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PubMed1987

Chromosomes of Onchocerca volvulus (Spirurida: Onchocercidae): a comparative study between Nigeria and Guatemala.

Chromosomes of Nigerian Onchocerca volvulus were compared with those of Guatemalan O. volvulus. Both parasites had basically the same chromosomal construct (2n = 8, XY type). Autosomes consisted of a pair of large and two smaller pairs. Sex chromosomes were made up of medium sized X chromosome and very small Y chromosome. It was not possible to infer the position of the centromeres.

Animals↗

PubMed1996

Cloning and characterization of cDNAs encoding putative glutamate transporters from Caenorhabditis elegans and Onchocerca volvulus.

We report the identification and partial characterization of cDNAs encoding for putative glutamate transporters from the free-living nematode Caenorhabditis elegans and the filarial parasite Onchocerca volvulus. Glutamate transporters can be used as reliable markers for identifying cells and neurons that synaptically release glutamate and aspartate. An amplified PCR fragment containing a highly conserved amino acid heptamer found in all vertebrate glutamate transporters was used to screen a C. elegans cDNA library. Two full-length cDNA sequences from C. elegans were deduced from the isolated cDNA clones and RT-PCR products with the splice leader. The two C. elegans cDNA sequences differ by only 97 nucleotides at the 5' end. The C. elegans glutamate transporter gene glt-1 spans at least 2.9 kb of chromosomal DNA and possesses nine exons and eight introns. Primers directed to the CeGlt cDNA were used with O. volvulus first-strand cDNA to amplify and isolate the O. volvulus cDNA homolog. The C. elegans and O. volvulus glutamate transporters are 98% identical over 492 amino acids to each other and 52 to 58% identical to the mammalian glutamate transporters. Antibodies generated against partial coding regions of the C. elegans glutamate transporter recognized a protein of approximately 66 kDa in C. elegans and O. volvulus protein extracts.

ATP-Binding Cassette Transporters↗

PubMedDate not supplied

Onchocerca volvulus: striking decrease in transmission in the Vina valley (Cameroon) after eight annual large scale ivermectin treatments.

The impact of repeated ivermectin treatments on the transmission of Onchocerca volvulus was evaluated in the Vina valley, northern Cameroon, by comparing the prevalence and intensity of infection observed in untreated 5-7 years old children living in the treated communities before and after 7-8 successive annual rounds of ivermectin treatment of the general population of those communities. The villages studied were Ngoumi and Babidan, where the initial community microfilarial loads (CMFL) were 83.7 and 216.4 microfilariae per skin snip, respectively. In 1995, after 8 annual treatments, the prevalence and intensity in Ngoumi had decreased by more than 90%, compared with the pretreatment values, and the prevalence continued to decrease between 1992 and 1995. In Babidan, after 7 annual treatments, the prevalence and intensity had also decreased significantly, but less than in Ngoumi. The study demonstrated that repeated treatments brought about a notable reduction in the transmission of O. volvulus in the Vina valley, despite unfavourable factors such as mean drug coverages below 60% and the good vectorial competence of Simulium damnosum s.s. and S. sirbanum.

Adult↗

PubMed1988

Isolation and characterization of expression cDNA clones encoding antigens of Onchocerca volvulus infective larvae.

The isolation of recombinant cDNA clones expressing antigens found in Onchocerca volvulus infective larvae is described. To isolate such clones, an expression cDNA library constructed from adult O. volvulus RNA was screened with antiserum raised against infective larvae. One clone, designated lambda RAL-1 was characterized further. The recombinant antigen produced by lambda RAL-1 stimulates proliferation of peripheral blood mononuclear cells from O. volvulus infected humans. Lambda RAL-1 is derived from a 1450 bases message that encodes a protein with an apparent molecular weight of 42,000 in adult O. volvulus. The inserted DNA of lambda RAL-1 contains an open reading frame of 1008 bp. The amino acid sequence predicted by this open reading frame contains three repeats of the sequence KKPEDWD. The identification of clones such as lambda RAL-1 will provide quantities of purified antigens sufficient to begin to study the immune response to and explore the development of immunity against the infectious form of the parasite.

Amino Acid Sequence↗

PubMed2002

Antigenic mimicry: Onchocerca volvulus antigen-specific T cells and ocular inflammation.

PURPOSE: Molecular mimicry has been suggested to play a role in the development of ocular onchocerciasis. The Onchocerca volvulus antigen Ov39 is cross-reactive with the retinal antigen hr44 and induces ocular inflammation in rats after immunization. This study was undertaken to determine whether Ov39-derived T-cell lines, which proliferate in response to stimulation with hr44, can transfer disease to naive Lewis rats. METHODS: Two separately derived IL-2-dependent CD4(+) T-cell lines, LKOV39 1.8 and LKOV39 4.5, specific to Ov39 were transferred to naïve Lewis rats. A T-cell line specific to the peripheral nerve protein P2 served as a positive control for transfer of disease. Ocular tissues were analyzed by immunohistology, and sera were tested for the presence of antibodies to hr44. RESULTS: Transfer of both T-cell lines caused inflammation of the limbus, iris, and choroid. In addition, LKOV39 1.8, which produced slightly more inflammation, induced activation of retinal microglia. LKOV39 4.5 induced a dose-dependent influx of CD8(+) cells into the limbus and the uvea. Sera from rats that received the T-cell lines had no significant antibody responses to hr44. CONCLUSIONS: These findings indicate that CD4(+) cell lines specific to the antigen Ov39 can induce ocular inflammation in naïve rats and suggest that recruitment of CD8(+) T cells may play a regulatory role. The inflammation is milder than that produced by immunization. The absence of antibody responses to hr44 in the animals receiving the T-cell lines may indicate a role for antibody in the development of ocular onchocerciasis.

Adoptive Transfer↗

PubMed1979

The use of an Onchocerca volvulus microfilarial antigen skin test in an epidemiological survey of onchocerciasis in Guatemala.

Onchocerca volvulus microfilariae obtained from onchocercal nodules were used as antigenic material for a skin test. This antigen is as specific as those previously employed in skin tests, detecting 85.2% of the persons with positive diagnostic signs of onchocerciasis in endemic areas. A low rate of false positives was found in the control group in nonendemic areas. The positive rate of reactions in the persons without positive signs in the endemic areas might be due to the presences of cases not detected as a result of palpation for nodules and two skin biopsies. In the examination of the residents of several populations, a close correlation was found between the positive rates of skin tests and the presence of infection. From these findings, we conclude that the skin test using the microfilarial antigen is a suitable procedure for the diagnosis of onchocerciasis. The skin test could also be useful as an epidemiological tool in assessing the effectiveness of control programmes.

Adolescent↗

PubMed1988

Cell-mediated immune responses in human infection with Onchocerca volvulus.

Mechanisms involved in modulation of the immune response in persons with chronic Onchocerca volvulus infection are poorly understood. In this study in vitro reactivity of PBMC to O. volvulus antigen (Ovag), streptolysin O (SL-O) and the mitogen PHA was tested in 62 infected individuals (INF), 17 persons living in the endemic area with exposure to the infection, but with no detectable infection (END), and 7 healthy controls (CTRL) in Liberia, West Africa. Mean blastogenic responses to Ovag were minimal and did not differ between the groups. There was, however, heterogenous reactivity to Ovag in the INF and END. For example, individuals with a history of therapy, and half of those less than 17 yr old who were tested, showed high responses. No significant differences in the response to SL-O or PHA were detected between the groups. IL-2 production in response to Ovag was minimal in the majority of infected subjects. Exogenous IL-2 was found to cause a significant increase in mean responses to Ovag and SL-O in INF and END only. Similarly, Ovag did not stimulate IL-1 production in most INF, whereas stimulation with LPS led to significantly greater production of IL-1. Depletion of plastic and nylon wool adherent cells did not increase responses to parasite-related antigen in INF, END or CTRL; however, responses to SL-O were augmented in INF, an effect that was also observed in CTRL. Finally, depletion of CD8 or CD16 cells in INF by C lysis did not increase blastogenic responses. These results indicate that cell-mediated immunity to parasite-related Ag as reflected in lymphocyte responses in vitro is diminished in infected individuals, and that this may be caused by defects in T cell activation.

Animals↗

PubMed1977

Lethal damage to Simulium metallicum following high intakes of Onchocerca volvulus microfilariae in Guatemala.

High intakes of microfilariae of a Guatemalan strain of Onchocerca volvulus by Simulium metallicum proved lethal to a considerable proportion of the flies within 24 hours of taking an infecting blood-meal. The gross histopathological changes resulting from infection were compared in groups of moribund and of apparently healthy flies during this period. The marked morbidity of the flies could be related to certain events occurring in the alimentary tract and haemocoele of the fly soon after ingestion of the parasitized blood. The rapid death of the flies may be ascribed to one or more of the following reasons: (i) reverse migration of numerous microfilariae from the expanded- to the tubular-part of the mid-gut, where they cause serious injury and disintegration of the gut epithelium; (ii) abrasive damage to the stomach epithelial cells by the invading microfilariae with occasional release of the gut contents into the haemocoele; (iii) interruption of the formation of the peritrophic membrane, particularly at its anterior and posterior ends, with subsequent failure of the flies to digest the blood in the stomach; (iv) passage of large amounts of parasitized blood from the stomach backwards into the hind-gut, leading to its mechanical obstruction and (v) invasion and injury of various organs of the fly, among them the ventral nerve-cord, brain, optic nerve, eye, halteres, fat-body and flight musculature by excessive numbers of microfilariae.

Animals↗

PubMed1993

Effect of ivermectin treatment on microfilarial load in patients with Onchocerca volvulus in Bebeka, Ethiopia.

In April 1990, one hundred ninety people from Bebeka Coffee Plantation, Kefa administrative region, western Ethiopia, all with moderate to heavy infection with Onchocerca volvulus were treated with ivermectin (150 mcg/kg body wt.). Clinical examination and microfilarial counts in skin snips were repeated at intervals for eight months. Therapy was associated with minimal side effects. Significant difference in mean microfilarial load was observed throughout the eight months post-treatment period. Difference in repopulation of microfilariae between four and eight months was not significant in either sex and all ages. Of all cases, 71 (37.5%) maintained zero microfilarial counts after four months, while only 38 (20%) remained at zero level until the end of eight months. The merit of a yearly treatment programme is discussed.

Adolescent↗

PubMed1996

Onchocerca volvulus and O. lienalis: the microfilaricidal activity of moxidectin compared with that of ivermectin in vitro and in vivo.

The activity of the veterinary drug moxidectin against Onchocerca volvulus and On. lienalis microfilariae (mf), both in vitro and in experimentally infected CBA/Ca mice, was compared with that of ivermectin. The in-vitro results demonstrated that both compounds (at a concentration of 10(-7) M) significantly reduced the mf motility index (MI) throughout the 7-day culture period and that this reduction was similar for the two compounds. Mice were treated with moxidectin and ivermectin by subcutaneous injection (sc) or orally (po); the two routes were equally efficacious. When mice infected with On. lienalis were treated with one of the drugs at 3.2, 1.6, 0.8, 0.4 or 0.2 micrograms/kg.day on days 3-7 post-infection, with necropsy on day 18, moxidectin cleared more mf than ivermectin at all of the doses examined. In mice treated with a single dose (on day 3 post-infection), 150 or 15 micrograms/kg moxidectin completely cleared the mf whereas 1.5 micrograms/kg produced a 90%-96% reduction in mf recoveries. Following ivermectin treatment at the same doses, mf were virtually cleared at 150 micrograms/kg, with a 98% reduction at 15 micrograms/kg but no significant effect at 1.5 micrograms/kg. When mice with On. volvulus infections were treated with a single dose of moxidectin at 15 or 1.5 micrograms/kg, there were reductions in mf recoveries of 96% and 23%, respectively, compared with only a 48% reduction with 15 micrograms ivermectin/kg and a 2% increase with 1.5 micrograms ivermectin/kg. In order to examine the persistence and activity of each drug, mice were treated with a single dose of 150 micrograms/kg up to 28 days before infection. Moxidectin was found to be more efficacious (with subsequent 99.9% reduction in mf when given 28 days pre-infection and a 100% reduction when give 16 or 4 days before or 3 days after infection) than ivermectin (giving reductions of 57.1%, 66.7%, 100% and 100%, respectively). The further evaluation of moxidectin and its potential usefulness for the treatment of human onchocerciasis are discussed.

Animals↗

PubMed1994

Identification and characterization of an Onchocerca volvulus cDNA clone encoding a highly immunogenic calponin-like protein.

The identification and characterization of a recombinant cDNA clone, designated OV9M, expressing an antigen present in Onchocerca volvulus infective larvae and adult stages is described. Clone OV9M was identified by screening a lambda gt11 cDNA expression library derived from adult O. volvulus mRNA using pooled rabbit antisera raised against the third (L3) and fourth (L4) stage larvae of the parasite. The cDNA clone encodes an open reading frame of 238 amino acids corresponding to a 27-kDa polypeptide. This polypeptide contains a series of five highly conserved repeats of 25 amino acids that are similar to repeats found in calponin, a protein previously only identified in vertebrate smooth muscle. Extension of the 5' end of the cDNA clone revealed two additional repeats extending the sequence to 378 amino acids, encoding a 41.8-kDa protein. Affinity purified antibodies, which bound specifically to the glutathione S-transferase-OV9M fusion polypeptide, recognize a series of antigens in extracts of O.volvulus microfilariae, L3, L4 and adult stages. The apparent molecular weight of the native OV9M protein in the adult is 45 kDa. Similar proteins are present in extracts of other nematodes including Caenorhabditis elegans, and antibodies from other filarial infections are cross reactive with glutathione S-transferase-OV9M fusion polypeptide. Immunoelectron microscopy revealed that the antigen encoded by this clone is present in the longitudinal muscles of the various larval stages and adult worms. Antibodies to the OV9M protein are present in 40-60% of both patently infected and non-patent individuals residing in onchocerciasis endemic areas.

Amino Acid Sequence↗

PubMed1999

Detection of Onchocerca volvulus infection in Simulium ochraceum sensu lato: comparison of a PCR assay and fly dissection in a Mexican hypoendemic community.

Detection of Onchocerca volvulus larvae in vector populations is of prime importance in the assessment of the effectiveness of onchocerciasis control programmes. Traditionally, detection of larvae is attained by the dissection of flies, but this time-consuming method cannot easily discriminate between species of Onchocerca. The genome of all Onchocerca species has a unique 150 bp repeat, which can be amplified by PCR, and O. volvulus-specific DNA probes can detect these products by Southern blot (SB). This study optimizes a PCR/SB assay, and compares it with fly dissection to estimate the prevalence (p) and intensity of infection (m) in the local vector population of a Mexican community that has become hypoendemic as a result of 7 years of treatment with ivermectin and nodulectomy. The PCR detected 1 infected fly in a pool of 99 uninfected flies, but the optimal pool size was 50 flies. At the community level, 1 out of 10,550 flies was positive (p = 0.0095%, 95% confidence intervals CI = 0.00024-0.05280%; m = 0.00027 larvae/parous fly, CI = -0.00026-0.00081) by PCR, and 4 out of 10,772 flies (p = 0.0371%, CI = 0.01012-0.09505%; m = 0.00107 larvae/parous fly, 95% CI = 0.00002-0.00212) by dissection (observed m = 0.0005). Both methods produce statistically similar estimates of the prevalence and intensity, indicating that pool screening is a viable alternative for entomological surveillance in areas where the intensity of transmission is becoming extremely low as a result of control interventions.

Animals↗

PubMed1994

Molecular characterisation and localisation of an Onchocerca volvulus pi-class glutathione S-transferase.

Glutathione S-transferases (GSTs) constitute a major detoxification mechanism in helminth organisms and are regarded vaccine candidates against helminth infections. Onchocerca volvulus glutathione-binding proteins were purified from the aqueous soluble fraction of homogenised adult females by affinity chromatography on glutathione-agarose. The eluted proteins had a specific GST activity of 1.6 mumol min-1 mg-1. Immunohistochemical studies localised these antigens in the hypodermis, the wall of the seminal receptacle and spermatozoa of adult worms. A lambda gt11 clone was isolated from an expression library of O. volvulus by immunoscreening. Sequence analysis revealed that it encoded a pi-class GST with 60% identity with Caenorhabditis elegans and up to 45% identity with mammalian pi-class GSTs. Antibodies affinity selected with recombinant GST demonstrated cross-reactivity between Litomosoides sigmodontis and O. volvulus GSTs.

Amino Acid Sequence↗

PubMed1997

Onchocerca volvulus: comparison of field collection methods for the preservation of parasite and vector samples for PCR analysis.

In recent years, methods for the identification of the filarial worm Onchocerca volvulus and its vector, blackflies of the Simulium damnosum complex (S. damnosum sensu lato (s.l.)), based on the amplification of parasite and vector DNA sequences with the polymerase chain reaction (PCR), have been developed. Routine application of these methods requires techniques for sample collection and preservation that are compatible with the limitations of field collection, yet preserve DNA in a form suitable for PCR. Two different methods for sample preservation were evaluated by the field collection teams and the DNA probe laboratory of the Onchocerciasis Control Programme in West Africa. The most successful involved the preservation of material from O. volvulus and its associated vectors in a dried state on microscope slides. Of over 1200 parasite samples preserved in this manner, more than 93% retained DNA yielding positive results in PCR analysis (1208/1291). Vector material (malpighian tubules and ovaries) preserved in the same manner on the same microscope slides also yielded DNA that was suitable for PCR.

Animals↗

PubMed1994

Epitopes of the Onchocerca volvulus RAL1 antigen, a member of the calreticulin family of proteins, recognized by sera from patients with onchocerciasis.

RAL1 is an antigen (Ag) encoded by the filarial nematode Onchocerca volvulus, the parasite causing onchocerciasis (river blindness). RAL1 shares 64.4% identity with the autoantigen calreticulin. The striking similarity of the parasite Ag and the human autoantigen has led to the hypothesis that RAL1 may induce a cross-reactive immune response to calreticulin, which in turn may be involved in the pathogenesis of onchocerciasis. To test this hypothesis, we explored the immune response to RAL1 recombinant Ag (RAL1 rAg) and human calreticulin in patients with O. volvulus infection. A total of 86% of the O. volvulus-infected individuals produced antibodies recognizing RAL1 rAg. Antibody reactivity to RAL1 rAg in patient sera was confined primarily to the central and carboxyl-terminal parts of the molecule. No significant correlations were found to associate recognition of RAL1 rAg, or any particular portion thereof, with a particular disease state. Antibodies against RAL1 thus appear to be produced as a general immune reaction to O. volvulus infection and do not necessarily lead to a cross-reacting response with the host protein. In contrast, 33% of the patient sera tested bound recombinant human calreticulin. All of these sera also recognized a polypeptide encompassing the carboxyl-terminal portion of the RAL1 rAg. These results suggest that recognition of an epitope encoded in the carboxyl-terminal portion of RAL1 is at least in part responsible for inducing a cross-reacting immune response to the host protein.

Amino Acid Sequence↗

PubMed2000

Identification of a stress-responsive Onchocerca volvulus glutathione S-transferase (Ov-GST-3) by RT-PCR differential display.

The effects of oxidative insult on gene transcript levels in the filarial nematode Onchocerca volvulus were investigated using differential display RT-PCR. Oxidative stress was applied with the reagents paraquat, plumbagin and xanthine-xanthine oxidase. In all three cases, a cDNA fragment encoding a novel glutathione S-transferase (GST) resembling members of the theta-class was identified as upregulated (PQ29, PG112, XOD26). The subsequently isolated full-length cDNA harbors a 753-bp open reading frame encoding a GST with 268 amino acid residues and a predicted molecular mass of 31 kDa. This stress-responsive GST (Ov-GST-3) possesses only 14 and 21% sequence identity with the other O. volvulus GSTs (Ov-GST-1 and Ov-GST-2, respectively). Interestingly, Ov-GST-3 shares higher sequence identity with GSTs that are upregulated due to environmental stress. In order to confirm the specific upregulation of the Ov-GST-3 transcripts identified by differential display and to analyze the mRNA levels of the other Ov-GSTs (Ov-GST-1 and Ov-GST-2) under elevated stress conditions, a semi-quantitative polymerase chain reaction-enzyme-linked immunosorbent assay was performed. The Ov-GST-3 gene transcript level increased dramatically in response to xanthine-xanthine oxidase and to a lesser extent with paraquat and plumbagin. In contrast, Ov-GST-1 and Ov-GST-2 did not show any significant alterations in their steady-state mRNA levels in response to oxidative stress when examining the same mRNA samples. The present study clearly demonstrates that Ov-GST-3 is a critical enzyme in the defense against oxidative stress.

Amino Acid Sequence↗

PubMed1996

Onchocerca volvulus: identification of cDNAs encoding a putative phosphatidyl-ethanolamine-binding protein and a putative partially processed mRNA precursor.

We have identified and sequenced cDNAs of the parasitic nematode Onchocerca volvulus which encode a homologue of phosphatidylethanolamine-binding proteins from mammals. These clones are also closely related to the O. volvulus Ov16 cDNA (Lobos et al., 1990). One identified cDNA clone appears to represent a partially processed precursor. These results suggest that these cDNAs are derived from a complex genomic locus, raising the possibility of polycistronic transcription in this nematode.

Amino Acid Sequence↗

PubMed1996

Molecular cloning, expression, and localization of E1, an Onchocerca volvulus antigen with similarity to brain ankyrin.

Protective immunity against human onchocerciasis may best be reflected by the existence of individuals who in spite of exposure to the filarial nematode Onchocerca volvulus do not develop disease (putatively immune). We observed preferential recognition of an O. volvulus antigen of approximately 90 kDa by sera from putatively immune individuals compared with sera from diseased individuals. Screening of an adult worm cDNA library with one serum recognizing this antigen almost exclusively led to the identification of a full length clone of 2043 base pairs designated E1. The open reading frame of 462 amino acid residues shows similarity to human brain ankyrin. E1 appears to represent a small transcript of the O. volvulus ankyrin gene. The nonfusion protein obtained by expression of the complete E1 cDNA exhibits an apparent molecular mass of 90 kDa on SDS-polyacrylamide gel electrophoresis. An antiserum against the recombinant protein reacts with the 90-kDa antigen in O. volvulus extract. In O. volvulus, E1 was localized in the neuronal cell bodies, the nerve ring, and the extracellular clefts of the basal labyrinth. These results identify an ankyrin-related O. volvulus protein as an immunogen to putatively immune individuals, suggesting that neuronal proteins may be important targets for immunity against O. volvulus in vivo.

Amino Acid Sequence↗