Tick infestation poses an important challenge to livestock in Pakistan. Farm management practices and environmental variables can influence tick infestation prevalence in animals. To this end, a cross-sectional survey of 96 farms in four different geographical districts (24 farms from each district) was conducted in Punjab, Pakistan, between October 2021 and January 2022. An epidemiological questionnaire was designed focusing on farm management practices and their impact on tick infestations at these farms. Data were collected via in-person interviews. Regional and farm-specific variables’ associations were evaluated using Pearson’s chi-square test and Fischer’s exact test, respectively. A multivariable logistic regression model was used to identify significant risk factors. This study identified that using soft bedding materials, e.g., wheat straw, leaf litter or plain soil posed a significant risk of tick infestation. Additionally, the absence of quarantine measures, open sheds and inadequate drainage were found as contributing factors in univariable analysis. Higher tick prevalence in the hotter seasons highlighted the influence of Punjab’s extreme weather on tick infestation. Despite regular veterinary visits and the use of acaricidal drugs, the prevalence of tick infestation at these farms suggested potential drug resistance in the ticks. The study recommended establishing quarantine practices, improving farms’ drainage systems and bedding and using a combination of chemical and traditional remedies to tackle drug resistance in ticks. Education and awareness programs on tick-borne diseases and control measures are advocated to reduce the tick infestation burden on animals. Further research on longitudinal studies to better understand tick population dynamics and develop effective acaricides is encouraged. This called for collaborative control efforts among farmers, veterinarians and research institutions.

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.