A highly pathogenic porcine reproductive and respiratory syndrome virus type 1 (PRRSV-1) strongly modulates cellular innate and adaptive immune subsets upon experimental infection

Highly pathogenic (HP) PRRSV isolates have been discovered within both PRRSV-1 and PRRSV-2 genotypes and investigated in recent years especially for their ability to cause extremely severe disease in conventional pig herds. The exacerbation of general and respiratory clinical signs has been attributed not only to an efficient replication (virulence) but also to the ability to dysregulate viral recognition and induce mechanisms of immune evasion or immune enhancement of humoral and cellular anti-viral responses differently from non-HP PRRSV isolates in terms of intensity and temporal onset. Thus, the understanding of the immunopathogenesis of HP PRRSV is a major concern for the study of virus biology and development of efficacious vaccines. The present study aims at addressing the modulation of relevant immune cell subsets by flow cytometry in the blood of 4- week-old pigs experimentally infected with the recently discovered PR40/2014 HP PRRSV-1.1 strain phenotypically characterized in Canelli et al. (2017) compared to pigs infected with a non-HP PRRSV isolate (PR11/ 2014) and uninfected controls. PR40 infected animals showed an early and marked reduction of pro-inflammatory CD172α+ CD14+CD16+ and CD14+CD163+ monocytes and TCRγδ+CD8α+/CD8α- lymphocytes when pigs were most infected, possibly due to a recruitment sustaining an acute inflammatory response in target tissues. The prolonged increased CD3+CD16+ NKT cell levels may sustain peripheral inflammation and/ or the anti-viral response. The late reduction (potential depletion) of γ/δ T lymphocytes and CD3+CD4+CD8α- naïve Th lymphocytes paralleled with the delayed increase of CD3+CD4+CD8α+ memory and CD3+CD4- CD8α/β+cytotoxic T lymphocytes. In addition, PR40 infection showed an early depletion of activated CD4+CD25+ T lymphocytes and Tregs together with an intense and lasting depletion of CD21+ B lymphocytes. Overall, these features demonstrate that the more severe clinical signs observed upon infection with the HP PR40 strain are sustained by remarkable changes in the peripheral blood distribution of immune cells and provide further insights into the immune regulation/immunopathogenesis induced by PRRSV-1 subtype 1 European isolates

Assessing the virucidal activity of essential oils against feline calicivirus, a non-enveloped virus used as surrogate of norovirus

Norovirus (NoV) causes serious gastrointestinal disease worldwide and is regarded as an important foodborne pathogen. Due the difficulties of in vitro cultivation for human NoV, alternative caliciviruses (i.e., feline calicivirus, FCV, or murine NoV) have long been used as surrogates for in vitro assessment of the efficacy of antivirals. Essential oils (EOs) are natural compounds that have displayed antimicrobial and antioxidant properties. We report in vitro the virucidal efficacy of four EOs, Melissa officinalis L. EO (MEO), Thymus vulgaris L. EO (TEO), Rosmarinus officinalis L. EO (REO), and Salvia officinalis L. EO (SEO) against FCV at different time contacts (10, 30 min, 1, 4 and 8 h). At the maximum non-cytotoxic concentration and at 10- and 100- fold concentrations over the cytotoxic threshold, the EOs did not decrease significantly FCV viral titers. However, MEO at 12,302.70 mu g/mL exhibited a significant efficacy decreasing the viral titer by 0.75 log10 Tissue Culture Infectious Dose (TCID50)/50 mu l after 10 min as compared to virus control. In this study, virucidal activity of four EOs against FCV, was investigated. A lack of virucidal efficacy of TEO, REO and SEO at different compound concentrations and time contacts against FCV was observed whilst MEO was able to significantly decrease FCV titer

EVALUATION OF INNATE AND ADAPTIVE IMMUNE RESPONSE IN PIGS NATURALLY INFECTED WITH MYCOPLASMA HYOPNEUMONIAE

Mycoplasma hyopneumoniae (MH) is the main causative agent of enzootic pneumonia of pigs. 49 pigs, presenting lung lesions, from 5 different herds were sampled at slaughterhouses. Lung lesions were individually scored according to Madec e Kobish (1982). MH was locally RT-PCR detected from each lung lesion according to Marois e coll. (2010) Lung samples were formalin-fixed. 5 μm paraffin sections were histologically evaluated with a score, ranging from 0 to 4, for BALT volume, severity of bronchi, bronchiolar disepithelization and subacute inflammation. Immunohistochemistry was carried out using commercial antibodies anti-MAC387 to detect macrophages, anti-CD3 for T-cells, anti CD79α for B-cells, Foxp3 for regulatory T-cells. The amount of antigen detected was scored from 0 to 4 based on the number of positive T and B cells foci per 4, 200 magnification fields. T-reg cells were individually counted in four BALT per pig. The average histological lung score ranged from 0.14 to 1.98. Results of RT-PCR were 32 positive and 17 negative samples. Lungs showed catarrhal bronco-interstitial pneumonia with increased volume of peribronchial, peribronchiolar and perivascular lymphoid tissue. 10 samples showed subacute inflammation and 22 showed lesions MH induced without secondary bacteria irruption. MAC387 positive cells were numerous in lung lesions (scored 4) affected by severe subacute inflammation while B, T and Treg cells were poorly represented. On the contrary, T cells were numerous in hyperplatic BALT MH induced (scored 3 to 4). B cells were scarcely represented in subacute inflammation; in MH induced BALT lesions were scored from 2 to 3. This study shows that Treg cells are very rare in hyperplastic BALT, suggesting a less active role of Treg cells in maintaining a low activation of immune system. Macrophages are numerous in subacute inflammation, while less in MH BALT, suggesting a low grade of apoptosis of cells of the follicle center

Immunoregulatory signal FoxP3, cytokine gene expression and IFN-γ cell responsiveness upon porcine reproductive and respiratory syndrome virus (PRRSV) natural infection

The study aims at evaluating gene expression of pro-inflammatory (IL-1β, IL-8, TNF-α), pro-immune (IFN-γ), anti-inflammatory (IL-10) cytokines and of the immunoregulatory signal FoxP3 in association with PRRSV-specific IFN-γ secreting cell (SC) responsiveness upon PRRSV natural infection. Forty PRRSV-negative pigs were assigned to two groups: 20 pigs were vaccinated at 3 weeks of age (weaning) against PRRSV (V-PRRSV) with a modified live virus vaccine (MLV) and 20 pigs were kept non-vaccinated (NV) as controls. Blood samples were collected at 3 (vaccination), 6, 8, 10, 12, 14, and 16 weeks of age. Natural infection occurred from 8 weeks of age onward in both groups and viremia lasted 8 weeks. In the early phase of infection, pro-inflammatory cytokines (IL-1β, IL-8, TNF-α) showed a delayed increase concomitant with the peak of viremia in both groups. In both groups, IL-10 peaked at 12 weeks in association with the increase of pro-inflammatory cytokines. Conversely, in vaccinated pigs (V-PRRSV), IFN-γ showed higher gene expression during the early phase of infection and a more intense secreting cell (SC) response in the late phase. Differently, gene expression of the transcription factor FoxP3, expressed by T regulatory lymphocytes (Tregs), increased significantly in controls only and was associated with the rise of the viral load. Moreover, FoxP3 levels remained significantly higher during the late phase of infection and paralleled with lower levels of IFN-γ SC detected by ELISPOT. The expression/production of immunoregulatory signals involved in Treg activation could be a promising marker to study the immunobiology of PRRSV infection