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The infectious bursal disease virus (IBDv) is widespread in poultry flocks all around the world. During the period between 2016- 2017, 22 IBD outbreaks were investigated in El-Minoufiya Governorate in different chicken sectors (11 broilers, 7 commercial layer pullets and 4 native baladi varieties). The disease occurred at 23-42 days of age. The mortality ranged between 3-9 % in commercial broilers farms, 5.8-12% in native breed varieties and 5-10 % in commercial layer replacement pullets. Two hundred one-day-old commercial male layer chicks was used for assessment of protection obtained after vaccination with HVT-IBDV vector vaccine "Vaxxitek" against challenged with vvIBDV local field isolate "Lay./Men.Egypt/17" at 46th-day of age. Maternal derived antibodies (MDA) was followed up by ELISA to determine MA waning and the suitable age of chicks for IBDV vaccination and/or challenge. Chicks divided into 4 groups of (50 birds/ of each), G1 (vaccinated with HVT-IBD vector vaccine at 1 day old of age and non-challeneged) and G2 (vaccinated with HVT-IBDV at one-day of age and challenged with vvIBDV local field isolate "Lay./Men.Egypt/017" at 46-day of age), G3 (non- vac-cinated and non-challenged) and G4 (non-vaccinated and challenged chickens). Our results revealed that the mortality percentage of commercial male layer chickens chal-lenged with recent vvIBDV local field isolate "lay./Men.Egypt/017" was (0%) in G2, while in G4 was (90%), at 7-days post challenge. The highest bursa body ratio observed was (5.32 and 5.31) in non- vaccinated and non-challenged birds (G3) and birds vac-cinated with the VAXXITEK HVT-IBDv vaccine (G1). Conclusion, the recombinant VAXXITEK HVT-IBDv vaccine has provided protection for commercial male layer chicks against challenge with recent vvIBDv isolate. Vaccination with vector vaccine in en-demic areas with vvIBDV, protect against mortality, partially against bursal atrophy and decreases immunosuppressive effect of vvIBDV.

Newcastle disease virus (NDV) outbreaks still occur and cause disastrous economic losses in broiler chickens despite intensive vaccination policies using genetically different vaccines from the circulating strains. This study aimed to compare protection conferred by different vaccination strategies using genotype matched (genotype VII) and mismatched vaccine (genotype I and II) against challenge with currently circulating viruses in broiler chickens. 175 one- day-old broiler chicks were divided in to 5 groups. Groups 1 to 4 (G1, G2, G3 and G4) were vaccinated using genotype I (Ulster 2C strain), genotype II (VG/GA strain) and genotype VII (KBNP-4152R2L strain) vaccines according to manufacturer's instructions and group 5 (G5) was kept as control. Protection assessed based on clinical protection, seroconversion, shedding and histopathology.Chickens vaccinated with live genotype II vaccine at 7 and 21 days of age and killed genotype VII vaccine at 7 days of age (G2) were completely protected clinically with no mortality compared with control non vaccinated chickens (G5) that showed typical NDV clinical picture with (100%) mortality. while, other vaccinated groups showed lower level of clinical protection with (2.8%) mortality for each group. Viral shedding was greatly reduced in groups vaccinated with inactivated recombinant genotype VII vaccine (G2 and G3) which recorded (0% & 0%) and (16.6% & 0%) at 7 & 10 days post challenge, respectively compared with groups vaccinated with inactivated genotype II vaccine (G1 and G4) regardless the genotype of the combined live vaccine (either genotype II or VII). Moreover, histopathology of control non vaccinated challenged group (G5) revealed severe lymphocytic depletion and necrosis, epithelial degeneration beside neurological lesions. It was concluded that using genotype matched NDV vaccine with the currently circulating field strains can provide adequate clinical protection and minimize virus shedding that can help in decreasing virus load in the environment.

Highly contagious Newcastle disease (ND) is associated with devastating outbreaks with highly variable clinical signs among gallinaceous birds. In this study we aimed to verify clinical ND suspicions in poultry holdings in Egypt suffering from respiratory distress and elevated mortality, comparing two groups of ND-vaccinated poultry holdings in three governorates. Besides testing for Newcastle disease virus (NDV), samples were screened for infectious bronchitis virus (IBV) and avian influenza virus (AIV) by RT-qPCR as well as by non-directed cell-culture approach on LMH-cells. Virulent NDV was confirmed only in group A (

In the present study, samples were collected from wild and domestic waterfowls to detect recently circulating HPAI H5Nx viruses. Partial HA gene sequencing followed by phylogenetic analysis of the obtained sequences of five selected isolates was performed. Phylogenetic analysis revealed clustering of the three H5N8 isolates in clade 2.3.4.4b, with other Egyptian H5N8 isolates with amino acid identity percent with each other ranging from (96.9% - 99.3%). The two H5N1 isolates clustered in clade 2.2.1.2 with other recent Egyptian isolates. The amino acid identity percent between vaccinal strains was ranging from (88.2% - 89.6%) with H5N8 field isolates and (96.5% - 99%) with H5N1 field isolates. The amino acid identity percent between H5N8 and H5N1 field isolates ranging from (86.3% - 87.8%). The antigenic relatedness between the two HPAI local isolates (H5N1 and H5N8) was low (R value 24.8%) indicating major antigenic difference between the two subtypes. Also, a major subtype difference in antigenicity (R value 20.7-31.0%) was also detected between the local HPAI H5N8 isolate and the three vaccinal strains. While, a minor subtype difference (R value 38.7-68.7%) was detected among the examined H5N1 local isolate and the three vaccinal strains. Our results support that, HPAI H5N8 virus is genetically and antigenically distinct from HPAI H5N1 virus. Also, some commercial vaccines which are used to control avian influenza in Egypt are genetically and antigenically distinct from HPAI H5N8 virus currently circulating in Egypt.

Toll-like receptors (TLRs), type I transmembrane pattern recognition receptors (PRRs), are composed of the extracellular domain that is implicated in the recognition of microbial products and initiates the innate and adaptive immune response. Previous reports on TLRs in birds showed significant levels of inter- and intraspecific genetic variation. Little is known about the structure and function of the avian immune system, especially waterfowl species. This work aimed to identify and clone Anas platyrhynchos (mallard duck) TLR-3 (dTLR-3) and its expression level following challenge with velogenic Newcastle disease virus (NDV) as a model for waterfowl species. The mallard duck TLR-3 full-length cDNA sequence had been cloned, which consisted of 2457 nucleotides. The translated amino acid sequence showed identity degree as 97% with Muscovy duck, 95% with geese, 89% with helmeted guineafowls, 88% with the chickens TLR-3 gene, 82% with turkey TLR-3, and 79% with zebra finch, while it showed 54% with human one; the analysis data suggested that the new sequence is probably homologous to vertebrates' TLR-3. The predicted protein encoded by the duck dTLR-3 mRNA sequence is composed of 819 amino acids. Analysis of the deduced amino acid sequence indicated that dTLR-3 has typical structural features and contains the main components of proteins in the TLR family. The dTLR-3 expressed in almost all examined tissues of mallard duck following quantitative real-time polymerase chain reaction (qPCR) analysis and using B-actin as a housekeeping gene. To check the functionality of the receptor and its role in viral infection, we evaluate the expression level in different tissues and its changes following NDV infection. The results showed significant (P0.05) upregulated in the brain at 24 h (1.84-fold), reached a peak at 48 h (4.82-fold), and recovered to normal levels at 72 h post-infection. These results indicate a complete and functional dTLR-3 that is orthologous to other vertebrate receptors with its potential role in early response against viral infection in mallard duck species.

The present study was undertaken to investigate and phenotypically characterize serotypes and antibiotic susceptibility of Salmonella isolates prevalent in broiler chicks. In this study a total of 26 broiler farms allocated in El-Gharbia and El-Menofia Governorates during the period of November 2015 to November 2016. The percentage of Salmonella positive samples were 15.4%, the following serotypes could be isolated and identified Salmonella Pullorum (n=1), Salmonella Havana (n=1), Salmonella Durham (n=1), and Salmonella Entritidis (n=1) from broiler chicks. In the present study, most of the tested Salmonella serovars were multi-drug resistant (MAR) and had high MAR indexes against the commonly used antibiotics in the poultry industry in Egypt.

In the present study, four very virulent infectious bursal disease virus (vvIBDV) isolates from flocks of chickens with vaccination failure in Egypt in 2003, 2007, 2010 and 2014 were characterized. The four viruses, designated USC2003, USC2007, USC2010 and USC2014, were detected by reverse transcription PCR, subjected to sequencing of both genomic segments (A and B) and compared with geographically and phylogenetically diverse IBDV strains. Phylogenetic analysis of segment A (complete) and B (partial) revealed a close relationship between Egyptian and vvIBDV reference strains of European and Asian origin. The sequences of segments of A and B the current Egyptian isolates were 96.1-98.2% and 96.5-98.7% identical, respectively, to those of other known vvIBDV isolates. The deduced amino acid sequences of VP1, polyprotein (pVP2-VP4-VP3) and VP5 revealed the presence of putative virulence determinants of Egyptian isolates compared with vvIBDV and less virulent (classical and variant) strains. The Egyptian isolates also possess unique amino acids substitutions within the hypervariable region of VP2 that differ from those of other reference IBDV strains. Further studies may be necessary to determine the pathogenic significance of these amino acid substitutions to fully understand the molecular epidemiology and evolution of IBDV.

Highlights • New prototype of emerging IBV variant recognized in Egypt. • New IBV strains form a forth subclade within S-genotype GI-23. • Mutations in the S1protein, including two defined neutralizing sites, point to antigenic variation. • Intra-genotypic recombination is recognized for genes S1, 3ab and E, inter-genotypic recombination for gene 6b (GI-19) • Specific gene 3ab genotype is linked to new IBV emergence of new variants., Infectious bronchitis virus (IBV), a gamma-coronavirus, causes infectious bronchitis (IB), a major respiratory disease of chicken. Its high mutation rate in conjunction with recombination of the RNA genome constantly creates IBV variants that are difficult to control by currently available vaccines. In this study, we addressed the question whether small-scale holdings might harbor IBV variants that serve as a reservoir for newly emerging variants. Egyptian IBV isolate EGY/NR725/2016 (NR725/16) from a small-scale broiler farm was assigned to genotype I, clade 23 (S1:GI-23), based on partial S1 gene sequences and corroborated by full genome sequencing. Analysis of the S1 gene established three subclades for historical IBV strains (S1:GI-23.1, S1:GI-23.2.1 and S1:GI-23.2.2) and confirmed NR725/16 as being part of a separate fourth subclade (S1:GI-23.3). Samples from the years 2018 and 2019 revealed that the new subclade prevails in Egypt, carrying fixed mutations within the hypervariable regions (HVR) 1-3 of the S1 protein that affect two neutralization sensitive epitopes at sites 294F, 297S and 306Y (48.2) and 329R (62.1). In addition, recombination was recognized in isolate NR 725/16, with intra-subtype mixing for the entire genes 3ab and E and inter-subtype mixing for the entire gene 6b with a close match to QX like viruses of genotype GI-19. Further analysis of gene 3ab detected the homologous gene pool to NR725/16 in samples from 2013 (3ab:C) and closely related 3ab genotypes in IBV Egyptian isolates from 2016, 2018 and 2019. These data prove a flourishing exchange between poultry holdings with a common gene pool. The continued circulation of viruses harboring genes S1:GI-23.3 and 3ab:C indicates an evolutionary advantage of this combination possibly by combining antigenic escape with modulated pathogenicity to facilitate IBV spread in the vaccinated poultry population in Egypt.

Low pathogenic avian influenza virus of subtype H9N2 is panzootic in multiple avian species causing respiratory manifestations and severe economic losses. H9N2 co-circulate simultaneously with high pathogenic avian influenza virus subtype H5N1 in Egyptian chicken farms suggesting the possibility of reassortment. The aim of the present study was to isolate and characterize H9N2 from the recent outbreaks in chicken farms. Also the diversity of amantadine-resistant mutants among these isolates was tested by in situ ELISA and sequence analysis. Three influenza H9N2 viruses, designated A/chicken/Egypt/SCU8/2014, A/chicken/Egypt/SCU9/2014 and A/chicken/Egypt/SCU20/2014 were isolated from commercial broiler and broiler breeder chickens in specific pathogen free embryonated chicken eggs. The eight gene segments were amplified by RT-PCR, cloned, and subjected to full length sequencing. Phylogenetic analysis of these viruses revealed a close relationship between Egyptian, Middle Eastern and Israel isolates with an average of 96-99 % nucleotide homology and identified an ancestor relationship to low pathogenic H9N2 Quail/HK/G1/1997 prototype. The internal segments of the currently isolated viruses were derived from the same sub-lineage with no new evidence of reassortment. The three isolates were sensitive to amantadine as suggested by absence of mutations of M2 and confirmed by a phenotypic assay. In conclusion, avian influenza H9N2 virus is circulating in Egyptian chicken farms causing respiratory manifestations. Continuous monitoring of the molecular epidemiology and its impact on the virulence as well as emergence of new strains are necessary.

Since November 2016, Europe witnesses another wave of incursion of highly pathogenic avian influenza (HPAI) A(H5) viruses of the Asian origin goose/Guangdong (gs/GD) lineage. Infections with H5 viruses of clade 2.3.4.4b affect wild bird and poultry populations. H5 viruses of clades 2.2, 2.3.1.2c and 2.3.4.4a were detected previously in Europe in 2006, 2010 and 2014. Clades 2.2.1.2 and 2.3.2.1.c are endemic in Egypt and Western Africa, respectively and have caused human fatalities. Evidence exists of their co-circulation in the Middle East. Subtype H5 viruses of low pathogenicity (LPAI) are endemic in migratory wild bird populations. They potentially mutate into highly pathogenic phenotypes following transmission into poultry holdings. However, to date only the gs/GD H5 lineage had an impact on human health. Rapid and specific diagnosis marks the cornerstone for control and eradication of HPAI virus incursions. We present the development and validation of five real-time RT-PCR assays (RT-qPCR) that allow sequencing-independent pathotype and clade-specific distinction of major gs/GD HPAI H5 virus clades and of Eurasian LPAI viruses currently circulating. Together with an influenza A virus-generic RT-qPCR, the assays significantly speed up time-to-diagnosis and reduce reaction times in a OneHealth approach of curbing the spread of gs/GD HPAI viruses.

Two truncated sequences (designated P1 and rHA1) of influenza A virus subtype H5 haemagglutinin (HA) were cloned and expressed in yeast Pichia pastoris (P. pastoris). These polypeptides were used in an indirect recombinant ELISA (rELISA) for detection of H5 antibodies in poultry. Serum samples obtained from broiler chickens vaccinated with commercial inactivated vaccine (H5N2) and control negative sera from non-vaccinated chickens against influenza were tested using rP1-ELISA, rHA1-ELISA, whole H5N1-ELISA, Western blot, agar gel immunodiffusion (AGID) and haemagglutination inhibition (HI) tests. The rHA1-ELISA proved to be highly sensitive and specific. To study the validity of rHA1-ELISA, a total of 179 serum samples obtained from commercial broiler chickens vaccinated previously with commercial H5N2 inactivated vaccines, were tested by rHA1-ELISA, commercial ELISA (cELISA) and HI. The relative sensitivity and specificity between rHA1-ELISA, and HI tests were 100% and 70%, respectively, and between cELISA and HI were 100% and 57%, respectively. The agreement ratio between rHA1-ELISA and HI was 84.9% and between cELISA and HI tests was 76.5%. Serum samples obtained from ducks vaccinated with commercial inactivated H5N2 were tested by rHA1-ELISA and the results showed significant reactivity with duck sera. In conclusion, the results demonstrate the potential applicability of the rELISA for the determination of antibodies to H5 influenza virus in chickens and ducks.

Fowl typhoid (FT), a systemic disease that results in septicemia in poultry, is caused by Salmonella enterica serovar Gallinarum biovar Gallinarum (SG). Mortality and morbidity rates from FT can reach up to 80%. Attenuated live Salmonella vaccine candidates have received considerable attention because they confer solid immunity, and they can produce systemic and mucosal immunity in the gut when administered orally. In the present study, five metabolic drift (MD) mutants with a single-(designated SG-Rif1, SG-Sm6) or double-attenuating marker (designated SG-Rif1-Sm4, SG-Sm6-Rif10, and SG-Rif1-Sm10) were isolated. The relative colony sizes to wild-type strain after 24 hr at 37 C incubation were 50%, 40%, 30%, 30%, and 20%, respectively. The probability of a back mutation can almost be excluded because the reduced colony sizes were stable after at least 50 passages on culture media. The safety and immunogenicity were evaluated in susceptible 1-day-old commercial layer chickens. After oral administration of 10(8) colony-forming units (CFU), all developed MD mutants proved to be safe and did not cause death of any infected birds during 15 days postvaccination, whereas chickens receiving 10(6) CFU SG wild-type strain showed a high mortality rate (40%). Vaccination of commercial layer chicks with SG-Rif1, SG-Sm6, SG-Rif1-Sm4, and SG-Sm6-Rif10 MD mutants could protect chickens against challenge by homologous wild-type strain; however, SG-Rif1-Sm10 could not protect against challenge, indicating hyperattenuation. In conclusion, vaccination with SG MD mutant vaccine appears to be safe and offers protection against FT in chickens.

Infectious laryngotracheitis virus (ILTV) continues to cause respiratory disease in Egypt in spite of vaccination. The currently available modified live ILTV vaccines provide good protection but may also induce latent infections and even clinical disease if they spread extensively from bird-to-bird in the field. Four field ILTV isolates, designated ILT-Behera2007, ILT-Giza2007, ILT-Behera2009, and ILT-Behera2010 were isolated from cross-bred broiler chickens. The pathogenicity based on intratracheal pathogenicity index, tracheal lesion score, and mortality index for chicken embryos revealed that ILT-Behera2007, ILT-Behera2009 and ILT-Behera2010 isolates were highly pathogenic whereas ILT-Giza2007 was non-pathogenic. To study the molecular epidemiology of these field isolates, the infected cell protein 4 gene was amplified and sequenced. Phylogenetic analysis revealed that ILT-Behera2007, ILT-Behera2009, and ILT-Behera2010 are chicken embryo origin (CEO) vaccine-related isolates while ILT-Giza2007 is a tissue culture origin vaccine-related isolate. These results suggest that CEO laryngotracheitis vaccine viruses could increase in virulence after bird-to-bird passages causing severe outbreaks in susceptible birds.