Ahmed A. El-Sanousi

Effect of subclinical lead toxicity on the immune response of chickens to Newcastle disease virus vaccine

The effect of lead acetate (20 and 40 mg kg-1 bodyweight daily) administered via the crop from day old to 56 days of age on the immune response to Newcastle disease virus vaccine (NDVV, La Sota strain) was studied in 354 Lohman chickens. Lead decreased the mitogenic response of peripheral blood lymphocytes (PBL) to phytohaemagglutinin-P (PHA-P) in birds vaccinated with NDVV. It also decreased the weights of the bursa of Fabricius, the thymus glands and the spleen relative to bodyweight. Lead administration decreased the antibody titre to NDVV in the vaccinated groups. The percentage mortality due to a challenge with a virulent velogenic Newcastle disease virus was higher in the lead intoxicated birds.

Molecular characterization of infectious bronchitis viruses isolated from broiler and layer chicken farms in Egypt during 2012

Abstract One of the major problems of avian infectious bronchitis virus (IBV) is the frequent emergence of new variants. In the present study 205 tracheal swabs and organs were collected from broilers and layers chicken farms during January to August 2012 from 19 governorates all over Egypt. The chickens demonstrated respiratory signs and mortality. Out of the examined samples, 130 of which (about 64%) of suspected farms were positive for IBV with real time RT-PCR. 13 IBV-positive samples were selected for further isolation and characterization. Isolation in specific pathogen free (SPF) embryos was carried out after studies three blind successive passages and the hypervariable region of spike protein1 (SP1) was amplified by RT-PCR and sequenced to study the genetic diversity between the isolated viruses. Phylogenetic analysis of the obtained sequences of 13 isolates compared with other IBV strains from the Middle East and worldwide reveled that 11 out of the 13 isolates had close relationship the Israeli variants (IS/885 and IS/1494/06) with nucleotide homology reached up to 89.9% and 82.3%, respectively. Only two isolates had close relationship with CR/88121 and 4/91 viruses with identities of 95% and 96%, respectively. This study indicates existence of two variant groups of IBV circulating in Egypt during 2012. Group I was similar but distinguishable from Israeli variant IS/885 and group II was related to 4/91 and CR/88121 vaccine strains. There was no geographical link between the 2 groups as they were distributed all over the country. These findings necessitate the need to revise the vaccination programs and control measures for IBV.

Isolation and molecular characterization of Newcastle disease virus genotypes II and VIId in Egypt between 2011 and 2012

The current study was conducted to isolate and characterize Newcastle disease virus (NDV) from recent outbreaks affecting poultry farms in Egypt between 2011 and 2012. Trachea, spleen, liver, proventriculus and caecal tonsils were collected from clinically infected NDV ten different vaccinated broiler farms in Fayoum, Behira and Giza Provinces. Inoculation of all the collected samples in 10-day-old embryonated chicken specific-pathogen-free eggs resulted in isolation of haemagglutinating agents in three samples. These haemagglutinating agents were confirmed as NDV by real-time reverse transcription polymerase chain reaction (rt RT-PCR) using matrix (M) gene-specific primers. The deduced amino acid sequences of the fusion protein revealed that one isolate possessed the motif 112RRQKRF117 at the cleavage site, indicating that this isolate is velogenic genotype, whereas the other two isolates carries the motif 112GRQGRL117 indicating they are lentogenic genotype. The phylogenetic analysis revealed that the velogenic genotype isolate clustered with published class II genotype VII sub genotype d NDVs and closely related to Middle East isolates, whereas the other two isolates clustered with published class II genotype II NDVs. The spread of velogenic genotype strain to Egypt via Middle Eastern countries is likely to be the source of infection.

Molecular and antigenic traits on hemagglutinin gene of avian influenza H9N2 viruses: Evidence of a new escape mutant in Egypt adapted in quails

The LPAI viruses of H9N2 subtype became widely distributed in Middle Eastern countries, causing great economic losses in poultry industry especially when complicated with other pathogens. The H9N2 viruses in Egypt have a wide spread nature since its first occurrence in 2011. In this study, we collected cloacal and tracheal samples from 19 flocks for detection and propagation of H9N2 virus using real-time RT-PCR and egg inoculation. We studied the molecular evolution of the Hemagglutinin gene of H9N2 viruses by full HA gene sequencing, then the antigenic characterization was implemented using the cross HI assay and analyzed using 3D Bioinformatics cartography software. The phylogenetic analysis of the HA gene of Egyptian H9N2 viruses clearly points out the presence of only one group (Egy/G1) of originally introduced viruses in 2011 related to the G1 lineage within group B, with the presence of multiple minor clusters includes viruses from 2011 to 2015. However, a new variant (Egy/G1var) cluster was detected in quails since 2012. Genetically, Egy/G1var viruses characterized by presence of 20 amino acid substitutions within and adjacent to the antigenic sites in comparison to other Egyptian viruses. In addition, two glycosylation sites at amino acid residues 127 and 189 were determined in close to the receptor binding and antigenic sites. The antigenic analysis based on 3D antigenic mapping showed that the Egy/G1var cluster was clearly distinct from the original Egy/G1 viruses. In conclusion, Egy/G1var is shown to be a new escape mutant variant cluster with an adaptive evolution in quails.

Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection.

The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused staggering economic damage. In addition, H5N1 avian influenza viruses represent a significant threat to public health. Therefore, the rapid detection of H5 viruses is very important in order to control the disease. In this study, a qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of hemagglutinin gene of H5 subtype influenza viruses was developed. The results were compared to the real-time reverse transcription polymerase chain reaction (RT-PCR). An in vitro transcribed RNA standard of 970 nucleotides of the hemagglutinin gene was developed and used to determine the assay sensitivity. The developed H5 RT-RPA assay was able to detect one RNA molecule within 7 min, while in real-time RT-PCR, at least 90 min was required. H5 RT-RPA assay did not detect nucleic acid extracted from H5 negative samples or from other pathogens producing respiratory manifestation in poultry. The clinical performance of the H5 RT-RPA assay was tested in 30 samples collected between 2014 and 2015; the sensitivity of H5 RT-RPA and real-time RT-PCR was 100%. In conclusion, H5 RT-RPA was faster than real-time RT-PCR and easily operable in a portable device. Moreover, it had an equivalent sensitivity and specificity.

Initiation and regulation of immune responses to immunization with whole inactivated vaccines prepared from two genetically and antigenically distinct lineages of Egyptian influenza A virus subtype H5N1.

Following the introduction of highly pathogenic avian influenza (HPAI) virus subtype H5N1, the Egyptian government implemented a massive poultry vaccination campaign as the cornerstone of its policies to control the virus. The efficacy of vaccination has been evaluated primarily by measuring titers of antibodies inhibiting the hemagglutinating activity of the viral hemagglutinin (HA). However, other aspects of the host response remain poorly understood. In the present study, in addition to hemagglutination inhibition (HI) titers, cytokine profiles were examined and IFNγ concentrations were measured in vivo after immunization with a whole inactivated virus (WIV) prepared from a classical strain of clade 2.2.1.2 (C121) and an antigenic drift variant of clade 2.2.1.1 (V1063). The results revealed an earlier response and higher HI titers and IFNγ levels in sera from chickens immunized with C121, accompanied by significantly higher expression of IL8, IL10, and IL18 in the spleen and IL6 and IL10 in the bursa, compared to those immunized with V1063. Furthermore, stimulation of the HD11 cell line with C121 induced gradual upregulation of pro-inflammatory cytokines, which was observed at 24 hours post-inoculation (hpi), and became more pronounced at 48 and 72 hpi, accompanied by upregulation of IFNα. Conversely, V1063 induced very early transient higher expression of pro-inflammatory cytokines at 3 and 6 hpi accompanied by upregulation of IL10, which then decreased at 24, 48 and 72 hpi. In summary, our results provide evidence of a correlation between adaptive immune responses induced by WIVs and higher expression of IL10 and IL18 in addition to early induction of IFNα. These findings could be used to improve immune responses induced by WIVs.

In-vitro assessment of differential cytokine gene expression in response to infections with Egyptian classic and variant strains of highly pathogenic H5N1 avian influenza virus

Abstract In Egypt, two distinct genetic groups of HPAI H5N1 viruses are co-circulating: classic 2.2.1/C sub-clade and antigenic drift variant 2.2.1.1 clade isolated from vaccinated poultry flocks. The response of chicken innate immunity to both genotypes is not investigated, so far. In this study, expression of immune related genes (IL1b, IL4, IL6, IL8, IL10, IL18, IFNα and IFNγ) after infecting chicken macrophage cell line (HD11) and chicken peripheral blood Mononuclear cells (PBMC) with a classic and a variant strains was assayed using quantitative reverse-transcription real-time polymerase chain reaction assays (qRT-PCR). In HD11, the variant strain induced higher levels of IL1b and IL8 at 6 hours post infection (hpi), IL4 at 24 / 48 hpi and IFNα at 48 hpi than the classic strain. Conversely, the classic strain induced about 10-fold increase of IFNγ at 24 and 48 hpi and the virus replicated at higher level than the variant strain. The results of PBMC infection were similar to that reported from HD11 except for IFNγ gene expression that was higher at variant strain infected cells than that infected with the classic strain. After 24hpi skewing the innate immune response toward anti-inflammatory (humoral-associated) cytokines was different between HD11 (through IL4) and PBMC (through IL10). To sum up, the classic strain produced less cytokines which may indicate adaptation to evade the recognition by the innate immune system and explain its higher pathogenicity.

Assessment of in vitro potency of inactivated Newcastle disease oil adjuvanted vaccines using hemagglutination test and blocking ELISA

Aim: The present study was aimed to establish a protocol for the evaluation of the in vitro potency of commercial inactivated Newcastle disease virus (NDV) oil-adjuvanted vaccines using hemagglutination test (HA) and blocking ELISA (B-ELISA) based on polyclonal antibodies. Materials and Methods: Aqueous phases from a total of 47 batches of inactivated NDV vaccines manufactured by 20 different companies were extracted with isopropyl myristate. The viral antigen in each sample was detected and quantified by a standard HA test and a B-ELISA assay. To verify the efficiency of the antigen extraction method used in the batches which showed HA and to test the validity of using in vitro antigen quantification by HA and B-ELISA tests, a subset of 13 batches (selected from the total 47 batches) was inoculated in groups of 3-4-week-old specific pathogen-free chickens using the recommended vaccine dose. The immunogenicity of the selected vaccine batches was assessed by the NDV-hemagglutination inhibition antibody titers in individual serum samples collected 4 weeks after vaccination. Further, the efficacy of the vaccines and their protection rates were determined by a challenge test carried out for the vaccinated chickens with the Egyptian 2012 isolate of the virulent NDV genotype VII. Results: A strong correlation was observed between HA titers and B-ELISA mean titers in the tested 47 batches (R2=0.817). This indicated the possibility of using the latter in vitro assays for vaccine potency assessment. The recommended protective NDV antigen titer measured by B-ELISA was determined to be 28 ELISA units per dose. The comparison between the HA titers of the aqueous extracts of test vaccines and the corresponding results of in vivo potency assays (i.e., immunogenicity and efficacy), including antibody titers in the serum of vaccinated birds, indicated that the efficiency of the antigen extraction used may interfere with obtaining a strong correlation between the in vitro and in vivo results. Conclusion: HA or B-ELISA tests can be used as rapid and cost-effective alternatives to traditional in vivo potency tests for vaccine potency assessment by quantifying the NDV antigen present in aqueous phase extracts of the tested vaccines. The latter in vitro protocol, however, requires efficient extraction of the antigen to be able to obtain good correlation with the traditional in vivo potency tests.

Molecular characterization of full fusion protein (F) of Newcastle disease virus genotype VIId isolated from Egypt during 2012-2016

Aim: The aim of this work was to study the full F gene sequence of Newcastle disease virus (NDV) in regard to pathotyping and genotyping and to study the evolution of this NDV in Egypt. Materials and Methods: The present study was conducted using samples from seven suspected NDV flocks of vaccinated chickens during 2012-2016 from six governorates in Egypt. The NDV was successfully isolated from pathological specimens through inoculation in specific pathogen-free embryonated chicken eggs. Results: Pathogenicity of the NDV isolates has been estimated through intracerebral pathogenicity index and ranged from 1.66 to 1.73 which indicates the velogenic type of NDV isolates. Pathotyping and genotyping of these isolates were done through sequencing of full-length F gene. Results indicated that the seven NDV isolates showed characteristic cleavage site motif (112RRQKRF117) for the velogenic strains of NDV. Phylogenetic analysis of the F gene clustered these isolates within Group I of genotype VIId within Israeli strains NDV/IS/2015, NDV-Ch/SD883, and most of the Middle East strains. Six of seven sequenced isolates have six potential N-linked glycosylation sites. The neutralization epitope on the five antigenic sites of fusion is conserved in all Egyptian strains of this study except NDV-KFR-B7-2012 which has a substitution at D 170 N in epitope A4. In all our strains, 10 cysteine residues are recorded, except one loss of cysteine at residue 370 in both NDV-EG-35-2014 and NDV-GHB-328F-2016. Conclusion: All viruses in this study have 52 amino acid substitutions within fusion gene in compared with Lasota strain that reveals importance for its antigenic and structural function. The present work highlights the important need to sequence F gene of NDV genotype VIId to investigate the evolution of this NDV in Egypt.

First report of fowl adenovirus 8a from commercial broiler chickens in Egypt: molecular characterization and pathogenicity

&NA; This study was performed to isolate fowl adenovirus (FAdV) circulating in commercial meat‐type chicken in Egypt during 2015 and to identify the pathogenicity of the isolated virus. Cloacal swabs were collected from 9 commercial broiler farms from chickens of 3‐5 wk of age in Behira province in Egypt during 2015. FAdV was isolated on chicken embryo liver cells. The virus was identified by conventional polymerase chain reaction targeting a conserved region in the hexon gene. Moreover, phylogenetic analysis of the L1 loop of the hexon gene revealed that the isolated viruses clustered with reference strains belonging to FAdV serotype 8a. This is the first record of FAdV from Egypt on the GenBank. The isolated virus is closely related to strains directly associated with inclusion body hepatitis (IBH) causing considerable economic losses. Pathogenicity study of the virus did not show any mortality, although necropsy and histopathological examination displayed severe hepatitis and degenerative changes in the immune system after 5 d from infection, proving that the virus can cause IBH with intermittent shedding.