Infection experiments in the Japanese flounder (Paralichthys olivaceus) with Edwardsiella tarda were conducted by intramuscular injection, intraperitoneal injection, immersion, and oral administration methods. Mortalities were produced by all the methods tested, LD50 being determined as 7.1×101CFU/fish by intramuscular injection, 1.7×102CFU/fish by intraperitoneal injection, 3.6 ×106CFU/ml by immersion, and 1.3×106CFU/fish by oral administration. These results show that the Japanese flounder has high susceptibility to E.. tarda. Mortalities continuously occurred during the observation period of 15 or 20 days showing a chronic nature of the disease.
The Japanese flounder (Paralichthys olivaceus) were vaccinated with formalin-killed cells (FKC) of Edwardsiella tarda by intramuscular injection, immersion and oral administration. Fish were also immunized by injection with diluted extracellular products (ECP) and intracellular components (ICC) of the bacterium, both of which were found lethal to flounder. As a result, mean serum agglutinating antibody titers against FKC rose in all the immunized fish groups except those vaccinated by immersion with FKC. When the immunized and control fish were challenged by injection and immersion with live cells, death was delayed in most of the immunized groups. However, clear protection was not observed in any group immunized either with FKC, ECP or ICC.
Japanese eel (Anguilla japonica) neutrophils underwent a striking increase in oxygen consumption by stimulation, which is usually called respiratory burst. This increased uptake of oxygen was insensitive to mitochondrial respiration inhibitor such as potassium cyanide. Superoxide was detected during the respiratory burst by measuring chemiluminescence (CL) enhanced by 2-methyl-6-phenyl-3, 7-dihydroimidazo [1, 2-a] pyrazine-3-one (CLA) which is highly specific and sensitive to superoxide. EDTA blocked while EGTA stilumated CLA-enhanced CL. These characters of the respiratory burst suggest that Japanese eel neutrophils have NADPH oxidase-like activity similar to the mammalian NADPH oxidase. Sodium azide, an inhibitor of myeloperoxidase (MPO) activity, did not affect luminol-enhanced CL, which is thought to be dependent on hypochlorous acid (HOCl) produced by catalysis of MPO. It is suggested that Japanese eel neutrophils do not generate HOCl during the respiratory burst because of lacking MPO in cytoplasm.
Rapid and confirmative diagnostic methods for the rod-shaped nuclear virus (RV-PJ) infection in the kuruma shrimp (Penaeus japonicus) by dark-field microscopy and transmission electron microscopy, respectively, were proposed. Under a dark-field microscope virus particles were seen as numerous fine particles (about 0.5μm) in the hemolymph. In stomach cuticular epidermis, virus-infected nuclei were seen as sharply outlined homogenous white bodies, 10-15μm in diameter, round or oblong in shape and hemocytic encapsulations of necrotic cells as small brown masses (about 20-50μm). Under a transmission electron microscope virus particles were seen as virions (400×150 nm) or capsids (390×85 nm) in negatively stained preparations from hemolymph and stomach.
A cDNA clone for coho salmon (Oncorhynchus kisutch) transferrin was isolated from a liver cDNA library. The total 2, 511 nucleotides contained a 5' non-coding region, an open reading frame encoded with a transferrin polypeptide consisting of 687 amino acids and 3' non-coding region. The amino acid sequence alignment of the coho salmon transferrin had the duplicated structure, conserved anion-binding, iron-binding, and cysteine residues and two glycosylation sites observed in the previously cloned cDNA transferrin of vertebrates. The coho salmon transferrin is similar in size to the corresponding nucleotides from medaka and Atlantic salmon. The deduced amino acid sequence of the transferrin cDNA of coho salmon had 48%, 46%, 67% and 85% amino acid identities with that of human, Xenopus, medaka and Atlantic salmon, respectively. The coho salmon gene was recognized to be transcribed only in the liver by Northern blot hybridization.
A method for estimating phagocytic activity of leucocytes using a bacterial thin-layer was applied for carp and rainbow trout leucocytes against Edwardsiella tarda. Blood was added onto petri dishes of which the buttom was coated with the bacteria. The number and area of plaques formed on the thin-layer were measured as indices of phagocytic activity at 10, 20 and 30°C using a computer program. Phagocytosis of carp leucocytes was most active at 30°C. In rainbow trout, the total plaque area and number of plaques were maximal at 20°C and average plaque area was largest at 30°C.
The duration of infectivity of the causative agent of “amyotrophia” of juvenile abalone, Haliotis discus, in sea water was examined by experimental infection. A filtrate (0.45μm) of diseased abalone homogenate was added to sterile sea water and stored at 10°C, 18°C or 25°C for 5, 10 or 20 days. After storage, juvenile abalones were exposed to the filtrate-containing sea water for 20 minutes, and reared for 80 days. The occurrence of infection was determined by examining abalones histopathologically. As a result, the infectivity of the agent was lost in 5 days at 25°C or 20 days at 18°C, but maintained for 20 days at 10°C.
The concentration of oxytetracycline in the plasma was compared between triploid hybrid of rainbow trout (Oncorhyncus mykiss) _??_and brook trout (Salvelinus fontinalis) _??_and diploid rainbow trout after a single oral administration at 50mg/kg body weight. The maximum concentration in the plasma was observed 48 h after the administration in triploid hybrid and diploid rainbow trout and their values did not differ significantly each other. These results suggest that there is no difference in changes in plasma of oxytetracycline concentration between the triploid hybrid and diploid rainbow trout.
Rod-shaped nuclear virus of Penaeus japonicus (RV-PJ) was purified from hemolymph of experimentally infected kuruma shrimp (P. japonicus). The hemolymph collected from infected shrimp was centrifuged at 1, 000×g for 15 min to remove hemocytes and debris. The supernatant was centrifuged at 30, 000×g for 45 min to collect viral particles. The pellet was suspended in 1ml of PBS (-), and the solution was layered onto a 25-50% (w/w) continuous sucrose gradient. After ultracentrifugation at 125, 000 ×g for 90min a single clear band was formed. Rod-shaped virions and nucleocapsids were observed in the band by electron microscopy.
The defensive effect of bovine lactoferrin on Cryptocaryon irritans infection of red sea bream, Pagrus major, was studied. Neither mortality nor clinical sign of white spot disease was observed in the fish orally administered with lactoferrin (40 mg/kg body weight/day) for 28 days, whereas most untreated controls died within 28 days. These facts suggest that the lactoferrin has a defensive effect on C. irritans infection of red sea bream.
The agglutinating antibody level against Pasteurella piscicida in cultured O-year-old yellowtail Seriola quinqueradiata was monitored during epizootics of pseudotuberculosis in1993 and 1994. In both years, the antibody titer slightly increased at the early period of epizootics and subsequently remained on low level. A rise of the titers was observed at the end of the epizootic, about 3 months after the first outbreak. This delayed high levels of antibody titer seem to be associated with the recent trend of prolonged epizootics of pseudotuberculosis.
Oral delivery of vaccines is the least effective method of vaccinating fish. However, anal intubation of antigens is very effective in inducing high serum antibody levels and protection against vibriosis and enteric redmouth. It is generally considered that degradation of antigen in the stomach and anterior intestine prevents immunostimulation taking place and that if antigens could be protected during passage through the foregut effective immunization could be achieved. Recent attempts to protect antigens have included the concomitant administration of antacids and antiproteases. Protective coating procedures have included encapsulation of particulated antigen with methacrylic acrylic acid polymers which resist acid but dissolve in the high pH of the intestine. Incorporation of antigen into microparticles of poly lactide co-glycolide polymers protects against enzyme degradation and allows increased uptake of intact antigen. Certain substances can enhance uptake of antigen by the intestine of fish and have adjuvant activity, such as cholera toxin β-subunit and Quil-A saponin. Overcoming problems of palatability of oral vaccines delivered to fish fry has been achieved by incorporating vaccine into live food such as Artemia. However, delivery of antigen to very young fish, before they are fully immunocompetent can induce immune suppression. Many of these methods have improved the level of antigen uptake by the intestine and the antibody response in fish and the prospects for improving the efficacy of oral vaccination appear optimistic.