Susceptible Rainbow Trout Research Articles

Characterisation of sRNAs enriched in outer membrane vesicles of pathogenic Flavobacterium psychrophilum causing Bacterial Cold Water Disease in rainbow trout.

Flavobacterium psychrophilum (Fp) causes Bacterial Cold Water Disease in salmonids. During host-pathogen interactions, gram-negative bacteria, such as Fp, release external membrane vesicles (OMVs) harbouring cargos, such as DNA, RNA and virulence factors. This study aimed to characterise the potential role of the OMVs' small RNAs (sRNAs) in the Fp-rainbow trout host-pathogen interactions. sRNAs carried within OMVs were isolated from Fp. RNA-Seq datasets from whole-cell Fp and their isolated OMVs indicated substantial enrichment of specific sRNAs in the OMVs compared to the parent cell. Many of the OMV-packaged sRNAs were located in the pathogenicity islands of Fp. Conservation of sRNAs in 65 strains with variable degrees of virulence was reported. Dual RNA-Seq of host and pathogen transcriptomes on day 5 post-infection of Fp -resistant and -susceptible rainbow trout genetic lines revealed correlated expression of OMV-packaged sRNAs and their predicted host's immune gene targets. In vitro, treatment of the rainbow trout epithelial cell line RTgill-W1 with OMVs showed signs of cytotoxicity accompanied by dynamic changes in the expression of host genes when profiled 24 h following treatment. The OMV-treated cells, similar to the Fp -resistant fish, showed downregulated expression of the suppressor of cytokine signalling 1 (SOCS1) gene, suggesting induction of phagosomal maturation. Other signs of modulating the host gene expression following OMV-treatment include favouring elements from the phagocytic, endocytic and antigen presentation pathways in addition to HSP70, HSP90 and cochaperone proteins, which provide evidence for a potential role of OMVs in boosting the host immune response. In conclusion, the study identified novel microbial targets and inherent characteristics of OMVs that could open up new avenues of treatment and prevention of fish infections.

Plasma proteomic profiling of bacterial cold water disease-resistant and -susceptible rainbow trout lines and biomarker discovery.

Genetic variation for disease resistance is present in salmonid fish; however, the molecular basis is poorly understood, and biomarkers of disease susceptibility/resistance are unavailable. Previously, we selected a line of rainbow trout for high survival following standardized challenge with Flavobacterium psychrophilum (Fp), the causative agent of bacterial cold water disease. The resistant line (ARS-Fp-R) exhibits over 60 percentage points higher survival compared to a reference susceptible line (ARS-Fp-S). To gain insight into the differential host response between genetic lines, we compared the plasma proteomes from day 6 after intramuscular challenge. Pooled plasma from unhandled, PBS-injected, and Fp-injected groups were simultaneously analyzed using a TMT 6-plex label, and the relative abundance of 513 proteins was determined. Data are available via ProteomeXchange, with identifier PXD041308, and the relative protein abundance values were compared to mRNA measured from a prior, whole-body RNA-seq dataset. Our results identified a subset of differentially abundant intracellular proteins was identified, including troponin and myosin, which were not transcriptionally regulated, suggesting that these proteins were released into plasma following pathogen-induced tissue damage. A separate subset of high-abundance, secreted proteins were transcriptionally regulated in infected fish. The highest differentially expressed protein was a C1q family member (designated complement C1q-like protein 3; C1q-LP3) that was upregulated over 20-fold in the infected susceptible line while only modestly upregulated, 1.8-fold, in the infected resistant line. Validation of biomarkers was performed using immunoassays and C1q-LP3, skeletal muscle troponin C, cathelcidin 2, haptoglobin, leptin, and growth and differentiation factor 15 exhibited elevated concentration in susceptible line plasma. Complement factor H-like 1 exhibited higher abundance in the resistant line compared to the susceptible line in both control and challenged fish and thus was a baseline differentiator between lines. C1q-LP3 and STNC were elevated in Atlantic salmon plasma following experimental challenge with Fp. In summary, these findings further the understanding of the differential host response to Fp and identifies salmonid biomarkers that may have use for genetic line evaluation and on-farm health monitoring.

Genome-wide identification of antisense lncRNAs and their association with susceptibility to Flavobacterium psychrophilum in rainbow trout.

Eukaryotic genomes encode long noncoding natural antisense transcripts (lncNATs) that have been increasingly recognized as regulatory members of gene expression. Recently, we identified a few antisense transcripts correlating in expression with immune-related genes. However, a systematic genome-wide analysis of lncNATs in rainbow trout is lacking. This study used 134 RNA-Seq datasets from five different projects to identify antisense transcripts. A total of 13,503 lncNATs were identified genome-wide. About 75% of lncNATs showed multiple exons compared to 36.5% of the intergenic lncRNAs. RNA-Seq datasets from resistant, control, and susceptible rainbow trout genetic lines with significant differences in survival rate following Flavobacterium psychrophilum (Fp) infection were analyzed to investigate the potential role of the lncNATs during infection. Twenty-four pairwise comparisons between the different genetic lines, infectious status, and time points revealed 581 differentially expressed (DE) lncNATs and 179 differentially used exons (DUEs). Most of the DE lncNATs strongly and positively correlated in expression with their corresponding sense transcripts across 24 RNA-Seq datasets. LncNATs complementary to genes related to immunity, muscle contraction, proteolysis, and iron/heme metabolism were DE following infection. LncNATs complementary to hemolysis-related genes were DE in the resistant fish compared to susceptible fish on day 5 post-infection, suggesting enhanced clearance of free hemoglobin (Hb) and heme and increased erythropoiesis. LncNATs complementary to hepcidin, a master negative regulator of the plasma iron concentration, were the most downregulated lncNATs on day 5 of bacterial infection in the resistant fish. Ninety-four DE lncNAT, including five complementary to hepcidin, are located within 26 QTL regions previously identified in association with bacterial cold water disease (BCWD) in rainbow trout. Collectively, lncNATs are involved in the molecular architecture of fish immunity and should be further investigated for potential applications in genomic selection and genetic manipulation in aquaculture.

Resistant and susceptible rainbow trout (Oncorhynchus mykiss) lines show distinctive immune response to Lactococcus garvieae

Lactococcosis is one of the main bacterial diseases affecting rainbow trout (Oncorhynchus mykiss), with significant economic and sanitary repercussion. Vaccination and antibiotic treatments are commonly used to prevent and control the infection outbreaks; however, these strategies have some drawbacks including limited coverage, handling costs, induction of antibiotic resistance and chemical residues in the environment. Selective breeding programs represent a promising complementary approach for increasing fish disease resistance in commercial farms and some immunological parameters may be tentatively used as indirect indicators for this purpose. The present study investigated for the first time some innate and adaptive immune responses in two groups of rainbow trout derived from selected lines (susceptible and resistant) showing a different “in field” phenotypical resistance to Yersinia ruckeri, Flavobacterium branchiophilum, F. psychrophilum, and Ichthyophthirius multifiliis, after an immersion-dilution based exposure to Lactococcus garvieae carried out in controlled experimental conditions. Twenty-six resistant and twenty-six susceptible female rainbow trout (mean body weight 80 g, 9 months aged, F5 generation) were obtained from an intensive farm considered L. garvieae free and were exposed to the pathogen. Moreover, 10 resistant and 10 susceptible fish were used as uninfected controls. After 5 days, blood and tissue samples were collected for immunological analyses. A significantly higher serum and mucus lysozyme activity was recorded in resistant rainbow trout compared to susceptible fish (P ≤ 0.05), both before and after exposure to L. garvieae. Similarly, respiratory burst activity of head kidney leukocytes resulted more intense in resistant fish (P ≤ 0.05), suggesting that phagocytes could more quickly activate their microbicidal mechanisms to counteract the bacterial spread. Resistant group displayed also an up-regulation of immunoglobulins M (IgM), major histocompatibility complex II (MHC-II) and interleukin 8 (IL-8) gene expression (P ≤ 0.05) and a significantly higher blood lymphocytes count (P ≤ 0.05), highlighting their potential better ability to trigger the recruitment of defensive cells and the initiation of specific immune processes such as antigen presentation to CD4+ T lymphocytes and IgM synthesis. The results herein presented might be useful for the identification of immunological markers to be used as indirect indicators in rainbow trout selective breeding programs.

Distinctive site preference of the fish parasite Myxobolus cerebralis (Cnidaria, Myxozoa) during host invasion.

Here, we experimentally studied the site preference of Myxobolus cerebralis, one of the most pathogenic myxozoan (Cnidaria, Myxozoa) fish parasites, which causes whirling disease in salmonids. Parasite invasion was examined in three fish species with various susceptibility levels: the type host brown trout, the highly susceptible rainbow trout, and the non-susceptible gibel carp, in which parasite spores do not develop. We investigated the first two hours of fish invasion, and measured the site preference of triactinomyxons (TAMs) during attachment and penetration of fish in three body parts (gills, fins, skin). Infection prevalence and intensity were estimated using a species-specific nested PCR, optimised in the present study. The highest infection prevalence was detected in the most susceptible fish species, rainbow trout. Interestingly, higher prevalence was observed in gibel carp than in the type host, brown trout (95.2% vs. 85.7%). Considering body locations, remarkable differences were detected in infection intensities. The highest intensity was observed in fins, whereas skin was the least infected body part in every fish species examined. Infection prevalence and intensity did not differ significantly among fish species. Thus, we confirmed that M. cerebralis TAMs cannot discern fish species. Furthermore, we proved experimentally that fish fin is significantly more attractive to fish-invading parasite TAMs than gills or skin.

Kinetics of local and systemic immune cell responses in whirling disease infection and resistance in rainbow trout

BackgroundWhirling disease (WD), caused by the myxozoan parasite Myxobolus cerebralis, is responsible for high mortalities in rainbow trout hatcheries and natural populations. To elucidate how resistant and susceptible rainbow trout strains respond to early invasion, a well-established model of WD was used to demonstrate the kinetics of local and systemic immune responses in two rainbow trout strains, the susceptible American Trout Lodge (TL) and the more resistant German Hofer strain (HO).MethodsParasite load and cellular immune responses were compared across several time points after M. cerebralis exposure to elucidate the kinetics of immune cells in resistant and susceptible rainbow trout in response to early invasion. In the course of the 20 days following exposure, leukocyte kinetics was monitored by flow cytometry in the caudal fin (CF), head kidney (HK) and spleen (SP). For the analysis of the leukocyte composition, cells were stained using a set of monoclonal antibodies with known specificity for distinct subpopulations of rainbow trout leukocytes.ResultsExperiments indicated general increases of CF, HK and SP myeloid cells, while decreases of B cells and T cells in the SP and HK were observed at several time points in the TL strain. On the other hand, in the HO strain, increases of T cells were dominant in CF, HK and SP at multiple time points. The differences between HO and TL were most distinct at 2, 4, 12 and 48 hours post-exposure (hpe) as well as at 4 days post-exposure (dpe), with the vast majority of innate immune response cells having higher values in the susceptible TL strain. Alteration of the leukocyte populations with augmented local cellular responses and excessive immune reactions likely lead to subsequent host tissue damage and supports parasite invasion and development in TL.ConclusionsThe findings of this study highlight the significance of effective local and systemic immune reaction and indicate proper activation of T lymphocytes critical for host resistance during M. cerebralis infection. The present study provides insights into the cellular basis of protective immune responses against M. cerebralis and can help us to elucidate the mechanisms underlying the variation in resistance to WD.

Genetic and transcriptomic analyses provide new insights on the early antiviral response to VHSV in resistant and susceptible rainbow trout

BackgroundThe viral hemorrhagic septicemia virus (VHSV) is a major threat for salmonid farming and for wild fish populations worldwide. Previous studies have highlighted the importance of innate factors regulated by a major quantitative trait locus (QTL) for the natural resistance to waterborne VHSV infection in rainbow trout. The aim of this study was to analyze the early transcriptomic response to VHSV inoculation in cell lines derived from previously described resistant and susceptible homozygous isogenic lines of rainbow trout to obtain insights into the molecular mechanisms responsible for the resistance to the viral infection.ResultsWe first confirmed the presence of the major QTL in a backcross involving a highly resistant fish isogenic line (B57) and a highly susceptible one (A22), and were able to define the confidence interval of the QTL and to identify its precise position. We extended the definition of the QTL since it controls not only resistance to waterborne infection but also the kinetics of mortality after intra-peritoneal injection. Deep sequencing of the transcriptome of B57 and A22 derived cell lines exposed to inactivated VHSV showed a stronger response to virus inoculation in the resistant background. In line with our previous observations, an early and strong induction of interferon and interferon-stimulated genes was correlated with the resistance to VHSV, highlighting the major role of innate immune factors in natural trout resistance to the virus. Interestingly, major factors of the antiviral innate immunity were much more expressed in naive B57 cells compared to naive A22 cells, which likely contributes to the ability of B57 to mount a fast antiviral response after viral infection. These observations were further extended by the identification of several innate immune-related genes localized close to the QTL area on the rainbow trout genome.ConclusionsTaken together, our results improve our knowledge in virus-host interactions in vertebrates and provide novel insights in the molecular mechanisms explaining the resistance to VHSV in rainbow trout. Our data also provide a collection of potential markers for resistance and susceptibility of rainbow trout to VHSV infection.

Serum IgM, MH class IIβ genotype and respiratory burst activity do not differ between rainbow trout families displaying resistance or susceptibility to the coldwater pathogen, Flavobacterium psychrophilum

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD) is a significant threat to global aquaculture. True to its name, BCWD tends to occur at temperatures between 8 and 12°C and presents as a systemic disease with characteristic skin ulcerations. Juvenile rainbow trout are particularly susceptible and in these fish the condition is referred to as rainbow trout fry syndrome (RTFS). Resistance to F. psychrophilum is heritable and is not adversely correlated with the growth of fish, thus selective breeding appears to be an achievable approach to its control. The current study explores the connection between resistance to BCWD and several immunological markers. After determining resistance/susceptibility to F. psychrophilum following experimental infection in 40 full-sibling families of rainbow trout, selected families were experimentally infected with F. psychrophilum and differences in antibody production, major histocompatibility (MH) class IIβ genotype and respiratory burst activity (RBA) throughout infection were compared. Serum IgM production increased over time but significant differences between resistant and susceptible families were not observed at either 28days or 120days. Of the six families that were genotyped for MH class IIβ, there did not appear to be specific genotypes that conferred resistance or susceptibility to F. psychrophilum. Further, the RBA of both head kidney leukocytes and whole blood was not significantly different between the resistant and susceptible rainbow trout families. Although the selected immune markers did not differ based on resistance status, the RBA of head kidney leukocytes in all families studied dramatically decreased seven days after infection while total blood RBA remained constant. Day seven was also when severe symptoms and/or mortality due to BCWD was first observed, thus these results may reveal information regarding the pathogenesis of the organism. A better understanding of appropriate immune defenses could provide the basis for breeding programs to effectively combat this costly pathogen, but further study of functional immune markers particularly during the fry stage of development is required.

Evolution of interleukin-1 receptor-like 1 and its role in rainbow trout (Oncorhynchus mykiss) resistance to Flavobacterium psychrophilum

Abstract Rainbow trout exhibit extensive phenotypic variation in innate disease resistance and we have divergently selected lines with either increased or reduced survival following exposure to the gram-negative bacterium, Flavobacterium psychrophilum (Fp). Following five generations of selection, gene expression was analyzed between the resistant and susceptible lines by RNA-seq and RT-PCR in either whole body or spleen tissue samples. Lower base-line expression of interleukin-1 receptor-like 1 (il1rl1) was identified in susceptible line fish as well as decreased expression in response to infection. Genomic analyses identified three tandem, il1rl1 genes on chromosome 3 located within a previously identified, bacterial cold water disease QTL. Here, we report the genomic organization and evolution of the three il1rl1 genes, and develop gene specific assays to measure the expression of the individual il1rl1 genes in susceptible and resistant rainbow trout lines. Sequence homology between these three putative genes is approximately 95%. Each gene contains a signal peptide, three IG/IG-like domains, a transmembrane region and a TIR domain. Two il1rl1 genes and one pseudogene are present in Atlantic salmon (Salmo salar) genome with 90–94% sequence identity. Phylogenetic analyses suggest that the salmonid il1rl1 genes expanded by tandem duplication within the salmonid linage. Our working model is that il1rl1 gene copy number or differential expression may contribute to specific disease resistance.

Temporal expression patterns of rainbow trout immune-related genes in response to Myxobolus cerebralis exposure

Infection of salmonids by the myxozoan parasite Myxobolus cerebralis can cause whirling disease, which is responsible for high mortalities in rainbow trout hatcheries and natural populations in the United States. Although considerable research has provided insight into disease pathology, host invasion, and inheritance patterns of resistance, the causal genetic variants and molecular mechanisms underlying host resistance or susceptibility remain elusive. A previous study found that expression changes of specific metallothionein genes following M. cerebralis infection are implicated in whirling disease resistance. The present study examines the dynamic transcriptional response to infection of several upstream regulators of the metallothionein gene family (IL-1β, KLF2, STAT3, STAT5), along with innate immune response genes (IFN-γ, IRF1 and iNOS). Pathogen loads and gene expression were compared across multiple time points after M. cerebralis exposure to elucidate how resistant and susceptible rainbow trout strains transcriptionally respond to early invasion. IL-1β, IFN-γ, IRF1, and iNOS all showed increased expression following M. cerebralis exposure for one or both strains across multiple time points. The interferon-related genes IFN-γ and IRF1 had consistently increased expression in the susceptible strain in comparison to the resistant strain, likely due to a less effective initial immune response. STAT3 was the only gene with consistently increased expression in the resistant strain following infection while remaining unchanged in the susceptible strain. Given its pleiotropic effects on immune response, STAT3 is an excellent candidate for future research of whirling disease resistance mechanisms.

Detection of Myxobolus cerebralis in the Lower Deschutes River Basin, Oregon

AbstractThe Deschutes River, located in central Oregon, is a tributary of the Columbia River. The migration of anadromous fish into the upper Deschutes River basin was blocked in the late 1960s by a series of dams constituting the Pelton Round Butte Hydroelectric Project (PRB). Plans to reintroduce anadromous fish runs above the PRB are complicated by concerns over the introduction of Myxobolus cerebralis (the agent of whirling disease), which is carried into the lower Deschutes River by adult salmon that stray on their return to enzootic regions of the upper Columbia River basin. To develop a fish passage strategy that minimizes the risk of parasite introduction into the upper Deschutes River basin, it is important to determine whether establishment of the M. cerebralis life cycle in waters below the PRB has occurred. Fry of susceptible rainbow trout Oncorhynchus mykiss and steelhead (anadromous rainbow trout) were exposed as sentinel fish at various locations in the Deschutes River and its tributaries between 1998 and 2007 to detect M. cerebralis. The parasite was found periodically at low prevalence by polymerase chain reaction (PCR) or quantitative PCR assay in fish held at several locations. However, in 2007, the parasite was detected from fish exposed in Trout Creek based on visual examination (pepsin–trypsin digest preparations and histological sections) and molecular methods. This result confirms that M. cerebralis is established in the Deschutes River basin below the PRB. Continued introduction of the parasite into the river system below the PRB via stray hatchery summer steelhead was also documented. These findings have implications for managing movement of M. cerebralis within the watershed.

A vaccinia-virus-free reverse genetics system for infectious hematopoietic necrosis virus

Reverse genetics system is a powerful tool to study the function of a particular gene. The currently available reverse genetics system for Novirhabdovirus is based on vaccinia-driven T7 RNA polymerase expression. An improved system for the recovery of infectious hematopoietic necrosis virus (IHNV) was developed which utilizes cellular RNA polymerase II machinery for transcription. A full-length cDNA clone of IHNV, flanked by hammerhead ribozyme and hepatitis delta ribozyme sequences, was assembled in an expression plasmid under the control of a cytomegalovirus promoter. Transfection of this full-length plasmid along with the supporting plasmids (N, P, NV and L) into epithelioma papulosum cyprini cells resulted in the recovery of a viable recombinant IHNV (rIHNV). The authenticity of rIHNV was confirmed by the presence of restriction sites introduced artificially in the genome. A recombinant IHNV expressing a foreign gene – enhanced green fluorescent protein – was also recovered. The recovered IHNVs showed similar growth characteristics as the parental IHNV in cell cultures. Challenge of susceptible rainbow trout with wild-type IHNV and rIHNV induced clinical disease signs indistinguishable from the parental strain and produced mortality. Thus, a vaccinia-virus-free reverse genetics system described for IHNV is applicable for the recovery of any Novirhabdovirus, which will facilitate studies on viral pathogenesis and the design of new generation of viral vectored vaccines.

Effects of Ceratomyxa shasta dose on a susceptible strain of rainbow trout and comparatively resistant Chinook and coho salmon

Ceratomyxa shasta infects salmon and trout, causing ceratomyxosis, a disease characterized by parasite proliferation in the intestine and death. We used laboratory challenges to investigate the infective dose for 3 fish species: a susceptible strain of rainbow trout Oncorhynchus mykiss and comparatively resistant Chinook O. tshawytscha and coho salmon O. kisutch. For susceptible rainbow trout, we determined the outcome of infection under conditions of varying parasite dose, fish size, and parasite concentration. A single actinospore was sufficient to cause a lethal infection in susceptible rainbow trout. The mean days to death (MDD) did not significantly decrease among doses causing 100% prevalence, indicating a minimum time required for parasites to replicate to a fatal level. When dose was constant, but delivered in a higher parasite concentration, higher infection prevalence and mortality resulted. One actinospore fish(-1) caused 57% infection and mortality in fish challenged in 0.5 1 of water, whereas 10 spores fish(-1) resulted in an average of 49% infection and mortality in 1 l challenges. This effect is most likely due to a higher encounter rate in the smaller water volume. Neither infection prevalence nor MDD was significantly different between large trout (84.9 g) and small trout (6.3 g). Chinook salmon did not become infected even when challenged with 5000 actinospores. One fatal infection occurred in coho salmon challenged with 1000 actinospores. This study confirms that even low doses of C. shasta cause severe infection in highly susceptible fish, describes the dose response on MDD, and demonstrates that parasite concentration influences infection prevalence.

In vivo exposure of susceptible and non-susceptible fish species to Myxobolus cerebralis actinospores reveals non-specific invasion behaviour

Due to controversial previous results, it has been unclear whether Myxobolus cerebralis (Myxosporea, Myxozoa) is able to specifically choose a salmonid host by selective attachment and penetration. Using a novel in vivo tracking assay that employs fluorescence staining of actinospore sporoplasms, we demonstrate a lack of host specificity of M. cerebralis actinospores during their initial invasion reactions. Fish were experimentally exposed to stained actinospores that could be detected as emitted sporoplasms in and on the fish integument of skin, gills and fins. There were no significant differences in the number of actively emerging sporoplasms found on epithelial surfaces of a susceptible and resistant strain of rainbow trout and common carp after experimental exposure. Numbers of parasite attachment rates to carp and trout gill tissue were also assessed using quantitative real-time PCR (qPCR). This method demonstrated that actinospore reactivity rate was not affected by the staining procedure. An even higher number of parasite stages was detected in carp than in trout gills. Subsequently, the ability of carp to lower the infection severity of susceptible rainbow trout by trapping the parasites under natural conditions was also investigated. Myxospore load was significantly reduced in hosts infected with actinospore samples that were preincubated with live carp. These results indicate the possibility of biological disturbance to the life cycle of the parasite in the wild by interceptor fish species as one measure to prevent whirling disease.

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(-1), 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.

Potential for Dissemination of the Nonnative Salmonid Parasite Myxobolus cerebralis in Alaska

Myxobolus cerebralis, the myxozoan parasite responsible for whirling disease in salmonids, was first introduced into the United States in 1958 and has since spread across the country, causing severe declines in wild trout populations in the intermountain western United States. The recent detection of the parasite in Alaska is further evidence of the species' capability to invade and colonize new habitat. This study qualitatively assesses the risk of further spread and establishment of M. cerebralis in Alaska. We examine four potential routes of dissemination: human movement of fish, natural dispersal by salmonid predators and straying salmon, recreational activities, and commercial seafood processing. Potential for establishment was evaluated by examining water temperatures, spatial and temporal overlap of hosts, and the distribution and genetic composition of the oligochaete host, Tubifex tubifex. The most likely pathway of M. cerebralis transport in Alaska is human movement of fish by stocking. The extent of M. cerebralis infection in Alaskan salmonid populations is unknown, but if the parasite becomes dispersed, conditions are appropriate for establishment and propagation of the parasite life cycle in areas of south-central Alaska. The probability of further establishment is greatest in Ship Creek, where the abundance of susceptible T. tubifex, the presence of susceptible rainbow trout Oncorhynchus mykiss, and the proximity of this system to the known area of infection make conditions particularly suitable for spread of the parasite.

Discovery of genes implicated in whirling disease infection and resistance in rainbow trout using genome-wide expression profiling

BackgroundWhirling disease, caused by the pathogen Myxobolus cerebralis, afflicts several salmonid species. Rainbow trout are particularly susceptible and may suffer high mortality rates. The disease is persistent and spreading in hatcheries and natural waters of several countries, including the U.S.A., and the economic losses attributed to whirling disease are substantial. In this study, genome-wide expression profiling using cDNA microarrays was conducted for resistant Hofer and susceptible Trout Lodge rainbow trout strains following pathogen exposure with the primary objective of identifying specific genes implicated in whirling disease resistance.ResultsSeveral genes were significantly up-regulated in skin following pathogen exposure for both the resistant and susceptible rainbow trout strains. For both strains, response to infection appears to be linked with the interferon system. Expression profiles for three genes identified with microarrays were confirmed with qRT-PCR. Ubiquitin-like protein 1 was up-regulated over 100 fold and interferon regulating factor 1 was up-regulated over 15 fold following pathogen exposure for both strains. Expression of metallothionein B, which has known roles in inflammation and immune response, was up-regulated over 5 fold in the resistant Hofer strain but was unchanged in the susceptible Trout Lodge strain following pathogen exposure.ConclusionThe present study has provided an initial view into the genetic basis underlying immune response and resistance of rainbow trout to the whirling disease parasite. The identified genes have allowed us to gain insight into the molecular mechanisms implicated in salmonid immune response and resistance to whirling disease infection.

Sequence analysis of OmNramp α and quantitative expression of Nramp homologues in different trout strains after infection with Myxobolus cerebralis

Salmonid whirling disease caused by the metazoan parasite Myxobolus cerebralis is an ongoing problem in wild and farmed rainbow trout Oncorhynchus mykiss populations. Rainbow trout from different strains vary in susceptibility to the parasite. Identification of underlying mechanisms could be a starting point for improved control of the disease. We conducted infection trials using 2 rainbow trout strains and brown trout Salmo trutta fario, a species not susceptible to the parasite, to investigate host immune response and resistance mechanisms. We compared expression levels of 2 natural resistance-associated macrophage proteins (Nramp alpha and beta) after infection with M. cerebralis. Total RNA was extracted from skin, muscle, kidney, head and spinal column, and gene expression was quantified by real-time PCR. Significant decreases in expression of both genes were observed at different time points in the infected susceptible rainbow trout compared to the non-infected group. Furthermore, the OmNramp alpha (O. mykiss natural resistance-associated macrophage protein alpha) sequences in 2 resistant and 1 non-resistant rainbow trout strain were analysed and compared for sequence aberrations.

Relative quantification of immune-regulatory genes in two rainbow trout strains, Oncorhynchus mykiss, after exposure to Myxobolus cerebralis, the causative agent of whirling disease

Expression of immune-regulatory genes that code for cyclooxigenase-2 (COX-2), transforming growth factor beta (TGF-beta), and two isoforms of interleukin-1beta (IL-1beta1 and IL-1beta2) was studied in susceptible and non-susceptible rainbow trout strains for 200 days after exposure to Myxobolus cerebralis. Expression of COX-2, IL-1beta1, and IL-1beta2 increased 5 min post exposure (p.e.) and was always more elevated in the susceptible strain than in the non-susceptible strain. In both strains, expression of COX-2 returned to the control level within a few hours p.e. Expression of IL-1beta1 and IL-1beta2 showed two elevated waves in both strains until 4 days p.e. Expression of TGF-beta in the non-susceptible strain was elevated at nearly all sampling points, but was decreased in the susceptible strain until up-regulation between 4 and 20 days p.e.; TGF-beta was the only gene where the expression in the non-susceptible strain was more elevated than in the susceptible strain. Rainbow trout of the non-susceptible strain appeared to resist infection by M. cerebralis with only minor transcriptional regulation of the genes investigated. Increased transcriptions of genes in the susceptible strain may be the result of an inability to antagonize the infection.

Spatial and Temporal Occurrence of the Salmonid Parasite Ceratomyxa shasta in the Oregon–California Klamath River Basin

AbstractThe parasite Ceratomyxa shasta has been implicated as a significant source of salmonid mortality in the lower Klamath River, California (i.e., below Iron Gate dam). A study of the prevalence of C. shasta and its geographic and temporal distribution throughout the Klamath River basin was conducted to determine when and where juvenile salmonids encounter lethal parasite doses. Susceptible rainbow trout Oncorhynchus mykiss were exposed to C. shasta 3–4 d at seven locations in the Klamath River between Beaver Creek and Keno Reservoir in April, June, July, September, and November 2003. Individuals from a Klamath River strain of fall Chinook salmon O. tshawytscha were held in three locations in the upper Klamath River in April, June, and July. In June 2004, rainbow trout were exposed to the parasite for 4 d at 18 locations from Klamath Lake to the mouth of the Klamath River, including several major spawning tributaries; one exposure occurred in the lower Klamath River. Rainbow trout mortality due to infection for groups exposed in the upper Klamath River was lower (<8.0%) and delayed (mean time to death, 40–110 d) in comparison with that in groups exposed in the lower Klamath River (>98%; mean time to death, 33–36 d). Experimental fall Chinook salmon did not become infected in the upper Klamath River, but infection was detected in Chinook salmon exposed in the lower Klamath River, nearly 50% of these succumbing to infection. These dramatic differences in mortality between the upper and lower Klamath River could not be explained by differences in water temperatures during exposure and are probably a result of differences in infectious dose. Lack of infection in groups exposed in tributaries supports the hypothesis that the parasite life cycle and the invertebrate host are largely confined to the main‐stem Klamath River.