Molecular screening, regional infection patterns and phylogenetic characterization of spotted fever group Rickettsia in ticks from domestic animals in Uzbekistan.

Spotted fever group (SFG) Rickettsia spp. are emerging zoonotic pathogens transmitted by ixodid and argasid ticks. Despite the recognized importance of ticks as vectors in Central Asia, data on the prevalence and genetic diversity of SFG Rickettsia in Uzbekistan remain scarce. This study aimed to determine minimum infection rates (MIR) and phylogenetic relationships of SFG Rickettsia circulating in ticks parasitizing domestic animals across five regions of Uzbekistan. Ticks (n = 12,769) were collected from 8632 domestic animals (cattle, sheep, goats, horses, camels, donkeys, and poultry) between 2021 and 2026. Ticks were screened either individually or in pools of 4-5 adult specimens, with each pool comprising ticks of the same species and sex collected from the same individual host, using real-time PCR. Among the qPCR-positive pools, 26 were randomly selected for conventional singleplex PCR targeting the gltA, ompA, ompB, and sca4 genes. Fourteen selected positive pools yielded amplicons suitable for sequencing, generating 25 partial gene sequences in total (gltA, n = 9; ompA, n = 3; ompB, n = 9; sca4, n = 4), which were subjected to bidirectional Sanger sequencing and phylogenetic analysis. Positive amplicons were sequenced and phylogenetic trees constructed using maximum likelihood with 1000 bootstrap replicates. Twelve ixodid and one argasid tick species were identified. Overall tick infestation prevalence in animals was 36.5% (95% CI: 35.5-37.6%). Rickettsia DNA was detected in 2331 ticks from positive pools (global MIR: 18.3%; 95% CI: 17.6-18.9%). Highest MIRs were found in Hyalomma scupense (25.1%), Rhipicephalus turanicus (23.4%) and Rhipicephalus pumilio (21.7%). Regional MIRs ranged from 0.77% (Syrdarya) to 31.8% (Jizzakh). gltA, ompA, ompB, and sca4 partial sequences of "Candidatus Rickettsia barbariae" and Rickettsia massiliae were identified for the first time in Uzbekistan. Phylogenetic analyses confirmed their placement within distinct SFG Rickettsia clades. This study provides baseline data on the diversity and distribution of SFG Rickettsia in Uzbekistan. Rickettsia DNA was detected in Hy. scupense, Hyalomma anatolicum, Hyalomma asiaticum, Rh. turanicus, Rhipicephalus sanguineus sensu lato, Rhipicephalus annulatus, Rh. pumilio, Argas persicus, and Haemaphysalis sulcata. The first detection of "Ca. R. barbariae" and R. massiliae in Uzbekistan underscores the need for integrated One Health surveillance of tick-borne rickettsioses in domestic animals and humans.