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.
