Molecular surveillance of tick-borne bacteria in hard ticks from Balochistan, Pakistan

Ticks are the most important arthropod vectors after mosquitoes and are responsible for transmitting a wide range of pathogens to both animals and humans. This study aimed to perform a molecular assessment of tick-associated pathogens belonging to the genera Rickettsia , Ehrlichia , Coxiella , and Anaplasma in hard ticks infesting various hosts in four districts of Balochistan, Pakistan. A total of 762 ticks were collected from 106 infested hosts including cattle, camels, goats and sheep. In this study, eight species of hard ticks were morphologically identified. Among these ticks, Hyalomma anatolicum ( n = 147/762, 19.29%) was the most abundant species followed by Hyalomma dromedarii ( n = 142/762, 18.63%), Rhipicephalus microplus ( n = 116/762, 15.22%), Rhipicephalus haemaphysaloides ( n = 82/762, 10.76%), Hyalomma turanicum ( n = 79/762, 10.36%), Hyalomma scupense ( n = 69/762, 9.06%), Hyalomma asiaticum ( n = 66/762, 8.66%), and Hyalomma isaaci ( n = 61/762, 8.00%). A total of 99 morphologically confirmed ticks were subjected to molecular analysis for the detection and genetic characterization of pathogens using genetic markers. Overall, 13/99 ticks (13.13%) were found positive for Ehrlichia minasensis ( n = 2/99, 2.02%), Coxiella burnetii ( n = 4/99, 4.04%), Rickettsia aeschlimannii ( n = 3/99, 3.03%), and Anaplasma sp. ( n = 4/99, 4.04%). By the BLAST analysis, the obtained gltA , ompA and ompB sequences of Rickettsia detected in H. anatolicum, H. isaaci and R. haemaphysaloides showed 100% identity with R. aeschlimannii . The obtained 16S rDNA sequences of Ehrlichia detected in H. scupense and H. isaaci showed 100% identity while dsb sequences displayed 99.38–100% identity with E. minasensis. Similarly, the groEL sequences of Coxiella detected in H. scupense , H. asiaticum and H. turanicum showed 100% identity with C. burnetii , while the 16S rDNA sequences for Anaplasma spp. detected in H. dromedarii, H. isaaci , and H. anatolicum revealed 100% identity with an undetermined Anaplasma sp. In phylogenetic trees, all obtained sequences clustered with corresponding species reported from different regions. To our knowledge, this study provides the first molecular evidence of R. aeschlimannii in H. anatolicum , H. isaaci , and R. haemaphysaloides ; E. minasensis in H. scupense and H. isaaci ; and C. burnetii in H. turanicum from Pakistan. These findings expand the known tick–pathogen associations in Balochistan and highlight the potential epidemiological relevance of livestock-associated ticks harboring pathogens of veterinary and zoonotic importance. The detection of multiple medically relevant pathogens emphasizes the need for broader molecular surveillance, integrated tick control, and One Health-based monitoring in the region.