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ARS Home » Northeast Area » Leetown, West Virginia » Cool and Cold Water Aquaculture Research » Research » Research Project #428108

Research Project: Integrated Research to Improve On-Farm Animal Health in Salmonid Aquaculture

Location: Cool and Cold Water Aquaculture Research

2018 Annual Report

1a. Objectives (from AD-416):
Objective 1: Define phenotypes and identify genetic markers to enhance selective breeding for disease resistance. Sub-objective 1.a. Selective breeding for improved CD resistance using the ARS-Fp-R line. Sub-objective 1.b. Evaluate approaches to exploit intra-family genetic variation for disease resistance to BCWD. Sub-objective 1.c. Fine-map the Omy19 BCWD QTL and determine mechanism of increased survival. Sub-objective 1.d. Evaluate survival, performance, environmental effects, and IHNV vaccination of ARS-Fp-R line in a 2015 large-scale field trial. Sub-objective 1.e. Evaluate ARS-Fp/Fc-R line in field trials. Sub-objective 1.f. Develop Fp and Fc isolate databases and elucidate genomic and virulence variation. Objective 2: Improve vaccine development through pathogen characterization. Sub-objective 2.a. Characterize expression of the Yr flagellar secretion phenotype during the infection process and characterize the role of flhDC in flagellar secretion regulation. Sub-objective 2.b. Identify flagellar regulatory elements and identify the flagellar secretion component(s) which antagonize virulence in Yr. Sub-objective 2.c. Evaluate strain TW32 as a live attenuated vaccine strain and as a novel carrier vaccine for en masse delivery of protein antigens to fish. Sub-objective 2.d. Delineate the molecular, structural and antigenic repertoire of the O-polysaccharides(O-PS) present in Fp and develop typing reagents. Objective 3: Genomic characterization of bacterial-host-environmental interactions leading to the disease state. Sub-objective 3.a. Metagenomic analysis of the aquaculture environment. Sub-objective 3.b. Determine the importance of Type III Secretion systems in mesophilic Aeromonads causing disease in rainbow trout.

1b. Approach (from AD-416):
Rainbow trout are a valuable finfish farmed in the U.S. and worldwide. Loss of trout from infectious disease is an important factor limiting production efficiency. Three prevalent bacterial diseases of rainbow trout are bacterial cold water disease (BCWD), enteric redmouth disease (ERM), and recently emerging, columnaris disease (CD). The goals of this project are to 1) develop well-characterized germplasm that exhibits dual on-farm resistance to both BCWD and CD, 2) utilize pathogen genomics to aid vaccine development and selective breeding, and 3)characterize both the host and aquaculture microbiome(s) associated with pathogen outbreaks. Our approach incorporates a comprehensive and multidisciplinary strategy that combines selective breeding, quantitative genetics, immunophenotyping, and functional genomics of pathogenic bacteria. This research builds on our previous studies in which we developed and released to industry, a BCWD resistant line (ARS-Fp-R) that has been extensively immunophenotyped, and have made progress in uncovering the genetic basis of disease resistance. In the first objective, we initiate selective breeding to improve CD survival, evaluate on-farm performance of single and double pathogen resistant lines and identify strategies for improving selective breeding for disease resistance. In the second objective, we characterize virulence factor regulation, develop serotyping tools, and evaluate new vaccine strategies to prevent disease. In the third objective, we utilize metagenomics and functional-genetic analyses to define the microbiome, identify virulence factors, and elucidate the contribution of these factors to disease outbreaks. The overall impact of this research is improved animal well-being, reduced antibiotic use and increased production efficiency.

3. Progress Report:
Progress was made on all three objectives and their sub-objectives in FY2018, all of which fall under National Program 106, Aquaculture (NP 106) Action Plan Components 1 and 3: Problem Statement 1B. Define Phenotypes and Develop Genetic Improvement Programs; Problem Statement 3A. Improve Understanding of Host Immunity, Immune System Evasion by Pathogens, and Disease-Resistant Phenotypes, and Problem Statement 3B. Control of Pathogens and Prevention of Disease. Sub-objective 1.a: We have previously shown that rainbow trout resistance to Flavobacterium columare, the causative agent of columnaris disease, is heritable and favorably correlated to resistance to Flavobacterium psychrophilum, the causative agent of bacterial cold water disease, in the USDA-NCCCWA (National Cool and Cold Water Aquaculture) nucleus population. Selection response after one generation of divergent selection for columnaris resistance was asymmetric; compared to a contemporary, unselected control line (ARS-Fp-R), survival of progeny from the resistant line (ARS-Fp/Fc-R) was only 2.5 percentage points higher, whereas survival of progeny from the susceptible line (ARS-Fc-S) was 16.6 percentage points lower. Broodstock have been selected based on family columnaris resistance breeding value estimates and retained from 40 ARS-Fp/Fc-R, 23 ARS-Fc-S, and 33 ARS-Fp-R families to produce second-generation families in 2019 to derive selection response for columnaris resistance through two generations. In addition, two consecutive generations of nucleus families from a commercial rainbow trout population were evaluated for resistance to columnaris and bacterial cold water disease, and data were analyzed using a bivariate animal threshold model to estimate heritabilities of, and genetic correlation between, these disease resistance traits. Similar to previous estimates from the USDA-NCCCWA nucleus population, resistance to columnaris and bacterial cold water disease were heritable (0.34 ± 0.09 and 0.43 ± 0.08, respectively) and favorably correlated (0.39 ± 0.18). Collectively, these data support the role of selective breeding in reducing mortalities and antibiotic use in rainbow trout aquaculture when fish are exposed to these pathogens. Sub-objective 1.c: This year we made progress toward characterizing two quantitative trait loci associated with resistance to bacterial cold water disease. These loci were previously mapped to rainbow trout chromosomes Omy 03 and Omy 19. Within the Omy 03 locus, we identified a candidate gene, interleukin-1 receptor-like 1 (Il1rl1 or ST2), a member of the interleukin-1 receptor family, that in mammals has pleiotropic roles including tissue homeostasis, inflammation, immune polarization, and disease resistance. A single orthologue was previously described in salmonid fish; however, the recently improved genome assembly of rainbow trout (Oncorhynchus mykiss) revealed three adjacent, tandem il1rl1 orthologues. We reported the genomic organization of the three il1rl1 genes (il1rl1alpha, il1rl1beta, il1rl1gamma), and used both RNA-seq and gene-specific qPCR methods to quantify expression patterns. Nucleotide sequence homology between the three genes was greater than 95% and each predicted protein contains three immunoglobulin-like domains, a transmembrane region and an intracellularToll/IL1 Receptor domain. In whole body lysates, il1rl1alpha was shown to have greater than 20-fold higher mRNA expression compared to il1rl1beta and il1rl1gamma. Furthermore, il1rl1alpha was more highly expressed in the ARS-Fp-R line fish as compared to the ARS-Fp-S line fish. Experiments are ongoing to test the association between gene expression and disease resistance in a separate commercial population of rainbow trout. Fish families predicted to be segregating for the Omy03 BCWD QTL were challenged with two strains of F. psychrophilum to measure the specificity of resistance. In collaboration with scientists from ARS research project project 8082-31000-012-00-D, current efforts are directed toward identifying the causative mutations on chromosomes Omy03 and Omy19 using whole genome resequencing. Sub-objective 1.e: In 2017 a large-scale farm-trial was initiated with a commercial producer located on the Columbia River where significant losses of large-sized rainbow trout during summer months have been attributed to columnaris disease. The trial compares the survival of a production lot of triploid, ARS-Fp-R line fish with the survival of an age-matched triploid commercial line. The early life-stage disease resistant phenotypes of both lines were confirmed at the NCCCWA using standardized BCWD and columnaris challenge procedures. Fish have been in net-pens since August 2017 and survival data has been and will continue to be collected through harvest, estimated to occur in early 2019. Sub-objective 1.f: A total of fifty F. columnare isolates from all known genomovars have been genome sequenced. Thirty-three of the isolates were associated with rainbow trout disease outbreaks at two farms. Phylogenetic analysis identified that isolates from the same farm were more similar than between farm sites. Sub-objective 2.c: We developed a live attenuated strain of Yersinia ruckeri and have tested utility as a novel carrier vaccine for delivery of proteins antigens to fish. We determined that expression of the gene encoding the green florescent protein in vaccine strain TW32 causes repression of flagellar synthesis causing this strain to partially regain virulence. This finding complicates the potential use of TW32 as an antigen delivery system and demonstrates that additional attenuating mutations will be necessary to ensure the safety of this approach. The role of filC (flagellin gene) in the virulence of Y. ruckeri and in the TW32 strain has also been determined. We have also initiated an experiment designed to determine the between-family variation in vaccine responsiveness among 50 ARS-Fp/Fc-R full-sib families (2017 year-class) and determine the heritability of this trait. This study will examine differences in response to injection vaccination against Yersinia ruckeri, a Gram-negative bacterium, and to injection vaccination against Lactococcus garvieae, a Gram positive bacterium. Poor response to vaccines has limited their development for aquaculture species and selective breeding for enhanced responsiveness could be beneficial. We also performed passive immunization studies showing that immune serum from L. garvieae-vaccinated fish confers protection against experimental challenge. This work shows that serum factors provide vaccination-induced protection and further establishes the correlation between L. garvieae- specific serum IgM and protection. Identification of specific serum IgM as a correlate of protection will allow us to monitor vaccine effectiveness indirectly by measuring antibody response and thus negate the need to perform experimental disease challenges. Sub-objective 2.d: We previously found that the o-polysaccharide (O-PS) gene clusters of F. psychrophilum strains 259-93 and 950106-1/1, which belong to different O-serotypes, are identical except for wzy, 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 (NMR), that the only structural difference between O-PS from these strains is the linkage between two sugars. The corresponding difference in O-serotype specificity was confirmed by the reactions of rabbit and trout anti-F. psychrophilum sera with purified O-PS as well as lipopolysaccharide (LPS). Moreover, differences in LPS antigenicity were noted among other F. psychrophilum strains with O-PS gene clusters that were identical to those of F. psychrophilum strains 259-93 or 950106-1/1, except for genes that are predicted to direct synthesis of different sugar modifications. The findings further our understanding of bacterial virulence factors by providing a framework for defining the genetic basis of O-PS structure and suggest that the repertoire of F. psychrophilum O-serotypes extends far beyond what is presently recognized from serological studies. Sub-objective 3.a: We expanded our planned 16S rDNA microbiome sampling scheme based on data collected in 2017. We analyzed over 150 samples collected at commercial production sites in Idaho and Washington. Our sampling scheme was modified to verify the detection of Flavobacterium columnare and F. psychrophilum on the surface of raceways and baffles. These results were critical for appropriately designing a replicate hatchery system that can be experimentally manipulated to study microbiome changes in aquaculture systems. Sub-objective 3.b: Type III secretion systems (T3SSs) are complex bacterial structures that provide gram-negative pathogens with a unique virulence mechanism enabling them to inject bacterial effector proteins directly into the host cell cytoplasm. Aeromonad fish pathogens contain genes for T3SSs but the contribution to disease is unclear. We succeeded in constructing multiple inactivating mutants of T3SSs effector system components. Aeromonas veronii strain Hm21 has two distinct T3SS; T3SS-1 and T3SS-2. We have mutants where either one or both of the T3SS are inactivated by deleting a key structural gene, ascV and yscV, respectively. In addition, we have mutated known and predicted effectors (toxins), aexT, aexU, aopX, ateA, ateB, aseG and tccC3; ascU was also inactivated in trout isolate BAQ71013-116. For selected effectors, we have also created double mutants. In addition, we sequenced the genomes of several fish pathogens, including Edwardsiella piscicida, F. columnare, F. psychrophilum, Lactococcus garvieae, and Weissella ceti.

4. Accomplishments

Review Publications
Duchaud, E., Rochat, T., Habib, C., Barbier, P., Loux, V., Guerin, C., Dalsgaard, I., Madsen, L., Nilsen, H., Sundell, K., Wiklund, T., Strepparava, N., Wahli, T., Caburlotto, G., Manfrin, A., Wiens, G.D., Fujiwara-Nagata, E., Avendano-Herrera, R., Bernardet, J., Nicolas, P. 2018. Genomic diversity and evolution of the fish pathogen Flavobacterium psychrophilum. Frontiers in Microbiology. 9(138).

Reichley, S.R., Ware, C., Steadman, J., Gaunt, P.S., Garcia, J.C., LaFrentz, B.R., Thachil, A., Stine, C.B., Waldbieser, G.C., Arias, C.R., Lock, T., Welch, T.J., Cipriano, R.C., Greenway, T.E., Khoo, L.H., Wise, D.J., Lawrence, M.L., Griffin, M.J. 2017. Comparative phenotypic and genotypic analysis of Edwardsiella spp. isolates from different hosts and geographic origins, with an emphasis on isolates formerly classified as E. tarda and an evaluation of diagnostic methods. Journal of Clinical Microbiology. 55:3466-3491.

Li, N., Zhu, Y., LaFrentz, B.R., Evenhuis, J., Hunnicutt, D.W., Conrad, R.A., Barbier, P., Gullstrand, C.W., Roet, J.E., Powers, J.L., Kulkami, S.S., Erbes, D.H., Garcia, J.C., Nie, P., McBride, M.J. 2017. The type IX secretion system is required for virulence of the fish pathogen Flavobacterium columnare. Applied and Environmental Microbiology. 83(23):e01769-17.

Talagrand-Reboul, E., Roger, F., Kimper, J., Colston, S.M., Graf, J., Laftif-Eugenin, F., Figueras, M., Petit, F., Marchadin, H., Jumas-Bilak, E., Lamy, B. 2017. Delineation of taxonomic species within complex of species: Aeromonas media and related species as a test case. Frontiers in Microbiology [serial online]. 8:621.

Nelson, M., Varney, J., Welch, T.J., Graf, J. 2016. Draft genome sequence of Lactococcus garvieae str. PAQ102015-99, an outbreak strain isolated from a commercial trout farm in the Northwestern United States. Genome Announcements. 4(4):e00781-16.

Bartelme, R.P., Barbier, P., Lipscomb, R.S., LaPatra, S.E., Evenhuis, J., McBride, M.J. 2018. Draft Genome Sequence of the Fish Pathogen Flavobacterium columnare Strain MS-FC-4. Genome Announcements. 6(20):e00429-18.

LaFrentz, B.R., Garcia, J.C., Waldbieser, G.C., Evenhuis, J., Loch, T.P., Liles, M.R., Wong, F.S., Chang, S.F. 2018. Identification of four distinct phylogenetic groups in Flavobacterium columnare with fish host associations. Frontiers in Microbiology. 9:452. doi: 10.3389/fmicb.2018.

Shaw, C.H., Gao, G., Wiens, G.D. 2018. Differential expression and evolution of three tandem, interleukin-1 receptor-like 1 genes in rainbow trout (Oncorhynchus mykiss). Developmental and Comparative Immunology. 87:193-203.

Kumar, G., Hummel, K., Welch, T.J., Razzazi-Fazeli, E., El-Matbouli, M. 2017. Global proteomic profiling of Yersinia ruckeri strains [online serial]. Veterinary Research. 48:55.