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The trematode Echinostoma paraensei is an intestinal parasite transmitted by ingestion of the infectious stage of metacercariae. For scientific purposes, its life cycle has been maintained in the laboratory, allowing analysis using various biological approaches. Different parasite isolates have revealed atypical patterns of migration and establishment in ectopic sites in Swiss-Webster mice. During the investigation of the biological life cycle of an E. paraensei isolate from the silvatic rodent Nectomys squamipes collected in the municipality of Rio Bonito (State of Rio de Janeiro), a bacterial coinfection with Pseudomonas aeruginosa was observed, which produced anatomopathological alterations, mainly in the liver, bile ducts, pancreas, and small intestine. The main macroscopic signs were the whitish suppurative pyogenic punctual lesions. The histological sections stained by hematoxylin-eosin showed an intense inflammatory reaction formed by mononuclear cells and macrophages surrounding the bile ducts, although the hepatic parenchyma still presented its normal aspect. Thus, pyogenic abscesses can be associated with E. paraensei infection depending on the strain and aggravating pathogenesis in the definitive host.KEY WORDS: Liver abscess; Swiss mice; trematode infection.

Angiostrongylus cantonensis is a metastrongylid nematode that has a heteroxenous cycle, where snails act as intermediate hosts and the rodents Rattus rattus and Rattus novergicus are the definitive hosts. However, humans may act as accidental hosts presenting an atypical form of parasitism. This fact has motivated research to better understand systems of relationships involving A. cantonensis, targeting the control of species of gastropods that act as intermediary hosts. For this, six groups were formed: three control groups (uninfected) and three infected groups, exposed to approximately 1200 L1 larvae of A. cantonensis. At the end of each week (1, 2, and 3 weeks), snails were dissected without anesthesia and the gonad–digestive gland (DGG) complex was separated for determination of oxygen consumption through high-resolution titration-injection respirometer (Oroboros, Oxygraph; Innsbruck, Austria). The results indicate suppression of mitochondrial oxidative metabolism of the host and compromised in different mitochondrial respiratory states. This effect, mainly observed in the group exposed to 1 week of infection, showed a decrease of approximately 38% (2.78 ± 0.37 pmol O2/mg of tissue; P

The immune system of snails is highly sensitive to pollutants, which can suppress its immune response. We investigated the effects of exposure to the glyphosate-based herbicide Roundup® Original on the snail Biomphalaria glabrata infected by the platyhelminth Echinostoma paraensei by evaluating changes in the snail's internal defense system. Four cohorts were studied: control group, infected snails, snails treated with Roundup®, and snails infected and treated with Roundup®. The hemocyte viability was assessed, morphological differentiation of cells was observed and flow cytometry was performed to determine the morphology, viability and the lectin expression profiles. The frequencies of dead hemocytes were lower in the infected group and higher in both pesticide treated groups. Three cell types were identified: blast-like cells, hyalinocytes and granulocytes. The highest number of all types of hemocytes, as well as the highest number of dead cells, were observed in the infected, pesticide-treated group. The association between infection and herbicide exposure greatly increased the frequency of dead hemocytes, suggesting that this condition impairs the internal defense system of B. glabrata making the snails more vulnerable to parasitic infections.

The terrestrial gastropod Bulimulus tenuissimus is widespread in South America. It is an intermediate host of many parasites, but there are no records of infection of this snail by Angiostrongylus cantonensis, despite the occurrence of this parasite and angiostrongyliasis cases in the same areas in which B. tenuissimus occurs. For this reason, it is important investigate the susceptibility of B. tenuissimus to A. cantonensis-infection, since it can be used as intermediate host of A. cantonensis, increasing the list of terrestrial gastropods that infect wild and domestic animals and humans with this parasite. The purpose of this study was to evaluate the susceptibility of B. tenuissimus to experimental infection with L1 larvae of A. cantonensis. The snails were exposed to 1200 L1 larvae and it was possible observe many developing larvae in the cephalopedal mass and mantle tissues, with intense hemocyte infiltration and collagen deposition, but no typical granuloma structures were formed. The glucose content and lactate dehydrogenase activity in the hemolymph varied, indicating an increase of anaerobic energy metabolism in the middle of infection, but with a tendency to return to normal values at the end of pre-patent period. This was corroborated by the marked reduction in the glycogen content in the cephalopedal mass and digestive gland in the first and second week after exposure, followed by a slight increase in the third week. The content of pyruvic acid in the hemolymph was 14.84% lower at the end of pre-patent period, and oxalic acid content was 41.14% higher. These results indicate an aerobic to anaerobic transition process. The PAS reaction showed a large amount of glycogen inside the developing larvae and muscular tissues of the cephalopedal mass, indicating that despite the high consumption of this polysaccharide by the parasite, the snail is able to maintain its energy metabolism based on carbohydrates. The results reveal that B. tenuissimus is a robust host, which can live with the developing larvae of A. cantonensis and overcome the metabolic damages resulting from parasitism.

Eosinophilic meningoencephalitis is an endemic zoonosis in Southeast Asia and the Pacific Islands, but in recent years, new cases have been reported in various countries outside these regions, including Brazil, where it is considered an emerging disease. In this study, the effect of infection by the nematode Angiostrongylus cantonensis, one of the main etiologic agent of this disease, on the reproductive biology of the planorbid snails Biomphalaria straminea and B. tenagophila was investigated during the pre-patent period. Alterations in the reproductive biology of B. straminea and B. tenagophila were analyzed in laboratory-reared specimens infected by A. cantonensis during 21 days; the number of eggs, number of egg masses, number of eggs/mass, number of eggs/snail, viable eggs/snail, survival and galactogen content in the albumen gland were measured. The results indicated the occurrence of initial compensation in reproductive effort in both snail species, but at different moments in the pre-patent period. More specifically, a reduction of 46.53% in the eggs/egg mass ratio in infected B. straminea was observed, a reflection of a 50% decline in the concentration of galactogen contained in the albumen gland. Changes in this parameter were also noted in B. tenagophila, but only at the end of the study period, with a reduction of 15.49%. Histological analyses indicate that changes observed can be explained by the tissue damages caused by the migration and development of the larvae. These results shed more light on the host-parasite relationship and indicate the importance of studying reproductive aspects for efforts to control infected snails. Considering that terrestrial snails can also transmit eosinophilic meningitis (in addition to aquatic mollusks), the data obtained expand knowledge of this host-parasite relationship and provide support for programs to control this zoonosis.

The impact of the Sao Francisco River Transposition on the helminth community of small mammals is unknown so far, particularly the existence of helminths of zoonotic origin. Therefore, the aim of the present study was to report the helminth fauna of small mammals in areas along the aqueduct of the Sao Francisco River in the Caatinga biome. Twenty-one small mammals were collected among seven species: Thrichomys laurentius (10), Necromys lasiurus (5), Cerradomys langguthi (1), Galea spixii (1), Rattus rattus (1), Oligoryzomys stramineus (1) and Didelphis albiventris (2). Five helminth species were identified: Heligmostrongylus tchekaprovae, Heligmostrongylus interrogans, Stilestrongylus freitasi, Syphacia alata and Viannia viannia. Among these, four species had their geographical distribution map expanded with this study. There was no evidence of zoonotic helminths in any of the animals examined. Thus, the present study contributes to increase the knowledge of the helminth fauna in small mammals in the Caatinga biome.

The exposure of wildlife and humans to toxic residues of Roundup(®) through agricultural practices or the food chain has been reported since the herbicide was found contaminating rivers. Glyphosate, N-(phosphonomethyl)glycine acid, is a nonselective post-emergent herbicide and is formulated as an isopropylamine salt with the surfactant taloamine polyethoxylate (POEA) representing the commercial formulation of Roundup(®). There is little knowledge about the effects of the herbicide on helminth parasites, particularly those whose life cycle is related to water bodies. Here we investigated the effects of the Roundup(®) on the food-borne trematode Echinostoma paraensei in experimental conditions using different developmental stages (eggs, miracidia, cercariae, metacercariae, newly excysted larvae (NEL), helminths at seven days and helminths at fourteen days). Three different herbicide concentrations were tested based on concentrations typically applied in the field: 225, 450 and 900 mg/L. Specimens were analyzed in vitro for hatching miracidia, mortality and excystment rate of metacercariae and in vivo for parasitic load and egg production. There was a significant difference in the hatching miracidia rate only for the newly embryonated eggs. The mortality of specimens and excystment rate of metacercariae were concentration-dependent. There was a significant difference in the miracidia mortality with respect to concentration until 56.3 mg/L. The same effect was observed for cercariae, and mortality was observed from 15 min onwards at concentrations of 225-900 mg/L. At low concentrations, mortality was detected after 30 min. The effects of the herbicide concentration on NEL and on helminths at seven and fourteen days showed a significant difference after 24 h. There was no significant difference in parasitic load and egg production after infection of rodents with exposed metacercariae. All developmental stages of the trematode E. paraensei were affected by Roundup(®) exposure under experimental conditions. These results suggest that dynamics of transmission of the trematode could be affected in the natural environments. The study also reinforces the usefulness of this trematode as a good model organism to test pesticides regarding human and environmental health.

The effect of infection by Echinostoma paraensei on the mitochondrial physiology of Biomphalaria glabrata was investigated after exposure to 50 miracidia. The snails were dissected one, two, three and four weeks after infection for collection and mechanical permeabilization of the gonad-digestive gland (DGG) complex. The results obtained indicate that prepatent infection by this echinostomatid fluke significantly suppresses the phosphorylation state (respiratory state 3) and basal oxygen consumption of B. glabrata, demonstrating that the infection reduces the ability of the intermediate host to carry out aerobic oxidative reactions. Additionally, relevant variations related to the uncoupled mitochondrial (state 3u) of B. glabrata infected by E. paraensei were observed. Four weeks after exposure, a significant reduction in mitochondrial oxygen consumption after addition of ADP (3.68±0.26pmol O2/mg proteins) was observed in the infected snails in comparison with the respective control group (5.14±0.25). In the uncoupled state, the infected snails consumed about 62% less oxygen than the infected snails (7.87±0.84pmol O2/mg proteins) in the same period. These results demonstrate a reduction in oxidative decarboxylation rate of the tricarboxylic acid cycle and faster anaerobic degradation of carbohydrates in the infected snails. The possible mechanisms that explain this new metabolic condition in the infected organisms are discussed.

Snails are essential to complete the life cycle of the metastrongylid nematode Angiostrongylus cantonensis, the causative agent of infections in domestic and wild animals, mainly rodents, and also of neural angiostrongyliasis or eosinophilic meningitis in humans. There are many reports of mollusks that can act as intermediate hosts of this parasite, especially freshwater snails and the African giant Achatina fulica. The terrestrial gastropod Bulimulus tenuissimus is widely distributed in Brazil and other species of the same genus occur in Brazil and other countries, overlapping regions in which there are reports of the occurrence of A. cantonensis and angiostrongyliasis. In spite of this, there are no records in the literature of this species performing the role of intermediate host to A. cantonensis. The present study analyzed the experimental infection with first-stage larvae of A. cantonensis, under laboratory conditions, of B. tenuissimus, by using histology and electron microscopy techniques. Three weeks after exposure to L1 larvae, it was possible to recover L3 larvae in small numbers from the infected snails. Developing larvae were observed in the cephalopedal mass (foot), ovotestis, and mantle tissues, being located inside a granulomatous structure composed of hemocyte infiltration, but there was no calcium or collagen deposition in these structures in significant amounts. In the third week post exposure, it was possible observe a sheath around the developing larvae. The infected snails presented reduction in the fibrous muscular tissue in the foot region, loss of the acinar organization in the digestive gland, with increase of amorphous material inside the acini and loss of epithelial pattern of nuclear organization in the acinar cells. However, the ovotestis seemed unaffected by the infection, since there was a large number of developing oocytes and spermatozoa in different stages of formation. The digestion of infected snails allows us the third-stage recovery rate of 17.25%, at 14 days post exposure to the L1. These L3 recovered from B. tenuissimus were used to infect rats experimentally, and 43 days post infection first-stage (L1) larvae of A. cantonensis were recovered from fresh feces. The results presented constituted the first report of the role of B. tenuissimus as an experimental intermediate host to A. cantonensis and shed some light on a possible problem, since the overlapping distribution of B. tenuissimus and A. cantonensis in Brazil and other countries where different species of Bulimulus occur enables the establishment and maintenance of the life cycle of this parasite in nature, with wild rodents as reservoirs, acting as a source of infection to humans, causing neural angiostrongyliasis. Resumo Os moluscos são um requisito essencial para a conclusão do ciclo de vida pelo nematoide metastrogilídeo Angiostrongylus cantonensis, o agente causador de infecções em animais domésticos e selvagens, principalmente roedores, e também de angiostrongilíase neural ou meningite eosinofílica em humanos. Há muitos relatos de moluscos que podem atuar como hospedeiro para este parasito, sendo o foco dado aos moluscos de água doce e no gigante africano Achatina fulica. O gastrópode terrestre Bulimulus tenuissimus é amplamente distribuído no território brasileiro e há outras espécies do mesmo gênero que ocorrem no Brasil e outros países, sobrepondo-se às regiões em que há relatos à ocorrência de A. cantonensis e angiostornigilíase. Apesar disso, não há registro na literatura, acerca desta espécie como hospedeiro intermediário para A. cantonensis. O presente estudo teve como objetivo verificar a possibilidade de infectar experimentalmente, utilizando larvas L1 de A. cantonensis, em condições laboratoriais, o molusco B. tenuissimus, utilizando técnicas de histologia e microscopia eletrônica. Três semanas após a exposição às larvas L1, foi possível recuperar larvas L3 dos moluscos infectados, em pequena quantidade. As larvas em desenvolvimento foram observadas na massa cefalopediosa (pé), ovotestis e nos tecidos do manto, sendo localizadas dentro de uma estrutura granulomatosa constituída por infiltração hemocitária, mas não houve deposição de cálcio ou colágeno nessas estruturas em quantidade significativa. Na terceira semana pós exposição, foi possível observar uma bainha ao redor das larvas em desenvolvimento. Os caracóis infectados apresentaram redução no tecido muscular fibroso na região do pé, perda da organização acinar na glândula digestiva, com aumento de material amorfo dentro dos ácinos e perda do padrão epitelial da organização nuclear nas células acinares. No entanto, o ovotestis, pareceu não ser afetado pela infecção, uma vez que houve um grande número de oócitos em desenvolvimento e espermatozóides em diferentes estágios de formação. A digestão dos moluscos infectados nos permitiu a recuperação de larvas de terceiro estágio (17,25%), aos 14 dias após a exposição à L1 de A. cantonensis . Estas L3 recuperadas de B. tenuissimus foram utilizados para infectar ratos experimentalmente, e 43 dias após a infecção, as larvas do primeiro estágio (L1) foram recuperadas de fezes frescas. Os resultados apresentados representam o primeiro registro do papel de B. tenuissimus como hospedeiro intermediário experimental de A. cantonensis e trazem alguma luz a um problema, até então silencioso, uma vez que a sobreposição da distribuição de B. tenuissimus e A. cantonensis no Brasil, e outros países, onde as diferentes espécies de Bulimulus ocorrem, torna possível o estabelecimento e manutenção do ciclo de vida deste parasito na natureza, com roedores selvagens como reservatório, agindo como fonte de infecção para humanos e causando a angiostrongilíase neural.

Investigation of co-infection by different helminths in snails can provide essential information about the biology of parasites co-existence under natural conditions. This study aimed to investigate the reproductive and histological changes of Biomphalaria glabrata experimentally co-infected with Echinostoma paraensei and Angiostrongylus cantonensis. Five groups of snails i.e. uninfected, with single and double infections, either E. paraensei first (E + A) or A. cantonensis first (A + E) were followed; three times a week during four weeks the numbers of egg masses, eggs and hatched snails were counted. Histological analysis of the ovotestis and albumen gland was performed after four weeks. The number of egg masses/snail, eggs/snail and hatched snails showed significative differences comparing the control group to all infected groups, especially in group E + A, with the majority of values of parameters analyzed lower than 50% of those observed for control snails. In addition, the mortality was higher in the group E + A than to control group. The histological analysis showed that presence of both parasites in the reproductive organs was associated with tissue damages.

For the first time, alterations in the oxidative metabolism of Achatina fulica experimentally infected with different parasite loads of Angiostrongylus cantonensis were determined. For this, the hemolymph activities of lactate dehydrogenase (LDH) and hexokinase and the glucose concentrations in the hemolymph, as well as the polysaccharide reserves in the digestive gland and cephalopedal mass, were assessed. Additionally, the contents of some carboxylic acids in the hemolymph of infected and uninfected snails were determined by high-performance liquid chromatography (HPLC), permitting a better understanding of the alterations related to the host's oxidative metabolism. As the main results, activation of oxidative pathways, such as the glycolytic pathway, was demonstrated in response to the increase in the activity of hexokinase. This tendency was confirmed by the decrease in the contents of glucose in the hemolymph of parasitized snails, indicating that the infection by A. cantonensis alters the host's metabolism, and that these changes are strongly influenced by the parasite load. This metabolic scenario was accompanied by activation of the anaerobic fermentative metabolism, indicated not only by an increase in the activity of (LDH), but also by a reduction of the content of pyruvic acid and accumulation of lactic acid in the hemolymph of parasitized snails. In this circumstance, maintenance of the host's redox balance occurs through activation of the fermentative pathways, and LDH plays a central role in this process. Together, the results indicate that A. cantonensis infection induces activation of the anaerobic metabolism of A. fulica, characterized not only by the accumulation of lactic acid, but also by a reduction in the pyruvic acid and oxalic acid contents in the hemolymph of the infected snails.

Angiostrongylus cantonensis is considered the main etiological agent of eosinophilic meningitis in humans. At present, this zoonosis is considered an emerging disease mainly in the Americas. The aim of this study was to determine the prevalence of Achatina fulica infected by Angiostrongylus cantonensis in restinga areas along beaches in the west zone of Rio de Janeiro city, Brazil. The study areas included the following beaches: Barra da Tijuca, Recreio dos Bandeirantes, Reserva, Prainha and Grumari. Ninety specimens of Achatina fulica were collected. Positive molluscs were found only in Barra da Tijuca. Infection prevalence was 5.5%. The presence of this parasite in the beachfront areas, in the west zone of Rio de Janeiro city demonstrates the potential risk of infection for visitors and the expansion of this helminth in the State of Rio de Janeiro.

The effect of experimental infection by different parasitic loads of Angiostrongylus cantonensis (Nematode, Metastrongylidae) on the activities of the aminotransferases and concentration of total proteins, uric acid and urea in the hemolymph of Achatina fulica (Mollusca, Gastropoda) were investigated. There was a significant decrease in the concentration of total proteins in the exposed snails to 5000 or more larvae. This change was accompanied by an increase in the concentrations of urea and uric acid in the hemolymph, suggesting a higher rate of deamination of the amino acids. Besides this, variations in the activities of the aminotransferases were also observed, with the highest values recorded in the groups exposed to greater parasite load. These results suggest an increase in the use of total proteins, since there was increased formation of nitrogenous catabolites, in conformity with an increase in the aminotransferase activities. Infection was verified by the fact that L3 larvae recovered from the snails was proportion to the exposure dose of L1 larvae. Histopathological results also indicated presence of an inflammatory cell infiltrate, favoring an increase of both transaminases.

Echinostoma paraensei (Digenea: Echinostomatidae) lives in the duodenum and bile duct of rodents and is reported as a useful model for studies on the biology of flatworms. Here, we compared the growth and development of pre and post ovigerous worms collected 3, 7, 14 and 21 days post infection from experimentally infected hamster (permissive host) and Wistar rat (less permissive hosts). Linear measurements and ratios were examined by light (morphology and morphometry) and confocal laser scanning microscopy. At day 3, either worm from hamsters or rats were small with poorly developed gonads. At seven day, worms increased in size and morphometric differences between hosts are statistically significant after this time. In addition, adult worms (14 and 21 days of age) harvested from hamster showed developed gonads and vitelline glands laterally distributed on the body, whereas worms from rat showed atrophied reproductive system characterized by underdeveloped vitelline glands and stunted ovary. The worm rate recovery in rat decreased from 29.3% (day 7) to 20.6% (day 14) and 8% (day 21), whilst it remained around 37% in hamster. In conclusion, this is the first appointment demonstrating that low permissiveness influences the reproductive system of echinostome since the immature stages of development. The phenotypic analysis evidenced that hamster provides a more favorable microenvironment for gonads development than rat, confirming golden hamster as a permissive host, whereas Wistar rat is less permissive host.

Different isolates of Biomphalaria tenagophila show a large spectrum of compatibility to the trematode Schistosoma mansoni, ranging from entirely refractory to highly susceptible. The aim of this study was to verify the pattern of compatibility of two B. tenagophila geographical isolates, resistant and susceptible to S. mansoni, when infected with Echinostoma paraensei. The snails were exposed to different numbers of miracidia, and mortality, histopathological characteristics and the number of cercariae released were evaluated. A correlation between the number of miracidia and the infectivity rate of B. tenagophila (TAIM) was observed. There was no correlation between the number of miracidia used and the number of cercariae released for both B. tenagophila isolates. Biomphalaria tenagophila (SJC) showed little susceptibility to the E. paraensei infection. The results demonstrate different degrees of compatibility for the two B. tenagophila isolates when infected with E. paraensei, and may contribute to studies about host-parasite relationships. Diferentes linhagens de Biomphalaria tenagophila mostram amplo espectro de compatibilidade para Schistosoma mansoni, que vai desde refratária a altamente suscetível. O objetivo deste estudo foi verificar o padrão de compatibilidade de duas linhagens geográficas de B. tenagophila, resistentes e suscetíveis ao S. mansoni, para a infecção com Echinostoma paraensei. Foram utilizadas diferentes cargas miracidianas onde se avaliou a mortalidade, características histopatológicas e emissão de cercárias. Observou-se correlação entre o número de miracídios e a taxa de infecção em B. tenagophila (TAIM). Não houve correlação entre a carga de miracídios utilizados e a emissão cercariana em ambas as linhagens de B. tenagophila. Biomphalaria tenagophila (SJC) mostrou pouca susceptibilidade à infecção por E. paraensei. Os resultados demonstraram diferentes graus de compatibilidade das duas linhagens de B. tenagophila ao E. paraensei, podendo contribuir para os estudos de relação parasito-hospedeiro.

The interaction between intermediate snail hosts and helminths can cause metabolic changes in the former. The snails use their reserves for maintenance of their vital processes, by activating the internal defense system and repairing tissue damage, while also supplying necessary energy for the parasites' development. Our aims were to evaluate the lactate dehydrogenase activity and the glucose concentration in the hemolymph of Biomphalaria glabrata experimentally coinfected by Angiostrongylus cantonensis and Echinostoma paraensei. Besides these aspects, the glycogen content in the digestive gland complex and cephalopedal mass along with histochemical changes in parasitized snails were analyzed. The snails were divided in group A (infected by 1200 L1 of A. cantonensis), group E (infected by 20 E. paraensei miracidia), group A + E (co-infected with A. cantonensis first and after a week by E. paraensei), group E + A (co-infected with E. paraensei first and then by A. cantonensis) and control group (not infected). During four weeks after exposure, samples were collected for biochemical and histochemical analyses. In the infected snails, glucose levels and glycogen content in the digestive gland complex and cephalopedal mass were significantly lower, in contrast with an increase of lactate dehydrogenase activity. These results indicate that the intense energy demand resulting from the presence of parasites causes the host snail to accelerate the anaerobic degradation of carbohydrates to obtain energy, in an attempt to maintain homeostasis. Both parasites were observed in histochemical analysis to cause tissue damages in the snails. So, although the snails were able to sustain the coinfection, several metabolic and tissue changes occurred, mainly in those infected with E. paraensei and then with A. cantonensis.

Angiostrongylus cantonensis is the etiologic agent of eosinophilic meningoencephalitis in humans. Cases have been recorded in many parts of the world, including Brazil. The aim of this study was to compare the differences in the biology and morphology of two different Brazilian haplotypes of A. cantonensis: ac8 and ac9. A significantly larger number of L1 larvae eliminated in the faeces of rodents at the beginning of the patent period was observed for ac9 haplotype and compared to the total of L1 larvae eliminated, there was a significant difference between the two haplotypes. The ac9 haplotype showed a significant difference in the proportion of female and male specimens (0.6:1), but the same was not observed for ac8 (1.2:1). The morphometric analysis showed that male and female specimens isolated from ac8 haplotype were significantly larger with respect to body length, oesophagus length, spicule length (male) and distance from the anus to the rear end (female) compared to specimens from ac9. The morphological analysis by light microscopy showed little variation in the level of bifurcations at the lateral rays in the right lobe of the copulatory bursa between the two haplotypes. The biological, morphological and morphometric variations observed between the two haplotypes agree with the observed variation at the molecular level using the cytochrome oxidase subunit I marker and reinforce the possible influence of geographical isolation on the development of these haplotypes.

Eosinophilic meningitis caused by Angiostrongylus cantonensis is a zoonosis endemic to Southeast Asia and the Pacific islands. It is considered an emerging disease because it has been expanding both geographically and in terms of the range of hosts. In South America, the first cases were reported in Brazil and were attributed to eating infected snails. In this study, 70 adult females of Rattus norvegicus (Wistar) were used to evaluate hematology, blood gases, cardiac markers and lung histopathology changes caused by this parasite. Of them, 60 were individually infected by orogastric gavage with 100 L(3) larvae and 10 uninfected animals formed the control group. The results obtained demonstrate that infection caused by A. cantonensis in R. norvegicus promotes significant hematological changes induced in the vertebrate host, manifested mainly in the form of regenerative anemia, thrombocytopenia and eosinophilia. Additionally, histopathological changes in the lung parenchyma demonstrated in rodents reveal the occurrence of areas of necrosis and extensive fibrosis, being directly related to the development of cellular hypoxia and enzyme cardiac changes. This study can contribute to a better understanding of the relationship between A. cantonensis and R. norvegicus.

The activity of lactate dehydrogenase and the concentrations of glucose in the hemolymph and of glycogen in the digestive gland and cephalopedal mass of Biomphalaria glabrata experimentally infected with Angiostrongylus cantonensis were evaluated. Additionally, high performance liquid chromatography (HPLC) was used to determine the hemolymph concentrations of some carboxylic acids (oxalic, piruvic, lactic and succinic). After one, two and three weeks of infection, the snails were dissected to collect the hemolymph and separate the tissues. A significant reduction of the levels of glucose in the hemolymph was observed as of the first week of infection in relation to the control group. The lactate dehydrogenase activity of the infected group was significantly higher than the average of the control group. This increase was accompanied by a reduction of the levels of piruvic acid and an increase in the levels of lactic acid in the hemolymph of the parasited snails, confirming the acceleration of the anaerobic metabolism, necessary for the host to obtain energy and maintain its redox balance. In parallel, there was a decrease in the glycogen content of the storage tissues, with that reduction being significantly greater in the cephalopedal mass than the digestive gland, demonstrating that in this interaction system, the mobilization of glycogen was not sufficient to maintain and reestablish the normal glycemia of the infected snails.

Eosinophilic meningitis is a disease characterized by increased eosinophils in the cerebrospinal fluid (CSF), which is the most commonly caused by invasion of the central nervous system by helminths, as occurs in Angiostrongylus cantonensis infections. The rodent Rattus norvegicus is the definitive natural host and humans act as accidental hosts and can become infected by eating raw or undercooked snails or food contaminated with infective L3 larvae. Recently in Brazil there have been four cases of eosinophilic meningitis due to ingestion of infected Achatina fulica. To evaluate biochemical and histopathological changes caused by this parasite, R. norvegicus were experimentally infected with 100 L3 larvae of A. cantonensis. After the anesthetic procedure, serum from the rodents was collected from the inferior vena cava for evaluation of the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyl transferase (GGT), total protein and its fractions. During the necropsy, the liver was collected and weighed. Then a 1-g fragment was extracted from the major lobe to quantify the hepatic glycogen and fragment remainder was taken from the same lobe and fixed in Milloning’s formalin for histopathological examination. Additionally, helminths were collected from the brain and lungs of the rodents. The activities of AST, ALT, ALKP and GGT in the serum and hepatic glycogen increased in response to infection, while the levels of globulin and total protein increased only in the eighth week of infection and there was a reduction in the levels of serum glucose. Albumin and bilirubin concentrations remained stable during the experiment. Infection with A. cantonensis caused metabolic and histopathological changes in the rodents. This study can contribute to a better understanding of the relationship between A. cantonensis and R. norvegicus.

Trypsin and bile salts have been identified as important triggers for excystation of Echinostoma metacercariae. Although excystation in trematodes is a well-known phenomenon, some morphological developmental changes remain to be elucidated. In order to gain further insight into the in vitro development of metacercariae, we assayed different cultivating conditions: 0.5% trypsin and 0.5% bile salts; 1% trypsin and 1% bile salts; 1% trypsin and 0.5% bile salts; 0.5% bile salts; or 0.5% trypsin. By means of light microscopy and confocal microscopy, we characterized each encysted, activated, breached and excysted stage based on the morphological features. However, breached and excysted stages were not revealed in both bile salts and trypsin-free medium. Excretory concretions (25 ± 3.9) were visualized within excretory tubules, close to the ventral sucker and genital anlage. The oral sucker armed with spines and digestive system was similar to those of adult worms. The reproductive system is composed of a genital anlage and the cirrus sac primordium. In short, trypsin and bile salts associated were fundamental for the in vitro metacercariae excystation of Echinostoma paraensei. This article presents the first detailed information of all stages of metacercariae excystation obtained through light and confocal microscopy.

The glucose content in the hemolymph and glycogen content in the digestive gland–gonad complex (DGG) and cephalopedal mass of Biomphalaria glabrata exposed to different parasite doses (5 and 50 miracidia) of Echinostoma paraensei as well as the activity of lactate dehydrogenase were evaluated. HPLC (high-performance liquid chromatography) analyses were also performed to determine the concentrations of four organic acids (oxalic, succinic, pyruvic and lactic) present in the hemolymph of infected and uninfected snails, to better understand the effect of infection on the host’s energetic/oxidative metabolism. The snails were dissected 1–4weeks after infection to collect the hemolymph and separate the tissues. There was alteration in the glycemia of the snails at both parasite doses, with a significant increase of glycemia from of the third week after infection in comparison to the control group. Changes were also observed in the lactate dehydrogenase activity, with increased activity as the infection progressed. In parallel, there was a decrease in the glycogen content in the storage tissues, with a markedly greater reduction in the digestive gland–gonad complex (larval development site) in comparison with the cephalopedal mass. Additionally, the infection by both miracidial doses resulted in an increase of oxalic and lactic acid levels, as well as in a decline of piruvic and succinic acid levels in B. glabrata, thus explaining the reduction of the oxidative decarboxylation rate in the tricarboxylic acid cycle and acceleration of the anaerobic degradation of carbohydrates in the snails, through lactic fermentation, which is essential to ensure energy supply and success of the infection.

Experimental infection of Biomphalaria glabrata by Angiostrongylus cantonensis induces significant changes in the concentrations of triacylglycerol and cholesterol in the hemolymph and of neutral lipids in the digestive gonad-gland (DGG) complex of the host snail. In this study, snails were dissected after 1, 2, and 3 weeks of infection to collect the hemolymph and DGG and to measure the levels of cholesterol and triacylglycerol in the hemolymph and neutral lipid fractions in the tissues. The results show that infection by this nematode resulted in a significant decrease in the concentrations of both cholesterol and triacylglycerol in the hemolymph of B. glabrata during the parasite's initial ontogenic development period. This reduction indicates the possible use of these molecules by both parasite and host not only as energy substrates but also as structural factors required during development of the parasite's larval stages. In parallel, changes in the neutral lipid profile in the DGG and lipase activity of the infected snails were observed, indicating the importance of these molecules for successful infection.

Chemotherapy of food-borne trematodes relies on two drugs, praziquantel and tricabendazole, and there is growing interest in finding alternative therapies. Plant oil extracts have long been used in traditional Chinese medicine as sources of bioactive compounds with antiparasitic activity. Species of the genus Echinostoma are used as good models to test effective compounds against food-borne trematodes. This study evaluated the anthelmintic activity of crude artesunate extracts in vitro on newly excysted metacercariae of Echinostoma paraensei by light and scanning electron microscopy (SEM). The flukes were incubated with 1 μg/mL, 10 μg/mL, 25 μg/mL, 50 μg/mL and 100 μg/mL of artesunate for 4, 12, 24, 48 and 72 h. When the exposure time and concentration of artesunate increased, there were changes in motor activity, tegument damage and death. Blebs and swelling were the most common damages quantified on the tegument. The in vitro study reproduced results described for other immature flukes incubated with artemisinin derivatives. Excysted metacercariae of E. paraensei constitute a good model to study in vitro drug effects.

The therapeutic effects of praziquantel (PZQ) against a Schistosoma mansoni isolate derived from Nectomys squamipes (isolate R) and a susceptible isolate (BH) were analyzed in Swiss mice by fecal egg counting, adult worm reduction and oogram pattern. Infected mice were orally administrated with 62.5mg/kg (group 1), 125mg/kg (group 2), 250mg/kg (group 3) and 500mg/kg (group 4), each dose divided over 3days (49, 50 and 51days after infection). The data were analyzed using one-way analysis of variance (ANOVA). In regard to isolate R, no fecal eggs were observed with 250mg/Kg and 500mg/kg (p0.05) affected the percentage of mature eggs, regardless of isolate. There was a large increase (p

The calcium content in the hemolymph and shell of Biomphalaria glabrata (Say, 1818) was determined after exposure to different parasite burdens (5 and 50 miracidia) of Echinostoma paraensei (Lie and Basch, 1967). The snails were dissected 1, 2, 3, and 4weeks after infection to collect the hemolymph and shell. An increase in calcemia was observed in snails infected with both miracidial doses. A significant decrease in the calcium ions in the shell was observed, coinciding with the calcemia peak in the hemolymph. This indicates greater mobilization of calcium between the shell and hemolymph to regulate the calcium content in the body when the snail is exposed to stress conditions, as has also been observed in some other infected snail species. The results obtained indicate that in this model, the calcium metabolism depends on the miracidial dose used.

This study was performed to gain insight into the maturation of the reproductive system of Echinostoma paraensei worms grown in an early infection of Mesocricetus auratus. Hamsters were infected with 100 metacercariae and necropsied on days 3, 5, 7, 10 and 14 post infection (dpi). Recovered flukes stained with hydrochloric carmine were preserved as whole mounts and analyzed by light and confocal scanning laser microscopy. The average worm recovery was 43.7 per host. Images of the male and female reproductive systems were taken. The ovary and anterior and posterior testis were evidenced on day 3, while the ootype and cirrus sac were present on day 5. Confocal imaging showed primordium testis and ovary as a cluster of primordial cells from day 3 onward. The testes, ovary, cirrus sac and uterus organs were already present during the first week of life. The two testes were seen as individual structures on 7dpi while the cirrus sac and vitelline glands were in development. The ovary was connected to the uterus while the ootype was adjacent to it. Both testes were larger than the ovary, showing cells at different stages of development, but with few bundles of functional spermatozoa in 10day-old worms. On day 14, eggs and spermatozoa were seen in the uterus and seminal vesicle, respectively, while oocytes appeared in the ootype as fertilized eggs. We conclude that the reproductive system of E. paraensei was functional on 14dpi in the hamsters.

Tests were performed to evaluate the biochemical alterations in Rattus norvegicus after infection by the intestinal trematode Echinostoma paraensei. The rodents received 150 metacercariae each, serum samples were collected and the parasite load was quantified weekly until the fifth week of infection. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALKP), gamma-glutamyl transferase (GGT), bilirubin, glucose, total proteins and fractions and hepatic glycogen were determined. All the animals exposed to the metacercariae were infected in the first week and worms were recovered up to the third week after infection. The levels of AST, ALT, GGT, bilirubin and globulin rose in the first and/or second week and declined thereafter to levels near those of the control group. In contrast, the level of total proteins in the plasma fell significantly in the first week while the ALKP activity went down only in the fourth and fifth weeks in relation to the control group. There was no significant difference in the levels of albumin, glycogen and glucose. Infection by E. paraensei in R. norvegicus causes changes in the hepatic function, possibly resulting from the cholestasis produced by the partial obstruction of the bile duct by the helminths.

Angiostrongylus cantonensis is considered the main agent responsible for human eosinophilic meningoencephalitis. This parasite has low specificity for mollusk hosts and it can also use aquatic snails as auxiliary hosts. Studies based on the metabolic profile of Biomphalaria spp. infected by A. cantonensis have been conducted to observe parasite-host interactions. In the present study, the glucose content in the hemolymph and glycogen content in the digestive gland and cephalopedal mass of Biomphalaria tenagophila and Biomphalaria straminea experimentally infected by A. cantonensis were evaluated, along with the activity of LDH. The snails were dissected from 6 to 21days after infection to collect the hemolymph and separate the tissues. Decreases of 96% and 6.4% in the glucose content triggered a transition from aerobic to anaerobic metabolism in the two infected snail species, B. straminea and B. tenagophila, respectively. That finding was confirmed by high-performance liquid chromatography. These results indicate that when infected, these snails are able to change their metabolic profile, suggesting a strategy to maintain their homeostatic balance.

• Rehydration solution (Na 3 PO 4 ) is not necessary to extract ancient DNA from desiccated archaeological samples.

To the Editor: The nematode Angiostrongylus cantonensis (rat lung worm), a zoonotic parasite that can accidentally infect humans and cause eosinophilic meningoencephalitis, has the Norway rat (Rattus norvegicus) as one of its most frequent definitive vertebrate hosts (1). Adult worms live in the pulmonary arteries of the definitive hosts, which excrete first-stage larvae in their feces. Intermediate hosts, such as snails and slugs, are infected by first-stage larvae, which reach the infective third stage after 2 molts. Third-stage larvae are then ingested by rats as they feed on the intermediate hosts, thus closing the life cycle. Humans become infected by eating raw or undercooked snails and slugs and through paratemic hosts and vegetables contaminated with infected snail mucus (2). In Brazil, the first 3 documented cases of eosinophilic meningoencephalitis occurred in 2007 in 2 cities in the southeastern state of Espirito Santo (3). In 2009, a new case was reported in Pernambuco in the northeast region (4). Only intermediate hosts have been found naturally infected with rat lung worm in Brazil. Infected terrestrial and freshwater snails of the species Achatina fulica, Sarasinula marginata, Subulina octona, and Bradybaena similaris in Espirito Santo; A. fulica and Pomacea lineata in Pernambuco; and A. fulica in Rio de Janeiro and Santa Catarina have been reported (3,5,6). Thus, because of the recent cases of eosinophilic meningoencephalitis in Brazil and the occurrence of naturally infected A. fulica snails in Rio de Janeiro, we investigated the existence of potential natural reservoirs for the parasite in Sao Goncalo. Sao Goncalo (22°48′26.7′′S, 43°00′49.1′′W) is a densely populated city (≈1 million inhabitants) with a tropical Atlantic climate (14°C–35°C) that is part of the metropolitan region of Rio de Janeiro. Two collections were made in March and June 2010. Forty live traps (20 Tomahawk [Tomahawk Live Trap Company, Tomahawk, WI, USA] and 20 Sherman [H.B. Sherman Traps Inc., Tallahassee, FL, USA] traps) were placed along two 30-m transects for 4 consecutive nights (Brazilian Institute of Environment and Renewable Natural Resources license no. 2227–1/2010) in an urban area where A. fulica snails had been collected in high numbers. Twenty-seven Norway rats (16 males) were captured. We collected 265 adult lung worms from the pulmonary arteries of the captured animals, fixed the worms in 70% ethanol, and taxonomically identified them as A. cantonensis on the basis of the large size of the spicules and the patterns of the bursal rays (7). Voucher specimens have been deposited in the Helminthological Collection of the Oswaldo Cruz Institute (no. 35712). Nineteen (74%) rats were infected; mean intensity and mean abundance were 13.52 ± 2.36 and 9.81 ± 1.96, respectively. To confirm the morphologic identification of the Angiostrongylus specimens obtained, a DNA bar coding approach was used. DNA was extracted from 3 ethanol-preserved adult worms previously recovered from the pulmonary arteries of a naturally infected Norway rat, PCR-amplified, sequenced for a partial region of the COI gene (8), and subsequently compared with available GenBank Angiostrongylus spp. sequences. The three 360-bp COI sequences obtained (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HQ440217","term_id":"330414605","term_text":"HQ440217"}}HQ440217) were Clustal-aligned (www.clustal.org) with homologous COI fragments of A. cantonensis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ398121","term_id":"254941204","term_text":"GQ398121"}}GQ398121), A. vasorum (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"EU493162","term_id":"187473267","term_text":"EU493162"}}EU493162, {"type":"entrez-nucleotide","attrs":{"text":"EU493163","term_id":"187473269","term_text":"EU493163"}}EU493163, {"type":"entrez-nucleotide","attrs":{"text":"EU493166","term_id":"187473275","term_text":"EU493166"}}EU493166, {"type":"entrez-nucleotide","attrs":{"text":"EU493167","term_id":"187473277","term_text":"EU493167"}}EU493167), and A. costaricensis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ398122","term_id":"254941217","term_text":"GQ398122"}}GQ398122) and subjected to phylogenetic analysis. Ancylostoma tubaeforme (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AJ407940","term_id":"18077725","term_text":"AJ407940"}}AJ407940) was used as the outgroup. Haplotypes for A. vasorum isolates from Brazil (A. vasorum 5421, 5641, and 5642) were reconstructed from published information (9) and included in the alignment. We used MEGA4 (www.megasoftware.net) to construct a neighbor-joining phylogenetic tree based on Kimura 2-parameter (K2-p) distances (Figure). The 3 A. cantonensis specimens from Sao Goncalo, Rio de Janeiro, yielded a single haplotype, which formed a clade with the A. cantonensis haplotype from the People’s Republic of China with low genetic distance (K2-p 0.038) and high bootstrap support (98), thus confirming the morphologic identification. Comparisons with the other 2 Angiostrongylus species yielded higher genetic distance values (K2-p 0.120, with A. vasorum, and 0.149, with A. costaricensis). Figure Neighbor-joining phylogenetic tree based on Kimura 2-parameter (K2-p) distances that includes all Angiostrongylus COI sequences in GenBank and the sequences obtained from 3 Angiostrongylus specimens recovered from the pulmonary arteries of a naturally ... These results indicate that A. cantonensis lung worm infection is enzootic among the exotic Norway rat population in the region studied. The natural infection rate of 74% is the second highest reported among 14 severely A. cantonensis infection–endemic regions (2). These findings, together with the observation of dense populations of A. fulica snails in urban areas of the country (10), call attention to the risk for disease transmission to humans, given that Norway rats also are likely to be present in these areas. Local residents should be informed about disease transmission and prevention, and physicians should consider A. cantonensis lung worm infection in the differential diagnosis when appropriate. Although public health authorities should consider implementation of surveillance and control strategies to reduce the populations of snail and rat hosts, a better understanding is needed of the epidemiologic significance of these findings, which can be attained through studies to identify human cases of eosinophilic meningitis in the region.

A new isolate of Echinostoma paraensei (LieBasch, 1967) was obtained from a natural vertebrate host, the water rat Nectomys squamipes. Relationships with sympatric snails, Biomphalaria glabrata; Physa marmorata and Lymnaea columella, as well as allopatric snails and the definitive hosts N. squamipes, Mus musculus, Rattus norvegicus, Mesocricetus auratus and birds were investigated. E. paraensei developed in all sympatric snail species following exposure to one or five miracidia. P. marmorata showed the highest infection rate (34% and 36%) followed by L. columella (18% and 24%) and B. glabrata (8% and 28%), respectively. Mortality was elevated in the patent period, with L. columella having the highest values. B. glabrata, P. marmorata, and L. columella did not differ in relation to cercarial encystment, and metacercariae from each species were able to infect N. squamipes with similar worm burdens. Recovered adult worms were found in clusters from the first to third sections of the small intestine. Miracidia of E. paraensei hatched after 10 days incubation in darkness in dechlorinated water at 28+/-1 micro C. Miracidia began to hatch 1 h after exposure to incandescent light.

Echinostoma paraensei is a trematode of the genus Echinostoma that causes echinostomiasis in humans. The objectives of this study were to: evaluate the ovicidal activity of the nematophagous fungus Pochonia chlamydosporia (VC1 and VC4) on a solid medium 2% water–agar (2% WA) against E. paraensei eggs (assay A); evaluate ovicidal effect (destruction of eggs) of the isolate VC4 in supplemented culture media (assay B); and evaluate the ovicidal ability of the crude extract (VC4) on E. paraensei eggs (assay C). Eggs of E. paraensei (assay A) were placed in Petri dishes containing 2% WA with an isolate of the fungus P. chlamydosporia (VC1 and VC4) grown for 10 days, and without fungus as a control and evaluated regarding their destruction. In assay B, eggs of E. paraensei were placed in Petri dishes with different supplemented culture media and with VC4 isolate and the destruction of eggs was examined at the end of 25 days of interaction. In assay C, effects of the crude extract of P. chlamydosporia (VC4) on eggs were evaluated at the end of 7 days. In assay A, there was no difference (p > 0.05) in ovicidal activity among the tested isolates (VC1 and VC4); however, the highest percentage for ovicidal activity (type 3 effect) was demonstrated by the isolate VC4. In assay B, the culture medium starch–agar showed the best results for the destruction of the eggs, with a percentage of 46.6% at the end of the assay. In assay C, the crude extract of VC4 was effective in the destruction of E. paraensei eggs, with a percentage reduction of 53%. The results of this study demonstrate that a rich culture medium with a greater availability of carbon and nitrogen may interfere directly in the predatory characteristics of ovicidal fungi.