PHYLOGENY OF A. CANTONENSIS IN THAILAND BASED ON CO1 PHYLOGENY OF ANGIOSTRONGYLUS CANTONENSIS IN THAILAND BASED ON CYTOCHROME C OXIDASE SUBUNIT I GENE SEQUENCE Apichat Vitta1,2,3, Narongrit Srisongcram1, Jittranuch Thiproaj1, Anucha Wongma1, Wilaiwan Polsut1, Chamaiporn Fukruksa1, Thatcha Yimthin1, Bandid Mangkit4, Aunchalee Thanwisai1,2,3 and Paron Dekumyoy5 1Department of Microbiology and Parasitology, Faculty of Medical Science, 2Center of Excellence in Medical Biotechnology (CEMB), 3Center of Excellence for Biodiversity Naresuan University, Phitsanulok; 4Department of Veterinary Technology, Faculty of Veterinary Technology, Kasetsart University, Bangkok; 5Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok, Thailand Abstract. Angiostrongylus cantonensis is an emerging infectious agent caus- ing eosinophilic meningitis or meningoencephalitis in humans with clinical manifestation of severe headache. Molecular genetic studies on classification and phylogeny of A. cantonensis in Thailand are limited. This study surveyed A. cantonensis larvae prevalence in natural intermediate hosts across Thailand and analyzed their phylogenetic relationships. A total of 14,032 freshwater and land snails were collected from 19 provinces of Thailand. None of Filopaludina sp, Pomacea sp, and Cyclophorus sp were infected with Angiostrongylus larvae, whereas Achatina fulica, Cryptozona siamensis, and Megaustenia siamensis col- lected from Kalasin, Kamphaeng Phet, Phetchabun, Phitsanulok, and Tak Provinces were infected, with C. siamensis being the common intermediate host. Based on morphology, larvae isolated from 11 samples of these naturally infected snails preliminarily were identified as A. cantonensis. Comparison of partial nucleotide sequences of cytochrome c oxidase subunit I gene revealed that four sequences are identical to A. cantonensis haplotype ac4 from Bangkok and the other seven to that of A. cantonensis isolate AC Thai, indicating two independent lineages of A. cantonensis in Thailand. Keywords: Angiostrongylus cantonensis, cytochrome c oxidase subunit I gene, phylogeny, snail, Thailand INTRODUCTION to the superfamily Metastrongylidae and family Angiostrongylidae (Wang et al, Angiostrongylus cantonensis (rat lung- 2008). A. cantonensis was first found in worm) is a zoonotic nematode belonging Rattus in China (Chen, 1935). At present, this worm is globally present, particularly Correspondence: Apichat Vitta, Department of Microbiology and Parasitology, Faculty of in Southeast Asia and the Pacific Islands Medical Science, Naresuan University, Phitsa- (Eamsobhana, 2006). Its life cycle requires nulok 65000, Thailand. gastropods as intermediate hosts and Tel: +66 (0) 55 964653; Fax: +66 (0) 55 964770 rodents as definitive hosts (Eamsobhana, E-mail [email protected] 2006). Larvae have been found in diverse Vol 47 No. 3 May 2016 377 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH families of mollusk intermediate hosts Hongladarom and Indarakoses, 1966; and adult worms reside in pulmonary Tangchai et al, 1967; Bunnag et al, 1969; Pu- arteries and right ventricle of rodent de- nyagupta et al, 1970; Nitidandhaprabhas finitive hosts. et al, 1975; Panamonta, 1985; Witoonpanich Humans are an accidental host, in et al, 1991; Laopornpichayanuwat, 2000; whom immature worms are not often Sawanyawisuth et al, 2007). found in infected organs although over In view of the number of reported 2,800 cases of human angiostrongyliasis cases of human angiostrongyliasis with have been recorded from some 30 countries eosinophilic meningitis, surveys of the (Wang et al, 2008). Nevertheless, A. canto- natural hosts of this worm have been con- nensis is considered as a primary causative ducted. In the early studies, a high preva- agent of eosinophilic meningitis or menin- lence (36.4-94.4%) of this rat lungworm in goencephalitis in humans (Eamsobhana, Achatina fulica was reported (Harinasuta 2006). Recently, this worm has been rec- et al, 1965; Setasubun et al, 1968; Pipitgool ognized as being an emerging infectious et al, 1997) while a lower prevalence (7.55- agent in North and South America, being 12.38%) was reported from recent studies found in both natural hosts and humans (Tesana et al, 2009; Vitta et al, 2011). Recent- (Maldonado et al, 2010; Thiengo et al, 2010; ly, Cryptozona siamensis was identified as a Espírito-Santo et al, 2013; Teem et al, 2013). new intermediate host for A. cantonensis in Transmission of A. cantonensis corresponds Thailand (Vitta et al, 2016). Prevalence of to the dispersal of intermediate and defini- A. cantonensis in rodent hosts was reported tive hosts. Invasive snails and rodents are to range from 1.4% to 42.1% from several implicated in an increase of the distribution parts of Thailand (Setasubun et al, 1968; of A. cantonensis in China (Lv et al, 2011; Namue and Wongsawad, 1997; Pipitgool Yang et al, 2013), Japan (Tokiwa et al, 2013) et al, 1997; Vitta et al, 2011). and Brazil (Graeff-Teixeira, 2007). Use of molecular genetics in iden- At least four species of the superfam- tification and analysis of phylogenetic ily Metastrongylidae, namely, A. cantonen- relationships of A. cantonensis is limited. sis, A. malaysiensis, A. siamensis, and Thais- Small subunit (SSU) ribosomal (r)DNA trongylus harinasuti, have been recorded has been used for identification of A. in Thailand (Chen, 1935; Bhaibulaya and cantonensis in Japan (Tokiwa et al, 2012). Cross, 1971; Ohbayashi et al, 1979). Only However, cytochrome c oxidase subunit A. cantonensis is the cause of angiostron- I (co1) is employed for study of phyloge- gyliasis in the country, the first being doc- netic relationships among closely related umented by Khwanmitra et al (1957), and species (Jefferies et al, 2009; Eamsobhana since then, over 2,000 cases of human an- et al, 2010a; Foronda et al, 2010; Monte giostrongyliasis have been reported from et al, 2012; Tokiwa et al, 2012; Lee et al, all areas of Thailand, but especially in the 2014). Thus, the latter technique was ad- northeast region of the country where opted to survey A. cantonensis in natural snails are commonly eaten (Khwanmi- intermediate hosts across Thailand. This tra et al, 1957; Tantibhedyangur, 1963; study provides a basic knowledge of the Benjapongse, 1964; Buranasin et al, 1965; genetic diversity and phylogeny related to Chularerk and Suyarnsethakorn, 1965; Jit- classification of isolates of A. cantonensis tayasothorn et al, 1965; Punyagupta, 1965; in Thailand. 378 Vol 47 No. 3 May 2016 PHYLOGENY OF A. CANTONENSIS IN THAILAND BASED ON CO1 MATERIALS AND METHODS Collection and isolation of An- giostrongylus from snails Fresh water snails (Filo- paludina sp and Pomacea sp) and land snails (Achatina fulica, Cryptozona siamensis, Cyclopho- rus spp, Megaustenia siamensis) were collected between July 2012 and January 2014 from 19 provinces of Thailand (Bueng Kan, Chaiyaphum, Chiang Mai, Kalasin, Kamphaeng Phet, Khon Kaen, Lampang, Lam- phun, Maha Sarakham, Mukda- han, Nakhon Ratchasima, Non- thaburi, Phetchabun, Phichit, Phitsanulok, Phrae, Sukhothai, Tak, and Uthai Thani) (Fig 1). Snails were identified at genus and species levels by external shell morphology according to Brandt (1974), Upatham et al (1983) and Panha (1996). Snails were digested with 0.7% pep- sin solution (Acros Organics, Geel, Belgium) for 1-2 hours at 37°C with shaking. Baermann’s technique was used for isolat- ing infective larvae (3rd stage larvae) of A. cantonensis (Vitta et al, 2016). In brief, a glass funnel joined with a piece of rubber tubing was used as the Baermann apparatus. A wire Fig 1–Map of snail sampling provinces across Thailand. Snail mesh covered with several lay- with (-) indicates snail negative and with (+) positive for Angiostrongylus larvae. Intensity of Angiostrongylus ers of gauze was placed at the larvae was 1.08 and 0.07 in A. fulica and C. siamensis bottom of the funnel, and with collected from Phetchabun Province, 1.65 and 0.07 in the outlet closed, digested snail A. fulica and C. siamensis from Kalasin Province, 4.33 content was poured into the and 0.01 in A. fulica and M. siamensis from Tak Province, funnel until the fluid level made 0.07 and 0.005 in A. fulica and C. siamensis collected from contact the wire mesh. In order Phitsanulok Province, and 0.1 and 23 in A. fulica and C. to allow Angiostrongylus larvae siamensis from Kamphaeng Phet Province. to migrate into the rubber tub- Vol 47 No. 3 May 2016 379 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH ing, the apparatus was left standing for seconds, and 72oC for 1 minute; with a 30-60 minutes. At the end of this period, final heating at 72oC for 5 minutes. Am- the rubber tubing was opened allowing plicons (450 bp) were analyzed by 1.2% the fluid containing enriched larvae to agarose gel-electrophoresis and ethidium be released into a petri dish. Larvae were bromide-stained bands were visualized collected under a stereomicroscope and and photographed under UV light. Am- identified as previously described (Eam- plicons were purified using a Gel/PCR sobhana, 2006) and stored at -20°C until DNA Fragments Extraction Kit (Geneaid used. Biotech, New Taipei City, Taiwan) and Amplification and sequencing ofA. can- sequenced (Macrogen, Seoul, Korea). tonensis cytochrome c oxidase subunit I Phylogenetic analysis gene (co1) Sequences were edited using SegMa- Genomic DNA from larvae was ex- nII software (DNASTAR, Madison, WI). tracted as previously described (Vitta et al, Sequences of 263-265 bp of Angiostron- 2016). In brief, 10-50 larvae were