DistinctLeishmania infantumStrains Circulate in Humans and Dogs in the Emilia–Romagna Region, Northeastern Italy

Abstract

Human leishmaniasis is an emerging problem in Italy and is on the increase in the Emilia-Romagna region, northeastern part of the country. Nevertheless, studies dealing with the molecular characterization of Leishmania spp. circulating in these areas are limited. In the present work, we explored the genetic polymorphism of Leishmania isolates from 28 cases of canine leishmaniasis and three cases of human visceral leishmaniasis (VL), which occurred in 2013-2014 in the Emilia-Romagna region. The characterization was carried out in comparison with nine human isolates of Leishmania from other VL endemic Italian regions and two reference strains. Nucleic acid from 31 Leishmania-positive phlebotomine sandfly pools, sampled in 2012-2013 in the Emilia-Romagna region, were also evaluated. DNA amplification and sequencing of the ribosomal internal transcribed spacer-1 and of a repetitive nuclear region on chromosome 31 were carried out for genotyping. Two size polymorphic targets were also analyzed by PCR, the cpb E/F-gene and the k26-gene. Altogether, the analysis showed the circulation of different Leishmania infantum genotypes in the Emilia-Romagna region: two genotypes found in dogs from public kennels were similar to VL isolates from other Italian regions, whereas a third genotype was detected in VL cases of the Emilia-Romagna region and in all but one of the sandfly pools. The combined molecular tools applied in this study can constitute a helpful support for parasite tracking (e.g., in outbreak investigations) and for a better understanding of the epidemiological evolution of leishmaniasis in northeastern Italy.

Multilocus microsatellite typing (MLMT) reveals host-related population structure in Leishmania infantum from northeastern Italy

“…DNA from all isolates was obtained as previously described [ 9 ]. DNA from canine bone marrow/lymph node aspirates and from sand fly samples was extracted using Qiagen DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, and the DNA was eluted in a final volume of 100 μl of elution buffer.…”

“…Twenty-five of the 65 samples included in the study were submitted to species identification by amplification and sequencing of the Internal Transcribed Spacer-1 (ITS-1) region according to El Tai et al [ 21 ], and following the protocols described by Rugna et al [ 9 ]. The results were compared to those of the other 40 samples that had been previously identified as L .…”

“…The evaluation of genetic markers, such as the repetitive nuclear region on chromosome 31, the cpb E/F-gene and the k26 -gene led to the observation that L . infantum strains circulating in the E-R region exhibit genetic polymorphism [ 9 ]. Molecular markers capable of resolving genetic differences to strain level are useful to understand parasite’s biology and dynamics of disease [ 10 ] and their employment can generate molecular epidemiology data, which may provide crucial information for public health authorities.…”

Isolation and Molecular Typing of Leishmania infantum from Phlebotomus perfiliewi in a Re-Emerging Focus of Leishmaniasis, Northeastern Italy

“…Sand flies were collected utilizing a carbon dioxide baited trap that operated overnight in a single site in the Valsamoggia municipality, Bologna province (latitude 44.480322 • , longitude 11.084237 • ) ( Figure 1). The sampling was started according to the appearance of the first Leishmania-positive pool, as detected in the previous surveillance seasons [20]. Collected samples were taken to the laboratory and sorted by the day of sampling.…”

“…When protozoa were detected by microscopy, isolation was performed by employing Evans modified Tobie's medium containing 15% defibrinated rabbit blood. Species identification of the isolated protozoa was performed by amplification and sequencing of the ribosomal DNA Internal Transcribed Spacer-1 (ITS-1) region of 320bp, using the primer pair LITSR/ L5.8S [25], and following the previously described protocols [20], (A.N. : MG969403).…”