[Show abstract][Hide abstract] ABSTRACT: We present the complete, closed, and finished chromosomal and extrachromosomal genome sequences of Yersinia ruckeri strain CSF007-82, the etiologic agent of enteric red mouth disease in salmonid fish. The chromosome is 3,799,036 bp with
a G+C content of 47.5% and encodes 3,530 predicted coding sequences (CDS), 7 ribosomal operons, and 80 tRNAs.
[Show abstract][Hide abstract] ABSTRACT: Weissellosis of rainbow trout is caused by the gram-positive bacteria Weissella ceti and has been reported in China, Brazil and the United States. This disease can result in high mortality in market-sized fish and thus can cause significant economic loss. Thus far, phenotypic characterization and 16S rRNA sequencing have been used to confirm a Weissellosis diagnosis. Here, we present the development of PCR-based diagnostic tools for the rapid identification and quantification of Weissella ceti within bacteriological culture and infected tissues. A duplex PCR, which amplifies both genus- and strain-specific targets, positively identifies isolates as Weissella ceti NC36. A qPCR assay was also developed to quantify pathogen load from infected tissues, using a Weissella ceti NC36 unique locus. A proof of concept study was performed to demonstrate that quantification using traditional plate count methods and qPCR were significantly correlated when assessed from infected brain and spleen tissue. These tools were also used to confirm diagnosis of Weissellosis in a commercial rainbow trout farm during an outbreak investigation. These are the first diagnostic tools developed for identification and quantification of Weissella ceti infection within rainbow trout, contributing to rapid Weissellosis diagnosis, enhanced pathogen surveillance, and epidemiological studies. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Abstract Text: Bacterial cold water disease (BCWD) causes significant economic losses in salmonids. In this study, we aimed to (1) predict genomic breeding values (GEBV) by genotyping training (n=583) and validation samples (n=53) with a SNP50K chip; and (2) assess the accuracy of genomic selection (GS) for BCWD resistance. For BCWD survival days and survival status, Bayes B method yielded higher accuracy of GS than Bayes C. For both disease phenotypes, accuracy of GS with Bayes B was moderate (0.45-0.50). The low accuracy of GS was due to using small training and validation samples. However, the accuracy of GS evaluated with cross-validation was variable (0.49-1.09) with a pooled accuracy of 0.77. The accuracy of GS for BCWD resistance will be increased using larger training and validation samples. To our knowledge, this is the first evaluation of GS accuracy with experimental data in salmonids.
10th World Congress on Genetics Applied to Livestock Production; 08/2014
[Show abstract][Hide abstract] ABSTRACT: 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 Yersinia 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 0.5μL 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.
[Show abstract][Hide abstract] ABSTRACT: 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.
Journal of Aquatic Animal Health 12/2013; 25(4):230-6. DOI:10.1080/08997659.2013.820225 · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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.
PLoS ONE 10/2013; 8(10):e75749. DOI:10.1371/journal.pone.0075749 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: All-female triploid fish are advantageous in rainbow trout (Oncorhynchus mykiss) aquaculture due to sterility and the consequent superior filet quality, growth, and feed conversion achieved at reproductive size. Triploid fish are commonly produced by pressure or temperature shock of the zygote (induced-triploids, 3NP), but can also be produced by mating a tetraploid parent with a diploid (2N) parent (intercross-triploids, 3NC). Little is known about the disease resistance of 3NC fish. In this study, we measured post-challenge survival of genetically-related 2N, 3NP and 3NC families after exposure to Flavobacterium psychrophilum the etiological agent of bacterial cold water disease (BCWD). The families used in this study originated from either an unselected commercial stock or BCWD resistant or susceptible line of rainbow trout. The overall survival of 2N fish was slightly higher than the respective 3NP and 3NC fish. Although 3NC fish tended to show higher survival than 3NP fish, this response was not sufficiently consistent to promote intercross production of triploids for increasing disease resistance. The survival of ten sets of 3NP, 3NC and 2N families, created from BCWD resistant and susceptible line parents, demonstrated overall similar inherent relative survival differences among the ploidy types both among families and between lines, although family differences in response to triploidization and triploidization strategy were observed. In summary, minimal survival difference between 3NP and 3NC families following experimental challenge suggests that innate resistance to F. psychrophilum is not substantially altered by the triploidization strategy and progress in family-based selective breeding of diploid fish for BCWD resistance will substantially translate into improved survival when triploids are derived either by shock treatment or intercross breeding. Published by Elsevier B.V.
[Show abstract][Hide abstract] ABSTRACT: Genetic variability in 16S rRNA gene sequences has been demonstrated among isolates of Flavobacterium columnare, and a restriction fragment length polymorphism (RFLP) assay is available for genetic typing of this important fish pathogen. Interpretation of restriction patterns can be difficult due to the lack of a formal description of the expected number and sizes of DNA fragments generated for each of the described genomovars. In this study, partial 16S rRNA gene sequences (ca. 1250-bp fragment) from isolates representing each described genomovar and isolates generating unique restriction patterns were cloned and sequenced. The results demonstrated that some isolates contained up to three different 16S rRNA genes whose sequences generate different RFLP patterns due to intragenomic heterogeneity within HaeIII restriction sites. The occurrence of HaeIII restriction sites within the portion of the 16S rRNA gene used for typing the F. columnare isolates and intragenomic heterogeneity within these sites explained the restriction patterns observed following RFLP analyses. This research provides a standard protocol for typing isolates of F. columnare by RFLP and a formal description of the expected restriction patterns for the previously described genomovars I, II, II-B and III. Additionally, we describe a new genomovar, I/II.
Journal of Fish Diseases 08/2013; 37(7). DOI:10.1111/jfd.12166 · 2.06 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 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 towards economical alternatives to costly fishmeal-based diets, such as grain-based formulations, and 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 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 and 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. Variation 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 only observed in evaluations of fin indices and 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.
[Show abstract][Hide abstract] ABSTRACT: Recent reports indicate that novel Weissella sp. bacteria have been associated with disease outbreaks in cultured rainbow trout (Oncorhynchus mykiss) at commercial farms in China and Brazil. In the summer of 2011, a severe disease outbreak displaying similar clinical signs occurred at a commercial rainbow trout farm in western North Carolina. Observed signs included dark skin coloration, lethargic swimming, bilateral exophthalmia, corneal opacity, ocular hemorrhage, occasional corneal rupture, and in some cases cerebral hemorrhage. Mortality was most severe in larger fish approaching market size. Bacteria isolated from moribund fish were identified to the genus level as Weissella sp. by 16S rRNA gene sequence analysis and were 99% identical to the sequences of isolates collected from the Chinese and Brazilian outbreaks. Laboratory-based disease challenge experiments using the isolated pathogen replicated both the disease signs and induction of mortality in exposed healthy rainbow trout. Aqueous vaccine formulations containing formalin-inactivated whole-cell Weissella sp. antigens conferred significant protection against experimental infection when both the vaccine and the pathogen were delivered by injection (relative percent survival [RPS] of 87.5% and 85% at 38 and 72 days after vaccination, respectively). The Weissella sp. vaccine was equally effective when combined with a commercially available Yersinia ruckeri vaccine, and this bivalent formulation did not alter the efficacy of the Y. ruckeri component of the vaccine. This is the first identification of this emerging rainbow trout disease, which we have termed Weissellosis, in the United States, and the spread of this pathogen might pose a significant threat to the domestic rainbow trout aquaculture industry. Our results also suggest that a bivalent Weissella/Y. ruckeri vaccine could be used as an effective and economical means for controlling this pathogen.
Aquaculture 04/2013; s 388–391(1):122–127. DOI:10.1016/j.aquaculture.2013.01.021 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Selective fish breeding programs for disease resistance comprise an increasingly important role in aquaculture production and offer an additional management tool for reducing bacterial-caused disease losses. Bacterial cold water disease (BCWD) is one of the most frequent causes of elevated mortality in juvenile salmonids, and we have selectively bred three genetic lines of rainbow trout for varying resistance to BCWD. These lines, designated ARS-Fp-R (resistant), ARS-Fp-C (control) and ARS-Fp-S (susceptible), differ in survival following standardized laboratory challenges with the causative agent of BCWD, Flavobacterium psychrophilum. This study evaluated survival of the genetic lines in laboratory challenges and in a production environment. Evaluations of disease resistance demonstrated a reproducible, 30% or greater, survival difference between ARS-Fp-R and ARS-Fp-S lines at body weights ranging from 0.7 to 13 g. Farm trials were performed to evaluate survival over an 80-day growth period starting after the trout began feeding. After a BCWD epizootic, the ARS-Fp-R line displayed significantly greater risk-adjusted survival (95.7%) than the ARS-Fp-S line (91.2%, P < 0.0001) and the ARS-Fp-C line (92.4%, P < 0.0001). Phenotype stability in farm-trial fish was also evaluated using laboratory challenges. The ARS-Fp-R line consistently displayed a higher, but not always statistically significant, survival percentage compared to the other lines and the data suggest that the magnitude of the survival phenotype difference is sensitive to environmental influence. In summary, the overall greater survival of the ARS-Fp-R line provides evidence of genetic improvement under production conditions.
Aquaculture 04/2013; s 388–391(1):128–136. DOI:10.1016/j.aquaculture.2013.01.018 · 1.88 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Novel Weissella sp. bacteria have recently been reported to be associated with disease outbreaks in cultured rainbow trout (Oncorhynchus mykiss) at commercial farms in China, Brazil, and the United States. Here we present the first genome sequence of this novel Weissella species, isolated from the southeastern United States.
[Show abstract][Hide abstract] ABSTRACT: Plasmid-mediated antibiotic resistance was first discovered in Edwardsiella ictaluri in the early 1990s, and in 2007 an E. ictaluri isolate harboring an IncA/C plasmid was recovered from a moribund channel catfish Ictalurus punctatus infected with the bacterium. Due to the identification of multidrug resistance plasmids in aquaculture and their potential clinical importance, we sought to determine whether the modified live E. ictaluri vaccine strain in AQUAVAC-ESC harbors such plasmids, so that the use of this vaccine will not directly contribute to the pool of bacteria carrying plasmid-borne resistance. Antimicrobial sensitivity testing of the E. ictaluri parent isolate and vaccine strain demonstrated that both were sensitive to 15 of the 16 antimicrobials tested. Total DNA from each isolate was analyzed by polymerase chain reaction (PCR) using a set of 13 primer pairs specific for conserved regions of the IncA/C plasmid backbone, and no specific products were obtained. PCR-based replicon typing of the parent isolate and vaccine strain demonstrated the absence of the 18 commonly occurring plasmid incompatibility groups. These results demonstrate that the vaccine strain does not carry resistance to commonly used antimicrobials and provide strong support for the absence of IncA/C and other commonly occurring plasmid incompatibility groups. Therefore, its use should not directly contribute to the pool of bacteria carrying plasmid-borne resistance. This work highlights the importance of thoroughly investigating potential vaccine strains for the presence of plasmids or other transmissible elements that may encode resistance to antibiotics.
Journal of Aquatic Animal Health 12/2011; 23(4):195-9. DOI:10.1080/08997659.2011.642093 · 0.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: To establish PCR-based assays for the rapid identification and differentiation of each of four known biotype 2 (BT2) phenotype-causing alleles in Yersinia ruckeri strains currently circulating in Europe and the United States.
Novel assays were developed relying on detection of mutant allele-specific changes in restriction enzyme cleavage sites within targeted PCR products. The developed assays were validated against isolates previously genotyped by DNA sequencing.
The described methods were specific, rapid and simple to perform and interpret.
The developed genotyping assays provide a valuable tool for identification and differentiation of specific BT2 strains of Y. ruckeri. These assays will be critical for the design and validation of new vaccines or other measures meant to control BT2 strains.
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: A study was undertaken to compare the virulence and serum killing resistance properties of Atlantic salmon and rainbow trout Yersinia ruckeri isolates. Five isolates, covering heat-stable O-antigen O1, O2 and O5 serotypes, were tested for virulence towards fry and juveniles of both species by experimental bath challenge. The sensitivity of 15 diverse isolates to non-immune salmon and rainbow trout serum was also examined. All five isolates caused significant mortality in salmon fry. Serotype O1 isolate 06059 caused the highest mortality in salmon (74% and 70% in fry and juveniles, respectively). Isolate 06041, a typical ERM-causing serotype O1 UK rainbow trout strain, caused mortalities in both rainbow trout and salmon. None of the salmon isolates caused any mortalities in 150-250 g rainbow trout, and only serotype O2 isolate 06060 caused any significant mortality (10%) in rainbow trout fry. Disease progression and severity was affected by water temperature. Mortality in salmon caused by the isolates 06059 and 05094 was much higher at 16 °C (74% and 33%, respectively) than at 12 °C (30 and 4% respectively). Virulent rainbow trout isolates were generally resistant to sera from both species, whereas salmon isolates varied in their serum sensitivity. Convalescent serum from salmon and rainbow trout that had been infected by serotype O1 isolates mediated effective classical pathway complement killing of serotype O1 and O5 isolates that were resistant to normal sera. Overall, strains recovered from infected salmon possess a wider range of phenotypic properties (relative virulence, O serotype and possession of serum-resistance factors), compared to ERM-causing rainbow trout isolates.
[Show abstract][Hide abstract] ABSTRACT: The bacterial pathogen Edwardsiella ictaluri is a primary cause of mortality in channel catfish raised commercially in aquaculture farms. Additional treatment and diagnostic regimes are needed for this enteric pathogen, motivating the discovery and characterization of bacteriophages specific to E. ictaluri.
The genomes of three Edwardsiella ictaluri-specific bacteriophages isolated from geographically distant aquaculture ponds, at different times, were sequenced and analyzed. The genomes for phages eiAU, eiDWF, and eiMSLS are 42.80 kbp, 42.12 kbp, and 42.69 kbp, respectively, and are greater than 95% identical to each other at the nucleotide level. Nucleotide differences were mostly observed in non-coding regions and in structural proteins, with significant variability in the sequences of putative tail fiber proteins. The genome organization of these phages exhibit a pattern shared by other Siphoviridae.
These E. ictaluri-specific phage genomes reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. Their genomic homogeneity is similarly observed among E. ictaluri bacterial isolates. The genomic analysis of these phages supports the conclusion that these are virulent phages, lacking the capacity for lysogeny or expression of virulence genes. This study contributes to our knowledge of phage genomic diversity and facilitates studies on the diagnostic and therapeutic applications of these phages.