Research Project: Reducing Impacts of Disease on Salmonid Aquaculture Production

Location: Office of The Director

2019 Annual Report

Objectives
Major impediments to production and profitability of U.S. aquaculture are the lack of genetically-defined species with traits for faster growth, greater feed efficiency/utilization, and improved disease resistance. Rainbow trout are important recreational and food fish species in the Great Lakes and it is thus important to improve productivity of this species in this region. Over these next 3 years we will focus on the following three Objectives and their supporting Sub-Objectives: Objective 1: Characterize mechanisms of innate immune response, and pathogen virulence, to control rhabdoviral diseases in salmonid aquaculture. • Sub-Objective 1.A.: Identify domains within viral proteins of infectious hematopoietic necrosis virus (IHNV) and viral hemorrhagic septicemia virus (VHSV) that interfere with the host virus recognition and response pathways in vitro. (Leaman, Stepien and Vakharia) • Sub-Objective 1.B.: Characterize the in vitro replication of recombinant IHNV and VHSV containing mutations designed to disrupt viral suppression of host recognition and response pathways. (Leaman and Vakharia) • Sub-Objective 1.C.: Assess the impact of IHNV and VHSV infection and subsequent innate immune suppression on the activation of dendritic cells (DCs). (Spear) • Sub-Objective 1.D.: Develop in vivo IHNV and VHSV challenge models in rainbow trout. (Spear and Shepherd) Objective 2: Use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture. • Sub-objective 2.A.: Develop genetic techniques for F. columnare strains that cause columnaris disease in rainbow trout. (McBride) • Sub-Objective 2.B.: Isolate and characterize F. columnare mutants and identify virulence factors associated with ability to cause disease in rainbow trout. (McBride) • Sub-Objective 2.C.: Develop improved genetic techniques for F. psychrophilum, the causative agent of bacterial coldwater disease. (McBride) Objective 3: Measure and modulate antimicrobial peptides (AMPs) as a means to control disease in salmonids. • Sub-Objective 3.A.: Characterize the environmental and endocrine contributions to regulation and expression of AMPs in rainbow trout. (Shepherd and Spear) • Sub-Objective 3.B.: Test the anti-viral and anti-bacterial activities of two synthetic trout AMPs in vitro. (Shepherd, Spear, Leaman and McBride)

Approach
For Objective 1: We will characterize the mechanisms of virulence for Viral Hemorrhagic Septicemia virus (VHSV) and Infectious Hematopoietic Necrosis virus (IHNV) in rainbow trout. Research will involve molecular analysis of viral diversity, mutational analysis of viral factors contributing to virulence in rainbow trout. These studies will utilize homologous in vitro systems (cell-lines and dendritic cells) to identify of host factors involved in recognition and response pathways to viral infection in rainbow trout. Lastly, disease challenge assays will be developed, and validated, to understand the virulence and the disease processes for IHNV and VHSV pathogens in rainbow trout. For Objective 2: This work will target mechanisms of pathogenesis of F. psychrophilum (causative agent in bacterial cold water disease) and F. columnare (causative agent in columnaris disease) in rainbow trout. To do this, we will use bacterial culture and genetic techniques to isolate mechanisms of pathogensis for Flavobacterium spp. in this species. Attenuated bacterial strains will be evaluated for pathogenesis using established disease challenge models, in this species. For Objective 3: We will characterize the physiological regulation of antimicrobial peptides (AMPs) and their actions in rainbow trout. To accomplish this, we will assess how environmental stressors, and hormones, affect expression (genes) and levels (proteins) of AMPs in this species. Additionally, we shall utilize in vitro techniques to evaluate biocidal actions of select AMPs against VHSV and IHNV and Flavobacterium spp.

Progress Report
For Objective 1, the aim is to characterize mechanisms of innate immune response, and pathogen virulence, to control rhabdoviral diseases in salmonid aquaculture. Comparative studies have been conducted between VHSV and IHNV genes, across four different fish cell lines, to enable transitioning of this work into using rainbow trout cell-lines. New mutational analyses have begun with the IHNV, NV gene/protein, by starting at the two ends of the protein (the N- and C-terminal ends). These deletions (5, 10 and 20 amino acids at one or both ends) are now completed and the resultant cDNAs are cloned into expression plasmids. To test these mutants, it was necessary to develop an assay with a quantifiable endpoint to screen new VHSV or IHNV NV mutants for gain or loss of activity, in lieu of the traditional apoptosis assay, which has limitations. This new assay involves characterization of the NV protein in regulating both protein processing through the endoplasmic reticulum (ER) and, ultimately, transcription and/or apoptosis through the cellular ER stress, or unfolded protein, response (UPR) via a key pro-apoptotic transcription factor mRNA called CHOP (CCAAT-enhancer-binding protein homologous protein). It was found that NV can alter CHOP mRNA induction by ER stressors in fish cells and that an N-terminal truncation of NV has enhanced activity, suggesting the presence of key regulatory domains within the first 20 amino acids. These results demonstrate development of an informative endpoint, and evidence for regulatory domains, that can allow hypothesis-driven experiments to be pursued to determine if NV promotes cell survival, and viral replication, by suppressing host attempts to slow ER protein synthesis. Using this assay, mutants that show reduced anti-transcriptional activity will progress to refinement and re-testing as efforts move towards development of attenuated NV therapeutics. Ongoing studies on the role of VHSV IVb NV implicate a role of either NV RNA or NV protein (or both) in regulating translation during VHSV infection. Removal of NV from VHSV reduced virus-induced interferon (IFN) expression as compared to rWT (recombinant wild-type, or unaltered) VHSV infection. Functional NV removal also significantly increased viral RNA accumulation, but decreased both viral protein expression and viral yield. NV deficient VHSV infection elicited a decreased mRNA abundance for a key translational factor (eIF2a mRNA), which is a marker for suppression of protein synthesis, as compared to rWT VHSV at variable multiples of virus infection (MOIs). Together, these data implicate a role of NV in host translation. Adding an inhibitor of protein kinase R (a kinase that regulates host innate responses), to EPC cells prior to infection decreased levels of eukaryotic initiation factor 2, and viral protein expression, like what was seen following infection with NV deficient virus. These findings suggest that IVb NV protein or RNA impact viral protein synthesis and host translation during VHSV infection and are consistent with the ER stress studies described above. Ongoing efforts will be directed toward a unifying hypothesis of NV action that should be of great interest to the researchers as it could aid in designing NV mutants for further study and potential use as vaccine candidates. Work has also begun to characterize the role of stress granules (SGs) in the host viral response pathway in rainbow trout. SGs are membraneless ribonucleoprotein (RNP) aggregates that contain an array of translationally stalled mRNAs and RNA-binding proteins that are required for their formation and stability. Recent research in mammals shows that viral infection activates formation of SGs, which coincides with initiation of antiviral responses by pattern recognition receptors (PRRs), but our understanding of these processes in fish is poor. In this last year, assays were developed to study the role of SG in teleost innate immune responses. One of the key proteins in human cells that is needed for SG formation is G3BP1 (Ras GTPase-activating protein SH3-domain-binding protein). Given similarities between human and trout G3BP1 sequences, we have determined that human antibodies to this protein are also cross-reactive to in cell lysates of RTG-2 and RTgill-W1 cells as determined by immunoblotting. Expanding on this, methods for immunofluorescence microscopy to monitor SG formation, in response to heat shock and oxidative stress, have been developed and used to show formation of SGs in rainbow trout cells. SG formation is triggered by phosphorylation of eIF2-alpha (eukaryotic initiation factor 2), which is a key regulator of protein translation in cells. Again, using human reactive antibodies this group has demonstrated specific activation of rainbow trout eIF2-alpha in response to stress inducers. Infection with VHSV remarkably induced SG formation concomitant with phosphorylation of eIF2-alpha. There is overlap in roles of stress pathways, particularly SGs, in viral and bacterial pathogenesis in mammals and the focus of this work is to characterize involvement of SGs in the fish host immune response using rainbow trout cell-lines. For Objective 2, the aim was to use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture. The genetic manipulation techniques developed in year-1, and the genome information and data regarding the importance of the Flavobacterium type IX protein secretion system (T9SS) for virulence generated in year-2, were used in year-3 to begin to identify secreted F. columnare proteins that are important for virulence. Sixteen gene deletion mutations were constructed. The genes targeted for deletion encode proteins that are secreted by the T9SS. Deletion of one of these genes, sprB, resulted in decreased virulence toward zebrafish and rainbow trout. sprB encodes the cell surface protein SprB that is involved in attachment to and movement over surfaces. The results suggest that SprB and cell movement are involved in the disease process. SprB may allow F. columnare cells to attach to and move over fish tissues. Deletion of genes encoding most other individual proteins, including enzymes (proteases, chondroitinases, nucleases) were fully virulent, but some mutants lacking multiple secreted enzymes exhibited decreased virulence. Experiments are ongoing to identify the most important secreted enzymes involved in columnaris disease. In the coming year additional gene deletion mutants will be constructed and examined for virulence in zebrafish and rainbow trout. Attenuated mutants will be examined to determine if they can function as protective vaccines. Genetic studies of F. psychrophilum, were postponed in year-3 due to lack of appropriate personnel. In years 1 and 2 a T9SS gene deletion mutant of F. psychrophilum strain THCO2-90 was generated and shown to be deficient in virulence against rainbow trout. Unfortunately, strain THCO2-90 is not the most relevant strain for salmonid aquaculture, and strains associated with disease outbreaks in rainbow trout have resisted genetic manipulation. In the coming year, efforts will be undertaken to overcome the gene transfer barriers for these strains of F. psychrophilum. This will allow genetic experiments like those performed above with F. columnare, to identify critical virulence factors, and to construct strains attenuated for virulence as potential vaccines. For Objective 3, the aim was to characterize new antimicrobial peptides (AMPs) in trout. AMPs are an ancient part of the innate immune system, which is a first line of defense against bacterial, viral and parasitic pathogens. Based on the new ARS rainbow trout genome assembly (Omyk_1.0), 6 new saposin-like antimicrobial protein genes were identified, and their chromosomal locations were mapped out. Protein sequence alignments, and in silico modeling, show that all proteins fall within the Nk-lysin AMP sub-family (termed: Nkl 1, 2, 3 4 and Nkl-like a & b). Transcriptomic data show that nkl2 & nkl4 mRNAs are primarily expressed in the central nervous system and gonads at low abundance. Expression of nkl1 & nkl3 mRNAs occurred in many tissues, with nkl1 mRNA showing the highest abundances among the nkl1-4 transcripts. By contrast, the nkl-like a & b mRNAs are mostly expressed in immune-related tissues (spleen and kidney) and at much higher abundances (10- to 50-fold higher) than all the nkl1-4 mRNAs. RNA sequencing was performed to measure gene expression for these AMPs in gill tissue of rainbow trout exposed to aquaculture stressors (handling stress, salinity, temperature and re-use water) and in juvenile trout challenged with Flavobacterium psychrophilum (Fp). In the stress study, abundances of nkl1, nkl2, nkl4, and nkl-like a were downregulated by high-temperature and salinity stress, and nkl3 and nkl-like b were downregulated by high-temperature only when compared with controls. In the bacterial challenge study, abundances of nkl3, nkl4, nkl-like a and nkl-like b, were significantly affected by genetic line (resistant vs non-resistant) and treatment (PBS or Fp), which were further verified by real-time quantitative PCR with spleen tissue sampled at 4 post-challenge time points (6 h, 24 h, 48 h and 144 h post-injection), but patterns differed substantially between individual transcripts. To further this work, synthetic peptide cores have been designed and synthesized for use in studies to assess their biocidal actions against flavobacterial and novirhabdoviral pathogens, in vitro. Additionally, polyclonal antibodies against the Nkl-family of trout AMPs have been developed and will be used to develop assays to determine where these proteins are produced within rainbow trout and how protein levels are affected by nutrition, stress and disease challenge.

Accomplishments
1. Algae-based diets have been developed and tested in yellow perch. Alternative protein and lipid sources are required to make aquafeeds sustainable and affordable. As opposed to terrestrial plant protein sources, seaweeds possess dispensable and indispensable amino acids, as well as micronutrients such as vitamins and minerals, which are required for proper animal development and growth. Seaweeds also possess pre-biotic components that may benefit production performance in finfish. ARS researchers in Milwaukee, Wisconsin, along with University of Wisconsin at Milwaukee scientists, investigated the potential of defatted microalgae meal, a co-product from the bio-energy industry, as a feed ingredient for yellow perch. Results of this study indicate that defatted microalgae meal, when blended with soy protein isolate, can be used to replace 25% of the fishmeal in a standard test diet while maintaining performance of yellow perch. Digestible algae-based diets can reduce nutrient outputs such as phosphorus and synergize with the bio-energy industry to improve the efficiency, profitability, and sustainability of yellow perch production.

2. The dose needed for rapid anesthesia has been identified for yellow perch. Anesthetics are an integral part of farmed fish production and research as they allow fish to be easily handled, and transported, in a way that reduces stress. It is well known that the responses to the same anesthetic can vary considerably within and among various species, and it is inappropriate to extrapolate optimal anesthetic doses between different species. For yellow perch, there are no published guidelines. To address this, ARS researchers in Milwaukee, Wisconsin, along with scientists from the University of Wisconsin at Milwaukee, performed studies in juvenile yellow perch and identified an optimal dose of the anesthetic tricaine methanesulfonate that enables rapid anesthesia and recovery for brief periods of time. The research also demonstrated that anesthesia induction and recovery times are affected by the size of the fish. These results give fish handlers an optimal combination for minimizing handling disturbance in juvenile yellow perch which could improve the welfare of animals subjected to frequent handling events in the research and commercial production settings.

3. The genome is sequenced for a commercially-important bacterial fish pathogen. Columnaris disease in rainbow trout is caused by the bacterial pathogen Flavobacterium columnare. ARS researchers in Milwaukee, Wisconsin and researchers from the University of Wisconsin-Milwaukee and Clear Springs Foods, Inc. report the sequencing and draft assembly of the Flavobacterium columnare strain MS-FC-4 genome. This F. columnare strain was isolated from a diseased rainbow trout and is currently being used as a reference strain to study host-pathogen interaction in trout. The availability of the genome sequence is aiding in the identification of virulence factors as new targets for vaccine development. Targeted vaccines will show less toxicity, and greater species protection, to limit use of antibiotics and reduce production losses to columnaris disease in the United States.

4. Bacterial secretion products contribute to virulence in Columnaris disease. The pathogen responsible for columnaris disease (Flavobacterium columnare) is responsible for large losses in multiple fish species important to the U.S. aquaculture industry. The mechanisms that F. columnare bacteria use to cause columnaris disease in freshwater fish are unknown. ARS researchers in Leetown, West Virginia and researchers from the University of Wisconsin-Milwaukee, St. Norbert College (De Pere, WI) and the Chinese Academy of Sciences (Wuhan, China) identified a component of the F. columnare protein secretion system that delivers protein toxins to the outside of the bacterial cell and found this component to be important in the disease process. Deletion of two genes (gldN and porV) that are required for secretion eliminated the ability of the bacterium to cause disease in multiple fish species. While secreted toxins from normal bacterial cells killed fish, the mutants failed to do this. This work helps us understand the virulence mechanisms of F. columnare and identifies potential target genes for vaccine development to prevent outbreaks of columnaris disease in commercial aquaculture systems.

Review Publications
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. https://doi.org/10.1128/AEM.01769-17.
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. https://doi.org/10.1128/genomeA.00429-18.
Ogawa, S., Selvarajah, G., Xiaochun, L., Shepherd, B.S., Parhar, I.S. 2018. Ghrelin stimulates growth hormone release from the pituitary via hypothalamic growth hormone-releasing hormone neurons in the cichlid, Oreochromis niloticus. Cell and Tissue Research. https://doi.org/10.1007/s00441-018-2870-6.
Zhai, S., Yang, S., Zhao, H.H., Jiang, M., Shepherd, B.S., Deng, D. 2018. Efficacy of tricaine methanesulfonate (MS-222) as an anesthetic agent for short-term anesthesia in juvenile yellow perch (Perca flavescens). Israeli Journal of Aquaculture. 70(2018):1515-1525.
Jiang, M., Zhao, H., Zhai, S., Shepherd, B.S., Wen, H., Deng, D.F. 2019. A defatted microalgae meal (Haematococcus pluvialis) as a partial protein source to replace fishmeal for feeding juvenile yellow perch Perca flavescens. Journal of Applied Phycology. 31:1197–1205. https://doi.org/10.1007/s10811-018-1610-3.