Epizootic Outbreaks of Bacterial Cold Water Disease among Populations of River Ayu, Plecoglossus altivelis, in Japan – a Review

Hisatsugu Wakabayashi

HW Fish Laboratory, 3－22－11－717 Sendagi, Bunkyo-ku, Tokyo, 113-0022, Japan

Correspondence: H. Wakabayashi ([email protected]

Abstract Since 1993, epizootics of bacterial cold water disease have been confirmed among populations of ayu, Plecoglossus altivelis, in many rivers. Outbreaks of the disease occurred in April through November, when water temperature was between 12 and 20 C. A sudden drop in water temperature after heavy raining had a major impact on the outbreaks. Carrier states for Flavobacterium psychrophilum were demonstrated in ayu populations in many rivers. Nation-wide surveys revealed that 20 out of 47 fish species carried F. psychrophilum. However, serotypes O-2 and O-2/4 were found only in isolates from ayu. In addition, genotype A based on PPIC gene was also unique to ayu.

Introduction Ayu or sweetfish, Plecoglossus altiveris is an amphidromous fish, the only species in the genus. It is a relative of smelts and in the order Osmeriforms. It occurs in rivers, lakes, and coastal waters of Hokkaido in Japan southward to Korea, China, and Taiwan. Typically, its life span is one year. In autumn, the female adults lay eggs on pebbles of the river bed. Then, the parent ayu die. The baby ayu are carried downstream into the sea. They stay near the bay area and feed planktons during the winter. In spring, the juvenile ayu start the upstream journey. In addition, the juveniles collected in Lake Biwa or the hatcheries are restocked into streams in order to increase ayu available for fishing in many rivers. In summer, they primarily feed on algae on underwater rocks. They show strong territorial behaviors.

Outbreaks of bacterial cold water disease in ayu In February 1987, Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) was isolated from diseased fingerling ayu at a farm in Tokushima Prefecture, 2-3 days after they were transported from Lake Biwa, Shiga Prefecture (Figure 1)1). This isolation is the first in Japan. In July through November of 1993, outbreaks of BCWD occurred in wild ayu in Gonokawa River, Hiroshima Prefecture (Figures 2)2). Since then, epizootics of BCWD have been confirmed in many rivers where ayu are grown. It is considered that the restocking of juvenile ayu distributed the pathogen carriers into many rivers and spread the disease over the country (Figure 3)3). The restocked ayu are checked free from F. psychrophilum before being released into rivers. However, outbreaks of BCWD still occur in most of the rivers every year. The outbreaks occur in river ayu populations in April through November 3). They usually occur at water temperature between 12 and 20 C, but sometimes down to 10 C and up to 24 C 3). The outbreaks are apt to occur 1 to 2 weeks after it rains heavily. An increase in an amount of water seems to have no impact. The effect of an increase in a turbidity of water is suspected, but no evidence has been provided for it with any experiments. A sudden drop in water temperature can have a major impact on the outbreaks. An experiment was conducted to show the effects of a sudden drop in water temperature on outbreaks of BCWD in ayu4). Fish group A and B were acclimated at 20 C. Then fish group A was exposed to F. psychrophilum and placed at 15 C. Fish group B was placed at 15 C without the exposure. Fish group C and D were acclimated at 15 C. Then fish group C was exposed to the pathogen and kept at the same temperature. Fish group D was kept at the same temperature without the exposure. The experiment was made double. As the results, fish group A showed significantly higher mortality than fish group C.

Carriers of F. psychrophilum Many investigations have been conducted to gain information on carriers of F. psychrophilum in natural waters. The land-locked ayu were collected in Lake Biwa in the spring of 19955). The homogenates of the kidney tissues were examined for F. psychrophilum with PCR. Thirty five percent of the samples (6/17) and 18 percent of the total fish examined (13/72) were positive. Examinations for the pathogen were conducted of different life cycle stages of ayu populations in 4 rivers6). These surveys indicated that 27.8 % of spawning adults, 5.0 % of eyed eggs, 5.9% of fry hatched out in rivers, 1.7% of ascending juveniles, and 2.6% of juveniles for restocking carried F. psychrophilum , but no carrier was found among juveniles collected in the sea. It was suspected that certain species of fish residents in the rivers provided source of infection for ayu migrating from the sea. In a nationwide survey, the pathogen carriers were found in ten out twelve of fish families examined, i.e. Anguillidae, Cottidae, Cyprinidae, Salmonidae, Cobitidae, Clupeidae, and Gobiidae. Totally, 20 out of 47 species examined were shown to carry the pathogen 6).

Serotypes and genotypes of F. psychrophilum Studies on the relationship between host species and serotypes of F. psychrophilum revealed that serotypes O-2 and O-2/4 were unique serotypes to ayu7). In other words, strains possessing the O-2 antigen have never been isolated from fishes other than ayu. In addition, genotype A that is based on peptidyl-prolyl cis-trans isomerase C (PPIC) gene have been found only in the isolates from ayu3)8) . An experiment showed that F. psychrophilum derived from diseased ayu might have no infectivity to other fish species6). In the experiment, three species of fish were exposed to outflow of a tank containing ayu with BCWD. The experiment was made double. Cumulative mortalities of ayu were 30~100% and 60~100%. In contrast, those of oikawa, Zacco platypus, and amago, Oncorhynchus rodurus were almost O %. The evidence that ayu has its own peculiar type of F. psychrophilum bring about a question where the type is originated. A hypothesis is that selection for the adapted mutants of F. psychrophilum might have occurred in Lake Biwa, the largest freshwater lake in Japan, because the single outlet of the lake, Seta River, is dammed and no ayu migrates from the sea. The landlocked ayu spawn in either rivers, approximately 340 inlets, or the lake shore, and the fry grow up in freshwater all their life. The reservoirs of the ayu-type of F. psychrophilum in rivers are unclear, especially in winter, when almost all ayu are absent from the rivers. Recently, it was found that over-wintering ayu in some rivers carried the ayu-type of the pathogen, suggesting a source of infection for new comers of ayu in the spring9).

References 1- Wakabayashi, H., T. Toyama and T. Iida (1994): A study on serotyping of Cytophaga psychrophila isolated from fishes in Japan. Fish Pathology, 29, 101-104. 2- Iida, Y. and A. Mizokami (1996): Outbreaks of coldwater disease in wild ayu and pale chub. Fish Pathology, 31, 157-164. 3- Council for ayu coldwater disease initiative, MAFF (2008): Final report, 22 pp. (in Japanese). 4- Kochi prefectural research group for control measure of ayu coldwater disease (2005): Annual Report for 2004, 55 pp.(in Jananese) 5- Izumi S. and H. Wakabayashi (1977): Use of PCR to detect Cytophaga psychrophila from apparently healthy juvenile ayu and coho salmon eggs. Fish Pathology, 32, 169-173. 6- Council for ayu coldwater disease initiative, MAFF (2001): Interim report, 63 pp. (in Japanese) 7- Izumi, S, H. Liu, F. Aranishi and H. Wakabayashi (2003): A novel serotype of Flavobactrium psychrophilum detected using antiserum against an isolate from amago, Oncorhunchus masou rodurus Jordan & Gilbert, in Japan. Journal of Fish Diseases, 26, 677-680. 8- Yoshiura, Y, T. Kamaishi, C.Nakayasu and M. Ototake (2006): Detection and genotyping of Flavobacterium psychrophilum by PCR targeted to peptidyl-prolyl cis-trans isomerase C gene. Fish Pathology, 41, 67-71 9- Miyazaki T. (2008): Flavobacterium psychrophilum isolated from over-wintering ayu Plecoglossus altivelis , Fish Pathology, 43, 167-169

Figure 1 BCWD in fingering ayu Figure 2 BCWD in wild adult river ayu

Figure 2 Spread of bacterial cold water disease (BCWD) among river ayu populations.