Performing Department
SCHOOL OF FISHERIES
Non Technical Summary
Largemouth bass is an up-and-coming cultured fish species in the United States, with greatpotential as a production foodfish. The early life stages remain to be better clarified for optimized production in both pond and indoor rearing systems. Largemouth bass are susceptible to bacterial diseases during the early rearing periods, and there is limited information on therapeutics for producers. A major bacterial pathogen is Aeromonasspp., which cause motileAeromonassepticemia. These bacteria are often synonymous with rearing stressors and can lead to high levels of mortality. As such, this dynamic project team aims to investigate disease susceptibility and immunological responses of largemouth bass during these critical production stages. Specifically, the outlined project objectives are to 1) Develop and evaluate an immersion-based challenge model for motileAeromonassepticemia (MAS;Aeromonas hydrophilaandAeromonas veronii) in largemouth bass; 2) Characterize disease susceptibility and immune responses to MAS infections in largemouth bass fry and fingerling across the early rearing stages; and 3) Assess the efficacy and potential for killed, immersion-basedA. hydrophilaandA. veroniibacterins when administered at various timepoints within the early life stages, along with potential cross-protection. The research team will translate research findings for commercial largemouth bass producers and the projectoutcomes will be used to enhance U.S. foodfish production for domestic food security.
Animal Health Component
80%
Research Effort Categories
Basic
20%
Applied
80%
Developmental
(N/A)
Goals / Objectives
The team's long-term goal is to develop disease mitigation strategies and define aspects of the LMB immune response to bacterial pathogens and vaccination during the critical early rearing periods. As such, we aim to 1)Develop and evaluate an immersion-based challenge model for MAS (AeromonashydrophilaandAeromonas veronii) in LMB; 2)Characterize disease susceptibility, gut microbiota, and immune responses to MASinfections in LMB fry and fingerling across the early rearing stages;and 3)Assess the efficacy and potential for killed, immersion-basedA. hydrophilaandA. veroniibacterins when administered at various time points within the early life stages, along with potential cross-protectionTogether, these approaches will give the aquaculture sector a scientific toolbox to combat diseases that are reoccurring in LMB farms.
Project Methods
Northern LMB embryos or sac fry will be transported to SFAAS, where they will be reared at Dr. Peatman's facility, in a land-based raceway system (4000 L tanks). This rearing strategy will optimize growth and development over the project period.LMB will be reared on standard commercial diets. For experimental challenges, a total of 4 virulent isolates (twoA. hydrophilaand twoA. veronii) obtained from Dr. Bruce's Lab or the USDA-ARS AAHRU will be revived from glycerol cryostock on tryptic soy agar (TSA) plates at 24°C. A single colony pick will be used to scale the culture to 1L volumes in tryptic soy broth (TSB) and a selected optical density at 600 nm (i.e., OD600), based on pilot culture work. Before initiating preliminary challenges, bacterial titration curves will be prepared to discern growth mechanics and colony-forming units per mL (CFU mL-1) for each of the four strains. For initial virulence and delivery testing, fish will be exposed to 4 dilutions (104to 107CFU mL-1) using triplicate tanks of 20 fish tank-1(65L tanks) for each dilution, with an initial screening size of 5-10 g, a tank volume of 10 L (100 mL of inoculum added), and a temperature of 24°C. The fish will be subjected to the static immersion challenge for 1h with aeration via air stones before flows (0.3-0.5 L min-1) are restored, and tank volumes are filled to 20 L. Triplicate tanks of mock-challenged fish (sterile TSB only) will also be included. Mortality will be assessed for a total of 10d post-initiation. Mortalities will be collected every 6-12 h, and spleen and posterior kidney tissues will be streaked on TSA for pathogen reisolation.Depending on the outcomes of the first round of immersion challenges, selected modifications to the challenge protocol will then be applied, as previously documented in virulentAeromonas hydrophilachallenges in channel catfish. First, skin mucus surrounding the dorsal fin will be removed with light abrasions using a flat spatula immediately prior to immersion (Li et al., 2013). The entire challenge process above will be repeated with varied inoculum doses. If this approach again proves unsuccessful in generating mortality, a clipping of a portion of the soft dorsal fin will be used, like the model developed by Zhang et al. (2016). For this procedure, LMB will be lightly anesthetized with 80 mg L-1of MS-222 and exposed to the pathogen immediately following the clip. If desirable cumulative percent mortality (CPM) of >50 % is achieved, a final modification step, incorporating the use of an iron-chelating xenosiderophore (deferoxamine mesylate; 0.4 mM), will be added during the bacterial inoculum incubation to increase pathogen virulence (Peatman et al., 2018). At the completion of this objective, the top candidate strains (one forA. hydrophilaand one forA. veronii) will be used in the following project aims. This stepwise approach to expanding the immersion-based model will allow for a more natural route of infection, which results in high and rapid mortality.The second project aim will build upon the optimized disease challenge model described in Objective 1 and discern aspects of the ontogenic LMB immune response to pathogens over 6 months. As described above, LMB fry and fingerling will be reared over a period of 6 months. The fish will be stocked into a total of three rearing tanks for propagation. Starting as 1g fry, LMB will be exposed toA. hydrophilaandA. veroniipathogen challenges every month throughout this period (6 times). Prior to the challenge, a subset of the fry or fingerling will be evaluated for morphometric indices by Dr. Butts. These include total length, eye diameter, body area or depth, jaw length, and developmental deformities. At the same time, samples of intestinal tissue (distal intestines from 3 fish per tank, across 6 tanks) will be aseptically collected and flash-frozen in liquid nitrogen for 16S profiling of the V3 and V4 regions. Diet and water samples will also be captured at these time points. In addition to the samples collected for microbiome analyses, kidney (target head kidney) and spleen samples will also be collected and preserved in Zymo DNA/RNA Shield for gene expression analysis. Following the pre-challenge sampling, fish will be subjected to thein vivoAeromonasspp. immersion challenges. At the challenge completion, gut microbiota (and associated rearing water) and immune tissues will again be sampled. All challenges will incorporate triplicate tanks for each pathogen type, including a shared group of triplicate tanks as a mock-challenged group.All gut microbiota samples will be sent to a contract laboratory (Zymo Research, Irvine, CA) for DNA extraction, library preparation, and sequencing on the MiSeq platform. Read files will be generated and shared with the team for analysis. Alpha and beta diversity parameters will be evaluated.For gene expression, a total of five immune genes will be selected for RT-qPCR analyses. RNA will be extracted from the tissues using a Zymo Quick-RNA MiniKit (Zymo Research Inc., Irvine, CA), and purified RNA will be evaluated for quality and quantity on a Nanodrop Onecspectrophotometer (ThermoFisher Scientific, Waltham, MA). RNA concentrations will be uniformly adjusted, and template cDNA will be created using a High-capacity RNA-to-cDNA Kit (ThermoFisher Scientific, Waltham, MA). The generated cDNA will then be used in 10mL reactions with 2x PowerUp SYBR MasterMix (ThermoFisher Scientific, Waltham, MA) and runs conducted in triplicate on a Quantstudio 5 instrument (Applied Biosystems, Waltham, MA). Primers used in the reactions will be incorporated from previous LMB gene expression studies or designed using the NCBI database's Primer Designing Tool. All primers will be optimized prior to conducting the assays. Targets of interest will include genes such as interleukin 1b(il-1b), interleukin 8 (il-8), and tumor necrosis factora(tnf-a) (Yang et al. 2020a). Two housekeeping genes, such as elongation factor 1a(ef1a) or18S,will be implemented. Negative template control (NTC) will also be included in the RT-qPCR runs.The final project aim will involve the assessment of immersion-based vaccination againstA. hydrophilaandA. veronii. This phase will include vaccinations starting at the 1g fish size (time 0) and then two additional vaccinations at both 2 months and 4 months following. Formalin-killed (0.5% v/v) bacterins for each pathogen will be prepared according to methods by Wu et al. (2021). The bacterins will be administered to LMB for 0.5 h in a static bath, and the fish will be challenged at 30d post-vaccination with challenge timing based on preliminary reports of increased specific antibody levels (Wu et al., 2021). A sham-vaccinated group will also be included for eachA. hydrophilaorA. veroniistrain. Blood will be collected at 15 d and 30 d post-vaccination (from five fish per treatment group) and sera will be extracted to discernAeromonassp.-specific antibody titers, using a mouse anti-LMB IgM monoclonal antibody in an ELISA assay. Morphometric analyses will again be conducted to observe any notable developmental changes in response to vaccination. At the time of the immersion challenge (as in Objective 1), both vaccinated and sham-vaccinated fish for each of the two bacterins will be infected with bothA. hydrophila andA. veronii, along with mock-challenged groups (sterile TSB), and the challenge will be conducted for a period of 10 days. This approach will allow the team to investigate the potential cross-protective ability of each bacterin. In addition to thein vivo pathogen challenge trials to assess protection, vaccine-enabled immune responses and the influence of vaccination on the gut microbiota will be characterized using gene expression and 16S rRNA analyses.From the five fish collected for sera, spleens, kidneys, and sections of the distal intestine will also be aseptically extracted for these downstream analyses.