Lack gene's immune response induced by cocktail dna vaccine with il-12 gene against cutaneous leishmaniasis in balb/c mice

Background: Leishmaniasis is caused by parasitic protozoa of the genus Leishmania which is an obligate intracellular parasite in the infected host. Individuals who have been recovered from clinical leishmaniasis develop strong immunity against reinfection. DNA vaccines are the new type of vaccines that induce expression of protein eukaryotic cells. DNA vaccines can be stimulated by the cellular and humoral immune responses using one or several genes

Methods: A DNA vaccine containing plasmids encoding the pcLACK+pcTSA genes of Leishmania major [L. major] [MHRO/IR/75/ER] in the vicinity of IL-12 gene expression was made and then its protective efficacy in comparison with single-gene of LACK was evaluated. Also, BALB/c mice were immunized intramuscularly three times. The humoral and cellular immune responses were evaluated after immunization with pcLACK, pcLACK+pcTSA+pCAGGS-IL12, and then challenged with L. major

Results: Humoral response and IFN-Gamma values were significantly higher than control groups after immunization with pcLACK, pcLACK+pcTSA+pCAGGS-IL12 and challenge with L. major [p

Conclusion: The survival time of the immunized mice with pcLACK, pcLACK+pcTSA+ pCAGGS-IL12 groups was higher than the control groups. Then, DNA vaccine of pcLACK appeared to be likely able to induce more protection against infection with L. major in mice. Therefore, cocktail DNA is effective to enhance specific immunity

Identification of sarcocystis species of slaughtered sheep in Gazvin Ziaran slaughterhouse using PCR-RFLP

Sarcocystosis is caused by species of Sarcocystis, an intracellular protozoan parasite in the phylum Apicomplexa. The intermediate hosts are the herbivores eating infected food or water containing sporocysts excreted by the carnivorous final host, and that results in tissue cysts in corpses. The present study was carried out to identify Sarcosystis species of sheep using PCR-RFLP. In the present study 60 specimens from the diaphragm, heart and esophagus muscles of sheep slaughtered in Qazvin Ziaran slaughterhouse were collected. 40 specimens contained macroscopic Sarcocystis cysts and the next 20 contained microscopic Sarcocystis cysts. They were examined by Dob Smear and digestion method. DNA extraction was carried out by a kit. PCR conditions were optimized for 18S rRNA amplification. Meanwhile, a comparative study was done to investigate the specific primers of the Toxoplasma and Neospora along with the Sarcocystis cases. According to the position of restriction sites, restricted enzymes were selected. Results indicated that the primers were completely unique and specific for detecting Sarcocystis. PCR-RFLP analysis showed that macroscopic cysts and microscopic cysts belonged to Sarcocystis gigantea and Sarcocystis arieticanis, respectively. Sarcocystis species of sheep can be recognized through PCR-RFLP technique using the designed specific primers. By this technique applying TaqI enzyme from microscopic cysts as well as TaqI and HincII enzymes for macroscopic cysts were found more efficient than the others

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

Protective role of antigens from peritoneal exudates of infected mice against toxoplasmosis

Toxoplasma gondii is an obligate intracellular parasite that infects all mammalian cells. Several antigens such as excreted/secreted antigens have been identified as a potential vaccine candidate. To determine how excreted/ secreted antigens from peritoneal exudates of infected mice [mESA] stimulate cell-mediated immune responses and induce protective immunity against toxoplasmosis in the murine model. The supernatants produced from the peritoneal fluids, were fractionated by precipitation in ammonium sulphate solution [30-80% saturated]. For induction of cell-mediated immune responses, delayed type hypersensitivity was measured, in injected footpad. Response to purified antigen was measured by lymphocyte proliferation assay. Nitric oxide was measured by Griess method. For immunization, Balb/c mice were immunized 2 times with mESA, mESA-40% and Toxoplasma Lysate Antigen [TLA]. The virulent RH strain of Toxoplasma gondii was used for challenging. The pattern of lymphocyte responsiveness was dependent on the antigen employed. In sensitized mice, those received mESA-40% displayed higher lymphocyte response than mice stimulated by mESA [p<0.05]. The highest amounts of nitric oxide were observed in macrophages, which received mESA-40% and mESA [p<0.05]. Mice immunized with mESA-40% survived longer than those immunized with mESA and other antigens [p<0.05]. As fraction 40% [mESA-40%] showed a good result in induction of cellmediated responses in the murine model, the purification and isolation of the mESA 40% is highly recommended for future study