支气管败血波氏杆菌的重组沙门氏菌基因工程疫苗研究
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摘要
支气管败血波氏杆菌(Bordetella bronchiseptica,Bb)可引起猪发生肺炎和萎缩性鼻炎(Atrophic rhinitis,AR),也是猪呼吸道疾病综合征(porcine respiratory diseasecomplex,PRDC)的重要致病因子之一。更重要的是,Bb的先期感染易于导致其它多种病原的继发感染,从而增加猪群呼吸道疾病的发病率和严重程度,造成严重经济损失。以AR为代表的猪波氏菌病现已遍布养猪业发达国家,已成为猪的重要呼吸道传染病之一。本研究对Bb的病原流行病学、生物学特性、免疫原性基因、诊断方法和重组沙门氏菌基因工程疫苗进行研究,为我国Bb的流行病学分布提供理论依据,为猪波氏菌病的临床诊断、预防与控制提供新方法。主要研究内容如下:
1.Bb的分离鉴定与病原流行病学研究
从全国十五个省市送检的2,057份有肺炎或AR症状猪的肺脏等病料组织中分离出190株Bb和不同数量的共感染菌。2003～2006年间的Bb总分离率为9.2%;不同省份Bb的总分离率介于7.5～14.1%之间;不同年份Bb的总分离率介于7.3～11.8%之间;Bb的分离率与猪日龄有一定关系。Bb最常见的共感染菌依次是链球菌(55.9%)、副猪嗜血杆菌(50.0%)、大肠杆菌(43.1%)、巴氏杆菌(25.5%)和绿脓杆菌(17.6%)。在43份有典型AR症状猪的病料中,分离出22株Bb、7株产毒素巴氏杆菌和6株绿脓杆菌;在分离出产毒素巴氏杆菌的7份病料中均同时分离出Bb,而其中6份又同时分离出绿脓杆菌。研究结果表明,Bb在我国猪群的感染十分普遍,Bb与其它病原菌的共感染情况非常严重,且在AR的发生中具有重要作用。
2.Bb的生长特性及强毒菌株的筛选
Bb在多种培养基中均生长良好,但加血液的鲍-姜氏培养基更易于BbⅠ相菌形态的维持,且在含绵羊血的鲍-姜氏培养基上产生更明显的β-溶血环。小鼠致死性试验结果表明Bb毒力普遍较弱,但少数菌株如1562、3331、HH0809等表现出很强的毒力。以HH0809株为出发菌株分别建立小鼠和仔猪的呼吸道感染模型。采用建立的感染模型测定HH0809株对小鼠的LD_(50)(the 50%lethal dose)为1.4×10~5 CFU,对猪的LD_(50)为2.0×10~(10) CFU,详细记录了感染小鼠和感染仔猪的症状和病理变化。Bb生长特性研究以及小鼠和仔猪的呼吸道感染模型的建立为猪波氏菌病快速诊断试剂及高效疫苗的研制、开发奠定基础。
3.Bb保护性抗原基因的筛选
以强毒菌株HH0809的基因组为模板,分段克隆表达Bb重要的保护性抗原丝状血凝素基因fhaB(6,260 bp)和百日咳杆菌粘附素基因prn(2040 bp)。fhaB基因分为5段,从N端到C端分别命名为F5(1,400 bp)、F4(1,200 bp)、F3(1,200 bp)、F2(1,800bp)和F1(660 bp);prn基因分为2段,从N端到C端分别命名为P1(1,190 bp)和P2(850 bp),以及prn基因全段(2,040 bp);将8段DNA片段分别克隆到pGEX-KG表达载体并在大肠杆菌BL21中进行诱导表达,纯化包涵体后使用30μg(与等体积弗氏完全佐剂混合)免疫BALB/c小鼠,于21 d后使用4×LD_(50)的Bb强毒株HH0809进行呼吸道的气雾攻毒。结果表明:fhaB基因各片段表达产物GST-F1、GST-F2、GST-F3、GST-F4和GST-F5免疫组小鼠的存活率分别为66.7%(6/9)、0(0/9)、0(0/9)、44.4%(4/9)和11.1%(1/9);prn基因各片段表达产物GST-P1、GST-P2和GST-PRN免疫组小鼠的存活率分别为88.9%(8/9)、100%(9/9)和100%(9/9)。小鼠保护力试验结果证实FHA和PRN均是Bb重要的保护性抗原成分。其中,FHA的C端F1片段(TypeⅠdomain)和PRN的P2段(RegionⅡdomain)分别为两种抗原最重要的免疫保护性抗原区域,有望作为波氏菌病的诊断抗原和新型疫苗的成分。
4.Bb抗体检测ELISA方法的建立与应用
利用已纯化的各段表达蛋白GST-F5、GST-F4、GST-F3、GST-F2、GST-F1、GST-P1、GST-P2和GST-PRN分别进行间接ELISA检测方法研究。结果显示,作为包被抗原,GST-PRN优于其它各段蛋白。进一步通过凝血酶Thrombin酶切GST-PRN并回收,获得纯的不含GST载体蛋白的PRN蛋白片段。以PRN蛋白片段为抗原建立检测PRN抗体的间接ELISA检测方法。结果表明,PRN-ELISA方法特异性良好,该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性;ELISA方法能够检测到人工感染仔猪14 d的血清抗体IgG,比乳胶凝集方法高4～128倍,但与其符合率为100%;用该ELISA方法检测2005～2007年间来自全国各地的1,229份猪血清样本,阳性率为32.7%。该方法对2个阳性猪场的检测结果表明保育期仔猪的合群导致猪群大量感染Bb。
5.表达Bb保护性抗原的重组沙门氏菌株的构建及生物学特性
将fhaB F1段和prn P2段依次与pMD18-T载体连接,然后将F1-P2融合片段转移连入沙门氏菌asd平衡表达质粒pYA3493中,制备重组平衡表达质粒pYA-F1P2。将pYA-F1P2和pYA3493分别电转化猪霍乱沙门氏菌C500的asd缺失株C501中,制备重组菌株C501(pYA-F1P2)和空载体菌株C501(pYA3493)。研究结果表明,重组菌株C501(pYA-F1P2)保留了亲本菌株C500的生化特性和抗原表型,能稳定遗传并高效分泌表达Bb的rF1P2抗原;C501(pYA-F1P2)的毒力较亲本菌C500降低了4.5倍。将C501(pYA-F1P2)分别高剂量接种仔猪和怀孕母猪观察其作为重组疫苗的安全性。结果表明,该重组菌株和亲本菌株C500接种的仔猪均没有异常临床表现;该重组菌株和亲本菌株C500接种的怀孕母猪也没有异常临床表现,与没做任何处理的对照组怀孕母猪所产仔数没有差异。猪霍乱沙门氏菌C500弱毒株是我国广泛使用的预防仔猪副伤寒的标准疫苗株。本研究利用猪霍乱沙门氏菌C500弱毒疫苗株的asd基因缺失株构建平衡载体表达系统菌株C501(pYA-F1P2)能高效分泌表达Bb免疫原性重组抗原蛋白;该活疫苗株C501(pYA-F1P2)的毒力较亲本菌株C500稍低,对断奶仔猪和怀孕母猪均是安全的,有望成为新型猪波氏菌病-副伤寒二价活疫苗的候选菌株。
6.支气管败血波氏杆菌的重组沙门氏菌基因工程疫苗研究
利用表达Bb免疫原性基因F1(fhaB的TypeⅠ)片段和prn基因的P2(prn的Region 2)片段重组猪霍乱沙门氏菌弱毒疫苗株C501(pYA-F1P2),制备支气管败血波氏杆菌重组沙门氏菌基因工程疫苗,检验该重组活疫苗对免疫BALB/c小鼠和猪针对猪霍乱沙门氏菌和Bb攻击的保护效力。将2.1×10~9 CFU和2.1×10~8 CFU重组菌株C501(pYA-F1P2)分别通过口服和皮下注射途径免疫BALB/c小鼠,结果显示两种免疫方法均能产生较高水平的血清IgG,并保护小鼠抵抗10×LD_(50)猪霍乱沙门氏菌强毒株C78-1的口服攻击(4/4)。但使用4×LD_(50) Bb强毒株HH0809进行呼吸道气雾攻毒后,口服免疫不能提供有效保护(4/22),而皮下免疫能够提供完全保护(22/22)。进一步检测结果表明,皮下免疫能产生较高水平的肺脏IgG,口服免疫则不能。同时,皮下免疫重组蛋白HIS-F1P2的小鼠也能完全抵抗4×LD_(50) Bb强毒株HH0809的攻击。将1.2×10~(10) CFU重组菌株C501(pYA-F1P2)通过颈部肌肉注射免疫20日龄仔猪,结果显示免疫仔猪能产生较高水平的针对沙门氏菌和Bb重组蛋白rF1P2的血清IgG,并能抵抗5×LD(Lethal dose)猪霍乱沙门氏菌强毒株C78-1的口服攻击(4/4)。同时,免疫仔猪也能抵抗4×LD_(50) Bb强毒株HH0809呼吸道途径的攻击(4/4),而PBS组(0/4)和C501(pYA3493)载体对照组(1/4)均不能提供有效保护。另外,重组菌株C501(pYA-F1P2)组诱导仔猪抵抗Bb攻击的保护力优于重组蛋白HIS-F1P2。该研究构建的支气管败血波氏杆菌重组沙门氏菌基因工程疫苗菌株C501(pYA-F1P2)能保护免疫仔猪完全抵抗猪霍乱沙门氏菌和Bb的致死性攻击,有望成为实用有效的新型双价基因工程活疫苗。
Bordetella bronchiseptica is an etiologic agent of atrophic rhinitis and bronchopneumonia in young pigs.B.bronchiseptica is also a contributory agent in porcine respiratory disease complex,a multifactorial disease state increasingly problematic for swine producers.Although the primary disease is important,more significant is the fact that this bacteria predisposes to colonization and disease with other viral and bacterial pathogens,resulting in higger morbidity and more serious diseases. This study reported here focused on the pathogen epidemiology,bio-characteristics, immunogenicity genes,diagnostic method,and new recombinate Salmonella vaccine, which provided very important materials for basic research and contributed for preventing and control of bordetellosis in China.The results are as follows.
1.Isolation and identification of B.bronchiseptica from pigs and pathogen epidemiology of bordetellosis in China
Up to 190 B.bronchiseptica strains were isolated from 2,057 lesion lungs of pigs with clinical signs of pneumonia or atrophic rhinitis,while different concurrent infection bacteria species were also separated from the same samples.The isolation ratios in the samples that were collected from different provinces and at the different time points(year) ranged from 7.5%to 14.1%and 7.3%to 11.8%,respectively.The average isolation ratio between the years of 2003 to 2006 was 9.2%.The isolation ratios of identified co-infection bacterial species were 55.9%(57 strains),50.0%(51 strains),43.1%(44 strains),25.5%(26 strains),17.6%(18 strains) and 11.8%(12 strains) for Streptococcus, Haemophillus parasuis,E.coli,Pasterella multocida,Pseudomonas aeruginosa and Salmonella,respectively.Twenty-two B.bronchiseptica strains were isolated from 43 samples of lesion lungs and nasal swabs of pigs with typical clinical signs of atrophic rhinitis,while 7 toxigenic Pasteurella multocida(T~+Pm) strains and 6 Pseudomonas aeruginosa strains were also separated from the same samples.The infection of swine caused by B.bronchiseptica,which was often accompanied with other co-infection bacterial species,is very prevalent in swine herds in China and B.bronchiseptica may play an important role in causing atrophic rhinitis of swine.
2.Bio-characteristics of B.bronchiseptica and selection of high virulence strains
The cultural characteristics of B.bronchiseptica have been evaluated in various kinds of medium.Good growth have been seen in several medium,but Bordet-Gengou(BG) agar medium supplemented with 15%(v/v) defibrinated blood showed to be better for maintaing I phase of B.bronchiseptica,andβ-zone of hemolysis on BG agar supplemented with sheep blood showed more obvious than that of swine blood.By virulence tests,we found that most of B.bronchiseptica showed low virulence,but several B.bronchiseptica strains,such as 1562,3331 and HH0809 strains showed very high virulence.In this study,a B.bronchiseptica aerosol infection model was established using BALB/c mice and piglets,respectively.The 50%lethal dose(LD_(50)) of HH0809 in mice have been tested to be 1.4×10~5 CFU in aerosol infection model.The LD_(50) of HH0809 in piglets had been tested to be 2.0×10~(10) CFU.In these two models,the symptom and pathological change have been recorded and summaried in detail.In this study,the determination of cultural characteristics and the development of mouse and piglets aerosol challenge modelsof B.bronchiseptica may contribute to the development of new vaccine and diagnostic method of bordetellosis.
3.Research on protective antigens of B.bronchiseptica
Using the genome of selected virulent Bb HH0809 strain as PCR template,fhaB(6,260 bp) and prn(2,040 bp) encoding the most important antigens of B.bronchiseptica filamentous haemogglutinin(FHA) and pertactin(PRN) were cloned and expressed in Escherichia coli BL21 segmentedly.The fhaB gene has been divided into 5 fragments,F5 (1,400 bp),F4(1,200 bp),F3(1,200 bp),F2(1,800 bp) and F1(660 bp) from N termination to C termination;The prn gene has been divided into 2 fragments,P1(1,190 bp) and P2(850 bp) from N termination to C termination,and the whole prn gene(2,040 bp).The all 8 DNA fragments were cloned into pGEX-KG and expressed in BL21, respectively.Then 30μg purified protein mixed with an equal volume of complete Freund's adjuvant was used to immunize BALB/c mice subcutaneously.Twenty-one days after immunization,the mice were challenged with 4×LD_(50) of HH0809 strain by using the aerosol challenge model to evaluate the protective efficacy of each protein fragments.The results showed that the expressed protein fragments of fhaB gene could provide groups of mice different protective efficacy:GST-F1 66.7%(6/9),GST-F2 0%(0/9),GST-F3 0% (0/9),GST-F4 44.4%(4/9) and GST-F5(1/9);the expressed protein fragments of prn gene have the following protective efficiency:GST-P1 88.9%(8/9),GST-P2 100%(9/9) and GST-PRN 100%(9/9).The expressed proteins could protect mice from B.bronchiseptica callenge with different proctective efficacy,which confirmed that FHA and pertactin antigens are important protective antigens of B.bronchiseptica.In addition,the results showed that the F1 fragment(TypeⅠdomain) of FHA and the P2 fragment(Region 2 domain) of PRN may be respectively the most important protective regions of FHA and pertactin,which may be used to develop new diagnostic antigens and new effective vaccines of bordetellosis.
4.Development of an indriect ELISA method based on expressed pertactin protein and it's application on diagnosis of bordetellosis
This study made a try to set up an indirect ELISA method for diagnosis of bordetellosis by using the expressed and purificated protein fragments,GST-F5,GST-F4,GST-F3, GST-F2,GST-F1,GST-P1,GST-P2 or GST-PRN.The results showed that GST-PRN was the best coating antigen than the others for ELISA method.The recombinant protein fragement of rPRN was purified from the GST-PRN fusion protein after digesting by thrombin protease.Then the rPRN-based indirect ELISA was developed for detection of antibodies against PRN.No false positive results were found in detection of seven antisera against porcine bacterial diseases,suggesting that PRN-ELISA had good specificity.The ELISA could pick up the positive samples in experimentally infected pigs 14 days postinoculation and the degree of sensitivity is 4 to 128 times higher than the latex agglutination test with the coating antigen of killed B.bronchiseptica,but with a 100%coincidence rate.Four hundred and two positive samples were picked up from 1,229 clinical serum samples with a positive rate of 32.7%in different provinces in China. Serum samples from two bordetellosis-positive pig fields were tested by the indirect ELISA method suggesting that most of pigs might be infected by B.bronchiseptica when the pig moved together during the nursery periods.
5.Construction and bio-characterics of Salmonella choleraesuis C500 strain expressing the recombinant FHA and pertactin antigens
The F1 DNA fragment specifying the important immunodominant typeⅠdomain of the fhaB gene,and the P2 DNA fragment specifying the main immunodominant regionⅡof the prn gene,were cloned into the pYA3493 vector,resulting in the recombinant plasmid pYA-F1P2.Then pYA-F1P2 and pYA3493 were electrotransformed toΔasdC500 strain (named as C501),resulting in the recombinant Salmonella strain C501(pYA-F1P2) and vector control C501(pYA3493) respectively.Both C501(pYA-F1P2) and C501(pYA3493) strains remained the serum type and energy source using characterics of parent C500 strain,and the recombinant heterologous antigens were highly expressed secretarily in C501(pYA-F1P2).The C501(pYA-F1P2) strain had 4.5 times lower virulence than the parent C500 strain in lethal test of BALB/c mice.In addition,the strain was safe to piglets and sows after being inocubated subcutaneously.Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid in China.In this study,the recombinant C501(pYA-F1P2) strain could express secretorily the recombinant heterologous antigens of B.bronchiseptica and was safe to piglets and sows,which might be a potential canditate strain as a new recombinant recombinate Salmonella vaccine against both bordetellosis and paratyphoid in swine.
6.A new recombinant Salmonella vaccine against both bordetellosis and paratyphoid in swine
The recombinant S.choleraesuis C501(pYA-F1P2) strain expressing the recombinant filamentous haemagglutinin typeⅠdomain and pertactin region 2 domain antigen(rF1P2) of B.bronchiseptica was used as a new recombinate vaccine against both bordetellosis and paratyphoid in swine.Both the subcutaneously(s.c.) and oral vaccines conferred complete protection(4/4) against fatal infection with 10×LD_(50) of virulent parent S. choleraesuis strain C78-1.All 20 mice vaccinated s.c.survived intranasal challenge with 4×LD_(50) of virulent B.bronchiseptica(HH0809) compared with 4 of 20 vector-treated controls and 1 of 18 phosphate-buffered saline(PBS)-treated controls that survived,but no significant protection against HH0809 was observed in orally vaccinated animals (4/22).Lung homogenates from s.c.vaccinated animals had detectably high levels of rF1P2-specific IgG.But only a much lower level of rF1P2-specific IgG was detected in samples from orally vaccinated mice.Simultaneously,mice immunized with the recombinant HIS-F1P2 protein survived the aerosol challenge of B.bronchiseptica. Piglets immunized s.c.with 1.2×10~(10) CFU C501(pYA-F1P2) produced robust Salmonella-and rF1P2-specific serum IgG and IgA antibodies.All piglets(4/4) survived the oral challenge with 5 times of lethal doses(LD) of wild-type S.choleraesuis C78-1.In addition,piglets survived the respiratory challenge with 4×LD_(50) of highly virulent B. bronchiseptica HH0809 strain compared with 1 of 4 vector-treated controls and 0 of 4 phosphate-buffered saline(PBS)-treated controls that survived.But the protective efficacy of HIS-F1P2 was inferior to the recombinant CS01(pYA-F1P2) vaccine. Subcutaneous immunization with the recombinant C501(pYA-F1P2) vaccine can provide piglets complete protection against infections with both S.choleraesuis and B. bronchiseptica,showing the potential as a new recombinant Salmonella vaccine against both piglets bordetellosis and paratyphoid.
1.Bb的分离鉴定与病原流行病学研究
从全国十五个省市送检的2,057份有肺炎或AR症状猪的肺脏等病料组织中分离出190株Bb和不同数量的共感染菌。2003～2006年间的Bb总分离率为9.2%;不同省份Bb的总分离率介于7.5～14.1%之间;不同年份Bb的总分离率介于7.3～11.8%之间;Bb的分离率与猪日龄有一定关系。Bb最常见的共感染菌依次是链球菌(55.9%)、副猪嗜血杆菌(50.0%)、大肠杆菌(43.1%)、巴氏杆菌(25.5%)和绿脓杆菌(17.6%)。在43份有典型AR症状猪的病料中,分离出22株Bb、7株产毒素巴氏杆菌和6株绿脓杆菌;在分离出产毒素巴氏杆菌的7份病料中均同时分离出Bb,而其中6份又同时分离出绿脓杆菌。研究结果表明,Bb在我国猪群的感染十分普遍,Bb与其它病原菌的共感染情况非常严重,且在AR的发生中具有重要作用。
2.Bb的生长特性及强毒菌株的筛选
Bb在多种培养基中均生长良好,但加血液的鲍-姜氏培养基更易于BbⅠ相菌形态的维持,且在含绵羊血的鲍-姜氏培养基上产生更明显的β-溶血环。小鼠致死性试验结果表明Bb毒力普遍较弱,但少数菌株如1562、3331、HH0809等表现出很强的毒力。以HH0809株为出发菌株分别建立小鼠和仔猪的呼吸道感染模型。采用建立的感染模型测定HH0809株对小鼠的LD_(50)(the 50%lethal dose)为1.4×10~5 CFU,对猪的LD_(50)为2.0×10~(10) CFU,详细记录了感染小鼠和感染仔猪的症状和病理变化。Bb生长特性研究以及小鼠和仔猪的呼吸道感染模型的建立为猪波氏菌病快速诊断试剂及高效疫苗的研制、开发奠定基础。
3.Bb保护性抗原基因的筛选
以强毒菌株HH0809的基因组为模板,分段克隆表达Bb重要的保护性抗原丝状血凝素基因fhaB(6,260 bp)和百日咳杆菌粘附素基因prn(2040 bp)。fhaB基因分为5段,从N端到C端分别命名为F5(1,400 bp)、F4(1,200 bp)、F3(1,200 bp)、F2(1,800bp)和F1(660 bp);prn基因分为2段,从N端到C端分别命名为P1(1,190 bp)和P2(850 bp),以及prn基因全段(2,040 bp);将8段DNA片段分别克隆到pGEX-KG表达载体并在大肠杆菌BL21中进行诱导表达,纯化包涵体后使用30μg(与等体积弗氏完全佐剂混合)免疫BALB/c小鼠,于21 d后使用4×LD_(50)的Bb强毒株HH0809进行呼吸道的气雾攻毒。结果表明:fhaB基因各片段表达产物GST-F1、GST-F2、GST-F3、GST-F4和GST-F5免疫组小鼠的存活率分别为66.7%(6/9)、0(0/9)、0(0/9)、44.4%(4/9)和11.1%(1/9);prn基因各片段表达产物GST-P1、GST-P2和GST-PRN免疫组小鼠的存活率分别为88.9%(8/9)、100%(9/9)和100%(9/9)。小鼠保护力试验结果证实FHA和PRN均是Bb重要的保护性抗原成分。其中,FHA的C端F1片段(TypeⅠdomain)和PRN的P2段(RegionⅡdomain)分别为两种抗原最重要的免疫保护性抗原区域,有望作为波氏菌病的诊断抗原和新型疫苗的成分。
4.Bb抗体检测ELISA方法的建立与应用
利用已纯化的各段表达蛋白GST-F5、GST-F4、GST-F3、GST-F2、GST-F1、GST-P1、GST-P2和GST-PRN分别进行间接ELISA检测方法研究。结果显示,作为包被抗原,GST-PRN优于其它各段蛋白。进一步通过凝血酶Thrombin酶切GST-PRN并回收,获得纯的不含GST载体蛋白的PRN蛋白片段。以PRN蛋白片段为抗原建立检测PRN抗体的间接ELISA检测方法。结果表明,PRN-ELISA方法特异性良好,该方法对猪巴氏杆菌病等7种常见细菌性疾病阳性血清的检测结果均为阴性;ELISA方法能够检测到人工感染仔猪14 d的血清抗体IgG,比乳胶凝集方法高4～128倍,但与其符合率为100%;用该ELISA方法检测2005～2007年间来自全国各地的1,229份猪血清样本,阳性率为32.7%。该方法对2个阳性猪场的检测结果表明保育期仔猪的合群导致猪群大量感染Bb。
5.表达Bb保护性抗原的重组沙门氏菌株的构建及生物学特性
将fhaB F1段和prn P2段依次与pMD18-T载体连接,然后将F1-P2融合片段转移连入沙门氏菌asd平衡表达质粒pYA3493中,制备重组平衡表达质粒pYA-F1P2。将pYA-F1P2和pYA3493分别电转化猪霍乱沙门氏菌C500的asd缺失株C501中,制备重组菌株C501(pYA-F1P2)和空载体菌株C501(pYA3493)。研究结果表明,重组菌株C501(pYA-F1P2)保留了亲本菌株C500的生化特性和抗原表型,能稳定遗传并高效分泌表达Bb的rF1P2抗原;C501(pYA-F1P2)的毒力较亲本菌C500降低了4.5倍。将C501(pYA-F1P2)分别高剂量接种仔猪和怀孕母猪观察其作为重组疫苗的安全性。结果表明,该重组菌株和亲本菌株C500接种的仔猪均没有异常临床表现;该重组菌株和亲本菌株C500接种的怀孕母猪也没有异常临床表现,与没做任何处理的对照组怀孕母猪所产仔数没有差异。猪霍乱沙门氏菌C500弱毒株是我国广泛使用的预防仔猪副伤寒的标准疫苗株。本研究利用猪霍乱沙门氏菌C500弱毒疫苗株的asd基因缺失株构建平衡载体表达系统菌株C501(pYA-F1P2)能高效分泌表达Bb免疫原性重组抗原蛋白;该活疫苗株C501(pYA-F1P2)的毒力较亲本菌株C500稍低,对断奶仔猪和怀孕母猪均是安全的,有望成为新型猪波氏菌病-副伤寒二价活疫苗的候选菌株。
6.支气管败血波氏杆菌的重组沙门氏菌基因工程疫苗研究
利用表达Bb免疫原性基因F1(fhaB的TypeⅠ)片段和prn基因的P2(prn的Region 2)片段重组猪霍乱沙门氏菌弱毒疫苗株C501(pYA-F1P2),制备支气管败血波氏杆菌重组沙门氏菌基因工程疫苗,检验该重组活疫苗对免疫BALB/c小鼠和猪针对猪霍乱沙门氏菌和Bb攻击的保护效力。将2.1×10~9 CFU和2.1×10~8 CFU重组菌株C501(pYA-F1P2)分别通过口服和皮下注射途径免疫BALB/c小鼠,结果显示两种免疫方法均能产生较高水平的血清IgG,并保护小鼠抵抗10×LD_(50)猪霍乱沙门氏菌强毒株C78-1的口服攻击(4/4)。但使用4×LD_(50) Bb强毒株HH0809进行呼吸道气雾攻毒后,口服免疫不能提供有效保护(4/22),而皮下免疫能够提供完全保护(22/22)。进一步检测结果表明,皮下免疫能产生较高水平的肺脏IgG,口服免疫则不能。同时,皮下免疫重组蛋白HIS-F1P2的小鼠也能完全抵抗4×LD_(50) Bb强毒株HH0809的攻击。将1.2×10~(10) CFU重组菌株C501(pYA-F1P2)通过颈部肌肉注射免疫20日龄仔猪,结果显示免疫仔猪能产生较高水平的针对沙门氏菌和Bb重组蛋白rF1P2的血清IgG,并能抵抗5×LD(Lethal dose)猪霍乱沙门氏菌强毒株C78-1的口服攻击(4/4)。同时,免疫仔猪也能抵抗4×LD_(50) Bb强毒株HH0809呼吸道途径的攻击(4/4),而PBS组(0/4)和C501(pYA3493)载体对照组(1/4)均不能提供有效保护。另外,重组菌株C501(pYA-F1P2)组诱导仔猪抵抗Bb攻击的保护力优于重组蛋白HIS-F1P2。该研究构建的支气管败血波氏杆菌重组沙门氏菌基因工程疫苗菌株C501(pYA-F1P2)能保护免疫仔猪完全抵抗猪霍乱沙门氏菌和Bb的致死性攻击,有望成为实用有效的新型双价基因工程活疫苗。
Bordetella bronchiseptica is an etiologic agent of atrophic rhinitis and bronchopneumonia in young pigs.B.bronchiseptica is also a contributory agent in porcine respiratory disease complex,a multifactorial disease state increasingly problematic for swine producers.Although the primary disease is important,more significant is the fact that this bacteria predisposes to colonization and disease with other viral and bacterial pathogens,resulting in higger morbidity and more serious diseases. This study reported here focused on the pathogen epidemiology,bio-characteristics, immunogenicity genes,diagnostic method,and new recombinate Salmonella vaccine, which provided very important materials for basic research and contributed for preventing and control of bordetellosis in China.The results are as follows.
1.Isolation and identification of B.bronchiseptica from pigs and pathogen epidemiology of bordetellosis in China
Up to 190 B.bronchiseptica strains were isolated from 2,057 lesion lungs of pigs with clinical signs of pneumonia or atrophic rhinitis,while different concurrent infection bacteria species were also separated from the same samples.The isolation ratios in the samples that were collected from different provinces and at the different time points(year) ranged from 7.5%to 14.1%and 7.3%to 11.8%,respectively.The average isolation ratio between the years of 2003 to 2006 was 9.2%.The isolation ratios of identified co-infection bacterial species were 55.9%(57 strains),50.0%(51 strains),43.1%(44 strains),25.5%(26 strains),17.6%(18 strains) and 11.8%(12 strains) for Streptococcus, Haemophillus parasuis,E.coli,Pasterella multocida,Pseudomonas aeruginosa and Salmonella,respectively.Twenty-two B.bronchiseptica strains were isolated from 43 samples of lesion lungs and nasal swabs of pigs with typical clinical signs of atrophic rhinitis,while 7 toxigenic Pasteurella multocida(T~+Pm) strains and 6 Pseudomonas aeruginosa strains were also separated from the same samples.The infection of swine caused by B.bronchiseptica,which was often accompanied with other co-infection bacterial species,is very prevalent in swine herds in China and B.bronchiseptica may play an important role in causing atrophic rhinitis of swine.
2.Bio-characteristics of B.bronchiseptica and selection of high virulence strains
The cultural characteristics of B.bronchiseptica have been evaluated in various kinds of medium.Good growth have been seen in several medium,but Bordet-Gengou(BG) agar medium supplemented with 15%(v/v) defibrinated blood showed to be better for maintaing I phase of B.bronchiseptica,andβ-zone of hemolysis on BG agar supplemented with sheep blood showed more obvious than that of swine blood.By virulence tests,we found that most of B.bronchiseptica showed low virulence,but several B.bronchiseptica strains,such as 1562,3331 and HH0809 strains showed very high virulence.In this study,a B.bronchiseptica aerosol infection model was established using BALB/c mice and piglets,respectively.The 50%lethal dose(LD_(50)) of HH0809 in mice have been tested to be 1.4×10~5 CFU in aerosol infection model.The LD_(50) of HH0809 in piglets had been tested to be 2.0×10~(10) CFU.In these two models,the symptom and pathological change have been recorded and summaried in detail.In this study,the determination of cultural characteristics and the development of mouse and piglets aerosol challenge modelsof B.bronchiseptica may contribute to the development of new vaccine and diagnostic method of bordetellosis.
3.Research on protective antigens of B.bronchiseptica
Using the genome of selected virulent Bb HH0809 strain as PCR template,fhaB(6,260 bp) and prn(2,040 bp) encoding the most important antigens of B.bronchiseptica filamentous haemogglutinin(FHA) and pertactin(PRN) were cloned and expressed in Escherichia coli BL21 segmentedly.The fhaB gene has been divided into 5 fragments,F5 (1,400 bp),F4(1,200 bp),F3(1,200 bp),F2(1,800 bp) and F1(660 bp) from N termination to C termination;The prn gene has been divided into 2 fragments,P1(1,190 bp) and P2(850 bp) from N termination to C termination,and the whole prn gene(2,040 bp).The all 8 DNA fragments were cloned into pGEX-KG and expressed in BL21, respectively.Then 30μg purified protein mixed with an equal volume of complete Freund's adjuvant was used to immunize BALB/c mice subcutaneously.Twenty-one days after immunization,the mice were challenged with 4×LD_(50) of HH0809 strain by using the aerosol challenge model to evaluate the protective efficacy of each protein fragments.The results showed that the expressed protein fragments of fhaB gene could provide groups of mice different protective efficacy:GST-F1 66.7%(6/9),GST-F2 0%(0/9),GST-F3 0% (0/9),GST-F4 44.4%(4/9) and GST-F5(1/9);the expressed protein fragments of prn gene have the following protective efficiency:GST-P1 88.9%(8/9),GST-P2 100%(9/9) and GST-PRN 100%(9/9).The expressed proteins could protect mice from B.bronchiseptica callenge with different proctective efficacy,which confirmed that FHA and pertactin antigens are important protective antigens of B.bronchiseptica.In addition,the results showed that the F1 fragment(TypeⅠdomain) of FHA and the P2 fragment(Region 2 domain) of PRN may be respectively the most important protective regions of FHA and pertactin,which may be used to develop new diagnostic antigens and new effective vaccines of bordetellosis.
4.Development of an indriect ELISA method based on expressed pertactin protein and it's application on diagnosis of bordetellosis
This study made a try to set up an indirect ELISA method for diagnosis of bordetellosis by using the expressed and purificated protein fragments,GST-F5,GST-F4,GST-F3, GST-F2,GST-F1,GST-P1,GST-P2 or GST-PRN.The results showed that GST-PRN was the best coating antigen than the others for ELISA method.The recombinant protein fragement of rPRN was purified from the GST-PRN fusion protein after digesting by thrombin protease.Then the rPRN-based indirect ELISA was developed for detection of antibodies against PRN.No false positive results were found in detection of seven antisera against porcine bacterial diseases,suggesting that PRN-ELISA had good specificity.The ELISA could pick up the positive samples in experimentally infected pigs 14 days postinoculation and the degree of sensitivity is 4 to 128 times higher than the latex agglutination test with the coating antigen of killed B.bronchiseptica,but with a 100%coincidence rate.Four hundred and two positive samples were picked up from 1,229 clinical serum samples with a positive rate of 32.7%in different provinces in China. Serum samples from two bordetellosis-positive pig fields were tested by the indirect ELISA method suggesting that most of pigs might be infected by B.bronchiseptica when the pig moved together during the nursery periods.
5.Construction and bio-characterics of Salmonella choleraesuis C500 strain expressing the recombinant FHA and pertactin antigens
The F1 DNA fragment specifying the important immunodominant typeⅠdomain of the fhaB gene,and the P2 DNA fragment specifying the main immunodominant regionⅡof the prn gene,were cloned into the pYA3493 vector,resulting in the recombinant plasmid pYA-F1P2.Then pYA-F1P2 and pYA3493 were electrotransformed toΔasdC500 strain (named as C501),resulting in the recombinant Salmonella strain C501(pYA-F1P2) and vector control C501(pYA3493) respectively.Both C501(pYA-F1P2) and C501(pYA3493) strains remained the serum type and energy source using characterics of parent C500 strain,and the recombinant heterologous antigens were highly expressed secretarily in C501(pYA-F1P2).The C501(pYA-F1P2) strain had 4.5 times lower virulence than the parent C500 strain in lethal test of BALB/c mice.In addition,the strain was safe to piglets and sows after being inocubated subcutaneously.Salmonella choleraesuis C500 strain was an attenuated vaccine strain to prevent piglet paratyphoid in China.In this study,the recombinant C501(pYA-F1P2) strain could express secretorily the recombinant heterologous antigens of B.bronchiseptica and was safe to piglets and sows,which might be a potential canditate strain as a new recombinant recombinate Salmonella vaccine against both bordetellosis and paratyphoid in swine.
6.A new recombinant Salmonella vaccine against both bordetellosis and paratyphoid in swine
The recombinant S.choleraesuis C501(pYA-F1P2) strain expressing the recombinant filamentous haemagglutinin typeⅠdomain and pertactin region 2 domain antigen(rF1P2) of B.bronchiseptica was used as a new recombinate vaccine against both bordetellosis and paratyphoid in swine.Both the subcutaneously(s.c.) and oral vaccines conferred complete protection(4/4) against fatal infection with 10×LD_(50) of virulent parent S. choleraesuis strain C78-1.All 20 mice vaccinated s.c.survived intranasal challenge with 4×LD_(50) of virulent B.bronchiseptica(HH0809) compared with 4 of 20 vector-treated controls and 1 of 18 phosphate-buffered saline(PBS)-treated controls that survived,but no significant protection against HH0809 was observed in orally vaccinated animals (4/22).Lung homogenates from s.c.vaccinated animals had detectably high levels of rF1P2-specific IgG.But only a much lower level of rF1P2-specific IgG was detected in samples from orally vaccinated mice.Simultaneously,mice immunized with the recombinant HIS-F1P2 protein survived the aerosol challenge of B.bronchiseptica. Piglets immunized s.c.with 1.2×10~(10) CFU C501(pYA-F1P2) produced robust Salmonella-and rF1P2-specific serum IgG and IgA antibodies.All piglets(4/4) survived the oral challenge with 5 times of lethal doses(LD) of wild-type S.choleraesuis C78-1.In addition,piglets survived the respiratory challenge with 4×LD_(50) of highly virulent B. bronchiseptica HH0809 strain compared with 1 of 4 vector-treated controls and 0 of 4 phosphate-buffered saline(PBS)-treated controls that survived.But the protective efficacy of HIS-F1P2 was inferior to the recombinant CS01(pYA-F1P2) vaccine. Subcutaneous immunization with the recombinant C501(pYA-F1P2) vaccine can provide piglets complete protection against infections with both S.choleraesuis and B. bronchiseptica,showing the potential as a new recombinant Salmonella vaccine against both piglets bordetellosis and paratyphoid.
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