Temporal dynamics and Leishmania infantum infection prevalence of Phlebotomus perniciosus (Diptera, Phlebotominae) in highly endemic areas of visceral leishmaniasis in Tunisia

Abstract

Phlebotomus perniciosus is one of the major vectors of Leishmania infantum in the Mediterranean basin. The aim of this work was (i) to provide information about abundance and temporal dynamics of this Larroussius species in a hot spot area of visceral leishmaniasis in Tunisia, (ii) to detect L. infantum DNA in wild caught female sandflies and (iii) to measure Phlebotomus perniciosus infection rate throughout the active season. Sandflies were collected monthly during one year using CDC miniature light-traps in house and in animal shelters. Male specimens were identified at species level according to morphological characters. Female specimens were conserved individually for molecular study. Leishmania infection was tested by kinetoplast DNA real-time PCR and ITS-1 PCR-sequencing. Subsequent sandfly species identification of infected specimens was done by mitochondrial cytochrome b sequencing. In one year period, overall 4,441 specimens (2230 males and 2211 females) were collected. Sandfly activity started in end-April and ended in early-November. Mean sandfly density in house was significantly lower than in animal shelters (51 ± 50 versus 504 ± 460 sandflies /CDC night, p<0.05). However, a higher proportion of females was found in house (58.4% versus 49.2%, p<0.001). Based on species identification of male specimens, Phlebotomus perniciosus was the dominant species (56% of the whole male sandfly fauna, p<0.0001). It showed two peaks of density in the active season, a sharp one in early May and a higher long lasting one from end-July to end-September. DNA was extracted from 190 female specimens randomly sampled and corresponding to 96 specimens from house and 94 from animal shelters. Twenty four female sandfly were infected by Leishmania infantum. All infected specimens were recognized as Phlebotomus perniciosus. Leishmania infantum infection rate in female sandflies was 2.3 fold higher in house than in animal shelters (17.7% versus 7.4%, p<0.05). In house, estimated number of infected specimens was the highest at the end of the active season. Abundance, dynamics of density and Leishmania infantum infection prevalence of Phlebotomus perniciosus in Tunisian hot spot of visceral leishmaniasis highlight the major role of this Phlebotominae species in L. infantum transmission.

Fig 1. Geographical situation and landscape of sample collection sites.

A: The map of Tunisia shows the governorate of Zaghouan (shaded grey) with a focus on the study site location ("El Khadhra"). B and C: two photographs show the house with close semi-open air shelters where are kept animals (mainly sheep). CDC traps were set in house and in animal shelters.

Fig 2. Leishmania identification by kDNA real time PCR.

A: Standard curve obtained from serial dilutions of Leishmania DNA expressed as the number of parasites per reaction tube. The standard curve was established from Leishmania DNA extracted from 10⁶ L. infantum promastigotes. One μl of serial dilutions, ranging from 1000 (P1) to 0.01 parasites (P6) was introduced into reaction tubes. P5 (0.01 parasites per μl) showed a Ct = 32.8. B: Real time PCR amplification curves showing qPCR positive sandfly female specimen (F) as well as negative sandfly male specimen (M). Amplification plots obtained from the serial dilution of Leishmania DNA (P1 to P6) are also shown.

Fig 3. Densities of total sandfly and Phlebotomus perniciosus male specimens according to period of capture.

Fig 4. Neighbor-joining tree based on 305 aligned base pairs of the ITS1gene.

The sequences from sandflies collected during this study (GenBank accession numbers MF597933 and MF597934) were compared to L. infantum, L. tropica and L. major ITS1 sequences available in GenBank. The percent bootstrap values are indicated on the branches.

Fig 5. In house male and female sandfly densities and percentage of infected females according to period of capture.

Fig 6. Neighbor-joining tree based on 279 aligned base pairs of the cytochrome b mitochondrial DNA (mtDNA).

The sequences from sandflies collected during this study (KHF8 and KHF12) were compared to Mediterranean haplotypes of P. perniciosus and P. longicuspis available in GenBank. The percent bootstrap values are indicated on the branches.