Pond-cultured Eels Research Articles

Studies on red spot disease of pond-cultured eels. An immunoelectrophoretic analysis of Pseudomonas anguilliseptica antigens.

Antigens of Pseudomonas anguilliseptica were investigated by immunoelectrophoresis using rabbit antisera. Fourteen distinct immunoelectrophoretic components were demonstrated in a representative strain belonging to Type I. Among them, one component having low electric mobility toward the anode corresponded to the heat-stable O antighen and ten different mobile components corresponded to 100°C-30 min heat-labile antigens which were thought to be H antigens. These eleven components and other two 100°C-30 min heat-stable components were all common to Type I and Type II. The remaining component which was 100°C-30 min heat-stable and migrated long toward the anode was specific to the Type I strain. This single component was thought to be the K antign reacting as agglutination-inhibitory substance in Type I strains. it was demonstrated by compara tive experiments that P. anguilliseptica shared some antigenic components with other fish-pathogenic bacterial strains belonging to the genera Pseudomonas, Vibrio, Aeromonas, and Edwardsiella.

Studies on red spot disease of pond-cultured eels - VI. Serological properties of Pseudomonas anguilliseptica in agglutination.

Serological properties of 96 strains of Pseudomonas anguillisepticu isolated from eels Anguilla japonica and A. anguilla were studied by agglutination tests with rabbit antisera. All strains tested were confirmed to possess a common heat-stable O-antigen. However, they could be divided into two antigenic types on the basis of the reaction to antisera produced by heat-killed cells (O-antisera). One type (Type I)was inagglutinable with O-antisera in unheated state, and this inagglutinability disappeared with a heat-treatment of 100°C-120 min or 121°C-30 min. Another (Type II) lacked such an inhibitory substance. This thermolabile agglutinationinhibitory antigen of P. anguilliseptica seems to be comparable to K-antigen known in the Coliform group. The existence of H-antigen was also demonstrated in both of the types. It was also confirmed that slide agglutination method was applicable to rapid identification of P. anguilliseptica.

Studies on Rapid Diagnosis System of Main Bacterial Diseases of Pond-cultured Eels in Japan-I

Epizootics of edwardsiellosis has been frequently occurring among pond-cultured eels, Anguilla japonica in Japan. Accurate diagnosis of the disease by means of conventional cultural method requires a day or more for completion. In this report, basic investigations for the application of direct immunofluorescence as rapid diagnostic tools of the disease were made.Antiserum was obtained from rabbit immunized with formalin-killed E. tarda (strain E-8) isolated from eel. The titer against formalin-killed homologous antigen was 1: 2048 by agglutination test. IgG of the antiserum was fractionated by ammonium sulfate precipitation and conjugated with FITC. The F/P molar ratio of labeled antibody was determined as 1.3.The labeled antibody had staining titer of 1:16 or 1:32 against 18 strains of E. tarda, including homologous strain E-8, isolated from pond-cultured eels at various regions in Japan. But no crossreaction was observed when 2 strains of V. anguillarum, 3 of Vibrio sp., 3 of A. liquefaciens, 8 of Aeromonas sp., one of Ps. anguilliseptica, one of Ps. fluorescens, one of Ps. putida, one of Escherichia coli, 2 of Salmonella sp., one of S. marcescens and one of F. columnaris were stained.The specific fluorescence of target antigen (E. tarda) was observed when acetone-fixed impression preparations of liver, kidney, spleen and injection site of muscle derived from artificially infected eel were stained with 4-fold dilution of labeled antibody. The specificity of the reaction on impression preparation was ascertained by one-step inhibition test. Stainability of E. tarda on impression preparation was stable for at least 11 days at 37°C. Stainability of E. tarda on impression preparation of liver tissue that had been preserved in 10% formalin for at least 7 days, though became slightly weaker, was not disappeared.

Studies on Rapid Diagnosis System of Main Bacterial Diseases of Pond-cultured Eels in Japan-II

Epizootics of edwardsiellosis has been frequently occurring among pond-cultured eels, Anguilla japonica in Japan. Accurate diagnosis of the disease by means of conventional cultural method requires a day or more for completion. In this report, basic investigations for the application of direct immunofluorescence as rapid diagnostic tools of the disease were made.Antiserum was obtained from rabbit immunized with formalin-killed E. tarda (strain E-8) isolated from eel. The titer against formalin-killed homologous antigen was 1: 2048 by agglutination test. IgG of the antiserum was fractionated by ammonium sulfate precipitation and conjugated with FITC. The F/P molar ratio of labeled antibody was determined as 1.3.The labeled antibody had staining titer of 1:16 or 1:32 against 18 strains of E. tarda, including homologous strain E-8, isolated from pond-cultured eels at various regions in Japan. But no crossreaction was observed when 2 strains of V. anguillarum, 3 of Vibrio sp., 3 of A. liquefaciens, 8 of Aeromonas sp., one of Ps. anguilliseptica, one of Ps. fluorescens, one of Ps. putida, one of Escherichia coli, 2 of Salmonella sp., one of S. marcescens and one of F. columnaris were stained.The specific fluorescence of target antigen (E. tarda) was observed when acetone-fixed impression preparations of liver, kidney, spleen and injection site of muscle derived from artificially infected eel were stained with 4-fold dilution of labeled antibody. The specificity of the reaction on impression preparation was ascertained by one-step inhibition test. Stainability of E. tarda on impression preparation was stable for at least 11 days at 37°C. Stainability of E. tarda on impression preparation of liver tissue that had been preserved in 10% formalin for at least 7 days, though became slightly weaker, was not disappeared.

Histopathological Studies on Gill-I

A bacterial gill disease named “Eragusare-byo” occurred among pond-cultured eels in a eel farm in Fukui Prefecture during March 1974. The pond-cultured eels with the disease included two species, Japanese eels (Anguilla japonica) and European eels (A. anguilla), and the water temperature of the ponds was above 20°C throughout the year. Histopathological studies on these diseased eels were done by the author and the following results were obtained.The eariest histopathological changes were observed on the gill lamellae of the two species of eels as inflammatory edema, swelling of the epithelial cells and excessive secretion of mucin. Column-like protrusions formed by a number of slender, rod-shaped Gram-negative bacteria were often found on the surfaces of the gill lamellae in these eels.In the subsequent inflammatory processes, there were marked differences regarding histological changes between Japanese and European eels. In the case of Japanese eels, there were three types of processes in the formation of “clubbing” of gill filaments. The type I was caused by hyperplasia of the lamellar epithelium at the distal tips of the lamellae. The type II was caused by hyperplasia of the epithelium in all the regions of the lamellae. The type III was caused by hyperplasia of the lamellar epithelium at the distal tips and basal parts of the lamellae. In many cases these three types of processes of the clubbing formation were recognized in the same individual. In a more aggravated case the clubbed filaments had gangrenous inflammation owing to combination of circulatory failure and putrifying bacterial infection, and finally they fell off from the gill arches. In European eels, the clubbing of the filaments was mainly caused by hyperplasia of mucous cells, but necrosis or defect of the clubbed filaments was never recognized.

Dermocystidium anguillae Found on the Gills of the European Eel, Anguilla anguilla Cultured in Japan

An epizootic of dermocystidiosis occurred among pond-cultured European eels, Anguilla anguilla for the first time at a fish farm in Shizuoka Prefecture. The fungus was identified as Dermocystidium anguillae which was first reported by SPANGENBERG (1975) in East Germany. The cysts were parasitic in the epithelial and connective tissues of the gills. Attempts were made to culture the parasite on some media, but without success.When the water temperature of tanks with the diseased eels was experimentally raised from 20°C to 30°C, the cysts disappeared from the gills of all the eels within a few days, which suggested that this disease could be cured by warm-water bathing.

Studies on red spot disease of pond-cultured eels. V. Immune response of the Japanese eel to the causative bacterium Pseudomonas anguilliseptica.

Immune response of the Japanese eel (Anguilla japonica; average weight: 70g) to formalinkilled Pseudomonas anguilliseptica cells administered intramuscularly was studied. Experimental fish had no specific agglutinin acquired naturally. Agglutinating antibody was detected 2 weeks after vaccination at water temperatures from 15°C to 28°C and the antibody response persisted over a period of 7 weeks (maximum titer: 256), and even at 10°C it was detected as late as 7 weeks after vaccination (titer: 2). And the antibody response in the eels immunized at about 20°C was maintained over 5 months through the low temperature period (minimum: 7°C). The use of Freund's complete adjuvant had a promotive effect on the development of agglutinin titer (maximum: 4096). Vaccinated eels were highly protected against experimental infection with virulent P. anguilliseptica, and this protective immune response was demonstrated even after wintering. These results suggest that this vaccination method will provide cultured eels with protective immunity against naturally occurring red spot disease.

Comparison of Intestinal Microflora between Healthy and Diseased Pond-cultured Eels

Quantitative and qualitative investigations of intestinal microflora were made on healthy and diseased eels collected from culturing ponds in Yoshida, Shizuoka Prefecture. The whole intestine of each eel was homogenized in sterile 0.85% NaCl solution. Tenfold dilutions were prepared and 0.1 ml of which was inoculated on the surface of nutrient agar (Difco). Plate cultures were incubated for five or seven days at 25°C and colonies were counted. Every colony within a given area of the plate of suitable dilutions was subcultured for identification.The numbers of viable bacteria per gram of intestinal tract with contents in healthy eels were ranged from 10 to 109 through the year. The percentage of Aeromonas spp. (mostly A. hydrophila) was relatively higher in winter, while that of Enterobacteriacea was higher from spring to fall. Vibrio sp. appeared only in October. Streptococcus sp. was always present 20% to 80% in the intestine of fed eels. Viable counts of intestinal bacteria of diseased eels were 10 to 106 higher than those of the healthy ones (Fig. 1 and Table 4). The percentage of A. hydrophila tended to be higher (Fig. 2), especially in the case of red disease. Moreover, virulent strains from eels suffered from red disease were greater in number than those from healthy ones or eels infected with Edwardsiella tarda (Table 5).

Seasonal changes of bacterial infections among pond-cultured eels, second series

Seasonal changes of bacterial infections among pond-cultured eels, second series

Studies on ostracoda in fishponds - I Two species in fishponds of the chiba prefectural freshwater fisgeries experimental station.

At the Chiba Prefectural Freshwater Fisheries Experimental Station some species of freshwater ostracods have often multiplied tremendously in fishponds and became a source of nuisance to pond-cultured eels. Two species of the ostracods were brought in for identification. Specimens were fixed with alcohol, and dissected and embedded in Canada balsam. The species proved to be the following: one called “Naga-Kaimijinko” was identi-fied as Candonocypris assimilis and is named “Hainaga-Kaimijinko”; the other, “Maru-Kaimijinko”, was allotted to Heterocypris incongruens attenuata and named “Kawari Ibo-Kaimijinko”, both of which are new to Japanese fauna. The former has hitherto been found only in Oceania in spite of its vital powers; its right valve is complicated in the marginal struc-ture. The latter has been confused with H. incongruens incongruens by some authors, but the scanning electron microscope photographs of valves may clarify the distinction between them.

Studies on Red Spot Disease of Pond-cultured Eels—II

Studies on Red Spot Disease of Pond-cultured Eels—II

Seasonal Changes of Bacterial Infections among Pond-Cultured Eels (Anguilla japonica)

Monthly investigations of bacterial infections among pond-cultured eels in the Yoshida area of Shizuoka prefecture were carried out in 1972 to 1973 (Table 1). Eels examined were collected from the ponds where mass mortalities had occurred just before each investigation. Isolation of bacteria from blood, liver, kidney, spleen and/or gill was attempted, using nutrient agar medium (Difco) and Cytophaga agar medium (modified).Among five established eel-pathogenic bacteria, Aeromonas hydrophila, Edwardsiella tarda, Pseudomonas anguilliseptica and Chondrococcus columnaris were isolated in these investigations, but not Vibrio anguillarum. Total incidences of the four bacterial infections and the mixed infections were calculated (Table 2 and 3). Seasonal distribution of each bacterial infection was shown in Fig. 1. Distribution of the eels of which any bacteria were not isolated from internal organs had a partiality for winter (Fig. 2). Most of the diseased eels in winter had symptoms of “branchionephritis”, from gill of which a myxobacterium was frequently isolated.

再び養殖池におけるヨーロッパウナギの性と成長について

Investigations were made on the sex ratio of European eels, Anguilla anguilla (L.), cultured on a commercial scale in fresh-water and salt water ponds. The following results were obtained: 1) No definite difference in sex ratio was observed between eels in freshwater ponds and those in salt water ponds. 2) The percentage of males based on examinations of 611 eels obtained from 3 eel farms including one reported in a previous paper was 92.4% for eels 30.0 to 39.9cm long, 56.0% for those 40.0 to 49.9cm long, and 0% for those over 50.0cm long. 3) Evidence was obtained which suggested that the growth rate of eels in culture ponds did not differ between the sexes until they reached about 40cm in length. Considering that growth rate becomes markedly different between the sexes in fish over 40cm, that sexes can be distinguished clearly in eels over 30cm, and that eels over 40cm are harvested, it seems that the sex ratio of pond-cultured eels is best determined with fish 30 to 40cm long. Hence the percentage of males in pond-cultured European eels was taken as 92.4%.

病鰻から分離された <i>Edwardsiella tarda (Paracolobactrum anguillimortiferum</i>) の性状

Morphological and biochemical characteristics of 22 strains of an enteric bacterium isolated from diseased pond-cultured eels in Shizuoka Prefecture in 1972 were examined. These organisms were gram-negative, peritrichously flagellated rods. They gave negative oxidase reaction, utilized glucose fermentatively in Hugh-Leifson's medium and reduced nitrate to nitrite. Some distinguishing features of the organisms were the produc-tion of hydrogen sulfide and indol, positive to methylted test, negative to Voges-Proskauer test, no growth on Simmons' citrate agar, growth on Christensen's citrate agar, decarboxi-lization of lysine and ornithine, and utilization of fructose, galactose, mannose, maltose, and glycerol (Table 1). All of the strains examined were sensitive to dihydrostreptomycin and kanamycin, but some of them were resistant to chloramphenicol, tetracycline and colistin (Table 2). The above properties suggest that the organism should be identified as Edwardsiella tarda, with which Paracolobactrum anguillimortiferum is thought to be synonymous.

Studies on red spot disease of pond-cultured eels—I

As previously reported (MUROGA et al. 1973), red spot disease (Pseudomonas anguilliseptica infection of cultured eels) prevails in brackish-water ponds when the water temperature is below 20°C, and ceases when it rises to 27°C.In this paper, physiological characteristics of the causative bacterium, P. anguilliseptica were investigated, and the results were discussed with respect to the epizootiological facts described above. The experimental results and conclusions are summerized as follows.1) Effects of pH, NaCl and temperature on the growth of the organism: It grew in nutrient broth at pH 5.3-9.7 (Optimum range 7-9), at NaCl 0-4% (Optimum 0.5-1%) and at temperatures from 5°C to 30°C (Optimum 15-20°C).2) Effects of NaCl or chlorinity on the survival of the organism: The period of survival was prolonged with the increase in concentration of NaCl or chlorinity, and the organism survived more than 200 days in sea water and in diluted sea waters (Cl≥1.9‰). This nature of the organism is considered to be closely related to the fact that the disease prevails not in freshwater ponds but in brackish-water ponds.3) Effects of temperature on the survival and the motility of the organism: The organism survived in diluted sea water (Cl 5.6‰) more than 40 days at 27°C and below, but it perished within 7 days at 30°C and above. The motility of the organism was gradually inactivated by increasing incubation temperature, and was poor above 25°C.4) The above-mentioned nature of the organism against temperature is considered to be the main cause of the temperature-dependence of the epizootic.

Branchiomycosis of pond-cultured eels

In July, 1972, branchiomycosis broke out among eels(Anguilla japonica) in a eel-culturing pond at Saijo, Ehime Pref., Japan, and caused considerable mortality.The causative agent was closely related to Branchiomyces sanguinis PLEHN.The width of the hyphae measured from 10 to 30 mThe spores in the hyphae were from 4.5 to 7m in diameter.The hyphae were located in the tissues of the gill filaments, not growing from the gills outwards.

Aerobic Bacteria in the Intestines of Pond-cultured Eels

Aerobic bacteria in the intestines of pond-cultured eels fed with raw fish were studied, with special reference to their gross quantitative changes with the progress of fasting, seasonal variations, and the appearance of fish pathogenic bacteria.In apparently healthy eels aerobic bacteria in the intestines reduced markedly in number with the progress of fasting and the intestines became “nearly” sterile after 3 week fasting in almost all fishes. Most of isolates were members of the genera Pseuomonas, Vibrio, Flavobacterium, Aeromonas, Achoromobacter and Alcaligenes. The occurrence of each genus changed with the season and pond, and no regular intestinal inhabitant was found.Part (9%) of isolates were demonstrated by experiment to be pathogenic to fish and all the fish pathogenic bacteria were members of the genus Aeromonas.

養殖ウナギの<i>Chondrococcus columnaris</i>感染症に関する研究-I

Since 1966, gill disease has been associated with serious mortality of pond-cultured eels during the summer months in many eel farms in Shizuoka prefecture. The disease was characterized by partial or extended losses of gill filaments and the occurrence of yellowish colonies of Chondrococcus-like bacteria on the gill surface. The cultures isolated from gill lesions were long flexible Gram-negative rods, which showed the movements and columnar formation peculiar to Chondrococcus columnaris. The formation of fruiting bodies and the production of microcysts were observed in the cultures maintained by BORG's method. The organism was therefore identified as Chondrococcus columnaris (DAVIS) ORDAL and LUCKER (1944). The biochemical characteristics of the organism (Table 2) were in full accord with those of C. columnaris isolated from Japanese loaches. The organism was occasionally isolated from skin lesions and the kidney of diseased fish. Eels were experimentally exposed to five strains of C. columnasis by means of pouring the broth cultures on the gill surface. Two preliminary experiments suggested that the pathogenicity of the organism might differ markedly with strains (Table 3). The same was perceived in the following experiments using three other strains (Table 4). In these experiments use was made of well-fed, fat and starved, thin fish, in which the opercula were removed and the surface of the gills on one side was lightly brushed with a tooth-brush prior to the experimentation. In the three strains typical yellowish colonies appeared on the gill two to three days after the exposure, and parts of the gill filaments began to degenerate and finally gill lesions of the same type as those that naturally occur were produced (Plate). Occasionally colonies once developed on the gill disappeared in course of time. Mechanical injuries on the gill surface did not necessarily facilitate the infection of the organism. The fatality of the organism to the eel varied considerably with the strain. As compared with fat fish, starved, thin fish seemed to be much resistant to the attack of the organism.