Shimaa M. G. Mansour

Natural infection with highly pathogenic avian influenza virus H5N1 in domestic pigeons (Columba livia) in Egypt

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 avian influenza virus transmission to domestic birds and humans remain questionable. In this study, an outbreak in domestic pigeons (1 to 18 months old) with 50% mortality was investigated. Pigeons exhibited nervous manifestations and greenish diarrhoea. Necropsy of the naturally infected pigeons revealed congestion of the internal organs, particularly the lungs and brain. The HPAIV subtype H5N1 designated A/Pigeon/Egypt/SHAH-5803/2011 was isolated from a 40-day-old pigeon. Sequencing of the haemagglutinin gene showed it to be closely related to viruses in group 2.2.1/C. Intravenous inoculation of the isolate in chickens induced 100% mortality within 2 days post inoculation and the intravenous pathogenicity index was 2.7. Virus pathogenicity and transmissibility was determined experimentally in 6-week-old domestic pigeons. Thirty per cent of pigeons inoculated oronasally with 106 median embryo infective dose showed congested beak, conjunctivitis, depression, and greenish diarrhoea. A mortality rate of 10% was recorded preceded by severe neurologic signs consisting of torticollis, incoordination, tremors, and wing paralysis. Pathological examination revealed a friable brain tissue and congested meningeal blood vessels. The lungs appeared oedematous and severely haemorrhagic. Subepicardial and petechial haemorrhages 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 HPAIVs and could be a source of infection to other birds and humans.

Loop-mediated isothermal amplification for diagnosis of 18 World Organization for Animal Health (OIE) notifiable viral diseases of ruminants, swine and poultry

Abstract Loop-mediated isothermal amplification (LAMP) is a simple, powerful state-of-the-art gene amplification technique used for the rapid diagnosis and early detection of microbial diseases. Many LAMP assays have been developed and validated for important epizootic diseases of livestock. We review the LAMP assays that have been developed for the detection of 18 viruses deemed notifiable of ruminants, swine and poultry by the World Organization for Animal Health (OIE). LAMP provides a fast (the assay often takes less than an hour), low cost, highly sensitive, highly specific and less laborious alternative to detect infectious disease agents. The LAMP procedure can be completed under isothermal conditions so thermocyclers are not needed. The ease of use of the LAMP assay allows adaptability to field conditions and works well in developing countries with resource-limited laboratories. However, this technology is still underutilized in the field of veterinary diagnostics despite its huge capabilities.

Pathogenicity of Highly Pathogenic Avian Influenza Virus H5N1 in Naturally Infected Poultry in Egypt.

Highly pathogenic avian influenza virus (HPAIV) H5N1 has been endemic in Egypt since 2006, and there is increasing concern for its potential to become highly transmissible among humans. Infection by HPAIV H5N1 has been described in experimentally challenged birds. However, the pathogenicity of the H5N1 isolated in Egypt has never been reported in naturally infected chickens and ducks. Here we report a 2013 outbreak of HPAIV H5N1 in commercial poultry farms and backyards in Sharkia Province, Egypt. The main symptoms were ecchymosis on the shanks and feet, cyanosis of the comb and wattles, subcutaneous edema of the head and neck for chickens, and nervous signs (torticollis) for ducks. Within 48-72 hrs of the onset of illness, the average mortality rates were 22.8-30% and 28.5-40% in vaccinated chickens and non-vaccinated ducks, respectively. Tissue samples of chickens and ducks were collected for analyses with cross-section immunohistochemistry and real-time RT-PCR for specific viral RNA transcripts. While viral RNA was detected in nearly all tissues and sera collected, viral nucleoprotein was detected almost ubiquitously in all tissues, including testis. Interestingly, viral antigen was also observed in endothelial cells of most organs in chickens, and clearly detected in the trachea and brain in particular. Viral nucleoprotein was also detected in mononuclear cells of various organs, especially pulmonary tissue. We performed phylogenetic analyses and compared the genomic sequences of the hemagglutinin (HA) and nonstructural proteins (NS) among the isolated viruses, the HPAIV circulated in Egypt in the past and currently, and some available vaccine strains. Further analysis of deduced amino acids of both HA and NS1 revealed that our isolates carried molecular determinants of HPAIV, including the multibasic amino acids (PQGERRRK/KR*GLF) in the cleavage site in HA and glutamate at position 92 (D92E) in NS1. This is the first report of the pathogenicity of the HPAIVH5N1 strain currently circulating in naturally infected poultry in Egypt, which may provide unique insights into the viral pathogenesis in HPAIV-infected chickens and ducks.

Cleavage site stability of Egyptian highly pathogenic avian influenza viruses in backyard chickens during 2009–2011

PURPOSE Two distinguishable subclades of H5N1 (classic and variant strains) are cocirculating among the poultry populations in Egypt despite the intensive vaccination programs. A study to investigate the genetic relationship between avian influenza virus (AIV) isolates from backyard chickens in Sharkia (2009-2011), subclades, and commercially available vaccines was carried out. METHODS Forty-eight suspected AIV infected birds were clinically examined and used for virus isolation followed by reverse transcription-polymerase chain reaction. Four H5N1 virus isolates were sequenced and analyzed. The intravenous pathogenicity index (IVPI) of three AIV isolates was determined. RESULTS Thirty-four hemagglutinating viral agents (30 AIV subtype H5N1 and 4 Newcastle disease virus) were detected. Both the nucleotide and amino acid sequence identities of four H5N1 virus isolates (SHZA-0412/2009, SHZA-0801/2010, SHMK-1903/2010, and SHAH-1403/2011) were high--98.4-99.7% and 100%, respectively--indicative of their genetic homogeneity. The hemagglutinin cleavage site characterization revealed the presence of multiple basic amino acids (-PQRERRRKKR/GL-) of the highly pathogenic phenotype. These results were supported by IVPI in chickens of 2.69-2.90. The similarity of our isolates with H5N1 AIV vaccine strains (93.9-95.1%) was higher than that with H5N2 strains (77.8-91.9%). The divergence of four sequences with classic and variant lineages is 2-2.7% and 2.3-3%, respectively, with two amino acid substitutions (A249P and N251Y). CONCLUSION Genetic characterization and IVPI data of backyard H5N1 isolates are indicative of a highly pathogenic avian influenza virus with hemagglutinin cleavage site constancy and two amino acids substitutions with Egyptian classic and variant lineages, suggesting a beginning of antigenic drift.

Co-circulation of paramyxo- and influenza viruses in pigeons in Egypt

ABSTRACT In recent years, avian influenza virus (AIV) and Newcastle disease virus (NDV) have caused large-scale outbreaks in many countries, including Egypt. The culling and vaccination strategies have failed to control both viruses in Egypt. In this study, we investigated the outbreaks of nervous manifestations and deaths in pigeons between 2013 and 2015. The H5N1 subtype of the highly pathogenic avian influenza virus and pigeon paramyxovirus-1, an antigenic variant of NDV, were found to be the cause; AIV and pigeon paramyxovirus-1 were isolated from 61.3% (19/31) and 67.8% (21/31) of tested pigeons, respectively. Co-infection with both viruses was detected in 51.6% of pigeons (16/31). The AIV sequences showed PQGEKRRKKR/GLF motif at the haemagglutinin gene cleavage site, which is typical of the highly pathogenic H5N1 subtype. The phylogenetic tree showed that the highly pathogenic avian influenza belonged to clade 2.2.1.2. The NDV sequences carried one of the three motifs, 112GKQGRL117, 112KRQKRF117 or 112RRQKRF117, at the fusion protein cleavage site and were classified as genotypes I, VI and II in NDV-class II, respectively. This indicated that different genotypes of NDV can circulate simultaneously among pigeons. Further analysis revealed the clustering of some sequences in sub-genotypes Ia and VIb.2. To the best of our knowledge, these sub-genotypes have not been previously reported from pigeons in Egypt. Our results should serve as a base for future studies on both viruses in Egypt.

Molecular characterization of circulating Foot and mouth disease virus (FMDV) serotype O topotype EA-3 and serotype A (African topotype) genotype IV in Egypt, 2016

In January-April 2016, cattle and buffalo farm owners and veterinarians reported clinical signs suggestive of foot and mouth disease virus (FMDV) outbreaks among non-vaccinated cattle and buffalo herds in Egypt. The clinical disease observed was either mild (small oral lesions and speedy recovery) or severe (extensive oral lesions and/or mortalities), and the form of the disease (either mild or severe) segregated by farm. This study aimed to confirm the presence of FMDV and to characterize the circulating strains associated with the outbreaks. Vesicular epithelia were collected from 41 animals representing 15 affected cattle and buffalo farms in five governorates (Behira, Cairo, Daqahlia, Giza and Ismailia), and tested by real time (rt) RT-PCR. Consequently, 92% (38/41) of examined samples were positive. Furthermore, the VP1 coding region of 60% (23/38) of positive specimens were amplified by RT-PCR and sequenced. The phylogenetic analysis identified two distinct strains characterized as serotype O topotype EA-3 and serotype A (African topotype) of genotype IV in the severe and mild disease forms, respectively. The newly identified strains clustered in distinct clades in the phylogenetic trees, indicating the likelihood of new incursions into Egypt. Those strains were most closely related to previously described Sudanese strains.

Co-infection of highly pathogenic avian influenza and duck hepatitis viruses in Egyptian backyard and commercial ducks

Abstract Highly pathogenic avian influenza (HPAI) H5N1 virus poses a major challenge to the poultry industry and human health in Egypt. Twenty one households and eight duck farms in Sharkia Province, Egypt were investigated for the presence of avian influenza virus (AIV) and/or duck hepatitis virus 1 (DHV-1). Mortality rates among the investigated farms and yards were, 18.9% (69/365) of native ducks, 60.9% (25/41) of Pekin ducks, 60.2% (6306/10473) of Muscovy ducks and 44.9% (1353/3015) of Mallard ducks. The RT-PCR revealed the circulation of HPAI-H5N1 virus (81/104) among the examined birds with a high percentage in Muscovy (83.7%) and Pekin (83.4%) ducks. Interestingly, co-infection of HPAI and DHV-1 viruses in three ducklings with age of 4–19 days was detected. Severe neurological signs with high mortality were observed in ducklings as early as 4 days of age. Influenza virus antigen was detected in the neurons and glial cells of the brain, hepatocytes, and the intestinal submucosal plexus. Although, genetic characterization of H5N1 isolates revealed HPAIV of clade 2.2.1.2, such increased mortalities and neurological signs regardless of the duck age might imply the natural selection of HPAI in ducks. Crucial monitoring of the disease situation in ducks is essential for the implementation of an effective prevention and control program.