DISCUSSION

Zoonotic relevance and biosafety considerations

NDV is not considered a major zoonotic pathogen; however, occasional cases of mild conjunctivitis and transient influenza-like illness have been reported in individuals exposed to infected birds. Although the zoonotic potential of NDV is very limited, appropriate biosafety practices, personal protective equipment, and proper handling of infected birds and clinical samples should be maintained during outbreak investigations and vaccination procedures [31, 46].

Despite intensive routine vaccination, poultry health remains compromised by a variety of viral diseases, many of which cause substantial mortality and economic losses [47]. In Egypt, numerous NDV outbreaks have been reported in domestic poultry, resulting in significant adverse effects on the poultry industry [48]. The poultry sector plays a major role in the dissemination of NDV among susceptible avian hosts, including ostriches, thereby facilitating disease transmission and environmental contamination. In addition, migratory birds may shed the virus and serve as a potential source of infection for domestic poultry [27, 49].

Figure 7: Amino acid sequence alignment of the Newcastle disease virus (NDV) F protein from the ostrich isolate compared with vaccine strains (LaSota, Hitchner, Clone 30, and VG/GA-avenue and representative reference isolates included in the phylogenetic analysis. The F protein cleavage site region (residues 112–117) is highlighted in a red box. The ostrich isolate exhibited the motif ¹¹²GRQGRL¹¹⁷, which is characteristic of lentogenic NDV strains. The upper row indicates the amino acid positions of the LaSota II fusion protein. Amino acid substitutions are represented by letters, identical residues by dots (.), and alignment gaps by dashes (−). The absence of multiple basic amino acids at the cleavage site further supports the molecular classification of the isolate as a low-virulence (lentogenic) NDV strain.

Economic importance of ostrich production and NDV impact

Ostrich farming has expanded considerably and is now well established in several countries, including Egypt. In Egypt, ostrich production has become an economically important component of the livestock and poultry sector because of the commercial value of ostrich meat, leather, feathers, and oil. Commercial ostrich farms are concentrated mainly in Ismailia, El-Menofia, El-Behera, Damietta, and El-Sharquia governorates, contributing to food security, rural employment, and export opportunities. Ostrich meat is particularly valued for its high protein content and low cholesterol, making it an attractive alternative to conventional red meat for health-conscious consumers and international markets [50–52].

The economic consequences of NDV infection in ostrich farms extend beyond direct mortality and include reduced growth performance, increased treatment and vaccination costs, impaired reproductive performance, trade restrictions, and reduced market value of ostrich-derived products. Even infection with lentogenic NDV strains may adversely affect meat production, leather quality, feather yield, and export potential. Considering the growing economic importance of ostrich farming in Egypt, continued circulation of NDV may represent an emerging threat to the sustainability and profitability of this industry [53, 54]. Although NDV infection has been reported previously in Egyptian ostrich farms [28, 30], the circulating viruses remain poorly characterized genetically, and only limited information is available regarding their genotypes [29]. Therefore, characterization of NDV isolates circulating among ostrich flocks is essential for improving the understanding of the epidemiology, evolution, and transmission dynamics of the disease. The present study addressed this knowledge gap through molecular characterization of NDV isolates by sequencing and phylogenetic analysis of the _F _gene together with comprehensive pathological evaluation of naturally infected tissues.

Clinical and gross pathological findings

The clinical signs and gross lesions observed in NDV-infected ostriches were generally consistent with those reported in both experimental and naturally infected ostriches and other avian species. Unlike previous reports describing velogenic genotype VII infection in ostrich embryos, the lentogenic genotype II isolate identified in the present study induced remarkable lesions in the respiratory, digestive, hepatic, and lymphoid tissues. Clinically, affected ostrich flocks from four Egyptian governorates exhibited mild respiratory signs, with an overall mortality rate of 4%. These observations agree with a previous study [55], which reported respiratory and neurological manifestations in experimentally infected ostriches, particularly among young birds, suggesting that disease severity is influenced by age and immune status.

Gross pathological examination revealed lesions involving the trachea, lungs, liver, proventriculus, and intestines that were compatible with infection by lentogenic NDV strains. The principal lesions included mild tracheitis, pulmonary congestion and edema, hepatomegaly with pallor and focal necrosis, and swelling of the proventricular papillae. Similar gross pathological findings have been described previously [56]. Mild hepatomegaly, accompanied by hepatic congestion, observed in the present study is also consistent with the previous findings [57], which reported multifocal hepatic necrosis and congestion in ostriches experimentally infected with NDV. Likewise, intestinal lesions were characterized primarily by mucosal congestion. Disease severity appeared to be age-dependent, with younger birds developing more severe systemic manifestations, as previously reported [29]. Furthermore, although many clinical and pathological findings resemble those observed in chickens and turkeys, ostriches generally develop milder respiratory lesions and more variable intestinal involvement, likely due to species-specific differences in host immune responses and viral replication dynamics [58, 59].

Histopathological changes and tissue tropism

Microscopic examination demonstrated that NDV infection induced substantial lesions in multiple organs. Degeneration and desquamation of the pseudostratified ciliated columnar epithelium of the trachea, accompanied by subepithelial edema and inflammatory cell infiltration, indicate direct viral injury to the respiratory mucosa. Similar lesions have been reported in chickens infected with lentogenic NDV strains, in which epithelial damage compromises mucociliary clearance and predisposes birds to secondary bacterial infections, including those caused by Escherichia coli [60, 61].

Pulmonary lesions consisted predominantly of interstitial pneumonia characterized by thickened interalveolar septa, intra-alveolar exudation, bronchiolar epithelial necrosis, and marked heterophilic infiltration. Comparable pulmonary lesions have been described in ostriches [30] and chickens infected with NDV, reflecting vascular injury and severe inflammatory responses. Peribronchiolar lymphoid depletion further supports the immunosuppressive nature of NDV infection [55].

Hepatic lesions, including sinusoidal lymphocytic infiltration and vascular congestion, were compatible with lesions previously associated with lentogenic NDV strains in several avian species [62]. Similar mild hepatic lesions have also been reported in a previous study [63]. Histopathological alterations observed in the proventriculus, including glandular epithelial degeneration, necrosis, mucosal ulceration, and lymphoid depletion, further demonstrate the affinity of NDV for glandular and lymphoid tissues. The eosinophilic material observed within glandular lumina most likely represented necrotic cellular debris derived from damaged epithelial cells. These lesions have been consistently reported in both natural and experimental NDV infections.

The intestinal lesions, particularly within the ileum and ceca, were characterized by villous atrophy, crypt necrosis, and marked lymphoid depletion. Destruction of GALT, including Peyer's patches and cecal tonsils, emphasizes the immunosuppressive characteristics of NDV infection and explains the increased susceptibility of affected birds to secondary enteric infections [64].

Virus isolation, biological pathotyping, and molecular detection

Following inoculation into SPF-ECE, infected embryos developed only mild congestion of the skin and internal organs, consistent with previous reports [26, 56, 65]. The recovered isolates were HA-positive, with an HA titer of 9 log₂ HA units/mL and an HI titer of 6 log₂. Biological pathotyping confirmed that all isolates were lentogenic, with an MDT of 96 h and an ICPI of 0.4. These findings agree with previous reports [26, 63, 73–75] but differ from a previous study [28], which described velogenic NDV infection associated with high embryonic mortality in Egyptian ostriches. Similar observations have also been reported elsewhere [13, 69].

Molecular analysis by rRT-PCR confirmed NDV infection in eight samples, of which four representative isolates were subjected to partial F gene sequencing. These findings are consistent with previous molecular investigations [26–29, 70]. Importantly, this study documents the circulation of genotype II lentogenic NDV in non-vaccinated commercial ostrich flocks distributed across multiple Egyptian governorates. The detection of genetically related genotype II viruses across geographically separated farms suggests ongoing environmental exposure and potential epidemiological connections among ostrich farms, backyard poultry, and commercial chicken production systems.

Pathogenicity of lentogenic NDV in young ostriches

Although the identified isolate was classified as lentogenic based on the F protein cleavage-site motif, MDT, and ICPI values, clinically affected ostriches still developed significant pathological lesions. This apparent discrepancy may be explained by several factors, including the high susceptibility of young ostrich chicks, immature immune responses, environmental and management stressors, infectious dose, and possible concurrent bacterial infections. Previous studies demonstrated that young ostriches are considerably more susceptible to NDV infection than adult birds, even following exposure to low-virulence strains [71]. Species-specific host–virus interactions and differences in innate immune responses may also contribute to the observed clinicopathological manifestations. Furthermore, free-range production systems, transportation stress, nutritional deficiencies, and secondary bacterial infections may exacerbate disease severity, allowing lentogenic strains to produce clinically significant disease [72]. Similar observations have recently been reported for lentogenic and vaccine-related NDV strains under field conditions [73].

Phylogenetic characterization and genotype distribution

Phylogenetic analysis based on the partial F gene demonstrated that the ostrich isolate belonged to genotype II, class II and possessed the characteristic lentogenic cleavage-site motif ¹¹²GRQGRL¹¹⁷. These findings agree with a previous report [29], which identified four major NDV genotypes (II, III–IV, VI, and VII) among ostrich isolates, indicating substantial genetic diversity. Similarly, Ren _et al. _[26] identified a lentogenic NDV strain from farmed ostriches in China using whole-genome sequencing, whereas Elboraay _et al. _[30] also reported a lentogenic cleavage-site motif in an ostrich NDV isolate. In contrast, Ghaly _et al. _[28] described a velogenic Egyptian ostrich isolate possessing the cleavage-site motif ¹¹²RRQKRF¹¹⁷. Collectively, these findings suggest that NDV strains circulating in Egyptian ostriches do not originate from a single ancestral lineage.

The present isolate exhibited very high genetic similarity to genotype II vaccine-related strains previously reported in Egyptian poultry, including LaSota-like and Clone 30-like viruses, with nucleotide identities of 98%–99% and amino acid identities of 97%–98%. These findings support previous reports demonstrating the protective efficacy of genotype II vaccines in ostriches [55, 66, 70]. At the same time, they suggest that ostriches may serve as reservoirs or silent carriers of lentogenic NDV strains that facilitate virus transmission between ostriches and domestic poultry.

Vaccine-related genotype II strains and epidemiological implications

The close phylogenetic relationship between the present isolate and genotype II vaccine strains raises concerns about the silent circulation of vaccine-derived NDV strains in the field. Live-attenuated vaccines, particularly LaSota-like viruses, may spread horizontally among susceptible birds and persist in poultry-dense environments, thereby contributing to viral maintenance and evolution [73, 74]. Although genotype II vaccines are considered safe, prolonged circulation under field conditions could facilitate viral adaptation, accumulation of mutations, or recombination with circulating virulent strains [75, 76].

Additional factors that may facilitate virus dissemination include environmental contamination from vaccine virus shedding, mechanical transmission via personnel, equipment, vehicles, feed, or water, and exposure to wild or feral birds that act as biological or mechanical carriers. Previous studies have suggested that vaccine-derived NDV strains may contribute to viral evolution and, under certain circumstances, may increase in virulence following repeated bird-to-bird transmission [74, 77]. Furthermore, several Egyptian studies have frequently detected genotype II vaccine-related viruses in vaccinated commercial poultry, particularly in broiler and backyard chicken flocks [52, 76, 78]. Collectively, these findings support the hypothesis that genotype II vaccine-related viruses circulate among multiple avian hosts in Egypt, including ostriches.

Comparison with genotype VII strains and recombination findings

The present isolate exhibited relatively low genetic similarity to genotype VII chicken isolates circulating in Egypt, sharing nucleotide identities of 80%–82% and amino acid identities of 82%–84%. These findings differ from those of Ghaly _et al. _[28], who reported very high similarity (98.9%–99.2%) between Egyptian ostrich isolates and genotype VIIb chicken strains. Moreover, no evidence of recombination was detected within the partial F gene sequence, whereas Yin et al. [29] previously reported recombination between genotype II and genotype VII NDV isolates obtained from ostriches and chickens.

Implications for ostrich farming and NDV surveillance

These observations suggest that NDV infection in Egyptian ostriches may originate from multiple epidemiological sources, including infected poultry populations. Because ostriches are commonly raised under open-air, free-range management systems, exposure to infected domestic poultry and wild birds is likely to facilitate the introduction and maintenance of viruses. Considering the high nutritional value, disease resistance, and international commercial importance of ostrich products [79], continuous molecular surveillance, strengthened biosecurity, and routine molecular characterization of circulating NDV strains are essential to minimize the impact of NDV on the expanding ostrich industry and to support effective disease prevention and control strategies.