ABSTRACT: BACKGROUND: Porcine circovirus type 2 (PCV2) is a dominant causative agent of postweaning multisystemic wasting syndrome (PMWS), a multifactorial disease complex with putative immunosuppressive characteristics. Little is known about adaptive PCV2-specific immune responses in infected pigs. Therefore, the T and B cell responses following PCV2 infection in 3-week old SPF piglets infected with PCV2 or PCV2 plus porcine parvovirus (PPV) were studied. RESULTS: All animals were asymptomatically infected. At 7 days post infection (d p.i.), B lymphocyte and T lymphocyte numbers decreased in the dual infected, but not in the single infected piglets. At this time point a transient PCV2 viraemia was noted in the PCV2 infected groups. Antibodies against the infecting virus were detectable at day 24-28 p.i. for anti-PCV2 antibodies and at day 10 p.i. for anti-PPV antibodies, with no apparent influence of PCV2 on the early PPV antibody development. In the animals infected with PPV alone, IFN-gamma secreting cells (SC) that were not specific for PCV2 were detected by ELISPOT assay at day 7p.i. Interestingly, this response was absent in the PCV2/PPV dual infected animals. PCV2-specific IFN-gamma SC were observed in the PCV2/PPV infected group at 7 d p.i. and in the PCV2 single infected group at 21 d p.i. A reduction in the numbers of IFN-gamma SC was observed following anti-CD4 and anti-CD8 antibody treatment, suggesting roles for both CD4+41 and CD8+42 T cells in the response against PCV2 infection. This was supported by an observed increase in the percentage of IFN-gamma positive CD8hi 43 cytotoxic T cells as well as IFN-gamma positive CD8-/low 44 helper T cells after PCV2 in vitro re-stimulation. CONCLUSIONS: Infection of weaned SPF piglets with PCV2 alone or combined with PPV does not induce disease and in both cases a relatively slow anti-PCV2 antibody response and weak lymphocyte responses were found. Knowledge on such immunologica characteristics is important for both PCV2 pathogenesis and vaccination.

What genetic determinants and phenotypic traits make a Staphylococcus aureus strain successful colonizer is largely unknown. Genetic diversity and population structure of 133 S. aureus isolates from healthy generally risk-free adult carriers was investigated using four different typing methods; MLST, AFLP, DLST (double locus sequence typing) and spa-typing were compared. Carriage isolates displayed great genetic diversity which could only be fully revealed by DLST. Results of AFLP and MLST were highly concordant in delineation of genotypic clusters of closely related isolates, roughly equivalent to clonal complexes. Spa-typing and DLST provided considerably less phylogenetic information. Resolution of spa-typing was similar to AFLP and inferior to DLST. AFLP proved to be the most universal method, combining phylogeny building capacity similar to MLST with much higher resolution. However, it had lower reproducibility than sequencing-based MLST, DLST and spa-typing. We found two cases of MRSA colonization, both most likely associated with employment at health service. Out of 21 genotypic clusters detected, two most prevalent, Cluster 45 and Cluster 30 each colonized 24% of the carrier population. The number of bacteria found in nasal samples varied significantly among the clusters, but the most prevalent clusters were not particularly numerous in the nasal samples. We did not find much evidence that genotypic clusters were associated with different carriers' characteristics such as age, sex, medical conditions or antibiotics use. This may provide an empirical support for the idea that genetic clusters in bacteria are maintained in the absence of adaptation to different niches. Alternatively, carrier characteristics other than evaluated here or factors other than human hosts may exert selective pressure maintaining genotypic clusters.

Porcine circovirus type 2 (PCV2) is the causative agent of a multifactorial disease associated with immunocompromisation and co-infections. In vivo, viral DNA and antigens are found in monocytic, epithelial and endothelial cells. Of these, PCV2 replication has only been studied in monocytic cells, in which little or no replication was identified. Accordingly, PCV2 infection was studied in the endothelial cell line PEDSV.15, aortic endothelial cells, gut epithelial cells, fibrocytes and dendritic cells (DC). In all cells except DC PCV2 replication was detectable, with an increase in the levels of capsid and replicase protein. Variations in endocytic activity, virus binding and uptake did not relate to the replication efficiency in a particular cell. Furthermore, replication did not correlate to cell proliferation, although a close association of viral proteins with chromatin in dividing cells was observed. No alteration in the division rate of PCV2-infected cultures was measurable, relating to replicase expression in only a small minority of the cells. In conclusion, the broad cell targeting of PCV2 offers an explanation for its widespread tissue distribution.

Circulating myeloid cells such as plasmacytoid dendritic cells (pDC), blood DC and monocytes act as blood sentinels detecting invading pathogens through a large repertoire of expression of toll-like receptors (TLRs). Activation of these receptors is crucial to detect invading pathogens by the innate immune system. In the present work, we analysed the TLR responsiveness of fibrocytes, a blood-derived cell type of myeloid origin. Fibrocytes efficiently responded to TLR2, TLR4, and TLR7 ligands as well as to poly (I:C) or viral stimulation by producing high amount of interleukin-6. Upon virus infection of fibrocytes, IFN type I was also induced. When compared to pDC or Flt3 ligand-derived DC, fibrocytes produced 5 times and 60 times more IL-6, respectively. This response was associated with a rapid and efficient translocation of the NF-kappaB transcription factor. Analysis of the expression and functionality of TLR7 in peripheral blood leukocyte subpopulations suggested that this receptor is expressed and functional in a CD163(+) monocytic cell subpopulation containing the fibrocyte precursors. Considering the rapid entry of fibrocytes into wounds, this efficient responsiveness to TLR danger signals, reflects a potentially important role of these cells in the first line of defence against pathogen invasion following traumata.

During in vitro investigations on the interaction of classical swine fever virus (CSFV)- an immunosuppressive viral pathogen - with monocyte-derived dendritic cells (MoDC) a soluble factor with a strong anti-proliferative activity for T lymphocytes was found. This activity, with an inhibitory dilution 50%(ID(50)) of 10(3)-10(7), was induced after virus infection of monocytes differentiating into DC. UV-inactivation of the supernatants and blocking experiments with a monoclonal antibody against the E2 envelope protein of CSFV initially indicated a virus-dependency. However, further investigations including filtration and centrifugation experiments as well as antibiotic treatment demonstrated the involvement of mycoplasma. This was confirmed by a Hoechst 33258 staining, PCR and mycoplasma cultures-Mycoplasma hyorhinis was identified as the contaminant. Elucidation of a mycoplasma presence occurred under conditions in which the original virus stocks prepared in SK6 cells were negative for mycoplasma using the above tests. Moreover, conventional passage of the virus on the SK6 cells used for this purpose did not reveal any mycoplasma. It was the passage of virus in MoDC rather than SK6 cells that was required to expose the contamination. Three passages of the anti-proliferative supernatants on MoDC cultures increased the ID(50) 10(3)-fold; only when these MoDC-derived supernatants were employed was the mycoplasma contaminant also detectable on SK6 cells. In conclusion, these data demonstrate that regular testing of cell lines and virus stocks for mycoplasma does not necessarily identify their presence, and that application of passage in MoDC cultures could prove an aid for identifying initially undetectable levels of mycoplasma contamination.

Porcine circovirus type 2 (PCV2) infection of natural interferon producing cells (NIPCs) impairs the induction of interferon (IFN)-alpha and tumour necrosis factor (TNF)-alpha by cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides (ODNs), thereby preventing both their autocrine maturation and the paracrine maturation of myeloid dendritic cells (DCs). The present study shows that the PCV2-mediated inhibition of NIPCs was mediated by viral DNA, although it was independent of virus replication. The inhibitory effect of PCV2 DNA was more diversified than if it had simply targeted CpG-ODN-induced cytokines (IFN-alpha, TNF-alpha, interleukin-6, IL-12). A broad spectrum inhibition was noted, affecting responses induced by toll-like receptor (TLR)-7 and TLR9 agonists, as well as viruses including pseudorabies virus, transmissible gastroenteritis virus and classical swine fever virus. From these results, it would appear that PCV2 DNA can induce a dominant negative signal influencing independent pattern recognition receptor-induced activation cascades. Despite a concomitant internalization of PCV2 DNA and CpG-ODNs, no colocalization was observed, indicating that PCV2 DNA and CPG-ODNs may not target the same receptor. This study describes a novel modulation of the innate immune response, which would render the host more susceptible to secondary or concomitant microbial infections.

Classical swine fever (CSF) is a highly contagious and often fatal disease of pigs characterised by fever, severe leukopenia and haemorrhages. With vaccines having an importance in disease control, studies are seeking improved protein-based subunit vaccine against the virus (CSFV). In this respect, recombinant viral NS3 protein was analysed for its immunopotentiating capacity, particularly in terms of cytotoxic immune responses. NS3 was effective at inducing in vitro responses, quantified by lymphoproliferation, IFN-γ ELISPOT, flow cytometric detection of activated T cell subsets, and cytotoxic T cell assays. Peripheral blood mononuclear cells from CSFV-immune pigs could be stimulated, but not cells from naïve animals. In addition to the IFN-γ responses, induction of both CD4+ T helper cell and CD8+ cytotoxic T cells (CTL) were discernible - activation of the latter was confirmed in a virus-specific cytolytic assay. Attempts were made to translate this to the in vivo situation, by vaccinating pigs with an E2/NS3-based vaccine compared with an E2 subunit vaccine. Both vaccines were similar in their abilities to stimulate specific immune responses and protect pigs against lethal CSFV infection. Although the E2/NS3 vaccine appeared to have an advantage in terms of antibody induction, this was not statistically significant when group studies were performed. It was also difficult to visualise the NS3 capacity to promote T-cell responses in vivo. These results show that NS3 has potential for promoting cytotoxic defences, but the formulation of the vaccine requires optimisation for ensuring that NS3 is correctly delivered to antigen presenting cells for efficient activation of CTL.

Vaccination of pigs against Classical swine fever virus (CSFV) by using live-virus vaccines induces early protection before detectable humoral immune responses. Immunological analyses indicate that this is associated with T-cell activation, underlining the importance of targeting cytotoxic T-lymphocyte (CTL) responses for vaccine improvement. Antigen-presenting cells (APCs) transfected with mRNA encoding structural protein E2 or non-structural viral proteins NS3-NS4A were used to identify viral genes encoding CTL epitopes. Monocyte-derived dendritic cells (DCs) and fibrocytes served as the APCs. In vitro translation of the mRNA and microscopic analysis of transfected cells demonstrated that E2 and NS3-NS4A could be identified. APCs transfected with either of the mRNA molecules restimulated CSFV-specific T cells to produce gamma interferon and specific cytotoxic activity against CSFV-infected target cells. The presence of CTL epitopes on E2 was confirmed by using d/d-haplotype MAX cells expressing E2 constitutively as target cells in d/d-haplotype CTL assays. A potent CTL activity against E2 was detected early (1-3 weeks) after CSFV challenge. This work corroborates the existence of CTL epitopes within the non-structural protein domain NS3-NS4A of CSFV. Furthermore, epitopes on the E2 protein can also now be classified as targets for CTLs, having important implications for vaccine design, especially subunit vaccines. As for the use of mRNA-transfected APCs, this represents a simple and efficient method to identify viral genes encoding CTL epitopes in outbred populations.

Natural interferon-producing cells (NIPC), also called plasmacytoid dendritic cells, are the most potent producers of IFN-alpha in response to viral and bacterial components, serving an important function in innate immune defences. The present work demonstrates that NIPC responsiveness can be primed by immunisation, increasing their capacity to produce IFN-alpha after viral infection. NIPC isolated from pigs immunised against classical swine fever virus (CSFV), a member of the Flaviviridae, were more receptive to viral infection and produced higher levels of IFN-alpha than NIPC from immunologically naive animals. This sensitisation of NIPC was maintained for at least 8 months after immunisation. IFN-alpha production was dependent on live virus and required replication, and the "immune" NIPC responded to lower infectious doses of virus. Co-localisation of the virus with Fc(gamma)RII (CD32) in polarised structures was observed with "immune" NIPC only. This Fc(gamma)RII-dependent virus capture and sensitisation of NIPC, evidently mediated through cytophilic CSFV-specific antibodies, was inhibited by non-specifically aggregated immunoglobulin as well as by pre-formed virus-antibody complexes. In conclusion, these results demonstrate that NIPC not only represent a major player in innate immunity but are also functionally linked to the immunological memory of the adaptive immune system.

Fibrocytes (Fb) are a population of circulating leukocytes reported to be capable of presenting antigen to CD4(+) T lymphocytes. In contrast, no information is available about their capacity to stimulate CD8(+) cytolytic T lymphocyte (CTL) responses. To this end, Fb were isolated from porcine blood to investigate their ability to stimulate CTL responses using a classical swine fever virus model. The isolated Fb (referred to as primary Fb) displayed the phenotype previously reported for mouse and human Fb, particularly in terms of the surface proteins necessary for antigen presentation, major histocompatibility complex (MHC) classes I and II, and CD80/86. These primary Fb endocytosed and degraded antigen efficiently. In absence of exogenous stimuli, endocytosis and MHC II expression were lost when the Fb were passaged and cultured. Treatment of such secondary Fb with interferon-gamma (IFN-gamma) restored the MHC II expression. The primary and secondary Fb were capable of stimulating antigen-specific CD4(+) T lymphocytes relating to previous reports. In addition, an efficient stimulation of virus-specific CD8(+)CTL was measured in terms of CD8(+) T cell proliferation, IFN-gamma production, and cytotoxic activity. This was noted even at low Fb/T lymphocyte ratios, at which dendritic cells were less efficient. Although IFN-gamma pretreatment of Fb was not necessary for this function, it could enhance the Fb activity. These results demonstrate that Fb are efficient, accessory cells for the presentation of viral antigen to specific CD8(+) CTL.