Since 1981, kuruma shrimp (about 11-14 cm in body length) exhibiting a few numbers of whitish-opaque patches in the abdominal muscles have been noticed at harvesting time, from autumn to winter, at culture farms in Yamaguchi Prefecture. These shrimp are rated a low grade, because they can not bear transportation to the market. Histopathological examination revealed that the skeletal muscles of the white areas were necrotic. The histopathological changes in the skeletal muscles were classified into three stages. Stage I; Muscle fibers, especially of the flexor of the 2nd to 5th abdominal segments, are nectrotic with fusion and cross splitting of myofibrils or separating from myoseptum. Hemocytic infiltration is often observed at the necrotic foci. Stage II; The necrotic muscle fibers are diminished. Proliferation of fibrocytes and production of collagenous fibers are extensive. Stage III; The necrotic muscle fibers have disappeared and are replaced by connective tissue. Environmental or physiological stress due to high water temperature, low dissolved oxygen and overcrowding has been suspected to produce muscle necrosis in shrimp. But no relation was found between the occurrence of this disease and the water quality of the kuruma shrimp culture ponds in the present study. Histopathological examination and etiological investigation suggest that the possible cause of this disease may be related to violent movement for escaping from electric net and other nets, or in the nets in overcrowding condition, used for harvest.
Direct fluorescent antibody technique (FAT) was evaluated as a differentiation method between α-hemolytic Streptococcus sp. (KUSUDA et al., 1976; KUSUDA et al., 1978) and β-hemolytic Streptococcus sp. (MINAMI et al., 1979; KITAO et al., 1981; OHNISHI and Jo, 1981; UGAJIN, 1981; NAKATSUGAWA, 1983) in this report. The staining titer of fluorescent antibodies were examined by using heat-fixed smears of α-and β-hemolytic Streptococcus sp., S. iniae, S. equisimilis and fish pathogens other than Streptococcus. The fluorescent antibody against α-hemolytic Streptococcus sp. had a titer of 1: 10, 000 and reacted only to α-hemolytic Streptococcus sp. The fluorescent antibody against β-hemolytic Streptococcus sp. had a titer of 1: 1, 000 and reacted only to β-hemolytic Streptococcus sp. and S. iniae. The data presented here suggest that direct FAT can be utilized for differentiation of α- and β-hemolytic Streptococcus spp., and that β-hemolytic Streptococcus sp. has a common antigen with S. iniae.
Whole cell suspensions from ten one-year-old carp were used for phagocytosis tests with opsonized sheep red blood cells (SRBC) within a duration of 24 hrs. The results were evaluated from the counts of smears stained for peroxidase activity and by Giemsa stain. The phagocytosing cells were of two lines : the macrophages and the neutrophils. A certain cell form resembling round thrombocytes could not be classified. The macrophages ingested the most SRBC/cell. The average number of ingested SRBC increased continuously in the macrophages, but remained low in the neutrophils. Increase in the total number of phagocytosed SRBC was the fastest in the first hour of incubation with a relatively slow progress in the succeeding hours. However, a second rise in the total number of phagocytosed SRBC occurred between the 12th and 24th hour of incubation. A strongly increasing number of juvenile neutrophil forms participating in the phagocytosis was observed during this period resulting in both the number of phagocytosing neutrophils and the number of phagocytosed SRBC by the neutrophils surpassing significantly those figures for the macrophages. Data on the phagocytosis by the neutrophils are assumed to reflect an important initial functional phase of the unspecific defence mechanism.
Red sea bream, Pagrus major, were injected intramuscularly once with Edwardsiella tarda crude lipopolysaccharide (LPS) and formalin killed cells (FKC) preparations. The immune response of red sea bream was studied 1, 2, 3 and 4 weeks after the immunization. The protein, albumin and glucose of the serum, agglutination antibody titer, passive hemagglutination titer, complement-like factor activity, bacteriolytic activity, hemagglutination activity of the serum and the skin mucus and the in vitro phagocytic index of the total blood of the immunized fish were determined. The results of the protein, albumin and glucose analysis of the immunized red sea bream serum did not show differences to the unimmunized fish. The agglutination and passive hemagglutination titers of the skin mucus did not show increase; however, the agglutination and passive hemagglutination titers of the serum showed increase from the first week after immunization, particularly in the FKC immunized fish. The activity of the serum complement-like factor showed apparently no increase of the immunized group, when compared to the unimmunized fish. The bacteriolytic activity of serum and skin mucus did not show differences between the immunized and unimmunized fish. The hemagglutination activity increases in both LPS and FKC immunized groups after two weeks from the immunization. The results of the in vitro phagocytic index of the total blood showed enhance from the first week after immunization in both LPS and FKC immunized groups, more than the unimmunized fish. The results suggest that the phagocytosis could be one important mechanism of red sea bream defence to E. tarda infection.
Ayu (Plecoglossus altivelis) were infected with Vibrio anguillarum by a water-born method. At 6, 12, 18, 36, 38-45 (moribund stage) and 48 h (dead) after infection, fish were sampled to determine the fate and location of the bacterium in various tissues by viable cell count and the enzyme-labelled antibody technique (ELAT). V. anguillarum was first detected in the skin at 12 h by bacterial isolation. It appeared in the muscle, spleen and liver at 24 h, but was not isolated from the gills or intestine until 36 h or 38-45 h. The same trend in the fate of the pathogen was confirmed by ELAT, and the cells were found in dermal layer of the skin from the early stage (12 h) of infection. Based on these observations, it was concluded that the first colonization site of V. anguillarum in ayu was the skin.
Immersion, subcutaneous injection and smear followed by immersion were studied in black sea bream fry in order to establish an effective method of infection with the gliding bacterium, Flexibacter maritimus. In addition, the occurrence or proliferation of the bacteria on the skin, gills and internal organs(kidney, liver and spleen)were determined by serial plate countdilution from separately homogenized samples. Immersion failed to infect the fish after 3d of observation whereas, fish infected by smear followed by immersion in 108, 106 and 104 cells/ml showed the symptoms of natural gliding bacteria infection in 23 h post infection. Subcutaneous injection resulted to serious injuries, in 30h post infection, on the site of injection like exposed musculature leading to heavy mortalities including the control fish. Therefore, subcutaneous injection did not serve a practical means of inducing gliding bacteria infection in this study.The bacteria were detected in greatest number in the skin from 15min until the 3rd hour, in the gills 1h up to 12h, and least detected in the internal organs from 1h until 12h post infection. In all cases, the bacteria could not be detected thereafter due to the successive invasion of other bacteria.