Emergence of the novel infectious bursal disease virus variant in vaccinated poultry flocks in Egypt.

RESEARCH HIGHLIGHTS: New variant IBDV which emerged in Egypt clustered with Chinese nVarIBDV.nVarIBDV spread subclinically across a wide geographic area.Mutation at 321 represents capsid's most exposed part, a defining feature.Antigenically modified vvIBDV still circulating in Egypt with typical lesions.

Differential replication characteristic of reassortant avian influenza A viruses H5N8 clade 2.3.4.4b in Madin-Darby canine kidney cell.

Late in 2016, multiple reassortant highly pathogenic (HP) avian influenza virus (AIVs) H5N8 was detected. AIVs infect different isolated hosts with a specific viral tropism. In the current study, the whole genome of the Egyptian A/chicken/NZ/2022 was genetically characterized. The H5N8-A/Common-coot/Egypt/CA285/2016, A/duck/Egypt/SS19/2017 previously isolated in Egypt, and the recently circulating A/chicken/Egypt/NZ/2022 reassortant viruses' replication, pathogenicity, and viral load in comparison to the H5N1-Clade 2.2.1.2 were investigated on Madin-Darby canine kidney cell (MDCK), by using the cytopathic effect (CPE) percent and matrix-gene reverse transcription quantitative real-time polymerase chain reaction to compute the virus titer at various points in time. The A/chicken/Egypt/NZ/2022 virus was similar to the reassortant strain clade 2.3.4.4b discovered in farms in 2016. The 2 sub-groupings of hemagglutinin (HA) and neuraminidase (NA) genes were identified (I and II); the A/chicken/Egypt/NZ/2022 HA and NA genes were associated with subgroup II. The subgroup II of the HA gene was further divided into A and B owing to acquired specific mutations. The A/chicken/Egypt/NZ/2022 in our study was associated with subgroup B. The M, NS, PB1, and PB2 genes were shown to be clustered into clade 2.3.4.4b by full genome sequence analysis; however, the PA and NP genes were found to be associated with H6N2 viruses, which had particular mutations that improved viral virulence and mammalian transmission. The current results showed that the circulating H5N8 viruses were more variable than previous viruses analyzed in 2016 and 2017. Compared to other reassortant HPAI H5N8, and HPAI H5N1, the growth kinetics of A/chicken/Egypt/NZ/2022 had a high CPE without the addition of trypsin and the most viral copies with a significant difference (P < 0.01) in comparison to HPAI H5N8 and HPAI H5N1 reassortant viruses. Accordingly, the effective viral replication of A/chicken/Egypt/NZ/2022 in the MDCK than other viruses may play a factor in the spread and maintenance of specific reassortant H5N8 influenza virus in the field.

Emergence of Highly Pathogenic Avian Influenza A Virus (H5N1) of Clade 2.3.4.4b in Egypt, 2021-2022.

Wild migratory birds have the capability to spread avian influenza virus (AIV) over long distances as well as transmit the virus to domestic birds. In this study, swab and tissue samples were obtained from 190 migratory birds during close surveillance in Egypt in response to the recent outbreaks of the highly pathogenic avian influenza (HPAI) H5N1 virus. The collected samples were tested for a variety of AIV subtypes (H5N1, H9N2, H5N8, and H6N2) as well as other pathogens such as NDV, IBV, ILT, IBDV, and WNV. Among all of the tested samples, the HPAI H5N1 virus was found in six samples; the other samples were found to be negative for all of the tested pathogens. The Egyptian HPAI H5N1 strains shared genetic traits with the HPAI H5N1 strains that are currently being reported in Europe, North America, Asia, and Africa in 2021-2022. Whole genome sequencing revealed markers associated with mammalian adaption and virulence traits among different gene segments, similar to those found in HPAI H5N1 strains detected in Europe and Africa. The detection of the HPAI H5N1 strain of clade 2.3.4.4b in wild birds in Egypt underlines the risk of the introduction of this strain into the local poultry population. Hence, there is reason to be vigilant and continue epidemiological and molecular monitoring of the AIV in close proximity to the domestic-wild bird interface.

First isolation of influenza a subtype H5N8 in ostrich: pathological and genetic characterization.

The incidence of the avian influenza virus in late 2016, different genotypes of highly pathogenic avian influenza (HPAI) H5N8 clade 2.3.4.4b have been reported among different domestic and wild bird species. The virus became endemic in the poultry population, causing a considerable economic loss for the poultry industry. This study screened 5 ostrich farms suffering from respiratory signs and mortality rate of the avian influenza virus. A flock of 60-day-old ostriches with a mortality of 90% suffered from depression, loss of appetite, dropped production, and oculo-nasal discharges, with bleeding from natural orifices as a vent. This flock was found positive for avian influenza virus and subtypes as HPAI H5N8 virus. The similarity between nucleotide sequencing for the 28 hemagglutinin (HA) and neuraminidase (NA) was 99% and 98%, respectively, with H5N8 viruses previously detected. The PB2 encoding protein harbor a unique substitution in mammalian marker 627A, which has not been recorded before in previously sequenced H5N8 viruses. Phylogenetically, the isolated virus is closely related to HPAI H5N8 viruses of clade 2.3.4.4b. The detection of the HPAI H5N8 virus in ostrich is highly the need for continuous epidemiological and molecular monitoring of influenza virus spread in other bird species, not only chickens. Ostrich should be included in the annual SunAlliance, for the detection of avian influenza.

Selenium nanoparticles enhance the efficacy of homologous vaccine against the highly pathogenic avian influenza H5N1 virus in chickens.

A proper vaccination against avian influenza viruses in chicken can significantly reduce the risk of human infection. Egypt has the highest number of recorded humans highly pathogenic avian influenza (HPAI)-H5N1 infections worldwide despite the widespread use of homologous vaccines in poultry. Enhancing H5N1 vaccine efficacy is ultimately required to better control HPAI-H5N1. The aim of this study is to boost chicken immunity by combined with inactivated HPAI-H5N1 with selenium nanoparticles (SeNPs). The chickens groups 1-3 were fed diets supplemented with SeNPs concentrations (0.25, 0.5, and 1 mg/kg) for 3 weeks and then vaccinated (inactivated HPAI-H5N1). while groups 4,5 and 6 were fed with SeNPs free diets and administered with 0.5 ml of the vaccine combined with 0.02, 0.06, and 0.1 mg/dose of SeNPs and then all groups were challenged with homologous virus 3 weeks post-vaccination (WPV). Group 7, 8 were used as control positive and negative respectively. At 4, 5, and 6 WPV, antibody titer was considerably higher in the group fed a meal supplemented with 1 mg SeNPs/kg. In contrast, both methods of SeNPs supplementation significantly increased the Interleukin 2 (IL2), Interleukin 6 (IL6), and Interferon γ (IFNγ) expressions in the blood cells in a dose-dependent manner, with a higher expression observed in the group that was vaccinated with 0.1 mg/dose. After the challenge, all groups that received SeNPs via diet or vaccines dose showed significant reduction in viral shedding and milder inflammation in lung, trachea, spleen, and liver in addition to higher expression of IL2, IL6, and IFNγ, with the highest expression observed in the group that was vaccinated with 0.1 mg/dose compared the plain vaccinated group. The groups of 1 mg SeNPs/kg and combined vaccinated with 0.1 mg/dose showed the best vaccine efficacy. However, the group vaccinated with 0.1 mg/dose showed the earliest reduction in viral shedding. Overall, SeNPs supplementation in the diet and the administration of the vaccine formula with SeNPs could enhance vaccine efficacy and provide better protection against HPAI-H5N1 in chickens by enhancing cellular immunity and reducing inflammation. We recommend using SeNPs as a vaccine combination or feeding with diet to increase the immunity and vaccine efficacy against H5N1.

Pathogenicity of three genetically distinct and highly pathogenic Egyptian H5N8 avian influenza viruses in chickens.

In late 2016, Egypt encountered multiple cases of the highly pathogenic avian influenza (HPAI) virus of the H5N8 subtype. In a previous study, three distinct genotypes, including A/common-coot/Egypt/CA285/2016 (H5N8) (CA285), A/duck/Egypt/SS19/2017 (H5N8) (SS19), and A/duck/Egypt/F446/2017 (H5N8) (F446), were isolated from wild birds, a backyard, and a commercial farm, respectively, during the first wave of infection. In this current study, we investigated the differences in the pathogenicity, replication and transmissibility of the three genotypes and A/chicken/Egypt/15S75/2015 (H5N1) (S75) was used as the control. The intravenous pathogenicity index was between 2.68 and 2.9. The chicken lethal dose 50 values of F446, SS19 and CA285 were 103.7, 103.7, an 104 with a natural route of infection, respectively. These strains took longer than S75 to cause death when infection was carried out through the natural route (HPAI H5N1). After inoculation with the original concentration of 105 and 106 egg infective dose 50 (EID50), F446 had a higher mortality rate with short mean death times of 4, and 7 days, respectively compared with the other H5N8 viruses. Chickens inoculated with F446 and contacted exposed chickens infected with F446 showed the highest viral titer with remarkable differences in all H5N8 tested swabs at 2-4 days postinfection (dpi) compared to S75 at 2 dpi. This indicates that F446 had a more efficient transmission and spread from contact exposed birds to other birds. All H5N8 viruses were able to replicate systematically in all organs (trachea, brain, lung, and spleen) of the chicken with high viral titer with significantly different and more pathological changes observed in F446 than in other H5N8 viruses at 2 and 4 dpi. Compared with H5N1, we recorded a significantly high viral titer in the samples obtained from the lung, brain and both cloacal and tracheal swabs at 2 and 4 dpi, respectively and in the samples obtained from the spleen at 2 and 4 dpi among the experimental chicken. The comparative pathogenesis study revealed that in comparison with the other HPAI H5N8 viruses, the genotype F446 was more pathogenic, and showed more efficient viral replication and transmissibility in chickens in Egypt. The genotype F446 also showed a high viral titer than HPAI H5N1 and short mean death time at the third day after inoculation with 106 and 105 EID50, which revealed a conservation of certain H5N8 genotypes and a decrease in the incidence of H5N1.

Comparison of genomic and antigenic properties of Newcastle Disease virus genotypes II, XXI and VII from Egypt do not point to antigenic drift as selection marker.

Newcastle disease (ND), caused by avian orthoavulavirus type-1 (NDV), is endemic in poultry in many regions of the world and causes continuing outbreaks in poultry populations. In the Middle East, genotype XXI, used to be present in poultry in Egypt but has been replaced by genotype VII. We investigated whether virus evolution contributed to superseding and focussed on the antigenic sites within the hemagglutinin-neuraminidase (HN) spike protein. Full-length sequences of an NDV genotype VII isolate currently circulating in Egypt was compared to a genotype XXI isolate that was present as co-infection with vaccine-type viruses (II) in a historical virus isolated in 2011. Amino acid differences in the HN glycoprotein for both XXI and VII viruses amounted to 11.7% and 11.9%, respectively, compared to the La Sota vaccine type. However, mutations within the globular head (aa 126-570), bearing relevant antigenic sites, were underrepresented (a divergence of 8.8% and 8.1% compared to 22.4% and 25.6% within the protein domains encompassing cytoplasmic tail, transmembrane part and stalk regions (aa 1-125) for genotypes XXI and VII, respectively). Nevertheless, reaction patterns of HN-specific monoclonal antibodies inhibiting receptor binding revealed differences between vaccine-type viruses and genotype XXI and VII viruses for epitopes located in the head domain. Accordingly, compared to Egyptian vaccine-type isolates and the La Sota vaccine reference strain, single aa substitutions in 6 of 10 described neutralizing epitopes of HN were found. However, the same alterations in neutralization sensitive epitopes were present in old genotype XXI as well as in newly emerged genotype VII isolates. In addition, isolates were indistinguishable by polyclonal chicken sera raised against different genotypes including vaccine viruses. These findings suggest that factors other than antigenic differences within the HN protein account for facilitating the spread of genotype VII versus genotype XXI viruses in Egypt.

Extensive Drug-Resistant Salmonella enterica Isolated From Poultry and Humans: Prevalence and Molecular Determinants Behind the Co-resistance to Ciprofloxacin and Tigecycline.

The emergence of extensive drug-resistant (XDR) Salmonella in livestock animals especially in poultry represents a serious public health and therapeutic challenge. Despite the wealth of information available on Salmonella resistance to various antimicrobials, there have been limited data on the genetic determinants of XDR Salmonella exhibiting co-resistance to ciprofloxacin (CIP) and tigecycline (TIG). This study aimed to determine the prevalence and serotype diversity of XDR Salmonella in poultry flocks and contact workers and to elucidate the genetic determinants involved in the co-resistance to CIP and TIG. Herein, 115 Salmonella enterica isolates of 35 serotypes were identified from sampled poultry (100/1210, 8.26%) and humans (15/375, 4.00%), with the most frequent serotype being Salmonella Typhimurium (26.96%). Twenty-nine (25.22%) Salmonella enterica isolates exhibited XDR patterns; 25 out of them (86.21%) showed CIP/TIG co-resistance. Exposure of CIP- and TIG-resistant isolates to the carbonyl cyanide 3-chlorophenylhydrazone (CCCP) efflux pump inhibitor resulted in an obvious reduction in their minimum inhibitory concentrations (MICs) values and restored the susceptibility to CIP and TIG in 17.24% (5/29) and 92% (23/25) of the isolates, respectively. Molecular analysis revealed that 89.66% of the isolates contained two to six plasmid-mediated quinolone resistance genes with the predominance of qepA gene (89.66%). Mutations in the gyrA gene were detected at codon S83 (34.62%) or D87 (30.77%) or both (34.62%) in 89.66% of XDR Salmonella. The tet(A) and tet(X4) genes were detected in 100% and 3.45% of the XDR isolates, respectively. Twelve TIG-resistant XDR Salmonella had point mutations at codons 120, 121, and 181 in the tet(A) interdomain loop region. All CIP and TIG co-resistant XDR Salmonella overexpressed ramA gene; 17 (68%) out of them harbored 4-bp deletion in the ramR binding region (T-288/A-285). However, four CIP/TIG co-resistant isolates overexpressed the oqxB gene. In conclusion, the emergence of XDR S. enterica exhibiting CIP/TIG co-resistance in poultry and humans with no previous exposure to TIG warrants an urgent need to reduce the unnecessary antimicrobial use in poultry farms in Egypt.

Dual Circulation of Duck Hepatitis A Virus Genotypes 1 and 3 in Egypt.

Duck hepatitis A virus (DHAV) causes acute hepatitis and mortality, resulting in high economic losses in the duck farm industry. The current study describes the outbreak of DHAV in vaccinated duck farms in North Egypt during 2019 and molecular characterization of the 3' untranslated region (UTR) and viral protein VP1 genes. The 30 samples were collected from 7- to 28-day-old commercial Pekin ducks that showed a history of nervous signs and sudden deaths and were on farms in 6 governorates. DHAV was typed by reverse transcription-polymerase chain reaction (RT-PCR) for 3' UTR and VP1 genes and revealed 20 positive farms, with the first detection of DHAV genotype 3 (DHAV-3) in 18 samples and the classic DHAV-1 in 2 samples. The phylogenetic analysis of VP1 and 3' UTR genes of the nine selected strains representative of six governorates revealed that seven strains were clustered with DHAV-3 Chinese and Korean-Vietnamese strains within different subgroups with 92.4%-93.7% amino acid identity; such strains were distinguishable from the vaccine strain of DHAV-1 used in Egypt with 74.4% amino acid identity. The other strains were closely related to the DHAV-1 Asian strain and the vaccine strain used in Egypt with 98.7%-99.6% amino acid identity for the VP1 gene with different clustering than that of recently isolated DHAV-1 Egyptian strains. The VP1 gene of DHAV-3 had 1 hypervariable region (HVR) with 10 amino acid mutations compared with DHAV3/DN2/Vietnam/2011, but DHAV-1 had 3 HVRs with 1 amino acid mutation in HVRII compared with the DHAV-1 vaccine strain. In conclusion, a new introduction of DHAV-3 with the classical DHAV-1 was recorded in Pekin duck farms in North Egypt that is genetically distant from the vaccinal strain.

Artículo regular­Circulacíon dual de los genotipos 1 y 3 del virus de la hepatitis A del pato en Egipto. El virus de la hepatitis A del pato (con las siglas en inglés DHAV) causa hepatitis aguda y mortalidad, lo que genera grandes pérdidas económicas en la industria de la críanza de patos. El estudio actual describe un brote del virus de la hepatitis A del pato en una granja de patos vacunados en el norte de Egipto durante el año 2019 y la caracterización molecular de los genes de la región no traducida 3' (3' UTR) y la proteína viral VP1. Las 30 muestras se recolectaron de patos Pekin comerciales de 7 a 28 días de edad que presentaban antecedentes de signos nerviosos y muerte súbita y se encontraban en granjas de seis gobernaciones. El virus de la hepatitis A del pato se tipificó mediante la transcripción inversa y reacción en cadena de la polimerasa (RT-PCR) para los genes 3' UTR y VP1 y reveló 20 granjas positivas, con la primera detección del genotipo 3 del virus de la hepatitis A del pato (DHAV-3) en 18 muestras y la detección del virus clásico de la hepatitis A del pato tipo1 en dos muestras. El análisis filogenético de los genes VP1 y 3' UTR de las nueve cepas seleccionadas representativas de seis provincias reveló que siete cepas se agruparon con cepas del virus de la hepatitis A del pato 3 chinas y coreano-vietnamitas dentro de diferentes subgrupos con una identidad de aminoácidos del 92.4% al 93.7%; dichas cepas se distinguían de la cepa vacunal del virus de la hepatitis A del pato tipo 1 utilizada en Egipto con 74.4% de identidad de aminoácidos. Las otras cepas estaban estrechamente relacionadas con la cepa asiática del virus de la hepatitis A del pato tipo 1 y la cepa de vacuna utilizada en Egipto con 98.7% -99.6% de identidad de aminoácidos para el gene VP1 con agrupaciones diferentes a las de las cepas egipcias de virus de la hepatitis A del pato tipo 1 aisladas recientemente. El gene VP1 del virus de la hepatitis A del pato tipo 3 tenía una región hipervariable (HVR) con 10 mutaciones en la secuencia de aminoácidos en comparación con la cepa DHAV3/ DN2/Vietnam/2011, pero el virus de la hepatitis A del pato tipo 1 tenía tres regiones hipervariables con una mutación de aminoácidos en la zona hipervariable II en comparación con la cepa de vacuna virus de la hepatitis A del pato tipo 1. En conclusión, se registró una nueva introducción del virus de la hepatitis A del pato tipo 3 con el virus de la hepatitis A del pato clásico tipo 1 en granjas de patos Pekín en el norte de Egipto, que está genéticamente distante de la cepa vacunal.

Genetic evolution of Marek's disease virus in vaccinated poultry farms.

BACKGROUND AND AIM: The Marek's disease virus (MDV) is a neoplastic disease causing serious economic losses in poultry production. This study aimed to investigate MDV occurrence in poultry flocks in the Lower Egypt during the 2020 breakout and genetically characterized Meq, gL, and ICP4 genes in field strains of MDV. MATERIALS AND METHODS: Forty samples were collected from different breeds from eight Egyptian governorates in 2020. All flocks had received a bivalent vaccine (herpesvirus of turkey FC-126 + Rispens CVI988). However, weight loss, emaciation, reduced egg production, paralysis, and rough/raised feather follicles occurred. Samples were collected from feather follicles, liver, spleen, and nerve tissue for diagnosis by polymerase chain reaction. MDV genetic characterization was then performed by sequencing the Meq, gL, and ICP4 genes of five positive samples representing different governorates and breeds. RESULTS: A total of 28 samples were positive for MDV field strains, while two were related to MDV vaccinal strains. All samples tested negative for ALV (A, B, C, D, and J) and REV. Phylogenetic analysis of the Meq gene of sequenced samples revealed that all MDVs were related to the highly virulent European viruses (Gallid herpesvirus 2 ATE and PC12/30) with high amino acid (A.A.) identity 99.2-100%. Alternatively, there was low A.A. identity with the vaccine strains CVI988 and 3004 (up to 82.5%). These results indicate that further investigation of the efficacy of current Egyptian vaccines is required. The Egyptian strains also harbor a specific mutation, allowing clustering into two subgroups (A and B). By mutation analysis of the Meq gene, the Egyptian viruses in our study had R101K, P217A, and E263D mutations present in all Egyptian viruses. Furthermore, R176A and T180A mutations specific to our strains contributed to the high virulence of highly virulent strains. There were no mutations of the gL or ICP4 genes. CONCLUSION: Further studies should evaluate the protection contributed by current vaccines used in Egypt.

Epidemiological and molecular analysis of circulating fowl adenoviruses and emerging of serotypes 1, 3, and 8b in Egypt.

Fowl adenoviruses (FAdVs) are a large group of viruses of different serotypes. They are responsible for inclusion body hepatitis, adenoviral gizzard erosion, and hepatitis hydropericardium syndrome. The present study presents a comprehensive overview of FAdVs in Egypt, with a focus on the epidemiological features of virus serotypes across the country. We conducted molecular investigation of multiple FAdV species based on the genetic signature of hypervariable regions 1-4 in the loop1 (L1) region of the hexon gene. Epidemiologically, the Nile Delta governorates showed high positivity of FAdVs, which were more commonly found in broilers than in layers. Genetically, species D and serotype 8a/E dominated, and the findings also revealed the emergence of new FAdV serotypes 1, 3, and 8b. The comparative analysis of hypervariable regions in the L1 region of the hexon gene revealed variables specific to each virus serotype. In silico predictions of L1 region revealed variations in the molecular structure and predicted the antigenic epitopes which may affect the cross-antigenicity between the different FAdV species and serotypes.

Detection of Novel Goose Parvovirus Disease Associated with Short Beak and Dwarfism Syndrome in Commercial Ducks.

Derzsy's disease causes disastrous losses in domestic waterfowl farms. A genetically variant strain of Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV) was named novel goose parvovirus (NGPV), which causes characteristic syndrome in young ducklings. The syndrome was clinically characterized by deformity in beaks and retarded growth, called short beaks and dwarfism syndrome (SBDS). Ten mule and pekin duck farms were investigated for parvovirus in three Egyptian provinces. Despite low recorded mortality rate (20%), morbidity rate was high (70%), but the economic losses were remarkable as a result of retarded growth and low performance. Isolation of NGPV was successful on primary cell culture of embryonated duck liver cells with a clear cytopathic effect. Partial gene sequence of the VP1 gene showed high amino acids identity among isolated strains and close identity with Chinese strains of NGPV, and low identity with classic GPV and MDPV strains. To the best of our knowledge, this can be considered the first record of NGPV infections in Egypt.

Genotyping of rabbit hemorrhagic disease virus detected in diseased rabbits in Egyptian Provinces by VP60 sequencing.

BACKGROUND AND AIM: Rabbit hemorrhagic disease (RHD) is an economically important disorder of rabbits, where infection results in severe losses to the meat and fur industries. Our goal was to characterize the RHD virus (RHDV) strains currently circulating in different regions of Egypt. MATERIALS AND METHODS: Fifty rabbits suspected of harboring RHDV from 15 Egyptian governorates were evaluated. Diseased rabbits were identified by clinical signs and postmortem lesions. RHDV was confirmed through hemagglutination assay (HA) and polymerase chain reaction (PCR). Partial sequencing of the VP60 gene was performed for genotyping. RESULTS: From 50 rabbits, we identified 16 cases of RHDV (32%) by HA and PCR, including seven males and nine females. We identified two distinct genotypes through sequencing of an amplified fragment of the virus VP60 gene. One group is composed of those circulating primarily in upper Egypt, which is closely related to the classical G3-G5 virus strains, and the second group, circulating predominantly in lower Egypt, was more closely related to the RHDV2 variant. The overall nucleotide sequence identity ranged from 78.4% to 100%, and identity with the vaccine strains ranged from 78.8% to 91.1%. CONCLUSION: Our results constitute important documentation of RHDV strains currently circulating in Egypt. The findings suggest that there may be a limit to the effectiveness of currently applied vaccine strains as this formulation may not cover all circulating strains. A wider investigation that includes both domestic and wild rabbits will be needed to identify appropriate control measures for this disease.

Wild Birds in Live Birds Markets: Potential Reservoirs of Enzootic Avian Influenza Viruses and Antimicrobial Resistant Enterobacteriaceae in Northern Egypt.

Wild migratory birds are often implicated in the introduction, maintenance, and global dissemination of different pathogens, such as influenza A viruses (IAV) and antimicrobial-resistant (AMR) bacteria. Trapping of migratory birds during their resting periods at the northern coast of Egypt is a common and ancient practice performed mainly for selling in live bird markets (LBM). In the present study, samples were collected from 148 wild birds, representing 14 species, which were being offered for sale in LBM. All birds were tested for the presence of AIV and enterobacteriaceae. Ten samples collected from Northern Shoveler birds (Spatula clypeata) were positive for IAV and PCR sub-typing and pan HA/NA sequencing assays detected H5N8, H9N2, and H6N2 viruses in four, four, and one birds, respectively. Sequencing of the full haemagglutinin (HA) gene revealed a high similarity with currently circulating IAV in Egypt. From all the birds, E.coli was recovered from 37.2% and Salmonella from 20.2%, with 66%-96% and 23%-43% isolates being resistant to at least one of seven selected critically important antimicrobials (CIA), respectively. The presence of enzootic IAV and the wide prevalence of AMR enterobacteriaceae in wild birds highlight the potential role of LBM in the spread of different pathogens from and to wild birds. Continued surveillance of both AIV and antimicrobial-resistant enterobacteriaceae in wild birds' habitats is urgently needed.

Pathological changes, shedding pattern and cytokines responses in chicks infected with avian influenza-H9N2 and/or infectious bronchitis viruses.

Avian influenza H9N2 (AIV-H9N2) and Infectious bronchitis (IB) viruses are the most commonly isolated viruses from poultry flocks suffering from respiratory signs with mortalities. The outcome of co-infection with both viruses hasn't been yet well understood. In this study, eighty 1-day-old specific pathogen free chicks were divided into four distinct groups. Group 1 remained uninfected as negative control group; groups 2, 3 and 4 were inoculated with either AIV-H9N2 or IBV or co infected with AIV-H9N2 followed by IBV three days post inoculation respectively. Chicks were monitored for clinical and pathological changes, virus shedding and both Interleukin-6 (IL6) and Interferon gamma (IFNγ) cytokines immune responses. Clinical signs varied from mild to moderate respiratory signs in all challenged groups but were more severe in group 4 with mortalities in groups 3 and 4. Tracheal shedding of both viruses washigher in group 4 than group 2 and 3. Mean AIV-H9 virus titer in lung and kidney was higher in group 4 than group 2 in all time points. IFNγ mRNA gene expression in lung was significantly lower in groups3 and 4. In conclusion, this study reports that co-infection of chicks with both viruses enhances the pathogenicity, increases both viruses shedding and extend AIV-H9 replication with impairment of IFNγ stimulation in lung.

Isolation of a Novel Reassortant Highly Pathogenic Avian Influenza (H5N2) Virus in Egypt.

Highly pathogenic avian influenza (HPAI) H5N1 and H5N8 have become endemic among domestic poultry in Egypt since 2006 and 2016, respectively. In parallel, the low pathogenic avian influenza H9N2 virus has been endemic since 2010. Despite the continuous circulation of these subtypes for several years, no natural reassortant has been detected so far among the domestic poultry population in Egypt. In this study, the HPAI (H5N2) virus was isolated from a commercial duck farm, giving evidence of the emergence of the first natural reassortment event in domestic poultry in Egypt. The virus was derived as a result of genetic reassortment between avian influenza viruses of H5N8 and H9N2 subtypes circulating in Egypt. The exchange of the neuraminidase segment and high number of acquired mutations might be associated with an alteration in the biological propensities of this virus.

The occurrence of disinfectant and antibiotic-resistant genes in Escherichia coli isolated from chickens in Egypt.

AIM: This work aimed to determine the occurrence of antibiotic and disinfectant resistance genes in Escherichia coli isolated from chickens in Egypt. MATERIALS AND METHODS: Organs (liver, lung, heart, yolk sac, and bone marrow) of 1500 chicken samples were collected from diseased chickens suffered from colibacillosis with PM findings as CRD, diarrhea and omphalitis from different governorates of Egypt as: Giza, EL-Bahira, Fayoum, El-Dakahlia, El-Ismalia, and El-Sharkia during 2015-2016. These samples were labeled and transported immediately on ice to the Reference laboratory for quality control on poultry production (RLQP). The samples were cultured onto MacConkey agar and Eosin Methylene Blue Agar. Isolation and identification of the E. coli were performed based on morphology, cultural, staining, and biochemical properties. Antimicrobial resistance test was carried out using disk diffusion method. The PCR employing tetA, qacED1 and qacA/B were carried out for detection of these genes in isolated E.coli. RESULTS: The prevalence of E. coli in chicken was 34%. Predominant serotypes of E. coli which serologically identified were O128, O111, O44, O158, and O2. Antibiotic susceptibility test of E. coli revealed that 100% of isolates were resistant to ampicillin, erythromycin, and sulfamethoxazole-trimethoprim, while 73.53% and 38.23% of them were sensitive for colistin sulfate and levofloxacin, respectively. Antibiotic resistance genes as tetA gene were tested for isolated E. coli and detected by incidence rate of 91.18%. qac resistance genes resembling as qacED1 and qacA/B genes were detected in isolated E. coli 70.6% and 14.7%, respectively. CONCLUSION: E. coli isolated from chickens in Egypt was carried qac and antibiotic-resistant genes that affect the poultry industry.

Chicken anaemia virus enhances and prolongs subsequent avian influenza (H9N2) and infectious bronchitis viral infections.

Immunosuppressive viral diseases have a great economic importance in the poultry industry due to the increased susceptibility to secondary infections. Chicken anaemia virus (CAV) is one of the major immunosuppressive diseases in chickens. In addition, low pathogenic avian influenza (LPAI) of subtype H9N2 and infectious bronchitis (IB) viruses are among the most frequently reported respiratory viral diseases in poultry worldwide. In the present study, specific pathogen free chickens were used to understand the impact of CAV on secondary infection with LPAI-H9N2 or IB viruses. Clinical outcomes, viral shedding dynamics, and cytokine levels wereassessed. The results exhibit that chickens previously infected with CAV produceconsiderablyhigher titresof LPAI-H9N2 or IB viruses in the oropharyngeal swabs (P < 0.05), tracheas and kidneys. In addition, the immunologic effect of CAV provokedthe development of clinical signs of LPAI-H9N2 and IB virus infections. Moreover, results suggested that pre-infection with CAV directly correlated with elevated levels of IL-6 and IFNγ. These findings underline the importance of CAV pre-infection on LPAI-H9N2 or IB infection in chickens, and indicate that co-circulation of CAV can contribute to the spread and evolution of LPAI H9N2 and IB viruses.

Cinnamon oil downregulates virulence genes of poultry respiratory bacterial agents and revealed significant bacterial inhibition: An in vitro perspective.

BACKGROUND AND AIM: Respiratory bacterial agents represent one of the most harmful factors that ordinarily threaten the poultry industry and usually lead to great economic losses. Meanwhile, there is a global demand to avoid the highly emerging antibiotic resistance and antibiotic residues in edible meat. Whereas, the use of alternatives became of great priority, especially for those substances extracted from natural plant origin. The study aimed to evaluate the antibacterial effect of cinnamon oil as a herbal extract on different respiratory bacterial agents. MATERIALS AND METHODS: One hundred and fifty biological samples were collected through targeted surveillance for respiratory diseased poultry farms representing three governorates, from which bacterial isolation and identification, DNA sequencing of representative strains were performed. Furtherly, phenotypic and genotypic evaluation of the antibacterial effect of cinnamon oil was performed by minimum inhibitory concentration, agar disk diffusion, and virulence genes expression real-time polymerase chain reaction. RESULTS: Cinnamon oil gave rise to acceptable degrees of virulence genes downregulation of 0.15, 0.19, 0.37, 0.41, 0.77, and 0.85 for Staphylococcus aureus sed gene, Escherichia coli stx1 gene, Avibacterium paragallinarum HPG-2 gene, Pasteurella multocida ptfA gene, Mycoplasma gallisepticum Mgc2 gene, and Ornithobacterium rhinotracheale adk gene, respectively. Phenotypically, using agar disk diffusion assay and broth microdilution susceptibility, cinnamon oil showed also tolerable results as it stopped the growth of S. aureus, E. coli, P. multocida, and A. paragallinarum with varying zones of inhibition. CONCLUSION: The encountered results declared the successful in vitro effect of cinnamon oil that recommends its application for living birds for future use as a safe antibacterial in the poultry industry.

Quantitative evaluation of viral interference among Egyptian isolates of highly pathogenic avian influenza viruses (H5N1 and H5N8) with the lentogenic and velogenic Newcastle disease virus genotype VII in specific pathogen-free embryonated chicken eggs model.

BACKGROUND AND AIM: Mixed infections of the highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are considered the most distressing problem of the poultry industry. The problem arises due to the influence of a hidden virus on the replication of another suspected virus. Consequently, misdiagnosis of the real cause of disease may become a source of infection for other healthy stock by transmission and dissemination of the hidden virus. This study aimed to determine the impact of HPAIV and NDV on each other in a specific pathogen-free embryonated chicken egg (SPF-ECE) model. MATERIALS AND METHODS: HPAIVs (H5N1 and H5N8) and NDVs [avirulent NDV [avNDV] and velogenic NDV [vNDV]) were inoculated into the allantois cavity of SPF-ECE with graded titers (2, 3, and 4 log10 EID50) at 24 and 48 h of incubation, followed by the collection of allantoic fluid. A quantitative reverse transcription real-time polymerase chain reaction was used to determine the viral RNA copies of both viruses. RESULTS: Obvious interference was reported on the growth of NDVs when co-inoculated with AIVs. NDV RNA titers reduction ranged from <3 to 5 log10 to complete suppression, but slight interference with the growth of AIVs occurred. H5N1 RNA titers showed <1-2 log10 reduction when co-inoculated with vNDV compared with the H5N1 control. The interference impact of H5N8 was more powerful than that of H5N1, while vNDV showed more resistance for interference than the avNDV strain. On the other hand, interference of AIVs was not observed except when vNDV was inoculated before H5N1. The interfering impact was increased after 48 h of inoculation, whereas no titer of avNDV was detectable. CONCLUSION: AIV strains had a powerful effect on NDV growth, regardless of which infection occurred first.