Potential of Onchocerca ochengi inosine-5'-monophosphate dehydrogenase (IMPDH) and guanosine-5'-monophosphate oxidoreductase (GMPR) as druggable and vaccine candidates: immunoinformatics screening.

Onchocerciasis is a vector-borne disease caused by the filarial nematode Onchocerca volvulus, which is responsible for most of the visual impairments recorded in Africa, Asia and the Americas. It is known that O. volvulus has similar molecular and biological characteristics as Onchocerca ochengi in cattle. This study was designed to screen for immunogenic epitopes and binding pockets of O. ochengi IMPDH and GMPR ligands using immunoinformatic approaches. In this study, a total of 23 B cell epitopes for IMPDH and 7 B cell epitopes for GMPR were predicted using ABCpred tool, Bepipred 2.0 and Kolaskar and Tongaonkar methods. The CD4+ Th computational results showed 16 antigenic epitopes from IMPDH with strong binding affinity for DRB1_0301, DRB3_0101, DRB1_0103 and DRB1_1501 MHC II alleles while 8 antigenic epitopes from GMPR were predicted to bind DRB1_0101 and DRB1_0401 MHC II alleles, respectively. For the CD8+ CTLs analysis, 8 antigenic epitopes from IMPDH showed strong binding affinity to human leukocyte antigen HLA-A*26:01, HLA-A*03:01, HLA-A*24:02 and HLA-A*01:01 MHC I alleles while 2 antigenic epitopes from GMPR showed strong binding affinity to HLA-A*01:01 allele, respectively. The immunogenic B cell and T cell epitopes were further evaluated for antigenicity, non-alllergernicity, toxicity, IFN-gamma, IL4 and IL10. The docking score revealed favorable binding free energy with IMP and MYD scoring the highest binding affinity at -6.6 kcal/mol with IMPDH and -8.3 kcal/mol with GMPR. This study provides valuable insight on IMPDH and GMPR as potential drug targets and for the development of multiple epitope vaccine candidates.Communicated by Ramaswamy H. Sarma.

Lipid profiling of the filarial nematodes Onchocerca volvulus, Onchocerca ochengi and Litomosoides sigmodontis reveals the accumulation of nematode-specific ether phospholipids in the host.

Onchocerciasis, a neglected tropical disease prevalent in western and central Africa, is a major health problem and has been targeted for elimination. The causative agent for this disease is the human parasite Onchocerca volvulus. Onchocerca ochengi and Litomosoides sigmodontis, infectious agents of cattle and rodents, respectively, serve as model organisms to study filarial nematode infections. Biomarkers to determine infection without the use of painful skin biopsies and microscopic identification of larval worms are needed and their discovery is facilitated by an improved knowledge of parasite-specific metabolites. In addition to proteins and nucleic acids, lipids may be suitable candidates for filarial biomarkers that are currently underexplored. To fill this gap, we present the phospholipid profile of the filarial nematodes O. ochengi, O. volvulus and L. sigmodontis. Direct infusion quadrupole time-of-flight (Q-TOF) mass spectrometry was employed to analyze the composition of phospholipids and their molecular species in the three nematode species. Analysis of the phospholipid profiles of plasma or serum of uninfected and infected hosts showed that nematode-specific phospholipids were below detection limits. However, several phospholipids, in particular ether lipids of phosphatidylethanolamine (PE), were abundant in O. ochengi worms and in bovine nodule fluid, suggesting that these phospholipids might be released from O. ochengi into the host, and could serve as potential biomarkers.

Stage-specific Proteomes from Onchocerca ochengi, Sister Species of the Human River Blindness Parasite, Uncover Adaptations to a Nodular Lifestyle.

Despite 40 years of control efforts, onchocerciasis (river blindness) remains one of the most important neglected tropical diseases, with 17 million people affected. The etiological agent, Onchocerca volvulus, is a filarial nematode with a complex lifecycle involving several distinct stages in the definitive host and blackfly vector. The challenges of obtaining sufficient material have prevented high-throughput studies and the development of novel strategies for disease control and diagnosis. Here, we utilize the closest relative of O. volvulus, the bovine parasite Onchocerca ochengi, to compare stage-specific proteomes and host-parasite interactions within the secretome. We identified a total of 4260 unique O. ochengi proteins from adult males and females, infective larvae, intrauterine microfilariae, and fluid from intradermal nodules. In addition, 135 proteins were detected from the obligate Wolbachia symbiont. Observed protein families that were enriched in all whole body extracts relative to the complete search database included immunoglobulin-domain proteins, whereas redox and detoxification enzymes and proteins involved in intracellular transport displayed stage-specific overrepresentation. Unexpectedly, the larval stages exhibited enrichment for several mitochondrial-related protein families, including members of peptidase family M16 and proteins which mediate mitochondrial fission and fusion. Quantification of proteins across the lifecycle using the Hi-3 approach supported these qualitative analyses. In nodule fluid, we identified 94 O. ochengi secreted proteins, including homologs of transforming growth factor-ß and a second member of a novel 6-ShK toxin domain family, which was originally described from a model filarial nematode (Litomosoides sigmodontis). Strikingly, the 498 bovine proteins identified in nodule fluid were strongly dominated by antimicrobial proteins, especially cathelicidins. This first high-throughput analysis of an Onchocerca spp. proteome across the lifecycle highlights its profound complexity and emphasizes the extremely close relationship between O. ochengi and O. volvulus The insights presented here provide new candidates for vaccine development, drug targeting and diagnostic biomarkers.

A histochemical study of the Nras/let-60 activity in filarial nematodes.

BACKGROUND: Control and elimination of filarial pathogens is a central focus of major global health efforts directed at parasitic diseases of developing countries. Accomplishment of these goals would be markedly enhanced by the enhanced destruction of the adult stage of filariae. The identification of new, more quantitative biomarkers that correlate with mortality or chemotherapeutic damage to adult filariae, would greatly facilitate, for example, the development of new macrofilaricides. METHODS: An immunocytochemical approach using an antibody against human Nras was used to identify and detect changes in the nematode homolog let-60 that is associated with cell growth and maintenance. Single Onchocerca volvulus nodules were removed from each of 13 patients treated with ivermectin (as part of a community-wide mass drug administration programme), and from each of 13 untreated individuals; these 26 nodules were stained with the anti-Nras antibody. The localization and degree of positivity of Nras/let-60 staining were assessed subjectively and compared between the two groups; the positivity of staining was also quantified, using image analysis, in a subgroup of these nodules. In addition, the specific morphological association between Nras/let-60 and the Wolbachia endosymbiont present in these parasites was also observed in 4 additional filarial species using an anti-Wolbachia surface protein (WSP) antibody under light and confocal microscopy. RESULTS: Nras/let-60 is present in many structures within the adult female worms. A statistically significant decrease in the general staining intensity of Nras/let-60 was observed in adult female O. volvulus treated with ivermectin when compared with parasites from untreated patients. Nras/let-60 staining was frequently observed to be co-localized with WSP in O.volvulus, Brugia malayi, Litomosoides sigmodontis and Dirofilaria immitis. Nras/let60 is also present in Onchocerca ochengi. CONCLUSION: Nras/let-60, as detected by immunocytochemical staining, is decreased in ivermectin-treated adult female O. volvulus relative to untreated control specimens, suggesting a suppressive effect of ivermectin on the overall biochemical activity of these parasites. Co-localization of Nras/let-60 and WSP suggests the possibility that the endosymbiont utilizes this nematode protein as part of a mutualistic relationship. Nras/let60 appears to be a useful biomarker for assessing the health of filariae.

Extracellular Onchocerca-derived small RNAs in host nodules and blood.

BACKGROUND: microRNAs (miRNAs), a class of short, non-coding RNA can be found in a highly stable, cell-free form in mammalian body fluids. Specific miRNAs are secreted by parasitic nematodes in exosomes and have been detected in the serum of murine and dog hosts infected with the filarial nematodes Litomosoides sigmodontis and Dirofilaria immitis, respectively. Here we identify extracellular, parasite-derived small RNAs associated with Onchocerca species infecting cattle and humans. METHODS: Small RNA libraries were prepared from total RNA extracted from the nodule fluid of cattle infected with Onchocerca ochengi as well as serum and plasma from humans infected with Onchocerca volvulus in Cameroon and Ghana. Parasite-derived miRNAs were identified based on the criteria that sequences unambiguously map to hairpin structures in Onchocerca genomes, do not align to the human genome and are not present in European control serum. RESULTS: A total of 62 mature miRNAs from 52 distinct pre-miRNA candidates were identified in nodule fluid from cattle infected with O. ochengi of which 59 are identical in the genome of the human parasite O. volvulus. Six of the extracellular miRNAs were also identified in sequencing analyses of serum and plasma from humans infected with O. volvulus. Based on sequencing analysis the abundance levels of the parasite miRNAs in serum or plasma range from 5 to 127 reads/per million total host miRNA reads identified, comparable to our previous analyses of Schistosoma mansoni and L. sigmodontis miRNAs in serum. All six of the O. volvulus miRNAs identified have orthologs in other filarial nematodes and four were identified in the serum of mice infected with L. sigmodontis. CONCLUSIONS: We have identified parasite-derived miRNAs associated with onchocerciasis in cattle and humans. Our results confirm the conserved nature of RNA secretion by diverse nematodes. Additional species-specific small RNAs from O. volvulus may be present in serum based on the novel miRNA sequences identified in the nodule fluid. In our analyses comparison to European control serum illuminates the scope for false-positives, warranting caution in criteria that should be applied to identification of biomarkers of infection.

Loa loa and Onchocerca ochengi miRNAs detected in host circulation.

A combination of deep-sequencing and bioinformatics analysis enabled identification of twenty-two microRNA candidates of potential nematode origin in plasma from Loa loa-infected baboons and a further ten from the plasma of an Onchocerca ochengi-infected cow. The obtained data were compared to results from previous work on miRNA candidates from Dirofilaria immitis and O. volvulus found in host circulating blood, to examine the species specificity of the released miRNA. None of the miRNA candidates was found to be present in all four host-parasite scenarios and most of them were specific to only one of them. Eight candidate miRNAs were found to be identical in the full sequence in at least two different infections, while nine candidate miRNAs were found to be similar but not identical in at least four filarial species.

Identification of in vivo released products of Onchocerca with diagnostic potential, and characterization of a dominant member, the OV1CF intermediate filament.

A sensitive and specific test for the routine diagnosis of active Onchocerca infection is currently lacking. A major drawback in the development of such a test has been the paucity of knowledge of suitable parasite antigens that can serve as targets in antigen-detection assays. In the present investigation, we employed mass spectrometry, bioinformatics and molecular techniques to identify and characterize several potentially diagnostic Onchocerca antigens in the in vivo nodular fluid, which is being investigated for the first time. The majority of the 27 identified antigens lacked a secretory signal. One of them, also identified and characterized in greater detail with the aid of previously developed monoclonal antibodies (Mabs), was a dominant circulating cytoplasmic intermediate filament protein, previously identified and named, OV1CF. Although OV1CF lacks a secretory signal in its amino acid sequence and is not detected in the pure 42 h in vitro released products, it is easily detected in the in vivo nodular fluid. We conclude that these in vivo released products offer promise as diagnostics markers in onchocerciasis.

Classical ligands interact with native and recombinant tubulin from Onchocerca volvulus with similar rank order of magnitude.

The alpha- and beta-tubulin genes from Onchocerca volvulus were individually expressed for the first time in Escherichia coli (DH5alpha). The recombinant tubulins were purified, renatured and reconstituted into oligomers, probably dimers, which were competent to bind three classical tubulin ligands: mebendazole (MBZ), taxol (TAX) and vinblastine (VBN). A new charcoal-dependent binding assay allowed accurate discrimination between specific and non-specific ligand binding in crude cell extracts. To compare the magnitude of binding of both native and recombinant forms of tubulin, we developed an ELISA assay for estimating the amount of tubulin in soluble protein extracts of O. volvulus. Binding assays were performed; both the maximum binding at saturating ligand concentrations (B(max)) and the equilibrium dissociation constants (K(d)) were determined. The B(max) values of the different ligands were significantly different from one another (P<0.05), but the order of the B(max) and K(d) for each drug were VBN > TAX > MBZ for both native and recombinant tubulin. Indeed, B(max) values for MBZ with native and recombinant tubulins were similar. On average, native tubulin had higher or similar binding capacity (B(max)) but a consistently higher affinity (lower K(d)) than the recombinant tubulin. We conclude that at least some of the recombinant molecules form receptors that are similar to those in native tubulin dimers. These data suggest that recombinant tubulin can be used to develop a molecular screen for novel anti-tubulin ligands to develop into drugs against onchocerciasis.

Effects of an Onchocerca-derived cysteine protease inhibitor on microfilariae in their simuliid vector.

A recombinant cysteine protease inhibitor, onchocystatin of the parasitic nematode Onchocerca volvulus, was tested for its role in microfilarial development in the simuliid vector. Onchocystatin was found to be present in female adults and skin microfilariae of the bovine parasite O. ochengi, the closest relative of O. volvulus. In addition the inhibitor could be detected as an excretory-secretory (E-S) product of the microfilariae. Co-injection of onchocystatin and the O. ochengi microfilariae into the surrogate vector Simulium ornatum s.l. significantly enhanced the recovery rates of the parasite within 24 h into the infection (P > 0.001). The findings suggest a possible role of onchocystatin in the evasion by the parasite of the immune response of its vector.

In-vitro uptake of ivermectin by adult male Onchocerca ochengi.

Ivermectin is not lethal to the adult worms of Onchocerca volvulus or to those of O. ochengi, a cattle parasite closely related to O. volvulus. Although ivermectin penetrates the nodules in which the adults of these nematodes live, it is not known what levels of the drug enter the worms. Adult male O. ochengi were incubated in [3H]ivermectin in a saturated solution of unlabelled ivermectin (11.44 microM), to measure uptake by the oral and transcuticular routes, and in [3H]inulin, to ascertain if oral ingestion occurs in vitro. Uptake of [3H]ivermectin was high [1040 disintegrations/min (d.p.m.) at 3 h, representing a mean total of 86 pmoles ivermectin/worm] and occurred predominantly by the transcuticular route. Viability of worms was not reduced by this exposure, and uptake continued for up to 12 h. Only low levels of [3H]inulin (four d.p.m.) were detected in worms, indicating that the gut is probably not functional in vitro. Scanning and transmission electron microscopy revealed that the epicuticle of both sexes had an irregular surface which was pitted with a honeycomb structure in males, and rough and abundantly folded in females. These structures greatly increased the absorptive surface of the worms. In conclusion, ivermectin is able to enter adult O. ochengi males at concentrations sufficient to kill non-filarial nematodes.

Onchocerca retinol- and ivermectin-binding protein activity.

The presence of retinol-binding protein (RBP) activity in Onchocerca cervicalis adult worms and interaction with ivermectin has been studied using high pressure size exclusion chromatography (HPSEC). Four distinct peaks of [3H]-retinol incorporation were obtained corresponding to approximate molecular weights of 150, 67, 19.7 and 4.6 kDa, the 2 smaller M(r) peaks accounting for most of the binding activity. Competition for binding using non-labelled retinol at 200-fold molar excess indicated that specific binding of retinol occurred only to the 19.7 kDa fraction. Competition by ivermectin also inhibited binding of [3H]-retinol to the third peak. Following incubation with [3H]-ivermectin 4 peaks of similar molecular weights were also detected by HPSEC in soluble adult worm homogenate. However, in this case the 150 kDa fraction was most prominent. Both non-labelled ivermectin and non-labelled retinol at 200-fold molar excess reduced binding of [3H]-ivermectin to all 4 fractions. These data indicate that the putative Onchocerca RBP has an approximate molecular weight of 19.7 kDa, that retinol also binds to 3 additional fractions non-specifically, that the pattern of binding of ivermectin to adult worm material is quantitatively and qualitatively different from the binding exhibited by retinol, and that ivermectin interferes with the binding of retinol to the 19.7 kDa Onchocerca protein.

Characterization of a novel non-muscle myosin-related protein from Onchocerca gibsoni.

A cDNA library was constructed in lambda gt11 using poly(A)+ mRNA from early larvae of Onchocerca gibsoni. Screening of the library using serum from a single onchocerciasis patient yielded several strongly immunoreactive clones, one of which (OGK2) was found to encode a novel myosin-related protein. cDNA clone OGK2 contained an insert of 2017 bp, consisting of continuous open reading frame in frame with the vector, hence this clone encodes 671 amino acid residues of a larger protein. A fragment (619 nt) of the OGK2 cDNA was subcloned into the expression vector pGEX-1N to generate a glutathione S-transferase fusion protein. Polyclonal antiserum raised to this fusion protein strongly recognised an O. gibsoni protein of approximately 220 kDa. Immunolocalization studies indicated that this protein was associated predominantly with the hypodermis and a number of other specific membrane layers in the adult parasite. Myosin-related proteins are frequently immunodominant parasite antigens and in a number of studies have been shown to confer a degree of protective immunity against the corresponding parasite. Evaluation of the protective potential of the OGK2 protein, therefore, appears to be warranted.

Onchocerca spp: a "family" of secreted acidic proteins expressed by infective larvae in blackflies.

Biosynthetic labeling of developing larvae of Onchocerca in blackflies has been used to characterize a group of stage-specific, secreted proteins produced by vector-stage parasites. These are highly acidic molecules (pI 4.4-5.1) present in at least three members of the genus (O. volvulus, O. lienalis, O. ochengi) that exhibit minor heterogeneity among species in apparent molecular mass (between 18 and 23 kDa). In O. volvulus, there are two polypeptides that run as a doublet of 18 and 20 kDa. In O. lienalis and O. ochengi, single polypeptides of 23 and 20 kDa were detected. The processes of synthesis and secretion appear to be temperature-sensitive and dissociated events. Experiments with O. volvulus in Simulium damnosum sl revealed that synthesis is initiated in second stage larvae and increases in infective-stage parasites: Secretion occurs when larvae leave the vector and enter the phase of development associated with the vertebrate host. Third-stage larvae of O. lienalis were shown to continue to express and accumulate the 23-kDa protein with age. The primary organ of secretion, as indicated by dissection, was the glandular esophagus. These data point to an important biological role for this group of molecules and suggest that they may belong to a family of related products. Because they have the distinctive characteristics of being secreted larval acidic proteins, we propose the acronym SLAP pending further insights into their functional properties.

The in vitro culture of Onchocerca spp.: II. The excretory/secretory products of the first and third stage larvae of Onchocerca lienalis and O. ochengi.

The developing first and third larval stages of two bovine Onchocerca species were maintained in vitro in the presence of 35S-methionine, following their initial development in the vector species Simulium ornatum s.1. to characterise the expression and secretion of their metabolites. The first larval stages of O. lienalis and O. ochengi did not release any E/S products. In contrast the supernatants of the third stage larvae of both species contained a double band of 21 and 20 kDa for O. ochengi and 23 and 22 kDa bands for O. lienalis when kept at room temperature. A temperature shift to 37 degrees C led to the increased expression of the 23 kDa protein with the infective larvae of O. lienalis. The possible role of these molecules is discussed.

Differential lectin binding of Onchocerca lienalis and Onchocerca ochengi infective larvae following their development in Simulium ornatum s.l.

Lectins have been used to investigate species specific differences in carbohydrate moieties on the surface of the infective larvae of two Onchocerca species following their development in Simulium ornatum. Of the seven FITC-labelled lectins used in this study only two, Arachis hypogea (PNA) and Helix pomatia (HPA), bound to the surface of O. lienalis, whereas no lectin binding could be detected on the surface of O. ochengi infective larvae. This indicates that in principal lectins might be a potential tool for the differentiation of more closely related Onchocerca species.

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.

Uptake of chloroquine by Onchocerca volvulus in vivo and in vitro.

Patients infected with Onchocerca volvulus in the Cayapa River focus in north-east Ecuador were given 500 mg chloroquine diphosphate (CQ) orally prior to nodulectomy. The concentrations of CQ were determined in parasite fragments and host tissue dissected from the nodules, in skin overlying the nodules, and in plasma at 3, 4, 7, and 24 hours after dosing. Onchocerca volvulus took up CQ rapidly, in some cases accumulating the drug to concentrations of over 600 pmol mg-1 worm tissue by three hours, and maintaining similar concentrations through 24 hours. These amounts were markedly higher than peak concentrations in plasma (3.16 pmol microliters-1) and in host tissues (78 pmol mgm-1) and skin (up to 93 pmol mg-1). In vitro uptake of CQ by females of O. volvulus was greater under alkaline conditions (pH 8.4) than at pH 6.8 and 7.4. Uptake reached equilibrium after one to two hours, with final concentrations being approximately 10 times lower than those reached in vivo. Inhibitory effects of chloroquine and its major metabolite desethylchloroquine on the motility of O. volvulus and other filariae have been observed previously in vitro; whether or not the drug had adverse effects on adult parasites in vivo was not determined in these experiments. However, the results illustrate the accessibility of O. volvulus to blood borne agents in vivo, and the potential importance of pharmacodynamic characteristics in the search for new macrofilaricidal agents.

Hallucinogenic and neuroleptic drug interactions with potential neurotransmitter receptors in parasitic nematodes.

Receptors potentially involved in neurotransmitting have been characterised in the muscle tissue and in whole worms of the nematodes Ascaris suum and Onchocerca volvulus, respectively. Binding studies revealed a high affinity for LSD with apparent KD values of 94 nM for A. suum and 120 nM for O. volvulus, whereas those of the neuroleptics haloperidol, spiperone and mianserin were found to be in the micromolar range. A variety of neurotransmitter antagonists, known to bind with high affinities either to mammalian D1/2 or to 5-HT1/2 receptors, were tested for their ability to bind to the nematode receptor. Results from these displacement experiments using tritiated LSD, mianserin, spiperone and haloperidol show distinct specificities of the nematode receptors compared to known receptor classes of mammals. With respect to this novel specificity, the nematode receptors seem to be unique and clearly distinct from those of the hosts.

Comparison of the cuticular structure of parasitic nematodes recognized by immunocytochemical and lectin binding studies.

The cuticle structure of some nematode species was studied by immunogold and lectin-gold techniques. The gold labelling made it possible to distinguish the cuticle layers by the distribution and/or the density of the marker. On the other hand, no labelling pattern was found which led to a clear grouping of the layers into larger 'zones', since there were no subunits consisting of more than one layer which reacted in a characteristic way as compared to the rest of the cuticle. The outer surface of the epicuticle of parasitic adult worms turned out to be highly inert; it did not react with any of the antibodies or lectins tested. The cuticle of parasitic nematodes seems to function as a protection against the host's defense mechanisms rather than as an interaction site. An immunogenic component on the surface was only found in infective larvae. All antibodies and lectins showed a preferential binding to the electron dense layers and fibrous structures (HPL/GalNAc, WGA/GlcNAc) or to the amorphous ground-substance (Con A/Glc, RCA I/Gal).

The use of cell-conditioned medium for the in vitro culture of Onchocerca spp. larvae (Nematoda: Filarioidea).

The effects of co-culture with monkey kidney cells (LLCMK2), cell-conditioned medium and decreased atmospheric oxygen on the in vitro molting and viability of infective stage larvae (L3) of Onchocerca lienalis and O. volvulus were examined. O. lienalis L3 were cultured in an RPMI 1640-based medium in the presence of an LLCMK2 cell monolayer or in medium which had been conditioned for three days by cells. In paired experiments cell conditioned medium alone in 95% air/5% CO2 produced molting levels of 54 +/- 14% which increased to 67 +/- 20% in treatments cultured under decreased oxygen; this value equalled the level of molting of worms cocultured with LLCMK2 cells. Worm survival in the three environments was similar. In seven additional experiments using O. lienalis (n = 186), overall levels of 74 +/- 12 percent molting and 75 +/- 7% viability on days 21-33 were obtained. Worms increased in length from 503 +/- 50 mu as L3 to 638 +/- 74 mu as L4 on day 21 (p = 0.0001, n = 42-44). Ultrastructural comparison of an in vitro derived L4, (39 days in culture) vs a vector-derived L3 revealed fewer annulations and decreased osmiophilia on the epicuticle of the L4 while the hypodermis showed increased morphogenetic definition. O. volvulus molted at an average rate of 74% (n = 40) with a mean viability on day 28 of 95%. L3 increased in length from a mean of 635 +/- 50 mu to 775 +/- 45 mu as L4 on day 28 (p = 0.0001). Larvae of both species were cultured under these conditions for periods of time exceeding 100 days.