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Report of Newcastle disease in peacocks

Article · January 2015

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Volume 19(5): 300-305, 2015.

Report of Newcastle disease in peacocks.

Zahra Broomand1, Mansour Mayahi1, Annahita Rezaie2, Somayeh Dibavand1, Elham Maleki1
1 2
Clinical sciences, Pathobiology, Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran

ABSTRACT

Broomand Z, Mayahi M, Rezaie A, DibavandS, Maleki E., Report of Newcastle disease in

peacocks 19(5): Onl J Vet Res., 300-305, 2015. Serological, histopathological and PCR assay
findings in 17 peacocks exhibiting clinical signs of Newcastle disease are described. Serum
antibody titers was determined by HI test, kidney, brain and caecal tonsils for virus detection
by RT-PCR and Proventriculus, liver, brain and intestinal tissue for histopathology. Hepatic
necrosis, proventricular bleeding and necrotic enteritis occurred in the digestive system.
Antibody titers varied 8-12. Neuronal degeneration, gliosis, perivascular cuffs and
perivascular edema was observed in the cerebrum with lymphoplasmacytic infiltration and
haemorrhage in the meninges. PCR confirmed the presence of Newcastle virus.

NDV was detected. This is the first report in peacocks to ND in Ahvaz, Iran.

Keywords: Newcastle disease, peacock, PCR, Histopathology

INTRODUCTION

Newcastle disease (ND) still continues to plague the poultry industry in spite of routine
vaccination of commercial poultry with a variety of live and killed vaccines. One possible
reason for this could be the existence of a susceptible non poultry avian population, which
do not come under regular vaccination cover. These susceptible populations include
backyard chicken, ducks, turkeys, pet birds and wild birds. These birds are not vaccinated for
various reasons and are highly susceptible to Newcastle disease virus (NDV) infections

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(Vijayarani et al, 2010). The severity of the disease is generally determined by the pathotype
of the virus involved and the species of bird infected. Although NDV has a very wide host
range, the severity of the disease caused by a given pathotype may depend on the species of
bird involved (Kaleta and Baldauf, 2000). We report suspected Newcastle disease virus in
peacocks based on clinical, histopathological, antibody and PCR test results.

MATERIALS AND METHODS

History
Seventeen peacoks were referred to the Veterinary Faculty in Ahvaz, Iran with signs of
anorexia, torticollis (Fig 1), and ataxia. Some of the birds died shortly after the onset of CNS
signs, but most stabilized and survived for prolonged periods with neurologic deficits.

Figure 1. Torticollis in a peacock.

Source and collection of specimens:

Tissue samples were used to assess gross lesions. Kidney, brain and caecal tonsils samples
were collected aseptically for virus detection by RT-PCR. Pro-ventriculus, liver, brain and
intestine samples were used for histopathology Antibody titers were assessed from blood.

Histopathological study:
Samples for histopathological evaluations were fixed in 10% neutral buffered formalin,
embedded in paraffin and sections of 5 μm in thickness, stained by haematoxylin and eosin
and assessed through light microscopy.

Serological Assay:
Beta-hemagglutination-inhibition method (HI) was used for determination of Serum antibody
titer against Newcastle disease virus. Sixteen peacocks were bled via wing vein (4 to 5 ml).
Blood samples were maintained at room temperature and allowed to clot, and kept in
refrigerator for 15 hours, then centrifuged for serum separation. Sera were separated and
stored at -20 0C until used. Sera were treated with heat inactivated at 56 0C for 30 min. The HI
assay was performed using 96 'U'-well micro titer plates, doubling dilution in PBS (0.01M)
and pH 7.2, 0.5% v/v Red Blood Cells (RBC), and 4 HA units of NDV antigen.

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RNA extraction: RNAs were extracted from the tissue samples. Briefly, 1 mL of RNX solution
(a commercial RNA extraction kit, CinnaGen, Iran) was added to 50-100 mg of each sample of
the homogenized tissue samples, then 200 μL of chloroform was add to the mixture. After
centrifugation of the samples at 12000 rpm for 15 min., the aqueous phases were transferred
to another tube. RNA was precipitated with the addition of an equal volume of isopropanol.
After washing the sediment with 75% ethanol, it was eluted in 50μL of distilled water and
stored at -70 °C until used.

Primer design, RT - PCR and gel electrophoresis: The respiratory virus detection primers used
in this study were previously evaluated in previous studies and are listed in Table 1.

Table 1. Polymerase chain reaction primers used for respiratory pathogen detection

oligonucleotide sequences gene PCR

product Reference
size/bp
NDV ndv1F -TT GAT GGC AGG CCT CTT G-<C> F 330 Mehrabanpour
et al. 2011
NDV ndv2R GG AGG ATG TTG GCA GCA T-<T> F

Table 2. RT-PCR program of NDV for 40 cycles

phase Temperature Time

Early denaturation 95°c 3 min
denaturation 95°c 30s
annealing 55 °c 1min
elongation 72 °c 1 min
Final elongation 72 °c 10 min

To identify NDV, by RT-PCR, specific primers based on F gene of NDV were used (5). The cDNA
was synthesized using AccuPowder RT PreMix kit (BioNeer Corporation, Republic of Korea)
according to the manufacturer’s instruction. The PCR condition for the amplification of F
gene is listed in table 2. These programs were performed in Quanta biotech cycler. The
products were analyzed in 1% agarose gel containing safe stain, using an ultraviolet
transilluminator. RT-PCR was performed to amplify a 330-bp fragment of the F gene of
Newcastle disease virus.

RESULTS AND DISCUSSION

Humoral immune responses: Results showed detection of antibody titers against newcastle
disease virus in serum of peacocks. The level of antibody titer was from 8 to 12. The mean Ab
in peacocks was 10.81. 8,8,10,10,10 ,11,11,11,11,11,12,12,12,12,12,12

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RT-PCR results: The presence of the virus in various organs of the dead bird's tissue samples
was determined by RT-PCR (Fig. 2).

Figure 2. Electrophoresis analysis (1% agarose gel) for Newcastle disease virus (NDV)
Lane M = 100-bp, lane 6= positive control (330 bp), lane 7= negative control, lane 1-5,
8 and 9= positive samples.

Post mortem findings: There were submucosal edema and ascites (Fig 3). Multifocal
petechial haemorrhages were seen on the mucosal surface of trachea, proventriculus, small
intestine and cecal tonsils. Air sacculitis were obvious. Macroscopic investigation of nervous
system revealed congestion and haemorrhage on brain.

Figure 3. Cerebrum. Perivascular cuffs and perivascular edema (arrows) are seen and
inflammatory cells infiltration is obvious in the meninge (H&E).

Histopathological results: the most remarkable histopathologic finding was observed in the
brain. Neuronal degeneration, gliosis, perivascular cuffs (mononuclear inflammatory cells
mostly lymphocyte and plasma cells accumulate around small capillaries) and perivascular

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edema was seen in cerebrum also lymphoplasmacytic infiltration and haemorrhage was
illustrated in meninge (Fig 3 and 4).

Figure 4. Cerebrum. Note to lymphoplasmacytic infiltration (Asterisk)) and edema

(Green edema) around capillary and neuronal deg

According to mentioned characteristics non suppurative meningoencephalitis were

diagnosed. In cerebellum, purkinje cell necrosis, perivascular cuffs, perivascular edema and
lymphoplasmacytic infiltration in meninge was detected as lymphoplasmacytic
meningocerebellitis (Fig 5).
eneration (Black arrow) (H&E).

Figure 5. Cerebellum. Purkinje cell necrosis (Black arrows) and lymphoplasmacytic infiltration
in meninges is obvious (H&E).

Necrosis of the superficial epithelium and haemorrhage were observed in the proventriculus
(Figure 6). Small foci of necrosis were seen in liver.

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 Figure 6. proventriculus. Note to epithelial cell necrosis (Black arrows) and
haemorrhage on the surface of proventriculus (Green arrows) (H&E).

NDV has been isolated from a peacock in India and China (Vijayarani et al, 2010, Dou and
Yang, 2007). As far as ND in Iran is concerned in wild and domestic avian there are no reports
of peacocks' ND. Here we report the identification of NDV from peacocks.

Newcastle disease (ND) is a highly contagious, generalized viral disease of domestic poultry,
cage and aviary birds, and wild birds. It is usually seen in domestic gallinaceous birds (poultry)
as a rapidly fatal, high-mortality condition characterized by gastrointestinal, respiratory
and/or nervous signs. In other avian species, the disease produced by virulent ND viruses
ranges clinically from in apparent to a rapidly fatal condition. Vijayarani et al, (2010) report
that the ND virus isolated from the peacock is a velogenic one. The interesting observation in
their study was the lack of clinical signs. Nevertheless, the necropsy results indicated the
involvement of pathogenic NDV with pneumotropic and viscerotropic lesions. We found
peacocks with clinical signs.

Acknowledgments

This research was supported by a grant of the Shiraz University Research Council.

REFERENCES

1. Dou F et al (2007), Isolation and identification of NDV from peacock, Journal of Economic
Animal, 11-9.
2. Kaleta EF et al (2000), Newcastle disease in free living and pet birds. In: Alexander DJ.
Newcastle disease.1st ed. Kluwer academic Publishers, Boston MA, pp 197–246
3. Mehrabanpour MJ et al (2011), Molecular Identification of Avian Respiratory Viral Pathogens
in Commercial Broiler Chicken Flocks with Respiratory Disease in Shiraz-Iran during 2009-2010,
International Journal of Animal and Veterinary Advances, 3(5): 300-304.
4. Vijayarani K et al (2010), Pathotyping of a Newcastle disease virus isolated from peacock
(Pavo cristatus), Trop Anim Health Prod, 42:415–419.

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