Blood Sample and Extraction of Total Genomic DNA

A subset of blood samples (n = 10) were randomly selected for metagenomic analysis from a larger cohort of horses suspected of having tick-borne diseases (TBD) recruited for studying prevalence of Anaplasma phagocytophilum and Borrelia burgdorferi infections during 2017–2019. In addition, blood samples (n = 5) from apparently healthy horses that were not part of the TBD study cohort were included in the metagenomic study. The horses with suspected tick-borne illnesses often had history of tick exposure and showed clinical signs such as fever, depression, petechiae, and inappetence. The reported clinical signs among the horses included in the study are listed in Table 1 and horses suspected with tick-borne infection with clinical signs are referred as “sick group.”

Demographic, clinical and laboratory analyses for horses included in the study.

NA, not available; PCR and Next-Generation Metagenomic analyses: 0 - negative and 1 - positive.

Blood samples were collected in EDTA vacutainer tubes (BD Bioscience, San Jose, CA). The total genomic DNA from blood samples were extracted using the Blood or Body Fluids Spin Protocol (QIAmp DNA extraction mini kit, Qiagen, Germantown, MD) following the manufacturer's instructions. The extracted DNA was not treated with RNAase and the quality and quantity of the DNA were analyzed by spectrophotometry (Nanodrop, Thermo Fisher Scientific, Waltham, MA) and fluorometric method (Qubit, Thermo Fisher Scientific, Waltham, MA), respectively.