Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Faeces Using a Rhodamine B Hydrazone Immunosensor on Antibody‐Coated Nitrocellulose Membranes

ABSTRACT Rapid, inexpensive and reliable detection of Mycobacterium avium subsp. paratuberculosis (MAP) is essential for the control of Johne's disease. This study developed a rhodamine B hydrazone nanoparticle‐based immunosensor coupled with antibody‐coated nitrocellulose membranes (IMS‐CNM) for the detection of MAP in bovine faecal samples. Anti‐MAP antibodies were purified from hyperimmune donkey and rabbit sera. Purified donkey IgG was immobilized on nitrocellulose membranes as the capture antibody, whereas rabbit anti‐MAP IgG was conjugated to rhodamine B hydrazone nanoparticles using EDC‐NHS chemistry to prepare the chromogenic immunosensor. The diagnostic performance of the assay was evaluated using 89 bovine faecal samples and compared with nested PCR. Nested PCR identified 62 positive and 27 negative samples, whereas the IMS‐CNM assay detected 60 positive and 29 negative samples. Using nested PCR as the reference method, the IMS‐CNM assay achieved a sensitivity of 90.32% (95% CI: 82.96%–97.68%) and a specificity of 85.19% (95% CI: 71.80%–98.58%). The analytical limit of detection was approximately 3 × 10 3 MAP cells/mL in PBS. No significant difference was observed between the IMS‐CNM assay and nested PCR in classifying positive and negative samples. The proposed IMS‐CNM assay provides a rapid, simple and cost‐effective approach for detecting MAP in bovine faeces with diagnostic performance comparable to nested PCR. Following further optimization and validation in larger field studies, this platform may serve as a practical screening tool for Johne's disease, particularly in resource‐limited settings.