Newcastle Disease 2016 Mohamed Nabeh

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‫العظيم‬ ‫هللا‬ ‫صدق‬
Dr.Mohamed Nabeh 2015

Surveillance Studies on Newcastle Disease Virus
strains circulating in The Poultry Field in Egypt
‫مرض‬ ‫فيروس‬ ‫عترات‬ ‫عن‬ ‫إستقصائية‬ ‫دراسات‬
‫مصر‬ ‫فى‬ ‫الدوجن‬ ‫مزارع‬ ‫فى‬ ‫النيوكاسيل‬

Supervision committee:
Prof. Dr. Hatem Salah Abd El –Hamid
Prof. of Poultry Diseases
Faculty of veterinary medicine
Damanhur University
Former Rector of Damanhur University
Prof. Dr. Hany Fawzy El Lakany
Prof. of Poultry Diseases
Dean Faculty of veterinary medicine
Damanhur University
Prof. Dr. Soad Abd El Aziz Abd El Wanis
Chief Researcher of poultry Diseases
National Laboratory for Veterinary Quality Control on Poultry Production.
Animal Health Researche Institute
Dr. Sherif Minshawy Nasr
Lecturer of Genetics
Fac. Vet. Med., Damanhur University.

Newcastle Disease
ND is a fatal and contagious disease of many
avian species predominantly the poultry sector
that creates a constant threat to the poultry
industry in the Egypt.
The disease is complicated due to different
pathotypes and strains of the virus that induce
variation in the severity of disease
characterized by fatal respiratory and
neurological pathogenesis

The NDV
Serotypes: 1
APMV-1
• Genetic analysis classify
NDV to
• Class I
a virulent strains
• Class II
both virulent and a virulent

Molecular structure of NDV
Main Structure genome of NDV
contains six genes
that code for the six
major structural
proteins
Genome sequence of
NDV are (15,186-
15,198 nt long)
•NDV is an enveloped,
single stranded non-
segmented, negative sense
RNA virus with helical
capsid symmetry
F M LNP P HN

The OIE definition for reporting an
outbreak of ND is:
Criteria for virulence
The virus has (ICPI) in day-old chicks (Gallus gallus) of 0.7 or greater.
Multiple basic amino acids have been demonstrated in the virus (either directly
or by deduction) at the C-terminus of the F2 protein and phenylalanine at
residue 117, which is the N-terminus of the F1 protein.three arginine or lysine
residues between residues 113 and 116. (OIE .,2012).

Snoeck et al. (2013)
NDV genotypes (I- XVIII)
Class II
Genotype I
Genotype II
Genotype
III
Genotype IV
Genotype V
Genotype VI
Genotype VII
VII a
VII b
VII g
VII B
VII f
VII c
VII d
VII e
Genotype VIII
Genotype IX
Genotype X
Genotype XI
Genotype XII
GENOTYPE XIII
Genotype IVX
Genotype XV
Genotype XVI
Genotype XVII
Genotype XVIII

NDV World Panazootics
Genotype
II,III,IV……
……. 1st
panazootic
(started in
southeast Asia
in the mid-
1920)
Genotype V
………………
……..2nd
panazootic
(started in the
Middle East
in the late
1960s and to
have spread
to most
countries by
the early
1970)
Genotype
VI……………
.ppmv-3rd
panazootic (
in the Middle
East in the
late 1970,
spread to
Europe in the
1980),
Genotype VII
and VIII
……. 4th
panazootic
(since the
early 1990s to
current date
in Asia,
Europe and
Africa
including
Egypt )
Genotype
VII(a-h) new
sub-genotypes
of vNDV
suggests that
a new, fifth,
panazootic of
NDV already
originated in
Southeast
Asia,
extended to
the Middle
East, and was
entering into
Eastern
Europe.
(Miller et al.,
2015)

Recent studies demonstrated that NDV of
genotype VIId had high level of virus
replication and cause intense innate
immune response lead to severe pathology
in the lymphoid tissues, severlymphocytic
depletion and necrosis of the spleen and
thymus compared with virulent viruses of
other genotypes
(Hu et al.,2015).

History OF NDV IN Egypt
disease
recorde
d in
Egypt
for the
first
time in
year
1942
(Daubn
ey and
Mancy,
1948).
El-
Nassari,
1957;
Eissa,.
1960
pigeon
(Ahmed
and
Reda,
1967),
Lancast
er and
Alexand
er,
.1975
(veloge
nic
NDV)
Khafagy
, 1983;
Amal
Eid,
1984;
and
Bakhit
and
Abd El-
Hamid,
1990)
Hassan
et al.,
2004).
(Abdel
monei
m et
al.,
2006;
Amer et
al.,
2006).
Mahmo
ud et
al.
(2006)
Ramzy
et al.
(2009)
Moham
ed et al.
(2009)
Saad et
al.
(2010)
Moham
ed et al.
(2011)
Radwan
et al.,
2013)
Hussien
et al.
(2014)
Ismail
et al.
(2014)
Shalaby
et al.
(2014)
Mostaf
a et al .
( 2014)
Awad
et al.
(2015)

Aim of The Thesis
1- Studying the epidemiology of NDV in
Gharbia, El Behera, Dakahlia and Kafer
EL Shiekh Governorates
2- Genetic characterization and evaluation
of the pathogenicity of NDV field isolates
3- Evaluation of protection afforded by
selected commercial vaccines used in the
poultry field

During outbreak between
(January 2014 –December 2015)
Mortality
rate
samplesFlocks
Types
ProvincesNo
of Flocks
Varies
between
(5%-85%)
(Tissue pool)
trachea,liver
,Spleen, lungs
Cecal tonsiles,
Proventriculus
Brain - only( birds
with nervous signs)
Broiler
Layers
Broiler
breeders
1. Gharbia
2. El Beherah
3. Kafer
ElShiek
4. Dakahlia
48

History of examined Chicken flocks
sample No Province Type of bird Age Flock
capacity
Daily Mortality
during the
disease course
Total
Mortality
%
1 Kafer el shiekh Saso Broiler 39 day 2000 20,25,34 15%
2 Gharbia Broiler 28 day 5000 28,35,40, 17%
3 Gharbia Broiler 35 day 4200 40,22,35 21%
5 Gharbia Broiler 30 day Not recorded Not recorded
7 Gharbia Broiler 26 day Not recorded Not recorded Not recorded
8 Gharbia Broiler 30day 5000 35,25,40 16%
11 Gharbia Balady 33 day 3000 23,20,15 23%
13 Gharbia Saso Broiler 35 day 6000 20,33,25 13%
17 Kafer elshiekh Layers pullets 40 day 30,000 25,30,70,100 5%
18 El Behera Broiler 28 days 10000 200/200/400 60%

26 Gharbia Broiler 26 day 5000 120,200,300,250,270,18
0
55%
27 Gharbia Broiler 30 day 3000 50,70,60,50 25%
28 El Behera Broiler 28 day 3500 55,50,70 ,100 60%
29 Gharbia Broiler 31 day 11000 100,120,90,65,100 30%
30 Gharbia Broiler 27 5000 40,20,55,35 20%
31 Gharbia Broiler 30 day 4500 22,50,65,70,100 40%
34 El Behera Broiler 28 day 2500 40,55,60,70 37%
35 El Behera Broiler 30 day 5000 55,70,90,85 31%
39 Kafer el shiekh Broiler 26 day 3600 20,35,40 20%
41 Gharbia Broiler 28 day 5000 50,70, 27%
43 Gharbia Saso Broiler 29 day 4500 16,25,40, 17%
44 Gharbia Balady 40 day 5000 33,45 22%
45 Dakahlia Saso broiler 44 day 17000 1500,1700,1800 85%
46 Gharbia breeders
pullets
60 day 15000 No no

Materials used for samples
collections and processing according
to (OIE, 2012).
Isolation in ECE ( 9-11 ) days age.
Virus was isolated according to the
protocol of OIE and European
standard (OIE, 2012).

Identification of NDV (HA and HI tests):
(Rapid HA Test) was performed according to
(Terregino and Capua, 2009).
HA test in Microtitre Plates (Micro HA Test ) carried
out according to (Terregino and Capua, 2009).
Micro HI Test (α technique) constant-serum diluted-
antigen (Thayer and Beard, 2008) .

Pathogenicity test
Intracerebral Pathogenicity Index ICPI in 1
day old chicks (OIE,2012).
Mean death time MDT in ECE (Alexander,
1989).

Polymerase Chain Reaction (one stepRT-
PCR) (conventional RT-PCR)
Thermo Scientific Gene JET Genomic RNA Purification Kit:
primers
Name Sequence (5′ - 3′ ) Annealing
temperatur
e
Amblicon
size
Reference
NDV F. 5′-TGGAGCCAAACCGCGCACCTGCGG-
3′) 55 co
766 bp
(Mase et al.,
2002).
R. 5'-GAGGATGTTGGCAGCAT-3′)

Material used for gene analysis
Sequencing the
purified PCR
product
• Using QIAquick
PCR Product
extraction kit.
(Qiagen Inc.
Valencia CA).
• Big dye Terminator
V3.1 cycle
sequencing kit.
• Applied
Biosystems 3130
genetic analyzer
(Hitachi, Japan).
Phylogenetic
analysis
The program
MEGA 6 using
the neighbor-
joining method.
BLAST on
NCBI Bioedite

Experiment
Evaluation of the protection of commercial live and
inactivated vaccines of NDV against an Egyptian very
virulent Newcastle Disease Virus isolates EG18/2015, EG
35/2014

Material used for experimental challenge:
Chickens : 265 commercial
broiler chicks
The viruses :
NDV/CH/ EG18/2015 strain,
NDV EG35 /2014strain with
titer ( EID50 106 /0.2 ml).
Reed and Muench, (1938).
Vaccines

Commercial vaccine name Strain Company Batch number
IZOVAC ND
(INACTIVATED)
La Sota IZO S.U.R.L 07741
HIPRAVIAR BI Strain HIPRA 68BJ-8
ND LASOTA La Sota MSD 14603CJ01
VECTOREMUNE® HVT
NDV
Newcastle Disease
Vaccine &Serotype 3
Live Mareks
BIOMUNE C0
Ceva Sante Animal
372-694
CEVA®VITAPEST L PHY LMV 42 ceva Sante Animal 1306C3S2C
AVINEW® VG/GA Strain Merial L419491
VOLVAC®ND LASOTA
MLV
La Sota BORENHIGHER 1409057A

The Expiremental Desigen
Groups Type of the
vaccine program
Day of
vaccination
Route
of
vaccination
Numbers of broiler chickens in groups
Vaccinated and challenged
group with isolate
EG18/2015*
Vaccinated and
challenged group with
isolate
EG 35/2014*
Vaccinatednon
challenged
A Vectoimmune ND
Hitchiner BI
LaSota
1 day
4 day
12 day
SC
O
ED
15 15 15
B Hitchiner BI
Vitapest
4 day
18day
O
ED
15 15 15
C Hitchiner BI
Avinew
4 day
18 day
O
ED
15 15 15
D Hitchiner BI
LaSota
4 day
18 day
O
ED
15 15 15
E Hitchiner BI
IZOVAC ND
(inactivated)
4 day
7 day
O
SC
15 15 15
F Non vaccinated
infected
- - 10 10 -
G Non vaccinated
Non infected
- - 10 10 -

Total
Mortality
rate
Total
Number
of flocks
Type of bird No of
flocks
Age by
days
Province
5-15%
10
broiler 4 26-35 Gharbia
Balady 2 46-50 Gharbia
Breeder pullets 1 38 Dakahlia
Broiler saso 2 39 Kafer El
Shiekh
Layer pullets 1 40 Kafer El
Shiekh
16-30%
24
Broiler
Saso
1 29 Gharbia
balady 2 33-40 Gharbia
31-60% 8
broiler 3 28-30 El Behera
61-85% 1 Broiler Saso 1 44 Dakahlia
Not
recorded
5 broiler 5 27-32 Gharbia

Nervous signs (torticollis, head shaking)

Ulceration in intestine Hemorrhage on cecal tonsil
Hemorrhagic Tracheaitis Hemorrhage on gland tips of Proventriculus
Gross Lesion of samples

Province No of
samples
No of positive samples in Rapid HA test
No percent
Gharbia 38 14 (36.84%)
El Behera 4 4 (100%)
Kafer ElShiekh 3 1 (33.3%)
Dakahlia 3 2 (66.7%)
Total 48 21 (43.75%)

Identification by one step RT-PCR
• The results of RT- PCR which were carried out to all RNA extracted from HA positive infectious allantoic fluids
samples to detect the positive samples of NDV and detect infection with AIV H9 , AIV H5 and mixed infection.
Sample number RT- PCR for NDV RT- PCR for AIV H5 RT- PCR for AIV H9
10 -ve -ve -
27 -ve -ve -
31 +ve -ve -ve
11 -ve -ve -
24 +ve -ve -ve
17 -ve -ve -
26 +ve -ve -ve
34 -ve -ve + ve
32 -ve -ve -
28 -ve +ve -
35 +ve -ve -ve
33 -ve -ve -
25 -ve +ve -ve
37 -ve -ve -
40 -ve -ve -
42 -ve -ve -
44 -ve -ve -
18 +ve -ve -ve
45 -ve +ve -
46 -ve -ve -
48 +ve -ve -ve
Total
Total
21 sample
6 3 1

L 10 11 31 17 24 25 2 6 37 2 7 28 35 34 33
766 bp
Fig. (3) Gel electrophoresis of 766 bp fragment from matrix and fusion gene o
NDV isolates in 1.5% agarose gel
L 18 2 3 4 5 6 7 8 9 10 11 12 13 14 15

Sample
No
ICPI value Virulence to Chicken
( Seal et al., 2000)
MDT
by hours
NDV Pathotype
18 1.89 Virulent 48 hr Velogenic
24 0.38 Avirulant 96 hr lentogenic
The ICPI values , MDT and pathotypes
of the 4 examined samples

GeneBank data of NDV isolates used in comparative analysis with the 4 Egyptian NDV
isolates in this study.
Strain Country Abbreviation Host
Accession
Number
Genotype
Lasota Lasota Chicken AF077761 II
Hitchiner USA HITCHINER BI Chicken JN872151 II
Avinew VG/GA USA VG/GA Chicken EU289028 II
VECTORIMMUNE HVT ND D26/76 Chicken M24692 I
VITAPEST PHY-LMV42 Chicken DQ097394 I
CLONE 30 Chicken Y18898 II
NDV/Chicken/2/2006 Egypt 93/EG/06 Chicken FJ969393 II
NDV/Chicken/ 3/2006 Egypt 94/EG/06 Chicken FJ969394 II
NDV/Chicken/4/2006, Egypt 95/EG/06 Chicken FJ969395 II
NDV/chicken/VRLCU138/
/2012
Egypt 68/EG/12 Chicken JX885868 VIId
NDV//CH/ SDWF07/2011 CHINA Chicken
JQ015295/KF93
5230
VIId

The nucleotides sequences of 4 NDV isolates were submitted to BLAST
on NCBI
NDV/EG/CH/18/2015 with identity 95 % with Newcastle disease
virus strain Chicken/China/SDWF07/2011
NDV/EG/CH/35/2014 with identity 91 %

Deduced amino acid sequences of the isolated NDV strains in
comparison to commonly used vaccine strains and reference isolates

Amino acid identity of 4 NDV isolates with NDV strains and NDV
vaccines

Phylogenetic tree for the 4 NDV isolates and related NDV strains based on the fusion
protein amino acids sequences.

Genotype II
Genotype I
Genotype IV
Genotype III
Genotype VI
Genotype V
Genotype VII

Molecular characterization of the sequenced
isolates of NDV
Serial
Number
Abbreviation Genotype Host Virulenc
e
Cleavage site of
F protein
112RRQKR116F117
Gene
Bank
Accession
No
18 NDV/EG/CH/18 /2015 VII d Chicken virulent RRQKRF Ku377781
26 NDV/EG/CH/26/2014 VII d Chicken virulent RRQKRF Ku377783

The mortality induced by both selected
isolates for protection experiment
No of infected
chicknes
Days PI NDV strain
EG/35/2014
NDV strain
EG/18/2015
daily
mortality
daily
mortality
10
1 0 0
2 1 2/10
3 0 2/10
4 0 3/10
5 1 0/10
6 3 3/10
7 2
8 0
9 0
10 3
Total mortality 100% 100%Dr.Mohamed Nabeh 2015

Gross lesion in internal organs of non vaccinated group after infection by NDV
strain EG/18/2015
Sever hemorrhages on tips of proventriculus glands and greenish contents
of gizzard Dr.Mohamed Nabeh 2015

1. Hemorrhages of intestinal cell wall intestinal ulceration
(button shaper of payer's patches ) in mucosa of small
intestine.
2.Intestinal ulceration and sever boudenal ulceration
(button shape of payer's patches ) in mucosa of small
intestine.
3. Ulceration in mucosa of small intestine ( elliptical
ulceration).
1 2
3

Petechial hemorrhage in the spleen
Sever congestion in trachea Sever hemorrhage of cecal tonsils

Protection Percent in Challenged Groups
Groups Vaccines Protection rate
NDV/EG/18/2015 NDV/EG/35/20
14
A Vectorimmune ND
Hitchner B1
LaSota
53.4% 100%
B Hitchner B1
Vitapest
60% 100%
C Hitchner B1
Avinew
60% 93.4%
D Hitchner B1
LaSota
93.4% 100%
E Hitchner B1
Inactivated ND IZA
100% 100%
F Non vaccinated infected 00% 00%
G Non vaccinated non infected 100% 100%

The protection percent in chicken groups infected by
NDV strain EG/18/2015 and NDV strain EG/35/2014

Results of HI titer by log 2 of serum samples collected from
non-infected groups during experiment after vaccination in
different ages
GROUPS A
Log 2
B
Log 2
C
Log 2
D
Log 2
E
Log 2
F
Log 2
G
Log 2DAYS
14 5 4.6 4.6 4.6 7 4.5 4.5
21 8.5 4.3 7.3 8.6 5.3 2.6 2.6
28 6.5 5.3 6 4.6 4 1 1
35 6 5 7.5 5 4.5 0.6 1

HI titer of vaccinated groups before and 7 days
post infection with NDV strain EG 18/2015 .
GROUPS A
Log 2
B
Log 2
C
Log 2
D
Log 2
E
Log 2
F
Log 2
G
Log 2
DAYS
Before
infection
6.5 5.3 6 4.6 4 1 1
7 day post
infection
9 9 5 12 9 Death
before 7
day
1

NDV strain EG 18/2015Dr.Mohamed Nabeh 2015

HA titer for allantoic fluid collected after inculation of ECE
9 day with cloacal swabs samples collected from the
experimentally challenged chicken groups
Groups
Shedding detection by micro plate HA TEST
NDV EG 18/2015
HA log2
NDV EG 35/2014
HA log2
3 dpc 5 dpc 7 dpc 3 dpc 5 dpc 7 dpc
A 3 1 1 1 3 0
B 6 2 1 1 4 3
C 3 0 9 1 3 0
D 5 4 10 0 1 0
E 3 0 0 2 1 0
F 5 3 3 5 6 4
G 0 0 0 0 0 0

the results of virus shedding in different groups post challenge.

Conclusion
Newcastle disease is still a major problem
for the poultry in Egyptian governorates .
NDV genotype VIId is currently endemic and more
spread in the country, induce high mortality rate in
vaccinated flocks.
The protection percent of the commercial available
live and inactivated vaccines gave different level of
protection and shedding, but the inactivated vaccine
with La Sota gave the best result followed by the
Vectorimmune vaccine
Not only protection varied according to thevaccination
program but also it was different according to the
pathogenicity of the challenge virus strains.

Newcastle Disease 2016 Mohamed Nabeh

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