시마연어, Oncorhynchus masou에서 분리된 아니사키스 속 선충 3기 유충의 분자생물학적 방법을 이용한 동정

전찬혁 ( Chan Hyeok Jeon ),김정호 ( Jeong Ho Kim ),( Eko Setyobudi ) 한국어병학회 2010 한국어병학회지 Vol.23 No.3

Anisakid nematodes third stage larvae were isolated from the muscles of masou salmon (Oncorhynchus masou). Fish were purchased from Jumunjin fishery market in Gangneung. Four Anisakid third stage larvae were isolated from 4 fish. Molecular identification of the isolated worms was conducted by PCR-RFLP analysis of ribosomal DNA internal transcribed spacer region and direct sequencing of mitochondrial DNA cox2 gene. As results, all the tested individual worms were identified as Anisakis simplex (sensu stricto). This is the first report of molecular detection of anisakid worms in salmonid fishes in Korea.

명태(Theragra chalcogramma) 종묘에서 분리된 Vibrio anguillarum의 생화학적, 분자생물학적 특성

전찬혁 ( Chan Hyeok Jeon ),남우화 ( U Hwa Nam ),김정호 ( Jeong Ho Kim ) 한국수산과학회(구 한국수산학회) 2016 한국수산과학회지 Vol.49 No.3

The health of Alaska pollock Theragra chalcogramma seedlings was monitored during February and April 2015. After microscopic examination for parasites, 20 samples sets were made by pooling 50 individuals for each sample set. Then, they were homogenized and examined for viral and bacterial pathogens. No parasites or viruses were detected using either microscopy or PCR. Colonies suspected of belonging to the genus Vibrio were isolated from Tryptic Soya Agar and Thiosulfate Citrate Bile Salts Sucrose Agar plate incubations, and identified as Vibrio anguillarum based on biochemical and physiological examinations and PCR amplification of the 16S rDNA, recA, and pyrH genes. Although there was no mortality during the sampling period, 65.0% (13/20) of the pooled samples were PCR-positive for V. anguillarum. To prevent possible outbreaks, the pathogenic potential of V. anguillarum should be investigated in the future.

청자갈치( Bothrocara hollandi)의 근육에 기생하는 점액포자충 Myxobolus aeglefini (Myxozoa: Myxobolidae)

전찬혁 ( Chan Hyeok Jeon ),김정호 ( Jeong Ho Kim ) 한국어병학회 2015 한국어병학회지 Vol.28 No.2

동해안에서 어획된 청자갈치 (Bothrocara hol-landi)의 체측 근육에 유백색의 불투명한 시스트를 형성하고 있는 점액포자충이 발견되었다. 시스트를 마쇄하여 광학현미경으로 관찰해본 결과, 원형에 가까운 점액포자충의 성숙 포자가 관찰되었다. 성숙 포자의 평균 길이는 11.9 (11.0~13.5) μm, 평균폭은 11.6 (10.7~13.6) μm, 평균 두께는 7.8 (6.9~8.8)μm이었다. 극낭의 평균 길이는 4.4 (3.2~5.3) μm이었으며, 평균 폭은 평균 3.3 (2.4~4.2) μm이었다. 감염숙주와 성숙포자의 형태학적 특징, 각 부위의 측정값으로부터 본 연구에서 발견된 점액포자충은 Myxobolus aeglefini Auerbach 1906으로 동정하였다. 또한 18S rDNA sequences를 이용한 계통분석결과 M. albi와 M. groenlandicus와 같은 분지에 속하는 것이 확인되었으며 각각 97.7%와 96.9%의 상동성을 나타내었다. A specimen of porous-head eelpout Bothrocara hollandi (Zoarcidae: Perciformes) caught from the East Sea was found to harbour a myxosporean parasite. Numerous whitish pseudocysts were scattered throughout the body musculature of this individual specimen. Fresh myxosporean spores were found from the squashed pseudocysts under light microscopy. They were subspherical in frontal view with a length of 11.9 (11.0~13.5) μm, width of 11.6 (10.7~13.6) μm, and thickness of 7.8 (6.9~8.8) μm. Two polar capsules were almost equally pyriform with a length of 4.4 (3.2~5.3) μm and width of 3.3 (2.4~4.2) μm. Morphometric and host ecology analysis revealed that this myxosporean parasite could be identified as Myxobolus aeglefini Auerbach 1906. Phylogenetic analysis based on 18S rDNA sequences also revealed that M. aeglefini was clustered with M. albi and M. groenlandicus in the same branch, sharing 97.7% and 96.9% sequence similarities with M. albi and M. groenlandicus, respectively.

어류 기생성 선충 Anisakis simplex sensu stricto와 Anisakis pegreffii 유충의 excretory-secretory products 및 somatic extracts의 가수분해효소 활성 비교

전찬혁 ( Chan Hyeok Jeon ),위성 ( Seong Wi ),김정호 ( Jeong Ho Kim ) 한국어병학회 2014 한국어병학회지 Vol.27 No.1

Hydrolase activities of excretory-secretory products (ESP) and somatic extracts (SE) from Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii larvae were investigated by using API ZYM kit. In esterase group, acid phosphatase showed high activity from both of A. simplex (s.s.) and A. pegreffii. Esterase (C4) showed activity only from SE and A. simplex (s.s.) showed higher activity than A. pegreffii. Alkaline phosphatase, acid phosphatase and naphthol-AS-BI-phosphohydrolase showed higher activity in 3rd stage larvae than in 4th stage larvae of both species. In aminopeptidase group, only leucine arylamidase showed remarkable activity in SE of both anisakid species, and A. simplex (s.s.) SE showed higher activity than A. pegreffii SE. In glycosidase group, N-acetyl-β-glucosaminidase, α-mannosidase, α-fucosidase showed higher activity in A. simplex (s.s.) than A. pegreffii, and 4th larvae showed higher activity than 3rd larvae. These differences in hydrolase activity of anisakid nematodes larvae are thought to be due to different metabolism such as growth, moulting, digestion and feeding.

양성 중인 명태(Gadus chalcogrammus)의 바이러스 모니터링

남우화 ( U-hwa Nam ),전찬혁 ( Chan-hyeok Jeon ),서현준 ( Hyun-joon Seo ),최다영 ( Da-young Choi ),서주영 ( Joo-young Seo ),권오남 ( O-nam Kwon ),김위식 ( Wi-sik Kim ),김정호 ( Jeong-ho Kim ) 한국어병학회 2017 한국어병학회지 Vol.30 No.1

2016년 2월에서 9월까지 강원도 고성, 양양, 강릉에서 각각 양성 중인 명태를 샘플링하여 RT-PCR법으로 바이러스(viral hemorrhagic septicemia virus, VHSV; nervous necrosis virus, NNV; marine birnavirus, MABV)의 검출을 시도하였다. 비장시료를 대상으로 한 one-step PCR에서 VHSV, NNV, MABV 모두 검출되지 않았으며, 뇌시료에서 NNV가 1.8%(1/55)의 검출률을 나타내었다. Two-step PCR에서는 VHSV가 51.6%(32/62 set), NNV가 1.6%(1/62 set)의 비장시료에서 검출되었으며 MABV는 검출되지 않았다. 뇌시료에서는 NNV가 3.6%(2/55)의 검출률을 나타내었다. 본 연구결과를 통해 양식산 명태에서 처음으로 VHSV와 NNV가 검출되었다. 그러나 거의 모든 양성개체에서 two-step PCR법으로 해당 바이러스의 유전자가 검출되었으며, 모니터링 기간 동안 바이러스 감염이 의심되는 외관증상을 보이는 개체 및 폐사 개체는 발견되지 않아 바이러스의 역가는 매우 낮을 것으로 생각된다. 차후 지속적인 모니터링 및 세포주를 사용한 바이러스의 분리, 병원성의 확인, PCR 양성개체의 캐리어 가능성 확인 등이 필요할 것으로 생각된다. This study was conducted to monitor the prevalence of viral hemorrhagic septicemia virus (VHSV), nervous necrosis virus (NNV) and marine birnavirus (MABV) in cultured walleye pollock Gadus chalcogrammus by RT-PCR. All of the viruses tested were not detected by one-step PCR in 62 spleen sample sets, except for NNV in one brain sample set (1/55). By two-step PCR, VHSV was detected in 51.6%(32/62) and NNV was detected in 1.6%(1/62) spleen sample set, but MABV was not detected. In the brain sample sets, the detection rate of NNV was 3.6%(2/55). VHSV and NNV were detected for the first time in cultured walleye pollock in this study. However, the titers of viruses in these sample sets are thought to be very low, because most of the positive sample sets were detected by two-step PCR and none of the fish showed any clinical symptoms of each virus. Continuous monitoring, subsequent virus isolation and validation of carrier fish will be necessary.

2010∼2012년 연안에서 서식하는 해산어에서 아니사키스 유충의 감염현황

김위식 ( Wi Sik Kim ),전찬혁 ( Chan Hyeok Jeon ),김정호 ( Jeong Ho Kim ),김도형 ( Do Hyung Kim ),오명주 ( Myung Joo Oh ) 한국어병학회 2012 한국어병학회지 Vol.25 No.3

A survey was conducted to investigate infection of anisakid nematode larvae in 243 wild marine fishes caught from the southern coastal area of Korea between 2010 and 2012. The samples comprised fishes from 9 orders, 30 families and 50 species. Total infection rate of anisakid nematode larvae was 10.7% (26/243 fish), which comprised from Yeosu, 7.4% (7/95) in 2010 and 22.7% (5/22) in 2011; from Jeju, 8.2% (5/61) in 2011; from Wando, 40.9% (9/22) in 2012. Anisakid nematode larvae were not detected in Tongyoung and Wando samples in 2011. Molecular identification of the 89 worms from 26 fish was conducted by PCR-RFLP and/or sequence analysis of internal transcribed spacer (ITS) region of ribosomal DNA. From the results, 6 kinds of anisakis species were identified: Anisakis pegreffii (infection rate: 53.9%, 48/89 worms), Hysterothylacium aduncum (38.2%, 34/89), H. fabri (3.4%, 3/89), hybird (A. simplex X A. pegreffii) (2.4%, 2/89), A. simplex (1.1%, 1/89) and Raphidascaris lophii (1.1%, 1/89). The rate of single infection was 80.8% (21/26 infected fish), while 19.2% (5/26) showed mixed infection with 2 to 3 different anisakis species.

Infectious hematopoietic necrosis virus (IHNV)-검출 Reverse transcriptase Loop-mediated isothermal amplification (RT-LAMP) 법의 평가

김위식 ( Wi Sik Kim ),전찬혁 ( Chan Hyeok Jeon ),김정호 ( Jeong Ho Kim ),오명주 ( Myung Joo Oh ) 한국어병학회 2012 한국어병학회지 Vol.25 No.3

A reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) assay was evaluated to monitor infectious hematopoietic necrosis virus (IHNV) from artificially infected rainbow trout Oncorhynchus mykiss. The cumulative mortalities of fish challenged with IHNV at 106.5 TCID50/fish, 105.5 TCID50/fish and 104.5 TCID50/fish were 40%, 0% and 0%, respectively. Dead fish and survivors at 16 and 28 d post-challenge in each group were employed for IHNV detection by RT-LAMP assay and virus isolation using BF-2 cells. IHNV from 104.3 to 106.8 TCID50/㎖ was isolated from all the dead fish and also detected in all of the examined dead fish by RT-LAMP assay. In survivors at 16 d, 60% (3/5 fish, 102.8-105.05 TCID50/㎖), 20% (1/5 fish, 101.05 TCID50/㎖) and 60% (3/5 fish, 101.05-104.8 TCID50/ ㎖) were found to be IHNV-positive by virus isolation in fish challenged with IHNV at 106.5 TCID50/fish, 105.5 TCID50/fish and 104.5 TCID50/fish, respectively, while 20% (1/5 fish), 0% (0/5 fish) and 20% (1/5 fish) were IHNV-positive by RT-LAMP assay. No IHNV was detected in the survivors at 28 d and control fish. These results indicate that the RT-LAMP assay is useful for detection of IHNV in diseased fish although it is not enough to monitor virus in IHNV-survivors.

단보 : 2014-2015년 남서해안 종묘장에서 생산된 넙치( Paralichthys olivaceus) 치어의 Kudoa septempunctata 감염실태 조사

김위식 ( Wi Sik Kim ),공경희 ( Kyoung Hui Kong ),정성주 ( Sung Ju Jung ),정명화 ( Myung Hwa Jung ),전찬혁 ( Chan Hyeok Jeon ),김정호 ( Jeong Ho Kim ),오명주 ( Myung Joo Oh ) 한국어병학회 2015 한국어병학회지 Vol.28 No.2

2014년 1월부터 2015년 5월까지 충청남도, 전라북도 및 전라남도에 위치한 6개지지역 (충청남도:태안, 보령, 전라북도: 고창, 전라남도: 무안, 영광, 완도) 총 11개소의 넙치 종묘장으로부터 채집한 넙치 치어 총 660마리 (132 pooling 시료)를 대상으로 Kudoa septempunctata의 감염현황을 조사하였다. 쿠도아 검사를 실시한 결과, 모든 넙치 종묘에서 PCR 음성 결과가 확인되었다. 이상의 결과로 쿠도아 진단 매뉴얼 방법에 의해 조사된 종묘장의 넙치에서는 K. septempunctata가 검출되지 않는 것으로 나타났다. A survey was conducted to investigate the infection of Kudoa septempunctata in 660 olive flounder (Paralichthys olivaceus) (132 pooling samples) cultured in 11 hatcheries in 6 different regions of Korea between 2014 and 2015. Polymerase chain reaction (PCR) results were negative for K. septempunctata for all samples. Based on the kudoa diagnostic manual, K. septempunctata was not detected in olive flounder hatcheries.