To control coccidiosis without using prophylactic medications, a DNA vaccine targeting the gametophyte antigen Gam56 from Eimeria maxima in chickens was constructed, and the immunogenicity and protective effects were evaluated. The ORF of Gam56 gene was cloned into an eukaryotic expression vector pcDNA3.1(zeo)+. Expression of Gam56 protein in COS-7 cells transfected with recombinant plasmid pcDNA-Gam56 was confirmed by indirect immunofluorescence assay. The DNA vaccine was injected intramuscularly to yellow feathered broilers of 1-week old at 3 dosages (25, 50, and 100 microg/chick). Injection was repeated once 1 week later. One week after the second injection, birds were challenged orally with 5x10(4) sporulated oocysts of E. maxima, then weighed and killed at day 8 post challenge. Blood samples were collected and examined for specific peripheral blood lymphocyte proliferation activity and serum antibody levels. Compared with control groups, the administration of pcDNA-Gam56 vaccine markedly increased the lymphocyte proliferation activity (P<0.05) at day 7 and 14 after the first immunization. The level of lymphocyte proliferation started to decrease on day 21 after the first immunization. A similar trend was seen in specific antibody levels. Among the 3 pcDNA-Gam56 immunized groups, the median dosage group displayed the highest lymphocyte proliferation and antibody levels (P<0.05). The median dosage group had the greatest relative body weight gain (89.7%), and the greatest oocyst shedding reduction (53.7%). These results indicate that median dosage of DNA vaccine had good immunogenicity and immune protection effects, and may be used in field applications for coccidiosis control.
Animals ; Antibodies, Protozoan/blood ; Antigens, Protozoan/genetics/*immunology ; Cell Proliferation ; Chickens ; Coccidiosis/immunology/pathology/*prevention & control ; Disease Models, Animal ; Eimeria/genetics/*immunology ; Injections, Intramuscular ; Lymphocytes/immunology ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Vaccination/methods ; Vaccines, DNA/administration & dosage/genetics/*immunology

OBJECTIVETo characterize the immune response induced by SAG1 encoding plasmid combined with IL-2 gene adjuvant in mice and to assess the protective effect of this vaccination against toxoplasmosis.

METHODSMice were co-injected intramuscularly with plasmid encoding Toxoplasma gondii SAG1 plus murine IL-2 expression vector at a dose of 100 microg. Booster immunizations were employed 2 more times at 3-week interval. As controls, mice were inoculated with PBS or empty plasmid pcDNA3. Humoral and cellular responses were assayed using ELISA for the determination of Ab, Ab isotype and IFN-gamma, as well as IL-4. To detect the integration and dissemination of DNA in the injected mice, PCR and in situ hybridization were performed. All mice were then infected with highly virulent RH tachyzoites of Toxoplasma gondii intraperitoneally.

RESULTSSignificant increases in specific IgG levels were observed in mice after immunization three times with SAG1 expression plasmid. With respect to the IgG isotype, co-inoculation of IL-2 expression plasmid enhanced the level of IgG2a and the production of IFN-gamma. Challenging mice by vaccinating with combined plasmids with RH tachyzoites resulted in prolonged survival.

CONCLUSIONHumoral and cytokine responses elicited by SAG1 DNA immunization can be modulated by co-inoculation with IL-2 expression plasmid. The use of DNA vaccine in combination with an appropriate cytokine gene to prevent T. gondii infection warrants further investigation.

Animals ; Antibodies, Protozoan ; blood ; Antigens, Protozoan ; Cytokines ; biosynthesis ; Female ; Immunization ; Immunoglobulin G ; blood ; classification ; Interleukin-2 ; genetics ; Mice ; Protozoan Proteins ; genetics ; Protozoan Vaccines ; immunology ; Toxoplasma ; immunology ; Toxoplasmosis, Animal ; prevention & control ; Vaccines, DNA ; immunology

Serologic tests are widely accepted for diagnosing Toxoplasma gondii but purification and standardization of antigen needs to be improved. Recently, surface tachyzoite and bradyzoite antigens have become more attractive for this purpose. In this study, diagnostic usefulness of 3 recombinant antigens (SAG1, SAG2, and SAG3) were evaluated, and their efficacy was compared with the available commercial ELISA. The recombinant plasmids were transformed to JM109 strain of Escherichia coli, and the recombinants were expressed and purified. Recombinant SAG1, SAG2, and SAG3 antigens were evaluated using different groups of sera in an ELISA system, and the results were compared to those of a commercial IgG and IgM ELISA kit. The sensitivity and specificity of recombinant surface antigens for detection of anti-Toxoplasma IgG in comparison with commercially available ELISA were as follows: SAG1 (93.6% and 92.9%), SAG2 (100.0% and 89.4%), and SAG3 (95.4% and 91.2%), respectively. A high degree of agreement (96.9%) was observed between recombinant SAG2 and commercial ELISA in terms of detecting IgG anti-Toxoplasma antibodies. P22 had the best performance in detecting anti-Toxoplasma IgM in comparison with the other 2 recombinant antigens. Recombinant SAG1, SAG2, and SAG3 could all be used for diagnosis of IgG-specific antibodies against T. gondii.
Antibodies, Protozoan/*blood ; Antigens, Protozoan/diagnostic use/*genetics ; Enzyme-Linked Immunosorbent Assay ; Humans ; Immunoglobulin G/blood ; Immunoglobulin M/blood ; Membrane Glycoproteins/*genetics ; Protozoan Proteins/*genetics ; Recombinant Proteins/diagnostic use/immunology ; Sensitivity and Specificity ; Toxoplasma/immunology ; Toxoplasmosis/blood/*diagnosis

A Cryptosporidium parvum sporozoite and oocyst lambda gt11 cDNA library was screened with a hyperimmune rabbit serum that was developed against insoluble fragments of ultrasonicated oocysts. A clone named Cp22.4.1 encoding a protein of 231 amino acids with 4 zinc-finger domains characterized by a Cys-X2-Cys-X4-His-X4-Cys motif was isolated and characterized. There was a complete match between the sequencing data of the coding region of Cp22.4.1 and the corresponding gene at chromosomal level. Cloning in a pBAD-TOPO-TA expression vector permitted to evaluate the antigenicity of the recombinant His-tagged antigen. This antigen was recognized by 2 out of 5 sera from Cryptosporidium immune calves and not by sera from parasite naive animals.
Amino Acid Sequence ; Animals ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/chemistry/*immunology ; Base Sequence ; Cattle/*immunology ; Cryptosporidium parvum/*immunology ; Molecular Sequence Data ; Protozoan Proteins/chemistry/genetics ; Rabbits ; Recombinant Proteins ; Zinc Fingers/genetics/*immunology

Recombinant antigenic proteins of Toxoplasma gondii are alternative source of antigens which are easily obtainable for serodiagnosis of toxoplasmosis. In this study, highly antigenic secretory organellar proteins, dense granular GRA2 and GRA3, rhoptrial ROP2, and micronemal MIC2, were analyzed by bioinformatics approach to express as water-soluble forms of antigenic domains. The transmembrane region and disorder tendency of 4 secretory proteins were predicted to clone the genes into pGEX-4T-1 vector. Recombinant plasmids were transformed into BL21 (DE3) pLysS E. coli, and GST fusion proteins were expressed with IPTG. As a result, GST fusion proteins with GRA225-105, GRA339-138, ROP2324-561, and MIC21-284 domains had respectively higher value of IgG avidity. The rGST-GRA225-105 and rGST-GRA339-138 were soluble, while rGST-ROP2324-561 and rGST-MIC21-284 were not. GRA231-71, intrinsically unstructured domain (IUD) of GRA2, was used as a linker to enhance the solubility. The rGST-GRA231-71-ROP2324-561, a chimeric protein, appeared to be soluble. Moreover, rGST-GRA231-71-MIC21-284 was also soluble and had higher IgG avidity comparing to rGST-MIC21-284. These 4 highly expressed and water-soluble recombinant antigenic proteins may be promising candidates to improve the serodiagnosis of toxoplasmosis in addition to the major surface antigen of SAG1.
Animals ; Antibodies, Protozoan/immunology ; Antibody Affinity ; Antigens, Protozoan/chemistry/*diagnostic use/genetics/immunology ; *Gene Expression ; Immunoglobulin G/blood/immunology ; Mice, Inbred BALB C ; Recombinant Proteins/chemistry/*diagnostic use/genetics/immunology ; Serologic Tests/methods ; Solubility ; Toxoplasma/genetics/immunology/*metabolism ; Toxoplasmosis/diagnosis

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

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

An ELISA was developed for the diagnosis of vivax malaria using multiple stage-specific recombinant antigens of Plasmodium vivax. The DNA from the whole blood of a malaria patient was used as template to amplify the coding regions for the antigenic domains of circumsporozoite protein (CSP-1), merozoite surface protein (MSP-1), apical merozoite antigen (AMA-1), serine repeat antigen (SERA), and exported antigen (EXP-1). Each amplified DNA fragment was inserted into pQE30 plasmid to induce the expression of His-tagged protein in Escherichia coli (M15 strain) by IPTG. His-tagged proteins were purified by Ni-NTA metal-affinity chromatography and used as antigens for ELISA with patient sera that were confirmed previously by blood smear examinations. When applied to patient sera, 122 (80.3%) out of 152 vivax malaria cases reacted to at least one antigen, while no reactions were observed with 128 uninfected serum samples. We applied this ELISA to the screening of 3, 262 civilian residents in endemic regions near the DMZ, which resulted in 236 positively detected (7.2%) cases. This method can be applied to serological diagnosis and mass screening in endemic regions, or can be used as a safety test for transfusion blood in endemic areas.
Animals ; Antibodies, Protozoan/*blood ; Antigens, Protozoan/genetics/*immunology ; *Endemic Diseases ; Enzyme-Linked Immunosorbent Assay/methods ; Humans ; Life Cycle Stages ; Malaria, Vivax/*diagnosis/epidemiology/parasitology ; Mass Screening ; Plasmodium vivax/*growth & development/immunology ; Recombinant Proteins/*immunology ; Serologic Tests

Among the panel of monoclonal antibodies (mAb) against Toxoplasma gondii, mAb of Tg621 (Tg621) clone blotted 38 kDa protein which localized in the cytoplasm of tachyzoites by immunofluorescence microscopy. The protein was not released into the parasitophorous vacuole during or after invasion. The cDNA fragment encoding the protein was obtained by screening a T. gondii cDNA expression library with Tg621. The full length cDNA sequence was completed with 5'-RACE as 1, 592 bp, which contained open reading frame of 942 bp. The deduced amino acid sequence of Tg621 consisted of a polypeptide of 313 amino acids, with significant homology to ribosomal P proteins (RPP) of other organisms especially high to those of apicomplexan species. The expressed and purified TgRPP was assayed in western blot with the sera of toxoplasmosis patients and normal sera, which resulted in the 74.0% of positive reactions in toxoplasmosis patients whereas 8.3% in normal group. Therefore, the antibody formation against TgRPP in toxoplasmosis patients was regarded as specific for T. gondii infection and suggested a potential autoantibody.
Amino Acid Sequence ; Animals ; Antibodies, Monoclonal/immunology ; Antigens, Protozoan/immunology ; Base Sequence ; Cloning, Molecular ; DNA, Protozoan/chemistry/genetics ; Human ; Molecular Sequence Data ; Protozoan Proteins/*genetics/immunology ; Recombinant Proteins/immunology ; Ribosomal Proteins/*genetics/immunology ; Sequence Alignment ; Support, Non-U.S. Gov't ; Toxoplasma/*genetics ; Toxoplasmosis/blood/*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