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author:("Lu, sangli")
1.  Molecular surveillance of pvdhfr, pvdhps, and pvmdr-1 mutations in Plasmodium vivax isolates from Yunnan and Anhui provinces of China 
Malaria Journal  2014;13(1):346.
Plasmodium vivax is the predominant species of human malaria parasites present in China. Although sulphadoxine-pyrimethamine (SP) and chloroquine (CQ) have been widely used for malaria treatment in China, the resistance profiles of these drugs are not available. Analysis of dihydrofolate reductase (dhfr), dihydropteroate synthase (dhps), and multidrug resistance (mdr-1) gene mutations in P. vivax isolates is a valuable molecular approach for mapping resistance to SP and CQ. This study investigates the prevalence of pvdhfr, pvdhps, and pvmdr-1 of P. vivax clinical isolates from China and provides baseline molecular epidemiologic data on SP- and CQ-associated resistance in P. vivax.
Plasmodium vivax clinical isolates were collected from two malaria-endemic regions of China, subtropical (Xishuangbanna, Yunnan province) and temperate (Bozhou, Anhui province), from 2009 to 2012. All isolates were analysed for single nucleotide polymorphism haplotypes in pvdhfr, pvdhps, and pvmdr-1 using direct DNA sequencing.
In pvdhfr, 15% of Xishuangbanna isolates carried wild-type (WT) allele, whereas the majority of isolates carried mutant genes with substitutions at five codons. Eight mutant haplotypes of pvdhfr were detected, while limited polymorphism of pvdhfr was found in Bozhou isolates. A size polymorphism was present in pvdhfr, with the three-repeat type being the most predominate in both Xishuangbanna (79%) and Bozhou (97%) isolates. In pvdhps, mutations at four codons were detected in Xishuangbanna isolates leading to six haplotypes, including WT allele, single-mutation, double-mutation, and triple-mutation alleles. All Bozhou isolates carried WT pvhdps. In pvmdr-1, isolates from Xishuangbanna carried mutations at codons Y976F and F1076L, whereas all isolates from Bozhou had only a single mutation at codon F1076L.
Plasmodium vivax isolates from subtropical and temperate zones of China are shown to have dramatically different frequencies and patterns of mutations in pvdhfr, pvdhps, and pvmdr-1. Whereas P. vivax populations in subtropical China are highly resistant to SP and CQ, those in the temperate zone may still be susceptible to SP and CQ. This information is useful for establishing treatment policy and provides a baseline for molecular surveillance of drug-resistant P. vivax in these areas.
Electronic supplementary material
The online version of this article (doi:10.1186/1475-2875-13-346) contains supplementary material, which is available to authorized users.
PMCID: PMC4161776  PMID: 25179752
Plasmodium vivax; Polymorphism; Drug resistance markers; China
2.  Genetic diversity of Plasmodium vivax population in Anhui province of China 
Malaria Journal  2014;13:13.
Although the numbers of malaria cases in China have been declining in recent years, outbreaks of Plasmodium vivax malaria were still being reported in rural areas south of the Yellow River. To better understand the transmission dynamics of P. vivax parasites in China, the extent of genetic diversity of P. vivax populations circulating in Bozhou of Anhui province of China were investigated using three polymorphic genetic markers: merozoite surface proteins 1 and 3α (pvmsp-1 and pvmsp-3α) and circumsporozoite protein (pvcsp).
Forty-five P. vivax clinical isolates from Bouzhou of Anhui province were collected from 2009 to 2010 and were analysed using PCR/RFLP or DNA sequencing.
Seven and six distinct allelic variants were identified using PCR/RFLP analysis of pvmsp-3α with HhaI and AluI, respectively. DNA sequence analysis of pvmsp-1 (variable block 5) revealed that there were Sal-I and recombinant types but not Belem type, and seven distinct allelic variants in pvmsp-1 were detected, with recombinant subtype 2 (R2) being predominant (66.7%). All the isolates carried pvcsp with VK210 type but not VK247 or P. vivax-like types in the samples. Sequence analysis of pvcsp gene revealed 12 distinct allelic variants, with VK210-1 being predominant (41.5%).
The present data indicate that there is some degree of genetic diversity among P. vivax populations in Anhui province of China. The genetic data obtained may assist in the surveillance of P. vivax infection in endemic areas or in tracking potential future disease outbreak.
PMCID: PMC3893497  PMID: 24401153
Plasmodium vivax; pvmsp-1; pvmsp-3α; pvcsp; Anhui; China
3.  Mast Cells Modulate Acute Toxoplasmosis in Murine Models 
PLoS ONE  2013;8(10):e77327.
The role of mast cells (MCs) in Toxoplasma gondii infection is poorly known. Kunming outbred mice were infected intraperitoneally with RH strain T. gondii, either treated with compound 48/80 (C48/80, MC activator) or disodium cromoglycate (DSCG, MC inhibitor). Compared with infected controls, infected mice treated with C48/80 exhibited significantly increased inflammation in the liver (P < 0.01), spleen (P < 0.05), and mesentery (P < 0.05) tissues, higher parasite burden in the peritoneal lavage fluids (P < 0.01), and increased levels of mRNA transcripts of T. gondii tachyzoite surface antigen 1 (SAG1) gene in the spleen and liver tissues (P < 0.01), accompanied with significantly increased Th1 cytokine (IFN-γ, IL-12p40, and TNF-α) (P < 0.01) and decreased IL-10 (P < 0.01) mRNA expressions in the liver, and increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.01) but decreased TNF-α (P < 0.01) and IL-4 (P < 0.01) in the spleens of infected mice treated with C48/80 at day 9-10 p.i. Whereas mice treated with DSCG had significantly decreased tissue lesions (P < 0.01), lower parasite burden in the peritoneal lavage fluids (P < 0.01) and decreased SAG1 expressions in the spleen and liver tissues (P < 0.01), accompanied with significantly increased IFN-γ (P < 0.01) and IL-12p40 (P < 0.05) in the liver, and decreased IFN-γ (P < 0.05) and TNF-α (P < 0.01) in the spleens; IL-4 and IL-10 expressions in both the spleen and liver were significantly increased (P < 0.01) in the infected mice treated with DSCG. These findings suggest that mediators associated with the MC activation may play an important role in modulating acute inflammatory pathogenesis and parasite clearance during T. gondii infection in this strain of mice. Thus, MC activation/inhibition mechanisms are potential novel targets for the prevention and control of T. gondii infection.
PMCID: PMC3797692  PMID: 24146978
4.  Correction: Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China 
PLoS ONE  2013;8(5):10.1371/annotation/2cc13197-e6fc-46eb-88d6-853be10b72c5.
PMCID: PMC3667983
5.  Genotypes and Mouse Virulence of Toxoplasma gondii Isolates from Animals and Humans in China 
PLoS ONE  2013;8(1):e53483.
Recent population structure studies of T. gondii revealed that a few major clonal lineages predominated in different geographical regions. T. gondii in South America is genetically and biologically divergent, whereas this parasite is remarkably clonal in North America and Europe with a few major lineages including Types I, II and III. Information on genotypes and mouse virulence of T. gondii isolates from China is scarce and insufficient to investigate its population structure, evolution, and transmission.
Methodology/Principal Findings
Genotyping of 23 T. gondii isolates from different hosts using 10 markers for PCR-restriction fragment length polymorphism analyses (SAG1, SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1 and Apico) revealed five genotypes; among them three genotypes were atypical and two were archetypal. Fifteen strains belong to the Chinese 1 lineage, which has been previously reported as a widespread lineage from swine, cats, and humans in China. Two human isolates fall into the type I and II lineages and the remaining isolates belong to two new atypical genotypes (ToxoDB#204 and #205) which has never been reported in China. Our results show that these genotypes of T. gondii isolates are intermediately or highly virulent in mice except for the strain TgCtwh6, which maintained parasitemia in mice for 35 days post infection although it possesses the uniform genotype of Chinese 1. Additionally, phylogenetic network analyses of all isolates of genotype Chinese 1 are identical, and there is no variation based on the sequence data generated for four introns (EF1, HP2, UPRT1 and UPRT7) and two dense granule proteins (GRA6 and GRA7).
A limited genetic diversity was found and genotype Chinese 1 (ToxoDB#9) is dominantly circulating in mainland China. The results will provide a useful profile for deep insight to the population structure, epidemiology and biological characteristics of T. gondii in China.
PMCID: PMC3538538  PMID: 23308233
6.  rROP2186–533: A Novel Peptide Antigen for Detection of IgM Antibodies Against Toxoplasma gondii 
Toxoplasma gondii infections are prevalent in a wide range of mammalian hosts including humans. Infection in pregnant women may cause the transmission of parasite to the fetus that makes serious problems. IgM antibodies against Toxoplasma (Toxo-IgM) have been believed to be significant indicators for both recently acquired and congenital toxoplasmosis. So far, however, there has not been any recognized protein of T. gondii that specifically reacts to IgM antibodies. Here, an antigen exclusively for detection of IgM antibodies screened by two-dimensional electrophoresis and mass spectrometry has been reported. The study identified 13 Toxoplasma proteins probed by IgG antibodies and one (rhpotry protein 2 [ROP2]) by IgM antibodies with human sera of Toxo-IgM–-IgG+ and -IgM+-IgG–, respectively, which had been prescreened by Toxo-IgM and -IgG commercial kits from the suspected cases. Following cloning, expression, and purification of the fragment of ROP2186–533, an enzyme-linked immunosorbent assay with rROP2186–533 to measure IgM and IgG antibodies was developed. As a result, 100%(48/48) of sera with Toxo-IgM+-IgG–showed positive Toxo-IgM but none of them (0%) showed positive Toxo-IgG when rROP2186–533 was used as antigen. Neither Toxo-IgG nor Toxo-IgM antibodies were found when tested with 59 sera of Toxo-IgM–-IgG+. These results indicate that rROP2186–533 could be used as an antigen that specifically capture Toxo-IgM antibodies and may have a high potential in the serological diagnosis of both acute acquired and congenital toxoplasmosis.
PMCID: PMC3250629  PMID: 22085219
7.  Identification and Characterization of Paramyosin from Cyst Wall of Metacercariae Implicated Protective Efficacy against Clonorchis sinensis Infection 
PLoS ONE  2012;7(3):e33703.
Human clonorchiasis has been increasingly prevalent in recent years and results in a threat to the public health in epidemic regions, motivating current strategies of vaccines to combat Clonorchis sinensis (C. sinensis). In this study, we identified C. sinensis paramyosin (CsPmy) from the cyst wall proteins of metacercariae by proteomic approaches and characterized the expressed recombinant pET-26b-CsPmy protein (101 kDa). Bioinformatics analysis indicated that full-length sequences of paramyosin are conserved in helminthes and numerous B-cell/T-cell epitopes were predicted in amino acid sequence of CsPmy. Western blot analysis showed that CsPmy was expressed at four life stages of C. sinensis, both cyst wall proteins and soluble tegumental components could be probed by anti-CsPmy serum. Moreover, immunolocalization results revealed that CsPmy was specifically localized at cyst wall and excretory bladder of metacercaria, as well as the tegument, oral sucker and vitellarium of adult worm. Both immunoblot and immunolocalization results demonstrated that CsPmy was highly expressed at the stage of adult worm, metacercariae and cercaria, which could be supported by real-time PCR analysis. Both recombinant protein and nucleic acid of CsPmy showed strong immunogenicity in rats and induced combined Th1/Th2 immune responses, which were reflected by continuous high level of antibody titers and increased level of IgG1/IgG2a subtypes in serum. In vaccine trials, comparing with control groups, both CsPmy protein and DNA vaccine exhibited protective effect with significant worm reduction rate of 54.3% (p<0.05) and 36.1% (p<0.05), respectively. In consistence with immune responses in sera, elevated level of cytokines IFN-γ and IL-4 in splenocytes suggested that CsPmy could induce combined cellular immunity and humoral immunity in host. Taken together, CsPmy could be a promising vaccine candidate in the prevention of C. sinensis regarding its high immunogenicity and surface localization.
PMCID: PMC3312334  PMID: 22470461
8.  Typing Plasmodium yoelii microsatellites using a simple and affordable fluorescent labeling method 
The rodent malaria parasite Plasmodium yoelii has been an important animal model for studying malaria pathology and host-parasite interactions. Compared with other rodent malaria parasites such as Plasmodium chabaudi, however, genetic mapping studies on P. yoelii have been limited, partly due to the absence of genetic markers and the lack of well characterized phenotypes. Taking advantage of the available genome sequence, we initiated a project to develop a high-resolution microsatellite (MS) map for P. yoelii to study malaria disease phenotypes. Here we report screening the P. yoelii genome for simple sequence repeats and development of an inexpensive method (modified from a previously reported procedure) for typing malaria parasite MS: instead of labeling individual polymerase chain reaction primers, a single fluorescently labeled primer was used to type the MS markers. We evaluated various polymerase chain reaction cycling conditions and M13-tailed/labeled M13 primer ratios to establish a simple and robust procedure for typing P. yoelii MS markers. We also compared typing efficiencies between individually labeled primers and the M13-tailed single labeled primer method and found that the two approaches were comparable. Preliminary analyses of seven P. yoelii isolates deposited at MR4 with 77 MS showed that the markers were highly polymorphic and that the isolates belonged to two groups, suggesting potential common ancestry or laboratory contaminations among the isolates. The MS markers and the typing method provide important tools for genetic studies of P. yoelii. There is a good possibility that this method can be applied to type MS from other malaria parasites including important human pathogens Plasmodium falciparum and Plasmodium vivax.
PMCID: PMC2039898  PMID: 17658627
Genetic mapping; Genotyping; Rodent malaria parasite; Simple sequence repeat
9.  cDNA sequences reveal considerable gene prediction inaccuracy in the Plasmodium falciparum genome 
BMC Genomics  2007;8:255.
The completion of the Plasmodium falciparum genome represents a milestone in malaria research. The genome sequence allows for the development of genome-wide approaches such as microarray and proteomics that will greatly facilitate our understanding of the parasite biology and accelerate new drug and vaccine development. Designing and application of these genome-wide assays, however, requires accurate information on gene prediction and genome annotation. Unfortunately, the genes in the parasite genome databases were mostly identified using computer software that could make some erroneous predictions.
We aimed to obtain cDNA sequences to examine the accuracy of gene prediction in silico. We constructed cDNA libraries from mixed blood stages of P. falciparum parasite using the SMART cDNA library construction technique and generated 17332 high-quality expressed sequence tags (EST), including 2198 from primer-walking experiments. Assembly of our sequence tags produced 2548 contigs and 2671 singletons versus 5220 contigs and 5910 singletons when our EST were assembled with EST in public databases. Comparison of all the assembled EST/contigs with predicted CDS and genomic sequences in the PlasmoDB database identified 356 genes with predicted coding sequences fully covered by EST, including 85 genes (23.6%) with introns incorrectly predicted. Careful automatic software and manual alignments found an additional 308 genes that have introns different from those predicted, with 152 new introns discovered and 182 introns with sizes or locations different from those predicted. Alternative spliced and antisense transcripts were also detected. Matching cDNA to predicted genes also revealed silent chromosomal regions, mostly at subtelomere regions.
Our data indicated that approximately 24% of the genes in the current databases were predicted incorrectly, although some of these inaccuracies could represent alternatively spliced transcripts, and that more genes than currently predicted have one or more additional introns. It is therefore necessary to annotate the parasite genome with experimental data, although obtaining complete cDNA sequences from this parasite will be a formidable task due to the high AT nature of the genome. This study provides valuable information for genome annotation that will be critical for functional analyses.
PMCID: PMC1978503  PMID: 17662120
10.  Experimental Ocular Toxoplasmosis in Genetically Susceptible and Resistant Mice  
Infection and Immunity  2005;73(8):5160-5165.
Genetic factors determining the pathogenesis and course of ocular toxoplasmosis are poorly understood. In this study, we explored the development of experimental ocular pathogenesis in genetically dissimilar mice infected with either the RH strain, the PLK strain, or the immunodominant surface antigen 1 (SAG1 [P30])-deficient mutant of the RH strain of Toxoplasma gondii. At 11 days postinfection, ocular infection of C57BL/6 mice with all of the strains of parasites resulted in severe inflammatory lesions and high numbers of parasites in eye tissue; less severe ocular lesions at earlier histopathology and prolonged survival were observed in this mouse strain infected with either the major surface antigen 1-deficient SAG1−/− strain or the less virulent PLK strain compared with RH infection. In contrast, both BALB/c and CBA/J mice had less severe lesions and low numbers of parasites in their eye tissue, and infection developed into the chronic stage in these mice. There were significantly higher serum levels of gamma interferon and tumor necrosis factor alpha in C57BL/6 mice than in BALB/c and CBA/J mice following ocular infection. These observations confirm earlier reports on systemic immunity to these parasites that the route of Toxoplasma infection markedly influences survival of mice. Our data indicate that genetic factors of the host as well as the parasite strain are critical in determining susceptibility to experimental ocular toxoplasmosis in murine models.
PMCID: PMC1201211  PMID: 16041033
11.  CD4+ T Cells in the Pathogenesis of Murine Ocular Toxoplasmosis  
Infection and Immunity  2004;72(9):4966-4972.
The role of CD4+ T cells in the pathogenesis of ocular toxoplasmosis was investigated in murine models utilizing inbred C57BL/6 mice deficient either in CD4+, CD8+, or B cells (μMT). Severe necrosis and inflammation with replicating parasites were observed in the eyes of control mice after primary ocular infection, and near-normal histology with few tachyzoites was observed in the eyes of mice immunized intraperitoneally with the avirulent ts-4 strain followed by intraocular challenge with the RH strain of Toxoplasma gondii. In contrast, mild inflammation without evidence of necrosis associated with increased parasite burdens were observed in the eyes of CD4 knockout (KO) mice after both primary ocular infection and challenge with RH tachyzoites. CD8 KO mice, as well as μMT mice, demonstrated increased ocular necrosis in response to either primary ocular infection or challenge. The parasite burden was increased in the eyes of both CD8 KO and μMT mice in which the parasite load was even higher. As expected, there were no increases in the levels of immunoglobulin G in serum or aqueous humor in μMT mice, and there was no increase in the levels of gamma interferon and tumor necrosis factor alpha in the sera of CD4 KO mice after both infection and challenge. These results suggest that the ocular inflammatory response to the parasite is mediated primarily by the CD4+-T-cell response. CD8+ T cells and B cells may play an important role in limiting tachyzoite proliferation in the eyes. Mice deficient in CD8+ CD4+ T cells or B cells exhibit diminished vaccine-induced resistance and increased ocular parasite burden after challenge.
PMCID: PMC517441  PMID: 15321988
12.  Interleukin-10 and Pathogenesis of Murine Ocular Toxoplasmosis 
Infection and Immunity  2003;71(12):7159-7163.
To understand the role of interleukin-10 (IL-10) in ocular toxoplasmosis, we compared C57BL/6 (B6) and BALB/c background mice lacking a functional IL-10 gene (IL-10−/−) and B6 transgenic mice expressing IL-10 under the control of the IL-2 promoter. Increased cellular infiltration and necrosis were observed in the eye tissue of IL-10−/− mice of both the B6 and BALB/c backgrounds with associated changes in the levels of cytokines in serum. In contrast, there was no evidence of necrosis in the eye tissue from IL-10 transgenic mice following parasite exposure. Our results demonstrate that IL-10 is important in the regulation of inflammation during acute ocular toxoplasmosis.
PMCID: PMC308941  PMID: 14638808

Results 1-12 (12)