Displaying publications 21 - 40 of 81 in total

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  1. Fulltext Efficacy, immunogenicity, and virus shedding in broiler chickens inoculated with live attenuated fowl adenovirus serotype 8b propagated a bioreactor
    Ugwu CC, Hair-Bejo M, Nurulfiza MI, Omar AR, Ideris A
    Open Vet J, 2024 Feb;14(2):617-629.
    PMID: 38549580 DOI: 10.5455/OVJ.2024.v14.i2.2
    BACKGROUND: Fowl adenovirus (FAdV) 8b causes huge economic losses in the poultry industry worldwide. Attenuated FAdV 8b could be useful in preventing FAdV infections globally and scale-up obstacles could be solved by bioreactor technology.

    AIM: This study was carried out to attenuate the FAdV 8b isolate, propagate it in a bioreactor, molecularly characterize the passage isolates, and determine the immunogenicity, efficacy, and shedding of the virus of chickens.

    METHODS: FAdV serotype 8b (UPM11142) isolate was passaged on chicken embryo liver (CEL) cells until attenuation and propagated in a bioreactor (UPM11142P20B1). Hexon and fiber genes of the isolates were sequenced and analyzed. UPM11142P20B1 was administered to 116-day-old broiler chickens divided into four groups, A (control), B (non-booster), C (booster with UPM11142P20B1), and D (booster with inactivated UPM11142P5B1). Eight chickens from each group were challenged. Body weight (BW) and liver weight (LW), liver: BW ratio (LBR), FAdV antibody titer, T lymphocyte sub-populations in the liver, spleen and thymus; and challenge virus load in the liver and shedding in cloaca were measured at weekly intervals.

    RESULTS: The isolate caused typical cytopathic effects on CEL cells typical of FAdV. Novel molecular changes in the genes occurred which could be markers for FAdV 8b attenuation. BW, LW, and LBR were similar among groups throughout the trial but the uninoculated control-challenged group (UCC) had significantly higher LBR than the inoculated and challenged groups at 35 dpi. Non-booster group had higher FAdV antibodies at all time points than the uninoculated control group (UCG); and the challenged booster groups had higher titer at 35 dpi than UCC. T lymphocytes increased at different time-points in the liver of inoculated chickens, and in the spleen and thymus as well, and was higher in the organs of inoculated challenged groups than the UCC. There was a significantly higher challenge virus load in the liver and cloaca of UCC chickens than in the non-booster chickens.

    CONCLUSION: UPM11142P20B1 was safe, efficacious, significantly reduced shedding, and is recommended as a candidate vaccine in the prevention and control of FAdV 8b infections in broiler chickens.

  2. Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus
    Hairul Aini H, Omar AR, Hair-Bejo M, Aini I
    Microbiol Res, 2008;163(5):556-63.
    PMID: 16971101
    The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.
  3. Infectious bursal disease virus tissue tropism and pathogenesis of the infection in chickens by application of in situ PCR, immunoperoxase and HE staining
    Hussein EA, Hair-Bejo M, Liew PS, Adamu L, Omar AR, Arshad SS, et al.
    Microb Pathog, 2019 Apr;129:195-205.
    PMID: 30738178 DOI: 10.1016/j.micpath.2019.01.049
    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.
  4. Velogenic newcastle disease virus tissue tropism and pathogenesis of infection in chickens by application of in situ PCR, immunoperoxase staining and HE staining
    Hussein EA, Hair-Bejo M, Omar AR, Arshad SS, Hani H, Balakrishnan KN, et al.
    Microb Pathog, 2019 Apr;129:213-223.
    PMID: 30771470 DOI: 10.1016/j.micpath.2019.02.017
    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.
  5. Fulltext Dietary zinc treatment for chronic copper intoxication in palm kernel cake (PKC) fed sheep
    Hair-Bejo M, Alimon A, Maria J, Hass M, Moonafizad M
    Malays J Nutr, 1996 Sep;2(2):196-206.
    PMID: 22692141
    Thirty, 4 month-old male Maim x Polled Dorset crossbred sheep were allocated into 6 groups of 5 animals each. Four groups of animals were stall-fed with basal diet of 90% palm kernel cake (PKC) and 10% grass (G) for 16 weeks. One group of the animal was slaughtered at the end of the 16 weeks feeding trial (PKC group), whilst the other three groups were further fed with either the same diet (PKC+PKC group) or fed with a new diet consisting of 30% corn and 10% fish meals (CF) and grass (60%) either with (PKC+CF+Zn group) or without (PKC+CF group) zinc supplementation (500 mg/g Zn as zinc sulfate) for another 16 weeks and were slaughtered at the end of the feeding trial, The other two groups which act as controls were fed with corn (30%) and fish meals (10%) and grass (60%), and were slaughtered at weeks 16 (CF group) and 32 (CF+CF group) of the trial. The blood, right and left liver, renal cortex and medulla, pancreas, bile and urine of all animals were analysed for copper and zinc contents using an atomic absorption spectrophotometer. The liver and kidney were also fixed in 10% buffered formalin for histopathological examination. The study showed that neither clinical signs nor gross lesions of copper or zinc toxicity were observed throughout the trial. However, the copper concentration in both the right and left liver of PKC fed sheep at weeks 16 and 32 rose to about 3 times that of the controls and remained high in both the PKC+CF and PKC+CF+Zn groups. A similar pattern of copper concentration was observed in the blood. The copper and zinc contents in the renal cortex and medulla, pancreas, bile and urine remained low in all groups. The zinc content in the liver of PKC+CF+Zn group was significantly increased. Histologically, moderate hepatic lesions were observed in the PKC fed sheep at week 32. The lesions were milder in the other groups especially in the PKC+CF+Zn group. No significant renal lesions was recorded in all groups. It was concluded that the usage of dietary zinc supplementation (500 mg/g) in the treatment of PKC toxicity in sheep was unsatisfactory. The ability of Malin x Polled Dorset crossbred sheep to tolerate the high copper content in PKC at least during the first 16 weeks of the feeding trial may provide more avenue in the utilization of PKC as a major feed ingredient in sheep.
  6. Fulltext The protective role of zinc in palm kernel cake (PKC) toxicity in sheep
    Hair Bejo M, Alimon A
    Malays J Nutr, 1995 Mar;1(1):75-82.
    PMID: 22692016
    Male Malin x Polled Dorset crossbred sheep were stall-fed with grass (10%) and PKC (90%) and supplemented with either zinc at 500 ug/g, as zinc sulfate (PKC+Zn group) or zinc (113 ug/g) and ammonium molybdate (500 ug/g) (PKC+Zn+Mo group) or unsupplemented diet (PKC group) for 20 weeks. Another group which acts as a control was fed with a diet consisting of corn and fish meal (2 0%) and grass (80%). The animals were monitored daily and the body weights were recorded at a period of two weeks intervals throughout the trial. Blood samples were also collected for mineral analysis. At the end of the trial the animals were slaughtered. The carcasses were examined for gross lesions, whilst the right liver lobes and renal cortex were isolated for histopathological evaluation and mineral analysis. All animals in the PKC group died before the end of the trial with the main clinical signs of generalised jaundice and haemoglobinuria. The kidneys were firm, enlarged and reddened or darkened. Histologically, the hepatocytes were swollen, vacuolated and necrotized, particularly at the periacinar zone. Hepatic fibrosis was observed at the periportal zone. Cellular swelling, vacuolation and necrosis were found in the tubular epithelial cells of the renal cortex. Neither clinical signs nor gross or remarkable histological lesions were observed in the other groups of animals. The hepatic, renal and blood copper levels In the PKC group were elevated when compared to the control. Addition of zinc either with or without ammonium molybdate in PKC diet inhibit the copper content in the organs, however the zinc contents were increased. The average daily gain of the PKC group was remained consistent to those of the other groups, except it was reduced starting at about 1 to 2 weeks prior to death. It was concluded that feeding PKC In excess in sheep can cause chronic copper toxicity. However, this effect can be prevented by dietary zinc supplementation either with or without ammonium molybdate.
  7. Fulltext In vitro fermentation of broiler cecal content: the role of lactobacilli and pH value on the composition of microbiota and end products fermentation
    Meimandipour A, Shuhaimi M, Hair-Bejo M, Azhar K, Kabeir BM, Rasti B, et al.
    Lett Appl Microbiol, 2009 Oct;49(4):415-20.
    PMID: 19725887 DOI: 10.1111/j.1472-765X.2009.02674.x
    To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products.
  8. Detection and differentiation of velogenic and lentogenic Newcastle disease viruses using SYBR Green I real-time PCR with nucleocapsid gene-specific primers
    Tan SW, Ideris A, Omar AR, Yusoff K, Hair-Bejo M
    J Virol Methods, 2009 Sep;160(1-2):149-56.
    PMID: 19447142 DOI: 10.1016/j.jviromet.2009.05.006
    SYBR Green I real-time PCR was developed for detection and differentiation of Newcastle disease virus (NDV). Primers based on the nucleocapsid (NP) gene were designed to detect specific sequence of velogenic strains and lentogenic/vaccine strains, respectively. The assay was developed and tested with NDV strains which were characterized previously. The velogenic strains were detected only by using velogenic-specific primers with a threshold cycle (C(t)) 18.19+/-3.63 and a melting temperature (T(m)) 86.0+/-0.28 degrees C. All the lentogenic/vaccine strains, in contrast, were detected only when lentogenic-specific primers were used, with the C(t) value 14.70+/-2.32 and T(m) 87.4+/-0.21 degrees C. The assay had a dynamic detection range which spans over a 5log(10) concentration range, 10(9)-10(5) copies of DNA plasmid/reaction. The velogenic and lentogenic amplifications showed high PCR efficiency of 100% and 104%, respectively. The velogenic and lentogenic amplifications were highly reproducible with assay variability 0.45+/-0.31% and 1.30+/-0.65%, respectively. The SYBR Green I real-time PCR assay detected successfully the virus from tissue samples and oral swabs collected from the velogenic and lentogenic NDV experimental infection, respectively. In addition, the assay detected and differentiated accurately NDV pathotypes from suspected field samples where the results were in good agreement with both virus isolation and analysis of the fusion (F) cleavage site sequence. The assay offers an attractive alternative method for the diagnosis of NDV.
  9. Development of SYBR green I based one-step real-time RT-PCR assay for the detection and differentiation of very virulent and classical strains of infectious bursal disease virus
    Kong LL, Omar AR, Hair Bejo M, Ideris A, Tan SW
    J Virol Methods, 2009 Nov;161(2):271-9.
    PMID: 19591873 DOI: 10.1016/j.jviromet.2009.06.023
    A SYBR Green I based one-step real-time reverse transcriptase polymerase chain reaction was developed for the detection and differentiation of very virulent (vv) and classical strains of infectious bursal disease virus (IBDV). The assay showed high PCR efficiency >93% and high reproducibility with coefficient of variation less than 0.5%. When tested on characterized IBDV strains, the very virulent and classical-specific primers detected accurately only vvIBDV and classical IBDV strains, respectively. The diagnostic efficacy of the assay was also tested on 140 bursal samples from experimental infection and 37 bursal samples from cases suspected of IBD. The assay was able to detect IBDV from bursal samples collected at days 3 and 5 post-infection with the vvIBDV strain UPM94/273 and the classical IBDV strain D78. The assay was also able to detect bursal samples infected dually with D78 and UPM94/273. The melting temperature values of the amplification products from the classical and very virulent viral infection were statistically significant (P<0.05). The specificity of the assay for detecting IBDV from suspected cases was confirmed by sequence analysis of the VP2 gene. The assay showed high sensitivity since bursal samples which were negative for IBDV were confirmed by virus isolation and PCR amplification. Hence, the new assay offers an attractive method for rapid detection of strains of IBDV.
  10. Fulltext Evaluation of the antigen relatedness and efficacy of a single vaccination with different infectious bronchitis virus strains against a challenge with Malaysian variant and QX-like IBV strains
    Ismail MI, Tan SW, Hair-Bejo M, Omar AR
    J Vet Sci, 2020 Nov;21(6):e76.
    PMID: 33263227 DOI: 10.4142/jvs.2020.21.e76
    BACKGROUND: The predominant infectious bronchitis virus (IBV) strains detected in chickens in Malaysia are the Malaysian variant (MV) and QX-like, which are associated with respiratory distress, nephropathy, and high mortality. On the other hand, the antigenic relatedness and efficacy of IBV vaccines against these 2 field IBV strains are not well characterized.

    OBJECTIVES: This study aimed to determine the antigen relatedness and efficacy of different IB vaccine strains against a challenge with MV and QX-like strains.

    METHODS: The antigen relatedness and the ability of different IB vaccine strains in conferring protection against MV and QX-like were assessed based on the clinical signs, macroscopic lesions, and ciliary activity.

    RESULTS: The MV strain IBS037A/2014 showed minor antigenic subtype differences with the vaccine virus Mass H120 and 4/91 strains but showed major antigenic subtype differences with the K2 strain. The Malaysian QX-like strain IBS130/2015 showed major antigenic subtype differences with the MV strain IBS037A/2014 and the vaccine strains except for K2. Chickens vaccinated once with Mass (H120) or with non-Mass (4/91 and K2) developed antibody responses with the highest antibody titer detected in the groups vaccinated with H120 and 4/91. The mean ciliary activities of the vaccinated chickens were between 56 to 59% and 48 to 52% in chickens challenged with IBS037A/2014 and IBS130/2015, respectively. The vaccinated and challenged birds showed mild to severe lesions in the lungs and kidneys.

    CONCLUSIONS: Despite the minor antigenic subtype differences, a single inoculation with Mass or non-Mass vaccines could not protect against the MV IBS037A/2014 and QX-like IBS130/2015.

  11. Immunochromatographic gold-based test strip for rapid detection of Infectious bursal disease virus antibodies
    Nurulfiza I, Hair-Bejo M, Omar AR, Aini I
    J Vet Diagn Invest, 2011 Mar;23(2):320-4.
    PMID: 21398455
    The immunochromatographic assay is an alternative method for simple and rapid detection of Infectious bursal disease virus (IBDV) in chickens using colloidal gold-antibody conjugate. The whole-virus antigen of IBDV (UPM04190 isolate) and the high-affinity polyclonal antibodies directed against IBDV were blotted onto nitrocellulose membranes for test and control lines, respectively. Evaluation of the strip was performed using serum samples from experimentally and naturally infected chickens. The results showed that the test strip was more sensitive than the commercial enzyme-linked immunosorbent assay (ELISA) because it could detect a dilution factor up to 120,000 (250 ELISA units) for positive samples. It was also specific, in that it detected IBDV antibodies and did not cross-react with antibodies to other chicken viruses. The method was rapid (2 min) in both clinical and field environments with samples needing only a minimum amount (50 µl) of blood to produce an acceptable detection signal. The pen-site test strip proved successful in monitoring the immune status of chickens against the IBDV infection.
  12. Fulltext Propagation and Molecular Characterization of Bioreactor Adapted Very Virulent Infectious Bursal Disease Virus Isolates of Malaysia
    Lawal N, Hair-Bejo M, Arshad SS, Omar AR, Ideris A
    J Pathog, 2018;2018:1068758.
    PMID: 30245887 DOI: 10.1155/2018/1068758
    Two Malaysian very virulent infectious bursal disease virus (vvIBDV) strains UPM0081 (also known as B00/81) and UPM190 (also known as UPM04/190) isolated from local IBD outbreaks in 2000 and 2004, respectively, were separately passaged for 12 consecutive times in 11-day-old specific pathogen free (SPF) chicken embryonated eggs (CEE) via the chorioallantoic membrane (CAM) route. The CEE passage 8 (EP8) isolates were passaged once in BGM-70 cell line yielding UPM0081EP8BGMP1 and UPM190EP8BGMP1, while the EP12 isolates were passaged 15 times in BGM-70 cell line yielding UPM0081EP12BGMP15 and UPM190EP12BGMP15 using T25 tissue culture flask. These isolates were all propagated once in bioreactor using cytodex 1 as microcarrier at 3 g per liter (3 g/L) yielding UPM0081EP8BGMP1BP1, UPM190EP8BGMP1BP1, UPM0081EP12BGMP15BP1, and UPM190EP12BGMP15BP1 isolates. The viruses were harvested at 3 days after inoculation, following the appearance of cytopathic effects (CPE) characterized by detachment from the microcarrier using standard protocol and filtered using 0.2 μm syringe filter. The filtrates were positive for IBDV by RT-PCR and immunofluorescence. Sequence and phylogenetic tree analysis indicated that the isolates were of the vvIBDV strains and were not different from the flask propagated parental viruses.
  13. Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia
    Subramaniam K, Shariff M, Omar AR, Hair-Bejo M, Ong BL
    J Fish Dis, 2014 Jul;37(7):609-18.
    PMID: 23952914 DOI: 10.1111/jfd.12152
    '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).
  14. Prevalence of feline coronavirus in two cat populations in Malaysia
    Sharif S, Arshad SS, Hair-Bejo M, Omar AR, Zeenathul NA, Hafidz MA
    J Feline Med Surg, 2009 Dec;11(12):1031-4.
    PMID: 19818660 DOI: 10.1016/j.jfms.2009.08.005
    The prevalence of feline coronavirus (FCoV) was studied in two catteries in Malaysia. Rectal swabs or faecal samples were collected from a total of 44 clinically healthy Persian purebred and mix-breed cats. RNA extracted from the faecal material was subjected to a reverse transcription-polymerase chain reaction (RT-PCR) using primers flanking for a conserved region of the virus genome. The overall prevalence of FCoV infection was 84% and the infection rate was higher in Persian purebred cats (96%) than mix-breed cats (70%). There was no significant association between the age or gender of tested cats and shedding the virus. This study is the first PCR-based survey for FCoV in Malaysia and showed the ubiquitous presence of FCoV in Malaysian cat colonies.
  15. Comparative Pathogenicity of Malaysian QX-like and Variant Infectious Bronchitis Virus Strains in Chickens at Different Age of Exposure to the Viruses
    Khanh NP, Tan SW, Yeap SK, Lee HJ, Choi KS, Hair-Bejo M, et al.
    J Comp Pathol, 2018 May;161:43-54.
    PMID: 30173857 DOI: 10.1016/j.jcpa.2018.04.006
    Infectious bronchitis viruses (IBVs) circulating in Malaysia are classified into two groups as Malaysian QX-like and variant strains. In this study, the pathogenicity of IBS130/2015 (QX-like) and IBS037A/2014 (variant) IBVs in 1-day-old and 30-day-old specific pathogen free (SPF) chickens was characterized. Both strains caused respiratory and kidney infections based on immunohistochemistry (IHC), real-time quantitative polymerase chain reaction (qPCR) and a ciliostasis study; however, the results showed that the QX-like strain was more pathogenic, caused higher mortality and showed higher tissue tropism for the kidney than the variant strain. In contrast, despite causing low or no mortality depending on the age of the infected chickens, the Malaysian variant strain showed high tissue tropism for the respiratory tract compared with the QX-like strain. IHC and qPCR indicated the presence of both IBV strains in the epithelial lining of villi in the jejunum and the caecal tonsil; however, no pathological changes were detected in these organs. Both the Malaysian QX-like and variant IBV strains are able to infect the respiratory tract and kidney of chickens irrespective of age.
  16. Fulltext Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris
    Abubakar MB, Aini I, Omar AR, Hair-Bejo M
    J Biomed Biotechnol, 2011;2011:414198.
    PMID: 21541235 DOI: 10.1155/2011/414198
    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).
  17. Isolation, identification and characterization of chicken anaemia virus in Malaysia
    Chowdhury SM, Omar AR, Aini I, Hair-Bejo M, Jamaluddin AA, Kono Y, et al.
    J. Biochem. Mol. Biol. Biophys., 2002 Aug;6(4):249-55.
    PMID: 12186740
    A study was conducted to isolate and identify chicken anaemia virus (CAV) from field samples of clinically infected broiler chickens in Malaysia. A total of 125 samples were collected from chickens aged 2-6 weeks with clinically depressed and retarded growth, of which five samples were found positive to CAV directly by polymerase chain reaction (PCR). Later, five isolates of CAV from the respective five PCR positive samples were isolated in MDCC-MSB1 cells at passage 4 based on cytopathic effects, PCR and indirect immunofluorescent antibody test. The isolates were identified as BL-1, BL-2, BL-3, BL-4 and BL-5. These CAV isolates were found to resist treatment with chloroform and heat at 37 degrees C for 2 h, 56 degrees C for 30 min and 70 degrees C for 5 min. One of the isolates, BL-5 produced significant reduction (p < 0.001) of hematocrit values (9-19%), pale bone marrow, thymus atrophy and haemorrhages in skin/muscle when inoculated into 1-day old SPF chickens. Restriction enzyme digestion of 926 bp genomic fragments of all the isolates including Cux-1 isolate with HindIII exhibited a similar pattern of bands in 2% agarose gel. The present findings confirmed the presence of CAV in Malaysia.
  18. Sequence and phylogenetic analysis of the VP2 gene of very virulent infectious bursal disease virus isolates
    Hoque MM, Omar AR, Hair-Bejo M, Aini I
    J. Biochem. Mol. Biol. Biophys., 2002 Apr;6(2):93-9.
    PMID: 12186763
    Previously we have shown that very virulent infectious bursal disease viruses (vvIBDV) that are SspI, TaqI and StyI positive (92/04, 97/61 and 94/B551) but not SspI and TaqI positive and StyI negative (94/273) cause high mortality, up to 80% in specific-pathogen-free chickens with significant damage of the bursal as well as nonbursal tissues. In this study, we sequenced the VP2 gene (1351 bp) of the 92/04, 94/273 and 94/B551 and compared them with other IBDV strains. All the isolates have the unique amino acid residues at positions 222A, 256I, 294I and 299S found in other vvIBDV strains. The deduced VP2 amino acids encoded by 92/04 is identical to the vvIBDV strains from Israel (IBDVKS), Japan (OKYM) and Europe (UK661), whereas the 94/273 and 94/B551 isolates have one to three amino acid substitutions. The 94/273 has two amino acid substitutions at positions 254 G to S and at 270 A to E that have not been reported before from vvIBDV strains. The 94/B551 also has one amino acid substitution at position 300 E to S, which is uncommon among other vvIBDV strains. However, phylogenetic analysis suggested that the isolates are very close to each other and all of them may have derived from the same origin as vvIBDV strains isolated from China, Japan and Europe. Even though antigenic index analysis of the 94/273 and 94/B551 indicated that the isolates are unique compared to other IBDV strains, their antigenic variation remain to be determined by monoclonal antibody study.
  19. Development of a modified method for sequencing high G:C regions in chicken anemia virus genome
    Chowdhury SM, Omar AR, Aini I, Hair-Bejo M, Jamaluddin AA, Wan KL, et al.
    J. Biochem. Mol. Biol. Biophys., 2002 Jun;6(3):229-32.
    PMID: 12186760
    Two areas in the chicken anemia virus (CAV) genome have high G:C content with secondary structures. These two G:C rich areas could not be sequenced with Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit. Several modifications were carried out to solve the problem. Finally, a package of modified method was developed to sequence the high G:C areas. The result showed that the Perkin Elmer's Big Dye Terminator Cycle Sequencing Kit with the normal procedures are not suitable for sequencing the high G:C regions of the CAV genome. The present developed method made the Perkin Elmer's Kit useful for the first time to sequence the G:C rich hairpin structures of the CAV genome. The system may be useful to sequence all other G:C rich DNA templates.
  20. Absorption and Bioavailability of Nano-Size Reduced Calcium Citrate Fortified Milk Powder in Ovariectomized and Ovariectomized-Osteoporosis Rats
    Erfanian A, Mirhosseini H, Rasti B, Hair-Bejo M, Bin Mustafa S, Abd Manap MY
    J Agric Food Chem, 2015 Jun 24;63(24):5795-804.
    PMID: 26022498 DOI: 10.1021/acs.jafc.5b01468
    The aim of this study was to evaluate the effects of fortification and nano-size reduction on calcium absorption and bioavailability of milk powder formula in sham, ovariectomized, and ovariectomized-osteoporosis rats as a menopause and menopause-osteoporosis model. Skim milk powder and skim milk powder fortified with calcium citrate and the suitable doses of inulin, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), and vitamins D3, K1, and B6 were formulated based on the North American and Western European recommended dietary allowances. Optimization on cycle and pressure of high-pressure homogenizer was done to produce nano-fortified milk powder. In vivo study demonstrated that fortification and calcium citrate nano-fortified milk powder increased absorption and bioavailability of calcium, as well as bone stiffness and bone strength in sham, ovariectomized, and ovariectomized-osteoporosis rats. This study successfully developed an effective fortified milk powder for food application.
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