Evaluation of Heat Inactivation of Human Norovirus in Freshwater Clams Using Human Intestinal Enteroids.

Foodborne disease attributed to the consumption of shellfish contaminated with human norovirus (HuNoV) is one of many global health concerns. Our study aimed to determine the conditions of the heat-inactivation of HuNoV in freshwater clams (Corbicula japonica) using a recently developed HuNoV cultivation system employing stem-cell derived human intestinal enteroids (HIEs). We first measured the internal temperature of the clam tissue in a water bath during boiling at 90 °C and found that approximately 2 min are required for the tissue to reach 90 °C. Next, GII.4 HuNoV was spiked into the center of the clam tissue, followed by boiling at 90 °C for 1, 2, 3, or 4 min. The infectivity of HuNoV in the clam tissue homogenates was evaluated using HIEs. We demonstrated that HuNoV in unboiled clam tissue homogenates replicated in HIEs, whereas infectivity was lost in all boiled samples, indicating that heat treatment at 90 °C for 1 min inactivates HuNoV in freshwater clams in our current HIE culture system. To our knowledge, this is the first study to determine the thermal tolerability of HuNoV in shellfish using HIEs, and our results could be informative for developing strategies to inactivate HuNoV in shellfish.

Susceptibility of Four Abalone Species, Haliotis gigantea, Haliotis discus discus, Haliotis discus hannai and Haliotis diversicolor, to Abalone asfa-like Virus.

Abalone amyotrophia is a viral disease that causes mass mortality of juvenile Haliotis discus and H. madaka. Although the cause of this disease has yet to be identified, we had previously postulated a novel virus with partial genome sequence similarity to that of African swine fever virus is the causative agent and proposed abalone asfa-like virus (AbALV) as a provisional name. In this study, three species of juvenile abalone (H. gigantea, H. discus discus, and H. diversicolor) and four species of adult abalone (the above three species plus H. discus hannai) were experimentally infected, and their susceptibility to AbALV was investigated by recording mortality, quantitatively determining viral load by PCR, and conducting immunohistological studies. In the infection test using 7-month-old animals, H. gigantea, which was previously reported to be insusceptible to the disease, showed multiplication of the virus to the same extent as in H. discus discus, resulting in mass mortality. H. discus discus at 7 months old showed abnormal cell masses, notches in the edge of the shell and brown pigmentation inside of the shell, which are histopathological and external features of this disease, while H. gigantea did not show any of these characteristics despite suffering high mortality. Adult abalones had low mortality and viral replication in all species; however, all three species, except H. diversicolor, became carriers of the virus. In immunohistological observations, cells positive for viral antigens were detected predominantly in the gills of juvenile H. discus discus and H. gigantea, and mass mortality was observed in these species. In H. diversicolor, neither juvenile nor adult mortality from infection occurred, and the AbALV genome was not increased by experimental infection through cohabitation or injection. Our results suggest that H. gigantea, H. discus discus and H. discus hannai are susceptible to AbALV, while H. diversicolor is not. These results confirmed that AbALV is the etiological agent of abalone amyotrophia.

Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major susceptibility to infectious hematopoietic necrosis virus (IHNV) isolate.

Infectious hematopoietic necrosis virus (IHNV) is known as a causative agent of heavy mortalities in farmed rainbow trout. However, there is limited information on its virulence for marine fish species. In this study, Japanese amberjack Seriola quinqueradiata and red sea bream Pagrus major were experimentally infected by intraperitoneal (IP) injection and immersion, with an IHNV isolate from rainbow trout in Japan, to evaluate the virulence of the virus for these fish species. The cumulative mortality for immersed rainbow trout was 15%. IHNV was isolated from all dead fish and 50% of the sequentially sampled rainbow trout. When Japanese amberjack were challenged by IP injection and immersion, the resulting cumulative mortality was 70% and 0%, respectively. The virus was isolated from all dead fish and 1 out of 3 Japanese amberjack sampled at 9 d post exposure. However, no mortality was observed in either of the red sea bream groups challenged with IHNV. IHNV was not isolated from any of the surviving red sea bream, or from any of the sequentially sampled fish. The viral titer on Japanese amberjack-derived YTF cells was in the same log range as that on FHM and RTH-149 cells, but the titers on the red sea bream cell lines SBK and GBRS were lower than the other cell lines, and were significantly different from the FHM and RTH-149 cell lines. These results suggest that Japanese amberjack has a low susceptibility to IHNV, and red sea bream has no or little susceptibility to the virus.

Coccidian Parasite in Sea Cucumber (Apostichopus japonicus) Ovaries.

We investigated an unknown ellipsoidal body that is sometimes found in the ovaries of the sea cucumber Apostichopus japonicus. Its external morphology, comprising an ellipsoidal dark central body (about 150 µm in length) and a surrounding transparent layer (about 50 µm in thickness), resembled that of a protozoan cyst, particularly an oocyst. Histological observations of the developing A. japonicus ovaries clarified that a small mass of organisms appeared in the cytoplasm of young oocytes, proliferated in these cells through budding, became rod shaped and arranged radially, and, finally, formed an outer layer. These processes were considered to be the formation of a cyst by a protozoan parasite. The small subunit ribosomal RNA (18S rRNA) gene was amplified from the DNA extracted from unknown ellipsoidal bodies by using polymerase chain reaction with universal primers for eukaryote 18S rRNA. The determined sequence was not identical to any of the known sequences in DNA databases, but it clustered in a clade of coccidian species belonging to Eucoccidiorida in phylogenetic analyses. From these results, we concluded that the unknown ellipsoidal body is a cyst (possibly an oocyst) of a coccidian parasite (order Eucoccidiorida) that is formed in the A. japonicus oocyte, though its lower taxonomic position is uncertain. In a survey of the gonads of wild A. japonicus at Esashi, Hokkaido, during the reproductive season, these cysts were detected in more than 50% of females but were never found in males. We consider that the cysts of this parasite can only be formed in A. japonicus ovaries.

A Spirochaete is suggested as the causative agent of Akoya oyster disease by metagenomic analysis.

Mass mortality that is acompanied by reddish browning of the soft tissues has been occurring in cultured pearl oyster, Pinctada fucata martensii. The disease is called Akoya oyster disease (AOD). Although spreading pattern of the disease and transmission experiments suggest that the disease is infectious, the causative agent has not yet been identified. We used shotgun and 16S rRNA-based metagenomic analysis to identify genes that are present specifically in affected oysters. The genes found only in diseased oysters were mostly bacterial origin, suggesting that the causative agent was a bacterial pathogen. This hypothesis was supported by the inhibition of AOD development in naïve oysters injected with the hemolymph of diseased animals followed immediately with penicillin bath-administration. Further analyses of the hemolymph and mantle specifically and universally detected genes of bacteria that belong to phylum Spirochaetes in diseased pearl oysters but not in healthy oysters. By in situ hybridization or immunostaining, a Brachyspira-like bacterium was observed in the smears of hemolymph from affected oysters, but not from healthy oysters. Phylogenetic analysis using 16S rRNA sequences showed that the presumptive causative bacterium was outside of but most closely related to family Brachyspiraceae. We propose 'Candidatus Maribrachyspira akoyae' gen. nov, sp nov., for this bacterium.

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.

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.

Ichthyobacterium seriolicida gen. nov., sp. nov., a member of the phylum 'Bacteroidetes', isolated from yellowtail fish (Seriola quinqueradiata) affected by bacterial haemolytic jaundice, and proposal of a new family, Ichthyobacteriaceae fam. nov.

A novel Gram-stain-negative, rod-shaped (0.3 × 4-6 µm), non-flagellated, aerobic strain with gliding motility, designated JBKA-6T, was isolated in 1991 from a yellowtail fish, Seriola quinqueradiata, showing symptoms of bacterial haemolytic jaundice. 16S rRNA gene sequence analysis showed that strain JBKA-6T was related most closely to members of the family Flavobacteriaceae in the phylum 'Bacteroidetes'. Furthermore, based on gyrB gene sequence analysis, JBKA-6T was classified into a single clade within the order Flavobacteriales, which was distinct from the known clades of the families Flavobacteriaceae, Blattabacteriaceae and Cryomorphaceae. The predominant isoprenoid quinone was identified as MK-6 (97.9 %), and the major cellular fatty acids (>10 %) were C14 : 0 and iso-C15 : 0. The main polar lipids were phosphatidylethanolamine, three unidentified phospholipids, two unidentified aminophospholipids and two unidentified polar lipids. The DNA G+C content of JBKA-6T, as derived from its whole genome, was 33.4 mol%. The distinct phylogenetic position and phenotypic traits of strain JBKA-6T distinguish it from all other described species of the phylum 'Bacteroidetes', and therefore it was concluded that strain JBKA-6T represents a new member of the phylum 'Bacteroidetes', and the name Ichthyobacterium seriolicida gen. nov., sp. nov. is proposed. The type strain of Ichthyobacterium seriolicida is JBKA-6T ( = ATCC BAA-2465T = JCM 18228T). We also propose that Icthyobacterium gen. nov. is the type genus of a novel family, Ichthyobacteriaceae fam. nov.

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.

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.

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.

The efficacy of five avirulent Edwardsiella tarda strains in a live vaccine against Edwardsiellosis in Japanese flounder, Paralichthys olivaceus.

We evaluated the tissue persistence and live vaccine efficacy of five avirulent Edwardsiella tarda strains (E22, SU100, SU117, SU138, and SU244) isolated from the Japanese eel (Anguilla japonica) and from the environment. The live vaccines, containing a single strain, were injected intraperitoneally into Japanese flounder (Paralichthys olivaceus). Viable bacteria from all the strains (excluding SU100) were recovered from trunk-kidney tissue 28 d post-injection. Japanese flounder inoculated with E22 had the highest relative percentage survival (RPS = 45%) in an artificial challenge with virulent E. tarda (NUF806). The serum of E22-vaccinated fish had a significantly higher agglutination titer against NUF806. In contrast, there was little or no increase in the agglutination titer of the fish that were inoculated with the remaining avirulent strains. Injection with avirulent E. tarda increased the expression of cytokine genes, including interleukin-1beta (IL-1beta), type 1 interferon (IFN), and IFN-gamma in head-kidney of the Japanese flounder.

Mass mortality of giant abalone Haliotis gigantea caused by a Francisella sp. bacterium.

In February 2005, a mass mortality of giant abalone Haliotis (Nordotis) gigantea Gmelin, 1791 occurred on a private abalone farm in Shimane Prefecture, Japan. The cumulative mortality rate reached about 84%. In histological observations, bacteria-like spherical particles were found in affected animals, suggesting a bacterial infection. Many of the bacteria-like particles were found in the cells that were presumably host phagocytes. DNA was extracted from the hemolymph of a diseased abalone and a bacterial 16S rRNA gene was amplified by PCR. The bacterium was classified within the genus Francisella by gene sequence analysis. A bacterial isolate was obtained by spreading hemolymph of a diseased abalone on modified Eugon agar dissolved in 70% seawater containing 1% (w/v) hemoglobin. A gene fragment of the expected size was amplified from the bacterial isolate by PCR using specific primers for the 16S rRNA gene obtained from the diseased abalone. Experimental infections were carried out by intramuscular injection with the bacterial isolate or by immersion in the bacterial suspension using 2 species of abalone, the giant abalone and the Japanese black abalone Haliotis (Nordotis) discus discus Reeve, 1846. Most (98.6%) of the abalone challenged with the bacterial isolate died in experimental infections. These results suggest that the Francisella sp. isolate was the causative agent for the mass mortality of giant abalone. This is the first report of a pathogenic Francisella sp. isolate for mollusks.

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.

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.

Microarray analyses of gene expression in Japanese flounder Paralichthys olivaceus leucocytes during monogenean parasite Neoheterobothrium hirame infection.

In this study, the gene expression patterns of peripheral blood leucocytes (PBL) from Japanese flounder Paralichthys olivaceus were analyzed during the course of monogenean parasite Neoheterobothrium hirame infection in order to select candidates for molecular biomarkers of infection. cDNA microarray analysis was performed to compare the gene expression patterns of PBL between infected and non-infected fishes. Among the 797 genes analyzed, 45 genes (5.6%) changed their expression levels. These genes included specific and non-specific immune-related genes (matrix metalloproteinase[MMP]-9, MMP-13, leukotriene B4 receptor, CD20 receptor, MHC [major histocompatibility complex] Class I, MHC Class II beta-chain, immunoglobulin light chain and immunoglobulin heavy chain). Significant up- and down-regulation of some unknown genes was also observed. Several candidates for infection-marker genes were selected for further study. These genes included MMP-9, MMP-13, leukotriene b4 receptor, CD20 receptor, immunoglobulin heavy chain, immunoglobulin light chain and unknown genes coded as B613, E25, LB3(8), WE2(3), WE8-18R and WF12-18R.

Gene expression of leucocytes in vaccinated Japanese flounder (Paralichthys olivaceus) during the course of experimental infection with Edwardsiella tarda.

In this paper, we focused on the detection of differentially expressed genes in peripheral blood leucocytes (PBL) during the course of Edwardsiella tarda infection in vaccinated and non-vaccinated Japanese flounder (Paralichthys olivaceus). cDNA microarray analysis was performed to compare the gene expression patterns of the PBL between the vaccinated and non-vaccinated fish in response to E. tarda inoculation. Fish were vaccinated twice, at a two-week interval and experimentally challenged with E. tarda two weeks after the second vaccination. Among the 1187 analyzed genes, 42 genes were up-regulated during the course of infection either in vaccinated or non-vaccinated fish. These genes included immune-related genes, such as MMP-9, MMP-13, CXC chemokine, CD20 receptor and hepcidin. Some immune-related genes were down-regulated after the E. tarda challenge, i.e. interferon inducible Mx protein, MHC class II-associated invariant chain, MHC class II alpha and MHC class II beta encoding genes, immunoglobulin light chain precursor, immunoglobulin light chain and IgM. These responses are thought to be a common reaction of Japanese flounder PBL in the course of edwardsiellosis, irrespective of immunized condition. Ten genes were significantly up-regulated only in vaccinated fish, and 11 genes were significantly up-regulated only in non-vaccinated fish. These genes may have a correlation with the efficacy of vaccination, although we have no evidence to link the different gene expression patterns and the efficacy of vaccination at present.

Virus-like particles associated with mass mortalities of the pen shell Atrina pectinata in Japan.

Mass mortalities of the pen shell Atrina pectinata occurred in the fishing grounds of Ariake Bay, in southwestern Japan, during late spring and summer in 2003 and 2004. Histological examination revealed extensive necrosis in the epithelial cells of the kidney and gill, and impairment of the endothelial cells of the mantle arteria. Although cestode larvae belonging to the genus Tylocephalum were found in the mantle, adductor muscle, kidney, and digestive gland, their prevalence and the intensity of infection were low. Examinations of moribund pen shells for Haplosporidium spp. infection using PCR analysis and for Perkinsus spp. infection using Ray's fluid thioglycollate medium were negative. Unenveloped virus-like particles were detected by transmission electron microscopy in the cytoplasm of affected kidney and gill cells of moribund pen shells. They were icosahedral spherical and 50 to 55 nm in diameter. These virus-like particles found in moribund pen shells are different from those described in other marine mollusks, and may be the causative agent of the mass mortalities of pen shells.

X-cells in fish pseudotumors are parasitic protozoans.

Bottom-dwelling teleosts, particularly flatfishes or cod living in temperate to cold seawater, sometimes develop tumor-like lesions on the body surface or in the branchial cavity. These lesions usually contain masses of so called 'X-cells' of unknown origin. We amplified a gene for small subunit ribosomal RNA (18S rRNA) from X-cell lesions of the flathead flounder Hippoglossoides dubius. Phylogenetic analysis clearly classified the obtained sequence as a protozoan, although the organism had no clear affinity with any known protistan groups. In situ hybridization showed that probes specific for the protozoan 18S rRNA hybridized only with X-cells, and not with the host-fish cells, indicating that X-cells harbor the protozoan rRNA. On the other hand, a probe specific for vertebrate 18S rRNA hybridized with the host-fish cells, but not with X-cells. This is conclusive evidence that X-cells are parasitic protozoans.