Simultaneous Detection of Flavobacterium psychrophilum, Pseudomonas plecoglossicida, and Vibrio anguillarum by a Multiplex PCR Targeting the gyrB Region

We have developed a multiplex PCR to detect bacterial disease agents of Flavobacterium psychrophilum, Vibrioanguillarum, and Pseudomonas plecoglossicida. Three primer pairs designed based on determined nucleotide sequences of thegyrB regions of these three bacteria were used. The detection limits and stringency of this method were higher enough forspecific detection of these three fish diseases. Using this multiplex PCR, the rapid and simultaneous diagnosis of three majorbacterial fish diseases caused by these bacteria from the organs of diseased fishes was successful.

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Actis, L. A., Tolmasky, M. E., & Crosa, J. H. (1999). Vibriosis. In P. T. K. Woo & D. W. Bruno (Eds.), Fish Diseases and Disorders, Vol. 3, Viral, Bacterial and Fungal Infections, (pp. 523–557). Wallingford, UK., CABI Publishing., 874 pp.

Ganzhorn, J. (1994). Vibriosis. In American Fisheries Society - Fish Health Section (Ed.) Blue Book 2016 Edition, Suggested Procedures for the Detection and Identification of Certain Finfish and Shellfish Pathogens., Maryland, USA., American Fisheries Society Book Series. Retrieved from http://www.afsfhs.org/perch/resources/14069220291.2.10vibriosis20 14.pdf

Izumi, S., & Wakabayashi, H. (1997). Use of PCR to detect Cytophaga psychrophila from apparently healthy juvenile ayu and coho salmon eggs. Fish Pathology, 3: 169-173. http://dx.doi.org/10.3147/jsfp.32.169

Izumi, S., & Wakabayashi, H. (2000). Sequencing of gyrB and their application in the identification of Flavobacterium psychrophilum by PCR. Fish Pathology, 35: 93-94. http://dx.doi.org/10.3147/jsfp.35.93

Izumi, S., & Aranishi, F. (2004). Relationship between gyrA mutations and quinolone resistance in Flavobacterium psychrophilum isolates. Applied and Environmental Microbiology, 70: 3968-3972. http://dx.doi.org/10.1128/AEM.70.7.3968-3972.2004

Izumi, S., Ouchi, S., Kuge, T., Arai, H., Mito, T., Fujii, H., Aranishi, F., & Shimizu, A. (2007). PCR-RFLP genotypings associated with quinolone resistance in Flavobacterium psychrophilum. Journal of Fish Diseases. 30: 141–147. http://dx.doi.org/10.1111/j.1365-2761.2007.00797.x

Izumi, S., Yamamoto, M., Suzuki, K., Shimizu, A., & Aranishi, F. (2007). Identification and detection of Pseudomonas plecoglossicida isolates with PCR primers targeting the gyrB region. Journal of Fish Diseases, 30: 391-397. http://dx.doi.org/10.1111/j.1365-2761.2007.00820.x

Izumi, S., & Suzuki, K. (2016). A Novel PCR detection method for major fish pathogenic bacteria of Vibrio anguillarum. Turkish Journal of Fisheries and Aquatic Sciences, 16: 571-578. http://dx.doi.org/10.4194/1303-2712-v16_3_09

Muroga, K., & Egusa, S. (1988). Vibriosis of Ayu: A Review. Journal of the Faculty of Applied Biological Science, Hiroshima University, 27: 1-17. http://dx.doi.org/0.15027/23482

Nishimori, E., Kita-Tsukamoto, K., & Wakabayashi, H. (2000). Pseudomonas plecoglossicida sp. Nov., the causative agent of bacterial haemorrhagic ascites of ayu, Plecoglossus altivelis. International Journal of Systematic and Evolutionary Microbiology, 50: 83-89. http://dx.doi.org/10.1099/00207713-50-1-83

Sako, H., & Kusuda, R. (1978). Chemotherapeutic studies on trimethoprim against vibriosis of pond-cultured ayu. I. Microbiological evaluation of trimethoprim and sulfonamides on thecausative agnent Vibrio anguillarum. Fish Pathology, 13: 91-96. http://dx.doi.org/10.3147/jsfp.13.91

Suzuki, K., Arai, H., Kuge, T., Katagiri, T., & Izumi, S. (2008). Reliability or PCR methods for the detection of Flavobacterium psychrophilum. Fish Pathology, 43: 124-127. http://dx.doi.org/10.3147/jsfp.43.124

Wakabayashi, H., Horinouchi, M., Bunya, T., & Hoshiai, G. (1991). Outbreaks of cold-water disease in coho salmon in Japan. Fish Pathology, 26: 211-212. http://dx.doi.org/10.3147/jsfp.26.211

Wakabayashi, H., Sawada, K., Ninomiya, K., & Nishimori, E. (1996). Bacterial hemorrhagic ascites of ayu caused by Pseudomonas sp. Fish Pathology, 31: 239-240. http://dx.doi.org/10.3147/jsfp.31.239

Walsh, P. S., Metzger, D. A., & Higuchi, R. (1991). Chelex100 as a medium for simple extraction of DNA for PCR based typing from forensic material. Biotechniques, 10: 507-513.