Location: Warmwater Aquaculture Research Unit
2015 Annual Report
Objectives
1) Investigate new and emerging diseases, and develop rapid diagnostic procedures for important pathogens of channel, hybrids and blue catfish.
Subobjective 1.1 Use diagnostic case submissions to identify changes in disease prevalence and emergence of new diseases.
Subobjective 1.2 Develop molecular diagnostic tests for simultaneous detection of multiple catfish pathogens.
This objective is non-hypothesis driven and serves as an essential component of the disease surveillance programs and helps define clinical and applied research programs that address critical fish health issues facing the catfish industry.
This research will aim to determine whether molecular markers can be used to identify multiple pathogens in a single PCR reaction.
2) Study the pathology and epidemiology of significant pathogens affecting cultured catfish.
Subobjective 2.1 Evaluate the susceptibility and pathology of catfish to Edwardsiella spp.
Subobjective 2.2 Evaluate the pathology of Drepanocephalus spathans in channel catfish.
This research will determine whether blue, channel, and blue x channel catfish hybrids have varying degrees of susceptibility to Edwardsiella tarda, Edwardsiella piscicida, and Edwardsiella piscicida–like spp and determine whether the metacercariae stage of the digenetic trematode Drepanocephalus spathans is short lived (< 6 months) in channel catfish.
3) Investigate the responses of channel, hybrid and blue catfish to therapeutic agents and oral vaccines for important bacterial pathogens.
Subobjective 3.1 Evaluate susceptibility of archived and clinical bacterial isolates to antimicrobials, and correlate antimicrobial susceptibility with treatment efficacy.
Sub-objective 3.2 Determine the optimal age for vaccinating catfish against ESC using an oral attenuated E. ictaluri vaccine.
Subobjective 3.3 Determine if certain stressors will affect vaccine efficacy and safety of catfish orally vaccinated with an attenuated ESC vaccine.
Subobjective 3.4 Evaluate mortality resulting from exposure of vaccinated fish to archived and year-end E. ictaluri isolates.
Data will establish uniform standardized interpretive criteria that can be used by all aquatic laboratories that are involved with clinical disease diagnosis of catfish. The research will also examine whether antimicrobial susceptibilities determined by minimum inhibitory concentration and zone diameters of inhibition will affect the treatment efficacy of medicated feeds.
4) Develop and implement practical management strategies to improve fish health and production efficiency.
Subobjective 4.1 Evaluate effects of hybrid catfish production on incidence and prevalence of Henneguya ictaluri in catfish ponds.
Subobjective 4.2 Field evaluation of an oral ESC vaccination platform for control of E. ictaluri infections in catfish.
This research will determine if the production of hybrid catfish reduces the incidence and severity of proliferative gill disease, caused by the myxozoan parasite H. ictaluri, in catfish ponds and whether oral vaccination will improve survival and production efficiency of channel catfish raised under commercial conditions.
Approach
Diagnostic records provide critical insight to changes in disease trends and emergence of new diseases affecting animal production systems, an essential component of population health management. This information provides clinicians, caregivers and researchers a good cross-section of disease occurrence across the industry. This information can then be used to prioritize the allocation of resources in the development of rapid diagnostic procedures, disease surveillance and treatment programs and implementation of biosecurity measures to stop the spread of emerging diseases when possible.
Edwardsiella tarda has been identified as an emerging disease as indicated by diagnostic case submissions and interaction with industry advisory groups. Recently, E. tarda has undergone a reclassification, suggesting many organisms previously thought to be E. tarda are actually E. piscicida, a newly described fish pathogen closely related to E. ictaluri. Research will focus on the comparative susceptibility of channel catfish, blue catfish and hybrid catfish to E. piscicida, E. tarda and the as yet classified E. piscicida-like sp. In addition, quantitative PCR assays will be developed to detect and quantify these various pathogens in fish tissues and the pond environment.
Non-hypothesis driven clinical research will be conducted to define minimum inhibitory concentration (MIC) and cut-off values for clinical bacterial pathogens against 3 commercially available antimicrobials. This information is critical to policy review concerning antibiotic approval in the catfish industry. Separately, hypothesis-driven research will be conducted to optimize the effectiveness of oral vaccination and determine environmental factors which may limit efficacy and safety.
Field studies will be conducted to evaluate practical management strategies for controlling two diseases which account for more than half of the diagnostic case submissions to the Aquatic Research and Diagnostic Laboratory (ARDL). Research will focus on development management strategies for controlling proliferative gill disease, caused by the myxozoan parasite Henneguya ictaluri and field evaluation of an oral vaccine for control of enteric septicemia of catfish (ESC) caused by the gram-negative bacteria Edwardsiella ictaluri. These management approaches have the potential to greatly reduce the impact of these diseases, that are estimated to cost the catfish industry $60-80 million annually.
Progress Report
This report documents research under Non Assistance Cooperative Agreement 58-6402-1-632, “Catfish Health Initiative” with Mississippi State University. The Aquatic Research and Diagnostic Laboratory (ARDL) received samples from catfish producers centered in Mississippi and surrounding states, including Alabama, Arkansas, Louisiana, and Texas. Routine diagnostic services included gross necropsy, histopathology, bacteriology, virology, mycology, parasitology, and water quality evaluation. A diagnostic summary was provided to farmers through newsletters and extension workshop. Diagnostic data was used by researchers to identify disease trends and possible emerging diseases. In 2014, the Aquatic Research & Diagnostic Laboratory (ARDL) received a total of 644 producer submitted fish diagnostic cases from 36 different farms. There were an additional 57 cases submitted by researchers for a total of 701 cases. The ARDL also analyzed 634 water samples. Most significant was the increase in antibiotic resistance. A total of 89 antibiotic resistant Edwardsiella (E.) ictaluri isolates were recovered (11 resistant to both Terramycin® and Aquaflor®, 75 resistant to Terramycin® with intermediate resistance to Aquaflor®, and 3 with intermediate resistance to Aquaflor®). All the E. ictaluri isolates were sensitive to Romet®. In addition, there were 2 Aeromonas hydrophila cases that were resistant to Terramycin®, one of the less virulent and one of the more virulent (atypical) strains. There were still 33 cases (4.7% of submissions) of the Bolbophorus trematode which is a decrease from the previous year (80 cases or 9.2% of submissions) but still above the 10-year average (2.7%). Work is in progress with 2 other laboratories to establish epidemiologic cutoff values using minimal inhibitory concentration (MIC) and disc diffusion values of 10 antibiotics against E. ictaluri. Florfenicol was shown equally effective in controlling mortality associated with E. ictaluri infection in channel, hybrid and blue catfish. Information is being used as supporting data to extend the use of florfenicol in other species of catfish. To aid diagnostic evaluations and disease monitoring programs real-time polymerase chain reaction (PCR) assays for catfish pathogens have been developed. This work led to the discovery that organisms classified as Edwardsiella tarda represented two phenotypically indistinguishable yet genetically distinct species, E. tarda and E. piscicida, a newly described pathogen from fish. A third tarda-like Edwardsiella species isolated from tilapia was also genetically characterized. Pathogenicity and infectivity doses of each bacterial species was determined in channel catfish. Edwardsiella piscicida was shown to cause significant mortality in channel catfish, while at equivalent doses E. tarda and the isolate recovered from tilapia did not cause death. Similar infectivity trials are being conducted in blue and blue x channel hybrid catfish. The digenetic trematode Drepanocephalus spathans was identified as being the cause of mortality in juvenile channel catfish. Histopathology revealed developing metacercariae concentrated in the cranial region especially within the branchial chamber with several at the base of the branchial arches within or adjacent to blood vessels, possibly the proximate cause of death. Infections, however, appear transient and resolve within 70 days. Data has been used in life cycle studies and to assess the disease potential and impact of this species in commercial catfish culture.
Research conducted under the previous project "Development of approaches to prevent and ameliorate diseases of catfish" led to the development of an oral live attenuated vaccine for control of enteric septicemia of catfish and a mechanized system for oral delivery. Continuation of this work under the current project focuses on refinement and validation of the delivery system, development of protocols for the commercial scale production and processing of vaccine serials, identification of factors potentially affecting vaccine safety and efficacy, genetic characterization of the vaccine isolate and development of a molecular based diagnostic assay to differentiate between the attenuated vaccine and wild type E. ictaluri isolate. In collaboration with Mississippi State University Department of Agricultural and Biological Engineering the mechanized delivery system has been refined and validated in field use. An invention disclosure is being developed and will be submitted by year's-end. In collaboration with the United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Biological Control Laboratory (NBCL), Stoneville, MS, fermentation and processing procedures for commercial scale vaccine production have been developed. Vaccine serials produced in 50 liters (L) fermentation batches were validated and are being used to conduct field trials on commercial catfish operations. Supporting work demonstrated the vaccine is effective against different E. ictaluri isolates collected between 1999 and 2014 from different geographic regions and low oxygen stress was not shown influence vaccine safety or efficacy. In 2014, commercial field trials demonstrated dramatic increases in production efficiency resulting in a 2-3 fold increase in gross sales. Additional field trials are currently underway involving approximately 60 million catfish fingerlings. The vaccine strain genome has been sequenced and unique regions have been identified. PCR primers and probes have been developed and are being validated in tissue and environmental samples. Other field production research focused on the development of management practices to control proliferative gill disease in commercially raised catfish. Research conducted under the previous research agreement evaluated the development of proliferative gill disease (PGD) in channel and channel x blue hybrid catfish. This work demonstrated much lower parasite loads in hybrid catfish compared to channel catfish. Histologically, mature parasites were only observed in channel catfish suggesting hybrid and blue catfish may serve as a dead-end host for the parasite. As a continuation of this research, pond trials are being conducted to determine if hybrid catfish can be used to break the life cycle of this parasitic disease. Channel and hybrid catfish have been raised under commercial culture conditions for the first production cycle. Actinospore have been estimated as well as disease severity in sentinel fish. The first year of production shows similar parasite levels and disease severity in sentinel fish in both channel and hybrid catfish production systems.
Accomplishments
1. Development of an oral vaccination platform for control of Enteric septicemia of catfish (ESC). ESC is a costly bacterial disease affecting channel catfish and is estimated to reduce production 25-30% at of cost of $60 million annually. Through this project, a live attenuated live vaccine and a mechanism for oral delivery was developed and tested in commercial scale field trials. In collaboration with the United States Department of Agriculture, Agricultural Research Service, National Biological Control Laboratory, fermentation and processing procedures for commercial scale vaccine production have been developed and validated. In 2014, vaccination resulted in significant improvements in production efficiency resulting in $2,100 and $3,700 per acre increase in gross sales in hybrid and channel catfish, respectively. Modifications to the vaccine delivery system have been completed and are being validated in field vaccination trials that are on-going. Based on data from field trials, implementation of this oral vaccination platform will greatly reduce disease related costs and substantially increase the net profitability of catfish farming and help to insure the economic viability of the U.S. catfish industry. This work is being conducted in collaboration with United States Department of Agriculture (USDA), Agricultural Research Service (ARS), National Biological Control Laboratory (Stoneville MS), USDA, ARS Warmwater Aquaculture Research Unit (Stoneville, MS), USDA, ARS, Poultry Research Unit (Starkville MS) Mississippi State University (MSU) College of Veterinary Medicine (Starkville, MS), MSU Department of Agricultural and Biological Engineering (Starkville, MS). A patent has been awarded on the vaccine isolate and vaccination process and apatent disclosure on the vaccine delivery system is being prepared.
Review Publications
Miller, R., Carson, J., Dalsgaard, I., Gaunt, P.S., Gieseker, C., Hawke, J., Reimschuessel, R., Smith, P., Somsiri, T., Wu, C. 2014. CLSI performance standards for antimicrobial susceptibility testing of bacteria isoloated from aquatic animals; second information supplement. CLSI document VET03/VET04-S2. Book Chapter. P. 56.
Miller, R., Carson, J., Dalsgaard, I., Gaunt, P.S., Gieseker, C., Hawke, J., Reimschuessel, R., Smith, P., Somsiri, T., Wu, C. 2014. Methods for broth dilution susceptibility testing of bacteria isolated from aquatic animals; approved guideline-second edition. Book Chapter. P. 56.
Leech, C.A., Thomas, M.W., Cooley, A.J., Waldbieser, G.C., Bosworth, B.G., Torrans, E.L., Khoo, L.H. 2014. Histological and computed tomographic evaluation of a parasitic conjoined twin in hybrid catfish (Ictalurus punctatus [rafinesque] X Ictalurus furcatus [lesueur]). Journal of Fish Diseases. 37:521-526.
Beecham, R., Thomas, T., Goa, D.X., Gaunt, P.S. 2014. The effects of a sublethal dose of botulinum serotype E on the swimming performance of channel catfish fingerlings. Journal of Aquatic Animal Health. 26:149-153.
Camus, A.C., Wise, D.J., Khoo, L.H., Jishu, S., Berghaus, R.D. 2014. Iron status of channel catfish Ictalurus punctatus affected by channel catfish anemia and response to parenteral iron. Diseases of Aquatic Organisms. 107:241-248.
Griffin, M.J., Reichley, S.R., Khoo, L.H., Ware, C., Greenway, T.E., Mischke, C.C., Wise, D.J. 2014. Comparative susceptibility of channel catfish, blue catfish, and their hybrid cross to experimental challenge with Bolbophorus damnificus (Digenea: Bolbophoridae) cercariae. Journal of Aquatic Animal Health. 26:149-153.
Keirstead, N.D., Brake, J.W., Griffin, M.J., Halliday-Simmonds, I., Soto, E. 2014. Fatal septicemia caused by the zoonotic bacterium Streptococcus iniae during an outbreak in Caribbean reef fish. Veterinary Pathology. 51:1035-1041.
Chou, L., Griffin, M.J., Fraites, T., Ware, C., Ferguson, H., Keristead, N., Brake, J., Wiles, J., Hawke, J.P., Kearney, M.T., Getchell, R., Gaunt, P., Soto, E. 2014. Phenotypic and genotypic heterogeneity among Streptococcus iniae isolates recovered from cultured and wild fish in North America, Central America and the Caribbean Islands. Journal of Aquatic Animal Health. 26:263-271.
Chatla, K., Gaunt, P., Petrie-Hanson, L., Hohn, C., Ford, L., Hanson, L. 2014. Zebrafish (Danio rerio) bioassay for visceral toxicosis of catfish and botulinum neurotoxin serotype E. Journal of Veterinary Diagnostic Investigation. 26:240-245.
Mischke, C.C., Tucker, C.S., Wise, D.J., Brown, T.W. 2015. DEET (N,N-diethyl-m-toluamide) toxicity to channel catfish Ictalurus punctatus sac fry. Journal of the World Aquaculture Society. 46:(3)344-347.
Griffin, M.J., Khoo, L.H., Steadman, J.M., Ware, C., Quiniou, S., Mischke, C.C., Terrence, G.E., Wise, D.J. 2014. Chronic pathology and longevity of Drepanocephalus spathans infections in juvenile channel catfish. Journal of Aquatic Animal Health. 26(4):210-218.
Rosser, T.G., Griffin, M., Quiniou, S., Khoo, L., Pote, L. 2014. 18S rRNA gene sequencing identifies a novel species of Henneguya parasitizing the gills of the channel catfish (Ictaluridae). Parasitology Research. 113(12):4651-4658.
