Decoding Leishmania in equines: A comparative analysis of molecular targets

Parasitic diseases caused by Leishmania spp. create considerable health concerns in animals, resulting in a considerable financial impact. They causes a complex infection in equines, affecting weight gain, skin, liver, and spleen. To date, there is a lack of reports on the occurrence of Leishmania in equines in Southern Punjab, Pakistan, highlighting the need for molecular epidemiological surveillance. The current study focused on determining the prevalence of Leishmania in the equine population from District Rahim Yar Khan, Southern Punjab, Pakistan, through amplification of mitochondrial (Cytochrome b) and nuclear (18S rRNA) genes of the parasite. For this purpose, a total of 384 equine - i.e. horses, mules, and donkeys - blood specimens, determined by calculation of the sample size formula, were obtained from District Rahim Yar Khan. The parasite was examined through the Microhematocrit method under the microscope. Leishmania was detected from the buffy coat layer after centrifugation of blood-filled microhematocrit tubes. To detect and characterize Leishmania spp.at the molecular level, DNA extraction from blood samples was carried out using standardized commercial kits, followed by PCR amplification. Information on potential risk factors was gathered through a structured questionnaire. The overall prevalence of Leishmania infection was observed to be 2.1 % via microscopy and 7.3 % and 8.8 % by amplification of the 18S rRNA and Cytochrome b genes using molecular methods. A significantly higher infection percentage was observed in female animals compared to males, and in older and underweight animals compared to younger and healthier ones. Additionally, the infection was non-significantly (P ≥ 0.05) more prevalent in gestating, non-dewormed, symptomatic, and poor body condition animals. Phylogenetic and sequence analyses confirmed that the identified gene sequences clustered within the Leishmania (Leishmania) infantum clade, consistent with strains reported in different animal hosts from various regions. In conclusion, the nuclear gene, i.e., 18S rRNA proved to be a more sensitive molecular marker for detecting Leishmania infection in equines compared to the mitochondrial gene, i.e., Cytochrome b.