Molecular Identification of A. marginale

For A. marginale identification, two unequivocal specie-specific genes were targeted: msp5, a single copy gene that encodes the outer major surface protein MSP5 and msp1β a three-copy gene that encodes the outer major surface protein MSP1b from A. marginale. For both target genes, PCR reactions were conducted using primers previously reported [26 (link),27 (link)]. The molecular amplifications were performed in a 50 µL reaction mixture (0.4 µmol of each primer, 0.2 mM of each deoxyribonucleotide triphosphate, 1.25 U of TopTaq DNA polymerase (QIAGEN. Hilden, Germany), 5 µL of 10× PCR buffer and purified water for 50 µL of final volume) using 200 ng of genomic DNA (from both blood or tick samples). Amplifications were carried out in a thermocycler (Bio-Rad MyCycler Thermal Cycler. Hercules, CA, USA) under previously described cycling conditions. For each amplification reaction, positive (DNA from A. marginale Mercedes strain) and negative (pure water) controls were included. An aliquot of 5 µL of each amplified product was analyzed by electrophoresis in 1.5% agarose gel stained with ethidium bromide. A molecular size marker (1 Kb Plus DNA Ladder, Invitrogen. Carlsbad, CA, USA) was used to determine PCR product size.