Filarial Hosts Research Articles

Molecular detection of Wolbachia endosymbiont in reptiles and their ectoparasites

Wolbachia, a maternally transmitted Gram-negative endosymbiont of onchocercid nematodes and arthropods, has a role in the biology of their host; thus it has been exploited for the filariasis treatment in humans. To assess the presence and prevalence of this endosymbiont in reptiles and their ectoparasites, blood and tail tissue as well as ticks and mites collected from them were molecularly screened for Wolbachia DNA using two sets of primers targeting partial 16S rRNA and Wolbachia surface protein (wsp) genes. Positive samples were screened for the partial 12S rRNA and cytochrome c oxidase subunit 1 (cox1) genes for filarioids. Of the different species of lizards (Podarcis siculus, Podarcis muralis and Lacerta bilineata) and snakes (Elaphe quatuorlineata and Boa constrictor constrictor) screened from three collection sites, only P. siculus scored positive for Wolbachia 16S rRNA. Among ectoparasites collected from reptiles (Ixodes ricinus ticks and Neotrombicula autumnalis, Ophionyssus sauracum and Ophionyssus natricis mites), I. ricinus (n = 4; 2.8%; 95% CI, 0.9–7) from P. siculus, N. autumnalis (n = 2 each; 2.8%; 95% CI, 0.9–6.5) from P. siculus and P. muralis and O. natricis (n = 1; 14.3%; 95% CI, 0.7–55.4) from Boa constrictor constrictor scored positive for Wolbachia DNA. None of the positive Wolbachia samples scored positive for filarioids. This represents the first report of Wolbachia in reptilian hosts and their ectoparasites, which follows a single identification in the intestinal cells of a filarioid associated with a gecko. This data could contribute to better understand the reptile filarioid-Wolbachia association and to unveil the evolutionary pattern of Wolbachia in its filarial host.

A rapid, parasite-dependent cellular response to Dirofilaria immitis in the Mongolian jird (Meriones unguiculatus)

BackgroundThe Mongolian jird (Meriones unguiculatus) has long been recognized as a permissive host for the filarial parasite Brugia malayi; however, it is nonpermissive to another filarial parasite, canine heartworm (Dirofilaria immitis). By elucidating differences in the early response to infection, we sought to identify mechanisms involved in the species-specific clearance of these parasites. We hypothesized that the early clearance of D. immitis in intraperitoneal infection of the jird is immune mediated and parasite species dependent.MethodsJird peritoneal exudate cells (PECs) were isolated and their attachment to parasite larvae assessed in vitro under various conditions: D. immitis and B. malayi cultured separately, co-culture of both parasites, incubation before addition of cells, culture of heat-killed parasites, and culture with PECs isolated from jirds with mature B. malayi infection. The cells attaching to larvae were identified by immunohistochemistry.ResultsIn vitro cell attachment to live D. immitis was high (mean = 99.6%) while much lower for B. malayi (mean = 5.56%). This species-specific attachment was also observed when both filarial species were co-cultured, with no significant change from controls (U(9, 14) = 58.5, p = 0.999). When we replicated these experiments with PECs derived from jirds subcutaneously infected with B. malayi, the results were similar (99.4% and 4.72% of D. immitis and B. malayi, respectively, exhibited cell attachment). Heat-killing the parasites significantly reduced cell attachment to D. immitis (mean = 71.9%; U(11, 14) = 7.5, p < 0.001) while increasing attachment to B. malayi (mean = 16.7%; U(9, 15) = 20, p = 0.002). Cell attachment to both species was reduced when larvae were allowed a 24-h pre-incubation period prior to the addition of cells. The attaching cells were identified as macrophages by immunohistochemistry.ConclusionsThese results suggest a strongly species-dependent response from which B. malayi could not confer protection by proxy in co-culture. The changes in cell attachment following heat-killing and pre-incubation suggest a role for excretory/secretory products in host immune evasion and/or antigenicity. The nature of this attachment is the subject of ongoing study and may provide insight into filarial host specificity.Graphical

Description, molecular characteristics and Wolbachia endosymbionts of Onchocerca borneensis Uni, Mat Udin & Takaoka n. sp. (Nematoda: Filarioidea) from the Bornean bearded pig Sus barbatus M\xfcller (Cetartiodactyla: Suidae) of Sarawak, Malaysia

BackgroundThe genus Onchocerca Diesing, 1841 includes species of medical importance, such as O. volvulus (Leuckart, 1893), which causes river blindness in the tropics. Recently, zoonotic onchocercosis has been reported in humans worldwide. In Japan, O. dewittei japonica Uni, Bain & Takaoka, 2001 from wild boars is a causative agent for this zoonosis. Many filarioid nematodes are infected with Wolbachia endosymbionts which exhibit various evolutionary relationships with their hosts. While investigating the filarial fauna of Borneo, we discovered an undescribed Onchocerca species in the bearded pig Sus barbatus Müller (Cetartiodactyla: Suidae).MethodsWe isolated Onchocerca specimens from bearded pigs and examined their morphology. For comparative material, we collected fresh specimens of O. d. dewittei Bain, Ramachandran, Petter & Mak, 1977 from banded pigs (S. scrofa vittatus Boie) in Peninsular Malaysia. Partial sequences of three different genes (two mitochondrial genes, cox1 and 12S rRNA, and one nuclear ITS region) of these filarioids were analysed. By multi-locus sequence analyses based on six genes (16S rDNA, ftsZ, dnaA, coxA, fbpA and gatB) of Wolbachia, we determined the supergroups in the specimens from bearded pigs and those of O. d. dewittei.ResultsOnchocerca borneensis Uni, Mat Udin & Takaoka n. sp. is described on the basis of morphological characteristics and its genetic divergence from congeners. Molecular characteristics of the new species revealed its close evolutionary relationship with O. d. dewittei. Calculated p-distance for the cox1 gene sequences between O. borneensis n. sp. and O. d. dewittei was 5.9%, while that between O. d. dewittei and O. d. japonica was 7.6%. No intraspecific genetic variation was found for the new species. Wolbachia strains identified in the new species and O. d. dewittei belonged to supergroup C and are closely related.ConclusionsOur molecular analyses of filarioids from Asian suids indicate that the new species is sister to O. d. dewittei. On the basis of its morphological and molecular characteristics, we propose to elevate O. d. japonica to species level as O. japonica Uni, Bain & Takaoka, 2001. Coevolutionary relationships exist between the Wolbachia strains and their filarial hosts in Borneo and Peninsular Malaysia.

Molecular detection of Wolbachia pipientis in natural populations of sandfly vectors of Leishmania infantum in endemic areas: first detection in Lutzomyia longipalpis.

A polymerase chain reaction-based method was used to screen sandflies for infection with Wolbachia (Rickettsiales: Rickettsiaceae), an intracellular bacterial endosymbiont found in many arthropods and filarial hosts. Positive results were obtained in five of 200 field-collected sandflies and were confirmed by sequencing. All sandflies were Lutzomyia longipalpis (Diptera: Psychodidae) captured in a region endemic for visceral leishmaniasis in Brazil. This is the first study to identify Wolbachia infection in this Lutzomyia species, which is the main vector of leishmaniasis in the study area. The low infection rate found in this study (2.5%), together with the lack of detection of Wolbachia in previous studies and the diversity found in the sequences analysed, suggests horizontal transmission to these sandflies.

Breakdown of coevolution between symbiotic bacteria Wolbachia and their filarial hosts.

Wolbachia is an alpha-proteobacterial symbiont widely distributed in arthropods. Since the identification of Wolbachia in certain animal-parasitic nematodes (the Onchocercidae or filariae), the relationship between arthropod and nematode Wolbachia has attracted great interest. The obligate symbiosis in filariae, which renders infected species susceptible to antibiotic chemotherapy, was held to be distinct from the Wolbachia-arthropod relationship, typified by reproductive parasitism. While co-evolutionary signatures in Wolbachia-arthropod symbioses are generally weak, reflecting horizontal transmission events, strict co-evolution between filariae and Wolbachia has been reported previously. However, the absence of close outgroups for phylogenetic studies prevented the determination of which host group originally acquired Wolbachia. Here, we present the largest co-phylogenetic analysis of Wolbachia in filariae performed to date including: (i) a screening and an updated phylogeny of Wolbachia; (ii) a co-phylogenetic analysis; and (iii) a hypothesis on the acquisition of Wolbachia infection. First, our results show a general overestimation of Wolbachia occurrence and support the hypothesis of an ancestral absence of infection in the nematode phylum. The accuracy of supergroup J is also underlined. Second, although a global pattern of coevolution remains, the signal is derived predominantly from filarial clades associated with Wolbachia in supergroups C and J. In other filarial clades, harbouring Wolbachia supergroups D and F, horizontal acquisitions and secondary losses are common. Finally, our results suggest that supergroup C is the basal Wolbachia clade within the Ecdysozoa. This hypothesis on the origin of Wolbachia would change drastically our understanding of Wolbachia evolution.

Wolbachia endosymbiont of Brugia malayi elicits a T helper type 17-mediated pro-inflammatory immune response through Wolbachia surface protein.

Wolbachia is an endosymbiotic bacterium of the filarial nematode Brugia malayi. The symbiotic relationship between Wolbachia and its filarial host is dependent on interactions between the proteins of both organisms. However, little is known about Wolbachia proteins that are involved in the inflammatory pathology of the host during lymphatic filariasis. In the present study, we cloned, expressed and purified Wolbachia surface protein (r-wsp) from Wolbachia and administered it to mice, either alone or in combination with infective larvae of B.malayi (Bm-L3) and monitored the developing immune response in infected animals. Our results show that spleens and mesenteric lymph nodes of mice immunized with either r-wsp or infected with Bm-L3 show increased percentages of CD4(+) T helper type 17 (Th17) cells and Th1 cytokines like interferon-γ and interleukin-2 (IL-2) along with decreased percentages of regulatory T cells, Th2 cytokines like IL-4 and IL-10 and transforming growth factor β (TGF-β) levels in culture supernatants of splenocytes. These observations were stronger in mice immunized with r-wsp alone. Interestingly, when mice were first immunized with r-wsp and subsequently infected with Bm-L3, percentages of CD4(+) Th17 cells and Th1 cytokines increased even further while that of regulatory T cells, Th2 cytokines and TGF-β levels decreased. These results for the first time show that r-wsp acts synergistically with Bm-L3 in promoting a pro-inflammatory response by increasing Th17 cells and at the same time diminishes host immunological tolerance by decreasing regulatory T cells and TGF-β secretion.

Immunological characterization of recombinant Wuchereria bancrofti cuticular collagen (COL-4) as putative vaccine candidate for human lymphatic filariasis

ObjectiveTo elucidate immunoprophylactic potential of recombinant Wuchereria bancrofti (W. bancrofti) cuticular collagen (COL-4) in BALB/c mice and filarial clinical samples. Methodscol-4 gene was PCR amplified from W. bancrofti L3 cDNA library and cloned in pRSET B vector. Recombinant COL-4 was over expressed in salt inducible system and was purified by nickel affinity chromatography. Humoral and cellular responses were measured by ELISA and peripheral blood mononuclear cells (PBMC) of various filarial clinical samples respectively using purified recombinant COL-4 antigen. Then the protective immune responses of COL-4 immunized BALB/c mice were characterized. ResultsSequence analysis of COL-4 with human host proteins reveals lack of homology. The recombinant COL-4 was found to be at 15 kDa fusion protein. The affinity purified COL-4 showed significant reactivity with putatively immune sera and in a similar fashion it demonstrated marked proliferation in PBMC samples. Immunization studies in experimental filarial host (mice) elicited significant titers with protective antibody isotype profile (IgM and IgG). Cellular immune responses were also significant in terms of splenocytes proliferation assay on mice samples. ConclusionsOur immunological findings in experimental host suggest Th2 mediated immune response. Hence, we propose that W. bancrofti COL-4 could be an efficacious vaccine candidate against lymphatic filariasis.

Localization of a filarial phosphate permease that is up-regulated in response to depletion of essential Wolbachia endobacteria

Wolbachia of filarial nematodes are essential, obligate endobacteria. When depleted by doxycycline worm embryogenesis, larval development and worm survival are inhibited. The molecular basis governing the endosymbiosis between Wolbachia and their filarial host is still being deciphered. In rodent filarial nematode Litomosoides sigmodontis, a nematode encoded phosphate permease gene (Ls-ppe-1) was up-regulated at the mRNA level in response to Wolbachia depletion and this gene promises to have an important role in Wolbachia-nematode endosymbiosis. To further characterize this gene, the regulation of phosphate permease during Wolbachia depletion was studied at the protein level in L. sigmodontis and in the human filaria Onchocerca volvulus. And the localization of phosphate permease (PPE) and Wolbachia in L. sigmodontis and O. volvulus was investigated in untreated and antibiotic treated worms. Depletion of Wolbachia by tetracycline (Tet) resulted in up-regulation of Ls-ppe-1 in L. sigmodontis. On day 36 of Tet treatment, compared to controls (Con), >98% of Wolbachia were depleted with a 3-fold increase in mRNA levels of Ls-ppe-1. Anti-Ls-PPE serum used in Western blots showed up-regulation of Ls-PPE at the protein level in Tet worms on day 15 and 36 of treatment. Immunohistology revealed the localization of Wolbachia and Ls-PPE in the embryos, microfilariae and hypodermis of L. sigmodontis female worms and up-regulation of Ls-PPE in response to Wolbachia depletion. Expression of O. volvulus phosphate permease (Ov-PPE) studied using anti-Ov-PPE serum, showed up-regulation of Ov-PPE at the protein level in doxycycline treated Wolbachia depleted O. volvulus worms and immunohistology revealed localization of Ov-PPE and Wolbachia and up-regulation of Ov-PPE in the hypodermis and embryos of doxycycline treated worms. Ls-PPE and Ov-PPE are upregulated upon Wolbachia depletion in same tissues and regions where Wolbachia are located in untreated worms, reinforcing a link between Wolbachia and this nematode encoded protein. The function of nematode phosphate permease in the endosymbiosis is unknown but could involve transportation of phosphate to Wolbachia, which encode all the genes necessary for de novo nucleotide biosynthesis. Electron microscopic localization of PPE and Wolbachia and RNAi mediated knock-down of PPE in filarial nematodes will bring further insights to the functions of PPE in the Wolbachia-nematode symbiosis.

A potential role for the interaction of Wolbachia surface proteins with the Brugia malayi glycolytic enzymes and cytoskeleton in maintenance of endosymbiosis.

The human filarial parasite Brugia malayi harbors an endosymbiotic bacterium of the genus Wolbachia. The Wolbachia represent an attractive target for the control of filarial induced disease as elimination of the bacteria affects molting, reproduction and survival of the worms. The molecular basis for the symbiotic relationship between Wolbachia and their filarial hosts has yet to be elucidated. To identify proteins involved in this process, we focused on the Wolbachia surface proteins (WSPs), which are known to be involved in bacteria-host interactions in other bacterial systems. Two WSP-like proteins (wBm0152 and wBm0432) were localized to various host tissues of the B. malayi female adult worms and are present in the excretory/secretory products of the worms. We provide evidence that both of these proteins bind specifically to B. malayi crude protein extracts and to individual filarial proteins to create functional complexes. The wBm0432 interacts with several key enzymes involved in the host glycolytic pathway, including aldolase and enolase. The wBm0152 interacts with the host cytoskeletal proteins actin and tubulin. We also show these interactions in vitro and have verified that wBm0432 and B. malayi aldolase, as well as wBm0152 and B. malayi actin, co-localize to the vacuole surrounding Wolbachia. We propose that both WSP protein complexes interact with each other via the aldolase-actin link and/or via the possible interaction between the host's enolase and the cytoskeleton, and play a role in Wolbachia distribution during worm growth and embryogenesis.

A new type F Wolbachia from Splendidofilariinae (Onchocercidae) supports the recent emergence of this supergroup

Wolbachia are vertically transmitted endosymbiotic bacteria of arthropods and onchocercid nematodes. It is commonly accepted that they co-evolved with their filarial hosts, and have secondarily been lost in some species. However, most of the data on the Wolbachia/Onchocercidae relationship have been derived from studies on two subfamilies, the Dirofilariinae and the Onchocercinae, which harbour parasites of humans and domestic animals. Within the last few years, analyses of more diverse material have suggested that some groups of Onchocercidae do not have Wolbachia, such as recently studied Splendidofilariinae from birds. This study takes advantage of the analysis of additional Splendidofilariinae, Rumenfilaria andersoni from a Finnish reindeer and Madathamugadia hiepei from a South African gecko, using PCR, immunohistochemical staining and whole-mount fluorescent analysis to detect Wolbachia and describe its strains. A DNA barcoding approach and phylogenetic analyses were used to investigate the symbiosis between Wolbachia and the Onchocercidae. A new supergroup F Wolbachia was demonstrated in M. hiepei, representing the first filarial nematode harbouring Wolbachia described in a non-mammalian host. In the adult, Wolbachia infects the female germline but not the hypodermis, and intestinal cells are also infected. The phylogenetic analyses confirmed a recent emergence of supergroup F. They also suggested several events of horizontal transmission between nematodes and arthropods in this supergroup, and the existence of different metabolic interactions between the filarial nematodes and their symbionts.

Interaction of a Wolbachia WSP-like protein with a nuclear-encoded protein of Brugia malayi

The Brugia malayi endosymbiont Wolbachia has recently been shown to be essential for its host’s survival and development. However, relatively little is known about Wolbachia proteins that interact with the filarial host and which might be important in maintaining the obligate symbiotic relationship. The Wolbachia surface proteins (WSPs) are members of the outer membrane protein family and we hypothesise that they might be involved in the Wolbachia– Brugia symbiotic relationship. Notably, immunolocalisation studies of two WSP members, WSP-0432 and WSP-0284 in B. malayi female adult worms showed that the corresponding proteins are not only present on the surface of Wolbachia but also in the host tissues, with WSP-0284 more abundant in the cuticle, hypodermis and the nuclei within the embryos. These results confirmed that WSPs might be secreted by Wolbachia into the worm’s tissue. Our present studies focus on the potential involvement of WSP-0284 in the symbiotic relationship of Wolbachia with its filarial host. We show that WSP-0284 binds specifically to B. malayi crude protein extracts. Furthermore, a fragment of the hypothetical B. malayi protein (Bm1_46455) was found to bind WSP-0284 by panning of a B. malayi cDNA library. The interaction of WSP-0284 and this protein was further confirmed by ELISA and pull-down assays. Localisation by immunoelectron microscopy within Wolbachia cells as well as in the worm’s tissues, cuticle and nuclei within embryos established that both proteins are present in similar locations within the parasite and the bacteria. Identifying such specific interactions between B. malayi and Wolbachia proteins should lead to a better understanding of the molecular basis of the filarial nematode and Wolbachia symbiosis.

Wolbachia infection and mitochondrial diversity in the canine heartworm (Dirofilaria immitis)

Background and aims: Many species of filarial nematodes are infected with Wolbachia pipientis, a maternally inherited endosymbiont. In addition to manipulating host reproduction, these bacteria also affect the evolution of the mitochondrial DNA with which they are transmitted. Selective sweeps can establish a single mitochondrial lineage within a Wolbachia-infected population and purge genetic diversity. While this phenomenon has been studied in insect model systems, it has not been thoroughly examined in a filarial nematode.Materials and methods: Patterns of mitochondrial diversity were examined in Dirofilaria immitis, a Wolbachia-infected species.Results: The levels of genetic diversity observed in canine heartworm were much lower than those in related species not known to be hosts of Wolbachia. Conclusion: Results suggest that a maternally inherited endosymbiont can depress mitochondrial diversity in a filarial host.

The Heme Biosynthetic Pathway of the Obligate Wolbachia Endosymbiont of Brugia malayi as a Potential Anti-filarial Drug Target

BackgroundFilarial parasites (e.g., Brugia malayi, Onchocerca volvulus, and Wuchereria bancrofti) are causative agents of lymphatic filariasis and onchocerciasis, which are among the most disabling of neglected tropical diseases. There is an urgent need to develop macro-filaricidal drugs, as current anti-filarial chemotherapy (e.g., diethylcarbamazine [DEC], ivermectin and albendazole) can interrupt transmission predominantly by killing microfilariae (mf) larvae, but is less effective on adult worms, which can live for decades in the human host. All medically relevant human filarial parasites appear to contain an obligate endosymbiotic bacterium, Wolbachia. This alpha-proteobacterial mutualist has been recognized as a potential target for filarial nematode life cycle intervention, as antibiotic treatments of filarial worms harboring Wolbachia result in the loss of worm fertility and viability upon antibiotic treatments both in vitro and in vivo. Human trials have confirmed this approach, although the length of treatments, high doses required and medical counter-indications for young children and pregnant women warrant the identification of additional anti-Wolbachia drugs.Methods and FindingsGenome sequence analysis indicated that enzymes involved in heme biosynthesis might constitute a potential anti-Wolbachia target set. We tested different heme biosynthetic pathway inhibitors in ex vivo B. malayi viability assays and report a specific effect of N-methyl mesoporphyrin (NMMP), which targets ferrochelatase (FC, the last step). Our phylogenetic analysis indicates evolutionarily significant divergence between Wolbachia heme genes and their human homologues. We therefore undertook the cloning, overexpression and analysis of several enzymes of this pathway alongside their human homologues, and prepared proteins for drug targeting. In vitro enzyme assays revealed a ∼600-fold difference in drug sensitivities to succinyl acetone (SA) between Wolbachia and human 5′-aminolevulinic acid dehydratase (ALAD, the second step). Similarly, Escherichia coli hemH (FC) deficient strains transformed with human and Wolbachia FC homologues showed significantly different sensitivities to NMMP. This approach enables functional complementation in E. coli heme deficient mutants as an alternative E. coli-based method for drug screening.ConclusionsOur studies indicate that the heme biosynthetic genes in the Wolbachia of B. malayi (wBm) might be essential for the filarial host survival. In addition, the results suggest they are likely candidate drug targets based upon significant differences in phylogenetic distance, biochemical properties and sensitivities to heme biosynthesis inhibitors, as compared to their human homologues.

Wolbachia endosymbionts of Onchocerca volvulus express a putative periplasmic HtrA-type serine protease

Wolbachia are intracellular bacteria of many filarial nematodes. A mutualistic interaction between the endobacteria and the filarial host is likely, because the clearance of Wolbachia by tetracycline leads to the obstruction of embryogenesis and larval development. Databases were searched for exported molecules to identify candidates involved in this mutualism. Fragments of a Wolbachia serine protease from the human filarial parasite Onchocerca volvulus were obtained ( Wol-Ov-HtrA) by the use of a PCR technique and primers based on the Rickettsia prowazekii genome. The deduced amino acid sequence exhibited 87% and 81% identity to the homologous Wolbachia proteases identified from Brugia malayi and Drosophila melanogaster, respectively. The full-length cDNA encodes 494 amino acids with a calculated mass of 54 kDa. Three characteristic features, (i) a catalytic triad of serine proteases, (ii) two PDZ domains and (iii) a putative signal peptide, classify the endobacterial protein as a member of the periplasmic HtrA family of proteases known to express chaperone and regulator activity of apoptosis. Using a rabbit antiserum raised against a recombinantly expressed 33-kDa fragment of Wol-Ov-HtrA, strong labelling of the antigen was found associated with endobacteria in hypodermis, oocytes, zygotes, all embryonic stages and microfilariae of O. volvulus. Staining of hypodermal cytoplasm surrounding the endobacteria indicated a possible release of the protein from the Wolbachia. The demonstration of Wol-Ov-HtrA-reactive IgG1 antibodies in sera of O. volvulus-infected persons indicated the exposure to the protein and its recognition by the human immune system. Wol-Ov-HtrA is a candidate for an exported Wolbachia protein that may interact with the filarial host metabolism.

Wolbachia in filarial nematodes: evolutionary aspects and implications for the pathogenesis and treatment of filarial diseases

The presence of intracellular bacteria in the body of various species of filarial nematodes, including important parasites such as Brugia malayi, Dirofilaria immitis, and Onchocerca volvulus, was observed as early as the mid-1970s. These bacteria were shown to be transovarially transmitted (from the female worm to the offspring) and to be present in significant amounts in the body of the nematode. As highlighted by their discoverers, the potential importance of these bacteria is fairly obvious: (1) bacteria-derived molecules should be considered as having an immunological and pathological role in filarial diseases; (2) the interaction between the bacteria and the filarial host deserves investigation, in view of the possibility that the bacteria are needed by the host nematode and could thus represent a target for therapy. Other authors, independently from the discovery of these intracellular bacteria, showed that the antibiotic tetracycline (which is well known for its efficacy on intracellular bacteria) had detrimental effects on two species of filarial nematodes ( Brugia pahangi and Litomosoides sigmodontis). It is therefore surprising that for more than 20 years, no further investigations focused on the bacteria of filarial nematodes, nor on the anti-filarial properties of tetracycline. Recently, the bacteria of filarial nematodes have been independently “rediscovered” by research groups from the schools of Hamburg, Liverpool and Milan. These bacteria are now classified as Wolbachia, and the basic aspects of their phylogenetic history and relationship with the Wolbachia of arthropods have been reconstructed. In addition, their implications for the pathogenesis and treatment of filarial diseases have started to be uncovered. This paper, which is authored by representatives of the three European schools who reopened this research area, reviews our present knowledge of these fascinating microorganisms, highlighting the complexity of a symbiotic system which involves, in addition to the nematode and its bacterium, the vertebrate host.