Molecular Characterization of IS1111-Positive Tick Pools from Punjab, Pakistan Reveals Clade C Coxiella-like Endosymbionts

Coxiella burnetii, the etiological agent of Q fever, is a significant zoonotic pathogen. Molecular detection of C. burnetii in ticks is challenging because ticks commonly harbor genetically related Coxiella-like endosymbionts (CLEs), which may yield positive results in assays targeting shared genetic markers. In Pakistan, where livestock populations are dense, robust data on C. burnetii in ticks are lacking. A retrospective molecular survey was conducted on 387 tick pools (Hyalomma spp. and Rhipicephalus spp.) collected from domestic ruminants in Punjab, Pakistan, between 2013 and 2014. A sequential diagnostic approach was applied, consisting of initial screening by IS1111 and icd qPCR followed by IS1111 and 16S rRNA gene PCR amplification and sequencing for phylogenetic analyses. In addition, co-detection with other tick-borne pathogens was assessed. The overall proportion of IS1111 qPCR-positive tick pools was 1.3% (5/386 evaluable pools; 95% CI: 0.5–3.0). All IS1111-positive tick pools were negative by icd qPCR. Sequencing of the IS1111 element was successful for three pools and of the 16S rRNA gene for two pools. Phylogenetic analysis of the 16S rRNA gene identified both sequenced pools as Clade C CLE, whereas the IS1111 sequences did not discriminate between C. burnetii and CLE. The remaining three IS1111 qPCR-positive pools could not be taxonomically resolved. Ehrlichia spp. DNA co-occurred in one CLE-confirmed pool (Hy. anatolicum) and Anaplasma marginale DNA in one unresolved IS1111 qPCR-positive pool (Rh. turanicus). No C. burnetii DNA was confirmed in the examined tick pools from the study region during the sampling period. The findings reinforce the importance of confirmatory sequencing and multilocus approaches for accurate discrimination between C. burnetii and CLEs. Furthermore, the results suggest that reports based solely on IS1111 detection should be interpreted with caution and highlight the need for further studies to clarify the occurrence of C. burnetii and the role of ticks in its ecology and transmission.