Publications by authors named "Gregory D Wiens"

52 Publications

In Vivo Experiments Provide Evidence That Flavobacterium psychrophilum Strains Belonging to Multilocus Sequence Typing Clonal Complex ST191 Are Virulent to Rainbow Trout.

J Aquat Anim Health 2021 Sep 30;33(3):190-195. Epub 2021 Aug 30.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources and Department of Pathobiology and Diagnostic Investigation, College of Veterinary Medicine, Michigan State University, East Lansing, Michigan, 48824, USA.

Flavobacterium psychrophilum, the causative agent of bacterial coldwater disease (BCWD), causes significant economic losses worldwide, particularly in farmed Rainbow Trout Oncorhynchus mykiss. Over the last decade, multilocus sequence typing has revealed >30 clonal complexes (CCs) globally, comprised of >320 F. psychrophilum sequence types (STs). Despite the large number of CCs worldwide, CC-ST10, which is currently the largest CC affecting Rainbow Trout, has been the primary focus of F. psychrophilum virulence studies, leaving the role of other CCs as primary causes of BCWD epizootics unclear. To this end, fingerling Rainbow Trout were experimentally challenged with F. psychrophilum strains belonging to the CC now recognized as the second largest in the world (CC-ST191) alongside CC-ST10 strains. Cumulative percent mortality was 100% in 7-month-old Rainbow Trout and between 27.8% and 61.1% in 8-month-old Rainbow Trout. All examined F. psychrophilum STs were virulent to Rainbow Trout, and no significant differences in virulence between CC-ST10 and CC-ST191 were detected. Due to their wide distribution and high pathogenic potential, both CC-ST191 and CC-ST10 F. psychrophilum strains are excellent candidates for further research aimed at preventing and controlling BCWD.
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http://dx.doi.org/10.1002/aah.10140DOI Listing
September 2021

Identification of High-Confidence Structural Variants in Domesticated Rainbow Trout Using Whole-Genome Sequencing.

Front Genet 2021 25;12:639355. Epub 2021 Feb 25.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture, Kearneysville, WV, United States.

Genomic structural variants (SVs) are a major source of genetic and phenotypic variation but have not been investigated systematically in rainbow trout (), an important aquaculture species of cold freshwater. The objectives of this study were 1) to identify and validate high-confidence SVs in rainbow trout using whole-genome re-sequencing; and 2) to examine the contribution of transposable elements (TEs) to SVs in rainbow trout. A total of 96 rainbow trout, including 11 homozygous lines and 85 outbred fish from three breeding populations, were whole-genome sequenced with an average genome coverage of 17.2×. Putative SVs were identified using the program Smoove which integrates LUMPY and other associated tools into one package. After rigorous filtering, 13,863 high-confidence SVs were identified. Pacific Biosciences long-reads of Arlee, one of the homozygous lines used for SV detection, validated 98% (3,948 of 4,030) of the high-confidence SVs identified in the Arlee homozygous line. Based on principal component analysis, the 85 outbred fish clustered into three groups consistent with their populations of origin, further indicating that the high-confidence SVs identified in this study are robust. The repetitive DNA content of the high-confidence SV sequences was 86.5%, which is much higher than the 57.1% repetitive DNA content of the reference genome, and is also higher than the repetitive DNA content of Atlantic salmon SVs reported previously. TEs thus contribute substantially to SVs in rainbow trout as TEs make up the majority of repetitive sequences. Hundreds of the high-confidence SVs were annotated as exon-loss or gene-fusion variants, and may have phenotypic effects. The high-confidence SVs reported in this study provide a foundation for further rainbow trout SV studies.
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http://dx.doi.org/10.3389/fgene.2021.639355DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7959816PMC
February 2021

Structure and regulation of the NK-lysin (1-4) and NK-lysin like (a and b) antimicrobial genes in rainbow trout (Oncorhynchus mykiss).

Dev Comp Immunol 2021 03 7;116:103961. Epub 2020 Dec 7.

USDA-ARS-School of Freshwater Sciences, 600 E. Greenfield Ave., Milwaukee, WI, 53204, USA. Electronic address:

Nk-lysin (Nkl), an antimicrobial peptide (AMP) product of natural killer cells and cytotoxic T cells in mammals, has recently been characterized in a number of finfish species. In this study, we identified six genes with sequence homology to Nkl and characterized their patterns of mRNA expression and abundances in rainbow trout (Oncorhynchus mykiss). The cDNA sequences for the six Nkls encoded precursor peptides of 128-133 aa in length, and mature peptides of 109-111 aa in length. Genomic DNA of the nkl1-4 genes consisted of five exons and four introns, whereas the nkl-like a & b genes consisted of four exons and three introns. Chromosomal locations of these peptides show that nkl1 was located on chromosome arm 25q, whereas the other five nkl genes were clustered on chromosome arm 19q. Phylogenetic analysis revealed a conserved structure of Nkls among the teleosts and further protein sequence analyses suggests that all six nkl genes fall within the Nkl sub-family of the Saposin family of proteins. Patterns of tissue-specific mRNA expression were asymmetric among the six trout Nkl homologues, with nkl1, nkl3, and nkl-like a & b occurring in immune competent organs such as spleen, gill, intestine and kidney, as well as pineal gland, brain and oocytes. However, nkl2 and nkl4, showed primary abundances in brain, pineal gland and oocyte tissues. Using mRNA sequencing, in whole-body pools of juvenile trout fry (1 g bw) exposed to Flavobacterium psychrophilum infection, we observed modest up-regulation (2-3 fold) of five (nkl 2-4 and nkl-like a & b) of the six nkl mRNAs over the five-day post-challenge time-course. However, no upregulation could be recorded in spleen tissue measured by qPCR in juvenile trout (270 g bw). Using mRNA sequencing again, mRNA abundances were determined in gill of juvenile trout (~57.7 g bw) exposed to various aquaculture stressors. The results indicated that all six nkls (nkl1-4 and nkl-like a and nkl-like b) were downregulated when exposed to high temperature, and that nkl1 was significantly downregulated following salinity challenge. Overall, these newly characterized AMPs may contribute to host innate immunity as they are modulated following pathogen challenge and by physiological stressors.
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http://dx.doi.org/10.1016/j.dci.2020.103961DOI Listing
March 2021

The Type IX Secretion System Is Required for Virulence of the Fish Pathogen Flavobacterium psychrophilum.

Appl Environ Microbiol 2020 08 3;86(16). Epub 2020 Aug 3.

Department of Biological Sciences, University of Wisconsin-Milwaukee, Milwaukee, Wisconsin, USA

causes bacterial cold-water disease in wild and aquaculture-reared fish and is a major problem for salmonid aquaculture. The mechanisms responsible for cold-water disease are not known. It was recently demonstrated that the related fish pathogen, , requires a functional type IX protein secretion system (T9SS) to cause disease. T9SSs secrete cell surface adhesins, gliding motility proteins, peptidases, and other enzymes, any of which may be virulence factors. The genome has genes predicted to encode components of a T9SS. Here, we used a SacB-mediated gene deletion technique recently adapted for use in the to delete a core T9SS gene, The Δ mutant cells were deficient for secretion of many proteins in comparison to wild-type cells. Complementation of the mutant with wild-type on a plasmid restored secretion. Compared to wild-type and complemented strains, the Δ mutant was deficient in adhesion, gliding motility, and extracellular proteolytic and hemolytic activities. The Δ mutant exhibited reduced virulence in rainbow trout and complementation restored virulence, suggesting that the T9SS plays an important role in the disease. Bacterial cold-water disease, caused by , is a major problem for salmonid aquaculture. Little is known regarding the virulence factors involved in this disease, and control measures are inadequate. A targeted gene deletion method was adapted to and used to demonstrate the importance of the T9SS in virulence. Proteins secreted by this system are likely virulence factors and targets for the development of control measures.
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http://dx.doi.org/10.1128/AEM.00799-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7414955PMC
August 2020

Comparative Structural and Antigenic Characterization of Genetically Distinct -Polysaccharides.

Front Microbiol 2019 8;10:1041. Epub 2019 May 8.

United States Department of Agriculture, Agricultural Research Service, National Center for Cool and Cold Water Aquaculture, Kearneysville, WV, United States.

Little is known about the underlying basis of serotype specificity among strains of , the agent of rainbow trout fry syndrome and bacterial cold-water disease. The identification of different heat-stable O-serotypes among strains of this gram-negative pathogen does, however, suggest structural variations in the -polysaccharide (O-PS) moiety of cell surface lipopolysaccharide (LPS). A trisaccharide composed of L-rhamnose (L-Rha), 2-acetamido-2-deoxy-L-fucose (L-FucNAc) and 2-acetamido-4-R-2,4-dideoxy-D-quinovose (D-Qui2NAc4NR), where R represents a dihydroxyhexanamido derivative, was previously identified as the repeating unit of CSF259-93 O-PS. Interestingly, the O-PS gene cluster of this strain and that of 950106-1/1, which belongs to a different O-serotype, are identical except for , which encodes the putative polymerase that links trisaccharide repeats into O-PS chains. We have now found from results of glycosyl composition analysis and high-resolution nuclear magnetic resonance, that the linkage of D-Qui2NAc4NR to L-Rha, which is α1-2 for CSF259-93 versus β1-3 for 950106-1/1, is the only structural difference between O-PS from these strains. The corresponding difference in O-serotype specificity was established from the reactions of rabbit and trout anti- antibody with purified O-PS and LPS. Moreover, LPS-based differences in antigenicity were noted between strains with O-PS loci identical to those of CSF259-93 or 950106-1/1, except for the genes predicted to direct synthesis of different R-groups in Qui2NAc4NR. The findings provide a framework for defining the genetic basis of O-PS structure and antigenicity and suggest that the repertoire of O-serotypes extends beyond what is presently recognized from serological studies of this important fish pathogen.
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http://dx.doi.org/10.3389/fmicb.2019.01041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6519341PMC
May 2019

Large-Scale Analysis of Flavobacterium psychrophilum Multilocus Sequence Typing Genotypes Recovered from North American Salmonids Indicates that both Newly Identified and Recurrent Clonal Complexes Are Associated with Disease.

Appl Environ Microbiol 2019 03 6;85(6). Epub 2019 Mar 6.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, Michigan, USA

, the etiological agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), causes significant economic losses in salmonid aquaculture, particularly in rainbow trout (). Prior studies have used multilocus sequence typing (MLST) to examine genetic heterogeneity within At present, however, its population structure in North America is incompletely understood, as only 107 isolates have been genotyped. Herein, MLST was used to investigate the genetic diversity of an additional 314 North American isolates that were recovered from ten fish host species from 20 U.S. states and 1 Canadian province over nearly four decades. These isolates were placed into 66 sequence types (STs), 47 of which were novel, increasing the number of clonal complexes (CCs) in North America from 7 to 12. Newly identified CCs were diverse in terms of host association, distribution, and association with disease. The largest CC identified was CC-ST10, within which 10 novel genotypes were discovered, most of which came from experiencing BCWD. This discovery, among others, provides evidence for the hypothesis that ST10 (i.e., the founding ST of CC-ST10) originated in North America. Furthermore, ST275 (in CC-ST10) was recovered from wild/feral adult steelhead and marks the first recovery of CC-ST10 from wild/feral fish in North America. Analyses also revealed that at the allele level, the diversification of in North America is driven three times more frequently by recombination than random nucleic acid mutation, possibly indicating how new phenotypes emerge within this species. is the causative agent of bacterial coldwater disease (BCWD) and rainbow trout fry syndrome (RTFS), both of which cause substantial losses in farmed fish populations worldwide. To better prevent and control BCWD and RTFS outbreaks, we sought to characterize the genetic diversity of several hundred isolates that were recovered from diseased fish across North America. Results highlighted multiple genetic strains that appear to play an important role in disease events in North American aquaculture facilities and suggest that the practice of trading fish eggs has led to the continental and transcontinental spread of this bacterium. The knowledge generated herein will be invaluable toward guiding the development of future disease prevention techniques.
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http://dx.doi.org/10.1128/AEM.02305-18DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6414368PMC
March 2019

Variance and covariance estimates for resistance to bacterial cold water disease and columnaris disease in two rainbow trout breeding populations1.

J Anim Sci 2019 Mar;97(3):1124-1132

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture, Kearneysville, WV.

Family-based selective breeding can be an effective strategy for controlling diseases in aquaculture. This study aimed to estimate (co)variance components for resistance to bacterial cold water disease (BCWD) and columnaris disease (CD) in two unrelated rainbow trout nucleus breeding populations: the USDA, ARS, National Center for Cool and Cold Water Aquaculture odd-year line (ARS-Fp-R), which has been subjected to five generations of selection for improved resistance to BCWD, and the Troutlodge, Inc., May-spawning odd-year line (TLUM), which has been selected for improved growth performance but not for disease resistance. A total of 46,805 and 27,821 pedigree records were available from both populations, respectively. Between 44 and 138 families per generation and population were evaluated under controlled BCWD and CD challenges, providing 32,311 and 17,861 phenotypic records for BCWD resistance, and 13,603 and 9,413 for CD resistance, in the ARS-Fp-R and TLUM populations, respectively. A two-trait animal threshold model assuming an underlying normal distribution for the binary survival phenotypes was used to estimate (co)variance components separately for each population. Resistance to BCWD (h2 = 0.27 ± 0.04 and 0.43 ± 0.08) and CD (h2 = 0.23 ± 0.07 and 0.34 ± 0.09) was moderately heritable in the ARS-Fp-R and TLUM populations, respectively. The genetic correlation between the resistance to BCWD and CD was favorably positive in the ARS-Fp-R (0.40 ± 0.17) and TLUM (0.39 ± 0.18) populations. These findings suggest that both disease resistance traits can be improved simultaneously even if genetic selection pressure is applied to only one of the two traits.
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http://dx.doi.org/10.1093/jas/sky478DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6396251PMC
March 2019

Analysis of the gut and gill microbiome of resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss).

Fish Shellfish Immunol 2019 Mar 1;86:497-506. Epub 2018 Dec 1.

University of New Mexico, Department of Biology, Center for Evolutionary and Theoretical Immunology (CETI), Albuquerque, NM, USA. Electronic address:

Commensal microorganisms present at mucosal surfaces play a vital role in protecting the host organism from bacterial infection. There are multiple factors that contribute to selecting for the microbiome, including host genetics. Flavobacterium psychrophilum, the causative agent of Bacterial Cold Water Disease in salmonids, accounts for acute losses in wild and farmed rainbow trout (Oncorhynchus mykiss). The U.S. National Center for Cool and Cold Water Aquaculture has used family-based selective breeding to generate a line of rainbow trout with enhanced resistance to F. psychrophilum. The goal of this study is to determine whether selective breeding impacts the gut and gill microbiome of the F. psychrophilum-resistant as compared to a background matched susceptible trout line. Mid-gut and gill samples were collected from juvenile fish maintained at high or low stocking densities and microbial diversity assessed by 16S rDNA amplicon sequencing. Results indicate that alpha diversity was significantly higher in the mid-gut of the susceptible line compared to the resistant line, while no significant differences in alpha diversity were observed in the gills. Mycoplasma sp. was the dominant taxon in the mid-gut of both groups, although it was present at a decreased abundance in the susceptible line. We also observed an increased abundance of the potential opportunistic pathogen Brevinema andersonii in the susceptible line. Within the gills, both lines exhibited similar microbial profiles, with Candidatus Branchiomonas being the dominant taxon. Together, these results suggest that selectively bred F. psychrophilum-resistant trout may harness a more resilient gut microbiome, attributing to the disease resistant phenotype. Importantly, interactions between host genetics and environmental factors such as stocking density have a significant impact in shaping trout microbial communities.
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http://dx.doi.org/10.1016/j.fsi.2018.11.079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8040288PMC
March 2019

Retrospective Evaluation of Marker-Assisted Selection for Resistance to Bacterial Cold Water Disease in Three Generations of a Commercial Rainbow Trout Breeding Population.

Front Genet 2018 3;9:286. Epub 2018 Aug 3.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture, Kearneysville, WV, United States.

Bacterial cold water disease (BCWD), caused by , is an endemic and problematic disease in rainbow trout () aquaculture. Previously, we have identified SNPs (single nucleotide polymorphisms) associated with BCWD resistance in rainbow trout. The objectives of this study were (1) to validate the SNPs associated with BCWD resistance in a commercial breeding population; and (2) to evaluate retrospectively the accuracy of MAS (marker-assisted selection) for BCWD resistance in this commercial breeding program. Three consecutive generations of the Troutlodge May breeding population were evaluated for BCWD resistance. Based on our previous studies, a panel of 96 SNPs was selected and used to genotype the parents and ten offspring from each of the 138 full-sib families of the 2015 generation, and 37 SNPs associated with BCWD resistance were validated. Thirty-six of the validated SNPs were clustered on chromosomes Omy3, Omy8 and Omy25. Thus, at least three QTL (quantitative trait loci) for BCWD resistance were validated in the 2015 generation. Three SNPs from each QTL region were used for haplotype association analysis. Three haplotypes, Omy3TGG, Omy8GCG and Omy25CGG, were found to be associated with BCWD resistance in the 2015 generation. Retrospective analyses were then performed to evaluate the accuracy of MAS for BCWD resistance using these three favorable haplotypes. The accuracy of MAS was estimated with the Pearson correlation coefficient between the total number of favorable haplotypes in the two parents and the family BCWD survival rates. The Omy8 and Omy25 haplotypes were positively correlated with the family BCWD survival rates across all three generations. The accuracies of MAS using these two haplotypes together were consistently around 0.5, which was equal or greater than the accuracy of the conventional family-based selection in the same generation. In conclusion, we have demonstrated that MAS for BCWD resistance is feasible in this commercial rainbow trout breeding population.
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http://dx.doi.org/10.3389/fgene.2018.00286DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6085459PMC
August 2018

Differential expression and evolution of three tandem, interleukin-1 receptor-like 1 genes in rainbow trout (Oncorhynchus mykiss).

Dev Comp Immunol 2018 10 15;87:193-203. Epub 2018 Jun 15.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, USDA, Kearneysville, WV 25430, USA. Electronic address:

Interleukin-1 receptor-like 1 (Il1rl1 or ST2), a member of the interleukin-1 receptor family, has pleiotropic roles including tissue homeostasis, inflammation, immune polarization, and disease resistance in mammals. A single orthologue was previously described in salmonid fish; however, a recently improved genome assembly of rainbow trout (Oncorhynchus mykiss) revealed three adjacent, tandem il1rl1 orthologues on chromosome Omy 03. Here, we report the genomic organization and evolution of the three il1rl1 genes (il1rl1α, il1rl1β, il1rl1γ), and use both RNA-seq and gene-specific qPCR methods to quantify expression patterns. Nucleotide sequence homology between the three genes is >95% and each predicted protein contains three IG/IG-like domains, a transmembrane region and a TIR domain. The amino acid sequence homology of the rainbow trout il1rl1 genes are highly related to two functional copies in Atlantic and Coho salmon (∼94%) but relatively low (22-26%) with avian and mammalian species. Transcript abundance measured by RNA-seq in 15 tissues of healthy adult rainbow trout indicate constitutive expression of each gene. In whole body lysates, il1rl1α was shown to have >20 fold mRNA expression compared to il1rl1β and il1rl1γ as measured by qPCR assays specific to il1rl1α or il1rl1γ, as well as a multi-gene qPCR assay (il1rl1α,β,γ). Unrooted phylogenetic trees grouped the rainbow trout il1rl1 genes apart from other interleukin-1 receptor family genes and genomic comparisons identify preserved synteny between mammals, birds and salmonids albeit a pseudogene is present in both Atlantic salmon and Coho salmon. Phylogenetic analyses suggest that the three genes arose by tandem duplication but are inconclusive whether these events occurred prior-to or after salmonid speciation. These findings further the understanding of interleukin receptor family evolution and their contribution to teleost immune function.
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http://dx.doi.org/10.1016/j.dci.2018.06.003DOI Listing
October 2018

Accurate genomic predictions for BCWD resistance in rainbow trout are achieved using low-density SNP panels: Evidence that long-range LD is a major contributing factor.

J Anim Breed Genet 2018 Jun 5. Epub 2018 Jun 5.

National Center for Cool and Cold Water Aquaculture, United States Department of Agriculture, Agricultural Research Service, Kearneysville, West Virginia.

Previously accurate genomic predictions for Bacterial cold water disease (BCWD) resistance in rainbow trout were obtained using a medium-density single nucleotide polymorphism (SNP) array. Here, the impact of lower-density SNP panels on the accuracy of genomic predictions was investigated in a commercial rainbow trout breeding population. Using progeny performance data, the accuracy of genomic breeding values (GEBV) using 35K, 10K, 3K, 1K, 500, 300 and 200 SNP panels as well as a panel with 70 quantitative trait loci (QTL)-flanking SNP was compared. The GEBVs were estimated using the Bayesian method BayesB, single-step GBLUP (ssGBLUP) and weighted ssGBLUP (wssGBLUP). The accuracy of GEBVs remained high despite the sharp reductions in SNP density, and even with 500 SNP accuracy was higher than the pedigree-based prediction (0.50-0.56 versus 0.36). Furthermore, the prediction accuracy with the 70 QTL-flanking SNP (0.65-0.72) was similar to the panel with 35K SNP (0.65-0.71). Genomewide linkage disequilibrium (LD) analysis revealed strong LD (r  ≥ 0.25) spanning on average over 1 Mb across the rainbow trout genome. This long-range LD likely contributed to the accurate genomic predictions with the low-density SNP panels. Population structure analysis supported the hypothesis that long-range LD in this population may be caused by admixture. Results suggest that lower-cost, low-density SNP panels can be used for implementing genomic selection for BCWD resistance in rainbow trout breeding programs.
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http://dx.doi.org/10.1111/jbg.12335DOI Listing
June 2018

Genomic Diversity and Evolution of the Fish Pathogen .

Front Microbiol 2018 7;9:138. Epub 2018 Feb 7.

Unité Mathématiques et Informatique Appliquées du Génome à l'Environnement (MaIAGE), Institut National de la Recherche Agronomique, Université Paris-Saclay, Jouy-en-Josas, France.

, the etiological agent of rainbow trout fry syndrome and bacterial cold-water disease in salmonid fish, is currently one of the main bacterial pathogens hampering the productivity of salmonid farming worldwide. In this study, the genomic diversity of the species is analyzed using a set of 41 genomes, including 30 newly sequenced isolates. These were selected on the basis of available MLST data with the two-fold objective of maximizing the coverage of the species diversity and of allowing a focus on the main clonal complex (CC-ST10) infecting farmed rainbow trout () worldwide. The results reveal a bacterial species harboring a limited genomic diversity both in terms of nucleotide diversity, with ~0.3% nucleotide divergence inside CDSs in pairwise genome comparisons, and in terms of gene repertoire, with the core genome accounting for ~80% of the genes in each genome. The pan-genome seems nevertheless "open" according to the scaling exponent of a power-law fitted on the rate of new gene discovery when genomes are added one-by-one. Recombination is a key component of the evolutionary process of the species as seen in the high level of apparent homoplasy in the core genome. Using a Hidden Markov Model to delineate recombination tracts in pairs of closely related genomes, the average recombination tract length was estimated to ~4.0 Kbp and the typical ratio of the contributions of recombination and mutations to nucleotide-level differentiation (r/m) was estimated to ~13. Within CC-ST10, evolutionary distances computed on non-recombined regions and comparisons between 22 isolates sampled up to 27 years apart suggest a most recent common ancestor in the second half of the nineteenth century in North America with subsequent diversification and transmission of this clonal complex coinciding with the worldwide expansion of rainbow trout farming. With the goal to promote the development of tools for the genetic manipulation of , a particular attention was also paid to plasmids. Their extraction and sequencing to completion revealed plasmid diversity that remained hidden to classical plasmid profiling due to size similarities.
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http://dx.doi.org/10.3389/fmicb.2018.00138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5808330PMC
February 2018

Similar Genetic Architecture with Shared and Unique Quantitative Trait Loci for Bacterial Cold Water Disease Resistance in Two Rainbow Trout Breeding Populations.

Front Genet 2017 23;8:156. Epub 2017 Oct 23.

National Center for Cool and Cold Water Aquaculture, United States Department of Agriculture, Agricultural Research Service, Kearneysville, WV, United States.

Bacterial cold water disease (BCWD) causes significant mortality and economic losses in salmonid aquaculture. In previous studies, we identified moderate-large effect quantitative trait loci (QTL) for BCWD resistance in rainbow trout (). However, the recent availability of a 57 K SNP array and a reference genome assembly have enabled us to conduct genome-wide association studies (GWAS) that overcome several experimental limitations from our previous work. In the current study, we conducted GWAS for BCWD resistance in two rainbow trout breeding populations using two genotyping platforms, the 57 K Affymetrix SNP array and restriction-associated DNA (RAD) sequencing. Overall, we identified 14 moderate-large effect QTL that explained up to 60.8% of the genetic variance in one of the two populations and 27.7% in the other. Four of these QTL were found in both populations explaining a substantial proportion of the variance, although major differences were also detected between the two populations. Our results confirm that BCWD resistance is controlled by the oligogenic inheritance of few moderate-large effect loci and a large-unknown number of loci each having a small effect on BCWD resistance. We detected differences in QTL number and genome location between two GWAS models (weighted single-step GBLUP and Bayes B), which highlights the utility of using different models to uncover QTL. The RAD-SNPs detected a greater number of QTL than the 57 K SNP array in one population, suggesting that the RAD-SNPs may uncover polymorphisms that are more unique and informative for the specific population in which they were discovered.
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http://dx.doi.org/10.3389/fgene.2017.00156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5660510PMC
October 2017

A BCWD-resistant line of rainbow trout exhibits higher abundance of IgT B cells and heavy chain tau transcripts compared to a susceptible line following challenge with Flavobacterium psychrophilum.

Dev Comp Immunol 2017 09 4;74:190-199. Epub 2017 May 4.

Department of Biology, The College of William and Mary, Williamsburg, VA 23185, USA.

Bacterial Cold Water Disease (BCWD) is a common, chronic disease in rainbow trout, and is caused by the gram-negative bacterium Flavobacterium psychrophilum (Fp). Through selective breeding, the National Center for Cool and Cold Water Aquaculture has generated a genetic line that is highly resistant to Fp challenge, designated ARS-Fp-R (or R-line), as well as a susceptible "control" line, ARS-Fp-S (S-line). In previous studies, resistance to Fp had been shown to correlate with naive animal spleen size, and further, naïve R-line trout had been shown to have a lower abundance of IgM and IgM cells compared to S-line fish. Here we wished to first determine whether the abundance of IgT and/or IgT cells differed between the two lines in naïve fish, and if so, how these patterns differed after in vivo challenge with Fp. Fp challenge was by intramuscular injection of live Fp and tissue collections were on days 5, 6, and/or 28 post-challenge, in two independent challenge experiments. Flow cytometric and gene expression analyses revealed that naïve R-line fish had a higher abundance of IgT B cells in their anterior kidney, spleen, and blood, compared to S line fish. Further, that after Fp challenge, this difference was maintained between the two lines. Lastly, abundance of IgT B cells and expression of secHCtau correlated with lower Fp pathogen loads in challenged fish. In the anterior kidney, IgM B cell abundance correlated with increased Fp loads. Together, these results suggest that IgT B lineage cells may have a protective function in the immune response to Fp.
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http://dx.doi.org/10.1016/j.dci.2017.04.019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5551897PMC
September 2017

Dataset of proinflammatory cytokine and cytokine receptor gene expression in rainbow trout () measured using a novel GeXP multiplex, RT-PCR assay.

Data Brief 2017 Apr 12;11:192-196. Epub 2017 Feb 12.

National Center for Cool and Cold Water Aquaculture, 11861 Leetown Rd, Kearneysville, WV 25430, USA.

A GeXP multiplex, RT-PCR assay was developed and optimized that simultaneously measures expression of a suite of immune-relevant genes in rainbow trout (), concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. The dataset includes expression values for , , , , , (e3-5), (e9-11), , , , , , , , , , , , and . Gene expression was measured at four time-points post-challenge in both a resistant line (ARS-Fp-R) and a susceptible line (ARS-Fp-S) of rainbow trout. In addition, fish body weight, spleen index and the load are reported. These data are an extension of information presented and discussed in Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with in resistant and susceptible lines of rainbow trout ()" (Kutyrev et al., 2016) [1].
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http://dx.doi.org/10.1016/j.dib.2017.02.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5315433PMC
April 2017

Genomic selection models double the accuracy of predicted breeding values for bacterial cold water disease resistance compared to a traditional pedigree-based model in rainbow trout aquaculture.

Genet Sel Evol 2017 02 1;49(1):17. Epub 2017 Feb 1.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture, Kearneysville, WV, USA.

Background: Previously, we have shown that bacterial cold water disease (BCWD) resistance in rainbow trout can be improved using traditional family-based selection, but progress has been limited to exploiting only between-family genetic variation. Genomic selection (GS) is a new alternative that enables exploitation of within-family genetic variation.

Methods: We compared three GS models [single-step genomic best linear unbiased prediction (ssGBLUP), weighted ssGBLUP (wssGBLUP), and BayesB] to predict genomic-enabled breeding values (GEBV) for BCWD resistance in a commercial rainbow trout population, and compared the accuracy of GEBV to traditional estimates of breeding values (EBV) from a pedigree-based BLUP (P-BLUP) model. We also assessed the impact of sampling design on the accuracy of GEBV predictions. For these comparisons, we used BCWD survival phenotypes recorded on 7893 fish from 102 families, of which 1473 fish from 50 families had genotypes [57 K single nucleotide polymorphism (SNP) array]. Naïve siblings of the training fish (n = 930 testing fish) were genotyped to predict their GEBV and mated to produce 138 progeny testing families. In the following generation, 9968 progeny were phenotyped to empirically assess the accuracy of GEBV predictions made on their non-phenotyped parents.

Results: The accuracy of GEBV from all tested GS models were substantially higher than the P-BLUP model EBV. The highest increase in accuracy relative to the P-BLUP model was achieved with BayesB (97.2 to 108.8%), followed by wssGBLUP at iteration 2 (94.4 to 97.1%) and 3 (88.9 to 91.2%) and ssGBLUP (83.3 to 85.3%). Reducing the training sample size to n = ~1000 had no negative impact on the accuracy (0.67 to 0.72), but with n = ~500 the accuracy dropped to 0.53 to 0.61 if the training and testing fish were full-sibs, and even substantially lower, to 0.22 to 0.25, when they were not full-sibs.

Conclusions: Using progeny performance data, we showed that the accuracy of genomic predictions is substantially higher than estimates obtained from the traditional pedigree-based BLUP model for BCWD resistance. Overall, we found that using a much smaller training sample size compared to similar studies in livestock, GS can substantially improve the selection accuracy and genetic gains for this trait in a commercial rainbow trout breeding population.
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http://dx.doi.org/10.1186/s12711-017-0293-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5289005PMC
February 2017

Differential expression of long non-coding RNAs in three genetic lines of rainbow trout in response to infection with Flavobacterium psychrophilum.

Sci Rep 2016 10 27;6:36032. Epub 2016 Oct 27.

Department of Biology and Molecular Biosciences Program, Middle Tennessee State University, Murfreesboro, TN, 37132, U.S.

Bacterial cold-water disease caused by Flavobacterium psychrophilum is one of the major causes of mortality of salmonids. Three genetic lines of rainbow trout designated as ARS-Fp-R (resistant), ARS-Fp-C (control) and ARS-Fp-S (susceptible) have significant differences in survival rate following F. psychrophilum infection. Previous study identified transcriptome differences of immune-relevant protein-coding genes at basal and post infection levels among these genetic lines. Using RNA-Seq approach, we quantified differentially expressed (DE) long non-coding RNAs (lncRNAs) in response to F. psychrophilum challenge in these genetic lines. Pairwise comparison between genetic lines and different infection statuses identified 556 DE lncRNAs. A positive correlation existed between the number of the differentially regulated lncRNAs and that of the protein-coding genes. Several lncRNAs showed strong positive and negative expression correlation with their overlapped, neighboring and distant immune related protein-coding genes including complement components, cytokines, chemokines and several signaling molecules involved in immunity. The correlated expressions and genome-wide co-localization suggested that some lncRNAs may be involved in regulating immune-relevant protein-coding genes. This study provides the first evidence of lncRNA-mediated regulation of the anti-bacterial immune response in a commercially important aquaculture species and will likely help developing new genetic markers for rainbow trout disease resistance.
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http://dx.doi.org/10.1038/srep36032DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5081542PMC
October 2016

Proinflammatory cytokine and cytokine receptor gene expression kinetics following challenge with Flavobacterium psychrophilum in resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss).

Fish Shellfish Immunol 2016 Nov 12;58:542-553. Epub 2016 Oct 12.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, USDA, 11861 Leetown Rd, Kearneysville, WV 25430, USA. Electronic address:

Flavobacterium psychrophilum (Fp) is the causative agent of bacterial cold water disease (BCWD) which causes appreciable economic losses in rainbow trout aquaculture. We previously reported development of a genetic line, designated ARS-Fp-R that exhibits higher survival relative to a susceptible line, designated ARS-Fp-S, following either laboratory or natural on-farm challenge. The objectives of this study were to determine the temporal kinetics of gene expression between experimentally-challenged ARS-Fp-R and ARS-Fp-S fish and the correlation between gene expression and pathogen load. We developed a GeXP multiplex RT-PCR assay to simultaneously examine expression of immune-relevant genes, concentrating on tumor necrosis factor and interleukin-1 ligand/receptor systems and acute phase response genes. Spleen tissue was sampled at 6 h, 24 h, 48 h and 144 h post-challenge and pathogen load quantified by qPCR. Transcript abundance of cytokine genes tnfa1, tnfa2, tnfa3, il1b1, il1b2, il11a; acute phase response genes saa and drtp1; and putative cytokine receptors il1r1-like-b, il1r2, tnfrsf1a, tnfrsf9, tnfrsf1a-like-b increased following challenge while the transcript abundance of il1r-like-1 and tnfrsf1a-like-a decreased compared to PBS-injected line-matched control fish. Principal component analysis identified transcript levels of genes il1r-like-1 and tnfrsf1a-like-a as exhibiting differential expression between genetic lines. In summary, Fp i.p. injection challenge elicited a proinflammatory cytokine gene expression response in the spleen, with ARS-Fp-R line fish exhibiting modestly higher basal expression levels of several putative cytokine receptors. This study furthers the understanding of the immune response following Fp challenge and differences in gene expression associated with selective breeding for disease resistance.
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http://dx.doi.org/10.1016/j.fsi.2016.09.053DOI Listing
November 2016

Evaluation of Genome-Enabled Selection for Bacterial Cold Water Disease Resistance Using Progeny Performance Data in Rainbow Trout: Insights on Genotyping Methods and Genomic Prediction Models.

Front Genet 2016 27;7:96. Epub 2016 May 27.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture Kearneysville, WV, USA.

Bacterial cold water disease (BCWD) causes significant economic losses in salmonid aquaculture, and traditional family-based breeding programs aimed at improving BCWD resistance have been limited to exploiting only between-family variation. We used genomic selection (GS) models to predict genomic breeding values (GEBVs) for BCWD resistance in 10 families from the first generation of the NCCCWA BCWD resistance breeding line, compared the predictive ability (PA) of GEBVs to pedigree-based estimated breeding values (EBVs), and compared the impact of two SNP genotyping methods on the accuracy of GEBV predictions. The BCWD phenotypes survival days (DAYS) and survival status (STATUS) had been recorded in training fish (n = 583) subjected to experimental BCWD challenge. Training fish, and their full sibs without phenotypic data that were used as parents of the subsequent generation, were genotyped using two methods: restriction-site associated DNA (RAD) sequencing and the Rainbow Trout Axiom® 57 K SNP array (Chip). Animal-specific GEBVs were estimated using four GS models: BayesB, BayesC, single-step GBLUP (ssGBLUP), and weighted ssGBLUP (wssGBLUP). Family-specific EBVs were estimated using pedigree and phenotype data in the training fish only. The PA of EBVs and GEBVs was assessed by correlating mean progeny phenotype (MPP) with mid-parent EBV (family-specific) or GEBV (animal-specific). The best GEBV predictions were similar to EBV with PA values of 0.49 and 0.46 vs. 0.50 and 0.41 for DAYS and STATUS, respectively. Among the GEBV prediction methods, ssGBLUP consistently had the highest PA. The RAD genotyping platform had GEBVs with similar PA to those of GEBVs from the Chip platform. The PA of ssGBLUP and wssGBLUP methods was higher with the Chip, but for BayesB and BayesC methods it was higher with the RAD platform. The overall GEBV accuracy in this study was low to moderate, likely due to the small training sample used. This study explored the potential of GS for improving resistance to BCWD in rainbow trout using, for the first time, progeny testing data to assess the accuracy of GEBVs, and it provides the basis for further investigation on the implementation of GS in commercial rainbow trout populations.
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http://dx.doi.org/10.3389/fgene.2016.00096DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4883007PMC
June 2016

Genetic Diversity of Flavobacterium psychrophilum Isolates from Three Oncorhynchus spp. in the United States, as Revealed by Multilocus Sequence Typing.

Appl Environ Microbiol 2016 06 16;82(11):3246-3255. Epub 2016 May 16.

Department of Fisheries and Wildlife, College of Agriculture and Natural Resources, Michigan State University, East Lansing, Michigan, USA

Unlabelled: The use of a multilocus sequence typing (MLST) technique has identified the intraspecific genetic diversity of U.S. Flavobacterium psychrophilum, an important pathogen of salmonids worldwide. Prior to this analysis, little U.S. F. psychrophilum genetic information was known; this is of importance when considering targeted control strategies, including vaccine development. Herein, MLST was used to investigate the genetic diversity of 96 F. psychrophilum isolates recovered from rainbow trout (Oncorhynchus mykiss), coho salmon (Oncorhynchus kisutch), and Chinook salmon (Oncorhynchus tshawytscha) that originated from nine U.S. states. The isolates fell into 34 distinct sequence types (STs) that clustered in 5 clonal complexes (CCs) (n = 63) or were singletons (n = 33). The distribution of STs varied spatially, by host species, and in association with mortality events. Several STs (i.e., ST9, ST10, ST30, and ST78) were found in multiple states, whereas the remaining STs were localized to single states. With the exception of ST256, which was recovered from rainbow trout and Chinook salmon, all STs were found to infect a single host species. Isolates that were collected during bacterial cold water disease outbreaks most frequently belonged to CC-ST10 (e.g., ST10 and ST78). Collectively, the results of this study clearly demonstrate the genetic diversity of F. psychrophilum within the United States and identify STs of clinical significance. Although the majority of STs described herein were novel, some (e.g., ST9, ST10, ST13, ST30, and ST31) were previously recovered on other continents, which demonstrates the transcontinental distribution of F. psychrophilum genotypes.

Importance: Flavobacterium psychrophilum is the causative agent of bacterial cold water disease (BCWD) and rainbow trout fry syndrome (RTFS) and is an important bacterial pathogen of wild and farmed salmonids worldwide. These infections are responsible for large economic losses globally, yet the genetic diversity of this pathogen remains to be fully investigated. Previous studies have identified the genetic diversity of this pathogen in other main aquaculture regions; however, little effort has been focused on the United States. In this context, this study aims to examine the genetic diversity of F. psychrophilum from the United States, as this region remains important in salmonid aquaculture.
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http://dx.doi.org/10.1128/AEM.00411-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4959235PMC
June 2016

Identification of single nucleotide polymorphism markers associated with bacterial cold water disease resistance and spleen size in rainbow trout.

Front Genet 2015 24;6:298. Epub 2015 Sep 24.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture Kearneysville, WV, USA.

Bacterial cold water disease (BCWD) is one of the frequent causes of elevated mortality in salmonid aquaculture. Previously, we identified and validated microsatellites on chromosome Omy19 associated with QTL (quantitative trait loci) for BCWD resistance and spleen size in rainbow trout. Recently, SNPs (single nucleotide polymorphism) have become the markers of choice for genetic analyses in rainbow trout as they are highly abundant, cost-effective and are amenable for high throughput genotyping. The objective of this study was to identify SNP markers associated with BCWD resistance and spleen size using both genome-wide association studies (GWAS) and linkage-based QTL mapping approaches. A total of 298 offspring from the two half-sib families used in our previous study to validate the significant BCWD QTL on chromosome Omy19 were genotyped with RAD-seq (restriction-site-associated DNA sequencing), and 7,849 informative SNPs were identified. Based on GWAS, 18 SNPs associated with BCWD resistance and 20 SNPs associated with spleen size were identified. Linkage-based QTL mapping revealed three significant QTL for BCWD resistance. In addition to the previously validated dam-derived QTL on chromosome Omy19, two significant BCWD QTL derived from the sires were identified on chromosomes Omy8 and Omy25, respectively. A sire-derived significant QTL for spleen size on chromosome Omy2 was detected. The SNP markers reported in this study will facilitate fine mapping to identify positional candidate genes for BCWD resistance in rainbow trout.
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http://dx.doi.org/10.3389/fgene.2015.00298DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4585308PMC
October 2015

Potential mechanisms of attenuation for rifampicin-passaged strains of Flavobacterium psychrophilum.

BMC Microbiol 2015 Sep 16;15:179. Epub 2015 Sep 16.

Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA, USA.

Background: Flavobacterium psychrophilum is the etiologic agent of bacterial coldwater disease in salmonids. Earlier research showed that a rifampicin-passaged strain of F. psychrophilum (CSF 259-93B.17) caused no disease in rainbow trout (Oncorhynchus mykiss, Walbaum) while inducing a protective immune response against challenge with the virulent CSF 259-93 strain. We hypothesized that rifampicin passage leads to an accumulation of genomic mutations that, by chance, reduce virulence. To assess the pattern of phenotypic and genotypic changes associated with passage, we examined proteomic, LPS and single-nucleotide polymorphism (SNP) differences for two F. psychrophilum strains (CSF 259-93 and THC 02-90) that were passaged with and without rifampicin selection.

Results: Rifampicin resistance was conveyed by expected mutations in rpoB, although affecting different DNA bases depending on the strain. One rifampicin-passaged CSF 259-93 strain (CR) was attenuated (4 % mortality) in challenged fish, but only accumulated eight nonsynonymous SNPs compared to the parent strain. A CSF 259-93 strain passaged without rifampicin (CN) accumulated five nonsynonymous SNPs and was partially attenuated (28 % mortality) compared to the parent strain (54.5 % mortality). In contrast, there were no significant change in fish mortalities among THC 02-90 wild-type and passaged strains, despite numerous SNPs accumulated during passage with (n = 174) and without rifampicin (n = 126). While only three missense SNPs were associated with attenuation, a Ser492Phe rpoB mutation in the CR strain may contribute to further attenuation. All strains except CR retained a gliding motility phenotype. Few proteomic differences were observed by 2D SDS-PAGE and there were no apparent changes in LPS between strains. Comparative methylome analysis of two strains (CR and TR) identified no shared methylation motifs for these two strains.

Conclusion: Multiple genomic changes arose during passage experiments with rifampicin selection pressure. Consistent with our hypothesis, unique strain-specific mutations were detected for the fully attenuated (CR), partially attenuated (CN) and another fully attenuated strain (B17).
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http://dx.doi.org/10.1186/s12866-015-0518-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4571129PMC
September 2015

Detection and Validation of QTL Affecting Bacterial Cold Water Disease Resistance in Rainbow Trout Using Restriction-Site Associated DNA Sequencing.

PLoS One 2015 16;10(9):e0138435. Epub 2015 Sep 16.

National Center for Cool and Cold Water Aquaculture, ARS-USDA, 11861 Leetown Rd., Kearneysville, West Virginia, 25430, United States of America.

Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. Using microsatellite markers in a genome scan, we previously detected significant and suggestive QTL affecting phenotypic variation in survival following challenge with Flavobacterium psychrophilum, the causative agent of BCWD in rainbow trout. In this study, we performed selective genotyping of SNPs from restriction-site associated DNA (RAD) sequence data from two pedigreed families (2009070 and 2009196) to validate the major QTL from the previous work and to detect new QTL. The use of RAD SNPs in the genome scans increased the number of mapped markers from ~300 to ~5,000 per family. The significant QTL detected in the microsatellites scan on chromosome Omy8 in family 2009070 was validated explaining up to 58% of the phenotypic variance in that family, and in addition, a second QTL was also detected on Omy8. Two novel QTL on Omy11 and 14 were also detected, and the previously suggestive QTL on Omy1, 7 and 25 were also validated in family 2009070. In family 2009196, the microsatellite significant QTL on Omy6 and 12 were validated and a new QTL on Omy8 was detected, but none of the previously detected suggestive QTL were validated. The two Omy8 QTL from family 2009070 and the Omy12 QTL from family 2009196 were found to be co-localized with handling and confinement stress response QTL that our group has previously identified in a separate pedigreed family. With the currently available data we cannot determine if the co-localized QTL are the result of genes with pleiotropic effects or a mere physical proximity on the same chromosome segment. The genetic markers linked to BCWD resistance QTL were used to query the scaffolds of the rainbow trout reference genome assembly and the QTL-positive scaffold sequences were found to include 100 positional candidate genes. Several of the candidate genes located on or near the two Omy8 QTL detected in family 2009070 suggest potential linkages between stress response and the regulation of immune response in rainbow trout.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0138435PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4574402PMC
June 2016

Whole-body transcriptome of selectively bred, resistant-, control-, and susceptible-line rainbow trout following experimental challenge with Flavobacterium psychrophilum.

Front Genet 2014 8;5:453. Epub 2015 Jan 8.

National Center for Cool and Cold Water Aquaculture, Agricultural Research Service, United States Department of Agriculture Kearneysville, WV, USA.

Genetic improvement for enhanced disease resistance in fish is an increasingly utilized approach to mitigate endemic infectious disease in aquaculture. In domesticated salmonid populations, large phenotypic variation in disease resistance has been identified but the genetic basis for altered responsiveness remains unclear. We previously reported three generations of selection and phenotypic validation of a bacterial cold water disease (BCWD) resistant line of rainbow trout, designated ARS-Fp-R. This line has higher survival after infection by either standardized laboratory challenge or natural challenge as compared to two reference lines, designated ARS-Fp-C (control) and ARS-Fp-S (susceptible). In this study, we utilized 1.1 g fry from the three genetic lines and performed RNA-seq to measure transcript abundance from the whole body of naive and Flavobacterium psychrophilum infected fish at day 1 (early time-point) and at day 5 post-challenge (onset of mortality). Sequences from 24 libraries were mapped onto the rainbow trout genome reference transcriptome of 46,585 predicted protein coding mRNAs that included 2633 putative immune-relevant gene transcripts. A total of 1884 genes (4.0% genome) exhibited differential transcript abundance between infected and mock-challenged fish (FDR < 0.05) that included chemokines, complement components, tnf receptor superfamily members, interleukins, nod-like receptor family members, and genes involved in metabolism and wound healing. The largest number of differentially expressed genes occurred on day 5 post-infection between naive and challenged ARS-Fp-S line fish correlating with high bacterial load. After excluding the effect of infection, we identified 21 differentially expressed genes between the three genetic lines. In summary, these data indicate global transcriptome differences between genetic lines of naive animals as well as differentially regulated transcriptional responses to infection.
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http://dx.doi.org/10.3389/fgene.2014.00453DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4288049PMC
January 2015

Reduction of rainbow trout spleen size by splenectomy does not alter resistance against bacterial cold water disease.

Dev Comp Immunol 2015 Mar 7;49(1):31-7. Epub 2014 Nov 7.

Department of Environmental and Aquatic Animal Health, Virginia Institute of Marine Science, The College of William and Mary, Williamsburg, VA 23185, USA.

In lower vertebrates, the contribution of the spleen to anti-bacterial immunity is poorly understood. We have previously reported a phenotypic and genetic correlation between resistance to Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) and spleen somatic index (spleen weight normalized to body weight, SI). Fish families with larger pre-challenge SI values were found to have greater BCWD survival (resistance) following intraperitoneal injection of a lethal dose of F. psychrophilum. Since the mammalian spleen is known to be crucial for capture and destruction of encapsulated bacteria, we tested the hypothesis that reduction of spleen size, by surgical splenectomy, should reduce the survival advantage of the larger-spleen, disease-resistant fish. Experiments were performed using two separate lines of fish that had previously been selected either based on BCWD survival (resistant and susceptible), or selected based on spleen size (high and low SI). Following 65 to 81 days post-surgical recovery, fish were challenged with F. psychrophilum and mortality monitored for a minimum of 21 days. No significant difference in the relative survival was detected between splenectomized or sham-operated groups, while SI of splenectomized fish was reduced to an average of 8-12% of control animals. A positive correlation was observed between the SI, measured at the time of splenectomy, and time-to-death post-challenge. In summary, these experiments argue that larger spleen size alone is not sufficient for greater BCWD resistance, but rather it is an indirect indicator of immunological status.
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http://dx.doi.org/10.1016/j.dci.2014.11.003DOI Listing
March 2015

Biochemical reference intervals and pathophysiological changes in Flavobacterium psychrophilum-resistant and -susceptible rainbow trout lines.

Dis Aquat Organ 2014 Oct;111(3):239-48

National Center for Cool and Cold Water Aquaculture, USDA-ARS, 11861 Leetown Rd, Kearneysville, WV 25430, USA.

Host genetic resistance against disease-causing pathogens can be enhanced through family-based selective breeding. At present, there is an incomplete understanding of how artificial selection of fish alters host physiology and response following pathogen exposure. We previously reported the generation of selectively-bred rainbow trout Oncorhynchus mykiss lines with either increased resistance (ARS-Fp-R) or susceptibility (ARS-Fp-S) to bacterial cold water disease (BCWD). This study (1) determined baseline reference-range intervals for packed cell volume (PCV) and 18 plasma biochemistry analytes, and (2) examined pathophysiological changes following infection between the genetic lines. PCV and biochemistry reference-range intervals did not significantly differ between genetic lines; thus data were pooled into a single reference-range population (n = 85). ARS-Fp-R and ARS-Fp-S line fish were intraperitoneally challenged with Flavobacterium psychrophilum, and plasma was collected on Days 1, 3, 6, and 9 post-challenge. Splenic bacterial load was measured using an F. psychrophilum-specific qPCR assay. In both genetic lines, changes were observed in mean PCV, total protein, albumin, glucose, cholesterol, chloride, and calcium, falling outside the established reference intervals and significantly differing from phosphate-buffered saline challenged fish, on at least 1d post-challenge. Mean PCV, total protein, and calcium significantly differed between ARS-Fp-R and ARS-Fp-S line fish on Day 9 post-infection, with values in the ARS-Fp-S line deviating most from the reference interval. PCV, total protein, cholesterol, and calcium negatively correlated with bacterial load. These findings identify divergent pathophysiological responses between ARS-Fp-R and ARS-Fp-S line fish following laboratory challenge that are likely associated with differential survival.
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http://dx.doi.org/10.3354/dao02777DOI Listing
October 2014

Complete Genome Sequence of Flavobacterium psychrophilum Strain CSF259-93, Used To Select Rainbow Trout for Increased Genetic Resistance against Bacterial Cold Water Disease.

Genome Announc 2014 Sep 18;2(5). Epub 2014 Sep 18.

Igenbio, Inc., Chicago, Illinois, USA.

The genome sequence of Flavobacterium psychrophilum strain CSF259-93, isolated from rainbow trout (Oncorhynchus mykiss), consists of a single circular genome of 2,900,735 bp and 2,701 predicted open reading frames (ORFs). Strain CSF259-93 has been used to select a line of rainbow trout with increased genetic resistance against bacterial cold water disease.
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http://dx.doi.org/10.1128/genomeA.00889-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4172266PMC
September 2014

Histopathologic Changes in Disease-Resistant-Line and Disease-Susceptible-Line Juvenile Rainbow Trout Experimentally Infected with Flavobacterium psychrophilum.

J Aquat Anim Health 2014 Sep;26(3):181-9

a National Center for Cool and Cold Water Aquaculture , 11861 Leetown Road, Kearneysville , West Virginia 25430 , USA.

Abstract A challenge to selectively breeding fish populations for improved disease resistance in aquaculture is an incomplete understanding of how artificial selection affects innate immunity at the host-pathogen level. The objective of this study was to determine whether Rainbow Trout Oncorhynchus mykiss bred for differential susceptibility to bacterial cold-water disease exhibited altered tissue damage and cellular inflammatory response following experimental challenge with Flavobacterium psychrophilum. Fish from disease-resistant (ARS-Fp-R) and disease-susceptible (ARS-Fp-S) lines were experimentally challenged as juveniles, and mortalities, as well as survivors, were sampled for histopathology during the acute phase of the disease. Microscopic lesions were quantified or semiquantified and statistically compared for changes over time and between genetic lines. Significant progression in the degree of perisplenitis, splenic necrosis, splenic inflammatory infiltrates, average splenic ellipsoid area, total splenic ellipsoid area, and peritonitis was present over time in both genetic lines on at least one postinfection time point. No differences were found between renal inflammatory infiltrates and renal hematopoietic cell depletion over time. Perisplenitis was significantly lower in fish from the ARS-Fp-R line on day 9 postinfection than in fish from the ARS-Fp-S line. The ARS-Fp-R line demonstrated a trend towards reduced splenic necrosis compared with the ARS-Fp-S line that approached significance, and fish from the ARS-Fp-S line were 3.6 times more likely than fish from the ARS-Fp-R line to have a higher splenic necrosis lesion score after day 3 postinfection. These findings support the hypothesis that differential survival is a result of divergence in disease magnitude and not altered disease course between genetic lines. Characterization of histopathologic changes between genetic lines and over time helps elucidate mechanisms of disease resistance and contributes to our understanding of disease pathogenesis in fish infected with F. psychrophilum. Received January 7, 2014; accepted March 10, 2014.
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http://dx.doi.org/10.1080/08997659.2014.920735DOI Listing
September 2014

B cell signatures of BCWD-resistant and susceptible lines of rainbow trout: a shift towards more EBF-expressing progenitors and fewer mature B cells in resistant animals.

Dev Comp Immunol 2015 Jan 4;48(1):1-12. Epub 2014 Aug 4.

Department of Biology, The College of William and Mary, Williamsburg, VA 23185, USA.

Bacterial cold water disease (BCWD) is a chronic disease of rainbow trout, and is caused by the Gram-negative bacterium Flavobacterium psychrophilum (Fp), a common aquaculture pathogen. The National Center for Cool and Cold Water Aquaculture has bred two genetic lines of rainbow trout: a line of Fp-resistant trout (ARS-Fp-R or R-line trout) and a line of susceptible trout (ARS-Fp-S, or S-line). Little is known about how phenotypic selection alters immune response parameters or how such changes relate to genetic disease resistance. Herein, we quantify interindividual variation in the distribution and abundance of B cell populations (B cell signatures) and examine differences between genetic lines of naive animals. There are limited trout-specific cell surface markers currently available to resolve B cell subpopulations and thus we developed an alternative approach based on detection of differentially expressed transcription factors and intracellular cytokines. B cell signatures were compared between R-line and S-line trout by flow cytometry using antibodies against transcription factors early B cell factor-1 (EBF1) and paired domain box protein Pax5, the pro-inflammatory cytokine IL-1β, and the immunoglobulin heavy chain mu. R-line trout had higher percentages of EBF(+) B myeloid/ progenitor and pre-B cells in PBL, anterior and posterior kidney tissues compared to S-line trout. The opposite pattern was detected in more mature B cell populations: R-line trout had lower percentages of both IgM(+) mature B cells and IgM-secreting cells in anterior kidney and PBL compared to S-line trout. In vitro LPS-activation studies of PBL and spleen cell cultures revealed no significant induction differences between R-line and S-line trout. Together, our findings suggest that selective resistance to BCWD may be associated with shifts in naive animal developmental lineage commitment that result in decreased B lymphopoiesis and increased myelopoiesis in BCWD resistant trout relative to susceptible trout.
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http://dx.doi.org/10.1016/j.dci.2014.07.018DOI Listing
January 2015

Transfer of serum and cells from Yersinia ruckeri vaccinated doubled-haploid hot creek rainbow trout into outcross F1 progeny elucidates mechanisms of vaccine-induced protection.

Dev Comp Immunol 2014 May 14;44(1):145-51. Epub 2013 Dec 14.

School of Biological Sciences and Center for Reproductive Biology, Washington State University, Pullman, WA 99164-4236, USA.

Yersinia ruckeri is a well-established bacterial pathogen for many salmonid species, against which a formalin-killed bacterin vaccine has been effective in reducing disease outbreaks. Previous studies have reported conflicting results about the protective value of the systemic humoral response to Y. ruckeri vaccination. Here we directly demonstrate that plasma contains the long-term protective component elicited by both immersion and intraperitoneal injection vaccination of rainbow trout. A total of 0.5 μL of plasma from vaccinated fish provided almost complete protection against experimental challenge. Conversely, the cells obtained from peripheral blood conferred little or no protection in naïve recipients. The protective component of immune sera was IgM based on size exclusion chromatography and recognition by monoclonal antibody Warr 1-14. Immune plasma generated against a Y. ruckeri biotype 1 strain protected equally against challenges with Y. ruckeri biotype 1 and 2 strains. These results illustrate the importance of the humoral IgM response against Y. ruckeri and the use of doubled haploid rainbow trout (Oncorhynchus mykiss) and transfer of plasma/serum and cells into F1 outcross progeny as a model system for dissection of the mechanism(s) of vaccine-induced protection.
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http://dx.doi.org/10.1016/j.dci.2013.12.004DOI Listing
May 2014
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