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Pathogenecity Trial of IBV in Diffferent Route of Inoculation in Embryonated Chichken Eggs
K. Devaki,Roshmi Mohan Kanchana,R. Narmadha
Research Journal of Poultry Sciences , 2012, DOI: 10.3923/rjpscience.2012.4.8
Abstract: Infectious Bronchitis Virus (IBV) which causes infectious bronchitis in poultry industry produces extensive economic loss to any poultry operation. Obvious failures in various vaccination programs necessitate the new vaccine development. Virus cultivation is the first step in vaccine production so, this study aimed in finding the best route of inoculation for virus cultivation in embryonated chicken eggs. The 5 and 9 days old embryonated chicken eggs were used and the inoculation of IBV is done in three routes namely yolk sac, chorio allantoic fluid and chorio allatoic membrane and the lesions were observed. More prominent observations like stunting embryo, Cashew nut appearance and expulsion of internal organs confirms that yolk sac route is the best route for cultivation of IBV.
ADAPTATION OF INDIGENOUS INFECTIOUS BURSAL DISEASE VIRUS (IBDV) IN EMBRYONATED CHICKEN EGGS
A. N. Ahmad, I. Hussain, M. Siddique and M. S. Mahmood
Pakistan Veterinary Journal , 2005,
Abstract: Infectious bursal disease virus was isolated from bursae of broilers suffering from Gumboro disease and was designated as field virus (FV). The virus was confirmed through agar gel precipitation test (AGPT) and counter current immunoelectrophoresis (CCIE). The virus was titrated by using reverse passive haemagglutination (RPHA) test and egg infective dose fifty (EID50). The FV was inoculated into 9-to 11-day-old embryonated chicken eggs through chorio-allantoic membrane (CAM). At each passage, the virus in the chorio-allantoic fluid (CAF) and embryos was confirmed by AGPT and titrated by RPHA test. Geometric mean titer (GMT) of the virus in CAF was 37 to 64 in 1-3rd passage, 111 to 239 in 4-7th passages. In 8 to 15th passages, virus titer remained from 294 to 588 and in 16-24th passages virus titer ranged from 675 to 2195. Similarly, virus titer in the embryos was 1024 to 512 in 1st -10th passages, while the virus titer in passages 11-24th ranged from 478 to 111. Embryos were monitored for lesions and mortality. Severe lesions were present on the CAM in 1st-7th passages, while moderate to mild haemorrhages were seen in 8th to 16th passages and in 17th _ 24th passages no lesions were observed.
Anti Marek’s Disease Virus Activity of Scurrula oortiana (Tea Mistletoe) Stem Extract in Embryonated Chicken Eggs*  [PDF]
S. Murtini,R. Murwani,F. Satrija,E. Handaryani
International Journal of Poultry Science , 2010,
Abstract: S. oortiana extract has been previously shown to have anti-tumour and antiviral activity in vitro. This study was carried out to investigate an antiviral effect of S. oortiana stem extract against Marek’s Disease Virus (MDV) in ovo. The antiviral effect was studied in embryonated chicken eggs (n = 5 per treatment) by inoculating 0, 0.1, 0.2, or 0.4 mg extract/egg before or after infection with MDV. In the “before” treatment, the extract was administered on day 9 with MDV infection on day 12. In the “after” treatment, the extract was administered on day 12 after infection with MDV on day 9. Histopathological examinations were performed on day 20 to determine the number of inclusion bodies, pock formation, macrophage infiltration and immunohistochemistry was used to determine the frequency and intensity of viral antigen expression. Inoculation of the extract before MDV infection significantly (p<0.05) reduced the frequency and intensity of viral antigen expression. There was also a significantly lower level of epithelial destruction on chorioallantoic membrane of the groups given 0.2 and 0.4 mg extract/egg than the control group given no extract. This study provides the first evidence of anti MDV viral activity of S. oortiana extract in vivo and may provide an alternative method to prevent or alleviate Marek’s disease.
High level expression of soluble glycoproteins in the allantoic fluid of embryonated chicken eggs using a Sendai virus minigenome system
Teresa Corral, Lorena S Ver, Geneviève Mottet, Olga Cano, Blanca García-Barreno, Lesley J Calder, John J Skehel, Laurent Roux, José A Melero
BMC Biotechnology , 2007, DOI: 10.1186/1472-6750-7-17
Abstract: Soluble forms of human respiratory syncytial virus (HRSV) and human metapneumovirus (HMPV) fusion (F) proteins, devoid of their transmembrane and cytoplasmic domains, were produced in allantoic fluids using the Sendai minigenome system. The first step was rescuing in cell cultures Sendai virus minigenomes encoding the proteins of interest, with the help of wild type Sendai virus. The second step was propagating such recombinant defective viruses, together with the helper virus, in the allantoic cavity of chicken embryonated eggs, and passage to optimize protein production. When compared with the production of the same proteins in the culture supernatant of cells infected with vaccinia recombinants, the yield in the allantoic fluid was 5–10 fold higher. Mutant forms of these soluble proteins were easily constructed by site-directed mutagenesis and expressed in eggs using the same approach.The simplicity and economy of the Sendai minigenome system, together with the high yield achieved in the allantoic fluid of eggs, makes it an attractive method to express soluble glycoproteins aimed for structural studies.Over the past decades different expression systems have been developed for production of recombinant proteins. Each of these systems has strengths and weaknesses concerning yield, cost, speed, ease of manipulation and folding and post-translational modifications of the target proteins. E. coli is the simplest and most widely used organism for protein expression due to low cost and ease of use but it has serious limitations for expression of mammalian gene products, particularly glycoproteins [1]. Unmodified yeasts, as eukaryotes, are suitable for the production of proteins that do not require mammalian-type glycosylation [2]. However, cultured animal cells still remain the best system in which to produce mammalian glycoproteins, although they have complex nutritional requirements and are sensitive to viral and bacterial contamination [1].A repertoire of animal viru
Evaluation of the Immunogenisity of Immune Complex Infectious Bursal Disease Vaccine Delivered In ovo to Embryonated Eggs or Subcutaneously to Day-Old Chickens  [PDF]
Mohammad Hassanzadeh,Mohammad Hassan Bozorgmehri Fard,Ali Tooluo
International Journal of Poultry Science , 2006,
Abstract: Immune complex vaccine of infectious bursal disease (IBD) were applied in ovo to embryonated eggs and subcutaneously to newly-hatched chickens in the hatchery, while the other group of chickens received a conventional IBD vaccine at days 12, 17 and 22 of age. At day 28 of age, ten chickens from each group were challenged with the field strain of IBD virus. Hatchability of eggs, survival of chicks, antibodies titres against Newcastle disease (ND) and IBD viruses were determined. Bursal index of post-challenged chickens were also measured. The present data indicates that in ovo IBD vaccination did not affect the hatchability of eggs or survival of hatched chicks. Vaccination with immune complex vaccine did not interfere with the degree of protection induced by ND vaccines. Moreover, this finding demonstrates that immune complex vaccine similar to that of conventional vaccine is able to provoke active immunity of birds and seem to protect chickens sufficiently from the IBD.
Highly Pathogenic Influenza A(H5N1) Virus Survival in Complex Artificial Aquatic Biotopes  [PDF]
Viseth Srey Horm, Ramona A. Gutiérrez, John M. Nicholls, Philippe Buchy
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034160
Abstract: Background Very little is known regarding the persistence of Highly Pathogenic Avian Influenza (HPAI) H5N1 viruses in aquatic environments in tropical countries, although environmental materials have been suggested to play a role as reservoirs and sources of transmission for H5N1 viruses. Methodology/Principal Findings The survival of HPAI H5N1 viruses in experimental aquatic biotopes (water, mud, aquatic flora and fauna) relevant to field conditions in Cambodia was investigated. Artificial aquatic biotopes, including simple ones containing only mud and water, and complex biotopes involving the presence of aquatic flora and fauna, were set up. They were experimentally contaminated with H5N1 virus. The persistence of HPAI H5N1 virus (local avian and human isolates) was determined by virus isolation in embryonated chicken eggs and by real-time reverse-polymerase chain reaction. Persistence of infectious virus did not exceed 4 days, and was only identified in rain water. No infectious virus particles were detected in pond and lake water or mud even when high inoculum doses were used. However, viral RNA persisted up to 20 days in rain water and 7 days in pond or lake water. Viral RNA was also detected in mud samples, up to 14 days post-contamination in several cases. Infectious virus and viral RNA was detected in few cases in the aquatic fauna and flora, especially in bivalves and labyrinth fish, although these organisms seemed to be mostly passive carriers of the virus rather than host allowing virus replication. Conclusions/Significance Although several factors for the survival and persistence of HPAI viruses in the environment are still to be elucidated, and are particularly hard to control in laboratory conditions, our results, along with previous data, support the idea that environmental surveillance is of major relevance for avian influenza control programs.
Isolation and characterization of highly pathogenic avian influenza virus subtype H5N1 from donkeys
Ahmed S Abdel-Moneim, Ahmad E Abdel-Ghany, Salama AS Shany
Journal of Biomedical Science , 2010, DOI: 10.1186/1423-0127-17-25
Abstract: Nasal swabs were collected from donkeys suffered from respiratory distress. The virus was isolated from the pooled nasal swabs in specific pathogen free embryonated chicken eggs (SPF-ECE). Reverse transcriptase polymerase chain reaction (RT-PCR) and sequencing of both haemagglutingin and neuraminidase were performed. H5 seroconversion was screened using haemagglutination inhibition (HI) assay on 105 donkey serum samples.We demonstrated that H5N1 jumped from poultry to another mammalian host; donkeys. Phylogenetic analysis showed that the virus clustered within the lineage of H5N1 from Egypt, closely related to 2009 isolates. It harboured few genetic changes compared to the closely related viruses from avian and humans. The neuraminidase lacks oseltamivir resistant mutations. Interestingly, HI screening for antibodies to H5 haemagglutinins in donkeys revealed high exposure rate.These findings extend the host range of the H5N1 influenza virus, possess implications for influenza virus epidemiology and highlight the need for the systematic surveillance of H5N1 in animals in the vicinity of backyard poultry units especially in endemic areas.Influenza A viruses belong to the family Orthomyxoviridae and have been isolated from a variety of different species. Further subtyping of influenza A viruses is based on antigenic differences between the two surface glycoproteins haemagglutinin (H1-H16) and neuraminidase (N1-N9) of the influenza A viruses [1,2]. The HA mediates the attachment of the virus to sialic-acid-containing receptors on the host cell surface, as well as the fusion of the virus envelope with the cellular membrane [3,4]. The specificity of the HA towards these molecules differs. Avian and equine influenza viruses preferentially bind the sialic acid α-2,3-galactose (SAα2,3Gal) linkage, while human influenza viruses preferentially bind the SAα2,6Gal linkage [5-7]. The highly pathogenic avian influenza virus H5N1 (HPAIV- H5N1) represents an important poultry pathog
Genetic drift evolution under vaccination pressure among H5N1 Egyptian isolates
Ahmed S Abdel-Moneim, Manal A Afifi, Magdy F El-Kady
Virology Journal , 2011, DOI: 10.1186/1743-422x-8-283
Abstract: H5N1 was isolated from the pooled organ samples of four different affected flocks in specific pathogen free embryonated chicken eggs (SPF-ECE). A reverse transcriptase polymerase chain reaction (RT-PCR) was performed to the haemagglutingin and neuraminidase. Sequencing of the full length haemagglutingin was performed. Sequence analyses of the isolated strains were performed and compared to all available H5N1 from Egyptian human and avian strains in the flu database. Changes in the different amino acid that may be related to virus virulence, receptor affinity and epitope configuration were assigned and matched with all available Egyptian strains in the flu database.One out of the four strains was found to be related to the B2 Egyptian lineage, 2 were related to A1 lineage and the 4th was related to A2 lineage. Comparing data obtained from the current study by other available Egyptian H5N1 sequences remarkably demonstrates that amino acid changes in the immune escape variants are remarkably restricted to a limited number of locations on the HA molecule during antigenic drift. Molecular diversity in the HA gene, in relevance to different epitopes, were not found to follow a regular trend, suggesting abrupt cumulative sequence mutations. However a number of amino acids were found to be subjected to high mutation pressure.The current data provides a comprehensive view of HA gene evolution among H5N1 subtype viruses in Egypt. Egyptian H5N1-AIVs are constantly undergoing genetic changes and reveal a complex pattern of drifts. These findings raise the concerns about the value of using influenza vaccines in correlation with the development of antigenic drift in influenza epidemics.The highly pathogenic H5N1 influenza virus is currently panzootic in Egyptian poultry populations and crosses species barriers to humans and animals. H5N1 viruses are classified into 10 distinct initial clades (numbered 0-9) based on the HA gene [1]. H5N1 virus strains from European-Middle Eastern-
Genetic characterization of 2008 reassortant influenza A virus (H5N1), Thailand
Alongkorn Amonsin, Jiradej Lapkuntod, Kamol Suwannakarn, Pravina Kitikoon, Sanipa Suradhat, Rachod Tantilertcharoen, Supanat Boonyapisitsopa, Napawan Bunpapong, Manoosak Wongphatcharachai, Trong Wisedchanwet, Apiradee Theamboonlers, Yong Poovorawan, Jiroj Sasipreeyajan, Roongroje Thanawongnuwech
Virology Journal , 2010, DOI: 10.1186/1743-422x-7-233
Abstract: H5N1 influenza A virus has caused avian influenza (AI) outbreaks worldwide. In Thailand, 7 major AI outbreaks have been reported since early 2004 [1-3]. In January 2008, outbreaks of H5N1 virus occurred in two provinces, Nakhon Sawan and Phichit. The outbreak in Nakhon Sawan affected 60,000 birds in a broiler farm and chicken in nearby backyards, while the outbreak in Phichit occurred among backyard chicken. In November 2008, H5N1 outbreaks were also reported in two provinces, Sukhothai and Uthai Thani. Both outbreaks occurred among backyard poultry in villages (Fig 1). Currently, at least two clades of influenza A virus (H5N1) have been reported in Thailand including clade1 viruses which are predominant in lower-north and central Thailand and clade2.3.4 viruses which are predominant in northeast Thailand [1,3,4]. Clade1 H5N1 viruses in Thailand have been further divided into 3 distinct subclades including the original clade1 (CUK2-like), clade1.p1 (PC168-like) and clade1.p2 (PC170-like) [3,5]. One study has documented evidence of genetic reassortment of H5N1 viruses in Thailand in 2007 [6]. In this study, we have comprehensively characterized the 2008 H5N1 viruses recovered during the 6th and 7th waves of AI outbreaks in Thailand. The 2008 H5N1 viruses were compared with H5N1 isolates obtained from each wave of AI outbreaks in Thailand. The whole genome sequences of the viruses were analyzed for nucleotide identity, genetic relatedness, virulence determinants, and possible sites of reassortment among H5N1 viruses.Eight H5N1 viruses were isolated from Nakhon Sawan (n = 3), Phichit (n = 1), Sukhothai (n = 2) and Uthai Thani (n = 2) (Table 1 and Fig 1). The viruses were isolated by embryonated egg inoculation [7]. All 8 viruses were confirmed as Influenza A virus subtype H5N1 by real-time-RT-PCR [8]. Whole genome sequences were obtained as previously described [9]. Phylogenetic and genetic relatedness analyses were conducted using the MEGA 4.0 program applying the nei
Establecimiento de una línea celular primaria a partir de huevos con embrión de Toxocara canis Establishing a primary cell line from Toxocara canis embryonated eggs  [cached]
Fernando Iván Marín,Jhon Carlos Casta?o
Infectio , 2011,
Abstract: Introducción. Toxocara canis es el segundo nematelminto más prevalente en perros a nivel regional y entre los tres más frecuentes en algunos países de la región. Debido a que la fuente de contaminación es el perro, éste se convierte en un nematodo con gran potencial zoonótico. Por esta razón, consideramos importante disponer de una línea celular de este helminto para el estudio de los aspectos básicos, así como para el desarrollo de técnicas diagnósticas. Objetivo. Obtener una línea celular primaria a partir de huevos con embrión de T. canis. Métodos. Los parásitos se extrajeron del intestino delgado de perros menores de un a o. Las células embrionarias se obtuvieron mediante la embriogénesis de los huevos de los nematodos adultos, en cuatro diferentes medios; dos ricos en sustancias nutritivas, el tercero con formol al 1 % y el cuarto con agua destilada. Las células se obtuvieron mediante disociación mecánica de los huevos con embrión mediante la utilización de jeringas 30G. Resultados. El tiempo estimado de obtención de la línea celular fue de 15 días, en los que siete eran utilizados en la embriogénesis de los huevos. Las células respondieron positivamente a los métodos de crioconservación luego de dos días, e inclusive dos meses después, permitiendo fases de replicación de cuatro pases. Conclusiones. Se logró obtener una línea celular de T. canis a partir de huevos con embrión de este helminto. Esta línea celular ayudará al entendimiento de las relaciones patógenas, posibles blancos terapéuticos y para el desarrollo de métodos diagnósticos. Introduction: Toxocara canis is the second most prevalent nemathelminthes in dogs at regional level and among the three most frequent in some countries in the region. Due to the fact that the dog is the contamination source, it becomes a nematode with a high zoonotic potential, so we consider it important to be able to use the cell line of this helminth to study the basic aspects, as well as the development of diagnostic techniques. Objective: To obtain a primary cell line from embryonated eggs of T.canis. Methods: The parasites were extracted from the small intestines of dogs under one year old. Embryonic cells were obtained by embryogenesis of the eggs secreted by adult worms in four different media; two were rich in nutrients, one was 1% formaldehyde, and the other was distilled water. The cells were obtained by mechanical dissociation of embryonated eggs using 30G needles. Results: The estimated time for obtaining the cell line was fifteen days, from which seven were used for egg embryogenesis. The cells resp
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