Discrimination of Borrelia persica and B.microiti the causative agents of relapsing fever using PCR-RFLP and specific primers

Utility of PCR-RFLP and species-specific PCR as novel and fast methods for identification and discrimination of causative agents of relapsing fever, Borrelia persica and B. microtii in infected blood were investigated. Genomic DNA of B.persica and B.microtii species were extracted from the highly infected blood samples. Two fragments of GlpQ and 16SrDNA genes were amplified using specific primers and then the PCR products were sequenced. Based on sequence variation between the two species, species-specific primers as well as restriction enzymes were respectively designed and selected for discrimination of these species. The results showed that using PCR technique we could easily amplify and detect the Borrelia species within the infected blood samples. Two different profiles of RFLP were produced when GlpQ PCR products of B.persica and B.microtii treated by Sspl, Taql, Dral, Hinfl, and EcoRV restriction enzymes. Also when 16SrDNA was digested with Taql enzyme we could discriminate between these two species. Based on GlpQ sequence variation, a set of primer 795r-BMGLPF produced specific band of 451 bp for B.microtii and a set of primer 128f-BPGLPR produced specific band of 252 bp for B.persica which could discriminate the both species well. In this study the discrimination of the two species of B.persica and B.microtii was investigated by PCR-RFLP and species-specific PCR methods for the first time. Both methods could easily distinguish the species from each other. Due to accuracy and speed of the molecular methods, they could be replaced with the classic methods. These fast and accurate diagnostic methods could be recommended for diagnosis laboratories in Iran and middle-east countries where both B.persica and B.microtii are prevalent

Detection and identification of Leishmania parasites sandflies using kDNA, rDNa and CPB loci

Molecular epidemiology of Visceral Leishmaniasis [VL] is currently used widely for different objectives such as vector incrimination studies. In this study three different loci including kinetoplast DNA [kDNA], ribosomal DNA [rDNA], cystein protease B [CPB[ of Leishmania parasite genome were used for detection and identification of natural infection of sand flies of Germi district of Ardebil province, the most important VL or Kala-azar foci in Iran. The results showed that the three loci of kDNA, rDNA and CPBs are respectively more appropriate for leptomonad infection/initial screening, identification of the L.donovani complex, and discrimination of the species complex. It was also verified that both members of the complex, L.donovani and L.infantum, are present in the study area and are transmitted to the hosts by Phlebotomus perfiliewi transcaucasicus sandflies. This is the first report on natural infection of sand flies to L.donovani in the country and since the ecology and biology of L.donovani differs extensively from L.infantum, it is necessary to perform further studies to highlight the role of L.donovani in epidemiology of VL in the region and country