Malacological survey of Biomphalaria snails in municipalities along the Estrada Real in the southeast of the State of Minas Gerais , Brazil

Introduction: The increasing practice of ecotourism and rural tourism in the State of Minas Gerais, Brazil, highlights the importance of studies concerning the occurrence of potential intermediate hosts of Schistosoma mansoni. This study aimed to identify species of Biomphalaria snails in municipalities along the Estrada Real, an important Brazilian tourism project. Methods: The specimens were collected in different water collections of 36 municipalities along the Estrada Real in the southeast of the State of Minas Gerais. Biomphalaria species were characterized using both morphological and molecular approaches. The research was conducted between August 2005 and September 2009 and all the sites visited were georeferenced using GPS. Results: Six Biomphalaria species were found in 30 of the 36 municipalities studied: glabrata, tenagophila, straminea, peregrina, occidentalis and schrammi. The first three species of Biomphalaria, recognized as intermediate hosts of S. mansoni, were present in 33.3%, 47.2% and 8.3% of the municipalities studied, respectively. The mollusks were found in different types of water collections and no infection by S. mansoni was detected. The highest occurrence of Biomphalaria concentration was verified in the area covered by the Caminho Novo route (Diamantina/MG to Rio de Janeiro/RJ). Conclusions: Considering the occurrence of schistosomiasis in the State of Minas Gerais and the socioeconomic repercussions involved in the Estrada Real Project, this work focuses on the vulnerability of water collections due to the presence of Biomphalaria mollusks and emphasizes the need for epidemiological surveillance and sanitary and educational measures integrated with the local community and tourism sectors.

Mollusks of the genus Biomphalaria infected with Schistosoma mansoni maintain the cycle of schistosomiasis in several Brazilian municipalities, despite recent improvements in the socioeconomic status of the population and education programs focusing on health, which were expected to reduce transmission of the disease.The relation between disease occurrence in human populations and the geographical presence of susceptible intermediate hosts is a well documented fact 1 .Poor rural communities continue to be major areas of schistosomiasis transmission 1 .With the phenomena of urbanization (migration to the outskirts of large cities), all aspects of the disease are reappearing, such as: neuroschistosomiasis, pulmonary hypertension, hepatosplenic schistosomiasis, acute episodes, glomerulonephritis and pyogenic liver abscesses [1][2][3][4][5][6][7][8][9][10][11][12][13] .
The State of Minas Gerais presents one of the highest prevalence rates for schistosomiasis in Brazil 14,15 .The presence of schistosomiasis in this state causes a number of concerns, not only for the healthcare sector, but also for economic sectors, such as tourism, which involve significant capital.
In the historical context of the economic development of State of Minas Gerais, the term Estrada Real refers to the routes taken by settlers from the time of the discovery of gold in Minas Gerais to the period of its exhaustion 16,17 .The Estrada Real was created by the Portuguese Crown in the 17 th century with the intention of controlling the flow of wealth, in the form of gold and diamonds and goods, which passed between the State of Minas Gerais and the coastal city of Rio de Janeiro, the capital of the colony at the that time 17   The State of Minas Gerais includes the majority of the municipalities of the Estrada Real Project with great potential for expanding ecotourism and rural tourism, because of the attraction represented by historical-cultural monuments, the diversity of the topography, fauna and flora and water bodies.Thus, mapping the geographical areas vulnerable to the transmission of schistosomiasis becomes a task not only of health surveillance, but also of encouraging productive sectors that drive the economy in this state.

Tibiriça SHC et al -Biomphalaria in municipalities along the Estrada Real
In order to contribute to the generation of georeferenced epidemiological data and the control of schistosomiasis in the municipalities of the Estrada Real Project in southeastern Minas Gerais, this work reports a malacological survey designed to verify the occurrence of mollusks of the genus Biomphalaria and the possibility of infection by S. mansoni.

Study area
The study municipalities are located in the southeast of the State of Minas Gerais, Brazil, and belong to the Regional Healthcare Administration of Juiz de Fora (Gerência Regional de Saúde, GRS/ JF).The state is administratively divided into 13 macroregions.Among these, the southeastern macroregion consists of eight microregions, of which the Juiz de Fora/Lima Duarte/Bom Jardim de Minas microregion is under the responsibility of the GRS/JF.This microregion is composed of 37 municipalities.Of these, 36 were included in the study.The core urban area of the City of Juiz de Fora was not included, because the results of a local malacological survey conducted prior to this study had already been published 18 .

Malacological survey
Mollusks of the Biomphalaria genus were collected from August 2005 to September 2009, by technicians of the GRS/JF.Catches were made in several areas of the municipalities in order to cover the largest possible area, with the aid of gloves, tongs and dip nets.In all 36 municipalities, the type and quantity of water collection sites surveyed were selected by the following criteria: reported local cases of human schistosomiasis, existence of water bodies (lotic and lentic) whose characteristics favored the occurrence of the Biomphalaria genus and the possibility of contact with human populations.Thus, based in these criteria, at least three water collections in each municipality were surveyed.The points were georeferenced using a Global Positioning System (GPS), Garmin Model II-12, and imported into a Geographic Information System (GIS) to permit visualization of their spatial distribution.Sites in which the presence of Biomphalaria was verified were photographed with a digital camera.

Mollusk identification and examination of Schistosoma mansoni infection
The captured snails were counted, transported in plastic containers and maintained in the laboratory until identification.Following collection, the snails were transported to the laboratory where they were measured and examined under artificial light.Some specimens from each sample were identified at the Parasitology Laboratory in the Federal University of Juiz de Fora and the Entomology Laboratory of the GRS/JF, Secretaria de Estado de Saúde de Minas Gerais (SES/MG), according to the protocol established by Paraense 19,20 .Some specimens were sent to the Laboratory of Helminthology and Malacology at the René Rachou Medical Research Center, Oswaldo Cruz Foundation (CPqRR/ FIOCRUZ), for confirmation of species by the technique of polymerase chain reaction (PCR) 21 .
Research on Biomphalaria was conducted in 36 municipalities, 19 of which form part of the Estrada Real Project, while the remainder are located within the coverage area (Table 1).As presented in Table 1, 30 municipalities were positive for snails of the genus Biomphalaria, while in six, the presence of these mollusks not observed.Six Biomphalaria species were identified: glabrata, tenagophila, straminea, peregrina, occidentalis and schrammi.B. tenagophila was the most prevalent species and was found in 17 municipalities, followed by B. peregrina, in 16 municipalities and by B. glabrata, in 12.A total of 3,772 specimens of mollusks were collected, all negative for cercariae of S. mansoni (Table 1).
Table 2 shows the water collections visited in the municipalities studied and the number of positive sites for Biomphalaria snails.The sites were classified according to the following definitions intentionally left in their categories as defined in Portuguese to preserve the distinctions between site types: stream, water flowing in a channel; dam, a barrier constructed to contain the flow of water; tank, a large artificial container used to hold water; irrigation ditch, a channel for water irrigation; bog, wet spongy ground containing clay; waterfall, a cascade of water; water tank, a container for supplying water under pressure; water trough, a container providing water for animals; canal, an artificial waterway; spring, water rising from the earth; well, a hole or shaft sunk into the earth to obtain water; and reservoir, a natural or artificial pond or lake used for storage and regulation of water.Among the various water collections, streams were the most positive sites for Biomphalaria species, with 35 locations, followed by dams.The greatest variety of species was found in the dams.Among the 12 water collections surveyed, 10 showed the presence of B. glabrata and/or B. tenagophila (Table 2).Figure 1 shows the coverage area of the Estrada Real Project in Brazil and highlights the positive and negative areas for Biomphalaria snails.A higher concentration of Biomphalaria occurs in the eastern region of the map, in the area covered by the Caminho Novo (Diamantina/MG to Rio de Janeiro/RJ), than in the western region of the map, which is closer to the Caminho Velho (Diamantina/MG to Paraty/RJ).In the eastern region, between the rivers Pinho and Paraibuna, belonging to the Paraíba do Sul river basin, all the species of mollusks reported in this work were found.The western region, between the rivers Grande and Aiuruoca, belonging to the Rio Grande water basin, is the area with just one positive collection dot for B. peregrina.In the southern region, between the rivers Grande and Preto, belonging to the Rio Grande and to the Rio Paraíba do Sul water basins, respectively, there are various collection dots for B. peregrina, but only one positive dot for B. tenagophila (Figure 1).

DISCUSSION
Increased ecotourism and rural tourism in the State of Minas Gerais has highlighted the importance of studies regarding the occurrence of potential intermediate hosts of S. mansoni.This study revealed the presence of Biomphalaria snails in 30 municipalities, in the southeast region of the state, belonging to the Estrada Real Project or to its coverage area.This is a pioneering work, undertaken to investigate Biomphalaria snails in municipalities of the Estrada Real route with the use of geographic coordinates (GPS) for mollusk location.
In Brazil, there are 11 species and one subspecies of mollusks of the genus Biomphalaria, but only B. glabrata, B. tenagophila and B. straminea are recognized as intermediate hosts of S. mansoni.B. peregrina proved to be experimentally susceptible to S. mansoni, while B. occidentalis and B. schrammi proved to be refractory to infection [22][23][24][25][26] .B. tenagophila was present in 47.2% of the municipalities surveyed, B. glabrata in 33.3% and B. straminea in 8.3%.Despite the undeniable importance of B. glabrata in Brazil, it is interesting to observe that B. tenagophila is the main species transmitting schistosomiasis in the State of São Paulo.These data reveal the importance of surveillance and the need for a control program for schistosomiasis in such areas, since B. glabrata showed significant occurrence and is of great importance due to its extensive geographic distribution, high infection indices and effectiveness in the transmission of schistosomiasis 27 .
Among the 36 municipalities studied, the absence of Biomphalaria snails was verified in only six.However, it is important to highlight that the present study was conducted over four years, which means that seasonal cycles, such as temperature and rainfall, for example, may have affected the abundance of mollusk populations 28,29 .
This study revealed several water collections colonized by mollusks of the Biomphalaria genus, including streams, dams and waterfalls, which could serve for sporting practices or for the construction of pools, providing further sources of infection by schistosomiasis among tourists 4,30 .
The Estrada Real Project is based on the exploration of the touristic potential of the region, with the implementation of regional ecotourism and the preservation of the natural patrimony 17 .These goals also culminate, unintentionally, in maintaining the habitat of the intermediate hosts of S. mansoni, which may promote outbreaks and the spread of schistosomiasis in the region, since in the majority of these sites sanitation conditions are poor, representing contamination risks for water collections in cases of infected individuals.
Little information exists regarding the prevalence of schistosomiasis in the municipalities studied, because the region is generally considered to be of low endemicity 18 .The presence of Biomphalaria species was previously reported in the municipalities of Piau, Rio Novo and Juiz de Fora 15,18,31 , although considering their geographical location, only Juiz de Fora has been surveyed 18 .Models to estimate the prevalence of schistosomiasis have been perfomed in the State of Minas Gerais 14,[32][33][34] and in some Estrada Real municipalities 16 , but in both studies, due to lack of georeferenced information for Biomphalaria, only data concerning the prevalence of human schistosomiasis and Biomphalaria species in relation to the municipality were used.In this study, all the sites where the occurrence of Biomphalaria was verified were mapped using GPS.Knowing the exact location of the breeding/water collections of the intermediate host participant in the chain of transmission of schistosomiasis permits greater flexibility in structuring measures for disease control.Moreover, these data may favor the use of geostatistical techniques, generating predictive values for the occurrence of Biomphalaria at sites in the region with no information regarding the intermediate hosts 34,35 , which could contribute to the drafting of a map of Biomphalaria snails in the State of Minas Gerais.
Considering the occurrence of schistosomiasis in the State of Minas Gerais and the socioeconomic repercussions that involve the Estrada Real Project, this work focuses on the vulnerability of the local water collections due to the presence of Biomphalaria mollusks and emphasizes the need for epidemiological surveillance and sanitary and education measures integrated with the local community and tourism sectors.

FIGURE 1 -
FIGURE 1 -Spatial distribution of the Biomphalaria genus in the municipalities of the Estrada Real, southeastern region of the State of Minas Gerais.The coverage area of the Estrada Real Project and highlighted, the Biomphalaria species collected in the municipalities of the Estrada Real in the southeast of the State of Minas Gerais.
Estrada Real Project.The Estrada Real Project consists of 193 municipalities, 162 of which are in the State of Minas Gerais, nine in the State of Rio de Janeiro and 22 in the State of São Paulo 17 .

TABLE 1 -Presence of Biomphalaria, the number of catch basins surveyed and snails collected in the municipalities of the Estrada Real in the southeast of the State of Minas Gerais. Water collections Species of Snails Municipalities studied (n) Biomphalaria* collected (n)
*Species of Biomphalaria: t: tenagophila, s: straminea, p: peregrina, o: occidentalis, sc: schrammi.g: glabrata, **Municipalities that belong to the Estrada Real Project.The other municipalities are part of the coverage area.

TABLE 2 -Type and quantity of water collection sites in which the occurrence of Biomphalaria was verified.
*The sites were classified according to the definitions intentionally left in their categories as defined in Portuguese to preserve the distinctions between site types (explanations in the results).**Refers to the presence of Biomphalaria, not necessarily infected with Schistosoma mansoni.***Species of Biomphalaria: g: glabrata, p: peregrina, t: tenagophila, s: straminea, o: occidentalis, sc: schrammi.