Scrapie (classical and atypical) susceptibility in sheep is strongly influenced by PRNP gene polymorphisms. In Portugal, limited data exist for native breeds such as Serra da Estrela, despite their relevance to animal conservation and food production. The full coding region of PRNP gene of 92 Serra da Estrela sheep was sequenced and SNP frequencies were analysed. The predicted functional impact of nonsynonymous SNPs was assessed using PolyPhen-2 and AMYCO. A total of 27 SNPs were identified, including 20 nonsynonymous variants. Thirteen major haplotypes were observed. The ARR allele, which provides resistance to classical scrapie, was present in 58.7% of the population, with 18.5% of animals being homozygous. Several previously unreported SNPs were identified, and their impact on prion protein aggregation propensity and structure was explored. The high frequency of the ARR allele without full ARR fixation suggests that no selective breeding for scrapie resistance has been applied. These results support the adoption of gradual selection strategies that preserve genetic variability and promote farmer compliance, while increasing classical and atypical scrapie resistance.

Sarcocystis spp. are cyst-forming protozoan parasites with a global distribution that infect a wide range of domestic and wild animals, impacting both animal health and livestock productivity. In sheep, infections can cause clinical disease, reproductive losses, and economic damage, particularly when pathogenic species such as Sarcocystis tenella are involved. Grazing sheep, including breeds such as the Serra da Estrela from central Portugal, are at increased risk due to frequent contact with shepherd dogs, which serve as definitive hosts. Despite their significance, data on the occurrence and distribution of Sarcocystis spp. in Portuguese sheep remain limited. This study analyzed 179 samples collected in central Portugal during 2024, including 41 brain tissues and 88 blood samples from sheep, and 50 stool samples from shepherd dogs, using conventional PCR and bidirectional Sanger sequencing. Sarcocystis sp. closely related to S. tenella was detected exclusively in sheep brain tissue, with a prevalence of 4.9% (2/41; 95% CI: 0.60–16.53), while no parasite DNA was found in blood or dog samples. These results provide the first molecular confirmation of Sarcocystis spp. closely related to S. tenella in Portuguese sheep raised for human consumption and establish baseline data for future epidemiological surveillance and control strategies.