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Scrapie is a neurodegenerative disorder belonging to the group of transmissible spongiform encephalopathies or prion diseases, which are caused by an infectious isoform of the innocuous cellular prion protein (PrPC) known as PrPSc. DNA methylation, one of the most studied epigenetic mechanisms, is essential for the proper functioning of the central nervous system. Recent findings point to possible involvement of DNA methylation in the pathogenesis of prion diseases, but there is still a lack of knowledge about the behavior of this epigenetic mechanism in such neurodegenerative disorders. Here, we evaluated by immunohistochemistry the 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) levels in sheep and mouse brain tissues infected with scrapie. Expression analysis of different gene coding for epigenetic regulatory enzymes (DNMT1, DNMT3A, DNMT3B, HDAC1, HDAC2, TET1, and TET2) was also carried out. A decrease in 5mC levels was observed in scrapie-affected sheep and mice compared to healthy animals, whereas 5hmC displayed opposite patterns between the two models, demonstrating a decrease in 5hmC in scrapie-infected sheep and an increase in preclinical mice. 5mC correlated with prion-related lesions in mice and sheep, but 5hmC was associated with prion lesions only in sheep. Differences in the expression changes of epigenetic regulatory genes were found between both disease models, being differentially expressed Dnmt3b, Hdac1, and Tet1 in mice and HDAC2 in sheep. Our results support the evidence that DNA methylation in both forms, 5mC and 5hmC, and its associated epigenetic enzymes, take part in the neurodegenerative course of prion diseases.

The non-toxic C-terminal fragment of the tetanus toxin (TTC) has been described as a neuroprotective molecule since it binds to Trk receptors and activates Trk-dependent signaling, activating neuronal survival pathways and inhibiting apoptosis. Previous in vivo studies have demonstrated the ability of this molecule to increase mice survival, inhibit apoptosis and regulate autophagy in murine models of neurodegenerative diseases such as amyotrophic lateral sclerosis and spinal muscular atrophy. 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. These diseases share different pathological features with other neurodegenerative diseases, such as amyotrophic lateral sclerosis, Parkinson’s disease or Alzheimer’s disease. Hitherto, there are no effective therapies to treat prion diseases. Here, we present a pilot study to test the therapeutic potential of TTC to treat prion diseases. C57BL6 wild-type mice and the transgenic mice Tg338, which overexpress PrPC, were intracerebrally inoculated with scrapie prions and then subjected to a treatment consisting of repeated intramuscular injections of TTC. Our results indicate that TTC displays neuroprotective effects in the murine models of prion disease reducing apoptosis, regulating autophagy and therefore increasing neuronal survival, although TTC did not increase survival time in these models.

Prion diseases are diagnosed in the symptomatic stage, when the neuronal damage is spread throughout the central nervous system (CNS). The assessment of biological features that allow the detection of asymptomatic cases is needed, and, in this context, scrapie, where pre-symptomatic infected animals can be detected through rectal biopsy, becomes a good study model. Neurogranin (Ng) and neurofilament light chain (NfL) are proteins that reflect synaptic and axonal damage and have been studied as cerebrospinal fluid (CSF) biomarkers in different neurodegenerative disorders. In this study, we evaluated Ng and NfL both at the protein and transcript levels in the CNS of preclinical and clinical scrapie-affected sheep compared with healthy controls and assessed their levels in ovine CSF. The correlation between these proteins and the main neuropathological events in prion diseases, PrPSc deposition and spongiosis, was also assessed. The results show a decrease in Ng and NfL at the protein and gene expression levels as the disease progresses, and significant changes between the control and preclinical animals. On the contrary, the CSF levels of NfL increased throughout the progression of the disease. Negative correlations between neuropathological markers of prion disease and the concentration of the studied proteins were also found. Although further research is needed, these results suggest that Ng and NfL could act as biomarkers for neurodegeneration onset and intensity in preclinical cases of scrapie.

Prion diseases are chronic and fatal neurodegenerative diseases characterized by the accumula-tion of disease-specific prion protein (PrPSc), spongiform changes, neuronal loss, and gliosis. Growing evidence shows that the neuroinflammatory response is a key component of prion diseases and contributes to neurodegeneration. Toll-like receptors (TLRs) have been proposed as important mediators of innate immune responses triggered in the central nervous system in other human neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and amyotrophic lateral sclerosis. However, little is known about the role of TLRs in prion dis-eases, and their involvement in the neuropathology of natural scrapie has not been studied. We assessed gene expression of ovine TLRs in four anatomically distinct brain regions in natural scrapie-infected sheep and evaluated possible correlations between gene expression and patho-logical hallmarks of prion disease. We observed significant changes in TLR expression in scra-pie-infected sheep that correlate with the degree of spongiosis, PrPSc deposition, and gliosis in each of the regions studied. Remarkably, TLR4 was the only gene upregulated in all regions, re-gardless of the severity of neuropathology. In the hippocampus, we observed milder neuropa-thology associated with a distinct TLR gene expression profile and the presence of a peculiar microglial morphology, called rod microglia, described here for the first time in the brain of scrapie-infected sheep. The concurrence of these features suggests partial neuroprotection of the hippocampus. Finally, comparison of findings in naturally-infected sheep versus an ovinized mouse model (tg338 mice) revealed distinct patterns of TLR gene expression. This research was funded by “Departamento de Ciencia, Universidad y Sociedad del Conocimiento” (Aragon Government) through the project “A05_20R: Enfermedades Priónicas, Vecto- riales y Zoonosis Emergentes”.

Metronidazole was until recently used as a first-line treatment for potentially life-threatening Clostridioides difficile (CD) infection. Although cases of metronidazole resistance have been documented, no clear mechanism for metronidazole resistance or a role for plasmids in antimicrobial resistance has been described for CD. Here, we report genome sequences of seven susceptible and sixteen resistant CD isolates from human and animal sources, including isolates from a patient with recurrent CD infection by a PCR ribotype (RT) 020 strain, which developed resistance to metronidazole over the course of treatment (minimal inhibitory concentration [MIC] = 8 mg L−1). Metronidazole resistance correlates with the presence of a 7-kb plasmid, pCD-METRO. pCD-METRO is present in toxigenic and non-toxigenic resistant (n = 23), but not susceptible (n = 563), isolates from multiple countries. Introduction of a pCD-METRO-derived vector into a susceptible strain increases the MIC 25-fold. Our finding of plasmid-mediated resistance can impact diagnostics and treatment of CD infections., Cases of C. difficile (CD) resistant to metronidazole have been reported but the mechanism remains enigmatic. Here the authors identify a plasmid, which correlates with metronidazole resistance status in a large international collection of CD isolates, and demonstrate that the plasmid can confer metronidazole resistance.

Prion diseases are transmissible spongiform encephalopathies (TSEs) caused by a conformational conversion of the native cellular prion protein (PrPC) to an abnormal, infectious isoform called PrPSc. Amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, and Huntington’s diseases are also known as prion-like diseases because they share common features with prion diseases, including protein misfolding and aggregation, as well as the spread of these misfolded proteins into different brain regions. Increasing evidence proposes the involvement of epigenetic mechanisms, namely DNA methylation, post-translational modifications of histones, and microRNA-mediated post-transcriptional gene regulation in the pathogenesis of prion-like diseases. Little is known about the role of epigenetic modifications in prion diseases, but recent findings also point to a potential regulatory role of epigenetic mechanisms in the pathology of these diseases. This review highlights recent findings on epigenetic modifications in TSEs and prion-like diseases and discusses the potential role of such mechanisms in disease pathology and their use as potential biomarkers.

Diagnosis of transmissible spongiform encephalopathies (TSEs), or prion diseases, is based on the detection of proteinase K (PK)-resistant PrPSc in post-mortem tissues as indication of infection and disease. Since PrPSc detection is not considered a reliable method for in vivo diagnosis in most TSEs, it is of crucial importance to identify an alternative source of biomarkers to provide useful alternatives for current diagnostic methodology. Ovine scrapie is the prototype of TSEs and has been known for a long time. Using this natural model of TSE, we investigated the presence of PrPSc in exosomes derived from plasma and cerebrospinal fluid (CSF) by protein misfolding cyclic amplification (PMCA) and the levels of candidate microRNAs (miRNAs) by quantitative PCR (qPCR). Significant scrapie-associated increase was found for miR-21-5p in plasma-derived but not in CSF-derived exosomes. However, miR-342-3p, miR-146a-5p, miR-128-3p and miR-21-5p displayed higher levels in total CSF from scrapie-infected sheep. The analysis of overexpressed miRNAs in this biofluid, together with plasma exosomal miR-21-5p, could help in scrapie diagnosis once the presence of the disease is suspected. In addition, we found the presence of PrPSc in most CSF-derived exosomes from clinically affected sheep, which may facilitate in vivo diagnosis of prion diseases, at least during the clinical stage.

Background: Antibiotic resistance and impaired wound healing are major concerns in S. aureus superficial skin infections, and new therapies are needed. Antimicrobial photodynamic therapy (aPDT) is a new therapeutic approach for infections, but it also improves healing in many wound models.Objective: To compare the antimicrobial activity and the effects on wound healing of aPDT based on Methylene Blue (MB-aPDT) with mupirocin treatment, either alone or in combination, in superficial skin wounds of S. aureus-infected mice. Additionally, to evaluate the clinical, microbiological, and cosmetic effects on wound healing.Materials and Methods: A superficial skin infection model of S. aureus was established in SKH-1 mice. Infected wounds were treated with MB-aPDT, MB-aPDT with a daily topical mupirocin or only with mupirocin. No treatment was carried out in control animals. Daily clinical and microbiological examinations were performed until complete clinical wound healing. Histopathological studies and statistical analysis were performed at the end of the study.Results: MB-aPDT treatment induced the best wound healing compared to mupirocin alone or to mupirocin plus MB-aPDT. Superficial contraction at 24 h and a greater reduction in size at 48 h, quicker detachment of the crust, less scaling, and absence of scars were observed. Histopathological studies correlated with clinical and gross findings. By contrast, mupirocin showed the highest logaritmic reduction of S. aureus.Conclusions: MB-aPDT and mupirocin treatments are effective in a murine superficial skin infection model of S. aureus. One session of MB-aPDT was the best option for clinical wound healing and cosmetic results. The addition of mupirocin to MB-aPDT treatment improved antimicrobial activity; however, it did not enhance wound healing. No synergistic antibacterial effects were detected.

Simple Summary Prion diseases are neurodegenerative disorders affecting humans and animals. The development of in vitro cellular models from naturally susceptible species like humans or ruminants can potentially make a great contribution to the study of many aspects of these diseases, including the ability of prions to infect and replicate in cells and therapeutics. Our study shows for the first time how ovine mesenchymal stem cells derived from bone marrow and their neural-like progeny are able to react to scrapie prion infection in vitro and assesses the effects of this infection on cell viability and proliferation. Finally, we observe that the differentiation of ovine mesenchymal stem cells into neuron-like cells makes them more permissive to prion infection. Abstract Scrapie is a prion disease affecting sheep and goats and it is considered a prototype of transmissible spongiform encephalopathies (TSEs). Mesenchymal stem cells (MSCs) have been proposed as candidates for developing in vitro models of prion diseases. Murine MSCs are able to propagate prions after previous mouse-adaptation of prion strains and, although ovine MSCs express the cellular prion protein (PrPC), their susceptibility to prion infection has never been investigated. Here, we analyze the potential of ovine bone marrow-derived MSCs (oBM-MSCs), in growth and neurogenic conditions, to be infected by natural scrapie and propagate prion particles (PrPSc) in vitro, as well as the effect of this infection on cell viability and proliferation. Cultures were kept for 48–72 h in contact with homogenates of central nervous system (CNS) samples from scrapie or control sheep. In growth conditions, oBM-MSCs initially maintained detectable levels of PrPSc post-inoculation, as determined by Western blotting and ELISA. However, the PrPSc signal weakened and was lost over time. oBM-MSCs infected with scrapie displayed lower cell doubling and higher doubling times than those infected with control inocula. On the other hand, in neurogenic conditions, oBM-MSCs not only maintained detectable levels of PrPSc post-inoculation, as determined by ELISA, but this PrPSc signal also increased progressively over time. Finally, inoculation with CNS extracts seems to induce the proliferation of oBM-MSCs in both growth and neurogenic conditions. Our results suggest that oBM-MSCs respond to prion infection by decreasing their proliferation capacity and thus might not be permissive to prion replication, whereas ovine MSC-derived neuron-like cells seem to maintain and replicate PrPSc.

Salmonellosis is a common subclinical infection in pigs and therefore apparently healthy animals may represent a reservoir of antibiotic-resistant Salmonella for humans. This study estimates and characterizes resistance to two classes of antimicrobials considered of the highest priority within the critically important antimicrobials for humans, i.e. colistin (CR) and 3rd generation cephalosporins (3GC), on a collection of Salmonella isolates from pigs from two periods: between 2008 and 09, when colistin was massively used; and in 2018, after three years under a National Plan against Antibiotic Resistance. Prevalence of CR was low (6 out of 625; 0.96%; 95%CI: 0.44–2.1) in 2008–09 and associated mostly to the mcr-1 gene, which was detected in four S. 4,5,12:i:- isolates. Polymorphisms in the pmrAB genes were detected in a S. 9,12:-:- isolate. No CR was detected in 2018 out of 59 isolates tested. Among 270 Salmonella isolates considered for the assessment of resistance to 3GC in the 2008–2009 sampling, only one Salmonella Bredeney (0.37%; 95%CI: 0.07–2.1) showed resistance to 3GC, which was associated with the blaCMY-2 gene (AmpC producer). In 2018, six isolates out of 59 (10.2%; 95%CI: 4.7–20.5) showed resistance to 3GC, but only two different strains were identified (S. 4,12:i:- and S. Rissen), both confirmed as extended-spectrum β-lactamases (ESBL) producers. The blaCTX-M-3 and blaTEM-1b genes in S. 4,12:i:- and the blaTEM-1b gene in S. Rissen seemed to be associated with this resistance. Overall, the prevalence of CR in Salmonella appeared to be very low in 2008–2009 despite the considerable use of colistin in pigs at that time, and seemed to remain so in 2018. Resistance to 3GC was even lower in 2008–2009 but somewhat higher in 2018. Resistance was mostly coded by genes associated with mobile genetic elements. Most serotypes involved in these antimicrobial resistances displayed a multidrug resistance pattern and were considered zoonotic.

Prion diseases are a group of neurodegenerative disorders that can be spontaneous, familial or acquired by infection. The conversion of the prion protein PrPC to its abnormal and misfolded isoform PrPSc is the main event in the pathogenesis of prion diseases of all origins. In spontaneous prion diseases, the mechanisms that trigger the formation of PrPSc in the central nervous system remain unknown. Several reports have demonstrated that the accumulation of PrPSc can induce endoplasmic reticulum (ER) stress and proteasome impairment from the early stages of the prion disease. Both mechanisms lead to an increment of PrP aggregates in the secretory pathway, which could explain the pathogenesis of spontaneous prion diseases. Here, we investigate the role of ER stress and proteasome impairment during prion disorders in a murine model of spontaneous prion disease (TgVole) co-expressing the UbG76V-GFP reporter, which allows measuring the proteasome activity in vivo. Spontaneously prion-affected mice showed a significantly higher accumulation of the PKR-like ER kinase (PERK), the ER chaperone binding immunoglobulin protein (BiP/Grp78), the ER protein disulfide isomerase (PDI) and the UbG76V-GFP reporter than age-matched controls in certain brain areas. The upregulation of PERK, BiP, PDI and ubiquitin was detected from the preclinical stage of the disease, indicating that ER stress and proteasome impairment begin at early stages of the spontaneous disease. Strong correlations were found between the deposition of these markers and neuropathological markers of prion disease in both preclinical and clinical mice. Our results suggest that both ER stress and proteasome impairment occur during the pathogenesis of spontaneous prion diseases., This research was funded by the project EFA148/16 REDPRION. The project EFA148/16 REDPRION was 65% co-financed by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra program (POCTEFA 2014–2020). POCTEFA aims to reinforce the economic and social integration of the French–Spanish–Andorran border. Its support is focused on developing economic, social and environmental cross-border activities through joint strategies favoring sustainable territorial development.

Neurotrophins constitute a group of growth factor that exerts important functions in the nervous system of vertebrates. They act through two classes of transmembrane receptors: tyrosine-kinase receptors and the p75 neurotrophin receptor (p75NTR). The activation of p75NTR can favor cell survival or apoptosis depending on diverse factors. Several studies evidenced a link between p75NTR and the pathogenesis of prion diseases. In this study, we investigated the distribution of several neurotrophins and their receptors, including p75NTR, in the brain of naturally scrapie-affected sheep and experimentally infected ovinized transgenic mice and its correlation with other markers of prion disease. No evident changes in infected mice or sheep were observed regarding neurotrophins and their receptors except for the immunohistochemistry against p75NTR. Infected mice showed higher abundance of p75NTR immunostained cells than their non-infected counterparts. The astrocytic labeling correlated with other neuropathological alterations of prion disease. Confocal microscopy demonstrated the co-localization of p75NTR and the astrocytic marker GFAP, suggesting an involvement of astrocytes in p75NTR-mediated neurodegeneration. In contrast, p75NTR staining in sheep lacked astrocytic labeling. However, digital image analyses revealed increased labeling intensities in preclinical sheep compared with non-infected and terminal sheep in several brain nuclei. This suggests that this receptor is overexpressed in early stages of prion-related neurodegeneration in sheep. Our results confirm a role of p75NTR in the pathogenesis of classical ovine scrapie in both the natural host and in an experimental transgenic mouse model.

MicroRNAs (miRNAs) may contribute to the development and pathology of many neurodegenerative diseases, including prion diseases. They are also promising biomarker candidates due to their stability in body fluids. We investigated miRNA alterations in a Tg501 mouse model of prion diseases that expresses a transgene encoding the goat prion protein (PRNP). Tg501 mice intracranially inoculated with mouse-adapted goat scrapie were compared with age-matched, mock inoculated controls in preclinical and clinical stages. Small RNA sequencing from the cervical spinal cord indicated that miR-223-3p, miR-151-3p, and miR-144-5p were dysregulated in scrapie-inoculated animals before the onset of symptoms. In clinical-stage animals, 23 significant miRNA alterations were found. These miRNAs were predicted to modify the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways including prion disease, extracellular matrix interactions, glutaminergic synapse, axon guidance, and transforming growth factor-beta signaling. MicroRNAs miR-146a-5p (up in cervical spinal cord) and miR-342-3p (down in cervical spinal cord, cerebellum and plasma), both indicated in neurodegenerative diseases earlier, were verified by quantitative real-time polymerase chain reaction (qRT-PCR). Minimal changes observed before the disease onset suggests that most miRNA alterations observed here are driven by advanced prion-associated pathology, possibly limiting their use as diagnostic markers. However, the results encourage further mechanistic studies on miRNA-regulated pathways involved in these neurodegenerative conditions.

Specific variations in the amino acid sequence of prion protein (PrP) are key determinants of susceptibility to prion diseases. We previously showed that an amino acid substitution specific to canids confers resistance to prion diseases when expressed in mice, and demonstrated its dominant-negative protective effect against a variety of infectious prion strains of different origins and characteristics. Here, we show that expression of this single amino acid change significantly increases survival time in transgenic mice expressing bank vole cellular prion protein (PrPC), which is inherently prone to misfolding, following inoculation with two distinct prion strains (the CWD-vole strain and an atypical strain of spontaneous origin). This amino acid substitution hinders the propagation of both prion strains, even when expressed in the context of a PrPC uniquely susceptible to a wide range of prion isolates. Non-inoculated mice expressing this substitution experience spontaneous prion formation, but showing an increase in survival time comparable to that observed in mutant mice inoculated with the atypical strain. Our results underscore the importance of this PrP variant in the search for molecules with therapeutic potential against prion diseases.

Phenotypic variability in prion diseases, such as scrapie, is associated to the existence of prion strains, which are different pathogenic prion protein (PrPSc) conformations with distinct pathobiological properties. To faithfully study scrapie strain variability in natural sheep isolates, transgenic mice expressing sheep cellular prion protein (PrPC) 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 (PrPres) banding patterns, lesion profiles and PrPSc distribution were studied. Inocula showed a remarkable homogeneity on banding patterns, all of them but one showing 19-kDa PrPres. However, a number of isolates caused accumulation of 21-kDa PrPres in TgShp XI. A different subgroup of isolates caused long survival periods and presence of 21-kDa PrPres 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 PrPC 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.

Background: Epigenetic changes in obstructive sleep apnea (OSA) have been proposed as a mechanism for end-organ vulnerability. In children with OSA, Forkhead Box P3 (FOXP3) DNA methylation were associated with inflammatory biomarkers, however, the methylation pattern and its effect in the expression of this gene have not been tested in adults with OSA. Methods: Plasma samples from subjects without comorbid conditions other than OSA were analyzed (the Epigenetics Status and Subclinical Atherosclerosis in Obstructive Sleep Apnea (EPIOSA) Study: NCT02131610). In 16 patients with severe OSA (Apnea-Hypopnea Index&mdash, AHI- &gt, 30 events/h) and seven matched controls (AHI &lt, 5), methylation of FOXP3 gen was evaluated by PCR of the promoter and by pyrosequencing of the intron 1 Treg-specific demethylated region (TSDR). In another 74 patients with OSA (AHI &gt, 10) and 31 controls, we quantified FOXP3 protein expression by ELISA and gene expression by quantitative real-time PCR. C-reactive protein (CRP) and plasma Treg cells were also evaluated. Results: Neither the levels of the promoter nor the TSDR demethylated region were different between controls and patients with OSA, whether they were grouped by normal or high CRP. FOXP3 protein and mRNA expression did not differ between groups. Conclusions: FOXP3 methylation or its expression is not altered in adults with OSA, whatever their inflammatory status.

Este artículo se encuentra disponible en la siguiente URL: https://onlinelibrary.wiley.com/doi/10.1111/tbed.13470 This is the peer reviewed version of the following article: Sevilla, E., Marín, C., Delgado-Blas, JF., González-Zorn, B., Vega, S., Kuijper, E., Bolea, R. & Mainar-Jaime, RC. Wild griffon vultures (Gyps fulvus) fed at supplementary feeding stations: Potential carriers of pig pathogens and pig-derived antimicrobial resistance?. Transbounddary and Emergency Diseases, vol. 67, i. 3 (may 2020), pp. 1295-1305, which has been published in final form at https://doi.org/10.1111/tbed.13470. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. Este es el post-print del siguiente artículo: Sevilla, E., Marín, C., Delgado-Blas, JF., González-Zorn, B., Vega, S., Kuijper, E., Bolea, R. & Mainar-Jaime, RC. Wild griffon vultures (Gyps fulvus) fed at supplementary feeding stations: Potential carriers of pig pathogens and pig-derived antimicrobial resistance?. Transbounddary and Emergency Diseases, vol. 67, i. 3 (may 2020), pp. 1295-1305, que se ha publicado de forma definitiva en https://doi.org/10.1111/tbed.13470 The carriage of two important pathogens of pigs, i.e. enterotoxigenic E. coli (ETEC) and Clostridioides difficile, was investigated in 104 cloacal samples from wild griffon vultures (Gyps fulvus) fed on pig carcasses at supplementary feeding stations (SFS), along with their level of antimicrobial resistance (AMR). E. coli was isolated from 90 (86.5%) samples but no ETEC was detected, likely because ETEC fimbriae confer the species specificity of the pathogen. Resistance to at least one antimicrobial agent was detected in 89.9% of E. coli isolates, being AMR levels extremely high (>70%) for tetracycline and streptomycin, and very high (>50%) for ampicillin and sulfamethoxazole-trimethoprim. Resistance to other critically important antimicrobials such as colistin and extended-spectrum cephalosporins was 2.2%, and 1.1%, respectively, and was encoded by the mcr-1 and blaSHV-12 genes. Multidrug resistance was displayed by 80% of the resistant E. coli and blaSHV-12 gene shared plasmid with other AMR genes. In general, resistance patterns in E. coli from vultures mirrored those found in pigs. C. difficile was detected in three samples (2.9%), two of them belonged to PCR-ribotype 078 and one to PCR-ribotype 126, both commonly found in pigs. All C. difficile isolates were characterized by a moderate to high level of resistance to fluoroquinolones and macrolides but susceptible to metronidazole or vancomycin, similar to what is usually found in C. difficile isolates from pigs. Thus, vultures may contribute somewhat to the environmental dissemination of some pig pathogens through their acquisition from pig carcasses and, more importantly, of AMR for antibiotics of critical importance for humans. However, the role of vultures would likely be much lesser than that of disposing pig carcasses at the SFS. The monitoring of AMR, and particularly of colistin resistant and ESLB-producing E. coli, should be considered in pig farms used as sources of carcasses for SFS.

Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE. info:eu-repo/semantics/acceptedVersion

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). To determine the relative amounts of PrP polymorphisms present in atypical scrapie, total PrP was digested with chymotrypsin to generate characteristic peptides spanning relevant polymorphisms at positions 136, 141, 154, 171, and 172 of sheep PrPC. A multiple reaction monitoring method (MRM), employing 15N-labeled internal standards, was used to detect and quantify these polymorphisms present in both the PrPSc and PrPC from heterozygous (ALRRY and ALHQY or ALRQD or AFRQY) atypical scrapie-infected or uninfected control sheep. Both polymorphisms of the full length and truncated (C1) natively expressed PrPC are produced in equal amounts. The overall amount of PrPC present in the infected or uninfected animals was similar. PrPSc isolated from heterozygotes was composed of significant amounts of both PrP polymorphis...

International audience; Multiple theories exist regarding the origin of bovine spongiform encephalopathy (BSE). An early and prominent theory proposed that BSE was the result of the adaptation of sheep scrapie to cattle. The reports to date indicate that the distribution of the pathological prion protein (PrPSc) in experimental bovine scrapie is largely restricted to the central nervous system (CNS). Here, we describe pathological findings in a calf intracerebrally inoculated with a Spanish classical scrapie isolate. While clinical disease was observed 30 months after inoculation and PrPSc was detected in the CNS, the corresponding phenotype differed from that of BSE. Immunohistochemistry and PMCA also revealed the presence of PrPSc in the peripheral nerves, lymphoid tissues, skeletal muscle and gastrointestinal tract, suggesting centrifugal spread of the scrapie agent from the brain. To the best of our knowledge, this is the first report describing the detection of PrPSc in tissues other than the CNS after experimental transmission of scrapie to cattle.

Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic peptides [M132LGSXMSRPL141 (X = A or V), Y153XENMY158 (X,= H or R), and Y166RPVDXY172 (X = H, K, Q, or R)] that could be used to detect and quantitate polymorphisms at positions 136, 154, and 171 of sheep PrPC or PrPSc. These peptides were used to develop a multiple reaction monitoring method (MRM) to detect the amounts of a particular polymorphism in a sample of PrPSc isolated from sheep heterozygous for their PrPC proteins. The limit of detection for these peptides was less than 50 attomole. Spinal cord tissue from heterozygous (ARQ/VRQ or ARH/ARQ) scrapie-infected Rasa Aragonesa sheep was analyzed using this MRM method. Both sets of heterozygotes show the presence of both polymorphisms in PrPSc. This was true fo...

The aim of this study was to estimate the prevalence of antimicrobial resistance (AMR) in Escherichia coli from a dog population in Spain and assess specific virulence factors. Susceptibility to 22 antimicrobials was tested along with the production of extended-spectrum β-lactamases (ESBLs) and AmpC in faecal isolates from 100 dogs. Virulence-related genes associated with attaching and effacing E. coli (eae, Stx1, Stx2) and extraintestinal pathogenic E. coli – ExPEC – (papC, hlyA and cnf1) were detected by PCR. At least one kind of AMR was observed in 73% of the isolates. The highest prevalences corresponded to penicillin (45%), aminoglycoside (40%) and non-extended spectrum cephalosporin (39%) classes. Multidrug resistance (MDR) was observed in 53.4% of the resistant isolates. No resistance to colistin was found. Production of ESBL/AmpC enzymes was detected in 5% of E. coli. Shiga toxin-producing E. coli were not observed, enteropathogenic E. coli were identified in only 12% of them, and ExPEC were found in 25%. Dog faeces can be a source of E. coli strains potentially presenting a threat to humans through their virulence factors or AMR. The non-hygienic keeping of animals may increase the risk of colonisation of such pathogens in humans.

Autophagy appears to play a role in the etiology and progress of misfolded protein disorders. Although this process is dysregulated in prion diseases, it is unknown whether this impairment is a cause or a consequence of prion neuropathology. The study of autophagy during the progress of the disease could elucidate its role. For this purpose, we have investigated its regulation at different stages of the disease in Tg338 mice, a transgenic murine model that overexpresses the highly susceptible ovine VRQ prion protein allele. Mice were intracerebrally inoculated with mouse-adapted classical scrapie and euthanized at the preclinical and clinical stages of the disease. Regulation of autophagy was investigated analyzing the distribution of LC3-B and p62 proteins by immunohistochemistry. Moreover, the expression of genes involved in autophagy regulation was quantified by real-time PCR. LC3-B and p62 proteins were downregulated and upregulated, respectively, in the central nervous system of infected mice with clinical signs of scrapie. Accumulation of p62 correlated with scrapie-related lesions, suggesting an impairment of autophagy in highly prion-affected areas. In addition, Gas5 (growth arrest-specific 5), Atg5 (autophagy-related 5), and Fbxw7 (F-box and WD repeat domain containing 7) transcripts were downregulated in mesencephalon and cervical spinal cord of the same group of animals. The impairment of autophagic machinery seems to be part of the pathological process of scrapie, but only during the late stage of prion infection. Similarities between Tg338 mice and the natural ovine disease make them a reliable in vivo model to study prion infection and autophagy side by side. This study shows a negative regulation of autophagy-related RNA transcripts and the upregulation and downregulation of p62 and LC3-B proteins, respectively, in the central nervous system of scrapie-infected transgenic mice at clinical stage. These findings likely reflect an impairment of the autophagic pathway at the late symptomatic stage of prion infection.