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Volume 17, Number 9—September 2011
Letter

Pathogenic Leptospira spp. in Wild Rodents, Canary Islands, Spain

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To the Editor: Leptospirosis is a major emerging infectious disease with a worldwide distribution (1). It is a systemic disease of humans and domestic animals (2). Regarded globally as a zoonosis because it is acquired by humans from contact with animals or from water contaminated with the urine of infected animals, it is presumed to be the most widespread zoonotic disease in the world (1,2). Species such as mice (Mus spp.) and rats (mainly Rattus norvegicus and R. rattus) serve as reservoirs for their host-related serovars (3).

Human patients usually exhibit a nonspecific self-limiting febrile illness; however, in 5%–10% of cases, severe forms of the disease develop, including Weil disease and severe pulmonary hemorrhagic syndrome. Case-fatality rates for Weil disease and severe pulmonary hemorrhagic syndrome are >10% and >74%, respectively (4,5).

Because leptospirosis has been found in humans in the Canary Islands (J. Alcoba-Flores, pers. comm.), detecting carrier animals is vital to the understanding of enzootic and epizootic leptospirosis in this environment. We examined the possible role of the rodent species found in the Canary Islands in the transmission of this pathogen to determine the risk for humans in these islands.

A total of 149 wild rodents (74 R. rattus and 75 Mus domesticus) were captured during 20009–2010 from 4 of the Canary Islands (Tenerife, El Hierro, La Gomera, Lanzarote). Urinary bladders of the animals were collected and preserved in 100% ethanol. Genomic DNA was extracted by using the Fast DNA Kit (BIO 101 Systems: MP Biomedicals, Santa Ana, CA, USA).

The lipL32 fragment (497 bp), which is present only in pathogenic leptospires, was amplified according to the method of Bomfim et al. (6) by using a MyCycler thermocycler (Bio-Rad, Hercules, CA, USA). L. interrogans serovar Icterohemorragiae (RGA strain) was used as a positive control.

Twenty-two samples were positive for Leptospira spp., indicating a general prevalence of 14.8% in the rodents. Although the prevalence was higher in rats (20.3%) than in mice (9.3%) (Table), the difference was not significant (χ2 test). Positive samples were obtained from all the studied islands and for both host species in all of them (Table), without significant differences in the prevalences between host species or between islands.

To confirm the amplified products belonged to pathogenic Leptospira spp., we sequenced some amplicons. Sequencing reactions were performed for both strands at the University of La Laguna Genomic Service. When the sequences were compared, 2 different sequences were obtained. The first sequence, L19 (GenBank accession no. HQ231747), from rats, clustered with L. interrogans serovar Copenhageni (GenBank accession no. AE016823) and different serovars of the same species by BLAST (99% identity). Previous results associate L. interrogans serovar Copenhageni with Rattus spp (7). However, the sequence obtained from mice (GenBank accession no. HQ231748), L47, showed a 100% BLAST identity with L. borgpetersenii (GenBank accession nos. DQ320625.1 and DQ286415.1).

New and published Leptospira sequences were aligned with the multiple alignment program ClustalW in MEGA3.1 (8), and minor corrections were made manually. The alignment for the 497-bp fragment starts at nt position 208, with respect to the complete sequence of the lipL32 (AY609332), and ends at nt position 705.

Phylogenetic relationships were inferred by using the neighbor-joining distance method with MEGA3.1. At least 1,000 bootstrap replicates were used to infer statistical support at branch nodes. The consensus tree yielded 3 monophyletic groups clearly separated by high bootstrap values. The first clade was formed by L. interrogans, L. kirschneri, and L. noguchii (93% bootstrap value). The sequence L19 was included in the L. interrogans node (92% bootstrap value). The second clade included L. borgpetersenii and L. weilii as a monophyletic group (97% bootstrap value). The sequence L47 clustered with L. borgpetersenii DQ286415 with a high bootstrap value (82%). These results are in accordance with those obtained by Haake et al. (9) based on lipL32. Finally, L. santarosai sequences formed the third separate clade (100% bootstrap) (data not shown).

Although the method we used does not enable specific identification, determining the most similar species by BLAST is needed for control programs. L. interrogans serovar Copenhageni is the predominant infecting serovar among patients with severe leptospirosis (7), and L. borgpetersenii is also commonly acquired from mice.

On the basis of these findings, the global distribution of Leptospira spp. must be revised to include the Canary Islands, with rodents as natural hosts. Because pathogenic Leptospira spp. were detected on every island studied and in both analyzed species, R. rattus and M. domesticus, the distribution of this pathogen likely extends to even the islands not studied. The high incidence found suggests that rodents play a role in transmission of human leptospirosis. Further studies are needed to identify other possible reservoir hosts and to determine the risk areas for acquiring pathogenic leptospires in the Canary Islands.

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Acknowledgments

We thank the Excmos Cabildos Insulares of Tenerife, La Gomera, El Hierro, and Lanzarote.

This work was supported by projects Red de Investigación Colaborativa de Centros de Enfermedades Tropicales (RD06/0021/0005) and CGL 2009-07759BOS.

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Pilar ForondaComments to Author , Aarón Martin-Alonso, Borja del Castillo-Figueruelo, Carlos Feliu, Horacio Gil, and Basilio Valladares

Author affiliations: Author affiliations: University of La Laguna, Canary Islands, Spain (P. Foronda, A. Martin-Alonso, B. del Castillo-Figueruelo, B. Valladares); University of Barcelona, Barcelona, Spain (C. Feliu); Instituto de Salud Carlos III, Madrid, Spain (H. Gil)

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References

  1. Levett  PN. Leptospirosis. Clin Microbiol Rev. 2001;14:296326. DOIPubMed
  2. Adler  B, de la Pena Moctezuma  A. Leptospira and leptospirosis. Vet Microbiol. 2010;140:28796. DOIPubMed
  3. Bharti  AR, Nally  JE, Ricaldi  JN, Matthias  MA, Diaz  MM, Lovett  MA, Leptospirosis: a zoonotic disease of global importance. Lancet Infect Dis. 2003;3:75771. DOIPubMed
  4. McBride  AJ, Athanazio  DA, Reis  MG. Ko Al. Leptospirosis. Curr Opin Infect Dis. 2005;18:37686. DOIPubMed
  5. Gouveia  EL, Metcalfe  J, de Carvalho  AL, Aires  TS, Villasboas-Bisneto  JC, Queirroz  A, Leptospirosis-associated severe pulmonary hemorrhagic syndrome, Salvador, Brazil. Emerg Infect Dis. 2008;14:5058. DOIPubMed
  6. Bomfim  MRQ, Barbosa-Stancioli  EF, Koury  MC. Detection of pathogenic leptospires in urine from naturally infected cattle by nested PCR. Vet J. 2008;178:2516. DOIPubMed
  7. de Faria  MT, Calderwood  MS, Athanazio  DA, McBride  AJ, Hartskeerl  RA, Pereira  MM, Carriage of Leptospira interrogans among domestic rats from an urban setting highly endemic for leptospirosis in Brazil. Acta Trop. 2008;108:15. DOIPubMed
  8. Kumar  S, Tamura  K, Nei  M. MEGA3: integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment. Brief Bioinform. 2004;5:15063. DOIPubMed
  9. Haake  DA, Suchard  MA, Kelley  MM, Dundoo  M, Alt  DP, Zuerner  RL. Molecular evolution and mosaicism of leptospiral outer membrane proteins involves horizontal DNA transfer. J Bacteriol. 2004;186:281828. DOIPubMed

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DOI: 10.3201/eid1709.101470

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Table of Contents – Volume 17, Number 9—September 2011

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Pilar Foronda, Institute of Tropical Diseases and Public Health of the Canary Islands, University of La Laguna, Avda. Fco. Sanchez s/n, 38203, Tenerife, Canary Islands, Spain

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Page created: September 06, 2011
Page updated: September 06, 2011
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