As the world population grows, the demand for food increases. Although vegetable oils provide an affordable and rich source of energy, the supply of vegetable oils available for human consumption is limited by the "fuel vs food" debate. To increase the nutritional value of vegetable oil, metabolic engineering may be used to produce oil crops of desirable fatty acid composition. We have isolated and characterized β-ketoacyl ACP-synthase II (KASII) cDNA from a high-oleic acid palm, Jessenia bataua. Jessenia KASII (JbKASII) encodes a 488-amino acid polypeptide that possesses conserved domains that are necessary for condensing activities. When overexpressed in E. coli, recombinant His-tagged JbKASII was insoluble and non-functional. However, Arabidopsis plants expressing GFP-JbKASII fusions had elevated levels of arachidic acid (C20:0) and erucic acid (C22:1) at the expense of stearic acid (C18:0) and oleic acid (C18:1). Furthermore, JbKASII failed to complement the Arabidopsis KASII mutant, fab1-2. This suggests that the substrate specificity of JbKASII is similar to that of ketoacyl-CoA synthase (KCS), which preferentially elongates stearic and oleic acids, and not palmitic acid. Our results suggest that the KCS-like JbKASII may elongate C18:0 and C18:1 to yield C20:0 and C22:1, respectively. JbKASII may, therefore, be an interesting candidate gene for promoting the production of very long chain fatty acids in transgenic oil crops.
Foot-and-mouth disease (FMD) is endemic in the countries of mainland Southeast Asia where it represents a major obstacle to the development of productive animal industries. The aim of this study was to use genetic data to determine the distribution of FMD virus (FMDV) lineages in the Southeast Asia region, and in particular identify possible sources of FMDV causing outbreaks in Malaysia. Complete VP1 sequences, obtained from 214 samples collected between 2000 and 2009, from FMD outbreaks in six Southeast Asian countries, were compared with sequences previously reported. Phylogenetic analysis of these sequences showed that there were two patterns of FMDV distribution in Malaysia. Firstly, for some lineages (O/SEA/Mya98 and serotype A), outbreaks occurred every year in the country and did not appear to persist, suggesting that these incursions were quickly eradicated. Furthermore, for these lineages FMD viruses in Malaysia were closely related to those from neighbouring countries, demonstrating the close epidemiological links between countries in the region. In contrast, for O/ME-SA/PanAsia lineage, viruses were introduced and remained to cause outbreaks in subsequent years. In particular, the recent incursion and maintenance of the PanAsia-2 sublineage into Malaysia appears to be unique and independent from other outbreaks in the region. This study is the first characterisation of FMDV in Malaysia and provides evidence for different epidemiological sources of virus introduction into the country.
Newcastle disease (ND), caused by Newcastle disease virus (NDV), is a highly contagious disease of birds and has been one of the major causes of economic losses in the poultry industry. Despite routine vaccination programs, sporadic cases have occasionally occurred in the country and remain a constant threat to commercial poultry. Hence, the present study was aimed to characterize NDV isolates obtained from clinical cases in various locations of Malaysia between 2004 and 2007 based on sequence and phylogenetic analysis of partial F gene and C-terminus extension length of HN gene.
Hepatitis B virus (HBV) and high liver iron deposits have both been associated with the development of cirrhosis. Among HBV factors, genotype and mutations in the basal core promoter (BCP) and precore regions have been most frequently studied but the evidence for a positive association with cirrhosis has been inconsistent. In this study, sera from persons with chronic HBV infection with and without cirrhosis were used for whole HBV genome analysis and for the estimation of serum iron marker (serum iron or ferritin) levels. Single codon analysis showed that the precore wild-type, TGG (nt 1,895-1,897), gave the highest accuracy (77.5%) for the identification of cirrhosis compared to other codons. When TGG was analyzed together with the precore start codon wild-type, ATG (nt 1,814-1,816), the accuracy was improved to 80.0% (odds ratio=35.29; 95% confidence interval=3.87-321.93; Phi=0.629; P<0.001). When the serum iron marker was included for analysis, it was clear that a combination of a precore wild-type and high serum iron marker gave a better accuracy (90.0%) (odds ratio=107.67; 95% confidence interval=10.21-1,135.59; Phi=0.804; P<0.001) for the identification of cirrhosis than either biomarker alone. It appeared that a combined use of both these biomarkers might help to predict the development of cirrhosis in a person with chronic HBV infection, but longitudinal studies are required to test this hypothesis.
Forty-seven Salmonella Typhimurium (33 zoonotic, 14 clinical) strains were tested for antimicrobial resistance using the standard disk diffusion method. Presence of relevant resistance genes and class 1 integrons were investigated by using PCR. Pulsed-field gel electrophoresis (PFGE) and plasmid profiling were carried out to determine the genomic diversity of Salmonella Typhimurium. Approximately 57.4% of S. Typhimurium were multidrug resistant (MDR) and showed high resistance rates to tetracycline (70.2%), sulphonamides (57.4%), streptomycin (53.1%), ampicillin (29.7%), nalidixic acid (27.6%), kanamycin (23.4%), chloramphenicol (21.2%) and trimethoprim (19.1%). Resistance towards cephalosporins was noted for cephalothin (27.6%), cephradine (21.2%), amoxicillin clavulanic acid (17.0%) and cephalexin (17.0%). Resistance genes, blaTEM, strA, aadA, sul1, sul2, tet(A), tet(B) and tet(C) were detected among the drug resistant strains. Thirty-three strains (70.2%) carried class 1 integrons, which were grouped in 9 different profiles. DNA sequencing identified sat, aadA, pse-1 and dfrA genes in variable regions on class 1 integrons. Thirty-five strains (74.4%) were subtyped to 22 different plasmid profiles, each with 1 - 6 plasmids (2.0 to 95 kb). PFGE subtyped the 47 strains into 39 profiles. In conclusion, high rates of multidrug-resistance were found among the Malaysian Salmonella Typhimurium strains. The emergence of multidrug-resistant Salmonella Typhimurium to cephalosporin antibiotics was also observed. The strains were very diverse and no persistent clone was observed. The emergence of MDR Salmonella Typhimurium is a worldwide problem and this report provides information for the better understanding of the prevalence and epidemiology of MDR S. Typhimurium in Malaysia.
Ninety (n = 90) imipenem-resistant Pseudomonas aeruginosa (IRPA) clinical isolates collected randomly during 2005 to 2008 from University Malaya Medical Center were assessed for the presence of different variants of metallo-beta-lactamase (MBL) genes. Polymerase chain reaction (PCR) assay detected 32 (n = 32) MBL gene PCR-positive isolates with the presence of bla(IMP) gene in 14 (n = 14) and bla(VIM) in 18 (n = 18) isolates. Four allelic variants, bla(IMP-7) (12 isolates), bla(IMP-4) (2 isolates), bla(VIM-2) (17 isolates), and bla(VIM-11) (1 isolate), of MBL genes were identified. This study is the first report of detection of bla(IMP-4), bla(VIM-2), and bla(VIM-11) MBL genes from IRPA clinical isolates in Malaysia.
Study site: University Malaya Medical Center (UMMC)
A bacterial strain, KM1S, was isolated from a Malaysian rainforest soil sample by using a defined enrichment medium that specifically facilitates selection of quorum quenching bacteria. KM1S was clustered closely to Bacillus cereus by 16S ribosomal DNA sequence analysis. It degraded N-3-oxo-hexanoyl homoserine lactone and N-3-oxo-octanoyl homoserine lactone in vitro rapidly at 4.98 and 6.56 microg AHL h(-1) per 10(9) CFU/ml, respectively, as determined by the Rapid Resolution Liquid Chromatography. The aiiA homologue, encoding an autoinducer inactivation enzyme catalyzing the degradation of N-acylhomoserine lactones, of KM1S was amplified and cloned. Sequence analysis indicated the presence of the motif (106)HXDH-59 amino acids-H(169)-21 amino acids-D(191) for N-acylhomoserine lactone lactonases.
Epidermal pseudotumours from Hippoglossoides dubius and Acanthogobius flavimanus in Japan and gill lesions in Limanda limanda from the UK have been shown to be caused by phylogenetically related protozoan parasites, known collectively as X-cells. However, the phylogenetic position of the X-cell group is not well supported within any of the existing protozoan phyla and they are currently thought to be members of the Alveolata.Ultrastructural features of X-cells in fish pseudotumours are somewhat limited and no typical environmental stages, such as spores or flagellated cells, have been observed. The life cycles for these parasites have not been demonstrated and it remains unknown how transmission to a new host occurs. In the present study, pseudobranchial pseudotumours from Atlantic cod, Gadus morhua, in Iceland and epidermal pseudotumours from the northern black flounder, Pseudopleuronectes obscurus, in Japan were used in experimental transmission studies to establish whether direct transmission of the parasite is achievable. In addition, X-cells from Atlantic cod were sequenced to confirm whether they are phylogenetically related to other X-cells and epidermal pseudotumours from the northern black flounder were analysed to establish whether the same parasite is responsible for infecting different flatfish species in Japan.
The structural gene of elastase strain K (elastase from Pseudomonas aeruginosa strain K), namely HindIII1500PstI, was successfully sequenced to contain 1497 bp. The amino acid sequence, deduced from the nucleotide sequence, revealed that the mature elastase consists of 301 amino acids, with a molecular mass of 33.1 kDa, and contains a conserved motif HEXXH, zinc ligands and residues involved in the catalysis of elastase strain K. The structural gene was successfully cloned to a shuttle vector, pUCP19, and transformed into Escherichia coli strains TOP10, KRX, JM109 and Tuner™ pLacI as well as P. aeruginosa strains PA01 (A.T.C.C. 47085) and S5, with detection of significant protein expression. Overexpression was detected from transformants KRX/pUCP19/HindIII1500PstI of E. coli and PA01/pUCP19/HindIII1500PstI of P. aeruginosa, with increases in elastolytic activity to 13.83- and 5.04-fold respectively relative to their controls. In addition, recombinant elastase strain K showed considerable stability towards numerous organic solvents such as methanol, ethanol, acetone, toluene, undecan-1-ol and n-dodecane, which typically pose a detrimental effect on enzymes; our finding provides further information to support the potential application of the enzyme in synthetic industries, particularly peptide synthesis.
Culture of Asian seabass, Lates calcarifer (Bloch) is a popular aquaculture activity in Malaysia. This fish is in high demand and fetches a good price in the local market. The seed for this fish is commercially produced by induced spawning in hatcheries. However, the seed supply is affected by frequent mass mortality of larvae aged between 15 and 60 dph. The clinical signs shown by the affected larvae include lethargy, loss of appetite, uncoordinated swimming, unusual spiral movement pattern and dark coloration. Histological examination of brain and eye of the affected specimens revealed extensive cell vacuolation in larvae aged 15-25 dph. Partial nucleotide sequence of the nervous necrosis virus coat protein gene of the affected larvae showed 94.0-96.1% homology to the nucleotide sequences of coat protein gene from nervous necrosis virus isolated from other countries in the Southeast Asia and Australia. This study provides scientific evidence based on molecular technique that many episodes of mass mortality in seabass larvae in Sabah is associated with the viral nervous necrosis. Because no effective treatment has been reported for this infection, stringent biosecurity measures must be adopted for exclusion of the pathogen from the culture system.
The majority of spinal muscular atrophy (SMA) patients showed homozygous deletion or other mutations of SMN1. However, the genetic etiology of a significant number of SMA patients has not been clarified. Recently, mutation in the gene underlying cat SMA, limb expression 1 (LIX1), has been reported. Similarity in clinical and pathological features of cat and human SMA may give an insight into possible similarity of the genetic etiology.
The ubiquitin extension protein (uep1) gene was identified as a constitutively expressed gene in oil palm. We have isolated and characterized the 5' region of the oil palm uep1 gene, which contains an 828 bp sequence upstream of the uep1 translational start site. Construction of a pUEP1 transformation vector, which contains gusA reporter gene under the control of uep1 promoter, was carried out for functional analysis of the promoter through transient expression studies. It was found that the 5' region of uep1 functions as a constitutive promoter in oil palm and could drive GUS expression in all tissues tested, including embryogenic calli, embryoid, immature embryo, young leaflet from mature palm, green leaf, mesocarp and meristematic tissues (shoot tip). This promoter could also be used in dicot systems as it was demonstrated to be capable of driving gusA gene expression in tobacco.
Nine 50-l surface water samples from a Malaysian recreational lake were examined microscopically using an immunomagnetisable separation-immunofluorescent method. No Cryptosporidium oocysts were detected, but 77.8% of samples contained low numbers of Giardia cysts (range, 0.17-1.1 cysts/l), which were genetically characterised by SSU rRNA gene sequencing. Genotype analyses indicated the presence of Giardia duodenalis assemblage A suggesting potential risk to public health. The present study represents the first contribution to our knowledge of G. duodenalis assemblages in Malaysian recreational water.
There are few reports of congenital disorders of glycosylation (CDGs) in the Asian population, although they have been reported worldwide. We identified a Malaysian infant female at 2 days of life with CDG type Ia. The diagnosis was suspected on the basis of inverted nipples and abnormal fat distribution. She had cerebellar hypoplasia and developed coagulopathy, hypothyroidism and severe pericardial effusion and died at 7 months of life. The diagnosis was supported by abnormal serum transferrin isoform pattern that showed elevated levels of the disialotransferrin isoform and trace levels of the asialotransferrin isoform. Enzyme testing of peripheral leukocytes showed decreased level of phosphomannomutase (PMM) activity (0.6 nmol/min per mg protein, normal range 1.6-6.2) and a normal level of phosphomannose isomerase activity (19 nmol/min per mg protein, normal range 12-25), indicating a diagnosis of CDG type Ia. Mutation study of the PMM2 gene showed the patient was heterozygous for both the common p.R141H (c.422T>A) mutation and a novel sequence change in exon 7, c.618C>A. The latter change is predicted to result in the replacement of the highly conserved phenylalanine residue at position 206 with a leucine residue (p.F206L) and occurs in the same codon as the previously reported p.F206S mutation. Analysis of 100 control chromosomes has shown that the p.F206L sequence change is not present, making it highly likely that this change is functionally important. To the best of our knowledge, this is the first report of CDG in the Malay population. Prenatal diagnosis was successfully performed in a subsequent pregnancy for this family.
Cryptosporidium fragile sp. n. (Apicomplexa) is described from black-spined toads, Duttaphrynus melanostictus (Schneider) (Amphibia, Anura, Bufonidae) from the Malay Peninsula. The parasitized animals were directly imported from Malaysia and harboured C. fragile at the time of arrival. Oocysts were subspherical to elliptical with irregular contour in optical section, measuring 6.2 (5.5-7.0) x 5.5 (5.0-6.5) microm. Oocyst wall was smooth and colourless in light microscopy. The endogenous development of C. fragile in the stomach of black-spined toad was analysed in detail using light and electron microscopy. Cryptosporidian developmental stages were confined to the surface of gastric epithelial cells. In transmission experiments, C. fragile has not been infective for one fish species, four amphibian species, one species of reptile and SCID mice. Full length small subunit rRNA gene sequence was obtained. Phylogenetic reconstruction revealed distinct status of C. fragile within the clade of species with gastric localisation including Cryptosporidium muris Tyzzer, 1907, Cryptosporidium serpentis Levine, 1980 and Cryptosporidium andersoni Lindsay, Upton, Owens, Morgan, Mead et Blagburn, 2000. Described characteristics differentiate C. fragile from the currently recognized Cryptosporidium species. Our experience with the description of C. fragile has led us to revise the recommended criteria for an introduction of a new Cryptosporidium species name. C. fragile is the first species described and named from an amphibian host. Its prevalence of 83% (15/18) in black-spined toads within the 3 months after importation calls for strict quarantine measures and import regulation for lower vertebrates.
Currently, the laboratory diagnosis of toxocariasis, caused by Toxocara canis or T. cati, mainly relies on serological tests. Unfortunately, however, the specificities of most of the commercial tests that are available for the serodiagnosis of this disease are not very high and this may cause problems, especially in tropical countries where co-infections with other helminths are common. In an effort to develop a serological assay with improved specificity for the detection of Toxocara infection, an IgG(4)-ELISA based on a recombinant version (rTES-30USM) of the 30-kDa Toxocara excretory-secretory antigen (TES-30) has recently been developed. To produce the antigen, the TES-30 gene was cloned via assembly PCR, subcloned into a His-tagged prokaryotic expression vector, and purified by affinity chromatography using Ni(2+)-nitrilotriacetic-acid (Ni-NTA) resin. The performance of the ELISA based on the recombinant antigen was then compared with that of commercial kit, based on an IgG-ELISA, for the serodiagnosis of toxocariasis (Toxocara IgG-ELISA; Cypress Diagnostics, Langdorp, Belgium). Both assays were used to test 338 serum samples, including 26 samples from probable cases of toxocariasis. Assuming that all the probable cases were true cases, the assay based on rTES-30USM demonstrated a sensitivity of 92.3% (24/26) and a specificity of 89.6% (103/115) whereas the commercial kit exhibited a sensitivity of 100% (26/26) but a specificity of only 55.7% (64/115). The high sensitivity and specificity exhibited by the new IgG(4)-ELISA should make the assay a good choice for use in tropical countries and any other area where potentially cross-reactive helminthic infections are common.
There have been three major rabies epidemics in China since the 1950s. To gain more insights into the molecular epidemiology of rabies viruses (RVs) for the third (the current) epidemic, we isolated RV from dogs and humans in major endemic areas, and characterized these isolates genetically by sequencing the entire glycoprotein (G) gene and the G-L non-coding region. These sequences were also compared phylogenetically with RVs isolated in China during previous epidemics and those around the world. Comparison of the entire G genes among the Chinese isolates revealed up to 21.8% divergence at the nucleotide level and 17.8% at the amino acid level. The available Chinese isolates could be divided into two distinct clades, each of which could be further divided into six lineages. Viruses in clade I include most of the Chinese viruses as well as viruses from southeast Asian countries including Indonesia, Malaysia, the Philippines, Thailand, and Vietnam. The viruses in the other clade were found infrequently in China, but are closely related to viruses distributed worldwide among terrestrial animals. Interestingly, most of the viruses isolated during the past 10 years belong to lineage A viruses within clade I whereas most of the viruses isolated before 1996 belong to other lineages within clades I and II. Our results indicated that lineages A viruses have been predominant during the past 10 years and thus are largely responsible for the third and the current epidemic in China. Our results also suggested that the Chinese RV isolates in clade I share a common recent ancestor with those circulating in southeast Asia.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
The influenza activity and circulation of influenza viruses in Lombardy (the most populous Italian region) were observed during two consecutive seasons (2005/2006 and 2006/2007) characterized by low influenza activity by the Italian Influenza Surveillance Network. The molecular characteristics of circulating viruses were analyzed to evaluate the introduction of new variants and emergence of vaccine-escape viruses. In both seasons, the epidemic in Lombardy was sustained almost exclusively by influenza A viruses, accounting for 80.5% and 93.6% of total detections, respectively, and the co-circulation of A/H3 viruses belonging to distinct phylogenetic groups was observed. The A/H1N1 viruses isolated during the 2005/2006 season were closely related to A/New Caledonia/20/99, while the hemagglutinin (HA) sequences of the A/H1N1 viruses from the 2006/2007 season exhibited a greater diversity. These viruses were A/Solomon Islands/3/2006-like and showed several variants. All B isolates were similar to B/Malaysia/2506/2004 belonging to the B/Victoria/2/87-lineage. Influenza B virus was the dominant virus in Europe in the 2005/2006 season and accounted for the 20% of total detections in Lombardy. Overall, the viruses studied presented heterogeneity in their HA sequences suggesting the circulation of a miscellaneous set of variants during the two seasons notwithstanding the medium-low activity of influenza. The importance of virological surveillance of influenza viruses is recognized widely and the molecular characterization of the viruses, especially in vaccinated subjects, is of particular importance to evaluate the introduction and circulation of new variants.
Two culture-independent methods, namely ribosomal DNA libraries and denaturing gradient gel electrophoresis (DGGE), were adopted to examine the microbial community of a Malaysian light crude oil. In this study, both 16S and 18S rDNAs were PCR-amplified from bulk DNA of crude oil samples, cloned, and sequenced. Analyses of restriction fragment length polymorphism (RFLP) and phylogenetics clustered the 16S and 18S rDNA sequences into seven and six groups, respectively. The ribosomal DNA sequences obtained showed sequence similarity between 90 to 100% to those available in the GenBank database. The closest relatives documented for the 16S rDNAs include member species of Thermoincola and Rhodopseudomonas, whereas the closest fungal relatives include Acremonium, Ceriporiopsis, Xeromyces, Lecythophora, and Candida. Others were affiliated to uncultured bacteria and uncultured ascomycete. The 16S rDNA library demonstrated predomination by a single uncultured bacterial type by >80% relative abundance. The predomination was confirmed by DGGE analysis.