Platt Ratree, Coutu Christopher, Meinert Todd, Roth James A
Department of Veterinary Microbiology and Preventive Medicine, College of Veterinary Medicine, Iowa State University, Ames, IA 50011, USA.
Vet Immunol Immunopathol. 2008 Mar 15;122(1-2):8-15. doi: 10.1016/j.vetimm.2007.11.009. Epub 2007 Dec 3.
The objective of this research project was to evaluate the antibody and cell-mediated immune responses to a multivalent vaccine containing killed bovine viral diarrhea virus (BVDV) types 1 and 2. Twenty castrated male crossbred beef cattle (350-420kg body weight) seronegative to BVDV were randomly divided into two groups of 10 each. Group 1 served as negative mock-vaccinated control. Group 2 was vaccinated subcutaneously twice, 3 weeks apart, with modified live bovine herpesvirus 1, parainfluenza 3 virus and bovine respiratory syncytial virus diluted in diluent containing killed BVDV type 1 (strain 5960) and type 2 (strain 53637) in an adjuvant containing Quil A, Amphigen, and cholesterol. Serum samples were collected from all cattle at days -21, 0, and days 21, 28, 35, 56 and 70 post-vaccination. Standard serum virus neutralization tests were performed with BVDV type 1 (strain 5960) and type 2 (strain 125C). Anticoagulated blood samples were collected at day 0, and days 28, 35, 56 and 70 post-vaccination. Peripheral blood mononuclear cells (PBMCs) were isolated, stimulated with live BVDV type 1 (strain TGAN) and type 2 (strain 890) and cultured in vitro for 4 days. Supernatants of cultured cells were collected and saved for interferon gamma (IFNgamma) indirect enzyme-linked immunosorbent assay (ELISA). Four-color flow cytometry was performed to stain and identify cultured PBMC for three T cell surface markers (CD4, CD8, and gammadelta TCR) and to detect the activation marker CD25 (alpha chain of IL-2 receptor) expression. The net increase in %CD25+ cells (Delta%CD25+) of each T cell subset of individual cattle was calculated. The results of all post-vaccination weeks of each animal were plotted and the areas under the curve of each T cell subset were statistically analyzed and compared between groups. The mean area under the curve of the Delta%CD25+ data for days 0-70 of all subsets, except CD4-CD8+gammadelta TCR- (cytotoxic) T cell subset of both BVDV types 1 and 2 stimulated cells, of the vaccinated group were significantly higher than the control group (P<0.05). IFNgamma production by PBMC from the vaccinated group showed significantly higher results (P<0.05) than the control group in the BVDV types 1 and 2 stimulated cells for at least some time points after vaccination. The vaccinated group also had significantly (P<0.0001) higher neutralizing antibody titers than the control group from day 28 onward.
本研究项目的目的是评估对一种包含1型和2型灭活牛病毒性腹泻病毒（BVDV）的多价疫苗的抗体和细胞介导的免疫反应。20头对BVDV血清学阴性的去势雄性杂交肉牛（体重350 - 420千克）被随机分为两组，每组10头。第1组作为阴性假疫苗接种对照。第2组在3周的间隔时间内进行两次皮下接种，接种用在含有Quil A、Amphigen和胆固醇的佐剂中稀释的1型（5960株）和2型（53637株）灭活BVDV、改良活牛疱疹病毒1型、副流感病毒3型和牛呼吸道合胞病毒。在接种前-21天、0天以及接种后第21天、28天、35天、56天和70天从所有牛采集血清样本。用1型（5960株）和2型（125C株）BVDV进行标准血清病毒中和试验。在接种前0天以及接种后第28天、35天、56天和70天采集抗凝血样本。分离外周血单个核细胞（PBMC），用1型（TGAN株）和2型（890株）活BVDV刺激并在体外培养4天。收集培养细胞的上清液并保存用于干扰素γ（IFNγ）间接酶联免疫吸附测定（ELISA）。进行四色流式细胞术以对培养的PBMC进行三种T细胞表面标志物（CD4、CD8和γδTCR）的染色和鉴定，并检测活化标志物CD25（IL - 2受体α链）的表达。计算每头牛每个T细胞亚群的CD25 +细胞百分比净增加量（Δ%CD25 +）。绘制每只动物接种后各周的结果，并对每个T细胞亚群曲线下面积进行统计分析和组间比较。在接种组中，除了1型和2型BVDV刺激细胞的CD4 - CD8 +γδTCR -（细胞毒性）T细胞亚群外，所有亚群在接种后0 - 70天的Δ%CD25 +数据的平均曲线下面积均显著高于对照组（P < 0.05）。在接种后至少一些时间点，接种组受1型和2型BVDV刺激的PBMC产生的IFNγ结果显著高于对照组（P < 0.05）。从第28天起，接种组的中和抗体滴度也显著高于对照组（P < 0.0001）。
