Field rodents involved in ecological food chains and which are the prey of carnivores in the natural environment may serve as reservoir hosts for Toxoplasma gondii infection in humans, however, no data has been published to date in Korea. A total of 1,008 Apodemus agrarius, a dominant species of field rodents in Korea, were trapped at various locations around the country, and their serum antibody (IgG) levels to T. gondii were examined by ELISA. The mean absorbance was 0.11, and fifteen samples (1.49%) showed positive titers from 0.18 to 0.59. The seropositive samples were analyzed by immunoblot. Five of them showed reactive bands to T. gondii water soluble antigens of 30, 35, and 43 kDa. This immunoblot analysis showed very similar patterns to that obtained using sera of experimentally infected mice with T. gondii. The present study presents indirect evidence of the existence of T. gondii in field rodents in Korea.
Animal ; Antibodies, Protozoan/blood* ; Antigens, Protozoan/immunology ; Enzyme-Linked Immunosorbent Assay ; Immunoblotting ; Molecular Weight ; Muridae/parasitology* ; Seroepidemiologic Studies ; Toxoplasma/isolation & purification* ; Toxoplasma/immunology

OBJECTIVETo develop a technology for production of recombinant SAG1 of Toxoplasma gondii (T.g) in batches.

METHODSThe rSAG1 of T.g was expressed in E.coli by high-density fermentation and purified by Sephadex G-75 column chromatography after Ni-NTA agarose at native condition. The activity of rSAG1 and its efficacy in T.g diagnosis were identified by Western blotting and ELISA, respectively.

RESULTSThe optical density (OD) of the bacteria reached 20.21 after induction, and 300 g bacteria were harvested from 11.5 L broth. The rSAG1 was highly expressed in E.coli as a fusion protein, accounting for about 25.82% of the total bacterial protein. The purity of rSAG1 reached 98.54% after purification by Ni-NTA combined with Sephadex G-75 column chromatography. Western blotting revealed a distinct band reacting with the sera of rabbits vaccinated by T.g. Twenty-four of the 25 sera of mice infected with T.g and 36 of the 38 sera of human subjects with IgG antibody against T.g were detected by rSAG1-ELISA.

CONCLUSIONA large-scale production of immunoreactive SAG1 of T.g is developed by high-density fermentation and purification with Ni-NTA combined with Sephadex G-75 column chromatography.

Animals ; Antigens, Protozoan ; biosynthesis ; genetics ; immunology ; Antigens, Surface ; immunology ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; metabolism ; Fermentation ; Protozoan Proteins ; biosynthesis ; genetics ; immunology ; Recombinant Fusion Proteins ; biosynthesis ; immunology ; isolation & purification ; Toxoplasma ; immunology

Experimental murine models with high, intermediate and low levels of genetically based susceptibility to Leishmania major infection reproduce almost entire spectrum of clinical manifestations of the human disease. There are increasing non-comparative studies on immune responses against isolated antigens of L. major in different murine strains. The aim of the present study was to find out whether there is an antigen that can induce protective immune response in resistant and susceptible murine strains. To do that, crude antigenic extract of procyclic and metacyclic promastigotes of L. major was prepared and subjected to SDS-PAGE electrophoresis. Western-blotting was used to search for antigen(s) capable of raising high antibody level of IgG2a versus IgG1 in the sera of both infected resistant and susceptible strains. Two novel antigens from metacyclic promastigotes of L. major (140 and 152 kDa) were potentially able to induce specific dominant IgG2a responses in BALB/c and C57BL/6 mice. The 2 antigens also reacted with IgG antibody of cutaneous leishmaniasis patients. We confirm that 140 and 152 kDa proteins of L. major promastigotes are inducing IgG production in mice and humans.
Protozoan Proteins/immunology/*isolation & purification ; Mice, Inbred C57BL ; Mice, Inbred BALB C ; Mice ; Life Cycle Stages/immunology ; Leishmaniasis, Cutaneous/immunology ; Leishmania major/*immunology ; Immunoglobulin G/*biosynthesis/blood ; Humans ; Female ; Blotting, Western/methods ; Antigens, Protozoan/immunology/*isolation & purification ; Animals

Babesia gibsoni is an intraerythrocytic apicomplexan parasite that causes piroplasmosis in dogs. B. gibsoni infection is characterized clinically by fever, regenerative anemia, splenomegaly, and sometimes death. Since no vaccine is available, rapid and accurate diagnosis and prompt treatment of infected animals are required to control this disease. Over the past decade, several candidate molecules have been identified using biomolecular techniques in the authors' laboratory for the development of a serodiagnostic method, vaccine, and drug for B. gibsoni. This review article describes newly identified candidate molecules and their applications for diagnosis, vaccine production, and drug development of B. gibsoni.
Animals ; Antigens, Protozoan/*diagnostic use/*immunology ; Antiprotozoal Agents/*isolation & purification/pharmacology ; Babesia/*drug effects/immunology/*isolation & purification ; Babesiosis/*diagnosis/drug therapy/prevention & control ; Dogs ; Drug Discovery/methods ; Protozoan Vaccines/*immunology

The protective effect of the Synadenium carinatum latex lectin (ScLL), and the possibility of using it as an adjuvant in murine model of vaccination against American cutaneous leishmaniasis, were evaluated. BALB/c mice were immunized with the lectin ScLL (10, 50, 100 microgram/animal) separately or in association with the soluble Leishmania amazonensis antigen (SLA). After a challenge infection with 10(6) promastigotes, the injury progression was monitored weekly by measuring the footpad swelling for 10 weeks. ScLL appeared to be capable of conferring partial protection to the animals, being most evident when ScLL was used in concentrations of 50 and 100 microgram/animal. Also the parasite load in the interior of macrophages showed significant reduction (61.7%) when compared to the control group. With regard to the cellular response, ScLL 50 and 100 microgram/animal stimulated the delayed-type hypersensitivity (DTH) reaction significantly (P < 0.05) higher than SLA or SLA plus ScLL 10 weeks after the challenge infection. The detection of high levels of IgG2a and the expression of mRNA cytokines, such as IFN-gamma, IL-12, and TNF-alpha (Th1 profiles), corroborated the protective role of this lectin against cutaneous leishmaniasis. This is the first report of the ScLL effect on leishmaniasis and shows a promising role for ScLL to be explored in other experimental models for treatment of leishmaniasis.
*Adjuvants, Immunologic ; Animals ; Antibodies, Protozoan/immunology ; Antibody Formation ; Antigens, Protozoan/immunology ; Cytokines/genetics/immunology ; Euphorbiaceae/*chemistry ; Hypersensitivity, Delayed/immunology ; Immunization ; Immunoglobulin G/immunology ; Latex/chemistry ; Leishmania/immunology ; Leishmaniasis, Cutaneous/*immunology/pathology ; Mice ; Mice, Inbred BALB C ; Nitric Oxide Synthase Type II/genetics/immunology ; Plant Lectins/*immunology/isolation & purification ; Protozoan Vaccines/immunology/pharmacology ; Skin/pathology

The identification and characterization of antigens that elicit human T cell responses is an important step toward understanding of Leishmania major infection and ultimately in the development of a vaccine. Micropreparative SDS-PAGE followed by electrotransfer to a PVDF membrane and elution of proteins from the PVDF, was used to separate 2 novel proteins from L. major promastigotes, which can induce antibodies of the IgG2a isotype in mice and also are recognized by antisera of recovered human cutaneous leishmaniasis subjects. Fractionation of the crude extract of L. major revealed that all detectable proteins of interest were present within the soluble Leishmania antigens (SLA). Quantitation of these proteins showed that their expression in promastigotes is relatively very low. Considering the molecular weight, immunoreactivity, chromatographic and electrophoretic behavior in reducing and non-reducing conditions, these proteins are probably 2 isoforms of a single protein. A digest of these proteins was resolved on Tricine-SDS-PAGE and immunoreactive fragments were identified by human sera. Two immunoreactive fragments (36.4 and 34.8 kDa) were only generated by endoproteinase Glu-C treatment. These immunoreactive fragments or their parent molecules may be ideal candidates for incorporation in a cocktail vaccine against cutaneous leishmaniasis.
Animals ; Antigens, Protozoan/*chemistry/*isolation & purification ; Blotting, Western ; Chromatography, High Pressure Liquid ; Electrophoresis, Polyacrylamide Gel/methods ; Humans ; Leishmania major/growth & development/*immunology ; Protein Isoforms/chemistry/isolation & purification

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

In Iran, Plasmodium vivax is responsible for more than 80% of the infected cases of malaria per year. Control interventions for vivax malaria in humans rely mainly on developed diagnostic methods. Recombinant P. vivax apical membrane antigen-1 (rPvAMA-1) has been reported to achieve designing rapid, sensitive, and specific molecular diagnosis. This study aimed to perform isolation and expression of a rPvAMA-1, derived from Iranian patients residing in an endemic area. Then, the diagnostic efficiency of the characterized Iranian PvAMA-1 was assessed using an indirect ELISA method. For this purpose, a partial region of AMA-1 gene was amplified, cloned, and expressed in pET32a plasmid. The recombinant His-tagged protein was purified and used to coat the ELISA plate. Antibody detection was assessed by indirect ELISA using rPvAMA-1. The validity of the ELISA method for detection of anti-P. vivax antibodies in the field was compared to light microscopy on 84 confirmed P. vivax patients and compared to 84 non-P. vivax infected individuals. The ELISA cut-off value was calculated as the mean+2SD of OD values of the people living in malaria endemic areas from a south part of Iran. We found a cut-off point of OD=0.311 that showed the best correlation between the sera confirmed with P. vivax infection and healthy control sera. A sensitivity of 81.0% and specificity of 84.5% were found at this cut off titer. A good degree of statistical agreement was found between ELISA using rPvAMA-1 and light microscopy (0.827) by Kappa analysis.
Antibodies, Protozoan/blood/immunology ; Antigens, Protozoan/*blood/genetics/immunology ; Diagnostic Tests, Routine/*methods ; Enzyme-Linked Immunosorbent Assay/*methods ; Female ; Humans ; Iran ; Malaria, Vivax/blood/*diagnosis/immunology/*parasitology ; Male ; Membrane Proteins/blood/genetics/immunology ; Plasmodium vivax/isolation & purification/*physiology ; Protozoan Proteins/blood/genetics/immunology ; Sensitivity and Specificity

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

The seroepidemiological status of toxoplasmosis was surveyed among the residents of Cheorwon-gun, Gangwon-do by means of ELISA using a crude extract antigen of Toxoplasma gondii. The sera of 1,661 adult residents (866 males and 795 females) were collected and checked for IgG antibody titers, which showed 17.0% positive rate (282 sera). The positive rate was significantly different between the sex; 20.6% for males and 13.1% for females (P<0.05). The positive rates were higher in fifties of males (28.7%) and forties of females (20.0%). This positive rate of toxoplasmosis in Cheorwon-gun residents is regarded as the highest among the surveys of different geographical regions of Korea. This high positive rate may due in part to peculiar geographical locality of the surveyed area near the naturally well preserved demilitarized zone (DMZ) or presumably consumption of the pork imported from high endemic nations. Therefore, it is necessary to study further the epidemiology of toxoplasmosis in Cheorwon-gun.
Adult ; Age Factors ; Aged ; Aged, 80 and over ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/isolation & purification ; Enzyme-Linked Immunosorbent Assay ; Female ; Humans ; Immunoglobulin G/blood ; Male ; Middle Aged ; Republic of Korea/epidemiology ; Seroepidemiologic Studies ; Sex Factors ; Toxoplasma/*immunology ; Toxoplasmosis/*epidemiology