Arnaldo Maldonado

The giant African snail Achatina fulica as natural intermediate host of Angiostrongylus cantonensis in Pernambuco, northeast Brazil

The human cases of eosinophilic meningitis recently reported from Brazil have focused the attention of the public health agencies on the role the introduced snail Achatina fulica plays as hosts of the metastrongylid nematodes. Determining the potential of this snail to host and develop infective larval stages of metastrongylids in the wild and identify the species harbored by them is crucial for designing effective control measures. Here we assess if A. fulica may act as intermediate host of A. cantonensis at the peridomiciliary areas of a patients house from state of Pernambuco (PE), who was diagnosed with eosinophilic meningitis and a history of ingesting raw molluscs. Larvae obtained from naturally infected A. fulica were orally administered to Rattus norvegicus. The worms were collected from the pulmonary artery and brain, and were morphologically characterized and compared to the Japan isolate of A. cantonensis. Adult worms and infective L(3) larvae (PE isolate) recovered from A. fulica specimens were also analyzed by polymerase chain reaction and restriction fragment length polymorphism of ITS2 region from rDNA and compared to A. cantonensis (ES isolate), A. vasorum (MG isolate) and A. costaricensis (RS isolate). The large size of the spicules (greater than those observed in other species of Angiostrongylus) and the pattern of the bursal rays agree with the original species description by Chen (1935). Furthermore, the morphology of the PE isolate was similar to that of Japan isolate. The PCR-RFLP profiles obtained were distinctive among species and no variation in patterns was detected among adult individuals from A. cantonensis isolates from PE and ES. The importance of A. fulica as an intermediate host of eosinophilic menigoencepahlitis in Brazil is emphasized.

Genetic and morphological characterisation of a new species of the genus Hysterothylacium (Nematoda) from Paralichthys isosceles Jordan, 1890 (Pisces: Teleostei) of the Neotropical Region, state of Rio de Janeiro, Brazil

Taking into account the difficulties of taxonomic identification of larval anisakid nematodes based on morphological characters, genetic analyses were performed, together with those usually applied, in order to identify anisakid larvae found in the flounder Paralichthys isosceles from the littoral of the state of Rio de Janeiro, Brazil. The analysis of 1,820 larvae revealed a new species, similar to Hysterothylacium MD, Hysterothylacium 2, Hysterothylacium KB and Hysterothylacium sp regarding the absence of the larval tooth, an excretory pore situated below the nerve ring level, and slender lateral alae. Moreover, the new species differs from Hysterothylacium fortalezae and Hysterothylacium reliquens with regard to the number and size of spines present on the tail end and from Hysterothylacium patagonicus by the absence of interlabia. The maximum parsimony and neighbour joining tree topologies based on the 18S ribosomal DNA gene, complete internal transcribed spacer region and cytochrome oxidase 2 (COII) gene demonstrated that the Brazilian larvae belong to Raphidascarididae and represent a unique genetic entity, confirmed as a new Hysterothylacium species. Furthermore, the new species presents COII genetic signatures and shares polymorphisms with Raphidascarididae members. This is the first description of a new anisakid species from Brazil through the integration of morphological and molecular taxonomy data.

Evaluation of the resistance to Schistosoma mansoni infection in Nectomys squamipes (Rodentia: Cricetidae), a natural host of infection in Brazil

The development of resistance in three stages throughout an active infection (pre-ovular, acute and initial chronic stages) was studied, comparing the total number of adult worms recovered from the reinfected group and the control groups. It was shown that Nectomys squamipes was unable to develop resistance in the tested conditions and, on the other hand, reinfection in the pre-ovular period of the parasite led the rodent to present the phenomenonacilitation, with reduction of natural resistance and an increase in the parasite load. These results suggest the existence of other forms of immunity diverse from the concomitant immunity in the host-parasite relationship, according to the employed model.

Endemic angiostrongyliasis in the Brazilian Amazon: Natural parasitism of Angiostrongylus cantonensis in Rattus rattus and R. norvegicus, and sympatric giant African land snails, Achatina fulica

Angiostrongylus cantonensis, the rat lungworm, is one etiological agent of eosinophilic meningoencephalitis in humans. This zoonosis is frequently found in Asia and, more recently, in North America, Caribbean Island and northeastern of South America. Until now, research of A. cantonensis in southern, southeastern and northeastern regions of Brazil has been found natural infections only terrestrial and freshwater intermediate snail hosts (Achatina fulica, Sarasinula marginata, Subulina octona, Bradybaena similaris and Pomacea lineate). In this study, we examined the occurrence of helminthes in the synantropic rodents Rattus rattus and Rattus norvegicus in northern Brazil, focusing on the role of these species as vertebrate hosts of A. cantonensis and A. fulica as intermediate host have found natural. Thirty specimens of R. rattus and twelve of R. norvegicus were collected in the Guamá and Jurunas neighborhoods of the city of Belém, in the Brazilian state of Pará, of which almost 10% harbored adult worms in their pulmonary arteries. Sympatric A. fulica were found to be infected by L(3) larvae, which experimental infection confirmed to be A. cantonensis. Natural infection of snails and rodents with A. cantonensis was confirmed through morphological and morphometrical analyses of adults and larvae using light microscopy, scanning electron microscopy and molecular sequences of partial Cytochrome Oxidase subunit I. Phylogenetic analyses showed that A. cantonensis isolated from Pará, Brazil is similar to Japan isolate; once these specimens produced a single haplotype with high bootstrap support with Rio de Janeiro isolate. This study confirms that A. cantonensis is now endemic in northern Brazil, and that R. rattus and R. norvegicus act as natural definitive hosts, and A. fulica as the intermediate host of the parasite in this region.

Activation of anaerobic metabolism in Biomphalaria glabrata (Mollusca: Gastropoda) experimentally infected by Angiostrongylus cantonensis (Nematoda, Metastrongylidae) by high-performance liquid chromatography.

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.

Biochemical profile of Biomphalaria glabrata (Mollusca: Gastropoda) after infection by Echinostoma paraensei (Trematoda: Echinostomatidae)

The effect of infection by Echinostoma paraensei on the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and the concentration of total proteins, uric acid and urea in the hemolymph of Biomphalaria glabrata were investigated after exposure to five or 50 miracidia. The biochemical concentrations were measured weekly until the end of the fourth week after exposure. There was a significant decrease in the concentrations of total proteins in the snails exposed both to five and 50 miracidia, as well as an increase in the nitrogenous products of excretion, ALT and AST activities. The higher ALT activity in the hemolymph of the snails after infection with 50 miracidia suggests highest energetic requirement in these snails in relation to snails exposed to five miracidia. The results also 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, frequently associated with tissue damages. This can be explained by damage due to penetration by the miracidia and subsequent development of intramolluscan sporocysts and rediae.

Ultrastructure of the Miracidium of Echinostoma paraensei Lie and Basch, 1967 (Trematoda, Echinostomatidae)

The morphology of Echinostoma paraensei was studied using transmission electron microscopy. The terebratorium region has many electrondense secretory granules and many folds on the surface. The epidermal cells that cover the larval body have unique nuclear morphology, many mitochondria and vesicles being attached to the interepidermal ridges by a septate junction. The cilia present the organization 9+2 and a typical structure with a shaft, axosome, basal body and rootlet. Below the epidermal cells there is a layer of circular muscle and, adjacent to it, a layer of longitudinal muscle fibers. The excretory system has two flame cells, with internal and external ribs and leptotriches at the barrel region, an excretory vesicle and an excretory pore.

ECHINOSTOMA LUISREYI N. SP. (PLATYHELMINTHES: DIGENEA) BY LIGHT AND SCANNING ELECTRON MICROSCOPY

The entire life cycle of the Brazilian Echinostoma luisreyi n. sp., of the 37–collar spine E. revolutum group, has been observed under experimental conditions. The snail Physa marmorata serves as its first natural intermediate host. This species and the planorbid Biomplalaria glabrata act as experimental second intermediate hosts. The worm recovery rate was 51.3 and 0.6% for mice and hamsters, respectively, but the infection did not develop in the quail Coturnix coturnix. The natural vertebrate host is not known. The most important morphological character of the new species separating it from the other Echinostoma species studied is the oral corner spines that increase in size from the latero-oral to the ventro-oral regions. The ratio of the sizes of the smaller oral spines and the larger aboral ones was 1.7:1.0. Also, the excretory pore is radially wrinkled and dorsally subterminal. Images obtained by scanning electron microscopy confirmed the characters that differentiate the new species and the most closely related species.

Aerobic to anaerobic transition in Biomphalaria glabrata (Say, 1818) infected with different miracidial doses of Echinostoma paraensei (Lie and Basch, 1967) by high-performance liquid chromatography

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 hosts energetic/oxidative metabolism. The snails were dissected 1-4 weeks 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.

Spirurids from Gracilinanus agilis (Marsupialia: Didelphidae) in Brazilian Pantanal wetlands with a new species of Physaloptera (Nematoda: Spirurida)

Gastrointestinal nematodes were recovered from thirty four Gracilinanus agilis from forty four collected in Pantanal, Mato Grosso do Sul State, Brazil. Two hundred seventy four spirurids were recovered from the esophagus, stomach and intestines, comprising three species from three different genera. These were identified as Pterygodermatites (Paucipectines) jägerskiöldi, Spirura guianensis and Physaloptera herthameyerae n. sp. is first described. This is the first record of nematodes of the genera Physaloptera and Spirura in hosts of the genus Gracilinanus. The high prevalence of spirurids in 72.3% of the G. agilis collected probably is influenced by the arboreal and diet behaviors.