We introduced eight fish pathogen phages and three Escherichia coli phages (T4, T7 and λ) into the intestinal tract of goldfish Carassius auratus by anal intubation (108.3 PFU/fish). All phages appeared in the kidney at mean PFUs ranging from 102.7 to 104.3 PFU/g 3 h after intubation. Anal intubation of a representative Pseudomonas plecoglossicida phage (PPpW-4) into goldfish revealed that the introduced phages transfer to the blood and kidney within 10 min and are retained there for at least 12 h. An intubation dose of 107.3 PFU/fish or higher is required for constant presence of the phages in the circulatory system. Our results indicate that phages have the ability to pass through the intestinal wall and spread to the circulatory system, suggesting the potential application of phage therapy with oral administration.
Aeromonas veronii is a Gram-negative opportunistic pathogen in fish. We have previously investigated the intestinal microbiota of carp Cyprinus carpio and revealed that most bacterial isolates with high mucus-binding capability were A. veronii. With regard to infection, mucosal adhesion of bacteria is regarded as a prerequisite for colonization and invasion. In the present study, we selected three A. veronii strains with different mucus-binding capability and pathogenicity, and compared the colonization capability and localization of these strains in the intestinal tract of carp, using an in vivo imaging system. We showed that only a pathogenic strain was mainly visible around the intestinal bulb and rectum of the intestinal tract at 48 h after administration. Frozen sections with fluorescence signals revealed that the administered bacteria were localized on the mucosal surfaces and in the lamina propria of the intestinal bulb, whereas they were mainly detected in the remaining intestinal contents in the rectum. These results suggest that the intestinal bulb is the primary region for A. veronii to invade and proliferate in healthy carp.
We inoculated yellowtail Seriola quinqueradiata with a commercial bivalent vaccine containing inactivated Vibrio anguillarum and Lactococcus garvieae to better understand the immunological responses to the vaccination. Serum samples were collected from vaccinated and control (PBS-injected) fish at 1, 3, 5, 7, 10, and 14 days post-vaccination (dpv). Compared to the controls, the serum from the vaccinated fish inhibited the proliferation of V. anguillarum from 7 dpv. However, vaccination had no effect on L. garvieae proliferation. The antibody titer against V. anguillarum obviously increased from 7 dpv, but no such increase was detected for L. garvieae. We confirmed that the protection conferred by the V. anguillarum vaccine was a result of the antibodies against the pathogen. However, humoral factors related to protection were not observed for L. garvieae. The expression of immune-related genes in the head kidney was profiled by microarray analysis. Twenty-two immune-related genes were up-regulated during the experimental period. Up-regulation of cellular immune-related genes, such as tumor necrosis factor, CC chemokine, and macrophage colony-stimulating factor receptor, was mostly observed from 1 to 3 dpv, whereas that of humoral immune-related genes was observed from 3 to 14 dpv. Notably, immunoglobulin-related genes showed continuous up-regulation during the 2-week experimental period.
We report the effect of Broussonetia kazinoki as a feed additive on disease resistance in olive flounder Paralichthys olivaceus, against Streptococcus parauberis. The cumulative mortalities were lower in fish fed with diets supplemented with 1.0% and 2.0% than in those fed with 0% and 0.1% diets. Phagocytic, complement and lysozyme activities were significantly enhanced by 0.1%, 1.0% and 2.0% B. kazinoki diets. Respiratory burst activity was significantly enhanced by 1.0% and 2.0% diets. This study suggests that diets supplemented with 1.0% and 2.0% B. kazinoki enhance the immune parameters concerning resistance against S. parauberis infection in P. olivaceus.
During the winter of 2009, we observed an outbreak of severe respiratory disease in Symphysodon discus in a fish farm. The disease began with anorexia, respiratory distress and behavioral disorders and caused the death of all affected juveniles (80%) within a few days. Gill filaments were inflamed, hyperplastic and necrotic, and were infected with Sciadicleithrum variabilum. We amplified the cytochrome oxidase I (COI) gene of the monogenean using PCR and sequenced the gene as a molecular marker to rapidly identify the species. We demonstrated successful sequencing of the COI gene for S. variabilum identification, together with the need to monitor this pathogen in fish farms.
We studied the effects of diet supplemented with 0% (control), 0.1%, 1.0% and 2.0% Zanthoxylum piperitum on the immune response and disease resistance in kelp grouper Epinephelus bruneus against Vibrio carchariae. The phagocytic and lysozyme activities were significantly enhanced in fish fed with diets supplemented with 1.0% and 2.0% Z. piperitum. The cumulative mortality of fish fed with 1.0% and 2.0% supplemented diets was lower than that of the controls. Therefore, it was suggested that diets supplemented with 1.0% and 2.0% Z. piperitum positively enhanced the innate immune system in E. bruneus against V. carchariae.
We evaluated the antiviral activity of several small interfering RNAs (siRNAs) corresponding to the Siniperca chuatsi rhabdovirus (SCRV) nucleoprotein (N) gene against the SCRV in a fish cell line, EPC. Among the siRNAs we examined, si983 was most effective at reducing virus titer compared to the mock group. Western blot analysis suggested that si983 inhibited N gene expression and virus replication in a dose-dependent manner. These results indicate that N gene 983 sites are potential targets for antiviral therapy. Furthermore, RNAi targeting of the N gene may represent a viable method of treating against SCRV infection.
A long, tubular “parasite” was found in the muscle of greater amberjack Seriola dumerili cultured in Kagoshima and Miyazaki prefectures. Infection with this parasite was very rare, only occurring in two amberjacks from Kagoshima and one from Miyazaki. Molecular analysis indicates the parasite is a cestode of the order Trypanorhyncha, most closely related to the subfamily Grillotiinae, family Lacistorhynchidae. The tubular structure was a blastocyst. There was no evidence of the scolex in the blastocysts, which may not have been sampled intact, making it impossible to identify the parasite. This is the first report of Trypanorhyncha infection in greater amberjack in Japan.