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 PCV2a was the first genotype detected with the highest frequency from the late 1990s to 2000, which was then superseded by PCV2b (first genotype shift).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.

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.

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.

Molecular detection of Theileria sergentii/orientalis/buffeli and Ehrlichia canis from aborted ovine and caprine products in Sardinia, Italy.

The economic impact and losses caused by abortion of small ruminants represent an important threat to livestock industry worldwide. Infectious agents are the most commonly causes of small ruminant abortion and many of which pose a serious threat to human health. The management of abortion outbreaks is essential to understand the transmission, prevention and control of the zoonotic diseases. This study aimed to increase the knowledge about the common known zoonotic pathogens causing abortion (Coxiella burnetii, Chlamydia abortus and Toxoplasma gondii) circulating in Sardinia. In addition, the occurrence of other infectious agents that, until now, had never been identified in abortion samples and which might be cocirculating during the abortion outbreaks were also considered. In this study, 125 abortion samples collected from 91 small ruminant farms were screened for the presence of Babesia/Theileria spp., Ehrlichia canis, Anaplasma spp., Chlamydia spp., C. burnetii and T. gondii by PCR analyses and sequencing. This is the first evidence on the presence of Theileria sergenti/orientalis/buffeli group and Eh. canis in 22 (22/125; 18%) and 26 (26/125; 21%) abortion products from small ruminants, respectively. Chlamydia abortus, C. burnetii and T. gondii were also detected in brain, liver, spleen and placentae at 46% (58/125), 34% (42/125) and 2% (2/125), respectively. This study highlights that pathogens with epizootic and zoonotic potential are circulating in the island and could be involved directly or in association with other pathogens as possible cause of ruminant abortion. Further studies are needed to fully assess the impact of Theileria sergenti/orientalis/buffeli group and Eh. canis on ruminant abortion and their real zoonotic risk in the island.

Identification of Echinococcus granulosus Genotypes G1 and G3 by SNPs Genotyping Assays.

Echinococcus granulosus sensu lato (s.l.) is the causative agent of cystic echinococcosis in animals and humans. Different E. granulosuss.l. genotypes exhibit great diversity in their life cycle, host selectivity and pathogenicity. For this reason, the study of genetic variation within Echinococcus species is of importance for their epidemiological implication. We employed two SNP genotyping technologies to distinguish G1 and G3 E. granulosus sensu stricto (s.s.). genotypes. The genotypes of DNA samples (n = 28) extracted from hydatid cysts of different animal species were identified by amplification and sequencing of a fragment of the mitochondrial nad5 gene. Two SYBR green and three TaqMan real time PCR assays were developed for targeting of three nad5 informative positions (SNP758, 1123, and 1380) known to be able to discriminate G1 from G3. Genotyping by SYBR Green PCR based on cycle threshold (Ct) with melting temperature (Tm) analysis and performed on SNP1123 and SNP1380 failed to identify one DNA sample. TaqMan assays for SNP758, 1123 and 1380 effectively confirmed genotype identification obtained by Sanger sequencing. Our results demonstrated that the combination of the three Taqman assays developed in this study represents a valuable and cost effective tool alternative to DNA sequencing for E. granulosus s.s. genotyping.

Coxiellaceae in Ticks from Human, Domestic and Wild Hosts from Sardinia, Italy: High Diversity of Coxiella-like Endosymbionts.

PURPOSE: Coxiella burnetii is known for its potential as veterinary and human bacterial pathogen. The bacteria have been described in ticks, but their role in transmission of Q fever in humans is considered low. Coxiella endosymbionts closely related to C. burnetii have been also isolated from an extensive range of tick species and evidence is growing that these endosymbionts could be linked to human bacteremia. The aim of this study was to get new information on the presence of Coxiella species in ticks infesting wild and domestic hosts in Sardinia, Italy. METHODS: Here, 138 ticks collected from the study area were analyzed for the presence of C. burnetii and Coxiella-like bacteria by polymerase chain reaction (PCR), sequencing and philogenetic analyses using a set of primers targeting the 16S rRNA gene. RESULTS: DNA of Coxiella species was detected in 69% of the total ticks examined. Based on phylogenetic analysis, the 16S rRNA Coxiella genotypes identified in this study grouped in strongly supported monophyletic clades with identified reference sequences of CLEs detected from Rhipicephalus, Dermacentor, Haemaphysalis and Ornithodoros species and with Coxiella burnetii strains isolated worldwide. CONCLUSION: This study reports the molecular detection of a high diversity of Coxiella-like bacteria in Sardinian ticks and confirms also the presence of C. burnetii in tick species previously identified in the island. The role that Coxiella-like endosymbionts play in Sardinian ticks and in their vertebrate hosts needs to be explored further.