Location: Warmwater Aquaculture Research Unit
2020 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
Clinical Research (Subobjectives 1.1.A, 1.1.B, 1.2.A, 1.3.A, 1.3.B): The Aquatic Research and Diagnostic Laboratory in Stoneville, Mississippi, received a total of 721 case submissions along with 556 water samples in 2019. Antibiotic resistant isolates and atypical bacterial pathogens have been archived for research. Significant trends included occurrence of Vibrio cholerae in hatchery fry, E. piscicida in hybrid catfish and trematode infestations with B. damnificus. Archived V. cholera and Y. ruckeri isolates are being evaluated for genetic variation and presence or absence of toxigenic genes. Real-time polymerase chain reaction assay for V. cholerae is in development for use in field and laboratory trials. A significant decline in atypical Aeromonas hydrophila infections were documented and the cause of this decline is being investigated. The USDA Food Safety Inspection Service has been charged with inspecting processed catfish for adulterated agents. A major concern in processed fish is the presence of violative residues of triphenylmethane dyes. Research has been initiated by requesting FDA for approval to conduct feed trials to determine if medicated feed types can cause false positive reactions for violative dyes in catfish tissue. Enzyme linked immunosorbent assay procedures for quantitating catfish antibody (total and specific) have been developed along with purification schemes for secondary antibody (rabbit, catfish) and antigen preparations for Edwardsiella ictaluri, Edwardsiella piscicida and Aeromonas hydrophila.
Bacterial Pathogens (Subobjectives 2.1.A, 2.1.B, 3.1, 3.2.A, 3.2.B, 3.4.A, 3.4.B, 3.5): Vaccination trials utilizing a live attenuated Edwardsiella ictaluri vaccine have been conducted to evaluate safety and efficacy under various conditions. Acute oxygen depredation, and multiple vaccine deliveries were not shown to increase negative vaccine reaction or compromise vaccine efficacy. Five discrete E. piscicida lineages have been identified from diseased channel and hybrid catfish. Immunization using the live-attenuated E. ictaluri vaccine and subsequent challenges using representatives of each E. piscicida phylogroup indicated the vaccine imparts cross-protective immunity in channel and hybrid catfish against all E. piscicida variants identified from catfish aquaculture. Cross-protection has also been demonstrated in on-farm vaccine trials. Production data from commercial operations is being collected to evaluate economic benefits of the current vaccination platform in fingerling and foodfish production. Information is being used to refine vaccination protocols. Multilocus sequence analysis and repetitive PCR fingerprinting have identified multiple genetic variants of Vibrio cholerae isolated from hatchery catfish fry. Preliminary infectivity trials evaluating have been conducted but have been unsuccessful in establishing infections following exposure of catfish eggs and fry to two V. cholerae genetic variants. It is currently believed that V. cholerae is an opportunistic pathogen in catfish fry and outbreaks in commercial catfish hatcheries are related to overcrowding in rearing vats. A haplotype survey and genome sequencing of archived atypical Aeromonas hydrophila (aAh) isolates from diagnostic case submissions confirm the existence of two clonal aAh haplotypes with spatial and temporal variation and differing pathogenic mechanisms. Field sampling identified aAh carrier states in asymptomatic populations of fish which is believed related to the rapid onset of mortality during epizootics. Immunization trials against the two primary atypical Aeromonas hydrophila pathotypes and epidemiological field studies using occupancy models are presently under investigation. Pond trials have been conducted and demonstrated iron supplements reflective of commercial practices do not increase iron concentrations in editable tissues. Experimental iron fortified diets at varied levels have been formulated to determine if iron supplements increase mortality following exposure to E. ictaluri, E. piscicida and A hydrophila. This information will be used to determine if iron supplements used to promote red blood cell production in anemic fish is resulting increased losses associated with bacterial infections.
Parasitic Pathogens (Subobjectives 2.2.A, 2.2.B, 2.2.C, 2.4): Pathophysiological responses of channel and hybrid catfish following exposure to Bolbophorus damnificus trematode cercaria were evaluated in laboratory challenge trials. Preliminary trials revealed the presence of a parasite-induced anemia associated with parasite encapsulation in the host. Surveys have been conducted revealing B. damnificus prevalence within populations of Planorbella trivolvis snails inhabiting ponds with confirmed trematode activity to be <2%. This work has led to identification of another trematode species associated with mortality and poor feed responses. The mitochondrial genomes and ribosomal RNA genes of adult trematodes collected from piscivorous birds endemic to catfish aquaculture have been sequenced to develop more resolute genetic markers to confirm trematode life stages in catfish and snail intermediate hosts. The mitochondrial genomes of Planorbella trivolvis and Biomphalaria havanensis have been sequenced for development of eDNA assays to detect presence of snail pests in catfish ponds. The influence of catfish host on myxozoan community composition within naturally infected gill tissues from clinical proliferative gill disease (PGD) cases and pond water from channel and hybrid monoculture systems was determined by metagenomic sequencing. Myxozoan community composition, diversity, species evenness, and individual taxa prevalence significantly differed between channel and hybrid systems in both gill and pond water datasets. Channel catfish systems had greater myxozoan diversity, species evenness, and Henneguya ictaluri abundance compared to hybrids. Hybrids exhibited year-to-year increases in H. exilis abundance with concurrent declines in H. ictaluri, myxozoan diversity, and species evenness. In nearly half of channel and hybrid PGD cases, H. ictaluri was present, but not the most prevalent taxa. The mitochondrial genome of Dero digitata was sequenced and primer sets are being evaluated to assess genetic diversity of D. digitata populations associated with PGD outbreaks. Experimental life cycle studies are underway to develop methods for laboratory propagation of catfish associated myxozoans. In situ hybridization probes have been developed and validated for 8 different Henneguya spp. associated with catfish aquaculture for evaluation of parasite development within the fish host and contributions of different Henneguya spp. in PGD pathogenesis. Histopathological assessments coupled with laser-capture microdissection and newly developed ISH assays have identified a potential emergent myxozoan gill disease attributed to massive burdens of H. exilis in hybrid catfish.
Viral Pathogens (Subobjectives 1.2.B, 1.4, 3.3.A, 3.3.B, 3.3.C): A Blue catfish herpes virus and an unknown virus isolated from channel catfish have been propagated in tissue culture on channel catfish and brown bullhead ovary cells. Cryostocks have been developed for future challenge studies and for fulfilling River’s postulates. Primary cell lines from multiple catfish tissues are being developed and tissue culture environments optimized to enhance viral replication. Assays will be used to screen suspected viral outbreaks that test negative for typical channel catfish herpes virus. In the first year of a two year survey, channel and hybrid catfish fry from 4 commercial catfish hatcheries have been collected and samples are being analyzed for viral latency. Information will be used to assess potential of viral outbreaks in hybrid catfish. The two unique channel catfish viral strains, along with blue catfish herpes virus isolated from diseased catfish have been archived and evaluated for differential susceptibility in channel blue and hybrid catfish. The genomes of ~30 temporally and geographically disparate channel catfish virus (CCV) isolates representing two discrete genetic groups were sequenced using long-read (Oxford Nanopore) and short read (Illumina MiSeq) platforms to assess the stability of the CCV genome over time. These studies are on-going.
Accomplishments
