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We studied on responses of neutrophils to E.tarda after an injection with the weakened-virulent bacteria and high-virulent bacteria with in the swimbladder in koi Cyprinus carpio, which were either unvaccinated or vaccinated by intramuscular injections with live bacteria of weakened-virulence. In the unvaccinated group challenge with the weakened-virulent bacteria（n=30）, infiltrated neutrophils phagocytized bacteria at 17-30 hs post challenge, and bacteria-laden neutrophils were decreased whereas normal cells were markedly increased at 48 hs. Electron microscopy（EM）revealed phagocytized bacteria were markedly damaged in the cell wall and cytoplasm with in the phagosomes, and formed secondary lysosomes in the cytoplasm. In the unvaccinated group challenged with the high-virulent bacteria（n=30）, neutrophils phagocytized bacteria at 17-30 hs, resulting in anincrease in fragmented cells at 48 hs. EM revealed neutrophils allowing propagation of undamaged bacteria in the cell. On the other hand in the vaccinated group challenge with the weakened-virulent bacteria（n=30）, many neutrophils phagocytized bacteria at 17-30 hs, and bacteria-laden neutrophils were decreased at 48hs. EM revealed phagocytized bacteria were markedly damaged with in the phagosomes and the cytoplasm of neutrophils. In another vaccinated group challenged with the high-virulent bacteria（n=30）, bacteria-laden neutrophils were increased between 30 and 48 hs whereas fragmented cells were markedly increased in just one fish in 48 hs. EM revealed phagocytized bacteria were markedly damaged within the phagosomes and the cytoplasm of neutrophils except for one fish that allowed bacterial propagation within neutrophils. These results determined that neutrophils were activated by immunization with live bacteria and successfully treated E. tarda of high-virulence as well as weak-virulence.

We performed active immunization by intramuscular injections with formalin-killed bacterin and live bacteria of E.tarda in koi Cyprinus carpio, and examined the specific antibody titer and the effectiveness by challenge with virulent bacteria. The mean titer of serum antibody was 214 in koi (n=5) vaccinated by bacterin, and 512 in koi (n=5) vaccinated by live bacteria with low virulence, indicating the successful immunization. After challenge with virulent bacteria, survival ratio was 32% in koi (n=19) vaccinated with bacterin whereas 70% in the control (n=20), resulted in no effectiveness. On the other hand survival ratio was 94% in koi (n=16) vaccinated with live bacteria whereas 27% in the control (n=15), resulted in definite effectiveness of immunization (RPS=91.8%). These results determined that the vaccination using live bacteria with low-virulence induced the definitely effective immunization in koi.