High agglutinating antibody titer serum of colored carp Cyprirus carpio was obtained after 4 times injection of formalin-killed Aeromonas hydrophila cells. The immunoglobulin of immunized carp was purified by gel filtration on Sepharose 6B and ion exchange chromatography on DEAE-Sephadex A-25. Agglutinating antibody activity of immunized carp was found in the second fraction using Sepharose 6B and DEAE-Sephadex A-25. The protein concentration of immunized carp was 39.84 mg/ml and the immunoglobulin accounts for about 5.5 % of the serum protein. The treatment of immunoglobulin with 2-ME reduced completely the agglutinating antibody activity. From the results of this study it is suggested that the immunoglobulin of colored carp resembles that of the IgM type in mammals.
When Japanese eels (Anguilla japonica) were injected intramuscularly (IM) with ferric ammonium citrate (FAC) at a sublethal dose of 10 μg/g and followed by IM-injection with various doses of Vibrio anguillarum, FAC injection enhanced greatly the virulence of the pathogen to eels, lowering the LD50 value from 107.9 to 104.2 CFU/100 g. Similar effects were obtained with ferrous sulfate and ferric chloride in eels. However, such a virulenceenhancing effect of FAC was scarcely observed in ayu (Plecoglossus altivelis), which has high susceptibility to the pathogen by nature. It was also found that addition of FAC (10 μg/ml) in fish sera accelerated the bacterial growth in vitro but the effect was much greater in eel serum than in ayu serum. The results of these in vivo and in vitro experiments demonstrated that the availability of free iron in host fish would have a significant influence on the pathogenesis of V. anguillarum infection.
The susceptibility of the fingerlings (mean weight 2g) of 5 different marine fish species to Yellowtail Ascites Virus (YAV) was studied by intraperitoneal (IP) injection or immersion (IS) infection methods: yellowtail Seriola quinqueradiata, hybrid of goldstriped amberjack Seriola aureovittata and amberjack Seriola dumerili, Japanese parrotfish Oplegnathus fasciatus, spotted parrotfish Oplegnathus punctatus and red sea bream Pagrus major. The challenge vir`us was grown in RTG-2 cells at 16°C for 3d and its titer was 2×107.63 TCID50/ml. The fingerlings were IP injected with 2×105.63 TCID50 of culture grown YAV/g fish or immersed for 50 min in seawater containing the same quantity of virus/ml. Results showed that yellowtail exhibited the highest susceptibility to YAV by both methods of infection followed by Japanese parrotfish, hybrid of goldstriped amberjack and amberjack, whereas spotted parrotfish and red sea bream were not sensitive to the virus. All dead fingerlings exhibited abdominal ascites and clinical symptoms differed among the species. Virus titers in the viscera were little different among the fish species.
The morphological, physiological, cultural and biochemical characteristics of nine strains of Vibrio isolated from diseased cultured fish in Italy were determined and compared with Japanese strains of Vibrio anguillarum. Comparative serological study of O-antigen was performed by agglutination method using rabbit anti-J-O-1 to J-O-8 sera. As a result, seven of the Italian isolates were identified as V. anguillarum biovar I and two strains as V. anguillarum biovar II. The serology showed that Italian strains have weak agglutination only with the antisera corresponding to the Japanese serotype classified as J-O-3, J-O-6 and J-O-8.
The present study determined the in vitro and in vivo antibacterial activities of florfenicol (FF) against the various fish pathogens; Pasteurella piscicida, Vibrio anguillarum, Edwardsiella tarda, Aeromonas hydrophila and A. salmonicida. FF demonstrated highly potent antibacterial activity against various fish pathogens inhibiting the growth of all of the test strains resistant to thiamphenicol (TP), chloramphenicol (CP) and other antimicrobials at the same MICs as drug-susceptible strains. In experimentally induced infections of P. piscicida in yellowtail, E.. trada in eel and V. anguillarum in goldflsh, FF proved to be very effective by the oral route and its efficacy was superior to that of TP, CP and oxytetracycline.
This study represents the first report of the development and use of an enzyme-linked immunosorbent assay (ELISA) and western blot analysis to monitor the production of Renibacterium salmoninarum soluble antigens (SA) in infected salmon. The sensitivity of the ELISA permitted detection of soluble antigens in sera samples to concentrations of 0.1 μg/ml. This assay demonstrates a clear resolution between infected and non-infected fish in experimental infections. The ELISA and western blot systems were used to assess the temporal progress of the disease in a quantitative and qualitative manner.
Flavobacterium sp. is a fastidious bacteria that it is often difficult to detect from the infected fish and its environment. The indirect fluorescent antibody technique (IFAT) was evaluated as a method for solving this problem. Rabbit antisera of 4 strains of Flavobacterium sp. and protein A-FITC conjugate (Pharmacia) were used in this study. Twenty six cultures from 18 species of bacteria other than Flavobacterium sp. were tested for cross reaction. The gills and the skin from experimentally or naturally infected fishes were investigated using IFAT, plating method and phase contrast microscope. The water from the fish ponds was also examined. Results revealed that the sensitivity and specificity of IFAT were capable of detecting the gill disease pathogen from the hosts and the environment.
Analysis of thermolabile antigens of the representative strains belonging to each O-serotype showed that the strains beloging to J-O-1, J-O-2 and J-O-3 possessed a common thermolabile antigen which was designated as k-1. In addition to k-1, the strains of all J-O-1 of phenon II (V. anguillarum biovar II) possessed another antigen which was designated as k-2. We subsequently examined the thermolabile antigens of the 103 strains (including three reference strains) of V. anguillarum of phenon I and 27 strains (including two reference strains) of V. anguillarum of phenon II belonging to J-O-1. All 103 strains of V. anguillarum of phenon I had negative reactions to the anti-k-2 factor serum. Conversely, V. anguillarum of phenon II all had positive reactions to the anti-k-2 factor serum. It became obvious that all the strains belonging to J-O-1 of phenon II possessed k-2 antigen but all of the strains belonging to J-O-1 of phenon I did not possess k-2 antigen.
The metacercaria of Clinostomum complanatum excysts from fish only after activation. The present study was conducted to determine whether activation was dependent on temperature, hydrogen ion concentration or on the stomach enzyme, pepsin. The data indicate that the metacercaria is very sensitive to a high temperature such as 39°C and 42°C. Warm water at 33°C and 36°C can also induce excystment of the metacercaria but not so powerful. Following the pretreatment of a low temperature, the metacercarial excystment can occur at a room temperature. Therefore, the excystment does not necessarily occur only at the temperature above 33°C but an increase in varying temperatures may be enough to trigger excystment. Histological observations suggest that the parasite-origin proteolytic enzyme(s) play a major role in the excystment.
In the previous paper (TAJIMA et al., 1987), we demonstrated that V. anguillarum had at least two thermolabile antigens, namely k-1 and k-2. All the strains belonging to somatic serotypes J-O-1 (phenon I), J-O-2 and J-O-3 possessed k-1 antigen only whereas all the strains belonging to J-O-1 of phenon II possessed both k-1 and k-2 antigens. In the present paper we examined the presence of these and other thermolabile antigens in the strains of other O-serotypes from J-O-4 to J-O-8 of V. anguillarum and in some strains of Vibrio and related genera, and then discussed the possibility of use of a thermolabile antigen in detection of V. anguillarum. The result of the present study indicated that k-1 antigen can be commonly found in all the strains of J-O-4, J-O-5, J-O-6, J-O-7 and J-O-8. It was also observed that k-1 antigen was present in some strains of V. metschnikovii, A. hydrophila and A. salmonicida. However, V. anguillarum can be distinguished from V. metschnikovii and A. hydrophila by the growth at 42°C and from A. salmonicida by the production of diffusible brown pigment. We therefore, concluded that any O-serotype of V. anguillarum can be detected by the k-1 antigen alone instead of examining their O-serotypes.
A freshwater life cycle of Hysterothylacium aduncum was demonstrated experimentally for the first time. Third-stage larvae of H. aduncum from naturally infected Japanese smelt, Hypomesus transpacificus nipponensis, developed to the adult stage in experimentally infected rainbow trout, Salmo gairdneri, in fresh water. Larvae in eggs developed to the second stage and hatched even in fresh water. Although hatched larvae died quickly in fresh water, unhatched second-stage larvae remained alive there within the egg for more than 10 days and developed to the third stage in the mysid, Neomysis intermedia. The worms recovered from experimental infections were described using light and scanning electron microscopy; the characteristic features of the different stags of H. aduncum are discussed