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 acute toxicity tests on juveniles of several fish species to develop a more complete understanding on the use of peracetic acid in aquaculture. We evaluated Flavobacterium columnare resistance/susceptibility in sunshine bass families. We found that survival varied by family, but not significantly, probably due to low sample size. We conducted lab experiments to evaluate the effect of copper sulfate incorporated in feed on channel catfish fingerlings to study their susceptibility to columnaris disease. Results indicate that resistance can be induced. A field trial to test these results on a larger scale is currently underway. We evaluated the influence of a variety of water chemistry parameters on the pathogenesis process of Flavobacterium columnare. Results suggest calcium plays a key role and further studies planned to elucidate the effects of individual chemical components. We determined the potential of copper sulfate treatments to prevent fungus on sunshine bass eggs. We concluded from the results of the study that 20 ppm copper sulfate is the target dose (Subordinate project 6225-32000-005-09N). We provided information to FDA as requested to support the previously submitted final study reports on use of copper sulfate to control fungus (saprolegniasis) infecting channel catfish eggs during the FDA review process (Subordinate project 6225-32000-005-07N).
1. Disease outbreak linked to water chemistry. Columnaris is a deadly fish disease that is caused by a bacteria (Flavo-bacterium columnare) that attaches to a fish's gills or skin (bacterial attachment). If we can stop the bacteria from attaching to the fish, we can prevent the fish from getting sick. Columnaris disease cannot be induced in some labs, and one possible explanation could be the water source. ARS scientists in Stuttgart, Arkansas, and Stoneville, Mississippi, found dramatic differences in catfish mortality after exposing fish to the columnaris bacteria in well-water from the two labs. Further investigation indicated bacterial attachment was 2000-fold less in softer waters with tannins. An additional study manipulated the calcium and tannin concentration in the waters to study their effects on the disease. The results suggest calcium is a key regulator of bacterial attachment. The study reveals the mechanisms of bacterial pathogenicity; this greater understanding will lead to further advances in controlling this disease.
2. Copper sulfate effectively controls fungus (Saprolegnia) and increases survival of sunshine bass eggs. Fungus in sunshine bass hatcheries can cause decreased hatch rates and decreased profits. A collaborative study with ARS scientists in Stuttgart, Arkansas, and Keo Fish Farm, Inc. (Keo, Arkansas) addressed sunshine bass egg mortality from fungal growth. This study determined the optimum concentration of copper sulfate needed to prevent fungus from destroying the eggs while allowing the hatched larvae to thrive. The impact of this accomplishment was to decrease larval mortality and reduce time and effort in the hatchery, thereby having an immediate economic benefit to the farmer with this inexpensive treatment. This provides direct benefits through increased survival rates in sunshine bass hatcheries which will economically benefit the industry; the majority of the industry is now using this treatment.
3. Analysis of the columnaris transcriptome reveals genes that mediate virulence. In lab studies, Flavobacterium columnare (the bacteria that causes columnaris disease) isolates from different fish species or different geographic regions have been shown to vary in their ability to cause disease, a characteristic termed virulence. To better understand this, ARS scientists in Stuttgart, Arkansas, and colleagues at Auburn University, Alabama, used a powerful, next-generation sequencing technology termed RNA-seq to examine thousands of genes in two isolates of the bacteria with known differences in virulence (i.e., one highly virulent and one weakly virulent). In the highly virulent isolate, genes previously shown to control virulence in human pathogens were highly upregulated. Genes and gene pathways identified were those involved in the acquisition of iron and carbohydrates, and genes that allow the bacterium to evade and protect itself from the host immune system. These findings can be used to make new columnaris disease vaccines, or improve existing vaccines, and provide scientists with a better understanding of the factors that lead to columnaris disease outbreaks in farmed fish.
4. Kaolinitic clay protects fish from columnaris disease. Columnaris disease, caused by the bacterium Flavobacterium columnare, is a costly disease of many commercially grown fish species, including channel catfish. Despite its importance, few preventatives or therapies exist for this disease. In this study, a type of clay called kaolin was evaluated for the prevention of columnaris disease. ARS scientists in Stuttgart, Arkansas, demonstrated that addition of kaolin to the water significantly improved the survival of channel catfish that were experimentally infected with the disease, and protected the gill from damage by the bacteria. Data suggests that kaolin works by binding to the bacteria, thereby preventing it from attaching to the fish and initiating disease. The impact of this accomplishment is to improve the economics of the industry by providing a novel, non-antibiotic treatment to increase survival rates in catfish hatcheries.
Marchand, P., Phan, T., Straus, D.L., Farmer, B.D., Stuber, A., Meinelt, T. 2012. Reduction of in vitro growth in Flavobacterium columnare and Saprolegnia parasitica by products containing peracetic acid. Aquaculture Research. 43:1861-1866.