TEAM 1
Investigation of bacteriophages of the
bird pathogen, Bordetella avium
Introduction
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
Hosts for B. avium
SO …what?
B. avium infections
result in severe
economic losses in all
poultry-producing
regions of the world.
What is a bacteriophage?


A bacteriophage is a virus that solely
infects bacteria.
What is a virus?


Obligate parasite, cannot replicate itself
Needs a host cell for survival
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
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
http://www.msu.edu/course/lbs/145/s02/graphics/campbell_18.5.gif
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
SEM of Phage
About 2/3 of the
chromosomes
are the same
Ends- enzymes
Center- structure
Goal
To determine which
phage(s) were in each
B. avium strain
Methods and Materials
Behavior in Live Bacteria

How did we check for
active phage?
Spontaneous lysis
 197N infection

Spontaneous Lysis



Grow single colonies of bacteria in
Brain Heart Infusion (BHI) broth
Add culture to melted BHI top agar
and spread over a BHI plate
Incubate at 30°C for 18-24 hours,
examine for plaques
Cartoon of a plate with plaques
plaque
bacteria
Testing bacterial strains for
phage that infect 197N



Add two drops chloroform to cultures
to kill bacteria, leaving only phage
Combine this culture with 197N
culture, plate and incubate
Examine for plaques
Polymerase Chain Reaction
Goal: amplification of a small
amount of DNA
 Ingredients: template DNA,
primers, nucleotides, and
thermostable Taq Polymerase

Polymerase Chain Reaction

Ingredients run through a series of
heating and cooling cycles
Denaturation – DNA separated into
two strands
 Annealing – Primers attach
 Polymerization – free bases attach

Our Primers
Beginning primers unique to Ba11 and Ba1-2 (left)
 Recombinase primers from Ba1-1
and Ba1-2 (right)
 Tail Fiber primer identical in both
strains (middle)

Gel Electrophoresis

Method used to analyze PCR
PCR product injected into gel wells
 Apply electrical field
 DNA travels from the negative electrode
to the positive


Travels through gel based on size
Gel Electrophoresis



Results read as dark lines in the gel
Fragment size read against a 1 kb (1000
base pair) ladder
Ethidium Bromide makes discrete bands
visible under UV
Agarose Gel & Gel
Electrophoresis
Results
Results for spontaneous
lysis and infection of 197 N
Strain
Spontaneous
Lysis
Infection of
197 N
Wampler
0
0
+
+
0
0
197 N
JBBA
Gel of Repressor Primer
Results for PCR
Strain
Unk-1
Rec 1
Tail fiber
Rep-2
Rec 2
Wampler
+
+
+
+
0
197 N
0
0
+
+
0
G24
0
0
0
0
0
Discussion
Discussion

Interpretation of results

Effectiveness of methods

Future applications and extensions of our work
Reasons for Tests

Behavior of phage in strain of B.avium
 Spontaneous lysis test
 197N infection test

Which part of phage DNA in bacterial DNA?
 PCR & gel electrophoresis using primers that
amplified pieces of DNA from different phages
Spontaneous Lysis
Which strains
made plaques?
What does that
mean?
What if there were
no plaques?
T4
Wampler
239

Phage present
and lytic


Phage absent
Phage highly
lysogenic
Not enough
phage
Infection of 197N
Which strains
made plaques?
What does that
mean?
What if there were
no plaques?
T4
Wampler
239
Ba011
Ba177
DBL260
ATCC

Phage present
and able to infect
197N


Phage absent
Phage unable to
infect 197N
Not enough
phage
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?
T4 and Wampler
•Positive in four primers
•Spontaneous Lysis, Infection
results also positive
•Probably contain both phages
D4, D10, and D27

All PCR primers yielded positive


Probably contains phages
Spontaneous Lysis, Infection results
negative
Could be phage debris
 May be in lysogenic cycle
 Conditions may not be right for lysis

G24
All PCR Primers yield negative
 Spontaneous Lysis, Infection
negative
 Probably does not contain
phages

197N and ATCC


Some negative, some positive
ATCC—Unk-1, Rec 1, TF positive
Infection positive
 Probably Ba 1-1, not Ba 1-2


197N—Rep-2, TF positive
Infection positive
 Probably Ba 1-2,not Ba 1-1

What’s next?

Experiments only the beginning

More PCR
Results not perfect, more PCR means
more accuracy
 Different primers


More Spontaneous Lysis and Infection

Don’t fully understand conditions for lysis
More Future Projects

Compare DNA/Genes of Ba 1-1 and
1-2
Similar genes code for proteins common
in both phages—head, tail fiber…
 Different genes will code for
differences—enzymes
 Researchers can discover what makes
the strains different

Acknowledgements

Thank you to…
Dr. Temple
 Holly Kuzmiak
 Kelly Prescott
 Octawia Wojcik
 Drew Biology Department
 Dr. Miyamoto
