Progress 10/01/09 to 09/30/14
Outputs
Target Audience: Target Audience: Our target audiences are fish disease diagnostic labs, for the research targeting detection of highly virulent A. hydrophila (VAh) and fish producers, for the control of A. hydrophila. We regularly communicate with Bill Hemstreet, director of the Fish Farming Center in Greensboro, AL, on the latest developments for detection and control of VAh. We are also working on ways in which we can achieve dissemination of vaccine strains to catfish farmers, through working with feed mills or commercial animal health companies. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This research has been accomplished primarily by the postdoctoral scientists Abel Carrias and Mohammad Jahangir Hossain, who have been provided the opportunities to publish their work and present their findings at regional and national conferences. Dr. Carrias has now taken a faculty position at the University of Belize, his native country. In the second year of support, PhD student Dawei Sun is playing a leading role in working with Dr. Hossain on the construction of mutants and evaluation of their virulence and their potential to serve as vaccine strains. Mr. Sun defending his PhD thesis on this research in 2014 and continues his research on Aeromonas hydrophila pathogenesis. Two undergraduate students, Shannon Wrenn and Laura Alexander have also been active research participants and have earned co-authorship on posters and manuscripts. How have the results been disseminated to communities of interest? The results of the research are also beeing published in peer-reviewed journals for communication with the wider scientific audience and by presentations by the PIs at regional, national and international meetings. Students and postdocs have actively attended meetings and communicated their findings. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have made good progress on our objectives: 1) Determine the structure of the Aeromonas hydrophila O-antigen and developing antibodies targeting this antigen. The structure of the A. hydrophila O-antigen has been completely determined, and represents a structure that has never been previously observed for any bacterial O-antigen. This unique O-antigen was then purified and was used to generate monoclonal antibodies. The mAbs were used to test for the specificity against a panel of A. hydrophila epidemic and non-epidemic isolates, and can be developed for multiple diagnostic applications. 2) Develop a system for genetic manipulation in A. hydrophila in order to rapidly create mutants that can be evaluated for their attenuated virulence and ability to serve as live, attenuated vaccines. We have now completed the construction of a plasmid-borne system that encodes the Lambda Red recombinase cassette, and my be introduced into bacterial pathogens such as A. hydrophila and E. ictaluri by conjugal transfer. This has been successfully demnstrated to work in both of these pathogens, and during the past year we have used this to generate 25 different mutants in A. hydrophila. 3) Test the virulence of A. hydrophila mutants in genes required for O-antigen synthesis. We have completed the construction of mutants in both the O-antigen ligase and the O-antigen polymerase, and observed the expected reduction in O-antigen size from bacterial cell extracts. These mutants have also been observed to be attenuated in catfish disease trials. We discovered that a genetic operon located in the O-antigen sysnthesis pathway, encoding the ymcA operon, was particularly necessary for virulence. Furthermore a plasmid complementing the ymcA operon has been completed and is currently being evaluated for its ability to complement the attenuated virulence of a ymcA mutant in order to formally demonstrate the contribution of this gene product to A. hydrophila virulence. Using other funding we are assessing the ability of these attenuated mutants to serve as live, attenuated vaccine strains.
Publications
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Mohammad J. Hossain, Dawei Sun, Shannon Wrenn, Laura Alexander, Christian Heiss, Parastoo Azadi, Jeffrey S. Terhune, and Mark R. Liles. Structural elucidation and virulence of a novel O-antigen of Aeromonas hydrophila isolates from a disease epidemic in catfish. American Society for Microbiology meeting, Boston, MA.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Mark R. Liles, Mohammad J. Hossain, Dawei Sun, Donald J. McGarey, Laura M. Alexander, Shannon Wrenn, Maria Elena Martino, Ye Xing, and Jeffrey S. Terhune. An Asian origin 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:
Accepted
Year Published:
2014
Citation:
Laura Alexander, Mohammad J. Hossain, and Mark R. Liles. 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.
- Type:
Conference Papers and Presentations
Status:
Accepted
Year Published:
2014
Citation:
Mohammad J. Hossain and Mark R. Liles. An epidemic of Aeromonas hydrophila in catfish: Has an emerging bacterial pathogen been introduced via invasive carp? Bacteriology 2013, November 2013, Baltimore, MD
- Type:
Journal Articles
Status:
Accepted
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:
Other
Year Published:
2015
Citation:
Mohammad J. Hossain, Dawei Sun, Shannon Wrenn, Laura Alexander, Christian Heiss, Parastoo Azadi, Jeffrey S. Terhune, and Mark R. Liles. Structural elucidation of a novel O-antigen of Aeromonas hydrophila isolates from a disease epidemic in catfish and the contribution of the ymcABC operon to virulence.
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Progress 01/01/13 to 09/30/13
Outputs
Target Audience: Our target audiences are fish disease diagnostic labs, for the research targeting detection of highly virulent A. hydrophila (VAh) and fish producers, for the control of A. hydrophila. We regularly communicate with Bill Hemstreet, director of the Fish Farming Center in Greensboro, AL, on the latest developments for detection and control of VAh. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? This research has been accomplished primarily by the postdoctoral scientists Abel Carrias and Mohammad Jahangir Hossain, who have been provided the opportunities to publish their work and present their findings at regional and national conferences. Dr. Carrias has now taken a faculty position at the University of Belize, his native country. In the second year of support, PhD student Dawei Sun is playing a leading role in working with Dr. Hossain on the construction of mutants and evaluation of their virulence and their potential to serve as vaccine strains. Mr. Sun is planning to defend his PhD thesis on this research in the summer of 2014. How have the results been disseminated to communities of interest? There has been regular communication via email, phone and in person between the personnel at Auburn (Prof. Liles and Prof. Terhune) and Bill Hemstreet who is the director of the diagnostic lab in Greensboro, AL that evaluate the vast majority of fish diseases cases in the State of Alabama. Bill Hemstreet in return has sent cultures of A. hydrophila from recent disease cases for evaluation by the Liles and Newton labs at Auburn. The results of the research are also beeing published in peer-reviewed journals for communication with the wider scientific audience and by presentations by the PIs at regional, national and international meetings. What do you plan to do during the next reporting period to accomplish the goals? The antibodies targeting the A. hydrophila O-antigen will be evaluated against an existing panel of strains, once the antibodies have been generated at the University of Georgia-Athens. The complementation of the O-antigen mutants is nearing completion, and once introduced into the respective mutant will be tested for restoration of the O-antigen and for complementation of virulence in a fish disease model. Furthermore, experiments to evaluate the efficacy of these mutants as vaccine candidates will be conducted in the Newton and Terhune labs, using fish that have been exposed to the respective mutant by IP injection followed by challenge with the wild-type A. hydrophila, and evaluation of protection by decreased mortality and by immune response as determined by ELISA assay.
Impacts
What was accomplished under these goals?
During the past year we have made good progress on our objectives: 1) Determine the structure of the Aeromonas hydrophila O-antigen and developing antibodies targeting this antigen. The structure of the A. hydrophila O-antigen has been completely determined, and represents a structure that has never been previously observed for any bacterial O-antigen. This unique O-antigen was then purified and is currently being used to generate monoclonal antibodies. In the second year of funding, the mAbs will be used to test for the specificity against a panel of A. hydrophila epidemic and non-epidemic isolates. 2) Develop a system for genetic manipulation in A. hydrophila in order to rapidly create mutants that can be evaluated for their attenuated virulence and ability to serve as live, attenuated vaccines. We have now completed the construction of a plasmid-borne system that encodes the Lambda Red recombinase cassette, and my be introduced into bacterial pathogens such as A. hydrophila and E. ictaluri by conjugal transfer. This has been successfully demnstrated to work in both of these pathogens, and during the past year we have used this to generate 25 different mutants in A. hydrophila. 3) Test the virulence of A. hydrophila mutants in genes required for O-antigen synthesis. We have completed the construction of mutants in both the O-antigen ligase and the O-antigen polymerase, and observed the expected reduction in O-antigen size from bacterial cell extracts. These mutants have also been observed to be attenuated in catfish disease trials. We are currently finishing the construction of plasmids that can complement the genetic deletions, in order to formally demonstrate the contribution of these respective genes to O-antigen synthesis and A. hydrophila virulence. In the following year we will also assess the ability of these attenuated mutants to serve as live, attenuated vaccine strains. 4) The Bacillus strains that have been selected for their ability to antagonize A. hydrophila have now been tested at different doses, and the optimal dose for prevention of mortality in catfish has been determined to be approximately 10^7 CFU/g of feed, when fed for 2 weeks prior to pathogen exposure.
Publications
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Hossain, M.J., Waldbieser, G.C., Sun, D., Capps, N.K., Hemstreet, W.B., Griffin, M.J., Khoo, L., Goodwin, A.E., Sonstegard, T., Schroeder, S., Hayden, K., Newton, J.C., Terhune, J.S., and Liles, M.R. (2013) Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in channel catfish. PLoS ONE 8(11): e80943. doi:10.1371/journal.pone.0080943
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Tekedar, H., Waldbieser, G., Karsi, A., Liles, M., Griffin, M., Vamenta, S., Sonstegard, T., Hossain, M., Schroeder, S., Khoo, L., and Lawrence, M. Complete genome sequence of channel catfish epidemic isolate Aeromonas hydrophila ML09-119. Genome Announcements, vol. 1 no. 5 e00755-13.
- Type:
Journal Articles
Status:
Accepted
Year Published:
2013
Citation:
Griffin, M.J., Goodwin, A.E., Merry, G., Williams, M.A., Liles, M.R., and Waldbieser, G. (2013) Rapid quantitative detection of Aeromonas hydrophila strains responsible for an epidemic in channel catfish. Journal of Veterinary Diagnostic Investigation, 25(4):473-481.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2014
Citation:
Hossain, M.J., Sun, D.G, McGarey, D.J., Wrenn, S.U, Alexander, L.M.U, Martino, M.E., Xing, Y., Terhune, J.S., Liles, M.R. An Asian origin of virulent Aeromonas hydrophila responsible for disease epidemics in United States-farmed catfish. Submitted for publication in mBio.
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Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: Diagnostic tests have been developed targeting the bacterial pathogens of channel catfish Edwardsiella ictaluri and Aeromonas hydrophila. The E. ictaluri research includes phage diagnostics and the development of a rapid molecular diagnostic in collaboration with the Lucigen Corporation (Middleton, WI). The Alabama Fish Farming Center has used the provided E. ictaluri-specific bacteriophages and routinely tests ESC cases using a spot lysis method. Six strains of E. ictaluri were provided to Auburn that did not exhibit significant phage lysis, and these strains have been sequentially infected by phage stocks, thereby producing new phage variants that these previously phage- resistant strains. These new phage variants have had their genome sequenced and the genetic loci responsible for improved strain infection have been identified. These phage variants have also been provided to the Alabama Fish Farming Center which is using them for E. ictaluri detection. In addition, a new National Science Foundation Phase II SBIR grant with the Lucigen Corporation has facilitated development of a molecular diagnostic assay for E. ictaluri. The diagnostic using a LAMP assay is sensitive to 10 E. ictaluri cells and can be used with pure cultures or cells in pond water. Tissue samples have proven inhibitory to the assay and there is cross-reactivity with a few other bacterial pathogens, so improvements are being developed and will be tested in 2013 to overcome these problems. The A. hydrophila diagnostic work has included an analysis of 12 A. hydrophila genomes sequenced by the USDA and analyzed in the Liles lab to identify virulent-strain specific primer sets. A epidemic A. hydrophila primer set has been validated, along with a rapid growth-based diagnostic assay, and these diagnostic tests are now being used by laboratories in AL, AR, and MS. These findings have been communicated to the Catfish Farmers Association meeting in 2012 (Demopolis, AL) and in the annual meeting of the American Society of Microbiology. Manuscripts are being prepared for submission. PARTICIPANTS: Graduate student Mohammad J. Hossain has completed his doctoral training based on his research concerning Aeromonas hydrophila comparative genomics and Dr. Hossain continues his research as a postdoctoral scientist from partial support from this Hatch project. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Diagnostic assays specific to E. ictaluri have been developed and are in use by the primary fish diagnostic laboratory in Alabama, the Alabama Fish Farming Center. The rapid molecular diagnostic remains in development but is expected to be evaluated with field samples in 2013. The epidemic Aeromonas hydrophila diagnostics (both PCR-based and growth-based) have been communicated to diagnostic laboratories in AL, AR, and MS and are currently in use for detection of the epidemic Aeromonas hydrophila.
Publications
- Hossain, M.J., Sun, D., McGarey, D.J., Terhune, J.S., Xing, Y., and Liles, M.R. 2012. The recent epidemic outbreak in channel catfish is caused by a novel subspecies of Aeromonas hydrophila. Southeastern Regional Branch Meeting of the American Society for Microbiology (SEB-ASM), Athens, GA.
- M.J. Griffin, A.E. Goodwin, G. Merry, M.A. Williams, M.R. Liles, and G. Waldbieser. 2012. A rapid genetic test for identification and quantification of an Aeromonas hydrophila strain associated with disease outbreaks in Alabama, Arkansas and Mississippi. American Fisheries Society meeting, Conway, AR.
- S. Jones, A. Goodwin, M. R. Liles, and M. Griffin. 2012. Tracking the Persistence of a New Strain of Aeromonas hydrophila in Catfish Tissues with Specific Quantitative Polymerase Chain Reaction. American Fisheries Society meeting, Conway, AR.
- M. J. Hossain, W. Hemstreet, G. C., Waldbieser, L. Khoo, M. J. Griffin, J. A. Bebak, J. C. Garcia, N. Capps, K. Hayden, J. S. Terhune, and M.R. Liles. 2012. Identification of genomic islands and unique functions in Aeromonas hydrophila strains associated with an epidemic in channel catfish. American Society for Microbiology meeting.
- M. Griffin, A. Goodwin, G. Merry, M. R. Liles, M. Williams and G. Waldbieser. 2012. A newly developed genetic test provides more rapid identification and quantification of an Aeromonas hydrophila strain associated with disease outbreaks in Alabama, Arkansas and Mississippi. Catfish Farmers of America Annual Convention and Research Symposium, Savannah, GA.
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Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: Diagnostic tests have been developed targeting the bacterial pathogens of channel catfish Edwardsiella ictaluri and Aeromonas hydrophila. The E. ictaluri research includes phage diagnostics and the development of a rapid molecular diagnostic in collaboration with the Lucigen Corporation (Middleton, WI). For continued work on expanding the phage-based diagnostic tests, the Alabama Fish Farming Center has been evaluating the provided E. ictaluri-specific bacteriophages and testing by phage spot lysis assay when new suspected ESC cases are identified. Six strains of E. ictaluri were provided to Auburn that did not exhibit significant phage lysis, and these strains have been sequentially infected by phage stocks, thereby producing new phage variants that these previously phage-resistant strains. These new phage variants have been provided to the Alabama Fish Farming Center which is using them for E. ictaluri detection. In addition, a new National Science Foundation Phase II SBIR grant with the Lucigen Corporation will facilitate further development of a molecular diagnostic assay for E. ictaluri. The A. hydrophila diagnostic work has included an analysis of 12 A. hydrophila genomes sequenced by the USDA and analyzed in the Liles lab to identify virulent-strain specific primer sets. A total of 26 primer sets were identified specific to virulent strains, and these have been provided to USDA researchers to evaluate against a large panel of A. hydrophila disease isolates. The best three primer sets have now been selected and are under further testing against virulent and less-virulent A. hydrophila strains. Future work will evaluate the nature of the virulence-associated genetic loci from A. hydrophila isolates in 2009 and 2010. These findings have been communicated to the Catfish Farmers Association meeting in 2011 (Demopolis, AL) and in the annual meeting of the American Society of Microbiology. A manuscript is being prepared for submission. PARTICIPANTS: Two PhD students are working on this project: Mohammad Jahangir Hossain, is working on the comparative analysis of the Aeromonas hydrophila genome and identification of the virulence factors that contribute to the pathogenicity of the epidemic A. hydrophila in catfish. Malachi Williams is working on the identification of epidemic strain-specific gene targets and development of diagnostic tests. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Identified bacteriophages that can infect primary disease isolates of E. ictaluri 2) Using bacteriophages as diagnostic tools in a fish diagnostic lab 3) Working collaboratively with Lucigen Corp. on a molecular diagnostic assay 4) Identified genetic loci associated with epidemic strains of A. hydrophila
Publications
- M. R. Liles, W. Hemstreet, G. C., Waldbieser, L. Khoo, M. J. Griffin, J. A. Bebak, J. C. Garcia, N. Capps, K. Hayden, and J. S. Terhune. 2011. Comparative genomics of Aeromonas hydrophila isolates from an epidemic in channel catfish. American Society for Microbiology meeting.
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: This project includes as an outcome research on developing diagnostic assays for E. ictaluri, using molecular sequences common to different E. ictaluri strains. In addition, this past year research has also focused on the bacterial pathogen Aeromonas hydrophila. Since in 2009 and 2010 more than 5 million pounds of catfish died from infection by new virulent strains of A. hydrophila, identifying molecular diagnostic tools to track these virulent A. hydrophila strains was a priority for our stakeholders. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: This project was initially focused on E. ictaluri diagnostics and included research on bacterial biological control agents (genus Bacillus) for ESC control. The bacterial biological control work has continued and expanded, but is now described in ALA080-051 as that project is focused on biological control. This project will in the future be focused on diagnostic research, continuing the work on E. ictaluri diagnostic assays and also including as of 2010 the research on A. hydrophila diagniostics. Considering the significant deleterious impact of A. hydrophila on channel catfish aquaculture in 2009 and 2010, it was an imperative that this project expand to include A. hydrophila.
Impacts
The E. ictaluri diagnostic tests included phage diagnostics and the development of a rapid diagnostic in collaboration with the Lucigen Corporation. For continued work on expanding the phage-based diagnostic tests, the Alabama Fish Farming Center has been evaluating the provided E. ictaluri-specific bacteriophages and testing by phage spot lysis assay when new suspected ESC cases are identified. Six strains of E. ictaluri were provided to Auburn that did not exhibit significant phage lysis, and these strains have been sequentially infected by phage stocks, thereby producing two new phage variants that could infect 4 of these E. ictaluri (previously phage-resistant) strains. These two new phage variants (passaged phage) have been provided to the Alabama Fish Farming Center and the final two E. ictaluri strains are still being evaluated for phage that can readily infect them. In addition, a new National Science Foundation Phase II SBIR grant with the Lucigen Corporation will facilitate further development of a molecular diagnostic assay for E. ictaluri. The A. hydrophila diagnostic work has included an analysis of 12 A. hydrophila genomes sequenced by the USDA (Dr. Geoffrey Waldbieser) and analyzed in the Liles lab to identify virulent-strain specific primer sets. A total of 26 primer sets were identified specific to virulent strains, and these have been provided to USDA researchers (Dr. Julie Bebak and Dr. Andy Goodwin) to evaluate against a large panel of A. hydrophila disease isolates. The best three primer sets have now been selected and are under further testing against virulent and less-virulent A. hydrophila strains. Future work will evaluate the nature of the virulence-associated genetic loci from A. hydrophila isolates in 2009 and 2010.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: 1) Test efficacy of E. ictaluri-specific phage for diagnosis of ESC 2) Use E. ictaluri strains that are less susceptible to phage infection to select for phage variants with expanded host range for diagnostic use 3) Supply the diagnostic laboratory with newly developed phage variants 4) Test for specificity of a LAMP assay for E. ictaluri PARTICIPANTS: Dr. Mark R. Liles Dr. Jeffery S. Terhune Mr. Jahangir Hossain Mr. William Hemstreet TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
1) Three naturally occurring bacteriophages specific to the catfish pathogen Edwardsiella ictaluri were previously identified from aquaculture ponds in the Southeastern United States. These three phage were provided to the Alabama Fish Farming Center for use in primary diagnosis of ESC infections in aquaculture farmed channel catfish. Approximately 100 freezer aliqouts were provided for each of the three phage, with greater than 1x10^8 PFU/ml titre in each microcentrifuge tube, and enough in each aliquot for 50 tests. During 2009 each of the presumed ESC infections were diagnosed by traditional methods (cultivation and biochemical tests) and also tested for phage susceptibility by spot lysis method. In the majority of cases phage lysis was observed, and for 6 E. ictaluri primary isolates no significant phage susceptibility was observed, but both biochemical and genetic tests confirmed the diagnosis of E. ictaluri. 2) Each of the phage-resistant E. ictaluri strains was used to select for phage variants that can better infect these strains. Phage were added at high multiplicity of infection to each bacterial culture (kept at low passage number by returning to the glycerol stock for each culture) and the soft agar overlay was extracted for phage. Each phage suspension was used to infect the same strain in this serial manner, and for 4 out of 6 strains a phage variant was produced that was capable of high efficiency infection of the previously phage-resistant E. ictaluri strain. The remaining two strains are still being actively pursued, and for one of these strains it has been determined that introduction of an E. ictaluri porin gene from a different (phage-susceptible) strain has now permitted phage infection. 3)Each of the 4 new phage variants was shipped to the diagnostic laboratory during the Fall of 2009 (in same aliquot format as described above)and when the next ESC season is expected to occur in Spring of 2010 these new phage variants will be evaluated for their respective ability to lyse primary E. ictaluri disease isolates. The eventual method will include a "cocktail" of different phage to make this method rapid, inexpensive, and effective as a diagnostic assay for diverse E. ictaluri strains. 4) In collaboration with the Lucigen Corporation, a diagnostic assay was evaluated that employs the LAMP assay for molecular detection of E. ictaluri. A primer set was developed that targets a repetitive element within the E. ictaluri genome. By comparison with other published E. ictaluri primer sets, this primer set provided much greater sensitivity of detection in a rapid format. The next series of experimental questions will relate to the robustness of this assay within catfish tissues, and development of a colorimetric output to allow the results of the assay to be observed without the need for expensive equipment that may not be available to primary diagnostic laboratories.
Publications
- No publications reported this period
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