Molecular characterization of Theileria spp. in livestock and the first report on the occurrence of Theileria sp. OT3 in Iran.

Theileria lestoquardi, T. ovis, and T. annulata are recognized as major causative agents for ovine and cattle theileriosis in Iran, respectively. Recently, there have been reports on the presence of Theileria spp. (Theileria sp. OT1, Theileria sp. OT3, and Theileria sp.). In this study, 37 blood samples were collected from sheep and cattle with clinically suspected Theileria infection in the Northwest of Iran. The samples were analyzed using a light microscope. DNA samples were amplified via nested-polymerase chain reaction (PCR) of 18S rRNA gene. The amplicons were digested with HpaII, following restriction fragment length polymorphism (RFLP) and sequenced to reconfirm Theileria species. The microscopic examination indicated that 4 out of 37 (10.8%) blood samples were infected with Theileria spp. Based on the nested PCR-RFLP and sequencing data, 5.4%, 13.5%, and 27% of blood samples were infected with Theileria sp. OT3, T. ovis, and T. annulata, respectively. The pairwise distance matrix of Theileria sp. OT3 showed 99.8-100% identity and 0-0.2% divergence in comparison with the registered sequences. The present study is the first report of Theileria sp. OT3 in Iran. To the evaluate evolution of Theileria spp. and providing resultant genetic data, further research with a larger sample is necessary in the region.

Molecular evaluation of pvdhfr and pvmdr-1 mutants in Plasmodium vivax isolates after treatment with sulfadoxine/pyrimethamine and chloroquine in Iran during 2001-2016.

The rising use of sulfadoxine/pyrimethamine (SP) in the treatment of chloroquine (CQ)-resistant Plasmodium falciparum has resulted in increased exposure to P. vivax isolates in Iran, where both species are being circulated. In this investigation, the frequency of pvdhfr and pvmdr-1 mutants was assessed in P. vivax strains during 2001-2016 after the introduction of SP/CQ in malarious areas of Iran. The P. vivax isolates (n, 52) were obtained from autochthonous samples in Southeast Iran during 2015-2016. The genomic DNA was extracted and examined using nested polymerase chain reaction-(PCR) and sequencing. Mutations were detected in pvdhfr codons P33L (21.2%), T61 M (25%), S93H (3.9%), and S117 T (1.9%) and 5 isolates showed double mutations (33 L/61 M, 7.7%; 33 L/117 T, 1.9%). No mutation was identified in pvdhfr codons F57 and S58. The pvmdr-1 1076 L mutation was detected in 93.3% of P. vivax isolates. The findings indicated that the frequency of three codons of pvdhfr F57/S58/S117 has decreased from 2001 (1.05%/7.0%/16.9%) to 2016 (0%/0%/1.9%). Genomic analysis of pvmdr-1 showed that the frequency of 1076 L has gradually increased from 2013 (93%) to 2016 (93.3%) (P > .05). The results demonstrated that P. vivax isolates are probably being exited under SP pressure, which reflects the appropriate level of training for field microscopists, as established by Iranian policymakers. Emergent pvdhfr codons 33L, 61M, and 93H should be noticed in plausible drug tolerance and treatment plans. The high prevalence of pvmdr-1 1076L mutation shows that efficacy of CQ combination with primaquine may be in danger of being compromised, however further investigations are needed to evaluate the clinical importance of CQ-resistant P. vivax isolates.

Genetic variability and transcontinental sharing of Giardia duodenalis infrapopulations determined by glutamate dehydrogenase gene.

Microevolutionary data of Giardia duodenalis sub-assemblages is a prerequisite for determining the invasion zoonotic patterns of the parasite. To infer transmission patterns that could not be differentiated by the phenotypic features, a population genetic investigation is crucial for the elucidation of the genetic structure of G. duodenalis among the continents. Forty G. duodenalis positive fecal samples were collected from different foci of Northwest Iran. The specimens were subjected to Trichrome staining and sucrose gradient flotation. DNA samples were extracted, amplified, and sequenced by targeting glutamate dehydrogenase (gdh) gene. The global gdh sequences of sub-assemblages AII and BIV retrieved from NCBI GenBank were analyzed to estimate diversity indices, neutrality indices, and gene migration tests. Sequencing analyses indicated various levels of genetic variability of sub-assemblages AII and BIV among the five continents. Sub-assemblage BIV had greater genetic variability (haplotype diversity: 0.975; nucleotide diversity: 0.04246) than sub-assemblage AII. The statistical Fst value demonstrated that the genetic structure of sub-assemblages AII and BIV are moderately differentiated between European-American populations (Fst: 0.05352-0.15182), whereas a significant differentiation was not seen among other geographical population pairs. We conclude that a high gene flow of G. duodenalis sub-assemblages AII and BIV is unequivocally sharing among the continents. The current findings strengthen our knowledge to assess the evolutionary patterns of G. duodenalis in endemic foci of the world and it will become the basis of public health policy to control human giardiasis.

The Correlation between Serum Levels of Anti-Toxoplasma gondii Antibodies and the Risk of Diabetes.

BACKGROUND: This study investigated the presence of specific antibodies against Toxoplasma gondii infection among people with diabetes (type I and II) in comparison with control group. METHODS: Overall 300 serum samples were collected equally from three groups including patients with type I and type II diabetes and non-diabetic healthy control that referred to Tabriz Central Laboratory in northwest Iran during July to Sep 2015. The level of specific IgG and IgM antibodies against T. gondii were measured using the chemiluminescence immunoassay (CLIA) method. Chi-square and One-Way ANCOVA were used for data analysis. RESULTS: Overall, 300 samples from diabetic patients (type I and type II) and control group were examined and results showed 3, 8 and 2 cases were seropositive for anti-T. gondii IgM respectively. Anti-T. gondii IgG seropositivity in type I and type II diabetes and control groups were 69%, 63% and 59% respectively. We did not observe any statistical differences among all studied groups in terms of toxoplasmosis. There was no statistically significant relationship between all variables and seropositivity for anti-T. gondii antibodies in type I and II diabetes and non-diabetic groups. CONCLUSION: Although there was no statistically significant relationship between diabetes and toxoplasmosis further investigations especially experimental studies using animal models are needed. Furthermore, these findings would not be contrary to the need for healthcare in order to the prevention of infectious disease in diabetic patients.

Loop-mediated isothermal amplification as a reliable assay for Toxocara canis infection in pet dogs.

Keeping of infected dogs as pet results in the potential transmission risk factors for shedding helminthic infections such as toxocariasis. Lack of accurate identification of Toxocara canis eggs in non-dewormed infected pet dogs remains a diagnostic concern among researchers. In this study, dog owners were asked to fill up a questionnaire regarding their pets and their attitude towards the deworming regimen. One hundred faecal samples were collected from pet dogs (Northwest Iran) and were subsequently identified by the ZnSo4 flotation technique, PCR and loop-mediated isothermal amplification (LAMP) assays. The DNA of the recovered T. canis eggs was then extracted and amplified by LAMP and PCR. Furthermore, ITS2 amplicons were sequenced for appraisal of the phylogenetic analysis. Nine, 5 and 11% of T. canis infections were identified by microscopy, PCR and LAMP, respectively. It was detected that LAMP was 10 times (10-10to 10-13 g/µl) more sensitive than PCR (10-10to 10-12 g/µl). The kappa value between LAMP and PCR indicated a faint concurrence (0.463). The kappa coefficient between LAMP and flotation technique indicated a strong agreement (0.667). The highest infection rate (n = 11) was detected in non-dewormed pet dogs, particularly those less than 3 months old (P < 0.05). None of the infected dogs had a history of walking and kennelled behaviours in public places. The LAMP assay can address as a simple, rapid and highly sensitive technique for detecting low burden of T. canis eggs in infected pet dogs. It was proposed that the dog holder's awareness is insufficient to implement regular deworming schedules. Additionally, regional policymakers should broadly revise anthelmintic treatment guidelines.

A PCR-Based Molecular Detection of Strongyloides stercoralisin Human Stool Samples from Tabriz City, Iran.

Strongyloides stercoralis is a nematode causing serious infections in immunocompromised patients. In chronically infected patients, the low parasitic content as well as the resemblance of the larvae to several other species make diagnosis basedonmorphology difficult. In the present study, a PCR-based method targeting the internal transcribed sequence 2 (ITS2) of the rDNA region was examined for the molecular detection of S. stercoralis infection from the stool samples. A total of 1800 patients were included. Three fresh stool samples were collected per patient, and S. stercoralis isolates were identified by the morphological method. A subset of isolates was later used in the PCR-based method as positive controls. Additionally, negative and no-template controls were included. Data analysis was accomplished using an x² test. Ap-value less than 0.05 was considered significant. In total, fivestool samples were found to be infected with S. stercoralis using the morphology method. PCR method detected S. stercoralis DNA target from all of the fiveDNA samples extracted from positive fecal samples. CONCLUSIONS: The PCR method used for amplifying a short fragment was successful for diagnosis of S. stercoralis in fecal samples and can be reliable for directly detecting the parasite bypassing morphological method.

Gene flow for Echinococcus granulosus metapopulations determined by mitochondrial sequences: A reliable approach for reflecting epidemiological drift of parasite among neighboring countries.

In genetic diversity and population structure of Echinococcus granulosus, the gene flow can illustrate how the Echinococcus isolates have epidemiologically drifted among endemic neighboring countries. 51 isolates of hydatid cysts were collected from human, dog, cattle and sheep in northwest Iran, where placed co-border with Turkey. DNA samples were extracted, amplified and subjected to sequence analysis of NADH dehydrogenase subunit 1 (nad1) and cytochrome oxidase subunit 1 (cox1) genes. As well, sequences of Echinococcus at east to the southeast regions of Turkey were retrieved from GenBank database for the cox1 gene. The confirmed isolates were grouped as G1 (n = 74) and G3 (n = 6) genotypes. 31 unique haplotypes were identified inferred by the analyzed sequences of cox1 among two distinct populations. A parsimonious network of the sequence haplotypes displayed star-like features in the overall population containing TUR1, IR15 and IR22 as the most common haplotypes. According to AMOVA test, the high value of haplotype diversity (0.94758-0.98901) of E. granulosus was reflected the total genetic variability within populations while nucleotide diversity was low (0.00727-0.01046) in Iranian and Turkish metapopulations. Neutrality indices of the cox1 were shown negative values (-15.078 to -10.057) in Echinococcus populations which indicating a significant divergence from neutrality. A pairwise fixation index (Fst) as a degree of gene flow was partially high value for all populations (0.151). The statistically Fst value indicates that E. granulosus sensu stricto (G1-G3) are genetically moderate differentiated among Iranian and Turkish isolates. The occurrence of TUR1 and IR15 elucidate that there is possibly the dawn of domestication due to transfer of alleles between populations through the diffusion of stock raising or anthropogenic movements. To evaluate the hypothetical evolutionary scenario, further exploration is necessitated to analyze isolates from various host species in rest Middle East countries.

Molecular Characterization of Trichomonas vaginalis Strains Based on Identifying Their Probable Variations in Asymptomatic Patients.

BACKGROUND: The aim of this study was to identify the Trichomonas vaginalis strains/haplotypes based on identifying their probable variations in asymptomatic patients referred to Tabriz health centers, northwestern Iran. METHODS: Sampling was taken from 50-suspected women to T. vaginalis in northwestern Iran. The obtained samples were smeared and cultured. Fifty DNA samples were extracted, amplified and identified by nested polymerase chain reaction and PCR-RFLP of actin gene using two endonuclease enzymes: MseI and RsaI. To reconfirm, the amplicons of actin gene were directly sequenced in order to identify the strains/haplotypes. RESULTS: PCR-RFLP patterns, sequencing and phylogenetic analyses revealed definitely the presence of the G (n=22; 73.4%) and E (n=8; 26.6%) strains. Multiple alignments findings of genotype G showed five haplotypes and two amino acid substitutions in codons 192 and 211 although, no remarkable unique haplotype was found in genotype E. CONCLUSION: The accurate identification of T. vaginalis strains based on discrimination of their unknown haplotypes particularly those which are impacted on protein translation should be considered in parasite status, drug resistance, mixed infection with HIV and monitoring of asymptomatic trichomoniasis in the region.

Genetic variability and discrimination of low doses of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification assay as a field-friendly molecular tool.

ABSTRACT: Aim: One of the main diagnostic problems of conventional polymerase chain reaction (PCR) is indiscrimination of low parasitic loads in soil samples. The aim of this study is to determine the genetic diversity and identification of Toxocara spp. from public areas soil inferred by loop-mediated isothermal amplification (LAMP) assay. MATERIALS AND METHODS: A total of 180 soil samples were collected from various streets and public parks of northwest Iran. The DNA of recovered Toxocara eggs were extracted and amplified by PCR and LAMP following ZnSO4 flotation technique. The amplicons of internal transcribed spacer-2 gene were sequenced to reveal the heterogeneity traits of Toxocara spp. In addition, Toxocara canis sequences of southwest Iran were directly retrieved to compare gene flow between two distinct populations. RESULTS: Toxocara spp. eggs were found in 57, 14 and 77 of soil samples using the microscopy, PCR and LAMP (detection limit 1-3 eggs/200 g soil), respectively. 7.7% of isolates were identified as T. canis by PCR method, while LAMP was able to detect 27.2%, 15.5% and 12.2% as Toxocara cati, T. canis and mixed infections, respectively. The kappa coefficient between LAMP and microscopy indicated a strong agreement (0.765) but indicated a faint agreement among LAMP-PCR (0.203) and PCR-microscopy (0.308) methods. A pairwise fixation index (Fst) as a degree of gene flow was generally low (0.02156) among Toxocara populations of northwest and southwest Iran. CONCLUSIONS: The statistically significant Fst value indicates that the T. canis populations are not genetically well differentiated between northwest and southwest Iran. This shows that here is possibly an epidemiological drift due to the transfer of alleles. The LAMP assay because of its shorter reaction time, more sensitivity, and simultaneous detection of environmental contamination to be appears as valuable field diagnosis compared to PCR. Therefore, the detection of low Toxocara spp. loads from public area soils will help to expand epidemiological understanding of toxocariasis and establishing preventive strategies in resource-limited endemic of Iran.

Molecular and Morphometric Characterization of Acanthamoeba spp. from Different Water Sources of Northwest Iran as a Neglected Focus, Co-Bordered With the Country of Iraq.

BACKGROUND: Acanthamoeba spp. is a free-living opportunistic protozoan parasites, which can be found in tap, fresh and bottled mineral waters, contact lens solutions, soil etc. OBJECTIVES: The present study is aimed to determine the Acanthamoeba spp. on the basis of their morpho-molecular aspects in different water sources of the West Azerbaijan province, Northwest of Iran. METHODS: In this cross-sectional study, 60 water samples were collected from rivers and tap waters during June to September 2015. The water samples were filtered through a cellulose nitrate filter and cultured on non-nutrient agar medium. The extracted DNAs were amplified and some ampliqons were sequenced using partial 18S rRNA for genotyping and phylogenetic analyses. RESULTS: Twenty-seven (45%) out of 60 water samples were positive to Acanthamoeba spp. using both culture and morphological examinations. In addition, 24 (40%) out of 27 positive samples in culture method were confirmed by PCR to be Acanthamoeba spp. CONCLUSIONS: A relatively high prevalence of Acanthamoeba spp. in rivers reflects a risk alert for threatening human health in the region. However, well hygienic status of the tap waters considering Acanthamoeba spp. cannot be ignored in western co-border regions of Iran-Iraq. This study can also serve as a platform for further explorations of water sources in Iran and neighboring countries.

The first morphometric and phylogenetic perspective on molecular epidemiology of Echinococcus granulosus sensu lato in stray dogs in a hyperendemic Middle East focus, northwestern Iran.

BACKGROUND: Hydatidosis is considered to be a neglected cyclo-zoonotic disease in Middle East countries particularly northwestern Iran which is caused by metacestode of tapeworm Echinococcus granulosus sensu lato. Human hydatidosis is a high public health priority in the area, however there is little known from a morphometric and phylogenetic perspective on molecular epidemiology of adult Echinococcus spp. in Iranian stray dogs. METHODS: 80 dogs (38 males and 42 females) were collected during June 2013 to April 2014 in northwestern Iran. The isolated parasites from each dog were distinguished by morphometric keys including small, large hook length and blade length. Subsequently, isolates were confirmed by sequencing of mitochondrial cytochrome oxidase subunit 1 gene. RESULTS: 16 (8 males and 8 females) (Prevalence 20%) out of 80 dogs were infected to genus Echinococcus. With regard to demographic factors, the frequency of parasitism in both male, female adults and their age groups showed no difference (P > 0.05). The phylogenetic analyses of cox1 sequences firmly revealed the 13 sheep strains (G1), one buffalo strain (G3), one camel strain (G6) and one mixed infection. The findings of rostellar hook morphology show an intraspecies variation range among G1 isolates. However, hook measurements in Echinococcus derived from G1 (sheep strain) were not a significant difference from those G6 and G3 strains. Six unique haplotypes were identified containing a high range of diversity (Haplotype diversity 0.873 vs. Nucleotide diversity 0.02). CONCLUSIONS: First presence of camel strain (G6) in this region seems to indicate that potential intermediate hosts play a secondary role in the maintenance of camel-dog biology. Current findings have heightened our knowledge about determination of Echinococcus prevalence, strains of taxonomy and genotypic trait of parasite in Iranian stray dogs which will also help in the development of strategies for monitoring and control of infected stray dogs in the area.

Trichostrongylus colubriformis: Possible Most Common Cause of Human Infection in Mazandaran Province, North of Iran.

BACKGROUND: Infection with Trichostrongylus spp. is common among human and herbivorous in most parts of Iran, especially in southern and northern areas. The aim of present study was to identify Trichostrongylus spp. among human population using excreted egg specimens, by the molecular method, in Mazandaran Province, northern Iran. METHODS: Overall, 33 positive fecal specimens were randomly sampled and examined. PCR amplification of ITS2-rDNA region was performed on the isolated egg and then a restriction fragment length polymorphism (RFLP) profile was considered to discriminate of Trichostrongylus spp. RESULTS: A total of 33 positive fecal specimens, 29(78.9%), 4(12.1%) were found T. colubriformis and T. axei respectively. Our data appear the molecular evidence of both human T. colubriformis and T. axei infections in North of Iran. CONCLUSION: T. colubriformis was the probable most common zoonotic species causing human trichostrongylosis infection in the area.

Designing and conducting in silico analysis for identifying of Echinococcus spp. with discrimination of novel haplotypes: an approach to better understanding of parasite taxonomic.

The definitive identification of Echinococcus species is currently carried out by sequencing and phylogenetic strategies. However, the application of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) patterns is not broadly used as a result of heterogeneity traits of Echinococcus genome in different regions of the world. Therefore, designing and conducting a standardized pattern should indigenously be considered in under-studied areas. In this investigation, an in silico mapping was designed and developed for eight Echinococcus spp. on the basis of regional sequences in Iran and the world. The numbers of 60 Echinococcus isolates were collected from the liver and lungs of 15 human, 15 sheep, 15 cattle, and 15 camel cases in Semnan province, Central Iran. DNA samples were extracted and examined by polymerase chain reaction of ribosomal DNA (rDNA) internal transcribed spacer 1 (ITS1) and PCR-RFLP via Rsa1 endonuclease enzyme. Moreover, 15 amplicons of cytochrome oxidase 1 (Cox1) were directly sequenced in order to identify the strains/haplotypes. PCR-RFLP and phylogenetic analyses revealed firmly the presence of the G1 and G6 genotypes with heterogeneity (three novel haplotypes) of Cox1 gene although no other expected genotypes were found in the region. Finding shows that the identification of novel haplotypes along with discrimination of Echinococcus spp. through regional patterns can unambiguously illustrate the real taxonomic status of parasite in Central Iran.

Seroprevalence of Toxoplasma Gondii Infection among High School Girls in Ajabshir from East Azarbaijan Province, Iran.

INTRODUCTION: Toxoplasmosis is a disease parasite which can infect human and animals. The infection may be serious if is transmitted to the fetus during pregnancy. The aim of this study was to determine the prevalence of specific antibodies and the associated risk factors for toxoplasmosis in students attending high-school in Ajabshir. METHODS: In this descriptive study, 549 blood samples were collected from high school girls. The samples divided into two groups (147 and 402 samples from rural and urban schools respectively). IgG and IgM specific antibodies to Toxoplasma gondii were detected using enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of study showed that from 402 urban samples, 50 (12.4) and 34(8.5) cases and from 147 rural samples, 38 (25.9) and 32 (21.8) cases were seropositive for anti-Toxoplasma IgG and IgM antibodies respectively. Of the risk factors studied, the significant association was found between T. gondii-specific antibodies with residency and age. CONCLUSION: Based on data found in our study, 87.6% of young girls from urban areas in Ajabshir did not have antibodies to Toxoplasma and this is a very important issue, because these young women were in fertile age. Therefore required Preventive and control programs especially in these cases in order to reduce the rate of disease.

Cryptosporidium infection in patients with gastroenteritis in sari, iran.

BACKGROUND: Cryptosporidiosis is a common coccidian parasite infection in patients with diarrhea that has worldwide distribution especially in developed countries. Therefore, the aim of this study was to determine the occurrence of Cryptosporidium infection in patients with gastroenteritis admitted to hospitals of Mazandaran University of Medical Sciences by parasitological and molecular methods in Sari, Iran. METHODS: Stool samples were collected from 348 patients with gastroenteritis admitted to the hospitals of Medical University in the Sari and Ghaemshahr cities in Mazandaran Province, Northern Iran in 2010-2011. Oocysts of Cryptosporidium identified using Formalin-Ether concentration method and stained by Aacid-fast staining (AFS) and Auramine phenol fluorescence (APF). Genomic DAN extracted from microscopically positive samples and nested PCR -RFLP by using SSU rRNA that identifies of the species of cryptosporidium. RESULTS: In 348 patients with gastroenteritis, the most clinical symptoms were diarrhea, nausea, vomiting, dehydration, fever and weight loss. 2.3% (8 cases) of diarrheal samples tested by both microscopy and molecular methods were positive for the presence of cryptosporidium. Nested PCR products yielded unique bands of 846 bp, correspond to cryptosporidium. Species diagnosis carried out by digesting the secondary PCR product with SspI restriction enzyme, which noted 3 clearly bands of 449, 254, and 108 bp correspond to Cryptosporidium spp. CONCLUSION: The results of present study on Cryptosporidium spp. in this area can make a background data for control programs and further molecular analyses. Thus, further work needs to determine the origin of Cryptosporidium species in this area.

Detection of Asymptomatic Carriers of Plasmodium vivax among Treated Patients by Nested PCR Method in Minab, Rudan and Bashagard, Iran.

BACKGROUND: Plasmodium vivax is the most widespread species of Plasmodium in humans and causing about 80 million clinical cases annually. This study was undertaken to detect P. vivax in asymptomatic treated vivax malaria patients to trace latent/sub-patent malaria infection. METHOD: The venous blood of all detected cases with P. vivax in Bashagard, Minab and Roodan Districts in Hormozgan Province from 2009 to 2010 was examined by microscopic and nested PCR methods for presence of the parasite. RESULTS: In microscopic examination of peripheral blood smears, all samples were negative for the presence of the parasites. But, we detected two P. vivax related bands in the electrophoresis of the nested PCR products (120 bp). CONCLUSION: Following up the malaria cases after treatment by a combination of methods, or new diagnostics such as RDTs can be included in the priorities of malaria elimination program in Iran.

Plasmodium vivax dhfr mutations among isolates from malarious areas of Iran.

The use of sulfadoxine and pyrimethamine (SP) for treatment of vivax malaria is uncommon in most malarious areas, but Plasmodium vivax isolates are exposed to SP because of mixed infections with other Plasmodium species. As P. vivax is the most prevalent species of human malaria parasites in Iran, monitoring of resistance of the parasite against the drug is necessary. In the present study, 50 blood samples of symptomatic patients were collected from 4 separated geographical regions of south-east Iran. Point mutations at residues 57, 58, 61, and 117 were detected by the PCR-RFLP method. Polymorphism at positions 58R, 117N, and 117T of P. vivax dihydrofolate reductase (Pvdhfr) gene has been found in 12%, 34%, and 2% of isolates, respectively. Mutation at residues F57 and T61 was not detected. Five distinct haplotypes of the Pvdhfr gene were demonstrated. The 2 most prevalent haplotypes were F57S58T61S117 (62%) and F57S58T61N117 (24%). Haplotypes with 3 and 4 point mutations were not found. The present study suggested that P. vivax in Iran is under the pressure of SP and the sensitivity level of the parasite to SP is diminishing and this fact must be considered in development of malaria control programs.