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

The Effect of Vitamin D3 Alone and Mixed With IFN-γ on Tachyzoites of Toxoplasma gondii (RH Strain) Proliferation and Nitric Oxide (NO) Production in Infected Macrophages of BALB/C Mice

Introduction: Toxoplasma gondii is an obligatory interacelullar parasite that infects nucleated cells in its intermediate hosts. The aim of the present study was to determine the effect of vitamin D3 on the multiplication of T. gondii in peritoneal macrophage of Balb/c mice and nitric oxide production by macrophages. Methods: According to usage of vitamin D3 (one dose or seven doses) and INFγ in vitro and in vivo, this study was divided into four experiments. In all experiments, the macrophages were col­lected from peritoneum and cultured in RPMI-1640. Then the supernatants were collected after 24 h and their nitric oxide was measure. After 96 h, the macrophages were collected and stained and the number of tachyzoites was measured. Results: The first experiment (the mice were infected with tachyzoites and after 2 h, got one dose vita­min D3 intraperitonealy) showed the best results. The mean of tachyzoites per macrophages was 2.37, and mean ± SD of nitric oxide was 187.8 ± 9.Discussion: High-level production of nitric oxide may be related to the only one injection of vita­min D3. The injection in long time might suppress the immune system

Diagnosis of liver and pulmonary Hydatid cyst by Dot-ELISA, IFA and IHA tests

History and Objectives: Although some serological tests are not highly sensitive in diagnosis of pulmonary hydatid cyst however Dot-ELISA is introduced as a simple, sensitive and a very efficient means of pulmonary hydatid cyst diagnosis. In the present study, we compared the validity of IHA, IFA and Dot-ELISA techniques in diagnosis of anti-echinococcal antibodies caused bu Echinococcus granulosis in cystic liver and lung hydatid disease. Materials and Methods: 244 sera of which 30 samples from hydatid patients, 104 sera from patients suffering a disease other than hydatidosis and 110 sera from healthy individuals were collected mainly from Tehran's hospitals. The hydatid patients were confirmed by surgery and consisted of 14, 10, 6 patients with liver, lung and liver-lung hydatid cysts respectively. The sera were tested by IHA, IFA and Dot-ELISA techniques. Sensitivity, specificity, predictive values, false positivity and false negativity of each test were evaluated in diagnosis of liver and lung hydatid cyst. Results: The results indicated that the sensitivities of IHA test for liver, lung and liver-lung cysts were 78.7, 60 and 100 respectively, while the sensitivities of IFA in detecting liver lung and liver-lung cysts were 93, 80 and 100 respectively. In this regard, the Dot-ELISA showed the best results whereas its sensitivities for liver, lung and liver-lung cysts were 100 each. Conclusion: These results indicate that detection of anti-echinococcal antibodies in human sera is superior by Dot-ELISA compared to IHA and IFA tests. Hence, the application of this test in medical laboratories for hydatid cyst diagnosis can be recommended

Characterization and expression of GRA7 gene of Toxoplasma gondii RH strain in eukaryotic pcDNA3 plasmid

Background: Toxoplasmosis, as a widespread and important disease, can cause severe complications in the congenital and HIV cases. Vaccination is one of the preventive approaches in humans and domestic animals. The GRA7, an excretory 29 kDa Toxoplasma gondii dense granule antigen released by the infected host cells , has been considered as a candidate to produce a vaccine . Materials and Methods: In this experimental study, the inserted plasmid containing GRA7 gene was extracted from TOP10 bacteria and digested with the BamH1 and EcoR1 enzymes. The isolated gene was inserted into the pcDNA3 plasmid . The cloning was confirmed by the PCR and sequencing. The protein expression was confirmed by the SDS-PAGE and Western blotting. Results: The results showed that the GRA7 gene was cloned into the pcDNA3 plasmid. The isolated gene cloned in pcDNA3 was confirmed by PCR and showed the 733bp band. The pcGRA7 plasmid expressed in the CHO cells showed the 29 kDa band using the SDS-PAGE and Western blotting. Conclusion: Considering that cloning of GRA7 gene has been done in the pcDNA3 plasmid and transformed in the eukaryotic cells, it can be used for the DNA vaccine researches

Determination and cloning of the gene encoding EG95 protein in Iranian isolate of Echinococcus granulosus

Background: Echinococcus granulosus is a cestode parasite that causes cystic hydatid disease in humans worldwide. The gene encoding EG95 protein may be a good candidate to design a DNA vaccine to prevent the disease. Considering the importance of EG95 gene and the scarceness of research on it in Iran, this study was carried out to determine and clone the gene encoding EG95 from Iranian isolate of E. granulosus. Materials and Methods: At the first stage, protoscoleces was isolated from hydatid cyst fluid and then RNA was extracted from protoscoleces and after performing RT-PCR, the amplified cDNA samples were detected by gel electrophoresis. In next stage, the obtained gene was cloned in pTZ57R/T vector. Two methods were used for conformation of cloning: colony PCR amplification and digestion with the EcoRI and XhoI restriction enzymes. Finally, the cloned EG95 gene in pTZ57R/T vector was sequenced.Results: Homological comparison of sequences showed that cDNA of EG95 in Iranian isolate of E. granulosus had 492 bp and was different from the standard strain of EG95 reported from New Zealand and Australia (X90928.1). Moreover, cloning of EG95 gene in pTZ57R/T plasmid was confirmed by digestion of this plasmid with the restriction enzymes. Conclusion: The EG95 gene was cloned in pTZ57R/T plasmid successfully and this plasmid can be used to design a DNA vaccine in further studies

Effect of aloe-emodin on growth and induction of apoptosis in Leishmania major promastigotes in vitro

Background: Cutaneous leishmaniasis is an endemic disease in some parts of Iran. The use of pentavalent antimony compounds as first therapeutic choice is associated with limitations and several adverse effects and complications. This study aimed to evaluate the effect of aloe-emodin on Leishmania major promastigote growth in vitro conditions and also the effect of aloe-emodin on induction of apoptosis in Leishmania major promastigotes. Materials and Methods: In this experimental study, different concentrations (40, 80, 120 and 160 µg/ml) of aloe-emodin were tested in three different times (24, 48 and 72h) on Leishmania promastigotes and the IC50 was calculated by counting the number of parasites. The MTT (3-4,5-dimethylthiazol-2-yl-2,5 diphenyl tetrazolium bromide) assay was used to determine the percentage of live promastigotes after adding aloe-emodin. Apoptosis at three different concentrations (40, 80 and 120 µg/ml) was evaluated by flowcytometry and also the percentages of early and late apoptosis and necrosis were determined. Results: Result showed that aloe-emodin has an inhibitory effect on the growth of Leishmania promastigotes. The IC50 value was 52.79µg/ml. Moreover, the results of flowcytometry showed that aloe-emodin induced the early and late apoptosis in Leishmania promastigotes. Conclusion: Aloe-emodin shows an antileishmanial effects in vitro condition and regarding its herbal origin, it can be tested as the new drug in vivo studies

Seroepidemiologic prevalence of Hydatid cyst in Hamadan 1991

History and Objectives: Due to side effect and varied prevalence and lack of information on the spread infection of the hydatid cyst in the region, the present study was conducted in urban and rural region of Hamadan in 1991. Materials and Methods: On 1530 inhabitants covered by urban and rural health and hygiene system, a descriptive study was conducted. Information related to age, sex, location of inhabitant and occupation were recorded. Blood samples (5ml) were obtained and Casoni skin test was performed on all individuals. Antibodies against parasite were measured by IFA method. Titration of 1/10 was taken as positive and for positive blood test higher titration were tested. IHA test was done on positive samples to confirm the results. Titration of 1/64 or higher was taken as positive in IHA test. Prevalence and confidence interval was determined. Results: From 1530 samples, 64 samples (4) were positive by IHA test (CI=2.2-3.8). Rate of infection in males and females were 2.9 and 3.1 percent respectively. Rate of infection for urban and rural regions were 3.4 (CI=2-4.8) and 2.7 (CI=1.6-3.8). Tests performed by IFA and IHA indicates that infection among females are more prevalent that in males. Conclusion: The present study indicates that the rate of hydatid cyst infection is rather high in the region. Therefore, attention should be paid especially to the female inhabitants. Preventive measures and control through institutions that are responsible and basic hygienic educational programs and control of runaway dogs are needed in order to reduce the rate of infection

A molecular beacon-based real time PCR assay for quantitative detection of Toxoplasma gondii in rat

Background: Application of quantitative real time PCR has evolved as a sensitive, specific, and rapid method for the detection of Toxoplasma gondii (T. gondii). The present study aimed to evaluate the efficacy of real time PCR method, using B1 gene, for the diagnosis of toxoplasmosis in the experimentally infected rats. Materials and Methods: Parasites were cultured in peritoneal cavity of mice and then the DNA was extracted in tachyzoite stage. The B1 gene of T. gondii was amplified by PCR and detected by real time PCR method based on the molecular beacon probe. Finally, real time PCR was evaluated for the quantization of T. gondii in the blood of the experimentally infected rats. Results: The B1 gene of T. gondii which was successfully amplified by PCR yielded an amplicon with an approximate length of 116 bp. Using this gene was evaluated highly appropriate for the quantization of T. gondii by real time PCR method. Conclusion: Application of real time PCR method is shown to be highly efficient in terms of sensitivity and rapidity for the detection of B1 gene as well as the quantization of T. gondii in blood of rat

Morphological and molecular characterization of Dicrocoelium isolated from sheep in the north and center of Iran

Background: Dicroceliosis is a hepatic parasitic disease of clinical and financial significance for both human health and animal breeding. Considering the health and economic importance of the disease, this study aimed to determine the morphological and molecular characterization of 28S rDNA for Dicrocoelium isolated from sheep in the north and center of Iran during 2010-11. Materials and Methods: A total number of 200 trematodes were collected during an abattoir inspection from livers of naturally infected sheep in East Azerbaijan, Razavi Khorasan, Mazandaran and Tehran provinces in Iran. Adult worms were morphologically identified based on morphometric characterization and 60 specimens were characterized molecularly by sequencing. For molecular study, DNA was extracted and 28S rDNA region was amplified by PCR. Then, Tru1I fastdigest restriction enzyme and also RFLP technique were used to identify the parasite species. Finally, the PCR product was sequenced.Results: A remarked morphological characteristic was that the orientation of testes in all isolates, were in tandem. The homological comparison of sequences showed that 28S rDNA in all isolates of Dicrocoelium had 963 bp and were similar to standard strain registrated in Genbank. RFLP pattern from D.dendriticum, which had 4 cut sites, produced 116, 145, 293 and 409 bp fragments. Although the morphological characterization in various provinces was significanly different, molecular identification showed that all specimens were identical (D.dendriticum) and there was not a significant difference between sequences of the collected parasites.Conclusion: Morphological and molecular assays show that Dicrocoelium dendriticum is the only species of Dicrocoelium among sheep in the north and center of Iran