An official website of the United States government.
The overall objectives are to define the pathogenicity of botulinum E (BoNT/E) in catfish and identify risk factors for visceral toxicosis of catfish (VTC). These findings will lead to prevention of VTC and decreased economic losses to catfish farmers.
<P> The specific aims are: <OL> <LI> Optimize the detection of Clostridium botulinum and BoNT/E in pond sediment, catfish tissues, dead fish, and pond invertebrates. This will be accomplished through: a. Mass spectrometric (MS) identification of BoNT/E by direct detection of intact toxin and its peptide fragments and indirectly by detection of fragments of a peptide substrate produced by peptidase activity of BoNT/E (Year 1: Method development for catfish serum, muscle, intestine; Year 2: Method development for invertebrates and dead catfish; Year 3: Monitor BoNT/E samples with optimized methodologies) b. Bacterial culture of C. botulinum and real-time PCR (QPCR) quantification of C. botulinum toxin E (bontE) gene (Method development: Year 1, assays samples Years 2-3) c. Onset of detection of the presence of C. botulinum, BoNT/E and bontE gene in catfish carcasses (Year 2) <LI> Identify environmental risk factors of VTC through monitoring diagnostic cases submitted to the MSUCVM Aquatic Diagnostic Laboratory in conjunction with evaluating data collected from catfish ponds and farming practices (e.g. water temperature and quality, fish stocking density, feeding regimens, etc). (Data collection Years 1-3) <LI> Evaluate the pathophysiologic responses of catfish to BoNT/E. a. Confirmation of the median lethal dosage (LD50) of BoNT/E in catfish injected intracoelomically (IC) (Year 1) b. Effects of chronic sublethal BoNT/E exposure on swimming activity (Year 2), mortality (Year 2) and disease resistance of catfish (Year 3) c. Effects of sublethal BoNT/E exposure on susceptibility of catfish to bacterial infection (such as Flavobacterium columnare). (Year 3) d. Serologic response and clinical efficacy in catfish of an experimental BoNT/E recombinant vaccine (Years 1-3)
</ol>
Expected Outputs: <OL> <LI> Define BoNT/E quantities lethal to catfish, the amount that is present in catfish fillets (to satisfy human food safety and workers (in catfish industry) concerns) <LI> Understanding the effects of chronic sublethal exposure to BoNT/E in catfish in terms of sensitivity to repeated low level exposures, synergistic effects with pathogenic bacteria such as Flavobacterium columnare, and interference with swimming ability. <LI> Identification of risk factors for botulism in catfish ponds and altering appropriate farm management practices to prevent VTC outbreaks <LI> Development of a vaccine to prevent VTC
NON-TECHNICAL SUMMARY: Visceral toxicosis of catfish (VTC) is a recently recognized syndrome in the catfish industry, that was first reported in the Mississippi Delta region in 1999. Farmers described the sudden death of larger catfish (market and brooder-size) when pond water surface temperatures were between 18-22 degrees C. Often entire ponds of market-ready fish were lost during outbreaks, which created huge economic losses for farmers. VTC is now linked to botulinum toxin (BoNT/E) which is produced by the anaerobic spore-forming bacterium Clostridium botulinum. The project has three main objectives: 1) Optimization of the diagnosis and detection of C. botulinum and BoNT/E in catfish serum, muscle, and intestinal tract, dead fish, pond sediments, and pond invertebrates through indirect and direct mass spectrometry detection methods, bacterial culture, and real time PCR; 2) Identification of environmental risk factors of VTC by evaluating data collected from catfish ponds and farming practices (e.g. water temperature and quality, fish stocking density, feeding regimens, etc); 3) Evaluation of the pathophysiologic responses of catfish to BoNT/E. These experiments include: determining the effects of sublethal BoNT/E exposures and swimming performance in catfish, assessing the effects of repeated sublethal injections of BoNT/E on survival in catfish, assessing the effect of a sublethal dose of BoNT/E on survival in Flavobacterium columnare-exposed catfish and assessing the immune response of a BoNT/E vaccine on VTC- or BoNT/E- challenged catfish.
<P>APPROACH: <BR> Aim 1 Diagnosis and detection of C. botulinum and BoNT/E in catfish, sediment, and pond invertebrates Aim 1a Optimize the diagnosis and detection of C. botulinum and BoNT/E via MS identification of BoNT/E by: 1. Indirect detection by endopeptidase assay coupled with MS detection of products of BoNT/E activity on the synthetic peptide substrate. 2. Direct detection via nano-LC nano-spray MSMS analysis to identify BoNT/E peptides. Software will be used for statistical analysis of MS data which will allow quantitation of the BoNT protein itself. Aim 1b Bacterial culture of C. botulinum and QPCR quantification of bontE gene. Catfish intestinal contents will be collected aseptically in anaerobic containers. Sediment and oligochetes from the same ponds will also be sampled. Specimens will be plated on C. botulinum isolation medium and incubated. DNA from isolated C. botulinum will be assayed for the presence (or absence) of C. botulinum Type E using QPCR with probes for bontE gene. <BR> <BR> Aim 2 Identify environmental risk factors of VTC by evaluating data collected from catfish ponds. Case-control methodology will assess risk factors for VTC. Cases will be confirmed VTC ponds. Two control ponds will be paired with each case pond. An investigative survey questionnaire on management practices will be developed for producers. At the time of investigation, pond water chemistries will be assayed, and fish, sediment, and invertebrates collected and tested for BoNT/E and bontE gene. Through statistical analysis this study should identify risk factors for VTC.<BR> <BR> Aim 3 Pathophysiologic responses of catfish to BoNT/E Aim 3a LD50 of BoNT/E in catfish Fish will be injected IC with various doses of BoNT/E to determine a statistical concentration that kills 50% of fish. The statistically determined LD50 will be pivotal for BoNT/E physiologic experiments. Aim 3b Sublethal BoNT/E exposure on mortality and swimming activity in catfish Sublethal BoNT/E quantities will be injected in fish every 3 days for 14 days. A regression model will test for significance between survival and dose. Another group will be injected with either a sublethal dose or sham injected. Swimming performance, respiration, and cost of transport will be measured. Fish will be placed in a swimming tunnel and water velocity increased every 30 minutes until fish fatigue. Data from treated and control fish will be analyzed. Aim 3c Disease resistance of catfish Three groups of fish will be compared. Groups 1 and 2 will be injected with sublethal doses of BoNT/E. Group 3 will be sham injected. Groups 1 and 3 will be challenged with F. columnare. Statistical analysis of mortality data will assess whether exposure to low doses of BoNT/E followed by exposure to F. columnare enhances catfish susceptibility to this bacterium. Aim 3d Serologic response and clinical efficacy of an experimental BoNT/E recombinant vaccine We will evaluate whether protection from a BoNT/E Hc subunit vaccine is provided against challenge with BoNT/E or VTC and whether cooler water affects the degree of protection. In all experiments susceptibility and antibody levels of vaccinated fish will be compared to sham-vaccinated controls.