Unraveling Leptospira spp genospecies from azorean rodents through new molecular tools: New Leptospira genus species in Azores

Eurolept20 I2 Poster presentations
PP2
UNRAVELING Leptospira spp. GENOSPECIES FROM AZOREAN RODENTS THROUGH NEW MOLECULAR TOOLS
Nunes M.1,2, Branca R.1, Vieira M. LY
1 Unidade de Ensino e lnvestiga9iio em Microbiologia Medica, Grupo de Leptospirose e Borreliose de Lyme, lnstituto de
Higiene e Medicina Tropical (IHMT), Universidade Nova de Lisboa (UNL), Lisboa, Portugal
2 Centro de Recursos Microbiol6gicos (CREM), Faculdade de Ciencias e Tecnologia, UNL, Lisboa, Portugal

Leptospirosis is a bacterial zoonosis that affects populations worldwide, mainly in tropical and temperate areas. The Azores Archipelago, a group of nine inhabited Portuguese islands in the North Atlantic Ocean, have been known as an endemic leptospirosis area since 1993; it was recognized as a major
health problem soon thereafter. The temperate and wet climate of these islands with mild winters and the high number of infected rodents favor the survival and transmission of these spirochetes. Conventional identification and diagnostic assays for Leptospira are based on serological methods. Although considerable difficulties are encountered in the classification ofleptospires at serovar level, this system remains as a standard to intra-species differentiation. Serovar identification has become faster with the introduction of genotyping methods that can serve as supplementary or alternative system to identify a set of serovars causing human and animal disease. These typing methods can identify differences at the serovar or serogroup level. Numerous PCR-based techniques such as random amplified polymorphic DNA (RAPD) and multiple-locus variable-number tandem repeat analysis (MLVA) have been developed and evaluated to study the population structure of bacteria and to charact~rize isolates even
from monomorphic populations. The RAPD and MLVA typing of pathogenic leptospires could permit the quick and reliable identification of strains during outbreaks. The aim of this study_$aS to genotype a large number of Leptospira isolates obtained from kidney from live-trapped roden1s on Sao Miguel
(SM, n=988) and Terceira (T, n=868) islands, through PCR-based assays. 1856 rodents were trapped and kidney cultured in EMJH media. The obtained isolates were genotyped by RAPD with Irep 1 primer and MLVA with eight variable-number tandem repeat (VNTR) loci. Leptospires were isolafodcin 60% (1102/1856) of rodents. 715 isolates were genotyped by RAPD and two species were identified as L. borgpetersenii, Ballum serogroup (82%) and L. interrogans sensu stricto, Ictbr~ha~morrhagiae serogroup (18%), both with a similar distribution pattern regarding the reservoir species. The VNTRs
analysis, specific for L. borgpetersenii species, was applied in 95 (9%) samples as an attempt to differentiate
at serovar level. With this approach it was possible to distinguish the serovar Castellonis from Ballum and Arborea serovars. However, so far, in the "new" isolates, Castellonis serovar was not identified. The RAPD approach proved to be a simple and rapid typing tool of Leptospira spp., at the serogroup level. Concerning the VNTR's analysis, this tool differentiates the L. borgpetersenii
serovars of Ballum serogroup. In conclusion these approaches demonstrated to be powerful tools in epidemiological studies and support the understanding the human leptospirosis prevalence in Azores.
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