Phylogenetic analysis of porcine circovirus type 2 in Sardinia, Italy, shows genotype 2d circulation among domestic pigs and wild boars

Abstract Porcine circovirus type 2 (PCV2) is associated with multi-factorial syndromes, commonly known as porcine-circovirus–associated diseases, which cause severe economic losses in the swine industry worldwide. Four genotypes (PCV2a, PCV2b, PCV2c, and PCV2d) have been identified. Lately, the prevalence of PCV2d has been increasing in many countries, thereby prefiguring a global replacement of PCV2b. Wild boars are also susceptible to PCV2 infection, with virus prevalence similar to that of domestic pigs. This work was aimed at expanding the knowledge about the molecular epidemiology of PCV2 in Italy. For this purpose, we analysed 40 complete ORF-2 sequences from PCV2 strains isolated from domestic pigs and wild boars in Sardinia (Italy) over a period of 5 years (2009–2013). Phylogenetic and Bayesian analyses were performed on three data sets compiled from DNA sequences over a large geographical area. PCV2b was found to be dominant in Sardinia, whereas no PCV2a and PCV2c were found. This study indicates the presence of genotype PCV2d-2 infecting both domestic and wild pigs, thus confirming its circulation in Italy. Sardinian sequences clustered mostly with Italian isolates and with strains from China, Belgium, Croatia, Taiwan, Korea, and Portugal. Genetic variability of PCV2 in Sardinia appears to be a result of both local viral evolution and different epidemic introduction events

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

In vitro phenotypic characterisation of two genotype I African swine fever viruses with genomic deletion isolated from Sardinian wild boars

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

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

IntroductionExtracellular vesicles (EVs) are nanometric-membrane-bound sub-cellular structures, which can be recovered from milk. Milk EVs have drawn increasing interest due to their potential biomedical applications, therefore it is important to investigate their impact on key immune cells, such as macrophages.MethodsIn this work, the immunomodulatory effects of goat milk EVs on untreated (moMФ) and classically activated (moM1) porcine monocyte-derived macrophages were investigated using flow cytometry, ELISA, and gene expression assays.ResultsThese particles were efficiently internalized by macrophages and high doses (60 mg protein weight) triggered the upregulation of MHC I and MHC II DR on moMФ, but not on moM1. In moMФ, exposure to low doses (0.6 mg) of mEVs enhanced the gene expression of IL10, EBI3, and IFNB, whereas high doses up-regulated several pro-inflammatory cytokines. These nanosized structures slightly modulated cytokine gene expression on moM1. Accordingly, the cytokine (protein) contents in culture supernatants of moMФ were mildly affected by exposure to low doses of mEVs, whereas high doses promoted the increased release of TNF, IL-8, IL-1a, IL-1b, IL-1Ra, IL-6, IL-10, and IL-12. The cytokines content in moM1 supernatants was not critically affected.DiscussionOverall, our data support a clinical application of these molecules: they polarized macrophages toward an M1-like phenotype, but this activation seemed to be controlled, to prevent potentially pathological over-reaction to stressors

Olive Mill Waste-Water Extract Enriched in Hydroxytyrosol and Tyrosol Modulates Host–Pathogen Interaction in IPEC-J2 Cells

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

Comparative Phenotypic and Functional Analyses of the Effects of IL-10 or TGF-β on Porcine Macrophages

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

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-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

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-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

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