Molecular and Next-Generation Sequencing Analysis of Tick-Borne Pathogens of Rhipicephalus Ticks (Acari: Ixodidae) in cattle and dogs.

Ticks are small and adaptable arachnid ectoparasites and global carriers of various pathogens that threaten both human and animal health. They are present in many parts of China. A total of 858 ticks were collected from various regions and hosts, then subjected to species identification based on morphological and molecular characteristics, as described in the authors' previous study. Eighty-three individual tick samples were selected for screening pathogens based on metagenomic next-generation sequencing (mNGS) and polymerase chain reaction (PCR) assays. The genomic DNA of tick species was extracted, and amplification of the bacterial 16S rRNA gene was carried out from DNA of individual ticks using V3-V4 hypervariable regions, before subjecting to metagenomic analysis. Each tick underwent specific PCR tests for identifying the bacterial species present, including Anaplasma, Ehrlichia, Coxiella, and Rickettsia, and also protozoans such as Babesia, Theileria, and Hepatozoon. Illumina NovaSeq sequencing results revealed that the dominant phylum and family in Rhipicephalus spp. were Bacteroidota and Muribaculaceae, respectively. Alpha diversity patterns varied depending on tick sex (R. linnaei only), species and location, but not on host. Furthermore, bacterial pathogens, including A. marginale (58%, 29/50), A. platys (6%, 3/50), E. minasensis (2%, 1/50), Ehrlichia sp. (10%, 5/50), T. sinensis (24%, 12/50), T. orientalis (54%, 27/50) and Coxiella-like bacteria (CLB) (80%, 40/50) were detected in R. microplus, while E. canis (33.33%, 10/30), H. canis (20%, 6/30) and CLB (100%, 30/30) were detected in R. linnaei. Also, Anaplasma sp. (33.33%, 1/3), A. marginale (33.33%, 1/3), R. felis (33.33%, 1/3) and CLB (100%, 3/3) were detected in R. haemaphysaloides. Dual and triple co-infections involving pathogens or CLB were detected in 84.00% of R. microplus, 66.66% of R. haemaphysaloides, and 33.00% of R. linnaei. The report on microbial communities and pathogens, which found from Rhipicephalus spp. in Hainan Island, is an important step towards a better understanding of tick-borne disease transmission. This is the first report in the area on the presence of Anaplasma sp., A. marginale, R. felis and Coxiella, in R. haemaphysaloides.