Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of Johne’s disease, which is an economically and clinically relevant pathogen for commercial deer production. The purpose of this study was to develop a method that could be used to rapidly detect MAP infection in deer using the Actiphage Rapid blood test. This test has previously been used to detect MAP in cattle blood following the purification of buffy coat using Ficoll gradients, however this method is quite laborious and costly. The purpose of this study was to develop a simpler method of blood preparation that was also compatible with deer blood and the Actiphage test. Initially differential lysis of RBCs using Ammonium Chloride-Potassium (ACK) blood lysis buffer was compared with the Ficoll gradient centrifugation method using cattle blood samples for compatibility with the Actiphage reagents, and it was found that the simpler ACK method did not have an impact on the Actiphage test reagents, producing an equivalent sensitivity for detection of low levels of MAP. When the two methods were compared using clinical blood samples from farmed deer, the ACK lysis method resulted in a cleaner sample. When a blinded test of 132 animals from 4 different production groups was carried out, the majority of the positive test results were found to be from animals in just one group, with a small number identified in a second group. The test results were found to be reproducible when a small set of positive animals were tested again 1 month after their initial testing. Finally a set of negative animals which had been previously screened using an ELISA test, all animals gave a negative Actiphage result. This study shows that this improved sample preparation method and Actiphage blood testing can be used to test blood samples from deer, and the full diagnostic potential of the method can now be evaluated.

中文翻译：

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开发一种使用 Actiphage ® Rapid 检测养殖鹿血液中副结核分枝杆菌的方法

鸟分枝杆菌亚种。副结核病 (MAP) 是约内病的病原体，约内病是商业鹿生产的经济和临床相关病原体。本研究的目的是开发一种方法，该方法可用于使用 Actiphage Rapid 血液测试快速检测鹿的 MAP 感染。该测试以前曾用于在使用 Ficoll 梯度纯化血沉棕黄层后检测牛血中的 MAP，但是这种方法非常费力且成本高昂。这项研究的目的是开发一种更简单的血液制备方法，该方法也与鹿血和 Actiphage 测试兼容。最初使用氯化铵 - 钾 (ACK) 血液裂解缓冲液对 RBC 进行差异裂解与使用牛血样品的 Ficoll 梯度离心方法进行比较，以便与 Actiphage 试剂兼容，发现更简单的 ACK 方法对Actiphage 检测试剂，对检测低水平 MAP 产生等效的灵敏度。当使用来自养殖鹿的临床血液样本对这两种方法进行比较时，ACK 裂解方法产生了更清洁的样本。当对来自 4 个不同生产组的 132 只动物进行盲法测试时，发现大多数阳性测试结果仅来自一组动物，第二组中发现了少数动物。当在最初测试后 1 个月再次测试一小组阳性动物时，发现测试结果是可重复的。最后一组之前已经使用ELISA测试筛选过的阴性动物，所有动物都给出了阴性的Actiphage结果。这项研究表明，这种改进的样品制备方法和 Actiphage 血液检测可用于检测鹿的血液样品，现在可以评估该方法的全部诊断潜力。