16S rRNA profiling of bacterial communities in the brown dog tick Rhipicephalus linnaei from stray dogs in Perak, Malaysia.

Rhipicephalus linnaei is a widespread tick species infesting dogs and capable of transmitting pathogens of veterinary and zoonotic concern. However, its associated bacterial communities remain poorly described in Malaysia. This study profiles the bacterial microbiome of R. linnaei collected from stray dogs in Kampar, Perak, using 16S rRNA gene amplicon sequencing targeting the V3-V4 region. A total of 360 ticks were collected from 13 dogs, of which 290 were pooled according to life stages and sex for microbial profiling. Shannon diversity indices indicated the mixed adult/nymph pool exhibited the highest richness and evenness (H'=5.4), whereas engorged adult females displayed the lowest diversity (H'=2.35), dominated by Gammaproteobacteria. Principal coordinate analysis revealed distinct microbial assemblages among pools, explaining 72% of total variance. Among 137 detected genera, Coxiella (0.6-34%), Staphylococcus (0.4-29%), Stenotrophomonas (0.3-5%), and Streptococcus (0.02-6%) were consistently found across all pools. Low-abundance but clinically relevant genera, including Ehrlichia (0.64%) and Nocardia (< 0.01%), were detected in adult males. The consistent presence of Coxiella-like endosymbionts across all stages suggests a likely symbiotic role in nutrient provisioning and reproduction. To our knowledge, this is the first 16S rRNA gene-based profiling of the bacterial communities associated with R. linnaei collected from stray dogs in Malaysia. This study highlights variation across pooled tick categories and contributes to improved understanding of tick-borne pathogen ecology within a One Health framework.