Tularemia in Denmark: Identification of a Francisella tularensis subsp. holarctica Strain by Real-Time PCR and High-Resolution Typing by Mul
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微生物临床杂志 2005年第10期
Department of NBC-Analysis, Swedish Defence Research Agency, Ume, Sweden
Department of Clinical Microbiology, Viborg Hospital, Viborg, Denmark
Department of Clinical Microbiology, Infectious Diseases, Ume University, Ume, Sweden
Department of Infectious Diseases, Sundsvall Hospital, Sundsvall, Sweden
ABSTRACT
We report ulceroglandular tularemia affecting an 8-year-old boy and the first recovery of Francisella tularensis in Denmark. A novel real-time PCR assay was used to identify the strain as F. tularensis subsp. holarctica (type B). Multiple-locus variable-number tandem repeat analysis demonstrated a close genetic relationship to strains from Norway.
TEXT
Human tularemia typically presents as an influenza-like illness including fever, generalized body aches, headache, chills, and malaise. The disease is caused by the facultative intracellular, gram-negative bacterium Francisella tularensis and is transmitted to humans by mosquitoes, deer flies, ticks, inhalation of F. tularensis-contaminated aerosols, or direct contact with an infected animal. Clinical forms of tularemia in humans largely depend on the infectious route (14). The most common clinical forms are ulceroglandular (infection through the skin) and respiratory (infection by inhalation). Tularemia is divided into two entities, type A and type B, corresponding to the causative subspecies F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively (14, 18). Type A tularemia is reported only from North America and may progress into a severe septic syndrome with mortality in the preantibiotic era in the range of 5 to 30% and today estimated to be 2% (4). Human or animal cases of type A tularemia have not been identified in Europe. Type B tularemia is endemic to countries of the Northern Hemisphere and is rarely life threatening but may lead to long-lasting complications (e.g., lymph node suppuration).
Due to its virulence and potential for aerosol infection, F. tularensis has received attention as a feared biological threat agent (15). Because of a high risk of airborne laboratory-acquired infection, fastidious growth, and low biochemical reactivity, the bacterium poses a challenge to microbiological laboratories (1, 14). Using traditional bacteriological methods, identification of F. tularensis is difficult and time-consuming. The few biochemical reactions that are available for typing to the subspecies level may be inconclusive, and definite distinction of type A and type B organisms requires virulence tests in animals (14, 18). Therefore, there is a need for more rapid and accurate diagnostic methods as well as for assays capable of distinguishing type A and type B tularemia. Here, we demonstrate the utility of DNA-based methods for unambiguous identification and high-resolution characterization of F. tularensis and describe for the first time culture recovery of this pathogen in Denmark.
In July 2003, at the island Fur in Limfjorden, Denmark, a tick was observed on the left thigh of an 8-year-old boy and removed 1 day later. On day 3, the boy was brought to a general practitioner with high fever, generalized body aches, tenderness in his left groin, and an erythematic skin reaction at the bite wound. On suspicion of borreliosis, oral treatment with penicillin V at 500 mg x 3 for 6 days was instituted. On day 5, the patient was hospitalized due to persistent high fever and a painful left inguinal lymph node enlargement, and on day 9, progression of lymphadenopathy was noted and antibiotic treatment was changed to oral dicloxacillin at 250 mg x 3, assuming a staphylococcal infection. By day 13 the boy still had fever, lymph nodes continued to enlarge, and there was a surrounding erythema. The bite wound was 1 cm in diameter without signs of healing. On day 17, ulceroglandular tularemia was suspected and a lymph node was surgically removed. Tissue specimens were put in saline and sent for laboratory analysis. Treatment was changed to intravenous gentamicin at 150 mg daily for 10 days, and the patient recovered quickly.
A section of lymph node tissue was used for bacterial culture. On day 21, growth of small gram-negative rods was detected in the aerobic part of a medium denoted semisolid agar plus pepsin blood plus thioglycolate, product no. 1133, which is further detailed at the website of Statens Seruminstitut, Copenhagen, Denmark (http://www.ssi.dk/sw965.asp). On day 23, bacterial growth occurred on chocolate blood agar (Statens Serum Institute). The same day, F. tularensis DNA was detected in lymph node specimens by PCR amplification of rRNA and the gene lpnA encoding a 17-kDa lipoprotein (17). The bacterial strain was sent to a biosafety level 3 laboratory, and F. tularensis was identified by PCR and a specific agglutination reaction (18). Histological examination of excised lymph node tissue showed a necrotizing granulomatous reaction consistent with tularemia. A patient blood sample obtained on day 16 was subsequently found positive in an agglutination test for tularemia (Statens Serum Institut).
We applied a novel real-time PCR assay with melting point analysis of the obtained PCR amplicon to the Danish strain and control strains of F. tularensis. The assay detects strains of F. tularensis subsp. holarctica (type B) by identification of a 30-bp deletion unique to the subspecies at a genomic locus designated Ft-M19 (11). F. tularensis DNA was isolated as described previously (9, 17), and triplicate samples of each strain were amplified using 0.8 μM of each of the primers 5'-CCAGTACAAACTCAATTTGGTTATCATC-3' and 5'-GTTTCAGAATTCATTTTTGTCCGTAA-3' and the SYBR green PCR master mix (Applied Biosystems) in a total reaction volume of 25 μl. An initial denaturation at 50°C for 2 min and 95°C for 10 min was followed by 40 cycles of 95°C for 15 s and 60°C for 60 s on an iCycler (Bio-Rad Laboratories, Hercules, CA). Reference strains (F. tularensis subsp. tularensis strain SCHU S4 and F. tularensis subsp. holarctica, live vaccine strain) and negative controls (water) were included in all PCR runs. After the final cycle, melting point analysis was performed with 0.5°C temperature increments, using software version 3.0A (Bio-Rad Laboratories). The novel real-time PCR assay was confirmed to successfully amplify DNA from a test panel of F. tularensis strains with a worldwide geographical origin. Each strain was tested at least twice. Strains of F. tularensis subsp. holarctica (type B; n = 22) consistently showed melting points of 73.5 to 74.0°C, whereas strains of F. tularensis subsp. tularensis (type A; n = 10) and F. tularensis subsp. novicida (n = 2) showed melting points of 71.5 to 72.5°C, and strains of F. tularensis subsp. mediasiatica (n = 3), showed melting points of 72.0 to 73.0°C (Fig. 1). In each PCR analysis, melting peaks corresponding to different subspecies were easily distinguished, with the exception of F. tularensis subsp. tularensis and F. tularensis subsp. novicida, which often coincided. The Danish strain was identified as F. tularensis subsp. holarctica (type B) based on melting point analysis of the real-time PCR product (Fig. 1). Results were confirmed by GeneScan analysis of marker Ft-M19 performed as described previously (11) and using the primers 5'-AGGCGGAGATCTAGGAACCTTT-3' and 5'-AGCCCAAGCTGACTAAAATCTTT-3' to amplify DNA of the test panel strains. All strains of F. tularensis subsp. holarctica (type B), including the Danish strain, showed PCR amplicon sizes at Ft-M19 of 220 bp. Strains of F. tularensis subsp. tularensis (type A), F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida showed sizes of 250 bp. The results are in agreement with a deletion of 30 bp at the genomic locus Ft-M19 in all type B strains, as was recently verified in a broader analysis of 192 F. tularensis strains (11). Two direct nucleotide repeats in the F. tularensis genome flank the deletion site at Ft-M19, and it is possible that these repeats mediated the deletion by homologous recombination of the two repeats in an ancestor to all strains of F. tularensis subsp. holarctica (type B) (Fig. 1). We recently showed that several other similar subspecies-specific excisions of sequence located between two direct repeats have occurred during the evolution of F. tularensis subsp. holarctica (19). The PCR amplicon melting point of F. tularensis subsp. mediasiatica strains was intermediate to that of type A and type B strains and is likely explained by two point mutations (AG) at the targeted genomic locus (Fig. 1). The difference in nucleotide composition causes a shift in melting point as compared to F. tularensis subsp. tularensis and F. tularensis subsp. novicida. Complete nucleotide sequences at Ft-M19 have previously been assigned accession no. AF247642, AF247685 to AF247690, and AF524865 in the GenBank database.
We here show that the real-time PCR assay enables immediate distinction of type B tularemia organisms. The assay might prove clinically useful. A rapid distinction of type A and type B tularemia may become crucial, particularly in urgent situations related to a possible intentional release of F. tularensis. For rapid identification of bacterial cultures suspected to be F. tularensis, we suggest the use of an F. tularensis-specific PCR assay that is targeting one or several genes, including lpnA (encoding a 17-kDa lipoprotein) (17, 21). The real-time PCR assay presented in this paper will add clinically useful information by rapid identification of type B organisms. It would be of putative interest to use the novel assay for direct detection of F. tularensis DNA in human specimens, but this has not been evaluated in the present study. The detection limit of the assay using water samples spiked with a type A or a type B strain was estimated by two different means. We determined the detectable number of CFU or genome equivalents (GE) per PCR. For CFU determinations, duplicate 10-fold serial dilutions of F. tularensis strains were prepared in phosphate-buffered saline and viable counts were calculated by a spread of 500 μl from each dilution in duplicate on modified Thayer-Martin plates. For determinations of GE, DNA concentrations in duplicate stock water solutions of each strain were determined in a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del.) and 10-fold serial dilutions were prepared. The detection limit of the assay was in the range of 10 to 100 viable bacteria as determined by CFU or 100 to 150 bacteria as determined by GE.
Multiple-locus variable-number tandem repeat analysis (MLVA), a highly discriminatory typing system based on detection of variable numbers of tandem repeats in the F. tularensis genome (11), was applied to investigate the genetic relationship of the Danish strain to other Eurasian strains. Six genomic loci (Ft-M3, Ft-M6, Ft-M20, Ft-M21, Ft-M22, and Ft-M24), previously found polymorphic among type B strains, were assayed in 16 of the 22 F. tularensis subsp. holarctica strains that were analyzed by the real-time PCR assay. Briefly, primers flanking each locus amplified DNA from each strain, and PCR fragments were size determined as described previously (11), using a 377XL DNA sequencer (PE Applied Biosystems). Primers were fluorescence labeled with 6-carboxyfluorescein or 6-carboxytetramethylrhodamine, and filter set A was applied. Typing data were analyzed using Bionumerics v. 3.5 (Applied-Maths, Saint-Martens-Latem, Belgium), the categorical coefficient, and the unweighted pair-group with arithmetic means algorithm.
Using MLVA, the most variable marker (Ft-M3) showed nine different PCR fragment sizes among 16 strains (Fig. 2). The observed size variation represents a difference in repeat copy numbers at the marker. Other markers showed less variability, with only two or three PCR fragment sizes being observed among the strains. Cluster analysis assigned the 16 strains into 15 genotypes (Fig. 2). The tree topology obtained indicated a correlation of geographical origin of a strain and its genotype. Clustering of the Danish strain with strains from Norway was consistent using the unweighted pair-group with arithmetic means (Fig. 2) or the neighbor-joining algorithm, and the obtained overall tree topologies were highly similar (not shown). Although this clustering is intriguing, analysis of larger numbers of strains will be required to make more firm conclusions regarding a geographic relationship. The Danish strain was genetically similar to several F. tularensis strains of European origin, which all share a relatively low variability at genomic loci exhibiting variable numbers of tandem repeats. Thereby, the present analysis supports a previous suggestion that F. tularensis subsp. holarctica is an evolutionarily young F. tularensis lineage with limited genetic diversity even at highly mutable sequences assayed by MLVA (11).
To our knowledge, this is the first report of isolation of F. tularensis in Denmark. Tularemia has been reported from all European countries except Great Britain, Iceland, and Portugal (20). According to the literature, Denmark was long considered devoid of the disease (3, 12). Only in recent years have a few patients been reported. During the time period 1987 to 2000, eight patients with serology-proven cases were assumed to have contracted tularemia in Denmark (16). In addition, two Swedes contracted tularemia on the Danish island Bornholm located in the Baltic Sea in the year 2000 (7). These reports and the present study validate that tularemia is endemic also to Denmark. There is a lack of knowledge of what factors govern the incidence and spread of tularemia. The present report of recovery of F. tularensis in Denmark contrasts with tularemia case counts reported from other Nordic countries. In 2003, 823 human cases of tularemia were reported in Finland, 698 in Sweden, and 22 in Norway according to surveillance data found at websites of the National Public Health Institute of Finland (http://www3.ktl.fi/stat/), the Swedish Institute for Infectious Disease Control (http://www.smittskyddsinstitutet.se/), and the Norwegian Institute of Public Health (http://www.msis.no/), respectively. The ecological correlates to observed differences in tularemia incidence in neighboring countries remain unknown. In the present report of a tularemia affecting a Danish boy, a tick bite preceded the appearance of a primary ulcer at the bite site and subsequent clinical manifestations. The tick species was not determined. In central Europe, ticks of the species Ixodes ricinus and Dermacentor reticularis have been shown to be naturally infected with F. tularensis, at a prevalence of 0.1 to 0.2% (6, 8, 22).
DNA detection in human specimens has proven a valuable tool for tularemia diagnosis in an acute setting (using wound swabs) as well as in retrospective analysis of archived samples (formalin-fixed tissues) (10, 13, 17). In this study, we showed a fresh lymph node specimen to be useful for detection of F. tularensis. This is in concordance with previous case reports on lymph node aspirates, even after the initiation of relevant antibiotic treatment (2, 5). In conclusion, our study demonstrates that new DNA-based methods for laboratory diagnosis and high-resolution characterization of F. tularensis make diagnostic work more rapid, more safe for laboratory personal, and more precise.
ACKNOWLEDGMENTS
We thank Linda Karlsson at the Swedish Defense Research Agency for performance of MLVA. We thank the Department of Clinical Microbiology and the Widal Laboratory, Statens Seruminstitut, for performance of F. tularensis DNA detection and serology.
This work was supported by funding from the Swedish Medical Research Council project no. 9485; the Medical Faculty, Ume University; the County Council of Vsternorrland; and the Swedish MoD, project no. A4854.
Present address: National Center for Antimicrobials and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
REFERENCES
Burke, D. S. 1977. Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J. Infect. Dis. 135:55-60.
Cristova, I., T. Velinov, T. Kantardjiev, and A. Galev. 2004. Tularaemia outbreak in Bulgaria. Scand. J. Infect. Dis. 36:785-789.
Dahlstrand, S., O. Ringertz, and B. Zetterberg. 1971. Airborne tularemia in Sweden. Scand. J. Infect. Dis. 3:7-16.
Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medical and public health management. JAMA 285:2763-2773.
Dolan, S. A., C. B. Dommaraju, and G. B. DeGuzman. 1998. Detection of Francisella tularensis in clinical specimens by use of polymerase chain reaction. Clin. Infect. Dis. 26:764-765.
Gurycova, D., E. Kocianova, V. Vyrostekova, and J. Rehacek. 1995. Prevalence of ticks infected with Francisella tularensis in natural foci of tularemia in western Slovakia. Eur. J. Epidemiol. 11:469-474.
Hansson, C., and T. Ingvarsson. 2002. Two cases of tularaemia after an orienteering contest on the non-endemic Island of Bornholm. Scand. J. Infect. Dis. 34:76.
Hubalek, Z., F. Treml, J. Halouzka, Z. Juricova, M. Hunady, and V. Janik. 1996. Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia. Med. Vet. Entomol. 10:241-246.
Ibrahim, A., L. Norlander, A. Macellaro, and A. Sjstedt. 1997. Specific detection of Coxiella burnetii through partial amplification of 23S rDNA. Eur. J. Epidemiol. 13:329-334.
Johansson, A., L. Berglund, U. Eriksson, I. Gransson, R. Wollin, M. Forsman, A. Trnvik, and A. Sjstedt. 2000. Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia. J. Clin. Microbiol. 38:22-26.
Johansson, A., J. Farlow, P. Larsson, M. Dukerich, E. Chambers, M. Bystrm, J. Fox, M. Chu, M. Forsman, A. Sjstedt, and P. Keim. 2004. Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis. J. Bacteriol. 186:5808-5818.
Jusatz, H. J. 1952. Tularemia in Europe, 1926-1951, p. 7-16. In E. Rodenwaldt (ed.), Welt-Suchen atlas, vol. 1. Falk-Verlag, Hamburg, Germany.
Lamps, L. W., J. M. Havens, A. Sjstedt, D. L. Page, and M. A. Scott. 2004. Histologic and molecular diagnosis of tularemia: a potential bioterrorism agent endemic to North America. Mod. Pathol. 17:489-495.
Penn, R. L. 2005. Francisella tularensis (Tularemia), p. 2674-2685. In G. L. Mandell, J. E. Bennet, and R. Dolin (ed.), Mandell, Douglas and Bennett's principles and practice of infectious diseases, 6th ed., vol. 2. Churchill Livingstone, Ltd., Edinburgh, Scotland.
Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential biological terrorism agents. Emerg. Infect. Dis. 8:225-230.
Schiellerup, P., and K. A. Krogfelt. 6 June 2001, posting date. National surveillance of communicable diseases, tularaemia. Epi-News, no. 23, 2001. Statens Serum Institut, Copenhagen, Denmark. [Online.] http://www.ssi.dk/sw2882.asp.
Sjstedt, A., U. Eriksson, L. Berglund, and A. Trnvik. 1997. Detection of Francisella tularensis in ulcers of patients with tularemia by PCR. J. Clin. Microbiol. 35:1045-1048.
Sjstedt, A. B. 2005. Francisella, p. 200-210. In D. J. Brenner, N. R. Krieg, J. T. Staley, and G. M. Garrity (ed.), The Proteobacteria, part B. Bergey's manual of systematic bacteriology, 2nd ed., vol. 2. Springer-Verlag, New York, N.Y.
Svensson, K., P. Larsson, D. Johansson, M. Bystrm, M. Forsman, and A. Johansson. 2005. Evolution of subspecies of Francisella tularensis. J. Bacteriol. 187:3903-3908.
Trnvik, A., H. S. Priebe, and R. Grunow. 2004. Tularaemia in Europe: an epidemiological overview. Scand. J. Infect. Dis. 36:350-355.
Versage, J. L., D. D. M. Severin, M. C. Chu, and J. M. Petersen. 2003. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens. J. Clin. Microbiol. 41:5492-5499.
Wicki, R., P. Sauter, C. Mettler, A. Natsch, T. Enzler, N. Pusterla, P. Kuhnert, G. Egli, M. Bernasconi, R. Lienhard, H. Lutz, and C. M. Leutenegger. 2000. Swiss Army survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks. Eur. J. Clin. Microbiol. Infect. Dis. 19:427-432.(Mona Bystrm, Sidsel Bcher)
Department of Clinical Microbiology, Viborg Hospital, Viborg, Denmark
Department of Clinical Microbiology, Infectious Diseases, Ume University, Ume, Sweden
Department of Infectious Diseases, Sundsvall Hospital, Sundsvall, Sweden
ABSTRACT
We report ulceroglandular tularemia affecting an 8-year-old boy and the first recovery of Francisella tularensis in Denmark. A novel real-time PCR assay was used to identify the strain as F. tularensis subsp. holarctica (type B). Multiple-locus variable-number tandem repeat analysis demonstrated a close genetic relationship to strains from Norway.
TEXT
Human tularemia typically presents as an influenza-like illness including fever, generalized body aches, headache, chills, and malaise. The disease is caused by the facultative intracellular, gram-negative bacterium Francisella tularensis and is transmitted to humans by mosquitoes, deer flies, ticks, inhalation of F. tularensis-contaminated aerosols, or direct contact with an infected animal. Clinical forms of tularemia in humans largely depend on the infectious route (14). The most common clinical forms are ulceroglandular (infection through the skin) and respiratory (infection by inhalation). Tularemia is divided into two entities, type A and type B, corresponding to the causative subspecies F. tularensis subsp. tularensis and F. tularensis subsp. holarctica, respectively (14, 18). Type A tularemia is reported only from North America and may progress into a severe septic syndrome with mortality in the preantibiotic era in the range of 5 to 30% and today estimated to be 2% (4). Human or animal cases of type A tularemia have not been identified in Europe. Type B tularemia is endemic to countries of the Northern Hemisphere and is rarely life threatening but may lead to long-lasting complications (e.g., lymph node suppuration).
Due to its virulence and potential for aerosol infection, F. tularensis has received attention as a feared biological threat agent (15). Because of a high risk of airborne laboratory-acquired infection, fastidious growth, and low biochemical reactivity, the bacterium poses a challenge to microbiological laboratories (1, 14). Using traditional bacteriological methods, identification of F. tularensis is difficult and time-consuming. The few biochemical reactions that are available for typing to the subspecies level may be inconclusive, and definite distinction of type A and type B organisms requires virulence tests in animals (14, 18). Therefore, there is a need for more rapid and accurate diagnostic methods as well as for assays capable of distinguishing type A and type B tularemia. Here, we demonstrate the utility of DNA-based methods for unambiguous identification and high-resolution characterization of F. tularensis and describe for the first time culture recovery of this pathogen in Denmark.
In July 2003, at the island Fur in Limfjorden, Denmark, a tick was observed on the left thigh of an 8-year-old boy and removed 1 day later. On day 3, the boy was brought to a general practitioner with high fever, generalized body aches, tenderness in his left groin, and an erythematic skin reaction at the bite wound. On suspicion of borreliosis, oral treatment with penicillin V at 500 mg x 3 for 6 days was instituted. On day 5, the patient was hospitalized due to persistent high fever and a painful left inguinal lymph node enlargement, and on day 9, progression of lymphadenopathy was noted and antibiotic treatment was changed to oral dicloxacillin at 250 mg x 3, assuming a staphylococcal infection. By day 13 the boy still had fever, lymph nodes continued to enlarge, and there was a surrounding erythema. The bite wound was 1 cm in diameter without signs of healing. On day 17, ulceroglandular tularemia was suspected and a lymph node was surgically removed. Tissue specimens were put in saline and sent for laboratory analysis. Treatment was changed to intravenous gentamicin at 150 mg daily for 10 days, and the patient recovered quickly.
A section of lymph node tissue was used for bacterial culture. On day 21, growth of small gram-negative rods was detected in the aerobic part of a medium denoted semisolid agar plus pepsin blood plus thioglycolate, product no. 1133, which is further detailed at the website of Statens Seruminstitut, Copenhagen, Denmark (http://www.ssi.dk/sw965.asp). On day 23, bacterial growth occurred on chocolate blood agar (Statens Serum Institute). The same day, F. tularensis DNA was detected in lymph node specimens by PCR amplification of rRNA and the gene lpnA encoding a 17-kDa lipoprotein (17). The bacterial strain was sent to a biosafety level 3 laboratory, and F. tularensis was identified by PCR and a specific agglutination reaction (18). Histological examination of excised lymph node tissue showed a necrotizing granulomatous reaction consistent with tularemia. A patient blood sample obtained on day 16 was subsequently found positive in an agglutination test for tularemia (Statens Serum Institut).
We applied a novel real-time PCR assay with melting point analysis of the obtained PCR amplicon to the Danish strain and control strains of F. tularensis. The assay detects strains of F. tularensis subsp. holarctica (type B) by identification of a 30-bp deletion unique to the subspecies at a genomic locus designated Ft-M19 (11). F. tularensis DNA was isolated as described previously (9, 17), and triplicate samples of each strain were amplified using 0.8 μM of each of the primers 5'-CCAGTACAAACTCAATTTGGTTATCATC-3' and 5'-GTTTCAGAATTCATTTTTGTCCGTAA-3' and the SYBR green PCR master mix (Applied Biosystems) in a total reaction volume of 25 μl. An initial denaturation at 50°C for 2 min and 95°C for 10 min was followed by 40 cycles of 95°C for 15 s and 60°C for 60 s on an iCycler (Bio-Rad Laboratories, Hercules, CA). Reference strains (F. tularensis subsp. tularensis strain SCHU S4 and F. tularensis subsp. holarctica, live vaccine strain) and negative controls (water) were included in all PCR runs. After the final cycle, melting point analysis was performed with 0.5°C temperature increments, using software version 3.0A (Bio-Rad Laboratories). The novel real-time PCR assay was confirmed to successfully amplify DNA from a test panel of F. tularensis strains with a worldwide geographical origin. Each strain was tested at least twice. Strains of F. tularensis subsp. holarctica (type B; n = 22) consistently showed melting points of 73.5 to 74.0°C, whereas strains of F. tularensis subsp. tularensis (type A; n = 10) and F. tularensis subsp. novicida (n = 2) showed melting points of 71.5 to 72.5°C, and strains of F. tularensis subsp. mediasiatica (n = 3), showed melting points of 72.0 to 73.0°C (Fig. 1). In each PCR analysis, melting peaks corresponding to different subspecies were easily distinguished, with the exception of F. tularensis subsp. tularensis and F. tularensis subsp. novicida, which often coincided. The Danish strain was identified as F. tularensis subsp. holarctica (type B) based on melting point analysis of the real-time PCR product (Fig. 1). Results were confirmed by GeneScan analysis of marker Ft-M19 performed as described previously (11) and using the primers 5'-AGGCGGAGATCTAGGAACCTTT-3' and 5'-AGCCCAAGCTGACTAAAATCTTT-3' to amplify DNA of the test panel strains. All strains of F. tularensis subsp. holarctica (type B), including the Danish strain, showed PCR amplicon sizes at Ft-M19 of 220 bp. Strains of F. tularensis subsp. tularensis (type A), F. tularensis subsp. mediasiatica, and F. tularensis subsp. novicida showed sizes of 250 bp. The results are in agreement with a deletion of 30 bp at the genomic locus Ft-M19 in all type B strains, as was recently verified in a broader analysis of 192 F. tularensis strains (11). Two direct nucleotide repeats in the F. tularensis genome flank the deletion site at Ft-M19, and it is possible that these repeats mediated the deletion by homologous recombination of the two repeats in an ancestor to all strains of F. tularensis subsp. holarctica (type B) (Fig. 1). We recently showed that several other similar subspecies-specific excisions of sequence located between two direct repeats have occurred during the evolution of F. tularensis subsp. holarctica (19). The PCR amplicon melting point of F. tularensis subsp. mediasiatica strains was intermediate to that of type A and type B strains and is likely explained by two point mutations (AG) at the targeted genomic locus (Fig. 1). The difference in nucleotide composition causes a shift in melting point as compared to F. tularensis subsp. tularensis and F. tularensis subsp. novicida. Complete nucleotide sequences at Ft-M19 have previously been assigned accession no. AF247642, AF247685 to AF247690, and AF524865 in the GenBank database.
We here show that the real-time PCR assay enables immediate distinction of type B tularemia organisms. The assay might prove clinically useful. A rapid distinction of type A and type B tularemia may become crucial, particularly in urgent situations related to a possible intentional release of F. tularensis. For rapid identification of bacterial cultures suspected to be F. tularensis, we suggest the use of an F. tularensis-specific PCR assay that is targeting one or several genes, including lpnA (encoding a 17-kDa lipoprotein) (17, 21). The real-time PCR assay presented in this paper will add clinically useful information by rapid identification of type B organisms. It would be of putative interest to use the novel assay for direct detection of F. tularensis DNA in human specimens, but this has not been evaluated in the present study. The detection limit of the assay using water samples spiked with a type A or a type B strain was estimated by two different means. We determined the detectable number of CFU or genome equivalents (GE) per PCR. For CFU determinations, duplicate 10-fold serial dilutions of F. tularensis strains were prepared in phosphate-buffered saline and viable counts were calculated by a spread of 500 μl from each dilution in duplicate on modified Thayer-Martin plates. For determinations of GE, DNA concentrations in duplicate stock water solutions of each strain were determined in a Nanodrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Del.) and 10-fold serial dilutions were prepared. The detection limit of the assay was in the range of 10 to 100 viable bacteria as determined by CFU or 100 to 150 bacteria as determined by GE.
Multiple-locus variable-number tandem repeat analysis (MLVA), a highly discriminatory typing system based on detection of variable numbers of tandem repeats in the F. tularensis genome (11), was applied to investigate the genetic relationship of the Danish strain to other Eurasian strains. Six genomic loci (Ft-M3, Ft-M6, Ft-M20, Ft-M21, Ft-M22, and Ft-M24), previously found polymorphic among type B strains, were assayed in 16 of the 22 F. tularensis subsp. holarctica strains that were analyzed by the real-time PCR assay. Briefly, primers flanking each locus amplified DNA from each strain, and PCR fragments were size determined as described previously (11), using a 377XL DNA sequencer (PE Applied Biosystems). Primers were fluorescence labeled with 6-carboxyfluorescein or 6-carboxytetramethylrhodamine, and filter set A was applied. Typing data were analyzed using Bionumerics v. 3.5 (Applied-Maths, Saint-Martens-Latem, Belgium), the categorical coefficient, and the unweighted pair-group with arithmetic means algorithm.
Using MLVA, the most variable marker (Ft-M3) showed nine different PCR fragment sizes among 16 strains (Fig. 2). The observed size variation represents a difference in repeat copy numbers at the marker. Other markers showed less variability, with only two or three PCR fragment sizes being observed among the strains. Cluster analysis assigned the 16 strains into 15 genotypes (Fig. 2). The tree topology obtained indicated a correlation of geographical origin of a strain and its genotype. Clustering of the Danish strain with strains from Norway was consistent using the unweighted pair-group with arithmetic means (Fig. 2) or the neighbor-joining algorithm, and the obtained overall tree topologies were highly similar (not shown). Although this clustering is intriguing, analysis of larger numbers of strains will be required to make more firm conclusions regarding a geographic relationship. The Danish strain was genetically similar to several F. tularensis strains of European origin, which all share a relatively low variability at genomic loci exhibiting variable numbers of tandem repeats. Thereby, the present analysis supports a previous suggestion that F. tularensis subsp. holarctica is an evolutionarily young F. tularensis lineage with limited genetic diversity even at highly mutable sequences assayed by MLVA (11).
To our knowledge, this is the first report of isolation of F. tularensis in Denmark. Tularemia has been reported from all European countries except Great Britain, Iceland, and Portugal (20). According to the literature, Denmark was long considered devoid of the disease (3, 12). Only in recent years have a few patients been reported. During the time period 1987 to 2000, eight patients with serology-proven cases were assumed to have contracted tularemia in Denmark (16). In addition, two Swedes contracted tularemia on the Danish island Bornholm located in the Baltic Sea in the year 2000 (7). These reports and the present study validate that tularemia is endemic also to Denmark. There is a lack of knowledge of what factors govern the incidence and spread of tularemia. The present report of recovery of F. tularensis in Denmark contrasts with tularemia case counts reported from other Nordic countries. In 2003, 823 human cases of tularemia were reported in Finland, 698 in Sweden, and 22 in Norway according to surveillance data found at websites of the National Public Health Institute of Finland (http://www3.ktl.fi/stat/), the Swedish Institute for Infectious Disease Control (http://www.smittskyddsinstitutet.se/), and the Norwegian Institute of Public Health (http://www.msis.no/), respectively. The ecological correlates to observed differences in tularemia incidence in neighboring countries remain unknown. In the present report of a tularemia affecting a Danish boy, a tick bite preceded the appearance of a primary ulcer at the bite site and subsequent clinical manifestations. The tick species was not determined. In central Europe, ticks of the species Ixodes ricinus and Dermacentor reticularis have been shown to be naturally infected with F. tularensis, at a prevalence of 0.1 to 0.2% (6, 8, 22).
DNA detection in human specimens has proven a valuable tool for tularemia diagnosis in an acute setting (using wound swabs) as well as in retrospective analysis of archived samples (formalin-fixed tissues) (10, 13, 17). In this study, we showed a fresh lymph node specimen to be useful for detection of F. tularensis. This is in concordance with previous case reports on lymph node aspirates, even after the initiation of relevant antibiotic treatment (2, 5). In conclusion, our study demonstrates that new DNA-based methods for laboratory diagnosis and high-resolution characterization of F. tularensis make diagnostic work more rapid, more safe for laboratory personal, and more precise.
ACKNOWLEDGMENTS
We thank Linda Karlsson at the Swedish Defense Research Agency for performance of MLVA. We thank the Department of Clinical Microbiology and the Widal Laboratory, Statens Seruminstitut, for performance of F. tularensis DNA detection and serology.
This work was supported by funding from the Swedish Medical Research Council project no. 9485; the Medical Faculty, Ume University; the County Council of Vsternorrland; and the Swedish MoD, project no. A4854.
Present address: National Center for Antimicrobials and Infection Control, Statens Serum Institut, Copenhagen, Denmark.
REFERENCES
Burke, D. S. 1977. Immunization against tularemia: analysis of the effectiveness of live Francisella tularensis vaccine in prevention of laboratory-acquired tularemia. J. Infect. Dis. 135:55-60.
Cristova, I., T. Velinov, T. Kantardjiev, and A. Galev. 2004. Tularaemia outbreak in Bulgaria. Scand. J. Infect. Dis. 36:785-789.
Dahlstrand, S., O. Ringertz, and B. Zetterberg. 1971. Airborne tularemia in Sweden. Scand. J. Infect. Dis. 3:7-16.
Dennis, D. T., T. V. Inglesby, D. A. Henderson, J. G. Bartlett, M. S. Ascher, E. Eitzen, A. D. Fine, A. M. Friedlander, J. Hauer, M. Layton, S. R. Lillibridge, J. E. McDade, M. T. Osterholm, T. O'Toole, G. Parker, T. M. Perl, P. K. Russell, and K. Tonat. 2001. Tularemia as a biological weapon: medical and public health management. JAMA 285:2763-2773.
Dolan, S. A., C. B. Dommaraju, and G. B. DeGuzman. 1998. Detection of Francisella tularensis in clinical specimens by use of polymerase chain reaction. Clin. Infect. Dis. 26:764-765.
Gurycova, D., E. Kocianova, V. Vyrostekova, and J. Rehacek. 1995. Prevalence of ticks infected with Francisella tularensis in natural foci of tularemia in western Slovakia. Eur. J. Epidemiol. 11:469-474.
Hansson, C., and T. Ingvarsson. 2002. Two cases of tularaemia after an orienteering contest on the non-endemic Island of Bornholm. Scand. J. Infect. Dis. 34:76.
Hubalek, Z., F. Treml, J. Halouzka, Z. Juricova, M. Hunady, and V. Janik. 1996. Frequent isolation of Francisella tularensis from Dermacentor reticulatus ticks in an enzootic focus of tularaemia. Med. Vet. Entomol. 10:241-246.
Ibrahim, A., L. Norlander, A. Macellaro, and A. Sjstedt. 1997. Specific detection of Coxiella burnetii through partial amplification of 23S rDNA. Eur. J. Epidemiol. 13:329-334.
Johansson, A., L. Berglund, U. Eriksson, I. Gransson, R. Wollin, M. Forsman, A. Trnvik, and A. Sjstedt. 2000. Comparative analysis of PCR versus culture for diagnosis of ulceroglandular tularemia. J. Clin. Microbiol. 38:22-26.
Johansson, A., J. Farlow, P. Larsson, M. Dukerich, E. Chambers, M. Bystrm, J. Fox, M. Chu, M. Forsman, A. Sjstedt, and P. Keim. 2004. Worldwide genetic relationships among Francisella tularensis isolates determined by multiple-locus variable-number tandem repeat analysis. J. Bacteriol. 186:5808-5818.
Jusatz, H. J. 1952. Tularemia in Europe, 1926-1951, p. 7-16. In E. Rodenwaldt (ed.), Welt-Suchen atlas, vol. 1. Falk-Verlag, Hamburg, Germany.
Lamps, L. W., J. M. Havens, A. Sjstedt, D. L. Page, and M. A. Scott. 2004. Histologic and molecular diagnosis of tularemia: a potential bioterrorism agent endemic to North America. Mod. Pathol. 17:489-495.
Penn, R. L. 2005. Francisella tularensis (Tularemia), p. 2674-2685. In G. L. Mandell, J. E. Bennet, and R. Dolin (ed.), Mandell, Douglas and Bennett's principles and practice of infectious diseases, 6th ed., vol. 2. Churchill Livingstone, Ltd., Edinburgh, Scotland.
Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential biological terrorism agents. Emerg. Infect. Dis. 8:225-230.
Schiellerup, P., and K. A. Krogfelt. 6 June 2001, posting date. National surveillance of communicable diseases, tularaemia. Epi-News, no. 23, 2001. Statens Serum Institut, Copenhagen, Denmark. [Online.] http://www.ssi.dk/sw2882.asp.
Sjstedt, A., U. Eriksson, L. Berglund, and A. Trnvik. 1997. Detection of Francisella tularensis in ulcers of patients with tularemia by PCR. J. Clin. Microbiol. 35:1045-1048.
Sjstedt, A. B. 2005. Francisella, p. 200-210. In D. J. Brenner, N. R. Krieg, J. T. Staley, and G. M. Garrity (ed.), The Proteobacteria, part B. Bergey's manual of systematic bacteriology, 2nd ed., vol. 2. Springer-Verlag, New York, N.Y.
Svensson, K., P. Larsson, D. Johansson, M. Bystrm, M. Forsman, and A. Johansson. 2005. Evolution of subspecies of Francisella tularensis. J. Bacteriol. 187:3903-3908.
Trnvik, A., H. S. Priebe, and R. Grunow. 2004. Tularaemia in Europe: an epidemiological overview. Scand. J. Infect. Dis. 36:350-355.
Versage, J. L., D. D. M. Severin, M. C. Chu, and J. M. Petersen. 2003. Development of a multitarget real-time TaqMan PCR assay for enhanced detection of Francisella tularensis in complex specimens. J. Clin. Microbiol. 41:5492-5499.
Wicki, R., P. Sauter, C. Mettler, A. Natsch, T. Enzler, N. Pusterla, P. Kuhnert, G. Egli, M. Bernasconi, R. Lienhard, H. Lutz, and C. M. Leutenegger. 2000. Swiss Army survey in Switzerland to determine the prevalence of Francisella tularensis, members of the Ehrlichia phagocytophila genogroup, Borrelia burgdorferi sensu lato, and tick-borne encephalitis virus in ticks. Eur. J. Clin. Microbiol. Infect. Dis. 19:427-432.(Mona Bystrm, Sidsel Bcher)