Tularemia Natural History of Francisella tularensis.

Tularemia

Natural History ofFrancisella tularensis

History● Leviticus 11:6-7

– 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

● Wherry WB, Lamb BH. Infection of man with Bacterium tularense. J Infect Dis 1914l 15:331-40

– 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.

– clinical and epidemiological study of >600 human cases

Pathogen – Francisella tularensis● Gram negative non-motile non-sporulating cocco-bacillus

● Reservoir unknown

– free living protozoa?

● Infects small mammals

– ground squirrels, rabbits, hares, voles, muskrats, water rats and other rodents

● Arthropod vectors

– ticks, biting flies, mosquitoes

● Uncommon zoonosis

– 125 cases/year in USA

– farmers, hunters, walkers, forest workers

– kills less than 50 people a year worldwide

Bioterrorism (1)

● Category A agents

– bacterial

● Francisella tularensis (tularemia)● Bacillus anthracis (anthrax)● Yersinia pestis (plague)● Clostridium botulinum (botulism)

– viral

● Variola major (smallpox)● Viral hemorrhagic fever (ebola, marburg, lassa, argentine)

Bioterrorism (2)

● Tested by Japan in WWII as potential bioweapon● Weaponised and stockpiled by USA and USSR

during Cold War● Epidemics (probably natural causes)

– Battle of Stalingrad 1942-1943

– Kosovo 1999 (327 cases, no fatalities)

– Recurrent outbreaks on Martha's vineyard (cause unknown)

Microbiology

Subspecies

● F. tularensis subsp. tularensis (Type A)

– LD50

~ 1 (mice)

– LD50

< 102 (humans)

● F. tularensis subsp. holarctica (Type B)

– LD50

> 106 (rabbits)

● F. tularensis subsp. mediasiatica● F. tularensis subsp. novicida

Taxonomy

Transmission

● Highly infectious– inhalation of 10 bacteria can cause disease

● Avenues– ingestion (water and food)

– inhalation

– direct contact

– arthropod intermediates

– animal bites

● No person to person spread

Vectors in America

Biting flyUtah, Nevada, California

TickEast of Rocky Mountains

Disease Forms

● Ulceroglandular (<5% mortality untreated)● Oculoglandular● Typhoidal syndrome (30-60% mortality

untreated)● Oropharyngeal● Gastrointestinal● Secondary pneumonia● Primary inhalational pneumonia

Clinical Disease● Ulceroglandular

– Incubation period 3-6 days

– Sudden onset flu like symptoms

– Ulcer at site of infection

– Enlargement of draining nodes

● Typhoidal syndrome

– septicemia without ulcer or lymphadenopathy

● Oropharyngeal

– sore throat, enlarged tonsils, yellow-white pseudomembrane

● Gastrointestinal

– persistent diarrhea, bowel ulceration -> acute fatal disease

● Pneumonia

Ulceroglandular Tularemia

Diagnosis

● Culture– high risk to laboratory personnel

● Non-culture methods– Serology

● ELISA● micro-agglutination● Western blot● flow cytometry● indirect immunofluorescence

– PCR

Treatment

● Recommended– Streptomycin

– Gentamicin

● Oral medication– Fluroquinolones (Ciprofloxacin)

● Mortality -> 0● Reduce debilitating features of disease

Vaccines ● Live vaccines

– F. tularensis LVS

– water rat -> USSR -> USAMRIID (1956)

– Use halted by FDA 2001

● Problems– protects against inhalational but not cutaneous disease

– protective response not well understood

– basis of attenuation not known

– potential for reversion

● No effective killed bacterial or subtype vaccine

Model Organism

● Animal models available● Cell culture models available● Live vaccine strain available● Grows rapidly in vivo and in vitro● Genome sequencing in progress● Public health interest

Host-pathogen (0)

Intracellular living1. Cell Entry2. Replication

●cytoplasmic●phagosomal●phagolysosomal

3. Cell Exit

Host-Pathogen (1)

● Obligate intracellular pathogen

– macrophages, hepatocytes, endothelial cells, fibroblasts

● Entry pathway unknown

– no respiratory burst

– complement/complement receptor dependent?

● Intracellular residence

– first 4 hours -> non-acidified phagosome

– after 8 hours -> cytoplasm

● Doubling time in macrophages is 4-6 hours

Host-Pathogen (2)

● Inhibits NF-kB and MAPK activation by TLR ligation

● Evade phagosome-lysosome fusion

● No evidence for secreted toxins

● Induces apoptosis in murine macrophages

Virulence Factors● Atypical LPS

– elicits 1000-fold less TNF and IL-1 than E. coli

● Capsule

– prevents serum-mediated lysis

● Pili

– adherence, cell-cell interactions, biofilm formation

● Proteins

– pathogenicity island (FPI) recently discovered

– mglA, mglB transcriptional regulator

– mglA positively regulates pdpA, pdpD, iglA, iglC, and iglD genes

– iglC (23 kD protein) disrupts TLR signaling

● Other components

– 'phosphoantigens' expand gd-T cells (significance unknown)

Francisella Pathogenecity Island (FPI)

1. 30 kb cluster of genes encoding virulence factors2. flanked by transposable elements3. G+C content significantly different from rest of chromosome

Immunology (1)● Early (<3 days) response is T cell independent

– infected macrophages -> TNF-a

– TNF-a -> NK cells -> IFN-g

– IFN-g -> infected macrophage -> NO

● Late (>3 days) and secondary response is T cell dependent

– Th1 response

– CD4+, CD8+ and Thy1+ CD4-CD8- T cells cells important

– IFN-g, TNF-a, RNS, ROS

– Role of perforin, Fas-FasL cell killing unknown

● Others– gd-T cells expanded

– Antibodies probably minor role

– Neutrophils can phagocytose opsonized bacteria but poor killing

Immunology (2)

● Infection confers long-lasting immunity – may be partial– Edward Francis infected at least 3 times

● Each immune individual recognises a mosaic of antigens

● No immunodominant antigens identified– Orchestration of the protective immune response to intracellular bacteria: Francisella tularensis

as a model organism. FEMS Immunol Med Microb, 1996, 13:221-225

Genome

● 1 partial annotation published– Preliminary analysis and annotation of the partial genome sequence of

Francisella tularensis strain Schu 4. J Appl Microbiol. 2001 Oct;91(4):614-20

– Predicted ORFs with no match in GenBank much higher than other microbial genomes

– No matches with known virulence genes in other pathogens

● 2 preliminary genome sequences completed– http://bbrp.llnl.gov/bbrp/html/microbe.html (Lawrence Livermore National

Laboratory)

– http://artedi.ebc.uu.se/Projects/Francisella/ (Uppsala University)

Transcriptome

● 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● Papers

– Hubalek M et al, Proteomics. 2004 Oct;4(10):3048-60.

– Hubalek M et al, J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Apr 5;787(1):149-77.

– Havlasova J et al, Proteomics. 2002 Jul;2(7):857-67.

– Kovarova H et al, Proteomics. 2002 Jan;2(1):85-93.

– Hernychova L et al, Proteomics. 2001 Apr;1(4):508-15.

● 2D PAGE Databases– http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi

● 3D protein structure– http://pat.sdsc.edu/perl/browser.pl?tax=Francisella%20tularensis&tid=263

References

● J Infect Dis, 2004, 189:1317-1331● Clin Microb Rev, 2002, 15:631-646● Trends Microb, 2003, 11:118-123● Curr Opin Microb, 2003, 6:66-71● Lancet, 2000, 356:1179-1182● Infect Immun, 2004 72:3204-3217● J Bacteriol, 2004,186:6430-6436