Research Project: Integrated Research to Improve Aquatic Animal Health in Warmwater Aquaculture

Location: Aquatic Animal Health Research

2023 Annual Report

Objectives
1. Identify virulence factors critical for pathogenesis of major catfish pathogens to guide the development of novel and cost-effective disease interventions. 1.A. Identify the genes (or their protein products) governing the virulence of Aeromonas hydrophila in catfish. 1.B. Characterize the environmental conditions of perturbations that influence the expression of virulence determinants in Aeromonas hydrophila. 1.C. Elucidate the capsular polysaccharide (CPS) antigenic diversity in Flavobacterium columnare and determine its role in pathogenesis. 2. Improve prevention and control strategies for bacterial and parasitic diseases of catfish and shrimp. 2.A. Evaluate the efficacy of next generation Flavobacterium columnare vaccines and identify the host immune responses that govern protection. 2.B. Determine the extent to which various feed additives (e.g. immunostimulants, toxin binders, etc.) modulate susceptibility of fish and shrimp to industry relevant pathogens. 2.C. Investigate host pathophysiology and performance following parasitic insult.

Approach
The catfish industry is the largest sector of U.S. aquaculture and shrimp production represents a growing and important sector. Improving the health of catfish, shrimp, and other warmwater species is important for long-term sustainability of these industries because losses due to disease are a significant impact to production. This project will take a multifaceted approach to accomplish two objectives that address the host, pathogen, and environmental interactions that are critical for improving aquatic animal health in aquaculture. Although Aeromonas (A.) hydrophila and Flavobacterium (F.) columnare have been studied for years, there are still gaps in our knowledge regarding the virulence factors of these pathogens and how environmental conditions alter their virulence. Therefore, Objective 1 will identify the genes governing the virulence of A. hydrophila, characterize environmental conditions that impact virulence amd elucidate the antigenic diversity of the capsular polysaccharide of F. columnare. Furthermore, prevention and control strategies for bacterial and parasitic diseases are limited and there are gaps in knowledge regarding host immune responses against pathogens. Research conducted under Objective 2 will develop new vaccines for F. columnare, determine the effect of feed additives on the susceptibility of fish and shrimp to disease, investigate the effect of parasitic insult on catfish performance and disease susceptibility, and determine the host immune mechanisms involved in protective immunity. The overall impact of this research is a reduction in disease related losses thereby increasing the profitability and production efficiency in the catfish, shrimp and other warmwater aquaculture industries.

Progress Report
This is the fourth year of a five-year project that has two major objectives. Objective 1 progress: Extracellular proteins, aerolysin (Arl) and hemolysin (Hly), produced by the bacterial pathogen Aeromonas hydrophila, were confirmed to be virulence factors that play important roles in causing disease and mortality of channel catfish. Mutants of A. hydrophila, depleted with either Arl or Hly gene were found to be significantly less virulent in causing disease. Research was completed to determine the virulence of columnaris causing bacteria (CCB) in channel catfish. Challenge experiments demonstrated Flavobacterium covae was highly virulent to channel catfish while F. columnare and F. davisii were on average less virulent. This data supported previous research demonstrating F. covae as the predominant CCB in the US catfish industry. Interestingly, F. oreochromis was highly virulent in channel catfish. In collaboration with university partners gene deleted mutants of Flavobacterium covae were developed to identify virulence factors of this pathogen. Several genes from the capsular polysaccharide biosynthetic operon were deleted and analyzed for phenotypic changes. One mutant exhibited no observable phenotypic or virulence differences from the parent isolate. Research is underway to determine the phenotype(s) associated with deletion of other genes and their virulence. The quorum sensing pathway may be involved in the virulence of CCB. To address this, a luxR deletion mutant and complemented mutant were generated and research indicated differences in biofilm formation between the mutant and parent strain. Research is underway to determine the phenotypic, transcriptomic, and virulence differences between the gene deleted mutant and parent strain. Aeromonas veronii strains were isolated from tissues of both healthy and diseased channel catfish obtained from Alabama farms and the genomes of seven strains were sequenced, assembled, and annotated. These genome sequences will enable comparative analyses to determine the roles these bacteria play in catfish aquaculture and the development of new preventative or management strategies. Comparative bacterial genomics is a powerful tool to discover genetic differences among strains of the same species of bacteria. Isolates of fish pathogens such as Streptococcus iniae and S. agalactiae were collected and the genomes were sequenced, assembled, and annotated. Comparative bacterial genomic analyses are underway to identify differences among strains and discover potential virulence factors involved in the emergence of these pathogens in aquaculture. Coinfections are common; however, there is a gap in knowledge of the effects of coinfections in catfish aquaculture. In collaboration with university partners, research was conducted to determine the impact of coinfection on mortality in catfish. A series of experiments were conducted in which catfish were experimentally infected with A. hydrophila, E. ictaluri, or F. covae alone or in combination with each other. Coinfections were observed to increase mortality in catfish when compared to fish infected with single pathogens which illustrates the need to further understand coinfection dynamics. White spot syndrome virus (WSSV), a circular and double stranded DNA virus, is a global concern for shrimp and crayfish producers, as it is responsible for white spot disease (WSD). In collaboration with university partners, crayfish exhibiting lethargy and mortality were sampled and WSSV was detected. Samples were also collected to sequence and annotate the WSSV genome. This is the first U.S. isolate having its whole genome sequenced and characterized. Objective 2 progress: Research was conducted to identify and develop additional recombinant F. covae proteins as vaccine candidates. Two different F. covae proteins were produced recombinantly and incorporated into experimental vaccines. Channel catfish were immunized with the vaccines and then experimentally infected with F. covae to determine efficacy. The results demonstrated varying levels of vaccine induced protection, providing a basis for further optimization of recombinant vaccines for F. covae. Research was conducted to determine the effects of three different types of flavonoids (catechin, quercetin, and hesperidin) on growth, feed utilization, hematological parameters, immune responses, and resistance of Nile tilapia to bacterial challenges. The results suggested that catechin and quercetin were ineffective at boosting growth and immune responses. However, hesperidin boosted immune responses and survival rates of fish following Streptococcus iniae challenge. Research was conducted to evaluate the gene expression of channel catfish fed diets containing different levels of frass as partial replacements for soybean meal, wheat short and corn meal. The results demonstrated the activation of many innate immune receptors in the systemic and mucosal tissues of fish fed medium to high frass levels. Further, the results showed a significant activation of the innate immune system which could suggest an added benefit to the frass than simply from its nutritional value. Research was conducted to evaluate gene expression of white bass fed diets containing various percentages of marine fish meal. A total of 1563 differently expressed genes were identified between all the comparisons, with most of these differences (95.9%) found in comparisons between the marine fish meal control and the no marine fish meal test diet or high soybean meal diets. Many genes involved in cellular stress, immune response, and apoptosis were differentially expressed with many unique signatures for each test diet, including the up regulation of immune response genes in many diets which could signal possible effects of anti-nutritional factors. In collaboration with university partners, snails were collected from catfish ponds in West Alabama and screened for infestations of Bolbophorus parasites. Snails that shed the parasite are currently being screened for positive cercariae and will be transported to the Unit for molecular diagnostics and catfish trials. PCR detection assays that discriminate only the infective Bolbophorus damnificus species have been designed and tested for sensitivity and specificity. Advanced imaging of parasites and histology of infested snails are also underway. Previous research in our laboratory suggested expression of glutathione-S transferase (GST) genes by channel catfish may contribute to protective immune responses against A. hydrophila. Genes coding for each of 6 GST families (alpha, kappa, mu, pi, rho, and theta) were cloned from cDNA prepared from skin and gill tissues of catfish. Recombinant GST proteins were produced recombinantly, and their enzymatic activities were quantified in vitro. Additionally, the relative expression of the GST genes in fish was also examined. Two toxins (PirA and PirB) produced by the plasmid of Vibrio parahaemolyticus help to manifest acute hepatopancreatic necrosis disease in shrimp. Research was conducted to evaluate the shrimp response to the recombinant toxins. Shrimp were administered the recombinant toxins and samples were subsequently taken at different time points for gene expression analysis. The analysis revealed that the recombinant toxins initiated an early host response involving several cell survival signaling and innate immune processes. Two disease phenotyping experiments with Streptococcus agalactiae and tilapia lake virus (TiLV) were completed using Nile tilapia families supplied by collaborators under a material transfer research agreement. Substantial additive genetic variation in resistance to S. agalactiae was demonstrated, and families bred for resistance or susceptibility, based on estimated breeding values, performed as anticipated following bacterial challenge. Substantial additive genetic variation in resistance to TiLV was also demonstrated suggesting that it is possible to improve resistance to TiLV through selective breeding. In collaboration with industry and university collaborators, research was conducted to identify pathological and gene expression differences in Nile tilapia with resistant or susceptible phenotypes. Resistant and susceptible fish were experimentally infected with S. inaie and samples were collected following infection for analysis. Lesions were observed in the anterior kidney, spleen and brain of susceptible tilapia consistent with S. iniae infection including histiocytic meningitis, splenitis, and nephritis. The severity of these lesions was greatly reduced in the resistant tilapia and no S. iniae were observed in the tissues. High thruput RNA sequencing has been performed on multiple tissues/individuals at multiple times post-infection to understand the genetic basis for the disease resistant phenotype. In collaboration with largemouth bass producers in the southeast region, the causes of disease outbreaks were determined. Columnaris disease and Aeromonas spp. were identified as the likely causative agents. Research is being conducted to determine which of the four species of columnaris causing bacteria are prevalent in the largemouth bass industry and developing methods for disease prevention. Research was conducted to improve the structural integrity of shrimp feed to minimize feed degradation and nutrient loss by using eight different agricultural-based biomass feedstocks Differences were observed in the ability of the experimental diets to improve pellet water stability, water absorption, and nutrient retention. Regarding water stability and nutrient (protein, lipid, and ash) retention, distiller’s dried gain with solubles, peanut hull fiber, cottonseed fiber, miscanthus, hemp fiber, and wheat straw were ranked as the highest bindings.

Accomplishments
1. Identification of a genetic marker for selectively breeding Nile tilapia for disease resistance. Streptococcus (S.) iniae is a causative agent of streptococcal disease in farm raised tilapia and contributes to large economic losses worldwide. ARS researchers Auburn, Alabama, and collaborating researchers from Bergen, Norway and Miami, Florida, previously demonstrated substantial genetic variation for resistance to S. iniae and confirmed the ability to improve resistance through selective breeding; however, it was unknown whether any genetic markers were associated with the resistant phenotype. The research team analyzed the genotypes of resistant and susceptible fish and identified a genetic marker significantly associated with resistance to the pathogen. The effectiveness of using the genetic marker for improving resistance of tilapia to S. iniae was confirmed experimentally. This was a significant milestone and demonstrates that tilapia families no longer need to be routinely infected with S. iniae to assess their level of resistance to this pathogen. Rather, their DNA can be analyzed and if they carry the favorable genetic marker, they will produce the more disease resistant offspring. This is the first genetic marker associated with resistance to a bacterial pathogen in Nile tilapia and will greatly contribute to breeding of fish with resistance to S. iniae and reduce losses associated with this pathogen in US and global tilapia aquaculture.

2. Development of recombinant monoclonal antibodies for use in catfish immunoassays. Research on functional adaptive immune responses in channel catfish is reliant upon the availability of monoclonal antibodies. Several monoclonal antibodies, originating from hybridoma technology, with specificity for channel catfish immunoglobulins have been used for many years; however, the hybridoma cell lines have the potential to lose their ability to express antibody. To safeguard these important research tools, ARS researchers in Auburn, Alabama, and collaborators at Auburn University developed a system to recombinantly express murine monoclonal antibodies (mAb). The system was used to generate a recombinant 9E1 mAb which specifically recognizes channel catfish immunoglobulin M (IgM). The recombinant 9E1 mAb was shown to effectively bind to soluble catfish IgM and was effective in a suite of commonly used immunological assays. The recombinant 9E1 mAb will be a valuable research tool for catfish immunology and the developed system provides a means to development additional recombinant mAbs to safeguard these important research tools.

3. Selective breeding of Nile tilapia for resistance to francisellosis is possible and does not impact other important commercial traits. Francisella (F.) orientalis is an important tilapia pathogen that causes francisellosis and results in substantial economic losses for the global tilapia industry. ARS researchers in Auburn, Alabama, and collaborating researchers from Bergen, Norway and Miami, Florida, conducted research to determine the potential to selectively breed tilapia for resistance to F. orientalis and to examine the relationship between other traits of economic importance. The results demonstrated moderate additive genetic variation in resistance to F. orientalis across four generations and families bred for resistance or susceptibility, based on estimated breeding values, performed as anticipated following bacterial challenge (i.e., resistant families had high survival and susceptible families had poor survival). No unfavorable genetic correlations were found between commercial traits such as resistance to other pathogens and growth. The results demonstrate that resistance to francisellosis in tilapia can be improved through selective breeding and this strategy will not impact other important commercial traits. This research has led to the development of a robust strain of tilapia that are resistant to disease and exhibit fast growth, important traits for the tilapia industry.

4. Thermal protocol developed to create triploid hybrid striped bass. ARS researchers Stuttgart, Arkansas, and Auburn, Alabama, in collaboration with an industry partner developed a protocol to produce triploid hybrid striped bass. Triploidly typically induces sterility, an issue of concern for producers as mature hybrid striped bass can become fertile through the normal production cycle. Temperature shock (3 warm and 3 cold temperatures at several times post-fertilization) on freshly fertilized eggs was used in small-scale, pilot trials. We identified the best cold and warm combination and then performed a full-scale, commercial production of larvae using optimal protocol and grew fish in ponds for 30 days. Results demonstrated that warm temperature shock was better than cold shock to induce a greater percentage triploid hybrid striped bass.

Review Publications
Shoemaker, C.A., Lafrentz, B.R., Beck, B.H., Paulson, M.D., Garcia, J.C., Heckman, T.I., Soto, E. 2022. Coinfection by Group C Streptococcus dysgalactiae subsp. equisimilis and Flavobacterium davisii in Nile Tilapia, from the United States. European Association of Fish Pathologists. https://doi.org/10.48045/001c.38695.
Hernandez, D.P., Abdelrahman, H.A., Galkanda-Arachchige, H.S., Kelly, A.M., Butts, I.A., Davis, D., Beck, B.H., Roy, L.A. 2022. Evaluation of aqueous magnesium concentration on performance of Pacific white shrimp (Litopenaeus vannamei) cultured in low salinity water of west Alabama, USA. Aquaculture. 565. Article 739133. https://doi.org/10.1016/j.aquaculture.2022.739133.
Lange, M.D., Abernathy, J.W., Rawles, A.A., Zhang, D., Shoemaker, C.A., Bader, T.J., Beck, B.H. 2023. Transcriptome analysis of Pacific white shrimp (Liptopenaeus vannamei) after exposure to recombinant Vibrio parahaemolyticus PirA and PirB proteins. Fish and Shellfish Immunology. 132. Article 108502. https://doi.org/10.1016/j.fsi.2022.108502.
Abernathy, J.W., Zhang, D., Liles, M., Lange, M.D., Shoemaker, C.A., Beck, B.H. 2023. Whole genome sequencing and annotation of seven strains of aeromonas veronii isolated from channel catfish. Microbiology Resource Announcements. 12(2):e01231-22. https://doi.org/10.1128/mra.01231-22.
Nguyen, K.Q., Bruce, T.J., Oluwafunmilola, E.A., Liles, M.R., Beck, B.H., Davis, A.D. 2022. Growth performance, survival, blood chemistry, and immune gene expression of channel catfish (Ictalurus punctatus) fed probiotic-supplemented diets. Veterinary Sciences. 9(12):701. https://doi.org/10.3390/vetsci9120701.
Wise, A.L., Lafrentz, B.R., Kelly, A.M., Liles, M.R., Griffin, M.J., Beck, B.H., Bruce, T. 2023. The infection dynamics of experimental Edwardsiella ictaluri and Flavobacterium covae coinfection in channel catfish (Ictalurus punctatus). Pathogens. 2023(12):462. https://doi.org/10.3390/ pathogens12030462.
Lange, M.D., Churchman, E.M., Wise, A.L., Bruce, T.J. 2023. A recombinant 9E1 monoclonal antibody binds membrane and soluble channel catfish immunoglobulin M. Fish and Shellfish Immunology Reports. 4:100086. https://doi.org/10.1016/j.fsirep.2023.100086.
Vela-Avitúa, S., Lafrentz, B.R., Lozano, C.A., Shoemaker, C.A., Ospina-Arango, J., Beck, B.H., Rye, M. 2023. Genome-wide association study for Streptococcus iniae in Nile Tilapia (Oreochromis niloticus) identifies a significant QTL for disease resistance. Frontiers in Genetics. 14:1078381. https://doi.org/10.3389/fgene.2023.1078381.
Jiménez-Bonilla, P., Zhang, J., Wang, Y., Blersch, D., Estela De-Bashan, L., Guo, L., Li, X., Zhang, D., Wang, Y. 2022. Polycationic surfaces promote whole-cell immobilization and induce micro-granulation of Clostridium saccharoperbutylacetonicum N1-4 for enhanced biobutanol production. ACS Applied Materials and Interfaces. 2022(14):49555-49567. https://doi.org/10.1021/acsami.2c14888.
Rawles, S.D., Fuller, S.A., Green, B.W., Abernathy, J.W., Straus, D.L., Deshotel, M.B., McEntire, M.E., Huskey Jr, G., Rosentrater, K., Beck, B.H., Webster, C.D. 2022. Growth, body composition, and survival of juvenile white bass (Morone chrysops) when dietary fish meal is partially or totally replaced by soybean meal, poultry by-product meal, an all-plant protein blend or a commercial plant-animal protein blend. Aquaculture Reports. 26. Article 101307. https://doi.org/10.1016/j.aqrep.2022.101307.
Strebel, L., Nguyen, K., Corby, T., Rhodes, M., Beck, B.H., Roy, L., Davis, A.D. 2023. On demand feeding and the response of Pacific white shrimp (Litopenaeus vannamei) to varying dietary protein levels in semi-intensive pond production. Aquaculture. https://doi.org/10.1016/j.aquaculture.2023.739698.
Tuttle, J., Bruce, T., Abdelrahman, H., Roy, L., Butts, I., Beck, B.H., Kelly, A. 2023. Persistence of a wild-type virulent aeromonas hydrophila isolate in pond sediments from commercial catfish ponds: a laboratory study. Veterinary Sciences. 10(3):236. https://doi.org/10.3390/vetsci10030236.
Tuttle, J., Bruce, T., Butts, I., Roy, L., Abdelrahman, H., Beck, B.H., Kelly, A. 2023. Investigating the ability of edwardsiella ictaluri and flavobac-terium covae to persist within commercial catfish pond sedi-ments under laboratory conditions. Pathogens. 12(7):871. https://doi.org/10.3390/pathogens12070871.
Shoemaker, C.A., Lozano, C.A., Lafrentz, B.R., Mumma, W.P., Vela-Avitua, S., Ospina-Arango, J., Yazdi, M., Rye, M. 2022. Additive genetic variation in resistance of Nile tilapia (Oreochromis niloticus) to Francisella orientalis and its genetic (co)variation to both harvest weight and resistance to Streptococcus agalactiae Ib. Aquaculture. 561:738736. https://doi.org/10.1016/j.aquaculture.2022.738736.
Mattison, C.P., He, Z., Zhang, D., Dupre, R., Lloyd, S.W. 2023. Cross-serological reaction of glandless cottonseed proteins to peanut and tree nut allergic IgE. Molecules. 28(4):1587. https://doi.org/10.3390/molecules28041587.
Abdelrahman, H.A., Hemstreet, W.B., Roy, L.A., Hanson, T.R., Beck, B.H., Kelly, A.M. 2022. Epidemiology and economic impact of disease-related losses on commercial catfish farms: a seven-year case study from Alabama, USA. Aquaculture. 566:739206. https://doi.org/10.1016/j.aquaculture.2022.739206.
Aksoy, M., Eljack, R.M., Beck, B.H., Peatman, E. 2022. Nutritional evaluation of frass from black soldier fly larvae as potential feed ingredient for Pacific white shrimp, Litopenaeus vannamei. Aquaculture. 27:101353. https://doi.org/10.1016/j.aqrep.2022.101353.
Aksoy, M., Eljack, R.M., Peatman, E., Beck, B.H. 2022. Immunological and biochemical changes in Pacific white shrimp, Litopenaeus vannamei, challenged with Vibrio parahaemolyticus. Microbial Pathogenesis. 172:105787. https://doi.org/10.1016/j.micpath.2022.105787.