Phylogenetic and morphological diversity of free-living diplomonads.

Diplomonadida is a lineage of anaerobic protists belonging to Fornicata, Metamonada. Most diplomonads are endobiotic or parasitic, such as Giardia intestinalis, which is a famous human pathogen, but several free-living species exist as well. Although it has been proposed that the free-living diplomonads are descendants of endobiotic organisms and thus interesting from the evolutionary point of view, they have been largely neglected. We obtained 58 cultures of free-living diplomonads belonging to four genera (Hexamita, Trepomonas, Gyromonas, and Trimitus) and six strains of endobiotic diplomonads and analyzed their SSU rRNA gene sequences. We also studied light-microscopic morphology of selected strains and the ultrastructure of Trepomonas rotans for the first time. Our phylogenetic analysis showed that the genus Hexamita, and, possibly, also the genus Trepomonas, are polyphyletic. Trepomonas rotans, which may represent a novel genus, is unique among Diplomonadida by having the cell covered in scales. Our results suggest that the evolution of the endobiotic life style and cell organization in diplomonads is more complicated than previously thought.

A chromosome-scale reference genome for Spironucleus salmonicida.

Spironucleus salmonicida is a diplomonad causing systemic infection in salmon. The first S. salmonicida genome assembly was published 2014 and has been a valuable reference genome in protist research. However, the genome assembly is fragmented without assignment of the sequences to chromosomes. In our previous Giardia genome study, we have shown how a fragmented genome assembly can be improved with long-read sequencing technology complemented with optical maps. Combining Pacbio long-read sequencing technology and optical maps, we are presenting here this new S. salmonicida genome assembly in nine near-complete chromosomes with only three internal gaps at long repeats. This new genome assembly is not only more complete sequence-wise but also more complete at annotation level, providing more details into gene families, gene organizations and chromosomal structure. This near-complete reference genome will aid comparative genomics at chromosomal level, and serve as a valuable resource for the diplomonad community and protist research.

VEuPathDB: the eukaryotic pathogen, vector and host bioinformatics resource center.

The Eukaryotic Pathogen, Vector and Host Informatics Resource (VEuPathDB, https://veupathdb.org) represents the 2019 merger of VectorBase with the EuPathDB projects. As a Bioinformatics Resource Center funded by the National Institutes of Health, with additional support from the Welllcome Trust, VEuPathDB supports >500 organisms comprising invertebrate vectors, eukaryotic pathogens (protists and fungi) and relevant free-living or non-pathogenic species or hosts. Designed to empower researchers with access to Omics data and bioinformatic analyses, VEuPathDB projects integrate >1700 pre-analysed datasets (and associated metadata) with advanced search capabilities, visualizations, and analysis tools in a graphic interface. Diverse data types are analysed with standardized workflows including an in-house OrthoMCL algorithm for predicting orthology. Comparisons are easily made across datasets, data types and organisms in this unique data mining platform. A new site-wide search facilitates access for both experienced and novice users. Upgraded infrastructure and workflows support numerous updates to the web interface, tools, searches and strategies, and Galaxy workspace where users can privately analyse their own data. Forthcoming upgrades include cloud-ready application architecture, expanded support for the Galaxy workspace, tools for interrogating host-pathogen interactions, and improved interactions with affiliated databases (ClinEpiDB, MicrobiomeDB) and other scientific resources, and increased interoperability with the Bacterial & Viral BRC.

PHI-base in 2022: a multi-species phenotype database for Pathogen-Host Interactions.

Since 2005, the Pathogen-Host Interactions Database (PHI-base) has manually curated experimentally verified pathogenicity, virulence and effector genes from fungal, bacterial and protist pathogens, which infect animal, plant, fish, insect and/or fungal hosts. PHI-base (www.phi-base.org) is devoted to the identification and presentation of phenotype information on pathogenicity and effector genes and their host interactions. Specific gene alterations that did not alter the in host interaction phenotype are also presented. PHI-base is invaluable for comparative analyses and for the discovery of candidate targets in medically and agronomically important species for intervention. Version 4.12 (September 2021) contains 4387 references, and provides information on 8411 genes from 279 pathogens, tested on 228 hosts in 18, 190 interactions. This provides a 24% increase in gene content since Version 4.8 (September 2019). Bacterial and fungal pathogens represent the majority of the interaction data, with a 54:46 split of entries, whilst protists, protozoa, nematodes and insects represent 3.6% of entries. Host species consist of approximately 54% plants and 46% others of medical, veterinary and/or environmental importance. PHI-base data is disseminated to UniProtKB, FungiDB and Ensembl Genomes. PHI-base will migrate to a new gene-centric version (version 5.0) in early 2022. This major development is briefly described.

Lateral Acquisitions Repeatedly Remodel the Oxygen Detoxification Pathway in Diplomonads and Relatives.

Oxygen and reactive oxygen species (ROS) are important stress factors for cells because they can oxidize many large molecules. Fornicata, a group of flagellated protists that includes diplomonads, have anaerobic metabolism but are still able to tolerate fluctuating levels of oxygen. We identified 25 protein families putatively involved in detoxification of oxygen and ROS in this group using a bioinformatics approach and propose how these interact in an oxygen detoxification pathway. These protein families were divided into a central oxygen detoxification pathway and accessory pathways for the synthesis of nonprotein thiols. We then used a phylogenetic approach to investigate the evolutionary origin of the components of this putative pathway in Diplomonadida and other Fornicata species. Our analyses suggested that the diplomonad ancestor was adapted to low-oxygen levels, was able to reduce O2 to H2O in a manner similar to extant diplomonads, and was able to synthesize glutathione and l-cysteine. Several genes involved in the pathway have complex evolutionary histories and have apparently been repeatedly acquired through lateral gene transfer and subsequently lost. At least seven genes were acquired independently in different Fornicata lineages, leading to evolutionary convergences. It is likely that acquiring these oxygen detoxification proteins helped anaerobic organisms (like the parasitic Giardia intestinalis) adapt to low-oxygen environments (such as the digestive tract of aerobic hosts).

Patterns of conservation of spliceosomal intron structures and spliceosome divergence in representatives of the diplomonad and parabasalid lineages.

BACKGROUND: Two spliceosomal intron types co-exist in eukaryotic precursor mRNAs and are excised by distinct U2-dependent and U12-dependent spliceosomes. In the diplomonad Giardia lamblia, small nuclear (sn) RNAs show hybrid characteristics of U2- and U12-dependent spliceosomal snRNAs and 5 of 11 identified remaining spliceosomal introns are trans-spliced. It is unknown whether unusual intron and spliceosome features are conserved in other diplomonads. RESULTS: We have identified spliceosomal introns, snRNAs and proteins from two additional diplomonads for which genome information is currently available, Spironucleus vortens and Spironucleus salmonicida, as well as relatives, including 6 verified cis-spliceosomal introns in S. vortens. Intron splicing signals are mostly conserved between the Spironucleus species and G. lamblia. Similar to 'long' G. lamblia introns, RNA secondary structural potential is evident for 'long' (> 50 nt) Spironucleus introns as well as introns identified in the parabasalid Trichomonas vaginalis. Base pairing within these introns is predicted to constrain spatial distances between splice junctions to similar distances seen in the shorter and uniformly-sized introns in these organisms. We find that several remaining Spironucleus spliceosomal introns are ancient. We identified a candidate U2 snRNA from S. vortens, and U2 and U5 snRNAs in S. salmonicida; cumulatively, illustrating significant snRNA differences within some diplomonads. Finally, we studied spliceosomal protein complements and find protein sets in Giardia, Spironucleus and Trepomonas sp. PC1 highly- reduced but well conserved across the clade, with between 44 and 62 out of 174 studied spliceosomal proteins detectable. Comparison with more distant relatives revealed a highly nested pattern, with the more intron-rich fornicate Kipferlia bialata retaining 87 total proteins including nearly all those observed in the diplomonad representatives, and the oxymonad Monocercomonoides retaining 115 total proteins including nearly all those observed in K. bialata. CONCLUSIONS: Comparisons in diplomonad representatives and species of other closely-related metamonad groups indicates similar patterns of intron structural conservation and spliceosomal protein composition but significant divergence of snRNA structure in genomically-reduced species. Relative to other eukaryotes, loss of evolutionarily-conserved snRNA domains and common sets of spliceosomal proteins point to a more streamlined splicing mechanism, where intron sequences and structures may be functionally compensating for the minimalization of spliceosome components.

Oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida.

BACKGROUND: Spironucleus salmonicida is an anaerobic parasite that can cause systemic infections in Atlantic salmon. Unlike other diplomonad parasites, such as the human pathogen Giardia intestinalis, Spironucleus species can infiltrate the blood stream of their hosts eventually colonizing organs, skin and gills. How this presumed anaerobe can persist and invade oxygenated tissues, despite having a strictly anaerobic metabolism, remains elusive. RESULTS: To investigate how S. salmonicida response to oxygen stress, we performed RNAseq transcriptomic analyses of cells grown in the presence of oxygen or antioxidant-free medium. We found that over 20% of the transcriptome is differentially regulated in oxygen (1705 genes) and antioxidant-depleted (2280 genes) conditions. These differentially regulated transcripts encode proteins related to anaerobic metabolism, cysteine and Fe-S cluster biosynthesis, as well as a large number of proteins of unknown function. S. salmonicida does not encode genes involved in the classical elements of oxygen metabolism (e.g., catalases, superoxide dismutase, glutathione biosynthesis, oxidative phosphorylation). Instead, we found that genes encoding bacterial-like oxidoreductases were upregulated in response to oxygen stress. Phylogenetic analysis revealed some of these oxygen-responsive genes (e.g., nadh oxidase, rubrerythrin, superoxide reductase) are rare in eukaryotes and likely derived from lateral gene transfer (LGT) events into diplomonads from prokaryotes. Unexpectedly, we observed that many host evasion- and invasion-related genes were also upregulated under oxidative stress suggesting that oxygen might be an important signal for pathogenesis. CONCLUSION: While oxygen is toxic for related organisms, such as G. intestinalis, we find that oxygen is likely a gene induction signal for host invasion- and evasion-related pathways in S. salmonicida. These data provide the first molecular evidence for how S. salmonicida could tolerate oxic host environments and demonstrate how LGT can have a profound impact on the biology of anaerobic parasites.

On the reversibility of parasitism: adaptation to a free-living lifestyle via gene acquisitions in the diplomonad Trepomonas sp. PC1.

BACKGROUND: It is generally thought that the evolutionary transition to parasitism is irreversible because it is associated with the loss of functions needed for a free-living lifestyle. Nevertheless, free-living taxa are sometimes nested within parasite clades in phylogenetic trees, which could indicate that they are secondarily free-living. Herein, we test this hypothesis by studying the genomic basis for evolutionary transitions between lifestyles in diplomonads, a group of anaerobic eukaryotes. Most described diplomonads are intestinal parasites or commensals of various animals, but there are also free-living diplomonads found in oxygen-poor environments such as marine and freshwater sediments. All these nest well within groups of parasitic diplomonads in phylogenetic trees, suggesting that they could be secondarily free-living. RESULTS: We present a transcriptome study of Trepomonas sp. PC1, a diplomonad isolated from marine sediment. Analysis of the metabolic genes revealed a number of proteins involved in degradation of the bacterial membrane and cell wall, as well as an extended set of enzymes involved in carbohydrate degradation and nucleotide metabolism. Phylogenetic analyses showed that most of the differences in metabolic capacity between free-living Trepomonas and the parasitic diplomonads are due to recent acquisitions of bacterial genes via gene transfer. Interestingly, one of the acquired genes encodes a ribonucleotide reductase, which frees Trepomonas from the need to scavenge deoxyribonucleosides. The transcriptome included a gene encoding squalene-tetrahymanol cyclase. This enzyme synthesizes the sterol substitute tetrahymanol in the absence of oxygen, potentially allowing Trepomonas to thrive under anaerobic conditions as a free-living bacterivore, without depending on sterols from other eukaryotes. CONCLUSIONS: Our findings are consistent with the phylogenetic evidence that the last common ancestor of diplomonads was dependent on a host and that Trepomonas has adapted secondarily to a free-living lifestyle. We believe that similar studies of other groups where free-living taxa are nested within parasites could reveal more examples of secondarily free-living eukaryotes.

Genetic diversity of Diplomonadida in fish of the genus Coregonus from Southeastern Siberia.

Diplomonadida are primitive flagellate protozoa, among which both commensals and pathogens have been recorded. To date, members of the genera Hexamita and Spironucleus have been reported in the digestive system of fish in the Baikal region. We determined the genetic diversity of Diplomonadida in fish of the genus Coregonus from south-eastern Siberia using molecular-genetic methods. Fish for analysis were caught in Lake Baikal and in the Barguzin, Nepa, Chechuy, and Kirenga rivers from 2010 to 2013. Gall bladders, hindguts and foreguts of 120 specimens of Coregonus migratorius representing three morpho-ecological groups, 25 specimens of Coregonus lavaretus baicalensis, 25 specimens of Coregonus tugun and 30 specimens of Coregonus lavaretus pidschian were analysed via amplification with primers specifically designed for eukaryotes. Amplicons positive for Diplomonadida were sequenced. A phylogenetic analysis revealed that diplomonad flagellates of whitefish from Southeastern Siberia belong to Spironucleus barkhanus. Positive Diplomonadida DNA samples were analysed with primers designed in the present study for the amplification of small subunits of ribosomal DNA fragments of S. barkhanus (about 1,430 bp) and sequenced. Phylogenetic analysis revealed inside the clade of S. barkhanus besides the cosmopolitan genotype from European salmon that was detected earlier in Baikalian grayling, a new genotype unique to the fish of the genus Coregonus from Lake Baikal.

Spironucleus meleagridis, an enteric diplomonad protozoan of cockatiels (Nymphicus hollandicus): preliminary molecular characterization and association with clinical disease.

A flagellated enteric diplomonad protozoan consistent with Spironucleus meleagridis (formerly Hexamita meleagridis) associated with gastrointestinal disease and mortality in psittacine birds including cockatiels (Nymphicus hollandicus) has been sporadically described in the literature. However, molecular characterization of psittacine protozoal isolates had not yet been performed. The 16S rRNA gene from a protozoan persistently shed in the feces in a small group of cockatiels demonstrated a 98% molecular identity with S. meleagridis isolated from turkeys. Based on these sequence data, a diagnostic PCR assay was developed to detect the presence of S. meleagridis. Nineteen privately owned pet cockatiels from unrelated households were clinically evaluated. All birds microscopically positive for this organism were PCR positive, with several additional birds microscopically negative but PCR positive. Many of the birds identified as positive for S. meleagridis by fecal PCR had signs of gastrointestinal disease such as diarrhea, soft feces, and melena, whereas none of the birds that tested negative had gastrointestinal signs. Examination of feces from two unrelated cockatiel breeding facilities revealed 70% and 86% PCR positive rates. Prevalence of infection and incidence of clinical disease, including factors that lead to clinical manifestation such as viral, bacterial, or mycotic coinfections, are not yet known and warrant further study, but spironucleosis is likely an under-recognized disease in cockatiels.

The genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments.

Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we identified energy-producing hydrogenosomes in S. salmonicida. Here we present a genome analysis of the fish parasite with a focus on the comparison to the more studied diplomonad Giardia intestinalis. We annotated 8067 protein coding genes in the ∼12.9 Mbp S. salmonicida genome. Unlike G. intestinalis, promoter-like motifs were found upstream of genes which are correlated with gene expression, suggesting a more elaborate transcriptional regulation. S. salmonicida can utilise more carbohydrates as energy sources, has an extended amino acid and sulfur metabolism, and more enzymes involved in scavenging of reactive oxygen species compared to G. intestinalis. Both genomes have large families of cysteine-rich membrane proteins. A cluster analysis indicated large divergence of these families in the two diplomonads. Nevertheless, one of S. salmonicida cysteine-rich proteins was localised to the plasma membrane similar to G. intestinalis variant-surface proteins. We identified S. salmonicida homologs to cyst wall proteins and showed that one of these is functional when expressed in Giardia. This suggests that the fish parasite is transmitted as a cyst between hosts. The extended metabolic repertoire and more extensive gene regulation compared to G. intestinalis suggest that the fish parasite is more adapted to cope with environmental fluctuations. Our genome analyses indicate that S. salmonicida is a well-adapted pathogen that can colonize different sites in the host.

Hydrogenosomes in the diplomonad Spironucleus salmonicida.

Acquisition of the mitochondrion is a key event in the evolution of the eukaryotic cell, but diversification of the organelle has occurred during eukaryotic evolution. One example of such mitochondria-related organelles (MROs) are hydrogenosomes, which produce ATP by substrate-level phosphorylation with hydrogen as a byproduct. The diplomonad parasite Giardia intestinalis harbours mitosomes, another type of MRO. Here we identify MROs in the salmon parasite Spironucleus salmonicida with similar protein import and Fe-S cluster assembly machineries as in Giardia mitosomes. We find that hydrogen production is prevalent in the diplomonad genus Spironucleus, and that S. salmonicida MROs contain enzymes characteristic of hydrogenosomes. Evolutionary analyses of known hydrogenosomal components indicate their presence in the diplomonad ancestor, and subsequent loss in Giardia. Our results suggest that hydrogenosomes are metabolic adaptations predating the split between parabasalids and diplomonads, which is deeper than the split between animals and fungi in the eukaryotic tree.

Repeat-enriched proteins are related to host cell invasion and immune evasion in parasitic protozoa.

Proteins containing repetitive amino acid domains are widespread in all life forms. In parasitic organisms, proteins containing repeats play important roles such as cell adhesion and invasion and immune evasion. Therefore, extracellular and intracellular parasites are expected to be under different selective pressures regarding the repetitive content in their genomes. Here, we investigated whether there is a bias in the repetitive content found in the predicted proteomes of 6 exclusively extracellular and 17 obligate intracellular protozoan parasites, as well as 4 free-living protists. We also attempted to correlate the results with the distinct ecological niches they occupy and with distinct protein functions. We found that intracellular parasites have higher repetitive content in their proteomes than do extracellular parasites and free-living protists. In intracellular parasites, these repetitive proteins are located mainly at the parasite surface or are secreted and are enriched in amino acids known to be part of N- and O-glycosylation sites. Furthermore, in intracellular parasites, the developmental stages that are able to invade host cells express a higher proportion of proteins with perfect repeats relative to other life cycle stages, and these proteins have molecular functions associated with cell invasion. In contrast, in extracellular parasites, degenerate repetitive motifs are enriched in proteins that are likely to play roles in evading host immune response. Altogether, our results support the hypothesis that both the ability to invade host cells and to escape the host immune response may have shaped the expansion and maintenance of perfect and degenerate repeats in the genomes of intra- and extracellular parasites.

Stable transfection of the diplomonad parasite Spironucleus salmonicida.

Eukaryotic microbes are highly diverse, and many lineages remain poorly studied. One such lineage, the diplomonads, a group of binucleate heterotrophic flagellates, has been studied mainly due to the impact of Giardia intestinalis, an intestinal, diarrhea-causing parasite in humans and animals. Here we describe the development of a stable transfection system for use in Spironucleus salmonicida, a diplomonad that causes systemic spironucleosis in salmonid fish. We designed vectors in cassette format carrying epitope tags for localization (3×HA [where HA is hemagglutinin], 2× Escherichia coli OmpF linker and mouse langerin fusion sequence [2×OLLAS], 3×MYC) and purification of proteins (2× Strep-Tag II-FLAG tandem-affinity purification tag or streptavidin binding peptide-glutathione S-transferase [SBP-GST]) under the control of native or constitutive promoters. Three selectable gene markers, puromycin acetyltransferase (pac), blasticidin S-deaminase (bsr), and neomycin phosphotransferase (nptII), were successfully applied for the generation of stable transfectants. Site-specific integration on the S. salmonicida chromosome was shown to be possible using the bsr resistance gene. We epitope tagged six proteins and confirmed their expression by Western blotting. Next, we demonstrated the utility of these vectors by recording the subcellular localizations of the six proteins by laser scanning confocal microscopy. Finally, we described the creation of an S. salmonicida double transfectant suitable for colocalization studies. The transfection system described herein and the imminent completion of the S. salmonicida genome will make it possible to use comparative genomics as an investigative tool to explore specific, as well as general, diplomonad traits, benefiting research on both Giardia and Spironucleus.

Multigene phylogenies of diverse Carpediemonas-like organisms identify the closest relatives of 'amitochondriate' diplomonads and retortamonads.

Diplomonads, retortamonads, and "Carpediemonas-like" organisms (CLOs) are a monophyletic group of protists that are microaerophilic/anaerobic and lack typical mitochondria. Most diplomonads and retortamonads are parasites, and the pathogen Giardia intestinalis is known to possess reduced mitochondrion-related organelles (mitosomes) that do not synthesize ATP. By contrast, free-living CLOs have larger organelles that superficially resemble some hydrogenosomes, organelles that in other protists are known to synthesize ATP anaerobically. This group represents an excellent system for studying the evolution of parasitism and anaerobic, mitochondrion-related organelles. Understanding these evolutionary transitions requires a well-resolved phylogeny of diplomonads, retortamonads and CLOs. Unfortunately, until now the deep relationships amongst these taxa were unresolved due to limited data for almost all of the CLO lineages. To address this, we assembled a dataset of up to six protein-coding genes that includes representatives from all six CLO lineages, and complements existing rRNA datasets. Multigene phylogenetic analyses place CLOs as well as the retortamonad Chilomastix as a paraphyletic basal assemblage to the lineage comprising diplomonads and the retortamonad Retortamonas. In particular, the CLO Dysnectes was shown to be the closest relative of the diplomonads + Retortamonas clade, with strong support. This phylogeny is consistent with a drastic degeneration of mitochondrion-related organelles during the evolution from a free-living organism resembling extant CLOs to a probable parasite/commensal common ancestor of diplomonads and Retortamonas.

3' processing in protists.

Molecular biologists have traditionally focused on the very small corner of eukaryotic evolution that includes yeast and animals; even plants have been neglected. In this article, we describe the scant information that is available concerning RNA processing in the other four major eukaryotic groups, especially pathogenic protists. We focus mainly on polyadenylation and nuclear processing of stable RNAs. These processes have--where examined--been shown to be conserved, but there are many novel details. We also briefly mention other processing reactions such as splicing.

Wild arctic char Salvelinus alpinus and trout Salmo trutta: hosts and reservoir of the salmonid pathogen Spironucleus salmonicida (Diplomonadida; Hexamitidae).

Spironucleus salmonicida is a diplomonad flagellate known to cause systemic infections in farmed salmonids. In northern Norway, outbreaks of spironucleosis in farmed Atlantic salmon Salmo salar have been a recurring problem. Common to all these outbreaks was the origin of smolts: all came from the same farm. In the present study, wild Arctic char Salvelinus alpinus and brown trout Salmo trutta were sampled from the lakes used as a water source for the smolt supplier. In addition, smolt and three-spined sticklebacks Gasterosteus aculeatus were sampled from the smolt farm. Bile and intestinal contents from the sampled fish were examined by light microscopy and PCR. Spironucleus salmonicida was identified in both wild Arctic char and brown trout from the lakes used as water sources by the smolt farm, suggesting that the farmed fish were exposed to this pathogen before transfer to the sea. Spironucleus barkhanus and Spironucleus salmonis were also identified in the sampled fish. The present study also demonstrated that infections with multiple Spironucleus species are present in wild salmonids. No indications of disease related to diplomonad infections were observed in the wild fish, suggesting that wild salmonids are reservoir hosts of Spironucleus salmonicida.

Risk factors and control of intestinal parasite infections in sled dogs in Poland.

Training and racing constitute serious challenges for working sled dogs. Attainment of the highest levels of stamina and speed are possible only by completely healthy dogs. Infections with nematodes as whipworm Trichuris sp. or hookworms Uncinaria/Ancylostoma can significantly reduce the fitness of working dogs leading to anemia or even to death. In the middle of the racing season, between December 2009 and April 2010, 108 individual fecal samples were collected from 25 sled dog kennels situated in different regions of Poland. Saturated salt flotation was performed for helminth egg detection. The immunofluorescent assay MeriFluor Cryptosporidium/Giardia and nested PCRs on 18S rRNA (Cryptosporidium spp.) and TPI gene (Giardia spp.) were carried out for detection of intestinal protozoa. Overall prevalence of 6 species of intestinal parasites was 68% in sled dogs (73/108). In 51 samples the eggs of a single species of helminth were detected (47%), two nematode species were detected in 13%, three species of nematodes were found in two dogs. The most prevalent helminths were the hookworms Uncinaria/Ancylostoma-identified in 36% of kennels, and in 34% of sled dogs. Toxocara eggs were detected in 36% of kennels, in 17% of dogs. Trichuris sp. eggs were found in 20% of kennels (5/25), in 13% of dogs. Cysts/oocysts of intestinal protozoa were detected in 31% of sled dogs. The most prevalent was Giardia spp. infection-in 54% of kennels [13/24], in 28% of dogs. Cryptosporidium spp. infections were identified in 37.5% of kennels [9/24], in 13% of dogs. Two sequenced Giardia isolates presented 100% homology with G. intestinalis Assemblage C isolate (AY228641.1), specific for dogs. A range of factors was shown to affect the prevalence of intestinal parasites in sled dogs. The highest prevalence of parasites was found among dogs from large kennels (housing >3 dogs), in dogs less than 2 years old, and in kennels, where prophylactic treatment was carried out 1-4 times a year. The present study has demonstrated a high prevalence of intestinal parasites in working sled dogs in Poland, including the zoonotic human pathogens Toxocara or Cryptosporidium.

Cell morphology and formal description of Ergobibamus cyprinoides n. g., n. sp., another Carpediemonas-like relative of diplomonads.

About 20 new isolates of Carpediemonas-like organisms (CLOs) have been reported since 2006. Small subunit rRNA gene phylogenies divide CLOs into six major clades: four contain described exemplars (i.e. Carpediemonas, Dysnectes, Hicanonectes, and Kipferlia), but two include only undescribed organisms. Here we describe a representative of one of these latter clades as Ergobibamus cyprinoides n. g., n. sp., and catalogue its ultrastructure. Ergobibamus cyprinoides is a bean-shaped biflagellated cell, 7-11.5 µm long, with a conspicuous groove. Instead of classical mitochondria there are cristae-lacking rounded organelles 300-400 nm in diameter. The posterior flagellum has a broad ventral vane and small dorsal vane. There are normally four basal bodies, two non-flagellated. There is one anterior root (AR), containing six microtubules. The posterior flagellar apparatus follows the "typical excavate" pattern of a splitting right root supported by fibres "I,""B," and "A," a "composite" fibre, a singlet root, and a left root (LR) with a "C" fibre. The B fibre originates against the LR--a synapomorphy of the taxon Fornicata--supporting the assignation of Ergobibamus to Fornicata, along with diplomonads, retortamonads, and other CLOs. Distinctive features of E. cyprinoides include the complexity of the AR, which is intermediate between Hicanonectes, and Carpediemonas and Dysnectes, and a dorsal extension of the C fibre.