CONTROL STRATEGIES FOR VIRULENT AEROMONAS HYDROPHILA IN CATFISH AQUACULTURE BY VACCINATION AND INFORMING POND MANAGEMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1012468
• Grant No.: 2017-67015-26794
• Cumulative Award Amt.: $500,000.00
• Proposal No.: 2016-09489
• Project Start Date: Aug 1, 2017
• Project End Date: Jul 31, 2021
• Grant Year: 2017
• Program Code: [A1221]- Animal Health and Production and Animal Products: Animal Health and Disease

Recipient Organization
MISSISSIPPI STATE UNIV
(N/A)
MISSISSIPPI STATE,MS 39762

Performing Department
CVM Basic Science Department

Non Technical Summary
Outbreaks caused by virulent A. hydrophila (VAh) from 2009 to 2014 resulted in the loss of more than 12 million pounds of catfish from U.S. aquaculture. In 2013, a regional coalition of scientists at Mississippi State University and Auburn University were awarded a USDA AFRI grant to address the VAh problem. As a result, we developed a candidate live attenuated vaccine and a recombinant vaccine strategy for VAh. We determined that pond environmental conditions significantly contribute to VAh outbreaks, and we determined that VAh has two distinct genomic subclades. Important work remains to be done to determine the efficacy of our vaccine approaches for cross-protection against different VAh subclades and to determine specific environmental conditions that predispose VAh outbreaks in catfish ponds. In this project renewal, we will determine antigenic and pathogenic similarity between two VAh subclades and evaluate efficacy of a live, attenuated vaccine strain. We will also determine efficacy of a recombinant Edwardsiella ictaluri vaccine carrier strategy to control VAh. Finally, we will identify risk factors and putative predictive indicators of VAh outbreaks in commercial catfish ponds. The target audiences of this research are catfish producers as well as fish pathologists, veterinarians, and extension personnel serving the industry. Information from this project will be disseminated through peer-reviewed publications, presentations at scientific meetings, and extension presentations and publications. The ultimate goals of this project are to develop an effective, practical vaccine strategy to control VAh and to inform producers on management practices to prevent VAh outbreaks.

Animal Health Component

40%

Research Effort Categories

Basic

30%

Applied

40%

Developmental

30%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	0810	1100	40%
311	0810	1040	10%
311	0810	1090	25%
311	0810	1160	10%
311	0810	1170	15%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1040 - Molecular biology; 1090 - Immunology; 1160 - Pathology; 1100 - Bacteriology; 1170 - Epidemiology;

Keywords

aeromonas hydrophila

aquaculture

catfish

g

risk

system dynamics modeling

vaccine

virulence

Goals / Objectives
Aeromonas hydrophila is a Gram-negative opportunistic pathogen that affects farm-raised catfish, which is the largest aquaculture industry in the U.S. Although it is historically considered a secondary pathogen, outbreaks caused by an emergent clade of virulent A. hydrophila from 2009 to 2014 have resulted in the loss of more than 16 million pounds of market-size catfish in Alabama and Mississippi. The virulent A. hydrophila isolates (referred to as VAh in this proposal) have unique biochemical and molecular phylogenetic features. In particular, comparative genomics revealed that the VAh isolates have approximately 55 unique and distinguishable genomic features. In 2013, a regional coalition of scientists at Mississippi State University (Delta Research and Extension Center and College of Veterinary Medicine) and Auburn University worked together and were awarded a cooperative USDA AFRI grant to address the VAh problem. In this previously funded USDA AFRI project, we determined the role of two unique VAh genomic islands in pathogenesis, one encoding myo-inositol utilization pathway, and one encoding O-polysaccharide biosynthesis. We identified unique VAh surface antigens with vaccine potential and developed a candidate live attenuated vaccine. We also used a validated VAh-specific quantitative PCR (qPCR) assay to investigate source and persistence of VAh during outbreaks; results suggested the existence of carrier fish and indicated pond environmental conditions significantly contribute to VAh outbreaks. Using comparative genomics, we recently determined that VAh is a distinct monophyletic clade within the A. hydrophila species, but it has at least two distinct subclades: one predominating in West Alabama and one occurring in the Mississippi Delta. Therefore, important work remains to be done to determine the efficacy of our vaccine approaches for cross-protection against different VAh subclades and to determine specific environmental conditions and reservoirs that predispose VAh outbreaks in catfish production ponds. The long-range goals of our working group are to 1) determine the molecular basis for the hypervirulent phenotype of VAh strains in catfish aquaculture and 2) to develop practical and reliable diagnostic and preventive methods to limit the disease's spread in the catfish aquaculture industry. Our hypothesis is that sufficient antigenic conservation between VAh subclades exists to enable development of an effective cross-protective vaccine; furthermore, there are specific environmental/water quality conditions that predispose VAh outbreaks in commercial catfish ponds. Our specific objectives for this proposal are:Determine antigenic and pathogenic similarity between Alabama and Mississippi VAh strains and efficacy of a live, attenuated vaccine strain prepared from an Alabama VAh isolate. Because there are at least two subclades among VAh strains isolated from diseased farmed catfish in Mississippi and Alabama, it is important to determine whether the vaccine based on the Alabama disease isolate provides protection against the diversity of isolates in the catfish aquaculture industry.Determine cross-protection of VAh surface antigens and ability of a recombinant Edwardsiella ictaluri vaccine strategy to control VAh. In this Aim, we will evaluate a patented E. ictaluri vaccine as a carrier for VAh surface antigens to provide protection against multiple VAh subclades.Identify risk factors and putative predictive indicators of outbreaks of VAh in commercial catfish ponds. In our previous USDA AFRI grant, we determined that VAh is detectable in carrier fish. Furthermore, there appears to be a significant environmental trigger to VAh outbreaks. In this Aim, we will investigate pond and water conditions that predispose VAh outbreaks.

Project Methods
We recently determined that VAh strains isolated from outbreaks in U.S. aquaculture are actually composed of two distinct subclades within the VAh group. VAh outbreaks in West Alabama are caused only by strains in the Alabama subclade. However, VAh outbreaks in the Mississippi Delta are caused by strains in the Mississippi subclade and in the Alabama subclade. We developed a candidate live attenuated VAh vaccine strain as a component of our 2013-2016 USDA AFRI grant that is derived from the Alabama subclade. It has demonstrated efficacy as a live vaccine in tank trials and as an injectable killed vaccine on a commercial catfish operation. Our efforts will focus on determining comparative pathology of Mississippi and Alabama VAh subclades in catfish using experimental immersion challenges. We will also determine antigenic cross-reactivity between the two subclades using enzyme-linked immunosorbent assay, and we will conduct efficacy evaluations of the candidate live attenuated vaccine against both VAh subclades. The feasibility of a single vaccine strain strategy will be evaluated by determining the vaccine candidate's ability to protect against VAh strains from both subclades by oral and injection delivery methods.Vaccination using recombinant proteins is an approach that is being used for protection of fish against A. hydrophila. In our 2013-2016 USDA-AFRI grant, we identified recombinant VAh surface proteins that are effective in protecting catfish against VAh disease. Furthermore, we have shown that a patented attenuated Edwardsiella ictaluri vaccine strain, ESC-NDKL1, is an effective carrier for these antigens using a recombinant plasmid-based expression system. Our efforts will be to construct stable recombinant attenuated Edwardsiella ictaluri with chromosomal integration of genes encoding VAh protective antigens. The recombinant vaccines will then be evaluated in experimental challenges to determine their efficacy against both Mississippi and Alabama subclades of VAh. Our efforts to develop a novel dual vaccine strategy to prevent VAh and enteric septicemia of catfish (ESC) will be evaluated by determining efficacy of the recombinant vaccine to prevent both diseases in catfish.In our 2013-2016 USDA AFRI grant, we detected non-clinical VAh carrier fish, and we determined that VAh is rarely detectable in the environment or fish except during an active outbreak where significant losses have occurred. In addition, there appears to be a significant environmental trigger associated with VAh outbreaks. Therefore, in the current grant our efforts will be to investigate pond and water conditions, as well as other environmental indicators, that putatively predispose ponds to outbreaks. Water, sediment, and fish samples will be collected from ponds with history of VAh outbreaks to determine impact of water quality conditions and plankton on prevalence of VAh in environment and fish. We will also analyze diagnostic VAh case submissions to fish diagnostic laboratories serving the catfish industry to determine prevalence of AL-VAh and MS-VAh subclades and develop an outbreak risk model. Our success in this study will be evaluated by the development of a reliable predictive model that can be practically used to help producers predict and manage VAh outbreaks.

Progress 08/01/17 to 07/31/21

Outputs
Target Audience:The target audience for this project is commercial catfish producers. The goal is to produce new knowledge and preventive methods to reduce prevalence and impact of disease caused by VAh. Another target audience is veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Changes/Problems:Due to state and university safety guidelines during the COVID-19 pandemic, research on this project stopped on March 16, 2020, and research began to restart on July 1, 2020. What opportunities for training and professional development has the project provided?At Mississippi State University, this project resulted in the training of two Ph.D. students (Bradley Richardson and Basant Gomaa) and a postdoctoral scientist (Dr. Hasan Tekedar). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Major activities completed Aim 1. In our previous USDA AFRI grant, a live attenuated vAh vaccine candidate strain (ML09-119 ΔymcA) was developed that is derived from the Alabama vAh subclade. Using intraperitoneal injection, ML09-119 ΔymcA resulted in protection against wild-type ML09-119 (AL subclade) and S14-452 (MS subclade). Relative percent survival (RPS) ranged from 80-100%, and the vaccine performed better against ML09-119 (97-100% RPS) than AL subclade strain S14-452 (80-85% RPS). IP injection of formalin-killed ML09-119 and S14-452 offered similar levels of protection against the live parent strains (96.5 and 87.4% RPS, respectively). Oral vaccination with ML09-119 ΔymcA in feed resulted in only marginal protection against the ML09-119 strain (27% RPS) and no protection against S14-452. There was no protective benefit from feeding fish formalin killed ML09-119 or S14-452. In summary, the live attenuated vaccine ML09-119 ΔymcA offers better protection against the Alabama vAh subclade than the Mississippi vAh subclade, and it offers some protection against the Alabama vAh subclade when delivery orally in feed. Aim 2. Recombinant vAh proteins Fim, FimA, FimMrfG, OmpAI, TonB, and ATPase have potential as vaccine antigens against vAh infection, and live attenuated Edwardsiella ictaluri vaccine strain ESC-NDKL1 is a potentially effective delivery vehicle for vAh antigens. We inserted genes encoding these vAh proteins into the deleted frdA gene site in ESC-NDKL1 using homologous recombination. This resulted in six strains derived from ESC-NDKL1, each with a different antigen-encoding gene inserted in the frdA deletion site. Based on the success of this strategy, we have constructed two additional plasmids, one containing the flanking regions for sdhC and one containing the flanking regions of gcvP. Using these plasmids, twelve ESC-NDKL1 strains expressing two vAh protein antigens were constructed, and eleven ESC-NDKL1 strains expressing three vAh protein antigens were constructed. ESC-NDKL1 strains expressing two vAh antigens showed improved protection over ESC-NDKL1 strains expressing a single vAh antigen. In immersion vaccination trials, four ESC-NDKL1 strains expressing two vAh antigens (ESC-NDKL::Fim/OmpA1, ESC-NDKL1::ATPase/FimMrfG, ESC-NDKL1::Fim/FimMrfG, and ESC-NDKL1::TonB/FimMrfG) provide significantly improved protection compared to ESC-NDKL1. Aim 3. We compared culture swabs taken from catfish gills and lower intestine to assess their efficacy as a rapid, field-based collection technique. Results showed that swabs taken from the lower intestine detected significantly more vAh-infected fish than gill swabs. We also used occupancy models and found that the majority of catfish in commercial aquaculture ponds can be vAh-positive with no clinical signs of disease, suggesting vAh can be present in catfish ponds in the absence of disease. Using field and clinical data from commercial catfish ponds with histories of annual vAh outbreaks, a systems model was built using empirical data and expert elicitation. Four potential disease vector mechanisms for vAh entry into a pond were investigated: infected fingerlings, bird vectors, pond environment (sediment and/or water), and latent infections of asymptomatic fish. Simulations using this model support the hypothesis that pond environment is the likely source of vAh infection, and outbreaks are stress-mediated. Timing of a given vAh outbreak has a significant impact on the resulting economic losses, as do antibiotic use and market demand. A compartmental Susceptible-Latent-Infectious-Removed (SLIR) model showed that scenarios where stocked fingerlings were the reservoir host caused the greatest profit loss, followed by bird vectors, latent infections in resident catfish, and an environmental (pond water) resident hypothesis. The additional cost of using medicated feed was offset by the reduced fish mortality from outbreaks, and medicated feed use increased profits compared to outbreaks not treated with antibiotics. The model shows the importance of evaluating different management strategies and monitoring programs to maintain profitability in the wake of emerging or unpredictable disease outbreaks. We validated a duplex end-point PCR to differentiate between the MS and AL subclades of vAh and tested approximately 200 vAh field isolates provided by three cooperating diagnostic labs in Mississippi and Alabama. In 2013, the majority of all isolates were the AL subclade. In 2015, vAh cases in Alabama were strictly due to the AL subclade; however, Mississippi cases were attributed to both AL and MS subclades. By 2017, 100% of vAh cases in western Mississippi were the MS subclade. A similar trend was shown in cases from eastern Mississippi. The subclade shift suggests increased fitness of the MS subclade in the host and/or environment, which may have important implications for management of vAh. Genomic analysis showed the two subclades are highly clonal across 113 genes investigated, but the presence of a type VI secretion system (T6SS) in the MS subclade is an important distinguishing feature that is not present in the AL subclade. Objectives met Aim 1. Efficacy trials of a live attenuated vaccine strain developed from an Alabama vAh subclade isolate (ML09-119 ΔymcA) are completed. The live attenuated vaccine is efficacious when catfish are vaccinated by injection, but it only provides marginal protection against the Alabama subclade of vAh by oral vaccination, and it does not protect against the Mississippi subclade by oral vaccination. Aim 2. Twenty-nine recombinant live attenuated vaccine candidate strains were constructed by inserting vAh antigen-encoding genes in the chromosome of live attenuated E. ictaluri vaccine ESC-NDKL1. Six express a single vAh antigen, twelve express two vAh antigens, and eleven express three vAh antigens. Vaccination by immersion showed that four of the vaccine candidate strains expressing two vAh antigens show significantly improved protection compared to ESC-NDKL1 Aim 3. Fish and pond samples were analyzed, revealing that vAh is carried by asymptomatic catfish in intestines. However, systems modeling suggests that pond environment, with contribution of host stress, is the likely source of vAh infection. In western and eastern Mississippi, the Mississippi vAh subclade is now isolated more frequently from MAS outbreaks in commercial catfish ponds than the Alabama subclade. ?Significant results achieved Injection vaccination of catfish using a live attenuated vAh vaccine candidate was effective in providing protection against vAh, but oral vaccination was not as effective. Four candidate live attenuated vaccine strains carrying vAh antigens in a live attenuated Edwardsiella ictaluri vaccine show potential as a dual vaccination strategy against MAS and ESC. vAh can be detected (prevalence of about 10%) in catfish from ponds with no clinical vAh disease, but prevalence increases to >90% during an outbreak. Systems modeling suggests pond environment is the likely source of vAh infections in commercial catfish ponds. We discovered that Alabama subclade of vAh has been replaced by Mississippi subclade in western Mississippi aquaculture productions from 2013-2017, and the same trend has now occurred in eastern Mississippi. Key outcomes ?Our results confirm the existence of latent carrier fish in ponds with no apparent clinical vAh; however, pond environment is the most likely source of vAh infections. The aquaculture industry in Mississippi has different vAh subclades in its two primary geographical regions. Since 2013, the Mississippi subclade of vAh has become the predominant subclade in West and East Mississippi, and the Alabama subclade predominates in Alabama. Four recombinant live attenuated E. ictaluri vaccine candidates carrying vAh antigens show potential for providing dual protection against ESC and MAS using immersion vaccination.

Publications

• Type: Journal Articles Status: Published Year Published: 2021 Citation: Richardson BM, Griffin MJ, Colvin ME, Wise DJ, Ware C, Mischke CC, Greenway TE, Byars TS, Hanson LA & Lawrence ML (2021). Using quantitative polymerase chain reaction (qPCR) and occupancy models to estimate atypical Aeromonas hydrophila (aAh) prevalence in catfish. Aquaculture, 530, 735687. https://doi.org/10.1016/j.aquaculture.2020.735687
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Tekedar, H. C., S. Kumru, J. Blom, A. D. Perkins, M. J. Griffin, H. Abdelhamed, A. Karsi, and M. L. Lawrence. 2019. Comparative genomics of Aeromonas veronii: identification of a pathotype impacting aquaculture globally. PLOS One 14(8):e0221018.
• Type: Journal Articles Status: Published Year Published: 2019 Citation: Tekedar, H. C., H. A. Abdelhamed, S. Kumru, J. Blom, A. Karsi, and M. L. Lawrence. 2019. Comparative genomics of Aeromonas hydrophila secretion systems and mutational analysis of tssD and tssI genes from T6SS. Frontiers in Microbiology 9:3216.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2022 Citation: Lawrence, M. L., H. Tekedar, B. Gomaa, H. Abdelhamed, S. Kumru, J. Blom, and A. Karsi. 2022. Use of comparative genomics to understand Aeromonas pathotypes and design a recombinant vaccine strategy. Aquaculture 2022 (World Aquaculture Society), San Diego, CA (abstract accepted).
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2021 Citation: Gomaa, B., H. Tekedar, and M. L. Lawrence. 2021. Developing a dual live attenuated vaccine to prevent motile aeromonas septicemia and enteric septicemia of catfish. Conference of Research Workers in Animal Diseases, Chicago, IL (abstract accepted).
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Richardson, B. M., M. J. Griffin, M. R. Liles, M. L. Lawrence, and D. J. Wise. 2020. Protective effects of prospective vaccine and formalin-killed cultures against atypical Aeromonas hydrophila (aAh) in channel catfish Ictalurus punctatus. Conference of Research Workers in Animal Diseases, virtual
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Richardson, B. M., G. C. Waldbieser, C. Ware, M. R. Liles, L. H. Khoo, P. S. Gaunt, L. A. Hanson, M. L. Lawrence, D. J. Wise and M. J. Griffin. 2020. Genomic analysis of atypical Aeromonas hydrophila (aAh) from catfish aquaculture in the southeastern United States with evidence of haplotype shifts form diagnostic case submissions. AFS-FHS Summer Seminar Series, virtual.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Lawrence, M. L., H. C. Tekedar, A. Karsi, and H. Abdelhamed. 2019. Vaccination against virulent Aeromonas hydrophila (vAh) using vAh surface antigens expressed in a recombinant Edwardsiella ictaluri vaccine. Aquaculture 2019 (World Aquaculture Society), New Orleans, Louisiana.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson, BM, MJ Griffin, CC Mischke, TE Greenway, DJ Wise and ME Colvin. 2019. Modeling atypical Aeromonas hydrophila (aAh) dynamics in catfish aquaculture ponds of the southeastern United States. Conference of Research Workers in Animal Diseases, Chicago, IL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson, BM, MJ Griffin, CC Mischke, TE Greenway, ME Colvin and DJ Wise. 2019. Atypical Aeromonas hydrophila (aAh) dynamics in catfish aquaculture ponds of the southeastern United States. Eastern Fish Health Workshop. Lake Placid, NY.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, Greenway TE. 2019. State boundaries draw hard lines in Aeromonas hydrophila disease strains of farm-raised channel catfish Ictalurus punctatus. Meeting of the Mississippi Chapter of the American Fisheries Society, Jackson, Mississippi
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, Greenway TE. 2018. Spatial and temporal profile of atypical Aeromonas hydrophila in catfish. Mississippi State University 3-Minute Thesis. Mississippi State, Mississippi.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, and Greenway TE. 2018. Evaluating Atypical Aeromonas hydrophila (aAh) in Catfish Aquaculture of the Mississippi Delta Region. 8th International Symposium on Aquatic Animal Health, Charlottetown, Prince Edward Island, Canada.
• Type: Theses/Dissertations Status: Other Year Published: 2020 Citation: Richardson, Bradley. "Improved monitoring and decision-making to manage atypical Aeromonas hydrophila in catfish aquaculture ponds." PhD diss., Department of Wildlife, Fisheries, and Aquaculture, Mississippi State University, 2020.

Progress 08/01/19 to 07/31/20

Outputs
Target Audience:The target audience for this project is commercial catfish producers. The goal is to produce new knowledge and preventive methods to reduce prevalence and impact of disease caused by VAh. Another target audience is veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Changes/Problems:Due to state and university safety guidelines during the COVID-19 pandemic, research on this project stopped on March 16, 2020, and research began to restart on July 1, 2020. What opportunities for training and professional development has the project provided?At Mississippi State University, this project resulted in the training of two Ph.D. students (Bradley Richardson and Basant Gomaa) and a postdoctoral scientist (Dr. Hasan Tekedar). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Infectivity trials evaluating the comparative pathology of Mississippi and Alabama vAh subclades and evaluation of antigenic variation between Mississippi and Alabama vAh subclades will be conducted. Additional vaccine candidates based on ESC-NDKL1 will be constructed that express a combination of three vAh antigens, and all strains will be evaluated. Fish and pond environment samples will continue to be collected along with data on pond conditions to improve a systems model to determine vAh outbreak drivers and continue building a Bayesian model to better predict future outbreaks.

Impacts
What was accomplished under these goals? Aim 1. In our previous USDA AFRI grant, a live attenuated vAh vaccine candidate strain (ML09-119 ΔymcA) was developed that is derived from the Alabama vAh subclade. In year 2 of our current project, we performed two vaccine trials of this candidate, one using vaccine delivery by intraperitoneal (IP) injection and one using an oral delivery method by vaccine/feed admix. By IP injection, using two different doses, the vaccine strain resulted in significant protection against wild-type strain ML09-119 (representing Alabama vAh subclade) and wild-type strain S14-452 (representing Mississippi vAh subclade). Relative percent survival (RPS) ranged from 80-100%, and the vaccine appeared to offer better protection against the parent strain ML09-119 (97-100% RPS) than Mississippi subclade strain S14-452 (80-85% RPS). Oral vaccination also resulted in significant protection against the ML09-119 strain, with ~50% RPS in the first 48 hours post-challenge. However, oral vaccination did not provide protection against challenge with S14-452 strain. In year 3, channel catfish fingerlings were again vaccinated by IP injection and oral delivery of the live vaccine, and they were also vaccinated with formalin killed ML09-119 or S14-452 culture. After 30 days, fish were challenged with IP injections of ML09-119 or S14-452. Our results revealed that oral vaccination of channel catfish provides less protection than IP injection against vAh. However, oral administration of the live attenuated ML09-119 vaccine provided significant protection against the ML09-119 parent strain. Comparably, oral delivery of the live attenuated ML09-119 strain did not protect against S14-452. This suggests the need for haplotype-specific or combinatory vaccines. In summary, the live attenuated vaccine offers better protection against the Alabama vAh subclade than the Mississippi vAh subclade, and we have demonstrated that it offers significant protection against the Alabama vAh subclade when delivery orally in feed. Aim 2. Recombinant vAh proteins Fim, FimMrfG, OmpAI, TonB, and ATPase have potential as vaccine antigens against vAh infection, and live attenuated Edwardsiella ictaluri vaccine strain ESC-NDKL1 is a potentially effective delivery vehicle for vAh antigens. We inserted genes encoding these vAh proteins into the deleted frdA gene site in ESC-NDKL1 using homologous recombination. This resulted in five strains derived from ESC-NDKL1, each with a different antigen-encoding gene inserted in the frdA deletion site (ESC-NDKL1::Fim, ESC-NDKL1::FimMrfG, ESC-NDKL1::OmpAI, ESC-NDKL1::Tdr, and ESC-NDKL1::ATPase). Based on the success of this strategy, we have constructed two additional plasmids, one containing the flanking regions for sdhC and one containing the flanking regions of gcvP. Using these plasmids, five strains expressing two protein antigens have been constructed: ESC-NDKL::Fim/OmpA1, ESC-NDKL1::FimMrfG/OmpA1, ESC-NDKL1::TonB/OmpA1, ESC-NDKL1::ATPase/OmpA1,and ESC-NDKL1::ATPase/TonB. Construction of triple antigen combinations is in progress. Aim 3. We compared culture swabs taken from catfish gills and lower intestine to assess their efficacy as a rapid, field-based collection technique. Results showed that swabs taken from the lower intestine detected significantly more vAh-infected fish than gill swabs. We also used occupancy models and found that the majority of catfish in commercial aquaculture ponds can be vAh-positive with no clinical signs of disease, suggesting vAh is part of the resident flora in healthy catfish. Using field and clinical data from commercial catfish ponds with histories of annual vAh outbreaks, a systems model was built using empirical data and expert elicitation that can be used to test an array of management strategies. Four potential disease vector mechanisms for vAh entry into a pond were investigated: infected fingerlings, bird vectors, pond environment (sediment and/or water), and latent infections of asymptomatic fish. Simulations using this model support the hypothesis that pond environment is the likely source of vAh infection, and outbreaks are stress-mediated. Timing of a given vAh outbreak has a significant impact on the resulting economic losses, as do antibiotic use and market demand. A Bayesian model was derived from on-farm observations using occupancy data, environmental data, disease dynamics data, diagnostic case submissions, and potentially genomics data. This decision network will support employment of best management practices by farmers to reduce number and severity of outbreaks. The end goal is to create a web-based application where farmers can input basic farm data (feeding activity, water quality, stocking rates, fish size/age, feed prices, etc.) to make informed management decisions. Specific objectives met Aim 1. Spatiotemporal trends of Alabama and Mississippi subclades of vAh in west and east Mississippi have been conducted. Efficacy trials of a live, attenuated vaccine strain prepared from an Alabama vAh isolate (ML09-119 ΔymcA) are completed. Infectivity trials evaluating the comparative pathology of Mississippi and Alabama vAh subclades and evaluation of antigenic variation between Mississippi and Alabama vAh subclades are in progress. Aim 2. Ten recombinant live attenuated vaccine candidate strains were constructed by inserting vAh antigen-encoding genes in the chromosome of live attenuated E. ictaluri vaccine ESC-NDKL1. Five express a single vAh antigen, and five express two vAh antigens. Efficacy trials are pending. Aim 3. Fish and pond samples were analyzed, revealing that vAh is carried by asymptomatic catfish in intestines. However, systems modeling suggests that pond environment, with contribution of host stress, is the likely source of vAh infection. Environmental data from production ponds is being evaluated to create a Bayesian model to inform catfish farmers of the risk of a vAh outbreak under specific pond conditions at the farm- and/or pond-level. Significant results achieved We discovered that Alabama subclade of vAh has been replaced by Mississippi subclade in West Mississippi aquaculture productions from 2013-2017, and the same trend has now occurred in East Mississippi (based on 2018 results). Alabama subclade still comprises almost all of the isolates from Alabama. Oral vaccination of catfish using a live attenuated vAh vaccine candidate was effective in providing protection against the Alabama subclade. Ten candidate live attenuated vaccine strains carrying vAh antigens in a live attenuated Edwardsiella ictaluri vaccine were constructed. vAh can be detected (prevalence of about 10%) in catfish from ponds with no clinical vAh disease, but prevalence increases to >90% during an outbreak. Systems modeling suggests pond environment is the likely source of vAh infections in commercial catfish ponds. Key outcomes or other accomplishments realized The aquaculture industry in Mississippi has different vAh subclades in its two primary geographical regions. Since 2013, the Mississippi subclade of vAh has become the predominant subclade in West and East Mississippi, and the Alabama subclade predominates in Alabama. Our results confirm the existence of latent carrier fish in ponds with no apparent clinical vAh; however, pond environment is the most likely source of vAh infections. Oral vaccination of catfish using a live attenuated vAh vaccine candidate provides significant protection against the Alabama subclade.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2020 Citation: Richardson, B. M., M. J. Griffin, M. E. Colvin, D. J. Wise, C. Ware, C. C. Mischke, T. E. Greenway, T. S. Byars, L. A. Hanson, and M. L. Lawrence. 2020. Using quantitative polymerase chain reaction (qPCR) and occupancy models to estimate atypical Aeromonas hydrophila (aAh) prevalence in catfish. Aquaculture, 735687.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Tekedar, H. C., S. Kumru, J. Blom, A. D. Perkins, M. J. Griffin, H. Abdelhamed, A. Karsi, and M. L. Lawrence. 2019. Comparative genomics of Aeromonas veronii: identification of a pathotype impacting aquaculture globally. PLOS One 14(8):e0221018.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Richardson, B. M., M. J. Griffin, M. R. Liles, M. L. Lawrence, and D. J. Wise. 2020. Protective effects of prospective vaccine and formalin-killed cultures against atypical Aeromonas hydrophila (aAh) in channel catfish Ictalurus punctatus. CRWAD 2020, virtual. December 2020.
• Type: Conference Papers and Presentations Status: Other Year Published: 2020 Citation: Richardson, B. M., G. C. Waldbieser, C. Ware, M. R. Liles, L. H. Khoo, P. S. Gaunt, L. A. Hanson, M. L. Lawrence, D. J. Wise and M. J. Griffin. Genomic analysis of atypical Aeromonas hydrophila (aAh) from catfish aquaculture in the southeastern United States with evidence of haplotype shifts form diagnostic case submissions. AFS-FHS Summer Seminar Series. May 15, 2020. Virtual.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Lawrence, M. L., H. C. Tekedar, A. Karsi, and H. Abdelhamed. 2019. Vaccination against virulent Aeromonas hydrophila (vAh) using vAh surface antigens expressed in a recombinant Edwardsiella ictaluri vaccine. Aquaculture 2019 (World Aquaculture Society), New Orleans, Louisiana.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Tekedar, H. C., H. A. Abdelhamed, S. Kumru, J. Blom, A. Karsi, and M. L. Lawrence. 2019. Comparative genomics of Aeromonas hydrophila secretion systems and mutational analysis of tssD and tssI genes from T6SS. Frontiers in Microbiology 9:3216.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson, BM, MJ Griffin, CC Mischke, TE Greenway, DJ Wise and ME Colvin. Modeling atypical Aeromonas hydrophila (aAh) dynamics in catfish aquaculture ponds of the southeastern United States. CRWAD 2019, Chicago, IL. November 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson, BM, MJ Griffin, CC Mischke, TE Greenway, ME Colvin and DJ Wise. Atypical Aeromonas hydrophila (aAh) dynamics in catfish aquaculture ponds of the southeastern United States. Eastern Fish Health Workshop. Lake Placid, NY. April 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, Greenway TE. State boundaries draw hard lines in Aeromonas hydrophila disease strains of farm-raised channel catfish Ictalurus punctatus. Meeting of the Mississippi Chapter of the American Fisheries Society, Jackson, Mississippi. February 2019.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, Greenway TE. Spatial and temporal profile of atypical Aeromonas hydrophila in catfish. Mississippi State University 3-Minute Thesis. Mississippi State, Mississippi. November 2018
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, and Greenway TE. Evaluating Atypical Aeromonas hydrophila (aAh) in Catfish Aquaculture of the Mississippi Delta Region. 8th International Symposium on Aquatic Animal Health, Charlottetown, Prince Edward Island, Canada. September 2018.
• Type: Theses/Dissertations Status: Other Year Published: 2020 Citation: Richardson, Bradley. "Improved monitoring and decision-making to manage atypical Aeromonas hydrophila in catfish aquaculture ponds." PhD diss., Department of Wildlife, Fisheries, and Aquaculture, Mississippi State University, 2020.

Progress 08/01/18 to 07/31/19

Outputs
Target Audience: The target audience for this project is commercial catfish producers. The goal is to produce new knowledge and preventive methods to reduce prevalence and impact of disease caused by VAh. Another target audience is veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? At Mississippi State University, this project resulted in the training of two Ph.D. students (Bradley Richardson and Basant Gomaa) and a postdoctoral scientist (Dr. Hasan Tekedar). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Infectivity trials evaluating the comparative pathology of Mississippi and Alabama vAh subclades and evaluation of antigenic variation between Mississippi and Alabama vAh subclades will be conducted, and efficacy of the live attenuated vaccine candidate using oral delivery will be continued. Four additional vaccine candidates based on ESC-NDKL1 will be constructed and evaluated. Fish and pond environment samples will continue to be collected along with data on pond conditions to improve a systems model to determine vAh outbreak drivers and continue building a Bayesian model to better predict future outbreaks.

Impacts
What was accomplished under these goals? Major activities completed Aim 1. In our previous USDA AFRI grant, a live attenuated vAh vaccine candidate strain (ML09-119 ΔymcA) was developed that is derived from the Alabama vAh subclade. In year 2 of our current project, we performed two vaccine trials of this candidate, one using vaccine delivery by intraperitoneal (IP) injection and one using an oral delivery method by vaccine/feed admix. By IP injection, using two different doses (1x106 colony forming units (CFU)/fish and 1x105 CFU/fish), the vaccine strain resulted in significant protection against wild-type strain ML09-119 (representing Alabama vAh subclade) and wild-type strain S14-452 (representing Mississippi vAh subclade). Relative percent survival (RPS) ranged from 80-100%, and the vaccine appeared to offer better protection against the parent strain ML09-119 (97-100% RPS) than Mississippi subclade strain S14-452 (80-85% RPS) when exposed to 1x106 CFU/fish by IP injection. Oral vaccination also resulted in significant protection against the ML09-119 strain, with ~50% RPS in the first 48 hours post-challenge. However, oral vaccination did not provide protection against challenge with S14-452 strain. In summary, the live attenuated vaccine offers better protection against the Alabama vAh subclade than the Mississippi vAh subclade, and we have demonstrated that it offers significant protection against the Alabama vAh subclade when delivery orally in feed. We validated a duplex end-point PCR to differentiate between Alabama and Mississippi vAh subclades. Using this PCR, we tested approximately 200 vAh field isolates collected from three aquaculture regions: Alabama, East Mississippi, and West Mississippi. Results suggest there is a shift in the predominant genetic strain within catfish aquaculture in Mississippi. In 2013, the majority of isolates were the Alabama subclade. However, in 2018, ~80% of isolates were the Mississippi subclade. We also sequenced the genomes of 25 Alabama subclade isolates and 25 Mississippi subclade isolates that are temporally and geographically discrete for multilocus sequence analysis (MLSA) to identify clonal relationships among these strains. Aim 2. Recombinant vAh proteins Fim, FimMrfG, OmpAI, TonB, and ATPase have potential as vaccine antigens against vAh infection, and live attenuated Edwardsiella ictaluri vaccine strain ESC-NDKL1 is a potentially effective delivery vehicle for vAh antigens. We inserted genes encoding these vAh proteins into the deleted frdA gene site in ESC-NDKL1 using homologous recombination. This resulted in five strains derived from ESC-NDKL1, each with a different antigen-encoding gene inserted in the frdA deletion site (ESC-NDKL1::Fim, ESC-NDKL1::FimMrfG, ESC-NDKL1::OmpAI, ESC-NDKL1::Tdr, and ESC-NDKL1::ATPase). Based on the success of this strategy, we have constructed two additional plasmids, one containing the flanking regions for sdhC and one containing the flanking regions of gcvP. These plasmids will be used to construct ESC-NDKL1 derivatives expressing a combination two or three vAh antigens. Aim 3. We compared culture swabs taken from catfish gills and lower intestine to assess their efficacy as a rapid, field-based collection technique. Results showed that swabs taken from the lower intestine detected significantly more vAh-infected fish than gill swabs. We also used occupancy models and found that the majority of catfish in commercial aquaculture ponds can be vAh-positive with no clinical signs of disease, suggesting vAh is part of the resident flora in healthy catfish. Using field and clinical data from commercial catfish ponds with histories of annual vAh outbreaks, a systems model was built using empirical data and expert elicitation that can be used to test an array of management strategies. Four potential disease vector mechanisms for vAh entry into a pond were investigated: infected fingerlings, bird vectors, pond environment (sediment and/or water), and latent infections of asymptomatic fish. Simulations using this model support the hypothesis that pond environment is the likely source of vAh infection, and outbreaks are stress-mediated. Timing of a given vAh outbreak has a significant impact on the resulting economic losses, as do antibiotic use and market demand. A Bayesian model was derived from on-farm observations using occupancy data, environmental data, disease dynamics data, diagnostic case submissions, and potentially genomics data. This decision network will support employment of best management practices by farmers to reduce number and severity of outbreaks. The end goal is to create a web-based application where farmers can input basic farm data (feeding activity, water quality, stocking rates, fish size/age, feed prices, etc.) to make informed management decisions. Specific objectives met Aim 1. Spatiotemporal trends of Alabama and Mississippi subclades of vAh in west and east Mississippi have been conducted. Infectivity trials evaluating the comparative pathology of Mississippi and Alabama vAh subclades, evaluation of antigenic variation between Mississippi and Alabama vAh subclades, and efficacy of a live, attenuated vaccine strain prepared from an Alabama vAh isolate (ML09-119 ΔymcA) are in progress. Aim 2. Five recombinant live attenuated vaccine candidate strains were constructed by inserting vAh antigen-encoding genes in the chromosome of live attenuated E. ictaluri vaccine ESC-NDKL1 (ESC-NDKL1::Fim, ESC-NDKL1::FimMrfG, ESC-NDKL1::OmpAI, ESC-NDKL1::TonB, and ESC-NDKL1::ATPase). Four additional vaccine candidates are being constructed, and evaluation is pending. Aim 3. Fish and pond samples were analyzed, revealing that vAh is carried by asymptomatic catfish in intestines. However, systems modeling suggests that pond environment, with contribution of host stress, is the likely source of vAh infection. Environmental data from production ponds is being evaluated to create a Bayesian model to inform catfish farmers of the risk of a vAh outbreak under specific pond conditions at the farm- and/or pond-level. Significant results achieved We discovered that Alabama subclade of vAh has been replaced by Mississippi subclade in West Mississippi aquaculture productions from 2013-2017, and the same trend has now occurred in East Mississippi (based on 2018 results). Alabama subclade still comprises almost all of the isolates from Alabama. Oral vaccination of catfish using a live attenuated vAh vaccine candidate was effective in providing protection against the Alabama subclade. Five candidate live attenuated vaccine strains carrying vAh antigens in a live attenuated Edwardsiella ictaluri vaccine were constructed. vAh can be detected (prevalence of about 10%) in catfish from ponds with no clinical vAh disease, but prevalence increases to >90% during an outbreak. Systems modeling suggests pond environment is the likely source of vAh infections in commercial catfish ponds. Key outcomes or other accomplishments realized The aquaculture industry in Mississippi has different vAh subclades in its two primary geographical regions. Since 2013, the Mississippi subclade of vAh has become the predominant subclade in West and East Mississippi, and the Alabama subclade predominates in Alabama. Our results confirm the existence of latent carrier fish in ponds with no apparent clinical vAh; however, pond environment is the most likely source of vAh infections. Oral vaccination of catfish using a live attenuated vAh vaccine candidate provides significant protection against the Alabama subclade.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Tekedar, H. C., S. Kumru, J. Blom, A. D. Perkins, M. J. Griffin, H. Abdelhamed, A. Karsi, and M. L. Lawrence. 2019. Comparative genomics of Aeromonas veronii: identification of a pathotype impacting aquaculture globally. PLOS One 14(8):e0221018.
• Type: Journal Articles Status: Accepted Year Published: 2019 Citation: Tekedar, H. C., H. A. Abdelhamed, S. Kumru, J. Blom, A. Karsi, and M. L. Lawrence. 2019. Comparative genomics of Aeromonas hydrophila secretion systems and mutational analysis of tssD and tssI genes from T6SS. Frontiers in Microbiology 9:3216.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Conference Papers and Presentations Richardson, BM, MJ Griffin, CC Mischke, TE Greenway, DJ Wise and ME Colvin. Modeling atypical Aeromonas hydrophila (aAh) dynamics in catfish aquaculture ponds of the southeastern United States. CRWAD 2019, Chicago, IL.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson, BM, MJ Griffin, CC Mischke, TE Greenway, ME Colvin and DJ Wise. Atypical Aeromonas hydrophila (aAh) dynamics in catfish aquaculture ponds of the southeastern United States. Eastern Fish Health Workshop. Lake Placid, NY.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, Greenway TE. State boundaries draw hard lines in Aeromonas hydrophila disease strains of farm-raised channel catfish Ictalurus punctatus. Meeting of the Mississippi Chapter of the American Fisheries Society, Jackson, Mississippi.
• Type: Conference Papers and Presentations Status: Other Year Published: 2019 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, Greenway TE. Spatial and temporal profile of atypical Aeromonas hydrophila in catfish. Mississippi State University 3-Minute Thesis. Mississippi State, Mississippi.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson BM, Colvin ME, Wise DJ, Griffin MJ, Mischke CC, and Greenway TE. Evaluating Atypical Aeromonas hydrophila (aAh) in Catfish Aquaculture of the Mississippi Delta Region. 8th International Symposium on Aquatic Animal Health, Charlottetown, Prince Edward Island, Canada.

Progress 08/01/17 to 07/31/18

Outputs
Target Audience:The target audience for this project is commercial catfish producers. The goal is to produce new knowledge and preventive methods to reduce prevalence and impact of disease caused by VAh. Another target audience is veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Changes/Problems:Aim 1. None to report. Aim 2.Instead of using a mini-Mu transposon to insert vAh antigen-encoding genes in the ESC-NDKL1 chromosome, we used a homologous recombination strategy to insert the genes in the frdA deletion site of ESC-NDKL1. Aim 3. None to report. What opportunities for training and professional development has the project provided?At Mississippi State University, this project resulted in the training of a Ph.D. student (Bradley Richardson) and a postdoctoral scientist (Dr. Hasan Tekedar). How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Infectivity trials evaluating the comparative pathology of Mississippi and Alabama vAh subclades, evaluation of antigenic variation between Mississippi and Alabama vAh subclades, and efficacy of a live, attenuated vaccine strain prepared from an Alabama vAh isolate (ML09-119 ΔymcA) will be conducted. Four additional vaccine candidates based on ESC-NDKL1 will be constructed and evaluated. Fish and pond environment samples will continue to be collected along with data on pond conditions to build a model to determine vAh outbreak drivers and better predict future outbreaks.

Impacts
What was accomplished under these goals? Major activities completed Aim 1. Over 180 isolates of Aeromonas hydrophila recovered from diagnostic labs in Mississippi, spanning from 2013-2018, were analyzed to determine spatiotemporal dynamics of two predominant vAh subclades: Alabama subclade (represented by strain ML09-119) and Mississippi subclade (represented by strain S14-452). Alabama subclade has rapidly declined in prevalence in west Mississippi (100% of cases in 2013; 20% of cases in 2015; 0% of cases in 2017). Comparatively, Mississippi subclade has increased in prevalence, accounting for ~80% of cases in 2015 and 100% of cases in 2017. Comparably, Alabama subclade comprised 85% of analyzed isolates from east Mississippi collected in 2016, compared to 15% for Mississippi subclade. Additional isolates from subsequent years collected in west Alabama and east Mississippi will be analyzed in years 2 and 3. Aim 2. At the conclusion of our previous USDA AFRI grant, we determined that recombinant proteins Fim, FimMrfG, OmpAI, TonB, and ATPase have potential as vaccine antigens against vAh infection, and live attenuated Edwardsiella ictaluri vaccine strain ESC-NDKL1 is a potentially effective delivery vehicle for vAh antigens. We proposed inserting the genes encoding these antigens into the ESC-NDKL1 chromosome using a mini-Mu transposon, but in our first year of the current project we developed an alternative strategy to insert the antigens into the deleted frdA gene site in ESC-NDKL1 using homologous recombination. We constructed a plasmid with sequences flanking frdA, then we constructed five derivatives of this plasmid, each with a different antigen-encoding gene in the deletion site. This strategy was successful, and we constructed and genetically confirmed five strains derived from ESC-NDKL1, each with a different antigen-encoding gene inserted in the frdA deletion site (ESC-NDKL1::Fim, ESC-NDKL1::FimMrfG, ESC-NDKL1::OmpAI, ESC-NDKL1::TonB, and ESC-NDKL1::ATPase). Based on the success of this strategy, we have constructed two additional plasmids, one containing the flanking regions for sdhC and one containing the flanking regions of gcvP. These plasmids will be used to construct ESC-NDKL1 derivatives expressing a combination two or three vAh antigens. Aim 3. We analyzed over 1200 samples collected from May-September 2018 to investigate vAh prevalence in fish (gill and rectal swabs), water, and sediment samples collected from a catfish farm in west Mississippi with annual outbreaks of vAh. vAh appears to have approximately 10% prevalence in catfish intestines during latent periods (no signs of disease). Once an outbreak is triggered (cause still unknown), prevalence rapidly increases to 90+% during active outbreak periods. We are currently building a model to determine vAh outbreak drivers and better predict future outbreaks. This model encompasses water quality, phytoplankton, zooplankton, and a range of management characteristics (stocking rates, species, co-stocking, etc.). High variation in pond-level variables and timing of sampling (compared to outbreak time) has made it difficult to detect key patterns in analyses, but there appears to be an environmental or immunosuppression trigger and/or possibly a threshold prevalence within the population. We are using case history data from cooperating aquaculture facilities and found that the odds of a pond having a vAh outbreak is 6.65 (95% CI: 1.94 - 22.79) times greater in ponds with a vAh outbreak the previous year compared to ponds with no outbreak in the previous year. In previous-year outbreak ponds, 85% of current outbreaks are attributable to the outbreak in the previous year. We are collecting real-time water temperature and light penetration, continuously, in specific ponds to help model biotic and abiotic processes within the ponds. This will help to evaluate pond conditions immediately preceding and during an active vAh outbreak. Catfish serum has been collected to evaluate vAh antibody titers in catfish from both outbreak and healthy ponds. At present it is unknown if catfish carriers possess antibodies that may protect against subsequent outbreaks, or if populations of catfish are truly naïve until outbreaks occur. Specific objectives met Aim 1. Spatiotemporal trends of Alabama and Mississippi subclades of vAh in west and east Mississippi have been conducted and are ongoing. Infectivity trials evaluating the comparative pathology of Mississippi and Alabama vAh subclades, evaluation of antigenic variation between Mississippi and Alabama vAh subclades, and efficacy of a live, attenuated vaccine strain prepared from an Alabama vAh isolate (ML09-119 ΔymcA) are in progress. Aim 2. Five recombinant live attenuated vaccine candidate strains were constructed by inserting vAh antigen-encoding genes in the chromosome of live attenuated E. ictaluri vaccine ESC-NDKL1 (ESC-NDKL1::Fim, ESC-NDKL1::FimMrfG, ESC-NDKL1::OmpAI, ESC-NDKL1::TonB, and ESC-NDKL1::ATPase). Four additional vaccine candidates are being constructed, and evaluation is pending. Aim 3. Fish and pond samples collected from May-September 2018 were analyzed, revealing that vAh has approximately 10% prevalence in catfish intestines during latent periods and >90% prevalence during active outbreak periods. Environmental data from production ponds is being collected and evaluated to create a Bayesian Belief Network to inform a risk model for catfish farmers that will use their specific pond conditions to determine risk of having a vAh outbreak in a given year at the farm- and/or pond-level. Significant results achieved We discovered that Alabama subclade of vAh has been replaced by Mississippi subclade in west Mississippi aquaculture productions from 2013-2017, while Alabama subclade comprises almost all of the isolates from east Mississippi collected in 2016. Five candidate live attenuated vaccine strains carrying vAh antigens in a live attenuated Edwardsiella ictaluri vaccine were constructed. We discovered that the odds of a pond having a vAh outbreak is 6.65 times greater in ponds with a vAh outbreak the previous year compared to ponds with no outbreak in the previous year. Furthermore, vAh can be detected (prevalence of about 10%) in catfish from ponds with no clinical vAh disease, but prevalence increases to >90% during an outbreak. ?Key outcomes or other accomplishments realized The aquaculture industry in Mississippi has different vAh subclades in its two primary geographical regions. Since 2013, the Mississippi subclade of vAh has become the predominant subclade in west Mississippi, and the Alabama subclade predominates in east Mississippi. Our results confirm the existence of latent carrier fish (about 10% prevalence) in ponds with no apparent clinical vAh. Ponds that have a history of vAh outbreaks in previous years have a significantly higher risk of experiencing outbreaks than ponds that have no prior history of vAh.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Kumru, S., H. Tekedar, M. J. Griffin, G. C. Waldbieser, M. J. Liles, T. Sonstegard, S. G. Schroeder, M. L. Lawrence, and A. Karsi. 2018. Draft Genome Sequence of Fish Pathogen Aeromonas bestiarum GA97-22. Genome Announcements 6(24). pii: e00524-18.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Abdelhamed, H., I. Ibrahim, S. Nho, M. M. Banes, R. W. Wills, A. Karsi, and M. L. Lawrence. 2017. Evaluation of three recombinant outer membrane proteins, OmpA1, Tdr, and TbpA, as potential vaccine antigens against virulent Aeromonas hydrophila infection in channel catfish (Ictalurus punctatus). Fish & Shellfish Immunology 66:480-486.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Tekedar, H. C., S. Kumru, J. Blom, H. Abdelhamed, A. Karsi, and M. L. Lawrence. 2018. Comparative genome analysis of the Aeromonas veronii strain ML09-123. ASM Microbe, Atlanta, Georgia.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Tekedar, H. C., S. Kalindamar, H. Abdelhamed, A. Karsi, and M. L. Lawrence. 2018. The role of selected secreted proteins and regulatory Hfq proteins in pathogenesis of virulent A. hydrophila. ASM Microbe, Atlanta, Georgia.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Tekedar, H. C., J. Blom, and M. L. Lawrence. 2018. Comparative mobilome analysis of Aeromonas hydrophila strains. MidSouth Computational Biology and Bioinformatics Society Conference, Starkville, Mississippi.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Abdelhamed, H., A. Karsi, and M. L. Lawrence. 2017. Aeromonas hydrophila Atpase protein stimulates protective immunity in catfish. American Fisheries Society Fish Health Section Annual Meeting, East Lansing, MI, USA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson, B. M., M. E. Colvin, D. J. Wise, M. J. Griffin, C. C. Mischke, and T. E. Greenway. 2018. Occupancy modeling to assess atypical Aeromonas hydrophila (aAh) prevalence in farm-raised catfish. 44th Annual Meeting of the Mississippi Chapter of the American Fisheries Society, Oxford, Mississippi.
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson, B. M., M. E. Colvin, D. J. Wise, M. J. Griffin, C. C. Mischke, and T. E. Greenway. 2018. Using occupancy models to investigate atypical Aeromonas hydrophila (aAh) prevalence in cultured catfish. Mississippi State University Graduate Student Research Symposium, Starkville, Mississippi. (1st Place - Doctoral Student Division)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson, B. M., M. E. Colvin, D. J. Wise, M. J. Griffin, C. C. Mischke, and T. E. Greenway. 2018. Status of atypical Aeromonas hydrophila (aAh) in Mississippi, Atlantic City, New Jersey. (Best Student Abstract Travel Award - AFS Fish Culture Section)
• Type: Conference Papers and Presentations Status: Other Year Published: 2018 Citation: Richardson, B. M., M. E. Colvin, D. J. Wise, M. J. Griffin, C. C. Mischke, and T. E. Greenway. 2018. Evaluating atypical Aeromonas hydrophila (aAh) in catfish aquaculture of the Mississippi Delta region. 8th International Symposium on Aquatic Animal Health, Prince Edward Island, Canada.