초록 ▼

○ 연구결과
1.JOL916를 2x106내시 2x107cfu를 근육 또는 피하로 5-6주령에 접종하면 SG9R보다 우수한 방어면역을 부여한다.1주령에 2x106cfu경구적으로 접종하여도 효과가 탁월하지만 3-4주령에서는 접종량을 올려야 하며 부작용이 나오기 쉬어 경구접종으로 실용화에 한계가 있다.
2.JOL916실용화 위한 실험실 내 시험을 시험백신을 제작하여 모두 수행하였다.
3.JOL969,JOL971은 안전성이 매우 높지만 방어력이 떨어진다.
4.JOL1229는 JOL916와 균수로 4:1로 혼합하여 1주

○ 연구결과
1.JOL916를 2x106내시 2x107cfu를 근육 또는 피하로 5-6주령에 접종하면 SG9R보다 우수한 방어면역을 부여한다.1주령에 2x106cfu경구적으로 접종하여도 효과가 탁월하지만 3-4주령에서는 접종량을 올려야 하며 부작용이 나오기 쉬어 경구접종으로 실용화에 한계가 있다.
2.JOL916실용화 위한 실험실 내 시험을 시험백신을 제작하여 모두 수행하였다.
3.JOL969,JOL971은 안전성이 매우 높지만 방어력이 떨어진다.
4.JOL1229는 JOL916와 균수로 4:1로 혼합하여 1주령 또는 6주령 병아리에 경구적으로 적용하여 안전성 및 방어력이 우수하다.

Abstract ▼

IV. Result and conclusions
1. Construction of a vaccine candidate by deletion of genes
Gene-deleted mutants were successfully constructed and the deletions are confirmed by PCR JOL916 vaccine is differentiated from wild type strains.
2. Construction of auxotrophic mutant as vaccine candidat

IV. Result and conclusions
1. Construction of a vaccine candidate by deletion of genes
Gene-deleted mutants were successfully constructed and the deletions are confirmed by PCR JOL916 vaccine is differentiated from wild type strains.
2. Construction of auxotrophic mutant as vaccine candidates that are lethal in hosts Successfully constructed.
3. Effect of gene deletion and suitability of JOL916 as a candidate
Deletion of lon resulted in mucoidal character and increase of fimbriae, increased invasiveness, and a little attenuation. Deletion of cpxR resulted in increase of fimbriae and a little attenuation of pathogenicity. Deletion of both genes resulted in increased invasiveness in phagocytes, mucoidal character, and increase of fimbrie. JOL916 was attenuated enough and better invasiveness in macrophage and longer duration of survival of the strain without significant pathogenicity. JOL916 appeared to be a potent candidate for a vaccine strain compared to JOL394, JOL914, or JOL915.
4. Determination of application condition of JOL916
Killed vaccine and live vaccine were prepared for experimental vaccination to compare efficacy of the candidate. Oral and parenteral routes of inoculation were performed and the efficacy was compared. Side-effect, immune-response, protection after challenge inoculation were observed for the comparison. Lie vaccination with JOL916 offered better protection compared to formalin-killed bacteria. Vaccination with JOL916 via the parenteral route offered better protection compared to the vaccination via the oral route. However, as vaccination with live cells via the oral route also offered reasonable protection, further research for oral application should be continued in the future.
Moderate to severe side effect including death was also observed in diseased or non healthy chickens in some experiment. Elevation of protection efficiency will be connected to reduction of vaccination dose, therefore, safety continuous effort for development of safety vaccine candidate is required.
5. Safety and protection efficacy of JOL916 as a vaccine candidate
After vaccination no detectable clinical signs are found in healthy chickens.
Detectable level of slight gross lesions are found or not found on the livers and spleens of vaccinated chickens, although the lesions are not likely to be related to side effect but likely to be included in immune responses. Vaccine candidate JOL916 invaded and proliferated at high efficiency to the spleen rather than the liver, diminish after the second week post vaccination the number of recovered bacteriae from the internal organs were neglectable after that.
Vaccination with 2 x 106 cfu per chicken via the parenteral route (intra muscular) induced significant levels of antigen specific immune responses and offered perfect protection against wild type challenge. Only a part of the vaccinated chickens showed slight and temporal clinical signs as an acute response (4th to 7th day post challenge) and demonstrated rapid recovery of general condition and body weight increase. Postmortem examination also demonstrated considerable protection offered by vaccination with 2 x 106 cfu per chicken via the intramuscular route.
Vaccine candidate JOL916 showed similar result compared to SG9R in the safety assay when inoculated with 2 x 106 cfu per chicken via the intramuscular route. However, better protection was offered by JOL916 based on the postmortem examination result such as gross lesion score and bacterial recovery.
6. Vaccination possibility in young chicks with candidates including JOL969 and JOL971
No live vaccine strain tested offered protection when vaccinated on the first day of birth. An auxotrophic strain JOL971 (high copy ori) did not offer reasonable protection even though it was safe after oral inoculation of live vaccine. Instead, oral inoculation of chicken with 2 x 107 cfu of JOL916 via the oral route showed a good efficacy no significant side effect such as reduced bodyweight increase, clinical sign, or significant gross lesions. For oral inoculation of young chicks with JOL916, required doses for reasonable protection were 2 x 107 cfu for the first week of age, 2 x 108 cfu for the second week of age, and more than 2 x 108 cfu for over the age. However, elevated dose is often related to side effect safety vaccine strain should be developed for better application for young chicken via the oral route.
7. Production of trial vaccine products and general and safety assay
Trial vaccine strain was successfully produced and submitted to general laboratory test and safety test.
8. Efficacy of the trial vaccine
Immunogenicity and protection against wild type challenge inoculation offered by trial vaccine products were monitored. Three vaccines including JOL916, PoulShot SG9R (39SG9R01), and Nobilis SG9R demonstrated reasonable immunogenicity even though SG9R antigen was used for the immunological test.
9. Long term experiment with the trial vaccine in a farm
No clinical sign was detected bodyweight increase, death rate, egg production rate were maintained at the level of normal and health model flock. Egg shell was not contaminated with the vaccine strain (JOL916). When he prime vaccination was performed on the 7 week of age, the reciprocal titer demonstrated to decrease since the 16 week of age. Therefore, the booster was inoculated on the 19 week of age that resulted in the elevation of the titer until 32 week of age, after the minimum level at 20 week of age.
10. Construction of live adjuvant strain
Successfully constructed and named JOL1229.
11. Application condition of JOL1229
For oral application of JOL1229 in a week age of chicken, JOL916 and JOL1229 should be mixed at rate of 4:1 on the basis of cell number, at a total cell number of 1 x 107 cfu this condition offers the best immune response and protection. However, for booster inoculation at 6 week of age via the oral route, JOL1229 should not be mixed inoculation with only JOL916 offers better protection. This prime-booster vaccine program is safe and offer good protection.
For oral inoculation at both 6 week of age prime vaccination and booster at 16 week of age, 1 x 108 cfu of mixture at 4:1 for JOL916 and JOL1229 can be applied. This prime-booster vaccine program is also safe and offer good protection.
12. Long termexperiment of JOL1229
Clinical sign, bodyseight increase, egg production rate, and egg contamination with vaccine strain, and immune responses were observed during and after prime vaccination at 6 week of age and booster at 16 week of age. Mixed vaccination of JOL916 and JOL1229 (totally 1 x 108 cfu, via the oral route) showed better cellular and mucosal immune responses compared to other vaccinated groups with JOL916 or SG9R. No significant differences were observed in egg production rates between all vaccinated (JOL1229+JOL916, JOL916, SG9R) and unvaccinated control group. No contamination with vaccine strains were observed in egg contents for 4 weeks after egg production after vaccination. The mixture of JOL1229 and JOL916 is suitable for oral vaccination. This mixture is a potent vaccine candidate for oral application to chickens.

목차 Contents

• 표지 ... 1
• 제 출 문 ... 2
• 요 약 문 ... 3
• SUMMARY ... 10
• CONTENTS(영 문 목 차) ... 17
• 목 차 ... 19
• 제 1 장 연구개발과제의 개요 ... 21
• 제 1 절 연구개발의 목적 ... 21
• 제 2 절 연구개발의 필요성 ... 21
• 제 3 절 연구개발의 범위 ... 23
• 제 2 장 국내외 기술개발 현황 ... 25
• 제 1 절 국내외 기술개발 현황 ... 25
• 제 2 절 본 연구팀의 선행기술 수준 ... 27
• 제 3 장 연구개발수행 내용 및 결과 ... 28
• 제 1 절 연차별 세부과제별 연구개발의 목표 및 개요 ... 28
• 1. 1차 연도 세부과제별 연구목표 및 연구내용 (표 4) ... 28
• 2. 2차 연도 세부과제별 연구목표 및 연구내용 (표 5) ... 30
• 3. 3차 연도 세부과제별 연구목표 및 연구내용 (표 6) ... 32
• 제 2 절 연구개발 수행방법 및 결과 ... 35
• 1. 병원성 관련 유전자 결실균주 작성 ... 35
• 2. 영양요구성 변이균주 작성 ... 43
• 3. 변이균주의 백신후보균주로서 가능성 시험 ... 48
• 4. 백신-도전 실험을 위한 예비실험 ... 57
• 5. 백신후보균주 JOL916의 접종형태 및 경로 모색 실험 ... 61
• 6. 백신후보균주 JOL916의 안전성 및 방어력 평가 실험 ... 80
• 7. 백신후보균주 JOL916와 SG9R균주의 비교 실험 ... 88
• 8. 어린 병아리에서 JOL916의 접종 조건 탐색 ... 94
• 9. 시험백신의 생산 및 일반, 안전 시험 ... 99
• 10. 시험 백신 효능성 평가 ... 102
• 11. 시험백신의 면역원성 시험 ... 105
• 12. 야외농장에서의 안전시험 ... 106
• 13. 면역증강 LTB 균주 JOL1229 제작 ... 115
• 14. 1주령 병아리에서 JOL1229의 혼합비율 결정 및 안전성 평가 ... 118
• 15. 1주령 병아리에서 JOL1229를 이용한 백신 program 시험 ... 126
• 16. 6주령 산란계에서 JOL1229의 혼합비율 결정 및 안전성 평가 ... 133
• 17. 6주령 산란계에서 JOL1229를 이용한 백신 program 실험 ... 143
• 18. JOL1229를 이용한 백신의 장기적 실험 ... 149
• 제 4 장 목표달성도 및 관련분야에의 기여도 ... 156
• 제 5 장 연구개발 성과 및 성과활용 계획 ... 158
• 1. 기술양도 및 산업화 계획 ... 158
• 2. 교육지도 홍보 등 기술확산 계획 등 ... 158
• 3. 특허, 품종, 논문 등 지식재산권 확보 계획 등 ... 158
• 제 6 장 연구개발과정에서 수집한 해외과학기술정보 ... 161
• 제 7 장 참고문헌 ... 162
• 끝페이지 ... 166