Characterization of recombinant baculovirus expressing envelope glycoprotein E of the local BoHV-1

A recombinant baculovirus expressing envelope glycoprotein E [gE] of the local Bovine Herpesvirus type-1 [BoHV-1] "Abu-Hammad strain" was characterized. PCR analysis was conducted on the genomic DNA of recombinant baculovirus to verify the presence of the inserted gE gene. Spodoptera frugiperda [Sf9] cells infected with the recombinant baculovirus expressed a high level of immunoreactive recombinant gE protein that was demonstrated by indirect immunoflurescence and Western blot analyses. The recombinant gE protein was used as a coating antigen in an indirect ELISA for detection of BoHV-1 anti-gE antibody to differentiate cattle vaccinated with the local of BoHV-1 "Abu-Hammad strain" from those vaccinated with gE negative marker vaccine. A limited number of bovine serum samples which previously tested by the indirect ELISA, virus blocking gE ELISA [IDEXX] were used to be tested by indirect ELISA. The obtained results proved the reactivity of the recombinant gE protein and its utility as a diagnostic antigen in a gE based indirect ELISA for diagnosis of BoHV-1 as well as distinguishing infected animals within a gE marker vaccinated herd

Molecular detection, isolation and characterization of avian rotavirus from broiler chicken

Chicken rotaviruses [CRVs] were detected, identified and characterized in broiler chicken with diarrhea for the first time in Egypt Fecal samples were collected from 85 naturally occurring diarrheal outbreaks in commercial chicken broilers fmis that are located in a wide range of geographical areas including many Egyptian govemorates mainly 6th Octobar, Elfayom, Giza, Qaluobea, Menofla and Elmansoura during year 2008. CRV was detected in the fecal samples by ELISA using monoclonal antibodies [Mabs] against VP6, Electron Microscopy [EM], Reverse Transcription-Polymerase Chain Reaction [RTPCR] and the virus was isolated using SPF chickens. The obtained results confirmed the isolation and identification of group A chicken rotavirus while the molecular characterization analysis using different primers sets suggested that the isolated chicken rotavirus does not belong to the same cluster of Chi rotavirus strain but most likely more related to Po-13 strain [mammalian like chicken rotavirus strains]. This study reports the importance of rotaviruses in broiler chickens with delayed growth and diarrhea

Improved quality control protocol for fowl pox virus vaccines based on the use of PCR and sequence analysis to detect REV and ALV as contaminants

In the present study, twenty one Fowl pox virus [FPV] vaccines were collected in the period between 2003-2005 and tested using the currently used and an improved quality control protocols. Results of currently used quality control protocol revealed negativity of all tested vaccines for the presence of contaminants and the results were satisfactory. Using PCR to detect Reticuloendotheliosis virus [REV] as contaminant in such vaccines revealed negative result except one suspected contaminated vaccine. Inoculation of this vaccine in egg, chicken embryo fibroblasts [CEFs], Specific pathogen free [SPF] chicks for three passages and testing of samples collected from inoculated host revealed positive amplification using REV specific primers. Sequence analysis of the obtained amplification fragment for REV revealed its negativity and confirmed the non specific amplification of such primers which were previously published in several PCR studies for REV. Using avian leucosis virus [ALV] sets of primers to detect groups A, B, C, D and J in a PCR reaction revealed positive amplification of ALV fragment and confirming the contamination of tested vaccine with ALV. The study proposes the importance of using PCR followed by sequence analysis of the amplified product to confirm the contaminations fonnd in the FPV vaccines

Effect of wheat germ oil and ascorbic acid supplementation on the immune response of broiler chicks to ND vaccines

In the present study, the effect of wheat germ oil as source of vitamin E and ascorbic acid as source of vitamin C on the immune response of broiler chicks to ND vaccine was determined. Four groups of chicks were used. Group 1 was vaccinated with 2 doses of ND vaccines [Hitchner then Lasota] and simultaneously supplemented with wheat germ oil via oral administration for 15 days. Group 2 was vaccinated with 2 doses of live ND vaccines [Hitchner then Lasota strains] and simultaneously supplemented with ascorbic acid via oral administration for 15 days. Group 3 was vaccinated with 2 doses of ND vaccines [Hitchner then Lasota strains]. Group 4 was kept non-vaccinated as control chicks. All birds were monitored weekly for the humeral and cellular immune response then challenged with the virulent NDV at 35 days of age. HI test for titration of antibodies for NDV, phagocytic activity, Nitric oxide, Lysozyme activity and total antioxidant in serum were used to determine the immune response of the chicks. Protective immunity induced in the vaccinated groups was varied. 80 and 60% protection were obtained in chicks vaccinated and supplemented with wheat germ [group 1] and ascorbic acid [group 2]; respectively. Live ND vaccinated group showed 60% protection. The present study reports the effect of supplementation of wheat germ [vitamin E] as immunostimulant in augmentation of the immune response ND vaccines

Protective immunity induced by co-injection of mixture of Mannan and B-glucan immunostimulant substances with the inactivated Bivalent Al-ND vaccine in broiler chickens

In the present study, the effect of a mixture of some immunostimulant substances on the immune response of broiler chicks to bivalent Al-ND vaccine was determined. Four groups of chicks were used. Group .1 was vaccinated with one dose of Al-ND vaccine and simultaneously injected with the mixture of immunostimulant substances. Group 2 was vaccinated with one dose of the bivalent AI.-ND vaccine. Group 3 was vaccinated with 2 doses of live ND vaccines [Hitchner then Lasota drains] and group 4 was kept none vaccinated control chicb. All birds were monitored weekly for the humoral and cellular immune response then challenged with the virulent NDV at 35 days of age. HI test was used for titration of antibodies for both AIV and NDV, phagocytic activity, Nitric oxide; Lysozyme activity and total antioxidant in serum were used to determine the cellular immune response of the chicks. Protective immunity induced in the vaccinated groups varied. The injected immunostimulant mixture demonstrates its effect on the immune response to the bivalent Al-ND vaccine in group 1 with 100% protection against the challenge NDV. Whereas, 80 and 60% protection were obtained in chicks vaccinated either. with Al-ND vaccine [group 2] or live ND vaccines [group 3]; respectively. The present study reports the effect of injection of some immunostimulant substances in augmentation of the immune response to the inactivated vaccines

Genetic transformation of Egyptian maize lines using the late embryogenesis abundant protein gene, HVA1, from barley

Maize genotype specificity has been one of the constraints limiting the transformation of many tropical and subtropical maize lines with desirable genes. In the present study callus induction and regeneration ability from immature embryos of four elite Egyptian maize lines were examined. Two media were assayed to investigate the effect of 2,4-D and dicamba on type II callus production and regeneration. Dicamba promoted fast differentiation in all inbred lines that led to increasing the number of shoots in comparison to 2,4-D.lnbreds Gz 650 and Sd 34 gave significantly high regeneration frequencies when maintained on callus induction medium containing dicamba. The late embryogenesis abundant [LEA] protein coding gene, HVA1, from barley [Hordeum vulgare L.] for abiotic stress tolerance along with the bar gene for herbicide resistance were introduced in three of these inbred lines using the biolistic mediated transformation method and independent transgenic events were obtained. Putative transgenic events have been tested by herbicide application. Moreover, molecular analysis using PCR and Southern blot hybridization proved the presence and integration of the transgenes in the genome of the putatively transgenic plants. The copy number of the transgenes ranged between 10 and 15 copies in individual events. This study suggests that LEA genes could hold considerable potential for use as molecular tools for genetic crop improvement toward stress tolerance

In vitro production of transgenic tomatoes expressing defensin gene using newly developed regeneration and transformation system

Tomato is considered one of the most important vegetable crops grown in Egypt. Fungal diseases are the most dangerous diseases of tomato on the basis of yield losses. Most previously published procedures for tomato transformation are genotype dependent and still far from routine and universal methods. This study was conducted to enhance regeneration and transformation efficiency of the local Egyptian tomato cultivar [Edkawy]. The developed regeneration system involves the culturing of decapitated seedlings [one cotyledon and a merstimatic shoot tip were removed and the rest of explants include radicals, hypocotyls and one cotyledonary leaf only] on basal MS medium. Multiple shoots per explant were developed after three weeks of cultivation on basal MS medium. The developed system was employed to transfer defensin gene, which renders plants resistance against fungal diseases to the Egyptian cultivar [Edkawy]. Pluronic acid and Agrobacterium concentration were found to be key factors for efficient Agrobacterium-mediated transformation of cultivar [Edkawy]. Transformation was carried out using disarmed A. tumefaciens strain LBA4404 harboring a binary vector pITB-AFP. The plasmid contains defensin gene [AFP] under the control of a CaMV35S promoter and nopaline synthase [NOS] terminator, hygromycin phosphotransferase gene [hpt] and beta-glucuronidase. The modfied developed regeneration/transformation system herein, which orignally rely on flmaingo bill-like explant and floral-dip transformation method, enabled us to produce transgenic tomato shoots without the necessity of a complicated tissue culture system. GUS expression was observed in transformed tomato shoots but never in non-transformed [control]. The possibility of false GUS positives was ruled out because the GUS gene was interrupted by intron. GUS positive explants reacted positively to polymerase chain reaction [PCR] and gave the expected amplicon [300bp] corresponding to AFP gene. The obtained results indicated that the gene of interest was introduced in tomato genome. The results of the present study can be seen as a step towards production of transgenic Egyptian tomtoes resistance to fungal diseases

utilization of monoclonal antibodies in immunocapture RT-PCR and dot blotting immunobinding assays for the detection of Cucumber mosaic virus

Reliable plant diagnostic techniques are necessary for good crop management. Occasionally, ELISA may not give a definitive result, requiring confirmation using different diagnostic methods. Immunocapture reverse transcription PCR [IC-RT-PCR] requires the direct confirmation of an ELISA result in a simple, cost-effective manner. We found that the use of captured antigens as a template in IC-RT-PCR can eliminate the need for traditional extraction methods and reduce the number of steps involved, costs incurred, and sampling variations. Experiments showed that a reliability of pathogens can be detected using ELISA in the IC-RT-PCR process. To date, a rapid assay for diagnosis of CMV has not been developed, and there is a critical need for a method that can be employed in both laboratory and field. Dot immunobinding assays [DIBA] are useful alternatives to microtitre plate enzyme-linked immunosorbent assay [ELISA]. It has the additional advantages of simplicity, and is completed quickly in the field or the laboratory on large numbers of samples, in a short time. The method proposed provides a sensitive quantitative technique for dot-immunobinding assay

Construction of a recombinant baculovirus expressing the major capsid protein [VP6] of bovine rotavirus

The present study reports the expression of VP6, the major inner capsid protein of bovine rotavirus Nebraska calf diarrhea virus [NCDV] strain in a baculovirus expression system. The full-length DNA copies of RNA segment 6 [coding for VP6 protein] of NCDV were inserted into a baculovirus expression vector. A recombinant baculovirus carrying the VP6 gene was constructed through homologous recombination between the baculovirus recombinant plasmid carrying the VP6 gene and Autographa californica nuclear polyhedrosis virus [AcNPV] under the control of the polyhedrin promotor. Infection of Spodoptera frugiperda [Sf9] cells with the recombinant baculovirus expressing VP6 protein revealed a high-level of expression when tested by immunoflurescence and solid phase ELISA tests using BRV-specific polyclonal antibodies. The VP6 expressed protein was detected in Coomassie blue stained SDS-PAGE and produced a detectable band in Western blot assay. The high degree of reactivity with BRV-specific polyclonal antibodies confirmed that the antigenic determinants of the expressed protein were unaltered. The use of the in vitro expressed VP6 protein in the field diagnosis and vaccine development to control rotavirus infection is of considerable intere

Expression of the bovine coronavirus spike glycoprotein subunits in insect cells using recombinant baculoviruses

In the present study, the bovine coronavirus [BCV] spike glycoprotein cleavage products [S1 and S2] were individually expressed in Spodoptera frugiperda [Sf9] insect cells, using a baculovirus expression system. The coding sequence of S1 and S2 gene fragments were amplified by RT-PCR and cloned into the baculovirus shuttle vector pBlueBac4.5/V5-His TOPO [R] TA. The cloned fragments were inserted into the genome of Autographa californica nuclear polyhydrosis virus [AcMNPV] under control of the polyhedrin promoter, through a process of homologous recombination between the shuttle vector and a linearized replication-defective baculovirus DNA [Bac-N-Blue[TM]]. Recombinant baculoviruses were selected by plaque purification; verified for the presence of target sequences using PCR and propagated for generation of high-titer viral stocks. Infection of insect cells with the recombinant baculoviruses revealed high-level expression of the target proteins as indicated by immunofluoresent test and solid phase ELISA using BCV-specific polyclonal antiserum

Molecular characterization and genetic relationships among cotton genotypes 2- AFLP Analysis

The high-resolution genotyping method of amplified fragment length polymorphism [AFLP] was used to study the genetic relationships among 21 cotton genotypes from two different species G. barbadense and G. hirsutum. Sixteen AFLP primer combinations were used to selectively amplify the DNA fragments that matches the primer-extension sequence to investigate the genetic polymorphism among the 21 cotton genotypes. The 16 AFLP primer combinations produced 940 bands among which 474 were polymorphic, thus, representing a level of polymorphism of 50.4% among the 21 cotton genotypes. The amplification of AFLP templates resulted in a number of reproducible fragments ranging from 31 to 90 per primer with a size range of 60 bp to 780 bp. Fifteen primer combinations detected unique specific markers identifying 8 out of the 21 genotypes. A dendrogram was generated from the AFLP information that revealed two main clusters. All the genotypes belonging to G. barbadense except one [Pima Early American] were grouped in one cluster, while the accessions representing G. hirsutum constituted the second cluster, thus, confirming the results previously obtained by RAPD, ISSR and SSR analysis on the same cotton genotypes. To evaluate the efficiency of the different marker systems, the sum effective number of alleles [SENA], the average expected heterozygosity for polymorphic markers [Hav[p]], the effective multiplex ratio [E] and marker index [MI] were calculated. The AFLP exhibited considerably high SENA [318.2] compared to RAPD, ISSR and SSR [127.7, 46.0 and 22.3, respectively]. The average heterozygosity values were comparable for the different marker systems [0.39, 0.36, 0.39 and 0.34 in AFLP, RAPD, ISSR and SSR, respectively]. The MI was 182.2 in AFLP's, while it was 73.6, 26.3 and 12.7 in RAPD, ISSR and SSR, respectively. Thus, the results indicated that AFLP is more effective in detecting high level of polymorphism. The correlation coefficient was considerably higher between SSR and ISSR [0.61], and it was lower between RAPD's and AFLP's [0.26] than that between AFLP and ISSR [0.44] and AFLP and SSR [0.49]. The results confirmed that different marker systems differ in the mechanism of detecting polymorphism, genome coverage and the ease of application. Therefore, they could complement each other to draw more accurate conclusions

Evidence for variant avian leucosis virus in cases of mixed infection with Marek's disease virus

In the present study, we report for the first time in Egypt the evidence for variants of avain leucosis virus subgroup J. ALV-J associated with 2 cases of mixed infection with Marek's disease virus, MDV [one case is associated with tumors and the other, from homogenates of blood and tissues of chicks experienced transient paralysis syndrome of MDV] was detected. The ALV-Js might be variants as indicated by histopathological behaviour; negativity of the ALV-J specific PCR; and sero-logical profile in the flock with mixed infection [tumor-cases of ALV and MDV] and in experimentally-infected chickens with blood and tissue homogenates of other mixed infection case of ALV-J and MDV was detected. Using specific PCR for REV and ALV groups A or B, or C and/ or, D all samples of both mixed infection cases revealed negative results for amplification. However, 28.3% of sera samples collected from chickens with tumor-cases at age of 38 weeks were positive for ALV-J and negative for ALV [groups A and B] by ELISA. Testing of sera collected from the flock with tumor-cases at 45 and 55 weeks of age revealed that 38.2% and 20% of sera were positive for ALV-J and 2.9 and 3% were positive for ALV [groups A and B], respectively. In experimentally-infected chickens with homogenates of blood and spleen, 14.2% of sera were positive to ALV-J and negative for ALV [groups A and B] when tested at 30 weeks post-inoculation. Histopathological examination revealed the occurrence of mixed infection of MDV and ALV-J with unique pathological lesions in eye and liver which were found to have heavily aggregations of myelocytes characteristic to ALV subgroup J

Transmissible viral proventriculitis and stunting syndrome in broiler chickens in Egypt: 1. isolation and characterization of variant infectious bursal disease virus [IBDV]

In the present study, 46 broiler chicken flocks [2-4 weeks of age in farms located in four governorates] were examined. They were affected with proventriculitis and stunting syndrome and were vaccinated with classical infectious bursal disease vaccines. The affected flocks generally showed stunting, reduced growth rate and uneven weight distribution. The virus was detected in the bursa at 72 hours PI by ELISA and electron microscope confirming the persistence of the variant IBDV in the chicks, despite the presence of IBDV classical antibodies. The present study reported, for the first time, the association of IBDV with stunting syndrome and proventriculitis in broilers in Egypt

Effect of combined treatments between gamma irradiation, spices and/or thermal treatment on the viability and enterotoxicity of Staphylococcus aureus

Heat-irradiation combination sequence decreased the thermal resistance of S. Aureus. The synergistic effect increased by increasing either the pre-irradiation thermal exposure time or by increasing the radiation dose. The complete inhibition of bacterial cell was recorded by pre-irradiation thermal treatment at 65, 60 and 55C for 5, 15 and 25 minutes, respectively, at the irradiation dose 0.5 kGy. Combination treatment between irradiation and species decreased the lethal species concentration from 2% to 1% at irradiation dose 0.25 kGy for all examined species and to 0.5% concentration with the irradiation dose 0.5 kGy. The combination treatments between species, temperature and radiation had a great synergistic effect on S. Aureus cells comparable with the individual treatment or with the combination treatment between two factors. There was no variation in the amounts of enterotoxin produced by the organism after treated by the sublethal doses of the combined three factors except in case of mint, which recorded 200% increase for untreated cells

Evaluation of different techniques used for diagnosis of Giardia lamblia in children

Twenty seven children presented to the pediatric clinic, Al-Hussein, University hospital with symptoms suggestive of chronic giardiasis were included in thisstudy. Their age ranged from 2 to 12 years. They were 12 males and 15 females. Selection of the cases was based upon the history and clinical evaluation. Investigations done for those children included simple stool analysis. stool analysis using zinc sulphate, duodenal aspiration and duodenal biopsy. Our results showed that the main presenting symptoms were recurrent abdominal pain, anorexia, vomiting, diarrhea and weight loss. Simple stool analysis [repeated] detected Giardia lumblia in 13 out of 27 cases [48-15%,] but after using zinc sulphate the parasite was detected in 16 cases [59.26%]. Zinc sulphate helps concentration and preservation of the parasite in the stool sample. Duodenal aspiration was the most sensitive method for diagnosis of Giardia Lamblia as it showed the parasite in 18 out of 27 cases [66.76%], Duodenal biopsy on the other hand showed the parasite in 3 cases only [11.1%] Although Giardia lamblia could not be visualized in most of auodenal biopsies yet, the pathological changes in the mucosa were very suggestive Suspected cases with negative stool results should have duodenal aspiration as it was found to be the most sensitive and the best method for diagnosis of Giardia lamblia

incidence and significance of optic atrophy in cases of pituitary tumours

A study was made on 18 cases with pituitary tumors to ascertain the incidence and significance of optic atrophy. Disc pallor was seen in 13 cases [22 eyes]. It was usually bilateral, but with a distinct difference between the sides. Disc pallor was commonly of the diffuse type [12 eyes]. Temporal pallor was seen in six eyes and in four eyes pallor was associated with atrophic excavation of and optic disc. In five cases ophthalmoscopic examination revealed no changes at the optic disc in spite of visual and field defects. Examination of the retinal fibers by red-free light in these five cases showed atrophy of the retinal fiber layer. Fluorescein anagiography showed signs of optic atrophy before ophthalmoscopic examination in three cases. Disc pallor was present even where the subjective duration of visual symptoms was as short as six months and it became increasingly frequent the longer the history. The optic atrophy usually lagged behind the loss of vision and the degree of disc damage was related to the degree of visual loss and to a less extent to the degree of field defect. The presence of optic atrophy did not preclude the possibility of rapid and full recovery of vision after extirpation of pituitary tumor. The outlook for recovery of visual function was generally good if pallor was not combined with atrophic excavation of the optic disc

Aspiration of soft cataract

54 patients with soft cataract were operated by lens aspiration technique. The operation is simple and safe, the incision is small and complications are significantly less than those resulting from the more conventional types of surgery. Successful results depend on preoperative pupil dilatation, wide excision of the anterior lens capsule, thorough removal of the lens cortex. avoidance of the posterior capsule and postoperative mydriasis

Experience with the Choyce anterior chamber lens implant

The rational, indications, contraindications and technique for the use of Choyce anterior chamber implant are discussed. The results of 18 operations are presented. About 90% achieved 6/12 or better. The Choyce anterior chamber implant is relatively easy to insert provided proper attention is given to surgical technique. The prerequisites are an established safe technique for routine cataract surgery and proper wound closure. No intraocular lens should be used unless the quality of routine cataract surgery is high. Proper selection of cases is vital for obtaining good results and the correct length of the implant is crucial for the success of surgery