Development of an RFLP-PCR assay for discrimination of three prevalent Old world Leishmania species in canine and human samples

Dogs in endemic regions are susceptible to leishmaniasis, a common vector-borne illness. Serological techniques and direct parasitological inquiry have been employed for diagnosis. However, molecular techniques are more sensitive and specific for identifying Leishmania infection in both clinically healthy and diseased dogs. This research aims to develop an approach for molecular identification of the parasite using restriction fragment length polymorphism (RFLP) analysis. Samples of skin and lymph nodes were taken from suspected dogs and humans and smears were prepared. DNA was extracted, and PCR reactions targeting the ribosomal internal transcribed spacer 1 (ITS1) region were used to identify the infection. Also, the PCR products were sequenced to provide control specimens. Before conducting an RFLP analysis, a bioinformatic study was performed on related sequences from the GenBank, as well as some previous data, to verify the results of RsaI and ApoI enzymatic digestion. The ITS1 PCR products were then subjected to digestion by these two enzymes, and the resulted pattern on the agarose gel were compared to those produced by the enzyme HaeIII, commonly used in other studies. Using the RsaI enzyme, fragments of approximately 95 and 146 bp were obtained for L. infantum, while L. tropica and L. major remained uncut. ApoI produced fragments of 101 and 140 bp for L. infantum and 108 and 157 bp for L. major. Based on these results, RFLP analysis with RsaI and ApoI proved to be a reliable tool for detecting different Leishmania species using a defined algorithm.