Application of molecular techniques in the study of natural infection of Leishmania infantum vectors and utility of sandfly blood meal digestion for epidemiological surveys of leishmaniasis.

Epidemiological studies on the distribution of leishmaniasis caused by Leishmania infantum Nicolle, 1908 (Kinetoplastida: Trypanosomatidae) have been based principally on serological surveys of the canine reservoir. This methodology is useful due to the facility of sampling, the rapidity in obtaining results, its consistency and because it allows the detection of heterogeneous foci of canine leishmaniasis (CanL) even in small areas. Other investigations have analysed Leishmania parasitism in sandflies (Diptera: Psychodidae: Phlebotominae) by using classical dissection techniques. These techniques allow the vector species to be incriminated in different foci, although they suffer from being very time consuming. Lately, studies in this field are increasingly using molecular techniques, which are faster and easier to perform. In the present work, we applied a nested-PCR in a study of natural infection of sandflies by Leishmania in three isolated farms where serological data on canine leishmaniasis of local dogs were also obtained. The analysis allowed the detection of 38.7% of females with positive nested-PCR (78%, 18% and 0%, respectively, in the different isolated farms). The positive Leishmania DNA samples were genotyped and identified as L. infantum. The results of this work provide new data for the vectorial capacity of Phlebotomus ariasi in a Pyrenean area, which can be considered at risk of becoming a new focus of CanL. The females with positive nested-PCR displayed blood in the midgut at different degrees of digestion, and/or were gravid. According to the multivariate logistic regression analysis, the risk of nested-PCR-positivity increased significantly with the degree of blood digestion (OR = 1.3; P value = 0.025). The Phlebotomus species and the presence of eggs were not statistically associated with nested-PCR positivity (P value of >0.05). The correlation of positive nested-PCR results with the presence of seropositive dogs in the farm confirms the utility of this technique in the study of the distribution and intensity of leishmaniasis foci. Also, the importance of sandfly blood-meal digestion for epidemiological surveys of leishmaniasis foci has been demonstrated.