Ferredoxin Gene Mutation in Iranian Trichomonas vaginalis Isolates

Background

Trichomonas vaginalis causes trichomoniasis and metronidazole is its chosen drug for treatment. Ferredoxin has role in electron transport and carbohydrate metabolism and the conversion of an inactive form of metronidazole (CO) to its active form (CPR). Ferredoxin gene mutations reduce gene expression and increase its resistance to metronidazole. In this study, the frequency of ferredoxin gene mutations in clinical isolates of T.vaginalis in Tehran has been studied.

Methods

Forty six clinical T. vaginalis isolates of vaginal secretions and urine sediment were collected from Tehran Province since 2011 till 2012. DNA was extracted and ferredoxin gene was amplified by PCR technique. The ferredoxin gene PCR products were sequenced to determine gene mutations.

Results

In four isolates (8.69%) point mutation at nucleotide position -239 (the translation start codon) of the ferredoxin gene were detected in which adenosine were converted to thymine.

Conclusion

Mutation at nucleotide -239 ferredoxin gene reduces translational regulatory protein''s binding affinity which concludes reduction of ferredoxin expression. For this reduction, decrease in activity and decrease in metronidazole drug delivery into the cells occur. Mutations in these four isolates may lead to resistance of them to metronidazole.     

Genetic Identification of Trichomonas vaginalis by Using the Actin Gene and Molecular Based Methods

Background

Trichomonas vaginalis is the agent of urogenital tract infection that causes human trichomoniasis with some serious health complications. More understanding about genetic features of the parasite can be helpful in the study of the pathogenesis, drug susceptibility and epidemiology of the infection. For this end, we conducted analysis of the actin gene of T. vaginalis by applying the PCR-SSCP (PCR-Single Stranded Conformational Polymorphism) and nucleotide sequencing method.

Methods

Fifty T. vaginalis samples were collected from 950 women attending gynecology clinics in two cities of Iran, Hamadan and Tehran, from November 2010 to July 2011. After axenisation of isolates, all samples subjected to PCR-SSCP and nucleotide sequencing.

Results

According to the SSCP banding patterns and nucleotide sequencing, seven sequence types were detected among the isolates. Alignment of the nucleotide sequences showed five polymorphic sites in the different strain types. Amino acid substitution was not observed in the nucleotide sequence translation of the all sequences.

Conclusion

The actin gene analysis represents genetic diversity of T. vaginalis and it suggests that various strains can be responsible for clinically different trichomoniasis in infected individuals. It is expected that further studies will be conducted to increase our knowledge about relationship between the actin gene polymorphism and different biological behavior of the parasite.     

Gene Diversity of Trichomonas vaginalis Isolates

Background

Trichomonas vaginalis is protozoan parasite responsible for trichomoniasis and is more common in high-risk behavior group such as prostitute individuals. Interest in trichomoniasis is due to increase one''s susceptibility to viruses such as herpes, human papillomavirus and HIV. The aim of this study was to find genotypic differences between the isolates.

Methods

Forty isolates from prisoners'' women in Tehran province were used in this study. The random amplified polymorphic DNA (RAPD) technique was used to determine genetic differences among isolates and was correlated with patient''s records. By each primer the banding pattern size of each isolates was scored (bp), genetic differences were studied, and the genealogical tree was constructed by using NTSYS software program and UPGMA method.

Results

The least number of bands were seen by using primer OPD8 and the most by using OPD3. Results showed no significant difference in isolates from different geographical areas in Iran. By using primer OPD1 specific amplified fragment with length 1300 base pair were found in only 8 isolates. All these isolates were belonged to addicted women; however, six belonged to asymptomatic patients and two to symptomatic ones.

Conclusion

There was not much genetic diversity in T vaginalis isolates from three different geographical areas.     

A preliminary investigation of microsatellite-based genotyping in Trichomonas vaginalis

The genetic epidemiology of Trichomonas vaginalis is poorly understood at present. The recent release of the organism's genome sequence opens the way to investigation of polymorphic markers allowing strain identification. We here report a preliminary analysis of microsatellite loci in T. vaginalis and show that this approach holds promise for future studies of infection transmission and organism diversity.     

Comparison of Resistant and Susceptible Strains of Trichomons vaginalis to Metronidazole Using PCR Method

Background

Metronidazole is drug of choice recommended by WHO for treatment of trichomoniasis, however, some reports claims drug resistance in Trichomonas vaginalis isolates recently. The objective of this study was to determine the minimum lethal concentration (MLC) of metronidazole in resistant and sensitive strains, as well as genetic patterns of these stains by PCR method.

Methods

From February 2006 to March 2007, in a cross sectional study, clinical and wet mount examination of vaginal smear along with culture were performed on 683 women attending to public and private outpatient clinics in Hamadan. Trichomoniasis marked based on major clinical symptoms. Diagnosis confirmed using wet mount microscopically and culture in Diamond medium. A serial concentration of metronidazole was provided and all isolated Trichomonas strains (resistant and sensitive) tested by standard method. Finally, all sensitive and resistant strains examined by PCR technique.

Results

Only 15/683, (2.2%) of patients clinically diagnosed trichomonal vaginitis were positive for T. vaginalis by wet smear and culture. The minimum lethal concentration (MLC) for clinically sensitive isolates was 25 µg/ml; however, this concentration for resistant isolates was 200 µg/ml after 24 h and 100 µg/ml after 50 h. The results of PCR examination of DNA from sensitive and resistant isolates had same pattern. The lanes appeared by two primers were 98 bp and 261 bp for both clinically sensitive and resistant strains.

Conclusion

Resistance to metronidazole in T. vaginalis has not relation to genetic variations and might be related to some physiologic pathways of organism.     

Molecular Diagnosis of Trichomoniasis in Negative Samples Examined by Direct Smear and Culture

Background

Trichomoniasis is an extremely common sexually transmitted infection (STI) worldwide and is associated with important public health problems, including amplification of HIV transmission. This disease is in forms of symptomatic and asymptomatic in women and may depend on host as well as parasite variables. Most of the studies reported from females are based on examination of vaginal secretions and urine samples by direct smear and culture in modified Diamond''s media. The aim of this study was checking the samples, which were negative by direct smear and culture, with PCR technique.

Methods

The urine samples and vaginal discharge of patients attending Gynecology Clinics of Mazandaran Province, Iran with different symptoms rechecked for Trichomonas vaginalis by PCR technique using primers targeting a conserved region of the beta-tubulin genes of the parasite. Data were analyzed by Epi Info software program

Results

Out of 161 negative samples by direct smear and culture, seven samples (4.3%) were positive by PCR technique.

Conclusion

Diagnosis of trichomoniasis by PCR is a sensitive and specific method that could play important role to help the physicians for properly treatment and control of infection.     

Molecular Identification and Differentiation of Fasciola Isolates Using PCR- RFLP Method Based on Internal Transcribed Spacer (ITS1, 5.8S rDNA,ITS2)

Background

In this study, we used both ITS1 and ITS2 for molecular identification of Fasciola species.

Methods

The region between 18S and 28S of ribosomal DNA was used in PCR-RFLP method for molecular identification of Fasciola species. Ninety trematodes of Fasciola were collected during abattoir inspection from livers of naturally infected sheep and cattle from Khorasan, East Azerbaijan, and Fars provinces in Iran. After DNA extraction, PCR was performed to amplify region ITS1, 5.8S rDNA, ITS2. To select a suitable restriction enzyme, we sequenced and analyzed the PCR products of F. hepatica and F. gigantica samples from sheep and cattle. Tsp509I fast digest restriction enzyme was selected for RFLP method that caused the separation specifically of Fasciola species.

Results

The fragment approximately 1000bp in all of the Fasciola samples was amplified and then digested with the Tsp509I restriction endonuclease. Seventy F. hepatica and 20 F. gigantica were identified of total 90 Fasciola isolates.

Conclusion

The new PCR-RFLP assay using Tsp509I restriction enzyme provides a simple, practical, fast, low cost, and reliable method for identification and differentiation of Fasciola isolates.     

Characterization of Eimeria Species in Commercial Broilers by PCR Based on ITS1 Regions of rDNA

Background

Coccidiosis is an intestinal disease of chickens caused by various species of protozoan parasites within the genus Eimeria. Diagnosis and genetic characterization of different species of Eimeria are central to the prevention, surveillance, and control of coccidiosis. The aim of this study was to detect different chicken Eimeria species from several areas in Khuzestan, southwest Iran.

Methods

From February to September 2008, PCR assay as well as parasitological examinations was applied for the identification of field isolates of Eimeria parasites around Ahvaz, center of Khuzestan, southwest Iran. Data were analyzed by the Kappa statistic test.

Results

Eimeria maxima, E. necatrix, E. tenella, E. acervulina and E. mitis were detected in this study. The prevalence of Eimeria spp. was 31.5% (126 of 400) and E. tenella was the most prevalent species in Khuzestan. Based on the Kappa statistical test, a good correlation between the results of PCR and traditional biometrical methods was only observed for E. maxima.

Conclusion

The present study is the first on the prevalence of Eimeria species in Khuzestan, based on the molecular findings. We believe that traditional methods are not sufficiently reliable for specific diagnosis of Eimeria species in chickens and PCR based amplification of DNA sequence of parasite, could resolve this problem.     

Genetic characterization of three Cuban Trichomonas vaginalis virus. Phylogeny of Totiviridae family

Trichomonas vaginalis can be infected with double stranded RNA (dsRNA) viruses known as T. vaginalis virus (TVV). This viral infection may have important implications for trichomonal virulence and disease pathogenesis. In this study we identified and genetic characterized three strains of TVVs isolated from T. vaginalis in Cuba. The three new predicted sequences of capsid protein and RNA-dependent RNA polymerase amounted to the previously determined 20 TVV sequences and other 21 viruses of Totiviridae family were used for a phylogenetic analysis. Four distinct monophyletic clades are shown in a phylogenetic tree. One corresponds with TVVs, other with Victorivirus, Leishmaniavirus and Eimeria brunetti virus and, other with viruses of the genus Totivirus and the last with Giardiavirus. The E. brunetti virus is identified in the phylogenetic tree as independent taxon between Leishmaniavirus and Victorivirus isolates, most closely related to Victorivirus. TVV constitute a monophyletic cluster distinguishable from all other viruses in Totiviridae family. This result suggested that TVV may be grouped in a separated genus and not inside of Giardiavirus. TVVs appear to be more closely related to protozoan viruses in the genus Leishmaniavirus and to fungal viruses in the genus Victorivirus than to other protozoan and fungal viruses in Giardiavirus and Totivirus. Among TVVs, four main groups can be recognized within Trichomonasvirus cluster, which correspond with the previous species classification proposed. Further studies, with more TVV strains, especially TVV3 and 4 strains, are needed in order to determine the phylogenetic relationship among Trichomonasvirus genus and specifically if TVV2 and 3 each also constitute a well-delimited group.     

Novel in Vitro Efficiency of Chitosan Biomolecule against Trichomonas gallinae

Background

Development of new natural agents for parasitic diseases treatment has unexpectedly increased to overcome effectively against emergence and re-emergence of parasitic diseases, the appearance of drug resistant organisms and toxic side effects of current agents. The aim of the study was to evaluate antiprotozoal activities of chitosan biomolecule on trophozoites of Trichomonas gallinae.

Methods

The antitrichomonal activity of various low molecular weight chitosan concentrations including 125, 250, 500 and 1250 µg ml⁻¹ against T. gallinae trophozoites cultured in trypticase-yeast extract-maltose medium supplemented with heat-inactivated cold horse serum was evaluated in vitro. Samples containing medium without chitosan were also assayed as controls.

Results

The mortality rates at 0, 3 and 6 h post treatment with all concentrations were significantly different from control group (P<0.05). Treated trophozoites showed more susceptibility to the highest concentration reaching mortality rate of 100% at 3h post inoculation. However, at this time, results for 125, 250 and 500 µg ml⁻¹ were 93%, 95% and 96.7%, respectively.

Conclusion

The results demonstrate that the application of chitosan biomolecule is a promising option for treatment of trichomoniasis in pigeons.     

Molecular Cloning and Expression of EG95 Gene of Iranian Isolates of Echinococcus granulosus

Background

Echinococcosis or hydatidosis is a chronic, zoonotic worldwide infection that occurs by the larval stages of taeniid cestodes of the genus Echinococcus. Iran is known as endemic region for this infection in the world. Vaccination has been considered as a good prevention method for this disease. Recombinant vaccines containing EG95 protein, against E. granulosus, has shown a high degree of protection against E. granulosus infection. In this study EG95 gene was extracted from Iranian isolates of E. granulosus and then cloned and expressed in expression vector.

Methods

Protoscoleces were collected from sheep hydatid cysts. Then DNA and RNA were extracted from protoscoleces, and amplified by PCR and RT-PCR with specific primer. Afterward the purified RT-PCR products were successfully ligated into pTZ57R/T plasmid vector. The pcDNA3 plasmid was used as expression vector and Eg95 fragment sub cloned into this plasmid. The pcEG95 plasmid was digested by restriction enzymes to confirm cloning of this gene in pcDNA3 plasmid. In last step, the subcloned gene was expressed in CHO as eukaryotic cell.

Results

EG95 fragment successfully was subcloned in pcDNA3 and EG95 protein was expressed by eukaryotic cell. The recombinant EG95 protein was confirmed by SDS-PAGE and Western blot.

Conclusion

Recombinant plasmid of pcEG95 was constructed successfully and express of recombinant EG95 protein was confirmed.     

The Problem of Mixing up of Leishmania Isolates in the Laboratory: Suggestion of ITS1 Gene Sequencing for Verification of Species

Background

Leishmaniasis is endemic in Iran. Different species of Leishmania (L.) parasites are causative agents of this disease. Correct identification of Leishmania species is important for clinical studies, prevention, and control of the diseases. Mix up of Leishmania isolates is possible in the laboratory, so there is need for verification of species for isolates of uncertain identity. Different methods may be used for this purpose including isoenzyme electrophoresis and molecular methods. The isoenzyme electrophoresis, due to its drawbacks, is feasible only in specialized laboratories while molecular methods may be more feasible. The aim of this research was to study the application of the internal transcribed spacer 1 (ITS1) sequencing method, in comparison to isoenzyme electrophoresis method, for verification of Leishmania species.

Methods

Six Leishmania isolates were received from different research institutions in Iran. The species of these isolates were known by donating institution according to their isoenzyme profile. The species of these isolates were re-identified in Pasteur Institute of Iran by PCR amplification of ITS1 followed by sequencing and comparison of these sequences with Leishmania sequences in GenBank. Isoenzyme electrophoresis was performed for confirmation of the results of ITS1.

Results

ITS1 sequence showed that some isolates were mixed up or contaminated with Crithidia. Isoenzyme electrophoresis confirmed the results of ITS1 sequences.

Conclusion

ITS1 sequencing is relatively more feasible than the traditional isoenzyme electrophoresis method and is suggested for verification of Leishmania species.     

不同pH值对阴道毛滴虫体外培养的影响

目的 观察阴道毛滴虫在不同的pH值培养基中的培养效果。方法 阴道毛滴虫在pH5.8,6.2,6.6,7.0四组不同培养基中37℃恒温培养，每隔24h观察虫数，活率并计算活虫数。结果 pH7.0组培养第2-4天虫数，活虫数，活率均明显低于其它三组，且在培养过程中无明显活虫数及活率高峰，阴道毛滴虫在培养基中能存活生长10天，其余三组均只生长存活6天，第1-4天，pH6.6组活虫数，活率均高于其它三组。结论 pH7.0不适宜阴道毛滴虫体外培养，pH6.6时，培养各项指标均较好。可能更适合阴道毛滴虫体外培养。     

Molecular Epidemiology of Cryptosporidiosis in Iranian Children,Tehran, Iran

Background

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.

Methods

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.

Results

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.

Conclusion

The predominancy of C. parvum species (specially, IId A20G1a subtype) in current study underlines the importance of zoonotic Cryptosporidium transmission in Iran.     

Investigation of Double-Band Electrophoretic Pattern of ITS-rDNA Region in Iranian Isolates of Leishmania tropica

Background

Leishmania tropica is a genetically divergent species. Amplification of entire internal transcribed spacer (ITS) region of L. tropica isolates obtained from Bam district, one of the well known focus of anthroponotic cutaneous leishmaniasis (ACL) in Iran, revealed a double-band pattern in agarose gel electrophoresis. This study explains how this pattern occurs.

Methods

Twenty seven L. tropica smear preparations were collected from Bam district, south east Iran, and eight L. major and one L. infantum smear preparations were gathered from Shiraz, south west Iran. Furthermore one L. major and one L. infantum cultured standard strains were tested using entire ITS-PCR to survey their electrophoretic pattern. The ITS sequences of L. tropica, L. major, and L. infantum already deposited in GenBank were analyzed. Analysis of GenBank sequences of L. tropica revealed two groups of sequences based on length size, one group having a 100 bp gap. Therefore, a new reverse primer namely LITS-MG was designed to exclude this gap in PCR products.

Results

Whole ITS fragment amplification resulted in a double-band pattern in all L. tropica cases, while a sharp single band was observed for L. infantum and L. major isolates. This result was corresponding to the result obtained from in silico analysis of GenBank sequences. Use of LITS-MG primer was expectedly resulted in a single band including ITS1, 5.8s and partial ITS2 product for L. tropica which is appropriate for following molecular studies such as sequencing or restriction analysis.

Conclusion

Sequences analysis of GenBank L. tropica sequences and following practical laboratory tests revealed at least two alleles in L. tropica which were confirmed in Bam isolates. This especial double-band pattern is because of a 100 bp fragment difference within ITS-rDNA alleles.     

Detection of Acanthamoeba and Toxoplasma in River Water Samples by Molecular Methods in Iran

Background:

Free-living amoebae such as Acanthamoeba species may act as carriers of Cryptosporidium and Toxoplasma oocysts, thus, may play an important role in the water-borne transmission of these parasites. In the present study, a loop mediated isothermal amplification (LAMP) method for detection of Toxoplasma and a PCR assay were developed for investigation of Acanthamoeba in environmental water samples.

Methods:

A total of 34 samples were collected from the surface water in Guilan Province. Water samples were filtrated with membrane filters and followed by DNA extraction. PCR and LAMP methods used for detection of the protozoan parasites Acanthamoeba and Toxoplasma respectively.

Results:

Totally 30 and 2 of 34 samples were positive for Acanthamoeba and Toxoplasma oocysts respectively. Two samples were positive for both investigated parasites.

Conclusion:

The investigated water supplies, are contaminated by Toxoplasma and Acanthamoeba (oo)cystes. Acanthamoeba may play an important role in water-borne transmission of Toxoplasma in the study area. For the first time in Iran, protocol of LAMP method was used effectively for the detection of Toxoplasma in surface water samples in Iran.     

Cloning and Expression of Recombinant Plasmid Containing P36/LACK Gene of Leishmania infantum Iranian Strain

Background:

There are several methods, such as vaccination, to control visceral leishmaniasis. Although there is no efficient vaccine, it seem DNA vaccination with stimulates both cellular and humoral immunity apparently is the best way. The aim of this study was cloning and expression of LACK gene, a 36kD protein, as a candidate protein for vaccination against Iranian L. infantum.

Methods:

Iranian strain of L. infantum [MCAN/IR/07/Moheb-gh] was used as a template for PCR to amplify LACK gene. The LACK gene was cloned in pTZ57R/T vector and after confirmation it was digested by restriction enzymes (BamH1) and cloned in pcDNA3.1 expression vector. Recombinant plasmid was extracted and analyzed by sequencing, restriction digestion analysis and PCR reaction. The pc- LACK recombinant plasmid was purified from transformed E.coli (DH5α) and its expression was analyzed by SDS-PAGE and Western blot.

Results:

The results of sequencing, restriction digestion analysis and PCR reaction revealed that LACK gene was cloned correctly in pcDNA3.1 vector and the results of SDS PAGE and Western blot emphasized that LACK protein of Iranian L. infantum is a well-expressed protein.

Conclusion:

We amplified, cloned and expressed Iranian L. infantum LACK gene successfully.     

Cloning,and Molecular Characterization of Polymorphic Iranian Isolate Theileria annulata Surface Protein (Tasp)

Background

Because of the strong immunologic responses of surface protein TaSp in Theileria annulata infected host, we tried to characterize this protein in a T. annulata isolate from Iran.

Methods

The RNA prepared from T. annulata infected cells was used to produce SMART-DS-cDNA. The Double strand cDNA was then amplified with primers derived from TaSp mRNA sequences. The PCR product was cloned in pTZ57R/T vector, sequenced and registered under accession no. {"type":"entrez-nucleotide","attrs":{"text":"JQ003240","term_id":"371927237"}}JQ003240 in GenBank.

Results

The sequence analysis showed 90%–94% nucleotide sequence identity and 68%–94% amino acid homology to the corresponding sequences of TaSp gene by T. annulata, T. sp. china I, T. sp. china and T. lestoquardi and three T. annulata reported from Iran respectively. Interestingly, the sequence analysis also showed small nucleotide sequence region near the 5‘ end in which the presented TaSp protein differed very strongly from the other known TaSp sequences. For the preparation of the recombinant protein, the cDNA was cloned in pQE-32 vector, the recombinant protein was prepared and assayed by Theileria infected bovine serum.

Conclusion

The polymorphism in TaSp gene could be detected in intra- as well as inter species. The different characterized TaSp proteins had a common identic region, which may be helpful for development of broad band vaccine based on the recombinant proteins. The polymorphism in this gene, make this protein also interesting for the diagnostic purposes.     

Sequence Diversity in tRNA Gene Locus A-L among Iranian Isolates of Entamoeba dispar

Background

A number of methods for detecting diversity in Entamoeba have been described over the years. In the present study the genetic polymorphism of noncoding locus A-L was analyzed using PCR and sequencing in order to clarify the genotypic differences among E. dispar isolates.

Methods

A total of 28 E. dispar from patients with gastrointestinal symptoms were determined and the genomic DNA was extracted directly from stool. For genotype analysis; Locus A-L was amplified by PCR and PCR products were sequenced. The sequences obtained were edited manually and aligned using Gene Runner software.

Results

With sequencing of PCR products a reliable genetic diversity in size, number and position of the repeat units were observed among the Iranian E. dispar isolates in locus A-L gene. Sequences showed variation in length from 448bp to 507bp and seven distinct types were identified.

Conclusion

The genetic diversity of loci like A-L shows them to be suitable for epidemiological studies such as the characterization of the routes of transmission of these parasites in Iran.