Progress 03/30/21 to 08/29/21
Outputs
Target Audience: Throughout the project period, growth trial and gut microbiome results were presented at the World Aquaculture Society's Aquaculture AmericaConferences in February 2020 and August 2021, as well as the Conference of Research Animal Disease Workers in December 2020. Two additional abstracts for oral presentations (growth and Aeromonas spp. virulence in burbot) were accepted for the 14th International Congress on the Biology of Fish (Burbot Symposium) in July 2020, but the conference was postponed until 2022 due to Covid-19. To date, research findings have been disseminated to students, academic and agency researchers, commercial aquaculture vendors, and representatives from the academic, government and private aquaculture sectors. Changes/Problems:Overall, this project accomplished the proposed objectives and Dr. Bruce had the opportunity to share research findings at a variety of scientific venues. There were delays and hurdles through the project due to complications due to Covid-19 and Dr. Bruce's transition into a faculty role in January 2021. Dr. Bruce had the opportunity to complete laboratory assays this summer and has been in communication with his previous mentor's laboratory (Dr. Ken Cain's lab at the University of Idaho) to finalize the antibody work with the newly developed monoclonal antibody.With this transition, Dr. Bruce has been delayed in submitting finalized manuscripts for peer-review, but is working to get these papers submitted to aquaculture journals. What opportunities for training and professional development has the project provided? Dr. Bruce has taken advantage of several on-campus training activities over the past year and has worked with his mentor, Dr. Cain, on progress evaluations of his Postdoctoral Fellowship IDP. At the University of Idaho, Dr. Bruce has participated in UI Postdoctoral Association meetings, funding search training with the UI Office of Sponsored Programs, grant funding information sessions with the Office of Research and Economic Development, and mock academic interview training with UI Career Services. Dr. Bruce also participated in four guest lectures for undergraduate classes and instructed an undergraduate lab involving burbot early life history. Dr. Bruce continues to contribute to his professional organizations and also presented a full lecture on the rainbow trout immune system/pathogens at the Broodstock Management workshop at the annual Idaho Chapter of the American Fisheries Society Meeting in March 2020. Lastly, Dr. Bruce completed an academic interview series this past summer and started as an Assistant Professor in January 2021 at Auburn University. The continuation of this postdoctoral fellowship allowed for Dr. Bruce to attend an additional national conference in August 2021 and complete the immune gene expression assays for Objective 3. How have the results been disseminated to communities of interest? Due to Covid-19, several research conferences had been postponed or canceled. Dr. Bruce has presented findings at three conferences to date (Aquaculture America 2020, Aquaculture America 2021,and CRWAD 2020), but has an upcoming platform presentation at Aquaculture America 2022in February 2022(discussing the stress trial results from this EWD project). Additionally, Dr. Bruce is finalizing the first growth performance manuscript to send out for peer review, with the second stress trial manuscript to be sent out shortly thereafter. A third manuscript, incorporating the virulence trial data is planned for submittal to a peer-reviewed journal, and will incorporate the vaccination trial findings from Objective 4 when trial antibodyresults can be incorporated. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
Objective 1 (Feeding Trial):Juvenile burbot (8.2±0.1g) were cultured in aerated flow-through tanks with conditions suitable for burbot culture (13°C; > 6 mg/L DO; 120 fish per 280L tank). An Atlantic cod marine-type (fishmeal-based control; REF) diet was formulated to approximately 48% crude protein and 16% lipid and fishmeal protein content was replaced at 25% with inclusions of de-hulled, solvent-extracted soybean meal (SBM), and dried distillers grains with solubles (DDGS).At 36d and 72d, the burbot was bulk weighed and the average fish weight was discerned (weights and lengths of 12 or 10 individuals per tank). Growth results from the trial endpoint indicate comparable performance for the FM, DDGS, and SBM diets, with respect to final weight per fish (P=0.123), specific growth rate (SGR; P=0.069), and thermal growth coefficient (TGC; P=0.052), but a different in tank biomass gain was noted (P=0.028), with the SBM-fed fish outperforming the DDGS group. For feed conversion ratio (FCR;P=0.019), the burbot fed the FM diet showed the best conversion (0.80±0.01), which was better than the SBM (0.98±0.03) and DDGS (0.96±0.05) diets. No morphological differences in the intestinal lamina propria cellularity (P=0.358), thickness (P=0.236), or the connective tissue thickness in the submucosa (P=0.304). Fecal material (trial start, 36d, and 72d) was collected for an assessment of the intestinal microbiota via 16S sequencing (V3 and V4 regions). Results indicate a difference in observed amplicon sequence variants (ASVs) across dietary treatments (P=0.030) and trial sampling timepoints (P=0.013), with the DDGS shifting constituents from 36d (17.9) to 72d (66.1;P=0.039). With respect to alpha diversity analyses, the FM diet displayed an increase in the Shannon index over time (P=0.031) and was found to be greater than the SBM group at 72d (P=0.034). With respect to beta diversity, an unweighted UniFrac analysis revealed diet-induced changes to the microbiome at d36, whereas the REF diet was found to be different from the SBM (P=0.12) and DDGS diets (P=0.006). No differences in diet were found at the 72d trial endpoint, but differences at all time points were found when compared to the beta diversity of the microbiome found at day 0. Objective 2 (Rearing Stressor Trial):Immediately following the 72d performance trial, a subset of the burbot (n=3 replicate tanks per diet; 30 fish per tank) were transferred to secondary flow-through systems and were subjected to both density and temperature stressors. Triplicate tanks in the original system served as a control group (i.e., no stressors). The high-temperature treatment was set for 20°C and the high-density treatment was set at 0.1 kg/L (adjusted with tank standpipe water volume and based on the d72 weights from the feeding trial). At the completion of the growth trial and at 10d post-stress initiation, burbot was sampled for baseline health metrics (n=5 per tank; blood for hematocrit/plasma and liver). Plasma glucose and lactate analyses were completed via commercial colorimetric assays. No differences in plasma glucose were discerned at 72d (P=0.191) or in the non-stressed control fish (P=0.159). In the temperature-stressed burbot, the DDGS group showed lower plasma glucose levels than the REF diet (P=0.007), while the density-stressed burbot fed DDGS had lower plasma glucose than both the REF (P=0.001) and SBM (P=0.015) groups. Plasma cortisol samples were analyzed via GC/MS and no differences were found across dietary treatment groups (P=0.755), and the experimental stressors did not appear to influence this metric (P=0.501). Liver tissues were processed for heat shock protein 70 (hsp70) expression levels via qPCR and no diet-related changes to hsp70 expression were observed in the temperature-stressed burbot (P=0.387), nor the density-stressed burbot (P=0.242). Post-stressor fecal material was also collected to assess the impact of these rearing stressors on the intestinal microbiota. Differences in ASV counts were found to be influenced by a stressor (P=0.021), but not by diet (P=0.736), and this same stress-related influence was also found for the Shannon (P=0.003) and Simpson (P=0.003) indices. Objective 3 (Virulence and Immune Response): Twotrials were conducted in September 2020 to assess the virulence of novel Aeromonas sp. isolates, including A. hydrophila. In Trial A, triplicate tanks of 25 fish per tank (mean weight of 40g) were challenged with a known virulent Aeromonas sp. isolate (A141)along with a newly recovered IR034 isolate at approximately 1 x 108CFU mL-1 in a 100 μl IP-injection. Mock controls were also challenged using culture media (tryptic soy broth). Endpoint cumulative percent mortality (CPM) at 21 days post-challenge (dpc) was 49.3±22.0% for A141 and 4.0±0.0% for the less virulent IR034. A similar trial was conducted with 55.0 g burbot, incorporating another recent isolate, IR004 (8 x 107CFU mL-1), along with a previously reported (in warm water species) virulent strain of A. hydrophila (ALG-15-097; 3 x 107CFU mL-1). Both isolates were found to be virulent, displaying a CPM of 85.3±6.1% for the IR004 and 97.3±4.6% for the ALG-15-097 at 21 dpc. Through the initial infection period (0h, 48h, and 120h post-challenge) spleen and kidney samples were collected from three fish per tank for immuneparameter monitoring. Trial results indicate that burbot is susceptible to an array of different Aeromonas sp. isolates,including A. hydrophila.Analysis of gene expression assays was completed in Summer 2021 and revealed differences in immune gene expression within the challenged burbot. In Trial A, Il-1b was found to be different at 48h post-challenge in the spleen (P=0.004) and head kidney tissues (P=0.009), with the A141 strains showing elevated expression compared to the control group. In Trial B, il-8was elevated at 48h post-challenge in the IR004-infected fish spleen (P=0.016), but not in the head kidney (P=0.550). tlr22 expression in the IR004 fish was also found to be reduced at 96h post-challenge in the spleen tissue (P=0.004) in comparison to the control group. Thus, differences in immune gene expression were characterized and related to the noted strain virulence observed during the in vivo pathogen challenge trial. Objective 4 (Bacterin Development): Based on the observed strain virulence in Objective 3, strains A141 and IR004 were chosen for bacterin development and use in the vaccination trial. As such, a 48h culture of each strain was formalin killed (1% v/v) and allowed to incubate for an additional 48h at 15oC, prior to confirming sterility/safety was washing for bacterin creation. For the burbot vaccination phase, four tanks were used (a sham-vaccinated and vaccinated tank for each vaccine strain, A141, and IR004) and stocked with 125, ~58g juvenile burbot and fish were bled biweekly for 8w for sera collection. At 8w post-vaccination, the burbot was challenged with their respective, virulent strains via IP injection for 28d. The CPM of the sham-vaccinated A141 group was 8±2% while the vaccinated group showed 0±0. The CPM of the sham-vaccinated IR004 burbot was 33±1%, while the vaccinates were 1±1%. The enzyme-linked immunosorbent assay (ELISA) to measure immunoglobulin M (IgM) specific for A141 and IR004 has been optimized. However, before the optimized ELISA can be used to determine specific titers in relation to vaccination and challenge the total IgM must be determined. The ELISA optimization, using serum from naïve and post-challenge burbot, suggests that burbot has the ability to mount a specific adaptive immune response to A141 and IR004. The initial attempts to quantify total IgM in burbot, using serum from naïve and post-challenge burbot, provide evidence that total IgM is elevated, relative to naive fish, after challenge with A141 and IR004.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2022
Citation:
Bruce, T.J., Bledsoe, J.W., Oliver, L.P., Ma, J., Jones, E.M., Vuglar, B.M, Welker, T.L., and Cain, K.D. Characterization of stress response and gut microbiota in cultured burbot (Lota lota maculosa) following feeding with plant-based diets. 2021. Oral presentation. Aquaculture America 2022,San Diego, CA.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Bruce, T.J., Oliver, L.P., Ma, J., Jones, E.M., Vuglar, B.M, Cain, K.D. Assessment of burbot (Lota lota maculosa) immune responses following experimental infection with Aeromonas spp. 2021. Oral presentation. Aquaculture America 2021, San Antonio, TX (August 2021).
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Bruce, T.J., Bledsoe, J.W., Cain, K.D. Investigation of growth performance and gut microbiota in culture burbot (Lota lota maculosa) fed plant-based diets. 2021. Oral presentation. Aquaculture America 2021, San Antonio, TX (August 2021).
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