Journal of the Faculty of Fisheries and Animal Husbandry, Hiroshima University Volume 14 Issue 1
1975-08-30 発行

Vibrio anguillarumおよびその感染症に関する研究

Studies on Vibrio anguillarum and V. anguillarum infection
Muroga, Kiyokuni
Part I
Vibrio anguillarum was identified as the causative organism of epizootics of vibriosis in Ayu (Plecoglossus altivelis) and in some other fishes cultured in Japan.
1) In every spring from 1965 to 1967, young Ayu, which were caught in Lake Hamana, a salt lake, as seed fish for pond-culture, showed heavy mortalities during the period of acclimatization to freshwater. A considerable part of the mortalities was caused by an infectious disease. The causative bacterium of this infection has been identified as Vibrio anguillarum.
2) In 1967 and 1970, similar disease occurred in stocked young Ayu caught in an estuary of the River Tone and along the sea-shore of some other districts, respectively. V. anguillarum was isolated from these diseased fish in each case.
3) In the summer of 1969, an epizootic occurred in Ayu in freshwater ponds in Shiga and Nagano prefectures. The causative organism was identified as V. anguillarum. Since these fish were caught as seed in Lake Biwa, a freshwater lake, this was the first time for the bacterium to be isolated as the etiological agent from Ayu which had exclusively inhabited freshwater environments.
4) In 1973, Vibrio anguillarum infection was prevalent in Ayu in freshwater ponds in various parts of Japan, and led to heavy losses due to the ineffectiveness of sulfa drugs and antibiotics, which had been frequently used before in controlling efficiently.
5) In 1971, V. anguillarum was isolated from the diseased eel (Anguilla japonica) cultured in freshwater ponds in Tokushima prefecture. The water of these ponds contained a slight amount of sea-water. Ever since then, this infection of cultured eel has repeatedly occurred in that district. In contrast, such infection has never been observed so far in eel ponds in other districts of Japan.
6) From 1966 to 1974, V. anguillarum was isolated occasionally also from diseased specimens of such fishes in sea- or brackish waters, as cultured rainbow trout (Salmo gairdneri), yellow tail (Seriola quinqueradiata) and Kampachi (S. purpurascens), and wild grey mullet (Mugil cephalus).
7) As described above, vibriosis has recently become a serious problem in fish culture, especially in Ayu culture in Japan. It is wished that the actual method of Ayu culture should be examined radically anew, in view of controlling the occurrence of this infectious disease.

Part II
The microbiological characteristics of V. anguillarum isolated by the present author were studied comparatively and comprehensively. The type description of V. anguillarum listed in Bergey's manual of determinative bacteriology (8th ed. 1974) was discussed on the basis of the characteristics of the strains isolated by the present author and some other strains reported by foreign workers. Furthermore, some comparative studies of the present strains of V. anguillarum with other fish-pathogenic vibrios reported in Japan, and also with Vibrio parahaemolyticus and Vibrio alginolyticus were carried out.
8) The characteristics of V. anguillarum isolated from various fishes as described in the previous part proved to be identical with each other (not serologically), and these isolates were reaffirmed to be classified as V. anguillarum in referrence to Bergey's new Manual.
9) From investigations of the present isolates, it seems necessary to revise the type description of V. anguillarum in the Manual for some items such as indole production, growth at 5℃ and some sugar utilizations.
10) It seems reasonable to combine V. anguillarum, Vibrio piscium, V. piscium var. japonicus and Vibrio ichthyodermis as a single species, under the name of V. anguillarum. At the same time, however, it is suggested that V. anguillarum should be divided into separate types on the basis of the differences in pathogenicity and in some biochemical characteristics.
11) It was indicated that some vibrios from rainbow trout and marine fishes in Japan, as reported by other authors, can be differentiated from V. anguillarum by certain biochemical characteristics and by their pathogenicity.
12) From comparative experiments, it was demonstrated that V. anguillarum differs from V. parahaemolyticus in the following points; arginine dihydrolation, lysine decarboxylation, sucrose fermentation, Voges-Proskauer reaction, NaCl-tolerance and pathogenicity for the eel. Likewise, V. anguillarum differs from V. alginolyticus by arginine dihydrolation, lysine decarboxylation, NaCl-tolerance and pathogenicity for the eel.

Part III
The Japanese eel (A. japonica) was selected as test-material for the evaluation of pathogenicity of V. anguillarum, and a method to confirm the pathogenicity was sought for. In the latter section of this part, some host-parasite relationships in experimental infection were investigated.
13) Susceptibilities of several freshwater and marine fishes against V. anguillarum (strain PB-15, from Ayu) were tested by means of intramuscular injection. As a result, it appeared that the Japanese eel, the European eel (A. anguilla) and loach (Misgurnus anguillicaudatus) possess a remarkable susceptibility; that rainbow trout, black sea bream (Mylio macrocephalus) and yellow tail have a relatively high susceptibility too, but that carp (Cyprinus carpio), goldfish (Carassius auratus) and crucian carp (C. carassius) have a low susceptibility.
14) On account of this high susceptibility and for practical convenience, the Japanese eel was chosen as the test-material fish, and a method for the evaluation of pathogenicity was sought. The conclusions were as follows: intramuscular injection proves to be the most reliable method and 1 mg of the bacterium in wet weight (8×108  cells) per 100 g of fish body weight is the adequate dose for inoculation. Even small eels (more than 10 g in body weight) can be used as materials. The experimental results can be obtained by a water temperature of 10℃, but show up more rapidly at 20℃.
15) Under the above mentioned experimental conditions, more than 90% of the Japanese eels that received a dose of 1 mg of cells of the bacterium died within a week, while none of the eels that received only 0.1 mg died. It was confirmed that the number of the bacterium in the blood and in some other tissues of the former eels augmented slowly up to 106 cells/ml or g; and that on the contrary, the bacterium in the latter eels diminished in number and at 72 hours after the inoculation the bacterium disappeared in every tissue.
16) From hematological studies of the inoculated eels, it was proved that no change had occurred in the hematocrit value, the hemoglobin content and the number of erythrocyte, even for the eels that had received a lethal dose (1 mg). But changes in number of leucocytes, especially of neutrophil, were markedly contrasted between the eels that received 1 mg and those received 0.1 mg.
17) From the histological studies, it could be shown that the eels which had received a lethal dose fell into a systemic septisemia.
18) A supernatant fluid of the sonicated cell suspension of the strain PB-15 was proved to have no toxic effect on the eel.
19) Most of the strains of V. anguillarum showed no pathogenicity for mouse, but in a few 'experiments some strains exhibited pathogenicity, though weaker than that of V. parahaemolyticus.

Supplementary part
Some immunological laboratory experiments were carried out, using the eels as test-material.
20) The eels produced agglutinins and protective immunity by receiving the merzonin-killed cells intramuscularly.
21) Water temperature played an important role on the formation of antibodies in the eel; within a temperature range from 15℃ to 23℃, the maximum titer, viz. 800~1,600, were attained more rapidly at higher temperature, but no difference was found between 23℃ and 27℃. No measurable antibody was produced at 11℃.
22) The agglutinating antibody induced by Freund's incomplete adjuvant vaccine were maintained for about 4 months at a low temperature of 7 to 15℃.
23) An attempt was made on oral immunization of the eel against V. anguillarum. The group fed on merzonin-killed cells did not produce antibodies, but the group fed on viable cells produced circulating agglutinating antibodies after three month's feeding. It was shown that the protective immunity of the eel fed on viable cells was very weak however, even after four month's feeding.