The first description of eggs in the male reproductive system of Physaloptera bispiculata (Nematoda: Spiruroidaea).

Physaloptera bispiculata (Nematoda: Spiruroidaea) is a parasite of Nectomys squamipes (Rodentia: Cricetidae), a water rat that only occurs in Brazil. Naturally infected rodents were captured in the municipality of Rio Bonito, Rio de Janeiro, Brazil. Adult P. bispiculata worms were collected, prepared and analysed by light and scanning electron microscopy. Under scanning electron microscopy, several eggs were seen glued by cement to the cloacal aperture. Light microscopy revealed that some male worms had an uncountable number of embryonated eggs in the ejaculatory duct, cloaca and also in the posterior portion of the intestine. The probable explanation is that the eggs developing in the female uterus are pumped by the female or sucked by the male to the cloacal opening and from this point to the intestine and ejaculatory duct. The male probably does not have the ability to expel the eggs and for this reason a large number were found in these organs. On the other hand, this could be an important adaptation for the parasite, i.e. male worms expelled by the host can carry a large number of eggs and spread them to intermediate hosts when ingested by these hosts. As far as we know this is the first record of a physalopterid nematode harbouring eggs in the cloacal region, ejaculatory duct or intestine.

Ultrastructural analysis of Wuchereria bancrofti (Nematoda: Filarioidea) body wall.

Bancroftian filariasis constitutes the principal mosquito-borne nematode infection of humans and the surface of adult of Wuchereria bancrofti seems to be especially important in the intricate interplay between host and parasite. The study of the parasite's surface structure might help to understand the localization and function of various organelles. W. bancrofti adult worms were recovered from untreated patients during hydrocele repair surgery and studied by transmission electron microscopy. The body wall of adult parasite is composed of cuticle, hypodermis and muscular layer. Cuticle is the external layer and shows transverse cuticular striation. It is composed by an epicuticle, cortical layers, median layer, fibrous layers and basal layer. The epicuticle is the most external cuticular layer and appears as a single laminar electron-dense layer. The cortical external region is more electron-dense and granular in appearance than the inner cortical layer. Electron-dense structures, called bosses are randomly distributed filling the cuticular striation. The median layer is formed by an electron-dense and continuous thick line. The fibrous layer is subdivided in inner and external layers connected by projections. The basal layer includes a large quantity of membranous projections directed toward the hypodermis. The hypodermis is a syncytium where some cellular organelles are observed. The somatic musculature is meromyarian. The muscle fibers consist of contractile and non-contractile regions and the contractile region is composed of myofilaments separated by dense body. This is the first study of W. bancrofti adult worms obtained from untreated patients and studied by transmission electron microscopy.

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 patient's 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.

Physiological changes in Lymnaea columella (Say, 1817) (Mollusca, Gastropoda) in response to Echinostoma paraensei Lie and Basch, 1967 (Trematoda: Echinostomatidae) infection.

The physiological interaction between the digenean Echinostoma paraensei larvae and the intermediate snail host Lymnaea columella was studied. The carbohydrate content was significantly altered in the digestive gland tissue of snails, decreasing after 20 days postinfection. At the end of the prepatent period, the carbohydrate content was reduced by 60% when compared to uninfected snails. The total protein was reduced by 80.01% and 76.42% in the hemolymph and digestive gland, respectively. The end products resulting from nitrogen degradation were also analyzed. The urea was significantly increased in both the hemolymph and digestive gland. In the former, the highest concentration was detected at day 10 postinfection and then decreased until the end of the prepatent period. In digestive glands from infected snails, urea was significantly higher than in uninfected ones after 20 days. The uric acid content was reduced by 94.72% in the hemolymph and 43.75% in digestive glands after 10 days postinfection. The ammonia was undetectable under the experimental conditions employed.

Further description of Cruzia tentaculata (Rudolphi, 1819) Travassos, 1917 (Nematoda: Cruzidae) by light and scanning electron microscopy.

Species of Cruzia are parasites of the large intestine of marsupials, reptiles, amphibians, and mammalians. Cruzia tentaculata specimens were collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Colombia (new geographical record) and from Brazil and analyzed by light and scanning electron microscopy. The morphology of males and females by light microscopy corroborated most of the previous description and the ultrastructure by scanning electron microscopy evidence: the topography of the cuticle, deirids, amphids, phasmids in both sexes, a pair of papillae near the vulva opening, and the number and location of male caudal papillae, adding new features for species identification only observed by this technique.

Nematode cysts and larvae found in Achatina fulica Bowdich, 1822.

This study describes the morphology of the nematode cysts and larvae found in Achatina fulica (giant African snail) in Brazil. Sixty snails were collected in Mesquita, Rio de Janeiro State. Fourteen of the snails were naturally infected. The cysts were spherical, pink colored and measured 0.97 to 1.57 mm in diameter. In the majority of cases they had a single larvae involved in amorphous material. A total of 222 encysted larvae were recovered. Of these, 30 were utilized in the morphological study. The length of the larvae varied from 2.57 to 5.8 mm and they were classified as small--up to 3.5 mm; medium--from 3.53 to 4.5 mm; and large--greater than 4.52 mm. The average length of the larvae in the three groups was 2.85, 3.87 and 5.23 mm, respectively. The larval cuticle was white, shiny and transversally striated until the posterior end of the body. At the anterior end there is a mouth with three lips, with amphids and papillae, followed by a muscular esophagus with average length of 0.61 mm, terminating in an esophageal bulb and having a nerve ring in the middle third of the esophagus, and an intestine with an opening near the posterior end. The tail begins from this opening and has two types of ends: short and abrupt or long and gradually tapering. The difference in the tail end can suggest sexual dimorphism, although no primordial reproductive structures were observed. These characteristics were not sufficient to identify the larvae, so there is a need for further study.

An atypical microfilaria in blood samples from inhabitants of Brazilian Amazon.

An unidentified microfilaria sharing characteristics with Mansonella ozzardi and Onchocerca volvulus was detected in blood samples from seven human volunteers, inhabitants of a community in the border of Amazonas and Acre State. They were detected during epidemiological studies carried out in some communities along Antimary, Acre, and Purus Rivers in the Brazilian Amazon. The most striking difference was presented in the shape of the cephalic space from this microfilaria which was different from those of M. ozzardi and with similarities to O. volvulus in this region, but no remarkable differences were observed at the caudal region. More accurate studies are being carried out in order to provide additional data and supporting evidences before establishment of a new species can be done.

Ultrastructure of reproductive system of female Litomosoides chagasfilhoi Moraes-Neto, Lanfredi and De Souza, 1997 (Nematoda: Filarioidea).

The ultrastructure of the female reproductive system of the filariid Litomosoides chagasfilhoi by transmission electron microscopy (TEM) is described for the first time. The ovary is composed of primary oocytes surrounded by a single layer of epithelial cells apposed on the basal laminae. The ovarian wall is completely filled with primary oocytes, which are arranged radially and are centrally connected around the rachis. The uterine wall consists of muscular fibers surrounded by a basal lamina and the epithelium underlying the lamina. Ameboid and aflagellate spermatozoa are present inside the distal portion of the uterus, some of them near oocytes, which present bacteria in its cytoplasm. An eletrondense well-defined eggshell covers the zygotes, which presents in its cytoplasm bacteria arranged in groups. These bacteria are also observed in embryos and in the hypodermal cord. These ultrastructural aspects of L. chagasfilhoi female worms presented herein contribute to the knowledge of the morphology and embryonary development of this filariid, providing means for further comparative analyses of the action of anti-filarial drugs. Besides this, the presence of bacteria Wolbachia-like is being reported for the first time in this species, showing the great importance of this experimental model of study.

Further description of Aspidodera raillieti (Nematoda: Aspidoderidae) from Didelphis marsupialis (Mammalia: Didelphidae) by light and scanning electron microscopy.

Nematodes of the family Aspidoderidae (Nematoda: Heterakoidea) Freitas 1956 are widely distributed from Americas. The species of the genus Aspidodera Railliet and Henry 1912 are parasites of mammals of the orders Edentata, Marsupialia, and Rodentia. In the present work, Aspidodera raillieti (L. Travassos, Mem Inst Oswaldo Cruz 5(3):271-318, 1913), collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Valle del Cauca, Colombia, is redescribed. The association of light and scanning electron microscopy (SEM) allowed a detailed analysis of the morphology and ultrastructure of this nematode. Some taxonomic features, such as cephalic region, topography of the cuticle, sucker, spicules, posterior end of males, localization of vulva, the anus, and posterior end of females were observed. Important structures such as amphid, details of cephalic region, phasmid, and number and localization of caudal papillae are documented by SEM, for the first time adding characters to identify this species. Colombia is a new geographical record for A. raillieti.

Comparative analysis of a chemotherapy effect on the cuticular surface of Wuchereria bancrofti adult worms in vivo.

Wuchereria bancrofti is the main species responsible for human lymphatic filariasis and remains a major public health problem in tropical countries around the world. Diethylcarbamazine (DEC) has been used for decades in control programs as an effective microfilaricide, although its efficacy in killing adult worms is only around 50% and its direct mode of action is unclear. Recently, in an attempt to control and eliminate lymphatic filariasis, WHO has recommended albendazole (ALB), a broad-spectrum anthelminthic combined with DEC or ivermectin for mass treatment. Some studies have shown that DEC alone blocks oogenesis, fertilization in adult worms, and loss of the microfilarial sheath of several filarial species, whereas ALB is thought to target nematode tubulin. So far, the direct effect of ALB in combination with DEC has not been described in W. bancrofti adult worms. Therefore, the purpose of this study is to investigate by scanning electron microscopy if DEC coadministered with ALB can induce in vivo morphological alterations of the W. bancrofti adult worm surface obtained from a patient in whom the adult worm remained alive, checked serially by ultrasonography for 2 months after antifilarial treatment. Our analysis demonstrates that worms presented morphologic alterations in some regions suggesting cuticular surface damage. On the other hand, adult worms that were recovered from a patient treated with DEC alone after a single dose did not show such any abnormalities.

Helminth communities of Nectomys squamipes naturally infected by the exotic trematode Schistosoma mansoni in southeastern Brazil.

The water rat Nectomys squamipes is endemic in Brazil and found naturally infected with Schistosoma mansoni. Helminth communities, their prevalences, intensity of infection and abundance in N. squamipes in an endemic area of schistosomiasis in the state of Rio de Janeiro, Brazil were studied. Four species of nematodes (Physaloptera bispiculata, Syphacia venteli, Hassalstrongylus epsilon and Litomosoides chagasfilhoi) were recovered in 85.3%, two trematodes (Schistosoma mansoni and Echinostoma paraensei) in 38.8% and one cestode species (Raillietina sp.) in 1.7% of rats examined. Rats were infected with up to five helminth species each, and these were highly aggregated in distribution. For H. epsilon and S. venteli, intensities and abundances were higher in adult male and subadult female hosts, respectively. Hassaltrongylus epsilon, P. bispiculata, S. venteli and S. mansoni were classified as dominant species, L. chagasfilhoi and E. paraensei as co-dominant and Raillietina sp. as subordinated. No significant correlation was found in the intensity of infecton between each pair of helminth species. Schistosoma mansoni was not related to any other helminth species according to their infection rates, althougth S. mansoni was well established in the natural helminth comunity of the water rat.

The taxonomy of some Poecilacanthoidea (Eucestoda: Trypanorhyncha) from elasmobranchs off the southern coast of Brazil.

Specimens of elasmobranchs, collected in the State of Santa Catarina, Brazil, were parasitized with Poecilacanthoidea trypanorhynch cestodes. Sharks of the species Prionace glauca harboured Callitetrarhynchus gracilis and Floriceps saccatus, and those of the species Sphyrna zygaena were infected with Callitetrarhynchus speciosus. Details of the proglottids of F. saccatus, provided by bright-field, and/or scanning electron microscopy, are described. Adults of F. saccatus are reported for the first time in the Brazilian coast.

The invasion of the midgut of the mosquito Culex (Culex) quinquefasciatus Say, 1823 by the helminth Litomosoides chagasfilhoi Moraes Neto, Lanfredi and De Souza, 1997.

The Litomosoides chagasfilhoi helminth was studied as a model for microfilaria invasion of the midgut of Culex quinquefasciatus mosquito, vector of Wuchereria bancrofti helminth, causative agent of the human filariasis. Histology and transmission and scanning electron microscopy were utilized to show the topography of mosquito midgut invasion by the helminth. An analysis of midguts dissected at different time points after a blood meal demonstrated that the microfilariae interacted and crossed the peritrophic matrix and the midgut epithelium of C. quinquefasciatus. The microfilariae invaded preferentially the mosquito abdominal midgut and the invasion process occurred between 2 and 3h after the blood feeding. In some cases, microfilariae caused an opening in the midgut that separated the epithelial cells, while in others cases, the worms caused the detachment of cells from the epithelium. Ultimately, L. chagasfilhoi crossing activity appeared to damage the midgut. It was also observed that the microfilariae lost their sheaths during their passage through the fibrous material of the peritrophic matrix, before they reached the midgut epithelium. Since the exsheathment process is necessary for the continuity of larvae development, it seems that the passage through the peritrophic matrix is an important step for the parasite's life cycle. This experimental model revealed details of the interaction process of helminthes within the vector midgut, contributing to the knowledge of factors involved in the vector competence of C. quinquefasciatus as a vector of filariasis.

Identification and location of albumin-like antigens in third-stage larva of W. bancrofti, in adult forms of Litomosoides chagasfilhoi and in the free-living nematode Caenorhabditis elegans.

Antigens resembling those of host proteins have been identified on the surface of several filarial parasites, such as immunoglobulins and serum albumins. The origin of albumin-like antigens on filarial parasites remains unclear. Several authors suggested that they have been adsorbed, or that they were metabolic waste products from nutritional utilization of human albumin, or perhaps a contamination with human products. This study searched for human albumin-like antigens by Western blot and ultrastructural analyses on filarial parasites, third stage of W. bancrofti and adult females of Litomosoides chagasfilhoi, and on the free-living Caenorhabditis elegans nematode. Our results showed approximately 67kDa proteins recognized by anti-human albumin antibodies on extracts and excretory-secretory (ES) products of the third-stage W. bancrofti. Similar albumin-like proteins were also detected on the filarial parasite L. chagasfilhoi and on C. elegans extracts. The immunocytochemistry analysis showed human albumin-like antigens on similar tissues of these nematodes. These results provide evidence that these proteins have antigenic similarity and similar distribution in nematodes tissues. Our observations suggest that albumin-like antigens presented on filarial parasites are not acquired from the host, but rather are shared antigenic determinants found even in the third-stage larvae recovered from the invertebrate host.

Biological variation between two Brazilian geographical isolates of Echinostoma paraensei.

The biological behaviour and morphometric data from two allopatric isolates of Echinostoma paraensei (Rio Bonito - RB and Sumidouro - SU) collected from naturally infected Nectomys squamipes from two secluded Atlantic Forest fragments were studied. Mice that had been experimentally infected with ten encysted metacercariae of each isolate were monitored weekly in two trials to analyse worm burden and the kinetics of worm distribution along the intestine. The total number of uterine eggs, wet weights and measurements of the worms and body, acetabulum, testes and ovaries were also analysed. The RB isolate showed a higher worm burden, 7.7+/-0.8, and a longer life span, 16 weeks, compared to a worm burden of 5.8+/-1.1 and life span of 9 weeks for the SU isolate. Worms of the RB isolate were clustered in the duodenum and in the bile duct while the SU isolate worms were dispersed along the small intestine of infected mice. Both isolates developed similarly as regards morphometric data and wet weight, although the total number of uterine eggs was greater in RB. The degree of intraspecific variation observed in the worm distribution along the intestine, worm burden and life span raises questions regarding the use of these criteria for species differentiation. These findings suggest that variation in biological parameters found between the E. paraensei isolates could result from geographical isolation and, in particular, the environmental conditions of transmission. Further studies on E. paraensei polulations from different forest fragments will contribute towards an understanding of the speciation of this parasite.

Ultrastructure of Procamallanus (Spirocamallanus) halitrophus (Nematoda: Camallanidae) parasite of flounder.

The ultrastructure of the camallanid nematode Procamallanus (Spirocamallanus) halitrophus, a parasite of flounder, is described for the first time by the use of transmission electron microscopy. The body wall is composed of an outer cuticle, a hypodermis, and a muscular layer. The cuticle comprises the epicuticle, the cortical, median, fibrous, and basal layers. The cortical layer is subdivided into an outer zone and an inner zone; the median layer is subdivided into an outer layer, rich in electrondense fibrils, and an inner layer, which does not contain these fibrils; the fibrous layer is subdivided into three regions delimited by electrondense lines; the basal layer presents electrondense sustaining structures. Underlying the basal layer is the hypodermis where many organelles are observed. The musculature is striated, and each muscle cell consists of individualized contractile and non-contractile regions. Inclusion bodies are present in the muscle fibers, hypodermis, hypodermal chord, and in the intestine.

Further description of Procamallanus (Spirocamallanus) halitrophus comb. n. (Nematoda: Camallanidae) from flounder off the Brazilian coast by light and scanning electron microscopy.

Procamallanus (Spirocamallanus) halitrophus Fusco and Overstreet, 1978 from the intestines of flounders (Syacium papillosum and Citharichthys macrops) from the coast of the State of Rio de Janeiro, Brazil, is redescribed. This study, which is based on results obtained by light and scanning electron microscopy, clarifies many morphological aspects of this species. Some taxonomic features, such as the cephalic structures, the topography of the cuticle, the vulva, the localization of caudal papillae on males, and the excretory pore, are observed for the first time. C. macrops represents a new host record, and Rio de Janeiro is a new geographical record for this species.

Taxonomic reports of Homeacanthoidea (Eucestoda: Trypanorhyncha) in Lamnid and Sphyrnid elasmobranchs collected off the coast of Santa Catarina, Brazil.

Elasmobranch specimens of lamnid and sphyrnid captured in 1999 in the State of Santa Catarina, Brazil, were parasitized with homeacanthoid trypanorhynch cestodes: Isurus oxyrinchus Rafinesque, 1810 with Nybelinia lingualis (Cuvier, 1817) Dollfus, 1929; Sphyrna zygaena (Linnaeus, 1758) with Heteronybelinia rougetcampanae (Dollfus, 1960) Palm, 1999. New details of internal morphology and/or scolex and/or proglottid surface ultrastructure are given. Adults of N. lingualis are reported for the first time in the Brazilian coast.

Morphology of the rediae of Echinostoma paraensei (Trematoda: Echinostomatidae) from its intermediate host Lymnaea columella (Mollusca, Gastropoda).

The morphology of the rediae of Echinostoma paraensei obtained from Lymnaea columella was studied using light, scanning and transmission electron microscopy. The measurements of the mature rediae differ from those described originally, and the taxonomic importance of the ambulatory buds and papilliform process is discussed. Uniciliated papillae were observed in the mouth region. The birth papilla is a bulb-like structure, well defined at the anterior end of the body of the rediae, which opens through a split. There are no microvilli in the tegument surface of the larvae, but numerous tegumental folds, varying according to the contraction of the body of the rediae. The outer syncytial layer is located on a thick basal lamina below which the circular and the longitudinal muscle fiber layers are located.

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.