Oligonucleotide based magnetic bead capture of Onchocerca volvulus DNA for PCR pool screening of vector black flies

Background

Entomological surveys of Simulium vectors are an important component in the criteria used to determine if Onchocerca volvulus transmission has been interrupted and if focal elimination of the parasite has been achieved. However, because infection in the vector population is quite rare in areas where control has succeeded, large numbers of flies need to be examined to certify transmission interruption. Currently, this is accomplished through PCR pool screening of large numbers of flies. The efficiency of this process is limited by the size of the pools that may be screened, which is in turn determined by the constraints imposed by the biochemistry of the assay. The current method of DNA purification from pools of vector black flies relies upon silica adsorption. This method can be applied to screen pools containing a maximum of 50 individuals (from the Latin American vectors) or 100 individuals (from the African vectors).

Methodology/Principal Findings

We have evaluated an alternative method of DNA purification for pool screening of black flies which relies upon oligonucleotide capture of Onchocerca volvulus genomic DNA from homogenates prepared from pools of Latin American and African vectors. The oligonucleotide capture assay was shown to reliably detect one O. volvulus infective larva in pools containing 200 African or Latin American flies, representing a two-four fold improvement over the conventional assay. The capture assay requires an equivalent amount of technical time to conduct as the conventional assay, resulting in a two-four fold reduction in labor costs per insect assayed and reduces reagent costs to $3.81 per pool of 200 flies, or less than $0.02 per insect assayed.

Conclusions/Significance

The oligonucleotide capture assay represents a substantial improvement in the procedure used to detect parasite prevalence in the vector population, a major metric employed in the process of certifying the elimination of onchocerciasis.     

Onchocerciasis-associated epilepsy: an update and future perspectives

Onchocerciasis-associated epilepsy (OAE) is an important neglected public health problem in areas with high ongoing onchocerciasis transmission. The risk that children in such areas develop epilepsy is related to their Onchocerca volvulus microfilarial (mf) load. Before the implementation of mass treatment with ivermectin, microfilariae were detected in cerebrospinal fluid (CSF). More recently, neither O. volvulus microfilariae nor DNA were detected in CSF or brain tissue; however, these samples were obtained years after seizure onset. It is possible that during fever-induced increased blood–brain barrier permeability, microfilariae enter the brain and, upon dying, cause an inflammatory reaction inducing seizures. Including OAE in the onchocerciasis disease burden estimation may mobilise extra resources for onchocerciasis disease elimination and treatment/care of OAE-affected persons/families.     

The Mbam drainage system and onchocerciasis transmission post ivermectin mass drug administration (MDA) campaign,Cameroon

BackgroundThe impact of large scale Mass Drug Adminstration (MDA) of ivermectin on active onchocerciasis transmission by Simulium damnosum, which transmits the parasite O. volvulus is of great importance for onchocerciasis control programmes. We investigated in the Mbam river system area, the impact of MDA of ivermectin on entomological indices and also verify if there are river system factors that could have favoured the transmission of onchocerciasis in this area and contribute to the persistence of disease. We compared three independent techniques to detect Onchocerca larvae in blackflies and also analyzed the river system within 9 months post-MDA of ivermectin.MethodSimulium flies were captured before and after 1, 3, 6 and 9months of ivermectin-MDA. The biting rate was determined and 41% of the flies dissected while the rest were grouped into pools of 100 flies for DNA extraction. The extracted DNA was then subjected to O-150 LAMP and real-time PCR for the detection of infection by Onchocerca species using pool screening. The river system was analysed and the water discharge compared between rainy and dry seasons.Principal findingsWe used human landing collection method (previously called human bait) to collect 22,274 adult female Simulium flies from Mbam River System. Of this number, 9,134 were dissected while 129 pools constituted for molecular screening. Overall biting and parous rates of 1113 flies/man/day and 24.7%, respectively, were observed. All diagnostic techniques detected similar rates of O. volvulus infection (P = 0.9252) and infectivity (P = 0.4825) at all monitoring time points. Onchocerca ochengi larvae were only detected in 2 of the 129 pools. Analysis of the river drainage revealed two hydroelectric dams constructed on the tributaries of the Mbam river were the key contributing factor to the high-water discharge during both rainy and dry seasons.ConclusionResults from fly dissection (Microscopy), real-time PCR and LAMP revealed the same trends pre- and post-MDA. The infection rate with animal Onchocerca sp was exceptionally low. The dense river system generate important breeding sites that govern the abundance of Simulium during both dry and rainy seasons.     

Onchocerca volvulus-specific antibody and cellular responses in onchocerciasis patients treated annually with ivermectin for 30 years and exposed to parasite transmission in central Togo

BackgroundAnnual mass drug administrations (MDA) of ivermectin will strongly reduce Onchocerca volvulus microfilariae (mf) in the skin and in the onchocerciasis patients’ eyes. Ivermectin treatment will also affect the expression of immunity in patients, such that activated immune defenses may help control and contribute to clearance of mf of O. volvulus. Longitudinal surveys are a prerequisite to determining the impact of ivermectin on the status of anti-parasite immunity, notably in risk zones where parasite transmission and active O. volvulus infections persist.Methodology/Principal findingsOnchocerciasis patients were treated annually with ivermectin and their Onchocerca volvulus antigen (OvAg) specific IgG and cellular responses were investigated before and at 30 years post initial ivermectin treatment (30yPT).Repeated annual ivermectin treatments eliminated persisting O. volvulus microfilariae (mf) from the skin of patients and abrogated patent infections. The OvAg-specific IgG1 and IgG4 responses were diminished at 30yPT to the levels observed in endemic controls. Prior to starting ivermectin treatment, OvAg-induced cellular productions of IL-10, IFN-γ, CCL13, CCL17 and CCL18 were low in patients, and at 30yPT, cellular cytokine and chemokine responses increased to the levels observed in endemic controls. In contrast, mitogen(PHA)- induced IL-10, IFN-γ, CCL17 and CCL18 cellular production was diminished. This divergent response profile thus revealed increased parasite antigen-specific but reduced polyclonal cellular responsiveness in patients. The transmission of O. volvulus continued at the patients’ location in the Mô river basin in central Togo 2018 and 2019 when 0.58% and 0.45%, respectively, of Simulium damnosum s.l. vector blackflies carried O. volvulus infections.Conclusions/SignificanceRepeated annual ivermectin treatment of onchocerciasis patients durably inhibited their patent O. volvulus infections despite ongoing low-level parasite transmission in the study area. Repeated MDA with ivermectin affects the expression of immunity in patients. O. volvulus parasite-specific antibody levels diminished to levels seen in infection-free endemic controls. With low antibody levels, antibody-dependent cellular cytotoxic responses against tissue-dwelling O. volvulus larvae will weaken. O. volvulus antigen inducible cytokine and chemokine production increased in treated mf-negative patients, while their innate responsiveness to mitogen declined. Such lower innate responsiveness in elderly patients could contribute to reduced adaptive immune responses to parasite infections and vaccines. On the other hand, increased specific cellular chemokine responses in mf-negative onchocerciasis patients could reflect effector cell activation against tissue invasive larval stages of O. volvulus. The annual Simulium damnosum s.l. biting rate observed in the Mô river basin was similar to levels prior to initiation of MDA with ivermectin, and the positive rtPCR results reported here confirm ongoing O. volvulus transmission.     

Diagnosis of Brugian Filariasis by Loop-Mediated Isothermal Amplification

In this study we developed and evaluated a Brugia Hha I repeat loop-mediated isothermal amplification (LAMP) assay for the rapid detection of Brugia genomic DNA. Amplification was detected using turbidity or fluorescence as readouts. Reactions generated a turbidity threshold value or a clear visual positive within 30 minutes using purified genomic DNA equivalent to one microfilaria. Similar results were obtained using DNA isolated from blood samples containing B. malayi microfilariae. Amplification was specific to B. malayi and B. timori, as no turbidity was observed using DNA from the related filarial parasites Wuchereria bancrofti, Onchocerca volvulus or Dirofilaria immitis, or from human or mosquito. Furthermore, the assay was most robust using a new strand-displacing DNA polymerase termed Bst 2.0 compared to wild-type Bst DNA polymerase, large fragment. The results indicate that the Brugia Hha I repeat LAMP assay is rapid, sensitive and Brugia-specific with the potential to be developed further as a field tool for diagnosis and mapping of brugian filariasis.     

Immunisation with a Multivalent,Subunit Vaccine Reduces Patent Infection in a Natural Bovine Model of Onchocerciasis during Intense Field Exposure

Human onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is controlled almost exclusively by the drug ivermectin, which prevents pathology by targeting the microfilariae. However, this reliance on a single control tool has led to interest in vaccination as a potentially complementary strategy. Here, we describe the results of a trial in West Africa to evaluate a multivalent, subunit vaccine for onchocerciasis in the naturally evolved host-parasite relationship of Onchocerca ochengi in cattle. Naïve calves, reared in fly-proof accommodation, were immunised with eight recombinant antigens of O. ochengi, administered separately with either Freund''s adjuvant or alum. The selected antigens were orthologues of O. volvulus recombinant proteins that had previously been shown to confer protection against filarial larvae in rodent models and, in some cases, were recognised by serum antibodies from putatively immune humans. The vaccine was highly immunogenic, eliciting a mixed IgG isotype response. Four weeks after the final immunisation, vaccinated and adjuvant-treated control calves were exposed to natural parasite transmission by the blackfly vectors in an area of Cameroon hyperendemic for O. ochengi. After 22 months, all the control animals had patent infections (i.e., microfilaridermia), compared with only 58% of vaccinated cattle (P = 0.015). This study indicates that vaccination to prevent patent infection may be an achievable goal in onchocerciasis, reducing both the pathology and transmissibility of the infection. The cattle model has also demonstrated its utility for preclinical vaccine discovery, although much research will be required to achieve the requisite target product profile of a clinical candidate.     

Repurposing Auranofin as a Lead Candidate for Treatment of Lymphatic Filariasis and Onchocerciasis

Two major human diseases caused by filariid nematodes are onchocerciasis, or river blindness, and lymphatic filariasis, which can lead to elephantiasis. The drugs ivermectin, diethylcarbamazine (DEC), and albendazole are used in control programs for these diseases, but are mainly effective against the microfilarial stage and have minimal or no effect on adult worms. Adult Onchocerca volvulus and Brugia malayi worms (macrofilariae) can live for up to 15 years, reproducing and allowing the infection to persist in a population. Therefore, to support control or elimination of these two diseases, effective macrofilaricidal drugs are necessary, in addition to current drugs. In an effort to identify macrofilaricidal drugs, we screened an FDA-approved library with adult worms of Brugia spp. and Onchocerca ochengi, third-stage larvae (L3s) of Onchocerca volvulus, and the microfilariae of both O. ochengi and Loa loa. We found that auranofin, a gold-containing drug used for rheumatoid arthritis, was effective in vitro in killing both Brugia spp. and O. ochengi adult worms and in inhibiting the molting of L3s of O. volvulus with IC₅₀ values in the low micromolar to nanomolar range. Auranofin had an approximately 43-fold higher IC₅₀ against the microfilariae of L. loa compared with the IC₅₀ for adult female O. ochengi, which may be beneficial if used in areas where Onchocerca and Brugia are co-endemic with L. loa, to prevent severe adverse reactions to the drug-induced death of L. loa microfilariae. Further testing indicated that auranofin is also effective in reducing Brugia adult worm burden in infected gerbils and that auranofin may be targeting the thioredoxin reductase in this nematode.     

Onchocerca spp: cryopreservation of microfilariae and subsequent development in the insect host.

Microfilariae of Onchocerca gutturosa, O. cervicalis and O. volvulus were successfully recovered after freezing, storage at ?196 C, and thawing. The technique that produced maximum viability involved a two-step cooling schedule consisting of an initial slow cool of 1 C min^?1 to an intermediate temperature of between ?14 and ?17 C, followed by a rapid cool into liquid nitrogen (taking about 1 sec). Upon rapid warming to 37 C, a high percentage of microfilariae showed normal motility. Following subcutaneous injection into T.O. mice, the microfilariae of O. gutturosa migrated to the skin of the ears and nose, and a proportion of them developed into third-stage larvae in the insect vector, Simulium ornatum. Microfilariae of O. volvulus also developed into third-stage larvae in this insect, while those of O. cervicalis developed similarly in their natural vector, Culicoides nubeculosus. This technique of preservation provides a good and reliable method for storage of viable microfilariae of these bovine, equine, and human Onchocerca spp.     

Identification of Human Semiochemicals Attractive to the Major Vectors of Onchocerciasis

Background

Entomological indicators are considered key metrics to document the interruption of transmission of Onchocerca volvulus, the etiological agent of human onchocerciasis. Human landing collection is the standard employed for collection of the vectors for this parasite. Recent studies reported the development of traps that have the potential for replacing humans for surveillance of O. volvulus in the vector population. However, the key chemical components of human odor that are attractive to vector black flies have not been identified.

Methodology/Principal Findings

Human sweat compounds were analyzed using GC-MS analysis and compounds common to three individuals identified. These common compounds, with others previously identified as attractive to other hematophagous arthropods were evaluated for their ability to stimulate and attract the major onchocerciasis vectors in Africa (Simulium damnosum sensu lato) and Latin America (Simulium ochraceum s. l.) using electroantennography and a Y tube binary choice assay. Medium chain length carboxylic acids and aldehydes were neurostimulatory for S. damnosum s.l. while S. ochraceum s.l. was stimulated by short chain aliphatic alcohols and aldehydes. Both species were attracted to ammonium bicarbonate and acetophenone. The compounds were shown to be attractive to the relevant vector species in field studies, when incorporated into a formulation that permitted a continuous release of the compound over time and used in concert with previously developed trap platforms.

Conclusions/Significance

The identification of compounds attractive to the major vectors of O. volvulus will permit the development of optimized traps. Such traps may replace the use of human vector collectors for monitoring the effectiveness of onchocerciasis elimination programs and could find use as a contributing component in an integrated vector control/drug program aimed at eliminating river blindness in Africa.     

In vitro haemagglutination and attenuation of microfilarial motility by haemolymph from individual blackflies (Simulium ornatum) infected with Onchocerca lienalis

Two in vitro tests were used to investigate the effect of Onchocerca lienalis Stiles infection on the haemolymph of Simulium ornatum Meigen. The first of these examined the effect of infected haemolymph on the motility of fresh O. lienalis or Brugia pahangi Buckley & Edeson microfilariae. Incubation of haemolymph from individual flies with fresh microfilariae was performed in the wells of Terasaki micro-tissue culture plates. Motility of both species of parasite was found to be significantly attenuated when compared to worms incubated in control haemolymph groups. The second assay was that of agglutination of cat erythrocytes in the presence of haemolymph from individual flies, also performed in Terasaki plates. This test demonstrated significant increases in the rates of haemagglutination in the haemolymph of O. lienalis infected blackflies. The titre appeared to increase during the initial 5 days of infection up to a level of 1/32+, but then fell between day 5 and 7 to a maximum level of 1/2. The proportion of flies exhibiting haemagglutination also rose following infection. Despite the apparent absence of melanization and encapsulation, simuliids may have at least two humoral haemolymph components available to them for parasite regulation; a fast-acting factor responsible for rapid parasite death, and more specific agglutinins, possibly lectins. The role of the latter in defence is as yet unclear.     

Lymphatic Vascularisation and Involvement of Lyve-1+ Macrophages in the Human Onchocerca Nodule

Onchocerciasis, caused by the filarial nematode Onchocerca volvulus, is a parasitic disease leading to debilitating skin disease and blindness, with major economic and social consequences. The pathology of onchocerciasis is principally considered to be a consequence of long-standing host inflammatory responses. In onchocerciasis a subcutaneous nodule is formed around the female worms, the core of which is a dense infiltrate of inflammatory cells in which microfilariae are released. It has been established that the formation of nodules is associated with angiogenesis. In this study, we show using specific markers of endothelium (CD31) and lymphatic endothelial cells (Lyve-1, Podoplanin) that not only angiogenesis but also lymphangiogenesis occurs within the nodule. 7% of the microfilariae could be found within the lymphatics, but none within blood vessels in these nodules, suggesting a possible route of migration for the larvae. The neovascularisation was associated with a particular pattern of angio/lymphangiogenic factors in nodules of onchocerciasis patients, characterized by the expression of CXCL12, CXCR4, VEGF-C, Angiopoietin-1 and Angiopoietin-2. Interestingly, a proportion of macrophages were found to be positive for Lyve-1 and some were integrated into the endothelium of the lymphatic vessels, revealing their plasticity in the nodular micro-environment. These results indicate that lymphatic as well as blood vascularization is induced around O. volvulus worms, either by the parasite itself, e.g. by the release of angiogenic and lymphangiogenic factors, or by consecutive host immune responses.     

Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite

Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.     

Interruption of Transmission of Onchocerca volvulus in the Southern Chiapas Focus,México

Background

The Southern Chiapas focus of onchocerciasis in Southern Mexico represents one of the major onchocerciasis foci in Latin America. All 559 endemic communities of this focus have undergone semi-annual mass treatment with ivermectin since 1998. In 50 communities of this focus, ivermectin frequency shifted from twice to four times a year in 2003; an additional 113 communities were added to the quarterly treatment regimen in 2009 to achieve a rapid suppression of transmission.

Methodology/Principal findings

In-depth epidemiologic and entomologic assessments were performed in six sentinel communities (which had undergone 2 rounds of ivermectin treatment per year) and three extra-sentinel communities (which had undergone 4 rounds of ivermectin treatment per year). None of the 67,924 Simulium ochraceum s.l. collected from this focus during the dry season of 2011 were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in an upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.06/2,000 flies examined. Serological assays testing for Onchocerca volvulus exposure conducted on 4,230 children 5 years of age and under (of a total population of 10,280 in this age group) revealed that 2/4,230 individuals were exposed to O. volvulus (0.05%; one sided 95% confidence interval = 0.08%).

Conclusions/Significance

The in-depth epidemiological and entomological findings from the Southern Chiapas focus meet the criteria for interruption of transmission developed by the international community.     

Elimination of Onchocerciasis from Mexico

Background

Mexico is one of the six countries formerly endemic for onchocerciasis in Latin America. Transmission has been interrupted in the three endemic foci of that country and mass drug distribution has ceased. Three years after mass drug distribution ended, post-treatment surveillance (PTS) surveys were undertaken which employed entomological indicators to check for transmission recrudescence.

Methodology/Principal findings

In-depth entomologic assessments were performed in 18 communities in the three endemic foci of Mexico. None of the 108,212 Simulium ochraceum s.l. collected from the three foci were found to contain parasite DNA when tested by polymerase chain reaction-enzyme-linked immunosorbent assay (PCR-ELISA), resulting in a maximum upper bound of the 95% confidence interval (95%-ULCI) of the infective rate in the vectors of 0.035/2,000 flies examined. This is an order of magnitude below the threshold of a 95%-ULCI of less than one infective fly per 2,000 flies tested, the current entomological criterion for interruption of transmission developed by the international community. The point estimate of seasonal transmission potential (STP) was zero, and the upper bound of the 95% confidence interval for the STP ranged from 1.2 to 1.7 L₃/person/season in the different foci. This value is below all previous estimates for the minimum transmission potential required to maintain the parasite population.

Conclusions/Significance

The results from the in-depth entomological post treatment surveillance surveys strongly suggest that transmission has not resumed in the three foci of Mexico during the three years since the last distribution of ivermectin occurred; it was concluded that transmission remains undetectable without intervention, and Onchocerca volvulus has been eliminated from Mexico.     

Emodepside targets SLO-1 channels of Onchocerca ochengi and induces broad anthelmintic effects in a bovine model of onchocerciasis

Onchocerciasis (river blindness), caused by the filarial worm Onchocerca volvulus, is a neglected tropical disease mostly affecting sub-Saharan Africa and is responsible for >1.3 million years lived with disability. Current control relies almost entirely on ivermectin, which suppresses symptoms caused by the first-stage larvae (microfilariae) but does not kill the long-lived adults. Here, we evaluated emodepside, a semi-synthetic cyclooctadepsipeptide registered for deworming applications in companion animals, for activity against adult filariae (i.e., as a macrofilaricide). We demonstrate the equivalence of emodepside activity on SLO-1 potassium channels in Onchocerca volvulus and Onchocerca ochengi, its sister species from cattle. Evaluation of emodepside in cattle as single or 7-day treatments at two doses (0.15 and 0.75 mg/kg) revealed rapid activity against microfilariae, prolonged suppression of female worm fecundity, and macrofilaricidal effects by 18 months post treatment. The drug was well tolerated, causing only transiently increased blood glucose. Female adult worms were mostly paralyzed; however, some retained metabolic activity even in the multiple high-dose group. These data support ongoing clinical development of emodepside to treat river blindness.     

Ongoing Transmission of Onchocerca volvulus after 25 Years of Annual Ivermectin Mass Treatments in the Vina du Nord River Valley,in North Cameroon

Background

Recent reports of transmission interruption of Onchocerca volvulus, the causing agent of river blindness, in former endemic foci in the Americas, and more recently in West and East Africa, raise the question whether elimination of this debilitating disease is underway after long-term treatment of the population at risk with ivermectin. The situation in Central Africa has not yet been clearly assessed.

Methods and findings

Entomologic data from two former endemic river basins in North Cameroon were generated over a period of 43 and 48 months to follow-up transmission levels in areas under prolonged ivermectin control. Moreover, epidemiologic parameters of animal-borne Onchocerca spp. transmitted by the same local black fly vectors of the Simulium damnosum complex were recorded and their impact on O. volvulus transmission success evaluated. With mitochondrial DNA markers we unambiguously confirmed the presence of infective O. volvulus larvae in vectors from the Sudan savannah region (mean Annual Transmission Potential 2009–2012: 98, range 47–221), but not from the Adamawa highland region. Transmission rates of O. ochengi, a parasite of Zebu cattle, were high in both foci.

Conclusions/significance

The high cattle livestock density in conjunction with the high transmission rates of the bovine filaria O. ochengi prevents the transmission of O. volvulus on the Adamawa plateau, whereas transmission in a former hyperendemic focus was markedly reduced, but not completely interrupted after 25 years of ivermectin control. This study may be helpful to gauge the impact of the presence of animal-filariae for O. volvulus transmission in terms of the growing human and livestock populations in sub-Saharan countries.     

Evaluation of a Community-Based Trapping Program to Collect Simulium ochraceum sensu lato for Verification of Onchocerciasis Elimination

Background

Collection of the black fly vectors of onchocerciasis worldwide relies upon human landing collections. Recent studies have suggested that the Esperanza Window Trap baited with a human scent lure and CO₂ had the potential to replace human hosts for the collection of Simulium ochraceum sensu lato in Southern Chiapas focus, Mexico. The feasibility of utilizing these traps in a community-based approach for the collection of S. ochraceum s.l. was evaluated.

Methodology/Principal findings

Local residents of a formerly endemic extra-sentinel community for onchocerciasis were trained to carry out collections using the traps. The residents operated the traps over a 60-day period and conducted parallel landing collections, resulting in a total of 28,397 vector black flies collected. None of the flies collected were found to contain parasite DNA when tested by a polymerase chain reaction assay targeting a parasite specific sequence, resulting in a point estimate of infection in the vectors of zero, with an upper bound of the 95% confidence interval 0.13 per 2,000. This meets the accepted criterion for demonstrating an interruption of parasite transmission.

Conclusions/Significance

These data demonstrate that Esperanza Window Traps may be effectively operated by minimally trained residents of formerly endemic communities, resulting in the collection of sufficient numbers of flies to verify transmission interruption of onchocerciasis. The traps represent a viable alternative to using humans as hosts for the collection of vector flies as part of the verification of onchocerciasis elimination.     

Purification of Onchocerca gibsoni microfilariae

Forsyth K. P., Copeman D. B. and Mitchell G. F. 1982. Purification of Onchocerca gibsoni microfilariae. International Journal for Parasitology12: 53–57. Viable microfilariae from the bovine parasite, Onchocerca gibsoni, were purified to a level suitable for immunochemical analysis and without appreciable loss in numbers by low speed centrifugation on Ficoll-paque. Microfilariae were obtained by incubation of intact O. gibsoni nodules or from worm fragments dissected from nodules. After purification, microfilariae were determined as viable by motility, ability to establish in mice and to incorporate ³⁵S-methionine into large numbers of proteins in vitro as determined by two-dimensional gel electrophoresis and fluorography.     

Epidemiology and control of onchocerciasis: The threshold biting rate of savannah onchocerciasis in Africa

Control of onchocerciasis currently focuses on community-directed treatment with the microfilaricide ivermectin which effectively kills Onchocerca volvulus microfilariae in the human host. The feasibility of elimination by this control strategy has recently been reported for some foci in Africa which has rekindled discussions on evaluating the threshold conditions of elimination of onchocerciasis. We developed a stochastic model based on a master equation which predicts, based on data from West and Central Africa, that elimination of savannah onchocerciasis can be expected around a threshold biting rate of 730 bites per person per year, ranging region-specifically roughly from 230 to 2300 bites per person and year. The threshold values give rise to optimism that elimination of onchocerciasis is feasible, but the associated measures of parasite prevalence and density suggest that onchocerciasis can remain endemic at very low infection intensities. Endemicity at a low level is a risk factor for elimination strategies, and we point to the necessity of investigating these issues on the basis of breakpoints which refer to threshold conditions based on parasite prevalence and density.     

Establishment of an in vitro culture system to study the developmental biology of Onchocerca volvulus with implications for anti-Onchocerca drug discovery and screening

BackgroundInfections with Onchocerca volvulus nematodes remain a threat in Sub-Saharan Africa after three decades of ivermectin mass drug administration. Despite this effort, there is still an urgent need for understanding the parasite biology especially the mating behaviour and nodule formation as well as the development of more potent drugs that can clear the developmental (L3, L4, L5) and adult stages of the parasite and inhibit parasite reproduction and behaviour.Methodology/Principal findingsPrior to culture, freshly harvested O. volvulus L3 larvae from dissected Simulium damnosum flies were purified by centrifugation using a 30% Percoll solution to eliminate fly tissue debris and contaminants. Parasites were cultured in both cell-free and cell-based co-culture systems and monitored daily by microscopic visual inspection. Exhausted culture medium was replenished every 2–3 days. The cell-free culture system (DMEM supplemented with 10% NCS) supported the viability and motility of O. volvulus larvae for up to 84 days, while the co-culture system (DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells) extended worm survival for up to 315 days. Co-culture systems alone promoted two consecutive parasite moults (L3 to L4 and L4 to L5) with highest moulting rates (69.2±30%) observed in DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells, while no moult was observed in DMEM supplemented with 10% NCS and seeded on LEC feeder cells. In DMEM supplemented with 10% FBS and seeded on LLC-MK₂ feeder cells, O. volvulus adult male worms attached to the vulva region of adult female worms and may have mated in vitro. Apparent early initiation of nodulogenesis was observed in both DMEM supplemented with 10% FBS and seeded on LLC-MK₂ and DMEM supplemented with 10% NCS and seeded on LLC-MK₂ systems.Conclusions/SignificanceThe present study describes an in vitro system in which O. volvulus L3 larvae can be maintained in culture leading to the development of adult stages. Thus, this in vitro system may provide a platform to investigate mating behaviour and early stage of nodulogenesis of O. volvulus adult worms that can be used as additional targets for macrofilaricidal drug screening.