Newcastle disease (ND) is a highly pathogenic viral infection of poultry with significant economic impacts worldwide. Despite the widespread use of vaccines, ND outbreaks continue to occur even within vaccinated poultry farms. Furthermore, novel Newcastle disease virus (NDV) genotypes are emerging in poultry, increasing the need for the development of rapid, accurate, and simple diagnostic methods. We therefore developed two novel sets of visual reverse transcription loop-mediated isothermal amplification (RT-LAMP) assays based on highly conserved regions of the HN and F genes. The limits of detection of the NDV-Common-LAMP assay, for all the NDV strains, were 10^3.0 EID₅₀/0.1 mL for Kr005 and 10^2.0 EID₅₀/0.1 mL for Lasota within 35 min. The sensitivity of the NDV-Patho-LAMP assay, used for the strain differentiation of virulent NDV, was 10^2.0 EID₅₀/0.1 mL for Kr005. No amplification was detected for the non-NDV templates. Next, we probed 95 clinical strains and 7 reference strains with the RT-LAMP assays to assess the feasibility of their use in diagnostics. We observed no cross-reactivity across the 102 strains. Furthermore, there was 100% congruence between the RT-LAMP assays and full-length sequencing of the target genes, indicating the potential for visual RT-LAMP in the identification and differentiation of NDV. These novel RT-LAMP assays are ideally suited for the field or resource-limited environments to facilitate the faster detection and differentiation of NDV, which can reduce or avoid further spread.

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. 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. 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.