M. W. Kennedy

The Ov20 Protein of the Parasitic Nematode Onchocerca volvulus A STRUCTURALLY NOVEL CLASS OF SMALL HELIX-RICH RETINOL-BINDING PROTEINS

Ov20 is a major antigen of the parasitic nematodeOnchocerca volvulus, the causative agent of river blindness in humans, and the protein is secreted into the tissue occupied by the parasite. DNA encoding Ov20 was isolated, and the protein was expressed in Escherichia coli. Fluorescence-based ligand binding assays show that the protein contains a high affinity binding site for retinol, fluorescent fatty acids (11-((5-dimethylaminonaphthalene-1-sulfonyl)amino)undecanoic acid, dansyl-dl-α-aminocaprylic acid, and parinaric acid) and, by competition, oleic and arachidonic acids, but not cholesterol. The fluorescence emission of dansylated fatty acids is significantly blue-shifted upon binding in comparison to similarly sized β-sheet-rich mammalian retinol- and fatty acid-binding proteins. Secondary structure prediction algorithms indicate that a α-helix predominates in Ov20, possibly in a coiled coil motif, with no evidence of β structures, and this was confirmed by circular dichroism. The protein is highly stable in solution, requiring temperatures in excess of 90u2009°C or high denaturant concentrations for unfolding. Ov20 therefore represents a novel class of small retinol-binding protein, which appears to be confined to nematodes. The retinol binding activity of Ov20 could possibly contribute to the eye defects associated with onchocerciasis and, because there is no counterpart in mammals, represents a strategic target for chemotherapy.

Genetic variation in sympatric Ascaris populations from humans and pigs in China

It has recently been shown using genetic markers that Ascaris in humans and pigs in Central America comprise reproductively isolated populations. We present a similar analysis for a region of China in which close association between pigs and humans has been the norm for thousands of years, and agricultural practices will result in frequent exposure to eggs from both sources. DNA fragments from selected regions of mitochondrial and ribosomal DNA were amplified by PCR and allelic forms identified following digestion with a panel of restriction enzymes, using DNA from a total of 115 individual worms from both people and pigs from 2 neighbouring villages. Significant frequency differences in both mtDNA haplotypes and the rDNA spacer were found between the 2 host-associated populations, indicating that they represented reproductively isolated populations. Mitochondrial haplotype frequencies were different from those observed in Guatemala and also from other Asian Ascaris populations, suggesting low levels of gene flow between populations. However, we found no evidence for significant heterogeneity in the genetic composition of Ascaris infrapopulations in either humans or pigs, possibly indicative of agricultural practices in China which have resulted in a random distribution of alleles within the parasite populations.

The ABA-1 allergen of Ascaris lumbricoides: sequence polymorphism, stage and tissue-specific expression, lipid binding function, and protein biophysical properties.

The ABA-1 protein of Ascaris lumbricoides (of humans) and Ascaris suum (of pigs) is abundant in the pseudocoelomic fluid of the parasites and also appears to be released by the tissue-parasitic larvae and the adult stages. The genes encoding the polyprotein precursor of ABA-1 (aba-1) were found to be arranged similarly in the two taxa, comprising tandemly repeating units encoding a large polyprotein which is cleaved to yield polypeptides of approximately 15 kDa which fall into 2 distinct classes, types A and B. The polyprotein possibly comprises only 10 units. The aba-1 gene of A. lumbricoides is polymorphic, and the majority of substitutions observed occur in or near predicted loop regions in the encoded proteins. mRNA for ABA-1 is present in infective larvae within the egg, and in all parasitic stages, but was not detectable in unembryonated eggs. ABA-1 mRNA was confined to the gut of adult parasites, and not in body wall or reproductive tissues. Recombinant protein representing a single A-type unit for the A. lumbricoides aba-1 gene was produced and found to bind retinol (Vitamin A) and a range of fatty acids, including the pharmacologically active lipids lysophosphatidic acid, lysoplatelet activating factor, and there was also evidence of binding to leukotrienes. It failed to bind to any of the anthelmintics screened. Differential Scanning Calorimetry showed that the recombinant protein was highly stable, and unfolded in a single transition at 90.4 degrees C. Analysis of the transition indicated that the protein occurs as a dimer and that the dimer dissociates simultaneously with the unfolding of the monomer units.

Rapid changes in the surface of parasitic nematodes during transition from pre- to post-parasitic forms

All mammalian-parasitic stages of a range of nematode species investigated (Brugia pahangi, Acanthocheilonema viteae, Strongyloides ratti, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi) labelled in a surface-restricted manner with the fluorescent lipid analogues 5-N-(octadecanoyl)aminofluorescein (AF18) or nitrobenzoxadiazole-cholesterol (NBD-chol), but failed to bind other similar probes. In contrast, the surfaces of the pre-parasitic infective stages of these species had affinity for neither AF18 nor NBD-chol. This exclusion of lipid analogues changed rapidly upon exposure of the larvae to tissue culture conditions which mimic the mammalian tissue environment (e.g. RPMI 1640/37 degrees C) such that the above probes could then insert into the surface layer of the larvae. The dauer larva of Caenorhabditis elegans also excluded the probes, but became permissive to labelling upon stimulation to emerge from the dauer state. The time taken for the surface transformation to occur ranged from less than 10 min in the vector-borne parasites to approximately 5 h in those which enter by the oral route, with direct skin-penetrators occupying an intermediate position. In all cases, the alteration proceeded too rapidly for it to have been associated with a moult. Fluorescence Recovery After Photobleaching (FRAP) studies of A. viteae larvae showed that approximately 50% of the AF18 probe was free to diffuse within the plane of the surface immediately after transformation. This is only a transitory state because AF18 was found to be highly restricted in its lateral diffusion on the surface of adult parasites. In the larvae of S. ratti, the change in affinity for AF18 was accompanied by the rapid shedding of an otherwise stable surface coat of polyanionic material, here visualized by labelling with fluorescein-conjugated cationized ferritin. Incubation of larvae in lipid-rich host serum during the induction of transformation inhibited subsequent labelling with AF18. This possibly reflects competition for insertion sites and an in vivo propensity towards the acquisition of host lipid by invading parasites.

Proteinases in the excretory/secretory products (ES) of adult Trichinella spiralis

Adult Trichinella spiralis were maintained in vitro using defined media and the material excreted/secreted (ES) during this time examined for proteolytic enzyme (proteinase) activity using an azocasein assay and gelatin-substrate gels. Several discrete proteinases in the size range 14-100 kDa were observed with optimal activity at pH 7.5. The use of a class-differentiating panel of proteinase inhibitors indicated that serine proteinases were predominant although some inhibition was evident in the presence of cysteine and metalloproteinase inhibitors. Of a panel of potential natural protein substrates tested, ES proteinases only degraded fibrinogen and plasminogen and degradation was, in part, susceptible to the action of serine, cysteine and aspartyl proteinase inhibitors. In addition, antibody harvested from immune but not normal mice inhibited ES proteinase activity, an observation of relevance to the immunobiology of Trichinosis.

The secreted and somatic proteinases of the bovine lungworm Dictyocaulus viviparus and their inhibition by antibody from infected and vaccinated animals

Proteinase activities were examined in extracts and excretory-secretory (ES) products of the infective and adult stages of the cattle lungworm, Dictyocaulus viviparus. Multiple enzyme activities were identified from each source, as defined by pH optima, substrate specificities, inhibitor effects and substrate gel electrophoresis. Serine-, cysteine- and metalloproteinases were identified, secreted materials being more active against protein substrates per unit protein than were extracts, and the particular proteinases produced varied with the developmental stage of the parasite. The antigenicity of these parasite proteinases was demonstrated by the inhibition of enzymic activity with Protein G-purified serum IgG antibody from both infected and vaccinated hosts and in the retardation of enzyme migration on electrophoresis of enzyme-antibody complexes. For the adult products, this confirmed that the enzymes concerned were of parasite origin, and not host-derived. These results argue for investigation of D. viviparus proteinases as targets for the antibody response in the limitation of parasite-mediated tissue damage and as the active principle behind the anti-D. viviparus vaccine.

Immunoepidemiology of Ascaris lumbricoides: relationships between antibody specificities, exposure and infection in a human community.

The serum antibody responses of 124 people naturally exposed to Ascaris lumbricoides infection were analysed by immunoprecipitation of radio-isotope labelled 3rd- and 4th-stage larval Ascaris suum excretory and secretory antigens (L3/4 ES). Profiles of antigens recognized were visualized by polyacrylamide gel electrophoresis (SDS-PAGE), and the band intensities of the 12 major precipitated antigens were individually scored. Most subjects were seropositive, but considerable variation was observed in the amount of total and individual ES antigens precipitated. The sex- and age-related profiles of antibody levels followed similar patterns to those of egg output. In addition, total antibody scores of individuals were closely correlated (r = 0.47-0.52) with their eggs per gram of faeces (e.p.g.) collected 4 months after blood samples were taken. These findings suggest that antibody levels against larval ES antigens reflect recent exposure and are consistent with the hypothesis that establishment of adult worms is proportional to the number of larvae that recently migrated through the lung.

Immunologically mediated, non-specific interactions between the intestinal phases of Trichinella spiralis and Nippostrongylus brasiliensis in the mouse

Interactions between infections of Trichinella spiralis and Nippostrongylus brasiliensis were studied in the NIH strain of mouse which is known to react strongly to T. spiralis. The course of N. brasiliensis infection in this strain of mouse is described and expulsion is shown to be accelerated in immunized mice and inhibited in cortisone-treated mice. There was no evidence of inter-specific competition between the two species of worm in concurrent infections; the number and location of adults of both species were normal and T. spiralis was able to grow and reproduce normally. No evidence was found of direct immunological cross-reaction between N. brasiliensis and T. spiralis as assessed by the kinetics of adult worm numbers on heterologous challenge of immunized mice 90 days after the initiation of the last of 3 immunizing infections. Interaction was observed only when the timing of concurrent infections was such that one species was established in the intestine immediately before the beginning of expulsion of the second species. Interaction was manifested as a premature loss of worms and, in addition, as impairment of growth and fecundity of T. spiralis. These effects on T. spiralis were similar to those observed as a consequence of a specific immune response to T. spiralis. The rapidity of appearance of these effects and the lack of direct cross-immunity between the two species of worm suggest that the events involved in interaction were non-specific in action and possibly due to environmental changes in the gut caused by the immune response. These non-specific effects are therefore analogous, but not necessarily homologous, to the expulsion of these parasites in single species infections.

Superoxide dismutase (SOD) activity of Dictyocaulus viviparus and its inhibition by antibody from infected and vaccinated bovine hosts

The presence of superoxide dismutase (SOD) activity in the bovine lungworm Dictyocaulus viviparus was examined using the xanthine-xanthine oxidase assay system and by non-denaturing PAGE followed by specific enzyme staining. High levels of activity were detected in excretory-secretory (ES) products of adult worms and in soluble extracts of both the L3 and adult stages of the parasite. Stage-specific and ES-specific activities were indicated by differences in SOD isoenzyme profiles between adult and larval parasite extracts and between adult extract and ES products, with a fast migrating activity being specific to ES products. All isoenzymes were sensitive to cyanide, indicating copper/zinc dependency. The antigenicity of ES SOD was demonstrated by a reduction in SOD activity in both the chemical assay and non-denaturing PAGE following incubation of parasite ES products with IgG antibody purified from serum of infected or vaccinated bovine hosts. The high level of SOD activity released by adult D. viviparus may be a reflection of the problems faced by a parasite occupying an oxygen-rich environment. Antibody inhibition of SOD may, therefore, be an important target of protective immunity.

Effects of the host immune response on the longevity, fecundity and position in the intestine of Trichinella spiralis in mice.

In female NIH strain mice, expulsion of a primary infection of the nematode Trichinella spiralis began on day 8 and was virtually complete by day 14 of infection. In secondary and tertiary infections, the number of larvae which established in the intestine was normal, but expulsion began on day 6 and was complete on day 10. In a primary infection the shedding of larvae by female worms began on day 5, reached its peak on days 6--7, began to decrease on day 8 and was minimal by day 10. In secondary and tertiary infections fecundity was depressed. The depression of fecundity occurred slightly in advance of worm loss. During the stable phase of infection, T. spiralis occurred in the anterior half of the small intestine. During expulsion, living worms were found increasingly in more posterior parts of the gut but their fecundity did not vary with position. After direct inoculation into the posterior ileum, adult and larval T. spiralis remained in the posterior half of the small intestine. In this position, larvae established in normal numbers, grew and reproduced normally. Therefore, any part of the small intestine was a suitable site for T. spiralis and expulsion is not merely due to a change in the position of the worms.