Immune Response in Young Thoroughbred Racehorses under Training

Training has a great impact on the physiology of an athlete and, like all stressful stimuli, can trigger an innate immune response and inflammation, which is part of a wider coping strategy of the host to restore homeostasis. The Thoroughbred racehorse is a valid animal model to investigate these changes thanks to its homogeneous training and highly selected genetic background. The aim of this study was to investigate modifications of the innate immune response and inflammation in young untrained Thoroughbred racehorses during the first training season through haematological and molecular investigations. Twenty-nine Thoroughbred racehorses were followed during their incremental 3-month sprint exercise schedule. Blood collection was performed at time 0 (T0; before starting the intense training period), 30 days after T0 (T30), and 90 days after T0 (T90). Haematological parameters (red and white blood cells, haemoglobin, and platelets) were evaluated and haematocrit (HCT), mean corpuscular haemoglobin concentration (MCHC), and red cells width distribution + standard deviation (RDW-SD) were calculated. Moreover, via RT-qPCR, we investigated the expression of, Interleukin 1β (IL-1β), Interleukin 4 (IL-4) Interleukin 6 (IL-6), Interleukin 2 (IL-2), Interleukin 3 (IL-3), Interleukin 5 (IL-5) Interleukin 8 (IL-8), Trasformig Growth Factor β and α (TGF-β), Tumor necrosis factor α (TNF-α), and Interferon γ (IFN-γ)genes. Main corpuscular volume (MCV) showed a significant (p = 0.008) increase at T90. Main corpuscular haemoglobin (MCH) and haemoglobin concentration (MCHC) values were significantly augmented at both T30 (p < 0.001) and T90 (p < 0.001). Basophils were significant increased at T30 (p = 0.02) and eosinophils were significantly increased at T90 (p = 0.03). Significant differences in gene expression were found for all the genes under study, with the exception of IFN-γ and TNF-α. In particular, IL-2 (T30, p = 0.011; T90, p = 0.015), IL-4 (T30, p = 0.009; T90, p < 0.001), and IL-8 (T30, p < 0.001; T90, p < 0.001) genes were significantly upregulated at both T30 and T90 with respect to T0, TGF-β was intensely downregulated at T30 (p < 0.001), IL-5 gene expression was significantly decreased at T90 (p = 0.001), while IL-1β (p = 0.005) and IL-3 (p = 0.001) expression was strongly augmented at the same time. This study highlighted long-term adjustments of O2 transport capability that can be reasonably traced back to exercise adaptation. Moreover, the observed changes of granulocyte numbers and functions and inflammatory cytokine gene expression confirm a major role of the innate immune system in the response to the complex of stressful stimuli experienced during the training period

Draft genome sequences of four Yersinia enterocolitica strains, isolated from wild ungulate carcasses

This study describes the draft genome sequences of four Yersinia enterocolitica strains, originally isolated from ungulate carcasses. These isolates were typed biochemically and two were determined to be highly virulent (biotype 1B). The draft genome sequences had a mean size of 4.77 Mb and a mean G+C content of 47.1%

Epithelial to Mesenchymal Transition (EMT) in a Laryngeal Squamous Cell Carcinoma of a Horse: Future Perspectives

Squamous cell carcinoma (SCC) is one of the most frequent tumors of skin and muco-cutaneous junctions in the horse. Equine papillomavirus type 2 (EcPV2) has been detected in equine SCC of the oral tract and genitals, and recently also in the larynx. As human squamous cell carcinoma of the larynx (SCCL), it is strongly etiologically associated with high-risk papillomavirus (h-HPV) infection. This study focuses on tumor cells behavior in a naturally occurring tumor that can undergo the so-called epithelial to mesenchymal transition (EMT). A SCCL in a horse was investigated by immunohistochemistry using antibodies against E-cadherin, pan-cytokeratin AE3/AE1, β-catenin, N-cadherin, vimentin, ZEB-1, TWIST, and HIF-1α. EcPV2 DNA detection and expression of oncogenes in SCC were investigated. A cadherin switch and an intermediate filaments rearrangement within primary site tumor cells together with the expression of the EMT-related transcription factors TWIST-1, ZEB-1, and HIF-1α were observed. DNA obtained from the tumor showed EcPV2 positivity, with E2 gene disruption and E6 gene dysregulation. The results suggest that equine SCCL might be a valuable model for studying EMT and the potential interactions between EcPV2 oncoproteins and the EMT process in SCCL

Cross-species infectivity of H3N8 influenza virus in an experimental infection in swine

Avian influenza A viruses have gained increasing attention due to their ability to cross the species barrier and cause severe disease in humans and other mammal species as pigs. H3 and particularly H3N8 viruses, are highly adaptive since they are found in multiple avian and mammal hosts. H3N8 viruses have not been isolated yet from humans; however, a recent report showed that equine influenza A viruses (IAVs) can be isolated from pigs, although an established infection has not been observed thus far in this host. To gain insight into the possibility of H3N8 avian IAVs to cross the species barrier into pigs, in vitro experiments and an experimental infection in pigs with four H3N8 viruses from different origins (equine, canine, avian, and seal) were performed. As a positive control, an H3N2 swine influenza virus A was used. Although equine and canine viruses hardly replicated in the respiratory systems of pigs, avian and seal viruses replicated substantially and caused detectable lesions in inoculated pigs without previous adaptation. Interestingly, antibodies against hemagglutinin could not be detected after infection by hemagglutination inhibition (HAI) test with avian and seal viruses. This phenomenon was observed not only in pigs but also in mice immunized with the same virus strains. Our data indicated that H3N8 IAVs from wild aquatic birds have the potential to cross the species barrier and establish successful infections in pigs that might spread unnoticed using the HAI test as diagnostic tool.We thank Jaime Maldonado and HIPRA (Spain) for the A/Swine/Spain/ 54008/2004 (H3N2) strain, Edward J. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) IAV strain, T. M. Chambers and the University of Kentucky for the A/Equine/OH/1/03 (H3N8) IAV strain, and Hon Ip and the U.S. Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbor Seal/New Hampshire/179629/2011 (H3N8) IAV strains. We thank Sergio López, David Solanes, Francisco X. Abad, Jordi Alberola, Jaume Martorell, and Eduard J. Cunilleras for help in providing different samples and during the experimental infections, as well as the personnel in Cat3 laboratories and the animal house. We thank Adolfo García-Sastre for providing materials and for support as the principal investigator of the NIAID-funded Center for Research in Influenza Pathogenesis (HHSN266200700010C). The research leading to these results received funding from the European Community’s Seventh Framework Programme (FP7, 2007-2013), the Research Infrastructures Action under grant FP7-228393 (a NADIR project), and projects AGL2010-22200-C02-01 and AGL2007-60274 of the Spanish Ministry of Science and Innovation

Antioxidant Marine Hydrolysates Isolated from Tuna Mixed Byproducts: An Example of Fishery Side Streams Upcycling

publishedVersio

Evaluation and validation of an alternative method to detect Campylobacter spp. in dairy products

Foods implicated in human campylobacteriosis include raw or undercooked poultry and raw dairy products. Because Campylobacter spp. are the most frequently reported cause of bacterial infection in the European Union and because conventional methods are cumbersome, rapid methods for Campylobacter detection and quantification in food are needed. With this study we sought to validate, according to the standard procedure (UNI EN ISO 16140:2003), an alternative to the reference analytical method (UNI EN ISO 10272- 1:2006) for official controls of Campylobacter spp. in raw milk and dairy products. Milk samples collected from 16 milk vending machines located throughout the Genoa metropolitan area were analyzed using two different methods, an enzymelinked fluorescent assay (ELFA) and a real-time PCR assay, and evaluated in parallel against the reference method. In addition, a total of 460 samples of raw milk collected from milk vending machines were analyzed by ELFA. Results obtained with ELFA showed it was compliant with UNI EN ISO 10272-1:2006 criteria and that the immunoassay had 100% sensitivity, specificity, and accuracy. Regarding samples of milk vending machines, 5.0% (23/460) tested positive at ELFA screening and were subsequently confirmed as C. jejuni. Validation according to UNI EN ISO 16140:2003 of the ELFA method suggests it may be a useful alternative to conventional methods for detecting Campylobacter spp. in official controls

Genetic Characterization of African Swine Fever Italian Clusters in the 2022–2023 Epidemic Wave by a Multi-Gene Approach

The first report of African swine fever virus (ASFV) genotype II in Italy in 2022 marked the beginning of a significant invasion in at least eight Italian regions with different infection clusters. In this study, we used the multi-gene approach to investigate the epidemiological associations between ASFV strains causing cases and outbreaks in wild boar and pigs in Italy from January 2022 to the end of 2023. Our results confirm that all the tested ASFV-positive Italian samples belonged to genotype II and show high homology with genotype II ASFV sequences previously collected in Eurasian countries. Molecular characterization revealed the presence of four genetic groups in Italy. The majority of African swine fever (ASF) samples analyzed in the current study (72%) belonged to genetic group 3, which was the most representative in Europe. The results also provide evidence of the prevalence of genetic group 19 (15.9%). In addition, we identified new putative genetic groups, genetic group 25 (9.1%) and genetic group 26 (3.0%), which have never been described before. This is the first detailed report on the molecular characterization of more than 130 ASFV strains circulating in Italy

Goat milk extracellular vesicles: immuno-modulation effects on porcine monocyte-derived macrophages in vitro

IntroductionExtracellular 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.MethodsIn 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.ResultsThese 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.DiscussionOverall, 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

Expression dynamics of innate immunity in influenza virus-infected swine

We would like to thank Dr. Jaime Maldonado and HIPRA, Spain for the A/swine/Spain/54008/2004 (H3N2) influenza virus; Dr. Dubovi and Cornell University for the A/Canine/NY/105447/08 (H3N8) influenza virus; Dr. Chambers and University of Kentucky for the A/Equine/OH/1/03 (H3N8) influenza virus; and Dr. Hon Ip and the US Geological Survey National Wildlife Health Center for the A/American black duck/Maine/44411-532/2008 (H3N8) and the A/Harbour Seal/New Hampshire/179629/2011 (H3N8) influenza viruses. The authors thank Sergio López, David Solanes and Francisco X. Abad for their help during the experimental infections as well as the personnel in Cat3 laboratories and animal house. The authors also wish to thank Dr. I. L. Archetti (IZSLER, Brescia, Italy) for the invaluable help in measuring some clinical immunology parameters, Dr. L. Fraile (UdL, Spain) for assistance in statistical analysis, Dr. J. Domínguez (INIA, Spain) for porcine antibodies, Dr. M. Gennari and Dr. M. Giunta (S.S. Genova, IZSPLV, Italy) for assistance in real-time PCR analyses. The skillful technical assistance of Mrs. C. Mantovani (IZSLER, Brescia, Italy) is also gratefully acknowledged. The research leading to these results has received funding from: the European Community's Seventh Framework Programme (FP7, 2007-2013), Research Infrastructures action, under the grant agreement No. FP7-228393 (NADIR project), and from the project AGL2010-22200-C02-01 of Spanish Ministry of Science and Innovation.Geological Survey National Wildlife Health Center/[u'duck/Maine/44411-532/2008', u'H3N8']The current circulating swine influenza virus (IV) subtypes in Europe (H1N1, H1N2, and H3N2) are associated with clinical outbreaks of disease. However, we showed that pigs could be susceptible to other IV strains that are able to cross the species barrier. In this work, we extended our investigations into whether different IV strains able to cross the species barrier might give rise to different innate immune responses that could be associated with pathological lesions. For this purpose, we used the same samples collected in a previous study of ours, in which healthy pigs had been infected with a H3N2 Swine IV and four different H3N8 IV strains circulating in different animal species. Pigs had been clinically inspected and four subjects/group were sacrificed at 3, 6, and 21 days post infection. In the present study, all groups but mock exhibited antibody responses to IV nucleoprotein protein. Pulmonary lesions and high-titered viral replication were observed in pigs infected with the swine-adapted virus. Interestingly, pigs infected with avian and seal H3N8 strains also showed moderate lesions and viral replication, whereas equine and canine IVs did not cause overt pathological signs, and replication was barely detectable. Swine IV infection induced interferon (IFN)-alpha and interleukin-6 responses in bronchoalveolar fluids (BALF) at day 3 post infection, as opposed to the other non-swine-adapted virus strains. However, IFN-alpha responses to the swine-adapted virus were not associated with an increase of the local, constitutive expression of IFN-alpha genes. Remarkably, the Equine strain gave rise to a Serum Amyloid A response in BALF despite little if any replication. Each virus strain could be associated with expression of cytokine genes and/or proteins after infection. These responses were observed well beyond the period of virus replication, suggesting a prolonged homeostatic imbalance of the innate immune system