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

Location: Aquatic Animal Health Research

2022 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 third year of a five-year project that has two major objectives. Objective 1 progress: Research was conducted on two hemolytic proteins, aerolysin (Arl) and hemolysin (Hly), secreted by the fish pathogen Aeromonas (A.) hydrophila (vAh). Characterization of both proteins demonstrated they form oligo structures with 6-8 subunits, are heat and detergent resistant, and can be detected by specific antibodies in sera from fish immunized with recombinant Arl and/or Hly monomers. Deletion of the Arl and Hly coding genes from the vAh genome was found to be highly attenuated compared to the virulent strain in experimental laboratory infections. Research demonstrated that the four genetic groups of Flavobacterium (F.) columnare previously identified in our laboratory represent four unique bacterial species. These are referred to as columnaris causing bacteria (CCB; i.e., F. columnare, F. covae, F. davisii, and F. oreochromis). Research was conducted to determine the virulence of the four CCB in channel catfish. The results confirmed previous research that F. covae is more virulent compared to F. columnare and F. davisii. CCB produce capsular polysaccharides (CPS) that are likely important for virulence and protective immunity. The biosynthetic genes responsible for CPS production were identified in a large panel of publicly available genomes. The analyses confirmed the presence of nine CPS types previously identified in our laboratory, and also indicate the presence of at least three additional CPS types. Putative CPS mutants were generated in F. covae by deletion of two CPS biosynthetic genes. Research is underway to determine the phenotype(s) associated with deletion of the genes and the ability of the CPS mutants to cause columnaris disease in channel catfish. In collaboration with university partners, the genomes of two F. covae isolates (LV-359-01 and LSU-066-04) were sequenced. The draft genomes are being utilized to identify virulence factors that might serve as potential recombinant protein vaccine targets for use under Objective 2. The clinical presentation of streptococcosis and columnaris coinfection in diseased Nile tilapia was investigated by examining phenotypic and genotypic characteristics of the isolated pathogens. Results demonstrated the streptococcal pathogen was Group C Streptococcus dysgalactiae subsp. equisimilis and the CCB was identified as Flavobacterium davisii. Objective 2 progress: One approach for developing an effective vaccine against F. covae is the incorporation of recombinant proteins into vaccines. Additional recombinant F. covae proteins were developed that will be tested as potential vaccine candidates. Research is also underway to continue to test the best route of administration for the recombinant vaccines which include the use of different commercial adjuvants. A 6-week experiment was conducted to determine if Akuapro®, a kaolinic clay, added to shrimp diets would increase growth, feed conversion, and immunological parameters in the Pacific white shrimp Litopenaeus vannamei. Shrimp were fed one of five isocaloric, isonitrogenous diets (35% crude protein) containing 0, 1, 2, 3, or 4% clay added to the diet. Shrimp in the 3% treatment had significantly higher weight and weight gain compared to the other treatments; however, survival of this treatment was also lower. A similar experiment was conducted on a commercial low salinity shrimp farm using a greenwater tank system and the results indicated no advantages of kaolin clay, likely due to the availability of natural food sources. A parasitic experimental lab site was constructed to harbor snails infected with the fish parasite Bolbophorus damnificus. In collaboration with Auburn University, catfish farmers were contacted in West Alabama that have had infestations with Bolbophorus parasite in their ponds. Snails that shed the parasite are currently being screened for positive cercariae and will be transported to the fish parasite lab for molecular diagnostics and catfish trials. In collaboration with ARS scientists at Stuttgart National Aquaculture Research Center (SNARC), Stuttgart Arkansas and the University of Arkansas at Pine Bluff, multiple databases/datasets were computationally screened and over 20 bacteriophage endolysin and peptidoglycan hydrolase genes were identified with potential lytic activity toward the fish bacterial pathogen Streptococcus (S.) iniae. Work is ongoing to recombinantly express these protein products and test them as potential therapeutants agents against S. iniae. Two disease phenotyping experiments with Francisella orientalis and S. agalactiae were completed using Nile tilapia families supplied by collaborators under a material transfer research agreement. Substantial additive genetic variation in resistance to both pathogens was confirmed. Estimated breeding values were used to produce resistant families and susceptible families for each pathogen and the groups performed as anticipated following experimental challenge (i.e., resistant families had high survival and susceptible families had poor survival). Collaborators are using the results for selectively breeding Nile tilapia for resistance to both important tilapia pathogens. Research was conducted to determine whether pre-exposure of catfish to A. hydrophila (vAh) stimulates a protective immune response. Channel catfish were exposed to a low concentration of vAh that resulted in no mortality for two weeks. Then, exposed and control fish were infected with a higher concentration of vAh and the results indicated that fish exposed to a low concentration of vAh exhibited 50% less mortality that the control fish that were not exposed to the bacterium. The primary difference between the exposed and non-exposed groups was quantitative differences in the concentration of six families of glutathione-S transferase (GST) proteins and Immunoglobulin M (IgM) antibodies. Fish exposed to low levels of vAh exhibited a greater concentration of GST-mu and contained GST-theta and IgM that were not present in the control fish. These differences likely contributed to the higher survival of fish exposed to low concentrations of vAh. In collaboration with a largemouth bass stakeholder, research demonstrated A. veronii and CCB as the causative agents of several epizootics. Research was conducted to develop laboratory infection models for use in identifying methods to prevent disease caused by the bacteria. A bivalent bacterin vaccine was developed and administered to largemouth bass fingerlings using different routes of administration such as immersion, injection, and through the feed. Vibrio (V.) parahaemolyticus is the causative agent of acute hepatopancreatic necrosis disease (AHPND), or early mortality syndrome (EMS), in shrimp. To better understand the defense mechanisms of shrimp to this problematic pathogen, changes in hematology, immunology and biochemistry of the hemolymph of Pacific white shrimp infected with V. parahaemolyticus were evaluated. Infected shrimp exhibited significantly decreased total hemocyte counts and lower hemocyanin compared to control shrimp. Hemocyte lysate phenoloxidase activity in infected shrimp was significantly increased compared to that of control shrimp. Although total plasma proteins levels were not different, plasma from infected shrimp significantly suppressed V. parahaemolyticus growth. The changes observed in hemolymph parameters may be useful indicators of the health status of shrimp. Additional research was conducted to further examine the host immune response of Pacific white shrimp to V. parahaemolyticus. Emerging strains of V. parahaemolyticus contain a plasmid containing genes that encode for two toxins, pirA and pirB. Experiments were conducted to determine the host response to recombinant PirA/B toxins. Following exposure to the toxins, samples were collected from treated and control shrimp, and then RNA sequencing was performed on individual shrimp samples over different time points. The results are being analyzed and the anticipated outcome of this research is to identify potential host mechanisms used to evade and or prevent the disease process during the production cycle. Research was conducted to evaluate the use of Frass as a potential protein source for shrimp feed. In this experiment, Pacific white shrimp were fed five diets containing frass at levels of 0, 5, 10, 20 and 30% as partial replacements of some other protein sources. No significant differences were observed in final weight gain, survival, and whole-body composition of shrimp fed up to 30% dietary frass. However, shrimp fed 20% and higher dietary frass level had significantly lower levels of lipid in the meat and plasma from shrimp fed 20% dietary frass could inhibit the growth of V. parahaemolyticus. The results suggest that Frass can be incorporated in shrimp diets up to 30% as a substitute for other protein sources without affecting their growth performance, body composition, and hemolymph parameters. The water stability of aquatic animal feeds is an important component of feed quality. A 10-week feeding experiment was performed to evaluate the effect of three feed binders derived from soybean hulls (SBH) and three control binders on the physiological and immunological status of Pacific white shrimp. During the feeding trial, growth, survival, body composition and plasma chemistry were not significantly affected by the diets. However, total hemocyte count and hemocyte lysate phenoloxidase activity were significantly higher in shrimp fed a diet incorporated with one of the SBH binders. The results suggest that use of SBH binder improves water stability and may improve the immune status of shrimp.

Accomplishments
1. Research reveals the fish pathogen Flavobacterium columnare is comprised of four unique bacterial species. Flavobacterium (F.) columnare is the causative agent of columnaris disease in freshwater fish. ARS researchers in Auburn, Alabama, previously demonstrated four discrete genetic groups exist within the species and reported associated host and virulence differences. The ARS researchers collaboratively determined the taxonomic status, phenotypic and chemotaxonomic traits, and DNA relatedness of the four genetic groups using polyphasic and phylogenomic approaches. Chemotaxonomic, matrix-associated laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) characterization and DNA relatedness afforded differentiation between the genetic groups, indicating each group is a discrete species. The names F. covae sp. nov., F. davisii sp. nov., and F. oreochromis sp. nov. were proposed to represent genetic groups 2, 3, and 4, respectively, while genetic group 1 isolates remain recognized as F. columnare. Since these pathogens are globally distributed and have significant impacts on wild and cultured fish species, research needs to target the correct bacterial species. Recognition of the four species will advance and improve research to define host-pathogen-environment relationships, epidemiology, and develop effective control and prevention measures to reduce the negative impact of columnaris disease in aquaculture.

2. Use of vaccine adjuvant reduces time needed for protective immune response in channel catfish. Flavobacterium covae is a causative agent of columnaris disease, which is one of the top diseases impacting the channel catfish industry. ARS researchers in Auburn, Alabama, previously developed a recombinant DnaK protein vaccine that provided protection against columnaris disease in laboratory challenges, but further research was needed to improve the effectiveness of the vaccine. ARS researchers evaluated the ability of an immersion adjuvant to boost and prime channel catfish for a more effective immune response. The results demonstrate significant protection of channel catfish at 6 to 8 weeks post vaccination and an overall benefit of using the immersion adjuvant to reduce the time required to stimulate a protective immune response. This research further confirmed the potential for developing an immersion vaccine based on the recombinant protein for the prevention of columnaris disease which is greatly needed for the catfish industry.

Review Publications
Lafrentz, B.R., Králová, S., Burbick, C.R., Alexander, T.L., Phillips, C.W., Griffin, M.J., Waldbieser, G.C., Garcia, J.C., De Alexandre, S., Soto, E., Loch, T.P., Liles, M.R., Snekvik, K.R. 2022. The fish pathogen Flavobacterium columnare represents four distinct species: Flavobacterium columnare, Flavobacterium covae sp. nov., Flavobacterium davisii sp. nov. and Flavobacterium oreochromis sp. nov. Systematic and Applied Microbiology. 45:126293. https://doi.org/10.1016/j.syapm.2021.126293.
Prior, B.S., Lange, M.D., Salger, S.A., Reading, B.J., Peatman, E., Beck, B.H. 2022. The effect of piscidin antimicrobial peptides on the formation of Gram-negative bacterial biofilms. Journal of Fish Diseases. 45(1):99-105. https://doi.org/10.1111/jfd.13540.
Wise, A.L., Lafrentz, B.R., Kelly, A.M., Khoo, L., Xu, T., Liles, M.R., Bruce, T.J. 2021. A review of bacterial co-infections in farmed catfish: Components, diagnostics, and treatment directions. Animals. 11:3240. https://doi.org/10.3390/ani11113240.
Churchman, E.M., Parello, G., Lange, M.D., Farmer, B.D., Lafrentz, B.R., Beck, B.H., Liles, M.R. 2022. Draft genome sequences of flavobacterium covae strains LSU-066-04 and LV-359-01. Microbiology Resource Announcements. 11(7):e00352-22. https://doi.org/10.1128/mra.00352-22.
Zhang, D., Lange, M.D., Shoemaker, C.A., Beck, B.H. 2022. Identification and characterization of differentially expressed channel catfish IgM transcripts after vaccination with antigens of virulent aeromonas hydrophila. Fishes. 2022(7):24. https://doi.org/10.3390/fishes7010024.
Lange, M.D., Abernathy, J.W., Farmer, B.D., Beck, B.H. 2021. Use of an immersion adjuvant with a Flavobacterium columnare recombinant protein vaccine in channel catfish. Fish and Shellfish Immunology. 117:136-139. https://doi.org/10.1016/j.fsi.2021.07.025.
Green, B.W., Rawles, S.D., Schrader, K., Mcentire, M.E., Abernathy, J.W., Ray, C.L., Gaylord, T.G., Lange, M.D., Webster, C.D. 2021. Impact of dietary phytase on tilapia performance and biofloc water quality. Aquaculture. 541:1-11. https://doi.org/10.1016/j.aquaculture.2021.736845.
He, Z., Zhang, D., Mattison, C.P. 2022. Quantitative comparison of the storage protein distribution in glandless and glanded cottonseeds. Agricultural and Environmental Letters. 7(1). Article e20076. https://doi.org/10.1002/ael2.20076.
Aksoy, B., Aksoy, M., Jiang, Z., Beck, B.H. 2022. Novel animal feed binder from soybean hulls -evaluation of binding properties. Animal Feed Science and Technology. 288:115292. https://doi.org/10.1016/j.anifeedsci.2022.115292.