Genetic categorization of Echinococcus granulosus from humans and herbivorous hosts in Iran using an integrated mutation scanning‐phylogenetic approach

Abstract

In the present study, we have extended earlier taxonomic, biochemical and experimental investigations to characterize Echinococcus granulosus from various hosts in Iran utilizing DNA regions (designated pcox1 and pnad1) within the cytochrome c oxidase subunit 1 and NADH dehydrogenase 1 mitochondrial genes, respectively. An emphasis was placed on the characterization of E. granulosus isolates (cyst material) from humans, sheep, goats, cattle and camels, and on assessing their genetic relationships. PCR-based SSCP analysis of pcox1 and pnad1 amplicons derived from individual isolates (n=148) of E. granulosus revealed five (pc1-pc5) and nine (pn1-pn9) electrophoretic profiles, respectively. Sequencing of pcox1 and pnad1 amplicons representing unique SSCP profiles demonstrated that each profile was linked unequivocally to a particular sequence and that single point mutations were readily detectable by SSCP. Phylogenetic analyses of pcox1 and/or pnad1 nucleotide sequence data were conducted using Bayesian inference and maximum likelihood tree-building methods. Following the phylogenetic analyses of concatenated pcox1+pnad1 sequence data, including representatives of all presently recognized Echinococcus species/genotypes as well as Taenia saginata (as the outgroup), the majority of cyst isolates (142 of 148; 95.9%) from humans, ruminants (sheep, goats and cattle) and camels were assigned to the G1-G3 complex of E. granulosus (or E. granulosus sensu stricto), whereas some E. granulosus cysts (6 of 19; 31.6%) from camels were assigned to the G6-G10 complex (or E. canadensis). The present study reinforces the advantages of the mutation scanning-sequencing-phylogenetic approach to explore variation in multiple mitochondrial loci within and among Echinococcus populations, which provides a platform for future, detailed studies of the molecular epidemiology of E. granulosus in Iran and other countries. (Note: The sequences determined in the present study have been deposited in the GenBank database under accession numbers: FJ796203-FJ796207 (pcox1) and FJ796208-FJ796216 (pnad1)).

Genetic Characterization of Echinococcus granulosus from a Large Number of Formalin-Fixed, Paraffin-Embedded Tissue Samples of Human Isolates in Iran

“…Previous studies have indicated the presence of the G6 genotype in different hosts such as sheep, goats, cattle, camels, and humans in Iran. 28,29,32,40,56 However, the high prevalence of the G6 genotype in this study is not in accordance with previous human CE studies in Iran (Table 1), because most of these studies indicated the G1 genotype as the only genotype found in humans. However, the sample sizes used within some of the previously mentioned studies were very low.…”

“…1,26 Several molecular epidemiological studies have been performed on E. granulosus isolates in Iran using sequence data of mitochondrial and nuclear genes. Overall, four different genotypes of E. granulosus (genotype G1, G3, and G6) have been reported from different livestock [27][28][29][30][31][32][33][34][35][36] and dogs (genotype G1, G2, and G3) 37 from Iran. To date, only a few human isolates of E. granulosus have been genetically characterized in Iran that indicated G1, G3, and G6 genotypes ( Table 1).…”

Molecular Characterization of Human and Animal Isolates of Echinococcus granulosus in the Thrace Region, Turkey

“…In the previous study, the pig strain G7 was reported in one human isolate and two isolates from sheep (13). Our results are consistent with the data obtained by other authors who used different techniques, such as PCR-RFLP (15,(32)(33)(34)(35)(36)(37) and SSCP (17,22,(38)(39)(40) analyses for the genotyping of E. granulosus. In addition, we examined 13 isolates of E. granulosus from different intermediate hosts using sequence analysis of the mitochondrial ND1 and CO1 genes.…”

“…The mitochondrial ND1 region was amplified by PCR using MS1 and MS2 primers (22 of PCRs, positive [G1: EU503084 (20), and G6-G10 complex: GU951511 (18)] and negative (no-DNA) controls were included. Four microliters of each PCR products were mixed with 12 µl of denaturating buffer.…”

“…Mitochondrial DNA sequencing and phylogenetic analysis Thirteen amplicons, representing each unique SSCP and RFLP profile, were selected, and fragments of amplicons mitochondrial CO1 and ND1 genes were amplified with primers published in Bowles et al (24) and Sharbatkhori et al (22), respectively. Then, PCR products were purified using the High Pure PCR Product Purification Kit (Roche, Germany).…”