New Insights Into the Transmissibility of Leishmania infantum From Dogs to Sand Flies: Experimental Vector-Transmission Reveals Persistent Parasite Depots at Bite Sites

Abstract

Canine leishmaniasis (CanL) is a chronic fatal disease of dogs and a major source of human infection through propagation of parasites in vectors. Here, we infected 8 beagles through multiple experimental vector transmissions with Leishmania infantum-infected Lutzomyia longipalpis. CanL clinical signs varied, although live parasites were recovered from all dog spleens. Splenic parasite burdens correlated positively with Leishmania-specific interleukin 10 levels, negatively with Leishmania-specific interferon γ and interleukin 2 levels, and negatively with Leishmania skin test reactivity. A key finding was parasite persistence for 6 months in lesions observed at the bite sites in all dogs. These recrudesced following a second transmission performed at a distal site. Notably, sand flies efficiently acquired parasites after feeding on lesions at the primary bite site. In this study, controlled vector transmissions identify a potentially unappreciated role for skin at infectious bite sites in dogs with CanL, providing a new perspective regarding the mechanism of Leishmania transmissibility to vector sand flies.

Keywords: Leishmania infantum; Lutzomyia longipalpis; bite site; canine leishmaniasis; dogs; infectivity; parasite-pickup; reservoir; skin; vector transmission.

Figure 1.

Eight beagles were subjected to 2 transmission clusters composed of 9 and 7 vector transmissions. A, Timeline of transmissions and follow-up. B, Evolution of lesions at bite sites in representative dog 1605 at month 3 M3, M12, and M17 after vector transmission. C, Lesions distal to the bite site in representative dogs 3080, 2912, and 1516. Stars denotes times of xenodiagnoses, spleen and bone marrow aspiration, and blood collection (plasma and peripheral blood mononuclear cells). Arrows point to lesions. Abbreviations: TC1, transmission cluster 1; TC2, transmission cluster 2.

Figure 2.

Pathology and leishmanin test reactions 22 months after experimental vector transmission to beagles. A, Spleen weight as a percentage of body weight (left) and parasite load in homogenized spleen tissue (right). B, The left panel shows leishmanin test indurations measured 48 hours after intradermal injection of soluble Leishmania antigen prior to necropsy. The mean of 2 perpendicular measurements delimiting the induration area is given. A mean induration of ≥5 mm (dotted line) was considered positive. The right panel shows a negative correlation between splenic parasite load and leishmanin test positivity. The solid line represents the mean. C, Parasite loads per lymph node for submandibular, prescapular, and popliteal lymph nodes. Parasite loads were determined by a limiting dilution assay. D, Hematoxylin and eosin–stained sections of the most affected part of the spleen in representative dogs 3080 and 5635. A section from the spleen of an uninfected dog was used as control. Images were taken at the magnification ×40.

Figure 3.

Development of Leishmania-specific immunity in beagles after experimental vector transmission. A, Eight beagles were followed serologically for 22 months by indirect fluorescence antibody testing (IFAT; A) and enzyme-linked immunosorbent assay (ELISA; B). Dotted lines indicate IFAT and ELISA cutoffs. A titer of 1:40 was used as a cutoff for the IFAT-positive response. The mean values ( + 2 SDs) for sera from 6 uninfected dogs were used to calculate the ELISA cutoff. C, Box plot of the ratio of immunoglobulin G2 (IgG2) to IgG1 for the 8 infected dogs. The plus signs denotes the means, the solid lines denote the medians, and the bars represent the 5th–95th percentiles. D, Dog peripheral blood mononuclear cells were stimulated for 6 days with soluble Leishmania antigen or left unstimulated. Supernatants were used to measure cytokine levels for interferon γ (IFN-γ), interleukin 10 (IL-10), interleukin 6 (IL-6), interleukin 12 (IL-12), and interleukin 2 (IL-2), using a 5-plex Luminex assay. Lines represent the mean. Symbols identify each dog. Abbreviation: M, month.

Figure 4.

Correlates of immunity to Leishmania infantum in beagles after experimental vector transmission. A, Leishmania skin test induration correlates positively to levels of interferon γ (IFN-γ). B, Correlation of splenic parasite load with IFN-γ, interleukin 10 (IL-10), and interleukin 2 (IL-2) levels. Symbols identify each dog. Solid lines in scatterplots represent mean values.