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Erik Sterud - One of the best experts on this subject based on the ideXlab platform.
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phylogenetic analysis of the ssu rrna gene from the piscine diplomonad Spironucleus torosus diplomonadida hexamitinae
Folia Parasitologica, 2007Co-Authors: Anders Jorgensen, Anders Alfjorden, Kristin Henriksen, Erik SterudAbstract:Previous studies have recorded Spironucleus torosus Poynton et Morrison, 1990 from several species of gadoid fishes, including the only freshwater gadoid, the burbot Lota lota (L.). Two morphologically different isolates of S. torosus have been described (elongate and pyriform). Both have been found in saltwater, while only the elongate has been found in freshwater. To address the conspecificity of the two morphs of S. torosus, and to identify the source of S. torosus in burbot in Norway, we have sequenced the small subunit ribosomal RNA (SSU rRNA) gene from 43 isolates of S. torosus from six species of gadoid fishes sampled at 15 localities in Norway, Sweden and the Baltic Sea. Phylogenetic analyses of the SSU rRNA gene sequence data recovered two major clades, one containing mainly isolates from burbot, while the other contained isolates from marine gadoid fishes only. The genetic distance (based on 25 nucleotide substitutions in 789 base pairs) separating the two assemblages was not large enough to consider the two groups separate species. Spironucleus torosus isolated from burbot displayed limited genetic variation in the small subunit ribosomal RNA (SSU rRNA) gene along the post-Pleistocene migration route of its host. The present study is the first report of S. torosus in tusk Brosme brosme (Ascanius), whiting Merlangius merlangus (L.), and fourbeard rockling Enchelyopus cimbrius (L.).
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phylogeny of Spironucleus eopharyngia diplomonadida hexamitinae
Protist, 2007Co-Authors: Anders Jorgensen, Erik SterudAbstract:Diplomonad flagellates from the genera Spironucleus and Hexamita (Hexamitinae) inhabit the digestive tract of a variety of animal hosts, some as important pathogens, and others as commensals. Species descriptions of many diplomonads have been based on light microscopy only, causing many taxa to be misidentified. Presently, electron microscopy is considered the appropriate tool for description of diplomonads. The existence of morphologically very similar genotypes/species has in addition prompted the need for molecular tools to resolve the true identity of many diplomonad taxa. To further explore the relationship within the Hexamitinae we have sequenced most of the SSU rRNA gene from Spironucleus torosa isolated from Atlantic cod, Spironucleus meleagridis isolated from turkey, Spironucleus vortens isolated from ide and Hexamita nelsoni isolated from oyster. Phylogenetic analyses recovered three non-monophyletic Spironucleus clades that may have originated separately in the sea, in freshwater or on land. Spironucleus torosa was identified as a sister taxon to Spironucleus barkhanus and Spironucleus salmonicida. Spironucleus vortens from ide appeared to be genetically very different from Spironucleus vortens isolated from angelfish.
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ultrastructure and molecular diagnosis of Spironucleus salmonis diplomonadida from rainbow trout oncorhynchus mykiss in germany
Diseases of Aquatic Organisms, 2007Co-Authors: Reza Saghari M Fard, Anders Jorgensen, Erik Sterud, Wilfrid Bleiss, Sarah L PoyntonAbstract:Diplomonad flagellates infect a wide range of fish hosts in aquaculture and in the wild in North America, Asia and Europe. Intestinal diplomonad infection in juvenile farmed trout can be associated with morbidity and mortality, and in Germany, diplomonads in trout are commonly reported, and yet are poorly characterised. We therefore undertook a comprehensive study of diplomonads from German rainbow trout Oncorhynchus mykiss, using scanning and transmission electron microscopy, and sequencing of the small subunit (ssu) rRNA gene. The diplomonad was identified as Spironucleus salmonis, formerly reported from Germany as Hexamita salmonis. Our new surface morphology studies showed that the cell surface was unadorned and a caudal projection was present. Transmission electron microscopy facilitated new observations of functional morphology, including vacuoles discharging from the body surface, and multi-lobed apices of the nuclei. We suggest the lobes form, via hydrostatic pressure on the nucleoplasm, in response to the beat of the anterior-medial flagella. The lobes serve to intertwine the nuclei, providing stability in the region of the cell exposed to internal mechanical stress. The ssu rRNA gene sequence clearly distinguished S. salmonis from S. barkhanus, S. salmonicida, and S. vortens from fish, and can be used for identification purposes. A 1405 bp sequence of the ssu rRNA gene from S. salmonis was obtained and included in a phylogenetic analysis of a selection of closely related diplomonads, showing that S. salmonis was recovered as sister taxon to S. vortens.
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the marine pathogenic genotype of Spironucleus barkhanus from farmed salmonids redescribed as Spironucleus salmonicida n sp
Journal of Eukaryotic Microbiology, 2006Co-Authors: Anders Jorgensen, Erik SterudAbstract:: There are two genotypes of the diplomonad Spironucleus barkhanus. Based on sequence data from the small subunit ribosomal RNA gene the conspecificity of these two genotypes has been questioned. Therefore, we have sampled Spironucleus from 27 fish, representing 14 populations, five species, and four genera. Partial nucleotide sequences from the three genes; small subunit ribosomal DNA, glutamate dehydrogenase 1 and alpha-tubulin were compared. The pathogenic isolates of S. barkhanus, which causes systemic spironucleosis in Atlantic salmon, Chinook salmon, and Arctic charr, all farmed in sea water, were genetically very different from the commensal isolate found in wild freshwater populations of Arctic charr and grayling. The genetic distances between the genotypes were of the same magnitude as those separating species of Giardia. Based on these genetic and ecological data, the pathogenic genotype from farmed salmonids is described as a new species, Spironucleus salmonicida n. sp. Scanning and transmission electron microscopy showed no specific morphological or ultrastructural features distinguishing S. salmonicida n. sp. from S. barkhanus. The present study clearly demonstrates the value of applying genetics in identification of Spironucleus species. Phylogenetic analyses that included the isolates of S. salmonicida n. sp. did not change the phylogenetic relationship within the genus Spironucleus.
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ssu rrna gene sequence reveals two genotypes of Spironucleus barkhanus diplomonadida from farmed and wild arctic charr salvelinus alpinus
Diseases of Aquatic Organisms, 2004Co-Authors: Anders Jorgensen, Erik SterudAbstract:Spironucleus barkhanus isolated from the blood of Arctic charr Salvelinus alpinus from a marine fish farm were genetically compared with S. barkhanus isolated from the gall bladder of wild Arctic charr. The wild Arctic charr were caught in the lake used as the water source for the hatchery from which the farmed fish originated. Sequencing of the small subunit ribosomal RNA gene (SSU rDNA) from these 2 populations showed that the isolates obtained from farmed and wild Arctic charr were only 92.7 % similar. Based on the sequence differences between these isolates, it is concluded that the parasites isolated from the farmed fish have not been transmitted from wild Arctic charr in the hatchery's fresh water source. It is therefore most likely that the farmed fish were infected by S. barkhanus after they were transferred to seawater. S. barkhanus isolated from diseased farmed Arctic charr were 99.7% similar to the isolates obtained from diseased farmed Chinook (Canada) and Atlantic salmon (Norway). The high degree of sequence similarity between S. barkhanus from farmed Arctic charr, Chinook and Atlantic salmon indicates that systemic spironucleosis may be caused by specific strains/variants of this parasite. The genetic differences between the isolates of farmed and wild fish are of such magnitude that their conspecificity should be questioned.
Anders Jorgensen - One of the best experts on this subject based on the ideXlab platform.
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Spironucleus species economically important fish pathogens and enigmatic single celled eukaryotes
Journal of Aquaculture Research and Development, 2013Co-Authors: Catrin Ffion Williams, Anders Jorgensen, Sarah L Poynton, Coralie Ode Millet, David Lloyd, Joanne CableAbstract:Diplomonads are aerotolerant anaerobic, binucleate flagellates, which are commonly found in the intestinal tract of wild and farmed fish. Of the diplomonad genera, Spironucleus, composed of opportunistic pathogens, poses the greatest threat to aquaculture. Immunocompromised hosts or fish without acquired immunity are thought to be more susceptible to parasitism by these otherwise commensal agents. Accumulation of flagellates along the intestinal tract often leads to systemic Spironucleosis causing high mortality of both ornamental and food fish in aquaculture. The life cycle of these piscine diplomonads is direct, consisting of a motile, parasitic trophozoite and a resilient encysted stage, which facilitates water-borne transmission. Confusion in the nomenclature, as well as numerous reassignments of taxa, hampers our understanding of host range and geographical distribution of fish diplomonads. Accurate identification requires transmission electron microscopy to characterise intricate ultrastructural features. Additionally, sequencing of the small subunit ribosomal RNA gene allows identification of cryptic Spironucleus spp. In vitro culture provides a convenient source of flagellates for biochemical and physiological research, allowing the identification of novel parasite-specific molecular pathways such as H2 production within Spironucleus sp. This provides insight into the pathogenicity of these organisms and offers potential new targets for chemotherapy. Restrictions on the administration of the current drug of choice, metronidazole, in aquacultural settings, as well as reported cases of drug resistance, means that control of Spironucleosis is especially difficult. Allium sativum (garlic)-derived compounds have proven highly effective at inhibiting parasite growth in vitro, showing great potential as a novel alternative therapy in the treatment of Spironucleosis. Further characterisation of the biochemistry, pathogenicity and taxonomy of fish diplomonads is required in order to fully appreciate the true impact and economic consequences of Spironucleus spp. in aquaculture.
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wild arctic char salvelinus alpinus and trout salmo trutta hosts and reservoir of the salmonid pathogen Spironucleus salmonicida diplomonadida hexamitidae
Diseases of Aquatic Organisms, 2011Co-Authors: Anders Jorgensen, Kari Torp, Marie A Bjorland, Trygve T PoppeAbstract:: 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.
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large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida
BMC Genomics, 2010Co-Authors: Katarina Roxstromlindquist, Anders Jorgensen, Jon Jerlstromhultqvist, Karin Troell, Staffan G Svard, Jan AnderssonAbstract:Background Microbial eukaryotes show large variations in genome structure and content between lineages, indicating extensive flexibility over evolutionary timescales. Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments. Approximately 5,000 expressed sequence tag (EST) sequences were generated from the fish commensal Spironucleus barkhanus and compared to sequences from the morphologically indistinguishable fish parasite Spironucleus salmonicida, and other diplomonads. The ESTs were complemented with sequence variation studies in selected genes and genome size determinations.
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Large genomic differences between the morphologically indistinguishable diplomonads Spironucleus barkhanus and Spironucleus salmonicida
BMC Genomics, 2010Co-Authors: Katarina Roxström-lindquist, Anders Jorgensen, Karin Troell, Staffan G Svard, Jon Jerlström-hultqvist, Jan O AnderssonAbstract:Background Microbial eukaryotes show large variations in genome structure and content between lineages, indicating extensive flexibility over evolutionary timescales. Here we address the tempo and mode of such changes within diplomonads, flagellated protists with two nuclei found in oxygen-poor environments. Approximately 5,000 expressed sequence tag (EST) sequences were generated from the fish commensal Spironucleus barkhanus and compared to sequences from the morphologically indistinguishable fish parasite Spironucleus salmonicida , and other diplomonads. The ESTs were complemented with sequence variation studies in selected genes and genome size determinations. Results Many genes detected in S. barkhanus and S. salmonicida are absent in the human parasite Giardia intestinalis , the most intensively studied diplomonad. For example, these fish diplomonads show an extended metabolic repertoire and are able to incorporate selenocysteine into proteins. The codon usage is altered in S. barkhanus compared to S. salmonicida . Sequence variations were found between individual S. barkhanus ESTs for many, but not all, protein coding genes. Conversely, no allelic variation was found in a previous genome survey of S. salmonicida . This difference was confirmed by sequencing of genomic DNA. Up to five alleles were identified for the cloned S. barkhanus genes, and at least nineteen highly expressed S. barkhanus genes are represented by more than four alleles in the EST dataset. This could be explained by the presence of a non-clonal S. barkhanus population in the culture, by a ploidy above four, or by duplications of parts of the genome. Indeed, genome size estimations using flow cytometry indicated similar haploid genome sizes in S. salmonicida and G. intestinalis (~12 Mb), whereas the S. barkhanus genome is larger (~18 Mb). Conclusions This study indicates extensive divergent genome evolution within diplomonads. Genomic traits such as codon usage, frequency of allelic sequence variation, and genome size have changed considerably between S. barkhanus and S. salmonicida . These observations suggest that large genomic differences may accumulate in morphologically indistinguishable eukaryotic microbes.
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phylogenetic analysis of the ssu rrna gene from the piscine diplomonad Spironucleus torosus diplomonadida hexamitinae
Folia Parasitologica, 2007Co-Authors: Anders Jorgensen, Anders Alfjorden, Kristin Henriksen, Erik SterudAbstract:Previous studies have recorded Spironucleus torosus Poynton et Morrison, 1990 from several species of gadoid fishes, including the only freshwater gadoid, the burbot Lota lota (L.). Two morphologically different isolates of S. torosus have been described (elongate and pyriform). Both have been found in saltwater, while only the elongate has been found in freshwater. To address the conspecificity of the two morphs of S. torosus, and to identify the source of S. torosus in burbot in Norway, we have sequenced the small subunit ribosomal RNA (SSU rRNA) gene from 43 isolates of S. torosus from six species of gadoid fishes sampled at 15 localities in Norway, Sweden and the Baltic Sea. Phylogenetic analyses of the SSU rRNA gene sequence data recovered two major clades, one containing mainly isolates from burbot, while the other contained isolates from marine gadoid fishes only. The genetic distance (based on 25 nucleotide substitutions in 789 base pairs) separating the two assemblages was not large enough to consider the two groups separate species. Spironucleus torosus isolated from burbot displayed limited genetic variation in the small subunit ribosomal RNA (SSU rRNA) gene along the post-Pleistocene migration route of its host. The present study is the first report of S. torosus in tusk Brosme brosme (Ascanius), whiting Merlangius merlangus (L.), and fourbeard rockling Enchelyopus cimbrius (L.).
Sarah L Poynton - One of the best experts on this subject based on the ideXlab platform.
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swarming and aggregation in the parasitic diplomonad flagellate Spironucleus vortens
Journal of Eukaryotic Microbiology, 2019Co-Authors: Sarah L Poynton, Lauren Ostrenga, Kenneth W WitwerAbstract:: 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.
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Spironucleus species economically important fish pathogens and enigmatic single celled eukaryotes
Journal of Aquaculture Research and Development, 2013Co-Authors: Catrin Ffion Williams, Anders Jorgensen, Sarah L Poynton, Coralie Ode Millet, David Lloyd, Joanne CableAbstract:Diplomonads are aerotolerant anaerobic, binucleate flagellates, which are commonly found in the intestinal tract of wild and farmed fish. Of the diplomonad genera, Spironucleus, composed of opportunistic pathogens, poses the greatest threat to aquaculture. Immunocompromised hosts or fish without acquired immunity are thought to be more susceptible to parasitism by these otherwise commensal agents. Accumulation of flagellates along the intestinal tract often leads to systemic Spironucleosis causing high mortality of both ornamental and food fish in aquaculture. The life cycle of these piscine diplomonads is direct, consisting of a motile, parasitic trophozoite and a resilient encysted stage, which facilitates water-borne transmission. Confusion in the nomenclature, as well as numerous reassignments of taxa, hampers our understanding of host range and geographical distribution of fish diplomonads. Accurate identification requires transmission electron microscopy to characterise intricate ultrastructural features. Additionally, sequencing of the small subunit ribosomal RNA gene allows identification of cryptic Spironucleus spp. In vitro culture provides a convenient source of flagellates for biochemical and physiological research, allowing the identification of novel parasite-specific molecular pathways such as H2 production within Spironucleus sp. This provides insight into the pathogenicity of these organisms and offers potential new targets for chemotherapy. Restrictions on the administration of the current drug of choice, metronidazole, in aquacultural settings, as well as reported cases of drug resistance, means that control of Spironucleosis is especially difficult. Allium sativum (garlic)-derived compounds have proven highly effective at inhibiting parasite growth in vitro, showing great potential as a novel alternative therapy in the treatment of Spironucleosis. Further characterisation of the biochemistry, pathogenicity and taxonomy of fish diplomonads is required in order to fully appreciate the true impact and economic consequences of Spironucleus spp. in aquaculture.
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intestinal ph profile in rainbow trout oncorhynchus mykiss and microhabitat preference of the flagellate Spironucleus salmonis diplomonadida
Diseases of Aquatic Organisms, 2007Co-Authors: Reza Saghari M Fard, Claudia Weisheit, Sarah L PoyntonAbstract:In farmed rainbow trout Oncorhynchus mykiss, the flagellate Spironucleus salmonis (Diplomonadida) is often found in the pyloric region of the intestine. While previous in vitro studies report a pH of 7.5 to 8.0 as optimal for presumed S. salmonis, no previous in vivo studies have inves- tigated the relationship between pH and microhabitat preference. Therefore, in 698 rainbow trout (75% were 5 to 6 mo old juveniles, 10 to 20 cm total length), we recorded occurrence and density of S. salmonis, and pH, in the pyloric, anterior, middle, and posterior intestine. There were no significant differences in total length or weight between infected and uninfected fish. S. salmonis preferred the pyloric region, with occurrence and density decreasing significantly from pyloric to posterior regions. In infected fish, pH in pyloric (6.8 to 7.9, mean 7.3) and posterior regions (6.5 to 8.0, mean 7.1) was significantly lower than in anterior (6.5 to 8.5, mean 7.7) and middle (6.8 to 8.2, mean 7.7) regions; in uninfected fish, the pH profile was similar. At the individual level, 90% of infected fish and 79% of uninfected fish showed this pH profile. In the pyloric region, pH was not significantly different among uninfected fish, and fish with light, moderate, or heavy infections. Our in vivo study suggests the opti- mal pH for S. salmonis is between 7.1 and 7.5, possibly close to 7.3 (the mean in pyloric region of infected fish). We conclude that while the presence of S. salmonis reflected tolerable pH, density of infection was not correlated with pH, and thus a causal relationship between microhabitat preference and pH is unlikely.
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ultrastructure and molecular diagnosis of Spironucleus salmonis diplomonadida from rainbow trout oncorhynchus mykiss in germany
Diseases of Aquatic Organisms, 2007Co-Authors: Reza Saghari M Fard, Anders Jorgensen, Erik Sterud, Wilfrid Bleiss, Sarah L PoyntonAbstract:Diplomonad flagellates infect a wide range of fish hosts in aquaculture and in the wild in North America, Asia and Europe. Intestinal diplomonad infection in juvenile farmed trout can be associated with morbidity and mortality, and in Germany, diplomonads in trout are commonly reported, and yet are poorly characterised. We therefore undertook a comprehensive study of diplomonads from German rainbow trout Oncorhynchus mykiss, using scanning and transmission electron microscopy, and sequencing of the small subunit (ssu) rRNA gene. The diplomonad was identified as Spironucleus salmonis, formerly reported from Germany as Hexamita salmonis. Our new surface morphology studies showed that the cell surface was unadorned and a caudal projection was present. Transmission electron microscopy facilitated new observations of functional morphology, including vacuoles discharging from the body surface, and multi-lobed apices of the nuclei. We suggest the lobes form, via hydrostatic pressure on the nucleoplasm, in response to the beat of the anterior-medial flagella. The lobes serve to intertwine the nuclei, providing stability in the region of the cell exposed to internal mechanical stress. The ssu rRNA gene sequence clearly distinguished S. salmonis from S. barkhanus, S. salmonicida, and S. vortens from fish, and can be used for identification purposes. A 1405 bp sequence of the ssu rRNA gene from S. salmonis was obtained and included in a phylogenetic analysis of a selection of closely related diplomonads, showing that S. salmonis was recovered as sister taxon to S. vortens.
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ultrastructure of Spironucleus salmonis n comb formerly octomitus salmonis sensu moore 1922 davis 1926 and hexamita salmonis sensu ferguson 1979 with a guide to Spironucleus species
Diseases of Aquatic Organisms, 2004Co-Authors: Sarah L Poynton, Reza Saghari M Fard, John Jenkins, H W FergusonAbstract:Diplomonad flagellates can be associated with significant morbidity and mortality in fishes, particularly in farmed salmonids. Diagnosis using only light microscopy is limited, and species cannot be confirmed. We therefore undertook a comprehensive transmission electron microscopy study of 20 trophozoites from the intestine of farmed juvenile rainbow trout Oncorhynchus mykiss from Northern Ireland (reported as Hexamita salmonis by Ferguson in 1979). Re-assignment to the genus Spironucleus was determined based on the anteriorly tapering and intertwined elongate nuclei, anterior-medial kinetosomes, and the presence of a flagellar pocket. At the species level we observed a tri-radiate pattern of microtubules in the opening of the asymetrical striated lamina, com- prising 3 to 5 microtubules following the edge of the flagellar pocket, 3 radiating away from the open- ing of the striated lamina, and 4 to 5 curving over the striated lamina. We observed electron-dense plaques adjacent to the kinetosomes, electron-dense bodies, numerous free ribosomes, aggregations of glycogen, bowl-shaped membranous structures, rough endoplasmic reticulum, and a novel dis- tinctive pyriform sac of densely packed free ribosomes at the posterior of the cell (8-shaped in trans- verse section, and delineated by furrowed endoplasmic reticulum). We now propose to rename H. salmonis sensu Ferguson (1979) Spironucleus salmonis. Our review of the morphology of Octomitus salmonis illustrated by Moore (1922a,b) and Davis (1926), has shown that this organism also belongs to the genus Spironucleus. We synonymise H. salmonis sensu Ferguson (1979) with O. salmonis sensu Moore (1922) and Davis (1926), and rename them S. salmonis. An expanded diagnostic guide, includ- ing new cytoplasmic elements, is presented for the 4 species of Spironucleus from fishes (S. barkhanus, S. salmonis, S. torosa and S. vortens). We recommend that type descriptions of diplo- monads be based on organisms taken directly from the host rather than from in vitro culture in order to reliably include the cytoplasmic organelles.
Staffan G Svard - One of the best experts on this subject based on the ideXlab platform.
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Proximity Staining Using Enzymatic Protein Tagging in Diplomonads.
mSphere, 2019Co-Authors: Asgeir Astvaldsson, Staffan G Svard, Kjell Hultenby, Jon Jerlström-hultqvistAbstract:ABSTRACT 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. intestinalis. IMPORTANCE 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.
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oxygen induces the expression of invasion and stress response genes in the anaerobic salmon parasite Spironucleus salmonicida
BMC Biology, 2019Co-Authors: Courtney W Stairs, Asgeir Astvaldsson, Jon Jerlstromhultqvist, Staffan G Svard, Anna Kokla, Thijs J G EttemaAbstract: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.
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dual transcriptomic analysis of Spironucleus salmonicida infected salmon cells identifies putative virulence factors and host responses
2019Co-Authors: Asgeir Astvaldsson, Courtney W Stairs, Feifei Xu, Lars Haag, Anders Alfjorden, Eva Jansson, Thijs J G Ettema, Staffan G SvardAbstract:Dual transcriptomic analysis of Spironucleus salmonicida-infected salmon cells identifies putative virulence factors and host responses
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experimental challenge of atlantic salmon salmo salar with the diplomonad parasite Spironucleus salmonicida to characterize the infection cycle
2019Co-Authors: Alfjorden Anders, Asgeir Astvaldsson, Staffan G SvardAbstract:Experimental infections were performed of Atlantic salmon (Salmo salar) from the Baltic Sea region with the Diplomonad fish parasite Spironucleus salmonicida in order to define the infection cycle, ...
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the genome of Spironucleus salmonicida highlights a fish pathogen adapted to fluctuating environments
PLOS Genetics, 2014Co-Authors: Feifei Xu, Asgeir Astvaldsson, Jon Jerlstromhultqvist, Staffan G Svard, Elin Einarsson, Jan AnderssonAbstract:Spironucleus salmonicida causes systemic infections in salmonid fish. It belongs to the group diplomonads, binucleated heterotrophic flagellates adapted to micro-aerobic environments. Recently we i ...
Trygve T Poppe - One of the best experts on this subject based on the ideXlab platform.
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wild arctic char salvelinus alpinus and trout salmo trutta hosts and reservoir of the salmonid pathogen Spironucleus salmonicida diplomonadida hexamitidae
Diseases of Aquatic Organisms, 2011Co-Authors: Anders Jorgensen, Kari Torp, Marie A Bjorland, Trygve T PoppeAbstract:: 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.
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intracellular infection with Spironucleus barkhanus diplomonadida hexamitidae in farmed arctic char salvelinus alpinus
Diseases of Aquatic Organisms, 2003Co-Authors: Erik Sterud, Trygve T Poppe, Geir BornoAbstract:A case of intracellular systemic infection with the diplomonad flagellate Spironucleus barkhanus in farmed Arctic char Salvelinus alpinus is described. The parasites were widely dissemi- nated throughout the vasculature and in most organs. Aggregates of the parasites were seen within well-defined structures regarded as host cells in capillaries and sinusoids of the liver, spleen and head kidney. Intracellular infection with Spironucleus spp. has never previously been reported. The prevalence of infection and mortality in the affected farm was low. In contrast to systemic spironu- cleosis in farmed Atlantic salmon, and despite huge numbers of flagellates in the vasculature, the tissues of the organs were remarkably unaffected. The relatively few gross and histopathological lesions may indicate that Arctic char are more tolerant to this parasite than Atlantic salmon.
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systemic spironucleosis in sea farmed atlantic salmon salmo salar caused by Spironucleus barkhanus transmitted from feral arctic char salvelinus alpinus
Diseases of Aquatic Organisms, 1998Co-Authors: Erik Sterud, Tor Atle Mo, Trygve T PoppeAbstract:Abstract A hexamitid flagellate was found in the gall bladder and intestine of Arctic char Salvelinus alpinus in northern Norway. Scanning and transmission electron microscopy showed this flagellate to be identical to Spironucleus barkhanus from grayling Thymallus thymallus and farmed Atlantic salmon Salmo salar. It is hypothesized that systemic spironucleosis in sea-caged Atlantic salmon was due to transmission of flagellates from feral char to the salmon.
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ultrastructure of Spironucleus barkhanus n sp diplomonadida hexamitidae from grayling thymallus thymallus l salmonidae and atlantic salmon salmo salar l salmonidae
Journal of Eukaryotic Microbiology, 1997Co-Authors: Erik Sterud, Tor Atle Mo, Trygve T PoppeAbstract:A hexamitid flagellate Spironucleus barkhanus n. sp., from the lumen of the gut and gall bladder of wild grayling Thymallus thymallus, and from muscle abscesses of farmed Atlantic salmon Salmo salar from Norway, is described by light, scanning and transmission electron microscopy. The flagellate was axenically cultured in trypticase, yeast extract, iron serum medium. Live trophozoites from axenic cultures incubated at 5° C, measure 11-20 X 6-14 μm. The flagellates show a typical bi-radial symmetry. Each recurrent flagellum is almost completely surrounded by a striated lamina. In the posterior end the lamina widens, appearing heart shaped in transverse section. Accompanying each recurrent flagellum are three narrow bands of microtubules, following the longitudinal groove created by the incomplete closure of the striated lamina. The recurrent flagella emerge posterio-medially through cytostome openings halfway surrounded by crescent-shaped ridges, oriented in opposite directions in the two openings. The position and adornment of the cytostome openings, and the arrangement and number of the microtubules accompanying the recurrent flagella, distinguish Spironucleus barkhanus n. sp. from previously described species of Spironucleus.
