Multilocus microsatellite typing (MLMT) reveals host-related population structure in Leishmania infantum from northeastern Italy

Abstract

Background: Visceral leishmaniasis (VL) caused by Leishmania infantum is an ongoing health problem in southern Europe, where dogs are considered the main reservoirs of the disease. Current data point to a northward spread of VL and canine leishmaniasis (CanL) in Italy, with new foci in northern regions previously regarded as non-endemic.

Methodology/principal findings: Multilocus microsatellite typing (MLMT) was performed to investigate genetic diversity and population structure of L. infantum on 55 samples from infected humans, dogs and sand flies of the E-R region between 2013 and 2017. E-R samples were compared with 10 L. infantum samples from VL cases in other Italian regions (extra E-R) and with 52 strains within the L. donovani complex. Data displayed significant microsatellite polymorphisms with low allelic heterozygosity. Forty-one unique and eight repeated MLMT profiles were recognized among the L. infantum samples from E-R, and ten unique MLMT profiles were assigned to the extra E-R samples. Bayesian analysis assigned E-R samples to two distinct populations, with further sub-structuring within each of them; all CanL samples belonged to one population, genetically related to Mediterranean MON-1 strains, while all but one VL cases as well as the isolate from the sand fly Phlebotomus perfiliewi fell under the second population. Conversely, VL samples from other Italian regions proved to be genetically similar to strains circulating in dogs.

Conclusions/significance: A peculiar epidemiological situation was observed in northeastern Italy, with the co-circulation of two distinct populations of L. infantum; one population mainly detected in dogs and the other population detected in humans and in a sand fly. While the classical cycle of CanL in Italy fits well into the data obtained for the first population, the population found in infected humans exhibits a different cycle, probably not involving a canine reservoir. This study can contribute to a better understanding of the population structure of L. infantum circulating in northeastern Italy, thus providing useful epidemiologic information for public health authorities.

Fig 1. Geographical distribution of human, canine and sand fly Leishmania-positive samples, 2013–2017, Emilia-Romagna region (northeastern Italy).

Colors depict the subpopulations inferred by STRUCTURE analysis: yellow for subPopA1; orange for subPopA2; green for subPopB1; brown for subPopB2; light blue for subPopB3; white for samples not submitted to cluster analysis. More than one sample per area are shown by numbers inside the icon. Map generated with Quantum-GIS (https://www.qgis.org/it/site/).

Fig 2. Estimated population structure for the 52 L. infantum strains from the Emilia-Romagna region (northeastern Italy), as inferred by STRUCTURE software on the basis of data for 15 microsatellite markers.

Each strain is represented by a single vertical line divided into K colors, where K is the number of populations that were assumed. Each color represents one population, and the length of the colored segment shows the strain’s estimated proportion of membership (Q) in that population. (A) The derived graph for ΔK shows a peak at K = 2, indicating the existence of two populations in the investigated strain set. (B) The derived graphs for ΔK indicated the existence of two subpopulations within PopA and three subpopulations within PopB.

Fig 3. Spatial distribution of 52 L. infantum strains from the Emilia-Romagna region (northeastern Italy) by factorial correspondence analysis (FCA).

Each square represents one microsatellite profile. The designations PopA (red circle), PopB (blue circle), subPopA1 (yellow circle), subPopA2 (orange circle), subPopB1 (green circle), subPopB2 (brown circle) and subPopB3 (light blue circle) correspond to the populations and subpopulations defined by STRUCTURE analysis as shown in Fig 2.

Fig 4. Bayesian phylogenetic tree (cladogram) obtained by the Sainudiin model [29] using data of 14 coincident microsatellite loci for the 62 L. infantum strains from Italy, 49 MLMT profiles available in literature and 3 WHO reference strains.

Posterior probabilities > 0.75 are showed near the nodes. Strains representing the different microsatellite profiles are listed in S1 and S2 Tables. Strains designations specify, respectively, the laboratory code, the zymodeme (ND, not defined), the geographical origin (IT, Italy; ER, Emilia-Romagna; FR, France; SP, Spain; GR, Greece; PT, Portugal; DZ, Algeria; TN, Tunisia; KE, Kenia; SD, Sudan; ET, Ethiopia; IN, India; NP, Nepal). Italian samples are differentiated by the color of STRUCTURE designation (K = 2): PopA samples in red, PopB samples in blue.