Phylogenetic study of Enterobius vermicularis isolates from Northwest of Iran

Abstract

Human infection with Enterobius vermicularis occurs worldwide, particularly in children. The Role of E. vermicularis in appendicitis is proven. was aimed at characterization and genotyping f this parasite from different samples inferred by mitochondrial DNA to get the parasitic gene flow among diverse populations. There were 40 appendectomies for acute clinical appendicitis, 40 positive scotch tape samples, and an adult female worm isolated from Urmia and Maragheh in the northwest of Iran. Genetic differentiation, haplotype network analysis, and isolates' population structure were analyzed based on cytochrome C oxidase subunit 1 (cox1) gene. It has been demonstrated that all isolations in the appendectomies specimens are similar, and the genetic difference divergence is seen in adult worm specimens. The neutral indices of the samples of Azerbaijan did not show a significant difference and show that there is no intraspecific and population distribution diversity. Our samples show different haplotypes in the B type of E. vermicularis population and add new information about genotyping of these parasites in Iran. In comparison with other studies, intraspecific variation of this parasite from Iran was observed.  

Introduction

Enterobius vermicularis is a cosmopolitan distributed intestinal nematode in children that cause pinworm disease Oxyuriase. Transmission of this parasite is directly in human population. The infection of this parasite is often asymptomatic. Other symptoms attributed to this parasite include itchy nose, appendicitis, vulvovaginitis, runny mouth, and gnashing of teeth at night with ectopic migration (Kucik et al. 2004; McDonald and Hourihane 1972). 

Relevant studies identified different prevalence rates for infection of E. vermicularis (Moosazadeh et al. 2016).  The prevalence of E. vermicularis in humans in different parts of Iran is 0.3% to 66.14% in various studies(Afshar et al. 2020; Moosazadeh et al. 2016; Tomanakan et al. 2020). In several countries, molecular and genotypic studies have been performed using mitochondrial markers (mt-DNA) to elucidate the genetic diversity of E. vermicularis, mainly to identify some patterns like types or specific haplotypes that may be associated with human isolates based on eggs and adult worms. Several phylogenetic studies from Asian and European countries have identified three different populations or subtypes: A, B, and C.

In addition, molecular studies of this E. vermicularis parasite previously performed in Iran have shown type B in humans. Genotypes A and B have been isolated from humans and animals like as chimpanzees, but genotype C has only been reported in chimpanzees. However, no study reports genotype C of E. vermicularis in humans.

Molecular approaches and the genetic diversity indices help us find the adaptation of parasites. Considering lake of adequate information on the genotype of E. vermicularis in Iran, this study was aimed at characterization and genotyping of this parasite from different samples inferred by mitochondrial DNA to get the parasitic gene flow among diverse populations and its relative comparison with other data in other studies.

Materials And Methods

Dna Extraction And Sequencing

According to the Ferrero study, for isolation of eggs, pieces of tape were crushed, and eggs were isolated for DNA extraction (Ferrero et al. 2013). Microdissected appendices tissue distinguished as E. vermicularis causing acute appendicitis were used to extract DNA by xylene treatment, which dissolves the paraffin from the tissue and then rehydrated using a series of ethanol washes (Pikor et al. 2011). DNA of obtained tissues was extracted with (ambio®, Sambio™, Iran), according to kit instructions. Total DNA from each worm was extracted with a High Pure PCR Template Preparation Kit (Dynabio®, Takapouzist, Iran), according to the instructions, and all of which were stored at − 20°C until use. The cox1 gene was amplified using primers based on Piperaki et al. study (Piperaki et al. 2011). DNA fragments of target region were amplified by polymerase chain reaction (PCR) using a pair primer forward (EVM1: 5 -TTTTTGGTCATCCTGAGGTTTATATTC-3) and reverse primers (EVM2: 5- CACATTATCCAAAATAGGATTAGCC-3). The total volume of reaction was 15 µl containing 1.5 µl DNA template, 5 µl distilled water, 10 pmol of each primer, and 7.5 µl master mix (amplicon).

Reaction cycles consisted of an initial denaturing step at 94°C for 5 min, followed by 35 cycles at 94°C for 90 s, 57°C for 60 s, and 72°C for 45 s, with a final extension at 72°C for 10 min using a gradient thermocycler. DNA fragments were analyzed by 1.5% agarose gel electrophoresis and sequenced by Bioneer Company using the same primers.

Phylogenetic Analysis

Sequences have been aligned and unlike the sequences from the region. In contrast to existing sequences from the area associated with E. vermicularis available in the GenBank, using Chromas 2.2 and multiple, some alignment was done with the data linked to E. vermicularis from Iran deposited in GenBank. Phylogenic analyses based on cox1 sequence data were conducted to the fullest possible using MEGAX (Kumar et al. 2018). All sequences were run unordered and equally weighted. Alignment gaps were treated as missing data, and bootstrap analyses were done using 1000 replicates.

Genetic Diversity Indices

The number of segregating sites, diversity indices (Haplotype diversity: Hd and Nucleotide diversity: π), and neutrality values (Tajima’s D and Fu’s Fs tests) were calculated by DnaSP software version 5.10(Rozas et al. 2003). The degree of gene flow among the E. vermicularis populations was evaluated using a pairwise fixation index (Fst: F-statistics) (Reynolds et al. 1983).

Results

Phylogenic analyses of E. vermicularis haplotypes based on the cox1 gene and type of nucleotides in Iran were conducted by Maximum likelihood (ML) using MEGAX with Syphacia obvelata designated as an outgroup shown in Figs. 2. The sequences obtained in this study were registered in GenBank under the following accession numbers: OL774836 to OL774839 and OL773357-58 and OL773361-62. The percentage of similarity and difference in genetic sequences in all three types of samples is shown in Table 1. It has been demonstrated that all isolations in the appendectomies specimens are similar, and the genetic difference divergence is seen in adult worm specimens.

Table 1. Percent Identity and Divergence of E. vermicularis from 3 types of samples in this study

Using DnaSP 5.10 software, haplotype differences (Hd), nucleotide differences (π = Nd), and neutrality indices of samples studied in this study and recorded from different parts of Iran in GeneBank were calculated (Table 2). The results show that the haplotype diversity in the samples of Azerbaijan is 0.712 and the number of haplotypes in this region is 3. On the other hand, the neutral indices of the samples of Azerbaijan did not show a significant difference and show that there is no intraspecific and population distribution diversity in the samples of Azerbaijan (this study).

Fst values between various populations of E. vermicularis were calculated by Dnasp5 software package with the nucleotide data set of cox1 gene. The results showed that there was the least difference in the genetic structure of the population in our studied samples such as Azerbaijan, Khorramabad, and Gilan north of Iran (Fst = 0) and the highest genetic population distance of Azerbaijan with the samples of Shiraz in southwest of Iran (Fst = 0.34524). This index (Table 3).

Discussion

Genotypic studies of worms have multiplied recently and provide valuable information on geographical distribution. Molecular studies from the field of genetics and population allow us to recreate the evolutionary relationships between parasites and hosts and understand their geographical distribution, diversity, and the etiology of the disease (Bozorgomid et al. 2020; Lymbery and Thompson 2012).

Based on mitochondrial DNA marker, three types of A, B, and C were identified from the E. vermicularis (Hasegawa et al. 2012; Yaguchi et al. 2014). Although this variety is in three groups in different parts of the world, type C is only seen in animals, and type B is dedicated to human specimens (Nakano et al. 2006). 

The cox1 tree topologies indicated that all samples were typed B. These results similar to Tavan et al. from Shiraz and Khoramabad in 2020 (Tavan et al. 2020). It seems that genotype B is the only type of E. vermicularis in Iran. However, a study from Iran showed that the B type is divided into two branches (Hagh et al. 2014), but our results indicate different haplotypes in the type B samples in Iran, and there are intraspecific variations in this type. Diversity indices in E. vermicularis population from 4 other locations from Iran show the variations of haplotypes. The findings could be attributed to high mutation rate in cox1 gene. Also, Tavan (2020) shows the genetic variability in the studied E. vermicularis population (Tavan et al. 2020). In a study, Piperaki et al. in 2011 showed that the single haplotypes in the studied human population were probably related to the principal and common source of infection (Piperaki et al. 2011). Like our study, they could not find any clear association between the haplotypes and the origin or location studied. 

Neutrality indices of the isolates from appendectomies did not show significantly different intraspecific diversities. In other words, despite the haplotype diversity in Iranian samples, these changes did not lead to intraspecific diversity.

Balloux describes genetic discrimination grade from infra-population in 2002 (Balloux and Lugon‐Moulin 2002). F_ST will seriously underestimate differentiation in highly structured populations. Fst values between the E. vermicularis population of this study and Gilan and Khorramabad are 0, indicating low genetic differentiation or same population. This finding could be attributed to little direct movement or interaction of infected human populations. The limitation of this study was that the number of sequences was related to isolates for more comprehensive details to compare the sequences with each other.

In conclusion, our samples show different haplotypes in the B type of E. vermicularis population and add new information about genotyping of these parasites in Iran. In comparison with other studies, intraspecific variation of this parasite from Iran was observed.  

Declarations

Acknowledgements

This article has been extracted from the M.sc thesis written by Kaveh Figh Shilanabadi in Department of biology, Urmia branch, Islamic Azad University, Urmia, Iran (Registration No.: 162413736).

The authors kindly thank Urmia Islamic Azad University and the vice-chancellor of research deputy of Maragheh University of Medical Sciences (MRGUMS) for supporting of the study. 

Authors' contributions

All authors contributed to the study design. FKH was the leader of the research. KF Sh and SR carried out experimental tests and prepared the Manuscript. All authors read and approved the final version of the manuscript.

Conflict of interest The authors declare that there is no conflict of interest.

Ethical standards This work approved by the Research Ethics Committee of Islamic Azad University- Urmia Branch (No.IR.IAU.URMIA.REC.1400.102).

References

1. Afshar MJA, et al. (2020) Prevalence and associated risk factors of human intestinal parasitic infections: a population-based study in the southeast of Kerman province, southeastern Iran. BMC infectious diseases 20(1):1–8
2. Balloux F, Lugon-Moulin N (2002) The estimation of population differentiation with microsatellite markers. Molecular ecology 11(2):155–165
3. Bozorgomid A, Rouhani S, Harandi MF, Ichikawa-Seki M, Raeghi S (2020) Genetic diversity and distribution of Fasciola hepatica haplotypes in Iran: Molecular and phylogenetic studies. Veterinary Parasitology: Regional Studies and Reports 19:100359
4. Ferrero MR, Röser D, Nielsen HV, Olsen A, Nejsum P (2013) Genetic variation in mitochondrial DNA among Enterobius vermicularis in Denmark. Parasitology 140(1):109–114
5. Hagh VRH, Oskouei MM, Bazmani A, Miahipour A, Mirsamadi N (2014) Genetic classification and differentiation of Enterobius vermicularis based on mitochondrial cytochrome c oxidase (cox1) in northwest of Iran. Journal of Pure and Applied Microbiology 8(5):3995–3999
6. Hasegawa H, Sato H, Torii H (2012) Redescription of Enterobius (Enterobius) macaci Yen, 1973 (Nematoda: Oxyuridae: Enterobiinae) based on material collected from wild Japanese macaque, Macaca fuscata (Primates: cercopithecidae). Journal of Parasitology 98(1):152–159
7. Kucik CJ, Martin GL, Sortor BV (2004) Common intestinal parasites. American family physician 69(5):1161–1168
8. Kumar S, Stecher G, Li M, Knyaz C, Tamura K (2018) MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular biology and evolution 35(6):1547
9. Lymbery A, Thompson R (2012) The molecular epidemiology of parasite infections: tools and applications. Molecular and biochemical parasitology 181(2):102–116
10. McDonald G, Hourihane DB (1972) Ectopic Enterobius vermicularis. Gut 13(8):621–626
11. Moosazadeh M, Abedi G, Afshari M, Mahdavi S, Farshidi F, Kheradmand E (2016) Prevalence of Enterobius vermicularis Among Children in Kindergartens and Primary Schools in Iran: A Systematic Review and Meta-Analysis. Osong public health and research perspectives
12. Nakano T, Okamoto M, Ikeda Y, Hasegawa H (2006) Mitochondrial cytochrome c oxidase subunit 1 gene and nuclear rDNA regions of Enterobius vermicularis parasitic in captive chimpanzees with special reference to its relationship with pinworms in humans. Parasitology Research 100(1):51–57
13. Pikor LA, Enfield KS, Cameron H, Lam WL (2011) DNA extraction from paraffin embedded material for genetic and epigenetic analyses. JoVE (Journal of Visualized Experiments)(49):e2763
14. Piperaki E-T, Spanakos G, Patsantara G, Vassalou E, Vakalis N, Tsakris A (2011) Characterization of Enterobius vermicularis in a human population, employing a molecular-based method from adhesive tape samples. Molecular and cellular probes 25(2–3):121–125
15. Reynolds J, Weir BS, Cockerham CC (1983) Estimation of the coancestry coefficient: basis for a short-term genetic distance. Genetics 105(3):767–779
16. Rozas J, Sánchez-DelBarrio JC, Messeguer X, Rozas R (2003) DnaSP, DNA polymorphism analyses by the coalescent and other methods. Bioinformatics 19(18):2496–2497
17. Tavan A, Mikaeili F, Mahmoudvand H, Sadjjadi SM, Bajelan S (2020) Prevalence and genotype distribution of Enterobius vermicularis among kindergarteners in Shiraz and Khorramabad cities, Iran. Asian Pacific Journal of Tropical Medicine
18. Tomanakan K, Sanpool O, Chamavit P, Lulitanond V, Intapan P, Maleewong W (2020) Genetic variation of Enterobius vermicularis among schoolchildren in Thailand. Journal of helminthology 94
19. Yaguchi Y, et al. (2014) Genetic analysis of Enterobius vermicularis isolated from a chimpanzee with lethal hemorrhagic colitis and pathology of the associated lesions. Parasitology research 113(11):4105–4109