Generation and protective efficacy of a cold-adapted attenuated genotype 2b porcine epidemic diarrhea virus

원호근,이동욱,장규환,노윤희,이승철,최환원,윤인중,유한상,이창희 대한수의학회 2019 Journal of Veterinary Science Vol.20 No.4

The recent emergence and re-emergence of porcine epidemic diarrhea virus (PEDV) underscore the urgent need for the development of novel, safe, and effective vaccines against the prevailing strain. In this study, we generated a cold-adapted live attenuated vaccine candidate (Aram-P29-CA) by short-term passage of a virulent PEDV isolate at successively lower temperatures in Vero cells. Whole genome sequencing identified 12 amino acid changes in the cold-adapted strain with no insertions and deletions throughout the genome. Animal inoculation experiments confirmed the attenuated phenotype of Aram-P29-CA virus in the natural host. Pregnant sows were orally administered P29-CA live vaccines two doses at 2-week intervals prior to parturition, and the newborn piglets were challenged with the parental virus. The oral homologous prime-boost vaccination of P29-CA significantly improved the survival rate of the piglets and notably mitigated the severity of diarrhea and PEDV fecal shedding after the challenge. Furthermore, strong antibody responses to PEDV were detected in the sera and colostrum of immunized sows and in the sera of their offspring. These results demonstrated that the cold-adapted attenuated virus can be used as a live vaccine in maternal vaccination strategies to provide durable lactogenic immunity and confer passive protection to litters against PEDV.

기니픽에서 Haemophilus parasuis 백신(글래써씨병 백신)의 항체가 측정을 위한 ELISA 기법 개발

원호근,김길동,신은경,강정무,김현일,조윤상,한태욱 한국수의공중보건학회 2005 예방수의학회지 Vol.29 No.2

PoulShotⓇ MG-F 백신의 마이코플라즈마 감염증에 대한 산란계 농장에서의 야외 효능 평가

전은옥,원호근,모인필,우창곡 한국가금학회 2010 韓國家禽學會誌 Vol.37 No.2

Mycoplasma gallisepticum (MG) infection results in severe economic loss in the poultry industry. In the previous reports, F strain, one of the MG live vaccine strains, could protect the bird from field infection of MG strains. In this study, efficacy of PoulShotⓇ MG-F vaccine againset mycoplasma gallisepticum infection was evaluated for filed application in commercial layers. Commercial layers from two different farms received with PoulShotⓇ MG-F, MG-F live vaccine at 9~14 weeks of age. Serological immune response to MG vaccine, the persistency of MG vaccine strain in the upper respiratory tracts and egg production rate were evaluated in the vaccinated, contacted or nonvaccinated flocks. The serological response was first detected at 3 weeks after vaccination (WAV) and persisted for 31 WAV. The MG vaccine strains were also persisted for 31 WAV based on the reisolation and PCR detection. There was no difference between the vaccinated or non-vaccinated flocks in the egg production rate but in the abnormality rate of eggs. Based on the above results, we suggested that the PoulShotⓇ, MG-F live vaccine was fully immunogenic and had characteristics of long persistence in the upper respiratory trachea which will reduce economic loss caused by MG infection in the layer farms 본 연구에서는 Mycoplasma gallisepticum F 주 생독 백신(PoulShotⓇ MG-F)의 안전성과 효능을 평가하였다. 충청북도 진천과 경기도 안성 지역의 산란계 농장을 선정하여, 백신과 야외주 공격 접종에 따른 혈청 역가 변화, 상부 호흡기에서의 마이코플라즈마균 재분리, 조직학적 병변과 백신 접종군 및 백신 미접종군 간의 산란율 및 오파란율의 차이를 농장 별로 평가하였다. 백신 접종에 의한 혈청 역가 변화는 백신 접종 후 3주부터 확인되었으며, 농장에 따라 접종 후 23주에서 31주까지 지속됨으로써 백신 항체가 오랫동안 유지되는 것을 확인할 수 있었다. 또한 상부 호흡기에서 MG-F 재분리 및 PCR에 의한 유전자 검출도 백신 접종 후 31주까지 양성이었다. 이러한 항체 및 항원의 지속적인 검출은 상부 호흡기에 MG-F 백신주의 집락 형성이 오랫동안 지속된다는 것을 의미하는 것이다. 동일한 방법으로 백신 접종군에 대한 야외주 공격 접종 후 상부 호흡기에서의 백신주 집락 형성을 분석한 결과, 공격 접종 후 3주까지 백신주의 집락 형성율이 야외주보다 동등하거나 높은 것으로 확인됨으로써 야외주 공격에 대한 백신주의 방어력이 입증되었다. MG-F의 안전성과 생산성 측면에서의 효능을 야외 농장에서 검증하기 위하여 두 실험 농장에서 백신 접종군과 백신 미접종군간의 산란율 및 오파란율을 비교하였다. 그 결과, MG-F 접종에 따른 임상적 부작용과 산란율 하락은 발견되지 않았으며, 오히려 백신 접종군의 오파란율이 백신 미접종군보다 평균 1~3% 낮은 것으로 분석됨으로써 백신 접종에 의한 난질 개선 효과가 있음이 확인되었다. 따라서, PoulShotⓇ MG-F 생균백신을 산란계에 접종하였을 때 임상적으로 안전하였으며, 오랜 기간 야외 감염에 방어할 수 있는 항체 형성과 상부 호흡기에서의 지속성이 확인됨으로써 마이코플라즈마 야외 감염을 효율적으로 방어할 수 있을 것으로 판단된다.

AlamarBlue™ assay를 이용한 닭 말초혈액에서의 림프구 증식 실험

홍영선,원호근,강정무,박선일,고필옥,채병조,한태욱 강원대학교 동물생명과학연구소(구 강원대학교 동물자원공동연구소) 2002 동물자원연구 Vol.13 No.-

Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency

정명환,원호근,신민경,오명환,심수진,윤인중,유한상 대한수의학회 2019 Journal of Veterinary Science Vol.20 No.2

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regionsamong the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Salmonella Enteritidis 분리주에서의 선발된 불활화 백신균주의 방어효과 및 면역원성

강정무,원호근,김은희,노윤희,최환원,한태욱,Kang, Zheng-Wu,Won, Ho-Keun,Kim, Eun-Hee,Noh, Yun-Hee,Choi, Hwan-Won,Hahn, Tae-Wook 대한수의학회 2011 大韓獸醫學會誌 Vol.51 No.1

Salmonella Enteritidis (SE) has been a major causative agent of food-borne human disease due to consumption of contaminated eggs and poultry meat. To prevent SE infection in poultry, and therefore minimize human infections, vaccination with either killed or live SE vaccine is suggested. We evaluated a newly developed killed bacterin using a representative SE isolate in Korea. Among pool of SE isolates, two highly virulent isolates (the one isolate from chicken, the other from human) were selected by measuring mortality in mouse and chickens administered. The chickens were injected intramuscularly with killed vaccine and were challenged with highly virulent SE strain 3 week after vaccination. The recovered colony count (cfu/g) of spleen and cecal content in the vaccinated groups was reduced compared with those of the unvaccinated control group. The antibody level in the vaccinated groups was higher at 3 week post vaccination. These results indicate that vaccination with killed vaccine was effective in preventing the infection of virulent SE. Further study for a large number of layers should be needed for the effect of egg production, SE shedding in feces, persistence of antibody level.

PoulShot^® MG-F 백신의 마이코플라즈마 감염증에 대한 산란계 농장에서의 야외 효능 평가

전은옥,우창곡,원호근,모인필,Jeon, Eun-Ok,Woo, Chang-Gok,Won, Ho-Keun,Mo, In-Pil 한국가금학회 2010 韓國家禽學會誌 Vol.37 No.2

본 연구에서는 Mycoplasma gallisepticum F 주 생독 백신(PoulShot$^{(R)}$ MG-F)의 안전성과 효능을 평가하였다. 충청북도 진천과 경기도 안성 지역의 산란계 농장을 선정하여, 백신과 야외주 공격 접종에 따른 혈청 역가 변화, 상부 호흡기에서의 마이코플라즈마균 재분리, 조직학적 병변과 백신 접종군 및 백신 미접종군 간의 산란율 및 오파란율의 차이를 농장별로 평가하였다. 백신 접종에 의한 혈청 역가 변화는 백신 접종 후 3주부터 확인되었으며, 농장에 따라 접종 후 23주에서 31주까지 지속됨으로써 백신 항체가 오랫동안 유지되는 것을 확인할 수 있었다. 또한 상부 호흡기에서 MG-F 재분리 및 PCR에 의한 유전자 검출도 백신 접종 후 31주까지 양성이었다. 이러한 항체 및 항원의 지속적인 검출은 상부 호흡기에 MG-F 백신주의 집락 형성이 오랫동안 지속된다는 것을 의미하는 것이다. 동일한 방법으로 백신 접종군에 대한 야외주 공격 접종 후 상부 호흡기에서의 백신주 집락 형성을 분석한 결과, 공격 접종 후 3주까지 백신주의 집락 형성율이 야외주보다 동등하거나 높은 것으로 확인됨으로써 야외주 공격에 대한 백신주의 방어력이 입증되었다. MG-F의 안전성과 생산성 측면에서의 효능을 야외 농장에서 검증하기 위하여 두 실험 농장에서 백신 접종군과 백신 미접종군간의 산란율 및 오파란율을 비교하였다. 그 결과, MG-F 접종에 따른 임상적 부작용과 산란율 하락은 발견되지 않았으며, 오히려 백신 접종군의 오파란율이 백신 미접종군보다 평균 1~3% 낮은 것으로 분석됨으로써 백신 접종에 의한 난질 개선 효과가 있음이 확인되었다. 따라서, PoulShot$^{(R)}$ MG-F 생균백신을 산란계에 접종하였을 때 임상적으로 안전하였으며, 오랜 기간 야외 감염에 방어할 수 있는 항체 형성과 상부 호흡기에서의 지속성이 확인됨으로써 마이코플라즈마 야외 감염을 효율적으로 방어할 수 있을 것으로 판단된다. Mycoplasma gallisepticum (MG) infection results in severe economic loss in the poultry industry. In the previous reports, F strain, one of the MG live vaccine strains, could protect the bird from field infection of MG strains. In this study, efficacy of PoulShot$^{(R)}$ MG-F vaccine againset mycoplasma gallisepticum infection was evaluated for filed application in commercial layers. Commercial layers from two different farms received with PoulShot$^{(R)}$ MG-F, MG-F live vaccine at 9~14 weeks of age. Serological immune response to MG vaccine, the persistency of MG vaccine strain in the upper respiratory tracts and egg production rate were evaluated in the vaccinated, contacted or nonvaccinated flocks. The serological response was first detected at 3 weeks after vaccination (WAV) and persisted for 31 WAV. The MG vaccine strains were also persisted for 31 WAV based on the reisolation and PCR detection. There was no difference between the vaccinated or non-vaccinated flocks in the egg production rate but in the abnormality rate of eggs. Based on the above results, we suggested that the PoulShot$^{(R)}$, MG-F live vaccine was fully immunogenic and had characteristics of long persistence in the upper respiratory trachea which will reduce economic loss caused by MG infection in the layer farms.

돼지단독 불활화 백신의 효능 시험

최환원 ( Hwan Won Choi ),원호근 ( Ho Keun Won ),윤인중 ( In Joong Yoon ) 한국수의공중보건학회 2008 예방수의학회지 Vol.32 No.2

Comparison of different primers and enrichment media for the rapid detection of Salmonella using polymerase chain reaction

김지주,박소연,조영재,원호근,한태욱 한국예방수의학회 2014 예방수의학회지 Vol.38 No.3

Salmonellosis is one of the most common food-borne diseases in both humans and animals. The recovery of Salmonella from fecal and environmental samples by bacteriological assays takes several days. Polymerase chain reaction (PCR) has become an important technique for the rapid detection of Salmonella in a variety of samples, including feces. For rapid identification of Salmonella by PCR, 1 ml of enrichment culture was harvested after overnight incubation and DNA was extracted by heat lysis. To investigate the optimal conditions for rapid PCR detection of Salmonella, three different primer sets and three different enrichment media were used on a panel of Salmonella strains and a panel of non-Salmonella strains. The results showed that selenite cysteine enrichment broth and a primer set designed for the invA gene provided the most specific and rapid detection of Salmonella by PCR after the enrichment step.

Salmonella enterica serovars Enteritidis의 온도감수성 변이주 및 폴리인산키나아제 변이주의 제작과 방어효과

김기주,박소연,조영재,곽정연,강정무,김은희,최환원,원호근,노윤희,한태욱,Kim, Kiju,Park, Soyeon,Cho, Youngjae,Kwak, Jeong-Yeon,Kang, Zheng-Wu,Kim, Eun-Hee,Choi, Hwan-Won,Won, Ho-Keun,Noh, Yun-Hee,Hahn, Tae-Wook 대한수의학회 2013 大韓獸醫學會誌 Vol.53 No.4

This study was focusing on evaluating the protection of polyphosphate kinase (ppk) deleted and/or temperature-sensitive (ts) Salmonella Enteritidis (SE) as an attenuated vaccine in chickens. We constructed SEppk, SEts and SEppk::ts mutants and screened those mutants by growth capability in vitro, protection study in mice model and antibody response in chickens. Among the mutants, SEppk::ts-3 was selected because it showed higher growth capability, good protection against highly virulent SE in mice model, and good antibody response in chickens. SEppk::ts-3 also showed good protection against highly virulent SE isolate because it decreased colonization of virulent SE challenge strain in spleen, liver and cecum compared with the non-vaccinated control. The SEppk::ts-3 mutant showed cross-protection against S. Gallinarum (SG) challenge although the its cross-protection rate was a little lower than that of SG9R, a commercial vaccine against SG infection. To use for live attenuated vaccine in chickens, it should further be characterized.