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United States Department of Agriculture

Agricultural Research Service

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Location: Warmwater Aquaculture Research Unit

2013 Annual Report

1a. Objectives (from AD-416):
Objective 1: Monitoring and surveillance of biotoxin, pathogen, pathogen/host interactions in aquatic environment. Objective 2: Develop methods to protect channel catfish and its hybrids with vaccines, antibiotics and other therapeutics against enteric septicemia of catfish, columnaris, proliferative gill disease, botulism, Bolbophorus and anemia. Objective 3: Effects of chemical and mycotoxin feed contaminants on growth and disease resistance of catfish.

1b. Approach (from AD-416):
Despite successful growth and prosperity of the past few decades, the U.S. catfish industry is threatened by increasing disease losses, low fish prices, high feed costs and foreign competition. Fish losses due to disease are estimated to cost the U.S. catfish industry $100 million in direct sales annually and are considered the largest impediment to increasing production efficiencies. Objective 1 will provide catfish farmers a better method to monitor biotoxins, pathogens, and pathogen/host interaction in the aquatic environment using new and better surveillance and monitoring procedures coupled with the development of experimental vaccines. Case submissions will also document the prevalence and the emergence of new diseases in the catfish industry. Objective 2 will develop new methods to protect catfish against known disease organisms including antibiotics and vaccines and evaluate the effectiveness of these products to improve disease resistance. New and improved on-farm management programs for the control of trematode infections will be developed. Objective 3 will investigate the prevalence of chemical and mycotoxin feed contaminants in fish feeds and develop methods to detect and control feed contaminants that affect disease resistance of catfish. The overall benefits of this project will be to improve fish health reduce losses due to infectious and non-infectious diseases. The reduction of losses to disease will make catfish farming a more profitable endeavor and increase the competitiveness of U.S. aquaculture.

3. Progress Report:
A mechanized system for mixing and delivering a live attenuated oral vaccine was developed in collaboration with United States Department of Agriculture-Agricultural Research Service (USDA-ARS), Poultry Unit, and Mississippi State University. Protocols for the commercial production of a live attenuated oral enteric septicemia of catfish (ESC) vaccine was developed in collaboration with USDA-ARS-National Biological Control Laboratory. Vaccine field trials conducted with the developed technologies showed excellent protection from disease and negated the need for medicated feeds. Field trials showed smallmouth buffalo did not alter oligochaete populations, intermediate host for proliferative gill disease, or reduce actinospore levels in pond water. As a result SMB are no longer recommended as a biological control for this disease. Increased pressure from the American white pelican has resulted in wide spread debilitating trematode infections on a majority of farms. Disease treatment regiments, developed from this project, have been implemented and were shown effective in controlling the intermediate host of the parasite. Real-time polymerase chain reactions (PCR) were developed to detect the trematode Bolbophorus damnificus in fish tissues and pond water. The assay differentiates between two morphologically similar pathogenic and non-pathogenic species of Bolbophorus and is critical to scientific studies. Development of real-time PCR assays for catfish pathogens led to the reclassification of Edwardsiella (E.) tarda. Further research identified this fish pathogen as E. piscicida, a newly described pathogen from fish. Assays for the detection of Flavobacterium columnare, E. tarda, E. piscicida and E. piscicida-like species are being validated. Work has been initiated into the development of a diagnostic PCR assay for the identification of the digenetic trematode Drepanocephalus spathans, a parasite of the double crested cormorant, and shown to be infective to channel catfish. In addition, work has also been initiated into the development of a discriminatory PCR assay for the identification of an unidentified echinostomatid cercaria, demonstrated to be infective to catfish. In 2012, the Aquatic Research & Diagnostic Laboratory received a total of 772 cases of which 635 were submitted by producers which is an increase from the previous year (599 cases) despite the contraction of the channel catfish industry. There were no antibiotic resistant isolates out of the 363 bacterial isolates cultured. Botulism Type E (BoNT/E) was identified as the causative agent of visceral toxicosis of catfish (VTC). Solid phase ELISA assays for the detection of anti-BoNT/E antibody were developed against recombinant peptide sequences of the heavy chain toxin. Antibody from convalescent fish showed low specificity to the peptide sequences employed in the assay. Fish immunized with a recombinant heavy chain (BoNT/E) yielded weak and negligible antibody responses and offered no protection following exposure to the toxin. The toxin was successfully inserted in channel catfish virus (CCV) and the recombinant BoNT/E CCV will be evaluated as a potential vaccine.

4. Accomplishments
1. Development of a live attenuated vaccine and in-pond vaccination platform to protect catfish against enteric septicemia of catfish. A new vaccine and an “in-pond" vaccine delivery platform has been developed resulting in exceptional protection against Enteric Septicemia of Catfish (ESC) and dramatic increases in production efficiency and economic returns. Commercial application of the developed vaccine technology was shown dependent on a system for mixing the vaccine with feed at the point of deliver. Mississippi State University (MSU) scientist working in collaboration with scientist with Mississippi State University, Department of Ag and Bio Engineering and USDA-ARS Poultry Unit, Starkville, MS, developed a mechanized on-demand vaccine delivery system shown to consistently deliver the target immunizing dose in experimental pond trials. Vaccination using the delivery system offered excellent protection from Edwardsiella (E.) ictaluri infection and increased production efficiency. The vaccine delivery system will be used in commercial field trials during the 2013 production season.

2. Commercial fermentation of a live attenuated vaccine for use in an oral delivery platform. Mississippi State University (MSU) scientist working in collaboration with the United States Department of Agriculture - Agricultural Research Service (USDA-ARS) National Biological Pest Control Laboratory at Stoneville, MS, developed fermentation protocols for the commercial production and storage of vaccine serials. Vaccine was fermented in 50 L floor model commercial ferementors, concentrated and frozen in 50 ml aliquots and validated for cell viability. Frozen vaccine serials were shown stable and effective for up to 1 year. Vaccine serials produced from protocols developed from this project will be used to in commercial field trials to assess field safety and efficacy, environmental fate, and establish proof of concept for the develop vaccine technologies. Safety studies on the developed master seed line will include a tissue persistence study evaluate clearance of the attenuated isolate and a backpassage study to assess the potential of reversion. Data collected from these trials will be used to pursue USDA licensing of an oral ESC vaccine for use in channel catfish.

3. Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as Edwardsiella (E.) tarda. Mississippi State University scientists working in collaboration with the United States Department of Agriculture – Warmwater Aquaculture Research Unit in Stoneville, MS, assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated polymerase chain reaction (PCR) and multi-locus sequence analysis of eight different housekeeping genes identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II represented a previously unrecognized, genetically distinct taxa of Edwardsiella, the newly described E. piscicida.

4. Edwardsiella piscicida identified in the southeastern United States. Mississippi State University (MSU) scientists working in collaboration with the United States Department of Agriculture – Warmwater Aquaculture Research Unit in Stoneville, MS, have identified a new taxa of Edwardsiella (E.) isolated from pond-raised catfish in the southeastern United States that is phenotypically indistinguishable from Edwardsiella tarda. This taxa has recently been described from fishes of Europe and Asia. Based on extensive genetic and phenotypic characterization, European researchers have determined this new strain does not belong to any established taxa within the genus Edwardsiella and have proposed the adoption of a new taxon, E. piscicida. Comparisons of gyrB sequences performed by MSU scientists at National Warmwater Aquacultural Center (NWAC), in Stoneville, MS, identified several isolates from the U.S. that demonstrated greater than 99.6% similarity to the the E. piscicida type strains from Europe and Asia, suggesting conspecificity. A discriminatory polymerase chain reaction (PCR) assay was developed by MSU researchers to differentiate between E. tarda, E. ictaluri and two genetic variants of E. piscicida. Using this novel PCR assay, a survey was conducted of 44 archived bacterial specimens isolated from diseased catfish from 2007-2012. Archived isolates were identified biochemically as E. tarda upon initial collection, all of which were confirmed by PCR to be misclassified E. piscicida. This supports previous claims from Europe and Asia, suggesting a cosmopolitan distribution of E. piscicida and a greater association with disease outbreaks in fish than E. tarda. This is the first report of E. piscicida in the United States.

5. The development of molecular test for the detection of significant catfish pathogens in environmental and tissue samples. Researchers from Mississippi State University (MSU) (Mississippi Agricultural and Forestry Experiment Station, MAFES and College of Veterinary Medicine (CVM) have developed and validated quantitative polymerase chain reaction assays for the detection and quantification of Aeromonas hydrophila, Edwardsiella ictaluri, Bolbophorus damnificus, Bolbophorus type II sp., and Henneguya ictaluri. Similar assays for Flavobacterium columnare, Edwardsiella tarda, Edwardsiella piscicida and Edwardsiella piscicida-like are in the final stages of validation. These assays provide a reliable method for the detection and quantification of pathogens in pond environments and are being used for rapid diagnostic evaluations and evaluation of treatment efficacies associated with recommended disease management strategies and research. Using this technology, risk assessment models for proliferative gill disease (PGD) have been developed and are being used by diagnostic services to mitigate losses during stocking and restocking programs. Additionally, the Aeromonas (A.) hydrophila assay is currently in use in our laboratory, as well as the Aquatic Diagnostic Laboratory of the University of Arkansas – Pine Bluff, the Aquatic Diagnostic Laboratory at the College of Veterinary Medicine in Starkville, MS, to confirm the presence/absence of the emerging virulent strain of A. hydrophila in suspect cases. In addition to their diagnostic benefits, these assays provide a means to conduct epidemiological and environmental studies and evaluate how management strategies alter pathogen loading rates in commercial catfish ponds. This information will be used to optimize disease management practices aimed at maximizing production efficiencies and economic returns.

6. Survey of myxozoan parasites associated with catfish aquaculture. Mississippi State University scientists have conducted a survey of myxozoan parasites associated with catfish aquaculture. Using techniques, actinospore stages of myxozoan life cycles have been isolated and genetically characterized by 18S small sub unit (SSU) ribosomal deoxyribonucleic acid (rDNA) sequence. This survey has identified at least 9 different myxozoan life cycles in catfish ponds. In addition, two myxozoan life cycles have been molecularly confirmed, linking the actinospore stage released by the benthic oligochaete to the myxospores stage in the catfish by 18S SSU rDNA sequence. Moreover, this work has also identified and genetically characterized a previously undescribed myxozoan parasite from catfish, although the actinospore stage associated with this myxospores has not been determined. The effects these previously uncharacterized myxozoan parasites have on catfish production is currently unknown and will be a focus of future research.

7. Pharmacokinetics of florfenicol in channel catfish. Researchers from Mississippi State University at Mississippi State, MS, conducted pharmacokinetic studies of florfenicol in catfish to extend label claims. Final study reports from a florfenicol pharmacokinetic study in catfish and minimal inhibitory concentration (MIC) studies were submitted to U.S. Food and Drug Administration (USFDA) for approval of Aquaflor® for control of mortality associated with Aeromonas hydrophila and Edwardsiella tarda in catfish.

8. The use of diagnostic case submissions to track disease trends in the catfish industry. The National Warmwater Aquaculture Center’s Aquatic Research & Diagnostic Laboratory (ARDL), administered by the Mississippi State University’s College of Veterinary Medicine is an American Association of Veterinary Laboratory Diagnosticians accredited laboratory. It provides comprehensive disease diagnostic service to catfish producers centered in Mississippi and surrounding states, including Alabama, Arkansas, Louisiana, and Texas. In 2012, the ARDL received a total of 635 producer submitted cases diagnostic cases from 41 different commercial farms. It also provided water quality analysis for the 1332 samples from 119 farms. Bacterial cases dominated the case submissions with 185 Columnaris disease cases and 165 Enteric Septicemia of Catfish cases. Unlike the previous year, no cases of the highly virulent Aeromonas hydrophilia cases were seen at the laboratory. Also no antibiotic resistant bacterial isolates were seen last year. There was an increase in the number of Bolbophorus trematode cases in 2012 (18 cases) and this upsurge appears to have continued in 2013 based on preliminary observations which may be due to increased loafing sites (abandoned farm ponds) for the final host, the American White Pelican. Hybrid catfish cases also increased corresponding to the expanded use of hybrid catfish by the industry.

9. Use of native smallmouth buffalo as a biological control for proliferative gill disease (PGD). Proliferative gill disease is caused by a myxozoan parasite that is transmitted by an aquatic oligochaete ubiquitous in pond environments. Preliminary studies conducted in simulated pond environments indicated smallmouth buffalo (SMB) may be an effective agent for biological control of benthic oligochaetes. As a continuation of this research, scientists with Mississippi State University conducted a two year field study evaluating the use of SMB to control PGD. Smallmouth buffalo were not shown to alter benthic oligochaete populations nor reducing myxozoan levels in pond water, as determined by PCR. As result SMB are no longer recommended for biological control of PGD.

Review Publications
Soto, E., Griffin, M., Wiles, J., Hawke, J. 2012. Genetic analysis and antimicrobial susceptibility of Francisella noatunensis subsp. orientalis (sun. F. asiatica) isolates from fish. Veterinary Microbiology. 154:407-412.

Chata, K., Gaunt, P.S., Hanson, L., Gao, D.X., Wills, R. 2012. Determination of the median lethal dose of botulinum serotype E in channel catfish fingerlings. Journal of Aquatic Animal Health. 24:105-109.

Soto, E., Griffin, M., Arauz, M., Martinez, A., Cabrejos, M.E. 2012. Edwardsiella ictaluri as the causative agent of mortality in cultured nile tilapia. Journal of Aquatic Animal Health. 24(2):81-90.

Gaunt, P.S., Langston, C., Gao, D., Adams, P., Crouch, L., Sweeny, D., Endris, R. 2012. Multidose pharmacokineetecs of orally administered florfenicol in channel catfish Ictalurus punctatus. Journal of Veterinary Pharmacology and Therapeutics. p. 1-5.

Mischke, C.C., Greenway, T.E., Griffin, M.J., Wise, D.J. 2012. Effects of fry age-at-stocking on growth and survival of channel catfish. Journal of the World Aquaculture Society. 43(1):135-139.

Gieseker, C.M., Mayer, T.D., Crosby, T.C., Carson, J., Dalsgaard, I., Darwish, A.M., Gaunt, P.S., Gao, D.X., Hsu, H., Lin, T.L., Oaks, J.L., Pyecroft, M., Teitzel, C., Somsiri, T., Wu, C.C. 2012. Quality control ranges for testing broth microdilution susceptibility of Flavobacterium columnare and F. psychrophilium to nine antimicrobials. Diseases of Aquatic Organisms. 101:207-215.

Soto, E., Kidd, S., Gaunt, P.S., Endris, R. 2012. Efficacy of florfenicol for control of mortality with Francisella noatunensis subsp. orientalis in Nile tilapia, oreochromis niloticus (L.). Journal of Fish Diseases. 36:411-418.

Soto, E., Lamon, V., Griffin, M., Keirstead, N., Beierschmitt, A., Palmour, R. 2012. Phenotypic and genotypic characterization of Klebsiella pneumonia recovered from nonhuman primates. Journal of Wildlife Diseases. 48(3):603-611.

Last Modified: 05/25/2017
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