的高度接触性传染病，临床上以高热、咳嗽、肺和胸膜发生浆液性和纤维蛋白性炎症为特点。广西近年时有疑似山羊传染性胸膜肺炎病例发生，但一直未能从病原学上确诊广西是否存在山羊传染性胸膜肺炎。
本研究首先从流行病学上对该病在广西的状况进行了调查。广西山羊传染性胸膜肺炎多发于7-9月，主要侵害一岁左右的羊只，发病率从26.67％至56.25％，平均发病率为35.8％；死亡率从16.67％到50％，平均死亡率为27.5％。用微量间接血凝试验(MIHA)检测了广西六个地区668份山羊血清中的抗体，以Y-98为抗原的MIHA试验检测结果显示，阳性反应89头份，阳性率为13.3％(89／668)。以PG3为抗原的MIHA试验进行检测结果为：阳性125头份，阳性率为18.7％(125／668)。其中有60份血清同时检测出以上两种支原体抗体，占被检血清的8.98％(60／668)。
采集南宁和柳州两地以呼吸道症状为主要特征的山羊病料进行了病原分离培养，在5份病料中有4份分离出支原体。分离物经纯培养、菌体和菌落形态的观察、生化试验、生长抑制试验、代谢抑制试验、动物感染试验，鉴定为丝状支原体山羊亚种(Macoplasma mycoides subsp. Capri)和绵羊肺炎支原体(Macoplasma ovipneumoniae)。首次在广西境内分离出山羊传染性胸膜肺炎病原，同时证明广西的山羊传染性胸膜肺炎由丝状支原体山羊亚种和绵羊肺炎支原体的混合感染引起。
根据文献合成了检测丝状支原体山羊亚种和绵羊肺炎支原体的引物，对4份分离物及8个菌株和20份病料进行了检测，4份分离物均扩增出状支原体山羊亚种和绵羊肺炎支原体的基因片段。菌株GX1~#-1、GX2~#-1、GX4~#-1、GX5~#-1扩增出丝状支原体山羊亚种的基因片段；菌株GX1~#-2、GX2~#-2、GX4~#-2、GX5~#-2扩增出绵羊肺炎支原体标准株Y98的基因片段。20份疑似病料中检测有14份为阳性，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，其中6份均扩增出两种支原体的基因片段。从分子生物学上对广西的传染性胸膜肺炎及其病原加以确诊。
以鹿寨传染性胸膜肺炎病羊的纯分离物为菌株制作了灭活菌苗，经无菌检验、安全性试验、免疫效果试验和田间应用试验，免疫效果好，可以转入中试。
Contagious caprine pleuropneumonia (CCPP) is an highly contagious disease ofgoats, caused by Mycoplasma spp., characterized by high fever, coughing, serous andfibrinous pleuropneumonia. In the recent years, suspectable CCPP cases always happenedin Guangxi. But agents couldn't be isolated.
In this project, firstly an epidemiological survey was performed on the status of CCPPin Guangxi. The results showed CCPP mainly happens between July to September andaffects young goats of one year old or so, the average incidence and mortality were35.8%(from 26.67% to 56.25%) and 27.5% (from 16.67% to 50%).,and 668 goat serumsamples were tested by Minim Indirect Haemagglutination test (MIHA) for detecting theAntibodies of M. ovipneumoniae and M. mycoides subsp, capri The antibody positiverate of M. ovipneumoniae and M. mycoides subsp, capri were 13.3%(89/668) and 18.7%(125/668) respectively,, and 60(8.98%) samples were both seropositive to M.ovipneumoniae and M. mycoides subsp, capri.
4 isolates of Mycoplasma spp. were isolated from 5 samples of diseased goats mainlycharacterised by respiratory signs in Nanning and Liu Zhou. The isolates were M.ovipneumoniae and M. mycoides subsp, capri identified by morphology, culture character,biochemical tests, growth inhibition test, metabolism inhibition test and animalexperiment, .It's first time that the pathogens of CCPP were identified in Guangxi. And atthe same time, it was confirmed that CCPP was caused by M. mycoides subsp, capri, and M.ovipneumoniae together.
PCR primers to M. mycoides subsp, capri, and M. ovipneumoniae(which gene?) werecomposed respectively according to the references. Four isolates, 8 strains and 20 sampleswere tested by PCR. Segments in genes of M. mycoides subsp, capri, and M.ovipneumoniae were amplified in all of the 4 isolates. A segment in gene of M. mycoidessubsp, capri, was amplified from strains GX1~#-1,GX2~#-1,GX4~#-1 and GX5~#-1. A segmentin gene of M. ovipneumoniae was amplified from strains GX1~#-2,GX2~#-2,GX4~#-2 andGX5~#-2. Fourteen of 20 samples were positive by PCR test. A segment in gene of M.ovipneumoniae was amplified in 10 samples and a segment in gene of M. mycoides subsp. and M. ovipneumoniae were amplified in 6 samples. CCPP in Guangxi and its agents werediagnosed by molecular biological techniques.
Inactive vaccine of isolate from CCPP infected goat was produced and was provenqualified by sterility test, safety test, potency test and field tests. And it got good feedbacks.So it can enters the experimental stage.
本研究首先从流行病学上对该病在广西的状况进行了调查。广西山羊传染性胸膜肺炎多发于7-9月，主要侵害一岁左右的羊只，发病率从26.67％至56.25％，平均发病率为35.8％；死亡率从16.67％到50％，平均死亡率为27.5％。用微量间接血凝试验(MIHA)检测了广西六个地区668份山羊血清中的抗体，以Y-98为抗原的MIHA试验检测结果显示，阳性反应89头份，阳性率为13.3％(89／668)。以PG3为抗原的MIHA试验进行检测结果为：阳性125头份，阳性率为18.7％(125／668)。其中有60份血清同时检测出以上两种支原体抗体，占被检血清的8.98％(60／668)。
采集南宁和柳州两地以呼吸道症状为主要特征的山羊病料进行了病原分离培养，在5份病料中有4份分离出支原体。分离物经纯培养、菌体和菌落形态的观察、生化试验、生长抑制试验、代谢抑制试验、动物感染试验，鉴定为丝状支原体山羊亚种(Macoplasma mycoides subsp. Capri)和绵羊肺炎支原体(Macoplasma ovipneumoniae)。首次在广西境内分离出山羊传染性胸膜肺炎病原，同时证明广西的山羊传染性胸膜肺炎由丝状支原体山羊亚种和绵羊肺炎支原体的混合感染引起。
根据文献合成了检测丝状支原体山羊亚种和绵羊肺炎支原体的引物，对4份分离物及8个菌株和20份病料进行了检测，4份分离物均扩增出状支原体山羊亚种和绵羊肺炎支原体的基因片段。菌株GX1~#-1、GX2~#-1、GX4~#-1、GX5~#-1扩增出丝状支原体山羊亚种的基因片段；菌株GX1~#-2、GX2~#-2、GX4~#-2、GX5~#-2扩增出绵羊肺炎支原体标准株Y98的基因片段。20份疑似病料中检测有14份为阳性，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，其中6份均扩增出两种支原体的基因片段。从分子生物学上对广西的传染性胸膜肺炎及其病原加以确诊。
以鹿寨传染性胸膜肺炎病羊的纯分离物为菌株制作了灭活菌苗，经无菌检验、安全性试验、免疫效果试验和田间应用试验，免疫效果好，可以转入中试。
Contagious caprine pleuropneumonia (CCPP) is an highly contagious disease ofgoats, caused by Mycoplasma spp., characterized by high fever, coughing, serous andfibrinous pleuropneumonia. In the recent years, suspectable CCPP cases always happenedin Guangxi. But agents couldn't be isolated.
In this project, firstly an epidemiological survey was performed on the status of CCPPin Guangxi. The results showed CCPP mainly happens between July to September andaffects young goats of one year old or so, the average incidence and mortality were35.8%(from 26.67% to 56.25%) and 27.5% (from 16.67% to 50%).,and 668 goat serumsamples were tested by Minim Indirect Haemagglutination test (MIHA) for detecting theAntibodies of M. ovipneumoniae and M. mycoides subsp, capri The antibody positiverate of M. ovipneumoniae and M. mycoides subsp, capri were 13.3%(89/668) and 18.7%(125/668) respectively,, and 60(8.98%) samples were both seropositive to M.ovipneumoniae and M. mycoides subsp, capri.
4 isolates of Mycoplasma spp. were isolated from 5 samples of diseased goats mainlycharacterised by respiratory signs in Nanning and Liu Zhou. The isolates were M.ovipneumoniae and M. mycoides subsp, capri identified by morphology, culture character,biochemical tests, growth inhibition test, metabolism inhibition test and animalexperiment, .It's first time that the pathogens of CCPP were identified in Guangxi. And atthe same time, it was confirmed that CCPP was caused by M. mycoides subsp, capri, and M.ovipneumoniae together.
PCR primers to M. mycoides subsp, capri, and M. ovipneumoniae(which gene?) werecomposed respectively according to the references. Four isolates, 8 strains and 20 sampleswere tested by PCR. Segments in genes of M. mycoides subsp, capri, and M.ovipneumoniae were amplified in all of the 4 isolates. A segment in gene of M. mycoidessubsp, capri, was amplified from strains GX1~#-1,GX2~#-1,GX4~#-1 and GX5~#-1. A segmentin gene of M. ovipneumoniae was amplified from strains GX1~#-2,GX2~#-2,GX4~#-2 andGX5~#-2. Fourteen of 20 samples were positive by PCR test. A segment in gene of M.ovipneumoniae was amplified in 10 samples and a segment in gene of M. mycoides subsp. and M. ovipneumoniae were amplified in 6 samples. CCPP in Guangxi and its agents werediagnosed by molecular biological techniques.
Inactive vaccine of isolate from CCPP infected goat was produced and was provenqualified by sterility test, safety test, potency test and field tests. And it got good feedbacks.So it can enters the experimental stage.
引文
[1] Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edition, 2004.
[2] 包惠芳 邓光明等。羊肺炎霉形体细胞蛋白多肽分析。中国兽医科技，1997年，27(10)：26～28
[3] Leach RH, Costas M, Mitchelmore DL. Relationship between Mycoplasma mycoides subsp. mycoides ('large-colony' strains) and M. mycoides subsp, capri, as indicated by numerical analysis of one-dimensional SDS-PAGE protein pattern, J Gen Microbiol. 1989, 135(11): 2993-3000.
[4] EDWARD DG. The pleuropneumonia group of organisms: a review, together with some new observations, J Gen Microbiol. 1954, 10(1): 27-64.
[5] 王栋等．我国山羊传染性胸膜肺炎病原的研究．中国兽医科技，1988，(9)：3～5
[6] 万一元，龙鳌，万晴姣等．贵州山羊传染性胸膜肺炎病原的分离鉴定．中国草食动物，2001，3(4)：44～46
[7] 邱立新，舒晓亮等，山羊支原体的分离与鉴定。中国动物检疫，2002，19(2)：21～23
[8] Mackay J M K, Nisbet D I, Foggie A. Isolation of pleuropneumonia-like organisms (Genus Mycoplasma) from case of sheep pulmonary adenomatosis(S. P A). Vet Rec, 1963, 75(21): 550～551
[9] Cottew G S. Characterisation of mycoplasmas isolated from sheep with pneumonia. Aust Vet J,1971,47(12): 591～696
[10] 胡景韶，蒋学良，胡诚隆，张道永．一种由支原体感染的绵羊增生性间质性肺炎的研究．中国兽医杂志，1982，(05)：2～6
[11] Linvingston, C. W. et al: Amer. J. Vet. Ret.,1979, (40) 3: 407～408
[12] 王栋等．从山羊分离出绵羊肺炎霉形体的报告．中国兽医杂志，1991(17)6：3～5
[13] Goltz JP, Rosendal S, McCraw BM, Ruhnke HL. Experimental studies on the pathogenicity of Mycoplasma ovipneumoniae and Mycoplasma arginini for the respiratory tract of goats. Can J Vet Res. 1986, 50(1): 59-67.
[14] 邓光明等．类山羊传染性胸膜肺炎诊断和防治的研究．中国兽医科技，1991，(21)6：6～9
[15] MacOwan KJ. Role of mycoplasma strain F38 in contagious caprine pleuropneumonia. Isr J Med Sci. 1984, 20(10): 979-81.
[16] Thiaucourt, F., Bo"lske, G., Libeau, G., Le Goff, C., Lefe vre, P.C.. The use of monoclonal antibodies in the diagnosis of contagious caprine pleuropneumonia (CCPP). Vet. Microbiol. 1994, 41, 191-203.
[17] March JB, Gammack C, Nicholas R. Rapid detection of contagious caprine pleuropneumonia using a Mycoplasrna capricolum subsp, capripneumoniae capsular polysaccharide-specific antigen detection latex agglutination test. J Clin Microbiol. 2000 Nov;38(11): 4152-9.
[18] Leach RH. The occurrence of Mycoplasma arginini in several animal hosts. Vet Rec. 1970 Sep 12; 87(11):319-320.
[19] Niang M, Rosenbusch RF, Lopez-Virella J, Kaeberle ML. Differential serologic response to Mycoplasma ovipneumoniae and MycopIasma arginini in lambs affected with chronic respiratory disease. J Vet Diagn Invest. 1999 Jan;11(1): 34-40.
[20] 方畴鑫，李志杰，邓光明，梁桂香．甘肃陇东地区羊肺炎病的流行病学调查．中国兽医杂志，1998，(03)：18～19
[21] 张道永，胡诚隆，林毅，王泽洲，王文贵，杨晓梅，李力．羊肺炎霉形体病研究[J]．中国兽医学报，1998，(1)．55～59
[22] 万一元，龙鳌，万晴姣等。贵州山羊传染性胸膜肺炎病原的分离鉴定。中国草食动物，2001，3(4)．44～46
[23] Thiaucourt, F., Bo"lske, G., 1996. Contagious caprine pleuropneumonia and other pulmonary mycoplasmoses of sheep and goats.
[24] Rurangirwa FR, McGuire TC, Kibor A, Chema S. A latex agglutination test for field diagnosis of contagious caprine pleuropneumonia. Vet Rec. 1987 Aug 29;121(9):191-3.
[25] Miserez R., Pilloud T., Cheng X., Nicolet J.,Griot C., Frey J., Development of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides SC, Mol. Cell. Probes 11 (1997) 103-111.
[26] Monnerat M. P., Thiaucourt F., Nicolet J., Frey J., Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. Capricolum and Mycoplasma capricolum subsp, capripneumoniae, Vet. Microbiol. 69 (1999), 157-172.
[27] Monnerat M. P., Thiaucourt F., Poveda J.B., Nicolet J., Frey J., Genetic and serological analysis of lipoprotein LppA in Mycopasma mycoides subsp, mycoides LC and Mycoplasma mycoides subsp, capri, Clin. Diagn. Lab. Immunol. 6 (1999) 224-230.
[28] Woubit S, Lorenzon S, Peyraud A, Manso-Silvan L, Thiaucourt F. A specific PCR for the identification of Mycoplasma capricolum subsp, capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP). Vet Microbiol. 2004 Nov 30;104(1-2):125-32.
[29] 张莉，杨壮省，王英珍。聚合酶链反应检测绵羊肺炎霉形体的研究．中国兽医科技(34)7：7～10
[30] Dominique LE GRAND, Estelle SARAS. Assessment of PCR for routine identification of species of the Mycoplasma mycoides duster in ruminants. Vet. Res. 35 (2004) 635-649
[31] Quinlan JR, Alley MR, Clarke JK. In vitro sensitivity of New Zealand strains of Mycoplasma ovipneumoniae to some antibiotics. N Z Vet J. 1975, 23(9): 188-9
[32] 万晴姣，万一元，潘淑惠，龙鳌，杨何，乐正中，吴立辉．PG_3支原体的药物敏感性试验[J]．贵州畜牧兽医，2001，(3)：9
[33] 蒋玉雯，黄安国，白安斌等．山羊传染性胸膜肺炎血清学调查．广西畜牧兽医，2000，16(2)：14～15
[34] 毛洪先．猪肺炎支原体霉形体固体培养基的制备及其培养的研究．家畜传染病，1981，(3)：41～44
[35] 万晴姣．山羊传染性胸膜肺炎病原的生化鉴定，贵州畜牧兽医，1999，(5)：22～231
[2] 包惠芳 邓光明等。羊肺炎霉形体细胞蛋白多肽分析。中国兽医科技，1997年，27(10)：26～28
[3] Leach RH, Costas M, Mitchelmore DL. Relationship between Mycoplasma mycoides subsp. mycoides ('large-colony' strains) and M. mycoides subsp, capri, as indicated by numerical analysis of one-dimensional SDS-PAGE protein pattern, J Gen Microbiol. 1989, 135(11): 2993-3000.
[4] EDWARD DG. The pleuropneumonia group of organisms: a review, together with some new observations, J Gen Microbiol. 1954, 10(1): 27-64.
[5] 王栋等．我国山羊传染性胸膜肺炎病原的研究．中国兽医科技，1988，(9)：3～5
[6] 万一元，龙鳌，万晴姣等．贵州山羊传染性胸膜肺炎病原的分离鉴定．中国草食动物，2001，3(4)：44～46
[7] 邱立新，舒晓亮等，山羊支原体的分离与鉴定。中国动物检疫，2002，19(2)：21～23
[8] Mackay J M K, Nisbet D I, Foggie A. Isolation of pleuropneumonia-like organisms (Genus Mycoplasma) from case of sheep pulmonary adenomatosis(S. P A). Vet Rec, 1963, 75(21): 550～551
[9] Cottew G S. Characterisation of mycoplasmas isolated from sheep with pneumonia. Aust Vet J,1971,47(12): 591～696
[10] 胡景韶，蒋学良，胡诚隆，张道永．一种由支原体感染的绵羊增生性间质性肺炎的研究．中国兽医杂志，1982，(05)：2～6
[11] Linvingston, C. W. et al: Amer. J. Vet. Ret.,1979, (40) 3: 407～408
[12] 王栋等．从山羊分离出绵羊肺炎霉形体的报告．中国兽医杂志，1991(17)6：3～5
[13] Goltz JP, Rosendal S, McCraw BM, Ruhnke HL. Experimental studies on the pathogenicity of Mycoplasma ovipneumoniae and Mycoplasma arginini for the respiratory tract of goats. Can J Vet Res. 1986, 50(1): 59-67.
[14] 邓光明等．类山羊传染性胸膜肺炎诊断和防治的研究．中国兽医科技，1991，(21)6：6～9
[15] MacOwan KJ. Role of mycoplasma strain F38 in contagious caprine pleuropneumonia. Isr J Med Sci. 1984, 20(10): 979-81.
[16] Thiaucourt, F., Bo"lske, G., Libeau, G., Le Goff, C., Lefe vre, P.C.. The use of monoclonal antibodies in the diagnosis of contagious caprine pleuropneumonia (CCPP). Vet. Microbiol. 1994, 41, 191-203.
[17] March JB, Gammack C, Nicholas R. Rapid detection of contagious caprine pleuropneumonia using a Mycoplasrna capricolum subsp, capripneumoniae capsular polysaccharide-specific antigen detection latex agglutination test. J Clin Microbiol. 2000 Nov;38(11): 4152-9.
[18] Leach RH. The occurrence of Mycoplasma arginini in several animal hosts. Vet Rec. 1970 Sep 12; 87(11):319-320.
[19] Niang M, Rosenbusch RF, Lopez-Virella J, Kaeberle ML. Differential serologic response to Mycoplasma ovipneumoniae and MycopIasma arginini in lambs affected with chronic respiratory disease. J Vet Diagn Invest. 1999 Jan;11(1): 34-40.
[20] 方畴鑫，李志杰，邓光明，梁桂香．甘肃陇东地区羊肺炎病的流行病学调查．中国兽医杂志，1998，(03)：18～19
[21] 张道永，胡诚隆，林毅，王泽洲，王文贵，杨晓梅，李力．羊肺炎霉形体病研究[J]．中国兽医学报，1998，(1)．55～59
[22] 万一元，龙鳌，万晴姣等。贵州山羊传染性胸膜肺炎病原的分离鉴定。中国草食动物，2001，3(4)．44～46
[23] Thiaucourt, F., Bo"lske, G., 1996. Contagious caprine pleuropneumonia and other pulmonary mycoplasmoses of sheep and goats.
[24] Rurangirwa FR, McGuire TC, Kibor A, Chema S. A latex agglutination test for field diagnosis of contagious caprine pleuropneumonia. Vet Rec. 1987 Aug 29;121(9):191-3.
[25] Miserez R., Pilloud T., Cheng X., Nicolet J.,Griot C., Frey J., Development of a sensitive nested PCR method for the specific detection of Mycoplasma mycoides SC, Mol. Cell. Probes 11 (1997) 103-111.
[26] Monnerat M. P., Thiaucourt F., Nicolet J., Frey J., Comparative analysis of the lppA locus in Mycoplasma capricolum subsp. Capricolum and Mycoplasma capricolum subsp, capripneumoniae, Vet. Microbiol. 69 (1999), 157-172.
[27] Monnerat M. P., Thiaucourt F., Poveda J.B., Nicolet J., Frey J., Genetic and serological analysis of lipoprotein LppA in Mycopasma mycoides subsp, mycoides LC and Mycoplasma mycoides subsp, capri, Clin. Diagn. Lab. Immunol. 6 (1999) 224-230.
[28] Woubit S, Lorenzon S, Peyraud A, Manso-Silvan L, Thiaucourt F. A specific PCR for the identification of Mycoplasma capricolum subsp, capripneumoniae, the causative agent of contagious caprine pleuropneumonia (CCPP). Vet Microbiol. 2004 Nov 30;104(1-2):125-32.
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