Porcine Immune Response Research Articles

Cloning and identification of porcine programmed death 1

Programmed death 1 (PD-1) is a member of the immunoglobulin (Ig) superfamily, which is expressed on activated T cells, B cells and monocytes. Many researches have demonstrated that a high level of PD-1 expression is closely related to persistent infection and immune evasion in some human infections. In order to study the relationship between PD-1 expression and persistent infections caused by some porcine viruses, we first cloned the porcine PD-1 from porcine PBMCs based on the blast result in the EST database using the human PD-1 sequence. Sequence analysis showed that the cloned PD-1 molecule shares 63 and 54% amino acid sequence identity with human and murine PD-1, respectively. Its molecular structure is also similar to that of human and murine PD-1, containing an IgV-like domain in the extracellular region and two immune regulatory motifs in its cytoplasmic tail. The in vitro T cell proliferation assay showed that the cloned PD-1 could inhibit porcine T cell proliferation by 71% and secretion of IFN-γ and IL-2 by 64 and 53%, respectively. These data suggest that porcine PD-1 negatively regulates the porcine immune response in a similar manner to that of its counterpart in the human and mouse immune system.

Influence of conjugated linoleic acid on the porcine immune response and morbidity: a randomized controlled trial

BackgroundConjugated linoleic acid (CLA) has diverse influences on the immune response in different experimental models. In the present study we investigated the effect of CLA feeding on inflammatory and immune responses in a piglet model. We studied the duration of this effect and possible detrimental effects of CLA feeding. After 12 weeks of CLA and control supplementation and washout, animals were sacrificed and parenchymal organs were histologically examined.ResultsIn activated peripheral mononuclear cells interferon-γ was significantly (p = 0.008) lower in the CLA group by the end of the feeding period. This effect disappeared as soon as supplementation was stopped. No differences were found in the tumour necrosis factor-α, interleukin-10 production, serum immunoglobulin-G levels and fat infiltration of the liver, except that fat storage cell infiltration was significantly (p < 0.04) higher in the CLA-fed group. The effect of time for interferon-γ, interleukin-10 and immunoglobulin-G levels was statistically significant.ConclusionAt the end of the feeding period the interferon-γ response was depressed. However, the maturation of the piglet immune system in our young pig model probably outweighs the impact of CLA feeding on the immune response, even though liver fat storage cell infiltration, which plays an important role in liver regeneration, increased during CLA feeding of the piglets.

Correlating blood immune parameters and a CCT7 genetic variant with the shedding of Salmonella enterica serovar Typhimurium in swine

The porcine response to Salmonella infection is critical for control of Salmonella fecal shedding and the establishment of Salmonella carrier status. In this study, 40 crossbred pigs were intranasally inoculated with Salmonella enterica serovar Typhimurium ( Salmonella Typhimurium) and monitored for Salmonella fecal shedding and blood immune parameters at 2, 7, 14 and 20 days post-inoculation (dpi). Using a multivariate permutation test, a positive correlation was observed between Salmonella Typhimurium shedding levels at 2 and 7 dpi and serum interferon-gamma (IFNγ) levels at 2 dpi ( p < 0.05), with Salmonella being shed in greater numbers from animals with higher IFNγ levels. A positive correlation was also observed between IFNγ levels and the number of banded neutrophils (2 dpi), circulating neutrophils (7 and 14 dpi), monocytes (7 dpi), and white blood cells (WBCs) (7, 14 and 20 dpi). We have further performed association studies on these immune response parameters as well as shedding status of the Salmonella-infected pigs with a single nucleotide polymorphism (SNP) in the porcine gene CCT7, previously shown by our group to be transcriptionally up-regulated in swine experimentally inoculated with Salmonella Typhimurium. Our analyses with the 40 pigs suggest a positive association ( p = 0.0012) of SNP genotype A/G at position AK240296.c1153G > A of the CCT7 gene with Salmonella shedding at 7 dpi compared to the G/G homozygote genotype. Linking specific genes and genetic polymorphisms with the porcine immune response to Salmonella infection and shedding may identify potential markers for carrier pigs as well as targets for disease diagnosis, intervention and prevention.

β-Glucan enhancement of T cell IFNγ response in swine

Beta-glucan has been shown to enhance anti-tumor and anti-infection functions in animals. Pigs at 4 months of age were infected with porcine reproductive and respiratory syndrome virus (PRRSV), and peripheral blood monocytes (PBMC) were isolated for the detection of interferon gamma (IFNgamma)-producing cells. We found that soluble high molecular weight beta-glucan could increase IFNgamma-producing cell frequency in a dose-dependent manner in the enzyme-linked immunospot assay (ELISPOT) in the absence of antigenic restimulation. A concentration as low as 1.6 microg/ml gave a significant increase and a similarly high enhancement was achieved at concentrations from 3.2 to 100 microg/ml. In PRRSV-specific IFNgamma ELISPOT, soluble beta-glucan elicited increased PRRSV-specific responses at concentrations from 3.2 to 50 microg/ml, but not at 100 microg/ml, whereas insoluble beta-glucan had no effect. Soluble beta-glucan augmented the porcine cellular immune response in an antigen-independent fashion, whereas insoluble beta-glucan had no activity. This finding suggests that soluble beta-glucan may enhance innate antiviral immunity against PRRSV.

Th-1/Th-2 type cytokine profiles of pig T-cells cultured with antigen-treated monocyte-derived dendritic cells

To examine the effects of cytokine environment at the time of antigenic exposure on T-cell cytokine profiles following T-cell–antigen presenting cell (APC) interaction, pig monocyte-derived dendritic cells (mDCs) were treated with hen egg white lysozyme (HEWL) or killed Mycobacterium tuberculosis (Mtb) alone or with a recombinant pig cytokine (TNF-α, interleukin (IL)-12, IL-10, interferon (IFN)-γ or IL-6) and then incubated with autologous T-cell-enriched lymphocytes. Messenger RNA was isolated from the T-cells and used to evaluate the effects of treatment on IL-12p35, IFN-γ, IL-4, IL-10 and IL-13 expression using RT-PCR. T-cells exposed to HEWL-treated mDCs expressed high IL-13 and moderate IL-10 and IFN-γ, suggesting T-helper 2 (Th-2) bias. Addition of any cytokine during HEWL treatment of mDCs reduced subsequent expression of IL-10 and IL-13 by T-cells. Added IL-12 increased IFN-γ mRNA. T-cells exposed to Mtb-treated mDCs expressed increased IFN-γ and decreased IL-10 suggesting Th-1 bias. Addition of cytokines to mDCs treated with Mtb altered T-cell cytokine mRNA expression such that TNF-α, IFN-γ or IL-12 increased IFN-γ; IL-12 and IFN-γ suppressed IL-10, while IL-10 and IL-12 enhanced IL-13. Messenger RNA for IL-4 and IL-12p35 was not detected in the T-cells. Results suggest Th-1/Th-2 type response bias in pigs T-cells as a function of antigen type and that cytokine environment at the time of antigen–mDC interaction alters cytokine profiles of T-cells responding to antigen-pulsed mDCs. Hence, cytokines may allow designed steering of porcine immune response.

Experimental schistosomiasis japonica in the pig: immunohistology of the hepatic egg granuloma

Use of the pig as an animal model in schistosomiasis research is increasing, but knowledge of the porcine immune response to schistosome infection is still very limited. We investigated the immunohistology of different maturation stages of the Schistosoma japonicum egg granuloma in pigs. Liver sections from pigs experimentally infected with S.japonicum for 9, 12 or 21 weeks were examined by immunohistochemistry using a three-step streptavidin-biotin-complex/immunoperoxidase method or a two-step alkaline phosphatase-mediated system. All granulomas showed marked expression of major histocompatibility complex (MHC) class II in epithelioid macrophages and were dominated by T lymphocytes, comprising both CD4+ and CD8+ phenotypes, with consistently higher proportions noted for CD8+ cells. B lymphocytes, as identified by expression of CD21, were confined to lymphoid nodular structures primarily associated with mature granulomas. Early and mature granulomas contained numerous immunoglobulin (Ig)G+ plasma cells. Significant differences in immunohistology related to duration of infection were not observed. The results indicate that all stages of the hepatic S.japonicum egg granuloma in the pig manifests MHC class II-dependent CD4+ T cell activity concomitant with infiltration of CD8+ T cells. B cell activity preceding the effector cell stage appears to occur in granuloma-associated lymphoid nodules, whereas antibody, mainly IgG, is produced within the granuloma.

Effect of chromium tripicolinate supplementation on porcine immune response during the postweaning period.

A total of 60 pigs from 15 litters were used during two experiments to assess the effects of maternal supplementation of Cr tripicolinate on performance and immune status of the offspring during the first 42 d after weaning. Gilts were raised on diets with either 0 (-Cr) or 200 (+Cr) ppb supplemental Cr from Cr tripicolinate. Their offspring were weaned at about 23 d of age. Pigs from dams fed supplemental Cr were fed Cr-supplemented diets after weaning and pigs from unsupplemented dams were not fed supplemental Cr. Pigs were housed in groups of three according to litter origin (six -Cr and nine +Cr) and BW. Feed and water were available for ad libitum consumption. Serum was collected 24 h after birth, the day after weaning (d 0), and, subsequently, every 7 d through d 28 for measurement of total immunoglobulin (Ig) G and IgM. For the 42-d nursery period, ADG (511 and 531 g/d for -Cr and +Cr, respectively), ADFI (827 and 851 g/d), and gain:feed (0.62 and 0.62) were not affected (P > 0.15) by Cr. Total IgG was not affected (P > 0.10) by Cr on d 0 (7.6 and 7.7 mg/mL for -Cr and +Cr, respectively) or on d 7 (6.3 and 6.1 mg/mL for -Cr and +Cr, respectively), when the lowest concentrations were observed. By d 28, total IgG had risen (11.7 and 8.9 mg/mL for -Cr and +Cr, respectively) and was affected by Cr (P = 0.03). Total IgM followed a similar pattern and was lowest on d 7. In each trial, IgG at 24 h after birth was inversely related to IgG from d 14 through 28 in the nursery. Additionally, the lowest Ig concentrations occurred between 4 and 5 wk of age, rather than at weaning (23 d of age); this may represent a vulnerable period for weanling pigs. Supplementation of the weanling pig diets with Cr tripicolinate did not significantly affect performance and immune status during the postweaning period.

Effect of chromium tripicolinate supplementation on porcine immune response during the periparturient and neonatal period

A total of 36 gilts were used to assess the effects of Cr tripicolinate supplementation on immune response in sows and their offspring during the periparturient and neonatal period. Gilts were raised from weaning to reproductive age on diets with either 0 (-Cr) or 200 (+Cr) ppb supplemental Cr from CrPic. Subsequently, 22 gilts (9 -Cr and 13 +Cr) in parity 1 and 16 sows in parity 2 (7 -Cr and 9 +Cr) underwent immune status testing. Only sows that completed all procedures in parity 1 were included in parity 2. Sows were immunized with ovalbumin about 3 wk (d 0), and again 14 d later for gilts, prior to anticipated farrowing, and serum was collected on d 0 and at 14-d intervals for a total of four samples. Serum was collected from five to six pigs/litter at 24 h after birth, three or six pigs/litter the day after weaning (25 d of age) in parity 1, and three pigs/litter the day of weaning (20 d of age) in parity 2. Milk was collected at 1 h (colostrum), 6.5 d (early), and 19 d (late) after farrowing. The only effect of Cr on total immunoglobulin (Ig) concentration was on sow serum IgG (21.7 and 24.1 mg/mL for -Cr and +Cr, respectively; P = 0.08) and IgM (11.0 and 12.5 mg/mL; P = 0.06) on d 0. No effect (P > 0.15) of Cr was observed on the IgG antibody response to ovalbumin, but Cr was associated (P < 0.10) with a decreased IgM antibody response to ovalbumin beginning on d 14. In parity 2, colostral total IgG increased (80.6 and 92.4 mg/mL for parity 1 and 2, respectively; P = 0.06), which was reflected in the neonates at 24 h after birth (33.6 and 39.7 mg/mL; P = 0.01) and at weaning (7.3 and 13.3 mg/mL; P < 0.001). Supplementation of Cr tripicolinate had minimal effects on humoral antibody response of the dam or its transfer to the neonate; however, parity greatly influenced the concentrations of immunoglobulins in the milk and their transfer to the neonate.

Detection of porcine interleukin-18 by sandwich ELISA and immunohistochemical staining using its monoclonal antibodies.

We describe here the development of sandwich enzyme-linked immunosorbent assay (sandwich ELISA) and immunohistochemical staining for porcine interleukin-18 (PoIL-18) and their application to detection of PoIL-18 in vivo. Ten anti-PoIL-18 monoclonal antibodies (mAb), all of which were reactive with recombinant PoIL-18 by Western blotting, were established. Four (2-C-4, 9-H-6, 11-H-5, and 12-C-12) of 10 neutralized the biologic activity of PoIL-18 to induce interferon-y (IFN-gamma) from porcine peripheral blood mononuclear cells (PBMC). Four (2-C-4, 5-F-6, 9-H-6, and 12-C-12) of 10 were shown to be useful in immunohistochemical staining and detected PoIL-18 in Kupffer cells and macrophages in hepatic focal necrosis and macrophages in interstitial pneumonia in piglets with experimental endotoxemia using formalin-fixed, paraffin-embedded sections. A sandwich ELISA was developed using mAb 7-G-8 as a capture antibody and biotinylated mAb 5-C-5 as a detection antibody. This ELISA detected PoIL-18 with a minimum detectable concentration of 20 pg/ml and did not show cross-reactivity against PoIL-1beta, IL-8, IL-12, and IFN-gamma or murine and human IL-18. Using this ELISA, PoIL-18 was detected in the plasma and the bronchoalveolar lavage fluid (BALF) of pigs experimentally infected with Actinobacillus pleuropneumoniae. The availability of this ELISA and immunohistochemical staining for PoIL-18 may contribute to a further understanding of the role of this cytokine in various porcine immune responses and diseases.

Characterization of porcine T lymphocytes and their immune response against viral antigens

T lymphocytes play a central role in the antigen-specific immune response against various pathogens. To detect and to characterize porcine T lymphocytes, monoclonal antibodies (mAb) against leukocyte differentiation antigens had been raised and classified for their specificity. Analyses of porcine T lymphocytes with specific mAb against CD4 and CD8 differentiation antigens revealed differences in the composition of the porcine T-lymphocyte population compared to other species. In addition to the known subpopulations, CD4 +CD8 − T helper cells and CD4 −CD8 + cytolytic T lymphocytes, extra-thymic CD4 +CD8 + T lymphocytes and a substantial proportion of CD2 −CD4 −CD8 − T cell receptor (TcR)-γδ + T cells could be detected in swine. Functional analyses of porcine T-lymphocyte subpopulations revealed the existence of two T-helper cell fractions with the phenotype CD4 +CD8 − and CD4 +CD8 +. Both were reactive in primary immune responses in vitro, whereas only cells derived from the CD4 +CD8 + T-helper-cell subpopulation were able to respond to recall antigen in a secondary immune response. With regard to T lymphocytes with cytolytic activities, two subsets within the CD4 −CD8 + T-cell subpopulation could be defined by the expression of CD6 differentiation antigens: CD6 − cells which showed spontaneous cytolytic activity and CD6 + MHC I-restricted cytolytic T lymphocytes including virus-specific cytolytic T lymphocytes. These results enable now a detailed view into the porcine T-cell population and the reactivity of specific T cells involved in the porcine immune response against pathogens. Furthermore this knowledge offers the possibility to investigate specific interactions of porcine T lymphocytes with virus-specific epitopes during vaccination and viral infections.

Antigen-specific immune response of porcine T lymphocytes to various pathogens

The importance of swine in agriculture has resulted in a substantial increase in research efforts on the swine immune system during the past few years. A better knowledge of the porcine immune system is required before improved vaccination strategies, the design of more efficient vaccines and breeding for disease resistance will be able to contribute to a reduction in the extensive economic losses caused by the disabling effects of viral, bacterial and parasitic infections. T lymphocytes play a central role in the antigen-specific immune response to the various pathogens. To detect and characterise porcine T lymphocytes, monoclonal antibodies (mAbs) were raised against different leucocyte differentiation antigens and classified for specificity in two international workshops. These mAbs have enabled detailed studies to be made on specific cell populations involved in the porcine immune response to pathogens, on T lymphocytes and on the peculiarities of porcine T lymphocyte sub-populations: extra-thymic CD4+CD8+ T lymphocytes and a substantial proportion of CD2-TCR gamma delta+ T cells. Furthermore, these reagents and the increased knowledge of the immune system have allowed studies of the interactions of T lymphocyte sub-populations with regard to different pathogens and the role which these play in infections.

Porcine CD3ε: its characterization, expression and involvement in activation of porcine T lymphocytes

The cloning, characterization and expression of porcine CD3 epsilon and establishment of its role in T-cell activation using an anti-porcine CD3 epsilon monoclonal antibody, as described here, provides a first step towards a greater understanding of the porcine immune response. Porcine CD3 epsilon was cloned from a porcine T-cell cDNA library by polymerase chain reaction and found to have up to 72% identity with other CD3 epsilon chains, retaining all the necessary primary structural motifs for correct functioning of porcine CD3 epsilon. When expressed in COS7 cells porcine CD3 epsilon was an intracellularly localized, monomeric 23,000 MW protein exhibiting no evidence of N-glycosylation. A monoclonal antibody, PPT3, recognized expressed porcine CD3 epsilon and activated porcine T cells as demonstrated by stimulation of calcium mobilization, an increase in protein tyrosine phosphorylation and proliferation. These results further reaffirm and identify CD3 epsilon as an important cell surface protein involved in signal transduction of activation signals in porcine T cells.

Porcine immune responses to African swine fever virus (ASFV) infection

Immune responses mediating protection against ASFV are poorly understood. Anti-ASFV antibodies may influence the course of the clinical disease but they have never been found to neutralize the virus. Recent developments on cellular defense mechanisms, using swine protection models, and on the induction and role of some cytokines warrant further investigation on these areas.

The effect of human recombinant interleukin-2 on the porcine immune response to a pseudorabies virus subunit vaccine

The effect of human recombinant interleukin-2 (rIL-2) as an immune enhancing agent was evaluated in pigs vaccinated with a pseudorabies virus subunit vaccine (SV). Two groups of three pigs received two 25 μg doses of SV given 3 weeks apart. One group received 10 5 kg −1 day −1 of rIL-2 subcutaneously over two 5-day periods beginning on the day of the first and second vaccine inoculation. Six other pigs were immunized with two 5 μg doses of SV. Three of these pigs were treated as above with rIL-2. The effect of treatment was evaluated by comparing: the humoral response; the cell-mediated immune (CMI) response as measured by lymphocyte blastogenesis before and after virus challenge; and the weight response and virus excretion pattern after challenge with virulent pseudorabies virus (PRV). The humoral antibody response as detected by the serum virus neutralization (SN) assay and the enzyme linked immunosorbent assay (ELISA) was consistently higher in rIL-2 treated pigs than in non-treated pigs. These differences were significant ( P < 0.05) among high vaccine dose pigs prior to virus challenge when measured by the SN assay and during the anamnestic response period between days 3 and 10 after challenge when measured by both the SN assay and the ELISA. No differences were detected between treatment groups in the weight response, virus excretion pattern or the CMI response. These results suggest that human rIL-2 may have enhanced the immune response of pigs to the subunit vaccine.

Antibody response in swine to individual transmissible gastroenteritis virus (TGEV) proteins.

Transmissible gastroenteritis virus (TGEV) is a coronavirus that produces a life-threatening diarrhea in neonatal pigs. Whereas sows infected with virulent virus produce protective colostral antibody, sows vaccinated with attenuated virus are significantly less effective in passively protecting baby pigs. Therefore, an understanding of the antigenic and biochemical properties of pathogenic TGEV and the pigs’ immune response to these antigens is a critical area of TGEV research. We are in the process of characterizing the porcine immune response to major TGEV antigens in a protein A immunoprecipitation assay. Protein A is known to bind approximately 90% of the swine immunoglobulin isotypes, IgG1. and IgG2, but is less efficient in binding either swine IgM or IgA.1 This study has led to the serological identification of a new TGEV-specific low molecular weight polypeptide.KeywordsNucleocapsid ProteinConvalescent SeronTransmissible Gastroenteritis VirusFeline Infectious Peritonitis VirusInfected Cell LysateThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Heat sensitivity of porcine IgG

The sensitivity to heat of porcine IgG was studied. The serum from immunized pigs was heated at 56°C for 30 min as for decomplementation. The elution pattern of the serum proteins on an agarose gel column showed a dramatic change with the appearance of a large peak of the gel-excluded material. This peak contained mainly IgG molecules which still remained its antibody activity. This fact points to misinterpretations which can easily occur in 7S and 19S antibody recognition during the porcine immune response. Correlation is suggested of this property with the large number of interheavy chain disulfide bridges present in porcine IgG.