Figure 1. Detection and characterization of the PrP^res of scrapie prion protein (PrP^Sc) from brain tissues of dromedary camels, Tunisia, 2019–2021. A) PrP^res detected using TeSeE Western blot kit (Bio-Rad Laboratories, https://www.bio-rad.com). Membrane probed with the Sha31 monoclonal antibody. Loading order (left to right): ovine scrapie control, Tunisian camel prion disease–positive cases (animal no. P81/9–65) and Tunisian camel prion disease–negative samples (animal nos. P81/64 and P81/15). All samples, except for P81/64 and P81/15, were diluted 1:4 prior to electrophoresis and loaded at 3.75 mg tissue equivalent per lane. The negative samples were loaded at 15 mg tissue equivalent per lane. Approximate molecular weights (expressed in kDa) are reported at top of blot. B) Representative Western blot analysis of different available brain regions from selected positive cases. Brain tissues analyzed using the ISS (Istituto Superiore di Sanità) discriminatory Western blot protocol. Case identifiers indicated below each blot. Corresponding brain regions labeled at the top: a, striatum; b, frontal cortex; c, prefrontal cortex; d, capsule; e, occipital cortex; f, parietal cortex; g, cerebellar peduncle; h, cerebellum; i, temporal cortex; j, basal ganglia; k, thalamus; l, hippocampus; m, midbrain; n, medulla oblongata; o, hypothalamus; p, pons; q, scrapie. All membranes probed with the 12B2 antibody, except for P81/17, which was probed with L42. Tissue equivalents loaded per lane: 2 mg for P81/9 and 0.5 mg for P81/13, P81/16, and P81/17. Molecular weights (in kDa) are indicated on the left of blot. C) Representative replica blots showing the epitope mapping analysis of PrP^res from brain homogenates of positive dromedary camel cases. A preliminary assessment of dromedary PrP^res reactivity with a panel of monoclonal antibodies was performed to identify suitable diagnostic tools for camel prion disease and to characterize PrP^res. Within each antibody group, the antibody with the best sensitivity toward dromedary PrP^Sc was chosen for epitope mapping (Appendix Table 2, Figure 1). In addition, the SAF32 antibody, which targets the octarepeat region, was included to enable comparison with scrapie, in which this epitope is partially lost. The brain areas analyzed for each sample were as follows: P81/9, prefrontal cortex; P81/13, frontal cortex; P81/14, thalamus; P81/16, parietal cortex; P81/17, prefrontal cortex; P81/65, not identifiable; scrapie, medulla oblongata. A scrapie-affected sheep sample was included as control in last lane of each blot. Molecular weights (expressed in kDa) reported on the left of blot. Membranes were probed with monoclonal antibodies indicated below each blot. D) Relative proportions of diglycosylated, monoglycosylated, and unglycosylated PrP^res bands in each available brain region of animal P81/13. Scrapie is shown on the right for comparison. Quantifications were performed on membrane probed with 12B2 antibody. Ctrl, control; PrP^res, proteinase K-resistant core.