| Avian pathogenic colibacillosis is a kind of important bacterial disease caused by avian pathogenic(APEC),which seriously harms global poultry industry.Poultry of all days of age are susceptible to APEC infection.APEC infection will seriously affect the growth performance of poultry and lead to significant economic losses.After APEC infection,it can cause sepsis,tissue and organ inflammation,including pericarditis,perihepatic inflammation,balloon inflammation,arthritis,peritonitis and encephalitis.APEC can also be transmitted to humans through contaminated poultry meat and produce contaminated with poultry faeces,leading to urinary tract infections and meningitis in newborns,posing a risk of zoonotic diseases with serious implications for public health security.Vaccination can have a certain protective effect against APEC,thus reducing economic losses in the poultry industry.At present,APEC vaccines mainly include inactivated vaccines,live attenuated vaccines and subunit vaccines.However,due to corresponding defects,APEC vaccines have not been widely used in clinical practice.In this study,based on a live vector vaccine candidate strain SG01 Δasd(pYA3342-APEC fim)of Salmonella Gallinarum expressing APEC type Ⅰ fimbria constructed in the laboratory,the culture conditions and animal challenge dose were screened,and the protective efficacy of challenge was tested.It provides the basis for developing a recombinant attenuated Salmonella Gallinarum vaccine with better challenge protection against APEC infection.1.Preliminary study on the culture conditions of recombinant live vector vaccine candidate strain SG01Δ asd(pYA3342-APEC fim)of Salmonella Gallinarum.In this study,the culture conditions of live vector vaccine SG01Δ asd(pYA3342-APEC fim)expressing type Ⅰ fimbriae of avian pathogenic Escherichia coli.In this experiment,a live vector vaccine candidate SG01Δ asd(pYA3342-APEC fim)expressing type Ⅰ fimbriae of avian pathogenic Escherichia coli was constructed in the laboratory,and the culture conditions were preliminarily explored.The optimal medium type and pH value of the media were screened and a linear equation was established between the growth OD600nm value of the vaccine strain and the corresponding number of live bacteria,in order to quickly know the number of living bacteria in the liquid.A linear equation was established between the OD600nm value of the vaccine strain and the corresponding number of live bacteria,in order to quickly obtain the live bacteria in the bacteria solution.The experimental results showed that SG01Δ?asd(pYA3342-APEC fim)vaccine candidate strains could obtain more viable bacteria in SOC medium with pH of about 7.5 by oscillating culture at 180 rpm for 12 h,and finally reached 3.5×10 CFU/mL to facilitate the development of subsequent experiments.2.Screening of challenge dose of avian pathogenic Escherichia coli APEC QD2(O78)virulent strain.In order to select the appropriate challenge infectious dose of APEC QD2(O78)virulent strain to 21-day-old chickens to meet the needs of subsequent challenge protection experiments.Forty 21-day-old SPF chickens were randomly divided into 4 groups:challenge group A,challenge group B,challenge group C and healthy control group with 10 chickens in each group.The chickens in group A,group B and group C were injected with virulent strain APEC QD2(O78)through the posterior chest air sac injection with a challenge dose of 1×109 CFU/chicken,1×108 CFU/chicken and 1×107 CFU/chicken respectively,and the healthy control group was inoculated with 0.2 mL sterile PBS solution by the same route.The chickens were observed continuously for 14 days after the challenge,and the mental state and diarrhea of the chickens were scored daily according to the established scoring criteria,and finally judged according to the disease criteria.According to the results,the morbidity of group A can reach 100%,and the mortality rate is 60%,while the morbidity of challenge group B and challenge group C was 80%and 60%,respectively,and no death occurred.After autopsy of dead chickens,it was found that severe pericarditis and perihepatic inflammation appeared in group A,and a yellow cellulose membrane appeared on the visceral surface.According to the scoring criteria,the average scores of the heart,liver and air sacs were 1.90 ± 1.10,1.40 ± 1.26 and 0.60 ± 0.84,respectively,which were significantly different from those of the healthy control group(p<0.05).Challenge group B and challenge group C had slight clinical symptoms,and challenge group C had no significant difference in clinical symptom score and lesion score at autopsy compared with healthy control group.The suitable antivirus dose for 21-day-old chickens is 1×109 CFU/chicken,which laid the foundation for the subsequent challenge protection test.3.Evaluation of protective efficacy of Salmonella Gallinarum vector born strain SG01Δasd(pYA3342-APEC fim).In order to explore the protective effect of Salmonella Gall inarum vector born strain SG01 asd(pYA3342-APEC fim)on the protective effect of challenge against APEC.In this study,40 five-day-old SPF chickens were randomly divided into 4 groups:immunization group,immunization attack group,challenge control group and healthy control group with 10 chickens in each group.The SG01 Δasd(pYA3342-APEC fim)strain was used to immunize each group by oral route with 0.2 mL each at an immune dose of 1 ×109 CFU.Two weeks and four weeks after the first immunization,the chickens were immunized with the same route and dose for three times.After immunization,blood samples were collected from immunized group every week,and the serum antibody agglutination titer was determined by S9HH(pBR322-APEC fim)agglutination antigen developed in the laboratory.The immunized attack group,the challenge control group and the healthy control group were weighed every two weeks after immunization and every week after challenge.Two weeks after the third immunization,APEC QD2(O78)virulent strain was injected into the posterior air sac to challenge.The dose was 1×109 CFU 0.2 ml per chicken.The chickens were observed continuously for 14 days.The mental and diarrhea scores of the three groups were scored every day according to the scoring standard.After 14 days of continuous observation,all live chickens were killed and observed.According to the experimental results,there was no significant difference in the average daily gain between the immunized chicks and the healthy control group.Through the observation of tissues and organs of chicks.The results showed no visible pathological changes,indicating that SG01Δ asd(pYA3342-APEC fim)strain had no significant effect on the growth performance of chicks and did not cause obvious disease.Serum antibody production was detected in the first week after immunization,and gradually increased.After the third week of immunization,more than half of the chicks had a titer of 1:32(7/10)of specific pili agglutination antibody,and maintained at a high level.In APEC QD2(O78)challenge,the related clinical symptoms and pathological changes were found in both the immunization attack group and the challenge control group.The average scores of clinical symptoms and organ pathological changes in the challenge control group were significantly higher than those in the immunization attack group and the healthy control group(p<0.05).The morbidity in the challenge control group were 100%.In contrast,the morbidity in the immunization attack group were 60%,and the morbidity were significantly decreased.The results showed that SG01 Δ asd(pYA3342-APEC fim)strain as a vector born vaccine candidate has the potential application to prevent APEC QD2(O78)infected chickens. |
PDF Full Text Request