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Ecology and Molecular Biology of BCWD- Pathogen

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Ecology and Molecular Biology of BCWD- Pathogen Genotyping and pathogenicity of Flavobacterium psychrophilum in Japan Erina Fujiwara-Nagata1,2, Kazuhiro Sugahara1,3, Mitsuru Eguchi1 1 Graduate School of Agriculture, Kinki University, Nara 631-8505, Japan 2 INRA, UR892 Virologie et Immunologie Molécularires, F-78350 Jouy-en-Josan, France 3 Fisheries Experimental Station of Shiga Prefecture, Shiga 522-0057, Japan Correspondence: E. Fujiwara-Nagata ([email protected]) Abstract Flavobacterium psychrophilum is a causative agent of ‘bacterial cold-water disease (BCWD)’ and ‘rainbow trout fry syndrome,’ which affect salmonid and other fish species. In Japan, BCWD causes serious damage to ayu, Plecoglossus altivelis. To prevent BCWD outbreaks in ayu, the precise distribution of F. psychrophilum, which infects ayu inhabiting natural water systems, needs to be determined. However, the infection route remains unclear partly because F. psychrophilum is isolated from not only Plecoglossidae and Salmonidae but also various other fish species, and its precise host range is unknown. In this study, we established a novel typing method for F. psychrophilum to distinguish the F. psychrophilum that infects ayu from the F. psychrophilum that infects other fish species. Using single nucleotide polymorphisms (SNPs) in the gyrA gene of F. psychrophilum, we classified isolated strains into 4 genotypes (ayu type, salmonid type, multi-fish type and new emerging type) and examined the pathogenicity of these strains against ayu. We used 232 strains of BCWD pathogens isolated from Plecoglossidae (ayu), Osmeridae (surf smelt), Cyprinidae (Japanese dace, pale chub, and crucian carp), Salmonidae (rainbow trout, coho salmon, etc). We clearly distinguished the strains on the basis of the SNPs present in their gyrA genes by using polymerase chain reaction-restriction fragment length polymorphism. We chose representative strains from these groups and examined their pathogenicity against ayu. Three strains with the ayu-type genotype exhibited strong pathogenicity for ayu, while 12 strains with the salmonid-type genotype exhibited no pathogenicity for ayu. Among 16 strains with the multi-fish-type genotype, only 8 exhibited pathogenicity for ayu. Further, 7 strains with the new emerging-type genotype did not exhibit pathogenicity for ayu. To examine the distribution of F. psychrophilum, water and fish samples were collected from a river flowing into Lake Biwa (Shiga, Japan) from May 2005 to January 2007. Modified cytophaga agar plate was used to isolate F. psychrophilum. Isolates were grouped into four clusters, “ayu”, “salmonid”, “multi-fish” and “new emerging” types using the gyrA-genotyping. For water samples, addition to the culture method, we applied a loop-mediated isothermal amplification method (LAMP) to enumerate the abundance of the F. psychrophilum cells1. We filtered water samples and extracted DNA. F. psychrophilum DNA (parE) in the water samples was amplified by LAMP. LAMP amplifies DNA with high specificity, sensitivity and rapidity under isothermal conditions2. From spring through autumn, juvenile ayu migrated to the upstream regions of the river. After autumn, which is the breeding season, all the adult ayu died. The results of parE-LAMP clarified that F. psychrophilum existed in the river throughout the year. Living F. psychrophilum with ayu-type and multi-fish-type genotypes were detected from early summer to autumn. After the disappearance of ayu from the river, F. psychrophilum with the ayu-type genotype was not detected. Biwa trout, a local trout, inhabited the river after the disappearance of the ayu. During this period, multi-fish and salmonid-type genotypes were detected in various fish species and water samples. Thus, the genotype of the F. psychrophilum detected in the samples from this river is closely associated with the host fish species inhabiting the river. References 1- Fujiwara-Nagata E. & Eguchi M., 2009. Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Flavobacterium psychrophilum. Journal of Fish Diseases, 32:873–881 2- Notomi T., Okayama H., Masubushi H., Yonekawa T., Watanabe K., Amino N. & Hase T., 2000. Loop-mediated isothermal amplification of DNA. Nucleic Acids Research, 28:e63 .
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