Diagnosis of bovine brucellosis by one-hour in situ hybridization in milk cytology specimens

One-hour in situ hybridization (ISH) was performed for the diagnosis of bovine brucellosis in raw milk and supramammary lymph nodes from naturally infected cattle. Specific primer set derived from the perosamine region of O-side chains related to survival of Brucella was designed and used for PCR assay. One-hour ISH using a homologous biotinylated DNA probe was performed on milk and lymph node impression smear and paraffin-embedded supramammary lymph node tissues. Twenty-nine (82.9%) out of 35 raw milk samples and 30 (85.7%) out of 35 supramammary lymph node of impression smear and histopathology tissue samples were positive by one-hour ISH. Positive signals were detected by ISH in the cytoplasm of macrophages in raw milk and lymph node impression smears and in the macrophages infiltrating ductal and alveolar lumina and interstitium of mammary gland and adjacent to hyaline trabeculae of deep cortex and lymphatic nodule of supramammary lymph node. These results suggested that one-hour ISH was rapid and reliable method to diagnose brucellosis in raw milk and supramammary lymph node samples.