OBJECTIVETo confirm the existence of point mutations in the SSU rDNA variable regions of 5 Leishmania donovani (L.d.) isolates from different epidemic foci in China.

METHODSSpecific SSU rDNA fragments from nuclear DNA of 7 Leishmania species/isolates were amplified by PCR and then cloned into pGEM(R)-T Easy Vectors. After that, the specific fragments were sequenced by an automated DNA sequencer.

RESULTSSequence analysis showed that the amplified DNA fragments of 7 Leishmania species/isolates were all 392 bp in length. All 5 point mutations were located in two unique sequence blocks (UQ-I and UQ-II), and no insertions or deletions were found. The identities of comparison of Leishmania in GeneBank were more than 98%.

CONCLUSIONFive point mutations exist in the SSU rDNA variable region of 5 L.d. isolates from different epidemic foci of visceral leishmaniasis (VL) in China. Sequence differences of the SSU rDNA variable region exist among L.d. isolates from different foci.

Animals ; DNA, Protozoan ; chemistry ; DNA, Ribosomal ; chemistry ; Humans ; Leishmania donovani ; genetics ; Leishmaniasis, Visceral ; parasitology ; Point Mutation ; Polymerase Chain Reaction

OBJECTIVETo identify the binding site on glycophorin A (GPA) for EBA-175 to provide clue for developing short peptide vaccine and therapeutic agents against Plasmodium falciparum.

METHODSWith the recombinant protein of EBA-175 as the target molecule, the mimetic peptides of GPA were screened from a 12-mer random peptide library. Three rounds of biopanning were carried out, and enzyme-linked immunosorbent assay (ELISA), competitive ELISA, Dot-ELISA and Western blotting used to evaluate the binding between the phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences deduced.

RESULTSThirty clones from the third round were randomly selected, of which 27 were found positive by sandwich ELISA. Competitive ELISA proved that most of the phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that 24 positive phage clones contained the conservative sequence of IRR, which was highly homologous with the 114-116 amino acids of GPA.

CONCLUSIONThese phage-displayed peptides can bind with EBA-175, and the amino acid sequence IRR might play an important role in the binding between EBA-175 and GPA.

Antigens, Protozoan ; metabolism ; Binding Sites ; Enzyme-Linked Immunosorbent Assay ; Glycophorin ; chemistry ; Humans ; Peptide Library ; Plasmodium falciparum ; Protozoan Proteins ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein

The disinfectant effects (DEs) of 10 types of chemicals, defined by their ability to destroy or inhibit oocysts and consequently prevent sporulation of Eimeria tenella field isolate, were evaluated in vitro. Correct species assignments and sample purities were confirmed by the singular internal transcribed spacer (ITS)-PCR analysis. A total of 18 treatments were performed, and the disinfection suppression levels were 75.9% for 39% benzene + 22% xylene (1:10 dilution), 85.5% for 30% cresol soup (1:1 dilution), and 91.7% for 99.9% acetic acid (1:2 dilution) group. The results indicate that acetic acid, cresol soup, and benzene+xylene are good candidates for suppression of E. tenella oocyst sporulation.
Animals ; Antiprotozoal Agents/*pharmacology ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal Spacer/chemistry/genetics ; Disinfectants/*pharmacology ; Eimeria tenella/*drug effects/*growth & development ; Microscopy ; Molecular Sequence Data ; Parasitic Sensitivity Tests ; Phylogeny ; Sequence Analysis, DNA ; Spores, Protozoan/*drug effects/*growth & development

Cryptosporidium and Giardia are 2 protozoan parasites responsible for waterborne diseases outbreaks worldwide. In order to assess the prevalence of these protozoans in drinking water samples in the northern part of Portugal and the risk of human infection, we have established a long term program aiming at pinpointing the sources of surface water, drinking water, and environmental contamination, working with the water-supply industry. Total 43 sources of drinking water samples were selected, and a total of 167 samples were analyzed using the Method 1623. Sensitivity assays regarding the genetic characterization by PCR and sequencing of the genes, 18S SSU rRNA, for Cryptosporidium spp. and beta,-giardin for G. duodenalis were set in the laboratory. According to the defined criteria, molecular analysis was performed over 4 samples. Environmental stages of the protozoa were detected in 25.7% (43 out of 167) of the water samples, 8.4% (14 out of 167) with cysts of Giardia, 10.2% (17 out of 167) with oocysts of Cryptosporidium and 7.2% (12 out of 167) for both species. The mean concentrations were 0.1-12.7 oocysts of Cryptosporidium spp. per 10 L and 0.1-108.3 cysts of Giardia duodenalis per 10 L. Our results suggest that the efficiency in drinking water plants must be ameliorated in their efficiency in reducing the levels of contamination. We suggest the implementation of systematic monitoring programs for both protozoa. To authors' knowledge, this is the first report evaluating the concentration of environmental stages of Cryptosporidium and Giardia in drinking water samples in the northern part of Portugal.
Animals ; Cryptosporidium/*isolation & purification ; Cytoskeletal Proteins/genetics ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Genes, rRNA ; Giardia lamblia/*isolation & purification ; Humans ; Molecular Sequence Data ; Polymerase Chain Reaction ; Portugal ; Protozoan Proteins/genetics ; RNA, Protozoan/genetics ; RNA, Ribosomal, 18S/genetics ; Risk Assessment ; Sequence Analysis, DNA ; Water/*parasitology

Pentatrichomonas hominis is considered a commensal protozoan in the large intestine of a number of mammalian hosts, such as cats, dogs, and non-human primates. The resulting infections, which can induce diarrhea, have been attributed to opportunistic overgrowth of P. hominis. This study was performed to confirm the P. hominis infection and its molecular characterization from the feces of puppies with diarrhea. Fecal samples were obtained from 14 German shepherd puppies with diarrhea over 1 week (7 females and 7 males, 2-9 months of age) residing on a dog farm in August 2007. Species-specific PCR assay identified P. hominis 18S rRNA genes in 3 of the 14 puppies (1 female and 2 males; 1 aged 2 months and 2 aged 9 months). This phylogenetic analysis established that P. hominis belonged to the 1st clade, which is comprised of Bos taurus and Felines.
Animals ; Base Sequence ; Cluster Analysis ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Diarrhea/parasitology/*veterinary ; Dog Diseases/*parasitology ; Dogs ; Feces/*parasitology ; Female ; Genes, rRNA ; Male ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction/methods ; Protozoan Infections, Animal/*parasitology ; RNA, Protozoan/genetics ; RNA, Ribosomal, 18S/genetics ; Republic of Korea ; Sequence Analysis, DNA ; Sequence Homology ; Trichomonadida/*classification/genetics/*isolation & purification

Toxoplasma gondii is a persistent protozoan parasite capable of infecting almost any warm-blooded vertebrates. SAG1 (p30) is the prototypic member of a superfamily of surface antigens called SRS (SAG1-related sequence). It constitutes the most abundant and predominant antigen. In this paper the primary structure of mature SAG1 gene of an Indonesian T. gondii isolate is described and sequence comparison is made with published sequence data of 7 other strains or isolates. Sequence comparison indicated that SAG1 is highly conserved through evolution and despite parasite spreading world-wide. Sequences may be divided into two major families, independent of the strain/isolate geographic origin. Variations were mainly localized at the C-terminal half or domain 2 and some clustered in restricted areas. Sequence comparison allowed us to define the Indonesian isolate as genuine virulent RH strain. A phylogenetic tree of Toxoplasma strains/isolates was constructed based on SAG1.
Amino Acid Sequence ; Animals ; Antigens, Protozoan/chemistry/*genetics ; Base Sequence ; Cloning, Molecular ; DNA, Protozoan/chemistry/genetics ; Goat Diseases/parasitology ; Goats ; Indonesia ; Molecular Sequence Data ; Phylogeny ; Polymerase Chain Reaction ; Protozoan Proteins/chemistry/*genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Toxoplasma/*genetics/*immunology/isolation&purification ; Toxoplasmosis/parasitology ; Zoonoses/parasitology

In order to develop tools for an early serodiagnosis of Plasmodium falciparum infection, we evaluated the usefulness of P. falciparum liver stage antigen-3 (LSA-3) as a serodiagnostic antigen. A portion of LSA-3 gene was cloned, and its recombinant protein (rLSA-3) was expressed in Escherichia coli and purified by column chromatography. The purified rLSA-3 and 120 test blood/serum samples collected from inhabitants in malaria-endemic areas of Mandalay, Myanmar were used for this study. In microscopic examinations of blood samples, P. falciparum positive rate was 39.1% (47/120) in thin smear trials, and 33.3% (40/120) in thick smear trials. Although the positive rate associated with the rLSA-3 (30.8%) was lower than that of the blood stage antigens (70.8%), rLSA-3 based enzyme-linked immunosorbent assay could detect 12 seropositive cases (10.0%), in which blood stage antigens were not detected. These results indicate that the LSA-3 is a useful antigen for an early serodiagnosis of P. falciparum infection.
Recombinant Proteins/biosynthesis/genetics/*immunology ; Plasmodium vivax/isolation & purification ; Plasmodium falciparum/*immunology ; Molecular Sequence Data ; Malaria, Falciparum/blood/*diagnosis ; Humans ; Genes, Protozoan/genetics/immunology ; Fluorescent Antibody Technique, Direct/methods ; Escherichia coli/genetics ; Enzyme-Linked Immunosorbent Assay/methods ; Early Diagnosis ; DNA, Protozoan/chemistry ; DNA Primers/chemistry ; Cloning, Molecular/methods ; Base Sequence ; Antigens, Protozoan/biosynthesis/chemistry/genetics/*immunology ; Animals ; Amino Acid Sequence

OBJECTIVETo confirm the genetic relation between Giardia lamblia (G. lamblia) isolates from different geographic regions of China and other countries.

METHODSGenomic DNA were extracted from the trophozoites or cysts of Giardia lamblia. The triose phosphate isomerase (tim) gene was amplified using polymerase chain reaction (PCR) technique. PCR products were digested with endonuclease and sequenced. The data of sequencing were analyzed with the DNAstar software and compared with that of the isolates acquired from GenBank.

RESULTSOf nine isolates of Giardia lamblia from China (C1, C2, CH2 and CH3), Cambodia (CAM), Australia (A1 and A2) and America (BP and CDC), respectively, 3 (A1, A2 and CAM) fit into Group 1 (WB), 2 (CH2 and CH3)) into Group 2, and 4 (C1, C2, BP and CDC) into Group 3 (GS). The results confirmed the genetic relatedness of G. lamblia isolates from all over the world.

CONCLUSIONGenotyping isolates of G. Lamblia provides important information for establishing the phylogenetic relationship or for the epidemiological evaluation of the spreading of this organism.

Amino Acid Sequence ; Animals ; Base Sequence ; DNA, Protozoan ; chemistry ; Genotype ; Giardia lamblia ; classification ; enzymology ; genetics ; Polymerase Chain Reaction ; Restriction Mapping ; Triose-Phosphate Isomerase ; chemistry ; genetics

In contrast to the gradual reduction in the number of locally transmitted malaria cases in China, the number of imported malaria cases has been increasing since 2008. Here, we report a case of a 39-year-old Chinese man who acquired Plasmodium ovale wallikeri infection while staying in Ghana, West Africa for 6 months in 2012. Microscopic examinations of Giemsa-stained thin and thick blood smears indicated Plasmodium vivax infection. However, the results of rapid diagnostic tests, which were conducted 3 times, were not in agreement with P. vivax. To further check the diagnosis, standard PCR analysis of the small-subunit rRNA gene was conducted, based on which a phylogeny tree was constructed. The results of gene sequencing indicated that this malaria is a variant of P. ovale (P. ovale wallikeri). The infection in this patient was not a new infection, but a relapse of the infection from the one that he had contracted in West Africa.
Adult ; Azure Stains ; Base Sequence ; China ; DNA, Protozoan/chemistry/genetics ; DNA, Ribosomal/chemistry/genetics ; Ghana ; Humans ; Malaria/*diagnosis/parasitology ; Male ; Molecular Sequence Data ; Phylogeny ; Plasmodium ovale/*classification/genetics/isolation & purification ; Polymerase Chain Reaction ; Recurrence ; Sequence Analysis, DNA ; Travel

The present study was designed to investigate polymorphism in Duffy binding protein (DBP) gene of Plasmodium vivax isolates of Korea. Thirty samples were obtained from P. vivax patients in Yonchon-gun, Kyonggi-do in 1998. The PCR products of the samples were subjected to sequencing and hybridization analyses of the regions II and IV of P. vivax DBP gene. Two genotypes, SK-1 and SK-2, were identified on the basis of amino acid substitution and deletion. The genotype of 10 isolates was SK-1 and that of 20 isolates was SK-2. Most of the predicted amino acids in the region II of DBP gene were conserved between the Korean isolates and Belem strain except for 4-5 amino acid substitutions. In the region IV of DBP, a 6-bp insert that was shown in the Sal-1 allele type was found in SK-1, and a 27-bp insert that was shown in the Papua New Guinea allele type was found in SK-2. In conclusion, the present findings suggest that two genotypes of P. vivax coexist in the endemic area of Korea.
Amino Acid Sequence ; Animals ; *Antigens, Protozoan ; Base Sequence ; Carrier Proteins/*analysis/chemistry/*genetics ; DNA, Protozoan/genetics ; Genotype ; Human ; Korea ; Malaria, Vivax/parasitology ; Molecular Sequence Data ; Plasmodium vivax/*genetics/isolation & purification ; Polymerase Chain Reaction ; *Polymorphism (Genetics) ; *Protozoan Proteins ; Receptors, Cell Surface/*analysis/chemistry/*genetics ; Support, Non-U.S. Gov't