Francisella tularensis

1. Francisella tularensis Tularemia

2. Francisella tularensis • Gram stain • Poorly staining, tiny Gram-negative coccobacilli

3. Francisella tularensis - One of the most infectious pathogenic bacteria known - Inoculation or inhalation of as few as ten organisms can cause disease - Extreme infectivity - Substantial capacity to cause illness and death + Humans cannot transmit infection to others

4. Can Survive For Weeks • Water • Soil • Moist hay • Straw • Decaying animal carcasses Because it is…. Hardy, non-spore forming organism

5. Reservoirs Small and medium sized mammals are the principal natural reservoirs for F. tularensis • Rabbits • Aquatic Rodents (Beavers, Muskrats) • Rats • Squirrels • Lemmings • Mice

6. Vectors • Ticks • Mosquitoes • Biting Flies

7. Also Known As… • Deer-fly fever (Utah) • Glandular tick fever (Idaho and Montana) • Market men’s disease (Washington D.C.) • Rabbit fever (Central States) • O’Hara’s disease (Japan)

8. History • First isolated in 1911 from a plague-like disease among ground squirrels in California • Its epidemic potential became apparent in the 1930’s and 1940’s when large waterborne outbreaks occurred in Europe and the Soviet Union and epizootic-associated cases occurred in the U.S.

9. Incidence across the Globe

10. Modern Worldwide Death Rate • Before antibiotics: pneumonic tularemia—50% localized tularemia—5% • After antibiotics: 2.3%

11. Reported Cases of Tularemia - 1990-1998

12. Four States Four states accounted for 56% of all reported tularemia cases • Arkansas (23%) • Missouri (19%) • South Dakota (7%) • Oklahoma (7%)

13. U.S. Outbreaks • Vermont, 1968 • 47 cases in people who handled muskrats four weeks before the onset of the illness • No fatalities, but 14 patients had severe prostrating illness that lasted an average of ten days • Utah, 1971 • 39 cases, most contracted from the bite of an infected deerfly • All patients recovered

14. U.S. Outbreaks, cont. • South Dakota, 1984 • 20 cases of glandular tularemia in children • Illness was mild • presumed to be caused by type B • Martha’s Vineyard, 2000 • 15 cases of tularemia • 11 patients had primary pneumonic disease • 1 fatality • Caused by type A

15. OUTBREAK!August, 2002—Prairie Dogs • Health officials were notified that some prairie dogs at a Texas pet distribution facility had died unexpectedly • After officials determined that they had died of Tularemia, further investigation found that several hundreds of potentially infected dogs were shipped to Ohio, West Virginia, Florida, Washington, Mississippi, Nevada, Illinois, and Virginia

16. It gets even worse, as… • Shipments also went out to Japan, the Czech Republic, the Netherlands, Belgium, Spain, Italy, and Thailand

17. Case Incidence • The highest incidence of cases was in 1939, when 2,291 cases were reported • The number remained high throughout the 1940’s • Declined in 1950’s to the relatively constant number of cases it is now—less than 200 per year • Most cases occur in rural environments; rarely do they occur in urban settings

18. Why the decrease in cases? • The development of effective antibiotics • Decrease in hunting in the U.S. and other developed nations reduced human exposure

19. Fransicella tularensisHistorical Background • First described by McCoy in 1912 as agent responsible for a tularemia outbreak in Tulare County in California and isolated the organism from infected squirrels. • Francis one of the premier researchers in the field elucidated the route of infection in man as: • RodentsBlood Sucking InsectsMan

20. Fransicella tularensisArthropod Vectors • Primary vectors are ticks (United States, former Soviet Union, and Japan), mosquitoes (former Soviet Union, Scandinavia, and the Baltic region), and biting flies (United States [particularly Utah, Nevada, and California] and former Soviet Union). Examples of specific species include: • Ticks: Amblyomma americanum (Lone Star tick), Dermacentor andersoni (Rocky Mountain wood tick), Dermacentor variabilis (American dog tick), Ixodes scapularis, Ixodes pacificus, and Ixodes dentatus • Mosquitoes: Aedes cinereus and Aedes excrucians • Biting flies: Chrysops discalis (deerfly), Chrysops aestuans, Chrysops relictus, and Chrysozona pluvialis

21. Francisella tularensisMorphology and Physiology I • Small, weakly staining gram-negative coccobacillus 0.2 to 0.2 – 0.7 um in size. • Nonmotile, displays bipolar staining with Giemsa stain, obligate anaerobe, and is weakly catalase positive. • Young cultures are relatively uniform in appearance while older cultures display extreme pleomorphism. • Carbohydrates are dissimilated slowly with the production of acid but no gas. • Displays a thick capsule whose loss is accompanied by loss of virulence.

22. Francisella tularensisMorphology and Physiology II • The lipid concentration in the capsule and cell wall (50 – 70%, respectively) is unusually high for a gram negative organism. • The lipid composition is unique with relatively large amounts of long-chain saturated and monoenoic C20 to C26 fatty acids as well as alpha and beta hydroxyl fatty acids. • Biochemical characterization is of little value in identification (other tests are utilized).

23. Francisella tularensisCulture Characteristics • Optimal growth at 370 C, growth range 240 to 390 C. Survival rate is best at lower temperatures. • Slow growing with a requirement for iron and cysteine or cystine. • No growth on routine culture media but small colony growth after 2 - 4 days on glucose-cysteine-blood agar or peptone-cysteine agar. • No true hemolysis on blood containing media only a greenish discoloration.

24. Francisella tularensisMicrobial Genomics-Introduction • Little is known about the cellular and molecular modes of infection, proliferation and immune response to tularemia. • Microbial genomics has begun to hopefully shed some light on the above mechanisms. • The lack of adequate genetic tools has hampered efforts to elucidate many questions about F. tularensis most importantly how it enters cells and the factors required for intracellular growth. • At present most of the genome of F. tularensis ShuS4 (high virulence) has been sequenced, compiled into ‘contigs’ and is available at the web site http://artedi.ebc.uu.se/Projects/Francisella/

25. Francisella tularensis Microbial Genomics-Intracellular Growth Genes I • Five genetic loci with the use of transposon mutagenesis have been identified in F. novicida that are associated with intracellular growth. • Gene 1: Alanine racemase catalyzes the reversible conversion of the L form of alanine to the D form. Potential effect: Alter bacterial cell wall making it more susceptible to microbiocidal agents produced by macrophages. • Gene 2: Glutamine phosphoribosylpyrophosphate amidotransferases (50% identity at a.a. level) which catalyzes the first step in de novo purine biosynthesis. Potential effect: Inhibition of de novo purine biosynthesis.

26. Francisella tularensis Microbial Genomics-Intracellular Growth Genes II • Gene 3: ClpB (60% identity to E.coli protein) an ATP-dependent protease stress response protein which hydrolyzes casein and is part of a system which hydrolyzes denatured proteins. Potential effect: Inhibit the removal of denatured proteins overwhelming cell. • Gene 4: 23Kd protein (99% identity) unique to Francisella as the dominantly induced protein after infection. Potential effect: Unknown. • Gene 5: AF374673 no significant similarity to any protein with a known function. Potential effect: Unknown.

27. Francisella tularensis Microbial Genomics-Intracellular Growth Genes III • The five genes found to be involved in intracellular growth all map using the available genomic sequence map to the intracellular growth locus iglABCD. • The iglABCD is a putative operon involved in intracellular growth and it is possible that all of the proteins encoded by the iglABCD operon are needed for intracellular growth and some are thought to be transcription factors. • The predicted molecular masses of the protein products from these genes corresponds to the masses of the observed proteins expressed during intracellular growth. • These observations suggest that these proteins play a critical role in the intracellular growth of F. tullarensis.

28. Francisella tularensisMicrobial Genomics-Tools • Yet another odd characteristic of F. tularensis is the absence of its own plasmids in any of the biovars. It is not clear whether this property is associated with the environment of the bacterium or with the specificity of its genetic apparatus. • It has been shown that heterologous plasmids can replicate in F. tularensis but must be maintained by antibiotic resistance selection. • One isolate, F. novidica-like strain F6168, is the only member of the genus that carries a native plasmid and this plasmid has no known function or gene products. • The 3990-bp cryptic plasmid from F6168 has been used to construct two recombinant plasmids, pFNL10 and pOM1. These plasmids were engineered to contain antibiotic selection genes, a polylinker for cloning, and the ori (origin of replication) from F6168. A third plasmid pKK214 has been designed to assay promoter activity. • These plasmid tools will hopefully help to elucidate some of the mechanisms of intracellular growth and virulence.

29. Francisella tularensis Microbial Genomics-Identification • Extensive allelic variation in the short sequence tandem repeat, SSTR, (5’-AACAAAGAC-3’) has been found among F. tularensis. • With the use of appropriately designed primers and conditions it is possible through the use of PCR to identify individual strains. • The analysis of the SSTR’s is a powerful tool for the discrimination of individual strains and epidemiological analysis.

30. Francisella tularensis Detection Methods • PCR is a rapid accurate detection method that can distinguish between strains. • ELISA has been used and various antibody labeling methods can be used for detection. • Time resolved flourometry (TRF) assay system is more accurate and sensitive than the ELISA method and requires at least two hours to perform. • Mass spectroscopy (MS) of whole bacteria and isolated coat proteins has also been developed. In a clinical lab it is feasible but new portable MS systems are still unreliable in the field.

31. Francisella tularensis NewDetection Methods I • New detection methods should be easy to use, practical, accurate, highly mobile and developed in a minimum amount of time. • Unfortunately development of instrumentation takes 2-5 years and costs millions of dollars. • The use of already tested, ‘off the shelf’ components would greatly reduce development time and cost, time being most important in light of recent events.

32. Francisella tularensis NewDetection Methods II • A cheap easy to use detection system could be assembled from the following existing products to perform quick accurate PCR analysis to identify individual Francisella strains. • Bacteria would be lysed in water at 940 C for 2 minutes PCR using a capillary light cycler( 25 cycles in less than 10 minutes) resolve products on either low percent pre-cast gel (visual identification) or fluorescent capillary electrophoresis (detection via labeled primer) (5-10 min) • Entire process less than 20 minutes and cost from 15-50 thousand dollars. • Requires power 120V, 10amps so can be transported and operated in a light truck or helicopter.

33. Francisella tularensisImmunology-I Internalization • The mode of infection, proliferation, and the immune response to tularemia are still not well defined. The cells targeted are the macrophages and parenchymal cells. • The mode of entry into cells is still unknown but it is thought to be similar to the Listeria monocytogenes, another intracellular bacteria. • The mode of entry utilized by L. monocytogenes, the ‘zipper-type’ mechanism in which bacterial surface proteins bind to host cell surface receptors and the bacteria are internalized. • In L. monocytogenes the E-cadherin has been identified as the host cell receptor involved, but to date no receptor has been identified for Francisella internalization.

34. Francisella tularensisImmunology-II Infection Overview • F. tularensis enters the cell. • Proliferation inside acidified compartments containing iron. • High levels of viable bacteria induce cytopathagenesis and apoptosis. • Inflammatory response due to pathogen entry attracts large numbers of macrophages. These macrophages are not activated and are easier to infect. • Due to bacterial capsule, immunity to the effect of neutrophils and complement. • Renewed infection in arriving macrophages.

35. Francisella tularensisImmunology-III Host Death • The accumulation of macrophages without removal of bacteria initiate granuloma formation and the continued activation of the immune system. • Host death due to complications due to pnuemonia and/or due to septic shock due to the large quantity of cytokines released. • Tularemia does not release or contain any known toxin that causes disease, but it does usurp the immune system and uses it against the host.

36. Francisella tularensisT-cell Activation Immunology-IV • In response to antigen CD4 and CD8 are activated and produce interferon gamma (IFN-gamma) activating macrophages. • The activated macrophages release tumor necrosis factor alpha (TNF-alpha). • IFN-gamma and TNF-alpha together act to up regulate phagocytosis by macrophages, cause them to sequester iron within activated macrophages, and to up regulate nitrous oxide release, levels of which are good indicators of the extent of action of this mechanism.

37. Francisella tularensisT-cell Activation Immunology-V • No individual antigen has yet to be identified. Hosts recognize a multide of antigens but no immuno-dominant antigen. • The presence of phosphoantigens have been identified in extracts of F. tularensis. • Phosphoantigens (alkyl-pyrophoshoesters) are potent inducers of the gamma/delta subset of T cells causing clonal expansion. • The role of the expansion of this subset of T cells and the relevance of phosphoantigens as vaccine candidates is still unclear.

38. Francisella tularensisImmunology-VI B-cell Involvement • B-cells play a role in the suppression of neutrophil mobilization. • B-cells are necessary to develop an immune response to future encounters with the antigen in F. tularensis infection. • It is not thought that the production of specific antibodies play a large part in the response. • IgM and low levels of IgG are detected early (3-10 days after infection) and are thought to confer early protective as well as long term immunity. • Immune responses appear primarily to be in response to the lipopolysaccharide (LPS) of the outer membrane of the bacterium which appears to be the major protective antigen.

39. Francisella tularensisImmunology-VII B-cell Involvement • This year the composition of the core LPS proteins have been uncovered. The composition of the core, lipid A and the O-side chain of F. tularensis have been found to have a unique compositions that does not confer host protection upon exposure. • Only the intact LPS has been found to induce a protective immune response.

40. Francisella tularensisConclusions • The ongoing sequencing of the SCHU S4 and LVS Francisella have resulted in a large increase in information included targets that can be used for the generation of attenuated strains. • Large scale proteomic work has begun. • Together the genomic and proteomic investigations will lead to the development of new strategies for genetic manipulation and hopefully lead to an understanding of the virulence mechanisms of this potent pathogen.

41. Francisella tularensis • Organisms are strict aerobes that grow best on blood-glucose-cysteine agar at 37°C • Facultative, intracellular bacterium that multiplies within macrophages • Major target organs are the lymph nodes, lungs, pleura, spleen, liver, and kidney

42. Tularemia • Contagious --- no • Infective dose --- 10-50 organisms • Incubation period --- 1-21 days (average=3-5 days) • Duration of illness --- ~2 weeks • Mortality --- treated: low untreated: moderate • Persistence of organism ---months in moist soil • Vaccine efficacy --- good, ~80%

43. Two subspecies • Type A –tularensis • Most common biovar isolated in North America • May be highly virulent in humans and animals • Infectious dose of less then 10 CFU • Mortality of 5-6% in untreated cutaneous disease • Type B—palaeartica (holartica) • Thought to cause all of human tularemia in Europe and Asia • Relatively avirulent • Mortality of less then .5% in untreated cutaneous disease

44. 7 Forms of Tularemia • Ulceroglandular • Glandular • Oropharyngeal (throat) • Oculoglandular (eye) • Typhoidal • Septic • Pneumonic

45. Mortality Rates • Overall mortality rate for Severe Type A strains is 5-15% • In pulmonic or septicemic cases without antibiotic treatment, the mortality rate has been as high as 30-60% • With treatment, the most recent mortality rates in the U.S. have been 2%

46. Infection • Routes of Infection • No human to human transmission • Inhalation (fewer than 30 organisms) • Ingestion • Incisions/Abrasions (fewer than 10 organisms) • Entry through unbroken skin • Example: Ulceroglandular Tularemia • Transmitted through a bite from an anthropod vector which has fed on an infected animal

47. Transmission • Organisms are harbored in the blood and tissues of wild and domestic animals, including rodents • In US chief reservoir hosts are wild rabbits and ground squirrels

48. Transmission

49. Infection • Incubation Period • 1-14 days, dependent on route and dose • Usually 3-5 days • Ulceroglandular and glandular tularemia are rarely fatal (mortality rate < 3%) • Typhoidal tularemia is more acute form of disease (mortality rate 30-60 %)

50. Symptoms • Immediate Symptoms: • Fever, headache, chills, rigors, sore throat • Subsequent Symptoms: • Loss of energy, appetite, and weight • Rare Symptoms: • Coughing, chest tightness, nausea, vomiting, diarrhea