| Avian colibacillosis is caused by avian pathogenic Escherichia coli(APEC),which results in serious economic losses and hinders the healthy development of poultry industry.Avian colibacillosis is a complex syndrome associated with pericarditis,perihepatic inflammation,air sac inflammation,yolk peritonitis and so on.Salmonella enterica serovar Pullorum(S.Pullorum)and Salmonella enterica.serovar Gallinarum(S.Gallinarum)are important bacterial pathogens in poultry industry,which caused pullorum and gallinarum disease.In addition,S.Pullorum caused diseases within 3 weeks of age,and also caused dysentery,egg production rate decreased in young and adult chicken.The pathogen can be both horizontally and vertically transmitted Therefore,a safe and effective way to control APEC and S.Pullorum infection in the poultry industry is urgent for the two kinds of diseases due to the limited usage of antibiotics.Based on our previous studies,the protective effect of the recombinant S.Gallinarum strain SG9R(pYA3342-fim)expressing type I fimbriae of APEC against APEC infections in chicken was good with 60%,but we noticed that the SG9R vaccine strain is limited to more than one-month-old chicken and not allow to be immunized within three-week-old chicken due to the residual virulence for commercial vaccine.In this study,the ?roA gene deletion strain was constructed based on the recombinant strain SG9R(pYA3342-fim)expressing type I fimbriae of APEC,and its safety and protective effect will be evaluated.It will help develop the attenuated Salmonella safety vaccine and serve as a safe vector for the new generation of multiple vaccines1.The construction of ?roA deletion of the recombinant strain SG9R(pYA3342-fim)expressing type I fimbriae of APEC and studying on its biological characteristics.Based on the original sequence of ?roA gene in the GenBank,we constructed the ?roA deletion of SG9R(pYA3342-fim)using two pairs of primers with a homologous 5' terminal to the target region and a homologous 3' terminal to the chloromycin-resistance cassette of pKD3 by Red recombination system.The ?roA deletion of SG9R(pYA3342-fim)expressing type I fimbriae of APEC was successfully constructed by Red recombination system.For generation of the SG9R(pYA3342-fim)??roA/p?roA complemented strain,the full-length ORF ?roA gene was PCR-amplified from SG9R genomic DNA,and further cloned into the expression plasmid vector pBR322 and transformed into the ?roA gene deletion strainThe results of biochemical reaction indicated that the SG9R(pYA3342-fim),SG9R(pYA3342-fim)??roA and SG9R(pYA3342-fim)A?roA/p?roA strains had the same biochemical characteristics.However,deletion of ?roA gene leads to a slower growth to the parent strains in vitro.The mannose-sensitive hemagglutination and hemagglutination inhibition assay results showed that the three strains could obviously agglutinate with chicken red blood cells,and the agglutination activity could be inhibited by D-mannose,and the results demonstrated that the SG9R(pYA3342-fim)successfully expressed type I fimbriae of APEC.The ?roA deletion of SG9R(pYA3342-fim)exhibited good genetic stability in genetic stability test of 30 passages in bacterial cultures.The minimal inhibit concentration(MIC)was determined by in a microdilution assay.The results showed that the three strains were sensitive to cephalosporins,streptomycin,gentamicin,doxycycline,florfenicol,polymyxin,meropenem,enrofloxacin and ciprofloxacin,and the results indicated that the deletion of ?roA gene does not affect the antibiotics resistance.The successful construction of ?roA deletion of SG9R(pYA3342-fim)will be beneficial for evaluating the safety of the live attenuated vaccine and multiple vaccine in further study.2.Evaluation of safety and protection effects of the ?roA deletion strain SG9R(pYA3342-fim)??roA expressing type I fimbriae of APEC.In order to explore the safety and potential application value of the ?roA deletion strain SG9R(pYA3342-fim)??roA expressing type I fimbriae of APEC as a live vaccine candidate for protection against APEC and S.Pullorum,safety and challenge protection of the deletion strain were evaluated in this study.In order to investigate the safety and potential application value of the recombinant S Gallinarum strain SG9R(pYA3342-fim)??roA as a candidate strain for live vaccine,the safety evaluation and immune protection of the deleted strain were carried out in this study One-day-old SPF chicken(n=50)were randomly divided into 10 groups.SG9R(pYA3342-fim),SG9R(pYA3342-fim)??roA and SG9R(pYA3342-fim)??roA p?roA were immunized orally at doses of 1×108cfu,1×109cfu,1×1010cfu,respectively,and PBS group was set up as control There were 5 chicken in each group,and the immune dose was 0.2mL per chicken.The results showed that the chicken in the prototype vaccine strain SG9R(pYA3342-fim)with 1×108cfu and 1×109cfu groups were healthy and had no adverse reactions,while two of the five chicken with 1×1010cfu had symptoms of feather loosening and mental depression within 24 hours.After 36 hours,the appetite returned to normal and mental state improved,as well as there was no death.The chicken of the deletion strain SG9R(pYA3342-fim)??roA with 1×108cfu,1×109cfu,1×1010cfu were healthy and had no adverse reactions.The chicken immunized with complementary strain SG9R(pYA3342-fim)??roA/p?roA with 1 × 108cfu and 1×109cfu were healthy and had no adverse reactions,while one of the five chicken in 1×1010cfu group showed symptoms of feather loosening and mental depression within 24 hours.After 36 hours,the appetite returned to normal and mental state improved,as well as there was no death.The results of safety evaluation of the three strains showed that ?roA gene deletion strain SG9R(pYA3342-fim)??roA was safer than wild strain SG9R(pYA3342-fim)and complementary strain SG9R(pYA3342-fim)??roA/paraoA,and 1×109cfu was a safer dose.Combined with the literature and the results of this experiment,the immune dose of the next immune protection test was temporarily determined to be 1×109cfu.One-day-old SPF chicken(n=75)were randomly divided into five groups:SG9R(pYA3342-fim)immunized group,SG9R(pYA3342-fim)??roA immunized group,SG9R(pYA3342-fim)??roA/p?roA immunized group,PBS control group with challenge and normal control group without challenge,there were 15 chicken in each group.The one-day-old chicken of each group were immunized orally at a dose of 1×109cfu as the first immunization,after two weeks,the chicken of each group were immunized with the same immunization dose and route as the second inmmunization,and after two weeks of secondary immunization,and the chicken of each group were immunized with the same dose and route for two weeks.The chicken of each group were immunized for three times with the same immune dose and route.The results showed that SG9R(pYA3342-fim),SG9R(pYA3342-fim)??roA and SG9R(pYA3342-fim)?arcoA/p?roA had no significant effect on the daily weight of chicken,nor affect the feed intake and mental status of chicken.After two weeks with three immunizations,1×1010cfu of the avian pathogenic Escherichia coli virulent strain QD2(serotype 078)was inoculated into posterior chest air sac to challenge chicken in each experimental group.The mortality rates of chicken immunized with SG9R(pYA3342-fim),SG9R(pYA3342-fim)??roA and SG9R(pYA3342-fim)??roA/p?roAand PBS challenge group were as follows:33.3%,20%,26.7%,66.7%.1×1010cfu of the virulent S.Pullorum strain SP03 was used to challenge chicken in each experimental group by oral administration.The incidence of SG9R(pYA3342-fim),SG9R(pYA3342-fim)??roA,SG9R(pYA3342-fim)??roA(/p?roA immunization group,PBS control group were 46.7%.33.3%,40%and 73.3%,respectively.Clinical examination and paraffin section observation of pathological tissue showed the clinical symptoms and degree of pathological changes in heart,liver and spleen of chicken immunized with SG9R(pYA3342-fim),SG9R(pYA3342-fim)??roA and SG9R(pYA3342-fim)??roA/p?roA were significantly less than that in the control group without challenge.The results of immune protection test in One-day-old SPF chicken showed that the recombinant S.Gallinarum deletion strain SG9R(pYA3342-fim)A?roA expressing type I fimbriae of APEC as a candidate of attenuated live vaccine could effectively prevent avian pathogenic Escherichia coli infection and play an effective cross-protective role in S.Pullorum infection. |
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