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Principal component analysis (PCA) showing the effects of the 47 significant discriminatory peaks on piglets positive or negative to PRRSV infection. The figure shows a projection of the measured peak intensities profiles onto the plane spanned by the three principal components (PCAs) that are the axes along which the data vary the most, for the 35 PRRSV-positive (blue) and the 35 PRRSV-negative (red) piglets of the validation study. PCA1, PCA2, and PCA3 accounted for 58.2%, 17.9%, and 12.9% of the variability in the data, respectively. PCA analysis illustrates a 3-dimentional plot comparison of PCA1, PCA2 and PCA3 in the three axes (A), as well as 2-dimentional score plot comparisons between PCA1 and PCA2 (B).
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Background
Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant swine diseases worldwide. Despite its relevance, serum biomarkers associated with early-onset viral infection, when clinical signs are not detectable and the disease is characterized by a weak anti-viral response and persistent infection, have not yet bee...
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Context 1
... analysis showed that 58.2% (PCA1), 17.9% (PCA2), and 12.9% (PCA3) of the total variability within the data was accounted for the X, Y, and Z axes, respectively. These axes were used to plot the data (Figure 2) and they provide an overview of the variation between the individual samples and show how samples grouped. Figure 2A showed three-dimensionally that the PCA peak profiles of piglets positive to PRRSV differed from piglets negative to PRRSV and revealed a good separation among the profiles of the two different groups, especially considering the high heterogeneity of the samples included in the study, as reported in the MM section and in [Additional file 1: Table S1 and Additional file 2: Table S2]. ...Context 2
... axes were used to plot the data (Figure 2) and they provide an overview of the variation between the individual samples and show how samples grouped. Figure 2A showed three-dimensionally that the PCA peak profiles of piglets positive to PRRSV differed from piglets negative to PRRSV and revealed a good separation among the profiles of the two different groups, especially considering the high heterogeneity of the samples included in the study, as reported in the MM section and in [Additional file 1: Table S1 and Additional file 2: Table S2]. Furthermore, with the exception of few The 785 total number of peaks detected and the 200 statistically significant (p < 0.05) discriminatory peaks associated with PRRS infection that were identified by the Ciphergen Express software are reported with the fraction, the array surface, and the acquisition focus mass (low: 1-20 kDa; high: 20-200 kDa). ...Context 3
... with the exception of few The 785 total number of peaks detected and the 200 statistically significant (p < 0.05) discriminatory peaks associated with PRRS infection that were identified by the Ciphergen Express software are reported with the fraction, the array surface, and the acquisition focus mass (low: 1-20 kDa; high: 20-200 kDa). outliers, PCA1 combined with PCA2 also separated well the two piglet populations ( Figure 2B). ...Similar publications
Porcine reproductive and respiratory syndrome virus (PRRSV) is an epidemic etiology in pigs of all ages causing reproductive failure and respiratory manifestation. PRRSV has been circulating in Chinese pig farms for almost 20 years. The aim of the present study was to fully understand the extent of the genetic diversity and molecular characteristic...
Porcine reproductive and respiratory syndrome virus (PRRSV) infects mainly the porcine alveolar macrophages (PAMs) and causes porcine reproductive and respiratory syndrome (PRRS). Previous studies have analyzed the global gene expression profiles of lung tissue in vivo and PAMs in vitro following infection with PRRSV, however, transcriptome-wide un...
Porcine Reproductive and Respiratory Syndrome (PRRS) is an important disease that causes severe economic losses in pig industry. PRRSV has two genotypes named as a European (EU) and North American (US). PRRSV appears globally a variety of countries including Canada, USA, South Korea, Germany, Spain, UK, Denmark and Greece. Furthermore, US genotype...
Contrary to pig farming in developed western countries, in a large part of the world, pigs are still traditionally kept, in small backyard farms, usually for family needs. Their main characteristics are low biosecurity, swill feeding, natural breeding and uncontrolled trade. Given the high number of backyard farms in Serbia and the risk they are th...
Porcine reproductive and respiratory syndrome (PRRS) is identified as one of the most important etiological agents in multifactorial respiratory disease of swine and can predispose pigs to secondary infections by other pathogens, usually bacteria. To understand the mechanism for an increased susceptibility to secondary bacterial infections, we inve...
Citations
... Some more reports have instead dealt with specific pathological conditions: these conditions included bacterial (Muk et al. 2019) and viral (Genini et al. 2008;Liu et al. 2011;Genini et al. 2012;Koene et al. 2012) infections; trauma as hypoxia-ischemia (Kyng et al. 2018), hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture (Cudjoe et al. 2019)), and diet-induced metabolic syndrome (Bell et al. 2010;Pas et al. 2018). The analytical approach was usually LC-MS/MS, but it was SELDI-TOF in a few cases (Genini et al. 2008;Genini et al. 2012;Koene et al. 2012). ...
... Some more reports have instead dealt with specific pathological conditions: these conditions included bacterial (Muk et al. 2019) and viral (Genini et al. 2008;Liu et al. 2011;Genini et al. 2012;Koene et al. 2012) infections; trauma as hypoxia-ischemia (Kyng et al. 2018), hemorrhagic shock, tissue injury, liver reperfusion, hypothermia, and comminuted bone fracture (Cudjoe et al. 2019)), and diet-induced metabolic syndrome (Bell et al. 2010;Pas et al. 2018). The analytical approach was usually LC-MS/MS, but it was SELDI-TOF in a few cases (Genini et al. 2008;Genini et al. 2012;Koene et al. 2012). ...
Acute phase proteins (APPs) reflect the health status of individuals and are important tools in diagnostics, as their altered levels are a sign of disturbed homeostasis. While, in most cases, quantitation of known serum APPs is routinely performed by immunoassays, proteomics is helpful in discovery of new biomarker candidates, especially in samples other than body fluids. Besides putting APP regulation into an overall context of differentially abundant proteins, this approach can detect further details or outright new features in protein structure or specific modifications, and help understand better their function. Thus, it can show up ways to make present diagnostic assays more sensitive and/or specific, or correlate regulations of disease-specific proteins. The APP repertoire is dependent on the species. The pig is both, an important farm animal and a model animal for human diseases, due to similarities in physiology. Besides reviewing existing literature, yet unpublished examples for two-dimensional electrophoresis in connection with pig APPs highlight some of the benefits of proteomics. Of further help would be the emerging targeted proteomics, offering the possibility to determine particular isoforms or proteoforms, without the need of specific antibodies, but this method is presently scarcely used in veterinary medicine.
... Quantitative translation initiation sequencing 339 (QTI-seq), a modified form of ribosome profiling which determines the location and approximate usage efficiency of initiation codons, could be used to assess whether initiation occurs in frame with ORF1b after prominent ORF1b sgRNA TRS bodies, and quantify any contribution this makes to overall ORF1b translation. Similarly to RNASeq, several proteomics datasets of PRRSV-infected cells are publicly available from studies which quantify changes in the host proteome in response to infection [719][720][721][722][723][724][725] . The viral proteome could be analysed in these datasets to confirm expression of ORF1b-overlapping ORFs and to determine whether the abundance of (ORF1b-frame) peptides from the 3′-proximal regions of ORF1b is greater than those at the 5′ end. ...
Porcine reproductive and respiratory syndrome virus (PRRSV) is an arterivirus which causes significant economic losses to the swine industry worldwide. Here, the viral translatome was characterised using ribosome profiling (RiboSeq), a high-throughput sequencing-based technique that allows the positions of translating ribosomes to be mapped to a genome with sub-codon precision. This was used in parallel with RNA sequencing (RNASeq) to analyse changes in viral gene expression over a timecourse of infection in MARC-145 cells. The PRRSV genome contains two programmed ribosomal frameshift (PRF) signals, at which conserved elements promote the backwards slippage of the ribosome by one or two nucleotides. PRRSV encodes a canonical −1 PRF site, at which frameshifting is directed by a downstream RNA pseudoknot structure, to facilitate expression of the ORF1b-encoded viral replicase. At a second PRF site, both −1 and −2 PRF are stimulated by a trans-acting complex of a viral (nsp1β) and cellular (PCBP) protein in a unique, non-canonical mechanism, generating alternative forms (nsp2N and nsp2TF) of a viral non-structural protein, nsp2. Frameshift efficiency at both sites was quantified and, at the non-canonical site, was found to increase with time, rendering this the second known example of temporally regulated PRF during infection. This novel aspect of viral gene expression regulation is likely facilitated by accumulation of the PRF-stimulatory viral protein, nsp1β, during the virus life cycle. Surprisingly, frameshift efficiency at the canonical ORF1ab PRF site was also found to increase over time, overturning the common assumption that RNA structure-directed sites operate at a fixed efficiency, with potential implications for the numerous other viruses which encode canonical PRF sites. Several highly translated additional ORFs were discovered in the PRRSV genome, the translation of which is potentially facilitated by multiple novel viral transcripts. For example, a 125-codon ORF overlapping nsp12 was discovered, which is expressed as highly as nsp12 itself in the late stages of replication and likely translated from novel subgenomic (sg) RNA transcripts overlapping the 3′ end of ORF1b. Similar transcripts were discovered for both PRRSV-1 and PRRSV-2, suggesting a potential conserved mechanism for temporal regulation of expression of the 3′-proximal region of ORF1b. In addition, a highly translated, short upstream ORF (uORF) was identified in the 5′ UTR, the presence of which is highly conserved amongst PRRSV-2 isolates. This work is the first application of RiboSeq to arterivirus-infected cells and reveals new features which add to our understanding of the complexity of gene expression programmes in this important family of nidoviruses.
... Prdx-2 plays a role in different metabolic pathways, including mediating insulin sensitivity (Fazakerley et al., 2018;Kim et al., 2018). Several oxidative stress challenges in livestock-including heat stress (Cruzen et al., 2015), LPS immune challenge (Outhouse et al., 2015), physiological stress (Marco-Ramell et al., 2016), and health challenge (Genini et al., 2012)-affect abundance and profile of Prdx-2. ...
... The Prdx-2 profile differs in response to a variety of oxidative stressors in pigs, including environmental heat stress (Cruzen et al., 2015), LPS-induced immune challenge (Outhouse et al., 2015), physiological stress (Marco-Ramell et al., 2016), and health challenge (Genini et al., 2012). Responses to these stresses include an increase in total Prdx-2, hyperoxidized Prdx-2, and potentially an increase in larger stacked decamers of hyperoxidized Prdx-2. ...
This study’s objective was to determine the impact of a dual respiratory and enteric bacterial health challenge on the antioxidant protein peroxiredoxin-2 (Prdx-2) profile and protein oxidation in the skeletal muscle of pigs from 2 lines that were divergently selected for residual feed intake (RFI). The hypotheses were that (1) differences exist in the Prdx-2 profile between 2 RFI lines and infection status and (2) muscle from less efficient high-RFI and health-challenged pigs have greater cellular protein oxidation. Barrows (50 ± 7 kg, N = 24) from the 11th generation of the high-RFI (n = 12) and low-RFI (n = 12) Iowa State University lines were used. Pigs (n = 6 per line) were inoculated with Mycoplasma hyopneumoniae and Lawsonia intracellularis (MhLI) on day 0 post infection to induce a respiratory and enteric health challenge. Uninoculated pigs served as controls (n = 6 per line). Necropsy was at 21 d post infection. Sarcoplasmic protein oxidation, various forms of Prdx-2, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) content were determined. Neither RFI line nor infection status significantly affected protein carbonylation. Under nonreducing conditions, MhLI pigs had a greater amount of a slower-migrating GAPDH band (P = 0.017), indicating oxidative modification. Regardless of health status, the low-RFI pigs had less total Prdx-2 (P = 0.035), Prdx-2 decamer (P = 0.0007), and a higher ratio of hyperoxidized peroxiredoxin relative to Prdx-2 (P = 0.028) than the high-RFI pigs. The increased pool of active Prdx-2 in high-RFI pigs suggests greater oxidative stress in muscle in high- versus low-RFI pigs. The increase in oxidized GAPDH seen in muscle from MhLI pigs—particularly the high-RFI MhLI pigs—may be a response to the greater oxidative stress in the high-RFI MhLI. This work suggests that antioxidant proteins are important in growth and health-challenge situations.
... Two-dimensional gel electrophoresis (2-DE) has been used to create reference serum proteomic maps for both healthy adult pigs [17] and growing piglets [18,19]. Furthermore, various proteomic tools such as surface-enhanced laser desorption/ionization time-offlight mass spectrometry (SELDI TOF-MS); two-dimensional and two-dimensional differential gel electrophoresis (2-DE/2D-DIGE) followed by matrix-assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS); or liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) have also been applied to evaluate serum proteomic profiles of (1) pigs with acute liver failure [20]; (2) pigs exposed to stress factors, such as insufficient space [21]; (3) pigs suffering from low-dose LPS-induced inflammation [22]; and (4) pigs experimentally or naturally infected with classical swine fever virus [23,24], food-and-mouth disease virus [25] or porcine reproductive and respiratory syndrome virus [26]. However, the vast majority of Trichinella and trichinellosis proteomic studies focus on the immunoproteomic approach, in which immunoreactive proteins from various life stages and different parts or organs of the parasite are subjected to in-depth proteomic analysis [27][28][29][30][31][32][33][34]. ...
... Serum contains thousands of proteins produced and released by cells and tissues, and, as a complex biological matrix, it reflects the general health status of the individual. Changes in the serum proteomic pattern were frequently observed during various pathophysiological abnormalities, such as viral, bacterial, or parasitic diseases, in certain food animal species, including pigs [20][21][22][23][24][25][26]. Therefore, farm animal serum proteomics has been developed in order to gain a deeper and more complete insight into the pathomechanism of these health disorders and to find potential biomarkers characteristic of their particular stages. ...
Although the available proteomic studies have made it possible to identify and characterize Trichinella stage-specific proteins reacting with infected host-specific antibodies, the vast majority of these studies do not provide any information about changes in the global proteomic serum profile of Trichinella-infested individuals. In view of the above, the present study aimed to examine the protein expression profile of serum obtained at 13 and 60 days postinfection (d.p.i.) from three groups of pigs experimentally infected with Trichinella spiralis, Trichinella britovi, and Trichinella pseudospiralis and from uninfected, control pigs by two-dimensional gel electrophoresis (2-DE) followed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. The comparative proteomic analysis of the T. spiralis group vs the control group revealed 5 differently expressed spots at both 13 and 60 d.p.i. Experimental infection with T. britovi induced significant expression changes in 3 protein spots at 13 d.p.i. and in 6 protein spots at 60 d.p.i. in comparison with the control group. Paired analyses between the group infected with T. pseudospiralis and the uninfected control group revealed 6 differently changed spots at 13 d.p.i. and 2 differently changed spots at 60 d.p.i. Among these 27 spots, 15 were successfully identified. Depending on the Trichinella species triggering the infection and the time point of serum collection, they were IgM heavy-chain constant region, antithrombin III-precursor, immunoglobulin gamma-chain, clusterin, homeobox protein Mohawk, apolipoprotein E precursor, serum amyloid P-component precursor, Ig lambda chains, complement C3 isoform X1, and apolipoprotein A-I. Our results demonstrate that various Trichinella species and different phases of the invasion produce a distinct, characteristic proteomic pattern in the serum of experimentally infected pigs.
... Proteomics analyses in swine have been used for potential clinical applications. These reports sought to identify novel biomarkers associated with swine viral diseases, 4-6 bacterial infection, [7][8][9] obesity/metabolic syndrome, 10,11 and stress. 12 The potential role of proteomics in swine has been addressed in several review articles. ...
Rationale:
There are no approved animal drugs for management of inflammation in swine due to lack of validated animal models. To assess efficacy, it was essential to develop proteomics approaches to identify suitable biomarkers of inflammation as presented in this study.
Methods:
Serum samples were collected from a group of 4 pigs prior to (baseline), and 24 and 48 h following lipopolysaccharide (LPS) stimulation to reveal proteomic changes during inflammation. Two other pigs served as untreated controls. Proteins were separated by either 1D SDS-PAGE or 2D gel electrophoresis (2DE) prior to analysis by nano flow liquid chromatography (nLC) coupled to tandem mass spectrometry.
Results:
We identified 165 proteins using SDS-PAGE, of which 47 proteins were also detected by 2DE prior to nLC-MS/MS. More than half (72%) of all characterized proteins were modulated as a result of LPS stimulation, many of which are known to be involved with innate and adaptive immunity. Pig serum samples obtained 24 h after LPS initiation of inflammation showed protein modulations of serum albumin, serotransferrin, light and heavy immunoglobulin chains (IGs), and major acute phase proteins including haptoglobin (HPT), serum amyloid A2 (SAA2), C-reactive protein (CRP), β-2-glycoprotein 1 (B-2GP1), alpha-2-HS-glycoprotein (A2HS), α-1-antitrypsin (A1AT), and α-1-acid glycoprotein (A1AG). SAA2 was distinguished from the other SAA isoforms by the unique peptide sequence of SAA2.
Conclusions:
The results provided proteomic analysis of swine serum due to LPS stimulation and indicated the importance of SAA2, which appears to be unique and may be regarded as a potential clinical diagnostic biomarker of inflammation.
... For example, Van Altena et al. has recently shown by proteomic profiling that 13 proteins exhibited enhanced expression in the milk of dairy cows with increased susceptibiltiy to bovine diseases [9]. In another study, Genini and coworkers described a panel of 14 differentially expressed proteins that could be used to distinguish piglets with porcine reproductive and respiratory syndrome from healthy controls with a sensitivity and specificity of 77% and 73%, respectively [10]. These studies highlighted the utility of proteomics in helping researchers interpret various complex pathophysiological processes on a molecular level. ...
The natural polysaccharides extracted from the pollen of Pinus massoniana (TPPPS) have been shown to be a promising immune adjuvant against several viral chicken diseases. However, the exact mechanism through which TPPPS enhances the host immune response in chicken remains poorly understood. In the current study, chicken peripheral blood lymphocytes were treated with varying concentrations of TPPPS and pro-inflammatory cytokines such as IFN-γ, iIL-2 and IL-6 were measured to determine the optimal dose of the polysaccharide. A comparative analysis was subsequently performed between the proteome of lymphocytes subjected to the best treatment conditions and that of untreated cells. Protein identification and quantitation revealed a panel of three up-regulated and seven down-regulated candidates in TPPPS-treated chicken peripheral blood lymphocytes. Further annotation and functional analysis suggested that a number of those protein candidates were involved in the regulation of host innate immune response, inflammation and other immune-related pathways. We believe that our results could serve as a stepping stone for further research on the immune-enhancing properties of TPPPS and other polysaccharide-based immune adjuvants.
... However, mRNA abundance is not always consistent with protein expression levels 23 . In recent years, proteomics analysis has been used to identify cellular protein expression profiles related to PRRSV infection [24][25][26] . Most reports of the proteomics analysis of PRRSV have described two-dimensional electrophoresis and mass spectrometry (MS) approaches 24,25,27 . ...
Abstract Porcine reproductive and respiratory syndrome is an infectious disease that causes serious economic losses to the swine industry worldwide. To better understand the pathogenesis of the porcine reproductive and respiratory syndrome virus (PRRSV), three PRRSV strains with different molecular markers and virulence were used to infect MARC-145 cells. A total of 1804 proteins were identified, and 233 altered proteins and 72 signaling pathways involved in the proteomic profiling of virus-infected MARC-145 cells increased with the virulence of the PRRSV strain. The three types of viral strains shared a common pathway—the electron transport reaction in mitochondria—in the infected-MARC-145 cells. Moreover, the antisense pathway was the most variable of all significant signaling pathways for the highly virulent SX-1 strain, indicating that this unique pathway may be connected to the high virulence of the SX-1 strain. Our study is the first attempt to provide a proteome profile of MARC-145 cells infected with PRRSV strains with different virulence, and these findings will facilitate a deep understanding of the interactions between this virus and its host.
... The second in-solution digestion using 20 μg of crude serum from the same sheep yielded 100 protein IDs. This result was considered substantial, as the number of protein IDs was comparable to those of other studies [10,[75][76][77][78][79][80][81]. Unexpectedly however, the third insolution that utilised 100 μg of pooled crude serum from six sheep under the same experimental conditions yielded only 32 IDs. ...
Background:
Unlike humans, there is currently no publicly available reference mass spectrometry-based circulating acellular proteome data for sheep, limiting the analysis and interpretation of a range of physiological changes and disease states. The objective of this study was to develop a robust and comprehensive method to characterise the circulating acellular proteome in ovine serum.
Methods:
Serum samples from healthy sheep were subjected to shotgun proteomic analysis using nano liquid chromatography nano electrospray ionisation tandem mass spectrometry (nanoLC-nanoESI-MS/MS) on a quadrupole time-of-flight instrument (TripleTOF® 5600+, SCIEX). Proteins were identified using ProteinPilot™ (SCIEX) and Mascot (Matrix Science) software based on a minimum of two unmodified highly scoring unique peptides per protein at a false discovery rate (FDR) of 1% software by searching a subset of the Universal Protein Resource Knowledgebase (UniProtKB) database (http://www.uniprot.org). PeptideShaker (CompOmics, VIB-UGent) searches were used to validate protein identifications from ProteinPilot™ and Mascot.
Results:
ProteinPilot™ and Mascot identified 245 and 379 protein groups (IDs), respectively, and PeptideShaker validated 133 protein IDs from the entire dataset. Since Mascot software is considered the industry standard and identified the most proteins, these were analysed using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) classification tool revealing the association of 349 genes with 127 protein pathway hits. These data are available via ProteomeXchange with identifier PXD004989.
Conclusions:
These results demonstrated for the first time the feasibility of characterising the ovine circulating acellular proteome using nanoLC-nanoESI-MS/MS. This peptide spectral data contributes to a protein library that can be used to identify a wide range of proteins in ovine serum.
... Additionally, Genini et al. 118 have used protein chip arrays and SELDI-TOF-MS for analysis of serum samples in large white piglets with and without porcine reproductive and respiratory syndrome (PRRS). They found 200 peaks that were signicantly different and among those, 47 peaks were conrmed during validation. ...
Proteomic methodologies for identification and analysis of biomarkers have gained more attention during recent years and have evolved rapidly. Identification and detection of disease biomarkers are important to foresee outbreaks of certain diseases thereby avoiding surgery and other invasive and expensive medical treatments for patients. Thus, more research into discovering new biomarkers and new methods for faster and more accurate detection is needed. It is often difficult to detect and measure biomarkers because of their low concentrations and the complexity of their respective matrices. Therefore it is hard to find and validate methods for accurate screening methods suitable for clinical use. The most recent developments during the last three years and also some historical considerations of proteomic methodologies for identification and validation of disease biomarkers are presented in this review.
... Porcine reproductive and respiratory syndrome 45 Serum Food and mouth 76 Serum Classical swine fever 104 Serum Stress 79 Serum Peritonitis-induced sepsis 109 Serum Small ruminants Paratubercolosis 120 Serum Horse ...
... Thus the main SPE protein bands in serum of cats with and without lymphoma, have been subjected to proteomic analysis. 45 The study confirmed established understanding of the content of the main bands but also revealed the presence of lower-abundance protein in particular fractions, such as inter-a (globulin) inhibitor 4 in the a 2 band of cats with lymphoma. ...
Advancement in electrophoresis and mass spectrometry techniques along with the recent progresses in genomics, culminating in bovine and pig genome sequencing, widened the potential application of proteomics in the field of veterinary medicine. The aim of the present review is to provide an in-depth perspective about the application of proteomics to animal disease pathogenesis, as well as its utilization in veterinary diagnostics. After an overview on the various proteomic techniques that are currently applied to veterinary sciences, the article focuses on proteomic approaches to animal disease pathogenesis. Included as well are recent achievements in immunoproteomics (ie, the identifications through proteomic techniques of antigen involved in immune response) and histoproteomics (ie, the application of proteomics in tissue processed for immunohistochemistry). Finally, the article focuses on clinical proteomics (ie, the application of proteomics to the identification of new biomarkers of animal diseases).
