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Zhiliang Wu

Publications and source records attributed to Zhiliang Wu.

13 recordsLinked to original sources
PubMed2004

Molecular cloning and characterisation of two kinds of proteins in excretory-secretory products of Trichinella pseudospiralis.

Two genes encoding Trichinella pseudospiralis excretory-secretory proteins related to the Trichinella spiralis glycoproteins were cloned and the excretory-secretory proteins were characterised. A cloned gene, designated Tp38 (Ts43), contained a cDNA transcript of 1035 bp, and the predicted amino acid sequence of the Tp38 (Ts43) pro-protein had a similarity of about 84% to that of the T. spiralis 43 kDa glycoprotein. A cloned gene, designated Tp53 (Ts53), contained a cDNA transcript of 1239 bp, and the predicted amino acid sequence of the Tp53 (Ts53) pro-protein had a similarity of about 68% to that of the T. spiralis 53 kDa glycoprotein. Southern blots indicated that the Tp38 (Ts43) and Tp53 (Ts53) genes were encoded in a single copy within the T. pseudospiralis genome. Western blots showed that T. pseudospiralis-infected sera recognised the Tp53 (Ts53) recombinant protein, but did not recognise the Tp38 (Ts43) recombinant protein. The Tp38 (Ts43) and Tp53 (Ts53) proteins in the excretory-secretory product were 3 and 9 kDa greater than the expected molecular mass, respectively, and had three isoforms with a similar molecular size. Reverse transcription polymerase chain reaction results showed that the production of the mRNA transcript for the Tp38 (Ts43) or Tp53 (Ts53) gene was restricted predominantly to muscle larvae. Western blots confirmed that the gene products were predominantly expressed by muscle-stage larvae. An immunolocalisation study showed the Tp38 (Ts43) and Tp53 (Ts53) proteins were present within the alpha-stichocyte and the beta-stichocyte of muscle larvae, respectively.

Amino Acid Sequence↗
PubMed2004

PCR-based identification of zoonotic isolates of Blastocystis from mammals and birds.

The genotype of Blastocystis isolated from humans and animals is highly polymorphic. Therefore, it is important to compare the genotypes of Blastocystis isolates from humans and animals to determine the zoonotic potential of animal isolates. PCR-based genotype classification using known sequence-tagged site (STS) primers allows identification of zoonotic isolates of animal origin. To this end, 51 isolates from monkeys, cattle, pigs, chickens, quails and pheasants were subjected to genotype analysis using seven kinds of STS primers. Out of the 51 isolates, 39 were identified as one of the known genotypes, four showed mixed genotypes, and eight were unknown genotypes as these were negative for all STS primers. When these results were combined with previous studies on 41 isolates from animals and compared with the diversity of genotypes of 102 human Blastocystis hominis isolates, 67.4 % (62/92) of isolates from mammals and birds were identical to human B. hominis genotypes. Since the unknown genotype of human origin had been placed into an additional clade in the small-subunit rRNA gene phylogeny, further molecular study on the eight isolates of unknown genotype from the present study will facilitate our understanding of their zoonotic potential.

Animals↗
PubMed2004

Molecular expression of a cysteine proteinase of Clonorchis sinensis and its application to an enzyme-linked immunosorbent assay for immunodiagnosis of clonorchiasis.

We produced a recombinant cysteine proteinase of Clonorchis sinensis and tested its value as an antigen for serologic diagnosis of C. sinensis infections. The predicted amino acid sequence of the cysteine proteinase of C. sinensis was 58, 48, and 40% identical to those of cathepsin L cysteine proteinases from Paragonimus westermani, Schistosoma japonicum, and Fasciola hepatica, respectively. Western blotting analysis showed that sera from patients infected with C. sinensis strongly reacted with the recombinant protein and that sera from patients infected with S. japonicum weakly reacted with the recombinant protein. Antibody against the recombinant protein stained proteins migrating at about 37 and 28 kDa in C. sinensis adult worm crude extracts. Immunostaining revealed that the cysteine proteinase of C. sinensis was located in the intestinal epithelial cells of the adult parasite and in intrauterine eggs. The specificity and sensitivity of the recombinant antigen or C. sinensis adult worm crude extracts were assessed by an enzyme-linked immunosorbent assay (ELISA) using serum samples from humans infected with different parasites, including 50 patients with clonorchiasis, and negative controls. The sensitivities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96 and 88%, respectively. The specificities of the ELISA with the recombinant antigen and C. sinensis adult worm crude extracts were 96.2 and 100%, respectively. The results suggested that the recombinant cysteine proteinase-based ELISA could provide a highly sensitive and specific assay for diagnosis of clonorchiasis.

Amino Acid Sequence↗
PubMed2004

Epidemiological survey of intestinal parasitic infections of diarrhoeal patients in Nepal and Lao PDR.

Intestinal parasitological survey was conducted to clarify the distribution of intestinal parasites in Nepal and Lao Peoples' Democratic Republic (Lao PDR) from 2001 to 2003. A total of 401 diarrheal fecal specimens were collected from patients aged one to 75 years (250 from Nepal and 151 from Lao PDR). The specimens were examined using the formalin-ether sedimentation (FES) and sucrose centrifugal flotation (SCF) techniques. Nine species (3 Nematoda, 1 Cestoda, and 5 Protozoa) of parasites were recovered from Nepal, whereas seven species (3 Nematoda, 1 Trematoda, and 3 Protozoa) from Lao PDR. Of the parasites detected, (14.4%) was the most common in Nepal, and was Opisthorchis viverrini (29.8%) in Lao PDR. Infection rates were markedly different among age groups in both countries; higher rates were observed in age groups of 10-29 years than in 0-9 years group. Scanning electron microscopic observation revealed that trematoda eggs recovered from Lao PDR were identified as O. viverrini, which could not be done by a light microscope.

Adolescent↗
PubMed2003

Polymerase chain reaction-based genotype classification among human Blastocystis hominis populations isolated from different countries.

Since the genotype of human Blastocystis hominis isolates is highly polymorphic, PCR-based genotype classification using known sequenced-tagged site (STS) primers would allow the identification or classification of different genotypes. Five populations of human B. hominis isolates obtained from Japan, Pakistan, Bangladesh, Germany, and Thailand were subjected to genotype analysis by using seven kinds of STS primers. Ninety-nine out of 102 isolates were identified as one of the known genotypes, while one isolate from Thailand showed two distinct genotypes and two isolates from Japan were negative with all the STS primers. The most dominant genotype among four populations, except for all four isolates from Thailand, was subtype 3 and it varied from 41.7% to 92.3%. The second most common genotype among four populations was either subtype 1 (7.7-25.0%) or subtype 4 (10.0-22.9%). Subtype 2, subtype 5, and/or subtype 7 were only rarely detected among the isolates from Japan and Germany, while subtype 6 was not detected. The phylogenetic position of the two isolates which were negative with all STS primers, was inferred from the small subunit rRNA (SSU rRNA) genes with the known sequence data of 20 Blastocystis isolates. Since the two isolates were positioned in an additional clade in the phylogenetic tree, this suggested they were a new genotype. These results demonstrated that PCR-based genotype classification is a powerful tool with which to analyse genotypes of Blastocystis isolates obtained from clinical samples. In addition, two groups of the isolates from 15 symptomatic and 11 asymptomatic patients in Bangladesh were compared with the PCR-based subtype classification. Since both groups were only classified into two distinct genotypes of subtype 1 or subtype 3 and no statistically significant difference was observed between the two groups, in this study it could not be shown that the specific genotype correlated with the pathogenic potential of B. hominis.

Animals↗
PubMed2003

Molecular characterization of Blastocystis isolates from primates.

Twelve Blastocystis isolates from primates were analyzed genetically by polymerase chain reaction (PCR) using diagnostic primers and PCR-restriction fragment length polymorphism (RFLP) of SSUrDNA. Two distinct genotypes, subtype 1 and a variant of subtype 1, were detected in two and six of the 12 isolates, respectively. The RFLP profiles of the isolates designated as subtype 1 were identical to the profile of ribodeme 1. The RFLP profiles of the six isolates designated as variants of subtype 1 were different from the profile of the variant of subtype 1 from a human reported previously. The other four isolates were not amplified with any diagnostic primers, but three of them showed the same RFLP profiles as ribodeme 6. This study was the first genomic analysis of Blastocystis isolates from primates, and showed the genetic similarity between the isolates from primates and the genotypes of Blastocystis hominis. However, it was unclear whether the isolates examined were zoonotic or not. Therefore, it is necessary to reveal the phylogenetic relationships between the isolates from primates and the multiple genotypes of B. hominis.

Animals↗
PubMed2003

Zoonotic genotypes of Blastocystis hominis detected in cattle and pigs by PCR with diagnostic primers and restriction fragment length polymorphism analysis of the small subunit ribosomal RNA gene.

Twenty two Blastocystis isolates from cattle and pigs were genotyped by PCR using diagnostic primers, and the homology among isolates was then confirmed by restriction fragment length polymorphism (RFLP) analysis of the small subunit ribosomal RNA gene. Two distinct genotypes, subtypes 1 and 3, were detected in one and two of the ten isolates from cattle, and three and one of the 12 isolates from pigs, respectively. The RFLP profiles of all isolates designated as subtype 1 and subtype 3 were identical to those of known ribodemes 1 and 2, respectively. We found that 31.8% (7/22) of isolates examined were zoonotic genotypes of Bastocystis hominis. However, 68.2% (15/22) of isolates were not amplified with any of the diagnostic primers, and the profiles of these isolates were different from any ribodemes of B. hominis reported previously. This study shows that Blastocystis organisms in cattle and pigs are a potential source of human infection.

Animals↗
PubMed2003

Intraspecies polymorphism of Cryptosporidium parvum revealed by PCR-restriction fragment length polymorphism (RFLP) and RFLP-single-strand conformational polymorphism analyses.

A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C. parvum isolates. Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat). These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15. The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates. The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively. Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively. Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie. The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc. The isolates from calves, a goat, and one Japanese human were identified as genotype II. Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc. All of the genotype I isolates were from humans. The Japanese isolate (code no. HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively. The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no. HJ2) was identified as genotype Ie. Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C. parvum.

Amino Acid Sequence↗
PubMed2003

Molecular comparative studies among Blastocystis isolates obtained from humans and animals.

This study compared specific polymerase chain reaction (PCR) primers and phylogenetic tree analysis of restriction fragment length polymorphism using the small subunit ribosomal RNA gene in various Blastocystis populations. A phylogenetic tree was constructed using 12 restriction enzymes and a sample pool of 22 isolates, including 2 reference strains and Proteromonas lacertae as an outgroup. The analysis showed that the 22 isolates could be separated into 7 clusters. Four of the 7 clusters were mixed groups that comprised isolates from both humans and nonhuman hosts. The other 3 clusters contained isolates from humans or nonhuman hosts only. The phylogenetic analysis also showed that B. hominis isolates from geographical separated areas did not necessarily cluster in the genetically different groups. The results of genetic homology and phylogenetic tree analysis among Blastocystis isolates from humans and animals indicated that all isolates from animals appear to be B. hominis. Polymerase chain reaction amplifications using previously described and newly defined specific primers mirrored the clusters obtained by the phylogenetic tree analysis. Our results show that primer PCR can be used as a powerful tool for the typing of Blastocystis populations.

Animals↗
PubMed2002

Molecular characterization of Blastocystis isolates from birds by PCR with diagnostic primers and restriction fragment length polymorphism analysis of the small subunit ribosomal RNA gene.

Seven Blastocystis isolates from birds were genotyped by polymerase chain reaction (PCR) using diagnostic primers, and the homology among isolates was then confirmed by restriction fragment length polymorphism (RFLP) analysis of the small subunit ribosomal RNA gene. A zoonotic genotype, subtype 4, was detected in three isolates by PCR, but the RFLP profiles of these isolates differed from those of the subtype 4 reported previously from a human. None of the other four isolates were amplified with any subtype-specific primer sets, and the profiles of these isolates were different from any ribodemes of Blastocystis hominis reported previously. In the present study, the seven isolates from birds were classified into three genetically distinct genotypes. Therefore, birds appear to be infected with multiple genotypes. It is unclear whether the isolates examined in the present study are zoonotic or not. Therefore, it is important to determine the phylogenetic relationships between unknown genotypes from birds and the multiple genotypes of B. hominis.

Animals↗
PubMed2002

Expression of excretory and secretory protein genes of Trichinella at muscle stage differs before and after cyst formation.

By adapting a semi-quantitative reverse transcriptase-PCR (RT-PCR) method, we investigated kinetics of gene expression at different developmental stages of Trichinella spiralis and T. pseudospiralis. The analyzed genes included four kinds of excretory and secretory (ES) proteins, a heat shock protein (HSP) and a DNA binding protein and showed that T. spiralis and T. pseudospiralis expressed ES proteins in a stage-specific manner. The gene encoding a 43 kDa ES protein was expressed by muscle larvae, either pre-cyst or post-cyst larvae. The genes encoding: the 53 kDa ES protein of T. spiralis; 53 kDa ES protein of T. pseudospiralis; and 19.6 kDa ES protein of T. spiralis were expressed by post-cyst larvae and adult worms, but not expressed by pre-cyst larvae or newborn larvae. The results showed that pre-cyst larvae and post-cyst larvae are similar but different in the expression of 53 and 19.6 kDa ES proteins. On the other hand, genes of housekeeping proteins, such as HSP and the DNA binding protein, were expressed at all stages although there were some differences in the expression level.

Animals↗