Influence of dietary Spirulin inclusion and lysozyme supplementation on the longissimus lumborum muscle proteome of newly weaned piglets

Research Areas: Biochemistry & Molecular BiologyArthrospira platensis (Spirulina) is a microalga with a high content of crude protein. It has a recalcitrant cell wall that limits the accessibility of the animal endogenous enzymes to its intracellular nutrients. Enzymatic supplementation aiming to degrade cell walls could benefit microalgae digestibility. The objective of this study was to evaluate the impact of dietary Spirulina and lysozyme supplementation over the muscle proteome of piglets during the post-weaning stage. Thirty piglets were randomly distributed among three diets: control (no microalga), SP (10% Spirulina) and SP + L (10% Spirulina +0.01% lysozyme). After 4 weeks, they were sacrificed and samples of the longissimus lumborum muscle were taken. The muscle proteome was analysed using a Tandem Mass Tag (TMT)-based quantitative approach. A total of 832 proteins were identified. Three comparisons were computed: SP vs Ctrl, SP + L vs Ctrl and SP + L vs SP. They had ten, four and twelve differentially abundant proteins. Glycogen metabolism and nutrient reserves utilization are increased in the SP piglets. Structural muscle protein synthesis increased, causing higher energy requirements in SP + L piglets. Our results demonstrate the usefulness of proteomics to disclose the effect of dietary microalgae, whilst unveiling putative mechanisms derived from lysozyme supplementation. Data available via ProteomeXchange with identifier PXD024083. Significance: Spirulina, a microalga, is an alternative to conventional crops which could enhance the environmental sustainability of animal production. Due to its recalcitrant cell wall, its use requires additional measures to prevent anti-nutritional effects on the feeding of piglets in the post-weaning period, during which they endure post-weaning stress. One of such measures could be CAZyme supplementation to help degrade the cell wall during digestion. Muscle proteomics provides insightful data on the effect of dietary microalgae and enzyme activity on piglet metabolism.info:eu-repo/semantics/publishedVersio

Biomarker and proteome analysis of milk from dairy cows with clinical mastitis: determining the effect of different bacterial pathogens on the response to infection

Antimicrobial usage (AMU) could be reduced by differentiating the causative bacteria in cases of clinical mastitis (CM) as either Gram-positive or Gram-negative bacteria or identifying whether the case is culture-negative (no growth, NG) mastitis. Immunoassays for biomarker analysis and a Tandem Mass Tag (TMT) proteomic investigation were employed to identify differences between samples of milk from cows with CM caused by different bacteria. A total of 94 milk samples were collected from cows diagnosed with CM across seven farms in Scotland, categorized by severity as mild (score 1), moderate (score 2), or severe (score 3). Bovine haptoglobin (Hp), milk amyloid A (MAA), C-reactive protein (CRP), lactoferrin (LF), α-lactalbumin (LA) and cathelicidin (CATHL) were significantly higher in milk from cows with CM, regardless of culture results, than in milk from healthy cows (all P-values <0.001). Milk cathelicidin (CATHL) was evaluated using a novel ELISA technique that utilises an antibody to a peptide sequence of SSEANLYRLLELD (aa49–61) common to CATHL 1–7 isoforms. A classification tree was fitted on the six biomarkers to predict Gram-positive bacteria within mastitis severity scores 1 or 2, revealing that compared to the rest of the samples, Gram-positive samples were associated with CRP < 9.5 μg/ml and LF ≥ 325 μg/ml and MAA < 16 μg/ml. Sensitivity of the tree model was 64%, the specificity was 91%, and the overall misclassification rate was 18%. The area under the ROC curve for this tree model was 0.836 (95% bootstrap confidence interval: 0.742; 0.917). TMT proteomic analysis revealed little difference between the groups in protein abundance when the three groups (Gram-positive, Gram-negative and no growth) were compared, however when each group was compared against the entirety of the remaining samples, 28 differentially abundant protein were identified including β-lactoglobulin and ribonuclease. Whilst further research is required to draw together and refine a suitable biomarker panel and diagnostic algorithm for differentiating Gram- positive/negative and NG CM, these results have highlighted a potential panel and diagnostic decision tree. Host-derived milk biomarkers offer significant potential to refine and reduce AMU and circumvent the many challenges associated with microbiological culture, both within the lab and on the farm, while providing the added benefit of reducing turnaround time from 14 to 16 h of microbiological culture to just 15 min with a lateral flow device (LFD)

Identification of novel biomarkers for treatment monitoring in canine leishmaniosis by high-resolution quantitative proteomic analysis

The objective of this study was to use the Tandem Mass Tag (TMT) isobaric label-based proteomic approach, in order to identify new potential biomarkers for the treatment monitoring of canine leishmaniosis that could not be identified by the use of gel-based techniques. For this purpose serum samples were obtained from 5 clinically diseased dogs before and one month after the treatment of canine leishmaniosis. The non-depleted serum samples were subjected to reduction, alkylation and trypsin digestion, and the resulting peptides were labeled using 6-plex TMT reagents. To obtain information about protein identities and relative quantification, liquid chromatography–MS analysis of multiplexed TMT-labeled peptides was employed. This gel-free, label-based quantitative proteomic approach enabled identification of 117 canine proteins. Among these, 23 showed significant difference (p < 0.05) in expression (two downregulated and 21 upregulated ranging from 1.25 to 2.5 fold change). Comparison of gel-free TMT-based quantification and a gel-based approach previously applied to the same samples resulted in the identification of some common markers (Apo-A1, vitamin D binding protein and RBP4). However, 20 additional differentially represented proteins were highlighted by the gel-free approach, 13 of which have not been previously reported in canine leishmaniosis. In conclusion, the TMT-based proteomic approach allowed identification of new serum proteins that significantly change in concentration after canine leishmaniosis treatment. These proteins are involved in various physiopathological processes such as inflammatory, coagulation or defense mechanisms, and could potentially be suitable biomarkers for treatment monitoring of this parasitic disease

Milk and serum proteomes in subclinical and clinical mastitis in Simmental cows

Bovine mastitis causes changes in the milk and serum proteomes. Here changes in both proteomes caused by naturally occurring subclinical and clinical mastitis have been characterised and quantified. Milk and serum samples from healthy dairy cows (n = 10) were compared to those of cows with subclinical (n = 12) and clinical mastitis (n = 10) using tandem mass tag (TMT) proteomics. Proteins that significantly increased or decreased in milk (n = 237) or serum (n = 117) were quantified and classified by the type of change in subclinical and clinical mastitis. A group of the proteins (n = 38) showed changes in both milk and serum a number of which decreased in the serum but increased in milk, suggesting a particular role in host defence for maintaining and restoring homeostasis during the disease. Proteins affected by bovine mastitis included proteins in host defence and coagulation pathways. Investigation of the modified proteomes in milk and serum was assessed by assays for haptoglobin, serum amyloid A and α acid glycoprotein validating the results obtained by quantitative proteomics. Alteration of abundance patterns of milk and serum proteins, together with pathway analysis reveal multiple interactions related to proteins affected by mastitis. Data are available via ProteomeXchange with identifier PXD022595. SIGNIFICANCE: Mastitis is the most serious condition to affect dairy cows and leads to reduced animal welfare as well as having a negative economic effect for the dairy industry. Proteomics has previously identified changes in abundance of milk proteins during mastitis, but there have been few investigations addressing changes that may affect proteins in the blood during the infection. In this study, changes in the abundance of proteins of milk and serum, caused by naturally occurring mastitis have been characterised by proteomics using a quantitative approach and both subclinical and clinical cases of mastitis have been investigated. In both milk and serum, change in individual proteins was determined and classified into varying types of altering abundance, such as increasing in subclinical mastitis, but showing no further increase in clinical mastitis. Of special interest were the proteins that altered in abundance in both milk and serum which either showed similar trends - increasing or decreasing in both biological fluids or showed reciprocal change decreasing in serum but increasing in milk. As well as characterising proteins as potential markers of mastitis and the severity of the disease, these results provide insight into the pathophysiology of the host response to bovine mastitis

Quantitative proteomics of cerebrospinal fluid using tandem mass tags in dogs with recurrent epileptic seizures

This prospective study included four dog groups (group A: healthy dogs, groups B: dogs with idiopathic epilepsy under antiepileptic medication (AEM), C: idiopathic epilepsy dogs without AEM administration, D: dogs with structural epilepsy). The purpose of the study was to compare the proteomic profile among the four groups. Samples were analyzed by a quantitative Tandem Mass Tags approach using a Q-Exactive-Plus mass-spectrometer. Identification and relative quantification were performed using Proteome Discoverer, and data were analyzed using R. Gene ontology terms were analyzed based on Canis lupus familiaris database. Data are available via ProteomeXchange with identifier PXD018893. Eighteen proteins were statistically significant among the four groups (P < 0.05). MMP2 and EFEMP2 appeared down-regulated whereas HP and APO-A1 were up-regulated (groups B, D). CLEC3B and PEBP4 were up-regulated whereas APO-A1 was down-regulated (group C). IGLL1 was down-regulated (groups B, C) and up-regulated (group D). EFEMP2 was the only protein detected among the four groups and PEBP4 was significantly different among the epileptic dogs. Western blot and SPARCL immunoassay were used to quantify HP abundance change, validating proteomic analysis. Both, showed good correlation with HP levels identified through proteomic analysis (r = 0.712 and r = 0.703, respectively)

Changes in saliva of dogs with canine leishmaniosis: A proteomic approach

In the present study, a quantitative proteomic approach to study changes in saliva proteins associated with canine leishmaniosis (CanL) was performed. For this, canine salivary proteins were analysed and compared between dogs before (TO) and after (T1) experimental infection with Leishmania infantum by high-throughput label-based quantitative LC-MS/MS proteomic approach and bioinformatic analysis of the in silico inferred interactome protein network was created from the initial list of differential proteins. More than 2000 proteins were identified, and of the 90 differentially expressed proteins between TO and T1, 12 were down-regulated with log2 fold change lower than -0.5849, and 19 were up-regulated with log2 fold change greater than 0.5849. This study provides evidence of changes in salivary proteome that can occur in canine leishmaniosis and revealed biological pathways in saliva modulated in canine leishmaniosis with potential for further targeted research.LFM was granted with predoctoral contract 'FPU' of University of Murcia, Spain. AT has a post-doctoral fellowship "Juan de la Cierva Incorporacion" supported by the "Ministerio de Economia y Competitividad", Spain. MCT and MCL were supported by grants RTC 2016 -50005-1 from the "Programa Estatal I+D+I" (MINECO). This work was supported by a grant from the Program for Research Groups of Excellence of the Seneca Foundation, Murcia, Spain (grant 19894/GERM/15) and European Commission ERA chair FP7 grant (VetMedZg #621394).Peer reviewe

Use of the WHO Access, Watch, and Reserve classification to define patterns of hospital antibiotic use (AWaRe) : an analysis of paediatric survey data from 56 countries

Background Improving the quality of hospital antibiotic use is a major goal of WHO's global action plan to combat antimicrobial resistance. The WHO Essential Medicines List Access, Watch, and Reserve (AWaRe) classification could facilitate simple stewardship interventions that are widely applicable globally. We aimed to present data on patterns of paediatric AWaRe antibiotic use that could be used for local and national stewardship interventions. Methods 1-day point prevalence survey antibiotic prescription data were combined from two independent global networks: the Global Antimicrobial Resistance, Prescribing, and Efficacy in Neonates and Children and the Global Point Prevalence Survey on Antimicrobial Consumption and Resistance networks. We included hospital inpatients aged younger than 19 years receiving at least one antibiotic on the day of the survey. The WHO AWaRe classification was used to describe overall antibiotic use as assessed by the variation between use of Access, Watch, and Reserve antibiotics, for neonates and children and for the commonest clinical indications. Findings Of the 23 572 patients included from 56 countries, 18305 were children (77.7%) and 5267 were neonates (22.3%). Access antibiotic use in children ranged from 7.8% (China) to 61.2% (Slovenia) of all antibiotic prescriptions. The use of Watch antibiotics in children was highest in Iran (77.3%) and lowest in Finland (23.0%). In neonates, Access antibiotic use was highest in Singapore (100.0%) and lowest in China (24.2%). Reserve antibiotic use was low in all countries. Major differences in clinical syndrome-specific patterns of AWaRe antibiotic use in lower respiratory tract infection and neonatal sepsis were observed between WHO regions and countries. Interpretation There is substantial global variation in the proportion of AWaRe antibiotics used in hospitalised neonates and children. The AWaRe classification could potentially be used as a simple traffic light metric of appropriate antibiotic use. Future efforts should focus on developing and evaluating paediatric antibiotic stewardship programmes on the basis of the AWaRe index. Copyright (C) 2019 The Author(s). Published by Elsevier Ltd