Expression of Hemagglutinin\u2013Neuraminidase and fusion epitopes of Newcastle Disease Virus in transgenic tobacco

Background: Newcastle disease is an important avian infectious disease that brings about vast economic damage for poultry industry. Transgenic plants represent a cost-effective system for the production of therapeutic proteins and are widely used for the production of poultry vaccines. In an attempt to develop a recombinant vaccine, a plant expression binary vector pBI121, containing the genes encoding Hemagglutinin\u2013Neuraminidase (HN) and Fusion (F) epitopes of Newcastle Disease Virus (NDV) under the control of CaMV35S promoter and NOS terminator was constructed and introduced into the tobacco ( Nicotiana tabacum ) plant by Agrobacterium -mediated transformation. Results: Putative transgenic plantswere screened in a selection medium containing 50 mg/L kanamycin and 30mg/L meropenem. Integration of the foreign gene in plant genome was confirmed by PCR. Expression of foreign gene was analyzed at transcription level by RT-PCR and at translation level by means of dot blotting and ELISA. All analyses confirmed the expression of recombinant protein. Conclusion: Developments in genetic engineering have led to plant-based systems for recombinant vaccine production. In this research, tobacco plant was used to express F and HN epitopes of NDV. Our results indicate that for the production of recombinant vaccine, it is a novel strategy to use concatenated epitopes without their genetic fusion onto larger scaffold structure such as viral coat protein

Genetic studies of beak and feather disease virus

The aim of the studies reported in this thesis was to genetically characterise the genome of beak and feather disease virus (BFDV), a circovirus causing psittacine beak and feather disease (PBFD). The objective was to utilise the sequence data to develop additional genetic-based tools for the detection of the virus and facilitate further investigation of the pathogenesis and epidemiology of the disease. To determine an optimal method of preparing purified virus DNA for cloning and sequencing, several methods for the extraction and purification of BFDV from tissues of PBFD-affected birds were compared. Maximum recovery of virus from tissues was achieved using a combination of solubilisation of virus with the chlorinated hydrocarbon Arklone™, differential centrifugation of the virus through a 45% sucrose cushion, followed by isopycnic ultracentrifugation of the virus in caesium chloride gradients. The circular, single-stranded DNA of one isolate of BFDV from a sulphur crested cockatoo with PBFD was cloned and sequenced. The genome of this strain was 1993 nucleotides in length. Analysis of the assembled double-stranded replicative form of the virus DNA demonstrated 7 open reading frames (ORF), 3 in the virion strand and 4 in the complementary strand, potentially encoding 7 viral proteins greater than 8.7 kDa. Fligh amino acid sequence identity was demonstrated between a potential 33.3 kDa protein product of ORF1 of BFDV and the replicase-associated protein (Rep) of porcine circovirus (PCV), and nanoviruses (plant circoviruses). No significant similarity was found between BFDV and chicken anaemia virus (CAV), another member of the family Circoviridae. A potential stem-loop structure and a nonanucleotide motif (TAGTATTAC) within it, similar to that found in PCV, nanoviruses and geminiviruses, was demonstrated in the virion strand of the BFDV genome. Comparison of the deduced amino acid sequences of ORF2 of BFDV and PCV demonstrated 29.1 % identity, and in both viruses ORF2 was located on the complementary strand, beginning close to or within the hairpin loop. The findings provided further evidence of that BFDV has a close relationship with PCV and the plant circoviruses but not with CAV. To determine the extent of genetic variability within BFDV, the complete nucleotide (nt) sequence of an additional 8 isolates and partial nucleotide sequence of one other isolate recovered from various psittacine species across Australia was determined. All isolates had the same basic structure including the position of the ORFs, the presence of hairpin or stem-loop structure between ORF1 and ORF2, the nonanucleotide motif (TAGTATTAC) therein, the 3 motifs of the Rep protein involved in rolling circle replication, and the P-loop motif. The size of the genome in the isolates ranged from 1992 to 2018 nt and overall nt identity of the isolates compared to the first strain that was sequenced ranged from 84% to 97%; the variation was due to a combination of point mutations and a number of deletions and insertions ranging from 1 to 17 nt in size detected in both the coding regions and non-coding regions. Phylogenetic analysis grouped the isolates into 4 clusters but there were no apparent regional differences or differences related to the psittacine species from which they were derived. While 7 ORFs with the potential to encode proteins greater than 8.7 kDa were detected in the original strain, only 3 of these ORFs were detected in all 10 BFDV isolates for which sequence data was obtained: ORF1, ORF2 and ORFS. The ORF1 and ORF2 probably encoded the Rep and capsid proteins, respectively, but the protein encoded by ORFS, if any, has to be determined. A universal PCR assay was designed that consistently detected BFDV in a range of psittacine species affected with PBFD from a variety of geographic regions within Australia. A Southern blot technique was also developed to detect BFDV genomic DNA in PBMC of infected birds, and also confirm the specificity of PCR products generated from feathers or peripheral blood mononuclear cells (PBMC) of PBFDaffected birds. A set of degenerate primers was designed to try and amplify genomic material of circoviruses related to BFDV and PCV and suspected to be present in other animal species. With these primers, PCR amplification of a region of the genome of a novel circovirus present in Senegal doves affected with a PBFD-like syndrome was achieved

Production of bacteriocins by Enterococcus spp. isolated from traditional, Iranian, raw milk cheeses, and detection of their encoding genes

Strong bacteriocins, or bacteriocins with a wide range of activity against pathogens and spoilage microorganisms, are actively sought for use as natural food preservatives. This work reports the inhibitory activity of 96 enterococcal isolates from two Iranian, raw milk cheeses against five indicator organisms (including Listeria innocua). Forty-eight isolates inhibited at least one indicator in spot agar assays. Of these, 20 isolates corresponding to 15 different strains were shown to produce bacteriocin-like substances in liquid cultures. PCR analysis revealed the genes coding for enterocins (enterococcal bacteriocins) A, B, P or X, or their combinations, in all but one of these 15 strains. In addition, the gene coding for enterocin 31 was detected in two strains. No amplification was obtained in one strain when using specific primers for all 13 bacteriocin genes sought. Three different enterocin genes were identified in most strains and four in one strain. Although the concomitant production of bacteriocins is still to be verified, producers of multiple enterocins could be of great technological potential as protective cultures in the cheese industry. © 2012 Springer-Verlag.This research was partially supported by a project from the Spanish Ministry of Science and Innovation (MICINN) (AGL2007-61869-ALI). A.A. was awarded a scholarship of the Severo Ochoa program from FICYT (BP08-053). S. Delgado was sup- ported by a research contract from MICINN under Juan de la Cierva program (JCI-2008-02391). The authors wish to thank the Iranian Ministry of Industries and Mines, as well as Razavi Dairy Industry (Mash- had, Iran) and the O Y ce of Industrial Relationships (OIR) of Ferdowsi University of Mashhad (FUM).Peer Reviewe

HIGHLY PATHOGENIC AVIAN INFLUENZA VIRUS H5N1 NEURAMINIDASE EXPRESSED IN SF9 INSECT CELLS WITH

This article reports heterologous expression in Sf9 insect cells of N1 neuraminidase derived from avian influenza virus A/chicken/Iran/53-3/2008(H5N1). A gene encoding the neuraminidase N1 was fused directly in-frame with the adipokinetic hormone (AKH) secretion signal and 6xHis-Tag coding sequence upstream of the cloning site for expressing fusion recombinant N1 protein with N-terminal tags in pIEx-3 vector. Recombinant N1 neuraminidase was expressed in Sf9 insect cells as a 80 kDa secreted protein. This insect-based Baculovirus-Free heterologous expression system provided functionally recombinant N1 neuraminidase that should be useful in anti-influenza drug screening, detection and diagnostic tests and also as a potential proteinbased vaccine

Effects of curcumin or nanocurcumin on blood biochemical parameters, intestinal morphology and microbial population of broiler chickens reared under normal and cold stress conditions

This study was conducted to evaluate the effect of curcumin/nanocurcumin on blood parameters, intestinal morphology and microbial population in broiler chickens reared under normal and cold stress conditions. The experiment was designed with two identical houses; each consisted of five diets with 5 replicates of 10 birds each. The diets were (1) control; (2) and (3) Control + 200 or 400 mg/kg curcumin; (4) and (5) Control + 200 or 400 mg/kg nanocurcumin, respectively. Birds in both houses were reared under commercial temperatures until day 14. The temperature in the first house was maintained according to the commercial practices, whereas the temperature in the second house dropped to 15°C on day 14 and maintained between 13–15°C until day 42. Total weight gain was decreased, but plasma malondialdehyde (MDA), liver enzymes activities and heterophils/lymphocytes ratio were increased in cold-stressed birds compared to those that grew in normal temperature. Supplementation of curcumin/nanocurcumin in diet improved the weight gain and villus surface area of birds in concomitance to lower their plasma MDA, liver enzymes activities, caecal E. coli population compared to those fed control diet. It is concluded that the addition of 200 mg/kg curcumin/nanocurcumin to diet may improve the performance, antioxidant status and jejunal tissue health in broiler chickens

Effects of curcumin and nanocurcumin on growth performance, blood gas indices and ascites mortalities of broiler chickens reared under normal and cold stress conditions

The aim of this study was to evaluate the effect of curcumin/nanocurcumin on performance, blood gases and ascites mortality in broiler chickens reared under normal and cold stress conditions. This experimental design was a split plot with 500 Ross 308 male chicks. Plots were two identical houses; each consisted of five diets (as sub-plots) with 5 replicates of 10 birds each. The diets were: (1) control; (2) control+ 200 mg/kg curcumin; (3) control+ 400 mg/kg curcumin; (4) control+ 200 mg/kg nanocurcumin; (5) control+ 400 mg/kg nanocurcumin. Birds in both houses reared under recommended temperatures until 14 d, when the temperature was dropped in one house from 28.5 to 13–15 °C and maintained at this level to induce ascites until 42 d. Whereas, in the second house the temperature was maintained according to the hybrid production guidelines. Weight gain was reduced and FCR was increased in birds reared under cold temperature compared to those in normal temperature condition. Cold stress increased blood pCO2, HTC and decreased pO2 and O2 saturation (p < .05) in birds at 42 d of age. Birds fed diets containing 200 mg/kg curcumin/nanocurcumin had higher weight gain compared to those fed control diet (p < .05). Moreover, supplementation of diets with curcumin/nanocurcumin alleviated the adverse effect of cold stress as reflected by a reduction in HTC and an increased O2 saturation at 42 d of age. It is concluded that the addition of curcumin/nanocurcumin to diet might be an effective feed additive to alleviate the adverse effect of cold stress on performance and ascites syndrome

Microbial diversity of the traditional Iranian cheeses Lighvan and Koozeh, as revealed by polyphasic culturing and culture-independent approaches

The microbiota of two traditional Iranian cheeses (Lighvan and Koozeh) made of raw ewe's milk or mixtures of ewe's and goat's milk without starter addition was explored by culture-independent and culture-dependent approaches. Three batches of Lighvan and one of Koozeh were subjected to culture-independent polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis and sequencing of dominant bands to assess the microbial structure and dynamics through manufacturing and ripening. In addition, culturing in elective media for lactic acid bacteria (M17, MRS and KAA), isolation of single colonies (n=130), molecular identification by PCR-amplified ribosomal DNA restriction analysis and sequencing, and differentiation at the strain level by repetitive extragenic palindromic PCR was also performed. DGGE analysis showed that the dominant amplicons in all four cheese batches belonged to Lactococcus lactis and Streptococcus parauberis. In addition, Escherichia coli and Lactococcus garvieae were frequently identified in both Lighvan and Koozeh, while Streptococcus thermophilus was found occasionally. In contrast, Enterococcus faecium and Enterococcus faecalis were found to be dominant among the isolates in all batches. These species showed a high genetic diversity. The discrepancy between culturing and DGGE results suggested that dominant populations were in a nonrecoverable state in the used media. This reinforces the idea that culture-dependent and cultureindependent techniques provide complementary data, ultimately affording a better description of cheese ecosystems. These data could be of help in the selection of commercial starters for industrial-scale manufacture of Lighvan and Koozeh cheeses using pasteurised milk. Alternatively, microbial analysis would allow the selection of appropriate strains for designing of specific starters for traditional cheese manufacture. © 2011 Springer-Verlag.This research was partially supported by a project from the Spanish Ministry of Science and Innovation (MICINN) to BM (Ref. AGL2007-61869-ALI). AA was awarded a scholarship of the Severo Ochoa programme from FICYT (Ref. BP08-053)Peer Reviewe

Vaccine adjuvants: past, current and future

Adjuvants are an essential component of modern vaccines. An adjuvant is an entity added to a vaccine formulation to ensure that robust immunity to the antigen is inoculcated. The adjuvant is typically vital for the efficacy of vaccines using subunit (pepdids, proteins and virus like particles) and DNA antigens. Furthermore, these components are used to reach the current new goals of preventing and/ or treating chronic infectious diseases and cancers. This review focuses on formulation aspects of adjuvants, safety considerations, progress in understanding their mechanisms of action and also their side effects with using 97 articles are acceceble in pubmed central and google scholar indexing which published during 1980-2016. Adjuvants can be broadly divided into two classes, based on their principal mechanisms of action; the first class are vaccine delivery systems that generally particulate and mainly function to target associated antigens into antigen presenting cells. The others are immunostimulatory adjuvants that predominantly derived from pathogens and often represent pathogen associated molecular patterns which activate cells of the innate immune system. Adjuvants induce cellular and humoral responses, in particular neutralizing antibodies that able to inhibit the binding of pathogens to their cellular receptors. Efficient Th1-immunity-inducing adjuvants are highly in demand. The adjuvants promote good cell-mediated immunity against subunit vaccines that have low immunogenicity themselves. However, attempts to develop a new generation of adjuvants, which are essential for new vaccines, is important, but their use is limited because, little is known about their mechanisms of action and health risks

Data on environmentally relevant level of aflatoxin B1-induced human dendritic cells' functional alteration

We assessed the effects of naturally occurring levels of AFB1 on the expression of key immune molecules and function of human monocyte-derived dendritic cells (MDDCs) by cell culture, RT-qPCR, and flow cytometry. Data here revealed that an environmentally relevant level of AFB1 led to remarkably weakened key functional capacity of DCs, up-regulation of key transcripts and DCs apoptosis, down-regulation of key phagocytic element, CD64, and creation of pseudolicensing direction of DCs. Flow cytometry data confirmed a damage towards DCs, i.e., increased apoptosis. The detailed data and their mechanistic effects and the outcome are available in this research article (Mehrzad et al., 2018) [1]. The impaired phagocytosis capacity with triggered pseudolicensing direction of MDDCs caused by AFB1 and dysregulation of the key functional genes could provide a mechanistic explanation for the observed in vivo immunotoxicity associated with this mycotoxin. Keywords: AFB1, Apoptosis, AFB1-detoxifying genes, Dendritic cells, Flow cytometry, Functional genes, Immunnoderegulation, Phagocytosis, RT-qPC