Plasma levels of innate immune mediators are associated with liver fibrosis in low parasite burden Schistosoma mansoni-infected individuals.

In the murine model, it was demonstrated that pro-inflammatory cytokines and chemokines are essential to the formation and modulation of Schistosoma-induced granulomatous inflammation. However, the relationship of these immune mediators and disease severity is hard to be established in naturally infected individuals. The current study evaluates the association between plasma concentrations of MIF, sTNF-R1, CCL3, CCL7 and CCL24 and schistosomiasis morbidity in Schistosoma mansoni-infected patients with a low parasite burden. For this propose, 97 S. mansoni-infected individuals were subjected to abdominal ultrasound analysis and clinical examination. Among them, 88 had plasma concentration of immune mediators estimated by ELISA assay. Multivariate linear regression models were used to evaluate the relationship between the plasma concentration of immune mediators and the variables investigated. Although most individuals presented low parasite burden, over 30% of them showed signs of fibrosis defined by ultrasound measurements and 2 patients had a severe form of schistosomiasis. No association between parasite burden and the plasma levels of chemokine/cytokines or disease severity was observed. There was a positive association between plasma concentration of CCL4, sTNF-R1, CCL3 and MIF with gall bladder thickness and/or with portal vein thickness that are liver fibrosis markers. In contrast, no association was found between CCL7 plasma concentrations with any of the schistosomiasis morbidity parameters evaluated. The data showed that CCL24, sTNFR1, MIF and CCL3 can be detected in plasma of S. mansoni-infected individuals and their concentration would be used as prognostic makers of Schistosoma-induced liver fibrosis, even in individuals with low parasite burden.

Ageing down-modulates liver inflammatory immune responses to schistosome infection in mice.

Ageing is associated with several alterations in the immune system. Our aim in this study was to compare the development of immunity to Schistosoma mansoni infection in young versus aged C57Bl/6 mice using the liver as the main organ to evaluate pathological alterations and immune responses. In the acute phase, young mice had large liver granulomas with fibrosis and inflammatory cells. Chronic phase in young animals was associated with immunomodulation of granulomas that became reduced in size and cellular infiltrate. On the other hand, aged animals presented granulomas of smaller sizes already in the acute phase. Chronic infection in these mice was followed by no alteration in any of the inflammatory parameters in the liver. In concert with this finding, there was an increase in activated CD4+ T, CD19+ B and NK liver cells in young mice after infection whereas old mice had already higher frequencies of activated B, NK and CD4+ T liver cells and infection does not change these frequencies. After infection, liver production of inflammatory and regulatory cytokines such as IFN-gamma, IL-4 and IL-10 increased in young but not in old mice that had high levels of IL-4 and IL-10 regardless of their infection status. Our data suggest that the unspecific activation status of the immune system in aged mice impairs inflammatory as well as regulatory immune responses to S. mansoni infection in the liver, where major pathological alterations and immunity are at stage. This poor immune reactivity may have a beneficial impact on disease development.

Regulation of immune responses to Strongyloides venezuelensis challenge after primary infection with different larvae doses.

Nematode infections are generally followed by high rates of reinfection, leading to elevated prevalence in endemic areas. Therefore, the effective control of nematode infections depends on understanding the induction and regulation of protective mechanisms. However, most experimental models for protective immune response against nematodes use high parasite exposure, not always reflecting what occurs naturally in human populations. In this study, we tested whether infecting mice with different Strongyloides venezuelensis larvae loads would affect protective responses against reinfection. Interestingly, we found that a previous infection with 10-500 larvae conferred high rate of protection against reinfection with S. venezuelensis in mice, by destroying large numbers of migrating larvae. However, low-dose priming did not abolish adult worm maturation, as detected in high-dose primed group. Results also indicated that a previous low-dose infection delayed the development of cellular infiltrate, while a high inoculum rapidly induced these inflammatory features. Cytokine production by splenocyte cultures of challenge infected mice demonstrated that low-dose priming had increased production of IL-4 and IFN-gamma, while high-dose induced IL-4 production but not IFN-gamma. Our data support the hypothesis that low-dose nematode infection does not induce a polarized type-2 immune response, allowing adult worm survival.

Flow cytometry analysis of the circulating haemocytes from Biomphalaria glabrata and Biomphalaria tenagophila following Schistosoma mansoni infection.

Aiming to further characterize the haemocyte subsets in Biomphalaria snails, we have performed a detailed flow cytometric analysis of whole haemolymph cellular components using a multiparametric dual colour labelling procedure. Ethidium bromide/acridine orange fluorescence features were used to first select viable haemocytes followed by flow cytometric morphometric analysis based on the laser scatter properties (forward scatter-FSC and side scatter-SSC). Our findings demonstrated that B. glabrata (BG-BH, highly susceptible to S. mansoni) and 2 strains of B. tenagophila (BT-CF, moderately susceptible and BT-Taim, resistant to S. mansoni) have 3 major circulating haemocyte subsets, referred to as small, medium and large haemocytes. The frequency of small haemocytes was higher in BG-BH, while medium haemocytes were the most abundant cell-type in both B. tenagophila strains. Schistosoma mansoni infection resulted in early reduction of large and medium circulating haemocytes followed by an increase of small haemocytes. Although parasite infection induced haemocyte alterations in all Biomphalaria strains, the response was particularly intense in BT-Taim, the parasite-resistant snail. Interestingly, the trematode infection induces changes in haemocytes with less granular rather than in those with more granular profile. The results indicated that, in B. tenagophila of Taim strain, circulating haemocytes, especially the medium and high subset with less granular profile, are very reactive cells upon S. mansoni infection, suggesting that this cell subset would participate in the early parasite destruction observed in this snail strain.

Increased susceptibility to Strongyloides venezuelensis in mice due to Mycobacterium bovis co-infection which modulates production of Th2 cytokines.

An estimated quarter of the world's population possesses an infection caused by gastrointestinal nematodes, which induce a Th2 type immune response. Concomitant infection of nematodes with Mycobacterium tuberculosis, which induces a predominantly Th1 type response, is very frequent in tropical and subtropical regions. This study examined immune responses of BALB/c mice infected with Strongyloides venezuelensis and then co-infected with Mycobacterium bovis. The number of worms in the intestine, eggs in feces, cytokine production in lungs and intestine and the expression of CD80, CD86, CTLA-4 and CD28 cell markers on pulmonary cells were analysed. Our results indicate that co-infected mice had an increased parasite burden, which correlates with elevated IFN-gamma and IL-10 cytokine production and decreased IL-4 and IL-13. Moreover, decreased expression of CD80 and increased expression of CTLA-4 were observed in co-infected mice. Our data point out that susceptibility to Strongyloides venezuelensis infection is increased by Mycobacterium bovis co-infection, resulting in higher parasite survival.

Participation of cell-free haemolymph of Biomphalaria tenagophila in the defence mechanism against Schistosoma mansoni sporocysts.

Biomphalaria tenagophila of Taim strain is able to completely destroy Schistosoma mansoni sporocyst few hours after parasite penetration, although the mechanism is still not well known. In this experimental work we show that passive transference of cell-free haemolymph, especially from B. tenagophila Taim, resulted in higher resistance of B. tenagophila Cabo Frio to S. mansoni infection. This effect was demonstrated in vivo, by the reduction in the infection rate, and the significantly lower production of sporocysts and cercariae of the parasite in snails treated with Taim cell-free haemolymph compared to CBSS-inoculated snails. The protective effect of Taim cell-free haemolymph was also observed during the in vitro interaction between haemocytes and sporocysts. In this system, addition of B. tenagophila cell-free haemolymph, especially from Taim strain, was responsible for significant increase in sporocyst mortality compared to B. glabrata cell-free haemolymph or culture medium. Moreover, the combination of Taim cell-free haemolymph and Cabo Frio haemocytes increased significantly the mortality of sporocysts. The results show that Taim cell-free haemolymph would act direct and indirectly on destruction of S. mansoni sporocysts. The results also suggest that cell-free haemolymph indirectly increases parasite recognition by the circulating granulocytes and it is species specific.

Evaluation of the immune response against Strongyloides venezuelensis in antigen-immunized or previously infected mice.

The present study was carried out to investigate the immune response against Strongyloides venezuelensis infection in Balb/c mice previously immunized with larva-antigens or primed with live-larvae. Our results indicate that all primed mice developed a strong protection against challenge infection that remained active for 45 days. In mice primed with live-larvae the challenge infection resulted in great reduction of migrating larvae and the worms were completely eliminated from the small intestine before maturation. The protection pattern did not alter when the primary infection was aborted by drug treatment. In these experimental groups, the challenge infection was accompanied by a type-2 predominant immune response, intense IgE and reactive IgG1 production, and granulocyte infiltration in skin, lungs and intestine. The challenge infection in antigen-immunized mice also resulted in great reduction of migrating larvae. However, the worms that reached the host intestine matured, produced eggs and were eliminated similarly to the ones from nonimmunized mice. Protective mechanisms after immunization with larva antigen were migrating larva-specific and associated with a strong and mixed Th1 and Th2 response, without tissue granulocyte infiltration. In conclusion, protective immunity induced by a previous infection or antigen-immunization are stage-specific and operate through different effector mechanisms.

Cytological and parasitological analysis of bronchoalveolar lavage fluid for the diagnosis of Angiostrongylus vasorum infection in dogs.

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of many pulmonary diseases. The aims of this work were to perform this procedure in dogs in the acute and chronic phases of an Angiostrongylus vasorum infection for cytological analysis and to evaluate the potential of this technique as a diagnostic method for this lung-heart worm. The BAL procedure was performed through the use of an endotracheal tube on seven A. vasorum infected dogs and on five non-infected dogs lined as a control group. Sixty days post-infection (dpi) active and live larvae were retrieved from the bronchoalveolar fluid (BALF) of all infected dogs. Furthermore, in one animal it was possible to retrieve larvae in its BALF before the pre-patent period. This work reports that the A. vasorum infection resulted in an increase of relative neutrophils and eosinophils counts. In contrast, there was a significant decrease in the alveolar macrophage relative count in infected animals from 60 to 330 dpi. This study shows that the BAL is an accurate technique for the diagnosis of canine angiostrongylosis. Moreover, the technique allows us to retrieve cells and other elements that line the lung surface for cytological evaluation, which provides information about inflammatory diseases, and the diagnosis and prognosis of pulmonary parasites such as A. vasorum.

Effect of Angiostrongylus vasorum infection on Biomphalaria tenagophila susceptibility to Schistosoma mansoni.

Concomitant infection with different parasites may be a helpful laboratorial strategy leading to the better understanding of the mechanisms used by the internal defense system (IDS) of Gastropoda to deal with helminth infection, such as Schistosoma mansoni. This work reports the effect of co-infection of Angiostrongylus vasorum and S. mansoni in hemocyte activity and in the outcome of infection. The simultaneous infection resulted in an increase of snail susceptibility to S. mansoni. In contrast, snails infected with both parasites, 15 days apart, did not show differences in the susceptibility compared to a single parasite infection. The increased susceptibility was measured by the significantly higher number of migrating sporocysts, higher percentage of snails shedding cercariae, higher number of cercariae shed and higher mortality in the experimental group that were simultaneously infected with A. vasorum and S. mansoni, when compared to snails infected only with S. mansoni. Snails simultaneously infected with A. vasorum and S. mansoni showed lower hemocyte activation during the first few days of infection, compared to activation induced only by A. vasorum infection. Between 5 and 15 days post-infection (dpi), granulocyte number and nitric oxide (NO) contents of simultaneously infected snails were lower than the S. mansoni-infected snails. Based on the results, we suggest that differences in the level of hemocyte response could explain the increased S. mansoni susceptibility observed in snails simultaneously infected with both parasites. However, when S. mansoni infection occurred after A. vasorum larvae are completely encapsulated, the response against S. mansoni was not altered, and therefore there were no differences in the susceptibility level.

Mechanisms underlying eosinophil trafficking and their relevance in vivo.

After their formation in the bone marrow, eosinophils circulate with a short half-life and are distributed throughout the body, especially in mucosal and sub-mucosal regions. Although a small amount of these cells are normally seen in healthy tissue, blood and tissue eosinophilia is a hallmark of helminthic and allergic diseases. The role of eosinophils in the normal physiology of mucosal tissues is not understood, but there is good evidence to demonstrate that these cells protect the host at least against some intestinal helminths, specially those with a lung cycle. In addition, there are now many data that support a role for eosinophils in the pathophysiology of allergic diseases, such as asthma. Because helminthic diseases have been largely controlled in developed countries, there has been much interest in the development of drugs which affect eosinophil migration and/or activation in the tissue and which may, thus, be useful in the treatment of allergic conditions. The understanding of the mechanisms controlling eosinophil trafficking and/or activation are essential in the development of anti-eosinophil-based therapeutic strategies. The present paper reviews aspects of eosinophil biology with emphasis on the role of eosinophils in parasitic infections and allergy, the basic mechanisms underlying the trafficking of eosinophils into tissue and how these can be modulated pharmacologically.

Importance of immunoglobulin E (IgE) in the protective mechanism against gastrointestinal nematode infection: looking at the intestinal mucosae.

This review discusses experimental evidences that indicate the IgE participation on the effector mechanisms that leads to gastrointestinal nematode elimination. Data discussed here showed that, for most experimental models, the immune response involved in nematode elimination is regulated by Th-2 type cytokines (especially IL-4). However, the mechanism(s) that result in worm elimination is not clear and might be distinct in different nematode species. Parasite specific IgE production, especially the IgE produced by the intestinal mucosae or associated lymphoid organs could participate in the intestinal elimination of Trichinella spiralis from infected rats. Intestinal IgE may also be important to the protective mechanism developed against other gastrointestinal nematodes that penetrate the murine duodenum mucosa tissue, such as Strongyloides venezuelensis and Heligmosomoides polygyrus. At least in Trichinella spiralis infected rats, the results indicated that intestinal IgE might work independently from mast cell degranulation for worm elimination.

Interaction of Schistosoma mansoni Sporocysts and Hemocytes of Biomphalaria.

Human infection by Schistosoma mansoni affects more than 100 million people worldwide, most often in populations of developing countries of Africa, Asia, and Latin America. The transmission of S. mansoni in human populations depends on the presence of some species of Biomphalaria that act as an intermediate host. The compatibility between S. mansoni and its intermediate host is influenced by behavioral, physiological, and genetical factors of the mollusc and the parasite. The susceptibility level of the mollusc has been attributed to the capacity of internal defense system (IDS)-hemocytes and soluble components of the hemolymph-to recognize and destroy the parasite, and this will be the center of interest of this paper. The schistosome-resistant Biomphalaria can be an alternative strategy for the control of schistosomiasis.

Development and pathology of Fasciola hepatica in CCL3-deficient mice.

Fasciola hepatica is a parasitic helminth that predominantly infects the liver and bile ducts of cattle and causes great losses of cattle production in the southern and southeastern regions of Brazil. The generation of liver lesions and the consequent inflammatory responses are intimately related to the migration of this parasite. The CC-group of chemokines plays a crucial role in the attraction of several cell types and in the recruitment of additional macrophages to an inflammatory focus in numerous diseases. In order to evaluate the role of CCL3 in the development of F. hepatica, we compared parasitological and pathological parameters in C57Bl/6J mice that were assigned to one of two experimental groups: the first group contained CCL3-producing mice (CCL3(+/+) mice) and the other group contained mice that were genetically deficient in CCL3 production (CCL3(-/-) mice). The mortality rate in the CCL3 non-deficient group was higher than of the deficient animals. In most animals from both experimental groups, the necropsied animals contained hemorrhages in their abdominal cavities. In the genetically modified animals, the lesioned liver areas were less extensive and presented focal and sub-capsular lesions. This work demonstrates that the development of F. hepatica is not affected by the absence of CCL3.

Differential lectin labelling of circulating hemocytes from Biomphalaria glabrata and Biomphalaria tenagophila resistant or susceptible to Schistosoma mansoni infection.

Lectins/carbohydrate binding can be involved in the Schistosoma mansoni recognition and activation of the Biomphalaria hemocytes. Therefore, expression of lectin ligands on Biomphalaria hemocytes would be associated with snail resistance against S. mansoni infection. To test this hypothesis, circulating hemocytes were isolated from B. glabrata BH (snail strain highy susceptible to S. mansoni), B. tenagophila Cabo Frio (moderate susceptibility), and B. tenagophila Taim (completely resistant strains), labelled with FITC conjugated lectins (ConA, PNA, SBA, and WGA) and analyzed under fluorescence microscopy. The results demonstrated that although lectin-labelled hemocytes were detected in hemolymph of all snail species tested, circulating hemocytes from both strains of B. tenagophila showed a larger number of lectin-labelled cells than B. glabrata. Moreover, most of circulating hemocytes of B. tenagophila were intensively labelled by lectins PNA-FITC and WGA-FITC, while in B. glabrata small hemocytes were labeled mainly by ConA. Upon S. mansoni infection, lectin-labelled hemocytes almost disappeared from the hemolymph of Taim and accumulated in B. glabrata BH. The role of lectins/carbohydrate binding in resistance of B. tengophila infection to S. mansoni is still not fully understood, but the data suggest that there may be a correlation to its presence with susceptibility or resistance to the parasite.

Local intestinal immune responses to infections with Trichinella spiralis. Real-time, continuous assay of cytokines in the intestinal (afferent) and efferent thoracic duct lymph of rats.

Levels of IL-4, IL-5, TNF-alpha, and IFN-gamma were quantitated in the intestinal (afferent) and efferent thoracic duct lymph of rats during the course (0 to 289 h) of an infection with Trichinella spiralis. Intestinal lymph was collected by cannulating thoracic ducts of mesenteric lymphadenectomized animals. These studies showed that cytokines typical of a Th2 type (IL-4 and IL-5) and a Th1 type (IFN-gamma) were simultaneously detected in the intestinal lymph during the first 8 days after infection. Worm expulsion (day 11 to 12) was associated with increased levels of IL-4 and IL-5 in the intestinal lymph. IL-5 levels rose as early as 15 to 20 h and remained elevated throughout the infection. IL-4 activity appeared in intestinal lymph 60 h after infection and reached peak levels during worm expulsion. Despite the predominantly Th2 nature of cytokine response, IFN-gamma levels showed several cycles of high and low production during the course of infection. A comparison of cytokine levels between intestinal and efferent lymph values showed no significant differences in IL-4 or IL-5 levels suggesting no contribution by the mesenteric node to efferent lymph. However, IFN-gamma and TNF-alpha levels were lower in efferent lymph compared with intestinal lymph suggesting mesenteric node consumption. Adoptive transfer experiments showed that protective CD4+CD45RC- cells primed the gut for a more rapid TH2-type response that was faster than in a primary infection. In contrast, adoptive transfer of CD4+CD45RC+ cells primed the gut for a more rapid Th1-type(IFN-gamma) response. These studies demonstrate a novel method for measuring real-time changes in cytokine levels in the gut during the course of an active infection.

Variability of the intestinal immunoglobulin E response of rats to infection with Trichinella spiralis, Heligmosomoides polygyrus or Nippostrongylus brasiliensis.

Total intestinal IgE level increased in rats infected with Trichinella spiralis or Heligmosomoides polygyrus (peak levels of 2.6 microg and 3.7 microg, respectively), but not in rats infected with Nippostrongylus brasiliensis. Intestinal implantation of young adult N. brasiliensis did not stimulate an intestinal immunoglobulin (Ig)E response, suggesting that mucosal penetration may be required for local intestinal IgE responses in rats. During a T. spiralis infection, total IgE levels in the intestinal lumen were consistently higher in LEWIS and LOU rats (rat strains that eliminate T. spiralis worms earlier in the infection) than in PVG, AO and WKA/H strain rats. There was no correlation in either the total level of serum IgE and IgA, or of intestinal IgA with differences between strains in the rate of worm elimination from the gut. Furthermore, the intestinal IgE immunoprecipitated from LEWIS rats 12 days after infection reacted with T. spiralis adult worm metabolic antigens, while intestinal IgE from PVG rats only became reactive with adult worm metabolic antigens from 14 days after infection. These data emphasize the significance of the intestinal IgE response and its unique features by comparison with serum IgE and IgA or intestinal IgA.

Intestinal transport and catabolism of IgE: a major blood-independent pathway of IgE dissemination during a Trichinella spiralis infection of rats.

Previous work has shown that Trichinella spiralis-infected rats transport IgE from plasma to intestinal tissue and fluids. In this study we quantitate IgE transport to the gut and circulation during T. spiralis infection in rats. Total IgE levels in intestinal fluid from infected rats were elevated by 4 days post-infection (dpi), but were not elevated in serum and lymph until 7 dpi. IgE levels in intestinal fluid ranged from 1 to 6 microg between 10 and 21 dpi, and serum and lymph IgE levels ranged from 100 to 200 ng/ml. Immunoprecipitation of intestinal fluid and enterocyte lysate at 11 dpi showed a protein of 190 kDa that was recognized by mouse anti-rat IgE-MARE-1 in Western blots. This protein was removed from intestinal wash samples with anti-IgE (A2)-Sepharose. The half-life of intact IgE in the intestinal lumen of rats 10 days after infection was 3.25 min. In serum, the half-life of IgE was 5 h. Analysis of IgE production and consumption in 10-day T. spiralis infected rats showed that about 4.67 microg IgE/day entered the serum, while 2570.00 microg IgE/day entered the intestinal lumen. The IgE present in serum 10 days after T. spiralis infection originated in the gut and/or associated lymphoid tissue and was transported to the circulation via thoracic duct lymph. However, most IgE produced in the intestine was transported to the gut lumen at a rate that exceeded transport to plasma by a factor of several-hundredfold.

Rapid expulsion of Trichinella spiralis in adult rats mediated by monoclonal antibodies of distinct IgG isotypes.

The role of IgG in rapid expulsion of Trichinella spiralis in adult rats was analysed. In this experimental model, rats were first infected with an unrelated nematode Heligmosomoides polygyrus, then 5-14 days later, immune serum, its fractions, or IgG monoclonal antibody (mAb) was transferred. Rats were challenged with T. spiralis muscle larvae 24 hr after antibody transfer and intestinal worms counted at various times, up to 24 hr, after challenge. Provided rats were exposed to H. polygyrus first, immune serum, affinity chromatography-isolated immune IgE, IgE-depleted immune serum, or monoclonal antibodies of IgG1, IgG2a and IgG2c isotypes were all able to transfer rapid expulsion. Protection varied from 40 to greater than 90% larval T. spiralis rejection and was dose dependent, requiring, for IgG1, a minimum of 5 mg of transferred protein. Antibody specificity was predominantly against the dominant larval secreted/cuticular antigen TSL-1 for IgE and was exclusively so for the mAb. A comparison of quantitative differences in effective amounts of transferred antibody as well as the distinct priming requirements suggest that IgE functions through an intestinal mechanism that is different from that for IgG1 and IgG2c. Whether or not IgG2a functions homocytotropically, or as the other IgG has not been resolved. Since neither the T-helper (Th) cell transfer or the H. polygyrus form of intestinal priming confers protection by itself, these data suggest that rapid expulsion is predominantly an antibody-mediated process albeit with a required intestinal element. The results support earlier data in showing that two steps are required for rapid expulsion to be expressed and this is so for both IgE- and IgG-mediated mechanisms. Finally, the results show that IgG of various isotypes and IgE have a functional role in the expression of intestinal immunity.

Brugia pahangi: quantitative analysis of infection in several inbred rat strains.

We report a comprehensive study of the infectivity of Brugia pahangi in male and female rats of eight different inbred strains. A single infection of any inbred rat strain will produce rats that become microfilaremic, have occult infection, or clear the primary infection. The proportion belonging to any category is determined by the basic susceptibility level of that strain. Patency rates (blood microfilaria+) ranged from 24% (AO rats) to 73% (WKA rats). The period for which microfilaria were in the circulation was directly related to microfilarial burden, with rats carrying less than 50 mf/ml of blood patent for 11.8 weeks +/- 12.2; for 50-499 mf/ml it was 37.6 +/- 14.8 and for 500+ mf/ml it was 63.3 +/- 34.2 weeks. Suckling rats were resistant to infection (0 patent) and weanlings were intermediate in resistance between suckling and adult rats. Female rats were highly resistant to infection. Approximately half of amicrofilaremic rats have occult infections. A high proportion of patent infections involve the testes or testicular lymphatics. In the most susceptible rat strains, more than 95% of the administered L3 or developing L4 parasites were killed within 28 days. During the course of the first 6 months, the ratio of males to females fell significantly, suggesting a shorter life span in male worms. The features of the infectivity/patency patterns in rats are compared with recognized patterns obtaining in human populations. We conclude that rats provide a valuable and underutilized model for the experimental analysis of filarial infections.

Anesthesia of Biomphalaria spp. (Mollusca, Gastropoda): sodium pentobarbital is the drug of choice.

The anesthetic effect of some water-soluble anesthesic or narcotic drugs currently used in mice was tested in molluscs of the Biomphalaria genus. Sodium thiopental was very toxic to the snails resulting in high rates of mortality in all the treatment schedules tested. Cetamine base, at concentration of 0.25 mg/ml of water, resulted in partial snail anesthesia (40% of snails were anesthetized) only after 20 h of exposition. The association of Cetamine base with Tiazine chloridrate did not improve the anesthesic effect, and higher concentrations of these drugs were toxic to the snails. Sodium pentobarbital at 0.4 mg/ml in water for 8 h was the best treatment schedule to anesthetize Biomphalaria snails. In this schedule, the snails were anesthetized without any toxic effect. The procedure provides a powerful tool for in vivo studies that demande a complete state of snail anesthesia.