Distribution of Lymnaeidae (Mollusca: Pulmonata), intermediate snail hosts of Fasciola hepatica in Venezuela

An extensive malacological survey was carried out between 2005-2009 in order to clarify the exact number of lymnaeid species which may be intermediate hosts of Fasciola hepatica in Venezuela. Four species were discovered during this survey, including two local species: Lymnaea cubensis and Lymnaea cousini and two exotic species: Lymnaea truncatula and Lymnaea columella. The most common local species was L. cubensis which was found at 16 out of the 298 sampling sites. This species has a large distribution area throughout the Northern part of Venezuela and was encountered from sea level to an altitude of 1,802 m in state of Trujillo. The second local species L. cousini was collected at only two sites of the Andean Region at altitudes of 3,550 m and 4,040 m, respectively. The European L. truncatula was found at 24 sites all located in the states of Mérida and Táchira at an altitude varying between 1,540-4,000 m. The respective distribution areas of L. cubensis and L. truncatula do not appear to overlap, but more detailed malacological surveys are needed. The fourth lymnaeid species, L. columella was collected in a canal from Mérida at an altitude of 1,929 m and in an irrigation canal from the state of Guárico, at an altitude of 63 m. The role of these four lymnaeid species in the transmission of fascioliasis in Venezuela is discussed.

Attraction to amino acids by Lymnaea acuminata, the snail host of Fasciola species

Adult Lymnaea acuminata (average length 20-22 mm) were collected locally from lakes and low-lying submerged fields from Gorakhpur. The chemoattraction studies were made in round glass aquaria measuring 30 cm in diameter and filled to a depth of 10 mm with 500 ml dechlorinated tap water. Each aquarium was divided into four concentric zones. At the starting time of the assay 10 snails were placed on the circumference of outermost zone 0. Snail attractant pellets (SAP) were added simultaneously in the center of central zone 3. SAP of different amino acids were prepared at concentrations of 10, 20, 50, 80 and 100 mM/2% agar solution and, subsequently, spread to a uniform thickness of 5 mm. After cooling, SAP were cut in small pieces of 5 mm in diameter. Lymnaea acuminata's attraction to amino acids was studied using different amino acid concentrations in SAP. Pellets containing amino acids with non-polar R groups (proline and tryptophan), a charged polar group (arginine) and uncharged polar R groups (serine, citrulline and asparagine) were tested. The snails were more attracted to the uncharged polar R group amino acid serine than to other groups of amino acids. The preferred amino acid concentration was 80 mM. The attraction of snails to different amino acids was concentration dependent. Snails could discriminate amongst the different amino acids at > or = 50 mM.

Biomphalaria tenagophila: genetic variability within intermediate snail hosts susceptible and resistant to Schistosoma mansoni infection

DNA analysis by molecular techniques has significantly expanded the perspectives of the study and understanding of genetic variability in molluscs that ere vectors of schistosomiasis. In tire present study, the genetic variability of susceptible and resistant B. tenagophila strains to S. mansoni infection was investigated using amplification of their genomic DNA by RAPD-PCR. The products were analyzed by PAGE and stained with silver. The results showed pdymorphism between tested strains with four different primers. We found two bonds of 1,900 and 3,420 bp that were characteristic of the susceptible strains with primer 2. The primers 9 end 10 identified a single polymorphic bond that was also characteristic of (3,136 and 5,041 bp, respectively) susceptible snails. Two polymorphic bonds were detected by primer 15: one with 1 800 bp was characteristic of the resistant strain and the other with 1,700 do in the susceptible one. These results provide additional evidence showing that the RAPD-PCR technique is adequate for the study of polymorphisms in intermediate hosts snails of S. mansoni. The obtained results are expected to expend the knowledge about the genetic variability of the snails and to permit the future identification of genomic sequences specifically related to the resistance/susceptibility of Biompholario to the larval forms of S. mansoni.

Altered gene expression in the host brain caused by a trematode parasite: Neuropeptide genes are preferentially affected during parasitosis

Schistosome parasites adjust the physiology and behavior of their intermediate molluscan hosts to their own benefit. Previous studies demonstrated effects of the avian-schistosome Trichobilharzia ocellata on peptidergic centers in the brain of the intermediate snail host Lymnaea stagnalis. In particular, electrophysiological properties and peptide release of growth- and reproduction-controlling neuroendocrine neurons were affected. We now have examined the possibility that the expression of genes that control physiology and behavior of the host might be altered during parasitosis. A cDNA library of the brain of parasitized Lymnaea was constructed and differentially screened by using mRNA from the brain of both parasitized and nonparasitized snails. This screening yielded a number of clones, including previously identified cDNAs as well as novel neuronal transcripts, which appear to be differentially regulated. The majority of these transcripts encode neuropeptides. Reverse Northern blot analysis confirmed that neuropeptide gene expression is indeed affected in parasitized animals. Moreover, the expression profiles of 10 transcripts tested showed a differential, parasitic stage-specific regulation. Changes in expression could in many cases already be observed between 1.5 and 5 hr postinfection, suggesting that changes in gene expression are a direct effect of parasitosis. We suggest that direct regulation of neuropeptide gene expression is a strategy of parasites to induce physiological and behavioral changes in the host.

Parasitic castration by the digenian trematode Allopodocotyle sp alters gene expression in the brain of the host mollusc Haliotis asinina

Infection of molluscs by digenean trematode parasites typically results in the repression of reproduction - the so-called parasitic castration. This is known to occur by altering the expression of a range of host neuropeptide genes. Here we analyse the expression levels of 10 members of POU, Pax, Sox and Hox transcription factor gene families, along with genes encoding FNIRFamide, prohormone convertase and P-tubulin, in the brain ganglia of actively reproducing (summer), non-reproducing (winter) and infected Haliotis asinina (a vetigastropod mollusc). A number of the regulatory genes are differentially expressed in parasitised H. asinina, but in only a few cases do expression patterns in infected animals match those occurring in animals where reproduction is normally repressed. (c) 2006 Published by Elsevier B.V. on behalf of the Federation of European Biochemical Societies.

Crab-eating fox (Cerdocyon thous), a South American canid, as a definitive host for Hammondia heydorni

Hammondia heydorni is a cyst forming coccidia closely related to other apicomplexans, such as Toxoplasma gondii, Neospora caninum and Hammondia hammondi with a two-host life cycle. Dogs and other canids as red foxes (Vulpes vulpes) and coyotes (Canis latrans) may serve as definitive hosts for H. heydorni. Sporulated oocysts are infective for cattle, sheep and goats, which may serve as intermediate hosts. Herein, we describe the ability of crab-eating fox (Cerdocyon thous), a wild carnivore that is commonly found from northern Argentina to northern South America, to serve as definitive host of H. heydorni. The whole masseter muscle and brain from two 2-year-old bovines were collected, minced and pooled together for the fox infection. The bovine pooled tissues were equally administered to four foxes, in two consecutive days. Two foxes shed subspherical unsporulated oocysts measuring 10-15 mu m, after 8 and 9 days post-infection, respectively. One of the foxes eliminated oocysts for 5 days, while the other fox shed oocysts for 9 days. A DNA sample of oocysts detected at each day of oocyst elimination was tested by two PCRs, one of them carried out employing primers directed to the common toxoplasmatiid 18S and 5.8S ribosomal RNA coding genes (PCR-ITS1) and the other based on heat-shock protein 70 kDa coding gene (PCR-HSP70). These samples were also submitted to a N. caninum specific nested-PCR protocol based on a N. caninum specific gene (Nc5-nPCR). All of them were positive by PCR-ITS1 and PCR-HSP70 but negative by Nc5-nPCR. The PCR-ITS1 and PCR-HSP70 nucleotide sequences amplified from the oocysts shed by the foxes revealed 100% identity with homologous sequences of H. heydorni. In conclusion, it is clear that H. heydorni also uses the crab-eating fox as a definitive host. The crab-eating fox is usually reported to live in close contact with livestock in several regions of Brazil. Therefore, it is reasonable to infer that such carnivores may play an important role in the sylvatic and domestic cycles of H. heydorni infection. (C) 2009 Elsevier B.V. All rights reserved.

Life cycle evolution in the Digenea: a new perspective from phylogeny

We use a new molecular phylogeny, developed from small and large subunit ribosomal RNA genes, to explore evolution of the digenean life cycle. Our approach is to map character states on the phylogeny and then use parsimony to infer how the character evolved. We conclude that, plesiomorphically, digenean miracidia hatched from eggs and penetrated gastropod first intermediate hosts externally. Fork-tailed cercariae were produced in rediae and emerged from the snail to be eaten directly by the teleost definitive host. These plesiomorphic characters are seen in extant Bivesiculidae. We infer that external encystment and the use of second intermediate hosts are derived from this behaviour and that second intermediate hosts have been adopted repeatedly. Tetrapod definitive hosts have also been adopted repeatedly. The new phylogeny proposes a basal dichotomy between 'Diplostomida' (Diplostomoidea, Schistosomatoidea and Brachylaimoidea) and 'Plagiorchiida' (all other digeneans). There is no evidence for coevolution between these clades and groups of gastropods. The most primitive life cycles are seen in basal Plagiorchiida. Basal Diplostomida have three-host life cycles and are associated with tetrapods. The blood flukes (Schistosomatoidea) are inferred to have derived their two-host life cycles by abbreviating three-host cycles. Diplostomida have no adult stages in fishes except by life cycle abbreviation. We present and test a radical hypothesis that the blood-fluke cycle is plesiomorphic within the Diplostomida.

dogs may be a reservoir host for Angiostrongylus costaricensis

Angiostrongylus costaricensis is a parasitic nematode of wild rodents. Several other vertebrate species including man may become infected by ingestion of the third stage larvae produced by the intermediate hosts, usually slugs from the family Veronicellidae. There is a report of the diagnosis of abdominal angiostrongyliasis in Canis familiaris with lesions resembling those found in human disease. As a preliminar evaluation of the adequacy of a canine model for pathogenetic studies, a dog was inoculated with 75 L3 of A. costaricensis. Infection was established and fist stage larvae were found in feces up to 88 days post infection, sometimes in very large numbers (9.5 x 10(4) L1/g). No clinical manifestations or significant lesions were detected. These are indications that dog may play a role as a reservoir host for A. costaricensis.

Polymerase chain reaction and restriction fragment length polymorphism analysis of the ITS2 region for differatiation of Brazilian Biomphalaria intermediate hosts of the Schistosoma mansoni

We sequenced the internal transcribed spacer 2 of the ribosomal DNA (ITS2-DNAr) from the three Schistosoma mansoni intermediate hosts in Brazil: Biomphalaria glabrata, Biomphalaria tenagophila and Biomphalaria straminea. Analysis of a restriction map from those sequences allowed us to select putative restriction enzymes able to identify the snail species under study. Four restriction enzymes were used and HpaII provided simple species-specific profiles easily visualized in polyacrylamide gels. The use of ITS2 is advantageous as it provides a small fragment of 460 bp which may be easily amplified by PCR. In the current work, we showed that the amplification of ITS2-DNAr together with HpaII enzyme restriction is an auxiliary molecular tool for the morphological identification of such snails as well as for taxonomic and phylogenetic studies of neotropical planorbids.

On the diversity of mollusc intermediate hosts of Angiostrongylus costaricensis Morera & Cespedes, 1971 in southern Brazil

Veronicellid slugs are considered the most important intermediate hosts of Angiostrongylus costaricensis, an intra-arterial nematode of rodents. Studies undertaken in three localities in southern Brazil led to identification of molluscs other than veronicellid slugs as hosts of A. costaricensis: Limax maximus, Limax flavus and Bradybaena similaris. These data indicate a low host specificity of larval stages of A. costaricensis, as it has been reported to other congeneric species.

DNA polymorphism of schistosomes and their snail hosts

Analysis of the genomes of schistosomes and one of their intermediate hosts, Biomphalaria glabrata, using Random Amplified Polymorphic DNA (RAPD) demonstrated that intraspecific genetic polymorphism in the parasite is limited but in the snail is highly pronounced. This suggests an important role for the snail in the determination of the epidemiology of the disease. In addition to their intraspecific stability, schistosome derived RAPDs exhibit a high level of interspecific polymorphism and are thus ideal for the construction of phylogenetic trees. For the detection of intraspecific polymorphisms extensive variation in the mitochondrial DNA is being exploited for the development of a PCR based test for Schistosoma mansoni. Gene level polymorphisms are being analyzed by Low Stringency Single Specific Primer PCR.

Schistosomiasis mansoni in Bananal (State of São Paulo, Brazil): II. Intermediate hosts

We conducted monthly snail captures in Bananal, State of São Paulo, Brazil, between March 1998 and February 2001, to identify Schistosoma mansoni vectors, estimate seasonal population changes, and delimit foci. We also evaluated the impact of improvements in city water supply and basic sanitation facilities. We identified 28,651 vector specimens, 28,438 as Biomphalaria tenagophila, 49 of them (0.2%) infected with S. mansoni, and 213 as B. straminea, none of the latter infected. Vectors predominated in water bodies having some vegetation along their banks. Neither population density nor local vegetation could be linked to vector infection. We found the first infected snails in 1998 (from March to May). Further captures of infected snails ocurred, without exception, from July to December, when rainfall was least. Irrespective of season, overall temperature ranged from 16.5ºC to 21ºC; pH values, from 6.0 to 6.8. Neither factor was associated with snail population density. Frequent contact of people with the river result from wading across it, extracting sand from its bottom, fishing, washing animals, etc. Despite a marked reduction in contamination, cercaria shedding persists. Whatever the location along its urban course, contact with river Bananal, particularly of the unprotected skin, entails risks of infection.

A Multiplex-PCR approach to identification of the Brazilian intermediate hosts of Schistosoma mansoni

Due to difficulties concerning morphological identification of planorbid snails of the genus Biomphalaria, and given a high variation of characters and in the organs with muscular tissue, we designed specific polymerase chain reaction (PCR) primers for Brazilian snail hosts of Schistosoma mansoni from available sequences of internal transcribed spacer 2 (ITS2) of the ribosomal RNA gene. From the previous sequencing of the ITS2 region, one primer was designed to anchor in the 5.8S conserved region and three other species-specific primers in the 28S region, flanking the ITS2 region. These four primers were simultaneously used in the same reaction (Multiplex-PCR), under high stringency conditions. Amplification of the ITS2 region of Biomphalaria snails produced distinct profiles (between 280 and 350 bp) for B. glabrata, B. tenagophila and B. straminea. The present study demonstrates that Multiplex-PCR of ITS2-DNAr showed to be a promising auxiliary tool for the morphological identification of Biomphalaria snails, the intermediate hosts of S. mansoni.

A comparative parasitologic study on Biomphalaria glabrata snail and C3H/He mice infected with human and murine isolates of Schistosoma mansoni derived from Sumidouro, Rio de Janeiro, Brazil

Experiments were carried out to analyze the biological characteristics of two sympatric isolates of Schistosoma mansoni derived from humans and murines in a low endemic transmission area (Sumidouro county, state of Rio de Janeiro, Brazil). Sympatric reared-laboratory Biomphalaria glabrata and C3H/He mice were used as experimental hosts. Parameters assessed comprised: precercarial period, infectivity and mortality (snails), prepatent period, infectivity (percentage of cercariae maturation into adult worm) and intestinal egg count (mice). The murine isolate showed a shorter precercarial period and higher infectivity than human isolate (p < 0.05). This biological heterogenicity did not correspond to the vertebrate data because any biological parameter presented significant difference (p > 0.05). These data suggest that both isolates are local sub-populations, providing support for the hypotheses that in a same biotope mixed populations or sub-populations circulate among their main host (human beings) and/or rodent as an anfixenous infection.

Field trials of low dose Bayluscide on snail hosts of schistosome and selected non-target organisms in sahelian Cameroon

More than 85% of all cases of schistosomiasis in Cameroon occur in the northern sahelian half of the country representing 20% of the population. Several workers have advocated the integrated approach to schistosomiasis control, including snail control, but the death and decay of aquatic organisms, and fish kill that often follows Bayluscide application at the dose of 1g/m³ decrease its acceptability. The present study was designed to assess the effect of lower Bayluscide doses on snail host and non-target fish, frog, the tadpole kill. Bayluscide was applied to study ponds at concentrations of 0, 0.25, 0.5, and 1 g/m³ (ppm). Pre and post application assessment of snails hosts of schistosomes, fish, frog, and tadpole kill were carried out. All 0.25, 0.5, and 1 g/m³ Bayluscide concentrations reduced snail population significantly. Bayluscide concentration of 0.50 g/m³ applied in two rounds of 0.25 g/m³ resulted in high snail mortality and low lethality to fish, frogs, and tadpoles. Further studies are needed to assess the cost-effectiveness of Bayluscide in the control of schistosomiasis following the simplified approach.