한국어병학회지 (Journal of fish pathology)

  • 제36권2호
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  • Pages.263-275
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  • 2023
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  • 1226-0819(pISSN)
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  • 2233-5412(eISSN)
한국어병학회 (The Korean Society of Fish Pathology)
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해수에 순치된 첨연어(Oncorhynchus keta)에서 분리된 정형 에로모나스 살모니시다(Aeromonas salmonicida)에 대한 특성 분석

Characterization of typical Aeromonas salmonicida isolated from Sea-Chum Salmon (Oncorhynchus keta)

  • 임종원 (강릉원주대학교 생명과학대학 수산생명의학과) ;
  • 고성재 (강릉원주대학교 생명과학대학 수산생명의학과) ;
  • 박영준 (강릉원주대학교 생명과학대학 수산생명의학과) ;
  • 안도일 (강릉원주대학교 생명과학대학 수산생명의학과) ;
  • 홍수희 (강릉원주대학교 생명과학대학 수산생명의학과)
  • Jongwon Lim (Department of Aquatic Life Medicine, Gangneung-Wonju National University) ;
  • Sungjae Ko (Department of Aquatic Life Medicine, Gangneung-Wonju National University) ;
  • Youngjun Park (Department of Aquatic Life Medicine, Gangneung-Wonju National University) ;
  • Do-il Ahn (Department of Aquatic Life Medicine, Gangneung-Wonju National University) ;
  • Suhee Hong (Department of Aquatic Life Medicine, Gangneung-Wonju National University)
  • 투고 : 2023.11.15
  • 심사 : 2023.12.14
  • 발행 : 2023.12.31
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초록

첨연어(Oncorhynchus keta)는 한국에서 회귀하는 연어로 종 보존을 위해 경상북도 울진군에 위치한 수산자원공단에서 해수 순치하며 종묘 생산하였다. 그러나, 사육 중이던 첨연어 치어에서 세균성 질병에 감염된 증상을 보이며 폐사하였다. 따라서, 본 연구에서는 2021년 10월경에 사육환경에 따라 구분된 첨연어로부터 원인체를 규명하고자 세균을 분리하였다. 분리된 세균은 16S rDNA, rpoD (RNA polymerase sigma factor σ70) 및 vapA (A-layer)의 염기서열을 기반으로 유전학적으로 동정하고, 추가로 염분에 따른 성장, 생화학적 특성 분석, 항생제 감수성 및 병원성 인자를 분석하여 균주를 특성화하였다. 그 결과, 분리된 4개의 균주는 모두 Aeromonas salmonicida subsp. salmonicida 아종으로 동정하였다. 또한, 분리된 균주는 염분의 농도가 증가할수록 배지에서 성장이 감소하였다. 생화학적 특성 분석에서 분리된 4개 균주는 용혈성이 확인되지 않았고, 모두 같은 생화학적인 성상을 나타냈다. 항생제 감수성 분석에서는 oxolinic acid, flumequine 그리고 florfenicol에 대한 항생제에서 40~44 mm 억제대를 형성하였다. 병원성 인자의 발현 분석은 mRNA 수준에서 RT-PCR로 확인되었으며, 4개 균주는 모두 A. salmonicida의 병원성에 크게 기여한다고 잘 알려진 outer membrane ring of T3SS (ascV), inner membrane ring of T3SS (ascC), vapA, enterotoxin (act) 및 lipase (lip) 유전자가 발현되었다. 이 연구에서 분리된 A. salmonicida subsp. salmonicida의 특성 분석은 해수에 순치된 첨연어에서 발병하는 절창병을 예방하기 위한 기초자료로 활용될 수 있을 것이다.

Chum salmon (Oncorhynchus keta) is a species which returns to Korea for spawning and was produced as seed production at the Fisheries Resources Agency located in Uljin-gun, Gyeongsangbuk-do to preserve the species. However, farmed chum salmon showed symptoms of bacterial infection. Therefore, in this study, bacteria were isolated to identify the causative agent from chum salmon in October 2021. The isolated bacteria were identified based on the sequences of 16S rDNA, rpoD (RNA polymerase sigma factor σ70), and vapA (A-layer) genes. Also, salinity-growth curve, biochemical characterization, antibiotic susceptibility test, and pathogenicity analysis were performed in four strains. As a result, four isolated strains were identified as Aeromonas salmonicida subsp. salmonicida. Additionally, the bacterial strains showed a decrease in growth as the salt concentration increased in the medium. All of the isolated strains exhibited γ-hemolysis, and the same biochemical properties. In the antimicrobial susceptibility test, all strains showed an inhibition zone of 40 to 44 mm for oxolinic acid, flumequine, and florfenicol. Pathogenic factors were assessed by RT-PCR at the mRNA level, and found that the four strains expresses the outer membrane ring of T3SS (ascV), inner membrane ring of T3SS (ascC), vapA, enterotoxin (act), and lipase (lip) genes which are well known to significantly contribute to the pathogenicity of A. salmonicida. The results of this study can be used as basic data to prevent A. salmonicida subsp. salmonicida occurring in sea-chum salmon in the future.

키워드

과제정보

이 논문은 2022학년도 강릉원주대학교 학술연구조성비 지원으로 수행되었습니다.

참고문헌

  1. Abbott, S. L., Cheung, W. K., and Janda, J. M.: The genus Aeromonas: biochemical characteristics, atypical re-actions, and phenotypic identification schemes. Journal of clinical microbiology, 41(6): 2348-2357, 2003. DOI: 10.1128/JCM.41.6.2348-2357.2003
  2. Amann, R. I., Ludwig, W., & Schleifer, K. H.: Phyloge-netic identification and in situ detection of individual microbial cells without cultivation. Microbiological reviews, 59(1), 143-169. 1995. DOI: 10.1128/mr.59.1.143-169.1995
  3. Beaz-Hidalgo, R., and Figueras, M. J.: Aeromonas spp. whole genomes and virulence factors implicated in fish disease. Journal of fish diseases, 36(4), 371-388. 2013. https://doi.org/10.1111/jfd.12025.
  4. Chacon, M. R., Soler, L., Groisman, E. A., Guarro, J., and Figueras, M. J.: Type III secretion system genes in clinical Aeromonas isolates. Journal of Clinical Microbiology, 42, 1285-1287, 2004. DOI: 10.1128/jcm.42.3.1285-1287.2004.
  5. Chopra, I., and Roberts, M.: Tetracycline antibiotics: mode of action, applications, molecular biology, and epidemiology of bacterial resistance. Microbiology and molecular biology reviews, 65(2), 232-260, 2001. DOI: 10.1128/mmbr.65.2.232-260.2001.
  6. Dallaire-Dufresne, S., Tanaka, K. H., Trudel, M. V., Lafaille, A., and Charette, S. J.: Virulence, genomic features, and plasticity of Aeromonas salmonicida subsp. salmonicida, the causative agent of fish furunculosis. Veterinary microbiology, 169(1-2):1-7, 2014. DOI: 10.1016/j.vetmic.2013.06.025.
  7. Daher, R. K., Filion, G., Tan, S. G. E., Dallaire-Dufresne, S., Paquet, V. E., and Charette, S. J.: Alteration of virulence factors and rearrangement of pAsa5 plasmid caused by the growth of Aeromonas salmo-nicida in stressful conditions. Veterinary Microbiology, 152(3-4), 353-360, 2011. DOI: 10.1016/j.vetmic.2011.04.034.
  8. Frey, J., and Origgi, F. C.: Type III secretion system of Aeromonas salmonicida undermining the Host's immune response. Frontiers in Marine Science, 3, 130, 2016. DOI: 10.3389/fmars.2016.00130.
  9. Gulla S, Lund V, Kristoffersen A. B, Sorum H, Colquhoun D. J. vapA (A-layer) typing differentiates Aeromonas salmonicida subspecies and identifies a number of previously undescribed subtypes. Journal of Fish Disease, 39(3): 329-42, 2016. DOI: 10.1111/jfd.12367.
  10. Guijarro, J. A., Cascales, D., Garcia-Torrico, A. I., GarciaDominguez, M., and Mendez, J.: Temperature-dependent expression of virulence genes in fish-pathogenic bacteria. Frontiers in microbiology, 6, 700, 2015. DOI: 10.1128/iai.43.1.195-201.1984
  11. Irshath, A. A., Rajan, A. P., Vimal, S., Prabhakaran, V. S., and Ganesan, R.: Bacterial pathogenesis in various fish diseases: Recent advances and specific challenges in vaccine development. Vaccines, 11(2), 470, 2003. DOI: 10.3390/vaccines11020470.
  12. Kingombe, C. I. B., Huys, G., Tonolla, M., Albert, M. J., Swings, J., Peduzzi, R., and Jemmi, T.: PCR detection, characterization, and distribution of virulence genes in Aeromonas spp. Applied and Environment Microbiology, 65, 5293-5302, 1999. DOI: 10.1128/AEM.65.12.5293-5302.199.
  13. Kim, D. H., Choi, S. Y., Kim, C. S., Oh, M. J., and Jeong, H. D.: Low-value fish used as feed in aquaculture were a source of furunculosis caused by atypical Aeromonas salmonicida. Aquaculture, 408:113-117, 2013. DOI: 10.1016/j.aquaculture.2013.05.014.
  14. Kim, J. H., Hwang, S. Y., Son, J. S., Han, J. E., Jun, J. W., Shin, S. P., ... & Park, S. C. (2011). Molecular characterization of tetracycline-and quinolone-resistant Aeromonas salmonicida isolated in Korea. Journal of Veterinary Science, 12(1), 41-48, 2011. DOI: 10.4142/jvs.2011.12.1.41.
  15. Kim, Y. S., Yoon, J. W., Han, H. J., Suebsing, R., and Kim, J. H.: Prevalence and characterization of typical Aeromonas salmonicida chum salmon isolates in Korea. Fisheries and aquatic sciences, 14(4):347-354, 2011. DOI: 10.5657/FAS.2011.0347.
  16. Lim, J., and Hong, S.: Characterization of Aeromonas salmonicida and A. sobria isolated from cultured salmonid fish in Korea and development of a vaccine against furunculosis. Journal of fish diseases, 43(5):609-620, 2020. DOI: 10.1111/jfd.13158.
  17. Lim, J., Koo, B., Kim, K. I., Jeong, H. D., and Hong, S.: Genetic identification of Aeromonas species using a housekeeping gene, rpoD, in cultured salmonid fishes in Gangwon-Do. Journal of Fish Pathology, 30, 79-88, 2017. DOI: 10.7847/jfp.2017.30.2.079.
  18. Maulu, S., Hasimuna, O. J., Haambiya, L. H., Monde, C., Musuka, C. G., Makorwa, T. H., ... & Nsekanabo, J. D.: Climate change effects on aquaculture production: sustainability implications, mitigation, and adaptations. Frontiers in Sustainable Food Systems, 5, 609097, 2021. DOI: 10.3389/fsufs.2021.609097
  19. Martinez-Murcia, A. J., Monera, A., Saavedra, M. J., Oncina, R., Lopez-Alvarez, M., Lara, E., and Figueras, M. J.: Multilocus phylogenetic analysis of the genus Aeromonas. Systematic and applied microbiology, 34(3), 189-199, 2011. DOI: 10.1016/j.syapm.2010.11.014.
  20. Miyata M, Inglis V, Aoki T.: Rapid identification of Aeromonas salmonicida subspecies salmonicida by the polymerase chain reaction. Aquaculture 141:13-24, 1996. DOI: 10.1016/0044-8486(95)01221-4.
  21. Nilsson, W. B., Gudkovs, N., and Strom, M. S.: Atypical strains of Aeromonas salmonicida contain multiple copies of insertion element ISAsa4, useful as a genetic marker and a target for PCR assay. Diseases of aquatic organisms, 70(3), 209-217, 2006. DOI: 10.3354/dao070209.
  22. Piotrowska, M., and Popowska, M.: Insight into the mobilome of Aeromonas strains. Frontiers in microbiology, 6, 494, 2015. DOI: 10.3389/fmicb.2015.00494.
  23. Reith M. E, Singh R. K, Curtis B, Boyd J. M, Bouevitch A, Kimball J, Munholland J, Murphy C, Sarty D, Williams J, Nash J. H, Johnson S. C, Brown L. L.: The genome of Aeromonas salmonicida subsp. salmonicida A449: insights into the evolution of a fish pathogen. BMC Genomics. Sep 18;9:427, 2008. DOI: 10.1186/1471-2164-9-427.
  24. Santos, J. A., Gonzalez, C. J., Otero, A., and Garcia-Lopez, M. L.: Hemolytic activity and siderophore production in different Aeromonas species isolated from fish. Applied and Environment Microbiology, 65, 5612-5614, 1999. DOI: 10.1128/AEM.65.12.5612-5614.1999.
  25. Sen, K., and Rodgers, M.: Distribution of six virulence factors in Aeromonas species isolated from US drinking water utilities: A PCR identification. Journal of Applied Microbiology, 97, 1077-1086, 2004. DOI: 10.1111/j.1365-2672.2004.02398.x.
  26. Sommerset, I., Krossoy, B., Biering, E., and Frost, P.: Vaccines for fish in aquaculture. Expert review of vaccines, 4(1), 89-101, 2005. DOI: 10.1586/14760584.4.1.89.
  27. Shivam, S., El-Matbouli, M., and Kumar, G.: Development of fish parasite vaccines in the OMICs era: Progress and opportunities. Vaccines, 9(2), 179, 2021. DOI: 10.3390/vaccines9020179.
  28. Stuber, K., Burr, S. E., Braun, M., Wahli, T., and Frey, J.: Type III secretion genes in Aeromonas salmonicida subsp. salmonicida are located on a large thermolabile virulence plasmid. Journal of clinical microbiology, 41(8), 3854-3856, 2003. DOI: 10.1128/jcm.41.8.3854-3856.2003.
  29. Wiklund T and Dalsgaard I.: Occurrence and significance of atypical Aeromonas salmonicida in non-salmonid and salmonid fish species: a review, 32: 49-69, 1998. DOI: 10.3354/dao032049.
  30. Wang, Y., and Gu, J.: Influence of temperature, salinity and pH on the growth of environmental Aeromonas and Vibrio species isolated from Mai Po and the Inner Deep Bay Nature Reserve Ramsar Site of Hong Kong. Journal of Basic Microbiology: An International Journal on Biochemistry, Physiology, Genetics, Morphology, and Ecology of Microorganisms, 45(1), 83-93, 2005. DOI: 10.1002/jobm.200410446.
  31. Wee, G. N., Lyou, E. S., Hong, J. K., No, J. H., Kim, S. B., & Lee, T. K.: Phenotypic convergence of bacterial adaption to sub-lethal antibiotic treatment. Frontiers in Cellular and Infection Microbiology, 12, 913415, 2022. DOI: 10.3389/fcimb.2022.913415.
  32. Wong, C. Y., Heuzenroeder, M. W., and Flower, R. L.: Inactivation of two haemolytic toxin genes in Aero-monas hydrophila attenuates virulence in a suckling mouse model. Microbiology, 144, 291-298, 1998. DOI: 10.1099/00221287-144-2-291.
  33. 명정구, 김병일, 이선병, 전길봉: 우리바다 어류도감, pp. 287, 예조원, 2010.