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
Compared the potential of static and flow-through hydrogen peroxide and copper sulfate treatments to increase channel catfish egg survival and prevent fungal growth on eggs; hydrogen peroxide static treatments proved best. Completed the development of a low-flow water delivery system for columnaris challenge testing. High levels of ammonia in water were shown to lower fish mortality in fish infected with bacteria, contrary to the generally accepted view that ammonia increases mortality in infected fish. Investigated the therapeutic value of copper sulfate against an acute columnaris infection in channel catfish; fish challenged with Flavobacterium columnare and treated by waterborne exposure to copper sulfate (4.2 and 2.1 mg/L) had reduced mortalities compared to non-treated fish. Evaluated the efficacy of florfenicol, copper sulfate and potassium permanganate in controlling infections of Aeromonas hydrophila and F. columnare in sunshine bass; florfenicol and copper sulfate were demonstrated to increase the survival rate. The microbiology laboratory participated by invitation in an international multi-laboratory study to establish the expected drug sensitivity ranges of the quality control bacteria, Escherichia coli (ATCC25922) and Aeromonas salmonicida (ATCC33658). Designed a primer set and fluorescent probe to amplify the 16S ribosomal gene of F. columnare to be used as an internal control for virulence gene expression. Completed the in-life phase of a safety study to demonstrate that the proposed recommended daily treatment of 17a-methyltestosterone (9 mg/kg fish body weight) and 3 or 5 times the recommended treatment for 28 days were safe for treating tilapia. Evaluated peracetic acid, copper sulfate, and potassium permanganate to control Ichthyobodo necator; copper sulfate was the best treatment. Completed testing on acute toxicity of CuSO4 to Ich theronts in reconstituted waters (Subordinate project 6225-32000-005-03N). Evaluated peracetic acid for fungus control on catfish egg masses in a flow-through system; 2.5 mg/L twice a day prevented fungus from growing and destroying the eggs (Subordinate project 6225-32000-005-04N).
1. FDA-required study on the safety of 17A-methyltestosterone (17MT) to tilapia. The histological safety of 17MT, used to produce an all-male population, is unknown, but is required as a major component of the FDA-approval process. A study was completed in collaboration with researchers at the US Fish & Wildlife Service in Bozeman, Montana, to demonstrate that the proposed recommended daily treatment (9 mg/kg fish body weight) and 3 or 5 times the recommended treatment for 28 days were safe for treating tilapia. Mortality data indicates an acceptable margin of safety. Approval will benefit the US tilapia industry by allowing farmers to use 17MT legally in tilapia hatcheries.
2. Peracetic acid (PAA) controls fungus on catfish eggs. Fungus in catfish hatcheries can cause decreased hatch rates. A collaborative study with a scientist at the Leibniz-Institute of Freshwater Ecology and Inland Fisheries (Berlin, Germany) and scientists at the HKD Stuttgart National Aquaculture Research Center addresses egg mortality from fungus in catfish hatcheries. This range-finding study determined that treating catfish eggs twice daily with 2.5 ppm PAA until hatching begins will prevent fungus from growing and destroying the eggs. This accomplishment will provide direct benefits through increased survival rates in catfish hatcheries, which will economically benefit the industry. In addition, we have provided the scientific community with more information on the uses of this novel compound.
3. FDA-Required study on copper sulfate safety to catfish eggs accepted by FDA. The safety of copper sulfate to catfish eggs was unknown. A report entitled 'A Target Animal Safety Study on the use of Copper Sulfate to Control Egg Mortality Associated with Saprolegniasis Infecting Channel Catfish Eggs' was submitted to the FDA in December 2009 and accepted in May 2010. This study was completed following Good Laboratory Practice guidelines and determined the safety of this compound to channel catfish eggs at 10, 30, and 50 mg/L when treated daily until embryos developed eyes. Hatch rate was not affected by copper toxicity, indicating this is an extremely safe treatment. This work completed a major component of the approval process, thereby improving the economics of the industry by providing an inexpensive treatment to increase survival rates in catfish hatcheries.
4. High levels of ammonia reduce mortality in channel catfish infected with columnaris. The interaction of ammonia and Flavobacterium columnare is poorly understood; however, it is generally accepted that elevated ammonia levels increase mortalities of diseased fish. A concentration of 15 ppm ammonia significantly increased survival of fish exposed to columnaris. Knowledge of this interaction will lead to a better understanding of the mechanisms involved in treating diseases. The use of high ammonia levels in fish tanks could produce an effective treatment for columnaris, which is one of the two most costly diseases to the channel catfish industry.
5. Hydrogen peroxide static treatments effective for fungus control on catfish eggs. Water molds or fungi can destroy entire channel catfish egg masses in commercial hatching troughs, costing fish producers several hundred thousands of dollars of losses and fish shortages. An experiment was designed to compare static and flow-through hydrogen peroxide and copper sulfate pentahydrate treatments for their ability to control fungus and increase egg survival. All treatments were observed to limit egg fungus and increase egg survival, but the highest egg survival and least fungal development were achieved using a static hydrogen peroxide treatment. The use of a static hydrogen peroxide treatment on fish eggs will potentially allow hatcheries to produce more juvenile channel catfish and realize greater economic benefits.
6. Copper sulfate is effective against acude columnaris infections in channel catfish. Approved antibiotics are costly and can promote bacterial resistance when treating columnaris. Copper sulfate was evaluated as a therapeutant against acute columnaris in channel catfish and significantly reduced the mortalities. The available resources to treat columnaris are limited, and having an economical treatment like copper sulfate will benefit the US aquaculture industry.
7. Florfenicol, copper sulfate, and potassium permanganate efficacy against a natural infection of Aeromonas hydrophila and Flavobacterium columnare in sunshine bass. Sunshine bass operations in the US have been plagued by pathogens like Aeromonas hydrophila and Flavobacterium columnare. Florfenicol medicated feed was shown to significantly increase the survival of sunshine bass infected with these bacteria. These results furnish the industry with potentially beneficial treatments against these common infections.
Mitchell, A.J., Straus, D.L., Farmer, B.D., Carter, R.R. 2010. Comparison of percent hatch and fungal infestation in channel catfish eggs after copper sulfate, diquat bromide, formalin, and hydrogen peroxide treatment. North American Journal of Aquaculture. 72:201-206.