Abstract The diagnosis of Johne’s Disease by isolating Mycobacterium avium subsp. paratuberculosis from feces requires 6 months and high-cost reagents, and therefore an indirect technique like ELISA, is often being used to avoid these obstacles. The aim of this work was the production of a rabbit anti-deer polyclonal antibody (anti-deer pAb) for use in an in-house ELISA, applying protoplasmic antigen paratuberculosis (PPA) as antigen. Two rabbits were immunized subcutaneously with three doses of deer semipurified gamma globulin emulsified with incomplete Freund's adjuvant. Rabbit serum was purified with protein A. Titers of anti-deer pAb against deer serum and cross-reaction against sera from other species were studied by ELISA and immunoblot. Samples of feces and sera from 16 deer were evaluated by fecal culture and the in-house PPA-ELISA. The results were analyzed statistically using ROC curves and the Kappa index to estimate sensitivity (Se), specificity (Sp) and the agreement level between both techniques. Subsequently, sera from three to five years old stags (n = 155) from a herd with reports of Johne’s Disease were evaluated using the in-house PPA-ELISA. Anti-deer pAb showed a specific titer of 2.56 × 105. Cross-reaction against cattle, sheep, llama and goat was detected by ELISA and immunoblot. The in-house PPA-ELISA showed a Se = 85.71 % and Sp = 88.89 %. The agreement level with fecal culture was substantial (κ = 0.62 ± 0.19) and the assay was able to identify 76.77 % of the deer in the suspected herd as positive. The anti-deer pAb obtained was not only useful in the development of the in-house PPA-ELISA, which allows us to reduce the costs of Johne’s Disease diagnosis, but it could also be used in other diagnosis tests in deer as well as other ruminant species.

中文翻译：

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兔抗鹿多克隆抗体在鸟分枝杆菌亚种诊断中的应用 马鹿的副结核病（Cervus elaphus）

摘要 通过分离鸟分枝杆菌亚种来诊断约内氏病。粪便副结核病需要 6 个月的时间和高成本的试剂，因此经常使用间接技术（如 ELISA）来避免这些障碍。这项工作的目的是生产用于内部 ELISA 的兔抗鹿多克隆抗体（抗鹿多克隆抗体），应用原生质抗原副结核 (PPA) 作为抗原。两只兔子用三剂用不完全弗氏佐剂乳化的鹿半纯化丙种球蛋白进行皮下免疫。用蛋白 A 纯化兔血清。通过 ELISA 和免疫印迹研究抗鹿血清抗鹿 pAb 的滴度和对其他物种血清的交叉反应。通过粪便培养和内部 PPA-ELISA 评估了 16 只鹿的粪便和血清样本。使用 ROC 曲线和 Kappa 指数对结果进行统计分析，以估计两种技术之间的敏感性 (Se)、特异性 (Sp) 和一致性水平。随后，使用内部 PPA-ELISA 评估了来自报告有约翰氏病的牛群的 3 至 5 岁雄鹿（n = 155）的血清。抗鹿 pAb 的特异性滴度为 2.56 × 105。ELISA 和免疫印迹法检测到与牛、绵羊、美洲驼和山羊的交叉反应。内部 PPA-ELISA 显示 Se = 85.71 % 和 Sp = 88.89 %。与粪便培养的一致性水平很高（κ = 0.62 ± 0.19），并且该测定能够将疑似鹿群中 76.77% 的鹿鉴定为阳性。获得的抗鹿 pAb 不仅可用于内部 PPA-ELISA 的开发，这使我们能够降低约尼氏病的诊断成本，