국내에서 분리한 Ornithobacterium rhinotracheale 균의 종란과 육계에서의 병원성

권용국(Yong Kuk Kwon),전우진(Woo Jin Jeon),강민수(Min Soo Kang),오재영(Jae Young Oh),안병기(Byung Ki An),송은아(Eun A Song),권준헌(Jun Hun Kwon),이청산(Cheong San Lee),김재홍(Jae Hong Kim) 韓國家禽學會 2010 韓國家禽學會誌 Vol.37 No.2

본 시험은 국내에서 분리한 Ornithobacterium rhinotracheale(OR) 병원성 세균이 부화중인 종란과 일반 육계에서 얼마만큼 병원성을 보이는지 알아보기 위하여 수행하였다. 첫 번째 9일령 부화란의 yolk sac에 국내에서 분리한 3종의 OR strain을 접종하여 12일 동안 관찰한 결과, 66% 이상의 폐사율이 관찰되어 높은 병원성이 인정되었다. 두 번째로 3주령 일반 육계를 대상으로 5가지 다른 공격접종법[Intratracheal, Intravenous, Intramuscular, Aerosol, Newcastle Disease Virus(NDV)와 혼합 Aerosol]으로 접종한 다음 병원성을 관찰하였다. NDV와 OR균을 동시에 분무 접종한 계군에서만 특이적인 임상증상인 침울, 기침, 안면 종대와 함께 부검시 치즈양 또는 요구르트와 비슷한 염증성 삼출물이 기낭에 관찰되었다. 조직학적으로도 기낭상피세포의 변성과 탈락, 대식세포와 다형태성 관립구의 침윤, 부종 등의 기낭염이 확인되었으며, 이들 기낭을 이용한 면역 조직 화학적 염색법을 적용한 결과 다량의 OR균의 항원이 검출되었다. 그러나 OR균만 단독 처리한 닭에서는 일시적이고 경미한 조직학적 병변만이 관찰되었다. 결론적으로 NDV가 OR균의 감염에 따른 임상 증상과 병리조직학적 병변 유발에 주요한 역할을 하는 것으로 판단된다. Field strains of Ornithobacterium rhinotracheale (OR) were tested on their virulence in specific pathogen free (SPF) embryonated chicken eggs and 3-week-old broilers. When infected with three different OR isolates (OR-161, OR-240 and OR-295) through yolk sac infection route, all strains appeared to be highly pathogenic with responsible mortality 66% and 100% within 12 days post infection (DPI). To test the virulence of OR in the commercial broilers, 3 week-old broilers were grouped depends on the inoculation route of OR isolate (OR-295) through five different infection routes; group 1 (IT: intratracheal), group 2 (IM: intramuscular), group 3 (Ⅳ: intravenous), group 4 (aerosol) and group 5 [Mixed: NDV (LaSota)+OR aerosol]. Within 5 to 7 days after inoculation, only broilers given NDV+OR were slightly depressed and coughing, and had mild facial redness. Grossly, foamy and yellow-white yogurt like exudate in the air sacs, predominantly in the abdominal air sacs was present. In histology, infiltration of the air sac epithelium and lamina propria by macrophage and polymorphonuclear granulocytes was seen with cell debris and inflammatory cells, correlated with the presence of OR antigen, as demonstrated by immunohistochemistry. Field strains of OR were able to induce high mortality in the embryonated chicken eggs, whereas broilers were less susceptible to OR infection. Interestingly, in the absence of NDV infection, the four groups of OR single infection only different route showed minimal and temporary microscopic air sac lesions. Thus, Newcastle disease virus (LaSota strain) showed triggering effects on the OR infection in chickens.

2003년 국내 원종계 및 종계의 추백리-가금티푸스 감염실태

김애란,김재홍,이영주,조영미,권준헌,권용국,이윤정,최준구,조성준,김민철,이은경,김창섭,양흥구,곽상익,성환우,모인필,Kim, Ae Ran,Kim, Jae Hong,Lee, Young Ju,Cho, Young Mi,Kwon, Jun Hun,Kwon, Yong Kuk,Lee, Youn Jeong,Choi, Jun Gu,Joh, Sung Jun,Kim, Min C 대한수의학회 2006 大韓獸醫學會誌 Vol.46 No.4

Serum samples of 30 chickens per flock from 6 grand parent stock (GPS) farms and 70 parent stock (PS) farms were collected for seroprevalent study of pullorum disease-fowl typhoid (PD-FT) infection by serum plate agglutination test (SPA). The incidence of PD-FT infection in GPS flocks and PS flocks were 0% and 15.7%, respectively. Especially PS flocks infected with PD-FT showed age dependent patterns that 22.2% of flocks between 20 to 30 weeks of age and 38.9% of flocks between 30 to 40 weeks of age were positive. The incidence of GPS flocks and PS flocks using Salmonella (S.) gallinarum 9R (SG9R) live vaccine were 33.3% and 58.6%, respectively. The sero-positive rate of 11 flocks were 6.7-83.3% by SPA and 2.9-55.6% by enzyme linked immunosorbent assay (ELISA), and ELISA showed more lower antibody levels than SPA. Furthermore, specific antibodies produced by SG9R vaccination were detectable by SPA using SG9R antigen without cross-reaction with the PD-FT infection.

국내 종계에서 난계대 전염병 감염 실태 보고

권용국(Yong Kuk Kwon),강민수(Min Soo Kang),오재영(Jae Young Oh),정병열(Byeong Yeal Jung),김혜령(Hye Ryoung Kim),김하영(Ha Young Kim),신소연(So Yeon Shin),권준헌(Jun Hun Kwon),정갑수(Gab Soo Chung) 韓國家禽學會 2010 韓國家禽學會誌 Vol.37 No.3

본 조사 연구는 종란을 생산하는 원종계, 종계, 백세미씨 알 생산 농장을 대상으로 수직 감염(난계대전파)되는 전염성 질병인 추백리/가금티푸스, 닭마이코플라즈마증(MG, MS), 전염성 빈혈증, 조류아데노바이러스 감염증에 대한 항원 및 항체 검사를 실시하였다. 조사 기간은 2009년 8월부터 12월까지 5개월간 원종계 45계군, 종계 1,018계군, 백세미씨알 생산 54계군에 대한 성적이다. 추백리/가금티푸스 항원 검사에서는 모든 계군이 음성으로 확인되었으나, 항체 검사결과 종계 3.2%, 백세미씨알 생산계군 3.0%의 항체 양성율이 관찰되었다. 계종별 가금티푸스의 발생률은 종계군의 항원 검사 결과와 상반되어 육계 44.3.7%, 백세미 26.2%, 산란계 15.7%, 토종닭 12.6%, 육용 종계 1.08%였다. MG 항체 검사 결과, 원종계 71.1%, 종계 및 백세미씨알 생산계군 각 88.7% 항체 양성율이 확인되었으며, MS 항체 검사 결과도 비슷한 수준으로 나타났다. 닭 전염성 빈혈 바이러스 검사 결과, 원종계 42.2%, 종계 18.0%가 바이러스를 갖고 있는 것으로 나타났으며, 항체 양성율도 86% 이상이었다. 이와 함께 조류 아데노바이러스항원검사결과에서는원종계4.4%, 종계2.7%, 백세미씨알 생산계군 9.35%가 바이러스를 보유하고 있는 것으로 조사되었다. 결론적으로 국내 종계군은 닭 마이코플라즈 마증과 닭 전염성 빈혈에 상당히 높은 수준으로 감염되어 있는 것으로 판명되어 질병별 적절한 예방책이 필요한 것으로 나타났다. A survey of transovarially transmitted diseases, including salmonellosis [(pullorum disease; PD)/(fowl typhoid; FT)], mycoplasmosis, avian infectious anemia (CIA), and fowl adenovirus infection was conducted in the breeder chickens from August to December in 2009. The numbers of flocks sampled out were: Grand Parents Stock (GPS), 45; Parents Stock (PS) 1,018, Baeksemi breeder (BSB) 54. The seroprevalence of salmonellosis (PD/FT) were 0% (GPS), 3.2% (PS), and 3% (BSB), respectively. A total of 983 chicken farms were affected with FT outbreaks between 2000 and 2008. The incidence of FT in commercial broilers, Baeksemi, commercial layers, native chickens, and broiler breeders was 44.3%, 26.2%, 15.7%, 12.6% and 1.08%, respectively. Of the affected broilers, over 90% birds were under 2 weeks of age, indicating it was possible that they were infected with S. gallinarum via vertical transmission. The sero-positive flocks against Mycoplasma gallisepticum (MG) were 71.1% (GPS), 88.7% (PS), 88.7% (BSB), while the rates of positive flocks against Mycoplasma synoviae (MS) were 86.0% (GPS), 77.0% (PS), and 98.0% (BSB). In GP and parent farms, the detection rates on specific genes of CIA virus were 19/45 (42.2%), and 169/1039 (18.0%), respectively, whereas the seroprevalence of CIA were 86.0% in GPS and 93.7% in PS flocks. In addition, positive flocks of fowl adenoviruses were 4.4% (GPS), 2.7% (PS) and 9.35% (BSB), respectively. As the results, avian mycoplasmosis and CIA have been more prevailing in chicken breeder than avian salmonellosis and fowl adenovirus infection in Korea.

흰뺨검둥오리(Anas poecilorhyncha)에서 분리된 뉴캣슬병 바이러스의 특성

최강석,이은경,전우진,권준헌,양창범,Choi, Kang-Seuk,Lee, Eun-Kyoung,Jeon, Woo-Jin,Kwon, Jun-Hun,Yang, Chang-Bum 대한수의학회 2008 大韓獸醫學會誌 Vol.48 No.2

Newcastle disease virus (NDV) is the causative agent of a highly contagious and devastating Newcastle disease of poultry. A NDV (isolate DK1/07) was isolated from apparently healthy wild spot-billed ducks (Anas poecilorhyncha) captured at upper branch of the SapGyo Creek in Chungbuk province, Korea during early 2007. The DK1/07 isolate of minimum chicken embryo lethal dose killed all SPF chicken embryos within 60 h. The cleavage site of the F protein possessed the amino acid sequence $^{112}R-R-Q-K-R-F^{117}$, which is a motif characteristic of virulent NDV strains. The F protein-based phylogenetic analysis revealed that the DK1/07 duck isolate was included in the cluster of genotype VIId and most closely related to recent NDV isolates obtained from chicken farms in Korea. Epidemiological importance of virulent NDV from wild duck is discussed.

한국호흡기형 닭전염성기관지염 생독백신주의 작성

최강석,전우진,이은경,계수정,박미자,권준헌,Choi, Kang-Seuk,Jeon, Woo-Jin,Lee, Eun-Kyoung,Kye, Soo-Jeong,Park, Mi-Ja,Kwon, Jun-Hun 대한수의학회 2011 大韓獸醫學會誌 Vol.51 No.3

An attenuated vaccine strain AVR1/08 of Korean respiratory type of infectious bronchitis virus (IBV) was developed by 89th passages of IBV D85/06 strain in chicken eggs. The AVR1/08 strain had higher virus titer at least 20 times ($10^{1.3}$) than the parent virus D85/06 by egg inoculation method. The AVR1/08 strain had a single point mutation (S to Y) at position 56 of spike protein of IBV compared to parent virus IBV D85/06 strain. The mutation was observed consistently at viruses after 47th passage in chicken eggs. The AVR1/08 strain showed no virulence even after 6 passages in chickens and all chickens inoculated induced anti-IBV antibody 14 days after vaccination. The AVR1/08 strain had broad protective efficacy against QX type Korean nephropathogenic virus (Q43/06 strain), KM91 type Korean nephropathogenic virus (KM91 strain) and Korean respiratory virus (D85/06 strain). In contrast, Massachusetts (Mass) type attenuated vaccine strain H120 showed protection of 37.5 to 50% against these three viruses. Our results indicate that the AVR1/08 strain has potential as an attenuated vaccine effective in controlling IBVs circulating in Korea.

배양세포에서 Semi-quantitative RT-PCR에 의한 조류인플루엔자 H9N2의 전사활성 분석 최적 시기 결정 및 전사체 분석

나기윤,이영민,변승준,전익수,박종현,조인수,주이석,이윤정,권준헌,구용범,Na, Gi-Youn,Lee, Young-Min,Byun, Sung-June,Jeon, Ik-Soo,Park, Jong-Hyeon,Cho, In-Soo,Joo, Yi-Seok,Lee, Yun-Jung,Kwon, Jun-Hun,Koo, Yong-Bum 한국미생물학회 2009 미생물학회지 Vol.45 No.3

The transcription of mRNA of avian influenza virus is regulated temporally during infection. Therefore, the measurement of transcript level in host cells should be performed before viral release from host cells because errors can occur in the analysis of the transcript levels if the viruses released from the infected cells re-infect cells. In this study, the timing of viral release was determined by measuring the level of viral RNA from viruses released from H9N2-infected chicken fibroblast cell line UMNSAH/DF-1 by semi-quantitative RT-PCR. The viral genomic RNA was isolated together with mouse total RNA which was added to the collected medium as carrier to monitor the viral RNA recovery and to use its GAPDH as an internal control for normalizing reverse transcription reaction as well as PCR reaction. It was found that viral release of H9N2 in the chicken fibroblast cell line UMNSAH/DF-1 took between 16 and 20 h after infection. We measured all 8 viral mRNA levels. Of the 8 transcripts, 7 species of viral mRNAs (each encoding HA, NA, PB1, PB2, NP, M, NS, respectively) except PA mRNA showed robust amplification, indicating these mRNA can be used as targets for amplification to measure transcript levels. These results altogether suggest that the method in this study can be used for screening antiviral materials against viral RNA polymerase as a therapeutic target. 조류인플루엔자 바이러스 mRNA의 전사는 감염 동안에 시간적으로 조절되어진다. 감염 후 세포 내에서 증식된 바이러스가 방출되어 세포를 다시 감염하게 되면 전사 수준 분석에 오차가 발생할 수 있으므로 바이러스가 방출 되기 이전에 전사 수준의 측정이 이루어져야 한다. 본 연구에서는 조류인플루엔자 H9N2를 감염시킨 닭의 섬유아세포주 UMNSAH/DF-1에서 바이러스 감염 후 증식된 바이러스의 방출까지의 시간을 측정하기 위하여 배양액 중에 방출되는 바이러스 RNA 게놈 수준을 semi-quantitative RT-PCR에 의해 측정하였다. 배양액 중의 바이러스 RNA 게놈의 분리 과정에서 발생할 수 있는 RNA 회수율의 오차를 보정하기 위해 mouse의 전체 RNA를 배양액 시료에 넣어서 바이러스 RNA 분리에 carrier로 사용하고 그 속에 포함된 mouse의 GAPDH를 RNA를 역전사 반응 및 PCR의 내부 대조 RNA로 사용하였다. 그 결과 감염 후 방출까지 16~20시간이 소요됨을 알 수 있었다. 따라서 감염 후 12시간 후에 세포 내에서 합성된 8종의 전사체의 수준을 측정하였다. 8종의 mRNA 중 PA를 제외한 7종의 mRNA (HA, NA, PB1, PB2, NP, M, NS를 암호화하는 mRNA)는 뚜렷한 증폭 band를 보였으며, 이것은 이들이 전사활성 측정에 사용될 수 있는 것으로 나타났다. 이상과 같은 전사수준의 측정 방법은 조류인플루엔자 바이러스의 RNA polymerase를 표적으로 한 항바이러스제의 검색에 사용할 수 있을 것으로 보인다.

닭 도축장에서의 뉴캣슬병 바이러스 오염 실태 조사

최강석(Kang-Seuk Choi),이은경(Eun-Kyoung Lee),전우진(Woo-Jin Jeon),권준헌(Jun-Hun Kwon),이진화(Jin-Hwa Lee),성환우(Haan-Woo Sung) 韓國家禽學會 2011 韓國家禽學會誌 Vol.38 No.2

국내 도축장 및 도축장 출하 닭을 대상으로 NDV 오염 실태를조사하였다. 조사결과, 닭출하농장, 닭수송용차량, 도축장 현수실, 도축장 냉각수에서 각각 조사 대상건수의 13.0%, 13.3%, 16.0%, 10.8%에서 NDV가 분리되었다. 시기별로 보면 차량, 현수실, 냉각수 모두에서 7월에 가장 많이 바이러스가 분리되었다. Pathotyping RT-PCR을 실시한 결과, 이들 분리된 NDV 분리주는 모두 저병원성 NDV 양성 반응을 나타내었으며, F 단백질 분절 부위에 모두 ¹¹²GKQGR/L¹¹? motif를 가지고 있었다. 본 연구에서 분리된 NDV 분리주의 F 단백질 유전자를 분석하여 보았을 때, 조사한 NDV 분리주 25주 중 24주가 NDV V4주와 같은 유전적 cluster를 형성하였으며, 나머지 1주는 NDV Ulster 2C주와 같은 유전적 cluster를 형성하였다. 이와 같은 연구 결과로 보아 국내에서는 출하 닭, 수송 차량, 현수실, 냉각수 등 생산에서 도축 단계까지 일련의 과정에서 높은 빈도로 저병원성 NDV 오염이 이루어지고 있음을 알 수 있었다. We conducted a 10-month (March to October 2009) surveillance of Newcastle disease virus (NDV) in 13 slaughterhouses in Korea. NDV was isolated in 13.0%, 13.3%, 16.0%, and 10.8% of chicken farms, transport vehicles, hang rooms, and chilling water, respectively. Of NDV isolates from slaughterhouses, 37% were isolated in July. All NDV isolates were determined to be lentogenic viruses by RT-PCR-based pathotyping, and all NDV isolates had the ¹¹²GKQGR/L¹¹? motif at the cleavage site of the F protein. Phylogenetic analysis based on the hypervariable region of the F protein gene classified all 25 NDV isolates examined into genotype I within class Ⅱ. Of these, 24 were clustered together with the NDV V4 strain, while the remaining isolate was placed in the cluster belonging to the NDV Ulster 2C strain. Our results indicate that lentogenic NDV was a high-frequency contaminant in the serial process of ranging live birds to slaughtering at slaughterhouses.

육계 농장 깔짚에서의 주요 바이러스 병원체 오염 실태 조사

최강석(Kang-Seuk Choi),전우진(Woo-Jin Jeon),이은경(Eun-Kyoung Lee),권준헌(Jun-Hun Kwon),이진화(Jin-Hwa Lee),성환우(Haan-Woo Sung) 韓國家禽學會 2011 韓國家禽學會誌 Vol.38 No.3

A 5-month (May to November in 2009) monitoring program for five viral pathogens in litter, such as avian influenza virus A (AIV), infectious bronchitis virus (IBV), infectious bursal disease virus (IBDV), fowl adenovirus (FAdV), and chicken infectious anemia virus (CIAV) was conducted in 62 flocks at 31 broiler farms (two flocks in each farm) in Korea in 2009. Viral pathogens were examined twice (before and at the end of the rearing period) at 31 broiler farms, and included fresh litter (n = 16) and recycled litter (n = 15) farms. Thirty-seven viruses (14 IBVs, 2 IBDVs, 9 FAdVs, and 12 CIAVs) were isolated from 75% (12/16) and 73% (11/15) of fresh litter and reused litter farms during the period, respectively, indicating no difference in viral contamination rate between farms using new and reused litter. Of these isolates, three (two CIAVs and one IBDV) were isolated from recycled litter samples collected before the rearing period at three broiler farms, whereas the others (n=34) were isolated from fresh and recycled litter samples collected at the end of the rearing period. When the performances, involving viability, body weight, and feed conversion ratio, were compared, no significant differences were found between farms using fresh and recycled litter during the period.

산란계군에서 뉴캣슬병 면역 수준 측정에 있어서 난황 항체 검사의 효용성

이은경 ( Eun Kyoung Lee ),전우진 ( Woo Jin Jeon ),박미자 ( Mi Ja Park ),권준헌 ( Jun Hun Kwon ),최강석 ( Kang Seuk Choi ) 한국수의공중보건학회 2008 예방수의학회지 Vol.32 No.3

2007~2008년 국내 H9N2 저병원성 조류인플루엔자 백신접종 농가 모니터링

강현미 ( Hyun Mi Kang ),정옥미 ( Ok Mi Jeong ),김민철 ( Min Chul Kim ),권지선 ( Ji Sun Kwon ),백마라 ( Mi Ra Paek ),권준헌 ( Jun Hun Kwon ),이윤정 ( Youn Jeong Lee ) 한국수의공중보건학회 2009 예방수의학회지 Vol.33 No.2