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First records of molluscs naturally infected with Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) in Northeastern Brazil, including new global records of natural intermediate hosts

ABSTRACT Human neural angiostrongyliasis is an emerging infectious disease caused by nematode Angiostrongylus cantonensis. The present study investigated the presence of Angiostrongylus spp. in terrestrial molluscs collected from the following areas in the Metropolitan Region of Aracaju, Sergipe State, Brazil: Barra dos Coqueiros, Nossa Senhora do Socorro, Sao Cristovao and Aracaju. In total, 703 specimens representing 13 mollusc species were screened for Angiostrongylus spp. Larvae of Angiostrongylus spp. were found in three species. Larvae recovered from Achatina fulica were used for experimental infection in Wistar rats (Rattus norvegicus). For specific identification of nematodes, the mitochondrial cytochrome c oxidase subunit I (COI) was sequenced from both larvae and adults recovered from molluscs and rats, respectively. Infection with A. cantonensis was detected in all municipalities and in the following three host species: Bulimulus tenuissimus, Cyclodontina fasciata (Barra dos Coqueiros), and A. fulica (Aracaju, Nossa Senhora do Socorro and Sao Cristovao). Co-infections were also found with Caenorhabditis sp. and Strongyluris sp. larvae. This is the first study of the helminth fauna associated with the terrestrial malacofauna in Sergipe State, and confirms that these three snail species are involved in the transmission of A. cantonensis in the state. In addition, B. tenuissimus and C. fasciata are newly reported natural hosts of the parasite.


INTRODUCTION
Achatina fulica Bowdich, 1822 is listed among 100 of the worst invasive species in the world and is present in almost all Brazilian states 1,2 . This species' rapid proliferation is the main reason it is considered an agricultural pest. It can also act as an intermediate host of Angiostrongylus cantonensis (Chen, 1935) and Angiostrongylus costaricensis Morera and Céspedes, 1971. These two species are the etiological agents of human neural angiostrongyliasis and human abdominal angiostrongyliasis, respectively [2][3][4] .
Eosinophilic meningitis is the principal clinical manifestation of human neural angiostrongyliasis, an emerging infectious disease that causes severe impairment of the central nervous system and can lead to death [5][6][7] . This nematode has low specificity for its intermediate hosts, which means that many mollusc species have already been found to be naturally infected globally 8,9 . However, A. fulica is an important intermediate host for A. cantonensis 4,8,[10][11][12] .
Human neural angiostrongyliasis occurs primarily by ingestion of snails that are infected with L 3 larvae 13 . These larvae reach the brain where they mature into L 4 and L 5 but are unable to migrate from the brain to the heart (as they do in the definitive rat hosts) but move around in the brain causing neurological damage and, especially when they die, cause intense inflammatory reactions that result in additional neurological damage and symptoms 5,7,14 .
More than 2,800 human cases of eosinophilic meningitis caused by A. cantonensis have been reported in more than 30 countries 11 since it was first reported in Taiwan

MATERIALS AND METHODS
This study was undertaken in urban environments of four municipalities that constitute the Metropolitan Region of Aracaju, Sergipe State ( Figure 1): Aracaju, Barra dos Coqueiros, Nossa Senhora do Socorro and Sao Cristovao (Table 1).
Each point of collection was georeferenced, followed by manual random sampling of terrestrial molluscs using forceps and gloves. The collections were carried out in the following three seasons: April (autumn), July (winter) and October (spring) of 2016, at three locations in each municipality. The samples were sent to the National Reference Laboratory for Schistosomiasis -Malacology (LRNEM) of the Instituto Oswaldo Cruz, Fundação Oswaldo Cruz (IOC/Fiocruz, Rio de Janeiro), where they were analyzed. The identification of the molluscs species was done based on conchological characteristics compared to photos and descriptions from catalogues [16][17][18][19] . We also compared our material with material deposited in the Instituto Oswaldo Cruz. Samples of each species and from each collection locality were deposited in the Mollusca Collection of the Instituto Oswaldo Cruz (CMIOC 10103 -10134, 11206).
To collect the nematode larvae, the molluscs were artificially digested by a modified Wallace and Rosen technique 20 . The recovered material was observed under a stereoscopic microscope following the LRNEM Identification Guide and based on the literature 4,21 . The Angiostrongylus sp. (Nematoda: Metastrongylidae) larvae were identified under a compound microscope according to Thiengo et al. 4 . Ten Angiostrongylus sp. larvae from each sample were collected and cryopreserved at -20 °C until DNA extraction for subsequent molecular analysis. Some rhabditiform larvae were observed and prepared for DNA extraction. In addition, Strongyluris sp. was morphologically identified, based on Thiengo 21 .
To assess the viability of the parasites, Angiostrongylus sp. larvae recovered from A. fulica from the municipality of Aracaju were used to infect two specimens of Rattus norvegicus through an orogastric tube at a concentration of 50 L 3 larvae per rodent. Fifty days after infection, the animals were killed and necropsied to confirm the presence of adult A. cantonensis 22 . This procedure was carried out following the Ethics Commission on Animal Use of the Oswaldo Cruz Foundation (LW-47/14).  23 ), and 0.25 μL of recombinant Taq DNA polymerase (Thermo Fisher Scientific). A total of 5 μL of the DNA sample was added to the mixture, producing a final volume of 25 μL for each reaction. For all the reactions, ultrapure water was used as a negative control template, and the positive control was performed with genomic DNA of A. cantonensis as template. The 700 bp fragment of the Angiostrongylus sp. larvae mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the following PCR conditions: initial denaturation at 94 °C for 1 min, five cycles at 94 °C for 40 s, 45 °C for 40 s, and 72 °C for 1 min, followed by 30 cycles at 94 °C for 40 s, 51 °C for 40 s, and 72 °C for 1 min and a final extension at 72 °C for 1 min.
For rhabditiform larvae, individual DNA from some isolates was used to amplify the 480 bp ribosomal 18S region 24 .
The PCR products were purified using the Illustra GFX PCR DNA and Gel Band Purification Kit (GE Healthcare, Little Chalfont, UK) following the manufacturer's protocol. Purified products after amplification were bidirectionally sequenced using BigDye Terminator v3.1 Cycle Sequencing kit (Applied Biosystems, California, USA) according to the manufacturer's instructions. Chromatograms of the sequences obtained were analyzed and edited using Geneious version R9 software (http://www.geneious. com), resulting in a consensus sequence (contig). Then, a comparative similarity search was performed on GenBank (www.ncbi.nlm.nih.gov/genbank/) 25  Angiostrongylus sp. larvae were found in specimens of A. fulica, B. tenuissimus and C. fasciata and were later confirmed by molecular analysis as A. cantonensis (Table 2).
Achatina fulica was found in all four municipalities, with snails harboring infection by A. cantonensis in three of them (Aracaju, Sao Cristovao and Nossa Senhora do Socorro). Bulimulus tenuissimus occurred in all municipalities, while specimens of C. fasciata were found only in Aracaju and Barra dos Coqueiros. Both B. tenuissimus and C. fasciata Two rats that were experimentally infected with Angiostrongylus sp. isolates displayed symptoms 50 days after infection. Forty A. cantonensis adult worms were recovered in the first rat (37.5% males and 62.5% females), and 42 adult worms were recovered in the second (40.4% males and 59.5% females).

DISCUSSION
The snail A. fulica has already been reported as being parasitized with A. cantonensis by several authors 2,4,8,10,19,26 . In Brazil, the species is considered one of the main Helicina sp. Several other terrestrial molluscs have also been found to be naturally infected with this nematode in Brazil 2,3,8,19 and in other countries 9,13,14 . Among these species are S. linguaeformis and S. octona 2,3,8,9 , which, however, were not found to be infected in the present study. A possible reason for this is the lower abundance of these species in the sampled localities. The three species found infected by A. cantonensis were also those that were more abundant in the environment.
The present study identifies, for the first time, the following two species as natural hosts of the parasite: C. fasciata, which had not yet been described in the literature as an intermediate host for A. cantonensis or for other nematodes of human and veterinary interest, and B. tenuissimus, described as a host of metacercariae of the genus Postharmostomum 32 . This interaction between exotic and native species has become a new concern in the ecology of the disease and may result in increased parasitic spread as well as pose a threat to native species 9 .
Studies indicate the need to implement control measures against molluscs associated with public health, particularly the exotic species A. fulica. However, passive geographic dispersal is the main form of spread of the giant African snail, which hinders its control 33 .
The present study reinforces the need to investigate the current situation of A. cantonensis dispersal in the Northeastern region of Brazil, since there are reports of