Identification of expressed protein profiles and antigenic determination are some of the most challenging aspects of proteomics. Two-dimensional gel electrophoresis (2-DE) combined with immunoblot analysis were employed to study the N. caninum proteome. Protein sample preparation was carried out by first conducting sonication, followed by adding lysis buffer containing 7M urea plus 2M thiourea to the purified tachyzoites in order to complete disruption. A total of 335 differentially expressed protein spots were detected using pH 4-7 IPG strip (7 cm) that were run in a 56 kVh isoelectric focusing (IEF) system. Of the spots analyzed, 64 were identified as antigenic spots on immunoblot profile. Major antigenic spots appeared at 65 kDa (pI 5.2-5.3), 51 kDa (pI 5.5), 38 kDa (pI 5.1), 33 kDa (pI 4.4), 29 kDa (pI 5.6) and 15.5 kDa (pI 5.0) were observed to be significantly distinct compared to the rest of the antigenic spots. The results indicate that combination of 2-DE and immunoblotting methods were thought as very useful tools in defining both proteins and antigens of N. caninum tachyzoites. Additionally, present 2-DE profiles may be valuable in further proteomic approaches and study of the pathogen.
Animals ; Antibody Formation ; Antigens, Protozoan/*analysis/isolation&purification ; Electrophoresis, Gel, Two-Dimensional/methods ; Electrophoresis, Polyacrylamide Gel ; Epitopes/analysis ; Image Processing, Computer-Assisted ; Immunoblotting/methods ; Isoelectric Focusing ; Neospora/*chemistry/immunology ; Proteome/analysis ; Proteomics ; Protozoan Proteins/*analysis/isolation&purification

Genotyping of Toxoplasma gondii has been performed in 23 PCR positive blood samples from stray cats in Korea. We used 2 separate PCR-restriction fragment length polymorphism (RFLP) patterns of SAG2 gene, amplifying the 5'and 3'ends of the locus. The results revealed that all samples belonged to the type I clonal lineage. Although T. gondii organisms were not isolated from the samples, the results of the present study represent that stray cats with T. gondii infection should be seriously concerned in our environment. Adequate and continuous control programs of stray cats are needed to reduce the risk of transmission of T. gondii as a zoonotic infection threatening the public health.
Animals ; Antigens, Protozoan/genetics ; Blood/*parasitology ; Cat Diseases/*parasitology ; Cats ; Cluster Analysis ; DNA Fingerprinting/methods ; DNA, Protozoan/genetics/isolation & purification ; Genotype ; Korea ; Polymerase Chain Reaction/methods ; Polymorphism, Restriction Fragment Length ; Protozoan Proteins/genetics ; Toxoplasma/*classification/*genetics/isolation & purification ; Toxoplasmosis, Animal/*parasitology

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

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

This study investigated the effect of breast-feeding in protection against protozoan infection in infants with persistent diarrhea. Infants were classified into 2 groups; 161 breast-fed infants and the same number of non-breast-fed infants. Microscopic examinations of stool were done for detection of parasites and measuring the intensity of infection. Moreover, serum levels of IgE and TNF-alpha were measured by ELISA. Cryptosporidium spp., Entamoeba histolytica/Entamoeba dispar, Giardia lamblia, and Blastocystis sp. were demonstrated in infants with persistent diarrhea. The percentage of protozoan infections was significantly lower in breast-fed infants than that in the non-breast-fed infants. The levels of IgE and TNF-alpha were significantly lower in the breast-fed group than in the non-breast-fed group. There were significant positive associations between the serum levels of IgE and TNF-alpha and the intensity of parasite infection in the breast-fed group. It is suggested that breast-feeding has an attenuating effect on the rate and intensity of parasite infection.
Antigens, Protozoan/analysis/*immunology ; Diarrhea, Infantile/*diagnosis/parasitology ; Entamoeba ; Entamoeba histolytica/*isolation & purification ; Entamoebiasis/*diagnosis/parasitology ; Enzyme-Linked Immunosorbent Assay ; Feces/parasitology ; Female ; Giardia lamblia ; Giardiasis/*diagnosis/parasitology ; Humans ; Infant ; Intestines/parasitology ; Protozoan Infections/*diagnosis/parasitology ; Tumor Necrosis Factor-alpha/metabolism

Toxoplasma gondii tachyzoites were isolated from the blood of an ocular patient, and have been successfully passaged in the laboratory, for over a year, by peritoneal inoculation in mice. The isolated parasite was designated the Korean Isolate-1 (KI-1) and its characteristics were compared with those of the RH strain, a wellknown virulent strain originating from a child who suffered from encephalitis. The morphology, pathogenicity, infectivity and cell culture characteristics of the KI-1 were similar to those of the RH strain. Both RH and KI-1 antigens were detected by an anti-T. gondii monoclonal antibody (mAb), Tg563, against the major surface protein SAG1 (30 kDa), whereas no reaction was observed against an anti-Neospora caninum mAb, 12B4. The KI-1 was confirmed as an isolate of T. gondii. A long-term laboratory maintenance and characterization of a local T. gondii isolate is reported for the first time in the Republic of Korea.
Animals ; Antigens, Protozoan/analysis ; Female ; Humans ; Korea ; Mice ; Mice, Inbred BALB C ; Microscopy, Electron ; Middle Aged ; Parasitemia/parasitology ; Sarcoma 180 ; Serial Passage ; Specific Pathogen-Free Organisms ; *Toxoplasma/classification/growth & development/isolation & purification/pathogenicity ; Toxoplasmosis, Ocular/*diagnosis/parasitology ; Tumor Cells, Cultured ; Virulence