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The surface antigens of Toxocara canis infective larvae have been identified by radio-iodination and compared with the excretory-secretory (ES) products released by the larvae in vitro. Common antigens, of molecular weight 32 000 and 120 000 are found on the larval surface, in the ES material and in culture supernatant following surface iodination of living T. canis larvae. The 120 000 antigens consist of three closely migrating bands in each of these preparations. However, one prominent ES component, of molecular weight 400 000, is not found on the larval surface. Additional molecules of 55 000 and 70 000 are present in the ES material, but while these may be discerned in surface preparations there appears to be more heterogeneity of surface molecules in this size range. Both sets of molecules are antigens to infected patients and experimental animals. A comparison of characterized human sera show that a radio-immunoprecipitation assay correlates with the established ELISA test (r = 0.89), and that all labelled molecules are antigenic to the infected host.

Previous reports have described profound effects on the function of the lymphoid system, especially the spleen, in mice infected with Trypanosoma brucei. This study provides further evidence of major change in the cell populations of the blood, peritoneum, and bone marrow, but shows that at least some of the stem cells of the bone marrow survive the damage caused by trypanosomes and retain their ability to repopulate the animal. In these infected mice the initial parasitemia was terminated by day 11 and was followed by a subpatent period of approximately 7 days before a final, lethal parasitemia occurred. Lymphopenia preceded the initial and final waves of parasites in the blood, and there was a marked increase in circulating neutrophils and large mononuclear cells for 1 week after the termination of the first wave of bloodstream parasitemia and during the final lethal parasitemia. Dividing macrophages were detected in the peritoneum only briefly during week 1 of infection, but the total number of peritoneal cells was increased from day 8 until the mice died. The bone marrow is severely stressed by the parasite infection. Total cell numbers and spleen colony-forming cells in the bone marrow were profoundly depleted during the resolution of the first parasitemia, but both these parameters largely recovered during the subpatent period before the mice were killed by the disease. Immune function was restored gradually after treatment with Berenil late in infection. We conclude that the progenitors of lymphocytes as well as the mature cells are affected by trypanosomes, but that some of the early bone marrow stem cells escape and rapidly repopulate the peripheral organs upon removal of the parasites.

Adult Onchocerca volvulus worms obtained by enzyme digestion from nodules of infected Mexicans were radio-isotope labelled by the chloramine-T or Bolton-Hunter methods. No antigenic determinants were detected in extracts of worms labelled by the chloramine-T method but 3 antigens were detected in extracts of the Bolton-Hunter labelled worms. Two were present in such small amounts that it was impractical to investigate them further, but a major component of mol. wt 20 kDa was purified by gel filtration and used in a serological survey of inhabitants of villages in Southern Mexico. Using the 20 kDa antigen, which is superficially located on both sexes of O. volvulus, sera from both non-endemic and endemic regions were analysed by radio-immunoprecipitation of this antigen. In Southern Mexico, the average sensitivity of the test was 92%, and the specificity 98%. Whilst the 20 kDa antigen did not detect antibodies in the sera of Trinidadians infected with Wuncheria bancrofti or Mansonella ozzardi, this antigen detected high levels of antibodies in Indians exposed to W. bancrofti.

A previous report showed that infection with the nematode Nippostrongylus brasiliensis stimulates a basophilia as well as an eosinophilia in the blood of August rats. The present study shows that blood levels of basophils and eosinophils were increased in two other rat strains, one inbred and one outbred, after infection with N. brasiliensis, and infection of two inbred rat strains with Trichinella spiralis also stimulated a basophilia as well as an eosinophilia. No increase occurred in basophils or eosinophils in athymic (nude) rats infected with N. brasiliensis, although both these cell types were found in the blood of control, specific pathogen free, nude rats in numbers comparable to those in specific pathogen free, heterozygote controls of the same strain. Rat basophils usually have few granules and in blood smears often appear as if they were partly degranulated. Basophils from uninfected nude rats contained more negative than positive staining granules compared with basophils from parasitized heterozygotes. The possession of small numbers of granules which vary in their reaction to stains of the Romanowski type is a normal feature of rat basophils in blood smears. Consequently rat basophils differ in these respects from those of other species.

African trypanosomiasis in mice leads to profound changes in lymphoid tissues. In an attempt to define the nature of the trypanosome stimulus, we have studied the effect of radio-attenuated trypanosomes and their subcellular fractions in vivo. We find that relatively low doses of irradiated Trypanosoma brucei S42 injected into (CBA/H x C57B1/6)F1 mice mimicked the previously reported effects of infective parasites. 2 x 10(7) irradiated trypanosomes caused a greater than two-fold increase in spleen weight accompanied by a roughly 10-fold increase in background plaque forming cells (PFC) to sheep red blood cells (SRBC). The primary response to SRBC was significantly enhanced when priming was carried out on the day of trypanosome injection, but significantly suppressed when carried out 3 days later. Disruption of trypanosomes by freeze-thawing did not destroy their mitogenic or immunosuppressive activities. A membrane fraction collected by high speed centrifugation (150 000 x g) after removal of larger organelles at 12 000 x g retained both mitogenic and suppressive activities. The high speed supernatant lost the ability to enhance background PFC, but still caused partial immunosuppression with a much lower potency than the membrane pellet. Whether immunosuppression and enhanced PFC levels relate to the same parasite product is not clear as yet, but both effects can be ascribed to a membrane fraction of the parasite.

B lymphocyte function was assessed in outbred nude mice and nu/+controls infected with Trypanosoma brucei brucei. On day 10 of the infection in outbred nu/nu mice in which the initial wave of parasites was strongly controlled, B cell function was unaltered on enhanced compared with uninfected animals or infected nu/+. In other nu/nu mice unable to control the initial parasitaemia, thymidine incorporation and Ig secretion by spleen cells were increased on day 10 and their response to lipopolysaccharide in vitro negated. By day 15 however, even the spleen cells of infected nu/nu which controlled the initial wave of parasites were proliferating and secreting Ig on removal from the mice and they were unable to respond to LPS in vitro. These experiments confirm results of a previous study of B cell function in T cell-depleted mice (Askonas et al. 1979). T. b. brucei infection of mice causes both enhanced Ig production and suppression of the ability of B cells to respond to mitogen even in the absence of T cells, but the presence of T cells may accelerate the changes which occur in B lymphocytes following this infection.

The adherence in vitro of leucocytes to the surface of various stages in the life cycle of T. spiralis and N. brasiliensis in the presence of serum was examined. Considerable differences were observed in the behaviour of mast cells, eosinophils, neutrophils and macrophages in this interaction. Mast cells adhered for a short time, did not flatten onto the surface and did not degranulate. Adherence ceased after 4-6 hr. Eosinophils adhered within minutes to the surface or worms, flattened and degranulated; only their cytoplasmic remnants could be seen on the worms' surface after 24 hr in culture. In contrast, only a small area of the cytoplasmic membrane of neutrophils flattened on the surface of the worms and adherence ceased after 2-24 hr. The NBT conversion reaction showed a positive deposit at the interface between neutrophils and parasites during neutrophil adherence. This deposit remained as "foot prints" on the surface of the nematodes following neutrophil detachment. This positive NBT reaction occurred only with neutrophils and not with eosinophils, mast cells or macrophages. Macrophages adhered permanently to the surface of these worms, they did not flatten and retained their integrity. Under the light microscope the cytoplasmic inclusions appeared to decrease in size during culture. Electron microscopy revealed the presence of fewer granules and an increased number of vacuoles in later cultures of macrophages. These findings are discussed in relationship to the immunopathology of nematode infection in vivo.

Young rats were not able to expel adult N. brasiliensis infections even when the worms were damaged by antibodies and the young rats were given all the cellular components (sensitized lymphocytes and bone marrow cells) shown to be necessary for the expulsion of antibody-damaged worms from adult rats. In contrast, most of the worms were expelled from young rats given sensitized lymph node cells on the day of a larval infection. These results show that the reduced ability of young rats to respond to infection by producing sensitized lymphocytes only partly explains their inability to expel the worms. It was not possible to explain the failure of young rats to expel adult worms by hypothesizing that they develop an active factor which prevents the cells from acting on the worms. It is also unlikely that worms persist in young rats because they differ in their susceptibility to cells compared with antibody-damaged worms from mature rats. This work suggests that the immune mechanism which affects the immature stages of this nematode may differ from that which controls the adult stages.

Peripheral blood lymphocytes of chickens and thoracic duct lymphocytes and mesenteric lymph node cells of rats, were labelled with 125I-deoxyuridine and injected intravenously into chickens or rats respectively. Sixteen to 18 h later the intestines of coccidia infected animals contained more radioactivity than those of uninfected controls. This result was obtained with cell suspensions from both infected and normal donors indicating that, as with nematode infected rodents, the increased homing of the cells to parasitised gut was not antigen specific. In chickens the stimulus which causes the increased homing of cells to the intestine was induced within hours of parasite inoculation. This reflects the rapid response of this host, previously described with other parameters, and which may be characteristic of birds.

Rats were infected with the nematode Trichinella spiralis and the primary serum antibody response to antigenic surface proteins of infective larvae, intestinal worms, and newborn larvae was studies. 1 wk after infection, the sera contained antibodies to surface antigens of both infective larvae and intestinal worms. These early sera, however, failed to react with newborn larvae surface antigens. In addition, adsorption of sera with living intestinal worms or infective larvae removed antibodies to surface antigens of the homologous stage only. Finally, the time-course of appearance of antibodies that mediate eosinophil adherence to the surface of each stage of the parasite. We concluded that in a primary infection in rats, the surface proteins of T. spiralis used in this study are antigenically stage specific. Furthermore, they could be targets for the stage-specific, antibody-dependent eosinophil-mediated destruction of this parasite, known to occur in vitro.

Antibody responses were measured in a volunteer infected four times with Necator americanus over a 27-month period. The main source of antigen was culture fluid in which living adult N. americanus had been maintained for several days. Antibodies to worm acetylcholinesterase and IgE antibodies were detected only with this material, but antibodies were identified by the enzyme-linked immunosorbent (ELISA) assay, with either adult worm secretions or extracts of third-stage infective larvae. The total serum IgE level fell after the first infection, but although it then increased during subsequent infections, it never rose above 600 U per ml. None of the antibody responses suppressed the rat of worm development to maturity, or reduced the fecundity of the parasites. However, it is suggested that the development of the immune response may be associated with the waning of the severe gastro-intestinal symptoms which were experienced in this infection, and which are frequently characteristic of hookworm infections.

The peripheral blood leucocyte (PBL) response to infection with two species of the intestinal protozoan Eimeria shows a remarkably similar pattern in both rats and chickens. Like resistance to these parasites, the PBL response to reinfection is species-specific and it coincides with the inhibition of development of the parasite and with cellular infiltration of the lamina propria. In primary infections, the numbers of PBL increased biphasically and significant changes were found in the count of polymorphonuclear cells (PMN), lymphocytes, and large mononuclear cells (LMN). A consistent pattern of two increases in PMN, some lymphopenia, and two increases in LMN was found in rats and chickens. The response to secondary infections was more rapid and much briefer, particularly in chickens in which significant changes were detected within hours of infection and consisted of an almost equal increase in PMN and in lymphocytes. This secondary response occurred only in animals challenged with the homologous species. It was not evoked by challenge with a related species which develops in approximately the same area of the intestine.

The functional changes in splenic lymphoid populations from mice infected with T. brucei strain S42 were studied throughout the 3 weeks of infection. Within a week of infection, proliferation of B and T cells profoundly increased as shown by 3H-labelled thymidine incorporation and fluorescent staining of surface Ig; the spleen cells secreted high levels of both IgM and IgG immediately cells were put into culture; but with progressing infection this Ig production declined. The early effect on T cells was reflected by lack of responsiveness to PHA. B-cell potential was studied in low-density cultures treated with lipopolysaccharide (E. coli). Normal spleen cells proliferate extensively in these cultures with subsequent secretion of IgG as well as IgM. The ability to proliferate and produce Ig in response to LPS was severely depressed by day 7 and almost totally absent by day 12 of infection. Removal of T cells from the spleen cells obtained early in infection partly restored the response to LPS but as the infection neared its fatal end, B-cell potential appeared to become exhausted. Macrophages obtained from infected mice even early in infection profoundly depressed the ability of normal spleen cells to proliferate and secrete immunoglobulin in LPS cultures. The general immunodepressing effect of trypanosomes can be attributed to clonal exhaustion of B-cell potential caused by an undefined blastogenic stimulus from the parasites which may operate at least in part by the generation of suppressive T cells and macrophages.

Nematode infections in the gut induce a strong immune response which is rapidly detected parenterally. The response is thymus-dependent and long-lasting and involves both antibodies and cell-mediated reactions. The immunological response to unrelated antigens, tumours and other infectious organisms is altered in animals infected with nematodes. Both antibodies and sensitized lymphocytes participate in the immune response which affects the nematodes themselves, and characteristically the lymphocyte-dependent step cannot act in lactating animals and is neither induced nor able to act in young animals. Present evidence suggests that, despite their well-known association with helminth infections, parasite rejection from the gut does not require the participation of IgE antibodies, mast cells or eosinophils. Homing of lymphoblasts from the mesenteric lymph node or thoracic duct lymph to the small intestine is increased in rats and mice infected with Nippostrongylus brasiliensis or Trichinella spiralis and the increase is antigenically non-specific. In mice infected with T. spiralis this increase is represented mainly by thymus-derived lymphoblasts.

The immunological response of rats to the intestinal phase of Trichinella spiralis was assessed using criteria derived from pervious studies with the nematode Nippostrongylus brasiliensis in rats and mice. In adult rats, the duration of infection with either parasite is similar and both infections are prolonged in young and lactating rats. As previously shown with N. brasiliensis, immunity to T. spiralis was transferred to recipients with antiserum or mesenteric lymph node cells from immune donors and antisera and cells given in combination had an additive effect. Signs of damage similar to that caused by antibodies in N. brasiliensis appeared in T. spiralis adult worms as the infection progressed and this damage occurred earlier in animals given antiserum or cells. On the basis of these results, it is concluded that the immunological control of adult T. spiralis requires both antibodies and cells, but the relative importance of these components and the way in which they affect T. spiralis requires further analysis.

The antibody response to antigens on the cuticular surface of Trichinella spiralis was compared in two strains of mice, NIH mice, which control the parasite relatively strongly, and C3H, which reject this nematode more slowly. The evolution of the antibody response to the nematode surface was monitored by the appearance of antibodies that mediate the adherence of eosinophils to the worm and the appearance of antibodies that recognize molecules on the surface that can be labeled with 125I. Antibody responses were measured using three life cycle stages; muscle stage larvae, immature and mature intestinal stages, and newborn larvae. We found that NIH mice responded immunologically to the four molecules found in extracts of surface-labeled T. spiralis adult worms, but that C3H mice did not produce antibodies to one of these molecules until much later in the infection. NIH mice produced antibodies against both infective larvae antigens by day 4 after infection, whereas C3H recognized only one before day 30. Variations in antibody response also occurred against antigens on the surface of newborn larvae: antibody to one of these antigens was not present in C3H immune sera until day 30. Sera from the two strains likewise differed in their ability to mediate eosinophil adherence to each parasite stage, and in some of the life cycle stages, these differences corresponded to the recognition of specific antigens found on the parasites' surface. The links between recognition of defined parasitic antigens and the fate of the parasite in genetically different strains of mice are discussed.

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.

These studies on eosinophils from beige mice have shown that such cells possess morphologically abnormal granules and that they also have an impaired capacity to interact in vitro with a non-phagocytosable target such as the infective larva of Trichinella spiralis. However, beige mice with these functionally abnormal eosinophils are able to control a T. spiralis infection as well as the normal heterozygote mice. A morphological study of beige eosinophils revealed the presence of structurally distinct lysosomal secretion granules. Some resembled granules in normal eosinophils while others were grossly enlarged and contained multiple crystalloids. When these peritoneal eosinophils were allowed to interact with T. spiralis in vitro in the presence of specific antibody and/or complement, they behaved differently. Cells containing only large granules adhered loosely and temporarily; they were not observed to degranulate. In contrast, cells containing a mixture of granules, or only small granules were able to adhere and degranulate. Despite the defects in the eosinophils of beige mice, the course of an infection with the nematode parasite T. spiralis in beige animals was similar to that in normal animals. Therefore, if eosinophils are crucial in the control of this infection as suggested from other studies, the defect in beige eosinophils is not sufficient to prevent an apparently normal response to the parasite.

Surface molecules of parasitic stages of the nematode Nippostrongylus brasiliensis can be readily iodinated by the chloramine T technique, and assessed for antigenic reactivity with humoral antibody from infected animals. Free-living infective larvae are less amenable to analysis by this, or similar methods, but within 18 hr of larvae entering the host, new macromolecular surface antigens can be detected. The parasites change their surface antigens twice more in the course of the maturation to the adult stage. Surface antigens are stage-specific: lung larvae (L3), intestinal larvae (L4) and gut-living adults each possess characteristic sets of cuticular molecules. Single stage infections result in antibody reactive only to the antigens from the homologous stage. The adult surface appears to bear the greatest number of antigens, one of which is found only on the male worm. The composition of these antigens does not differ grossly between adult worms from a naive or immune host, or worms established after the adaptation of a 'trickle' (multiple low dose) infection. There appears to be an interesting contrast between the rapidity and extent of changes in surface antigens in the early phases of infection, and the stability of adult antigens analysed at different points in the host immune response.

Proliferation of peripheral blood T lymphocytes from unsensitized donors in response to fractions of Trypanosoma brucei was observed to be monocyte-dependent. The activated T cells displayed enhanced 'helper' activity, but no 'suppressor' activity during pokeweed mitogen (PWM)-induced immunoglobulin (IgM and IgG) synthesis, and were resistant to the inhibitory effects of theophylline on E rosette formation. Whilst in vivo studies have failed to reveal excessive T cell proliferation in patients with sleeping sickness, these results suggest a possible role for T cells in the induction of hypergammaglobulinaemia characteristic of this disease. Trypanosome fractions were not inhibitory to PWM-induced proliferation, and actually enhanced immunoglobulin synthesis. Thus it is unlikely that direct inhibition by the parasite per se is a major factor in the generation of immunosuppression by the T. brucei subgroup.

A new technique is described for measuring antibodies to an enzyme which is not available in pure form. The secretions of the rat parasitic nematode, Nippostrongylus brasiliensis, were treated with tritiated diisopropylfluorophosphate. Only one component, an acetylcholinesterase, was radiolabelled. Antibodies to this enzyme in rat antisera were estimated by the Farr technique using the labelled enzyme as antigen. The acetylcholinesterase secreted by Necator americanus, the human hookworm, was similarly specifically labelled.

Surface antigens of three stages of three species of the filarial nematode genus Brugia have been analysed by radio-iodination and immunoprecipitation. These surface antigens have been shown to be characteristic for each stage by polyacrylamide gel electrophoresis. For example, infective larvae and adult worms have relatively complex patterns while microfilariae have few bands which are not found when other stages are radio-isotope labelled by the same technique. The surface antigens of Brugia malayi, B. timori and B. pahangi adult worms are all closely homologous, as are the surface antigens of infective larvae of the same three species, and of microfilariae of B. malayi and B. pahangi. Immunoprecipitation revealed that antibody raised in mice against one stage or species reacted with surface antigens from other stages and species. For example, sera raised against B. pahangi male adults reacted strongly with surface antigens from all three species. This cross-reactivity was dominant despite the apparent stage-specificity of the surface pattern seen on SDS-PAGE analysis. Moreover, in cross-immunization experiments, infective larvae were able to stimulate a secondary antibody response in mice previously primed with microfilarial surface antigens. The major microfilarial surface antigens (of mol. wt 65-70 000 Daltons) were recognized by serum antibody from microfilariae-, infective larvae- or adult-infected animals. Thus, although the dominant antigens from each stage are of different molecular weight, cross-reactions with stage-specific antisera suggest that there must be shared epitopes on Brugia surface antigens from each stage. Such shared antigenic determinants dominate the immune response, although other evidence, including the differences in molecular weight, indicates the existence of stage- and species-specific components.

The nature of complement binding to the surface to infective larvae of Trichinella spiralis and Nippostrongylus brasiliensis differs. When worms were incubated in serum from uninfected hosts, washed and incubated in fluorescent reagent the whole surface of T. spiralis fluoresced but in N. brasiliensis fluorescence was confined to the anterior end and some internal organs. The outer structure of the cuticle of the T. spiralis larvae was shown not to contain ATP-ase, thus differing from many cell membranes.

Most of the organisms of the phylum Nematoda are free living, but some are animal or plant parasites of major importance to man. During their life cycle all nematodes undergo a series of moults in which they shed an external cuticle, consisting of an outermost membrane-like layer of unknown composition and a series of fibrillar layers similar to collagens. Because of this structure, the cuticle has been viewed as an acellular exoskeleton with rather inert molecular components. However, observations have shown that it contains enzymes and sometimes haemoglobin, and that nutrients are absorbed through it in the infective larvae and adut stages of Brugia pahangi. It is bound by complement and antibody, resulting in the adherence of leukocytes, and antibody-dependent cell-mediated reactions damage the cuticle of newborn larvae of Trichinella spiralis and the microfilariae of Dipetalonema viteae and Litomosoides carinii. We report here that the surface of the cuticle of the parasitic nematode Trichinella spiralis expresses protein molecules which change qualitatively following the moulting process, and quantitatively during growth of the worms within one stage. Also, surface proteins are released in vitro at a rate which depends on the conditions of culture of the worms.