应用Dot-PPA-ELISA检测猪巴氏杆菌病抗体的研究
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摘要
本研究提取猪巴氏杆菌菌株的荚膜和外膜蛋白抗原，首次建立了检测猪巴氏杆菌病血清抗体的Dot-PPA-ELISA方法。本研究确定了抗原的最佳包被浓度为7.76ug／ml，酶标SPA的最佳稀释倍数为1：10。经动物试验初步确定了该方法的阳性标准。试验证明所建立的Dot-PPA-ELISA具有较好的特异性，与猪瘟、仔猪副伤寒、猪丹毒、猪细小病毒病、猪伪狂犬、猪布氏杆菌病、猪衣原体病阳性血清无交叉反应。试验所建立的Dot-PPA-ELISA灵敏度高，与琼扩试验比较，敏感度提高约128倍。诊断膜片具有很好的稳定性，4℃保存5个月，室温保存3个月，其特异性，灵敏度不变。本试验建立的Dot-PPA-ELISA方法操作方便、快速，结果客观、易于判定，重复性好，所用的相关试剂可以标准化。适用于养猪场猪巴氏杆菌病的诊断、免疫后抗体监测以及流行病学调查。
A Dot-PPA-ELISA method has been developed to detect the antibody in swine serum against pasteurellosis. The capsule polysaccharide and outer membrane protein of pasteurella multocida was prepared as the diaphragm antigen. Applying the x2 test,optimal concentration of the diaphragm antigen was determined,and the optimum dilution of enzyme HRP-SPA 1:10. The high specificity of the Dot-PPA-ELISA was confirmed by specific blocking test and also by cross-reaction test. The diaphragm did not react with the antibodies against Salmonellosis,Streptococcosis,Colibacillosis,Chlamydiosis,HCV,PPV,PRV,Brucellosis. Erysipelas,suis and Chlamydiosis in cross-reaction test.The diaphragm has good sensitivity and could detect some pasteurella-positive test serum which has been diluted to2-". In comprision with GDPT ,the sensetivity is improved by 128 times . When 10 positive serum was detected 3 times,and the result was reproducible. The diaphragm has good stability as well,stored at 4 for at least 5 months or at 10-25 for 3 mo
nths,the sensitivity and specificity of the diaphragm did not change.
This study endicates that the Dot-PPA-ELISA was a convenient,rapid,sensitive and specific method for the detection of antibody. Apart from these,all of the materials used in Dot-PPA-ELISA can be standardized. So it can be used for the surveillance of pasteurella multocida antibody in herd ,for the epidemiologic investigation of swine pasteurellosis ,and it provides a reference for a ratinal immunity schedule.
A Dot-PPA-ELISA method has been developed to detect the antibody in swine serum against pasteurellosis. The capsule polysaccharide and outer membrane protein of pasteurella multocida was prepared as the diaphragm antigen. Applying the x2 test,optimal concentration of the diaphragm antigen was determined,and the optimum dilution of enzyme HRP-SPA 1:10. The high specificity of the Dot-PPA-ELISA was confirmed by specific blocking test and also by cross-reaction test. The diaphragm did not react with the antibodies against Salmonellosis,Streptococcosis,Colibacillosis,Chlamydiosis,HCV,PPV,PRV,Brucellosis. Erysipelas,suis and Chlamydiosis in cross-reaction test.The diaphragm has good sensitivity and could detect some pasteurella-positive test serum which has been diluted to2-". In comprision with GDPT ,the sensetivity is improved by 128 times . When 10 positive serum was detected 3 times,and the result was reproducible. The diaphragm has good stability as well,stored at 4 for at least 5 months or at 10-25 for 3 mo
nths,the sensitivity and specificity of the diaphragm did not change.
This study endicates that the Dot-PPA-ELISA was a convenient,rapid,sensitive and specific method for the detection of antibody. Apart from these,all of the materials used in Dot-PPA-ELISA can be standardized. So it can be used for the surveillance of pasteurella multocida antibody in herd ,for the epidemiologic investigation of swine pasteurellosis ,and it provides a reference for a ratinal immunity schedule.
引文
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2. Zhao-GS, Pijoan-C, et al. Use of restriction endonuclease analysis and ribotyping to study epidemiology of Pasteurella multocida in closed swine herds. Infection-and-Immunity. 1992, 60(4), 1401-1405
3. Goodwin-RFW .Clinical and epidemioiogical aspects of respiratory disease in pigs. Proceedings of the Royal Veterinary College-Pfizer Ltd symposium: on respiratory diseases in cattle and pigs[M].1991,47-52.
4. Bowles-RE, Pahoff-JL, et al.Ribotype diversity of porcine Pasteurella multocida from Australia.Australian-Veterinary-Journal. 2000, 78(9):630-635.
5. Fussing-V, elsen-JP, et al. Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark. Veterinary-Microbiology. 1999, 65(1):61-74.
6. Pejsak-Z, Kolodziejczyk-P, et al. Incidence and drug sensitivity of bacteria isolated from diseased lungs in pigs. Medycyna-Weterynaryjna. 2001, 57(7): 480-485.
7. Vachev-B, Dobrev-V, et al. Outbreak of pasteurellosis among swine in Bulgaria. Veterinama-Sbirka.1981, 79(1): 20-22.
8. Zhao-GS, Pijoan-C,et al. Epidemiology of Pasteurella multocida in a farrow-to-finish swine herd[J].Canadian-Journal-of-Veterinary-Research. 1993, 57(2): 136-138.
9. Chang-CN ,Shen-YM, et aI.Epidemiological studies on Pasteurella pneumonia in pigs.Taiwan Sugar .1991,9. 133-141.
10．崔德凤，张永红．猪支原体和巴氏杆菌感染的诊断和防制[J]．中国兽医杂志，2001，37(5)：28
11．薛玉平．猪巴氏杆菌病的诊断及防治[J]．中国兽医科技，1999，29(2)：38
12．杨孚才．畜禽巴氏杆菌病爆发[J]．中国兽医杂志，1997，23(8)：35
13．吴铭锋．仔猪巴氏杆菌诊治[J]．福建畜牧兽医，1998，3：14
14．艾小生，艾水根．猪巴氏杆菌病的诊疗[J]．云南畜牧兽医，2000，2：46
15．唐荣华．猪群爆发巴氏杆菌的诊治报告[J]．广西畜牧兽医，1999，2：30-31
16．吴范庚．我国多杀性巴氏杆菌血清学鉴定及交互免疫试验[J]．中国兽医杂志1997，23(8)：14。15
17. Fussing-V, Nielsen-JP, et al.Development of a typing system for epidemiological studies of porcine toxin-producing Pasteurella multocida ssp. multocida in Denmark. Veterinary-Microbiology. 1999,65(1):61-74
18．中国农业科学院哈尔滨兽医研究所编著，兽医微生物学(第一版)[M]，中国农业出版社,1998
19. H.Kodama, DVM,PhD,et al Immunogenicity of capsular antigens of Pasteurella multocida in Turkeys[J].American-Journal-of-Veterinary-Research. 1981,42(10): 1838-1841
20. Chung-JingYeng, Wilkie-I, et al. Role of capsule in the pathogenesis of fowl cholera caused by Pasteurella multocida serogroup A.Infection-and-Immunity. 2001, 69 (4) :487-2492
21. Kodama. H, et al. Soluble fractions of Pasteurella multocida: their protective qualities against fowl cholera in turkeys[J]. Avian Diseases. 1983,27(1):283-291
22．高菘，刘秀梵等．一种快速提取大肠埃希氏菌外膜蛋白的方法[J]．微生物通报．1996,23(2)：122-123
23．黎世能，王浴生等．细菌β-内酰胺酶及外膜蛋白改变与耐药性关系的研究[J]．中国抗生素杂志 1993，18(4)：302-305
24．李忠花，刘晓霞．伤寒耐药性与外膜蛋白关系的研究[J]．中国传染病杂志．1995，13(2)：81-84
25．高菘，刘秀梵等．禽源大肠杆菌O_1、O_2、O_(78)分离株外膜蛋白的研究[J]．微生物学报 1997,37(6)：463-468
26．赵香汝，杨汉春，细菌外膜蛋白的研究现状[J]．中国兽药杂志．1997，23(12)：41-42
27．赵香汝．细菌外膜蛋白的结构与功能[J]．中国兽医科技．1999，29(10)：20-22
28. Thomas E.Zimmmerman, et al. Polypeptides associated with Pasteurella multocida infection in rabbits[J]. American-Journal-of-Veterinary-Research. 1992,53(7):1108-1112
29. U.S.Pati,et al. Immunogenicity of outer membrane protein of Pasteurella multocida in buffalo calves[J] .Veterinary microbiology. 1996,52:301-311
30. Knights,JM,et al.Characterization of envelope proteins from Pasteurella.haemolytica and Pasteurella multocida[J]. Journal-General-Microbiology. 1990,136:495-505
31. Vasfi-Marandi-M, MittaI-KR .Characterization of an outer membrane protein of Pasteurella multocida belonging to the OmpA family. Veterinary-Microbiology. 1996, 53 (3-4) : 303-314
32. Lugtenberg-B, et al. Atrophic rhinitis in swine: correlation of Pasteurella multocida pathogenicity with membrane protein and lipopolysaccharide patterns[J]. Infection and Immunity. 1984,46(1): 48-54
33. Lu.YS,Aguila HN, et al. Antibodies to outer membrane proteins but not lipopolysaccharide inhibit pulmnonary proliferation of Pasteureila multocida in mice[J] .Infection and Immunology. 1991,59:48-54
34. Luo-YuGang, Glisson-JR, et al. Cloning and characterization of the major outer membrane protein gene (ompH) of Pasteurella multocida X-73. Journal-of-Bacteriology. 1997, 179(24): 7856-7864
35. Ruffolo-CG;Adler-B cloning,sequencing, expression,and protective capacity of the oma87 gene encoding the Pasteurella multocida 87-kilodalton outer-membrane antigen[J]. Infection and immunity 1996,64:8,3161-3167
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