# Characterization of Leishmania infantum Isolates from Wild Leporids in the Community of Madrid (Spain)

## TL;DR

This study shows that a single Leishmania infantum isolate infects wild rabbits and hares in Madrid, supporting its role in the parasite's life cycle and human outbreaks.

## Contribution

The study proposes a reliable molecular typing method for Leishmania surveillance using PCR-RFLP and confirms a single isolate's dominance in wild leporids.

## Key findings

- PCR-RFLP analysis confirmed a single L. infantum isolate in wild leporids across Madrid.
- The same isolate has been circulating since at least the 1990s.
- Wild rabbits and hares sustain the L. infantum life cycle, both inside and outside outbreak areas.

## Abstract

Despite the public health implications of limited awareness regarding the biodiversity of Leishmania spp. and the persistence of human leishmaniasis outbreaks in endemic regions of Europe, there is still no consensus on a standardized molecular typing method for the surveillance of this parasite in humans, domestic animals, and wildlife reservoirs. Consequently, the development of a simple, rapid, effective, and cost-efficient typing approach remains an unmet need. The method proposed in this study, evaluated on a larger number of samples than in previous reports, may fulfill these criteria and contribute to improved molecular surveillance of Leishmania spp.

In this research, 59 samples from 31 animals (19 European rabbits, 11 Iberian hares, and 1 cat) and an axenic culture of the Leishmania infantum isolate (MCAN/ES/97/10445, zymodeme ZM/MON-1) used as a reference were studied based on the analysis of kinetoplast minicircle (kDNA) restriction fragments by combining polymerase chain reaction and length polymorphisms (PCR-RFLP). This analysis was performed in parallel with polyacrylamide gel electrophoresis (PAGE) and capillary electrophoresis (CE), as well as in silico digestion of the abovementioned reference. These analyses did not reveal differences between the L. infantum isolates detected in the different samples of wild lagomorphs (rabbits and hares) from various areas of the Community of Madrid or with the axenically cultured promastigotes of the L. infantum isolate (MCAN/ES/97/10445, zymodeme ZM/MON-1) used as a reference. Consequently, it was proven that with the implemented approaches, only one isolate of L. infantum was responsible for infection in wild leporids and that these animals sustained the pathogen’s life cycle, both in the area of the human leishmaniasis outbreak that has been occurring in the Community of Madrid since 2009 and outside of it. Additionally, this isolate has been circulating since at least the 1990s.