Download Wk4- Vectors

SPIROCHAETE BACTERIA
Treponema pallidum (syphilis)
http://www.geocities.com/CapeCanaveral/3504/gallery.htm
LEPTOSPIROSIS
• "rice-field fever" and "cane-cutters
fever" and "swine herder's disease“.
• SYMPTOMS: high fever, severe
headache, chills, muscle aches,
vomiting, jaundice (yellow skin and
eyes), red eyes, abdominal pain,
diarrhea, or a rash.
• If the disease is not treated, the
patient could develop kidney damage,
meningitis (inflammation of the
membrane around the brain and
spinal cord), liver failure, and
respiratory distress. In rare cases
death occurs.
http://www.vetmed.wisc.edu/pbs/zoonoses/Leptospira/leptoindex.html
http://nature.org/wherewework/northamerica/states/california/explore/
LEPTOSPIROSIS RESERVOIRS
Specific serovars are "hosted-adapted" to particular reservoir
species and generally do not cause disease in those hosts, e.g.:
- L. canicola/dogs
- L. icterohaemorrhagiae/rats
- L. grippotyphosa/voles, raccoons and other small mammals
- L. bratislava/pigs, and rats and other small mammals
Leptospires can persist in the renal tubules without causing
disease, and can be excreted in the urine for very prolonged
periods of time.
Rats are the most common source of infection for humans
worldwide.
In the U.S., however, the most significant sources of
infection for humans are dogs > livestock > rodents > wild
mammals.
NO VECTORS INVOLVED IN LIFE CYCLE!
http://www.vetmed.wisc.edu/pbs/zoonoses/Leptospira/leptoindex.html
http://www.leptospirosis.org/bacteria/
BORELLIA: LYME DISEASE
• SYMPTOMS: "bull's-eye" rash,
erythema migrans,
accompanied by nonspecific
symptoms such as fever,
malaise, fatigue, headache,
muscle aches (myalgia), and
joint aches (arthralgia).
The incubation period from
infection to onset of erythema
migrans is typically 7 to 14
days but may be as short as 3
days and as long as 30 days.
http://www.cdc.gov/ncidod/dvbid/lyme/
VECTOR LIFE CYCLE:
http://www.cdc.gov/ncidod/dvbid/lyme/
Are Lizards Lethal?
• Lizards, rodents, and birds are all hosts for
Ixodes ticks in Northern California.
• Borrelia burgdorferi infection is 3-4 times
greater in nymphal ticks than adult ticks.
• Borrelia are rarely identified in lizard
blood… are lizards lethal for Borrelia?
Eisen, Eisen, and Lane, 2004. The roles of birds, lizards, and rodents as hosts for the western black-legged tick Ixodes pacificus.
Journal of Vector Ecology 29(2): 295-308.
Lane and Quinstad, 1998. Borreliacidal factor in the blood of the western fence lizard (Sceloporus occidentalis). Journal of
Parasitology 84(1): 29-34.
Borreliacidal Factors in Lizard Blood!
• 10 lizards were exposed to 10 nymphs having 78%
Borrelia prevalence.
• After feeding and molting, the none of the adult ticks
contained Borrelia!
• In contrast, 55% of infected nymphs that fed on
rabbits passed Borrelia to adult ticks.
• Borrelia in lizard blood died within 1 hr as compared
to 72 hrs in mouse blood. Boiling the lizard blood
inactivated the borreliacidal response and the
Borrelia were then able to survive for the 72 hrs!
WHERE DOES LYME
DISEASE OCCUR?
Not your everyday bacteria!
-Inner and outer membranes.
-Periplasmic flagella.
-Flexible cell wall.
-No LPS.
-Linear chromosome.
-Many plasmids.
-Complete genome known.
-Difficult to genetically manipulate.
-In vitro different than in vivo?
-Mouse-tick experimental model
mimics natural infection exists.
Larvae and nymphs can also
be infected by Borrelia
exposure via capillary tube,
microinjection, or immersion.
OspA -> OspC expression.
Ticks eat for 3-7 days, and
only efficiently transmit
Borrelia after 2 days of
feeding.
LABORATORY MANIPULATION
• IN VIVO:
– Culture Borrelia within a dialysis membrane in
a rat abdomen.
– Borrelia can also be isolated from tissues of
infected animals in limited amounts.
• IN VITRO:
– Culture Borrelia using BSK growth media but
colonies take 1-2 weeks to appear and a
transformation experiment may take 1 month.
GENETIC STUDIES
• Selectable markers: antibiotic resistance
genes such as gyrB, ermC, kan. Avoid
generating ampicillin and tetracycline
resistant Borrelia since those antibiotics
are used in disease treatment.
• Strains: non-infectious strains utilized most
often because they are easier to
transform. Tradeoffs?
GENETIC MANIPULATION
• Allelic recombination:
– Electroporation with gene inactivation constructs and
antibiotic resistance reporter.
• Shuttle vectors:
– Broad host range vector for autonomous replication of
introduced DNA.
– Stability important.
– Green fluorescent protein (GFP) also used as reporter.
• Transposon mutagenesis:
– Random tagged mutagenesis in genome possible.
– Localized saturated mutagenesis still not possible.
Allelic Exchange
Shuttle Vectors
Transposon mutagenesis
TRANSFORMATION
• Electroporation or chemical methods.
• Low frequency and efficiency.
• Large amounts of DNA required.
• Plasmids may have restriction-modification
systems that can prevent transformation.
• Infectious clones are harder to work with than
non-infectious clones… so limited conclusions
can be drawn as to mutant Borrelia virulence
and pathogenicity.
BORRELIA PLASMIDS
• Largest number of plasmids in any known
bacterial genome.
• Encode essential functions such as resT
telomere resolvase on cp26 plasmid.
• Unstable plasmid loss of essential lp25 in
vitro -> reduced infectivity in vivo.
• Stable plasmids have non-essential genes such
as chb and OspC…only active under certain
conditions?
QUORUM SENSING?
• Borrelia has a luxS homologue.
• Borrelia luxS complemented E.coli luxS
deficiency and altered Borrelia protein
synthesis…
• But no autoinducers were detected in
supernatant of Borrelia cultures… is quorum
sensing only active under certain conditions?
It all began in Lyme, Connecticut!