Proteomic analysis of granulomas from cattle and pigs naturally infected with Mycobacterium tuberculosis complex by MALDI imaging.

Matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI) has recently gained prominence for its ability to provide molecular and spatial information in tissue sections. This technology has the potential to uncover novel insights into proteins and other molecules in biological and immunological pathways activated along diseases with a complex host-pathogen interaction, such as animal tuberculosis. Thus, the present study conducted a data analysis of protein signature in granulomas of cattle and pigs naturally infected with the Mycobacterium tuberculosis complex (MTC), identifying biological and immunological signaling pathways activated throughout the disease. Lymph nodes from four pigs and four cattle, positive for the MTC by bacteriological culture and/or real-time PCR, were processed for histopathological examination and MALDI-MSI. Protein identities were assigned using the MaTisse database, and protein-protein interaction networks were visualized using the STRING database. Gene Ontology (GO) analysis was carried out to determine biological and immunological signaling pathways in which these proteins could participate together with Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. Distinct proteomic profiles between cattle and pig granulomas were displayed. Noteworthy, the GO analysis revealed also common pathways among both species, such as "Complement activation, alternative pathway" and "Tricarboxylic acid cycle", which highlight pathways that are conserved among different species infected by the MTC. In addition, species-specific terms were identified in the current study, such as "Natural killer cell degranulation" in cattle or those related to platelet and neutrophil recruitment and activation in pigs. Overall, this study provides insights into the immunopathogenesis of tuberculosis in cattle and pigs, opening new areas of research and highlighting the importance, among others, of the complement activation pathway and the regulation of natural killer cell- and neutrophil-mediated immunity in this disease.

The scene of lung pathology during PRRSV-1 infection.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most economically important infectious diseases for the pig industry worldwide. The disease was firstly reported in 1987 and became endemic in many countries. Since then, outbreaks caused by strains of high virulence have been reported several times in Asia, America and Europe. Interstitial pneumonia, microscopically characterised by thickened alveolar septa, is the hallmark lesion of PRRS. However, suppurative bronchopneumonia and proliferative and necrotising pneumonia are also observed, particularly when a virulent strain is involved. This raises the question of whether the infection by certain strains results in an overstimulation of the proinflammatory response and whether there is some degree of correlation between the strain involved and a particular pattern of lung injury. Thus, it is of interest to know how the inflammatory response is modulated in these cases due to the interplay between virus and host factors. This review provides an overview of the macroscopic, microscopic, and molecular pathology of PRRSV-1 strains in the lung, emphasising the differences between strains of different virulence.

Effect of Feeding Saccharomyces cerevisiae boulardii CNCM I-1079 to Sows and Piglets on Piglets' Immune Response after Vaccination against Actinobacillus pleuropneumoniae.

The aim of this study was to assess the effect that feeding Saccharomyces cerevisiae boulardii CNCM I-1079 (LSB) to lactating sows and their progeny has on inflammatory response and mucosal immunity after vaccination against Actinobacillus pleuropneumoniae. Sixty-seven Danbred sows were allotted into two treatments when they entered the farrowing room seven days before the expected farrowing date: control (CON: lactation diet) and LSB (CON + 12 × 109 colony forming units (CFU)/d until weaning). At weaning, piglets were equally allotted into two experimental diets according to sow diet: control (CON: 2-phase post-weaning diets) and LSB (CON + 2 × 109 CFU/kg and 1 × 109 CFU/kg in phases 1 and 2, respectively). The piglets were vaccinated at days 26 and 49 post-weaning. Growth performance and number of IgA producing cells and cytokine's gene expression in lung, lymph node, and intestine samples at day 70 post-weaning were assessed and analyzed in SPSS Statistics 26: performance with a general linear model with sex, room, sow diet, piglet diet, and their interactions as main effects, and immunity with a Kruskal−Wallis test for k unrelated samples. Piglets from LSB-fed sows displayed a higher average daily gain (ADG; p < 0.01) and a heavier body weight (BW; p < 0.05) during lactation, tended (p < 0.1) to be heavier at day 49, and to have a higher ADG between days 26 and 49; had fewer number of IgA producing cells in the lymph node (p < 0.05); and all the cytokines studied were significantly under-regulated (p < 0.05) in the lung. It is concluded that feeding Saccharomyces cerevisiae boulardii CNCM I-1079 to sows improved piglet performance during lactation and showed a clear reduction in the inflammatory status of the lungs after vaccination against A. pleuropneumoniae, suggesting that there was a maternal imprinting effect on mucosal protection and a cross-talk between the gut microbiota and the lung.

PRRSV-1 induced lung lesion is associated with an imbalance between costimulatory and coinhibitory immune checkpoints.

Porcine reproductive and respiratory syndrome virus (PRRSV) induces a dysregulation on the innate and adaptive immune responses. T-cell activation requires a proper interaction and precise balance between costimulatory and coinhibitory molecules, commonly known as immune checkpoints. This study aims to evaluate the expression of immune checkpoints in lung and tracheobronchial lymph node from piglets infected with two PRRSV-1 strains of different virulence during the early stage of infection. Seventy 4-week-old piglets were grouped into three experimental groups: (i) control, (ii) 3249-infected group (low virulent strain), and (iii) Lena-infected group (virulent strain) and were euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi). Lung and tracheobronchial lymph node were collected to evaluate histopathological findings, PRRSV viral load and mRNA expression of costimulatory (CD28, CD226, TNFRSF9, SELL, ICOS, and CD40) and coinhibitory (CTLA4, TIGIT, PD1/PDL1, TIM3, LAG3, and IDO1) molecules through RT-qPCR. Our findings highlight a mild increase of costimulatory molecules together with an earlier and stronger up-regulation of coinhibitory molecules in both organs from PRRSV-1-infected animals, especially in the lung from virulent Lena-infected animals. The simultaneous expression of coinhibitory immune checkpoints could work in synergy to control and limit the inflammation-induced tissue damage. Further studies should be addressed to determine the role of these molecules in later stages of PRRSV infection.

Porcine reproductive and respiratory syndrome virus impacts on gut microbiome in a strain virulence-dependent fashion.

Porcine reproductive and respiratory syndrome (PRRS) is a viral disease defined by reproductive problems, respiratory distress and a negative impact on growth rate and general condition. Virulent PRRS virus (PRRSV) strains have emerged in the last years with evident knowledge gaps in their impact on the host immune response. Thus, the present study examines the impact of acute PRRS virus (PRRSV) infection, with two strains of different virulence, on selected immune parameters and on the gut microbiota composition of infected pigs using 16S rRNA compositional sequencing. Pigs were infected with a low virulent (PRRS_3249) or a virulent (Lena) PRRSV-1 strain and euthanized at 1, 3, 6, 8 or 13 days post-inoculation (dpi). Faeces were collected from each animal at the necropsy time-point. Alpha and beta diversity analyses demonstrated that infection, particularly with the Lena strain, impacted the microbiome composition from 6 dpi onwards. Taxonomic differences revealed that infected pigs had higher abundance of Treponema and Methanobrevibacter (FDR < 0.05). Differences were more considerable for Lena- than for PRRS_3249-infected pigs, showing the impact of strain virulence in the intestinal changes. Lena-infected pigs had reduced abundancies of anaerobic commensals such as Roseburia, Anaerostipes, Butyricicoccus and Prevotella (P < 0.05). The depletion of these desirable commensals was significantly correlated to infection severity measured by viraemia, clinical signs, lung lesions and immune parameters (IL-6, IFN-γ and Hp serum levels). Altogether, the results from this study demonstrate the indirect impact of PRRSV infection on gut microbiome composition in a strain virulence-dependent fashion and its association with selected immune markers.

Activation of T-bet, FOXP3, and EOMES in Target Organs From Piglets Infected With the Virulent PRRSV-1 Lena Strain.

Transcription factors (TFs) modulate genes involved in cell-type-specific proliferative and migratory properties, metabolic features, and effector functions. Porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most important pathogen agents in the porcine industry; however, TFs have been poorly studied during the course of this disease. Therefore, we aimed to evaluate the expressions of the TFs T-bet, GATA3, FOXP3, and Eomesodermin (EOMES) in target organs (the lung, tracheobronchial lymph node, and thymus) and those of different effector cytokines (IFNG, TNFA, and IL10) and the Fas ligand (FASL) during the early phase of infection with PRRSV-1 strains of different virulence. Target organs from mock-, virulent Lena-, and low virulent 3249-infected animals humanely euthanized at 1, 3, 6, 8, and 13 days post-infection (dpi) were collected to analyze the PRRSV viral load, histopathological lesions, and relative quantification through reverse transcription quantitative PCR (RT-qPCR) of the TFs and cytokines. Animals belonging to both infected groups, but mainly those infected with the virulent Lena strain, showed upregulation of the TFs T-bet, EOMES, and FOXP3, together with an increase of the cytokine IFN-γ in target organs at the end of the study (approximately 2 weeks post-infection). These results are suggestive of a stronger polarization to Th1 cells and regulatory T cells (Tregs), but also CD4+ cytotoxic T lymphocytes (CTLs), effector CD8+ T cells, and γδT cells in virulent PRRSV-1-infected animals; however, their biological functionality should be the object of further studies.

Effect of Feed Supplementation with Clostridium butyricum, Alone or in Combination with Carob Meal or Citrus Pulp, on Digestive and Metabolic Status of Piglets.

This work studied the effects of the inclusion of Clostridium butyricum on feed, alone or with carob meal or citrus pulp, on the digestive and metabolic status of weaned piglets. A total of 30 male piglets (weaned at 21 days) is used. There are five dietary treatments: negative without ZnO at high doses (C-), a positive control supplemented with ZnO at 2500 ppm of Zn (C+), supplemented with Clostridium butyricum as a probiotic (PRO), and supplemented with probiotic and 5% carob meal (PROC) or 5% citrus pulp (PROP). During the experiment (27 days), the piglets were periodically weighed and sampled for a serum biochemical, fecal microbiological, intestine histological, and digestive status analysis. The body weight, apparent ileal digestibility of dry matter (DM), and fecal microbiology were not affected by the treatments (p ≥ 0.05). However, the apparent fecal digestibility of DM was lower for the C- treatment than for C+ (p < 0.05), and the total concentration of volatile fatty acids (VFAs) in feces with C+ was lower than that for the PROC treatment (p < 0.05). The treatments with the probiotic had a higher molar proportion of butyric acid in feces than C+, and it was found that C- reached an intermediate value (p < 0.01). No general effects of diet were found on the histological measures performed on the jejunum and ileum, and in the serum biochemical analysis (p ≥ 0.05), only the concentration of interleukin-8 was lower for the PROC treatment compared to the C-, C+, and PRO treatments (p < 0.05). In conclusion, the intestinal wellness of piglets could be improved with the supplementation of Clostridium butyricum by increasing butyric acid, and this effect was not altered with the inclusion of carob meal or citrus pulp. More studies under commercial conditions are needed, as the effects might be different in more challenging environmental circumstances.

Activation of regulated cell death in the lung of piglets infected with virulent PRRSV-1 Lena strain occurs earlier and mediated by cleaved Caspase-8.

PRRSV-1 virulent strains cause high fever, marked respiratory disease and severe lesions in lung and lymphoid organs. Regulated cell death (RCD), such as apoptosis, necroptosis and pyroptosis, is triggered by the host to interrupt viral replication eliminating infected cells, however, although it seems to play a central role in the immunopathogenesis of PRRSV, there are significant gaps regarding their sequence and activation upon PRRSV-infection. The present study evaluated RCD events by means of caspases expression in the lung of PRRSV-1-infected pigs and their impact on pulmonary macrophage subpopulations and lung lesion. Conventional piglets were intranasally inoculated with the virulent subtype 3 Lena strain or the low virulent subtype 1 3249 strain and euthanised at 1, 3, 6, 8 and 13 dpi. Lena-infected piglets showed severe and early lung damage with a high frequency of PRRSV-N-protein+ cells, depletion of CD163+ cells and high viral load in the lung. The number of TUNEL+ cells was significantly higher than cCasp3+ cells in Lena-infected piglets during the first week post-infection. cCasp8 and to a lesser extent cCasp9 were activated by both PRRSV-1 strains after one week post-infection together with a replenishment of both CD163+ and Arg-1+ pulmonary macrophages. These results highlight the induction of other forms of RCD beyond apoptosis, such as, necroptosis and pyroptosis during the first week post-infection followed by the activation of, mainly, extrinsic apoptosis during the second week post-infection. The recovery of CD163+ macrophages at the end of the study represents an attempt to restore pulmonary macrophage subpopulations lost during the early stages of the infection but also a macrophage polarisation into M2 macrophages.

Proteome changes induced by a short, non-cytotoxic exposure to the mycoestrogen zearalenone in the pig intestine.

Intestinal epithelial homeostasis is regulated by a complex network of signaling pathways. Among them is estrogen signaling, important for the proliferation and differentiation of epithelial cells, immune signaling and metabolism. The mycotoxin zearalenone (ZEN) is an estrogen disruptor naturally found in food and feed. The exposure of the intestine to ZEN has toxic effects including alteration of the immune status and is possibly implicated in carcinogenesis, but the molecular mechanisms linked with these effects are not clear. Our objective was to explore the proteome changes induced by a short, non-cytotoxic exposure to ZEN in the intestine using pig jejunal explants. Our results indicated that ZEN promotes little proteome changes, but significantly related with an induction of ERα signaling and a consequent disruption of highly interrelated signaling cascades, such as NF-κB, ERK1/2, CDX2 and HIF1α. The toxicity of ZEN leads also to an altered immune status characterized by the activation of the chemokine CXCR4/SDF-1 axis and an accumulation of MHC-I proteins. Our results connect the estrogen disrupting activity of ZEN with its intestinal toxic effect, associating the exposure to ZEN with cell-signaling disorders similar to those involved in the onset and progression of diseases such as cancer and chronic inflammatory disorders. SIGNIFICANCE: The proteomics results presented in our study indicate that the endocrine disruptor activity of ZEN is able to regulate a cascade of highly inter-connected signaling events essential for the small intestinal crypt-villus cycle and immune status. These molecular mechanisms are also implicated in the onset and progress of intestinal immune disorders and cancer indicating that exposure to ZEN could play an important role in intestinal pathogenesis.

Activation of the extrinsic apoptotic pathway in the thymus of piglets infected with PRRSV-1 strains of different virulence.

In the last decade, the outbreaks caused by virulent porcine reproductive and respiratory syndrome virus (PRRSV) strains from both PRRSV-1 and PRRSV-2 have considerably increased. PRRSV is able to modulate the host's immune response through the induction of apoptosis of cells in lymphoid organs like thymus, increasing the susceptibility to secondary infectious agents. The present study aimed to compare the impact of two PRRSV-1 strains, a field low virulent strain (3249 strain) and a virulent strain (Lena strain), in the thymus of infected pigs, focusing on clinical signs, histological analysis, viraemia, thymus viral load and the study of the different routes of apoptosis phenomena by immunohistochemistry. Sera and thymus samples were collected from infected animals with 3249 strain, Lena strain and mock-infected animals at 1, 3, 6, 8 and 13 days post-infection (dpi). Lena-infected animals showed severe clinical disease, high sera and thymus viral loads with evident thymic atrophy since 6 dpi, matching with PRRSV-N protein, TUNEL and cCasp3 expression in the thymic cortex. In both infected groups, there was an increase in the number of cells expressing molecules related to the extrinsic pathway of apoptosis (cCasp8 and Fas) in cortex and medulla, showing an important role in the apoptosis induction produced in thymus of PRRSV-infected piglets. The extensive apoptosis in the thymus through this pathway would lead to a decrease in the number of mature T lymphocytes and the sustained release of viral particles, which may explain the greater severity of the clinical signs observed in Lena-infected pigs.

Ameliorative Effect of Spinach on Non-Alcoholic Fatty Liver Disease Induced in Rats by a High-Fat Diet.

The purpose of this work was to evaluate the effect of dietary carotenoids from spinach on the inflammation and oxidative stress biomarkers, liver lipid profile, and liver transcriptomic and metabolomics profiles in Sprague-Dawley rats with steatosis induced by a high-fat diet. Two concentrations of spinach powder (2.5 and 5%) were used in two types of diet: high-fat (H) and standard (N). Although rats fed diet H showed an accumulation of fat in hepatocytes, they did not show differences in the values of adiponectin, tumor necrosis factor alpha (TNF-α), and oxygen radical absorption (ORAC) in plasma or of isoprostanes in urine compared with animals fed diet N. The consumption of spinach and the accumulation of α and ß carotenes and lutein in the liver was inversely correlated with serum total cholesterol and glucose and the content of hepatic cholesterol, increasing monounsaturated fatty acids (MUFA), polyunsaturated fatty acids (PUFA) and reducing cholesterol in the livers of rats fed diet H and spinach. In addition, changes in the expression of genes related to the fatty liver condition occurred, and the expression of genes involved in the metabolism of fatty acids and cholesterol increased, mainly through the overexpression of peroxisome proliferator activated receptors (PPARs). Related to liver metabolites, animals fed with diet H showed hypoaminoacidemia, mainly for the glucogenic aminoacids. Although no changes were observed in inflammation and oxidative stress biomarkers, the consumption of spinach modulated the lipid metabolism in liver, which must be taken into consideration during the dietary treatment of steatosis.

Cytokines transcript levels in lung and lymphoid organs during genotype 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) infection.

Porcine Reproductive and Respiratory Syndrome (PRRS) is one of the most economically important diseases of swine. PRRS virus (PRRSV) infection in the pig is characterized by a weak or absent host innate immune response. The underlying mechanisms of PRRSV pathogenesis are still unclear. The analysis of transcript levels represents an alternative to immunoassays for the detection of cytokines that sometimes are difficult to detect due to their low amounts. This study sets out to determine the differences in pathogenesis and the immune response between lung, tonsil, tracheobronchial lymph node (Tb-LN) and retropharyngeal LN (Rf-LN) of PRRSV 2982 strain infected pigs. PRRSV strain 2982 avoided the onset of an effective innate immune response, especially in PRRSV main target (lung) and reservoir (tonsil) organs. PRRSV lead to an impaired expression of IFN-α and TNF-α gene expression, which finally induced a weak and delayed adaptive immune response through an inefficient IL-12 and IFN-γ expression. Finally, PRRSV replication favored the expression of the anti-inflammatory IL-10 cytokine in infected pigs.

Virulence and genotype-associated infectivity of interferon-treated macrophages by porcine reproductive and respiratory syndrome viruses.

The polarization into M1 and M2 macrophages (MΦ) is essential to understand MΦ function. Consequently, the aim of this study was to determine the impact of IFN-γ (M1), IL-4 (M2) and IFN-ß activation of MΦ on the susceptibility to genotype 1 and 2 porcine reproductive respiratory syndrome (PRRS) virus (PRRSV) strains varying in virulence. To this end, monocyte-derived MΦ were generated by culture during 72h and polarization was induced for another 24h by addition of IFN-γ, IL-4 or IFN-ß. MΦ were infected with a collection of PRRSV isolates belonging to genotype 1 and genotype 2. Undifferentiated and M2 MΦ were highly susceptible to all PRRSV isolates. In contrast, M1 and IFN-ß activated MΦ were resistant to low pathogenic genotype 1 PRRSV but not or only partially to genotype 2 PRRSV strains. Interestingly, highly virulent PRRSV isolates of both genotypes showed particularly high levels of infection compared with the prototype viruses in both M1 and IFN-ß-treated MΦ (P<0.05). This was seen at the level of nucleocapsid expression, viral titres and virus-induced cell death. In conclusion, by using IFN-γ and IFN-ß stimulated MΦ it is possible to discriminate between PRRSV varying in genotype and virulence. Genotype 2 PRRSV strains are more efficient at escaping the intrinsic antiviral effects induced by type I and II IFNs. Our in vitro model will help to identify viral genetic elements responsible for virulence, an information important not only to understand PRRS pathogenesis but also for a rational vaccine design. Our results also suggest that monocyte-derived MΦ can be used as a PRRSV infection model instead of alveolar MΦ, avoiding the killing of pigs.