보고서 정보
주관연구기관 |
농림축산검역본부 Animal and Plant Quarantine Agency |
연구책임자 |
조윤상
|
참여연구자 |
서현지
,
유미선
,
김근호
,
현방훈
,
김흥철
|
보고서유형 | 최종보고서 |
발행국가 | 대한민국 |
언어 |
한국어
|
발행년월 | 2017-12 |
과제시작연도 |
2017 |
주관부처 |
농림축산식품부 Ministry of Agriculture, Food and Rural Affairs(MAFRA) |
과제관리전문기관 |
농림축산검역본부 Animal and Plant Quarantine Agency |
등록번호 |
TRKO201800042125 |
과제고유번호 |
1545014844 |
사업명 |
농림축산검역검사기술개발 |
DB 구축일자 |
2018-10-20
|
키워드 |
말.파이로플라즈마증.실시간 중합효소연쇄반응법.equine.piroplasmosis.real-time PCR.diagnosis.prevalence.
|
DOI |
https://doi.org/10.23000/TRKO201800042125 |
초록
▼
가. 제1세부과제명: 말 파이로플라즈마증 실시간 PCR 진단법 개발
1) 말 파이로플라즈마증 표준진단법인 일반 PCR진단법 확립
- 세계동물보건기구 (OIE) 표준매뉴얼에 공인된 표준유전자진단법을 실험실내 확립
2) 진드기매개 말 파이로플라즈마증 실시간유전자진단법 국내 개발
B. caballi
Bc_18SF402 GTA ATT GGA ATG ATG GCG ACT TAA
Bc_18SR496 CGC TAT TGG AGC TGG AAT TAC C
Bc_18SP FAM-CCT CGC CAG AGT AA
가. 제1세부과제명: 말 파이로플라즈마증 실시간 PCR 진단법 개발
1) 말 파이로플라즈마증 표준진단법인 일반 PCR진단법 확립
- 세계동물보건기구 (OIE) 표준매뉴얼에 공인된 표준유전자진단법을 실험실내 확립
2) 진드기매개 말 파이로플라즈마증 실시간유전자진단법 국내 개발
B. caballi
Bc_18SF402 GTA ATT GGA ATG ATG GCG ACT TAA
Bc_18SR496 CGC TAT TGG AGC TGG AAT TAC C
Bc_18SP FAM-CCT CGC CAG AGT AA-MGB
T. equi
Be18SF GCG GTG TTT CGG TGA TTC ATA
Be18SR TGA TAG GTC AGA AAC TTG AAT GAT ACA TC
Be18SP VIC-AAA TTA GCG AAT CGC ATG GCT T
3) 진드기매개 말 파이로플라즈마증 2종 원인체의 유전자진단용 특정부위 후보 선정
- 바베시아 (Babesia spp.)의 18S rRNA 부위중 Babesia spp.와 Theileria spp. 전체 공통 염기서열 부분과 각각의 병원균 (Babesia caballi, Theileria equi)에 특이적인 유전자 부위를 선정하여 바베시아/타이레리아 스크리닝 qPCR 진단법과 말 파이로플라즈마증 원인체 감별용 duplex qPCR 진단법을 국내 개발하였음
- 각 병원균의 진단부위를 cloning하여 바베시아/타이레리아 스크리닝 및 Duplex quantitative PCR (Duplex qPCR)의 DNA standard control로 이용하였음
3) 바베시아/타이레리아 스크리닝 및 Bc/Te Duplex qPCR 유전자 증폭조건 설정
- Primer, probe 농도, annealing temperature 등을 농도별, 온도별 단계별로 조정하여 스크리닝 및 duplex qPCR 반응을 위한 최적 조건을 수립하였음
- Primer 농도 : 10 pmole; probe 농도 : 5 pmole
- PCR condition : 95℃ 5분, [95℃ 10초, 60℃ 30초] x 45 cycles
4) 말 파이로플라즈마증 신속정량진단법의 민감도 및 특이도 평가를 통한 유효성 평가
- 민감도: 스크리닝용 (Babesia spp./Theileria spp.) qPCR 검출한계는 10 copies 2종 원인체 감별용 duplex qPCR 검출한계는 10 copies
- 특이도: 큐열 (Coxiella burnetti), 리케치아 (Rickettsia conorii), 바토넬라 (Bartonella quintana, Bartonella henselae), 보렐리아 (Borrelia burgdorferi, Borrelia garnii, Borrelia afzelii), 아나플라즈마 (Anaplasma phagocytophlium)등의 공시병원체에서 음성
5) 개발된 말 파이로플라즈마증 실시간유전자진단법의 말 가검시료 적용시험
- 말 파이로플라즈마증 양성개체에 대한 바베시아/타이레리아 스크리닝 qPCR 적용 결과, 시기를 달리하여 2회 채혈한 시료 모두 양성으로 확인하였으며, 1차 채혈은 Ct값 20.93, 2차 채혈은 Ct 27.84로 확인됨
- 말 파이로플라즈마증 양성개체에 대한 Bc/Te duplex qPCR 적용 결과, 1차 및 2차 채혈 모두 T. equi에만 양성반응 보여 일반 PCR과 동일한 결과를 얻었으며, Ct값은 각각 22.37, 30.04로 확인되어 스크리닝 및 원인체감별용 duplex qPCR법은 야외 적용시에 신속정량진단법으로 유용할 것으로 사료됨
나. 제2세부과제명: 진드기매개 말 파이로플라즈마증 국내 감염실태 조사
1) 진드기매개 말 파이로플라즈마증 검사를 위한 말 혈액 및 혈청 시료확보
- 국내 말 사육농가, 승마장 및 한국마사회와 진드기매개 말 파이로플라즈마증 검사를 위한 시료채취를 협조요청하여 2016년 724두, 2017년 926두의 말 혈액 및 혈청을 확보함
2) 진드기매개 말 파이로플라즈마증의 전파 매개체인 말 진드기 채집 및 종동정
- 2016년도는 국내 말 사육초지와 축체로부터 총 6,946마리를 채집하였는바, 말 사육초지에서 5,656마리를 채집하였음 (한국마사회: 612마리, 개인목장: 2,079마리, 승마장: 3,658마리, 기타: 597마리)
- 2016년도 채집된 진드기 종은 2종이었으며 (Haemaphysalis longicornis, Ixodes nipponensis), 6,940마리(99.9%)가 Haemaphysalis longicornis로 우점 종으로 확인됨
- 2017년 현재까지 채집된 진드기는 19개 지역 36농가 2,867마리이었으며, 모두 Haemaphysalis longicornis로 동정됨 (100%)
3) 진드기매개 말 파이로플라즈마증 국내 감염실태 및 말 진드기내 병원체 조사
가) 국내 사육말에서 말 파이로플라즈마증 2종 병원체 감염현황 조사
- 2016년 확보된 724두의 말 혈액 및 혈청에 대한 검사결과 1두에서 Theileria equi 항원 및 항체 양성을 확인함 (항원 PCR 양성, 항체 ELISA 양성)
- PCR 양성으로 확인된 가검물의 유전자염기서열분석 결과, 2010년 한국에서 검출된 T. equi (Accession No. HM229407), 2012년 몽골에서 검출된 T. equi (Accession No. JQ657703), 2013년 중국에서 검출된 T. equi의 18S ribosomal RNA gene (Accession No. KF559357)과 100% 일치하였음
- 2017년확보된926두의말전혈및혈청에대한검사결과2두에서Theileria spp. 항원양성을 확인함
: 말 파이로플라즈마증 원인체인 T. equi는 아니었으며, 추후 종동정 필요
나) 국내 말 진드기의 말 파이로플라즈마증 2종 병원체 및 진드기매개 병원체 보유현황 조사
- 2016년 채집된 6,946마리의 진드기를 종별, 시기별, 채집지역별로 pooling하여 말 파이로플라즈마증 2종 병원체 및 진드기매개 병원체에 3종 대한 PCR 항원검사 결과 모두 음성으로 확인됨 (총 5종 병원체 검사: B. caballi, T. equi, Anaplasma phagocytophilum, Ehrlichia chaffeensis, Borrelia burdgoferi)
- 2017년 채집된 2,867마리의 진드기를 종별, 시기별, 채집지역별로 pooling하여 말 파이로플라즈마 병원체 및 진드기매개 질병에 대한 PCR 항원검사 결과 A. phagocytophilum 5건 양성으로 확인되었고 (경기 1건, 제주 4건), B. burgdorferi 4건 양성으로 확인 (전남 1건, 부산 1건, 제주 1건)되어, 국내 Anaplasmosis와 라임병의 발병위험이 상존하고 있음을 확인하였음
(출처 : 연구결과 요약 2p)
Abstract
▼
< Quantitative Duplex Real-Time PCR for Babesia caballi and Theileria equi >
Introduction: Babesia caballi and Theileria equi are two pathogens of equine piroplasmosis which caused large economic losses in global equine industry. It is one of OIE list diseases and is a tick-borne disease. The d
< Quantitative Duplex Real-Time PCR for Babesia caballi and Theileria equi >
Introduction: Babesia caballi and Theileria equi are two pathogens of equine piroplasmosis which caused large economic losses in global equine industry. It is one of OIE list diseases and is a tick-borne disease. The detection methods are monoplex PCR as molecular diagnosis and complement fixation test, ELISA, and IFA as serological diagnosis. But the previous detection methods have low sensitivity and are time-consuming. Therefore, we developed a highly sensitive, specific and rapid diagnostic method based B. caballi- and T. equi-specific gene segments to detect simultaneously and quantitatively.
Materials and Methods: Oligonucleotide primers and probes were used to amplify the genes encoding the 16S rRNA. The duplex quantitative PCR (qPCR) was used to detect each control plasmid DNA using specific primer, and probe. The duplex qPCR were tested using IQ super mix (Bio-rad) kit according to the manufacturer’s instructions. The reaction contained 5 ul DNA temples, 5 ul primer-probe mix (Final concentration of 3.2 uM for each primer and 200mM for each probe). All reaction were performed on an Bio-Rad CFX96 with the following cycling: 95 ℃ for 5 min, and then 45 cycles of 95 ℃ for 10 sec, 60 ℃ 30 sec. The sensitivity of duplex qPCR was evaluated 10-fold dilution of control DNA. The duplex qPCR for Babesia caballi and Theileria equi were confirmed to be specific with other tick-borne disease such as Anaplasma phagocytophilum, Borrelia burdorferi, Ricehttsia conorii and Coxiella burnetti.
Results: The detection limit of the duplex qPCR for B. caballi and T. equi were 10 copies. Various field samples such as horse whole blood and tick samples were analyzed using duplex qPCR, compared with previously published qPCRs. The test results of the field samples were identical for the duplex qPCR and other qPCRs, which showed all samples to be negative for B. caballi, except for one of 724 horse blood (0.14%) that was positive for T. equi.
Conclusion: The aim of this study was develop a highly sensitive and specific duplex qPCR assay for the differential diagnosis of B. caballi and T. equi. This technique may be useful for the diagnosis of equine piroplasmosis, which is needed to molecular surveillance in ROK because of increasing tick-borne disease outbreak factors, such as increase of import from currently occurred countries, climate change, increase vector population including tick and so on.
< Molecular Detection and Phylogenetic Analysis of Equine Piroplasmosis in the Republic of Korea >
Introduction: Equine piroplasmosis is caused by Babesia caballi and Theileria equi that is one of tick-borne protozoan diseases and infects equid species. Theses tick-borne protozoa are transmitted by blood-sucking ticks, such as genera Dermacentro, Hyalomma, and Rhipicephalus. Nowadays, horse population has been increasing in the Republic of Korea due to the implementation of promotion policy from 2009. Equine piroplasmosis is one of the most important tick-borne protozoan diseases of the racing industry worldwide. However, there is no available report on the disease in ROK. Therefore, we conducted a molecular survey of equine piroplasmosis in ROK, 2016
Materials and Methods: We investigate the presence of B. caballi and T. equi in blood collected from horse stable of 5 metropolitan cities and 8 provinces in Korea, which were Seoul, Deagu, Daejeon, Gwangju, Ulsan, Gyeonggi, Chungcheongbuk, Chungcheongnam, Gyeongsangbuk, Gyeongsangnam, Jeollabuk, Jeollanam, and Jeju.
Blood samples were collected and stored at -20 °C until used. Blood samples were tested for antigen detection of B. caballi, and T. equi using polymerase chain reaction (PCR). Consequently, the amplified PCR products were used for sequencing and phylogenetic analysis.
Results: The collected samples from April to September in 2016 were used for detection of two protozoa genetically. A total of 724 blood samples were tested for B. caballi, and T. equi PCR using 18S rRNA gene. All tested samples were negative for Babesia caballi. Meanwhile, 1 of 724 samples (0.14%) was tested positive for T. equi as confirmed by sequencing of 18S rRNA gene. The positive sample showed 100% degrees of similarity to T. equi (Accession No. HM229407, JQ657703, and KF55935).
Conclusion: Equine piroplasmosis is one of the most important protozoan diseases with economic impact on the horse industry worldwide. In this study, a molecular surveillance was conducted to detect the pathogen genetically of B. caballi and T. equi. One of 724 samples was diagnosed as Theileria equi by PCR. Horse infected with T. equi could be a carrier for a long period and passed to the foal in uterus.
To prevent emerging tick-borne protozoan equine piroplasmosis, continuous monitoring of susceptible equid species should be performed and pure isolation and characterization of B. caballi and/or T. equi from PCR positive horses are required.
< Serological Surveillance Equine Piroplasmosis in the Republic of Korea >
Introduction: Equine piroplasmosis is caused by Babesia caballi and Theileria equi that infects equid species, such as horses, mules, donkeys and zebras. These two protozoa are transmitted by tick of genera Dermacentro, Hyalomma, and Rhipicephalus. Horse population has been increasing with racing industry in the Republic of Korea (ROK). Equine piroplasmosis is one of the most important tick-borne diseases in the racing industry worldwide. However, there is no available information on the disease in ROK. Therefore, we conducted a serological survey of equine piroplasmosis in ROK from May to July in 2017.
Materials and Methods: We survey the antibody of equine piroplasmosis in sera collected from horse stable in 5 metropolitan cities and 8 provinces, which were Seoul, Incheon, Daejeon, Gwangju, Ulsan, Gyeonggi, Chungcheongbuk, Chungcheongnam, Gyeongsangbuk, Gyeongsangnam, Jeollabuk, Jeollanam, and Jeju.
Serum samples were collected, and stored at 4 °C until assayed. Serum samples were tested anti-B. caballi, and anti-T. equi antibodies, using competitive ELISA kits (VMRD, Pullman, USA). The cELISA tests were performed according to the manufacturer’s recommendation.
Results: The collected serum samples from May to July in 2017 were used for detection of antibody of equine piroplasmosis. A total of 926 serum samples were tested for antibody detection of B. caballi, and T. equi using cELISA kits for each pathogen. There is no B. caballi and/or T. equi antibody positive horse of 926 horses in 2017.
Conclusion: Equine piroplasmosis is one of the most important tick-borne protozoan diseases with economic impact on the horse industry worldwide. In this study, serological surveillance was conducted to detect the antibody to two pathgens of equine piroplasmosis. B. caballi and T. equi were not detected in the tested samples this year. However, continuing climate change, increasing the number and distribution of tick vectors, and increasing imported equid species from equine piroplasmosis-occurred countries will enhance the risk of introduction of equine piroplasmosis into ROK. Considering one health and environmental change, further studies are required continuous monitoring of tick-borne equine piroplasmosis in ROK.
< Tick Distribution at Horse Riding Course, Horse Ranch, and Horse Stables Site in the Republic of Korea >
Introduction: Tick is an important vector that transmits diseases to both humans and animals and nowadays increases pathogenic potential due to climate change, livestock population growth, increase of livestock import, especially equid species, such as donkeys, horses, and so on. Most of rickettsiae and protozoa, including Ehrlichia spp. Anaplasma spp., Bartonella spp., Rickettsia spp., and Theileria/Babesia spp., are transmitted by tick. Climate change, resulting in a habitat more favorable for vectors, may have raised the risk of potential introduction of exotic tick-borne diseases into the Republic of Korea. Horse population has been increasing in Republic of Korea (ROK), and then tick-borne diseases in horse could be increased in ROK. However, there is no information on the tick on horse raising environment. In this study, we selected the tick collected site in the area around horse stable. To examine the pathogenic potential of tick-borne diseases in horse, a monitoring project for horse tick has been started. Therefore, we monitored horse tick and identified the species level of ticks in the Republic of Korea from April 2016 to June 2017.
Materials and Methods: BioQuip's tick drag was used for monitoring ticks at 34 sites consisting of 8 Korea Racing Authority, 8 personal ranch, 15 horse riding course, and other 2 sites. Tick collection was operated from 2016 to 2017. Tick were removed from the traps, and transported to laboratory, where the collected ticks were stored at -70°C until identified at species level by microscopic morphological identification on a cold table using standard keys (Yamaguti et al., 1971). Ticks were placed, up to 30 specimens by species.
Results: A total of 7,344 ticks were collected from horse riding course, ranch, and horse stables from 2016 to 2017. The collected ticks (n = 7,344) were identified as 2 species of ticks. Interestingly, 99.94% of collected ticks was Haemaphysalis longicornis (n = 7,340), which was the predominant tick at horse raising environment in Korea.
Conclusions: Our study has showed the tick species in Korea Racing Authority, personal ranch, and horse riding course in ROK. Haemaphysalis longicornis was the dominant species in our selected site. Four of I. nipponensis were collected. Despite small number of collection, I. nipponensis might be important role in the transmission of tick-borne pathogens to humans, because human tick bites have been reported more frequently for this species among patients at medical clinics in Korea. This study is ongoing and the collected tick samples are to be tested for the presence of tick-borne disease such as anaplasmosis, ehrlichiosis, babesiosis, theileriosis, and rickettsiosis.
(출처 : 영문초록 28p)
목차 Contents
- 표지 ... 1
- 농림축산검역검사기술개발 연구과제 최종결과보고서 Final Report (FR) ... 2
- Ⅰ. 연구배경 및 목표 ... 5
- 1. 연구배경 ... 5
- 2. 연구최종목표 ... 6
- 3. 연차별 연구개발 목표 및 내용 ... 7
- Ⅱ. 연구방법 및 수행전략 ... 7
- 1. 재 료 ... 7
- 2. 연구전략 및 방법 ... 7
- Ⅲ. 연구결과 ... 8
- 1. 제1세부과제명: 말 파이로플라즈마증 실시간 PCR 진단법 개발 ... 8
- 2. 제2세부과제명: 말 파이로플라즈마증 감염실태 조사 ... 12
- Ⅳ. 연구결과요약 ... 24
- 1. 제1세부과제명: 말 파이로플라즈마증 실시간 PCR 진단법 개발 ... 24
- 나. 제2세부과제명: 말 파이로플라즈마증 감염실태 조사 ... 25
- V. 고찰 ... 26
- VI. 연구사업 추진상 문제점 및 건의사항 ... 26
- VII. 주요연구 변경사항 ... 26
- VIII. 참고자료 ... 26
- IX. 영문초록 ... 28
- 끝페이지 ... 31
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