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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.

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.

Papillomavirus (PV) infections may be related to anogenital lesions and cancer development in humans and several other animal species. To date, 11 different PVs have been reported in horses. Among them, a newly described PV named Equus caballus Papillomavirus Type9 (EcPV9) was thus far only reported in the semen of a stallion with penile lesions in Australia. This study reports for the first time the presence of EcPV9 in asymptomatic Italian horses. From July 2020 to January 2022, genital brush samples were collected from 209 horses with no apparent signs of neoplastic disease and no PV-associated lesions, clinically examined at the Didactic Veterinary University Hospital (OVUD) of Perugia and at the Veterinary University Hospital (OVU) of Turin. Brushes were submitted to real-time PCR targeting the EcPV9- L1 region. The first amplification targeted a region of ~116 bp, followed by the amplification and sequencing of ~533 bp of the positive samples. EcPV9- L1 DNA was found in eleven horses (5.3%), all female and mainly English Thoroughbred. Co-infection with EcPV2- L1 was found in 7 out of the 11 EcPV9- L1 positive horses (63.6%). This study contributes to the description of the prevalence of exposure or infection of EcPVs in the horse population in Italy, for which data are still limited. In this regard, here we provide a phylogenetic analysis and the completely reconstructed viral genomes of two Italian EcPV type 9 isolates, as well as four EcPV type 2 obtained from co-infected animals.

Equine Papillomavirus 2 (EcPV2) is responsible for squamous cell carcinomas (eSCCs) of external genitalia of both male and female horses. However, few studies report the EcPV2 prevalence among healthy horses. Currently, the lack of these data does not permit identifying at-risk populations and, thus, developing screening protocols aimed at the early detection of the infection, as for humans. The aim of our study was to estimate the genoprevalence of EcPV2 in clinically healthy horses in Italy and to evaluate their innate immune response. For this purpose, penile and vulvar swabs of 234 healthy horses were collected through sampling with sterile cytobrushes. Nucleic acids were isolated and EcPV2- L1 presence (DNA) and gene expression (RNA) were checked by RT-qPCR. Our results showed EcPV2- L1 DNA presence in 30.3% of the samples and L1 expression in 48% of the positive samples. No statistically significant differences were found in genoprevalence in relation to sex, age, and origin, while, concerning breeds, the Thoroughbred had the highest risk of infection. Concerning specifically the mares, 40.2% of them resulted in being positive for EcPV2; our findings show a major positivity in pluriparous ( p = 0.0111) and mares subjected to natural reproduction ( p = 0.0037). Moreover, samples expressing L1 showed an increased expression of IL1B ( p = 0.0139) and IL12p40 ( p = 0.0133) and a decreased expression of RANKL (p = 0.0229) and TGFB (p = 0.0177). This finding suggests the presence of an effective immune response, which could explain the low incidence of SCCs in positive horses, despite a high EcPV2 genoprevalence (30%).

Physical exercise has been associated with the modulation of micro RNAs (miRNAs), actively released in body fluids and recognized as accurate biomarkers. The aim of this study was to measure serum miRNA profiles in 18 horses taking part in endurance competitions, which represents a good model to test metabolic responses to moderate intensity prolonged efforts. Serum levels of miRNAs of eight horses that were eliminated due to metabolic unbalance (Non Performer-NP) were compared to those of 10 horses that finished an endurance competition in excellent metabolic condition (Performer-P). Circulating miRNA (ci-miRNA) profiles in serum were analyzed through sequencing, and differential gene expression analysis was assessed comparing NP versus P groups. Target and pathway analysis revealed the up regulation of a set of miRNAs (of mir-211 mir-451, mir-106b, mir-15b, mir-101-1, mir-18a, mir-20a) involved in the modulation of myogenesis, cardiac and skeletal muscle remodeling, angiogenesis, ventricular contractility, and in the regulation of gene expression. Our preliminary data open new scenarios in the definition of metabolic adaptations to the establishment of efficient training programs and the validation of athletes' elimination from competitions.

Milk extracellular vesicles (mEVs) seem to be one of the main maternal messages delivery systems. Extracellular vesicles (EVs) are micro/nano-sized membrane-bound structures enclosing signaling molecules and thus acting as signal mediators between distant cells and/or tissues, exerting biological effects such as immune modulation and pro-regenerative activity. Milk is also a unique, scalable, and reliable source of EVs. Our aim was to characterize the RNA content of cow, donkey, and goat mEVs through transcriptomic analysis of mRNA and small RNA libraries. Over 10,000 transcripts and 2000 small RNAs were expressed in mEVs of each species. Among the most represented transcripts, 110 mRNAs were common between the species with cow acting as the most divergent. The most represented small RNA class was miRNA in all the species, with 10 shared miRNAs having high impact on the immune regulatory function. Functional analysis for the most abundant mRNAs shows epigenetic functions such as histone modification, telomere maintenance, and chromatin remodeling for cow; lipid catabolism, oxidative stress, and vitamin metabolism for donkey; and terms related to chemokine receptor interaction, leukocytes migration, and transcriptional regulation in response to stress for goat. For miRNA targets, shared terms emerged as the main functions for all the species: immunity modulation, protein synthesis, cellular cycle regulation, transmembrane exchanges, and ion channels. Moreover, donkey and goat showed additional terms related to epigenetic modification and DNA maintenance. Our results showed a potential mEVs immune regulatory purpose through their RNA cargo, although in vivo validation studies are necessary.

Equine penile squamous cell carcinoma (epSCC) is the most frequent tumor of the external male genitalia, representing 67.5% of equine genital cancers. epSCC is associated with papilloma virus (PV) infection and has been recently proposed as a model for human PV-induced squamous cell carcinomas. It has already been suggested that epSCC might undergo epithelial-to-mesenchymal transition (EMT). This work aims to investigate in detail this process and the possible role of PV oncoproteins in epSCC. For this purpose, 18 penile SCCs were retrospectively selected and tested for both EcPV2 presence and oncoproteins (EcPV2 E6 and EcPV2 E7) expression. Moreover, immunohistochemical EMT characterization was carried out by analyzing the main epithelial markers (E-cadherin, β-catenin, and pan-cytokeratin AE3/AE1), the main mesenchymal markers (N-cadherin and vimentin), and the main EMT-related transcription factors (TWIST-1, ZEB-1). PCR analysis was positive for EcPV2 in 16 out of 18 samples. EMT was investigated in epSCC positive for EcPV2. The immunohistochemistry results suggested the presence of EMT processes in the neoplastic cells at the tumor invasive front. Moreover, the significant upregulation of RANKL , together with BCATN1 , LEF1 , and FOSL1 genes, might suggest a canonical Wnt pathway activation, similarly to what is reported in human penile squamous cell carcinomas.

Stress in livestock reduces productivity and is a welfare concern. At a physiological level, stress is associated with the activation of inflammatory responses and increased levels of harmful reactive oxygen species. Biomarkers that are indicative of stress could facilitate the identification of more stress-resilient animals. We examined twenty-one metabolic, immune response, and liver function biomarkers that have been associated with stress in 416 Italian Simmental and 436 Italian Holstein cows which were genotyped for 150K SNPs. Single-SNP and haplotype-based genome-wide association studies were carried out to assess whether the variation in the levels in these biomarkers is under genetic control and to identify the genomic loci involved. Significant associations were found for the plasma levels of ceruloplasmin ( Bos taurus chromosome 1-BTA1), paraoxonase (BTA4) and γ-glutamyl transferase (BTA17) in the individual breed analysis that coincided with the position of the genes coding for these proteins, suggesting that their expression is under cis-regulation. A meta-analysis of both breeds identified additional significant associations with paraoxonase on BTA 16 and 26. Finding genetic associations with variations in the levels of these biomarkers suggests that the selection for high or low levels of expression could be achieved rapidly. Whether the level of expression of the biomarkers correlates with the response to stressful situations has yet to be determined.

Equine genital squamous cell carcinomas (egSCCs) are among the most common equine tumors after sarcoids, severely impairing animal health and welfare. Equus caballus papillomavirus type 2 (EcPV2) infection is often related to these tumors. The aim of this study was to clarify the molecular mechanisms behind egSCCs associated with EcPV2 infection, investigating receptor activator of nuclear factor-kappa B ligand (RANKL) signaling in NF-kB pathway, together with the Wnt and IL17 signaling pathways. We analyzed the innate immune response through gene expression evaluation of key cytokines and transcription factors. Moreover, Ki67 index was assessed with immunohistochemistry. EcPV2- E6 DNA was checked, and viral presence was confirmed in 21 positive out to 23 cases (91%). Oncogene expression was confirmed in 14 cases (60.8%) for E6 and in 8 (34.7%) for E2 . RANKL , nuclear factor kappa-light-chain-enhancer of activated B cells ( NFKB )- p50 , NFKBp65 , interleukin (IL)-6 , IL17 , IL23p19 , IL8 , IL12p35 , IL12p40 , β-catenin (BCATN1) , FOS like 1 (FOSL1) , and lymphoid enhancer binding factor 1 (LEF1) showed a significant upregulation in tumor samples compared to healthy tissues. Our results describe an inflammatory environment characterized by the activation of RANKL/RANK and IL17 with the relative downstream pathways, and a positive modulation of inflammatory cytokines genes such as IL6 and IL8 . Moreover, the increase of BCATN1 , FOSL1 , and LEF1 gene expression suggests an activation of both canonical and non-canonical Wnt signaling pathway that could be critical for carcinogenesis and tumor progression.

Penile squamous cell carcinomas (SCCs) are common tumors in older horses, with poor prognosis mostly due to local invasion and recurrence. These tumors are thought to be mainly caused by Equus caballus papillomavirus type 2 (EcPV-2). The aim of this study is to characterize the tumor immune environment (TIME) in equine penile tumors. Equine penile epithelial tumors (17 epSCCs; 2 carcinomas in situ , CIS; 1 papilloma, P) were retrospectively selected; immune infiltrate was assessed by histology and immunohistochemistry; RT-qPCR tested the expression of selected chemokines and EcPV-2 DNA and RNA. The results confirmed EcPV-2-L1 DNA in 18/20 (90%) samples. L1 expression was instead retrieved in 13/20 cases (65%). The samples showed an increased infiltration of CD3 + lymphocytes, macrophages (MAC387; IBA1), plasma cells (MUM1), and FoxP3 + lymphocytes in the intra/peritumoral stroma when compared to extratumoral tissues ( p < 0.05). Only MAC387 + neutrophils were increased in EcPV-2 high viral load samples ( p < 0.05). IL12 / p35 was differentially expressed in EcPV high and EcPV low groups ( p = 0.007). A significant decrease of IFNG and IL2 expression was highlighted in TGFB1 -positive samples ( p < 0.05). IBA1 and CD20 were intratumorally increased in cases where IL-10 was expressed ( p < 0.005). EpSCCs may represent a good spontaneous model for the human counterpart. Further prospective studies are needed in order to confirm these preliminary results.

Physical exercise is universally recognized as stressful. Among the "sport species", the horse is probably the most appropriate model for investigating the genomic response to stress due to the homogeneity of its genetic background. The aim of this work is to dissect the whole transcription modulation in Peripheral Blood Mononuclear Cells (PBMCs) after exercise with a time course framework focusing on unexplored regions related to introns and intergenic portions. PBMCs NGS from five 3 year old Sardinian Anglo-Arab racehorses collected at rest and after a 2000 m race was performed. Apart from differential gene expression ascertainment between the two time points the complexity of transcription for alternative transcripts was identified. Interestingly, we noted a transcription shift from the coding to the non-coding regions. We further investigated the possible causes of this phenomenon focusing on genomic repeats, using a differential expression approach and finding a strong general up-regulation of repetitive elements such as LINE. Since their modulation is also associated with the "exonization", the recruitment of repeats that act with regulatory functions, suggesting that there might be an active regulation of this transcriptional shift. Thanks to an innovative bioinformatic approach, our study could represent a model for the transcriptomic investigation of stress.