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.

Immunological cross-reaction between an Onchocerca paramyosin-like molecule and a microfilaria surface antigen.

A monoclonal antibody (2A5B9), previously shown to be reactive with a 14 kD surface associated antigen of Onchocerca microfilariae, was found to recognise a 92 kD molecule present in an adult worm extract. The antibody was used to select cDNA clones with a coding capacity larger than 14 kD, from a lambda gt11 library of O. volvulus. Nucleotide sequencing of the cDNA of one such clone revealed extensive homology to the myosin (unc-54) and paramyosin (unc-15) genes of Caenorhabditis elegans, similarly to myosin and paramyosin genes of Onchocerca volvulus, Brugia malayi, Dirofilaria immitis and Schistosoma mansoni. The immunological implications of antigenic cross-reactivity between a surface molecule and paramyosin, a known protective antigen, are discussed.

Studies on a 14-kilodalton surface protein of Onchocerca microfilariae.

A 14-kDa antigen present on the surface of uterine microfilariae of Onchocerca spp. has been identified using monoclonal antibodies. The antigen was also found in skin microfilariae, but in a masked or cryptic form. A complementary DNA clone encoding the epitope recognised by one of the monoclonal antibodies was identified in a lambda gt11 library. Nucleotide sequencing revealed that the 233-bp cDNA fragment codes for the carboxy-terminus of the antigen. The deduced amino acid sequence consists of three hydrophobic domains with high potential for beta-sheet formation. The amino-terminal hydrophobic domain is followed by 4 positively charged residues (positions 22-25) which contribute to the rather basic character of the protein. Another interesting feature of the polypeptide is its richness in phenylalanine (12.7%). From the sequence information, a synthetic peptide was synthesised which was recognised by one of the monoclonal antibodies directed against the 14-kDa antigen and a small number of sera from patients with onchocerciasis. The relevance of this to vaccination is discussed.

Association between circulating antigen and parasite load in a model filarial system, Acanthocheilonema viteae in jirds.

Jirds (Meriones libycus) were infected with various numbers of Acanthocheilonema viteae L3 stage parasites. During the course of the ensuing 16 weeks, blood samples were collected at 2 weekly intervals and the amount of the major parasite excretory-secretory product (E-S 62) and antibodies directed against it measured. After 16 weeks, animals were sacrificed and the size of the mature worm burden established. In spite of interaction between E-S 62 and host antibody, a statistically significant relationship was found to exist between the amount of E-S 62 present in the bloodstream and the size of the parasite load. It is suggested that the detectable antigen level is more influenced by the size of the worm burden than the presence of antibody and that antibody is only likely to affect adversely antigen measurement in situations where the amount released is relatively low. Examples of this are early in infection and in low-level infections. These ideas are discussed in relation to the development and assessment of serological assays which attempt to predict parasite burden in human filarial infections.

The surface lipid of parasitic nematodes: organization, and modifications during transition to the mammalian host environment.

The biophysical properties of the surface lipid of a range of nematode species and their developmental stages were examined, using fluorescent lipid probes and fluorescence recovery after photobleaching (FRAP). These methods can be applied to living, intact parasites, and the analysis confined to lipid on the outermost surface. In all cases, surface lipid was unusual in its selectivity for the insertion of the lipid probes. In addition, a polar lipid probe was generally not free to diffuse in the plane of the surface, in contrast to a non-polar lipid probe which was free to diffuse. This is evidence that the surface lipid layer is heterogeneous, and possibly comprises lipid domains. The infective larvae of Acanthocheilonema viteae, Nippostrongylus brasiliensis, Trichinella spiralis and Ostertagia ostertagi were found to exhibit a rapid change in lipophilicity upon exposure to conditions simulating entry into a mammalian host environment. Parasitic nematodes, therefore, present their hosts not only with a highly unusual biological surface, but also one which can be rapidly re-organised upon a change of environment.

Origin, kinetics of circulation and fate in vivo of the major excretory-secretory product of Acanthocheilonema viteae.

The excretions-secretions (E-S) of Acanthocheilonema viteae consist mainly of one product, molecular weight 62kDa. This molecule is synthesized during the vertebrate phase of the parasite life-cycle and is first detectable in the E-S of L4 parasites. It is cross-reactive with E-S of human filarial parasites as a consequence of possessing a phosphorylcholine (PC) moiety. The 62 kDa molecule has been employed as a model for the study of the origin and fate of filarial E-S. Immunohistological analysis has shown the molecule to be located predominantly in the parasite gut. Transplantation of adult female [35S] methionine pulsed worms into uninfected jirds resulted in the radio-labelled secreted 62 kDa antigen being detected in the bloodstream within 4 h by SDS-PAGE/immunoprecipitation analysis. The systemic half-life of the molecule as estimated by clearance of injected, purified 125I-labelled material was measured in naive and infected jird hosts. It was reduced from 2-7 h in naive animals to less than 30 min in 4-10 week infected rodents, a finding which correlated with clearance of antigen by antibody in the infected group. In animals infected for longer time periods the serum half-life returned to the values observed in naive jirds. The idea that this change in half-life may reflect differences in the nature of 62 kDa antigen containing circulating immune complexes as infection progresses is discussed. The 125I-labelled antigen is predominantly removed from the circulation via the liver and ultimately excreted in the urine in a non-antigenic form. This work provides the first description of the origin, kinetics of circulation and fate of a defined filarial E-S product and may aid in determining the function and assessing the diagnostic utility of PC-bearing E-S components.

Evaluation of the potential of excretions-secretions (E-S) of Litomosoides carinii to substitute for human filarial E-S.

The antigenic cross-reactivity of the excretions-secretions (E-S) of Litomosoides carinii was investigated. Immunoprecipitation using pooled sera from a number of human filarial infections in combination with sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed variations in the cross-reactivity of individual molecules. Some were specific to L. carinii, the major examples in this category being two E-S components of 140 and 160 kDa released by day 40- to 42-day-old female worms. Another, a high-molecular-weight product of 26- to 28-day E-S, was broadly cross-reactive. A third group appeared to exhibit reactivity to antibody to some but not all human filarial parasites. The most striking examples of this were two distinct 14-kDa products that bound solely to antibodies in an onchocerciasis serum pool. These results are discussed in relation to the use of cross-reacting molecules in investigating the immunisation potential, defining the function, and evaluating the diagnostic utility of human filarial E-S.

Requirement for host Fc receptors and IgG antibodies in host immune responses against Rhipicephalus appendiculatus.

Guinea pigs sensitized by prior feeding of larval Rhipicephalus appendiculatus ticks expressed complete immunity to challenge feeding resulting in 100% tick rejection. Passive transfer of 1 ml of serum from animals expressing resistance into naive animals conferred recipients with significant protection (88% tick rejection). Successful transfer of resistance was blocked by pretreatment of recipients with rabbit IgG but not sheep IgG1. Passive transfer of IgG1 or IgG2 purified from tick-sensitized guinea pig serum by ion-exchange chromatography failed to confer resistance to naive guinea pigs. Furthermore, IgG1 from guinea pigs expressing resistance obtained from serum by passage through a heavy chain specific rabbit anti-guinea pig IgG1 column failed to confer resistance to naive guinea pigs, as did the eluate. These results suggest that both IgG subclasses are needed for the expression of resistance, or IgG1 in conjunction with IgE.

Variation in class-specific humoral immune responses of different mouse strains to microfilariae of Dipetalonema viteae.

The class-specific antibody responses of 3 strains of mice (C57/Bl10, BALB/C and CBA/N) known to vary in their ability to control the microfilaraemia which follows the subcutaneous transplantation of adult female Dipetalonema viteae has been investigated. The 3 mouse strains showed significant variation (a) in total levels of immunoglobulins and (b) in ability to recognize individual radio-isotope-labelled antigens as measured by coprecipitation. Within each mouse strain it was noted that antigens could vary with respect to the nature of the isotype of the antibody response which they elicited. Furthermore, by comparing results obtained from class-specific coprecipitation with surface ELISA it was found that a similar variation between responses to individual epitopes was also likely. No differences were observed in the humoral response of the 3 mouse strains which could explain the known resistance of the C57/Bl10 strain; reasons for this are discussed.

Quantitative and qualitative changes in production of excretions/secretions by Litomosoides carinii during development in the jird (Meriones unguiculatus).

Excretions and secretions (E-S) were collected from a series of developmental stages of Litomosoides carinii maintained in vitro. Measurement of the protein content of E-S obtained from each stage indicates that the rate of production of E-S varies enormously during development of the worm. E-S was iodinated using both Iodogen and the Bolton and Hunter Reagent and was also biosynthetically labelled by incubating worms in the presence of [35S]methionine and [3H]leucine. Attempts to biosynthetically label E-S of mature worms and microfilariae with [3H]glucose were unsuccessful. Examination of radio-isotope labelled E-S by SDS-PAGE revealed that some components were sex specific and that the differences in total E-S production during development were due to the existence of both stage-specific components and components whose rate of release varied during parasite maturation. Antigenic characterization of E-S, carried out by immunoprecipitation in combination with SDS-PAGE, indicated that E-S consists of immunogenic components, a molecule which is probably a non-immunogenic parasite product, and host albumin. The implications of these findings for the construction of diagnostic tests to detect products of human filarial parasites are discussed.

Surface proteins of a filarial nematode: a major soluble antigen and a host component on the cuticle of Litomosoides carinii.

The surface composition of three stages in the life cycle of Litomosoides carinii, a filarial parasite of rodents, has been studied using radio-iodination techniques. Confirmation that radiolabelled components were confined to the parasite surface was achieved using light and electron microscope autoradiography. Biochemical analysis of extracts of radiolabelled parasites by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) revealed that one major component (mol. wt. 55 000) could be solubilized with the aid of detergents. This component, which was present on male and female adult worms and on post-parasitic third stage larvae, accounted for about one-third of the total proteins available for surface iodination, and was antigenic in infected hosts. The remaining surface components could be solubilized only with urea and SDS under reducing conditions. The 55 000 mol. wt. surface antigens of male and female adult worms exhibited identical two-dimensional tryptic maps, but the similar 55 000 mol. wt. antigen of post-parasitic third stage larvae was different. There was, however, some sharing of antigenic determinants between adult and larval surface components. The principal protein present in detergent extracts of surface-radio-labelled blood microfilariae was host serum albumin.

Ornithodorus tartakovskyi: quantitation and ultrastructure of cutaneous basophil responses in the guinea pig.

Cutaneous lesions elicited in guinea pigs by primary and secondary feeding populations of the argasid tick, Ornithodorus tartakovskyi, were analyzed by light and electron microscopy. Small clusters of basophils appeared at primary bite sites within 24 hr of tick attachment, and by 72 hr constituted approximately 11% of the total leukocytes. Secondary feeding sites exhibited an augmented cellular infiltrate that was dominated by basophils at all times (48-56% of total cells). Eosinophil proliferation was minimal, however, and the remaining cells were of the mononuclear type. Despite mounting a strong cutaneous basophil response of the kind that mediates immune rejection of prolonged-feeding ixodid ticks, the guinea pigs showed no resistance to the fast-feeding Argasidae. It is suggested that argasid ticks probably complete their blood meal prior to basophil arrival at the bite site. Electron microscopy indicated that the number of epidermal Langerhans cells increased with time in both primary and secondary lesions; these cells were more numerous in challenge infections however, and seemed also to occur in the dermis. Basophils at secondary bite sites exhibited three kinds of structural alterations classified as: (1) piecemeal alterations--involving a vesicular degranulation mechanism; (2) an anaphylactic-type of alteration--involving single or compound exocytosis of whole granules; and (3) cytotoxic alterations culminating in complete disintegration. The majority of basophils in 72 hr secondary lesions exhibited cytotoxic alterations. It is suggested that such changes result from contact with tick-derived toxins or enzymes.

Cutaneous basophil associated resistance to ectoparasites (ticks). Electron microscopy of Rhipicephalus appendiculatus larval feeding sites in actively sensitised guinea pigs and recipients of immune serum.

Electron microscopy has been used to monitor cellular activity in dermal lesions elicited by larval Rhipicephalus appendiculatus feeding on actively sensitised guinea pigs and recipients of immune serum. The early primary response is characterised by mononuclear cells, many of which appear to be activated fibroblasts. Collagen deposition is enhanced as the reaction progresses. Granulocytes of all types appear in the lesion between 18 and 96 hr but they show no evidence of degranulation. Free, membrane-bounded eosinophil and basophil secretion granules may, however, be identified in the dermis at day 5 or 6, but they seem to be liberated as a consequence of cellular disruption, rather than active degranulation. Some feeding sites resume a normal morphology by day 7. Lesions induced in actively sensitised hosts by a secondary feeding tick population are dominated by basophils. These cells begin to infiltrate the dermis within 6 hr and they show evidence of anaphylactic degranulation at 12 hr. Maximal release of membrane-free secretion granules occurs at about 18 hr post-attachment, at which time eosinophils become prominent. Degranulating basophils show a reduction in numbers from 24 to 96 hr, and phagocytic macrophages ingest residual granules and cellular debris. Guinea pigs sensitised with immune serum and subjected to challenge exhibit lesions similar to but less dramatic than those of actively sensitised and challenged animals. Anaphylactic degranulation of basophils occurs both in the dermis and within blood vessels. The immunological consequences of these events are discussed in relation to other models of cutaneous basophil hypersensitivity.

Rhipicephalus appendiculatus: larval feeding sites in guinea pigs actively sensitized and receiving immune serum.

Histological analyses of larval Rhipicephalus appendiculatus feeding sites in naive and actively sensitized guinea pigs were made at 6, 24, 48, 72, 96 hr post-tick attachment. As primary feedings progressed the cavity at the entrance of the ticks mouthparts into the uppermost dermis, and the surrounding cellular infiltrate (lesion) both increased. Early (6 hr) lesions were dominated by eosinophils again predominated at 72 hr (44%), and finally basophils were dominant at 96 hr increased as tick feeding progressed and at each observation time was at least twice that observed in primary feedings. Dermal cavities at the site of entrance of the ticks mouthparts were occasional in occurrence and were reduced in size indicating altered tick feeding. Basophils were dominant at all observation times ranging from 61 to 91% of the infiltrate. The second cell type of significance was the eosinophil, ranging in abundance from 7 to 21%. Recipients of immune serum had a smaller cellular filtrate around feeding ticks, but basophils were also dominant. Basophils appear to be the principal host cell involved in acquired resistance to tick feeding as indicated by the profound cutaneous basophil reaction that characterized the immune response to larval ticks both in actively and passively sensitized hosts. The finding of significant eosinophil accumulations at tick feeding sites of both hosts indicates that these cells may also contribute to acquired resistance.

Macrophage-mediated damage to filarial worms (Dipetalonema setariosum) in vivo.

An adult female Dipetalonema setariosum (Mönnig 1926) recovered from the pleural cavity of Meriones libycus and bearing an adherent cell mass examined by means of electron microscopy. The host reaction consisted exclusively of macrophages and was associated with disruption of the cuticular membrane and invasion of the cuticle itself. There was no evidence of prior activity by other cell types. Initiation of damage appeared to be associated with the release of lysosomal enzymes by the macrophages. Within the reaction a localized breaching of the cuticle had occurred and cells had penetrated the internal tissues of the worm. It is suggested that macrophages may play a role in the elimination of effect worms from an established population.

Cutaneous basophil-associated resistance to ectoparasites (ticks). I. Transfer with immune serum or immune cells.

Immune resistance experiments were carried out in guinea-pigs employing two tick species that as adults are ectoparasites of cattle (Ixodes holocyclus and Rhipicephalus appendiculatus). These studies showed that susceptibility of non-immune guinea-pigs to infestation with tick larvae varies according to the species of tick and the strain of guinea-pig. With both tick species, greater than 90% acquired resistance was achieved in several guinea-pig strains. Immune resistance was evident within a week following primary infestation and lasted up to 9 months following a single sensitizing exposure to tick feeding. The strength and duration of resistance was influenced strongly by the size of the initial sensitizing dose. Immune resistance was readily transferred to naive recipients by intravenous administration of either peritoneal exudate cells or immune serum from donors sensitized by a single prior infestation with ticks. Doses of serum as small as 0.5 ml transferred resistance. These studies demonstrate that both sensitized cells and immune serum factors contribute significantly to acquired host resistance to ticks that as adults are ectoparasites of cattle.