1. Francisella tularensis
Tularemia
One of the most infective pathogenic bacteria known

2. Francisella tularensis
Gram stain
Poorly staining,
tiny Gram-negative
coccobacilli
This is a gram-stain of tularensis. And altho they are classically described as small, poorly staining gncb….they can, on occasion be seen as rods.
Non-motile
The gram stain of F. tularensis can be compared to a CSF specimen having an elevated protein level in which all you can see is a “proteinaceous matrix-like substance” adhering to the slide. I’d recommend an Acridine Orange to separate DNA/RNA from artifact.
Me or william?

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
It’s a zoonosis
Outbreaks of the disease in humans often parallel outbreaks of tularemia in wild animals.

6. Vectors Ticks Mosquitoes Biting Flies
Historically, most people contract tularemia in the summer, related to arthopod bites, and in the winter, related to hunters coming into contact with infected animal carcasses
Also, contaminated water is a problem, but chlorination of municipal drinking water has solved that problem.
I didn’t talk about how humans get infected—Kathie?people of all ages and sexes appear to be equally sussceptible—examination of an open lab plate can cause infectrion!

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.
Tulare County, CA
Dr. Edward Francis was one of the premier researchers –hence, francis-ella tular--emia
Shortly afterward, it was recognized as a potentially severe and fatal illness in humans

9. Incidence across the Globe
Country
Years
# of Cases
Japan
1,355
Slovakia
126
Turkey
205
Tularemia is not a disease which is notifiable to the WHO, and worldwide incidence is not known. However, some data on the incidence of disease is available.

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

11. Reported Cases of Tularemia - 1990-1998
Tularemia was first described in the United States in 1911, and has been reported in all states but Hawaii—as you can see, mostly in south-central and western states
There are approximately cases/yr in the US
Low level of natural transmission
In 1927, it was placed on the REPORTABLE DISEASE LIST
Removed in 1996

12. Four States
Four states accounted for 56% of all reported tularemia cases
Arkansas (23%)
Missouri (19%)
South Dakota (7%)
Oklahoma (7%)
1368 cases reported between about 17% of US counties reported at least one case
Arkansas—315 cases
Missouri—265 cases
S.D—96 cases
OK—90 cases
Kansas and Montana are the next two states with high recurring instances.

13. U.S. Outbreaks Vermont, 1968 Utah, 1971
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
--cutaneious ulcers at the site of the bite, fever, chronic malaise, weakness

14. U.S. Outbreaks, cont. South Dakota, 1984 Martha’s Vineyard, 2000
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
In children and young adults on COW CREEK INDIAN RESERVATIONS in the state
A similar outbreak occurred in 1979 on the Crow Indian reservation in Montana, where 12 cases were identified
Pateints weer more liekly to have used lawn mowers or brush cutters in the previous two weeks

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
During the 1930s, 2000 or more cases were reported annually
During the 1990s, mean number of cases is 124. This number does not reflect actual incidence, since many cases are either not reported or not accuratly diagnosed. Also, some strains have a benighn nature.
Rural or SEMIRURAL
Between 1985 and 1992, 1409 cases and 20 deaths were reported in the US, for a mean of 171 cases per year, and a case fatality of 1.4%

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
(streptomycin, 1944), provide doublic health and clinical responses to endemic tularemia
Tuleremia remians endemic throughout the northern hemisphere, but is not a large poblic health threat

19. Fransicella tularensis Historical 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 tularensis Arthropod 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 tularensis Morphology 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 tularensis Morphology 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 tularensis Culture 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 days on glucose-cysteine-blood agar or peptone-cysteine agar.
No true hemolysis on blood containing media only a greenish discoloration.

24. Francisella tularensis Microbial 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

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: AF 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 tularensis Microbial 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 New Detection 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 New Detection 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 thousand dollars.
Requires power 120V, 10amps so can be transported and operated in a light truck or helicopter.

33. Francisella tularensis Immunology-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 tularensis Immunology-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 tularensis Immunology-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 tularensis T-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 tularensis T-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 tularensis Immunology-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 tularensis Immunology-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 tularensis Conclusions
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 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%
BULLET #1: it is not transmissible from man to man, but can be acquired in lab. Third most commonly reported lab-acquired bacterial infection (Brucella is leading lab-assoc bacterial infection)
BULLET #2: very low infective dose… organisms.
LAST BULLET: vaccine has partial protection. Some individuals demonstrate protection (80%), while multiple episodes have occurred among others that have been vaccinated.
A PARADOX: Tularemia and brucellosis are not infectious from man to man…but can be acquired in the lab; whereas, pneumonic plague is readily acquired via man to man….but it is not infectious in the lab.

43. Two subspecies Type A –tularensis Type B—palaeartica (holartica)
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
Divided based on virulence, acid production from glycerol, citrulline ureidase activity (conversion of l-cittruline to ornithine)
10 CFU—50% lethal dose

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%
With Prompt antibiotic Treatment

46. Infection Routes of Infection Example: Ulceroglandular Tularemia
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
Ulceroglandular tularemia- most common form
Also transmitted when hunters and trappers handle infected meat and are infected via cuts or abrasions
Takes very small number of organisms to become infected
Entry through skin by unknown mechanism or mode

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 Route of Transmission Mode of Transmission
Skin or conjunctiva
Handling of infected animals
Skin
Bite of infected blood-sucking deer flies and wood ticks
GI tract
Ingestion of improperly cooked meat or contaminated water
Respiratory tract
Aerosol inhalation

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 %)
Wild animals are carriers – flies are infectious for up to 2 weeks, ticks are infectious for life, in blood 2 weeks, and in lesions 1 month
Rarely fatal even without treatment, although it may take significant amount of time to resolve
Typhoidal tularemia = septicemia without lymphadenopathy or the appearance of an ulcer, possibly have delirium and shock

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

51. Symptoms and Reaction
Symptoms are severe enough to immobilize people within first two days of infection.
Symptoms depend on route of infection.
Have localized reaction when there is a specific infection site (cut, tick bite).
Localized infection can develop into systemic infection through haematogenous spread.

52. Symptoms by Route of Infection
Aerosol or Ingestion
Systemic infections, no localized ulcers or lymph gland swelling
Aerosol
Pneumonia
Ingestion
Gastrointestinal irritation
Localized
Enlargement of local lymph glands, ulcer at infection site

53. Outbreaks
Chest X-ray of patient demonstrating complete whiteout of the left lung

54. Tularemia Lesion

55. Skin Ulcer of Tularemia

56. Diagnosis Confirmed by: Problems with above methods:
Successful culture of bacteria
Significant rise in specific antibodies
Problems with above methods:
Culture is difficult and dangerous
Response from antibody does not occur until several days after onset of disease

57. Future Diagnostic Techniques
New PCR based technique produces higher success level for identification than culturing currently does.
Future tests may allow for identification of the specific strain infecting a patient.
Could be useful for a bioterrorist attack.

58. Prevention Best Immunity (Permanent) Live Vaccine Strain (LVS)
Previous infection with a virulent strain
Dr. Francis
Live Vaccine Strain (LVS)
Best prophylactic
Foshay’s Vaccine (killed bacteria)
Provides lesser immunity towards systemic and fatal aspects of disease than LVS
Immunity to reinfection is permanent and is the result of a CMI mechanism in which macrophages activated by MAF released from antigen sensitized effector T cells destroy the phagocytized organisms
LVS

59. Prevention and Treatment
Vaccines take too long to have an effect, so can’t be used for treatment after exposure
Antibiotics are effective for treatment after exposure
Antibiotic treatment must begin several days post-exposure to prevent relapse
Without the gap in treatment, subject has onset of tularemia shortly after antibiotic regime ceases – reflects an inability to fully mobilize the immune system for several days, shows that idea that T cell independent mechanisms active initially can help contain the disease, but that T cell dependent mechanics are necessary to wipe out infection
Sufficiently high dose can overcome the immunity from the vaccine

60. Live Vaccine Live Virus Strain (LVS) Pros Cons
Only effective vaccine against tularemia
Cons
Doesn’t provide 100% immunity
Possibility of varying immunogenicity between different batches
Possibility of a spontaneous return to virulence
LVS available to lab workers

61. The incidence of acute inhalational Tularemia
Use of a killed vaccine
5.70 cases per 1000 people at risk
Use of a live vaccine
0.27 cases per 1000 people at risk
A retrospective study of civilians working with F. tularensis at a US army research facility
Although incidence of ulceroglandular disease remianed unchanged, symptoms were considered milder with the live vaccine
In volunteer studies, live attentuated vaccine did not protect all recipioents against aerosol f. tularensis

62. Antibiotics to Treat Tularemia
Streptomycin and aminoglycoside gentamicin
Pros
Effective against tularemia
Cons
Require intramuscular or intravenous administration
High toxicity profile
Can be relapses of tularemia on aminoglycosides
There exist streptomycin-resistant strains of F. tularensis
Beta lactams, cephlasporins, macrolides ineffective against F. tularensis
Strep and gent are considered to be the first line therapies
Cipro (a flouro) is effective but experience with them is limited. In one recent outbreak, cipro showed a lowere treatment failure rate then doxy or strep.
Tetracycline and chlorophenicol can be used, but relapses occur more b/c they are bacteriostatic
A study of type A isolates of F tularensis found them all to be highly susceptible to fluoroquinolones
Given the rarity of tularemia and non-specific nature of the disease, clinicians would not diagnose it and begin proper treatment until some time into the epidemic.

63. Antibiotics to Treat Tularemia
Tetracyclines and chloraphenicol
Pros
Effective against tularemia
Can be administered orally
Low toxicity
Cons
Higher relapse rate than aminoglycosides
Have a higher relapse rate probably because these drugs are bacteriostatic and aminoglycosides are bactericidal
These could be good in mass casualty situation cause suitable for milder cases and for prophylaxis, as long as treatment is continued for long enough time

64. Antibiotics to Treat Tularemia
Quinolines (including ciprofloxacin)
Pros
Generally works well
Low relapse rate
Can be administered orally
Cons
Has not been used extensively for treatment
Probably work well because of known capacity for these drugs to penetrate inside cells
Ciproflaxin and tetracycline doxycyline are effective against virulent Schu4 strain
Supported by AMAs Working Group on Civilian Biodefense as suitable for treatment and prophylaxis of tularemia

65. Outbreaks
No large recorded outbreaks of inhalational tularemia in United States
Single cases or small clusters including:
Laboratory exposures
Exposure to contaminated animal carcasses
Infective environmental aerosols

66. Outbreaks Laboratory Workers
Began with a fatal case of pulmonary tularemia in a 43 year old man
Total of 13 people in the microbiology laboratory and autopsy services used were exposed despite adhering to established laboratory protocol
Services should have been notified of possibility of tularemia
Tularemia ranks second in the US and third worldwide as a cause of laboratory associated infections
None developed signs or symptoms of tularemia

67. Outbreaks
Sweden
More than 600 patients infected with strains of milder European biovar of F. tularensis
Farm work created aerosols which caused inhalational tularemia
Cases peaked during the winter when rodent-infested hay was being sorted and moved from field storage sites to barns
No deaths were reported
Largest recorded airborne tularemia outbreak

68. Category A Agents
Based on probability of use, distribution, availability, and risk assessment, the CDC specified 6 agents that have the highest likelihood of successful use
Anthrax
Plague
Tularemia
Botulinum toxin
Smallpox
Viral Hemorrhagic Fevers
“CDC defines three categories of biologic agents with potential to be used as weapons, based on ease of dissemination, potential for major public health impact (e.g. high mortality), potential for public panic and social disruption, and requirements for public health preparedness”.

69. Bioterrorism Agents: Laboratory Risks
Agent BSL Laboratory Risk
B. anthracis Low
Y. pestis Medium
F. tularensis / High
Botulinum toxin Medium
Smallpox High
VHF High
What is the historical risk of acquiring these agents while working in a laboratory? Review each agent....
Anthrax: no lab-assoc cases since the late 1950’s, at which time the anthrax vaccine was introduced. USA prevalence: 3-5 cases reported in the last yrs.
Pestis: only 3 cases since the mid 1930’s. USA prevalence: cases/yr
Note: This information can be found on Table 7 (of the handouts), in the comment field.
Tularensis and brucella are the most frequently acquired lab-assoc infections.
Brucella being the most commonly reported lab-acquired bacterial infection, with tularensis as number 3. (typhoid is #2. TB#4, and hepatitis#5). Prevalence of tularemia: cases/yr, Brucella: 100 cases/yr
There has been only 1 documented Bot Tox case. (100 cases of bot/yr)
Smallpox: no cases for the last 20yr
And VHF….is not indigenous to USA.
Tuleremia is such a high risk as it may be present in virtually any human specimen, tissues from infected animals, and fluids from infected arthopods.

70. Why Use Tularemia? Col. Gerald Parker, director of USAMRIID
Ideal agent has availability, easy production, high rate of lethality or incapacitation, stability, infectivity, and aerosol deliverability
Tularemia and anthrax most potent by far, with the least amount necessary for a 50% kill in a 10 km area
Tularemia was removed from the list of nationally notafiable diseases in 1994, but increased concern about its use as a biological weapon led to its reinstatment in 2000.

71. However…
Col. Parker went on to prioritize smallpox and anthrax first for probable use, followed by plague and tularemia, followed by botulinum toxin and hemorrhagic fever viruses
Tularemia would be expected to have a slower progression of illness and a lower case-fatality rate than either inhalational plague or anthrax

72. Anthrax v. Tularemia
U.S. test involving dropping light bulbs on the subway tracks
Observed amount of bacteria seen throughout the system
Numbers of passengers per train
Average time per person spent on the subway
12,000 cases of anthrax
200,000 cases of Tularemia
12000 fatal anthrax (fatal without intensive support)
10,000 dead from tularemia, 190,000 heavily debilitated (pnuemonic kills 5% with antibiotic treatment and limited medical support
Also, a subway release hides bacteris from the sun and the air, so decay rate is like at the lab, 2% a minute—would be much higher in air and sun

73. “I know of no other infection of animals communicable to man that can be acquired from sources so numerous and so diverse. In short, one can but feel that the status of Tularemia, both as a disease in nature and of man, is one of potentiality.”
R. R. Parker

74. History of use as a Biological Weapon
During WWII, its potential use was studied both by Japan and by the U.S. and its allies
In the 1950’s and 1960’s, the U.S. developed weapons that could deliver aerosolized organisms of F. tularensis
It was stockpiled by U.S. military in the late 1960s, and the entire stock was destroyed by 1973
The Soviet Union continued weapons production of antibiotic and vaccine resistant strains of F. tularensis into the early 1990s.
Francisella tularensis has long been seen as a potential biological weapon due to its extreme virulence and potentially lethal course. It was one of a number of aganets studied at Japanese germ warfare research units operating in Manchuria between 1932 and 1945, and it was examined for military purposes in the West. Ken Alibeck, a former Soviet Union biological weapons scientist(deputy director of the program), has suggested that tularemia outbreaks affecting tens of thousand s of Soviet and German soldiers on the eastern European warfront during Woeld World II may have been the result of intentional use.(defense of stalingrad)
At the same time (as US dissemination), they conducted researd to better understand the pathophysiology of the disease, and tried to develop vaccines and antibiotic prophylaxis and treatment regimens.In some studies, volunteers were infected with F tularensis by direct aerosol and delivery systems, and by exposure in an aerosol chamber. It was then that a live attenuated vaccine with partial protection was developed
Soviet union—according to Ken Alibeck, this resulted in weapons production of F. tularensis strains engineered to be antibiotic and vaccine resistant.
By 1958, F tularensis was considered the most probable agent of retaliation employed by the US

75. High Exposure to Infection Rate
2500 spores to cause inhalational anthrax
Fewer than 10 organisms for intracutaneous tularemia infection
Fewer then 30 F. tularensis organisms through an aerosol
I THOUGH^T AN AEROSAL WAS INHALATIONAL!!

76. Indications of intentional release of biologic agent
An unusual clustering of illness (temporal or geographical)
An unusual age distribution for common diseases
Patients presenting with clinical signs or symptoms that suggest an infectious disease outbreak
Should alert authorities to a critical and unexpected public health event
Temporal/geographic– ie, people who attended the same event or public gathering
Age distribution—ie, adults that seem to have chicken pox, as it could be smallpox

77. “Outbreaks of pneumonic tularemia, particularly in low incidence areas, should prompt consideration of bioterrorism”

78. Can assume bioterrorism if
There is an abrupt onset and a single peak of cases
Among exposed people
Attack rates would be similar across age and sex groups
Risk would be related to degree of exposure to the point source
Rapid progression of a high proportion of cases from upper respiratory symptoms to life threatening pleuropneumonitis
An outbreak of inhalational tularemia in an urban setting
Onset of large numbers of acutely ill people, which would implicate a point-source exposure without secondary transmission
Attack rates…systematic infection of young healthy adults and children

79. World Health Organization Study
In the event that a tularemia mass casualty biological weapon was used against a modern city of 5 million people
an estimated 250,000 people would get sick, and 19,000 people would die
1970
Illness would be expected to persist for several weeks and disease relapses to occur for weeks or months to follow. It was assumed the vaccinated individuals would only be partially protected against aerosol exposure
sick=“incapacitating casualties”
An aerosol release would cause the greatest adverse medical and public health consequences
Inhalation or Inoculation of as few as ten organisms can cause disease
This survey was consercative, since it assumeed 250 organism were needed to cause the disease

80. Economic impact
Referring to this model, the CDC examined the expected economic impact of bioterrorist attacks and estimated the total base costs due to an F. tularensis aerosol attack to be $5.4 billion for every 100,000 people exposed
Recently, base costs to a society

81. Would expect Short half-life due to
Desiccation
Solar radiation
Oxidation
Other environmental factors
Limited risk from secondary dispersal
There is a lack of information on the survival of intentionally dispersed particles, but

82. Vaccine
A vaccine for Tularemia is under review by the FDA and is not currently available in the U.S.
Because of the 3-5 day incubation period, and post-vaccination immunity takes two weeks to develop, post exposure vaccination is not considered a viable public health strategy to prevent disease in the event of mass exposure
Live attenuated vaccine used for high risk workers until recently—now it is under review. (It has problems typical of a live vaccine, such as varying immunogenicity between batches, and the possibility of a sponteanious retuen to virulence)
Some properties of the LVS vaccine cause concern and difficultied for licensing—the protective response induced by the vaccine has not been characterized. Also, the basis of attenuation of the LVS strain is not known. LVS remains the only effective vaccine available to date, and work to fully license it is underway.

83. “Careful proactive initiation of post exposure prophylaxis should not be underestimated for its medical, public health, psychological and political merits in coping with a terrorist attack”.
Center for Infectious Disease

84. Postexposure Prophylaxis
One study involving volunteer subjects demonstrated that use of tetracycline within 24 hours after exposure can prevent disease occurrence

85. …for inhalational tularemia
If release of the agent becomes known during the incubation period, people in the exposed population should be placed on oral doxycycline or ciprofloxacin for 14 days
If release does not become apparent until the appearance of clinical cases, potentially exposed people should be placed on a fever watch
Working group on civilian biodefense has propsed
Doxy, 100 mg twice a day, cipro, 500 mg twice a day (adults). ORAL.
If release is covert

86. …what would happen?
Any person in whom fever or flu like illness develops should be evaluated and placed on appropriate antibiotic therapy for treatment of tularemia
Parenteral therapy in a contained casualty setting
Oral therapy in the mass casualty setting
Treatment should continue for fourteen days
(oral in a mass casualty setting b/c of obvious reasons. )
if an exposed population can be inferred with reasonable probability, strong consideration should be given to providing post exposure prophylaxis, even after the onset of clinical cases
As noted with anthrax in 2001 on capital hill, peoplecan discontinue use if they find out they are not at risk.

87. Where are these helpful items coming from?
Antibiotics for treating patients infected with tularemia in a bioterrorism scenario are included in a national pharmaceutical stockpile maintained by the CDC, as are ventilators and other emergency equipment
Needed to respond to situations of large numbers of critically ill people

88. Where antibiotics fail
There is a possibility that genetically induced antibiotic resistant strains could be used as weapons
Should be considered if patients deteriorate despite early initiation of antibiotic therapy
This increases the need to create a test which could rapidly identify the antibiotic susceptibility of tularemia strains
In this case, past experience may not be a valuble predictor of disease severity under such circumstances

89. Genetic Manipulation
Scientists generated a plasmid to insert resistance genes for tetracycline and chloraphenicol
Plasmid was capable of replicating within F. tularensis and E. coli
Allowing for the development of genes in the more comfortable environment of e coli before transferring them to F. tularensis

90. Genes to be worried about
Antibiotic resistance
Radiation resistance
Desiccation resistance
Genes that code for toxins from other bacteria
Genes that would decrease the incubation time of tularemia
What is dessication?

91. Possible Vaccines
The construction of a defined attenuated mutant of F. tularensis could provide a safe, effective, and licensable tularemia vaccine that could induce protective immunity
The construction of a vaccine that does not use a live pathogen
In a range of other pathogens, the introduction of defined mutations into genes in vivo has yeilded safe and effective vaccines.
However, work to date has failed to devise methods for construction of allelic replacement mutants. of F. tularensis, and such a vaccine would be about a decade away

92. It’s critical to develop…
Simple, reliable, and rapid diagnostic test to identify F. tularensis in people
Fast and accurate procedures to quickly detect F. tularensis in environmental samples
A system to monitor for the appearance of antibiotic resistant strains
New, effective antibiotic
Bullt 1:To identify people infected with it in the mass exposure setting
Bullet 4: b/c in the absence of an effective vaccine, antibiotics are the only available treatment or therapy

93. To Prevent Infection
Isolation would not be helpful given lack of human-to-human transmission
So….
Avoid infected animals
Wash your hands
Wear gloves, masks, face-shields, eye-protection, gowns
Handle patient equipment with care
And take proper precautions in handling animals
And proper clothing to protect from arthopod bites

94. Dialogue!!!
Local practitioners, national health organizations, and the international community must all communicate to control any outbreak
Whether natural or due to bioterrorism

95. Limitations of Commercial Identification Systems
Potential of generating aerosols
High probability of misidentification
Most common identification is Haemophilus influenzae and Actinobacillus species. The Vitek NHI panel may give as high as 99% confidence to the identification of Actinobacillus actinomycetemcomitans with strains of F. tularensis. Additionally, as it is classified as level A, it is considered a “dangerous, highly virulent organism that should not be manipulated at the bench”.

96. Who’s working for our safety?
1970- The US terminated its biological weapons development program by executive order
1973- The US had destroyed its entire biological arsenal
Since then, USAMRIID has been responsible for defensive medical research on potential biological warfare agents
The CDC operates a national program for bioterrorism preparedness and response
US army medical research institute of Infectious disease
To better protect the US military, including protocols on decontamination, prophylaxis, clinical recognition, laboratory diagnosis, and medical management
Cdc response incorporates a broad range of public health pertnerships