Metagenomic sequencing provides insight into pathogenic and related benign microbes in ticks collected from pastured cattle.

Biting arthropods (e.g., ticks and mosquitoes) feed on pastured cattle and may serve as sentinels for certain pathogens present in the environment, including those with the capacity to spillover from wildlife into managed herds and humans. Metagenomic next generation sequencing (mNGS)-enabled by applications such as Oxford Nanopore Technologies (ONT)-allows samples to be screened for a diverse array of known and unknown pathogens compared to traditional targeted methods reliant on polymerase chain reaction (PCR). This study examined the utility of mNGS to identify potential pathogens relevant to animal and human health in samples collected from pastured cattle in Arkansas. Twenty Angus calves (Bos taurus) were sampled by collecting whole blood, swabbing the nose, mouth, and peri-anal region, and collecting ticks during a three-minute search. Each sample type was processed according to published procedures, and samples were sequenced using ONT MinION flow cells. mNGS analysis identified potentially pathogenic Theileria, Ehrlichia, and Borrelia species in tick samples. Downstream PCR identified Theileria cervi in 28.89% (13/45) of tick pools and 5.00% (1/20) of blood samples, a Babesia sp. in 6.67% (3/45) tick pools, three Ehrlichia species (E. chaffeensis, E. ewingii, and Panola Mountain Ehrlichia) in 6.61% (8/121) of ticks, and Borrelia lonestari in 2.48% (3/121) of ticks. In conclusion, the mNGS approach illuminated a wide spectrum of suspected microorganisms down to the genus-level, which were further characterized and confirmed as pathogenic species with conventional molecular detection approaches, thereby demonstrating a rigorous approach for a broad-spectrum screen and confirmation framework for pathogen identification.