Humoral and cell-mediated immunity to Porcine Circovirus type 2 (PCV2) vaccination and natural infection

Porcine circovirus type 2 (PCV2) has been identified as the main causative agent of the postweaning multisystemic wasting syndrome (PMWS), one of the major swine diseases worldwide that is commonly referred, together with other relevant porcine diseases related to PCV2, as belonging to the porcine circovirus associated diseases (PCVD). The most important strategy to prevent and control PCV2 associated diseases, apart from management procedures and control of coinfections, is the vaccination of piglets or sows and gilts. Nowadays there are three commercial PCV2 vaccines available; even if their efficacy in reducing the viremia burden and viral-induced specific lymphoid lesions has been proved, the mechanisms by which they are able to elicit protective immunity have not been thoroughly clarified. Besides the development of humoral immunity that is generally characterised by the detection of total anti-PCV2 and virus-neutralizing antibodies, the mechanisms that allow the adaptive cell-mediated immune response to control PCV2 infection and the related diseases have not been clearly elucidated, particularly under field conditions. The present Thesis investigated the efficacy of a one-dose porcine circovirus 2 (PCV2) subunit vaccine based on the PCV2 Cap protein expressed in a baculovirus system in two different farms (farm1 and 2) at which a history of porcine circovirus-associated disease (PCVD) was present. Morbidity, mortality, average daily weight gain, carcass weight, PCV2 load in serum and vaccine immunogenicity, in terms of PCV2-specific antibodies, PCV2-specific IFN-γ secreting cell frequencies and mRNA expression profiles of relevant pro-inflammatory and immune cytokines, were assessed. Serology to potential coinfections due to porcine reproductive and respiratory syndrome virus (PRRSV) and Mycoplasma hyopneumoniae (M. hyo.) was also carried out. A double-blind, randomised, and controlled field trial was performed distributing 818 piglets in two treatment groups. At inclusion (weaning at 21±3 days of age), 408 animals received a 2-ml intramuscular dose of Porcilis PCV® (vaccinated group) suspended in a tocopherol-based adjuvant (Diluvac Forte®). Controls (410 piglets) received 2 ml of the same adjuvant alone intramuscularly. Weights were recorded at inclusion and at 12 and 26 weeks of age, and the average daily weight gain (ADWG) was calculated. The carcass weights of the pigs from farm 2 were recorded at slaughter (274 day-old pigs). All dead animals (died or culled) underwent autopsy to classify them as PMWS-affected or not. At each farm, blood samples were collected for serologic and cellular studies aimed at investigating the humoral (ELISA determination of PCV2-antibody titres in serum) and cell-mediated (ELISpot assay for the measurement of the PCV2-specific IFN-γ secreting cell frequencies in PBMC) immune response of pigs. The analyses of the present Thesis showed that vaccination with a single dose of a PCV2 Cap vaccine had beneficial effects against the PCVD, and especially PMWS. The vaccination reduced the mortality rate and morbidity, PCV2 viremia and viral load, and improved productive performances (e.g. ADWG: +70 g/day between 12 and 26 weeks of age when viremia and the specific disease occurred) as well as carcass weight at slaughter age (+4.5 kg). These effects were associated with virologic and clinical protection derived from the immunogenicity of the vaccine measured as activation of both humoral and a cellular immune responses. In this regard, ELISA quantification of PCV2-specific antibodies showed seroconversion (with exception of pigs with a titre of maternally derived antibodies >8 log2) and long lasting protective immunity in all vaccinated pigs. Furthermore, the increased frequency of IFN-γ secreting cells that was detected by the ELISpot assay during the post-vaccination period demonstrated the capability of a single dose of the PCV2 Cap-based vaccine to induce a virus-specific cell-mediated immune response. During the post-exposure period, vaccinated animals rapidly and efficiently counteracted virus spread since both humoral and cell-mediated immunity were associated with absent or low viremia and less severe clinical signs. In addition, in order to obtain more thorough information about the mechanisms of cellular immune reactivity, the evaluation of expression patterns of relevant pro-inflammatory (IL-8, TNF-α, IL-1β) and immune (IFN-γ, IL-10) cytokines was carried out. Cytokine modulation and course of viremia were assessed in 10 PCV2-vaccinated and 20 non-vaccinated pigs from farm 1. These analyses were performed by reverse transcriptional-quantitative PCR (RT-qPCR) before the onset of PCV2 viremia (16 weeks of age), upon PCV2 infection and after the onset of PMWS clinical signs (19 and 22 weeks of age, respectively). The cytokine response was evaluated with regards to evident clinical signs related to PMWS and course of viremia, grouping the animals into three groups: 1) vaccinated (PCV2-vac) pigs; 2) unvaccinated spontaneously infected/non-PMWS-affected (Ctrl) pigs; 3) unvaccinated spontaneously infected/PMWS-affected (Ctrl-PMWS+) pigs. Moreover, in order to establish an association between cytokine expression and viremia burden, each of the above mentionated groups was analysed dividing the animals in three different subgroups based on viremia: non-viremic pigs (NV), pigs with viremia <106 (V<106) and pigs with viremia ≥106 (V≥106) viral genome copy number / ml of serum. Higher IL-8, TNF-α and IFN-γ levels were detected in the PCV2-vac group, testifying a more efficient immune responsiveness, especially when compared to the Ctrl-PMWS+ group. In Ctrl-PMWS+ pigs, lower IFN-γ at 19 weeks of age was associated with high IL-10 at 19 weeks of age and low levels of pro-inflammatory cytokines at 22 weeks of age, namely IL-8 and TNF-α, a condition likely correlated with the onset of the disease. Contrarily, at 19 weeks of age, PCV2-vac and Ctrl pigs showed lower IL-10 expression, together with higher IFN-γ levels than the Ctrl-PMWS+ animals. At 22 weeks of age, vaccinated animals maintained higher levels of the pro-inflammatory cytokines. These evidences support that the outcome of PMWS could be associated with a reduction of the innate/pro-inflammatory response. Overall, the results show a different cytokine modulation in vaccinated and unvaccinated-infected pigs also developing PMWS. Vaccinated pigs coped with infection showing low or absent viremia burden, absence of PMWS disease and stronger inflammatory response and cellular IFN-γ-related reactivity

Platelet hyperaggregability in high-fat fed rats: A role for intraplatelet reactive-oxygen species production

<p>Abstract</p> <p>Background</p> <p>Adiposity greatly increases the risk of atherothrombotic events, a pathological condition where a chronic state of oxidative stress is reported to play a major role. This study aimed to investigate the involvement of (NO)-soluble guanylyl cyclase (sGC) signaling pathway in the platelet dysfunction from high fat-fed (HFF) rats.</p> <p>Methods</p> <p>Male Wistar rats were fed for 10 weeks with standard chow (SCD) or high-fat diet (HFD). ADP (10 μM)- and thrombin (100 mU/ml)-induced washed platelet aggregation were evaluated. Measurement of intracellular levels of ROS levels was carried out using flow cytometry. Cyclic GMP levels were evaluated using ELISA kits.</p> <p>Results</p> <p>High-fat fed rats exhibited significant increases in body weight, epididymal fat, fasting glucose levels and glucose intolerance compared with SCD group. Platelet aggregation induced by ADP (<it>n </it>= 8) and thrombin from HFD rats (<it>n </it>= 8) were significantly greater (<it>P </it>< 0.05) compared with SCD group. Platelet activation with ADP increased by 54% the intraplatelet ROS production in HFD group, as measured by flow cytometry (<it>n </it>= 6). N-acetylcysteine (NAC; 1 mM) and PEG-catalase (1000 U/ml) fully prevented the increased ROS production and platelet hyperaggregability in HFD group. The NO donors sodium nitroprusside (SNP; 10 μM) and SNAP (10 μM), as well as the NO-independent soluble guanylyl cyclase stimulator BAY 41-2272 (10 μM) inhibited the platelet aggregation in HFD group with lower efficacy (<it>P </it>< 0.05) compared with SCD group. The cGMP levels in response to these agents were also markedly lower in HFD group (<it>P </it>< 0.05). The prostacyclin analogue iloprost (1 μM) reduced platelet aggregation in HFD and SCD rats in a similar fashion (<it>n </it>= 4).</p> <p>Conclusions</p> <p>Metabolic abnormalities as consequence of HFD cause platelet hyperaggregability involving enhanced intraplatelet ROS production and decreased NO bioavailability that appear to be accompanied by potential defects in the prosthetic haem group of soluble guanylyl cyclase.</p

Foodborne Salmonellosis in Italy: Characterization of Salmonella enterica Serovar Typhimurium and Monophasic Variant 4,[5],12:i- Isolated from Salami and Human Patients.

Salmonella enterica serovar Typhimurium (STm) and its monophasic variant 4,[5],12:i:- (VMSTm) have been responsible for an increased number of foodborne infections in humans in Europe in recent years. The aim of this study was to investigate the origin of three foodborne salmonellosis outbreaks that occurred in Pavia Province (Lombardy region, northern Italy) in 2010. Phenotypic and genetic characteristics of the STm and VMSTm isolates from patients and from food that were recovered in the framework of the three outbreaks were evaluated through serotyping, phage typing, antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multiple-locus variable-number tandem repeat analysis (MLVA). Salami from three artisan producers, which had all purchased meat from the same slaughterhouse, was the food source of infection in outbreak I. STm isolates were recovered from salami and patients with symptoms of gastroenteritis. These isolates had the same PFGE type and the same rare MLVA profile (3-18-9-NA-211). The same molecular profiles were found in an STm isolate from a salami, which likely was the source of another family outbreak (II). A VMSTm strain with common phenotypic and molecular profiles was isolated from three hospitalized patients and identified as the cause of another putative outbreak (III). During the following 3 years (2011 through 2013), 360 salami produced in Pavia Province were monitored for the presence of S. enterica . In 2011, no STm and VMSTm isolates were recovered from 159 salami tested. During 2012 and 2013, 13.9% of 201 tested salami harbored S. enterica , and half of the isolates were VMSTm, mainly in salami from those artisan producers involved in the previous outbreaks. These isolates were genetically variable, especially in terms of MLVA profiles. The data collected suggest that from 2012, VMSTm has replaced STm in the environments of the salami producers monitored in this study, and these data confirm the dominance of this emergent serovar along the pork supply chain

Klebsiella pneumoniae carrying multiple alleles of antigen 43-encoding gene of Escherichia coli associated with biofilm formation

A clinical strain of Klebsiella pneumoniae typed as sequence type 307 carrying three different alleles of the flu gene encoding the Escherichia coli virulence factor antigen 43 associated with biofilm formation was detected and characterized. The flu alleles are located in the chromosome inside putative integrative conjugative elements. The strain displays the phenotypes associated with Ag43, i.e. bi-phasic colony morphology and enhanced biofilm production. Furthermore, the strain produces low amount of capsule known to affect Ag43 function. Analysis of 1431 worldwide deposited genomes revealed that 3.7% Klebsiella pneumoniae carry one or two flu alleles

Stereotactic body radiotherapy vs conventionally fractionated chemoradiation in locally advanced pancreatic cancer: A multicenter case‐control study (PAULA‐1)

The aim of this study was to compare two cohorts of LAPC patients treated with SBRT ± CHT vs CRT ± CHT in terms of local control (LC), distant metastases- free survival (DMFS), progression-free survival (PFS), overall survival (OS), and toxicity. Eighty patients were included. Patients in the two cohorts were matched ac- cording to: age ≤/&gt;65 years, tumor diameter (two cut-offs

Mutation of hilD in a Salmonella Derby lineage linked to swine adaptation and reduced risk to human health

Salmonella enterica variants exhibit diverse host adaptation, outcome of infection, and associated risk to food safety. Analysis of the distribution of Salmonella enterica serovar Derby (S. Derby) subtypes in human and swine identified isolates with a distinct PFGE profile that were significantly under-represented in human infections, consistent with further host adaptation to swine. Here we show that isolates with this PFGE profile form a distinct phylogenetic sub-clade within S. Derby and exhibit a profound reduction in invasion of human epithelial cells, and a relatively small reduction in swine epithelial cells. A single missense mutation in hilD, that encodes the master-regulator of the Salmonella Pathogenicity Island 1 (SPI-1), was present in the adapted lineage. The missense mutation resulted in a loss of function of HilD that accounted for reduced invasion in human epithelial cells. The relatively small impact of the mutation on interaction with swine cells was consistent with an alternative mechanism of invasion in this pathogen-host combination

Development and validation of an art-inspired multimodal interactive technology system for a multi-component intervention for older people: a pilot study

IntroductionThe World Health Organization (WHO) acknowledges the presence of a significant body of research on the positive effects of the arts on health, considering a variety of factors including physical well-being, quality of life, and social and community impact. The model that underlies cultural welfare puts the performing arts, visual arts, and cultural heritage at the service of people personal and societal well-being. The potential connections between movements of the body and artistic content have been extensively studied over time, considering movement as a non-verbal language with a universal character.MethodsThis pilot study presents the results of the validation of an innovative multimodal system, the DanzArTe-Emotional Wellbeing Technology, designed to support active and participative experience of older people providing physical and cognitive activation through a full-body physical interaction with a traditional visual work of art of religious subject. DanzArTe supports a replicable treatment protocol for multidimensional frailty, administered through a low cost and scalable technological platform capable of generating real-time visual and auditory feedback (interactive sonification) from the automated analysis of individual as well as joint movement expressive qualities. The study involved 45 participants, 23 of whom participated in the DanzArTe program and 22 who were included in the control group.ResultsThe two groups were similar in terms of age (p = 0.465) and gender (p = 0.683). The results showed that the DanzArTe program had a positive impact on participants' self-perceived psychological health and well-being (Mean Psychological General Well-Being Index—Short T1 = 19.6 ± 4.3 Vs. T2 = 20.8 ± 4.9; p = 0.029). The same trend was not observed in the control group (p = 0.389).DiscussionThe findings suggest that such programs may have a significant impact particularly on the mental and social well-being of older adults and could be a valuable tool for promoting healthy aging and improving quality of life

One dose of a porcine circovirus 2 subunit vaccine induces humoral and cell-mediated immunity and protects against porcine circovirus-associated disease under field conditions

International audienceThis study investigated the efficacy of a one-dose porcine circovirus 2 (PCV2) subunit vaccine based on the PCV2 Cap protein expressed in a baculovirus system on two different farms at which a history of porcine circovirus-associated disease (PCVD) was present. Morbidity, mortality, average daily weight gain, carcass weight, PCV2 load in serum and vaccine immunogenicity were assessed. Serology to porcine reproductive and respiratory syndrome virus (PRRSV) and was performed. A double-blind, randomised, and controlled field trial was performed distributing 818 piglets between two treatment groups. At inclusion (weaning at 21±3 days of age), 408 animals (group B) received a 2-mL intramuscular dose of Porcilis PCV (vaccinated group). Controls (group A, 410 pigs) received 2mL of the adjuvant Diluvac Forte intramuscularly. Weights were recorded at inclusion and at 12 and 26 weeks of age, and the average daily weight gain (ADWG) was calculated. The carcass weights of the pigs from farm 2 were recorded at slaughter (274 days old). All dead animals (died or culled) underwent autopsy to classify them as PMWS-affected or not. At each farm, blood samples were taken from 22 pigs/group for serologic studies. A beneficial effect was found after vaccination with a single dose of a PCV2 Cap vaccine against PCVD. The vaccination reduced the mortality rate and morbidity, reduced PCV2 viremia and viral load, improved productive performances (e.g. ADWG: +70g/day between 12 and 26 weeks of age when viremia and the specific disease occurred) as well as carcass weight at slaughter age (+4.5kg). These effects were associated with virologic and clinical protection from the immunogenicity of the vaccine measured as activation of both a humoral and a cellular immune response