Establishment of an in vitro culture system to study the developmental biology (growth, mating and nodule formation) of Onchocerca volvulus with implications for anti-onchocerca drug discovery and screening (original) (raw)
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Parasites & Vectors, 2012
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.
The study was carried out to verify reports of persistent transmission of onchocerciasis, despite long-term Community Directed Treatment with Ivermectin, (CDTI). Blackflies were collected along Asu River using human baits. A total of 91 female Simulium damnosum complex adults were caught and dissected-using a dissecting microscope-for microfilariae of O. volvulus. 58 flies representing (63.70%) of the total number of captured flies were caught in the month of June while 33 (36.20%) were caught in July. Despite ivermectin treatment, evidence of O. volvulus transmission was documented in Amagu Agba. A total of 27 larvae were recovered from the three body segments of the dissected flies, of this number, 17 larvae were recovered from the head region and a total of 10 were recovered from the thorax and abdomen representing 62.96 % and 37.04 % respectively of the total number of larvae recovered. The proportion of infected flies recorded was 8 (8.8 %) while the proportion of infective flies recorded was 17 (18.7%) of the total number of infected and infective flies. The high proportion of infective flies is indicative of active transmission in and around the study area. This should not be so as CDTI is currently going on in the area. Some militating factors such as: patterns of treatment coverage and compliance, parasite ivermectin susceptibility, parasite immigration in flies or people, may be responsible for the current unsuccessful ivermectin treatment strategy.There is therefore need for consistent and effective ivermectin distribution pattern and its resultant onchocerciasis prevalence, detected with blackfly dissection techniques and or more reliably detected in both blackfly population and human subjects through a combination of some molecular-based detection techniques and biocontrol approaches. Adopting these measures together with community-ownership participation concept, will successfully interrupt transmission in Amagu Agba community and other endemic areas.
PLoS Neglected Tropical Diseases, 2014
Background: For two decades, onchocerciasis control has been based on mass treatment with ivermectin (IVM), repeated annually or six-monthly. This drug kills Onchocerca volvulus microfilariae (mf) present in the skin and the eyes (microfilaricidal effect) and prevents for 3-4 months the release of new mf by adult female worms (embryostatic effect). In some Ghanaian communities, the long-term use of IVM was associated with a more rapid than expected skin repopulation by mf after treatment. Here, we assessed whether the embryostatic effect of IVM on O. volvulus has been altered following frequent treatment in Cameroonian patients.
Development of Onchocerca volvulus larvae in Simulium pintoi in the Amazonas region of Venezuela.
Am J Trop Med Hyg 33(3):414-9, 1984
Takaoka H, Suzuki H, Noda S, Tada I, Basanez MG, Yarzabal L. The intake and development of Onchocerca volvulus in Simulium pintoi from the Parima mountain region of the Federal Territory of Amazonas in Venezuela, were studied experimentally. When wild females fed on the lower half of the legs and lower third of the back of an onchocerciasis patient harboring 23 and 264 microfilariae per skin snip, respectively, at each site, an average (median) of 14 (range, 1-77) and 245 (range, 58-495) microfilariae was ingested. However, within 24 hours of microfilarial ingestion a mortality of 47% (16/34 flies) was observed in the group of flies which fed on the back, as compared with 2% (2/101 flies) in the other group which fed on the legs. At a temperature varying between 16 degrees C and 24 degrees C, the development of O. volvulus larvae in S. pintoi was synchronous and orderly; no abnormal nor deformed larvae were observed. Third-stage larvae were first seen in the head of flies dying between 8 and 9 days after microfilarial ingestion, and 98 of 100 larvae recovered from days 10-16 were in the third stage. The proportions of females harboring third-stage larvae among flies which lived through day 8 in the two groups which fed on the legs and back, respectively, were 55% (21/38 flies) and 63% (5/8 flies). Although only two of five positive flies in the latter group contained third-stage larvae in the head (1 and 12, respectively), 71% (15/21 positive flies) of the former group had an average of 2.7 third-stage larvae in the head (range, 1-10).(ABSTRACT TRUNCATED AT 250 WORDS) PMID: 6731673 [PubMed - indexed for MEDLINE]
Transactions of the Royal Society of Tropical Medicine and Hygiene, 1990
The effects of ivermectin at a concentration of 3-13~ 10w6~ used in combination with other antiparasitic drugs on the viability of adult Onchocerca in vitro were assessed using MTT calorimetry and worm motility levels. When ivermectin was used against male 0. gutturosa over a 7 d period in combination with suramin (5x 10m5hl), CGP 6140 (3.13~ ~O+M), CGP 20376 (1.95 x lo-'M), mefloquine (3.13~ 10-6~), levamisole (3.13~ 10e6h.l), mebendazole (5 x 10e5~) flubendazole (5 x ~O-'M) and albendazole (5~ lo-'MM), there was either no increased effect or only a marginally increased effect on motility levels when compared with the use of ivermectin alone. MTT calorimetry revealed that in most cases there was a cumulative effect of the 2 drugs used in combination but not a synergistic effect. In a trial extended to 26 d it was demonstrated that the combination of ivermectin and suramin did not produce a greater inhibition of motility than ivermectin alone. Using female 0. volvulus, the activity of ivermectin, CGP 6140 and the 2 drugs combined was examined. The motility of all 3 groups exposed to drug(s) was suppressed by 24 h compared with controls. MTT calorimetry performed on day 7, using the pre-weighed anterior end of each worm, illustrated that ivermectin alone produced a 43.4% inhibition of formazan formation compared with controls. CGP 6140 alone nroduced 50.6% inhibition. while thi drug combinatibn produced a 72% inhibition, equivalent to the heat-killed control. The cumulative effects seen with certain drug combinations in vitro should be examined in vivo, where they may have some practical value in the elimination of adult Onchocerca.
The American journal of tropical medicine and hygiene, 2004
The objective of this study was to examine nodules from Mexico, Guatemala, and Ecuador collected over a one-year period (2001) to determine the effects of semi-annual ivermectin treatments on Onchocerca volvulus macrofilarial populations. Nodules were sectioned, stained with hematoxylin and eosin, and histologic findings were compared between countries and with historical data prior to the introduction of ivermectin into the region. Nodules from Ecuador had 10 times more dead or moribund worms than the historical control (66.6% versus 6.5%); nodules from patients from Mexico and Guatemala did not differ from the control. More than 80% of the female worms in each country were uninseminated and producing unfertilized oocytes. Nodules containing males differed in each country from the historical control (P < 0.0001), with presence of males ranging from 19.7% in Mexico to 13.6% in Ecuador versus 73% in the control. Nodules with females producing active microfilariae ranged from 7.8% ...
Phosphoprotein Patterns in Onchocerca Volvulus Developmental Stages
Upsala Journal of Medical Sciences, 1994
Living females and microfilariae of Onchocerca volvulus incubated in culture medium containing [32P]orthophosphate were observed to phosphorylate their proteins rapidly. Patterns of phosphoproteins in extracts from these labelled parasites were compared after two dimensional electrophoresis and autoradiography. Protein extracts from eggs, microfilariae and adult females of 0. volvulus were phosphorylated in the presence of [ y -32P]ATP, magnesium acetate, and added cyclic AMPdependent protein kinase or the endogenous protein kinase present in the extracts. Patterns of phosphoproteins were compared after separation by single and twodimensional gel electrophoresis followed by autoradiography. Common phosphopeptide bands were observed when phosphorylated extracts from adult females, microfilariae and eggs were compared. However, extracts from eggs displayed unique phosphorylated polypeptides of M, 30 000 and 34 OOO that were absent from the extracts from microfilariae. Furthermore, two phosphorylated polypeptides of M, 47 OOO and 76 OOO were detected in extracts from microfilariae but not from eggs. These results indicate that 0. volvulus parasites may phosphorylate different proteins at different stages of their development. Onchocercu volvulus is a filarial parasite that infests about 17 million people living in the endemic tropical countries with a risk population of about 85 million (22). Onchocerciasis, the disease caused by this parasite, results in severe pruritus, dermatitis, subcutaneous nodules and eye involvement leading to blindness. Consequently, considerable effort has been employed to develop new and effective control measures for the disease (22). This notwithstanding, large scale eradication of onchocerciasis is not yet feasible. The microfilaricide ivermectin, at the normal dose regimen, 39 Ups J Med Sci Downloaded from informahealthcare.com by 91.187.117.6 on 05/20/14 For personal use only. Ups J Med Sci Downloaded from informahealthcare.com by 91.187.117.6 on 05/20/14 For personal use only. 42 Ups J Med Sci Downloaded from informahealthcare.com by 91.187.117.6 on 05/20/14 For personal use only.
American Journal …, 1984
The intake and development of Onchocerca volvulus in Simulium pintoi from the Parima mountain region of the Federal Territory of Amazonas in Venezuela, were studied experimentally. When wild females fed on the lower halfofthe legs and lower third of the back of an onchocerciasis patient harboring 23 and 264 microfllariae per skin snip, respectively, at each site, an average (median) of 14 (range, 1â€"77) and 245 (range, 58â€"495) microfilariae was ingested. However, within 24 hours of microfilarial ingestion a mortality of 47% (16/34 flies) was observed in the group of flies which fed on the back, as compared