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Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons in Egypt a
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Shimaa M.G. Mansour , Reham M. ElBakrey , Haytham Ali , David E.B. Knudsen & Amal A.M. Eid a
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Department of Virology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
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Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt c
Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt d
Department of Veterinary and Biomedical Sciences, Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD 57007, USA Accepted author version posted online: 27 May 2014.Published online: 27 May 2014.
To cite this article: Shimaa M.G. Mansour, Reham M. ElBakrey, Haytham Ali, David E.B. Knudsen & Amal A.M. Eid (2014): Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons in Egypt, Avian Pathology, DOI: 10.1080/03079457.2014.926002 To link to this article: http://dx.doi.org/10.1080/03079457.2014.926002
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Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology
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Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons in Egypt
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Shimaa M.G. Mansour1, Reham M. ElBakrey2, Haytham Ali3, David E.B.
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Knudsen4 and Amal A.M. Eid2*
Department of Virology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511,
Egypt 2 Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511, Egypt 3 Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511, Egypt 4 Department of Veterinary and Biomedical Sciences,
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DOI: http://dx.doi.org/10.1080/03079457.2014.926002
Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, 57007, USA
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Short Title: Avian influenza virus H5N1 in Pigeon in Egypt
* Corresponding author: Amal A.M. Eid E-mail address: [email protected] Telephone number: +201001747082
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Fax number: +20552284383
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Abstract
The highly pathogenic avian influenza virus (HPAIV) subtype H5N1 threatens animal and human health worldwide. Susceptibility of pigeons to HPAIV (H5N1) and their role in AIV transmission to domestic birds and humans remain questionable. In this study, an outbreak in domestic pigeons (1-18 months old) with 50% mortality was investigated. Pigeons exhibited
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nervous manifestations and greenish diarrhea. Necropsy of the naturally infected pigeons revealed congestion of the internal organs, particularly lungs and brain. A HPAIV subtype H5N1 designated A/Pigeon/Egypt/SHAH-5803/2011 was isolated from 40 days old pigeon. Sequencing
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of the hemagglutinin gene showed close relation to viruses in group 2.2.1/C. Intravenous inoculation of the isolate in chickens induced 100% mortality within 2 days postinoculation (dpi) and intravenous pathogenicity index was 2.7. Virus pathogenicity and transmissibility was determined experimentally in six-week-old domestic pigeons. Thirty percent of pigeons inoculated oronasally with 106 EID50 showed congested beak, conjunctivitis, depression and greenish diarrhea. A mortality rate of 10% was recorded preceded by severe neurologic signs consisting of torticollis, incoordination, tremors, and wing paralysis. Pathological examination
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Received: 11 April 2014
revealed a friable brain tissue and congested meningeal blood vessels. The lungs appeared edematous and severely hemorrhagic. Subepicardial and petechial hemorrhages on the coronary fat were observed. Both infected and contact pigeons shed virus via the oropharynx and cloaca. To our knowledge, this is the first description and characterization of HPAIV in naturally infected pigeons in Egypt. Our findings reveal that pigeons can indeed be susceptible to H5N1
HPAI viruses and could be a source of infection to other birds and human.
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Introduction
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Influenza A viruses belong to the family Orthomyxoviridae and have an enveloped segmented,
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single-stranded, and negative-sense RNA genome. Influenza A viruses of avian origin can be
hemagglutinin (H) and 9 different types of neuraminidase (N) (Fouchier et al., 2005). Avian
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influenza viruses are classified into two groups, highly pathogenic and low pathogenic based on the severity of the disease they cause in chickens (Swayne & Suarez, 2000). Highly pathogenic avian influenza viruses (HPAIV), which code for a furin-sensitive cleavage site in their HA
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protein are capable of inducing systemic infections with dramatic losses in poultry industry, particularly chickens and turkeys (Alexander, 1993).
The HPAI, an imperative transboundary disease poses a threat to both animal and public
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health. Controversial reports exist about the pigeon’s susceptibility to HPAIV. Their role as a link between wild and domestic birds as well as transmission and spread of HPAIV during epizootics over long distances is not yet clear (Kaleta & Hönicke, 2004). Although Asian lineage H5N1 HPAIV has been isolated from pigeons (Ellis et al., 2004), the existing evidence indicates that pigeons are only minimally susceptible or resistant to different subtypes of AIV infections and do not serve as efficient transmission host, even in the presence of immune dysfunction
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categorized into subtypes based on the two surface glycoproteins, 16 different types of
(Fang et al., 2006) like immunosuppression by cyclophosphamide (Panigrahy et al., 1996; Perkins & Swayne, 2002; Kaleta & Hönicke, 2004; Liu et al., 2007; Smietanka et al., 2011).
They probably played a minimal epidemiologic role in the perpetuation of the H5N1 Hong Kong-origin influenza viruses (Perkins & Swayne, 2002). Other experimental studies
demonstrated that pigeons are susceptible to highly pathogenic influenza viruses (Klopfleisch et al., 2006; Werner et al., 2007; Jia et al., 2008; Smietanka et al., 2011) and could be a source of infection to other animals (Songserm et al., 2006).
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The susceptibility of pigeons to H5N1 virus and their role in AIV transmission to domestic birds and humans remain questionable. Here we report for the first time in Egypt a case of natural infection with highly pathogenic avian influenza virus H5N1 in pigeons.
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Characterization, pathogenicity and transmissibility of the field AIV isolated from pigeon were
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Materials and methods
Samples and Virus isolation. In March 2011, two dead pigeon squabs (40 days old) were submitted to the Clinic of Avian and Rabbit Medicine Department, Faculty of Veterinary
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Medicine, Zagazig University, Egypt. According to the owner complain, pigeons exhibited greenish diarrhea, nervous manifestations and sudden deaths with mortality rate up to 50%
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within 10 days. The flock consisted of 50 non-vaccinated birds of variable ages (1-18 months) reared in backyard free range system. Pigeons were necropsied and respiratory pool samples (lung and trachea) were collected and injected into 10 days old specific pathogen free (SPF embryonated chicken eggs (ECE) via the allantoic route according to recommendations of the OIE (OIE, 2012). The collected allantoic fluid from dead embryos was screened by rapid
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investigated.
hemagglutination (HA) test. The infected allantoic fluid was then tested for the presence of AIV
by RT-PCR and further sequenced. Newcastle disease virus (NDV) infection was excluded in
HA positive allantoic fluids by negative results of both the Newcastle Disease Virus Antigen Rapid Test (Shenzhen Combined Biotech Co. Ltd, Shenzhen, China) and RT-PCR as described by Pang et al. (2002).
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RNA extraction and RT-PCR. Viral RNA was extracted from infected allantoic fluid using Gene JETTM RNA purification kit (Cat#K0731, Fermentas, EU) according to the manufacturer’s
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instructions. Five microlitres of extracted RNA was reverse transcribed to cDNA using
the manufacturer’s instructions. The PCR was performed with a set of primers specific for H5
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and N1 gene segments as described elsewhere (Njouom et al., 2008).
Viral sequencing and phylogenetic analysis. For characterization of the isolated virus, the HA gene was analyzed by PCR using a set of HA gene specific primers (Njouom et al., 2008). The PCR product was gel purified using Gene JETTM Gel Extraction Kit (Cat#K0691, Fermentas,
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EU) as recommended by the manufacturer and sequenced on both strands using the amplification primers (Solgent Co. ltd. Korea). The HA subtype was further identified by nucleotide BLAST
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(http://www.ncbi.nlm.nih.gov/BLAST) and submitted to GenBank. The obtained nucleotide sequence was aligned with other HA gene sequences available in GenBank and comparative alignment was performed by ClustalW method using MegAlign module of DNAStar software (Lasergene version 7.2, DNASTAR, Madison, WI, USA). Phylogenetic analysis was carried out on the HA gene region encompassing 1594 nucleotides. Forty-eight reference sequences retrieved from the GenBank database were used in this study to understand the relationship of the isolated virus with other co-circulating H5N1 viruses from birds and mammals. Phylogenetic
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RevertAidTM H Minus First Strand cDNA synthesis kit (Cat#K1611, Fermentas, EU) following
comparison of the aligned sequences was generated using neighbour-joining method employing the Kimura 2-parameter correction in MEGA version 5 (www.megasoftware.net). The reliability of internal branches was assessed by 1000 bootstrap replications and p-distance substitution model. The H5 numbering used throughout the study was based on the alignment with A/Goose/Guangdong/1/96 (H5N1) minus the 16 amino acids known as HA signal peptide.
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Chicken intravenous pathogenicity test. Pathogenicity test was performed and the intravenous pathogenicity index (IVPI) of the virus in chickens was determined according to the guidelines in the OIE manual (OIE, 2012).
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Pathogenicity and transmissibility of AIV isolated from naturally infected pigeons. To
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determine if the isolated H5N1 virus would produce infection under experimental condition, a
subtype were inoculated oronasally with 0.1 ml of virus fluid containing 106 EID50. Six pigeons
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were added as sentinels into the pigeons’ aviary to detect transmissibility. The sentinel pigeons had direct contact to the inoculated pigeons’ feces besides a common source of drinking water. Additionally, three sham inoculated control pigeons were housed separately from the other birds as a negative control group. All pigeons were monitored two or three times daily for a total of 14 days and clinical signs were recorded. Oropharyngeal and cloacal swabs were taken from
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pigeons at 3, 5 and 7 days post infection (dpi) for virus re-isolation and titration. The experimental studies were permitted by the Committee for Community Service and Environment
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Development, Zagazig University, Egypt.
Pathology. Pigeons were euthanized at 14 dpi, necropsied and internal organs were sampled for virus re-isolation. Selected organs were fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin (Prophet et al., 1994). Paraffin blocks were sectioned in duplicates at 5µm and routinely stained by hematoxylin and eosin (HE). Consecutive sections were stained by immunohistochemistry (IHC) to determine the AIV antigen distribution in
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group of 10 healthy six weeks old domestic pigeons and serologically negative for AIV H5N1
tissues. A monoclonal antibody against the influenza A virus nucleoprotein (ATCC HB-65 influenza A hybridoma cell line) was used as the primary antibody. Specific antigen-antibody reactions were visualized by 3,3´diaminobenzidine tetrahydrochloride treatment using the Dako EnVision system and counterstained with hematoxylin. Positive results appeared as brown precipitate localized at the site of binding (Perkins & Swayne, 2001).
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Virus re-isolation and identification. The collected tissue and swab samples were prepared and inoculated into 10 days old SPF-ECEs via allantoic cavity for virus isolation (OIE, 2012). Infectivity titres were calculated by the method of Reed and Muench (1938). Viral RNA was extracted and RT-PCR was performed to detect the presence of avian influenza virus as
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Nucleotide sequence accession number. The nucleotide sequence obtained from this study is available at GenBank under accession number JX965419.
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Results
Detection and isolation of AIV. Necropsied naturally infected pigeons showed congestion of
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the internal organs, particularly lungs and brain. The isolated virus in this study, designated as A/Pigeon/Egypt/SHAH-5803/2011 and determined as H5N1 by RT-PCR, caused embryo deaths within 3 dpi and showed a titer of 1/32 HA unit.
Molecular and biological feature of Pigeon AIV. Molecular characterization of the pigeon
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result of RT-PCR as described by Pang et al. (2002).
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previously described. NDV infection was excluded in HA positive allantoic fluids by negative
AIV isolate was performed by HA gene sequencing and nucleotide BLAST analysis. The Egyptian pigeon strain possessed multi-basic amino acids at the connecting peptide between HA1 and HA2, (PQGERRRKKR/GLF), typical for HPAIV of clade 2.2, as well as glutamine
and glycine (Q222–G224) at the receptor binding site besides and the presence of amino acid D387 (H5 HA numbering), characteristic to sub-clade 2.2.1. The HA gene of the investigated pigeon isolate revealed that it belonged to the 5J lineage according to the Influenza A Virus Genotype Tool (Lu et al., 2007).
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Virulence of A/Pigeon/Egypt/SHAH-5803/2011 was evaluated through intravenous challenge of chickens. Mortality in chickens was 100% within 48h. Chickens showed signs of depression, anorexia, edema and hemorrhages in the comb and shanks and demonstrated an
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intravenous pathogenicity index of 2.7.
99.4% with Egyptian AIV H5N1 subtypes isolated from birds and mammals respectively. The
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HA gene of the pigeon AIV was phylogenetically closely related to viruses in 2.2.1/C group
isolated from backyard chickens, ducks and human (Figure 1). A deletion within the receptor binding site at position 129S and additional seven amino acid substitutions (D43N, S120D, I151T, D154N, N155A, R162K and G272S) of the viral H5 protein were found in comparison to
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H5N1 virus introduced into Egypt in 2006 (Index/2006).
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Experimental infection
Pathology. Infected pigeons showed congested beak, conjunctivitis, slight depression at 2 dpi (3/10). At 4 dpi, severe neurologic signs consisting of torticollis (Figure 2A), incoordination, tremors, and wing paralysis followed by death at the 5th dpi were seen (1/10). Greenish diarrhea
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Phylogenetic analysis. The pigeon sequence had a nucleotide similarity of 95.2-99.6% and 97.6-
was seen at 7 dpi (3/10). No clinical signs were observed in the sham-inoculated control pigeons. Gross examination revealed a soft and friable brain tissue, besides congested meningeal
blood vessels (Figure 2B). The lungs appeared edematous and severely hemorrhagic (infected pigeon at 5 dpi) (Figure 2C). Subepicardial and petechial hemorrhages on the coronary fat were observed. Histologically, the liver showed focal aggregations of leukocytes in the portal areas
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and congestion of the blood vessels. Pulmonary edema, congestion of pulmonary blood vessels and occlusion of the alveolar spaces with erythrocytes were observed (Figure 2D). The brain suffered from vacuolation, spongiosis, degenerated neurons, microglial cell infiltrations and perivascular lymphocytic cuffing besides, congested blood vessels of the meninges (Figure 2E).
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Sporadic moderate positive staining of the pneumocytes, interstitial tissue and few intravascular
and intestinal tract as well. The sham-inoculated control pigeons showed no lesions and
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remained negative on immunostaining.
Virus excretion and transmission kinetics. Systemic infection through 5 dpi was confirmed in the infected pigeons. The virus titers ranged from 103.75 EID50/g to 104.5 EID50/g. Moreover, the
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virus was isolated from the brain and intestinal tissues of the sentinel pigeons at 14 dpi. Oropharyngeal and cloacal swabs were taken from pigeons to detect the virus shedding. The
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virus was re-isolated from collected swabs, with titers ranging from 101.6 to 103.16 EID50/ml and from 101.17 to 103.1 EID50/ml in infected and sentinel pigeons respectively (Figure 3). Swabs and tissue samples of sham-inoculated control pigeons were negative.
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mononuclear cells of the lungs were observed (Figure 2F). Viral antigens were detected in brain
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Discussion
Avian influenza H5 subtype viruses have caused destructive outbreaks in domestic poultry and several human infections raising the fear of a pandemic. In the present study, a H5N1 virus was
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isolated and identified from respiratory samples of naturally infected pigeons in Sharkia
virus clusters into group 2.2.1/C isolated from backyard birds, live bird market and human
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(Hafez et al., 2010; Abdelwhab et al., 2010). Since pigeons commonly reside with humans on
farms in Egypt, their role as potential carriers for H5N1 virus should not be neglected. Being the most abundant birds on backyard poultry farms, pigeons can get H5N1 virus infection during feeding when intermingle freely with domestic and aquatic birds.
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A major molecular determinant for pathogenicity of H5 and H7 viruses is the amino acid sequence specifying the proteolytic-cleavage site of HA. The HA mediates the attachment of the
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virus to sialic-acid-containing receptors and the fusion of the virus envelope with the endosomal membrane (Rott et al., 1995; Katz et al., 2000). The HA molecule of the pigeon isolate contains amino acid residues 222Q and 224G at the receptor-binding site, which determines the binding of HA molecule to avian-specific sialic acid α-2,3-galactose receptors (Matrosovich et al., 1999). Previously, it was proposed that isolates having isoleucine at residue 151 of the HA1 protein
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Province, Egypt, during 2011. Based on the phylogenetic analysis of the HA gene sequences, the
might be involved in different receptor binding (Claas et al., 1998). Recently, it was shown that deletion of RBD 129S combined with I151T increased α2-6 SA binding (Watanabe et al., 2011). It was observed that the pigeon strain isolated in this study has these substitutions. In addition,
specific amino acids positions Y94, S132, W149, H179, E186, K189, L190, E212, P217, K218, G221, Q222, S223, G224 (H5 influenza numbering) that are implicated in receptor specificity
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(Stevens et al., 2006), were further investigated. Interestingly, the substitutions at positions 94 (Y to N), 189 (K to R), 212 (E to K) and 217 (P to S) which were found in the examined pigeon isolate have also been observed in the HA gene sequence of A/equine/Egypt/av1/2009 isolated from donkeys in Egypt (Abdel-Moneim et al., 2010).
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The present study also demonstrates the ability of an Egyptian-origin HPAIV to replicate
transmitted to contact birds. These findings are different with previous research that had shown
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that pigeons were mostly resistant and probably played a minimal epidemiological role in the dissemination of the H5N1 Hong Kong-origin influenza viruses (Perkins & Swayne, 2002). Similar results were reported by Klopfleisch et al., (2006) in pigeons experimentally infected with a high infective dose (108 EID50) of HPAIV (A/chicken/Indonesia/2003), indicating that
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different H5N1 virus have distinctive biological properties. In this study, the aggressive outbreak recorded in the naturally infected pigeons could not be reproduced experimentally. This may also
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be attributed to co-infection with other pathogens, bad hygiene and/or stress of flying. Thus, our study further indicate that HPAI H5N1 viruses have established infection and pathogenicity among various aquatic and wild birds, suggesting that the viruses are continuously evolving compared to previously endemic HPAI viruses (Ellis et al., 2004; Chen et al., 2006). In experiments with a HPAI H5N1 virus in gallinaceous birds, the virus produced a
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in experimentally infected pigeons, inducing 30% morbidity and 10% mortality, and to be
fulminating and rapidly fatal systemic disease (Perkins & Swayne, 2001). The results from experimental infection performed in this study using oronasal inoculation in pigeons were similar to those found in previous study, with regards to systemic replication. The virus reached a titer of
104.5 EID50/g in the brains of experimentally infected pigeons, however, only one pigeon showed neurological manifestations and histologically showed severe vacuolisation, spongiosis and
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neuronal degeneration. A pronounced neurotropism of HPAIV A/chicken/Indonesia/2003 (H5N1) for the cerebrum and brainstem of pigeons was recorded (Klopfleisch et al., 2006). Moreover, our data showed that pigeons excreted infectious viruses via the oral or cloacal routes. In contrast to previous report (Werner et al., 2007), the virus was able to trigger an infection in
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contact non-inoculated pigeons. Strikingly, seventy percent of the infected pigeons had
virus silently and act as reservoir.
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In conclusion, this study is the first to report entire HA gene sequence of H5N1 virus
isolated from naturally infected pigeons in Egypt. The results of this study revealed that H5N1 HPAI viruses are continually evolving and changing their pathogenicity to be able to replicate and shed from infected pigeons consequently increasing the risk of transmission to other avian
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species and humans and emphasizing the importance of full genome sequencing of pigeon H5N1 isolates. Knowledge about the circulating genomes might increase the understanding about the
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spread and mechanisms of virulence and pathogenicity of H5N1 viruses to poultry and human. Further surveys, particularly continuing surveillance of pigeons, are necessary.
Acknowledgments
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considerable virus titer in their tissues and excreta without clinical signs and can thus pass the
We would like to thank the Department of Veterinary and Biomedical Sciences, Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, USA for supporting the material and equipment used in IHC staining and Prof. Dr. Christopher C.L Chase, SDSU, USA for his valuable scientific revision of the manuscript.
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Perkins, L.E.L. & Swayne, D.E. (2001). Pathobiology of A/chicken/Hong Kong/220/97 (H5N1) avian influenza virus in seven gallinaceous species. Veterinary Pathology, 38, 149-164.
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Perkins, L.E. & Swayne, D.E. (2002). Pathogenicity of a Hong Kong origin H5N1 highly pathogenic avian influenza virus for emus, geese, ducks, and pigeons. Avian Diseases, 46, 53-63. Prophet, E.B., Mills, B., Arrington, J.B. & Sobin, L.H. (1994). Laboratory methods in
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histotechnology. In: Armed Forces Institute of Pathology. American Registry of Pathology,
Reed, L. & Muench, H. (1938). A simple method of estimating fifty percent endpoints. American
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Journal of Hygiene, 27, 493-497.
Rott, R., Klenk, H.D., Nagai, Y. & Tashiro, M. (1995). Influenza viruses, cell enzymes, and pathogenicity. American Journal of Respiratory and Critical Care Medicine, 152, S16-S19. Smietanka, K., Minta, Z., Wyrostek, K., Józwiak, M., Olszewska, M., Domanska-Blicharz, A.K.,
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Reichert, A.M., Pikuła, A., Habyarimana, A. & van den Berg, T. (2011). Susceptibility of Pigeons to Clade 1 and 2.2 High Pathogenicity Avian Influenza H5N1 Virus. Avian
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Diseases, 55, 106-112.
Songserm, T., Amonsin, A., Jam-on, R., Sae-Heng, N., Meemak, N., Pariyothorn, N., Payungporn, S., Theamboonlers, A. & Poovorawan, Y. (2006). Avian influenza H5N1 in naturally infected domestic cats. Emerging Infectious Diseases, 12, 681-683. Stevens, J., Blixt, O., Tumpey, T.M., Taubenberger, J.K., Paulson, J.C. & Wilson, I.A. (2006).
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Structure and receptor specificity of the hemagglutinin from an H5N1 influenza virus. Science, 312, 404-410.
Swayne, D.E. & Suarez, D.L. (2000). Highly pathogenic avian influenza. Revue scientifique et technique (International Office of Epizootics), 19, 463-482.
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Watanabe, Y., Ibrahim, M.S., Ellakany, H.F., Kawashita, N., Mizuike, R., Hiramatsu, H., Sriwilaijaroen, N., Takagi, T., Suzuki, Y. & Ikuta, K. (2011). Acquisition of human-type receptor binding specificity by new H5N1 influenza virus sublineages during their emergence in birds in Egypt. PLoS Pathogens, 7:e1002068.
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Werner, O., Starick, E., Teifke, J., Klopfleisch, R., Prajitno, T.Y., Beer, M., Hoffmann, B. &
A/chicken/Indonesia/2003 (H5N1) from experimentally infected domestic pigeons
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88, 3089-3093.
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(Columbia livia) and lack of transmission to sentinel chickens. Journal of General Virology,
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Harder, T.C. (2007). Minute excretion of highly pathogenic avian influenza virus
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Figure 1. Phylogenetic relationship of Pigeon AIV, Egypt 2011 to other AIV H5N1 sequences available in GenBank. The tree was constructed on the basis of 1594 nucleotide sequence of the HA gene using the neighbor-joining method and the Kimura-2-parameter model in MEGA5 (www.megasoftware.net). Bootstrap values of 1000 resampling in percent (>70%) are indicated at key nodes. The virus isolated in this study is marked with solid square.
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Figure 2. Experimentally infected pigeon at 5 dpi. (A) Severe neurologic signs consisting of torticollis and wing paralysis. (B) Brain showing severely congested meningeal blood vessels. (C) Lungs showing edema and hemorrhage. (D) Pulmonary hemorrhage represented by occlusion of the alveolar spaces with erythrocytes. HE bar: 50µm. (E) Brain showing vacuolation, spongiosis, degenerated neurons (arrow heads), microglial cell infiltrations (arrows) and perivascular lymphocytic cuffing. HE bar: 50µm. (F) Immunostaining revealed sporadic moderate positive staining of the pneumocytes (asterisk), interstitial tissue and few intravascular mononuclear cells of the lungs (arrows). IHC bar: 50µm.
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Figure 3. Viral titers in oropharyngeal and cloacal swabs collected from infected and contact pigeons at 3, 5 and 7 dpi. The figure represents the mean value of virus titer (Log 10 EID50/ml) in swabs collected from 3 birds ± standard errors.
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Avian Pathology Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/cavp20
Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons in Egypt a
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Shimaa M.G. Mansour , Reham M. ElBakrey , Haytham Ali , David E.B. Knudsen & Amal A.M. Eid a
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Department of Virology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt
b
Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt c
Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt d
Department of Veterinary and Biomedical Sciences, Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, SD 57007, USA Accepted author version posted online: 27 May 2014.Published online: 27 May 2014.
To cite this article: Shimaa M.G. Mansour, Reham M. ElBakrey, Haytham Ali, David E.B. Knudsen & Amal A.M. Eid (2014): Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons in Egypt, Avian Pathology, DOI: 10.1080/03079457.2014.926002 To link to this article: http://dx.doi.org/10.1080/03079457.2014.926002
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Publisher: Taylor & Francis & Houghton Trust Ltd Journal: Avian Pathology
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Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons in Egypt
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Shimaa M.G. Mansour1, Reham M. ElBakrey2, Haytham Ali3, David E.B.
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Knudsen4 and Amal A.M. Eid2*
Department of Virology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511,
Egypt 2 Department of Avian and Rabbit Medicine, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511, Egypt 3 Department of Pathology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, 44511, Egypt 4 Department of Veterinary and Biomedical Sciences,
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DOI: http://dx.doi.org/10.1080/03079457.2014.926002
Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, 57007, USA
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Short Title: Avian influenza virus H5N1 in Pigeon in Egypt
* Corresponding author: Amal A.M. Eid E-mail address: [email protected] Telephone number: +201001747082
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Fax number: +20552284383
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Abstract
The highly pathogenic avian influenza virus (HPAIV) subtype H5N1 threatens animal and human health worldwide. Susceptibility of pigeons to HPAIV (H5N1) and their role in AIV transmission to domestic birds and humans remain questionable. In this study, an outbreak in domestic pigeons (1-18 months old) with 50% mortality was investigated. Pigeons exhibited
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nervous manifestations and greenish diarrhea. Necropsy of the naturally infected pigeons revealed congestion of the internal organs, particularly lungs and brain. A HPAIV subtype H5N1 designated A/Pigeon/Egypt/SHAH-5803/2011 was isolated from 40 days old pigeon. Sequencing
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of the hemagglutinin gene showed close relation to viruses in group 2.2.1/C. Intravenous inoculation of the isolate in chickens induced 100% mortality within 2 days postinoculation (dpi) and intravenous pathogenicity index was 2.7. Virus pathogenicity and transmissibility was determined experimentally in six-week-old domestic pigeons. Thirty percent of pigeons inoculated oronasally with 106 EID50 showed congested beak, conjunctivitis, depression and greenish diarrhea. A mortality rate of 10% was recorded preceded by severe neurologic signs consisting of torticollis, incoordination, tremors, and wing paralysis. Pathological examination
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Received: 11 April 2014
revealed a friable brain tissue and congested meningeal blood vessels. The lungs appeared edematous and severely hemorrhagic. Subepicardial and petechial hemorrhages on the coronary fat were observed. Both infected and contact pigeons shed virus via the oropharynx and cloaca. To our knowledge, this is the first description and characterization of HPAIV in naturally infected pigeons in Egypt. Our findings reveal that pigeons can indeed be susceptible to H5N1
HPAI viruses and could be a source of infection to other birds and human.
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Introduction
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Influenza A viruses belong to the family Orthomyxoviridae and have an enveloped segmented,
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single-stranded, and negative-sense RNA genome. Influenza A viruses of avian origin can be
hemagglutinin (H) and 9 different types of neuraminidase (N) (Fouchier et al., 2005). Avian
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influenza viruses are classified into two groups, highly pathogenic and low pathogenic based on the severity of the disease they cause in chickens (Swayne & Suarez, 2000). Highly pathogenic avian influenza viruses (HPAIV), which code for a furin-sensitive cleavage site in their HA
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protein are capable of inducing systemic infections with dramatic losses in poultry industry, particularly chickens and turkeys (Alexander, 1993).
The HPAI, an imperative transboundary disease poses a threat to both animal and public
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health. Controversial reports exist about the pigeon’s susceptibility to HPAIV. Their role as a link between wild and domestic birds as well as transmission and spread of HPAIV during epizootics over long distances is not yet clear (Kaleta & Hönicke, 2004). Although Asian lineage H5N1 HPAIV has been isolated from pigeons (Ellis et al., 2004), the existing evidence indicates that pigeons are only minimally susceptible or resistant to different subtypes of AIV infections and do not serve as efficient transmission host, even in the presence of immune dysfunction
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categorized into subtypes based on the two surface glycoproteins, 16 different types of
(Fang et al., 2006) like immunosuppression by cyclophosphamide (Panigrahy et al., 1996; Perkins & Swayne, 2002; Kaleta & Hönicke, 2004; Liu et al., 2007; Smietanka et al., 2011).
They probably played a minimal epidemiologic role in the perpetuation of the H5N1 Hong Kong-origin influenza viruses (Perkins & Swayne, 2002). Other experimental studies
demonstrated that pigeons are susceptible to highly pathogenic influenza viruses (Klopfleisch et al., 2006; Werner et al., 2007; Jia et al., 2008; Smietanka et al., 2011) and could be a source of infection to other animals (Songserm et al., 2006).
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The susceptibility of pigeons to H5N1 virus and their role in AIV transmission to domestic birds and humans remain questionable. Here we report for the first time in Egypt a case of natural infection with highly pathogenic avian influenza virus H5N1 in pigeons.
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Characterization, pathogenicity and transmissibility of the field AIV isolated from pigeon were
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Materials and methods
Samples and Virus isolation. In March 2011, two dead pigeon squabs (40 days old) were submitted to the Clinic of Avian and Rabbit Medicine Department, Faculty of Veterinary
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Medicine, Zagazig University, Egypt. According to the owner complain, pigeons exhibited greenish diarrhea, nervous manifestations and sudden deaths with mortality rate up to 50%
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within 10 days. The flock consisted of 50 non-vaccinated birds of variable ages (1-18 months) reared in backyard free range system. Pigeons were necropsied and respiratory pool samples (lung and trachea) were collected and injected into 10 days old specific pathogen free (SPF embryonated chicken eggs (ECE) via the allantoic route according to recommendations of the OIE (OIE, 2012). The collected allantoic fluid from dead embryos was screened by rapid
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investigated.
hemagglutination (HA) test. The infected allantoic fluid was then tested for the presence of AIV
by RT-PCR and further sequenced. Newcastle disease virus (NDV) infection was excluded in
HA positive allantoic fluids by negative results of both the Newcastle Disease Virus Antigen Rapid Test (Shenzhen Combined Biotech Co. Ltd, Shenzhen, China) and RT-PCR as described by Pang et al. (2002).
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RNA extraction and RT-PCR. Viral RNA was extracted from infected allantoic fluid using Gene JETTM RNA purification kit (Cat#K0731, Fermentas, EU) according to the manufacturer’s
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instructions. Five microlitres of extracted RNA was reverse transcribed to cDNA using
the manufacturer’s instructions. The PCR was performed with a set of primers specific for H5
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and N1 gene segments as described elsewhere (Njouom et al., 2008).
Viral sequencing and phylogenetic analysis. For characterization of the isolated virus, the HA gene was analyzed by PCR using a set of HA gene specific primers (Njouom et al., 2008). The PCR product was gel purified using Gene JETTM Gel Extraction Kit (Cat#K0691, Fermentas,
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EU) as recommended by the manufacturer and sequenced on both strands using the amplification primers (Solgent Co. ltd. Korea). The HA subtype was further identified by nucleotide BLAST
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(http://www.ncbi.nlm.nih.gov/BLAST) and submitted to GenBank. The obtained nucleotide sequence was aligned with other HA gene sequences available in GenBank and comparative alignment was performed by ClustalW method using MegAlign module of DNAStar software (Lasergene version 7.2, DNASTAR, Madison, WI, USA). Phylogenetic analysis was carried out on the HA gene region encompassing 1594 nucleotides. Forty-eight reference sequences retrieved from the GenBank database were used in this study to understand the relationship of the isolated virus with other co-circulating H5N1 viruses from birds and mammals. Phylogenetic
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RevertAidTM H Minus First Strand cDNA synthesis kit (Cat#K1611, Fermentas, EU) following
comparison of the aligned sequences was generated using neighbour-joining method employing the Kimura 2-parameter correction in MEGA version 5 (www.megasoftware.net). The reliability of internal branches was assessed by 1000 bootstrap replications and p-distance substitution model. The H5 numbering used throughout the study was based on the alignment with A/Goose/Guangdong/1/96 (H5N1) minus the 16 amino acids known as HA signal peptide.
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Chicken intravenous pathogenicity test. Pathogenicity test was performed and the intravenous pathogenicity index (IVPI) of the virus in chickens was determined according to the guidelines in the OIE manual (OIE, 2012).
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Pathogenicity and transmissibility of AIV isolated from naturally infected pigeons. To
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determine if the isolated H5N1 virus would produce infection under experimental condition, a
subtype were inoculated oronasally with 0.1 ml of virus fluid containing 106 EID50. Six pigeons
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were added as sentinels into the pigeons’ aviary to detect transmissibility. The sentinel pigeons had direct contact to the inoculated pigeons’ feces besides a common source of drinking water. Additionally, three sham inoculated control pigeons were housed separately from the other birds as a negative control group. All pigeons were monitored two or three times daily for a total of 14 days and clinical signs were recorded. Oropharyngeal and cloacal swabs were taken from
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pigeons at 3, 5 and 7 days post infection (dpi) for virus re-isolation and titration. The experimental studies were permitted by the Committee for Community Service and Environment
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Development, Zagazig University, Egypt.
Pathology. Pigeons were euthanized at 14 dpi, necropsied and internal organs were sampled for virus re-isolation. Selected organs were fixed in 10% neutral buffered formalin, routinely processed and embedded in paraffin (Prophet et al., 1994). Paraffin blocks were sectioned in duplicates at 5µm and routinely stained by hematoxylin and eosin (HE). Consecutive sections were stained by immunohistochemistry (IHC) to determine the AIV antigen distribution in
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group of 10 healthy six weeks old domestic pigeons and serologically negative for AIV H5N1
tissues. A monoclonal antibody against the influenza A virus nucleoprotein (ATCC HB-65 influenza A hybridoma cell line) was used as the primary antibody. Specific antigen-antibody reactions were visualized by 3,3´diaminobenzidine tetrahydrochloride treatment using the Dako EnVision system and counterstained with hematoxylin. Positive results appeared as brown precipitate localized at the site of binding (Perkins & Swayne, 2001).
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Virus re-isolation and identification. The collected tissue and swab samples were prepared and inoculated into 10 days old SPF-ECEs via allantoic cavity for virus isolation (OIE, 2012). Infectivity titres were calculated by the method of Reed and Muench (1938). Viral RNA was extracted and RT-PCR was performed to detect the presence of avian influenza virus as
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Nucleotide sequence accession number. The nucleotide sequence obtained from this study is available at GenBank under accession number JX965419.
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Results
Detection and isolation of AIV. Necropsied naturally infected pigeons showed congestion of
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the internal organs, particularly lungs and brain. The isolated virus in this study, designated as A/Pigeon/Egypt/SHAH-5803/2011 and determined as H5N1 by RT-PCR, caused embryo deaths within 3 dpi and showed a titer of 1/32 HA unit.
Molecular and biological feature of Pigeon AIV. Molecular characterization of the pigeon
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result of RT-PCR as described by Pang et al. (2002).
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previously described. NDV infection was excluded in HA positive allantoic fluids by negative
AIV isolate was performed by HA gene sequencing and nucleotide BLAST analysis. The Egyptian pigeon strain possessed multi-basic amino acids at the connecting peptide between HA1 and HA2, (PQGERRRKKR/GLF), typical for HPAIV of clade 2.2, as well as glutamine
and glycine (Q222–G224) at the receptor binding site besides and the presence of amino acid D387 (H5 HA numbering), characteristic to sub-clade 2.2.1. The HA gene of the investigated pigeon isolate revealed that it belonged to the 5J lineage according to the Influenza A Virus Genotype Tool (Lu et al., 2007).
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Virulence of A/Pigeon/Egypt/SHAH-5803/2011 was evaluated through intravenous challenge of chickens. Mortality in chickens was 100% within 48h. Chickens showed signs of depression, anorexia, edema and hemorrhages in the comb and shanks and demonstrated an
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intravenous pathogenicity index of 2.7.
99.4% with Egyptian AIV H5N1 subtypes isolated from birds and mammals respectively. The
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HA gene of the pigeon AIV was phylogenetically closely related to viruses in 2.2.1/C group
isolated from backyard chickens, ducks and human (Figure 1). A deletion within the receptor binding site at position 129S and additional seven amino acid substitutions (D43N, S120D, I151T, D154N, N155A, R162K and G272S) of the viral H5 protein were found in comparison to
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H5N1 virus introduced into Egypt in 2006 (Index/2006).
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Experimental infection
Pathology. Infected pigeons showed congested beak, conjunctivitis, slight depression at 2 dpi (3/10). At 4 dpi, severe neurologic signs consisting of torticollis (Figure 2A), incoordination, tremors, and wing paralysis followed by death at the 5th dpi were seen (1/10). Greenish diarrhea
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Phylogenetic analysis. The pigeon sequence had a nucleotide similarity of 95.2-99.6% and 97.6-
was seen at 7 dpi (3/10). No clinical signs were observed in the sham-inoculated control pigeons. Gross examination revealed a soft and friable brain tissue, besides congested meningeal
blood vessels (Figure 2B). The lungs appeared edematous and severely hemorrhagic (infected pigeon at 5 dpi) (Figure 2C). Subepicardial and petechial hemorrhages on the coronary fat were observed. Histologically, the liver showed focal aggregations of leukocytes in the portal areas
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and congestion of the blood vessels. Pulmonary edema, congestion of pulmonary blood vessels and occlusion of the alveolar spaces with erythrocytes were observed (Figure 2D). The brain suffered from vacuolation, spongiosis, degenerated neurons, microglial cell infiltrations and perivascular lymphocytic cuffing besides, congested blood vessels of the meninges (Figure 2E).
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Sporadic moderate positive staining of the pneumocytes, interstitial tissue and few intravascular
and intestinal tract as well. The sham-inoculated control pigeons showed no lesions and
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remained negative on immunostaining.
Virus excretion and transmission kinetics. Systemic infection through 5 dpi was confirmed in the infected pigeons. The virus titers ranged from 103.75 EID50/g to 104.5 EID50/g. Moreover, the
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virus was isolated from the brain and intestinal tissues of the sentinel pigeons at 14 dpi. Oropharyngeal and cloacal swabs were taken from pigeons to detect the virus shedding. The
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virus was re-isolated from collected swabs, with titers ranging from 101.6 to 103.16 EID50/ml and from 101.17 to 103.1 EID50/ml in infected and sentinel pigeons respectively (Figure 3). Swabs and tissue samples of sham-inoculated control pigeons were negative.
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mononuclear cells of the lungs were observed (Figure 2F). Viral antigens were detected in brain
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Discussion
Avian influenza H5 subtype viruses have caused destructive outbreaks in domestic poultry and several human infections raising the fear of a pandemic. In the present study, a H5N1 virus was
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isolated and identified from respiratory samples of naturally infected pigeons in Sharkia
virus clusters into group 2.2.1/C isolated from backyard birds, live bird market and human
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(Hafez et al., 2010; Abdelwhab et al., 2010). Since pigeons commonly reside with humans on
farms in Egypt, their role as potential carriers for H5N1 virus should not be neglected. Being the most abundant birds on backyard poultry farms, pigeons can get H5N1 virus infection during feeding when intermingle freely with domestic and aquatic birds.
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A major molecular determinant for pathogenicity of H5 and H7 viruses is the amino acid sequence specifying the proteolytic-cleavage site of HA. The HA mediates the attachment of the
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virus to sialic-acid-containing receptors and the fusion of the virus envelope with the endosomal membrane (Rott et al., 1995; Katz et al., 2000). The HA molecule of the pigeon isolate contains amino acid residues 222Q and 224G at the receptor-binding site, which determines the binding of HA molecule to avian-specific sialic acid α-2,3-galactose receptors (Matrosovich et al., 1999). Previously, it was proposed that isolates having isoleucine at residue 151 of the HA1 protein
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Province, Egypt, during 2011. Based on the phylogenetic analysis of the HA gene sequences, the
might be involved in different receptor binding (Claas et al., 1998). Recently, it was shown that deletion of RBD 129S combined with I151T increased α2-6 SA binding (Watanabe et al., 2011). It was observed that the pigeon strain isolated in this study has these substitutions. In addition,
specific amino acids positions Y94, S132, W149, H179, E186, K189, L190, E212, P217, K218, G221, Q222, S223, G224 (H5 influenza numbering) that are implicated in receptor specificity
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(Stevens et al., 2006), were further investigated. Interestingly, the substitutions at positions 94 (Y to N), 189 (K to R), 212 (E to K) and 217 (P to S) which were found in the examined pigeon isolate have also been observed in the HA gene sequence of A/equine/Egypt/av1/2009 isolated from donkeys in Egypt (Abdel-Moneim et al., 2010).
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The present study also demonstrates the ability of an Egyptian-origin HPAIV to replicate
transmitted to contact birds. These findings are different with previous research that had shown
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that pigeons were mostly resistant and probably played a minimal epidemiological role in the dissemination of the H5N1 Hong Kong-origin influenza viruses (Perkins & Swayne, 2002). Similar results were reported by Klopfleisch et al., (2006) in pigeons experimentally infected with a high infective dose (108 EID50) of HPAIV (A/chicken/Indonesia/2003), indicating that
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different H5N1 virus have distinctive biological properties. In this study, the aggressive outbreak recorded in the naturally infected pigeons could not be reproduced experimentally. This may also
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be attributed to co-infection with other pathogens, bad hygiene and/or stress of flying. Thus, our study further indicate that HPAI H5N1 viruses have established infection and pathogenicity among various aquatic and wild birds, suggesting that the viruses are continuously evolving compared to previously endemic HPAI viruses (Ellis et al., 2004; Chen et al., 2006). In experiments with a HPAI H5N1 virus in gallinaceous birds, the virus produced a
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in experimentally infected pigeons, inducing 30% morbidity and 10% mortality, and to be
fulminating and rapidly fatal systemic disease (Perkins & Swayne, 2001). The results from experimental infection performed in this study using oronasal inoculation in pigeons were similar to those found in previous study, with regards to systemic replication. The virus reached a titer of
104.5 EID50/g in the brains of experimentally infected pigeons, however, only one pigeon showed neurological manifestations and histologically showed severe vacuolisation, spongiosis and
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neuronal degeneration. A pronounced neurotropism of HPAIV A/chicken/Indonesia/2003 (H5N1) for the cerebrum and brainstem of pigeons was recorded (Klopfleisch et al., 2006). Moreover, our data showed that pigeons excreted infectious viruses via the oral or cloacal routes. In contrast to previous report (Werner et al., 2007), the virus was able to trigger an infection in
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contact non-inoculated pigeons. Strikingly, seventy percent of the infected pigeons had
virus silently and act as reservoir.
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In conclusion, this study is the first to report entire HA gene sequence of H5N1 virus
isolated from naturally infected pigeons in Egypt. The results of this study revealed that H5N1 HPAI viruses are continually evolving and changing their pathogenicity to be able to replicate and shed from infected pigeons consequently increasing the risk of transmission to other avian
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species and humans and emphasizing the importance of full genome sequencing of pigeon H5N1 isolates. Knowledge about the circulating genomes might increase the understanding about the
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spread and mechanisms of virulence and pathogenicity of H5N1 viruses to poultry and human. Further surveys, particularly continuing surveillance of pigeons, are necessary.
Acknowledgments
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considerable virus titer in their tissues and excreta without clinical signs and can thus pass the
We would like to thank the Department of Veterinary and Biomedical Sciences, Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Brookings, USA for supporting the material and equipment used in IHC staining and Prof. Dr. Christopher C.L Chase, SDSU, USA for his valuable scientific revision of the manuscript.
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Figure 1. Phylogenetic relationship of Pigeon AIV, Egypt 2011 to other AIV H5N1 sequences available in GenBank. The tree was constructed on the basis of 1594 nucleotide sequence of the HA gene using the neighbor-joining method and the Kimura-2-parameter model in MEGA5 (www.megasoftware.net). Bootstrap values of 1000 resampling in percent (>70%) are indicated at key nodes. The virus isolated in this study is marked with solid square.
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Figure 2. Experimentally infected pigeon at 5 dpi. (A) Severe neurologic signs consisting of torticollis and wing paralysis. (B) Brain showing severely congested meningeal blood vessels. (C) Lungs showing edema and hemorrhage. (D) Pulmonary hemorrhage represented by occlusion of the alveolar spaces with erythrocytes. HE bar: 50µm. (E) Brain showing vacuolation, spongiosis, degenerated neurons (arrow heads), microglial cell infiltrations (arrows) and perivascular lymphocytic cuffing. HE bar: 50µm. (F) Immunostaining revealed sporadic moderate positive staining of the pneumocytes (asterisk), interstitial tissue and few intravascular mononuclear cells of the lungs (arrows). IHC bar: 50µm.
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Figure 3. Viral titers in oropharyngeal and cloacal swabs collected from infected and contact pigeons at 3, 5 and 7 dpi. The figure represents the mean value of virus titer (Log 10 EID50/ml) in swabs collected from 3 birds ± standard errors.
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