Takashi Kamaishi

Mass mortality of cultured ascidians Halocynthia roretzi associated with softening of the tunic and flagellate-like cells.

Since 2007, mass mortalities of cultured ascidians Halocynthia roretzi (Drasche) have occurred in Miyagi Prefecture, Japan. The mortalities occur from November through August, and the tunics of affected animals become abnormally weak and soft. The number of farming areas where mass mortalities have occurred has increased rapidly: 3 in 2007, 6 in 2008, and 14 in 2009. When an outbreak of the disease occurred, mortality reached 17 to 100%. Prominent histopathological changes in the diseased ascidians were found in the tunics; the tunics of affected animals were usually much thinner than those of healthy individuals, and the tunic matrix showed marked disintegration with irregular arrangements of fiber layers or the presence of hollow spaces. In addition, flagellate-like cells (10-14 microm x 2-3 microm) stained with hematoxylin were observed in the tunics of 31 out of 36 diseased animals (86%), but not in apparently healthy animals (n=38). Experimental infection with the disease was successfully conducted by immersing small pieces of tunic samples from diseased ascidians into aquaria with healthy ascidians. The flagellate-like cells were confirmed in the tunics of all the experimentally infected animals. These results indicate that the mass mortalities of ascidians accompanied by abnormally softened tunics were caused by an infectious agent, and suggest the involvement of the flagellate-like cells in the disease.

Soft tunic syndrome in the edible ascidian Halocynthia roretzi is caused by a kinetoplastid protist

An etiological study was conducted to clarify whether the flagellate-like cells found in histological preparations of the tunic of diseased Halocynthia roretzi (Drasche) were the causative agent of soft tunic syndrome in this ascidian. When pieces of softened diseased tunic were incubated overnight in sterile seawater, live flagellated cells, which were actively swimming in the seawater, were observed in 47 out of 61 diseased ascidians (77%), but not in moribund or abnormal individuals with normal tunics (n = 36) nor in healthy animals (n = 19). The flagellate was morphologically very similar to those observed in histological sections of the diseased tunic. By contrast, flagellates were not found in tunic pieces of healthy, moribund, and abnormal individuals that did not exhibit softening of the tunic. Light and electron microscopy revealed that the flagellate has polykinetoplastic mitochondria with discoidal cristae. The cytomorphologies of the flagellate were the same as those of the flagellate-like cells in the diseased tunic. We cultured the flagellate from the softened tunic in vitro and confirmed that the tunics of healthy ascidians, which were immersion-challenged with suspensions of the subcultured flagellates, became softened 17 d after exposure, including the final 12 d in aerated, running seawater. The occurrence of flagellates was also confirmed by incubating pieces of soft tunic from experimentally infected animals in seawater overnight. These results indicate that the flagellate is the causative agent of soft tunic syndrome.

Identification of Major Antigenic Proteins of Edwardsiella Tarda Recognized by Japanese Flounder Antibody

Edwardsiella tarda is a fish pathogen that causes systemic infections in fresh water and marine fish. Determining the antigenic proteins is important for the development of an immunodiagnostic tests and a vaccine for effective infection control in fish. In the current study, antigens were detected by immunoblotting and affinity column chromatography using a Japanese flounder (Paralichthys olivaceus) antibody produced by experimental infection with E. tarda. GroEL, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), outer membrane protein A, filament protein, 30S ribosomal protein S6, 50S ribosomal protein L9, cold shock protein, and carbon storage protein were identified as antigens of E. tarda through biochemical analyses of the molecular weights, isoelectric points, and N-terminal amino-acid sequences. These proteins can be easily detected in flounder infected with E. tarda and are potential diagnostic markers.

Prevalence of red sea bream iridovirus among organs of Japanese amberjack (Seriola quinqueradiata) exposed to cultured red sea bream iridovirus

Red sea bream iridovirus (RSIV) is a representative of the genus Megalocytivirus which causes severe disease to aquaculture fish, mainly in Japan and South-east Asia. However, information to assess the viral kinetics of RSIV in fish is limited since reports on experimental infection by the immersion route, which is the natural infection route, are scarce. In this study, a method to evaluate the titre of RSIV was first developed. Experimental infections were continuously performed using RSIV cell culture as the inoculum to juvenile Japanese amberjack (Seriola quinqueradiata) (initial body weight 12.2 g) by immersion at three different concentrations. In addition, to investigate the prevalence of the virus among the organs of experimentally infected fish, viral DNA was measured at selected times by the real-time PCR method following viral inoculation by immersion. The developed titration method showed a 10(2) increase in sensitivity compared with the conventional method. We demonstrated that grunt fin cells can be used for continuous passage of RSIV. In the experimental infection, fish which were intraperitoneally injected with the RSIV cell culture or immersed with RSIV cell culture at 10(-2) and 10(-3) dilutions showed cumulative mortalities of 100 %. The results of measurements of the viral DNA of several organs from infected fish strongly suggest that the spleen is the target organ of RSIV in Japanese amberjack. Since the viral genome was detected from all the tested organs of two of five surviving fish which appeared to completely recover from the disease, it is suggested that these fish may become carriers.

Preliminary data on restriction mapping and detection of length variation in Japanese flounder mitochondrial DNA

Abstract We have mapped cleavage sites of ten restriction endonucleases in mitochondrial DNA (mtDNA) of Japanese flounder, Paralichthys olivaceus . Hybridization with cloned or PCR-amplified fragments determined the map position around structural genes of the mtDNA. Mapping experiments detected a high level of length variation including heteroplasmy near or within the D-loop containing region. These results provide some basic information for studies on genetic variation of the species important in fisheries and aquaculture. Individual flounders used in this study carry discrete types of mtDNA. The results implies mtDNA typing is useful for monitoring effectiveness or genetic impact of ongoing stocking of the species into the natural habitat.

Analysis of the complete genome sequence of Nocardia seriolae UTF1, the causative agent of fish nocardiosis: The first reference genome sequence of the fish pathogenic Nocardia species

Nocardiosis caused by Nocardia seriolae is one of the major threats in the aquaculture of Seriola species (yellowtail; S. quinqueradiata, amberjack; S. dumerili and kingfish; S. lalandi) in Japan. Here, we report the complete nucleotide genome sequence of N. seriolae UTF1, isolated from a cultured yellowtail. The genome is a circular chromosome of 8,121,733 bp with a G+C content of 68.1% that encodes 7,697 predicted proteins. In the N. seriolae UTF1 predicted genes, we found orthologs of virulence factors of pathogenic mycobacteria and human clinical Nocardia isolates involved in host cell invasion, modulation of phagocyte function and survival inside the macrophages. The virulence factor candidates provide an essential basis for understanding their pathogenic mechanisms at the molecular level by the fish nocardiosis research community in future studies. We also found many potential antibiotic resistance genes on the N. seriolae UTF1 chromosome. Comparative analysis with the four existing complete genomes, N. farcinica IFM 10152, N. brasiliensis HUJEG-1 and N. cyriacigeorgica GUH-2 and N. nova SH22a, revealed that 2,745 orthologous genes were present in all five Nocardia genomes (core genes) and 1,982 genes were unique to N. seriolae UTF1. In particular, the N. seriolae UTF1 genome contains a greater number of mobile elements and genes of unknown function that comprise the differences in structure and gene content from the other Nocardia genomes. In addition, a lot of the N. seriolae UTF1-specific genes were assigned to the ABC transport system. Because of limited resources in ocean environments, these N. seriolae UTF1 specific ABC transporters might facilitate adaptation strategies essential for marine environment survival. Thus, the availability of the complete N. seriolae UTF1 genome sequence will provide a valuable resource for comparative genomic studies of N. seriolae isolates, as well as provide new insights into the ecological and functional diversity of the genus Nocardia.

X-cells in pseudotumors of yellowfin goby Acanthogobius flavimanus: a protistan organism distinct from that in flathead flounder Hippoglossoides dubius

Yellowfin goby Acanthogobius flavimanus affected with X-cell pseudotumors were sampled from a river estuary in Tokyo Bay, Japan. We amplified the gene for small subunit ribosomal RNA (18S rRNA) of X-cells of the goby with PCR using universal primers. The gene that we obtained (DDBJ Accession no. AB451874) showed 91% sequence identity to that of the X-cells of the flathead flounder Hippoglossoides dubius. With in situ hybridization, the probes specific for the gene that we obtained hybridized with the goby X-cells but not with the flounder X-cells, whereas probes for the 18S rRNA gene of flounder X-cells hybridized with the flounder X-cells but not with goby X-cells. These findings indicate that, although the X-cells found in the goby are closely related to the protist found in flounder, the two are clearly distinct organisms.

Global Kinetoplastea phylogeny inferred from a large-scale multigene alignment including parasitic species for better understanding transitions from a free-living to a parasitic lifestyle

All members of the order Trypanosomatida known to date are parasites that are most likely descendants of a free-living ancestor. Trypanosomatids are an excellent model to assess the transition from a free-living to a parasitic lifestyle, because a large amount of experimental data has been accumulated for well-studied members that are harmful to humans and livestock (Trypanosoma spp. and Leishmania spp.). However, recent advances in our understanding of the diversity of trypanosomatids and their close relatives (i.e., members of the class Kinetoplastea) have suggested that the change in lifestyle took place multiple times independently from that which gave rise to the extant trypanosomatid parasites. In the current study, transcriptomic data of two parasitic kinetoplastids belonging to orders other than Trypanosomatida, namely Azumiobodo hoyamushi (Neobodonida) and Trypanoplasma borreli (Parabodonida), were generated. We re-examined the transition from a free-living to a parasitic lifestyle in the evolution of kinetoplastids by combining (i) the relationship among the five orders in Kinetoplastea and (ii) that among free-living and parasitic species within the individual orders. The former relationship was inferred from a large-scale multigene alignment including the newly generated data from Azumiobodo and Trypanoplasma, as well as the data from another parasitic kinetoplastid, Perkinsela sp., deposited in GenBank; and the latter was inferred from a taxon-rich small subunit ribosomal DNA alignment. Finally, we discuss the potential value of parasitic kinetoplastids identified in Parabodonida and Neobodonida for studying the evolutionary process that turned a free-living species into a parasite.