Agar Gel Precipitation Test Research Articles

Expression of VP2 protein of novel goose parvovirus in baculovirus and evaluation of its immune effect

Short-beak and dwarfism syndrome (SBDS) is a new disease caused by a genetic variant of goose parvovirus in ducks that results in enormous economic losses for the waterfowl industry. Currently, there is no commercial vaccine for this disease, so it is urgent to develop a safer and more effective vaccine to prevent this disease. In this study, we optimized the production conditions to enhance the expression of the recombinant VP2 protein and identified the optimal conditions for subsequent large-scale expression. Furthermore, the protein underwent purification via nickel column affinity chromatography, followed by concentration using ultrafiltration tube. Subsequently, it was observed by transmission electron microscopy (TEM) that the NGPV recombinant VP2 protein assembled into virus-like particles (VLPs) resembling those of the original virus. Finally, the ISA 78-VG adjuvant was mixed with the NGPV-VP2 VLPs to be prepared as a subunit vaccine. Furthermore, both agar gel precipitation test (AGP) and serum neutralization test demonstrated that NGPV VLP subunit vaccine could induce the increase of NGPV antibody in breeding ducks. The ducklings were also challenged with the NGPV, and the results showed that the maternal antibody level could provide sufficient protection to the ducklings. These results indicated that the use of the NGPV VLP subunit vaccine based on the baculovirus expression system could facilitate the large-scale development of a reliable vaccine in the future.

Antiviral efficacies of neem (Azadirachta indica J.) and scent (Ocimum sanctum L.) leaf meals against Newcastle and infectious bursal disease in broiler chickens

The effects of neem (Azadirachta indica) and scent (Ocimum santum) leaf meals fed singly or in combination on the growth response, haemato-biochemical parameters and their antiviral potencies in broiler chickens were investigated. Two hundred 1-day old Ross 308 unsexed broiler chicks were randomly allotted to 5 treatments, consisting of 5 replicates with 8 birds each. Treatments 1 and 2 were basal diets with no leaf meals, treatments 3, 4 and 5 had the basal diets plus 3% neem leaf meal (NLM), 3% scent leaf meal (SLM) and the blend of 2% NLM + 2% SLM, respectively. Birds in treatments 2-5 were unvaccinated against Newcastle disease and Infectious bursal disease while birds in treatment 1 were vaccinated. On days 21 and 42, blood samples were randomly collected from 2 birds per replicate for haemato-biochemical assay, haemagglutination inhibition and agar gel precipitation tests. Data were subjected to descriptive statistics and ANOVA at p<0.05. At the starter phase, an improved feed conversion ratio (p<0.05) was recorded in birds fed the blend at the starter (1.83) and finisher (1.92) phases and the overall performance (1.88) compared to other treatment groups. The least platelet counts (11.08 ×104/µL) were recorded in birds placed on the blend of 2% neem leaf meal + 2% scent leaf meal while the 3% scent leaf meal group recorded the lowest lymphocyte counts (53.60%). At the finisher phase, lymphocyte counts of birds that received the blend of 2% neem leaf meal + 2% scent leaf meal (63.00%) were higher than the other treatments. In conclusion, birds fed diets supplemented with 3% neem and 3% scent leaf meals singly had haemagglutination inhibition titre values and conferred immunity above the protective threshold against Newcastle disease and produced antibodies against Infectious bursal disease. Birds fed the blend of 2% neem + 2% scent leaf meal diets had improved overall performance.

Potential of goats’ milk from Aceh hybrid and Etawa goats in producing specific anti-S. mutans antibodies as candidate antibodies for caries immunotherapy

Background: Mother goats produce immunoglobulin G (IgG) as a specific antibody in serum, which is then transferred into milk as natural immunity for their children. Based on this, IgG from goat milk has the potential to replace the role of igg from mammalian blood in preventing caries. The goal of this study was to see if goat milk could be used to produce specific antibodies against Streptococcus mutans (S. mutans) as a candidate antibody for caries immunotherapy. Methods: In this study, pregnant goats were divided into two groups: control and treatment. The treatment group received an injection of S. mutans and then received the second injection, which was the administration of antigen added Freud adjuvant complete (booster 1) subcutaneously, and the third injection, which was the administration of antigen added Freud adjuvant complete (booster 1), and antigen added Freud adjuvant incomplete (booster 2). Blood serum was analyzed qualitatively using the Agar Gel Precipitation Test (AGPT) method to determine antibody formation. Furthermore, colostrum and goat milk were analyzed using the Sandwich ELISA method to determine the formation of anti-S mutans IgG in colostrum and goat milk. Results: Pregnant goats that had been given booster 1 and 2 with a dose of antigen + Freud Adjuvant 1.5:0.5 produced anti-S. mutans antibody titers. Normal levels of IgG in goat colostrum range from 40-60 mg/ml while in milk 0.6-7.5 mg/ml and there is a decrease in the concentration of IgG anti-S. mutans in each week. Conclusions: Colostrum and goat milk produced contain IgG-anti S. mutans antibodies that can be used to prevent dental caries.

Production of hyperimmune serum against genotype VII Newcastle disease virus in rabbits with several applications.

Objective:This study aimed to produce hyperimmune serum against genotype VII Newcastle disease virus (NDV) with several applications.Materials and Methods:Production of hyperimmune serum against genotype VII NDV was performed on eight New Zealand white rabbits divided into four groups. Rabbits were immunized three times on the 1st day, the 14th day, and the 30th day. Blood sampling was carried out on the 8th day after the third immunization.Results:All groups showed the same pattern of hemagglutination inhibition (HI) titer results. HI titers would peak on the 5th or the 9th day after the second immunization, then decrease until the 3rd day after the third immunization, and increase again on the 5th day after the third immunization. Rabbits immunized intravenously showed higher HI titers than the other groups. These results indicate that the intravenous route for hyperimmune serum production against genotype VII Newcastle disease virus greatly affects the immune response result.Conclusions:The production of hyperimmune serum by intravenous immunization three times was able to produce the highest titer of 210 at 38 days. The agar gel precipitation test and the Western blot assay showed that the hyperimmune serum was specific for the Newcastle disease antigen.

Development of SarS-Cov-2 antigen Detection Kit Based on Immunoglobulin Y (Igy) Using Surface Plasmon resonance (SPr)

SARS disease reappeared at the end of 2019 with a new name as Coronavirus Disease 2019 (COVID-19) caused by a new virus called SARS-CoV-2. This virus has spread throughout the world until recently and caused massive deaths and losses. The nucleic acid test in the form of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) is very important to diagnose COVID-19 in patients, but this method has several drawbacks such as operators who have to be trained, the diagnosis results appear in a relatively long time, and the examination price relatively expensive. This research was conducted to produce immunoglobulin Y (IgY) extracted from chicken egg yolk targeting the S-protein receptor-binding domain (RBD) on SARS-CoV-2 as a component of the surface plasmon resonance (SPR) SARS-CoV-2 antigen detection kit. This research was started by extracting IgY from hyperimmune chicken egg yolks with the polyethylene glycol (PEG) precipitation method and continued with dialysis. The extracted IgY was further purified using thiophilic adsorption chromatography and concentrated by using Amicon® Ultra-15 ultrafiltration. The IgY activity against SARS-CoV-2 RBD was tested qualitatively using the agar gel precipitation test (AGPT) technique and the total protein content was determined using the Lowry method. IgY was tested for its affinity against SARS-CoV-2 RBD using SPR. The IgY concentration obtained was 11 mg/mL. The AGPT result showed the presence of IgY activity against SARS-CoV-2 RBD isolated from egg yolk and chicken serum after 8 weeks after the first vaccination of chickens. The SDS-PAGE results showed a very clear band of IgY characters. The obtained IgY showed adequate interaction with commercial SARS-CoV-2 RBD on an SPR device. The purified IgY was able to bind with protein-S RBD and showed a fairly good affinity for the SARS-CoV-2 antigen sample. The results of these observations indicate that IgY anti-S-protein SARS-CoV-2 can be produced and purified from chicken egg yolk and can be used as a diagnostic component to detect SARS-CoV-2 antigen, especially on SPR.

Proposal of a subtype of serovar 4, K4b:O3, of Actinobacillus pleuropneumoniae based on serological and genotypic analysis

The aim of this study was to investigate an isolate of Actinobacillus pleuropneumoniae, named 14–760, which was serologically not classifiable among the recognised serovars of A. pleuropneumoniae. It reacted with the antisera raised against serovars 3, 6, 8, 15 and 17 in the agar gel precipitation (AGP) test, and was positive in the capsular serovar 4-specific PCR (cps4B PCR) assay. The isolate contains a type II capsule locus similar to serovar 4 but with variations in the length of four intergeneric regions (modF-cpxA, cpxD-cpsA, cpsC-a 114 bp orf, and lysA-ydeN), and three gene sequences (modF, cpsC and ydeN). The main difference found between the K4 and K4b cps genes is the additional 35 AAs found in type 4b due to a 4 bp insert in cps4bC. The LPS O-Ag locus is highly similar to that of reference strains of serovars 3, 6, 8, 15, 17 and 19. Isolate 14–760 is biovar 1 and contains solely the structural genes required for toxin ApxII production (apxIICA), and the type I secretion system (apxIBD) for the export of ApxII. Antiserum against isolate 14–760 adsorbed with antigen prepared from serovars 8, 15 or 17 reference strains remained reactive with isolate 14–760, but not with antigens prepared from serovars 1–18. Taken together, our results indicate the existence of a subtype of A. pleuropneumoniae, serovar 4, that we called “K4b:O3″, and we propose isolate 14–760 as the reference strain.

In-Vitro Identification of Infectious Bursal Disease Virus Isolates from Dakahlia Governorate, Egypt through the Period from 2017 to 2018.

Objective: Isolation, serological and molecular characterization of IBDV from field samples.Design: Descriptive study.Samples: A total of 55 chicken farms were studied during the period from March 2017 to August 2018. Three diseased birds were collected from each farm; their organs were pooled and considered as one sample. Procedures: The 55 collected samples were isolated on the chorioallantoic membrane (CAM) of embryonated chicken eggs (ECEs) for 3 passages and identified by changes in embryo and CAM. The embryo and CAM homogenates from the 3rd passage of 50 samples showing embryonic changes were tested by agar gel precipitation test (AGPT). Reverse transcriptase-polymerase chain reaction (RT-PCR) was done for IBDV confirmation in 10 selected isolates. PCR product of selected sample was sequenced, obtained nucleotide and deduced amino acid sequences were analyzed together with other sequences from GenBank.Results: Out of 55 tested samples, 43, 48 and 50 samples showed embryonic changes in the 1st, 2nd and 3rd passages respectively. In AGPT, 48 isolates showed precipitin lines. All RT-PCR tested isolates gave the expected band at 743 bp. Sequence analysis revealed that the studied isolate was closely related to Egyptian genogroup 3 vvIBDV with 95.8% to 98.1% identity. The studied isolate showed characteristic vvIBDV serine rich heptapeptides SWSASGS with characteristic vvIBDV VP2 hyper variable region conserved amino acids.Conclusion and clinical relevance:Nucleotide and amino acid sequences of IBDV-VP2 hyper variable region is a valuable tool for characterization of vvIBDV.

ISOLATION AND IDENTIFICATION OF ORNITHOBACTERIOSIS FROM BROILER CHICKENS IN NEW VALLEY AND ASSIUT GOVERNORATES

ABSTRACT Respiratory infections are the most dangerous diseases facing poultry industry. Ornithobacteriosis is a respiratory disease in chicken broiler farms, it causes severe economic losses (high condemnation rate at slaughter, high treatment costs). Scientific research papers should be focused on the causative agent in order to make a plan for the ideal steps for its isolation and identification, in addition to prevention and control of this infection. The present work was conducted to isolate and identify Ornithobacteriumrhinotracheale (ORT) in chicken broilers.A total of 195 samples (trachea, lungs and air sacs) were collected from dead and sacrificed broiler chickens aged (20 - 42 days old) from different broiler chickens farms in Assiut and New Valley Governorates for possible recovery of ORT. Samples were cultured on blood agar plates under 7.5 – 10 % CO2 tension at 37oC for 48 hours. Bacteriological examination used for isolation and identification of the ORT bacteria (cellular and colonial morphology, biochemical reactions, API 20 NE and growth on MacConkey's agar) showed 32 suspected isolates at percentage of 16.4%. Results of isolation by conventional method were confirmed by molecular identification using Polymerase Chain reaction technique (PCR); which revealed the positive detection of only 5 strains of ORT and produced the predicated 625 bp amplification product. Serotyping of the isolates by using agar gel precipitation test proved that all isolates belonged to serotype A. Challenge of broilers aged 14 days old with ORT strain alone or combined with live ND vaccine by spraying method caused mild respiratory manifestations with tracheitis, pneumonia and mild airsaculitis. While Infection of chicks with ORT via spray combined with prior administration of ND vaccine (stress factor) caused a moderate respiratory signs and more severe lesions. From the results obtained in this work, we can conclude that: The prevalence of ORT infection in broiler chickens in Assiut and New Valley governorates was 2.56% from living and scarified birds. The current investigation revealed that the isolates of ORT bacteria belonged to serotype A. Post mortem lesions especially yogurt like exudate in the abdominal air sac considered very pathognomonic in the tentative diagnosis of ORT infection. While PCR technique was proved to be more sensitive and specific for detection of ORT pathogen.

SEROLOGICAL SURVEY OF INFECTIOUS BURSAL DISEASE ANTIBODY IN LOCAL CHICKEN

Serological evidence for infectious bursal disease virus antibody in local chicken in Ago-Iwoye area of Ogun State was detected using agar gel precipitation test. 51 out of the 98 sera samples tested were positive for precipitating antibody against infectious bursal disease.

Recent advances in the laboratory diagnosis of peste des petits ruminants

Peste des petits ruminants (PPR) is a highly contagious and economically important, viral disease of small ruminants caused by the peste des petits ruminants virus (PPRV), which belongs to the genus Morbilivirus in the family Paramyxoviridae. PPR control is achieved mostly through vaccination and/or slaughter of susceptible animals coupled with clinical or laboratory-based diagnosis. Since clinical signs of PPR are not disease-specific and clinical diagnostics is not reliable, it should be confirmed by laboratory testing. Laboratory confirmation of clinical suspicions is made by detection of PPRV in blood, swabs or post-mortem tissues through classical virus isolation (VI), agar gel immunodiffusion (AGID)/agar gel precipitation test (AGPT), counter-immunoelectrophoresis (CIE), immunoperoxidase test (IPT) or enzyme-linked immunosorbent (ELISA) assays. However, these conventional methods have been superseded by more rapid, sensitive and accurate molecular diagnostic techniques based on the amplification of parts of either nucleocapsid (N) or fusion (F) protein gene, such as RT-PCR, real-time RT-PCR, reverse transcription loop-mediated isothermal amplification (RT-LAMP), reverse transcription recombinase polymerase amplification (RT-RPA) and Oxford nanopore MinION technology. Although these molecular diagnostic assays are accurate, rapid and sensitive, they have to be performed in laboratory settings, and samples must be transported under appropriate conditions from the field to the laboratory, which can delay the confirmation of PPRV infection. The recently developed immunochromatographic lateral flow device (IC-LFD) assay can be used in the field (“pen-side”) without the need for expensive equipment, so a well-established laboratory is not required. The control and eventual eradication of PPR is now one of the top priorities for the Food and Agriculture Organization (FAO) and the World Organization for Animal Health (OIE). In 2015, the international community agreed on a global strategy for PPR eradication, setting 2030 as a target date for elimination of the disease

Gambaran Mikroskopis Berbagai Organ Merpati yang Menunjukkan Gejala Tortikolis dan Identifikasi Virus Penyakit Tetelo Berdasarkan Uji Agar Gel Presipitasi

This study aimed to determine the microscopic conditions of organs of pigeons that suffered from torticollis and identify the cause of disease in torticollis pigeons. Three pigeons showed torticollis symptoms were obtained as sample and marked as pigeon A, B and C, respectively. Isolation of pathogen with inoculation into embryonated pigeon eggs obtained from parents with no history of vaccination and not indicated ND. Identification of ND with agar gel precipitation (AGP) test. Observation of microscopical changes with histopathologic preparation using hematoxylin and eosin staining. Histopathological examination showed that pigeons was done severe neuritis vagus, trakheitis, pneumonia, air sacculitis, hepatitis, pankreatitis, nefritis, jejunoileitis, ileocolitis and orchitis. Perivascular cuffing found in brain. Degenerative changes found in the hepar and ren. Cardiac severe necrotic lesion, and depletion in white pulp area of spleen. Proventricular tissue showed flattening of mucosal epithelium, congestion lesions found in pulmonary tissue. The results of slow hemagglutination test of pigeon egg allantoic fluid, which tested for hemagglutination (HA) showed positive HA result with titers varying between 2, 32, and 64. Serological identification carried out with the AGP test on all culture samples against ND antiserum showing positive results of ND virus. Based on these dara, it can be concluded that the pigeons with symptoms of torticollis is caused by ND virus.

Molecular Characterization of Infectious Bursal Disease Virus Isolated from Naturally Infected Broiler Chickens in Erbil, Iraq

The infectious bursal disease (IBD) is a highly contagious and immunosuppressive disease of broiler chickens and the development of a new genetic variant of the virus is responsible for major economic losses in the poultry industry. For this purpose, it was essential to isolate the molecular characterization of the virus from vaccinated broiler in Erbil, Iraq. Clinically, the infectious bursal disease is characterized by high mortality (10-15%) with hemorrhagic lesions on the breast and thigh muscles, hemorrhagic and edematous bursa of diseased chickens. In this study, the Bursa of Fabricus (BF) samples were collected between June 2018 and January 2019. Histopathological changes of the bursal sections showed existence of the cystic vacuolation of the lymphoid follicles with leukocytes infiltration as pathognomic features for IBD virus infection; and homogenates samples inoculated in chorioallantoic-membrane showed mortality in the second passage with varying degrees of hemorrhages. Agar gel precipitation test (AGPT) was positive with specific antisera. Reverse transcription polymerase chain reaction (RT-PCR) and nucleotide sequence analysis of five fragments in the hypervariable region of VP2 gene revealed transition and transversion changes. Among the five recent IBD virus isolates, the rate of identity was approximately 99% as compared with the very virulent IBD virus from Iran (ID: DQ785171.1). Phylogenetic analysis revealed that the five isolates were closely related to the Asian group with a different percentage ranged from 98-99% while it was 97% in the European group. The local isolate of the virus was registered in the Genebank under the accession number MN48052.1. In conclusion, the isolated IBDVs belong to a very virulent group. In addition, this study demonstrates the spread of this virulent virus to poultry industries in Erbil, Iraq. Further widespread surveys could help in delivering more information on the virus variability and might assist in designing novel vaccines for this pathogen.

Molecular differentiation between velogenic isolates and lentogenic LaSota strain of Newcastle disease virus

Objective: Isolation, serological and molecular identification and differentiation between velogenic and lentogenic of Newcastle disease virus (NDV) strains. Design: Descriptive study. Procedures: In this study, A total of 24 Pooled samples were collected from Dakahlia, Egypt, from 2017 to 2018 .The virus was isolated in 10 days old (ECEs) and serologically identified in third egg passage allantoic fluid by Haemagglutination (HA), Haemagglutination inhibition test (HIT) and agar gel precipitation test (AGPT). Molecular confirmation was done by reverse transcriptase-polymerase chain reaction (RT-PCR). Genetic diversity between velogenic and lentogenic NDV strains was done by RT-PCR amplification of 254bp F gene fragment from velogenic NDVs and sequence analysis of 362bp NDV F gene fragment. Results: Out of 24 tested samples, 20 samples were collected from allontoic fluid of 3rd passage positive by HA test, 18 samples by HIT, 16 samples by AGPT and 18 isolates were positive with RT-PCR amplification of 362bp F gene fragment. LaSota strain gave positive results in HA, HIT, AGPT and RT-PCR amplification of 362bp fragment. Interestingly, 254bp fragment failed to be amplified from LaSota strain. The phylogenic analysis revealed that our isolates were related to Egyptian velogenic genotype VII NDVs. The deduced amino acid sequence of the F protein cleavage site of our isolates was characteristic of velogenic NDVs (112R-R-Q-K-R↓F117) while LaSota strain had characteristic lentogenic NDVs cleavage site (112G-R-Q-G-R↓L117). Conclusion and clinical relevance: RT-PCR and sequence analysis in this study can be used for differentiation between velogenic and lentogenic NDV strains.

Conventional and Molecular Characterization of Sheep Poxvirus (SPV) in Dakahlia Governorate, Egypt

Objective: Isolation, conventional and molecular characterization of sheep poxvirus (SPV) from clinically diseased vaccinated sheep in Dakahlia Governorate, Egypt. Design: Descriptive study. Samples: Forty samples including 20 skin nodules and 20 from lung, liver, lymph node and tongue (5samples from each) were collected from sheep flock consisted of 25 adult and 5 lambs. Procedures: The suspected virus was isolated through embryonated chicken eggs (ECEs). SPV was detected by agar gel precipitation test (AGPT), indirect fluorescence antibody test (IFAT) and histopathology in field and egg passaged samples. Ten samples were selected for molecular characterization by conventional and real time polymerase chain reaction (PCR) and for sequence analysis based on P32 gene. Results: The disease prevalence was 16.7% (5 infected). Thirty-two samples (80%) recorded positive isolation of SPV (skin nodules, n=16; lung, n=5; lymph node, n=5; liver, n=3; tongue, n=3). The positive results percentage for AGPT and IFAT in field samples was 42.5% and 92.5%, respectively. In egg passaged samples, 82.5% of samples showed positivity by IFAT. Intra- cytoplasmic inclusions were detected in 75% of field samples, and in 62.5% of egg passaged samples. All selected samples for the virus molecular detection showed positive results. P32 gene sequence analysis revealed that our SPV was closely related to Saudi Arabia virus, 2019. Conclusion and clinical relevance: SPV detected from clinically diseased vaccinated sheep in Egypt. So further study is crucial to identify the reason for vaccination failure and also on precise determination of this virus origin as well as virus spread pattern analyses.

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates.

Two Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia in Japan were positive in the capsular serovar 15-specific PCR assay, but nontypeable (NT) in the agar gel precipitation (AGP) test. Nucleotide sequence analysis of gene clusters involved in the biosynthesis of capsular polysaccharide (CPS) and lipopolysaccharide O-polysaccharide (O-PS) revealed that both clusters contained transposable element ISApl1 of A. pleuropneumoniae belonging to the IS30 family. Immunoblot analysis revealed that these 2 isolates could not produce O-PS. We conclude that the ISApl1 of A. pleuropneumoniae can interfere in the biosynthesis of both CPS and O-PS.

The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells

Background: Pyocyanin (PCN), a highly pathogenic pigment produced by Pseudomonas aeruginosa, induces caspase 3-dependent human B cell (Raji cells) death. The aim of the present study, therefore, was to assess whether antigen-specific IgY antibodies may be protective on PCN-induced Raji cell death. Methods: Chickens were subcutaneously immunized with Freund's complete adjuvant containing PCN, and then given two boosted immunizations. Anti-PCN IgY antibodies were purified from egg yolk and detected using an agar gel precipitation test (AGPT) and ELISA. Protective effects of antigen-specific IgY on Raji cells were tested using a cell viability assay. Results: AGPT results showed the formation of strong immune complex precipitates, whilst ELISA further confirmed the presence of IgY antibodies specific to PCN at significant concentration. Further experiments showed that anti-PCN IgY antibodies significantly increased PCN-treated Raji cell viability in a dose-dependent fashion (p<0.05). Conclusions: The results of the present study suggest that anti-PCN IgY antibodies may be protective on PCN-induced Raji cell death.

Rapid, Sensitive, and Selective Detection of H5 Hemagglutinin from Avian Influenza Virus Using an Immunowall Device.

Avian influenza virus (AIV) infection, caused by influenza virus type A, is an infectious, acute respiratory disease of birds related to influenza outbreaks worldwide. The highly pathogenic AIV subtype H5N1 has crossed species barriers to infect mammals, including humans, with fatal outcomes and has received attention as a potential pandemic threat. A rapid and timely detection in poultry is vitally important to prevent the virus spread. Despite their great sensitivity, conventional detection methods such as real-time reverse transcription-polymerase chain reaction and the agar gel precipitation test are time-consuming and labor-intensive and require special training. In this work, an immunowall device was evaluated as an easier and faster way for detecting AIV H5-hemagglutinin (AIV H5-HA). For detection, fluorescence-labeled or enzyme-labeled antibody was employed as a labeling antibody in a sandwich immunoassay. Both were shown in this paper to be easier and faster assays for detection compared with the conventional enzyme-linked immunosorbent assay (ELISA) kit. In addition, high selectivity was achieved for AIV H5-HA detection after the evaluation of other different HA virus subtypes. The limit of detection was 0.23 ng/mL for the enzyme-labeled antibody. This value was equivalent to that of the conventional ELISA kit but 8 times faster (31 min compared to 260 min). The detection range was 0.23–100 ng/mL. The immunowall device with the enzyme-labeled antibody offers a rapid, sensitive, selective, and simple immunoassay system for future H5 AIV real sample detection.

The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells

Background: Pyocyanin (PCN), a highly pathogenic pigment produced by Pseudomonas aeruginosa, induces caspase 3-dependent human B cell (Raji cells) death. The aim of the present study, therefore, was to assess whether antigen-specific IgY antibodies may be protective on PCN-induced Raji cell death. Methods: Chickens were subcutaneously immunized with Freund's complete adjuvant containing PCN, and then given two boosted immunizations. Anti-PCN IgY antibodies were purified from egg yolk and detected using an agar gel precipitation test (AGPT) and ELISA. Protective effects of antigen-specific IgY on Raji cells were tested using a cell viability assay. Results: AGPT results showed the formation of strong immune complex precipitates, whilst ELISA further confirmed the presence of IgY antibodies specific to PCN at significant concentration. Further experiments showed that anti-PCN IgY antibodies significantly increased PCN-treated Raji cell viability in a dose-dependent fashion (p<0.05). Conclusions: The results of the present study suggest that anti-PCN IgY antibodies may be protective on PCN-induced Raji cell death.

The protective effects of antigen-specific IgY on pyocyanin-treated human lymphoma Raji cells

Background: Pyocyanin (PCN), a highly pathogenic pigment produced by Pseudomonas aeruginosa, induces caspase 3-dependent human B cell (Raji cells) death. The aim of the present study, therefore, was to assess whether antigen-specific IgY antibodies may be protective on PCN-induced Raji cell death. Methods: Chickens were subcutaneously immunized with Freund's complete adjuvant containing PCN, and then given two boosted immunizations. Anti-PCN IgY antibodies were purified from egg yolk and detected using an agar gel precipitation test (AGPT) and ELISA. Protective effects of antigen-specific IgY on Raji cells were tested using a cell viability assay. Results: AGPT results showed the formation of strong immune complex precipitates, whilst ELISA further confirmed the presence of IgY antibodies specific to PCN at significant concentration. Further experiments showed that anti-PCN IgY antibodies significantly increased PCN-treated Raji cell viability in a dose-dependent fashion (p<0.05). Conclusions: The results of the present study suggest that anti-PCN IgY antibodies may be protective on PCN-induced Raji cell death.

Isolation and identification of Marek’s disease virus (MDV) from feather follicle epithelium and internal organs of diseased chickens in Dakahlia Governorate, Egypt

In the present study, a total of 16 samples including feather follicle epithelium, ovary, spleen and kidney (4 samples for each organ) were collected from diseased chicken flocks suspected to be infected with Marek’s disease virus (MDV) at Dakahlia Governorate, Egypt during the period from October 2016 to October 2017. Each sample was pooled randomly from three to five birds (90 to 360 days old). The isolation of the suspected virus from the collected samples was carried out via chorioallantoic membranes (CAMs) of 12 days old embryonated chicken eggs (ECEs). Three egg passages were carried out for each sample. Hyperimmune serum was prepared against standard MDV. MDV in both field and egg passaged samples (after 3rd passage) was identified by agar gel precipitation test (AGPT) and indirect fluorescence antibody test (IFAT). Molecular identification of virus was carried out by conventional polymerase chain reaction (PCR) and real- time PCR in four selected samples. The results revealed that 14 samples (87.5%) including 4 (100%) samples from feather follicle epithelium, ovary and kidney and 2 (50%) samples from spleen, showed positive results in virus isolation after 3rd passage. The positive results percentage by AGPT for field samples were 50% (8 out of 16 samples), while after the 3rd passage in ECEs were 37.5% (6 out of 16 samples) and the positive results percentage by IFAT for field samples were 62.5% (10 out of 16 samples), while after the 3rd passage in ECEs were 81.25 % (13 out of 16 samples). Viral nucleic acid was detected in all selected samples by conventional and real- time PCR. The results indicate that feather follicle epithelium is the best organ for MDV detection. IFAT is superior over AGPT in virus detection. Conventional and real - time PCR could be efficiently used for molecular detection of the virus.