Morphological and molecular identification of two Paragonimus spp., of which metacercariae concurrently found in a land crab, Potamiscus tannanti, collected in Yenbai Province, Vietnam

Abstract

Paragonimosis is an important food-borne zoonosis especially in Asian countries. Among Paragonimus species, Paragonimus westermani followed by P. skrjabini complex are the major pathogens for human paragonimosis in Asia. In addition, P. heterotremus is an important pathogen in southern China and the Indochina Peninsula and is the only proven species to cause human paragonimosis in Vietnam. During a recent survey in Yenbai Province in northern Vietnam, we found small and large types of Paragonimus metacercariae often concurrently in mountainous crabs, Potamiscus tannanti. Adult worms from those small and large metacercariae were obtained separately by experimental infection in dogs and cats. Morphological and molecular phylogenetic study based on sequences of ITS2 and a part of CO1 genes were performed for the identification of small and large metacercariae and their adults. The results showed that small metacercariae and their adults are completely identical with P. heterotremus in morphology and molecular genetic profiles. In contrast, large metacercariae and their adults have some morphological similarities with P. skrjabini and P. harinasutai, but are unidentifiable from each other by morphology alone. Molecular phylogenetic tree analyses on ITS2 and CO1 genes revealed that large metacercariae and their adults were grouped in the same clade and different from any known Paragonimus species. Although they share the same ancestor with P. skrjabini complex, their genetic distance was considerably different from two other known subspecies, P. skrjabini skrjabini and P. skrjabini miyazakii. Our results provide a new insight on the phylogeny of the genus Paragonimus.

Appendix

Appendix 1: Accession numbers of sequences obtained from DNA database for reconstructing the phylogenetic trees

Sources of data: 1, Blair et al. (1999a); 2, Blair et al. (1997); 3, Iwagami et al. (2003); 4, Blair et al. (2005); 5, published only in database. Euparagonimus cenocopiosus ITS2: AF159601–159602 (Blair et al. 1999a); CO1: AF159594–159595 (Blair et al. 1999a). P. westermani ITS2: AF159604 (Blair et al. 1999a) and U96907 (Blair et al. 1997); CO1: U97205 and U97213 (Blair et al. 1997). P. ohirai ITS2: U96911 (Blair et al. 1997); CO1: U97214 (Blair et al. 1997). P. harinasutai ITS2: AF159609 (Blair et al. 1999a); CO1: AF159600 (Blair et al. 1999a). P. mexicanus ITS2: AF538945–538946 (Iwagami et al. 2003); CO1: AF538944 and AF538936 (Iwagami et al. 2003). P. bangkokensis ITS2: AB244621 and AB248091 (published only in database). P. macrorchis ITS2: AF159608 (Blair et al. 1999a); CO1: AF159598 (Blair et al. 1999a). P. heterotremus ITS2: AY618758 (Blair et al. 2005) and AF159603 (Blair et al. 1999a); CO1: AY618840–618841 (Blair et al. 2005) and AD159597 (Blair et al. 1999a). P. hokuoensis ITS2: AY618756 (Blair et al. 2005); CO1: AY618836 and AY618838 (Blair et al. 2005). P. skrjabini skrjabini ITS2: AY618729, AY618731, AY618734 and AY618751 (Blair et al. 2005); CO1: AY608762, AY618797, AY618783, AY618802 and AY618806 (Blair et al. 2005). P. skrjabini miyazakii ITS2: AY618740 and AY618757 (Blair et al. 2005); CO1: AY618814 and AY618832 (Blair et al. 2005).