RESULTS

Clinical findings, gross lesions, and histopathology

The NDV-positive ostrich farms in the four investigated governorates exhibited mild clinical signs, including depression, anorexia, coughing, nasal and ocular discharge, and sneezing in young birds, with a mortality rate of 4% and a morbidity rate of 30% during the course of the disease (Figure 2A). In a few cases, sudden death occurred without preceding clinical signs, particularly in young or immunocompromised birds.

Respiratory system histopathology

Gross examination of the trachea of infected ostriches revealed a hyperemic and congested mucosa with the lumen partially occluded by a thick, yellow-to-serosanguineous exudate. Microscopically, these lesions corresponded to degeneration and sloughing of the pseudostratified ciliated columnar epithelium, subepithelial edema, endothelial swelling, and dense infiltration of lymphocytes, plasma cells, and heterophils (Figures 2A, 3A, and 3B).

Meanwhile, the lungs and air sacs appeared grossly heavy, non-collapsed, and diffusely congested, with pulmonary edema, multifocal hemorrhages, fibrinous pleuritis, and air sacculitis. Microscopically, pronounced interstitial pneumonia was observed, characterized by thickened interalveolar septa due to mononuclear cell infiltration, intra-alveolar fibrin deposition and erythrocyte accumulation, bronchial epithelial desquamation, peribronchial lymphoid depletion, and perivascular heterophilic infiltration (Figures 2A, 3C, and 3D).

Digestive and hepatic pathology

Gross examination of the proventriculus revealed swollen glandular papillae. Histopathologically, the glandular epithelium exhibited degeneration, necrosis, and loss of normal cellular architecture with luminal eosinophilic debris, accompanied by mucosal ulceration and inflammation of the lamina propria (Figures 2C, 4B, and 4C).

Additionally, gross examination of the intestinal segments (duodenum, ileum, and ceca) revealed mucosal congestion, friability, and fluid, fetid, bile-stained greenish intestinal contents. Microscopically, the villi were shortened, blunted, or fused, with necrosis of the surface epithelium, dilated crypts containing cellular debris, and submucosal hemorrhages (Figures 2D and 4D).

Furthermore, the liver and gallbladder exhibited mild hepatomegaly with rounded borders, a friable consistency, a mottled parenchymal appearance characterized by pale necrotic foci alternating with congested areas, and a bile-distended gallbladder. Histopathological examination revealed disruption of the hepatic architecture due to multifocal-to-coalescing hepatocellular necrosis, sinusoidal dilation, Kupffer cell proliferation, and periportal lymphocytic infiltration (Figures 2B and 4A).

Lymphoid tissue depletion

Grossly, visceral lymphoid tissues, including Peyer's patches and GALT, were enlarged and congested. Microscopically, marked lymphoid depletion and extensive necrosis replaced the normal follicular architecture of GALT, including Peyer's patches and cecal tonsils, and were accompanied by lymphoid depletion within the proventricular lymphoid nodules (Figures 2D, 4B, 4C, and 4D).

Histopathological damage scoring

Semiquantitative lesion scoring of the trachea, lungs, liver, proventriculus, and intestine demonstrated that naturally infected ostriches consistently exhibited markedly higher lesion scores than healthy control birds, indicating severe histopathological damage associated with NDV infection. In contrast, the control group exhibited only minimal lesions, reflecting normal tissue morphology (Figure 5).

Trachea

The tracheal tissues of the control group had a mean lesion score of 0.5, indicating minimal histopathological alterations and the preservation of normal tissue architecture. Histological examination revealed intact pseudostratified ciliated columnar epithelium with well-preserved cilia and goblet cells.

In contrast, diseased ostriches exhibited a markedly elevated mean lesion score of 2.3, with lesion grades ranging from 2 to 3. These findings were associated with pronounced degenerative and inflammatory changes, including epithelial degeneration, ciliary loss, epithelial desquamation, and inflammatory cell infiltration.

Figure 2: Gross lesions of ostriches naturally infected with Newcastle disease virus. (A) Moribund ostrich showing paralysis of the neck. (B) Hepatomegaly with marked swelling and congestion of the liver (arrow). (C) Glandular papillae of the proventriculus showing prominent swelling and congestion at their tips (arrow). (D) Congestion of the intestinal serosal surface (arrow).

Figure 3: Photomicrographs of respiratory tissues from ostriches naturally infected with Newcastle disease virus. (A) Trachea showing degeneration of the pseudostratified ciliated columnar epithelium with areas of complete epithelial sloughing exposing the basement membrane (arrow). (B) Lamina propria of the trachea showing dense infiltration of lymphocytes and heterophils (arrows). (C and D) Lung showing interstitial pneumonia with marked heterophilic infiltration. The bronchi exhibited epithelial desquamation and necrosis, together with extensive perivascular heterophilic infiltration (arrows). (H&E stain; scale bar = 200 μm).

Lung

Lung tissues obtained from healthy ostriches exhibited a mean lesion score of 0.6, reflecting predominantly normal pulmonary histological architecture. Most of the sections examined displayed clear alveolar spaces, thin interalveolar septa, and patent bronchioles lined by simple columnar epithelium. In contrast, diseased ostriches demonstrated a significantly higher mean lesion score of 2.6, with lesion grades ranging from 2 to 3. Histopathological examination revealed severe disruption of the pulmonary architecture, alveolar deterioration, inflammatory cell infiltration, and vascular alterations.

Figure 4: Photomicrographs of different ostrich tissues naturally infected with Newcastle disease virus. (A) Liver showing severe congestion of the central veins (CV) and coagulative necrosis of the hepatic parenchyma (HP). (B) The glandular epithelium of the proventriculus exhibiting degeneration, necrosis (star), and dilated blood vessels within the submucosa (arrows). (C) The overlying mucosa of the proventriculus showing multifocal erosion and ulceration (stars). (D) The intestine showing severe mucosal damage, with the surface epithelium exhibiting erosion or complete necrosis (stars). The lamina muscularis was infiltrated with dense populations of mononuclear cells and heterophils (black arrow). Peyer's patches exhibited marked lymphoid depletion, with extensive areas of necrosis replacing the normal follicular architecture (black arrows). (H&E stain; scale bar = 200 μm).

Figure 5: Mean histopathological lesion scores in healthy and diseased ostriches illustrating differences in lesion severity among the examined organs. The intestine was the most severely affected organ in diseased ostriches, followed by the lung.

Liver

The liver tissues of the control group exhibited a mean lesion score of 0.25, indicating minimal pathological alterations and preservation of normal hepatic architecture. Histologically, hepatocytes appeared polyhedral and were arranged in cords separated by hepatic sinusoids surrounding the central veins. In comparison, diseased ostriches exhibited a significantly higher mean lesion score of 2.3, with lesion grades ranging from 2 to 3. The hepatic lesions were characterized by marked vacuolar degeneration, cytoplasmic alterations, and extensive inflammatory cell aggregates.

Proventriculus

The proventriculus of healthy ostriches exhibited a mean lesion score of 0.5, reflecting minimal histopathological alterations and preservation of normal tissue organization. Histological sections showed normal mucosal folds and well-developed proventricular glands arranged into lobules separated by delicate connective tissue septa. In contrast, diseased ostriches demonstrated a significantly increased mean lesion score of 2.8, with lesion grades ranging from 2 to 3. Histopathological findings included degeneration and necrosis of the glandular epithelium, dense aggregates of inflammatory cells, and vascular alterations.

Intestine

Intestinal tissues from healthy ostriches exhibited a mean lesion score of 0.5, indicating minimal histopathological abnormalities and preservation of normal intestinal morphology. Histological examination revealed intact villi, normal crypt architecture, and abundant goblet cells within the mucosa. In contrast, diseased ostriches demonstrated a markedly increased mean lesion score of 2.8, with lesion grades ranging from 2 to 3. The pathological alterations included severe villous blunting and degeneration, crypt distortion, goblet cell depletion, and inflammatory cell infiltration.

NDV isolation, identification, pathotyping, and coinfection screening

Following three successive passages of NDV in SPF-ECE, the inoculated embryos exhibited mild lesions, including slight congestion of the skin and internal organs. The recovered isolates were HA-positive, with an HA titer of 9 log₂ HA units/mL. Identification of the NDV isolates by the HI assay demonstrated a serum antibody titer of 6 log₂ in all positive samples.

Biological pathotyping confirmed that all NDV isolates belonged to the lentogenic pathotype, with an MDT of 96 h (>90 h indicates a lentogenic strain) and an ICPI of 0.4 (<0.5 indicates a lentogenic strain).

Molecular detection by rRT-PCR targeting the conserved M gene confirmed NDV RNA in 8 of the 60 tissue samples collected from eight ostrich farms. Positive samples were detected in farms located in Ismailia (four farms, with the highest prevalence), El-Menofia (two farms), El-Behera (one farm), and El-Sharquia (one farm). The prevalence rates and distribution of positive farms in each governorate are summarized in Table 1.

Furthermore, four representative NDV-positive samples were amplified by conventional RT-PCR for subsequent partial F gene sequencing and phylogenetic analysis. Among the eight NDV-positive samples identified by RT-qPCR, four representative isolates were selected for partial _F _gene sequencing based on lower cycle threshold values, higher RNA concentration and purity, and successful amplification by conventional RT-PCR.

All NDV-positive samples were additionally screened for AIV, IBV, and IBDV using specific RT-qPCR assays. No coinfection with these viruses was detected in the ostrich samples examined.

Sequencing and phylogenetic analysis

To unequivocally classify the current ostrich NDV isolate, phylogenetic analysis was performed using the partial F gene sequence together with representative reference and vaccine strains available in GenBank (Table 4, Figure 6).

The phylogenetic tree demonstrated that the ostrich NDV isolate, designated NDV-Fgene-Ismailia-ostrich-2024, clustered within genotype II, class II and was assigned GenBank accession number PX088918 (Figure 6).

Analysis of the F protein cleavage-site sequence revealed the motif ¹¹²GRQGRL¹¹⁷, which is characteristic of lentogenic NDV strains (Figure 7). In contrast, the polybasic cleavage-site motif ¹¹²RRQKRF¹¹⁷, which is typically associated with velogenic NDV strains, was absent. Therefore, NDV-Fgene-Ismailia-ostrich-2024 was classified as a lentogenic strain based on molecular pathotyping criteria.

Based on the phylogenetic analysis, the current isolate clustered closely with genotype II vaccine strains, including NDV-LaSota-II, NDV isolate Hitchner, NDV-Clone 30, and NDV-VG/GA, as well as genotype II field isolates NDV/chicken/Egypt/4/2006 (Egyptian isolate) and NDV/SRZ03 (Chinese isolate), exhibiting nucleotide identities ranging from 87% to 99% and amino acid identities ranging from 87% to 96%.

In contrast, NDV-Fgene-Ismailia-ostrich-2024 was genetically distinct from the genotype V velogenic strain NDV/turkey/USA(ND)/43084/92, the genotype I vaccine strain Vectormune ND, and the genotype I lentogenic strain NDV/chicken/N. Ireland/Ulster/67, exhibiting nucleotide identities ranging from 74% to 88% and amino acid identities ranging from 75% to 88%.

Similarly, the ostrich NDV isolate exhibited relatively low sequence homology with genotype VII reference strains circulating in China, Namibia, and Egypt, particularly within the hypervariable region of the amplified F gene fragment, with nucleotide identities of 80%–82% and amino acid identities of 82%–84% (Table 4).

Collectively, these findings represent the first phylogenetic characterization of a lentogenic genotype II NDV isolate from Egyptian ostriches. The isolate exhibited 97%–99% identity with LaSota-like vaccine strains while remaining genetically distinct from the velogenic genotype VII strains previously reported in ostrich embryos.

Multiple amino acid sequence alignment of the F protein demonstrated the absence of multiple basic amino acid insertions compared with published NDV sequences available in the GenBank database (Figure 7).

Regarding recombination, analysis using RDP5 software with multiple detection algorithms (RDP, BootScan, MaxChi, GENECONV, SiScan, Chimaera, LARD, Phyl-Pro, and 3Seq) revealed no statistically significant recombination events within the partial F gene sequence of NDV-Fgene-Ismailia-ostrich-2024. None of the applied methods identified supported recombination breakpoints or parental strains under the selected significance threshold (p > 0.05).

Novel aspects of the study

This study is the first to integrate virus isolation, comprehensive biological pathotyping (MDT and ICPI), real-time RT-PCR, partial F gene sequencing, and systematic histopathological examination of multiple tissues (trachea, lungs, liver, proventriculus, intestines, and other visceral organs) from naturally infected young ostriches. These complementary approaches provide a comprehensive understanding of the biological, molecular, and pathological characteristics of NDV infection in ostriches.

The study provides several novel contributions to the understanding of NDV infection in ratites. First, it presents the first detailed molecular and pathological characterization of a lentogenic genotype II NDV isolated from clinically affected young ostriches in Egypt, in contrast to previous reports that primarily described velogenic genotype VII infection in ostrich embryos. Second, it demonstrates that lentogenic NDV strains, traditionally considered to be of low pathogenicity, can induce significant histopathological lesions in naturally infected young ostriches. Third, the close genetic relatedness (97%–99%) between the identified isolate and commonly used poultry vaccine strains raises important questions regarding possible vaccine spillover and the potential role of ostriches as reservoirs for NDV circulation.

Sequence | A | B | C | D | E | F | G | H | I | J | K | L | M | N | O | P | Q | R | S | T | U
 |  |  |  |  |  |  |  |  |  | ** |  |  |  |  |  |  |  |  |  |  | 
A | ID | 90 | 98 | 99 | 96 | 98 | 98 | 99 | 98 | 97 | 82 | 82 | 82 | 82 | 84 | 79 | 83 | 75 | 83 | 37 | 82
B | 90 | ID | 89 | 90 | 90 | 89 | 89 | 89 | 89 | 88 | 81 | 81 | 81 | 80 | 84 | 76 | 81 | 74 | 80 | 36 | 80
C | 99 | 90 | ID | 99 | 95 | 100 | 100 | 99 | 100 | 98 | 82 | 82 | 82 | 81 | 84 | 79 | 82 | 74 | 82 | 37 | 81
D | 99 | 90 | 100 | ID | 96 | 99 | 99 | 99 | 99 | 97 | 82 | 82 | 82 | 82 | 84 | 79 | 82 | 75 | 82 | 37 | 82
E | 96 | 90 | 97 | 97 | ID | 95 | 95 | 96 | 95 | 94 | 83 | 83 | 83 | 82 | 84 | 79 | 83 | 74 | 83 | 38 | 82
F | 99 | 90 | 100 | 100 | 97 | ID | 100 | 99 | 100 | 98 | 82 | 82 | 82 | 82 | 84 | 79 | 82 | 75 | 82 | 37 | 82
G | 99 | 90 | 100 | 100 | 97 | 100 | ID | 99 | 100 | 98 | 82 | 82 | 82 | 81 | 84 | 79 | 82 | 74 | 82 | 37 | 81
H | 99 | 90 | 100 | 100 | 97 | 100 | 100 | ID | 99 | 98 | 82 | 82 | 82 | 81 | 84 | 79 | 82 | 75 | 82 | 36 | 81
I | 99 | 90 | 99 | 99 | 96 | 99 | 99 | 99 | ID | 98 | 82 | 82 | 82 | 81 | 84 | 79 | 82 | 74 | 82 | 37 | 81
J | 98 | 89 | 98 | 98 | 95 | 98 | 98 | 98 | 98 | ID | 81 | 81 | 81 | 80 | 83 | 78 | 81 | 73 | 82 | 36 | 80
K | 83 | 82 | 84 | 84 | 84 | 84 | 84 | 84 | 83 | 82 | ID | 99 | 99 | 98 | 88 | 75 | 88 | 87 | 96 | 37 | 98
L | 84 | 82 | 84 | 84 | 84 | 84 | 84 | 84 | 84 | 82 | 99 | ID | 100 | 99 | 89 | 75 | 89 | 88 | 97 | 37 | 99
M | 84 | 82 | 84 | 84 | 84 | 84 | 84 | 84 | 84 | 82 | 99 | 100 | ID | 99 | 89 | 75 | 89 | 88 | 97 | 37 | 99
N | 82 | 81 | 82 | 82 | 82 | 82 | 82 | 82 | 82 | 81 | 98 | 98 | 98 | ID | 88 | 74 | 88 | 87 | 96 | 37 | 98
O | 86 | 85 | 86 | 86 | 86 | 86 | 86 | 86 | 85 | 85 | 89 | 89 | 89 | 88 | ID | 77 | 94 | 79 | 88 | 37 | 88
P | 79 | 76 | 78 | 78 | 78 | 78 | 78 | 78 | 78 | 77 | 75 | 76 | 76 | 74 | 77 | ID | 76 | 82 | 76 | 33 | 74
Q | 82 | 81 | 81 | 81 | 82 | 81 | 81 | 81 | 81 | 80 | 88 | 88 | 88 | 86 | 94 | 74 | ID | 79 | 88 | 37 | 88
R | 76 | 76 | 76 | 76 | 76 | 76 | 76 | 76 | 76 | 74 | 87 | 88 | 88 | 86 | 78 | 83 | 77 | ID | 89 | 33 | 87
S | 85 | 83 | 85 | 85 | 84 | 85 | 85 | 85 | 84 | 84 | 96 | 97 | 97 | 95 | 88 | 76 | 86 | 90 | ID | 37 | 96
T | 12 | 13 | 12 | 12 | 13 | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 12 | 13 | 11 | 13 | 9 | 11 | ID | 37
U | 83 | 82 | 84 | 84 | 84 | 84 | 84 | 84 | 83 | 82 | 98 | 98 | 98 | 97 | 88 | 75 | 87 | 87 | 96 | 12 | ID
 |  |  |  |  |  |  | *** |  |  |  |  |  |  |  |  |  |  |  |  |  | 

Figure 6: Phylogenetic tree constructed from partial nucleotide sequences of the Newcastle disease virus (NDV) F gene together with representative reference strains and Egyptian isolates retrieved from GenBank. The current isolate, NDV-Fgene-Ismailia-ostrich-2024 (GenBank accession no. PX088918), is indicated in the tree. Phylogenetic analysis demonstrated that the ostrich isolate clustered within genotype II, class II, and was closely related to commonly used vaccine strains, including LaSota, Hitchner, Clone 30, and VG/GA. This clustering, together with the lentogenic F protein cleavage-site motif, supports the classification of the isolate as a lentogenic NDV strain. The phylogenetic tree was constructed using the Maximum-Likelihood method with 1,000 bootstrap replicates in MEGA version 7.0.