Tularemia: Microbiology, epidemiology, and pathogenesis

INTRODUCTION — Tularemia is the zoonotic infection caused by Francisella tularensis, an aerobic and fastidious gram-negative bacterium. Human infection occurs following contact with infected animals or invertebrate vectors. Synonyms include Francis disease, deer-fly fever, rabbit fever, market men disease, water-rat trappers disease, wild hare disease (yato-byo), and Ohara disease [1].

The microbiology, epidemiology, and pathogenesis of infection due to F. tularensis will be reviewed here. The clinical manifestations, diagnosis, treatment, and prevention of tularemia are discussed separately. (See "Tularemia: Clinical manifestations, diagnosis, treatment, and prevention".)

MICROBIOLOGY

Francisella species — The Francisella genus includes the potential human pathogens F. tularensis, F. philomiragia, F. hispaniensis, and F. opportunistica [2-4]. There are also the fish pathogens F. noatunensis, F. orientalis, F. salimarina, and F. halioticida and many environmental isolates [4-6]. Some environmental species previously placed in the Francisella genus have been reclassified in a new genus, Allofrancisella, including A. guangzhouensis (previously Francisella guangzhouensis) [7]. Other Francisella species, some not officially named, have also rarely caused human infections [6,8].

The subspecies of F. tularensis are [1,9,10]:

●F. tularensis subspecies tularensis (also called F. tularensis type A or Francisella neoarctica). In addition, molecular typing methods have identified distinct clades or genotypes of F. tularensis subspecies tularensis that differ in their geographic locations and virulence [11,12].

●F. tularensis subspecies holarctica (also called F. tularensis type B).

●F. tularensis subspecies novicida (also called Francisella novicida since its classification as a subspecies of F. tularensis has been controversial).

●F. tularensis subspecies mediasiatica.

The majority of infections in humans and animals are caused by F. tularensis subspecies tularensis (the more virulent subspecies) and F. tularensis subspecies holarctica. Specific strains of F. tularensis subspecies tularensis may be relatively restricted in their locations, although subspecies holarctica strains are more geographically dispersed [11]. Within a single region, animal and human outbreaks may involve multiple F. tularensis types [13,14].

Human disease is rarely associated with the subspecies novicida, F. philomiragia, F. hispaniensis, F. opportunistica, or other species [1,4,6,9,15]. (See 'Geographic distribution' below.)

Laboratory features — Francisella spp are small, pale-staining, slow-growing, aerobic, gram-negative coccobacilli. The organisms are fastidious and infrequently isolated from clinical specimens [9]. Most strains require cysteine or cystine for growth, but strains that are less fastidious or lack a cysteine requirement have been described [16]. F. tularensis grows on some commercial media, including chocolate agar, modified Thayer-Martin media, and buffered charcoal-yeast extract agar [9]. Culture material from nonsterile sites should be plated on supportive media supplemented with antibiotics. Growth in the laboratory is optimal at 35°C, with or without carbon dioxide supplementation.

Cultures on solid media may require 2 to 14 days of incubation to observe growth, and blood cultures may require 5 to 7 days of incubation [9].

Isolates suspicious for F. tularensis can be characterized as tiny, poorly staining, gram-negative coccobacilli that are slow growing, grow better on chocolate agar than on blood agar, do not grow on routine gram-negative selective media, are oxidase negative, are catalase negative or weakly catalase positive, are beta-lactamase positive, and are satellite or XV test negative [17]. These features should alert laboratory personnel that an isolate may be F. tularensis. In reference laboratories, F. tularensis can be differentiated from other Francisella species and from other bacteria by using direct fluorescent antibody staining, slide agglutination, polymerase chain reaction (PCR) assay, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF), sequencing, or cellular fatty acid composition analysis [9].

Laboratory precautions — Because F. tularensis is difficult to grow and dangerous to handle, laboratory personnel should be notified whenever tularemia is considered. Specimens from patients or animals suspected of having tularemia may be processed initially using Biosafety Level 2 practices, containment equipment, and facilities [9,18,19]. Any isolate suspected of being F. tularensis should be handled using Biosafety Level 3 practices, containment equipment, and facilities and, in the United States, immediately referred to the state public health laboratory or another appropriate Laboratory Response Network facility for further evaluation [18,19]. In particular, automated identification systems should not be used because they may generate aerosols and misidentify F. tularensis [17]. In addition, automated system misidentifications of Veillonella, Pseudomonas species, and Paenibacillus as F. tularensis have been reported [9,20,21].

Possession and shipment of F. tularensis is tightly restricted because of its potential as a bioterrorism agent, and appropriate federal regulations must be followed [1,17,19].

EPIDEMIOLOGY

Geographic distribution — Tularemia occurs throughout North America and Europe, as well as in Asia and the Middle East. Tularemia is relatively uncommon in Australia, England, and South American countries. Although tularemia has rarely been reported in African countries, there is increasing evidence of its presence in Africa [22-24].

North America — In North America, F. tularensis has been described in the United States, Canada, and Mexico. In these regions, F. tularensis subspecies tularensis, the most virulent subspecies, accounts for approximately 90 percent of human infections.

In the United States, the majority of cases traditionally occurred in the south-central states (figure 1) [25]. Over time, however, the southern border of tularemia has shifted northward [26]. As an example, the number of reported cases in Colorado, Nebraska, South Dakota, and Wyoming dramatically increased in 2015, and 74 percent of cases occurred in Arkansas, Colorado, Kansas, Missouri, Nebraska, Oklahoma, South Dakota, and Wyoming [27]. More recently, the majority of reported cases have occurred in the south-central states, Pacific Northwest, and parts of Massachusetts (figure 1) [25]. These changes are consistent with the predicted effects of climate change on the geographic distribution of tularemia [28,29].

In the United States, tularemia can occur throughout the year but more commonly occurs in the summer months (figure 2) (because of greater exposure to arthropods during this time) [25].

Europe and Asia — Tularemia is known to be endemic in most European countries, the former Soviet Union, Turkey, Iran, China, and Japan. Outside of North America, F. tularensis subspecies holarctica, a less virulent subspecies, is the most common cause of infection.

Sources of infection — The organism infects more than 100 species of wild and domestic vertebrates and over 100 species of invertebrates. The most important vertebrates associated with F. tularensis infection are lagomorphs (eg, rabbits and hares) and rodents. In the United States, important mammals include rabbits, beavers, muskrats, squirrels, and voles. In Europe, important mammals include hares, hamsters, mice, rats, beavers, lemmings, and voles.

Animals vary in their susceptibility and response to infection. Some lagomorphs, such as rabbits, and rodents tend to develop a fatal illness when infected, while many other species develop infection but do not succumb to the illness.

Francisella is hardy in nature (though it can be difficult to isolate in the laboratory). The organism can persist in animal carcasses, mud, or water for many weeks [30].

Routes of transmission — Humans can acquire infection by several routes (direct or indirect animal contact and arthropod bites) and manifest a variety of clinical syndromes depending on the route of transmission. Human-to-human spread does not occur [31]. However, transmissions during an autopsy and from solid organ transplantation have been documented [32,33]. (See "Tularemia: Clinical manifestations, diagnosis, treatment, and prevention", section on 'Clinical syndromes'.)

Arthropod bites — Transmission to humans can occur from any number of biting arthropods that have previously fed on an infected animal. Overall, tick exposure during the summer months is the most commonly recognized mode of transmission in the United States, particularly in children [34]. Other arthropod vectors include mosquitoes, horse flies, fleas, and lice [35]. In California, Nevada, and Utah, biting flies are the predominant arthropod vectors, whereas ticks are the predominant vectors in areas east of these states. In Finland, Sweden, and areas of the former Soviet Union, mosquitoes are the important insect vectors [36].

F. tularensis may be passed transstadially (ie, across life stages) in ticks. However, studies attempting to demonstrate transovarial transmission have found conflicting results [37].

Animal contact — Transmission to humans can occur from contact with an infected animal, such as when hunting or skinning mammals. The trade in wild animals as pets is another means of exposure to infected animals [38]. An animal bite or scratch can also transmit infection; carnivores, such as wild animals, domestic cats, and, in at least one instance, birds can transmit tularemia through carriage of the organism on claws or in the mouth [1,39,40]. Pet dogs may transmit infection by direct contact from bites, scratches, or face licking, or indirectly by facilitating exposure to contaminated carcasses or ticks [41,42]. Infection can also occur through splashing infected material from animals or ticks into the eyes or rubbing the eyes with contaminated fingers.

Contaminated food or water — Contaminated meat and water remain important environmental sources for transmission, and the disruptions caused by natural disasters or wars have been associated with tularemia outbreaks caused by the ingestion of contaminated food and water. As examples, transmission from unchlorinated drinking water occurred during several different outbreaks in Turkey, and an unusual transmission from a fish hook injury on a freshwater lake in the United States has been reported [43,44]. One outbreak of oropharyngeal tularemia in Germany was traced to grape must (freshly pressed juice that contains the solid parts of the fruit) that was likely contaminated with tissue from an infected rodent during fruit collection and processing [45]. Contaminated ice was responsible for an outbreak of illness in a prison caused by F. novicida [15].

F. tularensis can survive in water for long duration, even if the water is brackish or frozen [1,30,46,47]. Francisella species survival is enhanced in the presence of amoeba, and this may contribute to its persistence in water [48].

Airborne transmission — Transmission can occur from airborne spread of contaminated materials, including dust, hay, and water, as well as laboratory specimens. Activities that can increase the risk for airborne exposure to F. tularensis include lawn mowing or cutting brush. These were associated with infection during a 2000 outbreak of pneumonic tularemia in Martha's Vineyard, Massachusetts, which was thought to be mediated by airborne transmission [49].

In part because of this potential for airborne transmission, F. tularensis is a category A bioterrorism agent (ie, of highest concern for bioterrorism use), as classified by the United States Centers for Disease Control and Prevention. Clustered cases (mainly of pneumonic tularemia but also of typhoidal and other forms) occurring without the expected epidemiologic exposures to animals, arthropods, or environmental activities should raise the possibility of a bioterrorism event. Specific epidemiologic, clinical, and microbiologic clues should lead to early suspicion of intentional tularemia and rapid activation of the health alert system, since laboratory confirmation of the agent could be delayed. (See "Tularemia: Clinical manifestations, diagnosis, treatment, and prevention", section on 'Potential bioterrorism use'.)

Risk factors — The main risk factor for tularemia is exposure to the organism. Given the routes of transmission, farmers, veterinarians, hunters, landscapers, national park service employees, meat handlers, and laboratory workers are among the occupations that have an increased risk for tularemia. (See 'Routes of transmission' above.)

Tularemia may occur at any age, but in the United States, it is most common in children under 15 years of age and in middle-aged adults (figure 3). In all age groups, males are infected more often than females; perhaps they are more likely to participate in at-risk activities.

PATHOGENESIS — The pathogenesis of infection with F. tularensis and its virulence factors have been the subject of intense investigation, in part because of the organism's potential use as an agent of bioterrorism.

Initial infection — F. tularensis is a highly virulent organism; only 10 to 50 intradermal or inhaled organisms are required to produce clinical illness. F. tularensis subspecies tularensis (type A) in general causes a more severe clinical infection than subspecies holarctica (type B). Subspecies tularensis genotype A1b strains are more likely to be associated with invasive infections and have a higher mortality than type A1a, A2, or B strains [11].

After multiplying at the site of inoculation, F. tularensis spreads first to regional lymph nodes and may then spread systemically through a lymphohematogenous route. At sites of inoculation, there is an acute inflammatory reaction involving neutrophils, macrophages, and lymphocytes that results in tissue necrosis [50]. Granulomas may form and occasionally caseate, mimicking tuberculosis. Despite this host reaction, F. tularensis may remain alive in tissues for some time.

Cell entry and intracellular replication — F. tularensis is a facultative intracellular pathogen that replicates primarily within host macrophages, but may also infect many other cell types [51-53]. Adherence of F. tularensis to host cells is mediated by type IV pili and/or other bacterial surface proteins [54]. It has been shown that a type IV pilus subunit of subspecies holarctica is a ligand for ICAM-1 on rat vascular epithelial cells [55]. Macrophage uptake of F. tularensis is a unique process called "looping phagocytosis" that involves asymmetric and spacious pseudopod loops [56]. Entry into macrophages is dependent upon complement and complement receptors; alternative pathways involve mannose receptors, class A scavenger receptors, Fc gamma receptors, and cell surface nucleolin. F. tularensis also may be directly transferred from infected cells to uninfected macrophages during a contact-dependent exchange of cytosolic material [57]. After being taken up, phagosome maturation and phagosome-lysosome fusion are impaired, and F. tularensis quickly escapes into the cytosol. Francisella multiply in the cytosol, induce macrophage cell death, and are released to further spread the infection. Similar events occur in neutrophils [58]. After phagocytosis, human neutrophils are unable to kill F. tularensis; the organism inhibits NADPH oxidase, the oxidative burst is suppressed, and the organism escapes into the neutrophil cytoplasm [57,58]. F. tularensis also can inhibit apoptosis of neutrophils and prolong survival of infected neutrophils.

In a mouse model of F. tularensis infection, most organisms recovered from blood were not in leukocytes but, rather, free in plasma [59]. This was independent of the inoculum size, virulence, or route of administration. The organisms grew well in whole blood but not in plasma, suggesting a requirement for host cells. These observations suggest that F. tularensis in the blood may be taken up by leukocytes, in which it replicates, and then escapes into the plasma where it can begin a cycle of reinfection. It has also been shown that F. tularensis can infect erythrocytes and persist within them. Intraerythrocytic organisms are protected from killing by gentamicin, perhaps contributing to relapse after inadequate treatment [60]. Furthermore, intraerythrocytic F. tularensis are protected from the acidic pH of the tick gut; this enhances tick colonization by the organism [61].

Host immune response — An early innate host response functions to contain infection prior to the development of acquired immunity, but is not sufficient to clear the infection. Within the macrophage cytosol, organisms activate a host-protective multimolecular complex, the inflammasome, leading to the release of proinflammatory cytokines (interleukin-1 beta and interleukin-18) that trigger caspase-1 dependent cell death [62]. These effects require type I interferon signaling. Other mechanisms involved in the innate immune response include neutrophils, macrophages, dendritic cells, natural killer (NK) cells, lymphocytes, interleukin-12, tumor necrosis factor-alpha, interferon gamma, and Toll-like receptor (TLR) 2 with either TLR 1 or TLR 6 [52,63].

Natural antibody responses to carbohydrate antigens occur after the development of cellular immunity and are insufficient to protect against infection [64]. In contrast, recovery from infection is dependent upon the development of cell-mediated immunity. Cell-mediated immunity is directed against protein antigens and is dependent upon CD4+ and CD8+ T-cells [52,65]. As a consequence, macrophages are activated to kill intracellular F. tularensis through a process that involves tumor necrosis factor-alpha, interferon gamma, and the production of reactive nitrogen products [65]. Both humoral and cellular mechanisms are needed to optimize protective immunity in mice, but their relative importance in humans is unknown.

Virulence factors and evasion of immune response — The virulence of F. tularensis has been correlated with several of its phenotypic characteristics, including encapsulation, lipopolysaccharides (LPS), pili, and production of acid phosphatases and a siderophore [52,66].

Subsequently, genomic studies have identified specific genetic correlates of virulence, including a cluster of genes known as the Francisella pathogenicity island (FPI) [52]. FPI genes are involved in animal virulence and intracellular survival, including those encoding a type VI secretion system, and are induced by intracellular growth of the organism. FPI genes are under transcriptional regulation responsive to environmental factors, including intracellular growth, iron limitation, and oxidative stress.

In addition to preventing phagosome-lysosome fusion and intracellular killing, F. tularensis is capable of delaying, suppressing, or avoiding many other host immune responses to its presence. These include resistance to complement-mediated lysis, poor recognition of F. tularensis LPS by TLR4, repression of the host cell inflammasome, destabilization of host cell mRNA and impairment of proinflammatory cytokine production, the induction of prostaglandin E2 (PGE2) secretion that can inhibit interferon gamma production, and the alteration of host cell mitochondrial function [67-72].

SUMMARY

●Microbiology – Tularemia is a zoonotic infection caused by Francisella tularensis, a slow-growing aerobic gram-negative coccobacillus. The majority of infections in humans and animals are caused by F. tularensis subspecies tularensis (the more virulent subspecies) and F. tularensis subspecies holarctica. (See 'Microbiology' above.)

●Laboratory precautions – Because F. tularensis is difficult to grow and dangerous to handle, laboratory personnel should be notified whenever the possibility of tularemia is considered. (See 'Laboratory precautions' above.)

●Geographic distribution – Tularemia occurs throughout North America and Europe, as well as in Asia (eg, Turkey, states of the former Soviet Union, China, Japan) and the Middle East (eg, Iran). In the United States, the majority of reported cases occur in the south-central states, the Pacific Northwest, and Massachusetts (figure 1). (See 'Geographic distribution' above.)

●Routes of transmission:

•Arthropod vectors – These include ticks, mosquitoes, horse flies, fleas, and lice. Vector-borne disease (especially by ticks, but also by flies) has become the most common mode of transmission in the United States. In some European countries, mosquito-borne disease predominates. (See 'Arthropod bites' above.)

•Animal contact or consumption – Transmission can also occur through direct contact with infected animals (eg, hunting, skinning, scratches, or bites), contaminated water or meat, splashing infected material into the eyes, and rubbing the eyes with contaminated fingers. The most important vertebrates associated with infection are lagomorphs (eg, rabbits and hares) and rodents. The organism may survive in animal carcasses, mud, and water for many weeks. (See 'Animal contact' above and 'Contaminated food or water' above.)

•Airborne transmission – F. tularensis can also be transmitted through airborne spread of contaminated materials, including dust, hay, water, and laboratory specimens. (See 'Airborne transmission' above.)

●Bioterrorism potential – F. tularensis is a category A bioterrorism agent (ie, of highest concern for bioterrorism use), as classified by the United States Centers for Disease Control and Prevention, in part because of its low infectious dose, high associated mortality, and its potential for easy dissemination. Clustered cases (mainly of pneumonic tularemia, but also of typhoidal and other forms) occurring without the expected epidemiologic exposures to animals, arthropods, or environmental activities should raise the possibility of a bioterrorism event. (See "Tularemia: Clinical manifestations, diagnosis, treatment, and prevention", section on 'Potential bioterrorism use'.)