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Gerstmann-Sträussler-Scheinker disease (GSS) is a rare neurodegenerative illness that belongs to the group of hereditary or familial Transmissible Spongiform Encephalopathies (TSE). Due to the presence of different pathogenic alterations in the prion protein (PrP) coding gene, it shows an enhanced proneness to misfolding into its pathogenic isoform, leading to prion formation and propagation. This aberrantly folded protein is able to induce its conformation to the native counterparts forming amyloid fibrils and plaques partially resistant to protease degradation and showing neurotoxic properties. PrP with A117V pathogenic variant is the second most common genetic alteration leading to GSS and despite common phenotypic and neuropathological traits can be defined for each specific variant, strikingly heterogeneous manifestations have been reported for inter-familial cases bearing the same pathogenic variant or even within the same family. Given the scarcity of cases and their clinical, neuropathological, and biochemical variability, it is important to characterize thoroughly each reported case to establish potential correlations between clinical, neuropathological and biochemical hallmarks that could help to define disease subtypes. With that purpose in mind, this manuscript aims to provide a detailed report of the first Spanish GSS case associated with A117V variant including clinical, genetic, neuropathological and biochemical data, which could help define in the future potential disease subtypes and thus, explain the high heterogeneity observed in patients suffering from these maladies.

Mycobacterium tuberculosis comprises an unusual cell envelope dominated by unique lipids and glycans that provides a permeability barrier against hydrophilic drugs and is central for its survival and virulence. Phosphatidyl-myo-inositol mannosides (PIMs) are glycolipids considered to be not only key structural components of the cell envelope but also the precursors of lipomannan (LM) and lipoarabinomannan (LAM), important lipoglycans implicated in host-pathogen interactions. Here, we focus on PatA, a membrane-associated acyltransferase that transfers a palmitoyl moiety from palmitoyl coenzyme A (palmitoyl-CoA) to the 6-position of the mannose ring linked to the 2-position of inositol in PIM(1)/PIM(2). We validate that the function of PatA is vital for M. tuberculosis in vitro and in vivo. We constructed a patA conditional mutant and showed that silencing patA is bactericidal in batch cultures. This phenotype was associated with significantly reduced levels of Ac(1)PIM(2), an important structural component of the mycobacterial inner membrane. The requirement of PatA for viability was also demonstrated during macrophage infection and in a mouse model of infection, where a dramatic decrease in viable counts was observed upon silencing of the patA gene. This is reminiscent of the behavior of PimA, the mannosyltransferase that initiates the PIM pathway, also found to be essential for M. tuberculosis growth in vitro and in vivo. Altogether, the experimental data highlight the significance of the early steps of the PIM biosynthetic pathway for M. tuberculosis physiology and reveal that PatA is a novel target for drug discovery programs against this major human pathogen. IMPORTANCE Tuberculosis (TB) is the leading cause of death from a single infectious agent. The emergence of drug resistance in strains of M. tuberculosis, the etiologic agent of TB, emphasizes the need to identify new targets and antimicrobial agents. The mycobacterial cell envelope is a major factor in this intrinsic drug resistance. Here, we have focused on the biosynthesis of PIMs, key virulence factors and important components of the cell envelope. Specifically, we have determined that PatA, the acyltransferase responsible for the first acylation step of the PIM synthesis pathway, is essential in M. tuberculosis. These results highlight the importance of early steps of the PIM biosynthetic pathway for mycobacterial physiology and the suitability of PatA as a potential new drug target.

The resolution of the three-dimensional structure of infectious prions at the atomic level is pivotal to understand the pathobiology of Transmissible Spongiform Encephalopathies (TSE), but has been long hindered due to certain particularities of these proteinaceous pathogens. Difficulties related to their purification from brain homogenates of disease-affected animals were resolved almost a decade ago by the development of in vitro recombinant prion propagation systems giving rise to highly infectious recombinant prions. However, lack of knowledge about the molecular mechanisms of the misfolding event and the complexity of systems such as the Protein Misfolding Cyclic Amplification (PMCA), have limited generating the large amounts of homogeneous recombinant prion preparations required for high-resolution techniques such as solid state Nuclear Magnetic Resonance (ssNMR) imaging. Herein, we present a novel recombinant prion propagation system based on PMCA that substitutes sonication with shaking thereby allowing the production of unprecedented amounts of multi-labeled, infectious recombinant prions. The use of specific cofactors, such as dextran sulfate, limit the structural heterogeneity of the in vitro propagated prions and makes possible, for the first time, the generation of infectious and likely homogeneous samples in sufficient quantities for studies with high-resolution structural techniques as demonstrated by the preliminary ssNMR spectrum presented here. Overall, we consider that this new method named Protein Misfolding Shaking Amplification (PMSA), opens new avenues to finally elucidate the three-dimensional structure of infectious prions., Author summary Prion disorders are a group of devastating neurodegenerative diseases caused by an aberrantly folded isoform of the endogenous prion protein. The molecular mechanisms by which this proteinaceous pathogen is able to propagate in the central nervous system and cause neuronal death are poorly understood, partially due to the difficulties elucidating the three-dimensional structure of the aggregation-prone aberrant isoform or prion. Obtaining sufficient amounts of highly pure and homogeneous prions for high-resolution structural studies has been limited until now due to technical reasons. Here, we present a novel method for the production of large amounts of highly infectious recombinant prions suitable for solid state Nuclear Magnetic Resonance imaging, which could help to unveil the molecular pathogenesis of these particular pathogens.

Animal tuberculosis (TB) remains a major problem in some countries despite the existence of control programmes focused mainly on cattle. In this species, aerogenous transmission is accepted as the most frequent infection route, affecting mainly the respiratory system. Under the hypothesis that the oral route could be playing a more relevant role in transmission, diagnosis and disease persistence than previously thought, this study was performed to assess the course of TB infection in cattle and its effects on diagnosis depending on the route of entry of Mycobacterium bovis. Two groups of five calves each were either endotracheally (EC) or orally (OC) challenged. Necropsies were carried out 12 weeks after challenge except for three OC calves slaughtered 8 weeks later. All animals reacted to the tuberculin skin test and the entire EC group was positive to the interferon-gamma release assay (IGRA) 2 weeks after challenge and thereafter. The first positive IGRA results for OC calves (3/5) were recorded 4 weeks after challenge. Group comparison revealed significant differences in lesion and positive culture location and scoring. TB-compatible gross lesions and positive cultures were more frequently found in the thorax (p < 0.001) and lung (p < 0.05) of EC animals, whereas OC animals presented lesions (p = 0.23) and positive cultures (p < 0.05) mainly located in the abdomen. These results indicate that the infection route seems to be a determining factor for both the distribution and the time needed for the development of visible lesions. Our study suggests that confirmation of TB infection in some skin reactor animals can be problematic if current post-mortem examination and diagnostics are not improved., This study was supported with funds from the Spanish Ministry of Economy and Competitiveness (Research Project AGL2014-56305-C3-3-R) and the Department of Economic Development and Competitiveness of the Basque Government. MS holds a fellowship from the Department of Education of the Basque Government (PRE_2017_2_0043).

Deer species (family Cervidae) are often part of the Mycobacterium tuberculosis complex maintenance host community, and tuberculosis (TB) control in deer, including vaccination, is consequently an area of ongoing research. However, most research into deer vaccination against TB is focused on using the live bacillus Calmette Guerin (BCG). Oral inactivated vaccines represent an interesting alternative to either oral or parenteral BCG, since neither diagnostic cross-reactions nor vaccine strain survival are likely to occur. In order to describe the red deer response to heat-inactivated M. bovis (IV) as compared to BCG and to unvaccinated controls (n = 5/group), we ran an experiment with five month-old vaccinated red deer, which were challenged with a virulent M. bovis strain 70 days later and necropsied at 60 days post-challenge. A reduction in the IV group infection burden was discovered. There were significant differences between the IV group and the control group (53% lesion reduction) as regards to the TB lesion scores, but not between other pairs. Complement component 3 plasma levels increased after challenge, and there were no differences between groups. The plasma cytokines (IL-1β, TNFα, IFNγ, IL-10 and IL-12) levels did not change after vaccination, but IL-1β, TNFα and IL-10 did so following the challenge. The IL-1β level increased in all the groups while TNFα levels had a distinct response pattern in the IV group and IL-10 had a distinct response pattern in control group. The results showed that oral vaccination with IV reduces the TB lesion score in red deer challenged with a M. bovis field strain without interfering with the in vivo diagnosis of infection in this species., Research funding was provided by ‘Plan Nacional’ grant AGL2014-56305 (MINECO, Spain and FEDER). M.A. Risalde holds a ‘Juan de la Cierva program’ contract and F. Ruiz-Fons is funded by the ‘Ramón y Cajal’ program (Ministry of Economy and Competitiveness, Spain). J. Thomas was supported by a grant from the Indian Council of Agricultural Research-International Fellowship 2014–15 (ICAR-IF 2014–15). M. Serrano holds a pre-doctoral fellowship from the Department of Education of the Basque Government (PRE_2016_2_0155).

Summary Background Similarities between human inflammatory bowel disease (IBD) and ruminant paratuberculosis have fueled a heated discussion on the role of Mycobacterium avium subsp. paratuberculosis (MAP) in the etiology of IBD. Methods In order to determine microbiological and immunological evidence of an association between MAP and IBD, blood from 222 inflammatory bowel disease patients and 80 healthy donors from the Basque Country (Spain) were subjected to nested PCR for MAP-specific insertion sequence IS900, interferon-γ (IFN-γ) release test with PPA-3 MAP antigen (IFNMAP) or phosphate-buffered saline (IFNPBS), and antibody ELISA with PPA-3 MAP antigen (ABMAP). Results Highly significant differences in the proportion of PCR-positive IBD patients (17%) and healthy controls (43%) as well as lower IFNMAP and higher ABMAP and IFNPBS responses were observed. Treatment was associated with decreases in IFNMAP and PCR-positive frequency. Conclusions These results indicate the existence of immune responses and treatment interactions with MAP that strongly support an etiological role of this agent in IBD.

Johne's disease or paratuberculosis is a chronic granulomatous inflammation of the small intestine of ruminants caused by Mycobacterium avium subsp. paratuberculosis (MAP). Recent studies suggest an association between MAP and Crohn's disease in humans. MAP can become widely distributed within the tissues of infected animals, and meat may be a possible route of exposure of MAP to humans. In this study, 47 dairy and beef cattle were examined for the occurrence of viable MAP in diaphragm muscle. At the slaughterhouse, gut tissues, diaphragm muscle, blood, and feces of the 47 animals were collected for bacteriological culture, as well as gut samples for histopathological analysis. MAP was detected by bacteriological culture and conventional and real-time IS900 polymerase chain reaction in the diaphragm muscle of six infected cattle at slaughter (13%). The six animals showing evidence of MAP in diaphragm muscle had diffuse lesions and severe granulomatous inflammation in ileocecal lymph nodes, jejunal lymph nodes, ileocecal valve, and ileum. All six had heavy bacterial load in mesenteric lymph nodes, ileocecal valve, ileum, and jejunum, and four showed clinical signs of paratuberculosis. Two animals did not show clinical signs but had viable MAP in intestinal tissues and in diaphragm muscle as well. MAP was found in blood of only one of the six animals showing evidence of MAP in diaphragm muscle and in feces of three of them. In general, there was a positive association between enteric lesion severity, clinical signs of paratuberculosis, heavy bacterial load in intestinal tissues, fecal shedding of MAP, and the presence of disseminated MAP infection in diaphragm muscle. The results of this study demonstrated that MAP can be detected and cultured from muscle of MAP-infected cattle destined for human consumption and suggest a possible risk of exposure of humans to MAP via contaminated meat.

Background Mycobacterium avium subsp. paratuberculosis (Map) causes paratuberculosis in animals and is suspected of causing Crohn's Disease in humans. Characterization of strains led to classify paratuberculosis isolates in two main types, cattle type strains, found affecting all host species, and sheep type strains, reported affecting mainly sheep. In order to get a better understanding of the epidemiology of paratuberculosis a large set of Map isolates obtained from different species over the last 25 years have been characterized. Five-hundred and twenty isolates from different hosts (cattle, sheep, goats, bison, deer and wild boar) and origins had been cultured and typed by IS1311 restriction-endonuclease-analysis. Two-hundred and sixty-nine isolates were further characterized by pulsed-field gel electrophoresis (PFGE) using Sna BI and Spe I endonucleases. Differences in strain isolation upon various media conditions were also studied. Results All bovines, 4 and 26% of Spanish sheep and goats, respectively, and the deer and wild boar studied, carried IS1311-Cattle type strains. IS1311-Sheep type encompassed 96% and 74% of Spanish sheep and goats, and all three Portuguese sheep. Thirty-seven distinct multiplex PFGE profiles were found, giving 32 novel profiles. Profiles 2-1 and 1-1 accounted for the 85% of cattle isolates. Ten distinct profiles were detected in Spanish sheep, none of them with an incidence higher than 25%. Profile 16-11 (43%) and another three profiles were identified in Spanish caprine cultures. The hierarchical analysis, clustered all profiles found in cattle, "wild" hosts and some small ruminants within the same group. The other group included 11 profiles only found in Spanish sheep and goats, including Spanish pigmented profiles. Differences in growth requirements associated with isolate genotype were observed. Conclusion Cattle in Spain are infected with cattle type strains, while sheep and goats are mainly infected with sheep type strains. Although 7H9 broth based culture media seem to broadly cover the growth requirements of most Map strains, the use of various solid media is recommended to reduce any recovery biases. High genetic homogeneity of isolates from cattle, and heterogeneity of those from sheep and goats have been detected.

Background Paratuberculosis vaccination has been in use in some regions for many decades, but results have not been widely spread. A new Mycobacterium avium subsp. paratuberculosis (MAP) killed vaccine was studied in relationship with its effects on fecal shedding and milk production in four farms while other two were kept as controls submitted to a test and cull scheme. Findings Fecal detection (n = 1829) and milking records (n = 2413) have been analyzed after two (5 herds) and four (1 herd) years of the beginning of the intervention. Shedder prevalence was reduced by 100% in three of the four vaccinated farms, 68% in the total of vaccinated animals and 46% in the two control farms. Total amount of MAP shed was reduced 77% in the vaccinated farms and 94% in the control farms. Overall milk production increased up to 3.9% after vaccination, while there was no significant difference in production after intervention in the non-vaccinated farms. Conclusion MAP shedding reduction can be quickly accomplished both by vaccination and by testing and culling. However, vaccination appears to be a less expensive and more sustainable strategy since it required one single intervention and was also associated with an increase in milk production.

A study was carried out to compare the performance of enzyme-linked immunosorbent assay (ELISA) and blood polymerase chain reaction (PCR) for diagnosis of paratuberculosis in cattle and sheep. For cattle, a set of 278 samples from 1 paratuberculosis-affected Friesian farm was used; it included 80 ELISA-positive samples and 198 ELISA-negative samples from an age-matched group. Ninety-four samples were from heifers and 184 were from 2–5-year-old cows. The overall analysis showed a clear association (Fisher exact test [FET] P = 0.0049) but a weak negative agreement (45.3%, kappa = −0.1665 ± 0.0994) between the 2 tests. It reflected a moderate agreement among heifers (87.7%, kappa = 0.4471 ± 0.2435) and a moderate disagreement among cows (62.7%, kappa = −0.3670 ± 0.1057). For sheep, 496 blood samples from 53 Latxa dairy flocks were used; 180 of the blood samples were from dam/offspring pairs. The overall association between the 2 tests on ovine samples was strong (FET, P = 0.0005), whereas the agreement was low (kappa = 0.1622 ± 0.1188). There was slightly better agreement for ewes (kappa = 0.2135 ± 0.1992) than for lambs (kappa = 0.1193 ± 0.1301). There was also a highly unlikely proportion of dam/offspring positive results (FET, P < 0.0001, kappa = 0.6269 ± 0.1854). Four of 6 lambs that were necropsied 1 year after testing had paratuberculosis microscopic lesions in the ileocecal valve (3 lambs) or a PCR-positive result (4 lambs). These results suggest that blood PCR testing might be a potentially useful new approach in paratuberculosis diagnosis, especially in young animals.

Schmallenberg virus (SBV), an arthropod-transmitted pathogenic bunyavirus, continues to be a threat to the European livestock industry, causing morbidity and mortality among young ruminant livestock. Here, we describe a novel SBV subunit vaccine, based on bacterially expressed SBV nucleoprotein (SBV-N) administered with a veterinary-grade Saponin adjuvant. When assayed in an IFNAR-/- mouse model, SBV-N with Saponin induced strong non-neutralizing broadly virus-reactive antibodies, decreased clinical signs, as well as significantly reduced viremia. Vaccination assays also suggest that this level of immune protection is cell mediated, as evidenced by the lack of neutralizing antibodies, as well as interferon-γ secretion observed in vitro. Therefore, based on these results, bacterially expressed SBV-N, co-administered with veterinary-grade Saponin adjuvant may serve as a promising economical alternative to current SBV vaccines, and warrant further evaluation in large ruminant animal models. Moreover, we propose that this strategy may be applicable to other bunyaviruses.

Background Since 2002, an active surveillance program for transmissible spongiform encephalopathy in small ruminants in European Union countries allowed identification of a considerable number of atypical cases with similarities to the previously identified atypical scrapie cases termed Nor98. Case presentation Here we report molecular and neuropathological features of eight atypical/Nor98 scrapie cases detected between 2002 and 2009. Significant features of the affected sheep included: their relatively high ages (mean age 7.9 years, range between 4.3 and 12.8), their breed (all Latxa) and their PRNP genotypes (AFRQ/ALRQ, ALRR/ALRQ, AFRQ/AFRQ, AFRQ/AHQ, ALRQ/ALRH, ALRQ/ALRQ). All the sheep were confirmed as atypical scrapie by immunohistochemistry and immunoblotting. Two cases presented more PrP immunolabelling in cerebral cortex than in cerebellum. Conclusions This work indicates that atypical scrapie constitutes the most common small ruminant transmissible spongiform encephalopathy form in Latxa sheep in the Spanish Basque Country. Moreover, a new genotype (ALRQ/ALRH) was found associated to atypical scrapie.