MATERIALS AND METHODS

Ethical approval

The study was conducted in accordance with institutional guidelines for animal welfare and experimental procedures. Ethical approval was obtained from the Institutional Animal Care and Use Committee of the Faculty of Veterinary Medicine, New Valley University, Egypt (Approval No. 07-2025-24). Permission to collect samples was obtained from the owners of all participating ostrich farms before sample collection. All procedures were conducted and reported in accordance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) 2.0 guidelines.

Study period and location

The study was conducted from January to December 2024. Samples were collected from eight diseased ostrich farms located in four Egyptian governorates: Ismailia, El-Menofia, El-Behera, and El-Sharquia (Figure 1, Table 1).

Study design

A cross-sectional field investigation was conducted to determine the occurrence and molecular characteristics of NDV circulating in naturally infected young ostriches. The study included clinical assessment, sample collection, virus isolation, molecular detection, biological pathotyping, phylogenetic analysis, and histopathological examination of suspected NDV cases.

Sample collection and processing

A total of 60 tissue samples were collected from eight ostrich farms with a history of mild respiratory disease characterized by depression, ocular discharge, and mild eyelid edema. The flock size ranged from 50 to 800 birds per farm. None of the investigated flocks had been vaccinated against NDV. Tissue specimens, including the trachea, lungs, intestine, kidney, brain, liver, spleen, and proventriculus, were collected from ostriches aged 3 weeks to 2 months and stored at −80°C until virus isolation and PCR-based molecular detection.

All samples were collected from freshly dead ostriches that died naturally during disease outbreaks; therefore, no euthanasia or experimental sacrifice was performed. Farms were selected based on the presence of clinical signs compatible with NDV infection and recent mortality among young ostriches. Only non-vaccinated flocks with owner consent were included in the study. Freshly dead birds aged 3 weeks to 2 months that died during active outbreaks were selected for tissue collection, whereas severely decomposed carcasses and farms lacking sufficient epidemiological information were excluded.

The collected tissues were homogenized in sterile phosphate-buffered saline (PBS: pH 7.2) to prepare a 10% (w/v) tissue suspension supplemented with an antibiotic mixture containing penicillin (1000 IU/mL), streptomycin (2 mg/mL), and gentamicin (2 mg/mL) (Sigma Chemical Company, St. Louis, MO, USA). Following overnight incubation at 4°C, the homogenates were centrifuged at 3000 rpm for 10 minutes, and the clarified supernatants were collected for subsequent virus isolation.

Virus isolation and hemagglutination assay

Individual tissue homogenates (0.2 mL) were inoculated into the allantoic cavity of 9–11-day-old specific-pathogen-free (SPF)-embryonated chicken eggs (ECE) obtained from the Nile SPF Farm (Koom Oshiem, Fayoum, Egypt). The inoculated eggs were incubated at 37°C for 5 days and monitored daily by candling to evaluate embryo viability [38]. Three successive passages were performed for virus isolation. Embryos that died within the first 24 h post-inoculation were considered to have died from nonspecific causes and were excluded from further analysis. The allantoic fluid was harvested from the remaining embryos and tested for hemagglutination (HA) activity using freshly prepared 1% chicken RBCs.

HA-positive allantoic fluids were subsequently confirmed by hemagglutination inhibition (HI) testing with NDV reference antiserum, following previously described procedures [38]. The LaSota strain (inactivated NDV antigen) was included as a positive control. Both the HA and hemagglutination inhibition (HI) assays were performed according to the procedures described in the World Organization for Animal Health (WOAH) Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, Chapter: Newcastle disease (infection with Avian orthoavulavirus 1).

Figure 1: Distribution of Newcastle disease virus-positive ostrich farms in the Egyptian governorates of Ismailia, El-Menofia, El-Behera, and El-Sharquia, represented by red circles.

Flock location | Ostrich farms | Samples (n) | Flock age | No. of birds | Positive farms | Prevalence (%) | Trachea | Brain | Lung | Proventriculus | Intestine | Liver | Spleen | Kidney
Ismailia | 4 | 29 | 4–6 weeks | 50–800 | 4 | 13.7 | 4 | 3 | 3 | 8 | 5 | 2 | 1 | 1
El-Menofia | 2 | 12 | 3–5 weeks | 100–600 | 2 | 16.6 | 2 | 1 | 2 | 4 | 2 | 1 | 1 | 1
El-Behera | 1 | 9 | 4–7 weeks | 110–550 | 1 | 11.1 | 1 | 1 | 2 | 1 | 2 | – | 1 | 1
El-Sharquia | 1 | 10 | 4–8 weeks | 140–700 | 1 | 10.0 | 1 | 1 | 2 | 3 | 2 | 1 | - | -

Biological pathotyping

Biological pathotyping of the NDV isolates was performed by determining the mean death time (MDT) in SPF-ECE and the ICPI in 1-day-old SPF chicks according to established protocols [31]. The SPF-ECE and chicks were obtained from the Nile SPF Farm (Koom Oshiem, Fayoum, Egypt).

For the MDT assay, five 9–11-day-old SPF-ECE were inoculated with each virus dilution according to WOAH recommendations. The ICPI assay was performed by intracerebral inoculation of ten 1-day-old SPF chicks, which were observed daily for 8 consecutive days. All assays were conducted in duplicate.

Sterile PBS-inoculated SPF-ECE and SPF chicks served as negative controls and remained free of clinical signs, lesions, and mortality throughout the experimental period. MDT and ICPI values were interpreted according to the standard WOAH criteria for NDV pathotype classification. Because MDT and ICPI are standardized biological pathotyping assays, the results were interpreted descriptively without additional statistical analysis.

RNA extraction and rRT-PCR

Viral RNA was extracted from infected allantoic fluid obtained from ten NDV isolates using the QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The purity and concentration of the extracted RNA were determined spectrophotometrically prior to amplification to ensure RNA quality for downstream molecular analyses.

Detection of NDV was performed by rRT-PCR targeting the M gene using the QuantiTect® Probe RT-PCR Kit (Qiagen) according to the manufacturer's instructions. The primer and probe sequences used for amplification are presented in Table 2 [32]. Samples with cycle threshold values ≤35 were considered positive for NDV RNA. The rRT-PCR assay was performed according to previously validated protocols and the manufacturer's recommendations.

Assay type | Primer name | Sequence (5′–3′) | Target gene | Amplicon size | Thermal cycling conditions
rRT-PCR | NDV M-F | AGTGATGTGCTCGGACCTTC | Mgene | 121 bp | Reverse-transcription at 50°C for 30 min, followed by initial activation/denaturation at 95°C for 15 min. Amplification was performed for 40 cycles consisting of denaturation at 94°C for 15 s and annealing/extension at 60°C for 60 s.
rRT-PCR | NDV M-R | CCTGAGGAGAGGCATTTGCTA |  |  | 
rRT-PCR | NDV M-Probe | [FAM]TTCTCTAGCAGTGGGACAGCCTGC[TAMRA] |  |  | 
RT-PCR | M2 (Forward) | TGGAGCCAAACCCGCACCTGCGG | F gene | 766 bp | Reverse-transcription at 50°C for 30 min, followed by preliminary denaturation at 95°C for 15 min. PCR amplification consisted of 35 cycles of denaturation at 94°C for 30 s, annealing at 55°C for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min.
RT-PCR | F2 (Reverse) | GGAGGATGTTGGCAGCATT |  |  | 

Screening for coinfection by RT-qPCR

All NDV-positive samples were subsequently screened for coinfection with Avian influenza virus (AIV), Infectious bronchitis virus (IBV), and Infectious bursal disease virus (IBDV) using specific RT-qPCR assays. The primer and probe sequences used for consensus detection and subtype identification are presented in Table 3 [33–38].

F gene amplification, sequencing, and phylogenetic analysis

NDV-positive samples identified by RT-qPCR were subjected to partial amplification of the F gene using the QIAGEN OneStep RT-PCR Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Amplification was performed using the primer pair M2 (forward: 5′-TGGAGCCAAACCCGCACCTGCGG-3′; nucleotides 980–1003 of the M gene) and F2 (reverse: 5′-GGAGGATGTTGGCAGCATT-3′; nucleotides 503–485 of the F gene), generating a 766-bp amplicon as previously described [39].

The amplified PCR products were separated by electrophoresis on 1.5% agarose gels. The target bands were excised and purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's protocol. Purified PCR products were sequenced in both directions using the BigDye™ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Waltham, MA, USA) on an ABI 3500xL Genetic Analyzer (Life Technologies, Carlsbad, CA, USA).

The nucleotide sequence of the amplified F gene was deposited in GenBank under accession number PX088918. Nucleotide and deduced amino acid sequences were assembled and aligned using the ClustalW algorithm and compared with published NDV vaccine strains and representative reference strains belonging to classes I and II available in GenBank [40].

Phylogenetic analysis was performed using the maximum-likelihood method with 1000 bootstrap replicates in MEGA version 7.0, applying the Kimura two-parameter substitution model [41]. Raw forward and reverse chromatograms were manually inspected to verify sequence quality and ambiguous base calls before consensus sequence assembly. Low-quality terminal regions were trimmed before generating the final consensus sequences using BioEdit software. Final phylogenetic tree visualization and annotation were performed using MEGA version 7.0 and BioEdit.

Recombination analysis was conducted to investigate the presence of potential recombinant NDV strains using RDP5 software (version 4.97) with multiple detection algorithms, including RDP, BootScan, MaxChi, GENECONV, SiScan, Chimaera, LARD, PhylPro, and 3Seq [42, 43]. A recombination event was considered positive only when supported by at least four independent detection methods.

Target | Primer and probe sequences | Reference
AIV M gene | sep1: AGATGAGTCTTCTAACCGAGGTCG sep2: TGCAAAAACATCTTCAAGTCTCTG sep-probe: FAM-TCAGGCCCCCTCAAAGCCGA-TAMRA | [33]
AIV H5 subtype | H5LH1: ACATATGACTACCCACARTATTCAG H5RH1: AGACCAGCTAYCATGATTGC H5PRO: FAM-TCWACAGTGGCGAGTTCCCTAGCA-TAMRA | [34]
AIV H6 subtype | IAV-H6-1666F: CTTGGTGTGTATCAAATYCTTGC IAV-H6-1776R: CATTGARCCATTTGARCACATCCA IAV-H6-1693: FAM-TATAGTACGGTATCGAGCAGYCT-MGB | [35]
AIV H9 subtype | Forward: GGAAGAATTAATTATTATTGGTCGGTAC Reverse: GCCACCTTTTTCAGTCTGACATT Probe: FAM-AACCAGGCCAGACATTGCGAGTAAGATCC-TAMRA | [36]
AIV N1 subtype | Forward: TAYAACTCAAGGTTTGAGTCTGTYGCTTG Reverse: ATGTTRTTCCTCCAACTCTTGATRGTGTC Probe: FAM-TCAGCRAGTGCYTGCCATGATGGCA-TAMRA | [35]
AIV N2 subtype | Forward: TGGACAGGGAACAACACTAAAC Reverse: ACAAGCCTCCCATCGTAAAT Probe: TXRED-CAAATGAAATGGAACACCCAACTCAT-BHQ23 | [35]
AIV N8 subtype | N8-1296F: TCCATGYTTTGGGTTGARATGAT N8-1423R: GCTCCATCRTGCCAYGACCA Probe: FAM-TCHAGYAGCTCCATTGTRATGTGTGGAGT-TAMRA | [35]
IBV | AIBV-fr: ATGCTCAACCTTGTCCCTAGCA AIBV-as: TCAAACTGCGGATCATCACGT AIBV-TM: FAM-TTGGAAGTAGAGTGACGCCCAAACTTCA-TAMRA | [37]
IBDV | F/AUS GU: TCACCGTCCTCAGCTTACCCACATC R/AUS GL: GGATTTGGGATCAGCTCGAAGTTGC | [38]

Histopathological examination

Representative tissue samples were collected from naturally infected and recently deceased young ostriches suspected of NDV infection. Following necropsy under sterile conditions, specimens from the trachea, lungs, liver, proventriculus, and intestines were immediately fixed in 10% neutral-buffered formalin for 48 h.

After fixation, tissues were processed routinely for paraffin embedding. Samples were dehydrated through ascending grades of ethanol, cleared in xylene, embedded in molten paraffin wax, and sectioned at a thickness of 4–5 μm using a rotary microtome. Tissue sections were mounted on glass slides and stained with H&E. Histopathological examination was performed using a Leica DM 500 light microscope (Leica Microsystems, Wetzlar, Germany) [44].

Histopathological lesions were evaluated using a semiquantitative scoring system. Tissue sections without detectable lesions were assigned a score of 0, whereas slight, moderate, and severe lesions received scores of 1, 2, and 3, respectively [45].

Biosafety and containment

All procedures involving NDV-positive samples, virus isolation using SPF-ECE, and molecular analyses were conducted in a biosafety level 2 laboratory following institutional biosafety and biosecurity regulations for handling avian avulaviruses. Appropriate personal protective equipment was worn during all laboratory procedures, and infectious materials and biological waste were decontaminated in accordance with established institutional biosafety protocols.