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ARS Home » Southeast Area » Auburn, Alabama » Aquatic Animal Health Research » Research » Research Project #437658

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

Location: Aquatic Animal Health Research

2020 Annual Report

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.

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 first year of a five-year project that has two major objectives. Objective 1 progress: Protocols were optimized for the reliable and efficient gene deletion in virulent Aeromonas (A.) hydrophila (vAh). Several putative virulence genes were deleted using the CRISPR-Cas9 system and were analyzed for loss of virulence using both in vitro and in vivo strategies. Preliminary results indicated that deletion of one or two of these genes can reduce the bacterium’s virulence to various degrees. Flavobacterium (F.) columnare produces two carbohydrates in the outer membrane of cells, the lipopolysaccharide and the capsular polysaccharide (CPS). Both carbohydrates have been implicated to be important for virulence and protective immunity. Preliminary data demonstrated antigenic differences in the CPS among isolates. Rabbit polyclonal antisera was generated against nine isolates, suspected to have antigenically different CPS based on whole genome sequencing and analysis of the CPS biosynthetic gene cluster. Preliminary data indicates that antibodies generated against each isolate are CPS specific and do not cross react with the CPS of the other isolates. Research in ongoing to confirm the specificity of the antisera to additional isolates of the bacterium. Objective 2 progress: One approach for developing an effective vaccine against F. columnare is the incorporation of recombinant proteins into vaccines. Previous research demonstrated that vaccination of catfish with a recombinant DnaK protein of F. columnare provided significant protection following laboratory disease challenges. Further research was conducted to test commercial adjuvants to bolster the adaptive immune response to DnaK and expressing and testing additional recombinant F. columnare proteins to improve vaccine efficacy. The goal of this research to develop an effective and economical vaccine for the prevention of columnaris disease in the catfish industry. Culture conditions were evaluated for maintaining rams horn snails and inducing the release of Bolbophorus cercaria. Rams horn snails were collected from the edges of a commercial catfish pond with a history of pelican feeding activity and preliminary diagnosis of Bolbophorus. The snails were maintained in a tank filled with pond water and supplied with aquatic vegetation and/or lettuce as forage. Snails were placed individually into 6 to 12 well culture plates containing filtered pond water and incubated overnight at room temperature to assess presence or absence of Bolbophorus cercaria. Preliminary data suggested 104 snails were shedding putative Bolbophorus cercaria out of a total of 425 examined (24.5 % prevalence). The cercaria were identified morphologically as Bolbophorus sp. and confirmed to be Bolbophorus damnificus using published molecular protocols. This preliminary assessment confirms the ability to obtain cercaria needed to conduct the research on catfish pathophysiology and performance following parasitic insult. Acute hepatopancreatic necrosis disease (AHPND) is an emerging bacterial disease of shrimp. The disease is caused by Vibrio (V.) parahaemolyticus, which harbors a plasmid expressing PirA/B toxins that contribute to shrimp mortality. Recombinant protoxins PirA and PirB were expressed in Escherichia coli using the corresponding genes encoded by the plasmid of V. parahaemolyticus and tested for toxicity in Pacific white shrimp. The results showed that recombinant PirA and PirB in combination were toxic to shrimp. The recombinant toxins are being used to test for potential toxin inhibitors that may be used to block their activity and reduce mortality in shrimp due to AHPND. Additionally, research was conducted to gain a better understanding of the immune response of Pacific white shrimp against V. parahaemolyticus. Experiments were performed to investigate the host’s immune response through RNA sequencing of hepatopancreas samples from control shrimp and shrimp exposed to recombinant rPirA/rPirB proteins. A better understanding of these responses will assist in developing new therapies that can augment the shrimp defense response under disease conditions. In collaboration with a largemouth bass stakeholder, research implicated A. veronii and F. columnare as the causative agents of several bacterial epizootics. A panel of isolates were collected from the epizootics and experimental challenges were conducted and Koch’s postulates were fulfilled, indicating that both pathogens are virulent to largemouth bass. Additionally, the isolates are being characterized via sequencing of several housekeeping genes to understand the genetic diversity of the isolates involved in the epizootics. Research is ongoing to explore disease prevention strategies. In collaboration with university partners, we previously isolated F. inkyongense, an environmental bacterium, from a disease case in cichlids. Although the bacterium was found to be avirulent in tilapia, it was demonstrated that the bacterium persisted on the gills for up to 30 days. Given this observation and the close genetic relatedness of F. inkyongense to F. columnare, research was conducted to determine if vaccination with the avirulent F. inkyongense stimulates a protective immune response against F. columnare. Tilapia were vaccinated with live F. inkyongense and then experimentally challenged with F. columnare. The results demonstrated no difference in mortality between vaccinated and control fish, indicating live F. inkyongense is not a suitable vaccine for columnaris disease. Two disease phenotyping studies with Francisella orientalis and Streptococcus 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.

1. Rapid assay for genotyping flavobacterium columnare. Columnaris disease is caused by the bacterium Flavobacterium columnare, which has substantial economic impacts on almost all fin fish aquaculture industries in the United States including catfish, rainbow trout, tilapia, sport fish, baitfish, and ornamental fish. Previous research in our laboratory established the existence of four distinct genetic groups within the species F. columnare; however, there were no quick and easy methods to assign an unknown isolate to one of the four groups. ARS researchers in Auburn, Alabama, developed a molecular assay to quickly assign an isolate to genetic group. The results demonstrated that the assay is a rapid, sensitive, and specific molecular tool for genotyping F. columnare. It is inexpensive to perform and can be used by any laboratory with polymerase chain reaction capabilities. The assay is being used by stakeholders and university partners to determine which genetic group(s) are responsible for disease losses in different aquaculture industries impacted by columnaris disease. This knowledge is important because our research has indicated biological relevance to the identified genetic diversity, with some genetic groups isolated preferentially from columnaris disease cases in specific fish species. An increased understanding of this will allow for the development of improved targeted control and treatment measures for columnaris disease.

2. Alternative aquafeed protein source improves growth and immune responses in farmed fish. Frass is a by-product of the black soldier fly larval meal industry and is composed of larval excrement, shed exoskeletons and residual feed ingredients. ARS researchers in Auburn, Alabama, evaluated diets containing frass at levels of 0 to 30% as partial replacements for soybean meal, wheat short and corn meal. Experimental diets were fed to both fingerling catfish and tilapia and the results demonstrated that final weight gain was significantly increased in both fish species when fed feed containing frass. Additionally, catfish fed diets containing frass showed improvements in immune system machinery, while tilapia fed frass diets exhibited increased survival against two important pathogens, Flavobacterium columnare and Streptococcus iniae. Based on these findings, frass derived from the larvae of black solder flies has the potential as an alternative source of protein in aquafeeds or as an ingredient for enhancing palatability and growth. Further, use of frass in diets may prove beneficial for improving the resistance of catfish and tilapia against bacterial infections.

3. Omics tools provide practical insights on the host-pathogen-environment interactions triggering disease in the U.S. catfish industry. The Gram-negative bacterium, Aeromonas hydrophila, is responsible for nearly $100 million in losses to the catfish industry over the past decade. Usually regarded as a secondary pathogen, a virulent strain (vAh) has emerged with heightened pathogenicity linked to environmental conditions. USDA-ARS research has revealed that nutritional cues within the host catfish and water quality parameters in culture ponds together are critical for establishing conditions conducive to disease outbreaks. Research in Auburn, Alabama, has pointed to the importance of iron for vAh replication and survival, such that conditions of iron scarcity (common in ponds in some catfish farming regions), can trigger release of potent vAh virulence factors and lead to significantly elevated catfish mortality. ARS researchers in Auburn, Alabama, and Stuttgart, Arkansas, in collaboration with Auburn University, performed experiments that evaluated growth characteristics and protein expression of vAh following culture in iron-restricted conditions. Using cutting-edge proteomic analyses, they confirmed that iron acquisition is critically linked to virulence in this pathogen. Low iron conditions appear to trigger the release of factors that target catfish red blood cells and initiate the onset of disease. These findings are being applied in the ongoing development of an efficacious vaccine to protect against vAh as well as in practical pond applications/management strategies that seek to stabilize available iron levels.

Review Publications
Aksoy, M., Eljack, R.M., Beck, B.H. 2020. Nutritional value of frass from black soldier fly larvae, Hermetia illucens, in a channel catfish, Ictalurus punctatus, diet. Aquaculture Nutrition. 00:1-8.
Lange, M.D., Farmer, B.D., Abernathy, J.W. 2019. Vertebrate mucus stimulates biofilm development and upregulates iron acquisition genes in Flavobacterium columnare. Journal of Fish Diseases.
Lafrentz, B.R., Garcia, J.C., Shelley, J.P. 2019. Multiplex PCR for genotyping Flavobacterium columnare. Journal of Fish Diseases. 42:1531-1542.
Pomaranski, E.K., Griffin, M.J., Camus, A.C., Armwood, A.R., Shelley, J., Waldbieser, G.C., Lafrentz, B.R., Garcia, J.C., Yanong, R., Soto, E. 2019. Description of Erysipelothrix piscisicarius sp. nov., an emergent fish pathogen, and assessment of virulence using a Tiger Barb (Puntigrus tetrazona) infection model. International Journal of Systematic and Evolutionary Microbiology.
Aksoy, M., Eljack, R.M., Schrimsher, C., Beck, B.H. 2020. Use of dietary frass from black soldier fly larvae, Hermetia illucens, in hybrid tilapia (Nile x Mozambique, Oreocromis niloticus x O. mazambique) diets improves growth and resistance to bacterial diseases. Aquaculture Report. d.
Zhao, H., Beck, B.H., Fuller, S.A., Peatman, E. 2020. EasyParallel: a GUI platform for parallelization of STRUCTURE and NEWHYBRIDS analyses. PLoS One. 15(4):e0232110.
Roy, L., Teichert-Coddington, D., Beck, B.H., Dahl, S., James, J., Guo, J., Davis, A. 2020. Evaluation of stocking density and dietary fish meal inclusion for intensive tank production of Pacific White Shrimp (Litopenaeus vannamei) cultured in low salinity waters of west Alabama. North American Journal of Aquaculture. 82:345–353.
Bruce, T.J., Ma, J., Oliver, L.P., Jones, E.M., Lafrentz, B.R., Cain, K.D. 2020. Isolation and experimental challenge of cultured burbot (Lota lota maculosa) with Flavobacterium columnare and Aeromonas sp. isolates. Journal of Fish Diseases. 43:839–851.