Phylogenetic analysis of freshwater fish trypanosomes from Europe using ssu rRNA gene sequences and random amplification of polymorphic DNA.

The taxonomy and phylogenetic relationships of fish trypanosomes are uncertain. A collection of 22 cloned trypanosome isolates from 14 species of European freshwater fish and 1 species of African freshwater fish were examined by molecular phylogenetic analysis. The small subunit ribosomal RNA (ssu rRNA) genes of 8 clones were sequenced and compared with ssu rRNA gene sequences from a wider selection of vertebrate trypanosome isolates by phylogenetic analysis. All trypanosomes from freshwater fish fell in a single clade, subdivided into 3 groups. This clade sits within a larger, robust clade containing trypanosomes from marine fish and various amphibious vertebrates. All 22 trypanosome clones were analysed by random amplification of polymorphic DNA. The resulting dendrogram shows 3 groups, which are congruent with the groups identified in the ssu rRNA gene phylogeny. Two of the groups contain the majority of trypanosome isolates and within-group variation is slight. These groups do not separate purported trypanosome species distinguished by morphology or host origin, and thus these criteria do not appear to be reliable guides to genetic relationships among fish trypanosomes. However, we suggest that the 2 groups themselves may represent different species of fish trypanosomes. The polymorphic DNA markers we have identified will facilitate future comparisons of the biology of these 2 groups of fish trypanosomes.

Ovipleistophora gen. n., a new genus for Pleistophora mirandellae-like microsporidia.

Based on ultrastructural study and molecular analysis, a new genus, Ovipleistophora, is established for Pleistophora mirandellae-like microsporidia from roach and ruff oocytes. Unlike Pleistophora, Ovipleistophora has a thick additional envelope around the meront. This envelope breaks open to release the cells into the host cell cytoplasm. The cells, becoming multinuclear sporogonic plasmodia, already have a surface coat that transforms into the sporont wall and eventually into the sporophorous vesicle wall. The surface coat and its transformation differ from those of Pleistophora, but bear some resemblance to those of Trachipleistophora. In Trachipleistophora the sporonts, however, do not form plasmodia, as they do in Ovipleistophora and Pleistophora. Small subunit ribosomal DNA analysis supports the establishment of the new genus and assignment of P. mirandellae from 2 different fish hosts to the same species. The same small subunit ribosomal DNA analysis lends support for transferring P. ovariae into the genus Ovipleistophora.

Theloiania contejeani Henneguy, 1892: dimorphic life cycle and taxonomic affinities, as indicated by ultrastructural and molecular study.

Thelohania contejeani is a dimorphic species with two simultaneous routes for sporogony. In the first, diplokaryotic sporonts produce 8 uninucleate spores with 9-10 turns of polar tube, within a sporophorous vesicle wall. The episporontal space contains two kinds of tubules and a spongiform mass. In the second, single diplokaryotic sporonts produce small membrane-bound compartments in which they transform into mature diplokaryotic spores with 5-6 turns of polar tube. Analysis of the small subunit (SSU) rRNA revealed two copies of 16S-like rDNA, one of them 1,311 bp, the other 1,361 bp long, with an overall identity of 93%. The majority of sequence differences were located in a 120-bp stretch between positions 336 and 456, with only 40.5% identity between the sequences. Careful consideration suggests that the shorter sequence represents a pseudogene. According to the SSU rDNA sequence, T. contejeani is not closely related to any of the microsporidians where this sequence is available and could not be unambiguously placed in the 16S phylogenetic tree.

Redescription of Microsporidium takedai (Awakura, 1974) as Kabatana takedai (Awakura, 1974) comb. n.

Ultrastructural study of the microsporidian Microsporidium takedai from the muscles of masu salmon Oncorhynchus masou proved that this species can be assigned to the genus Kabatana Lom, Dyková and Tonguthai, 2000. The parasites develop within disintegrated sarcoplasm without any delimiting boundary or cyst. Cylindrical multinucleate meronts proliferate by serial constrictions into uninucleate stages which repeat the process. Eventually, the uninucleate stages transform into uninucleate sporonts, which divide once to produce sporoblasts, thus functioning as sporoblast mother cells. Spores, with a subterminally located anchoring disc and 3 to 4 turns of the polar tube coil, average 3.3 by 1.9 microm in size. The exospore is divided into small fields; the endospore frequently makes small invaginations into the spore inside. Phylogenetic analysis using SSU rDNA sequence consistently placed Kabatana takedai in a group consisting of Microgemma sp., Spraguea lophii and Glugea americanus. The K. takedai could easily be separated from the other species in the same group by 2 inserts in the SSU rDNA sequence.

Histopathology of Kabatana arthuri (Microspora) infection in sutchi catfish, Panagasius sutchi.

The microsporidian Kabatana arthuri (Lom, Dyková et Shaharom, 1990) induced severe regressive changes in trunk muscles of Pangasius sutchi (Fowler) from Thailand. Necrotic changes developed in muscle fibres around the developmental stages and on the periphery of giant aggregates of spores. The main feature of the host defence reaction was the phagocytic activity of macrophages. Inflammatory reaction was only exceptionally observed. Spore-laden macrophages were found in various tissues and organs; their infiltration in epidermis including its outermost layers may effectively enhance the spread of infection while the hosts still live.

Ultrastructural justification for the transfer of Pleistophora anguillarum Hoshina, 1959 to the genus Heterosporis Schubert, 1969.

This study presents the ultrastructure of the microsporidian infecting the trunk musculature of Anguilla japonica and originally described as Pleistophora anguillarum Hoshina, 1959. All stages develop within a special structure, the sporophorocyst (SPC), which is equipped with a thick dense wall. This wall grows along with the growth of the parasites within it. Meronts are uni- to binucleate, which divide and steadily give rise to sporonts. During transition to sporonts the cell coat of the meronts increases its thickness, temporarily featuring thick irregular projections. Eventually a uniformly thick sporont wall is formed, then the sporont cells detach themselves from the wall (= future wall of the sporophorous vesicle, SPV) and start a series of divisions to produce sporoblasts. The SPV wall is compact, has no pores and consists of 2 layers. The presence of the SPC justifies the transfer of the species into the genus Heterosporis. Spores from disrupted SPCs are ingested by macrophages and within them are spread into various body tissues including the outermost layers of the epidermis. From here, they can easily be released to the outside and can contaminate the environment while the host is still alive.

Kabataia gen. n., a new genus proposed for Microsporidium spp. infecting trunk muscles of fishes.

Based on a fine structural study, a new genus, Kabataia gen. n., is proposed for Microsporidium arthuri Lom, Dyková and Shaharom, 1990. It develops in trunk muscles of a South-East Asian freshwater fish, Pangasius sutchi. The genus has nuclei isolated throughout the cycle, merogony stages are multinucleate, sporogony proceeds in 2 steps: multinucleate sporont segments into sporoblast mother cells which produce 2 sporoblasts. Sporoblasts and early spores are characterized by a dense globule at the site of the posterior vacuole. Mature spores are of a rather variable shape. Their exospore is raised into small, irregular fields. The polaroplast is relatively small and its posterior part consists of flat vesicles with dense contents. The polar tube makes a small number (4 to 6) of turns. A congeneric species is Kabataia seriolae (Egusa, 1982) comb. nov. from cultured marine yellowtails Seriola quinqueradiata. Kabataia inflicts heavy damage on muscle tissue. The sarcoplasm within which Kabataia develops is reduced to an amorphous mass with tubule-like fibrils, microfibrils and small vesicles.

Fish trypanosomes: their position in kinetoplastid phylogeny and variability as determined from 12S rRNA kinetoplast sequences.

Fish trypanosomes have traditionally been classified according to the host species from which they were isolated, each isolate being regarded as a distinct species. To test the soundness of this practice, the genetic variabilities of the kinetoplast 12S rRNA-encoding genes of different fish trypanosomes isolates were compared. The DNAs were extracted from trypanosomes cloned from blood samples of 15 donors representing ten different fish species in four orders from waters of three major river systems of Central and Northern Europe. Comparison with other trypanosomatid sequences revealed that the fish trypanosomes form a monophyletic group with Trypanosoma brucei as a sister group. Pairwise comparisons of genetic distances yielded a wide range of continuous variation with no indication of any discontinuities attributable to barriers to gene flow. The genetic distances did not correlate with either the identity of the host species or geography. The host specificity of fish trypanosomes appears to be limited.

Nosema notabilis (Microsporidia), its ultrastructure and effect on the myxosporean host Ortholinea polymorpha.

Nosema notabilis Kudo, 1939 produces chain-forming meronts with a dense cell coat in direct contact with the host cell cytoplasm. Cytoplasmic microtubules and membranaceous whorls could be observed in meront cytoplasm. Sporonts differ in that they have a thicker cell wall and more conspicuous endoplasmic reticulum (ER) cisternae. Sporoblasts have an externally ridged cell wall. Spores have an apically located anchoring disc, an isofilar polar tube with 6 to 9 turns and polyribosomal strands in the sporoplasm. Diplokarya occur in all stages. Heavily infected plasmodia of Ortholinea polymorpha (Davis, 1917) reveal marked pathological signs. The most prominent are reduction of surface projections and/or pinocytosis, inflated mitochondria with altered inner structures, affected vegetative nuclei, damage to generative cells and occurrence of various anomalous formations in the plasmodium cytoplasm. The damage may result in complete disintegration of the plasmodium. However, the development of the microsporidian is affected by a remarkably high percentage of teratological stages revealing membranaceous and tubular structures.

Acanthamoeba strains isolated from organs of freshwater fishes.

Contrary to data on Acanthamoeba infections in humans, little is known about infections in fishes. The present study combines the description of strains isolated from fishes with presentation of an improved method for subgeneric classification. Acanthamoeba spp. were isolated aseptically from tissues of 14 (1.7%) of 833 asymptomatic fishes collected in rivers and streams in the Czech Republic. Acanthamoebae successfully cloned from 10 of the 14 isolated strains were examined here. Morphology of these isolates was evaluated using light optics plus scanning and transmission electron microscopy. Cyst morphology, which varied extensively within and among clones, was most like morphological group II, but species-level classification was considered impossible. A distance analysis based on 442 bases in an 18S rDNA polymerase chain reaction fragment of about 460 bp placed the isolates in a clade composed of sequence types T3, T4, and T11, the 3 subdivisions of morphological group II. Fluorescent in situ hybridization (FISH) using oligonucleotide probes indicated that all isolates belong to a single subdivision of group II, the T4 sequence type. It has been concluded that the fish isolates are most closely related to strains commonly isolated from human infections, especially Acanthamoeba keratitis. The shorter diagnostic fragment sequences have proved nearly as useful as complete 18S rDNA sequences for identification of Acanthamoeba isolates.

Cultivation of bloodstream forms of Trypanosoma carassii, a common parasite of freshwater fish.

Trypanosoma carassii (syn. T. danilewskyi) is a widespread parasite of carp and other cyprinid as well as some noncyprinid freshwater fish. It lives extracellularly in the blood and tissues of its hosts, causing chronic infections. In this paper the isolation of T. carassii from fish blood and the propagation and cloning of bloodstream forms in vitro are described. By several criteria, cultured and fish-derived trypomastigotes are indistinguishable. The culture system should be useful for the biochemical characterization of this trypanosome and its interaction with the fish immune system.

Vexillifera expectata sp. n. and other non-encysting amoebae isolated from organs of freshwater fish.

Four strains of non-encysting amoebae were isolated from organs of freshwater fishes and characterized using light and electron microscope. Morphology of three clonal strains was consistent with amoebae which had already been described from water habitats. Two strains, one isolated from kidney tissue of common goldfish, Carassius auratus (Linnaeus, 1758), and the second one from brain of chub, Leuciscus cephalus Linnaeus, 1758, were identified with Vannella platypodia (Gläser, 1912) Page, 1976. Both strains were identical, except for the length of glycostyles. The strain isolated from the liver of perch, Perca fluviatilis (Linnaeus, 1758), was assigned to the genus Vexillifera Schaeffer, 1926 as Vexillifera expectata sp. n. The taxonomic position of the fourth non-encysting strain could not be safely established, although it shares some trophic cell structures with protostelids (Protostelia, Eumycetozoea). We present its detailed description here also to demonstrate that amoeba stages of this type of organisms are capable to infect fishes.

Occurrence of Loma cf. salmonae in brook, brown and rainbow trout from Buford trout hatchery, Georgia, USA.

During a 6 mo study of moribund trout from Buford hatchery, Buford, Georgia, USA, a Loma cf. salmonae microsporidian parasite was studied in the gills of brook trout Salvelinus fontinalis, brown trout Salmo trutta, and rainbow trout Oncorhynchus mykiss. This parasite was morphologically similar to L. salmonae and L. fontinalis but differed in spore size. Scanning and transmission electron microscopy demonstrated that xenomas were embedded in gill filaments. Transmission electron micrographs prepared from fresh tissue showed mature spores with 12 to 15 turns of their polar tube. Spore diameters for the Georgia strain from formalin-fixed gill tissues measured 3.5 (SD +/- 0.1) by 1.8 (SD +/- 0.1) microns. Electron micrographs of formalin-fixed, deparaffinized tissues of rainbow trout from Pennsylvania and West Virginia show spores with a diameter of 3.5 (+/- 0.2) by 1.7 (+/- 0.1) microns and 3.4 (+/- 0.2) by 1.8 (+/- 0.1) microns, respectively. Transmission electron micrographs of spores from Pennsylvania and West Virginia show that mature spores from both states had 13 to 15 turns of their polar tubes. Measurements from transmission electron micrographs prepared from alcohol-fixed tissues from Virginia fish contained spores with a diameter of 3.0 (+/- 0.3) by 1.1 (+/- 0.3) microns and 12 to 15 turns of their polar tubes. These measurements are consistent with L. salmonae and therefore suggest that the parasite is present on the east coast of the United States. During the height of the Georgia epizootic, the percentage of fish with observed xenomas reached 62.2% (N = 87), and the highest number of xenomas counted per 10 gill filaments was 133 (N = 87). The microsporidian epizootic occurred either during the autumn months or when intake river water quality reached combined iron-manganese concentrations as high as 1.01 (mean 0.44, SD +/- 0.42) mg-1.

Cochliopodium minus, a scale-bearing amoeba isolated from organs of perch Perca fluviatilis.

A scale-bearing amoeba isolated from gills and other organs of perch Perca fluviatilis Linnaeus, 1758 was identified as Cochliopodium minus Page, 1976. This assignment was suggested by light microscopy and confirmed by ultrastructural study of both the strain isolated from perch and the environmental strain of C. minus (CCAP 1537/1A) serving as a control. This has been the first electron-microscopic definition of C. minus and, in addition, the first identification of an amoeba with a theca-like cover found to infect fish. The ability of the fish strain of C. minus to colonize gills and also internal body organs was proved experimentally.

Myxosporeans infecting the gills of bigmouth buffalo (Ictiobus bubalus) in Illinois, USA.

Four myxosporean species were found on the gills of Ictiobus bubalus from Illinois (USA). Myxobolus endovasus (Davis, 1947) Grinham et Cone, 1990 is revised. Three new species are recorded. Myxobolus enoblei sp. n. has spores ovoid in frontal view, 14.3 x 13 microns in size. Myxobolus morrisonae sp. n. has spores subcircular in frontal view, 10 x 9.5 microns in size; the surface of shell valves appears hairy when studied by SEM. Triangula illinoisensis sp. n. has spores rounded semicircular in frontal view, 10.2 x 12.8 microns in size. Triangula illinoisensis is the fourth species of its genus to be described from fishes.

Organization of mini-exon and 5S rRNA genes in the kinetoplastid Trypanoplasma borreli.

Mini-exon gene repeats from Trypanoplasma borreli, which belongs to the Cryptobiidae family of the Bodonina suborder of the Kinetoplastida, were isolated by PCR amplification and cloning. The presence of kinetoplastid-like mini-exon genes in T. borreli is consistent with the taxonomic status of this organism as a kinetoplastid protozoan. Two families of repeats were found: 597 nt (T1) and 794 nt (T2), each of which encodes an approximately 95-nt medRNA transcript. The T1 repeats also contain a complete 5S rRNA gene on the complementary strand. The T2 repeats contain a defective copy of a 5S gene, in which the 5' portion is absent. The intergenic regions between the 5'-ends of the mini-exon genes and the 5S rRNA genes in the T1 and T2 repeats are highly diverged. All or most mini-exon genes and 5S genes are located within either the T1 or the T2 repeats. The T1 repeats were localized to a megabase-size chromosome, while the T2 repeats were localized within at least 4 large chromosomes.