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The article examines the modulation of gene expression profiles in racing horses. It takes into account the chromosomal location of interleukin 8 (IL8) in Equus caballus. A genomic DNA fragment has been amplified using primers from the horse IL8 mRNA sequence. Overlapping clones 63-D22 and 163-L3 have been isolated. Through the use of bacterial artificial chromosome clone as probe in a fluorescence in situ hybridization, the location of IL8 is determined.

Bacteria-mediated treatments gained increasing attention as alternative therapies against tumors. An attenuated mutant strain of Salmonella enterica serovar Typhimurium (STMΔznuABC) has recently been considered as a potential new anti-cancer strategy. However, it is unclear whether this activity is tumor-induced or species-specific, and no data are available regarding STMΔznuABC on canine mammary tumors (CMTs). This study aimed to investigate the ability of STMΔznuABC in modulating the response of CMTs, focusing on cancer-associated fibroblasts. Four CMT cell lines (CF33, TM51, TM52 TM53) were treated with STMΔznuABC. Then, antiproliferative activity (MTT assay), bacterial invasion, and CMT cell lines gene expression analysis (RT-qPCR) of genes involved in immune response and cancer aggressiveness were evaluated. STMΔznuABC penetrated in TM51, TM52, TM53, and CF33 cell lines, causing a significant reduction of cell viability. Moreover, the expression of several genes was significantly modulated in all CMT cell lines: STMΔznuABC infection determined a significant up-regulation of CXCL8, IL18, IL10, TLR4 and RAD51, while CD14, IL6, CXCR4, P53, PTEN, STAT5, TLR5 and TGFB1 were downregulated in TM53. In CF33, CXCL8 and P53 were upregulated, while MYD88, MD2, IL18, TLR4,5, TGFB1 were downregulated. In TM52, CXCL8, CD44 and MD2 were upregulated and PTEN was downregulated, while in TM51 CXCL8, CD44 and ErbB2 were downregulated. We demonstrated the anti-proliferative and immuno-modulatory activity of STMΔznuABC in CMTs, paving the way for potential new anti-cancer treatments., Competing Interests: Declaration of competing interest The authors have no competing interests to declare. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results., (Copyright © 2024 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Recently, much interest has been raised for the characterization of signaling molecules carried by extracellular vesicles (EVs), which are particularly enriched in milk (mEVs). Such interest is linked to the capability of EVs to cross biological barriers, resist acidification in the gastric environment, and exert modulation of the immune system, mainly through their microRNA (miRNA) content. We characterized the small-RNA cargo of colostrum EVs (colosEVs) and mEVs from Italian Mediterranean buffalo through next generation sequencing. Colostrum (first milking after birth) and milk (day 50 of lactation) were sampled from seven subjects from five farms. ColosEVs and mEVs were subjected to morphological characterization, followed by high-depth sequencing of small RNA libraries produced from total RNA. The main difference was the amount of EV in the two samples, with colostrum showing 10 to 100-fold higher content than milk. For both matrices, miRNA was the most abundant RNA species (95% for colosEVs and 96% for mEVs) and three lists were identified: colosEV-specific, mEV-specific and shared most expressed. Gene ontology (GO) enrichment analysis on miRNA targets highlighted many terms related to the epigenetic, transcriptional and translational regulations across the three lists, with a higher number of enriched terms for colosEV-specific miRNAs. Terms specific to colosEVs were related to "cell differentiation" and "microvillus assembly", while for mEV "cardiac and blood vessel development" and "mitochondria" emergerd. Immune modulation terms were found for both sample-specific miRNAs. Overall, both matrices carry a similar molecular message in terms of biological processes potentially modulated into receiving cells, but there is significant difference in the abundance, with colostrum containing much more EVs than milk. Moreover, colosEVs carry molecules involved in signal transduction, cell cycle and immune response, as for mEVs and EVs of other previously characterized species, but with a special enrichment for miRNAs with epigenetic regulation capacities. These beneficial characteristics of colosEVs and mEVs are essential for the calf and could also be exploited for the therapeutic purposes in humans, although further studies are necessary to measure the sanitization treatment impact on EV conservation, especially in buffalo where milk is consumed almost exclusively after processing., (© 2024. The Author(s).)

Vulvo-vaginal epithelial tumors are uncommon in mares, and data on the epithelial-to-mesenchymal transition (EMT) and the tumor-immune microenvironment (TIME) are still lacking. This is a study investigating the equus caballus papillomavirus type 2 (EcPV2) infection state as well as the EMT process and the tumor microenvironment in vulvo-vaginal preneoplastic/ benign (8/22) or malignant (14/22) epithelial lesions in mares. To do this, histopathological, immunohistochemical, transcriptomic, in situ hybridization, and correlation analyses were carried out. Immunohistochemistry quantification showed that cytoplasmic E-cadherin and β-catenin expression as well as nuclear β-catenin expression were features of malignant lesions, while benign/preneoplastic lesions were mainly characterized by membranous E-cadherin and β-catenin expression. Despite this, there were no differences between benign and malignant equine vulvo-vaginal lesions in the expression of downstream genes involved in the canonical and noncanonical wnt/β-catenin pathways. In addition, malignant lesions were characterized by a lower number of cells with cytoplasmic cytokeratin expression as well as a slightly higher cytoplasmic vimentin immunolabeling. The TIME of malignant lesions was characterized by more numerous CD204 + M2-polarized macrophages. Altogether, our results support the hypothesis that some actors in TIME such as CD204 + M2-polarized macrophages may favor the EMT process in equine vulvo-vaginal malignant lesions providing new insights for future investigations in the field of equine EcPV2-induced genital neoplastic lesions., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The dietary supplementation of olive oil by-products, including olive mill waste-water (OMWW) in animal diets, is a novel application that allows for their re-utilization and recycling and could potentially decrease the use of antibiotics, antimicrobial resistance risk in livestock species, and the occurrence of intestinal diseases. Salmonella serovar typhimurium is one of the most widespread intestinal pathogens in the world, causing enterocolitis in pigs. The aim of this study was to investigate the effect of an OMWW extract enriched in polyphenols (hydroxytyrosol and tyrosol) in the immune response of an intestinal porcine epithelial cell line (IPEC-J2) following S. typhimurium infection. Cells were pre-treated with OMWW-extract polyphenols (OMWW-EP, 0.35 and 1.4 µg) for 24 h and then infected with S. typhimurium for 1 h. We evaluated bacterial invasiveness and assayed IPEC-J2 gene expression with RT-qPCR and cytokine release with an ELISA test. The obtained results showed that OMWW-EP (1.4 µg) significantly reduced S. typhimurium invasiveness; 0.35 µg decreased the IPEC-J2 gene expression of IL1B , MYD88 , DEFB1 and DEFB4A , while 1.4 µg down-regulated IL1B and DEFB4A and increased TGFB1. The cytokine content was unchanged in infected cells. This is the first study demonstrating the in vitro immunomodulatory and antimicrobial activity of OMWW extracts enriched in polyphenols, suggesting a protective role of OMWW polyphenols on the pig intestine and their potential application as feed supplements in farm animals such as pigs.

Training has a significant effect on the physiology of blood coagulation in humans and in horses. Several hemostatic changes have been reported after exercise in the horse but data available are inconclusive. The aim of this study was to investigate platelet activation and primary platelet-related hemostasis modifications in young never-trained Thoroughbreds in the first incremental training period in order to improve knowledge on this topic. Twenty-nine clinically healthy, untrained, 2-year-old Thoroughbred racehorses were followed during their incremental 4-month sprint exercise training. Blood collection was performed once a month, five times in total (T-30, T0, T30, T60, and T90). Platelet aggregation was measured by light transmission aggregometry in response to various agonists: adenosine diphosphate (ADP), collagen, and calcium ionophore A23187. Platelet function was evaluated using a platelet function analyzer (PFA-100 ® ) using collagen/ADP and collagen/adrenaline cartridges. Nitrite-nitrate (NOx) plasma concentrations were measured via a colorimetric assay to assess in vivo nitric oxide bioavailability. Platelet activation was also investigated through gene expression analyses (selectin P- SELP , ectonucleotidase CD39- ENTPD1 , prostaglandin I2 synthase- PTGIS , endothelial nitric oxide synthase 3- NOS3 ). Differences among the time points were analyzed and mean ± SEM were calculated. Significant modifications were identified compared with T-30, with an increase in platelet aggregation (collagen:32.6 ± 4.8 vs. 21.6 ± 4.9%; ADP: 35.5 ± 2.0 vs. 24.5 ± 3.1%; A23187: 30 ± 4.7 vs. 23.8 ± 4%) and a shorter closure time of C-ADP cartridges (75.6 ± 4.4 vs. 87.7 ± 3.4 s) that tended to return to the baseline value at T90. NOx concentrations in plasma significantly increased after 30 days of the training program compared with the baseline. The first long-term training period seems to induce platelet hyperactivity after 30 days in never-trained Thoroughbreds. Regular physical training reduces the negative effects of acute efforts on platelet activation.

With his bicentennial breeding history based on athletic performance, the Thoroughbred horse can be considered the equine sport breed. Although genomic and transcriptomic tools and knowledge are at the state of the art in equine species, the epigenome and its modifications in response to environmental stimuli, such as training, are less studied. One of the major epigenetic modifications is cytosine methylation at 5' of DNA molecules. This crucial biochemical modification directly mediates biological processes and, to some extent, determines the organisms' phenotypic plasticity. Exercise indeed affects the epigenomic state, both in humans and in horses. In this study, we highlight, with a genome-wide analysis of methylation, how the adaptation to training in the Thoroughbred can modify the methylation pattern throughout the genome. Twenty untrained horses, kept under the same environmental conditions and sprint training regimen, were recruited, collecting peripheral blood at the start of the training and after 30 and 90 days. Extracted leukocyte DNA was analyzed with the methylation content sensitive enzyme ddRAD (MCSeEd) technique for the first time applied to animal cells. Approximately one thousand differently methylated genomic regions (DMRs) and nearby genes were called, revealing that methylation changes can be found in a large part of the genome and, therefore, referable to the physiological adaptation to training. Functional analysis via GO enrichment was also performed. We observed significant differences in methylation patterns throughout the training stages: we hypothesize that the methylation profile of some genes can be affected early by training, while others require a more persistent stimulus., (© 2023. The Author(s).)

In recent years, the increased demand for agri-food products to feed livestock species has stimulated research to identify novel solutions for the valorization of natural waste, according to the modern concept of a circular economy. Numerous studies have shown the use of plant-derived and agro-industrial co-products that are sources of bioactive molecules for preparing animal feeds. Supplementation with co-products derived from the extraction of olive oil (i.e., olive pomace, olive mill wastewater, olive cake and olive leaf) in diet has been widely considered in recent decades, because these wastes are produced in high quantity and their re-use represents an innovative economic and environmental strategy. Olive oil co-products are characterized by various bioactive molecules such as polyphenols, carbohydrates, proteins, and lipids. Among them, polyphenols are the nutraceuticals most studied, showing to promote health effects in both humans and animals. Olive oil co-products and their phenolic extracts have shown many beneficial and promising effects when added to the diets of monogastric animals, by improving performance parameters and maintaining the oxidative status of meat and derived products. This review provides an update on the use of olive co-products in monogastric animal (swine, poultry and rabbit) diets and their effects on the productive performance, meat quality characteristics and gut health status., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2023 Ferlisi, Tang, Cappelli and Trabalza-Marinucci.)

Extracellular vesicles (EVs) are nanometric spherical structures, enclosed in a lipid bilayer membrane and secreted by multiple cell types under specific physiologic and pathologic conditions. Their complex cargo modulates immune cells within an inflammatory microenvironment. Milk is one of the most promising sources of EVs in terms of massive recovery, and milk extracellular vesicles (mEVs) have immunomodulatory and anti-inflammatory effects. The aim of this study was to characterize goat mEVs' immunomodulating activities on Toll-like receptors (TLRs) and related immune genes, including cytokines, using a porcine intestinal epithelial cell line (IPEC-J2) after the establishment of a pro-inflammatory environment. IPEC-J2 was exposed for 2 h to pro-inflammatory stimuli as a model of inflammatory bowel disease (IBD), namely LPS for Crohn's disease (CD) and H 2 O 2 for ulcerative colitis (UC); then, cells were treated with goat mEVs for 48 h. RT-qPCR and ELISA data showed that cell exposure to LPS or H 2 O 2 caused a pro-inflammatory response, with increased gene expression of CXCL8 , TNFA , NOS2 and the release of pro-inflammatory cytokines. In the LPS model, the treatment with mEVs after LPS determined the down-regulation of NOS2 , MMP9 , TLR5 , TGFB1 , IFNB , IL18 and IL12A gene expressions, as well as lower release of IL-18 in culture supernatants. At the same time, we observed the increased expression of TLR1 , TLR2 , TLR8 and EBI3 . On the contrary, the treatment with mEVs after H 2 O 2 exposure, the model of UC, determined the increased expression of MMP9 alongside the decrease in TGFB1 , TLR8 and DEFB1 , with a lower release of IL-1Ra in culture supernatants. Overall, our data showed that a 48 h treatment with mEVs after a pro-inflammatory stimulus significantly modulated the expression of several TLRs and cytokines in swine intestinal cells, in association with a decreased inflammation. These results further highlight the immunomodulatory potential of these nanosized structures and suggest their potential application in vivo.

Introduction: Extracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages., Methods: In this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays., Results: These particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected., Discussion: Overall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Franzoni, Mecocci, De Ciucis, Mura, Dell’Anno, Zinellu, Fruscione, De Paolis, Carta, Anfossi, Dei Guidici, Chiaradia, Pascucci, Oggiano, Cappelli and Razzuoli.)

Papillomaviruses (PVs) are small, non-enveloped viruses, ubiquitous across the animal kingdom. PVs induce diverse forms of infection, such as cutaneous papillomas, genital papillomatosis, and carcinomas. During a survey on the fertility status of a mare, a novel Equus caballus PV (EcPV) has been identified using Next Generation Sequencing, and it was further confirmed with genome-walking PCR and Sanger sequencing. The complete circular genome 7607 bp long shares 67% average percentage of identity with EcPV9, EcPV2, EcPV1, and EcPV6, justifying a new classification as Equus caballus PV 10 (EcPV10). All EcPV genes are conserved in EcPV10, and phylogenetic analysis indicates that EcPV10 is closely related to EcPV9 and EcPV2, genus Dyoiota 1. A preliminary EcPV10 genoprevalence study, carried out on 216 horses using Real Time PCRs, suggested a low incidence of this isolate (3.7%) compared to EcPVs of the same genus such as EcPV2 and EcPV9 in the same horse population. We hypothesize a transmission mechanism different from the one observed in the closely related EcPV9 and EcPV2 that particularly infect Thoroughbreds. This horse breed is usually submitted to natural mating, thus indicating a possible sexual diffusion. No differences were detected for breeds in terms of susceptibility to EcPV10. Further studies are needed to investigate the molecular mechanisms behind the host and EcPV10 infection to explain the reduced viral spread.

Introduction: The Italian peninsula is in the center of the Mediterranean area, and historically it has been a hub for numerous human populations, cultures, and also animal species that enriched the hosted biodiversity. Horses are no exception to this phenomenon, with the peculiarity that the gene pool has been impacted by warfare and subsequent "colonization". In this study, using a comprehensive dataset for almost the entire Italian equine population, in addition to the most influential cosmopolitan breeds, we describe the current status of the modern Italian gene pool. Materials and Methods: The Italian dataset comprised 1,308 individuals and 22 breeds genotyped at a 70 k density that was merged with publicly available data to facilitate comparison with the global equine diversity. After quality control and supervised subsampling to ensure consistency among breeds, the merged dataset with the global equine diversity contained data for 1,333 individuals from 54 populations. Multidimensional scaling, admixture, gene flow, and effective population size were analyzed. Results and Discussion: The results show that some of the native Italian breeds preserve distinct gene pools, potentially because of adaptation to the different geographical contexts of the peninsula. Nevertheless, the comparison with international breeds highlights the presence of strong gene flow from renowned breeds into several Italian breeds, probably due to historical introgression. Coldblood breeds with stronger genetic identity were indeed well differentiated from warmblood breeds, which are highly admixed. Other breeds showed further peculiarities due to their breeding history. Finally, we observed some breeds that exist more on cultural, traditional, and geographical point of view than due to actual genetic distinctiveness., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Capomaccio, Ablondi, Colombi, Sartori, Giontella, Cappelli, Mancin, Asti, Mantovani, Sabbioni and Silvestrelli.)

Gut represents a major immunological defense barrier with mucosal immune system and intestinal epithelial cells (IECs). In all intestinal diseases, in particular inflammatory bowel disease (IBD), both the absorption and the local immune system are compromised and alternative effective therapies are sought after. Extracellular Vesicles (EVs) have the capability to regulate immune cells within the inflammatory microenvironment, by dampening inflammation and restoring intestinal barrier integrity. Recently, the immune-modulatory role of EVs has also been confirmed for milk EVs (mEVs), notable for their easy production, high sample volumes, cost-effective scalable production and non-toxic and non-immunogenic behavior. In this context, the aim of this study was to evaluate goat mEV anti-inflammatory and immuno-modulating effects on an in vitro model (IPEC-J2) of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release dosage with ELISA test. After the establishment of a pro-inflammatory environment due to LPS stimuli, IL6, CXCL8, IL12p35, IL12p40, IFNB, IL18, TLR7 and NOS2 resulted significantly up-regulated in stimulated IPEC-J2 cells compared to those of the basal culture. After 48 h of mEV treatment in inflamed IPEC-J2 a partial restoration of initial conditions was detected, with the IL18 and IL12p40 significant down-regulation, and IL12p35, EBI3, TLR7, BD1 and BD3 up-regulation. IL-18 reduced protein production was also detected in supernatants. Moreover, a decrease of MMP9 and NOS2 together with a strong up-regulation of MUC2 indicated a recovery of cellular homeostasis and, therefore, potential beneficial effects on the intestinal mucosa. Nevertheless, 48 h post-treatment, an increased gene expression and protein release of IL-8 was observed. This paper is one of the firsts to assess the effect of goat mEVs and the first one, in particular, of doing this on an in vitro model of gut inflammation. The obtained results show a potential capability of goat mEVs to modulate inflammation and to play beneficial effects on the intestinal mucosa., Competing Interests: Declaration of Competing Interest None., (Copyright © 2022 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Extracellular Vesicles (EVs) are nano-sized double-lipid-membrane-bound structures, acting mainly as signalling mediators between distant cells and, in particular, modulating the immune response and inflammation of targeted cells. Milk and colostrum contain high amounts of EVs that could be exploited as alternative natural systems in antimicrobial fighting. The aim of this study is to evaluate cow colostrum-derived EVs (colosEVs) for their antimicrobial, anti-inflammatory and immunomodulating effects in vitro to assess their suitability as natural antimicrobial agents as a strategy to cope with the drug resistance problem. ColosEVs were evaluated on a model of neonatal calf diarrhoea caused by Escherichia coli infection, a livestock disease where antibiotic therapy often has poor results. Colostrum from Piedmontese cows was collected within 24 h of calving and colosEVs were immediately isolated. IPEC-J2 cell line was pre-treated with colosEVs for 48 h and then infected with EPEC/NTEC field strains for 2 h. Bacterial adherence and IPEC-J2 gene expression analysis (RT-qPCR) of CXCL8, DEFB1, DEFB4A, TLR4, TLR5, NFKB1, MYD88, CGAS, RIGI and STING were evaluated. The colosEVs pre-treatment significantly reduced the ability of EPEC/NTEC strains to adhere to cell surfaces (p = 0.006), suggesting a role of ColosEVs in modulating host−pathogen interactions. Moreover, our results showed a significant decrease in TLR5 (p < 0.05), CGAS (p < 0.05) and STING (p < 0.01) gene expression in cells that were pre-treated with ColosEVs and then infected, thus highlighting a potential antimicrobial activity of ColosEVs. This is the first preliminarily study investigating ColosEV immunomodulatory and anti-inflammatory effects on an in vitro model of neonatal calf diarrhoea, showing its potential as a therapeutic and prophylactic tool.