GENOMICS, VIRULENCE, AND DETECTION OF EMERGING PATHOGENIC AEROMONAS HYDROPHILA IN CHANNEL CATFISH

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: AFRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 1001248
• Grant No.: 2013-67015-21313
• Project No.: MISV- 081560
• Proposal No.: 2013-01070
• Program Code: A1221
• Project Start Date: Sep 1, 2013
• Project End Date: Aug 31, 2017
• Grant Year: 2013

Project Director
Lawrence, M.

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

Performing Department
College of Vet Med

Non Technical Summary
In 2009-2012, catfish producers lost more than 8 million pounds of market size catfish due to A. hydrophila epidemics. A multistate group of fish health specialists cooperated to determine that the outbreaks were caused by epidemic A. hydrophila isolates that have a unique biochemical and molecular phylogenetic profile. Genome sequencing and comparative genomics determined that epidemic A. hydrophila isolates have 54 unique genetic features that distinguish them from other A. hydrophila isolates. These features include unique biochemical pathways and genes that encode a unique polysaccharide envelope. Our multistate group has cooperated in designing and validating a quantitative PCR-based genetic method using these unique epidemic A. hydrophila sequences. Our hypothesis is that unique genes in epidemic A. hydrophila isolates enable these strains to be a primary pathogen for channel catfish; furthermore, these epidemic isolates are able to persist in the catfish pond environment and cause outbreaks under permissive host/environment conditions. In this project, we will determine roles of selected epidemic-associated genes in disease caused by epidemic A. hydrophila in channel catfish. Furthermore, we will determine efficacy of live attenuated and recombinant vaccine strategies to control epidemic A. hydrophila, and we will utilize a validated quantitative PCR assay for detection and quantification of epidemic A. hydrophila in catfish tissues and pond environments.

Animal Health Component

100%

Research Effort Categories

Basic

50%

Applied

50%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
311	0810	1100	100%

Knowledge Area
311 - Animal Diseases;

Subject Of Investigation
0810 - Finfish;

Field Of Science
1100 - Bacteriology;

Keywords

aeromonas hydrophila

channel catfish

transmission

vaccine

virulence

Goals / Objectives
In 2009-2012, catfish producers in Alabama and Mississippi lost more than 8 million pounds of market size (or slightly smaller) catfish due to Aeromonas hydrophila epidemics. Investigation of the outbreaks revealed that the epidemic A. hydrophila isolates had a unique biochemical and molecular phylogenetic profile. Comparative genomics analysis determined that epidemic A. hydrophila isolates have approximately 54 unique genomic features that distinguish them from historical A. hydrophila. Our group has validated a quantitative PCR (qPCR) assay based on unique sequences in epidemic isolates, completed and annotated the genome of epidemic isolate ML09-119, and developed mutagenesis methods for epidemic isolates. However, important work remains to be done to determine the roles of epidemic-associated genes in disease pathogenesis, develop effective control measures for epidemic A. hydrophila, and determine the environmental sources and conditions for epidemic A. hydrophila outbreaks in catfish production ponds. Our specific objectives for this project are: 1) Determine roles of selected epidemic-associated genes in pathogenesis of A. hydrophila septicemia in channel catfish. Epidemic A. hydrophila strains have unique genetic regions compared to RAh, and the goal of this aim will be to determine the role of some of these regions in EAh virulence. 2) Determine efficacy of live attenuated and recombinant vaccine strategies to control epidemic A. hydrophila. There is need for methods of preventing epidemic A. hydrophila in channel catfish. In this Aim, we will determine whether surface-expressed and extracellular proteins unique to epidemic A. hydrophila have potential for stimulating protective immunity as well as evaluate the potential for attenuated deletion mutants to serve as live, attenuated vaccine strains. 3) Utilize a validated qPCR assay for detection and quantification of the epidemic A. hydrophila strain in catfish tissues and pond environments. It is critical to determine the reservoir of epidemic A. hydrophila infection and how it is spread. With a specific, quantitative PCR method now available, we are positioned to address these questions.

Project Methods
There are multiple regions within the genomes of epidemic A. hydrophila strains that are consistently associated with their genomes but never found within the genomes of other A. hydrophila strains. These regions are implicated in the virulence of epidemic A. hydrophila due to their similarity with genes from other pathogens and/or their association with prophage elements. Therefore, we will generate targeted deletions in specific genetic loci of epidemic A. hydrophila. We will target three specific genetic regions for deletion: 1) the myo-inositol utilization pathway, 2) the epidemic A. hydrophila O-antigen biosynthesis region, and 3) epidemic A. hydrophila-associated prophage. We will then determine the contribution of specific genes to A. hydrophila virulence using fish challenges by bath immersion. Catfish producers are in need of an effective preventive method against epidemic A. hydrophila. Based on comparison between proteins encoded by epidemic A. hydorphila and other A. hydrophila isolates, several outer membrane proteins and secreted proteins are unique to the epidemic strains. Selected outer membrane or secreted proteins from epidemic A. hydrophila will be amplified by PCR and cloned into an expression vector. Vaccine efficacy of recombinant proteins will be determined in channel catfish. We will also determine the efficacy of attenuated deletion mutants as live attenuated vaccines. It is possible that a combination approach of live attenuated vaccine with recombinant protein may improve efficacy further. Finally, we will utilize a recently developed epidemic A. hydrophila-specific quantitative PCR assay to investigate the prevalence and bacterial burden of epidemic A. hyrophila in fish tissues and environmental samples collected from ponds during outbreaks, after outbreaks, and in ponds where outbreaks have never been detected. In addition, the dissemination of epidemic A. hydrophila and bacterial loads of epidemic A. hydrophila in catfish tissues over the course of infection will be determined in controlled disease challenges. To evaluate vaccine efficacy, mean percent mortalities for each treatment will be compared to non-vaccinated treatment by ANOVA for a randomized complete block design. If an effective vaccine strategy is identified, our effort will focus on partnering with a commercial vaccine company to develop and disseminate the technology to catfish producers as quickly as possible. Efficacy of the vaccine will be evaluated by determining the reduction of epidemic A. hydrophila in the catfish industry. Quantitative PCR data collected from commercial ponds (water, sediment, fish) will offer insight into the conditions that predispose ponds to an outbreak, in addition to determining the prevalence of epidemic A. hydrophila in different pond environments. In addition, data collected from challenges will identify the rate of dissemination, target organs, and rate of clearance in lethal and sub-lethal doses, providing information essential to pathogenesis of epidemic A. hydrophila in channel catfish. To allow changes in catfish production practices that will reduce transmission of epidemic A. hydrophila in the catfish industry, our effort will be to distribute information on epidemiology and transmission of epidemic A. hydrophila directly to producers through regularly held industry meetings. Information will also be distributed through extension and fish health diagnosticians/veterinarians that work directly with catfish producers. Impact of our efforts will be evaluated by the implementation of recommended practices and prevention of transmission of epidemic A. hydrophila in the industry.

Progress 09/01/13 to 08/31/17

Outputs
Target Audience:The target audience for this project is commercial catfish producers. Progress on vaccine development targets this audience and aims to reduce prevalence and impact of disease caused by virulent A. hydrophila. Other target audiences are veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Progress on generating new information related to disease pathogenesis and bacterial quantity/persistence in pond environment is primarily directed to this audience. Changes/Problems:Aim 1. We did not target the AH4 prophage genomic region given the lack of observation of the AH4 prophage and its associated genes among different virulent strains isolated from diseased fish in Alabama and Mississippi. Aim 2. None to report. Aim 3. After the first sampling season, we streamlined field sampling to exclude benthic oligochaetes because they do not appear to be a reservoir for the bacteria. What opportunities for training and professional development has the project provided?At Mississippi State University, this project resulted in the training of a PhD student (Jordan Smink) and a postdoctoral scientist (Dr. Hossam Abdelhamed). Graduate students (Graham Rosser, Neely Alberson, and Stephen Reichley) and veterinary interns (Madeliene Hendrix and Irene Yen) assisted in field sampling and were trained in fish anesthesia, blood collection, gill and rectal swabs, snag sampling, data recording, and general field sampling safety. At Auburn University, this project resulted in training for one postdoctoral scientist (Dr. M. Jahangir Hossain) and four PhD students (Charles Thurlow, Dawei Sun, Priscilla Barger, and Cody Rasmussen-Ivey). Dr. Hossain took a postdoctoral position at Johns Hopkins University. Two visiting scholars, Dr. Chao Ran (China) and Dr. Mohamed El-hady (Egypt) contributed to this project in the 2015-2016 time period. Four undergraduate students received training on this project at Auburn University (Shannon Wrenn, Alyson Childers, Laura Alexander [a NSF REU student from the University of Georgia] and Sara Odom [a NSF REU student from Auburn University]). How have the results been disseminated to communities of interest?Results of the 2015 pond studies and plans for future studies have been disseminated to the Alabama catfish industry through the Alabama Fish Farming Newsletter, prepared by Bill Hemstreet. Descriptive field sampling results and the potential existence of a carrier state in resident fish populations was presented at the Catfish Farmers of Arkansas annual meeting in Hot Springs, Arkansas in January 2016. 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. We made deletion mutants in five genes encoding predicted myo-inositol catabolism proteins and found that inability to use myo-inositol as a carbon source did not result in reduced virulence. However, mutation of a regulatory gene in the myo-inositol utilization locus (iolR) resulted in reduced virulence. We conducted a transcriptome analysis and identified putatively IolR-regulated genes, including a flagellin that is hypothesized to enhance Type IV pili expression. Thus, we have identified inositol as an environmental signal that induces virulence gene expression in vAh. We generated markerless in-frame deletions in multiple genes involved in O-antigen biosynthesis. Each of the genes in the ymcABC operon significantly contributed to A. hydrophila virulence. A ΔymcA mutant in particular was highly attenuated; no fish mortalities resulted from IP injection of this mutant. Moreover, a strong antibody response was elicited in vaccinated fish. To evaluate vaccine efficacy of the ΔymcA mutant, catfish fingerlings were IP injected with a formalin-inactivated ΔymcA mutant and stocked into floating in-pond raceways in catfish production ponds. Vaccinated fish showed a statistically significant increase in survival (99.6%) relative to control fish (94.4%) (P < 0.0001). The most significant protection in catfish fingerlings was provided by formalin-inactivated ΔymcA mutant by oral delivery. Our third molecular target was the AH4 prophage present within the A. hydrophila ML09-119 genome. Subsequent comparative genomics analysis indicated that the AH4 prophage is not universally conserved among all vAh strains, and we therefore did not move forward in evaluating the role of the AH4 prophage-associated genes in virulence. Aim 2. We cloned, expressed, and purified eight recombinant vAh proteins and assessed their ability to stimulate protective immunity in channel catfish fingerlings. Vaccination by IP injection with Fim, FimMrfG, ATPase, OmpA1, and TonB generated significant protection against vAh by immersion (relative percent survival of 95.41%, 85.72%, 89.16%, 98.59%, and 95.59%, respectively). A significant antibody response was generated in fish vaccinated with Fim, OmpA1, and TonB. Plasmids expressing three recombinant vAh proteins (Fim, FimMrfG, and ATPase) were transformed into a patented live attenuated E. ictaluri vaccine strain (ESC-NDKL1). Catfish were vaccinated by immersion exposure with 107 CFU/ml of recombinant ESC-NDKL1 for 1 h. At 21 days post-vaccination, catfish immunized with NDKL1::pETfim, ESC-NDKL1::pETmrfG, and ESC-NDKL1::pETATPase had significantly (p < 0.05) lower mortalities than sham-vaccinated fish. In summary, recombinant proteins Fim, FimMrfG, OmpAI, TonB, and ATPase have potential as vaccine antigens against vAh infection, and ESC-NDKL1 is a potentially effective delivery vehicle for vAh antigens. Aim 3. During the 2014 A. hydrophila season (June to September), water, sediment, benthic oligochaetes, zooplankton, and fish samples were periodically collected from two different commercial catfish farms. Several fish from ponds with a history of vAh were PCR positive for the bacteria from gills and anal swabs, but no fish showed outward signs of disease. This suggests the existence of a carrier state. In ponds where samples were collected during an active outbreak, vAh was detected in water, mud, and fish. However, once outbreaks subsided, vAh disappeared quickly from the system. Within 6 weeks, all sampled fish were PCR negative for vAh, and only trace amounts of vAh were detected in pond water. Ten weeks after a disease outbreak, bacteria could not be detected in pond water or pond muds. vAh was not found in benthic invertebrates. During 2015, an improved vAh qPCR assay was validated that includes an internal control to test for potential inhibition and reduce the incidence of false negatives. In summer 2015, we collected water, mud, and zooplankton, in addition to gill swabs, rectal swabs, and blood from 9 fish/pond at regular intervals from 12 ponds on two operations (six with a history of vAh and six without). qPCR results were incorporated with production data to build a system dynamics model to identify variables associated with disease outbreaks. In summer 2016, bi-monthly pond water samples and fish were collected from 22 ponds on a single operation in the Mississippi Delta with a history of vAh. Sampling consisted of blood, gill, and rectal swabs from 9 snagged fish/pond. Pond water was collected for vAh qPCR as well as zooplankton, phytoplankton, and water quality analysis. qPCR data suggests the existence of a carrier state in the resident fish population and an unidentified environmental trigger resulting in a compromised fish population leading to disease outbreaks. This data was incorporated into the system dynamics model to identify factors present in early summer that may be associated with outbreaks that occur in late summer. Specific objectives met Aim 1. We constructed deletion mutations in genes encoding myo-inositol catabolism proteins and O antigen biosynthesis proteins, and the mutants were evaluated for virulence in catfish. A vaccine candidate (ΔymcA mutant) was constructed and evaluated for efficacy. Orally delivered vaccine was used in a west Alabama production pond trial for efficacy evaluation. Aim 2. Eight vAh-specific proteins were cloned, purified, and evaluated in catfish fingerlings; five had good protective efficacy. Three were expressed in an E. ictaluri live attenuated vaccine carrier and demonstrated efficacy by immersion delivery. Aim 3. Sampling and quantitative PCR methods were established, and field sampling was completed. DNA isolation/qPCR analysis was completed for all field samples. Data was incorporated into a system dynamics model that is being used to identify risk factors associated with vAh and inform management decisions. Significant results achieved We discovered that mutations of the iolR gene (in the myo-inositol catabolism locus) in vAh cause attenuation of virulence. In the O-antigen capsule assembly locus, deletion of the ymcA gene causes complete attenuation of vAh. A formalin-killed ΔymcA mutant strain demonstrates efficacy in protecting catfish against vAh. We detected the existence of two vAh subclades based on genome sequencing, one that predominates in the Mississippi Delta and one that is in west Alabama. vAh strains from both subclades had no significant difference in virulence. A PCR method for differentiating the Alabama subclade and the Mississippi subclade was developed. We discovered that Fim, FimMrfG, ATPase, OmpAI, and TonB recombinant proteins produced significant protection in catfish and have potential for vaccine development against vAh. Immersion vaccination of catfish using a live attenuated E. ictaluri vaccine carrying these antigens showed efficacy in protecting against vAh. Field sampling identified that non-lethal collection of gill and rectal swabs is a better indicator of carrier state in catfish than lethal sampling and kidney culture. We identified the putative existence of a carrier state in fish that survive disease outbreaks without antibiotic intervention. After an outbreak subsides, vAh does not persist in the environment (or it persists at levels below our detectable limits). Collected data was incorporated into a systems dynamics model that is being used to identity risk factors associated with disease outbreaks. Key outcomes or other accomplishments realized A ymcA deletion mutation of vAh shows potential as a vaccine. The vaccine strain is being evaluated in commercial catfish production ponds that have a history of vAh outbreaks. Five recombinant vAh proteins (Fim, FimMrfG, ATPase, OmpA1, and TonB) show potential for vaccine development against vAh, and attenuated E. ictaluri serves as an effective vaccine carrier.

Publications

• 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: Journal Articles Status: Published Year Published: 2017 Citation: Tekedar, H. C., S. Kumru, S. Kalindamar, A. Karsi, G. C. Waldbieser, T. Sonstegard, S. G. Schroeder, M. R. Liles, M. J. Griffin, and M. L. Lawrence. 2017. Draft genome sequences of three Aeromonas hydrophila isolates from catfish and tilapia. Genome Announcements 5(3). pii: e01509-16.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Abdelhamed, H., S.W. Nho, G. Turaga, M. M. Banes, A. Karsi, and M. L. Lawrence. 2016. Protective efficacy of four recombinant fimbrial proteins of virulent Aeromonas hydrophila strain ML09-119 in channel catfish. Veterinary Microbiology 197:8-14.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Tekedar, H. C., S. Kumru, A. Karsi, G. C. Waldbieser, T. Sonstegard, S. G. Schroeder, M. R. Liles, M. J. Griffin, M. L. Lawrence. 2016. Draft genome sequences of four virulent Aeromonas hydrophila strains from catfish aquaculture. Genome Announcements 4(4):e00860-16.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Rasmussen-Ivey, C., Figueras, M.J., McGarey, D. and Liles, M.R. Virulence Factors of Aeromonas hydrophila in the Wake of Reclassification. Frontiers in Microbiology, 7:1337. http://dx.doi.org/10.3389/fmicb.2016.01337
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Tekedar HC, Kumru S, Karsi A, Waldbieser GC, Sonstegard T, Schroeder SG, Liles M, Griffin MJ, Lawrence ML. 2016. Draft genome sequence of Aeromonas hydrophila TN97-08. Genome Announcements 4(3): e00436-16
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hossain, M.J., Thurlow, C.M., Sun, D., Nasrin, S., and Liles, M.R. 2015. Genome modifications and cloning using a conjugally transferable recombineering system. Biotechnology Reports, doi:10.1016/j.btre.2015.08.005.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Tekedar, H. C., A. Karsi, A. Akgul, S. Kalindamar, G. C. Waldbieser, T. Sonstegard, S. G. Schroeder, and M. L. Lawrence. 2015. Complete genome sequence of fish pathogen Aeromonas hydrophila AL06-06. Genome Announcements 3(2) e00368-15.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Hossain, M.J., Sun, D., McGarey, D.J., Wrenn, S., Alexander, L.M., Martino, M.E., Xing, Y., Terhune, J.S., and Liles, M.R. 2014. An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish. mBio 5(3):e00848-14.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Tekedar, H.C., G. C. Waldbieser, A. Karsi, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegard, M. Hossain, S. G. Schroeder, L. Khoo, and M. L. Lawrence. 2013. Complete genome sequence of a channel catfish epidemic isolate, Aeromonas hydrophila strain ML09-119. Genome Announcements 1(5):e00755-13.
• 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: 2017 Citation: Tekedar, H. C., H. Abdelhamed, S. Kumru, J. Blom, A. Karsi, and M. L. Lawrence. 2017. Comparative genomics and mutational analysis of tssd and tssi genes in epidemic Aeromonas hydrophila isolate. ASM Microbe, New Orleans, Louisiana.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: Abdelhamed, H., I. Ibrahim, S. Nho, M. M. Banes, R. Wills, A. Karsi, and M. L. Lawrence. 2017. Protective efficacy of three recombinant outer membrane proteins against virulent Aeromonas hydrophila infection in channel catfish. ASM Microbe, New Orleans, Louisiana.
• Type: Conference Papers and Presentations Status: Other Year Published: 2017 Citation: C. M. Thurlow, M. J. Hossain, D. Sun, L. Foshee, C. Heiss, P. Azadi, J. C. Newton, J. S. Terhune and M. R. Liles. 2017. An O-Antigen Capsule Assembly Pathway is Involved in the Virulence of Aeromonas hydrophila in Channel Catfish. American Society for Microbiology international meeting, New Orleans, LA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Tekedar, H. C., S. Kumru, A. Karsi, and M. L. Lawrence. 2016. Extensive comparative genomics analysis of epidemic and reference fish isolates of Aeromonas hydrophila strains. ASM Microbe, Boston, Massachusetts.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Tekedar, H. C., S. Kalindamar, A. Karsi, and M. L. Lawrence. 2016. Comparative reverse vaccinology analysis of Aeromonas hydrophila ML09-119 genome. MCBIOS, Memphis, Tennessee.
• Type: Conference Papers and Presentations Status: Other Year Published: 2016 Citation: Tekedar, H. C., S. Kalindamar, A. Karsi, and M. L. Lawrence. 2016. Comparative genomics analysis of Aeromonas hydrophila strains. MCBIOS, Memphis, Tennessee.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Griffin, M., C. Ware, C. Mischke, L. Hanson, T. Greenway, T. Byars and D. Wise. 2015. Biotic and abiotic factors associated with outbreaks of an emergent strain of Aeromonas hydrophila in catfish aquaculture. 40th Eastern Fish Health Workshop. Charleston, SC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: SC. Thurlow, C.M., Hossain, M.J., Sun, D., Terhune, J.S., and Liles, M.R. 2015. An attenuated Aeromonas hydrophila mutant as a vaccine candidate for Motile Aeromonas Septicemia in Channel Catfish (Ictalurus punctatus). American Society for Microbiology meeting, New Orleans, LA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Tekedar, H. C., A. Karsi, and M. L. Lawrence. 2015. Comparative genomics analysis of fish pathogen Aeromonas hydrophila strain ML09-119. 115th General Meeting of the American Society for Microbiology, New Orleans, Louisiana.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Barger, P., Newton, J., and Liles, M.R. 2015. Differential Production and Secretion of Potentially Toxigenic ECPs from an Epidemic Strain of Aeromonas hydrophila. American Society for Microbiology meeting, New Orleans, LA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Liles, M. R., M. J. Hossain, D. Sun, D. J. McGarey, L. M. Alexander, S. Wrenn, M. E. Martino, Y. Xing, and J. S. Terhune. 2014. Identification of an Asian origin and toxic extracellular products of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish. 11th International Symposium on Aeromonas and Plesiomonas. Montpellier, France.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Tekedar, H. C., G. C. Waldbieser, A. Karsi, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegard, M. Hossain, S. G. Schroeder, L. Khoo, and M. L. Lawrence. 2014. Comparison of the genome sequence of channel catfish epidemic isolate Aeromonas hydrophila ML09-119 to the genome of strain ATCC 7966T. 11th International Symposium on Aeromonas and Plesiomonas. Montpellier, France.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Hossain, M. J., D. Sun, S. Wrenn, L. Alexander, C. Heiss, P. Azadi, J. S. Terhune, and M. R. Liles. 2014. Structural elucidation and virulence of a novel O-antigen of Aeromonas hydrophila isolates from a disease epidemic in catfish. General Meeting of the American Society for Microbiology, Boston, MA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Tekedar, H. C., A. Karsi, G. C. Waldbieser, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegard, M. Hossain, S. G. Schroeder, L. Khoo, and M. L. Lawrence. 2014. Complete genome sequence of Aeromonas hydrophila ML09-119. Seventh International Symposium on Aquatic Animal Health, Portland, Oregon.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Abdelhamed, H., A. Karsi, and M. L. Lawrence. 2014. Expression and purification of recombinant outer membrane proteins and secreted proteins of Aeromonas hydrophila strain ML09-119. Seventh International Symposium on Aquatic Animal Health, Portland, Oregon.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Alexander, L., M. J. Hossain, and M. R. Liles. 2014. Identification of a novel O-antigen and its role in the virulence of Aeromonas hydrophila isolated from diseased catfish. Center for Undergraduate Research Opportunities symposium, Athens, GA.

Progress 09/01/15 to 08/31/16

Outputs
Target Audience:The target audience for this project is commercial catfish producers. Progress on vaccine development targets this audience and aims to reduce prevalence and impact of disease caused by virulent A. hydrophila. Other target audiences are veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Progress on generating new information related to disease pathogenesis and bacterial quantity/persistence in pond environment is primarily directed to this audience. Changes/Problems:Aim 1. We are no longer targeting the AH4 prophage genomic region given the lack of observation of the AH4 prophage and its associated genes among different virulent strains isolated from diseased fish in Alabama and Mississippi. Aim 2. None to report. Aim 3. We have streamlined field sampling to exclude benthic oligochaetes because they do not appear to be a reservoir for the bacteria. We are evaluating a bath immersion protocol developed at the USDA ARS (Dr. Craig Shoemaker, personal communication) in which fingerling catfish have their adipose fin clipped while fish are immersed in a suspension of virulent A. hydrophila. We will compare this method to our current six hour immersion protocol to determine which method results in more consistent mortalities. What opportunities for training and professional development has the project provided?At Mississippi State University, this project is resulting in the training of a DVM-PhD student (Jordan Smink), and a postdoctoral scientist, (Dr. Hossam Abdelhamed). At Auburn University, this project is resulting in training for one postdoctoral scientist (Dr. M. Jahangir Hossain) and four PhD students (Charles Thurlow, Dawei Sun, Priscilla Barger, and Cody Rasmussen-Ivey). Dr. Hossain has now taken a postdoctoral position at Johns Hopkins University. Two visiting scholars, Dr. Chao Ran (China) and Dr. Mohamed El-hady (Egypt) contributed to this project in the 2015-2016 time period. Four undergraduate students have also received training on this project at Auburn University (Shannon Wrenn, Alyson Childers, Laura Alexander [a NSF REU student from the University of Georgia] and Sara Odom [a NSF REU student from Auburn University]). How have the results been disseminated to communities of interest?Results of the 2015 pond studies and plans for future studies have been dessiminated to the Alabama catfuish industry through the Alabama Fish Farming Newsletter, prepared by Bill Hemstreet. What do you plan to do during the next reporting period to accomplish the goals?Aim 1. Now that we have generated a plasmid (pIolR) that complements the iolR mutant, we will compare virulence of the ΔiolR, ΔiolR (pIolR) and PrpoD:iolR mutants relative to wild-type A. hydrophila in controlled aquarium challenges of channel catfish via IP injection. In addition to assessing virulence, we will also conduct a transcriptome study of these mutants relative to wild-type to identify putatively IolR-regulated genes that could be involved in A. hydrophila virulence. Our future research related to the contribution of the O-antigen in A. hydrophila pathogenesis will focus on the role of the O-antigen, which is specifically affected by mutations in the ymcABC operon. We have now identified media (e.g. tryptic soy broth containing a 5% catfish tissue extract) that induce O antigen capsule expression under laboratory conditions and will next be evaluating the role of the vAh O antigen capsule in mediating attachment to fish skin and in biofilm formation, as well as its other potential contributions to A. hydrophila virulence. Aim 2. Purification of five additional recombinant virulent A. hydrophila proteins will be completed followed by efficacy testing in catfish. Live attenuated bacteria or virus will be evaluated as carriers for promising recombinant proteins (Fim, FimMrfG and ATPase). Aim 3. Two years of sampling fish, water, and sediments from commercial catfish ponds have been completed, and DNA isolation/qPCR analysis has been initiated. This data will be analyzed to identify what factors, if any, predispose ponds to an outbreak. In addition, this analysis will determine what factors are relevant and what samples should be collected in future epidemiological studies to better establish risk factors associated with disease outbreaks. For the second part of this Aim, the experimental immersion exposure method to infect catfish fingerlings, and tissues will be collected at specific time points for bacterial quantification by qPCR.

Impacts
What was accomplished under these goals? Aim 1. We made deletion mutants in five genes encoding predicted myo-inositol catabolism proteins and demonstrated that inability to use myo-inositol as a carbon source did not result in a reduction in virulence when assessed by IP injection. One exception was found: mutation of a regulatory gene in the myo-inositol utilization locus (iolR) resulted in reduced virulence. Further studies with this mutant confirmed our hypothesized role of IolR as a repressor of transcription for genes involved in myo-inositol catabolism and uptake. We have mutated multiple genes involved in O-antigen biosynthesis. In particular, we generated markerless in-frame deletions for ymcA, ymcB, and ymcC, as well as the complete ymcABC operon. Each of the genes in the ymcABC operon significantly contributed to A. hydrophila virulence. The ΔymcA mutant in particular was highly attenuated, with multiple disease challenges being conducted and no fish mortalities resulting from IP injection of this mutant. Moreover, a strong antibody response was elicited in vaccinated fish. We therefore have advanced the ΔymcA mutant as a vaccine candidate. With additional funding sources, we have conducted vaccine trials in the summers of 2015 and 2016 in three catfish production ponds in western Alabama. In 2015, over 18,000 fingerling catfish were IP injected with a formalin-inactivated ΔymcA mutant, and these fish were stocked into three floating in-pond raceways and placed into three ponds with four replicates of the control and vaccine treatment groups in each pond. Vaccinated fish showed a statistically significant increase in survival (99.6%) relative to control fish (94.4%) in the same in-pond raceway unit (P < 0.0001). The 2016 trials have a similar design. Studies are ongoing to evaluate different vaccine delivery methods (feed or immersion). Our third molecular target was the AH4 prophage present within the A. hydrophila ML09-119 genome. Our subsequent comparative genomics analysis has indicated that the AH4 prophage present within vAH strain ML09-119 is not universally conserved among all vAh strains, and we have therefore decided to not move forward in evaluating the contribution of the AH4 prophage-associated genes to vAh virulence. Aim 2. We developed an experimental immersion challenge method that causes mortalities in catfish fingerlings, and it was used as the protocol for vaccine evaluation. We cloned, expressed, and purified eight recombinant VAh proteins [(P pilus assembly protein, pilin FimA (AGM42215.1), Fimbrial protein (AGM42222.1), Fimbrial protein MrfG (AGM42218.1), Fimbrial biogenesis outer membrane usher protein (AGM42220.1), ATPase (AGM45958 ), major outer membrane protein OmpAI (OmpA1), TonB-dependent receptor (TonB), and transferrin-binding protein A (TbpA)]. Their ability to protect and stimulate protective immunity in channel catfish fingerlings against A. hydrophila ML09-119 infection was assessed. Vaccination by IP injection with Fim, FimMrfG, and ATPase generated significant protection against virulent A. hydrophila by immersion compared to non-vaccinated groups (relative percent survival of 95.41%, 85.72%, and 89.16%). A strong antibody response was generated in fish vaccinated with rFim. Catfish fingerlings vaccinated by IP injection with OmpA1 and TonB were significantly (p<0.005) protected against subsequent A. hydrophila ML09-119 infection and had significant antibody response. TbpA stimulated the strongest antibody response but did not provide significant protection. The relative percent survival (RPS) for fish vaccinated with OmpA1, TonB, TbpA, and PBS-adjuvant were 98.59%, 95.59%, 47.89%, and 43.14%, respectively. Aim 3. During the 2014 A. hydrophila season (June to September), water, sediment, benthic oligochaetes, zooplankton and fish samples (gill swabs, rectal swabs and blood; 10 fish/pond) were collected from two different commercial catfish farms. Several fish from ponds with a history of virulent A. hydrophila were PCR positive for the bacteria (gills, anal swabs), with some fish culture positive in the blood, although no fish showed outward signs of disease. This suggests the existence of a carrier state. In ponds where samples were collected during an active outbreak, virulent A. hydrophila was detected in water, mud, and fish. However, once outbreaks subsided, the bacteria disappeared quickly from the system. Within 6 weeks, all sampled fish were PCR negative for the bacteria and only trace amounts of virulent A. hydrophila were detected in pond water. Ten weeks after a disease outbreak, bacteria could not be detected in pond water or pond muds. Moving forward, benthic invertebrates will not be sampled. In summer 2015, bi-monthly pond water samples were collected from 12 ponds on two operations (six with a history of virulent A. hydrophila and six without). In addition, once monthly (June-September) we collected water, mud, and zooplankton, in addition to gill swabs, rectal swabs, and blood from 9 fish/pond. Several of these ponds broke with virulent A. hydrophila during the study. Final results from PCR analysis are pending. Specific objectives met Aim 1. We have constructed deletion mutations in genes encoding myo-inositol catabolism proteins and O antigen biosynthesis proteins, and the mutants have been evaluated for virulence in catfish. A vaccine candidate (ΔymcA mutant) has been constructed and validated. In conjunction with other funding sources, the vaccine is currently in pond trials for further efficacy evaluation. Aim 2. Eight virulent A. hydrophila-specific proteins have been cloned, purified, and evaluated in catfish fingerlings. Aim 3. Sampling and quantitative PCR methods have been established, and field sampling has been completed. DNA isolation/qPCR analysis is underway. Significant results achieved We discovered that mutations of the iolR gene promoter region (in the myo-inositol catabolism locus) in virulent A. hydrophila cause attenuation of virulence. In the O-antigen capsule assembly locus, deletion of the ymcA gene causes complete attenuation of virulent A. hydrophila. Deletion of each of the genes in the ymcABC operon contribute to A. hydrophila virulence, implicating the importance of the O-antigen capsule in virulence. We discovered that Fim, FimMrfG, ATPase, OmpAI, and TonB recombinant proteins produced significant protection in catfish and have potential for vaccine development against virulent A. hydrophila. We identified the potential existence of a carrier state in fish that survive disease outbreaks without antibiotic intervention. We have also identified that once an outbreak subsides, the bacteria does not persist in the environment (or it persists at levels below our detectable limits). Further analysis is being conducted to confirm both of these findings. Benthic invertebrates are not a reservoir for virulent A. hydrophila. Key outcomes or other accomplishments realized A ymcA deletion mutation of virulent A. hydrophila shows potential as a live attenuated vaccine. It is completely attenuated and shows significant protection compared to non-vaccinated channel catfish. The vaccine strain is currently being evaluated in commercial catfish production ponds that have a history of virulent A. hydrophila outbreaks. A system was developed for mutagenesis of A. hydrophila based on Red-mediated recombination and flippase. These molecular tools have now been published and are publicly available. Three recombinant proteins (Fim, FimMrfG, and ATPase) show potential as novel targets for vaccine development against virulent A. hydrophila.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Rasmussen-Ivey, C., Figueras, M.J., McGarey, D. and Liles, M.R. Virulence Factors of Aeromonas hydrophila in the Wake of Reclassification. Frontiers in Microbiology, 7:1337. http://dx.doi.org/10.3389/fmicb.2016.01337
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Tekedar HC, Kumru S, Karsi A, Waldbieser GC, Sonstegard T, Schroeder SG, Liles M, Griffin MJ, Lawrence ML. 2016. Draft genome sequence of Aeromonas hydrophila TN97-08. Genome Announcements 4(3): e00436-16.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Tekedar, H. C., A. Karsi, and M. L. Lawrence. 2015. Comparative genomics analysis of fish pathogen Aeromonas hydrophila strain ML09-119. 115th General Meeting of the American Society for Microbiology, New Orleans, Louisiana.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Tekedar, H. C., A. Karsi, G. C. Waldbieser, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegard, M. Hossain, S. G. Schroeder, L. Khoo, and M. L. Lawrence. 2014. Complete genome sequence of Aeromonas hydrophila ML09-119. Seventh International Symposium on Aquatic Animal Health, Portland, Oregon.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Abdelhamed, H., A. Karsi, and M. L. Lawrence. 2014. Expression and purification of recombinant outer membrane proteins and secreted proteins of Aeromonas hydrophila strain ML09-119. Seventh International Symposium on Aquatic Animal Health, Portland, Oregon.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Griffin, M., C. Ware, C. Mischke, L. Hanson, T. Greenway, T. Byars and D. Wise. 2015. Biotic and abiotic factors associated with outbreaks of an emergent strain of Aeromonas hydrophila in catfish aquaculture. 40th Eastern Fish Health Workshop. Charleston, SC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Thurlow, C.M., Hossain, M.J., Sun, D., Terhune, J.S., and Liles, M.R. 2015. An attenuated Aeromonas hydrophila mutant as a vaccine candidate for Motile Aeromonas Septicemia in Channel Catfish (Ictalurus punctatus). American Society for Microbiology meeting, New Orleans, LA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Barger, P., Newton, J., and Liles, M.R. 2015. Differential Production and Secretion of Potentially Toxigenic ECPs from an Epidemic Strain of Aeromonas hydrophila. American Society for Microbiology meeting, New Orleans, LA.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Tekedar, H. C., A. Karsi, A. Akgul, S. Kalindamar, G. C. Waldbieser, T. Sonstegard, S. G. Schroeder, and M. L. Lawrence. 2015. Complete genome sequence of fish pathogen Aeromonas hydrophila AL06-06. Genome Announcements 3(2) e00368-15.

Progress 09/01/14 to 08/31/15

Outputs
Target Audience:The target audience for this project is commercial catfish producers. Progress on vaccine development targets this audience and aims to reduce prevalence and impact of disease caused by epidemic A. hydrophila. Other target audiences are veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Progress on generating new information related to disease pathogenesis and bacterial quantity/persistence in pond environment is primarily directed to this audience. Changes/Problems:Aim 1. We have altered our strategy that we had indicated in the USDA proposal because we discovered that there is substantial variability within the AH4 prophage genomic region among different epidemic strains. Therefore, there could be multiple genetic loci that contribute to virulence. As a result, we decided to delete the entire prophage region, including the prophage and associated genetic loci, from the A. hydrophila ML09-119 genome. Our original plan was to accomplish the prophage deletion using a two-stage process wherein we incorporate a genetic locus encoding chloramphenicol-resistance and recognized by a flippase on either side of the prophage-associated region, and then we screen for the loss of this region and an antibiotic resistance cassette. While the first stage of this worked well, there have been problems with use of the second resistance marker (encoding gentamicin resistance), and all of the mutants we obtained that were resistant to both of these antibiotics were not due to a second insertion at the correct site. We have therefore altered our strategy again, and we are using two different strategies to select for the loss of the complete AH4 prophage by 1) maintaining stronger selection for the gentamicin-resistance cassette and verifying the correct site of this insertion adjacent to the AH4 prophage, or 2) using a single-step strategy wherin we select for loss of the complete AH4 prophage while selecting for chloramphenicol-resistance. Aim 2. Expression of each recombinant protein requires optimization by testing different buffer conditions and parameters. This is being accomplished, and we anticipate production of the remaining five recombinant proteins in 2015-2016. Aim 3. We have streamlined field sampling to exclude benthic oligochaetes because they do not appear to be a reservoir for the bacteria. We are evaluating a bath immersion protocol developed at the USDA ARS (Dr. Craig Shoemaker, personal communication) in which fingerling catfish have their adipose fin clipped while fish are immersed in a suspension of epidemic A. hydrophila. We will compare this method to our current six hour immersion protocol to determine which method results in more consistent mortalities. What opportunities for training and professional development has the project provided?At Mississippi State University, this project is resulting in the training of a DVM-PhD student (Jordan Smink), and a postdoctoral scientist, (Dr. Hossam Abdelhamed). At Auburn University, this project is resulting in training for one postdoctoral scientist (Dr. M. Jahangir Hossain) and four PhD students (Charles Thurlow, Dawei Sun, Priscilla Barger, and Cody Rasmussen-Ivey). Dr. Hossain has now taken a postdoctoral position at Johns Hopkins University. Two visiting scholars, Dr. Chao Ran (China) and Dr. Mohamed El-hady (Egypt) will be contributing to this project in the 2015-2016 time period. Three undergraduate students have also received training on this project at Auburn University (Shannon Wrenn, Alyson Childers, and Laura Alexander [a NSF REU student from the University of Georgia]). 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?Aim 1. We will compare virulence of the ΔiolR and PrpoD:iolR mutants relative to wild-type A. hydrophila in controlled aquarium challenges of channel catfish via IP injection. In addition to assessing virulence, we will also conduct a transcriptome study of these mutants relative to wild-type to identify putatively IolR-regulated genes that could be involved in A. hydrophila virulence. Our future research related to the contribution of the O-antigen in A. hydrophila pathogenesis will focus on the role of the O-antigen, which is specifically affected by mutations in the ymcABC operon. We are investigating ways in which to induce expression of O-antigen and evaluate its role in mediating attachment to fish skin and in biofilm formation, as well as its other potential contributions to A. hydrophila virulence. We will continue to work toward deletion of the AH4 prophage, as described below. If deletion of the entire prophage region results in attenuated virulence, we will determine if virulence is restored by infecting the prophage deletion mutant with wild-type phage prepared by inducing ML09-119 with mitomycin C or UV radiation. Aim 2. Purification of five additional recombinant epidemic A. hydrophila proteins will be completed followed by efficacy testing in catfish. Live attenuated bacteria or virus will be evaluated as carriers for promising recombinant proteins (Fim, FimMrfG and ATPase). Aim 3. Two years of sampling fish, water, and sediments from commercial catfish ponds have been completed, and DNA isolation/qPCR analysis has been initiated. This data will be analyzed to identify what factors, if any, predispose ponds to an outbreak. In addition, this analysis will determine what factors are relevant and what samples should be collected in future epidemiological studies to better establish risk factors associated with disease outbreaks. For the second part of this Aim, the experimental immersion exposure method to infect catfish fingerlings, and tissues will be collected at specific time points for bacterial quantification by qPCR.

Impacts
What was accomplished under these goals? Major activities completed Aim 1. We have made deletion mutants in five genes encoding predicted myo-inositol catabolism proteins and demonstrated that inability to use myo-inositol as a carbon source did not result in a reduction in virulence when assessed by IP injection. One exception was found: mutation of a regulatory gene in the myo-inositol utilization locus (iolR) resulted in reduced virulence. Targeted deletion of iolR was accomplished as well as generating a mutant that has iolR transcription under the control of a strong promoter (rpoD). As predicted, the ΔiolR mutant has much more rapid growth compared to wild-type A. hydrophila ML09-119 when using myo-inositol as a sole carbon source, and the highly expressed PrpoD:iolR mutant had a moderate delay in growth relative to wild-type. These results are consistent with the hypothesized role of IolR as a repressor of transcription for genes involved in myo-inositol catabolism and uptake. We have mutated multiple genes involved in O-antigen biosynthesis. In particular, we generated markerless in-frame deletions for ymcA, ymcB, and ymcC, as well as the complete ymcABC operon. Each of the genes in the ymcABC operon was important for A. hydrophila virulence, with significant attenuation of virulence observed for each respective mutant as well as the ΔymcABC mutant. The ΔymcA mutant in particular was highly attenuated, with multiple disease challenges being conducted and no fish mortalities resulting from IP injection of this mutant. Moreover, a strong antibody response was elicited in vaccinated fish. We therefore have advanced the ΔymcA mutant as a vaccine candidate. With additional funding sources, we are now conducting vaccine trials in summer 2015 in three catfish production ponds in western Alabama. Over 18,000 fingerling catfish were IP injected with a formalin-inactivated ΔymcA mutant, and these fish were stocked into in-pond raceways permitting replicate trials. Our third molecular target was the AH4 prophage present within the A. hydrophila ML09-119 genome. We are using an alternative approach to our original proposed strategy (described in the Changes/Problems section). Aim 2. We performed a series of fish challenge trials to establish an experimental immersion challenge method that causes mortalities in catfish fingerlings. We found that immersion of catfish for 6 h at 30 ºC or 35 ºC caused 40-50% mortalities. Results had good repeatability; therefore, it was used as the protocol for vaccine evaluation. We assessed the ability of five previously purified recombinant proteins [(P pilus assembly protein, pilin FimA (AGM42215.1), Fimbrial protein (AGM42222.1), Fimbrial protein MrfG (AGM42218.1), Fimbrial biogenesis outer membrane usher protein (AGM42220.1), and ATPase (AGM45958 )] to protect and stimulate protective immunity in channel catfish fingerlings against A. hydrophila ML09-119 infection. Vaccination by IP injection with Fim, FimMrfG, and ATPase generated significant protection against epidemic A. hydrophila by immersion compared to non-vaccinated groups (relative percent survival of 95.41%, 85.72%, and 89.16%). A strong antibody response was generated in fish vaccinated with rFim. The five remaining proteins (transferrin-binding protein A, TonB-dependent receptor, major outer membrane protein OmpA, and two hypothetical proteins) have been cloned, and purification of the recombinant proteins is in progress. Once these recombinant proteins are purified, vaccine trials will be conducted. Aim 3. During the 2014 A. hydrophila season (June to September), water, sediment, benthic oligochaetes, zooplankton and fish samples (gill swabs, rectal swabs and blood; 10 fish/pond) were collected from two different commercial catfish farms. Several fish from ponds with a history of epidemic A. hydrophila were PCR positive for the bacteria (gills, anal swabs), with some fish culture positive in the blood, although no fish showed outward signs of disease. This suggests the existence of a carrier state. In ponds where samples were collected during an active outbreak, epidemic A. hydrophila was detected in water, mud, and fish. However, once outbreaks subsided, the bacteria disappeared quickly from the system. Within 6 weeks, all sampled fish were PCR negative for the bacteria and only trace amounts of epidemic A. hydrophila were detected in pond water. Ten weeks after a disease outbreak, bacteria could not be detected in pond water or pond muds. Moving forward, benthic invertebrates will not be sampled. In summer 2015, bi-monthly pond water samples were collected from 12 ponds on two operations (six with a history of epidemic A. hydrophila and six without). In addition, once monthly (June-September) we collected water, mud, and zooplankton, in addition to gill swabs, rectal swabs, and blood from 9 fish/pond. Several of these ponds broke with epidemic A. hydrophila during the study. DNA isolation is currently underway, and PCR analysis is pending. The second part of this Aim is to determine infectious and lethal doses of epidemic A. hydrophila in channel catfish during experimental infections by immersion. This experiment is currently in progress using the experimental immersion protocol established in Aim 2. Specific objectives met Aim 1. We have constructed deletion mutations in genes encoding myo-inositol catabolism proteins and O antigen biosynthesis proteins, and the mutants have been evaluated for virulence in catfish. A vaccine candidate (ΔymcA mutant) has been constructed and validated. In conjunction with other funding sources, the vaccine is currently in pond trials for further efficacy evaluation. Aim 2. Five of the ten proposed epidemic A. hydrophila-specific proteins have been cloned, purified, and evaluated in catfish fingerlings. The remaining five proteins are cloned, and recombinant protein purification is in progress. Aim 3. Sampling and quantitative PCR methods have been established, and field sampling has been completed. DNA isolation/qPCR analysis is underway. Significant results achieved We discovered that mutations of the iolR gene (in the myo-inositol catabolism locus) in epidemic A. hydrophila cause attenuation of virulence. In the O-antigen biosynthesis locus, deletion of the ymcA gene causes complete attenuation of epidemic A. hydrophila. Deletion of each of the genes in the ymcABC operon contribute to A. hydrophila virulence, implicating the importance of the O-antigen in virulence. We discovered that Fim, FimMrfG, and ATPase recombinant proteins produced significant protection in catfish and have potential for vaccine development against epidemic A. hydrophila. We have identified the potential existence of a carrier state in fish that survive disease outbreaks without antibiotic intervention. We have also identified that once an outbreak subsides, the bacteria does not persist in the environment (or it persists at levels below our detectable limits). Further analysis is being conducted to confirm both of these findings. Benthic invertebrates are not a reservoir for epidemic A. hydrophila. Key outcomes or other accomplishments realized A ymcA deletion mutation of epidemic A. hydrophila shows potential as a live attenuated vaccine. It is completely attenuated and shows significant protection compared to non-vaccinated channel catfish. The vaccine strain is currently being evaluated in commercial catfish production ponds that have a history of epidemic A. hydrophila outbreaks. A system was developed for mutagenesis of A. hydrophila based on Red-mediated recombination and flippase. These molecular tools have now been published and are publicly available. Three recombinant proteins (Fim, FimMrfG, and ATPase) show potential as novel targets for vaccine development against epidemic A. hydrophila.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Hossain, M.J., Thurlow, C.M., Sun, D., Nasrin, S., and Liles, M.R. 2015. Genome modifications and cloning using a conjugally transferable recombineering system. Biotechnology Reports, doi:10.1016/j.btre.2015.08.005.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Hossain, M.J., Sun, D., McGarey, D.J., Wrenn, S., Alexander, L.M., Martino, M.E., Xing, Y., Terhune, J.S., and Liles, M.R. 2014. An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish. mBio 5(3):e00848-14.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Griffin, M., C. Ware, C. Mischke, L. Hanson, T. Greenway, T. Byars and D. Wise. 2015. Biotic and abiotic factors associated with outbreaks of an emergent strain of Aeromonas hydrophila in catfish aquaculture. 40th Eastern Fish Health Workshop. Charleston, SC.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Thurlow, C.M., Hossain, M.J., Sun, D., Terhune, J.S., and Liles, M.R. 2015. An attenuated Aeromonas hydrophila mutant as a vaccine candidate for Motile Aeromonas Septicemia in Channel Catfish (Ictalurus punctatus). American Society for Microbiology meeting, New Orleans, LA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2015 Citation: Barger, P., Newton, J., and Liles, M.R. 2015. Differential Production and Secretion of Potentially Toxigenic ECPs from an Epidemic Strain of Aeromonas hydrophila. American Society for Microbiology meeting, New Orleans, LA.

Progress 09/01/13 to 08/31/14

Outputs
Target Audience: The target audience for this project is commercial catfish producers. Progress in 2013-2014 on vaccine development aims to reduce prevalence and impact of disease caused by epidemic A. hydrophila. Other target audiences are veterinarians, fish diagnosticians, and extension personnel serving the catfish industry. Progress in 2013-2014 on generating new information related to disease pathogenesis and bacterial quantity/persistence in pond environment is primarily directed to this audience. Changes/Problems: Aim 1. We have altered our strategy that we had indicated in the USDA proposal because we discovered that there is substantial variability within the AH4 prophage genomic region among different epidemic strains. Therefore, there could be multiple genetic loci that contribute to virulence. As a result, we decided to delete the entire prophage region, including the prophage and associated genetic loci, from the A. hydrophila ML09-119 genome. This will be a two stage process wherein we incorporate a genetic locus recognized by a flippase on either side of the prophage-associated region, and then we screen for the loss of this region and an antibiotic resistance cassette. We are currently in the second stage of this process. Aim 2 and Aim 3. We are experiencing difficulty in establishing an experimental infection method for epidemic A. hydrophila in channel catfish by the bath immersion or gastric gavage methods. Several experimental infection trials have been conducted at both Auburn University and Mississippi State University, but to date, only the intraperitoneal injection route of infection has been successful. Experiments for Aim 2 and Aim 3 depend on the establishment of this experimental infection method. What opportunities for training and professional development has the project provided? At Mississippi State University, this project is resulting in the training of a DVM-PhD student (Jordan Smink), and a postdoctoral scientist, (Dr. Hossam Abdelhamed). At Auburn University, this project is resulting in training for one postdoctoral scientist (Dr. M. Jahangir Hossain) and four PhD students (Charles Thurlow, Dawei Sun, Priscilla Barger, and Cody Rasmussen-Ivey). Three undergraduate students have also received training on this project at Auburn University (Shannon Wrenn, Alyson Childers, and Laura Alexander [a NSF REU student from the University of Georgia]). 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? Aim 1. Mutagenesis of myo-inositol catabolism genes and O-antigen biosynthesis genes has been accomplished. Complementation of ymcA gene will be completed and virulence testing will be completed. Deletion of the AH4 phage will be completed along with virulence testing. With deletion of the AH4 bacteriophage, all of the genes we proposed to delete will be completed. If deletion of the entire prophage region results in attenuated virulence, we will determine if virulence is restored by infecting the prophage deletion mutant with wild-type phage prepared by inducing ML09-119 with mitomycin C or UV radiation. Aim 2. Purification of recombinant proteins for epidemic A. hydrophila-specific outer membrane and secreted proteins will be completed. The majority of effort in 2014-2015 will be spent developing an effective experimental disease challenge model by immersion or gastric gavage exposure, followed by vaccine efficacy testing of recombinant proteins and attenuated deletion mutants. Aim 3. Sampling of fish, water, and sediments from commercial catfish ponds will be conducted and qPCR analysis will begin. For the second part of this Aim, the immersion exposure method for experimental infections will be developed.

Impacts
What was accomplished under these goals? 1) Major activities completed Aim 1. We have made deletion mutants in five genes encoding predicted myo-inositol catabolism proteins and demonstrated that mutation of any of these genes results in the inability of A. hydrophila ML09-119 to use myo-inositol as a sole carbon source. However, the inability to use myo-inositol as a carbon source has not been found to result in a reduction in virulence when assessed by IP injection. In fact, the only myo-inositol mutants that resulted in reduction in virulence were in the iolR promoter region. The iolR gene is predicted to encode a transcriptional regulator that negatively regulates multiple virulence factors in Salmonella enterica and in other bacteria, such as Type III secretion factors. Further experiments will need to be conducted to evaluate the potential role of the “IolR regulon” in the virulence of A. hydrophila, with the hypothesis being that IolR represses the transcription of multiple virulence factors and that the gene deletions upstream of the iolR gene result in de-repression of iolR transcription. We have mutated multiple genes involved in the O-antigen synthesis pathway. In particular, a ymcA mutant is completely attenuated compared to wild-type A. hydrophila. The ymcA gene encodes a protein that is part of the O-antigen synthesis pathway in other bacteria, but a specific function for YmcA has not been determined. Complementation of the ymcA mutant is ongoing. Challenges in fingerling catfish with the wild-type strain, ymcA mutant, and complemented ymcA mutant will formally determine the involvement of ymcA in A. hydrophila virulence. This mutant has been found to generate a strong antibody response in fingerling catfish, and the surviving fish after inoculation had a very significant (~65%) survival rate compared to naïve fish survival (~5%). Our third molecular target was the AH4 prophage present within the A. hydrophila ML09-119 genome. We are using an alternative approach to our original proposed strategy (described in the Changes/Problems section). Aim 2. We proposed to clone and express ten predicted epidemic A. hydrophila-specific outer membrane and extracellular proteins. Of these, the recombinant proteins are purified and confirmed for five. Recombinant proteins are expressed for two others, and they are undergoing confirmation. Two proteins have been cloned, but the recombinant proteins are not purified yet. For one protein, cloning is still in progress. The next stage for this Aim is to test for vaccine efficacy in channel catfish. Currently research is being conducted to establish an experimental infection procedure for epidemic A. hydrophila in channel catfish using either bath immersion or gastric gavage. Once the experimental infection protocol is established, vaccine trials will be conducted. Aim 3. In the first part of this Aim, we will determine quantity of epidemic A. hydrophila in catfish, catfish pond water, and catfish pond sediment using quantitative PCR. During the 2014 A. hydrophila season (June to August), commercial catfish ponds were identified that had potential for enrollment in this study, and producers are currently being approached to determine their willingness to participate. Procedures for sample collection and testing were established. The second part of this Aim is to determine infectious and lethal doses of epidemic A. hydrophila in channel catfish during experimental infections by immersion. This work will be conducted once the procedure for experimental immersion infections is established. 2) Specific objectives met Aim 1. We have constructed deletion mutations in genes encoding myo-inositol catabolism proteins and LPS biosynthesis proteins, and the mutants have been evaluated for virulence in catfish using the intraperitoneal injection exposure method. Aim 2. Five of the ten proposed epidemic A. hydrophila-specific outer membrane or secreted proteins have been cloned and purified. Four of the remaining five are cloned, and the recombinant proteins are being purified and confirmed. Aim 3. Sampling and quantitative PCR methods are established and commercial catfish producers are being enrolled in the study. 3) Significant results achieved We discovered that mutations upstream of the iolR gene (in the myo-inositol catabolism locus) in epidemic A. hydrophila cause attenuation of virulence. Mutation of other genes encoding myo-inositol catabolism proteins did not result in attenuation. In the O-antigen biosynthesis locus, deletion of the ymcA gene causes complete attenuation of epidemic A. hydrophila, but mutation of other O-antigen biosynthesis genes did not cause attenuation. 4) Key outcomes or other accomplishments realized A ymcA deletion mutation of epidemic A. hydrophila shows potential as a live attenuated vaccine. It is completely attenuated and shows significant protection compared to non-vaccinated channel catfish. A system was developed for mutagenesis of A. hydrophila based on Red-mediated recombination and flippase.

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Hossain, M.J., Sun, D., McGarey, D.J., Wrenn, S., Alexander, L.M., Martino, M.E., Xing, Y., Terhune, J.S., and Liles, M.R. 2014. An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish. mBio 5(3):e00848-14.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Tekedar, H.C., G. C. Waldbieser, A. Karsi, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegard, M. Hossain, S. G. Schroeder, L. Khoo, and M. L. Lawrence. 2013. Complete genome sequence of a channel catfish epidemic isolate, Aeromonas hydrophila strain ML09-119. Genome Announcements 1(5):e00755-13.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Liles, M. R., M. J. Hossain, D. Sun, D. J. McGarey, L. M. Alexander, S. Wrenn, M. E. Martino, Y. Xing, and J. S. Terhune. 2014. Identification of an Asian origin and toxic extracellular products of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish. 11th International Symposium on Aeromonas and Plesiomonas. Montpellier, France.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Tekedar, H. C., G. C. Waldbieser, A. Karsi, M. R. Liles, M. J. Griffin, S. Vamenta, T. Sonstegard, M. Hossain, S. G. Schroeder, L. Khoo, and M. L. Lawrence. 2014. Comparison of the genome sequence of channel catfish epidemic isolate Aeromonas hydrophila ML09-119 to the genome of strain ATCC 7966T. 11th International Symposium on Aeromonas and Plesiomonas. Montpellier, France.
• Type: Books Status: Awaiting Publication Year Published: 2014 Citation: Hanson, L., Liles, M.R., Hossain, M.J., Griffin, M. and Hemstreet, W. Motile Aeromonas Septicemia. In: Fish Health Section Blue Book 2014 Edition, section 1.2.9 (in press)
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Hossain, M. J., D. Sun, S. Wrenn, L. Alexander, C. Heiss, P. Azadi, J. S. Terhune, and M. R. Liles. 2014. Structural elucidation and virulence of a novel O-antigen of Aeromonas hydrophila isolates from a disease epidemic in catfish. General Meeting of the American Society for Microbiology, Boston, MA.
• Type: Conference Papers and Presentations Status: Other Year Published: 2014 Citation: Alexander, L., M. J. Hossain, and M. R. Liles. 2014. Identification of a novel O-antigen and its role in the virulence of Aeromonas hydrophila isolated from diseased catfish. Center for Undergraduate Research Opportunities symposium, Athens, GA.