Genetic variation of renibacterium salmoninarum genes in infected salmonids
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Genetic Variation of Renibacterium salmoninarum genes in infected salmonids. Jeffrey Burnett HHMI Summer Investigator Dr. Dan Rockey Laboratory Biomedical Sciences. Renibacterium salmoninarum. Causes bacterial kidney disease (BKD) Wild and farmed salmonid species. Introduction

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Genetic Variation of Renibacterium salmoninarum genes in infected salmonids

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Genetic variation of renibacterium salmoninarum genes in infected salmonids

Genetic Variation of Renibacterium salmoninarum genes in infected salmonids

Jeffrey Burnett

HHMI Summer Investigator

Dr. Dan Rockey Laboratory

Biomedical Sciences


Renibacterium salmoninarum

Renibacterium salmoninarum

  • Causes bacterial kidney disease (BKD)

    • Wild and farmed salmonid species

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Relevance

Relevance

  • Why is this a problem?

    • We eat salmonids

    • We depend on salmonids to keep an ecosystemic balance in our local rivers and streams

    • R. salmoninarum devastates whole populations; endangered fish stocks

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Relevance1

Relevance

  • All salmonids are susceptible to BKD

    • coho salmon (Oncorhynchus kisutch)

    • brook trout (Salvelinus fontinalis)

    • brown trout (Salmo trutta)

    • chinook salmon (Oncorhynchus tshawytscha)

    • rainbow trout (Oncorhynchus mykiss)

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Renibacterium salmoninarum1

Renibacterium salmoninarum

http://oregonstate.edu/dept/salmon/projects/images/4BKD.jpg

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Relevance2

Relevance

  • Prevalence

    • Found in majority of countries

    • Economic impact felt worldwide

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Relevance3

Relevance

  • Economics close to home

    • Local: Oregon Hatcheries

      • 2004 - $143,000

    • Largest Global Impact:

      • Chile,S.A. and Europe

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Big picture

Big Picture

  • Drug / Vaccine to eliminate bacteria

    • Difficult to treat

    • Current treatments ineffective

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Goal of my project

Goal of My Project

  • Genome analysis

    • American Tissue Culture Collection (ATCC) 33209

    • Accurate representation

    • ERGO by Integrated Genomics

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Specific goal

Specific Goal

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Hypothesis

Hypothesis

  • Due to extended laboratory culture, the genome of strain ATCC33209 has extensive mutations not representative of what is found in nature

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Genetic analysis

Genetic analysis

  • Fibronectin Binding Protein

  • Dipeptide Permease Protein

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

  • Citrate Synthase Protein

  • Tetracycline Resistance Protein P

Jeffrey Burnett - HHMI


Genomic dna

Genomic DNA

  • wt - 2 fish kidneys (A,B)

  • Mt239

  • ATCC33209

  • 684

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Primer design

Primer Design

  • Flank apparent frameshifts identified by ERGO

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

  • Tetracycline Resistance Protein P

Jeffrey Burnett - HHMI


Experiment

Experiment

  • PCR products inserted into expression vectors

  • Plasmids transformed into Escherichia coli

  • Plasmids purified from bacteria

  • Sent for sequencing

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Results

Results

  • Center for Genome Research and Biocomputing (CGRB) - OSU

  • 20 sequences in both directions

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Results1

Results

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Y = yes, the sequence is identical to the ATCC sequence

N = no, the sequence received is different from the ATCC sequence

Jeffrey Burnett - HHMI


Results2

Results

  • Verification of first round of results

  • Reconstruct plasmids from different samples of DNA strains

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Results3

Results

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

  • All of the samples marked “N” ran in duplicate, returned the same results

Jeffrey Burnett - HHMI


Discussion

Discussion

  • Findings are contrary to what we had originally hypothesized

  • Genes are actually more mutated in the other strain isolate DNA that we tested

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Conclusion

Conclusion

  • My research suggests that the ATCC sequence is representative of what is found in nature

  • The bacteria is acquiring more mutations in its genome than the original ATCC strain

Introduction

Hypothesis

Methods

Results

Discussion

Conclusion

Jeffrey Burnett - HHMI


Acknowledgements

Acknowledgements

  • Howard Hughes Medical Institute

    • Dr. Kevin Ahern

  • Dr. Dan Rockey Laboratory

    • Sara Weeks

    • Gina Capri

  • Integrated Genomics

Jeffrey Burnett - HHMI


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