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PubMed1998

Zoonotic Onchocerca (Nematoda:Filarioidea) in the cornea of a Colorado resident.

OBJECTIVE: A female patient, resident in the state of Colorado, presented with iritis of the right eye. Slit-lamp examination showed the presence of a thin, threadlike worm entwined in the cornea. The patient was taken to surgery for removal of the parasite. DESIGN: A case report. INTERVENTION: A 3-mm-long supertemporal incision was made in the cornea and further dissected until the worm could be grasped and removed by gentle traction. RESULTS: The worm, a filarial nematode, was identified as a member of the genus Onchocerca, most likely Onchocerca cervicalis, a natural parasite of horses. The patient had an uneventful recovery, and 1 week after surgery, her visual acuity, intraocular pressure, and corneal edema were all resolving. CONCLUSION: In the United States and elsewhere, most cases of zoonotic filarial infection involving the eye are caused by Dirofilaria or Dipetalonema-like worms. However, the current case was caused by a species of Onchocerca. This is the first case of zoonotic Onchocerca from the eye to be reported, only the second case of zoonotic Onchocerca in the United States, and the seventh case worldwide. The worm was removed surgically, and the patient had an uneventful recovery.

Animals↗
PubMed1989

Biochemical and immunochemical characterisation of a 20-kilodalton complex of surface-associated antigens from adult Onchocerca gutturosa filarial nematodes.

Surface radioiodination of adult Onchocerca parasites reveals a restricted range of proteins associated with the cuticle. We present data to show that prominent among these is a complex of low-molecular-weight proteins which can be released in soluble form by homogenisation of surface-labelled Onchocerca gutturosa in phosphate-buffered saline (PBS). One of this groups of proteins, designated gp20, has a molecular mass of 20,000, is glycosylated with two N-linked carbohydrate side chains, and has a basic pI. Other PBS-soluble, 125I-labelled proteins of similar size appear not to be glycosylated. A distinct group of molecules are released only in the presence of reducing agents, and are likely to be cuticular collagens. The low-molecular-weight components are antigenic and cross-reactive with Onchocerca volvulus infection sera. Cross-reactions are also observed in immunoprecipitation experiments using sera from Brugia-immunised animals and infected humans. Comparative two-dimensional analyses of these immunoprecipitates reveal at least two Onchocerca specific components. As an alternative to radiolabelling and PBS homogenisation, incubation of worms in medium containing the reducing agent 2-mercaptoethanol resulted in a similar set of molecules being released into the medium. Since surface antigens of O. gutturosa from bovines and O. volvulus from humans appear similar in size and are antigenically cross-reactive, the more readily available parasite is being used to study further the properties of these molecules and to provide reagents for raising antisera reactive to the equivalent O. volvulus antigens.

Animals↗
PubMed1993

Onchocerciasis in British cattle: a study of the transmission of Onchocerca sp. in north Wales.

The transmission of bovine Onchocerca spp. in Britain was investigated by studying the Ceratopogonidae and Simuliidae which attacked bait cattle between April and October at two sites in North Wales, where either Onchocerca lienalis and O. gutturosa (Cynwyd), or O. lienalis alone (Pentrefoelas) occurred. Based on the relative abundance of each species, their seasonal variation in abundance and preferential feeding sites on cattle, the presence of Onchocerca sp. infective larvae and the development of patent infections in bait cattle, vectors were identified as Simulium ornatum s.1. at Pentrefoelas and S. reptans at Cynwyd (0.5% and 1.5% of parous flies infective, respectively). There was no significant difference between the lengths of the infective larvae from vectors at either site (Pentrefoelas: mean = 543.5 microns, SD = 29.45; Cynwyd: mean = 550.86 microns, SD = 35.26; p > 0.1), which were consistent with descriptions of O. lienalis. The identity of a vector for O. gutturosa was not determined and the role of certain candidates is discussed. In this respect, the absence of Culicoides nubeculosus was notable. Transmission of Onchocerca sp. was limited to a short period of about 8 weeks, by a combination of vector abundance and parasite uptake from dermal skin layers, and a mean daily temperature requirement of 15 degrees C or more for complete parasite development in the vector. The effect of this temperature restriction in relation to the geographical distribution of the two species is discussed.

Animals↗
PubMed1991

The development of Onchocerca dukei and O. ochengi microfilariae to infective-stage larvae in Simulium damnosum s.l. and in members of the S. medusaeforme group, following intra-thoracic injection.

Neonate Simulium females from an endemic area of human and bovine onchocerciasis in North-Cameroon were injected in one set of experiments with microfilariae (mff) of Onchocerca dukei and in another with mff of O. ochengi. Onchocerca dukei was found to develop to infective third-stage larvae in Simulium flies: 148 third-stage larvae (28.1% of injected mff) were recovered in S. hargreavesi, seven (10.4% of injected mff) in S. vorax, and 12 (1.3%) in S. damnosum s.l. Onchocerca ochengi mff developed in S. hargreavesi at a rate of 17.3% and in S. damnosum s.l. at 16.1%, yielding a total of 77 and 32 third-stage larvae respectively. Onchocerca dukei infective larvae had a mean length of 717.3 microns and a mean maximum diameter of 17.5 microns, and were slightly shorter and conspicuously thinner than those of O. ochengi (748.3 microns x 20.4 microns). All larval stages of O. dukei typically had an attenuated anterior end. The implications of these findings for the epidemiology of human onchocerciasis are discussed.

Animals↗
PubMed1994

Natural vectors of three bovine Onchocerca species (Nematoda: Onchocercidae) and seasonal transmission by three blackfly species (Diptera: Simuliidae) in central Kyushu, Japan.

Eleven blackfly species were examined in relation to the transmission of bovine onchocerciasis in central Kyushu, Japan. A total of 19,005 out of 47,746 females captured from June 1989 to June 1990 at two cattle sheds each in Oita and Kumamoto were dissected for their parities, infections with Onchocerca larvae, and intakes of Onchocerca microfilariae. Overall results showed that Simulium bidentatum (Shiraki), the predominant species, is a natural vector of the two bovine Onchocerca species, i.e., type I (O. sp.) in both localities and type II (probably O. gutturosa Neumann), in Oita, whereas S. kyushuense Takaoka and S. arakawae Matsumura serve as the primary vector of type III (O. lienalis Stiles) in Kumamoto and Oita, respectively. Simulium arakawae also plays a secondary role in the transmission of type I in Oita and of type III in Kumamoto. Despite the biting activities and microfilarial intakes observed throughout most of the entire year, the transmission of these bovine Onchocerca was restricted to the mild or warm months between May and November. The natural infections with mermithid, trematode, ciliates, and fungi occurred but their influence on the vector Simulium species was apparently small, if at all.

Animals↗
PubMed2002

Orientation of Onchocerca lienalis stiles (Filarioidea: Onchocercidae) microfilariae to black fly saliva.

Black flies (Simulium spp.) are intermediate hosts and vectors of parasitic nematodes belonging to the genus Onchocerca (Filarioidea Onchocercidae). Infection and subsequent transmission of infective third-stage larvae occur at the vertebrate host-skin interface. Experimental evidence presented here demonstrates that Onchocerca lienalis Stiles microfilariae orient to one or more components (microfilarial orientation factor [s]; MOF) in black fly saliva. MOFs may serve as a means for microfilariae to find and infect black flies during the act of blood-feeding. Directed movement through the host's skin to the bite site is necessary because Onchocerca spp. microfilariae do not circulate in the blood. The substance directing microfilarial orientation appears to be a salivary protein, but it is not the Simulium vittatum Zetterstedt erythema protein (SVEP) described from New World Simulium spp. These results support earlier field observations that associated increased numbers of cutaneous microfilariae with black fly feeding and indicate that a fundamental molecular mechanism linked to vector saliva may be key for the maintenance of the life cycle of Onchocerca spp. Salivary molecules that induce orientation of microfilariae to the bite site are potential targets for use in transmission-blocking vaccines to uncouple this primary vector infection step.

Animals↗
PubMedDate not supplied

Identification of the forest strain of Onchocerca volvulus using the polymerase chain reaction technique.

Annual mass treatment with ivermectin for 12-15 years in endemic communities is the control strategy adopted by the African Programme for Onchocerciasis Control (APOC) for the control of onchocerciasis in Nigeria. This long-term treatment necessitates the use of Polymerase Chain Reaction (PCR) for the proper identification of the Onchocerca species and strains in endemic areas and also for monitoring recrudescence of infection in areas where infection has been controlled. This study, which forms part of a larger study on transmission of onchocerciasis identifies the Onchocerca volvulus strain in Ondo state using the Polymerase Chain Reaction (PCR) technique. Deoxyribonucleic acid (DNA) was extracted from the adult worm of Onchocerca parasite using the glass bead method of extraction. The repeated sequence family present in the genome of the parasite designated as 0-150bp was amplified by the polymerase chain reaction (PCR). The amplified parasites produced significant products visible as bands in a 2% agarose gel stained with ethidium bromide. Hybridization of the PCR products with specific DNA probe identified the products as forest strain of Onchocerca volvulus. The epidemiological implication of this is that there would be more of the skin lesions and low blindness rate in the area.

Animals↗
PubMed1989

On the possibility of bovine Onchocerca species infecting Simulium damnosum s.l. in the forest zone of Sierra Leone. II. Biting densities and filarial infections in Simulium spp. and Culicoides spp.

Previous studies of bovine Onchocerca spp. in cattle in Sierra Leone indicated that only O. gutturosa was transmitted in the forest zone at high intensity. To determine its vector(s) and the extent to which Onchocerca-like infections in Simulium damnosum were likely to be of bovine origin, three lines of investigation were pursued. Firstly, a study was made of the biting flies attacking an ox bait animal over a 14-month period at Njala University Campus, near Bo. Secondly, attempts were made to infect the dominant local forest cytospecies of S. damnosum s.l. with O. gutturosa by feeding them on an infected ox under a bed-net. Thirdly, S. damnosum s.l. were infected by intra-thoracic injection of O. gutturosa microfilariae (mff). In 113 collections made at dawn and dusk at weekly intervals from the ox bait, 624 simuliids and 7740 Culicoides spp. were collected. Almost all the simuliids were S. damnosum s.l. which, on the basis of iso-enzyme examination and knowledge of local breeding sites, were identified as S. soubrense 'B'. Although this cytospecies fed readily on the ox at ventral sites where O. gutturosa mff occurred and the bed-net experiments showed that 16.1% of engorged S. soubrense 'B' ingested an average of 3.3 O. gutturosa mff each, no development occurred. The refractoriness of S. damnosum s.l. to O. gutturosa was confirmed by the intra-thoracic injection experiments. The predominant Culicoides spp. attacking the ox bait were C. krameri, C. trifasciellus and C. fulvithorax, with smaller numbers of C. schultzei. In 5803 dissected Culicoides spp., natural infections of Onchocerca-like larvae, presumed to be O. gutturosa, were found in 0.3% of C. fulvithorax, 0.1% of C. trifasciellus and 0.06% of C. krameri. It was concluded that, in the forest zone of Sierra Leone, S. damnosum s.l. is not a vector of O. gutturosa and all Onchocerca-like larvae in S. damnosum are likely to be O. volvulus, while the natural vectors of O. gutturosa are the Culicoides species C. fulvithorax, C. trifasciellus and C. krameri.

Animals↗
PubMed1979

Studies on the Onchocerca (nematoda: Filarioidea) found in cattle in the United States. I. Systematics of O. gutturosa and O. lienalis with a description of O. stilesi sp. n.

Onchocerca gutturosa and O. lienalis are redescribed and comparative morphological descriptions provided. In addition, a third species of Onchocerca is described from cattle in the United States. This third species, Onchocerca stilesi sp. n., localizes in the connective tissues around the stifle joint, and can be distinguished morphologically from O. gutterosa and O. lienalis. Onchocerca gutturosa and O. lienalis apparently have widespread distribution in the eastern United States; however O. stilesi has been reported only from Minnesota.

Animals↗
PubMed1979

Bovine and equine onchocerciasis in eastern North America with a discussion on cuticular morphology of Onchocerca spp. in cattle.

Skin sections and/or the ligamentum nuchae and ligamentum gastrolienale were examined from twelve bovine carcasses obtained from southern and eastern Ontario and from Quebec. Of these, seven were shown to be infected with Onchocerca gutturosa and/or Onchocerca lienalis. The morphology of the adult female cuticle is discussed. Skin sections from 43 equine carcasses from a slaughter house in Grenville, Quebec were examined and microfilariae of Onchocerca sp. were recovered from 32 (74%). There are probably referable to Onchocerca cervicalis.

Animals↗
PubMed1998

Onchocerca ochengi: morphological identification of the L3 in wild Simulium damnosum s.l., verified by DNA probes.

In order to assess the prevalence of the cattle filaria Onchocerca ochengi in onchocerciasis vectors (Simulium damnosum s.l.) in North Cameroon, we searched for a means to morphologically identify its developing larvae, which closely resemble those of O. volvulus. To this end microfilariae of the 2 Onchocerca species were isolated from slaughter cattle in Ngaoundéré and injected into neonate Simulium species. Whereas the early developmental stages (sausage stage, L2 and pre-infective larva) were indistinguishable, the infective larvae (L3) of O. ochengi were longer (median: 740 microns), more slender (diameter = 19.3 microns = 2.6% of body length) and had a relatively shorter tail (4.9% of body length) than those of O. volvulus (680 microns, 20.5 microns, 3.0% and 5.8% respectively). The tail of O. ochengi L3 was thick and rounded, whereas it was slightly tapering in O. volvulus L3. O. ochengi L3 produced by feeding flies on infected cattle in a different area in North Cameroon (Sora Mboum) showed the same features as intrathoracically produced O. ochengi L3 from Ngaoundéré, but were even longer (785 microns). On the basis of the differences in length, relative diameter, length of the tail and shape of the tail, a simple key for the separation of O. volvulus and O. ochengi L3 was elaborated, and 248 L3 found in wild S. damnosum s.l. were separated into 'O. ochengi' (160 L3) and 'O. volvulus' (88 L3) following this key. Sequential dot blot hybridization of each of the 248 larvae with a DNA probe which reacts with O. ochengi and O. volvulus but not with other Onchocerca species (pOo5/1) and with an O. volvulus-specific DNA probe (pOv12) revealed that the morphological identification had been correct in 86-91% of the cases. Only a small proportion (6-9%) of the dot blots did not react with either probe. Since this proportion was equal in experiments using experimentally produced L3 and in experiments using wild L3, the nonhybridization was certainly due to a loss of L3 during washing of the filters and not due to the presence of other unknown L3 species resembling O. volvulus and O. ochengi. Our study shows that in Cameroon it is possible to identify O. volvulus and O. ochengi infective larvae during routine fly dissections by morphology alone.

Animals↗
PubMed1995

Stage-specific and species cross-reactive antibody responses in experimental Onchocerca infections of cattle.

Cattle experimentally infected with Onchocerca lienalis were examined by enzyme-linked immunosorbent assay and immunoblotting to determine the degree of stage- and species-specificity in the immune response to infection. Levels of serum antibodies to antigens derived from third-stage larvae increased little after the first three weeks of infection, and the range of antibody specificities remained limited following the appearance of microfilariae (mf) in the skin. In contrast, antibodies to antigens from adult worms of either sex exhibited a vigorous response, characterized by a series of peaks arising 15-30, 79, and > 266 days after infection that were coincident with the timings of larval molts and the onset of a patent infection. Antibody specificities to the adult worms included many directed to molecules that were shared with other life-cycle stages, but some were stage-specific and others were confined to one sex. A response cross-reactive with antigens from mf was initiated during the prepatent period, but antibody levels increased steeply after the infection became patent. This was followed by a major expansion of antibody specificities to products exclusively directed to mf, most notably in the range of 12-18 kilodaltons. Sera from O. lienalis-infected cattle cross-reacted extensively with antigens derived from O. volvulus adult worms and the profiles of antibody levels over time were indistinguishable from those obtained with O. lienalis extracts. The dominant response was of IgG1, although limited IgG2 and IgM reactivities were found, while no Onchocerca-specific IgA was detected. These results demonstrate that parasite development has a profound influence on the level and repertoire of antibodies produced during Onchocerca infections, and that extensive cross-reactivity exists between O. lienalis and O. volvulus, lending support to the role of cattle models in the study of human onchocerciasis.

Animals↗
PubMed1987

Onchocerca antigens in protection, diagnosis and pathology.

Characterization of the immune response to Onchocerca volvulus is important for the diagnosis, control and understanding of the disease it causes. The antibody response to surface, secreted and somatic antigens of the worm has therefore been examined at an individual immunoglobulin (Ig) class level, by using a panel of different human sera. Onchocerca-specific antigens tend to be of low molecular mass and preferentially recognized by IgG4 and IgE. There is considerable cross-reaction between O. volvulus and O. gibsoni, so that the latter may be an alternative source of material for use in diagnosis. A surface-enriched fraction of low molecular mass appears to be a most promising diagnostic tool. Amongst somatic antigens, two were uniquely recognized by IgG3 antibodies in sera from sowda patients, thereby providing a molecular correlate for a recognized pathological condition. Improved diagnosis is needed for detecting infection in both humans and the vector. Our target for detection in humans is a continuously released, nonimmunogenic product, which is ideally stage and parasite specific. The excretions of adult worms do contain components not recognized by antibodies in infected serum, but we cannot rule out that these are of host, rather than parasite origin. Excretions of Litomosoides carinii contain both host and parasite molecules and, in addition, stage-specific and sex-specific components. Unfortunately, however, the rate of production of excretions varies during the life of L. carinii. This finding may be relevant to the detection of Onchocerca excretions if they are produced at a similarly uneven rate. Finally, for detecting infective larvae in the vectors, we are currently screening a genomic library of O. volvulus for an appropriate probe. To date, one DNA sequence has been cloned that shows promising specificity.

Animals↗
PubMed1996

cDNAs from Onchocerca sp. encoding members of the MRS3/MRS4 class of mitochondrial solute carriers.

cDNA clones from the parasitic nematodes Onchocerca volvulus and Onchocerca gibsoni encode homologs of the yeast proteins MRS3 and MRS4. Together with an uncharacterised ORF on chromosome III of Caenorhabditis elegans, these constitute a new class of proteins belonging to the mitochondrial solute carrier protein superfamily. So far, five other members of this protein family have been identified in C. elegans, but levels of identity between these and the Onchocerca proteins were considerably lower. Consideration of cysteine content and overall charge implies that the natural substrates of the nematode proteins are small ions.

Adenosine Diphosphate↗
PubMed1989

Extracellular proteases of Onchocerca.

Two important events in infection by Onchocerca parasites involve cutaneous tissue migration by larval stages. L3 larvae migrate from the blackfly bite site to subcutaneous locations for adult development, and microfilariae from subcutaneous nodules to distant regions of the skin and sometimes the eye. By analogy to other tissue-invasive helminth larvae, it has been proposed that migration of Onchocerca larvae through cutaneous tissue is facilitated by secretion of proteolytic enzymes. To test this hypothesis, neutral protease activity capable of degrading a model of cutaneous extracellular matrix was assayed using live L3 larvae of O. lienalis and microfilariae of O. cervicalis and O. cervipedis. Five hundred L3 larvae degraded most of the matrix within 24 hr of incubation. Substrate gel electrophoresis and other protease assays showed a 43-kDa serine elastase was secreted by O. lienalis L3 larvae. Larvae and adults of the free-living nematode, Caenorhobditis elegans, by contrast, did not secrete neutral proteases and large numbers of motile C. elegans juveniles and adults produced no degradation of the extracellular matrix. Expression of Onchocerca neutral protease activity was stage specific. No protease activity corresponding to that seen in L3 larvae was found in adult worms. Microfilariae of O. cervicalis and O. cervipedis produced both a serine and a metalloprotease, but the level of protease activity of these microfilariae was substantially lower than that of L3 larvae, and no significant protease activity was detected in extracts of O. lienalis microfilariae. Uterine microfilariae of O. cervicalis had different protease species than skin microfilariae, suggesting that changes in protease expression parallel other morphologic and biochemical changes in the development of skin microfilariae. The serine protease of L3 larvae probably plays an important parasitic function, facilitating L3 migration from the blackfly bite site to distant regions of the body where adults will develop and form nodules. The protease activity of microfilariae, while individually considerably less than that of L3 larvae, may still contribute to the tissue destruction seen with heavy skin densities of microfilariae.

Animals↗
PubMed1989

Isolation of Onchocerca gibsoni tissue from frozen nodules for biochemical use.

A new procedure is described which enables gram quantities of adult Onchocerca tissue to be isolated from frozen connective tissue nodules, thus minimizing the risk of enzymatic degradation. Bovine connective tissue nodules containing adult Onchocerca gibsoni worms were obtained from Australia frozen at -70 degrees C and sectioned while still frozen into 3 mm thick slabs. The sections were thawed immediately before use, worm segments removed, rinsed, pelleted, and flash frozen in liquid nitrogen. Quality of the isolated material was demonstrated by the presence of an intact adult epicuticle as determined by electron microscopy, and by the presence of viable uterine larvae and cells. This procedure is applicable to other nodule-forming worms such as Onchocerca volvulus and is suitable for investigations which require the isolation of labile molecules or those present in minute quantities.

Animals↗
PubMed1991

Surface antigens of male worms and microfilariae of Onchocerca gibsoni.

Living adult males and microfilariae of the cattle filarial parasite Onchocerca gibsoni were externally labelled with radioactive iodine using the iodogen and Bolton-Hunter procedures. Characterization of labelled surface proteins by sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis revealed clear cut differences in the two life cycle stages. In addition, the two radiolabelling procedures yielded some differences in the profiles of radiolabelled surface proteins for both adults and microfilariae. Immunoprecipitation analysis revealed a number of labelled antigens recognized by antibodies in human onchocerciasis serum pools, thereby demonstrating the usefulness of O. gibsoni as a model in Onchocerca volvulus vaccine studies. The reactivity of microfilarial antigens extended to antibodies from other human nematode infections, whereas male surface antigens, particularly those of low molecular weight, were Onchocerca specific. This indicates that O. gibsoni can provide a convenient source of specific diagnostic antigen.

Animals↗
PubMed1990

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae.

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

Animals↗