Francisella tularensis

Francisella tularensis, a gram-negative, non-spore forming bacterium, is the causal agent of tularemia (rabbit fever, deerfly fever).  F. tularensis is endemic throughout North America and Eurasia and is one of the most infectious pathogenic bacteria known.  Due to the low infective dose of as few as 10 colony forming units (CFU), ease of dissemination and high capacity for illness/death, F. tularensis is considered a potential biological weapon and is classified as a select agent by the CDC

Potential Health Hazards

Five subspecies (subsp.) of F. tularensis have been identified; however, only two of those subspecies cause human disease, F. tularensis subsp. tularensis (Type A) and subsp. holarctica (Type B).  F. tularensis subsp. tularensis is further divided into two subpopulations, type A.I and A.II which are genetically distinct and differ in their geographic distribution.

F. tularensis can infect through the lungs, skin, mucus membranes and gastrointestinal tract.  Major target organs of this intracellular bacterium include the lymph nodes, lungs and pleura, spleen, liver, and kidney.  Once within the host, F. tularensis is believed to infect macrophages, avoid intracellular destruction and replicate within the cytosol.

Modes of Transmission

F. tularensis can be transmitted through aerosolization of the bacterium, contact with infected animal products and contact with or ingestion of contaminated water, food or soil.  The bacterium is able to pass through intact skin.  Blood-feeding arthropods, including ticks and biting flies, can also serve as a vector for F. tularensis.  Transmission rarely occurs through the bites of animals.  Direct person to person transmission of F. tularensis has not been documented.

Human infection can result from inhalation of as few as 10 CFU; the ease of aerosolization has lead to the classification of F. tularensis as a potential biowarfare agent.

Laboratory Acquired Infections

Laboratory acquired infections with F. tularensis are the third most commonly reported and are more frequently associated with cultures than with clinical materials or infected animals.  Infection can result from accidental inoculation, exposure to aerosols and infectious droplets, ingestion, and direct contact of skin or mucous membranes with infectious materials. Opening a culture plate on an open bench can create an exposure risk.

Sources of the bacterium include lesion exudates, respiratory secretions, cerebrospinal fluid, blood, urine, tissues and fluids from infected animals, and fluids from infected arthropods.

Host Range

F. tularensis can infect a wide host range, including over 200 species of mammals, birds, reptiles and fish.  Within the United States, important hosts involved in human infection include cottontail rabbits, hares, voles, mice, squirrels, muskrats and beavers.

Survival

While F. tularensis is a non-spore forming bacterium, this organism can survive for weeks in cold, moist environments including, water, soil, hay, straw and decaying animal carcasses.

Laboratory Practices

The appropriate biosafety level (BSL) practices and facilities will depend on the biological material that the laboratory is manipulating. 

Biosafety level 2 practices and facilities, at a minimum, must be used for activities involving diagnostic procedures for clinical material.  Additionally, two characterized strains of reduced virulence may be handled at BSL2, F. tularensis subsp. novicida (strain U112) and F. tularensis subsp. holarctica (strain LVS); however, if using high concentrations of these organisms, you must also use BSL3 practices.

  • Biohazard signs and labels must be displayed in areas and on equipment where bacteria are used and stored.  This includes, but is not limited to, laboratory entrance doors, biological safety cabinets, incubators, refrigerators, and freezers.
  • Use a biological safety cabinet (BSC) (a.k.a. tissue culture hood) for manipulations that can generate aerosols, such as pipetting, harvesting, infecting cells, filling tubes/containers, and opening sealed centrifuge canisters.  
  • Use aerosol containment devices when centrifuging.  These include sealed canisters that fit in the centrifuge bucket, covers for the centrifuge bucket, heat sealed tubes, or sealed centrifuge rotors.  Rotors should be removed and opened inside a BSC.  Centrifuge tubes should be filled and opened in a BSC.
  • Vacuum lines must be protected with liquid disinfectant traps and micron filters.
  • Policies for handling and disposing of sharps (needles, pipettes, broken glassware, etc.) must be implemented.  Work practice controls and engineering devices should be implemented to reduce sharps injuries.
  • Decontaminate work surfaces after completion of work and after any spill or splash of potentially infectious material with appropriate disinfectant.
  • All cultures, stocks and other potentially infectious materials should be decontaminated prior to disposal with an effective method, such as autoclaving or treatment with a bleach solution.
  • Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
  • Personal protective equipment (PPE) requirements are outlined below.
  • BSL3 practices must be implemented when working with high concentrations of strains that have reduced virulence:
    • All persons entering the laboratory must be advised of the potential hazards and meet specific entry/exit requirements.
    • Laboratory personnel must be provided medical surveillance (including serum storage) and offered appropriate immunizations for agents handled or potentially present in the laboratory.
    • A laboratory-specific biosafety manual must be prepared, adopted as policy, and available and accessible to staff.
    • The laboratory supervisor must ensure that lab personnel demonstrate proficiency in standard and special microbiological practices before working with such agents.
    • Potentially infectious materials must be placed in a durable, leak proof container during collection, handling, processing, storage, or transport within a facility.
    • Laboratory equipment should be routinely decontaminated, as well as, after spills, splashes or other potential contamination.
    • Spills involving infectious materials must be contained, decontaminated, and cleaned up by staff properly trained and equipped to work with infectious material.
    • Equipment must be decontaminated before repair, maintenance, or removal from the laboratory
  • Incidents that may result in exposure to infectious materials must be immediately reported to the supervisor and evaluated and treated according to procedures described in the laboratory’s biosafety manual. Medical evaluation, surveillance, and treatment should be provided and appropriate records maintained.
  • Animals and plants not associated with the work being performed must not be permitted in the laboratory.
  • All procedures involving the manipulation of infectious materials must be conducted within a BSC, or other physical containment devices. No work with open vessels is conducted on the bench.  
  • In addition, the laboratory should have directional airflow, so that air is drawn into the laboratory from “clean” areas toward “potentially contaminated” areas, with exhaust air dispersed outside and away from occupied areas and building air intake locations.

Biosafety level 3 practices and facilities must be used when preparing or manipulating cultures, animal necropsies and experimental animal studies with F. tularensis subsp. tularensis or subsp. holarctica

  • BSL3 facilities are specifically designed to prevent release of organisms using numerous controls. A primary containment feature is directional airflow, which entails drawing air into the laboratory from “clean” areas and toward “contaminated” areas.
  • A BSL3 specific biosafety manual and biosafety precautions are incorporated into standard operating procedures.
  • Respiratory protection may be required.
  • Policies and procedures are developed such that only persons who have been advised of the potential biohazards, who meet any specific entry requirements, and who comply with all entry and exit procedures may enter the lab.
  • BSL3 policies and standard operating procedures are outlined in manuals specific to the BSL3 laboratory.

Personal Protective Equipment

Personal protective equipment (PPE) for the BSL2 laboratory includes, but is not limited to:

  • Disposable gloves (nitrile, latex, etc.).
  • Lab coat when working in the area.  Remove when leaving the laboratory.
  • Face and eye protection must be used when potentially infectious material is handled outside of the biosafety cabinet or containment device.
  • Protective laboratory clothing must be laundered by the University and not taken home for cleaning.

PPE requirements for the BSL3 laboratory are outlined in the BSL3 specific manuals.

Precautions When Using Animals

Animal use requests are made to the Institutional Animal Care and Use Committee (IACUC) by submitting an Animal Care and Use Form (ACURF).
When animals are infected with F. tularensis, the animal biosafety level (ABSL) will be assigned to ABSL-3.  Animal projects involving F. tularensis subsp. novicida (strain U112) and F. tularensis subsp. holarctica (strain LVS) may be assigned to ABSL2, depending on a risk assessment.  

F. tularensis Research

F. tularensis is classified as a select agent; as a result, University faculty and staff working with this bacterium must register their work with the Responsible Official at the University (Haley Sinn, 100 EHS, 335-9553).  Strains exempt from the select agent regulations include F. tularensis subsp. novicida strain Utah 112, F. tularensis subsp. holarctica LVS, and F. tularensis strain B38 (ATCC 6223).

All protocols involving recombinant F. tularensis must be approved by the Institutional Biosafety Committee (IBC); complete an online rDNA Registration Document”.

Employee Exposure

Eye Exposure – Rinse eyes in an eyewash for at least 15 minutes.
Skin Exposure – Wash skin with soap and water.
Accidental Needlestick Injury – Scrub contaminated skin with soap and water.
Report Incidents and Seek Treatment – Report actual or suspected exposure incidents to your supervisor immediately and seek treatment at the Worker’s Health Clinic.  It is located on the first floor of Boyd Tower – General Hospital.  The clinic’s phone number is 353-8653.  If the incident occurs after 4:30 pm, during the weekend, or on a holiday, proceed to UIHC’s Emergency Treatment Center (ETC).  The phone number is 356-2233.

  • Incubation Period – The incubation period depends on the virulence of the infecting strain, the size of the inoculum (dose) and the route of infection.  Symptoms may appear between 1 to 14 days, usually within 3-5 days.
  • Symptoms – Symptoms include sudden onset of fever, headache, chills and rigors, generalized body aches and sore throat.  Cutaneous exposure presents as an ulcer at the site of infection and is accompanied by swelling of the regional lymph nodes; exposure through inhalation may be followed by a pneumonic disease.
  • Immunizations – An attenuated variant of F. tularensis subsp. holarctica has been used as a live vaccine strain (LVS); however, studies have shown immunization will not protect all recipients against inhalational exposures.  The vaccine is not currently available to research personnel.
  • Prophylaxis – Streptomycin and gentamicin are the preferred antibiotics when treating tularemia.  Tetracyclines and chloramphenicol may be prescribed; however, relapse and primary treatment failures have been reported more frequently with these antibiotics.  Postexposure prophylaxis with 14 days of doxycycline or ciprofloxacin is recommended as preventive therapy following potentially significant exposure to F. tularensis in an occupational or non-occupational setting. 

Spill and Disposal Procedures

For spills outside a biological safety cabinet, leave the area while holding your breath.  Once outside the area, wash hands and face with soap and water.  Do not allow anyone inside the area or room where the spill occurred.  Allow 30 minutes for the aerosols to settle.  Enter the room wearing required protective clothing, carefully cover the spill with paper towels, and apply disinfectant starting at the perimeter and working towards the center.  Allow the disinfectant to remain on the spill for at least 20 minutes before initiating spill clean up.  After initial clean up, disinfect the area a second time.

For spills inside a biological safety cabinet, cover the spill with paper towels or wipes.  Carefully pour disinfectant over the spill area.  Let the disinfectant soak for 20 minutes before cleaning up the spill.  After initial clean up, disinfect the area a second time.

  • Contaminated materials must be disposed of as biohazardous waste.
  • Decontaminate adjacent surfaces with a bleach solution.
  • For spills in the BSL3 laboratory, follow the specific procedures outlined in the BSL3 biosafety manual.

Disinfectants

Disinfectants should be allowed a minimum of 20-30 minutes contact time.  Use one of the following:

  • Sodium hypochlorite (1-10% dilution of fresh bleach).
  • Glutaraldehyde.
  • Formaldehyde.

Standard levels of chlorine used in municipal water sources should prevent waterborne transmission; however, all materials used in research are autoclaved prior to placement in the biohazardous waste containers. No live materials are disposed of in the sanitary sewer system.

Decontamination

Autoclave cultures and other laboratory waste for 60 minutes at 121° C or 250° F (15 lbs per square inch of steam pressure).

Infected animal carcasses must be autoclaved before disposing with the biohazardous waste.

Disinfect work surfaces using an effective germicide (see above).  This may be followed by an alcohol wipe to lessen the corrosive nature of the germicide.

Transport Requirements 

Shipments of F. tularensis (excluding exempted strains) must have prior CDC approval; both importation and domestic transport of the organism require CDC/USDA/APHIS permits.  Persons without prior CDC approval may not ship or receive a select agent.

In addition, materials must be appropriately contained and labeled for transport within the University.  Shipping infectious substances, diagnostic specimens, and/or shipping with dry ice off-campus require training and certification.  See EHS’s fact sheet “Transporting and Shipping Infectious Substances” for additional information.

If you have questions

Contact EHS’s Biological Safety staff at 335-8501.

Information and References

  • Dennis, D.T., et. al. (2001).  Tularemia as a biological weapon, medical and public health management.  Journal of the American Medical Association, 285: 2763-2773.
  • Farlow, J., et. al.  (2005).  Francisella tularensis in the United States.  Emerging Infectious Diseases, 11:1835-1841.
  • Health Canada Office of Laboratory Security. (2001, March). Infectious Substances SDS Web SiteFrancisella tularensis.  Retrieved May 2007.
  • McLendon, M., et. al. (2006).  Francisella tularensis: Taxonomy, genetics, and immunopathogenesis of a potential agent of biowarfare.  Annual Review of Microbiology, 60:167-185.
  • U.S. Department of Health and Human Services Centers for Disease Control and Prevention and National Institutes of Health. (2007,February) Biosafety in microbiological and biomedical laboratories (BMBL) 5th edition.  Retrieved February 2014.