Subolesin expression in questing or feeding adult ticks naturally infected with different pathogens. Subolesin expression was characterized in R. sanguineus (R.s.) and D. marginatus (D.m.) infected with R. conorii (A), R. bursa (R.b.), H. lusitanicum (H.l.) and H. m. marginatum (H.m.) infected with T. annulata, B. bigemina and T. buffeli, respectively (B), R. turanicus and R. bursa infected with A. ovis (C) and R. sanguineus infected with E. canis (D). In all cases, sex and collection-matching groups of uninfected tick samples were analyzed for comparison. Subolesin mRNA levels were analyzed by real-time RT-PCR and normalized against tick 16S rRNA using the comparative Ct method [9, 32]. The graph depicts the infected to uninfected subolesin mRNA ratio (± SD) calculated by dividing normalized subolesin mRNA levels in infected ticks by the average of the normalized subolesin mRNA level in uninfected control ticks. Normalized subolesin mRNA levels were compared between infected and uninfected ticks by Student's t-Test (*P < 0.05).