Incrimination of Phlebotomus kandelakii and Phlebotomus balcanicus as Vectors of Leishmania infantum in Tbilisi, Georgia

A survey of potential vector sand flies was conducted in the neighboring suburban communities of Vake and Mtatsminda districts in an active focus of visceral Leishmaniasis (VL) in Tbilisi, Georgia. Using light and sticky-paper traps, 1,266 male and 1,179 female sand flies were collected during 2006–2008. Five Phlebotomus species of three subgenera were collected: Phlebotomus balcanicus Theodor and Phlebotomus halepensis Theodor of the subgenus Adlerius; Phlebotomus kandelakii Shchurenkova and Phlebotomus wenyoni Adler and Theodor of the subgenus Larroussius; Phlebotomus sergenti Perfil'ev of the subgenus Paraphlebotomus. Phlebotomus sergenti (35.1%) predominated in Vake, followed by P. kandelakii (33.5%), P. balcanicus (18.9%), P. halepensis (12.2%), and P. wenyoni (0.3%). In Mtatsminda, P. kandelakii (76.8%) comprised over three fourths of collected sand flies, followed by P. sergenti (12.6%), P. balcanicus (5.8%), P. halepensis (3.7%), and P. wenyoni (1.1%). The sand fly season in Georgia is exceptionally short beginning in early June, peaking in July and August, then declining to zero in early September. Of 659 female sand flies examined for Leishmania, 12 (1.8%) specimens without traces of blood were infected including 10 of 535 P. kandelakii (1.9%) and two of 40 P. balcanicus (5.0%). Six isolates were successfully cultured and characterized as Leishmania by PCR. Three isolates from P. kandelakii (2) and P. balcanicus (1) were further identified as L. infantum using sequence alignment of the 70 kDa heat-shock protein gene. Importantly, the sand fly isolates showed a high percent identity (99.8%–99.9%) to human and dog isolates from the same focus, incriminating the two sand fly species as vectors. Blood meal analysis showed that P. kandelakii preferentially feeds on dogs (76%) but also feeds on humans. The abundance, infection rate and feeding behavior of P. kandelakii and the infection rate in P. balcanicus establish these species as vectors in the Tbilisi VL focus

Blood meal sources of <i>Phlebotomus</i> sand flies from VL case sites in Tbilisi, Georgia.

<p>Blood meal sources of <i>Phlebotomus</i> sand flies from VL case sites in Tbilisi, Georgia.</p

Natural <i>Leishmania</i> infections in wild-caught sand flies from VL case sites in Tbilisi, Georgia.

<p>Natural <i>Leishmania</i> infections in wild-caught sand flies from VL case sites in Tbilisi, Georgia.</p

Molecular characterization of <i>Leishmania</i> isolates from wild-caught sand flies in the Tbilisi VL focus.

<p>(A) Lanes 1 and 11, DNA size marker (100 base pair ladder); lane 2, <i>L. major</i> (MHOM/IL/80/Friedlin); lane 3, <i>L. infantum</i> (MHOM/ES/00/UCM-1); lanes 4–8, <i>Leishmania</i> isolates from <i>P. kandelakii</i>; lane 9, <i>Leishmania</i> isolate from <i>P. balcanicus</i>; lane 10, negative control. bp = basepairs. (B) Phylogenetic analysis of the <i>Leishmania</i> 70 kDa heat-shock protein (HSP70) gene. The sequences are represented by the <i>Leishmania sp.</i>, country of origin in parentheses and GenBank nucleotide accession numbers. Node values indicate branch support.</p

Diversity and relative abundance of sand fly species in Vake and Mtatsminda districts.

<p>Collections were made from May 2006 through September 2008. M = male; F = female; T = species total.</p

Seasonal distribution of sand fly species collected from Vake and Mtatsminda districts, 2006–2008.

<p>Seasonal distribution of sand fly species collected from Vake and Mtatsminda districts, 2006–2008.</p

Map of the visceral leishmaniasis (VL) focus in Tbilisi, Georgia.

<p>The districts of Vake, Mtatsminda and Krtsanisi are indicated. • = human VL case sites. Inset shows the geographical location of Georgia relative to neighboring countries and the position of its capital Tbilisi.</p

Diversity and relative abundance of sand fly species collected in Vake and Mtatsminda districts, 2006–2008. LT = light trap; ST = sticky trap.

<p>Diversity and relative abundance of sand fly species collected in Vake and Mtatsminda districts, 2006–2008. LT = light trap; ST = sticky trap.</p

Seasonal Dynamics of Phlebotomine Sand Fly Species Proven Vectors of Mediterranean Leishmaniasis Caused by Leishmania infantum

International audienceBackground: The recent geographical expansion of phlebotomine vectors of Leishmania infantum in the Mediterranean subregion has been attributed to ongoing climate changes. At these latitudes, the activity of sand flies is typically seasonal; because seasonal phenomena are also sensitive to general variations in climate, current phenological data sets can provide a baseline for continuing investigations on sand fly population dynamics that may impact on future scenarios of leishmaniasis transmission. With this aim, in 2011–2013 a consortium of partners from eight Mediterranean countries carried out entomological investigations in sites where L. infantum transmission was recently reported.Methods/Principal Findings: A common protocol for sand fly collection included monthly captures by CDC light traps, complemented by sticky traps in most of the sites. Collections were replicated for more than one season in order to reduce the effects of local weather events. In each site, the trapping effort was left unchanged throughout the survey to legitimate inter-seasonal comparisons. Data from 99,000 collected specimens were analyzed, resulting in the description of seasonal dynamics of 56,000 sand flies belonging to L. infantum vector species throughout a wide geographical area, namely P. perniciosus (Portugal, Spain and Italy), P. ariasi (France), P. neglectus (Greece), P. tobbi (Cyprus and Turkey), P. balcanicus and P. kandelakii (Georgia). Time of sand fly appearance/disappearance in collections differed between sites, and seasonal densities showed variations in each site. Significant correlations were found between latitude/mean annual temperature of sites and i) the first month of sand fly appearance, that ranged from early April to the first half of June; ii) the type of density trend, varying from a single peak in July/August to multiple peaks increasing in magnitude from May through September. A 3-modal trend, recorded for P. tobbi in Cyprus, represents a novel finding for a L. infantum vector. Adults ended the activity starting from mid September through November, without significant correlation with latitude/mean annual temperature of sites. The period of potential exposure to L.infantum in the Mediterranean subregion, as inferred by adult densities calculated from 3 years, 37 sites and 6 competent vector species, was associated to a regular bell-shaped density curve having a wide peak center encompassing the July-September period, and falling between early May to late October for more than 99% of values. Apparently no risk for leishmaniasis transmission took place from December through March in the years considered. We found a common pattern of nocturnal females activity, whose density peaked between 11 pm and 2 am.Conclusions: Despite annual variations, multiple collections performed over consecutive years provided homogeneous patterns of the potential behavior of leishmaniasis vectors in selected sites, which we propose may represent sentinel areas for future monitoring. In the investigated years, higher potential risk for L. infantum transmission in the Mediterranean was identified in the June-October period (97% relative vector density), however such risk was not equally distributed throughout the region, since density waves of adults occurred earlier and were more frequent in southern territories

Seasonal density of <i>Leishmania infantum</i> vectors recorded in the Mediterranean region in the 2011–2013 period.

<p>Densities of 6 vector species pooled from 37 sites are shown separately for each year and for the whole period. For a better presentation of data, density values are multiplied by 1000; annual and 3-year densities are shown on different scale of values.</p