Antigenic community between Schistosoma mansoni and Biomphalaria glabrata: on the search of candidate antigens for vaccines

We have previously confirmed the presence of common antigens between Schistosoma mansoni and its vector, Biomphalaria glabrata. Cross-reactive antigens may be important as possible candidates for vaccine and diagnosis of schistosomiasis. Sera from outbred mice immunized with a soluble Biomphalaria glabrata antigen (SBgA) of non-infected B. glabrata snails recognized molecules of SBgA itself and S. mansoni AWA by Western blot. Recognition of several molecules of the SBgA were inhibited by pre-incubation with AWA (16, 30, 36, 60 and 155 kDa). The only specific molecule of AWA, inhibited by SBgA, was a 120 kDa protein. In order to determine which epitopes of SBgA were glycoproteins, the antigen was treated with sodium metaperiodate and compared with non-treated antigen. Molecules of 140, 60 and 24 kDa in the SBgA appear to be glycoproteins. Possible protective effects of the SBgA were evaluated immunizing outbred mice in two different experiments using Freund's Adjuvant. In the first one (12 mice/group), we obtained a significant level of protection (46%) in the total worm load, with a high variability in worm recovery. In the second experiment (22 mice/group), no significant protection was observed, neither in worm load nor in egg production per female. Our results suggest that SBgA constitutes a rich source of candidate antigens for diagnosis and prophylactic studies.

Extra-cellular matrix changes in Schistosoma mansoni-infected Biomphalaria glabrata

Reactivity of snails against parasites exhibits a primitive focal reaction, with encapsulation, phagocytosis and destruction of parasite larvae by macrophage-like cells - the hemocytes. This reaction mimics granulomatous inflammation seen in higher animals. However, different from the latter, little is known about the participation of extra-cellular matrix in such snail defense reactions. Normal and Schistosoma mansoni-infected Biomphalaria glabrata of different strains were submitted to cytological, histological, ultrastructural and biochemical methods in order to investigate the behavior of extra-cellular tissues at the site of anti-parasite reactions. In spite of the presence of two cell-types in peripheral hemolymph, only one cell-type was present at the sites of tissue reactions. Although pre-existent collagen and elastic fibers and microfibrils sometimes appeared slightly compressed around focal reactions, no evidences of duplication, synthesis or deposition of connective-tissue extra-cellular components were observed within or around the zones of reactive cell accumulations. Thus, tissue reactions against S. mansoni in the snail B. glabrata appeared exclusively dependent on one specific population of hemocytes.

Apparent competition through facilitation between Melanoides tuberculata and Biomphalaria glabrata and the control of schistosomiasis

Interactions between two species that result in reduced growth rates for both and extinction of one of the species are generally considered cases of asymmetric interspecific competition. Exploitative or interference competition is the usual mechanism invoked. Here we describe another mechanism producing the same result, named apparent competition through facilitation (ACF), observed between Melanoides tuberculata and Biomphalaria glabrata populations. The superior competitor actually gives some benefit to the other species, whose population becomes unstable with progressively increasing oscillations, leading to extinction. A model of ACF using difference equations suggests initial dynamics distinct from traditional interspecific competition. The dynamics of two freshwater snails in the field and in laboratory experiments suggest ACF, and these relations should be considered in studies of schistosomiasis control. ACF could occur in natural populations, but might have gone undetected because the final result is similar to traditional interspecific competition.

Molluscicidal action of the latex of Euphorbia splendens var. hislopii N.E.B. ("Christ's Crown") (Euphorbiaceae) against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica Linnaeus, 1758 (Trematode: Fasciolidae): 1- test in laboratory

The latex action of Euphorbia splendens var. hislopii (Christ's Crown) against snails Lymnaea columella, intermediate host of Fasciola hepatica, derived from irrigation ditches of the Station of Pisciculture at Universidade Federal Rural do Rio de Janeiro, was studied in the laboratory. Lab bioassays, using aqueous solutions of the latex, varying between 0.1 and 10 mg/l, have proven molluscicidal activity of the product collected on the same day the tests were performed, during the four seasons of the year, finding the following lethal concentrations (LC90): 1.51 mg/l in the spring; 0.55 mg/l in the summer; 0.74 mg/l in the fall and 0.93 mg/l in winter, after 24 h exposure of the snails, showing significant differences among the seasons of the year (ANOVA test, F = 11.01, G.L.= 3/33, p < 0.05), as well as among the concentrations (ANOVA test, F = 27.38, G.L.= 11/33, p < 0.05). In the summer, mortality reached 100% from concentration at 0.6 mg/l, the same during fall and in winter as of 1 mg/l, while in spring it only reached 100% mortality as of 2 mg/l. Mortality in the controls was low, reaching 5% in the summer and winter and 10% in the fall and spring. None of the samples died. During the assay, with an aqueous solution of the latex at a concentration of 5 mg/l, in order to check the time of duration of the product effect, in the laboratory, it was observed that the molluscicidal activity remained stable up to the 15th day after the beginning of the test with 100% mortality of L. columella, gradually losing its effect until the 23rd day, when we no longer observed animal mortality. In the control group, there was a random daily variation in mortality rate ranging 0-50% after 48 h of observation for 30 days.

Acute schistosomiasis outbreak in the metropolitan area of Belo Horizonte, Minas Gerais: alert about the risk of unnoticed transmission increased by growing rural tourism

The present article describes the occurrence of 17 cases of acute schistosomiasis in the metropolitan area of Belo Horizonte, state of Minas Gerais, Brazil. All individuals affected took a bath in a swimming pool of a holiday resort that was provided with water from a nearby brook. The apparently clean water and the absence of snails in the pool gave the wrong impression that there was no risk for infection. During a malacological survey at the site snails of the species Biomphalaria glabrata were found and tested positive for Schistosoma mansoni. All the patients live in the middle-class area of Barreiro, metropolitan area of Belo Horizonte and have medium grade school education. The difficulties in establishing the right diagnosis is expressed by the search for medical attention in 17 different medical facilities, the wide range of laboratory test and the inadequate treatment administration. A lack of knowledge about the disease was found in all groups studied. The booming rural tourism in endemic areas is identified as a probable risk factor for infection, especially for individuals of the non-immune middle and upper class parts of the society in urban centers. Special attention is given to a multidisciplinary approach to the complex issue of disease control and prevention.

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.

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.

Activity of Euphorbia splendens var. hislopii N.E.B. (Euphorbiaceae) latex against Lymnaea columella (Say, 1817) (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica, Linnaeus, 1758 (Trematoda: Fasciolidae). 2: limited field-testing

The molluscicidal evaluation of Euphorbia splendens var. hislopii (Crown of thorns) against Lymnaea columella snails, intermediate host of Fasciola hepatica, in irrigation ditches of the Pisciculture Station at Universidade Federal Rural do Rio de Janeiro, was studied under limited field conditions. An aqueous solution of the latex at 5 mg/l was tested in two irrigation ditches (experimental and control ditches), after initial sampling of the snail population present. Twenty-four hours after application of the product, it was verified that 97.4% of free L. columella snails and 100% of snails of the same species captive in cages and used as sentinels at three points equidistant from the application site in the experimental ditch, died. For Biomphalaria tenagophila and Melanoides tuberculata snails, present in the experimental ditch, the mortality was 100%, for the species Pomacea spp. the mortality was 40%. No mortality was verified in the free mollusks, or in the sentinels in the ditch used as control. E. splendens var. hislopii latex is thus an efficient natural molluscicide, which may be used as an alternative control agent against L. columella.

Southernmost finding of Lymnaea viatrix orbigny, 1835 (Pulmonata: Lymnaeidae), intermediate host of Fasciola hepatica (Linnaeus, 1758) (Trematoda: Digenea), in urban and rural areas of Patagonia, Argentina

We report the first finding of Lymnaea viatrix south of parallel 41ºS, in rural and urban areas from Argentina. Ninety snails were collected during year 2000, from a concrete pond at a Public Square in El Bolsón Village, Río Negro province, and 811 snails in November 1999, and during 2000 from waterbodies within a farm at Cholila locality, Chubut province. Fasciola hepatica infection was detected in 0.9% snails from the rural area. We discuss the potential risk of L. viatrix to public health in urban areas and its epidemiological importance in rural areas of the Andean Patagonian region.

Analysis of the first and second internal transcribed spacer sequences of the ribosomal DNA in Biomphalaria tenagophila complex (Mollusca: Planorbidae)

The first and second internal transcribed spacer regions (ITS1 and ITS2) of the ribosomal DNA of Biomphalaria tenagophila complex (B. tenagophila, B. occidentalis, and B. t. guaibensis) were sequenced and compared. The alignment lengths of these regions were about 655 bp and 481 bp, respectively. Phylogenetic relationships among the Biomphalaria species were inferred by Maximum Parsimony and Neighbor-joining methods. The phylogenetic trees produced, in most of the cases, were in accordance with morphological systematics and other molecular data previously obtained by polymerase chain reaction and restriction fragment length polymorphism analysis. The present results provide support for the proposal that B. tenagophila represents a complex comprising B. tenagophila, B. occidentalis and B. t. guaibensis.

Molluscicidal effect of nicotinanilide and its intermediate compounds against a freshwater snail Lymnaea luteola, the vector of animal schistosomiasis

The molluscicidal effect of nicotinanilide was evaluated and compared with niclosamide (2',5-dichloro-4'-nitrosalicylanilide, ethanolamide salt) against different stages of the freshwater snail Lymnaea luteola i.e., eggs, immature, young mature, and adults. Calculated values of lethal concentrations (LC50 and LC90 ) showed that both nicotinanilide and niclosamide as toxic against eggs, immature, and adults. The young mature stage of the snails was comparatively more tolerant to both molluscicides than the other stages. The toxicity of the intermediate compounds of nicotinanilide against the young mature stage of the snails showed them as ineffective. The mortality pattern of the snails exposed to LC90 concentration of these molluscicides showed niclosamide to kill faster (within 8 to 9 h) than nicotinanilide (26 to 28 h). In view of the above studies it may be concluded that both molluscicides are toxic against all the stages of the L. luteola snails.

The use of the polymerase chain reaction and restriction fragment length polymorphism technique associated with the classical morphology for characterization of Lymnaea columella, L. viatrix, and L. diaphana (Mollusca: Lymnaeidae)

The specific identification of Lymnaeid snails is based on a comparison of morphological characters of the shell, radula, renal and reproductive organs. However, the identification is complicated by dissection process, intra and interspecific similarity and variability of morphological characters. In the present study, polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) techniques targeted to the first and second internal transcribed spacers (ITS1 and ITS2) rDNA and to the mitochondrial 16S ribosomal gene (16S rDNAmt) were used to differentiate the species Lymnaea columella, L. viatrix, and L. diaphana from some localities of Brazil, Argentina, and Uruguay as well as to verify whether the molecular results corroborates the classical morphological method.PCR-RFLP analysis of the ITS1, ITS2, and 16S using 12 restriction enzymes revealed characteristic patterns for L. columella and L. diaphana which were concordant with the classical morphology. On the other hand, for L. viatrix populations a number of 1 to 6 profiles were generated while morphology provided the species pattern results.

Distribution and Schistosoma mansoni infection of Biomphalaria glabrata in different habitats in a rural area in the Jequitinhonha Valley, Minas Gerais, Brazil: environmental and epidemiological aspects

This paper examines the distribution and infection of Biomphalaria glabrata with Schistosoma mansoni in all aquatic snail habitats in a rural area in the state of Minas Gerais, Brazil, in relation to physico/biotic and behavioral factors. Snail and environmental surveys were carried out semi-annually between July 2001 and November 2002 at 106 sites. Collected snails were examined in the laboratory for infection. B. glabrata densities were highest in overflow ponds, irrigation ponds, springs, canals and wells, and lowest in fishponds and water tanks. Snail densities were higher during the hot, rainy season except for streams and canals and were statistically associated with the presence of fish, pollution, and vegetation density. Tilapia fish and an unidentified Diptera larva were found to be predators of B. glabrata but ducks were not. Twenty-four of the 25 infected snails were collected in 2001(1.4% infection rate) and only one in 2002, after mass chemotherapy. The occurrence of B. glabrata in all 11 snail habitats both at and away from water contact sites studied indicates widespread risk of human infection in the study area. In spite of the strong association between B. glabrata and tilapia in fishponds we do not recommend its use in schistosomiasis control for ecological reasons and its relative inefficiency in streams and dams.

The initial epidemiological studies in the low endemicity schistosomiasis area in esteio, Rio Grande do Sul, the southernmost Brazilian State, 1997 to 2000

Nor Biomphalaria glabrata neither Schistosoma mansoni were reported from Rio Grande do Sul, the southernmost Brazilian state before 1997. Their detection next to the Sinos River, Esteio, confirmed predictions of schistosomiasis expansion to the south. Parasitological examinations both in snails and fecal samples from the human population were performed from 1997 to 2000. The last 3 out of 5 surveys were performed after a preliminar serological screening procedure in a risk group identified at a population census. A total of 11 infected individuals were found infected and snails from 2 different sites were positive for S. mansoni. Samples from these 2 and other sites were identified as B. glabrata. Egg counts in feces were below 1 per gram in 6 out of 11 patients. Some socio-cultural perceptions of water contact activities next to the Sinos River may cause difficulties to control efforts, but they also may be partially acting against a very rapid increase in transmission intensity. The southernmost schistomiasis mansoni foci in Americas rise the alert for its ongoing expansion.

Spatial distribution of schistosomiasis foci on Itamaracá Island, Pernambuco, Brazil

Acute cases of schistosomiasis have been found on the coastal area of Pernambuco, Brazil, due to environmental disturbances and disorderly occupation of the urban areas. This study identifies and spatially marks the main foci of the snail host species, Biomphalaria glabrata on Itamaracá Island. The chaotic occupation of the beach resorts has favoured the emergence of transmission foci, thus exposing residents and tourists to the risk of infection. A database covering five years of epidemiological investigation on snails infected by Schistosoma mansoni in the island was produced with information from the geographic positioning of the foci, number of snails collected, number of snails tested positive, and their infection rate. The spatial position of the foci were recorded through the Global Positioning System (GPS), and the geographical coordinates were imported by AutoCad. The software packages ArcView and Spring were used for data processing and spatial analysis. AutoCad 2000 was used to plot the pairs of coordinates obtained from GPS. Between 1998 and 2002 5009 snails, of which 12.2% were positive for S. mansoni, were collected in Forte Beach. A total of 27 foci and areas of environmental risk were identified and spatially analyzed allowing the identification of the areas exposed to varying degrees of risk.