Francisella Tularensis Subspecies Tularensis (Type A) Margie A. Scott Central Arkansas Veterans Healthcare System, and University of Arkansas for Medical Sciences Little Rock, AR

Click on each slide thumbnail image for an enlarged view

Background Francisella tularensis (FT), the causative agent of tularemia, is a fastidious gram negative bacterium found in North America as well as some parts of Europe and Asia. FT is divided into two subspecies: the more virulent F. tularensis subspecies tularensis (type A or Jellison type A) found in North America, and the less virulent F. tularensis subspecies palaearctica (type B) found in Europe and Asia. The agent has an extremely high infection rate with as few as 10 organisms resulting in human disease. Because of the high infection rate and the virulence of Type A strains, Francisella tularensis is considered a Category A select agent that could be used as a bioterrorism agent resulting in widespread disease with a high mortality rate, particularly if exposed through the respiratory route. Over the past 10 years there has been enhanced research, molecular characterization and development of rapid molecular diagnostic techniques for diagnosis of tularemia. Molecular diagnostic methods allow rapid confirmation of the agent and definitive alerting of the clinical microbiology staff that are at risk of infection while handling media plates laden with concentrated isolates. Although all slow growing gram negative bacteria should be handled with caution, isolation often requires extensive manipulation of the isolates, particularly with transferal of specimens from positive blood cultures to solid media. Because the clinical symptoms of tularemia are often non-specific, most clinicians and laboratory technologists are unaware that tularemia is the potential etiologic agent. It should be noted that the danger to laboratory personnel is due to the manipulation of concentrated isolates on media. No increase of infection is noted when handling direct samples using standard precautions and set-up techniques under a biological hood. Francisella tularensis is widely distributed throughout wildlife (domestic and wild mammals, birds, fish and reptiles) and can survive in water, mud and decaying animal carcasses for as long as 4 months. Tularemia was first described in the United States in 1911 and since that time tularemia has been documented within every state except Hawaii. Arkansas, with its rural terrain, abundant wildlife and avid hunting enthusiasts, has historically demonstrated the heaviest concentration of human cases each year. Awareness of the disease and it's transmission through animals and insect vectors has resulted in a decrease in disease incidence over the past 5-10 years. There are often two seasonal peaks of disease transmission; one during the summer corresponding to the peak of the tick season and the second during the winter during hunting season (more specifically Rabbit season). Common names used to describe this zoonotic disease include: Rabbit fever Rabbit plague Wild hare disease Deerfly fever Bubba and Spot disease Marketmen's disease Glandular tick fever O'Hara's disease Water rat trapper disease Francis' disease Slide 1 Click to view with ImageScope Click to view with a Web-Based Viewer Figure 1 Low power view of infected lymph node showing necrosis, foci of surrounding histiocytes and scattered granulomas at margin of lesion (H&E, 10x) Figure 2 High power view of lymph node giant cells demonstrating amber colored polarizable foreign material within cytoplasm (H&E, 40x) Figure 3 Discrete zonal necrosis with surrounding palisading histiocytes and adjacent giant cells mixed within nodal lymphocytes. (H&E, 10x). Figure 4 High power of interface between confluent zonal necrosis and histiocytic rim. (H&E, 40x). Spectrum of Clinical Disease: Tularemia can present via six distinctly different presentations. The severity of symptoms and type of presentation is dependent upon biologic load of organisms encountered, subspecies encountered (usually Jellison Type A in North America), route of infection and immune status of the host. Tularemia is often classified into six different clinical "types" based on the predominant clinical symptom, however, a great deal of overlap occurs. The incubation period is usually 3-5 days but may vary from 1 to 21 days. Symptoms generally start with an abrupt clinical fever followed by malaise, cough, abdominal pain, pharyngitis, vomiting and diarrhea. The six clinical types of tularemia are noted below (Table data extracted from Mandell's Principles and Practice of Infectious Diseases, 4th Edition, Churchill Livingstone, 1995): Type of Tularemia % of Cases Route of Exposure Clinical Presentation Ulceroglandular 20-85% Tick bites; animal contact Fever, skin lesion & enlarged tender node Glandular 5-20% Tick bites; animal contact Fever, enlarged lymph node Oculoglandular 0-5% Conjunctival direct contact (unilateral disease in 90%) Swollen lid, red conjunctiva, eye pain, sensitivity to light, enlarged lymph node. Pharyngeal 0-10% Oropharyngeal (contaminated water, food etc) Severe throat pain; exudative pharyngitis & tonsillitis (often multiple family members) +/-cervical adenopathy Typhoidal 5-30% Often undetermined Fever, diarrhea, vomiting, sore throat, cough, malaise may be severe; chronic Pneumonic 7-20% Inhalation (may be occupational exposure) Fever, cough, pleuritic chest pain, Xray infiltrate Diagnostic Methods: Culture remains a mainstay in the diagnosis of tularemia and appropriate samples include blood, sputum, bronchoscopy samples, wound drainage, lymph node aspirate/biopsy/drainage, and pleural exudates. Serologic studies are helpful if the disease has been ongoing for more than 3-4 weeks, or in retrospect by confirming the suspected diagnosis after antibiotic therapy. Molecular methods have become widely available through the upgrading molecular diagnostic capability of state public health laboratories in preparation for a potential bioterrorism event. Francisella tularensis grows slowly on nutrient rich sheep blood agar and in blood culture media. Optimum growth is obtained when a nutrient rich media such as chocolate agar, Thayer-Martin (TM) or buffered charcoal yeast extract (BCYE) agar is enriched with cysteine. Growth is usually detected in 48-72 hours with incubation at 35-37 degrees in ambient atmosphere or 5% CO2. If tularemia is suspected clinically and the patient has been on antibiotics, culture plates should be held for 7 days. Gram stain demonstrates faintly staining, minute (0.2-0.4 micron) coccobacillary gram negative pleomorphic rods. Bipolar staining is not detected. The CDC protocol for level A microbiology laboratories is noted below. LAB PROTOCOL FOR SUSPECTED Francisella tularensis If Francisella tularensis is suspected, DO NOT attempt to identify with commercial gram negative identification systems, as the probability of mis-identification is very high. [most commonly misidentified as H. influenzae or Actinobacillus sp.] If Francisella tularensis is suspected, limit manipulation of culture in the clinical microbiology laboratory to the presumptive identification algorithm as outlined by CDC above. This is due to the high risk of infection of laboratory staff while working with concentrated isolates on culture media. Histopathology of Tularemia: Most cases of tularemia diagnosed by tissue aspiration or biopsy involve evaluation of infected lymph nodes. The lymph nodes invariable show necrosis and abscess formation. The necrosis may vary from widespread complete obliteration of nodal architecture to focal subcapsular zonal necrosis. Necrotic foci may be surrounded by a zone of palisading histiocytes. Occasionally granulomas may be identified, but this is not a prominent feature. In patients who have completed multiple rounds of antibiotics, polarizable crystalline deposits may be seen within surrounding giant cells. Lung tissue shows abundant alveolar neutrophils, confluent areas of necrosis, fibrin depostion, edema and in some cases adjacent suppurative pleuritis. Autopsy studies have shown similar patterns of confluent necrosis and abscess formation in liver, spleen and gastrointestinal mucosa as well as suppurative leptomeningitis. References: Francis E. Tularemia Francis 1921. Hygienic Laboratory Bulletin No. 130, 1922. Guarner J, Saki SR. Histopathology and immunohistochemistry in the diagnosis of bioterroism agents. J. Histochem Cytochem. Jan;54(1):3-11, 2006. Jellison W L. Tularemia: Dr. Edward Francis and his hirst 23 isolates of Francisella tularensis. Bull. History Med. 96:477-85, 1972. Lamp LW, Havens JM, Sjostedt A, Page D, and Scott MA. Histologic and molecular diagnosis of tularemia: a potential bioterrorism agent endemic to North America. Mod Pathol. 17:489-495, 2004. Nelson A, Wilson ML. Biothreat agent and pathology laboratories. Semin Diagn Pathol. Nov; 24(4):209-216, 2007. Sjostedt A, Eriksson U, Berglund L and Tarnvik A. Detection of Francisella tularensis in Ulcers of Patients with Tularemia by PCR. J Clin Micro. Vol 35, No 5, p.1045-1048, 1997. Tarnvik A, Chu MC. New approaches to diagnosis and therapy of tularemia. Ann NY Acad Sci. Jun; 1105:378-404, 2007.