Sphaerospora epinepheli n. sp. (Myxosporea: Sphaerosporidae) observed in grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus, in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6-22.9 microns (length) x 8.4-21.6 microns (width). Spores were 7.8-10.0 microns (length) x 12.3-14.5 microns (thickness), and 7.0-9.5 microns (width) with two spherical polar capsules of equal size measuring 2.9-4.4 microns in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

Sphaerospora epinepheli N. Sp. (Myxosporea: Sphaerosporidae) Observed in Grouper (Epinephelus malabaricus)

Sphaerospora epinepheli n. sp. is described from grouper, Epinephelus malabaricus , in cage-cultured and wild fish collected from both coastal lines of southern Thailand. Subspherical to spherical spores and mono- or disporous pseudoplasmodia were observed in the lumen of kidney tubules. Pseudoplasmodia were round to elongate, size range 15.6–22.9 μm (length) × 8.4–21.6 μm (width). Spores were 7.8–10.0 μm (length) × 12.3–14.5 μm (thickness), and 7.0–9.5 μm (width) with two spherical polar capsules of equal size measuring 2.9–4.4 μm in diameter and containing polar filaments with six or seven windings. Two uninucleate sporoplasms showed iodine vacuoles. Blood stages, similar to C-blood protozoans observed from freshwater fish in Europe, were found from peripheral blood smears of grouper. Ultrastructural studies of blood stages showed a similar structure to unidentified mobile protozoans from the blood of carp. Electron dense bodies were observed in the cytoplasm of the primary cell blood stages. Infected proximal-tubular epithelial cells showed highly vacuolated cytoplasm and pycnotic nuclei.     

“Unidentified” Mobile Protozoans from the Blood of Carp and Some Unsolved Problems of Myxosporean Life Cycles1

Heavy infections with enigmatic mobile organisms have recently been found in the blood of carp (Cyprinus carpio) in Central Europe. The organisms measure up to 15 μm, are variable in shape, and exhibit an unceasing twitching or dancing movement. Their developmental cycle starts with a primary cell enclosing a secondary cell. The former grows while the latter produces inside itself by a series of binary fissions and internal cleavages up to eight secondary cells, each of which encloses an inner (tertiary) cell of its own. In addition, up to four tiny cells with compact nuclei (“residual bodies”) also result from divisions of the secondary cells. Primary cells containing the products of the division of secondary cells finally disintegrate, releasing the secondary cells, which in their turn become new primary cells and repeat the cycle all over again. The structure and behavior of these organisms were so incompatible with existing ideas on myxosporean development that their myxosporean affinity was at first unrecognized. The final proof of their identity–appearance of myxosporean spores in sterile, experimentally infected hosts–is still to be presented. The interpretation of the myxosporean features of their life cycle (i.e., [1] the pericyte nature of the primary cell, [2] proliferation by disintegration of the pseudoplasmodial primary cell, [3] no rigidly fixed pattern in vegetative development), their ultrastructure (i.e., [1] characteristic bundles of microtubules and numerous free ribosomes in secondary cells, [2] lack of centrioles, [3] membranes enclosing the secondary cells within the primary cells), and facts on their epizootiology (i.e., [1] no success at transmission via leeches, [2] the occurrence of these organisms along with Sphaerospora renicola Dykova and Lom) suggest that they are stages of S. renicola from the kidney of carp. Similar mobile organisms were found in the blood of fry of two other fishes (Gobio gobi and Tinca tinca) which are also hosts for a Sphaerospora that infects the kidney. This suggests that these organisms represent an early phase in the developmental cycle in the genus Sphaerospora. The existence of cells enveloped one within the other (secondary and tertiary cells) in the developmental cycle, a characteristic myxosporean feature itself, is an intriguing parallel to similarly enclosed cells in sporogenesis of Paramyxea (Ascetospora).     

PKX, the causative agent of proliferative kidney disease (PKD) in Pacific salmonid fishes and its affinities with the Myxozoa

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14 degrees C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dykovà & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental induction of Sphaerospora renicola (Myxosporea) infection in common carp (Cyprinus carpio) by transmission of SB-protozoans

The authors have experimental evidence that the protozoa causing the swimbladder inflammation (SBI) of the common carp (Cyprinus carpio) are indentical with presporogonic stages of Sphaerospora renicola Dyková et Lorn, 1982 parasitizing the renal tubules. Homogenates prepared from the thickened and inflamed swimbladder of naturally infected common carp, when injected into the abdominal cavity of fish, produced renal sphaerosporosis in the infection-free common carp if the homogenates contained the parasites described by Kovács -Gayer et al. (8). By intraperitoneal injection, the Unidentified Blood Organisms (UBOs) living in the blood of the common carp were transmissible to common carp, from the blood of which they were demonstrable for a long time. However, they were not transformed into Sphaerospora. To other cyprinids (gibel carp, silver carp, grass carp, tench, roach) neither the blood stages nor the swimbladder stages were transmissible from the common carp.     

PKX,the Causative Agent of Proliferative Kidney Disease (PKD) in Pacific Salmonid Fishes and Its Affinities with the Myxozoa1

Proliferative kidney disease (PKD), caused by an unclassified protozoan (PKX), is reported from Pacific salmon, Oncorhynchus tshawytscha (Walbaum) and O. kisutch (Walbaum), and steelhead trout, Salmo gairdneri Richardson, held at the Mad River Hatchery in California, USA. The cumulative mortality attributed to the disease was 95, 13, and 18% respectively. The mortalities were greatest at mean water temperatures of 12-14°C during July 1983. The ultrastructure of the PKX organism and its associated pathology during clinical disease in all three species were consistent with those of the parasite in rainbow trout (Salmo gairdneri) as described in European outbreaks. Significant mortalities did not occur after August, at which time the parasite could no longer be detected in the salmon species. The steelhead continued to exhibit parasites in the kidney interstitium and epithelium and lumens of the tubules. Myxosporidan trophozoites and developing spores were also observed in the lumens of the kidney tubules of these fish. Although a mixed infection with another parasite may have occurred, evidence suggests that the myxosporidans are later stages of PKX. They were only observed in fish exposed to water with the infective stage and were particularly prominent in recovering fish. The PKX organism is similar to UBO, an unclassified protozoan of carp suspected to be an early stage of Sphaerospora renicola Dyková & Lom. Both parasites infect the blood and kidney, divide by endogeny, and are released by disintegration of the primary mother cell. The intraluminal myxosporean forms show similarities to Sphaerospora spp. in that they are monosporous and sporoblasts are formed within pseudoplasmodia. It is possible that PKX migrates to the lumen of the kidney tubule and subsequently sporulates. If the myxosporean forms are later stages of PKX, then it would belong to the phlyum Myxozoa.     

Phylogenetic position of Sphaerospora testicularis and Latyspora scomberomori n. gen. n. sp. (Myxozoa) within the marine urinary clade

An amendment of the family Sinuolineidae (Myxosporea) is proposed in order to include a newly described genus Latyspora n. gen. The type species Latyspora scomberomori n. gen. n. sp. is a coelozoic parasite in the kidney tubules of Scomberomorus guttatus. In addition to the morphological and molecular characterization of L. scomberomori n. gen. n. sp., we also present novel SSU rDNA data on Sphaerospora testicularis, a serious parasite of Dicentrarchus labrax. Performed phylogenetic analyses revealed that both species cluster within the marine urinary clade encompassing the representatives with a shared insertion within their V4 SSU rRNA region and grouping according to the shape of their spores' sutural line and their similar tissue tropism in the host. Sphaerospora testicularis is the closest relative to Parvicapsula minibicornis within the Parvicapsula subclade and L. scomberomori n. gen. n. sp. is the basal species of the Zschokkella subclade. The phylogenetic position of S. testicularis, outwith the basal Sphaerospora sensu stricto clade, and its morphology suggest it being a non-typical Sphaerospora. The sequence data provided on S. testicularis can help in future revisions of the strongly polyphyletic genus Sphaerospora. We recommend re-sequencing of several sphaerosporids as an essential step before such taxonomic changes are accomplished.     

Species of the genus Chloromyxum Mingazzini (Myxozoa: Myxosporea) infecting burbot (Lota lota L.)

Three different species of the genus Chloromyxum Mingazzini were found in burbot (Lota lota L.) collected in south-west Bohemia (Czechoslovakia). Comparison with existing records revealed that one species could be identified as C. pseudomucronatum Kashkovskiy, 1966. Found in the urinary bladder, it had subspherical spores with fine surface ridges and polysporic plasmodia. The two other myxosporea were established as new species: C. lenorae n. sp. was found in the kidney, renal corpuscles, renal tubules and interstitium, and had ellipsoid spores with surface ridges barely perceptible in the light microscope but clearly revealed by transmission electron microscopy. In the polysporic plasmodia, spores developed in pansporoblasts. C. reticulatum n. sp. was found in the gall bladder. It had polysporic plasmodia and spherical spores (average size 8.1 m in diameter) with a unique surface structures: elevated crests marking off irregular fields which appeared as a reticulum. In five of the fish infected with C. lenorae, bloodstream myxosporean stages of an extrasporogonic cycle were found. Further research is needed to determine whether they are stages of Sphaerospora cristata Shulman, 1962, a species also found in two of the burbot examined, or stages belonging to the Chloromyxum life cycle.     

Kidney sphaerosporosis with extrasporogonic stages in the blood in Baryancistrus sp. (Loricaridae) from Amazonian Brazil

Examined fish of the genus Baryancistrus (Loricaridae) from Rio Xingu in central amazonian Brazil were found infected with extrasporogonic stages of Sphaerospora sp. (Myxosporea) in the blood and sporogonic stages in the glomeruli with the resulting escape of the sporogony products into the renal tubules, apparently when the mesangial tissue in the Bowman's capsule collapsed. Spores were seen evacuating via the urinary tubules. Blood stages were initially 2.1 x 2.8 to 2.8 x 2.8 microns in size, and when fully differentiated reached 5.6 x 5.6-5.6 x 7.0 microns; in blood smears they were 11.2 x 8.4 to 14 x 8.4 microns. Bisporoblasts in the glomerular taft were 9.8 x 7.0 microns, premature spores were 5.6 x 4.2-5.6 microns.     

Sporogony of Tetracapsuloides bryosalmonae in the brown trout Salmo trutta and the role of the tertiary cell during the vertebrate phase of myxozoan life cycles

Tetracapsuloides bryosalmonae is the myxozoan that causes the commercially and ecologically important proliferative kidney disease of salmonid fish species. Immunohistochemistry and electron microscopy were used to examine the development of this parasite within the kidney of the brown trout Salmo trutta. The main replicative phase of T. bryosalmonae is a cell doublet composed of a primary cell and a single secondary cell. Engulfment of one secondary cell by another to form a secondary-tertiary doublet (S-T doublet) heralded the onset of sporogony whereupon the parasite migrated to the kidney tubule lumen. Within the tubule, the parasite transformed into a pseudoplasmodium and anchored to the tubule epithelial cells via pseudopodial extensions. Within each pseudoplasmodium developed a single spore, composed of 4 valve cells, 2 polar capsules and 1 sporoplasm. The pseudoplasmodia formed clusters suggesting that large numbers of spores develop within the fish. This examination of T. bryosalmonae suggests that the main replicative phase of freshwater myxozoans within vertebrates is via direct replication of cell doublets rather than through the rupturing of extrasporogonic stages, while tertiary cell formation relates only to sporogony. Taken in conjunction with existing phylogenetic data, 5 distinct sporogonial sequences are identified for the Myxozoa.     

草鱼肾脏和脾脏血细胞发育过程的观察

鱼类肾脏、脾脏是机体造血的主要器官。印迹涂片显示:草鱼肾脏和脾脏内血细胞的发育过程大致经历了三个阶段,即原始阶段、幼稚阶段、成熟阶段。本文着重描述了各阶段细胞的形态特征井对草鱼血细胞的发育及命名等问题作了初步的探讨。       

Presence of Marteilia sp. (Paramyxea) in the razor clam Solen marginatus (Pennántt, 1777) in Galicia (NW Spain)

Protistan parasites of the genus Marteilia, phylum Paramyxea, cause the molluscs disease named Marteiliosis. Histological observations and transmission electron microscopy revealed the presence of life cycle stages of a Marteilia sp. in the bivalve mollusc Solen marginatus (Solenidae). Parasites occurred in epithelial cells of the digestive ducts and tubules. Early stages (primary cells) presented one or several nuclei while advances stages formed a complex of cells-within-cells (secondary and tertiary cells) culminating in spores. Refringent bodies were present inside the presporangia. This is the first report of a Marteilia sp. in S. marginatus.     

Ovarian structure and oogenesis of the extremophile viviparous teleost Poecilia mexicana (Poeciliidae) from an active sulfur spring cave in Southern Mexico

The structure of the ovary and oogenesis of Poecilia mexicana from an active sulfur spring cave is documented. Poecilia mexicana is the only poeciliid adapted to a subterranean environment with high hydrogen sulfide levels and extreme hypoxic conditions. Twenty females were captured throughout one year at Cueva del Azufre, located in the State of Tabasco in Southern Mexico. Ovaries were processed with histological techniques. P. mexicana has a single, ovoid ovary with ovigerous lamella that project to the ovarian lumen. The ovarian wall presents abundant loose connective tissue, numerous melanomacrophage centers and large blood vessels, possibly associated with hypoxic conditions. The germinal epithelium bordering the ovarian lumen contains somatic and germ cells forming cell nests projecting into the stroma. P. mexicana stores sperm in ovarian folds associated with follicles at different developmental phases. Oogenesis in P. mexicana consisted of the following stages: (i) oogonial proliferation, (ii) chromatin nucleolus, (iii) primary growth, subdivided into: (a) one nucleolus, (b) multiple nucleoli, (c) droplet oils‐cortical alveoli steps; (iv) secondary growth, subdivided in: (a) early secondary growth, (b) late secondary growth, and (c) full grown. Follicular atresia was present in all stages of follicular development; it was characterized by oocyte degeneration, where follicle cells hypertrophy and differentiate in phagocytes. The ovary and oogenesis are similar to these seen in other poeciliids, but we found frequent atretic follicles, melanomacrophage centers, reduced fecundity and increased of offspring size.     

Invasion and early development of Sarcocystis muris (Apicomplexa, Sarcocystidae) in tissue cultures

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30-45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Immunolocalization of metallothioneins in different tissues of turbot (Scophthalmus maximus) exposed to Cd

Metallothioneins (MT) were localized by immunochemistry in different organs and cell compartments of turbot exposed to sublethal concentrations (100 ppb) of Cd for 7 days. The polyclonal rabbit anti-cod MT antibody (NIVA, Norway) applied herein exhibited positive cross-reactivity with turbot MTs. Immunoreactive MTs were localized in the branchial epithelium, in the liver and in the kidney of turbot. In Cd exposed fishes MTs were demonstrated mainly in branchial chloride cells (CC) and to a lesser extend in the area where progenitor cells are located and in the cells of the respiratory epithelium (secondary lamellae). A higher staining intensity for MTs was observed in CC of the interlamellar space of the main branchial epithelium in comparison with control CC. MT-staining was also observed in the chondroblasts of the cartilage and in the erythrocytes within blood vessels both in control and Cd-exposed specimens. MT immunoreaction was high in the liver hepatocytes and weak in the epithelium of the proximal portion of the kidney in exposed turbot. The tegument, spleen and muscle were devoid of any immunolabelling in both treatments. Ultrastructural studies at the transmission electron microscope revealed that Cd-induced MTs were mainly located in the cytoplasm of gill CC, the lysosomes and the cytoplasm of hepatocytes and in the basal labyrinth of kidney proximal nephrocytes. The differential localization/induction of MTs in different cell types described hereby suggests that the quantification of the specific expression of MT may be used in biomonitoring programs as a biomarker of Cd exposure in aquatic environments.     

Invasion and Early Development of Sarcocystis muris (Apicomplexa,Sarcocystidae) in Tissue Cultures1

Ultrastructural observations on the invasion and early development of merozoites (bradyzoites) of Sarcocystis muris in Madin-Darby canine kidney (MDCK) cells are presented. Invading merozoites cause the host cell plasmalemma to invaginate; they form a membrane junction (moving junction) and move into the host cell where they are enclosed in a primary parasitophorous vacuole (PV). Within 30–45 min after becoming intracellular, merozoites begin to vacate the newly established primary PV and move, forming a new membrane junction, into a secondary PV. Simultaneously with the movement of the parasite, the contents of dense granules in the apical part of the merozoites are shed by exocytosis into the lumen of the developing secondary PV. A lamella of the endoplasmic reticulum of the host cell becomes attached to the PV membrane, forming a PV limited by three host cell membranes.     

Ortholinea alata n. sp. (Myxosporea: Ortholineidae) in the northern butterfly fish Chaetodon rainfordi

Ortholinea alata n. sp. is described from the northern butterfly fish, Chaetodon rainfordi collected at Heron Island, Great Barrier Reef, Australia. Spherical, disporous trophozoites (10-15 micrometers) and spores were observed in the lumina of kidney tubules and collecting ducts. Spores are broadly triangular with two short, broad processes that extend dorsoventrad from the posterior end of each of the two spore valves. Valves are bisected by a suture in the plane of the polar capsules. Spores are 12.6 micrometers (length) x 9.6 micrometers (width) x 9.9 micrometers (length), and at the anterior end contain two spherical, divergent polar capsules measuring 4.6 (4.1-5.1) micrometers. Sporogenesis is similar to that of renal Sphaerospora spp.; the intraluminal trophozoites of O. alata n. sp. correspond to pseudoplasmodia described for Sphaerospora spp. and no large, multinucleated plasmodia are formed. No significant histopathological changes were observed in the kidneys of infected fish.     

Various DNA content in myotube nuclei during myotomal myogenesis in Hymenochirus boettgeri (Anura: Pipidae)

During myotomal myogenesis in Hymenochirus boettgeri primary myoblasts differentiate into morphologically and functionally mature, mononucleate myotubes. Further muscle development in the studied species is due to fusion of mesenchymal cells with the latter, resulting in the presence of two classes of nuclei in the myotube: large of myotomal origin and small of mesenchymal origin. Densitometric measurements of DNA content revealed that the myotube nuclei at stages 35 reached values close to 4C DNA (3, 3C DNA), while at a later stage (42) the values were equal to 4C. Conversely, the secondary myoblast nuclei following the fusion with the myotube at stage 42 had 2C DNA--a content comparable to that found in erythrocyte nuclei. PCNA (Proliferating Cell Nuclear Antigen)--marker of S-phase of cell cycle, detected in the myotube nuclei (at stages 35, 42) appears during DNA replication.     

A New Candidate Type of Human Enterovirus (Drilon Strain) Isolated in the Philippines

A cytopathogenic agent was isolated in monkey kidney (MK) cell cultures from the stool specimen of a 3-month-old Filipina hospitalized with lower respiratory disease. The agent was designated the Drilon strain. It was characterized as an enterovirus on the basis of electron microscopic morphology, nucleic acid type (RNA), resistance to ether and acid (pH 3.0) treatments, stabilization by molar MgCl₂ against heat inactivation, and buoyant density in CsCl. The strain caused mild febrile illness in experimentally inoculated cynomolgus monkeys, but not in suckling mice. In addition to its effect in primary MK cells, the virus was cytopathogenic in primary and secondary human amnion or embryonic lung cell cultures and in WI-38 or HEp-2 cell lines, but not in primary bovine kidney, primary porcine kidney, primary embryonic mouse or primary embryonic chick cell cultures. The Drilon strain was not neutralized by reference antisera against the known enterovirus serotypes, and the antiserum prepared with the Drilon strain did not neutralize any of the recognized prototype enterovirus strains. Although the patient's sera were not available, antibodies against the Drilon strain were prevalent in normal Filipinos and Indonesians, but not in Japanese people. The Drilon strain fulfilled the criteria of human enterovirus and is considered a candidate for designation as a new type.     

Differential cell surface expression of four lysosomal integral membrane proteins (LIMPs) in normal rat kidney cells

Four monoclonal antibodies generated against rat, hepatocyte lysosomal integral membrane protein (LIMPs) (Barriocanal et al., 1986a, b) were used as probes to ascertain the distribution of similar proteins in normal rat kidney (NRK) cells. Comparison of immunoprecipitations of LIMPs 1-4 from hepatocytes and NRK cells revealed a marked similarity in the proteins, in both cell types, as determined by SDS-PAGE. Further, the LIMP epitopes recognized by the antibodies are situated intravesicularly. Ultrastructural immunocytochemistry, using pre-embedding peroxidase, revealed that primary and secondary lysosomes in NRK cells are readily stained with all four antibodies, as well as vesicles in the Golgi region. Immunofluorescence microscopy of non-permeabilized NRK cells with antibodies recognizing LIMPs 1 and 4 illustrated a limited but significant punctate staining pattern of the cell surface. Ultrastructural immunoperoxidase indicated these sites to be cell surface localized coated pits and vesicles. However, it is known that all LIMPs are expressed on the cell surface, albeit at different concentrations, although the total number of each LIMP per cell, respectively, is approximately the same (Barriocanal et al., 1987). Treatment of NRK cells with the acidotropic agent NH4Cl decreased the cell surface expression of LIMPs 1, 3 and 4, but had no effect on LIMP 2. Further, the relative diminution of the cell surface expression varied among the four LIMPs. These results are interpreted to suggest that not all lysosomes contain the same integral membrane proteins in their vesicle container.