Roberta Lima Caldeira

First record of molluscs naturally infected with Angiostrongylus cantonensis (Chen, 1935) (Nematoda: Metastrongylidae) in Brazil

Seeking the identification of Angiostrongylus cantonensis as a potential etiological agent of three clinical cases of eosinophilic meningitis, mollusc specimens were collected in the state of Espírito Santo, Brazil. The snails were identified as Sarasinula marginata (45 specimens), Subulina octona (157), Achatina fulica (45) and Bradybaena similaris (23). Larvae obtained were submitted to polymerase chain reaction and restriction fragment length polymorphism diagnosis. Their genetic profile were corresponded to A. cantonensis. Rattus norvegicus experimentally infected with third-stage larvae, developed menigoencephalitis, and parasites became sexually mature in the lungs. Additionally, larvae obtained from A. fulica snails, from São Vicente, state of São Paulo, also showed genetic profiles of this nematode. This is the first record of Brazilian molluscs infected with this nematode species.

The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil

The human cases of eosinophilic meningitis recently reported from Brazil have focused the attention of the public health agencies on the role the introduced snail Achatina fulica plays as hosts of the metastrongylid nematodes. Determining the potential of this snail to host and develop infective larval stages of metastrongylids in the wild and identify the species harbored by them is crucial for designing effective control measures. Here we assess if A. fulica may act as intermediate host of A. cantonensis at the peridomiciliary areas of a patients house from state of Pernambuco (PE), who was diagnosed with eosinophilic meningitis and a history of ingesting raw molluscs. Larvae obtained from naturally infected A. fulica were orally administered to Rattus norvegicus. The worms were collected from the pulmonary artery and brain, and were morphologically characterized and compared to the Japan isolate of A. cantonensis. Adult worms and infective L(3) larvae (PE isolate) recovered from A. fulica specimens were also analyzed by polymerase chain reaction and restriction fragment length polymorphism of ITS2 region from rDNA and compared to A. cantonensis (ES isolate), A. vasorum (MG isolate) and A. costaricensis (RS isolate). The large size of the spicules (greater than those observed in other species of Angiostrongylus) and the pattern of the bursal rays agree with the original species description by Chen (1935). Furthermore, the morphology of the PE isolate was similar to that of Japan isolate. The PCR-RFLP profiles obtained were distinctive among species and no variation in patterns was detected among adult individuals from A. cantonensis isolates from PE and ES. The importance of A. fulica as an intermediate host of eosinophilic menigoencepahlitis in Brazil is emphasized.

Further studies on the molecular systematics of Biomphalaria snails from Brazil

The polymerase chain reaction and restriction fragment length polymorphism (RFLP) of the internal transcribed spacer (ITS) region of the rRNA gene, using the enzyme DdeI were used for the molecular identification of ten species and one subspecies of Brazilian Biomphalaria. Emphasis is given to the analysis of B. oligoza, B. schrammi and B. amazonica. The RFLP profiles obtained using this enzyme were highly distinctive for the majority of the species and exhibited low levels of intraspecific polymorphism among specimens from different regions of Brazil. However, B. peregrina and B. oligoza presented very similar profiles that complicated their identification at the molecular level and suggested a very close genetic similarity between the two species. Others enzymes including HaeIII, HpaII, AluI and MnlI were tested for their ability to differentiate these species. For B. amazonica three variant profiles produced with DdeI were observed. The study demonstrated that the ITS contains useful genetic markers for the identification of these snails

Molecular Differentiation of Angiostrongylus costaricensis, A. cantonensis, and A. vasorum by Polymerase Chain Reaction- Restriction Fragment Length Polymorphism

Angiostrongylus cantonensis, A. costaricensis, and A. vasorum are etiologic agents of human parasitic diseases. Their identification, at present, is only possible by examining the adult worm after a 40-day period following infection of vertebrate hosts with the third-stage larvae. In order to obtain a diagnostic tool to differentiate larvae and adult worm from the three referred species, polymerase chain reaction-restriction fragment length polymorphism was carried out. The rDNA second internal transcribed spacer (ITS2) and mtDNA cytochrome oxidase I regions were amplified, followed by digestion of fragments with the restriction enzymes RsaI, HapII, AluI, HaeIII, DdeI and ClaI. The enzymes RsaI and ClaI exhibited the most discriminating profiles for the differentiation of the regions COI of mtDNA and ITS2 of rDNA respectively. The methodology using such regions proved to be efficient for the specific differentiation of the three species of Angiostrongylus under study.

Phylogenetic relationships among Brazilian Biomphalaria species (Mollusca: Planorbidae) based upon analysis of ribosomal ITS2 sequences

In spite of their abundance, widespread distribution and medical importance, the phylogenetic relationships among Biomphalaria snails have received relatively little attention. We have collected and studied 29 populations of snails obtained from different localities from Brazil. We have sequenced the ribosomal DNA second internal transcribed spacer (ITS2) from the following Biomphalaria species: B. glabrata, B. tenagophila tenagophila, B. occidentalis, B. straminea, B. peregrina, B. kuhniana, B. schrammi, B. amazonica, B. oligoza, B. intermedia and an outgroup species Helisoma duryi. The sequence from each species is unique. Three different methods of phylogenetic reconstruction were used (distance, maximum parsimony and maximum likelihood). The resulting phylogenetic trees obtained by these methods basically support current systematic relationships based on morphological characters alone. This study demonstrates that the ITS2 region contains markers useful for identification and determination of relationships among Biomphalaria species.

A bacterial artificial chromosome library for Biomphalaria glabrata, intermediate snail host of Schistosoma mansoni

To provide a novel resource for analysis of the genome of Biomphalaria glabrata, members of the international Biomphalaria glabrata Genome Initiative (http://biology.unm.edu/biomphalaria-genome.html), working with the Arizona Genomics Institute (AGI) and supported by the National Human Genome Research Institute (NHGRI), produced a high quality bacterial artificial chromosome (BAC) library. The BB02 strain B. glabrata, a field isolate (Belo Horizonte, Minas Gerais, Brasil) that is susceptible to several strains of Schistosoma mansoni, was selfed for two generations to reduce haplotype diversity in the offspring. High molecular weight DNA was isolated from ovotestes of 40 snails, partially digested with HindIII, and ligated into pAGIBAC1 vector. The resulting B. glabrata BAC library (BG_BBa) consists of 61824 clones (136.3 kb average insert size) and provides 9.05 x coverage of the 931 Mb genome. Probing with single/low copy number genes from B. glabrata and fingerprinting of selected BAC clones indicated that the BAC library sufficiently represents the gene complement. BAC end sequence data (514 reads, 299860 nt) indicated that the genome of B. glabrata contains ~ 63% AT, and disclosed several novel genes, transposable elements, and groups of high frequency sequence elements. This BG_BBa BAC library, available from AGI at cost to the research community, gains in relevance because BB02 strain B. glabrata is targeted whole genome sequencing by NHGRI.

Prevalência de helmintos intestinais em três mesorregiões do Estado de Minas Gerais

A parasitological assay of feces, through the Kato-Katz method, was undertaken in 18,973 schoolchildren (7 to 14 years old), at primary school level, from a public school of the State of Minas Gerais. Three mesoregions were studied: Tri ngulo Mineiro/ Alto Para ba ( 60 municipalities); northwest Minas Gerais (13) and south/south-west regions of Minas Gerais (144). Among the examined children, 15,545 (82%) were negative; 2,863 (15%) were infected with a single species of helminthes and 565 (3%) were infected by more than one species. The prevalence rates were: A. lumbricoides 10.3%; T. trichiura 4.7%; hookworm 2.9%; E. vermicularis 1.2%; H. nana 0.4% and Taenia sp 0.2%. The largest helminthes prevalence/mesoregion were for T. trichiura (24.2%) and A. lumbricoides (18.7%) in the south/south-west region; hookworm (12.1%) and Taenia sp (0.7%) in the northwest region of Minas Gerais. Intestinal helminthiasis is still regarded as a serious public health problem, including regions where the socio-economic conditions are more favorable.

Biomphalaria tenagophila/Schistosoma mansoni interaction: premises for a new approach to biological control of schistosomiasis

Biomphalaria tenagophila is very important for schistosomiasis transmission in Brazil. However its mechanisms of interaction with Schistosoma mansoni are still scantly studied. Since this snail displays strains highly susceptible or completely resistant to the parasite infection, the knowledge of that would be a useful tool to understand the mechanism of snail resistance. Particularly, the Taim strain consistently shows absolute resistance against the trematode, and this resistance is a dominant character. A multidisciplinary research group was created aiming at studying B. tenagophila/S. mansoni interaction. The possibility for applying the knowledge acquired to obtain a biological model for the control of S. mansoni transmission in endemic areas is discussed.

Molecular identification of similar species of the genus Biomphalaria (Mollusca: Planorbidae) determined by a polymerase chain reaction-restriction fragment length polymorphism

The freshwater snails Biomphalaria straminea, B. intermedia, B. kuhniana and B. peregrina, are morphologically similar; based on this similarity the first three species were therefore grouped in the complex B. straminea. The morphological identification of these species is based on characters such as vaginal wrinkling, relation between prepuce: penial sheath:deferens vas and number of muscle layers in the penis wall. In this study the polymerase chain reaction restriction fragment length polymorphism technique was used for molecular identification of these molluscs. This technique is based on the amplification of the internal transcribed spacer regions ITS1 e ITS2 of the ribosomal RNA gene and subsequent digestion of these fragments by restriction enzymes. Six enzymes were tested: Dde I, Mn1 I, Hae III, Rsa I, Hpa II e Alu I. The restriction patterns obtained with DdeI presented the best profile for separation of the four species of Biomphalaria. The profiles obtained with all the enzymes were used to estimate the genetic distances among the species through analysis of common banding patterns.

Rural tourism as risk factor for the transmission of schistosomiasis in Minas Gerais, Brazil

Recently, the booming rural tourism in endemic areas of the state of Minas Gerais was identified as a contributing factor in the dissemination of the infection with Schistosoma mansoni. This article presents data from six holiday resorts in a rural district approximately 100 km distant from Belo Horizonte, MG, Brazil, where a possibly new and until now unperceived way of transmission was observed. The infection takes place in swimming pools and little ponds, which are offered to tourists and the local population for fishing and leisure activities. The health authorities of the district reported cases of schistosomiasis among the local population after visiting these sites. As individuals of the non-immune middle class parts of the society of big urban centers also frequent these resorts, infection of these persons cannot be excluded. A malacological survey revealed the presence of molluscs of the species Biomphalaria glabrata and Biomphalaria straminea at the resorts. The snails (B. glabrata) of one resort tested positive for S. mansoni. In order to resolve this complex problem a multidisciplinary approach including health education, sanitation measures, assistance to the local health services, and evolvement of the local political authorities, the local community, the tourism association, and the owners of the leisure resorts is necessary. This evidence emphasizes the urgent need for a participative strategic plan to develop the local tourism in an organized and well-administered way. Only so this important source of income for the region can be ensured on the long term without disseminating the disease and putting the health of the visitors at risk.