Blood-feeding in the young adult filarial worms litomosoides sigmodontis

In this study with the filarial model Litomosoides sigmodontis, we demonstrate that the worms ingest host red blood cells at a precise moment of their life-cycle, immediately after the fourth moult. The red blood cells (RBC) were identified microscopically in live worms immobilized in PBS at 4 degrees C, and their density assessed. Two hosts were used: Mongolian gerbils, where microfilaraemia is high, and susceptible BALB/c mice with lower microfilaraemia. Gerbils were studied at 12 time-points, between day 9 post-inoculation (the worms were young 4th stage larvae) and day 330 p.i. (worms were old adults). Only the very young adult filarial worms had red blood cells in their gut. Haematophagy was observed between days 25 and 56 p.i. and peaked between day 28 and day 30 p.i. in female worms. In males, haematophagy was less frequent and intense. Similar kinetics of haematophagy were found in BALB/c mice, but frequency and intensity tended to be lower. Haematophagy seems useful to optimize adult maturation. These observations suggest that haematophagy is an important step in the life-cycle of L. sigmodontis. This hitherto undescribed phenomenon might be characteristic of other filarial species including human parasites

Onchocerca parasites and Wolbachia endosymbionts: evaluation of a spectrum of antibiotic types for activity against Onchocerca gutturosa in vitro

BACKGROUND: The filarial parasites of major importance in humans contain the symbiotic bacterium Wolbachia and recent studies have shown that targeting of these bacteria with antibiotics results in a reduction in worm viability, development, embryogenesis, and survival. Doxycycline has been effective in human trials, but there is a need to develop drugs that can be given for shorter periods and to pregnant women and children. The World Health Organisation-approved assay to screen for anti-filarial activity in vitro uses male Onchocerca gutturosa, with effects being determined by worm motility and viability as measured by reduction of MTT to MTT formazan. Here we have used this system to screen antibiotics for anti-filarial activity. In addition we have determined the contribution of Wolbachia depletion to the MTT reduction assay. METHODS: Adult male O. gutturosa were cultured on a monkey kidney cell (LLCMK 2) feeder layer in 24-well plates with antibiotics and antibiotic combinations (6 to 10 worms per group). The macrofilaricide CGP 6140 (Amocarzine) was used as a positive control. Worm viability was assessed by two methods, (i) motility levels and (ii) MTT/formazan colorimetry. Worm motility was scored on a scale of 0 (immotile) to 10 (maximum) every 5 days up to 40 days. On day 40 worm viability was evaluated by MTT/formazan colorimetry, and results were expressed as a mean percentage reduction compared with untreated control values at day 40. To determine the contribution of Wolbachia to the MTT assay, the MTT formazan formation of an insect cell-line (C6/36) with or without insect Wolbachia infection and treated or untreated with tetracycline was compared. RESULTS: Antibiotics with known anti-Wolbachia activity were efficacious in this system. Rifampicin (5 × 10(-5)M) was the most effective anti-mycobacterial agent; clofazimine (1.25 × 10(-5)M and 3.13 × 10(-6)M) produced a gradual reduction in motility and by 40 days had reduced worm viability. The other anti-mycobacterial drugs tested had limited or no activity. Doxycycline (5 × 10(-5)M) was filaricidal, but minocycline was more effective and at a lower concentration (5 × 10(-5)M and 1.25 × 10(-5)M). Inactive compounds included erythromycin, oxytetracycline, trimethoprim and sulphamethoxazole. The MTT assay on the insect cell-line showed that Wolbachia made a significant contribution to the metabolic activity within the cells, which could be reduced when they were exposed to tetracycline. CONCLUSION: The O. gutturosa adult male screen for anti-filarial drug activity is also valid for the screening of antibiotics for anti-Wolbachia activity. In agreement with previous findings, rifampicin and doxycycline were effective; however, the most active antibiotic was minocycline. Wolbachia contributed to the formation of MTT formazan in the MTT assay of viability and is therefore not exclusively a measure of worm viability and indicates that Wolbachia contributes directly to the metabolic activity of the nematode

Macrofilaricidal Activity after Doxycycline Only Treatment of Onchocerca volvulus in an Area of Loa loa Co-Endemicity: A Randomized Controlled Trial

The control of onchocerciasis in Africa relies on the sustained delivery of ivermectin. In certain areas, annual treatments delivered with high population coverage for at least 15–17 years can break transmission. In other endemic settings this strategy alone is thought to be insufficient to eradicate the disease. One of the major limitations occurs in areas that are co-endemic with another filarial infection caused by Loa loa, due to the risk of a rare severe adverse event associated with the rapid killing of L. loa microfilariae in heavily parasitized individuals. There are also concerns over recent evidence of reduced efficacy of ivermectin and the possible development of resistance. An alternative approach is to target the Wolbachia bacterial endosymbionts of Onchocerca volvulus with the antibiotic, doxycycline. In an area of Cameroon co-endemic for onchocerciasis and loiasis we conducted a trial comparing doxycycline with or without ivermectin treatment to ivermectin treatment alone. A six-week course of doxycycline delivers macrofilaricidal and sterilizing activities, which is not dependent upon co-administration of ivermectin. Doxycycline is well tolerated in patients co-infected with moderate intensities of L. loa microfilariae. The trial indicates that anti-wolbachial therapy is a feasible alternative to ivermectin in communities co-endemic for onchocerciasis and loiasis

Criteria for the differentiation between young and old Onchocerca volvulus filariae

Drugs exist that show long-lasting inhibition of embryogenesis and microfilaria production or macrofilaricidal activity against Onchocerca volvulus. Therefore, the patients have to be followed-up for several years. Clinical drug trials have to be performed in areas with ongoing transmission to assess the efficacy on younger worms. In addition, future vaccine trials may also require demonstrating efficacy against establishment of new worms. For the evaluation of the efficacy, it is necessary to differentiate between older worms, which were exposed to the drug, and younger worms newly acquired after drug treatment or vaccination. Here, we describe criteria for the differentiation between young and old filariae based on histological studies of worms with a known age from travellers, or from children, or patients living in areas with interrupted transmission in Burkina Faso, Ghana or Uganda. Older worms were larger and presented degenerated tissues. Gomori's iron stain showed that the worms accumulated more iron with increasing age, first in the gut and later in other organs. Using an antibody against O. volvulus lysosomal aspartic protease, the gut of young worms was stained only weakly; whereas, it was stronger labelled in older worms, accompanied by additional staining of hypodermis and epithelia. Using morphological and immunohistological criteria, it was possible to differentiate young (1–3 years old) from older females and to identify young males

The Wolbachia Genome of Brugia malayi: Endosymbiont Evolution within a Human Pathogenic Nematode

Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease

Alternative activation of macrophages by filarial nematodes is MyD88-independent

AbstractAlternative macrophage activation is largely defined by IL-4Rα stimulation but the contribution of Toll-like receptor (TLR) signaling to this phenotype is not currently known. We have investigated macrophage activation status under Th2 conditions in the absence of the core TLR adaptor molecule, MyD88. No impairment was observed in the ability of MyD88-deficient bone marrow derived macrophages to produce or express alternative activation markers, including arginase, RELM-α or Ym1, in response to IL-4 treatment in vitro. Further, we observed no difference in the ability of peritoneal exudate cells from nematode implanted wild type (WT) or MyD88-deficient mice to produce arginase or express the alternative activation markers RELM-α or Ym1. Therefore, MyD88 is not a fundamental requirement for Th2-driven macrophage alternative activation, either in vitro or in vivo

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18–24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks

Wolbachia endobacteria depletion by doxycycline as antifilarial therapy has macrofilaricidal activity in onchocerciasis: a randomized placebo-controlled study

In a randomized, placebo-controlled trial in Ghana, 67 onchocerciasis patients received 200-mg/day doxycycline for 4–6 weeks, followed by ivermectin (IVM) after 6 months. After 6–27 months, efficacy was evaluated by onchocercoma histology, PCR and microfilariae determination. Administration of doxycycline resulted in endobacteria depletion and female worm sterilization. The 6-week treatment was macrofilaricidal, with >60% of the female worms found dead, despite the presence of new, Wolbachia-containing worms acquired after the administration of doxycycline. Doxycycline may be developed as second-line drug for onchocerciasis, to be administered in areas without transmission, in foci with IVM resistance and in areas with Loa co-infections