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PubMed2004

Pathogenesis and host responses in human onchocerciasis: impact of Onchocerca filariae and Wolbachia endobacteria.

Onchocerca volvulus is a tissue-invasive parasitic nematode causing skin and eye pathology in human onchocerciasis. The filariae habour abundant intracellular Wolbachia bacteria, now recognised as obligatory symbionts, and therefore emerging as a novel target for chemotherapy. Recent research demonstrates that both the filariae and endobacteria contribute to the pathogenesis of onchocerciasis, and molecules have been identified that promote inflammatory or counter-inflammatory immune mechanisms, divert the host's immune response or procure evasion of the parasite.

Animals↗
PubMed2006

The filarial parasite Onchocerca volvulus generates the lipid mediator prostaglandin E(2).

Prostaglandins exhibit regulatory effects on the vascular and immune system. Prostaglandin E(2) (PGE(2)) modulates T helper (Th) cell and effector cell functional reactivity, thereby promoting Th2 responses. We found significant expression of PGE(2) in male and female Onchocerca volvulus. Using immunohistology, PGE(2) was predominantly detected in the hypodermis of adult O. volvulus, the metabolically most active tissue of the filaria. In contrast, the muscles were PGE(2)-negative and the epithelia of intestine and uterus and male genital tract showed only weak staining. Oocytes were well labeled whereas embryos and sperms did not react. Less pronounced PGE(2) staining was observed in some dermal microfilariae. The expression of PGE(2) was found independent of antifilarial (ivermectin) as well as anti-endobacterial (doxycycline) treatment of O. volvulus-infected patients. PGE(2) was also demonstrated in extracts of adult worms by high-performance liquid chromatography-mass spectrometry. Release of PGE(2) from live or moribund filariae can affect the host s metabolism and immune response in favor of the filarial parasite.

Animals↗
PubMed2006

Eosinophils contribute to killing of adult Onchocerca ochengi within onchocercomata following elimination of Wolbachia.

Many filarial nematodes, including Onchocerca volvulus (the cause of human 'River Blindness'), have a mutually dependent relationship with Wolbachia bacteria. There has been much interest in Wolbachia as a chemotherapeutic target, since there are no macrofilaricidal drugs (i.e., lethal to adult worms) of low toxicity. Using the bovine parasite O. ochengi, we previously demonstrated that combined intensive and intermittent (COM) oxytetracycline treatment induces a sustained depletion of Wolbachia and is macrofilaricidal, whereas a short intensive regimen (SIR) is non-macrofilaricidal. To understand how targeting Wolbachia with oxytetracycline can lead to worm death, O. ochengi nodules (onchocercomata) were sequentially excised from cattle administered COM or SIR therapy, and cell infiltrates were microscopically quantified. Pre-treatment, worms were surrounded by neutrophils, with eosinophils rare or absent. At 8-12weeks after either regimen, eosinophils increased around worms and were observed degranulating on the cuticle. However, with the SIR treatment, neutrophils returned to predominance by 48weeks, while in the COM group, eosinophilia persisted. These observations suggest that accumulation of degranulating eosinophils over a prolonged period is a cause rather than an effect of parasite death, and the macrofilaricidal mechanism of antibiotics may relate to facilitation of eosinophil infiltration around worms by ablation of Wolbachia-mediated neutrophilia.

Animals↗
PubMed2004

The Secreted Larval Acidic Proteins (SLAPs) of Onchocerca spp. are encoded by orthologues of the alt gene family of Brugia malayi and have host protective potential.

Onchocerca volvulus is a tissue-dwelling, vector-borne nematode parasite of humans and the causative agent of onchocerciasis, or 'River Blindness'. Resistance to infection is associated with immune responses to the infective, third-stage (L3) larvae. The antigens of greatest interest for their vaccine potential are surface and secreted molecules. We have previously identified a family of Secreted Larval Acidic Proteins (SLAPs) from the L3 larvae of O. volvulus by biosynthetic labelling. Here, we provide further characterisation of these molecules following cloning and expression of the corresponding cDNAs. Using protein sequencing, we show that SLAPs are members of the alt gene family, first described in the lymphatic filarial parasite, Brugia malayi. Ov-ALT-1 and Ov-ALT-2 correspond with 20 and 18kDa SLAPs. Both proteins are highly acidic and related by sequence, differing chiefly in an 8-amino acid deletion from Ov-ALT-2. By immunochemistry, we confirm that Ov-ALTs are highly stage-specific, being expressed exclusively in late L2 and L3 larvae during growth in the vector. They are synthesised and stored in the glandular oesophagus. Secretion is triggered by the resumption of development in the definitive host and occurs via the pseudocoelom and cuticle. Serological responses in humans to recombinant Ov-ALT-1 indicate that the level of IgG production may be governed by the force of transmission but does not overtly reflect infection status. Immunisation of mice with recombinant Ov-ALT-1 resulted in a modest level of protection against challenge with O. volvulus L3 larvae (P = 0.036). We conclude that Ov-ALT genes, like those of other filariae, are of interest from the standpoint of parasite transmission and infectivity. They may also offer promise as components of a future sub-unit vaccine should the means to enhance protection be achieved.

Amino Acid Sequence↗
PubMed2004

RNA interference targeting cathepsin L and Z-like cysteine proteases of Onchocerca volvulus confirmed their essential function during L3 molting.

We describe the successful use of RNA interference (RNAi) to investigate gene function in the human filarial parasite Onchocerca volvulus third-stage larvae (L3). We targeted two specific gene products, the O. volvulus cathepsin L (Ov-CPL) and cathepsin Z-like (Ov-CPZ) cysteine proteases, which were proposed to function during O. volvulus L3 molting. We show that fluorescent-labeled Cy3-dsRNA corresponding to cpl or cpz regions encoding the mature enzymes can enter the larvae. The molting rate of larvae treated overnight with 0.5 mg ml(-1) cpl was reduced by 92% and 86% in comparison to normal control worms. It appeared that although the larvae started the molting process the last stage of molting, ecdysis was inhibited. The effect was gene specific, as larvae that did not molt in the presence of cpl or cpz dsRNA expressed the other cysteine protease, CPZ and CPL, respectively. This was confirmed by immunoelectron microscopy using antibodies directed against each enzyme. Our present study validate conclusively that both enzymes are essential for the molting of O. volvulus L3 to fourth-stage larvae. We also confirmed that the activity of the enzymes is specific to the changes that occur during the molting process on days 1-3, when the separation between the cuticles is in progress. The development of RNAi in O. volvulus L3 could further help study many of the abundant L3 and molting L3 genes identified through the filarial genome project, many of which, although have no attributed function, were identified as vaccine candidates or potential drug targets.

Animals↗
PubMed2005

A comparison of genetic polymorphism in populations of Onchocerca volvulus from untreated- and ivermectin-treated patients.

An analysis of the polymorphism of 16 genes from Onchocerca volvulus was undertaken, in two populations of worms from either ivermectin-naïve patients or patients who had been repeatedly treated with ivermectin, in Ghana. Six genes were selected for analysis because studies in other nematodes had suggested a possible association with ivermectin resistance. The other 10 genes were included as control genes and have not been associated with ivermectin resistance. Twelve of the 16 genes were polymorphic, including five of the candidate genes and seven of the control genes. In all of the control genes and four of the candidate genes, there were no differences in genetic polymorphism between the untreated and ivermectin treatment worms. However, there were statically significant differences (chi2=0.05) in allelic frequencies between the untreated and treatment derived worms for P-glycoprotein and beta-tubulin genes; both genes which have been previously associated with ivermectin resistance in other nematodes. These genes were in Hardy-Weinberg equilibrium in the untreated population. However, the P-glycoprotein alleles, in the worms from the patients under treatment were not in Hardy-Weinberg equilibrium, and analysis of the allele frequencies of beta-tubulin suggested that this gene may have also been under selection in the worms from the ivermectin-treated patients. This data provides evidence of genetic selection by ivermectin on O. volvulus and indicates that investigations should be made to determine whether ivermectin resistance is developing. The beta-tubulin and P-glycoprotein genes may prove useful for monitoring for possible development of ivermectin resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Wolbachia endosymbiont levels in severe and mild strains of Onchocerca volvulus.

Epidemiological, clinical and genetic data have all suggested that the filarial parasite Onchocerca volvulus, the causative agent of onchocerciasis (or river blindness) exists as two strains in West Africa. The severe strain induces severe ocular disease in a large proportion of the infected population, while the mild strain induces little ocular disease. Although DNA probes based upon a non-coding repeat sequence family can distinguish the two strains, the underlying basis for this difference in pathogenicity is not understood. Recently, several studies have implicated products produced by the Wolbachia endosymbiotic bacterium of O. volvulus in the pathogenesis of onchocerciasis. This suggested the hypothesis that differences in the Wolbachia endosymbiont population might be responsible for the pathogenic differences noted in the two strains. To test this hypothesis, quantitative PCR assays were used to measure the amount of Wolbachia DNA per nuclear genome in a collection of well characterized samples of mild and severe strain O. volvulus. The median ratio of Wolbachia DNA to nuclear DNA was significantly greater in severe strain parasites than in mild strain parasites. These data support the hypothesis that the pathogenic differences seen in severe and mild strain O. volvulus may be a function of their relative Wolbachia burden and provide additional support to the hypothesis that Wolbachia products may play a central role in the pathogenesis of ocular onchocerciasis.

Animals↗
PubMed2005

Genomic organization and effects of ivermectin selection on Onchocerca volvulus P-glycoprotein.

Ivermectin (IVM) was first developed for use with livestock. It is now the only drug used for mass treatment of onchocerciasis. It is difficult to prove whether reports of sub-optimal responses to IVM in some Onchocerca volvulus infected patients are a result of drug resistance, as procedures typically used to examine IVM efficacy in livestock can not be performed on humans. To determine the effects of IVM on O. volvulus, one approach is to examine allele frequencies before and after treatment. Allele(s) linked to resistance may increase in frequency after repeated treatment. Mass treatment of large human populations to reduce transmission of O. volvulus will impose selection pressure for resistance. P-glycoprotein has been implicated as a candidate IVM resistance gene in nematodes. In this study, the intron-exon structure of O. volvulus P-glycoprotein (OvPGP) has been defined. The gene spans 10.6 kb, is AT-rich, contains 24 exons and a high proportion of class 0 introns. The genetic diversity of 28 loci spanning the entire OvPGP gene was examined in four O. volvulus populations from the Volta Region of Ghana. Worms collected in 1999 and 2002 from IVM treated patients showed reduced genetic polymorphism and an increase in the number of loci not in Hardy-Weinberg equilibrium. Changes in allelic patterns and a reduction in diversity at many loci in P-glycoprotein in the parasites from IVM treated patients in 1999 and 2002 suggest that IVM is imposing selection on this gene, consistent with a possible development of IVM resistance.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2005

Characterization of a half-size ATP-binding cassette transporter gene which may be a useful marker for ivermectin selection in Onchocerca volvulus.

ATP-binding cassette (ABC) transporters comprise a large paralogous protein family and several confer drug resistance. Ivermectin (IVM) is the only drug approved for treatment of onchocerciasis and is a substrate for some ABC transporters. Furthermore, there is accumulating evidence that IVM selects on some ABC transporter genes in Onchocerca volvulus and other parasitic nematodes. The onchocerciasis control programs rely on community based treatment with IVM each year to reduce morbidity and decrease parasite transmission. This appears to be imposing selection pressure on O. volvulus. A half-size ABC transporter cDNA has previously been reported for O. volvulus, however, the full length gene has not been previously characterized and investigated for possible selection by IVM. Genes under selection may be identified by patterns of linkage disequilibrium (LD) and a loss of genetic polymorphism at physically linked loci. Twelve genetic markers spanning the full gene were examined in O. volvulus from non-treated and IVM treated populations in Ghana. Analysis of the genomic organization of the half-size ABC transporter (OvPLP) indicates that it spans approximately 3.8 kb comprising nine exons. Worms from treated people showed a reduction in gene diversity, a loss of genetic polymorphism at several markers, a selection for specific alleles, and a reduction in the number of regions in LD; these effects were more pronounced as treatment increased. These changes suggest that IVM is imposing selection pressure on this gene. The region between transmembrane domains four and five may be a useful marker for IVM selection in O. volvulus.

ATP-Binding Cassette Transporters↗
PubMed2006

Ivermectin selection on beta-tubulin: evidence in Onchocerca volvulus and Haemonchus contortus.

Ivermectin resistance is common in trichostrongylid nematodes of livestock, such as Haemonchus contortus. This anthelmintic is the only drug approved for mass administration to control onchocerciasis caused by the nematode parasite, Onchocerca volvulus. In parts of West Africa up to 18 rounds of ivermectin treatment have been administered to communities and there are reports of poor parasitological responses to treatment. Understanding ivermectin resistance and ivermectin selection is an important step to reduce selection pressure for resistance, and to develop molecular markers which can be used to monitor the development of resistance and its spread. Here we report evidence that ivermectin selection changes the frequency of beta-tubulin alleles in both the sheep parasite, H. contortus, and the human parasite, O. volvulus. In O. volvulus we have been able to look at the frequency of beta-tubulin alleles in O. volvulus obtained before any ivermectin was used in humans in Africa, and following its widespread use. In H. contortus, we have been able to look at the frequency of beta-tubulin alleles in a strain which has not seen any anthelmintic selection and in an ivermectin selected strain derived from the unselected strain. We have found ivermectin selects on beta-tubulin in both of these nematode species. In the case of O. volvulus, we had previously reported that ivermectin selects for specific single nucleotide polymorphisms in the O. volvulus beta-tubulin gene. This polymorphism results in three amino acid changes in the H3 helix of beta-tubulin, as well as deletions in an associated intron. We report a simple PCR assay to detect the amplicon length polymorphism, resulting from these intronic deletions, which can be used to monitor the frequency of the beta-tubulin allele selected for by ivermectin in O. volvulus.

Africa, Western↗
PubMed2004

Impact of four years of large-scale ivermectin treatment with low therapeutic coverage on the transmission of Onchocerca volvulus in the Mbam valley focus, central Cameroon.

The extent to which ivermectin treatments have an impact on onchocerciasis transmission is a matter of some concern. We investigated this issue in the Mbam valley, a hyperendemic focus located in a forest-savannah mosaic area of Cameroon. Parasitological examinations of 5-9-year-old children, who had never received any antifilarial drug, were conducted before the first distribution of ivermectin in 1991-1993 and again in 2002, after four annual rounds of mass treatments. After matching for gender, age and village of residence, the prevalence and intensity of microfilaridermia corresponded respectively, in 2002, to 66.2 and 42.0% of the initial values. The decrease was more marked among the youngest children who, compared with the older ones, were submitted to the reduced force-of-infection earlier in their life. The results of the present study suggest that the specific vectorial competence of Simulium squamosum cytotype B, the vector of Onchocerca volvulus in the Mbam valley, allows a significant decrease in onchocerciasis transmission after several years of treatment, despite low therapeutic coverage. Though these results are encouraging, efforts should be made to improve the therapeutic coverage in the area.

Animals↗
PubMed1997

Differential immune response to Onchocerca volvulus: IgG4 antibody responses differ in onchocerciasis patients from Guatemala and Ghana.

Geographical differences exist in the clinical features of onchocerciasis in Central America and West Africa, which could be due in part from variations in the antigenic composition of the infecting organism. In an attempt to address this question, adult female worms of Onchocerca volvulus derived from nodules of patients from Guatemala and Ghana were compared in terms of polypeptide composition and the IgG4 antibody responses induced in patients. It was shown that a Tris-buffer soluble extract from the worms obtained in the two regions differ in polypeptide composition. Furthermore, the diagnostic polypeptides were found to be in the 30 kDa region but the recognition of these antigens was less intense and less frequently observed in the sera of microfilaria (mf) positive patients from Ghana than equivalent age and sex matched patients from Guatemala.

Adolescent↗
PubMed1998

Detection of a chemotactic factor for neutrophils in extracts of female Onchocerca volvulus.

Neutrophilic granulocytes and macrophages are the dominant inflammatory cell types observed in the vicinity of and attached to adult Onchocerca volvulus in the subcutaneous nodules. Crude extract from female O. volvulus was examined for chemotactic activity for peripheral neutrophils from healthy individuals by use of an endogenous component chemotactic assay in Boyden chambers. Significant chemotactic responses of neutrophils were detected using O. volvulus extracts at > or = 15 microg/ml in a dose-dependent manner. Checkerboard analysis demonstrated low chemokinetic in addition to chemotactic activity. Neutrophil migration was also elicited by excretory-secretory products of vital females. Fractionation of the female worm extract by FPLC revealed two components with chemotactic activity, one with a molecular mass less than 12 kDa and another with a molecular mass of > 200 kDa. Immunohistological examination of onchocercomas containing only one adult alive filarial worm demonstrated that neutrophils were accumulated near and attached to the cuticle of immature females, females producing microfilariae and males.

Africa, Western↗
PubMed2000

Ivermectin: concentration-dependent effects on adenosine triphosphatases in adult worms of Onchocerca volvulus.

The effect of increasing concentrations of ivermectin on adenosine triphosphatase (ATPase) activity was investigated in adult worms of Onchocerca volvulus. Mean Mg- and Na,K-ATPase activities decreased significantly (F ratio = 29.82, P < 0.01 and F ratio = 28.54, P < 0.01, respectively) with increasing concentrations of ivermectin (0-100 ng/ml) in the female worms. When male and female worms were mixed with equal amounts of proteins from each, only the Na,K-ATPase activity was significantly decreased (F ratio = 56.61, P < 0.01) over a similar range of ivermectin concentrations. Since ivermectin exhibits concentration-dependent effects on both ATPases in female adult worms, this might provide an insight into other effects of the drug. However, the adjustment of the dose of ivermectin to obtain a nodular concentration of at least 40 ng/ml is therefore recommended in the complete chemotherapy of onchocerciasis.

Adenosine Triphosphatases↗
PubMedDate not supplied

Onchocerca volvulus keratitis (river blindness) is exacerbated in BALB/c IL-4 gene knockout mice.

To determine the outcome of Onchocerca volvulus keratitis in IL-4(-/-) BALB/c mice, animals were immunized subcutaneously and injected into the corneal stroma with soluble O. volvulus antigens. IL-4(-/-) BALB/c mice had a deviated cellular response, with decreased serum IgE and IgG1 and elevated IgG2a compared with control BALB/c mice. In marked contrast to control BALB/c, C57BL/6, and IL-4(-/-) C57BL/6 mice, IL-4(-/-) BALB/c mice developed severe corneal opacification and neovascularization that was associated with a pronounced neutrophil infiltrate to the corneal stroma. STAT-6(-/-) BALB/c mice had the same phenotype as IL-4(-/-) BALB/c mice, and complement depletion had no effect on the severity of O. volvulus keratitis in these mice. These findings indicate that on a BALB/c background, IL-4 has a critical role in regulating neutrophil recruitment to the cornea and development of O. volvulus keratitis.

Animals↗
PubMed1995

Characterization of native pathogenic antigens of Onchocerca volvulus: identification of high molecular mass protein antigens eliciting interstitial keratitis in a guinea pig model.

Sclerosing keratitis is the predominant cause of blindness due to onchocerciasis which is a major human parasitic disease caused by the filarial parasite Onchocerca volvulus. In the present investigation, native pathogenic antigens of O. volvulus which are particularly potent in causing interstitial keratitis were characterized utilizing a guinea pig model. Following demonstration of the protein nature of these antigens using pronase digestion, the crude O. volvulus antigen extract was subjected to stepwise procedures of protein purification. At each stage of purification, pooled antigen fractions were injected into one cornea of presensitized guinea pigs followed by clinical evaluation of stromal inflammation and vascularization at different intervals of time after intrastromal challenge. Initial purification of the pathogenic antigens was carried out in the following order: molecular sieve chromatography on Bio-gel A-5m. anion exchange chromatography on Mono Q followed by DEAE-Sepharose CL-6B and cation exchange chromatography on Mono S. Two out of six different pools from the Mono S column (pool a eluted unbound at 10 mM-NaCl and pool e eluted between 130 mM and 475 mM-NaCl) were found to be most pathogenic. Further purification of Mono S pool a and pool e separately by gel filtration chromatography using Superose 12 demonstrated that the fractions which were most potent in inducing interstitial keratitis contained proteins with approximate molecular masses between 100 and 200 kDa. These results show that minor subfractions of total crude antigens of O. volvulus are largely responsible for induction of experimental interstitial keratitis. We have demonstrated the presence of these antigens in O. volvulus microfilariae by their cross-reactivities with anti-microfilarial antibodies, and hence the relevance of the purified antigens to ocular onchocerciasis in man since sclerosing keratitis is associated with invasion of the cornea by O. volvulus microfilariae. Isolation of these two pathogenic antigen pools represents the practical limits of purification and subsequent animal experiments possible with the available amounts of native parasite material obtained from infected human individuals in the absence of a suitable non-human host or of an in vitro culture system for O. volvulus.

Animals↗
PubMed2002

Expression and characterization of Ov-47, a dominant antigen of Onchocerca volvulus.

The expression and characterization of a recombinant antigen termed Ov-47 are described. Ov-47 was identified and isolated from a lambda gt-11 cDNA expression library derived from adult female Onchocerca volvulus mRNA using rabbit antiserum raised against the surface proteins of O. volvulus female worms. The antiserum was earlier found to mediate, in vitro, cytoadherence and cytotoxicity reactions to microfilariae in the presence of heat-labile serum factors. The deduced amino acid sequence of the gene was assigned the EMBL GenBank Accession No. Y15993. The open reading frame (1077 bp) of the gene was then subcloned into pQE-60 and expressed in Escherichia coli JM109 cells. The gene encodes a protein with an apparent molecular weight of 47,000 Da as revealed by SDS-PAGE. Up to 100 micrograms/ml pure Ov-47 recombinant protein could be isolated from E. coli cultures by Ni-agarose affinity chromatography. The 47-kDa protein was recognized by sera from both infected and endemic normal subjects. The parent protein was found to have a molecular weight of 60 kDa. IgG3 subclass responses to Ov-47 were significantly higher in endemic normals than in infected subjects (P < 0.05). In contrast, IgG4 responses were higher in infected subjects than in endemic normals (P < 0.05). IgG2 response exhibited marked age dependency with lower responses in younger patients, which rose to higher levels in elderly patients. IgG1, IgG3, and IgG4 responses did not show any age dependency. This study clearly shows that Ov-47 is a dominant antigen of O. volvulus adult worms with an important role in the host-parasite-interplay.

Adolescent↗
PubMed2000

Regional genetic variation in the major sperm protein genes of Onchocerca volvulus and Mansonella ozzardi (Nematoda: Filarioidea).

Onchocerca volvulus and Mansonella ozzardi are two human filarial parasites present in South and Central America. In the Brazilian Amazonia they are found in sympatry, and the lack of clear morphological diagnostic characters in the microfilariae hinders their identification. The major sperm protein (MSP) gene of both species has been sequenced and characterised to determine its potential as a molecular diagnostic character. The length of the MSP gene is different in each species, and this could be used to detect and differentiate them by running the polymerase chain reaction (PCR) product in an agarose gel. Two major gene groups were identified in O. volvulus with a genetic distance of 6% between them. In M. ozzardi only one major group of genes was observed. The high similarity between the protein amino acid sequence of both filarial species confirms that the MSP has been highly conserved through nematode evolution.

Animals↗