Khosravi Mohammad, Haji Hajikolaei MohammadRahim, Nouri Mohammad, Kalanter Shayan
Department of Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Department of Clinical Sciences, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Ahvaz, Iran.
Vet Med Sci. 2025 May;11(3):e70346. doi: 10.1002/vms3.70346.
Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease in ruminants, is typically transmitted through the faecal-oral route. This study aimed to optimize and evaluate an immunoelectric (IE) method for detecting MAP in faecal samples from infected cows. The necessary polyclonal antibodies were extracted from hyperimmune donkeys and rabbits using affinity chromatography. Furthermore, cross-reactive antibodies were eliminated through absorption with Mycobacterium phlei. The binding of donkey antibodies to a polystyrene filter and rabbit antibodies to Fe nanoparticles was facilitated by a diethylenetriaminepentaacetic acid (DTPA) linker. The trapping of bacteria on the filter and the fixation of Fe nanoparticles attached to specific antibodies led to a modification in the electrical resistance of the filter. This alteration in electrical resistance can be quantified using a high-precision electrical meter. In this research, MAP was identified through both polymerase chain reaction (PCR) and the IE methods in faecal samples from dairy cows, producing varied outcomes in the enzyme-linked immunosorbent assay (ELISA) test. The sensitivity and specificity of the ELISA approach for detection of the serum antibodies in comparison to the PCR technique were determined to be 75% and 72%, respectively. In contrast, the sensitivity and specificity of the IE method relative to the PCR approach were found to be 96% and 95%, respectively. With a detection time of less than 60 min, cost-effectiveness per sample, user-friendly operation utilizing an IE device, no requirement for specialized machinery and applicability in farm or dairy settings, this technique emerges as a promising alternative to traditional bacterial detection methods.
副结核分枝杆菌（MAP）是反刍动物约翰氏病的病原体，通常通过粪-口途径传播。本研究旨在优化和评估一种免疫电（IE）方法，用于检测感染奶牛粪便样本中的MAP。使用亲和色谱法从超免疫驴和兔中提取所需的多克隆抗体。此外，通过用草分枝杆菌吸收来消除交叉反应性抗体。二乙烯三胺五乙酸（DTPA）接头促进了驴抗体与聚苯乙烯滤膜的结合以及兔抗体与铁纳米颗粒的结合。细菌在滤膜上的捕获以及附着在特异性抗体上的铁纳米颗粒的固定导致滤膜电阻的改变。这种电阻变化可以使用高精度电表进行量化。在本研究中，通过聚合酶链反应（PCR）和IE方法在奶牛粪便样本中鉴定出MAP，在酶联免疫吸附测定（ELISA）试验中产生了不同的结果。与PCR技术相比，ELISA方法检测血清抗体的灵敏度和特异性分别确定为75%和72%。相比之下，IE方法相对于PCR方法的灵敏度和特异性分别为96%和95%。该技术检测时间不到60分钟，每个样本具有成本效益，使用IE设备操作方便，无需专门仪器，适用于农场或奶牛场环境，是传统细菌检测方法的一种有前途的替代方法。
