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

Location: Cool and Cold Water Aquaculture Research

2019 Annual Report

Objectives
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.

Approach
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.

Progress Report
Progress was made on three objectives and their sub-objectives in FY2019, 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: Second-generation families from the ARS-Fp/Fc-R (100 families), ARS-Fp-R (pool of 35 families), and ARS-Fc-S (23 families) were produced and evaluated for resistance to columnaris challenge using our standard laboratory-based disease challenge model. Average survival of second-generation families was 19.1% (ARS-Fp/Fc-R), 19.7% (ARS-Fp-R), and 6.2% (ARS-Fc-S), and these differences among the three lines suggest that there was no additional selection response in the second generation. The limited and asymmetric selection response in the first generation was plausible given the high survival rates in the base population (generation 0), but the lack of selection response in the second generation was unexpected. We have convincing evidence that resistance to columnaris disease is a heritable trait that can be improved by selective breeding from: 1) estimates of favorable genetic correlation (0.35 – 0.40) with bacterial cold water disease resistance in two unrelated nucleus rainbow trout populations and a favorable correlated response in columnaris resistance when selection was practiced for increased bacterial cold water resistance; and 2) comparative analysis of columnaris disease resistance among three rainbow trout lines divergently selected for resistance to bacterial cold water disease. The lack of response to direct selection for columnaris disease resistance in our current population suggests fundamental issues with our genetic model, the repeatability of our disease challenge model, or both. Current and future efforts are aimed at understanding the lack of response to direct selection for columnaris resistance in our population. Sub-objective 1.c.: This year, in collaboration with scientists from Project 8082-31000-012-00D, we continued study of quantitative trait loci (QTL) associated with resistance to bacterial cold water disease (BCWD). Previously, a QTL was identified on trout chromosome Omy03 and three tandem interleukin-1 receptor-like 1 genes were proposed as potential candidate genes. One of the three genes, il1rl1-alpha exhibited higher expression in the BCWD resistant line, ARS-Fp-R, and lower expression in the BCWD susceptible line, ARS-Fp-S. A single nucleotide polymorphism (SNP) was identified that correlated with differential expression and association mapping was performed using several families from an independent population, TLUM17. Variable SNP association results were obtained using two individual families and thus the relationship between the candidate genes and the Omy03 QTL is uncertain. Whole genome resequencing has also been performed on fish carrying the Omy03 and Omy19 QTL’s and analyses are ongoing. 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 and genotyping is ongoing. Additional crosses have been made this year to further aid QTL mapping efforts. Sub-objective 1.f: This year in collaboration with researchers at Michigan State University, we published the analysis of 314 Flavobacterium psychrophilum isolates obtained from ten fish species originating from twenty US states and one Canadian province. These isolates were collected over a nearly four-decade period. We utilized multilocus sequence typing (MLST) to compare the relatedness of the isolates. We identified 66 different sequence types (ST) of which 47 were new. The most common type associated with disease was ST10, and combined with related variants, this sequence type had the widest geographical distribution and infected the most different types of fish. We also found the same sequence type in both female broodstock and progeny suggesting that egg disinfection practices are not fully effective. This information is being used to more appropriately develop control measures to monitor and reduce the severity of BCWD. Sub-objective 2.c: We previously demonstrated that unregulated flagellin expression in Yersinia ruckeri mutant strain TW32 causes inappropriate flagellin expression during infection, likely triggering host recognition and immune stimulation. We believe that these unique properties of strain TW32 make it an ideal live-attenuated vaccine because its attenuation is dependent on stimulation of an immune response. We have tested this by comparing the response elicited by exposure to TW32 to that of a standard auxotrophic (metabolically disabled) aroA mutant and to a TW32/aroA double mutant. Immersion vaccination with TW32 elicited a strong anti-Y. ruckeri LPS IgM response, while the aroA and TW32/aroA double mutant were less immunogenic. These results demonstrated that the TW32 mutant is a superior live-attenuated vaccine strain. In addition, the observation that the TW32/aroA double mutant was also poorly immunogenic suggests that the increased expression of flagellin in TW32 alone does not explain its increased immunogenicity. We have also continued to assist with a Weissella ceti outbreak occurring at a trout aquaculture facility in Canada. We have demonstrated that application of an autogenous vaccine against this strain confers protection under laboratory conditions and are in the process of confirming the efficacy and duration of protection of this vaccine when applied under farm conditions. Sub-objective 3.a: We expanded our sampling scheme based on our data collected in 2018 focusing on Clear Spring Foods, CSF, in Idaho. We analyzed 142 samples focusing especially on the raceways in the hatchery. In addition, water samples were spiked with Flavobacterium columnare and F. psychrophilum to verify their detection using Next Generation sequencing and droplet digital PCR. In addition, biofilms were observed using Fluorescence in situ hybridization. Different surfaces were tested for biofilm formation at the NCCWA. Sub-objective 3.b: We developed a bioinformatic pipeline to discover previously unreported toxins in Aeromonas. In a comparison of 105 genomes we discovered 21 putative toxins of which 13 had not been reported in Aeromonas. To assess the toxicity of them we cloned them into expression vectors and tested them for cytotoxicity in yeast. 15 of the 21 tested were cytotoxic including 9 that had not been previously reported in Aeromonas. We also succeeded in constructing a number of mutants. Aeromonas veronii strain Hm21 has two distinct T3SS (T3SS-1 and T3SS-2). We have mutants where either or both of the T3SS is inactive by deleting a key structural gene, ascV and yscV, respectively. In addition, we have mutated known and predicted effectors (toxins) and tested aexT, aexU, and aopX for virulence in zebra fish. When infected with the aexT/aexU/aopX triple mutant, 60% of the animals survived while 30% survived when infected with the wild type. The same strains were tested in cytotoxicity assays with EPC cells (derived from Pimephales promelas) and RTGill-W1 cells (derived from Oncorhynchus mykiss). No effects were noted with RTGill-W1 cells but a slight delay in cytotoxicity was observed in EPC cells when exposed to the triple effector mutant.

Accomplishments
1. Selective breeding improves resistance to bacterial cold water disease and columnaris disease. Bacterial cold water disease and columnaris disease are important diseases that affect rainbow trout aquaculture. Antibiotics are routinely used to control these diseases because there are limited alternative control strategies currently available. ARS researchers in Leetown, West Virginia, evaluated the genetics of resistance to both diseases in two rainbow trout populations. Resistance to both diseases was found to be heritable and favorably genetically correlated, suggesting that a rainbow trout’s resistance to both diseases is due, at least in part, to shared genes. Based on these studies, molecular genetic approaches are now being used to identify the actual genes that affect disease resistance. Commercial breeders that select for improved resistance to only one of the diseases can expect to reduce the impacts of both diseases in their population.

2. The pathogen Yersinia ruckeri can sense its host. Disease-causing bacteria have evolved clever systems to recognize their hosts, and they respond by turning off functions that might trigger an immune response. ARS researchers in Leetown, West Virginia, demonstrated that the pathogen Yersinia ruckeri shuts off production of the flagellum when it senses its rainbow trout host. The flagellum is a whip-like structure that bacteria use for locomotion, but it also is a potent immune stimulator. By creating a mutant Yersinia ruckeri strain that cannot shut off flagellum expression, the researchers demonstrated that absence of the flagellum during infection is critical for the bacteria to avoid being recognized, and subsequently killed, by the fish’s immune system. This work provides a better understanding of the factors leading to infection and will guide development of new vaccines for disease control.

Review Publications
Brown, R., Wiens, G.D., Salinas, I. 2018. Analysis of the gut and gill microbiome of resistant and susceptible lines of rainbow trout (Oncorhynchus mykiss). Fish and Shellfish Immunology. 86:497-506. https://doi.org/10.1016/j.fsi.2018.11.079.
Wiens, G.D., Palti, Y., Leeds, T.D. 2018. Three generations of selective breeding improved Rainbow trout (Oncorhynchus mykiss) disease resistance against natural challenge with Flavobacterium psychrophilum during early life-stage rearing [abstract]. Aquaculture. 497(2018):414-421.
Knupp, C., Wiens, G.D., Faisal, M., Call, D.R., Cain, K.D., Nicolas, P., Van Vliet, D., Yamashita, C., Ferguson, J.A., Meuninck, D., Hsu, H., Baker, B.B., Shen, L., Loch, T.P. 2019. Large-scale analysis of Flavobacterium psychrophilum MLST genotypes recovered from North American salmonids indicates both newly identified and recurrent clonal complexes are associated with disease. Applied and Environmental Microbiology. Mar 6;85(6)e02305-18.
Gulla, S., Barnes, A.C., Welch, T.J., Romalde, J.L., Rydere, D., Ormsby, M.J., Carson, J., Lagesen, K., Verner-Jeffreys, D.W., Davies, R.L., Colquhoun, D.J. 2018. Multi-Locus Variable number of tandem repeat Analysis (MLVA) of Yersinia ruckeri 2 confirms the existence of host-specificity, geographic endemism and anthropogenic 3 dissemination of virulent clones. Applied and Environmental Microbiology. doi:10.1128/AEM.00730-18.
Kumar, G., Hummel, K., Noebauer, K., Welch, T.J., Razzazi-Fazeli, E., El-Matboul, M. 2019. Proteome analysis reveals a role of rainbow trout lymphoid organs during the Yersinia ruckeri infection process. Scientific Reports. 8:13998. https://doi.org/10.1038/s41598-018-31982-6.
Jozwick, A., La Patra, S., Graf, J., Welch, T.J. 2019. Flagellar regulation mediated by the Rcs pathway is required for virulence in the fish pathogen Yersinia ruckeri. Fish and Shellfish Immunology. 91:306.314. https://doi.org/10.1016/j.fsi.2019.05.036.
Goldstein, S., Beka, L., Graf, J., Klassen, J. 2019. Evaluation of strategies for the assembly of diverse bacterial genomes using MinION long-read sequencing. BMC Bioinformatics. 20(23):1-17. https://doi.org/10.1186/s12864-018-5381-7.
Silva, R., Evenhuis, J., Vallejo, R.L., Tsuruta, S., Wiens, G.D., Martin, K., Parsons, J., Palti, Y., Lourenco, D., Leeds, T.D. 2018. Variance and covariance estimates for resistance to bacterial cold water disease and columnaris disease in two rainbow trout breeding populations. Journal of Animal Science. 97(3):1124-1132. https://doi.org/10.1093/jas/sky478.
Cisar, J.O., Bush, A., Wiens, G.D. 2019. Comparative structural and antigenic characterization of genetically distinct Flavobacterium psychrophilum O-polysaccharides. Frontiers in Microbiology. 10:1041. https://doi.org/10.3389/fmicb.2019.01041.
Rangel, L.T., Marden, J., Sestubal, J.C., Graf, J., Gogarten, J.P. 2019. Identification and characterization of putative Aeromonas spp. T3SS effectors. PLoS One. 14(6):e0214035. https://doi.org/10.1371/journal.pone.0214035.
Talagrand-Reboul, E., Latif-Eugenin, F., Beaz-Hidalgo, R., Colston, S., Figueras, M., Graf, J., Jumas-Bilak, E., Lamy, B. 2018. Genome-driven evaluation and redesign of PCR tools for improving the detection of virulence-associated genes in aeromonads. PLoS One. 13(8):e0201428. https://doi.org/10.1371/journal.pone.0201428.