Tularemia: Emergence/Re-Emergence Jeannine Petersen, Martin Schriefer

Tularemia: emergence/re-emergence Jeannine Petersen, Martin Schriefer

To cite this version:

Jeannine Petersen, Martin Schriefer. Tularemia: emergence/re-emergence. Veterinary Research, BioMed Central, 2005, 36 (3), pp.455-467. 10.1051/vetres:2005006. hal-00902975

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Tularemia: emergence/re-emergence

Jeannine M. PETERSEN*, Martin E. SCHRIEFER

Bacterial Zoonoses Branch, Division of Vector-Borne Infectious Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Foothills Campus, PO Box 2087, Ft. Collins, CO 80522, USA

(Received 28 May 2004; accepted 9 August 2004)

Abstract – Francisella tularensis is a gram-negative coccobacillus and the etiologic agent of the zoonotic disease tularemia. First described in 1911 in Tulare County, California, it has since been reported throughout the Northern Hemisphere, with natural infections reported among an unusually wide range of vertebrates and invertebrates. In recent years, tularemia has emerged in new geographic locations, populations, and settings. This review will serve to highlight mechanisms contributing to the recent emergence of tularemia as well as a repertoire of diagnostic tools useful for detecting and diagnosing disease. tularemia / zoonosis / factors of emergence / Francisella tularensis

Table of contents

1. History ...... 456 2. Epidemiology...... 456 3. Natural emergence and associated factors ...... 457 3.1. Spain (1997) ...... 459 3.2. Kosovo (2000)...... 460 3.3. United States (2002)...... 460 3.4. Australia (2002) ...... 461 4. Bioterrorism and emergence...... 461 5. Diagnosis and detection ...... 462 5.1. Culture...... 462 5.2. Serology ...... 463 5.3. PCR ...... 463 5.4. 16S rDNA sequencing...... 463 5.5. Molecular subtyping...... 464 6. Conclusion ...... 464

* Corresponding author: [email protected] 456 J.M. Petersen, M.E. Schriefer

1. HISTORY trappers who skinned water-rats for their pelts [62, 71]. Soon thereafter, tularemia was also Tularemia is a zoonotic disease caused by reported in Norway (1929), Canada (1930), the small, gram-negative bacterium, Fran- Sweden (1931) and Austria (1935) [31]. cisella tularensis, one of the most infectious bacteria known, with < 10 organisms capa- ble of causing severe disease in both humans 2. EPIDEMIOLOGY and animals [23, 60]. First described as a disease of significance nearly a century ago, Today, tularemia is recognized as a widely tularemia has recently emerged in areas with dispersed disease throughout the Northern no previous known risk and re-emerged in Hemisphere with foci in certain parts of research and public health circles due to its North America, Europe, and northern Asia biothreat potential. [23, 43]. Few, if any, zoonotic diseases have The complexity of F. tularensis and its a broader or more complex host distribution associated disease can be illustrated by a and epizootiology. F. tularensis infection brief review of the early history. In 1911, has been evidenced in a staggering number McCoy and Chapin described a plague-like of wildlife species including various lago- illness of rodents in Tulare County, Califor- morphs, rodents, insectivores, carnivores, nia, and soon after (1912) cultured F. tula- ungulates, marsupials, birds, amphibians, fish rensis from squirrels in the area [49]. Two and invertebrates [8, 42, 43, 50]. Arthro- years later, the first human illness attributed pods, including ticks, biting flies, and pos- to F. tularensis was described by Wherry sibly mosquitoes, serve as vectors and poten- and Lamb in Ohio, who isolated the bacte- tially, long-term reservoirs [8, 24, 42, 44, rium from two patients with confirmed wild 50]. Despite the complexity of the global rabbit contact [68]. Subsequently, Edward picture of tularemia, the main components Francis, for whom the genus is named, of regional disease cycles are much more established that several clinical syndromes narrow, typically involving only one to a throughout the United States were caused few, key mammalian and arthropod species. by F. tularensis and proposed the name “tularemia” to describe them [29, 30]. These Two disease cycles, terrestrial and aquatic, syndromes included “rabbit fever”, “market’s have been described [44, 50]. In the terres- men disease” and “meat-cutter’s disease”, trial cycle, rabbits and hares typically serve all of which were used to describe the illness as amplifying hosts and ticks or biting flies associated with dressing rabbits for meat. are arthropod vectors. In the aquatic cycle, “Deer-fly fever” was used to describe the beaver, muskrat and voles serve as impor- disease following a deerfly bite, with most tant mammalian hosts and appear to shed early observations made by Pierce in Utah. live organisms into their environments. In “Glandular type of tick fever” was used by Sweden, mosquitoes have been strongly physicians in Idaho who noted enlargement implicated as vectors of tularemia and may of lymph nodes in response to a tick bite. acquire infection from other components of Until 1925, it was widely believed that the aquatic cycle. Curiously, mosquitoes tularemia was a disease with risk limited to the are not thought to be significant contribu- United States. This perception soon changed. tors to disease transmission in the United Ohara, studying hare disease (Yato-byo) in States, despite their sharing of the same Japan, recognized the similarity of the disease environments as other components of the to tularemia and sent specimens to Francis, aquatic system. Recently, protozoa have been who confirmed the presence of F. tularensis shown to harbor F. tularensis and may play [52]. In the USSR (1928), F. tularensis was an important role in aquatic cycles [1]. The recognized as the causative agent of “water- interaction between aquatic and terrestrial rat-trappers’ disease”, an illness acquired by cycles is largely unknown. Emergence/re-emergence of tularemia 457

F. tularensis has been classified into four (described below). Glandular tularemia is distinct subspecies, tularensis, holarctica, quite similar to ulceroglandular disease but mediasiatica, and novicida [60]. The global lacks the ulcerated site of infection. Ocu- distribution of disease caused by each sub- loglandular tularemia occurs when the species is shown in Figure 1; clearly, subsp. conjunctiva is the initial site of infection, tularensis and holarctica are responsible usually a result of mechanical transfer of for the majority of described human and organisms from an infectious source to the animal illness. Of the four subspecies, subsp. eye by the fingers. This form of disease is tularensis, also known as Type A, has the characterized by the appearance of ulcers and highest mortality rate. Infections due to nodules on the conjunctiva and regional lymph Type A have been limited to North America. node swelling. Oropharyngeal tularemia In comparison, F. tularensis subsp. holarc- results from ingestion of contaminated water tica, also known as Type B, has been doc- or food and is characterized by a severe sore umented throughout the Northern Hemi- throat with enlargement of the tonsils and sphere. Thus, in North America, both swollen cervical lymph nodes. Most human Type A and Type B are present and often cases in the recent outbreak of tularemia in over-lap within a given sub-region. The Kosovo (described below) were of this subsp. novicida and mediasiatica have form. Pneumonic tularemia, the most severe more focal distributions with mediasiatica form of disease, occurs by direct inhalation isolated only from the Central Asian of the organism, or may develop secondar- regions of the former USSR, and novicida ily by septicemic spread of infection from isolated from North America and more a primary site of infection. Historically, farm- recently Australia [18, 41, 60, 69]. These two ing, and more recently landscaping, have subspecies are infrequently associated with been significant occupational risk-factors human disease. associated with pneumonic tularemia in Risk factors associated with human dis- certain endemic areas [26, 63]. Typhoidal ease are linked to local disease ecology. For tularemia is used to describe a systemic dis- example, in the Western United States, bit- ease with fever and other constitutional signs ing fly exposures, tick bites and animal con- in the absence of lymphadenitis, cutaneous tact are all significant risks factors [43, 44]. lesions, or primary pulmonary involvement. In contrast, human cases in the central United States are rarely linked with biting flies and most often associated with tick bites 3. NATURAL EMERGENCE and animal exposure [43, 44]. Similarly, AND ASSOCIATED FACTORS mosquito exposure is an important risk fac- tor in Sweden, whereas tabanid exposure is Perhaps Roy Parker best voiced the poten- more strongly linked to cases in Russia [8]. tial for emergence of tularemia when he Clinical presentation of tularemia is varia- said: “I know of no other infection of ani- ble and dependent on the route of infection mals communicable to man that can be [22, 23]. Presentations include ulceroglandu- acquired from sources so numerous and so lar, glandular, oculoglandular, oropharyn- diverse. In short, one can but feel that the geal, pneumonic and typhoidal forms of status of tularemia, both as a disease in tularemia. Ulceroglandular tularemia gen- nature and of man, is one of potentiality” erally arises from contact with an infected [54]. With F. tularensis being so wide- animal or by the bite of an infected vector spread in wildlife species and the environ- and is characterized by the presence of an ment, it is poised to appear in new places ulcerated lesion and enlargement of regional and populations at any time. lymph nodes. This type of exposure is exem- As examples of natural emergence or re- plified by cases in a recent outbreak in Spain emergence of tularemia, three outbreaks 458 J.M. Petersen, M.E. Schriefer shadings represent the dis- ( Note that in North America, disease ). subspecies. The different subspecies. The different mediasiatica (), and and (), F. tularensis novicida of disease caused by p. Type A and Type B. A and p. Type The global distribution Figure 1. Figure 1. tributions of Type A and Type B ( B Type A and of Type tributions ( B ), Type ), is caused by both subs by is caused Emergence/re-emergence of tularemia 459

Table I. Emergence of naturally occurring F. tularensis (1997–2002).

Site of emergence Subspecies Year Source of infection Likely basis for emergence

Spain holarctica 1997 Hares Importation of hares from (Humans) 1998 Crayfish endemic countries Kosovo holarctica 2000 Food and water War conditions/rodent (Humans) population increase United States holarctica 2002 Prairie dogsExotic Pet trade/unregulated (Prairie Dogs) commercial sales of wild-caught animals Australia novicida 2002 Water Enhanced detection/ (Human) laboratory skills

and an isolated case will be highlighted in from the outbreak the previous year [4]. this review (Tab. I) and include: the first Nineteen cases of ulceroglandular tularemia evidence of tularemia in Spain (1997) and were identified in persons who had contact in Kosovo (2000), the first large-scale out- with crayfish. This outbreak was most unu- break of tularemia in prairie dogs (United sual in that tularemia had not previously States, 2002), and the finding of F. tularen- been associated with fishing. Waterborne sis in the Southern Hemisphere (2002). outbreaks of tularemia have occurred in the While other examples of tularemia emer- past, but most have been associated with gence exist, the cases detailed here serve to contaminated drinking water [35, 43]. Tran- highlight a variety of mechanisms for dis- sient contamination of the river and crayfish ease emergence. was implicated as the cause of the outbreak, with most patients incurring crayfish-related 3.1. Spain (1997) scratches, cuts, and abrasions while fishing. A sewage plant, which intermittently dis- Although tularemia has been reported charged water into the river, was linked to throughout much of Europe, it first emerged the cause of the outbreak. Type B, PCR pos- as a human disease in Spain in 1997–1998, itive samples were obtained from the river, when a large outbreak occurred [21, 37, 55]. crayfish, and human lymph node aspirates. A total of 559 cases of tularemia were reported It is not clear when tularemia was intro- with 519 cases from the community of Cas- duced into Spain. Since tularemia was not tille-Leon in northwestern Spain. A study recognized as a disease of Spain it would of 142 patients in this region indicated that not have been considered in a clinical diag- 97.2% had previous contact with hares; nosis prior to 1997. Nonetheless, evidence 83.8% had prepared hare carcasses and 13.3% suggests that F. tularensis was present in had handled hare meat [55]. As expected Spain before the first outbreak. Retrospec- based on contact with infected animals, tive studies have shown the presence of ulceroglandular tularemia was the most anti-F. tularensis antibodies in humans prior common form of clinical disease observed to 1997 and tularemia as the cause of a large (87% of patients). Type B was isolated from hare die-off in the Castille-Leon area in patients and also from hares in the region. 1994 [27, 34, 37]. In 1998, a second outbreak of tularemia The basis for emergence of tularemia in in humans was reported, this time in the Spain remains speculative, with a likely central province of Cuenca, a region distant explanation that infected hares were imported 460 J.M. Petersen, M.E. Schriefer from tularemia endemic countries. Every The emergence of tularemia in Kosovo year, hares are imported from Central is linked to the conditions resulting from Europe for hunting purposes and in 1996 war and political crisis [59]. The principal tularemia infected hares from France, Italy, populations affected by the outbreak lived Austria, Finland, Sweden and Slovakia in rural farming villages and had fled their were described [5]. villages in 1999 as a result of ongoing con- Importation of infected rabbits has been flict, leaving behind unprotected food stor- attributed to the emergence of tularemia age areas, unharvested crops and plowed, elsewhere in the Northern Hemisphere. In unseeded fields. Untended wells were dam- the United States in the 1930s, infected rab- aged and contaminated. Returning villagers bits from the tularemia endemic states, Mis- noted a population explosion of rodents with souri and Arkansas, were imported by game increased rodent activity apparent both in clubs into Massachusetts and liberated [7]. and around homes. Thus, it is likely that the Soon thereafter the first locally acquired war time conditions led to increase in the cases of tularemia were reported [3, 7]. local rodent population which allowed for epizootic spread of tularemia in rodents and consequent widespread environmental con- 3.2. Kosovo (2000) tamination. The consequences of war, damaged pub- The first report of tularemia in Kosovo lic health care systems, unsound hygiene, occurred in early 2000, at the end of 10 years and rodent increase, set the stage for the of political crisis and warfare in the region, emergence and spread of a variety of infec- when a cluster of patients was identified tious diseases. Conflict conditions, during with an unusual syndrome of fever, phar- World War II and in Bosnia in 1995, have yngitis and pronounced cervical lymphad- led to previous outbreaks of tularemia enopathy [59, 70]. Active case-finding and [53, 59]. serology testing confirmed 327 cases of tularemia in 21 Kosovo municipalities, with most cases presenting as oropharyngeal 3.3. United States (2002) tularemia. Ingestion of F. tularensis was Tularemia is a recognized disease of the suspected as the cause of the outbreak and United States, yet in 2002 it emerged in a a follow-up case-control study indicated rather unusual and unexpected setting [6, that the outbreak was food and water related 15, 56]. The site of the outbreak was a Texas [59]. exotic pet facility where a variety of animal Retrospective analysis of public health species were housed together and sold to records back to 1946 showed no prior evi- domestic and international distributors. Thou- dence of tularemia in Kosovo [59]. None- sands of wild-caught prairie dogs were sup- theless, it is possible that tularemia had plied to this facility for commercial purposes been present in Kosovo prior to the out- and in July 2002 a large die-off (~ 250 prai- break, but unrecognized in the absence of a rie dogs) occurred. Type B was isolated from large number of human cases. Both animal the dead prairie dogs at the Texas facility as and human tularemia had been reported in well as prairie dogs distributed to the Czech neighboring countries and independent prov- Republic and to a Texas pet shop. inces [59]. In 1976, a five-year ecological In this outbreak, the trade of trapping and study in neighboring Croatia showed that selling wild animals as exotic pets allowed meadow and common voles were foci of disease emergence. Although both tularemia tularemia in the region [10]. In addition, and plague had previously been identified tularemia was reported in Turkey in the in wild-caught prairie dogs, there were no 1990s, with the majority of human cases quarantine restrictions or testing require- being waterborne (83% of cases) [36, 39]. ments for wild-captured animals, prior to Emergence/re-emergence of tularemia 461 their worldwide distribution as pets [2, 13, ish waters in the Northern Territory of Aus- 47]. Apparently, one or more infected ani- tralia. The toe wound yielded an isolate of mals were wild-captured, and the disease F. tularensis subsp. novicida. Because spread by cannibalism as a result of the F. tularensis was not known to be present unnaturally close contact at the exotic pet in the Southern Hemisphere, molecular facility. The primary mode of transmission comparison with other Type A, Type B, and was ingestion of F. tularensis, as all infected novicida isolates was required before this prairie dogs displayed enlarged submandib- isolate could be appropriately identified. ular lymph nodes, a hallmark of oropharyn- As in the case of tularemia in Spain, it’s geal tularemia. unlikely that this case represents true emer- In the years prior to this outbreak, the gence of F. tularensis in Australia. F. tula- exotic pet trade had experienced growing rensis subsp. novicida is a less virulent sub- popularity in the United States with unreg- species of F. tularensis with only a few human ulated importation and exportation. Wild cases of tularemia-like illness described prairie dogs, found throughout the Great worldwide. In addition, tularemia was not Plains of North America from southern recognized as a disease of Australia and Canada to just inside Mexico, were col- would not have been considered in a clinical lected in the United States between the diagnosis. As a result, the identification of months of April and July every year and dis- infection due to F. tularensis relied heavily tributed to pet stores throughout the country on the laboratory’s skill and persistence in as well as exported internationally. identifying the isolate. The availability of molecular tools and national and interna- In 2003, the Centers for Disease Control tional collaboration contributed to the accu- and Prevention (CDC) and the Food and Drug rate identification of F. tularensis in the Administration (FDA) issued an interim Southern Hemisphere. final rule to establish new restrictions on the commercial sale of prairie dogs and the Interesting questions remain to be eluci- importation of certain exotic species into dated regarding F. tularensis in Australia, the United States as the result of a monkey- including how long the organism has been pox outbreak in prairie dogs and subsequent present in Australia and whether there might transmission to human owners [16, 17, 58]. have been previous unrecognized cases. Both the tularemia and monkeypox out- Nonetheless, this example serves to under- breaks in prairie dogs served to highlight score the likelihood that F. tularensis is the public health dangers associated with more widespread than previously thought the exotic pet trade, including the speed and elicits the question of whether other with which infected animals can be trans- F. tularensis subspecies are also present in ported and the potential for the exotic pet Australia and elsewhere in the Southern trade to rapidly introduce non native path- Hemisphere. ogens worldwide.

3.4. Australia (2002) 4. BIOTERRORISM AND EMERGENCE Although F. tularensis has long been considered to cause disease in the Northern Perhaps the most unique aspect of F. tula- Hemisphere, in 2002 F. tularensis subsp. rensis emergence relates to recent world- novicida emerged in the Southern Hemi- wide concerns regarding the potential use sphere with the first report of human infec- of biological weapons [40]. Whereas the tion [69]. A 53-year old man presented with previous examples all discussed emergence a swollen toe and swollen inguinal lymph with respect to the appearance of the disease nodes as a result of a cut received in brack- in a new geographic location or population, 462 J.M. Petersen, M.E. Schriefer emergence here is used to describe the gen- contributed to the first identification of eral public and scientific community aware- F. tularensis in the Southern Hemisphere. ness of tularemia. Due primarily to concerns over the use of F. tularensis as a biological weapon, tularemia has emerged as a widely 5. DIAGNOSIS AND DETECTION recognized disease throughout the world. This is in stark contrast to 10 years prior Identification of natural cases of disease when tularemia was considered to be of neg- emergence is limited by our diagnostic tools, ligible public health significance, removed as well as by the skills and attention of cli- from the United States nationally notifiable nicians and laboratorians. In this section we list of diseases [14] and recognized only by will provide a review of the current diag- a few subject matter experts around the nostics for F. tularensis and their impor- world. tance in understanding disease emergence, F. tularensis has been classified as a Cat- as well as some recent advances in diagnos- egory A select agent, because of its extreme tics that may aid in future investigations. infectivity, ease of dissemination, and sub- stantial capacity to cause illness and death 5.1. Culture [20]. Heightened concern regarding the intentional use of F. tularensis as a bio- Culture recovery and characterization weapon is also due to its previous history remains the “gold standard” for laboratory [22]. It was studied by Japanese germ war- confirmation of tularemia infection accord- fare units between 1932 and 1945 and the ing to the CDC. However, this approach has United States military stockpiled F. tula- historically proven itself challenging, par- rensis for use as a biological weapon in the ticularly with F. tularensis subsps., Type A 1960’s. In the 1990s, the Soviets were and Type B. F. tularensis subsps., Type A reported to have produced F. tularensis and Type B, are slow-growing, fastidious strains with engineered resistance to vac- organisms requiring sulfhydryl compounds cines and antibiotics. and 24–72 h for growth on artificial media In the scientific research community, at 37 °C [19, 60]. F. tularensis is also noto- massive amounts of bioterrorism funding rious for causing laboratory acquired infec- are being directed at Category A agents. As tions and has to be handled under BSL-3 a result, more researchers are working with conditions [19, 60]. Despite these concerns, F. tularensis than ever before. Numerous culture provides a conclusive diagnosis of laboratories in the United States are study- infection and an invaluable resource for ing the biology of this organism, with respect molecular epidemiology, subtyping and dis- to vaccine development, new diagnostics, covery of novel species and subspecies. and virulence factors. F. tularensis from primary clinical sources In public health laboratories around the grows well on several media, including world, laboratorians are now on the lookout enriched chocolate agar (CA), cysteine heart for potential bioterrorism agents. In coun- agar with 9% chocolatized blood (CHAB), tries where F. tularensis has not previously and buffered charcoal yeast extract (BYCE) been found, microbiologists are now becom- [19, 60]. F. tularensis can be isolated from ing familiar with the protocols to isolate and nutrient enriched specimens (tissues) on identify this organism. Awareness of the sheep blood agar (SBA), but CHAB is disease has also increased among the med- strongly recommended for subculture as the ical community. With this enhanced world- organism will fail to thrive with continued wide surveillance, we may expect to dis- passage on SBA. Additonally, growth on cover F. tularensis in new and unexpected CHAB provides for presumptive identifica- places. No doubt bioterrorism awareness tion of F. tularensis as the organism shows Emergence/re-emergence of tularemia 463 characteristic growth on this media (green, 5.3. PCR opalescent, raised, shiny colonies at 24– 48 h). Once a pure isolate has been recov- A variety of PCR methods have been ered, glycerol fermentation can be used to described for the detection of F. tularensis differentiate Type A (glycerol fermentation DNA in both clinical and environmental positive) and Type B (glycerol fermenta- specimens. PCR can be an invaluable diag- tion negative). F. tularensis subsp. novicida nostic tool when organisms are noncultiva- is non-fastidious and can be isolated on gen- ble or nonviable. The majority of PCR tests eral microbiological agars, including SBA. for F. tularensis have been gel-based PCR Contaminated specimens pose an espe- assays targeted at the genes encoding the cially difficult challenge when attempting outer membrane proteins, fopA or tul4 [32, to isolate F. tularensis. Evidence suggests 48, 61]. These PCR assays show good spe- that some bacteria inhibit F. tularensis growth cificity and allow for rapid detection of F. [57]. This finding has important implica- tularensis in specimens. The tul4 PCR assay displays a sensitivity of 75% when tions when attempting to isolate F. tularensis from a variety of samples that contain applied to wound specimens from patients other bacterial species. Recently, an antibi- with ulceroglandular tularemia [45]. otic supplemented CHAB media (CHAB-A) Advances in PCR detection have been was shown to significantly improve recov- made with the development of real-time Taq- ery rates of F. tularensis from tissue sources Man PCR assays. These assays have increased contaminated or overgrown by other flora specificity and rapidity over gel-based PCR [57]. In clinical or environmental speci- and can provide added sensitivity when mens where mixed flora is likely, the use of testing both clinical and environmental CHAB-A should be considered. specimens in which the number of organisms is expected to be quite low. A real-time F. tularensis multitarget TaqMan PCR assay 5.2. Serology based on three targets, the ISFtu2 element, Serology is the most commonly used lab- 23kDa and tul4 genes was recently described oratory approach for confirmation of sus- [66]. In addition to increased sensitivity, a pected disease. However, specific antibody detection limit of ~ 1 CFU, the multitarget responses are typically not detectable prior aspect of this assay has the added advantage to two weeks of infection with currently of decreasing the likelihood of false posi- available tests [9]. Nonetheless, retrospec- tives. tive serosurveys can be extremely useful for understanding the timing of disease emer- 5.4. 16S rDNA sequencing gence in a particular area or population. IgM, IgA and IgG antibodies appear simul- Several studies have shown the useful- taneously after initial infection and IgM ness of 16S rDNA sequence identification antibodies can last for many years. Agglu- in the diagnostic laboratory, especially as tination based on formalin-killed whole cells relates to slow-growing, unusual, and fas- is the standard serology test used for deter- tidious bacteria [38, 64]. For identifying mining the presence of antibody against true emergence of F. tularensis, in areas F. tularensis [12]. ELISAs based on LPS or where it has not previously been reported, outer membrane carbohydrate-protein frac- 16S rDNA sequencing is a particularly use- tions have also been utilized [9, 67]. With ful diagnostic test. 16S rDNA sequencing renewed interest in F. tularensis due to bio- played an important role in the first identi- terrorism, we may well expect the identifi- fication of F. tularensis in the Southern cation of new diagnostic antigens useful for Hemisphere and also in the tularemia out- early detection and subtyping. break associated with crayfish in Spain [4, 464 J.M. Petersen, M.E. Schriefer

69]. For identification of recovered bacte- of this bacterial agent. Ironically, tularemia rial isolates, the universal 16S rDNA prim- was dropped from the list of nationally ers as well as the Francisella specific 16S reportable diseases in the United States in rDNA primers, provide good sequence data 1994 only to be reinstated in 2000. Although [28]. For diagnostic identification of Fran- actively investigated in the early 1900’s, cisella spp. in contaminated samples (ticks, many of the questions posed during that era water, field specimens), the Francisella are still valid. What are the differences that specific 16S rDNA primers should be con- account for virulence between Type A and sidered [4, 51]. Type B strains? Where does tularemia per- sist during quiescent periods? Is there spill- 5.5. Molecular subtyping over between aquatic and terrestrial disease cycles? What are the environmental factors PCR subtyping assays have been devel- that lead to outbreaks in animals and humans? oped that allow for discriminating F. tula- Natural factors influencing the ecology and rensis subsp., Type A and Type B, in the emergence of tularemia are poorly under- absence of a culture [46, 56]. These assays stood but undoubtedly include climate, num- are gel-based and center on the detection of bers of suitable hosts and vectors, and resist- differences in amplified product sizes. More ance of hosts to infection. Although these recently, a gel-based PCR assay targeted at parameters may be influenced by man, more the region of difference 1 (RD1), has been immediate outcomes are affected by activ- reported to distinguish between all four sub- ities such as the introduction or movement species of F. tularensis, Type A, Type B, of diseased and susceptible hosts. Recent novicida, and mediaasiatica, although eval- and novel human outbreaks have served to uation of a larger panel of isolates, especially heighten the awareness, importance, and novicida , will be required before this assay interest in this complex zoonotic disease. can be employed routinely for F. tularensis With the aid of current molecular diagnos- subtyping [11]. tic tools, tried-and-true bacteriologic meth- Advances have recently been made in ods, increased clinical recognition, epide- strain differentiation using a variety of molec- miologic investigations and an infusion of ular techniques including restriction frag- research funding, the answers to these ques- ment linked polymorphism (RFLP) South- tions are well within our grasp. ern blot, pulsed-field gel electrophoresis (PFGE) and multi-locus variable number tandem repeat assays (MLVA) [25, 33, 65]. ACKNOWLEDGEMENTS These techniques will be extremely important for detailed molecular epidemiology We thank Pedro Anda and Raquel Escudero studies in the future. With continued devel- for information regarding the emergence of opment and refinement of these techniques, tularemia in Spain. We also thank Charles B. we can well expect molecular strain differ- Beard, David Withum, Barbara J. Johnson, and entiation of F. tularensis to play an impor- Kiersten Meacham for critically reading the tant role in understanding the true basis of manuscript. disease emergence.

REFERENCES 6. CONCLUSION [1] Abd H., Johansson T., Golovliov I., Sandström G., Forsman M., Survival and growth of Fran- Renewed scientific and public interest in cisella tularensis in Acanthamoeba castella- the field of tularemia has evolved recently nii, Appl. Environ. Microbiol. 69 (2003) 600– in large part due to the biothreat potential 606. Emergence/re-emergence of tularemia 465

[2] Alexander J.L., Plague, pet prairie dogs – [13] Centers for Disease Control and Prevention, USA (Texas), ProMed. July 10, 1998, Fatal human plague – Arizona and Colorado, http://www.promedmail.org, archive number: 1996, Morb. Mortal. Wkly. Rep. 46 (1997) 19980710.1303. 618–620. [3] Ayres J.C., Feemster R.F., Epidemiology of [14] Centers for Disease Control and Prevention, tularemia in Massachusetts with a review of Tularemia-United States, 1990–2000, Morb. the literature, N. Engl. J. Med. 238 (1948) 187– Mortal. Wkly. Rep. 51 (2002) 182–184. 193. [15] Centers for Disease Control and Prevention, [4] Anda P., Segura del Pozo J., Diaz Garcia J.M., Outbreak of tularemia among commercially Escudero R., Garcia Pena F.J., Lopez Velasco distributed prairie dogs, 2002, Morb. Mortal. M.C., Sellek R.E., Jimenez Chillaron M.R., Wkly. Rep. 51 (2002) 688–699. Sanchez Serrano L.P., Martinez Navarro J.F., [16] Centers for Disease Control and Prevention, Waterborne outbreak of tularemia associated Multistate outbreak of monkeypox–Illinois, with crayfish fishing, Emerg. Infect. Dis. 7 Indiana, and Wisconsin, 2003, Morb. Mortal. (2001) 575–582. Wkly. Rep. 52 (2003) 537–540. [5] Anonymous, Diseases of lagomorphs: tularemia, [17] Centers for Disease Control and Prevention, Office International des épizooties, Sanidad Food and Drug Administration, Department animal mundial en 1996, tomo 1, p. 28. of Health and Human Services, Control of communicable diseases; restrictions on Afri- [6] Avashia S., Petersen J.M., Lindley C., can rodents, prairie dogs, and certain other Schriefer M.E., Gage K., Cetron M., Demarcus animals. Interim final rule; opportunity for T.A., Kim D., Buck J., Monteneiri J.A., Lowell public comment, Fed. Regist. 68 (2004) J., Antolin M., Kosoy M., Carter L.G., Chu 62353–62369. M.C., Hendricks K., Dennis D.T., Kool J.L., Prairie dog to human tularemia transmission, [18] Clarridge J.E. 3rd, Raich T.J., Sjöstedt A., Texas, 2002, Emerg. Infect. Dis. 10 (2004) Sandström G., Darouiche R.O., Shawar R.M., 483–486. Georghiou P.R., Osting C., Vo L., Character- ization of two unusual clinically significant [7] Belding D.L., Merrill B., Tularemia in Francisella strains, J. Clin. Microbiol. 34 imported rabbits in Massachusetts, N. Engl. J. (1996) 1995–2000. Med. 224 (1941) 1085–1087. [19] Chu M.C., Weyant R., Francisella and Bru- [8] Bell J.F., Tularemia – a review, CRC Hand- cella, in: Murray P.R., Baron E.J., Jorgensen book Series in Zoonoses, CRC Press, Boca J.H., Pfaller M.A., Yolken R.H. (Eds.), Man- Raton, Section A, 1977, p. 161. ual of Clinical Microbiology, 8th ed. Ameri- [9] Bevanger L., Maeland J.A., Kvan A.I., Com- can Society for Microbiology, Washington parative analysis of antibodies to Francisella DC, 2003, pp. 789–797. tularensis antigens during the acute phase of [20] Darling R.G., Catlett C.L., Huebner K.D., Jarrett tularemia and eight years later, Clin. Diagn. D.G., Threats in bioterrorism. I: CDC cate- Lab. Immunol. 1 (1994) 238–240. gory A agents, Emerg. Med. Clin. North Am. [10] Borcic B., Hrabar A., Dulic B., Tvrtkovic N., 20 (2002) 273–309. Bilic V., Mikacic D., Ecological features of [21] De Mateo S., Cosin C.R., Outbreak of the tularemia natural focus in central Posavina tularaemia in Castilla y Leon, Spain, Euro- (Croatia), Folia Parasitol. (Praha) 23 (1976) surveillance Weekly, [on line] (1998) www. 257–265. eurosurveillance.org/ew/1998/980122.asp. [11] Broekhuijsen M., Larsson P., Johansson A., [22] Dennis D.T., Inglesby T.V., Henderson D.A., Bystrom M., Eriksson U., Larsson E., Prior Bartlett J.G., Ascher M.S., Eitzen E., Fine R.G., Sjöstedt A., Titball R.W., Forsman M., A.D., Friedlander A.M., Hauer J., Layton M., Genome-wide DNA microarray analysis of Lillibridge S.R., McDade J.E., Osterholm M.T., Francisella tularensis strains demonstrates O'Toole T., Parker G., Perl T.M., Russell P.K., extensive genetic conservation within the spe- Tonat K., Working Group on Civilian Biode- cies but identifies regions that are unique to fense, Tularemia as a biological weapon: med- the highly virulent F. tularensis subsp. tula- ical and public health management, JAMA rensis, J. Clin. Microbiol. 41 (2003) 2924– 285 (2001) 2763–2773. 2931. [23] Ellis J., Oyston C.F., Green M., Titball R.W., [12] Brown S.L., McKinney F.T., Klein G.C., Tularemia, Clin. Microbiol. Rev. 15 (2002) Jones W.L., Evaluation of a safranin-O-stained 631–646. antigen microagglutination test for Francisella [24] Eliasson H., Lindback J., Nuorti J.P., Arneborn tularensis antibodies, J. Clin. Microbiol. 11 M., Giesecke J., Tegnell A., The 2000 tularemia (1980) 146–148. outbreak: a case-control study of risk factors 466 J.M. Petersen, M.E. Schriefer

in disease-endemic and emergent areas, Swe- Western Black Sea region of Turkey, Yonsei den, Emerg. Infect. Dis. 8 (2002) 956–960. Med. J. 29 (2004) 17–22. [25] Farlow J.K., Smith L., Wong J., Abrams M., [37] Gutierrez M.P., Bratos M.A., Garrote J.I., Lytle M., Keim P., Francisella tularensis strain Duenas A., Almaraz A., Alamo R., Rodriguez typing using multiple-locus, variable-number Marcos H., Rodriguez Recio M.J., Munoz tandem repeat analysis, J. Clin. Microbiol. 39 M.F., Orduna A., Rodriguez-Torres A., Sero- (2001) 3186–3192. logic evidence of human infection by Fran- [26] Feldman K.A., Enscore R.E., Lathrop S.L., cisella tularensis in the population of Castilla Matyas B.T., McGuill M., Schriefer M.E., y Leon (Spain) prior to 1997, FEMS Immunol. Stiles-Enos D., Dennis D., Petersen L.R., Med. Microbiol. 35 (2003) 165–169. Hayes E.B., An outbreak of primary pneu- [38] Hall L., Doerr K.A., Wohlfiel S.L., Roberts monic tularemia on Martha’s Vineyard, N. G.D., Evaluation of the MicroSeq system for Engl. J. Med. 345 (2001) 1601–1606. identification of mycobacteria by 16S ribos- [27] Fernández de Luco et al., Diagnóstico de tula- omal DNA sequencing and its integration into remia por Francisella tularensis en la liebre a routine clinical mycobacteriology labora- ibérica (Lepus granatensis) en los años 1994– tory, J. Clin. Microbiol. 41 (2003) 1447–1453. 98, Abstract book of the X Reunión de la [39] Helvaci S., Gedikoglu S., Akalin H., Oral Sociedad Española de Anatomía Patológica H.B., Tularemia in Bursa, Turkey: 205 cases Veterinaria, Lugo, 1998, Abstract No. 58. in ten years, Eur. J. Epidemiol. 16 (2000) 271– [28] Forsman M., Sandström G., Sjöstedt A., Anal- 276. ysis of 16S ribosomal DNA sequences of Francisella strains and utilization for determi- [40] Henderson D.A., The looming threat of bio- nation of the phylogeny of the genus and for terrorism, Science 283 (1999) 1279–1282. identification of strains by PCR, Int. J. Syst. [41] Hollis D.G., Weaver R.E., Steigerwalt A.G., Bacteriol. 44 (1994) 38–46. Wenger J.D., Moss C.W., Brenner D.J., Fran- [29] Francis E., Tularaemia-Francis 1921. The cisella philomiragia comb. nov. (formerly Yers- occurrence of tularaemia in nature as a disease inia philomiragia) and Francisella tularensis of man, Public Health Rep. 36 (1921) 1731– biogroup novicida (formerly Francisella novi- 1738. cida) associated with human disease, J. Clin. Microbiol. 27 (1989) 1601–1608. [30] Francis E., Tularemia, JAMA 84 (1925) 1243– 1250. [42] Hopla C.E., The ecology of tularemia, Adv. Vet. Sci. Comp. Med. 18 (1974) 25–53. [31] Francis E., Sources of infection and seasonal incidence of tularaemia in man, Public Health [43] Hopla C.E., Hopla A.K., Tularemia, in: Beran Rep. 52 (1937) 103–113. G.W., Steele J.H. (Eds.), Handbook of Zoon- oses, 2nd ed., CRC Press, Inc., Boca Raton, [32] Fulop M., Leslie D., Titball R., A rapid, highly 1994, pp. 113–126. sensitive method for the detection of Fran- cisella tularensis in clinical samples using the [44] Jellison W.L., Tularemia in North America, polymerase chain reaction, Am. J. Trop. Med. University of Montana, Missoula, Montana, Hyg. 54 (1996) 364–366. 276 p. [33] Garcia Del Blanco N., Dobson M.E., Vela A.I., [45] Johansson A., Berglund L., Eriksson U., De La Puente V.A., Gutierrez C.B., Hadfield Göransson I., Wollin R., Forsman M., Tärnvik T.L., Kuhnert P., Frey J., Dominguez L., A., Sjöstedt A., Comparative analysis of PCR Rodriguez Ferri E.F., Genotyping of Fran- versus culture for diagnosis of ulceroglandu- cisella tularensis strains by pulsed-field gel lar tularemia, J. Clin. Microbiol. 38 (2000) electrophoresis, amplified fragment length 22–26. polymorphism fingerprinting, and 16S rRNA gene sequencing, J. Clin. Microbiol. 40 (2002) [46] Johansson A., Ibrahim A., Goransson I., 2964–2972. Eriksson U., Gurycova D., Clarridge J.E. 3rd, Sjöstedt A., Evaluation of PCR-based meth- [34] García-Peña F.J., Tularemia: description y sit- ods for discrimination of Francisella species uación en España de una enfermedad que and subspecies and development of a specific afecta a 256 especies, Vet. Madrid 42 (1998) PCR that distinguishes the two major subspe- 20–29. cies of Francisella tularensis, J. Clin. Micro- [35] Greco D., Allegrini G., Tizzi T., Ninu E., biol. 38 (2000) 4180–4185. Lamanna A., Luzi S., A waterborne tularemia [47] La Regina M., Lonigro J., Wallace M., Fran- outbreak, Eur. J. Epidemiol. 3 (1987) 35–38. cisella tularensis infection in captive, wild- [36] Gurcan S., Otkun M.T., Otkun M., Arikan caught prairie dogs, Lab. Anim. Sci. 36 (1986) O.K., Ozer B., An outbreak of tularemia in 178–180. Emergence/re-emergence of tularemia 467

[48] Long G.W., Oprandy J.J., Narayanan R.B., I., Tularemia outbreak investigation in Kos- Fortier A.H., Porter K.R., Nacy C.A., Detec- ovo: case control and environmental studies, tion of Francisella tularensis in blood by Emerg. Infect. Dis. 8 (2002) 69–73. polymerase chain reaction, J. Clin. Microbiol. [60] Sjöstedt A., Family XVII. Francisellaceae, 31 (1993) 152–154. Genus I. Francisella, in: Brenner D.J. (Ed.), [49] McCoy G.W., Chapin C.W., Further observa- Bergey’s manual of systemic bacteriology, tions on a plague-like disease of rodents with Springer-Verlag, New York, 2005. a preliminary note on the causative agent, Bacterium tularense [61] Sjöstedt A., Eriksson U., Berglund L., Tärnvik , J. Infect. Dis.10 (1912) A., Detection of Francisella tularensis in 61–72. ulcers of patients with tularemia by PCR, J. [50] Mörner T., The ecology of tularaemia, Rev. Clin. Microbiol. 35 (1997) 1045–1048. Sci. Tech. Off. Int. Epizoot. 11 (1992) 1123– 1130. [62] Suvarov S., Volfertz V., Voronkova M.M., Plague like lymphadentitis of the Astrikhansky [51] Noda H., Munderloh U.G., Kurtti T.J., Endo- Region, Vestnik Mikrobiol. Epidemiol. Parazi- symbionts of ticks and their relationship to tol. 7 (1928) 293. Wolbachia spp. and tick-borne pathogens of humans and animals, Appl. Environ. Micro- [63] Syrjala H., Kujala P., Myllyla V., Salminen biol. 63 (1997) 3926–3932. A., Airborne transmission of tularemia in farmers, Scand. J. Infect. Dis. 17 (1985) 371– [52] Ohara H., Human inoculation experiment 375. with a disease of wild rabbits, with a bacteriologic study, Kinsei Igaku 12 (1925) 401. [64] Tang Y.W., Ellis N.M., Hopkins M.K., Smith D.H., Dodge D.E., Persing D.H., Comparison [53] Olsulfiev N.G., Rudne G.P., Tularemia, Pub- of phenotypic and genotypic techniques for lishing House for Medical Literature, Mos- identification of unusual aerobic pathogenic cow, 1960, p. 459. gram-negative bacilli, J. Clin. Microbiol. 36 [54] Parker R.R., Recent studies of tick-borne dis- (1998) 3674–3679. ease made at the United States Public Health [65] Thomas R., Johansson A., Neeson B., Isherwood Service laboratory at Hamilton, Montana, D., Sjöstedt A., Ellis J., Titball R.W., Discrim- in: Proceedings, 5th Pac. Sci. Cong., 1934, ination of human pathogenic subspecies of pp. 3367–3374. Francisella tularensis by using restriction [55] Perez-Castrillon J.L., Bachiller-Luque P., Martin- fragment length of polymorphism, J. Clin. Luquero M., Mena-Martin F.J., Herreros V., Microbiol. 41 (2003) 50–57. Tularemia epidemic in northwestern Spain: [66] Versage J.L., Severin D., Chu M.C., Petersen clinical description and therapeutic response, J.M., Development of a multitarget real-time Clin. Infect. Dis. 15 (2001) 573–576. TaqMan PCR assay for enhanced detection of [56] Petersen J.M., Schriefer M., Carter L.G., Zhou Francisella tularensis in complex specimens, Y., Sealy T., Bawiec D., Yockey B., Urich S., J. Clin. Microbiol. 41 (2002) 5492–5499. Zeidner N., Avashia S., Kool J., Buck J., Lindley [67] Viljanen M.K., Nurmi T., Salminen A., C., Celeda L., Montenieri J., Gage K., Chu M.C., Enzyme-linked immunosorbent assay (ELISA) Laboratory analysis of tularemia in wild with bacterial sonicate antigen for IgM, IgA, trapped, commercially distributed prairie dogs, and IgG antibodies to Francisella tularensis: Texas, 2002, Emerg. Infect. Dis. 10 (2004) comparison with bacterial agglutination test 419–425. and ELISA with lipopolysaccharide antigen, [57] Petersen J.M., Schriefer M., Gage K.L., J. Infect. Dis. 148 (1983) 715–720. Montenieri J.A., Carter L.G., Stanley M., Chu [68] Wherry W.B., Lamb B.H., Infection of man M.C., Methods for the enhanced recovery of with Bacterium tularense, J. Infect. Dis. 15 Francisella tularensis cultures, Appl. Envi- (1914) 331–340. ron. Microbiol. 70 (2004) 3733–3735. [69] Whipp M.J., Davis J.M., Lum G., de Boer J., [58] Reed K.D., Melski J.W., Graham M.B., Regnery Zhou Y., Bearden S.W., Petersen J.M., Chu R.L., Sotir M.J., Wegner M.V., Kazmierczak M.C., Hogg G.G., Characterisation of a novi- J.J., Stratman E.J., Li Y., Fairley J.A., Swain cida-like subspecies of Francisella tularensis G.R., Olson V.A., Sargent E.K., Kehl S.C., isolated in Australia, J. Med. Microbiol. 52 Frace M.A., Kline R., Foldy S.L., Davis J.P., (2003) 839–842. Damon I.K., The detection of monkeypox in humans in the Western Hemisphere, N. Engl. [70] World Health Organization, Tularemia, Kos- J. Med. 350 (2004) 342–350. ovo, Wkly. Epidemiol. Rec. 75 (2000) 133– 134. [59] Reintjes R., Dedushaj I., Gjini A., Jorgensen T.R., Cotter B., Lieftucht A., D'Ancona F., [71] Zarkai G.I., Tularemia among water rats; Dennis D.T., Kosoy M.A. Mulliqi-Osmani G., methods of studying them, Bull. Hyg. 5 (1930) Grunow R., Kalaveshi A., Gashi L., Humolli 875.