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.

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.

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.

Retortamonads from vertebrate hosts share features of anaerobic metabolism and pre-adaptations to parasitism with diplomonads.

Although the mitochondria of extant eukaryotes share a single origin, functionally these organelles diversified to a great extent, reflecting lifestyles of the organisms that host them. In anaerobic protists of the group Metamonada, mitochondria are present in reduced forms (also termed hydrogenosomes or mitosomes) and a complete loss of mitochondrion in Monocercomonoides exilis (Metamonada:Preaxostyla) has also been reported. Within metamonads, retortamonads from the gastrointestinal tract of vertebrates form a sister group to parasitic diplomonads (e.g. Giardia and Spironucleus) and have also been hypothesized to completely lack mitochondria. We obtained transcriptomic data from Retortamonas dobelli and R. caviae and searched for enzymes of the core metabolism as well as mitochondrion- and parasitism-related proteins. Our results indicate that retortamonads have a streamlined metabolism lacking pathways for metabolites they are probably capable of obtaining from prey bacteria or their environment, reminiscent of the biochemical arrangement in other metamonads. Retortamonads were surprisingly found do encode homologs of components of Giardia's remarkable ventral disk, as well as homologs of regulatory NEK kinases and secreted lytic enzymes known for involvement in host colonization by Giardia. These can be considered pre-adaptations of these intestinal microorganisms to parasitism. Furthermore, we found traces of the mitochondrial metabolism represented by iron­sulfur cluster assembly subunits, subunits of mitochondrial translocation and chaperone machinery and, importantly, [FeFe]­hydrogenases and hydrogenase maturases (HydE, HydF and HydG). Altogether, our results strongly suggest that a remnant mitochondrion is still present.

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.

The Phylogenetic Position of Spironucleus sp. (Diplomonadida: Hexamitidae) from the Intestine of Chinese Sleeper Perccottus glenii Dybowski, 1877 (Actinopterygii: Odontobutidae).

INTRODUCTION: The Chinese (Amur) sleeper (Perccottus glenii Dybowski, 1877) (Actinopterygii: Odontobutidae) is a freshwater fish species with high invasive potential. Diplomonads have been detected in the intestines of Chinese sleepers using light microscopy. AIM: The aim of this study was to identify the diplomonads in Chinese sleepers using molecular-genetic methods. MATERIALS AND METHODS: The fish used in this analysis were caught in the following bodies of water in Russia between 2014 and 2016: Lake Dolgoe, the floodplain of the Ingoda River (Amur River basin), the Tsna River (the Oka River basin), and the littoral of the Kotlin Island (Gulf of Finland). Partial sequences of small subunit rRNA genes were obtained for the intestinal diplomonads of Chinese sleeper. RESULTS: The analysis of all sequenced samples revealed the presence of Spironucleus salmonis Moore, 1922; other Spironucleus species were not found in the sampled fish. With 82% probability, the sampled sequences of diplomonads from Chinese sleeper formed a separate cluster in the clade of S. salmonis on the phylogenetic tree. CONCLUSION: This is the first record of S. salmonis in fish in the family Odontobutidae.

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.

Proximity Staining Using Enzymatic Protein Tagging in Diplomonads.

The diplomonads are a group of understudied eukaryotic flagellates whose most prominent member is the human pathogen Giardia intestinalis Methods commonly used in other eukaryotic model systems often require special optimization in diplomonads due to the highly derived character of their cell biology. We have optimized a proximity labeling protocol using pea ascorbate peroxidase (APEX) as a reporter for transmission electron microscopy (TEM) to enable the study of ultrastructural cellular details in diplomonads. Currently available TEM-compatible tags require light-induced activation (1, 2) or are inactive in many cellular compartments (3), while ascorbate peroxidase has not been shown to have those limitations. Here, we have optimized the in vivo activities of two versions of pea ascorbate peroxidase (APXW41F and APEX) using the diplomonad fish parasite Spironucleus salmonicida, a relative of G. intestinalis We exploited the well-known peroxidase substrates, Amplex UltraRed and 3,3'-diaminobenzidine (DAB), to validate the activity of the two tags and argue that APEX is the most stable version to use in Spironucleus salmonicida Next, we fused APEX to proteins with established localization to evaluate the activity of APEX in different cellular compartments of the diplomonad cell and used Amplex UltraRed as well as antibodies along with superresolution microscopy to confirm the protein-APEX localization. The ultrastructural details of protein-APEX fusions were determined by TEM, and we observed marker activity in all cellular compartments tested when using the DAB substrate. Finally, we show that the optimized conditions established for S. salmonicida can be used in the related diplomonad G. intestinalisIMPORTANCE The function of many proteins is intrinsically related to their cellular location. Novel methods for ascertainment of the ultrastructural location of proteins have been introduced in recent years, but their implementation in protists has so far not been readily realized. Here, we present an optimized proximity labeling protocol using the APEX system in the salmon pathogen Spironucleus salmonicida This protocol was also applicable to the human pathogen Giardia intestinalis Both organisms required extraneous addition of hemin to the growth medium to enable detectable peroxidase activity. Further, we saw no inherent limitation in labeling efficiency coupled to the cellular compartment, as evident with some other proximity labeling systems. We anticipate that the APEX proximity labeling system might offer a great resource to establish the ultrastructural localization of proteins across genetically tractable protists but might require organism-specific labeling conditions.

Swarming and Aggregation in the Parasitic Diplomonad Flagellate Spironucleus vortens.

Pathogenicity, evolutionary history, and unusual cell organization of diplomonads are well known, particularly for Giardia and Spironucleus; however, behavior of these aerotolerant anaerobes is largely unknown. Addressing this deficit, we studied behavior of the piscine diplomonad Spironucleus vortens (ATCC 50386) in in vitro culture. Spironucleus vortens trophozoites from Angelfish, Pterophyllum scalare, were maintained axenically in modified liver digest, yeast extract, and iron (LYI) medium, at 22 °C in the dark, and subcultured weekly. Cultures were monitored every 1-2 d, by removing an aliquot, and loading cells into a hemocytometer chamber, or onto a regular microscope slide. We observed three distinct swimming behaviors: (i) spontaneous formation of swarms, reaching 200 µm in diameter, persisting for up to several min in situ, (ii) directional movement of the swarm, via collective motility, and (iii) independent swimming of trophozoites to form a band (aggregation), presumably at the location of optimal environmental conditions. These behaviors have not previously been reported in Spironucleus. The observation that flagellate motility can change, from individual self-propulsion to complex collective swarming motility, prompts us to advocate S. vortens as a new model for study of group behavioral dynamics, complementing emerging studies of collective swimming in flagellated bacteria.

Exploring the cellular uptake and localisation of phosphorescent rhenium fac-tricarbonyl metallosurfactants as a function of lipophilicity.

A systematic study of the cellular uptake of emissive complexes as a function of their lipophilicity is presented. Here a series of amphiphilic rhenium fac-tricarbonyl bisimine complexes bearing axial substituted imidazole or thiazole ligands, [Re(bpy)(CO)3(ImCnHm)]+ {n = 1 m = 3 (1+), n = 4 m = 9 (2+), n = 8 m = 17 (3+), n = 12 m = 25 (4+), n = 16 m = 33 (5+), n = 2 m = 3 (6+); bpy = 2,2'-bipyridine, Im = imidazole} and [Re(bpy)(CO)3(L)]+ {L = 1-mesitylimidazole, ImMes (7+), 4,5-dimethylthiazole, dmt (8+) and 4-methyl-5-thiazole-ethanol, mte (9+)} is reported. The X-ray crystal structures of 2+, 8+ and 9+ confirm the geometry and expected distribution of ligands and indicated that the plane of the imidazole/thiazole ring is approximately parallel to the long axis of the bipy ligand. Luminescence studies revealed excellent properties for their use in cell imaging with visible excitation and broad emission profiles. Their uptake in two distinct species has been examined by fluorescence imaging of the diplomonad fish parasite Spironucleus vortens (S. vortens) and rod-shaped yeast Schizosaccharomyces pombe (Schiz. pombe) as a function of their lipophilicity. The uptake of the complexes was highest for the more lipophilic 2+-5+ in both S. vortens and Schiz. pombe in which the long alkyl chain aids in crossing bilipid membranes. However, the increased lipophilicity of longer chains also resulted in greater toxicity. Localisation over the whole cell varied with differing alkyl chain lengths with complex 2+ preferentially locating to the nucleus of S. vortens, 3+ showing enhanced nuclear partitioning in Schiz. pombe, and 4+ for the remaining cell wall bound in the case of S. vortens. Interestingly, complexes of intermediate lipophilicity such as 7+ and 8+ showed reasonable uptake, proved to be non-toxic, and were capable of crossing exterior cell walls and localising in the organelles of the cells.

Comparative Ultrastructure of Fornicate Excavates, Including a Novel Free-living Relative of Diplomonads: Aduncisulcus paluster gen. et sp. nov.

The Fornicata (Excavata) is a group of microbial eukaryotes consisting of both free-living lineages (e.g., Carpediemonas) and parasitic lineages (e.g. Giardia and Retortamonas) that share several molecular and ultrastructural traits. Carpediemonas-like organisms (CLOs) are free-living lineages that diverged early within the Fornicata, making them important for inferring the early evolutionary history of the group. Molecular phylogenetic analyses of free-living fornicates, including sequences from environmental PCR surveys, have demonstrated that CLOs form six different lineages. Representatives from five of these lineages have been studied at the ultrastructural level. The sixth lineage has been labeled "CL2" but has yet to be described with ultrastructural data. Improved understanding of CL2 is expected to help elucidate character evolution within the Fornicata. Therefore, we comprehensively characterized CL2 (NY0171) in order to understand the ultrastructural traits in this lineage, especially the organization of the microtubular root system (i.e., the flagellar apparatus). CL2 shared several morphological features with other fornicates, including reduced mitochondria and an arched B fiber bridging flagellar roots 1 and 2. The molecular phylogenetic position combined with some distinctive ultrastructural traits (e.g., a curved ventral groove) in CL2 required us to establish a new genus and species, Aduncisulcus paluster gen. et sp. nov.

Arginine deiminase pathway enzymes: evolutionary history in metamonads and other eukaryotes.

BACKGROUND: Multiple prokaryotic lineages use the arginine deiminase (ADI) pathway for anaerobic energy production by arginine degradation. The distribution of this pathway among eukaryotes has been thought to be very limited, with only two specialized groups living in low oxygen environments (Parabasalia and Diplomonadida) known to possess the complete set of all three enzymes. We have performed an extensive survey of available sequence data in order to map the distribution of these enzymes among eukaryotes and to reconstruct their phylogenies. RESULTS: We have found genes for the complete pathway in almost all examined representatives of Metamonada, the anaerobic protist group that includes parabasalids and diplomonads. Phylogenetic analyses indicate the presence of the complete pathway in the last common ancestor of metamonads and heterologous transformation experiments suggest its cytosolic localization in the metamonad ancestor. Outside Metamonada, the complete pathway occurs rarely, nevertheless, it was found in representatives of most major eukaryotic clades. CONCLUSIONS: Phylogenetic relationships of complete pathways are consistent with the presence of the Archaea-derived ADI pathway in the last common ancestor of all eukaryotes, although other evolutionary scenarios remain possible. The presence of the incomplete set of enzymes is relatively common among eukaryotes and it may be related to the fact that these enzymes are involved in other cellular processes, such as the ornithine-urea cycle. Single protein phylogenies suggest that the evolutionary history of all three enzymes has been shaped by frequent gene losses and horizontal transfers, which may sometimes be connected with their diverse roles in cellular metabolism.

Pathogenesis of mixed infection by Spironucleus sp. and Citrobacter freundii in freshwater angelfish Pterophyllum scalare.

The present study was carried out to identify and describe the pathology of the freshwater angelfish Pterophyllum scalare during chronic mortality in an in-door aquaculture system. Scraping of the integument and gills and the collection of intestinal contents to search for external and internal parasites were performed. Kidneys were collected aseptically for the microbiological analysis and the isolates were subjected to antibiotics to test for susceptibility. Subsequently, necropsy for macroscopic assessment and collection of internal organs for histopathology were performed. The fish exhibited lethargy, lip tumor, hemorrhage and liver granuloma. No ectoparasites were diagnosed. Endoparasites of the genus Spironucleus were found in large numbers in the intestine of the affected fish. In the microbiological analysis, Citrobacter freundii was isolated from the kidney and identified by colony PCR. This bacterium showed susceptibility to three of the eight antibiotics evaluated: ciprofloxacin, cefoxitin and tetracycline. For the pathological analysis, liver and spleen granulomas were present. In the intestinal tissue, a large and unusual amount of mast cells and their free granules were described and discussed in detail. The present study showed that mast cells play an important role during the chronic infection of freshwater angelfish.

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.

The redox-active drug metronidazole and thiol-depleting garlic compounds act synergistically in the protist parasite Spironucleus vortens.

Spironucleus vortens is a protozoan parasite associated with significant mortalities in the freshwater angelfish, Pterophyllum scalare. Control of this parasite is especially problematic due to restrictions on the use of the drug of choice, metronidazole (MTZ), on fish farms. Use of garlic (Allium sativum) is undergoing a renaissance following experimental validations of its antimicrobial efficiency. Ajoene ((E,Z)-4,5,9-trithiadodeca-1,6,11-triene 9-oxide), is a stable transformation product of allicin, the primary biologically active component of garlic. In the current study, an ajoene oil crude extract had a minimum inhibitory concentration (MIC) of 40µg/ml against S. vortens. GC-MS and NMR spectroscopy revealed this ajoene extract contained a mixture of the (E) and (Z)-ajoene isomers along with diallyl disulphide (DADS) and diallyl trisulphide (DATS). The only component of the ajoene crude oil found to substantially inhibit S. vortens growth by optical density monitoring (Bioscreen C Reader) was (Z)-ajoene (MIC 16µg/ml). Ajoene oil acted in synergy with MTZ in vitro, reducing the individual MIC of this drug (4µg/ml) by 16-fold, and that of ajoene oil by 200-fold with a fractional inhibitory concentration (FIC) index of 0.263. This synergistic interaction was confirmed in vivo. S. vortens-infected Pterophyllum scalare angelfish dosed orally with 0.5% (v/w) MTZ combined with 0.05% (v/w) ajoene displayed a significant reduction in faecal trophozoite count, whilst those fed on 0.5% MTZ flakes (half the recommended oral dose) alone did not. This study demonstrates for the first time the synergistic interaction between the synthetic drug MTZ and natural ajoene oil both in vitro and in vivo. Future work should evaluate the potential synergy of ajoene and MTZ against MTZ-resistant bacteria and protists.

Treatment of diplomonad intestinal parasites with magnesium sulphate at a commercial rainbow trout (Oncorhynchus mykiss) facility.

Rainbow trout (average weight of 2 g) in fresh water experienced high mortality and were infected with a diplomonad intestinal parasite. Tanks of fish experienced an immediate reduction in mortality after an in-feed treatment with 3% Epsom salts for 2 d. Treatments had to be applied several times, but in each case there was a similar reduction in mortality.

Traitement des parasites intestinaux diplomonades à l'aide de sulfate de magnésium dans une installation commerciale de truites arc-en-ciel(Oncorhynchus mykiss). Des truites arc-en-ciel (poids moyen de 2 g) élevées en eau douce ont connu une mortalité élevée et ont été infectées par un parasite intestinal diplomonade. Une réduction immédiate de la mortalité a été observée dans les bassins de poissons après un traitement dans l'alimentation de 3 % de sel d'Epsom pendant 2 jours. Les traitements ont dû être appliqués plusieurs fois, mais, dans chaque instance, il s'est produit une réduction semblable de la mortalité.(Traduit par Isabelle Vallières).

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.

Motility of the diplomonad fish parasite Spironucleus vortens through thixotropic solid media.

Investigation of a series of nutrient-supplemented thixotropic gels at successive dilutions that impede the trajectories of a highly vigorous motile flagellated protist, Spironucleus vortens, provides insights into both its swimming characteristics and a means for its immobilization. The progress of movement of this organism through the solidified growth medium was monitored by the in situ reductive production of a formazan chromophore from a dissolved tetrazolium salt. The physical properties of the gels were measured using an Anton Paar rheometer. The test parameters and measurements included: angular frequency, complex viscosity, complex shear modulus, shear rate and rotational recovery. These rheological characteristics affected the forward velocity of the organism through the gels, during and after multiple resetting, information potentially useful for determination of the dynamic characteristics of flagellar movement and propulsion rates of the organism. Application to separation of single cells, individuals of distinct sizes or the differing species from mixed cultures of motile and non-motile organisms or less actively swimming species was evident. These applications can be used when isolating the parasite from the intestinal contents of its host or from faecal pellets.