1a. Objectives (from AD-416)
The long-term objective of this project is to characterize the pathogenesis of fish diseases and to develop approaches to control diseases in warm water fishes. Over the next 5 years, our project at the Stuttgart National Aquaculture Research Center (SNARC) will focus on the following objectives: Objective 1. Characterize genetic variation of white bass and sunshine bass resistance to columnaris and develop strategies to control columnaris in warm water aquaculture. Subobjective 1A. Develop a Flavobacterium columnare disease model in white bass (Morone chrysops) and sunshine bass (female Morone chrysops x male M. saxitalis). Determine whether there is genetic variation for survival of white bass and sunshine bass in response to columnaris infection (Darwish and Mitchell). Subobjective 1B. Characterize Flavobacterium columnare virulence genes using real-time PCR to evaluate gene expression (Straus). Objective 2. Evaluate compounds and methods to treat bacterial and fungal (columnaris, streptococcosis, and saprolegniasis) fish diseases. Subobjective 2A. Control columnaris in channel catfish with copper sulfate (Darwish, Mitchell, and Straus). Subobjective 2B. Control saprolegniasis on channel catfish eggs and improving hatch rate by chemical means (Mitchell and Straus). Subobjective 2C. Evaluate caprylic acid to control streptococcosis in sunshine bass (Darwish). These objectives are designed to address current research concerns of stakeholders and will help in the ultimate goal of better treatments for disease epizootics resulting in higher profits for producers.
1b. Approach (from AD-416)
Objective 1. An infection method will be developed for white bass and sunshine bass to produce consistent mortalities similar to a natural infection of the bacterial disease columnaris caused by F. columnare. Resistance to columnaris will be characterized in families of sunshine bass and white bass, and maternal and paternal effects will be determined. The expression level of the putative virulence genes (identified by sequence comparison) in F. columnare will be characterized to provide insight into bacterial pathogenesis. Reverse-transcriptase real-time PCR methods will be developed and used to profile the transcript level of selected genes in isolates of high and low pathogenicity. Objective 2. The efficacy of CuSO4 against columnaris will be tested in vitro using micro-dilution assays and in vivo by using four treatment levels of CuSO4 against a columnaris disease model simulating a natural infection. The scenarios used to expose channel catfish to CuSO4 prior to bacterial challenge based on previous work will consist of either pre-exposure to CuSO4 dissolved in the water or pre-exposure to CuSO4 incorporated in feed. Research will be designed to improve the effectiveness of CuSO4, H2O2, and formalin to fungi on channel catfish eggs and on the hatch rate of these eggs. A new aquaculture therapeutant (peracetic acid) will be used to treat saprolegniasis on intact channel catfish eggs in hatching troughs. The concentration of caprylic acid that inhibits the bacteria S. iniae will be determined in vitro and its potential efficacy will be assessed against an experimental S. iniae infection in sunshine bass.
3. Progress Report
We compared the potential of static and flow-through peracetic acid (PAA) treatments to increase channel catfish egg survival and prevent fungal growth on eggs in a hatchery setting; static treatments required the lowest concentration of PAA, but turning water off in hatcheries would not be a practical application. We determined the acute toxicity of PAA to channel catfish fry; PAA was safer to use on newly hatched fry than on fry that were about 9 days old, but there was very low toxicity. We collaborated on a study to develop data on the use of PAA to reduce the growth rates of Flavobacterium columnare and Saprolegnia parasitica in culture. (Subordinate project 6225-32000-005-04N). We used the previously developed low-flow water delivery system to study the disease progression of columnaris in sunshine bass to produce consistent infections in order to study immune response characteristics; samples were taken to understand the molecular mechanisms of the disease and to observe any cellular changes. We determined that copper sulfate was more effective than potassium permanganate in lowering fish mortality in catfish naturally infected with columnaris, contrary to the generally accepted view that potassium permanganate is the best treatment. The efficacy of Aquaflor (c), copper sulfate, and potassium permanganate in controlling infections of Aeromonas hydrophila and F. columnare in sunshine bass was evaluated; Aquaflor (c) and copper sulfate were demonstrated to increase survival. We determined the length of time a parasite (Bolbophorus damnificus) can persist in channel catfish; this study outlined the potential severity of an infection. We collaborated on a study to determine the bioaccumulation of copper in red drum tissues after prescribed treatments to control external parasites (e.g., Amyloodinium sp.); this study went further to define the withdrawal period so the fish can be safely returned to the food supply. We used molecular methods (i.e., real-time PCR) to measure virulence gene expression in cultured Flavobacterium columnare strains and will compare this expression to samples from diseased fish; this test will predict which strain of the bacteria will cause the most severe disease.
1. Copper sulfate beats potassium permanganate in treating columnaris in channel catfish. Columnaris is known to be a severe bacterial disease in catfish culture. Treatment with approved antibiotics (such as potassium permanganate) is costly and can promote bacterial resistance. ARS scientists at the Stuttgart National Aquaculture Research Center, Stuttgart, AR, used fish with a natural infection of columnaris to evaluate the effectiveness of copper sulfate and potassium permanganate. The copper sulfate treatment was superior and significantly reduced mortalities. Since available resources to treat columnaris are limited, having a proven, economical treatment like copper sulfate will benefit the US aquaculture industry.
2. Immune response during simulated disease progression of columnaris in sunshine bass. Columnaris is one of the most lethal bacterial diseases of farmed fish. In an effort to identify factors that confer resistance to the disease, ARS scientists at the Stuttgart National Aquaculture Research Center, Stuttgart, AR, conducted a study to examine how the sunshine bass immune system responds to a laboratory-induced infection. Using different doses, researchers determined the optimum concentration of bacteria that reliably produced columnaris disease. Fish surviving the infection had more white blood cells in certain immune tissues than uninfected fish. White blood cells isolated from survivors responded more vigorously than those from fish that were not exposed to the bacteria. These findings are being used to develop screening techniques to predict whether certain fish may be more resistant to columnaris disease, which would ultimately improve profit margins for sunshine bass producers.
3. Longevity of Bolbophorus damnificus infections in channel catfish. The worm-like parasite Bolbophorus damnificus has a complex life cycle and infects commercial catfish, causing mortality, lower feed consumption, and reduced growth in surviving fish. In a collaborative study with scientists at Mississippi State University and ARS, scientists at the Stuttgart National Aquaculture Research Center, Stuttgart, AR, determined the length of time it remains viable in catfish. Our results indicate that the parasite can survive during the standard 18 to 30 month production cycle in the catfish industry. Live parasites impact catfish growth, and infected fish can serve as intermediate hosts for these parasites for at least 2.5 years. This study demonstrates to the catfish industry how important it is to use treatments to prevent the introduction of this parasite into the pond.
4. Peracetic acid has low toxicity to channel catfish fry. For peracetic acid (PAA) to be used to treat fungus on catfish eggs, its toxicity to hatching fry needed to be demonstrated. A collaborative study with a scientist at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany, and ARS scientists at the Stuttgart National Aquaculture Research Center, Stuttgart, AR, determined that PAA was safer to use on newly hatched fry than on fry that were about 9 days old, but there was very low toxicity. This research demonstrates the safety and usefulness of this novel compound and may result in widespread use.
5. Small amounts of peracetic acid reduce growth in aquatic pathogen cultures. Commercial products containing peracetic acid (PAA) are strong disinfectants with a wide spectrum of antimicrobial activity. A collaborative study with the Leibniz-Institute of Freshwater Ecology and Inland Fisheries, Berlin, Germany, the Université Catholique de Louvain, Louvain-la-Neuve, Belgium, and the Stuttgart National Aquaculture Research Center, Stuttgart, AR, compared the in vitro reduction of growth of Flavobacterium columnare and Saprolegnia parasitica using seven commercial PAA-containing products. Results showed that the products with a low concentration of PAA and a low PAA: hydrogen peroxide-ratio were generally more effective against pathogens. This research provides data for a new use of a widely used disinfectant that may also be useful in U.S. and international aquaculture.
6. Bioaccumulation of copper in red drum tissues after treatment. Red drum are cultured for food fish, and copper is routinely used to control external parasites. In a collaborative study, scientists at the Harbor Branch Oceanographic Institute of Florida Atlantic University, Fort Pierce, FL, and ARS scientists at the Stuttgart National Aquaculture Research Center, Stuttgart, AR, suspected that it tends to accumulate in the tissue of the fish. Healthy red drum were exposed to a chelated copper product for 8 months to control external parasites. Various tissues were analyzed for copper content, and we found that liver and intestine accumulated a high amount, while skin, gill, and muscle tissue did not accumulate substantial amounts. We also found that red drum cultured in recirculating systems had lower copper concentrations in muscle tissue relative to wild-caught red drum. Data indicates a 21-day withdrawal period is sufficient to meet the standards for human consumption. This research will significantly benefit the safety of the U.S. food supply while helping fish-farmers maintain a healthy fish population.
7. Treating common bacterial diseases in sunshine bass with Aquaflor (R), copper sulfate and potassium permanganate. Sunshine bass operations in the US have been plagued by pathogens like Aeromonas hydrophila and Flavobacterium columnare. This was a collaborative study with ARS scientists at the Aquatic Animal Health Research Laboratory, Auburn, AL, the Thad Cochran Research Center,University, MS, and the Stuttgart National Aquaculture Research Center, Stuttgart, AR. Aquaflor (R) - medicated feed (active ingredient is the antibiotic florfenicol) was shown to significantly increase the survival of sunshine bass infected with these bacteria better than copper sulfate and potassium permanganate. These results furnish the industry with potentially beneficial treatments against common infections.
Bowker, J., Trushenski, J., Tuttle-Lau, M., Straus, D.L., Gaikowski, M., Goodwin, A., Sprague, L., Bowman, M. 2011. Guide to Using Drugs, Biologics, and Other Chemicals in Aquaculture. American Fisheries Society Book Series. Available: http://www.fishculturesection.org/DrugGuide/Files/GUIDE_FEB_2011.pdf.