Last updated September 6, 2013
Tularemia is caused by Francisella tularensis (formerly Pasteurella tularensis). Key microbiological characteristics include the following (ASM 2013, Cross 2000, Penn 2010, Sneath 1986, Wong 1999).
- Tiny, faintly staining, pleomorphic gram-negative coccobacillus (0.2-0.5 mcm x 0.7-1.0 mcm); smaller in patient samples than in culture; may be confused with Haemophilus or Actinobacillus species
- Difficult to see by light microscopy in blood, tissue samples, or other specimens that contain significant background material
- Nonsporulating, nonmotile
- Aerobic (obligatory)
- Requires cysteine (or cystine or another sulfhydryl source) for growth (although atypical strains that lack this requirement have been identified [Bernard 1994])
- Grows on commercial blood culture media, but does not grow (or grows unreliably) on most other standard agar media
- Visible growth on appropriate media requires 2 to 5 days
- Weakly catalase-positive (although may be negative); oxidase-negative
- Thin, lipid-rich capsule
- Distinctive cellular fatty-acid profile
Other characteristics of F tularensis include the following:
- Wild-type F tularensis strains generally are susceptible to aminoglycosides (streptomycin, gentamicin, kanamycin), tetracyclines, chloramphenicol, and fluoroquinolones.
- F tularensis strains generally are resistant to beta-lactam antibiotics, owing in part to beta-lactamase activity.
- Ingestion of F tularensis by environmental amoebas may affect the bacterial ecology by:
- Increasing environmental resistance
- Increasing virulence (Berdal 1996)
- Organisms can persist for long periods in moist environments like water, mud, and decaying animal carcasses.
- Both type A and B can survive for approximately 30 days in brackish water (Berrada 2011).
There are three subspecies of F tularensis. These subspecies (subsp) can be differentiated by biochemical and molecular tests, and the current taxonomy is as follows (Ellis 2002, Kugeler 2006, Morner 1993, Whipp 2003):
- F tularensis subsp tularensis (type A) (Farlow 2005, Johansson 2004, Petersen 2006, Svensson 2005):
- Highly infectious, generally more virulent, and more genetically diverse than subsp holarctica
- Found almost exclusively in North America
- Demonstrates citrulline ureidase activity
- Produces acid from glycerol fermentation
- Two distinct genetic clades have been identified. The geographic distribution of the two clades in human cases correlates with the distribution of arthropod vectors and rabbit hosts (Farlow 2005, Kugeler 2009):
- Clade 1 (aka subpopulation 1, A.I, A1, type A-east) occurs primarily in the central United States, is associated with the distribution of Amblyomma americanum (the Lone Star tick) and Dermacentor variabilis (the American dog tick), and appears to have a high case-fatality rate.
- Genotypes A1a and A1b have been identified more recently.
- Infection with A1b is associated with higher mortality than A1a or A2.
- Clade A1b is more virulent than other clades of F tularensis, as demonstrated by survival curves, gross anatomy, and bacterial burden in mice (Molins 2010).
- Clade 2 (aka subpopulation 2, A.II, A2, type A-west) occurs primarily in the western United States, is associated with the distribution of Dermacentor andersoni (the Rocky Mountain wood tick) and Chrysops discalis (the deer fly), and has a low case-fatality rate.
- Genotypes A2a and A2b have been identified more recently.
- No difference in mortality has been observed between infections caused by A2a and A2b.
- Strains of both major clades have been fully sequenced (Beckstrom-Sternberg 2007, Larsson 2005).
- F tularensis subsp holarctica (type B):
- Generally considered less virulent than subsp tularensis
- Does not demonstrate citrulline ureidase activity, and does not produce acid from glycerol fermentation
- Found throughout the Northern Hemisphere and accounts for approximately 30% of cases found in North America.
- Tularemia cases in Europe, Russia, and Japan are almost exclusively caused by type B.
- Three biovariants (biovars) have been identified:
- Biovar I: erythromycin sensitive; primarily found in North America, France, Spain, Italy, Switzerland, Siberia, the Far East, and Kazakhstan
- Biovar II: erythromycin resistant; primarily found in Eurasia
- Biovar japonica: found in Japan
- Biovar I contains the phylogenic group B.FTNF002-00, and Biovar II contains the B.13 group and several geographically defined subclades (Gyuranecz 2012).
- F tularensis subsp mediasiatica: Found in the Central Asian republics of the former Soviet Union (virulence is similar to subsp holarctica infections are extremely rare); produces acid from glycerol and thus may be confused with subsp tularensis (Sandstrom 1992).
- With multiple methods available for comparing the genomes of the different subspecies of F tularensis, the current method, which is based on phenotypic traits, appears outdated (Johansson 2010).
Other Francisella Species
- Other Francisella species may be confused with F tularensis in clinical or environmental samples.
- F novicida: To date, eight cases have been reported; three primarily had an ulceroglandular or glandular presentation, and five had bacteremia and/or pneumonia. Two case-patients died, and the outcome for one is unknown. Three had an underlying immunosuppressive condition (Birdsell 2009, Breett 2012, Clarridge 1996, Hollis 1989, Leelaporn 2008, Whipp 2003).
- F tularensis and F novicida traditionally have been considered separate species. Some consider F novicida as a subspecies of F tularensis; however, this does not take into account recent advances in comparative genomics (Busse 2010, Huber 2010, Johansson 2010).
- F philomiragia is the only other taxonomically defined species in the genus other than F tularensis. It is halophilic, rarely associated with human disease, and difficult to identify by conventional methods (Ellis 2002, Friis-Moller 2004, Whipp 2003).
- Undefined Francisella-like bacteria appear to be common in the environment (Barns 2005).
- A novel Francisella species, proposed as Francisella hispaniensis, was isolated from a patient in Spain in 2003 (Huber 2010).
- Investigators also have identified a novel Francisella species in ixodid ticks in some regions. This discovery highlights the need for careful analysis of polymerase chain reaction (PCR)-based identification (Sreter-Lancz 2008).
Virulence factors that contribute to the pathogenesis of F tularensis have not been well defined, and further studies are needed; however, key points on pathogenesis are outlined below (Ellis 2002, Sjostedt 2003, Titball 2003):
- F tularensis is a facultative intracellular pathogen that multiplies predominantly within macrophages. The organisms initially enter macrophages through phagocytosis by a novel process of engulfment within asymmetric pseudopod loops and then disrupt the phagosomal membrane to gain direct access to the cytoplasm (Clemens 2004).
- F tularensis virulence is determined in part by the ability of the organisms to replicate within macrophages. Bacteria are released from the macrophage following cell death by apoptosis. A study using a murine model demonstrated high levels of F tularensis in the plasma of infected mice. On the basis of this finding, the authors suggest that F tularensis in the blood of infected hosts is taken up by and replicates within leukocytes and eventually escapes in to the plasma, where it propagates a cycle of infection, escape, and reinfection (Forestal 2007).
- The capsule appears to be necessary to protect against serum-mediated lysis but is not required for survival following phagocytosis.
- The lipopolysaccharide (LPS) does not exhibit the properties of a classic endotoxin and demonstrates low toxicity in vivo and in vitro, although LPS may have a role in macrophage growth.
- The presence of type IV pili appears to be a virulence factor and may be particularly important for F tularensis infections that occur via the peripheral route. Direct repeat-mediated deletion of genes coding for type IV pili results in major virulence attenuation (Forslund 2006).
- Investigators have demonstrated that F tularensis can invade erythrocytes during infection. This feature may contribute to relapses of tularemia after short-duration antibiotic therapy, since erythrocytes have a relatively long life span (~120 days) (Horzempa 2011).
- Funding for work to further elucidate the pathogenesis of F tularensis is disappearing, leaving many unanswered questions (Conlan 2011: Francisella tularensis: a red-blooded pathogen).
Pathologic features for the various clinical syndromes caused by F tularensis have been described and are briefly summarized below.
Glandular and Ulceroglandular Tularemia
In both glandular and ulceroglandular tularemia, organisms enter the skin through the bite of infective arthropods, direct contact with infectious materials (such as contaminated carcasses), or percutaneous inoculation with a sharp object (such as a bone fragment from a contaminated carcass).
- Organisms can enter through inapparent breaks in the skin surface.
- The infectious dose for humans following percutaneous or inhalational inoculation is 10 to 50 organisms (Cross 2000, Penn 2010).
- In the ulceroglandular form, the organisms proliferate locally and cause a papule to develop at the site of inoculation within 3 to 5 days after initial exposure (Cross 2000, Penn 2010).
- The papule develops as a result of a localized inflammatory response that involves fibrin, neutrophils, macrophages, and T lymphocytes.
- The initial inflammatory nidus becomes necrotic and degenerates over the next several days, thereby forming a tender ulcerated lesion at the site of the papule.
- The ulcer is typically 2 to 4 cm in diameter and has an irregular and raised border.
- A dark scab (which may resemble the characteristic eschar of anthrax) may occur over the area of ulceration.
- Organisms spread from the site of inoculation to regional lymph nodes, where they cause necrotizing lymphadenitis surrounded by a neutrophilic and granulomatous inflammatory infiltrate (CDC 2004). Granulomas may develop in lymph nodes as the inflammatory process progresses; these may eventually coalesce to form abscesses. Follicular hyperplasia and inflammatory cell infiltrates involving predominantly granulocytes often are noted (Sutinen 1986).
- Affected lymph nodes may become fluctuant, rupture, and sometimes create draining sinus tracts in the skin.
- Organisms may disseminate via hematogenous spread to involve multiple organs, and sepsis syndrome can occur.
- In glandular tularemia, regional lymph node involvement occurs, but ulceration at the site of inoculation is absent.
Organisms enter the lungs either through inhalation of infectious aerosols or through hematogenous spread. The infectious dose by the respiratory route is 10 to 50 organisms (Franz 1997, Saslow 1961).
Once in the lungs, the organisms enter pulmonary macrophages within minutes and begin replicating. The explosive replicative capacity of F tularensis appears to be an important factor in virulence associated with pulmonary infection (Malik 2006). An intense accumulation of inflammatory cells, particularly neutrophils and macrophages, can be seen at sites of bacterial replication. The influx of neutrophils appears to play more of a destructive than protective role in the host response.
- Ulcerative bronchitis and bronchiolitis
- Hemorrhagic edema with a nonspecific inflammatory response consisting of lymphocytes, plasma cells, and eosinophils (early in the clinical course)
- Discrete nodules with acute suppurative necrosis of lung parenchyma
- Alveolar exudates involving mononuclear cells, fibrin, and red blood cells
- Nodular, segmental, or lobar consolidation
- Caseous or cavitary lesions (late in the clinical course)
- Granuloma formation (late in the clinical course)
- Pleural fibrinous, fibrinocellular, or fibrinocaseous exudation
- Hilar lymphadenopathy
- Organisms gain entry via the conjunctiva.
- Superficial necrosis and ulceration of the conjunctiva occur, often accompanied by lymphocytic infiltration. Papules also may be noted (Lillie 1937).
- Granulomatous nodules may develop over time (Lillie 1937).
- Organisms spread from the conjunctiva to the preauricular, submandibular, or cervical lymph nodes, where they cause focal necrosis and lesions similar to those noted with ulceroglandular tularemia.
- Infection most commonly is unilateral.
- Organisms enter the mucous membrane of the oropharynx following ingestion or inhalation of organisms.
- Exudative pharyngitis or tonsillitis usually occurs, and ulcers may develop.
- Organisms spread to the cervical lymph nodes, where necrosis and suppuration may occur.
- Typhoidal tularemia involves a systemic illness without anatomic localization of infection.
- Organisms enter the bloodstream through breaks in the skin or through mucous membranes and may affect the lungs and reticuloendothelial organs (ie, lymph nodes, liver, spleen, bone marrow).
- Necrotic foci can occur in any involved organ, and caseating granulomas may develop.
- Sepsis may occur, leading to shock, organ system failure, adult respiratory distress syndrome, and disseminated intravascular coagulation.
Small and medium-sized mammals are the principal natural reservoirs for F tularensis. Examples include (Dennis 1998, Gelman 1961, Hopla 1974):
- Lagomorphs (rabbits, hares) (predominantly North America, Europe, Japan)
- Aquatic rodents (beaver, muskrats, water voles)
- Field voles
- Water and wood rats
- Lemmings (former Soviet Union, Sweden, Norway)
- Meadow and field mice (predominantly former Soviet Union)
Humans, other mammalian species (eg, cats, dogs, cattle, non-human primates), and some species of birds, fish, and amphibians are incidental hosts.
- A serologic survey of 91 cats in Connecticut and New York found that 12% had antibodies to F tularensis (Magnarelli 2007).
- An outbreak of tularemia in commercially distributed prairie dogs was recognized in the United States in 2002 (CDC 2002: Outbreak of tularemia among commercially distributed prairie dogs, 2002; Petersen 2004). Serologic testing of potentially exposed persons demonstrated that one person (an animal handler) had a positive F tularensis titer of 1:128 on initial testing that subsequently declined to 1:32 on follow-up testing at 4 and 6 months, suggesting prairie dog–to-human transmission (Avashia 2004).
- Outbreaks also have occurred in nonhuman primates housed outdoors. In a German primate facility, 18 of 35 cynomolgus monkeys (Macaca fasicularis) contracted tularemia within a 2-year period; 6 of the animals died (Matz-Rensing 2007).
- Incidental hosts may play a larger role than previously thought in maintaining a reservoir for F tularensis (Franke 2010).
- Occasionally outbreaks in hares are associated with high mortality rates in the affected population (Decors 2011).
Information from studies conducted on Martha's Vineyard suggests that F tularensis can persist in the environment and that persons can acquire infection by engaging in activities that lead to aerosolization of the organisms (such as lawn mowing, weed-whacking, and using a power blower) (Feldman 2001, Feldman 2003). An analysis of sera from a variety of mammals on Martha’s Vineyard found that skunks and raccoons were frequently seroreactive (49% of skunks tested and 52% of raccoons), whereas white-footed mice, cottontail rabbits, deer, rats, and dogs were much more likely to be seronegative (Berrada 2006).
In Martha’s Vineyard, the American dog tick is a stable reservoir and a vector for F tularensis. Ticks infected with F tularensis appear to have a higher mortality rate than ticks not infected with F tularensis. Furthermore, ticks infected with the common genotype of F tularensis do not have increased mortality, whereas ticks infected with a unique genotype of F tularensis do. These findings highlight the complex ecology of F tularensis in Martha’s Vineyard (Goethert 2011).
During the fall and winter of 2003, F tularensis was identified on several filters from a biodetection air-monitoring system in Houston, Texas (see Oct 10, 2003, CIDRAP News story). An investigation conducted at that time supported contamination of the filters by naturally occurring F tularensis organisms, although the environmental reservoir was not definitively identified. Similar types of detections have occurred in other areas of the country, suggesting that these organisms (or organisms that are near-neighbors to F tularensis) are not uncommon in the environment (see Apr 5, 2010, CIDRAP News story).
Diverse bacteria, including select agents, were detected in urban air by DNA arrays during a 17-week study of airborne bacterial composition and dynamics in Austin and San Antonio, Texas. Although taxonomic clusters containing organisms closely related to Francisella were found, F tularensis was never encountered (Brodie 2007).
In a study of natural outbreaks of human tularemia in two locations in Sweden, genotyping of F tularensis subsp holarctica isolates was used in conjunction with information obtained from patients to investigate the epidemiology and geographic spread of disease. Strong, highly localized spatial associations were found between F tularensis subpopulations and places of disease transmission, indicating likely point sources of infection. Most patients were infected from mosquito bites. Disease clusters were linked to recreational areas near water, and identical subtypes were present throughout the tularemia season and persisted over different years (Svensson 2009). Results from one study suggest that mosquitoes come into contact with the organisms during the larvae stage, while they are still in their original aquatic habitat (Lundstrom 2011). As with the studies on Martha's Vineyard, these findings support persistence of F tularensis in the environment over time.
F tularensis appears to survive within Acanthamoeba (relatively ubiquitous protozoa), suggesting that these organisms may serve as a reservoir for F tularensis. Survival within Acanthamoeba may provide a mechanism for F tularensis to persist in the environment (Abd 2003, El-Etr 2009).
- A number of different arthropod vectors that transmit F tularensis have been identified (Dennis 1998, Hopla 1974).
- 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: A americanum (lone star tick), D andersoni (Rocky Mountain wood tick), D variabilis (American dog tick), Ixodes scapularis, I pacificus, I ricinus, and I dentatus
- Mosquitoes: Aedes cinereus and A excrucians
- Biting flies: C discalis (deer fly), C aestuans, C relictus, and Chrysozona pluvialis
In one study in Missouri, investigators found that the density of tick populations correlated with an increased incidence of tularemia at the country level (Brown 2011). Ticks also may play a major role in the ecology of F tularensis in the environment (Gyuranecz 2011).
In southwestern Germany, I ricinus ticks are the primary vector for the introduction and maintenance of a new clone of F tularensis associated with an increase in human cases (Gehringer 2012). Identifying regions with a high density of infected ticks should be a priority for surveillance in Europe (Reye 2013).
Modes of Transmission
F tularensis can be transmitted to humans via various mechanisms:
- Bites by infected arthropods (Klock 1973, Markowitz 1985)
- Direct contact with infected animals
- Handling of infectious animal tissues or fluids (Young 1969)
- Transplanted organs (Ozkok 2012)
- Ingestion of contaminated food, water, or soil (Barut 2009, Greco 1987, Mignani 1988, Reintjes 2002, Willke 2009, Djordjevic-Spasic 2011, Komitova 2010, Larssen 2011); murine models have confirmed that F tularensis is an effective oral pathogen and may pose a hazard, particularly to immunocompromised individuals, if ingested in contaminated food or water (KuoLee 2007)
- Possibly direct contact with contaminated soil or water
- Inhalation of infectious aerosols, including dust from contaminated hay (Dahlstrand 1971) and aerosols generated by lawn mowing and brush cutting (Feldman 2001, Feldman 2003)
- Exposure in the laboratory setting (eg, inhalation of infectious aerosols, handling cultures or other infectious materials, accidental percutaneous exposure) (Overholt 1961, Pike 1976)
Person-to-person transmission has not been documented.
- Colloquial terms for human illness associated with F tularensis include "rabbit fever," "deerfly fever," and "lemming fever."
- Most US cases in recent years have been associated with bites from infected arthropods, although rabbits, hares, and other small mammals continue to be major sources of exposure for cases in the southeastern United States (Kugler 2009).
- During the 1930s, 2,000 or more cases were reported annually. Since that time, reported case numbers have gradually declined.
- Approximately 120 cases are reported each year—although this number likely does not reflect actual incidence since many cases either are not reported or are not accurately diagnosed (CDC 2011: Reported tularemia cases by state, United States, 2001-2010). Tularemia was reported in all US states except Hawaii. States with the highest number of reported cases during these years were Arkansas, Missouri, Oklahoma, Kansas, South Dakota, and Montana. Martha's Vineyard also had a high number of identified cases. Reported incidence rates were higher in males than in females, with the highest rates reported for children 5 to 9 years old and persons 75 years of age or older.
- Traditionally, most cases have occurred in rural or semirural environments; cases rarely occur in urban settings.
- In the United States, cases occur most commonly between May and September, although cases can occur during any time of year (Kugeler 2009).
- Data from a study of cases in the United States from 1964 through 2004 indicate that type A and type B infections differ in terms of affected populations, anatomic site of isolation, and geographic distribution. Molecular subtyping and pulsed-field gel electrophoresis (PFGE) defined two clades of type A organisms (A1 [type A-east] and A2 [type A-west]) that differ in geographic distribution, disease outcome, and transmission (Staples 2006).
- Of 316 F tularensis isolates obtained from 39 states, 208 (66%) were type A and 108 (34%) were type B.
- Type B infections clustered along major waterways, including the upper Mississippi River, and areas with high rainfall, such as the Pacific Northwest.
- Type A1 infections occurred in Arkansas, Missouri, Oklahoma, and along the Atlantic Coast east of the Appalachians.
- Type A2 infections occurred predominantly in the arid regions of the southwestern United States.
- Type A2 infections were less severe than either type B or type A1 infections. The case-fatality rate for type A1 was 14%, for type B was 7%, and for type A2 was 0%.
- In the previously described study by Staples et al, only 20% of the type A isolates were genotyped. Subsequently, PFGE was performed on all of the type A (n=302) and a portion of the type B (n=61) human and animal isolates. Molecular subtyping of these isolates identified four distinct type A genotypes (A1a, A1b, A2a, and A2b) as well as type B. Significantly higher mortality was observed for human infections caused by genotype A1b (24%) compared with genotypes A1a (4%) and A2 (0%) (Kugeler 2009).
- Building on the work of Staples et al, ecological niche modeling has been conducted on human and animal isolates obtained from 1964 through 2005 to identify the geographic niches in which the six genotypes (A, B, A1, A2, A1a, and A1b) are expected to be present (Nakazawa 2010). According to the models, types A and B have broad overlapping ranges; however, type B is absent in most southeastern states and type A is absent in most northeastern states. A1 occurs in the central and southeastern states along with California, Oregon, and parts of Utah and Idaho. Type A2 occurs in in the western states except for parts of Arizona, California, Oregon, Washington, and Idaho. A1a and A1b largely overlap, covering the central and southeastern states and the western coastline.
- Occasionally, more than one subspecies or subtype can be identified in a localized outbreak. In a deer-fly associated outbreak involving five human cases in Utah in 2007, human infections arose from either type A1 or A2 (F tularensis subsp tularensis). Animal carcasses collected in the area harbored type A1, A2, or B organisms (F tularensis subsp holarctica) (Petersen 2008).
- Climate change may affect future distribution of tularemia. Modeling studies suggest that, as the climate warms, the southern border of tularemia distributions in the United States will shift northward about 600 km. This could mean a reduced incidence in the south central United States (eg, Louisiana, Mississippi) and a greater incidence in north central states (eg, Michigan to North Dakota) (Nakazawa 2007). Combined environmental and tularemia-incidence data have been used to develop a multivariate logistic regression model for predicting areas that have increased disease risk (Eisen 2008).
- Thirty-eight documented cases of tularemia were reported from 1946 through 2010 in Alaska (Hansen 2011). Of these, detailed information is available for 19. Ulceroglandular tularemia was documented in 70% of cases; 79% of those for whom information was available had contact with animals, 84% of which were known reservoirs for F tularensis.
Outbreaks of tularemia occasionally have been recognized in the United States; examples include the following:
- Martha's Vineyard, 2000: Fifteen cases of tularemia were reported; 11 patients had primary pneumonic disease and 1 patient died (Feldman 2001). Illness was caused by F tularensis, type A. Patients were more likely than controls to have used a lawn mower or brush cutters in the 2 weeks before illness onset.
- South Dakota, 1984: Twenty cases of glandular tularemia were reported in children and young adults on Crow Creek Indian reservations in the state (Markowitz 1985). Illness was mild (fever, headache, and lymphadenopathy) and was presumably caused by F tularensis, type B. A similar outbreak occurred in 1979 on the Crow Indian Reservation in Montana, where 12 cases were identified (Schmid 1983: Clinically mild tularemia associated with tick-borne Francisella tularensis).
- Utah, 1971: Thirty-nine cases of tularemia were reported; most patients contracted illness from the bite of an infected deerfly (Klock 1973). Clinical features included cutaneous ulcers at the site of a bite, lymphadenopathy, fever, chronic malaise, and weakness; all patients recovered. Strain type was not reported.
- Vermont, 1968: Forty-seven cases of tularemia were diagnosed in persons who had handled muskrats in the 4 weeks before illness onset (Young 1969). No fatalities were reported, but 14 patients had a severe prostrating illness that lasted an average of 10 days. Strain type was not reported.
- Nearly all tularemia cases reported outside of North America are cause by F tularensis subsp holarctica (type B).
- Outside the United States, the incidence of disease is highest in Scandinavian countries and Russia (Dennis 1998). In central Europe, analysis of populations in low-risk areas suggests that the disease incidence may be seriously underestimated (Splettstoesser 2009).
- Tularemia is endemic in neoarctic and paleoarctic regions between the latitudes of 30° and 71° N (ie, North America, Europe, states of the Russian Federation, China, and Japan [Dennis 1998]).
- In 2011, investigators identified one case of type B infection in a woman who was bitten by a ringtail possum in a forest in Tasmania, Australia (Jackson 2012). This report may signal the emergence of tularemia in the Southern Hemisphere.
- Outbreaks involving a number of countries in Europe and nearby regions have been reported (Christenson 1984, Dahlstrand 1971, Eliasson 2002, Greco 1987, Gurycova 2006, Kantardjiev 2006, Perez-Castrillon 2001, Reintjes 2002, Siret 2006, Syrjala 1985). Highlights include the following:
- Sweden periodically has years of heavy tularemia activity. In 2003, 698 cases were reported, with a peak in August (300 cases) and September (164 cases). Other years of heavy activity include 1967, 1970, 1981, and 2000 (Payne 2005). A large outbreak (90 cases) occurred in central Sweden in 2006 (Wik 2006).
- An outbreak in the Castilla y Leon region of Spain resulted in more than 500 cases of tularemia in the fall of 2007. The outbreak is thought to have arisen from unusual climatic and environmental circumstances, which produced suitable conditions for propagation of F tularensis. Many of the case-patients were involved in harvesting and related farm work, which likely contributed to production of aerosols that transported the bacteria (Allue 2008, Martin 2007).
- Outbreaks occurred in six provinces in northwestern Turkey between 2004 and 2006. Another outbreak was observed in the Middle Black Sea Region of Turkey in 2005, and was confined to a large, extended family living in the same house. Microagglutination assays were performed on serum samples from all members of the family. Eight of the 16 family members were found to be positive for tularemia serologically, resulting in an attack rate of 50%. Epidemiologic and environmental findings suggested that contaminated water or food was the cause of these outbreaks (Barut 2009, Celebi 2006, Gurcan 2006, Willke 2009, Meric 2010).
- During a recent large tularemia outbreak in Bulgaria, a hunter acquired tularemia through a scratch from a buzzard at a location approximately 100 km from the epidemic focus. No other human or animal cases had been recorded in the hunting region. The isolate from the hunter was found to be identical to one of the four outbreak genotypes. These findings suggest bird-to-human transmission of F tularensis and the role of birds in dissemination of the disease (Padeshki 2010).
- During a 4-year period in France (2006 through 2010), 101 confirmed cases of tularemia were identified (Maurin 2011). Of these, 34% of patients were diagnosed with ulceroglandular disease, 24% with glandular disease, 17% with oropharyngeal disease, and 10% with pneumonic disease. Animal contact was implicated in 27% of the cases and 45% of the cases had unknown exposures. Treatment failures were observed in 23 patients who began antibiotics before being diagnosed with tularemia.
- An endurance athlete developed ulceroglandular tularemia with a primary lesion on his right ankle after running in the forests around Paris. Because his illness occurred during the winter, when insects were unlikely to be viable vectors, the authors suggested that the source of his exposure may have been contact with plants contaminated by a tularemia-infected animal (Meckenstock 2013).
F tularensis is considered a category A bioterrorism agent (CDC: Select agents and toxins). The following information supports the use of F tularensis as a potential biological weapon (CDC: Key facts about tularemia, Christopher 1997, Dennis 2001).
- F tularensis is highly infectious, occurs widely in nature, and can be isolated and grown in quantity in the laboratory.
- During World War II, the Japanese conducted research on F tularensis as a biological weapon.
- During the 1950s and 1960s, the United States developed weapons that could deliver aerosolized F tularensis organisms. The United States government stockpiled weaponized tularemia until stockpiles were destroyed in 1973.
- The former Soviet Union also weaponized F tularensis; the Soviet program included development of antibiotic- and vaccine-resistant strains.
- In 1969, the World Health Organization estimated that an aerosol dispersal of 50 kg of virulent F tularensis over a metropolitan area with 5 million inhabitants in a developed country would result in 250,000 illnesses, including 19,000 deaths.
The most likely form of intentional release for F tularensis organisms would be via infectious aerosols. An aerosol release would be expected to cause the following clinical syndromes:
- Many of the patients would present with primary pneumonic tularemia; however, some would present with a nonspecific febrile illness of varying severity (ie, typhoidal tularemia).
- Cases of oculoglandular tularemia could occur from eye contamination.
- Cases of glandular or ulceroglandular disease could occur through exposure of broken skin to infectious aerosol.
- Cases of oropharyngeal disease also could occur through inhalation of organisms.
An outbreak of tularemia caused by a bioterrorist attack would be expected to have the following features:
- The incubation period generally correlates with the virulence of the infecting strain; in a bioterrorist attack, a highly virulent strain with a relatively short incubation period likely would be used. Illness onsets would generally occur 3 to 5 days after the initial release but could occur as soon as 7 hours and up to 14 days later (Eliasson 2007).
- Illness would probably occur in an urban area and not in rural regions (where naturally occurring tularemia would be more prevalent).
- Patients would not have risk factors for tularemia exposure (eg, outdoor field work, recent outdoor recreational activity, agricultural exposures, exposure to tissues of potentially infected animals).
In the event of a bioterrorist attack, use of F tularensis strains with enhanced virulence or antimicrobial resistance is of concern; therefore, past experience may not be a valuable predictor of disease severity under such circumstances.
A variety of surveillance systems, in addition to traditional pathogen-specific disease reporting, are in place to detect unusual events impacting humans, such as what would be expected in a bioterrorism attack (Kmam 2012). For example, syndromic surveillance involves monitoring symptom-based patterns for patients reporting to outpatient or hospital settings such as emergency departments. Other electronic surveillance systems monitor data from non-traditional sources, such as school absenteeism rates, 911 calls, or sales of over-the-counter medications. In some areas, these systems are supplemented with air monitoring for select biological agents through the BioWatch program or through other military-based systems (Kmam 2012).
Some animals might serve as sentinels of certain bioterrorism agents, including Bacillus anthracis and Yersinia pestis. While animals are not likely to provide early warning for a bioterrorist event caused by F tularensis, one review indicates that certain animals (such as prairie dogs, other rodents, raccoons, skunks, and cats) could serve as markers for ongoing environmental exposure risk following a tularemia bioterrorist event or could propagate or maintain an epidemic (Rabinowitz 2006).
F tularensis infection can cause the following clinical syndromes (Dennis 1998):
- Glandular tularemia (10% to 25% of naturally occurring cases)
- Ulceroglandular tularemia (45% to 85% of naturally occurring cases)
- Pneumonic tularemia (<5% of naturally occurring cases, although outbreaks following inhalational exposure have been reported; secondary pneumonia [often associated with the typhoidal form] occurs relatively frequently and results from hematogenous spread to the lungs)
- Oculoglandular tularemia (<5%)
- Oropharyngeal tularemia (<5%)
- Typhoidal tularemia (<5%, although in outbreaks caused by aerosol exposure, this percentage may be much higher)
Tularemia can range from a mild infection to a life-threatening illness.
- Before antibiotic therapy was available, the overall case-fatality rate was approximately 7%, although rates as high as 80% were seen with pneumonia and other forms of severe infection (Dennis 2001, Dienst 1963, Pullen 1945).
- Most patients respond rapidly to appropriate antibiotic therapy, with fever and generalized symptoms improving in 24 to 48 hours.
- Historically, type A has been considered to cause more severe disease than type B; however, recent genotyping studies in the United States have demonstrated that type A1b strains are more virulent than type A1a, type A2, or type B. In one analysis of US isolates submitted to the Centers for Disease Control and Prevention (CDC) between 1964 and 2004, investigators found that the mortality rate for genotype A1b (24%) was significantly higher than the mortality rates for A1a (4%), A2 (0%), or type B (7%) (Kugeler 2009).
- One study identified the following factors as associated with a poor outcome (ie, death, relapse, prolonged recovery) (Penn 1987):
- Underlying comorbidity (eg, alcoholism, diabetes)
- Delay in seeking medical care
- Delay in instituting appropriate antibiotic therapy
Clinical features for the major syndromes caused by F tularensis are outlined in the tables below. All of the clinical syndromes can be caused by type A or type B; however, these tables do not distinguish between various subtypes and genotypes.
Initial signs and symptoms can be relatively nondescript, and the diagnosis may be missed (Dembek 2003). One study describes two cases of human infection with F tularensis that were initially diagnosed as herpes simplex or varicella zoster infection (Byington 2008).
Clinical Features of Glandular and Ulceroglandular Tularemia
3-5 days (range, <1-14 days)
—Local painful cutaneous lesion at site of inoculation (papule that ulcerates within a few days) in ulceroglandular form; no cutaneous lesion in glandular form
—In one series of 88 patients with tularemia, admission WBCs ranged from 5,000 to 22,000/mm3 (median, 10,400/mm3)d; differential usually normal early in clinical course
—Suppuration of involved lymph nodes
—4.4% of 181 patients with ulceroglandular tularemia and 4.3% of 23 patients with glandular tularemia among case series of 225 patients reported from pre-antibiotic eraf
Clinical Features of Pneumonic Tularemiaa
3-5 days (range, <1-14 days)
—Patients often present with community-acquired atypical pneumonia nonresponsive to conventional antibiotic therapy
—Radiographic findings for 50 tularemia patients with pleuropulmonary involvementd:
—Lung abscesses or cavitary lesions
—Fatalities rare with appropriate antibiotic therapy (reported as 2.3% in one case series of 88 patients with tularemia, about half of whom had pulmonary involvement; both deaths occurred in patients with pneumoniae)
Clinical Features of Oculoglandular Tularemia
3-5 days (range, 1-14 days)
—Multiple painful yellow conjunctival nodules
—Suppuration of affected lymph nodes
—1 (14.3%) of 7 patients with oculoglandular tularemia among case series of 225 patients reported from pre-antibiotic erac
Clinical Features of Oropharyngeal Tularemia
3-5 days (range, 1-14 days)
—The most common symptoms among 145 patients in Turkeyc:
~Swelling of the neck (92%)
—Generally unremarkable, although leukocytosis may be present
—Fatalities rare with appropriate antibiotic therapy
Clinical Features of Typhoidal Tularemiaa
3-5 days (range, 1-14 days)
—In one series of 88 patients with tularemia, admission WBCs ranged from 5,000 to 22,000/mm3 (median, 10,400/mm3); differential usually normalc
—Secondary pneumonia (83% of patients with typhoidal disease in one case seriesc and 50% in anotherd)
—50% in one series of 14 patients with typhoidal tularemia among case series of 225 patients reported from pre-antibiotic erai
In general, clinical manifestations of tularemia are similar in children and adults. One report from Arkansas compared type of illness and clinical symptoms between children and adults with naturally occurring tularemia identified in 1983; findings are noted in the following table.
Comparison Between Cases of Tularemia in Children and Adults, Arkansas 1983
Type of Disease or Symptoms
Percentage of Children
Percentage of Adults
Type of Disease
These findings are also supported by a recent review of pediatric cases of tularemia in Turkey (Kaya 2012). Oropharyngeal disease was diagnosed in 85% of the cases and glandular disease in the other 15%. The most common symptoms included a sore throat, fever and myalgias, and the most common clinical signs were lymphadenopathy, pharyngeal hyperemia, tonsillitis, and rash.
Tularemia is rarely reported in pregnant women; as of 2013, only seven cases had been identified in the literature (Yesilyurt 2013). Two of the cases, both involving ulceroglandular disease, occurred in the United States in the pre-antibiotic era; one resulted in a spontaneous abortion and the other a premature birth. A 2008 ulceroglandular case was successfully treated with doxycycline and josamycin without any complications. Four oropharyngeal cases occurred in 2010. These women were between 18 and 30 weeks gestation and were successfully treated with gentamicin and ciprofloxacin without complications. These findings suggest that, with proper treatment, tularemia can be managed effectively in pregnant women.
Differential Diagnosis for Glandular Tularemia
Bubonic plague (Yersinia pestis)
—Clinical course often fulminant
Cat-scratch disease (Bartonella henselae)
—History of contact with cats; usually history of cat scratch
Mycobacterial infection, including scrofula (Mycobacterium tuberculosis and other Mycobacterium species)
—With scrofula, adenitis occurs in cervical region
Sporotrichosis (Sporothrix schenckii)
—Lymph nodes generally painless and nontender
Streptococcal or staphylococcal adenitis (Staphylococcus aureus, Streptococcus pyogenes)
—Site of initiating infection often present distal to involved nodes (ie, pustule, infected traumatic lesion)
Chancroid (Haemophilus ducreyi)
—Adenitis occurs in inguinal region only
Lymphogranuloma venereum (Chlamydia trachomatis)
—Adenitis occurs in inguinal region only
Primary genital herpes
—Herpes lesions in genital area
Secondary syphilis (Treponema pallidum)
—Enlarged lymph nodes in inguinal region only
Differential Diagnosis for Ulceroglandular Tularemia
Anthrax (Bacillus anthracis)
—Painless ulcer that develops into black eschar over several days
Orf (orf virus, a parapox virus)
—Occurs in farm workers
Pasteurella infections (Pasteurella multocida)
—History of dog or cat exposure (animal bite or licking of open wound)
Primary syphilis (Treponema pallidum)
—Characterized by painless ulcer (chancre) in genital area
Rat-bite fever (Spirillum minus)a
—Infection caused by S minus occurs in Asia
Rickettsialpox (Rickettsia akari)
—Initial presentation involves painless papule which forms black eschar
Scrub typhus (Orientia tsutsugamushi; formerly Rickettsia tsutsugamushi)
—Zoonotic infection from chigger bites
Staphylococcal or streptococcal cellulitis (Staphylococcus aureus, Streptococcus pyogenes)
—May be history of trauma or preexisting lesion at site of infection
Differential Diagnosis for Pneumonic Tularemia
Community-acquired bacterial pneumonia
—Legionellosis and many other bacterial agents (S aureus, S pneumoniae, H influenzae, K pneumoniae, M catarrhalis) usually occur in persons with underlying pulmonary or other disease or in elderly
Inhalational anthrax (Bacillus anthracis)
—Widened mediastinum and pleural effusions seen on CXR or chest CT
Pneumonic plague (Yersinia pestis)
—Hemoptysis commonly occurs
Q fever (Coxiella burnetii)
—Exposure to infected parturient cats, cattle, sheep, goats
Tuberculosis (Mycobacterium tuberculosis)
—More common among elderly or among persons who have lived in tuberculosis-endemic countries (ie, developing world, countries of the former Soviet Union)
—Influenza generally seasonal (October-March in United States) or involves history of recent cruise ship travel or travel to tropics
Differential Diagnosis for Oculoglandular Tularemia
Adenoviral infection (adenovirus)
—Generally not associated with regional lymphadenopathy
Cat-scratch disease (Bartonella henselae)
—Watery discharge, granulomatous conjunctival nodule, chemosis
Coccidioidomycosis (Coccidioides immitis)
—Granulomatous conjunctival nodule with small areas of necrosis
Herpes infection (herpes simplex virus)
—Causes characteristic dendritic keratitis in addition to conjunctivitis
Pyogenic bacterial infections
—Mild cases not associated with regional lymphadenopathy
Sporotrichosis (Sporothrix schenckii)
—Firm chancre in skin of eyelid, yellow conjunctival nodules, subcutaneous nodules along lymphatics
Syphilis (Treponema pallidum)
—Conjunctival ulcer with indurated margin and gray base
Tuberculosis (Mycobacterium tuberculosis)
—Small conjunctival ulcer embedded in nodule
Differential Diagnosis for Oropharyngeal Tularemia
Streptococcal pharyngitis (Streptococcus pyogenes)
—Responds to penicillin therapy
Infectious mononucleosis (Epstein-Barr virus)
—Most common in young adults
Adenoviral infection (adenovirus)
—Occurs mostly in children and young adults
Diphtheria (Corynebacterium diphtheriae)
—Primarily occurs in nonimmune children under 15 yr of age
Differential Diagnosis for Typhoidal Tularemia
Typhoidal tularemia without sepsisa
Brucellosis (Brucella abortus and other Brucella species)
—Usually history of contact with tissues, blood, aborted fetuses of infected animals (cattle, swine, goats, sheep)
Disseminated mycobacterial or fungal infection
—Underlying illness usually present
—Features of endocarditis (eg, cardiac murmur, embolic phenomenon) often present
Leptospirosis (Leptospira interrogans)
—History of exposure to infected animals or to water or soil contaminated with urine from infected animals
Pontiac fever (Legionella pneumophila)
—Often mild clinical illness; does not require antibiotic therapy
Malaria (Plasmodium species)
—History of travel to malaria-endemic area is typical
Q fever (Coxiella burnetii)
—Exposure to infected parturient cats, cattle, sheep, goats
Typhoid fever (Salmonella typhi)
—Symptoms of enterocolitis and abdominal pain may be more prominent with typhoid fever than with typhoidal tularemia
Typhoidal tularemia with sepsis
Meningococcemia (Neisseria meningitidis)
—Rapid progression to shock and often death
Septicemic plague (Yersinia pestis)
—Often secondary to bubonic plague (characteristic bubo present in groin, axilla, or cervical region)
Septicemia caused by other gram-negative bacteria
—Underlying illness usually present
Staphylococcal or streptococcal TSS (Staphylococcus aureus, Streptococcus pyogenes)
—Streptococcal TSS may be associated with necrotizing fasciitis
As of July 2013, only eight cases of F novicida infection had been reported in the literature. These cases are summarized below.
Case Reports for Francisella novicida Infection
Age and Gender
Year and Location
Presentation and Underlying Illness
Clinical Course and Outcome
—Presented with cervical lymphadenopathy
—Lymph node biopsy yielded F novicida
—Presented with fever, nausea, and vomiting
—Blood cultures yielded F novicida
Galveston, Texas, 1991
—Experienced 3 weeks of progressive weakness, dyspnea, and chest pain
—Blood cultures yielded F novicida
Rural Texas, 1995
—Described a 1-week history of fever, chills, cough, dyspnea, and nausea
—Blood cultures yielded F novicida
Australia, 2003 (date of publication; date of occurrence is not recorded)
—Presented with a swollen toe and inguinal lymphadenopathy 1 day after cutting foot in brackish water
—A swab of the toe yielded F novicida
—Had 2-3 weeks of swelling of the neck and face
—Biopsy in the area of the right parotid gland yielded F novicida
—History of advanced ovarian cancer
—Blood cultures yielded F novicida
South Carolina, 2012 (date of publication; date of occurrence is not recorded)
—Near drowning with severe neck trauma and resultant quadriplegia
—Blood cultures yielded F novicida
Specimen Collection and Transport
The Laboratory Response Network (LRN)
Standard Tests for Detection of F tularensis
Additional Tests for Detection, Confirmation, and Characterization of F tularensis
Selected Tests Approved for LRN National Laboratories
- Because F tularensis may grow in conventional culture media, thus posing danger to laboratory personnel, the ordering physician should immediately notify the laboratory if the diagnosis of tularemia is suspected. Delay in such notification can lead to unnecessary exposure of laboratory personnel (Shapiro 2002).
- Advance notification also may increase the likelihood of detection, as use of special culture media can improve recovery of the organism.
Collection and Transport of Clinical Specimens for Diagnosis of Tularemia
Specimen Collection and Transporta,b
—Collect volume and number of sets per established laboratory protocol.
Ulcer, other tissue specimens
—Collect biopsy (preferred), scraping, swab, or aspirate of ulcer, lymph node, or other involved tissue.
—Respiratory specimens (pleural fluid, sputum), gastric washings, or corneal scraping (oculoglandular tularemia) can be sent for culture.
—Environmental samples should only be collected in context of epidemiologic investigation.
c Walker 2010.
Guidelines have been published for packing and shipping of infectious substances, diagnostic specimens, and biological agents from suspected bioterrorism (ASM 2011).
F tularensis remains an important potential cause of laboratory-acquired infections (Pike 1976), owing in part to the very low infectious dose (ie, as low as 10 organisms by aerosol exposure or inoculation). Tularemia was the most common laboratory-acquired infection during the 25 years of the US Biological Warfare Program (Rusnak 2007). More recently, in November 2009, a researcher at the US Army Medical Research Institute of Infectious Diseases (USAMRIID) in Fort Detrick, Maryland, contracted tularemia while working on a vaccine for the disease (Eckstein 2009).
- The agent may be present in virtually any human specimen, tissues from infected animals, and fluids from infected arthropods.
- Recognized laboratory hazards include the following (CDC 2009: Biosafety in microbiological and biomedical laboratories):
- Exposure to infectious aerosols or droplets through manipulation of cultures
- Direct contact of skin or mucous membranes with infectious materials
- Accidental parenteral inoculation
- Accidental ingestion
- Manipulation of cultures presents the greatest risk to laboratory workers. In Missouri from 2000 through 2007, specimens for 33 (85%) of 39 culture-confirmed cases were received by the laboratory without any indication of suspicion of a tularemia diagnosis. Laboratory workers should take appropriate precautions when handling culture specimens from tularemia-endemic regions (CDC 2009: Tularemia--Missouri).
- Recommended laboratory precautions include the following (CDC 2009: Biosafety in microbiological and biomedical laboratories):
- Biosafety level 2 (BSL-2) practices, containment equipment, and facilities (which are present in most hospital clinical laboratories) are recommended for activities with clinical materials of human or animal origin containing or potentially containing F tularensis.
- Biosafety level 3 (BSL-3) practices, containment equipment, and facilities are recommended for all manipulations of cultures and for experimental animal studies.
- Vaccination is not recommended for Laboratory Response Network (LRN) sentinel laboratory personnel (CDC: Emergency Preparedness and Response > Preparedness for All Hazards > Labs > Biosafety > Vaccines).
- Laboratory safety practices associated with F tularensis and other potential agents of bioterrorism have been reviewed elsewhere (Sewell 2003).
- One report demonstrated that delay in appropriate identification of F tularensis led to the potential exposure of 12 microbiology laboratory workers (11 of whom received prophylactic antibiotics; the other was pregnant and was placed on fever watch) (Shapiro 2002). The authors suggested that when an organism is initially recognized as having the following properties, further work with the organism should be performed within a biological safety cabinet by a technologist wearing gloves and a gown until the organism is further identified:
- Small, poorly staining, Gram-negative rods seen mostly as single cells that yield mostly pinpoint colonies on chocolate agar and often sheep agar at 48 hours
- No growth on MacConkey or eosin-methylene blue agar
- Oxidase-negative with a negative or weakly positive catalase test
- A negative satellite test
- Isolates that grow only on buffered chocolate yeast extract and chocolate agar
- F tularensis is classified as a select agent and therefore is regulated under 42 CFR part 73 (Possession, Use, and Transfer of Select Agents and Toxins), which was published as a Final Rule in the Federal Register in March 2005 and amended in October 2012 (HHS 2012)As specified in the Public Health Security and Bioterrorism Preparedness and Response Act of 2002, 42 CFR part 73 provides requirements for laboratories that handle select agents (including registration, security risk assessments, safety plans, security plans, emergency response plans, training, transfers, record keeping, inspections, and notifications). Effective April 3, 2013, F tularensis will be considered a Tier 1 agent and subject to additional security requirements (HHS 2012). Select agents are biological agents designated by the US government to be major threats to public health and safety. A current list of select agents is published on the CDC Web site under information about the Select Agent Program (CDC/APHIS 2008). In addition, the CDC has published additional guidelines for enhancing laboratory security for laboratories working with select agents (CDC 2002: Laboratory security and emergency response guidance for laboratories working with select agents).
- Cultures of F tularensis must be transported as "Category A infectious substances." F tularensis is classified under World Health Organization (WHO) infectious agent risk group 4. Isolates that are reasonably suspected to contain F tularensis must be transported as "infectious substances." The US Department of Transportation regulations and International Air Transport Association (IATA) rules require training of all individuals involved in the transport of dangerous goods, including infectious substances (DOT and IATA 2012).
- Chain-of-custody should be documented for material that may constitute evidence of criminal activity. Samples collected for forensic analysis typically are stored in PBS. However, 1% ammonium chloride is comparable to PBS for preserving samples for additional analysis and will allow for mass spectrometry, making it an ideal sample storage solution if mass spectrometry will be used (Valentine 2011).
The LRN is a network of more than 150 national and international laboratories. The network includes federal, state, and local public health, military, food testing, environmental, veterinary, and international laboratories (CDC: Facts about the Laboratory Response Network, CDC: Laboratory Response Network).
- The LRN structure for bioterrorism designates laboratories as sentinel, reference, or national. Designation depends on the types of tests a laboratory can perform and how it handles infectious agents to protect workers and the public.
- Sentinel laboratories, formally called "level A laboratories,"represent an estimated 25,000 hospital-based laboratories that have direct contact with patients. In an unannounced or covert terrorist attack, sentinel laboratories could be the first facilities to encounter suspicious specimens. These laboratories generally have at least BSL-2 containment capabilities. A sentinel laboratory's responsibility is to rule out B anthracis or refer a suspicious sample to the nearest LRN reference laboratory. Sentinel laboratories use the ASM Sentinel Level Clinical Microbiology Laboratory Guidelines to rule out microorganisms that might be suspected as agents of bioterrorism (ASM: Sentinel level clinical microbiology laboratory guidelines).
- Reference laboratories, sometimes referred to as "confirmatory reference," can perform tests to detect and confirm the presence of a threat agent. These laboratories ensure a timely local response in the event of a terrorist incident. Rather than having to rely on confirmation from laboratories at the CDC, reference laboratories are capable of producing conclusive results; this allows local authorities to respond quickly to emergencies. These are mostly state or local public health laboratories but also include military, international, veterinary, agriculture, and food- and water-testing laboratories. Reference laboratories operate with BSL-3 containment facilities that have been given access to nonpublic testing protocols and reagents. One of the roles of the LRN reference laboratories is to provide guidance, training, outreach, and communications to the sentinel laboratories in their jurisdictions.
- National laboratories have unique resources to handle highly infectious agents and the ability to identify specific agent strains through molecular characterization methods. These laboratories also are responsible for methods development, bioforensics, and select-agent activity.
No commercial "kit" system is available for F tularensis, but a number of methods are available for diagnostic purposes (Shapiro 2009). Tularemia diagnosis traditionally has been based on serology, with a definitive diagnostic criterion of a fourfold or greater rise in antibody titers between acute-phase and convalescent-phase sera. As with other infections, there are limitations to serologic diagnosis of tularemia, including difficulty in obtaining acute and convalescent specimens (especially with the nonspecific clinical presentation), cross-reactivity with other bacteria, blunting of the serologic response by antiobiotic therapy, and long-term persistence of antibody levels (Hepburn 2007).
Direct Stains for Bacterial Micromorphology
- LRN-approved Gram stain (ASM 2013, Cross 2000, Sneath 1986, Wong 1999):
- Tiny, faintly staining, pleomorphic Gram-negative rods (0.2-0.5 mcm X 0.7-1.0 mcm) are noted; cells are smaller than those of Haemophilus species.
- The sensitivity of Gram stain is limited in samples that contain background material (eg, blood, sputum, biopsy tissue).
- Organisms are nonsporulating.
- Tissue stains: F tularensis may be visualized in fixed tissues or impression smears with May-Grunwald-Giemsa, phenol thionin, or modified Dieterle spirochete stain (Gallivan 1980, OIE 2004).
- Approved for LRN sentinel laboratories.
- Standard procedures for culture are as follows (ASM 2013, Wong 1999):
- Culture setup procedure: Use standard routine protocols for specimen type.
- In addition, add cysteine-supplemented media, such as chocolate agar (CA) with IsoVitaleX or buffered charcoal yeast extract agar (BCYE).
- Cysteine heart agar supplemented with 9% heated sheep red blood cells (CHAB) was mentioned in earlier CDC protocols but was excluded from current level A protocols (ASM 2013).
- Selective, enriched media such as Thayer-Martin (TM) media improves isolation from contaminated samples.
- Organisms do not grow on MacConkey or eosin-methylene blue (EMB) agars; they may grow initially on sheep blood agar (SBA) but not on subculture.
- Tape plates shut if F tularensis is suspected.
- Optimal growth conditions include 35oC to 37oC at ambient atmosphere in contrast to Y pestis, which grows faster at 28oC. Use of 5% CO2 is acceptable.
- Incubation time generally is 5 days (7 days if the patient has been treated with a bacteriostatic antibiotic).
- Colony morphology at 24 hours is too small to be seen.
- At 48 hours, colonies are 1 to 2 mm in diameter, white to gray to bluish-gray, opaque, flat, and smooth, with an entire edge and shiny surface.
- F tularensis typically grows slowly in broth media, requiring a minimum of 10 days of incubation without shaking for detection (Ellis 2002).
- Attempts at identification of organisms with the above microscopic, colony, and biochemical characteristics should not be attempted. [Note: This is extremely important because of the potential for generation of infectious aerosols and because of the potential for misidentification.]
- Commercial identification tests:
- The Vitek NHI card may identify F tularensis as Actinobacillus actinomycetemcomitans with as high as a 99% confidence level.
- Other considerations for LRN sentinel laboratories:
- If tularemia is initially suspected, the sentinel laboratory should consult with the state public health laboratory director (or designate) prior to or concurrent with testing.
- The laboratory should communicate immediately with the state public health laboratory director (or designate), the infection control department at the hospital, and the attending physician if F tularensis cannot be ruled out.
- If a bioterrorist release is suspected, the original specimens should be preserved pursuant to a potential criminal investigation. In such situations, the state public health department, in conjunction with the Federal Bureau of Investigation (FBI) or local law enforcement as necessary, will arrange for transport of specimens and/or isolates to a higher-level LRN laboratory.
The CDC has approved the following tests for detection of F tularensis at various levels of the LRN.
Selected Tests Approved for LRN Reference Laboratories
- Direct fluorescent antibody (DFA) stain
- Slide agglutination
- Antimicrobial susceptibility testing
- Biochemical identification/characterization
- Serology (antibody detection)
- Environmental specimen evaluation
- Mouse inoculation
- Cellular fatty acid analysis
- Molecular-based subtyping tests
- Immunohistochemistry (IHC)
Other Described Tests for F tularensis
A wide variety of other tests for F tularensis detection have been developed. These include new serologic tests, PCR tests (including multiplex PCR), 16S rRNA sequencing, time-resolved fluorometry (TRF) assay, and molecular subtyping.
Hand-held immunochromatographic assays have been described and are commercially available, but detection limits appear to be higher than PCR or competitive enzyme-linked immunoabsorbent assay (cELISA) (Alexeter Technologies, Grunow 2000, New Horizons Diagnostics Inc).
Antimicrobial Susceptibility Studies
Two studies have examined antimicrobial susceptibilities of F tularensis strains to various antibiotics. Data from these studies are shown in the table below. The studies were conducted prior to the development of standard methods and interpretive criteria and should be used for comparison purposes only. Only LRN laboratories classified as reference or national should perform susceptibility testing.
The major conclusions from each of the two studies are as follows:
- Ikaheimo 2000, using 38 human and animal F tularensis subsp holarctica strains; tests conducted using Etest (Biodisk; Solna,Sweden):
- All strains were susceptible to the antibiotics traditionally used to treat tularemia (streptomycin, tetracycline, chloramphenicol) as well as other aminoglycosides (tobramycin, gentamicin) and fluoroquinolones (ciprofloxacin, levofloxacin, grepafloxacin, trovafloxacin).
- All strains were resistant to beta-lactam antibiotics and azithromycin.
- The strains used in this study were F tularensis subsp holarctica (type B), which may not reflect susceptibility patterns of F tularensis subsp tularensis (type A), the more likely agent in a bioterrorism event.
- Baker 1985, using 13 "glycerol-positive" and 2 "glycerol-negative" strains of F tularensis; tests conducted using broth dilution:
- Aminoglycosides (streptomycin, gentamicin, tobramycin), tetracycline, chloramphenicol, and erythromycin were effective in vitro.
- The authors concluded that several cephalosporins (ceftazidime, cefotaxime, ceftriaxone) were effective in vitro, while other beta-lactam antibiotics were not. They acknowledged, however, that all strains produced beta-lactamase.
MICs of Various Antibiotics for Francisella tularensis Isolates as Identified in Two Studies
Additional antimicrobial susceptibility studies include the following:
- A study of eight isolates of F tularensis subsp tularensis (type A, the type most likely to be involved in a bioterrorism event) from the United States found them all to be highly susceptible to fluoroquinolones at 0.125 mcg/mL or less (Johansson 2002).
- Resistance of F tularensis to chloramphenicol and tetracycline has been experimentally created in the laboratory by the use of transformed plasmids (Pavlov 1996).
- In Austria, F tularensis subsp holarctica biovar II strains from 47 hares and three humans were found to be susceptible by Etest to tetracyclines, aminoglycosides, quinolones, chloramphenicol, and rifampicin. Isolates were generally resistant to macrolides, penicillins, azetreonam, cephalosporins, and carbapenems (Tomaso 2005).
- The Clinical and Laboratory Standards Institute provided a susceptibility testing method with breakpoints. In total, they tested 169 isolates (92 type A, 77 type B) against seven antimicrobial agents (streptomycin, gentamicin, tetracycline, doxycycline, ciprofloxacin, levofloxacin, and chloramphenicol) used for treating tularemia. The minimum inhibitory concentrations (MICs) for all of the isolates were within the susceptible range. All isolates had MICs for erythromycin between 0.5 and 4 mcg/mL, in contrast to greater than 256 mcg/mL for the common laboratory live vaccine strain (LVS) (Urich 2008).
- A study of 39 F tularensis subsp holarctica isolates from central Turkey found that they were all highly susceptible fluoroquinolones, with levofloxacin reporting the best in vitro results (Yesilyurt 2011).
Images of F tularensis Gram stain, direct fluorescent antibody stain, and colony morphology can be viewed on the CDC's Web site (CDC: Public Health Image Library).
Available data support the following statements about treatment of tularemia:
- Streptomycin and gentamicin have been shown to be effective and are considered first-line therapies (AAP 2006, Enderlin 1994, Evans 1985, Hassoun 2006, Mason 1980). Susceptibility testing supports their use as first-line therapies for all four subspecies of F tularensis as well as for F philomiragia and F hispaniensis (Georgi 2012).
- Ciprofloxacin and other fluoroquinolone antibiotics have been used effectively to treat tularemia, although experience with these medications is somewhat limited to date (Johansson 2000, Limaye 1999). In one outbreak, ciprofloxacin showed a lower treatment failure rate than streptomycin or doxycycline (Perez-Castrillon 2001).
- Tetracycline and chloramphenicol can be used to treat tularemia; however, because these drugs are bacteriostatic, relapses occur more often than with use of aminoglycosides (Evans 1985, Overholt 1961).
The Working Group on Civilian Biodefense recommends the following for treating bioterror-related tularemia (Dennis 2001):
- In a contained casualty setting in which the medical care delivery system can effectively manage the number of patients, parenteral antibiotics should be administered according to the table below to all patients whenever possible.
- In a mass casualty setting in which the medical care delivery system is not able to meet the demands for patient care, use of oral antibiotics may be necessary. In such a situation, the medications listed in the table below on antibiotic postexposure prophylaxis should be used and therapy should be continued for 14 days.
- Supportive care of patients also is critical, including fluid management and hemodynamic monitoring as indicated. Some patients would require intensive care with respiratory support owing to complications of Gram-negative sepsis (eg, shock, adult respiratory distress syndrome, multisystem failure, disseminated intravascular coagulation).
- Bioterrorist use of an F tularensis strain resistant to conventional antibiotic therapy is of concern and should be considered, particularly if patients deteriorate despite early initiation of antibiotic therapy.
Recommendations for Treatment of Tularemia During a Bioterrorism Event
Choices by Patient Category
Adults: Preferred choices
Streptomycin: 1 g IM twice daily for 10 daysc,d,e
Adults: Alternative choices
Doxycycline: 100 mg IV twice daily for 14-21 dayse
Children: Preferred choices
Streptomycin: 15 mg/kg IM twice daily (maximum daily dose, 2 g) for 10 daysc
Children: Alternative choices
Some have suggested that doxycycline is a good first choice for treatment, because it has greater efficacy than ciprofloxacin, has less potential for development of resistance, and is less expensive. Patient relapse is possible with this drug, however (Brouillard 2006, Tarnvik 2007). Initial data from a mouse study suggest that doxycycline is most effective in keeping mice alive if administered within 24 hours after exposure and that its effectiveness drops to 30% if not administered until 48 hours after exposure (Rotem 2012). Ciprofloxacin remained 70% effective when administered up to 72 hours after exposure.
A study of 145 patients with oropharyngeal tularemia showed that 55 (38%) of those treated experienced initial therapeutic failure (defined as persistence or recurrence of fever, persistence of constitutional symptoms, or increase in size or appearance of new lymphadenopathies). Those patients required a second course of antibiotics, usually using an alternative agent. Logistic regression analysis found that initial therapeutic failure was significantly associated with delay of treatment exceeding 14 days and was not associated with the type of antibiotic used initially (Meric 2008). Treatment failure has been associated with gentamicin use, suggesting that streptomycin may be the most appropriate first-line drug for treating possible tularemia infections (Kaya 2011).
According to the American Academy of Pediatrics (AAP), streptomycin, gentamicin, and amikacin are recommended for treatment of children (AAP 2006). Other therapies cited by the AAP include tetracycline and chloramphenicol, although relapse rates are higher with these agents.
Ciprofloxacin has been shown to be an effective therapy for tularemia in children (Johansson 2000, Johansson 2002). The drug has been approved only for specific indications in patients younger than 18 years old (AAP 2006).
The newer fluoroquinolones trovafloxacin and grepafloxacin were studied in mice challenged via the subcutaneous route with 1 x 103 colony-forming units of F tularensis. Antibiotics were orally administered once a day for 7 days, commencing at 24 and 48 hours postchallenge. Mice were observed for an additional 14 days. Trovafloxacin provided complete protection against experimental F tularensis whether administered at 24 or 48 hours following infection. Grepafloxacin had more limited therapeutic activity, with survival rates of 50% and 80% when treatment was initiated at 24 hours and 48 hours, respectively. No untreated control mice survived. Both of these antibiotics have since been withdrawn from the market; however, these data confirm the efficacy of newer-generation fluoroquinolones for postexposure prophylaxis against tularemia. Furthermore, the data provide additional evidence of the likely therapeutic windows for the use of these antibiotics (Kenny 2009).
One challenge study involving volunteer subjects demonstrated that antibiotic postexposure prophylaxis using tetracycline can prevent disease occurrence among exposed persons if given within 24 hours after challenge (Sawyer 1966).
The Working Group on Civilian Biodefense has made the following recommendations for postexposure prophylaxis of inhalational tularemia during a bioterrorist attack (Dennis 2001):
- If the release of F tularensis becomes known before clinical cases occur (ie, during the incubation period), persons in the exposed population should be placed on prophylactic oral antibiotics (doxycycline or ciprofloxacin) for 14 days, according to the regimens outlined in the table below.
- If the release is covert and does not become apparent until after clinical cases begin to occur, then potentially exposed persons should be placed on a fever watch. Any person in whom a fever or flulike illness develops should be evaluated and placed on appropriate antibiotic therapy (either parenteral [in the contained casualty setting] or oral [in the mass-casualty setting]) for treatment of presumptive tularemia.
Decision making around use of postexposure prophylaxis would likely be extremely difficult in situations involving a suspected bioterrorist attack. If an exposed population can be inferred with reasonable probability from the ongoing investigation, strong consideration should be given to providing postexposure prophylaxis, even after clinical cases begin to occur. As noted during the anthrax investigation on Capitol Hill in late 2001, patients initiating prophylaxis may later discontinue it if the investigation subsequently determines that they were not at risk. Careful proactive initiation of postexposure prophylaxis should not be underestimated for its medical, public health, psychological, and political merits in coping with a terrorist attack.
Recommendations for Antibiotic Postexposure Prophylaxis During an Outbreak of Tularemia Following a Bioterrorism Eventa
Adults (including pregnant women)
Doxycycline: 100 mg PO twice daily for 14 daysc
- No licensed vaccine against tularemia currently is available in the United States.
- Until relatively recently, a vaccine incorporating an attenuated, type B derivative LVS was used in the United States to protect laboratory workers at high risk for F tularensis exposure. The vaccine was available at USAMRIID as an investigational new drug, but it has since been withdrawn and is no longer being manufactured (Rusnak 2007). The cell-mediated immune response to this vaccine has been shown to remain high up to 34 years post vaccination, with the immune response being similar to that seen in persons who have recently been vaccinated (Eneslatt 2011).
- An improved LVS preparation has been tested for preclinical safety, tolerability, and immunogenicity in rabbits and is currently being evaluated in human clinical studies (Pasetti 2008). A major barrier for licensure of an LVS vaccine is the need to rely on the animal rule, becausea correlate of protection for the LVS vaccine does not exist. To date, no new vaccine has been approved using the animal rule (Conlan 2011: Tularemia vaccines: recent developments and remaining hurdles).
- Four collections of human sera, which included sera from persons who had natural infections and those who had been vaccinated, were evaluated to identify commonly reactive proteins. Seven proteins were found to react with more than 60% of the sera samples, raising the possibility that one of these proteins could be used as a correlate of protection (Fulton 2011).
- Because the incubation period for tularemia is usually 3 to 5 days and immunity following vaccination takes about 2 weeks to develop, postexposure vaccination is not considered a viable public health strategy to prevent disease in the event of a mass exposure.
- Mice that received LVS immune serum and were subsequently challenged with F tularensis over the next 48 hours survived the exposure. Previously investigators had believed that the protection provided to mice by LVS was primarily cellular, but these results suggest that a humoral component may be essential for protection, particularly in the first few hours following infection (Mara-Koosham 2011).
- Future work to develop vaccines against F tularensis likely will focus on subunit vaccines and use of recombinant technology (Jia 2009, Sjostedt 2003). However, developing a new, effective vaccine against F tularensis poses several challenges:
- The immunodominant antigens have not been fully identified and characterized.
- The virulence determinants are not well known.
- Generation of both humoral and cellular immune responses appears to be necessary for protection against infection.
Person-to-person transmission of tularemia has not been documented; therefore, Standard Precautions are considered adequate for patients with tularemia (CDC/HICPAC 2007).
- Commercially available bleach or a 1:10 dilution of household bleach and water is considered adequate for cleaning contaminated surfaces. After 10 minutes, a 70% solution of alcohol can be used to further clean the area and reduce the corrosive action of the bleach (Dennis 2001).
- Following direct exposure to powder or liquid aerosols containing F tularensis, body surfaces and clothing should be washed with soap and water.
- Water contamination can be eradicated through standard chlorination. Normal chlorination levels in drinking water will reduce F tularensis strains by 4 log10 in less than 2 hours (O'Connell 2011). Chlorinating natural water sources is generally not feasible, so contaminated natural water sources would need to be contained to prevent public and animal use (Hodges 2010).
- The risk of environmental contamination following an intentional release of F tularensis is expected to be minimal, and no special environmental decontamination procedures are recommended (Dennis 2001).
- The most current guidelines from the CDC indicate that Standard Precautions should be used for postmortem care. These include using a surgical scrub suit, surgical cap, impervious gown or apron with full sleeve coverage, a form of eye protection (eg, goggles or face shield), shoe covers, and double surgical gloves with an interposed layer of cut-proof synthetic mesh (CDC 2004).
- In addition, autopsy personnel should wear N-95 respirators during all autopsies, regardless of suspected or known pathogens. Powered air-purifying respirators (PAPRs) equipped with N-95 or high-efficiency particulate air (HEPA) filters should be considered.
- Procedures that induce aerosols or splashing should be avoided if possible.
- Contact with corpses should be limited to trained personnel, and routine precautions should be implemented when transporting corpses.
- Bodies infected with biological terrorism agents should not be embalmed (CDC 2004). Bodies infected with F tularensis can be directly buried without embalming.
Tularemia was removed from the list of Nationally Notifiable Diseases in 1994 but was reinstated in 2000 because of concerns about tularemia as a biological weapon (CDC 2002: Summary of Notifiable Diseases—United States, 2000). Cases of tularemia should be reported immediately to public health authorities, according to disease reporting rules within each state or local jurisdiction.
In 1997, the Council of State and Territorial Epidemiologists (CSTE) and the CDC revised the public health case definitions for conditions under public health surveillance (CDC 1997). The current public health case definitions for tularemia include the following.
An illness characterized by several distinct forms, including the following:
- Glandular (regional lymphadenopathy with no ulcer)
- Ulceroglandular (cutaneous ulcer with regional lymphadenopathy)
- Pneumonic (primary pleuropulmonary disease)
- Oculoglandular (conjunctivitis with preauricular lymphadenopathy)
- Oropharyngeal (stomatitis or pharyngitis or tonsillitis and cervical lymphadenopathy)
- Intestinal (intestinal pain, vomiting, and diarrhea)
- Typhoidal (febrile illness without early localizing signs and symptoms)
Clinical diagnosis is supported by evidence or history of a tick or deerfly bite, exposure to tissues of a mammalian host of F tularensis, or exposure to potentially contaminated water.
- Elevated serum antibody titer(s) to F tularensis antigen (without documented fourfold or greater change) in a patient with no history of tularemia vaccination or
- Detection of F tularensis in a clinical specimen by fluorescent assay
- Isolation of F tularensis in a clinical specimen or
- Fourfold or greater change in serum antibody titer to F tularensis antigen
Probable:A clinically compatible case with laboratory results indicative of presumptive infection
Confirmed:A clinically compatible case with confirmatory laboratory results
Early disease outbreak recognition may significantly modify the outcome of a biological attack. A list has been developed of potential epidemiologic clues or "red flags" for an unusual event. Although these clues may be associated with natural outbreaks and bioterrorism events, their occurrence should heighten suspicion. Potential clues include the following (Dembek 2007):
- Highly unusual event with large numbers of casualties
- Higher morbidity or mortality than is expected
- Uncommon disease
- Unusual disease manifestation
- Lower attack rates in protected persons
- Point-source outbreak
- Multiple epidemics
- Downwind plume pattern
- Dead animals
- Reverse or simultaneous spread of human and animal cases
- Direct evidence
Several naturally occurring outbreaks have had characteristics of an unusual event; one of these was the 2000 Martha's Vineyard tularemia outbreak. Of the clues listed above, the first four were present in the Martha's Vineyard outbreak. After investigation, the proposed exposure scenario for this outbreak was that F tularensis was shed in animal excreta, persisted in the environment, and infected humans via mechanical aerosolization and inhalation.
In 2006, an animal control contractor observed a jackrabbit die-off near a metropolitan airport in Texas. Inspection of the area also revealed a large number of ticks. Specimens from several mammals (jackrabbits, a skunk, and a coyote) and ticks tested positive for F tularensis. Further investigation determined the die-off most likely was due to epizootic tularemia, and suspicion of a bioterrorism event was low. A coordinated, multiagency response was organized, however, because of proximity to human traffic areas and fears of transmission of tularemia to humans via contact with infected animals, vectors, or aerosolization of live bacteria. No human tularemia cases occurred, and the tularemia epizootic subsequently ended; the favorable outcome may have been due to the response efforts or spontaneous resolution. During an after-action analysis, the following were identified as lessons learned (Pierce 2009):
- The importance of animal illness surveillance
- The usefulness of pre-event response planning, training, and exercises
- The importance of familiarity with the CDC category A bioterrorism agents for rapid response
- The usefulness of an established, effective communication system with the healthcare community
- The possibility that environmental control attempts may result in perturbations in animal populations with unintended consequences
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