Combining PCR and Metagenomic Approaches to Reveal Tick-Borne Pathogens in Ticks Collected from Livestock and Companion Animals in Cambodia

In Cambodia, livestock production plays an important role in the national economy and food security, yet tick-borne diseases remain an underrecognized constraint on animal health and productivity. Domestic animals may also serve as reservoirs of zoonotic pathogens in this predominantly rural setting. To address the lack of baseline molecular data on tick-borne pathogens in Cambodia, we conducted a cross-sectional study of ticks collected from November 2022 to April 2023 across 24 provinces. Ticks were collected from various hosts and environments, including cats, cattle, dogs, goats, pangolins, pythons, wild pigs, and bat cave floors, representing urban, rural, farm, wildlife rescue center, and forest fringe habitats. A total of 1526 ticks belonging to nine species were pooled into 352 samples and screened using conventional PCR (cPCR) targeting Anaplasma, Ehrlichia, Babesia, and Coxiella. Additionally, a subset of Rhipicephalus microplus ticks was analyzed using metatranscriptomic next-generation sequencing (NGS). Rhipicephalus microplus ticks collected from cattle tested positive for Anaplasma marginale (1.1% of pools) and Ehrlichia minasensis (0.9% of pools), whereas Rhipicephalus linnaei ticks collected from dogs were positive for Anaplasma platys (0.3% of pools) and Babesia canis (2.0% of pools). A high prevalence of Coxiella-like endosymbionts (15.6% of pools) was found in R. microplus from both cattle and goats. Metatranscriptomic analysis also identified six tick-associated viruses in R. microplus from cattle; with Guangdong tick manly virus being the most dominant (32.5% of samples); followed by Zhangzhou Totiv tick virus 1 (15.0%), Jingmen tick virus (5.0%), and Mogiana tick virus; Rhipicephalus-associated rhabdo-like virus; and Rhipicephalus-associated flavi-like virus; each at 2.5%. These findings provide the first molecular evidence of numerous bacterial, protozoal, and viral pathogens circulating in R. microplus and R. linnaei in Cambodia. The study highlights the need for integrated One Health surveillance to better understand, prevent, and control tick-borne diseases in the region.