副猪嗜血杆菌病Neu、Lipo、OMP5蛋白的原核表达及免疫原性分析
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摘要
副猪嗜血杆菌(Haemophilus parasuis, HPS)属于巴斯德菌科的嗜血菌属的革兰氏阴性菌,能引起猪的Glasser病,主要特征为纤维素性浆膜炎、关节炎和脑膜炎。HPS感染猪,影响2周龄到4月龄的青年猪,主要在断奶前后和保育阶段发病,发病率一般在10%-15%,严重时患病猪死亡率可达50%,给养殖业造成巨大经济损失。目前预防该病注射的疫苗为灭活苗,但是副猪嗜血杆菌血清型多样且很大比例不能进行分型,菌株间毒力差异也较大,目前还没有一种灭活苗能同时对所有致病菌产生交叉保护。随着分子生物学技术的快速发展,疫苗的研究方向也转向基因工程疫苗。本实验通过将副猪嗜血杆菌的Lipo、Neu、Omp5基因克隆及原核表达,并将三个蛋白分别免疫昆明小鼠研究其免疫原性。现将主要的工作及结果报道如下：
(一)、根据GenBank中的副猪嗜血杆菌序列(登陆号：CP001321.1)设计特异性引物扩增Lipo基因,再将Lipo基因克隆到pMD18-T载体上。以pMDLipo为模板用PCR方法扩增Lipo成熟肽编码区。通过EcoRⅠ和SalⅠ双酶切处理Lipo PCR回收产物与表达载体pBV220,将Lipo基因连接至pBV220中。SDS-PAGE电泳检测显示重组质粒pBV-Lipo在大肠杆菌Rosetta (DE3)中实现高效表达,lipo蛋白为可溶性表达,并通过硫酸铵沉淀纯化lipo蛋白。
(二)、根据GenBank中的副猪嗜血杆菌鸟枪序列(登陆号：NZ-ABKM01000006)设计特异性引物扩增Neu全基因,再将Neu全基因克隆到pMD18-T载体上。以pMDNeu为模板PCR方法扩增Neu部分基因。通过EcoRⅠ和SalⅠ双酶切处理Neu PCR回收产物与表达载体pET-28a(+),将Neu基因连接至pET-28a(+)中。将连接产物转入大肠杆菌DH5α,通过PCR和酶切挑选阳性质粒,再将阳性质粒转入Rosetta (DE3)进行诱导。SDS-PAGE检测显示pET-Neu在大肠杆菌Rosetta (DE3)中实现高效表达, Neu蛋白以包涵体形式表达,用tritonX-100等洗涤包涵体。
(三)、根据GenBank中的副猪嗜血杆菌鸟枪序列(登陆号：NC-011852)设计引物扩增OMP5全基因,再将OMP5全基因克隆到pMD18-T载体上。通过摸索酶切条件,用EcoRⅠ和SalⅠ将pMDOMP5质粒与表达载体pET-28a(+)进行双酶切,将OMP5基因连接至载体质粒pET-28a(+)中。连接产物转入至大肠杆菌DH5α通过PCR和酶切挑选阳性质粒,再将阳性质粒转入Rosetta (DE3)进行诱导。SDS-PAGE检测显示pET-OMP5在大肠杆菌Rosetta (DE3)中实现高效表达,OMP5蛋白以包涵体形式表达,用tritonX-100等洗涤包涵体。
(四)、将重组纯化的Lipo、Neu、Omp5蛋白分别分两组,一组使用弗氏佐剂；另一组使用氢氧化铝佐剂。初次免疫后的14天和21天进行加强免疫,总共免疫3次,每次剂量为50μg/只；对照组未免疫任何蛋白,使用生理盐水。以致死量HPs对免疫小鼠和对照组小鼠进行攻毒。各实验组的攻毒保护结果如下：Neu-弗氏佐剂组的保护率为60%(6/10),Neu-氢氧化铝佐剂组的保护率为10%(1/10)；OMP5-弗氏佐剂组的保护率为30%(3/10),OMP5-氢氧化铝佐剂组的保护率为11.1%(1/9); Lipo-弗氏佐剂组的保护率为86.6%(8/9),免疫Lipo和氢氧化铝佐剂组的保护率为50%(5/10)；未免疫蛋白采用致死量攻毒的对照组小鼠,在攻毒后全部死亡；而未免疫蛋白注射灭活HPs的对照组小鼠,在注射4h内部分小鼠精神萎靡,但4h后很快恢复正常,精神转好。结果显示了这三个重组蛋白都能对小鼠起到保护作用,证明Lipo、Neu、OMP5都具有一定的免疫源性,其中Lipo蛋白明显高于Neu、OMP5,但是效果均不如灭活组。
Haemophilus parasuis (H.parasuis) is a Gram-negative bacterium belonging to the Pasteurellaceae family. The H. parasuis can cause the Glasser's disease which is mainly characterzed by polyserositis,arthritis and meningitis. H. parasuis usually affects young pigs from 2 weeks to 4 months in the nursery.The incidence rate of H. parasuis is generally from 10% to 15%.when serious illness come the mortality of pigs are up to 50%. H. parasuis leads to increasing economic losses in the swine industry worldwide.Control of H. parasuis infections can be achieved by use of vaccination using bacterins,but a major variability has been reported when testing the development of cross-protection,depending on H. parasuis strains and serotypes.With the rapid development of molecular biology techniques,vaccine reseach has turn to genetic engineering vaccine.Prokaryotic expression of Neu、Lipo and OMP5 gene of H.parasuis and analysis of immunogenicity of the expressed protein.
According to GenBank in Haemophilus parasuis shotgun sequence, the part of Lipo gene was amplified from pMT18-Lipo by PCR with a pair of specific primers which primers designed in the upstream EcoRⅠsites upstream and downstream primers SalⅠsites. By EcoRⅠand SalⅠrestriction enzyme digestion treatment Lipo PCR product and expression vector recovery pBV220, the Lipo gene connected to the pBV220. SDS-PAGE electrophoresis analysis showed that the recombinant plasmid pBV-Lipo in E. coli Rosetta (DE3) to achieve high expression, lipo soluble protein expression and purification of lipo protein by ammonium sulfate precipitation.
According to GenBank in Haemophilus parasuis shotgun sequence, the part of Neu gene was amplified from pMT18-Neu by PCR with a pair of specific primers which primers designed in the upstream EcoRⅠsites upstream and downstream primers SalⅠsites. By EcoRⅠand SalⅠrestriction enzyme digestion treatment Neu PCR product and expression vector recovery pET-28a (+), the Neu gene connected to the pET-28a(+).SDS-PAGE analysis showed that pET-Neu in E. coli Rosetta (DE3) to achieve high expression, Neu protein expressed in the form of inclusion bodies and purified according to literature Neu protein.
Through exploring the digestion conditions, with EcoRⅠand SalⅠwill pMT18-OMP plasmid expression vector pET-28a (+) for restriction enzyme digestion, the OMP5 gene connected to the plasmid pET-28a (+). The products were transferred to E. coli DH5a by PCR and restriction enzyme selection plasmid. Then positive plasmids were transformed into Rosetta (DE3),37℃shaking culture to OD600 of about 0.5 to 0.6 plus IPTG to a final concentration of 1mmol/L to shaking culture 4h. SDS-PAGE analysis showed that pET-OMP5 in E. coli Rosetta (DE3) to achieve high expression, OMP5 protein expressed in the form of inclusion bodies and purified according to literature OMP5 protein.
The recombinant purified Lipo, Neu,Omp5 proteins were divided into two groups, one group with Freund's adjuvant; another group using aluminum hydroxide adjuvant.14 days after the first immunization 21 days to strengthen the immune, immune 3 times in total, each dose of 50μg/only; control group were not immune to any protein, using saline. To lethal HPs and a control group of mice immunized with mouse poison attack. Virus challenge in each experimental group protection was as follows:Neu-Freund's adjuvant group protection was 60%(6/10), Neu-protected aluminum hydroxide adjuvant group was 10%(1/10); OMP5-Freund's adjuvant, the protection rate 30%(3/10), OMP5-protected aluminum hydroxide adjuvant was 11.1%(1/9); Lipo-protection Freund adjuvant was 86.6%(8/9), immune Lipo and the protection of aluminum hydroxide adjuvant group was 50%(5/10); not lethal attack by the immune protein drug control group mice all died in the attack drug; and Immunity protein is not inactivated HPs of the control group injected mice, in mice injected with some of the spirit of malaise within 4h, but soon returned to normal after 4h, the spirit of better. The results showed that these three recombinant proteins can play a protective effect in mice, that Lipo, Neu, OMP5 has certain immunogenic, which Lipo protein was significantly higher than Neu, OMP5, but the results were inferior to inactivated group.
(一)、根据GenBank中的副猪嗜血杆菌序列(登陆号：CP001321.1)设计特异性引物扩增Lipo基因,再将Lipo基因克隆到pMD18-T载体上。以pMDLipo为模板用PCR方法扩增Lipo成熟肽编码区。通过EcoRⅠ和SalⅠ双酶切处理Lipo PCR回收产物与表达载体pBV220,将Lipo基因连接至pBV220中。SDS-PAGE电泳检测显示重组质粒pBV-Lipo在大肠杆菌Rosetta (DE3)中实现高效表达,lipo蛋白为可溶性表达,并通过硫酸铵沉淀纯化lipo蛋白。
(二)、根据GenBank中的副猪嗜血杆菌鸟枪序列(登陆号：NZ-ABKM01000006)设计特异性引物扩增Neu全基因,再将Neu全基因克隆到pMD18-T载体上。以pMDNeu为模板PCR方法扩增Neu部分基因。通过EcoRⅠ和SalⅠ双酶切处理Neu PCR回收产物与表达载体pET-28a(+),将Neu基因连接至pET-28a(+)中。将连接产物转入大肠杆菌DH5α,通过PCR和酶切挑选阳性质粒,再将阳性质粒转入Rosetta (DE3)进行诱导。SDS-PAGE检测显示pET-Neu在大肠杆菌Rosetta (DE3)中实现高效表达, Neu蛋白以包涵体形式表达,用tritonX-100等洗涤包涵体。
(三)、根据GenBank中的副猪嗜血杆菌鸟枪序列(登陆号：NC-011852)设计引物扩增OMP5全基因,再将OMP5全基因克隆到pMD18-T载体上。通过摸索酶切条件,用EcoRⅠ和SalⅠ将pMDOMP5质粒与表达载体pET-28a(+)进行双酶切,将OMP5基因连接至载体质粒pET-28a(+)中。连接产物转入至大肠杆菌DH5α通过PCR和酶切挑选阳性质粒,再将阳性质粒转入Rosetta (DE3)进行诱导。SDS-PAGE检测显示pET-OMP5在大肠杆菌Rosetta (DE3)中实现高效表达,OMP5蛋白以包涵体形式表达,用tritonX-100等洗涤包涵体。
(四)、将重组纯化的Lipo、Neu、Omp5蛋白分别分两组,一组使用弗氏佐剂；另一组使用氢氧化铝佐剂。初次免疫后的14天和21天进行加强免疫,总共免疫3次,每次剂量为50μg/只；对照组未免疫任何蛋白,使用生理盐水。以致死量HPs对免疫小鼠和对照组小鼠进行攻毒。各实验组的攻毒保护结果如下：Neu-弗氏佐剂组的保护率为60%(6/10),Neu-氢氧化铝佐剂组的保护率为10%(1/10)；OMP5-弗氏佐剂组的保护率为30%(3/10),OMP5-氢氧化铝佐剂组的保护率为11.1%(1/9); Lipo-弗氏佐剂组的保护率为86.6%(8/9),免疫Lipo和氢氧化铝佐剂组的保护率为50%(5/10)；未免疫蛋白采用致死量攻毒的对照组小鼠,在攻毒后全部死亡；而未免疫蛋白注射灭活HPs的对照组小鼠,在注射4h内部分小鼠精神萎靡,但4h后很快恢复正常,精神转好。结果显示了这三个重组蛋白都能对小鼠起到保护作用,证明Lipo、Neu、OMP5都具有一定的免疫源性,其中Lipo蛋白明显高于Neu、OMP5,但是效果均不如灭活组。
Haemophilus parasuis (H.parasuis) is a Gram-negative bacterium belonging to the Pasteurellaceae family. The H. parasuis can cause the Glasser's disease which is mainly characterzed by polyserositis,arthritis and meningitis. H. parasuis usually affects young pigs from 2 weeks to 4 months in the nursery.The incidence rate of H. parasuis is generally from 10% to 15%.when serious illness come the mortality of pigs are up to 50%. H. parasuis leads to increasing economic losses in the swine industry worldwide.Control of H. parasuis infections can be achieved by use of vaccination using bacterins,but a major variability has been reported when testing the development of cross-protection,depending on H. parasuis strains and serotypes.With the rapid development of molecular biology techniques,vaccine reseach has turn to genetic engineering vaccine.Prokaryotic expression of Neu、Lipo and OMP5 gene of H.parasuis and analysis of immunogenicity of the expressed protein.
According to GenBank in Haemophilus parasuis shotgun sequence, the part of Lipo gene was amplified from pMT18-Lipo by PCR with a pair of specific primers which primers designed in the upstream EcoRⅠsites upstream and downstream primers SalⅠsites. By EcoRⅠand SalⅠrestriction enzyme digestion treatment Lipo PCR product and expression vector recovery pBV220, the Lipo gene connected to the pBV220. SDS-PAGE electrophoresis analysis showed that the recombinant plasmid pBV-Lipo in E. coli Rosetta (DE3) to achieve high expression, lipo soluble protein expression and purification of lipo protein by ammonium sulfate precipitation.
According to GenBank in Haemophilus parasuis shotgun sequence, the part of Neu gene was amplified from pMT18-Neu by PCR with a pair of specific primers which primers designed in the upstream EcoRⅠsites upstream and downstream primers SalⅠsites. By EcoRⅠand SalⅠrestriction enzyme digestion treatment Neu PCR product and expression vector recovery pET-28a (+), the Neu gene connected to the pET-28a(+).SDS-PAGE analysis showed that pET-Neu in E. coli Rosetta (DE3) to achieve high expression, Neu protein expressed in the form of inclusion bodies and purified according to literature Neu protein.
Through exploring the digestion conditions, with EcoRⅠand SalⅠwill pMT18-OMP plasmid expression vector pET-28a (+) for restriction enzyme digestion, the OMP5 gene connected to the plasmid pET-28a (+). The products were transferred to E. coli DH5a by PCR and restriction enzyme selection plasmid. Then positive plasmids were transformed into Rosetta (DE3),37℃shaking culture to OD600 of about 0.5 to 0.6 plus IPTG to a final concentration of 1mmol/L to shaking culture 4h. SDS-PAGE analysis showed that pET-OMP5 in E. coli Rosetta (DE3) to achieve high expression, OMP5 protein expressed in the form of inclusion bodies and purified according to literature OMP5 protein.
The recombinant purified Lipo, Neu,Omp5 proteins were divided into two groups, one group with Freund's adjuvant; another group using aluminum hydroxide adjuvant.14 days after the first immunization 21 days to strengthen the immune, immune 3 times in total, each dose of 50μg/only; control group were not immune to any protein, using saline. To lethal HPs and a control group of mice immunized with mouse poison attack. Virus challenge in each experimental group protection was as follows:Neu-Freund's adjuvant group protection was 60%(6/10), Neu-protected aluminum hydroxide adjuvant group was 10%(1/10); OMP5-Freund's adjuvant, the protection rate 30%(3/10), OMP5-protected aluminum hydroxide adjuvant was 11.1%(1/9); Lipo-protection Freund adjuvant was 86.6%(8/9), immune Lipo and the protection of aluminum hydroxide adjuvant group was 50%(5/10); not lethal attack by the immune protein drug control group mice all died in the attack drug; and Immunity protein is not inactivated HPs of the control group injected mice, in mice injected with some of the spirit of malaise within 4h, but soon returned to normal after 4h, the spirit of better. The results showed that these three recombinant proteins can play a protective effect in mice, that Lipo, Neu, OMP5 has certain immunogenic, which Lipo protein was significantly higher than Neu, OMP5, but the results were inferior to inactivated group.
引文
[1]Nicolet, J.1986. Haemophilus infection, p.426-436. In A. D. Leman et al.(ed.), Diseases of swine,6th ed. Iowa State University Press, Ames, Iowa.
[2]Little T.W.A. (1970):Haemophilus parasuis infection in pigs. Veterinary Record,87, 399-402.
[3]Peet R.L., Fry J., Lloyd J., Henderson J., Curran J., Moir D. (1983):Haemophilus parasuis septicemia in pigs. Australian Veterinary Journal,60,187
[4]Oliveira S, Batista L, TorremorellM, et al. Experimental colonization of piglets and giltswith systemic strains of Haem ophilus parasuis and streptococcus suis to p revent disease. Can J Vet Res,2001,65 (3):161-167
[5]Little T.W.A. (1970):Haemophilus parasuis infection in pigs. Veterinary Record,87, 399-402.
[6]Lewis P.A., Shope R.E. (1931):Swine influenza. Ⅱ. Haemophilic bacillus from the respiratory tract of infected swine. Journal of Experimental Medicine,54,361-371.
[7]Biberstein E.L., White D.C. (1969):A proposal for the establishment of two new Haemophilus species. Journal of Medical Microbiology,2,75-77.
[8]Morozumi, T., Nicolet, J.,1986. Morphological variations of Haemophilus parasuis. J. Clin. Microbiol.23,138-142.
[9]Pohl, S.,1981. DNA relatedness among members of Haemophilus, Pasteurella and Actinobacillus. In:Killian, M., Frederiksen, W., Biberstein, E.L. (Eds.), Haemophilus, Pasteurella and Actinobacillus. Academic Press, London, pp.245-253.
[10]De Ley, J., Mannheim, W., Mutters, R., Piechulla, K., Tytgat,R., Segers, P., Bisgard, M., Frederiksen, W., Hinz, K.M.,Vanhoucke, M.,1990. Inter-and intrafamilial similarities of rRNA cistrins of the Pasteurellaceae. Int. J. Syst. Bacteriol.39,258-266.
[11]Dewhirst, F.E., Paster, B.J., Olsen, I., Fraser, G.J.,1992. Phylogeny of 54 representative strains of species in the family Pasterurellaceae as determined by comparison of 16S rRNA sequences. J. Bacteriol.174,2002-2013.
[12]Dewhirst, F.E., Paster, B.J., Olsen, I., Fraser, G.J.,1992.Phylogeny of 54 representative strains of species in the familyPasterurellaceae as determined by comparison of 16S rRNA sequences. J. Bacteriol.174,2002-2013.
[13]Kielstein, P., Wuthe, H., Angen, O., Mutters, R., Ahrens, P.,2001. Phenotypic and genetic characterization of NAD-dependent Pasteurellaceae from the respiratory tract of pigs and their possible pathogenetic importance. Vet. Microbiol.81,243-255.
[14]Morozumi, T., Pauki, U.R.S., Braun, R., Nicolet, J.,1986. Deoxyribonucleic acid relatedness among strains of Haemophilus parasuis and other Haemophilus spp. of swine. Int. J. Syst. Bacteriol.36,17-19.
[15]Moller, K., Fussing, V., Grimont, P.A.D., Paster, B.J., Dewhirst, F.E., Kilian, M. Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Actinobacillus indolicussp. nov., three new V-factor dependent species from the respiratory tract of pigs. Int. J. Syst. Bacteriol.1996,46,951-956.
[16]Nicolet J. (1992):Haemophilus parasuis. In:Diseases of Swine.7th ed. Iowa State University.526-528.
[17]Straw BE,Allaire SD,Meigeling WL,Taylor DJ.Diseases of swine.8th Edition.1999,475-481
[18]Harris DL, Ross RF, Switzer WP:1969, Incidence of certain microorganisms in nasal cavities of swine in Iowa. Am J Vet Res 30:1621-1624.
[19]Rapp-Gabrielson VJ, Gabrielson DA:1992, Prevalence ofHaemophilus parasuis serovars among isolates from swine. Am J Vet Res 53:659-664.
[20]Oliveira S., Batista L., Torremorell M., Pijoan C. (2001):Experimental colonization of piglets and gilts with systemic strains of Haemophilus parasuis and Streptococcus suis to prevent disease. Canadian Journal of Veterinary Research,65,161-167.
[21]Kielstein P, Leirer R:1990, Zur glasserschen Krankheit des Schweines-atiologisch-epizootiologische Untersuchungen zum Erregerspektrum. Mh Vet-Med 45:577-582.
[22]Kielstein P,Wuthe H,Angen O,Mutters R,Ahrens P.Phenotypic and genetic characterization of NAD-dependent Pasteurellanceae from the respiratory tract of pigs and their possible pathogenetic importance.Vet Microbiol,2001,81:243-255.
[23]Kielstein, P., and V. J. Rapp-Gabrielson.1992. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. J. Clin. Microbiol.30:862-865.
[24]Moller, K., Kilian, M. (1990):V-factor dependent members of the family Pasteurellaceae in the porcine upper respiratory tract. Journal of Clinical Microbiology,28,2711-2716.
[25]Kielstein P., Rapp-Gabrielson V.J. (1992):Designation of 15 serovars of. Haemophilus parasuis based immunodifusion using heatstable antigen extracts. Journal of Clinical Microbiology,30,862-865.
[26]Rapp-Gabrielson V.J., Gabrielson D.A. (1992):Prevalence of Haemophilus parasuis serovars among isolates from swine. American Journal of Veterinary Research,53, 951-956.
[27]Rubies X., Kielstein P., Costs L.I., Riera P., Artigas C., Espuna E. (1999):Prevalence of Haemophilus parasuis serovars isolated in Spain from 1993 to 1997. Veterinary Microbiology,66,245-248.
[28]Tadjine M., Mittal K.R., Bourdon S., Gottschalk M. (2004):Development of new serological test for serotyping Haemophilus parasuis isolates and determination of their prevalence in North America. Journal of Clinical Microbiology,42,839-840.
[29]Cai X., Chen H., Blackall P.J., Yin Z., Wang L., Liu Z., Jin M. (2005):Serological characterization of Haemophilus parasuis isolates from China. Veterinary Microbiology, 111,231-236.
[30]Rafiee M., Bara M., Stephens C.P., Blackall P.J. (2000):Application of ERIC-PCR for the comparison of isolates of Haemophilus parasuis. Australian Veterinary Journal,78, 846-849.
[31]Angen O., Svensmark B., Mittal K.R. (2004):Serological characterization of Danish Haemophilus parasuis isolates. Veterinary Microbiology,103,255-258.
[32]Oliveira S, Blackall P J, Pijoan C. Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping [J].Am J Vet Res,2003,64:435-442.
[33]Min Yue, Fan Yang, Jian Yang. Complete Genome Sequence of Haemophilus parasuis SH0165. Journal of Bacteriology, Feb.2009, p.1359-1360
[34]Rafiee, M., Blackall, P.J.,2000. Establishment, validation and use of the Kielstein-Rapp-Gabrielson serotyping scheme for Haemophilus parasuis. Aust. Vet. J.78, 172-174.
[35]A. Olvera, M. Calsamiglia, and V. Aragon.2006, Genotypic Diversity of Haemophilus parasuis Field Strains. Applied and environmental microbiology, June 2006, p.3984-3992
[36]Del Rio M L,Gutierrez B,Rodriguez Ferri E F.Value of indirect hemagglutination and coagglutination tests for serotyping Haemophilus parasuis.J Clin Microbiol,2003,41:880-882
[37]Tadine M,Mittal K P,Bourdon S, GottsehalkM, GottsehalkM.Development of a new serological test for seroytping Haemophilus parasuis isolates and determination of their prevalence in North America. J Clin Microbiol,2004,42:839-840
[38]Smart, N.L., Miniats, O.P., McInnes, J.I.,1988. Analysis of Haemophilus parasuis isolates from southern Ontario swine by restriction endonuclease fingerprinting. Can. J. Vet. Res. 52,319-324.
[39]Smart, N.L., Hurnik, D., MacInnes, J.I.,1993. An investigation of enzootic Glasser's disease in a specific pathogen-free grower-finisher facility using restriction endonuclease analysis.Can. Vet. J.34,487-490.
[40]Blackall, P.J., Trott, D.J., Rapp-Gabrielson, V., Hampson, D.J.,1997. Analysis of Haemophilus parasuis by multilocus enzyme electrophoresis. Vet. Microbiol.56, 125-134.
[41]Rafiee, M., Bara, M., Stephens, C.P., Blackall, P.J.,2000.Application of ERIC-PCR for. the comparison of isolates of Haemophilus parasuis. Aust. Vet. J.78,846-849.
[42]De la Puente Redondo, V.A., Me'ndez, J.N., Blanco, N.G., Boronat, N.L., Mart'n, C.B.G., Ferri, E.F.R.,2003. Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene. Veterinary Microbiology 2511,1-10.
[43]Lin, B.,2003. Identification and differentiation of Haemophilus parasuissero-nontypeable strains using a species specific PCR and the digestion of PCR products with Hindlll endonuclease. In:Proceedings of the American Association of Swine Veterinarians Annual Meeting, Orlando, FL, USA, p.299.
[44]Del Rio, M.L., Martin, C.B., Navas, J., Gutierrez-Muniz, B., Rodriguez-Barbosa, J.I., Rodriguez Ferri, E. F.,2006. aroA gene PCR-RFLP diversity patterns in Haemophilus parasuis and Actinobacillus species. Research in Veterinary Science 80, 55-61.
[45]RedondoVA, Mendez J N, Blnaeo N G, Bornat N L, Mart C B, Ferri E F.Typing of Haempohlius parasuis strain byPCR-RFLP nalysis of the tbpA gene. Vet Mierobiol,2003, 2511:1-10
[46]Jun-xing Li, Ping Jiang. Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens. Preventive Veterinary Medicine 91 (2009) 274-279
[47]李郁,陈申秒,王桂军等.副猪嗜血杆菌安徽分离株的致病性与RELP基因型的相关性研究[J].中国预防兽医学报.第31卷第8期
[48]Alex Olvera, Marta Cerda'-Cue'llar, Virginia Aragon. Study of the population structure of Haemophilus parasuis by multilocus sequence typing. Microbiology (2006),152, 3683-36house-keeping gene
[49]Stackebrandt, E. G., and B. M. Goebel.1994. Taxonomic note:a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol.44:846-849.
[50]蔡旭旺,刘正飞,陈焕春等.副猪嗜血杆菌的分离培养和血清型鉴定[J].华中农业大学学报,2005,24(1)：55-58.
[51]尹秀凤,姜平等.副猪嗜血杆菌的分离与鉴定[J]. Animal Husbandry & Veterinary Medicine.2004,Vol.36 No.7
[52]John L. Vahle, Joseph S. Haynes, John J. Andrews 1995 Experimental reproduction of Haemophilus parasuis infection in swine:clinical, bacteriologic, and morphologic findings. J Vet Diagn Invest 7:476-480 (1995)
[53]Ghyslaine Vanier, Anna Szczotka, Peter Friedl. Haemophilus parasuis invades porcine brain microvascular endothelial cells. Microbiology,2006,152,135-142
[54]Narita M,Kaeashima K,Matsuura S.Pneumonia in pigs infected with pseudorabies virus ans Haemophilus parasuis serovar 4.J Comp Pathol,1994,110:329-339
[55]Solano GI,Bautista,E.Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis. Vet Micribiol.1997,55:247-257
[56]VahleJ L, Hyanes J S, Anderws J J.Interaction of Haemophilus parasuis with nasal and tracheal mueosa of fllowing intarnasa inoculation of cesarean and derived colostrum deprived (CDCD) swine.Can J Vet Res,1997,61:200.206
[57]Amano H, Shibata M, Kajio N, Mooruzmi T. Pathologic observations of pigs intranasally inoeulated with serovarl,4 and 5 of Haemophilus Parasuis using immunoperoxidase method.J Vet Med Sci,1994,56:639-644
[58]Segales J, Domingo M, Solnao G I, pjioan C. Immunohistochemical deteetion of Haempohilus parasuis serovar 5 in formalin-fixed, paraffin-embedded tissues of experimentally inefeted swine.J Vet Digan Invest,1997,9:237-243
[59]Kirkwood R N, Rawluk S A, Cegielske A C, Otto A J. Eeffet of Pig age and autogenous sow vaceination on nasal mucosal colonization of pigs by Haempohilus parasuis.J Swine Health Prod,2001,9:77-79
[60]Olvera, A., M. Cerda'-Cue'liar, M. Nofrarias, E. Revilla, J. Segale's, and V.Aragon.2007. Dynamics of Haemophilus parasuis genotypes in a farm recovered from an outbreak of Glasser's disease. Vet. Microbiol.123:230-237.
[61]Hoefling D.C.The various forms of Haemophilus parasuis. Journal of Swine Health Production.1994.2,1
[62]Rapp-Gabrielson V.J.Heamophilus parasuis.In:Diseases of Swine.8th ed.Low State University,PP.475-482
[63]Peet R.L., Fry J., Lloyd J., Henderson J., Curran J., Moir D. Haemophilus parasuis septicemia in pigs. Australian Veterinary Journal1983,60,187.
[64]Vahle J.L., Haynes J.S., Andrews J.J. Interaction of Haemophilus parasuis with nasal and tracheal ucosa following intranasal inoculation of cesarean-derived colostrum deprived (CDCD) swine. Canadian Journal of Veterinary Research.1997,61,200-206.
[65]Nielsen R.Pathogenicity and immunity studies of Haemophilus parasuis serotypes. Acta Vet Scand,1993,34:193-198
[66]Takahashi K,Nagai S,Yagihashi T.A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovar 2 ans 5 bacterins,and evaluation of a bivalent vaccine under laboratory and field conditions.J Vet Med Sci,2001,63:487-491
[67]Solnano-Aguilar G I,Pjiona C, Rapp-Gabrielson V, Collins J, Cavralho L. Protective role of maternal antibodies against Haemophilus parasuis inefetion.Am J Vet Res.1999,60:81-87
[68]Miniats O P,Smart N L,Ewert E.Vaccination of gnotobiotic primary specific pathofen-free pigs anainsr Haemophilus parasuis.Can J Vet Res,1991,55:33-36
[69]Miniats O P,Smart N L,Rosendal S.Cross protection among Haemophilus parasuisstrains in immunized gnotobiotic pigs. Can J Vet Res,1991,55:37-41
[70]王艳,夏万田.副猪嗜血杆菌间接EL ISA抗体检测方法的建立.畜牧与兽医,2006.第38卷第3期
[71]王老七,陶海静.副猪嗜血杆菌间接血凝诊断液的制备.兽医畜牧科学.2007,Vo123,No2
[72]冯小明,储岳峰.副猪嗜血杆菌抗体间接ELISA检测方法的建立与应用.中国兽医科学.2009,39(07)：597-60
[73]魏子贡,蔡旭旺.副猪嗜血杆菌抗体间接血凝检测方法的建立及应用.中国兽医科学.2006,36(09)：7132718
[74]石碧,崔耀文.副猪嗜血杆菌血清5型间接血凝试验和间接ELISA抗体检测方法的建立.中国兽医科学2007,37(11)：964-96
[75]Simone Oliveira, Lucina Galina. Development of a PCR test to diagnose Haemophilus parasuis infections.2001, J Vet Diagn Invest 13:495-501
[76]Kwonil Jung,Yoonchul H A.Development of polymerase Chain Reaction and Comparison with In situ Hybridization for the Detection of Haemophilus paraduis in Formalin-Fixed,Paraffin-Embedded Tissues.2004,J.Vet.Med.Sci.66(7):841-845
[77]万世平,王建.副猪嗜血杆菌PCR检测方法的建立与初步应用.动物医学进展,2009,30(1)：9212
[78]尹秀凤,王艳.副猪嗜血杆菌PCR检测方法的建立与应用.中国兽医学报,2007,第27卷第2期
[79]张盼锋,仇微.副猪嗜血杆菌PCR快速诊断方法的建立.广东畜牧兽医科技2009年(第34卷)第1期
[80]Kielstein P,Rosner R.Zur Glasserchen Krankheit des Schweines-Atioligisch-epizootiologische Untersuchungen zum Erregerspektrum.Mh Vet Med,1990,45:577-582
[81]Xueli Zhou, Xiaojuan Xu. Distribution of antimicrobial resistance among different serovars of Haemophilus parasuis isolates. Vet. Microbiol.2009.05.012
[82]Clark L K,Hill M A.An evaluation of the components of medicated early weaning.Swine Health and Production,1994,2:5-11
[83]Baunann G,Valenza F,Effect of vaccinating sows and their piglets on the development of Glassers disease induced by a virulent strain of Haemophilus parasuis serovar 5. Vet Rec,2002,151:18-21
[84]Smart N L,Miniats O P,Rosendal S,Frindship P.Glassers disease and prevalence of subclinical infection with Haemophilus parasuis in swine in southern Ontario.Can Vet J,1989,30:339-343
[85]Rapp-Gabrielson V J,Kocus G J,Haemophilus parasuis immunity in swine following vaccination.Vet Med,1997,92:83-90
[86]Ruiz A,Simone Oliveira.Outer Membrane Proteins and DNA Profiles in Strains of Haemophihlus parasuis Recovered from Systemic and Respiratory Site.2001,Journal of Clinical Microbiology,May 2001, JCM.39.5.1757-1762
[87]Oliveira S,Pijoan C.Computer-based analysis of Haemophilus parasuis protein fingerprints.Canadian Journal of Veterinary Research,2004,68:161-167
[88]A.J.Martin de la Fuente,E.F.Rodriguez-Ferri,R.Frandoloso.Systemic antibody response in colostrum-deprived pigs experimentally infexted with Haemophilus paraduis.Research in Veterinary Science 2009,86:248-253
[89]A.J.Martin de la Fuente,E.F.Rodriguez-Ferri,R.Frandoloso.Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis. Research in Veterinary Science.2009,86:230-234
[90]Tadjine, M., Mittal, K.R., Bourdon, S., Gottschalk, M.. Production and characterization of murine monoclonal antibodies against Haemophilus parasuis and study of their protection role in mice. Microbiology,2004,150,3935-3945.
[91]Mingguang Zhoub, Yi Guo, Jianping Zhao. Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5. Vaccine 27 (2009) 5271-5277
[92]Michael A. Mullins, Karen B. Register, Darrell O. Bayles. Characterization and Comparative Analysis of the Genes Encoding Haemophilus parasuis Outer Membrane Proteins P2 and P5. Journal of Bacteriology.2009, p.5988-6002
[93]Bin Zhang, Cheng Tang. Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis. Veterinary Microbiology.2009,136: 408-410
[94]Alex Olveral, Sonia Pina. Virulence associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo. Vet. Res.2010,41:26
[95]Sonia Pina, Alex Olvera. Trimeric Autotransporters of Haemophilus parasuis:Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains. Journal of Bacteriology, Jan.2009, p.576-587
[96]李军星,姜平.副猪嗜血杆菌热休克蛋白70基因的序列分析及抗原性鉴定.中国预防兽医学报.2009,31(7)
[97]Charlnad N,D'Silva C QDumont R A,et al.Contact-dependent acquisition of transferring-bound iron by two strains of Haemophilus parasuis.Can J Microbiol.1995,41:70-74
[98]Lichtensteiger C A, Vimr E R. Neuraminidase (sialidase) activity of Haemophilus parasuis [J]. FEMS Microbiol Lett,1997,152:269-274.
[99]朱小宁,余兴龙,李润成等.副猪嗜血杆菌的分离与鉴定及其16S rRNA生物信息学分析[J].湖南农业大学学报,2009.
[100]萨姆布鲁克著.黄培堂等译.分子克隆实验指南(第三版)[M].北京：科学出版社,2002.
[101]颜真,张英起蛋白质研究技术[M].第四军医大学出版社.27-35
[102]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2000,42-46.
[103]Simone Oliveira, Carlos Pijoanl. Haemophilus parasuis:new trends on diagnosis, epidemiology and control.Veterinary Microbiology.2004.99,1-12
[104]熊盛,王一飞.BALB/C小鼠对不同佐剂SARS灭活疫苗的早期免疫反应.中国免疫学杂志.2004,第20卷,413-417
[105]高鹏程,储岳峰.副猪嗜血杆菌对部分常用实验动物的致病性试验.畜牧兽医学报.2009,40(1)：138-141
[2]Little T.W.A. (1970):Haemophilus parasuis infection in pigs. Veterinary Record,87, 399-402.
[3]Peet R.L., Fry J., Lloyd J., Henderson J., Curran J., Moir D. (1983):Haemophilus parasuis septicemia in pigs. Australian Veterinary Journal,60,187
[4]Oliveira S, Batista L, TorremorellM, et al. Experimental colonization of piglets and giltswith systemic strains of Haem ophilus parasuis and streptococcus suis to p revent disease. Can J Vet Res,2001,65 (3):161-167
[5]Little T.W.A. (1970):Haemophilus parasuis infection in pigs. Veterinary Record,87, 399-402.
[6]Lewis P.A., Shope R.E. (1931):Swine influenza. Ⅱ. Haemophilic bacillus from the respiratory tract of infected swine. Journal of Experimental Medicine,54,361-371.
[7]Biberstein E.L., White D.C. (1969):A proposal for the establishment of two new Haemophilus species. Journal of Medical Microbiology,2,75-77.
[8]Morozumi, T., Nicolet, J.,1986. Morphological variations of Haemophilus parasuis. J. Clin. Microbiol.23,138-142.
[9]Pohl, S.,1981. DNA relatedness among members of Haemophilus, Pasteurella and Actinobacillus. In:Killian, M., Frederiksen, W., Biberstein, E.L. (Eds.), Haemophilus, Pasteurella and Actinobacillus. Academic Press, London, pp.245-253.
[10]De Ley, J., Mannheim, W., Mutters, R., Piechulla, K., Tytgat,R., Segers, P., Bisgard, M., Frederiksen, W., Hinz, K.M.,Vanhoucke, M.,1990. Inter-and intrafamilial similarities of rRNA cistrins of the Pasteurellaceae. Int. J. Syst. Bacteriol.39,258-266.
[11]Dewhirst, F.E., Paster, B.J., Olsen, I., Fraser, G.J.,1992. Phylogeny of 54 representative strains of species in the family Pasterurellaceae as determined by comparison of 16S rRNA sequences. J. Bacteriol.174,2002-2013.
[12]Dewhirst, F.E., Paster, B.J., Olsen, I., Fraser, G.J.,1992.Phylogeny of 54 representative strains of species in the familyPasterurellaceae as determined by comparison of 16S rRNA sequences. J. Bacteriol.174,2002-2013.
[13]Kielstein, P., Wuthe, H., Angen, O., Mutters, R., Ahrens, P.,2001. Phenotypic and genetic characterization of NAD-dependent Pasteurellaceae from the respiratory tract of pigs and their possible pathogenetic importance. Vet. Microbiol.81,243-255.
[14]Morozumi, T., Pauki, U.R.S., Braun, R., Nicolet, J.,1986. Deoxyribonucleic acid relatedness among strains of Haemophilus parasuis and other Haemophilus spp. of swine. Int. J. Syst. Bacteriol.36,17-19.
[15]Moller, K., Fussing, V., Grimont, P.A.D., Paster, B.J., Dewhirst, F.E., Kilian, M. Actinobacillus minor sp. nov., Actinobacillus porcinus sp. nov., and Actinobacillus indolicussp. nov., three new V-factor dependent species from the respiratory tract of pigs. Int. J. Syst. Bacteriol.1996,46,951-956.
[16]Nicolet J. (1992):Haemophilus parasuis. In:Diseases of Swine.7th ed. Iowa State University.526-528.
[17]Straw BE,Allaire SD,Meigeling WL,Taylor DJ.Diseases of swine.8th Edition.1999,475-481
[18]Harris DL, Ross RF, Switzer WP:1969, Incidence of certain microorganisms in nasal cavities of swine in Iowa. Am J Vet Res 30:1621-1624.
[19]Rapp-Gabrielson VJ, Gabrielson DA:1992, Prevalence ofHaemophilus parasuis serovars among isolates from swine. Am J Vet Res 53:659-664.
[20]Oliveira S., Batista L., Torremorell M., Pijoan C. (2001):Experimental colonization of piglets and gilts with systemic strains of Haemophilus parasuis and Streptococcus suis to prevent disease. Canadian Journal of Veterinary Research,65,161-167.
[21]Kielstein P, Leirer R:1990, Zur glasserschen Krankheit des Schweines-atiologisch-epizootiologische Untersuchungen zum Erregerspektrum. Mh Vet-Med 45:577-582.
[22]Kielstein P,Wuthe H,Angen O,Mutters R,Ahrens P.Phenotypic and genetic characterization of NAD-dependent Pasteurellanceae from the respiratory tract of pigs and their possible pathogenetic importance.Vet Microbiol,2001,81:243-255.
[23]Kielstein, P., and V. J. Rapp-Gabrielson.1992. Designation of 15 serovars of Haemophilus parasuis on the basis of immunodiffusion using heat-stable antigen extracts. J. Clin. Microbiol.30:862-865.
[24]Moller, K., Kilian, M. (1990):V-factor dependent members of the family Pasteurellaceae in the porcine upper respiratory tract. Journal of Clinical Microbiology,28,2711-2716.
[25]Kielstein P., Rapp-Gabrielson V.J. (1992):Designation of 15 serovars of. Haemophilus parasuis based immunodifusion using heatstable antigen extracts. Journal of Clinical Microbiology,30,862-865.
[26]Rapp-Gabrielson V.J., Gabrielson D.A. (1992):Prevalence of Haemophilus parasuis serovars among isolates from swine. American Journal of Veterinary Research,53, 951-956.
[27]Rubies X., Kielstein P., Costs L.I., Riera P., Artigas C., Espuna E. (1999):Prevalence of Haemophilus parasuis serovars isolated in Spain from 1993 to 1997. Veterinary Microbiology,66,245-248.
[28]Tadjine M., Mittal K.R., Bourdon S., Gottschalk M. (2004):Development of new serological test for serotyping Haemophilus parasuis isolates and determination of their prevalence in North America. Journal of Clinical Microbiology,42,839-840.
[29]Cai X., Chen H., Blackall P.J., Yin Z., Wang L., Liu Z., Jin M. (2005):Serological characterization of Haemophilus parasuis isolates from China. Veterinary Microbiology, 111,231-236.
[30]Rafiee M., Bara M., Stephens C.P., Blackall P.J. (2000):Application of ERIC-PCR for the comparison of isolates of Haemophilus parasuis. Australian Veterinary Journal,78, 846-849.
[31]Angen O., Svensmark B., Mittal K.R. (2004):Serological characterization of Danish Haemophilus parasuis isolates. Veterinary Microbiology,103,255-258.
[32]Oliveira S, Blackall P J, Pijoan C. Characterization of the diversity of Haemophilus parasuis field isolates by use of serotyping and genotyping [J].Am J Vet Res,2003,64:435-442.
[33]Min Yue, Fan Yang, Jian Yang. Complete Genome Sequence of Haemophilus parasuis SH0165. Journal of Bacteriology, Feb.2009, p.1359-1360
[34]Rafiee, M., Blackall, P.J.,2000. Establishment, validation and use of the Kielstein-Rapp-Gabrielson serotyping scheme for Haemophilus parasuis. Aust. Vet. J.78, 172-174.
[35]A. Olvera, M. Calsamiglia, and V. Aragon.2006, Genotypic Diversity of Haemophilus parasuis Field Strains. Applied and environmental microbiology, June 2006, p.3984-3992
[36]Del Rio M L,Gutierrez B,Rodriguez Ferri E F.Value of indirect hemagglutination and coagglutination tests for serotyping Haemophilus parasuis.J Clin Microbiol,2003,41:880-882
[37]Tadine M,Mittal K P,Bourdon S, GottsehalkM, GottsehalkM.Development of a new serological test for seroytping Haemophilus parasuis isolates and determination of their prevalence in North America. J Clin Microbiol,2004,42:839-840
[38]Smart, N.L., Miniats, O.P., McInnes, J.I.,1988. Analysis of Haemophilus parasuis isolates from southern Ontario swine by restriction endonuclease fingerprinting. Can. J. Vet. Res. 52,319-324.
[39]Smart, N.L., Hurnik, D., MacInnes, J.I.,1993. An investigation of enzootic Glasser's disease in a specific pathogen-free grower-finisher facility using restriction endonuclease analysis.Can. Vet. J.34,487-490.
[40]Blackall, P.J., Trott, D.J., Rapp-Gabrielson, V., Hampson, D.J.,1997. Analysis of Haemophilus parasuis by multilocus enzyme electrophoresis. Vet. Microbiol.56, 125-134.
[41]Rafiee, M., Bara, M., Stephens, C.P., Blackall, P.J.,2000.Application of ERIC-PCR for. the comparison of isolates of Haemophilus parasuis. Aust. Vet. J.78,846-849.
[42]De la Puente Redondo, V.A., Me'ndez, J.N., Blanco, N.G., Boronat, N.L., Mart'n, C.B.G., Ferri, E.F.R.,2003. Typing of Haemophilus parasuis strains by PCR-RFLP analysis of the tbpA gene. Veterinary Microbiology 2511,1-10.
[43]Lin, B.,2003. Identification and differentiation of Haemophilus parasuissero-nontypeable strains using a species specific PCR and the digestion of PCR products with Hindlll endonuclease. In:Proceedings of the American Association of Swine Veterinarians Annual Meeting, Orlando, FL, USA, p.299.
[44]Del Rio, M.L., Martin, C.B., Navas, J., Gutierrez-Muniz, B., Rodriguez-Barbosa, J.I., Rodriguez Ferri, E. F.,2006. aroA gene PCR-RFLP diversity patterns in Haemophilus parasuis and Actinobacillus species. Research in Veterinary Science 80, 55-61.
[45]RedondoVA, Mendez J N, Blnaeo N G, Bornat N L, Mart C B, Ferri E F.Typing of Haempohlius parasuis strain byPCR-RFLP nalysis of the tbpA gene. Vet Mierobiol,2003, 2511:1-10
[46]Jun-xing Li, Ping Jiang. Genotyping of Haemophilus parasuis from diseased pigs in China and prevalence of two coexisting virus pathogens. Preventive Veterinary Medicine 91 (2009) 274-279
[47]李郁,陈申秒,王桂军等.副猪嗜血杆菌安徽分离株的致病性与RELP基因型的相关性研究[J].中国预防兽医学报.第31卷第8期
[48]Alex Olvera, Marta Cerda'-Cue'llar, Virginia Aragon. Study of the population structure of Haemophilus parasuis by multilocus sequence typing. Microbiology (2006),152, 3683-36house-keeping gene
[49]Stackebrandt, E. G., and B. M. Goebel.1994. Taxonomic note:a place for DNA-DNA reassociation and 16S rRNA sequence analysis in the present species definition in bacteriology. Int. J. Syst. Bacteriol.44:846-849.
[50]蔡旭旺,刘正飞,陈焕春等.副猪嗜血杆菌的分离培养和血清型鉴定[J].华中农业大学学报,2005,24(1)：55-58.
[51]尹秀凤,姜平等.副猪嗜血杆菌的分离与鉴定[J]. Animal Husbandry & Veterinary Medicine.2004,Vol.36 No.7
[52]John L. Vahle, Joseph S. Haynes, John J. Andrews 1995 Experimental reproduction of Haemophilus parasuis infection in swine:clinical, bacteriologic, and morphologic findings. J Vet Diagn Invest 7:476-480 (1995)
[53]Ghyslaine Vanier, Anna Szczotka, Peter Friedl. Haemophilus parasuis invades porcine brain microvascular endothelial cells. Microbiology,2006,152,135-142
[54]Narita M,Kaeashima K,Matsuura S.Pneumonia in pigs infected with pseudorabies virus ans Haemophilus parasuis serovar 4.J Comp Pathol,1994,110:329-339
[55]Solano GI,Bautista,E.Effect of porcine reproductive and respiratory syndrome virus infection on the clearance of Haemophilus parasuis. Vet Micribiol.1997,55:247-257
[56]VahleJ L, Hyanes J S, Anderws J J.Interaction of Haemophilus parasuis with nasal and tracheal mueosa of fllowing intarnasa inoculation of cesarean and derived colostrum deprived (CDCD) swine.Can J Vet Res,1997,61:200.206
[57]Amano H, Shibata M, Kajio N, Mooruzmi T. Pathologic observations of pigs intranasally inoeulated with serovarl,4 and 5 of Haemophilus Parasuis using immunoperoxidase method.J Vet Med Sci,1994,56:639-644
[58]Segales J, Domingo M, Solnao G I, pjioan C. Immunohistochemical deteetion of Haempohilus parasuis serovar 5 in formalin-fixed, paraffin-embedded tissues of experimentally inefeted swine.J Vet Digan Invest,1997,9:237-243
[59]Kirkwood R N, Rawluk S A, Cegielske A C, Otto A J. Eeffet of Pig age and autogenous sow vaceination on nasal mucosal colonization of pigs by Haempohilus parasuis.J Swine Health Prod,2001,9:77-79
[60]Olvera, A., M. Cerda'-Cue'liar, M. Nofrarias, E. Revilla, J. Segale's, and V.Aragon.2007. Dynamics of Haemophilus parasuis genotypes in a farm recovered from an outbreak of Glasser's disease. Vet. Microbiol.123:230-237.
[61]Hoefling D.C.The various forms of Haemophilus parasuis. Journal of Swine Health Production.1994.2,1
[62]Rapp-Gabrielson V.J.Heamophilus parasuis.In:Diseases of Swine.8th ed.Low State University,PP.475-482
[63]Peet R.L., Fry J., Lloyd J., Henderson J., Curran J., Moir D. Haemophilus parasuis septicemia in pigs. Australian Veterinary Journal1983,60,187.
[64]Vahle J.L., Haynes J.S., Andrews J.J. Interaction of Haemophilus parasuis with nasal and tracheal ucosa following intranasal inoculation of cesarean-derived colostrum deprived (CDCD) swine. Canadian Journal of Veterinary Research.1997,61,200-206.
[65]Nielsen R.Pathogenicity and immunity studies of Haemophilus parasuis serotypes. Acta Vet Scand,1993,34:193-198
[66]Takahashi K,Nagai S,Yagihashi T.A cross-protection experiment in pigs vaccinated with Haemophilus parasuis serovar 2 ans 5 bacterins,and evaluation of a bivalent vaccine under laboratory and field conditions.J Vet Med Sci,2001,63:487-491
[67]Solnano-Aguilar G I,Pjiona C, Rapp-Gabrielson V, Collins J, Cavralho L. Protective role of maternal antibodies against Haemophilus parasuis inefetion.Am J Vet Res.1999,60:81-87
[68]Miniats O P,Smart N L,Ewert E.Vaccination of gnotobiotic primary specific pathofen-free pigs anainsr Haemophilus parasuis.Can J Vet Res,1991,55:33-36
[69]Miniats O P,Smart N L,Rosendal S.Cross protection among Haemophilus parasuisstrains in immunized gnotobiotic pigs. Can J Vet Res,1991,55:37-41
[70]王艳,夏万田.副猪嗜血杆菌间接EL ISA抗体检测方法的建立.畜牧与兽医,2006.第38卷第3期
[71]王老七,陶海静.副猪嗜血杆菌间接血凝诊断液的制备.兽医畜牧科学.2007,Vo123,No2
[72]冯小明,储岳峰.副猪嗜血杆菌抗体间接ELISA检测方法的建立与应用.中国兽医科学.2009,39(07)：597-60
[73]魏子贡,蔡旭旺.副猪嗜血杆菌抗体间接血凝检测方法的建立及应用.中国兽医科学.2006,36(09)：7132718
[74]石碧,崔耀文.副猪嗜血杆菌血清5型间接血凝试验和间接ELISA抗体检测方法的建立.中国兽医科学2007,37(11)：964-96
[75]Simone Oliveira, Lucina Galina. Development of a PCR test to diagnose Haemophilus parasuis infections.2001, J Vet Diagn Invest 13:495-501
[76]Kwonil Jung,Yoonchul H A.Development of polymerase Chain Reaction and Comparison with In situ Hybridization for the Detection of Haemophilus paraduis in Formalin-Fixed,Paraffin-Embedded Tissues.2004,J.Vet.Med.Sci.66(7):841-845
[77]万世平,王建.副猪嗜血杆菌PCR检测方法的建立与初步应用.动物医学进展,2009,30(1)：9212
[78]尹秀凤,王艳.副猪嗜血杆菌PCR检测方法的建立与应用.中国兽医学报,2007,第27卷第2期
[79]张盼锋,仇微.副猪嗜血杆菌PCR快速诊断方法的建立.广东畜牧兽医科技2009年(第34卷)第1期
[80]Kielstein P,Rosner R.Zur Glasserchen Krankheit des Schweines-Atioligisch-epizootiologische Untersuchungen zum Erregerspektrum.Mh Vet Med,1990,45:577-582
[81]Xueli Zhou, Xiaojuan Xu. Distribution of antimicrobial resistance among different serovars of Haemophilus parasuis isolates. Vet. Microbiol.2009.05.012
[82]Clark L K,Hill M A.An evaluation of the components of medicated early weaning.Swine Health and Production,1994,2:5-11
[83]Baunann G,Valenza F,Effect of vaccinating sows and their piglets on the development of Glassers disease induced by a virulent strain of Haemophilus parasuis serovar 5. Vet Rec,2002,151:18-21
[84]Smart N L,Miniats O P,Rosendal S,Frindship P.Glassers disease and prevalence of subclinical infection with Haemophilus parasuis in swine in southern Ontario.Can Vet J,1989,30:339-343
[85]Rapp-Gabrielson V J,Kocus G J,Haemophilus parasuis immunity in swine following vaccination.Vet Med,1997,92:83-90
[86]Ruiz A,Simone Oliveira.Outer Membrane Proteins and DNA Profiles in Strains of Haemophihlus parasuis Recovered from Systemic and Respiratory Site.2001,Journal of Clinical Microbiology,May 2001, JCM.39.5.1757-1762
[87]Oliveira S,Pijoan C.Computer-based analysis of Haemophilus parasuis protein fingerprints.Canadian Journal of Veterinary Research,2004,68:161-167
[88]A.J.Martin de la Fuente,E.F.Rodriguez-Ferri,R.Frandoloso.Systemic antibody response in colostrum-deprived pigs experimentally infexted with Haemophilus paraduis.Research in Veterinary Science 2009,86:248-253
[89]A.J.Martin de la Fuente,E.F.Rodriguez-Ferri,R.Frandoloso.Blood cellular immune response in pigs immunized and challenged with Haemophilus parasuis. Research in Veterinary Science.2009,86:230-234
[90]Tadjine, M., Mittal, K.R., Bourdon, S., Gottschalk, M.. Production and characterization of murine monoclonal antibodies against Haemophilus parasuis and study of their protection role in mice. Microbiology,2004,150,3935-3945.
[91]Mingguang Zhoub, Yi Guo, Jianping Zhao. Identification and characterization of novel immunogenic outer membrane proteins of Haemophilus parasuis serovar 5. Vaccine 27 (2009) 5271-5277
[92]Michael A. Mullins, Karen B. Register, Darrell O. Bayles. Characterization and Comparative Analysis of the Genes Encoding Haemophilus parasuis Outer Membrane Proteins P2 and P5. Journal of Bacteriology.2009, p.5988-6002
[93]Bin Zhang, Cheng Tang. Molecular cloning, sequencing and expression of the outer membrane protein A gene from Haemophilus parasuis. Veterinary Microbiology.2009,136: 408-410
[94]Alex Olveral, Sonia Pina. Virulence associated trimeric autotransporters of Haemophilus parasuis are antigenic proteins expressed in vivo. Vet. Res.2010,41:26
[95]Sonia Pina, Alex Olvera. Trimeric Autotransporters of Haemophilus parasuis:Generation of an Extensive Passenger Domain Repertoire Specific for Pathogenic Strains. Journal of Bacteriology, Jan.2009, p.576-587
[96]李军星,姜平.副猪嗜血杆菌热休克蛋白70基因的序列分析及抗原性鉴定.中国预防兽医学报.2009,31(7)
[97]Charlnad N,D'Silva C QDumont R A,et al.Contact-dependent acquisition of transferring-bound iron by two strains of Haemophilus parasuis.Can J Microbiol.1995,41:70-74
[98]Lichtensteiger C A, Vimr E R. Neuraminidase (sialidase) activity of Haemophilus parasuis [J]. FEMS Microbiol Lett,1997,152:269-274.
[99]朱小宁,余兴龙,李润成等.副猪嗜血杆菌的分离与鉴定及其16S rRNA生物信息学分析[J].湖南农业大学学报,2009.
[100]萨姆布鲁克著.黄培堂等译.分子克隆实验指南(第三版)[M].北京：科学出版社,2002.
[101]颜真,张英起蛋白质研究技术[M].第四军医大学出版社.27-35
[102]汪家政,范明.蛋白质技术手册[M].北京：科学出版社,2000,42-46.
[103]Simone Oliveira, Carlos Pijoanl. Haemophilus parasuis:new trends on diagnosis, epidemiology and control.Veterinary Microbiology.2004.99,1-12
[104]熊盛,王一飞.BALB/C小鼠对不同佐剂SARS灭活疫苗的早期免疫反应.中国免疫学杂志.2004,第20卷,413-417
[105]高鹏程,储岳峰.副猪嗜血杆菌对部分常用实验动物的致病性试验.畜牧兽医学报.2009,40(1)：138-141