E.J.L. Soulsby

Antigenic analysis of developmental stages of Ascaris scum I. comparison of eggs, larvae and adults

Antigenic analysis of developmental stages of Ascaris suum I. Comparison of eggs, larvae and adults. Experimental Parasitology, 27, 150–162. Antisera, produced in rabbits, against extracts of adult Ascaris suum were used in gel-diffusion, immunoelectrophoresis, and passive hem agglutination tests to compare the antigenic composition of eggs, second stage larvae, and third stage larvae of this parasite. A considerable sharing of antigens between larval stages and the adult worm was observed as well as a progressive acquisition of antigens during development. Antisera absorbed with extracts of adults of Toxocara cams and Toxascaris leonina detected “genus-specific” antigens in third stage larvae and in the adults of A. suum, but not in eggs and second stage larvae. Antisera from rabbits which had been infected orally several times with eggs of A. suum demonstrated antigens in second and third stage larvae that were absent in adults. Those present in third stage larvae were antigenically and electrophoretically distinct from those in second stage larvae, and were successfully separated by ammonium sulfate fractionation. The significance of these findings is discussed in terms of immunodiagnosis of ascariasis and the origin of protection-inducing antigens during infection with A. suum.

Granuloma formation to Capillaria hepatica eggs I. Descriptive definition

Abstract The course of the cellular response in the liver to nonembryonated Capillaria hepatica (Bancroft, 1893) eggs given by intravenous injection into the portal circulation of unsensitized and sensitized mice was studied qualitatively and quantitatively. A gradual infiltration of predominantly mononuclear cells occurred around the eggs in the liver, leading to the formation of distinct granulomatous lesions characterized by macrophages and lymphocytes. This was followed by an infiltration of eosinophils. Previous intraperitoneal sensitization led to an earlier and an enhanced reaction to an intravenous challenge with eggs. Specificity of the cellular response was suggested by the lack of an enhanced reaction to presensitization with eggs of a closely related species, Trichuris muris . These studies indicate that granuloma formation to C. hepatica eggs has an immunological basis and furthermore the cell composition of the granuloma would suggest that a cell-mediated component may be involved as part of the specific response.

Lymphoid cell kinetics in guinea pigs infected with Trichostrongylus colubriformis: Tritiated thymidine uptake in gut and allied lymphoid tissue, humoral IgE and hemagglutinating antibody responses, delayed hypersensitivity reactions, and in vitro lymphocyte transformations during primary infections

Abstract The kinetics of the lymphocyte responses of Trichostrongylus colubriformis -infected and normal guinea pigs were measured by the in vivo uptake of tritiated thymidine either as dpm 3 H/mg tissue or as the percentage change in [ 3 H] -labeled lymphoblasts in autoradiographs of tissue impression smears and sections. The lymphoid response was predominantly a local one centering on the infected area of the small intestine. The greatest lymphocyte reactions as assessed by counts of labeled lymphoblasts occurred in the Peyers patches and mesenteric lymph nodes where the peak responses took place 11 and 6 days after infection, respectively. The local nature of the responses was exemplified by the fact that the mesenteric lymph nodes of the anterior small intestine showed a peak response on the sixth day but the response from the posterior small intestine peaked 7 days later. A similar but less dramatic relationship existed among the Peyers patches. In addition no labeled lymphoblast response was elicited in the inguinal lymph nodes or cecal lymphoid patches throughout the infection and the first increased responsiveness of the spleen did not take place until after Day 13, by which time the lymphoid proliferations associated with the infected intestine had subsided. Initially, the spleen showed a marked depletion of labeled blast cells during the first 7 days of the infection. This was taken as indicating at the time the infection was being established the export of cells capable of transformation in response to parasite antigen. This was supported by the observation that large numbers of phytohemagglutinin responsive lymphocytes were found in the peripheral circulation at this time. The in vitro responsiveness of peripheral lymphocytes to T. colubriformis antigen was also studied. Positive lymphocyte transformations first occurred 6 days after infection but thereafter declined to the normal level by Day 13; the peak transformation ratio was found 25 days after infection but by Day 38 it had declined to a low but persistently positive level. There was a correlation between the circulation of specifically sensitized cells, probably of thymic origin, IgE antibody titers, and the development of positive dermal delayed hypersensitivity reactions in infected guinea pigs, suggesting a close relationship among these three immunological phenomena. All lymphoblast responses in Peyers patches, mesenteric lymph nodes, and lamina propria of the intestine were completed before the immune elimination of the parasite commenced 10 days after infection. During the first 10 days of infection specifically sensitized lymphocytes appeared and disappeared from the circulation. The loss of circulating sensitized lymphocytes at the time immune elimination of the parasite was taking place in the gut suggested that the sensitized cells were “homing-in” on the local area of infection. After the immune elimination of the parasite had commenced, the level of sensitized lymphocytes and IgE antibodies then increased rapidly in the blood. Evidence from the kinetics of the hemagglutinating antibodies indicated that stage specific antigens occur in T. colubriformis .

Fate of Litomosoides carinii adults transplanted into the pleural or peritoneal cavity of infected and naive multimammate rats (Mastomys natalensis).

In each of 4 experiments, 58 multimammate rats (Mastomys natalensis) were subdivided into 5 groups. Thirty-four rats were infected with Litomosoides carinii and infections were allowed to become patent. Ten days after patency adult worms were surgically transferred from donor rats to each of 1 group of infected rats and 1 group of naive rats. Groups of infected and naive rats served as controls. Transfers were made either intrapleurally or intraperitoneally. Samples of blood and tissues were taken from each of 2 animals necropsied from each group at intervals to 31 days. At necropsy, the transferred worms and the original population (if any) were examined and samples were fixed. Infected rats accepted new worms with a minimum of reaction while naive rats rejected worms beginning at day 10 (intrapleural) of 17 (intraperitoneal). Rejections were complete leaving a white fibrous mass by day 24 or 31, respectively. Hemagglutination antibody titers fell into 2 groups: infected and naive. IHA titers of naive recipient rats increased into the infected range by day 3 (intrapleural) or day 10 (intraperitoneal). Microfilaria counts presented a more varied pattern but a similar lag in the intraperitoneal recipient group was observed. It was concluded that a preparation period is necessary for successful residence of adult worms, and that this preparation is not restricted to the pleural cavity.

Ascaris suum: Cuticular binding of the third component of complement by early larval stages

Abstract Binding of the third component of complement (C3) to infective or parasitic larvae of Ascaris suum was evaluated by the direct fluorescent antibody technique and by detection of radiolabeled-C3 binding. Ensheathed infective-stage larvae bound C3 as detected by both techniques. Fluorescence studies indicated C3 binding was over the entire surface of the infective-stage sheath. Day 1 parasitic larvae bound C3 spottily only over the head and tail regions. Day 2 parasitic larvae however bound C3 over the entire surface. C3 uptake as detected by fluorescence was inhibited in 56 C-heated serum, but not in 50 C-heated serum, when either normal or C4-deficient guinea pig serum (C4D) was used as the C3 source. Radiolabeled-C3 binding was enhanced in the presence of unheated C4D serum and inhibited by Ca 2+ chelation. C3 binding appeared dependent on neither classical nor alternative pathway activation of complement in this study.

Capillaria hepatica: Granuloma formation to eggs: II. Peripheral immunological responses

Abstract Peripheral immunological responses were assessed by indirect hemagglutination, passive cutaneous anaphylaxis, gel diffusion, and delayed dermal reactivity in mice with experimental primary and secondary Capillaria hepatica egg granulomas. Agglutinating and homocytotropic antibodies as well as delayed dermal reactivity, but not precipitating antibodies, were detected in animals with primary and secondary granulomas. The demonstration of circulating antibody during the course of granuloma formation indicates a possible role for antibody in the response and is cause for a reassessment of the etiology of experimental helminth egg granulomas.

Isolation and continuous cultivation of Trypanosoma theileri in media containing tissue culture fluids

Abstract Tissue culture fluids, supplemented with newborn calf serum and bovine blood or with broth, newborn calf serum and blood or blood products were, respectively, suitable for the isolation and continuous cultivation of Trypanosoma theileri at 37 °C. Culture of the parasite at 37 °C in large volumes was accomplished in a tissue culture fluid-broth-blood coagulum medium. The organism was unaffected by high concentrations of penicillin or dihydrostreptomycin but was susceptible to nystatin. Culture forms of T. theileri remained viable for at least 50 days at −70 °C. An infection rate of 88% was observed in 17 cattle examined.

Antigenic analysis of the developmental stages of Ascaris suum: II. Host components☆

Abstract Antisera against host serum and tissues were used in gel-diffusion and hemagglutination tests to detect host components in extracts of eggs, larvae, and adults of Ascaris suum . The perienteric fluid of adult A. suum reacted with antiserum to normal porcine serum, but no reactions were observed with extracts of the eggs or the larval stages. Extracts of third stage larvae from the lungs of rabbits reacted with antisera to rabbit serum proteins as did the supernatant fluid from in vitro cultures of these larvae. No reactions were observed between antisera to rabbit serum and extracts of eggs or second stage larvae, and antisera to rabbit liver and lung tissues did not react wth extracts of larvae derived from these sources. These findings are discussed in the light of recent reports on the sharing of antigenic determinants between host and parasite.

Litomosoides carinii: effect of splenectomy on the ability of naive Mastomys natalensis to accept transplanted adults.

Abstract Fifty-eight male rats Mastomys natalensis , were used in each of two experiments to study the effect of splenectomy on the rejection of transplnted nematodes, Litomosoides carinii . The rats were divided into five groups: splenectomized recipients (SR), normal recipients (NR), splenectomized (S), normal (N) and donor (D). Each group had 12 animals except for the D group which had 10. After patency of the D group (tank infected with Litomosoides carinii ), groups SR and S were splenectomized. Fourteen days later, (three to five L. Carinii derived from the D group) were surgically transplanted into the peritoneal cavity of the SR and NR groups. In the first experiment, weekly sacrifices were made starting at Day 3. In the second experiment, all groups were sacrificed at Day 32. Worms transplanted into the SR group were accepted while those transplanted into the NR group were rejected. Sequentially examined antibody titers after Day 3 fell into two groups, those that were recipients of transplants (SR and NR) and those that were not (NR and SR). After becoming positive on Day 3, the microfilaremia of the SR group rose by Day 31 while that of the NR group fell to near 0. It was concluded that the spleen is necessary for the rejection of transplanted L. carinii by naive M. natalensis .

Ascaris suum: immune response in the guinea pig. I. Lymphoid cell responses during primary infections.

Abstract The lymphoid cell responses of the mesenteric, hepatic, and mediastinal lymph nodes, of the spleen, and of the Peyers patches of normal guinea pigs that (a) were orally infected with 10,000 infective eggs of Ascaris suum (Group I), (b) received 10,000 artificially hatched second-stage larvae of A. suum via the mesenteric vein (Group II), or (c) received 1500 third-stage larvae of A. suum via the saphenous vein (Group III) were assessed by antigen-induced lymphocyte-transformation, rosette-formation, and rosette-plaquing techniques. Antigen-induced lymphocyte transformation measured qualitatively the antigen-sensitive cell population, rosette formation measured quantitatively the immunoglobulin-receptored cell population and rosette-plaquing measured quantitatively the receptored antibody-producing and the nonreceptored antibody-producing cell populations. Antigen-induced lymphocyte-transformation and rosette-formation studies showed that the immune response to A. suum was local in character at least for the first 11–12 days of infection as exemplified by the marked responses of the draining lymph nodes at the time the parasite was migrating through the tissues of the host. Rosette-plaquing studies in conjunction with the rosette-inhibition studies showed that the lymphoid cell responses of the draining lymph nodes progressed from an antigen-sensitive state to an immunoglobulin-receptored state, then to a receptored state capable of antibody production and finally to a nonreceptored antibody-producing state. In animals of Group I, the lymphoid cells of the mesenteric, hepatic, and mediastinal lymph nodes formed predominantly IgM and IgE antigen-specific rosettes (peak responses at Days 2, 5, and 7 for the mesenteric, hepatic, and mediastinal nodes, respectively), rosette-plaques (peak responses at Days 5, 7, and 9 for the mesenteric, hepatic, and mediastinal nodes, respectively), and plaques (peak responses at Days 7, 9, and 12 for the mesenteric, hepatic, and mediastinal nodes, respectively). The IgE-responsive cells were appreciably higher in the mediastinal lymph nodes than in the other lymphoid organs studied. The responses of the spleen were predominantly IgM in character and they increased gradually as the infection progressed without showing detectable peak responses. The lymphocytes of the Peyers patches did not exhibit antigen-specific rosetting, rosette-plaquing, or plaquing responses. In Group II animals (elimination of the bowel phase of the infection) only the hepatic and mediastinal nodes exhibited peak responses and these were similar to those of Group I animals in the type and pattern of the responses and in the time of their sequential appearance. In animals of Group III (elimination of both the bowel and the hepatic phase of the infection) only the mediastinal nodes exhibited peak responses and these were similar to those of Groups I and II in the type (predominantly IgM and IgE) and their sequential pattern but differed in the time of their appearance: Peak antigen-specific rosettes, rosetteplaques, and plaques occurred in the mediastinal nodes of Group III animals at Days 2, 5, and 7 after infection, respectively. In each of the responsive lymphoid organs studied, there occurred a certain population of lymphoid cells which was producing immunoglobulins that were not necessarily specific for the A. suum antigen.