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Classical scrapie affects sheep and goats. To control prevalence in sheep, the European Union initiated breeding programs targeting resilient genotypes. Although certain goat polymorphisms, such as Q K, are linked to resistance, specific breeding programs have not been implemented. Hemizygous transgenic mice carrying the goat K cellular prion protein (PrP) allele (K -Tg516) exhibited resistance to several classical scrapie isolates. We inoculated homozygous K -Tg516 and Q -Tg501 mice with various scrapie isolates. Homozygous K -Tg516 mice reached the end of their lifespan without exhibiting clinical signs; we observed brain proteinase K-resistant PrP accumulation in those mice that was lower than in Q -Tg501 mice. Histologically, K -Tg516 brains lacked prion-related lesions, except for the presence of few isolated scrapie PrP plaques in cases of isolates highly adapted to the K -PrP environment. Our findings caution against including that polymorphism in breeding programs, because it could lead to emergence of asymptomatic silent prion carriers of classical scrapie among goat populations.

The agents of prion diseases have the capacity to efficiently infect susceptible hosts by peripheral routes and to project to clinical target areas of the central nervous system (CNS) via peripheral nerves. Understanding the process of prion spread from the site of infection to the CNS may allow us to identify novel therapeutic strategies. To investigate the mechanism involved in the intranerval transit of 263K scrapie prions in golden Syrian hamsters (GSHs), we transected the sciatic nerve at increasing times post-footpad injection and recorded the incubation periods as estimates of the efficiency of infection. We calculated that intranerval transit of this strain of scrapie is at least 10 times faster than previously reported and may reach 50 mm/day, similar to other neurotropic viruses. By in vivo exposure/injection of sciatic nerves to 263K infectivity, we have also shown that prion entry likely occurs via nerve terminals rather than by direct contact with the sciatic nerve. Application of this experimental approach in other forms of prion diseases could allow verification of the timing of neuroinvasion, a relevant parameter for the definition of therapeutic interventions.

Classical scrapie is a prion disease that affects goats and sheep and is transmitted mainly horizontally. However, infection can also occur vertically, from mother to offspring, but the exact mechanism is unclear. The possibilities include transmission during pregnancy, birth, or after birth. Some studies have reported that prions are present in various reproductive tissues and fluids, suggesting that vertical transmission could occur through the germ line. However, the presence of prions in female reproductive tissues and oocytes has not been extensively studied, so their role in vertical transmission is unknown. Oocytes and ovarian tissues from 25 naturally infected ewes (ARQ/ARQ and VRQ/VRQ genotypes) and healthy controls were analysed. The samples were subjected to PMCA for prion detection. Histopathological and immunohistochemical evaluations of ovarian tissues were also performed. We demonstrated that PrP Sc can accumulate in the oocytes and ovaries of scrapie-infected ewes, indicating a potential germline route of transmission. Prion accumulation appears to be influenced by the host genotype and prion strain, emphasizing the need for ultrasensitive detection techniques. Further research under controlled conditions is necessary to elucidate the mechanisms and implications for disease control and breeding programs.

Scrapie is a contagious neurodegenerative disease of sheep and goats sustained by prions. The aim of this study was to assess the effectiveness of selective interventions in Italy by describing the temporal trends and geographical distribution (a) of scrapie prevalence and (b) of the application of the National Genetic Selection Plan (NGSP). Poisson models were used to study temporal trends of disease prevalence (in terms of prevalence ratios, PR), whereas NGSP genotyping data from rams tested between 2005 and 2022 were used to study the temporal trend and geographical distribution of the percentage of susceptible animals. In addition, change-point regression analysis was used to identify changes in the trend. The trend in prevalence at the national level shows an increase in the period 2005–2015 (PR = 1.09, 95% CI 1.04–1.15), followed by a sharp decline between 2016 and 2022 (PR = 0.88, 95% CI 0.83–0.93). The proportion of susceptible rams shows a decreasing temporal trend, both along 2005–2022 (PR 0.97, 95% CI 0.96–0.98) and in the two periods (respectively, 2005–2015: PR 0.92, 95% CI 0.89–0.96; 2016–2022: PR 0.94, 95% CI 0.89–0.98). Change-point regression analysis identified a nationwide trend shift between 2015 and 2020, with Sardinia showing an earlier change between 2012 and 2015. The analysis of temporal trends showed geographical heterogeneity associated with different control strategies, especially in Sardinia, where the early targeted interventions in 2009 anticipated most of the provisions of the second NGSP.

Prions are unorthodox infectious agents that replicate by templating misfolded conformations of a host-encoded glycoprotein, collectively termed PrPSc. Prion diseases are invariably fatal and currently incurable, but oral drugs that can prolong incubation times in prion-infected mice have been developed. Here, we tested the efficacy of combination therapy with two such drugs, IND24 and Anle138b, in scrapie-infected mice. The results indicate that combination therapy was no more effective than either IND24 or Anle138b monotherapy in prolonging scrapie incubation times. Moreover, combination therapy induced the formation of a new prion strain that is specifically resistant to the combination regimen but susceptible to Anle138b. To our knowledge, this is the first report of a pathogen with specific resistance to combination therapy despite being susceptible to monotherapy. Our findings also suggest that combination therapy may be a less effective strategy for treating prions than conventional pathogens.

After the detection of bovine spongiform encephalopathy (BSE), and a zoonotic transmissible spongiform encephalopathy (TSE) caused by the pathological prion protein (PrPSc) in two goats, the investigation of goat prions became of greater interest. Therefore, a broad collection of European goat TSE isolates, including atypical scrapie, CH1641 and goat BSE as reference prion strains were biochemically characterised and subsequently inoculated into seven rodent models for further analysis (already published results of this comprehensive study are reviewed here for comparative reasons). We report here the histopathological and immunohistochemical data of this goat TSE panel, obtained after the first passage in Tgshp IX (tg-shARQ) mice, which overexpress the ovine prion protein. In addition to the clear-cut discrimination of all reference prion strains from the classical scrapie (CS) isolates, we were further able to determine three categories of CS strains. The investigation further indicates the occurrence of sub-strains that slightly resemble distant TSE strains, such as BSE or CH1641, reinforcing the theory that CS is not a single strain but a mixture of sub-strains, existing at varying extents in one isolate. This study further proved that Tgshp IX is a potent and reliable tool for the in-depth characterisation of prion strains.

The role of microRNAs (miRNAs) in neurodegenerative diseases has gained significant attention because of their involvement in gene regulation and potential as biomarkers. In prion diseases, including scrapie, miRNAs may modulate pathogenesis and disease progression. This study investigated circulating miRNA profiles in the blood of sheep naturally affected by scrapie at preclinical and clinical stages using small RNA sequencing and RT-qPCR validation. While only one novel miRNA was dysregulated in preclinical blood samples, 66 previously annotated miRNAs were significantly dysregulated in clinical sheep compared with healthy sheep. These miRNAs are associated with pathways commonly altered in neurodegenerative diseases, such as autophagy, ubiquitin-mediated proteolysis, and endoplasmic reticulum protein processing. Notably, miR-1271-5p, let-7f-5p, miR-186-5p, and miR-425-5p were consistently upregulated in the central nervous system of clinical animals, replicating the results observed in blood, with an increasing trend already in the preclinical stage and a strong correlation with neuropathological prion features. Additionally, predicted target genes such as UBQLN2 , PGK1 , KRAS , and CLTC were inversely expressed relative to these miRNAs, supporting their regulatory roles. These findings highlight the relevance of circulating miRNAs in prion neuropathology and support further research into the specific functional roles of these miRNAs and their predictive capacity for disease progression.

Phenotypic variability in prion diseases, such as scrapie, is associated to the existence of prion strains, which are different pathogenic prion protein (PrP Sc ) conformations with distinct pathobiological properties. To faithfully study scrapie strain variability in natural sheep isolates, transgenic mice expressing sheep cellular prion protein (PrP C ) are used. In this study, we used two of such models to bioassay 20 scrapie isolates from the Spain-France-Andorra transboundary territory. Animals were intracerebrally inoculated and survival periods, proteinase K-resistant PrP (PrP res ) banding patterns, lesion profiles and PrP Sc distribution were studied. Inocula showed a remarkable homogeneity on banding patterns, all of them but one showing 19-kDa PrP res . However, a number of isolates caused accumulation of 21-kDa PrP res in TgShp XI. A different subgroup of isolates caused long survival periods and presence of 21-kDa PrP res in Tg338 mice. It seemed that one major 19-kDa prion isoform and two distinct 21-kDa variants coexisted in source inocula, and that they could be separated by bioassay in each transgenic model. The reason why each model favours a specific component of the mixture is unknown, although PrP C expression level may play a role. Our results indicate that coinfection with more than one substrain is more frequent than infection with a single component.

Scrapie is a transmissible spongiform encephalopathy affecting sheep and goats. The prion protein-encoding gene ( PRNP ) plays a crucial role in determining susceptibility and resistance to scrapie. At the European level, surveillance of scrapie is essential to prevent the spread of the disease to livestock. According to the Regulation EU 2020/772 polymorphisms K222, D/S146 could function as resistance alleles in the genetic management of disease prevention. In Italy, a breeding plan for scrapie eradication has not been implemented for goats. However, surveillance plans based on the PRNP genotype have been developed as a preventive measure for scrapie. This research aimed to describe the polymorphisms at 7 positions within the PRNP gene in 956 goats of the Alpine, Saanen and mixed populations farmed in the Lombardy Region in Italy. PRNP polymorphisms were detected using single nucleotide polymorphism markers included in the Neogen GGP Goat 70 k chip. The K222 allele occurred in all populations, with frequencies ranging from 2.1 to 12.7%. No animals carried the S/D146 resistance allele. However, it has been demonstrated that polymorphisms in the other positions analysed could influence resistance or susceptibility to scrapie outbreaks in different ways. Ten potentially distinct haplotypes were found, and the most prevalent of the three populations was H2, which differed from the wild type (H1) in terms of mutation (S vs P) at codon 240. This study provided additional information on the genetic variability of the PRNP gene in these populations in the Lombardy region of Italy, contributing to the development of genetic control measures for disease prevention.

Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrP Sc  accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8–9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrP Sc  accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie.