Molecular detection of toxoplasma gondii oocytes in the soil from the public parks of the Arak City, Iran

Soil structure is mainly composed of sand, silt, clay, and organic materials. Organisms can live in the soil. The large number of stray cats in the cities of Iran is a major environmental and health problem. Toxoplasma oocysts are shed with the feces of cats, so soil is known as a potential source of transmission of toxoplasmosis. The aim of this study was to determine the soil contamination with Toxoplasma gondii oocyst in the public parks of the Arak city. Sixty soil samples were collected from 15 main parks of Arak city. Four soil samples from the children's playground, a potting place, around trash bins, and around toilets were taken. Oocyst was isolated from soil by floatation in saturated sucrose. Floating debris was tested by two methods: staining by the modified Ziehl-Neelsen technique and PCR. The target of PCR was the 122 bp fragment of the B1 gene. From 60 soil samples of public parks of Arak city, 8 samples [13%] were suspected to Toxoplasma oocyst contamination in staining smears. Only 3 samples [5%] of 60 samples were positive in PCR. The results showed that the staining method is not a good method to detect oocysts in the soil because the diversity of oocyst in soil is very high and similar in appearance. This study showed soils of public parks in the Arak city were contaminated to oocyst of Toxoplasma. Also molecular method for the detection of parasites in the soil was more suitable than staining method

Identification of Malassezia species isolated from patients with Pityriasis versicolor using PCR-RFLP method in Markazi province, central Iran

The lipophilic yeasts of Malassezia species are members of the normal skin microbial that are cause of pityriasis versicolor. Pityriasis versicolor is a common superficial fungal infection with worldwide distribution. The phenotypic methods for identification of Malassezia species usually are time consuming and unreliable to differentiate newly identified species. But DNA-based techniques rapidly and accurately identified Malassezia species. The purpose of this study was isolation and identification of Malassezia Species from patients with pityriasis versicolor by molecular methods in Markazi Province, Central Iran in 2012. Mycologic examinations including direct microscopy and culture were performed on clinical samples. DNA extraction was performed from colonies. The ITS1 region of rDNA from isolates of Malassezia species were amplified by PCR reaction. The PCR were digested by Cfo I enzyme. From 70 skin samples, were microscopically positive for Malassezia elements, 60 samples were grown on culture medium [85.7%]. Using PCR-RFLP method, that was performed on 60 isolates, 37[61.6%] M. globosa, 14[23.3%] M. furfur, 5[8.4%] M. sympodialis and 4[6.7%] M. restrictawere identified. In one case was isolated M. globosa along with M. restricta. The PCR-RFLP method is a useful and reliable technique for identification of differentiation of Malassezia species

Cloning of 1183 bp fragment from rhoptry protein I [ROPI] gene of toxoplasma gondii [RH] in expression prokaryote plasmid PET32a

Toxoplasma gondii is an obligatory intracellular protozoan. Considering to high prevalence of this disease the best way to reduce the raised loses is prevention of human and animal infection, rapid diagnosis, differentiation between acute and chronic disease. Rhoptry protein 1 of Toxoplasma gondii is an excretory-secretory antigen that exists in the most stages of life cycle. According to specifications of excretory-secretory antigen that seems this antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit. The main object of the present work was cloning rhoptry protein 1 [ROP1] gene of Toxoplasma gondii [RH] in a cloning vector for further production of rhoptry proteins. Genomic DNA was extracted by phenol-chloroform method. The ROP1 fragment was amplified by PCR. This product was approved by sequencing and was cloned between the EcoR1 and Sal1 sites of the pTZ57R/T vector. Then transformed into Escherichia coli DH5 alpha strain and screened by IPTG and X-Gal. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. The plasmid was purified and approved by electrophoresis, enzyme restriction and PCR. After isolating of this gene from pTZ57R/T, it was subcloned into pET32a plasmid. After enzyme restriction and electrophoresis a fragment about 1183bp was separated from pET32a. Recombinant plasmid of ROP1 gene was constructed and ready for future study. That seems the antigen is a suitable candidate to produce recombinant vaccine and diagnostic kit