Emerging Bacterial disease- Tularemia

1. Name: Purshotam Kumar Sah Kanu
Roll No.: MB 1318/075
Level: M.Sc Microbiology (3rd Sem)
Central Department of Microbiology
Tribhuvan University, Kirtipur
Kathmandu, Nepal
MB 607 Systemic and Diagnostic Bacteriology-2

2. Emerging Bacterial Diseases
-Tularemia
Also called -
Tularaemia, Pahvant Valley
plaque, rabbit fever, deep fly
fever, Ohara’s fever
(Also known as rabbit fever)

3. Scientific Classification
Domain : Bacteria
Phylum: Proteobacteria
Class: Gammaproteobacteria
Order: Thiotrichales
Family: francisellaceae
Genus: Francisella
Species: F. tularensis
Binomial name
Francisella tularensis
(McCoy and chapin)
Dorofe’ev 1947

4. Position in risk Category
• Category A agent
• Definition of Category A agent:
can be easily disseminated or transmitted from person to person;
result in high mortality rates and have the potential for major public health
impact;
might cause public panic and social disruption; and
require special action for public health preparedness

5. Laboratory Safety For Handling Tularemia
• F. tularensis is a common cause of laboratory-acquired infection from aerosol
exposure.
• However, most cases occur in research facilities that handle large quantities of
liquid cultures. The agent may be present in nearly all clinical specimens.
• Laboratory hazards include direct contact of skin or mucous membranes with
infectious material, accidental parenteral inoculation or ingestion, and exposure to
infectious aerosols or droplets through manipulation of cultures.
• The greatest risk to laboratory personnel is associated with manipulation of
cultures.
• The recommended laboratory precautions for handling clinical specimens are
BSL2 practices, containment equipment, and facilities.
• BSL3 conditions are recommended for all activities involving cultures.

6. Use as a biological weapon
• It is easy to aerosolize
• It is highly infective; between 10 and 50 bacteria are sufficient to infect victims
• It is nonpersistent and easy to decontaminate (unlike anthrax)
• It is highly incapacitating to infected persons
• It has comparatively low lethality, which is useful where enemy soldiers are in
proximity to noncombatants, e.g. civilians

7. Introduction
• Tularaemia is caused by the bacterium Francisella tularensis, which is found in
animals, especially rabbits, hares and rodents.
• Many species, including humans, are susceptible to infection.
• Francisella tularensis is one of the most infectious bacteria known and it is,
therefore, considered as a potential biological weapon.
• Even small doses of these bacteria (10–50 bacteria) have the potential to cause
severe disease.
• The organism is capable of surviving for weeks at low temperatures in water,
moist soil, or decaying plant and animal matter.

8. Geographical distribution
• Tularaemia is endemic in many parts of the world including North America,
Eastern Europe, China, Japan and Scandinavia.
• It has been reported from Thailand.

10. Agent
• Currently there are four recognized subspecies F. tularensis (F. tularensis, F.
holarctica, F. mediasiatica, and F. novicida).
• Francisella tularensis is a Gram-negative coccobacillus, an aerobic bacterium.
• It is nonspore forming, nonmotile,
• It is a fastidious, facultative intracellular bacterium, which requires cysteine for
growth
• Appears as small rods (0.2 by 0.2 µm), and is grown best at 35–37 °C

11. Reservoir
• Rabbits, hares and rodents are natural reservoirs and often die in large numbers
during outbreaks.

12. Transmission:
• People can get tularemia many different ways:
• being bitten by an infected tick, deerfly or other insect
• handling infected animal carcasses
• eating or drinking contaminated food or water
• breathing in the bacteria, F. tularensis
• Tularemia is not known to be spread from person to person. People who have
tularemia do not need to be isolated. People who have been exposed to the
tularemia bacteria should be treated as soon as possible. The disease can be fatal if
it is not treated with the right antibiotics

13. Usual sources of infection are
Tick or deer fly bites
 In the United States, ticks that transmit tularemia
to humans include the dog tick ( Dermacentor
variabilis ), the wood tick ( Dermacentor
andersoni), and the lone star tick ( Amblyomma
americanum ).
 Deer flies (Chrysops spp. ) have been shown to
transmit tularemia in the western United States.
 Infections due to tick and deer fly bites usually
take the form of ulceroglandular or glandular
tularemia . American dog tick (Dermacentor
variabilis)

14. Handling infected animals
 F. tularensis bacteria can be transmitted to humans
via the skin when handling infected animal tissue.
 In particular, this can occur when hunting or
skinning infected rabbits, muskrats, prairie dogs and
other rodents.
 Domestic cats are very susceptible to tularemia and
have been known to transmit the bacteria to humans.
 Care should be taken when handling any sick or
dead animal.
 Infection due to handling animals can result in
glandular, ulceroglandular and oculoglandular
tularemia .
 Oropharyngeal tularemia can result from eating
under-cooked meat of infected animals.
Lone Star tick (Amblyomma
americanum)

15. Other exposures
• Humans can acquire tularemia by inhaling dust or aerosols contaminated with F.
tularensis bacteria.
• This can occur during farming or landscaping activities, especially when
machinery (e.g. tractors or mowers) runs over infected animals or carcasses.
• Although rare, this type of exposure can result in pneumonic tularemia, one of the
most severe forms of the disease.
• on Workers who report sniffing a culture plate or conducting procedures that
generate aerosols are probably at greater risk than those who simply worked with
the organism the bench.

16. Risk factors
• An underlying disease that suppresses immunity is an important factor for
contracting tularaemia.
• Hunters, pet owners and farmers are high-risk groups for exposure to infection

17. Pathogenesis
• The cell wall of F. tularensis possesses high levels of fatty acids, and wild strains
have an electron-transparent, lipid-rich capsule. Loss of this capsule may result in
loss of serum resistance and virulence; however, the capsule exhibits no innate
immunogenicity or toxicity.
• A subcutaneous inoculum of 10 organisms is sufficient to induce disease, whereas
an inhalational exposure of 25 organisms may cause a severely debilitating or fatal
disease.
• Over the first 3-5 days after cutaneous exposure, the organism multiplies locally
and a papule forms.

18. Cont…..
• During the next 2-4 days, the site ulcerates.
• Organisms spread from the entry site to regional lymph nodes and may
disseminate lympho-hematogenously to involve multiple organs.
• Pulmonary findings may be primary after direct inhalation of aerosolized bacteria
or may be present in up to half of all tularemia cases from hematogenous spread
(secondary pneumonia).
• Patients are most likely bacteremic at this time, although this is not usually
detected
• Infection produces an acute inflammatory response initially involving local
macrophages, neutrophils and fibrin.
• T lymphocytes, epithelioid cells and giant cells then migrate into local necrotic
tissues.

19. Virulence factors
• The virulence mechanisms for F. tularensis have not been well characterized. Like
other intracellular bacteria that break out of phagosomal compartments to replicate
in the cytosol.
• A hemolysin activity, named NlyA- facilitate degradation of the phagosome
• Type VI secretion system (T6SS)
• ATP Binding Cassette (ABC) proteins that may be linked to the secretion of
virulence factors.
• Type IV pili to bind to the exterior of a host cell and thus become phagocytosed.
• The expression of a 23-kD protein known as IglC is required for F. tularensis
phagosomal breakout and intracellular replication

20. Clinical signs and symptoms
• Incubation period: 3–5 days (range 1–14 days)
• The signs and symptoms of tularemia vary depending on how the bacteria enter
the body.
• Illness ranges from mild to life-threatening.
• Tularaemia is characterized by a sudden onset of high fever(104℉), headache,
muscle and joint pains and swollen, painful lymph nodes.
• Fever, headache and body aches resemble other diseases.
• Slow healing sores and lesions develop at the entry site of bacteria.
• The organism may spread widely, causing major organs to fail.
• Pneumonia is common after inhalation.

21. • In humans, tularaemia is observed in six distinct clinical syndromes depending on
the route of exposure:
I. Ulceroglandular:
This is the most common form of tularemia and usually occurs following a tick or
deer fly bite or after handing of an infected animal.
A skin ulcer appears at the site where the bacteria entered the body. The ulcer is
accompanied by swelling of regional lymph glands, usually in the armpit or groin.
Ulceroglandular tularemia on an
extremity
Ulceroglandular type of tularemia on the
hand

22. Cont…
II. Glandular:
Similar to ulceroglandular tularemia but without an ulcer.
Also generally acquired through the bite of an infected tick or deer fly or from handling sick
or dead animals.
III. Typhoidal:
• This is rare and serious form of the disease usually causes:
• High fever and chills
• Muscle pain
• Sore throat
• Vomiting and diarrhea
• Enlarged spleen
• Enlarged liver
• Pneumonia

23. IV. Oculoglandular:
This form occurs when the bacteria enter through the eye.
This can occur when a person is butchering an infected animal and touches his or
her eyes.
• This form affects the eyes and may cause:
• Eye pain
• Eye redness
• Eye swelling and discharge
• An ulcer on the inside of the eyelid
• Sensitivity to light

24. V. Oropharyngeal:
This form results from eating or drinking contaminated food or water.
• This form affects the mouth, throat and digestive tract. Signs and symptoms
include:
• Fever
• Throat pain
• Mouth ulcers
• Abdominal pain
• Vomiting
• Diarrhea
• Inflamed tonsils
• Swollen lymph nodes in the neck

25. VI. Pneumonia:
This is the most serious form of tularemia.
Symptoms include cough, chest pain, and difficulty breathing.
This form results from breathing dusts or aerosols containing the
organism.
It can also occur when other forms of tularemia (e.g. ulceroglandular)
are left untreated and the bacteria spread through the bloodstream to the
lungs

26. Diagnosis
• Specimen
• Scrapings from infected ulcers, lymph node biopsies, and sputum, whole
blood.
• Serum is generally collected from all patients early in disease and during
convalescence.
• To minimize the loss of viable organisms, samples should be transported to
the laboratory within 24 hours.
• If specimens are to be held longer than 24 hours, specimens should be
refrigerated in Amie’s transport medium.
• F. tularensis should remain viable for up to 7 days stored at ambient
temperature in Amie’s medium.
• Specimens for molecular testing should be placed in guanidine
isothiocyanate buffer.

27. Direct detection method
• Gram staining of clinical material is of little use with primary
specimens unless the concentration of organisms is high, as in swabs
from wounds or ulcers, tissues, and respiratory aspirates.
• Gram negative coccobacilli are seen in Gram staining procedure.
• A more sensitive and specific approach is direct staining of the clinical
specimen with fluorescein labeled antibodies directed against the
organism.
• Fluorescent antibody stains and immunohistochemical stains are
commercially available for direct detection of the organism in lesion
smears and tissues and are typically available in reference
laboratories.

28. Culture
• The organism is strictly aerobic and is enhanced by enriched media containing
sulfhydryl compounds (cysteine, cystine, thiosulfate or IsoVitaleX) for primary
isolation.
• Two commercial media for cultivation of the organism are available: glucose
cystine agar and cystine-heart agar, both require the addition of 5% sheep or
rabbit blood.
• However, F. tularensis can grow on chocolate agar or buffered charcoal yeast
extract (BCYE) agar, media supplemented with cysteine that are used in most
laboratories.
• They are slow-growing organisms and require 2 to 4 days for maximal colony
formation.
• They are weakly catalase positive and oxidase negative.
• Identification is done by growth on chocolate agar but not blood agar (blood agar
is not supplemented with cysteine) is also helpful.
• The identification is confirmed by demonstrating the reactivity of the bacteria
with specific antiserum (i.e., agglutination of the organism with antibodies
against Francisella).

30. Antibody detection
• Because of the risk of infection to laboratory personnel and other inherent
difficulties with culture, diagnosis of tularemia is usually accomplished
serologically by whole-cell agglutination (febrile agglutinins or newer
enzyme-linked immunosorbent assay techniques).
• Serum antibody detection is useful for all forms of tularemia.
• After the initial specimen, a convalescent sample should be collected at 14
days and preferable up to 3 to 4 weeks after the appearance of symptoms.
• Tularemia is diagnosed in most patients by the finding of a fourfold or
greater increase in the titer of antibodies during the illness or a single titer
of 1:160 or greater.
• However, antibodies (including IgG, IgM, and IgA) can persist for many
years, making it difficult to differentiate between past and current disease.

31. Molecular methods
• Conventional and real-time polymerase chain reaction (PCR) assays
have been developed to detect F. tularensis directly in clinical
specimens.
• Of significance, several patients with clinically suspected tularemia
with negative serology and culture had detectable DNA by PCR.

32. Basic Characteristics Properties (Francisella tularensis subsp. tularensis)
Capsule Positive (+ve)
Catalase Positive (+ve)
Flagella Negative (-ve)
Gas Negative (-ve)
Gelatin Hydrolysis Negative (-ve)
Gram Staining Faintly stained Gram-negative
H2S (in cysteine-supplemented medium) Positive (+ve)
Indole Negative (-ve)
Motility Negative (-ve)
Nitrate Reduction Negative (-ve)
NH3 produced in liquid media Negative (-ve)
Oxidase Negative
Shape Short, rod-shaped or coccoid
Spore Negative (-ve)
Sodium ricinoleate solubility Positive (+ve)
Urease Negative (-ve)

33. Fermentaton of
Glucose Positive (+ve)
Glycerol Positive (+ve)
Lactose Negative (-ve)
Maltose Positive (+ve)
Sucrose Negative (-ve)

34. Vaccination
• In the United States, a live attenuated vaccine derived from avirulent
F. tularensis biovar palaearctica (type B) has been used to protect
laboratorians routinely working with the bacterium.
• Until recently, this vaccine was available as an investigational new
drug. It is currently under review by the Food and Drug
Administration

35. Treatment
• Tularaemia is treated with intramuscular injection of antibiotics.
• If untreated, tularaemia causes prolonged fever and fatigue and is often fatal.
• Treatment usually lasts 10 to 21 days depending on the stage of illness and the
medication used.
• Adult:
• Preferred choices:
Streptomycin, 1g IM twice daily
Gentamicin, 5 mg/kg IM or IV once daily†
• Alternative choices:
Doxycycline, 100 mg IV twice daily
Chloramphenicol, 15 mg/kg IV 4 times daily
Ciprofloxacin, 400 mg IV twice daily†

36. • Children:
• Preferred choices:
Streptomycin, 15 mg/kg IM twice daily (should not exceed 2 gm/d)
Gentamicin, 2.5 mg/kg IM or IV 3 times daily†
• Alternative choices:
Doxycycline,
If weight >= 45 kg, 100 mg IV
If weight < 45 kg, give 2.2 mg/kg IV twice daily
Chloramphenicol, 15 mg/kg IV 4 times daily†
Ciprofloxacin, 15 mg/kg IV twice daily‡

37. Cont…..
• Pregnant Women:
• Preferred choices:
Gentamicin, 5 mg/kg IM or IV once daily†
Streptomycin, 1 g IM twice daily
• Alternative choices:
Doxycycline, 100 mg IV twice daily
Ciprofloxacin, 400 mg IV twice daily†

39. Prevention and control
• Avoid drinking, bathing, swimming, or working in untreated water
where infection may be common among wild animals.
• Use impervious gloves and clothes when skinning, handling, or
dressing wild animals, especially rabbits.
• Cook the meat of wild rabbits and rodents thoroughly.
• Use insect repellents.
• Protect food warehouses from contact with vector animals.
• Wear protective masks against infected dust and aerosols if you are a
member of the professional group, such as farmers or gardeners.

40. • Avoid being bitten by deer flies and ticks.
• Check your clothing often for ticks climbing toward open skin.
• Wear white or light-colored long-sleeved shirts and long pants so the
tiny ticks are easier to see.
• Tuck long pants into your socks and boots.
• Wear a head covering or hat for added protection.
• Walk in the center of trails so weeds do not brush against you.

41. Cases of Tularemia in Nepal
• https://www.researchgate.net/publication/330266640_Cross-
sectional_seroprevalence_of_tularemia_among_murine_rodents_of_
Nepal reported by Acharya, Narayan & Acharya, Krishna & Dhakal,
Ishwari. (2019).
• Tularemia: a re-emerging tick-borne infectious disease reported by
Yeni, D. K., Büyük, F., Ashraf, A., & Shah, M. (2021).

42. References
• Centers for Disease Control and Prevention. 2000. Biological and chemical
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• Acharya, Narayan & Acharya, Krishna & Dhakal, Ishwari. (2019). Cross-sectional
sero-prevalence of tularemia among murine rodents of Nepal. Comparative
Clinical Pathology. 019. 10.1007/s00580-019-02895-1.
• www.Wikipedia.com/tularema
• www.cdc.gov/tularemia
• www.who.int/ a brief guide to emerging infectious diseases and zoonoses
• www.microbesnotes.com/tularemia

43. • Yeni, D. K., Büyük, F., Ashraf, A., & Shah, M. (2021). Tularemia: a re-
emerging tick-borne infectious disease. Folia microbiologica, 66(1), 1–
14. https://doi.org/10.1007/s12223-020-00827-z