Proteomic approach to identify candidate effector molecules during the in vitro immune exclusion of infective Teladorsagia circumcincta in the abomasum of sheep

International audienceIn the present study we have employed an in vitro organ challenge model to study the post-challenge responses in parasite naïve and immune gastric tissue of sheep, in an attempt to identify the host derived factors involved in immune exclusion of Teladorsagia circumcincta larvae. Proteins present in the epithelial cells and mucus from ovine abomasa following parasite challenge in previously naïve and immune animals were analysed through Matrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-Tof)-MS and shotgun proteomics. MALDI-ToF analysis of epithelial cell lysates revealed that a number of proteins identified were differentially expressed in naïve and immune cells. These included intelectin and lysozymes, which were present at higher levels in epithelial cell lysates derived from immune samples. A large number of proteins were identified in the mucosal wash from immune tissue which were not present in the mucosal wash of the naïve tissue. Some of these proteins were present in washes of immune tissue prior to the parasite challenge including immunoglobulin A, galectin 14 and 15 and sheep mast cell protease 1. However, other proteins, such as calcium activated chloride channel and intelectin were only detected in the washings from the challenged tissue. The latter may be related to an enhanced mucus release, which may result in entrapment of infective larvae and thus reduced establishment in tissue that has been previously challenged with the parasite. In conclusion, several proteins have been identified which may be involved, either directly or indirectly, in the exclusion and immune elimination of incoming infective larvae. In the present study, the usefulness of the in vitro model has been confirmed, and the global proteomic approach has identified proteins that had not previously been associated with parasite exclusion from abomasal mucosa, such as the calcium activated chloride channel

Unveiling the influence of adaptation time on xylanase and arabinoxylan-oligosaccharide efficacy: a study on nutrient digestibility, viscosity, and scanning electron microscopy in the small and large intestine of growing pigs fed insoluble fiber

The experiment objective was to evaluate the impact of xylanase over time on viscosity and digestibility in growing pigs fed corn-based fiber. Twenty gilts with an initial body weight of 30.6 ± 0.2 kg (n = 5 per dietary treatment) were fitted with t-cannulae in the medial jejunum and terminal ileum, housed individually, and randomly assigned to one of four dietary treatments: low-fiber control (LF) with 10.4% total dietary fiber (TDF), 30% corn bran high-fiber control (HF; 26.4% TDF), HF + 100 mg xylanase/kg (XY; Econase XT 25P; AB Vista, Marlborough, UK), and HF + 50 mg arabinoxylan-oligosaccharide/kg (AX). Gilts were limit fed for three 17 d periods (P1, P2, P3); each included 5 d adaptation, 2 d fecal collection, 3 d ileal collection, 3 d jejunal collection, and 4 d related rate of passage study. Data were analyzed as repeated measures using a linear mixed model with surgery date as a random effect, and dietary treatment, period, and their interaction as fixed effects. Jejunal and ileal digesta viscosity did not differ among dietary treatments or periods (P > 0.10). There was a dietary treatment × period interaction for the apparent jejunal digestibility (AJD) of dry matter (DM), gross energy (GE), insoluble dietary fiber (IDF), neutral detergent fiber (NDF), total arabinoxylan (T-AX), total non-starch polysaccharide (T-NSP), and TDF (P 0.05). In P1, LF had the greatest AJD of DM (15.5%), and relative to HF and AX, XY decreased it (9.3%, 10.1 %, and 6.3%, respectively). In P2, the AJD of DM in XY was greater than HF (11.7% vs. 9.1%) but did not differ from AX (10.5%). Relative to HF, in P3, XY increased AJD of DM (11.7 vs 15.3%), and AX decreased it (7.2%). For the AJD of NDF, AX performed intermediately in P1; in P2, relative to HF, XY, and AX increased the AJD of NDF (8.4%, 13.1%, and 11.7%, respectively), and in P3, XY, and LF did not differ (13.6 vs. 14.4%). A similar response was observed for the AJD of IDF and TDF, except for XY having the greatest AJD of IDF, T-AX, T-NSP, and TDF in P3 (P This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited

Meiosis-specific gene discovery in plants: RNA-Seq applied to isolated Arabidopsis male meiocytes

<p>Abstract</p> <p>Background</p> <p>Meiosis is a critical process in the reproduction and life cycle of flowering plants in which homologous chromosomes pair, synapse, recombine and segregate. Understanding meiosis will not only advance our knowledge of the mechanisms of genetic recombination, but also has substantial applications in crop improvement. Despite the tremendous progress in the past decade in other model organisms (e.g., <it>Saccharomyces cerevisiae </it>and <it>Drosophila melanogaster</it>), the global identification of meiotic genes in flowering plants has remained a challenge due to the lack of efficient methods to collect pure meiocytes for analyzing the temporal and spatial gene expression patterns during meiosis, and for the sensitive identification and quantitation of novel genes.</p> <p>Results</p> <p>A high-throughput approach to identify meiosis-specific genes by combining isolated meiocytes, RNA-Seq, bioinformatic and statistical analysis pipelines was developed. By analyzing the studied genes that have a meiosis function, a pipeline for identifying meiosis-specific genes has been defined. More than 1,000 genes that are specifically or preferentially expressed in meiocytes have been identified as candidate meiosis-specific genes. A group of 55 genes that have mitochondrial genome origins and a significant number of transposable element (TE) genes (1,036) were also found to have up-regulated expression levels in meiocytes.</p> <p>Conclusion</p> <p>These findings advance our understanding of meiotic genes, gene expression and regulation, especially the transcript profiles of MGI genes and TE genes, and provide a framework for functional analysis of genes in meiosis.</p

Dietary intake of xylose impacts the transcriptome and proteome of tissues involved in xylose metabolism in swine

Xylose is a primary component of arabinoxylan in swine diets. As arabinoxylan is a significant component of fiber, and fiber is generally rising in practical pig diets globally, the study of arabinoxylan and xylose is of increasing interest. However, the mechanisms by which free xylose may be absorbed and the pathways impacted by xylose have yet to be elucidated in pigs. The objective of this study was to determine the impact of xylose supplementation on gene expression and protein abundance in jejunum, kidney, liver, and muscle tissues which have previously been identified as possible sites of xylose absorption or metabolism. This study aimed to expand the preliminary understanding of dietary xylose metabolism and utilization in pigs. One study, replicated twice with 24 crossbred gilts, was used to assess two dietary treatments: a xylose-free (0%) control and 8% D-xylose. The impact of xylose on growth was monitored by measuring initial and final body weight, serum IGF-1, and liver glycogen concentrations. The rate and efficiency of weight gain were reduced on the xylose diet but not to a level that would occur if xylose was not used at all; the detection of xylose systemically further supports this conclusion. This study confirmed that pigs can utilize dietary xylose. To determine the impact of xylose on tissue metabolism, samples were collected from all four tissues for gene expression analysis by RNA-sequencing, and kidney and liver samples were subjected to proteomic analysis using 2D-DIGE and mass spectrometry. The majority of differentially expressed (DE) genes were identified in the kidney samples (n = 157), with a few identified in the jejunum (n = 16), liver (n = 1), and muscle (n = 20) samples. The DE genes in the kidney were mainly identified as being involved in lipid biosynthesis and fatty acid metabolism. Proteomic results corroborated these findings. Although the inclusion of xylose in a diet at practical levels is shown to impact energy metabolic processes, it has been confirmed that this five-carbon sugar can support levels of growth only slightly below those of glucose, a six-carbon sugar that is more commonly utilized as an energy source in pig diets

Persistence of immunity to Nematodirus battus infection in lambs

Fifty-four Greyface Suffolk lambs aged 3 months were allocated to six groups of seven and one group of 12. Three groups were infected continuously with Nematodirus battus larvae (L3) over a 7-week period and three groups remained worm-free. One week after the last larval dose all six groups were treated with anthelmintic and challenged with a single dose of 30 000 N. battus L3 either 1, 6 or 12 weeks post-treatment (PT) and killed 10 days later. A seventh continuously infected and treated group (n = 12) was segregated into four sub-groups of three lambs which were used as tissue cell count controls and provided data on local cellular responses prior to challenge. Lambs in the first sub-group were killed immediately after anthelmintic treatment and those in the other sub-groups were killed on the same day that the lambs in the other main groups were challenged. Overall post-challenge worm burdens did not differ significantly between previously infected and challenge control groups although they were significantly reduced in both treatment groups by Week 12 PT. The principal manifestation of acquired immunity that was maintained throughout 12 weeks without further infection was retardation in larval development. There was also evidence of preferential rejection of male worms from immune lambs. Local mast cell, but not eosinophil, responses were significantly enhanced by previous infection and persisted up to Week 12 PT. The numbers of bone marrow eosinophils were significantly increased as a result of previous infection and this response persisted up to Week 12 PT. During primary infection anti-L4 and anti-adult worm IgG responses were significantly increased in the previously infected lambs by Day 42 post-infection. Eosinophil responses during this period did not differ between groups. The inflammatory cell responses, coupled with the parasitological observations, suggest that immunity to previous infection is maintained for up to 12 weeks PT without further antigenic stimulation. This 'immunological memory' may have waned partially after 6 weeks PT although the superimposition of age resistance may have masked the effect

Suppression of ovine lymphocyte activation by Teladorsagia circumcincta larval excretory-secretory products

Teladorsagia circumcincta is an important pathogenic nematode of sheep. It has been demonstrated previously that stimulation of murine T lymphocytes with excretory-secretory (ES) products derived from fourth stage larvae of T. circumcincta (Tci-L4-ES) results in de novo expression of Foxp3, a transcription factor intimately involved in regulatory T cell function. In the current study, Foxp3(+) T cell responses in the abomasum and the effects of Tci-L4-ES on ovine peripheral blood mononuclear cells (PBMC) following T. circumcincta infection were investigated. T. circumcincta infection resulted in a significant increase in numbers of abomasal Foxp3(+) T cells, but not an increase in the proportion of T cells expressing Foxp3. Unlike in mice, Tci-L4-ES was incapable of inducing T cell Foxp3 expression but instead suppressed mitogen-induced and antigen-specific activation and proliferation of ovine PBMC in vitro. This effect was heat labile, suggesting that it is mediated by protein(s). Suppression was associated with up-regulation of interleukin-10 (IL-10) mRNA, and specific monoclonal antibody neutralisation of IL-10 resulted in a 50% reduction in suppression, indicating involvement of the IL-10 signaling pathway. Suppression was significantly reduced in PBMC isolated from T. circumcincta infected vs. helminth-naïve lambs, and this reduction in suppression was associated with an increase in Tci-L4-ES antigen-specific T cells within the PBMC. In conclusion, we have identified a mechanism by which T. circumcincta may modulate the host adaptive immune response, potentially assisting survival of the parasite within the host. However, the impact of Tci-L4-ES-mediated lymphocyte suppression during T. circumcincta infection remains to be determined

The use of a Psoroptes ovis serodiagnostic test for the analysis of a natural outbreak of sheep scab

<p>Abstract</p> <p>Background</p> <p>Sheep scab is a highly contagious disease of sheep caused by the ectoparasitic mite <it>Psoroptes ovis</it>. The disease is endemic in the UK and has significant economic impact through its effects on performance and welfare. Diagnosis of sheep scab is achieved through observation of clinical signs e.g. itching, pruritis and wool loss and ultimately through the detection of mites in skin scrapings. Early stages of infestation are often difficult to diagnose and sub-clinical animals can be a major factor in disease spread. The development of a diagnostic assay would enable farmers and veterinarians to detect disease at an early stage, reducing the risk of developing clinical disease and limiting spread.</p> <p>Methods</p> <p>Serum samples were obtained from an outbreak of sheep scab within an experimental flock (n = 480 (3 samples each from 160 sheep)) allowing the assessment, by ELISA of sheep scab specific antibody prior to infestation, mid-outbreak (combined with clinical assessment) and post-treatment.</p> <p>Results</p> <p>Analysis of pre-infestation samples demonstrated low levels of potential false positives (3.8%). Of the 27 animals with clinical or behavioural signs of disease 25 tested positive at the mid-outbreak sampling period, however, the remaining 2 sheep tested positive at the subsequent sampling period. Clinical assessment revealed the absence of clinical or behavioural signs of disease in 132 sheep, whilst analysis of mid-outbreak samples showed that 105 of these clinically negative animals were serologically positive, representing potential sub-clinical infestations.</p> <p>Conclusions</p> <p>This study demonstrates that this ELISA test can effectively diagnose sheep scab in a natural outbreak of disease, and more importantly, highlights its ability to detect sub-clinically infested animals. This ELISA, employing a single recombinant antigen, represents a major step forward in the diagnosis of sheep scab and may prove to be critical in any future control program.</p

Development of a cDNA microarray for the measurement of gene expression in the sheep scab mite Psoroptes ovis

Background: Sheep scab is caused by the ectoparasitic mite Psoroptes ovis which initiates a profound cutaneous inflammatory response, leading to the development of the skin lesions which are characteristic of the disease. Existing control strategies rely upon injectable endectocides and acaricidal dips but concerns over residues, eco-toxicity and the development of acaricide resistance limit the sustainability of this approach. In order to identify alternative means of disease control, a deeper understanding of both the parasite and its interaction with the host are required. Methods: Herein we describe the development and utilisation of an annotated P. ovis cDNA microarray containing 3,456 elements for the measurement of gene expression in this economically important ectoparasite. The array consists of 981 P. ovis EST sequences printed in triplicate along with 513 control elements. Array performance was validated through the analysis of gene expression differences between fed and starved P. ovis mites. Results: Sequences represented on the array include homologues of major house dust mite allergens and tick salivary proteins, along with factors potentially involved in mite reproduction and xenobiotic metabolism. In order to validate the performance of this unique resource under biological conditions we used the array to analyse gene expression differences between fed and starved P. ovis mites. These analyses identified a number of house dust mite allergen homologues up-regulated in fed mites and P. ovis transcripts involved in stress responses, autophagy and chemosensory perception up-regulated in starved mites. Conclusion: The P. ovis cDNA microarray described here has been shown to be both robust and reproducible and will enable future studies to analyse gene expression in this important ectoparasite

The Long Term Response of Birds to Climate Change: New Results from a Cold Stage Avifauna in Northern England

The early MIS 3 (55–40 Kyr BP associated with Middle Palaeolithic archaeology) bird remains from Pin Hole, Creswell Crags, Derbyshire, England are analysed in the context of the new dating of the site’s stratigraphy. The analysis is restricted to the material from the early MIS 3 level of the cave because the upper fauna is now known to include Holocene material as well as that from the Late Glacial. The results of the analysis confirm the presence of the taxa, possibly unexpected for a Late Pleistocene glacial deposit including records such as Alpine swift, demoiselle crane and long-legged buzzard with southern and/or eastern distributions today. These taxa are accompanied by more expected ones such as willow ptarmigan /red grouse and rock ptarmigan living today in northern and montane areas. Finally, there are temperate taxa normally requiring trees for nesting such as wood pigeon and grey heron. Therefore, the result of the analysis is that the avifauna of early MIS 3 in England included taxa whose ranges today do not overlap making it a non-analogue community similar to the many steppe-tundra mammalian faunas of the time. The inclusion of more temperate and woodland taxa is discussed in the light that parts of northern Europe may have acted as cryptic northern refugia for some such taxa during the last glacial. These records showing former ranges of taxa are considered in the light of modern phylogeographic studies as these often assume former ranges without considering the fossil record of those taxa. In addition to the anomalous combination of taxa during MIS 3 living in Derbyshire, the individuals of a number of the taxa are different in size and shape to members of the species today probably due to the high carrying capacity of the steppe-tundra