Chlamydia suis is an obligate intracellular bacterium endemic in pig populations and is detected in the gastrointestinal tract, suggesting that the intestine may be an important site of chlamydial colonization. Despite this, intestinal chlamydial infections remain poorly understood, largely due to the lack of models that accurately mimic the interaction of C. suis with the gut epithelium. The aim of this study was to evaluate whether porcine jejunum-derived enteroids constitute a suitable in vitro model to investigate intestinal infection by C. suis and to compare infection dynamics with the closely related human pathogen Chlamydia trachomatis. Porcine enteroid monolayers were exposed to C. suis and C. trachomatis, and bacterial uptake, inclusion formation, and replication were assessed using microscopy-based and molecular approaches. Both C. suis and C. trachomatis efficiently attached to primary intestinal epithelial cells and formed intracellular inclusions, indicating successful bacterial uptake and early intracellular survival. Infection levels increased in a dose-dependent manner, confirming that enteroid-derived monolayers are suitable for studying early host-pathogen interactions. Furthermore, treatment with tetracycline reduced C. suis inclusion formation and extracellular bacterial release, thereby functionally confirming that an active infection had been established and that the model responded as expected to antibiotic treatment. However, unlike the McCoy cell model which supports productive bacterial replication, enteroid cultures did not exhibit a significant increase in intracellular bacterial load over time. Despite this limited replication in enteroids, chlamydial DNA accumulated in the culture supernatants, suggesting extracellular release of bacterial material. In conclusion, porcine intestinal epithelial cells permit chlamydial attachment, internalization, and early inclusion formation. However, under the tested conditions, efficient completion of the chlamydial developmental cycle was not observed. Nevertheless, porcine enteroid-derived cultures represent a physiologically relevant in vitro platform to investigate the early stages of intestinal chlamydial infection and provide a valuable system for studying host-pathogen interactions at the intestinal interface.

The current classification of porcine immunoglobulin G (IgG) subclasses relies on phylogenetic analysis of gene sequences, resulting in a nomenclature that is inapt to predict biological function. Here, we propose a revised, functionality-based nomenclature derived from a comprehensive re-analysis of Fc domain interaction motifs and in vitro effector function assays. We produced twelve recombinant porcine IgG subclasses and evaluated their ability to induce reactive oxygen species (ROS) production in porcine monocytes and neutrophils. Our results reveal a striking functional dichotomy: only the subclasses re-classified here as the pIgG4 family (IgG4a, IgG4b, IgG4c and IgG4d) induced a significant respiratory burst. In silico analysis suggests this unique ability is linked to a conserved Glycine-Proline (“GP”) motif in the lower hinge, which is absent in other subclasses. Furthermore, we predicted the structural determinants of complement activation, identifying a putative role for a “PAP” motif and Lysine-320 in the predicted C1q binding site. Subclasses with a K320E mutation (IgG4d, IgG5 family) or sequence deviations in the “PAP” motif displayed reduced (IgG5 family) or abolished (pIgG3) complement activity. Additionally, the ancestral pIgG3 Fc domain was confirmed to be functionally inert, a phenotype most-likely driven by a P329L mutation that disrupts the hydrophobic Fc domain/Fcγ receptor interface. These findings challenge the previous assumption that pIgG1 is a potent activator of all porcine myeloid cells and highlight that discrete amino acid motifs play a role in IgG effector functions. The proposed functional nomenclature provides a predictive framework for porcine immunology that enables a better understanding of IgG subclass diversification in health and disease.

Vaccination is an important control measure against infections with Mycoplasma hyopneumoniae (M. hyopneumoniae), the primary pathogen of enzootic pneumonia in pigs. The present study investigated local and systemic humoral and cell-mediated immune responses and efficacy upon vaccination of piglets with a combined vaccine containing inactivated M. hyopneumoniae and porcine circovirus 2d (Cirbloc® M Hyo, Ceva), followed by experimental M. hyopneumoniae infection (D21). Forty-five piglets were allocated to three groups, namely the not-vaccinated and infected (C) (n = 20), vaccinated and infected (V) (n = 20), and not-vaccinated and not-infected (NC) groups (n = 5). Vaccination was applied once (D0) intramuscularly. Four pigs from the V and C groups were euthanized 2 weeks post-infection (D35), and the remaining pigs 2 weeks later (D49). Blood, lung tissue, bronchoalveolar lavage fluid (BALF), and bronchial lymph nodes were collected at different timepoints. Piglets in the V group exhibited seroconversion on days 21 and 35, with markedly elevated levels of specific IgA and IgG in BALF on D35. Furthermore, the V group showed activation of interferon (IFN)-γ+/tumor necrosis factor (TNF)-α+ CD4+CD8α+ T cells and T cell proliferation on day 7, with these responses remaining elevated through D21. The concentrations of interleukin (IL)-1β, TNF-α, C–X–C motif chemokine ligand 8 (CXCL-8), and IL-17A in BALF of the C group were higher on D49, correlating with higher M. hyopneumoniae DNA loads. Clinical signs and lung lesions were less severe in vaccinated pigs. The combined vaccine elicited robust M. hyopneumoniae-specific humoral and cellular responses in blood and enhanced post-infection cellular immunity in draining lymph nodes, underscoring its protective efficacy.

Self-amplifying mRNA (saRNA) is emerging as a leading platform for both vaccination and gene therapy. However, saRNA and the lipid nanoparticles (LNPs) used for their delivery activate the innate immune system, which may complicate pulmonary delivery of saRNA-LNPs. In this study, we demonstrated that intratracheal administration of saRNA-LNPs can cause significant side effects that can be linked to innate immune cell recruitment and cytokine release in the lungs. However, through systematic optimization of the LNP formulation and the saRNA dose, we could restrict the inflammation in the lungs to an acceptable level. With these optimized saRNA-LNPs, high levels of transgene expression were achieved in the lungs of mice that lasted for at least 21 days. Moreover, intratracheal delivery of optimized saRNA-LNPs encoding SARS-CoV-2 nanobodies resulted in detectable nanobody levels in the lungs.

Mesenchymal stromal cells (MSCs) are a promising alternative cell source for cultured meat production, but no robust protocols exist for farm animal MSCs to efficiently induce myogenic differentiation. Differentiation efficiency was evaluated under different conditions: normoxia versus hypoxia, with or without 5-Aza-2 '-deoxy-cytidine (5-Aza) for 24 h versus 21 days, horse serum versus fetal bovine serum (FBS), and supplementation with galectin-1 (Gal-1) versus the combination dexamethasone + hydrocortisone. A protocol was established by supplementing 10 mu M 5-Aza, 100 nM Gal-1, and 10% FBS in normoxia, as confirmed by increased mRNA levels of MYF5, MYOD1, MRF4 and MYOG, decreased PAX3, increased desmin, tropomyosin, and MyHC protein levels, and formation of myotube-like structures, as indicated by the presence of multinucleated cells. To study breed effects, bovine MSCs from Holstein Friesian (HF) and Belgian Blue (BB) were myogenically differentiated. Differences were observed with MYOG expression and MyHC expression being higher in BB-MSCs, although fusion into multinucleated myotubes was limited. Additionally, immunophenotypic profiles of HF-MSCs and BB-MSCs were assessed before and after myogenic differentiation. Following myogenic differentiation, significant reduction of MSC markers (CD29, CD44, CD90, and CD34) was observed. Our findings lay the foundation for further studies aimed at optimizing myogenic differentiation of MSCs.

This manuscript reviews current knowledge on Mycoplasma hyopneumoniae, the primary agent of enzootic pneumonia in pigs, integrating advances in epidemiology, pathogenesis, immunity, diagnostics, and control. The pathogen is globally endemic, with transmission driven mainly by direct contact within a herd, and by purchase of animals and airborne transmission between herds. Infection dynamics are shaped by production systems, sow-piglet transmission, and co-infections, while strain diversity and subclinical infections complicate disease expression and control. Pathogenesis is understood as a complex, multifactorial phenomenon rather than being determined by single virulence factors. Adhesion, biofilm formation, extensive posttranslational processing, metabolic adaptation, and immune evasion collectively enable persistent colonization. A major conceptual advance is the discovery of migrasome-mediated dissemination, which may explain chronic infection and reduced antimicrobial efficacy. Despite significant advances, important knowledge gaps remain in multiple areas of Mycoplasma hyopneumoniae research. Uncertainties persist in transmission and epidemiology, including the role of environmental spread, wild boar reservoirs, and the extent of subclinical infections. The impact of strain diversity on virulence, transmission, persistence, and vaccine performance is not yet fully understood, and predictive markers are lacking. Key aspects of host–pathogen interactions, such as immune evasion, occasional intracellular survival, macrophage polarization, and the in vivo relevance of migrasome-mediated dissemination, require further clarification. Protective immunity remains poorly defined, with unresolved questions on correlates of protection and the roles of mucosal and systemic responses. Diagnostic challenges include distinguishing active from residual infection and improving specificity and Differentiating Infected from Vaccinated Animals (DIVA) compatibility. Evidence on antimicrobial effects on transmission and resistance monitoring is limited. In addition, variability in vaccine efficacy and the influence of maternal immunity remain unclear. Finally, scalable elimination strategies, particularly for very large production systems and reduced antimicrobial use, are still needed.

Chlamydia suis, a close relative of the human pathogen C. trachomatis, can be detected in the porcine gut, yet its prevalence and viability across intestinal segments remain poorly defined. This study aimed to assess the segment-specific prevalence, isolation success, and tetracycline susceptibility of C. suis in grower-finisher pigs. Jejunal, ileal, and colonic samples (n = 200 per intestinal segment) were collected from 600 pigs at slaughter and analyzed using C. suis-specific real-time PCR and culture. PCR revealed significantly higher detection rates in the colon (40%) than in the jejunum or ileum (both 4.5%), accompanied by significantly higher calculated bacterial loads in colonic samples. In contrast, viable C. suis was most frequently isolated from ileal material, indicating that the ileum may provide a more favorable condition for active bacterial replication. Among 24 culture-confirmed isolates, 75% were susceptible to tetracycline (MIC/MBC < 2 µg/mL), 12.5% exhibited an intermediate phenotype (2 µg/mL < MIC/MBC < 4 µg/mL) and another 12.5% were resistant (MIC/MBC > 4 µg/mL). Intermediate phenotypes were recovered from the jejunum and ileum, whereas resistant isolates were found in the ileum and colon. These findings suggest that the porcine colon may serve as an intestinal reservoir for C. suis, while the ileum supports more robust bacterial replication. Overall, these data contribute to our understanding of the intestinal ecology of C. suis under field conditions and its tetracycline susceptible patterns.

Post-weaning diarrhea in piglets is frequently caused by enterotoxigenic Escherichia coli (ETEC) F4+. The objective of this study was to examine the possible protective effect of galacto-oligosaccharides (GOS) on ETEC F4+-induced intestinal injury. Growth inhibition of ETEC F4+ in the presence of 2% GOS was assessed, as well as the ability of GOS to reduce pathogen adhesion and invasion in the intestinal Caco-2 cell line. GOS ability to counteract ETEC F4+ adhesion was also assessed ex vivo in piglet small intestinal villi. Protective activity of GOS against ETEC F4+-induced membrane damage in Caco-2 cells was evaluated through transepithelial electrical resistance (TEER), phenol red apparent permeability (Papp) and immunolocalization of tight junction proteins occludin and ZO-1. Inflammation was assessed by quantification and immunolocalization of phosphorylated-p65 protein, indicative of NF-κB activation. Finally, GOS prebiotic activity on probiotic strains Lactobacillus amylovorus: ATCC 33198 and DSM 16698, as well as Limosilactobacillus reuteri subsp. porcinus DSM 110571, was also investigated. The results showed that GOS significantly reduced ETEC F4+ adhesion and invasion in Caco-2 cells, as well as adhesion to piglet intestinal villi. Furthermore, GOS markedly decreased ETEC F4+ induced membrane damage, as evidenced by improvement of TEER, phenol red Papp and tight junction protein immunolocalization. A reduction of p65 phosphorylation and nuclear translocation in the presence of GOS indicated a diminished activation of the NF-κB pathway. GOS also exhibited promising prebiotic activity toward the tested probiotic strains. Taken together, these in vitro findings suggest the potential of including GOS in piglet weaner diets to prevent ETEC F4+-induced intestinal injury.

In livestock species such as pigs, antibiotics are frequently used during weaning, a period of increased susceptibility to infections. However, the emergence and spread of antimicrobial resistance necessitate alternative strategies to improve animal health. Immune potentiating polysaccharides, including chitin, chitosan, and their derivatives, show promise as functional feed ingredients, yet their direct effects on porcine innate immune cells remain unclear. Here, ex vivo porcine primary neutrophils, monocytes, and monocyte-derived macrophages were used to assess the immunomodulatory properties of structurally distinct chitin-based polymers. In peripheral blood mononuclear cells, neither chitin nor chitosan induced significant secretion of IL-1β, TNF-α, or IL-6, suggesting that only specific subpopulations mediate these effects. Following fractionation, cytokine production was restricted to the CD14⁺ monocyte compartment, where high concentrations of chitosan elicited strong pro-inflammatory responses. To further investigate innate immune programming, trained immunity assays were performed. In monocytes, chitin did not induce training or tolerance, whereas low-molecular-weight, highly deacetylated chitosan promoted a trained phenotype, evidenced by enhanced cytokine production upon lipopolysaccharide restimulation. In contrast, in monocyte-derived macrophages, chitin pretreatment induced a tolerance-like phenotype, characterized by reduced cytokine responses following subsequent stimulation, while chitosan had no significant effect. These findings demonstrate that structural differences in chitin-based polymers determine the magnitude and direction of innate immune responses, supporting their targeted use as functional immunomodulators to enhance health and resilience in livestock.

Lactoferrin (LF) is a glycoprotein found in neutrophils, milk, and various mammalian secretions that plays a crucial role in host defense by modulating the immune response. Previous studies have shown that LF is taken up by human monocytes and can be present in their nucleus. However, it is unclear whether the iron saturation levels or the protease activity of LF are involved in this uptake and nuclear translocation. In addition, the activation of monocytes might influence these processes. The present study investigated the uptake and nuclear translocation of bovine LF (bLF) and porcine LF (pLF) in porcine blood-derived monocytes and how this affects monocyte function. Both bLF and pLF were internalized by porcine monocytes. Their uptake was not affected by increased iron saturation levels or by inactivation of the proteolytic activity of LF. Similarly, LPS-induced activation of monocytes did not affect LF internalization. We further investigated whether bLF could modulate LPS-induced cytokine responses by monocytes. Across all tested LPS serotypes and concentrations, bLF failed to decrease the LPS-induced TNF-α secretion by porcine monocytes. Besides internalization, we found that bLF and pLF translocated to the nucleus in a subset of porcine monocytes. This nuclear translocation was not affected by the iron saturation level and proteolytic activity of LF, nor by LPS-induced monocyte activation. Together, these findings further deepen our understanding of LF's interaction with porcine innate immune cells and provide insights into its immunomodulatory properties.

Porcine Shiga toxin-producing Escherichia coli (STEC) strains pose significant challenges to the pig industry. The toxins produced by these strains, particularly Shiga toxin subtype 2e (Stx2e), are associated with a range of clinical symptoms such as diarrhoea and oedema disease, which in severe cases result in death. Understanding the factors that influence the production and secretion of Stx2e is crucial to elucidate porcine STEC pathogenesis and to develop effective therapeutic strategies. Therefore, this study aimed to characterize the variability in Stx2e production among different porcine STEC strains and assess the effect of several external factors, including bile acids and antibiotics. Our results highlighted a substantial variation in extracellular Stx2e levels by porcine STEC strains. In addition, bile acids, especially the bile acid deoxycholate, exerted strain-specific effects on these extracellular Stx2e levels. Antibiotics also affected extracellular Stx2e levels with ciprofloxacin and enrofloxacin inducing a substantial increase in toxin production in certain strains. Genome analysis revealed that these strains encode a holin gene downstream of the Stx2e operon. Deleting this holin gene abolished the antibiotic-induced increase in extracellular Stx2e levels, while introducing holin expression in unresponsive strains increased the presence of Stx2e in the extracellular environment. These findings unravel a role for phage holins in Stx2e secretion and highlight the intricate interplay between genetic and environmental factors in regulating Stx2e production in porcine STEC strains. Together, our results offer insights into STEC pathogenesis.

Increased micro- and nanoplastic (MNP) pollution poses significant health risks, yet the mechanisms of their accumulation and effects on absorptive tissues remain poorly understood. Addressing this knowledge gap requires tractable models coupled to dynamic live cell imaging methods, enabling multi-parameter single cell analysis. We report a new method combining adult stem cell-derived small intestinal organoid cultures with live fluorescence lifetime imaging microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an ‘apical-out’ topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles interacted differently with apical and basal membranes of the organoids and showed a species-specific pattern of cellular uptake. Using a phasor analysis approach, we demonstrate improved sensitivity of FLIM over conventional intensity-based microscopy. The resulting ‘fluorescence lifetime barcoding’ enabled distinguishing of different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 day)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total cell energy budget and epithelial inflammation. We found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and enable the tracing of other fluorescent nanoparticles in live organoid and 3D ex vivo systems.

CD94 is a natural killer (NK) cell receptor that also marks subsets of T cells, referred to as NKT cells. In humans, the role of CD94 as both an immune checkpoint and a potential therapeutic target has gained increasing attention. However, data about its expression in leukemia and lymphoma in dogs remain limited. This study aimed to explore CD94 expression in canine leukemia and nodal lymphoma, using a newly available anti-canine CD94 monoclonal antibody in a multicolor flow cytometry panel. Surplus blood and lymph node aspirate samples from eleven client-owned dogs (leukemia: n = 7, lymphoma n = 4) and two clinically healthy controls, were analyzed. The control dogs as well as most cases showed low CD94+ lymphocyte frequencies, consistent with a non-neoplastic population. However, markedly expanded CD94+ populations were identified in two out of four of the T cell chronic lymphocytic leukemia (T-CLL) cases. In one of them, the neoplastic population was uniformly CD3+CD8+CD94+, while the other showed a heterogeneous mixture of CD3+CD8+CD94+ and CD3−CD8+CD94+ lymphocytes. Our findings demonstrate that the canine-specific CD94 antibody can be applied to both blood and lymph node samples in a diagnostic flow cytometry setting. While CD94 expression was infrequent overall, its detection in a subset of T-CLL cases highlights the need for larger studies to determine its diagnostic and therapeutic value in canine leukemia and lymphoma.

Enterotoxigenic Escherichia coli (ETEC) is a common cause of diarrhea in humans and animals, including pigs. Enterotoxins are important virulence factors for ETEC. Although much is known about the mechanism of enterotoxin-induced diarrhoea, less is known about its effects on innate immune cells such as monocytes. Monocytes can differentiate into macrophages and dendritic cells and play a pivotal role in bridging the innate and adaptive immune systems. Understanding the interaction between ETEC enterotoxins and monocytes can help in the development of more effective preventive and therapeutic strategies to combat this disease. In this study, we aimed to investigate the effects of heat labile enterotoxin (LT) and heat stable enterotoxin a (STa) produced by ETEC on porcine monocytes. Our results showed that STa did not affect the viability or effector functions of monocytes. LT, on the other hand, decreased the viability of monocytes. While LT did not alter the production of reactive oxygen species (ROS) by monocytes, it significantly reduced the production of ROS induced by phorbol 12-myristate 13-acetate (PMA). In addition, LT decreased the phagocytosis of E. coli by monocytes and enhanced the survival of intracellular ETEC. Furthermore, LT triggered the production of the cytokines IL-1β, IL-6 and TNF-α as well as the chemokines CCL-3 and CXCL-8. Together, our results show that, in contrast to STa, LT can cause monocyte death and disrupt monocyte immune effector functions, potentially acting as an immune evasion strategy to establish infection.

Tick-borne encephalitis (TBE) is the most frequent tick-borne viral disease transmitted by ticks in Europe and Asia. In Belgium, autochthonous cases of TBE have been reported, but even though some tick collection was carried out in the past, no TBEV-positive ticks have been found thus far. In this study, questing ticks were collected by flagging at the precise location where a patient was reported to have been bitten by a tick before developing TBE in Belgium in 2020. In total, 350 ticks were pooled by life stage (nymphs, adult females, adult males) and collection date, lysed, and RNA extracted. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed to detect tick-borne encephalitis virus (TBEV) and Ixodes 18S rRNA, followed by Oxford nanopore amplicon sequencing. TBEV was detected in all three types of pools. Out of 69 nymph pools, 2 were positive, in adult female pools, 2 out of 16 were positive, and 1 of the 14 adult male pools was positive. A complete sequence was retrieved through sequencing. This sequence shares greater similarity with a strain found in Finland than the neighboring Salland strain (the Netherlands) and the Neudoerfl reference strain. These findings confirm that TBE can be acquired from tick bites within the country. It is therefore necessary to increase awareness of the disease among healthcare professionals.

Vaccination against Mycoplasma hyopneumoniae is still carried out worldwide, but unfortunately current commercial vaccines only provide partial protection. Therefore, two M. hyopneumoniae strains were genetically modified by transposon-mediated gene disruption of mmsA and mnuA, encoding methylmalonate semialdehyde dehydrogenase and membrane nuclease A, respectively. We investigated how immune responses elicited by these genetically modified M. hyopneumoniae strains protected pigs against challenge infection. An endotracheal single dose vaccination with genetically modified M. hyopneumoniae strain 1 (ΔmmsA) or 2 (ΔmnuA), or physiological saline solution (Control) was followed by challenge infection. Piglets from ΔmnuA had a higher respiratory disease score post-vaccination, but this group coughed significantly less after challenge. Significantly fewer DNA copies of the challenge strains were observed in broncho-alveolar lavage fluid (BAL) from ΔmnuA after challenge. Two weeks post-challenge, significantly more BAL IgG and BAL IgA was observed in ΔmnuA, but at euthanasia significantly more IgA and less pro-inflammatory cytokines were detected in BAL from both vaccinated groups. Furthermore, a significantly lower percentage of IFN-γ+ and TNF-α+IFN-γ+ CD8+ T cells was observed after administration of ΔmnuA. The percentage of IFN-γ+ CD8+ T cells was significantly lower in ΔmmsA at euthanasia. To conclude, the results of this exploratory study show that a single endotracheal administration of ΔmnuA resulted in coughing post-vaccination, but reduced clinical signs post-challenge and challenge strain DNA load in BAL. Therefore, a strain mutated in the mnuA gene might be an interesting mutant strain that could be promising as a potential live vaccine candidate strain as it can reduce M. hyopneumoniae infection burden under field conditions.

Ticks are important vectors of zoonotic pathogens, and their presence can be influenced by the composition of the tick microbiome. In turn, this microbiome is shaped by environmental and ecological factors, as demonstrated in several studies conducted under controlled conditions. However, the extent of these influences under natural ecological conditions remains underexplored. In this study, we investigated the diversity of the microbiome and the prevalence of pathogens in Ixodes ricinus nymphs across three distinct Belgian ecoregions: Sandy Loam, Condroz, and Ardennes. Using real-time quantitative PCR (qPCR) and Oxford Nanopore 16S rRNA sequencing, we assessed how geography and pathogen presence influence tick-associated microbial communities. Our results revealed significant regional differences in microbiome composition and pathogen prevalence. Borrelia burgdorferi sensu lato (s.l.) was most prevalent in the Ardennes (9% (7.4-10.9) vs 3.8% (2.8-5.2) in the Condroz and 2.1% (1.4-3.2) in Sandy Loam) while Anaplasma phagocytophilum was more common in the Sandy Loam region (21.1% (18.7-23.8) vs 4% (3-5.4) in the Condroz and 3.2% (2.2-4.4) in the Ardennes). Endosymbionts such as Midichloria mitochondrii and Spiroplasma ixodetis also exhibited distinct geographic distributions. Network analysis identified potential pathogen-microbiota interactions, with certain bacterial taxa showing positive or negative associations with specific pathogens. Moreover, microbiome composition was influenced not only by ecoregion but also by microorganisms such as Rickettsia helvetica, suggesting that its colonization may actively shape microbial community structure, potentially through competition or facilitation mechanisms. Additionally, microbiome network robustness varied across ecoregions, highlighting the role of ecological context in shaping microbial interactions within ticks. These findings underscore the complex interplay between geography, pathogen presence, and microbial diversity in ticks, highlighting the importance of integrating these interactions to inform microbiome-based strategies for vector control and disease prevention.

Xenogeneic tumour origin and batch-to-batch variability of Engelbreth-Holm-Swarm sarcoma tumour cell-derived hydrogels (Matrigel, Cultrex) limit the biomedical application of organoids in tissue engineering. The gelatin-methacryloyl (GelMA) hydrogels represent a defined, tunable, and GMP-friendly alternative, but they are rarely studied as alternative to Matrigel. Here, we studied effects of mechanical properties of GelMA and addition of laminin-111 on encapsulation and growth of small intestinal organoids. GelMA-embedded organoids displayed polarity reversion, resulting in apical-out and apical-basal phenotypes, independent from the matrix stiffness. Addition of laminin-111 softened hydrogels and also resulted in a partial restoration of the basal-out phenotype. Interestingly, despite the incomplete polarity restoration, GelMA-organoids still showed minor growth. GelMA stiffness and concentration influenced the transition from 3D to 2D organoid cultures. Collectively, our study confirms that tuning of GelMA mechanical properties alone cannot recapitulate the basal membrane matrix. However, controlled polarity reversion offers a tool for engineering organoids and enabling apical membrane access.

The increasing micro- and nanoplastic (MNP) pollution poses significant risks to human and animal health, yet the mechanisms of their accumulation and effects on absorptive tissues such as the gastrointestinal tract remain poorly understood. Addressing these knowledge gaps requires tractable models coupled to dynamic live cell imaging methods, to enable multi-parameter analysis at single cell resolution. Here we report a new method combining adult stem cell-derived small intestinal organoid cultures with multi-parameter live Fluorescence Lifetime Imaging Microscopy (FLIM) to study MNP interactions with gut epithelium. To facilitate this, we optimized live imaging of porcine and mouse small intestinal organoids with an apical-out topology. Subsequently, we produced a set of pristine MNPs based on PMMA and PS (<200 nm, doped with deep-red fluorescent dye) exhibiting different surface charges, and evaluated their interaction with organoids displaying controlled epithelial polarity. We found that nanoparticles differently interacted with apical and basal membranes of the organoids and even showed a species-specific pattern of cellular uptake. Using a phasor-FLIM approach, we demonstrate better sensitivity of FLIM over conventional intensity-based microscopy. The fluorescence lifetime barcoding enabled distinguishing different types of MNP and their interaction sites within organoids. Finally, we studied short (1 day)- and long (3 days)-term exposure effects of PMMA and PS-based MNPs on mitochondrial function, total energy budget and epithelial inflammation and found that even pristine MNPs could disrupt chemokine production and mitochondrial membrane potential in intestinal epithelial cells. The presented FLIM approach will advance the study of MNP toxicity, their biological impacts on gastrointestinal tissue and help tracing other types of fluorescent nanoparticles in live organoid and 3D ex vivo systems. ### Competing Interest Statement The authors have declared no competing interest.

BACKGROUND:CD4 +CD8 + double-positive (DP) T cells are present in low numbers in the peripheral blood of both healthy and sick humans and dogs. In humans, these cells play cytotoxic or suppressive roles depending on the disease, but their function in dogs remains unclear. OBJECTIVES:This study aims to investigate the presence of DP T cells in a cohort of dogs with adverse food reactions (AFR), compare their frequency among AFR, non-food-induced atopic dermatitis (NFICAD), and healthy dogs (HTY), and evaluate whether DP T cells could serve as a diagnostic tool to differentiate between AFR and NFICAD and identify the culprit allergens in AFR dogs. METHODS:Peripheral blood samples were collected from dogs with AFR, NFICAD, and healthy controls. PBMCs were isolated and analyzed by flow cytometry to assess T cell subpopulations. AFR dogs were grouped by their specific culprit allergens, and DP T cell proliferation in response to each allergen was compared across groups. An overall comparison of DP T cell proliferation was made between the three groups (AFR, NFICAD, HTY) under both stimulated and non-stimulated conditions. The mean percentage of proliferating DP T cells in healthy dogs was used as a cut-off to correlate with oral food challenge (OFC) results. RESULTS:DP T cells proliferated in all groups, with the greatest proliferation observed in the AFR group when stimulated with food allergens. Statistically significant differences were found between AFR and NFICAD groups, with AFR dogs showing more proliferation. The test identified the culprit allergens in 28.57 % of cases, with false positives occurring in 17.86 %. CONCLUSIONS:DP T cells showed greater proliferation in food-allergic dogs compared to those with other allergic conditions like NFICAD. Despite these differences, overlapping results indicate that DP T cells are not a reliable screening test for distinguishing allergic from healthy dogs. While the test holds potential for identifying allergic phenotypes, it lacks sufficient diagnostic value for pinpointing specific allergens. Future studies with larger sample sizes and refined methods are needed to improve diagnostic accuracy.