The mechanism of immune dysregulation caused by porcine reproductive and respiratory syndrome virus (PRRSV).

PRRSV is capable of evading the effective immune response, thus persisting in piglets and throughout the swine herd. We show here that PRRSV invades the thymus and causes depletion of T-cell precursors and alteration of the TCR repertoire. Developing thymocytes are affected during negative selection when they transit from the triple-negative to triple-positive stages at the corticomedullary junction just before entering the medulla. The restriction of repertoire diversification occurs in both helper and cytotoxic αß-T cells. As a result, critical viral epitopes are tolerated, and infection becomes chronic. However, not all viral epitopes are tolerated. Infected piglets develop antibodies capable of recognizing PRRSV, but these are not virus neutralizing. Further analysis showed that the lack of an effective immune response against the critical viral structures results in the absence of a germinal center response, overactivation of T and B cells in the periphery, robust production of useless antibodies of all isotypes, and the inability to eliminate the virus. Overall, the results show how a respiratory virus that primarily infects and destroys myelomonocytic cells has evolved strategies to disrupt the immune system. These mechanisms may be a prototype for how other viruses can similarly modulate the host immune system.

Characterization of Porcine Monocyte-Derived Macrophages Cultured in Serum-Reduced Medium.

The aim of this study was to establish a cell culture system for the generation of porcine monocyte-derived macrophages (MDMs) under reduced-serum conditions. Cultures based on either the Nu-Serum™ Growth Medium Supplement (NUS) or a conventional fetal bovine serum (FBS) were compared, which included the assessment of FBS from two different providers (FBS1 and FBS2). The data obtained confirmed the significant impact of culture conditions on in vitro-generated MDMs. The MDMs cultured under reduced-serum conditions showed increased levels of IL-1ß and CD86 mRNA and a proinflammatory cytokine profile, characterized by the increased mRNA expression of IL-23p19, CXCL10, and CCL5. Phagocytic and respiratory burst activities were not adversely affected. Surprisingly, the difference between the two FBSs was much more pronounced than the effect of the reduced-serum supplement. The FBS1 culture conditions gave rise to macrophages with higher surface levels of CD14, CD16, and CD163, a lower CD80 mRNA expression, and an increased induction of IL-10 gene expression. In contrast, none of these trends were observed in macrophage cultures supplemented with FBS2. Instead, the FBS2 culture showed increased levels of IL-1b and CD86 mRNA. In conclusion, reduced-serum culture is a useful tool for in vitro porcine MDM generation, in line with the current research trend of reducing FBS use in biological research.

Experimental Evidence of the Long-Term Survival of Infective African Swine Fever Virus Strain Ba71V in Soil under Different Conditions.

The survival of African swine fever virus (ASFV) on different matrices and its infectivity in wild as well as domestic swine is still a matter of interest. ASFV is resistant to environmental effects; this fact is enhanced by the presence of organic material. Therefore, the aim of this work was to determine the ability of laboratory ASFV to survive in soil at different temperatures (4 and 22 °C) and with and without the presence of blood using culture procedures. The suitability of the procedure for determining the viability and titre of the ASFV field strain by the hemadsorption method was also verified, when a higher decrease in virus infectivity in the case of clay compared with peat was demonstrated. The stability of the virus was clearly temperature-dependent, the infectious virus was detected after 112 days, and the viral DNA was still detected in the matrix 210 days after inoculation in a relatively high and stable concentration (between 106 and 107 genome equivalents/mL). Based on this knowledge, soil and other environmental samples could provide rapid and reliable information on the disease outbreak and serve as indicators of the risk posed by the affected locality.

Study of Biological Activities and ADMET-Related Properties of Novel Chlorinated N-arylcinnamamides.

A series of eighteen 4-chlorocinnamanilides and eighteen 3,4-dichlorocinnamanilides were designed, prepared and characterized. All compounds were evaluated for their activity against gram-positive bacteria and against two mycobacterial strains. Viability on both cancer and primary mammalian cell lines was also assessed. The lipophilicity of the compounds was experimentally determined and correlated together with other physicochemical properties of the prepared derivatives with biological activity. 3,4-Dichlorocinnamanilides showed a broader spectrum of action and higher antibacterial efficacy than 4-chlorocinnamanilides; however, all compounds were more effective or comparable to clinically used drugs (ampicillin, isoniazid, rifampicin). Of the thirty-six compounds, six derivatives showed submicromolar activity against Staphylococcus aureus and clinical isolates of methicillin-resistant S. aureus (MRSA). (2E)-N-[3,5-bis(trifluoromethyl)phenyl]- 3-(4-chlorophenyl)prop-2-enamide was the most potent in series 1. (2E)-N-[3,5-bis(Trifluoromethyl)phenyl]-3-(3,4-dichlorophenyl)prop-2-enamide, (2E)-3-(3,4-dichlorophenyl)-N-[3-(trifluoromethyl)phenyl]prop-2-enamide, (2E)-3-(3,4-dichloro- phenyl)-N-[4-(trifluoromethyl)phenyl]prop-2-enamide and (2E)-3-(3,4-dichlorophenyl)- N-[4-(trifluoromethoxy)phenyl]prop-2-enamide were the most active in series 2 and in addition to activity against S. aureus and MRSA were highly active against Enterococcus faecalis and vancomycin-resistant E. faecalis isolates and against fast-growing Mycobacterium smegmatis and against slow-growing M. marinum, M. tuberculosis non-hazardous test models. In addition, the last three compounds of the above-mentioned showed insignificant cytotoxicity to primary porcine monocyte-derived macrophages.

The Role of Antibodies Against the Crude Capsular Extract in the Immune Response of Porcine Alveolar Macrophages to In Vitro Infection of Various Serovars of Glaesserella (Haemophilus) parasuis.

In Glässer's disease outbreaks, Glaesserella (Haemophilus) parasuis has to overcome the non-specific immune system in the lower respiratory tract, the alveolar macrophages. Here we showed that porcine alveolar macrophages (PAMs) were able to recognize and phagocyte G. parasuis with strain-to-strain variability despite the presence of the capsule in virulent (serovar 1, 5, 12) as well in avirulent strains (serovar 6 and 9). The capsule, outer membrane proteins, virulence-associated autotransporters, cytolethal distending toxins and many other proteins have been identified as virulence factors of this bacterium. Therefore, we immunized pigs with the crude capsular extract (cCE) from the virulent G. parasuis CAPM 6475 strain (serovar 5) and evaluated the role of the anti-cCE/post-vaccinal IgG in the immune response of PAMs to in vitro infection with various G. parasuis strains. We demonstrated the specific binding of the antibodies to the cCE by Western-blotting assay and immunoprecipitation as well as the specific binding to the strain CAPM 6475 in transmission electron microscopy. In the cCE, we identified several virulence-associated proteins that were immunoreactive with IgG isolated from sera of immunized pigs. Opsonization of G. parasuis strains by post-vaccinal IgG led to enhanced phagocytosis of G. parasuis by PAMs at the first two hours of infection. Moreover, opsonization increased the oxidative burst and expression/production of both pro- and anti-inflammatory cytokines. The neutralizing effects of these antibodies on the antioxidant mechanisms of G. parasuis may lead to attenuation of its virulence and pathogenicity in vivo. Together with opsonization of bacteria by these antibodies, the host may eliminate G. parasuis in the infection site more efficiently. Based on these results, the crude capsular extract is a vaccine candidate with immunogenic properties.

Glucan particles as suitable carriers for the natural anti-inflammatory compounds curcumin and diplacone - Evaluation in an ex vivo model.

Natural compounds offer a wide spectrum of potential active substances, but often they have a poor bioavailability. To increase the bioavailability and bioactivity of the natural anti-inflammatory molecules curcumin and diplacone, we used glucan particles (GPs), hollow shells from Saccharomyces cerevisiae composed mainly of ß-1,3-d-glucan. Their indigestibility and relative stability in the gut combined with their immunomodulatory effects makes them promising carriers for such compounds. This study aimed to determine how curcumin and diplacone, either alone or incorporated in GPs, affect the immunomodulatory activity of the latter by assessing the respiratory burst response and the secretion of pro-inflammatory cytokines by primary porcine innate immune cells. Incorporating curcumin and diplacone into GPs by controlled evaporation of the organic solvent substantially reduced the respiratory burst response mediated by GPs. Incorporated curcumin in GPs also reduced GPs mediated secretion of IL-1ß and TNF-α by innate immune cells. The obtained results indicate a potentially beneficial effect of the incorporation of curcumin or diplacone into GPs against inflammation.

Effect of cysteine peptidase inhibitor of Eudiplozoon nipponicum (Monogenea) on cytokine expression of macrophages in vitro.

The gills of the common carp, whose mucosal surface belongs to the key defence mechanisms of piscine immunity, can be infested with both the larval and adult stage of Eudiplozoon nipponicum (Monogenea). Although on their own, monogeneans do not considerably compromise their hosts' health status, fish with epithelial barriers damaged in parasite feeding and attachment sites are at an increased risk of bacterial challenge with possible harmful consequences. Several studies suggest that helminth parasites of teleost fish evade and manipulate host immune system via their excretory-secretory products, but our knowledge of these processes in the monogeneans is limited. Cysteine peptidase inhibitors (CPI), which are found in the secretions of numerous parasites, often induce immunosuppression by subverting Th1 mechanisms and drawing the immune system towards a Th2/Treg response. We employed the qPCR to test the effect of recently characterised CPI of E. nipponicum (rEnStef) on the mRNA expression of pro-inflammatory cytokine TNF-α and anti-inflammatory cytokine IL-10 produced by porcine macrophages in vitro. After an initial preincubation with rEnStef, we stimulated the macrophages using LPS. By inducing a Th1 pro-inflammatory response, we imitated the immune reaction during a bacterial challenge in tissue damaged by the feeding and attachment of E. nipponicum. We observed a significant dose-dependent downregulation of the expression of TNF-α and IL-10 cytokines. The observed suppression of TNF-alpha expression by rEnStef could result in decreased pathogen control, which might in turn lead to increased rates of secondary bacterial infections in fish infected by E. nipponicum.

Dynamics and Differences in Systemic and Local Immune Responses After Vaccination With Inactivated and Live Commercial Vaccines and Subsequent Subclinical Infection With PRRS Virus.

The goals of our study were to compare the immune response to different killed and modified live vaccines against PRRS virus and to monitor the antibody production and the cell mediated immunity both at the systemic and local level. In the experiment, we immunized four groups of piglets with two commercial inactivated (A1-Progressis, A2-Suivac) and two modified live vaccines (B3-Amervac, B4-Porcilis). Twenty-one days after the final vaccination, all piglets, including the control non-immunized group (C5), were i.n., infected with the Lelystad strain of PRRS virus. The serum antibody response (IgM and IgG) was the strongest in group A1 followed by two MLV (B3 and B4) groups. Locally, we demonstrated the highest level of IgG antibodies in bronchoalveolar lavages (BALF), and saliva in group A1, whereas low IgA antibody responses in BALF and feces were detected in all groups. We have found virus neutralization antibody at DPV 21 (days post vaccination) and higher levels in all groups including the control at DPI 21 (days post infection). Positive antigen specific cell-mediated response in lymphocyte transformation test (LTT) was observed in groups B3 and B4 at DPV 7 and in group B4 at DPV 21 and in all intervals after infection. The IFN-γ producing lymphocytes after antigen stimulation were found in CD4-CD8+ and CD4+CD8+ subsets of all immunized groups 7 days after infection. After infection, there were obvious differences in virus excretion. The virus was detected in all groups of piglets in serum, saliva, and occasionally in feces at DPI 3. Significantly lower virus load was found in groups A1 and B3 at DPI 21. Negative samples appeared at DPI 21 in B3 group in saliva. It can be concluded that antibodies after immunization and infection, and the virus after infection can be detected in all the compartments monitored. Immunization with inactivated vaccine A1-Progressis induces high levels of antibodies produced both systemically and locally. Immunization with MLV-vaccines (Amervac and Porcilis) produces sufficient antibody levels and also cell-mediated immunity. After infection virus secretion gradually decreases in group B3, indicating tendency to induce sterile immunity.

Concurrent infection of monocyte-derived macrophages with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis: A role of IFNα in pathogenesis of co-infections.

Porcine reproductive and respiratory syndrome virus (PRRSV) predisposes pigs to secondary bacterial infection caused by Haemophilus parasuis. The aim of the present study was to analyse the immune response of monocyte-derived macrophages (MDMs), serving as a model of macrophages accumulating at the site of inflammation. The second part of the study was focused on the role of IFNα in the production of inflammatory cytokines in co-infected MDMs. Concurrent infection with PRRSV and H. parasuis decreased gene expression of pro-inflammatory cytokines (IL-1ß, IL-8) in MDMs in comparison with MDMs infected with PRRSV or H. parasuis alone. Our data showed that MDMs express IFNα after PRRSV infection. Thereafter, we exposed cells to the experimental addition of IFNα and a subsequent infection with H. parasuis, and detected a decreased expression/production of pro-inflammatory cytokines. Thus, we assume that IFNα, produced after PRRSV infection, could affect the immune response of monocyte-derived macrophages. Down-regulation of pro-inflammatory cytokine expression in inflammatory macrophages may allow the development of secondary bacterial infections in pigs.

Monocyte derived macrophages as an appropriate model for porcine cytomegalovirus immunobiology studies.

Porcine cytomegalovirus (PCMV) causes lifelong latent infections in swine. The pathogen is occasionally associated with inclusion body rhinitis and pneumonia in piglets, reproductive disorders in pregnant sows and respiratory disease complex in older pigs. Immunosuppressive potential of PCMV infection is discussed. Macrophages were recognised as one of target cell types where propagation of virus occurs. The aim of present study was to set up model PCMV infection of monocyte derived macrophages (MDMs) in vitro for PCMV immunobiology research. Obtained results showed that PCMV is able to infect and propagate in MDMs. Possible immunosuppressive effect of PCMV on infected macrophages was evaluated by measurement of immune relevant gene expression in MDMs. Infection decreased expression of IL-8 and TNF-α (pro-inflammatory cytokines) and increased expression of IL-10 (anti-inflammatory cytokine) on mRNA transcription level. Obtained data support hypothesis that higher sensitivity of animals to coinfection with other swine pathogens and its more severe clinical manifestations could potentially be the consequence of PCMV infection.

Concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in two types of porcine macrophages: apoptosis, production of ROS and formation of multinucleated giant cells.

Porcine reproductive and respiratory syndrome (PRRS) is one of the most significant and economically important infectious diseases affecting swine worldwide and can predispose pigs to secondary bacterial infections caused by, e.g. Haemophilus parasuis. The aim of the presented study was to compare susceptibility of two different types of macrophages which could be in contact with both pathogens during infection with PRRS virus (PRRSV) and in co-infection with H. parasuis. Alveolar macrophages (PAMs) as resident cells provide one of the first lines of defence against microbes invading lung tissue. On the other hand, monocyte derived macrophages (MDMs) represent inflammatory cells accumulating at the site of inflammation. While PAMs were relatively resistant to cytopathogenic effect caused by PRRSV, MDMs were much more sensitive to PRRSV infection. MDMs infected with PRRSV increased expression of pro-apoptotic Bad, Bax and p53 mRNA. Increased mortality of MDMs may be also related to a higher intensity of ROS production after infection with PRRSV. In addition, MDMs (but not PAMs) infected with H. parasuis alone formed multinucleated giant cells (MGC); these cells were not observed in MDMs infected with both pathogens. Higher sensitivity of MDMs to PRRSV infection, which is associated with limited MDMs survival and restriction of MGC formation, could contribute to the development of multifactorial respiratory disease of swine.

Immune response of porcine alveolar macrophages to a concurrent infection with porcine reproductive and respiratory syndrome virus and Haemophilus parasuis in vitro.

Porcine reproductive and respiratory syndrome virus (PRRSV) can predispose pigs to secondary respiratory infection with bacteria such as Haemophilus parasuis. Animals infected with both pathogens develop more severe clinical disease. The immune response of porcine alveolar macrophages (PAMs) to simultaneous infection with PRRSV and H. parasuis was analysed in vitro, describing cytokine production, expression of cell surface molecules, and production of reactive oxygen species (ROS). Concurrent infection with PRRSV and H. parasuis increased gene expression of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-8) in PAMs in comparison with PAMs infected with PRRSV or H. parasuis alone. An additive effect of dual infection on IL-1ß production was confirmed at the protein level. PAMs infected with PRRSV showed increased production of ROS compared to controls. Conversely, simultaneous infection of PAMs with PRRSV and H. parasuis decreased production of ROS, indicating the presence of an H. parasuis defence mechanism against respiratory burst. Concurrent infection of PAMs with PRRSV and H. parasuis was shown to elicit a pro-inflammatory immune response represented by significant IL-1ß production. Severe multifactorial respiratory disease in natural conditions caused by both pathogens could be the consequence of pro-inflammatory mediated immunopathology.