Molecular Characterisation Of Infectious Bursal Disease Virus And Expression Of Vp2 Protein For The Development Of Diagnostic Kit And Recombinant Vaccine

Outbreak of infectious bursal disease (IBD) in chickens due to highly pathogenic strain of IBD virus (vvIBDV) was first reported in Europe in late 1980’s and in Malaysia in 1991. The disease caused significant economic losses, estimated more than RM72 million per year in Malaysia alone due to high mortality and immunosuppression. Treatment of IBD is of no value and the disease can only be controlled and prevented by proper vaccination programme and biosecurity. It was the objectives of the study to determine the molecular characteristic of Malaysian field isolates of IBDV and expression of the VP2 gene of the isolate for the development of diagnostic kit and recombinant vaccine. Three IBDV isolates identified as UPM04178, UPM04190 and UPM04238 were characterised. Based on their pathogenicity and sequence characteristic, the highest similarity (98%) concerning both nucleotide and amino acid sequences, the IBDV isolates were characterized as vvIBDV strains. Evolutionary relatedness of the isolates to vvIBDV strains was demonstrated by three phylogenetic methods: bootstrap values of 100%, 95% and 90% for nucleotide sequences and those of 58%, 86% and 96% for amino acid sequences were obtained by the distance, maximum parsimony and maximum likehood methods, respectively. Phylogenetic analysis revealed clustering of the isolates with vvIBDV strains of serotype 1, which originate from a common ancestor of IBDV strains present in Malaysia. Using informative characteristics of the isolate, both diagnostic kit and recombinant vaccine were successfully developed using a new wild-type field vvIBDV strain of UPM04190 isolate. A safe and effective recombinant IBD vaccine was developed base on the construction of recombinant VP2 gene of the isolate cloned into an Escherichia coli expression system. The VP2 gene was inserted into pRSET B vector as a fusion protein with histidine tag, which can be easily purified. The recombinant VP2 protein bands were expressed to their expected sizes of ~50 kDa from cell lysate. The pRSET vectors are pUC-derived expression vectors and expression of the gene of interest from pRSET is controlled by the strong phage T7 promoter that drives expression of gene 10 (Φ10) which provides protein stability and help to maintain the original structure of the protein. High-level production (3 mg/ml) of soluble product of VP2 recombinant protein was achieved with modified techniques of expression conditions and approaches. Efficacy test demonstrated that the recombinant vaccine of various fractions could provide protection ranging from 75% to 100% in highly susceptible chickens (specific pathogen free chickens) when challenged with vvIBDV (B00/81) at 104.25 EID50/ml per chicken following vaccination. One-step-immunostrip kit which is highly specific and sensitive was developed using whole virus as capture antigen and high-affinity polyclonal IBD antibodies coated with gold particles. Rapid detection of IBD antibody can be achieved as fast as two minutes in a clinical or field environment. The kit is highly sensitive as it can detect as low as 250 ELISA units compared to commercial ELISA kit that only goes to 391 ELISA units for positive samples. The specificity of the kit was evaluated against antibody of other chicken viruses. No signal of reactivity or cross react exists among the antibodies tested. Thus, it was highly specific to IBDV. It was concluded that the local IBDV isolates were proven to be vvIBDV strain, the constructed recombinant vaccine provide a safe and effective protection and, the developed one-step-immunostrip kit is rapid, specific, sensitive, safe and economic in detection of IBDV infection and monitoring immune status of chicken against IBD

Identification, characterisation and phylogenetic analysis of commensal bacteria isolated from human breast milk in Malaysia

Human breast milk microbiota is essential for infant immune system development, maturation and protection against infection. However, there is scarce information on the fluid’s microbiological composition from Malaysia. The objective of the study was to isolate, identify and characterise commensal bacterial population present in human breast milk from Malaysia. One hundred bacteria were isolated from the human breast milk of healthy lactating women (n=30). After preliminary screening, 20 isolates were characterised using both phenotypic and molecular techniques. The results indicated that most frequently identified bacteria in this study were E. faecalis and S. hominis. These organisms alongside E. cloacae were all metabolised D-Maltose, Sucrose, D-Turanose, α-D-Glucose, D-Fructose, D-Mannose, D-Galactose, D-sorbitol and D-Mannitol and were able to grow at pH 5 and 6, 1% sodium lactate, 1%, 2% and 8% NaCl. BLAST showed over 99% similarity to those deposited in Genbank. Phylogenetic-relatedness was depicted using neighbour-joining method and had two clades with 100% bootstrap. These findings provided insight into the nature, characteristics and also phylogenetic-relatedness of bacteria present in human milk from Malaysia. Isolation and identification of commensal bacteria from human milk are considered the first step for future studies on the benefit of these organisms towards human health

Anti-breast cancer effects of live, heat-killed and cytoplasmic fractions of Enterococcus faecalis and Stuphylococcus hominis isolated from human breast milk

Development of tumour that is resistant to chemotherapeutics and synthetic drugs, coupled with their life-threatening side effects and the adverse effects of surgery and hormone therapies, led to increased research on probiotics' anticancer potentials. The current study investigated the potential of live, heat-killed cells (HKC) and the cytoplasmic fractions (CF) of Enterococcus faecalis and Staphylococcus hominis as anti-breast cancer agents. MCF-7 cell line was treated with 25, 50, 100 and 200 μg/mL each of live, HKC and CF of the bacteria; and cytotoxicity was evaluated for 24, 48 and 72 h using MTT assay. The morphological features of the treated cells were examined by fluorescence microscopy. The stage of cell cycle arrest and apoptosis were quantified by flow cytometry. The bacterial effect on non-malignant breast epithelial cell line, MCF-10A, was assessed using MTT assay for 24, 48 and 72 h. All the three forms of the bacteria caused a significant decrease in MCF-7 (up to 33.29%) cell proliferation in concentration- and time-dependent manner. Morphological features of apoptosis like cell death, cell shrinkage and membrane blebbing were observed. Flow cytometry analyses suggested that about 34.60% of treated MCF-7 was undergoing apoptosis. A strong anti-proliferative activity was efficiently induced through sub-G1 accumulation (up to 83.17%) in treated MCF-7 and decreased number in the G0/G1 phase (74.39%). MCF-10A cells treated with both bacteria showed no significant difference with the untreated (>90% viability). These bacteria can be used as good alternative nutraceutical with promising therapeutic indexes for breast cancer because of their non-cytotoxic effects to normal cells

Analysis of bacterial communities in rhizosphere soil of symptomless and basal stem rot (BSR)-infected oil palm using terminal restriction fragment length polymorphism (T-RFLP)

Basal Stem Rot (BSR) disease caused by pathogenic fungi known as Ganoderma boninense has been identified as a major threat in oil palm plantation. Previously used methods to control this disease have been ineffective while method using chemical treatment is not environmentally friendly. An inadequate knowledge on the core microbiome of oil palm rhizosphere and the relationship between BSR disease incidences hinders effective controls against this pathogenic disease. Hence, the objectives of this project are to determine the bacterial communities of symptomless and BSR-infected oil palm using T-RFLP analysis, to perform cluster analysis of the samples based on the T-RFLP data and to analyze the relationship between soil bacterial communities and soil pH. The rhizosphere samples of symptomless and BSR-infected oil palm were collected at different microsites (bulk soil from harvesting path and rhizosphere soil from weeded circle and frond pile) and at different depths (10 cm and 30 cm from upper soil surface) from Oil Palm Plantation, Seberang Perak. In T-RFLP analysis, 16s rRNA region of the bacterial DNA were amplified by using 8F forward primer labelled with 6-FAM fluorescent dye and unlabelled 1492R reverse primer. The PCR products were then digested either with restriction enzyme AluI or HhaI or double digestion using AluI and HhaI. The raw fragments data were aligned and analyzed in T-REX (T-RFLP Analysis Expedited Software). The ordination analysis of Additive Main Effects and Multiplicative Interaction Model (AMMI) analysis from T-REX software revealed higher percentages of signal compared to noise interaction effects for frond pile at depth 30 cm with interaction difference of 21.28% (analysis based on relative abundance). The higher difference between signal and noise indicates that there are larger differences in microbial community between symptomless and BSR-infected oil palm. Meanwhile, cluster analysis showed that the sample obtained from harvesting path (bulk soil) at 10 cm and 30 cm depth clustered closely together indicating that there are small differences in microbial community at these microsites. Clustering analysis based on relative abundance shows that there is larger difference in microbial abundance between symptomless and BSR-infected sample at Frond Pile (30 cm depth). These results will provide preliminary knowledge in selecting representative samples of symptomless and BSR-infected oil palm for further microbial interaction analysis using Next Generation Sequencing

Soil pH analysis in relation to basal system rot (BSR) disease in oil palm at different depths and microsites

In agricultural industry, soil is known as important indicator for plant growth and productivity. Variations in soil conditions will alter soil ecosystem and communities that exist within the environment. Significant changes in soil environment sometimes favor the growth of plant pathogen and thus affecting plant health. Basal Stem Rot (BSR) disease caused by pathogenic fungi known as Ganoderma boninense has been identified as a major threat in oil palm plantation. The establishment of different microsites (weeded circle, harvesting path and frond pile) in oil palm management may induce the spatial differences of soil properties within a field including soil pH. Hence, our aim is to study the adverse changes in soil pH in relation to BSR disease of oil palm at different microsites (harvesting path, weeded circle and frond pile) and soil depth (10 cm and 30 cm from upper soil surface). In general, the soil samples of oil palm obtained from FELCRA Seberang Perak have shown acidic nature in which soil from infected oil palm has lower pH value (pH range 2.93-5.94) as compared to soil pH from healthy oil palm (pH range 3.39-6.29). The statistical results showed that soil depth has no significant effect to the soil pH while soil from different microsites has a significant effect to the soil pH regardless of healthy or infected oil palm. At different microsites, weeded circle give the most acidic value as compared to harvesting path and frond pile. Besides, the interaction between healthiness, depth and microsites did not have a significant effect to the soil pH. Together, these results suggest that BSR disease are likely to occur at soil with lower pH (pH below 6) and soil pH are strongly correlates with different microsites but less affected by soil depth regardless of the healthiness of oil palm

Kids array

Children have limitless imagination, and drawing can be one the ways in which they can be creative. In order to compare the effect of psychology and environment, between urban and rural children, forty 10-year-old schoolchildren were assigned to draw their interpretation of “bird” on a long paper scroll. A drawing could reveal how children feel about their environment. Interestingly, the patterns of drawing of each group were relatively consistent. A majority of female schoolchildren from urban school drew imaginative forms of bird, flying freely

Molecular characterization of fowl adenoviruses isolated from inclusion body hepatitis outbreaks in commercial broiler chickens in Malaysia

Fowl adenoviruses (FAdVs), belonging to the Aviadenovirus genus of the family Adenoviridae, have been classified into five species (A to E) and further divided into 12 serotypes. The objective of this study was to identify the serotype classification of five Malaysian FAdV isolates obtained from field outbreaks of IBH in commercial broiler chickens. Hexon-based polymerase chain reactions (PCR), combined with restriction enzyme analysis (REA), were applied. Viral DNA reacted positively with H1/H2 and H3/ H4 primer pairs which hybridised to highly conserved regions of the hexon genes. The restriction enzyme profiles of the H1/H2 fragment digested with HaeII and the H3/H4 fragment digested with HpaII revealed that all five isolates shared identical patterns and are characterised as being FAdV-8b, species E. Meanwhile, sequence analysis of the L1 loop region of the hexon gene revealed 98.1% identity with FAdV-8b strain 764. High bootstrap values in phylogenetic analysis supported the clustering of the Malaysian FAdV isolates into FAdV species E. The present study has provided a very useful reference for further studies of FAdVs in Malaysia. Vaccination strategies should be developed against FAdVs infection in commercial broiler chickens to prevent IBH outbreaks in the country

Hexon and fiber gene changes in an attenuated fowl adenovirus isolate from Malaysia in embryonated chicken eggs and its infectivity in chickens

Fowl adenovirus (FAdV) is distributed worldwide and causes economic losses in the poultry industry. The objectives of this study were to determine the hexon and fiber gene changes in an attenuated FAdV isolate from Malaysia in specific pathogen-free chicken embryonated eggs (SPF CEE) and its infectivity in commercial broiler chickens. SPF CEE were inoculated with 0.1 mL FAdV inoculum via the chorioallantoic membrane (CAM) for 20 consecutive passages. The isolate at passage 20 (E20), with a virus titer of 108.7TCID50/mL (TCID50, 50% tissue culture infective dose), was inoculated (0.5 mL) into one-day-old commercial broiler chicks either via oral or intraperitoneal routes. The study demonstrated that 100% embryonic mortality was recorded from E2 to E20 with a delayed pattern at E17 onwards. The lesions were confined to the liver and CAM. Substitutions of amino acids in the L1 loop of hexon at positions 49 and 66, and in the knob of fiber at positions 318 and 322 were recorded in the E20 isolate. The isolate belongs to serotype 8b and is non-pathogenic to broiler chickens, but it is able to induce a FAdV antibody titer. It appears that molecular changes in the L1 loop of hexon and the knob of fiber are markers for FAdV infectivity

Transcriptomic analysis on susceptibility of different inbred chicken lines towards very virulent infectious bursal disease virus infection

Infectious bursal disease virus (IBDV) is an economically important virus which affects the poultry industry worldwide. The virus is a causative agent for Gumboro disease which can cause high mortality rate in young chickens by infecting and destroying actively dividing IgM-bearing B lymphocytes in the bursa of Fabricius leading to immunosuppression. Previous studies have identified differential expression of immune-mediated genes related to inflammatory response in chickens with different genetic susceptibility to IBDV infections. However, the mechanisms of genetic resistance against IBD are not known. RNA sequencing through next-generation sequencing (NGS) technologies provide an excellent platform to study differentially expressed genes of known or unknown function to better define effective mechanism of host resistance. Therefore, this study is aimed at investigating susceptibility of different inbred chicken lines toward very virulent IBDV through transcriptomic analysis. This analysis allows for quantification of gene expression and identification of possible single nucleotide polymorphisms (SNPs), indels, and novel protein-coding sequence. RNA isolated from bursa of day 3 IBDV-infected and control chickens were used for Illumina sequencing. Bioinformatics analysis of this data will allow function annotation of differentially expressed genes, indicating possible roles in the response to infection. Gene of interest, virus detection and copy number variation between different lines will be validated using qPCR. Genes of interest that exhibit novel SNP and/or indels will be validated using siRNA experiments. This study is expected to provide information that able to decipher the genetic resistance of chickens towards IBDV infection

Biochemical and molecular aspects of 1,2-dimethylhydrazine (DMH)-induced colon carcinogenesis: a review

1,2-dimethylhydrazine (DMH) is a member in the class of hydrazines, strong DNA alkylating agent, naturally present in cycads. DMH is widely used as a carcinogen to induce colon cancer in animal models. Exploration of DMH-induced colon carcinogenesis in rodent models provides the knowledge to perceive the biochemical, molecular, and histological mechanisms of different stages of colon carcinogenesis. The procarcinogen DMH, after a series of metabolic reactions, finally reaches the colon, there produces the ultimate carcinogen and reactive oxygen species (ROS), which further alkylate the DNA and initiate the development of colon carcinogenesis. The preneolpastic lesions and histopathological observations of DMH-induced colon tumors may provide typical understanding about the disease in rodents and humans. In addition, this review discusses about the action of biotransformation and antioxidant enzymes involved in DMH intoxication. This understanding is essential to accurately identify and interpret alterations that occur in the colonic mucosa when evaluating natural or pharmacological compounds in DMH-induced animal colon carcinogenesis