Vector-borne pathogen profiling and phylogeny of ticks infesting domestic dogs in Chad, Africa, using targeted next-generation sequencing and DNA barcoding

BACKGROUND: Understanding the range of vectors and pathogen diversity in sub-Saharan Africa is crucial for efficient disease prevention and control efforts. Domestic dogs are susceptible to many vector-borne pathogens (VBPs) and serve as sentinels for several human pathogens. Yet, critical surveillance gaps in canine VBPs persist in Central Africa, where the tropical climate and pastoral agro-livelihoods create unique transmission risks. METHODS: We conducted a cross-sectional survey from September to October 2021, collecting 4238 ticks from 1254 domestic dogs across 56 villages in Chad, Africa. All ticks were identified morphologically and/or by sequence analysis of 16S rDNA, 12S rDNA, and cytochrome c oxidase subunit I (COI) gene regions. A representative subset (n = 618) was subsequently screened for comprehensive, simultaneous detection of 21 VBPs using a targeted next-generation sequencing (tNGS) assay. Logistic regression models were used to assess associations between potential risk factors and pathogen detection. RESULTS: Five tick species belonging to four genera were identified: Rhipicephalus linnaei (89.8%; 3805/4238), Rhipicephalus muhsamae (9.4%; 400/4238), Amblyomma variegatum (0.6%; 26/4238), Haemaphysalis leachi (0.1%; 4/4238), and Hyalomma truncatum (0.1%; 3/4238). Overall, 59.1% (365/618; 95% CI 55.1 - 62.9%) of the ticks were positive for at least one pathogen. We detected 14 pathogens, with the most prevalent being Hepatozoon canis (47.6%, n = 294/618), followed by Ehrlichia canis (8.4%, n = 52/618), Coxiella-like endosymbionts (5.3%, n = 33/618), Rickettsia spp. (4.7%, n = 29/618), Anaplasma platys (4.4%, n = 27/618), Anaplasma spp. (3.7%, n = 23/618), hemotropic Mycoplasma spp. (1.5%, n = 9/618), and Anaplasma phagocytophilum (1.1%, n = 7/618). Babesia caballi, Hepatozoon felis, Rickettsia conorii, and Rickettsia massiliae were each identified in < 1% of the ticks. The highest pathogen diversity was detected in R. linnaei, although R. muhsamae harbored the highest diversity of zoonotic Rickettsia species. Independent predictors for VBP infections included tick species for E. canis (P = 0.02) and H. canis (P = 0.001), and geographic region for E. canis (P = 0.01). CONCLUSIONS/SIGNIFICANCE: This study provides a comprehensive molecular characterization of canine VBPs in ticks from Central Africa using deep sequencing. The high pathogen prevalence and diversity highlight the need for expanded surveillance and control strategies in Chad, including educating dog owners on ectoparasite prevention.