Onchocerca Species Research Articles

An Onchocerca Species from Cattle on Kyushu Island is O. suzukii, a Transfuge Parasite from the Japanese Endemic Bovid, Capricornis crispus.

In 1988-1989, microfilariae of an unidentified Onchocerca species were found in the skin of eight of the 16 cattle aged at least 5 years that were examined in Oita, Kyushu, Japan. Adult worms were not found in cattle, but the microfilariae taken from skin snips, which have coiled posterior parts, were grown to the infective stage in simuliid. In 1997, both adults and microfilariae of 0. suzukii were recovered from Japanese serows (Capricornis crispus) in Yamagata, Honshu. Such microfilariae from serows, reported here for the first time, had coiled posterior parts and measurements {130-160 (mean, 140) pm long and 4-6 (5) μm wide} close to those from cattle. The findings showed that the Onchocerca sp. in cattle in Kyushu is O. suzukii, a transfuge parasite from the Japanese endemic bovid, C. crispus.

PCR and DNA Hybridization Indicate the Absence of Animal Filariae from Vectors of Onchocerca volvulus in Uganda

In order to identify Onchocerca volvulus larvae from vectors, DNA of filaria larvae from dissected blackflies was isolated, and a 150-bp long tandemly repeated DNA sequence (0-150), which occurs in many Onchocerca species, was amplified using polymerase chain reaction (PCR). Subsequently, the PCR product was blotted onto a nylon membrane and hybridized with DNA probes specific for O. volvulus or Onchocerca ochengi. Filaria larvae from 395 infected Simulium neavei were examined and 259 samples produced detectable PCR products. Among these samples, 239 (92%) reacted with an O. volvulus-specific oligonucleotide. A sample of 69 PCR products was tested using an O. ochengi DNA probe, but all failed to hybridize. Filaria larvae from 64 infected Simulium damnosum, presumably of the cytotypes "Nyamagasani" and "Nkusi" were studied and 0-150 was amplified from 38 samples. From these samples, 35 (92%) hybridized specifically with an O. volvulus probe but none with the O. ochengi-specific DNA sequence. Nonamplified samples were obtained mainly from blackflies that contained only 1 or 2 filaria larvae, and therefore, an insufficient DNA extraction was assumed. It can be concluded that few, if any, filaria species of animal origin were transmitted by S. neavei and S. damnosum s.l. in Kabarole and Kasese districts in Uganda.

Distribution of mast cells and their correlation with inflammatory cells around Onchocerca gutturosa, O. tarsicola, O. ochengi, and O. flexuosa.

In recent years, bovine Onchocerca species have been used as models for human onchocerciasis in drug screens. They have been suggested for immunology studies and evaluation of vaccine candidates. Therefore, mast cells and their association with other inflammatory cells were studied in five onchocercal species of cattle and deer using immunohistology. Intact mast cells occurred in large numbers in the capsule and septae of nodules, in fibrous tissue adjacent to nonnodular worms, and perivascularly. Inactive and, more frequency, activated and degranulating mast cells were observed within infiltrates in the nodule center or around nonnodular filariae. They were not detected in direct contact with the cuticle of adult worms or of microfilariae or among the macrophages, giant cells, and neutrophils forming the innermost layer around the worms. Eosinophils, but not mast cells, were obviously associated with microfilariae-producing females. The distribution, frequency, and activity of mast cells were similar for all five species and O. volvulus.

Immunohistological studies on an Onchocerca volvulus ankyrin (EI).

The distribution of an Onchocerca volvulus ankyrin, designated E1, was studied in different O. volvulus stages and other helminths by immunohistochemistry using rabbit antibodies raised against the recombinant E1 protein. In adult O. volvulus the protein designated E1 was localized to the extracellular clefts as well as to the cytoplasm adjacent to the cell membrane in the area of the basal labyrinth in hypodermis, intestine and uterus and to a lesser extent in oviduct and vas deferens. Neuronal cell bodies were also labelled. No labelling of the basal laminae, muscles or epithelia of ovary or testis was observed. Detection of the E1 protein was associated with embryonic development. Germ cells and early morulae showed no reaction; labelling was first seen in late morulae, corresponding to the stage of gastrulation, and increased in the following embryonic stages. In microfilariae the nerve ring and the cephalic space, which represents the anterior nerve-enriched portion of the body, were labelled. In third-stage larvae of O. volvulus labelling was associated with the hypodermis, and in those of Anisakis sp. the cytoplasm adjacent to the membrane of the excretory gland cell and the basal labyrinth of the hypodermis were labelled. Following anthelminthic treatment a disruption of the labelling pattern of the E1 protein was observed in adult O. volvulus with leakage of the protein into neighbouring areas. Damage to the worm was associated with reduction and finally loss of E1 protein labelling. No E1 protein was detected in dead adult worms, embryos or microfilariae. Labelling of the same organs was observed in 8 other Onchocerca species and in several other nematodes, but no reaction was seen in trematodes. The results indicate that the EI protein is associated with neuronal structures of O. volvulus, that its presence is developmentally regulated and that it has cross-reactive homologues in other nematodes. The results suggest that E1 is a functional protein. It may be useful for the assessment of parasite damage and death as well as in the characterization of the filarial nervous system.

Identification of a Common Filarial Larva in Simulium damnosum s.l. (Type D, Duke, 1967) as Onchocerca ramachandrini from the Wart Hog

Filarial larvae resembling Type D (Duke, 1967), which are common in the Simulium damnosum s.i. vectors of human onchocerciasis ("riverblindness") in several parts of West Africa, were dissected from wild-caught flies in north Cameroon and examined morphologically. This was done in order to establish a possible synonymy with infective larvae (L3) of 2 recently discovered Onchocerca species of wart hogs (Onchocerca ramachandrini Bain, Wahl, and Renz, 1993 and Onchocerca sp. Wahl and Bain, 1995), which had been found to resemble Type D. After dissection of approximately 1,700 S. damnosum s.1., 13 Type D-like larvae were recovered from 12 infected flies. Their morphology corresponded to O. ramachandrini.

Prevalence of Onchocerca species and Thelazia lacrimalis in horses examined post mortem in Normandy.

The umbilical skin and ligamentum nuchae of 368 horses were examined post mortem for Onchocerca species. Only four of the horses were infected and pathological changes were observed on the skin of two of them. Thelazia lacrimalis was recovered from 38 (10.3 per cent) of the horses, and animals aged six months to two years were more frequently infected. No ocular lesions were observed. The prevalences of these two nematodes were low when compared with the infection rates reported in the United Kingdom and North America.

Development by injection in Simulium damnosum s.l. of two Onchocerca species from the wart hog to infective larvae resembling type D larvae (Duke, 1967).

Four wart hogs (Phacochoerus aethiopicus) examined in the Sudan savanna of North-Cameroon were all found infected with two types of skin microfilariae. One was O. ramachandrini Bain, Wahl and Renz, 1993, the adult worms of which live in the subcutaneous tissues of the feet. The other, smaller type belongs to a new Onchocerca species, the adult worms of which were not yet found. O. ramachandrini-microfilariae were evenly distributed across the whole body surface, those of Onchocerca sp. were concentrated on the back. The two species of microfilariae were isolated from an infected hide separated under the dissecting microscope and injected into the thorax of pupae-hatched S. squamosum and S. damnosum s.slr. females. Both filariae developed in both flies at high rates (33-47% of injected microfilariae) and without pathological forms to infective larvae L3). Both L3-species had a caudal tip, were long, slender and very motile and had a conspicuous glandular oesophagus. L3 from O. ramachandrini-microfilariae had a long glandular oesophagus (55% of total L3 length), a round head and measured an average of 955 microns long and 19.2 microns wide. L3 from the other microfilaria-species were shorter (845 microns, P < 0.001) and thinner (16.7 microns, P < 0.001) and had a shorter glandular oesophagus (36%, P < 0.001), a shorter tail (P < 0.01) and a conical head. Both L3-species, by their caudal tip, their long and slender silhouette, their great motility and their conspicuous glandular oesophagus resemble non-O. volvulus filarial L3 known, since many years, to occur in "wild" S. damnosum s.l. in Cameroon (Type D larvae, Duke, 1967) and in Liberia (Agamofilaria Type VI, Voelker and Garms, 1972). During our study, L3 such larvae were found in 12 wild S. damnosum s.l. from two geographically different areas of North Cameroon and all identified as O. ramachandrini. The excellent development of the two Onchocerca species from the wart hog in S. damnosum s.l. after artificial infection, and the identification of all recently examined wild Type D larvae as O. ramachandrini suggest that S. damnosum s.l. is a natural vector of O. ramachandrini and that most (if not all) of the Type D larvae in onchocerciasis vectors in North-Cameroon originate from wart hogs.

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.

Light and electron microscopy studies onOnchocerca jakutensis andO. flexuosa of red deer show different host-parasite interactions

Adult filariae of two intranodular Onchocerca species of red deer (Cervus elaphus) were examined at the ultrastructural level. In all, 90 nodules of O. flexuosa and 20 nodules of O. jakutensis were studied by histology. O. jakutensis caused cystic and pus-filled nodules in which the female and male worms were motile. Female worms possessed a thick cuticle and a well-developed somatic musculature. The basal lamina of the muscular syncytia was connected with the transhypodermal fibers through long protrusions. The epicuticle had only few protuberances and no visible surface coat. Female O. flexuosa showed morphological features similar to those of other intranodular filariae. A surface coat could not be detected. In 24% of 33 nodules with young live female worms and 40% of 38 nodules with older live female O. flexuosa the worms were surrounded by giant cells apparently attacking a cuticle. This was found even in nodules obtained from animals no older than 9 months. It is probable that the lack of a surface coat protecting the female worm and the inability to move due to the reduced somatic muscles lead to an early elimination of O. flexuosa by the host's immune system.

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.

Bovine onchocercosis in North Cameroon

Ventral skin biopsies from 204 Gudali cattle of the Vina division in the Adamawa highlands revealed microfilariae of Onchocerca gutturosa, O. ochengi and O. dukei in 85%, 51% and 8% of the animals, respectively. In 60 Fulani cattle from the Tcholliré division in the Sudan savanna, the same microfilaria species were detected in 92%, 83% and 47% of the animals. Onchocerca armillata adult worms were found in 67% of the Gudalis and in 100% of the Fulanis. In areas of high transmission the prevalences declined in old animals, possibly indicating acquired resistance. For all species no significant difference in prevalence was found between male and female cattle. The microfilariae of O. ochengi and O. dukei were concentrated in the skin of the posterior and anterior belly, respectively. Onchocerca gutturosa microfilariae had highest densities on the hump and near the umbilicus, whereas those of O. armillata were distributed more evenly across the body surface. In infected hides the mean microfilarial densities of O. gutturosa, O. ochengi, O. dukei and O. armillata were respectively 3.1 microfilariae (mff) mg −1, 0.6 mff mg −1, 0.7 mff mg −1 and 0.092 mff mg −1 for the whole body surface and 9.3 mff mg −1, 3.8 mff mg −1 and 1.9 mff mg −1 for the sites of highest density ( O. armillata had no predilection site). Ninety-five per cent of the microfilariae were located in the uppermost skin layer of 2 mm depth, 5% were in the corium and none were found in the subcutis. Two cattle had skin microfilariae of a hitherto unknown Onchocerca species.

Isolation of new markers to detect genetic variation inOnchocerca volvulus

Newly developed techniques (RADP: Random Amplified Polymorphic DNA and microsatellite DNA sequences) were used to study genetic variation in Onchocerca volvulus. RAPD technique, derived from the Polymerase Chain Reaction (PCR), allow clear distinction between the different species of the genus Onchocerca.

Entomological evaluation of ivermectin mass treatment against onchocerciasis

To evaluate the impact of onchocerciasis control programmes on the annual transmission potential, it is customary to monitor the man-biting rate of vector Siniuliuin (Diptera: Simuliidae) and the number of Oizclzocerca (Nematoda: Onchocercidae) infective larvae found in them (Walsh et al., 1978). In addition to the transmission of Onchocerca volvulus (Leuckart) causing human onchocerciasis, Simuliunz females may also be the vectors of various other species of Onchocerca which parasitize wild or domestic animals. The larvae of other Oizclzocerca spp. are difficult to distinguish from those of O. volvulus. Therefore, using routine methods in the field, it is not easy to determine the efficiency of a mass host chemotherapy campaign targeting only one Oizclzocerca species. To evaluate the impact of ivermectin on natural transmission of O. volvulus, we tried to improve the usual entomological index of infection by measuring lengths of Onchocerca larvae found in female blackflies (Simuliidae) collected on human bait. Annual mass treatment with ivermectin has been carried out since 1987 in the Vina Valley, a savanna region of North Cameroon, involving about 20,000 people (Prod'hon et al., 1991). Ivermectin was given to eligible people at the target dosage of 150 kg per kilogram of body weight. Coverage with the treatment was higher than 60% (Prod'hon et al., 1991). According to Traore-Lamizana & Lemasson (1987), the principal vectors of onchocerciasis in Vina valley are Simulium damnosunz Theobald sensu stricto and Xsirbaizurn (Vajime & Dunbar). Entomological surveys of man-biting Simuliidae were performed 1 month before treatment; then done again during the 2 months following the campaign. Simiilium females were dissected and larvae of Onchocerca were observed and counted as described by Philippon (1977). All third stage larvae found in the blackfly head, considered to be infective larvae, were stretched by heat exposure, then measured using an eye-piece micrometer. Infective larvae were recorded per 1000 parous blackflies. Infective larva lengths were repre-

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.

Glutamine-Supported Motility of Adult Filarial Parasites In vitro and the Effect of Glutamine Antimetabolites

The survival in culture of adult female Brugia pahangi, Acanthocheilonema viteae, and Onchocerca volvulus and adult male Onchocerca gibsoni was assessed by measuring parasite motility. Survival of all species was maximal in a nutritionally complex medium (RPMI-1640). All species survived for up to 48 hr in a simpler medium in which the only energy source was 10 mM glutamine; motility in this medium was dependent upon pH. For the species of Onchocerca, motility was maintained better in the presence of glutamine as the sole energy source than in glucose-only medium. Motility of B. pahangi incubated in 10 mM succinate was equivalent to that seen with 10 mM glutamine, but no other tricarboxylic acid intermediate supported this parasite in vitro. Antimycin A (1 microM) and potassium cyanide (KCN, 100 microM) paralyzed B. pahangi incubated in 10 mM glutamine, an effect antagonized by glucose. KCN at 10 or 100 microM was effective also against Onchocerca gutturosa in glutamine-only medium. Several glutamine antimetabolites reduced motility of B. pahangi by 72 hr. This inhibition was prevented by 2 mM glutamine. However, the inhibition of motility in the species of Onchocerca caused by these compounds was attenuated only partially by glutamine. These data demonstrate that, under certain conditions, filarial nematodes can utilize non-sugar substrates as energy sources. The differential sensitivity seen among these organisms to mitochondrial toxins and glutamine antimetabolites may be related to the extent to which they can use these alternative substrates to generate energy.

Onchocerciasis in Minnesota Cattle

Umbilical hides from 536 dairy cattle in Minnesota were tested for the microfilarial stage of Onchocerca species to determine the distribution of onchocerciasis in the state. The infection was widespread as microfilariae were obtained from 214 (40%) of the animals, representing nearly all areas of the state. Adult Onchocerca parasites were collected primarily from nodules associated with tibial bones but also were found to a lesser extent within the gastrosplenic ligament. Specific identity of these organisms is unclear as they exhibit certain morphological features previously described as being characteristic of either Onchocerca gutturosa, Onchocerca lienalis, or Onchocerca stilesi.

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

Catmull J., Dan Z. and Miller D. J. 1992. Nucleotide sequence of a cDNA from Onchocerca gibsoni encoding a novel repetitive antigen. International Journal for Parasitology 22: 685–687. 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.

Onchocerciasis in a California Dog

A 15-year-old dog that had lived all of its life on ranches and at the Wildlife Waystation on the western edge of the San Gabriel mountains near Los Angeles, California, developed an extensive granulomatous lesion involving the right eye and associated tissues requiring removal of both the eye and the lesion. Microscopic examinations of the tissues revealed the presence of living and dead gravid female worms and male worms belonging to the genus Onchocerca. Unsheathed microfilariae presumed to be Onchocerca species were found in the skin as well. Because Onchocerca species are not natural parasites of dogs, it is presumed that this infection was acquired accidentally from bovine, equine, or other animal host sharing the environment. This appears to be the first published record of patent onchocerciasis in a dog.

The murid filariaMonanema martini: a model for onchocerciasis

A study of the anatomo-pathological lesions induced by Monanema martini, a filaria with skin-dwelling microfilariae, was performed using 65 Lemniscomys striatus fixed from 30 minutes to 36 months after inoculation of the infective larvae, 5 Arvicanthis niloticus and 3 Meriones unguiculatus fixed during the patent phase, and controls. Attempts at quantification of lesions in L. striatus was made. Approximately 20% of L. striatus had microfilariae in the eyeballs, and many more presented ocular lesions. The delay of the patent period seems to have more effects on the gravity of lesions than repeated inoculations. The location of the lesions and parasites presuppose that microfilariae enter the eyeball through the lymphatic capillaries of the irido-corneal angles. Cutaneous lesions were often severe: there is a parallel between the importance of lesions and the abundance of microfilariae. Larvae are responsible for damage to various structures of the lymphatic system (thrombo-lymphangitis, acute or granulomatous lymphadenitis...) into which they migrate, explaining the mechanism of elephantiasis. These rodent lesions appear similar to those observed in human onchocerciasis and lymphatic filariasis. Whatever the M. martini stage and the organ examined, major lesions belonged to the inflammatory process. Various types of inflammatory reaction (acute, subacute, or chronic inflammation, scarring sclerosis etc.) can co-exist within a single tissue area. The accidental escape of a microfilaria from a lymphatic capillary into the connective tissue (including the corneal stroma) induces an inflammatory reaction. Thus M. martini, as human Onchocerca species, causes a chronic disease, associating recent lesions to old ones.

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—fOGl—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. fOGl can detect as little as 100–200 pg of O. gibsoni DNA. It is thus concluded that fOG 1 has the sensitivity to detect microfllariae of O. gibsoni found in the skin of cattle and the specificity to differentiate them from closely related species living in the same environment.