Development Of A Recombinant Salmonella Vaccine Against Both B. Bronchiseptica And Salmonella Infections In Swine

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 ChinaUp 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 strainsThe 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(LD₅₀) of HH0809 in mice have been tested to be 1.4×10⁵ CFU in aerosol infection model.The LD₅₀ of HH0809 in piglets had been tested to be 2.0×10¹⁰ 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.bronchisepticaUsing 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×LD₅₀ 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(Typeâ… domain) 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 bordetellosisThis 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 antigensThe F1 DNA fragment specifying the important immunodominant typeâ… domain of the fhaB gene,and the P2 DNA fragment specifying the main immunodominant regionâ…¡of 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 swineThe recombinant S.choleraesuis C501(pYA-F1P2) strain expressing the recombinant filamentous haemagglutinin typeâ… domain 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×LD₅₀ of virulent parent S. choleraesuis strain C78-1.All 20 mice vaccinated s.c.survived intranasal challenge with 4×LD₅₀ 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 1.2×10¹⁰ 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×LD₅₀ 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.