Redescription of Cercopithifilaria rugosicauda (Böhm & Supperer, 1953) (Spirurida: Onchocercidae) of roe deer, with an emended diagnosis of the genus Cercopithifilaria and a genetic characterisation.

Newly collected material of Cercopithifilaria rugosicauda from roe deer Capreolus capreolus was analysed and compared to descriptions of C. rugosicauda from Austria and Hungary. The present specimens were assigned to the genus Cercopithifilaria using both morphological and molecular analyses. Complementary morphological data on the males and microfilariae of C. rugosicauda were described. The main morphological characters of different species of Cercopithifilaria were outlined and an emended generic diagnosis proposed. A genetic characterisation based on the analyses of cox1 and 12S rDNA sequences was reported supporting that C. rugosicauda was included in the clade of the genus Cercopithifilaria distinctly from other congeneric species available. However, these molecular analyses did not solve the relationships between the species of Cercopithifilaria. These could be approached using morphological characters that might be representative of their evolutionary history. In addition, Wolbachia was not seen in C. rugosicauda, either by immunohistological or by molecular approaches.

Inhibition of cathepsin B by E-64 induces oxidative stress and apoptosis in filarial parasite.

BACKGROUND: Current available antifilarial drug strategies only eliminate the larval stages of filarial parasites. Therefore, there is an urgent need of drugs which are macrofilaricidals. Identification of molecular targets crucial for survival of parasite is a prerequisite for drug designing. Cathepsin B, a cysteine protease family member is known to play crucial role in the normal growth, digestion of nutrients, exsheathment of the helminth parasites. Therefore, we targeted this enzyme in the filarial parasite using its specific inhibitor, E-64. METHODS AND FINDINGS: We have exposed the parasites to E-64 and observed their motility and viability at various time intervals. It caused marked decrease in the motility and viability of the parasites ultimately leading to their death after 8 hours. It is well known that E-64 protects the cell from apoptosis, however, it causes apoptotic effect in carcinoma cell lines. To understand the mechanism of action of E-64 on parasite survival, we have measured levels of different apoptotic markers in the treated parasites. E-64 significantly reduced the level of ced-9 and activity of tyrosine phosphatases, cytochrome c oxidase. It also activated ced-3, homolog of mammalian caspase 3 suggesting initiation of an apoptotic like event in the filarial parasites. Different antioxidant enzymes were also evaluated to further explore the mechanism behind the death of the parasites. There was marked decrease in the level of GSH and activity of Glutathione reductase and glutathione-s-transferase leading to increased generation of reactive oxygen species. This led to the induced oxidation of fatty acids and protein which might alter the mitochondrial membrane permeability. CONCLUSION: This study suggests that inhibition of cathepsin B by E-64 generates oxidative stress followed by mitochondrial mediated apoptotic like event in filarial parasites leading to their death. Hence, suggesting filarial cathepsin B as a potential chemotherapeutic target for lymphatic filariasis.

Species diversity of dermal microfilariae of the genus Cercopithifilaria infesting dogs in the Mediterranean region.

Following the recent description of microfilariae of a Cercopithifilaria sp. in a dog from Sicily, Italy, (herein after referred to as Cercopithifilaria sp. I), numerous skin samples were collected from dogs in the Mediterranean region. In addition to Cercopithifilaria sp. I (185·7 ± 7·2 µm long), microfilariae of 2 other species were identified, namely Cercopithifilaria grassii (651·7 ± 23·6 µm long) and a yet undescribed microfilaria, Cercopithifilaria sp. II (264·4 ± 20·2 µm long, with evident lateral alae). The morphological differentiation among the 3 species of dermal microfilariae was confirmed by differences in cytochrome c oxidase subunit 1 and ribosomal 12S sequences examined (mean level of interspecific pairwise distance of 11·4%, and 17·7%, respectively). Phylogenetic analyses were concordant in clustering these with other sequences of Cercopithifilaria spp. to the exclusion of Dirofilaria spp., Onchocerca spp. and Acanthocheilonema spp. Dermal microfilariae collected (n = 132) were morphologically identified as Cercopithifilaria sp. I (n = 108, 81·8%), Cercopithifilaria sp. II (n = 17, 12·9%), whereas only 7 (5·3%) were identified as C. grassii. Mixed infestations were detected in all sites examined. The great diversity of these neglected filarioids in dogs is of biological interest, considering the complex interactions occurring among hosts, ticks and Cercopithifilaria spp. in different environments.

Evidence for bovine parafilariosis in Italy: first isolation of Parafilaria bovicola (Tubangui, 1934) from autochthonous cattle.

In May and July 2010 and in March 2011, the presence of multiple bleeding nodules at the level of the neck, shoulders, withers, back and rump in five cattle from three herds in north-central Italy were observed. A diagnosis of parafilariosis was made. Fragments of adult females of Parafilaria bovicola could be identified from the derma of three to five bioptic extirpates. Larvated eggs and free larvae were observed in serohemorrhagic exudates. This paper is the first report of the occurrence of parafilariosis in Italy and subsequent to the various outbreaks reported, it can now be said that bovine parafilariosis is also currently present in Italy.

New insights into the evolution of Wolbachia infections in filarial nematodes inferred from a large range of screened species.

BACKGROUND: Wolbachia are intriguing symbiotic endobacteria with a peculiar host range that includes arthropods and a single nematode family, the Onchocercidae encompassing agents of filariases. This raises the question of the origin of infection in filariae. Wolbachia infect the female germline and the hypodermis. Some evidences lead to the theory that Wolbachia act as mutualist and coevolved with filariae from one infection event: their removal sterilizes female filariae; all the specimens of a positive species are infected; Wolbachia are vertically inherited; a few species lost the symbiont. However, most data on Wolbachia and filaria relationships derive from studies on few species of Onchocercinae and Dirofilariinae, from mammals. METHODOLOGY/PRINCIPAL FINDINGS: We investigated the Wolbachia distribution testing 35 filarial species, including 28 species and 7 genera and/or subgenera newly screened, using PCR, immunohistochemical staining, whole mount fluorescent analysis, and cocladogenesis analysis. (i) Among the newly screened Onchocercinae from mammals eight species harbour Wolbachia but for some of them, bacteria are absent in the hypodermis, or in variable density. (ii) Wolbachia are not detected in the pathological model Monanema martini and in 8, upon 9, species of Cercopithifilaria. (iii) Supergroup F Wolbachia is identified in two newly screened Mansonella species and in Cercopithifilaria japonica. (iv) Type F Wolbachia infect the intestinal cells and somatic female genital tract. (v) Among Oswaldofilariinae, Waltonellinae and Splendidofilariinae, from saurian, anuran and bird respectively, Wolbachia are not detected. CONCLUSIONS/SIGNIFICANCE: The absence of Wolbachia in 63% of onchocercids, notably in the ancestral Oswaldofilariinae estimated 140 mya old, the diverse tissues or specimens distribution, and a recent lateral transfer in supergroup F Wolbachia, modify the current view on the role and evolution of the endosymbiont and their hosts. Further genomic analyses on some of the newly sampled species are welcomed to decipher the open questions.

Combination of DEC plus aspirin induced mitochondrial mediated apoptosis in filarial parasite Setaria cervi.

Diethylcarbamazine (DEC) is the main drug used against lymphatic filariasis but it is only microfilaricidal. Hence there is an urgent need for adulticidal drug. Aspirin is known nonsteroidal anti-inflammatory drugs which can inhibit prostaglandin H synthase and also induces apoptosis. Studies presented in this paper demonstrated that exposure of worms to the combination of DEC plus aspirin (DEC + A) at 100 microM concentration irreversibly paralyzed adult worms as well as microfilariae within 2 h. Some of the apoptosis markers viz; DNA fragmentation with accompanying ladder formation, upregulation of Bax expression and decrease in Bcl-2 have suggested that the parasite may be killed due to mitochondrial mediated apoptosis. The levels of several apoptosis regulating proteins and enzymes have also shown to be altered. DEC + A treated worms showed significant decrease in prostaglandin H synthase activity (PGHS) and increase in the level of nitric oxide (NO) and cysteine proteases while glutathione (GSH) and peroxidase level was found to be decreased. NO is known inducer of mitochondrial mediated apoptosis and acts by increasing the permeability of mitochondrial membrane through Bax and allowing cytochrome c to release in cytosol, inducing caspases leading to apoptosis. The DEC + A concentration used in this study is much lower than recommended dose so its intake is safe. Here we report for the first time that combination of DEC and aspirin is more effective and could be used as an adulticidal for control of human filarial infections.

[Ultrastructural basis of interactions between prokaryotes and eukaryotes in different symbiotic models]. / Basi ultrastrutturali delle interazioni tra procarioti ed eucarioti in differenti modelli simbiotici.

This paper reviews the Author's contribution to the knowledge of the ultrastructural basis of the prokaryote-eukaryote interactions in different models assessed by an ultrastructural approach. In agreement with the hypothesis of the origin of eukaryotic cells, which are chimeras of several prokaryotes with different morpho-functional specializations, symbiosis had major consequence for evolution of life. In Arthropods, one of the most successful lifestyles, the presence of endosymbiotic prokaryotes, plays an important role in their metabolism. In some cases, genome integration has occurred in the endosymbiotic relationships with the host, proving that intracellular symbiosis is not merely a nutritional supplement. Intracellular symbiotic bacteria are also described in nematodes. In particular, the presence of intracellular Wolbachia in filariae, even if its function is not yet completely known, influences positively the reproductive biology and the survival of the host, as proved by antibiotic treatment against this bacterium. The ultrastructural images reported in this review were obtained using different species of cockroaches, termites, ticks and filarial nematodes. The traditional methods of transmission (TEM), scansion (SEM) and immuno electron microscopy were used. In addition, also freeze-fracture and deep-etching techniques were employed. The cockroaches and the primitive termite Mastotermes darwiniensis host symbiotic bacteria in the ovary and in specialized cells (bacteriocytes) of the fat body. These bacteria have the typical cell boundary profile of gram-negative bacteria and are enveloped in a vacuolar membrane produced by the host cell. Molecular sequence data of 16S rDNA of endosymbionts of five species of cockroaches and M. darwiniensis indicate that they are members of the Flavobacteria-bacteroides group and that the infection occurred in an ancestor common to cockroaches and termites probably after the end of the Paleozoic (250 Ma BP). The symbiotic bacteria are transmitted transovarially and, during embryogenesis, they are integrated into the morphogenetic processes. In particular, we were able to demonstrate that the origin of the bacteriocyte should be looked for in the cells of the haemocyte line (embryonic plasmatocytes). The eggs are infected by the bacteria emerging from the bacteriocytes of the ovaric fat body and, at the end of the vitellogenesis, they are actively phagocytized by the egg membrane. In filarial nematodes, intracellular bacteria belonging to the genus Wolbachia have been described: they have evolved an obligatory mutualistic association with their host. In fact, antibiotic treatments lead to the clearance of bacteria and this loss produces a negative impact on reproduction and survival of the filarial host. We evidenced, by TEM, the degenerative events occurring during the embriogenesis of Brugia pahangi and Dirofilaria immitis after tetracycline treatment. The data suggest that the Wolbachia play a direct role in worm metabolism. Finally, a new additional model of the prokaryote-eukaryote interaction has been described: we have recently discovered a new intracellular alpha-proteobacterium, named Iric ES1, which resides in the ovarian tissues of the tick Ixodes ricinus. The intriguing characteristic of this bacterium is its ability to invade and consume the ovaric mitochondria. From an evolutionary perspective, it is interesting to note that Iric ES1 enters mitochondria in a similar way to that employed by the "predatory" bacterium Bdellovibrio bacteriovorus.

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.

Wolbachia endosymbiotic bacteria of filarial nematodes. A new insight into disease pathogenesis and control.

Filarial nematodes are parasitic worms that cause some of the most devastating of all tropical diseases such as elephantiasis and river blindness. Studies on the inflammatory pathogenesis of filarial disease have shown that endotoxin-like activity derived from endosymbiotic Wolbachia bacteria is the major inflammatory stimulus of filarial nematodes. Wolbachia appear to have evolved as essential symbionts of their filarial nematode hosts. Antibiotic depletion of bacteria shows that they are required for normal fertility and development of the worm and may even protect the parasites from host immunity. In addition to the uncovering of a fascinating symbiotic relationship, this discovery means we can now consider using antibiotics as a new approach to the treatment of filarial diseases.

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.

Cytomorphology of filariasis revisited. Expansion of the morphologic spectrum and coexistence with other lesions.

OBJECTIVE: To review the cytomorphologic spectrum of the filarial worm and associated tissue response in 33 cases. STUDY DESIGN: Retrospective analysis was carried out in clinically unsuspected cases of filariasis diagnosed on cytology over a period of 10 years. Twenty-nine aspirate smears from 28 patients were air dried and stained with May-Grünwald-Giemsa stain. Four routine cervical smears and one centrifuged smear of urine were stained with Papanicolaou stain. RESULTS: Microfilariae alone and along with adult gravid females were present in 25 and 4 cases, respectively. In one case both adult male and female worms with microfilariae and eggs were seen. The diagnosis was based on the presence of eggs alone in one case and fragments of female worms in two. Four of these cases were neoplastic lesions, and microfilariae were found incidentally. In one case of splenomegaly microfilariae were seen along with Leishman-Donovan bodies. CONCLUSION: Filariasis can be diagnosed on cytology by demonstrating microfilariae, a male or female worm, or eggs alone. It can be seen in association with neoplastic lesions and rarely with other parasitic infections.

Lungworm, Filaroides osleri, infection in a dog in Japan.

A 7-month-old male Pomeranian had severe dyspnea for 2 weeks. The lateral bronchogram showed a stenosis of the trachea. Inspite of supportive therapy including supplemental oxygen, the dog died 5 days later. Six pedunculated nodules were recognized in the mucosal surface of the trachea at necropsy. The tracheal nodules were histopathologically granuloma characterized by many coiled parasites containing a little collagen fibers, lymphocytes, plasma cells and macrophages. Female parasites had a lot of embryonated eggs in the uterus. Immature worms were observed in the dilated lymph vessels of bronchial and bronchiolar wall in the lungs. The worms were identified as Filaroides osleri based on the parasitological examinations.

Laboratory diagnosis of filariasis.

In this article, the authors describe how to detect blood-borne and skin-dwelling filarial infections parasitologically. Considerable space is devoted to identifying and separating microfilarial from nonparasite objects, and the evaluation of morphologic features, including size, shape, and internal anatomy is stressed.

[A new filaria, Chabfilaria jonathani n.gen, n.sp., an onchocercid parasite of edentates]. / Une nouvelle filaire, Chabfilaria jonathani n. gen., n. sp., Onchocercidae parasite de xénarthre.

Chabfilaria jonathani n. gen., n. sp. is described from Choloepus didactylus (family Bradypodidae) from French Guyana. This species and C. freitaslenti (Yeh, 1957) n. comb., constitute a genus interesting because of its primitive characters, which could represent a link between the Onchocercinae and the Setariinae. Eleven species of filaria are known from the Xenarthra. They are primitive members of the Onchocercinae, the Dirofilariinae and if our notions on Chabfilaria are correct, the Setariinae; thus, the three major groups which occur in mammals. The neotropical region with its primitive fauna (marsupials, Xenarthra and protoungulates) may be of particular importance in understanding the origins of the filaria of mammals.