Molecular Characterization of Velogenic Newcastle Disease Virus (Sub-Genotype VII.1.1) from Wild Birds, with Assessment of Its Pathogenicity in Susceptible Chickens

Abstract

Newcastle disease (ND) is considered to be one of the most economically significant avian viral diseases. It has a worldwide distribution and a continuous diversity of genotypes. Despite its limited zoonotic potential, Newcastle disease virus (NDV) outbreaks in Egypt occur frequently and result in serious economic losses in the poultry industry. In this study, we investigated and characterized NDV in wild cattle egrets and house sparrows. Fifty cattle egrets and fifty house sparrows were collected from the vicinity of chicken farms in Kafrelsheikh Governorate, Egypt, which has a history of NDV infection. Lung, spleen, and brain tissue samples were pooled from each bird and screened for NDV by real-time reverse transcriptase polymerase chain reaction (RRT-PCR) and reverse transcriptase polymerase chain reaction (RT-PCR) to amplify the 370 bp NDV F gene fragment. NDV was detected by RRT-PCR in 22 of 50 (44%) cattle egrets and 13 of 50 (26%) house sparrows, while the conventional RT-PCR detected NDV in 18 of 50 (36%) cattle egrets and 10 of 50 (20%) of house sparrows. Phylogenic analysis revealed that the NDV strains identified in the present study are closely related to other Egyptian class II, sub-genotype VII.1.1 NDV strains from GenBank, having 99.7-98.5% identity. The pathogenicity of the wild-bird-origin NDV sub-genotype VII.1.1 NDV strains were assessed by experimental inoculation of identified strains (KFS-Motobas-2, KFS-Elhamoul-1, and KFS-Elhamoul-3) in 28-day-old specific-pathogen-free (SPF) Cobb chickens. The clinical signs and post-mortem changes of velogenic NDV genotype VII (GVII) were observed in inoculated chickens 3 to 7 days post-inoculation, with 67.5-70% mortality rates. NDV was detected in all NDV-inoculated chickens by RRT-PCR and RT-PCR at 3, 7, and 10 days post-inoculation. The histopathological findings of the experimentally infected chickens showed marked pulmonary congestion and pneumonia associated with complete bronchial stenosis. The spleen showed histocytic cell proliferation with marked lymphoid depletion, while the brain had malacia and diffuse gliosis. These findings provide interesting data about the characterization of NDV in wild birds from Egypt and add to our understanding of their possible role in the transmission dynamics of the disease in Egypt. Further research is needed to explore the role of other species of wild birds in the epidemiology of this disease and to compare the strains circulating in wild birds with those found in poultry.

Keywords: Egypt; NDV sub-genotype VII.1.1; RT-PCR; phylogenetic analysis; wild birds.

Figure 1

The diagram illustrates RT-PCR targeting 370 bp F gene fragment (nt no. 330–700) with F primer containing a characteristic virulent Newcastle disease virus (NDV) F gene cleavage site (nt no. 334–351). The low-virulent NDV cleavage site showed seven nucleotide substitutions (guanine instead of adenine at positions 334, 342, 343, 344, and 345 and cytosine instead of thiamine at positions 348 and 349) when compared to the forward primer nucleotide sequence, making it difficult to amplify the target fragment from low-virulent NDV.

Figure 2

Phylogenetic maximum-likelihood tree of NDV F gene fragment sequences with 1000 bootstrap repeats, including GVII sub-genotypes. Our strains KFS-Motobas-2, KFS-Elhamoul-1, and KFS-Elhamoul-3 (red triangles) were aligned with other GVII.1.1 strains obtained from GenBank.

Figure 3

BioEdit Clustal W algorithm alignment of the Egyptian GVII 1.1 strain’s nucleotide sequences showed G609A and T675C nucleotide substitutions in our three identified strains, T523C and A540G nucleotide substitutions in KFS-Elhamoul-1 and KFS-Elhamoul-3 strains, and T634C nucleotide substitution in KFS-Motobas-2.

Figure 4

BioEdit Clustal W algorithm alignment of the Egyptian GVII.1.1 strain’s deduced amino acid sequences showing no amino acid substitution in our strains (KFS-Motobas-2, KFS-Elhamoul-1, and KFS-Elhamoul-3).

Figure 5

Results of NDV GVII.1.1 isolation in the allantoic cavities of 10-day-oldembryonated chicken eggs. (A) NDV sub-genotype VII.1.1 KFS-Motobas-2-strain-inoculated embryo showing dwarfism, abnormal feathering, and congestion, with sub-cutaneous hemorrhages on the head and legs and (B) the negative control embryo inoculated with the negative control sample.

Figure 6

Post-mortem changes in experimentally infected chickens: (A) hemorrhages on the tips of proventricular glands (white arrow) and greenish proventricular contents (yellow arrow); (B) greenish intestinal contents (blue arrow) and hemorrhagic enteritis (green arrow).

Figure 7

Histopathological lesions in the lungs of experimentally infected Cobb chickens 5 days post-infection (dpi): (A) lung of the negative control bird (G4) showing normal parabronchi (arrowhead) and normal air capillaries (arrow); (B) lung of an experimentally infected bird in G1 showing congestion of blood capillaries and bronchial obstruction (arrowhead) attributed to peribronchial inflammatory cells infiltration (arrows); (C) lung of an experimentally infected bird in G1 showing marked endodermal hyperplasia around the parabronchi (arrows), associated with obvious inflammatory cells infiltration; (D) lung of an experimentally infected bird in G2 showing focal pneumonia associated with inflammatory cell infiltration (arrow); (E) lung of an experimentally infected bird in G2 showing mild congestion (arrowhead) and mostly patent bronchi and air capillaries; and (F) lung of an experimentally infected bird in G3 showing mild endodermal hyperplasia (arrows) and increase in the functional respiratory spaces stained by Hematoxylin and eosin (H&E X200).

Figure 8

Histopathological lesions in the brains of experimentally infected Cobb chickens 5 dpi: (A) brain of the negative control bird (G4) showing normal neuronal cells (arrow); (B) brain of an experimentally infected bird in G1 showing spongiosis of the nerve fibers (arrowhead) and focal malacia associated with diffuse and focal conglomerate aggregation of glia cells (arrow); (C) brain of an experimentally infected bird in G1 showing gliosis associated with neuronophagia (arrows); (D) brain of an experimentally infected bird in G2 showing ischemic neuronal injury (arrows); (E) brain of an experimentally infected bird in G3 showing mild to moderate degree of ischemic neuronal injury associated with marked neuronal tigrolysis (arrow); and (F) brain of an experimentally infected bird in G3 showing a mild degree of ischemic neuronal injury (arrow) (H&E, X200).

Figure 9

Histopathological lesions in the splenic tissues of experimentally infected Cobb chickens 5 dpi: (A) Scheme 4 showing a normal lymphoid follicle with normal lymphocytes around the central arteriole (arrow); (B) spleen of an experimentally infected bird in G1 showing marked lymphoid depletion associated with marked histocytic cell proliferation (arrows); (C) spleen of an experimentally infected bird in G2 showing marked histocytic cell proliferation (arrows); (D) spleen of an experimentally infected bird in G2 showing a normal lymphoid nodule (arrow); (E) spleen of an experimentally infected bird in G3 showing an increase in lymphoid cell proliferation within the white pulp (arrow); and (F) spleen of an experimentally infected bird in G3 showing increased lymphoid cell proliferation (arrow) (H&E, X200).