Molecular Detection of

Diphyllobothrium
nihonkaiense in
Humans, China
Shanhong Chen, Lin Ai, Yongnian Zhang, Jaixu Chen, Weizhe
Zhang, Yihong Li, Maki Muto, Yasuyuki Morishima, Hiromu
Sugiyama, Xuenian Xu, Xiaonong Zhou, and Hiroshi Yamasaki
Reported by: Chan, Fernandez, Lansang, Ortega, San Antonio, Vivo

Introduction

Fish-borne cestodiasis

Diphyllobothriosis
Genus
Diphyllobothrium

D. latum and D. nihonkaiense

Humans are definitive hosts

Image from:
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nt/uploads/2015/05/seared-salmon-raw.jpg

Clinical Symptoms
Can be absent or mild
Mild abdominal pain
Watery diarrhea
Abdominal discomfort

Epidemiology
Japan
South Korea
Far Eastern Federal District of Russia (originally reported
as D. klebanovskii infection)
Europe
North America
New Zealand
China

Misidentification of the
species
Use of morphologic characteristics is
inadequate
Diphyllobothriosis initially attributed to D.
latum was confirmed to be caused by D.
nihonkaiense using
molecular analysis of expelled proglottids

Misidentification of the
species
D. nihonkaiense infection
4 cases in Shanghai (2008-2011)
1 case in the moderately populous city of
Harbin in Heilongjiang Province

Methodology

 5 recent cases of Diphyllobothrium spp. infections in human were examined

Each case originally reported D. latum infection on the basis of morphologic
identification
Case 12 - Harbin City, Heilongjang Province
Cases 16-19 - Shanghai; eating of raw salmon
D. latum is associated with consumption of freshwater fish such as
perch (Perca spp.), not Pacific salmon (Oncorhynchus keta, O. masou) and
Atlantic salmon (Salmo salar) in the Northern Hemisphere

 Diphyllobothrium spp. was reidentified by examining

morphologic features of the passed strobili and using
molecular markers
proglottids embedded in
Case 12 - stained with acetic acid
paraffin
Case 16 - preserved in 10% formalin
sagittal sections prepared
Case 17-19 - preserved in 70% formalin
for microscopy

Molecular Identification
Genomic DNA sample extraction
DNeasy Blood & Tissue Kit

Gene Amplification
Polymerase Chain Reaction
Mitochondrial cytochrome c oxidase subunit 1 gene (cox1, 1566 bp)
3 Cases (17 -19): whole gene using Ex Taq DNA polymerase
2 Cases (12 & 16): shorter cox1 fragment (249 bp: bp 880-1128)
using KOD FX DNA polymerase

DNA sequencing of amplicons

Genetic Analyzer

Phylogenetic Analysis
Maximum Likelihood Method using bootstrap resampling
Bayesian Inference using posterior probability
Basic Local Alignment Search Tool (BLAST) Analysis for cases 12 & 16
(sequence identity %)

Results

Identification of
Diphyllobothrium spp.
Cases 1 to 11

Morphological
identification only
Identity: D. latum

Cases 12 to 19

Morphological and
molecular
identification
Identity: 5 cases (12, 16,
17, 18, 19) were found
to be D. nihonkaiense

Identification of Diphyllobothrium spp. using

both morphological and molecular methods

Discussion

Morphological Identification
Criterion: angle formed by the cirrus sac and
the anterior-posterior axis of the
proglottids
D. latum: horizontal angle
D. nihonkaiense: oblique angle
In another study (Jeon et al, 2009): Longitudinal sections of the
gravid proglottids showed an obtuse angle of about 150
degree between the cirrus sac and seminal vesicle

This criterion is not considered definitive

Jeon, H.-K., Kim, K.-H., Huh, S., Chai, J.-Y., Min, D.-Y., Rim, H.-J., & Eom, K. S. (2009). Morphologic and Genetic Identification of Diphyllobothrium nihonkaiense in
Korea. The Korean Journal of Parasitology, 47(4), 369375. http://doi.org/10.3347/kjp.2009.47.4.369

Morphological Identification

Saggital sections of the proglottids from case-patients 16-19

Case 12.
D. latum

Case 16.
D. latum

Case 17.
D. latum

Case 18.
D. nihonkaiense

Case 19.
D. latum

Recall: Reading phylogenetic

trees
A phylogenetic tree represents the
evolutionary relationships among a
set of organisms or groups of
organisms, called taxa.
Tips of the tree: groups of
descendent taxa
Nodes: common ancestor
Sister groups and Outgroups

Reference: How to read a phylogenetic tree. Retrieved 29 March 2016 from http://epidemic.bio.ed.ac.uk/how_to_read_a_phylogeny

Molecular Identification
Cytochrome c oxidase subunit 1 (cox 1)
DNA-Based Identification System
Sequence divergences at cox1 regularly
enable the discrimination of closely allied
species in all animal phyla except the
Cnidaria (Hebert et al, 2003)
Hebert, P. D. N., Ratnasingham, S., & deWaard, J. R. (2003). Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species.
Proceedings of the Royal Society B: Biological Sciences, 270(Suppl 1), S96S99. http://doi.org/10.1098/rsbl.2003.0025

Molecular Identification
Phylogenetic tree based on the complete cox 1 nucleotide
sequences showed the same topologies in maximum
likelihood and Bayesian inference analyses.
Case patients 17, 18, 19 (GenBank accession numbers
AB674621-AB648623) are D. nihonkaiense
Case patients 12 and 16 were identified as D.
nihonkaiense on the basis of their 99-100% sequence
identity to D. nihonkaiense

The Chinese isolates

(AB684621-23) are sister
groups with the D.
nihonkaiense isolate
(AM412559)

Bootstrap values and

posterior probabilities

Number of base
substitutions per site

Diphyllobothrium
nihonkaiense

First described by Yamane in 1986 in Japan

Cases were also reported in South Korea and in the
Far Eastern Federal District of Russia
Autochthonous and linked to the consumption of
wild Pacific Salmon in these regions
Oncorhynchus masou, O. keta, and Hucho perryi (Jeon et al, 2009)

Discrepancies in the identification

of Diphyllobothrium species
Morphologic similarities between the
species of of D. latum and D. nihonkaiense
Century-long confusion between the parasite
D. latum and the parasite that caused
human diphyllobothriasis associated with
the consumption of Pacific Salmon in Japan

Conclusion

Diphyllobothriosis in Mainland
China
Case caused by D. nihonkaiense
Confirmed using a mitochondrial DNA Marker

Diphyllobothriosis Cases
Thought to be caused by D. latum
Misdiagnosis due to morphologic similarities

Molecular Analysis as the best

tool
No more diagnostic confusion
Reliable epidemiologic and epizootic information
Improve clinical relevance and preventive controls
for diphyllobothriosis

Recommendations

1 Dissemination Information
For food handlers, restaurant owners, physicians,
and consumers

2 Careful Identification of
Species
Note:
Causes of previous Diphyllobothriasis cases
in china can longer be determined

3 Conduct Studies
Studies on distribution
Identify sources of infection

References

Shanhong Chen, Lin Ai, Yongnian Zhang, Jiaxu Chen, Weizhe Zhang, Yihong Li, Maki Muto, Yasuyuki
Morishima, Hiromu Sugiyama, Xuenian Xu, Xiaonong Zhou, and Hiroshi Yamasaki. (2014). Molecular
Detection of Diphyllobothrium nihonkaiense in Humans, China. Emerging Infectious Diseases, 20(2),
315-318.
Jeon, H.-K., Kim, K.-H., Huh, S., Chai, J.-Y., Min, D.-Y., Rim, H.-J., & Eom, K. S. (2009). Morphologic and
Genetic Identification of Diphyllobothrium nihonkaiense in Korea. The Korean Journal of Parasitology,
47(4), 369375. http://doi.org/10.3347/kjp.2009.47.4.369
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