African Swine Fever (ASF) is a highly contagious and deadly disease that causes a devastating impact on the swine industry, threatening food security worldwide. The current outbreak affecting 10% of the hog population in China and recent occurrences in the Caribbean islands (Dominican Republic and Haiti) highlight the risk of its potential emergence in the U.S. This disease is difficult to eradicate when the virus establishes a sylvatic life cycle involving argasid (soft) ticks and the warthog/feral swine populations. The USDA-APHIS has geared up an emergency response preparedness, and, the National Animal Health Laboratory Network (NAHLN) has been expanding its diagnostic infrastructure to meet any eventuality of ASF introduction into the U.S. Of the 30 soft tick species in the U.S., three species of Ornithodoros (O. turicata, O. coriaceus, and O. puertoricensis) were proven experimentally as competent vectors. The threat of three soft tick species acting as biological vectors in the U.S. is active; however, O. turicata poses a significant risk for sylvatic establishment as it occurs in the geographical range where common warthogs exist. We hypothesize that the microbiome of O. turicata must be the determinant of vector competence for the ASF virus, including genome alterations and virulence trait selection. In this pilot study, we will evaluate the gut and salivary gland microbiome composition of O. turicata (known competent vector) and O. tartakovskyi (not a proven vector). Apart from examining colony-reared ticks of both species, we will study field-collected ticks of the two distinct populations of O. turicata in the U.S. [O. turicata (Texas), O. turicata americanus (Florida)]. From these ticks the gut and salivary gland tissues will be dissected, and extracted DNA will be subjected to 16S rRNA PCR for bacteria and sequenced through the Oxford Nanopore MinION platform. The deep amplicon sequencing reads will be analyzed using the custom-designed bioinformatic pipeline for 16S to determine microbial composition. Organ-/collection site-associated microbiomes will be classified for individual ticks and compared between distinct tick populations/species. This study will provide the baseline for further exploring the role microbiomes play in the vector competence of O. turicata. A proactive approach to evaluate the determinants linked to the success of the ASF virus in the competent vector is the first step toward understanding the sylvatic potential of this virus if introduced in the U.S. Determining the microbiome signatures of Ornithodoros ticks, their association with the ASF virus, its virulence, and the disease process will deepen our knowledge on molecular epidemiology aiming to contain the looming threat of ASF in this continent.