Progress 10/01/07 to 09/30/12
Outputs
OUTPUTS: This research project has resulted in the identification of multiple probiotic bacteria within the genus Bacillus that have the ability to control disease due to bacterial pathogens in catfish and tilapia. Bacillus strains isolated from catfish intestine and a collection of 160 Bacillus strains were evaluated for their antimicrobial activity against multiple pathogens of channel catfish. In particular, strains were selected for their potent antagonism against Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), and against a recent epidemic strain of Aeromonas hydrophila. Each of the top 22 strains that inhibited both E. ictaluri and A. hydrophila showed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 22 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore supplemented feed (109 CFU/g feed) for 7 days followed by normal feed for 3 days. Several Bacillus strains with populations over 108 CFU/g of intestine were observed. Seven Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8 x 107 CFU/g and fed to channel catfish for 5 days before they were challenged by E. ictaluri in replicate aquaria. All seven strains conferred benefit in reducing catfish mortality compared to the control, with Bacillus strain AP193 showing a 84% reduction in mortality. Similar benefits were observed when strain AP193 was fed to tilapia and challenged with A. hydrophila by gavage, with a significant reduction in mortality observed from 71 +/- 15 % mortality in the control group compared to 27 +/- 9 % mortality in the probiotic-fed fish (p < 0.05). No changes in fish growth or performance were observed as a result of probiotic feeding, and the results of genome sequencing analysis have not indicated the presence of plasmids or potentially toxic gene products. These probiotic strains have good potential for application as a feed additive for biological control of multiple pathogens in aquaculture farmed fish. These results have been disseminated by presentations at scientific conferences, to catfish producers, and by publication of the research in peer-reviewed journals. PARTICIPANTS: Mark R. Liles was the PI of this award along with co-PIs Jeffery S. Terhune and Joseph C. Newton. Graduate students received training in molecular microbiology and fish disease challenges, including Abel Carrias, Ph.D. and Mohammad J. Hossain, Ph.D., both of whom are currently funded as postdoctoral scientists in the Liles laboratory. In addition the current Ph.D. student Chao Ran has conducted his thesis research on these Bacillus probiotic strains and will defend his Ph.D. thesis in 2013. Collaborative research has been conducted with the Lucigen Corporation, who have expertise in Bacillus fermentation and genetics, and a NSF Phase I and Phase II SBIR award has been awarded to Lucigen and Auburn scientists to continue this research both in terms of probiotics for biological control of disease and in advancing rapid diagnostics for aquaculture pathogens. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
The direct impact of this research is in the identification of specific beneficial bacterial strains, in particular Bacillus strain AP193, with the ability to control disease in fish species. Our specific trials have focused on the major bacterial pathogens in aquaculture farmed fish in the State of Alabama, Edwardsiella ictaluri and Aeromonas hydrophila, but these probiotic strains have the potential to control a wide diversity of bacterial and fungal pathogens. This project enabled the animal trials to extend the research from the laboratory to aquarium models of disease, and to select the probiotic strains that performed the best in animal studies. Furthermore, the safety of the probiotic strains was determined by conducting longer term feeding studies, which demonstrated the lack of any problem associated with direct feeding of Bacillus spores within the fish feed.
Publications
- Ran, C., Carrias, A., Williams, M.A., Capps, N., Dan, B.C.T, Newton, J.C., Kloepper, J.W., Ooi, E.L., Browdy, C.L., Terhune, J.S., and Liles, M.R. (2012) Identification of Bacillus strains for biological control of catfish pathogens. PLoS ONE 7(9): e45793. doi:10.1371/journal.pone.0045793.
- Carrias, A., Ran, C., Terhune, J., and Liles, M.R. (2012) Biological agents for control of disease in aquaculture, pp. 353-393. In: Infectious Diseases in Aquaculture, S. Austin (Ed.). London: Woodhead Publishing.
- Hossain, M.J., Rahman, K.S., Terhune, J.S., and Liles, M.R. (2012) An Outer Membrane Porin Protein Modulates Phage Susceptibility in Edwardsiella ictaluri. Microbiology, 158:488-497.
- Carrias, A., Welch, T.J., Waldbieser, G.C., Mead, D.A., Terhune, J.S., and Liles, M.R. (2011) Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri. Virology Journal 8:6, doi:10.1186/1743-422X-8-6.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Bacillus strains isolated from catfish intestine and a collection of 160 Bacillus strains were evaluated for their antimicrobial activity against multiple pathogens of channel catfish. In particular, strains were selected for their potent antagonism against Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), and against a recent epidemic strain of Aeromonas hydrophila. Each of the top 22 strains that inhibited both E. ictaluri and A. hydrophila showed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 22 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore supplemented feed (109 CFU/g feed) for 7 days followed by normal feed for 3 days. Several Bacillus strains with populations over 108 CFU/g of intestine were observed. Seven Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8 x 107 CFU/g and fed to channel catfish for 5 days before they were challenged by E. ictaluri in replicate aquaria. All seven strains conferred benefit in reducing catfish mortality compared to the control, with one Bacillus strain showing a 84% reduction in mortality. These findings have been shared with the Catfish Farmers of America at their annual meeting in 2011, and by publication in peer-reviewed journals (see below). PARTICIPANTS: Two graduate students are working on this project: Mohammad Jahangir Hossain, PhD student, is planning to graduate with his PhD in 2012 and is working on submission of 3 more manuscripts this year. Chao Ran, PhD student, is working on submission of a manuscript focusing on this project within the next month. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Screened a collection of beneficial bacteria isolated from soils and catfish to identify probiotic bacteria that have the potential to promote fish health and protect against disease 2) Identified specific strains that produce antimicrobials that are active against the major bacterial and fungal pathogens of channel catfish 3) Some Bacillus strains have been found to protect channel catfish from disease due to the major bacterial pathogen Edwardsiella ictaluri 4) Ongoing studies evaluating specific strains and their dose-response and safety
Publications
- Carrias, A., Ran, C., Terhune, J., and Liles, M.R. 2012. Biological agents for control of disease in aquaculture. In: Infectious Diseases in Aquaculture, S. Austin (Ed.). London: Woodhead Publishing, in press.
- Zhou, Z., Yao, B., Waines, P., Ringo, E., Emery, M., Liles, M.R., and Merrifield, D.L. 2012. Methodological approaches to assess microbial communities. In: Probiotics, Prebiotics and Gut Health in Aquaculture Nutrition, Merrifield, D. L.; Ringo, E. (Eds.). Hoboken, N.J.: Wiley-Blackwell, in press.
- Hossain, M.J., Rahman, K.S., Terhune, J.S., and Liles, M.R. 2012. An Outer Membrane Porin Protein Modulates Phage Susceptibility in Edwardsiella ictaluri. Accepted for publication in Microbiology.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The bacteriophages specific to E. ictaluri have now been evaluated for their potential as therapeutic agents in channel catfish in preventing ESC. In addition to the ongoing work with bacteriophages specific to E. ictaluri, this project now includes the evaluation of Bacillus strains (of species B. subtilis, B. amyloliquefaciens, and B. pumilus) for biological control of disease in channel catfish. The outputs include in vitro screening for antibiosis against catfish pathogens, evaluation of the in vivo survival of Bacillus strains within the catfish intestine, and initial evaluation of Bacillus strains efficacy in preventing mortality due to ESC. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: This project has progressed from the evaluation of bacteriophages as biological control agents to the use of bacterial cultures within the genus Bacillus. While in each challenge assay the bacteriophages have shown some degree of protective effect, the magnitude of the therapeutic effect is expected to be greater for the Bacillus biological control agents and due to their ability to form endospores they have much greater potential application due to their stability and lower cost of production.
Impacts
Bacillus strains isolated from catfish intestine and a collection of 160 Bacillus strains were evaluated for their antimicrobial activity against multiple pathogens ofchannel catfish. In particular, strains were selected for their potent antagonism against Edwardsiella ictaluri, the causative agent of enteric septicemia of catfish (ESC), and against a recent virulent isolate of Aeromonas hydrophila. Each of the top 22 strains that inhibited both E. ictaluri and A. hydrophila showed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 22 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore supplemented feed (10^9 CFU/g feed) for 7 days followed by normal feed for 3 days. Several Bacillus strains with populations over 10^8 CFU/g of intestine were observed. Seven Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8 x 10^7 CFU/g and fed to channel catfish for 5 days before they were challenged by E. ictaluri in replicate aquaria. All seven strains conferred benefit in reducing catfish mortality compared to the control, with Bacillus strain AP193 showing a 84% reduction in mortality.
Publications
- Carrias, A., Welch, T.J., Waldbieser, G.C., Mead, D.A., Terhune, J.S., and Liles, M.R. (2011) Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri. Virology Journal 8:6, doi:10.1186/1743-422X-8-6.
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 1) Improve E. ictaluri-specific phage titers 2) Evaluate phage stability. 3) Identify E. ictaluri genetic determinants of phage infection. 4) Complete the sequencing of the genomes of three E. ictaluri-specific bacteriophages. 5) Annotate and compare the bacteriophage genomes. 6) Evaluate the efficacy of E. ictaluri-specific phage to control ESC in channel catfish fingerlings PARTICIPANTS: Dr. Mark R. Liles Dr. Jeffery S. Terhune Dr. Joseph C. Newton Mr. Abel Carrias Mr. Jahangir Hossain Mr. Koichiro Tsuji Mrs. Nancy Capps TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) By using a multiplicity of infection of 0.01 and an altered homogenization protocol, phage titers exceeding 10^12 PFU/ml can now be produced, enabling much more concentrated application of phage onto catfish feed. 2) Phage stability on catfish feed was monitored at different temperatures over time. No significant drop in phage titer was observed at either 4oC or at room temperature, and phage titers only decreased when stored at 37oC after 12 days at this temperature. 3) Over 180,000 transposon mutants were generated in 3 E. ictaluri strains, and screened by selecting for phage-resistant mutants. Twelve stable phage-resistant mutants were obtained. The genetic loci that have been inactivated were identified by inverse PCR or shotgun subcloning. Several different E. ictaluri gene products appear to be required for phage infection, including a negative transcriptional regulator known to be involved in hemolysin expression, a putative antibiotic efflux pump, and an outer membrane porin. The porin may be the receptor for phage infection, and changes in the porin amino acid sequence have been mapped to phage infectivity. Phage that have been adapted to infection of a specific E. ictaluri strain appear to be adapting to a specific porin structure, as complementation of the phage-resistant porin mutant with porin genes from different E. ictaluri strains is strain-specific. 4) Phage genome sequencing has been completed for the three unique bacteriophages eiAU, eiDWF, and eiMSLS, using a combination of Sanger sequencing and primer walking to complete each contiguous phage genome. 5) Each phage genome has been annotated by 1)identifying open reading frames (ORFs), 2) comparing each predicted ORF against the GenBank and VIROME databases. A comparative genomic analysis is nearing completion. 6)A catfish challenge with E. ictaluri was used to evaluate the efficacy of phage-amended feed in reducing mortality due to ESC. Lyophilized feed was spray-coated with phage solution (~3x10^8 PFU/mL) at a rate of 8 % of the dry/lyophilyzed weight of the feed. Fish received normal or phage-impregnated feed, that contained approximately 3x10^7 PFU per gram of feed for 27 days starting 3 days prior to challenging fish with bacteria. For challenge, E. ictaluri were added directly into each aquarium at a final dosage of approximately 1x10^5 CFU/ml aquarium water. Upon outbreak of ESC, fish were observed for signs of disease, and moribund fish were necropsied with tissue samples streaked on TSA for bacterial isolation. Percent survival was 70.7 % for treatment 1 (feed alone) and 79.3 % for treatment 2 (phage-amended feed). The survival for the negative control aquaria was 100 %. Treatment 1 and treatment 2 are significantly different (p < 0.05). Results indicate that the use of phage administered through feed offers protection against ESC in channel catfish. The use of phage resulted in 8 % higher final survival compared to non treated fish. Future studies in year 3 of funding will include the use of different phage dosages, types of phage, and combinations of phage to optimize treatment.
Publications
- No publications reported this period
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Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: Substantial progress has been made in understanding the genetics, host interactions, stability, and host range of bacteriophages specific to the catfish pathogen Edwardsiella ictaluri. In collaboration with USDA scientists Dr. Geoff Waldbieser and Dr. Tim Welch, the genome sequences for three unique E. ictaluri-specific bacteriophages have been determined. The genome sequences indicate that these bacteriophages are closely related to each other, are within the Family Siphoviridae, and are distantly related to virulent phages that infect Salmonella species. Importantly, the genomic data supports the conclusion that these phage are not lysogenic, and are capable of rapid infection and lysis of E. ictaluri. The bacteriophages have been supplied to the Alabama Fish Farming Center to determine the efficacy of phage lysis as a diagnostic tool for E. ictaluri primary isolates. The phage-based typing assay is inexpensive, rapid, and effective. Of the dozens of E. ictaluri isolates in collections and primary isolates that have been tested, only six isolates show decreased susceptibility to phage infection and/or lysis. These six E. ictaluri strains were used to serially passage bacteriophage eiAU, and the resulting passaged phage has shown dramatic increases in lysis for the strains in whcih they were passaged (over 10,000% in one case). To date every E. ictaluri strain that has been used to serially passage phage have allowed generation of highly infective phage variants, thereby permitting coverage of the diversity of E. ictaluri genotypes present among primary disease cases. In addition the genetic determinants of phage infection in E. ictaluri are being investigated. Three E. ictaluri strains were subjected to transposon mutagenesis (plasmid pLOF-Km), and the resulting transposon mutants were screened for phage-resistance. Inverse PCR was used to amplify the genetic regions surrounding the transposon insertion, and preliminary sequence data have indicated multiple genetic loci putatively involved as phage infection. Phage-resistant mutants will be tested in virulence assays to determine if phage-resistance impacts E. ictaluri virulence. PARTICIPANTS: Dr. Mark R. Liles Dr. Jeffery S. Terhune Dr. Joseph C. Newton Mr. Abel Carrias Mr. Jahangir Hossain Mr. Koichiro Tsuji Mrs. Nancy Capps Mr. John Walakira Ms. Eileen Jones TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Identification of naturally occurring microorganisms, including both bacterial and viral microflora, that can ameliorate the severity or incidence of disease can be an important alternative to antibiotic treatment or dietary restriction for control of ESC in channel catfish. The tools provided by identifying bacteriophages specific to E. ictaluri include both diagnostic and therapeutic applications. For diagnostic applications, the E. ictaluri-specific bacteriophages have already been provided to personnel involved in diagnosis of ESC among catfish farms in western Alabama. The feedback being given from these studies of bacteriophage infection of primary E. ictaluri isolates is enabling selection for phage variants that can infect a diversity of E. ictaluri genotypes. The next focus of this research is in the therapeutic application of bacteriophages and/or bacterial cultures as biological control for E. ictaluri disease in channel catfish. Preliminary studies have indicated long-term survival of bacteriophages and/or bacterial cultures when applied to catfish feed, and ongoing studies to determine the efficacy of biological control strategies for disease prevention will establish the best formulations for application in aquaculture pond studies.
Publications
- Walakira, J., Carrias, A., Hossain, M., Jones, E., Terhune, J.S., and Liles, M.R. (2008) Identification and characterization of bacteriophages specific to the catfish pathogen Edwardsiella ictaluri. Journal of Applied Microbiology, 105(6):2133-2142.
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Progress 01/01/07 to 12/31/07
Outputs
OUTPUTS: This project aims to use naturally-occurring bacteriophages specific to the catfish pathogen Edwardsiella ictaluri for biological control of catfish disease. Several experimental outputs have been achieved during the short funding time of this work. 1) Genomic libraries have been constructed for the two unique E. ictaluri-specific bacteriophages. 2) These genomic libraries have been shipped to the USDA labs in Stoneville, MS (under supervision of Dr. Geoff Waldbieser), where they are currently being sequenced to ~5x coverage. 3) Work has been completed on the initial characterization of these two bacteriophages, in terms of their host range (specific to E. ictaluri), response to calcium and magnesium, and other characteristics necessary for publication of this work 4) An additional E. ictaluri bacteriophage has been discovered from an aquaculture pond with a history of catfish disease, and is currently being characterized and compared to the previously isolated
bacteriophages. 5) A recirculating system for catfish challenges has been established at the AU Vet School (lab of co-PI Dr. Joe Newton) for purposes of experimental control of catfish infection
PARTICIPANTS: Mr. Abel Carrias (Ph.D. student, co-advised with Drs. Liles and Terhune), working on comparative phage genomics and catfish challenge models of phage biocontrol Mr. John Walakira (M.S. student, co-advised with Drs. Liles and Terhune), working on characterization of already isolated bacteriophages, first-author on manuscript, anticipated graduation May 2008. Mr. Jahangir Hossain (Ph.D. student, Liles lab), just joined team in 2008, working on characterization of phage lysins and directed evolution of phage for long-circulating phenotype in catfish Mr. Koichiro Tsuji (undergrad student, Liles lab), newly joined the team, working with Mr. Carrias as mentor. Would like to pursue career in veterinary pathology, will contribute to projects on catfish challenges and directed evolution of phage for identification of phage mutants with greater infectivity for less-susceptible E. ictaluri strains Mrs. Nancy Capps (technician, Liles lab, AU Biological Sciences), support for the
project in terms of media preparation, phage preps, is working on improving phage stability through addition of amendments to phage storage buffer, and discovery of additional phages specific to E. ictaluri. 75% support from this funding Mrs. Priscilla Barger (technician, Newton lab, AU Vet School), providing support for catfish challenge models at Vet School. Maintains catfish aquariums and feeds fish. Dr. Geoff Waldbieser (USDA labs, Stoneville, MS), providing technical support and sequencing of phage genomes at the USDA labs in Stoneville, MS. Dr. Joe Newton (AU Vet School). Provides technical support, mentoring of technicain, and help in design and execution of catfish challenge experiments at Vet School. Contributes to manuscript and grant writing and editing. Dr. Jeff Terhune (AU Dep. Fisheries and Allied Aquacultures). Provides technical support, mentoring of grad students, editing of manuscripts, and execution of catfish challenge experiments and E. ictaluri pathogenesis
aspects of the project. Also leading efforts to coordinate with aquaculture community and involve catfish feed industry. Contributes to manuscript and grant writing and editing. Dr. Mark Liles (AU Dep. Biological Sciences). Project leader, involved with grad student mentoring, coordination of team members on different projects, supervision of technician, manuscript and grant writing, submission of annual reports, submission of intellectual property disclosure(s).
TARGET AUDIENCES: Contacts have been made with aquaculture farmers in Alabama to solicit feedback on attitudes toward using bacteriophage biocontrol. Feedback has been very positive from aquaculture farmers, and these efforts will continue and increase in frequency as the work reaches the next phase of implementation. Additional contacts in the catfish feed industry are being cultivated by Dr. Terhune and Liles, as well as other biotech companies with interest in phage biocontrol in other agricultural systems.
PROJECT MODIFICATIONS: No significant major changes to date.
Impacts
This work is still at a preliminary stage in terms of our understanding of how these bacteriophages may be used experimentally and in aquaculture ponds for biological control of catfish disease. 1) The discovery of another bacteriophage from a catfish aquaculture ponds contributes to our ability to use a "cocktail" of different phages in treatment regimes for catfish biological control strategies 2) Preliminary sequence data indicates significant genetic similarity between the different phage genomes, with also regions of apparent genetic divergence. The collaboration with USDA labs in MS and WV has enabled this project to use a previously isolated E. ictaluri bacteriophage (which unfortunately produced turbid plaques) as a reference genome for comparison with newly isolated bacteriophages. 3) The phage genome sequences will indicate putative phage lysin gene(s), which may then be subcloned and tested as alternative biological control agent(s). 4) Progress in
developing experimental catfish challenge systems will enable testing of phage biocontrol under controlled conditions.
Publications
- Identification and characterization of bacteriophages specific to the catfish pathogen Edwardsiella ictaluri (2008) John Walakira, Abel Carrias, Mohammad J. Hossain, Eileen Jones, Jeffery S. Terhune, and Mark R. Liles, Manuscript in preparation.
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