Evaluation of immune responses induced by GRA7 and ROP2 genes by DNA vaccine cocktails against acute toxoplasmosis in BALB/c mice

Background: The severe damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with plasmid DNA is a promising vaccination technique. Therefore, GRA7 plasmid was prepared to be used as a vaccine. The purpose of this study was evaluation of immunization with cocktail DNA vaccine including plasmids encoding Toxoplasma gondii ROP2 and GRA7 in BALB/c mice

Methods: In this study, 733 bp of GRA7 gene was cloned in pCDNA3.1 plasmid as an expression vector. The plasmids containing GRA7 and ROP2 genes were administered via IM according to immunized mice three times with a 3 week interval. For lymphocyte proliferation and cytokine assay, splenocytes of immunized mice were cultured for proliferation and cytokine assay. The other mice in each group were inoculated by the parasite and mortality of the mice was evaluated on a daily basis

Results: The cytokine assay results and lymphocyte proliferation response in cocktail DNA vaccines showed that IFN-gamma levels were significantly higher than controls [p<0.05], whereas IL-4 expression level in BALB/c mice immunized with cocktail was lower than that in control groups and these results are confirmed by MTT test. Predominance of the levels of IgG2a over IgG1 was observed in sera of the immunized mice. Furthermore, IgG2a values in cocktail DNA vaccines pcGRA7 were significantly higher than control group [p<0.01]. In contrast, IgG1 antibodies were similar between the two groups [p>0.5]. So, survival time in the immune groups was significantly prolonged in comparison to control ones [p<0.01]

Conclusion: The immunized mice by DNA vaccine produce higher titration of IFNgamma, indicated with Th1 response which is confirmed by high level of IgG2a. These data demonstrate that the cocktail GRA7/ROP2 is a potential vaccine candidate against toxoplasmosis

[Evaluation of immunization of BALB/c mice by plasmid encoding complete ROP2 gene of Toxoplasma gondii]

Toxoplasma gondii is an obligate intracellular protozoan that causes Toxoplasmosis in human and animal. In recent years, significant progress has been made in the identification of vaccine candidates which can induce protective responses. In this study we used complete Rhoptry protein 2 gene of Toxoplasma gondii as a single DNA vaccine and evaluated its immune responses in comparison with control groups. BALB/c mice were immunized intramuscularly with three weaks time interval with pcROP2 [as case group] and pc-DNA3 and PBS [as control groups]. After immunization, we evaluated the immune response using cytokine and antibody measurements. The results of cytokine [IFN-gamma, IL-4] assays showed that mice immunized with pcROP2, elicited stronger Th1-type cellular immune responses than those immunized with empty plasmid, or PBS [high level of IFN-gamma and low-level of IL-4]. Also Anti-T. gondii IgG titres [OD] increased markedly in the pcROP2 group, which was significantly higher than those of control groups [P<0.05]. When challenged with the highly virulent Toxoplasma gondii RH strain, mice immunized with pcROP2 had siginificantly higher survival rates compared to control groups [P<0.05]. This study showed that pc-ROP2 as a single DNA vaccine is effective to prime enhanced and balanced cellular and humeral immunity responses, and relatively improved mice survival time against toxoplasmosis

Application of PCR-ELISA method based on RE domain for diagnosis of toxoplasmosis in experimentally infected rat [Rattus norvegicus]

Toxoplasmosis may cause significant damage to the developing fetus and is as life-threatening opportunistic infection in immunocompromised persons. Molecular methods are known to be more sensitive and more specific than serological assays for diagnosis of toxoplasmosis. Application of quantitative PCR has evolved sensitive, specific, and rapid method for the detection of RH strain of Toxoplasma gondii DNA. In the present study, quantitative PCR-ELISA [Polymerase Chain Reaction- enzyme linked immunosorbent assay] was used for quantization of Taxoplasma gondii in the blood of 15 rats [Rattus norvegicus] infected experimentally with the parasite. In this regard Polymerase PCR - ELISA was developed for rapid detection of Toxoplasma gondii. DIG-labeled [digoxigenin-labeled] amplicons were hybridized with a specific biotinylated oligonucleotide probe in solution phase and subsequently transferred to streptavidin coated plates. The captured DNA-DNA hybrids were colorimetrically detected by the addition of anti-digoxigenin antibody peroxidase conjugate and substrate. DNA of Toxoplasma gondii were efficiently detected within 4 hours and no interference was encountered in the amplification and detection of the parasite. Efficiency of PCR-ELISA system was evaluated we found with several advantages in terms of sensitivity, rapidity and simplicity in this system

[Cloning and sequencing of L. major leishmania homologue of receptors for activated C kinase [LACK] gene]

Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which in the infected host is obligate intracellular parasite. LACK gene is conserved among related Leishmania species. LACK is the immuno-dominant antigen of L.major which is considered as the most promising molecule for a recombinant or DNA vaccine against leishmaniasis. In this study the genomic DNA of an Iranian standard strain of Leishmania major [MRHO/IR/75/ER] was ertracted and the LACK gene was amplilified by PCR. Then the PCR product was cloned into pTZ57R/T cloning vector, The PT-LACK recombinant plasmid was extracted from transformed E.coli bacteria [TG1 strain] and sequenced. The LACK gene [Accession no LmjF28.2740] of MRHO/IR/75/ER and L. major was amplified using PCR method. LACK gene was cloned into pTZ57R/T coloning vector. Sequence analysis of the cloned LACK gene showed high homology 89% with LmjF28.2740 [LACK gene]. The LACK gene of L.major was cloned in pTZ57R/T vector successfully. Recombinant plasmid was confirmed and could be used for the production of recomiomort antigens is DNA vaccines, for further studies

[ study on single nucleotide polymorphisms in Plasmodium fakiparum chloroquine resistance during two years in Chabahar]

Plasmodium falciparum chloroquine resistance is a major problem in malaria endemic areas. Single nucleotide polymorphisms in pfcrt and pfmdr1 genes are known to be associated with chloroquine resistance in some parts of the world. The major goal of the present study was to detect the five single nucleotide polymorphisms in pfmdr1 gene and one single nucleotide polymorphisms in pfcrt gene. Total of 26 blood samples were collected from falciparum malaria infectious person with chloroquine failure in Chabahar, a harbor located in Sistan baluchestan during 2 years. Detection of single nucleotide polymorphisms were carried out by Real-Time PCR using Light CyclerTM hybridization probe assay. Our data showed that the pfmdr1 N86Y mutation was detected in 6[23%] samples. Although this mutation was not observed in the first year but in the second year it was substancial. In addition the pfcrt K76T mutation was detected in 11 samples [42.3%] of CVMNT haplotype, 7 samples [26.9%] of CVIET haplotype, 5 samples [19.2%] of SVMNT haplotype and 2 samples [7.6%] of SVIET haplotype. The mutations considerably have increased during 2 years. Our results showed single nucleotide polymorphisms in pfmdr1 and pfcrt genes. This could be considered as chloroquine resistance markers for malaria control in Chabahar

[Cloning and sequencing of granular antigen 7 [GRA7] in TOPO vector and transformation in TOP10]

Toxoplasmosis, caused by an intracellular protozoan parasite, and the Toxoplasma gondii, is widespread throughout the world. In recent years, significant progress has been made in the identification of vaccine candidates which could induce a protective response. GRA7, an excretory 29 kDa Toxoplasma gondii a dense granular antigen released by infected host cells. In tachyzoite-infected cells, p29 accumulates within the parasitophorous vacuole and co-localizes with its delimiting membrane. In the present work, first genomic DNA of Toxoplasma gondii was extracted and used for amplifying of GRA7 gene as a template. Then PCR product was extracted from agarose gel and cloned into TOPO vector. The plasmid containing GRA7 gene was extracted from the transformed bacteria [TOP10 strain] and sequenced. Sequence analysis of GRA7 gene cloned into TOPO vector showed only one base difference when composed with the gene bank sequence for RH strain was only one base. The results indicated that this clone is suitable for subcloning in Prokaryotic and Eukaryotic plasmid

Seroepidemiological Survey of Hydatid cyst by ELISA in Kordestan Province

This study was conducted to evaluate the frequency of human hydatidosis in Kurdestan province by ELISA technique. In this study the sera of 1979 individuals were collected from different area [cities and villages] of Kordestan province. The serum dilution of 1/400 was selected for ELISA test. The results indicated that 1.12% of the individuals from Kordestan province showed positive sera. The results also showed that in Kordestan 0.9% and 1.42% of the people who live in the cities and villages had positive sera respectively. In this study 1.65% of female and 0.45% of male were positive. From the obtained result we found maximum number of infected people were in the range of 30-40 years [1.59%]. According to the results obtained from this study the highest percent of infection was found in the city of Ivandarre, the reason for this difference [1.69%] is due to the fact that most of the people who are involved in animal husbendary in the province live in this city