Leishmania

Detection of Leishmania View in CoL , Trypanosoma , and Bartonella DNA in sand flies

To detect pathogens in sand flies, primer sets on the basis of three genes were selected: Leishmania sp. (ITS1), Trypanosoma sp. (SSU rRNA), and Bartonella sp. (gltA). Tese primer sets were chosen because of their high sensitivity and specificity, as well as their suitability for phylogenetic analysis. Conventional polymerase chain reaction (PCR) was carried out using primers LeF (5 ′ - TCCGCCCGAAAGTTCACCGATA-3 ′) and LeR (5 ′ - CCAAGTCATCCATCGCGACACG-3 ′) that targeted the ITS1 region of the ribosomal RNA gene for the detection of Leishmania [ 43]. PCR reagents and amplification conditions were described by Sunantaraporn et al. [ 24]. For Trypanosoma detection, PCR amplification of the Trypanosoma sp. SSU rRNA gene was performed using primers TRY927F (5 ′ -GAAACAAGAAACACGGGA G-3 ′) and TRY927R (5 ′ -CTACTGGGCAGCTTGGA- 3 ′) [ 44]. Te PCR reaction and amplification were performed in accordance with those of Srisuton et al. [ 17]. Te estimated product size for Leishmania and Trypanosoma was approximately 379 and 900 bp, respectively. Te amplified products were separated on a 1.5% (W/V) agarose gel electrophoresis. Te expected products were imaged with Quantity One Quantification Analysis Software Version 4.5.2 (Gel DocEQ System; Bio-Rad, Hercules, CA, USA), after staining with ethidium bromide.

Te presence of Bartonella DNA in sand flies was tested in all DNA samples targeting the citrate synthase (gltA) gene, using the primers BhCS871p (5 ′ -GGGGAC CAGCTCATGGTGG-3 ′) and BhCS1137n (5 ′ -AAT GCAAAAAGAACAGTAAACA-3 ′) [ 45]. Conventional PCR was performed following the methods previously described by Promrangsee et al. [32]. Te presence of an expected band of 379 bp was determined by 1.5% (W/V) agarose gel electrophoresis.

DNA extracted from L. martiniquensis promastigotes, Trypanosoma evansi DNA from blood-infected dogs, and Bartonella sp. detected from cattle lice DNA were used as positive controls and deionized distilled water was used as a negative control.