A novel recombinant Bacille Calmette-Guerin (rBCG) vaccine co-expressed Eimeria tenella rhomboid and cytokine chicken IL-2 (chIL-2) was constructed, and its efficacy against E. tenella challenge was observed. The rhomboid gene of E. tenella and chIL-2 gene were subcloned into integrative expression vector pMV361, producing vaccines rBCG pMV361-rho and pMV361-rho-IL2. Animal experiment via intranasal and subcutaneous route in chickens was carried out to evaluate the immune efficacy of the vaccines. The results indicated that these rBCG vaccines could obviously alleviate cacal lesions and oocyst output. Intranasal immunization with pMV361-rho and pMV361-rho-IL2 elicited better protective immunity against E. tenella than subcutaneous immunization. Splenocytes from chickens immunized with either rBCG pMV361-rho and pMV361-rho-IL2 had increased CD4+ and CD8+ cell production. Our data indicate recombinant BCG is able to impart partial protection against E. tenella challenge and co-expression of cytokine with antigen was an effective strategy to improve vaccine immunity.
Adjuvants, Immunologic/genetics/*metabolism ; Administration, Intranasal ; Animals ; Antigens, Protozoan/genetics/*immunology ; BCG Vaccine/administration & dosage/*genetics ; CD4-Positive T-Lymphocytes/immunology ; CD8-Positive T-Lymphocytes/immunology ; Chickens ; Coccidiosis/*prevention & control ; Disease Models, Animal ; Drug Carriers/administration & dosage ; Eimeria tenella/genetics/*immunology ; Genetic Vectors ; Injections, Subcutaneous ; Interleukin-2/genetics/*metabolism ; Protozoan Vaccines/administration & dosage/genetics/*immunology ; Spleen/immunology ; Vaccines, Synthetic/administration & dosage/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

The vir genes are antigenic genes and are considered to be possible vaccine targets. Since India is highly endemic to Plasmodium vivax, we sequenced 5 different vir genes and investigated DNA sequence variations in 93 single-clonal P. vivax isolates. High variability was observed in all the 5 vir genes; the vir 1/9 gene was highly diverged across Indian populations. The patterns of genetic diversity do not follow geographical locations, as geographically distant populations were found to be genetically similar. The results in general present complex genetic diversity patterns in India, requiring further in-depth population genetic and functional studies.
Antigens, Protozoan/*genetics ; Humans ; India/epidemiology ; Malaria, Vivax/epidemiology/parasitology ; Phylogeny ; Plasmodium vivax/*genetics ; *Polymorphism, Genetic ; Protozoan Proteins/genetics/*metabolism

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

The dense granule of Toxoplasma gondii is a secretory vesicular organelle of which the proteins participate in the modification of the parasitophorous vacuole (PV) and PV membrane for the maintenance of intracellular parasitism in almost all nucleated host cells. In this review, the archives on the research of GRA proteins are reviewed on the foci of finding GRA proteins, characterizing molecular aspects, usefulness in diagnostic antigen, and vaccine trials in addition to some functions in host-parasite interactions.
Animals ; Antigens, Protozoan/*metabolism ; Cytoplasmic Granules/metabolism ; *Host-Parasite Interactions ; Humans ; Protozoan Proteins/*metabolism ; Toxoplasma/*metabolism ; Toxoplasmosis/*parasitology ; Vacuoles/*metabolism

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

OBJECTIVETo identify the binding site on glycophorin A (GPA) for EBA-175 to provide clue for developing short peptide vaccine and therapeutic agents against Plasmodium falciparum.

METHODSWith the recombinant protein of EBA-175 as the target molecule, the mimetic peptides of GPA were screened from a 12-mer random peptide library. Three rounds of biopanning were carried out, and enzyme-linked immunosorbent assay (ELISA), competitive ELISA, Dot-ELISA and Western blotting used to evaluate the binding between the phage-borne peptides and EBA-175. The insert DNA sequences of positive clones were determined and their amino acid sequences deduced.

RESULTSThirty clones from the third round were randomly selected, of which 27 were found positive by sandwich ELISA. Competitive ELISA proved that most of the phage-borne peptides could competitively inhibit the binding of antibody (EBA-175 Ab) with EBA-175. Analysis of DNA and amino acid sequences indicated that 24 positive phage clones contained the conservative sequence of IRR, which was highly homologous with the 114-116 amino acids of GPA.

CONCLUSIONThese phage-displayed peptides can bind with EBA-175, and the amino acid sequence IRR might play an important role in the binding between EBA-175 and GPA.

Antigens, Protozoan ; metabolism ; Binding Sites ; Enzyme-Linked Immunosorbent Assay ; Glycophorin ; chemistry ; Humans ; Peptide Library ; Plasmodium falciparum ; Protozoan Proteins ; metabolism ; Sequence Analysis, DNA ; Sequence Analysis, Protein

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.
Animals ; Antigens, Protozoan/genetics/*immunology/metabolism ; Bacterial Proteins/genetics/immunology/metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Immunoglobulin G/immunology ; Interferon-gamma/metabolism ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/metabolism ; Protozoan Proteins/genetics/immunology/metabolism ; Recombinant Fusion Proteins/genetics/immunology/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Saponins/*immunology ; Toxoplasma/growth & development/*immunology ; Toxoplasmosis, Animal/*immunology/metabolism/microbiology ; Vaccination/*methods

PURPOSE: Surface antigen 3 (SAG3) of Toxoplasma gondii is very similar in structure to the major surface antigen 1 (SAG1). Although numerous studies have supported the importance of SAG1 in protection against T. gondii infection, few reports exist on SAG3. MATERIALS AND METHODS: Glutathione-S-transferase (GST)-fused SAG3 of T. gondii (rSAG3) were immunized into BALB/c mice alone or in combination with Quil A (rSAG3/Quil A), and then evaluated the protective immunity in vivo and in vitro against murine toxoplasmosis. RESULTS: Immunization with rSAG3 or rSAG3/Quil A resulted in significantly more survival days and fewer brain cysts after challenge with T. gondii compared to an infected control group. Mice immunized with rSAG3 alone or in combination with Quil A produced significantly more specific IgG2a antibody, whereas specific IgG1 antibody titers did not increase. The percentage of CD8+ T cells, IFN-gamma mRNA expression, and nitric oxide production significantly increased in rSAG3- and rSAG3/Quil A-immunized mice. CONCLUSION: These results indicate that vaccination with Toxoplasma rSAG3 results in partial protective immunity against T. gondii infection through induction of a Th1-type immune response, and that protective immunity is accelerated by the modulating effects of Quil A.
Animals ; Antigens, Protozoan/genetics/*immunology/metabolism ; Bacterial Proteins/genetics/immunology/metabolism ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Female ; Flow Cytometry ; Immunoglobulin G/immunology ; Interferon-gamma/metabolism ; Mice ; Mice, Inbred BALB C ; Nitric Oxide/metabolism ; Protozoan Proteins/genetics/immunology/metabolism ; Recombinant Fusion Proteins/genetics/immunology/metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Saponins/*immunology ; Toxoplasma/growth & development/*immunology ; Toxoplasmosis, Animal/*immunology/metabolism/microbiology ; Vaccination/*methods

Two-dimensional electrophoresis (2-DE) was employed to compare the proteome of Diclazuril-resistance Eimeria tenella with that of sensitive strains for identifying unique proteins of these stains. 5 protein spots were found to express differentially. Four spots which remarkably were measured by matrix assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS). The data obtained from peptide mass fingerprinting were used in NCBInr database search, two protein spots in gel were identified as Eimeria tenella sporulated oocyst TA4 antigen protein, Heat shock 70kD protein, two protein spots were functional proteins of Eukaryote. These proteins are potentially basic work for finding molecular mechanism about drug-resistance of Eimeria tenella and new marker in the detection of resistance of Eimeria tenella.
Animals ; Antigens, Protozoan ; analysis ; genetics ; Chickens ; Coccidiostats ; pharmacology ; Drug Resistance ; genetics ; Eimeria tenella ; drug effects ; genetics ; metabolism ; Electrophoresis, Gel, Two-Dimensional ; HSP70 Heat-Shock Proteins ; analysis ; genetics ; Nitriles ; pharmacology ; Oocysts ; metabolism ; Proteome ; analysis ; genetics ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; Triazines ; pharmacology