[Prevalence of human adenovirus 36 and its association with overweight/obese and lipid profiles in the Tehran lipid and glucose study]

Nowadays, obesity is a major public health problem in the most developed countries and its persistence impacts the incidence of cardiovascular diseases and diabetes. Compared to genetical and other behavioral factors, the viral origin of obesity has been less studied, which is why we undertook to assess the prevalence of human adenovirus 36 [Adv36] antibody and its association with obesity and lipid profiles in a Tehranian population. In this cross-sectional study, 348 individuals were selected randomly from among participants of the Tehran Lipid and Glucose Study [TLGS]. Anthropometric, blood pressure, and biochemical factors were measured and the human Adv36 antibody was determined using the ELISA method. The prevalence of seropositive Adv36 was 61.8% [N =215], and that of anti-Adv36 was lower in overweight and/or obese subjects in comparison to non-obese ones [57.3 vs. 68.6%; p<0.05]. Children and adolescents with Adv36 seropositive had higher mean height, weight, waist, TC, LDL-C, TG, DBP, and SBP and lower HDL-C. Adv36 seropositive adults had higher mean height, weight, and TG and lower HDL-C. Despite the high prevalence of Adv36 in this Tehranian population, no significant correlation was found between Adv36 seropositivity and BMI, although, it has been associated with lipid disorders. Therefore, further research using neutralization confirmatory methods is recommended

Molecular detection and identification of Anaplasma species in sheep from Ahvaz, Iran

Ovine anaplasmosis is a tick-borne rickettsial disease, widespread in tropical and subtropical areas. In the present study, a PCR-RFLP method based on major surface protein 4 [MSP4] gene, was utilized for the detection of Anaplasma infection in 119 sheep blood samples collected from different parts of Ahvaz in the southwest of Iran. PCR identified Anaplasma infections in 87.4% [104/119] of the samples in contrast to the routine blood smear examination, which revealed inclusion bodies in only 33.6% [40/119] of samples. RFLP assessment revealed that all PCR positive samples were A. ovis, while for the first time in Iran, a mixed infection with A. marginale was seen in 50% [52/104] of Anaplasma infected samples. These results suggest higher sensitivity of PCR method over the conventional microscopic technique for diagnosis of anaplasmosis, particularly in carrier animals. It also revealed that ovine anaplasmosis caused by A. ovis and A. marginale is present and highly prevalent in Ahvaz and appears to be the first report from this region

Double-stranded RNA viral infection in Tehran trichomonas vaginalis isolates

Trichomonas vaginalis is a pathogenic protozoon and may be contaminated with dsRNA virus called Trichomonas vaginalis virus [TVV]. The viral infection is an important factor for its pathogenesis and sensitivity to metronidazole. The presence of TVV is associated with qualitative and quantitative expression of cysteine proteinases and surface immunogenic; P270. The purpose of this study was to determine TVV frequency in T. vaginalis clinical isolates in Tehran, Iran. The 46 T. vaginalis isolates were collected from Tehran Province and cultured in TYI-S-33 culture medium. Viral RNA was extracted and RT-PCR was done. Of 46 T. vaginalis isolates, 8 isolates [17.39%] were infected with TVV-1. There was not any association between patient age and TVV- infected T. vaginalis. There were 17.39% viral infection in T. vaginalis isolates which was lower than that reported by other researchers. This is the first report on T. vaginalis isolates infection by TVV-1 in Iran

Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 micro g of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

Inhibition of leishmania major PTR1 gene expression by antisense in escherichia coli

Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase [DHFR]. Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid

Expression and purification of P43 Toxoplasma gondii surface antigen

Toxoplasma gondii is an obligate intracellular protozoan parasite, capable of infecting all species of mammals including man. Congenital toxoplasmosis is more important during pregnancy for the first time. In this study we expressed and purified P43 Toxoplasma gondii tachyzoite and bradyzoite specific surface antigen. The recombinant pGEMEX-1 contained Toxoplasma P43 coding sequence was transformed into E. coli and mass cultured in LB medium contained 100 micro g/ml ampicillin at 37 [degree sign]C over night .The T7 promoter was induced by 1mM isopropyl-1-thio-beta-D-galactopyranoside [IPTG. Recombinant protein was purified by affinity chromatography and confirmed by gel diffusion dot blot and western blot,-using specific anti Toxoplasma antibodies. Recombinant plasmid was induced by IPTG and analyzed by SDS-PAGE. Recombinant protein was confirmed by Western-blot and dot blot using anti human Toxoplasma antibody. Recombinant Toxoplasma P43 was produced successfully

[Detection of drug resistance gene in trichomonas vaginalis by PCR]

Trichomoniasis is a worldwide protozoan parasitic disease. Considering the importance of the disease in public health and the controversial ideas about the prevalence of drug resistance, this study was designed to determine the prevalence of metronidazole resistance gene in trichomonas vaginalis [T. vaginalis] with PCR-RFLP method in Tehran and in Kashan. In this descriptive study 140 samples of T. vaginalis in patients with T. vaginalis infections were collected and assessed microscopically. Then they were isolated and examined by culturing in dorset's medium, DNA extraction and PCR amplification. The PCR products were analyzed using RFLP and suspected samples were sequenced. All but 7 samples were T. vaginalis positive by PCR. Sixty-two samples [44.4%] were examined by microscopic, culture and PCR techniques; 12 samples [8.5%] by microscope and PCR, 56 samples [40%] by culture and PCR and other 3 samples [2.1%] were positive only by PCR. Two samples [1.5%] were also examined for detection of mutation in 18S rRNA gene with RFLP in Tehran. This study shows that T. vaginalis infections in the female population living in Tehran are metronidazole-resistant. Since metronidazole is considered as the drug choice for T. vaginalis infections, more studies are recommended for identification of the drug resistance mechanisms and prevention of the disease

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

Study on ITS1 gene of Iranian Trichomonas vaginalis by molecular methods

Trichomoniasis is a worldwide protozoan parasitic disease and metronidazole is a choice drug for its treatment. Because of disease importance in public health and its controversial ideas about the prevalence of drug resistance, this study was carried out. Fifty-two suspected vaginal samples were collected from 2006 to 2007 in Gynecology Maryam Hospital, Tehran, Iran. All isolates were examined by microscopic, culture and PCR techniques. The PCR products were analyzed by RFLP and CSGE methods and two suspected samples were sequenced. Trichomonas vaginalis was identified from all 52 samples. Of 52 isolates, 45 samples were successfully cultured and amplified by PCR except one. Seven were positive only by PCR. Finally, ITS1 fragment was successfully amplified in 51 of 52. CSGE analysis and PCR products digestion by MspI followed by sequencing showed nucleotide mutation at position 209 [C209T] of the ITS1 fragment in two [3.9% of them. The results showed mutation in ITS1 fragment of T. vaginalis in two [3.9%] of Iranian isolates which may be related to metronidazole resistance

Serological evaluation of EgAgB16 kDa, a recombinant antigen from echinococcus granulosus for diagnosis of human hydatidosis

Regarding that accurate diagnosis of human hydatidosis still needs more investigations, the present study was conducted to clone, express, and evaluate the gene encoding AgB subunits [EgAgB16 kDa] from Echinococcus granulosus [Iranian G1 strain] and its evaluation by ELISA test. DNA was extracted from protoscoleces and was utilized by PCR for strain identification. Total RNA was prepared with RNeasy protect mini kit from E. granulosus [Iranian G1 strain] protoscoleces collected from naturally infected sheep with hydatid cyst. Recombinant AgB16 kDa was produced using pETDuet as vector and evaluated by ELISA method. A panel of sera including hydatid cyst-infected individuals [n=72], healthy individual [n=48], toxoplasmosis [n=4], strongyloidosis [n=4], kala-azar [n=5] and tuberculosis [n=5] were examined using this recombinant antigen. Recombinant protein was purified by affinity chromatography using His-Tag column. After purification, recombinant protein was confirmed by western blot analysis using His Tag monoclonal anti body or hydatid positive human serum. The sensitivity, specificity; positive and negative predictive values were calculated as 93.5%, 95.6%, 96% and 92.9%, in that order. The cut-off point was detected 0.3 for rAgB16. While the produced recombinant AgB16 kDa showed promising results in diagnosing human hydatidosis, but more investigations should be implemented to reach an accurate gold standard

Cloning and expression of human vascular endothelial growth factor gene and inhibition of its expression by antisense in prokaryotic system

Angiogenesis is an important process in physiology and disease pathogenesis and is controlled in a healthy body by a number of stimulatory and inhibitory factors. The aim of this study was to determine the effect of antisense transcript on the sense transcript of the endothelial growth factor [EGF] gene in bacterial system as an approach for the gene regulation in tumors. The hepatoma cell line [HepG2] was stimulated by PMA. VEGF mRNA was used for RT-PCR. VEGF cDNA was synthesised and cloned into T- vector pTZ57R, then sense fragment of VEGF subcloned into pACYC Duet-1 expression vector and antisense VEGF subcloned into pCDNAS expression vector. Recombinant plasmids were transforemed into BL2 1 bacterial cells. Expression of recombinant plasmid was analysed by western blot technique. The recombinant pCDNA3-VEGF [pYZantiVEGF] was successfully expressed in BL21 cells. Western blot analysis showed that the expression of VEGF decreased significantly in the cells transfected with VEGF antisense RNA compared with the pACYCDUET-1 - VEGF [pYZsenseVEGF] transfected and control. The expression of VEGF in BL21 cells was strong. In vitro, antisense of VEGF inhibited VEGF expression significantly in BL21 cells

Gene cloning of Iranian Leishmania major mannose-1-phosphate guanyltransferase

Leishmania is an obligatory intracellular protozoan parasite, which infects human beings when infected sand fly vector takes a blood meal. Most efforts are towards designing an effective vaccine to prevent leishmaniasis. In this way, development of candidate antigen for vaccine has special important. In this study, we cloned mannose-1-phosphate guanyltransferase gene of Iranian L .major in pET32a expression vector. Primers based on L. major mannose-1-phosphate guanyltransferase sequence gene was designed and synthesized. DNA of Leishmania promastigotes was extracted and PCR reaction was done. PCR product was cloned into pTZ57R and sub cloned into pET32a expression vector. Recombinant plasmid containing 1140 bp as L. major mannose-1-phosphate guanyltransferase gene was extracted and confirmed by restriction analysis. PCR product was sequenced and deposited to GenBank. There were some differences in amino acid sequences between Iranian L. major mannose-1-phosphate guanyltransferase and others previously accepted in GenBank. We amplified and cloned Iranian L. major mannose-1-phosphate guanyltransferase successfully

Molecular identification of anaplasmosis in goats using a new PCR-RFLP method

In this study blood samples were collected from 193 goats in north and northeastern Iran with the aim to develop a PCR-RFLP assay, as a specific and sensitive diagnostic tool enabling direct and concurrent identification of two Anaplasma species [A. ovis, A. marginale] in goats. A polymerase chain reaction [PCR] for amplification of a fragment of the major surface protein 4 [msp4] gene from A. ovis and A. marginale was developed. The results revealed that 123 out of 193 blood samples were positive for Anaplasma spp. infection. All 43 positive samples detected by microscopic examination were approved as positive by PCR, whereas no rickettsials were seen through light microscopy in the other 80 PCR positive cases. All positive samples were A. ovis as confirmed by restriction fragments length polymorphism [RFLP] method. Our results showed that PCR-RFLP of the msp4 gene could be a useful method for the detection of A. ovis in goats

[Comparison between serology and PCR methods for the diagnosis of visceral Leishmaniasis]

Canine visceral leishmaniasis [CVL] caused by Leishmania Infantum is endemic in most Mediterranean basin and its seroprevalence ranges from 10 to 37%. Diagnosis of Infection is very important especially in asymptomatic dogs for control of human leishmaniasis for control of human visceral leishmaniasis. This study was aimed to compare three methods for detection of canine visceral leishmaniasis. In this research process study, 71 dogs were selected from 4 endemic villages in Meshkin-Shahr district. Peripheral blood samples were tested by serologic [DAT and Dipstick rK39] and molecular [PCR] methods. Skin samples were tested by molecular [PCR] methods. Twelve samples of PCR products were sequenced that all of them were identified as Leishmania infantum and 2 nucleotide sequence data submitted to the GenBank database. From 71 dogs that were studied, 21.1% were symptomatic and others were asymptomatic[78.9%]. 17 dogs [23.9%] had >/= 1:320 titer of antibody by direct agglutination test [DAT]. Twenty two dogs[31%] were positive by Dipstick rK39 test, 21 dogs [29.6%] were positive by PCR on skin samples, 31 dogs [43.7%] were positive in blood PCR and 38 dogs [53.5%] were positive by skin/blood PCR. The highest correlation was between DAT and Dipstick test [76%].According to the results of this study, we can diagnose infection in symptomatic and asymptomatic dogs by DAT as a suitable method and PCR is suitable to follow parasite DNA in skin and other tissues of dogs

Gene cloning of 30 kDa toxoplasma gondii tachyzoites surface antigen [SAG1]

Toxoplasma gondii is an obligate intracellular parasite and its sexual and asexual cycles, respectively take place in the intestinal epithelial cell of definitive host and tissue of intermediate hosts. Congenital toxoplasmosis is more important when the mother acquired the infection during pregnancy period for the first time. Serological tests are the only methods for diagnosis of toxoplasmosis. Among serological tests, ELISA has specific value and availability of parasite specific antigen increases the specificity of test. This study has designed and performed in the aim of availability to specific antigen of Toxoplasma. A pair of forward and reverse primers was designed based on published sequence of T. gondii SAG1 gene. PCR reaction was performed and PCR product was cloned in the pQE-30 expression vector. The gene of 30 kDa protein of Toxoplasma tachyzoites was cloned in expression vector successfully. Recombinant plasmid was confirmed and is ready to express recombinant protein for further studies. In this research we cloned P30 gene of T. gondii tachyzoites surface protein successfully and is ready to express the recombinant protein