초록 ▼

5. 최종 연구결과 요약
○ M41 HI 진단액 비교 및 안전성
-자체제조, 상업용 및 VLA HI 진단액 각각을 이용하여 국내 5개업체 및 수입업체의 닭전염성기관지염 백신면역 항항혈에 대한 HI 역가 비교는 거의 동일함을 확인
-자체제조 HI 진단액은 4℃와 -20℃(50% glycerol)에서 7개월간 역가 유지 확인
○ 국가검정기준 검사법 VN test(VNT)와 HI 및 ELISA법과의 상관관계 시험
-국가검정 닭전염성기관지염 백신주 M41, H120, KM91, K2에 대한 기존검사법 VNT는 E

5. 최종 연구결과 요약
○ M41 HI 진단액 비교 및 안전성
-자체제조, 상업용 및 VLA HI 진단액 각각을 이용하여 국내 5개업체 및 수입업체의 닭전염성기관지염 백신면역 항항혈에 대한 HI 역가 비교는 거의 동일함을 확인
-자체제조 HI 진단액은 4℃와 -20℃(50% glycerol)에서 7개월간 역가 유지 확인
○ 국가검정기준 검사법 VN test(VNT)와 HI 및 ELISA법과의 상관관계 시험
-국가검정 닭전염성기관지염 백신주 M41, H120, KM91, K2에 대한 기존검사법 VNT는 ELISA 보다 HI 시험법이 더 높은 상관관계를 보임
-M41 HI 진단액을 이용하여 M41, H120, KM91, K2 백신면역 항혈청에 대한 HI 결과 M41과 H120은 상관관계를 보였고, KM91과 K2는 상관관계가 낮음을 확인
○ 공격접종 및 방어효능 측정
-M41 백신접종 후 HI 역가 5 log₂에 대한 공격접종 실시 90% 방어효능 확인
○ 국가검정기준 닭전염성기관지염 역가시험법 개선 및 설정
-백신주 M41 및 H120의 역가시험법은 M41 HI 진단액을 이용한 HI 시험
:HI 시험 결과 개체별 HI 역가 5 log₂ 이상이며, 전체의 80% 이상인 경우 합격
-백신주 KM91 및 K2 역가시험법은 기존 역가시험법 VNT 수행

Abstract ▼

Agreement between Haemagglutination Inhibition and Virus Neutralization test for Infectious Bronchitis virus vaccine potency test
Infectious bronchitis virus (IBV) is a causative agent causing respiratory and urogenital disorder in chicken. To prevent the disease, live (L) and killed (K) vaccines

Agreement between Haemagglutination Inhibition and Virus Neutralization test for Infectious Bronchitis virus vaccine potency test
Infectious bronchitis virus (IBV) is a causative agent causing respiratory and urogenital disorder in chicken. To prevent the disease, live (L) and killed (K) vaccines have widely been used in the fields. The antibody formation after vaccinations has been tested by haemagglutination inhibition (HI) test, virus neutralization (VN) test and ELISA. Among them, VN and HI are generally being used to detect erotype-specific antibody because ELISAs are sensitive but type or strain specific. In general, VN test is the golden method but there have been shortcomings for laborious work, cost and animal welfare. Therefore, the aim of the study was to compare the HI and VN test for alternative IBV vaccine potency test.
IBV vaccine antisera were obtained from the specific pathogen free (SPF) chickens given intramuscular and oral inoculation of IBV monovalent to pentavalent vaccines. The sera were collected at 3 weeks after postinnoculation and subjected to HI and VN test against vaccine strain M41. IBV M41 was propagated in 10 day-old SPF chicken egg. Allantoic fluid was centrifuged at 18,000 rpm for 2.5 h and the pellet was suspended to PBS. Phospholipase C was added at a final concentration of 1U/mL, which was used as haemagglutination inhibition (HI) antigen. Commercial and Veterinary Laboratories Agency (VLA) antigen were used for controls. The HI test was done by adding 4HA units of HA antigen to kaolin-treated antisera, according to the OIE manual. The VN test was conducted using embryonated chicken eggs by inoculating a mixture of vaccine strain M41 and pooled vaccine antisera by the usual procedure alpha method. The r2, as a measure for correlation between VN and HI titers, was calculated using Excel software (Microsoft Office) and Cohen kappa values between HI and VN test were calculated to determine the concordance.
There were no differences in HI titers using IBV M41 antigens such as commercial and VLA antigen. Vaccine sera were examined by VN and HI test. The r2 values for correlation between VN and HI titers were 0.7641 (Fig. 1). Cohen kappa was K (HI 2⁶) = 0.77 - 0.47/1- 0.47 = 0.56, K (HI 2⁴) = 0.86 - 0.5/1 - 0.5 = 0.72, respectively (Table 2). Conclusion The r2 values between VN and HI titers were 0.76 (Fig. 1). Cohen kappa value was K (HI 2⁶) = 0.56 and K (HI 2⁴) = 0.72, respectively (Table 2). We found that K (HI 24) showed agreement between VN and HI compared to K (HI 2⁶). The results indicate that there is a moderate agreement between HI and VN test. Although VN test is the golden method, there have been shortcomings for laborious work, cost and animal welfare. As a result, HI test with M41 antigen could be alternative to VN test which is standard test for IBV vaccine potency test.
Agreement between Haemagglutination Inhibition and Virus Neutralization test for Infectious Bronchitis virus vaccine potency test and protection in chickens
Avian infectious bronchitis (IB) is a major disease in the poultry industry worldwide. IB is a highly contagious disease caused by infectious bronchitis virus (IBV) in the respiratory and urogenital tracts of chickens. To prevent the disease, live and killed vaccines have widely been used in the fields. Among them, IB M41 vaccine is one of the most prevalent IB vaccines worldwide including Korea. The antibody titer has been tested by haemagglutination inhibition (HI), virus neutralization (VN) and ELISA . In Korea, VN has only been used as potency test in Korean Standard Assay of Veterinary Biological Products. Although VN test is the golden method, there have been shortcomings for laborious work, cost and animal welfare. Therefore, the aims of the study were to compare the HI and VN test for alternative IB vaccine potency test and protection of chickens against IBV after IB M41 vaccination.
Specific pathogen free (SPF) chickens were given with IB M41 vaccine via intramuscular and tracheal inoculation and sera were collected at 3 weeks. It was subjected to HI and VN test. IBV was propagated in 10 day-old SPF chicken eggs. Allantoic fluids were centrifuged and the pellet was suspended to PBS. Neuraminidase was added at a final concentration of 200U/mL. The virus suspension was used for HA antigen and its stability was determined at 4℃, -20℃ and -20℃ in 50% glycerol for 8 months using M41 HA antigens. The HI was done by adding 4HA units of HA antigen to kaolin-treated antisera. The VN test was conducted by the usual procedure alpha method using embryonated chicken eggs which were inoculated by a mixture of IBV M41 and pooled antisera. Thecorrelation (r²) between VN and HI titers was determine dusing Excel software (Microsoft Office) and Cohen kappa values between HI (2⁴and2⁶) and VN were calculated to determine the concordance. To determine the protection efficacy, chickens showing HI titer 2⁴ after vaccination were challenged with IBV 10^4.5 EID₅₀ through eye drop and were observed daily for 5days. Trachea and kidney were collected for virus isolation. The number of IBV isolation was determined by RT-PCR analysis.
HA antigen stored at 4℃ showed the highest titer for 7 months (Fig. 1). Vaccine sera were examined by VN and HI test. The r² values for correlation between VN and HI were 0.8587. Cohen kappa was K(HI2⁴) = 0.91-0.5/1-0.5 = 0.82 and K(HI2⁶) = 0.77-0.47/1-0.47 = 0.56, respectively. To evaluate the protective efficacy, chickens showing HI titer 2⁴ afte rvaccination were challenged with IBV M41. Protection rates were 90% for chickens given the inactivated IBM41 vaccine and 0% for chickens treated with PBS.
The r² values for correlation between VN and HI titers were 0.8587 (Fig. 2). The results indicated that correlation was seen between HI and VN test. Cohen kappa were K (HI 24) = 0.82 and K (HI 26) = 0.56, respectively. It shows greater similarity between VN and HI in the HI 24, compared to HI 26 titer. Protective efficacy of chicken having HI titer 24 was 90%. Although VN test is the golden method, there have been shortcomings for laborious work, cost and animal welfare. As a result, HI could be used for alternative to VN which is used in Korean Standard Assay of Veterinary Biological Products potency test.

목차 Contents

• 총괄요약표 ... 1
• I. 연구배경 및 목표 ... 2
• 1. 연구배경 ... 2
• 2. 연구최종목표 ... 3
• 3. 연차별 연구개발 목표 및 내용 ... 3
• II. 연구방법 및 수행전략 ... 4
• 1. 재료 ... 4
• 2. 연구전략 및 방법 ... 4
• III. 연구결과 ... 5
• IV. 연구결과요약 ... 13
• V. 결론 ... 13
• VI. 연구결과 활용실적 및 계획 ... 14
• VII. 연구사업 추진상 문제점 및 건의사항 ... 14
• VIII. 주요연구과제 변경사항 ... 14
• IX. 참고자료 ... 14
• X. 영문초록 ... 16
• 끝페이지 ... 17