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PubMed1991

Onchocerca volvulus: in vitro killing of microfilaria by neutrophils and eosinophils from experimentally infected chimpanzees.

Peripheral blood leukocytes from chimpanzees experimentally infected with third stage larvae, or with nodular microfilaria (mf) of Onchocerca volvulus, were evaluated for their ability to kill nodular mf in vitro. Inoculation of chimpanzees with third stage larvae, and prospective follow up, permitted each animal to be classified as patent, infected but not patent, or post-patent. Neither lymphocytes nor monocytes significantly adhered to, immobilized, or killed mf. However, neutrophils or eosinophils killed mf effectively in the presence of immune serum. Neutrophil and eosinophil enriched cell preparations from control animals were able to kill mf with serum from infected animals, but to a significantly lesser degree than when both cells and serum were taken from infected animals. Chimpanzees inoculated with mf showed a progressive increase in cellular killing of mf after a second inoculation which surpassed that seen with animals inoculated with third stage larvae. Antibody-mediated killing of mf by neutrophils and eosinophils may be important in the termination of patency in this infection.

Animals↗
PubMed1980

Quantitative aspects of the infection of Simulium ochraceum by Onchocerca volvulus: the relation of skin microfilarial density to vector infection.

In Guatemala, 16 wild Simulium ochraceum were permitted to feed on a 15" x 15" grid on the back of each of 13 persons infected with Onchocerca volvulus. Based on 6 skin biopsies, the mean microfilarial density (mfd) in these study participants ranged from 1.256 mf/mg of skin to 111.824 mf/mg. The files were dissected 6 to 10 hours after feeding and the number of microfilariae in the blood meal and escaping the midgut into the thoracic musculature were counted. A nearly linear relationship was found between the mean skin mfd and the mean microfilarial uptake by S. ochraceum (rs = 0.868). An even stronger relation existed between the mean mfd and the mean number of microfilariae escaping the midgut (rs = 0.915). Because the approximate 1:1 relationship observed between the number of microfilariae escaping the midgut and the subsequent number of infective (L3) larvae has been previously demonstrated, it can be concluded that a similar linear relationship exists between microfilarial skin densities and the number of L3 larvae available for transmission of O. volvulus by the Guatemalan vector.

Animals↗
PubMed1984

Morphogenesis of larval Onchocerca volvulus in the Panamanian black fly, Simulium quadrivittatum.

Simulium quadrivittatum Loew (Diptera: Simuliidae), a man-biting black fly, was shown, for the first time, to be capable of supporting development of Onchocerca volvulus Leuckart (Nematoda: Filarioidea) from microfilariae to third-stage (infective) larvae. The black flies were collected in Chiriqui Province, Panama and transported alive to Guatemala, where they were allowed to feed on a human subject infected with O. volvulus. Samples of these flies were dissected over an 11-day period to assess morphogenesis of the parasite. Vigorously motile microfilariae were recovered from the mid-gut during the first 24 hours postfeeding; second-stage larvae were found in the thoracic musculature on day 4; and fully developed third-stage larvae were obtained from the cephalic capsule by day 10. This rate of larval development is similar to that observed in Guatemalan S. ochraceum. Onchocerciasis is not known to occur in Panama. The results of the present study direct attention to a potential public health hazard there and possibly elsewhere in Central America.

Animals↗
PubMed1991

Histochemical localization of gene expression in Onchocerca volvulus: in situ DNA histohybridization and immunocytochemistry.

We report here the development of in situ hybridization and immunohistochemistry protocols which permit the histological identification of gene expression of a cloned antigen of Onchocerca volvulus, OI5, in the parasite. Skin nodules containing female adult worms were fixed in a modified Carnoy's fixative and embedded in paraffin. Histological staining of tissue sections revealed uniformly excellent morphology and RNA preservation. To localize mRNA by in situ hybridization, tissue sections were incubated with biotin-labeled pOI5, the plasmid containing the genomic sequence of the antigen, and hybridization signals were histochemically visualized using a streptavidin-enzyme conjugate and chromogenic substrates. The protein antigen was localized immunohistochemically by incubating the sections with specific antibodies prepared against a recombinant fusion protein containing the OI5 sequence (OI3), and visualized via a secondary antibody-biotin-enzyme conjugate procedure. The results reported here showed distinct localization of the OI5 mRNA and OI3 antigen in specific cellular and tissue regions of the adult parasite, and in microfilariae located within the uteri and in the surrounding host tissue. The specificity and high sensitivity of these histological detection methods should be generally applicable for the characterization of gene expression in the filarial parasite, particularly the insect-borne, infective filarial larvae, which are severely limited in quantity.

Animals↗
PubMed1991

Experimental ocular onchocerciasis in cynomolgus monkeys. IV. Chorioretinitis elicited by Onchocerca volvulus microfilariae.

Onchocerciasis is a major cause of blindness worldwide, and much of the blindness is caused by onchocercal chorioretinitis. In an experimental animal model for ocular onchocerciasis, intravitreal injections of 10,000 live Onchocerca volvulus microfilariae isolated from infected humans into the eyes of cynomolgus monkeys (Macaca fascicularis) resulted in patchy, progressive loss of retinal pigment with pigment clumping. Areas of pigment loss were less extensive in animals that had been sensitized with microfilariae. Intravitreal injections of dead O. volvulus microfilariae resulted in mild vitritis with relatively less clinical change noted in the retina and choroid. Histopathologic examination revealed thinning and loss of outer retinal layers with pigment migration into the retina, and inflammation was more pronounced in eyes that received live microfilariae. Clinical changes appeared in eyes receiving live microfilariae before the development of significant antibody or cell-mediated immune responses. O. volvulus microfilariae appear to be more suitable than O. lienalis microfilariae in producing lesions which resemble human onchocerciasis in the primate model.

Animals↗
PubMed1991

An immunogenic Onchocerca volvulus antigen: a specific and early marker of infection.

Onchocerciasis (river blindness) is a serious health problem and a severe obstacle to social and economic development, especially in Africa. A complementary DNA fragment coding for an Onchocerca volvulus antigen (OV-16) was cloned and expressed in the plasmid vector pCG808fx. Immune responses to this O. volvulus-specific recombinant antigen were detectable in patients with documented onchocerciasis; the antibody response was also detectable at 3 months and at more than 1 year before infection could otherwise be detected in humans and in chimpanzees experimentally infected with O. volvulus third-stage larvae.

Animals↗
PubMed1975

Observations on filarial infections and parous rates of anthropophilic blackflies in Guatemala, with reference to the transmission of Onchocerca volvulus.

In Guatemala, in areas of different onchocerciasis endemicity and outside the endemic zone, the main anthropophilic blackfly species Simulium ochraceum, S. metallicum, S. callidum and S. gonzalezi were examined for infections with Onchocerca volvulus, other filariae, and non-filarial parasites. Third stage larvae, indistinguishable from those of O. volvulus were found only in 3 females of S. ochraceum out of 3,513 examined. In S. metallicum, 2 females out of 3,121 dissected harboured infective larvae of another filaria species, morphologically different from O. volvulus. Thoracic infections were encountered in all four Simulium species. Only in S. ochraceum was there a correlation between the infection rates in the human and in the fly populations. In the endemic zone, up to 15% of the parous flies were infected, against none in the non-endemic area. Parous S. metallicum showed low over-all infection rates of 0.6 to 2.6%, and had infections also outside the endemic zone, but these could not have been of human origin. Non-filarial infections, including fungi of the ovaries, ciliates, mermithids, nematodes, were commonly observed in Guatemalan blackflies. Parous rates were 40% in S. ochraceum, 23% in S. metallicum, 39% in S. callidum, and 49% in S. gonzalezi. The size of the follicular relics showed a daily cycle in S. ochraceum. They were usually large in flies biting man in the afternoon, but small in flies biting in the morning. This suggests that the flies found a new host the same day, or the morning after they had deposited their eggs.

Animals↗
PubMed1991

Experimental Onchocerca volvulus infections in mangabey monkeys (Cercocebus atys) compared to infections in humans and chimpanzees (Pan troglodytes).

Three chimpanzees, three mangabey monkeys (Cercocebus atys), and 14 patas monkeys (Erythrocebus patas) were inoculated with L3 Onchocerca volvulus of Guatemalan origin. One chimpanzee and two mangabey monkeys developed antibody activity to at least three different antigens. Both mangabey monkeys recognized a 20 kDa antigen 3.5-5 months post-inoculation, and the monkeys and the chimpanzee developed antibody activity to 14 and 22 kDa antigens 7.5-13 months post-inoculation. One mangabey monkey and the chimpanzee became microfilaria-positive in skin snips at 16 and 21 months post-inoculation, respectively. Antibody activity to the 20 kDa antigen in the mangabey monkeys is noteworthy because of the prominence of this antigen among putatively immune persons living in onchocerciasis-endemic areas. The two mangabey monkeys responded parasitologically in a manner comparable to immune humans. No microfilariae were detected in one monkey and only scant numbers of microfilariae were observed in the second. The mangabey monkey may be a good animal model for the study of onchocerciasis.

Animals↗
PubMed1982

Histochemical enzyme variation in Onchocerca volvulus microfilariae from rain-forest and Sudan-savanna areas of the Onchocerciasis Control Programme in West Africa.

Histochemical staining methods for acid phosphatase were used to study the differences among microfilariae of various West African strains of Onchocerca volvulus in both forest and Sudan-savanna onchocerciasis zones. The results have shown statistically significant differences in the staining patterns of microfilarial populations in the two zones. In the rain-forest areas, where onchocerciasis is transmitted by Simulium yahense, S. sanctipauli, S. soubrense and S. squamosum, there were no significant differences of microfilarial staining patterns in patients, by age and sex, between the three Simulium-Onchocerca complexes studied. There was a close relationship between the "strain differences", as revealed morphoenzymatically, and the clinical picture of the disease in both the forest and the Sudan-savanna zones. The present findings are in favour of the hypothesis that there are intrinsic differences in the strains of the parasite occurring in the two areas. The application of the histochemical means of parasite characterization appears to be a useful tool in differentiating strains of O. volvulus and could contribute towards a better understanding of the epidemiology of human onchocerciasis in different bioclimatic zones where the disease is endemic.

Adolescent↗
PubMed1990

Morphological alterations of male Onchocerca volvulus after in vitro exposure to mel w and milbemycin a confirming the results of viability tests.

The suitability of two viability parameters used for screening of antifilarial activities of new compounds was examined by parallel observation of the morphology. Male Onchocerca volvulus were exposed in vitro to 10 mumol mel w and milbemycin a and examined by light and transmission electron microscopy. The viability was assessed by measurement of the motility, using a micromotility meter and by determination of tetrazolium reduction. Already twelve hours after exposure to mel w the muscles of the body wall showed severe damage. After 36 hours the other tissues revealed degenerative changes and after 60 hours disintegration of all tissues was observed. Effects on the morphology caused by milbemycin a were seen earliest after 60 hours. Condensed cytoplasm in the hypodermal layer and beginning degeneration of spermatogenic stages indicated drug activity. The time-point of appearance of these drug induced morphological alterations was in accordance with the decrease of the motility indices and the degree of tetrazolium reduction. Morphological alterations indicating irreversible damage of worm tissues are a reliable parameter to detect macrofilaricidal activity. The good agreement between the results of the morphological examination and the assessment of the motility and the tetrazolium reduction confirms the suitability of the latter two assays for in vitro drug screening with O. volvulus.

Animals↗
PubMed1989

Electron microscopic studies on the effects of CGP 6140 and CGP 20376 on microfilariae and third stage larvae of Onchocerca volvulus.

Electron microscopic examination was used to assess the effects of CGP 6140 (proposed generic name: amocarcine) and CGP 20376 (proposed generic name: metobethiamide) on the fine structure of microfilariae and third stage larvae of Onchocerca volvulus after in vitro exposure to different concentrations of these compounds. The range of concentrations was selected according to the expected plasma levels that may be reached with these drugs. The microfilariae showed distinct effects on the muscle cells as myelin figures, vacuolisation and disintegration of the cytoplasm after both compounds. The third stage larvae showed the same effects on the muscle cells including changes in the mitochondria and the hypodermal tissue. In addition, the glandular esophagus demonstrated degenerative alterations of the cytoplasm and loosening of the cuticular lining after exposure to CGP 6140. CGP 20376 led to severe damage of the fibres of the nerve ring. No degenerative changes of the microtubuli were observed. Comparison of the morphological effects observed by electron microscopy with the reduction rates of motility and moulting recorded in the in vitro tests showed good correlation between these different parameters. The observed effect on the fine structure was the most sensitive parameter.

Animals↗
PubMed1991

The reproductive lifespan of Onchocerca volvulus in West African savanna.

The epidemiological model ONCHOSIM--a model and computer simulation program for the transmission and control of onchocerciasis--has been used to determine the range of plausible values for the reproductive lifespan of Onchocerca volvulus. Model predictions based on different lifespan quantifications were compared with the results of longitudinal skin-snip surveys undertaken in 4 reference villages during 13 to 14 years of successful vector control in the Onchocerciasis Control Programme in West Africa. Good fits between predicted and observed trends in skin microfilarial loads could be obtained for all villages. It is concluded that the reproductive lifespan of the savanna strain of O. volvulus lies between 9 and 11 years, and that 95% of the parasites reach the end of reproduction before the age of 13 to 14 years.

Africa, Western↗
PubMed1991

Characterization and molecular cloning of a Cu/Zn superoxide dismutase from the human parasite Onchocerca volvulus.

Evidence suggests that the helminth antioxidant enzyme superoxide dismutase (SOD) may play a role in parasite's defense against the cellular immune mechanisms of the host. In order to investigate this for the human parasite Onchocerca volvulus, the enzyme activity was characterized, the release of SOD by the parasite was examined, and a complete cDNA encoding the O. volvulus SOD was identified. The SOD activity in adult O. volvulus was found to be 8.1 +/- 4.2 U/mg of protein. A Cu/Zn-containing enzyme was demonstrated by its sensitivity towards cyanide, azide, and hydrogen peroxide. Isoelectric focusing, combined with an enzyme activity assay, revealed two activities at pI 6.8 and 7.6, with both activities inhibited by KCN. Adult parasites, maintained in vitro, released SOD into the culture medium, which was detected by enzyme activity. In parallel, lactate production was measured to ensure the viability of the parasite. Oligonucleotides (based upon conserved sequences in the SOD genes of other organisms) and the polymerase chain reaction were used to identify a portion of the SOD gene from O. volvulus genomic DNA. A cDNA library was constructed in lambda unizapII and screened with the genomic polymerase chain reaction fragment. A complete cDNA encoding the Cu/Zn SOD was identified, and its nucleotide sequence was determined. Southern blot hybridization experiments indicated that the Cu/Zn SOD is encoded by a single-copy gene with at least one intron.

Amino Acid Sequence↗
PubMed1979

[The epidemiological significance of neo-natal parasitism with microfilariae of Onchocerca volvulus (author's transl)].

Between April 1977 and June 1978, 214 babies born of 210 mothers infected with Onchocerciasis, were examined at the maternity of Po Hospital (Upper Volta). In four of them (1.9%) dermal microfilariae of Onchocerca volvulus were found during the first week after birth. In two other cases microfilariae were seen in the tissue of the umbilical cord. All specimens from placentas and amniotic fluid gave negative results. The infected babies were reexamined monthly monthly from 2 to 18 months, depending on the cases. One child remained positive until the 6th month follow-up. The base-line examination of 5.757 children less than 2 years old from different villages in the WHO Onchocerciasis Control Programme in the Volta River Basin Area showed that 1% of children less than one year old and 2% of children of 12 to 23 months old were carriers of dermal microfilariae. These results indicate that in meso- or hyperendemic areas children less than one year old and carriers of microfilariae of Onchocerca volbulus may have been infected in utero. The existence of such cases, in an area of vector control, does not prove a residual vector borne transmission.

Burkina Faso↗
PubMed1979

The development of Onchocerca volvulus in two temperate blackfly species, Simulium ornatum Meigen and Simulium lineatum Meigen.

In view of the promise shown by temperate simuliids for laboratory colonization, the ability of Simulium ornatum and S. lineatum to support the development of Onchocerca volvulus was investigated. An injection technique is described for the introduction of O. volvulus microfilariae from a chimpanzee into female simuliids in the absence of a bloodmeal. RPMI 1640 medium, with penicillin, streptomycin and foetal calf serum, proved satisfactory as an inoculation medium. Third stage larvae were obtained in both S. ornatum and S. lineatum after seven days at 27 degrees C. The rate of development of the parasite in S. ornatum is recorded.

Animals↗
PubMed1991

Onchocerca volvulus, O. gutturosa, Brugia malayi, and Dirofilaria immitis: a comparative study of the immunochemical properties of cuticular proteins from filarial parasites.

We compared the chemical and immunological properties of cuticular collagens from four species of filarial nematodes, Onchocerca volvulus, O. gutturosa, Brugia malayi, and Dirofilaria immitis. The electrophoretic mobility of the major polypeptides extracted from adult worms is characteristic for each species studied. Cuticular collagens from adult worms and infective larvae differ in their susceptibility to proteases that cleave vertebrate collagens and to collagenases prepared from different developmental stages of filarial parasites. The overall amino acid composition of filarial collagens resembles that of vertebrate interstitial collagens and differs from that reported for collagens from free-living or intestinal nematodes. However, cuticular proteins of the four filarial species studied significantly differed in amino acid composition and in their reactivity with antisera to interstitial and basement membrane collagens of vertebrates.

Amino Acids↗