Angiostrongylus cantonensis and A. malaysiensis Broadly Overlap in Thailand, Lao PDR, Cambodia and Myanmar: A Molecular Survey of Larvae in Land Snails

Abstract

Angiostrongylus cantonensis is a zoonotic nematode parasite causing human eosinophilic meningitis (or meningoencephalitis) worldwide. A closely related species, Angiostrongylus malaysiensis, might also be a human pathogen. Larvae were obtained from land snails in Lao PDR, Cambodia, Myanmar and Thailand. We sequenced two nuclear gene regions (nuclear ribosomal ITS2 and SSU rRNA) and a portion of one mitochondrial gene (COI) from these larvae. Angiostrongylus cantonensis and A. malaysiensis were identified. This is the first report of the molecular identification of the two Angiostrongylus species in Lao PDR, Cambodia and Myanmar. The regional distributions of the two species broadly overlap. Phylogenetic relationships were inferred including data from Angiostrongylus species deposited in public databases. All the gene regions we sequenced have potential value in distinguishing between species of Angiostrongylus. The COI gene exhibited the greatest intraspecific variation in the study region (five haplotypes in A. cantonensis and four in A. malaysiensis) and might be suitable for more detailed phylogeographic studies.

Fig 1. Map of sampling locations of Angiostrongylus cantonensis and Angiostrongylus malaysiensis showing their overlapping distributions in six countries.

Stars indicate A. malaysiensis; circles indicate A. cantonensis; the dotted line shows inferred range of A. malaysiensis; the dashed line shows range of A. cantonensis in the region. 1, Mongla Township in Myanmar (present study); 2, Vientiane capital city in Lao PDR (present study); 3, Siem Reap Province of Cambodia (present study); 4–21, Provinces in Thailand (4, Chiang Rai (KP721449); 5, Nan (KC995246); 6, Chiang Mai (KP721447); 7, Mae Hong Sorn (KP732099); 8, Phrae (present study); 9, Tak (KP732097); 10, Phitsanulok (KP721445); 11, Khon Kaen (present study); 12, Maha-Sarakham (KC995223); 13, Lopburi (KC995212); 14, Kanchanaburi (KC995206); 15, Bangkok (KC995188); 16, Samut Prakan (KP721442); 17, Prachuap Khiri Khan (KC995262); 18, Surat Thani (present study); 19, Nakhon Si Thammarat (KC995260); 20, Phang Nga (KP732100); 21, Phattalung (present study)); 22, Cao Bang Province of Vietnam (De et al [18]); 23, Peninsular Malaysia (JN663729).

Fig 2. Maximum-likelihood phylogenetic tree of five species of Angiostrongylus (A. cantonensis, A. malaysiensis, A. dujardini, A. costaricensis and A. vasorum), Aelurostrogylus abstrusus and Nippostronglus braziliensis (outgroups) based on partial SSU rRNA sequences.

Support values (ML bootstrap/MP bootstrap/NJ bootstrap/Bayesian posterior probabilities) are shown above the branches. A dash (-) instead of a numerical support value indicates that a particular grouping was not found by that method of analysis. Bold letters indicate sequences obtained in the present study. Numbers in parentheses following each newly obtained sequence indicate the number of larvae possessing that sequence.

Fig 3. Maximum-likelihood phylogenetic tree of Angiostrongylus cantonensis, A. malaysiensis, A. vasorum, A. chabaudi, A. costaricensis and A. dujardini based on the partial ITS2 sequences.

Support values (ML bootstrap/MP bootstrap/NJ bootstrap/Bayesian posterior probabilities) are shown above the branches. A dash (-) instead of a numerical support value indicates that a particular grouping was not found by that method of analysis. Bold letters indicate sequences obtained in the present study. Numbers in parentheses following each newly obtained sequence indicate the number of larvae possessing that sequence.

Fig 4. Maximum-likelihood phylogenetic tree of Angiostrongylus cantonensis, A. malaysiensis, A. vasorum, A. costaricensis, Aelurostrogylus abstrusus and Metastrongylus salmi (the last two as outgroups) based on partial COI sequences.

Support values (ML bootstrap/MP bootstrap/NJ bootstrap/Bayesian posterior probabilities) are shown above the branches. A dash (-) instead of a numerical support value indicates that a particular grouping was not found by that method of analysis. Bold letters indicate sequences obtained in the present study. AC1-AC13 are the 13 distinct haplotypes of A. cantonensis; AM1-AM4 are the 4 distinct haplotypes of A. malaysiensis. Numbers in parentheses following each newly obtained sequence indicate the number of larvae possessing that sequence.