Malaysia, experienced two epidemic waves of HPAI; its fi rst outbreak of HP H5N1 in August 2004 that occurred in the state of Kelantan and the second and subsequent outbreaks in February–March 2006 in three states on the west coast of Malaysia namely Wilayah Persekutuan
Kuala Lumpur, Perak and Penang. Five outbreaks occurred in village chickens and one in a multi-species enclosure of birds in a bird park resort facility. Molecular epidemiological studies by genomic sequencing and phylogenetic analyses of the viruses isolated showed that the
virus isolated from WP Kuala Lumpur is of the V-genotype and it originated from Hunan China, two viruses were found to be similar to the Fujian/Hunan strains and other viruses were similar to the Vietnam/ Thailand strains.
Two hundred and sixty four samples of ready -to-eat foods (RTE) were obtained over a period of six months (April to September 2005) from 33 school hostel kitchens and canteens previously implicated in food poisoning outbreaks from 2000 to 2004. Sampling was done by food technologists and assistant environmental health ofhcers from various districts in Pahang while microbiological analysis was carried out at the Mentakab Food Quality Control Laboratory. The objective of the study was to obtain a comprehensive picture on the microbiological status of the foods that may have accounted for food poisoning outbreaks at school hostels and canteens in Pahang. Each food sample was analyzed for Total Plate Count (TPC), Salmonella, Coliform, Escheria coli, Staphylococcus aerus and Bacillus cereus. lt was found that none of the ready-to-eat foods sampled contained Salmonella although hve samples contained Bacillus cereus , four samples contained Staphylococcus aerus. High Coliform Counts were found in 15 food samples while Escheria coli was detected in two samples. Overall, it was found that 10.2 % of the samples had unsatisfactory counts.
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.
Three isolates of Infectious bursal disease virus (IBDV), designated UPM04178, UPM04190 and UPM04238, were obtained from severe outbreaks of infectious bursal disease (IBD) in Malaysia in 2004. The hypervariable region (HPVR) of VP2 gene of these isolates was sequenced. The obtained sequences were compared with those of other isolates. The highest similarity (98%) concerning both nucleotide and amino acid sequences was found to very virulent IBDV (vvIBDV) strains. Phylogenetic analysis revealed clustering of the three isolates with vvIBDV strains. Evolutionary relatedness of the three 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. It is concluded that UPM04178, UPM04190 and UPM04238 are vvIBDV isolates of serotype 1, which originate from a common ancestor of IBDV strains present in Malaysia.
Two Infectious bursal disease virus (IBDV) isolates, NP1SSH and NP2K were obtained from a severe infectious bursal disease (IBD) outbreak in Nepal in 2002. The hypervariable (HV) region of VP2 gene (1326 bp) of the isolates was generated by RT-PCR and sequenced. The obtained nucleotide sequences were compared with those of twenty other IBDV isolates/strains. Phylogenetic analysis based on this comparison revealed that NP1SSH and NP2K clustered with very virulent (vv) IBDV strains of serotype 1. In contrast, classical, Australian classical and attenuated strains of serotype 1 and avirulent IBDV strains of serotype 2 formed a different cluster. The deduced amino acid sequences of the two isolates showed a 98.3% identity with each other and 97.1% and 98.3% identities, respectively with very virulent IBDV (vvIBDV) isolates/strains. Three amino acids substitutions at positions 300 (E-->A), 308 (I-->F) and 334 (A-->P) within the HV region were common for both the isolates. The amino acids substitutions at positions 27 (S-->T), 28 (I-->T), 31 (D-->A), 36 (H-->Y), 135 (E-->G), 223 (G-->S), 225 (V-->I), 351 (L-->I), 352 (V-->E) and 399 (I-->S) for NP1SSH and at position 438 (I-->S) for NP2K were unique and differed from other IBDV isolates/strains. NP1SSH and NP2K showed highest similarity (97.8%) with the BD399 strain from Bangladesh as compared with other vvIBDV isolates/strains. We conclude that the NP1SSH and NP2K isolates of IBDV from Nepal represent vvIBDV of serotype 1.
Nine Newcastle disease virus (NDV) isolates from Newcastle disease (ND) outbreaks in different regions of Iran were characterized at molecular level. Sequence analysis revealed that the isolates shared two pairs of arginine and a phenylalanine at the N-terminus of the fusion (F) protein cleavage site similarly to other velogenic isolates of NDV characterized earlier. Eight of the nine isolates had the same amino acid sequence as VOL95, a Russian NDV isolate from 1995. However, one isolate, MK13 showed 5 amino acid substitutions, of which 3 have been reported for other velogenic NDV isolates. These results suggest that the origin of the outbreaks of ND in different parts of Iran in 1995-1998 is VOL95.
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.
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.
The VP2 hypervariable region of P97/302 local infectious bursal disease virus (IBDV) isolate was amplified by the reverse transcriptase (RT) nested polymerase chain reaction (PCR) and cloned. This region of P97/302 local isolate was sequenced and compared with eight other reported IBDV sequences. The result showed that P97/302 IBDV was most identical to the reported very virulent IBDV strains because it has amino acid substitutions at positions 222, 256, 294, and 299, which encode alanine, isoleucine, isoleucine, and serine, respectively. This region can be digested with restriction enzymes of Taq1, Sty1, Ssp1 but not with Sac1. The P97/302 isolate was then used for the optimization of RT nested PCR enzyme-linked immunosorbent assay (ELISA). The RT nested PCR ELISA was able to detect 10(-4) dilution of the infected bursa homogenates and was 10 times more sensitive when compared with the agarose gel detection method. The RT nested PCR ELISA can detect up to 0.48 ng of the PCR product. The specificity of this nested PCR ELISA was also high (100%).
The food pellet vaccine has been shown to be effective in trials conducted under laboratory and simulated field conditions. The village chickens vaccinated with the food pellet vaccine during the field trial were protected against virulent Newcastle disease virus. The efficacy of the food pellet vaccine in the field was evaluated by challenge trial in which 60 per cent protection was obtained, or by monitoring the incidence of Newcastle disease in vaccinated and unvaccinated birds. There was no report of Newcastle disease outbreaks in the vaccinated birds during the two-year period of the field trial. The ease in administering the food pellet vaccine makes it readily accepted by the farmers.
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).
A series of plasmids containing the HSP70 gene of Mycobacterium tuberculosis fused to the hemagglutinin (H5) gene of H5N1 avian influenza virus (AIV) (H5-HSP70 (heat shock protein 70) vaccine) or individual H5 gene (H5 vaccine) or HSP70 gene (HSP70 vaccine) were constructed based on the plasmid pcDNA3.1. Expression of H5 gene in Vero cells in vitro and in chickens in vivo was confirmed following their transfection and immunization with H5 or H5-HSP70 vaccines. Controls consisted of HSP70 vaccine, empty plasmid pcDNA3.1 and co-administered H5 and HSP70 vaccines. H5-HSP70 vaccine produced in chicken higher hemagglutination inhibition (HI) antibody titer than H5 vaccine. However, the increase was not statistically significant. We have demonstrated for the first time that the H5 DNA vaccine with fused HSP70 gene may produce an enhanced induction of humoral immune response to AIV in chickens.
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.
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.
Anhydrous milkfat (AMF) was fractionated to obtain a series of high-melting milkfat fractions (HMF). Solid fat content (SFC) of HMF as determined by nuclear magnetic resonance (NMR) was in the range 37.6-43.6% and 21.2-27.5% measured at 20 and 30 degrees C, respectively. The HMF have a higher melting characteristic compared to AMF as analyzed by differential scanning calorimetry (DSC) with melting enthalpies of 92.2-105.0 J/g and melting peak temperatures of 39.3-41.5 degrees C. The AMF was also blended with soft palm stearin (SPOs and/or hard palm stearin (HPOs)) according to a three conventional component mixture design which providing suitable formulations for HMF. This represented three selected blends of AMF:SPOs:HPOs at three different proportions (70:15:15, 60:30:10 and 50:45:5), having SFC and DSC melting characteristics of HMF. The study revealed that higher-melting characteristics of AMF could be achieved equally well by using both fractionation and blending techniques.
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.
Field isolates of herpesviruses recovered from falcon, pigeon, and psittacine birds were compared by restriction endonuclease (RE) analysis using four separate enzymes. Pigeon and falcon herpesviruses had strikingly similar DNA cleavage patterns, while DNA cleavage pattern of virus isolates from a double-yellow headed Amazon and an African grey parrot had different genomic patterns to both the pigeon and falcon herpesviruses. These findings support the field observations that pigeon herpesvirus causes a fatal herpesviral infection in the livers of pigeon-eating falcons.
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.