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.
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.
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.
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-1?, 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.
Abstract A challenge for improving disease resistance in fish through genetics is to understand specificity of resistance and whether selection for one pathogen alters the response to unrelated pathogenic microorganisms. Adult Rainbow Trout Oncorhynchus mykiss that had been bred for differential susceptibility to Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) and designated ARS-Fp-R (resistant), ARS-Fp-S (susceptible), and ARS-Fp-C (control line), as well as a pool of commercial-stock Rainbow Trout, were intraperitoneally challenged with Weissella sp. NC36. Clinical signs, survival, and innate mechanisms affecting disease resistance were monitored over 9 d. Acute disease signs included exophthalmia associated with retrobulbar inflammation and hemorrhage, cerebral hemorrhage, and mild to moderate granulomatous pericarditis. The ARS-Fp-R line did not demonstrate significant survival differences over a 9-d period compared with the ARS-Fp-C and ARS-Fp-S lines (P ? 0.09) indicating that during the acute phase of disease, the resistance factors that limit BCWD do not confer cross protection against Weissella sp. NC36. The linear effect of body weight at challenge was statistically significant, as each 10-g increase in body weight increased the hazard of death by 1% (P = 0.02). Bacterial loads on day 3, assessed by splenic and cerebral CFU counts, did not differ between ARS-Fp-R and ARS-Fp-S trout and there was no correlation between CFU counts and body weight. These findings help elucidate the specificity of disease resistance in selectively bred lines and contribute to our understanding of disease caused by Weissella sp., a recently described pathogen found in cultured Rainbow Trout. Received June 19, 2013; accepted June 23, 2013.
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?L 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.
Bacterial cold water disease (BCWD) causes significant economic loss in salmonid aquaculture. We previously detected genetic variation in survival following challenge with Flavobacterium psychrophilum (Fp), the causative agent of BCWD in rainbow trout (Oncorhynchus mykiss). A family-based selection program to improve resistance was initiated in 2005 at the USDA National Center for Cool and Cold Water Aquaculture. Select crosses were made in 2007 and 2009 to evaluate family-based disease survival using Fp injection challenges. From each putative F2/BC1 family generated in 2009, 200-260 fish were challenged in 4-7 replicates per family. Whole genome QTL scans of three F2/BC1 families were conducted with about 270 informative microsatellite loci per family spaced at an average interval size of 6 cM throughout the rainbow trout genome. Markers on chromosomes containing QTL were further evaluated in three additional F2/BC1 families. The additional F2/BC1 families were sire or dam half-sibs (HS) of the initially genome scanned families. Overall, we identified nine major QTL on seven chromosomes that were significant or highly significant with moderate to large effects of at least 13 % of the total phenotypic variance. The largest effect QTL for BCWD resistance explaining up to 40 % of the phenotypic variance was detected on chromosome OMY8 in family 2009070 and in the combined dam HS family 2009069-070. The nine major QTL identified in this study are candidates for fine mapping to identify new markers that are tightly linked to disease resistance loci for using in marker assisted selection strategies.
Rapid detection and quantification of Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD) in rainbow trout, are crucial to disease surveillance and encompass an essential component of BCWD research. Real-time, or quantitative polymerase chain reaction (qPCR) assays that have previously targeted the 16S rRNA gene of F. psychrophilum are complicated by polymorphisms and off-target amplification. Insignia primer and probe development software were used to identify a conserved single-copy signature sequence in the F. psychrophilum genome that codes for a hypothetical protein with unknown function. Primer and probes were used in a TaqMan qPCR assay that amplified 210 F. psychrophilum isolates with a lower limit of linear detection at 3.1 genome equivalents per reaction, with no amplification of 23 nontarget bacterial isolates. The assay was not inhibited by host spleen DNA or spleen homogenate. Methods were successfully applied to detect F. psychrophilum in rainbow trout from naturally occurring BCWD outbreaks and to quantify bacterial loads in experimentally infected rainbow trout. This assay will be applied to future studies to characterize disease pathogenesis in fish selectively bred for BCWD resistance.
As global aquaculture fish production continues to expand, an improved understanding of how environmental factors interact in fish health and production is needed. Significant advances have been made toward economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and toward defining the effect of rearing density on fish health and production. Little research, however, has examined the effects of fishmeal- and grain-based diets in combination with alterations in rearing density. Moreover, it is unknown whether interactions between rearing density and diet impact the composition of the fish intestinal microbiota, which might in turn impact fish health and production. We fed aquacultured adult rainbow trout (Oncorhynchus mykiss) fishmeal- or grain-based diets, reared them under high- or low-density conditions for 10 months in a single aquaculture facility, and evaluated individual fish growth, production, fin indices, and intestinal microbiota composition using 16S rRNA gene sequencing. We found that the intestinal microbiotas were dominated by a shared core microbiota consisting of 52 bacterial lineages observed across all individuals, diets, and rearing densities. Variations in diet and rearing density resulted in only minor changes in intestinal microbiota composition despite significant effects of these variables on fish growth, performance, fillet quality, and welfare. Significant interactions between diet and rearing density were observed only in evaluations of fin indices and the relative abundance of the bacterial genus Staphylococcus. These results demonstrate that aquacultured rainbow trout can achieve remarkable consistency in intestinal microbiota composition and suggest the possibility of developing novel aquaculture strategies without overtly altering intestinal microbiota composition.
Renibacterium salmoninarum is the causative agent of bacterial kidney disease, a major pathogen of salmonid fish species worldwide. Very low levels of intra-species genetic diversity have hampered efforts to understand the transmission dynamics and recent evolutionary history of this Gram-positive bacterium. We exploited recent advances in the next-generation sequencing technology to generate genome-wide single-nucleotide polymorphism (SNP) data from 68 diverse R. salmoninarum isolates representing broad geographical and temporal ranges and different host species. Phylogenetic analysis robustly delineated two lineages (lineage 1 and lineage 2); futhermore, dating analysis estimated that the time to the most recent ancestor of all the isolates is 1239 years ago (95% credible interval (CI) 444-2720 years ago). Our data reveal the intercontinental spread of lineage 1 over the last century, concurrent with anthropogenic movement of live fish, feed and ova for aquaculture purposes and stocking of recreational fisheries, whilst lineage 2 appears to have been endemic in wild Eastern Atlantic salmonid stocks before commercial activity. The high resolution of the SNP-based analyses allowed us to separate closely related isolates linked to neighboring fish farms, indicating that they formed part of single outbreaks. We were able to demonstrate that the main lineage 1 subgroup of R. salmoninarum isolated from Norway and the UK likely represent an introduction to these areas ?40 years ago. This study demonstrates the promise of this technology for analysis of micro and medium scale evolutionary relationships in veterinary and environmental microorganisms, as well as human pathogens.The ISME Journal advance online publication, 31 October 2013; doi:10.1038/ismej.2013.186.
Selective breeding of animals for increased disease resistance is an effective strategy to reduce mortality in aquaculture. However, implementation of selective breeding programs is limited by an incomplete understanding of host resistance traits. We previously reported results of a rainbow trout selection program that demonstrated increased survival following challenge with Flavobacterium psychrophilum, the causative agent of bacterial cold water disease (BCWD). Mechanistic study of disease resistance identified a positive phenotypic correlation between post-challenge survival and spleen somatic-index (SI). Herein, we investigated the hypothesis of a genetic correlation between the two traits influenced by colocalizing QTL. We evaluated the inheritance and calculated the genetic correlation in five year-classes of odd- and even-year breeding lines. A total of 322 pedigreed families (n?=?25,369 fish) were measured for disease resistance, and 251 families (n?=?5,645 fish) were evaluated for SI. Spleen index was moderately heritable in both even-year (h(2) ?=?0.56±0.18) and odd-year (h(2) ?=?0.60±0.15) lines. A significant genetic correlation between SI and BCWD resistance was observed in the even-year line (rg ?=?0.45±0.20, P?=?0.03) but not in the odd-year line (rg ?=?0.16±0.12, P?=?0.19). Complex segregation analyses of the even-year line provided evidence of genes with major effect on SI, and a genome scan of a single family, 2008132, detected three significant QTL on chromosomes Omy19, 16 and 5, in addition to ten suggestive QTL. A separate chromosome scan for disease resistance in family 2008132 identified a significant BCWD QTL on Omy19 that was associated with time to death and percent survival. In family 2008132, Omy19 microsatellite alleles that associated with higher disease resistance also associated with increased spleen size raising the hypothesis that closely linked QTL contribute to the correlation between these traits. To our knowledge, this is the first estimation of spleen size heritability and evidence for genetic linkage with specific disease resistance in a teleost fish.
The tumor necrosis factor superfamily (TNFSF) and the TNF receptor superfamily (TNFRSF) have an ancient evolutionary origin that can be traced back to single copy genes within Arthropods. In humans, 18 TNFSF and 29 TNFRSF genes have been identified. Evolutionary models account for the increase in gene number primarily through multiple whole genome duplication events as well as by lineage and/or species-specific tandem duplication and translocation. The identification and functional analyses of teleost ligands and receptors provide insight into the critical transition between invertebrates and higher vertebrates. Bioinformatic analyses of fish genomes and EST datasets identify 14 distinct ligand groups, some of which are novel to teleosts, while to date, only limited numbers of receptors have been characterized in fish. The most studied ligand is TNF of which teleost species possess between 1 and 3 copies as well as a receptor similar to TNFR1. Functional studies using zebrafish indicate a conserved role of this ligand-receptor system in the regulation of cell survival and resistance to infectious disease. The increasing interest and use of TNFSF and TNFRSF modulators in human and animal medicine underscores the need to understand the evolutionary origins as well as conserved and novel functions of these biologically important molecules.
Rainbow trout infected with Yersinia ruckeri, the causative agent of enteric redmouth disease (ERM), produce a pro-inflammatory and acute-phase response attributed in part to the innate recognition of bacterial-produced flagellin. Recently, variants of Y. ruckeri have been identified that lack flagella and associated motility. These strains are classified as biotype 2 (BT2) Y. ruckeri and these are considered an emerging problem in salmonid aquaculture. Little is known about the salmonid immune response to these variants. Herein, we report temporal and quantitative changes in rainbow trout immune response parameters following a primary challenge with BT2 Y. ruckeri strain YRNC10. Fish were injection-challenged with ten-fold dilutions of viable bacteria and sampled on days 1, 3, 5 and 7 post-challenge. TNFalpha1 and IL1-beta1 transcripts were increased by day 1 post-challenge, and on days 3, 5 and 7 maximal gene transcript up-regulation occurred at a threshold of approximately 64-256CFU per mg spleen tissue. Infection induced robust SAA gene up-regulation that was significantly correlated with increased gene expression of IL-1beta1 (r=0.81, P<0.0001) and TNFalpha1 (r=0.55, P<0.0001). Y. ruckeri infection induced modest changes in INFgamma and Mx-1 gene transcript abundance at intermediate or high challenge doses and the expression patterns of both genes were positively correlated with pro-inflammatory gene and acute-phase gene transcription patterns. TNF superfamily 13b (BAFF) gene expression was significantly down-regulated in response to infection on days 3, 5 and 7 at the highest challenge doses. The spleen somatic index was significantly increased on days 3, 5 and 7 post-infection and positively correlated with spleen colony forming units and abundance of gene transcripts SAA, TNFalpha1, and IL1-beta1. In summary, rainbow trout had a strong innate response following challenge with BT2 Y. ruckeri strain YRNC10 indicating that flagellin expression is not required for production of a robust pro-inflammatory and acute-phase gene transcription response. This study further supports the use of SAA transcript abundance and spleen somatic index as general measures of immunological status and fish health.
Induction of innate immune pathways is critical for early anti-microbial defense but there is limited understanding of how teleosts recognize microbial molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 1 and 2 form a heterodimer involved in recognizing peptidoglycans and lipoproteins of microbial origin. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR1 gene ortholog and its mRNA expression. Two TLR1 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA sequencing and genetic linkage analyses. Full length cDNA clone and direct sequencing of four BACs revealed an intact omTLR1 open reading frame (ORF) located on chromosome 14 and a second locus on chromosome 25 that contains a TLR1 pseudogene. The duplicated trout loci exhibit conserved synteny with other fish genomes that extends beyond the TLR1 gene sequences. The omTLR1 gene includes a single large coding exon similar to all other described TLR1 genes, but unlike other teleosts it also has a 5 UTR exon and intron preceding the large coding exon. The omTLR1 ORF is predicted to encode an 808 amino-acid protein with 69% similarity to the Fugu TLR1 and a conserved pattern of predicted leucine-rich repeats (LRR). Phylogenetic analysis grouped omTLR1 with other fish TLR1 genes on a separate branch from the avian TLR1 and mammalian TLR1, 6 and 10. omTLR1 expression levels in rainbow trout anterior kidney leukocytes were not affected by the human TLR2/6 and TLR2/1 agonists diacylated lipoprotein (Pam(2)CSK(4)) and triacylated lipoprotein (Pam(3)CSK(4)). However, due to the lack of TLR6 and 10 genes in teleost genomes and up-regulation of TLR1 mRNA in response to LPS and bacterial infection in other fish species we hypothesize an important role for omTLR1 in anti-microbial immunity. Therefore, the identification of a TLR2 ortholog in rainbow trout and the development of assays to measure ligand binding and downstream signaling are critical for future elucidation of omTLR1 functions.
Induction of the innate immune pathways is critical for early anti-viral defense but there is limited understanding of how teleost fish recognize viral molecules and activate these pathways. In mammals, Toll-like receptors (TLR) 7 and 8 bind single-stranded RNA of viral origin and are activated by synthetic anti-viral imidazoquinoline compounds. Herein, we identify and describe the rainbow trout (Oncorhynchus mykiss) TLR7 and TLR8 gene orthologs and their mRNA expression. Two TLR7/8 loci were identified from a rainbow trout bacterial artificial chromosome (BAC) library using DNA fingerprinting and genetic linkage analyses. Direct sequencing of two representative BACs revealed intact omTLR7 and omTLR8a1 open reading frames (ORFs) located on chromosome 3 and a second locus on chromosome 22 that contains an omTLR8a2 ORF and a putative TLR7 pseudogene. We used the omTLR8a1/2 nomenclature for the two trout TLR8 genes as phylogenetic analysis revealed that they and all the other teleost TLR8 genes sequenced to date are similar to the zebrafish TLR8a, but are distinct from the zebrafish TLR8b. The duplicated trout loci exhibit conserved synteny with other fish genomes extending beyond the tandem of TLR7/8 genes. The trout TLR7 and 8a1/2 genes are composed of a single large exon similar to all other described TLR7/8 genes. The omTLR7 ORF is predicted to encode a 1049 amino acid (aa) protein with 84% similarity to the Fugu TLR7 and a conserved pattern of predicted leucine-rich repeats (LRR). The omTLR8a1 and omTLR8a2 are predicted to encode 1035- and 1034-aa proteins, respectively, and have 86% similarity to each other. omTLR8a1 is likely the ortholog of the only Atlantic salmon TLR8 gene described to date as they have 95% aa sequence similarity. The tissue expression profiles of omTLR7, omTLR8a1 and omTLR8a2 in healthy trout were highest in spleen tissue followed by anterior and then posterior kidney tissues. Rainbow trout anterior kidney leukocytes produced elevated levels of pro-inflammatory and type I interferon cytokines mRNA in response to stimulation with the human TLR7/8 agonist R848 or the TLR3 agonist poly I:C. Only poly I:C-induced IFN2 transcription was significantly suppressed in the presence of chloroquine, a compound known to block endosomal acidification and inhibit endosomal maturation. The effect of chloroquine on R848-induced cytokine expression was equivocal and so it remains questionable whether rainbow trout recognition of R848 requires endosomal maturation. TLR7 and TLR8a1 expression levels in rainbow trout anterior kidney leukocytes were not affected by poly I:C or R848 treatments, but surprisingly, TLR8a2 expression was moderately down-regulated by R848. The down-regulation of omTLR8a2 may imply that this gene has evolved to a new or altered function in rainbow trout, as often occurs when the two duplicated genes remain active.
The 57 kDa protein (p57) is an important diagnostic antigen that is implicated in the pathogenesis of salmonid bacterial kidney disease. Little is known about the nature and extent of antigenic variation in p57. Previously, we reported that p57 produced by Renibacterium salmoninarum Strain 684 contains a mutation that disrupts monoclonal antibody (MAb) 4C11 binding. In the present study, we examined MAb binding to a panel of 23 additional R. salmoninarum isolates obtained from diverse geographic locations to examine the prevalence of this variant and whether additional variability exists within other p57 epitopes. Six p57-specific MAbs (4C11, 4D3, 3H1, 4H8, 4D10 and 1A1) were used to probe dot and western blots to determine the relative expression, size and cellular association of p57. Full-length p57 was produced by all isolates, and for each isolate, the protein was associated with the bacterial cell surface. The epitopes recognized by 4 MAbs, 4D3, 4H8, 3H1 and 1A1, were conserved among all strains tested. The 4C11 epitope was absent in 5 of 8 strains originating from Norway, while the 4D10 epitope was partially disrupted in one isolate from British Columbia, Canada. The 5 Norwegian antigenic-variant strains appeared to be clonally related as they shared the following characteristics: one tandem repeat in the ETRA locus, a Sequovar-4 16-23S rRNA intervening DNA sequence, a larger XhoI fragment in the msa1 5 region, and absent msa3 gene. These results indicate that limited antigenic and genomic variation exists between strains and this variation appears geographically restricted in distribution.
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