Cercarial Challenge Research Articles

Localized skin changes at the site of immunization with highly irradiated cercariae of Schistosoma mansoni are associated with enhanced resistance to a challenge infection.

The level of immunity to a percutaneous cercarial challenge with Schistosoma mansoni was assayed 4-6 weeks after immunization of mice with highly irradiated (20 krad.) cercariae or schistosomula. When immunization and challenge occurred through the same skin site, resistance, particularly that which occurred in the skin, was greater than that observed when immunization and challenge occurred in different sites. The enhanced resistance is believed to be due to localized changes in the skin; 4 weeks after exposure to irradiated cercariae, abdominal skin is characterized by a thickened epidermis, changes in the ground substance and a cellular infiltration of the dermis. A convenient mouse model is described in which one or both ear pinnae are exposed to irradiated cercariae and a percutaneous challenge is given via the abdomen, thus eliminating the effects of local skin changes. In this model, the majority of the challenge infection which succumbs to the immune response appears to be killed in the skin.

Decreasing immunogenicity of developing schistosome larvae.

Cercariae, skin stage schistosomula, and lung stage schistosomula of Schistosoma mansoni were attenuated by gamma irradiation and tested for their ability to induce protective immunity against cercarial challenge in C57Bl/6J mice. The highest levels of resistance were induced by cercariae administered either percutaneously or intramuscularly. Skin stage schistosomula inoculated intramuscularly gave less protection while lung stage schistosomula from syngeneic donors were the least immunogenic. A similar ranking in immunogenicity was observed when the anti-skin stage schistosomular antibody responses induced by the different parasite stages were compared. In contrast, none of the immunization protocols were found to stimulate antibodies capable of recognizing lung stage schistosomula. These results suggest that, as schistosome larvae mature from the cercarial to the lung stage, they undergo a substantial loss in immunogenicity. This change may help explain the failure of older larvae to be immunologically destroyed in infected hosts.

Enzyme-linked immunosorbent assay with egg antigens of Schistosoma japonicum.

A micromethod of the enzyme-linked immunosorbent assay (ELISA) was applied to infections with Schistosoma japonicum in humans and mice with egg antigens. In mouse infections, antibody responses were detected between 6 and 8 weeks after the cercarial challenge. In human infections, a circum oval precipitin positive group showed the highest reaction values in ELISA. ELISA with egg antigens did not show any cross reaction with sera of humans infected with intestinal parasites (Ascaris, Trichuris and hookworms). Fractionation of the egg antigen was attempted using gel filtration, ion-exchange and lectin affinity chromatographies. Antigenic fractions were detected in two molecular weight groups of glycoprotein, which showed major pI value at 5-6. However, ELISA with partially purified antigen (JEA-1) did not give better sensitivity or specificity than the crude extract.

Amphotericin B-induced resistance to Schistosoma mansoni.

Amphotericin B (AmB) increases host resistance against several infectious agents and tumors. We evaluated its effect on infections with the helminth Schistosoma mansoni. A single dose of 500 microgram i.p. or 25 microgram i.v. increased resistance in mice to a challenge with 500 or 100 S. mansoni cercariae. Schistosomula recovery from the lungs, 5 days after challenge, was reduced 33 to 40%. Adult worm recovery 8 wk after infection was reduced 36 to 43%, AmB was effective in protecting mice when given from 9 days before until the day of cercarial challenge but was ineffective 2 wk before or at all intervals after S. mansoni infection. To examine the mechanism of this resistance, we obtained peritoneal exudate cells (PEC) from animals treated with AmB and assessed their ability to kill schistosomula, the migrating larval stage of the parasite. Killing by adherent PEC 1 or 2 days after AmB treatment was the same as that of resident macrophages from untreated animals. Adherent PEC from animals 3 to 7 days after AmB treatment showed enhanced killing of schistosomula (38.0 plus or minus 3.0% at day 4, which exceeded control of 8.1 plus or minus 4.0%, p less than 0.01). These PEC also displayed increased killing of Staphylococcus aureus. When AmB was added to cultures of resident peritoneal adherent cells from untreated mice at 0.5 microgram/ml to 2.0 microgram/ml (concentrations not toxic to the organism), schistosomula killing was increased 1- to 6- fold. These findings demonstrate activation of murine macrophages by AmB in vitro and in vivo, which results in greater killing of this multicellular parasite and potentially contributes to enhanced resistance to infection.

The response of mice immune to Schistosoma mansoni to a challenge infection which bypasses the skin: evidence for two mechanisms of immunity

Mice which had developed immunity to reinfection with Schistosoma mansoni following exposure to 20 cercariae and mice which had been immunized against S. mansoni by exposure to 400 highly irradiated (20 krad) cercariae, were tested for their ability to resist a percutaneous cercarial challenge and an intravenous challenge with 5-day-old lung-stage schistosomula derived from the same cercariae. Although both types of immune mice showed a marked resistance to a cercarial challenge, only the infected mice showed a comparable immunity to an intravenous challenge with lung schistosomula. These results confirm earlier studies which suggest that the major attrition of a cercarial challenge in infected mice occurs at the post-lung stage, whilst the attrition of a challenge infection in mice immunized with highly irradiated cercariae takes place in the skin. They provide further evidence for two separate mechanisms of immunity against S. mansoni in mice.

The Eosinophil in the Inflammatory Response to Cercarial Challenge of Sensitized and Chronically Infected CBA/J Mice *

Sensitized or chronically infected CBA/J mice were challenged percutaneously with cercariae of Schistosoma mansoni. Early inflammatory response was characterized by edema and neutrophil infiltration directed toward penetration tracks; chief pathology associated with schistosomula was localized disruption of epithelial cell relationships, except in areas of desmosomes. By 24 hours eosinophils were the prominent cells in the inflammatory reaction, with large numbers replacing neutrophils in penetration tracks. Extensive local eosinophilia occurred in areas of widespread epidermal destruction and collagen damage. Electron microscopy revealed degranulation of eosinophils in microabscesses and in areas of collagen damage and metabolically active fibroblasts. Eosinophils were rarely observed in contact with schistosomula. No marked difference could be observed between sensitized (but unprotected) mice and chronically infected mice in their response to cercarial challenge; a similar eosinophil response took place whether the mice were partially protected or totally unprotected. The data indicate that although eosinophils may be involved in this protection their presence alone is not sufficient to afford acquired resistance to murine schistosomiasis mansoni.

Resistance to cercarial challenge upon transfer of Schistosoma mansoni into mice.

Various numbers of 25- to 28-day-old schistosomes of both sexes were surgically transferred into the mesenteric veins of mice. Recipient animals (plus sham-operated controls) were percutaneously challenged with cercariae at various times after transfer, and the number of surviving worms was subsequently determined by portal perfusion. Significantly fewer challenge parasites were recovered from mice which had received a worm transfer than from sham controls; the reduction was comparable to that observed in mouse recipients of a percutaneous primary infection with cercariae. Resistance of mice to cercarial challenge was dependent upon the number of schistosome pairs transferred, and upon the time interval between transfer and challenge.

Schistosoma mansoni: Vaccination of mice with cryopreserved irradiated schistosomules

In our earlier experiments, NIH/Nmri (CV) mice developed protective immunity to a Schistosoma mansoni cercarial challenge when previously exposed percutaneously to highly 60Co-irradiated homologous cercariae. Experiments reported here were conducted to assess the immunogenicity of unfrozen and frozen and thawed schistosomules derived from 60Co-irradiated cercariae (irradiated schistosomules). Immunization of NIH/Nmri (CV) mice by 60Co-irradiated unfrozen schistosomules reduced worm burdens from a subsequent percutaneous challenge with normal cercariae by 41 to 72%. Immunogenicity was not narrowly dependent on irradiation dose rates between 1 and 8 kR/min, or on the total dose of irradiation given the schistosomules between 25 and 50 kR. Comparable protective immunity developed after injection of irradiated schistosomules which had been frozen to −196 C in liquid nitrogen and thawed. Cryopreservation appears to offer a solution to the problem of storage of attenuated, immunogenic S. mansoni schistosomules.

Adoptive Transfer of Protective Immunity in Experimental Schistosomiasis in the Mouse

Passive transfer of immune serum alone did not confer protection to recipient mice irrespective of the routes of serum transfer or cercarial challenge of Schistosoma mansoni. Mice that received both sensitized cells and immune serum were protected against challenge by subcutaneous injection of cercariae but not by percutaneous exposure. The immune serum could be transferred as late as 8 days after subcutaneous challenge, suggesting that the protection was afforded in part by a late parasite killing mechanism which functions after the schistosomula have migrated through the lungs.

Immunology of Schistosoma Mansoni Infection: Workshop Report III

The third annual informal Elvio H. Sadun Memorial Workshop on the “Immunology of Schistosomiasis” was held on 9 November 1977 in conjunction with the meeting of the American Society of Tropical Medicine and Hygiene in Denver, Colorado. The following report presents a brief synopsis of that meeting.The first area of discussion concerned methods and mechanisms of inducing protective responses against cercarial challenge. Dr. Shirley Maddison described her work with rhesus monkeys which received intramuscular injections of viable bacillus Calmette-Guerin Mycobacterium tuberculosis (BCG) organisms, in conjunction with passive transfer of immune monkey serum. When these monkeys were then exposed to 1,500 cercariae, strong protection was observed. Comparable effects were obtained by pretreatment with Corynebacterium parvum and whole worm extract (1 m KCl:pH 3.0). These regimens induced approximately a 35% reduction in worm burdens upon challenge.

Effect of transferred adult Schistosoma mansoni on resistance of mice to cercarial challenge.

Four or eight adult Schistosoma mansoni surgically transfered to the mesenteric veins of mice survive well and may play a role in resistance to later cercarial challenge. After receiving transfers of single sex or paired flukes, animals exposed to 50or 100 cercariae showed decreased numbers of parasites derived from the challenge infection when compared with control animals 30 days later. Although statistical analysis of the data indicated that the presence of transferred S. mansoni probably had an adverse effect on the survival of worms in a subsequent challenge, the results were interpreted as inconclusive.

The Role of Eosinophils in Immunity to Parasites: An Introduction *

Although the association of eosinophils with many helminthic infections is not new, their role as bona fide effector cells in the immune attack on the parasite is. New evidence comes from studies discussed earlier in which it was shown, in vitro, that human eosinophils will mediate antibody-dependent damage to schistosomula of Schistosoma mansoni; the evidence was supported by in vivo studies which demonstrated that depletion of eosinophils by anti-eosinophil serum would abolish established immunity to cercarial challenge in mice. Other studies have shown that eosinophils are intimately involved in the inflammatory reactions around schistosomula at the site of cercarial challenge in an immune animal and also around schistosomula in the lung when these organisms have been injected intravenously into immunized mice. In addition to their active role against the larvae, eosinophils have been shown recently to destroy Schistosoma mansoni eggs in vitro, and are found in abundance in granulomas induced by eggs from Schistosoma mansoni.

Serum from CBA/Ca mice vaccinated with irradiated cercariae of Schistosoma mansoni protects naive recipients through the recruitment of cutaneous effector cells.

Passive transfer experiments showed that 76% of the resistance induced in CBA/Ca mice by exposure to radiation-attenuated cercariae of Schistosoma mansoni could be transferred to naive recipients by administration of donor serum. The level of protection achieved depended on the volume of serum administered and immunity was demonstrated most consistently with serum harvested from thrice vaccinated donors. The serum was twice as effective when given to the recipients at the time of cercarial challenge as compared to administration 5 days after challenge, a result which indicates that serum-dependent challenge elimination is probably accomplished in the cutaneous tissues. This view was confirmed by the observation that passive protection could be ablated by administration of a monoclonal antibody which we have shown elsewhere to deplete the cutaneous inflammatory reaction to cercarial penetration. Histopathological studies revealed that vaccine serum induced subdermal inflammatory reactions in challenged recipient mice which were identical both in induration and kinetics to those seen in conventionally vaccinated individuals; on days 4 and 5 post-challenge, the reactions comprised 60% mononuclear cells and 40% eosinophils. Challenge larvae, which had transformed from skin-stage to lung-stage parasites, became trapped within such reactions and eventually showed generalized vacuolation consistent with the onset of damage. Some foci were seen to contain degenerated leucocytes, free eosinophil granules and debris which is thought to represent the remnants of dead parasites. Small focal reactions were identified on occasion in naive challenged mice and in recipients of normal mouse serum, but these reactions comprised predominantly mononuclear cells and were rarely seen to encompass challenge parasites. These data show that serum-transferred resistance in the vaccinated CBA/Ca mouse model involves the induction of a cutaneous inflammatory response in the recipients.

In vitro detection of cytotoxic antibodies to Schistosoma mansoni schistosomules.

A simplified in vitro technique was utilized to detect cytotoxic antibodies in serum from hosts exposed to cercariae of Schistosoma mansoni. Significant destruction of cultured schistosomules occurred in the presence of immune rat, rabbit, mouse, and monkey serum. Immune sera induced heavy precipitate formation around the schistosomules. The cytotoxic activity resided in the gamma globulin fraction of immune serum and was shown to be almost wholly complement dependent. Immune serum adversely affected the in vivo viability of schistosomules before detection by the dye exclusion technique was possible. Rabbit gamma globulin that exhibited strong in vitro cytotoxicity failed to protect mice against a challenge infection. Serum from rabbits immunized with adult worm culture antigen did not possess significant in vitro cytotoxicity, although this same antigen effectively stimulated resistance in mice to cercarial challenge. These results, coupled with the findings of other investigators, make it difficult to equate the cytotoxic titer of a host's serum with its immune status.