Research Project: New Approaches to Managing Catfish Health in Aquaculture

Location: Warmwater Aquaculture Research Unit

2021 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
ARS researchers in Stoneville, Mississippi, received a total of 763 case submissions along with 429 water samples in 2020. Antibiotic resistant isolates and atypical bacterial pathogens have been archived for research purposes. Significant trends included increased occurrence of Vibrio cholerae in hatchery fry, the persistence of Edwardsiella piscicida in hybrid catfish and increased incidence of trematode infestations with Bolbophorus damnificus. A significant decline in atypical Aeromonas hydrophila (aAh) infections were documented and the cause of this decline is being investigated. Yersinia ruckeri was isolated from diseased hybrid catfish from multiple ponds on a single operation. Channel and hybrid catfish were challenged with Y. ruckeri by intraperitoneal injection as well as bath immersion to fulfill Koch’s postulates. Results suggest hybrids may be more susceptible to Y. ruckeri. However, since 2016, no Y. ruckeri cases have been reported. This milestone has been fully met and continued research is low priority. Sequences from >50 V. cholerae isolates from ornamental fish and catfish hatcheries have been sequenced to identify genetic regions that can be exploited by PCR. Fecal swabs, pond water and catfish eggs have been collected and samples stored for microbiome analysis to identify the source of contagion. Sequence data demonstrates all V. cholerae isolates from catfish lack the CTX toxin genes associated with cholera epidemics in humans. Isolates recovered from fish are likely opportunistic and can be controlled through maintaining proper environmental quality and minimizing crowding in hatcheries. Atypical A. hydrophila isolates and metadata from clinical diagnostic submissions were provided by three cooperating diagnostic labs in Mississippi and Alabama. Clinical isolates were identified by duplex polymerase chain reaction (PCR). Draft genomes were sequenced from a subset of isolates using Oxford Nanopore technology. Results revealed that as of 2015, aAh cases in Alabama were strictly due to the ML09-119 haplotype; however, Mississippi cases were attributed to both the ML09-119 and S14-452 haplotypes. Approximately 20% of aAh cases in the Mississippi Delta were caused by the S14-452 haplotype in 2014. This percentage increased rapidly to 100% of diagnostic cases in 2017. The presence of a complete type VI secretion system (T6SS) in S14-452 is an important difference between the two haplotypes. The haplotype shift shown in this study suggest increased fitness of S14-452 in the host and/or environment which may have important implications for the management of this pathogen. This study provides a spatial and temporal overview of two aAh haplotypes (ML09-119-like; S14-452-like) from catfish aquaculture with evidence of a shift in haplotype dominance in Mississippi. A prospective vaccine derived from the live-attenuated ML09-119 haplotype was evaluated for protective effects against the second aAh haplotype. Channel catfish fingerlings vaccinated by IP injection were protected against both haplotypes. No mortality differences were observed in non-vaccinated fish exposed to both haplotypes. Intraspecific genetic variability of E. piscicida isolates recovered from diseased channel and hybrid catfish in Mississippi was assessed and virulence associations among genetic variants were determined. Repetitive extragenic palindromic sequence-based polymerase chain reaction (rep-PCR) was used to screen 158 isolates. A subsample of 39 E. piscicida isolates was further characterized by multilocus sequence analysis (MLSA) targeting housekeeping genes. The MLSA provided greater resolution than rep-PCR, revealing 5 discrete phylogroups that correlated similarly with virulence gene profiles. Across all phylogroups, mortality was higher in hybrid catfish supporting previous work indicating E. piscicida is an emerging threat to hybrid catfish aquaculture. Immunization of channel and hybrid catfish using a live attenuated Edwardsiella ictaluri vaccine was shown to cross-protect against E. piscicida variants from each phyletic group. The reciprocal trial demonstrate exposure to heterologous E. piscicida isolates significantly improved survival during E. ictaluri challenge, regardless of variant. These findings indicate the presence of shared and conserved antigens among E. piscicida and the currently available E. ictaluri vaccine offers a viable management tool to mitigate losses associated with E. piscicida infection until more targeted E. piscicida vaccine candidates can be identified. Research has revealed the existence of two discrete genetic variants of channel catfish virus (CCV) identified by restriction length polymorphism analysis (RFLP). The genomes of representative strains have been determined and real-time PCR assays exploiting differences within these genomes have been developed and validated. Genomic DNA was isolated from 60 individual fry from each of 6 catfish hatcheries and analyzed for latency in each RFLP group. Analysis is expected to be completed in September 2021. Analysis of an archived virus isolated from blue catfish identified a new alloherpesvirus identified as blue catfish alloherpes virus (BCAHV). Virulence challenge trials among the RFLP groups and BCAHV demonstrated BCAHV was pathogenic to blue catfish, with limited pathogenicity in channel and hybrid catfish. There was no difference in mortality between CCV RFLP groups in channel and hybrid catfish. Fish surviving exposure to BCAHV were refractive to subsequent exposure to both RFLP groups suggesting a cross-protective immune response and the potential for BCAHV as a vaccine candidate against CCV. The influence of fish host on myxozoan community composition and diversity was assessed from naturally infected gill tissues from diagnostic case submissions from 2017 to 2019. Metagenomic amplicon analysis from gill tissues revealed myxozoan community composition differed between channel and hybrid catfish diagnosed with Proliferative Gill Disease. Myxozoan community structure was more diverse in channel catfish. Interestingly, Henneguya ictaluri, cited as the causative agent of PGD was not the most abundant myxozoan in almost half the cases examined, indicating other myxozoan species may also contribute to PGD pathology. The detection of numerous known and unclassified myxozoan sequences in addition to H. ictaluri provides evidence PGD may involve mixed species infections. Furthermore, the presence of numerous unclassified myxozoan sequences in gill samples from clinical PGD cases indicates the number of described species from U.S. farm-raised catfish vastly underestimates the true myxozoan diversity present within the varied pond microcosms associated with catfish aquaculture. Species specific in situ hybridization (ISH) in experimental and natural infections. In the experimental challenge, plasmodia of H. ictaluri and Henneguya postexilis formed in channel catfish but no mature plasmodia were observed in hybrid catfish, suggesting impaired development of multiple myxozoan life cycles in hybrid catfish. ISH investigations of both experimental and naturally infected tissues confirmed the presence of mixed infections with characteristic PGD lesions associated with H. ictaluri. In addition to confirming the development timeline of H. ictaluri, presporogonic stages of other myxozoans were associated with secondary lesions and, rarely, PGD-like pathology. These findings suggest other Henneguya spp. may contribute to PGD lesions and supports previous research implying hybrid catfish are likely a dead-end host in the H. ictaluri life cycle. These works have also led to the increased understanding of Henneguya postexilis. Draft genomes of H. ictaluri, H. exilis, H. adiposa and H. bulbosus have been used to create more resolute molecular markers targeting the myxozoan Elongation Factor 2 gene, which in addition to 18S and 28S genes, provides better discrimination among closely related myxozoan species. These EF2 primers have been shown to amplify EF2 from a multitude of myxozoan species in addition to those found in catfish, providing a tool for myxozoan researchers working with other fish systems. The mitochondrial genome of the oligochaete host (Dero digitata) was sequenced and preliminary investigations into the genetic diversity of the oligochaete host Dero digitata indicate limited variability among individuals, suggesting populations in catfish ponds are largely clonal. Mortality in juvenile channel and hybrid catfish exposed to B. damnificus was evaluated in two discrete, replicated infectivity challenges. Mortality curves in Channel and hybrid catfish were consistent with trends previously established for B. damnificus in Channel Catfish, although in both challenges cumulative mortality was significantly lower in hybrids. All exposed fish presented clinical signs consistent with B. damnificus infection—namely lethargy, exophthalmia, distended abdomens, and metacercariae grossly visible below the skin. The biological and economic implications of these findings on catfish production are unknown but suggest that hybrid catfish are less susceptible to the deleterious effects and reduced productivity elicited by B. damnificus in Channel Catfish. The mitochondrial genomes of Planorbella trivolvis and Biomphalaria havanensis have been sequenced and will be used to develop discriminatory PCR primers that can be employed to detect snail environmental DNA (eDNA) in pond systems.

Accomplishments
1. Use of vaccines to control Edwardsiollosis in catfish aquaculture. Catfish aquaculture is plagued by two closely related bacterial species in the genus Edwardsiella. Edwardsiella ictaluri is the most detrimental bacterial disease affecting catfish aquaculture. The disease is most problematic in the fingerling stage of channel and hybrid catfish production. Historically, disease control has relied on feed restriction and use of medicated feed, both of which have production consequences. With increased adoption of hybrid catfish as a production fish, E. piscicida has emerged as a serious threat to hybrid catfish aquaculture. In contrast to E. ictaluri, E. piscicida primarily affects market sized fish incurring significant production costs. In efforts to combat E. ictaluri infections, ARS researchers in Stoneville, Mississippi, developed a live attenuated vaccine under the previous project agreement as a method of prevention. Experimental and on-farm vaccination trials have shown significant improvements in survival, feeding rates and economic returns. Recent research demonstrated the presence of shared and conserved antigens among E. piscicida and E. ictaluri conferring protection against cross infections. The vaccine and vaccine delivery system are currently being used to commercially vaccinate channel and hybrid catfish resulting in increased yield and economics returns in fingerling and food fish production systems. To date, over 300 million catfish fingerlings are vaccinated each year representing approximately 90% of catfish raised in the southeastern United States.

2. Potential of stock rotations to mitigate proliferative gill disease in catfish aquaculture. Proliferative gill disease (PGD) in channel and hybrid catfish is a devastating disease associated with myxozoan parasitism. The causative agent is cited as Henneguya ictaluri, a myxozoan parasite present in nearly all catfish ponds during the spring of the year and can cause catastrophic losses (>90% mortality) in severe outbreaks. The life cycle involves the benthic oligochaete Dero digitata, which is ubiquitous in aquatic environments and is often present in very high numbers in the nutrient rich pond sediments of catfish ponds. Previous research has indicated the hybrid catfish may be a dead-end host in the Henneguya ictaluri life cycle and submissions to ARS researchers in Stoneville, Mississippi, point to reduced incidence of PGD in hybrid systems. Controlled experimental challenges coupled with newly developed in situ hybridization probes, which target pathogen specific DNA sequences in tissue sections, revealed maturation of Henneguya ictaluri myxospores in channel catfish from 14-20 weeks post-infection, but no myxospore development in concurrently exposed hybrid catfish. This supports previous work by our research group demonstrating the suppression of H. ictaluri life stages in hybrid pond systems. This research suggests hybrid catfish are a dead-end host in the H. ictaluri life cycle, or that development in hybrids is significantly arrested/delayed. This work implies strategic crop rotations between channel and hybrid catfish could be a viable management strategy to minimize incidence of PGD on commercial catfish operations.