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.     

Whitespotted puffer Arothron hispidus, a new host for lymphocystis in Qingdao Aquarium of China

In April 2004 white nodular lesions were found on the fins of whitespotted puffer Arothron hispidus (Linnaeus). Diagnostic studies were carried out to confirm the disease using light and electron microscopy, histochemical methods and PCR. The results revealed that the nodules were composed of giant cells up to 400 microm in diameter. These cells were surrounded by a periodic acid-Schiff (PAS)-positive hyaline capsule containing dot-shaped, Feulgen-positive inclusion bodies in the cytoplasm and an irregular nucleus. Numerous virus particles 200 nm in diameter and with hexagonal profiles were observed in the cytoplasm. These features were consistent with those of lymphocystis disease. Additionally, based on the gene sequences of major capsid protein (MCP) of lymphocystis disease virus (LCDV) from Japanese flounder Paralichthys olivaceus, 2 pair primers were designed; after a nested PCR was performed for detection of LCDV in A. hispidus, a positive amplified product was obtained showing the presence of LCDV. Therefore, the white nodules were the lymphocystis lesions caused by LCDV infection and A. hispidus was demonstrated to be a new host for LCDV.     

Lymphocystis disease in the winter flounder, Pseudopleuronectes americanus.

Nodular lesions on the fins of winter flounder, Pseudopleuronemphocystis disease. Hypertrophied, encapsulated connective tissue cells contained cytoplasmic inclusions composed of icosahedral virus particles. The winter flounder is a new host for lymphocystis disease and is the second flatfish in the western North Atlantic to have the disease.     

The frequency, distribution, and pathology of three diseases of demersal fishes in the Bering Sea

During September and October, 1975, bottom-dwelling fish from the Bering Sea were examined for diseases. The three most commonly observed diseases and their frequency of occurrence were epidermal papillomas of rock sole ( Lepidopsetta bilineata Ayres), 1-0%; lymphocystis of yellowfln sole ( Limanda aspera Pallas), 2-1 %; and epidermal tumours of the pseudobranch of adult Pacific cod ( Gadus macrocephalus Tilesius), 7-4%. Both the appearance and the histological characteristics of epidermal papillomas on rock sole resembled those found on several species of flatfish in Puget Sound. The frequency of tumour-bearing rock sole ranged from 0-23%, with the highest occurrence at sampling stations around the periphery of the Bering Sea in water depths of 20-30 fathoms (36-54 m). The virus-caused lymphocystis lesions were usually on the 'blind' side fins and skin surfaces of yellowfin sole, the largest lesions being found on the operculum, and the most common site of infection was the pectoral fin. The frequency of lymphocystis was 0-15 %, with a gradual increase of prevalence from the northern to southern sampling stations. The Pacific cod tumours were always bilateral, and contained remnants of normal-appearing pseudobranchial tissue on the surface or deep inside the tumours. Portions of some tumours were necrotic and liquefied. Tumour-bearing cod were most commonly found in the south and south-eastern Bering Sea and the frequency of affected cod ranged from 0-100%.     

Characteristics of lymphocytoid cells detected during post-radiation bone marrow recovery]

As a result of complex study of lymphocyte-like cells which arise in the regenerating bone marrow of sublethally irradiated mice, it was established that: (1) X-cells precipitate in the 2nd and, partially, 3rd fractions of the albumin gradient, i. e. in the same fractions as the stem cells of intact animals; (2) X-cells do not belong to polypotent hemopoietic stem cells; (3) X-cells belong to B-lymphocytes, i. e. to thymus-independent lymphocytes.     

Lymphocystis in wild plaice Pleuronectes platessa (L.), and flounder, Platichthys flesus (L.), in British coastal waters: A histopathological and serological study

Wild plaice and flounders with lymphocystis were sampled and their lesion distribution noted. Regression of the lymphocystis cells was seen by histology and a host response including macrophages and immune cells occurred. Antibodies were detectable by a double diffusion precipitin test. The epidemiology is discussed with reference to these findings and to the life histories of the two species.     

X-cells in the gills of an Antarctic teleost, Pagothenia borchgrevinki

X-cell tumours have been described previously from teleost fish of the Northern Hemisphere, in which they occurred as lesions of the skin, pseudobranchs or gills. The present study describes X-cell tumours from the gills of an Antarctic teleost, Pagothenia borchgrevinki , thus extending the range to the Antarctic and also the Southern Hemisphere. Gills of affected fish were distinctly swollen and white in appearance, indicating that the gills were not functional as gas exchange organs. The affected gill tissue contained large numbers of X-cells, large spherical cells with a distinct extracellular coat and many densely staining membrane-bound granules. The possible origin of the cells is discussed.     

A histopathological report of some epidermal anomalies of Dover sole, Solea solea L., and other flatfish species in coastal waters off south-east England

The gross pathological and histological changes of typical examples of epidermal anomalies of Dover sole, Solea solea L., and other flatfish from the sewage dumping areas of the Thames Estuary and two 'control' areas are described. Five per cent of flatfish showed anomalies including trawl damage abrasions, fin-rot, ulcers, nodules and tumours, lymphocystis and skeletal anomalies. Many of the anomalies suggested a traumatic aetiology and were often chronic. There was no significant difference in disease incidence between the areas sampled, except more evidence of lymphocystis in one of the control areas.     

Annual variation in lymphocystis infection frequency in flounder, Platichthys flesus (L.)

The infection frequency of the virus induced lymphocystis disease has been registered in flounder from the Oslofjord, Norway, during a 4 year period. The disease has a clear regular seasonal variation, and also an annual variation. The percentage infection in the summer was 1–10%, and in the winter up to 57%. The possibility that the high pollution level of the inner Oslofjord could be the reason for the high frequency of lymphocystis disease, is discussed.     

Iridovirus infections in farm-reared tropical ornamental fish

A systemic viral infection in both gourami Trichogaster spp. and swordtail Xiphophorus hellerii and an outbreak of lymphocystis in scalare Pterophyllum scalarae and gourami are reported to have occurred in fish reared in ornamental fish farms in Israel. The systemic infection developed in endothelial cells that became hypertrophic and their contents were modified. The presence of such cells in light-microscopically examined stained smears and sections provides an initial indication for this systemic viral infection. Infection in gourami caused hemorrhagic dropsy. Transmission electron microscopic (TEM) images of iridovirus-like particles recovered from gouramies showed them to be 138 to 201 nm from vertex to vertex (v-v); those from swordtails were 170 to 188 nm v-v. TEM images of lymphocystis virions from scalare were 312 to 342 nm v-v and from gourami 292 to 341 nm v-v. Lymphocystis cells from the gourami were joined by a solid hyaline plate, which was lacking in the infection in scalare where the intercellular spaces between the lymphocystis cells consisted of loose connective tissue.     

X-cell disease in Antarctic fishes

A number of Antarctic fish species are affected by an unusual gill condition known as X-cell disease, named in reference to morphologically similar lesions of unknown aetiology reported from northern hemisphere fishes. Despite the disease being first recorded in Antarctic fishes over 25 years ago, no progress has been made in identifying its cause or in confirming any possible relationship with northern fishes. Although once thought to be a neoplasm, observations of lesions in non-Antarctic fishes point towards a parasitic origin. The life cycle of the proposed causal organism is unknown, however, and the only stages identified are those of the eponymous cells in the lesions. Here, we show X-cells in diseased gills of the Antarctic nototheniid Trematomus bernacchii represent multinucleate cysts of an unknown parasitic organism. Furthermore, we use molecular genetic methodology to show that the organism responsible is closely related to that identified in X-cell lesions of the common European dab, Limanda limanda and that the disease thus has a global distribution. Phylogenetic tree construction based on 18S rDNA sequences confirms that X-cell organisms form a group of closely related parasites, but robust positioning of the X-cell clade in the tree awaits more extensive genetic sequencing.     

Reversible alteration of morphology in an invertebrate erythrocyte: properties of the natural inducer and the cellular response

The normal shape of the erythrocytes of the bivalves known as blood clams is maintained by a marginal band (MB) of microtubules. When hemolymph (or "blood") is withdrawn from the animal, its erythrocytes change, within minutes, from the normal smooth-surfaced, flattened ellipsoids (N-cells) to spheroids with folded surfaces (X-cells). This alteration can be prevented by rapidly diluting the hemolymph with physiological medium, yielding N-cells for use in studying the transformation to X-cells. Bioassays showed that shape transformation was induced by a hemolymph activity (Hx) and was a function, in part, of cell responsiveness to this activity. Eventually the shape of the cells spontaneously returned to normal, at a rate dependent upon the concentration of the cells and of Hx; recovery was correlated with loss of Hx. The X-cells contained an intact but highly deformed MB, but this was not the effector of the transformation. Erythrocytes made to lack MBs still changed shape, although they did not recover as completely as did the MB-containing controls. When clams were cooled before hemolymph was withdrawn, the concentration of Hx was reduced. Hx was retained after dialysis of hemolymph, and initial filtration and chromatography indicated that its Mr was greater than 500,000. Shape transformation was blocked by EGTA, by serine protease inhibitors, and by sodium azide; the last indicates ATP-dependence. Although the mechanism responsible for shape transformation remains to be determined, the data suggest that the change is triggered by a coagulation-related activity in response to the removal of hemolymph from the animal.     

Methylation pattern of fish lymphocystis disease virus DNA.

The content and distribution of 5-methylcytosine in DNA from fish lymphocystis disease virus was analyzed by high-pressure liquid chromatography, nearest-neighbor analysis, and with restriction endonucleases. We found that 22% of all C residues were methylated, including methylation of the following dinucleotide sequences: CpG to 75%, CpC to ca. 1%, and CpA to 2 to 5%. Comparison of relative digestion of viral DNA with MspI and HpaII indicated that CCGG sequences were almost completely methylated at the inner C. The degree of methylation of GCGC was much lower. The methylation pattern of fish lymphocystis disease virus DNA differed from that of the host cell DNA.     

Seasonal changes in frequency of diseases in dab,Limanda limanda,from the southern North Sea

Infestation rates of flatfishLimanda limanda from the southern North Sea (German Bight) were investigated in 1981 and 1982. At three stations, including a dumping area of acid-iron wastes northwest of Helgoland, dabs were examined for the occurrence of lymphocystis and epidermal papillomas. Marked seasonal changes of infestation rates were observed with maxima of diseased fish in March and May (epidermal papillomas 2 % in 1981, 4.1 % in 1982; lymphocystis 7.5 % in 1981, 17.2 % in 1982). Infestation minima were found from June to October. Differences in incidence rates were observed between stations in winter and spring, but not in summer. Both diseases turned out to be size-dependent. Seasonality of the two diseases is discussed in relation to spawning cycle and water quality, including pollution.     

Seasonal prevalence of skin tumors from walleye (Stizostedion vitreum) from Oneida Lake, New York

A seasonal survey of skin tumor prevalence in walleyes (Stizostedion vitreum) was conducted during the ice-free period on Oneida Lake, New York in 1986. During the survey, 1,028 walleyes were collected and examined for the presence of lymphocystis disease, dermal sarcoma, discrete epidermal hyperplasia and diffuse epidermal hyperplasia. Skin growths were high in prevalence in early spring, low in prevalence during the summer, and again high in prevalence in the fall. Lymphocystis disease and dermal sarcoma were more frequently observed than either discrete or diffuse epidermal hyperplasia. Histologically, a moderate to severe inflammatory response was associated with dermal sarcoma in the early spring and late spring but not in the fall. Regardless of the time of year, varying degrees of inflammatory response were seen associated with lymphocystis disease. Samples were inadequate to assess seasonal trends in incidence of discrete and diffuse epidermal hyperplasia.     

Metabolic-morphologic characteristics of the integument of teleost fish with mature lymphocystis nodules

Summary Normal and virus-infected (lymphocystis disease) integument from five species of teleosts was examined by light and TEM autoradiography and SEM to establish metabolic-morphologic characteristics of integument with mature lymphocystis cells (LC's). LC's with numerous morphologic attributes of a late developmental stage showed highest incorporation of [³H]-thymidine in vivo (1–91 h) above the intracytoplasmic inclusion body (ci) with little radiolabel in nuclei, cytoplasmic icosahedral deoxyriboviruses (ICDVs) or capsule. Analysis by quantitative autoradiography revealed that the % total cell label in ci and cytoplasm did not vary appreciably from 1–91 h and was corroborative with morphologic criteria of maturity. A possible phylogenetic difference was noted between teleosts, wherein normal integument showed uptake of [³H]-thymidine in vivo (1 h) by cells at all levels of the epidermis, and cyclostomes (Spitzer et al. 1979) wherein labeling was confined to the basal third of the epidermis. Among four infected teleost species, the mean diameters of the ICDVs measured under the same conditions, ranged from 259.5 nm to 290.0 nm with the mean for each species differing significantly (p < 0.01) from each of the other means. Ruptured LC's were shown by TEM and SEM to have released ICDVs onto the lesions and integument. Various stages of LC degeneration, host response, and integumental repair processes were documented. An evaluation of labeling in vivo of the capsular matrix was compatible ([³H]-D-galactose> [³H]-L-lysine [³H]-L-fucose) with a glycosaminoglycan-protein structure.     

Linkage Mapping of Toll-Like Receptors (TLRs) in Japanese Flounder, <Emphasis Type="Italic">Paralichthys olivaceus</Emphasis>

Toll-like receptors (TLRs) are responsible for the recognition of specific pathogen-associated molecular patterns and consequently activate signal pathways leading to inflammatory and interferon responses. The region surrounding several TLRs was previously found to be associated with resistance to specific disease. Hence, we determined the location of 11 TLRs in Japanese flounder (Paralichthys olivaceus) using polymorphic microsatellite markers. TLR1 and TLR3 were located on linkage group (LG) 21 and 7, respectively. Membrane TLR5 and soluble TLR5 were mapped to LG22. TLR7 and TLR8 were mapped to LG3. TLR9 was found on LG1 and TLR14 and TLR21 were located on the same linkage group, LG10. TLR22 was found on LG8. Interestingly, TLR2 was mapped with the previously reported Poli9-8TUF microsatellite marker which is tightly associated with lymphocystis virus disease resistance. Therefore, TLR2 is a candidate gene for resistance to lymphocystis disease. These results imply that the location of a TLR associated with a particular disease may be valuable for the research on the relationship between host immune response and disease resistance.     

Ergasilus rotundicorpus n.sp. (Copepoda: Ergasilidae) from Siganus guttatus (Bloch) in the Philippines

Summary A new species of Ergasilus is described and illustrated from the gills of juvenile (c. 70 mm total length) Siganus guttatus raared in a Philippine fish farm. Of 15 fish examined, all were infested and nine had concurrent lymphocystis infections. ac]19820901