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PubMed2000

Onchocerca volvulus: comparative analysis of antibody responses to recombinant antigens in two animal models of onchocerciasis.

Experimental infections of chimpanzees with Onchocerca volvulus and cattle with Onchocerca ochengi provide model systems for research in human onchocerciasis. These infections share many similarities from the standpoint of parasite biology, but little is known about the comparability of immune responses in the two systems. To make a direct comparison between the models in terms of immune responsiveness to defined parasite products, three recombinant antigens of O. volvulus (Ov7, Ov103, and B20) were used to analyze the kinetics of antibody production following experimental infection. Each of the antigens was derived from adult cDNA libraries following immunoscreening with sera from chimpanzees (Ov7, Ov103) or cattle (B20). All chimpanzees (n = 12) and cattle (n = 8) displayed responses to Ov7 and Ov103, and all cattle, but only 33% of chimpanzees, showed responses to B20. The dynamics of the response to individual antigens showed further similarities between the chimpanzees and the cattle, with responses to Ov7 and Ov103 peaking after, and B20 before, the onset of patent infections. We conclude that there is good preliminary evidence of concordance in the kinetics of serological responses in the two models. However, individual antigens many be more or less immunogenic in the two systems, making it inadvisable to extrapolate between models concerning the relative immunodominance of specific parasite products.

Animals↗
PubMed1997

Electron microscope observations on Onchocerca ochengi and O. fasciata (Nematoda:Filarioidea).

The fine structure of the females and males of Onchocerca ochengi (parasitizing zebu and cattle) and of the females of O. fasciata from camels were described and compared to other filariae of the genus Onchocerca. It was shown that O. ochengi resembles O. volvulus of humans in its degree of development, while being more primitive than O. gibsoni. Besides other similarities O. ochengi attracts inflammatory cells in the way of O. volvulus and these could be a model for chemotherapeutic trials.

Animals↗
PubMed1991

The conditions required for the maintenance of Onchocerca lienalis microfilariae in vitro.

Microfilariae of the bovine parasite Onchocerca lienalis were maintained in vitro using a tick cell line as a feeder layer, and under the conditions provided would develop to the sausage stage. A number of media supported this achievement with CMRL 1066 producing the highest yields, particularly when used in conjunction with either medium 199, HAM's F-12, RPMI 1640 or Mark's M20. The addition of inactivated foetal calf serum (iFCS) suppressed development; on the other hand, the addition of tryptose phosphate broth (TPB) enhanced it. The pH range of the medium preferred by the developing worms was 7.35-7.85, and the most favourable osmolality lay in the range 360 to 390 mosM/kg. Insect-derived hormones did not improve yields of developed larvae nor promote moulting to the second larval stage. The culture conditions described in this work, which favour parasite survival and development, provide further insights into the physiological requirements of filariae as well as guide lines to achieving successful in vitro maintenance of Onchocerca sp. A morphological examination of the developing larvae was carried out at electron microscopical level.

Animals↗
PubMed1993

Molecular variation in Onchocerca spp.

Over the past two decades there has been an upsurge of interest in defining morphological, immunological, biochemical, biological and genetic differences between species of Onchocerca to provide solutions to practical problems associated with finding models and epidemiological tools to assist with control of human onchocerciasis. The information gathered has confirmed the close relationship between species of Onchocerca and provided highly sensitive and specific probes to distinguish species and even strains of the same species. It has also identified pathways, especially using sequences from common DNA repeat units, that may lead to a better understanding of the progression of divergence of species of this genus than has previously been possible.

Animals↗
PubMed1991

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.

Animals↗
PubMed1992

Experimental onchocerciasis in chimpanzees. Antibody response and antigen recognition after primary infection with Onchocerca volvulus.

Nine of 18 chimpanzees inoculated with 250 infective third-stage larvae (L3) each developed patent (i.e., positive for microfilariae) Onchocerca volvulus infection. Four of 6 infected chimpanzees that received 200 micrograms/kg ivermectin at 28 days postinfection (pi) became patent, whereas, when ivermectin was given concurrently with L3 challenge only 1 of 6 infected animals developed patent infection. The antibody response to O. volvulus adult worm-derived antigens (OvAg) showed clear differences between patent and nonpatent chimpanzees. Three months pi, all sera detected several OvAg in the range of M(r) 35-120 k. Sera collected 6 mo pi from later patent animals recognized increasing numbers of OvAg, especially in the lower MW range of M(r) 13 to 33 k. Beginning 10 months pi Onchocerca-antigens of M(r) 21, 24, 26, and 28 k were detected only by patent chimpanzee's sera. The antibody response in nonpatent chimpanzees consistently recognized fewer OvAg, most of which were limited to the higher M(r) range (35-120 k). The reactivity of sera from infected chimpanzees to a low molecular weight fraction (LMW) of total OvAg doubled within 6 months pi, and increased continuously in patent animals from 13 until 30 months pi. Serological reactivity of nonpatent animals to LMW-OvAg remained low. The titers of circulating IgG directed against total OvAg increased in all infected chimpanzees, and continued to rise with patency. In nonpatent chimpanzees the antibody production gradually returned to preinfection values. Total and OvAg-specific IgE increased in patent and nonpatent chimpanzees. Also, during prepatency the granulocyte and antibody-mediated in vitro killing of microfilariae of O. volvulus increased in subsequently patent chimpanzees. The in vitro immobilization of L3 remained low.

Animals↗
PubMed1990

Cloning of a species-specific DNA probe from Onchocerca gibsoni.

A genomic library of Onchocerca gibsoni has been prepared in the vector lambda-gt10 and has been screened for specific DNA sequences by hybridization with radiolabelled total genomic DNA from a number of Onchocerca species. A clone--fOGI--has been isolated which does not interact with DNA prepared from O. gutturosa, O. lienalis, O. ochengi, O. cervicalis or O. volvulus (both Liberian and Mexican isolates). In addition, no hybridization is observed with host (cattle) DNA. fOGI can detect as little as 100-200 pg of O. gibsoni DNA. It is thus concluded that fOGI has the sensitivity to detect microfilariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.

Animals↗
PubMed1992

Nucleotide sequence of a cDNA from Onchocerca gibsoni encoding a novel repetitive antigen.

mRNA from uterine microfilariae of the cattle parasite Onchocerca gibsoni was used for the construction of cDNA libraries. A cDNA clone encoding an antigen recognized by serum from human individuals infected with O. volvulus was found to contain five copies of an 87 bp unit. These 87 bp units were present in the genome in high copy number as long tandem arrays. These are the first cDNA sequence data obtained directly from larvae of any Onchocerca species.

Amino Acid Sequence↗
PubMed1994

Biosynthetic radiolabelling of excretions-secretions of adult male Onchocerca gibsoni.

Maintenance of adult male worms of the bovine filarial parasite Onchocerca gibsoni in vitro, in the presence of radioactive precursors, resulted in the time-dependent excretion-secretion of radiolabelled parasite macromolecules (ES). Molecules labelled with amino acids ([35S] methionine, [3H] leucine) covered a wide range of molecular weights, whereas labelling with [3H] glucosamine produced predominantly molecules of high molecular weight. Many of the products were recognized by antibodies in two serum pools from humans infected with Onchocerca volvulus. O. gibsoni ES may therefore provide a substitute source of material for studies on the ES of the less readily available human parasite.

Animals↗
PubMedDate not supplied

Onchocerca gutturosa and O. volvulus: studies on the viability and drug responses of cryopreserved adult worms in vitro.

The viability and drug responses of cryopreserved adult Onchocerca have been examined in vitro. Male worms were cryopreserved in liquid nitrogen (-196 degrees C) using ethanediol as a cryoprotectant in a 2-step incubation procedure. After thawing, 85-90% of O. gutturosa males were normally motile. These motile worms were evaluated for viability using 4 measurements (long-term motility/survival in culture; [U-14C]adenine uptake and leakage; glucose utilization; MTT-formazan colorimetry) and were no different from unfrozen controls. Subsequent experiments demonstrated that the motility responses of cryopreserved worms exposed to the antifilarial drugs ivermectin, CGP 6140 and levamisole were virtually identical to unfrozen controls. Some success was also obtained with this technique in cryopreserving O. volvulus males, with 2 thawed specimens surviving in culture for 93 and 106 d respectively. Following collagenase isolation, female worms were cryopreserved in medium +10% serum without protectant at -79 degrees C. A batch of 8 female O. gutturosa were all motile when thawed 14 d later, with a mean survival time (based on 5 specimens) of 71 d (range 60-90). However, a batch of worms transferred from -79 degrees C to -196 degrees C were badly damaged when thawed. Female O. volvulus were cryopreserved at -79 degrees C in Guatemala and sent by air freight on solid CO2 to the UK. Most specimens were active when thawed. Survival of motile specimens ranged from 7 to 272 d in culture. It is concluded that these techniques are of practical value for the storage and transportation of adult Onchocerca.

Adenine↗
PubMedDate not supplied

Morphometric differentiation of Onchocerca volvulus and O. ochengi infective larvae.

By microinjection of cryopreserved microfilariae (mf) into nulliparous flies, a comparison of the lengths of the infective larvae (L3) of Onchocerca volvulus and O. ochengi from the head of Simulium damnosum s.l. (presumed S. sirbanum) has been made. The suitability of S. sirbanum as a host was similar for both Onchocerca spp. The mean length +/- standard deviation of O. ochengi infective larvae measured in aqueous medium after storage of infected flies in liquid nitrogen was 762 +/- 63 microns (n = 39), significantly longer (P much less than 0.0001) than those of a savanna isolate of O. volvulus (676 +/- 56 microns, n = 26). Although the frequency distributions of the lengths of larvae of the 2 species overlapped, a critical value for discrimination of 719 microns applied to normally distributed populations with means and standard deviations of these samples would result in correct classification of 78% of true O. volvulus and 75% of true O. ochengi. A discriminant function analysis incorporating width measurements did not usefully improve the level of accuracy of discrimination. Larvae from flies stored in 70% ethanol and stained with acid haemalum were about 10% shorter, but O. ochengi infective larvae were still proportionately longer than those of O. volvulus (693 +/- 40 microns, n = 45 compared to 580 +/- 38 microns, n = 6, respectively). These data show that the infective L3 of O. volvulus and O. ochengi differ morphologically. Although the population length distributions overlap, by classifying larvae greater than 719 microns long as O. ochengi and those less than 719 microns long as O. volvulus a more accurate estimation of true O. volvulus infection rates in S. damnosum s.l. can be derived than is currently possible.

Animals↗
PubMed1989

Onchocerca volvulus heat shock protein 70 is a major immunogen in amicrofilaremic individuals from a filariasis-endemic area.

Infestation with organisms causing lymphatic filariasis (i.e. Wuchereria bancrofti and Brugia malayi) results in a variety of clinical presentations. It is possible that some of the variation is due to differences in host response to parasite. To determine whether individuals who live in an endemic area but differ in their clinical manifestations respond to different filarial antigens, we screened Onchocerca volvulus expression libraries with sera from a number of individuals belonging to different clinical groups. The results of the study demonstrate that there are indeed differences in the recognition of three cloned filarial antigens and that this differential recognition is related to clinical symptomatology. The most striking finding is that an Onchocerca volvulus protein homologous to the 70 kDa Xenopus laevis heat shock protein is primarily recognized by individuals who are amicrofilaremic. Further analysis is required to determine whether these antigens play any role in the pathogenesis of filarial infection or have any potential value in protective immunity.

Adolescent↗
PubMed1991

Characterization of an Onchocerca volvulus cDNA clone encoding a genus specific antigen present in infective larvae and adult worms.

The isolation and characterization of a recombinant cDNA clone (OV7) expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Using chimpanzee antiserum generated against irradiated infective larvae, we isolated a cDNA clone from a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA. The open reading frame encodes 131 amino acids corresponding to a 15.2-kDa protein. Affinity purified antibodies which bound specifically to OV7 fusion polypeptide recognized a single antigen with an apparent molecular weight of 17,000 in extracts of L3, L4 and adult worms. Immunoelectron microscopy established that the antigen encoded by this clone is present in the hypodermis and the basal layer of the cuticle of L3 and female adult worm, and in the egg shell around developing microfilariae. Since the OV7 fusion polypeptide is onchocerca-specific and is recognized specifically by sera from onchocerciasis patients, and sera from non-patent but infected chimpanzees, and not by sera from patients with other filarial parasites, it may have potential as an antigenic component in a test for detection of non-patent and patent infections of O. volvulus. The OV7 amino acid sequence contains residues that have a probable homology with the cysteine proteinase inhibitor superfamily.

Amino Acid Sequence↗
PubMed1995

Characterisation of an immunodominant glycoprotein antigen of Onchocerca volvulus with homologues in other filarial nematodes and Caenorhabditis elegans.

The full-length cDNA corresponding to an Onchocerca volvulus antigen, OvMBP/11, which had been selected as a serodiagnostic tool was isolated, sequenced, and the native antigen encoded by the cDNA characterised. The cDNA encodes a protein of 20.5 kDa (termed Ov 20) containing a putative signal peptide. Southern blot analysis indicates that there is only a single O. volvulus gene corresponding to Ov 20 but it has significant sequence similarity to genes corresponding to two 20.5-kDa predicted proteins of Caenorhabditis elegans. Homologues of the Ov 20 gene were detected at high stringency by Southern blot in the other Onchocerca species O. gibsoni, and O. gutturosa and at lower stringency in the related filarial nematodes Brugia malayi and Acanthocheilonema viteae. The Ov 20 native antigen has two molecular mass forms, 20 and 22 kDa, in all the life cycle stages studied. These isoforms have different levels of N-linked glycosylation on a peptide backbone of 17.5 kDa. Immunolocalisation and in situ hybridisation studies demonstrated that Ov 20 is transcribed and translated in the body wall of adult females and also in microfilariae, third and fourth stage larvae. Antigen was detected in the supernatant of in vitro cultured adult female nematodes. The B. malayi and A. viteae homologues are antigenically cross-reactive to Ov 20, share the same size peptide backbone but differ in their degree of glycosylation.

Amino Acid Sequence↗
PubMedDate not supplied

OvB20, an Onchocerca volvulus-cloned antigen selected by differential immunoscreening with vaccination serum in a cattle model of onchocerciasis.

A cDNA of Onchocerca volvulus has been isolated by differential immunoscreening of an adult worm expression library using sera raised in cattle against the related species, O. lienalis. It was selected because of its recognition by antibodies from cattle immunized with irradiated third-stage (L3) larvae and not by antibodies from animals infected with non-irradiated larvae. The original 311-bp clone was used to isolate a 1478-bp cDNA. Designated OvB20, this codes for 460 amino acid residues, hybridizes with a approximately 1.6 kBp transcript and appears to be transcribed from a filarial-specific, single copy gene. It is expressed in developing stages from embryo to L4 larva, but not in the adult. The product of OvB20 appears to undergo co- or post-translational processing: in vitro transcription and translation give rise to a polypeptide consistent with the deduced amino acid sequence (approximately 52 kDa), whilst products of 52 and 65 kDa are detected in larvae by immunoblotting and following in vitro translations to which exogenous microsomes have been added. A 42-kDa protein was also detected in all in vitro translations. No homologous genes were found in the computer databases, although there are regions of weak sequence similarity with C-reactive proteins. The functional role of OvB20 may warrant further attention, as it has recently been shown that the recombinant protein confers host protection against a related rodent filaria following active immunization (Taylor, M.J., Abdel-Wahab, N., Wu, Y., Jenkins, R.E. and Bianco, A.E. (1995) Onchocerca volvulus larval antigen, OvB20 induces partial protection in a rodent model of onchocerciasis. Infect. Immun. 63, 4417-4422).

Amino Acid Sequence↗
PubMed1989

Distribution of Onchocerca spp. microfilariae in the dermis of cattle.

A grid was drawn on a lateral half of 10 hides, from cattle infected with both Onchocerca gutturosa and Onchocerca lienalis, which were obtained from an abattoir in central Florida. Paired 7-mm diameter skin cores were taken from 24 grid sites on each hide and microfilariae (mff) extracted to determine their distribution. The only consistent pattern distribution was that mff were recovered from all 10 hides at four sample sites along the ventral midline near the umbilicus. Microfilariae were also frequently found in large numbers along the dorsal cervical midline and in the region of the tarsal and carpal-metacarpal joints.

Abattoirs↗
PubMed2006

Molecular phylogeny of the filaria genus Onchocerca with special emphasis on Afrotropical human and bovine parasites.

Filarial parasites of the genus Onchocerca are found in a broad spectrum of ungulate hosts. One species, O. volvulus, is a human parasite that can cause severe disease (onchocerciasis or 'river blindness'). The phylogenetic relationships and the bionomics of many of the nearly 30 known species remain dubious. Here, the phylogeny of 11 species representing most major lineages of the genus is investigated by analysing DNA sequences from three mitochondrial genes (ND5, 12S and 16S rRNA) and portions of the intergenic spacer of the nuclear 5s rRNA. Special emphasis is given to a clade containing a yet unassigned specimen from Uganda (O. sp. 'Siisa'), which appears to be intermediate between O. volvulus and O. ochengi. While the latter can be differentiated by the O-150 tandem repeat commonly used for molecular diagnostics, O. volvulus and O. sp.'Siisa' cannot be differentiated by this marker. In addition, a worm specimen from an African bushbuck appears to be closely related to the bovine O. dukei and represents the basal taxon of the human/bovine clade. At the base of the genus, our data suggest O. flexuosa (red deer), O. ramachandrini (warthog) and O. armillata (cow) to be the representatives of ancient lineages. The results provide better insight into the evolution and zoogeography of Onchocerca. They also have epidemiological and taxonomic implications by providing a framework for more accurate molecular diagnosis of filarial larvae in vectors.

Africa South of the Sahara↗
PubMedDate not supplied

Onchocerca volvulus: expression and immunolocalization of a nematode cathepsin D-like lysosomal aspartic protease.

The N-terminal region of the cathepsin D-like aspartic protease from the human filarial parasite Onchocerca volvulus was expressed as His-tag fusion protein. Light and electron microscopic immunohistology using antibodies against the recombinant protein showed labeling of lysosomes in the hypodermis and epithelia of the intestine and the reproductive organs of Onchocerca. While developing oocytes were negative, mature oocytes and early morulae showed strong labeling. In older embryos and mature microfilariae, stained lysosomes were only found in a few cells. Cell death in degenerating microfilariae of patients untreated and treated with microfilaricidal drugs was associated with strong expression of aspartic protease. IgG1, IgG4, and IgE antibodies reactive with the recombinant protein were demonstrated in sera from onchocerciasis patients indicating exposure and recognition of the enzyme by the host's defence system. The aspartic protease of O. volvulus appears to function in intestinal digestion and tissue degradation of the filaria.

Animals↗