Project : USDA ARS

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

Location: Aquatic Animal Health Research

2024 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 final year of a five-year project that has two major objectives. Objective 1 progress: Hemolytic proteins identified from annotated genomes of virulent Aeromonas hydrophila were analyzed for their molecular structure variation, hemolytic activity in vitro, cytotoxicity in cultured cells, and pathogenicity in fish. Mutants of A. hydrophila lacking hemolytic protein secretion were found to be attenuated in catfish. The potential use of these mutants as live-attenuated vaccines for prevention of A. hydrophila is under study. In collaboration with Auburn University (AU), research was conducted to determine the persistence of A. hydrophila, Edwardsiella ictaluri, and columnaris-causing bacteria in pond sediments and to determine whether antimicrobial resistance is present in bacterial communities of pond sediment. Of the three pathogens analyzed, A. hydrophila had the greatest ability to persist in pond sediments, and the results demonstrated the presence of antimicrobial resistance in bacteria isolated from pond sediment. In collaboration with AU, research was conducted to improve the ability to make targeted genetic deletions in Flavobacterium covae. Use of a different Escherichia coli strain increased the transfer of DNA into F. covae by about 75-fold. Research is ongoing to further improve this genetic manipulation system to enhance its efficacy in diverse F. covae strains and allow for more rapid genetic mutants to be generated. A predicted sialidase protein that is produced by F. covae strains was identified that is expected to be an important determinant of F. covae binding to, and interactions with, fish mucus. Research suggests that this sialidase protein is secreted and may be involved in the initial stages of F. covae mucus interactions. Future studies will discern how the sialidase is involved in F. covae virulence through targeted genetic deletion and characterization. In collaboration with Tuskegee University research continued to examine the effects of feed status (fed or not fed) on disease susceptibility. Challenges of channel catfish with virulent A. hydrophila either fed or not fed were completed and histopathology is being conducted. Gene expression analysis among the challenged groups is currently underway. In collaboration with AU, research was conducted to determine the impact of coinfections (bacterial-bacterial and bacterial-viral) on mortality and gene expression in catfish. A series of experiments were conducted in which catfish were experimentally infected with F. covae, virulent A. hydrophila, or channel catfish virus (CCV) alone or in combination with each other. Results indicated elevated mortality levels in catfish when pathogens were combined to simulate coinfections. Tissue samples from these fish are being analyzed to identify differentially expressed genes among disease groups to elucidate mechanisms occurring during coinfection dynamics. The genome of the highly virulent isolate of CCV, 2013-CCV-DRB, used for these experiments was also sequenced and annotated. Objective 2 progress: Research was conducted to further develop recombinant protein vaccines for bacterial pathogens impacting warmwater aquaculture. Research is being performed to evaluate the most efficient route of administration, establish the optimal delivery method(s), and evaluate the use of adjuvants. Adaptive immune responses to these vaccines are being examined to understand the host components that confer protection. In collaboration with AU, research was conducted to examine gene expression of catfish vaccinated against F. covae. Skin explants from catfish have been collected throughout vaccination trials and a custom gene expression quantitative polymerase chain reaction panel has been developed to examine temporal innate and adaptive immune gene expression changes. To further examine the immune system, single-cell RNA sequencing on nuclei isolated from whole catfish spleens was performed. Single nuclei sequencing libraries were prepared from multiple individual spleens, sequenced, aligned to the reference genome assembly, and gene features counted. This allowed for the identification and characterization of multiple cell types within this tissue, including erythroid cells, B-cells, T-cells, Natural Killer cells, myeloid cells, and hematopoietic stem cells. Each cell type was found to express specific genes for use in developing genetic markers. In collaboration with AU, an antibiotic difficidin produced by a beneficial Bacillus velezensis strain was found to inhibit the growth and viability of F. covae. The difficidin has been purified and shown to inhibit a diverse collection of F. covae strains, and research is underway to identify the mode of action of difficidin in inhibiting bacterial growth. Glutathione (GSH) is a natural product and recognized as a beneficial antioxidant. Research demonstrated GSH can kill A. hydrophila at low dosages and may have potential as an antimicrobial peptide against A. hydrophila and other fish pathogens. In an international collaborative effort, a lytic bacteriophage infecting A. hydrophila was identified. Therapeutic application of this phage to fish showed up to 100% survival when challenged with A. hydrophila suggesting it may have potential as an alternative to antibiotics. In collaboration with AU, snails from catfish ponds in West Alabama have been screened for infestations of Bolbophorus parasites. Polymerase chain reaction detection assays that discriminate the infective B. damnificus species have been designed and tested for sensitivity and specificity. Advanced imaging of parasites and histology of infested snails is underway, as well as collection of samples for whole genome sequencing. Research efforts are ongoing to characterize global gene expression of tilapia selectively bred for resistance or susceptibility to Streptococcus iniae. High throughput RNA sequencing has been performed on multiple tissues/individuals at multiple times post-infection to understand the genetic mechanisms responsive to this bacterial resistance. A challenge model for Streptococcus agalactiae Ia was developed. The challenge model was used to determine the susceptibility of Nile tilapia families provided by industry partners. Substantial additive genetic variation was demonstrated indicating that it is possible to improve resistance to S. agalactiae Ia through selective breeding. Research is underway to continue to investigate the effects of various levels of phytase addition to channel catfish diets on gene expression. Preliminary results indicate that there are no growth differences, gene responsive differences and little physiological differences in catfish despite the level of phytase included in the diet. In collaboration with AU and Harbor Branch Oceanographic Institute, the genome of a Florida pompano from the Gulf of Mexico was sequenced and assembled. Functional annotation is ongoing. Results from this collaboration will be useful in further domestication and commercialization efforts for this species. In collaboration with AU, a variety of lab and field-based experiments were conducted to fill critical knowledge gaps regarding proper dosing of copper sulfate for control of harmful algal blooms. Research results showed (1) general strong negative effects on cyanobacteria regardless of copper sulfate concentration, (2) low dose copper promoted beneficial chlorophytes, (3) standard copper sulfate doses lead to the development of resistance to copper in remaining phytoplankton, and (4) standard copper doses harm zooplankton, which are natural grazers of phytoplankton. These findings highlight the importance of correct dosing when treating ponds with chemicals to avoid unintended, negative effects. Research with AU was conducted to determine the impact of removing small planktivorous fishes from catfish production ponds (biomanipulation) on water quality and production. The results showed that biomanipulation improved water quality by reducing phytoplankton and cyanobacteria and improved catfish production. Other methods for water quality management were investigated by characterizing the ability of cost-effective materials such as biochar and activated carbon to remove phosphorus from catfish ponds. Local strains of five cyanobacteria genera were isolated and techniques were developed to obtain optimal growth in laboratory culture conditions. This laboratory system is being used to characterize the effect of herbicides commonly used in Alabama aquaculture ponds on cyanobacteria to further the ability to control harmful algal blooms. In collaboration with AU, research was conducted to address issues regarding big fish in catfish aquaculture. The age structure of hybrid catfish that exceed market size was determined in a pond field study and found that fish ranged in age from 1-11 years, with some fish weighing more than 50 pounds. This research confirmed the inefficiency of existing commercial seining practices on catfish farms. In collaboration with AU, research was conducted to improve production techniques for shrimp cultured in ponds, biofloc, and recirculating aquaculture systems. Acoustic-based feeding technology was tested and found effective at decreasing production time and improving survival and growth of shrimp. Adding aqueous magnesium to improve magnesium to calcium ratios in shrimp ponds was found to be effective at improving shrimp growth but may not be cost effective. The effect of rearing shrimp in ponds with different dissolved oxygen (DO) concentrations on production metrics and gene expression was tested. Preliminary results indicate there are no differences in any groups between the highest and lowest dissolved oxygen, indicating that long-term low DO rearing may be feasible.

Accomplishments
1. Flavobacterium covae is the predominant species of columnaris-causing bacteria in the United States catfish industry. Columnaris disease is one of the largest contributors to disease losses for catfish farmers in the southeastern United States. The term ‘columnaris-causing bacteria’ (CCB) has been coined in reference to the four recently described species that cause columnaris disease, Flavobacterium columnare, F. covae, F. davisii, and F. oreochromis. Historically, F. columnare, F. covae, and F. davisii have been isolated from columnaris disease cases in the catfish industry. However, the CCBs most severely impacting the industry, as well as their virulence in channel catfish were not known. ARS researchers in Auburn, Alabama, and university collaborators determined the species of CCB implicated in 259 columnaris disease cases across Mississippi and Alabama. The results demonstrated that F. covae is the predominant CCB impacting the catfish industry and this data was supported by laboratory infection experiments which demonstrated F. covae causes more mortality in catfish compared to F. columnare and F. davisii. Collectively, these results demonstrate F. covae is the predominant CCB in the US catfish industry and research aimed at developing new prevention and control strategies should target this bacterial species.

2. Inclusion of frass into catfish diets stimulates the expression of immune related genes. The larval waste, exoskeleton shedding, and leftover feed components of the black soldier fly and its larvae make up the by-product known as frass. Despite the advancement in using frass as an alternative protein in feed, studies related to the immune response and transcriptomes in the channel catfish are scarce. ARS researchers in Auburn, Alabama, fed channel catfish with different dietary amounts of frass and examined the expression of growth and immune-related genes. The results demonstrated that a series of metabolic and immune-related genes were differentially regulated after being fed either a low- or high-frass diet for 10 weeks. This data supports a beneficial role of frass when included into catfish feed and potential to boost the catfish immune system and enhance disease resistance.

3. Genetic mechanisms of resistance to columnaris disease in moronid basses. Columnaris is a common disease that is devastating aquaculture-raised fish populations. Columnaris-causing bacteria are present in essentially all freshwater environments, making it particularly difficult to prevent and treat infection. Hybrid striped bass (cross between white bass females and striped bass males) are a prominent aquaculture product in the United States that, with their parent species, are often raised in fresh water and therefore can be exposed to columnaris-causing bacteria. ARS researchers in Auburn, Alabama, and Stuttgart, Arkansas, demonstrated that hybrid striped bass are more susceptible to columnaris disease than the maternal white bass parental species. Gene expression of gill tissue before infection and at three different time-points after infection with Flavobacterium covae were examined to elucidate the mechanisms behind moronid disease resistance. The results of this study showed that the white bass immune response mechanisms are activated earlier than those of the hybrid striped bass in the presence of this pathogen. Further, immune-related genes identified through this study can be the focus of future research and selective breeding efforts to produce a hybrid with greater disease resistance like that of the white bass.

4. Bacterial coinfections in channel catfish amplify disease dynamics. In the United States catfish production pond environment, fish are simultaneously exposed to multiple aquatic pathogens and bacterial coinfections are commonly reported. However, their role in augmenting pathogen and host interactions are not well known. University partners in collaboration with ARS researchers in Auburn, Alabama, assessed the effects of bacterial coinfections of Flavobacterium covae and Aeromonas hydrophila in juvenile channel catfish. The results demonstrated an additive effect on mortality (i.e., increased mortality) when these two pathogens were used to infect catfish simultaneously, thus illustrating a negative impact of coinfections for the industry. Reducing disease outbreaks in catfish farming is critical to enhancing production yields and quality products, and an increased understanding of coinfection dynamics will provide more insight into targeted control measures for catfish health.

5. Unexpected negative consequences of gypsum applications to catfish ponds. Gypsum applications to aquaculture ponds are a reliable method for increasing hardness (dissolved calcium and magnesium) that is important for fish development and growth. Other benefits of gypsum treatments in ponds include a small reduction in pH and adsorption of phosphorus that is an important nutrient for phytoplankton. Although commonly used, other potential impacts of gypsum application to ponds were unknown. University partners in collaboration with ARS researchers in Auburn, Alabama, conducted a six-month, replicated, whole pond experiment at a farm in west Alabama where catfish production ponds were not or were treated with 500 mg/L gypsum (calcium sulfate). The results showed large increases in soluble reactive phosphorus (7x) and cyanobacteria (3.5x) after gypsum addition, which was unexpected. The “new” phosphorus appears to be released from the sediment once gypsum is applied. Although gypsum is a common additive to ponds to elevate water hardness, the negative impacts of gypsum on water quality could lead to algal blooms and fish death.

U.S. DEPARTMENT OF AGRICULTURE

Aquatic Animal Health Research: Auburn, AL