Lau Yee Ling

Abstract: Detection of Toxoplasma gondii infection is essential in pregnant women and immunosuppressed patients. Numerous studies have shown that the recombinant production of several Toxoplasma antigens, including dense granule antigens (GRAs) has high potential as diagnostic reagents. In the present study, we produced GRA2 using Pichia pastoris system. RNA of T. gondii RH strain tachyzoite was used as a template to produce cDNA clones of full-length GRA2 via reverse transcriptase PCR. Amplicons were inserted into pPICZ alpha A and the recombinant plasmid transformed into P. pastoris, X-33 strain. The expressed recombinant protein was identified by SDS-PAGE and Western blotting. A recombinant protein of similar to 28 kDa was produced, which could be detected by toxoplasmosis positive human sera indicating that the recombinant protein retained its antigenicity. The present study indicates that P. pastoris-expressed GRA2 should be useful for detection of Toxoplasma infection.

Abstract: Brugia malayi is one of the parasitic worms which causes lymphatic filariasis in humans. Its geographical distribution xD;includes a large part of Asia. Despite its wide distribution, very little is known about the genetic variation and molecular xD;epidemiology of this species. In this study, the internal transcribed spacer 1 (ITS1) nucleotide sequences of B. malayi from xD;microfilaria-positive human blood samples in Northeast Borneo Island were determined, and compared with published xD;ITS1 sequences of B. malayi isolated from cats and humans in Thailand. Multiple alignment analysis revealed that xD;B. malayi ITS1 sequences from Northeast Borneo were more similar to each other than to those from Thailand. xD;Phylogenetic trees inferred using Neighbour-Joining and Maximum Parsimony methods showed similar topology, with 2 xD;distinct B. malayi clusters. The first cluster consisted of Northeast Borneo B. malayi isolates, whereas the second consisted xD;of the Thailand isolates. The findings of this study suggest that B. malayi in Borneo Island has diverged significantly from xD;those of mainland Asia, and this has implications for the diagnosis of B. malayi infection across the region using ITS1-based xD;molecular techniques.

Abstract: The cytochrome oxidase subunit I (COXI) gene sequences of three recent (20072008) clinical Plasmodium knowlesi isolates from Klang Valley, peninsular Malaysia, were determined and compared with those of older (1960's) peninsular Malaysia, recent isolates from Sarawak (on Borneo Island), and an isolate from Thailand. Multiple alignment of the sequences showed that the three clinical isolates were more similar to the older peninsular Malaysia isolates than to those from Sarawak and Thailand. Phylogenetic tree based on the COXI sequences revealed three distinct clusters of P. knowlesi. The first cluster consisted of isolates from peninsular Malaysia, the second consisted of Sarawak isolates and the third composed of the Thailand isolate. The findings of this study highlight the usefulness of mitochondrial COXI gene as a suitable marker for phylogeographic studies of P. knowlesi.

Abstract: Background: In Malaysia, researchers and medical practitioners are unfamiliar with Naegleria infections. Thus little is known about the existence of pathogenic Naegleria fowleri, and the resultant primary amoebic meningoencephalitis (PAM) is seldom included in the differential diagnosis of central nervous system infections. This study was conducted to detect the presence of Naegleria species in various environmental samples. Methods/Findings: A total of 41 Naegleria-like isolates were isolated from water and dust samples. All these isolates were subjected to PCR using two primer sets designed from the ITS1-ITS2 regions. The N. fowleri species-specific primer set failed to produce the expected amplicon. The Naegleria genus-specific primers produced amplicons of 408 bp (35), 450 bp (2), 457 bp (2) or 381 bp (2) from all 41 isolates isolated from aquatic (33) and dust (8) samples. Analysis of the sequences from 10 representative isolates revealed that amplicons with fragments 408, 450 and 457 bp showed homology with nonpathogenic Naegleria species, and 381 bp showed homology with Vahlkampfia species. These results concurred with the morphological observation that all 39 isolates which exhibited flagella were Naegleria, while 2 isolates (AC7, JN034055 and AC8, JN034056) that did not exhibit flagella were Vahlkampfia species. Conclusion: To date, pathogenic species of N. fowleri have not been isolated from Malaysia. All 39 isolates that produced amplicons (408, 450 and 457 bp) from the genus-specific primers were identified as being similar to nonpathogenic Naegleria. Amplicon 408 bp from 5 representative isolates showed 100% and 99.7% identity to Naegleria philippinensis isolate RJTM (AM167890) and is thus believed to be the most common species in our environment. Amplicons 450 bp and 457 bp were respectively believed to be from 2 new species of Naegleria, since representative isolates showed lower homology and had a longer base pair length when compared to the reference species in the Genbank, Naegleria schusteri (AJ566626) and Naegleria laresi (AJ566630), respectively.

Abstract: Rhoptry protein 2 (ROP2) of Toxoplasma gondii is a rhoptry-secreted protein that plays a critical role in parasitophorous vacuole membrane formation during invasion. In previous studies, ROP2 has been shown to be efficient in triggering humoral and cell-mediated responses. High immunogenicity of ROP2 makes it a potential candidate for diagnosis and vaccination against toxoplasmosis. In this study, the ROP2 gene was cloned into pPICZ alpha A expression vector and extracellularly expressed in the yeast Pichia pastoris, which has numerous advantages over other expression systems for eukaryotic proteins expression. The effectiveness of the secreted recombinant ROP2 as a diagnosis agent was assessed by Western Blot with 200 human serum samples. Recombinant ROP2 reacted with toxoplasmosis-positive human serum samples and yielded an overall sensitivity of 90% and specificity of 95%. However, recombinant ROP2 is a better marker for detection of IgG (91.7%) rather than IgM (80%).

Abstract: Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30 C, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

Abstract: Seven stains were studied to determine the best color and contrast for staining the developmental stages of free living pathogenic Acanthamoeba and Naegleria species. The acid-fast bacilli stain (AFB) produced a blue color without contrast; trichrome-eosin and modified Field's showed various color contrasts; Giemsa, iron-hematoxylin, modified AFB and Gram produced only one color which distinguished the nucleus, nucleolus, cytoplasm, food- and water-vacuoles. The motile organs (acanthopodia, pseudopodia, lobopodia and flagella) were also clearly differentiated but produced a similar color as the cytoplasm. These motile organelles were first induced by incubating at 37 C for at least 15 minutes and then fixing with methanol in order to preserve the protruding morphology prior to staining. The trichrome-eosin and iron-hematoxylin stains showed good color contrast for detecting all three stages, the trophozoite, cyst and flagellate; Giemsa and Gram stained the trophozoite and flagellate stages; the modified Field's and modified AFB stains stained only the trophozoite stage. Depending on the purpose, all these stains (except the AFB stain) can be used to identify the developmental stages of Acanthanweba and Naegleria for clinical, epidemiological or public health use.

Abstract: In this study, we successfully expressed a chimerical surface antigen 1 and 2 (SAG1/2) of Toxoplasma gondii in Pichia pastoris. Eighty human serum samples, including 60 from confirmed cases of toxoplasmosis, were tested against the purified recombinant SAG1/2 in Western blots. Results of Western blots targeted at Toxoplasma IgG and IgM showed that the recombinant SAG1/2 reacted with all sera from the toxoplasmosis cases but none with the Toxoplasma-negative serum samples. These results showed that the P. pastoris-derived recombinant SAG1/2 was sensitive and specific and suitable for use as antigen for detecting anti-Toxoplasma antibodies. To further investigate the immunological characteristic of the recombinant protein, the recombinant SAG1/2 was injected subcutaneously into BALB/c mice, and their serum was tested against total protein lysate of T. gondii. Mice immunized with the recombinant SAG1/2 reacted specifically with the native SAG1 and SAG2 of T. gondii. Significant proliferation of splenocytes stimulated with tachyzoite total protein lysate was observed in vaccinated BALB/c mice but not in those from negative control mice. Specific production of IFN-gamma, the Th1-type cytokines, was also found in stimulated splenocytes from vaccinated mice. These results show that the chimeric protein recombinant SAG1/2 can elicit a Th1-associated protection against T. gondii infections in mice. Finally, vaccinated mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.005), and their survival time increased significantly compared to the negative control.

Abstract:

Abstract: Toxoplasma gondii is an important parasite in pregnant women. This case-controlled study assessed the seroprevalence of toxoplasmosis in 640 pregnant women in southern Thailand and identified their associated risk factors. The overall seroprevalence of toxoplasmosis was 181 (28.3%). Of this, 138 (21.6%) were positive for only anti- Toxoplasma immunoglobulin G (IgG) antibody, 43 (6.7%) were positive for both IgG and IgM antibodies, and none were positive for IgM antibody. Multivariate analysis revealed that increasing age (adjusted odds ratio [OR] = 1.64, 95% confidence interval [CI] = 1.01–2.67), living outside Songkhla province (adjusted OR = 1.56, 95% CI = 1.08–2.24), parity (adjusted OR = 1.65, 95% CI = 1.01–2.68), contact with cats (adjusted OR = 1.70, 95% CI = 1.20–2.43), and drinking of unclean water (adjusted OR = 1.70, 95% CI = 1.08–2.68) were factors associated with Toxoplasma seroprevalence. On the basis of the results obtained, a health surveillance program should be initiated as a primary preventive measure for congenital toxoplasmosis and focus on educating women of the child-bearing age group to avoid contact with cats and to strictly practice personal hygiene.

Abstract: Background: The emergence of Plasmodium knowlesi in humans, which is in many cases misdiagnosed by microscopy as Plasmodium malariae due to the morphological similarity has contributed to the needs of detection and differentiation of malaria parasites. At present, nested PCR targeted on Plasmodium ssrRNA genes has been described as the most sensitive and specific method for Plasmodium detection. However, this method is costly and requires trained personnel for its implementation. Loop-mediated isothermal amplification (LAMP), a novel nucleic acid amplification method was developed for the clinical detection of P. knowlesi. The sensitivity and specificity of LAMP was evaluated in comparison to the results obtained via microscopic examination and nested PCR. Methods: LAMP assay was developed based on P. knowlesi genetic material targeting the apical membrane antigen-1 (AMA-1) gene. The method uses six primers that recognize eight regions of the target DNA and it amplifies DNA within an hour under isothermal conditions (65 degrees C) in a water-bath. Results: LAMP is highly sensitive with the detection limit as low as ten copies for AMA-1. LAMP detected malaria parasites in all confirm cases (n = 13) of P. knowlesi infection (sensitivity, 100%) and none of the negative samples (specificity, 100%) within an hour. LAMP demonstrated higher sensitivity compared to nested PCR by successfully detecting a sample with very low parasitaemia (< 0.01%). Conclusion: With continuous efforts in the optimization of this assay, LAMP may provide a simple and reliable test for detecting P. knowlesi malaria parasites in areas where malaria is prevalent.

Abstract: Five local Malaysian patients with clinical manifestations consistent with lymphatic filariasis were referred to our medical centre between 2003 and 2006. Although no microfilariae (mf) were detected in their nocturnal blood samples, all were diagnosed to have lymphatic filariasis on the basis of clinical findings and positive serology results. PCR on their blood samples revealed that two of the patients were infected with Brugia pahangi, an animal filarial worm hitherto not known to cause human disease in the natural environment. All the patients were successfully treated with anti-filarial drugs: four patients were treated with a combination of diethylcarbamazine (DEC) and albendazole, and one with doxycycline. Four of them were residents of Petaling Jaya, a residential suburbia located 10 km southwest of Kuala Lumpur city, Malaysia. The fifth patient was a frequent visitor of the suburbia. This suburbia has no history or record of B. malayi infection. The most likely vector of the worm was Armigeres subalbatus as extensive entomological surveys within the suburbia revealed only adult females of this mosquito species were infected with B. pahangi larvae. Wild monkeys caught in the suburbia were free from B. pahangi mf, but domestic cats were mf positive. This suggests that infected cats might be the source of the zoonotic infection in the suburbia. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Abstract: Objective: The objective of this study was to investigate the antioxidant of methanolic extracts of Lantana camara various parts and the determination of their total phenolics content. xD;Methods: The extract was screened for possible antioxidant activities by free radical scavenging activity (DPPH), xanthine oxidase inhibition (XOI) activity and Griess-Ilosvay Method. xD;Results: The results showed that all the plant parts possessed antioxidant properties including radical scavenging, Xanthine oxidase inhibition and nitrites scavenging activities. The antioxidative activities were correlated with the total phenol. The leaves extract was more effective than of other parts. Conclusion: This study suggests that L. camara extracts exhibit great potential for antioxidant activity and may be useful for their nutritional and medicinal functions.

Abstract: Background and objective: Cassia fistula is widely used in traditional medicine to treat various types of ailments. The evaluation of toxic properties of C. fistula is crucial when considering public health protection because exposure to plant extracts can result in undesirable effects on consumers. Hence, in this study the acute oral toxicity of C. fistula seeds extract was investigated in mice. Results: Oral administration of crude extract at the highest dose of 5000 mg/kg resulted in no mortalities or evidence of adverse effects, implying that C. fistula in nontoxic. Throughout 14 days of the treatment no changes in behavioural pattern, clinical sign and body weight of mice in both control and treatment groups. Also there were no any significant elevations observed in the biochemical analysis of the blood serum. Further, histopathological examination revealed normal architecture and no significant adverse effects observed on the kidney, heart, liver, lung and spleen. Conclusions: Overall, the results suggest that, the oral administration of C. fistula methanolic seeds extract did not produce any significant toxic effect in mice. Hence, the extract can be utilized for pharmaceutical formulations.

Abstract: Severe combined immunodeficiencies (SCID) are a group of rare inherited disorders with profound defects in T cell and B cell immunity. From 2005 to 2010, our unit performed testing for IL2RG, JAK3, IL7R, RAG1, RAG2, DCLRE1C, LIG4, AK2, and ZAP70 mutations in 42 Chinese and Southeast Asian infants with SCID adopting a candidate gene approach, based on patient's gender, immune phenotype, and inheritance pattern. Mutations were identified in 26 patients, including IL2RG (n = 19), IL7R (n = 2), JAK3 (n = 2), RAG1 (n = 1), RAG2 (n = 1), and DCLRE1C (n = 1). Among 12 patients who underwent hematopoietic stem cell transplantation, eight patients survived. Complications and morbidities during transplant period were significant, especially disseminated bacillus Calmette-Gu,rin disease which was often difficult to control. This is the first cohort study on SCID in the Chinese and Southeast Asian population, based on a multi-centered collaborative research network. The foremost issue is service provision for early detection, diagnosis, management, and definitive treatment for patients with SCID. National management guidelines for SCID should be established, and research into an efficient platform for genetic diagnosis is needed.

Abstract: Background and objective: Cassia fistula L belongs to the family Leguminosae, and it is one of the most popular herbal products in tropical countries. C. fistula seeds have been used as a herbal medicine and have pharmacological activity which includes anti-bacterial, anti-fungal, and antioxidant properties. The goal of this study was to identify compounds from C. fistula seeds which are responsible for anti-Candida albicans activity using bioassay-directed isolation. Results: The preliminary phytochemical screening of the plant seed revealed the presence of anthraquinones, flavonoids, saponins, tannins and terpenoids. The isolation of active compounds was carried out in four steps: multiple extractions, fractionation using column chromatography and purification using preparative thin-layer chromatography (TLC) and liquid chromatography/mass spectrometry (LC/MS). The structure of separated compounds was determined on the basis of mass spectrometry data. One compound was identified is roseanone. Conclusions: The MS analysis on the active fraction from seed extract of C. fistula confirmed the presence of roseanone with antiyeast activity.

Abstract: Loop-mediated isothermal amplification (LAMP), a rapid nucleic acid amplification method, was developed for the clinical diagnosis of toxoplasmosis. Three LAMP assays based on the SAG1, SAG2, and B1 genes of Toxoplasma gondii were developed. The sensitivities and specificities of the LAMP assays were evaluated by comparison with the results of conventional nested PCR. The LAMP assays were highly sensitive and had a detection limit of 0.1 tachyzoite, and no cross-reactivity with the DNA of other parasites was observed. Blood was collected from 105 individuals to test the LAMP assays: 40 patients with active toxoplasmosis, 40 negative controls, and 25 patients with other parasitic infections. The SAG2-based LAMP (SAG2-LAMP) had a greater sensitivity (87.5%) than the SAG1-LAMP (80%), B1-LAMP (80%), and nested PCR (62.5%). All the LAMP assays and nested PCR were 100% specific. This is the first report of a study which applied the LAMP method to diagnose toxoplasmosis from human blood samples. Due to its simplicity, sensitivity, and specificity, LAMP is suggested as an appropriate method for routine diagnosis of active toxoplasmosis in humans.

Abstract: SAG2 is one of the major surface antigens of the intracellular protozoan parasite Toxoplasma gondii. In the present study, truncated recombinant SAG2(S) and full length recombinant SAG2(T) of T. gondii were optimally produced (~15 mg/liter) in Pichia pastoris expression system using BMMY medium at pH 3, 25ºC in 0.5-1% methanol and a time-course of 1-2 days. The recombinant proteins were purified using a commercial gel filtration purification system obtaining ~33% recovery. The purified SAG2(S) and SAG2(T) showed molecular masses of 45 and 36 kDa by SDS-PAGE, respectively. The recombinant proteins were evaluated by Western blotting with patients’ sera and demonstrated 90% sensitivity and 100% specificity for detection of toxoplasmosis. This study provided a means for largescale expression and purification of SAG2, which should be useful for diagnosis of toxoplasmosis.

Abstract: Detection of Toxoplasma gondii in blood by means of the polymerase chain reaction (PCR) may facilitate early diagnosis of toxoplasmosis in different groups of patients. We evaluated this approach in 42 patients presenting with ocular or psychotic diseases by comparing the sensitivity and specificity of PCR after heat treatment using a microwave oven with a standard genomic DNA extraction method for paired serum and whole blood samples. The presence of serum IgM and IgG antibodies against T. gondii was detected using a standard commercial enzyme-linked immunosorbent assay and enzyme immunoassay for IgG avidity test. Of 42 whole blood samples, PCR after microwave treatment was positive in 8 samples with a sensitivity of 73% and specificity of 100% compared to 11 samples positive by the extraction method. Although none of 42 sera samples was PCR positive by the extraction method, 7 specimens were positive after microwave treatment. This is the first study to use a microwave heat treatment, which is simple, rapid and a promising alternative method, in detecting small amounts of T. gondii DNA in human blood. Furthermore, irradiation of blood samples with microwaves allows incorporation of PCR into a practical tool for routine clinical assessment of patients with Toxoplasma infection.

Abstract: GRA4 of Toxoplasma gondii has been shown to prompt IgG, IgM and IgA responses in previous studies and is thus considered one of the major immunogenic proteins from T. gondii that can be used for both diagnostics purposes and vaccine development. This study seeks to clone and express the GRA4 in Pichia pastoris, which has numerous advantages over other systems for expression of eukaryotic proteins. In order to achieve this, the gene was cloned into the pPICZ alpha A expression vector, which was then incorporated into the P. pastoris genome via insertional integration for expression of the recombinant protein, under the AOX1 promoter. The antigen was expressed along with the prepro sequence of the alpha-factor of yeast so that it could be excreted out of the P pastoris cells and obtained from the medium. Upon SDS-PAGE analysis it was found that the recombinant protein was expressed optimally as a 40 kDa protein after 96 hours of induction with 0.75% of methanol. The expressed GRA4 protein showed discrepancy in size with the calculated molecular mass. This may be attributed to the various posttranslational modifications including glycosylation and phosphorylation. Despite the difference in molecular weight, the recombinant protein was able to detect toxoplasmosis in Western blot format. The recombinant GRA4 was expressed with an intact polyhistidine-tag, which could be used for future purification of the antigen.

Abstract: This study reports the detection of Acanthamoeba and Naegleria species in 14 swimming pools around Petaling Jaya and Kuala Lumpur, Malaysia. Sampling was carried out at 4 sites (the platforms (P), wall (W), 1 meter from the wall (1) and middle (2)) of each swimming pool. These free living amoebae (FLA) were detected under light and inverted microscopes after being cultured on the surface of non-nutrient agar lawned with Escherichia coli. Acanthamoeba species were detected in higher number of culture plates from all sampling sites of all the swimming pools. While Naegleria, were detected in fewer culture plates at 3 sampling sites (absent at site P) of 8 swimming pools. This suggested that the thick double-walled cysts of Acanthamoeba were more resistant, thus remaining viable in the dry-hot areas of the platforms and in chlorinated water of the swimming pools whereas Naegleria cysts, that are fragile and susceptible to desiccation, preferred watery or moist areas for growth and proliferation. The prevalence of both FLA was highest at site W (76.2%), followed by site 1 (64.7%), lowest at site 2 (19.4%), and could be detected at all 3 sampling levels (top, middle and bottom) of these 3 sites. The surface of site W might act as a bio-film that accumulated all kinds of microbes providing sufficient requirement for the FLA to develop and undergo many rounds of life cycles as well as moving from top to bottom in order to graze food. Other factors such as human activities, the circulating system which was fixed at all swimming pools, blowing wind which might carry the cysts from surroundings and the swimming flagellate stage of Naegleria, could also contribute to the distribution of the FLA at these sampling sites. Both FLA showed highest growth (80.4%) at room temperature (25-28 degrees C) and lesser (70.0%) at 37 degrees C which might be due to the overgrowth of other microbes (E. coli, fungi, algae, etc). While at 44 degrees C, only Acanthamoeba species could survive thus showing that our swimming pools are free from potentially pathogenic Naegleria species. However, further study is needed in order to confirm the virulence levels of these amoebae isolates.

Abstract: Children aged 11 to <24 months received 2 intranasal doses of live attenuated influenza vaccine (LAIV) or placebo, 35 +/- 7 days apart. Dose 1 was administered concomitantly with a combined measles, mumps, and rubella vaccine (Priorix). Seroresponses to measles and mumps were similar between groups. Compared with placebo, response rates to rubella in LAIV+Priorix recipients were statistically lower at a 15IU/mL threshold (83.9% vs 78.0%) and the prespecified noninferiority criteria were not met. In a post hoc analysis using an alternate widely accepted threshold of 10IU/mL, the noninferiority criteria were met (93.4% vs 89.8%). Concomitant administration with Priorix did not affect the overall influenza protection rate of LAIV (78.4% and 63.8% against antigenically similar influenza strains and any strain, respectively). (C) 2009 Elsevier Ltd. All rights reserved.

Abstract: Elaeis guineensis (Arecaceae) is widely used in West African traditional medicine for treating various ailments. An evaluation on the toxicity of extracts of this plant is crucial to support the therapeutic claims. The acute oral toxicity and brine shrimp lethality of a methanolic extract of this plant was tested. Oral administration of crude extract at the highest dose of 5,000 mg/kg resulted in no mortalities or evidence of adverse effects, implying that E. guineensis is nontoxic. Normal behavioral pattern, clinical signs and histology of vital organs confirm this evidence. The E. guineensis extracts screened for toxicity against brine shrimp had 50% lethal concentration (LC(5)(0)) values of more than 1.0 mg/mL (9.00 and 3.87 mg/mL, at 6 and 24 h, respectively), confirming that the extract was not toxic. Maximum mortalities occurred at 100 mg/mL concentration while the least mortalities happened to be at 0.195 mg/mL concentration. The results of both tests confirm that E. guineensis is nontoxic and hence safe for commercial utilization.

Abstract: We report a case of unusual cutaneous toxoplasmosis manifestation in a HIV-positive patient. He presented with hard and painful nodular lesions on the arms, hands and chest. Serology tests for anti-Toxoplasma antibody were negative. However, histopathologic examination of the lesion revealed foci of macrophages containing crescent-shaped organisms resembling the zoites of the protozoan parasite Toxoplasma gondii. Ultrastructure examination under electron microscopy and PCR confirmed the organism as T. gondii.

Abstract: The full length surface antigen 2 (SAG2) gene of the protozoan parasite Toxoplasma gondii was cloned and intracellularly expressed in the Pichia pastoris expression system. The molecular weight of the expressed recombinant SAG2 (36 kDa) was much larger than the native SAG2 (22 kDa). This discrepancy in size was due to hyperglycosylation, as deglycosylation assay reduced the size of the recombinant SAG2 to 22 kDa. Despite being hyperglycosylated, the recombinant SAG2 reacted strongly with pooled anti-Toxoplasma human serum, pooled anti-Toxoplasma mouse serum and a SAG2-specific monoclonal antibody. The glycosylated recombinant SAG2 was further evaluated in Western blot and in-house enzyme-linked immunosorbent assay (ELISA) using 80 human serum samples, including confirmed early acute (IgM positive, IgG negative; n = 20), acute (IgM positive, IgG positive; n = 20) and chronic (IgM negative, IgG positive; n = 20) toxoplasmosis patients, and toxoplasmosis negative control patients (n = 20). Results of the Western blot showed that the recombinant SAG2 reacted with all 60 samples of the toxoplasmosis cases but not with the Toxoplasma-negative samples. The sensitivity of in-house ELISA was 80%, 95% and 100% for early acute, acute and chronic patients' serum samples, respectively. Vaccination study showed that serum from mice immunised with the glycosylated recombinant SAG2 reacted specifically with the native SAG2 of T. gondii. The mice were significantly protected against lethal challenge with live T. gondii RH strain tachyzoites (P < 0.01) and their survival time was increased compared to controls. Therefore, the present study shows that the A pastoris-derived recombinant SAG2 was specific and suitable for use as antigen for detecting anti-Toxoplasma IgG and IgM antibodies. The vaccination study showed that recombinant SAG2 protein was immunoprotective in mice against lethal challenge. (c) 2008 Elsevier Inc. All rights reserved.

Abstract: This paper presents the first reported use of 18S rRNA gene sequence to determine the phylogeny of Brugia pahangi. The 18S rRNA nucleotide sequence of a Malaysian B. pahangi isolate was obtained by PCR cloning and sequencing. The sequence was compared with 18S rRNA sequences of other nematodes, including those of some filarial nematodes. Multiple alignment and homology analysis suggest that B. pahangi is closely related to B. malayi and Wuchereria bancrofti. Phylogenetic trees constructed using Neighbour Joining, Minimum Evolution and Maximum Parsimony methods correctly grouped B. pahangi with other filarial nematodes, with closest relationship with B. malayi and W. bancrofti. The phylogeny of B. pahangi obtained in this study is in concordance with those previously reported, in which the 5S rRNA gene spacer region and cytochrome oxidase subunit I (COI) sequences were used.

Abstract: The surface antigen 2 (SAG2) gene of the protozoan parasite, Toxoplasma gondii, was cloned and extracellularly expressed in the yeast Pichia pastoris. The effectiveness of the secreted recombinant SAG2 (rSAG2-S) as a serodiagnosis reagent was assessed by western blots and ELISA. In the western blot assay, rSAG2-S reacted with all Toxoplasma-antibody positive human serum samples but not with Toxoplasma-negative samples. In the ELISA, rSAG2-S yielded sensitivity rates ranging from 80% (IgG negative, IgM positive) to 100% (IgG positive, IgM negative). In vivo experiments showed that serum from mice immunized with rSAG2-S reacted specifically with the native SAG2 of T. gondii. These mice were protected when challenged with live cells of T. gondii.

Abstract: An analysis of a turbine blade fatigue life that includes the physics of fluid-structure interaction on the high cycle fatigue (HCF) life estimate of turbine blades is carried out. The rotor wake excitation is modeled by rows of Karman vortices superimposed on an inviscid uniform flow. The vortex-induced vibration problem is modeled by a linear cascade composed of five turbine blades and the coupled Euler and structural dynamics equations are numerically solved using a time-marching boundary element technique. The analysis can be applied to any blade geometries; it is not limited to the blade geometry considered here. Two major design parameters have been identified; the ratio of blade spacing to blade chord length s/c of the stator, and the normalized frequency parameter c/d which is related to the wake passing frequency of the rotor. For a rigid cascade, it is found that aerodynamic resonance prevails at the resonant c/d values corresponding to an isolated blade while s/c is responsible for the level of the aerodynamic response. If the central blades were elastic, the parameter s/c plays a different role in the fluid-structure interaction problem. With a c/d that could lead to structural resonance for an isolated blade, changing s/c would stabilize the aerodynamic and structural response of the elastic blade in a cascade. On the contrary, an improper choice of s/c might turn the elastic blade response into structural resonance even though the oncoming c/d is non-resonant. The results of the nonlinear effects of c/d and s/c could be used together with the Campbell diagram to obtain an improved HCF design of rotor-stator pair. (C) 2007 Elsevier Ltd. All rights reserved.

Abstract: SAG2 is one of the major surface antigens of the intracellular protozoan parasite Toxoplasma xD;gondii. In this study, we used the Pichia pastoris yeast expression system to produce a truncated form xD;of the SAG2 and determined the serological characteristics of this recombinant antigen. We chose the xD;Pichia system because of its high efficiency in expressing recombinant genes, and its ability to modify xD;and secrete the recombinant proteins. Our strategy was to clone and express the part of the SAG2 gene xD;that encodes the carboxyl half of the antigen. The recombinant antigen (recSAG2-C) secreted by the xD;Pichia cells was harvested, and then evaluated in Western blot and enzyme-linked immunoassays (ELISA). xD;Sixty human serum samples, including 45 from confirmed cases of toxoplasmosis, were tested against xD;recSAG2-C. Results from the Western blot assay showed that recSAG2-C reacted with all 45 sera from the xD;toxoplasmosis cases but none with the Toxoplasma-negative serum samples. Similar results were obtained xD;for ELISA. These results indicated that recSAG2-C was specific for Toxoplasma antibody. To investigate xD;the immunological characteristic of recSAG2-C, this recombinant antigen was injected subcutaneously xD;into mice, and their serum was tested against total protein of T. gondii. It was observed that the serum xD;specifically detected the native SAG2 (22 kDa) of T. gondii. This demonstrates that the recSAG2-C could xD;evoke the production of antibody in mice, which readily recognized the native SAG2

Abstract: A truncated form of surface antigen 2 (SAG2) of the protozoan parasite Toxoplasma gondii was cloned and expressed in the methylotrophic yeast Pichia pastoris. This recombinant antigen, designated as recSAG2-N, contained only the N-terminal half of the native SAG2. The recSAG2-N was secreted by the Pichia pastoris into the culture supernatant, and it was harvested by using the trichloroacetic acid precipitation method. Specificity of recSAG2-N was evaluated in western blot assays. Fifty human serum samples, including 32 from confirmed cases of toxoplasmosis, were tested. Results from the assays showed that recSAG2-N reacted with sera from the toxoplasmosis cases only. In vivo experiments showed that serum from mice which received recSAG2-N reacted with the native SAG2 of T. gondii.

Abstract: Aeromonas hydrophila is a gram-negative opportunistic pathogen of animals and humans. The pathogenesis of A. hydrophila is multifactorial. Genomic subtraction and markers of genomic islands (GIs) were used to) identify putative virulence genes in A. hydrophila PPD134/91. Two rounds of genomic subtraction led to the identification of 22 unique DNA fragments encoding 19 putative virulence factors and seven new open reading frames, which are commonly present in the eight virulence strains examined. In addition, four GIs were found, including O-antigen, capsule, phage-associated, and type III secretion system (TTSS) gene clusters. These putative virulence genes and gene clusters were positioned on a physical map of A. hydrophila PPD134/91 to determine their genetic organization in this bacterium. Further in vivo study of insertion and deletion mutants showed that the TTSS may be one of the important virulence factors in A. hydrophila pathogenesis. Further. more, deletions of multiple virulence factors such as S-layer, serine protease, and metalloprotease also increased the 50% lethal dose to the same level as the TTSS mutation (about 1 log) in a blue gourami infection model. This observation sheds light on the multifactorial and concerted nature of pathogenicity in A. hydrophila. The large number of putative virulence genes identified in this study will form the basis for further investigation of this emerging pathogen and help to develop effective vaccines, diagnostics, and novel therapeutics.

Abstract: A gene encoding the larval excretory-secretory antigen TES-120 of the dog ascarid worm Toxocara canis was cloned into the methylotrophic yeast Pichia pastoris. Specificity of the recombinant TES-120 antigen produced by the yeast was investigated. Forty-five human serum samples from patients infected with differentparasitic organisms, including 8 cases of toxocariasis, were tested against the recombinant antigen in immunoblot assays. Results from the assays showed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

Abstract: The gene encoding the excretory-secretory antigen TES-120 of dog ascarid worm Toxocara canis was cloned into the bacterium Escherichia coli. The specificity of the recombinant TES-120 antigen produced by the bacterium was investigated. A total of 45 human serum samples from patients infected with differenthelminthes and protozoa, including 8 cases of toxocariasis, were tested against the recombinant antigens in immunoblot assays. The results from the assays revealed that the recombinant TES-120 antigen reacted with sera from toxocariasis patients only. This highly specific recombinant TES-120 antigen can potentially be used for the development of an inexpensive serodiagnostic assay for human toxocariasis.

Abstract: The genetic organization and sequences of the group II capsule gene cluster of Aeromonas hydrophila PPD134/91 have been determined previously. The purified capsular polysaccharides can increase the ability of avirulent strain PPD35/85 to survive in naive tilapia serum but have no inhibitory effect on the adhesion of PPD134/91 to carp epithelial cells. In this study, the presence of group II capsules among 33 randomly chosen A. hydrophila strains was examined by electron microscopy and genetic analysis. Ten strains were found to produce group II capsules. A PCR detection system was developed to identify two types of group II capsules (IIA and IIB) based on their genetic organization in the region II gene clusters. Group IIA capsules in the authors' collection of A. hydrophila strains are mainly found in the O:18 and O:34 serogroups, while group IIB capsules are found in the O:21 and O:27 serogroups. The presence of group II capsules in A. hydrophila strongly correlates with the serum and phagocyte survival abilities (seven out of ten strains). The results indicate that the authors' PCR detection system can constitute a reliable assay for the classification of group II capsules in A. hydrophila.