Research Project: Reducing Impacts of Disease on Salmonid Aquaculture Production

Location: Office of The Director

2017 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
Objective 1 in the first year of this new project was to characterize mechanisms of innate immune response and pathogen virulence in order to control rhabdoviral diseases in salmonid aquaculture. We have completed two of the critical components. The first component was to clone and express all of the infectious hematopoietic necrosis virus (IHNV) gene products in cells to determine their impact on host cell gene expression in three ways using Epithelioma Papulosum Cyprini (EPC) cells and varying constitutively-active promoter constructs. The resulting data were interesting and clear, wherein the IHNV matrix (M) protein was by far the most potent inhibitor of gene induction, mirroring what we have previously observed and reported for viral hemorrhagic septicemia virus (VHSV). Interestingly, IHNV nucleoprotein (N) protein exhibited potent inhibition that was not true in VHSV. Nonvirion (N) protein showed variable effects, augmenting some responses while inhibiting others. To further assess virulence of M, the gene protein in IHNV, we used sequence information to identify conserved regions of IHNV M protein to mutate for structure/function studies in cell-based transcriptional inhibition studies. The second component was to use studies already done with VHSV M protein to inform our choices of amino acid residues to mutate in IHNV M protein for these same types of studies. Using a similar approach, we made a variety of alterations in IHNV M gene to mimic the single- and double-mutants made in VHSV M. Unfortunately, none of those mutants exhibited activity that was significantly different from the wild type IHNV M protein in SV40-luciferase co-transfection studies. Thus, we will revisit the idea of identifying regions within less pathogenic strains of IHNV to target for M mutagenesis. Work in the coming year will transition into rainbow trout (RT) cells and expand our analyses of M- and N- protein functions. We have made good progress on transitioning our cell-based reporter systems into RT cells. We will also use RTG-2 (gonad) cells in future studies. Work has also been done to test RTgill-W1 cells for transfection efficiency and responses. Among several options, we have found one transfection reagent that works for both cell types under optimized conditions, allowing us to generate data more quickly with fewer cells. We are now replicating the EPC data in the RT cells. If the interesting NV results obtained in the EPC cells repeat in the RT cells, there will be a clear path toward dissection of NV function. Further efforts are underway to sequence VHSV isolates from wild-caught fish, including new isolates from 2017 outbreaks. These sequences are being mapped into haplotype maps for assessment of amino acid changes that might correlate with distribution, function, and evolution. Objective 2 of this project is to use genetic techniques to characterize mechanisms of Flavobacterium virulence and identify potential strategies to control bacterial disease in salmonid aquaculture. Forty-three Flavobacterium columnare strains have been tested; 11 were identified that could be genetically manipulated in the laboratory setting. Two F. columnare strains were selected for future genetic studies because of ease of genetic transfer, demonstrated virulence for rainbow trout, and availability of genomic sequence data. One of the strains is currently being used by USDA-ARS collaborators for breeding of resistant rainbow trout. Plasmids were constructed to delete the gliding motility protein (gldN) gene from both F. columnare strains cited above. This protein is required for secretion of protein toxins and virulence in Flavobacterium spp. The deletion plasmid was introduced into both F. columnare strains, and gldN deletion mutants were obtained. These were deficient in protein secretion and in gliding motility. A plasmid carrying wild type gldN was constructed to complement (restore function to) the gldN deletion mutants; it restored protein secretion and gliding motility to the mutants. The mutants will be examined in Year 2 for virulence against rainbow trout. Preliminary results were also obtained regarding identification of secreted virulence proteins. We deleted genes encoding several of these proteins; the roles of these proteins in columnaris disease will be characterized in Year 2. Studies that were not initially proposed, but have been conducted, involve construction of F. columnare deletion mutants (defective for secretion, but not motility) and complemented strains. These mutants will allow us to separate motility defects and secretion defects as causes for decreased virulence of F. columnare mutants. In addition, we have developed techniques to insert DNA of interest at a neutral site on the F. columnare chromosome. As a test, we introduced the gene encoding the green fluorescent protein into the chromosome. The resulting fluorescent strains will not only serve as a proof of principle, but will be useful to study how F. columnare interacts with fish. Attachment of the fluorescent bacteria to fish and progression of infection can then be easily monitored. We also began genetic manipulation of F. psychrophilum. We transferred plasmid into two F. psychrophilum strains. Transfer into one strain was efficient. However, transfer into the other strain was inefficient, and optimization will be attempted in Year 2. We constructed a gldN deletion mutant of a F. psychrophilum strain and complemented the mutant. We will analyze these strains for virulence in Year 2 to determine the importance of protein secretion in bacterial cold water disease of rainbow trout. Objective 3 of this project is to measure and modulate antimicrobial peptides (AMPs) as a means to control disease in salmonids. Synthetic antimicrobial peptides were designed for in vitro testing of their biocidal effects on rainbow trout Rhabdoviral and Flavobacterial pathogens. Polyclonal antibodies were also developed to enable detection of the homologous proteins in rainbow trout tissues via Western blotting. In collaboration with ARS researchers at Leetown, West Virginia, samples have been obtained from rainbow trout that have been exposed to various aquaculture stressors, including salinity challenge. Total ribonucleic acid (RNA) has been extracted from liver and gill tissues and we will measure messenger RNA (mRNA) abundance of known AMPs using real-time quantitative polymerase chain reaction assays. Evaluation of the short-term effects of lipopolysaccharide (LPS: a pathogen mimetic) and cortisol on the liver immune response in rainbow trout was recently conducted using the in vitro liver slice system. While further optimization is required, two antimicrobial peptide mRNAs (hepcidin and LEAP-2A) were elevated with LPS and suppressed with cortisol over an 8-h period. This may be one of the first results showing a direct effect of cortisol on the liver immune response in a teleost.

Accomplishments
1. Development of efficient techniques for genetic modification of Flavobacterium columnare. Columnaris disease is an emerging problem for rainbow trout farmers, causing losses in both fry and harvest-size fish. University of Wisconsin (Milwaukee, Wisconsin) scientists and ARS researchers at Leetown, West Virginia systematically explored genetic components that contribute to the virulence of Flavobacterium columnare. By using gene transfer, gene deletion, and complementation, an F. columnare mutant deficient for protein secretion was created; this mutant failed to cause columnaris disease in rainbow trout. The understanding of the genetic components that contribute to virulence and toxicity of this pathogen in commercially bred finfish will enable development of safe candidate vaccine strains to treat columnaris disease and reduce losses.

2. First study to show that the stress steroid, cortisol, affects the immune response of rainbow trout liver in vitro. Stress-induced cortisol levels negatively influence growth and immunity in finfish, but no studies have assessed the direct effects of cortisol on liver immune function in finfish. More importantly, the immune response of the liver in finfish is poorly understood. ARS researchers in Milwaukee, Wisconsin, along with scientists from the University of Calgary (Alberta, Canada) and the University of Toledo (Toledo, Ohio), used a novel in vitro technique involving the culture of liver slices in controlled conditions. They evaluated the short-term effects of an immune stimulant and cortisol (a stress steroid) on the liver immune response in rainbow trout. The abundance of two antimicrobial peptides was elevated with the immune stimulant and suppressed by cortisol. It is believed that this is among the first reports showing a direct suppressive effect of cortisol on the liver immune response in a finfish. Understanding how cortisol affects host, immunity will improve husbandry practices that reduce stress levels to improve animal health, thus reducing the use of antibiotics and chemotherapeutics in aquaculture operations.

3. A key protein of the VHS virus has been shown to suppress the fish immune response. Viral hemorrhagic septicemia virus (VHSV) is a pathogenic fish virus found in locales throughout the northern hemisphere that leads to up-regulation of the host’s virus detection response, but the virus quickly suppresses interferon production and antiviral gene expression. Using systematic screening methods, scientists from the University of Toledo (Toledo, Ohio), the University of Maryland (College Park, Maryland), and ARS researchers in Milwaukee, Wisconsin identified matrix (M) protein as the most potent host immunosuppressive protein. This protein indicated one mechanism by which the pathogen suppresses the host immune response. This work demonstrates that small changes in the sequence of the VHSV M gene can dramatically affect the interaction of the virus with the host cell, suggesting the individual involvement of the VHSV genome with pathogen virulence. A greater understanding of how viral proteins impact cellular antiviral recognition and response pathways, and how the virus might evade or suppress the host immune response, will enable rational design of methods to combat VHSV infection in commercial finfish species.