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.     

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

Onchocerca volvulus: application of the polymerase chain reaction to identification and strain differentiation of the parasite

Previous studies have demonstrated that the genome of Onchocerca volvulus contains a variable tandemly repeated DNA sequence family with a unit length of 150 bp. The variability of the 150-bp family has been exploited to develop O. volvulus strain and species specific DNA probes. Application of these DNA probes to the study of the epidemiologically most significant life cycle stages of the parasite has been confounded by several obstacles. These include the relative insensitivity of some of the DNA probes and the difficulty in releasing genomic DNA from infective larvae and skin microfilariae in a form that may be directly detected by hybridization to the probes. DNA sequence comparison of 18 known examples of the 150-bp repeat has been used to develop two populations of degenerate oligonucleotides. These oligonucleotides have been shown to support the amplification of the 150-bp repeat family from Onchocerca DNA, using the polymerase chain reaction. Both strain and species specific members of the repeat family are faithfully amplified, allowing characterization of a parasite on the basis of hybridization of the PCR amplification products to the previously developed DNA probes. This method is shown to be applicable to all diagnostically important forms of the parasite, including adults, infective larvae, and skin microfilariae. In addition, the method is capable of detecting O. volvulus infective larvae directly in extracts of blackfly vectors.     

Infective larvae of five Onchocerca species from experimentally infected Simulium species in an area of zoonotic onchocerciasis in Japan

Microfilariae of five Onchocerca species, O. dewittei japonica (the causative agent of zoonotic onchocerciasis in Oita, Kyushu, Japan) from wild boar (Sus scrofa), O. skrjabini and O. eberhardi from sika deer (Cenus nippon), O. tienalis from cattle, and an as yet unnamed Onchocerca sp. from wild boar, were injected intrathoracically into newly-emerged black flies of several species from Oita to search the potential vector(s) of these parasites and identify their infective larvae. Development of O. dewittei japonica microfilariae to the infective larvae occurred in Simulium aokii, S. arakowae, S. bidentatum, S. japonicum, S. quinquestriatum, and S. rufibasis while development of infective larvae of O. skrjabini, O. eberhardi, and the unnamed Onchocerca sp. was observed in S. aokii, S. arakawae, and S. bidentatum. Development of O. lienalis microfilaria to infective larvae occurred in S. arakawae. Based on the morphology of infective larvae obtained, we proposed a key of identification of Onchocerca infective larvae found in Oita. We also reconsider the identification of three types of infective larvae previously recovered from Simulium species captured at cattle sheds: the large type I larvae that may be an undescribed species; the small type III identified as O. lienalis may include O. skrjabini too; the intermediary type II that may be O. gutturosa, or O. dewittei japonica, or the unnamed Onchocerca sp. of wild boar.     

Fate of developing larvae of Onchocerca lienalis and O. volvulus in micropore chambers implanted into laboratory hosts

Infective larvae of Onchocerca lienalis and O. volvulus implanted subcutaneously within micropore chambers into laboratory hosts moulted to the fourth stage (L4) and underwent limited development and growth. Similar recoveries of O. lienalis L4 larvae in the range of 33-66% were obtained from chambers implanted into CBA and BALB/c strains of mice, jirds, and the natural bovine host. A relatively constant proportion of larvae survived up to 24 days post implantation and thereafter recoveries declined, although some worms were still alive after 96 days. Recoveries of O. volvulus L4 larvae from chambers given to normal or T-cell deprived mice were equivalent to one another and to those obtained with O. lienalis. Moulting of O. lienalis in chambers was observed on days 3 and 5, in close accordance with the timing of the third moult in cattle following systemic infection. Moulting of O. volvulus occurred between days 3-6. Morphological changes in developing larvae included a small but significant increase in length, a transient increase in width, and early development of the spicular primordia and genital tube. L4 larvae of O. lienalis, but not those of O. volvulus, exhibited 3 distinct caudal papillae not present on infective larvae.     

Microfilarial surface carbohydrates as a function of developmental stage and ensheathment status in six species of filariids

The lectin-binding properties of microfilariae of Onchocerca volvulus, O. lienalis, Brugia pahangi, Wuchereria bancrofti, Dirofilaria immitis, and Monanema (= Ackertia) marmotae share a number of characteristics. Carbohydrates specific for lectins are associated with the egg shell or sheath. N-acetyl-D-glucosamine is the predominant carbohydrate associated with the ensheathed forms with lesser quantities of D-galactose and/or alpha-lactose and D-galactosamine. The density of these carbohydrates on the sheath surface diminishes as the larvae undergo normal growth and development. Similar carbohydrates are not found on the cuticle as exsheathed microfilariae show virtually no ability to bind lectins.     

Seasonal changes in density and tissue distribution of Onchocerca cervicalis microfilariae in ponies and related changes in Culicoides variipennis populations in Louisiana

Seasonal changes in density and spatial distribution of Onchocerca cervicalis microfilariae were studied in ventral-midline skin of 15 infected pony mares in southern Louisiana. Triple running mean analysis of data over a 13-mo period indicated that a distinct pattern exists in total microfilariae population density and in microfilariae occurrence in different levels of the dermis. Microfilariae density reaches peak levels in the spring followed by a 58% decrease in the summer, a 19% increase in the fall, and a decrease to the lowest numbers in the winter. Microfilariae were found in all levels of the skin during the spring, summer, and fall but were not found in the superficial layers of the dermis during the winter months. The population density of Culicoides variipennis, a demonstrated vector of O. cervicalis, appeared to have seasonal fluctuations similar to the changes in microfilarial density. Harmonic wave analysis of microfilariae density data in individual ponies showed that all individuals did not follow the population trend.     

Dirofilaria immitis: proteases produced by third- and fourth-stage larvae.

A model of cutaneous extracellular matrix was used to determine if live Dirofilaria immitis larvae secrete proteases which are active at physiological pH and capable of degrading macromolecules found in cutaneous tissue. After 72 hr, 100 third-stage larvae (L3) degraded 24% of the total matrix, while fourth-stage larvae (L4) degraded 10%. A sharp increase in the amount of matrix degraded by L3 corresponded with the onset of the molting process. L3 and L4 degraded comparable amounts of the glycoprotein and elastin components of the matrix, but molting L3 degraded nearly twice the amount of the collagen component (62% vs 35%). Characterization of proteases present in larval-soluble extracts and excretory-secretory products using synthetic substrates and protease inhibitors demonstrated cysteine-protease and metalloprotease activity. Cysteine protease activity was found in whole worm extracts of both L3 and L4. Metalloprotease was secreted at higher levels by molting L3, but was also secreted by L4. Partial separation of the metalloprotease by size-exclusion chromatography indicated that the molecular weight of the native enzyme was in the 49-54 kDa range. The cysteine protease activity was demonstrated in fractions corresponding to 34-39 kDa. The biological function of the D. immitis larval proteases remains to be conclusively determined; however, these data suggest that they are involved in degradation of components of cutaneous tissue and in the molting process.     

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.     

Identification and characterization of onchoastacin, an astacin-like metalloproteinase from the filaria Onchocerca volvulus

The tissue-invasive nematode Onchocerca volvulus causes skin and eye pathology in human onchocerciasis. While the adult females reside sessile in subcutaneous nodules, the microfilariae are abundantly released from the nodules, males and juvenile worms migrate through the host tissue. Matrix-degrading metallo- and serine proteinases have been detected in excretory-secretory worm products that may be essential for migration of the mobile stages. In this study, a 1713bp long cDNA encoding for a putative proteinase of O. volvulus has been isolated. The predicted protein sequence includes a signal peptide indicating secretion to the extracellular space, a propeptide, an astacin-like protease domain, an EGF-like and a CUB-domain, thereby identifying the protein as a member of the astacin family of zinc endopeptidases. Onchoastacin, Ov-AST-1, is most closely related to a subfamily comprising nematode astacins including Caenorhabditis and Ancylostoma. Ov-AST-1 was expressed as a recombinant protein in baculovirus-infected insect cells and exhibited enzymatic activity. The exposure of onchoastacin to the host immune system is indicated by demonstration of IgG reacting with the recombinant Ov-AST-1 and with two peptides of the protein. Since a homologous metalloproteinase is part of a promising hookworm vaccine, Ov-AST-1 may be a candidate for intervention strategies in filarial infections.     

Surface carbohydrate changes on Onchocerca lienalis larvae as they develop from microfilariae to the infective third-stage in Simulium ornatum

Use was made of seven FITC labelled lectins as tools to investigate the surface of Onchocerca lienalis larvae as they develop through to the infective third-stage in a natural vector, Simulium ornatum. The lectins were derived from Canavalia ensiformis (Con A), Lens culinaris (lentil), Triticum vulgaris (wheat germ), Arachis hypogaea (peanut), Helix pomatia, Phaseolus vulgaris (kidney bean) and Tetragonolobus purpureus (asparagus pea). Between 70 and 100 living parasites were examined for each developmental stage; i.e. skin microfilariae, late first-stages, second-stages, preinfective third-stages and infective third-stages isolated from the mouth parts of the flies. None of the lectins used bound to the surface of the microfilariae. However, progressive binding to the cuticle of the first- and second-stages was observed using Con. A, lentil lectin and wheat germ agglutinin (WGA). Following moulting to the third-stage, binding of these three lectins declined. Furthermore, as these lectins decreased, peanut and Helix pomatia lectins progressively increased in their binding, despite the fact that they showed little or no binding to the first- and second-stages; stages at which Con A, lentil and WGA were at their maximum. Asparagus pea and kidney bean lectins failed completely to bind to any of the larvae examined. Carbohydrate inhibition tests showed that the lectin was indeed binding specifically to glycoconjugates on the parasite surface. WGA binding was not inhibited by prior incubation with N-acetyl-D-glucosamine, even at high concentrations, but neuraminic acid did completely inhibit its binding. Judging from the patterns of binding on the nematodes themselves, the carbohydrates may not be vector in origin, but derive from the worms. The lectin specificities indicate that initially mannose/glucose type derivatives are present on the surface. Following moulting to the third-stage these are progressively replaced, or overlaid with galactosamine type derivatives, also present on the infective third-stage as it enters the bovine host. The availability of these surface glycoconjugates to attack mediated by natural insect lectins may be of importance in the parasite regulatory mechanisms of the blackfly. Variability in these surface carbohydrates, and in the response to them could well be a contributing factor in the cytospecific variation in S. damnosum susceptibility to geographical variants of O. volvulus.     

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.     

In vitro haemagglutination and attenuation of microfilarial motility by haemolymph from individual blackflies (Simulium ornatum) infected with Onchocerca lienalis

Two in vitro tests were used to investigate the effect of Onchocerca lienalis Stiles infection on the haemolymph of Simulium ornatum Meigen. The first of these examined the effect of infected haemolymph on the motility of fresh O. lienalis or Brugia pahangi Buckley & Edeson microfilariae. Incubation of haemolymph from individual flies with fresh microfilariae was performed in the wells of Terasaki micro-tissue culture plates. Motility of both species of parasite was found to be significantly attenuated when compared to worms incubated in control haemolymph groups. The second assay was that of agglutination of cat erythrocytes in the presence of haemolymph from individual flies, also performed in Terasaki plates. This test demonstrated significant increases in the rates of haemagglutination in the haemolymph of O. lienalis infected blackflies. The titre appeared to increase during the initial 5 days of infection up to a level of 1/32+, but then fell between day 5 and 7 to a maximum level of 1/2. The proportion of flies exhibiting haemagglutination also rose following infection. Despite the apparent absence of melanization and encapsulation, simuliids may have at least two humoral haemolymph components available to them for parasite regulation; a fast-acting factor responsible for rapid parasite death, and more specific agglutinins, possibly lectins. The role of the latter in defence is as yet unclear.     

Vitellogenin sequestration by Simulium oocytes: the effect of Onchocerca infection

Abstract. The effects of Onchocerca infection on Simulium ornatum Meigen vitellogenesis were investigated. Vitellogenesis is triggered by ingestion of blood and is therefore linked to disease transmission. Vitellin subunits were described for the first time in the Simuliidae. In the British blackfiy S.ornatum , vitellin in the ovaries consists of two subunits, with a large subunit of molecular weight 200 kDa and a small subunit of 68 kDa. Simulium ornatum vitellin was recognized by polyclonal antibodies produced from Aedes aegypti (L.) vitellin. When blackflies were injected with Onchocerca lienalis (Stiles) microfilariae, there was a significant reduction in the amount of vitellin in the ovaries at 24, 34 and 50 h post blood-feeding.     

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)     

Expression and secretion of a larval-specific chitinase (family 18 glycosyl hydrolase) by the infective stages of the parasitic nematode, Onchocerca volvulus

A recently reported chitinase gene, expressed in the infective, third-stage (L3) larvae of the human filarial parasite Onchocerca volvulus, belongs to the family 18 glycosyl hydrolases and has been designated Ov-chi-1. The gene product of Ov-chi-1 is chitinolytic. Allosamidin ablates activity of the native enzyme in a dose-dependent manner but did not significantly inhibit the moulting of L3 larvae. Mono-specific antibodies were used to characterize Ov-CHI-1 as a 60-kDa protein expressed almost exclusively in L3 stages. Immunoelectron microscopy showed that Ov-CHI-1 expression is initiated in late L2 larvae and increases markedly in infective, L3 larvae. It is synthesized exclusively in the glandular esophagus and stored within discrete secretory granules. Secretion occurs through de-granulation during post-infective development, and the primary route of transport appears to be via the pseudo-coelom. An orthologue of Ov-chi-1 was detected in Caenorhabditis elegans by BLAST analysis. It is constitutively expressed at a low level and is overexpressed in dauer larvae and embryonated eggs. It is chitinolytic. We conclude that Ov-CHI-1 is a highly stage-specific enzyme that may have a role in infectivity of the parasite, aiding escape from the vector or participating in early post-infective migration and/or development. The identification of an orthologue in C. elegans opens the way for further studies into the biological function(s) of this intriguing parasite product.     

Strongyloides stercoralis: identification of a protease that facilitates penetration of skin by the infective larvae

Host invasion and tissue migration of several helminths have been linked to the expression and release of parasite-derived proteases. One of the most remarkable examples of tissue migration is that of larvae of the nematode parasite Strongyloides stercoralis, which can move through tissue at speeds of up to 10 cm per hour. We have shown the Strongyloides L3 larvae secrete a potent histolytic metalloprotease to facilitate their rapid migration. This protease has elastase activity and catalyzes the degradation of a model of dermal extracellular matrix. The importance of this enzyme in the pathogenesis of strongyloidiasis is underscored by the observation that invasion by larvae of skin in vitro is prevented by metalloprotease inhibitors. These results substantiate the role of proteases as virulence factors in strongyloidiasis, as well as other related parasitic infections, and suggest new approaches to therapy.     

Onchocerca eberhardi n. sp. (Nematoda: Filarioidea) from sika deer in Japan; relationships between species parasitic in cervids and bovids in the Holarctic region

Onchocerca eberhardi n. sp. from the sika deer, Cervus nippon, in Japan is described. Adult worms lived in the carpal ligament; infection reached high levels (up to 25 female and 16 male worms in a single carpal limb). Skin dwelling microfilariae were mainly found in the ears. Prevalence of infection was 81% at the type locality, Mt. Sobo, in Kyushu. The new material was compared to the 31 species of Onchocerca presently known. Onchocerca eberhardi n. sp. females were characterized by a long slender anterior end and a thin esophagus < or =1 mm long with no or only a slight glandular region. The vulva was located near the level of the mid-esophagus and the cuticle had transverse external ridges and internal striae (two striae between adjoining ridges). The most similar species were O. stilesi (re-examined), O. lienalis, and to a lesser extent O. gutturosa, all from bovids (cattle). Two main lineages of Onchocerca are recognized in cervids with either primitive or with derived characteristics (as exemplified by the new species). The species in both lineages are not restricted to cervids but are also found in bovids in the Holarctic region, suggesting that the species diversified in the two host groups simultaneously, when these host groups lived in the some geographic area.