Transmissible spongiform encephalopathies (TSE) are fatal neurodegenerative diseases known to affect several mammalian species, including humans and ruminants. TSEs in ruminants include scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cows and chronic wasting disease (CWD) in cervids. Scrapie is not regarded as a human health risk, whereas BSE has been recognized as the cause of a new TSE in humans defined as variant Creutzfeldt-Jakob disease (vCJD) [1, 2].

The second largest Italian small ruminants population is present in Sicily with about 880 thousands sheep and 132 thousands goats reared in 11,416 farms [3]. A significant biodiversity is represented by three native dairy sheep breeds (Comisana, Valle del Belice and Pinzirita) and five native dairy goat breeds (Girgentana, Messinese, Argentata dell’Etna, Maltese and Rossa Mediterranea or Derivata di Siria), in addition to other Italian breeds. These breeds have an important role in the production of high quality milk requested for typical dairy products.

Prion protein (PrP) is encoded by PRNP gene and is strongly conserved among mammals [4]. Mutations and polymorphisms of PRNP sequences may influence disease susceptibility through modified protein conformation. The PrP polymorphic forms are equally expressed, however in prion material the deposition of the polymorphic-variant PrP^Sc can differ between two allotypes in a heterozygous individual [5–7].

In various sheep breeds the level of risk for scrapie is associated with PRNP genotype [8] particularly at the three codons: 136 (alanine or valine; A, V), 154 (histidine or arginine; H, R), and 171 (glutamine, arginine or histidine; Q, R, H). PRNP haplotypes VRQ and ARQ are considered the most susceptible to classical scrapie, whereas the ARR allele has been linked to resistance [9, 10].

In goats the analysis on PRNP polymorphisms showed that some polymorphic variants can be protective for the disease but their prevalence in different breeds are still under investigation. The polymorphisms that might be related to TSE resistance are present at PRNP codon 222 with a lysine (K) instead of glutamine (Q) [11–16] and at codon 146 with a serine (S) or an aspartic acid (D) instead of asparagine (N) [17, 18]. Strong evidence by experimental challenges in goats, rodents had shown that with the K222 allele a high level of TSE-resistance can be reached even if present in heterozygous animals [19–22].

The active surveillance by rapid diagnosis in sheep and goats started in EU in 2002 on random sampled animals. Fallen and healthy slaughtered animals over 18 months of age were included for a total number of tests that has varied each year. In Italy from 2002 to 2014, 656 positive flocks were detected on a total 620,000 tested animals (Italian Reference Centre for Animal Encephalopathy, personal communication).

The overall results of TSE surveillance and PRNP polymorphisms studies in native small ruminant breeds of Sicily are reported in this paper to evaluate the biodiversity of PRNP gene related to TSE resistance in autochthonous breeds.

The results of passive and active surveillance had shown how 57 scrapie outbreaks have occurred in the period from 1997 to 2014 in Sicily. The disease probably became endemic in the island when an infected vaccine against Mycoplasma agalactiae was used in small ruminant flocks [24]. Scrapie is not regarded as a human health risk, but to reduce the risk of scrapie or BSE in sheep the EU decided to implement selection against susceptible PRNP alleles (Commission Regulation EC 999/2001 and Commission Decisions 2002/1003/EC3 and 2003/100/EC4). Since then, several member states established breeding programs to enrich for resistant alleles. In Sicily a regional public plan for genetic selection in sheep started in 2005 for pure breed animals and was extended to cross breed flocks with more than 200 animals in 2013. In addition, genotyping of several flocks had started in 2005 on voluntary basis particularly to manage scrapie outbreaks. A previous study on limited number of pure breed sheep flocks (Valle del Belice and Comisana) and cross breed animals from five outbreaks showed already a good level of ARR haplotype frequency [25]. The results of a more extended analysis on reproductive males and females and young animals from pure breed sheep flocks in different Sicilian provinces confirmed the high frequency of the resistant haplotypes (Table 2). It is widely recognized that stamping out in sheep is not an efficient strategy for TSEs eradication probably because the pathogenic prion protein is highly persisting in the environment and naïve animals can acquire the infection from the soil [26]. In contrast the selective breeding program is a good way to manage scrapie outbreaks and the best alternative to stamping out of animals which represents a great economic loss for the farmers and a serious threat of extinction for the endangered breeds [27, 28]. The application of breeding programs in goats could also aid the ongoing EU efforts to reduce the chance of scrapie in sheep populations since, goats may represent a possible scrapie reservoir for sheep. This would theoretically also apply if goats were positive for the zoonotic BSE strain [29–31]. In Europe, very few cases of goat scrapie had been recorded prior to 2002 but in Sicily the first clinical case in goat was reported in a mixed flock in 1997 and 27 outbreaks have been diagnosed with 9 including pure breed animals. Any breeding program should consider the endangered status of each goat population to preserve the genetic variability and the biodiversity also when dealing with disease control. For these reasons, the investigation into the relationships between caprine PRNP haplotypes and resistance to scrapie in goat breeds is important [11–17, 31]. With the K222 allele a high level of TSE-resistance can be reached even if present in heterozygous animals [19–22]. Except for Maltese, all other Sicilian goats presented K222 variant in frequency higher than most commercial breeds although more analysis are necessary for Argentata dell’Etna and Rossa Mediterranea.

Our results suggest that a breeding program to select for ARR haplotype in sheep and K222 resistant allele in goat is feasible in the autochthonous breeds of Sicily.

Small ruminants are bred worldwide, particularly in difficult and marginal geographical territories (mountains or dry areas) to which they are well adapted. The native breeds are usually confined but important in low-income rural areas even in developed countries. The focus on increasing biosecurity and quality of local animal production can stimulate the breeding and protection of autochthonous animals through a revival of their economic value as suggested by FAO [32].

The high prevalence of the two TSE resistance related ARR haplotype and K222 allele present in Sicilian sheep and goat breeds respectively, shows how the genetic biodiversity could serve as a resource for the selection of resistance to disease, in our case for TSE eradication. It is very probable that enhancing the frequencies of resistant polymorphisms could be sufficient to reach a sufficiently high level of heterozygosis with the advantage to maintain both biodiversity and resistance. Genetic selection for resistance could assure the full eradication of scrapie in small ruminants. These results may increase interest in the breeding of Sicilian autochthonous breeds preserving the biodiversity on the island. Additionally the promotion of typical dairy and meat products with high quality and biosecurity from autochthonous animals might represent a driving force for the economy in rural areas.

amino acid code, A, alanine; V, valine; R, arginine; Q, glutamine; H, histidine; K, lysine; S, serine; D, aspartic acid; N, asparagine; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; DNA, deoxyribonucleic acid; ELISA, enzyme linked immunosorbent assay; EU, European Union; FAO, Food and Agriculture Organization; PCR, polymerase chain reaction; PRNP, prion protein gene; PrP, prion protein; PrPs, scrapie prion protein; TSE, transmissible spongiform encephalopathy; vCJD, vCreutzfeldt-Jakob disease