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

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. 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. 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. 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

[Evaluating the immunogenicity for plasmid encoding GRA5 antigen of Toxoplasma gondii in BALB/c mice]

Severe or lethal damages of toxoplasmosis clearly indicate the need for the development of a more effective vaccine. Immunization with recombinant plasmid encoding protective proteins is a promising vaccination technique. Therefore, this study aimed to evaluate the immunization with plasmid encoding GRA5 antigen of Toxoplasma gondii in BALB/c mice. In this experimental study, three groups of BALB/c mice [n=10 in each group] were selected using simple random sampling. GRA5 gene was cloned into pcDNA3 plasmid and purified by plasmid purification kits and then the product was injected [IM]. To determine the status of cellular and hurnoral immunity, the 11-4, IFN- gamma and IgG; IgG2a, IgG subtypes were evaluated respectively using the ELISA-based assay. The group immunized with pcGRA5 indicated a significant augmented response in humoral and cellular immunity [P

[Morphological and molecular characterization of dicrocoelium isolated from sheep in the north and center of Iran]

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. 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. A remarked morphological characteristic was that the orientation of testes in all isolates, were in tandem. Position 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. Morphological and molecular assays show that Dicrocoelium dendriticum is the only species of Dicrocoelium among sheep in the north and center of Iran

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

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.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. 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. The EG95 gene was cloned in pTZ57R/T plasmid successfully and this plasmid can be used to design a DNA vaccine in further studies

[Cloning and sequencing the plasmid encoding Toxoplasma gondii Microneme 3 protein]

Toxoplasma gondii, an obligatory intracellular protozoan parasite, causing toxoplasmosis in human and animal with worldwide spread. Microneme 3 [MIC3] protein, a 90 kDa parasite factor attaching to the host cells in the beginning of the invasion, is secreted in all stages of parasite development [e.g. sporozoite, tachyzoite and bradyzoite] and also is considered as a potent antigen. Therefore, besides the immunogenicity and the candidacy for vaccine design, the protein is used for diagnostic purposes, as well. The aim of the present study was to transfer MIC3 gene into plasmid vector [PTZ57R/T] for subcloning in eukaryotic and prokaryotic plasmids. Toxoplasmia genomic DNA extracted using phenol-chloroform method and MIC3 gene was then amplified by PCR with specific primers. Electrophoresis was performed by using agarose gel and PCR product was purified by T4 DNA ligase enzyme into a cloning vector. Finally, recombinant plasmid was transformed into E.coli [Top10 strain]. The extracted clone was verified with PCR, digestion enzymes and sequencing. The PCR product was seen as a 1052bp band in agarose gel [1%]. The recombinant plasmids was restricted by HindIII and EcoRV enzymes and two obtained 2886 and 1052bp bands showed that the MIC3 gene was cloned in PTZ57R/T plasmid. The results revealed that the cloning and transformation of MIC3 gene in pTZ57R/T was done successfully

effect of vitamin D3 alone and mixed with IFN-gamma on tachyzoites of toxoplasma gondii [RH strain] proliferation and nitric oxide [NO] production in infected macrophages of BALB/C mice

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. According to usage of vitamin D3 [one dose or seven doses] and INF gamma 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. The first experiment [the mice were infected with tachyzoites and after 2 h, got one dose vita min 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. High-level production of nitric oxide may be related to the only one injection of vita min D3. The injection in long time might suppress the immune system

[Identification of Sarcocystis gigantea by PCR- RFLP]

Sarcocyst is one the most important protozoa belonged to apicomplexa. This parasite is prevalent among warm blooded animals throughout the world. In the present work, Sarcocystis gigantean was identified by amplification of 18SrRNA gene using PCR- RFLP. In this regard macroscopic cysts of sarcocystis were collected from esophagus and intra costal muscles of sheep in Shahriar slaughterhouse. Then genomic DNA of the parasites was extracted from specimens using phenol-chloroform method. 18S rRNA gene was amplified with specific primers. For RFLP two restricted enzymes of MspI and MobI were used and the patterns were analyzed accordingly. According to the resulted band, all the specimens were identified as Sarcocystis gigantean. This is the first report of molecular identification of Sacrocystis gigantean in Iran

Evaluation of Schistosoma haematobium adult and egg antigens by ELISA in diagnosis of urinary schistosomiasis

The aim of this study was using ELISA for detection of anti-Schistosoma haematobium antibodies in both sera and urine of patients with urinary schistosomiasis. Thirty three sera and urine samples were collected from patients with acute schistosomiasis in Diyala Province, east of Iraq in 2006. Their diseases were confirmed by finding S. haematobium ova in urine examination. Sera and urines of 10 healthy individuals as well as 5 patients with hydatidosis and 5 patients with acute toxoplasmosis were examined as well. Samples were examined for antibody detection by ELISA method. The antigens used in this study were egg and adult antigens. All positive samples [sera and urines] showed positivity by using egg antigen whereas the negative control samples were negative; only two samples with hydatidosis were positive with using serum sample whereas with urine sample only one sample was positive. In this study, the best sensitivity and specificity obtained when using urine and adult antigen. Antibody detection by using urine is a useful, simple, and sensitive method for diagnosis of schistosomiasis

Photodynamic therapy as a new treatment of cutaneous leishmaniasis

We assessed the effectiveness of photodynamic therapy in the treatment of cutaneous leishmaniasis in 5 patients. Delta-aminolevulinic acid in a water- in-oil emulsion was applied to the lesions and irradiation was performed. The treatment was repeated once a week for a month. Each time, direct smears of the lesions were prepared and cultured in NNN media. In direct staining, smears showed no amastigotes after 1 or 2 sessions. Healing and cosmetic outcome after photodynamic therapy was excellent. Only mild local inflammatory reaction was noted with no scarring and 4 months after the last treatment session, there were no clinical signs of recurrence

In vitro effect of folic acid and cobalamin [vitamin B12] on adhesion and growth of Giardia lamblia

Giardia lamblia is one of the most common intestinal protozoan parasites infecting human in the world. The goal of this study was searching for in-vitro effect of folic acid and cobalamin on adhesion and growth of G. lamblia as two important mechanisms in the pathogenesis in TYI-S-33 medium. G. lamblia trophozoites were obtained by in- vitro excystation procedure. Three groups of Giardia trophozoites were analyzed: control group, G.lamblia was cultured in TYI-S-33 without any vitamin, 2nd group with 0.1 micro g/ml vitamin B12 or folic acid, and 3rd group with 0.5 micro g/ml of vitamin B12 or folic acid. All culture media tubes incubated at 37 °C. After 2 h of incubation, the adherence into borosilicate culture tubes, and after 24 h the growth of trophozoites were measured .The results showed that in vitamin B12 groups, the growth was increased significantly [P? 0.05] but the adherence decreased significantly [P