Evidence of Porcine Circovirus Type 2 (PCV2) Genetic Shift from PCV2b to PCV2d Genotype in Sardinia, Italy.

Porcine Circovirus type 2 (PCV2) is the etiological agent of a disease syndrome named Porcine Circovirus disease (PCVD), representing an important threat for the pig industry. The increasing international trade of live animals and the development of intensive pig farming seem to have sustained the spreading of PCVD on a global scale. Recent classification criteria allowed the identification of nine different PCV2 genotypes (PCV2a-i). PCV2a was the first genotype detected with the highest frequency from the late 1990s to 2000, which was then superseded by PCV2b (first genotype shift). An ongoing genotype shift is now determining increasing prevalence rates of PCV2d, in replacement of PCV2b. In Italy, a complete genotype replacement was not evidenced yet. The present study was carried out on 369 samples originating from domestic pigs, free-ranging pigs, and wild boars collected in Sardinia between 2020 and 2022, with the aim to update the last survey performed on samples collected during 2009-2013. Fifty-seven complete ORF2 sequences were obtained, and the phylogenetic and network analyses evidenced that 56 out of 57 strains belong to the PCV2d genotype and only one strain to PCV2b, thus showing the occurrence of a genotype shift from PCV2b to PCV2d in Sardinia.

The Synergic Role of Emerging and Endemic Swine Virus in the Porcine Respiratory Disease Complex: Pathological and Biomolecular Analysis.

Porcine respiratory disease complex (PRDC) represents a significant threat to the swine industry, causing economic losses in pigs worldwide. Recently, beyond the endemic viruses PRRSV and PCV2, emerging viruses such as TTSuV, PCV3, and PPV2, have been associated with PRDC, but their role remains unclear. This study investigates the presence of PCV2 and PRRSV and emerging viruses (PCV3, TTSuV, and PPV2) in the lungs of swine belonging to different age groups by histopathology and real-time PCR. The prevalent lung lesion was interstitial pneumonia with increased severity in post-weaning pigs. PRRSV was detected in 33% of piglets' lungs and in 20% of adults and post-weaning pigs with high Ct, while PCV2 was found in 100% of adult pigs, 33% of post-weaning pigs, and 22% of piglets, with low Ct in post-weaning pigs. PCV3 was present in all categories and coexisted with other viruses. TTSuV was detected in all swine in combination with other viruses, possibly influencing the disease dynamics, while PPV2 was detected in 100% of adults' and 90% of piglets' lungs. The detection of TTSuV, PCV3, and PPV2 in affected pigs prioritizes the need for comprehensive approaches in implementing appropriate control measures and minimizing economic losses associated with PRDC.

Evaluation of Haematological and Immunological Parameters of the ASFV Lv17/WB/Rie1 Strain and Its Derived Mutant Lv17/WB/Rie1/d110-11L against ASFV Challenge Infection in Domestic Pigs.

African swine fever virus (ASFV) is the etiological agent of a haemorrhagic disease that threatens the global pig industry. There is an urgency to develop a safe and efficient vaccine, but the knowledge of the immune-pathogenetic mechanisms behind ASFV infection is still very limited. In this paper, we evaluated the haematological and immunological parameters of domestic pigs vaccinated with the ASFV Lv17/WB/Rie1 strain or its derived mutant Lv17/WB/Rie1/d110-11L and then challenged with virulent Armenia/07 ASFV. Circulating levels of C-reactive protein (CRP), 13 key cytokines and 11 haematological parameters were evaluated throughout the study. Lv17/WB/Rie1 triggered an inflammatory response, with increased levels of CRP and pro-inflammatory cytokines, and induced lymphopenia, thrombocytopenia and a decline in red blood cell (RBC) parameters, although this was transitory. Lv17/WB/Rie1/d110-11L triggered only transitory thrombocytopenia and a mild inflammatory reaction, with no increase in serum levels of pro-inflammatory cytokines, but it raised IL-1Ra levels. Both strains counteracted several adverse reactions elicited by virulent challenge, like thrombocytopenia, a decline in RBC parameters, and inflammation. Within this paper, we provided a deep portrayal of the impact of diverse ASFV strains on the domestic pig's immune system. A better understanding of these immune-pathological mechanisms would help to design suitable vaccines against this disease.

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

[This corrects the article DOI: 10.3389/fimmu.2023.1209898.].

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

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.

Heterogeneity of Phenotypic and Functional Changes to Porcine Monocyte-Derived Macrophages Triggered by Diverse Polarizing Factors In Vitro.

Swine are attracting increasing attention as a biomedical model, due to many immunological similarities with humans. However, porcine macrophage polarization has not been extensively analyzed. Therefore, we investigated porcine monocyte-derived macrophages (moMΦ) triggered by either IFN-γ + LPS (classical activation) or by diverse "M2-related" polarizing factors: IL-4, IL-10, TGF-ß, and dexamethasone. IFN-γ and LPS polarized moMΦ toward a proinflammatory phenotype, although a significant IL-1Ra response was observed. Exposure to IL-4, IL-10, TGF-ß, and dexamethasone gave rise to four distinct phenotypes, all antithetic to IFN-γ and LPS. Some peculiarities were observed: IL-4 and IL-10 both enhanced expression of IL-18, and none of the "M2-related" stimuli induced IL-10 expression. Exposures to TGF-ß and dexamethasone were characterized by enhanced levels of TGF-ß2, whereas stimulation with dexamethasone, but not TGF-ß2, triggered CD163 upregulation and induction of CCL23. Macrophages stimulated with IL-10, TGF-ß, or dexamethasone presented decreased abilities to release proinflammatory cytokines in response to TLR2 or TLR3 ligands: IL-10 showed a powerful inhibitory activity for CXCL8 and TNF release, whereas TGF-ß provided a strong inhibitory signal for IL-6 production. While our results emphasized porcine macrophage plasticity broadly comparable to human and murine macrophages, they also highlighted some peculiarities in this species.

Development of molecular and antigenic-based rapid tests for the identification of African swine fever virus in different tissues.

African swine fever (ASF) is a severe haemorrhagic infectious disease affecting suids, thus representing a great economic concern. Considering the importance of the early diagnosis, rapid point of care testing (POCT) for ASF is highly demanded. In this work, we developed two strategies for the rapid onsite diagnosis of ASF, based on Lateral Flow Immunoassay (LFIA) and Recombinase Polymerase Amplification (RPA) techniques. The LFIA was a sandwich-type immunoassay exploiting a monoclonal antibody directed towards the p30 protein of the virus (Mab). The Mab was anchored onto the LFIA membrane to capture the ASFV and was also labelled with gold nanoparticles for staining the antibody-p30 complex. However, the use of the same antibody for capturing and as detector ligand showed a significant competitive effect for antigen binding, so required an experimental design to minimize reciprocal interference and maximize the response. The RPA assay, employing primers to the capsid protein p72 gene and an exonuclease III probe, was performed at 39 °C. The limit of detection of the method was assessed using a plasmid encoding the target gene and resulted in 5 copy/µL. The new LFIA and RPA were applied for ASFV detection in the animal tissues usually analysed by conventional assays (i.e., real-time PCR), such as kidney, spleen, and lymph nodes. A simple and universal virus extraction protocol was applied for sample preparation, followed by DNA extraction and purification for the RPA. The LFIA only required the addition of 3% H2O2 to limit matrix interference and prevent false positive results. The two rapid methods (25 min and 15 min were needed to complete the analysis for RPA and LFIA, respectively) showed high diagnostic specificity (100%) and sensitivity (93% and 87% for LFIA and RPA, respectively) for samples with high viral load (Ct < 27). False negative results were observed for samples with low viral load (Ct > 28) and/or also containing specific antibodies to ASFV, which decreased antigen availability and were indicative of a chronic, poorly transmissible infection. The simple and rapid sample preparation and the diagnostic performance of the LFIA suggested its large practical applicability for POC diagnosis of ASF.

Origin, Genetic Variation and Molecular Epidemiology of SARS-CoV-2 Strains Circulating in Sardinia (Italy) during the First and Second COVID-19 Epidemic Waves.

Understanding how geography and human mobility shape the patterns and spread of infectious diseases such as COVID-19 is key to control future epidemics. An interesting example is provided by the second wave of the COVID-19 epidemic in Europe, which was facilitated by the intense movement of tourists around the Mediterranean coast in summer 2020. The Italian island of Sardinia is a major tourist destination and is widely believed to be the origin of the second Italian wave. In this study, we characterize the genetic variation among SARS-CoV-2 strains circulating in northern Sardinia during the first and second Italian waves using both Illumina and Oxford Nanopore Technologies Next Generation Sequencing methods. Most viruses were placed into a single clade, implying that despite substantial virus inflow, most outbreaks did not spread widely. The second epidemic wave on the island was actually driven by local transmission of a single B.1.177 subclade. Phylogeographic analyses further suggest that those viral strains circulating on the island were not a relevant source for the second epidemic wave in Italy. This result, however, does not rule out the possibility of intense mixing and transmission of the virus among tourists as a major contributor to the second Italian wave.

Host Response of Syrian Hamster to SARS-CoV-2 Infection including Differences with Humans and between Sexes.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has highlighted the importance of having proper tools and models to study the pathophysiology of emerging infectious diseases to test therapeutic protocols, assess changes in viral phenotypes, and evaluate the effects of viral evolution. This study provided a comprehensive characterization of the Syrian hamster (Mesocricetus auratus) as an animal model for SARS-CoV-2 infection using different approaches (description of clinical signs, viral load, receptor profiling, and host immune response) and targeting four different organs (lungs, intestine, brain, and PBMCs). Our data showed that both male and female hamsters were susceptible to the infection and developed a disease similar to the one observed in patients with COVID-19 that included moderate to severe pulmonary lesions, inflammation, and recruitment of the immune system in the lungs and at the systemic level. However, all animals recovered within 14 days without developing the severe pathology seen in humans, and none of them died. We found faint evidence for intestinal and neurological tropism associated with the absence of lesions and a minimal host response in intestines and brains, which highlighted another crucial difference with the multiorgan impairment of severe COVID-19. When comparing male and female hamsters, we observed that males sustained higher viral RNA shedding and replication in the lungs, suffered from more severe symptoms and histopathological lesions, and triggered higher pulmonary inflammation. Overall, these data confirmed the Syrian hamster as a suitable model for mild to moderate COVID-19 and reflected sex-related differences in the response against the virus observed in humans.

The Long-Jumping of African Swine Fever: First Genotype II Notified in Sardinia, Italy.

African swine fever (ASF) is a devastating infectious disease of domestic pigs and wild boar that is spreading quickly around the world and causing huge economic losses. Although the development of effective vaccines is currently being attempted by several labs, the absence of globally recognized licensed vaccines makes disease prevention and early detection even more crucial. ASF has spread across many countries in Europe and about two years ago affected the Italian susceptible population. In Italy, the first case of ASF genotype II in wild boar dates back to January 2022, while the first outbreak in a domestic pig farm was notified in August 2023. Currently, four clusters of infection are still ongoing in northern (Piedmont-Liguria and Lombardy), central (Lazio), and southern Italy (Calabria and Campania). In early September 2023, the first case of ASFV genotype II was detected in a domestic pig farm in Sardinia, historically affected by genotype I and in the final stage of eradication. Genomic characterization of p72, p54, and I73R/I329L genome regions revealed 100% similarity to those obtained from isolates that have been circulating in mainland Italy since January 2022 and also with international strains. The outbreak was detected and confirmed due to the passive surveillance plan on domestic pig farms put in place to provide evidence on genotype I's absence. Epidemiological investigations suggest 24 August as the most probable time of ASFV genotype II's arrival in Sardinia, likely due to human activities.

A Naturally Occurring Microhomology-Mediated Deletion of Three Genes in African Swine Fever Virus Isolated from Two Sardinian Wild Boars.

African swine fever virus (ASFV) is the etiological agent of a lethal disease of domestic pigs and wild boars. ASF threatens the pig industry worldwide due to the lack of a licensed vaccine or treatment. The disease has been endemic for more than 40 years in Sardinia (Italy), but an intense campaign pushed it close to eradication; virus circulation was last detected in wild boars in 2019. In this study, we present a genomic analysis of two ASFV strains isolated in Sardinia from two wild boars during the 2019 hunting season. Both isolates presented a deletion of 4342 base pairs near the 5' end of the genome, encompassing the genes MGF 360-6L, X69R, and MGF 300-1L. The phylogenetic evidence suggests that the deletion recently originated within the Sardinia ecosystem and that it is most likely the result of a non-allelic homologous recombination driven by a microhomology present in most Sardinian ASFV genomes. These results represent a striking example of a genomic feature promoting the rapid evolution of structural variations and plasticity in the ASFV genome. They also raise interesting questions about the functions of the deleted genes and the potential link between the evolutionary timing of the deletion appearance and the eradication campaign.

Assessment of the Impact of a Toll-like Receptor 2 Agonist Synthetic Lipopeptide on Macrophage Susceptibility and Responses to African Swine Fever Virus Infection.

Toll-like receptor 2 (TLR2) ligands are attracting attention as prophylactic and immunopotentiator agents against pathogens, including viruses. We previously reported that a synthetic diacylated lipopeptide (Mag-Pam2Cys_P48) polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. Here, we investigated its role in modulating monocyte-derived macrophage (moMΦ) responses against African swine fever virus (ASFV), the etiological agent of one of the greatest threats to the global pig industry. Two ASFV isolates were compared: the attenuated NH/P68 and the virulent 26544/OG10. No effect on virus infection nor the modulation of surface markers' expression (MHC I, MHC II DR, CD14, CD16, and CD163) were observed when Mag-Pam2Cys_P48 treated moMΦ were infected using a multiplicity of infection (MOI) of 1. Mag-Pam2Cys_P48 treated moMΦ released higher levels of IL-1α, IL-1ß, IL-1Ra, and IL-18 in response to infection with NH/P68 ASFV compared to 26544/OG10-infected and mock-infected controls. Surprisingly, when infected using a MOI of 0.01, the virulent ASFV 26544/OG10 isolate replicated even slightly more efficiently in Mag-Pam2Cys_P48 treated moMΦ. These effects also extended to the treatment of moMΦ with two other lipopeptides: Mag-Pam2Cys_P80 and Mag-Pam2Cys_Mag1000. Our data suggested limited applicability of TLR2 agonists as prophylactic or immunopotentiator agents against virulent ASFV but highlighted the ability of the virulent 26544/OG10 to impair macrophage defenses.

A Review on Pathological and Diagnostic Aspects of Emerging Viruses-Senecavirus A, Torque teno sus virus and Linda Virus-In Swine.

Swine production represents a significant component in agricultural economies as it occupies over 30% of global meat demand. Infectious diseases could constrain the swine health and productivity of the global swine industry. In particular, emerging swine viral diseases are omnipresent in swine populations, but the limited knowledge of the pathogenesis and the scarce information related to associated lesions restrict the development of data-based control strategies aimed to reduce the potentially great impact on the swine industry. In this paper, we reviewed and summarized the main pathological findings related to emerging viruses, such as Senecavirus A, Torque teno sus virus, and Linda virus, suggesting a call for further multidisciplinary studies aimed to fill this lack of knowledge and better clarify the potential role of those viral diseases in swine pathology.

Changes in Estimating the Wild Boar Carcasses Sampling Effort: Applying the EFSA ASF Exit Strategy by Means of the WBC-Counter Tool.

African swine fever (ASF) is a devastating disease, resulting in the high mortality of domestic and wild pigs, spreading quickly around the world. Ensuring the prevention and early detection of the disease is even more crucial given the absence of licensed vaccines. As suggested by the European Commission, those countries which intend to provide evidence of freedom need to speed up passive surveillance of their wild boar populations. If this kind of surveillance is well-regulated in domestic pig farms, the country-specific activities to be put in place for wild populations need to be set based on wild boar density, hunting bags, the environment, and financial resources. Following the indications of the official EFSA opinion 2021, a practical interpretation of the strategy was implemented based on the failure probabilities of wrongly declaring the freedom of an area even if the disease is still present but undetected. This work aimed at providing a valid, applicative example of an exit strategy based on two different approaches: the first uses the wild boar density to estimate the number of carcasses need to complete the exit strategy, while the second estimates it from the number of wild boar hunted and tested. A practical free access tool, named WBC-Counter, was developed to automatically calculate the number of needed carcasses. The practical example was developed using the ASF data from Sardinia (Italian island). Sardinia is ASF endemic from 43 years, but the last ASFV detection dates back to 2019. The island is under consideration for ASF eradication declaration. The subsequent results provide a practical example for other countries in approaching the EFSA exit strategy in the best choices for its on-field application.

Cell Lines for the Development of African Swine Fever Virus Vaccine Candidates: An Update.

African swine fever virus (ASFV) is the etiological agent of a highly lethal disease in both domestic and wild pigs. The virus has rapidly spread worldwide and has no available licensed vaccine. An obstacle to the construction of a safe and efficient vaccine is the lack of a suitable cell line for ASFV isolation and propagation. Macrophages are the main targets for ASFV, and they have been widely used to study virus-host interactions; nevertheless, obtaining these cells is time-consuming and expensive, and they are not ethically suitable for the production of large-scale vaccines. To overcome these issues, different virulent field isolates have been adapted on monkey or human continuous cells lines; however, several culture passages often lead to significant genetic modifications and the loss of immunogenicity of the adapted strain. Thus, several groups have attempted to establish a porcine cell line able to sustain ASFV growth. Preliminary data suggested that some porcine continuous cell lines might be an alternative to primary macrophages for ASFV research and for large-scale vaccine production, although further studies are still needed. In this review, we summarize the research to investigate the most suitable cell line for ASFV isolation and propagation.

Analyses of the Impact of Immunosuppressive Cytokines on Porcine Macrophage Responses and Susceptibility to Infection to African Swine Fever Viruses.

African swine fever viruses (ASFV), currently a serious threat to the global pig industry, primarily target porcine macrophages. Macrophages are characterized by their remarkable plasticity, being able to modify their phenotype and functions in response to diverse stimuli. Since IL-10 and TGF-ß polarize macrophages toward an anti-inflammatory phenotype, we analyzed their impact on porcine monocyte-derived macrophages' (moMΦ) susceptibility to infection and their responses to two genotype I ASFV strains, virulent 26544/OG10 and attenuated NH/P68. At a low multiplicity of infection (MOI), NH/P68, but not 26544/OG10, presented a higher ability to infect moM(IL-10) compared to moMΦ and moM(TGF-ß), but no differences were appreciated at a higher MOI. Both strains replicated efficiently in all moMΦ subsets, with no differences at later times post-infection. Both strains downregulated CD14 and CD16 expression on moMΦ, irrespective of the activation status. ASFV's modulation of CD163 and MHC II DR expression and cytokine responses to NH/P68 or 26544/OG10 ASFV were not affected by either IL-10 or TGF-ß pre-treatment. Our results revealed little impact of these anti-inflammatory cytokines on moMΦ interaction with ASFV, which likely reflects the ability of the virus to effectively modulate macrophage responses.

Cadmium and wild boar: Environmental exposure and immunological impact on macrophages.

Cadmium (Cd2+) is regarded as one of the most toxic heavy metals, which can enter the food chain through environmental contamination and be bioaccumulated. Its exposure in Ligurian wild boars was monitored between 2016-2020 and revealed high level of this heavy metal in different provinces. In one of these polluted area, 21 wild boars were additionally sampled and the relationship between hepatic and renal Cd2+ concentration suggested that majority of these animals presented chronic intoxication. Cd2+ exposure of wild boar might lead to an immunosuppression status, thus in vitro experiments on wild boar monocyte-derived macrophages (moMФ) were carried out. Effects of Cd2+ scalar doses were evaluated through viability and adsorption assays, ELISA, qPCR. Moderate doses of this environmental pollutant (20 µM) were absorbed by moMФ, with subsequent reduction of their viability. This heavy metal did not trigger release of either IFN- ß, anti-inflammatory or pro-inflammatory cytokines by moMФ, instead 24 h treatment with 20 µM of Cd2+ resulted in down-regulated expression of TNF-α, IL-12p40, several TLRs, CD14, MD2, BD2, MyD88, p65, and NOS2. The results of our monitoring activity suggested that wild boar can be useful to monitor environmental exposure of this heavy metal and can help in understanding the type of contamination. In addition, in vitro experiments on wild boar moMФ revealed that Cd2+ exposure negatively affected the immune function of these cells, likely leading to increased susceptibility to infection.

First Genomic Evidence of Dual African Swine Fever Virus Infection: Case Report from Recent and Historical Outbreaks in Sardinia.

African swine fever virus (ASFV) is one of the pathogens of highest concern worldwide. Despite different virus lineages co-circulating in several areas, dual infections in the same animal have been rarely observed, suggesting that ASF superinfections are infrequent events. Here we present the first genome-wide detection and analysis of two intragenotype dual ASFV infections. The dual infections have been detected in a hunted wild boar and in a pig carcass, both infected by ASFV genotype I in Sardinia in 1984 and 2018, respectively. We characterize the genetic differences between the two sequences, their intra-host frequency, and their phylogenetic relationship among fully sequenced ASFV strains from Sardinia. Both dual infections involve pairs of closely related but different viruses that were circulating in Sardinia in the same period. The results imply that dual ASFV infections or similar ASFV strains are more common than expected, especially in ASF endemic areas, albeit difficult to detect.

A Deeper Insight into Evolutionary Patterns and Phylogenetic History of ASFV Epidemics in Sardinia (Italy) through Extensive Genomic Sequencing.

African swine fever virus (ASFV) is the etiological agent of the devastating disease African swine fever (ASF), for which there is currently no licensed vaccine or treatment available. ASF is defined as one of the most serious animal diseases identified to date, due to its global spread in regions of Africa, Europe and Asia, causing massive economic losses. On the Italian island of Sardinia, the disease has been endemic since 1978, although the last control measures put in place achieved a significant reduction in ASF, and the virus has been absent from circulation since April 2019. Like many large DNA viruses, ASFV mutates at a relatively slow rate. However, the limited availability of whole-genome sequences from spatial-localized outbreaks makes it difficult to explore the small-scale genetic structure of these ASFV outbreaks. It is also unclear if the genetic variability within outbreaks can be captured in a handful of sequences, or if larger sequencing efforts can improve phylogenetic reconstruction and evolutionary or epidemiological inference. The aim of this study was to investigate the phylogenetic patterns of ASFV outbreaks between 1978 and 2018 in Sardinia, in order to characterize the epidemiological dynamics of the viral strains circulating in this Mediterranean island. To reach this goal, 58 new whole genomes of ASFV isolates were obtained, which represents the largest ASFV whole-genome sequencing effort to date. We provided a complete description of the genomic diversity of ASFV in terms of nucleotide mutations and small and large indels among the isolates collected during the outbreaks. The new sequences capture more than twice the genomic and phylogenetic diversity of all the previously published Sardinian sequences. The extra genomic diversity increases the resolution of the phylogenetic reconstruction, enabling us to dissect, for the first time, the genetic substructure of the outbreak. We found multiple ASFV subclusters within the phylogeny of the Sardinian epidemic, some of which coexisted in space and time.

African Swine Fever in Smallholder Sardinian Farms: Last 10 Years of Network Transmission Reconstruction and Analysis.

African swine fever (ASF) is a viral disease of suids that frequently leads to death. There are neither licensed vaccines nor treatments available, and even though humans are not susceptible to the disease, the serious socio-economic consequences associated with ASF have made it one of the most serious animal diseases of the last century. In this context, prevention and early detection play a key role in controlling the disease and avoiding losses in the pig value chain. Target biosecurity measures are a strong strategy against ASF virus (ASFV) incursions in farms nowadays, but to be efficient, these measures must be well-defined and easy to implement, both in commercial holdings and in the backyard sector. Furthermore, the backyard sector is of great importance in low-income settings, mainly for social and cultural practices that are highly specific to certain areas and communities. These contexts need to be addressed when authorities decide upon the provisions that should be applied in the case of infection or decide to combine them with strict preventive measures to mitigate the risk of virus spread. The need for a deeper understanding of the smallholder context is essential to prevent ASFV incursion and spread. Precise indications for pig breeding and risk estimation for ASFV introduction, spread and maintenance, taking into account the fact that these recommendations would be inapplicable in some contexts, are the keys for efficient target control measures. The aim of this work is to describe the 305 outbreaks that occurred in domestic pigs in Sardinia during the last epidemic season (2010-2018) in depth, providing essential features associated with intensive and backyard farms where the outbreaks occurred. In addition, the study estimates the average of secondary cases by kernel transmission network. Considering the current absence of ASF outbreaks in domestic pig farms in Sardinia since 2018, this work is a valid tool to specifically estimate the risk associated with different farm types and update our knowledge in this area.