Discrepancies in genetic identification of fish-derived Aeromonas strains

한현자,김도형 한국어병학회 2009 한국어병학회지 Vol.22 No.3

Genetic identification of 17 fish-derived Aeromonas strains was attempted using 5 housekeeping genes. 16S rRNA, gyrB, rpoD, dnaJ and recA genes from the 17 strains were amplified, and total of 85 amplicons were sequenced. DNA sequences of the strains and type strains of the 17 Aeromonas homology groups were used for genetic identification and phylogenetic analyses. None of the strains was identified as a single species using the 16S rRNA gene, showing the same identities (average = 99.7%) with several Aeromonas species. According to gyrB, rpoD, dnaJ, and recA, 9 strains and RFAS-1 used in this study were identified as A. hydrophila and A. salmonicida, respectively. However, the other strains were closely related to 2 or more Aeromonas species (i.e., A. salmonicida, A. veronii, A. jandaei, A. media and A. troda) depending on the genetic marker used. In this study, gyrB, rpoD, dnaJ and recA gene sequences proved to be advantageous over 16S rRNA for the identification of field Aeromonas isolates obtained from fish. However, there are discrepancies between analyses of different phylogenetic markers, indicating there are still difficulties in genetic identification of the genus Aeromonas using the housekeeping genes used in this study. Advantages and disadvantages of each housekeeping gene should be taken into account when the gene is used for identification of Aeromonas species. Genetic identification of 17 fish-derived Aeromonas strains was attempted using 5 housekeeping genes. 16S rRNA, gyrB, rpoD, dnaJ and recA genes from the 17 strains were amplified, and total of 85 amplicons were sequenced. DNA sequences of the strains and type strains of the 17 Aeromonas homology groups were used for genetic identification and phylogenetic analyses. None of the strains was identified as a single species using the 16S rRNA gene, showing the same identities (average = 99.7%) with several Aeromonas species. According to gyrB, rpoD, dnaJ, and recA, 9 strains and RFAS-1 used in this study were identified as A. hydrophila and A. salmonicida, respectively. However, the other strains were closely related to 2 or more Aeromonas species (i.e., A. salmonicida, A. veronii, A. jandaei, A. media and A. troda) depending on the genetic marker used. In this study, gyrB, rpoD, dnaJ and recA gene sequences proved to be advantageous over 16S rRNA for the identification of field Aeromonas isolates obtained from fish. However, there are discrepancies between analyses of different phylogenetic markers, indicating there are still difficulties in genetic identification of the genus Aeromonas using the housekeeping genes used in this study. Advantages and disadvantages of each housekeeping gene should be taken into account when the gene is used for identification of Aeromonas species.

2013-2016년 양식 조피볼락(Sebastes schlegeli)의 해역별 선충(Clavinema mariae) 및 병원체 감염 현황

한현자,송준영,조미영,최혜승,정승희,서한길 한국수산과학회 2020 한국수산과학회지 Vol.53 No.3

Diagnostic monitoring in Korean rockfish cages was performed to survey the prevalence of pathogens in culturedKorean rockfish Sebastes schlegeli from May 2013 to July 2016. A total of 1,945 fish samples collected from thewestern (Cheonsu Bay and Heuksando), southern (Tongyeong and Namhae), and eastern coasts (Pohang) of Koreawere tested for parasites, viruses, and bacteria. In this study, 1,264 and 334 fishes were infected with Microcotylesebastis and Clavinema mariae, respectively. The prevalence rates of C. clavinema in fishes from Cheonsu Bay,Heuksando, and Tongyeong were 35.3%, 3.9% and 1.9%, respectively. No C. clavinema infection was detected incultured rockfish from Namhae and Pohang. Furthermore, bacteria including Photobacterium damselae (8.9%), Photobacteriumpiscicola (2.3%), Photobacterium spp. (8.9%), Aeromonas salmonicida (1.8%), Aeromonas spp. (0.9%),Vibrio scophthalmi (1.5%), Vibrio spp. (3.3%), Streptococcus iniae (1.2%), and others (8.0%) were detected in 373of 1,364 fishes. No virus was detected in any fish investigated in this study.

Determination of epidemiological tetracycline MIC cut-off value for Vibrio ichthyoenteri

한현자,김도형,이덕찬,원경미,이순정,조미영,지보영,김진우 한국어병학회 2011 한국어병학회지 Vol.24 No.2

Normalized resistance interpretation (NRI) analysis for tetracycline was applied to generate information on the epidemiological cut-off value for Vibrio ichthyoenteri isolated from diseased olive flounder (Paralichthys olivaceus)larvae. Thus, 42 strains of V. ichthyoenteri were used to determine minimum inhibitory concentration (MIC) values of tetracycline using Etest. Also, 11 tetracycline resistance related genes were investigated by PCR method. Most tetracycline-resistant strains harbored both tetB and tetM with a few ceptions. NRI-derived mean and 2 SD above the mean of theoretical normal distributions of susceptible isolates were 0.33 mg/L and 1.66 mg/L, respectively. The epidemiological cut-off value for V. ichthyoenteri from the calculations could be set to S ≤ 2 mg/L. Of the 42 strains, 15 were classified as non-wild type (NWT), and MIC values of the NWT strains vary regardless of tetB and tetM detection, suggesting that there may be other mechanisms involved in tetracycline resistance in this Vibrio species.

Phenotypic Diversity of Vibrio ichthyoenteri Isolated from the Gastrointestinal Tract of Larval Olive Flounder Paralichthys olivaceus

한현자,김도형,이덕찬,최혜성,정성희,김진우 한국수산과학회 2013 Fisheries and Aquatic Sciences Vol.16 No.2

Vibrio ichthyoenteri is a facultatively anaerobic gram-negative bacterium with straight or slightly curved rod morphology. The bacterium is an etiological agent of bacterial enteritis of Olive flounder Paralichthys olivaceus. Only a handful of studies, using a limited number of isolates, have investigated the phenotypic and genetic characteristics of V. ichthyoenteri. We isolated 40 V. ichthyoenteri strains, identified based on the 16S rRNA gene sequence, from the diseased flounder larvae and investigated the API 20E and ZYM profiles. The isolates exhibited highly divergent phenotypic characteristics regardless of sampling time point and location, and fish age. Essential enzymes produced by V. ichthyoenteri seemed to be alkaline phosphatase, leucine arylamidase, and N-acetyl-β-glucosaminidase. This study reveals a much greater enzymatic and biochemical phenotype diversity than has been evident to date. These results suggest that a given population of V. ichthyoenteri could be heterogeneous in terms of its phenotypic characteristics.

국내 수산용 백신 개발 현황 및 발전 방향

한현자,김수진,김태호,김명석,조미영,최혜승 한국수산과학회 2021 한국수산과학회지 Vol.54 No.4

In Korea, fish is a major food source, and sustainable production of fish is critical for the aquaculture industry. Recently, infectious diseases have become an unavoidable problem in aquaculture and have caused serious economic losses. Therefore, there is an urgent need to overcome this challenge to increase productivity in aquaculture. Vaccination is the most effective and long-term measure for improving the management of a fish farm. Sales of fish vaccines in Korea have been steadily increasing, with a confirmed increase from 600 million won in 2007 to 3.4 billion won in 2018. As of 2021, 30 licensed fish vaccines, including both single and combination vaccines containing more than two inactivated pathogens, have been approved in Korea for 10 fish pathogens. Twenty-eight of these are used in olive flounder Paralichthys olivaceus; however, only two vaccines are used in starry flounder Platichthys stellatus, red seabream Ragrus major and rock bream Oplegnathus fasciatus. Therefore, vaccine development is required not only for the olive flounder but also for other cultured fish species. The development of effective vaccines and vaccination programs for fish is a continuous requirement for a sustainable aquaculture industry.

양식 넙치에서 연쇄구균 혼합백신 접종 후 항체가 변화 조사

한현자,이덕찬,김수진,김태호,정선명,김재휘,최윤재,조경용,조미영 한국어병학회 2024 한국어병학회지 Vol.37 No.1

Streptococcosis, caused by Streptococcus iniae and Streptococcus parauberis is an important bacte-rial disease that affects in olive flounder in Korea. In Korea, multivalent bacterial vaccines are used to prevent streptococcal diseases in aquaculture. In this study, commercial vaccines containing formal- in-inactivated bacterial cells of S. iniae and S. parauberis were administered at six fish farms and one unvaccinated fish farm were designated for investigation (Wando; 4 sites and Jeju; 3 sites). Blood was collected from vaccinated and unvaccinated olive flounders, and titers of antibodies against S.iniae and S. parauberis in serum were analyzed using ELISA. After a one shot vaccination in the farms at Jeju (farm A) and Wando (farm D), the proportion of individuals with specific antibodies against S. parauberis OD values of 0.4 or higher was 60% and 53.5%, respectively. But after booster vaccination, the proportion of individuals with serum OD values of 0.4 or higher was higher sub-stantially increased to 96.6% (farm A) and 100% (farm D). The levels of S. parauberis specific antibodies of olive flounder were increased after vaccination in three fish farms (farm D, E, and F), but not S. iniae specific antibodies.

An outbreak of Lactococcus garvieae Infection in Cage-cultured Red Lip Mullet Chelon haematocheilus with Green Liver Syndrome

한현자,이남실,김명석,정성희 한국수산과학회 2015 Fisheries and Aquatic Sciences Vol.18 No.3

Red lip mullet Chelon haematocheilus (body weight = 468 ± 91 g) which became sick during an outbreak of disease at mariculture facilities at Cheonsu Bay, Korea, during July–August 2013, were examined to identify the cause of the disease. Diseased mullets displayed green liver syndrome, and Lactococcus garvieae were isolated from their internal organs. Argulus sp., Trichodina sp., and/or Vibrio spp. were also discovered in some infected fish. Histopathological examination revealed that fatty liver syndrome with hepatocyte degeneration, reflected in heterokaryons, inflammatory lesions, and melanomacrophage centers (MMCS), had caused fibrosis around the kidney, spleen, and blood vessels. After the outbreak, visceral fat and green liver syndrome in the mullets were consistently observed throughout the year in the same mariculture facilities, indicating that the cultured mullets suffered a chronic metabolic disorder. Although Vibrio spp. were also isolated from some individuals, L. garvieae, which is known to be a causative agent of red lip mullet mortality, was isolated from all diseased individuals. This is the first report of L. garvieae infection in cultured red lip mullet.

Molecular and Histopathological Evidence of Mycobacteriosis in Paradise Fish Macropodus opercularis Imported into Korea

한현자,오명주,김정호,전찬혁,김위식,김도형,정성주 한국수산과학회 2013 Fisheries and Aquatic Sciences Vol.16 No.3

We report on mycobacteriosis in an imported tropical ornamental fish Macropodus opercularis commonly known as the paradise fish. Mass mortality occurred in paradise fish imported to Korea from Southeast Asia in 2008. The affected fish did not show any outward clinical signs, but enlargement of the spleen, kidneys, and liver was observed on dissection. Histopathological examination revealed numerous granulomas in the spleen, and acid-fast bacilli were observed in the centers of the granulomas. About 65% of spleen DNA samples were PCR positive using mycobacteria-specific primers targeting the 16S rRNA and hsp65 genes. The nucleotide identities of the 16S rRNA and hsp65 genes with those of Mycobacterium marinum were 99.5% and 99.4%, respectively. Although the bacterium was not cultured, the molecular diagnosis and histopathological findings were consistent with mycobacteriosis in paradise fish.

한국과 일본의 담수어에서 분리되는 운동성 aeromonads의 표현형적 특성과 약제감수성

한현자 ( Hyun Ja Han ),고빈다사미비빅카난한 ( Vivekanandhan Govindasami ),히로노이쿠오 ( Ikuo Hirono ),아오키타카하시 ( Takahashi Aoki ) 한국어병학회 2007 한국어병학회지 Vol.20 No.3

Prevalence and Characterization of Typical Aeromonas salmonicida Chum Salmon Isolates in Korea

김용석,윤장원,한현자,Rungkarn Suebsing,김정호 한국수산과학회 2011 Fisheries and Aquatic Sciences Vol.14 No.4

Aeromonas salmonicida is an important fish pathogen commonly associated with furunculosis in salmonids. Typical A. salmonicida strains have the surface virulence A-layer protein, a major virulence determinant encoded by the vapA gene. In this study, 880 chum salmon Oncorhynchus keta were collected from the east coast of Korea during 2006-2011, including 560 wild adults and 320 artificially hatched fry pools, and the presence of typical A. salmonicida was examined by PCR using the typical A. salmonicida-specific vapA gene primers. The results demonstrated that 34.5% of the samples (304/880 samples) were PCR positive, implying that a typical A. salmonicida infection is highly prevalent among chum salmon in Korea. Twenty typical A. salmonicida isolates were recovered based on their brown pigmentation on Trypticase Soy Agar (TSA) plates, which indicates the existence of the A-layer protein. Further biochemical analyses with the four randomly selected typical A. salmonicida isolates revealed some variations in their amino acid decarboxylation and carbohydrate fermentation activity. A phylogenetic analysis based on the entire vapA gene sequence suggested that the A. salmonicida isolates from chum salmon were clustered with those isolated from Atlantic salmon in Europe. Further study is needed to resolve such an interesting relationship in detail.