Aspects of the maintenance of the life cycle of Fasciola hepatica in Lymnaea columella in Minas Gerais,Brazil

Fascioliasis is a parasitic disease of domestic ruminants that occurs worldwide. The lymnaeid intermediate hosts of Fasciola hepatica include Lymnaea columella, which is widely distributed in Brazil. A colony of L. columella from Belo Horizonte, MG, was reared in our laboratory to be used in studies of the F. hepatica life cycle, the intermediate host-parasite relationship and development of an anti-helminthic vaccine. In the first experiment 1,180 snails were exposed to miracidia of F. hepatica eggs removed from the biliary tracts of cattle from the State of Rio Grande do Sul. In the second and third experiments the snails were exposed to miracidia that had emerged from F. hepatica eggs from Uruguay, maintained in rabbits. The rates of infection in the first, second and third experiments were 0, 42.1 and 0% respectively. Over 15,806 metacercariae were obtained and stored at 4 degrees C. Four rabbits weighing 1.5 kg each were infected with 32-44 metacercariae and two with 200. Three rabbits begin to eliminate eggs of the parasite in the feces from 84 days after infection onwards. The biological cycle of F. hepatica in L. columella and the rabbit was completed within 124 days.     

A nucleic acid-based test for detection of Fasciola hepatica.

The use of nucleic acid techniques in the diagnosis of parasitic infection has become increasingly widespread. An oligonucleotide probe derived from a rRNA sequence was developed for the detection of Fasciola hepatica in its intermediate snail host Pseudosuccinea columella. Total RNA obtained from whole adult liver flukes was used in a polymerase chain reaction to isolate and amplify a region of approximately 650 base pairs in the small subunit rRNA. This portion of the ribosomal cDNA, which contains highly conserved regions as well as variable regions, was subcloned and sequenced. In comparison to known small subunit rRNA sequences, a sequence unique to F. hepatica was identified and an oligonucleotide probe (CS4) for detection of F. hepatica was developed. A northern blot analysis using CS4 successfully identified small subunit rRNA from F. hepatica. Slot-blot analysis determined that RNA derived from 5 miracidia can be detected with CS4. Moreover, a slot blot utilizing CS4 distinguished RNA derived from snails infected with F. hepatica from RNA of uninfected snails.     

Population dynamics of Lymnaea columella and its natural infection by Fasciola hepatica in the State of Minas Gerais,Brazil

Studies on the population dynamics and natural infection of Lymnaea columella by Fasciola hepatica were carried out from September 1999 to December 2000 in a low-lying area near Itajubá in the Brazilian State of Minas Gerais. A total of 626 snails were collected monthly at nine different sites, counted, and dissected to search for larvae of F. hepatica. The highest populations of L. columella were reached in October of 1999 and August of 2000, and the highest natural infection rates of snails by F. hepatica were reached in September 1999 (5.2%) and July 2000 (3.9%). The removal by farmers of aquatic plants from the drainage furrows caused a drastic reduction in this snail population.     

Unusual snail species involved in the transmission of Fasciola hepatica in watercress beds in central France

Four freshwater pulmonate species (Lymnaea ovata, L. stagnalis, Physa acuta, Planorbis leucostoma) were living in several watercress beds known for their relationships with human cases of fasciolosis, whereas L. truncatula was never found. The aims of these studies were to determine the prevalence of natural infections with Fasciola hepatica in snails and to verify if these species might ensure the full larval development of this trematode (with cercarial shedding) when they were experimentally subjected to F. hepatica only, or to co-infections with an other trematode species. Investigations were so carried out in six snail populations living in watercress beds (including three for P. acuta) and in four others originating from three brooks or a pond (as controls). Snails naturally infected with F. hepatica were found in two watercress beds inhabited by L. ovata (prevalence of infection: 1.4%) and P. leucostoma (0.1%), respectively. The L. ovata from the watercress bed could be infected at a higher size than those from the control population and the prevalence of this infection was greater in the bed population. Similar findings were noted for L. stagnalis. Despite single or dual infections, the results obtained with the four populations of P. acuta were unsuccessful. In contrast, the co-infections of young P. leucostoma with Paramphistomum daubneyi and F. hepatica resulted in the shedding of some F. hepatica cercariae. According to the authors, the occurrence of fasciolosis in these watercress beds would be the consequence of frequent natural encounters between parasite and snails (L. ovata, L. stagnalis), or of co-infections with P. daubneyi and F. hepatica (P. leucostoma). In watercress beds only colonized by P. acuta, a lymnaeid species would have ensured the larval development of F. hepatica but it would have been eliminated by P. acuta, as this last species was known to be invasive and could colonize open drainage ditches on siliceous soil.     

Development of Fasciola hepatica in Lymnaea columella infected with miracidia derived from cattle and marmoset infections

The development of Fasciola hepatica from two species of definitive hosts, i.e. cattle (Bos taurus) and a marmoset (Callithrix penicillata) in the snail Lymnaea columella was determined based on the production of rediae and cercariae and snail survival rate. More rediae and cercariae at 60-74 days post-infection were produced by snails infected by cattle-derived miracidia (cattle group) than by those infected by marmoset-derived miracidia (marmoset group). Among the L. columella parasitized by the marmoset group, the survival rate and the percentage of positive snails were higher than among those parasitized by the cattle group. Eggs of F. hepatica released in cattle faeces were significantly bigger than those released in marmoset faeces. Miracidia originating from parasites that completed their development in cattle were more efficient in infecting the intermediate host. These results suggest that vertebrate-host origin influences the eggs produced by the parasite and the infection rates in the snail host L. columella.     

Role of the prosegment of Fasciola hepatica cathepsin L1 in folding of the catalytic domain

The N-terminal propeptides of cysteine proteinases play regulatory roles in the folding and stability of their catalytic domains, as well as being potent and highly specific inhibitors of their parental mature enzymes. Cysteine proteinases play a major role in the biology of the parasitic trematode Fasciola hepatica; in particular, this organism secretes significant amounts of cathepsin L enzymes. The isolated propeptide of F. hepatica cathepsin L1 functioned as a chaperone for the mature enzyme in renaturation experiments. A double point mutation (N701/F721) within the GxNxFxD motif of the propeptide affected its conformation and markedly decreased its affinity for the mature enzyme. When this mutation was introduced into preprocathepsin L1 expressed in yeast, the secretion of active enzyme dropped dramatically. However, significant enzyme activity was recovered from the culture supernatants after denaturation and renaturation in the presence of native propeptide. Thus, the variant prosegment gave rise to an enzyme with altered conformation, which could be refolded to the active form with the assistance of the native propeptide.     

Fascioliasis and other plant-borne trematode zoonoses

Fascioliasis and other food-borne trematodiases are included in the list of important helminthiases with a great impact on human development. Six plant-borne trematode species have been found to affect humans: Fasciola hepatica, Fasciola gigantica and Fasciolopsis buski (Fasciolidae), Gastrodiscoides hominis (Gastrodiscidae), Watsonius watsoni and Fischoederius elongatus (Paramphistomidae). Whereas F. hepatica and F. gigantica are hepatic, the other four species are intestinal parasites. The fasciolids and the gastrodiscid cause important zoonoses distributed throughout many countries, while W. watsoni and F. elongatus have been only accidentally detected in humans. Present climate and global changes appear to increasingly affect snail-borne helminthiases, which are strongly dependent on environmental factors. Fascioliasis is a good example of an emerging/re-emerging parasitic disease in many countries as a consequence of many phenomena related to environmental changes as well as man-made modifications. The ability of F. hepatica to spread is related to its capacity to colonise and adapt to new hosts and environments, even at the extreme inhospitality of very high altitude. Moreover, the spread of F. hepatica from its original European range to other continents is related to the geographic expansion of its original European lymnaeid intermediate host species Galba truncatula, the American species Pseudosuccinea columella, and its adaptation to other lymnaeid species authochthonous in the newly colonised areas. Although fasciolopsiasis and gastrodiscoidiasis can be controlled along with other food-borne parasitoses, fasciolopsiasis still remains a public health problem in many endemic areas despite sustained WHO control programmes. Fasciolopsiasis has become a re-emerging infection in recent years and gastrodiscoidiasis, initially supposed to be restricted to Asian countries, is now being reported in African countries.     

First report of Lymnaea columella Say, 1817 (Pulmonata: Lymnaeidae) naturally infected with Fasciola hepatica (Linnaeus,1758) (Trematoda: Digenea) in Argentina

We report the first evidence of natural infection of Lymnaea columella with Fasciola hepatica in Argentina. A sample of 601 snails was collected in May 2003 in northeastern Corrientes, a province bounded on the north by Paraguay, on the east by Brazil and on the southeast by Uruguay. Among 500 examined snails, 44 (8.8%) were exclusively infected with F. hepatica. Parasite identification was based on morphological features of cercariae from snails, and of eggs and adult flukes from Wistar rats. We discuss the events suggesting that an enzootic transmission cycle of F. hepatica has been recently established in northeastern Corrientes.     

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.     

Effect of the exposure to Fasciola hepatica (Trematoda: Digenea) on life history traits of Lymnaea cousini and Lymnaea columella (Gastropoda: Lymnaeidae)

The snails Lymnaea columella and Lymnaea cousini have both been reported as intermediate hosts of Fasciola hepatica in Colombia. The effect of the exposure to the parasite on survival, fecundity and size of these snails was evaluated by means of experimental infections and the life history traits of control and exposed groups were compared. Infection rates were 82.2 and 34% for L. columella and L. cousini, respectively. A reduction in fitness was observed in both species when exposed to the parasite: fecundity alone was reduced in L. columella whereas in L. cousini there was also a decline in survival rate. Unlike other studies, increased size was not observed in either species. On the contrary, a reduction in growth rate was observed in L. columella.     

The hemoglobins of the trematodes Fasciola hepatica and Paramphistomum epiclitum: a molecular biological, physico-chemical, kinetic, and vaccination study

The trematode Fasciola hepatica (Fa.he.) is a common parasite of human and livestock. The hemoglobin (Hb) of Fa.he., a potential immunogen, was chosen for characterization in the search for an effective vaccine. Characterization of trematode Hbs show that they are intracellular single-domain globins with the following remarkable features: (1) Fa.he. expresses two Hb isoforms that differ at two amino acid sites (F1: 119Y/123Q; F2: 119F/123L). Both isoforms are monoacetylated at their N-termini; (2) the genes coding for Fa.he. and Paramphistomum epiclitum (Pa.ep.) Hbs are interrupted by two introns at the conserved positions B12.2 and G7.0.; (3) UV/VIS and resonance Raman spectroscopy identify the recombinant Fa.he. HbF2 as a pentacoordinated high-spin ferrous Hb; (4) electron paramagnetic resonance spectroscopy of cyano-met Fa.he. HbF2 proves that the endogenously bound imidazole has no imidazolate character; (5) the major structural determinants of the globin fold are present, they contain a TyrB10/TyrE7 residue pair on the distal side. Although such distal-site pair is a signature for high oxygen affinity, as shown for Pa.ep. Hb, the oxygen-binding rate parameters for Fa.he. Hb are intermediate between those of myoglobin and those of other trematode Hbs; (6) the three-dimensional structure of recombinant Fa.he. HbF2 from this study closely resembles the three-dimensional structure of Pa.ep. determined earlier. The set of distal-site polar interactions observed in Pa.ep. Hb is matched with small but significant structural adjustments; (7) despite the potential immunogenic character of the fluke Hb, vaccination of calves with recombinant Fa.he. HbF2 failed to promote protection against parasitic infection.     

Metacercarial aggregation in Digenea (Fasciola hepatica and Paramphistomum daubneyi): environmental or species determinism?

Metacercarial aggregation of Fasciola hepatica and Paramphistomum daubneyi was studied under experimental conditions to determine if the formation of these aggregates was influenced by environmental factors, or it was a characteristic of trematode species. This process was studied using the confinement of infected snails on the bottom of Petri dishes (diameter, 14 cm) for 3 days. The formation of metacercarial aggregates of F. hepatica was not significantly modified by environmental factors such as intensity and duration of lighting, quality and volume of water. Metacercariae of F. hepatica were more numerous on the Petri dish walls and 63.9% of them constituted aggregates. In contrast, most metacercariae of P. daubneyi were found on the Petri dish bottoms and 78.3% of them were isolated or in groups of two metacercariae each. The mean number of metacercariae per aggregate ranged from 6.7 to 12.2 in the case of F. hepatica, and from 2.7 to 4.5 in the case of P. daubneyi. However, these mean numbers were independent of the site of cercarial attachment. The tendency of cercariae to form metacercarial aggregations was a characteristic of F. hepatica and was species determined.     

A recombinant antigen recognized by Fasciola hepatica-infected hosts

This work reports the detection of specific immunoglobulins (Ig) against rFh8, a recombinant Fasciola hepatica adult worm excretion-secretion antigen, in sera from experimentally (rabbit, Wistar rat, cattle, and sheep), or naturally (human) infected hosts. In the case of laboratory experimental models the study revealed significant differences between rabbits, which recognized the recombinant antigen all along the infection, and Wistar rats, which showed high anti-rFh8 Ig levels only for a short period of the infection. Available sera from experimentally infected cattle and sheep, as well as sera from naturally F. hepatica-infected humans, also contained significant levels of Ig against rFh8, suggesting that Fh8 was produced by F. hepatica at a very early stage of infection in all hosts so far analyzed and that the rFh8 antigen could be used as a tool for the diagnosis of F. hepatica infections.     

Multiplex PCR for both identification of Brazilian Biomphalaria species (Gastropoda: Planorbidae) and diagnosis of infection by Schistosoma mansoni (Trematoda: Schistosomatidae)

A simple and single-step technique based on multiplex PCR (multiplex polymerase chain reaction) has been developed for simultaneous identification of Brazilian Biomphalaria species, the intermediate hosts of Schistosoma mansoni, and their diagnosis of infection by the trematode. We used species-specific primers directed both to the internal transcribed spacer 2 (ITS2) of ribosomal DNA from 3 of the S. mansoni host species and to the mitochondrial DNA (mtDNA) from the trematode. Those primers were used simultaneously in a single multiplex-PCR reaction, and template DNA was obtained from S. mansoni-infected and noninfected snails. The results were visualized in silver stained polyacrylamide gels, revealing the presence of specific bands. The methodology has shown to be efficient, fast, and reproducible for Biomphalaria species identification and diagnosis of snails infected by S. mansoni during prepatent periods.     

Characterisation of a beta-tubulin gene from the liver fluke, Fasciola hepatica

This study represents the first beta-tubulin sequence from a trematode parasite, namely, the liver fluke, Fasciola hepatica. PCR of genomic DNA showed that at least one beta-tubulin gene from F. hepatica contains no introns. A number of amino acids in the primary sequence of fluke tubulin are different from those described previously in various nematode species and the cestode, Echinococcus multilocularis. beta-Tubulin is an important target for benzimidazole anthelmintics, although (with the exception of triclabendazole) they show limited activity against F. hepatica. The amino acid differences in fluke beta-tubulin are discussed in relation to the selective toxicity of benzimidazoles against helminths and the mechanism of drug resistance.     

Variation of snail's abundance in two water bodies harboring strains of Pseudosuccinea columella resistant and susceptible to Fasciola hepatica miracidial infection, in Pinar del Río Province, Cuba

The abundance of freshwater snails in two rural sites of Pinar del Río, Cuba, which harbor Pseudosuccinea columella susceptible and resistant to miracidia of Fasciola hepatica was followed for one year. Susceptible snails were found in the most anthropic site (IPA) whereas the resistant population inhabited the most preserved one (El Azufre). Only two snail species coexisted with P. columella at IPA site (Physa cubensis and Tarebia granifera) while five species were found at El Azufre, including an endemic from that province (Hemisinus cubanianus). Populations of both resistant and susceptible snails showed stable densities throughout the year, although the susceptible strain attained higher abundance. The highest densities were observed in April-May 2004 for the susceptible population whereas the resistant strain attained its highest abundance in January 2004. No record of Fossaria cubensis was made and the thiarid T. granifera occurred only at low densities. One of the sampled sites (IPA) meets all the conditions for the first report of P. columella naturally infected with larvae of F. hepatica.     

Search of natural occurrence of xiphidiocercariae (trematoda) in fresh water snails of nine countries from São Paulo State,Brazil

Xiphidiocercariae, aquatic larval stages of some trematodes are considered a potential instrument for biological control of mosquitoes. In this study we evaluated its natural occurrence in Campinas region and two places in Vale do Ribeira (Registro and Miracatu), S?o Paulo State. Snails were obtained from fresh water collections from September 1996 to February 1999. The species collected were Lymnaea columella, Physa marmorata, Biomphalaria tenagophila, Biomphalaria sp., Drepanotrema cimex, D. lucidum and Drepanotrema sp. Fasciola hepatica, xiphidiocercariae (Haematoloechidae) and echinostomatid cercariae were detected in the lymnaeids snails from Miracatu, SP. In the same locality were found planorbids snails parasitized by furcocercariae, echinostomatid cercariae and xiphidiocercariae. The xiphidiocercariae found in the planorbids were different from those obtained from lymnaeids. One Biomphalaria sp. infected with furcocercariae was found in Louveira, SP. In the ROSA place (Campinas, SP) an individual of Biomphalaria sp. and one of L. columella were found infected by the furcocercariae and echinostomatid cercariae, respectively. In the place UNI-I, in Campinas, one L. columella was infected by furcocercariae. Double infection in snails from Miracatu was also observed.     

Isoenzymes of lactate dehydrogenase (EC 1.1.1.27) in Dicrocoelium dendriticum and Fasciola hepatica (Trematoda)

The isoenzymes of LDH (EC 1.1.1.27) were studied in Dicrocoelium dendriticum and Fasciola hepatica by horizontal electrophoresis on polyacrylamide gel. Six isoenzymes in D. dendriticum and four in F. hepatica were detected. Densitometric scans demonstrated that the enzyme pattern of LDH in D. dendriticum parasitizing hepatic tissue of Capra hircus was clearly different from the one obtained when the trematode parasitized other hosts such as Bos taurus or Ovis aries.