Subtype-specific multiplex reverse transcription-polymerase chain reaction (RT-PCR) was developed to simultaneously detect three subtypes (H5, H7 and H9) of avian influenza virus (AIV) type A. The sensitivity of the multiplex RT-PCR was evaluated and compared to that of RT-PCR-enzyme-linked immunosorbent assay (ELISA) and conventional RT-PCR. While the sensitivity of the multiplex RT-PCR is as sensitive as the conventional RT-PCR, it is 10 times less sensitive than RT-PCR-ELISA. The multiplex RT-PCR is also as sensitive as the virus isolation method in detecting H9N2 from tracheal samples collected at day 3 and 5 post inoculation. Hence, the developed multiplex RT-PCR assay is a rapid, sensitive and specific assay for detecting of AIV subtypes.
The ability of a heat-inactivated whole virus from a highly virulent infectious bursal disease virus (hvIBDV) and VP2 protein from hvIBDV expressed in E. coli provided protection against a hvIBDV challenge in specificpathogen-free (SPF) chickens. Six out of seven chickens that were injected three times with crude VP2 protein developed significant antibody titer against IBDV. However, only four out of the seven chickens survived the hvIBDV challenge. Despite showing low antibody titer profiles, all chickens immunized with the heat-inactivated whole virus also survived the challenged with hvIBDV. However, all of these chickens had bursal atrophy and mild to moderate depletion of lymphocytes. Thus, antibodies raised against IBDV VP2 protein expressed in E. coli and denatured IBDV proteins induced some degree of protection against mortality but not against bursal damage following challenge with hvIBDV.
It has been shown that a chicken anemia virus (CAV) isolates which had undergone 60 passages in MSB-1 cells (SMSC-1/P60, 3-1/P60) acquired 33-66 nucleotide substitutions at the coding region resulting in 13-16 amino acid changes as compared to the CAV isolates passaged only 5 times in MSB-1 cells (SMSC-1 and 3-1) (Chowdhury et al., Arch. Virol. 148, 2437-2448, 2003). In this study we found that a low CAV (BL-5) and a high CAV passage (BL-5/P90) differed by only 15 nucleotide substitutions resulting in 11 amino acid changes. Phylogenetic analysis based on VP1 also revealed that both isolates were close to each other but not to other CAV isolates from Malaysia, namely SMSC-1 and 3-1.
Specific-pathogen-free (SPF) chickens infected with very virulent (vv) infectious bursal disease virus (IBDV) UPM94/273 developed lower pathogenicity compared to UPM97/61. Sequence analysis indicated that UPM94/273 is an exceptional vvIBDV. In this study, a SYBR Green I based real-time reverse transcriptase reaction assay was developed to measure viral RNA in the bursae of SPF chickens infected with IBDV. Specificity of the amplified products was confirmed by melting temperature analysis. A linear relationship was observed between the amount of input viral RNA and the threshold values for IBDV-specific product over five log10 dilutions. The viral RNA level following infection with UPM94/273 was significantly higher at day 1 and 2 post-inoculation (p.i.) compared to UPM97/61 infected chickens. However, chickens infected with UPM97/61 had significantly higher numbers of bursal cells undergoing apoptosis compared to UPM94/273 infected chickens. In both groups, the number of apoptotic cells and viral RNA levels peak at day 3 p.i. This study indicates that UPM97/61 and UPM94/273 have different efficiency of replication and percentage of apoptotic cells in bursae during the acute phase of IBDV infection.
Dysregulated metabolism is implicated in obesity and other disease conditions like type 2 diabetes mellitus and cardiovascular diseases, which are linked to abnormalities of peroxisome proliferator-activated receptor gamma (PPARγ). PPARγ has been the focus of much research aimed at managing these diseases. Also, germinated brown rice (GBR) is known to possess antidiabetic, antiobesity and hypocholesterolemic effects. We hypothesized that GBR bioactive compounds may mediate some of the improvements in metabolic indices through PPARγ modulation. Cultured HEP-G2 cells were treated with 50 ppm and 100 ppm of extracts from GBR (GABA, ASG and oryzanol) after determination of cell viabilities using MTT assays. Results showed that all extracts upregulated the expression of the PPARγ. However, combination of all three extracts showed downregulation of the gene, suggesting that, in combination, the effects of these bioactives differ from their individual effects likely mediated through competitive inhibition of the gene. Upregulation of the gene may have therapeutic potential in diabetes mellitus and cardiovascular diseases, while its downregulation likely contributes to GBR's antiobesity effects. These potentials are worth studying further.
Newcastle disease virus (NDV) strain AF2240 is a viscerotropic velogenic strain that is used as a vaccine challenge virus in Malaysia. The identification of the full length genome will be a crucial platform for further studies of this isolate. In this study, we fully sequenced the genome of a derivative of this strain named AF2240-I. The 15,192 nt long genome contains a 55-nt leader sequence at the 3' whereas the trailer region consists of 114 nt at the 5'. The intergenic sequences between the NP-P, P-M, M-F, F-HN, and HN-L genes comprise 1, 1, 1, 31, and 47 nt, respectively. The acknowledged cleavage site of fusion protein showed amino acid sequence of 112-R-R-Q-K-R-F-117, which corresponds to those of virulent NDV strains. Phylogenetic analysis of the whole virus genome shows that the strain AF2240-I belongs to genotype VIII and is more closely related to velogenic strains QH1, QH4, Fontana, Largo, and Italienas compared to other strains of NDV. Differences are noticed in the hemagglutinin-neuraminidase (HN) and matrix (M) gene between AF2240 and its derivative AF2240-I. This is the first report of a complete genome sequence of an NDV strain isolated in Malaysia.
In order to develop a systemically administered safe and effective nonviral gene delivery system against avian influenza virus (AIV) that induced cytokine expression, the hemagglutinin (H5) gene of AIV, A/Ck/Malaysia/5858/04 (H5N1) and green fluorescent protein were cloned into a coexpression vector pIRES (pIREGFP-H5) and formulated using green synthesis of silver nanoparticles (AgNPs) with poly(ethylene glycol) and transfected into primary duodenal cells taken from 18-day-old specific-pathogen-free chick embryos. The AgNPs were prepared using moderated temperature and characterized for particle size, surface charge, ultraviolet-visible spectra, DNA loading, and stability. AgNPs and AgNP-pIREGFP-H5 were prepared in the size range of 13.9 nm and 25 nm with a positive charge of +78 ± 0.6 mV and +40 ± 6.2 mV, respectively. AgNPs with a positive surface charge could encapsulate pIREGFP-H5 efficiently. The ultraviolet-visible spectra for AgNP-pIREGFP-H5 treated with DNase I showed that the AgNPs were able to encapsulate pIREGFP-H5 efficiently. Polymerase chain reaction showed that AgNP-pIREGFP-H5 entered into primary duodenal cells rapidly, as early as one hour after transfection. Green fluorescent protein expression was observed after 36 hours, peaked at 48 hours, and remained stable for up to 60 hours. In addition, green fluorescent protein expression generally increased with increasing DNA concentration and time. Cells were transfected using Lipocurax in vitro transfection reagent as a positive control. A multiplex quantitative mRNA gene expression assay in the transfected primary duodenal cells via the transfection reagent and AgNPs with pIREGFP-H5 revealed expression of interleukin (IL)-18, IL-15, and IL-12β.
Acute coronary syndrome (ACS) patients with positive troponin T (TnT) test are at higher risk for death and myocardial reinfarction. They would significantly benefit from early aggressive pharmacologic and invasive therapy. However, TnT test is not widely available. This retrospective study of 173 patients with ACS showed: that prolonged or repetitive episodes of angina at rest in the previous 24 hours (p = 0.01) and evidence of myocardial ischaemia on ECG (p < 0.001) were associated with positive TnT tests (> or = 0.1 ng/mL). The two variables in combination showed 100% positive predictive value, facilitating early identification and streamlining of therapy.
This study was conducted to determine whether early age feed restriction improves heat tolerance in female broiler chickens. Chicks were brooded for 3 wk and then maintained at 24+/-1 C. On Day 0, chicks were assigned to one of four feeding regimens; each regimen was applied to four cages of chicks. The feeding regimens were 1) ad libitum feeding (ALF); 2) 40% feed restriction at 4, 5, and 6 d of age (F40); 3) 60% feed restriction at 4, 5, and 6 d of age (F60); and (4) 80% feed restriction at 4, 5, and 6 d of age (F80). From 35 to 41 d of age, all birds were exposed to 38+/-1 C for 2 h/d. Serum concentrations of glucose were elevated by the heat challenge, but were not affected by the feeding regimen. The heat treatment resulted in hypocholesteremia among ALF and F80 chicks, whereas the concentrations increased and remained constant in the F60 and F40 birds, respectively. Subjecting chicks to F60 improved growth and survivability and reduced heterophil to lymphocyte ratios (H/L) in response to the heat treatment as compared with the ALF and F80 regimens. The survivability rate and H/L of F40 chicks were similar to those attained by chicks on other regimens. Newcastle disease antibody titer of ALF birds declined with duration of heat treatment. It is concluded that the F60 regimen is beneficial for alleviating, at least in part, the detrimental effects of heat stress in female broiler chickens.
The complete nucleotide sequences encoding precursor polyprotein (VP2-VP3-VP4) and VP5 of a highly virulent (hv) infectious bursal disease virus (IBDV), UPM97/61 was determined. Comparison of the deduced amino acid sequences with the published ones revealed 8 common amino acid substitutions, which were found only in the hv IBDV including the UPM97/61 strain. Three of the amino acid substitutions (222 Ala, 256 Ile and 294 Ile) were used as a marker for determining hv IBDV strains. The other five substitutions (685 Asn, 715 Ser, 751 Asp, 990 Val and 1005 Ala) were also conserved in hv IBDV strains isolated in various countries. UPM97/61 strain demonstrated also 8 unique amino acid substitutions of which 3 were in VP2, 4 in VP3 and 1 in VP4. There was 1 unique amino acid substitution in VP5 at position 19 (Asp-->Gly) not found in other strains. However, all the strains have a conserved 49 Arg. The amino acid sequence of UPM97/61 strain differed by 1.09% from the Japanese (OKYM) and Hong Kong (HK46) strains, and by 1.48% from the Israeli (IBDVKS) and European (UK661) strains. Hence, UPM97/61 is more closely related to the hv strains from Asia. However, phylogenetic analysis indicated that the origin of UPM97/61 might be the same as that of other hv strains isolated from other parts of the world.
The nucleocapsid (NP) protein of Newcastle disease virus (NDV) self-assembled in Escherichia coli as ring-like and herringbone-like particles. Several chimeric NP proteins were constructed in which the antigenic regions of the hemagglutinin-neuraminidase (HN) and fusion (F) proteins of NDV, myc epitope, and six histidines (a hexa-His tag) were linked to the C-terminus of the NP monomer. These chimeric proteins were expressed efficiently in soluble form in E. coli as detected by Western blot analysis. Electron microscopy of the purified products revealed that they self-assembled into ring-like particles. These chimeric particles exhibited antigenicity of the myc epitope, suggesting that the foreign sequences were exposed on the surface of the particles. Chickens inoculated with the chimeric particles mounted an immune response against NDV, suggesting the possibility of use of the ring-like particle as a carrier of immunogens in subunit vaccines and immunological reagents.
A model of pneumonic pasteurellosis has been established in goats using Pasteurella multocida harvested from pneumonic lungs of goats (types A and D), rabbits (type A) and sheep (type D). The resultant infections were acute, subacute or chronic. The gross and histological lesions of the subacute and chronic infections were typical of pneumonic pasteurellosis. P. multocida type D produced significantly (P < 0.01) more severe lesions when compared with other isolates. There were strong correlations between the clinical signs and the severity of lesions.
Pulsed field gel electrophoresis analysis of genomic DNA was used to investigate genetic diversity among Dichelobacter nodosus from footrot in sheep in Malaysia. Twelve Dichelobacter nodosus strains isolated from lesion materials from infected sheep were confirmed as Dichelobacter nodosus by polymerase chain reaction technique using the species-specific Dichelobacter nodosus 16S RNA sequence Ac and C as primers. Pulsed field gel electrophoresis banding profiles using restriction enzymes ApaI (5'GGGCCC3'), SfiI (5'GGCCNNNNNGGCC3') and SmaI ('5CCCGGG3') enabled the 12 Dichelobacter nodosus strains to be differentiated into eight different PFGE patterns and thus genome-types, with F (coefficient of similarity) values ranging from 0.17 to 1.0 (ApaI), 0.14 to 1.0 (SfiI) and 0.22 to 1.0 (SmaI). Strains with origin in different farms were shown to have different PFGE patterns (two strains, M7 and M8 were the only exception). On the basis of their PFGE, all field strains used in the study differed from the reference strains. Our data revealed that there are several clonal types of Dichelobacter nodosus isolates and indicated that there is probably more than one source of this pathogen on the farms studied. The study showed that strains of D. nodosus exhibited considerable genetic diversity using this method and that genomic analysis by pulsed field gel electrophoresis was useful in discriminating the D. nodosus strains.
A 16-year-old female from Rantau Panjang, Kelantan reported having diarrhoea for three months. During this period, she lost 15 lb in weight and was treated with antibiotics and anti-spasmodic tablets with no improvement. Stool examinations by private laboratories revealed "worm-like eggs". She was treated for worms with mebendazole which helped to reduce the symptoms but not completely. The patient continued passing out the abnormal "worm-like eggs" which were later identified as pollen grains.
Sixteen goats either subjected to transport stress or without transport stress were treated with dexamethasone for 3 days prior to infection with P. haemolytica serotype A2 intranasally. The transport-stressed and dexamethasone-treated goats in the first group had various degrees of pulmonary lesions and the organism was re-isolated from the nasal cavity, lymph nodes and lungs. None of the goats treated with dexamethasone only were infected with P. haemolytica and had no lesions of pneumonic pasteurellosis. Treatment with dexamethasone alone failed to induce experimental infection by P. haemolytica except in combination with another stress factor.
A two-step SYBR Green I real time polymerase chain reaction (PCR, real time PCR) for the detection of Newcastle disease virus (NDV) was developed. A melting curve analysis was performed to distinguish specific from non-specific products and primer dimers. Regardless of different virus pathotypes the melting temperature (Tm) ranged from 86 degrees C to 87 degrees C. The sensitivity of the real time PCR was compared with the reverse transcription (RT)-nested PCR enzyme-linked immunosorbent assay (ELISA, RT-nested PCR ELISA). Whereas the detection limit of the real time PCR was 10 pg DNA, the RT-nested PCR ELISA and conventional PCR could only detect up to 1 ng and 10 ng DNA, respectively. Thus the real time PCR offers a sensitive, rapid and convenient method for screening large number of NDV specimens.
The deduced amino acid sequences of segment A and B of two very virulent Infectious bursal disease virus (vvIBDV) isolates, UPM94/273 and UPM97/61 were compared with 25 other IBDV strains. Twenty amino acid residues (8 in VP1, 5 in VP2, 2 in VP3, 4 in VP4, 1 in VP5) that were common to vvIBDV strains were detected. However, UPM94/273 is an exceptional vvIBDV with usual amino acid substitutions. The differences in the divergence of segment A and B indicated that the vvIBDV strains may have been derived from genetic reassortment of a single ancestral virus or both segments have different ability to undergo genetic variation due to their different functional constraints.
The effects of early age feed restriction and heat conditioning on heat shock protein (HSP) 70 expression, antibody production, resistance to infectious bursal disease (IBD), and growth of heat-stressed male broiler chickens were investigated. Chicks were divided into 4 groups: 60% feed restriction on d 4,5, and 6 (FR); exposure to 36 +/- 1 degrees C for 1 h from d 1 to 21 (HT); combination of FR and HT (FRHT); and control. From d 35 to 50, heat stress was induced by exposing birds to 38 +/- 1 degrees C and 80% RH for 2 h/d. On d 36, each bird was administered 10 times the normal dose of live IBD vaccine. After heat exposure, the FRHT birds had higher HSP 70 density (d 41) and weight gain (from d 35 to 49) and lower bursal histological score (BHS) (d 51) than their HT and control counterparts. The HSP 70 expression and BHS of FR birds were not significantly different from those of the other 3 groups during the heat exposure period. Heat shock protein 70 and BHS data were negatively correlated (r = -0.33, P = 0.0008). We concluded that FRHT could improve weight gain and resistance to IBD in male broiler chickens under heat stress conditions. The improved heat tolerance and disease resistance in FRHT birds could be attributed to better HSP 70 response.
Newcastle disease virus (NDV) exerts its naturally occurring oncolysis possibly through the induction of apoptosis. We hypothesized that the binding of the virus to the cell via the hemagglutinin-neuraminidase (HN) glycoprotein may be sufficient to not only induce apoptosis but to induce a higher apoptosis level than the parental NDV AF2240 virus. NDV AF2240 induction of apoptosis in MCF-7 human breast cancer cells was analyzed and quantified. In addition, the complete HN gene of NDV strain AF2240 was amplified, sequenced and cloned into the pDisplay eukaryotic expression vector. HN gene expression was first detected at the cell surface membrane of the transfected MCF-7 cells. HN induction of apoptosis in transfected MCF-7 cells was analyzed and quantified. The expression of the HN gene alone was able to induce apoptosis in MCF-7 cells but it was a less potent apoptosis inducer compared to the parental NDV AF2240 strain. In conclusion, the NDV AF2240 strain is a more suitable antitumor candidate agent than its recombinant HN gene unless the latter is further improved by additional modifications.
Newcastle disease virus (NDV) AF2240 Malaysian strain is a very virulent avian virus. NDV strain AF2240 was previously demonstrated to induce apoptosis in human breast carcinoma MCF-7 cells. However, at which stage of the NDV life cycle apoptosis is induced and whether NDV replication and protein synthesis are involved in apoptosis induction have yet to be determined. In the present study, we investigated the time course of NDV strain AF2240 nucleoprotein (NP) gene expression and the early apoptotic signs in the form of activation of caspase-8 and mitochondrial transition pore opening. In addition, the induction of apoptosis by both ultraviolet-inactivated and cycloheximide-treated NDV-infected MCF-7 cells were examined. Our findings showed that NDV strain AF2240 induced apoptosis at 1 h post-infection (pi) through activation of mitochondrial transition pore opening and at 2 h through activation of caspase-8, while the NP gene was expressed at 6 h pi. The induced apoptosis was independent of both virus replication and protein synthesis. In conclusion, NDV strain AF2240 induces apoptosis at an early stage of its life cycle, possibly during virus binding or fusion with the cell membrane. The mitochondrial-related pathway may be the central activator in NDV strain AF2240-induced apoptosis.