Lymphatic filariasis and onchocerciasis are parasitic helminth diseases that constitute a serious public health issue in tropical regions. The filarial nematodes that cause these diseases are transmitted by blood-feeding insects and produce chronic and long-term infection through suppression of host immunity. Disease pathogenesis is linked to host inflammation invoked by the death of the parasite, causing hydrocoele, lymphoedema, and elephantiasis in lymphatic filariasis, and skin disease and blindness in onchocerciasis. Most filarial species that infect people co-exist in mutualistic symbiosis with Wolbachia bacteria, which are essential for growth, development, and survival of their nematode hosts. These endosymbionts contribute to inflammatory disease pathogenesis and are a target for doxycycline therapy, which delivers macrofilaricidal activity, improves pathological outcomes, and is effective as monotherapy. Drugs to treat filariasis include diethylcarbamazine, ivermectin, and albendazole, which are used mostly in combination to reduce microfilariae in blood (lymphatic filariasis) and skin (onchocerciasis). Global programmes for control and elimination have been developed to provide sustained delivery of drugs to affected communities to interrupt transmission of disease and ultimately eliminate this burden on public health.

Endosymbiotic bacteria living in plasmodia or worm parasites are required for the homoeostasis of their host and should be excellent targets for chemotherapy of certain parasitic diseases. We show that targeting of Wolbachia spp bacteria in Onchocerca volvulus filariae by doxycycline leads to sterility of adult worms to an extent not seen with drugs used against onchocerciasis, a leading cause of blindness in African countries.

Ivermectin is the drug used for mass chemotherapy of onchocerciasis within the WHO African Programme for Onchocerciasis Control. This approach aims to eliminate the disease as a public health problem but using one dose per year may not completely interrupt transmission since it does not suppress microfilaridermia thoroughly enough. Here we show that additional treatment with doxycycline, previously shown to sterilise adult female worms for a few months by depletion of symbiotic wolbachia endobacteria, significantly enhances ivermectin-induced suppression of microfilaridermia, rendering anti-wolbachia treatment a promising basis for blocking transmission by a drug-based approach.

Filarial nematodes are important and widespread parasites of animals and humans. We have been using the African bovine parasite Onchocerca ochengi as a chemotherapeutic model for O. volvulus, the causal organism of 'river blindness' in humans, for which there is no safe and effective drug lethal to adult worms. Here we report that the antibiotic, oxytetracycline is macrofilaricidal against O. ochengi. In a controlled trial in Cameroon, all adult worms (as well as microfilariae) were killed, and O. ochengi intradermal nodules resolved, by nine months' post-treatment in cattle treated intermittently for six months. Adult worms removed from concurrent controls remained fully viable and reproductively active. By serial electron-microscopic examination, the macrofilaricidal effects were related to the elimination of intracellular micro-organisms, initially abundant. Analysis of a fragment of the 16S rRNA gene from the O. ochengi micro-organisms confirmed them to be Wolbachia organisms of the order Rickettsiales, and showed that the sequence differed in only one nucleotide in 858 from the homologous sequence of the Wolbachia organisms of O. volvulus. These data are, to our knowledge, the first to show that antibiotic therapy can be lethal to adult filariae. They suggest that tetracycline therapy is likely to be macrofilaricidal against O. volvulus infections in humans and, since similar Wolbachia organisms occur in a number of other filarial nematodes, against those infections too. In that the elimination of Wolbachia preceded the resolution of the filarial infections, they suggest that in O. ochengi at least, the Wolbachia organisms play an essential role in the biology and metabolism of the filarial worm.

Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hosts immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-alpha and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.

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.

BACKGROUND: Wolbachia endosymbionts of filarial nematodes are vital for larval development and adult-worm fertility and viability. This essential dependency on the bacterium for survival of the parasites has provided a new approach to treat filariasis with antibiotics. We used this strategy to investigate the effects of doxycycline treatment on the major cause of lymphatic filariasis, Wuchereria bancrofti. METHODS: We undertook a double-blind, randomised, placebo-controlled field trial of doxycycline (200 mg per day) for 8 weeks in 72 individuals infected with W bancrofti from Kimang'a village, Pangani, Tanzania. Participants were randomly assigned by block randomisation to receive capsules of doxycycline (n=34) or placebo (n=38). We assessed treatment efficacy by monitoring microfilaraemia, antigenaemia, and ultrasound detection of adult worms. Follow-up assessments were done at 5, 8, 11, and 14 months after the start of treatment. Analysis was per protocol. FINDINGS: One person from the doxycycline group died from HIV infection. Five (doxycycline) and 11 (placebo) individuals were absent at the time of ultrasound analysis. Doxycycline treatment almost completely eliminated microfilaraemia at 8-14 months' follow-up (for all timepoints p<0.001). Ultrasonography detected adult worms in only six (22%) of 27 individuals treated with doxycycline compared with 24 (88%) of 27 with placebo at 14 months after the start of treatment (p<0.0001). At the same timepoint, filarial antigenaemia in the doxycycline group fell to about half of that before treatment (p=0.015). Adverse events were few and mild. INTERPRETATION: An 8-week course of doxycycline is a safe and well-tolerated treatment for lymphatic filariasis with significant activity against adult worms and microfilaraemia.

Recently, experts have warned that mass treatment with ivermectin alone may not interrupt the transmission of Onchocerca. Hence, additional drugs are needed, such as antibiotics acting on symbiotic endobacteria of the filariae, the causative agents of onchocerciasis. Based on animal experiments, human onchocerciasis was treated with doxycycline, and preliminary observations published in 2001 in The Lancet showed sterility in female worms by depletion and marked reduction in symbiotic Wolbachia endobacteria from the filariae. Here, a detailed kinetic analysis of the features of the worms, following administration or not of doxycycline to the patients is reported. Sixty-three onchocerciasis patients in Ghana were treated with 100 mg doxycycline daily for 6 weeks and 2 or 6 months later with ivermectin. Onchocercomas were extirpated 2, 6, 11 and 18 months after the onset of treatment and the filariae were examined by immunohistology and PCR. The analysis showed:(i) progressive depletion of Wolbachia from adult worms and microfilariae by doxycycline over a period of 6 months;(ii) inhibition of embryogenesis by doxycycline after 6 months with respect to all embryo stages followed by decline in microfilariae after 11 months;(iii) reduction in spermatozoa in the female genital tract by doxycycline, whereas spermiogenesis was only partly reduced after 11 and 18 months;(iv) no relevant macro- or microfilaricidal activity;(v) depletion/marked reduction in endobacteria and inhibition of embryogenesis were sustained until 18 months after doxycycline and 12 months after co-administration of ivermectin;(vi) no severe adverse side effects were seen. Due to its long-lasting inhibition of embryogenesis, doxycycline presents an additional strategy for the treatment of onchocerciasis and control of Onchocerca microfilariae transmission. Extension of the existing registration will not require much time or high cost. Treatment of individual patients can be considered immediately.

Inherited microorganisms that disturb the reproduction of their host have been characterized from a number of host taxa. To understand the general principles underlying the genetic and mechanistic basis of interactions, study of different agents in model host species is required. To this end, the nature and genetics of the maternally inherited sex-ratio trait of Drosophila bifasciata were investigated. Successful curing of affected lines with antibiotics demonstrated this trait was associated with the presence of a bacterium, and molecular systematic analysis demonstrated an association between the presence of the trait and infection with an A group Wolbachia. The penetrance and heritability of the trait did not vary with maternal age. Exposure to elevated temperatures did reduce trait penetrance but did not affect heritability. Examination of the effect of temperature on bacterial density in eggs revealed a decrease in bacterial density following exposure of the parent to elevated temperature, consistent with the hypothesis that male killing in D. bifasciata requires a threshold density of Wolbachia within eggs. The male offspring produced following exposure to elevated temperatures were infected with Wolbachia on emergence as adults. Crossing studies demonstrated a weak cytoplasmic incompatibility phenotype exhibited by Wolbachia in these males. The results are discussed with respect to the incidence of male killing within the clade Wolbachia, the general nature of Wolbachia-host interactions, and the prospects for using this association to investigate the mechanism of male killing.

Chemotherapy of onchocerciasis by doxycycline, which targets symbiotic Wolbachia endobacteria, has been shown to result in a long-term sterility of adult female worms and corresponding absence of microfilariae. It represents an additional chemotherapeutic approach. The aim of this study was to determine whether a similar regimen would also show efficacy against Wuchereria bancrofti. Ghanaian individuals ( n=93) with lymphatic filariasis and a minimum microfilaremia of 40 microfilariae/ml were included in a treatment study consisting of four arms:(1) doxycycline 200 mg/day for 6 weeks;(2) doxycycline as in (1), followed by a single dose of ivermectin after 4 months;(3) ivermectin only; or (4) no treatment during observation period of 1 year (ivermectin at the end of the study). Doxycycline treatment resulted in a 96% loss of Wolbachia, as determined by real time PCR from microfilariae. After 12 months, doxycycline had led to a 99% reduction of microfilaremia when given alone, and to a complete amicrofilaremia together with ivermectin. In contrast, after ivermectin treatment alone a significant presence of microfilariae remained (9% compared to pretreatment), as known from other studies. This study shows that doxycycline is also effective in depleting Wolbachia from W. bancrofti. It is likely that the mechanism of doxycycline is similar to that in other filarial species, i.e., a predominant blockade of embryogenesis, leading to a decline of microfilariae according to their half-life. This could render doxycycline treatment an additional tool for the treatment of microfilaria-associated diseases in bancroftian filariasis, such as tropical pulmonary eosinophilia and microfiluria.