[Identification of Fasciola species by PCR-RFLP assay in northern Iran]

Background and Objective: Identification ofFasciola species is important. Fascioliasis is one of the important diseases in animals and humans caused by genus Fasciola. This study was done to determine the identification of Fasciola species with RFLP-PCR in animal liver in Gorgan City, northern Iran

Methods: In this descriptive study, worms were obtained from the livers of infected sheep and cattle in Gorgan slaughterhouse in northern Iran. DNA of worms was extracted with phenol- chloroform method. Fragment of ITS-1 genome was amplified and TasI enzyme was utilized for amplified fragments then 8 samples were sequenced

Results: A total of 49 Fasciola worms were isolated from infected cattle and sheep. The PCR products of all specimens were affected by the TasI enzyme, and F.hepatica species showed two fragments and F.gigantica species indicated three fragments. The enzyme in F.hepatica species showed a fragment of 151 bp and a fragment of 312, but in the F.gigantica, three fragments were 151, 93 and 219 bp. 36 [73.46%] worms were identified as Fasciola gigantica and 13 [26.53%] worms were identified as Fasciola hepatica. Out of the six infected sheep liver, 22 were isolated from the Fasciola worms, 13 [59.1%] of which were F.hepatica and 9 [40.9%] of them were F.gigantica. Out of the six infected cattle liver, 27 Fasciola worms were identified, all of which were identified as Fasciola gigantica [100%]

Conclusion: This study showed that Fasciola gigantica is the dominant species in infected livers of the cattle in Gorgan city

[Antibiotic resistance pattern of E.coli isolated from urine culture in 660 army clinical laboratory center in Tehran 2008]

Urinary tract infections are the second most common type of body infection. E.coli is the most common cause of urinary tract infection both sex. For this descriptive study a total 4494 urine samples were examined during one year [2008-2009] in 660 Army Medical Laboratories. The urine cultures and Antibiogram profiles were performed by Kirby- Bauer method according to NCCLS standards. Data analyzed by SPSS software ver. 16. A total of 4494 urine samples were analyzed that out of each 456 samples were showed the significance growth. The most frequently detected gram negative bacterium was E.coli in 257 cases [10.1%]. The most antibiotic resistance rates of E.coli detected from urine culture were to Ampicillin, Co-trimoxazol and Gentamycine with 80%, 37%, 27.7% frequencies respectively. Furthermore, Norofoloxazin and Nitroforantoin had the highest sensitivity 89.6% and 89% respectively. Present findings demonstrated the significance of resistance E.coli that was detected from urine culture to various group of antibiotic drugs caused by the irregular use of antibiotics

[Molecular determination of Echinococcus granulosus isolated from hydatid cyst using mitoconderial atp6 gene]

Several strains of the Echinococcus granulosus have been described based on morphological characters, intermediate host specificity and/or genetic analysis of mitochondrial and nuclear DNA. The aim of this study was to characterize different E.granulosus isolates by using sequences of mitochondrial atp6 gene. In this study, Sixty infected liver and lungs of cattle, sheep and goats were collected from the abattoir of Varamin city-Iran during 2008. Protoscoleces were removed from each fertile cyst and DNA extracted. New and specific primers were designed for two existing genotypes [G1 and G6] of E. granulosus known to occur in Iran and applied in PCR reactions. The new primers selectively amplified the G1 and G6 genotypes of E. granulosus with specific bands of 708 and 705 bp respectively. The G1 genotype was identified in all fertile cyst samples. This study showed that the new primer pairs which specifically amplify portions of the mitochondrial atp6 gene of the G1 and G6 strains of Echinococcus granulosus are proper molecular marker for investigating genetic variation in a number of isolates of E. granulosus from a range of hosts [sheep, goats, cattle] in Iran. The result of sequenced samples showed that our sequences were the same as those reported previously for these strains

Development of sensitive detection of cryptosporidum and giardia from surface water in Iran

The protozoan parasites Cryptosporidium spp. and Giardia are known to occur widely in both raw and drinking waters. They are two of the causative agents of waterborne outbreaks of gastroenteritis throughout the world. In the present study, a PCR assay and FA were developed for detection of Cryptosporidium oocysts and Giardia cyst in environmental samples. We have detected Cryptosporidium spp. oocysts and Giardia cysts in seeded and unseeded environmental water samples by PCR method. Water samples were spiked with oocysts [50, 100, 300, 500] and filtrated with a 1.2- micro m pore size cellulos nitrate and follow by DNA extraction and purification by QIA amp DNA mini kit. Nested -PCR assay amplified an 850 bp fragment of 18s rDNA gene specific for Cryptosporidium and 453 bp fragment of glutamate dehydrogenase [GDH] target genre for Giardia. Also many river water from north of Iran, be checked by these methods. Cryptosporidium and Giardia DNAs were detected in seeded water sample and Giardia was detected in all 5 water samples from river in north of Iran by nested-PCR and FA. Also in one river water sample, Cryptosporidium was detected. This protocol if effective for detection of these waterborne parasites in treated and untreated water samples. This study can also serve as a platform for further investigations and research water source in Iran

Molecular epidemiology of cryptosporidiosis in Iranian children, Tehran, Iran

Cryptosporidium is a worldwide protozoan parasite and one of the most common causes of infection and diarrhea in humans and cattle. The aim of the present study was determination of subtypes of Cryptosporidium among children with diarrhea in Tehran by sequence analysis of the highly polymorphic 60-kDa glycoprotein [GP60] gene. Fecal samples were collected from 794 diarrheic children. Initial identification of Cryptosporidium was carried out on stool samples by Ziehl-Neelsen acid-fast staining method. DNA was extracted from positive microscopically samples and Cryptosporidium genotypes and subtypes were determined, accordingly. Out of 794 collected samples, 19 [2.40%] were positive for Cryptosporidium oocysts. Sequences analysis of GP60 gene showed that 17 [89.47%] of the positive isolates were Cryptosporidium parvum and 2 [10.52%] were C. hominis. All subtypes of C. parvum isolates belonged to allele families IIa [6/17] and IId [11/17]. The most common allele in all 17 isolates belonged to IId A20G1a [41.18%]. A22G1 [IF] subtype was detected in two C. hominis isolates of the children. The predominancy of C. parvum species [specially, IId A20G1a subtype] in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran

[Cryptosporidium genetic diversity based on analysis of COWP, SSU-rRNA and TRAP-C2 polymorphic genes in children with diarrhea in Tehran and qazvin provinces: a survey from Iran]

Since Cryptosporidium is a worldwide distributed protozoan parasite and is considered as one of the most common causes of infection and diarrhea in humans with autoimmune deficiency, as well as in young live stock, molecular epidemiologic studies of cryptosporidiasis will be helpful for underlying transmission and molecular pathogenesis of Cryptosporidium in humans. The aim of the present study was to determine the species and genotypes of Cryptosporidium among children with diarrhea in Tehran and Qazvin provinces by PCRRFLP using the three polymorphic regions of SSU-rRNA, COWP and TRAP-C2 genes. 1263 stool samples were collected from the children less than 12 years with diarrhea who referred to Pediatrics Medical Centers in Gazvin and Tehran Provinces, Iran, during 2005-2007. After determination of the presence of Cryptosporidium oocytes by ZiehlNeelsen acid, fast staining genomic DNA was extracted. Nested PCR-RFLP was performed by -rRNA, COWP and TRAP-C2 genes. Results of microscopically positive samples showed that the overall prevalence of infection in children was 31 [2.5%]. Results of nested PCR amplification showed that of 31 isolates of children, all of three targeted gene were successfully amplified. Our results indicated that the zoonotic transmission is the main mode of infection in Iran and indicates that direct or indirect contact with animals, especially calf, is possibly the main route of human infection

Identification of Leishmania species isolated from human cutaneous lesihmaniasis using PCR method

To determine the epidemiological status of cutaneous leishmaniasis outbreak, isolation and identification of the agent parasite, Leishmania, using PCR method in Gonbad-e Qabus County, north Iran, during 2006-2007. Data were collected on the prevalence of scars and ulcers over a period of 3 months among 6990 inhabitants of five villages around Gonbad-e Qabus County, north Iran, during 2006-2007. Cultured promastigotes were identified using PCR technique. Itsl and its2 of Non Coding Transcribed region at ribosomal DNA of 46 Leishmania isolates wre amplified and the PCR products were separated by electrophoresis in 1.5% agarose gel [200 mA, 140 V], visualized by staining with ethidium bromide, and photographed. Among 6990 inhabitants of 5 villages, 62.9% were identified as scars and 1.5% as active lesions, Individuals 11 to 20 years were the most highly infected age group. All the parasite isolates were Leishmania major. Cutaneous leishmaniasis due to L. major is endemic in Gonbad-e Qabus County, north Iran

Molecular characterization of dihydrofolate reductase-thymidylate synthase gene concerning antifulate resistance of Plasmodium vivax

The recently reported resistance to antimalarials contributes to making the control of malaria more difficult. There is a need to evaluate the current antimalaria regiments to prevent this emerging problem. The aim of this study was to determine dihydrofolate reductase-thymidylate synthase gene mutation [pvdhfr] regarding antifulate resistance in Plasmodium vivax. From 2007 to 2009, 117 P.vivax infected blood samples collected from two regions of Hormozgan Province, south of Iran were analyzed using PCR, semi-nested-PCR and RFLP methods. Eighty four isolates [71.8%] showed no mutation in pvdhfr gene of P. vivax known as wild type and 33 [28.2%] of the samples revealed nine single [7.7%], twenty two double [18.8%] and two [1.7%] triple mutations. Genetic diversity was observed by molecular methods in pvdhfr gene of p.vivax in Hormozgan Province suggests that the antifolate falciparum malaria drug [fansidar] is proportionally affecting P.vivax dhfr mutation. Therefore, more studies to evaluate antimalarial drugs that should preferably be effective against both P.vivax and P.falciparum are recommended