Molecular differentiation and phylogenetic relationships of three Angiostrongylus species and Angiostrongylus cantonensis geographical isolates based on a 66-kDa protein gene of A. cantonensis (Nematoda: Angiostrongylidae) Praphathip Eamsobhana a, * , Phaik Eem Lim b,c , Hongman Zhang d , Xiaoxian Gan e , Hoi Sen Yong b a Department of Parasitology, Faculty of Medicine Siriraj Hospital, Mahidol University, Bangkok 10700, Thailand b Institute of Biological Sciences, University of Malaya, 50603 Kuala Lumpur, Malaysia c Institute of Ocean and Earth Science, University of Malaya, 50603 Kuala Lumpur, Malaysia d Guangxi Zhang Autonomous Region, Center for Disease Control and Prevention, Nanning 530028, Guangxi, PR China e Institute of Parasitic Diseases, Zhejiang Academy of Medical Sciences, Hangzhou 310013, PR China article info Article history: Received 15 January 2010 Received in revised form 28 May 2010 Accepted 1 June 2010 Available online 8 June 2010 Keywords: Angiostrongylus cantonensis Angiostrongylus costaricensis Angiostrongylus malaysiensis Molecular differentiation Phylogenetic relationships DNA sequences abstract The phylogenetic relationships and molecular differentiation of three species of angiostrongylid nem- atodes (Angiostrongylus cantonensis, Angiostrongylus costaricensis and Angiostrongylus malaysiensis) were studied using the AC primers for a 66-kDa protein gene of A. cantonensis. The AC primers successfully amplified the genomic DNA of these angiostrongylid nematodes. No amplification was detected for the DNA of Ascaris lumbricoides, Ascaris suum, Anisakis simplex, Gnathostoma spinigerum, Toxocara canis, and Trichinella spiralis. The maximum-parsimony (MP) consensus tree and the maximum-like- lihood (ML) tree both showed that the Angiostrongylus taxa could be divided into two major clades – Clade 1 (A. costaricensis) and Clade 2 (A. cantonensis and A. malaysiensis) with a full support bootstrap value. A. costaricensis is the most distant taxon. A. cantonensis is a sister group to A. malaysiensis; these two taxa (species) are clearly separated. There is no clear distinction between the A. cantonen- sis samples from four different geographical localities (Thailand, China, Japan and Hawaii); only some of the samples are grouped ranging from no support or low support to moderate support of boot- strap values. The published nucleotide sequences of A. cantonensis adult-specific native 66 kDa pro- tein mRNA, clone L5–400 from Taiwan (U17585) appear to be very distant from the A. cantonensis samples from Thailand, China, Japan and Hawaii, with the uncorrected p-distance values ranging from 26.87% to 29.92%. Ó 2010 Elsevier Inc. All rights reserved. 1. Introduction Angiostrongylus cantonensis, a nematode parasite inhabiting the pulmonary arteries of rats, is a primary cause of human eosino- philic meningitis or eosinophilic meningoencephalitis (cerebral angiostrongyliasis) in Asia and the Pacific Islands (Eamsobhana and Tungtrongchitr, 2005; Eamsobhana, 2006). Angiostrongylus costaricensis which inhabits the mesenteric arteries of rats pro- duces abdominal angiostrongyliasis in humans in Central and South America (Morera, 1985). Angiostrongylus malaysiensis is very similar to A. cantonensis and also inhabits the lung of rats. It has been shown to produce neurologic abnormality in infected rodent host (Cross, 1979), but the potential of being pathogenic to humans need further elucidation. Biochemical and immunological approaches had been used to discriminate antigenic variability in Angiostrongylus species (A. cantonensis, A. costaricensis and A. malaysiensis)(Eamsobhana et al., 1998; Sawabe and Mikiya, 1994). More recently, molecular analysis has been used to differentiate various Angiostrongylus spe- cies. Restriction fragment length polymorphisms (RFLPs) have proved valuable in differentiating A. cantonensis, A. costaricensis, and Angiostrongylus vasorum (Caldeira et al., 2003). The small-sub- unit ribosomal DNA sequences have been used for constructing phylogenetic tree of five species of Angiostrongylus, viz. A. canton- ensis, A. costaricensis, Angiostrongylus dujardini, A. malaysiensis and A. vasorum (van Megan et al., 2009; Fontanilla and Wade, 2008). Phylogenetic tree has also been constructed with internal tran- scribed spacer 2 (ITS-2) for A. cantonensis, A. costaricensis (from Costa Rica and Brazil) and A. vasorum (from Brazil and Europe) (Jefferies et al., 2009). In addition other nucleotide sequences, including the mitochondrial cytochrome-c oxidase subunit I (COI) have been deposited in the GenBank. 0014-4894/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.exppara.2010.06.001 * Corresponding author. E-mail address: sipes@mahidol.ac.th (P. Eamsobhana). Experimental Parasitology 126 (2010) 564–569 Contents lists available at ScienceDirect Experimental Parasitology journal homepage: www.elsevier.com/locate/yexpr