Occurrence of multiple genotype infection caused by Leishmania infantum in naturally infected dogs

Genetic polymorphisms in naturalLeishmaniapopulations have been reported in endemic areas. Microsatellite typing is a useful tool to elucidate the genetic variability of parasite strains, due to its capability for high-resolution mapping of genomic targets. The present study employed multilocus microsatellite typing (MLMT) to explore the genotypic composition ofLeishmania infantumin naturally infected dogs by genotyping parasites infecting different tissues with or withoutin vitroexpansion. Eighty-six samples were collected from 46 animals in an endemic region of visceral leishmaniasis (VL). MLMT was performed for 38 spleen samples and 48L.infantumcultures isolated from different tissues. Of the 86 samples, 23 were effectively genotyped by MLMT, identifying nine multilocus genotypes (MLG; referred to as MLG A-I). MLGs A, B and C were detected in more than one type of tissue and in more than one sample. Conversely, MLG D-I were uniquely detected in one sample each. The results showed that multiple genotype infections occur within a single host and tissue. Paired sample analysis revealed the presence of different MLMT alleles in 14 dogs, while the same MLG allele was present in 15 animals. STRUCTURE analysis demonstrated the presence of two populations; 13 samples displayed a similar admixture of both ancestral populations, and these were not assigned to any population. Only samples for which Q >= 0.70 after CLUMPP alignment were considered to be part of Population 1 (POP1) or Population 2 (POP2). POP2 comprised the majority of samples (n = 54) compared to POP1 (n = 19). This study presents evidence of multiple genotype infections (caused byL.infantum) in dogs in an area with high VL transmission. Further investigations must be undertaken to determine the multiple effects of infection on the host immune response and disease dynamics and treatment. Author summary American visceral leishmaniasis (VL) is a parasitic disease caused by the protozoanLeishmania infantum. This parasite can infect humans and animals and is transmitted by sand flies. Domestic dogs are considered an important host, and like humans, they can manifest the disease or present asymptomatic infections. Studies have identified genetic variations amongL.infantumparasites from different endemic regions in the American continent. For other parasitic diseases (e.g., malaria), studies have suggested that multigenetic infection predicts the development of symptoms and can lead to a high level of transmission. However, the effects of the genetic composition ofLeishmaniaparasites on VL need to be ascertained. This study used highly variable microsatellite markers to investigate multigenotypeL.infantumpopulations among naturally infected dogs living in an area in which VL is highly prevalent. Samples obtained from different tissues were examined to identify the occurrence of multiple genotypes in the same animal and even within the same tissue.