Physiological responses to social isolation stress were compared in 56-day-old male Japanese quail. Birds were fed pretreated diets for 3 days as follows: (i) Basal diet (control); (ii) Basal diet+1500 mg/kg metyrapone (BM); (iii) Basal diet+30 mg/kg corticosterone (BCO); (iv) Basal diet+250 mg/kg ascorbic acid (BC); (v) Basal diet+250 mg/kg α-tocopherol (BE); (vi) Basal diet+250 mg/kg ascorbic acid and 250 mg/kg α-tocopherol (BCE). The birds were subsequently socially isolated in individual opaque brown paper box for 2 hours. Plasma corticosterone (CORT) concentration and heart and brain heat shock protein 70 (Hsp 70) expressions were determined before stress and immediately after stress. Two hours of isolation stress elevated CORT concentration significantly in the control and BE birds but not in the BC, BCE and BM birds. There was a significant reduction in CORT concentration after isolation stress in the BCO group. Isolation stress increased Hsp 70 expression in the brain and heart of control and BM birds. However, brain and heart Hsp 70 expressions were not significantly altered in the isolated BC, BCE and BE birds. Although, the CORT concentration of BM birds was not affected by isolation stress, Hsp70 expression in both brain and heart were significantly increased. Moreover, exogenous corticosterone supplementation did not result in elevation of Hsp 70 expression. It can be concluded that, although Hsp 70 induction had not been directly affected by CORT concentration, it may be modulated by the HPA axis function via activation of ACTH.
Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).
Feline infectious peritonitis (FIP) is a lethal systemic disease caused by FIP virus (FIPV). There are no effective vaccines or treatment available, and the virus virulence determinants and pathogenesis are not fully understood. Here, we describe the sequencing of RNA extracted from Crandell Rees Feline Kidney (CRFK) cells infected with FIPV using the Illumina next-generation sequencing approach. Bioinformatics analysis, based on Felis catus 2X annotated shotgun reference genome, using CLC bio Genome Workbench is used to map both control and infected cells. Kal's Z test statistical analysis is used to analyze the differentially expressed genes from the infected CRFK cells. In addition, RT-qPCR analysis is used for further transcriptional profiling of selected genes in infected CRFK cells and Peripheral Blood Mononuclear Cells (PBMCs) from healthy and FIP-diagnosed cats.
The influenza virus (IV) is known to be a resistant virus with frequent mutations, causing severe respiratory diseases in the upper respiratory system. Public health concerns about clinical efficacy of all conventional drugs are ambiguous; therefore, finding additional therapeutic agents is critical to prevent and control influenza outbreaks. Influenza is associated with the induction of proinflammatory cytokines. Scientists have reported that anti-inflammatory drugs, with pleiotropic effects, reduce the burden of severe influenza diseases. Therefore, statins, which are cardioprotective drugs with anti-inflammatory and immunomodulatory effects, may help patients suffering from influenza virus (IV). This review delineates the potential use of statins as an alternative therapy in treating influenza related illness.
Influenza virus is still a severe respiratory disease affecting human and other species. As conventional drugs are not recommended for long time because of side effects and drug resistance occurrence, traditional medication has been focused as alternative remedy. HESA-A is a natural compound from herbal-marine origin. Previous studies have reported the therapeutic properties of HESA-A on psoriasis vulgaris and different types of cancers and we also showed its anti-inflammatory effects against influenza A infection.
Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.
'Gold standard' OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).
Germinated brown rice (GBR) is rich in bioactive compounds, which confer GBR with many functional properties. Evidence of its hypocholesterolemic effects is emerging, but the exact mechanisms of action and bioactive compounds involved have not been fully documented. Using type 2 diabetic rats, we studied the effects of white rice, GBR, and brown rice (BR) on lipid profile and on the regulation of selected genes involved in cholesterol metabolism. Our results showed that the upregulation of apolipoprotein A1 and low-density lipoprotein receptor genes was involved in the hypocholesterolemic effects of GBR. Additionally, in vitro studies using HEPG2 cells showed that acylated steryl glycoside, gamma amino butyric acid, and oryzanol and phenolic extracts of GBR contribute to the nutrigenomic regulation of these genes. Transcriptional and nontranscriptional mechanisms are likely involved in the overall hypocholesterolemic effects of GBR suggesting that it may have an impact on the prevention and/or management of hypercholesterolemia due to a wide variety of metabolic perturbations. However, there is need to conduct long-term clinical trials to determine the clinical relevance of the hypocholesterolemic effects of GBR determined through animal studies.
Poorly controlled diabetes mellitus can result in serious complications. Gene therapy is increasingly being considered as an alternative approach to treat diabetes, because of its ability to induce physiological insulin secretion and it allows patients to escape insulin injections. The properties of gut K and L-cells, including glucose sensitivity, the ability to process insulin and a regulated secretion pathway support their use as surrogate β-cells. Previous in vitro studies have provided sufficient evidence supporting the use of these cells for gene therapy studies. Therefore, we examined the ability of K and L-cells to produce insulin in diabetic mice. Chitosan nanoparticles were used to transfer the insulin gene into intestinal cells via oral administration. The efficiency of chitosan as a gene vehicle was investigated through the use of reporter gene. Insulin mRNA and protein expression levels were measured by RT-PCR and ELISA, respectively. Blood glucose testing revealed that this treatment reduced glucose levels in diabetic mice. The decrease in blood glucose level in the first week of treatment was greater in mice with K-cell specific insulin expression compared with mice with L-cell-specific insulin expression. These results indicate that inducing insulin secretion in K-cells conferred a quicker response to gene therapy.
The objectives of this study were to determine the psychological fatigue and analyze muscle activity of production workers who are performing processes jobs while standing for prolonged time periods.
Environmental stressors may influence chicken performance and susceptibility to pathogens, such as Salmonella enteritidis. This study was conducted to determine the effects of heat shock protein (Hsp)70 expression on resistance to Salmonella enteritidis infection in broiler chickens subjected to heat exposure. Chicks were divided into 3 feeding regimens: ad libitum feeding (control); 60% feed restriction on d 4, 5, and 6 (FR60); and 60% feed restriction on d 4, 5, and 6 plus 1,500 mg/kg of quercetin (FR60Q). On d 35, all of the chickens were individually inoculated with 1 mL of Salmonella enteritidis (1.5 × 10(8) cfu/bird) and exposed to an ambient temperature of 37 ± 1°C and 70% RH for 3 h/d. The FR60 and FR60Q chickens had significantly lower Salmonella enteritidis colonization and lower Hsp70 expression than that of the control chickens following the heat exposure period. The least colonization was observed in the FR60Q group (1.38 log(10) cfu/g in the spleen and 1.96 log(10) cfu/g in the cecal content) and the highest was in the control group (2.1 log(10) cfu/g in the spleen and 4.42 log(10) cfu/g in the cecal content). It appears that neonatal feed restriction can enhance resistance to Salmonella enteritidis colonization in heat-stressed broiler chicks, and the underlying mechanism could be associated with the lower expression of Hsp70.
In the present study, we describe the development of a DNA vaccine against chicken anemia virus. The VP1 and VP2 genes of CAV were amplified and cloned into pBudCE4.1 to construct two DNA vaccines, namely, pBudVP1 and pBudVP2-VP1. In vitro and in vivo studies showed that co-expression of VP1 with VP2 are required to induce significant levels of antibody against CAV. Subsequently, the vaccines were tested in 2-week-old SPF chickens. Chickens immunized with the DNA-plasmid pBudVP2-VP1 showed positive neutralizing antibody titer against CAV. Furthermore, VP1-specific proliferation induction of splenocytes and also high serum levels of Th1 cytokines, IL-2 and IFN-γ were detected in the pBudVP2-VP1-vaccinated chickens. These results suggest that the recombinant DNA plasmid co-expressing VP1 and VP2 can be used as a potential DNA vaccine against CAV.
Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene.
Studies have shown that the VP22 gene of Marek's Disease Virus type-1 (MDV-1) has the property of movement between cells from the original cell of expression into the neighboring cells. The ability to facilitate the spreading of the linked proteins was used to improve the potency of the constructed DNA vaccines against chicken anemia virus (CAV).
Hemagglutinin-neuraminidase (HN) protein of Newcastle disease virus (NDV) plays a vital role in the viral infectivity, host immunity, and disease diagnosis. A portion of the HN gene encoding the ectodomain (nt 142-1739) was cloned and expressed in Escherichia coli yielding an insoluble HN protein and a soluble NusA-HN protein containing N-utilization substance A (NusA) fusion component. Both recombinant proteins were purified and used for immunization of chickens. The recombinant HN protein induced higher antibody titers as compared to the recombinant NusA-HN protein. These antibodies were able to react in immunoblot analysis with the corresponding recombinant proteins as well as with the HN protein of NDV.
A series of plasmids containing the HSP70 gene of Mycobacterium tuberculosis fused to the hemagglutinin (H5) gene of H5N1 avian influenza virus (AIV) (H5-HSP70 (heat shock protein 70) vaccine) or individual H5 gene (H5 vaccine) or HSP70 gene (HSP70 vaccine) were constructed based on the plasmid pcDNA3.1. Expression of H5 gene in Vero cells in vitro and in chickens in vivo was confirmed following their transfection and immunization with H5 or H5-HSP70 vaccines. Controls consisted of HSP70 vaccine, empty plasmid pcDNA3.1 and co-administered H5 and HSP70 vaccines. H5-HSP70 vaccine produced in chicken higher hemagglutination inhibition (HI) antibody titer than H5 vaccine. However, the increase was not statistically significant. We have demonstrated for the first time that the H5 DNA vaccine with fused HSP70 gene may produce an enhanced induction of humoral immune response to AIV in chickens.
The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p<0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.
Chicken anemia virus (CAV) is the causative agent of chicken infectious anemia (CIA). Study on the type of CAV isolates present and their genetic diversity, transmission to their progeny and level of protection afforded in the breeder farms is lacking in Malaysia. Hence, the present study was aimed to detect CAV from commercial broiler breeder farms and characterize CAV positive samples based on sequence and phylogenetic analysis of partial VP1 gene.
Two hundred thirty-five 1-d-old broiler chickens showing short or long tonic immobility responses were classified as low fear (LF) or high fear (HF) responders, respectively. On d 41, they were subjected to either crating or heat challenge (34 +/- 1 degrees C) for 3 h and its effect on plasma corticosterone concentration, heterophil/lymphocyte ratios, and heat shock protein (HSP) 70 expression in brain tissue were determined. Crating and heat exposure elevated heterophil/lymphocyte ratios in both LF and HF birds. Circulating corticosterone, however, was greater in HF than LF birds after crating and heat challenge. Although differences between fear responder group for HSP 70 were negligible before heat challenge, after 3 h of heat exposure, the response was greater for the HF than the LF group. Both LF and HF showed similar increases in HSP 70 after crating.
The haemagglutinin-neuraminidase (HN) gene ofNewcastle disease virus (NDV) strain AF2240, amplifiedfrom the viral genomic RNA ( approximately 1.8 kb) was directionallycloned and inserted into a baculovirus expressionvector system. The recombinant glycoprotein expressedin Spodoptera frugiperda (Sf9) cellsshowed haemagglutinin (HA), neuraminidase (NA) andhemadsorption activities. HA activity was detected inboth extra- and intra-cellular recombinant HN(recHNAF2240) samples. In addition, both HA andhemadsorption activities were inhibited by polyclonalanti-NDV sera. Furthermore, significant expression ofthe recombinant protein was observed on the surface ofinfected cells. SDS-PAGE analysis revealed thepresence of visually distinguishable bands between the70 and 80 kDa in size that were absent in thewild-type samples. Western blot analysis showed thatthe distinct approximately 63 kDa band and a approximately 75 kDa bandcorresponded to the unglycosylated and glycosylated HNglycoprotein respectively as reported in anotherstudy. These observations indicated that the HNrecombinant protein was not only expressed on thesurface of the infected cells as well as with theviral coat protein, but also appears to be functional.