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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.

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