First report of molecular genotyping, pathotyping, and histopathological characterization of lentogenic genotype II Newcastle disease virus circulating in young ostrich flocks in Egypt.

Background and Aim: Newcastle disease virus (NDV) is one of the most economically important avian pathogens worldwide, yet information regarding its molecular epidemiology and pathological characteristics in ostriches remains limited. This study investigated the prevalence, molecular characteristics, pathotype, phylogenetic relationships, and histopathological changes associated with NDV circulating in young ostrich flocks in Egypt. Materials and Methods: A total of 60 tissue samples were collected from diseased, non-vaccinated ostriches aged 3 weeks to 2 months from eight farms across four Egyptian governorates during 2024. Virus isolation was performed using specific-pathogen-free embryonated chicken eggs, followed by hemagglutination and hemagglutination inhibition assays. Pathogenicity was determined using the mean death time (MDT) and intracerebral pathogenicity index (ICPI). Molecular detection was conducted by real-time reverse-transcription polymerase chain reaction targeting the matrix gene, followed by partial fusion gene amplification, sequencing, and phylogenetic analysis. Histopathological examination was performed on major organs, and positive samples were additionally screened for avian influenza virus, infectious bronchitis virus, and infectious bursal disease virus to exclude coinfections. Results: Eight of 60 samples tested positive for NDV, with the highest prevalence detected in Ismailia governorate. All positive samples were negative for the tested coinfecting viruses. The isolates exhibited a hemagglutination titer of 9 log₂ hemagglutination units/mL and a hemagglutination inhibition titer of 6 log₂. Biological pathotyping confirmed a lentogenic pathotype with an MDT of 96 h and an ICPI of 0.4. Phylogenetic analysis classified the isolates within genotype II, class II, possessing the characteristic lentogenic fusion protein cleavage motif ¹¹²GRQGRL¹¹⁷. The isolates shared 97%-99% nucleotide identity with commonly used vaccine strains, including LaSota, Hitchner, and Clone 30. Histopathological examination revealed marked lesions in the respiratory and digestive systems, including epithelial degeneration, hemorrhage, inflammatory infiltrates, lymphoid depletion, hepatic necrosis, and intestinal villous damage, despite the lentogenic nature of the virus. Conclusion: This study provides the first comprehensive molecular, pathotyping, phylogenetic, and histopathological characterization of lentogenic genotype II NDV circulating in young ostriches in Egypt. The findings demonstrate that lentogenic vaccine-related genotype II strains can induce clinically relevant pathological alterations in ostriches, emphasizing the need for continuous molecular surveillance, host-specific vaccination strategies, enhanced biosecurity, and integrated monitoring of ostriches, poultry, and wild birds to reduce NDV transmission and evolution.