| Summary: | Molecular assays and capillary electrophoresis sequencing have been used to identify parasites in livestock. The low sample capacity, which increases labor and processing time, is one drawback. Targeted amplicon sequencing (Ampliseq) uses the fast and large sample capacity platform to identify parasites in the target host, overcoming this limitation. DNA was extracted from 162 whole blood samples collected from cattle in three provinces in the Philippines. Using Illumina’s Miseq platform, the V4 hypervariable region of the piroplasma 18S rRNA gene was amplified and sequenced. The AMPtk pipeline was used to obtain distinct amplicon sequence variants (ASVs) and the NCBI BLAST non-redundant database was used to assign taxonomy. In total, 95 (58.64%) samples were positive for piroplasma. Using the AMPTk pipeline, 2179 ASVs were obtained. A total of 79 distinct ASVs were obtained after clustering and filtering, which belonged to genera <i>Babesia</i> (n = 58), <i>Theileria</i> (n = 17), <i>Hepatozoon</i> (n = 2), and <i>Sarcocystis</i> (n = 2). The ASV top hits were composed of 10 species: <i>Babesia bovis</i>, <i>B. bigemina</i>, <i>Theileria orientalis</i>, <i>Babesia</i> sp., <i>Hepatozoon canis</i>, <i>Sarcocystis cruzi</i>, <i>T. annulata</i>, <i>T. equi</i>, <i>T. mutans</i>, and <i>Theileria</i> sp. Thung Song. The results generated in this study demonstrated the applicability of Ampliseq in detecting piroplasmid parasites infecting cattle in the Philippines.
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