Animals ; Antibodies, Protozoan ; analysis ; Antigens, Protozoan ; analysis ; Asthma ; blood ; parasitology ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Toxoplasma ; immunology ; Toxoplasmosis ; blood

The antigen location of Cryptosporidium parvum, which stimulates antibody formation in humans and animals, was investigated using infected human sera. Immuno-electron microscopy revealed that antigenicity-inducing humoral immunity was located at various developmental stages of parasites, including asexual, sexual stages, and oocysts. The amount of antigen-stimulating IgG antibodies was particularly high on the oocyst wall. The sporozoite surface was shown to give stimulation on IgG and IgM antibody formation. Trophozoites implicated the lowest antigenicity to humoral immunity, both IgG and IgM, by showing the least amount of gold labeling. Immunogold labeling also provided clues that antigens were presented to the host-cell cytoplasm via feeder organelles and host-parasite junctions.
Animals ; Antibodies, Protozoan/*immunology ; Antigens, Protozoan/*analysis ; Cryptosporidium parvum/*chemistry/*immunology/ultrastructure ; Female ; Humans ; Immunoglobulin G/immunology ; Immunoglobulin M/immunology ; Mice ; Microscopy, Immunoelectron ; Sporozoites/chemistry/immunology/ultrastructure ; Staining and Labeling/methods ; Trophozoites/chemistry/immunology/ultrastructure

To evaluate the seroprevalence against circumsporozoite protein (CSP) of Plasmodium vivax in sera of Korean patients, the central repeating domain (CRD) of CSP was cloned and analyzed. From the genomic DNA of patient's blood, 2 kinds of CSPs were identified to belong to a VK210 type, which is the dominant repeating of GDRA(D/A)GQPA, and named as PvCSPA and PvCSPB. Recombinantly expressed his-tagged PvCSPA or PvCSPB in Escherichia coli reacted well against sera of patients in western blot, with the detecting rate of 47.9% (58/121), which included 15 cases positive for PvCSPA, 6 cases positive for PvCSPB, and 37 cases for both. The mixture of PvCSPA and PvCSPB was loaded to a rapid diagnostic test kit (RDT) and applied with the same set of patient sera, which resulted in detection rates of 57.0% (69/121). When the protein sequences of PvCSPA were compared with those of P. vivax in endemic regions of India and Uganda, they were compatibly homologous to PvCSPA with minor mutations. These results suggested that the recombinant PvCSPA and PvCSPB loaded RDT may be a milestone in latent diagnosis which has been a hot issue of domestic malaria and important for radical therapy in overlapped infections with P. falciparum in tropical and subtropical areas. During the biological process of malarial infection, exposure of CSP to antigen-antibody reaction up to 57.0% is the first report in Korea.
Amino Acid Sequence ; Antibodies, Protozoan/*blood/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Base Sequence ; Humans ; India ; Malaria, Vivax/*diagnosis/*epidemiology/immunology ; Merozoite Surface Protein 1/genetics/*immunology ; Plasmodium vivax/genetics/immunology ; Protozoan Proteins/genetics/*immunology ; Reagent Kits, Diagnostic ; Recombinant Proteins/diagnostic use/immunology ; Republic of Korea/epidemiology ; Sequence Analysis, DNA ; Seroepidemiologic Studies ; Uganda

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

This experiment was focused on the characterization of anti-Toxoplasma monoclonal antibodies (mAbs) and the effect of mAbs on the parasite invasion of mouse peritoneal macrophages. Twenty eight mAbs including M110, M556, R7A6 and M621 were characterized by Ab titer, immunoglobulin isotyping and western blot pattern. Antibody titer (optical density) of 4 mAbs, M110, M556, R7A6 and M621, were 0.53, 0.67, 0.45 and 0.39 (normal mouse serum; 0.19) with the same IgG1 isotypes shown by Enzyme-linked immunosorbent assay (ELISA). Western blot analysis showed that M110, M556, R7A6 and M621 reacted with the 33 kDa (p30), 31 kDa (p28), 43 kDa and 36 kDa protein. Immunogold labelling of mAbs M110, M556, R7A6 and M621 reacted with the surface membrane, dense granules and parasitophorous vacuolar membrane (PVM), rhoptries and cytoplasm of tachyzoite, respectively. For in vitro assay, preincubation of tachyzoites with four mAbs, M110, M556, R7A6 and M621 resulted in the decrease of the number of infected macrophages (P < 0.05) and the suppression of parasite multiplication at 18 h post-infection. Four monoclonal antibodies including M110 (SAG1) were found to have an important role in the inhibition of macrophage invasion and T. gondii multiplication in vitro, and these mAbs may be suitable for vaccine candidates, diagnostic kit and for chemotherapy.
Animals ; *Antibodies, Monoclonal/pharmacology ; *Antibodies, Protozoan/pharmacology ; Antigens, Protozoan/*analysis/immunology ; Cells, Cultured ; Depression, Chemical ; Macrophages, Peritoneal/parasitology ; Mice ; Mice, Inbred ICR ; Support, Non-U.S. Gov't ; Toxoplasma/growth & development/*immunology

Acanthamoeba and Naegleria are widely distributed in fresh water, soil and dust throughout the world, and cause meningoencephalitis or keratoconjunctivitis in humans and other mammals. Korean isolates, namely, Naegleria sp. YM-1 and Acanthamoeba sp. YM-2, YM-3, YM-4, YM-5, YM-6 and YM-7, were collected from sewage, water puddles, a storage reservoir, the gills of a fresh water fish, and by corneal washing. These isolates were categorized into three groups based on the mortalities of infected mice namely, highly virulent (YM-4), moderately virulent (YM-2, YM-5 and YM-7) and nonpathogenic (YM-3). In addition, a new species of Acanthamoeba was isolated from a freshwater fish in Korea and tentatively named Korean isolate YM-4. The morphologic characters of its cysts were similar to those of A. culbertsoni and A. royreba, which were previously designated as Acanthamoeba group III. Based on experimentally infected mouse mortality, Acanthamoeba YM-4 was highly virulent. The isoenzymes profile of Acanthamoeba YM-4 was similar to that of A. royreba. Moreover, an anti-Acanthamoeba YM-4 monoclonal antibody reacted only with Acanthamoeba YM-4, and not with A. culbertsoni. Random amplified polymorphic DNA marker analysis and RFLP analysis of mitochondrial DNA and of a 18S small subunit ribosomal RNA, placed Acanthamoeba YM-4 in a separate cluster based on phylogenic distances. Thus Acanthamoeba YM-4 was identified as a new species, and assigned Acanthamoeba sohi. Up to the year 2002 in Korea, two clinical cases were found to be infected with Acanthamoeba spp. These patients died of meningoencephalitis. In addition, one case of Acanthamoeba pneumonia with an immunodeficient status was reported and Acanthamoeba was detected in several cases of chronic relapsing corneal ulcer, chronic conjunctivitis, and keratitis.
*Acanthamoeba/classification/genetics/immunology/pathogenicity ; Amebiasis/diagnosis/epidemiology/*parasitology/therapy ; Animals ; Antigens, Protozoan/analysis/genetics/immunology ; DNA, Mitochondrial/analysis ; DNA, Protozoan/analysis ; Korea/epidemiology ; Life Cycle Stages ; *Naegleria/classification/genetics/immunology/pathogenicity ; Phylogeny ; Polymorphism, Restriction Fragment Length ; Random Amplified Polymorphic DNA Technique/veterinary ; Virulence/genetics

Toxoplasma gondii is an intracellular protozoan parasite that causes a Th1 cellular immunity. Our previous study showed that T. gondii lysate antigen (TLA) treatment in S180 tumor-bearing mice resulted in tumor reduction by suppressing CD31 expression, a marker of angiogenesis. In the present study, to investigate tumor suppressive effect of TLA under the absence of T lymphocytes, athymic nude mice were compared with euthymic mice in the anti-tumorigenic effect triggered by TLA in CT26 tumors. According to the results, intratumorally injected TLA reduced tumor growth and TIMP-1 level, a metastatic marker, in both euthymic and athymic mice. TLA treatment led to a sharp increase in IL-12 expression in serum cytokine profiling of athymic mice, and increased MyD88 signals in macrophages derived from the bone marrow, implying the activation of innate immunity. The selective induction of IL-12 by TLA treatment had an anti-tumorigenic effect.
Animals ; Antigens, Protozoan/*immunology ; Disease Models, Animal ; Immunity, Innate ; Immunotherapy/*methods ; Interleukin-12/*blood ; Macrophages/immunology ; Mice, Inbred BALB C ; Mice, Nude ; Myeloid Differentiation Factor 88/analysis ; Neoplasms/pathology/*therapy ; Toxoplasma/*immunology ; Treatment Outcome

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