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.

Intraperitoneal development of the filarial nematode Brugia malayi in the Mongolian jird (Meriones unguiculatus).

In the present study, we describe intraperitoneal development of the FR3 strain of Brugia malayi in Mongolian jirds (Meriones unguiculatus). The third molt for male worms occurred between 4 and 7 days postinfection (dpi) and between 4 and 8 dpi for females. The fourth and final molt occurred between days 21 and 29 for males and 25 and 34 for females, considerably earlier than the times reported for subcutaneous infection models using cats and jirds. The timing of the third molt coincided largely with reports for subcutaneous Brugia pahangi infections of cats and jirds, but the final molt occurred considerably later and lasted longer than those reported for subcutaneous B. pahangi models. Spermatogenesis occurred by at least 50 dpi in adult males, and insemination of females likely occurred between 50 and 60 dpi. Microfilariae were observed in the uteri and ovejectors of adult females at 65 dpi.

Filarial parasites develop faster and reproduce earlier in response to host immune effectors that determine filarial life expectancy.

Humans and other mammals mount vigorous immune assaults against helminth parasites, yet there are intriguing reports that the immune response can enhance rather than impair parasite development. It has been hypothesized that helminths, like many free-living organisms, should optimize their development and reproduction in response to cues predicting future life expectancy. However, immune-dependent development by helminth parasites has so far eluded such evolutionary explanation. By manipulating various arms of the immune response of experimental hosts, we show that filarial nematodes, the parasites responsible for debilitating diseases in humans like river blindness and elephantiasis, accelerate their development in response to the IL-5 driven eosinophilia they encounter when infecting a host. Consequently they produce microfilariae, their transmission stages, earlier and in greater numbers. Eosinophilia is a primary host determinant of filarial life expectancy, operating both at larval and at late adult stages in anatomically and temporally separate locations, and is implicated in vaccine-mediated protection. Filarial nematodes are therefore able to adjust their reproductive schedules in response to an environmental predictor of their probability of survival, as proposed by evolutionary theory, thereby mitigating the effects of the immune attack to which helminths are most susceptible. Enhancing protective immunity against filarial nematodes, for example through vaccination, may be less effective at reducing transmission than would be expected and may, at worst, lead to increased transmission and, hence, pathology.

CCL17 controls mast cells for the defense against filarial larval entry.

Filarial parasites have to trespass many barriers to successfully settle within their mammalian host, which is equipped with mechanical borders and complex weaponry of an evolved immune system. However, little is known about mechanisms of early local events in filarial infections. In this study, bone marrow-derived dendritic cells not only upregulated activation markers CD40 and CD80 upon in vitro stimulation with filarial extracts, but also secreted CCL17, a chemokine known to be produced upon microbial challenge. Mice deficient for CCL17 had an up to 4-fold higher worm burden compared with controls by day 10 of infection with the murine filaria Litomosoides sigmodontis. Also, numbers of mast cells (MCs) invading the skin and degranulation were significantly increased, which was associated with enhanced vascular permeability and larval establishment. This phenotype was reverted by inhibition of MC degranulation with disodium cromoglycate or by blockade of histamine. In addition, we showed that CCL17-mediated vascular permeability was dependent on the presence of Wolbachia endosymbionts and TLR2. Our findings reveal that CCL17 controls filarial larval entry by limiting MC-dependent vascular permeability.

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.

A new species of Rhabdias Stiles et Hassall, 1905 (Nematoda: Rhabdiasidae) from Blommersia domerguei (Guibé) (Amphibia: Mantellidae) in Madagascar.

Rhabdias blommersiae sp. n. (Nematoda: Rhabdiasidae) is described from the lungs of Domergue's Madagascar frog, Blommersia domerguei (Guibé) (Amphibia: Mantellidae), in Madagascar. The new species differs from congeners parasitizing amphibians in having a smaller body and buccal capsule, six equal lips, large excretory glands of unequal length and a posteriorly inflated body vesicle. A combination of characters distinguishes it from Afromalagasy species of Rhabdias Stiles et Hassall, 1905. Rhabdias blommersiae is the third species of the genus described from amphibians in Madagascar. Close similarities in the number and shape of circumoral structures in two Rhabdias species described from mantellid hosts in Madagascar suggest a close relationship and common origin of the two species, with subsequent adaptation to separate hosts within the Mantellidae.

Tracking the vector of Onchocerca lupi in a rural area of Greece.

During a hot Mediterranean summer, an expedition brought parasitologists from Brazil, France, Greece, Italy, and Serbia to a wooded area near Xanthi, Thrace, northeastern Greece, near the Turkish border, on the track of the vector of the little-known nematode Onchocerca lupi. The scientific purposes of the expedition blended then with stories of humans, animals, and parasites in this rural area.

An assessment of genetic variability in the mitochondrial cytochrome c oxidase subunit 1 gene of Cercopithifilaria sp. (Spirurida, Onchocercidae) from dog and Rhipicephalus sanguineus populations.

This study investigates sequence variation in mitochondrial cytochrome c oxidase subunit 1 gene within Cercopithifilaria sp. recorded recently in Italy. Fourteen sequence types (haplotypes) were characterized for 163 (7.7%) amplicons from 2111 Genomic DNA samples prepared from skin samples from dogs and from Rhipicephalus sanguineus (ticks) from different geographical areas of the Mediterranean basin (i.e., Italy, Spain and Greece). The most prevalent sequence types represented haplotypes I (70.5%) and X (16.0%), followed by haplotype VIII (4.9%) and other 11 haplotypes (8.6%). Three haplotypes (II, V and VI) were found exclusively in ticks. The overall intraspecific nucleotide variation among pcox1 haplotypes ranged from 0.4 to 3.5% (mean = 1.6%), whereas a mean interspecific difference of 9.5% was detected as compared with other onchocercids. Phylogenetic analysis of the nucleotide sequence data showed a clustering of Cercopithifilaria sp. with the other Cercopithifilaria species (with strong statistical support) to the exclusion of other onchocercids. The number of haplotypes identified here might be explained by complex ecology and transmission patterns as well as the high mutation rate of mitochondrial DNA and/or inbreeding associated with hosts and their vectors.

New insights into the ecology and biology of Acanthocheilonema reconditum (Grassi, 1889) causing canine subcutaneous filariosis.

In spite of its wide distribution among dogs and the evidence of its implication as a zoonotic agent, scant information is available on the biology of Acanthocheilonema reconditum (Spirurida, Onchocercidae). In this study, blood samples from 152 Sicilian dogs were examined for A. reconditum microfilariae at the beginning of the study and 1 year later. The periodicity of microfilaraemia was investigated by bleeding 2 highly microfilaraemic dogs twice a day for 10 days and, later on, every 2 weeks for 1 year and a third animal every 3 h for 96 h. Fleas and ticks infesting dogs were collected and dissected for the detection of A. reconditum larvae. The prevalence of infestation was 11·2% (17/152) and 13·3% (16/120) at the beginning and at the end of the study, with a 1 year cumulative incidence of 5·9%. Although dogs bled twice a day showed a higher number of microfilariae in most of the morning samples, the absence of any circadian rhythm was suggested by data of the third experiment conducted by bleeding a dog every 3 h for 4 days. A. reconditum developing forms were detected in 5·1% (4/78) of dissected fleas, but not in any of the 272 ticks. The study provides new insights into the biology and ecology of this dog filarioid in its definitive and intermediate hosts.

Human intraocular filariasis caused by Dirofilaria sp. nematode, Brazil.

A case of human intraocular dirofilariasis is reported from northern Brazil. The nematode was morphologically and phylogenetically related to Dirofilaria immitis but distinct from reference sequences, including those of D. immitis infesting dogs in the same area. A zoonotic Dirofilaria species infesting wild mammals in Brazil and its implications are discussed.

Human intraocular filariasis caused by Pelecitus sp. nematode, Brazil.

A male nematode was extracted from iris fibers of a man from the Brazilian Amazon region. This nematode belonged to the genus Pelecitus but was distinct from the 16 known species in this genus. Similarities with Pelecitus spp. from neotropical birds suggested an avian origin for this species.

A worm's best friend: recruitment of neutrophils by Wolbachia confounds eosinophil degranulation against the filarial nematode Onchocerca ochengi.

Onchocerca ochengi, a filarial parasite of cattle, represents the closest relative of the human pathogen, Onchocerca volvulus. Both species harbour Wolbachia endosymbionts and are remarkable in that adult female worms remain viable but sessile for many years while surrounded by host cells and antibodies. The basis of the symbiosis between filariae and Wolbachia is thought to be metabolic, although a role for Wolbachia in immune evasion has received little attention. Neutrophils are attracted to Wolbachia, but following antibiotic chemotherapy they are replaced by eosinophils that degranulate on the worm cuticle. However, it is unclear whether the eosinophils are involved in parasite killing or if they are attracted secondarily to dying worms. In this study, cattle infected with Onchocerca ochengi received adulticidal regimens of oxytetracycline or melarsomine. In contrast to oxytetracycline, melarsomine did not directly affect Wolbachia viability. Eosinophil degranulation increased significantly only in the oxytetracycline group; whereas nodular gene expression of bovine neutrophilic chemokines was lowest in this group. Moreover, intense eosinophil degranulation was initially associated with worm vitality, not degeneration. Taken together, these data offer strong support for the hypothesis that Wolbachia confers longevity on O. ochengi through a defensive mutualism, which diverts a potentially lethal effector cell response.

A new species of Litomosoides (Nematoda: Onchocercidae), parasite of Nectomys palmipes (Rodentia: Cricetidae: Sigmodontinae) from Venezuela: description, molecular evidence, Wolbachia pipientis screening.

Abstract: The onchocercid filaria Litomosoides taylori sp. n. is described from the sigmodontine cricetid Nectomys palmipes Allen et Chapman in northeast Venezuela. A voucher specimen of the new species was used for molecular analysis of the coxI and 12S rDNA genes, and screened for the presence of the endobacterium Wolbachia pipientis. Litomosoides taylori belongs to the "sigmodontis group" of Litomosoides and a combination of characters can be used to distinguish it from the remaining 18 species forming this group. Among the five Nectomys species, all living near running water, N. squamipes also harbours Litomosoides species, L. khonae in Brazil and L. navonae in Argentina. These three Litomosoides species of the "sigmodontis group" do not share any particular characters. Gene sequences of L. taylori differ from those of the five Litomosoides species available, the three of the "carinii group" being the most distant. The new species harbours W pipientis, which is concurrent with the great majority of Litomosoides species screened to date.

[Nematodes of the genus Oesophagostomum: an emerging risk for humans and apes in Africa?]. / Les nématodes du genre Oesophagostomum. Un risque émergent pour l'homme et les grands singes en Afrique?

Nematodes of the genus Oesophagostomum are common intestinal parasites found in cattle, pigs and primates. They can cause severe illness, resulting from the formation of granulomas, caseous lesions and abscesses in the intestinal wall. Human oesophagostomosis is endemic in northern Ghana and Togo. In these regions, epidemiological investigations have been conducted to determine the biological characteristics, transmission dynamics and optimal management of clinical cases. Nodular oesophagostomosis has also been described in free-ranging chimpanzees and gorillas. Clinical signs associated with nodules have been observed in great apes raised in sanctuaries, while the health status of their wild counterparts does not seem to be significantly affected It has been suggested that some nonhuman primates may act as reservoirs for human oesophagostomosis. In Ghana, identification of genetic differences among Oesophagostomum nematodes infecting different primate hosts suggests that oesophagostomosis is a rare zoonosis. In Uganda, where the situation is diferent, cross-infection is probably more frequent.

Early recruitment of natural CD4+ Foxp3+ Treg cells by infective larvae determines the outcome of filarial infection.

Human helminth infections are synonymous with impaired immune responsiveness indicating suppression of host immunity. Using a permissive murine model of filariasis, Litomosoides sigmodontis infection of inbred mice, we demonstrate rapid recruitment and increased in vivo proliferation of CD4(+)Foxp3(+) Treg cells upon exposure to infective L3 larvae. Within 7 days post-infection this resulted in an increased percentage of CD4(+)T cells at the infection site expressing Foxp3. Antibody-mediated depletion of CD25(+) cells prior to infection to remove pre-existing 'natural' CD4(+)CD25(+)Foxp3(+) Treg cells, while not affecting initial larval establishment, significantly reduced the number of adult parasites recovered 60 days post-infection. Anti-CD25 pre-treatment also impaired the fecundity of the surviving female parasites, which had reduced numbers of healthy eggs and microfilaria within their uteri, translating to a reduced level of blood microfilaraemia. Enhanced parasite killing was associated with augmented in vitro production of antigen-specific IL-4, IL-5, IL-13 and IL-10. Thus, upon infection filarial larvae rapidly provoke a CD4(+)Foxp3(+) Treg-cell response, biasing the initial CD4(+) T-cell response towards a regulatory phenotype. These CD4(+)Foxp3(+) Treg cells are predominantly recruited from the 'natural' regulatory pool and act to inhibit protective immunity over the full course of infection.

New species of Rhabdias (Nematoda: Rhabdiasidae) from afrotropical anurans, including molecular evidence and notes on biology.

Despite the small sample size the diversity of Rhabdias Stiles et Hassall, 1905 from anurans in the Afrotropical region was found to be high. Four species were collected from four localities, one in South Africa, two on Cameroonese mountains and one in Madagascar: Rhabdias picardiae sp. n. from the bufonid Amietophrynus gutturalis (Power); Rhabdias ohlerae sp. n. and Rhabdias tanyai sp. n. from the arthroleptids Leptopelis brevirostris (Werner) and Astylosternus rheophilus Amiet, respectively; and Rhabdias vencesi sp. n. from the mantellid Boophis madagascariensis (Peters). Distinctive characters between these species are numerous and obvious, based on body size, shape and size of the buccal capsule, arrangement of head papillae, and shape and size of the oesophagus and intestinal apex. Molecular data based on 500 bp of 12S rDNA and 600 bp of coxl of three of the four species are presented. Rhabdias vencesi resembles Rhabdias madagascariensis Chabaud, Brygoo et Petter, 1961 from an African ptychadenid introduced on Madagascar, but differs in body size and head morphology. The remaining new species are clearly distinct from those previously known from Afrotropical anurans. Outside the Afrotropics, some Rhabdias species present characters similar to those observed in the new species, but they all differ in various other characters. No clear correlation was seen between Rhabdias species and families of anuran hosts in this region. However, the narrow buccal capsule seen in Rhabdias species from Afrotropical lissamphibians opposes them to the majority of Rhabdias parasitic in chamaeleonids. Furthermore, the infective larva of R. vencesi has a conical pointed tail, while those of Rhabdias from chameleons have a rounded tail tip ornated with a few buds.

Adjuvant-free immunization with infective filarial larvae as lymphatic homing antigen carriers.

Controlled infection with intestinal nematodes has therapeutic potential for preventing the symptoms of allergic and autoimmune diseases. Here, we engineered larvae of the filarial nematode Litomosoides sigmodontis as a vaccine strategy to induce adaptive immunity against a foreign, crosslinked protein, chicken egg ovalbumin (OVA), in the absence of an external adjuvant. The acylation of filarial proteins with fluorescent probes or biotin was not immediately detrimental to larval movement and survival, which died 3 to 5 days later. At least some of the labeled and skin-inoculated filariae migrated through lymphatic vessels to draining lymph nodes. The immunization potential of OVA-biotin-filariae was compared to that of an OVA-bound nanoparticulate carrier co-delivered with a CpG adjuvant in a typical vaccination scheme. Production of IFNγ and TNFα by restimulated CD4+ cells but not CD8+ confirmed the specific ability of filariae to stimulate CD4+ T cells. This alternative method of immunization exploits the intrinsic adjuvancy of the attenuated nematode carrier and has the potential to shift the vaccination immune response towards cellular immunity.

Integrated taxonomy: traditional approach and DNA barcoding for the identification of filarioid worms and related parasites (Nematoda).

BACKGROUND: We compared here the suitability and efficacy of traditional morphological approach and DNA barcoding to distinguish filarioid nematodes species (Nematoda, Spirurida). A reliable and rapid taxonomic identification of these parasites is the basis for a correct diagnosis of important and widespread parasitic diseases. The performance of DNA barcoding with different parameters was compared measuring the strength of correlation between morphological and molecular identification approaches. Molecular distance estimation was performed with two different mitochondrial markers (coxI and 12S rDNA) and different combinations of data handling were compared in order to provide a stronger tool for easy identification of filarioid worms. RESULTS: DNA barcoding and morphology based identification of filarioid nematodes revealed high coherence. Despite both coxI and 12S rDNA allow to reach high-quality performances, only coxI revealed to be manageable. Both alignment algorithm, gaps treatment, and the criteria used to define the threshold value were found to affect the performance of DNA barcoding with 12S rDNA marker. Using coxI and a defined level of nucleotide divergence to delimit species boundaries, DNA barcoding can also be used to infer potential new species. CONCLUSION: An integrated approach allows to reach a higher discrimination power. The results clearly show where DNA-based and morphological identifications are consistent, and where they are not. The coherence between DNA-based and morphological identification for almost all the species examined in our work is very strong. We propose DNA barcoding as a reliable, consistent, and democratic tool for species discrimination in routine identification of parasitic nematodes.

Inherent biomechanical traits enable infective filariae to disseminate through collecting lymphatic vessels.

Filariases are diseases caused by arthropod-borne filaria nematodes. The related pathologies depend on the location of the infective larvae when their migration, the asymptomatic and least studied phase of the disease, comes to an end. To determine factors assisting in filariae dissemination, we image Litomosoides sigmodontis infective larvae during their escape from the skin. Burrowing through the dermis filariae exclusively enter pre-collecting lymphatics by mechanical disruption of their wall. Once inside collectors, their rapid and unidirectional movement towards the lymph node is supported by the morphology of lymphatic valves. In a microfluidic maze mimicking lymphatic vessels, filariae follow the direction of the flow, the first biomechanical factor capable of helminth guidance within the host. Finally, non-infective nematodes that rely on universal morpho-physiological cues alone also migrate through the dermis, and break in lymphatics, indicating that the ability to spread by the lymphatic route is an ancestral trait rather than acquired parasitic adaptation.

Pleural cellular reaction to the filarial infection Litomosoides sigmodontis is determined by the moulting process, the worm alteration, and the host strain.

The filarial nematode Litomosoides sigmodontis model was used to decipher the complex in vivo relationships between filariae, granulomas and leukocytes in the host's pleural cavity. The study was performed from D5 p.i.: to D47 p.i. in resistant C57BL/6 mice, to D74 p.i. in susceptible BALB/c mice, and to D420 p.i. in permissive jirds. We showed that, during the first month, leukocytes only clustered as granulomas around shed cuticles (exuviae) and with eosinophils as the major constituents. In addition, carbohydrates residues became abundant on exuviae only, suggesting a glycan-dependent mechanism of eosinophil attachment. Neutrophils were absent from the pleural cavity of all rodents and from the murine granulomas, but they made up 25% of the granuloma cell population in jirds. After the first month of infection granulomas formed around developed adult worms and morphological evidence suggested that leukocytes preferentially clustered around altered, but still motile, worms. No carbohydrates were detected on these worms and neutrophils were abundant in those granulomas. Finally, a rare third type of granuloma was observed in the resistant mice only; they contained young newly moulted adult worms; typically these granulomas were attached to the lateral lines of the worm via eosinophils; this feature correlated with the persistence of carbohydrate residues on the worms' lateral lines. Neutrophils were always in low proportion in all granulomas from resistant mice, suggesting difference in their adhesive properties in these mice. In vitro neutrophil recruitment in resistant mice was similar to that observed in susceptible mice although they expressed less cell surface CD11b.