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 continued using molecular methods (i.e., real-time quantitative 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(s) of bacteria will cause the most severe disease. We initiated acute toxicity tests on juveniles of several species to develop a more complete understanding on the use of peracetic acid in aquaculture. We evaluated Flavobacterium columnare concentrations and fish densities required to produce consistent columnaris infections in white bass and in hybrid striped bass. We found that white bass genetically selected for growth are less susceptible to F. columnare than hybrid striped bass. We used molecular methods to study the mechanisms of columnaris disease. We also examined the role of nutritional status on RBL regulation. We conducted experiments to evaluate the effects of pre-exposure of channel catfish fingerlings to copper sulfate on susceptibility to columnaris disease. Results indicate that resistance can be induced but is strongly correlated to the time between copper sulfate exposure and bacterial challenge. We compared the potential of copper sulfate and peracetic acid treatments to prevent fungus on hybrid striped bass eggs. Preliminary data suggests that copper sulfate is more effective than peracetic acid and results in higher survival of fry through the hatching stage (Subordinate project 6225-32000-005-09N). We prepared final study reports for completed effectiveness dose confirmation studies on using copper sulfate to control egg mortality associated with saprolegniasis infecting channel catfish eggs and are submitting these to the sponsor who will submit them to FDA (Subordinate project 6225-32000-005-07N).
1. Pre-exposure to copper can protect fish from columnaris disease. Columnaris is a major fish bacterial disease causing significant losses to the aquaculture industry. ARS researchers at the Stuttgart National Aquaculture Center in Stuttgart, Arkansas, demonstrated that challenging fish with columnaris bacteria immediately after a 24-hour copper sulfate exposure produced significantly lower survival than fish that were allowed an additional 24 hours in flow-through water before bacterial challenge; resistance was determined to last at least 9 days after the copper sulfate exposure. This research can be used by producers to develop best management practices for implementation when risk of disease is greatest (e.g., seining ponds, grading fingerlings, hauling fish to grow-out facilities), since available treatments for columnaris are limited. As there is a trend towards reducing expensive antibiotic treatments, having a proven, economical treatment like copper sulfate benefits the US aquaculture industry. More widespread adoption of this technology is expected in the next few years.
2. Bacterial binding sites can be altered to control columnaris disease. Columnaris is a major fish bacterial disease causing significant losses to the aquaculture industry. Evidence suggests a part of the fish immune system that assists in killing bacteria, called rhamnose-binding lectins, are increased in the gill of catfish infected with columnaris and can be measured using molecular tools. Using these tools, ARS scientists in Stuttgart, Arkansas, demonstrated dramatic evidence of increased immunity to columnaris after exposing fish to the bacteria and examining the quantity of lectins in the gills of two different families of channel catfish, and determined that one family was susceptible to the disease, while the other was completely resistant. The quantity of lectins in the gill was strongly increased in the columnaris-susceptible family, but by reducing the number of lectin binding sites on the gill, the catfish could be protected from columnaris disease. The nutritional status of the fish also had an effect, with a far greater amount of lectins in the gill of unfed fish relative to well-fed fish. Application of these findings to develop screening techniques for enhanced resistance to columnaris would substantially reduce losses of farm-raised catfish and increase industry profitability, while potential future application to other farm-raised fish species would benefit additional U.S. aquaculture producers and consumers.
Farmer, B.D., Mitchell, A.J., Straus, D.L. 2011. The effect of high total ammonia concentration on the survival of channel catfish experimentally infected with Flavobacterium columnare. Journal of Aquatic Animal Health. 23:162-168.