It has been a serious problem of eel culturists in Tanegashima, Kagoshima Prefecture that mass mortality of eel elvers was brought about by an uncertain disease. The introduction of elvers (50 kg) of eels, Angilla japonica into a pond was conducted for initial investigation on the disease in February, 1980. Intermittent deaths (number in mortality: about 500-1000 per day) lasted till April resulting in mortality over one half of all the elvers for about two months. Most of surviving elvers also died at the end of August, showing abnormal and retarded growth. The second investigation to determine the cause was done using new elvers since January, 1981. By microscopic examination on various tissue sections of the elvers, we found that metacercariae of Centrocestus formosanus (NISHIGORI, 1924) infected in the gills of them. We considered that the parasitism of metacercariae in gills was the primary phase of the disease and the following mass mortality was caused by high cultur temperature (25°C) and secondary bacterial infection. Further, abnormal behaviour of the host eel elvers (weighing 0.25 g in average), which was characterized by such signs as (1) abnormal swimming (2) non-swimming and reposing at the bottom, and (3) climbing up pond walls, was also observed with a notable inter-relationship to the degree of parasitism.
Edwardsiella tarda has been known as the causative bacterium of paracolo-disease (edwardsiellosis)in cultured eels in Japan. However, the ecology of the pathogen in eel culture ponds has not been fully investigated so far. Field surveys on the presence and abundance of E. tarda in water and mud of eel culture ponds in Tokushima Prefecture were made at four seasons from 1981 to 1982 by a modification of the detection method described by WYATT et al.(1979). The incidence of E. tarda in water(W)and mud(M)samples from some 30 ponds were 90%(W)and 91%(M)in summer, 97%(W)and 100%(M)in autumn, 48%(W)and 25%(M)in winter, and 73%(W)and 75%(M)in spring. The number of the organism also increased during the warmer seasons. From the results of intramuscular injections into Japanese eels(Anguilla japonica), 37 strains out of 159 strains of E. tarda isolated from water and mud samples were proved virulent. Although these virulent strains shared common O-antigens with each other, they were divided into 2 serotypes based on a bsorption tests. About one-third of the avirulent strains also agglutinated with rabbit O-antisera against the virulent strains.
Susceptibility in fry of several salmonid to Oncorhynchus masou virus (OMV) was studied. Each 100 fry of chum salmon (Oncorhynchus keta; 0, 1, 2, 3, 4, 5, 6 and 7-month-old), masu salmon (O. masou; 0, 3 and 5-month-old), kokanee salmon (O. nerka; 0 and 1-month-old), coho salmon (O. kisutch; 0 and 1-month-old) and rainbow trout (Salmo gairdneri; 1.6-month-old) were used. The fry were immersed for one hour in 10°C water containing 100 TCID50/ml of culture grown OMV, and then held in running water at 10°-15°C. The cumulative mortality of just hatching chum salmon, observed in ensuing 4 months was 35%, but between 1-month-old and 5-month-old fry, the cumulative mortality was more than 80%, in particular, at 3-month-old, the fry exhibited 98%mortality, the greatest sensitivity, At 6-and 7-month-old, the frys'susceptibility was reduced and only 7 and 2% fish had succumbed. On the other hand, masu salmon fry, at 1-month-old, was the most sensitive and the cumulative mortality reached 87%. In 3-month to 5-month-old fry, cumulative mortality decreased from 65% to 24%. Comparing the five different salmonid fry, at the age of 1-month-old, for relative sensitivity to OMV, kokanee salmon exhibited the greatest sensitivity, and 100% of them died. Masu salmon and chum salmon exhibited high sensitivity at 87% and 83% mortality respectively. Coho salmon and rainbow trout were shown less sensitive to OMV infection at 39% and 29% mortality respectively.
The application of a coagglutination test using staphylococci sensitized with specific antibody for the diagnosis of furunculosis causing by A. salmonicida in salmonids was studied. This test proved to be a simple, rapid and reliable diagnostic procedure of furunculosis in the field as well as in the laboratory without any special apparatus. The procedure of this method is summarized as follows; 1) Furuncle or kidney sample from diseased fish is homogenized with nine volumes of PBS, and heated in a boiling water for 30 min. 2) The supernatant is collected after centrifugation at 4000 rpm for 20 min. This may be omitted if a centrifuge is unavailable. 3) One drop of the supernatant and one drop of anit-A. salmonicida antibody-sensitized staphylococci suspension are mixed on a glass slide and incubate at room temperature. The results is read after 30 and 60 min. 4) If positive results was observed by means of the coagglutination test, the existence of A. salmonicida in the same samples which were employed on the coagglutination test should be reconfirmed by using a conventional cultural procedure.
In the previous paper (1981), this author indicated that the most characteristic cytological change of this disease producing high mortality of kuruma shrimp larvae and postlarvae was hypertrophied nuclei resulting in chromatolysis of the affected mid-gut gland epitherial cells. SANO et al. (1981) also pointed out the hypertrophy of affected nuclei as the characteristic symptom of this disease, demonstrated Baculoviral infection in such hypertrophied nuclei and the epizootic in the intensive culture farm of kuruma shrimp. In the present study, the author examined to diagnose this epizootic by detecting hypertrophied nuclei in squash and stained preparations of the affected mid-gut gland, or in squash unstained preparations under central illumination, dark field illumination, phase contrast and interference microscope. These results obtained were as follows; 1. Squash and stained preparations: hypertrophied nuclei were clearly seen due to probably increased DNA. 2. Squash unstained preparations: 1) under central illumination hypertrophied nuclei were observed as if containing many granules, but not clear. 2) under dark field illumination hypertrophied nuclei were clearly seen in white. 3) under phase contrast a lot of rod-like particles (about 1μam length) were often observed in the hypertrophied nuclei. These rod-like particles were arranged almost vertically to the nuclear membrane at the marginal area, and also they scattered throughout in the nuclei, or sometimes they lined up parallely to each other. 4) under interference surface of the hypertrophied nuclei was somewhat rough. Three out of these methods, squash and stained preparations, squash unstained preparations under dark field illumination and phase contrast microscope seemed to be available as presumptive diagnostic techniques for Baculoviral Mid-Gut Gland Necrosis of kuruma shrimp.
Periodic Acid-Shiff (PAS) reaction of neurtophils in the peripheral blood of the eel, Anguilla japonica was investigated. PAS-positivity of neutrophils in healthy fish was characterized by diffused reddish stainning in the entire cytoplasm. The PAS positive substance of this cell was identified as glycogen, because it was completely spoiled by salivary digestion. Following injection of eels with bacteria, the number of neurtophil in the peripheral blood was increased, and there was an apparent change in the PAS-positive substance. Based on the morphology of the PAS-positive substance in the cytoplasm, neutrophils were divided into five types: Type I-weak positive with diffused stainning. Type II-strong positive with diffused stainning. Type III-strong positive with numerous fine granules. Type IV-strong positive with numerous coarse granules. Type V-strong positive with massive blocks. Distribution of the adove mentioned types in the peripheral blood was examined at regular intervals after injection of the whole cells of bacteria, sonic extract of bacteria and culture filtrates. In the fish inoculated with bacteria, there was a remarkable increase in type III and type IV cells after 24 hours of injection and type V cells increased sharply in the moribund fish. In the case of eels inoculated with sonic extract of bacteria there was an increase in type III and type IV cells, whereas in the eels inoculated with culture filtrates, type III cells only increased. Eels injected with irritant substances other than bacteria did not show any significant changes in the type distribution of neutrophils.
From October 1981 to January 1982, a bacterial disease occurred among O-age cultured flounder Paralichthys olivaceus in the Kunda Bay, Kyoto Prefecture, Typical symptoms of the diseased fish were hemorrhage of the opercle, corneal opacity, exophthalmos and hemorrhage of the eyes. The causative organism isolated from diseased fish was found to be a β-hemolytic Streptococcus. This bacterium could not be identified with any known Streptococcus species listed in Bergey's Manual 8th ed., however, there was a close resemblance between this isolate and Streptococcus sp. reported by KITAO et al. (1981), OHNISHI and Jo (1981), and UGAJIN (1981) from various freshwater fishes.