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Levels of infection with the lungworm Angiostrongylus cantonensis in terrestrial snails from Thailand, with Cryptozona siamensis as a new intermediate host

Published online by Cambridge University Press:  15 January 2016

A. Vitta*
Affiliation:
1Department of Microbiology and Parasitology, Faculty of Medical Science 2Centre of Excellence in Medical Biotechnology (CEMB) 3Center of Excellence for Biodiversity, Naresuan University, Phitsanulok 65000, Thailand
W. Polsut
Affiliation:
1Department of Microbiology and Parasitology, Faculty of Medical Science
C. Fukruksa
Affiliation:
1Department of Microbiology and Parasitology, Faculty of Medical Science
T. Yimthin
Affiliation:
1Department of Microbiology and Parasitology, Faculty of Medical Science
A. Thanwisai
Affiliation:
1Department of Microbiology and Parasitology, Faculty of Medical Science 2Centre of Excellence in Medical Biotechnology (CEMB) 3Center of Excellence for Biodiversity, Naresuan University, Phitsanulok 65000, Thailand
P. Dekumyoy
Affiliation:
4Department of Helminthology, Faculty of Tropical Medicine, Mahidol University, Bangkok 10400, Thailand
*
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Abstract

Angiostrongylus cantonensis is primarily considered an emerging infectious agent of eosinophilic meningitis or meningoencephalitis with a worldwide distribution. Rodents and snails are important invasive hosts for transmission and expansion of A. cantonensis. The objective of this study was to investigate infection levels of A. cantonensis in snails, the most important natural intermediate host. Our study location was Mueang Kamphaeng Phet district, Kamphaeng Phet Province, and was undertaken between October and December 2012. A total of 2228 freshwater and terrestrial snails were collected, comprising 1119 Filopaludina spp., 409 Pomacea caniculata, 275 Achatina fulica and 425 Cryptozona siamensis. Angiostrongylus larvae were isolated by artificial digestion methods following Baermann's techniques. A low prevalence and intensity of A. cantonensis were observed in A. fulica, while higher numbers were found in C. siamensis. None of the Filopaludina spp. and Pomacea caniculata were infected with A. cantonensis. Molecular characterization was performed by analysing the 264 bp of cytochrome c oxidase subunit I (COI). Three COI sequences of Angiostrongylus were identical to A. cantonensis with 91–99% identity. Cryptozona siamensis has not previously been recorded as an intermediate host for A. cantonensis in Thailand. The infection of A. cantonensis identified in the natural intermediate hosts is new and important information to assist in the prevention and control of human angiostrongyliasis.

Type
Research Papers
Copyright
Copyright © Cambridge University Press 2016 

Introduction

Angiostrongylus cantonensis or the rat lungworm is the causative agent of human angiostrongyliasis. It is known to be a cause of eosinophilic meningitis and eosinophilic meningoencephalitis. Human angiostrongyliasis has been reported in more than 30 countries on all continents, but especially in Taiwan, China and Thailand (Wang et al., Reference Wang, Lai, Zhu, Chen and Lun2008). Recently, infections in both natural hosts and human cases have been reported from North and South America (Maldonado et al., Reference Maldonado, Simões, Oliveira, Motta, Fernandez, Pereira, Monteiro, Torres and Thiengo2010; Thiengo et al., Reference Thiengo, Maldonado, Mota, Torres, Caldeira, Carvalho, Oliveira, Simões, Fernandez and Lanfredi2010; Espírito-Santo et al., Reference Espírito-Santo, Pinto, Mota and Gryschek2013; Teem et al., Reference Teem, Qvarnstrom, Bishop, da Silva, Carter, White-McLean and Smith2013). In general, humans are an accidental host where the worms develop into the young adult stage without being able to develop into adult stages. Infection with A. cantonensis occurs following the ingestion of improperly cooked food from intermediate hosts such as Pila snails and Filopaludina snails, or from eating improperly cooked food from paratenic hosts such as shrimp, crab and yellow tree monitor, or from eating vegetables contaminated with infective larvae. In Thailand, the infection in patients usually occurs in communities that eat raw or undercooked Pila snails, often as an accompaniment to drinking alcohol (Eamsobhana, Reference Eamsobhana2013). The most obvious clinical symptoms are severe headache and neck stiffness. Diagnosis can be made by several methods, such as knowing the history of eating intermediate or paratenic hosts, laboratory findings and clinical symptoms (Wang et al., Reference Wang, Lai, Zhu, Chen and Lun2008; Vitta, Reference Vitta2012). There are no specific drugs available for the treatment of this infection.

Angiostrongylus cantonensis requires both an intermediate and a definitive host to complete its life cycle. Previously, gastropods from 44 families, 99 genera and 140 species, identified as intermediate hosts, naturally or experimentally infected with A. cantonensis, were found to have worldwide distribution (Kim et al., Reference Kim, Hayes, Yeung and Cowie2014). In Thailand specifically, several species of freshwater snails have been reported as natural intermediate hosts, namely, Pila ampullacea, P. polita, P. gracilis, P. scutata, P. pesmei, P. turbinis, Pomacea canaliculata, Clea helena, Bithynia siamensis goniomphalos, Melanoides tuberculata, Filopaludina martensi martensi and F. sumartrensis polygramma (Punyagupta, Reference Punyagupta1965; Crook et al., 1968; Setasubun et al., 1968; Pipitgool et al., Reference Pipitgool, Sithithaworn, Pongmuttasaya and Hinz1997; Tesana et al., Reference Tesana, Srisawangwong, Sithithaworn, Laha and Andrews2009). The terrestrial snails and slugs included Achatina fulica, Hemiplecta distincta, H. siamensis, Macrochlamys (Sarika) resplendens and Veronicella siamensis, all of which have been documented as natural intermediate hosts for the rat lungworm in Thailand, including Cryptozona siamensis as a new intermediate host (Crook et al., 1968; Pipitgool et al., Reference Pipitgool, Sithithaworn, Pongmuttasaya and Hinz1997; Tesana et al., Reference Tesana, Srisawangwong, Sithithaworn, Laha and Andrews2009; Vitta et al., Reference Vitta, Polseela, Nateeworanart and Tattiyapong2011). Rodents are definitive hosts for A. cantonensis. Five species of rat, namely Rattus rattus, R. exulans, R. norvegicus, Bandicota indica and B. bengalensis, have been found to be naturally infected with A. cantonensis (Setasubun et al., 1968; Namue & Wongsawad, Reference Namue and Wongsawad1997; Pipitgool et al., Reference Pipitgool, Sithithaworn, Pongmuttasaya and Hinz1997; Vitta et al., Reference Vitta, Polseela, Nateeworanart and Tattiyapong2011). Paratenic hosts observed naturally were the reptile yellow tree monitor (Varanus bengalensis) (Radomyos et al., Reference Radomyos, Tungtrongchitr, Praewanich, Khewwatchan, Kantangkul, Junlananto and Ayudhya1994), prawns and the clawed frog (Xenopus laevis) and its tadpoles (Cross & Chen, Reference Cross, Chen, Murrell and Fried2007).

The high susceptibility of its hosts is considered to be an important factor in the distribution of A. cantonensis throughout these areas. Thus the aim of the present study was to investigate the infection levels of A. cantonensis in natural intermediate hosts from Kamphaeng Phet Province, Thailand. As part of this survey, Cryptozona siamensis, a terrestrial snail, was collected and helminth larvae were isolated. This snail is found in several regions of Thailand because the environment is suitable for its development. Molecular characterization of the A. cantonensis worm was also performed to confirm the morphological identification.

Materials and methods

Collection of larval nematodes from snails

Freshwater snails Filopaludina spp. and Pomacea caniculata and terrestrial snails Achatina fulica and Cryptozona siamensis were collected randomly in the Trai Trueng sub-district, Mueang Kamphaeng Phet district, Kamphaeng Phet Province, between October and December 2012. Snails were maintained in aerated boxes and transported to the Department of Microbiology and Parasitology at the Faculty of Medical Science, Naresuan University, where they were fed on lettuce leaves for a short time in the laboratory. The shell of each snail was cracked open with a hard stone and the entire soft parts removed, dissected and minced in a blender with 0.7% pepsin solution containing 7 g of pepsin powder (Acros Organics, Geel, Belgium), 10 ml of HCl and 990 ml of distilled water. The snail contents and pepsin solution were then incubated in a water bath at 37°C for 90 min. The Baermann technique was used for isolating the Angiostrongylus larvae, which were identified morphologically by the following characteristics: possessing a long, thin body with two chitinous rods at the anterior end, transverse striations on the cuticle and a cone-shaped, slightly curved and pointed tail (Eamsobhana, Reference Eamsobhana2006).

DNA extraction

Genomic DNA was extracted from the larvae. Approximately 50 larvae were put in a 1.5-ml microcentrifuge tube containing 5 μl ATL buffer (Qiagen, Germany). The worms were ground with a sterile tip. Another 45 μl of ATL buffer was added. The tube was vortexed vigorously for about 1 min. The lysate was frozen at − 20°C for 1 h, thawed at room temperature and digested with 15 μl of 10 mg/ml proteinase K. The mixture was incubated at 65°C overnight and then at 95°C for 1 h. Subsequently, the tube was centrifuged at 12,000 rpm for 2 min. The supernatant containing DNA was precipitated with 1/10 volume of 5 m NaCl followed by 2 volumes of cool absolute ethanol and centrifuged at 12,000 rpm for 30 min. The pellet was washed by centrifugation with 100 μl 70% ethanol, dried in air and dissolved with 15 μl of distilled water. One microlitre of purified genomic DNA was examined on 0.8% agarose gel electrophoresis running in 0.5 ×  TBE (Tris–borate–EDTA) buffer at 100 V. The gel was stained with 10 μg/ml ethidium bromide for 1 min and destained with distilled water for 30 min. The DNA band was visualized and compared to a 100 bp molecular size marker (GeneDireX, Taoyuan, Taiwan). The DNA was kept at − 20°C until used.

Molecular analysis

The polymerase chain reaction (PCR) was performed to amplify a partial region of cytochrome c oxidase subunit I (COI) according to Jefferies et al. (Reference Jefferies, Shaw, Viney and Morgan2009). Specific primers were: CO1_F 5′-TAAAGAAAGAACATAATGAAAATG-3′ and CO1_R 5′-TTTTTTGGGCATCCTGAGGTTTAT-3′. The PCR reaction was carried out in 30 μl volumes which consisted of 3 μl of 10 ×  buffer, 4.2 μl of 25 mm MgCl2, 0.6 μl of 200 mm deoxynucleoside triphosphates (dNTPs), 1.2 μl of of each primer (5 μm), 0.6 μl of 100 units Taq DNA polymerase (Sigma, St. Louis, Missouri, USA), 11.7 μl of distilled water and 7.5 μl of DNA template. PCR was performed using a thermal cycler (Applied Biosystems Life Technologies, Carlsbad, California, USA) with an initial denaturation at 94°C for 5 min; followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 30 s and extension at 72°C for 1 min; and a final extension at 72°C for 5 min. One microlitre of PCR products was analysed by 1.2% agarose gel electrophoresis in 0.5 ×  TBE buffer at 100 V and compared to a 100 bp standard ladder. The gels were stained with ethidium bromide for 1 min and destained with distilled water for 30 min. The bands were visualized and photographed under UV light.

PCR products were purified using a Gel/PCR DNA Fragments Extraction Kit (Geneaid Biotech Ltd, Taiwan). The PCR product (30 μl) was transferred into a 1.5-ml microcentrifuge tube. Five volumes of Gel/PCR buffer were added to the tube and vortexed. The mixture was then transferred into a DFH column in a 2-ml collection tube and centrifuged at 12,000 rpm for 30 s and the flow-through was discarded. The tube containing the DFH column was centrifuged at 12,000 rpm for 3 min to dry the column matrix. The DFH column was transferred to a new 1.5-ml microcentrifuge tube. Twenty microlitres of elution buffer were added into the centre of the column matrix. The tube was allowed to stand for 2 min in order to completely absorb the elution buffer. To elute the purified DNA, the tube was then centrifuged at 12,000 rpm for 2 min. The purified PCR product was checked by running 1.2% agarose gel electrophoresis in 0.5 ×  TBE buffer at 100 V and comparing to a 100 bp standard ladder. The gels were stained with ethidium bromide for 1 min, destained with distilled water for 30 min and visualized and photographed under UV light. Purified PCR product was sent to Korea for sequencing by Macrogen Inc. (Korea).

To characterize species of Angiostrongylus, a BLASTN search against a nucleotide database was performed (http://blast.ncbi.nlm.nih.gov/Blast.cgi). All sequences were edited using SegManII software (DNASTAR, Madison, Wisconsin, USA). A 264 bp region of the COI gene was used for analysing the species, using ClustalW (Thompson et al., Reference Thompson, Higgins and Gibson1994) and MEGA software version 5.0 (Tamura et al., Reference Tamura, Peterson, Peterson, Stecher, Nei and Kumar2011).

Results

Infection levels of Angiostrongylus cantonensis in snails

Of the freshwater snails, a total of 1119 Filopaludina spp. and 409 P. caniculata were collected but neither snail species was infected, in contrast to both the terrestrial species, A. fulica and C. siamensis. Nevertheless the majority of these two terrestrial species were free of infection with A. cantonensis, and up to 3 of 275 (1.1%) of A. fulica were infected with a range of 5–15 larvae, representing an average intensity of 0.1. Higher levels of infection were found in C. siamensis, where 45 of 425 (10.6%) snails were infected with a range of 1–4858 larvae. An average intensity of 23.0 was recorded, where larval numbers of 1–10 and 11–100 were found in 3.5% and 4.2% of infected snails, respectively.

Molecular characterization of Angiostrongylus cantonensis

A partial region of COI sequence from Angiostrongylus larvae from C. siamensis was determined by PCR and sequenced. PCR products were approximately 450 bp in size when compared with the standard DNA ladder. Two COI sequences of Angiostrongylus larvae collected from two C. siamensis and one COI sequence from an adult worm were obtained. After a BLASTN search of the trimmed sequence of 264 bp, the two sequences from larvae were found to be closely related to A. cantonensis from Miyagi, Sendai, Japan (GenBank accession number AB684375) with 91–92% identity, and the sequence of the adult worm was 99% identical to A. cantonensis from Bangkok, Thailand (GenBank accession number AB684368).

Discussion

Based on both morphological and molecular characterization of the larvae, A. cantonensis was identified and then isolated from A. fulica and C. siamensis. We demonstrated the prevalence and intensity of A. cantonensis in C. siamensis collected from Kamphaeng Phet Province. Having not been previously reported, this land snail is considered to be a novel intermediate host for this worm in Thailand. At present, over 140 species of molluscs have been reported as natural or experimental intermediate hosts for A. cantonensis (Kim et al., Reference Kim, Hayes, Yeung and Cowie2014). This indicates that A. cantonensis occurs in a wide range of intermediate hosts. The terrestrial snail is also distributed in Laos, Cambodia and Malaysia. Two species of this genus, Cryptozona imperator and Cryptozona bistrialis, have previously been reported as hosts of A. cantonensis (Ko, Reference Ko1991; Cross & Chen, Reference Cross, Chen, Murrell and Fried2007). This terrestrial snail can transmit larvae to natural hosts when eaten and, although not a favourite food for Thai people, the snail is found in close proximity to humans in agricultural areas, thereby being a source of infection.

The low prevalence of A. cantonensis in A. fulica was shown in the present study, which is in contrast to previous studies in which a high prevalence of A. cantonensis in A. fulica collected from Thailand was recorded. The high level of prevalence previously reported ranged from 36.4% to 94.4% (Harinasuta et al., Reference Harinasuta, Setasuban and Radomyos1965; Setasubun et al., 1968; Pipitgool et al., Reference Pipitgool, Sithithaworn, Pongmuttasaya and Hinz1997). The prevalence of A. cantonensis in A. fulica in this study was lower than that found by Vitta et al. (Reference Vitta, Polseela, Nateeworanart and Tattiyapong2011) (12.38%) and Tesana et al. (Reference Tesana, Srisawangwong, Sithithaworn, Laha and Andrews2009) (7.55%).

Achatina fulica is considered to be an important intermediate host for transmission of A. cantonensis. Recently, this giant African land snail was introduced to Brazil and was considered as the source of the spread of human angiostrongyliasis (Graeff-Teixeira, Reference Graeff-Teixeira2007). On the other hand, freshwater snails, namely P. polita, F. martensi martensi, F. sumatrensis polygramma, P. pesmei, C. helena and B. siamensis goniomphalos have been reported to have a low prevalence of less than 5% (Setasubun et al., 1968; Pipitgool et al., Reference Pipitgool, Sithithaworn, Pongmuttasaya and Hinz1997; Tesana et al., Reference Tesana, Srisawangwong, Sithithaworn, Laha and Andrews2009), while P. ampullacea was negative for larval infection (Pipitgool et al., Reference Pipitgool, Sithithaworn, Pongmuttasaya and Hinz1997). Our results agree with those above, as no P. canliculta and Filopaludina spp. specimens in our sample were infected with A. cantonensis. This may be due to these two snails being hosts with low susceptibility.

The average intensity of A. cantonensis in C. siamensis was 23 larvae/snail and the highest intensity was 4858 larvae in one snail. It is possible that larvae of A. cantonensis are restricted to the faecal pellets of the definitive host. In addition the dispersion of the larval stage within the snail is typically overdispersed or aggregated within the host population and likely to conform to a negative binomial distribution, where only a small number of snail hosts are infected with high numbers of larvae (Pal & Lewis, Reference Pal and Lewis2004). This was also observed in several species of parasites, such as A. cantonensis in intermediate hosts (Tesana et al., Reference Tesana, Srisawangwong, Sithithaworn, Laha and Andrews2009) and Opisthorchis viverrini in secondary intermediate hosts (Haswell-Elkins et al., Reference Haswell-Elkins, Elkins, Sithithaworn, Treesarawat and Kaewkes1991).

In conclusion, the present study has recorded a new, previously unidentified, intermediate host for A. cantonensis in Thailand. The terrestrial snail C. siamensis should be included in the list of intermediate hosts for A. cantonensis. Prevalence and intensity of A. cantonensis infective larvae were reported to be low in the studied area. However, this investigation provides us with basic knowledge about the biology of A. cantonensis which is new and important information to assist in the prevention and control of human angiostrongyliasis.

Acknowledgements

We would like to thank the Department of Microbiology and Parasitology, Faculty of Medical Science, Naresuan University, Thailand for provision of facilities. We are very grateful to Mr Roy Irvine Morien, Naresuan University Language Centre (NULC), Naresuan University, for assistance in editing the English of the manuscript.

Financial support

We would like to thank the Thailand Research Fund for financial support (grant number MRG5580044).

Conflict of interest

None.

References

Crook, J.R., Fulton, S.E. & Supanwong, K. (1968) Ecological studies on the intermediate and definitive hosts of Angiostrongylus cantonensis (Chen, 1935) in Thailand. Annals of Tropical Medicine and Parasitology 62, 2744.Google Scholar
Cross, J.H. & Chen, E.R. (2007) Angiostrongyliasis. pp. 263290 in Murrell, K.D. & Fried, B. (Eds) Food-borne parasitic zoonoses: Fish and plant-borne parasites. New York, Springer Science and Business Media LLC.Google Scholar
Eamsobhana, P. (2006) The rat lungworm Parastrongylus (Angiostrongylus ) cantonensis: parasitology, immunology, eosinophilic meningitis, epidemiology and laboratory diagnosis. 156 pp. Bangkok, Wankaew (IQ) Book Center.Google Scholar
Eamsobhana, P. (2013) Angiostrongyliasis in Thailand: epidemiology and laboratory investigations. Hawaii Journal of Medicine and Public Health 72, 2832.Google ScholarPubMed
Espírito-Santo, M.C., Pinto, P.L., Mota, D.J. & Gryschek, R.C. (2013) The first case of Angiostrongylus cantonensis eosinophilic meningitis diagnosed in the city of São Paulo, Brazil. Revista do Instituto de Medicina Tropical de São Paulo 55, 129132.CrossRefGoogle ScholarPubMed
Graeff-Teixeira, C. (2007) Expansion of Achatina fulica in Brazil and potential increased risk for angiostrongyliasis. Transactions of the Royal Society of Tropical Medicine and Hygiene 101, 743744.CrossRefGoogle ScholarPubMed
Harinasuta, C., Setasuban, P. & Radomyos, P. (1965) Observations on Angiostrongylus cantonensis in rats and mollusks in Thailand. Journal of the Medical Association of Thailand 48, 158172.Google Scholar
Haswell-Elkins, M.R., Elkins, D.B., Sithithaworn, P., Treesarawat, P. & Kaewkes, S. (1991) Distribution patterns of Opisthorchis viverrini within a human community. Parasitology 103, 97101.Google Scholar
Jefferies, R., Shaw, S.E., Viney, M.E. & Morgan, E.R. (2009) Angiostrongylus vasorum from South America and Europe represent distinct lineages. Parasitology 136, 107115.Google Scholar
Kim, J.R., Hayes, K.A., Yeung, W.N. & Cowie, R.H. (2014) Diverse gastropod hosts of Angiostrongylus cantonensis, the rat lungworm, globally and with a focus on the Hawaiian Islands. Plos One 9, e94969.CrossRefGoogle ScholarPubMed
Ko, R.C. (1991) Current status of food-borne parasitic zoonoses in Hong Kong. The Southeast Asian Journal of Tropical Medicine and Public Health 22, 4247.Google Scholar
Maldonado, A. Jr, Simões, R.O., Oliveira, A.P., Motta, E.M., Fernandez, M.A., Pereira, Z.M., Monteiro, S.S., Torres, E.J. & Thiengo, S.C. (2010) First report of Angiostrongylus cantonensis (Nematoda: Metastrongylidae) in Achatina fulica (Mollusca: Gastropoda) from Southeast and South Brazil. Memórias do Instituto Oswaldo Cruz 105, 938941.Google Scholar
Namue, C. & Wongsawad, C. (1997) A survey of helminth infection in rats (Rattus spp.) from Chiang Mai moat. The Southeast Asian Journal of Tropical Medicine and Public Health 28, 179183.Google Scholar
Pal, P. & Lewis, J.W. (2004) Parasite aggregations in host populations using a re-formulated negative binomial distribution. Journal of Helminthology 78, 5761.CrossRefGoogle Scholar
Pipitgool, V., Sithithaworn, P., Pongmuttasaya, P. & Hinz, E. (1997) Angiostrongylus infections in rats and snails in northeast Thailand. The Southeast Asian Journal of Tropical Medicine and Public Health 28, 190193.Google Scholar
Punyagupta, S. (1965) Eosinophilic meningoencephalitis in Thailand: summary of nine cases and observations on Angiostrongylus cantonensis as a causative agent and Pila ampullacea as a new intermediate host. The American Journal of Tropical Medicine and Hygiene 14, 370374.Google Scholar
Radomyos, P., Tungtrongchitr, A., Praewanich, R., Khewwatchan, P., Kantangkul, T., Junlananto, P. & Ayudhya, S.I. (1994) Occurrence of the infective stage of Angiostrongylus cantonensis in the yellow tree monitor (Varanus bengalensis) in five provinces of Thailand. The Southeast Asian Journal of Tropical Medicine and Public Health 25, 498500.Google Scholar
Setasuban, P., Vairasthira, S. & Harinasuta, C. (1968) The preliminary observation on natural infection of rat lung worm (Angiostrongylus cantonensis) in rodents and intermediate host in Thailand. Journal of the Medical Association of Thailand 51, 156157.Google Scholar
Tamura, K., Peterson, D., Peterson, N., Stecher, G., Nei, M. & Kumar, S. (2011) MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Molecular Biology and Evolution 28, 27312739.CrossRefGoogle ScholarPubMed
Teem, J.L., Qvarnstrom, Y., Bishop, H.S., da Silva, A.J., Carter, J., White-McLean, J. & Smith, T. (2013) The occurrence of the rat lungworm, Angiostrongylus cantonensis, in nonindigenous snails in the Gulf of Mexico region of the United States. Hawaii Journal of Medicine and Public Health 72, 1114.Google Scholar
Tesana, S., Srisawangwong, T., Sithithaworn, P., Laha, T. & Andrews, R. (2009) Prevalence and intensity of infection with third stage larvae of Angiostrongylus cantonensis in mollusks from Northeast Thailand. The American Journal of Tropical Medicine and Hygiene 80, 983987.Google Scholar
Thiengo, S.C., Maldonado, A., Mota, E.M., Torres, E.J., Caldeira, R., Carvalho, O.S., Oliveira, A.P., Simões, R.O., Fernandez, M.A. & Lanfredi, R.M. (2010) The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil. Acta Tropica 115, 194199.Google Scholar
Thompson, J.D., Higgins, D.G. & Gibson, T.J. (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Research 22, 46734680.Google Scholar
Vitta, A. (2012) Diagnosis of human angiostrongyliasis. Asian Biomedicine 6, 141150.Google Scholar
Vitta, A., Polseela, R., Nateeworanart, S. & Tattiyapong, M. (2011) Survey of Angiostrongylus cantonensis in rats and giant African land snails in Phitsanulok province, Thailand. Asian Pacific Journal Tropical Medicine 4, 597599.CrossRefGoogle ScholarPubMed
Wang, Q.P., Lai, D.H., Zhu, X.Q., Chen, X.G. & Lun, Z.R. (2008) Human angiostrongyliasis. Lancet Infectious Diseases 8, 621630.Google Scholar