Albert E Bianco

Immunodiagnosis of Strongyloides stercoralis infection: a method for increasing the specificity of the indirect Elisa

Indirect enzyme-linked immunosorbent assay (ELISA) allows sensitive detection of serum immunoglobulin (Ig) G against a soluble extract of Strongyloides stercoralis infective larvae. In this study, 40/40 (100%) human strongyloidiasis sera had high levels of anti-S. stercoralis IgG, but 30/40 (75%) filariasis sera, and 12/40 (30%) necatoriasis sera also had higher levels than control sera from UK residents. In attempts to increase the assay specificity by absorption of cross-reactive IgG, the effectiveness of pre-incubation of sera with extracts of different parasitic nematodes was investigated. One hour of incubation with 20 micrograms/ml aqueous extract of Onchocerca gutturosa absorbed cross-reactive IgG in most filariasis and necatoriasis sera, reducing the proportion with IgG levels above the positivity threshold by more than one-half. Preliminary results suggest that absorption with extracts of other filarial nematodes is equally effective, and that some of the cross-reactive IgG is directed against phosphorylcholine. Cross-reactive IgG in most necatoriasis sera was effectively absorbed with 20 micrograms/ml extract of Necator americanus. Cross-reactive IgG was not effectively absorbed with an extract of Ascaris lumbricoides. Absorption of cross-reactive IgG is an effective means of increasing the specificity of the indirect ELISA, for use in the immunodiagnosis and immuno-epidemiology of S. stercoralis infection.

An improved technique for the cryopreservation of Onchocerca microfilariae.

Experimental describe the use of ethanediol to store Onchocerca microfilariae in liquid nitrogen. The technique involves a 2-stage incubation of the parasites at 37 degrees C and 0 degrees C in ethanediol, before rapid cooling to -196 degrees C. Viability has been assessed by motility, by migration in a proxy host and by development to the infective stage in the insect vector. A total of 71-79% of the cryopreserved microfilariae was shown to be viable compared to unfrozen controls. The technique is simple, inexpensive and very effective when compared to previous cryopreservation procedures for microfilariae and should be particularly suited for use under field conditions.

Humoral immune responses in human infection with the whipworm Trichuris trichiura

Summary The humoral immune response to infection with Trichuris trichiura was investigated by ELISA and immunoblotting using human sera from the Caribbean island of St Lucia. Immunoblot analysis of the degree of cross‐reactivity with the related trichuroid Trichinella spiralis and with the other commonly co‐existent nematodes. Ascaris lumbricoides and Toxocara canis, was carried out using selected sera. The IgM, IgA, IgE, and IgG subclass antibody levels were measured in ELISA using a detergent solubilized extract of adult T. trichiura. The IgG and IgE responses were highly Trichuris specific. Anti‐T. trichiura IgM responses were totally cross‐reactive with A. lumbricoides and were completely ablated by pre‐incubation of sera with Ascaris antigen. The IgG response was predominantly of the IgG1 subclass with a minimal IgG3 response. Only 1 person out of 130 tested had a delectable IgG3 response. The IgG2 response appeared to be directed primarily against carbohydrate or polysaccharide antigens as pre‐treatment of the ELISA plates with poly‐L‐lysine was necessary before a response could be detected. These data are the first demonstration of human isotypic responses to infection with T. trichiura.

Extracellular proteases of Onchocerca

Two important events in infection by Onchocerca parasites involve cutaneous tissue migration by larval stages. L3 larvae migrate from the blackfly bite site to subcutaneous locations for adult development, and microfilariae from subcutaneous nodules to distant regions of the skin and sometimes the eye. By analogy to other tissue-invasive helminth larvae, it has been proposed that migration of Onchocerca larvae through cutaneous tissue is facilitated by secretion of proteolytic enzymes. To test this hypothesis, neutral protease activity capable of degrading a model of cutaneous extracellular matrix was assayed using live L3 larvae of O. lienalis and microfilariae of O. cervicalis and O. cervipedis. Five hundred L3 larvae degraded most of the matrix within 24 hr of incubation. Substrate gel electrophoresis and other protease assays showed a 43-kDa serine elastase was secreted by O. lienalis L3 larvae. Larvae and adults of the free-living nematode, Caenorhobditis elegans, by contrast, did not secrete neutral proteases and large numbers of motile C. elegans juveniles and adults produced no degradation of the extracellular matrix. Expression of Onchocerca neutral protease activity was stage specific. No protease activity corresponding to that seen in L3 larvae was found in adult worms. Microfilariae of O. cervicalis and O. cervipedis produced both a serine and a metalloprotease, but the level of protease activity of these microfilariae was substantially lower than that of L3 larvae, and no significant protease activity was detected in extracts of O. lienalis microfilariae. Uterine microfilariae of O. cervicalis had different protease species than skin microfilariae, suggesting that changes in protease expression parallel other morphologic and biochemical changes in the development of skin microfilariae. The serine protease of L3 larvae probably plays an important parasitic function, facilitating L3 migration from the blackfly bite site to distant regions of the body where adults will develop and form nodules. The protease activity of microfilariae, while individually considerably less than that of L3 larvae, may still contribute to the tissue destruction seen with heavy skin densities of microfilariae.

The relationship between Trichuris trichiura transmission intensity and the age-profiles of parasite-specific antibody isotypes in two endemic communities

The present study compares parasite-specific antibody responses in two Caribbean communities with high and low levels of Trichuris trichiura transmission. The age-dependency of antibody levels suggest that IgG1 and IgG2 levels relate to the current intensity of infection (as assessed by density of eggs in stool (e.p.g.) and reflect the age-intensity profile at the population level. IgG4, IgE and IgA levels persist into early adulthood and the subsequent decline is gradual. In the low transmission area, lower infection levels are reflected in lower parasite-specific antibody levels (of all isotypes) in the community as a whole. Despite a significantly greater past experience of infection in the high transmission area, antibody levels are not maintained at significantly higher levels throughout adulthood. The production of IgA appears to require a threshold for triggering, and a vigorous IgA response is maintained into early adulthood only in the high transmission village where peak intensity is greatest and the age-convexity of intensity is most marked. Experimental and theoretical studies focusing on the dynamic nature of host-helminth interactions in hosts exposed to high and low infection levels, and the putative role of acquired immunity, are discussed in relation to the data presented.

Age-dependency of infection status and serum antibody levels in human whipworm (Trichuris trichiura) infection

Summary This study examines the age‐dependency of the relationships between human infection with whipworm (Trichuris trichiura) and parasite‐specific antibody level measured by ELISA against an extract of adult worms after preincubation of the sera with Ascaris lumbricoides adult worm extract. The convex age‐profile of parasite infection intensity is shown to be mirrored ban ase‐dependent change in age‐class mean levels of IgG (all subclasses except IgG3). IgA. IgM and IgE. Mean antibody levels rise with increasing acquisition of infection in childhood and decline as the intensity of infection falls in adulthood. Immunobiot analysis of selected sera from different age‐classes indicates that antigen recognition is simitath dependent on infection intensity. In individual children, antibody levels correlate positively with acquisition of infection, consistent with a simple model of antigen dosage specifying the magnitude of the humoral immune response. In adults, Igd correlates positively and IgA negatively with intensity of infection, suggesting involvement of these isolypes in functional roles of immune blockade or effector mechanisms, respectively.

Developmentally regulated expression and secretion of a polymorphic antigen by Onchocerca infective-stage larvae

In order to analyse the developmental biology of Onchocerca spp. with a view to identifying molecules with specialised functions, we have devised a novel method for labelling proteins synthesised by larvae during growth in the vectors. Pulse labelling of Onchocerca lienalis by micro-injections of [35S]methionine into blackflies have revealed a major acidic protein of 23 kDa which is developmentally expressed almost exclusively by infective, third-stage larvae. The protein appears to be antigenically conserved between O. lienalis and Onchocerca volvulus, but exhibits size polymorphisms both among species and among individual organisms. It continues to be elaborated after terminal differentiation of the parasite in flies, but not by post-infective larvae entering the phase of development in the vertebrate host. A shift in temperature from 26 degrees C to 37 degrees C triggers secretion of the 23-kDa molecule as a discrete event 24-72 h after transmission. The labelling technique has been successfully employed with filarial species that develop in mosquitoes, and in principle should be widely applicable to the study of endoparasite gene expression within arthropods.

Immunization of calves against the microfilariae of Onchocerca lienalis.

Three Jersey bull calves were immunized by subcutaneous injections of sonicated Onchocerca lienalis microfilariae suspended in phosphate buffered saline, and three control animals were injected with medium only. All calves received booster injections 27 days later, and were challenged with live microfilariae 44 days after the booster. Following challenge, only the immunized animals developed an elevated level of circulating eosinophils. When necropsied ten days after challenge there was a 97% reduction in recoveries of microfilariae from immunized animals compared to challenge controls. In human onchocerciasis it is the microfilariae which are the principal cause of pathology, and we believe that studies on O. lienalis in both the natural bovine host and in inbred rodents provide a promising model to investigate immunity to Onchocerca microfilariae.

Characterization of peptidases of adult Trichuris muris

Excretory/secretory (E/S) material of Trichuris muris was found to contain 2 major peptidases, M(r) 85 and 105 kDa, which degrade gelatin optimally at pH 6.0 in sodium dodecyl sulphate-polyacrylamide gels. The peptidases were inactivated by diisopropylfluorophosphate, leupeptin and soybean trypsin inhibitor, but were unaffected by inhibitors of aspartic-, cysteine- and metallo-peptidases, indicating that they are serine peptidases. Both enzymes were detectable within 5 h after incubation of worms in culture medium and showed a time-dependent increase in levels. Neither peptidase was detected in worm extracts, suggesting that they are activated during or following secretion from worms. Live worms degraded a radio-isotope labelled extracellular matrix protein substratum derived from mammalian cells. Aminopeptidase activities capable of catalysing hydrolysis of amino acyl aminomethylcoumarin (MCA) substrates and a Z-Phe-Arg-MCA-hydrolysing cysteine peptidase activity, were detected in extracts of adult worms but not in E/S material.

Biochemical and immunochemical characterisation of a 20-kilodalton complex of surface-associated antigens from adult Onchocerca gutturosa filarial nematodes

Surface radioiodination of adult Onchocerca parasites reveals a restricted range of proteins associated with the cuticle. We present data to show that prominent among these is a complex of low-molecular-weight proteins which can be released in soluble form by homogenisation of surface-labelled Onchocerca gutturosa in phosphate-buffered saline (PBS). One of this groups of proteins, designated gp20, has a molecular mass of 20,000, is glycosylated with two N-linked carbohydrate side chains, and has a basic pI. Other PBS-soluble, 125I-labelled proteins of similar size appear not to be glycosylated. A distinct group of molecules are released only in the presence of reducing agents, and are likely to be cuticular collagens. The low-molecular-weight components are antigenic and cross-reactive with Onchocerca volvulus infection sera. Cross-reactions are also observed in immunoprecipitation experiments using sera from Brugia-immunised animals and infected humans. Comparative two-dimensional analyses of these immunoprecipitates reveal at least two Onchocerca specific components. As an alternative to radiolabelling and PBS homogenisation, incubation of worms in medium containing the reducing agent 2-mercaptoethanol resulted in a similar set of molecules being released into the medium. Since surface antigens of O. gutturosa from bovines and O. volvulus from humans appear similar in size and are antigenically cross-reactive, the more readily available parasite is being used to study further the properties of these molecules and to provide reagents for raising antisera reactive to the equivalent O. volvulus antigens.