'Who's who' in renal sphaerosporids (Bivalvulida: Myxozoa) from common carp, Prussian carp and goldfish--molecular identification of cryptic species, blood stages and new members of Sphaerospora sensu stricto.

Myxozoans are a group of diverse, spore-forming metazoan microparasites bound to aquatic environments. Sphaerospora dykovae (previously S. renicola) causes renal sphaerosporosis and acute swim bladder inflammation (SBI) in juvenile Cyprinus carpio carpio, in central Europe. A morphologically similar species with comparably low pathogenicity, S. angulata has been described from C. c. carpio, Carassius auratus auratus and Carassius gibelio. To clarify uncertainties and ambiguities in taxon identification in these hosts we decided to re-investigate differences in spore morphology using a statistical approach, in combination with SSU and LSU rDNA sequence analyses. We found that developing spores of S. angulata and S. dykovae cannot be distinguished morphologically and designed a duplex PCR assay for the cryptic species that demonstrated S. dykovae is specific to C. c. carpio, whereas S. angulata infects C. a. auratus and C. gibelio. The molecular identification of myxozoan blood stages in common carp and goldfish, which had previously been ascribed to Sphaerospora spp. showed that approximately 75% of blood stages were from non-sphaerosporid coelozoic species infecting these cyprinids and more than 10% were from an alien species, Myxobilatus gasterostei, developing in sticklebacks. We hereby report non-selective myxozoan host invasion and multi-species infections, whose role in SBI still requires clarification.

Phylogeny and morphology of Glugea hertwigi from rainbow smelt Osmerus mordax found in Prince Edward Island, Canada.

Infection of rainbow smelt Osmerus mordax with the microsporidian Glugea hertwigi was diagnosed for the first time in Prince Edward Island, Canada. The prevalence of infection was 24%, 45 infected out of 187 examined fish captured in February and March 2009. Both large and small xenomas of G. hertwigi observed within the submucosa of the gastrointestinal tract and along the mesentery of the host contained only mature spores. Advanced and degraded xenomas associated with host reaction were described using light and transmission electron microscopy. The first rDNA sequence of G. hertwigi prepared in the present study completed the set of sequences of Glugea spp. available for comparison. The high level of rDNA sequence identity between Glugea spp. suggests that these may be variants of a single species.

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.

Neoparamoeba branchiphila n. sp., and related species of the genus Neoparamoeba Page, 1987: morphological and molecular characterization of selected strains.

A total of 18 Neoparamoeba strains were characterized both morphologically and using the SSU rRNA gene sequences as molecular markers. Nine were isolated from gills of farmed Atlantic salmon, Salmo salar L., six from sediments sampled in areas of sea-cage farms and three from net material of sea-cages. The newly obtained sequences extended substantially the dataset of Neoparamoeba strains available for phylogenetic analyses, which were used to infer taxonomic relatedness among 32 strains morphologically assigned to this genus. In addition to the N. pemaquidensis and N. aestuarina clades, phylogenetic analyses clearly distinguished a third clade with sequences from six strains. Members of this clade are characterized as representatives of a new species, N. branchiphila n. sp. The diagnostic primers for the identification of this species are introduced.

Identity of Naegleria strains isolated from organs of freshwater fishes.

Eighteen Naegleria strains were isolated from organs of freshwater fishes belonging to 5 species. Morphometric study allowed the separation of the Naegleria strains from the non-vahlkampfiid amoeboflagellates, but was inadequate for species determination. Six strains, representatives of groups that had a slightly different cyst size, were selected and corresponding derived clones were subjected to sequence analysis and riboprinting restriction fragment length polymorphism (RFLP)-PCR analysis of the small subunit (SSU) rRNA genes. One strain isolated from the brain of a fish with systemic infection was characterised by an intronless 2 kb long SSU rRNA gene and was identified as N. australiensis. Another 5 strains had a 1.3 kb long group I intron in their SSU rRNA gene and, based on the SSU rRNA sequences and riboprints, RFLP-PCR patterns appeared in phylogenetic trees to be closely related to Naegleria clarki.

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.

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.

Analysis of kinetoplast DNA of freshwater fish trypanosomes.

The sequences of 10 conservative regions (CR) of minicircles of 6 selected isolates of freshwater fish trypanosomes have typical organization of this region with high degree of sequence conservation. The comparison with CRs of other trypanosomatids showed that freshwater fish trypanosomes represent a compact separate group within the genus Trypanosoma. The alignment of all sequences obtained revealed, however, the existence of 2 types of CRs in sequenced minicircles, with the differences concentrated in a short region. Taxonomic consequences of these results are discussed.

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.

Growth, morphology and division of flagellates of the genus Trypanoplasma (Protozoa, Kinetoplastida) in vitro.

Nine strains of trypanoplasms were grown in axenic culture. Cultures of Trypanoplasma borreli Laveran and Mesnil, 1901 from fish hosts Blicca bjoerkna, Cyprinus carpio, Scardinius erythrophthalmus and Tinca tinca and of T. guerneyorum Minchin, 1909 from Esox lucius and Trypanoplasma spp. from the leech Piscicola geometra were maintained in biphasic blood-agar medium SNB-9 supplemented with vitamins and antibiotics. In culture, the flagellates transformed into smaller, elongated stages with a little-developed undulating membrane and into short flagella that were morphologically similar to stages in the leech vector. The cultures were passaged weekly at 17-20 degrees C, but they also grew at 4 degrees C. The flagellates divided by binary fission, which was initiated by the formation of two new anterior flagella. The original anterior flagellum of the mother individual was gradually apposed to the cell surface and became the recurrent flagellum of one of the daughter individuals. In the meantime, nuclear division took place, followed by transverse cleavage of the kinetoplast. The division was completed by longitudinal fission of the mother individual into two offspring. Multiple fission that resulted in rosettes, which then cleaved into several daughter cells, was also observed, as well as some dyskinetoplastic and other anomalous forms. In cultures isolated from tenches with high parasitaemia, non-dividing, long filiform stages were observed. Culture stages were not infective for susceptible fishes.

The interaction of fish trypanosome culture forms with some lectins.

The interactions of fish trypanosome culture forms with 11 purified lectins were compared using the agglutination test in microwell plates. Altogether, ten stocks of ten different freshwater fish species were examined. Three basic types of cell-lectin interactions were observed on the microscopical level. The strong agglutination of all stocks regardless their original host was found in the presence of Con A, PSA, RCA60, and RCA120, which implies the presence of relatively high amounts of sugar residues of D-mannose and D-galactose in the surface of culture forms of these parasites. Weak agglutinations of some stocks were observed in the presence of LCA, PNA, SBA, and WGA lectins, but their low intensity makes them not sufficiently reliable for stock characterization. The lectins UEA I, HPA, and PHA caused no agglutination. In conclusion, in case of unequivocal results no remarkable differences in the interactions of various stocks of trypanosomes culture forms with used lectins were observed. These results imply the high degree of similarity of their main cell surface saccharide structures.