p.Asn176Lys and p.Met137Thr dimorphisms of the PRNP gene significantly decrease the susceptibility to classical scrapie in ARQ/ARQ sheep.

In this study, we investigated the susceptibility to scrapie of Sarda breed sheep carrying the genotype ARQ/ARQ with additional polymorphisms at the PRNP gene. To do this, we examined 256 scrapie-affected sheep and 320 flock-mate negative controls from 24 flocks. Logistic regression analysis demonstrated that sheep carrying the ARQ/ARQ genotype with additional dimorphisms had lower risk of becoming scrapie affected when compared with those with ARQ/ARQ(wildtype) genotype. ARQ/ARQ genotypes that were detected with heterozygous or homozygous p.Asn176Lys and p.Met137Thr dimorphisms were associated with the lowest susceptibility to the disease. A significant lower risk was also associated with the p.Arg154His dimorphism, while p.Leu141Phe had a protective effect that was not statistically significant.

Characterization of sheep (Ovis aries) palatine tonsil innervation.

Palatine tonsils (PTs), together with ileal Peyer's patches, rank among the first colonization sites for infectious prions. After replicating in these lymphoid tissues, prions undertake the process of "neuroinvasion," which is likely mediated by the peripheral nerves connecting lymphoid tissues to the central nervous system (CNS). To study the connections between the tonsils and the CNS, we injected fluorescent tracers into the PTs of lambs; the highest number of Fast Blue (FB)-labeled neurons was found in cranial cervical ganglia (CCG), whereas a progressively decreasing number of cells were detected in proximal glossopharyngeal, proximal vagal, trigeminal, pterygopalatine, and cervicothoracic ganglia. Immunohistochemistry was carried out on tonsil and ganglia cryosections. Immunoreactivity (IR) for tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DBH), neuronal nitric oxide synthase (nNOS), calcitonin gene-related peptide (CGRP), substance P (SP), and calcium-binding protein S100 (S100), was observed in the fibers around and within PT lymphoid nodules. In the trigeminal, proximal glossopharyngeal and vagal ganglia the retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR. In all ganglia some retrogradely-labeled neurons showed nNOS-, SP- and CGRP-IR co-localization. It is worth noting that only 66+/-19% and 75+/-13% of retrogradely-labeled neurons in CCG showed TH- and DBH-IR, respectively. The present results allow us to attribute PT innervation mainly to the sympathetic component and to the glossopharyngeal, vagal and trigeminal cranial nerves. Furthermore, these data also provide a plausible anatomic route through which infectious agents, such as prions, may access the CNS, i.e. by traveling along several cranial and sympathetic nerves, as well as by migration via glial cells.

Ileal tract and Peyer's patch innervation in scrapie-free versus scrapie-affected ovines.

Ileal Peyer's patches (PPs) are involved early during sheep scrapie infection. This study qualitatively and semi-quantitatively evaluated ileal tract and PP innervation in 29 Sarda ovines of different age, PrP genotype and scrapie status. A prominent network of fibres was detected within PPs, mainly located in interfollicular lymphoid and stromal components. Intrafollicular fibres were rarely observed, with no apparent differences between scrapie-free and scrapie-affected animals, or among ovines carrying different PrP genotypes. In adult sheep, independent of their scrapie status, nerve fibres could be detected infrequently, close to the follicle-associated epithelium. Fibres were also detected within newly formed follicles and intrafollicular microgranulomas.

A survey of Taenia multiceps coenurosis in Sardinian sheep.

A survey was carried out to assess the occurrence of Coenurus cerebralis infection in Sardinian sheep. A prevalence of 0.35% was observed when 566 regularly slaughtered sheep were examined. However, in 120 sheep with suspected symptoms of coenurosis examined from November 2001 to October 2002, a total of 299 cerebral coenurosis lesions were observed with an incidence of 1% per year. Lesions were classified as migratory, cystic and secondary. Most migratory lesions were found in sheep aged 3-6 months. Cavitary lesions containing cysts in different developing stages were found with high incidence per year in sheep aged 7-12 months. Secondary lesions due to the development of Coenurus were most frequent in sheep aged 19-36 months. Most sheep were found infected in spring and in early summer, between March and June. Most lesions were located in the cortex. The mean number of protoscolices per cyst was 149 (range 10-370).

PrP(Sc) deposition in nervous tissues without lymphoid tissue involvement is frequently found in ARQ/ARQ Sarda breed sheep preclinically affected with natural scrapie.

The pathogenesis of natural scrapie in Sarda breed sheep was investigated in 1050 asymptomatic and 49 sick sheep from scrapie-affected flocks. Central and peripheral nervous system, along with lymphoreticular system (LRS) tissues, were subjected to immunohistochemistry (IHC) and Western-blotting (WB) for detection of pathological isoform of the prion protein (PrP(Sc)). A total of 69 of the 1050 clinically healthy sheep were found to be infected with scrapie, with PrP(Sc) being detected in both the central nervous system (CNS) and enteric nervous system (ENS) plexuses of 60 of the sheep, while IHC and WB yielded evidence of (PrP(Sc)) deposition only in lymphoid tissues of the remaining 9 clinically healthy sheep. PrP(Sc) was also detected in the CNS, as well as in ENS plexuses from all of the 49 clinically affected sheep. Nevertheless, 18 of the 69 clinically healthy animals (26%, 17 ARQ/ARQ and 1 ARQ/AHQ sheep), along with 3 ARQ/ARQ sheep (6%) of the clinically affected group, showed no IHC or WB evidence of PrP(Sc) in lymphoid tissues, but PrP(Sc) could be still detected in their CNS and ENS plexuses. The study demonstrates dual CNS and ENS PrP(Sc) deposition in Sarda sheep with scrapie, in spite of an apparent lack of lymphoid tissue involvement in a number of cases.

Distribution of vascular amyloid in scrapie-affected sheep with different genotypes.

Vascular amyloidosis in the brain is a pathological feature of ovine scrapie. Its occurrence varies between sheep, but whether this variation reflects differences in the host or the infecting scrapie strain (or both) is not clear. To investigate whether amyloidosis, like vacuolation and PrPsc distribution, is associated with genotype, the brains from 131 sheep representing a range of genotypes commonly associated with scrapie were examined histologically and immunohistochemically. Vascular amyloidosis was absent in 66 sheep, 59 of which were of the ARQ/ARQ genotype and seven the ARQ/AHQ genotype. In contrast, it was found in four of 39 ARQ/VRQ sheep (10.2%) and in 10 of 26 VRQ/VRQ sheep (38.4%). The distribution of amyloid was highly variable, but the most severely affected areas were the lateral geniculate nuclei (five cases) and the ventral thalamic nuclei (four cases). No amyloidosis was found in the medulla or in the basal nuclei. From this preliminary study it was concluded that amyloidosis is relatively rare in sheep with scrapie. Moreover, its occurrence appeared to depend on the presence of at least one valine at codon 136.

Clinicopathological findings in sheep from Sardinia showing neurological signs of disease.

Histopathological and bacteriological examinations were performed on 178 brains from Sardinian sheep which were showing neurological signs. The sheep represented the total number of sheep with neurological syndromes submitted for diagnostic investigations over a three-year period in Sardinia. Scrapie was detected in 57 cases, cerebrocortical necrosis in 25, intoxication by a typical Mediterranean plant (Cistus species) was suspected in 25, coenurosis was detected in 11 cases, Listeria monocytogenes in eight cases and focal symmetrical encephalomalacia in six cases. Non-suppurative inflammatory changes were observed in three of the brains and suppurative changes were noted in two. Lesions restricted to the spinal cord were found in three cases. In the remaining 38 cases there were no significant neuropathological changes.

Animal transmissible spongiform encephalopathies and genetics.

The genotype of the host plays a crucial role in the pathogenesis of transmissible spongiform encephalopathies (TSEs). In this respect, the most important factor is represented by the gene of the prion protein (PrP). The present work summarizes the currently available knowledge on the genetic basis of TSEs focusing, in particular, on sheep scrapie. Interest in this disease has grown markedly following the discovery of bovine spongiform encephalopathy, both for scientific and health reasons. In Italy, specific research grants from the Ministry of Health and the National Research Council (CNR), together with cooperation between the Istituto Superiore di Sanità and the Istituti Zooprofilattici Sperimentali, have allowed us to study the PrP genotype and to investigate the genetic susceptibility to scrapie in the most important Italian sheep breeds, with special reference to Sarda, Comisana and Massese. The PrP genotype in relation to scrapie susceptibility was also studied in goats of Ionica breed.

Distinction of scrapie phenotypes in sheep by lesion profiling.

Major determinants of the pathological phenotype of natural scrapie are considered to be the agent strain and host prion protein (PrP) genotype, but the relationship between these is far from clear. Little is known about the strains that produce natural scrapie. A method of brain vacuolation profiling was developed which enables this aspect of disease phenotype to be characterized in detail. This method distinguished at least two distinct pathological phenotypes in sheep of a single genotype (ARQ/ARQ) from different flocks in the UK. Great similarity was also demonstrated between one of these phenotypes and the phenotype of sheep from a flock in Sardinia. The profile of four sheep of the same ARQ/ARQ genotype experimentally infected with bovine spongiform encephalopathy (BSE) was determined for comparison. It would appear from these preliminary observations that the application of lesion profiling techniques to ovine transmissible spongiform encephalopathy (TSE) may contribute to the definition of a particular scrapie phenotype within a flock. It may, therefore, have potential for improving our understanding of current TSE phenotypes in sheep, with regard to the possibility of identifying those of bovine origin.

PrP genotype in Sarda breed sheep and its relevance to scrapie. Brief report.

Several PrP gene polymorphisms modulate sheep scrapie susceptibility. Recently, an increase of scrapie outbreaks has been reported in Italy. A vaccine containing sheep brain homogenate was used in most of the outbreaks. We investigated PrP gene polymorphisms in scrapie-affected and clinically healthy Sarda breed sheep from a flock exposed to the aforementioned vaccine, and in affected Sarda sheep from unexposed flocks. All affected animals were (Gln/Gln)171 homozygous. Moreover, we observed no variation for Ala136 and a new polymorphism (Lys to Asn) at codon 176. Our findings confirm the correlation between scrapie and (Gln/Gln)171 in breeds with no variation for Ala136.