Egg Drop Syndrome Virus Research Articles

Outbreak of Egg Drop Syndrome in Bangladesh

An incident alike to egg-drop syndrome (EDS) occurred in commercial egg laying chickens in Gazipur district,Bangladesh from July to November 2005. The chickens showed decreased egg production along with production ofshell less and soft shelled eggs when they were at predicted peak production age. Nutritional status, especially crudeprotein (16.65-18.50%) and calcium (2.15-3.62%) content of the supplied feed was within the range. Virological andserological investigations on causes of decreased egg production and production of soft-shelled and shell less egg by thelaying hens revealed that the responsible causative agent is EDS virus. Total seven EDS viruses were isolated fromcloacal swab (4), soft-shelled eggs (2) and feces (1). The isolation rate 71.43% (5/7) was higher in second passage thanin first passage 28.57% (2/7) in duck embryos. None of the 7 viruses grew in chicken embryo even after second passage.All five flocks tested, were vaccinated against infectious bronchitis and Newcastle disease along with other commonvaccines generally used in Bangladesh but not against EDS. Serological investigation revealed that birds in all testedfarms (100%) had antibody against EDS virus (in 2 farms in 100% samples and in 3 farms in 80% samples). The sourceof the virus remained to be determined. To the best of our knowledge, this is the first report of EDS outbreak inBangladesh. To save poultry industry in Bangladesh from harm of this disease the biosecurity measures should bestrictly followed along with vaccination against the disease.

Study on the areca nut for its antimicrobial properties

This study evaluates the antibacterial, antifungal, and antiviral properties of the areca nut in vitro using isolated organisms. A variety of human and veterinary isolates, both gram positive and gram negative, were tested against areca nut extract by measuring the growth of the organisms using the spectrophotometric method. It was found that both gram negative and gram positive organisms were susceptible to the areca nut extract. The concentration needed for 100% inhibition of growth was found to be 3.3-7 µg/ml for gram negative organisms and 16 µg/ml for gram positive bacteria. The extract was also found to inhibit the growth of Candida albicans at a concentration of 16 µg/ml and inhibited as atoxin production by Aspergillus fl avus . The extract was also found to inhibit the viral growth of the New Castle Disease Virus (NDV) and Egg Drop Syndrome Virus (EDS) grown in embryo cultures. These results indicate that betel nut chewing may have signiÞ cant disinfective properties. Key words: Areca nut, antibacterial activity, antifungal, antiviral activity

Recombinant egg drop syndrome subunit vaccine offers an alternative to virus propagation in duck eggs

Egg drop syndrome (EDS) virus vaccines are routinely produced in embryonated duck eggs (Solyom et al., 1982). This procedure poses the risk of dissemination of pathogens, such as avian influenza virus, as the eggs used are not from specific pathogen free birds. To address this problem, the knob and part of the shaft domain of the fibre protein of the EDS virus (termed knob-s) were expressed in Escherichia coli and assessed as a subunit vaccine. A single vaccination with the recombinant protein induced the production of anti-EDS virus antibodies, as detected by haemagglutination inhibition, enzyme-linked immunosorbent assay and virus neutralization tests, for at least 20 weeks. A positive correlation was demonstrated between these three assays. A dose–response assessment showed that the vaccine was effective over the range of 2 to 64 µg protein per dose. Two vaccinations with the recombinant protein, administered before the onset of lay, induced high haemagglutination inhibition antibody titres, comparable with those induced by an inactivated whole-virus vaccine. The vaccine did not have any adverse effects on egg production, quality or weight. The present study has shown that two vaccinations with the recombinant knob-s protein elicited high neutralizing antibody titres that persisted for more than 50 weeks of lay.

Application of polymerase chain reaction in detection of egg drop syndrome (EDS) virus.

Egg drop syndrome (EDS) is caused by a hemagglutinating adenovirus which has become a major cause of lost egg production and sever economic losses in breeder and layer chicken flocks throughout the world. A PCR assay was optimized for detection of EDS virus in inoculated allantoic fluids (AFs) of duck and chicken emberyonated eggs. Two strains of EDS viruses were propagated in allantoic cavity of eggs. Then the virus DNA was extracted. PCR test was designed and carried out by specific primers. The 1900 bp band was detected in agar gel electrophoresis. Serial two fold dilutions of infected allantoic fluid were prepared. The HA test and PCR assay were carried out for each dilution and finally results were compared to each other. The PCR assay could detect some negative HA titers. This study clearly indicates the superiority of PCR assay over HA test for detection of EDS virus in AFs.

Identification and Characterization of Hemagglutinating Viruses in Native Chickens in Bangladesh

2 Abstract: A total number of 224 cloacal swabs were examined for hemagglutinating activity from 4 different poultry farms of Noakhali district in Bangladesh. Out of the total samples 15 exhibited positive reactions with chicken erythrocytes. Among the 15 positive samples, 11 were found to be positive for Newcastle disease virus. The highest (100%) prevalence of Newcastle disease virus was found in Ram Nagar union and the lowest (66.66%) were found in Rajapur union. Seventy five percent prevalence was found in Jaylashker and Matubhuyian union. The overall prevalence of Newcastle disease virus was found to be 79.17% in the four unions. No egg drop syndrome virus and avian influenza viruses were identified in the samples those gave positive result in hemaggluntination test.

Detection of Antibodies to Egg Drop Syndrome Virus in Chicken Serum Using a Field-Based Immunofiltration (Flow-Through) Test

A simple, user-friendly, and rapid method to detect the presence of antibodies to egg drop syndrome 76 (EDS) virus in chicken sera based on an immunofiltration (flow-through) test was developed. Purified EDS virus antigen was coated onto nitrocellulose membranes housed in a plastic module with layers of absorbent filter pads underneath. Following addition of serum to be tested and washing, monoclonal antibodies or polyclonal serum to chicken immunoglobulin G (IgG) was used as a bridge antibody to mediate binding between EDS virus-specific IgG and protein A gold conjugate. The appearance of a pink dot indicated the presence of antibodies to EDS virus in the sample tested. The results could be obtained within 5-10 min. The developed immunofiltration test could detect antibodies in the sera of experimentally vaccinated chickens from 2 wk postvaccination. With field sera samples, this test was positive in samples having hemagglutination inhibition titers of 8 and above. This test has the potential to be used as a field-based kit to assess seroconversion in EDS-vaccinated flocks.

Naturally Occurring Egg Drop Syndrome Infection in Turkeys

A decrease in the egg quality, production, fertility and hatchability without serious clinical signs of illness was recorded in turkey fl ocks in Croatia at the beginning of 2002. It was assumed that the egg drop syndrome virus might be one of the etiological agents responsible for the abnormalities in the egg production. The systematic serological monitoring, using a haemagglutination inhibition test, showed that the antibodies to the egg drop syndrome virus existed in 94.4 and 55.1% of the sera analysed in 2002 and 2003, respectively. The haemagglutination inhibition titres ranged from 16 to 128. The sera samples were randomly collected from 11 - to 46-week-old hens from the affected fl ocks. The serological evidence of the egg drop syndrome virus infection was confirmed by detection of the presence of the virus genome in the turkey sera by the polymerase chain reaction. Vaccination of the 18- and 25-week-old turkey hens against the egg drop syndrome virus started in March 2003. After this period, the presence of antibodies to the egg drop syndrome virus (the haemagglutination inhibition titres between 16 and 256) was found in 96.7% of the analysed sera, while the egg production reached normal or higher values for the Nicholas hybrid line of turkeys.

Dynamics of Macrophages in Laying Hens during Second and Third Production Cycles after Zinc Induced Molting

The influence of a non-feed removal molting program on certain parameters of cell mediated and humoral immune response of the laying birds during 2nd and 3rd production cycles were recorded. Birds at the end of their 1st production cycle were induced to molt through the supplementation of high dietary zinc oxide (3000mg/Kg) for their 2nd and 3rd production cycles. Macrophage engulfment (%) and engulfment/cell were significantly increased after the use of opsonized sheep red blood cells (SRBC) for co-incubation during 2nd production cycle. Lipopolysacchrides (LPS) greatly enhanced the production of nitric oxide, as compared to the SRBC stimulation during the 2nd production cycle. Zinc feeding strengthened the antibody response against Newcastle disease virus (NDV) and egg drop syndrome virus (EDS) at the peak of the 2nd and 3rd production cycles, but decreased at 5% production stages. NDV also provoked a better antibody response when compared to EDS. Zn concentration in plasma showed a positive correlation with an increase in egg production. Our results validate the strengthened immune response during the 2nd production cycle after zinc-induced molting.

Phylogenetic analysis of the hexon loop 1 region of an adenovirus from psittacine birds supports the existence of a new psittacine adenovirus (PsAdV)

Adenovirus infections in psittacine birds have been well known. Most of these infections were caused by fowl adenoviruses (FAdV). In this study, liver samples showing typical histological signs of an adenovirus infection were collected from Poicephalus spp. with acute disease. A PCR amplifying the variable loop 1 region of the hexon gene was developed using primers located in two conserved pedestal regions. A PCR product of approximately 590 bp in size was amplified and sequenced. The sequence obtained grouped outside of the FAdV reference strains of the 12 serotypes as well as egg drop syndrome virus and turkey adenovirus 3 indicating that a new avian adenovirus was detected. In comparison to the FAdV reference strains, the percentage of identical nucleotides ranged between 60.3 and 67.0 and that of identical amino acids (aa) between 51.3 and 61.0. Furthermore, 37 unique aa exchanges were observed; out of these, 27 are located in the 4 hypervariable regions of loop 1, which encode the serotype-specific epitopes. The g/c content, the isoelectric point and the charge of the amplified fragment, however, are in the range as those of group I avian adenoviruses. It was proposed, therefore, to designate this new adenovirus as psittacine adenovirus (PsAdV).

Comarative studies on attenuated and inactivated oil emulsion egg DROP syndrome (EDS) virus vaccine prepared on chicken liver cell culture and duck eggs vaccine

Comarative studies on attenuated and inactivated oil emulsion egg DROP syndrome (EDS) virus vaccine prepared on chicken liver cell culture and duck eggs vaccine

An Aluminium Hydroxide Gel Adsorbed Inactivated Egg Drop Syndrome Vaccine

An inactivated vaccine was prepared from local isolates of egg drop syndrome (EDS) virus using aluminum hydroxide as an adjuvant. A total of 30 commercial layers at the age of 16 weeks were procured and divided in to two groups, A and B having 20 and 10 birds, respectively. The birds of group A were vaccinated with 0.5ml aluminium hydroxide gel adsorbed EDS vaccine. The birds of group B were kept as unvaccinated control. The serum samples were collected at a 14-day interval till 6 weeks post vaccination. At 22 weeks of age group A was again divided into two groups A and A Birds of 1 2. group A and group B were challenged with a virulent EDS virus. The results revealed that birds of group 2 A had HI titer ranging from 1:128 to 1:512 at different times of sample collection with a geometric mean titer 116. Highest antibody response appeared on 4th week post vaccination. Results also revealed that the vaccination protected the birds against challenge with the virulent EDS virus. Where there was a significant drop in egg production in unvaccinated challenged birds. The egg quality was also deteriorated as miss-shapened, soft-shelled and shell-less eggs were laid in EDS unvaccinated layers.

Isolation and characterization of virus of highly pathogenic avian influenza H5 subtype of chicken from outbreaks in Indonesia

A study on the isolation and characterization of Highly Pathogenic Avian Influenza of chicken from outbreaks in Indonesia was conducted at Indonesian Research Institute for Veterinary Science. Outbreaks of avian disease had been reported in Indonesia since August 2003 affecting commercial layer, broiler, quail, and ostrich and also native chicken with showing clinical signs such as cyanosis of wattle and comb, nasal discharges and hypersalivation, subcutaneous ptechiae on foot and leg, diarre and sudden high mortality. The aim of this study is to isolate and characterize the causal agent of the disease. Samples of serum, feather follicle, tracheal swab, as well as organs of proventriculus, intestine, caecal tonsil , trachea and lungs were collected from infected animals. Serum samples were tested haemaglutination/haemaglutination inhibition to Newcastle Disease and Egg Drop Syndrome viruses. Isolation of virus of the causal agent of the outbreak was conducted from samples of feather follicle, tracheal swab, and organs using 11 days old specific pathogen free (SPF) embryonated eggs. The isolated viruses were then characterised by agar gel precipitation test using swine influenza reference antisera, by haemaglutination inhibition using H1 to H15 reference antisera, and by electron microscope examination. The pathogenicity of the viruses was confirmed by intravenous pathogenicity index test and its culture in Chicken Embryo Fibroblast primary cell culture without addition of trypsin. The study revealed that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus based upon serological tests, virus isolation and characterization using swine influenza reference antisera, and electron microscope examination. While subtyping of the viruses using H1 to H15 reference antisera suggested that the virus is very likely to be an avian influenza H5N1 subtype virus. The pathogenicity test confirmed that the viruses are highly pathogenic to experimental animals. It is concluded that the causative agent of the outbreaks of avian disease in Indonesia was avian influenza H5 subtype virus. The result has been the basis of further study such as development serological tests and vaccine production. The decission of Indonesian Government to conduct vaccination program using homolog vaccine in order to control the disease is regarded as the correct choice. However, it should be accompanied by conducting surveillance and monitoring of the disease as well as the possibility of mutation of virus. The program should be coordinated nationally. Key words: Virus isolation, characterization, chicken, outbreak, highly pathogenic avian influenza (HPAI), H5 subtype, Indonesia

No evidence for adaptation of current egg drop syndrome 1976 viruses to chickens.

In order to determine whether the current field strains of egg drop syndrome (EDS) 1976 viruses adapt to chickens, we compared the growth efficiency of three Japanese field strains (PA-1/79, AWI/98, Gifu/01) in chicken and duck embryo liver cells. The growth efficiency in chicken or duck embryo liver cells was almost similar in these strains. The fiber protein may carry the type-specific antigen and the hemagglutination activity, and hexon protein may contain the subgroup-specific antigenic determinants. Therefore, the fiber head and hexon loop 1 DNA domain sequences of the six Japanese field strains UPA-1/79, ME/80, 44/81, Kyoto/91, AWI/98, Gifu/01) were compared, but these DNA domains were identical among the six field strains. Our data suggested that the EDS virus was maintained without discernible changes for the last two decades in the field.

Detection of egg drop syndrome virus antigen or genome by enzyme-linked immunosorbent assay orpolymerase chain reaction

Mouse monoclonal antibodies (mAbs) were produced against an Indian isolate of egg drop syndrome (EDS)virus and characterized. Four hybridoma clones were secreting mAbs that bound to a 100 kDa protein,presumably the hexon protein. These mAbs were found to cross-react with two other Indian isolates of EDSvirus and to the reference UK 127 strain. Three of these mAbs were mapped to the same epitope compared withthe other mAb (F8), which bound to a different epitope. An antigen-capture enzyme-linked immunosorbentassay (AC-ELISA) was developed using the F8 mAbs as capture antibody and polyclonal chicken serum againstEDS virus as detection antibody. A polymerase chain reaction (PCR) was used to detect the EDS viral genome.Following experimental infection of oestrogen-treated chickens with EDS virus, cloaca1 swabs, oviduct, uterusand spleen were collected at different days post-infection and used in both AC-ELISA and PCR, directly andafter a single passage in embryonated duck eggs. The sensitivity and specificity of antigen detection by ACELISAor PCR was 95% and 98%, respectively. For diagnosis of EDS viral infections, PCR is recommendeddue to its ease and the lack of requirement of prepared reagents such as mAbs or conjugates. We recommendthat PCR be performed directly on boiled tissue homogenates. Any negative samples may be passaged inembryonated duck eggs and the allantoic fluids tested by PCR before a conclusive negative diagnosis is given.

A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein

In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested. The fiber protein is responsible for binding the virus to the target cell. The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein. The hexon, fiber capsid protein and knob-s were produced in E. coli and injected into chickens. Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity. Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control—whole virus vaccine. We assume that production of only part of the fiber enables the protein produced in E. coli to fold correctly. Antibodies produced against the hexon protein showed no SN activity. In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber. The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.

Genetic organization and sequence analysis of pVIII, fiber and early region 4 of bovine adenovirus type 7.

The DNA sequence of 8,810 nucleotides at the right end of bovine adenovirus type 7 (BAV7) genome was determined and compared with similar regions of other adenoviruses. This genomic region of BAV7 consists of sequences encoding partial 33K, pVIII, fiber, putative early region 4 (E4) proteins and other unassigned proteins. However, BAV7 E3 region is not present in the expected location between pVIII and fiber as BAV7 intergenic region between pVIII and fiber genes is only 183 nucleotides. The predicted pVIII and fiber demonstrates highest homology to corresponding proteins of ovine adenovirus 287 (OAV287), bovine adenovirus-4 (BAV4) and egg drop syndrome virus (EDSV). The E4 region encodes three ORFs, which shows significant homology only to corresponding proteins encoded by E4 region of OAV287 and BAV4. Sequence comparisons, phylogenetic analysis and overall genome organization in this region of BAV7 provide further evidence for the inclusion of BAV7 together with OAV287, BAV4, and EDSV in the proposed genus 'Atadenovirus'.

The role of egg drop syndrome virus in acute respiratory disease of goslings

An outbreak of severe acute respiratory disease characterized by tracheitis and bronchitis was observed in young goslings on a large-scale goose farm in Hungary. Histological examination revealed amphophilic intranuclear inclusion bodies in the superficial epithelial cells of the trachea and bronchi. Adenovirus-like particles were detected by electron microscopy, and the virus isolated from the trachea and the lungs was identified as egg drop syndrome (EDS) virus by serological and genomic examination. The clinical and pathological signs were reproduced by intratracheal administration of the virus isolate to 1-day-old goslings free of EDS antibodies. The presence of EDS virus DNA in different organs of the naturally and experimentally infected goslings was detected by polymerase chain reaction. This is the first report on the involvement of EDS virus in severe respiratory disease of geese.

An in vitro and in vivo evaluation of the virulence of egg drop syndrome virus for the chicken reproductive tract

The virulence of strains of egg drop syndrome (EDS) 1976 virus for the female reproductive tract of chickens was assessed in vitro using oviduct organ cultures (OOC) prepared from precociously induced oviducts in young chicks by oestrogen treatment. Ciliostasis, haemagglutination and virus titres in infected OOC supernatants, histology and immunoperoxidase test results indicated the pathogenic ability of the four viruses for the precocious oviducts. One of the isolates, EDS TN4, produced higher virus titres in the supernatants of infected OOC and more severe glandular atrophy and necrosis, but caused slightly delayed ciliostasis. When this isolate was used in vivo, virus could not be detected by haemagglutination, but was detected in a few birds using a polymerase chain reaction on the allantoic fluids of infected duck embryos. Ciliostasis of OOC and histological lesions were confined to early stages of infection. This technique could be a pointer to possible variations in virulence of EDS virus isolates, and warrants further investigation. The potential value of OOC from young chickens for EDS diagnosis is emphasized.

The complete genomic sequence of egg drop syndrome virus strain AAV-2

In the search for the genome of egg drop syndrome virus (EDSV-76) Chinese strain AAV-2, part of restriction endonuclease physical map is analyzed, the complete genomic library is organized. On basis of this, the complete genome nucleotide sequences (32 838 bp in length, including terminal structures) are determined. The data analysis shows: compared with the other Adenoviruses, strain AAV-2 has more disparity on genomic structure and the distribution of open reading frame (ORF). There are no clear E1, E3 and E4 regions in AAV-2 genome. Two segments located at both ends of genome (1.1 kb and 8.3 kb in length respectively) have no homology with the other adenovirus genomes. In addition, strain AAV-2 genome lacks ORFs encoding ElA, pV and pIX, which are common ORFs encoding early, lately proteins in Adenovirus. This reveals differences between EDSA-76, the sole standard strain of group III Avian Adenoviruses, and the other Avian Adenoviruses for the first time. It will help the search for Avian Adenovirus and will also help the search of all Adenoviruses.

Hexon based PCRs combined with restriction enzyme analysis for rapid detection and differentiation of fowl adenoviruses and egg drop syndrome virus

Three different polymerase chain reactions (PCRs), two of them combined with restriction enzyme analysis (REA), were developed for detection and differentiation of all 12 fowl adenovirus (FAV) serotypes and the egg drop syndrome (EDS) virus. For primer construction FAV1, FAV10 and EDS virus hexon proteins were aligned and conserved and variable regions were determined. Two primer sets (H1/H2 and H3/H4) for single use were constructed which hybridize in three conserved regions of hexon genes. Each primer pair amplifies approximately half of the hexon gene including two loop regions. An amplification product was detected with both primer sets using purified DNA from all FAV1-12 reference strains. Viral EDS DNA was negative using the H1/H2 or H3/H4 primer pair. HaeII digestion of the H1/H2 amplification products differentiates between all viruses except FAV4 and FAV5. In comparison, much more clustering among genomic closely related FAV serotypes was seen after HpaII digestion of the H3/H4 PCR products. Oligonucleotides H5/H6 located in the variable regions of EDS virus hexon gene do not detect any of the FAV serotypes. The PCRs and REA described are suitable to detect all avian adenoviruses infecting chickens, to distinguish all 12 FAV reference strains and to differentiate FAVs from the EDS virus.