Interferon-gamma response of PBMC indicates productive pseudorabies virus (PRV) infection in swine

In Chinese Meishan/German Landrace cross-bred swine F2 generation interferon gamma (IFN-gamma) production by peripheral blood mononuclear cells (PBMC) was determined directly ex vivo at different time points after survival of a virulent pseudorabies virus (PRV) infection. This reactivity was compared with the reactivity of na?ve PBMC. Significant IFN-gamma production was determined in ELISA and ELISPOT only after in vitro PBMC re-stimulation with PRV and not with the closely related bovine herpesvirus BHV-1. The PRV-specific IFN-gamma secretion from re-stimulated PBMC showed high levels 6 days after infection, before the presence of serum antibodies, and it persisted at a high level over a 3 months period. The response of a group of eight piglets infected intranasally with PRV varied. Only two animals showed the expected typical fever response. PRV specific IFN-gamma production by PBMC clearly indicated that infection had occurred. Early significant IFN-gamma production by primed PBMC turned out to be a reliable and specific ex vivo marker for cellular response against productive PRV infection in swine before antibody formation.     

Virus antigen expression and alterations in peripheral blood mononuclear cell subpopulations after classical swine fever virus infection.

Depletion in the number of lymphocytes and viral persistence are thought to be the most important outcomes of classical swine fever virus (CSFV) infection. To define the change in peripheral blood mononuclear cells (PBMC) and virus replication in leukocytes after CSFV infection, 8-week old pigs were infected with the LPC vaccine strain or virulent CSFV (HCV-YL strain). Changes in the relative number of PBMCs were analyzed by flow cytometry. The results showed a significant increase in the relative percentage of monocytes in PBMCs during acute CSFV infection of naive pigs (p < 0.05). Monocyte frequencies were not changed in LPC-vaccinated pigs and control pigs. There was also a significant decrease in the number of IgM+ cells (p < 0.05) and a slight decrease in the number of CD4+ lymphocytes after 5 days of infection. There was no change in the frequency of CD8+ lymphocytes in PBMCs after infection. To define which subpopulation of PBMCs was the target for CSFV infection, PBMC populations from CSFV infected pigs were separated and stained for virus antigen expression. Alveolar macrophages (AM) were also studied. The results showed that CSFV replicated in all PBMC subpopulations: CD4+, CD8+, and IgM+ lymphocytes, and monocytes as well as AMs. However, virus antigen expression was more intense in monocytes and AMs. The infection of lymphocytes may, therefore, contribute to the depletion in their numbers after infection and lead to defective antibody production during virulent CSFV infection.     

Immunomodulation of caprine lentiviral infection by interleukin-16

Interleukin-16 (IL-16) is a proinflammatory cytokine produced by a variety of cells including lymphocytes, macrophages, mast cells, and eosinophils. We have shown in our previous studies increased expression of IL-16 mRNA and protein in caprine arthritis-encephalitis virus (CAEV)-infected goats blood. In this study, we determined the immunomodulatory effects of IL-16 in vitro using cells derived from CAEV infected and uninfected goats. Human recombinant IL-16 (rhIL-16) significantly increased chemotaxis of peripheral blood mononuclear cells (PBMCs) of both control and CAEV-infected goats. Pretreatment of PBMC with anti-goat CD4 monoclonal antibody inhibited IL-16-induced chemotaxis of PBMC of control and infected goats suggesting that IL-16 exerts its action in goats primarily by binding to CD4. The CAEV proviral DNA was less in caprine monocytes treated with rhIL-16 infected in vitro with CAEV. These data suggest inhibitory effect of IL-16 on viral integration. Flow cytometric studies indicated a trend toward IL-16-induced increased expression of lymphocyte activation markers. Combined with our previously reported data, these experiments suggest that increased IL-16 expression during CAEV infection may inhibit viral integration.     

猪外周血T淋巴细胞增殖反应MTT检测方法的建立

T细胞增殖反应是宿主T细胞识别病原的结果,也是宿主细胞免疫应答的重要指标之一。为了便于检测猪群在病原感染或者疫苗免疫过程中产生的细胞免疫应答,本研究应用MTT法建立了体外检测猪外周血T细胞增殖反应的研究方法。通过密度梯度离心法从外周血分离得到外周血单个核细胞（PBMC）,然后利用单核细胞和淋巴细胞不同的生长特性（贴壁与否）,弃掉贴壁的单核细胞,获得外周血淋巴细胞(PBL)。外周血淋巴细胞的流式分析结果显示,分离获得的PBL中T细胞所占比例达到了80%以上。应用MTT法分析了非特异性刺激物刀豆蛋白A（ConA）的浓度和细胞培养密度对T细胞增殖的影响。结果显示,ConA的工作浓度为5 μg/mL、细胞培养密度为2×106/mL时T细胞的增殖反应最强烈。本研究所建立的猪外周血T细胞增殖反应检测法可以为研究猪针对病原或疫苗的细胞免疫反应提供参考。     

Classical swine fever virus in plasma and peripheral blood mononuclear cells of acutely infected swine

The distribution of classical swine fever virus (CSFV) in plasma, monocytes, T and B lymphocytes in peripheral blood was monitored during experimentally induced acute classical swine fever infection in piglets. Six piglets were infected with 10(3.8) TCID50 of virus and blood samples taken up to 18 days post-inoculation (p.i.). Infectious virus was detected in monocytes, T and B lymphocytes to similar titres in five of the six infected piglets. Infectious virus was detected earlier in plasma than in any of the mononuclear cell subpopulations. No significant difference was observed in the period of time in which virus could be isolated from the three cell subpopulations. While a progressive lymphopenia developed, a marked B cell depletion was observed. However, B cells were apparently replaced by non-IgM-bearing mononuclear cells, as the proportion 'total lymphocyte/total leucocytes' remained unaltered throughout the experiment. Virus titres in plasma and peripheral blood mononuclear cells showed a tendency to increase as the disease progressed to its outcome.     

Increased susceptibility of peripheral blood mononuclear cells to equine herpes virus type 1 infection upon mitogen stimulation: a role of the cell cycle and of cell-to-cell transmission of the virus

Equine herpesvirus-1 (EHV-1) is an important pathogen of horses, causing abortion and nervous system disorders, even in vaccinated animals. During the cell-associated viremia, EHV-1 is carried by peripheral blood mononuclear cells (PBMC), mainly lymphocytes. In vitro, monocytes are the most important fraction of PBMC in which EHV-1 replicates, however, mitogen stimulation prior to EHV-1 infection increases the percentage of infected lymphocytes. The role of the cell cycle in viral replication and the role of cluster formation in cell-to-cell transmission of the virus were examined in mitogen-stimulated PBMC. Involvement of the cell cycle was examined by stimulating PBMC with ionomycin/phorbol dibutyrate (IONO/PDB) during 0, 12, 24 and 36 h prior to inoculation. Cell cycle distribution at the moment of inoculation and the percentage of EHV-1 antigen-positive PBMC at 0, 12 and 24 hours post inoculation (hpi) were determined by flow cytometry and immunofluorescence microscopy, respectively. The role of clusters was examined by immunofluorescence staining within clusters of stimulated PBMC using antibodies against EHV-1. Significant correlations were found between the increase of cells in the S- or G2/M-phase after a certain time interval of prestimulation and the increase of EHV-1 antigen-positive cells. The percentage of clusters with adjacent infected cells significantly increased from 3.3% at 8 hpi to 23.7% at 24 hpi and the maximal number of adjacent infected cells increased from 2 to 7. Addition of anti-EHV-1 hyperimmune serum did not significantly alter these percentages. Mitogen stimulation favours EHV-1 infection in PBMC by: (i) initiating cell proliferation and (ii) inducing formation of clusters, thereby facilitating direct cell-associated transmission of virus.     

In vivo and in vitro effects of moderately virulent African swine fever virus on mitogenesis of pig lymphocytes

Six pigs were infected oro-nasally with a moderately virulent African swine fever (ASF) virus from the Dominican Republic (DR II). The effect of virus infection on the pig's immune system was tested by measuring peripheral leucocyte numbers and the ability of mononuclear leucocytes (MNL) to respond by lymphocyte proliferation (LP) to the mitogens phytohemagglutinin-P (PHA-P), concanavalin-A (Con-A), and pokeweed mitogen (PWM). All 6 pigs developed high viremias between 4 and 18 days post-inoculation (DPI) which became undetectable by 32 to 46 DPI. Virus was found in erythrocytes, plasma, and mononuclear leucocytes from peripheral blood. Overall, virus infection had only minor effects on the number of circulating leucocytes, lymphocytes, monocytes and granulocytes. At the early acute phase of infection slight neutrophilia and lymphocytopenia were observed with mildly elevated monocyte numbers and slightly depressed neutrophil numbers that continued from the time of evident reduction in viremia to beyond the period of viral clearance. The infected pigs readily produced high titers of ASF virus antibody shortly after the onset of viremia. No significant differences in LP responses of MNL from the 6 pigs to PHA-P, Con-A and PWM were observed after infection when compared to those obtained with MNL from normal pigs. The in vitro addition of infectious ASF virus to MNL from normal pigs did not affect LP responses to any of the three mitogens. These results do not support the hypothesis that immunosuppression is a consequence of ASFV infection of pigs.     

Suppression of the proliferation of mouse splenocytes by pseudorabies virus

Pseudorabies virus (PRV) infection in resistant swine caused immunosuppression which sometimes resulted in secondary infection by other viruses or bacteria. However the mechanism of the immunosuppression is not well understood. In this study, the effect of PRV on the immune system was examined in the mouse model. Splenocytes or lymphocytes prepared from the spleen of BALB/c mice were incubated in vitro with mitogen, and the ability of cells to proliferation was measured. When the cells were incubated with PRV, the ability of cells to proliferate was inhibited, although PRV did not multiply in the lymphocytes. UV-inactivated PRV also suppressed the proliferation of mice splenocyte. This result suggests that the structural component of PRV virion might cause the immunosuppression.     

Are Low‐Density Granulocytes the Major Target Cells of Classical Swine Fever Virus in the Peripheral Blood?

The target cells of classical swine fever (CSF) virus in the peripheral blood of pigs infected with recent field isolates from Germany were studied. Eight weaned pigs were inoculated oronasally with the CSF virus field isolate Visbek/Han 95 and three weaners were inoculated with the isolate Losten/Freese 98. All pigs showed severe clinical signs typical of CSF and died or had to be euthanized between 9 and 24 days post‐infection (dpi). The first cells in the peripheral blood which became infected with CSF virus were mixed granulocytes (a combination of low‐ and high‐density granulocytes). These cells yielded the highest infectivity for PK 15 cell cultures. On day 7 post‐infection, the peripheral blood mononuclear cell (PBMC) fraction was virus positive, while the peripheral blood leucocyte (PBL), peripheral blood T lymphocyte (PBT) and high‐density granulocyte fractions were either negative or their infectivity was lower than the infectivity of the PBMC fraction. These results indicate that PBMC contain more virus‐positive cells than other fractions of leucocytes. These findings may also have diagnostic implications for the detection of CSF virus in blood samples. Because PBMC showed the highest infectivity in the early stages of CSF, it should be the sample of choice for CSF virus isolation.     

Humoral immune response to immediate-early protein of pseudorabies virus in swine with induced or naturally acquired infection

Pseudorabies virus (PRV) immediate-early (IE) protein is a nonglycosylated polypeptide localized in the nuclei of infected cells. The IE protein is a regulatory protein that is only synthesized during viral replication and is presented to the immune system of PRV-infected swine. Antibodies to the IE protein were demonstrated in swine with induced or naturally acquired infection. However, antiserum raised against purified IE protein could not neutralize PRV in vitro.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.     

The immune response against Chlamydia suis genital tract infection partially protects against re-infection

The aim of the present study was to reveal the characteristic features of genital Chlamydia suis infection and re-infection in female pigs by studying the immune response, pathological changes, replication of chlamydial bacteria in the genital tract and excretion of viable bacteria. Pigs were intravaginally infected and re-infected with C. suis strain S45, the type strain of this species. We demonstrated that S45 is pathogenic for the female urogenital tract. Chlamydia replication occurred throughout the urogenital tract, causing inflammation and pathology. Furthermore, genital infection elicited both cellular and humoral immune responses. Compared to the primo-infection of pigs with C. suis, re-infection was characterized by less severe macroscopic lesions and less chlamydial elementary bodies and inclusions in the urogenital tract. This indicates the development of a certain level of protection following the initial infection. Protective immunity against re-infection coincided with higher Chlamydia-specific IgG and IgA antibody titers in sera and vaginal secretions, higher proliferative responses of peripheral blood mononuclear cells (PBMC), higher percentages of blood B lymphocytes, monocytes and CD8⁺ T cells and upregulated production of IFN-γ and IL-10 by PBMC.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-014-0095-6) contains supplementary material, which is available to authorized users.     

Functional impairment of PRRSV-specific peripheral CD3+CD8high cells

The replication of porcine reproductive and respiratory syndrome virus (PRRSV) in lungs and lymphoid tissues of PRRSV-infected pigs is already strongly reduced before the appearance of neutralizing antibodies, indicating that other immune mechanisms are involved in eliminating PRRSV at those sites. This study aimed to determine whether PRRSV Lelystad virus (LV)-specific cytotoxic T-lymphocytes (CTL) can efficiently eliminate PRRSV-infected alveolar macrophages. Therefore, CTL assays were performed with PRRSV-infected alveolar macrophages as target cells and autologous peripheral blood mononuclear cells (PBMC) from PRRSV-infected pigs as a source of PRRSV-specific CTL. PBMC of 3 PRRSV-infected pigs were used either directly in CTL assays, or following restimulation in vitro. CTL assays with pseudorabies virus (PRV) Begonia-infected alveolar macrophages and autologous PBMC, from 2 PRV Begonia-inoculated pigs, were performed for validation of the assays. In freshly isolated PBMC, derived from PRRSV-infected pigs, CTL activity towards PRRSV-infected macrophages was not detected until the end of the experiment (56 days post infection – dpi). Restimulating the PBMC with PRRSV in vitro resulted in proliferation of CD3⁺CD8^high cells starting from 14 dpi. Although CD3⁺CD8^high cells are generally considered to be CTL, CTL activity was not detected in PRRSV-restimulated PBMC of the 3 pigs until 49 dpi. A weak PRRSV-specific CTL activity was observed only at 56 dpi in PRRSV-restimulated PBMC of one pig. In contrast, a clear CTL activity was observed in PRV Begonia-restimulated PBMC, derived from PRV Begonia-infected pigs, starting from 21 dpi. This study indicates that PBMC of PRRSV-infected pigs contain proliferating CD3⁺CD8^high cells upon restimulation in vitro, but these PBMC fail to exert CTL activity towards PRRSV-infected alveolar macrophages.     

Decreased expression of L‐selectin (CD62L) on lymphocytes in enzootic bovine leukaemia

Expression of L‐selectin was determined by single‐ and two‐colour immunofluorescence on granulocytes, peripheral blood mononuclear cells (PBMC) and blasts of bovine origin by means of a monoclonal antibody IVA94 which recognizes bovine L‐selectin (CD62L). Cells were separated from peripheral blood of healthy cattle and colleagues infected with bovine leukaemia virus (BLV). BLV‐infected animals comprised lymphocytotic and non‐lymphocytotic cows. L‐selectin was expressed on 90–98 % of granulocytes in all tested animals. The percentage of PBMC expressing L‐selectin was lower in cattle with persistent lymphocytosis than in non‐lymphocytotic or BLV‐free cattle, and inversely correlated with lymphocyte counts. The ratio of B lymphocytes stained for L‐selectin was significantly decreased from 60.2 ± 1.9 % in BLV‐free cattle to 43.8 ± 3.6 and 22.5 ± 5.7 % in non‐lymphocytotic and lymphocytotic cattle, respectively. B‐lymphocytes stained for L‐selectin exhibited about 50 % reduction in L‐selectin expression in BLV‐infected cattle compared with BLV‐free cattle, as judged by the mean fluorescence intensity (MFI). The percentage of L‐selectin‐positive PBMC not bearing surface immunoglobulin M (predominantly T lymphocytes) was comparable in BLV‐free and BLV‐infected cattle. However, L‐selectin expression on T lymphocytes was reduced (about 50 %) in BLV‐infected cattle, as judged by the MFI. We suppose that BLV infection results in a decreased L‐selectin expression on lymphocytes, and accordingly, it may contribute to deregulation of the host immune system.     

Differential effects of heparin on NO and tumor necrosis factor-alpha production in bovine blood mononuclear cells stimulated with Salmonella typhimurium lipopolisaccharide.

We investigated the influence of heparin, one of the extracellular matrix (ECM) components, on nitric oxide (NO) and tumor necrosis factor-alpha (TNF-alpha) production by bovine peripheral blood mononuclear cells (PBMC) and monocytes left to adhere for 2 (freshly adherent monocytes) and 48 h (resting monocytes), activated with Salmonella typhimurium lipopolysaccharide (LPS). After 24-h stimulation with LPS, heparin (100 microg/ml) increased (by about 40%) NO production by peripheral blood mononuclear cells and by freshly adherent monocytes. However, it did not change NO synthesis by the resting monocytes. Unlike its influence on NO level, heparin diminished TNF-alpha production by PBMC and monocytes stimulated with LPS. Microscopical examination of PBMC stained with biotin-labeled heparin, showed that both lymphocytes and monocytes were able to bind this glycosaminoglycan. We suggest that heparin, as a component of ECM, modulates the early response of monocytes to exogenous stimuli.     

Effect of infections with swine fever virus on immune functions I. Response of lymphocytes from blood and lymphoid organs from infected and normal pigs to anti-immunoglobulin serum and protein A

Pigs exposed to a low-virulent strain of swine fever virus (SFV) developed an inapparent infection. At times when a transient leucopenia occurred, the peripheral blood lymphocytes (PBL) were unresponsive to the mitogenic stimulus of anti-immunoglobulin serum (anti-Ig) and protein A.Pigs lethally infected with a virulent SFV showed leucopenia and unresponsiveness of PBL to anti-Ig and protein A from 2 days post infection until death.This suggests a defect in B lymphocyte function in pigs infected with SFV. The unresponsiveness to anti-Ig appeared not to be caused by a reduced ability of lymphocytes to redistribute their receptors into caps, the presence of suppressor cells or absence of surface immunoglobulin bearing lymphocytes in the peripheral blood. A direct action of the virus itself also seemed unlikely.Lymphocytes from spleen reacted as PBL. However, lymph node cells did not lose their capability to respond to anti-Ig.These data suggest that a change in the migration pattern of anti-Ig responsive lymphocytes could account for the observed unresponsiveness of PBL and spleen lymphocytes to anti-Ig.     

Induction of proinflammatory cytokines and caspase-1 by leptin in monocyte/macrophages from holstein cows

The proliferation of peripheral blood mononuclear cells (PBMC) containing both monocyte/macrophages and T lymphocytes increased after treatment with T-cell mitogen (concanavalin A: Con A). PBMC treated with either leptin alone or combination of leptin and ConA showed enhanced proliferative activity by 10-40%, compared with those treated with ConA alone. In contrast, isolated T lymphocytes treated with leptin and ConA showed lowered proliferative activity than the ConA-treated alone, indicating that leptin induced production of some cytokines from monocyte/macrophages, that subsequently resulted in enhancement of T lymphocytes proliferation in PBMC. Among the cytokines examined, monocyte/monocytes constitutively expressed interleukin (IL)-1beta, IL-12p35, IL-18 mRNA, and faintly expressed tumor necrosis factor (TNF)-alpha and IL-12p40 mRNA. Leptin treatment augmented the monocyte/macrophages mRNA expression of only TNF-alpha and IL-12p40 to comparable levels of cells treated with lipopolysaccharide (LPS). However, leptin treatment increased monocyte/macrophages production of IL-1beta as well as TNF-alpha, and induced the mRNA expression of caspase-1, which is shown to mediate the conversion of latent pro-IL-1beta and pro-IL-18 to active forms. These results suggest that leptin directly acts on monocyte/macrophages to produce factors that induce T lymphocytes proliferation such as IL-12p35/p40 complex through IL-12p40 induction and IL-1beta/IL-18 production through caspase-1 induction.     

Effect of infection by bovine viral diarrhea virus (BVDV) in vitro on interleukin-1 activity of bovine monocytes

The effect of bovine viral diarrhea virus (BVDV) infection in vitro on the interleukin-1 (IL-1) activity of bovine monocytes was studied. Supernatants from BVDV-infected monocytes suppressed IL-1-stimulated proliferation of mouse thymocytes and masked lipopolysaccharide-stimulated IL-1 activity of bovine monocytes in the mouse comitogen thymocyte assay. Suppression of mouse thymocyte proliferation was restored by the addition of IL-1. IL-1 inhibitory activity was induced both by the prototype variants BVDV/NADL cytopathic and BVDV/NY-1 noncytopathic and by BVDV variants isolated from persistently infected cattle. Suppressed IL-1 activity was also found in supernatants from monocytes from persistently infected cattle following infection with BVDV in vitro. No differences in levels of IL-1 mRNA synthesis were detected between BVDV-infected and uninfected monocytes by RNA-cDNA hybridization. These results suggest that infection of bovine monocytes with BVDV results in the production and/or activation of a soluble inhibitor of IL-1 activity.     

A monoclonal antibody specific for bovine T cell surface antigen associated with the E-rosette receptor

From mice immunized with T lymphocyte-enriched bovine peripheral blood mononuclear cells (PBMC), a monoclonal antibody termed BLMo-12 was obtained. BLMo-12 reacted with the antigen of Mr 56,000 in lysate of T lymphocytes. This mAb was found to inhibit spontaneous rosette formation by T-bovine lymphocytes with sheep red blood cells but it did not react with B lymphocytes, monocytes, neutrophils or eosinophils. In frozen section of the thymus, BLMo-12 showed a positive staining both the cortex and the medulla. In lymph nodes, the mAb stained the T-dependent paracortex. BLMo-12 reacted with 49.9% of PBMC and 82.5% of thymocytes. Recognition of the bovine homologue of CD2 on the T lymphocyte surface by this mAb was discussed.