Location: Harry K. Dupree Stuttgart National Aquaculture Research Cntr
2019 Annual Report
Objectives
Objective 1. Characterize the cellular and molecular immune responses governing resistance/susceptibility to infectious disease (Flavobacterium columnare), and develop strategies to enhance immune protection through immunomodulatory compounds, nutritional status, or water chemistry.
Subobjective 1A. Describe the cellular, transcriptional, and pathological responses of mucosal tissues in healthy and Flavobacterium columnare-challenged fish.
Subobjective 1B. Investigate the effects of copper sulfate, short-term feed deprivation, and water chemistry on the mucosal immune system.
Objective 2. Assess microbial community diversity, prevalence, and distribution under homeostatic conditions, after columnaris disease challenge, and following environmental perturbation.
Subobjective 2A. Profile the composition and population dynamics of microbes that reside on the skin and gill of healthy and columnaris-challenged fish.
Subobjective 2B. Investigate the effects of copper sulfate, short-term feed deprivation, and water chemistry on microbial community composition.
Approach
In Objective 1 our research team will utilize next-generation sequencing to establish temporal profiles of transcriptional responses to Flavobacterium (F.) columnare in gill and skin; use flow cytometry to monitor the type and abundance of immune cells trafficking to the gill of challenged fish; and characterize the histopathological changes to an F. columnare challenge and draw parallels to the shifts in immune-related transcripts and cell phenotypes. Studies will also examine the transcriptional and immunological consequences to alterations in water chemistry and to chemical compounds such as copper sulfate. In Objective 2 the core microbiota associated with mucosa in healthy fish and F. columnare infected fish will be characterized using high-throughput 16S v4 amplicon sequencing. This work will identify the core microbial communities commonly residing in the mucosal barriers of both warmwater bass and channel catfish; vital information towards identifying bacterial species that can be exploited to improve fish health.
Progress Report
ARS scientists in Stuttgart, Arkansas, have previously determined that sunshine hybrid striped bass, and its maternal parent white bass, display differences in their susceptibility to the bacterium, Flavobacterium (F.) columnare, whereby hybrid striped bass incurs significantly higher mortality when challenged under laboratory conditions compared to white bass. Studies to evaluate the transcriptome through RNA sequencing of white bass and hybrid striped bass gill tissue after F. columnare challenge are currently being conducted. This research will provide a better understanding of the host-pathogen interactions that are important in the production of Morone spp. in order to improve treatments and aid in the development of disease-resistance genetic markers.
ARS scientists in Stuttgart, Arkansas, continued their work to characterize factors which stimulate F. columnare biofilms, which are a critical part of the disease process. Biofilms are an important stage of pathogenesis which requires a sustained host-pathogen interaction at mucosal sites (gill, skin). ARS scientists have identified that skin mucus from different teleost fish species can stimulate the formation of in vitro biofilms. Studies conducted to evaluate iron acquisition systems indicate these systems are upregulated in biofilm cells during their transition from being planktonic cells. This work will help benefit the development of new vaccines and novel therapies to treat infectious diseases.
ARS scientists in Stuttgart, Arkansas, continued a collaboration with researchers at the University of Arkansas at Pine Bluff in Pine Bluff, Arkansas, and Keo Fish Farm (Keo, AR) to develop the use of heat- and cold-shock on eggs to produce triploid (sterile) hybrid striped bass. We will also determine growth rates and morphology of these triploid fish as compared to diploid (normal) fish. Preliminary studies found the temperature methods we used can produce majority triploid populations.
ARS scientists in Stuttgart, Arkansas, continued collaborating with researchers in Denmark, Germany and the U.S., to establish the importance of the potent disinfectant peracetic acid to the global aquaculture industry. After evaluating our research, the major manufacturer of peracetic acid in the U.S. (PeroxyChem) and another company that retails fishery products (AquaTactics) have EPA Registrations to use their products in the aquaculture industry as a disinfectant. ARS researchers also continued their collaboration with researchers at Bowling Green State University (Bowling Green, Ohio) and the Freshwater Institute (Shepherdstown, West Virginia) to quantify the amount of aquatic fungus in recirculating aquaculture systems and to treat these systems with peracetic acid to eliminate pathogens.
ARS scientists in Stuttgart, Arkansas, completed copper sulfate toxicity studies to various species of fish in 5 reconstituted waters ranging from very soft to very hard. These tests determined the LC50 (acute toxicity) as well as the highest concentration of copper sulfate that does not cause mortality. With the range of reconstituted waters, this study is vital for all farmers in the industry to understand the amount copper sulfate they can use to treat their fish. This is the first comprehensive research to demonstrate safety of copper across multiple finfish species in the same waters.
Accomplishments
1. Elucidation of molecular mechanisms behind catfish resistance to Flavobacterium columnare when treated with copper sulfate. ARS scientists im Stuttgart, Arkansas, and Auburn, Alabama, had previously determined that timing of copper sulfate treatment could either confer protection or make channel catfish more susceptible to the pathogen Flavobacterium (F.) columnare. Most recently, researchers examined catfish gill tissues of copper sulfate-induced resistance and susceptibility to F. columnare using high-throughput RNA sequencing. ARS researchers determined significant gene expression differences as well as functional differences in transcript processing and exon usage were induced over a time-course of infection. These discoveries could aid in the development of new treatments to this problematic bacterial pathogen.
2. Microbial profiling of tilapia gut reared in either biofloc or flow-through aquaculture systems reveals dynamic populations associated to each environment. ARS researchers in Stuttgart, Arkansas, in collaboration with ARS researchers in Auburn, Alabama, and Gainesville, Florida, reared juvenile tilapia in each system for 154 days on two test diets and a control diet. Water, feed and gut contents were sampled at multiple time points over the course of the study, including day 0, day 7, day 60, and at the study endpoint. DNA was isolated from a total of 440 tilapia gut, feed, and water samples including biological replicates. This genetic material was used for amplicon sequencing of the 16S ribosomal DNA gene and the Nuclear Large Ribosomal RNA gene to assess the composition of bacteria, fungi, and protozoa in each sample. Data was processed using the QIIME2 software. After statistical analyses between replicates, it was revealed that the prokaryotic and eukaryotic communities were strongly structured by the sample type (gut, feed, and water) and by the environment (outdoor, biofloc systems vs. indoor, flow-through systems). Since diet and the environment affect the intestinal microbiota of fish and microbial communities are highly relevant to fish health, these findings provide for a better understanding of the dynamics in rearing tilapia in biofloc technology.
3. Use of a recombinant Flavobacterium columnare DnaK protein vaccine to protect catfish against columnaris disease. Flavobacterium columnare is the causative agent of columnaris disease which severely impacts channel catfish production in the United States. ARS scientists in Stuttgart, Arkansas have identified F. columnare proteins which activate the channel catfish adaptive immune response. A recombinant DnaK protein vaccine was developed for testing for immune protection against columnaris disease. To evaluate the immunological effect of bath immunization on catfish with a recombinant F. columnare DnaK protein vaccine, ARS scientists inf Stuttgart, Arkansas performed RNA sequencing on skin explant samples from control and rDnaK immunized catfish at different time intervals. We evaluated the different gene expression patterns between the groups with a focus on identifying differences in innate and adaptive immune function. Significant differential gene expression was observed between rDnaK vaccinated and control catfish indicates an adaptive immune response was activated in the skin of immunized catfish. The fundamental role of developing a skin cell-mediated immune response and its contribution to overall immune protection against different fish pathogens will need to be studied more thoroughly. We will continue to investigate the adaptive immune response in fish using conventional immunological assays as well as using genomics technologies to determine optimal conditions for long lasting immunization procedures. Research that will further enhance the catfish immune response to F. columnare rDnaK is underway; as this protein remains a promising candidate for additional optimization and experimental trials in a production setting.
Review Publications
Lange, M.D., Abernathy, J.W., Farmer, B.D. 2018. Catfish mucus alters the Flavobacterium columnare transcriptome. FEMS Microbiology Letters. 365(22)1-13. https://doi.org/10.1093/femsle/fny244.
Davidson, J., Summerfelt, S., Straus, D.L., Schrader, K.K., Good, C. 2019. Evaluating the effects of prolonged peracetic acid dosing on water quality and rainbow trout Oncorhynchus mykiss performance in recirculation aquaculture systems. Aquacultural Engineering. 89:117-127. https://doi.org/10.1016/j.aquaeng.2018.12.009.
Lange, M.D., Waldbieser, G.C., Lobb, C.J. 2019. The proliferation and clonal migration of B cells in the systemic and mucosal tissues of channel catfish suggests there is an interconnected mucosal immune system. Fish and Shellfish Immunology. 84:1134-1144. https://doi.org/10.1016/j.fsi.2018.11.014.
Lange, M.D., Abernathy, J.W., Farmer, B.D. 2019. Evaluation of Flavobacterium columnare DnaK recombinant protein vaccine as a means of protection against columnaris disease in channel catfish, Ictalurus punctatus. Frontiers in Immunology. p. 1-13. https://doi.org/10.3389/fimmu.2019.01175.
Straus, D.L., Ledbetter, C.K., Heikes, D.L. 2019. Inhibiting fungus on largemouth bass eggs with copper sulfate and its toxicity to fry and juveniles. Journal of the World Aquaculture Society. p. 1-10. https://doi.org/10.1111/jwas.12631.
Declercq, A., Cai, C., Naranjo, W., Thongda, W., Eeckhaut, V., Bauwens, E., Arias, C., De La Fuente, L., Beck, B.H., Lange, M.D., Peatman, E., Aerts, J., Haesebrouck, F., Decostere, A. 2019. Evidence that the stress hormone cortisol regulates biofilm formation differently among Flavobacterium columnare isolates. Veterinary Research. 50(1):24. https://doi.org/10.1186/s13567-019-0641-3.
