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Tularemia
Natural History of
Francisella tularensis
History
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Leviticus 11:6-7
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Wherry WB, Lamb BH. Infection of man with Bacterium
tularense. J Infect Dis 1914l 15:331-40
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But you shall not eat any of the following that only
chew the cud or only have hoofs ... the hare, which
indeed chews the cud, but does not have hoofs and is
therefore unclean for you
first description of human tularemia with culture of
causative organism
Francis E. A summary of the present knowledge of
tularemia. Medicine (Baltimore) 1928;7:411-32.
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clinical and epidemiological study of >600 human
cases
Pathogen – Francisella tularensis
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Gram negative non-motile non-sporulating cocco-bacillus
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Reservoir unknown
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Infects small mammals
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ground squirrels, rabbits, hares, voles, muskrats, water rats and
other rodents
Arthropod vectors
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free living protozoa?
ticks, biting flies, mosquitoes
Uncommon zoonosis
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125 cases/year in USA
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farmers, hunters, walkers, forest workers
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kills less than 50 people a year worldwide
Bioterrorism (1)
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Category A agents
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bacterial
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Francisella tularensis (tularemia)
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Bacillus anthracis (anthrax)
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Yersinia pestis (plague)
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Clostridium botulinum (botulism)
viral
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Variola major (smallpox)
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Viral hemorrhagic fever (ebola, marburg, lassa, argentine)
Bioterrorism (2)
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Tested by Japan in WWII as potential bioweapon
Weaponised and stockpiled by USA and USSR
during Cold War
Epidemics (probably natural causes)
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Battle of Stalingrad 1942-1943
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Kosovo 1999 (327 cases, no fatalities)
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Recurrent outbreaks on Martha's vineyard (cause
unknown)
Microbiology
Subspecies
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F. tularensis subsp. tularensis (Type A)
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LD50 ~ 1 (mice)
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LD50 < 102 (humans)
F. tularensis subsp. holarctica (Type B)
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LD50 > 106 (rabbits)
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F. tularensis subsp. mediasiatica
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F. tularensis subsp. novicida
Taxonomy
Transmission
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Highly infectious
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inhalation of 10 bacteria can cause disease
Avenues
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ingestion (water and food)
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inhalation
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direct contact
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arthropod intermediates
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animal bites
No person to person spread
Vectors in America
Biting fly
Utah, Nevada, California
Tick
East of Rocky Mountains
Disease Forms
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Ulceroglandular (<5% mortality untreated)
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Oculoglandular
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Typhoidal syndrome (30-60% mortality
untreated)
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Oropharyngeal
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Gastrointestinal
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Secondary pneumonia
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Primary inhalational pneumonia
Clinical Disease
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Ulceroglandular
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Incubation period 3-6 days
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Sudden onset flu like symptoms
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Ulcer at site of infection
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Enlargement of draining nodes
Typhoidal syndrome
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Oropharyngeal
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sore throat, enlarged tonsils, yellow-white pseudomembrane
Gastrointestinal
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septicemia without ulcer or lymphadenopathy
persistent diarrhea, bowel ulceration -> acute fatal disease
Pneumonia
Ulceroglandular Tularemia
Diagnosis
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Culture
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high risk to laboratory personnel
Non-culture methods
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Serology
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ELISA
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micro-agglutination
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Western blot
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flow cytometry
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indirect immunofluorescence
PCR
Treatment
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Recommended
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Streptomycin
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Gentamicin
Oral medication
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Fluroquinolones (Ciprofloxacin)
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Mortality -> 0
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Reduce debilitating features of disease
Vaccines
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Live vaccines
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F. tularensis LVS
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water rat -> USSR -> USAMRIID (1956)
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Use halted by FDA 2001
Problems
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protects against inhalational but not cutaneous
disease
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protective response not well understood
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basis of attenuation not known
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potential for reversion
No effective killed bacterial or subtype vaccine
Model Organism
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Animal models available
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Cell culture models available
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Live vaccine strain available
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Grows rapidly in vivo and in vitro
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Genome sequencing in progress
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Public health interest
Host-pathogen (0)
Intracellular living
1. Cell Entry
2. Replication
●cytoplasmic
●phagosomal
●phagolysosomal
3. Cell Exit
Host-Pathogen (1)
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Obligate intracellular pathogen
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macrophages, hepatocytes, endothelial cells, fibroblasts
Entry pathway unknown
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no respiratory burst
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complement/complement receptor dependent?
Intracellular residence
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first 4 hours -> non-acidified phagosome
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after 8 hours -> cytoplasm
Doubling time in macrophages is 4-6 hours
Host-Pathogen (2)
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Inhibits NF-kB and MAPK activation by TLR ligation
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Evade phagosome-lysosome fusion
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No evidence for secreted toxins
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Induces apoptosis in murine macrophages
Virulence Factors
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Atypical LPS
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Capsule
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prevents serum-mediated lysis
Pili
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elicits 1000-fold less TNF and IL-1 than E. coli
adherence, cell-cell interactions, biofilm formation
Proteins
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pathogenicity island (FPI) recently discovered
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mglA, mglB transcriptional regulator
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mglA positively regulates pdpA, pdpD, iglA, iglC, and iglD genes
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iglC (23 kD protein) disrupts TLR signaling
Other components
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'phosphoantigens' expand gd-T cells (significance unknown)
Francisella Pathogenecity Island (FPI)
1. 30 kb cluster of genes encoding virulence factors
2. flanked by transposable elements
3. G+C content significantly different from rest of chromosome
Immunology (1)
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Early (<3 days) response is T cell independent
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infected macrophages -> TNF-a
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TNF-a -> NK cells -> IFN-g
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IFN-g -> infected macrophage -> NO
Late (>3 days) and secondary response is T cell dependent
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Th1 response
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CD4+, CD8+ and Thy1+ CD4-CD8- T cells cells important
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IFN-g, TNF-a, RNS, ROS
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Role of perforin, Fas-FasL cell killing unknown
Others
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gd-T cells expanded
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Antibodies probably minor role
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Neutrophils can phagocytose opsonized bacteria but poor killing
Immunology (2)
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Infection confers long-lasting immunity – may be
partial
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Edward Francis infected at least 3 times
Each immune individual recognises a mosaic of
antigens
No immunodominant antigens identified
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Orchestration of the protective immune response to intracellular bacteria: Francisella
tularensis as a model organism. FEMS Immunol Med Microb, 1996, 13:221-225
Genome
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1 partial annotation published
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Preliminary analysis and annotation of the partial genome sequence of
Francisella tularensis strain Schu 4. J Appl Microbiol. 2001 Oct;91(4):61420
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Predicted ORFs with no match in GenBank much higher than other
microbial genomes
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No matches with known virulence genes in other pathogens
2 preliminary genome sequences completed
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http://bbrp.llnl.gov/bbrp/html/microbe.html (Lawrence Livermore National
Laboratory)
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http://artedi.ebc.uu.se/Projects/Francisella/ (Uppsala University)
Transcriptome
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Samrakandi MM et al, Genome diversity among regional populations of
Francisella tularensis subspecies tularensis and Francisella tularensis
subspecies holarctica isolated from the US, FEMS Microbiol Lett. 2004 Aug
1;237(1):9-17.
Broekhuijsen M et al, Genome-wide DNA microarray analysis of Francisella
tularensis strains demonstrates extensive genetic conservation within the species
but identifies regions that are unique to the highly virulent F. tularensis subsp.
tularensis, J Clin Microbiol. 2003 Jul;41(7):2924-31.
Proteome
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Papers
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Hubalek M et al, Proteomics. 2004 Oct;4(10):3048-60.
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Hubalek M et al, J Chromatogr B Analyt Technol Biomed Life Sci. 2003
Apr 5;787(1):149-77.
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Havlasova J et al, Proteomics. 2002 Jul;2(7):857-67.
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Kovarova H et al, Proteomics. 2002 Jan;2(1):85-93.
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Hernychova L et al, Proteomics. 2001 Apr;1(4):508-15.
2D PAGE Databases
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http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi
3D protein structure
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http://pat.sdsc.edu/perl/browser.pl?tax=Francisella%20tularensis&tid=263
References
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J Infect Dis, 2004, 189:1317-1331
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Clin Microb Rev, 2002, 15:631-646
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Trends Microb, 2003, 11:118-123
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Curr Opin Microb, 2003, 6:66-71
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Lancet, 2000, 356:1179-1182
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Infect Immun, 2004 72:3204-3217
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J Bacteriol, 2004,186:6430-6436