Immunity to vector saliva is compromised by short sand fly seasons in endemic regions with temperate climates.

Individuals exposed to sand fly bites develop humoral and cellular immune responses to sand fly salivary proteins. Moreover, cellular immunity to saliva or distinct salivary proteins protects against leishmaniasis in various animal models. In Tbilisi, Georgia, an endemic area for visceral leishmaniasis (VL), sand flies are abundant for a short period of ≤3 months. Here, we demonstrate that humans and dogs residing in Tbilisi have little immunological memory to saliva of P. kandelakii, the principal vector of VL. Only 30% of humans and 50% of dogs displayed a weak antibody response to saliva after the end of the sand fly season. Likewise, their peripheral blood mononuclear cells mounted a negligible cellular immune response after stimulation with saliva. RNA seq analysis of wild-caught P. kandelakii salivary glands established the presence of a typical salivary repertoire that included proteins commonly found in other sand fly species such as the yellow, SP15 and apyrase protein families. This indicates that the absence of immunity to P. kandelakii saliva in humans and dogs from Tbilisi is probably caused by insufficient exposure to sand fly bites. This absence of immunity to vector saliva will influence the dynamics of VL transmission in Tbilisi and other endemic areas with brief sand fly seasons.

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

BACKGROUND: 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.

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.

Epidemiologic aspects of an emerging focus of visceral leishmaniasis in Tbilisi, Georgia.

BACKGROUND: Over the last 15 years, visceral leishmaniasis (VL) has emerged as a public health concern in Tbilisi, the capital of Georgia. METHODOLOGY/PRINCIPAL FINDINGS: Seroepidemiological surveys were conducted to determine the prevalence and incidence of infection in children and dogs within the main focus of VL, and to identify risk factors associated with human infection. Of 4,250 children investigated, 7.3% were positive by direct agglutination test in a baseline survey; an apparent incidence rate of 6.0% was estimated by one year follow-up. None of the seropositive children progressed to VL during the survey. Increased seropositivity at one year was predicted by presence at baseline of clustered flying insects (OR = 1.49; P = 0.001), perceived satisfactory sanitation (OR = 1.65; P<0.001), stray dogs (OR = 1.33; P = 0.023), and by persistent fever during the 6 months prior to baseline survey (OR = 14.2; P<0.001). Overall, 18.2% (107/588) of domestic and 15.3% (110/718) of stray dogs were seropositive by the rk39 dipstick test. Clinical VL signs were found in 1.3% of domestic and 2.9% of stray, seropositive dogs. Parasites isolated from human and dog samples were identified by PCR and phylogenetic analysis of the Leishmania 70 kDa heat-shock protein (HSP70) gene as Leishmania infantum. CONCLUSIONS/SIGNIFICANCE: There is an active focus of L. infantum transmission in Tbilisi with a high prevalence of human and canine infections.