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Background: Thirty-two-day-old broiler chickens at a farm located in northwestern South Korea displayed adverse neurological symptoms including limping, lying down, and head shaking. Approximately 2.1% of chickens died or were culled due to severe symptoms. Five carcasses were submitted to the Avian Disease Division of the Animal and Plant Quarantine Agency (APQA) for disease diagnosis. Results: Broilers displayed severe pericarditis and perihepatitis associated with gross lesions. Broilers also displayed microscopic lesions in the cerebrum and in the granular layer of the cerebellum, which were associated with multifocal perivascular cuffing and purulent necrosis in the cerebrum, and severe meningitis with heterophil and lymphocyte infiltration. Staphylococcus spp. were identified in the liver and heart using bacteriological culture. PCR/RT-PCR assays revealed that broilers were negative for avian Clostridium botulinum, Newcastle disease virus, and avian encephalomyelitis virus. Bacterial and viral metagenomic analysis of brain sample further revealed the presence of Pseudomonas spp. and Marek's disease virus, which are known etiological agents of chicken meningoencephalitis. Conclusions: This study reports a diagnostic analysis of gross and histopathological lesions from 32-day-old broilers displaying unique neurological symptoms that revealed the presence of the several neurological diseases including meningoencephalitis. The causative agents associated with meningoencephalitis of broilers that had not been identified by routine diagnostic methods could be diagnosed by metagenomics, which proves the usefulness of metagenomics as a diagnostic tool for unknown neurological diseases in broilers. [ABSTRACT FROM AUTHOR]