To identify sequences of Entamoeba histolytica associated with the development of amebic liver abscess (ALA) in hamsters, subtractive hybridization of cDNA from E. histolytica HM-1:IMSS under 2 growth conditions was performed: 1) cultured in axenic medium and 2) isolated from experimental ALA in hamsters. For this procedure, 6 sequences were obtained. Of these sequences, the mak16 gene was selected for amplification in 29 cultures of E. histolytica isolated from the feces of 10 patients with intestinal symptoms and 19 asymptomatic patients. Only 5 of the 10 isolates obtained from symptomatic patients developed ALA and amplified the mak16 gene, whereas the 19 isolates from asymptomatic patients did not amplify the mak16 gene nor did they develop ALA. Based on the results of Fisher's exact test (P<0.001), an association was inferred between the presence of the mak16 gene of E. histolytica and the ability to develop ALA in hamsters and with the patient's symptoms (P=0.02). The amplification of the mak16 gene suggests that it is an important gene in E. histolytica because it was present in the isolates from hamsters that developed liver damage.
Adolescent ; Animals ; Cricetinae ; Entamoeba histolytica/*genetics ; Gene Expression ; *Genes, Protozoan ; Genetic Association Studies ; Humans ; Liver Abscess, Amebic/*genetics/*parasitology ; Male ; Virulence Factors/*genetics ; Young Adult

Autophagy-related protein 8 (Atg8) is an essential component of autophagy formation and encystment of cyst-forming parasites, and some protozoa, such as, Acanthamoeba, Entamoeba, and Dictyostelium, have been reported to possess a type of Atg8. In this study, an isoform of Atg8 was identified and characterized in Acanthamoeba castellanii (AcAtg8b). AcAtg8b protein was found to encode 132 amino acids and to be longer than AcAtg8 protein, which encoded 117 amino acids. Real-time PCR analysis showed high expression levels of AcAtg8b and AcAtg8 during encystation. Fluorescence microscopy demonstrated that AcAtg8b is involved in the formation of the autophagosomal membrane. Chemically synthesized siRNA against AcAtg8b reduced the encystation efficiency of Acanthamoeba, confirming that AcAtg8b, like AcAtg8, is an essential component of cyst formation in Acanthamoeba. Our findings suggest that Acanthamoeba has doubled the number of Atg8 gene copies to ensure the successful encystation for survival when 1 copy is lost. These 2 types of Atg8 identified in Acanthamoeba provide important information regarding autophagy formation, encystation mechanism, and survival of primitive, cyst-forming protozoan parasites.
Acanthamoeba castellanii/cytology/*genetics/physiology ; Amebiasis/*parasitology ; Amino Acid Sequence ; Autophagy ; Cell Membrane/metabolism ; DNA, Protozoan/chemistry/genetics ; Gene Dosage ; Gene Silencing ; Genes, Reporter ; Humans ; Molecular Sequence Data ; Phagosomes/metabolism ; Protein Isoforms ; Protozoan Proteins/*genetics/metabolism ; RNA, Messenger/genetics ; RNA, Protozoan/genetics ; RNA, Small Interfering/chemical synthesis/genetics ; Recombinant Fusion Proteins ; Sequence Alignment

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

OBJECTIVETo establish an tachyzoite-brachyzoite interconversion system for Toxoplasma gondii RH strain in vitro.

METHODSCOS-7 cells were inoculated with purified tachyzoites of T.gondii RH strain and cultured in vitro. The morphology of the cultured cells and parasites was observed and the total cellular RNA extracted on days 1 to 6 following the inoculation for detecting the expression of tachyzoite-specific protein (SAG1) and bradyzoite-specific proteins (BAG1 and SAG2C) using RT-PCR.

RESULTSWith the passage of time, the number of parasites in COS-7 cells increased but the proliferation rate was lowered gradually. The intracellular tachyzoites proliferated by means of budding and binary fission, which led to the changes in the alignment of the parasites in the cells from curved pairs, rosette or clustered, and semi-circular patterns to spherical encapsulation-like structures. These changes indicated the gradual transformation of the tachyzoites into bradyzoites. The expressions of the tachyzoite-specific SAG1 gene were detected throughout the 6 days of in vitro culture. The expression of the bradyzoite-specific BAG1 gene had been detected since the second day after the inoculation and SAG2C gene since the fifth day. Alteration of the culture condition resulted in gradual transformation of the bradyzoites into tachyzoites.

CONCLUSIONAn in vitro tachzoites-bradyzoite interconversion system for T.gondii has been successfully established, which provides the basis for further study of the mechanism of interconversion.

Animals ; COS Cells ; Cell Culture Techniques ; Cercopithecus aethiops ; Cysts ; Female ; Genes, Protozoan ; genetics ; Host-Parasite Interactions ; Mice ; Protozoan Proteins ; biosynthesis ; genetics ; Toxoplasma ; growth & development ; physiology

Animals ; Chemotaxis ; physiology ; Dictyostelium ; genetics ; metabolism ; physiology ; Genes, Protozoan ; genetics ; physiology ; Genes, ras ; genetics ; physiology ; Phosphatidylinositol 3-Kinase ; genetics ; metabolism ; Protein-Serine-Threonine Kinases ; genetics ; metabolism ; Proto-Oncogene Proteins ; genetics ; metabolism ; Protozoan Proteins ; genetics ; metabolism ; Signal Transduction ; Transcription Factors ; genetics ; metabolism

The objective of this study was to construct and express recombinant prokaryotic plasmid pET32a (+)- ast1 in E. coli BL21(DE3). Amastin gene was amplified from genomic DNA of Leishmania Donovani and its transmembran region was predicted by the methods of SOSUI and Tmpred; astl located in N-terminus of amastin gene was amplified and cloned into prokaryotic plasmid pET32a(+), which was named pET32a(+)-ast1, and then rAST1 was expressed in E. coli BL21(DE3). The results of SDS-PAGE and immunobloting assay showed that a fusion protein rAST1 (relative molecular mass about 27 kDa) was able to express in BL21. The recombinant prokaryotic plasmid pET32a(+)- ast1 was successfully constructed, and noted to be efficiently expressed in E. coli BL21(DE3).
Animals ; Cloning, Molecular ; Escherichia coli ; genetics ; metabolism ; Extracellular Space ; Genes, Protozoan ; Leishmania donovani ; genetics ; Plasmids ; genetics ; Protozoan Proteins ; biosynthesis ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; genetics

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

Two species of Cryptosporidium are known to infect man; C. hominis which shows anthroponotic transmission between humans, and C. parvum which shows zoonotic transmission between animals or between animals and man. In this study, we focused on identifying genotypes of Cryptosporidium prevalent among inhabitants and domestic animals (cattle and goats), to elucidate transmittal routes in a known endemic area in Hwasun-gun, Jeollanam-do, Republic of Korea. The existence of Cryptosporidium oocysts was confirmed using a modified Ziehl- Neelsen stain. Human infections were found in 7 (25.9%) of 27 people examined. Cattle cryptosporidiosis cases constituted 7 (41.2%) of 17 examined, and goat cases 3 (42.9%) of 7 examined. Species characterizations were performed on the small subunit of the rRNA gene using both PCR-RFLP and sequence analysis. Most of the human isolates were mixtures of C. hominis and C. parvum genotypes and similar PCR-RFLP patterns were observed in cattle and goat isolates. However, sequence analyses identified only C. hominis in all isolates examined. The natural infection of cattle and goats with C. hominis is a new and unique finding in the present study. It is suggested that human cryptosporidiosis in the studied area is caused by mixtures of C. hominis and C. parvum oocysts originating from both inhabitants and domestic animals.
Rural Health ; Prevalence ; Polymorphism, Restriction Fragment Length ; Polymerase Chain Reaction/methods ; Mutation/genetics ; Molecular Sequence Data ; Korea/epidemiology ; Humans ; Goats ; Goat Diseases/epidemiology/*parasitology ; Genotype ; Genes, rRNA/genetics ; DNA, Protozoan/chemistry ; DNA Primers/chemistry ; Cryptosporidium parvum/genetics/isolation & purification ; Cryptosporidium/classification/*genetics ; Cryptosporidiosis/*epidemiology/*parasitology ; Cattle Diseases/epidemiology/*parasitology ; Cattle ; Base Sequence ; Animals

A shuttle vector for Escherichia coli and Giardia lamblia was modified to produce a reporter plasmid, which monitors the expression of prescribed gene in G. lamblia by measuring its luciferase activity. Promoter regions of the gap2 gene, one of the genes induced during encystation, were cloned into this plasmid, and the resultant constructs were then transfected into trophozoites of G. lamblia. Transgenic trophozoites containing one of the 3 gap2-luc reporters were induced to encystation, and characterized with respect to gap2 gene expression by measuring their luciferase activities. Giardia containing a gap2-luc fusion of 112-bp upstream region showed full induction of luciferase activity during encystation.
Transfection/methods ; Time Factors ; Recombinant Fusion Proteins/analysis/biosynthesis ; Promoter Regions (Genetics)/*physiology ; Plasmids ; Luciferases/genetics/metabolism ; Life Cycle Stages/physiology ; Giardia lamblia/*genetics ; Genetic Engineering/methods ; Genes, Reporter/genetics ; Genes, Protozoan/genetics/*physiology ; Gene Order ; Gene Expression/genetics/*physiology ; GTPase-Activating Proteins/*genetics ; Blotting, Southern/methods ; Animals