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ARS Home » Southeast Area » Stoneville, Mississippi » Warmwater Aquaculture Research Unit » Research » Research Project #418787


Location: Warmwater Aquaculture Research Unit

2012 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:
In 2011, the Aquatic Research & Diagnostic Laboratory saw a total of 852 cases of which 599 were cases submitted by producers. In addition, a total of 1,060 water quality samples were submitted and analyzed. With regards to antibiotic resistance, there were no resistant isolates seen in the over 400 bacterial isolates cultured. Only 1 of 191 isolates of Edwardsiella (E.) ictaluri and 2 out of 19 isolates of Edwardsiella tarda had intermediate sensitivities. Oral vaccination of fish with an attenuated E. ictaluri vaccine was shown very effective in preventing mortality associated with enteric septicemia of catfish. The vaccine was shown to negate the need for medicated feeds to control mortality. Preliminary studies indicated smallmouth buffalo (SMB) may be an effective agent for biological control of benthic oligochaetes, which transmit myxozoan parasites, including the causative agent of proliferative gill disease. Ponds stocked with juvenile SMB (2-3”) had significantly lower oligochaete populations compared to ponds without SMB. Primer and probe combinations have been developed for specific amplification for Edwardsiella ictaluri, Flavobacterium (F.) columnare, Bolbophorus (B.) damnificus and Henneguya (H.) ictaluri and an emerging, highly virulent strain of Aeromonas hydrophila. A similar assay for E. tarda is being developed. Assays for H. ictaluri, B. damnificus and E. ictaluri have been validated and will be used to evaluate infections rates and integrated into field monitoring programs. Assessment of antigen specific antibody levels in channel catfish is based on a detection system employing a mouse monoclonal antibody (9E1) developed against channel catfish immunoglobulinm (IgM). Validation studies show 9E1 binds equally with IgM antibodies of blue and blue x channel hybrid catfish. Immunoassays assessing humoral immunity in blues and hybrids have been developed to key catfish pathogens. The LD50 for botulinum type-E toxin (BoNT/E) was determined to establish vaccination/challenge protocols for catfish. The 96 hour LD50 for 5.3 grams channel catfish was determined to be 13.7 pico grams (pg). Immunization trials with recombinant heavy chain BoNT/E have been conducted to hyper-immunize channel catfish to create a reference serum to determine optimal antigen coating concentrations for enzyme-linked immunosorbent assay (ELISA). Based on endpoint titers, generated from hyper-immunized fish (IP injection), the recombinant antigen appears poorly immunogenic. Studies have been conducted evaluating the effects of mycotoxin contaminated feed on growth and disease resistance of catfish. Catfish fed diets formulated with alfatoxin contaminated feed showed alfatoxin levels of up to 160 ug/kg feed had no effect on growth or disease resistance. Similar results were also observed in catfish fed diets containing elevated levels of deoxynivalenol (DON) toxin.

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). Based on experimental pond studies conducted under conditions similar to the commercial production setting, vaccination increased survival, fish size, and feed consumption while decreasing the feed conversion ratio by approximately 40.4%. Improved feed conversion ratio dramatically decreased feed costs which is the largest variable cost of production. At a given feed cost of $350/ton, the cost of feed to produce 1 lb of vaccinated fish was $0.23 compared to a feed cost of $0.36 to produce the same weight of non-vaccinated fish. Improved production efficiencies in vaccinated fish increased gross sales from $1,294 to $3,726 on a per acre basis, representing a 187.9% increase. On a commercial scale vaccination could easily increase gross sales to over $6,000/acre. Similar results were obtained in the second year of field testing showing 125% increase in total production and a 44% improvement in feed conversion ratio resulting in a 42% reduction in feed costs. Field and laboratory research is being conducted as part of the USDA licensing process for live attenuated vaccines.

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, characterized fermentation and storage protocols for the commercial production of a live attenuated vaccine. The developed protocols were shown to produce a stable product resulting in a deliverable oral immunization dose. This information is being used to develop protocols for the commercialization of a new live attenuated vaccine to protect fish against enteric septicemia of catfish (ESC).

3. The development of molecular test for the detection of significant catfish pathogens in environmental and tissue samples. Researchers from 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 (A.) hydrophila, Edwardsiella ictaluri, Bolbophorus damnificus, Bolbophorus type II sp., and Henneguya ictaluri. Similar assays for Flavobacterium columnare and Edwardsiella tarda are in the final stages of validation. The 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 A. hydrophila assay is currently in use in our laboratory, as well as the Aquatic Diagnostic Laboratory of the University of Arkansas – Pine Bluff, 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.

4. Pharmacokinetics of florfenicol in channel catfish. Researchers from Mississippi State University, College of Veterinary Medicine (MSU-CVM) at Stoneville, Mississippi, conducted pharmacokinetic studies of florfenicol in catfish. These studies along with laboratory and field efficacy studies were pivotal in obtaining Food and Drug Administration (FDA) approval for the use of florfenicol medicated feed to treat Flavobacterium (F.) columnare and Edwardsiella ictaluri infection in catfish. In addition, these studies along with one conducted by the United States Fish and Wildlife Service in Bozeman, MT, cleared the way for approval of florfenicol to treat F. columnaris infection infections in all freshwater-reared warmwater fish. The drug is prescribed under a veterinary feed directive and has been shown extremely effective in controlling bacterial infections in warmwater fish.

5. The National Warmwater Aquaculture Center’s Aquatic Research and Diagnostic Laboratory (ARDL), administered by the Mississippi State University, College of Veterinary is an American Association of Veterinary Laboratory Diagnosticians accredited laboratory that provides a comprehensive disease diagnostic service to catfish producers centered in Mississippi and surrounding states, including Alabama, Arkansas, Louisiana, and Texas. The ARDL received a total of 599 producer submitted cases diagnostic cases from 54 commercial farms and provided water quality analysis for 1,060 samples in 2011. A highly virulent strain of Aeromonas hydrophila has been identified as the cause of significant losses in Alabama and East Mississippi. There were 3 cases of the virulent strain of Aeromonas in the Delta region in MS but unlike cases in AL and east MS, the losses were limited to the affected 25 case submissions coming from 2 farms.

6. Channel catfish anemia continues to be a problem in farm-raised catfish. The disease primarily affects high value production size fish and is a primary cause of decreased production efficiencies in harvestable size fish. In controlled laboratory studies, oral supplementation with iron dextran and ferrous sulfate were shown to significantly elevate pack cell volumes of anemic fish. Similar results were observed in field trials where iron supplementation was shown to increase red blood cell production of anemic fish. As a result, ferrous sulfate concentrations have been elevated in commercial diets on a limited basis as supportive therapy for this disease with apparent success.

7. Identification of a new species of trematode affecting production efficiencies of catfish. In response to a suspected Bolbophorus damnificus infestation, rams-horn snails were collected from ponds demonstrating significantly reduced feeding activity within the resident fish population. Very few snails were actively releasing Bolbophorus-type cercaria (<1%), however a disproportionate number of snails were releasing a xiphidio-type cercariae, previously thought inconsequential to catfish health. Preliminary cohabitation challenges with actively shedding snails demonstrated this cercaria can infect channel catfish, resulting in mortality in some fish. Follow up challenges supported this initial finding, with the developing metacercaria predominately localized in the vessels of the branchial arches. Molecular analysis identified this parasite as Drepanocephalus (D.) spathans, a digenetic trematode known to parasitize the double-crested cormorant. The apparent predilection of this parasite for the branchial arches suggests the parasite could significantly impact gill function, reducing overall production during the early stages of infection. Based on the high numbers of D. spathans infected snails collected from commercial catfish ponds, this trematode species is likely a significant pathogen of channel catfish and has been included in disease monitoring efforts.

8. Development of Romet® antibiotic sensitivity discs for use in aquatic diagnostic laboratories. When Romet® discs needed for disc diffusion susceptibility testing of pathogenic bacteria from catfish were no longer commercially available, scientist at Mississippi State University aquatic diagnostic laboratory (ADL) at Stoneville, MS, worked in collaboration with the drug manufacturer to produce replacement discs. The MSU ADL is the sole source of Romet® sensitivity discs and currently provides these discs to 14 other national and international aquatic labs.

Review Publications
Khoo, L.H., Goodwin, A.E., Wise, D.J., Holmes, W.E., Hanson, L.A., Steadman, J.M., Mcintyre, L.M., Gaunt, P.S. 2011. The pathology associated with visceral toxicosis of catfish. Journal of Veterinary Diagnostic Investigation. 23(6):1217-1221.

Gaunt, P.S., Gao, D.X., Wills, R. 2011. Preparation of ormetoprim-sulfadimethoxine-medicated discs for disc diffusion assay. North American Journal of Aquaculture. 73:17-20.

Griffin, M.J., Goodwin, A.E. 2011. Theohanellus toyamai infecting the gills of Koi cyprinus carpio in the Eastern United States. Journal of Parasitology. 97(3):493-502.

Griffin, M.J., Mauel, M.J., Greenway, T.E., Khoo, L.H., Wise, D.J. 2011. A real time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi. Journal of Aquatic Animal Health. 23(4):178-188.

Gaunt, P.S., Langston, C., Wrzesinski, C.L., Gao, D., Adams, P., Couch, L.S. 2011. Single intravenous and oral dose pharmacokinetics of florfenicol in the channel catfish Ictalurus punctatus. Journal of Veterinary Pharmacology and Therapeutics. P.1365-2885.