International Journal of Animal and Veterinary Advances 4(2): 125-129, 2012
ISSN: 2041-2908
© Maxwell Scientific Organization, 2012
Submitted: January 27, 2012
Accepted: February 16, 2012
Published: April 20, 2012
Serological and Molecular Assays for Detection of Avian Influenza Virus in Broiler
Chickens Flocks of Fars Province-Iran
1
E. Kariminejhad and 2M.J. Mehrabanpour
Islamic Azad University Jahrom Branch, Jahrom, Iran
2
Department of Virology, Razi Vaccine and Serum Research Institute, Shiraz, Iran
1
Abstract: H9N2 Avian Influenza Virus (AIV) is endemic in many Eurasian countries. Since 1998, this virus
is pathogen of poultry industry in Iran. The aim of this study was to serological and molecular assays of H9N2
AIVs exposure of broiler chickens by the heamagglutination inhibition test (HI), Enzyme Linked
Immunosorbent Assay (ELISA) test and RT-PCR technique in Fras province between November 2010 and
March 2011. In the present study, tracheal tissues, cloacal swabs and serum samples collected from 30 broiler
chicken flocks with respiratory disease symptoms in Fars province-Iran. The result of RT-PCR test showed that
out of 24 flocks, for H9N2 AIVs were positive. The overall 300 serum samples were examined by HI and
ELISA tests. In these tests, H7N7 and H5N1 AIV antibody (Ab) titers were negative in all flocks. Also,
seroprevalence of H9 AIV subtype was positive in 263 and 274 serum samples by HI and ELISA test,
respectively. The difference of Ab titers related to avian's immune system, age and many other factors. The
results revealed the important role of H9N2 AIV in respiratory disease outbreak in commercial broiler farms
of Fars province. In addition, the infectious flocks with high degree of mortality had high Ab titers against
H9N2 AIV and it is show that low protective effects of Ab.
Key words: Avian influenza virus, Elisa, H9N2, Iran
viruses isolated in these cases still produce little or no
disease in experimentally infected chickens (Pazani et al.,
2008). During 50 years ago, HPAI viruses such as H5N1
and H7N7 had major importance in economic damages,
because they could cause severe disease with high
mortality. The other hands, LPAI viruses are widely
distributed in wild and domestic avian species around the
world and also due to a contagious mild viral disease
(Hadipour, 2011). Infections with the H9N2 subtype of
AIV that named as LPAI in poultry industry, especially in
chickens, have been reported frequently in many Asian
countries. More recently, H9N2 viruses have been
reported in Middle Eastern countries and have been
responsible for widespread and serious disease problems
in commercial chickens in Iran, Pakistan, Saudi Arabia
and United Arab Emirates (Mehrabanpour et al., 2011).
This virus was first reported in 1998 in a layer farm in
Tehran and also caused widespread outbreaks in
commercial broiler chickens in Iran (Mosleh et al., 2009).
Fars province is an active pole of the poultry industry in
Iran, and where majority of broiler, layer and broiler
breeder farms of Iran are located. To control infection and
transmission of AIVs in the operation, an intensive
vaccination program was implemented in the following
years with inactivated vaccine (Zhang et al., 2008).
Despite the vaccination effort and the strict bio security
INTRODUCTION
Influenza is a highly contagious, acute illness that has
afflicted humans, animals and birds since ancient times.
Influenza viruses are part of the Orthomyxoviridae family
and are grouped into types A, B and C according to
antigenic characteristics of the core proteins (Hadipour,
2010). Type A Influenza viruses are further divide into
subtypes based on H and N antigens, some of these
subtypes are highly or lowly virulent in poultry (Woo and
Park, 2008). Based on the pathogenicity of Avian
Influenza Virus (AIV) to domestic poultry, these viruses
are sub classified into two pathotype groups of Highly
Pathogenic Avian Influenza (HPAI) viruses, causing rapid
mortality in poultry which often approaches 100% and
non-Highly Pathogenic Avian Influenza (n HPAI) viruses
including Mildly Pathogenic (MP), Low Pathogenic
Avian Influenza (LPAI) viruses and Non Pathogenic
Avian Influenza (NPAI) viruses, causing unapparent
diseases with mild respiratory sings, egg production
losses and sometimes with slightly elevated mortality and
increased cost for vaccination in the commercial poultry
farms (Pazani et al., 2008). However when exacerbation
of this influenza infection is caused by other bacterial and
viral organisms or environmental conditions, severe
disease with high mortality may be seen, although the
Corresponding Author: M.J. Mehrabanpour, Department of Virology, Razi Vaccine and Serum Research Institute, Shiraz, Iran
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Int. J. Anim. Veter. Adv., 4(2): 125-129, 2012
Table 1: Primers used for RT-PCR reaction
Name
Primer
Primer sequences
A
IVNP-1200F
5'-CAG (A/G) TACTGGGC (A/T/C) (A/G) AC
AIV
NP-152R
5'-GCATTGTCTCCGAAGAAATAAG
AIV
H5-155F
5'-ACACATGCYCARGACATACT
AIV
H5-699R
5'-CTYTGRTTYAGTGTTGATGT
AIV
H9-F
5'-CT(C/T) CACACAGA (A/G) CACAAATGG
AIV
H9-9R
5'-GTCACACTTGTTGTTGT (A/G) TC
AIV
H7-12F
5'-GGGATACAAAATGAAYACTC
AIV
H7-645R
5'-CCATABARYYTRGTCTGYTC
R: A/G; Y: C/T; B: G/C (Codes for mixed bases position)
measures imploded, these viruses were still isolated from
chickens (Zhang et al., 2008). The aim of this study was
to serological and molecular assays of AIVs exposure of
broiler chickens by the HI test, ELISA test and RT-PCR
technique in Fras province, between November 2010 and
March 2011.
PCR product
330 bp
545 bp
488 bp
634 bp
Serological analysis:
Heamagglutination inhibition (HI) test: This test was
used for all of the serums according to methods
recommended by the World Organization for Animal
Health (2009) for antibody (Ab) detection against H9, H5
and H7 virus. The amount of antigen used in each well
was 4HA unit for each subtype. The test serum samples
were heated at 56ºC for 30 min to inactivate
complements. HI test was performed in 96 well round
bottomed microtiter plates. After making serial dilutions
of the tested serum, antigen was added, incubated for 30
min and 1% chicken erythrocyte suspension was added.
The plates were left at room temperature for 30 min. At
least, the maximum dilution of each serum sample causing
inhibition of heamagglutination was used as endpoint. The
HI titer of each serum sample was expressed as reciprocal
of the serum dilution (top to bottom) (Alkhalaf, 2010).
MATERIALS AND METHODS
Collection and processing of samples: The study was
carried out in 30 broiler poultry farms with respiratory
problems around Shiraz city between November 2010 and
March 2011. Also, all of the flocks received of AIV
vaccines. The following types of samples were collected
from the diseased flocks:
Serum samples were collected from sick broiler
chickens flocks with respiratory disease symptoms. In
addition, blood samples were collected from wing vein
and kept at 4ºC for an overnight after which serum was
separated by centrifugation at 5000 rpm for 2 min.
Collected serum samples were kept in a separate vial and
stored at -70ºC.
The other hands, tracheal tissues and cloacal swabs
were collected from dead and morbid birds showing signs
of respiratory distress or other symptoms for laboratory
investigations. In order to check bacterial and fungal
contamination, swabs and tracheal tissue samples were
placed into tubes containing 1 mL PBS solution (PH 7.2)
and antibiotics (10.000 IU/mL penicillin, 1 mg/mL
streptomycin sulphate, 1 mg/mL gentamicin sulphate)
(Noroozian et al., 2007). Then, taken directly to the
laboratory in a cold box and stored at -70ºC until used.
Frozen tissues were thawed, grinded and squashed in a
sterile pestle and a 10% suspension with transport
medium were made (Numan et al., 2008). Homogenate
was centrifuged at 3000 rpm for 15 min and supernatant
was collected. Frozen cloacal swabs were thawed,
votrexed and pooled from each flock and centrifuged at
3000 rpm for 15 min (Numan et al., 2008; Noroozian
et al., 2007). Supernatants were collected and filtered
through 0.22 :m syringe filter (Gelman, USA). A part of
filtered material was used for extraction of the RNA
required for PCR identification.
Enzyme Linked Immunosorbent Assay (ELISA): The
harvested serums were heat inactivated at 56ºC for 30
min. The test serum samples screened with the
competitive ELISA for the qualitative detection of
antibody (Ab) to the most common and prevalent AIV in
chicken serum by used ANIGEN AIV Ab ELISA kit,
according to the manufacturer's instruction. The
colorimetric readings of ELISA plates that coated with the
antigen (Nucleoprotein) were performed by using a
spectrophotometer at 450 nm.
Molecular analysis:
RNA extraction: Viral RNA was extracted from all
filtered material samples using viral Gene-spin TM viral
DNA/RNA extraction kit (Vetek, Korea) according to the
manufacturer's instructions.
Polymerase Chain Reaction (PCR): The PCR was
carried out following the standard method of reverse
transcription-PCR (Lee et al., 2001). We used from
specific primers based on conserved sequences of the NP
gene and HA gene of avian virus (Table 1), respectively
as described previously (lee et al., 2001). RT-PCR was
carried out in 8 :L reaction mixture containing (dNTP,
enzyme mix, reaction buffer and stabilizing buffer), 1 :L
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Int. J. Anim. Veter. Adv., 4(2): 125-129, 2012
from each forward and reverse primer, 2 :L of template
RNA, 0.25 RNase inhibitor and 8 :L of RNase free water
was added to make a reaction volume of 20 :L (Lee et al.,
2001). The tube was than vortexes and spin for 3-5 sec
and before being placed over the thermalcycler for
reaction. The one step PCR condition for the
amplification was: RT at 45ºC for 45 min, one cycle at
95ºC for 3 min, 40 cycles of heat denaturation at 95ºC for
30 sec, primer annealing at 55ºC for 1 min, primer
extension at 72ºC for 1 min and one cycle of final
extension step at 72ºC for 10 min in automated thermal
cycler (Ahmed et al., 2009; Lee et al., 2001;
Mehrabanpour et al., 2011). The PCR condition for the
amplification of NP, H9 and H7 was the same as above,
except that the annealing temperature for H5 was 58ºC
(Lee et al., 2001). The RT-PCR products were checked by
1.5% agarose gel electrophoresis and analyzed under UV
light (Kang et al., 2006).
Fig. 1: RT-PCR products of NP gene (AIV), An expected size
PCR product, 330 bp of NP gene was detected. Lane 1:
Marker 100 bp DNA ladder; Lane 2: Positive control;
Lane 3: Negative control; Lane 4-9: Field samples
RESULTS AND DISCUSSION
The result of this study, reveled that the H9N2 Avian
Influenza Virus (AIV) viruses have important role in the
respiratory disease outbreak in broiler flocks of Fars
province. In the present research, HI, ELISA and RT-PCR
tests were applied for the detection of AIV infections. At
the beginning, HI test used for AIV detection and
subtyping of chicken serum samples. H7N7 and H5N1
AIV Ab titres were negative in all flocks, but H9N2 AIV
antibody (Ab) titers between 3 to 9 log2 were found in all
of the flocks. The cut-off point for seropositivity in HI test
was determined as HI Ab titers $5 in all vaccinated
broiler flocks and titres #5 were considered as negative
result. Thus, a total of 300 serum samples from 30 disease
flocks were examined by this test and the results show
that out of 263 serum samples for H9N2 were positive and
non of serum samples were positive for H7N7 and H5N1
(Table 2). The other method for detection of Abs against
AIV was ELISA test. In this test, PI value $51 were
positive, thus, 274 serum samples were positive for AIV
Ab between 300 serum samples (Table 2).
At last, between 30 commercial broilers flocks with
a history of respiratory disease, out of 24 flocks, for AIV
were positive by RT-PCR technique (Fig. 1). Further the
serotyping of 24 AIV isolates showed that all of them
were H9 and we didn't find any H5 or H7 (Fig. 2).
Avian influenza is one of the highly contagious
Office of International Epizootics (OIE) lists "A"
diseases. It leads to high mortality in chicken, resulting in
extensive losses. Seroepidemiological and molecular
studies to determine the mode of transmission of the virus
and the risk factor associated with infection are deemed
necessary (Alkhalaf, 2010). Even for control strategic
point of view, this monitoring in mandatory.
Fig. 2: RT-PCR products of HA gene for AIV, An expected
size PCR product for 488bp for H9. Lane 1: Marker
100bp DNA ladder; Lane 2: Positive control; Lane 3:
Negative control; Lane 4-9: Field samples
Table 2: Results of HI and ELISA for detection of antibodies against
AIV in chicken serum samples collected form 30 fields in Fars
province
Sample\ kind of test
Tasted samples
Positive samples
ELISA
300
274
HI-H9
300
263
HI-H5
300
0
HI-H7
300
0
HI test is common and current serological test for
suspension fields. The advantage of this test is low cost as
compared with other diagnostic test. The competitive
ELISA is an accurate test amenable to semi-automation
and the rapid survey of large number of samples. Most of
the examined flocks showed high level of Ab titers to AIV
by ELISA technique. The findings of the current study
indicate a high infectious AIV activity among commercial
broilers of Fars province. In fact, AIV is responsible for
the majority of respiratory disease outbreaks in vaccinated
broiler flocks (Mehrabanpour et al., 2011). Generally,
AIV infections and the process of vaccination cause to
stimulate immune system. The intensity of avian immune's
respond is different and depends on many agents such as
age, environmental factors and etc. In total, HI and
ELISA tests are more subsidy than RT-PCR technique.
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Int. J. Anim. Veter. Adv., 4(2): 125-129, 2012
Also, in most of the times, these serological methods are
applied in order to evaluate extreme viral infections. It is
common practice in Iran, as in other countries, to
vaccinate broiler flocks against AIV. Despite the use of
AIV vaccines, it is common to find AIV infections in
vaccinated broiler flocks (Roussan et al., 2008).
Vaccinated flocks normally have Ab titers, but higher
seroprevalence rate was founded in flocks with high
infections and high mortality. Thus, the low protective
effects of Ab have reported in this study.
Avian influenza is the major case of acute respiratory
and reproductive tract infection of chickens and every
year bring about high morbidity and mortality worldwide
(Siddique et al., 2008). Clinically it is impossible to detect
AIV’s subtypes from each other. Therefore, it is necessary
to develop a diagnostic test for the rapid identification of
these viruses directly from clinical specimens (Siddique
et al., 2008). Further, we and other researchers concluded
that molecular techniques such as RT-PCR help in rapid
and accurate identification of the etiological agents
responsible for an infection (Mehrabanpour et al., 2011).
RT-PCR has the ability to even detect a single virus
particle, whether active or inactive (Siddique et al., 2008).
It also provides better detection of the virus from the
clinical samples which might otherwise appear negative
due to inappropriate sampling or less of infectivity during
shipment (Mehrabanpour et al., 2011).
The high percentage of avian influenza reported in
this study addresses the strong need for more aggressive
monitoring and vaccination of the susceptible and already
vaccinated poultry flocks. Our data showed that H9N2
AIVs were the most important causes of respiratory
disease in broiler chicken in Iran in this period. Further
studies are necessary to assess circulating strains for
prepare suitable vaccine, economic losses caused by
infections and co infections of these pathogens. In fact, it
was concluded from the study that AIV is prevalent in
various fields of Fars province (Alkhalaf, 2010).
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CONCLUSION
In this study, AIV viruse of nHPAI isolated from
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respiratory disease in this study.
ACKNOWLEDGMENT
This study has been supported by of Razi vaccine and
serum research institute Shiraz -Iran and we are grateful
to the Dr. Rezaeian, for sampling assistance.
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