Molecular Investigation of Tularemia Outbreaks, Spain, 1997–2008 - Volume 20, Number 5—May 2014 - Emerging Infectious Disease journal - CDC

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Molecular Investigation of Tularemia Outbreaks, Spain, 1997–2008 - Volume 20, Number 5—May 2014 - Emerging Infectious Disease journal - CDC

Volume 20, Number 5—May 2014

Research

Molecular Investigation of Tularemia Outbreaks, Spain, 1997–2008

Article Contents

• Methods
• Results
• Discussion
• Acknowledgment
• References
• Figure 1
• Figure 2
• Figure 3
• Figure 4
• Table
• Technical Appendix - 27 KB 
• Suggested Citation

Jaime Ariza-Miguel, Anders Johansson, María Isabel Fernández-Natal, Carmen Martínez-Nistal, Antonio Orduña, Elías F. Rodríguez-Ferri, Marta Hernández, and David Rodríguez-Lázaro 

Author affiliations: Instituto Tecnológico Agrario de Castilla y León, Valladolid, Spain (J. Ariza-Miguel, M. Hernández, D. Rodríguez-Lázaro); Umeå University, Umeå, Sweden (A. Johansson);Complejo Asistencial Universitario de León, León, Spain (M.I. Fernández-Natal); Laboratorio Regional de Sanidad Animal León, Valladolid (C. Martínez-Nistal); Universidad de Valladolid, Valladolid (A. Orduña); Universidad de León, León (E.F. Rodríguez-Ferri); Universidad de Burgos, Burgos, Spain (D. Rodríguez-Lázaro)

Suggested citation for this article

Abstract

Tularemia outbreaks occurred in northwestern Spain in 1997–1998 and 2007–2008 and affected >1,000 persons. We assessed isolates involved in these outbreaks by using pulsed-field gel electrophoresis with 2 restriction enzymes and multilocus variable number tandem repeat analysis of 16 genomic loci of Francisella tularensis, the cause of this disease. Isolates were divided into 3 pulsotypes by pulsed-field gel electrophoresis and 8 allelic profiles by multilocus variable number tandem repeat analysis. Isolates obtained from the second tularemia outbreak had the same genotypes as isolates obtained from the first outbreak. Both outbreaks were caused by genotypes of genetic subclade B.Br:FTNF002–00, which is widely distributed in countries in central and western Europe. Thus, reemergence of tularemia in Spain was not caused by the reintroduction of exotic strains, but probably by persistence of local reservoirs of infection.

Tularemia is a zoonosis caused by the gram-negative bacterium Francisella tularensis. F. tularensis is a highly contagious facultative intracellular pathogen and has infectious doses as low as 10–50 bacteria; it is transmitted by inhalation, direct contact with infected animals, or ingestion of contaminated water or food. The number of species susceptible to infection by this agent is higher than for any other known zoonotic pathogen (1). Because of its potential to cause adverse public health effects and mass casualties by bioterrorist attack, the pathogen is 1 of 6 agents listed as a Tier 1 agent by the US Department of Health and Human Services (2).

F. tularensis includes 4 subspecies (F. tularensis subsp. tularensis, F. tularensis subsp. holarctica,F. tularensis subsp. novicida, and F. tularensis subsp. mediasiatica), which show marked differences in many epidemiologic features, including geographic distribution, virulence, and genetic diversity (3). F. tularensis subsp. tularensis (Jellison type A) and F. tularensis subsp.holarctica (Jellison type B) are major clinical pathogens. F. tularensis subsp. tularensis is the most virulent subspecies and can cause life-threatening disease; its distribution seems to be restricted to North America, although a single report indicated its presence in Europe (4–7). F.tularensis subsp. holarctica causes a less severe disease, and although widespread throughout the Northern Hemisphere, it has restricted genetic diversity, which suggests recent emergence and successful geographic spread (5,7–9).

Tularemia was first reported in Spain in 1997, when it caused one of the largest outbreaks in humans ever described (10). Overall, 559 cases were confirmed during June 1997–April 1998 in 10 provinces. The outbreak was associated with hunting and handling of hares (Lepus europaeus) in northwestern Spain. The most common clinical form was ulceroglandular tularemia (55.4%); glandular (15.3%) and typhoid forms (6.6%) of the disease also occurred frequently. A second major human outbreak in humans, which affected 507 persons, occurred in the same area in 2007 and 2008, but in a different epidemiologic context. Its timing coincided with a population peak of the common vole (Microtus arvalis), and the most frequent clinical forms of the disease were typhoidal and pneumonic (65% of the cases), which is consistent with infection being acquired through inhalation of F. tularensis (11–13). Sporadic tularemia cases and small outbreaks were reported during 2000–2006 in the interval between the 2 major outbreaks in northwestern Spain (13,14).

We report comparative genetic analyses of F. tularensis cultured from humans and animals during the 2 main tularemia outbreaks (1997–1998 and 2007–2008). We also studied F. tularensis isolates circulating in Spain during outbreaks with different epidemiologic patterns and investigated whether reemergence of the pathogen after 10 years of no epidemiologic activity was caused by introduction of exotic strains or by establishment of the pathogen in local reservoirs of infection.

Acknowledgment

This study was supported by project PEP 2009/1422 of the Junta de Castilla y León (Spain). A.J. was supported by the Laboratory for Molecular Infection Medicine Sweden within the Nordic European Molecular Biology Laboratory Partnership for Molecular Medicine and by the Västerbotten County Council.

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Figures

• Figure 1. Genetic relationships among 108 Francisella tularensis isolates based on comparison of pulsed-field gel electrophoresis (PFGE) profiles obtained with restriction enzyme BamHIThe dendrogram was produced by using a Dice similarity...
• Figure 2. Minimum-spanning tree based on multilocus variable number of tandem repeat analysis (MLVA) genotypes, showing genetic relationships among 98 Francisella tularensissubspholarctica isolates from Spain (white circles), 10 Ftularensis subspholarctica...
• Figure 3. A) Geographic distribution of 98 Francisella tularensis subspholarctica isolates from SpainColor codes represent geographic origin, and black circles represent number of isolates recovered per provinceB) Minimum-spanning tree based on...
• Figure 4. Minimum-spanning tree based on multilocus variable number tandem repeat (MLVA) analysis of genotypes showing genetic relationships among 98 Francisella tularensissubspholarctica isolates from Spain with reference to 2 human...

Table

• Table. Multilocus variable number tandem repeat analysis of 98 Francisella tularensis isolates from Spain and 11 reference isolates

Technical Appendix

• Technical Appendix. Information on Francisella tularensis subspholarctica isolates used in this study and results of PFGE and MLVA.  27 KB 

Suggested citation for this article: Ariza-Miguel J, Johansson A, Fernández-Natal MI, Martínez-Nistal C, Orduña A, Rodríguez-Ferri EF, et al. Molecular investigation of tularemia outbreaks, Spain, 1997–2008. Emerg Infect Dis [Internet]. 2014 May [date cited].http://dx.doi.org/10.3201/eid2005.130654

DOI: 10.3201/eid2005.130654