Location: Warmwater Aquaculture Research Unit
2016 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
Annual reports generated from diagnostic case submissions documented changes in the prevalence of diseases afflicting cultured catfish. The predominate diseases were associated with infection caused by Flavobacterium (F.) columnare and Edwardsiella (E.) ictaluri but several emerging diseases have been identified. Most notably is E. piscicida which has increased in prevalence due to an increase in the use of hybrid catfish as a culture species. Atypical Aeromonas (A.) hydrophila, associated with high mortality in west Alabama and east Mississippi has been sporadically recovered from disease case submissions in the Delta region of west Mississippi. Molecular methods have been developed to differentiate E. piscicida from E. ictaluri as well as the atypical A. hydrophila from the typical A. hydrophila that is predominantly an opportunistic pathogen secondary to other disease agents or poor water quality. More recently, Yersinia ruckeri the causative agent of enteric redmouth disease in salmonids was isolated from diagnostic submissions and preliminary studies are underway to determine if this is an emerging pathogen of concern. There has been some antibiotic resistance observed in E. ictaluri case isolates, although this resistance is mostly to Terramycin with some intermediate resistance to Aquaflor. With regards to parasites, Proliferative Gill Disease caused by the myxozoan Henneguya (H.) ictaluri continues to be the most prevalent parasitic disease based on case submissions, although there was a drop in 2015 which could be related to an increased acreage used in the production of hybrid catfish. Comparatively, although the number of digenetic trematode Bolbophorus (B.) damnificus cases are relatively low (10 year average of less than 3% of total submissions) it appears to be a significant problem in 2016, as case submissions currently exceed the 10 year average. Drepanocephalus (D.) auritis is another digenetic trematode infective to catfish showing significant pathology in catfish fingerlings. At present a multiplex real-time polymerase chain reaction (PCR) assay has been developed and validated for the four Edwardsiella spp. considered to be fish pathogens (E. ictaluri, E. piscicida, E. tarda, E. anguillarum), allowing for the simultaneous detection and quantification of all four pathogens in a single reaction. Similar assays have been evaluated for detecting multiple catfish pathogens for use in field surveillance, coupled with an internal reference standard to identify the presence/absence of inhibitors in field samples and are in the final stages of development.
Scientists at the Thad Cochran National Warmwater Aquaculture Center, Stoneville, MS, are cooperating with researchers at the U.S. Food and Drug Administration (FDA) and Louisiana State University in establishing uniform criteria to determine whether bacteria isolated from catfish are susceptible or resistant to antibiotics in medicated feed. Research is continuing on establishing the breakpoints for E. ictaluri. The Clinical and Laboratory Standards Institute (CLSI) states the minimal inhibitory concentration (MIC) are determined at 24 hours (h). Work is being conducted to modify CLSI requirements to extend test limits to 48 h to account for slow growing bacterial species such as E. ictaluri. In addition, all three laboratories are currently researching the breakpoints for A. hydrophila and F. columnare. This information will provide uniform measurements to determine the susceptibility of bacteria to antimicrobials and improve diagnosticians' abilities throughout the world to choose appropriate antimicrobial agents to control mortality associated with bacterial agents.
Malacological surveys have identified a second snail host (Biomphalaria sp.) that transmits B. damnificus and D. auritus, with parasites released by this host showing infectivity to catfish. Experimental life cycle studies are ongoing, but preliminary data has shown double crested cormorants fed catfish experimentally infected with D. auritus develop patent trematode infections, suggesting catfish can serve as an intermediate host in the D. auritus life cycle. Similarly, malacological surveys have linked cercariae released by Biomphalaria sp. to several trematode species in the family Diplostomidae, which cause ocular diplostomiasis in fish worldwide. Experimental challenges have demonstrated at least one of these species (Austrodiplostomum ostrowkiae) causes ocular diplostomiasis in channel catfish. Moreover, research has identified a parasite induced anemia associated with the encapsulation process of B. damnificus in channel catfish, which coincided with mortality in experimentally challenged fish. While infected fish that survived the encapsulation process eventually recovered, packed cell volumes of parasitized fish were significantly lower than their non-parasitized cohorts, which offers some explanation to the reduced production seen in trematode infected fish. Studies on the acute stages of D. auritus development in channel catfish are ongoing.
Research into the effects of H. ictaluri, the causative agent of proliferative gill disease (PGD) in channel and hybrid catfish continues. Studies investigating the in vivo development of H. ictaluri have shown the parasite undergoes arrested or stunted development in hybrid catfish, as mature H. ictaluri spores are typically observed 6-12 weeks post infection in channel catfish, but have not been observed, or observed only as a rare event, in hybrid catfish as much as 14 weeks post challenge. It is thought the hybrid catfish is a dead end host in the H. ictaluri life cycle. This supports field observations that suggest incidence and prevalence of PGD in hybrid production ponds is less than observed in channel production systems. Comparative pond studies have demonstrated H. ictaluri densities in hybrid catfish ponds are less than channel catfish ponds managed under similar conditions after only two years of production.
Similar work is ongoing comparing relative susceptibility of channel, blue and hybrid catfish to several Edwarsdiella spp. formerly classified as E. tarda. Our work has demonstrated E. piscicida is more pathogenic to catfish than E. tarda or E. anguillarum, while hybrid and blue catfish are more susceptible to E. piscicida than channels. This is in line with anecdotal reports from industry that suggest E. piscicida is of greater concern in hybrids than channels.
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 has been filed and a third prototype has been developed and currently being validated for field use. 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 100 liter (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 2016 from different geographic regions and low oxygen stress was not shown influence vaccine safety or efficacy. In 2015, over 90 million fingerlings were vaccinated on 6 commercial operations. Data is being used to conduct time-in-motion studies to determine resources needed for commercial application.
Accomplishments
1. Development and validation of protocols for oral vaccination of catfish against enteric septicemia of catfish. Under this agreement a patented oral vaccination platform and mechanism for delivering vaccine to pond fish was developed and are being used to conduct commercial field trials. Vaccination was shown to dramatically improve production efficiency increasing the value of fish production by approximately $3,000 per acre for channel catfish and $2,000 for hybrid catfish. In 2015, over 90 million fingerlings were vaccinated on 6 commercial operations. Data is being used to conduct time-in-motion studies to determine resources needed for commercial application. A patent was awarded for the vaccination process and an invention disclosure has been filed on the delivery system developed in collaboration with Mississippi State University, Department of Agricultural and Biological Engineering.
None.
Review Publications
Allen, P.J., Wise, D., Greenway, T., Khoo, L., Griffin, M.J., Jablonsky, M. 2015. Using 1-D 1H and 2-D 1H J-resolved NMR metabolomics to understand the effects of anemia in channel catfish (Ictalurus punctatus). Metabolomics. 11(5):1131-1143.
Reichley, S.R., Ware, C., Greenway, T.E., Wise, D.J., Griffin, M.J. 2015. Real-time PCR assays for detection and quactification of Edwardsiella tarda, Edwardsiella piscicida, Edwardsiella piscicida-like sp. in catfish tissues and pond water. Journal of Veterinary Diagnostic Investigation. 27(2):130-139.
Wise, D.J., Greenway, T.E., Byars, T.S., Griffin, M.J., Khoo, L.H. 2015. Oral vaccination of channel catfish against enteric septicemia of catfish (ESC) using a live attenuated Edwardsiella ictaluri isolate. Journal of Aquatic Animal Health. 27(2):135-143.
Wolf, J.C., Baumgartner, W.A., Blazer, V.S., Camus, A.C., Englhardt, J.A., Fournie, J.W., Frasca, S., Groman, D.B., Kent, M.K., Khoo, L.H. 2015. Nonlesions, misdiagnoses, missed diagnoses, and other interpretive challenges in fish histopathology studies-A guide for investigators, authors, reviewers, and readers. Toxicologic Pathology. 43(3):297-325.
Wise, D.J., Greenway, T.E., Byars, T.S., Griffin, M.J., Khoo, L.H. 2015. Oral vaccination of channel catfish against enteric septicemia of catfish using a live attenuated Edwardsiella ictaluri isolate. Journal of Aquatic Animal Health. 27(2):135-143.
