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Introduction and Objectives: Dipeptidyl peptidase III (DPPIII) member of M49 peptidase family is a zinc-dependent metallopeptidase that cleaves dipeptides sequentially from the N-terminus of its substrates. It is absent in trypanosomes, however, in Leishmania, it was reported that the DPPIII with other peptidases, could be key to the parasites' growth and survival. In a previous study, we used a coding sequence annotated as DPPIII to develop a PCR assay that is specific to dermotropic Old World (OW) Leishmania species. Thus, our objective was to further assess use of this gene to identify Leishmania species or to study their evolution. Material and Methods: Orthologous DDPIII genes were searched in all Leishmania genomes and aligned to design primers and to search restriction enzymes. A PCR-RFLP scheme was designed and tested on 51 OW typed Leishmania strains belonging to 10 Leishmania species (L. major (L.m), L. infantum (L.i), L. tropica (L.t), L. donovani (L.d), L. aethiopica (L.aet), L. arabica (L.ar), L. turanica (L.tu), L. chagasi (L.ch), L. archibaldi (L.arch) and L. tarentolae (L.tar)), 7 strains isolated from CL patients, and 50 biological samples taken for diagnosis of suspected Tunisian CL patients. Seventy-one PCR products were further studied by sequence analysis using MEGA and DNA sp softwares to infer evolution and phylogenetic relationship of studied species and strains. Results: DPPIII gene is present in all genomes. Inter- and intra-specific genetic diversity was used to develop universal amplification of a 662bp fragment followed by a double digestion with HaeIII and KpnI. Overall, this PCR-RFLP yielded distinct profiles for each of the species L.m, L.t, L.aet, and L.tar. L.d, L.arch and L.ishared their profile with L.ara and L.tur. Sequence analysis showed 145/589 variable sites (116 being parsimony informative), high haplotype diversity (H=0.81; SD=0.026), and low nucleotide diversity (p=0.04; SD=0.005). Test of natural selection (dN/dS=4.7, Fu and Li's D test (2.79; p<0.02)) inferred a positive selection confirming importance of DPPIII for parasite adaptation to its environment. Phylogenetic relationships confirmed occurrence of 6 clusters congruent to L.m, L.t, L.aet, L.ar, L.tu, L. tar species, and one to the L.i, L.d and L.arch group of species. Conclusion: DPPIII gene is suitable to investigate Leishmania epidemiology and evolution and can contribute to timely disease control. [ABSTRACT FROM AUTHOR]

Introduction and Objectives: Rodents and dogs are the confirmed leishmaniases reservoir hosts in Tunisia. Recently, we described hedgehog Leishmania (L.) major and L. infantum infection in an L. infantum endemic area in the North-West. In order to assess if the observation could extend to other endemic areas and to highlight the potential role of hedgehogs as reservoir, we aimed here at investigating their Leishmania infection in different foci in Tunisia located along a North-South transect, during and outside transmission seasons. Material and Methods: Twelve hedgehog specimens were trapped from 3 endemic foci. DNA extraction were made from different samples and swabs taken from different body parts and fluids (N=104). Smears were made from viscera of 6 animals for amastigote search upon cytologic analysis. Leishmania infection was assessed using 3 PCR assays targeting repeated DNA fragments using specific primers. Leishmania species identification was inferred from RFLP analysis and confirmed using sequence analyses and phylogeny. Results and Conclusions: Based on morphological criteria, 2 hedgehogs' species, Atelerix algirus and Paraechinus aethiopicus were identified. Cytologic analysis showed presence of amastigotes in 9/22 samples corresponding to 4 Atelerix algirus specimens. Also, by combining 3 PCR tests targeting repeated DNA fragments using specific primers, all hedgehogs (N=12) showed a Leishmania infection. The infection rates were very high on spleen (91.66%), kidney (91.66%), blood (90.90%), liver (83.33%) and eye swabs (100%). Parasites were also detected in peritoneum. Three hedgehogs were found infected with L. infantum and the only Paraechinus aethiopicus specimen with L. major. A mixed L. major and L. infantum infection was identified in 8 animals, while the last one also had an L. tropica infection. Interestingly, 2 animals had skin lesions infected with L. major while all others appeared asymptomatic. There was a correlation between infected status and epidemiological profiles of the localities. Sequences and phylogeny indicated micro-heterogeneity and lack of correlation with sampling, season, or localities. We confirmed natural infection of Atelerix algirus and originally of Paraechinus aethiopicus in Tunisia. High rate of asymptomatic infection, parasitemia, proximity to transmission cycles, epidemiological patterns of infection together with hedgehogs' abundance, lifespan and lifestyle corroborate the hypothesis they constitute reservoir hosts for leishmaniases. [ABSTRACT FROM AUTHOR]