Immune response to ASFV and other pathogens.
Macrophage polarization in pigs and other farm animals.
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Scopus Publications
Scopus Publications
Dynamics of leukocyte populations, immune-regulatory cytokines, and biochemical parameters in wild boar and domestic pigs experimentally infected with a virulent African swine fever virus genotype II strain Giulia Franzoni, Fabian Zhi Xiang Lean, Emanuela Giaconi, Giuseppe Tedde, Susanna Zinellu, Paola Nicolussi, Mireille Le Dimna, Marie-Frédérique Le Potier, Elliot Steedman, Helen Rachel Crooke, Cecilia Righi, Stefano Petrini, Noemí Rayón, Dolores Gavier-Widen, Alejandro Núñez, Pedro Jose Sanchez-Cordon Frontiers in Immunology, 2026 African Swine Fever Virus (ASFV) is the causative agent of devastating disease affecting domestic and wild pigs globally. A previous study of the intranasal inoculation of domestic pigs (DP) and wild boar (WB) with the ASFV genotype II strain “Armenia 2007” demonstrated distinct disease outcomes. This study aims to compare the leukocyte, cytokine and biochemical profiles in experimentally infected DP and WB. Blood and serum samples were collected before infection (day 0), from animals euthanized in groups of six (comprising 3 DP and 3 WB) on days 1, 2, 3 and 5 post infection (pi) and from animals that reached a humane endpoint. Both DP and WB developed severe lymphopenia, occurring earlier in WB. Inflammatory response occurred earlier in WB, as evident from day 3 pi by the increased levels of TNF, followed by IL-6 and, to a lesser extent, IL-1β. IL-8 concentrations only increased in some WB, but not in DP. No modulation of Th1-associated cytokines (IFN-γ, IL-12 and IL-18) was detected in DP, whereas WB had a moderate increase in IL-12 and IFN-γ levels from day 5 pi, which peaked at humane endpoint. C-reactive protein levels increased in concomitant with the release of pro-inflammatory cytokines, as early as day 5 pi in WB, reaching its maximum in both DP and WB at the humane endpoint. A delayed but significant increase in the levels of anti-inflammatory mediators IL-1Ra and IL-10 was observed in both groups, but earlier in some WB from day 5 pi. Biochemical analysis revealed potential perturbations of the liver function in both subspecies, characterized by changes in serum AST and triglycerides levels, in addition to renal alterations in DP evidenced by changes in creatinine and urea levels. These findings underscore earlier immune activation in WB, potentially contributing to the different subspecies-specific disease outcomes following ASFV inoculation.
Targeting Toll-like Receptor 2: synthetic diacylated lipopeptides polarize equine macrophages towards a pro-inflammatory phenotype Chiara Grazia De Ciucis, Floriana Fruscione, Filippo Dell’Anno, Susanna Zinellu, Emanuela Giaconi, Simone Loi, Nicolò Columbano, Giulia Franzoni, Elisabetta Razzuoli Frontiers in Immunology, 2026 Toll-like receptors (TLRs) are a group of pattern recognition receptors (PRRs), that play critical roles in initiating host immune defenses. TLR-2 agonists can activate innate immune cells and thus are attracting increasing attention as prophylactic and/or therapeutic agents against infectious diseases or in cancer immunotherapy. In this work, the impact of three synthetic diacylated lipopeptides (Mag-Pam2Cys_P48, MagPam2Cys_P80, and Mag-Pam2Cys_MAG1000) on equine monocyte-derived macrophages (moMΦ) phenotype and functionality was thoroughly investigated. MoMΦ were generated in vitro from circulating monocytes, and they were stimulated with these TLR-2 agonists, alongside untreated controls. The immunomodulatory effect was evaluated by RT-qPCR (expression of key immune genes) and ELISA multiplex (release of cytokines). Subsequently, the impact of MagPam2Cys_P80 on the phenotype of cells stimulated with IL-4 or IL-10 (‘M2-related’ cytokines) was investigated. We observed that stimulation with the three synthetic diacylated lipopeptides polarizes moMΦ towards a pro-inflammatory phenotype, with enhanced induction/release of pro-inflammatory cytokines, but with lower intensity compared to classical activation (IFN-γ + LPS). No differences between these agonists were detected, thus one of them (Mag-Pam2Cys_P80) was selected for further experiments with moM(IL-4) or moM(IL-10). Our data revealed that MagPam2Cys_P80 triggered increased release of IL-8, but not IL-1β, from moM(IL-10) 24 h after stimulation. In addition, TNF release was not observed when cells were simultaneously stimulated with IL-10. These data suggest that the inflammatory activity evoked by those agonist compounds could be partially mitigated in vivo by the release of anti-inflammatory molecules (e.g. IL-10), avoiding a potentially harmful dysregulated inflammatory response.
A dual-gene-deleted ASFV Lv17/WB/Rie1-ΔCD candidate administered orally to wild boar confers DIVA-compatible protection against virulent challenge Jose A. Barasona, Aleksandra Kosowska, Gabriela González-García, Marta Díaz-Frutos, Giulia Franzoni, Paola Nicolussi, Nestor Porras, Mónica Sánchez-Segovia, Daniel De Antonio-Gómez, Paloma Rueda, Sandra Barroso-Arévalo Veterinary Quarterly, 2026 TCID₅₀). Animals were challenged intramuscularly with the virulent Armenia07 genotype II strain. The ΔCD vaccine was well tolerated, inducing only transient low-grade fever. Prime-re-exposure vaccination induced earlier seroconversion (mean 12 ± 4 dpv) and sterilizing immunity in 5/6 animals in the high dose group. Overall protection reached 90%, while all unvaccinated controls died within 7 days. Quantitative PCR revealed >10³-fold reductions in viral genome copies in blood and tissues versus controls. DIVA ELISA reliably distinguished vaccine-induced antibodies from infection-derived responses. These findings identify Lv17/WB/Rie1-ΔCD as a safer oral ASFV vaccine candidate, addressing concerns raised with the parental Lv17/WB/Rie1 by increasing attenuation and supporting multi-gene deletion strategies. Further studies on safety, transmission, genetic stability, and environmental behaviour are required before large-scale field trials.
Correlating milk cytokines and somatic cell counts to intramammary infections in dairy sheep Giulia Franzoni, Susanna Zinellu, Vittoria D’Ascenzo, Emanuela Giaconi, Giovanni Vito Denti, Marina Adele Lucia Manai, Angelo Fiori, Silvia Dei Giudici, Riccardo Bazzardi, Sara Casu, Antonello Carta, Ilaria Fadda, Ciriaco Ligios, Simone Dore Frontiers in Veterinary Science, 2026 Mastitis represents an important health problem in dairy sheep and somatic cell count (SCC) is frequently used as indicator of subclinical intra-mammary infection. Cytokines might represent another reliable and sensitive tool for defining the inflammatory status in relation with the SCC, thus, the levels of 12 key immune cytokines were monitored in ovine milk samples with different SCC values. First, samples were divided into five SCC-groups: group 1 (0–300 cell/mL*1000, n = 16), group 2 (300–500 cell/mL*1000, n = 16), group 3 (500–1,000 cell/mL*1000, n = 16), group 4 (1000–2000 cell/mL*1000, n = 15), group 5 (> 2000 cell/mL*1000, n = 16). Samples belonging to group 5 presented the highest values of IL-1α, IL- β , IL-6, MIP-1α, MIP-1β, IFN- γ , IL-17, IL-10. Samples belonging to group 1 presented IL-4 levels higher than to those belonging to groups 2–3-4, and lower IL-1α and MIP-1α values compared to groups 3–4. Next, cluster analysis was performed. Three clusters were defined: cluster 1 (samples with low SCC values negative to bacterial culture), cluster 2 (samples positive to culture isolation with intermediate SCC values), cluster 3 (samples positive to culture isolation and with high SCC levels). Samples in cluster 1 were characterised by low levels of IFN- γ , IL-1α, IL-1β, IL-6, MIP-1α, whereas samples in cluster 3 presented the highest values of these five cytokines. Samples in cluster 2 presented higher levels of IL-1α and MIP-1α compared to cluster 1, as well as lower levels of IL-4. MIP-1α and IL-1α showed the strongest correlation with SCC values. Overall, our data revealed that intra-mammary infection in dairy sheep was correlated with high levels of pro-inflammatory cytokines in milk samples, which reflect the presence of an inflammatory reaction and confirm the utility of SCC determination in the diagnosis of subclinical mastitis.
Exploring alternative cytokines as potential biomarkers for Mycobacterium bovis infection in cattle Giulia Franzoni, Federica Signorelli, Grazia Carbotti, Anna Donniacuo, Lorena Schiavo, Susanna Zinellu, Emanuela Giaconi, Pasqualino Cappuccio, Mauro Nitti, Orlando Paciello, Giuseppe Iovane, Francesco Napolitano, Maria Beatrice Boniotti, Alessandra Martucciello Frontiers in Immunology, 2026 Mycobacterium bovis ( M. bovis ) is the primary agent of Bovine tuberculosis (bTB) in cattle. It represents both a threat to human health and the cattle industry worldwide. Improving bTB diagnostic performance in cattle represents a key step in eradicating the disease. The interferon-gamma (IFN-γ) release (IGRA) blood assay is routinely used in the diagnosis of M. bovis infection, but additional cytokines might be useful as biomarkers of this infection in cattle. In our study, we evaluated the utility of sixteen immune cytokines as diagnostic biomarkers of M. bovis infection. Fifty-five cattle were used in this study: healthy animals (N = 19), infected (IFN-γ test positive, no post-mortem lesions; N = 17), and affected (IFN-γ test positive, visible post-mortem lesions; N = 19). Heparin blood samples were stimulated in vitro with bovine purified protein derivative (PPD-B), alongside controls. After 18–24 h of incubation, plasma were collected and levels of 16 key cytokines were measured: IL-1α, IL-4, IL-6, IL-10, IL-17, IL-36Ra, MIP-1α, IP-10, MCP-1, TNF, VEGF-A, IFN-γ, IL-23, IL-27, IL-35, and THBS-1. We observed that both M. bovis exposed cattle (both infected and affected) released higher levels of PPD-B specific IFN-γ and IP-10. On the contrary, only cattle belonging to the affected group released higher levels of PPD-B specific IL-4, IL-17, and TNF compared to healthy subjects. Canonical discriminant analyses (CDA) indicated that IP-10, IL-4, IL-17, and TNF could be useful biomarkers for infection status. In particular, our data suggest that the parallel measurement of IFN-γ and IP-10 might improve the diagnosis of M. bovis infection in cattle in terms of sensitivity and specificity, although this should be validated on a larger set of animals. In the CDA analysis, only a modest separation between infected and affected cattle was observed. Nevertheless, our data suggested that IL-4, IL-10, and TNF might improve, at least in part, the differentiation of cattle in diverse stages of TB infection. Overall, the data generated in our study provide a foundation to improve the diagnosis and staging of M. bovis in cattle.
Characterisation of Cell-Mediated Immunity Against Bovine Alphaherpesvirus 1 (BoAHV-1) in Calves Giulia Franzoni, Cecilia Righi, Immacolata De Donato, Giovanna Cappelli, Giovanna De Matteis, Eleonora Scoccia, Giulia Costantino, Emanuela Giaconi, Susanna Zinellu, Carlo Grassi, Alessandra Martucciello, Francesco Grandoni, Stefano Petrini Vaccines, 2025 Background: Bovine alphaherpesvirus 1 (BoAHV-1) is a major respiratory and reproductive pathogen in cattle worldwide. Both innate and adaptive immune responses contribute to protection against this virus; however, virus-host interactions remain partly undefined. In this study, the impact of BoAHV-1 infection on calves’ immune responses was investigated in detail. Methods: Six calves were intranasally infected with wild-type BoAHV-1, and blood samples were collected longitudinally. Leukocyte subset dynamics were assessed by complete haematological assay and flow cytometry, while multiplex ELISA was used to quantify serum levels of ten cytokines. For each parameter, post-infection values (days 2, 4, 8, 10, and 14) were compared with pre-infection baseline values (day 0). Results: Infection induced an initial phase of immunosuppression, reflected by decreased circulating αβ and γδ-T cells. However, infected animals rapidly developed a protective immune response, characterised by increased circulating classical and intermediate monocytes and elevated levels of the related chemokine MIP-1β. Early post-infection, rises in serum IFN-γ and IL-10 were also detected. Conclusions: Our data suggest that monocyte recruitment and increased serum levels of IFN-γ and IL-10 are positively associated with the ability to overcome infection. A better understanding of the immunopathogenic mechanisms underlying BoAHV-1 infection will support the development of more effective vaccines against this virus.
Dynamics of Biochemical Parameters, Inflammatory and Stress Markers in Lambs Undergoing Caudectomy Using Two Different Methods Giovannantonio Pilo, Giuseppe Tedde, Angelo Peli, Pier Attilio Accorsi, Gavina Carta, Loredana Secchi, Giulia Franzoni, Paola Nicolussi Animals, 2025 Zootechnical practices such as tail docking are still in use in dairy sheep farming, performed in the first week of life, mainly by rubber ring and only rarely by surgical methods. In this study, we evaluated the impact of caudectomy on ovine stress levels, inflammation, and health status by comparing tail docking carried out using rubber rings or surgical amputation. Twenty-one lambs were randomly selected and equally allocated into three groups: controls (n = 7), lambs with tail cut by rubber rings (n = 7), and lambs with caudectomy performed by surgical practice (n = 7). Several biochemical parameters and inflammatory markers were monitored at different times post-caudectomy, as well as wool levels of the stress marker cortisol. Our data revealed that lambs that underwent tail docking by rubber rings, but not by surgical procedure, presented inflammation and stress, as well as a moderate increase in muscular damage markers. These results are useful for the evaluation of animal welfare in dairy sheep that underwent caudectomy, highlighting the need to re-evaluate this procedure, as well as the ways in which it is performed.
Biocompatibility of Water-Dispersible Pristine Graphene and Graphene Oxide Using a Close-to-Human Animal Model: A Pilot Study on Swine Paola Nicolussi, Giovannantonio Pilo, Maria Giovanna Cancedda, Guotao Peng, Ngoc Do Quyen Chau, Alejandro De la Cadena, Renzo Vanna, Yarjan Abdul Samad, Tanweer Ahmed, Jeremia Marcellino, Giuseppe Tedde, Linda Giro, Acelya Ylmazer, Federica Loi, Gavina Carta, Loredana Secchi, Silvia Dei Giudici, Simona Macciocu, Dario Polli, Yuta Nishina, Ciriaco Ligios, Giulio Cerullo, Andrea Ferrari, Alberto Bianco, Bengt Fadeel, Giulia Franzoni, Lucia Gemma Delogu Advanced Healthcare Materials, 2025 Graphene‐based materials (GBMs) are of considerable interest for biomedical applications, and the pilot study on the toxicological and immunological impact of pristine graphene (GR) and graphene oxide (GO) using swine as a close‐to‐human provides valuable insights. First, ex vivo experiments are conducted on swine blood cells, then GBMs are injected intraperitoneally (i.p.) into swine. Hematological and biochemical analyses at various intervals indicate that neither GO nor GR cause systemic inflammation, pro‐coagulant responses, or renal or hepatic dysfunction. Importantly, no systemic toxicity is observed. Analysis of a panel of 84 immune‐related genes shows minimal impact of GO and GR. The animals are sacrificed 21 days post‐injection, and transient absorption imaging and Raman mapping show the presence of GO and GR in the mesentery only. Histological evaluation reveals no signs of alterations in other organs. Thus, clusters of both materials are detected in the mesentery, and GO aggregates are surrounded only by macrophages with the formation of granulomas. In contrast, modest local reactions are observed around the GR clusters. Overall, these results reveal that i.p. injection of GBMs resulted in a modest local tissue reaction without systemic toxicity. This study, performed in swine, provides essential guidance for future biomedical applications of graphene.
Modulation of antiviral genes by African swine fever isolates of diverse virulence Giulia Franzoni, Lorena Mura, Susanna Zinellu, Pedro J. Sanchez-Cordon, Miriam Pedrera, Silvia Dei Giudici Frontiers in Veterinary Science, 2025 African swine fever virus (ASFV), the aetiological agent of a devastating swine disease, has developed several strategies to replicate in porcine macrophages, its main target cells. In this work, we investigated the expression of 84 antiviral genes in macrophages infected with the virulent strain 26544/OG10 or the attenuated strain NH/P68. Infection with both strains caused an early activation of antiviral defenses, with up-regulation of RNA-sensing molecules and interferon-stimulating genes. However, as viral replication progresses, down-regulation of key inflammatory genes was observed, especially during infection with NH/P68, suggesting an impairment of macrophages' inflammatory response. Data generated provide a better portrait of ASFV immune evasion strategies.
Polyfunctionality of CD4+ T lymphocytes in buffaloes and cattle: comparative antigen-specific cytokine responses in bovine tuberculosis infection Susana Flores-Villalva, Giovanna De Matteis, Francesco Grandoni, Maria Carmela Scatà, Anna Donniacuo, Lorena Schiavo, Giulia Franzoni, Piera Mazzone, Mahmoud Elnaggar, Esterina De Carlo, Giorgio Galiero, William C. Davis, Alessandra Martucciello Frontiers in Immunology, 2025 IntroductionBovine tuberculosis (BTB), caused by Mycobacterium bovis, is a chronic infectious disease of major veterinary and public health concern. It affects a broad range of domestic and wild animals, including water buffalo, and poses a risk to humans due to its zoonotic nature. The economic consequences of BTB, arising from production losses and trade restrictions, further underline its global importance. While cattle immune responses to BTB are well characterized, the immune mechanisms in buffalo remain poorly understood, despite their increasing role as livestock in endemic regions. Given that buffaloes and cattle, although closely related, display notable immunological differences, comparative studies are essential. This study aimed to investigate and compare antigen-specific cytokine responses in CD4+ T lymphocytes from buffaloes and cattle exposed to or infected with M. bovis.MethodsA multicolor flow cytometry assay was established to enable high-resolution analysis of cytokine-expressing CD4+ T cells. Blood samples were obtained from 35 buffaloes (17 IGRA-positive from BTB outbreak farms and 18 IGRA-negative, including animals from both outbreak and Officially Tuberculosis-Free [OTF] herds) and 10 cattle (6 IGRA-positive from a BTB outbreak farm and 4 IGRA-negative from an OTF herd). Following six hours of in vitro stimulation with PPD-B or PBS, intracellular cytokine staining was performed. This approach allowed simultaneous quantification of single and polyfunctional CD4+ T cell subsets producing IFN-γ, TNF-α, and IL-17A. Data were analyzed using factor analysis of mixed data (FAMD) to explore species- and infection-related immune response patterns.ResultsThe multicolor flow cytometry approach successfully identified distinct cytokine-producing CD4⁺ T cell populations in both species. Overlapping immune profiles were observed between buffaloes and cattle; however, specific subsets—including IL-17A+, IFN-γ+IL-17A+, and TNF-α+IL-17A+ cells—contributed to interspecies differences. Importantly, the frequency of IFN-γ+ and TNF-α+ producing CD4+ T cells correlated with IGRA test status, enabling discrimination between infected/exposed and non-infected animals. These results demonstrate the ability of cytokine expression patterns to reflect both infection status and host species.DiscussionThe findings indicate that buffaloes and cattle share broadly similar antigen-specific cytokine responses, although subtle differences in CD4⁺ T cell subsets exist. The study highlights the value of multicolor flow cytometry as a high-resolution tool for dissecting immune responses in veterinary immunology. These insights enhance understanding of buffalo immune mechanisms against BTB and may contribute to improved disease control strategies.
First Molecular Characterisation of Porcine Parvovirus 7 (PPV7) in Italy Silvia Dei Giudici, Lorena Mura, Piero Bonelli, Luca Ferretti, Salwa Hawko, Giulia Franzoni, Pier Paolo Angioi, Anna Ladu, Graziella Puggioni, Elisabetta Antuofermo, Maria Luisa Sanna, Giovanni Pietro Burrai, Annalisa Oggiano Viruses, 2024
The Long-Jumping of African Swine Fever: First Genotype II Notified in Sardinia, Italy Silvia Dei Giudici, Federica Loi, Sonia Ghisu, Pier Paolo Angioi, Susanna Zinellu, Mariangela Stefania Fiori, Francesca Carusillo, Diego Brundu, Giulia Franzoni, Giovanni Maria Zidda, Paolo Tolu, Ennio Bandino, Stefano Cappai, Annalisa Oggiano Viruses, 2024
Host Response of Syrian Hamster to SARS-CoV-2 Infection including Differences with Humans and between Sexes Martina Castellan, Gianpiero Zamperin, Giulia Franzoni, Greta Foiani, Maira Zorzan, Petra Drzewnioková, Marzia Mancin, Irene Brian, Alessio Bortolami, Matteo Pagliari, Annalisa Oggiano, Marta Vascellari, Valentina Panzarin, Sergio Crovella, Isabella Monne, Calogero Terregino, Paola De Benedictis, Stefania Leopardi Viruses, 2023
