Rapid identification of nine species of diphyllobothriidean tapeworms by pyrosequencing

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.

Figure 1. Multiple sequence alignments for indicating primer annealing positions and diagnostic target region.

Nucleotide alignment of the mitochondrial cox1 gene derived from D. dendriticum, D. ditremum, D. latum, D. nihonkaiense, D. stemmacephalum, Di. balaenopterae, A. pacificus, S. decipiens and Sp. proliferum. The solid arrows indicate the position of Psedo_F (forward primer) and biotinylated Psedo_R (reverse primer). The broken arrow indicates Psedo_S (sequencing primer). The boxed region shows the position (881–921) of the target area used for species-level identification. Dots indicate identical nucleotides between sequences. AM712906 (D. latum) is the reference sequence for nucleotides positions.

Figure 2. Theoretical pyrograms and pyrograms of control plasmids or analytical sample raw reads of 9 diphyllobothriid species.

Pyrograms of the 41-base fragment of the cox1 gene from D. dendriticum (A) D. ditremum (B) D. latum (C) D. nihonkaiense (D) D. stemmacephalum (E) Di. balaenopterae (F) A. pacificus (G) S. decipiens (H) and Sp. proliferum (I). Theoretical pyrogram patterns (top of each panel) and representative raw data of control plasmids or analytical samples from pyrosequencing (bottom of each panel) are shown. The actual sequence obtained by pyrosequencing is displayed below the panels following “Seq”. The Y-axis shows the level of fluorescence emitted by the incorporation of a nucleotide base, and the X-axis shows total number of bases added at that point in time; G, C, T, A, nucleotide bases. The underlined letters indicate the nucleotides used for identification of diphyllobothriidean tapeworms.