Survival of Onchocerca volvulus in nodules implanted in immunodeficient rodents.

Onchocerca volvulus is an obligate human parasite, and its study has been difficult due to an inability to maintain it outside the human host. We report the successful transplantation of onchocercomata containing living adult O. volvulus worms into immunodeficient C.B.-17.scid/scid (scid) mice or athymic rnu/rnu (nude) rats. Living, motile worms containing viable microfilariae were present in onchocercomata recovered from scid mice or nude rats for up to 20 wk, establishing a novel animal model for future investigation of O. volvulus.     

Development of Dipetalonema viteae third-stage larvae (Nematoda: Filarioidea) in micropore chambers implanted into jirds, hamsters, normal and immunized mice

Development of third-stage larvae of Dipetalonema viteae within subcutaneously implanted micropore chambers proceeded in all hosts tested up to the fourth-stage larvae and occasionally to adolescent worms. In the jird the timing of development was comparable to a natural infection. Although the mouse is an insusceptible host, larval development could take place, but was very slow. Two intraperitoneal inoculations of living third-stage larvae into mice induced the production of antibodies against the larval cuticle and against common antigens. In such immune mice the development of third- and fourth-stage larvae within micropore chambers was significantly inhibited, larval mortality was increased, and the larval motility was impaired.     

Identification of surrogate rodent hosts for larval Onchocerca lienalis and induction of protective immunity in a model system.

The objectives of this project were to screen a variety of inbred rodent species and strains to determine their usefulness as surrogate hosts for the study of the early larval development of Onchocerca lienalis and then to use a selected model to study the induction of protective immunity. In the primary screen, 6 strains of mice, 5 strains of rats, jirds, and multimammate rats were tested. Animals were infected with fresh O. lienalis by subcutaneous implantation of third-stage larvae (L3) contained in diffusion chambers covered with 5.0-microns pore-size membranes. After 7 days the chambers were recovered, and larval viability and growth were assessed. Approximately one-half of inoculated larvae were recovered alive regardless of the host tested. Larvae were implanted in CBA/J and DBA/2J mice in chambers covered with membranes that prevented host cells from entering; survival and growth rates of the larvae were not altered by the absence of cells from the chambers. Cryopreserved larvae were implanted in chambers with 5.0-microns pore-size membranes in CBA/J and DBA/2J mice and Wistar Furth rats for 3-28 days. No statistically significant difference was seen in the larval recoveries on days 3-28 in all 3 hosts. Statistically significant increases in length were seen in the 3 strains from day 3 to day 14, after which growth appeared to cease. Molting from L3 to fourth-stage larvae was observed in all 3 hosts beginning on day 3, with most larvae completing the molt by day 7.(ABSTRACT TRUNCATED AT 250 WORDS)     

Onchocerciasis in British cattle: a study of Onchocerca gutturosa and O. lienalis in North Wales

Onchocerca gutturosa and O. lienalis infections in British cattle were studied by examination of cattle post-mortem originating from North Wales and Cheshire (north west England). In 463 adult animals, the microfilarial (mf) prevalence was 28.5%. In 95.3% of the mf infected animals, gravid worms could not be found at either the ligamentum nuchae or the gastro-splenic omentum. Dermal mf at the head were identified as O. gutturosa on the basis of their highly significant association with the presence of gravid O. gutturosa at the ligamentum nuchae, which were found in only 3.2% of cattle. Mfs were isolated from different skin sites and from adult worms and a minimum of 10 mfs from each isolate were examined for width and acid phosphatase (AP) staining pattern. The width of O. gutturosa dermal mf was less than 4 micron (4 isolations), narrower than that of putative O. lienalis mf isolated from umbilical skin of cattle without evidence of O. gutturosa, which were in 20/22 isolations greater than 4 micron wide. The dermal mf were also distinguished on the basis of different AP staining patterns which, for each species, correlated closely with that of hatched intrauterine mf from their respective adult female worms. Based on the criteria of morphology and AP staining patterns the mf species prevalences in the survey population were estimated as O. lienalis 24.1% and O. gutturosa 2.2%, with a further 2.2% of cattle infected with both species. The results indicate that the predilection site of adult O. lienalis is not the gastro-splenic omentum. In North Wales, the distribution of the two species was different; O. lienalis was widely distributed in all cattle rearing areas both lowland and upland, whereas O. gutturosa was largely restricted to valleys close to major rivers.     

Onchocerca volvulus: immunization of chimpanzees with X-irradiated third-stage (L3) larvae.

To provide a theoretical basis for the potential development of vaccines against Onchocerca volvulus (Ov) a trial has been conducted to assess the protective efficacy of immunization of chimpanzees with X-irradiated L3 larvae. Approximately 1000 larvae were injected at 0, 1, and 7 months. The immunized animals, and unimmunized controls, were then challenged with 250 live L3. In order to provide possibly protective exposure to the immunologically distinct L4 epicuticle, a radiation dose (45 krad) was chosen which preserved about 50% of the molting ability of unirradiated larvae. Despite the presence of a strong immune response to crude adult worm extracts, and to cloned Ov antigens, at the time of challenge little or no significant protection against patent infection was observed: three of four immunized animals developed patent infection as compared to four of four controls. One immunized animal failed to become patent or to manifest the late antibody response to adult worm antigens seen in both subpatent and patent infections in this model, and may have been protected from infection. The implications of these studies for future attempts to immunize against O. volvulus are discussed.     

Onchocerca volvulus: biochemical and morphological characteristics of the surface of third- and fourth-stage larvae

The annulated cuticles of third- and fourth-stage larvae of Onchocerca volvulus have the typical structure of other nematodes but the cuticle of fourth-stage larvae was thinner. The surface of the third-stage larva was wrinkled and fuzzy, while that of the fourth-stage was smooth. Intermediate stages in the formation of the new cuticle and epicuticle beneath the old basal layer and of the separation of the cuticles are shown. Monoclonal antibodies specific to the surface of third-stage larvae did not react with the surface of the fourth-stage larvae. Binding of the monoclonal antibodies to the third-stage larvae was abrogated by treatment of the worms with trypsin and proteinase K, but was unaffected by treatment with periodate or the detergents sodium deoxycholate and SDS. The lectins RCA120 and WGA, but not any of the other lectins tested, bound only to the surface of fourth-stage larvae, and not to that of third-stage larvae. The surfaces of third- and fourth-stage larvae were shown to be different and contained stage-specific surface epitopes.     

Immunity to Onchocerca lienalis microfilariae in mice. II. Effects of sensitization with a range of heterologous species

The model of Onchocerca lienalis microfilariae (mff) injected into CBA and T.O. strains of mice has been used to examine immunity to skin-dwelling microfilariae following exposure to a range of species of helminths. Mice which had received a primary infection with O. cervicalis mff were significantly resistant to challenge with O. lienalis mff (58% reduction relative to challenge controls). Immunization with the uterine contents (eggs and mff) of O. lienalis, O. gutturosa or O. volvulus conferred equivalent levels of protection against challenge with O. lienalis mff (66 to 75%). Similar results were obtained with immunizations in mice that employed either fresh or freeze-killed eggs of O. gutturosa. Significant reductions in the recoveries of O. lienalis mff were also demonstrated following the intraperitoneal implantation of adult male worms of O. gutturosa (30 to 52%), the adults of either sex of Dipetalonema viteae (60%), or after infection with Trichinella spiralis (27 to 81%). Infections with the trematode, Schistosoma mansoni, had a negligible effect on mff recoveries. It is concluded that partial resistance in mice to Onchocerca mff may be stimulated by factors, yet to be determined, that are neither stage nor species-specific.     

Chromosome numbers in Onchocerca dukei and O. tarsicola

Testicular examination of male Onchocerca dukei and German O. tarsicola revealed five pairs of chromosomes, which is typical of most filariae. In contrast to German O. tarsicola, specimens from Sweden had only three pairs of chromosomes.     

CD4+-dependent immunity to Onchocerca volvulus third-stage larvae in humans and the mouse vaccination model: common ground and distinctions

Onchocerciasis is a major filarial disease and is the second most common cause of infectious blindness in the world. Disease development after infection with Onchocerca volvulus varies widely and is determined by the host's immune response to the parasite. Vector control and administration of ivermectin has reduced infection and disease rates significantly. However, limitations of these programmes, including ivermectin's selective activity on microfilariae, the need for 10-15 years of annual treatments, logistical obstacles and the potential emergence of drug-resistant strains demand alternative strategies. A vaccine that targets O. volvulus infective third-stage larvae (L3) could provide an additional tool to guarantee successful elimination of infection with O. volvulus. An essential step in the development of immunoprophylactic procedures and reagents is the identification of host immune responses toward antigens of O. volvulus L3 and L3 developing to the fourth-stage larvae that are associated with protection against these stages of the parasite. This review summarises the recent advancements in understanding the immune mechanisms in particular the CD4(+) responses to L3 stages in humans and in the mouse vaccination model. Comparison between the two uncovered common immunological elements in naturally exposed humans and mice vaccinated with radiation attenuated L3 or recombinant O. volvulus antigens, as well as significant differences. These studies promisingly suggest that the O. volvulus mouse model is a very useful adjunct to the studying of natural infection in humans and could provide us with the tools to identify the target molecules and the effector immune correlates of protection in humans responsible for attrition of L3 stages. Since some of these antigens may exist in other nematodes, any insight gained into the mechanisms of vaccine-induced anti-O. volvulus L3 protective immunity in both humans and mice could be applicable to the development of vaccines against other nematode infections.     

Effect of diethylcarbamazine on the concentration of Onchocerca volvulus microfilariae in hydrocoele fluid and urine.

The effect of graded doses of diethylcarbamazine on the concentration of microfilariae in the hydrocoele fluid, urine and skin of patients suffering from onchocerciasis was studied. The results showed that a significant number of microfilariae migrated into hydrocoele fluid and urine immediately after the drug was given and thereafter returned to pre-treatment levels rather more quickly in the former than the latter. The increase was found to be roughly directly proportional to the intensity of infection in the skin. By contrast the skin concentration of microfilariae fell sharply after treatment and remained significantly lower than pre-treatment levels for over four weeks. The potential use to which these observations could be put with respect to clinical screening of drugs in onchocerciasis is discussed.     

Fate of H2-activated T lymphocytes in syngeneic hosts. III. Differentiation into long-lived recirculating memory cells.

Memory to H2 determinants was studied with an adoptive transfer system using a population of H2-activated blast T cells (T.TDL) obtained from thoracic duct lymph of irradiated F₁ hybrid mice injected with parental strain T cells. CBA T.TDL activated either to DBA/2 or C57BL determinants were transferred to syngeneic “B” mice. Thoracic duct lymphocytes (TDL) were obtained from the recipients 4–6 weeks later and tested for their capacity to produce (a) a graft-versus-host (GVH) reaction, (b) a mixed lymphocyte reaction (MLR) (measured by an in vivo technique) and (c) allograft rejection (suppression of the growth of allogeneic tumour cells in vivo). Control experiments involved testing the function of TDL obtained from “B” mice preinjected with TDL or no cells.TDL from “B” mice injected with TDL (passaged TDL) gave strong MLR and GVH reactions to both DBA/2 and C57BL determinants. Passaged T.TDL activated to C57BL antigens gave intermediate MLR and GVH reactions to the specific (C57BL) determinants but only very low responses to third-party (DBA/2) determinants; reciprocal results were obtained with passaged T.TDL activated to DBA/2 determinants. TDL from “B” mice given no cells failed to respond to either set of determinants.Since the responses by the passaged T.TDL did not exceed those by passaged TDL there was no evidence that adoptive transfer of T.TDL had conferred to the recipients a state of memory to either MLR or GVH determinants. Adoptive transfer did, however, lead to qualitative changes in the properties of T.TDL since, before transfer, they were unable to evoke GVH reactions or produce an MLR of normal kinetics.Passaged T.TDL were far superior to passaged TDL at suppressing the growth of allogeneic tumour cells. The protection was specific since protection against DBA/2 tumour cells was, cell for cell, 5–10 fold more effective with passaged T.TDL activated to DBA/2 determinants than with cells activated to C57BL determinants. No protection was observed with cells treated with anti-θ serum. The protective cells appeared to be precursors of effector cells rather than effector cells per se since they failed to lyse the tumour cells in vitro. These data suggest therefore that the descendants of T.TDL which survived after transfer to “B” mice were highly enriched in long-lived recirculating T lymphocytes reactive to determinants expressed by specific tumour allografts.     

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

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

Molecular cloning and characterization of onchocystatin, a cysteine proteinase inhibitor of Onchocerca volvulus.

A cDNA clone designated OV7 encodes a polypeptide that corresponds to a highly antigenic Onchocerca volvulus protein. OV7 has significant amino acid sequence homology to the cystatin superfamily of cysteine proteinase inhibitors. In this report we establish that the OV7 recombinant protein is active as a cysteine proteinase inhibitor, and we have named it onchocystatin. It contains a cystatin-like domain that inhibits the activity of cysteine proteinases at physiological concentrations. Recombinant glutathione S-transferase-OV7 (GST-OV7, 1 microM) and maltose-binding protein-OV7 (MBP-OV7, 4 microM) fusion polypeptides inhibit 50% of the enzymatic activity of the bovine cysteine proteinase cathepsin B. Neither fusion polypeptide inhibits serine or metalloproteinases activity. The Ki for GST-OV7 fusion polypeptide is 170 nM for cathepsin B and 70 pM or 25 nM for cysteine proteinases purified from a protozoan parasite Entamoeba histolytica or the free living nematode Caenorhabditis elegans, respectively. The 5' end of the OV7 clone was isolated by polymerase chain reaction and sequenced, thus extending the previous cDNA clone to 736 base pairs. This represents the complete coding sequence of the mature onchocystatin (130 amino acids). A hydrophobic leader sequence of 32 amino acids was found, indicating a possible extracellular function of the onchocerca cysteine proteinase inhibitor.     

Incorporation of radioactive precursors into filarial larvae of Brugia developing in susceptible and refractory mosquitoes.

The incorporation of tritiated precursors injected into mosquito hosts parasitized by developing filarial larvae of Brugia patei has been studied by autoradiography in 2 species of mosquito, Aedes togoi in which filarial development was normal and Anopheles labranchiae atroparvus in which filarial development was abnormal. In both mosquito hosts there was significant incorporation into 4--5-day-old developing larvae of uridine and amino acids (isoleucine, leucine, valine, arginine, lysine, cystine, methionine, phenylalanine, tyrosine, tryptophan, histidine, and proline), although lower incorporation of methionine, tyrosine, and tryptophan was found during abnormal development. No incorporation of thymidine, hydroxytryptophan, dopa, or carbohydrate was found at this stage of larval development. Some incorporation of glucose and dopa was found in or around earlier stages of development in An. l. atroparvus. Mosquito flight muscle showed lower incorporation of glucose, but not of amino acids, around the site of filarial parasite development. The flight muscle of An. l. atroparvus showed a higher level of incorporation of lysine compared to that in A. togoi and higher levels of lysine and valine were found in the abnormally developing filarial larvae in the refractory mosquito.     

Molecular systematics of five Onchocerca species (Nematoda: Filarioidea) including the human parasite, O. volvulus, suggest sympatric speciation

The genus Onchocerca (Nematoda: Filarioidea) consists of parasites of ungulate mammals with the exception of O. volvulus, which is a human parasite. The relationship between O. volvulus, O. ochengi and O. gibsoni remains unresolved. Based on morphology of the microfilariae and infective larvae, vector transmission and geographical distribution, O. ochengi and O. volvulus have been placed as sister species. Nevertheless, the cuticle morphology and chromosomal data (O. volvulus and O. gibsoni have n=4 while O. ochengi is n=5) suggest that O. gibsoni could be more closely related to O. volvulus than O. ochengi. Sequences from the 12S rRNA, 16S rRNA and ND5 mitochondrial genes have been used to reconstruct the phylogeny of five Onchocerca species including O. volvulus. Analyses with maximum likelihood and maximum parsimony showed that O. ochengi is the sister species of O. volvulus, in accordance with the classification based on morphology and geographical location. The separate specific status of the species O. gutturosa and O. lienalis was supported, although their phylogenetic relationship was not well resolved. The analyses indicated that the basal species was O. gibsoni, a South-East Asian and Australasian species, but this result was not statistically significant. The possible involvement of sympatric speciation in the evolution of this group of parasites is discussed.     

Onchocerca volvulus: molecular cloning, primary structure, and expression of a microfilarial surface-associated antigen.

Although the filarial nematode parasite Onchocerca volvulus is an important human pathogen in large areas of Africa and Latin America, little is known of the molecular interactions that govern the clinical status of patients with this chronic, debilitating disease. As a step toward defining the parasite molecules important to the immunobiology of host-parasite interactions, we have identified and cloned a major surface-associated antigen expressed by O. volvulus microfilariae. Radiolabeling experiments demonstrated that O. volvulus microfilariae have a limited repertoire of peptides at the surface. Prominent among these labeled peptides is an 18-kDa component. Immunological cross-reactivity between a surface-associated component of Dirofilaria immitis microfilariae and the 18-kDa surface-associated molecule from O. volvulus was exploited in a strategy to clone this potentially important O. volvulus microfilarial antigen. The cross-reacting antibodies were used to immunoscreen O. volvulus cDNA expression libraries. One clone, M2f.e, contained an open reading frame of 495 bp encoding an 18.1-kDa protein (OVMS18). Antibodies produced against the expression product of M2f.e recognized an 18-kDa component in extracts of O. volvulus microfilariae and bound to the surface of intact O. volvulus and D. immitis microfilariae. Southern blot analyses showed that M2f.e-like sequences are present in the genomic DNA of a number of filarial nematode species, but not in DNAs from nonfilarial nematode species.