Molecular Detection of Trypanosoma kaiowa in Tabanus triangulum (Diptera: Tabanidae) from the Coastal Plain of Rio Grande do Sul, Southern Brazil

Molecular of Anaplasma marginale Theiler (Rickettsiales: Anaplasmataceae) in horseflies (Diptera: Tabanidae) in Uruguay

“…This species accounted for 22% of the specimens tested, showing no detection of the pathogen, despite being one of the most frequent species in NZI traps in the department of Colonia 15 . Recently, studies detected the presence of Trypanosoma kaiowa 27 in 33% of specimens of T. triangulum in the coastal plain of Rio Grande do Sul and did not find other parasites of veterinary importance, despite the high abundance of this horsefly species in southern Rio Grande do Sul, Brazil 25 , 28 .…”

“…The 25 µl PCR mixture consisted of 2 µl of DNA template (100 ng total input), 12.5 µl of GoTaq ® Green Master Mix 2 ×, 1 µl each of forward and reverse primer (10 µM each primer) and enough nuclease-free water to reach the total volume. Electrophoresis of PCR products was carried out at a constant voltage of 10 V/cm by using a Bio-Rad electrophoresis assembly with a 1.2% agarose gel in 0.5 × Tris Borate EDTA (TBE) buffer 28 . Products were purified by excluding unwanted components from the PCR using a PureLink ® PCR Purification Kit (Life Technologies, USA) following the manufacturer's instructions 28 .…”

“…Electrophoresis of PCR products was carried out at a constant voltage of 10 V/cm by using a Bio-Rad electrophoresis assembly with a 1.2% agarose gel in 0.5 × Tris Borate EDTA (TBE) buffer 28 . Products were purified by excluding unwanted components from the PCR using a PureLink ® PCR Purification Kit (Life Technologies, USA) following the manufacturer's instructions 28 .…”

“…Purified PCR products were quantified by using a UV spectrophotometer and inserted into the pCR™2.1-TOPO ® vector using the TOPO ® TA Cloning ® Kit (Life Technologies, USA). The cloning reaction with a 6 µl volume containing 4 µl of purified PCR products, 1 µl of salt solution and 1 µl of pCR™2.1-TOPO ® vector was incubated for 5 min at room temperature 28 . Each cloning reaction was used to transform electrocompetent Escherichia coli DH5α cells.…”

“…The transformed cells were poured into Luria–Bertani agar (LB agar) plates with 100 mg/ml ampicillin and incubated overnight at 37 °C. The automatic sequencing of the positive clones was performed on a Biosystems 3500 Genetic Analyser ® (Life Technologies, USA) by using a Big Dye ® v3.1 Terminator Kit (Applied Biosystems, USA) (Life Technologies, USA) 28 .…”