Retarded Onchocerca volvulus L1 to L3 larval development in the Simulium damnosum vector after anti-wolbachial treatment of the human host

Abstract

Background: The human parasite Onchocerca volvulus harbours Wolbachia endosymbionts essential for worm embryogenesis, larval development and adult survival. In this study, the development of Wolbachia-depleted microfilariae (first stage larvae) to infective third stage larvae (L3) in the insect vector Simulium damnosum was analysed.

Methods: Infected volunteers in Cameroon were randomly and blindly allocated into doxycycline (200 mg/day for 6 weeks) or placebo treatment groups. After treatment, blackflies were allowed to take a blood meal on the volunteers, captured and dissected for larval counting and DNA extraction for quantitative real-time PCR analysis.

Results: PCR results showed a clear reduction in Wolbachia DNA after doxycycline treatment in microfilariae from human skin biopsies with > 50% reduction at one month post-treatment, eventually reaching a reduction of > 80%. Larval stages recovered from the insect vector had similar levels of reduction of endosymbiotic bacteria. Larval recoveries were analysed longitudinally after treatment to follow the kinetics of larval development. Beginning at three months post-treatment, significantly fewer L3 were seen in the blackflies that had fed on doxycycline treated volunteers. Concomitant with this, the proportion of second stage larvae (L2) was significantly increased in this group.

Conclusions: Doxycycline treatment and the resulting decline of Wolbachia endobacteria from the microfilaria resulted in retarded development of larvae in the insect vector. Thus, anti-wolbachial treatment could have an additive effect for interrupting transmission by reducing the number of L3 that can be transmitted by blackflies.

Figure 1

Study timeline of recruitment, treatment and analysis. Patients were recruited in November/December 2006. After the informed consent was signed by each volunteer, skin biopsies were taken from which Mf were isolated and counted. Volunteers were allocated into two treatment groups. Before treatment with doxycycline or placebo (pre-treatment, month 0), volunteers were brought to the capture side to allow Simulium damnosum flies to take a blood meal. Flies were captured and raised in an insectarium for seven days. After dissecting the flies Onchocerca volvulus larvae were counted. This was repeated monthly for five months after doxycycline treatment was completed.

Figure 2

Depletion of Wolbachia from Onchocerca volvulus larvae reduces the number of larvae that develop into the infective stage in the Simulium vector. The proportion of larvae at each stage was calculated by dividing the number of larvae (stage L1+L2; L3) by the total larvae collected from the captured blackflies after they had fed on the doxycycline (n = 7) or placebo (n = 5) treated volunteers. The proportion of L1+L2 larvae in flies that had fed on doxycycline treated individuals (black bars) was higher beginning at 3 months post-treatment compared to placebo. The proportion of L3 larvae in flies that had fed on doxycycline treated volunteers (white bars) was lower than that in the placebo volunteers at 3, 4 and 5 months post-treatment. Proportions are given as percentages of median larval recovery. 100% corresponded to the total number of larvae recovered at each indicated time.