Saurian-associated Leishmania tarentolae in dogs: Infectivity and immunogenicity evaluation in the canine model

Abstract

In canine leishmaniosis endemic areas, Leishmania infantum may occur in sympatry with the non-pathogenic Leishmania tarentolae, which is associated to reptiles. The potential infectivity of L. tarentolae for mammals raises questions about the interactions between the two Leishmania species, and the potential cross-immune protection in dogs. This study aimed to assess the outcome of experimental L. tarentolae infection in dogs, determining: i) the anti-L. tarentolae antibody production, ii) the duration of the immunity and cytokine expression, and iii) the possible pathogenic effect in the canine host. Twelve purpose-bred beagle dogs were randomly allocated to three groups (intravenous inoculation, G1; intradermal inoculation, G2; negative control, G3). G1 and G2 dogs were inoculated twice (day 0, day 28) with 108 promastigotes of L. tarentolae strain (RTAR/IT/21/RI-325) isolated from a Tarentola mauritanica gecko. The animals were followed until day 206. Blood, serum, conjunctival swabs and lymph node aspirate samples were collected monthly and bone marrow, liver and spleen biopsies on day 91. Hematological and biochemical parameters were assessed monthly, as well as serology (IFAT and ELISA) and molecular identification of L. tarentolae. Mononuclear cells (PBMC) were obtained to assess the cytokine expression through in vitro stimulation or (re-) infection. Data from this study demonstrated that DNA from L. tarentolae is detectable up to 3 months post-infection, with seroconversion after day 28. Moreover, the non-pathogenic nature of L. tarentolae was confirmed, with a neutral Th1/Th2 polarization, and a possible shift to Th1 phenotype after derived macrophages (re-) infection, as demonstrated by the expression of IFN-gamma. Therefore, L. tarentolae demonstrated a great potential as a surrogate pathogen and/or immune-prophylaxis/immune-therapy against Leishmania infections in dogs and humans.

Fig 1. Molecular detection and quantitation cycle (Cq) values of Leishmania tarentolae in different tissues and timepoints.

Asterisks (*) represent obtained sequences for ITS1 gene.

Fig 2. Anti-Leishmania tarentolae and Leishmania infantum antibody titers at indirect fluorescence immunoassay (IFAT).

Results are shown according to different groups of dogs (G1 intravenous, G2 intradermic and G3 control) and time points.

Fig 3. Specific anti-Leishmania tarentolae IgG antibody levels, detected by in-house enzyme-linked immunosorbent assay (ELISA).

Each bar represents mean ± standard deviation. (A) Asterisk indicates the significant difference as determined after Tukey’s multiple comparisons test (p<0.05 denoted as *, p<0.01 denoted as **, p<0.001 denoted as ***). Dots represent original data points; (B) Symbols indicate mean ± standard deviation.

Fig 4. Relative gene expression of cytokines in peripheral blood mononuclear cells (PBMC).

Stimulation after 24h with Leishmania spp. soluble antigen (LSA) of Leishmania tarentolae (Lt) or Leishmania infantum (Li), or with Concanavalin (Conc; positive control) or unstimulated (medium). Each bar represents mean ± standard deviation.

Fig 5. Relative gene expression (relative units) of pro-inflammatory cytokines IFN-gamma and IL-12 and anti-inflammatory cytokine IL-10.

Data are shown according to each in vitro re-infection condition on G1 (intravenous), G2 (intradermic) and G3 (control) (standard deviation shown).

Fig 6. Study design of the experimental infection with Leishmania tarentolae.

Routes of administration (G1 intravenous, G2 intradermic and G3 control), the type of samples and tissues collected, diagnostic techniques and cytokine expression tests employed. Created in BioRender.