Demonstration of lateral transmission of scrapie between sheep kept under natural conditions using lymphoid tissue biopsy

Scrapie free adult sheep were introduced to a sheep flock specifically maintained to maximise scrapie infection. Native born sheep of the highly susceptible VRQ/VRQ genotype in this flock show highly efficient transmission, evidenced by 100% infection, with an age at death of less than 2 years. Infection in introduced sheep was identified by biopsy of tonsilar and nictitating membrane lymphoid tissue. Progeny of these sheep were monitored and clinical disease confirmed by examination of the brain using routine diagnostic methods. Na?ve sheep of New Zealand origin introduced to the flock in adulthood became infected, demonstrating that lateral transmission had occurred. Lambs born to introduced ewes became infected and died at the same age as lambs born to native ewes, consistent with lateral transmission of scrapie to lambs. Although maternal transmission cannot be totally excluded for the lambs in this study, the data are consistent with lateral transmission being the most important means of spread leading to the high incidence of scrapie observed in this flock.     

Susceptibility of transgenic mice expressing chimeric sheep,bovine and human PrP genes to sheep scrapie

The use of Transgenic (Tg) mice expressing chimeric sheep/mouse (Sh/Mo) prion protein (PrP) and chimeric bovine/mouse (Bo/Mo) PrP genes was evaluated as a sheep scrapie model. We also investigated the potential for the transmission of sheep scrapie to a human/mouse (Hu/Mo) PrP Tg mouse line. The Sh/Mo PrP and Bo/Mo PrP Tg Prnp(+/+) or Prnp(0/0) mouse lines were inoculated intracerebrally with brain homogenates from three sheep with natural scrapie (KU, Y5 or S2). Incubation periods were slightly shorter in Sh/Mo PrP Tg Prnp(+/+), than in non-Tg mice inoculated with KU brain homogenate. In contrast, the incubation period was significantly prolonged (p<0.05) in Bo/Mo PrP Tg Prnp(+/+) mice inoculated with KU brain homogenate. The incubation period was significantly longer in all Tg Prnp(+/+) and Prnp(0/0), than in non-Tg mice (p<0.01) inoculated withY5 brain homogenate. None of the Tg Prnp(0/0) mice inoculated with S2 brain homogenate developed clinical signs and PrP(Sc) was undetectable in their brains. These results suggested that expression of the Sh/Mo PrP or Bo/Mo PrP transgenes does not confer susceptibility to sheep prions upon mice, and thus none of the Tg mouse lines could be a suitable model of sheep scrapie. Hu/Mo PrP Tg Prnp(0/0) mice inoculated with natural and experimental scrapie or mouse prions did not develop clinical signs of scrapie and PrP(Sc) was undetectable. These results suggested that neither sheep nor mouse strains of scrapie are highly transmissible to humans.     

Accumulation of pathogenic prion protein (PrPSc) in nervous and lymphoid tissues of sheep with subclinical scrapie

All sheep older than 1 year of age from a flock of the Rygja breed in which clinical scrapie was detected for the first time in two animals (4%) were examined for accumulation of pathogenic prion protein (PrPSc) by immunohistochemistry in the obex, the cerebellum, and the medial retrophayngeal lymph node. In addition, six lambs, 2-3 months old, all offspring of PrPSc-positive dams, were examined for PrPSc in the ileal Peyers' patch (IPP), the distal jejunal lymph node, the spleen, and the medial retropharyngeal lymph node (RPLN). In this flock, 35% (17/48) of the adult sheep showed accumulation of PrPSc, an eightfold increase compared with clinical disease. All positives carried susceptible PrP genotypes. Three sheep had deposits of PrPSc in the RPLN and not in the brain, suggesting that this organ, easily accessible at slaughter, is suitable for screening purposes. Two 7-year-old clinically healthy homozygous V136Q171 ewes showed sparse immunostaining in the central nervous system and may have been infected as adults. Further, two littermates, 86-days-old, showed PrPSc in the IPP. Interestingly, one of these lambs had the intermediate susceptible PrP genotype, VA136QR171. In addition to early immunolabeling in the dorsal motor nucleus of the vagal nerve, a few of the sheep had early involvement of the cerebellum. In fact, a 2-year-old sheep had sparse deposits of PrPSc in the cerebellum only. Because experimental bovine spongiform encephalopathy (BSE) in sheep seems to behave in a similar manner as natural scrapie, these results, particularly regarding spread of infectivity, may have implications for the handling of BSE should it be diagnosed in sheep.     

Experimental transmission of sheep scrapie by intracerebral and oral routes to genetically susceptible Suffolk sheep in the United States.

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent on the genetic makeup of the host. This study documents clinicopathological findings and the distribution of abnormal prion proteins (PrPres) by immunohistochemical and Western blot techniques, in tissues of genetically susceptible sheep inoculated with US sheep scrapie agents. Four-month-old Suffolk lambs (QQ or HQ at codon 171) were inoculated (5 intracerebrally and 19 orally) with an inoculum (#13-7) consisting of a pool of scrapie-affected sheep brains. Intracerebrally inoculated animals were euthanized when advanced clinical signs of scrapie were observed. Orally inoculated animals were euthanized at predetermined time points (4, 9, 12, 15, and 21 months postinoculation [PI]) and thereafter when the animals had terminal signs of disease. All intracerebrally inoculated animals exhibited clinical signs of scrapie and were euthanized between 13 and 24 months PI. Spongiform lesions in the brains and PrPres deposits in central nervous system and lymphoid tissues were present in these sheep. In orally inoculated sheep, clinical signs of scrapie were seen between 27 and 43 months PI in 5/9 animals. The earliest detectable PrPres was observed in brainstem and lymphoid tissues of a clinically normal, orally inoculated sheep at 15 months PI. Three of the 4 clinically normal sheep were positive at 15, 20, and 49 months PI by PrPres immunohistochemistry.     

PrP genotype frequencies in German breeding sheep and the potential to breed for resistance to scrapie

Genetic susceptibility to scrapie is associated with polymorphisms in three different codons of the ovine prion protein (PrP) gene (136, 154, 171). Studies of PrP genotypes linked to scrapie have revealed the resistance of homozygous PrPARR/PrPARR animals and the high risk of PrPVRQ/PrPVRQ and PrPvRQ/PrPARQ animals in scrapie-affected flocks. The selection of PrPARR/PrPARR genotypes may therefore provide a strategy for controlling clinical scrapie. The genotypes of 1361 German breeding sheep from 15 different breeds in northern Germany were determined. Apart from the wildtype allele PrPARQ, at least four mutually exclusive allelic variants were found. The greatest variability within the PrP gene was encountered in texel sheep, in which 14 PrP genotypes were found. In the important meat breeds, Suffolk, German whiteheaded mutton and German blackheaded mutton, the PrPARR allele was predominant, and in these breeds the breeding of scrapie-resistant pedigree flocks within four generations seems to be a feasible option. In the texel sheep, the German merino, the German milk and the German land sheep breeds, the frequency of the PrPARR allele was much lower, and in several breeds no homozygous rams were available for breeding purposes. In these breeds the breeding strategy would depend on the number of heterozygous rams available, but resistant pedigree flocks could be achieved within nine generations.     

Transcriptional profiling of inflammatory cytokine genes in African buffaloes (Syncerus caffer) infected with Theileria parva

Theileria parva (T. parva) causes East Coast fever (ECF), which is of huge economic importance to Eastern and Southern African countries. In a previous bovine model, inflammatory cytokines were closely associated with disease progression in animals experimentally infected with T. parva. The African Cape buffalo (Syncerus caffer), the natural reservoir for T. parva, is completely resistant to ECF despite a persistently high parasitaemia following infection with T. parva. Characterizing basic immunological interactions in the host is critical to understanding the mechanism underlying disease resistance in the African Cape buffalo. In this study, the expression level of several cytokines was analyzed in T. parva-infected buffaloes. There were no significant differences in the expression profiles of inflammatory cytokines between the infected and uninfected animals despite a remarkably high parasitaemia in the former. However, the expression level of IL-10 was significantly upregulated in the infected animals. These results indicate a correlation between diminished inflammatory cytokines response and disease resistance in the buffalo.     

Clinical and pathological features of experimental scrapie in Irish Blackface Mountain sheep

There have been no reports of natural scrapie in Irish Blackface Mountain (BM) sheep which account for approximately 16% of the Irish national sheep flock. The aim of this study was to determine if Irish BM sheep had unusual clinical and/or pathological features of scrapie which would account for failure to diagnose the disease in this breed. BM (n=7), Texel (n=3) and Suffolk sheep (n=1) of scrapie-susceptible PrP genotypes (ARQ/ARQ and VRQ/ARQ) were orally challenged with scrapie-infected brain inoculum. The incubation period, clinical signs, pathology and distribution of disease specific prion protein (PrP(d)) in scrapie-affected BM sheep were similar to scrapie in the Texel and Suffolk sheep. It was concluded that there was no evidence to suggest that scrapie in BM sheep differs clinicopathologically from scrapie in other breeds of sheep.     

Breeding with resistant rams leads to rapid control of classical scrapie in affected sheep flocks

ABSTRACT: Susceptibility to scrapie, a transmissible spongiform encephalopathy in sheep, is modulated by the genetic make-up of the sheep. Scrapie control policies, based on selecting animals of resistant genotype for breeding, have recently been adopted by the Netherlands and other European countries. Here we assess the effectiveness of a breeding programme based on selecting rams of resistant genotype to obtain outbreak control in classical scrapie-affected sheep flocks under field conditions. In six commercially-run flocks following this breeding strategy, we used genotyping to monitor the genotype distribution, and tonsil biopsies and post-mortem analyses to monitor the occurrence of scrapie infection. The farmers were not informed about the monitoring results until the end of the study period of six years. We used a mathematical model of scrapie transmission to analyze the monitoring data and found that where the breeding scheme was consistently applied, outbreak control was obtained after at most four years. Our results also show that classical scrapie control can be obtained before the frequency of non-resistant animals is reduced to zero in the flock. This suggests that control at the national scale can be obtained without a loss of genetic polymorphisms from any of the sheep breeds.     

Susceptibility to scrapie and disease phenotype in sheep: cross-PRNP genotype experimental transmissions with natural sources

It has long been established that the sheep Prnp genotype influences the susceptibility to scrapie, and some studies suggest that it can also determine several aspects of the disease phenotype. Other studies, however, indicate that the source of infection may also play a role in such phenotype. To address this question an experiment was set up in which either of two different natural scrapie sources, AAS from AA₁₃₆ Suffolk and VVC from VV₁₃₆ Cheviot sheep, were inoculated into AA₁₃₆, VA₁₃₆ and VV₁₃₆ sheep recipients (n = 52). The immunohistochemical (IHC) profile of disease-associated PrP (PrP^d) accumulation in the brain of recipient sheep was highly consistent upon codon 136 homologous and semi-homologous transmission, but could be either similar to or different from those of the inoculum donors. In contrast, the IHC profiles were highly variable upon heterologous transmission (VVC to AA₁₃₆ and AAS to VV₁₃₆). Furthermore, sheep of the same Prnp genotype could exhibit different survival times and PrP^d profiles depending on the source of infection, and a correlation was observed between IHC and Western blot profiles. It was found that additional polymorphisms at codons 112 or 141 of AA₁₃₆ recipients resulted in a delayed appearance of clinical disease or even in protection from infection. The results of this study strongly suggest that the scrapie phenotype in sheep results from a complex interaction between source, donor and recipient factors, and that the Prnp genotype of the recipient sheep does not explain the variability observed upon codon 136 heterologous transmissions, arguing for other genetic factors to be involved.     

Rapid testing leads to the underestimation of the scrapie prevalence in an affected sheep and goat flock

To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrP(Sc) in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrP(Sc) depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrP(Sc) was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrP(Sc) exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy.     

Detection and clinical evolution of scrapie in sheep by 3rd eyelid biopsy

The goal of this article was to characterize the clinical evolution of scrapie in naturally affected sheep. Eighteen sheep with scrapie diagnosed by examination of 3rd eyelid biopsy and 12 control ewes were studied throughout the duration of their disease. Diagnosis was confirmed postmortem by histopathologic, immunohistochemical, and Western blot analysis of nervous tissue. Complete clinical examinations were performed every 2 weeks for each animal, of which 3 clinical examinations per animal are reported. Those clinical signs that showed a significant frequency within the corresponding clinical examination were considered representative of each stage of the disease (ie, early, middle, and late). The representative clinical signs for the early stage were hypoesthesia in the limbs, alteration of mental status, and a body condition score <3. Remarkably, hypoesthesia in the limbs was one of the 1st signs appearing during the early clinical stage in the affected animals, even before the appearance of other signs. For the middle stage, representative signs were the same as those for the early stage, together with hyporreflexia in the limbs, cardiac arrhythmia, pruritus/wool loss, and the appearance of the nibbling reflex. Representative clinical signs for the late stage were the same as those for the early and middle stage, together with head tremors, hyperexcitability to external stimuli, ataxia or gait abnormalities, and teeth grinding. On the basis of these results, we propose the calculation of an objective clinical index that allows the differentiation among clinical stages and that could be useful for further studies. The usefulness of 3rd eyelid lymphoid tissue biopsies for sequential clinical studies in naturally scrapie-affected sheep is demonstrated.     

湖羊和巴什拜羊FSHR基因多态性分析

本文旨在了解绵羊(湖羊和巴什拜羊)促卵泡激素受体(FSHR)基因3'-UTR特征,分析高、低繁殖力绵羊品种FSHR基因3'-UTR突变位点多态性差异,并探索其机制。研究获得788 bp的湖羊和巴什拜羊FSHR基因3'-UTR序列,两者一致性为98.17%,且均含有加尾信号、保守的ARE元件和多个miRNA结合位点;池DNA测序在FSHR基因终止密码子后325nt处发现一个碱基T插入/缺失,命名为*325del T;多态性分析发现绵羊FSHR基因*325del T位点有3种基因型(TT型、T-型和--型),在湖羊群体中T为优势等位基因(频率为0.604),而在巴什拜羊群体中-为优势等位基因(频率为0.635);荧光素酶活性分析发现*325del T突变对绵羊FSHR转录活性无显著影响。发现FSHR基因*325del T的多态性可能与绵羊繁殖性能有关。