Infectious Disease Research

Veterinary Pathology
50(4) 638-647
Comparative Study on the Pathogenesis ª The Author(s) 2012
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of the Generated 9a5b Newcastle DOI: 10.1177/0300985812467470
vet.sagepub.com
Disease Virus Mutant Isolate Between
Chickens and Waterfowl

Z. Anis1,3,4, T. Morita1, K. Azuma1, H. Ito2, T. Ito2, and A. Shimada1

Abstract
Lentogenic Newcastle disease viruses (NDVs), circulating among waterfowl, have the potential to become highly pathogenic by
replication in chickens. The pathological studies that compare NDV infections in chickens and waterfowl are rare. The virulent
9a5b mutant NDV isolate was generated by passaging the lentogenic Goose/Alaska/415/91 NDV isolate in chickens. The
pathogenesis of the virulent 9a5b mutant isolate is unknown in both chickens and waterfowl. In this study, the virulent 9a5b
mutant NDV isolate was inoculated intranasally in 32-day-old specific pathogen-free white Leghorn chickens and Japanese
commercial ducks. Unlike ducks, which remained clinically normal throughout the study, chickens had depression, gasping, oral
discharges, and greenish-white soft feces. Gross and histologic lesion patterns as well as viral replication supported the differing
clinical outcome. Ducks had slight inflammation mainly in respiratory and digestive tracts, whereas slight nonpurulent encephalitis,
necrotizing pancreatitis, tubulointerstitial nephritis, and mild inflammation in respiratory and digestive tracts were detected in
chickens. In agreement, interferon-beta (IFN-b)–immunopositive signals were more intense in lung tissue of ducks than that of
chickens, and NDV replications were detected intensively in chicken tissues. These results suggest that the 9a5b mutant NDV
isolate is more virulent in chickens, and slight histological lesions were induced in ducks even with virulent NDVs.

Keywords
chickens, ducks, histopathology, Newcastle disease virus, 9a5b isolate

Newcastle disease (ND) is a highly contagious disease affect-
ing many domestic and wild avian species, causing serious
economic losses to the poultry industry worldwide.4 ND is
caused by ND virus (NDV), synonymous with avian paramyx-
ovirus–1 (APMV-1).2,3 NDV is an enveloped virus containing
a negative-sense, single-stranded RNA genome, which encodes
for 6 structural and 2 nonstructural proteins.11,21 NDVs have
3 major pathotypes: lentogenic (low virulence), mesogenic
(moderate virulence), and velogenic (high virulence). In chick-
ens, lentogenic strains produce mild or inapparent respiratory
infections. Mesogenic strains are associated with low mortality,
acute respiratory disease, and neurologic signs in some birds.
The velogenic strains are further divided into neurotropic
velogenic NDVs (causing respiratory and neurologic signs with
high mortality) and viscerotropic velogenic NDVs (causing
acute lethal infections with necro-hemorrhagic lesions that are
most obvious in the gastrointestinal tract).3
The virulent 9a5b NDV mutant isolate was generated
from the lentogenic waterfowl Goose/Alaska/415/91 (original)
isolate by 9 consecutive passages in chicken air sacs, followed
by 5 passages in chicken brain. Goose/Alaska/415/91 strain
has been classified phylogenetically into class I lineage 6,
which includes viruses from wild waterfowl and US live-bird
markets.37 Although the original isolate has an intracerebral
pathogenicity index (ICPI) equal to 0 and an avirulent fusion
(F) gene cleavage site, the 9a5b NDV isolate has an ICPI equal
to 1.88 and a virulent F gene cleavage site.32
Nonvirulent NDVs have the typical avirulent cleavage motif,
112
R/G-R/ K-Q-G-R116, with a leucine (L117) at the N terminus
of the F protein after cleavage (F1), which is only susceptible
to trypsin-like enzymes found in limited tissues such as those
in respiratory and digestive tracts, causing localized infection.
On the other hand, the virulent NDVs have a virulent cleavage
motif, 112R/K-R-Q-R/K-R116, and phenylalanine (F117) at the
1
Department of Veterinary Pathology, Tottori University, Minami, Koyama-cho,
Tottori, Japan
2
Department of Veterinary Public Health, Tottori University, Minami,
Koyama-cho, Tottori, Japan
3
The United Graduate School of Veterinary Science, Yamaguchi University,
Yamaguchi, Japan
4
Faculty of Veterinary Medicine, Menoufiya University, Sadat City, Egypt
Corresponding Author:
T. Morita, Tottori University, Koyama-cho Minami4-101, Tottori, 680-8553,
Japan.
Email: morita@muses.tottori-u.ac.jp
Anis et al
N terminus of F1, which enables them to infect the host systemi-
cally by acquiring susceptibility to furin or other ubiquitous
intracellular host cell proteases.10,12–14,18,27,30–32,42
Moreover, a genetic comparison between the original and
9a5b isolates demonstrated 3–amino acid substitutions in the
hemagglutinin-neuraminidase (HN) proteins, changing the
inactive HN0 precursor (found in avirulent viruses) to a biolo-
gically active HN protein (found in virulent viruses and is
responsible for the attachment of virus particles to the host cell
and promotes fusion activity of the F protein).16,24,29,36,38
Among poultry, chickens are the most susceptible to NDV
infection, whereas ducks and geese are the least susceptible.17
Furthermore, wild birds are considered to be the natural reservoir
of NDV.43 Avirulent viruses, maintained in wild waterfowl in
nature and bearing the consensus avirulent-type sequence, have
the potential to become velogenic after transmission and circula-
tion in chicken populations.32 Therefore, investigating the differ-
ences in the pathogenesis of NDV infection between chickens
and waterfowl can help us to determine why waterfowl is a suit-
able reservoir for these viruses and why ND outbreaks occur in
chickens rather than in waterfowl.
This study was initiated to compare the clinical signs,
lesions, and viral distribution in specific pathogen-free (SPF)
chickens and commercial ducks infected with the mutant
9a5b NDV isolate. This isolate was chosen because (1) the
original isolate was isolated from migratory waterfowl and
was lentogenic; (2) mutation in its genome occurred by
serial passages in chickens, which changed it to a virulent
isolate; (3) and the pathogenicity index was previously
determined before and after mutation. The data reported
here provide, to the best of the authors’ knowledge, the first
histopathological comparative study between chickens and
ducks after experimental NDV infection.
Materials and Methods
Virus
The 9a5b NDV mutant isolate was obtained from the Depart-
ment of Public Health, Tottori University, Japan.32 After pro-
pagation in SPF eggs, 0.1 ml of the viral suspension, which
contained 108.75 egg infective dose (EID)50, was inoculated
intranasally in both chickens and ducks.
Chickens and Ducks
Twenty-three 32-day-old male white Leghorn SPF chickens
and twenty-three 32-day-old male Japanese commercial ducks
were obtained from the Nippon Institute for Biological Science
and divided into 4 groups. The first (n ¼ 5) and the second
(n ¼ 18) groups comprised control and infected SPF chickens,
respectively. The third (n ¼ 5) and the fourth (n ¼ 18) groups
comprised control and infected Japanese commercial ducks,
respectively. All groups were reared separately in bird-bred
isolators in biohazard rooms and given water and food ad
libitum and monitored clinically.
639
After a 1-week acclimatization period, the infected groups
were inoculated intranasally with the virus. Three birds from
each infected group were slaughtered on the 1st, 2nd, 4th,
6th, 8th, and 10th day postinoculation (dpi). Control birds were
slaughtered on the last day of the experiment (10th dpi). To
reduce the pain, the birds were slaughtered under Halothane
inhalation anesthesia. The ethics committee on animal experi-
ments from Tottori University approved the experimental pro-
tocol, and the experiments were carried out in accordance with
the guidelines for animal experiments in the same facility.
Necropsy Examination and Sampling
Necropsy examination was performed immediately after the
birds were killed. The brain, eyelid, nostrils, larynx, trachea,
lung, air sac, heart, esophagus, proventriculus, gizzard, duode-
num, jejunum, ileum, cecum, colon, liver, pancreas, and kidney
were collected and fixed in 10% neutral buffered formalin
(NBF). Tracheal samples were taken and fixed in 2.5% glutaral-
dehyde for scanning electron microscopy (SEM) investigation.
Histopathology and Immunohistochemistry
After 72 hours of fixation in NBF, samples from the above-
mentioned organs were dehydrated, embedded in paraffin wax,
and sectioned for hematoxylin and eosin (HE) staining (3-mm-
thick sections) or immunohistochemistry (IHC; 5-mm-thick
sections on positively charged slides). To detect the nucleopro-
tein (NP) of NDV by IHC, we followed the method described in
Kommers et al19 for making the primary antibody. Briefly, a
peptide antigen was used to produce antisera with the sequence
TAYETADESETRRIC. This sequence represents residues 181
to 194 of the NP protein with a C addition for coupling. The
peptide was coupled to keyhole limpet hemocyanin and used
to immunize a rabbit by the standard procedures. The immuno-
globulin G fraction was purified by affinity-column chromato-
graphy with the coupled peptide (Hokudo Bioscience, Tokyo,
Japan). For IHC staining, the following protocol was used: after
deparaffinization, tissue sections were treated with 3% H2O2
for 15 minutes at room temperature (RT) for endogenous per-
oxidase inactivation and then subjected to antigen retrieval
by microwaving for 10 minutes at full power in citrate buffer
(pH 5.4), followed by blocking with 10% normal goat serum
(NGS) for 5 minutes in the microwave. Tissues were incubated
with the primary antibody (X8000) overnight at 4 C. After
stringent washing, sections were incubated with a labeled poly-
mer (ChemMate Dako EnVision/HRP [DAP]; Dako, Carpin-
teria, CA) for 30 minutes at RT and then subjected to
stringent washing again. The substrate was diaminobenzidine
(DAB; Dako), and the sections were stained with hematoxylin
and covered with DPX mounting media (Sigma Life Science,
Steinheim, Germany).
To detect interferon-beta (IFN-b) in lung tissues by IHC, we
followed the above-mentioned protocol with the following
modification: microwaving antigen retrieval was done for 20
minutes before H2O2 treatment, primary antibody (X400) was
640 Veterinary Pathology 50(4)

Table 1. Histopathological Findings in Chickens and Ducks Infected With the Mutant 9a5b Newcastle Disease Virus (NDV) Isolate.

Organs Infected Chickens Infected Ducks

Brain Slight nonpurulent encephalitis (P6–P10)a No significant changes

Eyelid Mild conjunctivitis (P4–P6) Slight conjunctivitis (P6–P8)
Nostrils Moderate lymphocytic rhinitis with deciliation, degeneration, Slight lymphocytic rhinitis with heterophilic infiltration (P1–P2)
and necrosis of epithelial cells (P2–P4)
Larynx Moderate lymphocytic laryngitis (P2–P4) Infiltration of lymphocytes and macrophages in the mucosa
(P2–P6)
Trachea Mild tracheitis with deciliation of some parts (P4–P8) Proliferation of goblet and lymphoid cells in the mucosa
(P4–P6)
Lung Moderate interstitial pneumonia (P2–P8) Mild interstitial pneumonia (P2–P8)
Air sacs Moderate airsacculitis (P8–P10) Slight airsacculitis (P8–P10)
Heart Multifocal areas of mild lymphocytic myocarditis (P6–P8) No significant changes
Esophagus Slight esophagitis (P4–P8) Proliferation of lymphoid aggregates in lamina propria
(P2–P10)
Proventriculus Mild proventriculitis with heterophilic infiltration (P2–P6) Infiltration of macrophages and lymphoid cells (P4–P6)
Gizzard Mild infiltration of macrophages and lymphocytes in mucosa Slight heterophilic infiltration in tunica mucosa (P2–P6)
and tunica muscularis (P6–P8)
Duodenum Mild duodenitis (P2–P6) Heterophilic infiltration and increased mitotic figures (P4–P6)
Jejunum Mild jejunitis (P4–P6) Proliferation of lymphoid cells in the mucosa (P4–P8)
Ileum Proliferation of mucosal lymphoid and goblet cells (P4–P6) Proliferation of macrophage, goblets cells, and increased
mitotic figures in mucosal epithelium (P2–P8)
Cecum Mild hemorrhagic tonsillitis (P4–P10) Proliferation of the cecal tonsils (P2–P10)
Colon Slight proliferation of lymphoid cells in lamina propria As chickens plus heterophilic infiltration and increased mitotic
(P4–P10) figures in epithelium (P4–P8)
Liver Marked proliferation of ectopic lymphoid tissue, bile pigment Mild proliferation of ectopic lymphoid tissue with heterophilic
in dilated bile canaliculi (P4–P8) infiltration (P4–P8)
Pancreas Severe necrotizing pancreatitis (P6–P10) No significant changes
Kidney Marked proliferation of ectopic lymphoid tissue, multifocal Slight proliferation of ectopic lymphoid tissue (P4–P10)
areas of necrotizing interstitial nephritis (P8–P10)
a
Px–Pz means the peak period of histological findings, and P means the postinfection day.

polyclonal rabbit anti-human IFN Beta (AbD Serotec, Kidling- in the infected duck group or in any of the 2 control groups. No
ton, UK), and a weak wash was applied with this antibody. deaths were recorded in this experiment.

Scanning Electron Microscopy Gross Lesions

Tracheal samples were processed for SEM by the standard
method. Briefly, tissue specimens were fixed in 2.5% glutaral-
dehyde in 0.1M phosphate buffer (PB; pH 7.3) at 4 C over-
night, washed in PB, postfixed in 1% osmium tetraoxide (pH
7.4) for 1 hour at RT, washed in PB, and dehydrated in increas-
ing concentrations of ethanol and finally in t-butyl alcohol.
Samples were glued with carbon cement on aluminum stubs
and coated with silver in a vacuum evaporator. Observation
was made using an SEM (Hitachi Co. Ltd., Tokyo, Japan).
Results
Clinical Signs
Clinical signs, which appeared only in infected chickens,
started from the third dpi and included a sleeping appearance
and greenish-white soft feces, and they peaked at the fourth and
the fifth dpi, with symptoms such as depression, gasping, and
oral discharges. All of these symptoms decreased from the
sixth dpi, and the chickens showed only slight depression on
the last day of the experiment. No clinical signs were detected
The infected chickens had mild congestion and hemorrhages in
the nasal mucosa and lung, besides swelling of the 2 ceca. The
gallbladder was swollen and engorged with bile, and the con-
tent of the digestive tracts was stained with the bile pigment.
The air sacs were slightly thick and opaque. In addition, small
hemorrhages were detected in the myocardium and cecal ton-
sils. Furthermore, the pancreatic white necrotic foci were the
most prominent gross change by the 10th dpi. On the other
hand, the infected ducks basically had no gross lesions, but dis-
tended ceca and engorged gallbladder were observed. In addi-
tion, the content of the digestive tracts was stained with bile
pigment. No gross lesions were detected in control chickens
and ducks.
Histopathology and Immunohistochemistry
Histopathological and immunohistochemical findings are sum-
marized in Tables 1, 2, and 3. Differences between chickens
and ducks infected with the 9a5b NDV isolate are described
below.
Anis et al 641

Table 2. Immunolabelingsa for Newcastle Disease Viral Nucleoprotein Antigen by Immunohistochemistry in Both Chickens and Ducks.

Infected Chickens Infected Ducks

Organs P1 P2 P4 P6 P8 P10 P1 P2 P4 P6 P8 P10

Brain – – – – – – – – – – – –
Eyelid – – – – – – – – – – – –
Nostrils þþþ þþþ þþþ þþ þþ þ þ þ þ þ þ þ
Larynx – – þ þ þ þ þ þ þ – – –
Trachea – – þ þ þ þ – þ þ – – –
Lung þ þþ þþ þþ þþ þ þ þ þ þ þ þ
Air sacs – – þ þ þ þ – – – – þ þ
Heart – – þ þ þ – – – – – – –
Esophagus – – – – – – – – – – – –
Proventriculus – – þ þ þ þ – – – þ þ –
Gizzard – – þ þ þþ þ – – – – – –
Duodenum þ þ þþ þ þþ þþ þ þ þ þ þ þ
Jejunum þ þ þ þ þ þ – – þ þ þ þ
Ileum þ þ þþ þ þþ þþ – – þ – – –
Cecum þ þ þþ þ þþ þþ – – þ – – –
Colon – – þ þ þ þ – – – – þ þ
Liver – – þ þ þ þ – – – – – –
Pancreas – – – – þþ þþ – – – – – –
Kidney – – – – – þ – – – – – –
a
No labeling (–); rare positive cells (þ); frequent positive cells (þþ); regular clusters of positive cells (þþþ).

observed in the infected ducks (Fig. 2a), and the NDV-NP–

Table 3. Immunohistochemical Demonstration of Interferon-b in
Lung Tissue at Early Stage After Infection With 9a5b Newcastle
Disease Virus Isolate in Chickens and Ducks.a
Infected Chickens Infected Ducks
Control – –
P1 + þ
P2 þ þ
P4 þ þþ
a laryngeal and tracheal mucosa.
No staining (–); minimal (+); mild (þ); moderate clusters of positive cells (þþ).
Brain. Brain lesions were observed in the infected chickens
only. Slight nonsuppurative lymphocytic encephalitis was
detected, and it was characterized by focal areas of microgliosis
and lymphocytic perivascular cuffing in the cerebrum, cerebel-
lum, and medulla oblongata. In addition, vacuolation and
demyelination in the cerebellar white matter, as well as mild
loss of Purkinje cells, were also observed. In contrast, the
NDV-NP antigen could not be detected in the brain by IHC.
Conjunctiva. Although lymphocytic conjunctivitis with het-
erophilic infiltration was mild in chickens and slight in ducks,
the NDV-NP antigen could not be detected in both.
Respiratory system. The infected chickens had moderate
lymphocytic rhinitis with slight hemorrhages, deciliation,
and degeneration and necrosis of epithelial cells (Fig. 1a)
with associated NDV-NP–immunopositive signals in the
epithelial cells and mononuclear cells at the same site (Fig. 1b).
On the other hand, slight lymphocytic rhinitis with proli-
feration of goblet cells and heterophilic infiltration were
immunopositive signals detected in the epithelial cells and
mononuclear cells (Fig. 2b) were less intensive than those
of chickens. Moderate lymphocytic laryngitis and mild tra-
cheitis with loss of cilia and erosion were detected in the
infected chickens, whereas laryngitis and tracheitis in the
infected ducks were milder than those of chickens, and
hyperplasia of goblet cells and infiltration of heterophils,
lymphocytes, and macrophage-like cells were prominent in
The infected chickens had moderate lymphocytic interstitial
pneumonia, which was characterized by slight hemorrhages,
proliferation of periparabronchial lymphoid tissues, and infil-
tration of macrophages, lymphoid cells, and few heterophils
(Fig. 3) with associated NDV-NP antigen in macrophage-
like cells and epithelial cells (inset of Fig. 3). Furthermore,
moderate airsacculitis was developed at the late stage of the
experiment. On the other hand, mild lymphocytic interstitial
pneumonia, which was characterized by heterophilic infiltra-
tion and proliferation of periparabronchial and air passages
submucosal lymphoid tissues, was developed in the infected
ducks (Fig. 4). NDV-NP–positive signals were detected in
macrophage-like cells and epithelial cells (inset of Fig. 4).
In addition, slight airsacculitis was developed at the late stage
of the infection.
Weak IFN-b–immunopositive signals were detected in
the lung tissues of the infected chickens, especially in the
epithelial cells of the parabronchi (Fig. 5). On the other
hand, IFN-b–immunopositive staining in the infected ducks
was detected in cells morphologically consistent with fibro-
blasts and macrophages in lung tissues, especially in the air
passages subepithelial tissues and in the epithelial cells of
642 Veterinary Pathology 50(4)

Figure 1. Nostrils; chicken, P1. (a) Mild lymphocytic rhinitis with degeneration and necrosis of the mucosal epithelial cells (arrows). Hematox-
ylin and eosin (HE). (b) Positive signals for Newcastle disease virus–nucleoprotein (NDV-NP) in mucosal epithelial cells (arrows) and mono-
nuclear cells (arrowhead). Immunohistochemistry (IHC), hematoxylin counterstained. Figure 2. Nostrils; duck, P1. (a) Hyperplasia of
goblet cells (arrow) and infiltration of mononuclear cells (arrowhead). HE. (b) Positive signals for NDV-NP in mucosal epithelial cells and in
mononuclear cells (arrowhead). IHC, hematoxylin counterstained. Figure 3. Lung; chicken, P2. Moderate interstitial pneumonia with infiltration
Anis et al 643

the parabronchi (Fig. 6), and it was more intensive than
those of chickens, as described in Table 3.
Heart. The heart lesion, which developed in the infected
chickens only, was mild and appeared at the first dpi as scat-
tered areas of lymphocytic myocarditis, and then degeneration
of myofibers, slight hemorrhages, and heterophilic infiltration
were detected later (Fig. 7a) with associated NDV-NP–immu-
nopositive macrophages and lymphocytes in the myocardium
(Fig. 7b).
Digestive tract. The infected chickens had slight esophagitis
and mild hemorrhagic proventriculitis with infiltration of
lymphocytes, macrophages, and heterophils in the mucosa
and lamina propria. In addition, infiltration of macrophages
and lymphocytes in the mucosa and tunic muscularis of the giz-
zard, mild duodenitis with scattered areas of degeneration, and
necrosis in the duodenal mucosa (Fig. 8a) were detected.
Furthermore, mild jejunitis, proliferation of lymphoid and gob-
let cells in the ileum mucosa, proliferation of cecal tonsils with
slight hemorrhages, and proliferation of lymphoid tissues of the
colon lamina propria were also found. NDV-NP–immunoposi-
tive signals were detected in macrophages and epithelial cells
in many parts of the digestive tract (Table 2) and, as described
in Fig. 8b, in the necrotic area and epithelial cells of the duode-
nal mucosa. On the other hand, in the infected ducks, the mito-
tic figures were increased in the epithelial cells of the mucosal
gland throughout the digestive tract (Fig. 9). In addition, hyper-
plasia of goblet cells, proliferation of lymphoid cells, and
heterophilic infiltration in the mucosa and submucosa of the
digestive tract were observed. NDV-NP–immunopositive
signals in the digestive tract of the infected ducks were less
frequent and fainter than those of chickens, as described in
Table 2 and as shown in macrophage-like cells between duode-
nal glands (Fig. 9).
Liver. The infected chickens had proliferated ectopic lym-
phoid tissues, and the bile canaliculi were dilated and engorged
with bile (Fig. 10a) with associated NDV-NP–immunopositive
macrophages and Kupffer cells (Fig. 10b). In turn, in the
infected ducks, mild proliferation of ectopic lymphoid tissues
and moderate heterophilic migration in hepatic sinusoids were
observed (Fig. 11).
Pancreas. Only in the infected chickens was the pancreatic
lesion developed. Slight necrotizing pancreatitis was observed
at the first dpi and dramatically increased at the sixth dpi and
did not regress until the end of the experiment (Fig. 12a), with
associated NDV-NP–immunopositive signals in necrotic
pancreatic acinar cells (Fig. 12b).
Kidney. Proliferations of the ectopic lymphoid tissues were
observed in the infected chickens at the second dpi, whereas
tubulointerstitial nephritis was detected by the sixth dpi and
increased in severity until the end of the experiment (Fig. 13),
with associated NDV-NP–immunopositive staining in the
degenerated renal tubules (inset of Fig. 13). On the other hand,
renal tissues of the infected ducks had slight proliferation of
ectopic lymphoid tissue.
The histological features in both control chickens and ducks
were within the normal limits, and no NDV-NP–immunoposi-
tive signals were detected in these groups.
Tracheal Ultrastructural Changes
Infected chickens had excessive secretion of globular mucous
particles on the epithelial surface at the first dpi. By the fourth
dpi, a marked increase in the proportion of nonciliated to
ciliated epithelium was observed, and disorientation of the cilia
as well as adherence of the cilia to each other and to the under-
lying surface was seen. Deciliation of large parts of the tracheal
surface and erosions in the mucosal epithelial cells were also
detected (Fig. 14). In addition, scanty mucous was found on the
tracheal surface by the 10th dpi. In turn, the infected ducks had
mild globular mucous particles, slight disorientation of the
cilia, and a fine mucous network on the cilial tips (Fig. 15), and
blankets of mucous were covering the underlying epithelium.
Ultrastructural features of tracheal mucosa in both control
chickens and ducks were within normal limits.
Discussion
In this study, the 9a5b NDV isolate (which has a virulent F gene
cleavage site and a high ICPI of 1.88)32 induced clinical signs
in the infected chickens only, and they were mild. In agree-
ment, the chicken’s lesions were more severe than the ducks’
lesions, and the distribution and intensity of the NDV-NP–pos-
itive signals were greater in chickens than in ducks. In our pre-
vious study on the immune organs from the same experiment,
the results showed that the replication and dissemination of the
9a5b NDV isolate were wider in chickens than in those of
ducks and that the spleen was the most affected organ.6
NDV virulence groups (lentogen, mesogen, and velogen)
are very broad categories, and the degree of clinical disease

Figure 3. (continued) of lymphocytes (arrows) and macrophages (arrowhead). HE. Inset: Positive signals for NDV-NP in macrophage (arrow)
and in epithelial cell (arrowhead). IHC, hematoxylin counterstained. A, atria; B, air capillaries. Figure 4. Lung; duck, P2. Mild interstitial pneumonia
with infiltration of lymphocytes (arrows) and heterophils (arrowhead). HE. Inset: Positive signals for NDV-NP in macrophage (arrowhead). IHC,
hematoxylin counterstained. A, atria; B, air capillaries. Figure 5. Lung; chicken, P4. Positive signals for interferon-beta (IFN-b) in epithelial cells of
parabronchi (arrow). Inset: lung, chicken negative control. PL, parabronchial lumen. IHC, hematoxylin counterstained. Figure 6. Lung; duck, P4.
Positive signals for IFN-b in macrophage-like cell (arrow), fibroblast-like cells (arrowhead), and epithelial cells of parabronchial lumen (PL). Inset:
lung, duck negative control. IHC, hematoxylin counterstained. Figure 7. Heart; chicken, P4. (a) Multifocal myocardial degeneration with infiltration
of inflammatory cells. HE. (b) Positive signals for NDV-NP in lymphocytes and macrophages in degenerated area of the myocardium. IHC,
hematoxylin counterstained.
644 Veterinary Pathology 50(4)

Figure 8. Duodenum; chicken, P4. (a) Degeneration and necrosis in the mucosal epithelium (asterisk). Hematoxylin and eosin (HE). (b) Positive
signals for Newcastle disease virus–nucleoprotein (NDV-NP) in degenerated mucosa (asterisk) and in epithelial cells (arrows) in the main fig. and
inset. Immunohistochemistry (IHC), hematoxylin counterstained. Figure 9. Duodenum; duck, P4. Positive signals for NDV-NP in macrophage
(arrow) and mitosis in the epithelial cells of the duodenal gland (arrowhead). IHC, hematoxylin counterstained. Figure 10. Liver; chicken, P4.
(a) Proliferation of the ectopic lymphoid tissues (asterisk), with dilated bile caniliculi (arrows) and portal bile duct (BD, inset). Blood sinusoid
Anis et al 645

Figure 14. Trachea; chicken, P4. Excessive globular mucous particles (white arrowhead) and erosions of the tracheal epithelial cells (white
arrows). Scanning electron microscopy (SEM). Figure 15. Trachea; duck, P4. Globular mucous particles (white arrowhead), fine mucous net-
work (white arrows), and slight disorientation of the cilia. SEM.

does not always segregate with ICPI. There are cases, for
example, in which a strain is considered ‘‘virulent’’ by ICPI but
does not cause much in the way of clinical disease.35,39,40 In
addition, the pathogenic effects of NDV are not dependent on
the fusion cleavage site alone.39 As described previously, NDV
isolates that had a virulent fusion protein cleavage site and high
ICPI differed in their ability to cause clinical signs, in their
lesions, and in their viral distribution in SPF chickens.28,35
Moreover, chickens and ducks vary in their response to virulent
NDV infection.1
In the present study, the respiratory and digestive lesions
started at an early stage (except airsacculitis) and subsided at
a late stage in both chickens and ducks. On the other hand,
necrotizing pancreatitis and tubulointerstitial nephritis were
detected only in chickens; they dramatically increased at the
late stage and did not subside until the 10th dpi. We are una-
ware of what the sequelae of these lesions might have been if
the birds were studied for a longer period. Moreover, mild lym-
phocytic myocarditis and slight nonpurulent encephalitis were
detected only in chickens, and these lesions were described pre-
viously.19,20 Immunohistochemical NDV-NP–positive signals
were detected in infected chickens at a late stage in the degen-
erated pancreatic acinar cells and degenerated renal tubules, at
the middle of the experimental period in the myocardium, but
not in the brain, indicating no or too low virus replication in the
brain tissue to be detected by IHC in addition to the direct
effect of NDV on the pancreatic acinar cells, renal epithelial
cells, and myocardial fibers. This finding is consistent with a
previous observation in SPF chickens infected with NDV
isolates of different virulence.8
In our previous study, the pattern of apoptosis in the spleen
was distinct between chickens and ducks after 9a5b NDV
infection. Although apoptotic cells were abundant in the periel-
lipsoidal white pulp, periellipsoidal lymphoid sheath, periarter-
iolar lymphoid sheath, and perivenous lymphoid sheath
(PVLS) of infected chickens, apoptosis was concentrated in the
germinal centers (GCs) of the infected ducks.6
Ducks in the present study had no lesions in the brain, heart,
pancreas, or kidney; the NP-positive signals were not detected
in these organs either. These findings indicate wider dissemina-
tion of the 9a5b isolate in tissues of chicken than in those of
duck. Chickens, turkeys, and ducks vary in their response to
virulent NDV infection. Although asymptomatic infection
occurred in ducks and turkeys even at the highest dose (106
EID50) without any deaths noted, chickens were shown to be
extremely susceptible, and all died even with a low dose
(101.5 EID50).1 In line with that, in SPF chickens, the meso-
genic Anhinga NDV isolate induced eyelid edema, multifocal
myofiber disruption with infiltrates of lymphocytes, and
macrophages in the heart. The cerebellum had multifocal glio-
sis with Purkinje cell loss and epithelial necrosis in the comb as
well as NDV-NP–positive signals in some organs, including
brain.20 Chickens are the most susceptible to NDV infection
among poultry species, whereas ducks and geese are the least
susceptible;17 for this reason, wild birds are considered to be
the natural reservoir of NDV.43 Unfortunately, pathological

Figure 10. (continued) (arrowhead); PA, portal artery. HE. (b) Signals for NDV-NP in macrophages (arrows) and in Kupffer cells (inset). IHC,
hematoxylin counterstained. Figure 11. Liver; duck, P4. Accumulation of heterophils in hepatic sinusoids (arrow). HE. Figure 12. Pancreas;
chicken, P10. (a) Necrotizing pancreatitis with marked infiltration of inflammatory cells (asterisk). HE. (b) Signals for NDV-NP in the degenerated
pancreatic acinar cells in the main fig. and inset. IHC, hematoxylin counterstained. Figure 13. Kidney; chicken, P10. Tubulointerstitial nephritis.
HE. Inset: Positive signals for NDV-NP in degenerated renal tubules. IHC, hematoxylin counterstained.
646
studies of Newcastle disease in ducks are rare, so it is difficult
to compare our results with previous data.
IFN-b appears to be protective during the early stage of
influenza infection and cannot be compensated by IFN-a, and
therefore superior innate immunity might protect the duck dur-
ing this critical period.7 Retinoic acid–inducible gene-like
receptors (RLRs), including retinoic acid–inducible gene I
(RIG-I) and melanoma differentiation–associated gene 5
(Mda-5), are cytoplasmic RNA sensors41 that can detect influ-
enza virus25 and NDV,33 leading to production of IFN-b and
expression of downstream IFN-stimulated antiviral genes.25
RLRs play central roles in viral recognition and subsequent
induction of antiviral immune responses in conventional den-
dritic cells (cDCs), macrophages, and fibroblasts.22,34 RIG-I
is present in ducks and absent in chickens,7 whereas the V pro-
teins of paramyxoviruses directly interact with Mda-5 to block
RLR signaling,5 and this indicates that chickens after NDV
infection cannot produce IFN-b through the RLR pathway,
which gives the duck an advantage in this point.5,7,25,33,41 In
this study and in our previous study,6 immunohistochemical
IFN-b expression in lung tissues and in immune organs, respec-
tively, was earlier and stronger in ducks compared with chick-
ens after infection with the 9a5b NDV isolate. Therefore, ducks
may have rapid and strong innate immunity against NDV
infection.
In our study, histopathological lesions were more severe in
infected chickens. On the other hand, heterophilic infiltration,
proliferation of lymphoid tissues, and hyperplasia of goblet
cells were more often observed in the infected ducks. Avian
heterophils are highly phagocytic and capable of a broad spec-
trum of antimicrobial activity, and they form the first line of
cellular defense against invading microbial pathogens in the
lungs and air sacs, where resident macrophages are typically
lacking.9,15 In this study, SEM investigation of tracheal tissues
revealed that 9a5b NDV infection induced rapid and abundant
mucous secretion at the 1st dpi in chickens, but scanty mucous
was observed at the 10th dpi, possibly indicating severe exhaus-
tion of goblet cells, which is in line with previous studies.23,26
In infected ducks, the mucous secretion was mildly increased
from the 1st to the 10th dpi. Moreover, deciliation and mucosal
destruction were greater in chickens than in ducks. Therefore,
in field conditions, secondary infection may occur more easily
in chickens than in ducks.
As described previously, chicken eyelid inoculation with 3
virulent NDV isolates did not cause any lesions at the first
dpi.35 In contrast, in the present study, at the first dpi, the
lesions in the infected chickens were not restricted to the
respiratory and digestive tracts. A few localized areas of lym-
phocytic myocarditis and pancreatitis were detected even
though NDV-NP could not be detected by IHC in the heart and
pancreas on that day (Table 2). Therefore, the early detectable
lesions in our experiment may depend on the route of inocu-
lation as well as virulence differences. Further studies to
detect the virus by in situ hybridization or viral isolation and
to explain the development of early lesions in the heart and
pancreas are needed.
Veterinary Pathology 50(4)
Finally, the 9a5b mutant isolate induced a mild infection in
SPF chickens and asymptomatic infection in Japanese commer-
cial ducks. Accordingly, the lesions and virus dissemination
were greater in chickens than in ducks. In contrast, IFN-b
immunopositivity was more intensive in lung tissue of ducks,
which may indicate stronger duck innate immunity against NDV
infection. Further studies to compare chickens and waterfowl are
needed, which may help us to elucidate the natural mechanisms
underlying waterfowl resistance to NDV infection.
Acknowledgements
We appreciate the financial support rendered by the Egyptian govern-
ment to the first author (Anis Zaid). We are grateful to all members of
the Department of Veterinary Pathology and to Ms E. Kawahara, Tot-
tori University, for assistance.
Declaration of Conflicting Interests
The author(s) declared no potential conflicts of interest with respect to
the research, authorship, and/or publication of this article.
Funding
This work was supported by the Egyptian government and the Labora-
tory of Veterinary Pathology, Tottori University, Japan.
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