Search

Mycobacterium bovis (M. bovis) is the primary agent of bovine tuberculosis (TB) in Mediterranean buffalo, which has a negative economic impact on buffalo herds. Improving TB diagnostic performance in this species represents a key step to eradicate efficiently this disease. We have recently shown the utility of the IFN-γ assay in the diagnosis of M. bovis infection in Mediterranean buffaloes (Bubalus bubalis), but other cytokines might be useful immunological biomarkers of this infection. We therefore investigated the utility of key immune cytokines as diagnostic biomarkers of M. bovis infection in this species. Thirty-six Italian Mediterranean buffaloes were used in this study: healthy animals (N = 11), infected (IFN-γ test positive, no post-mortem lesions, no M. bovis detection; N = 14), and affected (IFN-γ test positive, visible post-mortem lesions; N = 11). Heparin blood samples were stimulated with bovine purified protein derivative (PPD-B), alongside controls, and 18–24 h later plasma were collected. Levels of 14 key cytokines were measured: IFN-γ, IL-17, IL-4, IL-10, TNF, IL-1α, IL-1β, IL-6, IP-10, MIP-1α, MIP-1β, MCP-1, IL-36Ra, and VEGF-A. We observed that both infected and affected animals released higher levels of IFN-γ, IL-17, IL-10, TNF, IL-1α, IL-6, MIP-1β, in response to PPD-B compared to healthy subjects. Mycobacterium bovis infected animals released also higher levels of IL-1β and IP-10 in response to PPD-B compared to healthy subjects, whereas only tendencies were detected in affected animals. Affected animals only released MIP-1α in response to PPD-B compared to healthy subjects and in this group higher values of PPD-B specific TNF was also observed. Finally, canonical discriminant analysis (CDA) was used to generate predictive cytokine profiles by groups. Our data suggest that IL-10, TNF, IL-1α, IL-6, MIP-1β could be useful biomarkers of TB in Mediterranean Buffalo and can improve the TB diagnostic performance in this specie.

Low temperature is the main strategy to preserve fruit quality post-harvest, in the supply chain. Low temperatures reduce the respiration, ethylene emission, and enzymatic activities associated with senescence. Unfortunately, peaches are sensitive to low temperatures if exposed for long periods, resulting in physiological disorders that can compromise commercial quality. Maximum damage occurs at 5 °C while at 1 °C damage is reduced. Therefore, rapid early detection methods for the distribution chain to monitor length and temperature of fruit storage are needed. The aim of this work was to identify candidate genes to develop an antibody-based marker system in peach fruit to monitor chilled storage. Two cultivars were tested: ‘Sagittaria’, an early ripening peach, and ‘Big Top’ a mid-season ripening nectarine, with delayed softening and resistance to supply-chain conditions. Both cultivars were subjected to 1 or 5 °C chilled storage for different times to simulate typical supply-chain conditions. Identification and expression of potential marker genes was assessed using a previous transcriptomic study following storage at 1 °C. Fifteen candidate genes were selected, however only seven proteins encoded were suitable as protein markers as they lack a transmembrane domain. Real-time qPCR using fruit from the subsequent year to the transcriptome was used to assess expression at both 1 and 5 °C chilled storage of five candidate genes. Four genes and the related proteins were identified that would be suitable for the development of molecular markers: a Pathogenesis-Related Bet v I family protein, a dehydrin, a Glycosyl hydrase family 18 protein and a Late Embryogenesis abundant protein.

Swiss chard is a rich dietary source of bioactive betalain pigments responsible of the red colour of the plant's organs and of phenolic compounds having recognized antioxidant properties. Vegetable colour and appearance, as the first characteristics perceived, are important quality attributes that influence the consumer's acceptance. Currently, elicitation based on growth plant regulator supply is considered in most cases a useful strategy to enhance the sensorial and nutritional value of dietary plants. Thus, this study aimed to assess the impact of salicylic acid application on Swiss chard (Beta vulgaris L. ssp. cicla) grown, isolated and under water shortage, in relation to physiological and qualitative parameters throughout cultivation and at harvest. Different effects were observed depending on the type and interaction of stress-inducing treatments. Positive effects consisted in highest chlorophyll (r.u.) (+19–40 %) carotenoids (+34–48 %), and phenolic contents (+2–42 %) in leaves when treatments were applied isolated or in combination, which also led to high sugar levels in plants (up to 380 %). Negative effects included the reduction of biomass production (−40 %), leaf size (−38 %), and betalain content (-7-27 %), especially with combined treatments. Nitrate accumulation was not affected but salicylic acid induced a reduction in proline levels probably due to increased carbohydrate and nitrate metabolisms in regulating water deficit.

Abstract African swine fever virus (ASFV) causes a devastating disease affecting domestic and wild pigs. ASF was first introduced in Sardinia in 1978 and until 2019 only genotype I isolates were identified. A remarkable genetic stability of Sardinian ASFV isolates was described, nevertheless in 2019 two wild boar isolates with a sustained genomic deletion (4342 base pairs) were identified (7303WB/19, 7212WB/19). In this study, we therefore performed in vitro experiments with monocyte-derived macrophages (moMФ) to unravel the phenotypic characteristics of these deleted viruses. Both 7303WB/19 and 7212WB/19 presented a lower growth kinetic in moMФ compared to virulent Sardinian 26544/OG10, using either a high (1) or a low (0.01) multiplicity of infection (MOI). In addition, flow cytometric analysis showed that both 7303WB/19 and 7212WB/19 presented lower intracellular levels of both early and late ASFV proteins. We subsequently investigated whether deleted virus variants were previously circulating in wild boars in Sardinia. In the four years preceding the last genotype I isolation (February 2015–January 2019), other eight wild boar isolates were collected, all belonging to p72 genotype I, B602L subgroup X, but none of them presented a sustained genomic deletion. Overall, we observed the deleted virus isolates in Sardinia only in 2019, at the end of a strong eradication campaign, and our data suggest that it might possess an attenuated phenotype in vivo. A better understanding of ASFV evolution in endemic territories might contribute to development of effective control measures against ASF.

The use of biostimulants in agriculture has been rapidly increasing in recent years, because of their positive effects on crop yield, product quality, and tolerance to abiotic stresses. In the present study the efficacy of multiple applications of a plant-derived biostimulant, obtained from the aqueous maceration of borage (Borago officinalis, L.) flowers on two lettuce cultivars, namely a green (Lactuca sativa L. cv. Expertise RZ) and red (Lactuca sativa L. cv. Codex RZ) Salanova® was evaluated. The treatment was applied at 10 mL L−1 as foliar spray three times, once a week starting from two weeks after transplant. Control plants were treated with water. Non-destructive measurements (pigments, leaf nitrogen index, chlorophyll a fluorescence) were taken during plant growth after at each treatment application. At the end of the experiment, destructive analyses were performed to assess qualitative traits. The research work was focused on the evaluation of physiological parameter changes during plant growth, and on primary and secondary metabolism. Foliar applications did not affect the accumulation of total sugars (4.56 mg g−1 in Expertise, 3.5 mg g−1 in Codex) in either cultivar. However, the lettuce head weight was negatively affected by the extract application in red cultivar (-10 g/plant), while no changes were observed in the green lettuce. The nitrogen-flavonol index (NFI) increased after the third application of borage extract in green cultivar (+67 %), suggesting an improvement of nitrogen nutrition status or a reduced stress condition. A different response resulted in term of maximum quantum efficiency of PSII (FV/FM), performance index (PI), nitrate, and anthocyanin accumulation in leaves. The FV/FM ratio significantly increased in green cultivar after the first application (from 0.80 to 0.84) and at harvest (from 0.79 to 0.84). The PI showed a slight but not significant increase at the same time points. On the contrary, the PI was significantly higher in red cultivar after the third application (+9.4 %). Interestingly, the borage extract induced a significant decrease of nitrate accumulation in lettuce leaves of the red cultivar (from 4149.7 to 2711.6 mg/kg, −34 %). At the same time a positive variation of anthocyanin content was observed in red lettuce (+24.7 %). The application of biostimulant products might improve the quality of some lettuce varieties as regards the accumulation of metabolites useful for the plant to overcome stress conditions and fundamental in human healthy diet, increasing the leaf concentration of Ca, Na, and Mg.

Plastic containers in the horticulture sector largely rely on plastics of fossil origin. Although these plastics have excellent mechanical properties, resilience towards chemical/microbiological degradation, durability, and affordable price, they have a high environmental impact due to their inherent non-biodegradability. In line with the most recent EU strategies on a trans-sectorial transition to sustainable systems, the horticultural sector is seeking for new materials to produce plant nursery plugs as an alternative to conventional plastics. The present work is a part of the project "BBPlug”, which aims to add value to agri-food industry wastes, reducing plastics and fertilizers in horticulture. Here, we propose a new material made of pectin extracted from citrus peel as a green and biodegradable substrate to produce plant nursery plugs. To this purpose, pectin-based films were fabricated by solvent-casting from film-forming solutions with increasing amounts of glycerol as plasticizer (6.7 – 33.3 gGlycerol/gPectin), microfibrillated cellulose (MFC) as reinforcing agent (2.7 – 8.1 mgMFC/gPectin), and at two different pH values (3.5 and 7.0). Puncture resistance, water solubility, and oxygen-barrier properties of the films were then investigated. Films from formulations at pH = 3.5 exhibited an overall better mechanical behavior over their counterpart at pH = 7. The best puncture resistance and water solubility were displayed by films from the least glycerol-loaded formulations. The addition of MFC to the film-forming solution improved the oxygen-barrier properties of the films but led to a reduction in their water solubility. In other tests, a selection of different plant growth-promoting (PGP) bacteria was demonstrated to have a boosting effect on the development of a model vegetable (i.e., lettuce), thus offering creative opportunities for the advancement of the “BBPlug” project.

Abstract Storage or transportation temperature is very important for preserving the quality of fruit. However, low temperature in sensitive fruit such as peach can induce loss of quality. Fruit exposed to a specific range of temperatures and for a longer period can show chilling injury (CI) symptoms. The susceptibility to CI at low temperature varies among cultivars and genetic backgrounds. Along with agronomic management, appropriate postharvest management can limit quality losses. The importance of correct temperature management during postharvest handling has been widely demonstrated. Nowadays, due to long-distance markets and complex logistics that require multiple actors, the management of storage/transportation conditions is crucial for the quality of products reaching the consumer. Peach fruit exposed to low temperatures activate a suite of physiological, metabolomic, and molecular changes that attempt to counteract the negative effects of chilling stress. In this review an overview of the factors involved, and plant responses is presented and critically discussed. Physiological disorders associated with CI generally only appear after the storage/transportation, hence early detection methods are needed to monitor quality and detect internal changes which will lead to CI development. CI detection tools are assessed: they need to be easy to use, and preferably non-destructive to avoid loss of products. Graphical Abstract

Porcine parvoviruses (PPVs) are among the most important agents of reproductive failure in swine worldwide. PPVs comprise eight genetically different species ascribed to four genera: Protoparvovirus (PPV1, PPV8), Tetraparvovirus (PPV2-3), Copiparvovirus (PPV4-6), and Chaphamaparvovirus (PPV7). In 2016, PPV7 was firstly detected in the USA and afterwards in Europe, Asia, and South America. Recently, it was also identified in Italy in pig farms with reproductive failure. This study aimed to evaluate the circulation of PPV7 in domestic and wild pigs in Sardinia, Italy. In addition, its coinfection with Porcine Circovirus 2 (PCV2) and 3 (PCV3) was analysed, and PPV7 Italian strains were molecularly characterised. PPV7 was detected in domestic pigs and, for the first time, wild pigs in Italy. The PPV7 viral genome was detected in 20.59% of domestic and wild pig samples. PPV7 detection was significantly lower in domestic pigs, with higher PCV2/PCV3 co-infection rates observed in PPV7-positive than in PPV7-negative domestic pigs. Molecular characterisation of the NS1 gene showed a very high frequency of recombination that could presumably promote virus spreading.

IntroductionBiostimulants exert positive functions in plants, improving yield and quality, and alleviating the negative effects of abiotic stresses. Among them, the application of herbicides may cause damage to nontarget plants. At present, limited information is available regarding the interaction between biostimulants and herbicides.MethodsThe purpose of the present study was to assess the effect of an herbicide’s mixture (Harmony® 50 SX® + Tuareg® + Zetrola®), used in combination with several biostimulant raw materials (BRM), on the agronomic and physiologic characteristics of soybean. The experiments were conducted in two seasons, 2020 and 2021, applying the herbicides mixture alone or associated with 10 BRM.ResultsDifferences emerged between the two years, considering nitrate, total sugars concentration, flavonol, and chlorophyll a fluorescence-related parameters. Chlorophyll content significantly declined (−45% in 2020) in plants treated with the herbicides mixture alone but, in combination with potassium silicate, the chlorophyll values were restored to control levels. The same positive effect observed in response to the combination of potassium silicate and the herbicide mixture has been confirmed in the second year of experiment. At the same time, chlorophyll content and Nitrogen Index were positively increased (up to 8% and 30%, respectively) depending on the application of some BRM and the year. A significant effect of biostimulants on yield was confirmed by the application of Ascophyllum nodosum (+16%) and humic acids (+7%), in 2020. However, the combined use with the herbicides nullifies the increase. DiscussionThe results obtained from these experiments support the use of biostimulants in improving specific quality traits (chlorophyll content, leaves nitrogen status, and secondary metabolites accumulation). At the same time, the potential use of biostimulants in combination with herbicides needs to be further explored since of external factors (environment, year…) still have a strong effect on their efficacy.

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.

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.

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.

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- β, 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.

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.

Bovine Alphaherpesvirus 1 (BoHV-1) is one of the major respiratory pathogens in cattle worldwide. Infection often leads to a compromised host immune response that contributes to the development of the polymicrobial disease known as “bovine respiratory disease”. After an initial transient phase of immunosuppression, cattle recover from the disease. This is due to the development of both innate and adaptive immune responses. With respect to adaptive immunity, both humoral and cell-mediated immunity are required to control infection. Thus, several BoHV-1 vaccines are designed to trigger both branches of the adaptive immune system. In this review, we summarize the current knowledge on cell-mediated immune responses directed against BoHV-1 infection and vaccination.

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.

African swine fever (ASF) is a hemorrhagic viral disease of domestic pigs and wild suids (all Sus scrofa) caused by the ASF virus (ASFV). The disease is spreading worldwide without control, threatening pig production due to the absence of licensed vaccine or commercially available treatments. A thorough understanding of the immunopathogenic mechanisms behind ASFV infection is required to better fight the disease. Cytokines are small, non-structural proteins, which play a crucial role in many aspects of the immune responses to viruses, including ASFV. Infection with virulent ASFV isolates often results in exacerbated immune responses, with increased levels of serum pro-inflammatory interleukins (IL-1α, IL-1β, IL-6), TNF and chemokines (CCL2, CCL5, CXCL10). Increased levels of IL-1, IL-6 and TNF are often detected in several tissues during acute ASFV infections and associated with lymphoid depletion, hemorrhages and oedemas. IL-1Ra is frequently released during ASFV infection to block further IL-1 activity, with its implication in ASFV immunopathology having been suggested. Increased levels of IFN-α and of the anti-inflammatory IL-10 seem to be negatively correlated with animal survival, whereas some correlation between virus-specific IFN-γ-producing cells and protection has been suggested in different studies where different vaccine candidates were tested, although future works should elucidate whether IFN-γ release by specific cell types is related to protection or disease development.

Drought stress is one of the main factors limiting horticultural crops, especially in environments such as the Mediterranean basin, which is often characterized by sub-optimal water availability [...]

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.

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-1β, 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.

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.

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.

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.

African swine fever virus (ASFV) remains a threat to global pig populations. Infections with ASFV lead to a hemorrhagic disease with up to 100% lethality in Eurasian domestic and wild pigs. Although myeloid cells are the main target cells for ASFV, T cell responses are impacted by the infection as well. The complex responses remain not well understood, and, consequently, there is no commercially available vaccine. Here, we review the current knowledge about the induction of antiviral T cell responses by cells of the myeloid lineage, as well as T cell responses in infected animals, recent efforts in vaccine research, and T cell epitopes present in ASFV.

Extracellular vesicles (EVs) are lipid bilayer nano-dimensional spherical structures and act mainly as signaling mediators between cells, in particular modulating immunity and inflammation. Milk-derived EVs (mEVs) can have immunomodulatory and anti-inflammatory effects, and milk is one of the most promising food sources of EVs. In this context, this study aimed to evaluate bovine mEVs anti-inflammatory and immunomodulating effects on an in vitro co-culture (Caco-2 and THP-1) model of intestinal inflammation through gene expression evaluation with RT-qPCR and cytokine release through ELISA. After establishing a pro-inflammatory environment due to IFN-γ and LPS stimuli, CXCL8, IL1B, TNFA, IL12A, IL23A, TGFB1, NOS2, and MMP9 were significantly up-regulated in inflamed Caco-2 compared to the basal co-culture. Moreover, IL-17, IL-1β, IL-6, TNF-α release was increased in supernatants of THP-1. The mEV administration partially restored initial conditions with an effective anti-inflammatory activity. Indeed, a decrease in gene expression and protein production of most of the tested cytokines was detected, together with a significant gene expression decrease in MMP9 and the up-regulation of MUC2 and TJP1. These results showed a fundamental capability of mEVs to modulate inflammation and their potential beneficial effect on the intestinal mucosa.

Biostimulants are agronomic tools that have been gaining importance in the reduction of fertilizer applications. They can improve the yield of cropping systems or preventing crop yield losses under abiotic stresses. Biostimulants can be composed of organic and inorganic materials and most of the components are still unknown. The characterization of the molecular mechanism of action of biostimulants can be obtained using the omics approach, which includes the determination of transcriptomic, proteomic, and metabolomic changes in treated plants. This review reports an overview of the biostimulants, taking stock on the recent molecular studies that are contributing to clarify their action mechanisms. The omics studies can provide an overall evaluation of a crop’s response, connecting the molecular changes with the physiological pathways activated and the performance with or without stress conditions. The multiple responses of plants treated with biostimulants must be correlated with the phenotype changes. In this context, it is also crucial to design an adequate experimental plan and statistical data analysis, in order to find robust correlations between biostimulant treatments and crop performance.

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-β 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-β), 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-β 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.

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.

The efficacy of plant-based biostimulants lays on the extraction of bioactive compounds that can trigger positive biological responses in plants. Their use has been increasing for lowering the production costs, enhance the yield and the stress tolerance, and improve the cropping systems’ sustainability. The aim of this work was to investigate the effect of maceration time on borage extracts, subsequently sprayed on rocket plants hydroponically grown. A preliminary test was also assessed to evaluate the extracts’ effect on seeds germination. Borage leaves and flowers were separated and macerated in the same conditions for different times. The obtained borage extracts were applied two times during the cultivation of rocket plants. The yield was not affected by the treatments, while the quality of leafy vegetables was improved. The results indicated that the flower extracts (FE) were more active than leaf extracts (LE) in stimulating the crop response. Promising results were observed on the nitrate assimilation, with reduced accumulation in the edible and commercial parts. Non-destructive analysis revealed that electron transport rates were significantly higher in treated plants. Biostimulant effects were also confirmed by the lowering of sucrose and sugars content in the treated plants and by a general increment of secondary metabolites.

Toll-like receptor 2 (TLR2) ligands are attracting increasing attention as prophylactic and immunotherapeutic agents against pathogens and tumors. We previously observed that a synthetic diacylated lipopeptide based on a surface protein of Mycoplasma agalactiae (Mag-Pam2Cys) strongly activated innate immune cells, including porcine monocyte-derived macrophages (moMΦ). In this study, we utilized confocal microscopy, flow cytometry, multiplex cytokine ELISA, and RT-qPCR to conduct a comprehensive analysis of the effects of scalar doses of Mag-Pam2Cys on porcine moMΦ. We observed enhanced expression of activation markers (MHC class I, MHC class II DR, CD25), increased phagocytotic activity, and release of IL-12 and proinflammatory cytokines. Mag-Pam2Cys also upregulated the gene expression of several IFN-α subtypes, p65, NOS2, and molecules with antimicrobial activities (CD14, beta defensin 1). Overall, our data showed that Mag-Pam2Cys polarized porcine macrophages towards a proinflammatory antimicrobial phenotype. However, Mag-Pam2Cys downregulated the expression of IFN-α3, six TLRs (TLR3, -4, -5, -7, -8, -9), and did not interfere with macrophage polarization induced by the immunosuppressive IL-10, suggesting that the inflammatory activity evoked by Mag-Pam2Cys could be regulated to avoid potentially harmful consequences. We hope that our in vitro results will lay the foundation for the further evaluation of this diacylated lipopeptide as an immunopotentiator in vivo.

Macrophages are phagocytic cells involved in maintaining tissue homeostasis and defense against pathogens. Macrophages may be polarized into different functionally specialized subsets. M2c macrophages arise following stimulation with IL-10 or TGF-β and mediate anti-inflammatory and tissue repair functions. M2c macrophages remain poorly characterized in the pig, thus we investigated the impact of these regulatory cytokines on porcine monocyte-derived macrophages (moMΦ). The phenotype and functionality of these cells was characterized though confocal microscopy, flow cytometry, ELISA, and RT-qPCR. Both cytokines induced CD14 and MHC II DR down-regulation and reduced IL-6, TNF-α, and CD14 expression, suggestive of an anti-inflammatory phenotype. Interestingly, neither IL-10 or TGF-β were able to trigger IL-10 induction or release by moMΦ. Differences between these cytokines were observed: stimulation with IL-10, but not TGF-β, induced up-regulation of both CD16 and CD163 on moMΦ. In addition, IL-10 down-regulated expression of IL-1β and IL-12p40 4h post-stimulation and induced a stronger impairment of moMΦ ability to respond to either TLR2 or TLR4 agonists. Overall, our results provide an overview of porcine macrophage polarization by two immunosuppressive cytokines, revealing differences between IL-10 and TGF-β, and reporting some peculiarity of swine, which should be considered in translational studies.

The need for animal welfare definition and assessment is increasing worldwide, and several studies have been conducted to help fill the knowledge gaps regarding the welfare of cattle. However, further studies are needed to provide valid synthetized measures for welfare evaluation. The aim of this study was to assess the welfare status of 16 Sardinian dairy cattle farms, based on the developed Animal Welfare and Biosecurity Evaluation checklist (AWB-EF) and the corresponding hematological, biochemical, and electrophoretic profiles of these animals. Considering the AWB-EF as gold standard, blood samples were collected from 230 Holstein breed dairy cattle, aged between 3 and 8 years, out of the periparturient period, and with no clinical signs of specific pathologies. Principal Component (PC) and correlation analyses were performed to simplify phenomena interpretation and assess positive/negative associations. Four PCs were able to explain 76% of the total variability, and six laboratory parameters were strongly associated with the AWB-EF score (Spearman’s correlation coefficient ≥ 0.40, p-Value < 0.05), reflecting the real health status of the animals. Given the complexity of animal welfare as a multidimensional concept and the need to include both animal-based and non-based measures in welfare evaluation, the present work represents a sound basis for future evaluation and veterinary health planning.

African swine fever (ASF) severely threatens the swine industry worldwide, given its spread and the absence of an available licensed vaccine, and has caused severe economic losses. Its persistence in wild boar (WB), longer than in domestic pig farms, and the knowledge gaps in ASF epidemiology hinder ASF virus (ASFV) eradication. Even in areas where disease is effectively controlled and ASFV is no longer detected, declaring eradication is difficult as seropositive WBs may still be detected. The aim of this work was to estimate the main ASF epidemiological parameters specific for the north of Sardinia, Italy. The estimated basic (R0) and effective (Re) reproduction numbers demonstrate that the ASF epidemic is declining and under control with an R0 of 1.139 (95% confidence interval (CI) = 1.123–1.153) and Re of 0.802 (95% CI = 0.612–0.992). In the last phases of an epidemic, these estimates are crucial tools for identifying the intensity of interventions required to definitively eradicate the disease. This approach is useful to understand if and when the detection of residual seropositive WB is no longer associated with any further ASFV circulation.

African swine fever virus (ASFV), the cause of a devastating disease affecting domestic and wild pigs, has been present in Sardinia since 1978. In the framework of the regional ASF eradication plan, 4484 illegal pigs were culled between December 2017 and February 2020. The highest disease prevalence was observed in the municipality with the highest free-ranging pig density, and culling actions drastically reduced ASFV circulation among these animals. ASFV-antibody were detected in 36.7% of tested animals, which were apparently healthy, thus, the circulation of low-virulence ASFV isolates was hypothesized. ASFV genome was detected in 53 out of 2726 tested animals, and virus isolation was achieved in two distinct culling actions. Two ASFV haemadsorbing strains were isolated from antibody-positive apparently healthy pigs: 55234/18 and 103917/18. Typing analysis revealed that both isolates belong to p72 genotype I, B602L subgroup X; phylogenetic analysis based on whole genome sequencing data showed that they were closely related to Sardinian ASFV strains collected since 2010, especially 22653/Ca/2014. Our data suggested the absence of immune-escaped ASFV variants circulating among free-ranging pigs, indicating that other elements contributed to virus circulation among these animals. Understanding factors behind disease persistence in endemic settings might contribute to developing effective countermeasures against this disease.

Porcine circovirus 3 (PCV3) is a recently discovered member of the Circoviridae family. So far, its presence has been reported in North America, Asia, South America, and Europe. In this study, blood and tissue samples from 189 Sardinian suids (34 domestic pigs, 115 feral free ranging pigs, and 39 wild boars) were used to genetically characterize the PCV3 strains from Sardinia. PCV3 infection in the animals was confirmed by real time PCR. The detection rate in the three groups analyzed was l7.64% in domestic pigs, 77.39% in free ranging pigs, and 61.54% in wild boars. Moreover, our results showed that co-infection of PCV3 with other viruses is quite a common occurrence. Molecular characterization of Sardinian PCV3 strains was performed by sequencing 6 complete genomes and 12 complete cap genes. Our results revealed that there is a high similarity between our strains and those identified in different countries, confirming the genetic stability of PCV3 regardless of geographical origin. Haplotype network analysis revealed the presence of 6 whole genomes or 12 unique ORF2 haplotypes and a nonsynonymous mutation in ORF2 that leads to an R14K amino acid substitution. Phylogenetic analysis of whole genome and ORF2 was also conducted. The Sardinian strains were allocated in three different clusters of phylogenetic trees of both complete genome and ORF2. With this study, we have provided a snapshot of PCV3 circulation in Sardinia. Our findings might help to achieve a deeper understanding of this emerging porcine virus.

African Swine Fever Virus (ASFV) has tropism for macrophages, which seems to play a crucial role in disease pathogenesis and viral dissemination. Previous studies showed that ASFV developed mechanisms to evade type I interferon (IFN) responses. Hence, we analyzed the ability of ASFV strains of diverse virulence to modulate IFN-β and IFN-α responses. Porcine monocyte-derived macrophages un-activated (moMΦ) or activated with IFN-α (moMΦ + FN-α) were infected with virulent (22653/14) or attenuated (NH/P68) ASFV strains, and expressions of IFN-β and of 17 IFN-α subtypes genes were monitored over time. ASFV strains of diverse virulence induced different panels of IFN genes: infection of moMΦ with either strains caused statistically significant up-regulation of IFN-α3, -α7/11, whereas only attenuated NH/P68 determined statistically significant up-regulation of IFN-α10, -α12, -α13, -α15, -α17, and IFN-β. Infection of activated moMΦ with either strains resulted in up-regulation of IFN-β and many IFN-α subtypes, but statistical significance was found only for IFN-α1, -α10, -α15, -α16, -α17 in response to NH/P68-infection only. These data revealed differences in type I IFNs expression patterns, with differences between strains of diverse virulence. In addition, virulent 22653/14 ASFV seems to have developed mechanisms to suppress the induction of several type I IFN genes.

African swine fever (ASF) poses a severe threat to the global pig industry for which currently there is no available vaccine. The aetiological ASF virus (ASFV) has a predilection for cells of the myeloid lineage, however little is known about its interaction with polarised macrophages. This study focused on the in vitro interactions of porcine monocyte-derived un-activated (moMΦ), classically (moM1), alternatively (moM2), and IFN-α-activated macrophages with two genotype I ASFV strains: virulent 22653/14 and attenuated NH/P68. At a high multiplicity of infection, NH/P68, but not 22653/14, presented a reduced ability to infect moM1 and IFN−α-activated moMΦ compared to moMΦ. IFN-α activation resulted in a dose-dependent reduction in the proportion of ASFV-infected cells. Both strains replicated efficiently in all the subsets. While higher levels of IL-1α, IL-1β, and IL-18 were secreted by NH/P68-infected moM1 compared to 22653/14, both strains negatively affected moMΦ ability to release IL-6, IL-12, TNF-α in response to classical activation or stimulation with a TLR2 agonist. Our results suggest that ASFV 22653/14 covertly replicates in macrophages, compromising the development of effective immune responses. Attenuated NH/P68 has partially lost these mechanisms, which may enhance immune surveillance. A better understating of these mechanisms should aid the rational design of live attenuated ASFV vaccines.

Salinity is one of the major abiotic stress causing yield losses and decreasing product quality. The beneficial effects of biostimulant products to enhance plant tolerance to abiotic stresses have been reported in several crops, but their mode of action is poorly understood. This work aims to better understand the effect of salt stress on wild rocket treated with a borage extract. The expression of some of the transcription factors (TFs) typically involved in salt stress response was studied within a 24 h period. Physiological parameters such as chlorophyll, chlorophyll a fluorescence, carotenoids, phenols, and anthocyanin were analyzed. Results obtained showed that salt stress induced a general increase in the expression levels of almost all TFs studied, whereas the treatment with the plant-base extract only induced an increase at specific time points. Moreover, the approach adopted allowed indagating the change in gene expression during time. Different pathways such as sugars metabolism, cuticular wax biosynthesis, and brassinosteroids signaling took part in plant responses.

Abiotic stresses strongly affect plant growth, development, and quality of production; final crop yield can be really compromised if stress occurs in plants’ most sensitive phenological phases. Additionally, the increase of crop stress tolerance through genetic improvements requires long breeding programmes and different cultivation environments for crop performance validation. Biostimulants have been proposed as agronomic tools to counteract abiotic stress. Indeed, these products containing bioactive molecules have a beneficial effect on plants and improve their capability to face adverse environmental conditions, acting on primary or secondary metabolism. Many companies are investing in new biostimulant products development and in the identification of the most effective bioactive molecules contained in different kinds of extracts, able to elicit specific plant responses against abiotic stresses. Most of these compounds are unknown and their characterization in term of composition is almost impossible; therefore, they could be classified on the basis of their role in plants. Biostimulants have been generally applied to high-value crops like fruits and vegetables; thus, in this review, we examine and summarise literature on their use on vegetable crops, focusing on their application to counteract the most common environmental stresses.

Several viral infections of swine are responsible for major economic losses and represent a threat to the swine industry worldwide. New tools are needed to prevent and control endemic, emerging, and re-emerging viral diseases. Dendritic cells (DC) play a central role in linking the innate and adaptive arms of the immune system, so knowledge regarding their interaction with pathogens is necessary to understand the mechanisms underlying diseases pathogenesis and protection. In the first part of this review, we provide an update on the heterogeneous cell subsets that comprise the porcine DC family. In the second part of this review, we provide an overview of how three viruses, affecting pork production at a global level, African swine fever virus (ASFV), classical swine fever virus (CSFV), and porcine circovirus 2 (PCV2), modulate DC function.

Vaccination with live attenuated classical swine fever virus (CSFV) vaccines induces a rapid onset of protection which has been associated with virus-specific CD8 T cell IFN-γ responses. In this study, we assessed the specificity of this response, by screening a peptide library spanning the CSFV C-strain vaccine polyprotein to identify and characterise CD8 T cell epitopes. Synthetic peptides were pooled to represent each of the 12 CSFV proteins and used to stimulate PBMC from four pigs rendered immune to CSFV by C-strain vaccination and subsequently challenged with the virulent Brescia strain. Significant IFN-γ expression by CD8 T cells, assessed by flow cytometry, was induced by peptide pools representing the core, E2, NS2, NS3 and NS5A proteins. Dissection of these antigenic peptide pools indicated that, in each instance, a single discrete antigenic peptide or pair of overlapping peptides was responsible for the IFN-γ induction. Screening and titration of antigenic peptides or truncated derivatives identified the following antigenic regions: core₂₄₁₋₂₅₅ PESRKKLEKALLAWA and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY, or minimal length antigenic peptides: E2₉₉₆₋₁₀₀₃ YEPRDSYF, NS2₁₂₂₃₋₁₂₃₀ STVTGIFL and NS5A₃₀₇₀₋₃₀₇₈ RVDNALLKF. The epitopes are highly conserved across CSFV strains and variable sequence divergence was observed with related pestiviruses. Characterisation of epitope-specific CD8 T cells revealed evidence of cytotoxicity, as determined by CD107a mobilisation, and a significant proportion expressed TNF-α in addition to IFN-γ. Finally, the variability in the antigen-specificity of these immunodominant CD8 T cell responses was confirmed to be associated with expression of distinct MHC class I haplotypes. Moreover, recognition of NS₁₂₂₃₋₁₂₃₀ STVTGIFL and NS3₁₉₀₂₋₁₉₁₂ VEYSFIFLDEY by a larger group of C-strain vaccinated animals showed that these peptides could be restricted by additional haplotypes. Thus the antigenic regions and epitopes identified represent attractive targets for evaluation of their vaccine potential against CSFV.

Salicylidene acylhydrazides identified as inhibitors of virulence-mediating type III secretion systems (T3SSs) potentially target their inner membrane export apparatus. They also lead to inhibition of flagellar T3SS-mediated swimming motility in Salmonella enterica serovar. Typhimurium. We show that INP0404 and INP0405 act by reducing the number of flagella/cell. These molecules still inhibit motility of a Salmonella ΔfliH-fliI-fliJ/flhB((P28T)) strain, which lacks three soluble components of the flagellar T3S apparatus, suggesting that they are not the target of this drug family. We implemented a genetic screen to search for the inhibitors' molecular target(s) using motility assays in the ΔfliH-fliI/flhB((P28T)) background. Both mutants identified were more motile than the background strain in the absence of the drugs, although HM18 was considerably more so. HM18 was more motile than its parent strain in the presence of both drugs while DI15 was only insensitive to INP0405. HM18 was hypermotile due to hyperflagellation, whereas DI15 was not hyperflagellated. HM18 was also resistant to a growth defect induced by high concentrations of the drugs. Whole-genome resequencing of HM18 indicated two alterations within protein coding regions, including one within atpB, which encodes the inner membrane a-subunit of the F(O)F(1)-ATP synthase. Reverse genetics indicated that the alteration in atpB was responsible for all of HM18's phenotypes. Genome sequencing of DI15 uncovered a single A562P mutation within a gene encoding the flagellar inner membrane protein FlhA, the direct role of which in mediating drug insensitivity could not be confirmed. We discuss the implications of these findings in terms of T3SS export apparatus function and drug target identification.

Bluetongue virus (BTV) is the causative agent of a major disease of livestock (bluetongue). For over two decades, it has been widely accepted that the 10 segments of the dsRNA genome of BTV encode for 7 structural and 3 non-structural proteins. The non-structural proteins (NS1, NS2, NS3/NS3a) play different key roles during the viral replication cycle. In this study we show that BTV expresses a fourth non-structural protein (that we designated NS4) encoded by an open reading frame in segment 9 overlapping the open reading frame encoding VP6. NS4 is 77-79 amino acid residues in length and highly conserved among several BTV serotypes/strains. NS4 was expressed early post-infection and localized in the nucleoli of BTV infected cells. By reverse genetics, we showed that NS4 is dispensable for BTV replication in vitro, both in mammalian and insect cells, and does not affect viral virulence in murine models of bluetongue infection. Interestingly, NS4 conferred a replication advantage to BTV-8, but not to BTV-1, in cells in an interferon (IFN)-induced antiviral state. However, the BTV-1 NS4 conferred a replication advantage both to a BTV-8 reassortant containing the entire segment 9 of BTV-1 and to a BTV-8 mutant with the NS4 identical to the homologous BTV-1 protein. Collectively, this study suggests that NS4 plays an important role in virus-host interaction and is one of the mechanisms played, at least by BTV-8, to counteract the antiviral response of the host. In addition, the distinct nucleolar localization of NS4, being expressed by a virus that replicates exclusively in the cytoplasm, offers new avenues to investigate the multiple roles played by the nucleolus in the biology of the cell.