Molecular Characterization of Cutaneous Leishmaniasis and Canine Leishmaniasis Strains Isolated from Northern Cyprus and Their Clinical Reflections in Mice Models

Abstract

Leishmaniasis is a parasitic infection caused by Leishmania species in tropical and subtropical regions. Cyprus, located in the Eastern Mediterranean basin, is considered endemic for leishmaniasis. In our study, we aimed to investigate the origins of two Leishmania strains isolated from Turkish Republic of Northern Cyprus by sequencing and to establish in vivo animal models by examining the clinical reflections of these strains in mice. A biopsy material was taken from a seven-year-old girl with a lupoid lesion on her forehead and amastigotes were detected in the smears. Following culture of the specimen on enriched NNN media and detection of promastigotes, the patient was diagnosed with cutaneous leishmaniasis (CL) (no: 504). A four-year-old boxer dog was taken to a veterinary clinic with complaints of weight loss, weakness, hair loss and lymphadenopathy. A blood sample was taken from the dog, cultured in enriched NNN media and canine leishmaniasis (CanL) was diagnosed with the growth of promastigotes (no: 753). Leishmania promastigotes obtained from CL and CanL cases were cryopreserved and stored in the Parasite Bank, Manisa Celal Bayar University. When the isolates were to be used, they were transferred to RPMI-1640 medium and grown to the concentration of 10(7) promastigotes/mL required for the study. The isolates were identified to the species level using primers specific for ITS-1 and cpb E/F regions of Leishmania. A phylogenetic tree was constructed according to the data obtained from the sequence analysis. In addition to the Northern Cyprus Leishmania isolates for in vivo studies, one each of Leishmania donovani (MLd) and Leishmania tropica (MLt) isolates from Manisa were included for comparison. Eight study groups and two control groups (uninfected) were formed with five Mus musculus (Balb/c) mice in each group (total 10 groups and 50 Balb/c mice). Four mouse study groups were separately used with MLt, 753, MLd and 504 strains to establish the CL animal model. The other four mice groups were used for the VL animal model with the same strains. In addition, one group each for the CL and VL models was used as a negative control group. Mice in the CL groups were infected with 100 mu L of the prepared promastigote suspension on the sole of the right foot intradermally and the width and height of the sole of the foot were measured weekly for six weeks. Mice in VL groups were infected with 100 mu L promastigote suspension intraperitoneally and VL symptoms were monitored. Thirty days after the injection, the spleen and liver were removed by autopsy and their sizes were measured. In our study, one of the two Northern Cyprus Leishmania isolates molecularly characterised was Leishmania donovani (human strain, no: 504) and the other was Leishmania tropica (canine strain, no: 753). Mice in the CL and VL groups developed symptoms of CL and VL, respectively, and both Manisa and Northern Cyprus isolates had similar reflections in mice. In the literature, it is reported that Leishmania infantum is the causative agent of CanL cases in Northern Cyprus. However, according to our study, L.tropica may also be the source of CanL infections in the island. In addition, it was observed that Northern Cyprus isolates caused both CL and VL infections in mice and it was concluded that they can be used in future in vivo studies.