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

Location: Aquatic Animal Health Research

2021 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 second year of a five-year project that has two major objectives. Objective 1 progress: Pathogenic bacteria produce many proteins that are detrimental to the host and are involved in the ability of the bacteria to cause disease. It has been suggested that the lethality of virulent Aeromonas (A.) hydrophila (vAh) in catfish may be due to the production of enzymatic proteins that destroy red blood cells (hemolysins). Six genes with presumptive roles in hemolysis were identified in the genome of vAh and were individually deleted using the optimized CRISPR-Cas9 system. In vitro research indicated that two of the mutants (lacking individual hemolysis genes) failed to produce their characteristic proteins in the extracellular products of the bacterium and one of these was also deficient in hemolytic activity. Experimental infection of catfish with these two mutants indicated that the mutant lacking hemolytic activity caused significantly lower mortality (approximately 57% less) and the other mutant resulted in approximately 16% lower mortality compared to the virulent wildtype strain. A double deletion mutant was generated in which both genes were deleted from the genome. Experimental infection of catfish with this mutant resulted in approximately 86% less mortality compared to the virulent wildtype strain. The results demonstrated that these hemolysins are virulence factors of vAh; however, there are other mechanisms used by the bacterium to induce disease in catfish. Columnaris disease, caused by Flavobacterium (F.) columnare, is an important pathogen impacting the catfish industry. Our laboratory established the existence of four phylogenetically distinct genetic groups in this bacterium. An analysis of F. columnare from the catfish industry was completed and demonstrated that greater than 90% of disease cases are caused by isolates belonging to genetic group 2. Research is currently being conducted to determine the virulence of isolates belonging to the four genetic groups in channel catfish to validate the association identified between genetic group 2 isolates and columnaris disease cases in the catfish industry. Whole genome sequencing of isolates belonging to the four genetic groups of F. columnare suggests that each group may represent a unique species of bacteria. A polyphasic approach was completed to characterize the four genetic groups including phylogenetic analyses, whole genome sequencing, biochemical and physiological characterization, antimicrobial susceptibility, chemotaxonomic analyses, and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). The data are currently being analysed to determine if each genetic group can be described as a new bacterial species. Flavobacterium (F.) columnare produces a capsular polysaccharide (CPS) that is suggested to be important for virulence and protective immunity. Research was initiated to generate gene deleted mutants lacking the CPS. The genes involved in the biosynthesis of the CPS were identified in the genome of a virulent genetic group 2 isolate. Primers were designed and polymerase chain reaction (PCR) products were cloned into a suicide vector to enable the deletion of two critical biosynthetic genes of the CPS. Currently, protocols for the conjugal transfer of the suicide vectors into F. columnare are being optimized to allow for efficient construction of the CPS mutants. Once constructed, the mutations will be verified and the ability of the CPS mutants to cause columnaris disease will be determined through experimental infections with catfish. An understanding of the role of the CPS in the virulence of F. columnare will improve our ability to develop prevention strategies and reduce the impact of this disease. Objective 2 progress: One approach for developing an effective vaccine against F. columnare is the incorporation of recombinant proteins into vaccines. Research was conducted to generate new recombinant F. columnare proteins to be tested as potential vaccine candidates. Once generated, catfish will be vaccinated with the recombinant proteins to determine if they provide protection from columnaris disease, and the best adjuvants and route of administration will be identified. The goal of this research is to develop an effective and economical vaccine for the prevention of columnaris disease in the catfish industry. In collaboration with university partners, an assay to screen a panel of Bacillus velezensis probiotic strains for growth inhibition activity against F. columnare was optimized. Research is ongoing to test the ability of the identified probiotics to decrease the severity of columnaris disease in catfish and to evaluate their ability to enhance the adaptive immune response during vaccination. Previous research in our laboratory demonstrated that catfish immunized with extracellular proteins of vAh developed immunity against subsequent infection with the bacterium. Analysis of the antiserum from immunized fish revealed that antibodies in the serum recognized multiple vAh antigens that likely contributed to the protection observed. To further understand the immune response to vAh antigens, immunoglobulin (Ig) M transcripts were analyzed and compared in tissues of the kidney and liver from immunized and control fish. The antigen receptor (the variable segment, VH) diversity of the IgM heavy chain was uncovered. Sequence analysis identified a total of 79 VH genes, 48 of which were unique VHs, consisting of 10 VH families. The unique VHs were differentially expressed in the kidney and liver between immunized and control fish. Phylogenetic analysis demonstrated that most of family 2 VHs were associated with IgM in immunized fish. This research heightens our understanding of the humoral antibody responses that govern protection of the catfish host to vAh. These data will assist in evaluating vaccine responses to this problematic pathogen. Two disease phenotyping experiments with Francisella orientalis and Streptococcus (S.) agalactiae were completed using Nile tilapia families supplied by collaborators. Substantial additive genetic variation in resistance to both pathogens was observed. Collaborators utilized this data for selectively breeding Nile tilapia for resistance to both important tilapia pathogens. Additionally, research was conducted to identify the immune mechanisms responsible for resistance to S. iniae that has been obtained through family based and subsequently genomic assisted selection. Groups of tilapia that were selectively bred for resistance or susceptibility to S. iniae were infected with the bacterium, and tissues samples were collected at different timepoints post-infection. The tissues were processed to identify differentially expressed genes, histopathological changes, bacterial loads, and serum immune factors between the two groups of fish. The samples are currently being analyzed and the results from this research will provide an understanding of the immune mechanisms responsible for resistance to S. iniae obtained through selective breeding.

Accomplishments
1. Bioeconomics of Flavobacterium columnare vaccine in pond trials with channel catfish. Flavobacterium columnare is a well-known fish pathogen that has been studied for over 100 years but, columnaris disease remains poorly controlled in farmed catfish. Outside of therapeutants (chemicals and antibiotics), the disease remains relatively unchanged in the U.S. catfish industry. Attempts to improve the management of this disease have led to the development of a live-attenuated genetic group 2 F. columnare vaccine (hereafter 17-23). ARS in Auburn, Alabama, and Auburn University scientists investigated the efficacy of the 17-23 vaccine delivered by immersion to catfish fingerlings that were grown to food size in earthen ponds. Data were collected from the two treatments (vaccinated vs non-vaccinated) to evaluate impacts on survival, growth, feed conversion, antibody development, and economic benefit. There were no obvious natural outbreaks of columnaris disease observed during the trial and no difference in survival of vaccinated versus control fish. The vaccinated fish had significantly elevated antibodies at 4 weeks post-vaccination but not at 12 weeks. However, vaccinated fish were significantly larger at harvest (0.78 ± 0.07 lbs) than control fish (0.64 ± 0.04 lbs), and the feed conversion ratio (FCR) of vaccinated fish (1.35) was significantly better than control fish (2.13). Partial budget analysis demonstrated use of 17-23 vaccinated fingerlings resulted in a net benefit of US $600 per acre. The vaccine showed economic benefit to producers, but the results should be further substantiated under both research and commercial settings.

2. Selectively breeding Nile tilapia for disease resistance does not negatively impact harvest weight. Fish growth is of high economic importance for farmers; therefore, the relationship between growth and other traits that affect performance is paramount. ARS scientists in Auburn, Alabama, in collaboration with industry stakeholders have demonstrated that resistance to Streptococcus (S.) iniae and S. agalactiae is heritable and improved lines of tilapia have been produced that are more resistant to disease. However, the impact of selective breeding for disease resistance on the harvest weight of tilapia was unknown. Data from eight generations of selective breeding including survival following S. iniae/S. agalactiae infection and harvest weight was analyzed. The results demonstrated there were no significant relationships, favorable or unfavorable, between harvest weight and survival to both Streptococcus species. This means that selectively breeding for disease resistance will not negatively impact harvest weight of the fish. Thus, multi-trait selection is recommended to balance growth and disease resistance. The goal of this research is to provide fish farmers with a robust stock of tilapia that are resistant to disease and exhibit fast growth to improve the profitability of tilapia aquaculture.

3. Bioactive and potent recombinant toxins (PirA and PirB) were produced from Vibrio parahaemolyticus. Acute hepatopancreatic necrosis disease (AHPND), caused by emerging strains of V. parahaemolyticus (pirA/pirB plasmid positive isolates), in shrimp aquaculture is of concern not only in Asia but also in Central and North America. Toxins produced by the plasmid of V. parahaemolyticus, residing in shrimp, are the culprit of AHPND. The two secreted proteins from the V. parahaemolyticus culture were subsequently identified and found to be similar with known binary insecticidal toxins, Photorhabdus insect related proteins A and B (PirA and PirB). In this study, recombinant PirA and PirB (rPirA and rPirB) were produced in Escherichia coli and the intra- and inter-molecular interaction between rPirA and rPirB was analyzed. The relative toxicity of rPirA and rPirB was also assessed in shrimp. Results demonstrated bioactive and potent recombinant PirA and PirB were produced. Use of recombinant proteins rPirA and rPirB will enable toxin testing independent of the bacterium. These recombinant proteins may also aid the search for antitoxin strategies against AHPND.

Review Publications
Lafrentz, B.R., Lafrentz, S.A., Beck, B.H., Arias, C. 2020. Draft genome sequences of Cetobacterium somerae 2G large and two novel Cetobacterium isolates from intestines of channel catfish (Ictalurus punctatus). Microbiology Resource Announcements. 9:e01006-20. https://doi.org/10.1128/MRA.01006-20.
Lafrentz, B.R., Lozano, C.A., Shoemaker, C.A., Garcia, J.C., Ospina-Arango, J.F., Rye, M. 2020. Genetic (co)variation between harvest weight and resistance to both Streptococcus iniae and S. agalactiae capsular type Ib in Nile tilapia (Oreochromis niloticus). Aquaculture. 529:735726. https://doi.org/10.1016/j.aquaculture.2020.735726.
Shahin, K., Pirezan, F., Rogge, M., Lafrentz, B.R., Shrestha, R.P., Hildebrand, M., Lu, F., Hogenesch, H., Soto, E. 2020. Development of IglC and GroEL recombinant vaccines for francisellosis in Nile tilapia, Oreochromis niloticus. Fish and Shellfish Immunology. 105:341-349. https://doi.org/10.1016/j.fsi.2020.07.045.
Xu, D., Zhang, D., Shoemaker, C.A., Beck, B.H. 2020. Dose effects of a DNA vaccine encoding the immobilization antigen on the immune response of channel catfish against Ichthyophthirius multifiliis. Fish and Shellfish Immunology. 106: 1031-1041. https://doi.org/10.1016/j.fsi.2020.07.063.
Heckman, T.I., Griffin, M.J., Camus, A.C., Lafrentz, B.R., Morick, D., Smirnov, R., Ofek, T., Soto, E. 2020. Multilocus sequence analysis of diverse Streptococcus iniae isolates indicates an underlying genetic basis for phenotypic heterogeneity. Diseases of Aquatic Organisms. 141:53-69. https://doi.org/10.3354/dao03521.
Lange, M.D., Abernathy, J.W., Shoemaker, C.A., Zhang, D., Kirby, A., Peatman, E., Beck, B.H. 2020. Proteome analysis of virulent Aeromonas hydrophila reveals the upregulation of iron acquisition systems in the presence of a xenosiderophore. FEMS Microbiology Letters. 367(20):1-8. https://doi.org/10.1093/femsle/fnaa169.
Alamdari, N.E., Aksoy, B., Aksoy, M., Beck, B.H., Jiang, Z. 2021. A novel paper-based and pH-sensitive intelligent detector in meat and seafood packaging. Food Control. 224. https://doi.org/10.1016/j.talanta.2020.121913.
Demaar, T.W., Lafrentz, B.R., Garner, M.G. 2021. Three cases of acute bacterial sepsis in pygmy hippopotamus (Choeropsis Liberiensis) calves siblings. Journal of Zoo and Wildlife Medicine. 52(2): 755-762. https://doi.org/10.1638/2020-0097.
Zhang, D., Bader, T.J., Lange, M.D., Shoemaker, C.A., Beck, B.H. 2021. Toxicity of recombinant PirA and PirB derived from Vibrio parahaemolyticus in shrimp. Microbial Pathogenesis. 155:104886. https://doi.org/10.1016/j.micpath.2021.104886.
Barger, P., Liles, M., Newton, J., Beck, B.H. 2021. Differential production and secretion of potentially toxigenic extracellular proteins from hypervirulent Aeromonas hydrophila under biofilm and planktonic culture. BMC Microbiology. 21:8. https://doi.org/10.1186/s12866-020-02065-2.
He, Z., Mattison, C.P., Zhang, D., Grimm, C.C. 2021. Vicilin and legumin storage proteins are abundant in water and alkali soluble protein fractions of glandless cottonseed. Scientific Reports. 11:9209. https://doi.org/10.1038/s41598-021-88527-7.
Malecki, J.K., Roy, L., Arias, C.R., Lange, M.D., Beck, B.H., Truong, N., Hanson, T.R. 2021. Bioeconomics of Flavobacterium columnare vaccine pond trials with channel catfish Ictalurus punctatus. North American Journal of Aquaculture. 83: 207-217. https://doi.org/10.1002/naaq.10191.
Lewis, M.R., Silliman, K.E., Beck, B.H., Sammons, S.M., Peatman, E.J. 2021. Failure of phenotypic markers to accurately identify black bass species and associated hybrids in Mobile River Basin, Alabama. North American Journal of Fisheries Management. 41(5):1591-1601. https://doi.org/10.1002/nafm.10673.