Divalent cation requirements for the hemolysis of rabbit red blood cells (RaRBC) by the alternative complement pathway (ACP) of fish (rainbow trout Salmo gairdneri, ayu Plecoglossus altivelis, carp Cyprinus carpio, tilapia Tilapia nilotica, yellowtail Seriola quinqueradiata, porgy Pagrus major and flounder Paralichthys olivaceus) were investigated using sera freed of divalent cations and natural antibody. Among divalent cations tested (Mg2+, Ca2+, Mn2+, Co2+, Ni2+, Zn2+, Sr2+, Cd2+ and Ba2+), Mg2+, Co2+ and Ni2+ activated the ACP of all fishes, on the other hand, Ca2+, Mn2+, Zn2+, Sr2+ and Cd2+ activated the ACP of one to four fishes. It is of great interest that carp serum hemolyzed RaRBC in the presence of Ca2+ alone, while mammalian ACP is known to be activated by Mg2+, but not with Ca2+. The fish sera hemolyzed sheep red blood cells (SRBC), which are not hemolyzed by mammalian sera, in the presence of Mg2+ and EGTA (ethylene glycol-bis (2-aminoethyl ether) tetraacetic acid). In particular, the degree of hemolysis (%) of SRBC by yellowtail and flounder sera were 60-85% of that of RaRBC. In addition, neuraminidase-treatment of SRBC raised the degree of hemolysis of the cells. This indicates that the sialic acid content of the membrane of a target cell influences the activation of the fish ACP.
The bactericidal activities and factors affecting bactericidal activities were studied for five disinfectants, sodium hypochlorite, benzalkonium chloride, povidone iodine, saponated cresol and formaldehyde solutions, using killing-curve test on Aeromonas salmonicida, Vibrio anguillarum and V. ordalii. Three bacteria showed similar response to respective disinfectants. The dose to reduce viable cells of three bacteria from 105 cells/ml to less than 10 cells/ml within a minute (decimal reduction time=15 sec)at 20°C were 10 ppm for sodium hypochlorite solution, 10-30 ppm for povidone iodine solution, 0.03-0.1% for benzalkonium chloride solution, 0.3% for saponated cresol solution and≥1.0% for formaldehyde solution. Cell number of bacteria and organic contamination in the solution strongly affected bactericidal activities of sodium hypochlorite and povidone iodine solutions. Bactericidal activities of benzalkonium chloride, saponated cresol and formaldehyde solutions were decreased remarkably by lowering the temperature of the solution. Water hardness reduced bactericidal activity of saponated cresol solution and seawater also reduced that of povidone iodine solution, when used as solvents.
The survival of Flexibacter columnaris was studied in a variety of waters containing different concentrations of 4 cations. The survival of F. columnaris over a period of 7 days was found to be optimal at the concentrations of 0.03% NaCl, 0.01% KCl, 0.002% CaCl2·2H2O, and 0.004% MgCl2·6H2O, respectively. The best survival was obtained in the formulated water medium containing the combination of the four cations at their respective level of optimum concentration. The survival of F. columnaris in the formulated water was very high and almost similar to the survival in tap water; whereas, distilled water did not support the long-term survival of F. columnaris. The survival of F. columnaris in the media containing single salt at their optimal concentrations were similar to each other but notably lower than the survival in the formulated water and tap water.
The experimental infection ofFlexibacter columnaris to juvenile common carp (Cyprinus carpio) and loach (Misgurnus anguillicaudatus) was studied in respect of the influence of water media containing 4 cations, Na+, K+, Ca++ and Mg++. The occurrence of columnaris disease varied with the different environmental waters as well as with the fish species. In the case of common carp, infection occurred in every fish in both the formulated water (0.03% NaCl, 0.01 % KCl, 0.002% CaCl2·2H2O2 and 0.004% MgCl2·6H2O) and tap water at the exposure density of 4-6 × 108 CFU/ml of F. columnaris. At a lower exposure density of 4-6 × 107 CFU/ml, an 80% inflection was observed in the formulated water but no infection occurred in tap water. In loach, infection occurred in every fish at the exposure density of 4-6 × 106 CFU/ml in both the formulated water and tap water. No infection was observed in distilled water and the percentage of infection in the individual salt solutions were lower than the occurrence of infection in either the formulated water or tap water. Overall, the formulated water was detected as the most suitable water among the tested media for F. columnaris infection in both fish species.
The fourth complement component (C4) of carp Cyprinus carpio was partially purified from serum by affinity chromatography on Blue-Cellulofine, ion-exchange chromatography on DEAEToyopearl 650M and gel filtration on Sepharose CL-6B using ammonia-inactivated carp serum as a reagent for detecting C4. About 1, 000-fold purification was achieved in a yield of 25% of the initial hemolytic activity. This partially purified C4 was free of C1 and C2, and it contained trace amounts of C3. The molecular weight of carp C4 was estimated to be 170, 000 by gel filtration on Sepharose CL-6B. The protein migrated to a slow β-globulin region on agarose gel electrophoresis. Carp C4 was stable on heating at 58°C for 60 min. Incubation with ammonia or hydrazine led to inactivation of the component, while carrageenan or zymosan had no effect. These characteristics of carp C4 were in fair agreement with those reported for mammalian C4. Carp C4 was, however, incompatible with C1 or C2 of the guinea pig complement.
Amoxicillin(2S, 5R, 6R)-6-[(R)-(-)-2-Amino-2-(p-hydroxyphenyl)acetamido]-3, 3-dimethyl-7-oxo-4-thia-1-azabicyclo 3.2.0 heptane-2-carboxylic acid trihydrate)is a penicillin class antibiotic and efficacious against Pasteurella piscicida infection of cultured yellowtail(Seriola quinqueradiata). This study was carried out to investigate toxicity of amoxicillin at doses of 80mg/kg B.W. and 400mg/kg B.W. for 10 days administration in young yellowtail(about 100g body weight). The toxicity was evaluated by appetite, abnormal movement, growth, external and internal views, hematology and histopathology. The fish administered at a dose of 400mg/kg B.W. showed no abnormality of appetite, movement, external and internal views and hematology excepting increased erythrophagocytosis of splenic macrophages. Results obtaind in this study indicated that doses under 400mg/kg B.W. of amoxicillin was safe for young yellowtail.
Surface membrane immunoglobulin(smlg)on yellowtail blood cells was detected by fluorescent antibody technique(FAT)using anti-yellowtail immunoglobulin M rabbit IgG. For the detection of smIg cells, direct FAT was more convenient than the indirect method since it was relatively simple and gave less nonspecific staining. Yellowtail smIg cells were capable of cap formation at 4°C and an optimum of 18-25°C. Antigen-bound smIg disappeared within 3 to 4.5hr at 25°C, and appeared again after 6 to 24 hr in RPMI 1640 medium withoutanti-yellowtail immunoglobulin M rabbit IgG. However, it did not appear again in an excess of the anti-yellowtail immunoglobulin M rabbit IgG. Cap formation was inhibited by iodoacetamide, sodium azide, dinitrophenol and potassium cyanid. Cap formation did not occurwith monovalent antibody. The data show that the smIg-bearing cells of yellowtail are similarto mammalian B lymphocytes.
“Beko” disease in red sea bream juveniles was characterized by formation of masses of a microsporidian parasite and muscle liquefaction around the masses in the lateral muscle in a similar manner to “Beko” disease in yellowtail juveniles, Seriola quinqueradiata caused by Microsporidium seriolae. In most of the diseased fish studied masses of the parasite were composed of multi-nucleated cells in the outer layer, sporoblast mother cells in the middle layer and sporoblasts and spores in the inner layer. Neither pansporoblast membrane nor sporophorous vesicle was observed. These cells were much the same in arrangement and morphology as the cells in sporulation stages of M. seriolae; this fact indicated that the present organism is closely related to M. seriolae. Although the present organism differed in spore size and host species from M. seriolae, the distinction between the two must wait the results of cross-infection experiments. For this reason the present organism was tentatively designated as Microsporidium sp.