Location: Warmwater Aquaculture Research Unit
2023 Annual Report
Objectives
1. Identify emergent pathogens in catfish aquaculture and develop disease diagnostic methodologies for use in field surveillance studies.
1.1. Identify emergent pathogens in catfish aquaculture and develop disease diagnostic methodologies for field surveillance studies.
1.2. Characterize and evaluate pathogenesis of emergent diseases and fulfillment of Koch’s postulates or River’s postulate for newly recognized or emergent pathogens.
1.3. Develop rapid diagnostic tests and ELISA procedures to determine total and antigen specific antibody for epidemiological studies.
1.4. Develop primary catfish cell lines for identification and confirmation of fish viruses.
2. Optimize treatments and management strategies to minimize infectious diseases in catfish aquaculture.
2.1. Optimize Edwardsiella (E.) ictaluri vaccine delivery, evaluate cross protective potential of E. ictaluri vaccine against E. piscicida.
2.2. Role of other myxozoans (non-H. ictaluri) and intraspecific variability of oligochaete hosts in occurrence of proliferative gill disease (PGD) in channel and hybrid catfish.
2.3. Role of iron fortified diets and occurrence of bacterial infections in channel and hybrid catfish.
2.4. Evaluation of the pathophysiological effects of Bolbophorus damnificus (trematode) in hybrid and channel catfish.
3. Determine the epidemiology of infectious diseases in catfish aquaculture and conduct economic evaluations of disease management strategies.
3.1. Significance of genetic E. piscicida variants recovered from commercially cultured hybrid and channel catfish.
3.2. Epidemiology of A. hydrophila infections in catfish aquaculture; predictive modeling to determine risk factors.
3.3. Spatio-temporal survey of channel catfish virus (CCV) isolates and evaluation of trends in the occurrence and virulence of different genetic strains of CCV in channel and hybrid catfish.
3.4. Evaluate disease transmission of emergent Vibrio spp. infections in hatchery fry.
3.5. Economic evaluation of a live, attenuated E. ictaluri vaccine in commercial fingerling and foodfish production.
Approach
In the United States, pond production of catfish ranks as the leading aquaculture species in terms of farm gate value. Health management strategies, technologies, and bio-security plans that are environmentally safe are necessary to help mitigate disease-related losses. There is presently a lack of validated technologies for early and rapid detection of pathogens, disease prevention, and treatment of diseases in catfish aquaculture, which has hindered the growth and profitability of the industry. Validated diagnostic tools for use in production systems to detect the disease agents in a rapid fashion are needed. In addition to the need for diagnostics, developing effective control strategies to manage disease is a priority, given only a few drugs are available for the treatment of sick fish. Further research will develop molecular based diagnostic tools used in to monitor potential emergent pathogens, optimize vaccination strategies for control of bacterial infections, determine the epidemiology of priority infectious diseases and assess costs and benefits of disease management strategies in hybrid and channel catfish aquaculture.
We will identify emergent pathogens in catfish aquaculture and develop disease diagnostic methodologies for use in field surveillance studies. Specifically, we will characterize and evaluate pathogenesis of emergent diseases and fulfillment of Koch’s postulates or River’s postulate for newly recognized or emergent pathogens, develop rapid diagnostic tests and ELISA procedures to determine total and antigen specific antibody for epidemiological studies, and develop primary catfish cell lines for identification and confirmation of fish viruses. To improve disease management strategies in catfish aquaculture we will optimize Edwardsiella (E.) ictaluri vaccine delivery and evaluate cross protective potential of E. ictaluri vaccine against E. piscicida, determine the role of other myxozoans (non-H. ictaluri) and intraspecific variability of oligochaete hosts in occurrence of proliferative gill disease (PGD) in channel and hybrid catfish, determine the role of iron fortified diets and occurrence of bacterial infections in channel and hybrid catfish, and evaluate the pathophysiological effects of Bolbophorus damnificus (trematode) in hybrid and channel catfish.
In order to determine the epidemiology of infectious diseases in catfish aquaculture and conduct economic evaluations of disease management strategies we will determine the significance of genetic E. piscicida variants recovered from commercially cultured hybrid and channel catfish, determine the epidemiology of atypical Aeromonas hydrophila (aAh) infections in catfish aquaculture, perform a spatio-temporal survey of channel catfish virus (CCV) isolates and evaluate trends in the occurrence and virulence of different genetic strains of CCV in channel and hybrid catfish, evaluate disease transmission of emergent Vibrio spp. infections in hatchery fry, and evaluate economic impact of a live, attenuated E. ictaluri vaccine in commercial fingerling and foodfish production.
Progress Report
The Aquatic Research and Diagnostic Laboratory (ARDL) provides diagnostic support for the catfish industry and researchers in the southeastern United States and a mechanism to identify emergent pathogens and disease trends. The ARDL received 451 case submission along with 419 water samples submitted for analysis. Bacterial diseases were the predominant diagnoses with 205, 179, and 49 cases associated with Flavobacterium coveae, Edwardsiella ictaluri, and E piscicida, respectively. Antibiotic resistance continues to be a growing threat to the utility of approved medicated feeds. However, compared to previous years there was a 26% reduction in antibiotic resistance which is likely related to the recent adoption of an E. ictaluri vaccine in catfish fingerling production, which also provides some cross- protection against E. piscicida infections in hybrid catfish. Vibrio cholerae, was being monitored as a potential emergent pathogen in hatchery fry, however, there have been no reported outbreaks over the last several years. Clinical work has determined outbreaks are related to poor environmental quality related to overstocking and crowding that can result from holding fish too long. The genomes of 66 Vibrio spp. isolated from diseased fish have been sequenced and all lack the toxin producing genes (CTX toxin) related to human illness. Similarly, Yersinia ruckeri was isolated from diseased hybrids and identified as another potential emergent pathogen but no additional outbreaks have been diagnosed over the past 5 years. Erysipelothrix piscisicarius, an emerging disease in ornamental aquaculture, was detected in mosquitofish collected from catfish ponds during a survey of myxozoan parasitism in non-catfish pond inhabitants. Challenge trials showed catfish are not susceptible to this pathogen. As such, this bacterial species is not presently considered a pathogen of concern in catfish aquaculture. Molecular tests for detection and quantification of these isolates have been developed in support of research.
A variant of channel catfish virus (CCV) was isolated from blue catfish from a farm in the mid 1990’s. As part of a separate project investigating the evolution of catfish alloherpesviruses, the archived sample was characterized and is proposed as a new virus species, tentatively referred to as blue catfish alloherpesvirus (BCAHV). Molecular assays to differentiate CCV variants have been developed and used in monitoring programs to assess the prevalence and impact of CCV variants in catfish aquaculture. This work revealed previously unknown diversity of CCV, identifying multiple variants within geographically and temporally discrete isolates. The newly characterized virus from blue catfish showed differential mortality in channel and hybrid catfish. Survey data from hatcheries showed CCV latency to be dominated by a single variant. In addition, BCAHV was not detected in any of the survey data and has been shown to cause minimal losses in channel catfish and hybrid catfish. Interestingly, BCAHV conferred protection against the other two CCV variants in fish surviving initial challenge with BCAHV, indicating BCAHV could be modified as a vaccine to protect channel catfish against CCV infection. Since hybrid catfish production require large blue catfish populations, BCAHV is considered a potential emergent pathogen even though no clinical outbreaks have been reported since initially isolated. Viral isolates associated with diagnostic cases are currently being characterized to assess the true prevalence of BCAHV. In addition, an ictalurid catfish-originated cell line was established for the detection, isolation, and replication of viruses to evaluate host-cell interactions and modes of infection.
Experimental laboratory and pond trials, along with field application of a recently developed live attenuated E. ictaluri vaccine was shown safe in channel and hybrid catfish following multiple vaccine deliveries. Information was used to better define vaccination protocols, allowing for the safe application of multiple vaccine deliveries in situations where fish require re-vaccination due to poor feed performance or delivered below an immunizing dose. A series of studies explored the protective effects of an E. ictaluri vaccine against different variants of the closely related E. piscicida in channel and hybrid catfish. Initially, immunization baths with wild-type E. piscicida protected channel catfish from immersion challenge with wild-type E. ictaluri. Similarly, channel catfish immunized orally with a live-attenuated E. ictaluri vaccine were protected against injections of high and low doses of E. piscicida. These findings suggest the potential for developing polyvalent or cross-protective vaccines utilizing the shared antigens of E. piscicida and E. ictaluri.
Additional work was conducted to clarify ambiguity regarding the pathology of E. tarda, E. piscicida, and E. anguillarum infections in US farm-raised catfish, channel catfish, blue catfish, and catfish hybrids. The most severe pathology and mortality occurred in fish challenged with E. piscicida, supporting previous reports of increased pathogenicity in commercially important ictalurids, while E. anguillarum and E. tarda warrant only minimal concern. Hybrid catfish were shown significantly more susceptible to E. piscicida infection than channel catfish supporting diagnostic reports from hybrids on commercial farms. Serologically, fish vaccinated against E. ictaluri produced elevated serum IgM antibodies against E. ictaluri and E. piscicida. Additional work also led to the discovery of 5 E. piscicida genetic variants with each variant showing some level of preference towards different fish groups. The discovery of genetic variants is critical to vaccine development, in that vaccines must be constructed to protect against a the most prevalent genetic variants. Differential immune-gene expression was demonstrated following infection providing insight into mechanisms responsible for the development of a protective immune response. These studies highlight the influence of bacterial infection, vaccination, and fish type on the host response to E. ictaluri and E. piscicida infection in channel and hybrid catfish.
The myxozoan parasite Henneguya ictaluri is cited as the cause of proliferative gill disease (PGD) in channel and hybrid catfish, causing a destructive branchitis which leads to impaired gill function. The parasite has a complex, two-host life cycle that involves catfish and a benthic oligochaete. The oligochaete sheds the waterborne stage of the parasite, which subsequently infects the fish. Research indicates both hybrid and channel catfish are susceptible to the initial infection, but hybrid catfish appear to be a dead-end host, which may break the life cycle in ponds. However, over the past several years there has been an increase in PGD diagnostic cases in hybrid catfish indicating the parasite can persist in hybrid systems, contradicting experimental pond and laboratory results. These observations led to several possible conclusions; the presence of a disease vector other than channel catfish to propagate the parasite, adaptation of the parasite to infect hybrid catfish, delayed life cycle development in hybrid catfish or the presence of other myxozoan species that can infect hybrid catfish and cause similar gill pathology. Case studies using molecular diagnostic procedures indicate H. ictaluri is the predominant species in hybrid and channel catfish suffering from PGD, but life cycle studies in hybrid catfish showed no developing life stages up to 5 months post-exposure. Molecular assessment of PGD cases revealed the presence of multiple myxozoan species which could be contributing to associated gill pathology. Piscivorous birds are currently being investigated as a vector allowing for dissemination of myxozoan parasites in catfish production systems.
Bolbophorus damnificus is a digenetic trematode causing substantial economic losses. The life cycle involves the American White Pelican (final definitive host), snails (first intermediate host) and fish (second intermediate host). In experimental trials, infectivity rates between hybrid and channel catfish were similar, however hybrid catfish experienced lower mortality when exposed to comparable numbers of trematode cercariae compared to channel catfish. The parasite induces a severe anemia in channel and hybrid catfish, but hybrid catfish do not suffer the same level of mortality as channel catfish cohorts. Although infection rates are similar, hybrid catfish appear more resistant to the deleterious effect of B. damnificus infestations. Treatment focuses on breaking the parasite life cycle by eradicating snails from ponds. Copper sulfate is the most effective chemical for killing snails and previous work demonstrated a single application rate greater than 2.5 ppm was effective in killing over 90% of the snail population in ponds. However, copper is also an effective algaecide and at the targeted treatment rate often causes a bloom die-off leading to oxygen depletions and fish losses. Protocols were modified to increase treatment safety by applying multiple low dose copper applications which were shown just as effective as a single high copper treatment. To aid in delivery, a mechanized delivery system for applying copper sulfate crystals was developed allowing for precise application along the margins of the pond in a single pass. Experimental pond trials have been completed and we are currently evaluating treatment protocols on commercial farms.
Accomplishments
1. Enteric septicemia of catfish vaccine effective against both E. ictaluri and E. piscicida infection. Enteric septicemia of catfish, caused by the gram-negative bacteria Edwardsiella ictaluri, poses a significant threat to the profitability of channel catfish fingerling production. In recent years, Edwardsiella piscicida, a closely related bacterial pathogen, has emerged as a major concern during the food-fish phase of hybrid catfish production. ARS researchers in Stoneville, Mississippi, via previous research agreement (Project No. 6066-31320-004-D) yielded groundbreaking results, leading to the development of a live, attenuated vaccine (Patent No. 8,999,319; April 7, 2015) and a mechanized deliver system (Patent No. 11,330,833; May 2, 2022) allowing for in-pond oral vaccination of catfish. Work related to the current project validated the use of the vaccine against E. piscicida infection in hybrid catfish allowing for the immunization against both bacterial pathogens. The oral vaccination platform is the first effective vaccine used in catfish aquaculture and is commercially available under veterinary prescription. An estimated 300 million fingerlings are vaccinated annually, which are the source of fish used in food-fish production in the southeastern United States. The impact of the vaccine has been remarkable, as vaccination has increased yield (fish size and survival) and net revenues exceeding $3,500 per hectare. Consequently, nearly all commercial fingerling producers have adopted this vaccination strategy. Vaccination has also led to decreased antibiotic use and reduced antibiotic resistance, a growing problem in all sectors of animal production. This novel delivery approach not only allowed for the successful immunization of catfish against Edwardsiellosis but also paves the way for the administration of other vaccines in aquaculture where immersion delivery is not feasible. The value and adoption of the oral vaccination platform underscores the crucial role this project plays in supporting U.S. catfish aquaculture.
2. Proliferative gill disease (PGD) risk assessment model otherwise good to go. PGD is caused by a ubiquitous myxozoan parasite causing substantial losses in commercially raised catfish. Most losses occur when catfish fingerlings are introduced in grow-out ponds for food fish production. Research by ARS researchers in Stoneville, Mississippi, related to the current and research agreement (Project No. 6066-31320-004-D), a PGD risk assessment model was developed to determine the likelihood of fish losses in newly stocked production ponds or when fish are understocked for food fish production. The risk assessment model relies on comprehensive water analysis, utilizing eDNA methodologies and a quantitative polymerase chain reaction (PCR) assay specifically developed and validated through this project. By determining the levels of infectious life stages present in the pond water, we have established correlations between parasite levels and mortality events observed in experimental pond trials. This valuable data allows us to accurately assess the probability of fish losses upon stocking. This program has been implemented as a demonstration project on farms where water samples are collected concurrently with sentinel fish exposures to identify ponds that can be safely stocked with minimal risk to fish health.
