Effect of interleukin-22 on immunogenicity of DNA vaccine encoding TSA gene of leishmania major in BALB/c mice

Background and purpose: Previous Research shows the use of plasmids containing genes TSA to be useful as vaccines for Leishmania major. Recently, the role of interleukin-22 (IL-22) in tissue repair has been demonstrated. In this research, the effect of IL-22 on encoding TSA gene of Leishmania major in BALB/c mice was assessed

Expression of complete rhoptry protein 2 (ROP2) gene of Toxoplasma gondii in eukaryotic cell

Toxoplasma gondii is the intracellular protozoan parasite responsible for animal and human toxoplasmosis. In immunodeficient patients, chronic infection with T. gondii can reactivate and produceencephalitis, which is often lethal. ROP2 (rhoptry protein of T. gondii) is one of the most important interferer in organelle and PVM blending. ROP2 protein is recognized by clone T-cell (Tcc32) in humanbody and also has epitope for B-cell. All of these characteristics of ROP2 makes it a candidate for cocktail vaccine and recombinant vaccine against toxoplasmosis. We described the expression of thegene which encodes the complete rhoptry protein 2 (ROP2) of T. gondii in CHO cells and confirmed it by SDS-PAGE and Western blot analysis. In the present work, genomic DNA of T. gondii was extractedand used for amplifying of ROP2 gene as a template. Then PCR product was cloned into pTZ57R/T vector, and plasmid containing ROP2 gene (pT-ROP2) was extracted from transformed bacteria andsequenced. We hope to use from this recombinant plasmid (pT-ROP2) to make DNA vaccine against toxoplasmosis

A Molecular Study on Hepatozoon canis Infection in Dogs in Tehran (Iran)

Hepatozoonosis is a protozoal disease caused by various species of Hepatozoon. This parasite is transmitted from tick; the main vector of Hepatozoon canis is usually the brown dog tick (Rhipicephalus sanguineus). However, several species of ticks are disposed as the alternative vectors. Dogs are usually infected by eating the tick or a part of the tick organ infected by the mature oocysts containing infectious sporozoite. In the current study, a total of 145 blood samples were collected from the cephalic vein of pet, stray, and shelter dogs in Tehran. To conduct this study, first thin blood smears were prepared from all the samples and stained with the Giemsa method. Then, after extraction of DNA from the blood samples, in order to trace Hepatozoon canis, the 18S rRNA gene segment of the parasite was amplified using polymerase chain reaction (PCR). To confirm the PCR-positive results, five randomly selected PCR-positive samples were sequenced. According to the results, through direct observation of microscopic slides, no infection of H. canis parasite was observed, but according to the PCR results, 32 out of the 145 blood samples were found to be infected by H. canis.  In this study, infection to H. canis in older dogs was higher than in young dogs, and more male dogs were found to be infected by the parasite compared to female dogs; but no significant difference was observed in this regard (P > 0.05). Moreover, stray dogs showed a significantly higher rate of infection, compared to the pet and shelter ones (P < 0.05)

Effect of IL-22 on DNA vaccine encoding LACK gene of Leishmania major in BALB/c mice

In the present study, the effect of IL-22 together with the plasmid encoding LACK (Leishmania homolog of receptors for activated C-kinase) gene of Leishmania major on the trend of leishmaniasis in BALB/c mice was evaluated.Evaluation of the cellular and humoral immunity was performed by measurement of IL-4 and IFN-γ, culture of splenocytes and MTT assay, and measurement of total IgG, IgG1, and IgG2a in the control and immunized groups. Clinical evaluations were also carried out by measurement of the lesion size, survival rate, and body weight of mice.Comparison of the mean size of lesions in the LACK and LACK. +. IL-22 groups demonstrated that the mean size of lesions of the two groups was significantly different from week four (p<. 0.05).The survival rate at day 170 after challenge for the PBS, pcDNA3 (empty plasmid), pcLACK (pcDNA3 containing LACK gene), and pcLACK. +. IL-22 groups were 20%, 40%, 60%, and 80%, respectively.According to the results of IFN-γ, IL-4, total IgG, IgG1, and IgG2a measurement and the MTT assay, IL-22 obviously caused an increase in IFN-γ production and a decrease in IL-4 production before and after the challenge (p<. 0.05). The results showed the effectiveness of IL-22 in DNA vaccine. It showed that IL-22 brought about Th1 cytokine responses and high survival rate of mice. © 2013 Elsevier Inc

Partial immunity in murine by immunization with a toxoplasmic DNA vaccine

Toxoplasma gondii is an obligate intracellular protozoan that is causative agent of atoxoplasmosis, a disease which may result in a spectrum of consequences. Previous studies have reported that DNA vaccine can be effective in partial protection against this parasite. In this study, we constructed a single DNA vaccine containing rhoptry protein 1 (ROP1) and evaluated its immune response in Balb/c mice. We used alum as an adjuvant to enhance the immune response. After intramuscular injection, we evaluated the immune response using cytokine and antibody assay and mortality rate. The results show that mice immunized by pcROP1 with or without alum produced high Th1 immune response compared with the control groups. This kind of DNA vaccine prolonged survival time. The current study showed that ROP1 DNA vaccine could induce partial protective response against toxoplasmosis.Key words: Toxoplasma gondii, DNA, vaccine, Rhoptry protein 1 (ROP1)

DNA vaccination with a plasmid encoding LACK-TSA fusion against Leishmania major infection in BALB/c mice

Vaccination would be the most important strategy for the prevention and elimination of leishmaniasis. The aim of the present study was to compare the immune responses induced following DNA vaccination with LACK (Leishmania analogue of the receptor kinase C), TSA (Thiol-specific-antioxidant) genes alone or LACK-TSA fusion against cutaneous leishmaniasis (CL). Cellular and humoral immune responses were evaluated before and after challenge with Leishmania major (L. major). In addition, the mean lesion size was also measured from 3th week post-infection. All immunized mice showed a partial immunity characterized by higher interferon (IFN)-gamma and Immunoglobulin G (IgG2a) levels compared to control groups (p< 0.05). IFN-gamma/Interleukin (IL)-4 and IgG2a/IgG1 ratios demonstrated the highest IFN-gamma and IgG2a levels in the group receiving LACK-TSA fusion. Mean lesion sizes reduced significantly in all immunized mice compared with control groups at 7th week post-infection (p< 0.05). In addition, there was a significant reduction in mean lesion size of LACK-TSA and TSA groups than LACK group after challenge (p< 0.05). In the present study, DNA immunization promoted Th1 immune response and confirmed the previous observations on immunogenicity of LACK and TSA antigens against CL. Furthermore, this study demonstrated that a bivalent vaccine can induce stronger immune responses and protection against infectious challenge with L. major

Molecular assessment of Neospora caninum and Toxoplasma gondii in hooded crows (Corvus cornix) in Tehran, Iran

Abstract Neospora caninum and Toxoplasma gondii are two closely related protozoan parasites that have been detected from various species of bird hosts. However, little is known about the prevalence of N. caninum and T. gondii in crows. Hence, we examined the molecular frequency of N. caninum and T. gondii in the brain samples of hooded crows (Corvus cornix) that collected from different public parks of Tehran, Iran by nested-PCR method. We used the primers targeting the Nc5 and GRA6 genes for detection of N. caninum and T. gondii, respectively. From a total of 55 brain samples, 5 (9.9%) and 9 (16.36%) samples were positive for N. caninum and T. gondii, respectively. Sequencing of a N. caninum isolate revealed 95%–100% identity with the deposited N. caninum in GenBank. Genotyping of T. gondii isolates by PCR-RFLP analysis of the GRA6 gene revealed type III genotype in 8 isolates. The results of this study indicate that hooded crows may have a putative role in transmission of N. caninum and T. gondii to canines and felines definitive hosts, respectively

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA, ITS2)

Background: In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.Methods: The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and ana-lyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifi-cally of Fasciola species. Results: The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.Conclusion: The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates