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.
Edwardsiella ictaluri is the etiologic agent of enteric septicemia of catfish, which causes substantial losses in catfish aquaculture. To determine pathogen-host interactions, previous studies have used the green fluorescence protein (GFP) gene. Here, the pEI2 plasmid of E. ictaluri isolate I49 was tagged using a Tn10-GFP-kan cassette to create the green fluorescence-expressing derivative I49-gfp. The Tn10-GFP-kan insertion site was mapped by plasmid sequencing to 663 bp upstream of open reading frame 2 and appeared to be at a neutral site in the plasmid. Purification of the pEI2::GFPKan plasmid and mobilization into E. coli resulted in GFP expression. The isolated pEI2::GFPkan plasmid was used to retransform the wild type I49 isolate (ensuring a single Tn10-GFP-kan insertion) and an independent E. ictaluri isolate, S97-73-3. The wild type and the green fluorescent-tagged strains were compared for modulation of pathogenicity in channel catfish Ictalurus punctatus by immersion challenge. A significant reduction in mortalities occurred for the I49GFPkan strain as compared to its isogenic parent, but no difference was observed between the S97-73-3GFPkan strain and the S97-73-3 wild type. This GFP-tagged plasmid will be useful for determining the effects that the pEI2::GFPkan plasmid has on virulence and host-pathogen interactions between E. ictaluri isolates.
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.
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.
Biotype 2 (BT2) variants of the bacterium Yersinia ruckeri are an increasing disease problem in U.S. and European aquaculture and have been characterized as serovar 1 isolates that lack both peritrichous flagella and secreted phospholipase activity. The emergence of this biotype has been associated with an increased frequency of enteric redmouth disease (ERM) outbreaks in previously vaccinated salmonid fish. In this study, four independent specific natural mutations that cause the loss of both motility and secreted lipase activity were identified in BT2 strains from the United States, United Kingdom, and mainland Europe. Each of these was a unique mutation in either fliR, flhA, or flhB, all of which are genes predicted to encode essential components of the flagellar secretion apparatus. Our results demonstrate the existence of independent mutations leading to the BT2 phenotype; thus, this phenotype has emerged separately at least four times. In addition, BT2 strains from the United Kingdom were shown to have the same mutant allele found in U.S. BT2 strains, suggesting a common origin of this BT2 lineage. This differentiation of distinct BT2 lineages is of critical importance for the development and validation of alternative vaccines or other treatment strategies intended for the control of BT2 strains.
The characteristic increase in protein catabolism during muscle atrophy is largely the result of an increase in E3 ubiquitin ligase expression, specifically that of atrogin-1, or FBXO32, which functions to polyubiquitinate proteins. In rainbow trout, the cDNA sequences of two E3 ubiquitin ligase F-box proteins, FBXO32 and FBXO25, were characterized and their expression across tissues in response to feed deprivation was determined. The cDNA sequence for FBXO32 encodes a protein 355 amino acids long and is 97% identical to the homologous protein in salmon, 85% to zebrafish and 72% identical to both human and mouse. The cDNA for FBXO25 encodes a protein 356 amino acids in length that is 98% identical to the homologous protein in salmon, 84% to zebrafish, and 75% to human. After 28days of feed deprivation, FBXO32 expression increased by approximately 13-fold, 3-fold, and 5-fold in white muscle, red muscle, and intestine, respectively (P<0.05). Expression of FBXO32 and FBXO25 in kidney decreased 0.3-fold and 0.2-fold, respectively, and FBXO25 expression decreased by 0.2-fold in liver (P<0.05). These results indicate that these protein sequences are conserved and suggest that the up-regulation of FBXO32 is associated with skeletal and smooth muscle atrophy that occurs during fasting.
Here we demonstrate that flagellar secretion is required for production of secreted lipase activity in the fish pathogen Yersinia ruckeri and that neither of these activities is necessary for virulence in rainbow trout. Our results suggest a possible mechanism for the emergence of nonmotile biotype 2 Y. ruckeri through the mutational loss of flagellar secretion.
The mucosal immune systems of fishes are still poorly understood, and defined models for studying natural host-pathogen interactions are lacking. The objective of this study was to evaluate different challenge models and pathogens to examine the magnitude of change in intestinal immune gene expression. Rainbow trout were exposed by immersion to Yersinia ruckeri or by intraperitoneal injection with Flavobacterium psychrophilum. At 3, 9, or 10 days post-challenge, pathogen load was quantified by plate count and intestinal tissue was removed and immune gene expression measured by real-time PCR. In general, the magnitude of infection was correlated with change in immune gene transcript abundance. We found that messages for the innate immune molecules, SAA, IL-8, INF-? and TNF-?, as well as the message for IgM, were up-regulated in intestinal tissue in both challenge paradigms. A >250-fold increase was observed in SAA and 20-fold increase of IL-8 gene transcript abundance occurred on day 10 following challenge with F. psychrophilum. Within individual fish, there was a positive correlation between bacteria load in the spleen and the increase of immune gene message between 3 and 10 days post-infection. These findings demonstrate that measurable changes in immune gene expression occur in the intestine of rainbow trout following bath challenge with Y. ruckeri or injection challenge with F. psychrophilum.
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