This paper describes a short-amplicon-based TaqMan probe quantitative real-time PCR (qPCR) assay for the quantitative detection of canine meat in chicken nuggets, which are very popular across the world, including Malaysia. The assay targeted a 100-bp fragment of canine cytb gene using a canine-specific primer and TaqMan probe. Specificity against 10 different animals and plants species demonstrated threshold cycles (Ct) of 16.13 ± 0.12 to 16.25 ± 0.23 for canine DNA and negative results for the others in a 40-cycle reaction. The assay was tested for the quantification of up to 0.01% canine meat in deliberately spiked chicken nuggets with 99.7% PCR efficiency and 0.995 correlation coefficient. The analysis of the actual and qPCR predicted values showed a high recovery rate (from 87% ± 28% to 112% ± 19%) with a linear regression close to unity (R(2) = 0.999). Finally, samples of three halal-branded commercial chicken nuggets collected from different Malaysian outlets were screened for canine meat, but no contamination was demonstrated.
Cinnamon deodorised aqueous extract (CinDAE) was prepared and evaluated for its total phenolic (315.3 ± 35.4 mg GAE/g) and flavonoid (99.3 ± 9.6 mg RE/g) contents. Stabilizing efficiency of CinDAE, for chicken meatballs, was measured against oxidative deterioration as function of storage time under chilled conditions. For this purpose, oxidative stability [2-thiobarbituric acid reactive substances (TBARS); peroxide value (PV)], colour and sensory acceptability were measured in the control meatballs (C), and those stabilized with 200 ppm of: CinDAE (T1), ascorbic acid (T2), BHA/BHT (50/50; w/w) (T3). In comparison to "C", induction period (IP) and redness (a* value) of the stabilized samples (T1, T2 and T3) were increased, while PV and TBARS were decreased throughout storage (8 ± 1 °C) significantly (p
The effects of supplementing different dietary selenium (Se) sources on intestinal histomorphology, caecal bacterial proliferation, and caecum short-chain fatty acid in layer chickens were studied. A total of 120 of 54-week-old Lohman Brown Classic laying hens were subjected to four dietary treatments: control group with no Se supplementation (T1), 0.3 mg/kg of sodium selenite (T2), 0.3 mg/kg of selenium yeast (T3), and 0.3 mg/kg of bacterial Se (Stenotrophomonas maltophilia, ADS18) (T4). All treatments were also supplemented with 250 mg/kg of vitamin E. Results showed significantly (p < 0.05) higher villus height in different small intestine parts in hens fed with diets containing organic Se sources (T3 and T4), as compared to inorganic sources (T2) and control (T1). A greater crypt depth was observed for the T1 group, as compared to T3 and T4 groups. The total bacterial populations of Bifidobacteria spp. and Lactobacilli spp. were significantly increased (p < 0.05), and tEscherichia coli population was significantly decreased (p < 0.05) in T3 and T4 groups. No effect on the total caecal short-chain fatty acid (SCFAs) content was observed. However, there was a significant increase (21.5%) of the butyric acid content in T4 group. In conclusion, organic Se supplementation, particularly bacterial organic Se, enhances intestinal histomorphology, increases the beneficial caecal bacterial proliferation, and increases butyric acid content.
The characteristics of the pathogenic infectious bursal disease virus (IBDV) that infected avian species other than commercial chickens were largely unknown. In this study, by using in vivo and molecular methods, we had characterized an IBDV isolate (named 94268) isolated from an infectious bursal disease (IBD) outbreak in Malaysian village chickens--the adulterated descendant of the Southeast Asian jungle fowl (Gallus bankiva) that were commonly reared in the backyard. The 94268 isolate was grouped as the very virulent IBDV (vvIBDV) strain because it caused severe lesions and a high mortality rate in village chickens (>88%) and experimentally infected specific-pathogen-free chickens (>66%). In addition, it possessed all of the vvIBDV molecular markers in its VP2 gene. Phylogenetic analysis using distance, maximum parsimony, and maximum likelihood methods revealed that 94268 was monophyletic with other vvIBDV isolates and closely related to the Malaysian vvIBDV isolates. Given that the VP2 gene of 94268 isolate was almost identical and evolutionarily closely related to other field IBDV isolates that affected the commercial chickens, we therefore concluded that IBD infections had spread across the farm boundary. IBD infection in the village chicken may represent an important part of the IBD epidemiology because these birds could harbor the vvIBDV strain and should not be overlooked in the control and prevention of the disease.
This study was carried out to investigate the modulatory effects of dietary methionine and n-6/n-3 polyunsaturated fatty acids (PUFA) ratio on immune response and performance of infectious bursal disease (IBD)-challenged broiler chickens. In total, 350 one-day-old male broiler chicks were assigned to 1 of the 6 dietary treatment groups in a 3 × 2 factorial arrangement. There were 3 n-6/n-3 PUFA ratios (45, 5.5, and 1.5) and 2 levels of methionine (NRC recommendation and twice NRC recommendation). The results showed that birds fed with dietary n-6/n-3 PUFA ratio of 5.5 had higher BW, lower feed intake, and superior FCR than other groups. However, the highest antibody response was observed in birds with dietary n-6/n-3 PUFA ratio of 1.5. Lowering n-6/n-3 PUFA ratio reduced bursa lesion score equally in birds fed with n-6/n-3 PUFA ratio of 5.5 and 1.5. Supplementation of methionine by twice the recommendation also improved FCR and reduced feed intake and bursa lesion score. However, in this study, the optimum performance (as measured by BW, feed intake, and FCR) did not coincide with the optimum immune response (as measured by antibody titer). It seems that dietary n-3 PUFA modulates the broiler chicken performance and immune response in a dose-dependent but nonlinear manner. Therefore, it can be suggested that a balance of moderate level of dietary n-6/n-3 PUFA ratio (5.5) and methionine level (twice recommendation) might enhance immune response together with performance in IBD-challenged broiler chickens.
The bursa of Fabricius is critical for B cell development and differentiation in chick embryos. This study describes the production in vitro, from dissociated cell suspensions, of cellular agglomerates with functional similarities to the chicken bursa. Co-cultivation of epithelial and lymphoid cells obtained from embryos at the appropriate developmental stage regularly led to agglomerate formation within 48 hours. These agglomerates resembled bursal tissue in having lymphoid clusters overlaid by well organized epithelium. Whereas lymphocytes within agglomerates were predominantly Bu-1a(+), a majority of those emigrating onto the supporting membrane were Bu-1a(-) and IgM(+). Both agglomerates and emigrant cells expressed activation-induced deaminase with levels increasing after 24 hours. Emigrating cells were actively proliferating at a rate in excess of both the starting cell population and the population of cells remaining in agglomerates. The potential usefulness of this system for investigating the response of bursal tissue to avian Newcastle disease virus (strain AF2240) was examined.
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.
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.
Newcastle disease (ND) is a highly contagious avian disease and one of the major causes of economic losses in the poultry industry. The emergence of virulent NDV genotypes and repeated outbreaks of NDV in vaccinated chickens have raised the need for fundamental studies on the virus-host interactions. In this study, the profiles of B and T lymphocytes and macrophages and differential expression of 26 immune-related genes in the spleen of specific-pathogen-free (SPF) chickens, infected with either the velogenic genotype VII NDV strain IBS002 or the genotype VIII NDV strain AF2240, were evaluated. A significant reduction in T lymphocyte population and an increase in the infiltration of IgM+ B cells and KUL01+ macrophages were detected in the infected spleens at 1, 3 and 4 days post-infection (dpi) (P<0.05). The gene expression profiles showed an up-regulation of CCLi3, CXCLi1, CXCLi2 (IL-8), IFN-γ, IL-12α, IL-18, IL-1β, IL-6, iNOS, TLR7, MHCI, IL-17F and TNFSF13B (P<0.05). However, these two genotypes showed different cytokine expression patterns and viral load. IBS002 showed higher viral load than AF2240 in spleen at 3 and 4dpi and caused a more rapid up-regulation of CXCLi2, IFN-γ, IL-12α, IL-18, IL-1β, iNOS and IL-10 at 3dpi. Meanwhile, the expression levels of CCLI3, CXCLi1, IFN-γ, IL-12α, IL-1β and iNOS genes were significantly higher in AF2240 at 4dpi. In addition, the expression levels of IL-10 were significantly higher in the IBS002-infected chickens at 3 and 4dpi. Hence, infection with velogenic genotype VII and VIII NDV induced different viral load and production of cytokines and chemokines associated with inflammatory reactions.
Ticks are vectors in the transmission of many important infectious diseases in human and animals. Ticks can be readily found in the semi-forested areas such as the settlements of the indigenous people in Malaysia, the Orang Asli. There is still minimal information available on the bacterial agents associated with ticks found in Malaysia. We performed a survey of the bacterial communities associated with ticks collected from domestic animals found in two Orang Asli villages in Malaysia. We collected 62 ticks, microscopically and molecularly identified as related to Haemaphysalis wellingtoni, Haemaphysalis hystricis and Haemaphysalis bispinosa. Bacterial 16s rRNA hypervariable region (V6) amplicon libraries prepared from the tick samples were sequenced on the Ion Torrent PGM platform. We detected a total of 392 possible bacterial genera after pooling and sequencing 20 samples, indicating a diverse bacterial community profile. Dominant taxa include the potential tick endosymbiont, Coxiella. Other dominant taxa include the tick-associated pathogen, Rickettsia, and environmental bacteria such as Bacillus, Mycobacterium, Sphingomonas and Pseudomonas. Other known tick-associated bacteria were also detected, including Anaplasma, Ehrlichia, Rickettsiella and Wolbachia, albeit at very low abundance. Specific PCR was performed on selected samples to identify Rickettsia and Coxiella. Sequence of Rickettsia felis, which causes spotted fever in human and cats, was identified in one sample. Coxiella endosymbionts were detected in three samples. This study provides the baseline knowledge of the microbiome of ticks in Malaysia, focusing on tick-associated bacteria affecting the Orang Asli communities. The role of the herein found Coxiella and Rickettsia in tick physiology or disease transmission merits further investigation.
Interaction of an anticancer drug, vandetanib (VDB) with a ligand transporter, lysozyme (LYZ) was explored using multispectroscopic techniques, such as fluorescence, absorption and circular dichroism along with computational analysis. Fluorescence data and absorption results confirmed VDB-LYZ complexation. VDB-induced quenching was characterized as static quenching based on inverse correlation of KSV with temperature as well as kq values. The complex was characterized by the weak binding constant (Ka=4.96-3.14×103M-1). Thermodynamic data (ΔS=+12.82Jmol-1K-1; ΔH=-16.73kJmol-1) of VDB-LYZ interaction revealed participation of hydrophobic and van der Waals forces along with hydrogen bonds in VDB-LYZ complexation. Microenvironmental perturbations around tryptophan and tyrosine residues as well as secondary and tertiary structural alterations in LYZ upon addition of VDB were evident from the 3-D fluorescence, far- and near-UV CD spectral analyses, respectively. Interestingly, addition of VDB to LYZ significantly increased protein's thermostability. Molecular docking results suggested the location of VDB binding site near the LYZ active site while molecular dynamics simulation results suggested stability of VDB-LYZ complex. Presence of Mg2+, Ba2+ and Zn2+ was found to interfere with VDB-LYZ interaction.
This study demonstrates that cell wall treatment of Lactococcus lactis harbouring the internal ribosome entry site-incorporated lactococcal bicistronic vector pNZ:VIG mediated the delivery of genes into an eukaryotic cell line, DF1 cells, through bactofection. Bactofection analysis showed that the pNZ:VIG plasmid in L. lactis can be transferred into DF1 cells and that both the VP2 and gfp genes cloned in the plasmid can be transcribed and translated. The protein band relative to the Mr of VP2 protein (49 kDa) was successfully detected via Western blot analysis, while green fluorescence was successfully detected using a fluorescence microscope. The intensity of the bands detected increased for samples treated with both 1.5% (w/v) glycine and 10 μg/mL of lysozyme when compared to L. lactis treated with glycine alone and without treatment. Cell wall treatment of L. lactis with a combination of both glycine and lysozyme was not only shown to mediate plasmid transfer to DF1 cells, but also to increase the plasmid transfer efficiency.
Thiol-functionalized magnetic carbon nanotubes (TMCNTs) were employed as the sorbent in the magnetic micro-solid phase extraction (M-µ-SPE) of sulfonamide antibiotics (SAs) in water, milks and chicken meat products prior to high performance liquid chromatography-diode array detector (HPLC-DAD) analysis. The synthesized sorbent was characterized by several spectroscopic techniques. Optimum conditions were: 20 mg of TMCNTs at pH 4, 2 min extraction time, 10% addition of salt and 30 mL of sample volume. Under the optimized TMCNTs-M-µ-SPE and HPLC-DAD conditions, the method showed good linearity in the range of 0.1-500 µg L-1 (r2 ≥ 0.9950), low limits of detection (0.02-1.5 µg L-1), good analytes recovery (80.7-116.2%) and acceptable RSDs (0.3-7.7%, n = 15). The method was applied to tap water (1), milks (15) and commercial chicken meat products (35), SAs were detected in five chicken meat samples (3.0-25.7 µg L-1). The method is critically compared to those reported in the literature.
Infectious bursal disease is one of an OIE list of notifiable diseases. Chicken is the only host that manifests clinical signs and its pathogenicity is correlated with the distribution of antigens in organs. This study was conducted to determine disease pathogenesis and virus tissue tropism by in situ PCR, immunoperoxidase staining (IPS), and HE staining. Twenty four chickens were infected with very virulent Infectious Bursal Disease Virus (vvIBDV). Fifteen chickens were kept as a control group. Infected chickens were sacrificed at hrs 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). While, control chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Different tissues were collected, fixed in 10% buffered formalin, and processed. At hr 2 pi, virus was detected in intestinal, junction of the proventriculus and gizzard, cecal tonsil, liver, kidney, and bursa of Fabricius. At hr 4 pi, virus reached spleen, and at hr 6 pi, it entered thymus. At hr 12 pi, virus concentration increased in positive tissues. The latest invaded tissue was muscle on day 1 pi. Secondary viraemia occurred during 12-24 h pi. In situ PCR was the most sensitive technique to highlight obscure points of infection in this study.
Limited deep studies are available in the field of early stages of pathogenesis of Newcastle disease virus (NDV) infection and tissue tropism of NDV. In this study, 24 specific pathogen free (SPF) chickens of white leghorn breed were infected with Newcastle disease (ND) by intranasal administration of 10⁵ 50% EID50/0.1 mL of velogenic NDV (vNDV). A second group of 15 chickens were kept as a control group. Chickens were monitored every day to record clinical signs. Infected chickens were euthanized by cervical dislocation at successive times, namely at hours (hrs) 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). Whereas, control group chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining, immunoperoxidase staining (IPS) and in situ PCR were applied. It was concluded that at hr 2 pi, virus seemed to be inclined to trachea and respiratory tract. Meanwhile, it attacked caecal tonsils, intestine and bursa of Fabricus. While primary viraemia was ongoing, virus created footing in kidney and thymus. At hr 4 pi, proventriculus, liver, and spleen were attacked. However, at hr 6 pi, brain and heart were involved. Secondary viraemia probably started as early as hr 12 pi since all collected tissues were positive. Tissue tropism was determined in trachea, caecal tonsil, liver, bursa of Fabricius, intestine, proventriculus, lung, spleen, thymus, kidney, heart, and brain.
Organotypic slice culture is a living cell research technique which blends features of both in vivo and in vitro techniques. While organotypic brain slice culture techniques have been well established in rodents, there are few reports on the study of organotypic slice culture, especially of the central nervous system (CNS), in chicken embryos. We established a combined in ovo electroporation and organotypic slice culture method to study exogenous genes functions in the CNS during chicken embryo development. We performed in ovo electroporation in the spinal cord or optic tectum prior to slice culture. When embryonic development reached a specific stage, green fluorescent protein (GFP)-positive embryos were selected and fluorescent expression sites were cut under stereo fluorescence microscopy. Selected tissues were embedded in 4% agar. Tissues were sectioned on a vibratory microtome and 300 μm thick sections were mounted on a membrane of millicell cell culture insert. The insert was placed in a 30-mm culture dish and 1 ml of slice culture media was added. We show that during serum-free medium culture, the slice loses its original structure and propensity to be strictly regulated, which are the characteristics of the CNS. However, after adding serum, the histological structure of cultured-tissue slices was able to be well maintained and neuronal axons were significantly longer than that those of serum-free medium cultured-tissue slices. As the structure of a complete single neuron can be observed from a slice culture, this is a suitable way of studying single neuronal dynamics. As such, we present an effective method to study axon formation and migration of single neurons in vitro.
Nanofiber membrane chromatography integrates liquid membrane chromatography and nanofiber filtration into a single-step purification process. Nanofiber membrane can be functionalised with affinity ligands for promoting binding specificity of membrane. Dye molecules are a good affinity ligand for nanofiber membrane due to their low cost and high binding affinity. In this study, a dye-affinity nanofiber membrane (P-Chitosan-Dye membrane) was prepared by using polyacrylonitrile nanofiber membrane modified with chitosan molecules and immobilized with dye molecules. Reactive Orange 4, commercially known as Procion Orange MX2R, was found to be the best dye ligand for membrane chromatography. The binding capacity of P-Chitosan-Dye membrane for lysozyme was investigated under different operating conditions in batch mode. Furthermore, desorption of lysozyme using the P-Chitosan-Dye membrane was evaluated systematically. The recovery percentage of lysozyme was found to be ~100%. The optimal conditions obtained from batch-mode study were adopted to develop a purification process to separate lysozyme from chicken egg white. The process was operated continuously using the membrane chromatography and the characteristic of the breakthrough curve was evaluated. At a lower flow rate (i.e., 0.1 mL/min), the total recovery of lysozyme and purification factor of lysozyme were 98.59% and 56.89 folds, respectively.
Recent foodborne outbreaks in multiple locations necessitate the continuous development of highly sensitive and specific biosensors that offer rapid detection of foodborne biological hazards. This work focuses on the development of a reduced graphene oxide‑titanium dioxide (rGO-TiO2) nanocomposite based aptasensor to detect Salmonella enterica serovar Typhimurium. A label-free aptamer was immobilized on a rGO-TiO2 nanocomposite matrix through electrostatic interactions. The changes in electrical conductivity on the electrode surface were evaluated using electroanalytical methods. DNA aptamer adsorbed on the rGO-TiO2 surface bound to the bacterial cells at the electrode interface causing a physical barrier inhibiting the electron transfer. This interaction decreased the DPV signal of the electrode proportional to decreasing concentrations of the bacterial cells. The optimized aptasensor exhibited high sensitivity with a wide detection range (108 to 101 cfu mL-1), a low detection limit of 101 cfu mL-1 and good selectivity for Salmonella bacteria. This rGO-TiO2 aptasensor is an excellent biosensing platform that offers a reliable, rapid and sensitive alternative for foodborne pathogen detection.
Production of Scenedesmus sp. biomass in chicken slaughterhouse wastewater (CSWW) is a promising alternative technique for commercial culture medium due to the high nutritional content of the generated biomass to be used as fish feeds. The current work deals with optimising of biomass production in CSWW using response surface methodology (RSM) as a function of two independent variables, namely temperature (10-30 °C) and photoperiod (6-24 h). The potential application of biomass yield as fish feeds was evaluated based on carbohydrate, protein and lipid contents. The results revealed that the best operating parameters for Scenedesmus sp. biomass production with high contents of carbohydrates, proteins and lipids were determined at 30 °C and after 24 h. The actual and predicted values were 2.47 vs. 3.09 g, 1.44 vs. 1.27 μg/mL, 29.9 vs. 31.60% and 25.75 vs. 28.44%, respectively. Moreover, the produced biomass has a high concentration of fatty acid methyl ester (FAME) as follows: 35.91% of C15:1; 17.58% of C24:1 and 14.11% of C18:1N9T. The biomass yields have 7.98% of eicosapentaenoic acid (EPA, C20:5N3) which is more appropriate as fish feeds. The Fourier transform infrared (FTIR) analysis of biomass revealed that the main functional groups included hydroxyl (OH), aldehyde (=C-H), alkanes and acyl chain groups. Scanning electron micrograph (SEM) and energy-dispersive X-ray spectroscopic analysis (EDS) indicated that the surface morphology and element distribution in biomass produced in BBM and CSWW were varied. The findings have indicated that the biomass produced in CSWW has high potential as fish feeds.
Sonic hedgehog (SHH) is a vertebrate homologue of the secreted Drosophila protein hedgehog and is expressed by the notochord and floor plate in the developing spinal cord. Sonic hedgehog provides signals relevant for positional information, cell proliferation and possibly cell survival, depending on the time and location of expression. Although the role of SHH in providing positional information in the neural tube has been experimentally proven, the underlying mechanism remains unclear. In this study, in ovo electroporation was employed in the chicken spinal cord during chicken embryo development. Electroporation was conducted at stage 17 (E2.5), after electroporation the embryos were continued incubating to stage 28 (E6) for sampling, tissue fixation with 4% paraformaldehyde and frozen sectioning. Sonic hedgehog and related protein expressions were detected by in situ hybridization and fluorescence immunohistochemistry and the results were analysed after microphotography. Our results indicate that the ectopic expression of SHH leads to ventralization in the spinal cord during chicken embryonic development by inducing abnormalities in the structure of the motor column and motor neuron integration. In addition, ectopic SHH expression inhibits the expression of dorsal transcription factors and commissural axon projections. The correct location of SHH expression is vital to the formation of the motor column. Ectopic expression of SHH in the spinal cord not only affects the positioning of motor neurons, but also induces abnormalities in the structure of the motor column. It leads to ventralization in the spinal cord, resulting in the formation of more ventral neurons forming during neuronal formation.