An interlaboratory proficiency test using metagenomic sequencing as a diagnostic tool for the detection of RNA viruses in swine fecal material

Metagenomic shotgun sequencing (mNGS) can serve as a generic molecular diagnostic tool. An mNGS proficiency test (PT) was performed in six European veterinary and public health laboratories to detect porcine astroviruses in fecal material and the extracted RNA. While different mNGS workflows for the generation of mNGS data were used in the different laboratories, the bioinformatic analysis was standardized using a metagenomic read classifier as well as read mapping to selected astroviral reference genomes to assess the semiquantitative representation of astrovirus species mixtures. All participants successfully identified and classified most of the viral reads to the two dominant species. The normalized read counts obtained by aligning reads to astrovirus reference genomes by Bowtie2 were in line with Kraken read classification counts. Moreover, participants performed well in terms of repeatability when the fecal sample was tested in duplicate. However, the normalized read counts per detected astrovirus species differed substantially between participants, which was related to the different laboratory methods used for data generation. Further modeling of the mNGS data indicated the importance of selecting appropriate reference data for mNGS read classification. As virus- or sample-specific biases may apply, caution is needed when extrapolating this swine feces-based PT for the detection of other RNA viruses or using different sample types. The suitability of experimental design to a given pathogen/sample matrix combination, quality assurance, interpretation, and follow-up investigation remain critical factors for the diagnostic interpretation of mNGS results. IMPORTANCE: Metagenomic shotgun sequencing (mNGS) is a generic molecular diagnostic method, involving laboratory preparation of samples, sequencing, bioinformatic analysis of millions of short sequences, and interpretation of the results. In this paper, we investigated the performance of mNGS on the detection of porcine astroviruses, a model for RNA viruses in a pig fecal material, among six European veterinary and public health laboratories. We showed that different methods for data generation affect mNGS performance among participants and that the selection of reference genomes is crucial for read classification. Follow-up investigation remains a critical factor for the diagnostic interpretation of mNGS results. The paper contributes to potential improvements of mNGS as a diagnostic tool in clinical settings.</p

Enrichment of in vivo transcription data from dietary intervention studies with in vitro data provides improved insight into gene regulation mechanisms in the intestinal mucosa

Abstract Background Gene expression profiles of intestinal mucosa of chickens and pigs fed over long-term periods (days/weeks) with a diet rich in rye and a diet supplemented with zinc, respectively, or of chickens after a one-day amoxicillin treatment of chickens, were recorded recently. Such dietary interventions are frequently used to modulate animal performance or therapeutically for monogastric livestock. In this study, changes in gene expression induced by these three interventions in cultured “Intestinal Porcine Epithelial Cells” (IPEC-J2) recorded after a short-term period of 2 and 6 hours, were compared to the in vivo gene expression profiles in order to evaluate the capability of this in vitro bioassay in predicting in vivo responses. Methods Lists of response genes were analysed with bioinformatics programs to identify common biological pathways induced in vivo as well as in vitro. Furthermore, overlapping genes and pathways were evaluated for possible involvement in the biological processes induced in vivo by datamining and consulting literature. Results For all three interventions, only a limited number of identical genes and a few common biological processes/pathways were found to be affected by the respective interventions. However, several enterocyte-specific regulatory and secreted effector proteins that responded in vitro could be related to processes regulated in vivo, i.e. processes related to mineral absorption, (epithelial) cell adherence and tight junction formation for zinc, microtubule and cytoskeleton integrity for amoxicillin, and cell-cycle progression and mucus production for rye. Conclusions Short-term gene expression responses to dietary interventions as measured in the in vitro bioassay have a low predictability for long-term responses as measured in the intestinal mucosa in vivo. The short-term responses of a set regulatory and effector genes, as measured in this bioassay, however, provided additional insight into how specific processes in piglets and broilers may be modulated by “early” signalling molecules produced by enterocytes. The relevance of this set of regulatory/effector genes and cognate biological processes for zinc deficiency and supplementation, gluten allergy (rye), and amoxicillin administration in humans is discussed

Effects of Digested Onion Extracts on Intestinal Gene Expression: An Interspecies Comparison Using Different Intestine Models.

Human intestinal tissue samples are barely accessible to study potential health benefits of nutritional compounds. Numbers of animals used in animal trials, however, need to be minimalized. Therefore, we explored the applicability of in vitro (human Caco-2 cells) and ex vivo intestine models (rat precision cut intestine slices and the pig in-situ small intestinal segment perfusion (SISP) technique) to study the effect of food compounds. In vitro digested yellow (YOd) and white onion extracts (WOd) were used as model food compounds and transcriptomics was applied to obtain more insight into which extent mode of actions depend on the model. The three intestine models shared 9,140 genes which were used to compare the responses to digested onions between the models. Unsupervised clustering analysis showed that genes up- or down-regulated by WOd in human Caco-2 cells and rat intestine slices were similarly regulated by YOd, indicating comparable modes of action for the two onion species. Highly variable responses to onion were found in the pig SISP model. By focussing only on genes with significant differential expression, in combination with a fold change > 1.5, 15 genes showed similar onion-induced expression in human Caco-2 cells and rat intestine slices and 2 overlapping genes were found between the human Caco-2 and pig SISP model. Pathway analyses revealed that mainly processes related to oxidative stress, and especially the Keap1-Nrf2 pathway, were affected by onions in all three models. Our data fit with previous in vivo studies showing that the beneficial effects of onions are mostly linked to their antioxidant properties. Taken together, our data indicate that each of the in vitro and ex vivo intestine models used in this study, taking into account their limitations, can be used to determine modes of action of nutritional compounds and can thereby reduce the number of animals used in conventional nutritional intervention studies

Ontwikkeling van een meetlat voor immuuncompetentie in varkens, vleeskuikens en vleeskalveren

Het doel van dit project is om een “meetlat” te ontwikkelen die de effecten van (voedings)interventies gericht op de verbetering van de immuuncompetentie van varkens, pluimvee en vleeskalveren kan vaststellen. Immuuncompetentie is binnen dit project gedefinieerd als het vermogen van dieren om effectieve responsen van het immuunsysteem te tonen op het moment dat de gezondheid van het dier onder druk wordt gezet. Een meetlat voor immuuncompetentie kan in de toekomst door de diervoedingssector gebruikt worden bij de ontwikkeling en evaluatie van nieuwe voerconcepten, ingrediënten en additieven gericht op de verbetering en ondersteuning van diergezondheid. Het is bekend dat de samenstelling van de voeding van jonge dieren invloed heeft op de functionele ontwikkeling van het maagdarmkanaal en op de samenstelling van de daarin aanwezige microbiota. De interacties tussen de microbiota en de weefsels van het darmkanaal (cross talk) hebben een belangrijke invloed op de ontwikkeling van immuuncompetentie. Daarom wordt in dit project gefocust op de effecten van (voedings)interventies op de microbiota, genexpressie veranderingen in darmweefsel, en morfologische en immunologische veranderen in de darm. De hier gepresenteerde meetlat voor immuuncompetentie is gebaseerd op de resultaten van onderzoek binnen het VDI programma van Feed4Foodure (projecten VDI-11; vleeskuikens, VDI-12; biggen, VDI- 13; gespeende biggen en kalveren) waarin m.b.v. model interventies de effecten van variatie in voersamenstelling op de microbiota samenstelling in het darmkanaal, de biologische responsen van darmweefsel en de zoötechnische dierprestaties zijn onderzocht. In de hier gepresenteerde meetlat worden gemeten effecten in deze studies aan elkaar gerelateerd en functioneel inzichtelijk gemaakt. Dit rapport beschrijft de ontwikkeling en totstandkoming van een eerste versie van de meetlat. Hierbij worden gemaakte keuzes, beperkingen en mogelijkheden van de meetlat bediscussieerd. Tenslotte wordt inzicht gegeven in de mogelijkheden tot verdere verfijningen en de toepasbaarheid van de meetlat

Experimental and field investigations of exposure, replication and transmission of SARS-CoV-2 in pigs in the Netherlands

In order to assess the risk of SARS-CoV-2 infection, transmission and reservoir development in swine, we combined results of an experimental and two observational studies. First, intranasal and intratracheal challenge of eight pigs did not result in infection, based on clinical signs and PCR on swab and lung tissue samples. Two serum samples returned a low positive result in virus neutralization, in line with findings in other infection experiments in pigs. Next, a retrospective observational study was performed in the Netherlands in the spring of 2020. Serum samples (N =417) obtained at slaughter from 17 farms located in a region with a high human case incidence in the first wave of the pandemic. Samples were tested with protein micro array, plaque reduction neutralization test and receptor-binding-domain ELISA. None of the serum samples was positive in all three assays, although six samples from one farm returned a low positive result in PRNT (titers 40-80). Therefore we conclude that serological evidence for large scale transmission was not observed. Finally, an outbreak of respiratory disease in pigs on one farm, coinciding with recent exposure to SARS-CoV-2 infected animal caretakers, was investigated. Tonsil swabs and paired serum samples were tested. No evidence for infection with SARS-CoV-2 was found. In conclusion, Although in both the experimental and the observational study few samples returned low antibody titer results in PRNT infection with SARS-CoV-2 was not confirmed. It was concluded that sporadic infections in the field cannot be excluded, but large-scale SARS-CoV-2 transmission among pigs is unlikely.info:eu-repo/semantics/publishedVersio

Early transcriptional response in the jejunum of germ-free piglets after oral infection with virulent rotavirus

Germ-free piglets were orally infected with virulent rotavirus to collect jejunal mucosal scrapings at 12 and 18 hours post infection (two piglets per time point). IFN-gamma mRNA expression was stimulated in the mucosa of all four infected piglets, indicating that they all responded to the rotavirus infection. RNA pools prepared from two infected piglets were used to compare whole mucosal gene expression at 12 and 18 hpi to expression in uninfected germ-free piglets (n = 3) using a porcine intestinal cDNA microarray. Microarray analysis identified 13 down-regulated and 17 up-regulated genes. Northern blot analysis of a selected group of genes confirmed the data of the microarray. Genes were functionally clustered in interferon-regulated genes, proliferation/differentiation genes, apoptosis genes, cytoskeleton genes, signal transduction genes, and enterocyte digestive, absorptive, and transport genes. Down-regulation of the transport gene cluster reflected in part the loss of rotavirus-infected enterocytes from the villous tips. Data mining suggested that several genes were regulated in lower- or mid-villus immature enterocytes and goblet cells, probably to support repair of the damaged epithelial cell layer at the villous tips. Furthermore, up-regulation was observed for IFN-γ induced guanylate binding protein 2, a protein that effectively inhibited VSV and EMCV replication in vitro (Arch Virol 150:1213–1220, 2005). This protein may play a role in the small intestine’s innate defense against enteric viruses like rotavirus

Chromatic periodic activity down to 120 MHz in a Fast Radio Burst

Fast radio bursts (FRBs) are extragalactic astrophysical transients whose brightness requires emitters that are highly energetic, yet compact enough to produce the short, millisecond-duration bursts. FRBs have thus far been detected between 300 MHz and 8 GHz, but lower-frequency emission has remained elusive. A subset of FRBs is known to repeat, and one of those sources, FRB 20180916B, does so with a 16.3 day activity period. Using simultaneous Apertif and LOFAR data, we show that FRB 20180916B emits down to 120 MHz, and that its activity window is both narrower and earlier at higher frequencies. Binary wind interaction models predict a narrower periodic activity window at lower frequencies, which is the opposite of our observations. Our detections establish that low-frequency FRB emission can escape the local medium. For bursts of the same fluence, FRB 20180916B is more active below 200 MHz than at 1.4 GHz. Combining our results with previous upper-limits on the all-sky FRB rate at 150 MHz, we find that there are 3-450 FRBs/sky/day above 50 Jy ms at 90% confidence. We are able to rule out the scenario in which companion winds cause FRB periodicity. We also demonstrate that some FRBs live in clean environments that do not absorb or scatter low-frequency radiation.Comment: 50 pages, 14 figures, 3 tables, submitte

A High-resolution Mosaic of the Neutral Hydrogen in the M81 Triplet

We present a 3° × 3°, 105-pointing, high-resolution neutral hydrogen (H I) mosaic of the M81 galaxy triplet, (including the main galaxies M81, M82, and NGC 3077, as well as dwarf galaxy NGC 2976) obtained with the Very Large Array C and D arrays. This H I synthesis mosaic uniformly covers the entire area and velocity range of the triplet. The observations have a resolution of ̃20″ or ̃420 pc. The data reveal many small-scale anomalous velocity features highlighting the complexity of the interacting M81 triplet. We compare our data with Green Bank Telescope observations of the same area. This comparison provides evidence for the presence of a substantial reservoir of low-column density gas in the northern part of the triplet, probably associated with M82. Such a reservoir is not found in the southern part. We report a number of newly discovered kpc-sized low-mass H I clouds with H I masses of a few times 106 M ☉. A detailed analysis of their velocity widths show that their dynamical masses are much larger than their baryonic masses, which could indicate the presence of dark matter if the clouds are rotationally supported. However, due to their spatial and kinematical association with H I tidal features, it is more likely that the velocity widths indicate tidal effects or streaming motions. We do not find any clouds that are not associated with tidal features down to an H I mass limit of a few times 104 M ☉. We compare the H I column densities with resolved stellar density maps and find a star formation threshold around 3-6 × 1020 cm-2. We investigate the widths of the H I velocity profiles in the triplet and find that extreme velocity dispersions can be explained by a superposition of multiple components along the line of sight near M81 as well as winds or outflows around M82. The velocity dispersions found are high enough that these processes could explain the linewidths of damped-Lyα absorbers observed at high redshift.</p