2012 Annual Report
1a.Objectives (from AD-416):
The objective of this cooperative research project is to conduct aquaculture research addressing fish health problems limiting the production of channel catfish, Ictalurus punctatus.
1b.Approach (from AD-416):
All research conducted under this agreement will address practical, production oriented fish health or disease problems facing the farm-raised catfish industry in the southeastern United States and will address components of the USDA-ARS action plan for National Program 106-Aquaculture. Research projects will be conducted using pond and laboratory facilities at the Delta Research and Extension Center (National Warmwater Aquaculture Center, Stoneville, MS). These facilities are under the direction of the Mississippi Agricultural and Forestry Experiment Station (MAFES) and Mississippi State University College of Veterinary Medicine (CVM). Research will address problems in the following broad subject areas: fish health management, fish diagnostics, epidemiology, and risk assessment modeling. On-farm demonstration projects, scientific articles, technical bulletins and educational workshops will be used to disseminate information to stakeholders.
Develop methods to protect channel catfish and its hybrids with vaccines, antibiotics and other therapeutants against enteric septicemia of catfish, columnaris, proliferative gill disease, botulism, Bolbophorus damnificus and anemia. Bacterial challenges have been conducted comparing mortality of fish fed bactericidal and bacteriostatic antibiotics before and after exposure to Edwardsiella (E.) ictaluri. The bactericidal drug was shown more effective in treating infection but could interfere with the development of acquired immunity if used prior to infection. 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. Experimental trials were conducted by administering the anti-parasitic drugs, amprolium, salinomycin, and albendazole with the feed during and after exposure to Henneguya (H.) ictaluri actinospores. Salinomycin was not palatable to channel catfish and albendazole was shown to be highly toxic. As a result, these drugs will no longer be evaluated for use in catfish. Amprolium was not shown effective in reducing the acute stages of disease determined by gross microscopic examination, however, it is anticipated that the drug will interfere with the developing life stages associated with chronic H. ictaluri infection. Trials have been conducted to evaluate development stages of the parasite in fish tissues by quantitative polymerase chain reaction (qPCR) analysis and histopathology to be used in evaluation of drug efficacy. Initial studies indicated smallmouth buffalo may be an effective agent for biological control of benthic oligochaetes, which transmit myxozoan parasites, including the causative agent of proliferative gill disease. A three year study is being conducted to evaluate the use of small mouth bass (SMB) to control benthic macro invertebrates and oligochaetes in 1 acre experimental ponds. Ponds stocked with juvenile SMB (2-3”) had significantly lower oligochaete populations compared to ponds without SMB. No difference, however, were observed after 9 months. This observation may have related to changes in prey preference as the SMB matures. Ponds have been restocked with juvenile SMB to validate this observation. Primer and probe combinations have been developed for specific amplification for Bolbophorus damnificus. The assay has been validated for field use and will be integrated in 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. Challenge protocols to evaluate susceptibility of blue, channel and hybrid catfish to E. ictaluri, E. tarda and H. ictaluri have been developed and are being used to support the USDA/ARS/CGRU genetic selection program.
Monitoring and surveillance of biotoxin, pathogen, pathogen/host interactions in aquatic environments. Primer and probe combinations have been developed for specific amplification for Edwardsiella ictaluri, Flavobacterium 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. In 2011, the Aquatic Research & Diagnostic Laboratory at Stoneville, MS, 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. Most of these were the typical cases seen in the laboratory except that we continued to see a few cases (3) of steatitis (inflammation of the fat). The causes of steatites in these cases are unknown but Vitamin E deficiency has been implicated in other fish species. 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 ictaluri and 2 out of 19 isolates of Edwardsiella tarda had intermediate sensitivities. Mississippi State University College of Veterinary Medicine (MSU-CVM) was one of nine laboratories that participated in a multi-laboratory broth microdilution method trial to standardize the specialized test conditions required for the fish pathogens Flavobacterium (F.) columnare and F. psychrophilum. These data and their associated quality control (QC) ranges have been approved by the Clinical and Laboratory Standards Institute (CLSI), and will be included in the next edition of the CLSI’s M49-A Guideline. To aide in disease monitoring studies, a toxin-free enzyme-linked immunosorbent assay (ELISA) procedure has been developed to measure the level of anti-BoNT/E antibody in catfish sera. A peptide library has been constructed based on the known primary structure of the heavy chain subunit of BoNT/E. Archived channel catfish sera collected from fish exposed to BoNT/E has been screened for antibody binding with a panel of 69 synthetic peptides composed of 18 amino acid residues. Archived sera were highly reactive to cryptic and non-cryptic amino acid residues suggesting a high degree of non-specific binding. This indicates the primitive nature of catfish antibody production, exhibiting a high degree of natural non-antigen driven production of poly-reactive antibodies. This may limit the use the peptide library in discriminating specific antibody responses. Immunization (BoNT/E recombinant heavy chain) trails have been conducted to hyper-immunize channel catfish to create a reference serum used to determine optimal antigen coating concentrations for ELISA. Based on endpoint titers, generated from hyper-immunized fish (IP injection), the recombinant antigen appears poorly immunogenic. The LD50 for BoNT/E was determined to establish vaccination/challenge protocols for catfish. The 96 hour LD50 for 5.3 g channel catfish was determined to be 13.7pg. In support of this research, zebrafish are being evaluated as a model for bioassays. The adult zebrafish is a small biomedical research animal that is available year round and will help standardized challenge protocols. The utility of the adult zebrafish and channel catfish fingerlings as BoNT/E and VTC bioassay organisms were compared. The zebrafish was found to be more sensitive than channel catfish for detection of BoNT/E.
Chemical and mycotoxin feed contaminants affect growth and disease resistance of catfish. Studies have been conducted evaluating the effects of mycotoxin contaminated feed on growth and disease resistance of catfish. Catfish fed diet formulated with alfatoxin contaminated feed showed alfatoxin levels of up to 160 ug/kg feed had no effect on growth or disease resistance. These results are in agreement with other studies conducted in our laboratory showing catfish are resistant to relatively high levels of alfatoxin. Similar results were also observed in catfish fed diets containing elevated levels of deoxynivalenol (DON) toxin. No differences in growth were observed among any treatments, however, in one trial fish fed DON had higher survival resulting from E. ictaluri infection compared to fish fed DON free diets. These results are being investigated to determine the effects of DON on non-specific immunity. An unknown cause of feed related anemia was identified in limited quantities of bagged catfish feed. The condition appears to mimic the characteristics of channel catfish anemia observed in pond raised channel catfish. A feed contaminant is thought to be the cause of decreased hematocrit. Analysis indicates the feed in question was nutritionally complete and was negative for mycotoxins and other potential contaminants (CHOP analysis). Research is continuing to investigate the link between channel catfish anemia (CCA) and feed induced anemia.