PCR법을 이용한 국내 분리 캠필로박터균에서 Guillain-Barre Syndrome 관련 galE 유전자 검출에 관한 연구

구복경 ( Bok Kyung Ku ),김혜지 ( Hae Ji Kim ),김영일 ( Young Il Kim ),최정수 ( Jung Soo Choi ),박미영 ( Mi Young Park ),권진욱 ( Jin Wook Kwon ),김용환 ( Yong Hwan Kim ),김종현 ( Jong Hyun Kim ),이영주 ( Young Joo Lee ),김은경 ( E 한국수의공중보건학회 2010 예방수의학회지 Vol.34 No.3

Guillain-Barre syndrome (GBS) is the most common cause of acute flaccid paralysis and autoimmune polyradiculo-neuropathy. Campylobacter jejuni is the most commonly identified infectious trigger for GBS. A sialic-acid containing lipopolysaccharide (LPS) of Campylobacter is thought to be involved in the triggering of GBS. The galE (UDP-galactoset-epimerase) gene of Campylobacter spp. is involved in the synthesis of LPS. In this study, we detected the galE gene in Campylobacter spp. responsible for triggering the onset of GBS. The PCR assay detected the presence of the gene in 14 of the 25 (56%) Campylobacter isolates from domestic chicken, 20 of the 28 (71.4%) Campylobacter isolates from imported chicken and 50 of the 51 (98%) Campylobacter isolates from human clinical samples. Also, the specific 497-bp region of galE sequence in Campylobacters responsible for triggering the onset of GBS was amplified from GBS patient. These results could provide evidence of the first GBS-related C. jejuni infection in Korea.

국내 발생 구제역 바이러스(foot-and-mouth disease virus)의 특성과 전파력에 관한 연구

서정향,신진호,구복경,최강석,권병준,손현주,고영준,최정업,권창희,김종염,안수환,김기석,문운경,김재훈,최상호,이홍길,황의경,김순복,강신석,김옥경,Sur, Jung-hyang,Shin, Jin-ho,Loubroth, Juan,Yeh, Max,Ku, Bok-kyung,Choi, Kang-seuk,Kweon, Byung-joon,Sohn, Hyun-joo,Ko, 대한수의학회 2000 大韓獸醫學會誌 Vol.40 No.4

A study was conducted to determine the susceptibility of swine to Korean foot-and-mouth disease virus (FMDV; subtype O, isolated from Chungju province) in April, 2ooo. One holstein cow was inoculated intradermolingually with suspension of homogenized tissue from a Korean native cow naturally infected with Korean FMDY. Infected cow was housed with one susceptible cow and one susceptible pig (contact sentinels). Four additional susceptible pigs were housed in the same room but caged separately (non-contacted sentinels). The contacted pig and cow as well as non-contact pigs developed typical clinical signs after 2, 3, and 7 days post exposure, respectively. We compared neutralizing antibody from the animals to FMDV $O_1$ Lombardy, O Taiwan, $O_1$ Campos, and $O_1$ Manisa after 0, 4, 7, 10, 14, 21, 28 days post challenge and post-exposure. The highest viral neutralization titer could be interpreted that serotype O Korea (Chungju isolate) is antigenically more related to $O_1$ Manisa. In addition, immunohistochemistry was used to further characterize Korean FMDV from tissues of infected pigs. Korean FMDV antigen was observed in the tongue, hoof, esophagus, and tonsil tissues of sentinel pigs. These findings suggest that Korean FMD virus isolated from cattle can be rapidly transmitted to pigs both directly and indirectly contrast field observation in which only cattle were clinically ill.

국내 우군에서 소 결핵 진단을 위한 피내검사법과 Interferon-γ 생성 검사의 비교

신승원,신민경,차승빈,우종태,이성모,구복경,조윤상,정석찬,유한상,Shin, Seung Won,Shin, Min Kyoung,Cha, Seung Bin,Woo, Jong Tae,Lee, Sung Mo,Ku, Bok Kyung,Cho, Yun Sang,Jung, Suk Chan,Yoo, Han Sang 대한수의학회 2011 大韓獸醫學會誌 Vol.51 No.2

Bovine tuberculosis (bTB), caused primarily by Mycobacterium bovis, continues to exert an economic loss, even in countries with active control measures, and is one of zoonotic diseases enable to be transmitted to human. The control and eradication of bTB are mainly based on a test and slaughter policy and/or abattoir surveillance. Various factors including limitation of diagnostic tests have been considered as major constraints to eradication. Single intradermal test (SIT) is the official diagnostic test. New diagnostic methods are needed to be developed, because of limitations of the test. In the present study SIT was compared with single intradermal comparative cervical test (SICCT) and interferon (IFN)-${\gamma}$ assay. There was very low correlation between SIT and SICCT. However, high correlation was shown between SIT and IFN-${\gamma}$ assay while no correlation was observed between SICCT and IFN-${\gamma}$ assay. Therefore, our results suggest the possibility of replacement of SIT with IFN-${\gamma}$ assay for the diagnosis of bovine tuberculosis.

소 림프절에서 Mycobacterium bovis DNA의 신속 검출과 M. bovis와 M. tuberculosis 감별을 위한 real-time PCR 개발

고바라다 ( Ba Ra Da Koh ),장영부 ( Young Boo Jang ),구복경 ( Bok Kyung Ku ),조호성 ( Ho Seong Cho ),배성열 ( Seong Yeol Bae ),나호명 ( Ho Myung Na ),박성도 ( Seong Do Park ),김용환 ( Yong Hwan Kim ),문용운 ( Yong Un Mun ) 한국가축위생학회 2011 韓國家畜衛生學會誌 Vol.34 No.4

Mycobacterium bovis, a member of the M. tuberculosis complex (MTC), is the causative agent of bovine tuberculosis. Detection of M. bovis and M. tuberculosis using conventional culture- and biochemical- based assays is time-consuming. Therefore, a simple and sensitive molecular assay for rapid detection would be of great help in specific situations such as faster diagnosis of bovine tuberculosis (bTB) infection in the abattoirs. We developed a novel multiplex real-time PCR assay which was applied directly to biological samples with evidence of bTB and it was allowed to differentiate between M. bovis and M. tuberculosis. The primers and TaqMan probes were designed to target the IS1081 gene, the multi-copy insertion element in the MTC and the 12.7-kb fragment which presents in M. tuberculosis, not in the M. bovis genome. The assay was optimized and validated by testing 10 species of mycobacteria including M. bovis and M. tuberculosis, and 10 other bacterial species such as Escherichia coli, and cattle lymph nodes (n=113). The tests identified 96.4% (27/28) as M. bovis from the MTC-positive bTB samples using conventional PCR for specific insertion elements IS1081. And MTC-negative bTB samples (n=85) were tested using conventional PCR and the real-time PCR. When comparative analyses were conducted on all bovine samples, using conventional PCR as the gold standard, the relative accuracy of real-time PCR was 99.1%, the relative specificity was 100%, and the agreement quotient (kappa) was 0.976. The detection limits of the real-time PCR assays for M. bovis and M. tuberculosis genomic DNA were 10 fg and 0.1 pg per PCR reaction, respectively. Consequently, this multiplex real-time PCR assay is a useful diagnotic tool for the identification of MTC and differentiation of M. bovis and M. tuberculosis, as well as the epidemiologic surveillance of animals slaughtered in abattoir.

Cis-acting Replication Element Variation of the Foot-and-mouth Disease Virus is Associated with the Determination of Host Susceptibility

Hyo Rin Kang(강효린),Mi So Seong(성미소),Bok Kyung Ku(구복경),JaeHun Cheong(정재훈) 한국생명과학회 2020 생명과학회지 Vol.30 No.11

구제역바이러스(FMDV)는 피코나바이러스 과에 속하는 바이러스로서 야생과 가축화된 소와 돼지에 감염된다. FMDV는 제어되기 어려워서 가축의 생산과 국제통상에 큰 장애가 되고 있다. FMDV RNA 게놈의 복제 과정에서 3D 중합효소가 특이적인 복제 기능을 담당하는데 게놈에 결합하는 부위가 매우 중요하다. 이 사실은 FMDV 게놈의 비코딩영역 내에서 3D 중합효소에 의해 인지되는 특이 RNA 구조가 관여함을 제시한다. 이 과정에서 cis-acting replication element (CRE)는 RNA 복제를 위한 개시에 필요하다. FMDV CRE는 15-17 뉴클레오티드의 고리와 이를 지지하는 이중가닥으로 형성된 줄기-고리 모양을 가지며 이들을 구성하는 RNA 뉴클레오티드 서열의 차이가 다른 RNA 이차구조를 생성한다. CRE 이외에 FMDV 복제를 위해서 많은 바이러스와 세포 인자들이 단백질-단백질 결합과 단백질-RNA 결합을 통해 협조적인 네트워크를 만들어낸다. 이 연구에서 국내에서 2010년부터 2017년 까지 구제역이 발생한 소와 돼지에서 FMDV를 분리하여 CRE 서열을 분석하였으며 이들 FMDV들은 A형과 O형의 유전자형을 가졌다. 흥미롭게 국내 FMDV들의 CRE RNA 이차구조의 변이들은 바이러스 간의 계통유전학적 상관관련성과 일치하며 특정 숙주 동물종의 FMDV 감염의 특이성을 밝혀주었다. 이를 토대로 국내 FMDV의 각 유전군의 분류는 독특한 기능적 CRE에 의해 특징지을 수 있으며 새로운 유전적 계통의 진화학적 연속성은 특징있는 CRE 모티프의 구분과 연관지을 수 있다. 그러므로 CRE의 특이적 RNA 구조는 숙주 동물종 의존적인 FMDV 부류의 부가적인 단서로 활용할 수 있음을 제안한다. 이들 연구 결과들은 향후 FMDV 대량감염이 발생할때 숙주동물종의 특이성을 CRE 서열로 조기에 정확히 분석하는데 도움이 될 것이다. The foot-and-mouth disease virus (FMDV), a member of the Aphthovirus genus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. During replication of the FMDV RNA (ribonucleic acid) genome, FMDV-encoding RNA polymerase 3D acts in a highly location-specific manner. This suggests that specific RNA structures recognized by 3D polymerase within non-coding regions of the FMDV genome assist with binding during replication. One such region is the cis-acting replication element (CRE), which functions as a template for RNA replication. The FMDV CRE adopts a stem-loop conformation with an extended duplex stem, supporting a novel 15-17 nucleotide loop that derives stability from base-stacking interactions, with the exact RNA nucleotide sequence of the CRE producing different RNA secondary structures. Here, we show that CRE sequences of FMDVs isolated in Korea from 2010 to 2017 exhibit A and O genotypes. Interestingly, variations in the RNA secondary structure of the Korean FMDVs are consistent with the phylogenetic relationships between these viruses and reveal the specificity of FMDV infections for particular host species. Therefore, we conclude that each genetic clade of Korean FMDV is characterized by a unique functional CRE and that the evolutionary success of new genetic lineages may be associated with the invention of a novel CRE motif. Therefore, we propose that the specific RNA structure of a CRE is an additional criterion for FMDV classification dependent on the host species. These findings will help correctly analyze CRE sequences and indicate the specificity of host species for future FMDV epidemics.

단보 : 소에서의 비결핵성 마이코박테리아 분리 현황

장영부 ( Young Boo Jang ),김평희 ( Ryung Hee Kim ),김재명 ( Jae Myung Kim ),정석찬 ( Suk Chan Jung ),구복경 ( Bok Kyung Ku ) 한국수의공중보건학회 2010 예방수의학회지 Vol.34 No.4

Nontuberculous mycobacteria (NTM) often confounds the interpretation of skin test in tuberculosis diagnosis, resulting in false-positive reaction. And, NTM are emerging pathogens that cause opportunistic infections in both humans and animals. The prevalence of NTM on human disease has been well investigated, whereas that of NTM on animal disease is not well established in Korea. The aim of this study was to determine the prevalence of NTM on cattle, which did not have symptoms of tuberculosis disease. A total of 426 isolates were collected in Korean native cattle and dairy cattle from 2007 to 2010. The most frequently isolated organisms were Mycobacterium peregrimum (n=5, 28%), Mycobacterium fortuitum (n=3, 17%), Mycobacterium intracellulare (n=2, 11%), Mycobacterium phlei (n=1, 5.6%), Mycobacterium terrae (n=1, 5.6%), Mycobacterium llatzerense (n=1, 5.6%), and Norcadia spp. (n=1, 5.6%). It seems to be necessary to further study the prevalence of NTM on environment (water, soil, feces) as well as animals.

구제역바이러스의 FMDV 2C 단백질은 소포체 스트레스를 통해서 염증 유도 사이토카인 TNFα의 발현을 증가시킴

강효린(Hyo Rin Kang),성미소(Mi So Seong),나진주(Jin Ju Nah),류소연(Soyoon Ryoo),구복경(Bok Kyung Ku),정재훈(JaeHun Cheong) 한국생명과학회 2020 생명과학회지 Vol.30 No.3

구제역바이러스(FMDV)는 Picornaviridae 과의 Aphthovirus 속의 한 종류이며, 야생과 가축의 소와 돼지에 감염한다. FMDV는 감염 조직에서 중증의 염증반응을 포함한 다양한 임상적 증후들을 일으킨다. FMDV 게놈 RNA는 약 8.3 kb 길이의 양성-단일 가닥을 가지고 있으며, 하나의 긴 단백질 번역틀(ORF)을 만든다. 이 ORF는 바이러스의 단백질가수분해효소에 의해서 구조단백질과 비구조단백질로 나누어진다. FMDV의 FMDV 2C 단백질은 FMDV 유전자에서 만들어지는 비구조단백질로서 염증과 세포사를 포함한 FMD 병리 과정과 바이러스 복제에서 중요한 역할을 한다. 이 연구에서 우리는 FMDV 2C가 염증 유도 사이토카인인 tumor necrosis factor alpha (TNFα)의 세포내 발현을 유도하는 가능성을 검토하였다. FMDV 2C의 돼지 세포인 IBRS-2 세포내 발현은 TNFα의 유전자 발현 조절 부위인 프로모터의 활성화를 이용하여 전사수준에서 TNFα의 mRNA와 단백질 생성을 증가시켰다. 추가적으로, 소포체 스트레스를 감소시키는 화학물질인 4-phenylbutyric acid (4-PBA) 처리는 FMDV 2C에 의해 유도된 TNFα 발현을 감소시켰다. 소포체 스트레스 반응을 매개하는 전사인자의 한 종류인 ATF4는 TNFα 프로모터의 활성을 유도하고, TNFα의 mRNA와 단백질 발현을 증가시켰다. 하지만, ATF4의 기능 결핍 돌연변이체 단백질의 발현은 FMDV 2C에 의한 TNFα 생성을 유도하지 못하였다. 이들 결과들은 FMDV FMDV 2C 단백질이 ATF4-매개 TNFα 발현을 통해 임상적 염증반응을 증가시키고, 이는 소포체 스트레스의 유도와 연관되어있음을 제시한다. Foot-and-mouth disease virus (FMDV), a member of the genus Aphthovirus in the Picornaviridae family, affects wild and domesticated ruminants and pigs. FMDV causes various clinical symptoms, including severe inflammation in infected tissue. Genome RNA of FMDV shows a positive single-strand chain approximately 8.3 kb long and encodes a single long open reading frame (ORF). The ORF is translated into structural and non-structural proteins by viral proteases. The FMDV 2C protein is one of the non-structural proteins encoded by FMDV and plays a critical role in FMD pathogenesis, including inflammation, apoptosis, and viral replication. In this study, we examined whether FMDV 2C induces intracellular expression of pro-inflammatory cytokine tumor necrosis factor alpha (TNFα). FMDV 2C expression in pig IBRS-2 cells increased mRNA and protein expression of TNFα at the transcriptional level via activation of TNFα promoter. Treatment with 4-phenylbutyric acid, an endoplasmic reticulum (ER) stress reducer, decreased TNFα expression induced by FMDV 2C. Activating transcription factor 4 (ATF4), a transcription factor mediating ER stress response, induced transactivation of TNFα promoter and expression of mRNA and protein of TNFα. However, the dominant negative mutant of ATF4 did not induce FMDV 2C–mediated TNFα expression. The results indicate that FMDV 2C protein increases clinical inflammation via ATF4-mediated TNFα expression and is associated with ER stress induction.

구제역바이러스 신속진단을 위한 pan-serotype reverse transcription loop-mediated isothermal amplification (RT-LAMP) 진단법

임다래 ( Da-rae Lim ),박유리 ( Yu-ri Park ),박선영 ( Sun-young Park ),김혜령 ( Hye-ryung Kim ),박민지 ( Min-ji Park ),구복경 ( Bok-kyung Ku ),나진주 ( Jin-ju Nah ),유소윤 ( So-yoon Ryoo ),위성환 ( Sung-hwan Wee ),전효성 ( Hyo-sung 한국동물위생학회(구 한국가축위생학회) 2018 韓國家畜衛生學會誌 Vol.41 No.1

In this study, we developed a sensitive and specific reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid visual detection of foot-and-mouth disease virus (FMDV) circulated in Korea. The RT-LAMP was completed in 40 min at 62°C and the results of the assay were directly detected by naked eye without any detection process. The assay specifically amplified all 7 serotypes of FMDV RNAs but not amplified other viral and cellular nucleic acids. The sensitivity of the RT-LAMP was 10², 10³ and 10³ TCID₅₀/mL for serotype O, A and Asia 1 FMDV, respectively, which was comparable to conventional reverse transcription polymerase chain reaction (RT-PCR) and relatively lower than that of real time quantitative RT-PCR (qRT-PCR). Clinical evaluation of the RT-LAMP using different serotypes of Korean and foreign FMDV strains showed a 100% (35/35) agreement with the results of the RT-PCR and qRT-PCR. These results indicated that RT-LAMP assay developed in this study could be a valuable diagnostic method for FMDV monitoring and surveillance.

국내산 및 수입산 식육에서의 Campylobacterjejuni 및 Campylobactercoli오염도 조사 (2005~2009)

박현정 ( Hyun Jung Park ),김영조 ( Young Jo Kim ),김지호 ( Ji Ho Kim ),송시욱 ( Si Wook Song ),허은정 ( Eun Jeong Heo ),김혜지 ( Hae Ji Kim ),구복경 ( Bok Kyung Ku ),이수화 ( Su Wha Lee ),이지연 ( Ji Youn Lee ),문진산 ( Jin San Mo 한국수의공중보건학회 2010 예방수의학회지 Vol.34 No.3

To determine the prevalence of Campylobacter jejuni and Campylobacter coli in meats, a total of 4,161 samples (1,953 domestic and 2,208 imported) were collected from 304 slaughterhouses nationwide and registered cold storages for imported meats in Korea during 2005~2009. The isolation rates of C. jejuni and C. coli in domestic beef, pork, chicken and duck meats were 0.1% (1/630), 0% (0/630), and 0.1% (1/644), 0% (0/644) and 20.5% (125/609), 10.2% (62/609) and 25.7% (18/70), 20.0% (14/70), respectively. In the case of imported meats, C. jejuni were isolated from 0.1% (1/943) and 15.2% (83/546) of pork and chicken meats, respectively, and C. coli were detected only from 4.8% (26/546) of chicken meats. Neither C. jejuni nor C. coli were detected from imported beef, and C. coli were also not detected from imported pork. In conclusion, chicken meats had much higher rate of contamination with Campylobacter compared to beef and pork. Therefore, HACCP system that is now mandatory for slaughterhouses should be actively practiced for safe and sanitary processing, handling, and marketing of chicken meats. In addition, all critical control points should be determined by processing procedures at processing plants as well as farms and slaughterhouses, and monitoring should be carried out at regular intervals.

Multiplex PCR을 이용한 Mycobacterium bovis와 Mycobacterium tuberculosis의 동정

장영부 ( Young Boo Jang ),김재명 ( Jae Myung Kim ),최재영 ( Jae Yeong Choe ),이햇님 ( Haet Nim Lee ),정석찬 ( Suk Chan Jung ),박영길 ( Young Kil Park ),구복경 ( Bok Kyung Ku ) 한국예방수의학회(구 한국수의공중보건학회) 2011 예방수의학회지 Vol.35 No.4

Tuberculosis (TB) is a significant disease for both humans and animals worldwide. The genus Mycobacterium includes several species that cause TB disease in humans and other animals. Amongst the members of the Mycobacterium tuberculosis complex (MTC), M. tuberculosis is mainly a human pathogen, whereas M. bovis has a broad host range and is the principal agent responsible for TB in domestic and wild mammals. M. bovis also infects humans, causing zoonotic TB through ingestion, inhalation. M. bovis accounts for only a small percentage of the reported cases of TB in humans. In recent years, TB in farmed deer has become a disease as public health importance in several countries. Nowadays, there has been rapid outbreak of bovine TB in cattle and deer in Korea. Investigations are needed to elucidate the relative importance of M. bovis on TB incidence in humans, especially in Korea where bovine TB remains a problem. Also, the human sources as the cause of animal infection, M. tuberculosis from the farm workers also important for TB control of animals in Korea. Differentiation between the causative organisms may only be achieved by sophisticated laboratory methods involving bacteriological characteristics, biochemical properties, and routine resistance to pyrazinamide (PZA). M. bovis shows inherently resistant to PZA whereas M. tuberculosis is susceptible to PZA. In this study, we developed a multiplex-PCR assay based on a 12.7-kb fragment for the differential detection of M. bovis and M. tuberculosis. A total of 131 M. tuberculosis complex isolates were randomly obtained from cattle and deers that were PPD positive. they all yielded M. bovis. M. tuberculosis was not isolated from animals. and, a total of 25 M. tuberculosis complex isolates which is resistant to PZA were obtained from patient. PZA resistant MTC in humans was caused entirely by M. tuberculosis. The multiplex-PCR protocol was highly species-specific and time saving for identification of M. bovis and M. tuberculosis. This multiplex-PCR assay will be easily used as a routine monitoring tool in veterinary and medical laboratories.