First Year of the Highly Pathogenic Avian Influenza H5N1 Outbreak in Egypt: Rapid Antigenic/Molecular Diagnosis and Virus Isolation
In mid-February 2006, an outbreak of Highly Pathogenic Avian Influenza (HPAI) H5N1 affected the commercial poultry production sector and backyards in Egypt and resulted in magnificent socioeconomic losses. The present study was designed for detection, isolation and characterization of Avian Influenza Viruses (AIV) circulating among different poultry species during the first year of the outbreak. A total of 22 commercial poultry farms (16 chicken, 5 ducks and 1 quails) and 5 zoo birds were examined for such purpose. While, no influenza viruses were detected in zoo birds, 68% of the commercial poultry farms located in seven Egyptian governorates were positive to AIV and 53.3% of them were subtyped as H5. Virus isolation in SPF chicken embryos was successful for nine samples from the first egg passage. Molecular characterization of the viral RNA, in the harvested allantoic fluid by multiplex RT-PCR, confirmed the virus identity. On the basis of the OIE criteria of pathogenicity and the observed mortality profile, the AIV isolated during the first wave of the outbreak was identified as highly pathogenic, where the Intravenous pathogenicity index (IVIP) score was 2.83 and the mortality rate was 100%. In conclusion, this study outlines the major implication of subtype H5 HPAI in field affections during the first year of the outbreak. Additional involvement of other influenza A viruses and probably haemagglutinating respiratory agents can not be excluded.
to cite this article:
W.H. Mady, A.A. Sanousi, H.M. Amer, I.M. El-Sabagh, A.M. Khafagy and M.S. Saber, 2010. First Year of the Highly Pathogenic Avian Influenza H5N1 Outbreak in Egypt: Rapid Antigenic/Molecular Diagnosis and Virus Isolation. International Journal of Virology, 6: 73-81.
Influenza A viruses are negative-sense, single-stranded enveloped RNA viruses
with a segmented genome belonging to the family Orthomyxoviridae (Cox
et al., 2000). On the antigenic bases of their surface glycoproteins;
particularly hemagglutinin (HA) and neuraminidase (NA), influenza A viruses
are divided into distinct subtypes. To date, sixteen HA (H1-H16) and nine NA
(N1-N9) subtypes, which are found in many different combinations have been identified
(Horimoto and Kawaoka, 2005).
Influenza A viruses have been isolated from a wide variety of hosts, including
more than 90 bird species and various mammalian species, such as humans, pigs,
horses, cats, minks and marine mammals. However, ecological studies suggest
that their natural reservoirs are wild aquatic birds belonging to the orders
Anseriformes (ducks, geese and swan species) and Charadriiformes
(shorebird species, including gulls) (Webster et al.,
Avian influenza viruses of all 16 subtypes can cause Low Pathogenicity Avian
Influenza (LPAI) in susceptible birds, with mild respiratory symptoms and low
mortality rates. However, in some cases, this infection may cause significant
mortality rates, generally when it occurs in a combination with other bacterial
or viral infections. Highly Pathogenic Avian Influenza (HPAI) is a systemic
disease with high mortality rates approaching 100% in chickens and Turkeys.
This disease is restricted to strains belonging to H5 and H7 subtypes (Johnson
and Maxfield, 1976; Alexander and Gough, 1986; Alexander,
The outbreaks of HPAI H5N1 viruses have been recognized in Asia since 1996,
when a fulminating and rapidly fatal systemic disease appeared and spread massively
in domestic poultry (Shortridge, 1999). Although, the
poultry depopulation policy adopted at that time had been succeeded in controlling
the virus spread, it was not sufficient to prevent frequent episodes of the
disease outbreaks in several Asian, African and European countries over the
following few years. The current outbreak of HPAI H5N1 affected an unprecedented
number of countries (No. = 61) extending over a wide geographical zone (OIE
In mid-February, 2006 a devastating HPAI H5N1 infected the commercial poultry
production sectors and backyards in Egypt (Aly et al.,
2006 a, b). The outbreak caused great socioeconomic
losses in poultry industry (Safwat, 2006) and still considered
a renewable problem warning with a possible endemic.
Laboratory diagnosis of Influenza A virus infections is routinely performed
by isolation and identification of the virus either in Embryonated Chicken Eggs
(ECE) or in cell culture followed by typing the isolated virus by haemagglutination-inhibition
test and/or RT-PCR. Virus pathogenicity is determined by estimating the intravenous
pathogenicity index or by sequencing the multiple basic amino acids located
at the cleavage site of haemagglutinin protein (OIE Manual,
In this study, we report the isolation and identification of AIV from the tracheal and cloacal swabs of infected chickens, ducks and quails located in seven different Egyptian governorates during the first year of the HPAI H5N1 outbreak.
MATERIALS AND METHODS
Tracheal and cloacal swab specimens were collected from suspected cases
of Avian Influenza in 22 different commercial poultry flocks located in seven
Egyptian governorates (Sharkia, Giza, Dakahlia, Gharbia, Menofia, Ismailia and
Menia) as well as from 5 flocks in Giza Zoo between October 2005 and March 2007.
Affected birds included chickens (broilers, layers and broiler breeders); ducks
(parents and meat type); Quails (parents) and zoo birds (White and black chickens,
white and colored ducks and swans). Cotton swabs were used for specimens' collection
and were subsequently immersed in 2 mL phosphate buffered saline, pH 7.2 containing
4000 IU penicillin, 400 μg streptomycin and 1000 IU mycostatin. The swab
suspension was clarified by centrifugation at 2000 rpm for 10 min and the supernatant
was stored at -20°C till use.
Rapid Antigen Detection Tests for Typing and Subtyping
Chromatographic immunoassay test kits for influenza type A and subtype H5
antigens (Anigen, Korea) were used for rapid detection and subtyping of avian
influenza viruses in swab specimens (Gavin and Thomson,
2003). The test device combines a sample well with a reading well that contains
a membrane strip for influenza A or H5 viral antigens. The tests were performed
according to the manufacturer's instructions where, each sample was mixed with
a specimen diluent containing buffered saline, detergent, mucolytic agent and
preservative. Then, 200 μL were transferred by pipette into the middle
of the test well of the device. A positive test was indicated by two color bands
in the reading well, one in the Test (T) region and one in the Control (C) region.
A negative test was indicated by only one color band in the C region. The absence
of any color bands in the T or C regions represented an invalid test. Test readings
were performed and recorded after 20-30 min of incubation.
The supernatant fluid of each swab suspension was inoculated into the allantoic
cavity of five 9-11 days-old embryonating Specific-Pathogen-Free (SPF) chicken
eggs. The eggs were incubated at 33-34°C for 4-7 days and were examined
daily. Eggs containing dying embryos after 24 h of incubation and those remaining
alive till the end of incubation period were chilled to 4°C and the allantoic
fluid was harvested for further evaluation by haemagglutination test. Fluids
that produced negative haemagglutination results passaged two further times
before considered negative for virus isolation (OIE Manual,
Twenty-five microliters of PBS were added to all wells of a plastic V-bottomed
microtiter plate. Twenty-five microliters of the infected allantoic fluid samples
were added to the first wells and diluted serially two-fold across the plate.
After dispensing, a further 25 μL of PBS to all wells of the plate, 25
μL of 1% (v/v) washed chicken RBCs were added. The plate was incubated
at room temperature for 40 min by which the control RBCs usually settles to
a distinct button. The HA titers of allantoic fluid samples were expressed as
the reciprocal of the highest antigen dilution showing complete haemagglutination
(Hierholzer et al., 1969).
Extraction of the total RNA from infected allantoic fluid samples was performed
using TRIzol reagent (Invitrogen, San Diego, CA) according to the manufacturer's
recommendations. Briefly, 400 μL of the allantoic fluid were mixed with
1 mL of the TRIzol reagent and incubated at Room Temperature (RT) for 10 min.
After addition of 200 μL Chloroform, the tube was shacked vigorously before
reincubated at RT for 15 min. Centrifugation of the tube at 14,000 rpm for 15
min at 4°C resulted in separation of the mixture into 3 phases. The upper
phase containing the RNA was transferred to another tube, where 500 μL
of isopropanol were added and the tube was incubated at RT for 10 min. After
another cycle of centrifugation at 14,000 rpm for 10 min, RNA precipitated in
the form of gel-like pellet. RNA was washed by ethanol 75%, air-dried for 10
min and dissolved in 30 μL nuclease-free water.
First Strand (cDNA) Synthesis
Reverse Transcription (RT) of the total RNA extract was carried out using
RevertAidTM first strand cDNA synthesis kit (Fermentas, Germany).
A reaction mixture of 5 μL RNA extract, 200 ng random hexamer primers and
6 μL nuclease-free water was prepared and incubated at 70°C for 5 min.
The mixture was placed on ice for 5 min; then 4 μL of 5x reaction buffer;
1 μL of RNase inhibitor; 2 μL of 10 mM dNTP mix and 1 μL of MMuLV
reverse transcriptase were added. The RT reaction was conducted in a thermal
cycler (Gene-Amp 9700, Applied Biosystem Inc., CA) for one cycle at 25°C
for 10 min; 42°C for 60 min and 70°C for 10 min. The synthesized cDNA
was used for PCR amplification of influenza specific genes.
||Oligonucleotide primers for multiplex PCR
The PCR amplification of type A and H5 subtype AIV-specific sequences was
optimized and performed using the oligonucleotide primers shown in Table
1. Multiplex PCR was carried out in a 50 μL reaction volume containing
1x PCR reaction mixture (Fermentas, Germany); 0.2 μM of each M-specific
primer; 0.4 μM of each H5-specific primer and 5 μL of cDNA. The used
thermal cycling profile was initial denaturation for 5 min at 94°C followed
by 35 cycles of 94°C for 30 sec; 50°C for 30 sec; and 72°C for 1
min with a final extension of 10 min at 72°C. The PCR products were resolved
by electrophoresis in 1.2% agarose gel stained with ethidium bromide.
Intravenous Pathogenicity Index (IVPI) Test
The test was performed as described by OIE Manual (2005),
where fresh infective allantoic fluid samples of 4 log2 HA titers were diluted
1:10 in PBS. Ten eight-weeks-old SPF chickens were injected intravenously with
0.2 mL of the diluted allantoic fluid. Birds were examined for clinical signs
and death at 24 h intervals for 10 days. At each observation, each bird is scored
0 if normal; (1) if sick (showing a single clinical sign); (2) if severely sick
(showing multiple signs) and (3) if dead. The IVPI is the mean score per bird
per observation over the 10 days period. An index of 3.00 means that all birds
died within 24 h and an index of 0.00 means that no bird showed any clinical
signs during the 10 days period. In general, any influenza virus regardless
of the subtype, giving a value greater than 1.2 is considered to be highly pathogenic.
Rapid Detection and Subtyping of Avian Influenza Viruses
All the pooled tracheal and cloacal swab specimens, that represent 22 different
poultry flocks (16 chickens, 5 ducks and 1 Quail) and 5 Zoo birds, were tested
using the rapid Avian Influenza A antigen detection strips. With the exception
of Zoo birds that was proved negative, the different tested domestic poultry
species developed positive reactions in 13/16 (81%) of the chicken flocks, 1/4
(25%) of the duck flocks and 1/1 (100%) of the quail flocks. The affected flocks
were distributed in 6 different Egyptian governorates including: Gharbia 2/2
(100%), Menia 2/2 (100%); Ismailia 1/1 (100%); Menofia 1/1 (100%); Giza 8/12
(66.7%) and Sharkia 1/3 (33.3%).
Only positive samples were subtyped as H5 using the rapid H5 antigen detection strip tests. While, 7 out of 13 of the chicken flocks (53.8%) and the sole quail flock reacted positively, the only AI-positive duck flock was not an H5 influenza virus (Table 2).
||A Collective data on the test samples and their reactivity
in the different assays performed
|*ND: Not done, **Haemagglutination unit/25μL of the virus
Isolation of Avian Influenza Viruses in Embryonated Chicken Eggs
The different collected samples were propagated in the allantoic cavity
of SPF chicken embryos for virus isolation. Only 9 samples (7 from chickens,
one from ducks and one from quails) could be isolated. Most of the isolated
samples were recovered from the first egg passage. The isolated virus identity
was further confirmed by the hae magglutination (HA) test, which showed variable
HA titers ranging from 4 log2 to 12 log2 (Table 2).
Simultaneous Identification and Subtyping of the Isolated Viruses Using
A multiplex RT-PCR assay was optimized for use in detection and subtyping
of the H5N1 strains of Avian influenza virus. The oligonucleotide primers were
chosen for amplification of fragments of the M and H5 gene sequences; analyzed
and optimized in a standard reaction. The test was applied on 18 samples showed
positive reactivity either in antigen detection or in virus isolation. Only
six samples proved their identity as H5 virus strains by amplification of 2
specific bands of 244 and 456 bp (Table 2, Fig.
Application of IVPI test on ten eight-weeks-old SPF chicken showed that
the isolated viruses have a mean score of 2.83 (Table 3).
This result along with the death of all inoculated chicken confirmed the high
pathogenicity nature of the isolated H5 influenza A viruses.
||Multiplex RT-PCR products of selected samples for detection
of HPAI H5N1 viruses as shown in ethidium bromide stained agarose gel electrophoresis:
Lane M represents 100 bp DNA molecular weight ladder; Lanes 3 and 6 show
positive amplification of a 244 bp fragment of M gene only; Lanes 2, 4 and
5 show positive amplification of 244 and 456 bp fragments of the M and H5
genes, respectively; Lane 7 represents a standard strain of Avian Influenza
H9N2 and Lane 1 and 8 represents negative samples with no amplification
||Intravenous pathogenicity index (IVPI) scores throughout the
|Scoring; 0: Normal, 1: Sick 2: Very sick (more than one sign)
3: Dead. IVPI: Sum/Total = 283/100 = 2.83
Influenza A viruses containing HA of subtypes H5 and H7 are well known as highly
pathogenic viral agents causing severe outbreaks in poultry worldwide (Alexander
and Gough, 1986; Alexander, 2000). During the last
few months of 2005, there was a rising anticipation of an H5N1 attack towards
the Egyptian poultry industry following by a similar situation occurred in Turkey
and Romania (Nicoll, 2005). At mid-February 2006, the
expectations were changed into a reality, when the first isolates of highly
pathogenic Avian Influenza H5N1 viruses were recovered from backyard birds.
Soon, after the poultry farms of intensive breeding systems were affected (Aly
et al., 2006a, b). By the end of March 2006,
twenty Egyptian governorates officially reported the occurrence of HPAI H5N1
infection inside their range, a matter that caused great socioeconomic losses
in poultry industry (Kilany, 2006; Safwat,
This study was started so early at October 2005, five months before the announcement of outbreak in Egypt and extended till March 2007 to cover the entire first year of the outbreak. It planned for detection, isolation and characterization of avian influenza viruses among domestic and wild birds in different Egyptian governorates and determination of their pathogenicity. During the course of the study, a total of 22 commercial poultry flocks (including chicken, ducks and quails), located into seven Egyptian governorates, were examined. All flocks showed either significant drop in production and/or high mortality rates. In addition, five wild birds located at Giza Zoo, suddenly died with no clinical symptoms, were also included in the study. Only tracheal or cloacal swabs were collected and used for virological examination.
Detection of influenza A viral antigens in clinical specimens by rapid immunochromatographic
assays are widely used for diagnosis and typing of avian influenza viruses;
because of their simplicity and ability for rapid diagnosis. However, the usefulness
of such assays seems somewhat limited due to their low sensitivity. It is generally
recommended that the negative reactions should be confirmed by virus isolation
and/or RT-PCR (Gavin and Thomson, 2003; Peiris
et al., 2004). In the current study, all the collected samples were
tested using a rapid influenza A antigen detection kit. The positive samples
were subtyped using a parallel H5 detection kit. Only, 15/27 (55.6%) of the
samples were identified as influenza A viruses, from which 8 samples (53.3%)
were subtyped as H5 (Table 2). These levels of infection justify
the role of avian influenza viruses, particularly H5N1, in the field outbreaks
among poultry flocks in Egypt during the period of study. Besides, the results
of rapid antigen test kits indicate a sufficient level of detection for the
positive cases and their ability to give fast response to the potential field
problems (Davison et al., 1998). Further testing
of the negative samples, by virus isolation and haemagglutination test, identified
two more samples that are possibly containing avian influenza virus or a similar
respiratory virus of potential haemagglutinating activity (e.g., Newcastle disease
Virus isolation still remains the gold standard of diagnosis and indispensable
for virus characterization (De Jong and Hien, 2006).
In this study, avian influenza viruses were successfully recovered from 9 tested
samples (four of H5 subtype and five of other subtypes). Only 4 samples failed
in the isolation process even after 3 successive blind passages. This lower
percentage of positivity comparing with the rapid antigen detection may demonstrate
a degree of false positive reactivity in antigen detection assays, or due to
the presence of inactivated viral antigens in such samples. Titration of the
isolated viruses by haemagglutination test exhibited a widely variable titers
ranging from 4 log2 to 12 log2 that reflect a distinct difference of viral load
in the original inoculums.
In the last few years, RT-PCR was established as the most accurate and reliable
assay for detection of H5N1 in clinical samples (Payungporn
et al., 2004; Tran et al., 2004;
Ng et al., 2006). This test combines the speed
and simplicity of rapid antigen detection assays with the higher sensitivity
and specificity of virus isolation. In this context, a one-step multiplex RT-PCR
was developed and optimized for specific detection of H5 avian influenza viruses
in the allantoic fluid collected from inoculated eggs. Unexpectedly, lower range
of positive samples (6/18) was detected by the developed assay, which may indicate
the necessity of further validation. However, several samples were detected
by the multiplex RT-PCR although, they did not show any reactivity in antigen
detection assays and/or virus isolation. Such degree of specificity and sensitivity
promises with encouraging outputs of such assay, after complete validation,
in rapid and accurate diagnosis of field outbreaks.
Pathogenicity assessment of the isolated avian influenza virus strains is usually
indicated by the Intravenous pathogenicity index (IVIP), according to the OIE
Manual (2005) and by the induced mortality level. To date, all HPAI viruses
generate IVPI scores greater than 1.2 and induce mortalities exceeding 75%.
In the current study, the recovered viruses showed clinical signs, gross lesions
and death of 10 out of 10 of the inoculated chickens with a calculated IVPI
of 2.83 during the observation period (Table 3). Based on
this assessment, avian influenza virus isolates, obtained from infected poultry
in Egypt during this study, are highly pathogenic.
Different virological tests were utilized for detection and characterization of avian influenza viruses that were circulating among poultry population in seven Egyptian governorates during the first year of the outbreak. These tests lead to an affirmed diagnosis of H5 HPAI in 50% of tested samples with a subsidiary existence of other subtypes of influenza A and probably respiratory haemagglutinating viruses. Therefore, the use of multiple diagnostic approaches is strongly recommended for an accurate and definite diagnosis of HPAI especially during the periods of the outbreak.
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