Prion protein gene variability in Spanish goats. Inference through susceptibility to classical scrapie strains and pathogenic distribution of peripheral PrP(sc.)

Abstract

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrP(sc)) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrP(sc) distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.

Figure 1. Immunohistochemical distribution of PrP^sc through peripheral tissues.

mAb L42. A) PrP^scdeposits detected in the medullar region of the adrenal gland. B) Detail of PrP^sc distribution in the fascicular region of the adrenal gland. C) and D) Caudal vestibular region of the nasal mucosa. PrP^sc deposits in a spin bundle of the submucosa. E) and F) Tongue. PrP^sc deposits in the lamina propria of the mucosa.

Figure 2. Intraneuronal deposition of PrP^sc by immunohistochemistry.

Maintenance of intraneuronal deposits with the different epitope mapping antibodies. Lateral cunneate nucleus. A) and B) mAb P4; C) and D) mAb R-522; E) and F) mAb 12B2 G) and H) mAb 6C2.

Figure 3. Intraneuronal PrP^sc immunohistochemical profile in the obex, a representative profile is shown (C2).

Maintenance of PrP^sc intraneuronal profile through all analysed antibodies. DMNV: Dorsal Motor Nucleus of the Vagus; Hypo: Hypoglossal nucleus; Cun: Lateral cunneate nucleus; STV: Spinal tract of the trigeminal nerve; RF: Reticular formation; Raphe and Oliv: Olivary nuclei.

Figure 4. VLA Discriminatory western blot (WB) showing samples from C2 to C5 (brain stem) in duplicate lanes.

A) The antibody used to detect PrP^sc was mAb 6H4. Lane 1: Empty; Lanes 2 and 3: Biotinylated molecular weight marker and MagicMark™ XP western protein standard respectively; Lane 4: Classical scrapie +ve; Lanes 5, 8, 13 and 16: BSE +ve; Lanes 6 and 7: C2 classical scrapie +ve; Lanes 9 and 10: C3 classical scrapie +ve; Lanes 11 and 12: C4 classical scrapie +ve; Lanes 14 and 15: C5 classical scrapie +ve. B) The antibody used to detect PrP^sc was mAb P4. Lane 1: Empty; Lanes 2 and 3: Biotinylated molecular weight marker and MagicMark™ XP western protein standard respectively; Lane 4: Classical scrapie +ve; Lanes 5, 8, 13 and 16: BSE +ve; Lanes 6 and 7: C2 classical scrapie +ve; Lanes 9 and 10: C3 classical scrapie +ve; Lanes 11 and 12: C4 classical scrapie +ve; Lanes 14 and 15: C5 classical scrapie +ve.

Figure 5. Bio-Rad western blot (WB) showing samples from C2 to C9 (brain stem) in duplicate lanes.

The antibody used to detect PrP^sc was mAb Sha31 as provided by the protocol test. Relative molecular mass (Mr) for MagicMark markers are shown on the right. A) Lane 1: Kaleidoscope; Lane 2: Classical scrapie +ve; Lane 3: Atypical scrapie +ve; Lanes 4 to 15: C2 to C7 classical scrapie +ve in duplicate lanes; Lanes 16 and 17: Scrapie −ve; Lane 18: MagicMark XP western standard. B) Lane 1: Kaleidoscope; Lane 2: Classical scrapie +ve; Lanes 3, 6 and 7: Atypical scrapie +ve; Lanes 4 and 5: C8 classical scrapie +ve; Lanes 8 and 9: C9 classical scrapie +ve; Lanes 10 to 17: Empty; Lane 18: MagicMark XP western standard.