Age Matters: Community Assembly in the Pig Fecal Microbiome in the First Month of Life

Despite the wealth of research into strategies for microbiome modulation, studies of microbiome management in pig hosts have found mixed results. A refined understanding of the patterns of microbiome assembly during the host’s early life, when management strategies are most commonly applied, is necessary for the development of successful management practices. Here, we study the development of the pig gut microbial community in a monitoring experiment, sampling the microbiome of pigs in a commercial farm intensively during the first month of life. We found that the community’s taxonomic richness increased linearly with host age. Furthermore, rapid changes across communities occurred in stages, and non-linear patterns in relative abundance were commonly observed among dominant taxa across host age, consistent with primary succession. Our results highlight the importance of understanding the patterns of microbiome assembly during host development, and identify successional stages as windows of opportunity for future research

Gene expression profiling en association with prion-related lesions in the medulla oblongata of symptomatic natural scrapie animals.

The pathogenesis of natural scrapie and other prion diseases remains unclear. Examining transcriptome variations in infected versus control animals may highlight new genes potentially involved in some of the molecular mechanisms of prion-induced pathology. The aim of this work was to identify disease-associated alterations in the gene expression profiles of the caudal medulla oblongata (MO) in sheep presenting the symptomatic phase of natural scrapie. The gene expression patterns in the MO from 7 sheep that had been naturally infected with scrapie were compared with 6 controls using a Central Veterinary Institute (CVI) custom designed 4×44K microarray. The microarray consisted of a probe set on the previously sequenced ovine tissue library by CVI and was supplemented with all of the Ovis aries transcripts that are currently publicly available. Over 350 probe sets displayed greater than 2-fold changes in expression. We identified 148 genes from these probes, many of which encode proteins that are involved in the immune response, ion transport, cell adhesion, and transcription. Our results confirm previously published gene expression changes that were observed in murine models with induced scrapie. Moreover, we have identified new genes that exhibit differential expression in scrapie and could be involved in prion neuropathology. Finally, we have investigated the relationship between gene expression profiles and the appearance of the main scrapie-related lesions, including prion protein deposition, gliosis and spongiosis. In this context, the potential impacts of these gene expression changes in the MO on scrapie development are discussed

Prion protein self-peptides modulate prion interactions and conversion

<p>Abstract</p> <p>Background</p> <p>Molecular mechanisms underlying prion agent replication, converting host-encoded cellular prion protein (PrP^C) into the scrapie associated isoform (PrP^Sc), are poorly understood. Selective self-interaction between PrP molecules forms a basis underlying the observed differences of the PrP^Cinto PrP^Scconversion process (agent replication). The importance of previously peptide-scanning mapped ovine PrP self-interaction domains on this conversion was investigated by studying the ability of six of these ovine PrP based peptides to modulate two processes; PrP self-interaction and conversion.</p> <p>Results</p> <p>Three peptides (octarepeat, binding domain 2 -and C-terminal) were capable of inhibiting self-interaction of PrP in a solid-phase PrP peptide array. Three peptides (N-terminal, binding domain 2, and amyloidogenic motif) modulated prion conversion when added before or after initiation of the prion protein misfolding cyclic amplification (PMCA) reaction using brain homogenates. The C-terminal peptides (core region and C-terminal) only affected conversion (increased PrP^resformation) when added before mixing PrP^Cand PrP^Sc, whereas the octarepeat peptide only affected conversion when added after this mixing.</p> <p>Conclusion</p> <p>This study identified the putative PrP core binding domain that facilitates the PrP^C-PrP^Scinteraction (not conversion), corroborating evidence that the region of PrP containing this domain is important in the species-barrier and/or scrapie susceptibility. The octarepeats can be involved in PrP^C-PrP^Scstabilization, whereas the N-terminal glycosaminoglycan binding motif and the amyloidogenic motif indirectly affected conversion. Binding domain 2 and the C-terminal domain are directly implicated in PrP^Cself-interaction during the conversion process and may prove to be prime targets in new therapeutic strategy development, potentially retaining PrP^Cfunction. These results emphasize the importance of probable PrP^C-PrP^Cand required PrP^C-PrP^Scinteractions during PrP conversion. All interactions are probably part of the complex process in which polymorphisms and species barriers affect TSE transmission and susceptibility.</p

Scrapie prevalence in sheep of susceptible genotype is declining in a population subject to breeding for resistance

<p>Abstract</p> <p>Background</p> <p>Susceptibility of sheep to scrapie infection is known to be modulated by the PrP genotype of the animal. In the Netherlands an ambitious scrapie control programme was started in 1998, based on genetic selection of animals for breeding. From 2002 onwards EU regulations required intensive active scrapie surveillance as well as certain control measures in affected flocks.</p> <p>Here we analyze the data on genotype frequencies and scrapie prevalence in the Dutch sheep population obtained from both surveillance and affected flocks, to identify temporal trends. We also estimate the genotype-specific relative risks to become a detected scrapie case.</p> <p>Results</p> <p>We find that the breeding programme has produced a steady increase in the level of genetic scrapie resistance in the Dutch sheep population. We also find that a significant decline in the prevalence of scrapie in tested animals has occurred a number of years after the start of the breeding programme. Most importantly, the estimated scrapie prevalence level per head of susceptible genotype is also declining significantly, indicating that selective breeding causes a population effect.</p> <p>Conclusions</p> <p>The Dutch scrapie control programme has produced a steady rise in genetic resistance levels in recent years. A recent decline in the scrapie prevalence per tested sheep of susceptible prion protein genotype indicates that selective breeding causes the desired population effect.</p

Eradication of scrapie with selective breeding: are we nearly there?

<p>Abstract</p> <p>Background</p> <p>Following EU decision 2003/100/EC Member States have recently implemented sheep breeding programmes to reduce the prevalence of sheep with TSE susceptible prion genotypes. The present paper investigates the progress of the breeding programme in the Netherlands. The PrP genotype frequencies were monitored through time using two sets of random samples: one set covers the years 2005 to 2008 and is taken from national surveillance programme; the other is taken from 168 random sheep farms in 2007. The data reveal that although the level of compliance to the breeding programme has been high, the frequency of susceptible genotypes varies substantially between farms. The 168 sheep farms are a subset of 689 farms participating in a postal survey inquiring about management and breeding strategies. This survey aimed to identify how much these strategies varied between farms, in order to inform assessment of the expected future progress towards eradication of classical scrapie.</p> <p>Results</p> <p>On the one hand, we found that compliance to the national breeding program has been high, and the frequency of resistant genotypes is expected to increase further in the next few years. On the other hand, we observed a large variation in prevalence of the scrapie resistant PrP genotype ARR between farms, implicating a large variation of genetic resistance between farms. Substantial between-flock differences in management and breeding strategies were found in the postal survey, suggesting considerable variation in risk of scrapie transmission between farms.</p> <p>Conclusions</p> <p>Our results show that although there has been a good progress in the breeding for scrapie resistance and the average farm-level scrapie susceptibility in the Netherlands has been significantly reduced, still a considerable proportion of farms contain high frequencies of susceptible genotypes in their sheep population. Since 2007 the breeding for genetic resistance is voluntarily again, and participation to selective breeding can decrease as a result of this. This, together with the patterns of direct and indirect contact between sheep farms, might present a challenge of the aim of scrapie eradication. Communication to sheep owners of the effect of the breeding programme thus far, and of the prospects for classical scrapie eradication in The Netherlands might be essential for obtaining useful levels of participation to the voluntary continuation of the breeding programme.</p

Rapid and discriminatory diagnosis of scrapie and BSE in retro-pharyngeal lymph nodes of sheep

BACKGROUND: Diagnosis based on prion detection in lymph nodes of sheep and goats can improve active surveillance for scrapie and, if it were circulating, for bovine spongiform encephalopathy (BSE). With sizes that allow repetitive testing and a location that is easily accessible at slaughter, retropharyngeal lymph nodes (RLN) are considered suitable organs for testing. Western blotting (WB) of brain homogenates is, in principle, a technique well suited to both detect and discriminate between scrapie and BSE. In this report, WB is developed for rapid diagnosis in RLN and to study biochemical characteristics of PrP(res). RESULTS: Optimal PrP(res )detection in RLN by WB was achieved by proper tissue processing, antibody choice and inclusion of a step for PrP(res)concentration. The analyses were performed on three different sheep sources. Firstly, in a study with preclinical scrapie cases, WB of RLN from infected sheep of VRQ/VRQ genotype – VRQ represents, respectively, polymorphic PrP amino acids 136, 154, and 171 – allowed a diagnosis 14 mo earlier compared to WB of brain stem. Secondly, samples collected from sheep with confirmed scrapie in the course of passive and active surveillance programmes in the period 2002–2003 yielded positive results depending on genotype: all sheep with genotypes ARH/VRQ, VRQ/VRQ, and ARQ/VRQ scored positive for PrP(res), but ARQ/ARQ and ARR/VRQ were not all positive. Thirdly, in an experimental BSE study, detection of PrP(res )in all 11 ARQ/ARQ sheep, including 7 preclinical cases, was possible. In all instances, WB and IHC were almost as sensitive. Moreover, BSE infection could be discriminated from scrapie infection by faster electrophoretic migration of the PrP(res )bands. Using dual antibody staining with selected monoclonal antibodies like 12B2 and L42, these differences in migration could be employed for an unequivocal differentiation between BSE and scrapie. With respect to glycosylation of PrP(res), BSE cases exhibited a greater diglycosylated fraction than scrapie cases. Furthermore, a slight time dependent increase of diglycosylated PrP(res )was noted between individual sheep, which was remarkable in that it occurred in both scrapie and BSE study. CONCLUSION: The present data indicate that, used in conjunction with testing in brain, WB of RLN can be a sensitive tool for improving surveillance of scrapie and BSE, allowing early detection of BSE and scrapie and thereby ensuring safer sheep and goat products

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom

The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection

The shufflon of IncI1 plasmids is rearranged constantly during different growth conditions

One of the factors that can affect conjugation of IncI1 plasmids, amongst others, is the genetic region known as the shufflon. This multiple inversion system modifies the pilus tip proteins used during conjugation, thus affecting the affinity for different recipient cells. Although recombination is known to occur in in vitro conditions, little is known about the regulation and the extent of recombination that occurs. To measure the recombination of the shufflon, we have amplified the entire shufflon region and sequenced the amplicons using nanopore long-read sequencing. This method was effective to determine the order of the segments of the shufflon and allow for the analysis of the shufflon variants that are present in a heterogeneous pool of templates. Analysis was performed over different growth phases and after addition of cefotaxime. Furthermore, analysis was performed in different E. coli host cells to determine if recombination is likely to be influenced. Recombination of the shufflon was constantly ongoing in all conditions that were measured, although no differences in the amount of different shufflon variants or the rate at which novel variants were formed could be found. As previously reported, some variants were abundant in the population while others were scarce. This leads to the conclusion that the shufflon is continuously recombining at a constant rate, or that the method used here was not sensitive enough to detect differences in this rate. For one of the plasmids, the host cell appeared to have an effect on the specific shufflon variants that were formed which were not predominant in another host, indicating that host factors may be involved. As previously reported, the pilV-A and pilV-A' ORFs are formed at higher frequencies than other pilV ORFs. These results demonstrate that the recombination that occurs within the shufflon is not random. While any regulation of the shufflon affected by these in vitro conditions could not be revealed, the method of amplifying large regions for long-read sequencing for the analysis of multiple inversion systems proved effective.</p

State-of-the-art review of goat TSE in the European Union, with special emphasis on PRNP genetics and epidemiology

Scrapie is a fatal, neurodegenerative disease of sheep and goats. It is also the earliest known member in the family of diseases classified as transmissible spongiform encephalopathies (TSE) or prion diseases, which includes Creutzfeldt-Jakob disease in humans, bovine spongiform encephalopathy (BSE), and chronic wasting disease in cervids. The recent revelation of naturally occurring BSE in a goat has brought the issue of TSE in goats to the attention of the public. In contrast to scrapie, BSE presents a proven risk to humans. The risk of goat BSE, however, is difficult to evaluate, as our knowledge of TSE in goats is limited. Natural caprine scrapie has been discovered throughout Europe, with reported cases generally being greatest in countries with the highest goat populations. As with sheep scrapie, susceptibility and incubation period duration of goat scrapie are most likely controlled by the prion protein (PrP) gene (PRNP). Like the PRNP of sheep, the caprine PRNP shows significantly greater variability than that of cattle and humans. Although PRNP variability in goats differs from that observed in sheep, the two species share several identical alleles. Moreover, while the ARR allele associated with enhancing resistance in sheep is not present in the goat PRNP, there is evidence for the existence of other PrP variants related to resistance. This review presents the current knowledge of the epidemiology of caprine scrapie within the major European goat populations, and compiles the current data on genetic variability of PRNP

Farm dust resistomes and bacterial microbiomes in European poultry and pig farms

Background: Livestock farms are a reservoir of antimicrobial resistant bacteria from feces. Airborne dust-bound bacteria can spread across the barn and to the outdoor environment. Therefore, exposure to farm dust may be of concern for animals, farmers and neighboring residents. Although dust is a potential route of transmission, little is known about the resistome and bacterial microbiome of farm dust. Objectives: We describe the resistome and bacterial microbiome of pig and poultry farm dust and their relation with animal feces resistomes and bacterial microbiomes, and on-farm antimicrobial usage (AMU). In addition, the relation between dust and farmers' stool resistomes was explored. Methods: In the EFFORT-study, resistomes and bacterial microbiomes of indoor farm dust collected on Electrostatic Dust fall Collectors (EDCs), and animal feces of 35 conventional broiler and 44 farrow-to-finish pig farms from nine European countries were determined by shotgun metagenomic analysis. The analysis also included 79 stool samples from farmers working or living at 12 broiler and 19 pig farms and 46 human controls. Relative abundance of and variation in resistome and bacterial composition of farm dust was described and compared to animal feces and farmers' stool. Results: The farm dust resistome contained a large variety of antimicrobial resistance genes (ARGs); more than the animal fecal resistome. For both poultry and pigs, composition of dust resistomes finds (partly) its origin in animal feces as dust resistomes correlated significantly with fecal resistomes. The dust bacterial microbiome also correlated significantly with the dust resistome composition. A positive association between AMU in animals on the farm and the total abundance of the dust resistome was found. Occupational exposure to pig farm dust or animal feces may contribute to farmers' resistomes, however no major shifts in farmers resistome towards feces or dust resistomes were found in this study. Conclusion: Poultry and pig farm dust resistomes are rich and abundant and associated with the fecal resistome of the animals and the dust bacterial microbiome