Advances in Animal and Veterinary Sciences
research Article
Efficacy of commercial Vaccines against H9n2 Avian Influenza
challenge in chickens
HOSSAM A. GADO1,2, IBRAHIM A. GHANEM1, ABDULLAH A. SELIM3, MOUNIR M. ELSAFTY4, REEM A.
SOLIMAN4, AMAL A.M. EID1*
1
Avian and Rabbit Medicine Department, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig,
Egypt; 2Reference Laboratory for Quality Control on Poultry Production (RLQP). Animal Health Research Institute,
Sharkia Branch, 44516, Zagazig, Egypt; 3Reference Laboratory for Quality Control on Poultry Production (RLQP).
Animal Health Research Institute, Dokki, Giza, 12618, Egypt; 4Central laboratory for evaluation of Veterinary
Biologics (CLEVB), Cairo, 11381, Egypt.
Abstract | Avian influenza (AI) is a contagious disease caused by type A influenza viruses. Despite the mild nature
of H9N2 low pathogenic AIV, the virus was isolated from frequent disease outbreaks with high mortality in different
parts of the world. Nine chicken flocks of different breeds and ages during 2018 were examined in Sharkia Province.
The flocks were tested by real time PCR for determination of the H9N2 subtype in each sample. Likewise; they were
tested for the presence of other viruses (H5, H7, NDV and IBV). Four flocks were positive for H9AIV, negative for
other avian pathogens; two were positive for IBV, and three flocks were negative. Sequence analysis was carried out
for HA and NA genes of two H9N2 isolates. Phylogenetic analysis revealed that the HA and NA of tested H9N2
isolates are clustered to the prototype G1-like viruses that were circulating in the Middle Eastern and forming a
distinct cluster. Protection efficacy of commercial H9N2 vaccines was evaluated by two field available vaccines (vaccine
1 A/chicken/Egypt/ME543V/2016 and vaccine 2 A/chicken/Iran/Av1221/1998) through groups each (15 birds) of
SPF one day old chicks then challenged them with a field isolate A/chicken/Egypt/S6/2018. Protection was evaluated
by clinical signs, histopathology, weekly immune response by ELISA and HI tests and tracheal swabs were used to
monitor virus shedding at 3, 5 and 7 days post-challenge by qRT-PCR test. From this study, it is concluded that H9N2
vaccines are efficient to decrease and prevent AI shedding in specific pathogen free (SPF) chickens and give very good
antibodies response against H9N2 after single vaccination with oil emulsion inactivated vaccines.
Keywords | AIV, LPAI H9N2, Sequence, SPF chickens, inactivated vaccine, Egypt
received | April 28, 2021; Accepted | August 01, 2021; Published | December 01, 2021
*correspondence | Amal A.M. Eid, Avian and Rabbit Medicine Department, Faculty of Veterinary Medicine, Zagazig University, 44511, Zagazig, Egypt; Email:
amalaeidvet@gmail.com
citation | Gado HA, Ghanem IA, Selim AA, Elsafty MM, Soliman RA, Eid AAM (2022). Efficacy of commercial vaccines against h9n2 avian influenza
challenge in chickens. Adv. Anim. Vet. Sci. 10(1): 35-48.
doI | http://dx.doi.org/10.17582/journal.aavs/2022/10.1.35.48
ISSn (online) | 2307-8316; ISSn (Print) | 2309-3331
copyright © 2022 Gado et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.
IntroductIon
Avian influenza viruses are divided into two pathotypes:
highly pathogenic avian influenza (HPAI) viruses, which
n Egypt, the poultry industry is considered one of the cause severe and fatal infections in chickens, and low
most profitable national income (El-Nagar and Ibrahim, pathogenic avian influenza (LPAI) viruses, which are
2007). Avian influenza virus (AIV) is one of the most generally much less virulent. LPAI viruses may contain any
devastating viral diseases in poultry. It has a worldwide haemagglutinin antigen, but to date all HPAI viruses have
distribution that infects many domesticated and wild birds contained either H5 or H7 (OIE, 2019).
(Swayne and Glisson, 2013).
I
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The H9N2 avian influenza virus (AIV) was reported to
be low pathogenic in chickens (Bano et al., 2003). Still,
its infection may result in significant losses in the poultry
industry when complained with other pathogens (Naeem
et al., 1999). In Egypt, H9N2 virus was first detected in
May 2011 from bobwhite quail (El-Zoghby et al., 2012).
Recently, simultaneous detection of the three subtypes
(H5N1, H5N8, and H9N2) has been described in a poultry
farm in Egypt (Shehata et al., 2019). The emerged HPAI
(H5N2) virus from a commercial duck farm was the first
natural reassortment in Egypt resulted from a reassortant
between HPAI H5N8 of clade 2.3.4.4 (group B) virus and
the Egyptian LPAI (H9N2) G1-like lineage virus (Hagag
et al., 2019).
Several experimental studies have reported that inactivated
AI vaccines can induce antibody response, which helps
protect the infected birds from mortality and egg
production decline (Capua and Alexander, 2008). The
monitoring of H9N2 virus shedding in an infected bird is
a tool for evaluating AI vaccine effectiveness. An effective
vaccine prevents clinical disease and reduces viral shedding
(Subtain et al., 2011). In this study, recent LPAI H9N2
viruses were isolated and molecularly characterized from
Sharkia Province. Besides, the analysis of inactivated oil
emulsion Avian Influenza H9N2 vaccines efficacy in
specific-pathogen-free chickens in terms of reduction of
viral replication and shedding, antibody response, and
histopathological examination.
MAtErIAlS And MEtHodS
SAMPLING AND SAMPLE PROCESSING
The current investigation was done to detect and isolate
field H9 subtype AI virus to be used in challenge studies
in nine suspected flocks (broilers, balady, and layers; 3/
each). Eighty tracheal and cloacal swabs, besides seven
respiratory tissues, were collected for this purpose at Sharkia
Province during 2018.The history of the investigated flocks
(breed, age, vaccination, and flock capacity) was illustrated
(Supplementary Table S1). The samples were kept at -80oC
until examination. Tissue samples were pooled (each pool
consists of trachea and lungs from three birds), then each
was homogenized in an adequate volume (1:10 w/v) of
sterile phosphate-buffered saline. The tissue homogenates
were centrifuged at 1000×g for 10 min. Penicillinstreptomycin mixture (Lonza Group AG, Walkersville,
MD) was added to each supernatant (1000 IU penicillin
and 1000 µg streptomycin per milliliter). Tracheal swabs
were pooled (5/each) in 2 ml of PBS solution containing
antibiotics, followed by centrifugation at 1000×g for 10
min, and the supernatants were collected into cryotubes
(OIE manual, 2015).
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Advances in Animal and Veterinary Sciences
AVIAN INFLUENZA H9 DETECTION USING REAL-TIME
RT-PCR
All collected swabs and tissue homogenates were subjected
to Real-time RT-PCR. Samples tested for Matrix and H9
genes and other suspected co-infections (H5, H7, IBV, and
NDV). RNA was extracted from samples using QIAamp
Viral RNA Mini Kit (Qiagen, Valencia, Calif., USA).
Cat. No.52904.The procedure was done according to the
company’s work instructions. The amplification process was
performed using Quantitect RT-PCR Kit reaction buffer
(Qiagen. 204443- USA). Stratagene MX3005P real-time
PCR machine (Stratagene, USA) was used. Primers and
probes were supplied from Metabion (Germany) (Table
1). The reaction mix volumes for each reaction of the
Matrix, H9, H5 and H7 genes of avian influenza viruses,
IBV and NDV, were 5ul RNA template; 0.25 ul for RTenzyme 12.5 ul for RT-PCR buffer; 0.5 ul of each forward
and reverse primers; 0.125ul for probe, and 6.125 uL of
RNase-free water. Thermal cycling RT-PCR conditions
included a reverse transcription at 50°C for 30 min, then
an inactivation of reverse transcription enzyme and initial
denaturation at 95°C for 15 min, followed by 40 cycles at
(Table 1).
GENETIC CHARACTERIZATION OF HA AND NA GENES
Amplification of HA and NA genes for two selected
positive samples (S2 and S6): it was implemented by
one-step RT-PCR using Qiagen kit (QIAGEN, Hilden,
Germany) with specific primers in (Table 2), The RT-PCR
cycling as follows: one cycle at 50°C for 30 min, one cycle
at 95 °C for 15 min and 40 cycles of 94°C for 45 Sec, 56°C
for 45 Sec and 72°C for 2 min., a final extension at 72°C
for 10 min. in thermocycler 2720 ABI.
SEQUENCING FOR HA AND NA GENES
Both HA and NA gene sequencing was done by using
Big dye Terminator V3.1 cycle sequencing kit. (PerkinElmer, Foster City, CA). The cycling protocol for sequence
reactions is: one cycle at 96° C for one min, 25 Cycles
repeated at 96° C for 10 Sec, 50° C for 5 Sec. and 60°C for
2 min. The sequencing reactions were purified using a spin
column Centrisep kit (Applied Biosystems, USA), then
loaded in a sequencer plate of ABI (Applied Biosystems
3130 genetic analyzers, USA).
Nucleotide and amino acid identities were determined
using the Meg Align module of Lasergene DNA Star
software (Madison, WI). The nucleotide sequences
were submitted to GenBank with accession numbers
MT463345, MT463385, MT463346, MT463386,
respectively. Nucleotide sequences were aligned, and
phylogenetic analysis was performed using the neighborjoining method in the Molecular Evolutionary Genetics
Analysis (MEGA7) at 1000 Bootstrap (Kumar et al., 2016).
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table 1: Primers, probes and conditions applied in real time PCR for detection of AI, ND and IB viruses and Mycoplasma
species supplied from Metabion (Germany).
Virus/ target Primer/ probe sequence 5'-3'
bacteria*
dena- Anneal- Exten- ref
turation ing
sion
AI
94˚C
30 sec.
M
Sep1 AGATGAGTCTTCTAA CCGAGGTCG
Sep 2 TGCAAAAACATCTTC AAGTCTCTG
Slomka et
al., 2007
60°C
20 sec
SEPRO [FAM]TCAGGCCCC CTCAAAGCCGA [TAMRA]
H5
H5LH1 ACATATGACTAC CCACARTATTCA G
H5RH1 AGACCAGCT AYC ATGATTGC
54˚C
30 sec.
72˚C
10 sec.
Londt et
al., 2008
H5PRO[FAM]TCWACA GTGGCGAGT TCCCTAGCA[TAMRA]
H9
H9F GGAAGAATTAATTATTATTGGTCGGTAC
H9R GCCACCTTTTTCAGTCTGACATT
60°C
45sec
Ben
Shabat et
al., 2010
H9 Probe[FAM]AACCAGGCCAGACATTGCGAGTAAGATCC[BHQ]
H7
LH6H7 : GGC CAG TAT TAG AAA CAA CAC CTA TGA
RH4H7 : GCC CCG AAG CTA AAC CAA AGT AT
54˚C
30 sec.
72˚C
10 sec.
Slomka et
al., 2009
H7pro11 : [HEX]CCG CTG CTT AGT TTG ACT GGG TCA
ATCT [TAMRA]
IB
N
AIBV-fr ATGCTCAACCTTGTCCCTAGCA
AIBV-as TCAAACTGCGGATCATCACGT
Meir et al.,
2010
60˚C
60 sec.
AIBV-TM[FAM]TTGGAAGTAGAGTGACGCCCAAACTTCA
[TAMRA]
ND
f
F+4839 TCCGGAGGATACAAGGGTCT
F-4939 AGCTGTTGCAACCCCAAG
52˚C
30 sec.
72˚C
10 sec.
Wise et
al., 2004
49˚C
30 sec.
72˚C
10 sec.
Callison et
al., 2006
58˚C
30 sec.
72˚C
10 sec.
Sprygin et
al., 2010
F+4894 [FAM]AAGCGTTTCTGTCTCCTTCCTCCA [TAMRA]
MG
MG
CTA GAG GGT TGG ACA GTT ATG
A0319 GCT GCA CTA AAT GAT ACG TCA AA
{FAM} CAG TCA TTA ACA ACT TAC CAC CAG AAT CTG
{TAMRA}
MS
vlhA
CCA GGA GGT GGT ACA GTT GAC
TTA ATG CTT CTT TAA CT(G/A) AAT CTG A
{FAM}CTG CTA AAA CAG AAG CTA AAA C(C/T)G CTA T
{BHQ-2}
Avian Influenza= AI, Newcastle disease virus= NDV, infectious bronchitis virus= IBV, Mycoplasma Gallisepticum = MG and
Mycoplasma Synoviae= MS.*
table 2: Primers used in Reverse Transcriptase-PCR for sequencing of HA and NA genes of AIV H9 subtype.
Prime Id
Gene nucleotide Sequence 5'-3'
reference
F1-6
H9
Adel et al., 2016
TAG CAA AAG CAG GGG AAT TTC TT (1-23)
H9- R940
GCC ACC TTT TTC AGT CTG ACA TT (898-920)
H9-For
GGA AGA ATT AAT TAT TAT TGG TCG GTA C (738-765)
HT7R
TAA TAC GAC TCA CTA TAA GTA CAA ACA AGG GTG
(1731-1763) (1731-1763)
N2-F1
N2
ATG AAT CCA AAT CAA AGG ATA A
N2-R950
CGC CAA CAA GTC CTG AGC ACA CAT
N2-F630
CAT GGG ATG CTT ACC GAC AGT ATT
Designed by RLQPManufactured by (MetabionGermany) RLQP
N2-R1410
GAA AGC TTA TAT AGG CAT GAA AT
HA, Hemagglutinin and NA, Neuraminidase.
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table 3: Experimental design for Evaluation of H9N2 inactivated oil emulsion commercial vaccines in SPF chickens.
Group no. of Vaccine regime*
no.
birds vaccine type
inactivated vaccine
challenge at 28-day of age
Age/days
Dose/bird
G1
15
vaccine 1 A/chicken/Egypt/ME543V/2016 7 day
batch No.1908080101 350 HA unit
0.5 ml S/C
---
G2
15
vaccine 2 A/chicken/Iran/Av1221/1998
batch No. B10335 ≥10 HI.U
7 day
0.3 ml S/C
---
G3
15
vaccine 1 A/chicken/Egypt/ME543V/2016 7 day
batch No.1908080101
350 HA unit
0.5 ml S/C
++ 1 x106 EID50 per 0.1 ml per bird.
G4
15
vaccine 2 A/chicken/Iran/Av1221/1998
batch No. B10335 ≥10 HI.U
7 day
0.3 ml S/C
++ 1 x106 EID50 per 0.1 ml per bird .
G5
15
---
---
---
++ 1 x106 EID50 per 0.1 ml per bird .
G6
15
--------*Assessment of protection was done by virus shedding monitoring at 3, 5, 7 days post challenge by qRT-PCR, serological by ELISA
and HI tests and histopathological findings.
CHICKEN EMBRYO INOCULATION
The two selected viruses (S2 and S6) were propagated
on 10 days old SPF embryonated chicken eggs (ECE)
through intra-allantoic inoculation, and then incubated
at 37°C for 3-5 days. The SPF-ECEs were obtained from
the National SPF egg project, Kom Oshim, EL-Fayoum
Province. The allantoic fluids were harvested from the
inoculated embryos and tested for virus hemagglutination
properties (OIE Manual, 2015). The titration of each virus
was calculated (Reed and Muench, 1938) to determine the
50% embryo infectious dose titer (EID50).
PURITY OF H9 AIV VIRUSES
Samples were subjected to test the purification after virus
isolation and titration to make sure that they were negative
for other common avian pathogens, such as Newcastle
disease virus (NDV), infectious bronchitis virus (IBV),
AIV (H5, H7), Mycoplasma gallisepticum (MG) and
Mycoplasma synoviae (MS) (Table 1).
CHALLENGE VIRUS
The used H9N2 LPAIV Egyptian isolate (A/chicken/
Egypt/S6/2018) was selected based on its purity, low CT
(CT=11). The virus was diluted to adjust the amount of
inoculum to 1 x106 EID50 per 100 µl per bird).
IN VIVO EVALUATION OF H9 VACCINES
shedding, serological and histopathological findings were
carried out for the vaccine evaluation.
CLINICAL AND PM EXAMINATION
The chickens were observed daily for signs of disease and
mortality for 14 days post-challenge. The PM examination
was done for the euthanized birds for histopathology as well.
VIRUS SHEDDING
A total of 90 tracheal swabs were collected (5 /each group)
on viral transporting media; consisting of 10% glycerol, 200
U of penicillin per mL, 200 mg of streptomycin per mL,
250 mg of gentamicin per mL, and 50 U of nystatin per mL.
The collection was at the 3rd, 5th and 7th days post -challenge.
The samples were kept at -80oC till being examined by
qRT-PCR for detection and titration of shedders of AIV
H9N2 challenge virus. A Standard curve was calculated
using A/chicken/Egypt/S6/2018 by serial dilutions of the
virus from 10−1 to 10−6. Since Ct. is defined as the point
at which the curve crosses the horizontal threshold line.
The virus log10 titers of was plotted against the Ct value,
and the best fit line was constructed. AIV-H9N2 quantity
in unknown samples was derived by plotting the CT of
an unknown against the standard curve and was expressed
in log 10 EID50/mL equivalents. RNA extraction from
swabs was carried out by QIAamp viral RNA extraction kit
(Qiagen, Valencia, Calif., USA) as per the manufacturer’s
protocol.
One hundred one-day-old chicks were kept in biosafety
isolators previously cleaned and disinfected in Central Lab
for Evaluation of Veterinary Biologics (CLEVB); feed and
water were supplied ad libitum. The experimental design
for evaluation of the effectiveness of H9N2 avian influenza
inactivated vaccines was shown in (Table 3).
Blood samples were collected separately from 5 birds /
group at the 1st, 7, 14, 21, 28, 35 and 42 days of age and
sera were separated and kept in sterile tubes at -20oC.
VACCINE EFFICACY EVALUATION
ENZYME-LINKED IMMUNOSORBENT ASSAY
Daily observation and recording of clinical findings, virus
To monitor the immune response after vaccination the
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SEROLOGY
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authors used the commercially available H9 AIV antibody
test kit (ID-screen Influenza H9 indirect, Grabels,
France) lot number G11. The kit was used according to
the manufacturer’s instructions. Samples were tested in
duplicates. Controls; antigen, known positive and negative
sera were included. Optical density values were read at 450
nm using an ELISA microplate reader (Sunrise, Tecan,
Grodig, Austria).
HEMAGGLUTINATION INHIBITION (HI) ASSAY
The HI test was carried out by using 4 HA units of avian
influenza using two antigens (reference imported antigen
VLDIA113-INFH9, supplied from GD lab Lot No.
19660-040419 and Local antigen provided kindly from
ME-VAC company batch No. RD200101AG), (OIE
Manual, 2015).
HISTOPATHOLOGICAL EXAMINATION
Three chickens from each group were humanly euthanized
and subjected to necropsy at the 5th-day post-challenge.
Trachea, lungs, bursa, cecal tonsils, thymus, and kidney
were collected and kept in 10% buffered formalin for 24
hours (Suvarna et al., 2018).
homology of the two H9 isolates was 97.2% with each
other. The HA gene amino acid identities of A/chicken/
Egypt/S2/2018 and A/chicken/Egypt/S6/2018 with
other contemporary Egyptian isolates (2012-2019) were
94-98.6 95.6-98.8%, respectively. The HA gene of the
current two isolates shared amino acid homology of 88.8
and 90 %, respectively, with the prototype HK-G1. In
comparison with the four H9N2 vaccine strains used in
commercial vaccines in Egypt (A/chicken/Saudi Arabia/
CP7/1998, A/ chicken/Iran/av1221/1998, A chicken
Egypt ME543V 2016 and A chicken UAE AG53799), ranges of deduced amino acid identities were 91.4
to 92.6 %, 91.2 to 92.3%, 97.4 to 99.3% and, 91.9 to
93% respectively. Our isolates were closely related to the
Israeli viruses, with a high identity percentage ranged
from 94.4-96.7%
table 4: Molecular detection of avian influenza viruses
and other suspected coinfections (IBV and NDV) by realtime PCR.
Sample Sample
results
no.
Id
M CT H5 H7 H9 CT ND IB CT
1
S1
+
26
-
-
+
27
-
-
>40
STATISTICAL ANALYSIS
2
S2
+
24
-
-
+
25
-
-
>40
The obtained data were subjected to one-way analysis of
variance using SPSS (version 22, Armonk, NY, USA).
Differences within means of treatments were tested at p
< 0.5.
3
S3
-
>40 -
-
-
>40 -
-
>40
4
S4
-
>40 -
-
-
>40 -
+
16
5
S5
-
>40 -
-
-
>40 -
+
15
6
S6
+
22
-
-
+
23
-
-
>40
rESultS And dIScuSSIon
7
S7
-
>40 -
-
-
>40 -
-
>40
8
S8
-
>40 -
-
-
>40 -
-
>40
CLINICAL AND PM FINDINGS
9
S9
+ 27 +
28 - >40
CT >40 is considered negative result; M, Matrix gene; Newcastle
disease virus, NDV; infectious bronchitis virus, IBV.
The naturally affected broiler flocks revealed respiratory
signs: sneezing, swelling of periorbital tissues with
conjunctivitis, nasal and ocular discharges. Whitish
diarrhea also was noticed. The postmortem findings of
the examined broilers revealed tracheitis, congestion of
lungs, and air-sacculitis. Swollen Kidney was also detected.
Mortality rates ranged from 3% to 5% in examined broiler
flocks. Drop-in egg production ranged from 25% to 30%
in affected layer flocks with thin-shelled, rough, and
misshapen eggs.
MOLECULAR DETECTION OF AIV, IBV AND NDV
Real time RT-PCR was positive in four samples for
Matrix and H9 genes and negative for other viruses. IBV
was detected in two samples. Neither of H5, H7 subtypes,
and NDV was detected (Table 4).
SURFACE
GLYCOPROTEINS
HA
AND
NA
AMINO ACID
IDENTITIES
Regarding sequencing of HA and NA genes in two
selected H9 subtype isolates, the HA gene amino acid
January 2022 | Volume 10 | Issue 1 | Page 39
However, The NA gene amino acid homology was 97.9%
between two of our isolates. The percentages of NA gene
amino acid identities of A/chicken/Egypt/S2/2018 and
A/chicken/Egypt/S6/2018 with Egyptian isolates (20122016) were 94.5-97.6 and 94.8-97.4 %, respectively. The
NA gene shared amino acid homology of 90.5 and 90.8%
with the HK-G1 prototype. Compared with the H9N2
vaccine strains used in commercial vaccines in Egypt (A/
chicken/Saudi Arabia/CP7/1998 and A/ chicken/Iran/
av1221/1998), ranges of amino acid identities were 92.2%
and 91.5, respectively (Table 5).
PHYLOGENETIC ANALYSIS
Phylogenetic analysis of the HA, NA genes showed
that our Egyptian isolates of H9N2 AIV were grouped
in the Quail/HK/G1/97 lineages, similar to the viruses
circulating in the Middle East close phylogeny to the
Israeli viruses (Figure 1).
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Figure 1: Phylogenetic analysis of H9 and N2 genes nucleotide sequences of AIV (S2andS6) isolates from Sharkia
Province, Egypt and other sequences available in GenBank. (A) The tree was constructed via multiple alignments of 1465
bp nucleotide sequence of HA gene. (B) The tree was constructed via multiple alignments of 1265 bp of NA gene, using
the neighbor-joining method and the Kimura-2-parameter models in MEGA7. Strain A/Turkey/Wisconsin/1966 was
used as the root of the tree at 1000 bootstrap replicates. Isolates in this study are marked with solid circle. Vaccine seeds
are marked triangle.
VIRUS ISOLATION
The H9 virus isolation trials in SPF ECE revealed survival
of the embryos within (2-4) days after inoculation. The
January 2022 | Volume 10 | Issue 1 | Page 41
HA titer for the harvested allantoic fluid was (128 and
512) for S2 and S6, respectively.
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table 5: Percent amino acid identity comparison between
our study isolates with other viruses and vaccinal seeds.
Viruses
GROSS LESIONS
There were no detectable gross lesions in all organs of the
chickens for different groups except G5 two birds showed
lesions of mild pneumonia, congestion in the small
intestine, and mildly swollen kidneys.
A/chicken/
Egypt/
S2/2018
A/chicken/
Egypt/
S6/2018
HA
HA
NA
Each other
97.2% 97.9% ----
----
TRACHEAL SHEDDING
A-Quail-HK-G1-97
88.8% 90.5% 90%
90.8%
There was no tracheal shedding in G1, G2, G4, and
G6 throughout the experiment. One bird only showed
tracheal shedding in G3 at the 3rd day post-infection
(DPI) (1/5) with titer (1.511 x 105). All challenged birds
in G5 showed shedding H9N2 AIV at the 3rdDPI (5/5)
with amount (2.657 x 107, 4.474 x 107, 6.049 x 107, 1.135
x 107and 1.514 x 107, respectively). At the 5th day postchallenge, three birds in G5 were positive (3/5) with titer
(1.184x108, 6.658x107, and 6.515 x 106, respectively). The
shedding in infected non vaccinated birds stopped at 7th
day post-infection (DPI) (Table 6).
NA
A/quail/Egypt/113413v/2011
96.5% 96.9% 98.1% 97.4%
A-Duck-HongKong-Y280-97
88.6% 89.8% 89.3% 89.8%
A-chicken-Egypt-ME543V-2016 97.4% nd
99.3% nd
Achicken-SaudiArabia-CP7-1998 91.4% 92.2% 92.6% 92.2%
A-chicken-Iran-av1221-1998
91.2% 91.5% 92.3% 91.5%
A-chicken-UAE-AG537-99
91.9% nd
93%
A/quail/Egypt/122313v/2012
94%
nd
95.6% Nd
A/chicken/Egypt/D5490B/2012
nd
97.6% nd
A/turkey/Egypt/1386s/2013
96.5% nd
A/chicken/Egypt/F7297B/2013
nd
A/chicken/Egypt/1474v/2014
95.1% nd
A/Quail/Egypt/14864v/2014
nd
A/chicken/Egypt/152537v/2015
96.5% nd
nd
97.4% nd
A/chicken/Egypt/F12168B/2016 nd
97%
94.5% nd
A/chicken/Egypt/1685SL/2016
97.4%
98.4% nd
96.7% nd
A/chicken/Egypt/1560VG/2015
Nd
97.2%
nd
SEROLOGICAL RESPONSE
96%
The mean antibody titers detected by HI for H9 AIV in
different groups of SPF chickens are represented in Figure
2. The antibodies were detected by ELISA in 50% and
80% of vaccinated birds at two weeks post-vaccination but
increased to 100% at the 3rd week P (Table 7).
98.4% nd
97.2% nd
96.4%
98.8% nd
94.3% nd
94.8%
A/chicken/Egypt/17159v/2017
98.6% nd
97.7% Nd
A/chicken/Egypt/V1527/2018
98.6% nd
97.7% Nd
A/chicken/Egypt/V2692/2019
97.2% nd
97.7% Nd
A-Turkey-israel-1608-2006
95.3% 96.4% 96.7% 96.9%
A-Chicken-israel1067-2010
94.4% nd
96.3% nd
Nd, not done.
PURITY OF H9 AIV ISOLATES
Real time PCR of isolates 2 and 6 after isolation in SPF
ECE were positive for H9 AIV only with CT= 18 and 11,
respectively and negative for (H5 and H7 subtypes, IBV,
MG, MS and ND).
VIRUS TITRATION
The pure strain (A/chicken/Egypt/S6/2018) was titrated
in SPF ECE and revealed 10 9.5/ 100 µL EID50.
CHALLENGE STUDY FINDINGS
CLINICAL SIGNS
No mortalities were recorded in all groups along the period
of experimentation. All birds in the five groups (G1, G2,
G3, G4 and G6) did not develop any signs of clinical illness
all over the 14 days of monitoring on a daily basis. The
G5 (positive control group) birds developed clinical illness
starting at the 3rd day post-challenge, which completely
stopped at the 7th-day post-challenge. The commonly
observed clinical manifestations were; reduction in the
feed consumption rate, general depression, and diarrhea.
January 2022 | Volume 10 | Issue 1 | Page 42
HISTOPATHOLOGICAL EXAMINATION
Histopathological findings revealed mild desquamation of
tracheal epithelium, low degree of interstitial pneumonia
and interstitial inflammatory cells infiltrations in the
kidneys of the positive control group (G5) as shown in
(Figure 3).
Avian influenza (AI) is a respiratory disease in poultry caused
by influenza A viruses of the family Orthomyxoviridae.
Several virus subtypes are classified according to the
two major surface glycoprotein: Hemagglutinin (H) and
neuraminidase (N) (Luczo et al., 2015).
In the Middle East, H9N2 AIV has been reported
for several years, indicating additional risk to poultry
production. It is a significant concern the spread of H9N2
in Egypt can negatively affect poultry health overall and
increase the risk of H5N1andH5N8 HPAI infections
(Hegazy et al., 2019).
The current study throws light on the isolation and
molecular characterization of recent circulating LPAI
H9N2 at Sharkia Province for more accurate identification.
As well as to evaluate the commercial in SPF chickens
through viral replication and shedding by qRT-PCR,
serological response using ELISA and HI tests, and
clinical and histopathological examination.
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Figure 2: Geometric mean log2 of HI titers in experimental SPF chickens using two antigens, G1: vaccine 1 non
challenged, G2: vaccine 2 non challenged, G3: vaccine 1 challenged, G4: vaccine 2 challenged, G5: challenged non
vaccinated (Pos control), G6: non treated (Neg control). (A) HI test results using imported antigen: representing vaccine
2 seed virus from day 1 to day 28 before challenge, (B) HI test results using local antigen: Local antigen similar to vaccine
1 seed virus from day 1 to day 28 before challenge, (C) HI test results using imported antigen: representing vaccine 2
seed virus day 35 (1st week post challenge) and day 42 (2nd week post challenge), (D)HI test results using local antigen:
Local antigen similar to vaccine 1 seed virus day 35 (1st week post challenge) and day 42 (2nd week post challenge).
Figure 3: Histopathologic findings of chickens 5 days after challenge with AIV H9N2. a, Normal pulmonary tissues in
G6; b, Interstitial pneumonia (arrow) in G5; c,Normal tracheal tissues in G6; d, Mild desquamated tracheal epithelium
(arrow) in G5; e, Normal lymphoid follicles in G6; f, Necrotic some lymphoid follicles (arrow) in G5; g, Normal cortical
and medullary lymphoid population in G6; h, congested blood vessels with thickening of septae in G5; i, normal
lymphoid aggregations in G6; j, partial depletion (arrow) in G5; k, normal renal glomerular and tubular structure in G6;
l, interstitial inflammatory cells infiltrations (arrow) in G5 H and E stain.
January 2022 | Volume 10 | Issue 1 | Page 43
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table 6: Mean titer* of virus shedding and number of shedder in vaccinated and non-vaccinated challenged with H9N2
virus in SPF chickens.
Group Group treatment
no
h9n2 virus shedding in tracheal swabs post challenge
rd
5th day post challenge
3 day post challenge
7th day post challenge
Titer
No. shedders
(mean ±SD) (pos/total)
Titer
(mean ±SD)
No. shedders
(pos/total)
Titer
(mean ±SD)
No. shedders
(pos/total)
G1
Vaccinated non challenged
(Vaccine 1)
0
0/5
0
0/5
0
0/5
G2
Vaccinated non challenged
(Vaccine 2)
0
0/5
0
0/5
0
0/5
G3
Vaccinated challenged
(Vaccine 1)
0.230
x105±0.06
1/5
0
0/5
0
0/5
G4
Vaccinated challenged
(Vaccine 2)
0
0/5
0
0/5
0
0/5
G5
Non Vaccinated challenged 3.165 x
107±0.26
5/5
3.829 x
107±0.5
3/5
0
0/5
0/5
0
0/5
0
0/5
G6
Non treated
*Titers expressed as EID50/ml ±SD.
0
The nine flocks under current investigation were allocated
at different regions in Sharkia Province during 2018. The
examined birds experienced respiratory distresses or a dropin egg production. The present results revealed that the
frequently observed clinical signs among examined broiler
flocks were respiratory: Sneezing, periorbital swelling with
conjunctivitis, nasal and ocular discharges. Swollen kidneys
were also noticed. While, the layer flocks showed clinical
signs in the form of depression accompanied by a decrease
in feed consumption with the drop in egg production
ranging from 25% to 30% with thin-shelled, rough and
misshapen eggs. Comparable clinical signs were recorded
among H9N2 infected birds (Naeem et al., 2007; Capua
and Terregino, 2009; Qi et al., 2016).
Concerning real time PCR results for determination of the
H9N2 subtype as well as other viruses H5, H7, NDV, and
IBV), which revealed only four flocks(broiler/layer) were
positive for H9AIV, with no detection of other examined
viruses; however, two were positive for IBV, and three
flocks were negative.
On the other hand, The NA identity % among our isolates
shows (97.9%), also with other Egyptian isolates in gene
bank (2012-2016) shows the percent of (94.5-97.6 and
94.8-97.4%, respectively) and, were closely related to the
Israeli viruses with high identity% (96.4-96.9%). Similar
results were obtained by (Kandeil et al., 2014) who
reported the homology of the NA gene among H9N2
isolates ranged from 94.7 to 99.9%.
By browsing the range of identity among amino acids in
both HA and NA genes either between our isolates or
with the previously isolated viruses in Egypt showed high
similarity with minor changes along with the history of
isolation from 2011 (Abdel-Latif et al., 2020). Upon the
results mentioned above related to molecular identity of
H9N2 viruses, the chance of commercial vaccine success
to minimize the risk of the virus infection. However,
the biological behavior of the virus and the vaccine in
experimental birds will be an added value to support this
conclusion (Talat et al., 2020).
Detection of H9 subtype AI and IB viruses was reported
among chicken congregations suffering from respiratory
or egg production problems in natural infections (Sultan
et al., 2015a; Abd El-Hamid et al., 2018).
Phylogenetic analysis revealed that the HA and NA of our
Egyptian H9N2 isolates are related to the Middle East
H9N2 isolates and maintained a direct out-group of the
G1-like viruses, forming a distinct cluster. The same result
was obtained by (Kandeil et al., 2017; Abdel-Latif et al.,
2020).
The HA amino acid identity % among our isolates shows
a percent (97.2 %). While, other Egyptian isolates (20122019) in the gene bank was (94-98.6, and 95.6-98.8%,
respectively). Our detected viruses and the other Egyptian
viruses were closely related to two Israeli viruses with high
identity% (94.9-96.7%). These results were consistent with
that of (Adel et al., 2017), who stated that the identity
with the Israeli viruses was 95-97%.
The samples were isolated and propagated in the SPF
embryonated chicken eggs. Then, the collected allantoic
fluids were tested by HA assay to detect the virus titers
that produce titers ranging from 27 to 29. The isolate (A/
chicken/Egypt/S6/2018) revealed 10 9.5/ 100 µL EID50
(Reed and Muench, 1938).
January 2022 | Volume 10 | Issue 1 | Page 44
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The protection efficacy of commercial H9N2 vaccines was
evaluated by two field available vaccines (vaccine 1 A/
chicken/Egypt/ME543V/2016 and vaccine 2(A/chicken/
Iran/Av1221/1998) SPF chicks at 7th day of age and
challenged three weeks later.
table 7: Serological response by ELISA ID-screen H9 kit
in experimental chickens.
Group Age/
name days
Mean**
Std.
Deviation
Std.
Error
Percentage
G1
1.40 C
0.70
0.22
0
1.40
C
0.70
0.22
0
1.10
C
0.32
0.10
0
1.10 C
0.32
0.10
0
1.30 C
0.48
0.15
0
1.50 C
0.71
0.22
0
C
1.58
0.50
0
12.89
4.07
0
Day1
G2
G3
G1
G2
G3
G1
G2
G3
G1
G2
Day7
Vaccination day
Day14
1st week
PV
1.50
1.10
0.32
0.10
0
Day21
2nd week
PV
1051.50b
1075.19
340.01
50%
1470.00b
1194.18
377.63
80%
4.40c
G3
G1
G2
G3
ElISA results*
Day28
3rd week
PV
6.50c
c
3736.20
6.31
2.00
0
a
51.34
16.23
100%
a
857.87
271.28
100%
3184.40
c
0.67
0.21
0
a
111.60
49.91
100%
a
366.89
164.08
100%
a
3765.40
82.54
36.91
100%
G4
3825.40a
88.21
39.45
100%
G5
b
1439.31
643.68
100%
G1
G2
G3
Day 35
7 days
PC
2.00
3787.40
3533.80
2321.40
c
G6
0.32
0.10
0
a
122.80
54.92
100%
a
22.35
9.99
100%
a
4847.40
3448.01
1542.00 100%
G4
4097.00a
2020.47
903.58
100%
G5
b
1293.93
578.66
100%
G1
G2
G3
1.10
Day 42
2nd week
PC
3906.20
3812.40
2642.20
G6
2.00c
0.67
0.21
0
Wells of ELISA plate is coated with recombinant H9
hemagglutinin *; ** Anova one way used to test Significance
difference between group, the same letters mean nonSignificance difference, significant difference at p value (p ≤
0.05); PV= post vaccination, PC= Post challenge. G1: vaccine
1 non challenged, G2: vaccine 2 non challenged, G3: vaccine
1 challenged, G4: vaccine 2 challenged, G5: challenged non
vaccinated (Pos control), and G6: non treated (Neg control).
Our findings indicated that only some depression and
diarrhea were apparent in G5 only and these results were
consistent with the previous report of H9 AIV infectious
January 2022 | Volume 10 | Issue 1 | Page 45
in an experimental situation (Bijanzad et al., 2013; Elfeil
et al., 2018), who stated that single H9N2 experimental
infection couldn’t introduce significant clinical signs. There
was no any mortality in all groups during the experiment,
which confirmed the low pathogenic nature of H9N2
AIV. This agreed with reported with (Elfeil et al., 2018).
In contrast, one study revealed 20% cumulative mortality
in experimentally infected chickens with LPAI H9N2
infection (Abd El-Hamid et al., 2016). Thus, variation in
the severity of the symptoms and mortalities could be due
to the mentioned experiment done in commercial broiler
chickens.
Since one of the most critical challenges among H9N2
vaccination is preventing field virus shedding to avoid wild
reassortment, the quantitative shedding was carried in the
current study.
Consequently, the vaccine must reduce the virus shedding
compared with the group that has challenge virus only
(G5 positive control group). It has been shown that
H9N2 vaccine one stopped shedding of the field virus at
the 3rd-day post-challenge and vaccine two succeeded in
preventing any field virus shedding completely. Our results
are in agreement with the recommendation of (OIE,
2019). Moreover, the results of the H9N2 AIV shedding
pattern revealed that there is demarked reduction in virus
shedding in tracheal shedding in the 7th DPI in the
positive control group. These results agreed with (Khalil
et al., 2015).
Regarding serological monitoring, the present study
indicated that there is the proper amount of antibodies
against H9N2 after single vaccination with oil emulsion
inactivated vaccine with both vaccines, which support
the earlier findings (Lee et al., 2011; Sultan et al., 2015b;
Ibrahim and Seioudy, 2020) who mentioned that although
a single administration of oil-based inactivated H9N2
LPAI vaccine is very immunogenic and highly protective
in laboratory trials using SPF chickens.
Both HI and ELISA were comparable in antibody
evaluation. But variation in antibody titers post-challenge
may be attributed either to the HA antigen or coated
antigen identity with vaccinal and field viruses. In turn, the
interpretation of serological results the aforementioned
factors should be given to evaluate vaccines. Upon the
current serological findings, the threshold of antibody 24
in HI and 1800 in ELISA could be protective, particularly
against field virus challenge.
Histopathological findings of our study pointed that the
H9N2 AIV is epitheliotropic, especially toward respiratory,
lymphoid organs, and urinary; the results were obtained
(Elfeil et al., 2018).
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The trachea showed mild desquamation of the epithelial
cells in infected non-vaccinated G5 and less desquamation
in vaccinated challenge G3 and normal tracheal tissues in
the other groups at the 5th day post challenge. These results
were close to the results of (Awadin et al., 2018).
The examination of the pulmonary tissue revealed normal
pulmonary tissues; however, G5 showed interstitial
pneumonia similar finding reported by (Subtain et al.,
2011). Contradictories about pulmonary lesions were
recorded by (Slemons et al., 1991), who stated no pulmonary
lesions. Mild or less significant lung lesions were reported
by (Hablolvarid et al., 2004). These variations could be
referred to either the experimental birds were commercial
or SPF and the strain of challenge virus.
The renal tissue revealed marked interstitial inflammatory
cells infiltrations in G5, while moderate local interstitial
inflammatory cells infiltrations in G3 and normal renal
glomerular and tubular tissues in other groups. These results
were close to the results of (El Miniawy et al., 2014), who
reported renal lesions in the infected group with Egyptian
isolate of LPAI H9N2 in the form of tubule-interstitial
nephritis.
In the present study, the predominantly histologic changes
in the bursa of Fabricius and cecal tonsils were necrotic
some lymphoid follicles. Moreover, congested blood vessels
with thickening of septae were seen in the thymus.
These pathological changes in the immune organs could
indicate of the involvement of the immune system as well
as the immunosuppressive effect of H9N2 subtype on
chickens in Egypt. Our results were similar to the lesions
observed in the thymus and bursa of Fabricius in chickens
inoculated with 106 EID50 intra-nasally per bird of H9N2
avian influenza (Hadipour et al., 2011; Abd El-Hamid et
al., 2016). As well as atrophy in the bursa was reported
after the inoculation of H9N2 in broiler chickens (Qiang
and Youxiang, 2011).
concluSIonS And
rEcoMMEndAtIonS
It could be concluded from the presented study that The
LPAI H9N2 virus is still circulating in Sharkia Province
among both broiler and layer flocks. Under the current
experimental circumstances, the H9N2 AIV did not cause
severe disease as a single pathogen in SPF chickens and
is still low pathogenic. However, concurrent viral and
bacterial infections can exacerbate the disease condition.
The available commercial vaccines can limit the spread of
the field H9N2 virus based on virus shedding.
January 2022 | Volume 10 | Issue 1 | Page 46
AcKnowlEdGMEntS
This research was supported by grant #27667 from the
Newton Mosharafa, “Improving Vaccines and Diagnostics
for Avian influenza Viruses.” The authors would also
appreciate the STDF, Egypt and Dr Amal AM Eid,
professor of Avian and Rabbit Diseases and PI of the
project.
noVElty StAtEMEnt
The available commercial vaccines in Egypt can limit the
spread of the field H9N2 virus based on virus shedding.
Author’S contrIbutIon
Hossam A. Gado conducted the experimental procedures,
collected samples and wrote the manuscript. Ibrahim A.
Ghanem contributed to design the experimental work,
provided guidance, technical support. Abdullah A. Selim
helped supervise the molecular studies and participated in sequence analysis. Reem A.Soliman performed the
Experimental sample preparations for laboratory analysis. Mounir M. Elsafty supervised the Experimental work
and analysis the data. Amal A.M. Eid planned the work,
conducted the statistical analyses and critically revised the
manuscript.
CONFLICT OF INTEREST
The authors have declared no conflict of interest.
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Supplementary table S1: Descriptive data of examined chicken flocks that suspected to be infected with avian influenza
virus subtype H9.
Flock locality
no
h9n2 AI virus
vaccination*
date of examination
breed
1
Menia-ElKamh 70 and 112 day
28/11/2018
2
Menia-ElKamh 15, 45 and 120 day
3
Age
(day)
Flock
no of examcapacity ined birds
no of collect- lab
ed swabs
code
Decalb 245
5000
Nd
5T+5C
S1
1/12/2018
Bovans 305
3750
Nd
5T+5C
S2
Menia-ElKamh 5 day
10/12/2018
Ross
23
7000
3
5T+5C
S3
4
Meet Abo Ali
No
12/12/2018
Balady 52
6500
3
5T+5C
S4
5
Menia-ElKamh No
16/12/2018
Balady 85
2000
3
5T+5C
S5
6
Menia-ElKamh No
18/12/2018
Cobb
3500
3
5T+5C
S6
7
Menia-ElKamh No
24/12/2018
Balady 300
5000
3
5T+5C
S7
8
Tal Rak
28/12/2018
H & N 365
2000
3
Nd
S8
35 and 109 day
28
9
Menia-ElKamh No
30/12/2018 Cobb 18
700
3
5T+5C
Nd, not done * routine vaccination against NDV, IBV, IBD and AIV subtype H5 were given to these flocks.
January 2022 | Volume 10 | Issue 1 | Page 48
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