New morphological data of Litomosoides chagasfilhoi (Nematoda: Filarioidea) parasitizing Nectomys squamipes in Rio de Janeiro, Brazil.

Litomosoides chagasfilhoi, originally described by Moraes Neto, Lanfredi & De Souza (1997) parasitizing the abdominal cavity of the wild rodent, Akodon cursor (Winge, 1887), was found in the abdominal cavity of Nectomys squamipes (Brants, 1827), from the municipality of Rio Bonito, Rio de Janeiro State, Brazil. This study led to addition of new morphological data and a new geographical distribution for this filarioid in Brazil. Several characters were detailed and emended to previous records of L. chagasfilhoi in N. squamipes, and confirming the original description in A. cursor: buccal capsule longer than wide with walls thinner than the lumen, right spicule slightly sclerotized, with membranous distal extremity slender, with a small tongue-like terminal portion, left spicule with handle longer than the blade, whose edges form large membranous wings folded longitudinally.

New morphological data of Litomosoides chagasfilhoi (Nematoda: Filarioidea) parasitizing Nectomys squamipes in Rio de Janeiro, Brazil / Novos dados morfológicos de Litomosoides chagasfilhoi (Nematoda: Filarioidea): parasitando Nectomys squamipes no Rio de Janeiro, Brasil

Abstract Litomosoides chagasfilhoi, originally described by Moraes Neto, Lanfredi & De Souza (1997) parasitizing the abdominal cavity of the wild rodent, Akodon cursor (Winge, 1887), was found in the abdominal cavity of Nectomys squamipes (Brants, 1827), from the municipality of Rio Bonito, Rio de Janeiro State, Brazil. This study led to addition of new morphological data and a new geographical distribution for this filarioid in Brazil. Several characters were detailed and emended to previous records of L. chagasfilhoi in N. squamipes, and confirming the original description in A. cursor: buccal capsule longer than wide with walls thinner than the lumen, right spicule slightly sclerotized, with membranous distal extremity slender, with a small tongue-like terminal portion, left spicule with handle longer than the blade, whose edges form large membranous wings folded longitudinally.

Resumo Litomosoides chagasfilhoi, originalmente descrito por Moraes Neto, Lanfredi & De Souza (1997) parasitando a cavidade abdominal do roedor silvestre Akodon cursor (Winge, 1887), foi encontrado na cavidade abdominal de Nectomys squamipes (Brants, 1827), no município de Rio Bonito, Estado do Rio de Janeiro, Brasil. Este estudo propiciou a adição de novos dados morfológicos e uma nova distribuição geográfica deste filarídeo no Brasil. Vários caracteres foram detalhados e adicionados ao registro anterior de L. chagasfilhoi em N. squamipes, e confirmando a descrição original em A. cursor: cápsula bucal mais alta do que larga com paredes mais finas que o lúmen, espículo direito ligeiramente esclerotizado, com extremidade distal membranosa mais estreita, com uma pequena porção terminal em forma de língua, espículo esquerdo com cabo mais longo do que a lâmina, cujas bordas formam grandes asas membranosas dobradas longitudinalmente.

Breinlia (Breinlia) jittapalapongi n. sp. (Nematoda: Filarioidea) from the Asian house rat Rattus tanezumi Temminck in Lao PDR.

A new species of filarioid nematode of the genus Breinlia Yorke & Maplestone, 1926 (Nematoda: Filarioidea) is described from rodents in Lao PDR and according to its morphology, is placed in the subgenus Breinlia. Breinlia (Breinlia) jittapalapongi n. sp. occurs in the Asian house rat (Rattus tanezumi Temminck) and the Sikkim rat (Rattus andamanensis Blyth) and is reported from two localities (Luang Prabang and Champasak). The new species can be distinguished from all other congeners, which are mostly distributed in Australasia (twenty-two species), South East Asia (four species) and India (two species), by the following characters of the males: shape and size of gubernaculum, length of spicules, pattern of cloacal papillae and presence of sclerotised ring in the buccal capsule. This is the fifth species of Breinlia described from South East Asia.

Microscopic and molecular characterization of Hepatozoon domerguei (Apicomplexa) and Foleyella furcata (Nematoda) in wild endemic reptiles from Madagascar.

Madagascar is one of the world's top twelve "megadiversity" hot spots hosting unique and threatened flora and fauna. Parasites are a major component of biodiversity but remain largely uncharacterized in wildlife. In this study we combine microscopic and molecular assessment of hemoparasites in endemic reptile species from Madagascar. We detected three distinct parasites: the apicomplexans Hepatozoon and Sarcocystis, and filarial nematodes. The prevalence and intensity of these apicomplexans were low overall, while microfilarial infections in chameleons were relatively high. We detected mixed infections of two Hepatozoon haplotypes in Madagascarophis colubrinus, and of Hepatozoon and microfilariae in a Furcifer sp. Phylogenetic analyses of Hepatozoon showed evidence of prey-predator transmission, with identical sequences found in the snakes M. colubrinus and Ithycyphus oursi, and their prey Furcifer sp. Based on previous studies regarding the life cycle of Hepatozoon domerguei Landau, Chabaud, Michel, and Brygoo, 1970 in these hosts and due to their morphological similarity, we propose that this Hepatozoon haplotype is Hepatozoon domerguei. Future studies, including the examination of invertebrate hosts, are needed to verify this preliminary taxonomic identification. A distinct hemogregarine haplotype was found in Oplurus sp., which displayed morphologically different gametocytes, some of which were apparently inside leukocytes. The Sarcocystis identified from Tracheloptychus petersi was identical to that reported in a North African snake, indicating that the same lineage is found in geographically distinct regions. By combining morphological and genetic information, Foleyella furcata (Linstow, 1899) filarial nematodes were identified in several Furcifer chameleons. This study provides insights into the distribution, diversity and host-parasite interactions of hemoparasites in wild reptile populations from Madagascar.

Acanthocheilonema delicata n. sp. (Nematoda: Filarioidea) from Japanese badgers (Meles anakuma): description, molecular identification, and Wolbachia screening.

Acanthocheilonema delicata n. sp. (Filarioidea: Onchocercidae: Onchocercinae) is described based on adult filarioids and microfilariae obtained from subcutaneous connective tissues and skin, respectively, of Japanese badgers (Meles anakuma) in Wakayama Prefecture, Japan. No endemic species of the genus had been found in Japan. Recently, some filarioids (e.g., Acanthocheilonema reconditum, Dirofilaria spp., and Onchocerca spp.) have come to light as causative agents of zoonosis worldwide. The new species was readily distinguished from its congeners by morphologic characteristics such as body length, body width, esophagus length, spicule length, and the length of microfilariae. Based on the molecular data of the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene, A. delicata n. sp. was included in the clade of the genus Acanthocheilonema but differed from two other congeneric species available for study, A. viteae and A. reconditum. Acanthocheilonema delicata n. sp. did not harbor Wolbachia. It is likely that the fauna of filarioids from mammals on the Japanese islands is characterized by a high level of endemicity.

Effects of albendazole on Litomosoides chagasfilhoi (Nematoda: Filarioidea) females in vivo.

Gerbils (Meriones unguiculatus) experimentally infected with Litomosoides chagasfilhoi were treated with a single oral dose of 40 or 80 mg of albendazole, respectively. Observation of the microfilaremia after the treatment showed that both single oral doses of albendazole decreased the microfilaremia in L. chagasfilhoi infection. The body wall was composed of a cuticle, a hypodermis, and a muscular layer, and treated nematodes showed no morphological alterations. The ultrastructural alterations produced by treatment with 40 mg of albendazole included a higher number of membrane invaginations in the basal labyrinth of the uterine epithelium and the presence of myelin figures in this region. Inside the uterus, most embryos and microfilariae were disintegrated. The treatment with 80 mg of albendazole did not produce alterations in the uterine wall, and the number of vesicles near the microfilariae sheath was smaller than that observed in the untreated and in the 40-mg treatment groups. However, all the microfilariae observed in the uterus were extensively damaged with cytoplasmic vacuolization and cellular degeneration. No alterations in the intestinal cells were observed after treatment with 40 or 80 mg of albendazole. The present study contributes to the knowledge of albendazole's effects in filariids and demonstrates the potential embryotoxic and microfilaricidal consequences of this drug.

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.

Filaria martis Gmelin 1790 (Spirurida, Filariidae) affecting beech marten (Martes foina): morphological description and molecular characterisation of the cytochrome oxidase c subunit I.

Filaria martis causes a poorly known subcutaneous filariosis in mustelids. Few information is available about lesions that F. martis causes in beech martens, on its morphology, biology and the occurrence of the infection. From 1997 to 2006, 29 beech martens from two sites of southern Italy (Sites A and B) have been necropsied. Ectoparasites and nematodes were collected and morphologically identified. A variable region of the cytochrome c oxidase subunit 1 (cox1) of F. martis has been characterised to compare females presenting caudal tips smooth without spines (i.e. Morphotype 1-Mrph. 1) and with spines (i.e. Mrph. 2). All ticks collected were identified as Haemaphysalis erinacei. Eleven animals from Site A were found infected by F. martis nematodes in subcutaneous tissue in both membranous capsules or free under the inner skin surface. The most important morphological characters of F. martis have been reported and discussed. The molecular analysis showed 100% homology among cox1 sequences from Mrph. 1 and 2 thus indicating that the shape of female posterior edge may vary among specimens of F. martis. The results here presented provide new insights into the biology, ecology and morphological characteristics of this scantly known nematode.

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.

Fine structure and cytochemical analysis of the intestinal wall along the body of adult female of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

Litomosoides chagasfilhoi is a filariid nematode parasite of the abdominal cavity of the wild rodent Akodon cursor (Winge, 1887), that has been described and used in Brazil as a new model for human filariasis. The fine structure of the intestine of this nematode was analyzed based on observations made by light and transmission electron microscopies of serial sections along the body. Cytochemical analysis was carried out to investigate the composition of the intestinal wall. This structure consisted of a basal lamina and an epithelium of variable thickness, composed of cells that have an irregular shape. The cytoplasm of intestinal cells contains few organelles: vacuoles, lysosomal bodies, spheroid bodies, endoplasmic reticulum, and many large lipid droplets. In the anterior portion of the intestine, the lysosomal bodies, spheroid bodies, and vacuoles presented positive reaction for acid phosphatase, and carbohydrates were detected in lysosomal bodies. The midbody and posterior regions presented less organelles and lipid droplets, and nuclei were more abundant. Residues of L-fucose were detected by Ulex europaeus lectin binding in the midbody sections. Basic proteins were associated to lipid droplets, in the posterior region. In the whole extension of the intestine, carbohydrates were detected on tight junctions. These results indicate that the metabolized material in the epithelium can contribute to the microfilariae development and also probably can be involved with the excretory/secretory mechanism of these nematodes.

Trans-species transfer of Wolbachia: microinjection of Wolbachia from litomosoides sigmodontis into Acanthocheilonema viteae.

Intracellular bacteria of the genus Wolbachia are found in most filarial nematodes, but are lacking in some species like Acanthocheilonema viteae. Due to their symbiotic nature and their role in the pathology of filarial infections they are considered to be potential targets for intervention against filarial infections in man. Infection of A. viteae (a species which does not naturally carry Wolbachia) with Wolbachia bacteria could allow comparative studies on the effect of the endobacterium on the parasite and on the host's immune systems. As a step towards such studies we microinjected adult female A. viteae with Wolbachia obtained from Litomosoides sigmodontis. The bacteria were isolated from L. sigmodontis by density-gradient centrifugation, microinjected into A. viteae worms and bacterial DNA detected by PCR with Wolbachia specific primers (ftsZ gene). Microinjected worms were cultured in vitro, and 81% survived for 10 days. Implantation of microinjected worms into Meriones unguiculatus, the rodent host of A. viteae resulted in 38% survival. The DNA of the microinjected worms recovered from jirds 8 weeks after implantation contained Wolbachia DNA as shown by PCR, suggesting that Wolbachia of L. sigmodontis can be horizontally transmitted to A. viteae.

Further studies on the structural analysis of the cuticle of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

In order to obtain further information on the structural organization of the cuticle of nematodes, this structure was isolated from adult forms of the filariid Litomosoides chagasfilhoi. The purity of the fraction was determined by light and transmission electron microscopy, deep-etching, high resolution scanning electron microscopy, atomic force microscopy, immunocytochemistry, gel electrophoresis (SDS-PAGE) and Western blot. The epicuticle presented a rugous surface with parallel rows and several globular particles that could be involved in the absorption of nutrients and secretion of products. Analysis by SDS-PAGE of purified cuticles revealed five major polypeptides corresponding to 151, 41, 28, 13 and 11 kDa. A polyclonal antibody against a synthetic 18 amino-acid peptide that corresponds to the sequence of domain E of the Haemonchus contortus3A3 collagen gene recognized several protein bands on the Western blot of purified cuticle, and labeled all cuticular layers, as shown by immunocytochemistry.

Deep-etched view of the cuticle of adults of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

The fine structure of the cuticle of the filariid nematode Litomosoides chagasfilhoi is described, based on observations made by transmission electron microscopy and deep-etched replicas. The cuticle consists of a trilaminate epicuticle, the outermost layer that interfaces with the host and four other layers: cortical, intermediate, fibrous and basal. In deep-etched replicas, the cortical layer is formed by a meshwork of globular particles and fibers with a thickness of 4-8 nm. The intermediate layer is electron-lucid and contains a densely-stained line. In deep-etched replicas, it is composed by a meshwork of fibers with longitudinal orientation. The fibrous layer is the thickest and most electron-dense and consists of two types of fibers: thick (9-26 nm) and thin (3-6 nm). The innermost basal layer is intimately associated with the hypodermis. In common with other nematodes, the cuticle of L. chagasfilhoi presents channels that are probably involved in nutrient acquisition and transport.

[Redescription of Litomosoides brasiliensis Almeida 1936 (Nematoda: Filariiidae) from a new host Anoura caudifera (Chiroptera: Phyllostomidae)]. / Redescrição de Litomosoides brasiliensis Almeida, 1936 (Nematoda: Filariidae) Parasito de Anoura caudifera (Chiroptera: Phyllostomidae).

The study of the surface topography added details regarding the disposition of male caudal papillae, spicules and area rugosa apart from vulva and oral aperture. The occurrence of this nematode in the state of Amapá represents a new geographical distribution.

Coexistence of five Cercopithifilaria species in the Japanese rupicaprine bovid, Capricornis crispus.

The Japanese serow, Capricornis crispus (Bovidae, Caprinae, Rupicaprini), is parasitized by five Cercopithifilaria species: C. shohoi, recently described, and reexamined in this paper, C. multicauda n. sp., C. minuta n. sp., C. tumidicervicata n. sp., and C. bulboidea n. sp. Coinfections are frequent. The location (skin or subcutaneous regions) in the host of adult worms differed between the species, as did many morphological characters of both adults and microfilariae. The location (limbs, trunk, etc.) in the host of adult worms and dermal microfilariae seemed to differ depending on the species. Male and female worms of the same species had similar head shapes, buccal capsules, and, in four species, swellings in the anterior region of the body (because of the presence of a giant ventral pseudocoelomocyte). The Cercopithifilaria spp. from C. crispus were related to the primitive forms of the genus, parasites of Bovidae and Cervidae, presently recorded in Africa and Europe. C. bulboidea was particularly close to the most primitive species, C. ruandae and C. dermicola, in Africa, with the pairs of caudal papillae numbered 8 and 9 being distant from each other, but the species also had several specialized characters. Like C. rugosicauda in a European cervid, the four other species had pairs 8 and 9 close to each other; they reflect an evolutionary trend (hypertrophy of pair 6, and reduction and posterior migration of pair 7) that suggests diversification in the host.

Fine structure, freeze-fracture and deep-etch views of the sheath and cuticle of microfilariae of Litomosoides chagasfilhoi (Nematoda: Filarioidea).

The fine structure of the sheath and the cuticle of microfilariae of the filariid Litomosoides chagasfilhoi is described based on observations made using transmission electron microscopy (TEM) and especially on deep-etched replicas of fully developed intrauterine microfilariae and mature stretched microfilariae released by adult females through cultivation in vitro. TEM showed that the sheath was trilaminated. In contrast, in deep-etching replicas the sheath presented two layers: an inner layer composed of tightly arranged globular material, and an outer layer whose external surface was relatively smooth. Both in thin sections and in classical freeze-fracture and deep-etched replicas, the cuticle presented two distinct regions: an external one, corresponding to the trilaminated epicuticle, and an inner one, corresponding to the inner cuticle. Deep-etching replicas revealed that the epicuticle presented several structures on the annulations of the microfilariae and that the inner region was composed by two parallel rows of globular structures.

Redescription of Parafilaria bovicola Tubangui, 1934 (Nematoda: Filarioidea) from Swedish cattle.

Parafilaria bovicola was introduced into Sweden and identified for the first time in Swedish cattle in 1978. Since then the parasite has become well established and has been responsible for substantial economic losses in Swedish beef production. Although studies have been undertaken on serodiagnosis, life cycle, vector and geographical distribution within Sweden this is the first full description of P. bovicola from Swedish cattle and the first study to use scanning electron microscopy. P. bulgarica Daskalov, 1944 is regarded as a synonym of P. bovicola Tubangui, 1934. Tooth-like thickenings described on the anterior lobes of the oesophagus for the first time may assist the parasite in its migration through the host tissues.

Cercopithifilaria shohoi n. sp. (Nematoda: Filarioidea) from the relict Bovidae, Capricornis crispus, in Japan.

Cercopithifilaria shohoi n. sp. was found in the relict bovid, Capricornis crispus, in Japan, and is described and compared with other species in the genus. Adult male and female worms were found in subcutaneous tissues of the trunks of 6 serows shot in Mt. Zao, Yamagata Prefecture, in the northern part of Honshu, the largest island of Japan. The one complete male found was 19.7 mm long, and the five females were 31.6-50.9 mm long. Unsheathed or sheathed microfilariae 104-122 microns long were taken from the females. One microfilaria was found in the sediment of the preservation solution of the tissues, but none were found in the blood of the infected serows, so microfilariae may be limited to the skin. Males of this species had one pair of papillae between perianal and subterminal groups of caudal papillae. In having this intermediate pair, C. shohoi n. sp. resembled species such as C. faini from an African bovid and C. rugosicauda from a European deer. From its morphological characteristics, C. shohoi n. sp. seems to be one of the more primitive species in the genus Cercopithifilaria.

Litomosoides chagasfilhoi sp. nov. (Nematoda:Filarioidea) parasitizing the abdominal cavity of Akodon cursor (Winge, 1887) (Rodentia:Muridae) from Brazil.

Litomosoides chagasfilhoi sp. nov., a parasite of the abdominal cavity of the wild rodent Akodon cursor (Winge. 1887), is described herein according to investigations conducted by light and scanning electron microscopy. The leading morphological characteristics of the new species are as follows: the buccal capsule is higher than it is wide and has walls thinner than the lumen, and the left spicule presents a handle longer than the blade, whose edges from large membranous wings folded longitudinally. This new species is different from L. silvai Padilha and Faria, 1977, living in the thoracic cavity of the same host.