Improving the potency of DNA vaccine against Chicken Anemia Virus (CAV) by fusing VP1 protein of CAV to Marek's Disease Virus (MDV) Type-1 VP22 protein

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). The VP1 and VP2 genes of CAV isolate SMSC-1 were amplified and inserted into eukaryotic co-expression vector, pBudCE4.1 to construct pBudVP2-VP1. We also constructed pBudVP2-VP1/VP22 encoding CAV VP2 and the VP22 of MDV-1 linked to the CAV VP1. In vitro expression of the genes was confirmed by using RT- PCR, Western blot and indirect immunofluorescence. The vaccines were then tested in 2-week-old SPF chickens which were inoculated with the DNA plasmid constructs by the intramuscular route. After in vivo expression studies, immune responses of the immunized chickens were evaluated pre- and post-immunization. Chickens vaccinated with pBudVP2-VP1/VP22 exhibited a significant increase in antibody titers to CAV and also proliferation induction of splenocytes in comparison to the chickens vaccinated with pBudVP2-VP1. Furthermore, the pBudVP2-VP1/VP22-vaccinated group showed higher level of the Th1 cytokines IL-2 and IFN-g. This study showed that MDV-1 VP22 gene is capable of enhancing the potency of DNA vaccine against CAV when fused with the CAV VP1 gene

Neonatal feed restriction modulates circulating levels of corticosterone and expression of glucocorticoid receptor and heat shock protein 70 in aged Japanese quail exposed to acute heat stress.

This study aimed to determine the effect of neonatal feed restriction on plasma corticosterone concentration (CORT), hippocampal glucocorticoid receptor (GR) expression, and heat shock protein (Hsp) 70 expression in aged male Japanese quail subjected to acute heat stress. Equal numbers of chicks were subjected to either ad libitum feeding (AL) or 60% feed restriction on d 4, 5, and 6 (FR). At 21 (young) and 270 (aged) d of age, birds were exposed to 43 ± 1°C for 1 h. Blood and hippocampus samples were collected to determine CORT and Hsp 70 and GR expressions before heat stress and following 1 h of heat stress, 1 h of post-heat stress recovery, and 2 h of post-heat stress recovery. With the use of real-time PCR and enzyme immunoassay, we examined the hippocampal expression of GR and Hsp 70 and CORT. The GR expression of the young birds increased following heat stress and remained consistent throughout the period of recovery. Conversely, no significant changes were noted on GR expression of aged birds. Although both young and aged birds had similar CORT before and during heat stress, the latter exhibited greater values following 1 and 2 h of recovery. Within the young group, feeding regimens had no significant effect on Hsp 70 expression. However, neonatal feed restriction improved Hsp 70 expression in aged birds. Neonatal feed restriction, compared with the AL group, resulted in higher CORT on d 21 but the converse was noted on d 270. Neonatal feed restriction appears to set a robust reactive hypothalamo-pituitary-adrenal response allowing the development of adaptive, healthy, and resilient phenotypes in aged quail as measured by a higher hippocampal Hsp 70 expression along with lower CORT

Effects of agitation speed, temperature, carbon and nitrogen sources on the growth of recombinant Lactococcus lactis NZ9000 carrying domain 1 of aerolysin gene

Lactococcus lactis is a Gram-positive bacterium widely used in the production of buttermilk and cheese. Recently, the bacterium becomes famous as the genetically modified organism can be used alive for the treatment of disease. In this study, different cultural conditions based on agitation speed and temperature on the growth of recombinant L. lactis NZ9000 harboring domain 1 of aerolysin gene (NHD1Aer) were investigated using shake flask experiment. The effect of different carbon (glucose, sucrose and lactose) and nitrogen (yeast extract, peptone, NH4Cl, (NH4)2SO4, and urea) sources in M17 medium on the cell accumulation were also tested. The results showed that the highest cell concentration (3.22 g/L, μm = 0.58 h-1) was obtained when the cultivation was incubated at 27°C and at agitation of 100 rpm. The cells growth was markedly improved when utilizing glucose and peptone/yeast extract as carbon and nitrogen sources, respectively. The aerolysin gene in the cells after four generation time was extracted and then analyzed using agarose gel electrophoresis. The results obtained showed a 250 bp band amplified of domain 1 of the aerolysin gene.Keywords: Aerolysin, Lactococcus lactis, fermentation, one-factor-at-a-timeAfrican Journal of Biotechnology Vol. 9(33), pp. 5392-5398, 16 August, 201

Physiological responses of 3 chicken breeds to acute heat stress.

Domestic animals have been modified by selecting individuals exhibiting desirable traits and culling the others. To investigate the alterations introduced by domestication and selective breeding in heat stress response, 2 experiments were conducted using Red Jungle Fowl (RJF), village fowl (VF), and commercial broilers (CB). In experiment 1, RJF, VF, and CB of a common chronological age (30 d old) were exposed to 36 ± 1°C for 3 h. In experiment 2, RJF, VF, and CB of common BW (930 ± 15 g) were subjected to similar procedures as in experiment 1. Heat treatment significantly increased body temperature, heterophil:lymphocyte ratio, and plasma corticosterone concentration in CB but not in VF and RJF. In both experiments and irrespective of stage of heat treatment, RJF showed lower heterophil:lymphocyte ratio, higher plasma corticosterone concentration, and higher heat shock protein 70 expression than VF and CB. It can be concluded that selective breeding for phenotypic traits in the domestication process has resulted in alterations in the physiology of CB and concomitantly the ability to withstand high ambient temperature compared with RJF and VF. In other words, domestication and selective breeding are leading to individuals that are more susceptible to stress rather than resistant. It is also apparent that genetic differences in body size and age per se may not determine breed or strain variations in response to heat stress

Improved Protection from Velogenic Newcastle Disease Virus Challenge Following Multiple Immunizations With Plasmid DNA Encoding for F And HN Genes

Specific-pathogen free (SPF) chicken were inoculated with the plasmid combination and challenged with velogenic NDV. The antibody level against NVD was measured using commercial enzyme linked immunosorbent assay (ELISA). In the first immunization regimen, SPF chickens inoculated twice with NDV-F or NDV-HN constructs elicited antibody responses 1 week after the second injection. However, the levels of the antibody were low and did not confer significant protection from the lethal challenge. In addition, admininistration of the plasmid constructs with Freund’s adjuvant did not improve the level of protection. In the second immunization regimen, chickens inoculsted trice with the plasmid constructs emulsified with Freund”s adjuvant induced significant antibody titers after the third injection. Three out of nine (33.3 %) chickensvaccinated with pEGFP-HN, five of ten (50.0%) chicken vaccinated with pEGFP-F and nine of ten (90.0%) chicken vaccinated with combined pEGFP-F and pEGFP-HN were protected from the challenge. No significant differences in the levels of protection were observed when the chicken were vaccinated with linearized pEGFP-F. the results suggested that more than two injections with both F and HN encoding plasmid DNA were required to induce level of antibodies for protection against velogenic NDV in chickens

Lactobacillus acidophilus as a live vehicle for oral immunization against chicken anemia virus.

The AcmA binding domains of Lactococcus lactis were used to display the VP1 protein of chicken anemia virus (CAV) on Lactobacillus acidophilus. One and two repeats of the cell wall binding domain of acmA gene were amplified from L. lactis MG1363 genome and then inserted into co-expression vector, pBudCE4.1. The VP1 gene of CAV was then fused to the acmA sequences and the VP2 gene was cloned into the second MCS of the same vector before transformation into Escherichia coli. The expressed recombinant proteins were purified using a His-tag affinity column and mixed with a culture of L. acidophilus. Whole cell ELISA and immunofluorescence assay showed the binding of the recombinant VP1 protein on the surface of the bacterial cells. The lactobacilli cells carrying the CAV VP1 protein were used to immunize specific pathogen-free chickens through the oral route. A moderate level of neutralizing antibody to CAV was detected in the serum of the immunized chickens. A VP1-specific proliferative response was observed in splenocytes of the chickens after oral immunization. The vaccinated groups also showed increased levels of Th1 cytokines interleukin (IL)-2, IL-12, and IFN-γ. These observations suggest that L. acidophilus can be used in the delivery of vaccines to chickens

Inhibition of adrenal steroidogenesis and heat shock protein 70 induction in neonatally feed restricted broiler chickens under heat stress condition.

This study was conducted to determine the roles of plasma corticosterone concentration (CORT) and heat shock protein 70 (Hsp70) expression in the acquisition of thermotolerance in broiler chickens subjected to neonatal feed restriction.Equal numbers of chicks were divided into one of the four feeding regimens: ad libitum (control), 60% feed restriction on days 4, 5 and 6 (FR60), 60% feed restriction on days 4, 5 and 6 + 1500 mg/kg metyrapone (FR60M) and 60% feed restriction on days 4, 5 and 6 + 1500 mg/kg quercetin (FR60Q). From 35 to 42 days of age, all birds were exposed to 37 ± 1°C and 70-80% relative humidity for 3 h/day. The neonatal feed restriction elevated heterophil to lymphocyte ratios (HLR), CORT and Hsp70 expression in FR60 chicks. The FR60M and FR60Q treatments suppressed CORT and Hsp70 expression, respectively. Subjecting birds to FR60 improved feed conversion ratio (FCR) and body weight gain, and reduced HLR when compared to controls in response to heat exposure. Following heat exposure, the HLR and weight gain of FR60M and control birds were not significantly different. Although FR60Q chickens had improved FCR and weight gain, their HLR were similar to controls following heat exposure. The control birds had significantly lower CORT than other groups in response to heat exposure. The Hsp70 expression of FR60 and FR60Q birds was lower than those of control and FR60M groups. It is concluded that Hsp70 expression didnot appear to be a major indicator of thermotolerance acquisitionin neonataly manipulated broiler chickens

Education and communicating positive young minds in creating sustainable environment and development

Purpose: Proposed course content for each group age is outlined from the early age of a child to the university education system. However, these course contents are a recommendation which indicates the importance of continuity to ensure that the young generations will subsequently not only aware of preserving the environment and its surroundings but, will also work hard to improve the existing situation of the world for a quality environment and a better place to live. Design/methodology/approach: These programs and activities include the awareness and sensitivity to the depleting greens, flora and fauna, the climatic change due to human activities and the pollution to soil and groundwater. Humans, animals and other living things rely greatly on soil, water air and the sun. Findings: Thus, it is critical to inculcate better understanding and creating a more caring society to ensure the sustainability of the earth for the future existence of mankind. Research limitations/implications: In this paper, a discussion on the needs of introducing environmental geotechnics to young generations as early as in the pre-school education is deemed necessary as being conducted by most developed countries. Practical implications: Developing countries such as Malaysia must start now to ensure that existing resources such as its soils and groundwater quality are preserved as the country continue to strive in becoming a developed and modern country by the year 2020. Originality/value: This paper is original. Paper type: Research paper

Development of a DNA vaccine against chicken anemia virus by using a bicistronic vector expressing VP1 and VP2 proteins of CAV.

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

Coupled plasma filtration adsorption (CPFA) plus continuous veno-venous haemofiltration (CVVH) versus CVVH alone as an adjunctive therapy in the treatment of sepsis

To compare the efficacy of Coupled Plasma Filtration and Adsorption (CPFA) plus Continuous Veno-Venous Haemofiltration (CVVH) versus CVVH alone as an adjunct treatment of sepsis in terms of haemodynamic stability, inotropic requirement and inflammatory mediators. Design and Methods: Prospective randomized controlled trial involving septic patients with/without acute kidney injury (AKI) whom were randomized to receive CPFA + CVVH or CVVH alone. Haemodynamic parameters including inotropic requirements and inflammatory mediators [procalcitonin (PCT) and C reactive protein (CRP)] were measured. Results: Twenty-three patients [CPFA + CVVH (n = 11), CVVH (n = 12)] were enrolled. Haemodynamic stability occurred earlier and sustained in the CPFA + CVVH group with an increase in diastolic blood pressure (p = 0.001 vs. p = 0.226) and mean arterial pressure (p = 0.001 vs. p = 0.575) at the end of treatment with no increment in inotropic requirement. Both groups had a reduction in PCT and CRP (CPFA + CVVH: p = 0.003, p = 0.026 and CVVH: p = 0.008, p = 0.071 respectively). The length of intensive care unit stay, hospital stay and 30 day outcomes were similar between the groups. There was an inverse association between serum albumin and CRP (p = 0.018). Serum albumin positively correlated with systolic blood pressure (p = 0.012) and diastolic blood pressure (p = 0.009). We found a trend between CRP and length of hospital stay (p = 0.056). Patients with a lower PCT at 24 h had a better outcome (survival) than those with a higher PCT (p = 0.045). Conclusion: CPFA is a feasible, albeit expensive adjunctive extracorporeal treatment that may be superior to CVVH alone in the treatment of severe sepsis