Study locations and Samples collection
Urmia city (37°32′55″N 45°04′03″E) is the center of West Azerbaijan province in Iran, and It is situated at an altitude of 4,360 ft above sea level. Also, Maragheh city (37°23′21″N 46°14′15″E) is located at the south west of East Azarbaijan province on the south hillside of Sahand mountain and situated at an altitude of 4,845 ft above sea level. The climate of the two cities is temperate disposed to cold and humid (Fig. 1). 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.
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.
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).