Development of monoclonal antibodies against the myxosporean, ceratomyxa shasta, and characterization of the salmonid host response

Monoclonal antibodies (Mabs) were produced against the myxosporean parasite, Ceratomyxa shasta, and were characterized by indirect immunofluorescent techniques, western blot analysis, and immunoelectron microscopy. The resulting Mabs reacted specifically with the trophozoite and sporoblast stages of the parasite, but not with the mature spore, indicating that certain antigens are stage-specific. Two of the Mabs produced also reacted with trout white blood cells as evidenced by indirect fluorescent antibody techniques (IFAT) and Western blot analysis. Further characterization of these cross-reacting antibodies showed that they were directed against carbohydrate epitopes on both the parasite and trout immunoglobulin heavy chain. One hybridoma produced antibodies of high specificity for prespore stages of C. shasta. Ultrastructural analysis of the parasite using immunogold labeling showed that the Mab specifically bound antigen located in the cytoplasm of the trophozoites. The C. shasta-specific antibodies produced by monoclonal technology, standard histological methods, and scanning electron microscopy were used to investigate the host response of Sa lmo gairdneri against infection by the parasite. In infected fish the parasite was first observed in the mucosal epithelium of the posterior intestine using IFAT. As the trophozoites proliferated, the infection spread anteriorally and into the submucosa, muscularis, and serosa of the intestine. Hepatic tissues were also infected early in the disease process. In the terminal stages of the infection, trophozoites had penetrated the stomach, pyloric caeca, pancreas, and adipose tissues and were also observed in the blood and kidney. In fish refractory to infection, the parasite was observed in the lumen of the intestine. Examination of infection in a moribound fish by scanning electron microscopy showed extensive destruction of the mucosal folds of the posterior intestine. Although a vigorous tissue response was evident in infected tissues, trout antibodies specific for the parasite were not detected by either Western blot analysis or IFAT. The distribution of the infective stage of C. shasta in the Columbia River basin was also investigated. The range of the parasite was extended in the Columbia River to its confluence with the Snake River and in the Snake River to Oxbow Dam

Myxozoan Adhesion and Virulence: Ceratonova shasta on the Move

Motility factors are fundamental for parasite invasion, migration, proliferation and immune evasion and thus can influence parasitic disease pathogenesis and virulence. Salmonid enteronecrosis is caused by a myxozoan (Phylum Cnidarian) parasite, Ceratonova shasta. Three parasite genotypes (0, I, II) occur, with varying degrees of virulence in its host, making it a good model for examining the role of motility in virulence. We compare C. shasta cell motility between genotypes and describe how the cellular protrusions interact with the host. We support these observations with motility gene expression analyses. C. shasta stages can move by single or combined used of filopodia, lamellipodia and blebs, with different behaviors such as static adhesion, crawling or blebbing, some previously unobserved in myxozoans. C. shasta stages showed high flexibility of switching between different morphotypes, suggesting a high capacity to adapt to their microenvironment. Exposure to fibronectin showed that C. shasta stages have extraordinary adhesive affinities to glycoprotein components of the extracellular matrix (ECM). When comparing C. shasta genotypes 0 (low virulence, no mortality) and IIR (high virulence, high mortality) infections in rainbow trout, major differences were observed with regard to their migration to the target organ, gene expression patterns and proliferation rate in the host. IIR is characterized by rapid multiplication and fast amoeboid bleb-based migration to the gut, where adhesion (mediated by integrin-β and talin), ECM disruption and virulent systemic dispersion of the parasite causes massive pathology. Genotype 0 is characterized by low proliferation rates, slow directional and early adhesive migration and localized, non-destructive development in the gut. We conclude that parasite adhesion drives virulence in C. shasta and that effectors, such as integrins, reveal themselves as attractive therapeutic targets in a group of parasites for which no effective treatments are known.Fil: Alama Bermejo, Gema. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. Oregon State University; Estados Unidos. Universidad Nacional del Comahue. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". - Provincia de Río Negro. Ministerio de Agricultura, Ganadería y Pesca. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Centro Nacional Patagónico. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni"; ArgentinaFil: Holzer, Astrid Sybylle. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Bartholomew, Jerri. Oregon State University; Estados Unido

The effects of water velocity on the Ceratomyxa shasta infectious cycle

Ceratomyxa shasta is a myxozoan parasite identified as a contributor to salmon mortality in the Klamath River, USA. The parasite has a complex life cycle involving a freshwater polychaete, Manayunkia speciosa and a salmonid. As part of ongoing research on how environmental parameters influence parasite establishment and replication, we designed a laboratory experiment to examine the effect of water flow (velocity) on completion of the C. shasta infectious cycle. The experiment tested the effect of two water velocities, 0.05 and 0.01 m/s, on survival and infection of M. speciosa as well as transmission to susceptible rainbow trout and comparatively resistant Klamath River Chinook salmon. The faster water velocity facilitated the greatest polychaete densities, but the lowest polychaete infection prevalence. Rainbow trout became infected in all treatments, but at the slower velocity had a shorter mean day to death, indicating a higher infectious dose. Infection was not detected in Chinook salmon even at a dose estimated to be as high as 80,000 actinospores per fish. The higher water velocity resulted in lower C. shasta infection prevalence in M. speciosa and decreased infection severity in fish. Another outcome of our experiment is the description of a system for maintaining and infecting M. speciosa in the laboratory.This is an author's peer-reviewed final manuscript, as accepted by the publisher. The published article is copyrighted by the author(s) and published by John Wiley & Sons Ltd. The article can be found at: http://onlinelibrary.wiley.com/journal/10.1111/%28ISSN%291365-2761.Keywords: Ceratomyxa shasta, Water velocity, Salmonids, Manayunkia speciosa, Infection, Parasite ecolog

Predicted Redistribution of Ceratomyxa shasta Genotypes with Salmonid Passage in the Deschutes River, Oregon

Aseries of dams on the Deschutes River, Oregon, act as migration barriers that segregate the river system into upper and lower basins. Proposed fish passage between basins would reunite populations of native potamodromous fish and allow anadromous fish of Deschutes River origin access to the upper basin. We assessed the potential redistribution of host-species-specific genotypes (O, I, II, III) of the myxozoan parasite Ceratomyxa shasta that could occur with fish passage and examined the influence of nonnative fish on genotype composition. To determine the present distribution of the parasite genotypes, we exposed eight salmonid species—three native and five stocked for sport fishing—in present and predicted anadromous salmonid habitats. We monitored fish for infection by C. shasta and sequenced a section of the parasite ribosomal DNA gene from fish and water samples to determine parasite genotype. Genotype O was present in both upper and lower basins and detected only in steelhead Oncorhynchus mykiss. Genotype I was spatially limited to the lower basin, isolated predominately from Chinook salmon O. tshawytscha, and lethal for this species only. Genotype II was detected in both basins and in multiple species, but only as a minor component of the infection. Genotype III was also present in both basins, had a wide host range, and caused mortality in native steelhead and multiple nonnative species. Atlantic salmon Salmo salar and kokanee O. nerka were the least susceptible to infection by any genotype of C. shasta. Our findings confirmed the host-specific patterns of C. shasta infections and indicated that passage of Chinook salmon would probably spread genotype I into the upper Deschutes River basin, but with little risk to native salmonid populations.Keywords: Chinook, Basin, Myxozoa, Rainbow trout, Mortality, Assay, Infection, Myxosporean parasite, Host, Oncorhynchus tshawytsch

A new mitochondrial gene order in the banded cusk-eel Raneya brasiliensis (Actinopterygii, Ophidiiformes)

The complete mitochondrial genome of the banded cusk-eel, Raneya brasilensis (Kaup, 1856), was obtained using next-generation sequencing approaches. The genome sequence was 16,881 bp and exhibited a novel gene order for a vertebrate. Specifically, the WANCY and the nd6–D-loop regions were re-ordered, supporting the hypothesis that these two regions are hotspots for gene rearrangements in Actinopterygii. Phylogenetic reconstructions confirmed that R. brasiliensis is nested within Ophidiiformes. Mitochondrial genomes are required from additional ophidiins to determine whether the gene rearrangements that we observed are specific to the genus Raneya or to the subfamily Ophidiinae.Fil: Fromm, Amir. Tel Aviv University George S. Wise Faculty Of Life Sciences; IsraelFil: Atkinson, Stephen D.. State University of Oregon; Estados UnidosFil: Alama Bermejo, Gema. Universidad Nacional del Comahue. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". - Provincia de Río Negro. Ministerio de Agricultura, Ganadería y Pesca. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Centro Nacional Patagónico. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni"; ArgentinaFil: Cartwright, Paulyn. University of Kansas; Estados UnidosFil: Bartholomew, Jerri. State University of Oregon; Estados UnidosFil: Huchon, Dorothée. Tel Aviv University George S. Wise Faculty Of Life Sciences; Israe

Relationship Between Temperature and Ceratomyxa shasta–Induced Mortality In Klamath River Salmonids

Water temperature influences almost every biological and physiological process of salmon, including disease resistance. In the Klamath River (California), current thermal conditions are considered sub-optimal for juvenile salmon. In addition to borderline temperatures, these fish must contend with the myxozoan parasite Ceratomyxa shasta, a significant cause of juvenile salmonid mortality in this system. This paper presents 2 studies, conducted from 2007 to 2010, that examine thermal effects on C. shasta induced mortality in native Klamath River Chinook (Oncorhynchus tshawytscha) and coho (Oncorhynchus kisutch) salmon. In each study, fish were exposed to C. shasta in the Klamath River for 72 hr and then reared in the laboratory under temperature-controlled conditions. The first study analyzed data collected from a multi-year monitoring project to asses the influence of elevated temperatures on parasite-induced mortality during the spring/summer migration period. The second study compared disease progression in both species at 4 temperatures (13, 15, 18, and 21 C) representative of spring/summer migration conditions. Both studies demonstrated that elevated water temperatures consistently resulted in higher mortality and faster mean days to death. However, analysis of data from the multi-year monitoring showed that the magnitude of this effect varied among years and was more closely associated with parasite density than with temperature. Also, there was a difference in the timing of peak mortality between species; Chinook incurred high mortalities in 2008 and 2009, whereas coho was greatest in 2007 and 2008. As neither temperature nor parasite density can be easily manipulated, management strategies should focus on disrupting the overlap of this parasite and its obligate hosts to improve emigration success and survival of juvenile salmon in the Klamath River.Keywords: Rainbow trout, Water temperature, Myxozoa, Myxosporean parasite, Coho salmon, Steelhead, California, Assay, Chinook salmo

Dam Removal and Implications for Fish Health: Ceratomyxa shasta in the Williamson River, Oregon, USA

The removal of dams on a river is one potential tool for the ecological restoration of native salmonid fishes. However, the removal of barriers also introduces risks, such as the introduction of fish pathogens into previously isolated populations. The proposed removal of four dams on the Klamath River, Oregon–California, provides an opportunity for examining the disease risks associated with dam removal. A salmonid pathogen endemic to the region, Ceratomyxa shasta, is responsible for high mortality in juvenile Chinook salmon Oncorhynchus tshawytscha and coho salmon O. kisutch below the dams. Above the dams, parasite densities are lower and not implicated in salmonid mortality, except in the Williamson River tributary, where high parasite densities raise concerns over the restoration of anadromous fish that are likely to take advantage of spawning habitat in that river. In the current study, baseline information on parasite density, distribution, and genotype composition in the Williamson River was gathered to determine how salmonid reintroduction might be affected by parasite dynamics. Assay of water samples highlighted two areas of high parasite density: between the mouth of the Williamson River and the confluence of the Sprague River tributary, and above the Spring Creek confluence. Despite these high parasite densities, mortality did not occur in sentinel coho or Chinook salmon. Genetic analyses of parasites from water samples and infected fish demonstrated that C. shasta genotype II was dominant and was associated with stocked nonnative rainbow trout O. mykiss. The absence of pathogenicity of this parasite genotype for Chinook and coho salmon suggests that reintroduction plans will not initially be adversely affected by the high parasite densities in the Williamson River. However, following dam removal, returning adult salmon will transport parasite genotypes present below the dams upstream. These genotypes are likely to become established and may reach densities that could affect juvenile Chinook and coho salmon.This is the publisher’s final pdf. The published article is copyrighted by Taylor & Francis for the American Fisheries Society and can be found at: http://www.tandfonline.com/toc/ujfm20/current

Transcriptome-Wide Comparisons and Virulence Gene Polymorphisms of Host-Associated Genotypes of the Cnidarian Parasite Ceratonova shasta in Salmonids

Ceratonova shasta is an important myxozoan pathogen affecting the health of salmonid fishes in the Pacific Northwest of North America. Ceratonova shasta exists as a complex of host-specific genotypes, some with low to moderate virulence, and one that causes a profound, lethal infection in susceptible hosts. High throughput sequencing methods are powerful tools for discovering the genetic basis of these host/virulence differences, but deep sequencing of myxozoans has been challenging due to extremely fast molecular evolution of this group, yielding strongly divergent sequences that are difficult to identify, and unavoidable host contamination. We designed and optimized different bioinformatic pipelines to address these challenges. We obtained a unique set of comprehensive, host-free myxozoan RNA-seq data from C. shasta genotypes of varying virulence from different salmonid hosts. Analyses of transcriptome-wide genetic distances and maximum likelihood multigene phylogenies elucidated the evolutionary relationship between lineages and demonstrated the limited resolution of the established Internal Transcribed Spacer marker for C. shasta genotype identification, as this marker fails to differentiate between biologically distinct genotype II lineages from coho salmon and rainbow trout. We further analyzed the data sets based on polymorphisms in two gene groups related to virulence: cell migration and proteolytic enzymes including their inhibitors. The developed single-nucleotide polymorphism-calling pipeline identified polymorphisms between genotypes and demonstrated that variations in both motility and protease genes were associated with different levels of virulence of C. shasta in its salmonid hosts. The prospective use of proteolytic enzymes as promising candidates for targeted interventions against myxozoans in aquaculture is discussed. We developed host-free transcriptomes of a myxozoan model organism from strains that exhibited different degrees of virulence, as a unique source of data that will foster functional gene analyses and serve as a base for the development of potential therapeutics for efficient control of these parasites.Fil: Alama Bermejo, Gema. Universidad Nacional del Comahue. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". - Provincia de Río Negro. Ministerio de Agricultura, Ganadería y Pesca. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni". Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet Centro Nacional Patagónico. Centro de Investigación Aplicada y Transferencia Tecnológica en Recursos Marinos "Almirante Storni"; Argentina. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. State University of Oregon; Estados UnidosFil: Meyer, Eli. Oregon State University; Estados UnidosFil: Atkinson, Stephen D.. Oregon State University; Estados UnidosFil: Holzer, Astrid S.. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Wiśniewska, Monika M.. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República ChecaFil: Kolísko, Martin. Academy of Sciences of the Czech Republic. Biology Centre. Institute of Parasitology; República Checa. University of South Bohemia; República ChecaFil: Bartholomew, Jerri. Oregon State University; Estados Unido

The cnidarian parasite Ceratonova shasta utilizes inherited and recruited venom-like compounds during infection

Background Cnidarians are the most ancient venomous organisms. They store a cocktail of venom proteins inside unique stinging organelles called nematocysts. When a cnidarian encounters chemical and physical cues from a potential threat or prey animal, the nematocyst is triggered and fires a harpoon-like tubule to penetrate and inject venom into the prey. Nematocysts are present in all Cnidaria, including the morphologically simple Myxozoa, which are a speciose group of microscopic, spore-forming, obligate parasites of fish and invertebrates. Rather than predation or defense, myxozoans use nematocysts for adhesion to hosts, but the involvement of venom in this process is poorly understood. Recent work shows some myxozoans have a reduced repertoire of venom-like compounds (VLCs) relative to free-living cnidarians, however the function of these proteins is not known. Methods We searched for VLCs in the nematocyst proteome and a time-series infection transcriptome of Ceratonova shasta, a myxozoan parasite of salmonid fish. We used four parallel approaches to detect VLCs: BLAST and HMMER searches to preexisting cnidarian venom datasets, the machine learning tool ToxClassifier, and structural modeling of nematocyst proteomes. Sequences that scored positive by at least three methods were considered VLCs. We then mapped their time-series expressions in the fish host and analyzed their phylogenetic relatedness to sequences from other venomous animals. Results We identified eight VLCs, all of which have closely related sequences in other myxozoan datasets, suggesting a conserved venom profile across Myxozoa, and an overall reduction in venom diversity relative to free-living cnidarians. Expression of the VLCs over the 3-week fish infection varied considerably: three sequences were most expressed at one day post-exposure in the fish’s gills; whereas expression of the other five VLCs peaked at 21 days post-exposure in the intestines, coinciding with the formation of mature parasite spores with nematocysts. Expression of VLC genes early in infection, prior to the development of nematocysts, suggests venoms in C. shasta have been repurposed to facilitate parasite invasion and proliferation within the host. Molecular phylogenetics suggested some VLCs were inherited from a cnidarian ancestor, whereas others were more closely related to sequences from venomous non-Cnidarian organisms and thus may have gained qualities of venom components via convergent evolution. The presence of VLCs and their differential expression during parasite infection enrich the concept of what functions a “venom” can have and represent targets for designing therapeutics against myxozoan infections