| Summary: | Tick-borne rickettsioses are mainly caused by obligate intracellular bacteria belonging to the spotted fever group (SFG) of the <i>Rickettsia</i> genus. So far, the causative agents of SFG rickettsioses have not been detected in cattle ticks from Tunisia. Therefore, the aim of this study was to investigate the diversity and phylogeny of ticks associated with cattle from northern Tunisia and their associated <i>Rickettsia</i> species. Adult ticks (<i>n</i> = 338) were collected from cattle in northern Tunisia. The obtained ticks were identified as <i>Hyalomma excavatum</i> (<i>n</i> = 129), <i>Rhipicephalus sanguineus</i> sensu lato (<i>n</i> = 111), <i>Hyalomma marginatum</i> (<i>n</i> = 84), <i>Hyalomma scupense</i> (<i>n</i> = 12) and <i>Hyalomma rufipes</i> (<i>n</i> = 2). After DNA extraction from the ticks, 83 PCR products based on the mitochondrial 16S rRNA gene were sequenced and a total of four genotypes for <i>Rh. sanguineus</i> s.l., two for <i>Hy. marginatum</i> and <i>Hy. excavatum</i> and only one for <i>Hy. scupense</i> and <i>Hy. rufipes</i> were recorded, with the occurrence of one, two and three novel genotypes, respectively, for <i>Hy. marginatum</i>, <i>Hy. excavatum</i> and <i>Rh. sanguineus</i> s.l. mitochondrial 16S rRNA partial sequences. The tick DNA was tested for the presence of <i>Rickettsia</i> spp. by using PCR measurements and sequencing targeting three different genes (<i>ompB</i>, <i>ompA</i> and <i>gltA</i>). Of the 338 analyzed ticks, 90 (26.6%), including 38 (34.2%) <i>Rh. sanguineus</i> s.l., 26 (20.1%) <i>Hy. excavatum</i>, 25 (29.8%) <i>Hy. marginatum</i> and one (50%) <i>Hy. rufipes</i> tick, were positive for <i>Rickettsia</i> spp. Based on 104 partial sequences of the three analyzed genes, the BLAST analysis and phylogenetic study showed the infection of <i>Hy. excavatum</i>, <i>Hy. marginatum</i> and <i>Rh. sanguineus</i> s.l. tick specimens with <i>R. massiliae</i>, <i>R. aeschlimannii</i> and <i>R. sibirica</i> subsp. <i>mongolitimonae</i> and one <i>Hy. rufipes</i> tick specimen with <i>R. aeschlimannii</i>. In addition, coinfection with <i>R. massiliae</i> and <i>R. aeschlimannii</i> was reported in one <i>Hy. marginatum</i> and one <i>Rh. sanguineus</i> s.l. tick specimen, while a coinfection with <i>R. massiliae</i> and <i>R. sibirica</i> subsp. <i>mongolitimonae</i> was recorded in one <i>Rh. sanguineus</i> s.l. tick specimen. In conclusion, our study reports, for the first time in Tunisia, the infection of cattle ticks belonging to <i>Hyalomma</i> and <i>Rhipicephalus</i> genera with zoonotic <i>Rickettsia</i> species belonging to the SFG group.
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