Location: Warmwater Aquaculture Research Unit
2019 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
The Aquatic Research and Diagnostic Laboratory (ARDL) in Stoneville, Mississippi, which provides services to producers as well as fish health and production researchers in southeastern United States received on average over 700 case submissions case submissions since 2015. Research funds were used to investigate disease potential of emergent pathogens, trends in antibiotic susceptibility patterns and develop molecular based rapid diagnostic assays for the detection of primary catfish pathogens and potential emergent pathogens from environmental and tissue samples.
Annual reports generated from diagnostic case submissions documented changes in the prevalence of diseases afflicting cultured catfish. The predominant diseases were associated with Flavobacterium (F.) columnare and Edwardsiella (E.) ictaluri, but several emerging diseases have been identified. Most notably is E. piscicida which has increased in prevalence concomitant with increased use of hybrid catfish as a culture species. Comparative susceptibility studies involving channel, blue and hybrid catfish demonstrated hybrid catfish are more susceptible to E. piscicida than channel catfish and supports anecdotal reports from industry that E. piscicida is more burdensome in hybrid catfish. More recently, Yersinia (Y.) ruckeri, the causative agent of enteric redmouth disease in salmonids, was isolated from diseased hybrid catfish and a putative, non-toxigenic Vibrio (V.) cholera strain has been associated with losses in hatchery fry. Research is being conducted to generically characterize archived isolates and determine the pathobiology of these potential emergent disease agents.
Overall antibiotic resistance across the industry has remained relatively constant although there has been a distinct increase in antibiotic resistance on a few isolated farms. In efforts to improve antibiotic therapy, scientists at the Thad Cochran National Warmwater Aquaculture Center, Stoneville, Mississippi, 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 continues on the establishment of clinical breakpoints for E. ictaluri. In addition, all three laboratories have research to establish epidemiological cutoff values (ECOVs) for Aeromonas (A.) hydrophila and F. columnare. This information will provide uniform measurements to determine susceptibility of bacteria to antimicrobials and improve diagnosticians' abilities throughout the world to choose appropriate antimicrobial agents to control mortality associated with bacterial agents. The results of the A. hydrophila and F. columnare work will be published in 2020 in the newly revised Clinical and Laboratory Standard Institute VET04-53.
Malacological surveys have identified an emergent second snail host (Biomphalaria havanensis) that transmits Bolbophorus (B.) damnificus and a newly recognized trematode pathogen of catfish, Drepanocephalus (D.) auritus, with larval stages released by these hosts showing infectivity to catfish. Experimental life cycle studies have shown the double crested cormorants (Phalocrocorax auritus) 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. This work established developmental timelines associated with ontogenetic stages of the parasite life cycle, providing critical baseline information for management strategies to reduce digenetic trematodes in catfish aquaculture. Survey data has also linked cercariae released by Biomphalaria (Bi) 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 coincides with mortality in experimentally infected fish. While infected fish that survived parasite encapsulation 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.
In addition, four previously unknown trematode life cycles associated with piscivorous birds endemic to catfish aquaculture in Mississippi have been identified. The significance to catfish aquaculture is being investigated. Treatment recommendations are the same for controlling B. damnificus trematode infections which has decreased over the past several years. A decrease in diagnostic cases was likely related to increased disease recognition by producers, prompt treatment to reduce the intermediate host presence in catfish ponds and possibly increased use of hybrid catfish. Laboratory trials show hybrid catfish appear refractive to the deleterious effects of B. damnificus infection under equivalent infestation levels.
Research into the effects of Henneguya (H.) ictaluri, the causative agent of proliferative gill disease (PGD) in channel and hybrid catfish has demonstrated the parasite undergoes arrested or stunted development in hybrid catfish. This evidence indicates hybrid catfish are 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 significantly reduced compared to channel catfish ponds managed under similar conditions after only two years of production.
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 developed for A. hydrophila, B. damnificus, H. ictaluri, and F. columnare for use in field surveillance studies and to aide in diagnostic evaluations. The genomes of multiple Y. ruckeri isolates recovered from diseased hybrid catfish, as well as a panel of V. cholerae from spontaneous mortality events in catfish hatcheries and an archived Streptococcus ictaluri isolate have been sequenced to identify molecular markers that can be exploited by qPCR.
An experimental live attenuated oral E. ictaluri vaccine and vaccine delivery system has been developed under this project and validated in large scale commercial field trials. Research involved validation of a mechanized delivery system development of protocols for 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 isolate. Genomic sequencing demonstrated the live attenuated E. ictaluri vaccine and the parent strain do not carry multi-drug resistant plasmids that have been reported from other field strains of E. ictaluri. Research supporting vaccine development demonstrated vaccination protected catfish against 22 archived field isolates regardless of host species, geographic region or isolation year. The homogenous nature of E. ictaluri isolates revealed by repetitive sequence medicated PCR fingerprinting and virulence gene amplification negates the need for multivalent vaccine development. Plasmid profiling of archived isolates heterogeneity of at least 4 bacterial isolates but were not shown to affect vaccine efficacy. In addition other supporting research demonstrated acute oxygen deprivation and confinement stress did not affect vaccine safety. Commercial field trials involving over 350 million vaccinated fish demonstrated vaccination greatly improved survival, growth, feed conversion and yield in hybrid and channel catfish. Economic analysis of field data showed a net economic benefit of $3,868 and $7,063/hectare (ha) in channel and hybrid catfish, respectively. Collaborators on the project included Mississippi State University (MSU), Department of Agricultural and Biological Engineering (Starkville, Mississippi), United States Department of Agriculture (USDA), Agriculture Research Service (ARS), National Biological Control Laboratory (Stoneville, Mississippi), USDA, ARS, Warmwater Aquaculture Research Unit (Stoneville, Mississippi) and MSU college of Veterinary Medicine (Starkville, Mississippi). Vaccination technologies have been licensed to a locally owned fish health company for commercialization.
Accomplishments
1. Clinical research in fish health management. Catfish aquaculture represents the largest aquaculture industry in the United States. In 2018, U.S. catfish aquaculture produced approximately 340 million pounds from over 60,000 acres, valued at an estimated $350 million (USD). However as catfish production has intensified infectious disease threatens the economic viability of the catfish industry. The development of cost effective disease management strategies are dependent on accurate disease diagnoses and clinical research to understand the pathobiology of specific diseases, identify disease vectors and develop biosecurity measures. The College of Veterinary Medicine Aquatic Research and Diagnostic Laboratory through a cooperative agreement with ARS scientists in Stoneville, Mississippi, offers a comprehensive disease diagnostic service to catfish producers centered in Mississippi and surrounding states, including Alabama, Arkansas, Louisiana, and Texas. These services allow for the construction of an accurate database allowing for the surveillance and mapping of disease and disease trends which is available to other research institutions conducting fish health research. Through this project Vibrio cholerae, Yersinia ruckeri, Edwardsiella piscicida and a host of digenetic parasites have been identified as emergent pathogens of catfish. To aide in field monitoring studies quantitative polymerase chain reaction assays have been developed for all important catfish pathogens in tissue and environmental samples. Assays are critical to disease surveillance programs are being used to determine disease vectors, evaluate treatment efficacies and monitor pathogen loading rates in catfish production systems.
2. Myxozoan parasites associated with catfish aquaculture. Henneguya (H.) ictaluri is the etiologic agent of proliferative gill disease (PGD) in farm raised channel and hybrid catfish and is attributed to significant annual losses. The previous project investigated the development of H. ictaluri in channel catfish, blue catfish, and their hybrid cross. H. ictaluri DNA was found in greater abundance throughout multiple organ systems in channel catfish compared to hybrid and blue catfish. Despite copious myxospores present in channel catfish throughout the study, no myxospores were observed grossly in blue or hybrid catfish. Myxospores in hybrids were only observed histologically and were sparse (<1% of examined fish), suggesting significant arrested development in the hybrid host. This data generated through a cooperative agreement with ARS scientists in Stoneville, Mississippi, indicates rotating culture fish between channel and hybrid catfish could be an effective management strategy to reduce parasite loading rates in catfish production systems by breaking the parasites life cycle. This work also lead to the characterization of 9 previously undocumented myxozoan parasites from catfish and other temperate freshwater fish (H. mississippiensis; H. sutherlandi; H. laseeae; H. bulbosus; Myxobolus (M.) ictiobus; M. minutus; M. lepomis; M. axelrodi; Unicauda fimbrithelae). The pathobiology and impact on catfish production of these previously uncharacterized parasites is being investigated.
3. Development of management strategies to control digenetic trematode infestations. The digenetic trematode, Bolbophorus damnificus, has become a significant threat to the viability of the commercial catfish industry. The parasite life cycle sequentially involves the Ram’s horn snail, Planorbella trivolvis, which shed cercarial life stages infectious to fish. The life cycle is complete when the American White Pelican consumes infected fish harboring the adult trematode. Field research and disease monitoring programs through a cooperative agreement with ARS scientists in Stoneville, Mississippi, demonstrated approximately 30% of the acreage in catfish production contain trematode infected fish. Economic analysis of this data showed even light infestation decreased incomes with severe infestations resulting in a complete loss of production. This analysis showed even mild infestations can reduce net return land by 61%. Sales receipts from pond populations with moderate to severe infestations did not cover the costs of production, resulting in a negative net return to land of $1,929/acre and $2,774/acre respectively. This work led to the development of best management practices for the control of trematode infestations in commercially raised catfish and has been disseminated to catfish producers in Mississippi through extension workshops and direct farmer contacts. To date, all catfish operations in Mississippi have adopted some form of a disease surveillance and treatment program for the control of trematode infestations. Implementation of treatment strategies derived from this work has prevented production losses costing the industry approximately $30.6 million and potential farm closures resulting from lost production. These management practices are also effective for other trematode species that have been recently identified.
4. Identification Drapanocephalus spathans as a catfish pathogen. Digenetic trematodes have been recognized as a significant threat to catfish production since the discovery of Bolbophorus (B.) damnificus in the late 1990s. Economic analysis has demonstrated even low infection prevalence of B. damnificus can have deleterious effects on the profitability of commercial operations. Through a cooperative agreement with ARS scientists in Stoneville, Mississippi, this work identified another digentic trematode, Drapanocephalus (D.) spathans, as the cause of mortality in catfish. Developing metacerariae were shown to concentrate in the cranial regions, often occluding blood vessels at the base of the branchial arch is postulated as being the resultant cause of death. While the severe infestations can be the cause of death, the infections appear short lived and resolve within 2 months. Management practices effective for B. damnificus infestations are also effective in control D. spathans infections since both trematode species utilize planorbid snails as the first intermediate host. Recognition of D. spathans as a cause of catfish mortality is critical to diagnostic evaluations and effective treatment recommendations.
5. Identification of definitive and intermediate hosts involved in trematode infestations in catfish aquaculture. The Ram’s horn snail, Planorbella trivolvis, was initially recognized as the only snail species transmitting the deleterious trematode, Bolbophorus (B.) damnificus. Research involved with trematode life cycle development through a cooperative agreement with ARS scientists in Stoneville, Mississippi, identified a second planorbid snail species, Biomphalaria (Bi.) havanensis, capable of shedding two trematode species, Bolbophorus (B.) damnificus and Drepanocephalus (D.) spathans. Planrobid species are involved in the development of schistosomiasis in humans but shown refractory to Schistomsoma mansoni infection, suggesting they do not pose a risk to human health. These snails were found shedding D. spathans and B. damnificus, respectively. Infectivity trials of channel catfish exposed to D. spathans and B. damnificus released from Bi. havanensis were successful, providing evidence that naturally occurring B. damnificus and D. spathans from Bi. havanensis are infective to catfish. Furthermore, Bi. havanensis snails were also found shedding cercariae of an Austrodiplostomum sp. reported from inland silversides inhabiting catfish ponds. Channel catfish fingerlings exposed to these cercariae developed metacercariae in both the eyes and brain, the long-term impacts of which are unknown. In addition, the great egret Ardea (A.) alba is often found foraging on commercial catfish operations. During a recent survey, two new species of Clinostomum (C.) were described from A. alba, known hereafter as C. album n. sp. and C. poteae, although their effects on catfish production are unknown. Recognition of host species is critical to surveillance and treatment programs effective in controlling digenetic trematode infestations in catfish production system.
6. Use of iron fortified diets to control catfish anemia. Catfish anemia (CCA) is a disease of unknown etiology that has plagued the catfish industry since its inception. While normal packed cell volumes (PCV) of catfish range from approximately 20-40%, anemic catfish often have extremely low PCV of 5% or less. Clinically, these fish are often lethargic and show signs of respiratory distress in the face of adequate dissolved oxygen concentrations. Clinical and applied research has led to a cost effective treatment that is being employed throughout the catfish industry. Initial clinical trials demonstrated diets fortified with iron increase red blood cell production in anemic fish. The use of iron supplements were validated in field trials and were shown effective in stopping and preventing the development of catfish anemia. As a result of this work through a cooperative agreement with ARS scientists in Stoneville, Mississippi, catfish operations with recurrent anemia have begun using diets fortified with modest ferrous sulfate levels to promote RBC production. To date this practice has resulted in a dramatic decrease in the incidence of CCA which has traditionally cost the industry between $5-10 million annually.
7. Development of a live attenuated oral vaccine to control Enteric septicemia of catfish. Enteric septicemia of catfish (ESC) is a bacterial disease caused by a gram negative enteric identified as Edwardsiella ictaluri and is the most prevalent bacterial disease affecting channel catfish fingerling production, resulting in significant economic losses. In efforts to develop more effective disease management programs through a cooperative agreement with ARS scientists in Stoneville, Mississippi, a live-attenuated ESC vaccine and delivery method facilitating in-pond oral vaccine delivery was developed. The oral vaccination platform has been validated in experimental and commercial field trials demonstrating significant increases in survival, growth, feed efficiency and yield. Economic analysis of field data showed a net economic benefit of $3,868 and $7,063/hectare (ha) in channel and hybrid catfish, respectively. Whole-farm models showed additional profit ranging from $71,758 to $133,887/400-ha on farms that integrate fingerling production to their production strategies due to the appropriation of more of the otherwise incumbent fingerling production acreage into food fish production. The patented vaccine and patent-pending oral delivery system has been licensed to a locally owned private company for commercialization.
Review Publications
Griffin, M., Khoo, L., Reichley, S., Ware, C., Rosser, T., Alberson, N., Woodyard, E., Pote, L., Greenway, T., Tiwari, A., Mischke, C., Wise, D. 2018. Encapsulation of Bolbophorus damnificus (Digenea: Bolbophoridae) metacercariae in juvenile channel catfish Ictalurus punctatus is linked to delayed onset mortality. Journal of the World Aquaculture Society. 49(3):601-611.
Li, M., Wise, D., Mischke, C., Kumar, G., Lucas, P. 2018. Response of pond-raised fingerling hybrid catfish, Ictalurus punctatus x Ictalurus furcatus, to dietary protein concentrations and sources. Journal of the World Aquaculture Society. 49(5):912-918.
Stilwell, J., Griffin, M., Rosser, T., Leary, J., Hagen-Frei, K., Mischke, C., Pomaranski, E., Soto, E., Camus, A. 2019. First detection of Erysipelothrix sp. infection in western mosquitofish, Gambusia affinis, inhabiting channel catfish, Ictalurus punctatus, production ponds in Mississippi. Diseases of Aquatic Organisms. 133:39-46.
Griffin, M., Reichley, S., Baumgartner, W., Aarattuthodiyil, S., Ware, C., Steadman, J., Lewis, M., Gaunt, P., Khoo, L., Wise, D. 2018. Emergence of Edwardsiella piscicida in farmed channel female, Ictalurus punctatus × Blue male, Ictalurus furcatus, hybrid catfish cultured in Mississippi. Journal of the World Aquaculture Society. 50(2):420-432.
Griffin, M., Khoo, L., Reichley, S., Ware, C., Rosser, G., Alberson, N., Woodyard, E., Pote, L., Greenway, T., Tiwari, A., Mischke, C., Wise, D. 2018. Encapsulation of Bolbophorus damnificus (Digenea: Bolbophoridae) Metacercariae in juvenile channel catfish, Ictalurus punctatus, is linked to delayed-onset mortality. Journal of the World Aquaculture Society. 49(3):601-611.
Stilwell, J., Camus, A., Leary, J., Mohammed, H., Griffin, M. 2019. Molecular confirmation of Henneguya adiposa (Cnidaria: Myxozoa) and associated histologic changes in adipose fins of channel catfish, Ictalurus punctatus (Teleost). Parasitology Research. 118:1639.
Subramaniam, K., Venugopalan, A., Davison, A., Griffin, M., Ford, L., Waltzek, T., Hanson, L. 2019. Complete genome sequence of an ictalurid herpesvirus 1 strain isolated from blue catfish (Ictalurus furcatus). Microbiology Resource Announcements. 8(15):e00082-19. https://doi.org/10.1128/MRA.00082-19.
Rosser, G., Khoo, L., Wise, D., Mischke, C., Griffin, M., Alberson, N., Reichley, S., Woodyard, E., Steadman, J., Ware, C., Pote, L., Greenway, T. 2019. Arrested development of Henneguya ictaluri (Cnidaria: Myxobolidae) in female channel catfish X male blue catfish hybrids. Journal of Aquatic Animal Health. 31:201-213.
