P1251328618sFKbh

1
(No Transcript)
2
SPIROCHAETE BACTERIA
Treponema pallidum (syphilis)
http//www.geocities.com/CapeCanaveral/3504/galler
y.htm
3
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/nor
thamerica/states/california/explore/
4
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 gt livestock gt rodents gt wild mammals.
NO VECTORS INVOLVED IN LIFE CYCLE!
http//www.vetmed.wisc.edu/pbs/zoonoses/Leptospira
/leptoindex.html http//www.leptospirosis.org/bact
eria/
5
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/
6
(No Transcript)
7
(No Transcript)
8
VECTOR LIFE CYCLE
http//www.cdc.gov/ncidod/dvbid/lyme/
9
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.
10
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!

11
WHERE DOES LYME DISEASE OCCUR?
12
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.
13
(No Transcript)
14
Larvae and nymphs can also be infected by
Borrelia exposure via capillary tube,
microinjection, or immersion.
OspA -gt OspC expression. Ticks eat for 3-7 days,
and only efficiently transmit Borrelia after 2
days of feeding.
15
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.

16
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?

17
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.

18
Allelic Exchange
Shuttle Vectors
Transposon mutagenesis
19
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.

20
(No Transcript)
21
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 -gt reduced infectivity in vivo.
• Stable plasmids have non-essential genes such as
  chb and OspConly active under certain conditions?

22
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?

23
It all began in Lyme, Connecticut!