Diagnosis in Scrapie: Conventional Methods and New Biomarkers.

Scrapie, a naturally occurring prion disease affecting goats and sheep, comprises classical and atypical forms, with classical scrapie being the archetype of transmissible spongiform encephalopathies. This review explores the challenges of scrapie diagnosis and the utility of various biomarkers and their potential implications for human prion diseases. Understanding these biomarkers in the context of scrapie may enable earlier prion disease diagnosis in humans, which is crucial for effective intervention. Research on scrapie biomarkers bridges the gap between veterinary and human medicine, offering hope for the early detection and improved management of prion diseases.

Novel polymorphisms in the prion protein gene (PRNP) and stability of the resultant prion protein in different horse breeds.

Prion diseases are fatal neurodegenerative disorders in which the main pathogenic event is the conversion of the cellular prion protein (PrPC) into an abnormal and misfolded isoform known as PrPSc. Most prion diseases and their susceptibility and pathogenesis are mainly modulated by the PRNP gene that codes for PrP. Mutations and polymorphisms in the PRNP gene can alter PrPC amino acid sequence, leading to a change in transmission efficiency depending on the place where it occurs. Horses are animals that are considered to be highly resistant to prions. Several studies have attempted to identify polymorphisms in the PRNP gene that explain the reason for this high resistance. In this study, we have analysed 207 horses from 20 different breeds, discovering 3 novel PRNP polymorphisms. By using computer programmes such as PolyPhen-2, PROVEAN, PANTHER, Meta-SNP and PredictSNP, we have predicted the possible impact that these new polymorphisms would have on the horse prion protein. In addition, we measured the propensity for amyloid aggregation using AMYCO and analysed the lack of hydrogen bridges that these changes would entail together with their electrostatic potentials using Swiss-PdbViewer software, showing that an increased amyloid propensity could be due to changes at the level of electrostatic potentials.

Heterogeneity of pathological prion protein accumulation in the brain of moose (Alces alces) from Norway, Sweden and Finland with chronic wasting disease.

Prion diseases are a group of neurodegenerative, transmissible, and fatal disorders that affect several animal species. They are characterized by the conformational conversion of the cellular prion protein (PrPC) into the pathological prion protein (PrPSc). In 2016, chronic wasting disease (CWD) gained great importance at European level due to the first disease detection in a wild reindeer (Rangifer tarandus) in Norway. The subsequent intensive CWD surveillance launched in cervids resulted in the detection of CWD in moose (Alces alces), with 11 cases in Norway, 3 in Finland and 4 in Sweden. These moose cases differ considerably from CWD cases in North American and reindeer in Norway, as PrPSc was detectable in the brain but not in lymphoid tissues. These facts suggest the occurrence of a new type of CWD. Here, we show some immunohistochemical features that are clearly different from CWD cases in North American and Norwegian reindeer. Further, the different types of PrPSc deposits found among moose demonstrate strong variations between the cases, supporting the postulation that these cases could carry multiple strains of CWD.

Peripheral neuropathy caused by fenugreek (Trigonella foenum-graecum) straw intoxication in cattle and experimental reproduction in sheep and goats.

Trigonella foenum-graecum (fenugreek) is a legume widely used as a food supplement in humans and less frequently in ruminants. Toxicity has been described sporadically in ruminants grazing mature fenugreek plants or stubble; however, the pathological features are unclear. This report describes a natural outbreak of intoxication in cattle fed fenugreek straw and the experimental reproduction using 8 sheep and 8 goats. Affected cattle presented clinical signs approximately 1 month after consuming the straw and 100 of 400 cattle (25%) were affected, of which 60 of 100 (60%) died or were euthanized. Clinical signs were characterized by proprioceptive positioning defects with abnormal postures and weakness of hindlimbs. Forelimbs were also affected in severely affected animals, and cattle became recumbent. Locomotion was characterized by trembling, and some cattle showed high-stepping movements of their forelimbs and knuckled over in their fetlocks. Experimental intoxication induced clinical signs only in sheep and were similar to cattle, although with signs starting in the forelegs. Gross and microscopic lesions were similar in spontaneous and experimental intoxications. Macroscopic changes corresponded with muscular hemorrhages and edema, mainly surrounding the peripheral nerves. Microscopic examination only demonstrated lesions in the distal peripheral nerves, which included edema, hemorrhages, and Wallerian degeneration. Neurofilament immunohistochemistry revealed altered axon labeling and S100 showed a decrease in myelin intensity and loss of its typical compact arrangement around axons. Biochemical and hematological abnormalities included elevated levels of muscle and liver enzymes and thrombocytopenia. These findings indicate that fenugreek straw induces peripheral neuropathy in cattle and sheep, but not in goats.

Classical and Atypical Scrapie in Sheep and Goats. Review on the Etiology, Genetic Factors, Pathogenesis, Diagnosis, and Control Measures of Both Diseases.

Prion diseases, such as scrapie, are neurodegenerative diseases with a fatal outcome, caused by a conformational change of the cellular prion protein (PrPC), originating with the pathogenic form (PrPSc). Classical scrapie in small ruminants is the paradigm of prion diseases, as it was the first transmissible spongiform encephalopathy (TSE) described and is the most studied. It is necessary to understand the etiological properties, the relevance of the transmission pathways, the infectivity of the tissues, and how we can improve the detection of the prion protein to encourage detection of the disease. The aim of this review is to perform an overview of classical and atypical scrapie disease in sheep and goats, detailing those special issues of the disease, such as genetic factors, diagnostic procedures, and surveillance approaches carried out in the European Union with the objective of controlling the dissemination of scrapie disease.

SARS-CoV-2 Outbreak on a Spanish Mink Farm: Epidemiological, Molecular, and Pathological Studies.

Farmed minks have been reported to be highly susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and may represent a risk to humans. In this study, we describe the first outbreak of SARS-CoV-2 occurred on a mink farm in Spain, between June and July 2020, involving 92,700 animals. The outbreak started shortly after some farm workers became seropositive for SARS-CoV-2. Minks showed no clinical signs compatible with SARS-CoV-2 infection throughout the outbreak. Samples from 98 minks were collected for histopathological, serological, and molecular studies. Twenty out of 98 (20.4%) minks were positive by RT-qPCR and 82 out 92 (89%) seroconverted. This finding may reflect a rapid spread of the virus at the farm with most of the animals overcoming the infection. Additionally, SARS-CoV-2 was detected by RT-qPCR in 30% of brain samples from positive minks. Sequencing analysis showed that the mink sequences were not closely related with the other mink SARS-CoV-2 sequences available, and that this mink outbreak has its probable origin in one of the genetic variants that were prevalent in Spain during the first COVID-19 epidemic wave. Histological studies revealed bronchointerstitial pneumonia in some animals. Immunostaining of viral nucleocapsid was also observed in nasal turbinate tissue. Farmed minks could therefore constitute an important SARS-CoV-2 reservoir, contributing to virus spread among minks and humans. Consequently, continuous surveillance of mink farms is needed.

Fatal stagger poisoning by consumption of Festuca argentina (Speg.) Parodi in goats from Argentine Patagonia.

The present study describes the spontaneous and experimental poisoning of goats by Festuca argentina in Argentine Patagonia. In April 2017, eight seven-month-old Creole male goats were accidentally introduced into a paddock that contained F. argentina. After four days, two of the goats were found dead and four out of the six remaining goats were clinically affected. Two of the latter had to be later euthanized in extremis. The main clinical signs were progressive nervous signs, starting with moderate muscle tremors, wide-based stance and ataxia. Postmortem examination was performed on the two euthanized goats. Epidermal fragments of F. argentina were found in the rumen samples from the necropsied goats and the fecal samples from the four affected goats. For the experimental poisoning, fresh sheaths of F. argentina collected from the paddock were offered to two goats at 10 g/kg body weight for 3 days. After 24-36 h, both animals exhibited severe muscle tremors, reluctance to move, tetanic convulsions, and opisthotonus. In both the spontaneously and experimentally poisoned goats, gross lesions were similar and consisted of dehydration, petechial hemorrhages in the epicardium and congestion. The main microscopic findings consisted of degeneration and loss of Purkinje cells and torpedoes in the granular layer of the cerebellum. The F. argentina sheaths collected from the pasture were found to contain tremorgenic indole-diterpene alkaloids. Taken together, the results of the present study suggest that the tremorgenic syndrome observed in the spontaneously poisoned goats was due to poisoning by F. argentina.

Characterization of goat prions demonstrates geographical variation of scrapie strains in Europe and reveals the composite nature of prion strains.

Bovine Spongiform Encephalopathy (BSE) is the only animal prion which has been recognized as a zoonotic agent so far. The identification of BSE in two goats raised the need to reliably identify BSE in small ruminants. However, our understanding of scrapie strain diversity in small ruminants remains ill-defined, thus limiting the accuracy of BSE surveillance and spreading fear that BSE might lurk unrecognized in goats. We investigated prion strain diversity in a large panel of European goats by a novel experimental approach that, instead of assessing the neuropathological profile after serial transmissions in a single animal model, was based on the direct interaction of prion isolates with several recipient rodent models expressing small ruminants or heterologous prion proteins. The findings show that the biological properties of scrapie isolates display different patterns of geographical distribution in Europe and suggest that goat BSE could be reliably discriminated from a wide range of biologically and geographically diverse goat prion isolates. Finally, most field prion isolates showed composite strain features, with discrete strain components or sub-strains being present in different proportions in individual goats or tissues. This has important implications for understanding the nature and evolution of scrapie strains and their transmissibility to other species, including humans.

Four types of scrapie in goats differentiated from each other and bovine spongiform encephalopathy by biochemical methods.

Scrapie in goats has been known since 1942, the archetype of prion diseases in which only prion protein (PrP) in misfolded state (PrPSc) acts as infectious agent with fatal consequence. Emergence of bovine spongiform encephalopathy (BSE) with its zoonotic behaviour and detection in goats enhanced fears that its source was located in small ruminants. However, in goats knowledge on prion strain typing is limited. A European-wide study is presented concerning the biochemical phenotypes of the protease resistant fraction of PrPSc (PrPres) in over thirty brain isolates from transmissible spongiform encephalopathy (TSE) affected goats collected in seven countries. Three different scrapie forms were found: classical scrapie (CS), Nor98/atypical scrapie and one case of CH1641 scrapie. In addition, CS was found in two variants-CS-1 and CS-2 (mainly Italy)-which differed in proteolytic resistance of the PrPres N-terminus. Suitable PrPres markers for discriminating CH1641 from BSE (C-type) appeared to be glycoprofile pattern, presence of two triplets instead of one, and structural (in)stability of its core amino acid region. None of the samples exhibited BSE like features. BSE and these four scrapie types, of which CS-2 is new, can be recognized in goats with combinations of a set of nine biochemical parameters.

Low sequence diversity of the prion protein gene (PRNP) in wild deer and goat species from Spain.

The first European cases of chronic wasting disease (CWD) in free-ranging reindeer and wild elk were confirmed in Norway in 2016 highlighting the urgent need to understand transmissible spongiform encephalopathies (TSEs) in the context of European deer species and the many individual populations throughout the European continent. The genetics of the prion protein gene (PRNP) are crucial in determining the relative susceptibility to TSEs. To establish PRNP gene sequence diversity for free-ranging ruminants in the Northeast of Spain, the open reading frame was sequenced in over 350 samples from five species: Iberian red deer (Cervus elaphus hispanicus), roe deer (Capreolus capreolus), fallow deer (Dama dama), Iberian wild goat (Capra pyrenaica hispanica) and Pyrenean chamois (Rupicapra p. pyrenaica). Three single nucleotide polymorphisms (SNPs) were found in red deer: a silent mutation at codon 136, and amino acid changes T98A and Q226E. Pyrenean chamois revealed a silent SNP at codon 38 and an allele with a single octapeptide-repeat deletion. No polymorphisms were found in roe deer, fallow deer and Iberian wild goat. This apparently low variability of the PRNP coding region sequences of four major species in Spain resembles previous findings for wild mammals, but implies that larger surveys will be necessary to find novel, low frequency PRNP gene alleles that may be utilized in CWD risk control.

Protein misfolding cyclic amplification corroborates the absence of PrPSc accumulation in placenta from foetuses with the ARR/ARQ genotype in natural scrapie.

Ovine scrapie is a worldwide spread prion disease that is transmitted horizontally under field conditions. Placenta from scrapie-infected ewes is an important source of infection, since this tissue can accumulate high amounts of PrPSc depending on the foetal genotype. Therefore, placentas carrying susceptible foetuses can accumulate PrPSc but there is not PrPSc accumulation in presence of foetuses with at least one ARR haplotype. In scrapie eradication programs, ARR/ARR males are used for breeding to increase the resistant progeny and reduce the horizontal transmission of the disease through the placenta. The development of highly sensitive techniques, that allow the detection of minimal amounts of PrPSc, has caused many secretions/excretions and tissues that had previously been deemed negative to be relabeled as positive for PrPSc. This has raised concerns about the possible presence of minimal amounts of PrPSc in placentas from ARR foetuses that conventional techniques had indicated were negative. In the present study we examined 30 placentas from a total of 23 gestations; 15 gestations resulted from naturally ARQ/ARQ scrapie-infected ewes mated with ARR/ARR rams. The absence of PrPSc in placentas carrying the foetal ARR haplotype (n=19) was determined by IDEXX HerdChek scrapie/BSE Antigen EIA Test, Prionics®-Check WESTERN and corroborated by the highly sensitive Protein Misfolding Cyclic Amplification technique (PMCA). By immunohistochemistry, several unspecific stainings that might mislead a diagnosis were observed. The results of the present study support that using ARR/ARR males in scrapie eradication programs efficiently decreases the spreading of the agent in the environment via shed placentas.

Transmission of sheep-bovine spongiform encephalopathy to pigs.

Experimental transmission of the bovine spongiform encephalopathy (BSE) agent has been successfully reported in pigs inoculated via three simultaneous distinct routes (intracerebral, intraperitoneal and intravenous). Sheep derived BSE (Sh-BSE) is transmitted more efficiently than the original cattle-BSE isolate in a transgenic mouse model expressing porcine prion protein. However, the neuropathology and distribution of Sh-BSE in pigs as natural hosts, and susceptibility to this agent, is unknown. In the present study, seven pigs were intracerebrally inoculated with Sh-BSE prions. One pig was euthanized for analysis in the preclinical disease stage. The remaining six pigs developed neurological signs and histopathology revealed severe spongiform changes accompanied by astrogliosis and microgliosis throughout the central nervous system. Intracellular and neuropil-associated pathological prion protein (PrP(Sc)) deposition was consistently observed in different brain sections and corroborated by Western blot. PrP(Sc) was detected by immunohistochemistry and enzyme immunoassay in the following tissues in at least one animal: lymphoid tissues, peripheral nerves, gastrointestinal tract, skeletal muscle, adrenal gland and pancreas. PrP(Sc) deposition was revealed by immunohistochemistry alone in the retina, optic nerve and kidney. These results demonstrate the efficient transmission of Sh-BSE in pigs and show for the first time that in this species propagation of bovine PrP(Sc) in a wide range of peripheral tissues is possible. These results provide important insight into the distribution and detection of prions in non-ruminant animals.

Prion protein gene variability in Spanish goats. Inference through susceptibility to classical scrapie strains and pathogenic distribution of peripheral PrP(sc.).

Classical scrapie is a neurological disorder of the central nervous system (CNS) characterized by the accumulation of an abnormal, partially protease resistant prion protein (PrP(sc)) in the CNS and in some peripheral tissues in domestic small ruminants. Whereas the pathological changes and genetic susceptibility of ovine scrapie are well known, caprine scrapie has been less well studied. We report here a pathological study of 13 scrapie-affected goats diagnosed in Spain during the last 9 years. We used immunohistochemical and biochemical techniques to discriminate between classical and atypical scrapie and bovine spongiform encephalopathy (BSE). All the animals displayed PrP(sc) distribution patterns and western blot characteristics compatible with classical scrapie. In addition, we determined the complete open reading frame sequence of the PRNP in these scrapie-affected animals. The polymorphisms observed were compared with those of the herd mates (n = 665) and with the frequencies of healthy herds (n = 581) of native Spanish goats (Retinta, Pirenaica and Moncaina) and other worldwide breeds reared in Spain (Saanen, Alpine and crossbreed). In total, sixteen polymorphic sites were identified, including the known amino acid substitutions at codons G37V, G127S, M137I, I142M, H143R, R151H, R154H, R211Q, Q222K, G232W, and P240S, and new polymorphisms at codons G74D, M112T, R139S, L141F and Q215R. In addition, the known 42, 138 and 179 silent mutations were detected, and one new one is reported at codon 122. The genetic differences observed in the population studied have been attributed to breed and most of the novel polymorphic codons show frequencies lower than 5%. This work provides the first basis of polymorphic distribution of PRNP in native and worldwide goat breeds reared in Spain.

Medulla oblongata transcriptome changes during presymptomatic natural scrapie and their association with prion-related lesions.

BACKGROUND: The pathogenesis of natural scrapie and other prion diseases is still poorly understood. Determining the variations in the transcriptome in the early phases of the disease might clarify some of the molecular mechanisms of the prion-induced pathology and allow for the development of new biomarkers for diagnosis and therapy. This study is the first to focus on the identification of genes regulated during the preclinical phases of natural scrapie in the ovine medulla oblongata (MO) and the association of these genes with prion deposition, astrocytosis and spongiosis. RESULTS: A custom microarray platform revealed that 86 significant probes had expression changes greater than 2-fold. From these probes, we identified 32 genes with known function; the highest number of regulated genes was included in the phosphoprotein-encoding group. Genes encoding extracellular marker proteins and those involved in the immune response and apoptosis were also differentially expressed. In addition, we investigated the relationship between the gene expression profiles and the appearance of the main scrapie-associated brain lesions. Quantitative Real-time PCR was used to validate the expression of some of the regulated genes, thus showing the reliability of the microarray hybridization technology. CONCLUSIONS: Genes involved in protein and metal binding and oxidoreductase activity were associated with prion deposition. The expression of glial fibrillary acidic protein (GFAP) was associated with changes in the expression of genes encoding proteins with oxidoreductase and phosphatase activity, and the expression of spongiosis was related to genes encoding extracellular matrix components or transmembrane transporters. This is the first genome-wide expression study performed in naturally infected sheep with preclinical scrapie. As in previous studies, our findings confirm the close relationship between scrapie and other neurodegenerative diseases.

Changes in HSP gene and protein expression in natural scrapie with brain damage.

Heat shock proteins (Hsp) perform cytoprotective functions such as apoptosis regulation and inflammatory response control. These proteins can also be secreted to the extracellular medium, acting as inflammatory mediators, and their chaperone activity permits correct folding of proteins and avoids the aggregation of anomalous isoforms. Several studies have proposed the implication of Hsp in prion diseases. We analysed the gene expression and protein distribution of different members of the Hsp27, Hsp70, and Hsp90 families in the central nervous system of sheep naturally infected with scrapie. Different expression profiles were observed in the areas analysed. Whereas changes in transcript levels were not observed in the cerebellum or medulla oblongata, a significant decrease in HSP27 and HSP90 was detected in the prefrontal cortex. In contrast, HSP73 was over-expressed in diencephalons of scrapie animals. Western blotting did not reveal significant differences in Hsp90 and Hsp70 protein expression between scrapie and control animals. Expression rates identified by real-time RT-PCR and western blotting were compared with the extent of classical scrapie lesions using stepwise regression. Changes in Hsp gene and protein expression were associated with prion protein deposition, gliosis and spongiosis rather than with apoptosis. Finally, immunohistochemistry revealed intense Hsp70 and Hsp90 immunolabelling in Purkinje cells of scrapie sheep. In contrast, controls displayed little or no staining in these cells. The observed differences in gene expression and protein distribution suggest that the heat shock proteins analysed play a role in the natural form of the disease.

An assessment of the efficiency of PrPsc detection in rectal mucosa and third-eyelid biopsies from animals infected with scrapie.

In classical scrapie, detection of PrPsc on lymphoreticular system is used for the in vivo and post mortem diagnosis of the disease. However, the sensitivity of this methodology is not well characterised because the magnitude and duration of lymphoid tissue involvement can vary considerably. The aim of the present study was to evaluate the efficiency of detecting PrPsc in rectal mucosa and third-eyelid biopsies. A total of 474 genetically susceptible sheep and 24 goats from three scrapie infected flocks were included in this study. A sample from rectal mucosa and a sample from third-eyelid lymphoid tissue were collected from each animal. Biopsy samples were fixed in formaldehyde and processed for immunohistochemical examination. Animals with negative biopsy results were studied more closely through a post mortem examination of central nervous and lymphoreticular systems and if there was a positive result, additional biopsy sections were further tested. The sensitivity of rectal mucosa and third-eyelid assays were 36% and 40% respectively on initial examination but increased to 48% and 44% respectively after retesting. The results of this field study show a high percentage of infected animals that do not have detectable levels of PrPsc in the biopsied lymphoid tissue, due mainly to the relatively high number of animals with minimal or no involvement of lymphoid tissue in the pathogenesis of the disease.

Variability in disease phenotypes within a single PRNP genotype suggests the existence of multiple natural sheep scrapie strains within Europe.

Variability of pathological phenotypes within classical sheep scrapie cases has been reported for some time, but in many instances it has been attributed to differences in the PRNP genotype of the host. To address this issue we have examined by immunohistochemistry (IHC) and Western blotting (WB) for the disease-associated form of the prion protein (PrP(d)), the brains of 23 sheep from five European countries, all of which were of the same ARQ/ARQ genotype. As a result of IHC examinations, sheep were distributed into five groups with different phenotypes and the groups were the same regardless of the scoring method used, 'long' or 'short' PrP(d) profiling. The groups made did not respond to the geographical origin of the cases and did not correlate with the vacuolar lesion profiles, which showed a high individual variability. Discriminatory IHC and WB methods coincided to detect a 'CH1641-like' case but otherwise correlated poorly in the classification of disease phenotypes. No other polymorphisms of the PRNP gene were found that could account for the pathological differences, except perhaps for a sheep from Spain with a mutation at codon 103 and a unique pathological phenotype. Preliminary evidence indicates that those different IHC phenotypes correlate with distinct biological properties on bioassay, suggesting that they are indicative of strain diversity. We therefore conclude that natural scrapie strains exist and that they can be revealed by detailed pathological examinations, which can be harmonized between laboratories to produce comparable results.