Rapid identification of nine species of diphyllobothriidean tapeworms by pyrosequencing.

Tongjit Thanchomnang,Wanchai Maleewong,Somjintana Tourtip,Oranuch Sanpool,Viraphong Lulitanond,Chairat Tantrawatpan,Pewpan M Intapan,Hiroshi Yamasaki

doi:10.1038/srep37228

Abstract

The identification of diphyllobothriidean tapeworms (Cestoda: Diphyllobothriidea) that infect humans and intermediate/paratenic hosts is extremely difficult due to their morphological similarities, particularly in the case of Diphyllobothrium and Spirometra species. A pyrosequencing method for the molecular identification of pathogenic agents has recently been developed, but as of yet there have been no reports of pyrosequencing approaches that are able to discriminate among diphyllobothriidean species. This study, therefore, set out to establish a pyrosequencing method for differentiating among nine diphyllobothriidean species, Diphyllobothrium dendriticum, Diphyllobothrium ditremum, Diphyllobothrium latum, Diphyllobothrium nihonkaiense, Diphyllobothrium stemmacephalum, Diplogonoporus balaenopterae, Adenocephalus pacificus, Spirometra decipiens and Sparganum proliferum, based on the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene as a molecular marker. A region of 41 nucleotides in the cox1 gene served as a target, and variations in this region were used for identification using PCR plus pyrosequencing. This region contains nucleotide variations at 12 positions, which is enough for the identification of the selected nine species of diphyllobothriidean tapeworms. This method was found to be a reliable tool not only for species identification of diphyllobothriids, but also for epidemiological studies of cestodiasis caused by diphyllobothriidean tapeworms at public health units in endemic areas.

Highlights

Human diplogonoporiasis refers to a tapeworm infection caused by Diplogonoporus balaenopterae
The pyrosequencing reproducibility was confirmed for the amplicons by Sanger sequencing conducted at First BASE Laboratories Sdn Bhd (Selangor, Malaysia) using the BigDye terminator v3.1 cycle-sequencing kit (Applied Biosystems (ABI), Carlsbad, CA), and both strands were directly sequenced using the polymerase chain reaction (PCR) primers as sequencing primers (Model 310 or 3100, ABI), which yielded identical sequence data
The taxonomy of the diphyllobothriidean tapeworms generally depends on morphological classification, but morphological characteristics are not always decisive criteria, in larval and immature worms, or inadequately preserved specimens

Summary

Introduction

Human diplogonoporiasis refers to a tapeworm infection caused by Diplogonoporus balaenopterae Larvae and adult tapeworms of the genera Diphyllobothrium, Adenocephalus and Spirometra are morphologically similar, their identification in human tissues is exceedingly difficult and requires special expertise. One study has successfully used morphometric and ultrastructural (surface morphology) egg features to distinguish among the 8 species of diphyllobothriids[30] For these reasons, many molecular methods using different genetic markers have been developed for the identification of diphyllobothriid tapeworms: cox[1] for Diphyllobothrium[31,32,33,34] and S. erinaceieuropaei[16,35], sdhB for Sp. proliferum and S. erinaceieuropaei[36], and nad[3] for Sp. proliferum[5]. We developed a pyrosequencing methodology that characterizes cox[1] gene amplicons for the identification of diphyllobothriidean tapeworms in humans and intermediate hosts

Methods

Results

Conclusion