Filarial Nematode Species Research Articles

Pyrosequencing Using SL and 5S rRNA as Molecular Markers for Identifying Zoonotic Filarial Nematodes in Blood Samples and Mosquitoes.

Lymphatic filariasis is principally caused by Wuchereria bancrofti, and Brugia malayi. The other two filarial nematode species, Brugia pahangi and Dirofilaria immitis, possibly cause human zoonotic diseases. We propose the development of a PCR assay linked with DNA pyrosequencing as a rapid tool to identify W. bancrofti, B. malayi, B. pahangi, and D. immitis in blood samples and mosquitoes. Primers targeting the fragment of the 5S ribosomal RNA and spliced leader sequences were newly designed and developed to identify these four filarial nematodes. Analytical sensitivity and specificity were evaluated. Pyrosequencing determination of nucleotide variations within 36 nucleotides for B. malayi and B. pahangi, and 32 nucleotides for W. bancrofti and D. immitis is sufficient for differentiation of those filarial nematodes, and for detection of intraspecies genetic variation of B. malayi. This analysis could detect a single B. malayi, B. pahangi, W. bancrofti, and D. immitis microfilaria in blood samples. Overall, the PCR-linked pyrosequencing-based method was faster than direct sequencing and less expensive than real-time PCR or direct sequencing. This is the possibility of choice that can be applied in a high-throughput platform for identification and surveillance of reservoirs and vectors infected with lymphatic filaria in endemic areas.

Hemoparasites in a wild primate: Infection patterns suggest interaction of Plasmodium and Babesia in a lemur species.

Hemoparasites can cause serious morbidity in humans and animals and often involve wildlife reservoirs. Understanding patterns of hemoparasite infections in natural populations can therefore inform about emerging disease risks, especially in the light of climate change and human disruption of natural ecosystems. We investigated the effects of host age, sex, host group size and season on infection patterns of Plasmodium sp., Babesia sp. and filarial nematodes in a population of wild Malagasy primates, Verreaux's sifakas (Propithecus verreauxi), as well as the effects of these infections on hematological variables. We tested 45 blood samples from 36 individuals and identified two species of Plasmodium, one species of Babesia and two species of filarial nematodes. Plasmodium spp. and Babesia sp. infections showed opposite patterns of age-dependency, with babesiosis being prevalent among young animals, while older animals were infected with Plasmodium sp. In addition, Babesia sp. infection was a statistically significant negative predictor of Plasmodium sp. infection. These results suggest that Plasmodium and Babesia parasites may interact within the host, either through cross-immunity or via resource competition, so that Plasmodium infections can only establish after babesiosis has resolved. We found no effects of host sex, host group size and season on hemoparasite infections. Infections showed high prevalences and did not influence hematological variables. This preliminary evidence supports the impression that the hosts and parasites considered in this study appear to be well-adapted to each other, resulting in persistent infections with low pathogenic and probably low zoonotic potential. Our results illustrate the crucial role of biodiversity in host-parasite relationships, specifically how within-host pathogen diversity may regulate the abundance of parasites.

Canine filarial infections in Liguria, north-west Italy.

This paper reports the findings of a study on the presence of various species of filarial nematodes in dogs in Liguria, north-west Italy, a region traditionally considered free from the disease. Between 2009 and 2012 blood samples were taken from 365 dogs in rural areas in Liguria. The blood samples were then submitted to Knott's test, histochemical staining, polymerase chain reaction (PCR) and the enzyme-linked immunosorbent assay (ELISA) for Dirofilaria immitis antigens. Overall, 35 of the 365 dogs were positive using Knott's test for microfilariae (prevalence 9.6%; 95% confidence interval (CI): 6.6-12.6%). Acanthocheilonema reconditum was the most prevalent species (8.0%), while Dirofilaria repens (1.4%) and Dirofilaria immitis (0.6%) were less common. One co-infection by D. repens and A. reconditum was observed. All morphological identifications were confirmed by histochemical staining and PCR. In addition, a retrospective analysis of data on D. immitis antigens in 11,363 samples of canine sera was carried out. Sera were collected and analysed for D. immitis antigens by the Istituto Zooprofilattico Sperimentale (IZS) of Piedmont, Liguria and Aosta Valley (Imperia section) between 2004 and 2013 during annual tests for leishmaniasis on autochthonous dogs throughout Liguria. Serological data from IZS showed an overall seroprevalence of 0.65% (95% CI: 0.50-0.80%) for D. immitis throughout the region. The present study updates the epidemiological map of canine filarial infections in Italy and suggests the need for surveillance and prophylaxis in Liguria.

Comparing the mitochondrial genomes of Wolbachia-dependent and independent filarial nematode species

BackgroundMany species of filarial nematodes depend on Wolbachia endobacteria to carry out their life cycle. Other species are naturally Wolbachia-free. The biological mechanisms underpinning Wolbachia-dependence and independence in filarial nematodes are not known. Previous studies have indicated that Wolbachia have an impact on mitochondrial gene expression, which may suggest a role in energy metabolism. If Wolbachia can supplement host energy metabolism, reduced mitochondrial function in infected filarial species may account for Wolbachia-dependence. Wolbachia also have a strong influence on mitochondrial evolution due to vertical co-transmission. This could drive alterations in mitochondrial genome sequence in infected species. Comparisons between the mitochondrial genome sequences of Wolbachia-dependent and independent filarial worms may reveal differences indicative of altered mitochondrial function.ResultsThe mitochondrial genomes of 5 species of filarial nematodes, Acanthocheilonema viteae, Chandlerella quiscali, Loa loa, Onchocerca flexuosa, and Wuchereria bancrofti, were sequenced, annotated and compared with available mitochondrial genome sequences from Brugia malayi, Dirofilaria immitis, Onchocerca volvulus and Setaria digitata. B. malayi, D. immitis, O. volvulus and W. bancrofti are Wolbachia-dependent while A. viteae, C. quiscali, L. loa, O. flexuosa and S. digitata are Wolbachia-free. The 9 mitochondrial genomes were similar in size and AT content and encoded the same 12 protein-coding genes, 22 tRNAs and 2 rRNAs. Synteny was perfectly preserved in all species except C. quiscali, which had a different order for 5 tRNA genes. Protein-coding genes were expressed at the RNA level in all examined species. In phylogenetic trees based on mitochondrial protein-coding sequences, species did not cluster according to Wolbachia dependence.ConclusionsThus far, no discernable differences were detected between the mitochondrial genome sequences of Wolbachia-dependent and independent species. Additional research will be needed to determine whether mitochondria from Wolbachia-dependent filarial species show reduced function in comparison to the mitochondria of Wolbachia-independent species despite their sequence-level similarities.

Onchocerciasis: the Role of Wolbachia Bacterial Endosymbionts in Parasite Biology, Disease Pathogenesis, and Treatment

The discovery of Wolbachia intracellular bacteria within filarial nematodes, including Onchocerca volvulus, the causative agent of onchocerciasis or "river blindness," has delivered a paradigm shift in our understanding of the parasite's biology, to where we now know that the bacterial endosymbionts are essential for normal development of larvae and embryos and may support the long-term survival of adult worms. The apparent mutualistic dependency has also offered a novel approach to the treatment of onchocerciasis through the use of antibiotics to eliminate Wolbachia, delivering for the first time a treatment which has significant macrofilaricidal efficacy. Studies with other filarial nematode species have also highlighted a role for Wolbachia in transmission and infection of the mammalian host through a fascinating manipulation of mast cell-mediated vasodilation to enhance infectivity of vector-borne larvae. Wolbachia has also been identified as the principal driver of innate and adaptive Th1 inflammatory immunity, which can either contribute to disease pathogenesis or, with the Wolbachia-mediated recruitment of mast cells, enhance infectivity. The Wolbachia activation of innate inflammation also drives inflammatory adverse events in response to chemotherapy with either diethylcarbamazine (DEC) or ivermectin. In this review we summarize the experimental and field trial data which have uncovered the importance of Wolbachia symbiosis in onchocerciasis.

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.

Endosymbiont DNA in endobacteria-free filarial nematodes indicates ancient horizontal genetic transfer.

Background Wolbachia are among the most abundant symbiotic microbes on earth; they are present in about 66% of all insect species, some spiders, mites and crustaceans, and most filarial nematode species. Infected filarial nematodes, including many pathogens of medical and veterinary importance, depend on Wolbachia for proper development and survival. The mechanisms behind this interdependence are not understood. Interestingly, a minority of filarial species examined to date are naturally Wolbachia-free.Methodology/Principal FindingsWe used 454 pyrosequencing to survey the genomes of two distantly related Wolbachia-free filarial species, Acanthocheilonema viteae and Onchocerca flexuosa. This screen identified 49 Wolbachia-like DNA sequences in A. viteae and 114 in O. flexuosa. qRT-PCR reactions detected expression of 30 Wolbachia-like sequences in A. viteae and 56 in O. flexuosa. Approximately half of these appear to be transcribed from pseudogenes. In situ hybridization showed that two of these pseudogene transcripts were specifically expressed in developing embryos and testes of both species.Conclusions/SignificanceThese results strongly suggest that the last common ancestor of extant filarial nematodes was infected with Wolbachia and that this former endosymbiont contributed to their genome evolution. Horizontally transferred Wolbachia DNA may explain the ability of some filarial species to live and reproduce without the endosymbiont while other species cannot.

The bacteriaWolbachiain filariae, a biological Russian dolls’ system: new trends in antifilarial treatments

Filarial nematode species can host Wolbachia bacterial endosymbionts. To understand the symbiosis, a higher level of complexity should be considered, taking in account the tripartite association between Wolbachia, filariae and mammals. This overview article discusses the biology of Wolbachia in filariae, including their distribution and phylogeny, mechanisms of action, inflammatory consequences on mammal host and biological control implications for filariases. Potential directions for future research are also discussed.

Highly sensitive multiplex PCR for simultaneous detection and discrimination of Dirofilaria immitis and Dirofilaria repens in canine peripheral blood

Dirofilaria immitis and Dirofilaria repens are the most common species of filarial nematodes described in the dogs with increasing spread into new geographical areas. The diagnosis of canine dirofilariosis is usually based upon the microscopical detection and identification of circulating microfilariae together with ELISA detection of serum circulating heartworm antigens or antibodies. The identification of the parasite species using the traditional approaches sometimes can be difficult and can lead to misdiagnosis especially on samples from areas where both Dirofilaria are present. In this paper we report a new molecular method based on single-step multiplex PCR to detect and differentiate simultaneously and unequivocally D. immitis and D. repens on DNA extracted from canine peripheral blood. The amplification was performed using a set of primers designed on a portion of the small subunit ribosomal RNA gene of the mitochondrion (12S rDNA). The single-step multiplex PCR here described ensured high (4 mf/ml) sensitivity and specificity with reduced cost and time saving. The multiplex PCR assay represents an additional tool for epidemiological studies and routine disease assessment in areas co-endemic for the two Dirofilaria species.

INOs expression is stimulated by the major surface protein (rWSP) from Wolbachia bacterial endosymbiont of Dirofilaria immitis following subcutaneous injection in mice

The bacterial endosymbiont Wolbachia of several species of filarial nematodes plays an important role in the inflammatory pathology of filariasis. Nitric oxide (NO) production has also been implicated in the immune response during filarial infections. Here we present data indicating that a recombinant Wolbachia surface protein (rWSP) induces iNOs mRNA expression and NO production, as well as IFN-γ and a Th1-type antibody response, in inoculated BALB/c mice. This effect is not observed when mice are inoculated with a recombinant heat shock protein from Wolbachia (GroEL).

Efficient Procedure for Purification of ObligateIntracellular Wolbachia pipientis and RepresentativeAmplification of Its Genome by Multiple-DisplacementAmplification

Bacteria belonging to the genus Wolbachia are obligatory microendocytobionts that infect a variety of arthropods and a majority of filarial nematode species, where they induce reproductive alterations or establish a mutualistic symbiosis. Although two whole genome sequences of Wolbachia pipientis, for strain wMel from Drosophila melanogaster and strain wBm from Brugia malayi, have been fully completed and six other genome sequencing projects are ongoing (http://www.genomesonline.org/index.cgi?want=Prokaryotic+Ongoin), genetic analyses of these bacteria are still scarce, mainly due to the inability to cultivate them outside of eukaryotic cells. Usually, a large amount of host tissue (a thousand individuals, or about 10 g) is required in order to purify Wolbachia and extract its DNA, which is often recovered in small amounts and contaminated by host cell DNA, thus hindering genomic studies. In this report, we describe an efficient and reliable procedure to representatively amplify the Wolbachia genome by multiple-displacement amplification from limited infected host tissue (0.2 g or 2 x 10(7) cells). We obtained sufficient amounts (8 to 10 microg) of DNA of suitable quality for genomic studies, and we demonstrated that the amplified DNA contained all of the Wolbachia loci targeted. In addition, our data indicated that the genome of strain wRi, an obligatory endosymbiont of Drosophila simulans, shares a similar overall architecture with its relative strain wMel.

Evidence for a Global Wolbachia Replacement in Drosophila melanogaster

Wolbachia are maternally inherited intracellular alpha-Proteobacteria found in numerous arthropod and filarial nematode species. They influence the biology of their hosts in many ways. In some cases, they act as obligate mutualists and are required for the normal development and reproduction of the host. They are best known, however, for the various reproductive parasitism traits that they can generate in infected hosts. These include cytoplasmic incompatibility (CI) between individuals of different infection status, the parthenogenetic production of females, the selective killing of male embryos, and the feminization of genetic males. Wolbachia infections of Drosophila melanogaster are extremely common in both wild populations and long-term laboratory stocks. Utilizing the newly completed genome sequence of Wolbachia pipientis wMel, we have identified a number of polymorphic markers that can be used to discriminate among five different Wolbachia variants within what was previously thought to be the single clonal infection of D. melanogaster. Analysis of long-term lab stocks together with wild-caught flies indicates that one of these variants has replaced the others globally within the last century. This is the first report of a global replacement of a Wolbachia strain in an insect host species. The sweep is at odds with current theory that cannot explain how Wolbachia can invade this host species given the observed cytoplasmic incompatibility characteristics of Wolbachia infections in D. melanogaster in the field.

The bacterial catalase from filarial DNA preparations derives from common pseudomonad contaminants and not from Wolbachia endosymbionts.

Wolbachia are obligatory endosymbionts in many species of filarial nematodes. Certain bacterial molecules induce antibody responses in mammalian hosts infected with filariae, while others activate inflammatory responses that contribute to pathology. These findings, coupled with antibiotic studies demonstrating the dependence of filarial embryogenesis on the presence of Wolbachia, have intensified research on Wolbachia-nematode interactions, and the effects of Wolbachia molecules on the mammalian immune system. By amplification and sequencing of 16S rDNA and catalase sequences, we show that filarial DNA samples prepared from nematodes collected under typical conditions are frequently contaminated with Pseudomonas DNA. Analysis of a published DNA fragment containing a catalase attributed to the Wolbachia of Onchocerca volvulus showed it to be most like Pseudomonas, both in terms of sequence similarity and genomic organization. Additionally, there was no obvious catalase in either of two available Wolbachia genome sequences. Contamination of filarial DNA with bacterial sequences other than Wolbachia can complicate studies of the role of these symbionts in filarial biology.

Evidence against Wolbachia symbiosis in Loa loa

BackgroundThe majority of filarial nematode species are host to Wolbachia bacterial endosymbionts, although a few including Acanthocheilonema viteae, Onchocerca flexuosa and Setaria equina have been shown to be free of infection. Comparisons of species with and without symbionts can provide important information on the role of Wolbachia symbiosis in the biology of the nematode hosts and the contribution of the bacteria to the development of disease. Previous studies by electron microscopy and PCR have failed to detect intracellular bacterial infection in Loa loa. Here we use molecular and immunohistological techniques to confirm this finding.MethodsWe have used a combination of PCR amplification of bacterial genes (16S ribosomal DNA [rDNA], ftsZ and Wolbachia surface protein [WSP]) on samples of L. loa adults, third-stage larvae (L3) and microfilariae (mf) and immunohistology on L. loa adults and mf derived from human volunteers to determine the presence or absence of Wolbachia endosymbionts. Samples used in the PCR analysis included 5 adult female worms, 4 adult male worms, 5 mf samples and 2 samples of L3. The quality and purity of nematode DNA was tested by PCR amplification of nematode 5S rDNA and with diagnostic primers from the target species and used to confirm the absence of contamination from Onchocerca sp., Mansonella perstans, M. streptocerca and Wuchereria bancrofti. Immunohistology was carried out by light and electron microscopy on L. loa adults and mf and sections were probed with rabbit antibodies raised to recombinant Brugia malayi Wolbachia WSP. Samples from nematodes known to be infected with Wolbachia (O. volvulus, O. ochengi, Litomosoides sigmodontis and B. malayi) were used as positive controls and A. viteae as a negative control.ResultsSingle PCR analysis using primer sets for the bacterial genes 16S rDNA, ftsZ, and WSP were negative for all DNA samples from L. loa. Positive PCR reactions were obtained from DNA samples derived from species known to be infected with Wolbachia, which confirmed the suitability of the primers and PCR conditions. The quality and purity of nematode DNA samples was verified by PCR amplification of 5S rDNA and with nematode diagnostic primers. Additional analysis by 'long PCR' failed to produce any further evidence for Wolbachia symbiosis. Immunohistology of L. loa adults and mf confirmed the results of the PCR with no evidence for Wolbachia symbiosis.ConclusionDNA analysis and immunohistology provided no evidence for Wolbachia symbiosis in L. loa.

Wolbachia and river blindness

The filarial nematode Onchocerca volvulus is the causative agent of river blindness. Onchocerca volvulus can result in a range of disease states, one of which is eye disease, but the aetiology of this eye disease is unclear. It has been thought that microfilariae (the first larval stage progeny of adult worms) can enter the host eye and (perhaps following microfilarial death) generate immune responses which can cause eye disease. Other work has suggested that the immunological cross-reaction between O. volvulus antigens and human eye antigens are the reason for this immune damage.Recently, it has been found that O. volvulus and a range of other filarial nematodes (all transmitted by arthropods) have obligate bacterial endosymbionts, Wolbachia. Similar endosymbionts are known from arthropods, where they can have one of several effects on their host, ranging from generating cytoplasmic incompatibility at mating to the induction of parthenogenetic reproduction. Endosymbionts can be eliminated from infected animals by treatment with antibiotics. When filarial nematodes are treated with antibiotics, the nematodes appear stunted and they become sterile suggesting that the endosymbiont in filarial nematodes has become an obligate part of the nematode's ‘normal’ biology.Andre et al. have investigated the role of Wolbachia in the pathology of onchocerciasis using a murine model of corneal inflammation in which worm extracts are injected into the corneal stroma [1xThe role of endosymbiotic Wolbachia bacteria in the pathogenesis of river blindness. Andre et al. Science. 2002; 295: 1892–1895Crossref | PubMed | Scopus (260)See all References][1]. When extracts from O. volvulus are used in this model, there is an increase in the thickness of the corneal stoma, greater stromal haze and neutrophil infiltration, compared with the controls, all of which are measures of eye pathology. However, when extracts of O. volvulus treated with antibiotics (therefore free of Wolbachia) are used, there is no eye pathology when using these measures. These results suggest that immune responses against the Wolbachia component of O. volvulus might be responsible for the eye disease in onchocerciasis. Andre et al. extended this study to two other species of filarial nematodes, one with Wolbachia endosymbionts (Brugia malayi) and one without (Acanthocheilonema viteae). This showed that B. malayi extracts induced eye pathology, whereas A. viteae did not, as would be predicated if the Wolbachia component of the filarial nematodes is responsible for the induction of the eye pathology.Immunological responses to bacterial lipopolysaccharide (LPS) require a host Toll-like receptor (TLR4). Immune responses to Wolbachia are likely to involve this receptor, whereas those against nematode antigens per se will not. Mice hypo- or hyper-responsive to LPS (as a result of mutations in TLR4) were compared in their responses to O. volvulus. These results showed that mice hyper-responsive to LPS had greater eye pathology compared with hypo-responsive mice, further suggesting that immune responses against Wolbachia are responsible for the observed eye disease.Antibiotic therapy reduces the reproduction of these filarial worms and therefore reduces host microfilaraemia. The disease, due to this microfilaraemia, might therefore be susceptible to treatment simply by the administration of antibiotics. The discovery that the eye pathology of river blindness could similarly be due to the bacterial endosymbiont raises the prospect that this aspect of the disease might also be susceptible to such treatment.

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.

Tetracycline Inhibits Development of the Infective-Stage Larvae of Filarial Nematodes in Vitro

Smith, H. L., and Rajan, T. V. 2000. Tetracycline inhibits development of the infective-stage larvae of filarial nematodes in vitro. Experimental Parasitology95, 265–270. In recent years, studies have linked tetracycline treatment of filaria-infected animals with reduced adult worm burdens and decreased levels of microfilaremia. These observations are believed to be attributable to clearance of Wolbachia, intracellular rickettsial-like organisms found within filarial tissues. Although maximal worm reductions were observed when treatment was initiated early in infection, it is not known whether tetracycline inhibits development of infective-stage larvae. To address this issue, we studied the effect of tetracycline on three different species of filarial nematodes, Brugia malayi, Brugia pahangi, and Dirofilaria immitis, in a serumfree in vitro system supporting molting to the fourth larval stage. Tetracycline was capable of inhibiting L3 to L4 molting within a dosage range similar to that reported for susceptible rickettsial organisms. However, Wolbachia DNA could still be detected in nematodes from tetracycline-treated cultures. In addition, three other antibiotics with anti-rickettsial and anti-chlamydial activity (chloramphenicol, erythromycin, and ciprofloxacin) failed to inhibit L3 to L4 molting. Although tetracycline is capable of completely blocking molting of infective-stage larvae, it remains possible that this effect is due to pharmacological activities unrelated to its anti-rickettsial functions.

Tetracycline therapy targets intracellular bacteria in the filarial nematode Litomosoides sigmodontis and results in filarial infertility

Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.

Phylogeny of Wolbachia in filarial nematodes.

Intracellular bacteria have been observed in various species of filarial nematodes (family Onchocercidae). The intracellular bacterium of the canine filaria Dirofilaria immitis has been shown to be closely related to Wolbachia, a rickettsia-like micro-organism that is widespread among arthropods. However, the relationships between endosymbionts of different filariae, and between these and the arthropod wolbachiae, appear not to have been studied. To address these issues we have examined ten species of filarial nematodes for the presence of Wolbachia. For nine species, all samples examined were PCR positive using primers specific for the ftsZ gene of Wolbachia. For one species, the examined samples were PCR negative. Sequences of the amplified ftsZ gene fragments of filarial wolbachiae fall into two clusters (C and D), which are distinct from the A and B clusters recognized for arthropod wolbachiae. These four lineages (A-D) are related in a star-like phylogeny, with higher nucleotide divergence observed between C and D wolbachiae than that observed between A and B wolbachiae. In addition, within each of the two lineages of filarial wolbachiae, the phylogeny of the symbionts is consistent with the host phylogeny. Thus, there is no evidence for recent Wolbachia transmission between arthropods and nematodes. Endosymbiont 16S ribosomal DNA sequences from a subset of filarial species support these findings.

Molecular cloning of a gene expressed during early embryonic development in Onchocerca volvulus

Little attention has been paid to the reproductive biology of filarial nematode parasites as a possible target for immunological or chemotherapeutic intervention. An interruption of the reproductive process would, in addition to breaking the cycle of transmission, reduce the morbidity associated with certain filarial infections. As part of our efforts to define molecules that have important functions during filarial embryogenesis, antibodies against embryo-associated proteins were used to identify a 6308-bp cDNA sequence (ovt1) from an Onchocerca volvulus cDNA expression library. The ovt1 cDNA contained an open reading frame that coded for 2022 amino acids. The deduced amino acid sequence was highly hydrophilic, α-helical in nature and included two leucine zipper domains. OVT1 also contained a single Arg-Gly-Asp (RGD) site. The results of Southern blot analyses demonstrated that an ovt1-like gene occurs in a number of different species of filarial nematodes. In situ hybridization experiments to identify tissues that contain ovt1 transcripts showed that ovt1 was transcribed at high levels in the late morula/early blastocyst stage of embryonic development. Transcripts for ovt1 were also detected in O. volvulus larvae and in the hypodermal cells of adult parasites. Two fragments of ovt1 were expressed as fusion proteins and the fusion proteins were used to produce antibodies in rabbits. Both antibodies recognized a native protein with an apparent molecular mass of 230 kDa in extracts from gravid female O. volvulus. In addition, the antibodies reacted with a restricted number of lower-molecular mass bands which may represent the products of post-transcriptional or post-translational processing. The predicted coiled-coil structure and the sites of transcription suggest that OVT1 may be a component of the extracellular matrix.