Inés Ruedas-Torres

Antibiotic restrictions in swine have increased interest in alternative feed additives to support intestinal health and immunity during the post-weaning period. Based on previous findings, this study evaluated the effects of heat-killed Mycolicibacterium manresensis (hkMm) on cellular, humoral, and innate immunity in weaned piglets. Four-week-old piglets were randomly allocated into three groups (n=5) receiving a control diet or diets supplemented with 10 or 50 ppm hkMm. At day 70, duodenal and jejunal samples were collected and analyzed by immunohistochemistry using markers for T cells (CD3, TCRδ, and FoxP3), IFN-γ⁺ cells, B cells (CD20), IgA-producing cells, and macrophages (CD163). Supplemented animals showed increased numbers of CD3⁺, particularly TCRδ⁺ and FoxP3⁺ subsets, resulting in higher TCRδ/CD3 and FoxP3/CD3 ratios. hkMm also enhanced humoral and mucosal immune responses, as indicated by increased numbers of CD20⁺ B cells and IgA+ cells, especially at 10 ppm (p = 0.04). CD163⁺ macrophages showed slight increases, suggesting enhanced regulatory and homeostatic mechanisms, while a moderate increase in jejunal IFN-γ⁺ cells was observed at 10 ppm. Correlation analysis revealed positive associations between CD3 and TCRδ (r = 0.53; p = 0.05), and betweeen TCRδ and CD163 in the jejunum (r = 0.70; p = 0.01), whereas CD163 was negatively associated with IFN-γ in the duodenum (r = −0.53; p = 0.04). Overall, the 10 ppm dose elicited the most consistent responses, promoting coordinated changes in cellular, humoral, and innate immunity. These findings suggest that hkMm may enhance mucosal immune competence while supporting intestinal homeostasis during the post-weaning period.

Yersinia pseudotuberculosis is a food-borne pathogen responsible for a self-limiting mesenteric lymphadenitis. Yersinia pseudotuberculosis lineages 1 and 8 are also associated to the Far East scarlet-like fever (FESLF) syndrome, characterized by skin rash, hyperemic tongue, and desquamation. Genome sequencing of lineage 8 FESLF strains previously revealed the presence in the plasmid pVM82 of dot/icm genes, homologous to those known to encode a Type 4B Secretion System (T4BSS) in the intracellular pathogens Legionella pneumophila and Coxiella burnetii. We validated the presence of all essential dot/icm genes required for the structure of a T4BSS and we identified acidic pH and 37°C as the main signals triggering yT4BSS expression. Putative yT4BSS effectors were also identified using the S4TE algorithm but their secretion was not confirmed in the studied conditions. The yT4BSS is also expressed in epithelial cells and macrophages as well as in vivo during mouse infection, but it is not required for bacterial intracellular survival. Nevertheless, the yT4BSS is implicated in Y. pseudotuberculosis FESLF pathogenicity in mice when bacteria are orally inoculated. The yT4BSS does not influence organ colonization and is rather implicated in the induction of important necrosis lesions in mesenteric lymph nodes and ceca of mice. Cytokine profile analyses in mouse bone marrow-derived macrophages revealed a yT4BSS-dependent induction of innate immunity related cytokines and chemokines. In conclusion, we identify the yT4BSS as a virulence factor of Y. pseudotuberculosis strains from lineage 8 responsible for FESLF which modulates cytokine responses of the host innate immune system.

African swine fever (ASF) is a devastating disease with a high impact on the pork industry worldwide. ASF virus (ASFV) is a very complex pathogen, the sole member of the family Asfaviridae, which induces a state of immune suppression in the host through infection of myeloid cells and apoptosis of lymphocytes. Moreover, haemorrhages are the other main pathogenic effect of ASFV infection in pigs, related to the infection of endothelial cells, as well as the activation and structural changes of this cell population by proinflammatory cytokine upregulation within bystander monocytes and macrophages. There are still many gaps in the knowledge of the role of proteins produced by the ASFV, which is related to the difficulty in producing a safe and effective vaccine to combat the disease, although few candidates have been approved for use in Southeast Asia in the past couple of years.

Yersinia pseudotuberculosis is a food-borne pathogen responsible for a self-limiting gastrointestinal disease in humans known as mesenteric lymphadenitis. A phylogenetically distinct Y. pseudotuberculosis cluster from lineages 1 and 8 is associated to a specific syndrome called the Far East scarlet-like fever (FESLF), characterized by skin rash, hyperemic tongue and desquamation. Genome sequencing of FESLF strains previously revealed the presence in the plasmid pVM82 of dot/icm genes, homologous to those known to encode a T4BSS in the intracellular pathogens Legionella pneumophila and Coxiella burnetii. In the present article, we characterized the genomic features and functionality of the Y. pseudotuberculosis T4BSS ( y T4BSS). We found higher dot/icm gene identity between Y. pseudotuberculosis and Pseudomonas putida genes than with those of L. pneumophila or C. burnetii . We validated the presence of all essential dot/icm genes required for the structure of a T4BSS. We then evaluated the conditions required for y T4BSS gene expression in vitro and identified an influence of temperature, with higher expression at 37°C, which mimicks the mammalian host temperature. The y T4BSS is also expressed in cellulo during the Y. pseudotuberculosis intracellular life cycle and in vivo during mouse infection. Although T4BSS functions are well characterized in the intracellular life cycle of L. pneumophila and C. burnetii , the y T4BSS appears to not be required for the intracellular survival nor for the establishment of a replication niche within cells of Y. pseudotuberculosis . Interestingly, the y T4BSS is implicated in Y. pseudotuberculosis FESLF strain pathogenicity when orally inoculated to mice but not during intravenous inoculation. Despite a role in virulence during oral infection, the y T4BSS does not influence organ colonization. However, the y T4BSS appears to be implicated in induction of important necrosis lesions in mesenteric lymph nodes and cæca of mice. Cytokine profil analyses revealed an induction of production of innate immunity related cytokines and chemokines depending on the y T4BSS in cellulo using a mouse bone marrow-derived macrophages infection model. Thus, the y T4BSS modulates cytokine responses of the host innate immune system during oral infection. In conclusion, the y T4BSS is a newly characterized virulence factor implicated in pathogenicity of Y. pseudotuberculosis strains from lineage 8 responsible for FESLF.

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.

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, andCD40) and coinhibitory (CTLA4,TIGIT,PD1/PDL1,TIM3,LAG3, andIDO1) 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.

The diagnosis of bovine tuberculosis (bTB) is based on the single intradermal tuberculin test (SIT), interferon gamma, and compulsory slaughter of reactor animals. Culture and PCR from fresh tissue are regarded as gold standard techniques for post-mortem confirmation, with the former being time-consuming and presenting moderate to low sensitivity and the latter presenting promising results. Histopathology has the advantage to identify and categorize lesions in both reactor and non-reactor animals. Therefore, this study aims to highlight the role of histopathology in the systematic diagnosis of bTB to shorten the time to disclose positive animals. Blood (212) and lymph node (681) samples were collected for serological, bacteriological, and histopathological analyses from a total of 230 cattle subjected to the Spanish bTB eradication program. Seventy-one lymph nodes and 59 cattle yielded a positive result to bacteriology, with 59 lymph nodes and 48 cattle presenting a positive result in real-time PCR from fresh tissue. Roughly 19% (40/212) of sera samples gave a positive result to ELISA. Tuberculosis-like lesions (TBLs) were observed in 11.9% (81/681) of the lymph nodes and 30.9% (71/230) of cattle. Noteworthy, TBLs were evidenced in 18 out of 83 SIT⁻ and real-time PCR and bacteriology negative animals, with 11/18 disclosing a positive result to Ziehl-Neelsen technique and two of them to ddPCR from paraffin blocks targeting IS6110. Six out of these 11 ZN⁺ corresponded with mesenteric LN and were confirmed positive to paratuberculosis. Histopathology yielded a sensitivity of 91.3% (CI₉₅ 83.2–99.4%) and a specificity of 84.4% (CI₉₅ 78.6–89.3%) with good agreement (κ = 0.626) when compared with real-time PCR. Our results confirm that histopathology allows a rapid confirmation of real-time PCR and bacteriology, emphasizing its contribution to bTB control and monitoring.

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.