Prevalence and distribution of Angiostrongylus cantonensis ( Nematoda , Angiostrongylidae ) in Achatina fulica ( Mollusca , Gastropoda ) in Baixada Santista , São Paulo , Brazil

Introduction: Angiostrongylus cantonensis causes eosinophilic meningoencephalitis in humans. Worldwide expansion of this nematode is linked to the dispersion of their hosts. This study aimed to determine the prevalence of A. cantonensis infection in Achatina fulica in the nine municipalities that make up Baixada Santista, São Paulo, Brazil. Methods: Angiostrongylus cantonensis larvae were analyzed using optical microscopy. We performed polymerase chain reaction and restriction fragment length polymorphism using restriction endonuclease ClaI, directed to the internal transcribed spacer region 2 of A. cantonensis larval DNA. Results: Of the 540 snails analyzed, 117 (21.7%) were infected by A. cantonensis. For morphological and morphometric analyses, 60 larvae were used. Second-stage larvae were, on average, 358.2μm long and 26.4μm wide, while third-stage larvae were, on average, 450μm long and 21.12μm wide. The tails of the larvae ended in a fine tip. Conclusions: All municipalities comprising Baixada Santista had A. fulica that were naturally infected with A. cantonensis. All of the observed characteristics were typical of the species.

Achatina fulica plays a crucial role in the global dispersion of A. cantonensis 1,8,22,25,26 , since it is present in most areas where this nematode is endemic.These mollusks are associated with an anthropic environment, and once established, their population 27 .Remains of human activity favor the adaptation of this mollusk, as such remains provide food and shelter 28 .In Brazil, this mollusk has high potential to be involved in the transmission of A. cantonensis owing to its wide distribution, including to different ecosystems [29][30][31] .
In the present study, the role of A. fulica as an intermediate host for A. cantonensis in the municipalities comprising Baixada Santista, São Paulo State, Brazil, was investigated.

METHODS
Samples were collected from January to July, 2012.Specimens were captured in vacant lots in urban areas or where there were forest fragments or waste remains from 90 sites in the nine municipalities comprising Baixada Santista, São Paulo State: Bertioga, Cubatão, Guarujá, Itanhaém, Mongaguá, Santos, São Vicente, Peruíbe, and Praia Grande (Figure 1).Six adult snails were collected from ten sites in each municipality, for a total of 540 individuals.All 90 sites were characterized as of the snail, performed in accordance to Simone 32 , the digestion procedure of mollusks was individually performed in accordance with methods of Wallace and Rosen 33 , followed by the Baermann method 34 .A. cantonensis larvae were then counted and subjected to molecular analysis.The DNA was extracted from the pool of larvae from each snail using the to the manufacturer's instructions.The deoxyribonucleic acid (DNA) was subjected to polymerase chain reaction associated with restriction fragment length polymorphism (PCR-RFLP), and the primers used were directed to the internal transcribed spacer region 2 (ITS2) of ribossomal DNA (rDNA).NC1 (forward; 5'ACGTCTGGTTCAGGGTTGTT-3') and NC2 primers (reverse: 5'-TTAGTTTCTTTTCCTCCGCT-3') were designed by Gasser 35 of subunit 5.8S and the initial portion of subunit 28S.Further, cleavage of this amplicon was performed with endonuclease ClaI (Biolabs) and the profiles were compared to those of A. cantonensis and A. costaricensis established by Caldeira 36 .For morphological and morphometric analysis, 60 larvae were used, and analyzed (Leica Application Suite LAS V 3.8 Software and DMB 5000 Leica ® microscope, Leica Microsystems, Wetzlar, on morphological and morphometric parameters established by Ash 37 and Lv 10 .The SADIE index 38 was used to analyze the spatial patterns of the percentage of infected specimens from geographical coordinates and the percentage of infected A. fulica.

RESULTS
Achatina fulica was detected in anthropogenic environments, especially in those with great availability of food and shelter (82% of evaluated sites).Of the 90 sites analyzed, 73 (81.1%) had mollusks with nematode larvae, and, of these, 52 (71.2%)

A B
C D E were infected with A. cantonensis.Of the 540 mollusks, 204 (37.7%) had nematode larvae, of which, 117 (57.3%) were infected with A. cantonensis (21.6% of the total) (Table 1).The prevalence of A. cantonensis infection in A. fulica for each municipality and the absolute number of parasite loads per mollusk are shown in Table 2.
The results were negative for the presence of A. costaricensis.Spatial analysis showed that the percentage of A. fulica infected with A. cantonensis in Baixada Santista had a random distribution, characterized by the absence of areas with much higher or much smaller infection percentages within the region (I = 1:38; p = 0.0957).
Morphological and morphometric analyses revealed that the larvae showed filiform bodies, striated cuticles in the transverse direction with rounded anterior ends showing two well-developed structures in the form of buttons and another in the form of a rod, followed by a long esophagus (Figure 2).The results of the morphological analyses of second-stage larvae (L2) and L3 of A. cantonensis are shown in Table 3.

Several snails play roles as intermediate hosts for
A. cantonensis.Among them, the giant African snail A. fulica is one of the most important due to its abundance and occupation in different ecosystems.In this study, among 540 A. fulica specimens analyzed, 204 (37.8%) were found to contain nematodes, a value similar to that obtained by Rocco 39 , who reported a rate of 34.2%.In both studies, specimens were obtained in anthropic environments where snails probably lived with small rodents, which is critical for the maintenance of parasites in the environment.
Recovered A. cantonensis larvae presented two morphotypes as larval stages 2 and 3 (L2 and L3).Although the detail of factor 37 ; however, L3 presented measures compatible with those obtained by Ash 37 and Thiengo 11 (Table 3).
Lv 10 found that, before the second molting, the main characteristics of L2 were similar to those of L3, as shown in Figure 2, which were two structures, similar to buttons and rods in shape.The founding of these two larval stages in the same snails is probably due to constant reinfections of the mollusk in the natural environment and to the method by which the analyzed material was obtained, in which the entire contents of the soft parts were processed.
Molecular analysis revealed the presence of A. fulica that were naturally infected with A. cantonensis in urban areas of the nine municipalities of the Baixada Santista region, with an infection rate of 21.7%.The variation of this rate is broad and has been observed in several municipalities, such as São Gonçalo (35.4%) and Barra do Piraí (10.3%), both in the State of Rio de Janeiro and Joinville/SC (27.4%) 12 , China (13.4% and 28.4%) 40,41 , Pernambuco (42%) 11 , and Japan (52.79%) 42 .The climatic characteristics of Baixada Santista are appropriate for the development of A. fulica and A. cantonensis.In fact, Ishii 43 has observed that the L3 of A. cantonensis develop better at temperatures ranging from 20°C to 30°C.In addition to environmental factors such as temperature, variations in the the parasite in its hosts, by the population density of mollusks and rodents, and by biological characteristics 22,42,44 .
These results indicate the need for more attention to this emerging parasite through awareness campaigns for local and medical communities, the development of a health surveillance system, improved health education, and the distribution of information about the management action adapted to each reality, since 82% of the analyzed wastelands had some type of garbage or rubble.Studies on the distribution of intermediate and paratenic hosts in areas near houses and the parasite-host compatibility should be investigated to improve understanding of transmission dynamics.In Brazil, there have been few   9,[11][12][13][14] .Most animal populations have aggregate spatial distribution patterns, generally owing to the distribution and supply of resources in the environment 45 .In this study, a regular spatial distribution pattern was observed, which is quite rare.The probability is that this distribution was due to the presence of high populations of A. fulica in urban areas related to their high adaptability, which makes it not a limiting resource of the A. cantonensis distribution.Furthermore, as the parasite can be found in different species of intermediate hosts, its spatial distribution becomes more regular.These data are especially useful and can be used by public health authorities to establish policies related to surveillance and planning of preventive actions.Isolated cases of eosinophilic meningoencephalitis have recently been reported in Brazil 9,11,14,15,46,47 .Thus, it is plausible that A. cantonensis continues to spread to new regions, increasing the risk of eosinophilic meningoencephalitis in humans.

FIGURE 1 -
FIGURE 1 -A: Location of the study area and B: collection points in the municipalities comprising Baixada Santista, São Paulo, Brazil.

TABLE 2
Prevalence of infection by Angiostrongylus cantonensis in Achatina fulica by each municipality and the absolute number of parasitic loads per snail.

TABLE 3
Measurements (µm) of second-and third-stage larvae and tail characteristics of Angiostrongylus cantonensis retrieved from naturally infected Achatina fulica.
L2: second-stage larvae; L3: third-stage larvae.studieson the action of A. fulica and other species of mollusks as intermediate hosts of A. cantonensis and their role in public health.For example, previous studies have shown the presence of other species naturally infected with A. cantonensis in addition to A. fulica in Brazil, such as Bradybaena similares, Subulina octona, Sarasinula marginata, and Sarasinula linguaeformis