Fig 2: PLD3 expression analysis in the central nervous system of healthy, preclinical and clinical scrapie-affected sheep. A High-magnification image of PLD3 immunoreactivity showing a striated cytoplasmic staining pattern (arrowhead) in neurons and granular deposits in the neuropil (asterisks). B, C Grouped bar plots representing semiquantitative scores (from 0 to 5) of immunohistochemical (IHC) analysis across six brain regions (B; CSc: cervical spinal cord, Mo: medulla oblongata at the level of the obex, Cb: cerebellum, Th: thalamus, Hc: hippocampus, Fc: frontal cortex) and their specific subregions (C; Wm: white matter, Gm: grey matter, CN: cuneate nucleus, DMNV: dorsal motor nucleus of vagus, ON: inferior olive nucleus, Ml: molecular layer, Pl: Purkinje cell layer, Gl: granular layer, VT: ventral thalamus, CT: central thalamus, DT: dorsal thalamus, Dg: dentate gyrus, CA4-1: cornu ammonis 4–1, SLM: stratum lacunosum-moleculare). Data presented as the mean and standard deviation. Significant differences between groups were measured using Kruskal–Wallis followed by Dunn’s test. *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ^p-value < 0.1 (tendency). D Representative IHC images from healthy, preclinical and clinical sheep in the dorsal motor nucleus of vagus of the obex. E Western blot analysis in thalamic tissue, with molecular weights (in kDa) represented on the left. F Grouped bar plot with overlaid scatter plot showing individual 2−∆∆Ct values of gene expression, relative to the healthy group, measured by RT-qPCR in the medulla oblongata at the level of the obex (Mo) and thalamus (Th). Bars and error lines indicate the mean of individual values and standard deviation or the median and interquartile range, for normally and non-normally distributed data, respectively. PLD3 gene expression was normalised to the mean of SDHA and GAPDH in the obex and to SDHA and G6PD in the thalamus. Significant differences between groups were measured using one-way ANOVA test followed by Tukey HSD (for normally distributed data) or Kruskal–Wallis followed by Dunn’s test (for non-normally distributed). *p-value < 0.05.

Fig 3: Serum concentrations of SYNCRIP, PLD3, CTSD, SPP1 and C4 measured by ELISA. Box plots represent concentrations in healthy controls (green, n = 8) and preclinical (blue, n = 5) and clinical (red, n = 8) scrapie-affected sheep. Boxes span from the first to the third quartile and the median is indicated by the horizontal line inside each box. Whiskers extend to the most extreme data point within 1.5 times the interquartile range; data points beyond this range are considered potential outliers. Statistical significance between groups was assessed using one-way ANOVA test followed by Tukey HSD for normally distributed data, or Kruskal–Wallis followed by Dunn’s test for non-normally distributed data. *p-value < 0.05, **p-value < 0.01.

Fig 4: Correlation matrix between SYNCRIP, PLD3, CTSD, SPP1 and C4 and prion neuropathology, in obex (A) and thalamus (B). Spearman correlation coefficients and significance between the semiquantitative scores of immunohistochemical analysis of the five proteins in healthy (n = 7) and preclinical (n = 5) and clinical (n = 7) naturally scrapie-affected sheep and their neuropathological scores: PrPSc deposits (PrPSc), spongiosis (HE), astrogliosis measured with the glial fibrillary acidic protein (GFAP), and microgliosis measured using the ionised calcium-binding adaptor molecule 1 (Iba1). *p-value < 0.05, **p-value < 0.01, ***p-value < 0.001, ^p-value < 0.1 (tendency).