[ 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

[Primary research on gastro-intestinal cryptosporidium incidence rate in lambs and calves in amol city, Iran]

Cryptosporidium is an ubiqutous enteric protozoan parasite that infects a wide range of vertebrate hosts. In humans and many other mammals, cryptosporidium is recognized as a significant pathogen primarily as a cause of acute, severe diarrheal illness. At this investigation animal samples [stool] were collected from 708 heads of lambs [in the beginning of the birth to three months] and 713 heads of calves [in the beginning of the birth to six months] in spring, summer, autumn and winter seasons at amol city in 1374. the samples were examinated after staining using modified zihil - nelson technique. Results showed, 29 samples of lambs [4.09%] and 28 samples of calves [3.92%] were positive, also in winter season infestation rate was higher than the other seasons [4.65%]. whereas infestation rate in animals without clinical signs is high, so this subject is a important problems for public health