Quantitative analysis of the antigen-specific IFNgamma+ T cell-mediated immune response in conventional outbred pigs: kinetics and duration of the DNA-induced IFNgamma+ CD8+ T cell response

It is now well established that antigen-specific CD8⁺ T cells play a major role in vaccine-induced immunity against intracellular pathogens and tumor cells. The detection of these immune cells in outbred animals has been hampered mainly by the need to generate individual autologous antigen-presenting cells (APCs) due to the high degree of polymorphism of the major histocompatibility complex (MHC) Class I loci. We used individually derived immature porcine dendritic cells infected with a pox-based recombinant viral vector to ex vivo stimulate PBMCs from vaccinated conventional pigs. The frequencies of antigen-specific T cells was determined by the number of IFNγ-secreting cells in a quantitative enzyme-linked immune spot (ELISPOT) assay. Using this approach we were able to rank different pseudorabies virus (PRV) vaccines strategies for their ability to prime viral-specific IFNγ⁺ T cells. Plasmid DNA has recently emerged as a promising tool with multiple applications in the field of infectious diseases, allergy and cancer. We showed for the first time in this study that DNA immunization induced a long-lived antigen-specific IFNγ⁺ T cells response in conventional pigs. Additional studies allowed us to show that these virus-specific IFNγ⁺ responding cells detected in this ELISPOT assay were MHC-restricted and comprised in the CD8^bright pig T cell subset. These new data confirm the usefulness of DNA vaccines to control diseases requiring cellular immunity in pigs.     

Analysis of T lymphocyte subsets proliferating in response to infective and UV-inactivated African swine fever viruses.

The proliferative response to infective and UV-inactivated African swine fever virus was analyzed in cells from pigs surviving an experimental infection with attenuated virus. All the pigs showed strong dose-dependent proliferative responses to both infective and UV-inactivated virus. This response was also observed when nitrocellulose-bound solubilized virus proteins were used in the assay. Heterologous isolates also induced proliferation, however it was significantly lower than that induced by the isolate used to infect the animals. The response to infective virus was blocked equally by anti-CD4 and anti-CD8 monoclonal antibodies (mAb); the response to UV-inactivated virus was almost abolished by anti-CD4 and 60% inhibited by anti-CD8 mAb. FACS analysis of 28-day T cell lines derived from peripheral blood mononuclear cells demonstrated the progressive increase of the CD8+ subset when the cells were stimulated with infective virus, whereas the stimulation with UV-inactivated virus induced the increase of both CD4+ and CD8+ subsets. In this case, the sum of CD4+ and CD8+ percentages was higher than the total percentage of T cells, suggesting the presence of cells positive for both CD4+ and CD8+.     

纳米铝胶佐剂增强猪细小病毒灭活疫苗猪体免疫的效果

将20头35日龄断奶仔猪随机分为4组,将制备的纳米铝胶佐剂猪细小病毒(Porcine parvovirus,PPV)灭活疫苗肌肉注射免疫接种,并以常规铝胶佐剂PPV灭活疫苗组、市售PPV油乳剂灭活疫苗组为疫苗对照,生理盐水组做阴性对照,应用HI和ELISA检测接种猪的体液免疫水平,流式细胞仪检测免疫猪的细胞免疫水平。体液免疫检测结果显示,PPV纳米铝胶佐剂疫苗组能显著提高机体产生抗体的速度,在首免后第2周产生有效保护抗体水平显著高于油乳剂疫苗和常规铝胶佐剂疫苗组(P0.05),在第3周其抗体水平达到峰值;细胞免疫检测结果显示,3种疫苗均能诱导机体产生细胞免疫应答,但各组之间差异不显著(P0.05)。结果表明,以纳米铝胶为佐剂制备的猪细小病毒灭活疫苗,能显著提高机体的免疫水平,并较早刺激机体产生抗体,具有较好的免疫保护效果。     

Development of the neonatal B and T cell repertoire in swine: implications for comparative and veterinary immunology

Birth in all higher vertebrates is at the center of the critical window of development in which newborns transition from dependence on innate immunity to dependence on their own adaptive immunity, with passive maternal immunity bridging this transition. Therefore we have studied immunological development through fetal and early neonatal life. In swine, B cells appear earlier in fetal development than T cells. B cell development begins in the yolk sac at the 20th day of gestation (DG20), progresses to fetal liver at DG30 and after DG45 continues in bone marrow. The first wave of developing T cells is gammadelta cells expressing a monomorphic Vdelta rearrangement. Thereafter, alphabeta T cells predominate and at birth, at least 19 TRBV subgroups are expressed, 17 of which appear highly homologous with those in humans. In contrast to the T cell repertoire and unlike humans and mice, the porcine pre-immune VH (IGHV-D-J) repertoire is highly restricted, depending primarily on CDR3 for diversity. The V-KAPPA (IGKV-J) repertoire and apparently also the V-LAMBDA (IGLV-J) repertoire, are also restricted. Diversification of the pre-immune B cell repertoire of swine and the ability to respond to both T-dependent and T-independent antigen depends on colonization of the gut after birth in which colonizing bacteria stimulate with Toll-like receptor ligands, especially bacterial DNA. This may explain the link between repertoire diversification and the anatomical location of primary lymphoid tissue like the ileal Peyers patches. Improper development of adaptive immunity can be caused by infectious agents like the porcine reproductive and respiratory syndrome virus that causes immune dysregulation resulting in immunological injury and autoimmunity.     

Immune response to an Actinobacillus pleuropneumoniae vaccine in swine

Piglets vaccinated with an inactivated tetravalent vaccine (Pleurovac 4) against Actinobacillus pleuropneumoniae serotypes 1, 2, 5 and 7, produced circulating antibodies after a first intramuscular injection and showed an anamnestic reaction after a second. The rise in antibody levels in vaccinated piglets was statistically significant when compared with those of the control group. The administration of 1 or 2 mL of vaccine did not lead to significantly different antibody levels. The specificity of the immune response is demonstrated by an increase in titer to all four serotypes in pigs given the tetravalent vaccine, but an increase in titer to only two serotypes in pigs given a bivalent vaccine (Pleurovac).     

运用BLUP选种效果的探讨

应用多性状动物模型BLUP法，经四年选育湖北省畜牧良种场外种猪的产肉性能从表型和遗传上得到显著提高，长白猪、约克夏和杜洛克体重达100千克的背膘厚分别为1997年的16.5、19.7和19.1毫米下降到2000年的13.2、15.1和15.3毫米，体重达100千克日龄分别从1997年的183.5、188.7和194.3天下降到2000年的164.3、179.7和179.5天；背膘厚的年度平均育值分别由1995年的1.24、0.95和0.92甜言蜜语米下降到2000年的-0.48、-0.29和-0.21毫米，达100千克日龄的年均育种值分别为1995年3.84、0.84和5.69天下降到2000年的-0.38、-0.30和-0.55天，两性状的育种值指数分别从1995年的67.9、67.3和60.3点提高到2000年的110.5、106.2和105.9点。     

Flow cytometric analysis of T cell response in mice infected with Cowdria ruminantium.

A 3-fold increase in the numbers of Lyt-2+ T cells in the circulating blood of mice infected and re-infected with the Welgevonden stock of Cowdria ruminantium, as determined by flow cytometry, is supportive evidence that immunity in heartwater is cell-mediated. The rise in Lyt-2+ cells only after re-infection of the mice is further evidence that the development of immunity in heartwater is dependent on the unhindered and adequate replication of C. ruminantium.     

黄芪多糖对猪瘟弱毒疫苗的免疫增强作用

本研究旨在探讨黄芪多糖对猪瘟疫苗免疫的影响.把21日龄断奶仔猪分成4组,在2～4组仔猪基础日粮中分别添加浓度为2×10-4、3×10-4、4×10-4的黄芪多糖,连续饲喂7d;第1组日粮中不添加任何药物,作为空白对照组.用药结束后用猪瘟弱毒疫苗免疫,并于免疫后第1、3、5周分别用正向间接血凝抑制试验和流式细胞术检测外周血猪瘟抗体效价及淋巴细胞亚群动态.结果显示:与对照组相比,黄芪多糖能提高仔猪外周血猪瘟抗体水平1个滴度以上,具有提高CD3+、CD4+T淋巴细胞百分数的趋势.说明黄芪多糖能提高仔猪特异性免疫力,是良好的免疫增强荆.     

Mucosal and systemic T cell response in mice intragastrically infected with Neospora caninum tachyzoites

The murine model has been widely used to study the host immune response to Neospora caninum. However, in most studies, the intraperitoneal route was preferentially used to establish infection. Here, C57BL/6 mice were infected with N. caninum tachyzoites by the intragastric route, as it more closely resembles the natural route of infection through the gastrointestinal tract. The elicited T-cell mediated immune response was evaluated in the intestinal epithelium and mesenteric lymph nodes (MLN). Early upon the parasitic challenge, IL-12 production by conventional and plasmacytoid dendritic cells was increased in MLN. Accordingly, increased proportions and numbers of TCRαβ⁺CD8⁺IFN-γ⁺ lymphocytes were detected, not only in the intestinal epithelium and MLN, but also in the spleen of the infected mice. In this organ, IFN-γ-producing TCRαβ⁺CD4⁺ T cells were also found to increase in the infected mice, however later than CD8⁺ T cells. Interestingly, splenic and MLN CD4⁺CD25⁺ T cells sorted from infected mice presented a suppressive activity on in vitro T cell proliferation and cytokine production above that of control counterparts. These results altogether indicate that, by producing IFN-γ, TCRαβ⁺CD8⁺ cells contribute for local and systemic host protection in the earliest days upon infection established through the gastrointestinal tract. Nevertheless, they also provide substantial evidence for a parasite-driven reinforcement of T regulatory cell function which may contribute for parasite persistence in the host and might represent an additional barrier to overcome towards effective vaccination.     

Intradermal testing of swine to monitor changes in delayed hypersensitivity response

Pigs were tested (intradermal injection) on the abdomen with either phytohemagglutinin (PHA) or PHA and tuberculin. The response to PHA was consistent; double-skin thickness averaged 247% of the preinjection thickness during daily testing. Pigs that were primed with Freund's complete adjuvant had a mean double-skin thickness of 208% for tuberculin and 236% for PHA, indicating that the mitogen induced an antigen-like response. Unexpectedly, injections of dexamethasone (0.1 mg/kg of body weight on 2 days) increased the reactivity to both tuberculin and PHA, indicating that the lymphocyte and macrophage response to dexamethasone in swine may be slightly different than the response reported in other species.     

Immunoblot analysis of IgG antibody response to Sarcoptes scabiei in swine

This study was performed to determine the frequencies and specificities of IgG antibodies binding to component of Sarcoptes scabiei extracts in swine with hypersensitive and chronic mange. The hypersensitive form is characterised by pruritus and the presence of small red papules over the flanks and belly. The chronic form is characterised by crusts, which contain large numbers of mites and are attached to the skin; the lesions are most commonly found on the internal pinna extending into the auditory canal. S. scabiei mite extract was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis with subsequent immunoblotting. IgG-binding proteins were detected with individual sera from 30 hypersensitive and 21 chronically infected pigs; eight "Specific Pathogen Free" pigs were used as negative controls. Seven protein bands with molecular weights ranging from >220 to 30 Kilodalton (KDD) (>220, 218, 110, 80, 66, 52, 36 KDD) strongly bound with IgG antibodies; five out of these seven components (218, 110, 80, 66, 52 KDD) bound also with sera from negative pigs. There is a statistically significant difference in the antigenic recognition spectra between hypersensitive and chronically infected pigs; component of >220 KDD is more frequently recognized by chronically infected pigs (P=0.0006, chi(2)=11.74), in contrast component of 36 KDD is more frequently recognized by hypersensitive pigs (P=0.001, chi(2)=10). Our results clearly indicate there is a difference in the reactivity to antigenic peptides/proteins of S. scabiei mite between hypersensitive and chronically infected pigs, and revealed that only two antigens may be considered S. scabiei-specific and used for diagnostic purposes in swine.     

Immune response in swine given soluble antigens from group E Streptococcus

Swine were given a series of injections of soluble or whole cell antigens of group E Streptococcus (GES). In experiment 1, swine given autoclaved extracts developed greater amounts of antibody to antiphagocytic factor (as detected with bactericidal and long-chain tests) than did swine given pepsin-extracted or whole cell antigens, and the swine given extract developed fewer abscesses when challenge exposed with live virulent GES added to their feed. In experiment 2, swine given injections of concentrated autoclaved extract develped higher antiphagocytic factor antibody titers than did control swine given injections of physiologic saline solution. When challenge exposed with live virulent GES by exposure to carrier swine, swine that were given extract developed 71.4% fewer abscesses than did the control swine. Furthermore, 58.3% of the abscesses in the swine that were given extrac" were less than 1 cm in diameter as compared with 38.2% of the abscesses in the controls, suggesting that the development of some abscesses was arrested in vaccinated swine. Data indicate that a degree of immunity to streptococcal lymphadenitis of swine can be induced by vaccinating swine with nonliving GES antigen.     

微创与开腹膀胱造口手术对小型猪应激反应的影响

对14头健康小型猪分别进行开腹(open cystostomy,OC)、腹腔镜膀胱造口手术(laparoscopic cystostomy,LC),通过检测小型猪术后不同时间点血清皮质醇(cortisol,COR)、白介素-6(Interleukin-6,IL-6)和C反应蛋白(C-reactive protein,CRP)的变化,比较OC手术与LC手术对机体应激反应的影响。结果表明,LC、OC2组分别在不同时间点所检测到的的COR、IL-6和CRP与试验前相比,都有极显著性差异(P<0.01)。在术后即刻和4h后OC组对应激指标的影响要明显高于LC组,并且术后3dLC组都恢复到试验前水平,而OC组术后5d才恢复。说明LC和OC都可导致血清中COR、IL-6、CRP水平的升高,但OC较LC对COR、IL-6和CRP的影响更为显著,即腹腔镜膀胱造口手术比开腹膀胱造口手术对机体应激反应的影响更小。     

Cytokines and synthetic double-stranded RNA augment the T helper 1 immune response of swine to porcine reproductive and respiratory syndrome virus

Immunization of pigs with a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine initially elicits a weak interferon (IFN)-gamma response. To improve the immune response, an adjuvant consisting of plasmid encoding either porcine interleukin (IL)-12 or IFN-alpha was co-administered during vaccination. In the presence of either adjuvant, at least a three-fold increase in the primary virus-specific IFN-gamma response was observed. While this enhancement was only transient (1 week) when the IL-12 expressing plasmid was used, the effect was not only still apparent at 6 weeks after vaccination in the presence of the IFN-alpha expressing plasmid but even after challenge with a virulent genetically divergent PRRSV. In contrast, no effect of either adjuvant on the production of anti-virus antibodies was noticed throughout the study. Despite the apparent augmentation of a T helper (Th) 1 type response by the inclusion of IFN-alpha or IL-12 during vaccination, this modulation did not necessarily correlate with a reduction in viremia. Since a similar increase in the degree of the IFN-gamma response to the PRRSV vaccine could be achieved by substituting polyinosinic-polycytidylic acid in lieu of either cytokine, exposure to PRRSV in the presence of a variety of Th 1 polarizing molecules can positively influence the development of the cell-mediated immune response of swine to this pathogen. Conceivably, such intervention could be applied to improve the formulation of anti-PRRSV vaccines.