1. TEAM 1 Investigation of bacteriophages of the bird pathogen, Bordetella avium

2. Introduction

3. What is Bordetella avium? Bacteria that causes upper respiratory disease bordetellosis in avian species (chicken, turkey, etc.) Gram negative, non-fermentable, aerobic and motile Infects commercially grown turkeys throughout the world

4. Hosts for B. avium

5. SO …what? B. avium infections result in severe economic losses in all poultry-producing regions of the world.

6. What is a bacteriophage? A bacteriophage is a virus that solely infects bacteria. A bacteriophage is a virus that solely infects bacteria. What is a virus? What is a virus? Obligate parasite, cannot replicate itself Obligate parasite, cannot replicate itself Needs a host cell for survival Needs a host cell for survival

7. Structure of Bacteriophage Phage head: composed of coat protein and genome in the core Genome: DNA codes for enzymes and proteins necessary to replicate more viruses Tail Sheath: DNA travels from head to bacteria through sheath Tail fiber: helps anchor the phage on the cell membrane

8. Life cycles of a Temperate Phage Two life cycles: Lytic cycle – viruses lyse the cell after replicating in the host cell Lysogenic cycle – viral DNA integrates into host cell DNA to replicate, but no new viruses are synthesized

9. http://www.msu.edu/course/lbs/145/s02/graphics/campbell_18.5.gif

10. Ba1-1 and Ba1-2 First identified as Ba1 After sequencing, two phage chromosomes were found Ba1-1, Ba1-2 Under electron microscope, look exactly the same

11. SEM of Phage

12. About 2/3 of the chromosomes are the same Ends- enzymes Center- structure

13. Goal To determine which phage(s) were in each B. avium strain

14. Methods and Materials

15. Behavior in Live Bacteria How did we check for active phage? How did we check for active phage? Spontaneous lysis Spontaneous lysis 197N infection 197N infection

16. Spontaneous Lysis Grow single colonies of bacteria in Brain Heart Infusion (BHI) broth Grow single colonies of bacteria in Brain Heart Infusion (BHI) broth Add culture to melted BHI top agar and spread over a BHI plate Add culture to melted BHI top agar and spread over a BHI plate Incubate at 30°C for 18-24 hours, examine for plaques Incubate at 30°C for 18-24 hours, examine for plaques

17. Cartoon of a plate with plaques plaque bacteria

18. Testing bacterial strains for phage that infect 197N Add two drops chloroform to cultures to kill bacteria, leaving only phage Add two drops chloroform to cultures to kill bacteria, leaving only phage Combine this culture with 197N culture, plate and incubate Combine this culture with 197N culture, plate and incubate Examine for plaques Examine for plaques

19. Polymerase Chain Reaction Goal: amplification of a small amount of DNA Goal: amplification of a small amount of DNA Ingredients: template DNA, primers, nucleotides, and thermostable Taq Polymerase Ingredients: template DNA, primers, nucleotides, and thermostable Taq Polymerase

20. Polymerase Chain Reaction Ingredients run through a series of heating and cooling cycles Ingredients run through a series of heating and cooling cycles Denaturation – DNA separated into two strands Denaturation – DNA separated into two strands Annealing – Primers attach Annealing – Primers attach Polymerization – free bases attach Polymerization – free bases attach

22. Our Primers Beginning primers unique to Ba1- 1 and Ba1-2 (left) Beginning primers unique to Ba1- 1 and Ba1-2 (left) Recombinase primers from Ba1-1 and Ba1-2 (right) Recombinase primers from Ba1-1 and Ba1-2 (right) Tail Fiber primer identical in both strains (middle) Tail Fiber primer identical in both strains (middle)

24. Gel Electrophoresis Method used to analyze PCR Method used to analyze PCR PCR product injected into gel wells PCR product injected into gel wells Apply electrical field Apply electrical field DNA travels from the negative electrode to the positive DNA travels from the negative electrode to the positive Travels through gel based on size Travels through gel based on size

25. Gel Electrophoresis Results read as dark lines in the gel Results read as dark lines in the gel Fragment size read against a 1 kb (1000 base pair) ladder Fragment size read against a 1 kb (1000 base pair) ladder Ethidium Bromide makes discrete bands visible under UV Ethidium Bromide makes discrete bands visible under UV

26. Agarose Gel & Gel Electrophoresis

27. Results

28. Results for spontaneous lysis and infection of 197 N Strain Spontaneous Lysis Infection of 197 N Wampler0+ 197 N 00 JBBA+0

29. Gel of Repressor Primer

30. Results for PCR StrainUnk-1 Rec 1 Tail fiber Rep-2 Rec 2 Wampler++++0 197 N 00++0 G2400000

31. Discussion

32. Discussion Interpretation of results Interpretation of results Effectiveness of methods Effectiveness of methods Future applications and extensions of our work Future applications and extensions of our work

33. Reasons for Tests Behavior of phage in strain of B.avium Behavior of phage in strain of B.avium Spontaneous lysis test Spontaneous lysis test 197N infection test 197N infection test Which part of phage DNA in bacterial DNA? Which part of phage DNA in bacterial DNA? PCR & gel electrophoresis using primers that amplified pieces of DNA from different phages PCR & gel electrophoresis using primers that amplified pieces of DNA from different phages

34. Spontaneous Lysis Which strains made plaques? What does that mean? What if there were no plaques? T4 T4 Wampler Wampler 239 239 Phage present and lytic Phage present and lytic Phage absent Phage absent Phage highly lysogenic Phage highly lysogenic Not enough phage Not enough phage

35. Infection of 197N Which strains made plaques? What does that mean? What if there were no plaques? T4 T4 Wampler Wampler 239 239 Ba011 Ba011 Ba177 Ba177 DBL260 DBL260 ATCC ATCC Phage present and able to infect 197N Phage present and able to infect 197N Phage absent Phage absent Phage unable to infect 197N Phage unable to infect 197N Not enough phage Not enough phage

36. The plates don’t tell… If plaques… which phage active in that strain? If no plaques… does the bacterial strain still contain some phage DNA? Which pieces?

37. T4 and Wampler Positive in four primers Spontaneous Lysis, Infection results also positive Probably contain both phages

38. D4, D10, and D27 All PCR primers yielded positive All PCR primers yielded positive Probably contains phages Probably contains phages Spontaneous Lysis, Infection results negative Spontaneous Lysis, Infection results negative Could be phage debris Could be phage debris May be in lysogenic cycle May be in lysogenic cycle Conditions may not be right for lysis Conditions may not be right for lysis

39. G24 All PCR Primers yield negative All PCR Primers yield negative Spontaneous Lysis, Infection negative Spontaneous Lysis, Infection negative Probably does not contain phages Probably does not contain phages

40. 197N and ATCC Some negative, some positive Some negative, some positive ATCC—Unk-1, Rec 1, TF positive ATCC—Unk-1, Rec 1, TF positive Infection positive Infection positive Probably Ba 1-1, not Ba 1-2 Probably Ba 1-1, not Ba 1-2 197N—Rep-2, TF positive 197N—Rep-2, TF positive Infection positive Infection positive Probably Ba 1-2,not Ba 1-1 Probably Ba 1-2,not Ba 1-1

41. What’s next? Experiments only the beginning Experiments only the beginning More PCR More PCR Results not perfect, more PCR means more accuracy Results not perfect, more PCR means more accuracy Different primers Different primers More Spontaneous Lysis and Infection More Spontaneous Lysis and Infection Don’t fully understand conditions for lysis Don’t fully understand conditions for lysis

42. More Future Projects Compare DNA/Genes of Ba 1-1 and 1-2 Compare DNA/Genes of Ba 1-1 and 1-2 Similar genes code for proteins common in both phages—head, tail fiber… Similar genes code for proteins common in both phages—head, tail fiber… Different genes will code for differences—enzymes Different genes will code for differences—enzymes Researchers can discover what makes the strains different Researchers can discover what makes the strains different

43. Acknowledgements Thank you to… Thank you to… Dr. Temple Dr. Temple Holly Kuzmiak Holly Kuzmiak Kelly Prescott Kelly Prescott Octawia Wojcik Octawia Wojcik Drew Biology Department Drew Biology Department Dr. Miyamoto Dr. Miyamoto