Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India.
Department of Food Microbiology, CSIR-Central Food Technological Research Institute, Mysore 570 020, India.
Vet Microbiol. 2014 Jun 4;170(3-4):358-67. doi: 10.1016/j.vetmic.2014.02.041. Epub 2014 Mar 12.
Leukotoxin M/F'-Panton Valentine (LukM/F'-PV), a beta pore-forming toxin secreted by Staphylococcus aureus, is a major virulence factor involved in the pathogenesis of bovine mastitis. The present study was aimed to determine immunogenicity of two recombinant subunits of LukM/F'-PV, rLukM (MW 38 kDa) and rLukF (MW 39 kDa), develop and validate an indirect enzyme linked immunosorbent assay (ELISA) using polyclonal antibodies raised in rabbits, and evaluate applicability of the assay to diagnose clinical and subclinical bovine mastitis. Additionally, in vitro assays were conducted to determine abilities of antibodies to neutralize cytotoxicity of the native leukotoxin. A total of 87 bovine milk samples (healthy, subclinical and clinical mastitis) were evaluated for the presence of toxin determinants. Receiver-operator characteristic curve for the experimental ELISA values statistically interpreted a cut-off score of >0.109 OD405, with an assay specificity of 100% and sensitivity in the range of 80-87.5%. In addition, area under curve of 0.93-0.98 revealed the test was accurate in categorizing samples from infected and non-infected bovine. The rLukF IgG-ELISA was more sensitive than rLukM IgG-ELISA. Furthermore, it was evident from MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium) dye reduction, indirect immunofluorescence and lactate dehydrogenase assays that anti-rLukM/rLukF antibodies, with high neutralizing titers, inhibited in vitro leukotoxic activity and protected bovine neutrophil membrane integrity from cytotoxicity of native leukotoxin. The findings demonstrated that antibodies produced from recombinant subunits contribute to specific and sensitive immunodiagnosis and may also have the potential to provide passive therapeutic benefit in the management of bovine mastitis.
白细胞毒素 M/F'-Panton Valentine（LukM/F'-PV）是由金黄色葡萄球菌分泌的一种β孔形成毒素，是参与牛乳腺炎发病机制的主要毒力因子。本研究旨在确定两种重组 LukM/F'-PV 亚单位 rLukM（MW 38 kDa）和 rLukF（MW 39 kDa）的免疫原性，用兔多克隆抗体开发和验证间接酶联免疫吸附试验（ELISA），并评估该试验在诊断临床和亚临床牛乳腺炎中的适用性。此外，还进行了体外试验以确定抗体中和天然白细胞毒素细胞毒性的能力。评估了 87 份牛牛奶样（健康、亚临床和临床乳腺炎）中毒素决定簇的存在情况。实验 ELISA 值的接收者操作特性曲线统计解释了 >0.109 OD405 的截断值，该试验具有 100%的特异性和 80-87.5%的敏感性。此外，曲线下面积为 0.93-0.98 表明该试验能够准确地对感染和未感染牛的样本进行分类。rLukF IgG-ELISA 比 rLukM IgG-ELISA 更敏感。此外，从 MTT（3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑）染料还原、间接免疫荧光和乳酸脱氢酶试验中明显看出，具有高中和效价的抗-rLukM/rLukF 抗体抑制了体外白细胞毒素活性，并保护牛中性粒细胞膜完整性免受天然白细胞毒素的细胞毒性。研究结果表明，来自重组亚单位的抗体有助于特异性和敏感性免疫诊断，并且在牛乳腺炎的管理中也可能具有提供被动治疗益处的潜力。
