Elevated dietary zinc oxide levels do not have a substantial effect on porcine reproductive and respiratory syndrome virus (PPRSV) vaccination and infection

Background Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important infectious agents for the swine industry worldwide. Zinc (Zn) salts, which are widely used as a dietary supplement in swine nutrition, have shown antiviral effects in vitro as well as in vivo. The purpose of this study was to determine the influence of dietary zinc oxide supplementation on vaccination and challenge infection with PRRSV. Findings The clinical course of PRRS and the success of vaccination with an experimental inactivated vaccine were compared between animals receiving a conventional diet (50 ppm Zn, control group) and diets supplemented with Zn oxide (ZnO) at final Zn concentrations of 150 or 2,500 ppm. Pigs receiving higher dietary Zn levels showed a tendency towards higher neutralizing antibody levels after infection, while dietary Zn levels did not substantially influence the number of antiviral IFN-gamma secreting cells (IFN-gamma-SC) or percentages of blood immune cell subsets after infection. Finally, feeding higher dietary Zn levels reduced neither clinical symptoms nor viral loads. Conclusions Our results suggest that higher levels of dietary ZnO do not have the potential to stimulate or modulate systemic immune responses after vaccination and heterologous PRRSV infection to an extent that could improve the clinical and virological outcome

Allelic variations in coding regions of the vitamin D receptor gene in dairy cows and potential susceptibility to periparturient hypocalcaemia

Periparturient hypocalcaemia (milk fever) is a disorder of Ca metabolism in dairy cattle primarily affecting multiparous cows. The major reasons for the rapid decrease of blood Ca concentration after calving are the prompt increase of Ca secretion into the colostrum and the delayed activation of Ca regulation mechanisms including calcitriol, a metabolite of vitamin D. In man, vitamin D receptor (VDR) gene polymorphisms are reported to be associated with disturbances of Ca metabolism, whereas data confirming the same in dairy cows are still missing. Moreover, polymorphisms that only affect non-coding regions are sometimes difficult to ascribe to a specific disorder as pathways and unequivocal links remain elusive. Therefore, the idea of the present study was to investigate in a small group of dairy cows with documented clinical records whether polymorphisms in the coding regions of the VDR gene existed and whether these potentially found variations were correlated with the incidence of periparturient hypocalcaemia. For this purpose, blood DNA was isolated from 26 dairy cows in their 4th to 6th lactation, out of which 17 had experienced hypocalcaemia at least once, whereas 9 cows had never undergone periparturient hypocalcaemia in their lifetime. The 10 VDR exons and small parts of adjacent introns were sequenced and compared with the Bos taurus VDR sequence published on NCBI based on the DNA of one Hereford cow. In total, 8 sequence alterations were detected in the fragments, which were primarily heterozygous. However, only 4 of them were really located on exons thereby potentially causing changes of the encoded amino acid of the VDR protein, but were not correlated with the incidence of periparturient hypocalcaemia. Certainly, this lack of statistical correlation could be due to the small number of animals included; anyhow, it was not encouraging enough to initiate a larger study with hundreds of cows and document blood Ca levels post partum for at least four lactations

Dietary Enterococcus faecium NCIMB 10415 and Zinc Oxide Stimulate Immune Reactions to Trivalent Influenza Vaccination in Pigs but Do Not Affect Virological Response upon Challenge Infection

Swine influenza viruses (SIV) regularly cause significant disease in pigs worldwide. Since there is no causative treatment of SIV, we tested if probiotic Enterococcus (E.) faecium NCIMB 10415 or zinc (Zn) oxide as feed supplements provide beneficial effects upon SIV infection in piglets. Seventy- two weaned piglets were fed three different diets containing either E. faecium or different levels of Zn (2500 ppm, Zn(high); 50 ppm, Zn(low)). Half of the piglets were vaccinated intramuscularly (VAC) twice with an inactivated trivalent SIV vaccine, while all piglets were then infected intranasally with H3N2 SIV. Significantly higher weekly weight gains were observed in the E. faecium group before virus infection, and piglets in Zn(high) and E. faecium groups gained weight after infection while those in the control group (Zn(low)) lost weight. Using ELISA, we found significantly higher H3N2-specific antibody levels in the E. faecium+VAC group 2 days before and at the day of challenge infection as well as at 4 and 6 days after challenge infection. Higher hemagglutination inhibition (HI) titers were also observed in the Zn(high)+VAC and E. faecium+VAC groups at 0, 1 and 4 days after infection. However, there were no significant differences in virus shedding and lung lesions between the dietary groups. Using flow cytometry analysis significantly higher activated T helper cells and cytotoxic T lymphocyte percentages in the PBMCs were detected in the Zn(high) and E. faecium groups at single time points after infection compared to the Zn(low) control group, but no prolonged effect was found. In the BAL cells no influence of dietary supplementation on immune cell percentages could be detected. Our results suggest that feeding high doses of zinc oxide and particularly E. faecium could beneficially influence humoral immune responses after vaccination and recovery from SIV infection, but not affect virus shedding and lung pathology

The repetitive landscape of the 5100 Mbp barley genome

While transposable elements (TEs) comprise the bulk of plant genomic DNA, how they contribute to genome structure and organization is still poorly understood. Especially in large genomes where TEs make the majority of genomic DNA, it is still unclear whether TEs target specific chromosomal regions or whether they simply accumulate where they are best tolerated.Peer reviewe

Simultaneous Identification of DNA and RNA Viruses Present in Pig Faeces Using Process-Controlled Deep Sequencing

Background: Animal faeces comprise a community of many different microorganisms including bacteria and viruses. Only scarce information is available about the diversity of viruses present in the faeces of pigs. Here we describe a protocol, which was optimized for the purification of the total fraction of viral particles from pig faeces. The genomes of the purified DNA and RNA viruses were simultaneously amplified by PCR and subjected to deep sequencing followed by bioinformatic analyses. The efficiency of the method was monitored using a process control consisting of three bacteriophages (T4, M13 and MS2) with different morphology and genome types. Defined amounts of the bacteriophages were added to the sample and their abundance was assessed by quantitative PCR during the preparation procedure. Results: The procedure was applied to a pooled faecal sample of five pigs. From this sample, 69,613 sequence reads were generated. All of the added bacteriophages were identified by sequence analysis of the reads. In total, 7.7 % of the reads showed significant sequence identities with published viral sequences. They mainly originated from bacteriophages (73.9%) and mammalian viruses (23.9%); 0.8 % of the sequences showed identities to plant viruses. The most abundant detected porcine viruses were kobuvirus, rotavirus C, astrovirus, enterovirus B, sapovirus and picobirnavirus. In addition, sequences with identities to the chimpanzee stool-associated circular ssDNA virus were identified. Whole genome analysis indicates that this virus, tentatively designated as pig stool-associated circular ssDNA virus (PigSCV), represents a novel pi

Evidence for Regulated Interleukin-4 Expression in Chondrocyte-Scaffolds under In Vitro Inflammatory Conditions

OBJECTIVE: To elucidate the anti-inflammatory and anabolic effects of regulated expression of IL-4 in chondrocyte-scaffolds under in vitro inflammatory conditions. METHODS: Mature articular chondrocytes from dogs (n = 3) were conditioned through transient transfection using pcDNA3.1.cIL-4 (constitutive) or pCOX-2.cIL-4 (cytokine-responsive) plasmids. Conditioned cells were seeded in alginate microspheres and rat-tail collagen type I matrix (CaReS®) to generate two types of tissue-engineered 3-dimensional scaffolds. Inflammatory arthritis was simulated in the packed chondrocytes through exogenous addition of recombinant canine (rc) IL-1β (100 ng/ml) plus rcTNFα (50 ng/ml) in culture media for 96 hours. Harvested cells and culture media were analyzed by various assays to monitor the anti-inflammatory and regenerative (anabolic) properties of cIL-4. RESULTS: cIL-4 was expressed from COX-2 promoter exclusively on the addition of rcIL-1β and rcTNFα while its expression from CMV promoter was constitutive. The expressed cIL-4 downregulated the mRNA expression of IL-1β, TNFα, IL-6, iNOS and COX-2 in the cells and inhibited the production of NO and PGE(2) in culture media. At the same time, it up-regulated the expression of IGF-1, IL-1ra, COL2a1 and aggrecan in conditioned chondrocytes in both scaffolds along with a diminished release of total collagen and sGAG into the culture media. An increased amount of cIL-4 protein was detected both in chondrocyte cell lysate and in concentrated culture media. Neutralizing anti-cIL-4 antibody assay confirmed that the anti-inflammatory and regenerative effects seen are exclusively driven by cIL-4. There was a restricted expression of IL-4 under COX-2 promoter possibly due to negative feedback loop while it was over-expressed under CMV promoter (undesirable). Furthermore, the anti-inflammatory /anabolic outcomes from both scaffolds were reproducible and the therapeutic effects of cIL-4 were both scaffold- and promoter-independent. CONCLUSIONS: Regulated expression of therapeutic candidate gene(s) coupled with suitable scaffold(s) could potentially serve as a useful tissue-engineering tool to devise future treatment strategies for osteoarthritis

A chromosome conformation capture ordered sequence of the barley genome

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Shifting the limits in wheat research and breeding using a fully annotated reference genome

Introduction: Wheat (Triticum aestivum L.) is the most widely cultivated crop on Earth, contributing about a fifth of the total calories consumed by humans. Consequently, wheat yields and production affect the global economy, and failed harvests can lead to social unrest. Breeders continuously strive to develop improved varieties by fine-tuning genetically complex yield and end-use quality parameters while maintaining stable yields and adapting the crop to regionally specific biotic and abiotic stresses. Rationale: Breeding efforts are limited by insufficient knowledge and understanding of wheat biology and the molecular basis of central agronomic traits. To meet the demands of human population growth, there is an urgent need for wheat research and breeding to accelerate genetic gain as well as to increase and protect wheat yield and quality traits. In other plant and animal species, access to a fully annotated and ordered genome sequence, including regulatory sequences and genome-diversity information, has promoted the development of systematic and more time-efficient approaches for the selection and understanding of important traits. Wheat has lagged behind, primarily owing to the challenges of assembling a genome that is more than five times as large as the human genome, polyploid, and complex, containing more than 85% repetitive DNA. To provide a foundation for improvement through molecular breeding, in 2005, the International Wheat Genome Sequencing Consortium set out to deliver a high-quality annotated reference genome sequence of bread wheat. Results: An annotated reference sequence representing the hexaploid bread wheat genome in the form of 21 chromosome-like sequence assemblies has now been delivered, giving access to 107,891 high-confidence genes, including their genomic context of regulatory sequences. This assembly enabled the discovery of tissue- and developmental stage–related gene coexpression networks using a transcriptome atlas representing all stages of wheat development. The dynamics of change in complex gene families involved in environmental adaptation and end-use quality were revealed at subgenome resolution and contextualized to known agronomic single-gene or quantitative trait loci. Aspects of the future value of the annotated assembly for molecular breeding and research were exemplarily illustrated by resolving the genetic basis of a quantitative trait locus conferring resistance to abiotic stress and insect damage as well as by serving as the basis for genome editing of the flowering-time trait. Conclusion: This annotated reference sequence of wheat is a resource that can now drive disruptive innovation in wheat improvement, as this community resource establishes the foundation for accelerating wheat research and application through improved understanding of wheat biology and genomics-assisted breeding. Importantly, the bioinformatics capacity developed for model-organism genomes will facilitate a better understanding of the wheat genome as a result of the high-quality chromosome-based genome assembly. By necessity, breeders work with the genome at the whole chromosome level, as each new cross involves the modification of genome-wide gene networks that control the expression of complex traits such as yield. With the annotated and ordered reference genome sequence in place, researchers and breeders can now easily access sequence-level information to precisely define the necessary changes in the genomes for breeding programs. This will be realized through the implementation of new DNA marker platforms and targeted breeding technologies, including genome editing

The Piggeldat database as a basis for the analysis of the effects of nutritional factors on piglets

Die Auswirkung eines Nahrungsfaktors beim Hausschwein, Sus scrofa domestica, hängt von einem komplexen Zusammenspiel zwischen den Bestandteilen der Ernährung, der gastrointestinalen Mikrobiota und der Reaktion des Wirts ab. Die Untersuchung der Auswirkung eines Nahrungsfaktors profitiert hierdurch besonders von der Integration von Messdaten aus den Bereichen Metagenomics, Metaproteomics, Metatranscriptomics und Metabolomics. Der Sonderforschungsbereich 852 führte in den Jahren 2010 bis 2013 sieben Tierversuche durch, um die Auswirkung des Probiotikums Enterococcus faecium NCIMB 10415 und des Zusatzes von Zink auf die Entwicklung des Ferkels zu untersuchen. Die Projektgruppen des SFB 852 bestimmten dabei unter anderem die Zusammensetzung der gastrointestinalen Mikrobiota, die Expression immunrelevanter Gene, die Zusammensetzung von Immunzellpopulationen und physiologische Parameter. Im Rahmen dieser Arbeit erfolgte die Installation und die Administration der Piggeldat-Datenbank zur Verwaltung und zur Archivierung der diversen Forschungsdaten des SFB 852. Die Piggeldat-Datenbank enthält die Metadaten von insgesamt 490 Ferkeln, 57 Messungen, 1244 Merkmalen und sie enthält insgesamt 81060 Messwerte. Damit bietet die Piggeldat- Datenbank den Zugriff auf die Forschungsdaten von 17 wissenschaftlichen Veröffentlichungen an. Die dokumentierten Daten sind innerhalb des Forschungsprojekts frei verfügbar und können bei Bedarf über die Piggeldat- Website veröffentlicht werden. Die zentrale Speicherung der Daten unterstütze die Durchführung von mehreren statistischen und bioinformatischen Datenanalysen, womit vielfältige Auswirkungen der Fütterung der beiden Nahrungsfaktoren gezeigt werden konnten. Die integrative Analyse der Messdaten aus einem Fütterungsversuch zeigte das veränderte Zusammenspiel zwischen gastrointestinaler Mikrobiota und dem Immunsystem des Wirts durch die Fütterung mit dem Probiotikum Enterococcus faecium NCIMB 10415. Zur Installation der Piggeldat-Datenbank erfolgte die Programmierung der Datenbankanwendung Catria. Catria basiert auf dem Content-Management-System Drupal und dient zur Verwaltung von Forschungsdaten aus Fütterungsversuchen über eine Website. Hierbei bietet Catria die vier Funktionen der Nutzerverwaltung, Datenintegration, Datenverwaltung und Datenansicht an. Das Catria-Datenmodell unterstützt die Integration von allgemeinen Multi-Omics Messdaten aus Fütterungsversuchen und liefert damit ein allgemeines Muster zur strukturierten Publikation von Forschungsdaten aus Fütterungsversuchen. Nach der Integration der Forschungsdaten können die Nutzer die Daten mit anderen Nutzern teilen oder die eigenen Messdaten öffentlich publizieren. Catria ist frei verfügbar und lässt sich in zukünftigen Projekten zur Datenverwaltung einsetzen. Die Integration von Messdaten aus unterschiedlichen biologischen Organisationsebenen diente zur explorativen Untersuchung der Auswirkung des Probiotikums Enterococcus faecium NCIMB 10415 auf das Zusammenspiel zwischen Ernährung, gastrointestinaler Mikrobiota und Wirt beim Ferkel. Insgesamt war die Auswirkung der Fütterung des Probiotikums auf die gemessenen Merkmale relativ gering. Die Anwendung einer Hauptkomponentenanalyse zeigte, dass starke Alterseffekte die Varianz im gesamten Datensatz dominierten. Hierbei konnten vier verschiedene Muster der Altersentwicklung identifiziert werden. Nichtsdestotrotz zeigte die Fütterung des Probiotikums einen signifikanten Effekt auf die Gesamtheit der gemessenen Merkmale. Bei den 26 Tage alten Ferkeln und 34 Tage alten Ferkeln konnte der deutlichste Unterschied zwischen den Behandlungsgruppen beobachtet werden. Dabei unterschieden sich die Auswirkungen des Probiotikums zwischen den vier Messzeitpunkten deutlich. Besonders ausgeprägt war der Effekt der Probiotikum-Fütterung auf die Zusammensetzung und die Aktivität der gastrointestinalen Mikrobiota und auf die Reaktion des Immunsystems. Die Untersuchung der Korrelationen zwischen den gemessenen Merkmalen zeigte ein verändertes Zusammenspiel zwischen der gastrointestinalen Mikrobiota und der Reaktion des Immunsystems. Des Weiteren erhöhte Enterococcus faecium NCIMB 10415 den Zusammenhang der gastrointestinalen Mikrobiota zwischen Ileum und Colon.The effect of a nutritional factor on piglets depends on a complex cross-talk between nutrition, intestinal microbiota and the host reaction. The analysis of nutritional factors benefits especially from the integration of measurement data from the fields of metagenomics, metaprotomics, metatranscriptomics and metabolomics. The collaborative research group SFB 852 performed seven animal trials between 2010 and 2013 to analyse the effects of the probiotic Enterococcus faecium NCIMB 10415 and of the addition of zinc to growing piglets. The project groups of the SFB 852 performed diverse measurements to determine the composition and activity of the intestinal microbiota, expression of immune-related genes, composition of immune cells and physiological parameters. In the context of this work the Piggeldat database was installed and administered in order to manage and archive diverse measurement data from the SFB 852. The Piggeldat database contains metadata about 490 piglets, 57 measurements, 1244 attributes and it contains 81060 measurement values. With this, the Piggeldat database provides access to measurement data from 17 scientific publications. The documented data is freely available to all members of the research group and may be published online on the Piggeldat website on demand. The central storage of measurement data supported the application of several statistical and bioinformatical data analyses, which resulted in the identification of diverse effects of the two nutritional factors. For instance, an integrative analysis of measurement data from one feeding trial identified an effect of the probiotic Enterococcus faecium NCIMB 10415 on the interplay between microbial community and immune system. The database software Catria was implemented in order to install the Piggeldat database. Catria is based on the content management system Drupal and allows for the management of research data from feeding trials via a website. Thereby, Catria provides the separate functions of user administration, data integration, data management and data view. The Catria data-model permits the integration of diverse types of multi-omics data from feeding trials. With this, Catria provides a general pattern for the structured publication of measurement data from feeding trials. After data integration, users may share their data with other users or give public access to their data sets. Catria is freely available and may be used for data management in other projects. A comprehensive data set with measurements from different levels of biological areas was used for an exploratory data analysis to elucidate effects of the probiotic Enterococcus faecium NCIMB 10415 on the interplay between nutrition, intestinal microbiota and host in growing piglets. The overall effect of the probiotic treatment on the whole dataset was relatively low. The application of a principle component analysis indicated dominant age effects that were separated into four distinct patterns. Nevertheless, the analysis identified a significant probiotic effect. The difference between the treatment groups was strongest for piglets between 26 and 34 days old but the effect of the probiotic treatment varied between piglets from different age groups. Enrichment analysis indicated that the effect of the probiotic treatment was most recent on the activity and composition of the intestinal microbiota as well as on the reaction of the immune system. Correlation analyses identified altered associations between intestinal microbial communities and immune system reactions by probiotic supplementation. Furthermore, Enterococcus faecium NCIMB 10415 increased the association of the intestinal microbiota between ileum and colon

The general composition of the faecal virome of pigs depends on age, but not on feeding with a probiotic bacterium.

BACKGROUND:The pig faecal virome, which comprises the community of viruses present in pig faeces, is complex and consists of pig viruses, bacteriophages, transiently passaged plant viruses and other minor virus species. Only little is known about factors influencing its general composition. Here, the effect of the probiotic bacterium Enterococcus faecium (E. faecium) NCIMB 10415 on the pig faecal virome composition was analysed in a pig feeding trial with sows and their piglets, which received either the probiotic bacterium or not. RESULTS:From 8 pooled faecal samples derived from the feeding trial, DNA and RNA virus particles were prepared and subjected to process-controlled Next Generation Sequencing resulting in 390,650 sequence reads. In average, 14% of the reads showed significant sequence identities to known viruses. The percentage of detected mammalian virus sequences was highest (55-77%) in the samples of the youngest piglets and lowest (8-10%) in the samples of the sows. In contrast, the percentage of bacteriophage sequences increased from 22-44% in the youngest piglets to approximately 90% in the sows. The dominating mammalian viruses differed remarkably among 12 day-old piglets (kobuvirus), 54 day-old piglets (boca-, dependo- and pig stool-associated small circular DNA virus [PigSCV]) and the sows (PigSCV, circovirus and "circovirus-like" viruses CB-A and RW-A). In addition, the Shannon index, which reflects the diversity of sequences present in a sample, was generally higher for the sows as compared to the piglets. No consistent differences in the virome composition could be identified between the viromes of the probiotic bacterium-treated group and the control group. CONCLUSION:The analysis indicates that the pig faecal virome shows a high variability and that its general composition is mainly dependent on the age of the pigs. Changes caused by feeding with the probiotic bacterium E. faecium could not be demonstrated using the applied metagenomics method