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DNA samples were diluted with nuclease-free water.Amplifications were performed by using AM-For 16S forward primer (5′-TTGGCAAGGCAGCAGCTT-3′) and AM-Rev 16S reverse primer (5′-TTCCGCGAGCATGTGCAT-3′) at a concentration of 0.6 μmol/L each, and AM-Pb probe (5′-6-FAM/TCGGTCTAACATCTCCAGGCTTTCAT/3BHQ_1-3′) at a concentration of 0.2 μmol/L.Subsequent blood samples were collected during May 2018 from 7 animals within the index herd.Two of these animals previously had intracellular piroplasms; 5 of these animals were sampled randomly.

Affected cattle included bulls, cows, and steers ranging in age from 3 months to 13 years. In September 2017, an additional cow from the index farm was examined for weakness, icterus, and anemia (packed cell volume [PCV] 12.0%).

Amplicons with sizes of 700–800 bp were submitted to the Virginia Biocomplexity Institute (Blacksburg, VA, USA) for bidirectional Sanger sequencing.