Babesia bovis: successful vaccination against homologous challenge in splenectomised calves using a fraction of haemagglutinating antigen

A soluble extract from lysed bovine erythrocytes infected with Babesia bovis, was separated by preparative agarose electrophoresis. A fraction with a mobility of plasma β globulins was shown to contain babesial antigen confined mainly to the infected erythrocyte and was used to vaccinate a group of four calves. Following challenge with homologous B. bovis all four calves vaccinated with the antigen survived the infection whereas all the calves in a control group of four died from infection.     

ABO Blood Groups Influence Macrophage-mediated Phagocytosis of Plasmodium falciparum-infected Erythrocytes

Erythrocyte polymorphisms associated with a survival advantage to Plasmodium falciparum infection have undergone positive selection. There is a predominance of blood group O in malaria-endemic regions, and several lines of evidence suggest that ABO blood groups may influence the outcome of P. falciparum infection. Based on the hypothesis that enhanced innate clearance of infected polymorphic erythrocytes is associated with protection from severe malaria, we investigated whether P. falciparum-infected O erythrocytes are more efficiently cleared by macrophages than infected A and B erythrocytes. We show that human macrophages in vitro and mouse monocytes in vivo phagocytose P. falciparum-infected O erythrocytes more avidly than infected A and B erythrocytes and that uptake is associated with increased hemichrome deposition and high molecular weight band 3 aggregates in infected O erythrocytes. Using infected A₁, A₂, and O erythrocytes, we demonstrate an inverse association of phagocytic capacity with the amount of A antigen on the surface of infected erythrocytes. Finally, we report that enzymatic conversion of B erythrocytes to type as O before infection significantly enhances their uptake by macrophages to observed level comparable to that with infected O wild-type erythrocytes. These data provide the first evidence that ABO blood group antigens influence macrophage clearance of P. falciparum-infected erythrocytes and suggest an additional mechanism by which blood group O may confer resistance to severe malaria.     

Babesia argentina: intraerythrocytic location of babesial antigen extracted from parasite suspensions

Goodger B.V. 1973. Babesia argentina: Intraerythrocytic location of babesial antigen extracted from parasite suspensions. International Journal for Parasitology3:387–391. A crude soluble antigen obtained from a mixture of Babesia argentina parasites and infected erythrocyte stromata has been partly purified and characterized by immunological procedures and its intraerythrocytic site demonstrated by fluorescent antibody techniques. This product contained at least two distinct antigens which were species specific. One had an electrophoretic mobility. similar to serum prealbumin, had little HA activity, and was found in or on the internal rim of the erythrocytic membrane. The other had an electrophoretic mobility similar to serum β₁ globulins, was highly active in HA tests, and was found in granules on the internal stromata of the infected erythrocytes. The evidence suggests that both antigens are produced either from the parasite and are associated closely with erythrocytic components or are produced by digestion of erythrocytic components and represent metabolites of the parasite. In either case the antigenic compounds detected are specific to B. argentina.     

Plasmodium lophurae: Antibody-induced movement and capping of surface membranes of erythrocyte-free malarial parasites

Surface antigens of the avian malarial parasite, Plasmodium lophurae, and its host cell, the duckling erythrocyte, were visualized at the ultrastructural level using rabbit antisera and ferritin-labeled goat anti-rabbit IgG. Rabbit antisera to P. lophurae caused an aggregation of parasite and parasitophorous vacuole surface membrane antigens, a phenomenon known as capping. Capping required living plasmodia and did not occur if parasites had been fixed with glutaraldehyde prior to exposure to antisera. Antisera against duckling erythrocytes did not cross-react with erythrocyte-free malarial parasites, and did not form caps on the surface of the red blood cell. Antiplasmodial sera did not react with normal or malaria-infected red cells. These results suggest that surface membrane proteins of the intracellular plasmodium are capable of lateral movement.     

Proteinases in the lysate of bovine erythrocytes infected with Babesia bovis: initial vaccination studies

The lysate of erythrocytes infected with Babesia bovis was tested for proteinases using an electrophoretic method in which substrate was included in the acrylamide matrix. Two babesial proteinases, which seemingly exist in both free and complexed forms, were detected. One of the proteinases was prepared by chromatography and preparative electrophoresis and used to vaccinate four splenectomized calves. The latter, along with a group of control splenectromized calves, were challenged with a strain of B. Bovis from which the proteinase was obtained. All the control calves died whereas only one of the vaccinates died. The protection was evident as a suppression of parasitaemia.     

Bovine babesiosis: Partial purification and characterization of blood culture-derived Babesia bovis

Morphologic, biologic and immunologic properties of corpuscular and soluble fractions of Babesia bovis purified from in vitro blood cultures were studied. Supernatant fluids obtained during routine medium exchange were studied. Supernatant fluids obtained during routine medium exchange were submitted to differential centrifugation to separate soluble and corpuscular babesial antigens from erythrocyte stroma. Extracellular babesiae were sedimented with infected and uninfected erythrocyte ghosts. The majority of babesiae were found in erythrocyte ghosts. Clumps of extracellular parasites were sometimes formed in vitro and generally could not be separated from uninfected erythrocytes. Centrifugation over a discontinuous Ficoll density gradient did not improve separations. Parasites remained viable throughout the purification procedure but were killed by freezing and rapid thawing. Both corpuscular and soluble antigen fractions elicited the production of specific anti-babesial antibodies when injected into calves. Electron microscopy of corpuscular antigen revealed the presence of intra- and extraerythrocytic babesial merozoites. A surface coat was visible loosely adhering to the plasma membrane of the parasites. Parasite suspensions and cell-free supernatant fluids obtained from in vitro cultures of B. bovis should provide a variety of unique antigens for further in vitro and in vivo studies.     

Fowl cholera: Cross-protective turkey antisera and IgG antibodies induced with Pasteurella multocida-infected tissue bacterins

Turkey antisera induced with formolized Pasteurella multocida-infected tissues (T antisera) passively cross-immunized 48 of 55 chickens against a challenge dose of P. multocida organisms, from which 0 of 15 controls survived. However, turkey antisera induced with formalin-killed, agar-cultured P. multocida cells (A antisera) passively cross-immunized only 4 of 30 chickens. Cross-immunity refers to protection against a different immunologic type of P. multocida. Quantitative precipitin reactions of the A and T antisera with antigens from agar-cultured cells showed that more antibody was present in the A than in the T antisera. However, antigens extracted from the infected tissues reacted with the T and not with the A antisera in the Ouchterlony procedure, demonstrating qualitative differences between the agar-cultured antigens and those extracted from the infected tissue. The gel precipitins isolated from the A and T antisera were characterized as 7S immunoglobulins, which behaved in immunoelectrophoresis as would be expected for a IgG immunoglobulin. The IgG fraction from the T antiserum passively cross-immunized chickens almost as well as the whole antiserum; hence, the IgG antibody is a major factor in cross-immunity.     

Babesia bovis: vaccination trial with a dominant immunodiffusion antigen in splenectomised calves

The dominant immunodiffusion antigen of Babesia bovis was prepared from the lysate of infected erythrocytes by cation exchange chromatography, gel filtration and preparative native acrylamide electrophoresis. It was seemingly free of other babesial antigens and tested as a vaccine. In vaccinated calves, compared to controls, there was a delay in parasitaemia and at times a statistically significant difference in parasite numbers. However, the vaccinates showed little difference in pathophysiological parameters or survival rates from the controls. It was concluded that serodominance cannot necessarily be correlated with protection.     

Trypanosoma cruzi: Effect on B-cell-responsive and -responding clones

During the course of Trypanosoma cruzi infection in C57BL/6 mice, which are relatively resistant to the parasite, the hosts developed antibody activity against previously unencountered antigens. The anti-sheep erythrocyte and antitrinitrophenyl antibody levels increased rapidly from Day 7 of infection, reached a peak by the 21st day, and were maintained at this level through 120 days postinfection in these mice. In contrast, highly susceptible C3H(He) mice did not have demonstrable antibody responses to SRBC or TNP during the 24-day infection period. Autoantibody activity against the selfantigens presented on isologous erythrocytes or thymocytes, however, were reduced in infected C57BL/6 mice. No significant reduction in autoreactivity to the self-antigens on erythrocytes or thymocytes was observed in C3H(He) mice infected with T. cruzi although a trend of reduced autoresponsiveness toward erythrocytes appeared to be developing by the time of death. C57BL/6 mice immunized with sheep erythrocytes as neonates and infected with T. cruzi as adults, or adult mice primed with low doses of sheep erythrocytes prior to infection, had elevated antibody responses to sheep erythrocytes unless the mice were immunized with sheep erythrocytes during the course of infection, in which case suppression of the response against sheep erythrocytes resulted. The nonspecific synthesis of immunoglobulins in infected C57BL/6 mice was, in part, a result of the lymphocyteactivating properties of T. cruzi-associated antigens. The T. cruzi-associated antigens induced proliferative and differentiative responses in spleen cells in vitro. It is proposed that the T. cruzi-associated antigens differentially affect lymphocytes capable of responding to antigen and those lymphocytes previously stimulated by antigen.     

Plasmodium falciparum antigens on the surface of the gametocyte-infected erythrocyte

Background

The asexual blood stages of the human malaria parasite Plasmodium falciparum produce highly immunogenic polymorphic antigens that are expressed on the surface of the host cell. In contrast, few studies have examined the surface of the gametocyte-infected erythrocyte.

Methodology/Principal Findings

We used flow cytometry to detect antibodies recognising the surface of live cultured erythrocytes infected with gametocytes of P. falciparum strain 3D7 in the plasma of 200 Gambian children. The majority of children had been identified as carrying gametocytes after treatment for malaria, and each donated blood for mosquito-feeding experiments. None of the plasma recognised the surface of erythrocytes infected with developmental stages of gametocytes (I–IV), but 66 of 194 (34.0%) plasma contained IgG that recognised the surface of erythrocytes infected with mature (stage V) gametocytes. Thirty-four (17.0%) of 200 plasma tested recognised erythrocytes infected with trophozoites and schizonts, but there was no association with recognition of the surface of gametocyte-infected erythrocytes (odds ratio 1.08, 95% C.I. 0.434–2.57; P = 0.851). Plasma antibodies with the ability to recognise gametocyte surface antigens (GSA) were associated with the presence of antibodies that recognise the gamete antigen Pfs 230, but not Pfs48/45. Antibodies recognising GSA were associated with donors having lower gametocyte densities 4 weeks after antimalarial treatment.

Conclusions/Significance

We provide evidence that GSA are distinct from antigens detected on the surface of asexual 3D7 parasites. Our findings suggest a novel strategy for the development of transmission-blocking vaccines.     

Contribution of glycolipids to species-specific antigens on erythrocytes of several animal species as to recognition of antigens with rabbit anti-glycolipids and anti-erythrocyte antisera

Because anti-glycolipid antibodies are involved in the onset of several neurological diseases, the reactivity of glycolipids on erythrocytes and the probability of generating the antibodies were determined to clarify the contribution of glycolipids as antigens. Anti-erythrocyte antisera reacted with the following glycolipids in a species-specific manner, i.e. blood group A-active glycolipid for man, Forssman glycolipid for sheep, Gg₃Cer for guinea pig, and Gg₄Cer and fucosyl GM1 for rat, and the hemolytic activities of the anti-erythrocyte antisera were attenuated by absorption of the antisera with liposomes prepared from the lipids of erythrocytes to the following levels, 94.5% for man, 24.5% for sheep, 17.5% for guinea pig, and 54.5% for rat. These species-specific glycolipids on erythrocytes reacted well with the respective anti-glycolipid antisera, but Gb₄Cer in man and GM1 in rat were shown to be cryptic on immunization with erythrocytes, indicating that the contribution of glycolipids as erythrocyte antigens differs among animal species. The glycolipid nomenclature is based on the recommendations of the IUPAC-IUB Commission on Biochemical Nomenclature [1]. The ganglioside nomenclature of Svennerholm is employed throughout [2].     

Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi.

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals.Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi.Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection.Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.     

Serological relationships among antigens of the BoLA and the bovine M blood group systems

Alloimmunizations with either lymphocytes or red cells from donor cows positive for BoLA w16 and blood group M' antigens into recipients negative for these antigens produced antisera reactive in the cytotoxic test with w16-positive lymphocytes and in the haemolytic test with M'-positive erythrocytes. Similarly, alloimmunizations of blood group M1-negative recipients with either lymphocytes or red cells from donor cows possessing the M1 blood group factor produced antisera specifically reactive with lymphocytes and erythrocytes from M1-positive cattle. Absorptions with either lymphocytes or erythrocytes from individual animals of the same M antigenic type as the donor removed all haemolytic and cytotoxic reactivity. The results indicate that blood group M' and BoLA w16 share a similar antigenic structure. Likewise, blood group M1 has an antigenically similar counterpart which is also part of the BoLA system.     

Induction of protective immunity against Chlamydia muridarum intravaginal infection with the chlamydial immunodominant antigen macrophage infectivity potentiator

We previously reported that 5 Chlamydia muridarum antigens reacted with antisera from >90% mice urogenitally infected with C. muridarum and they are TC0660 (ABC transporter or ArtJ), TC0727 (outer membrane complex protein B or OmcB), TC0828 (macrophage infectivity potentiator or MIP), TC0726 (inclusion membrane protein or Inc) & TC0268 (hypothetical protein or HP). The orthologs of these antigens in Chlamydia trachomatis were also highly reactive with antisera from women urogenitally infected with C. trachomatis. In the current study, we evaluated these C. muridarum antigens for their ability to induce protection against a C. muridarum intravaginal challenge infection in mice. We found that only MIP induced the most pronounced protection against C. muridarum infection. The protection correlated well with robust C. muridarum MIP-specific antibody and Th1-dominant T cell responses. The MIP-immunized mice displayed significantly reduced live organism shedding from the lower genital tract and highly attenuated inflammatory pathologies in the upper genital tissues. These results demonstrate that MIP, an immunodominant antigen identified by both human and mouse antisera, may be considered a component of a multi-subunit chlamydial vaccine for inducing protective immunity.     

Serological and immunological studies with a hexane extract of Babesia bovis-infected erythrocytes.

Antigenic and immunogenic activities of a hexane extract from Babesia bovis-infected erythrocytes were investigated. Positive ELISA and IFAT reactions were obtained with bovine antisera to B. bovis and B. bigemina produced by natural infection and rabbit antisera to the hexane extract, respectively. In contrast, negative ELISA reactions were obtained with Anaplasma marginale antisera indicating that the antigen(s) is specific for the genus Babesia. The IFAT clearly demonstrated that the antigen was associated with the parasite and the infected erythrocyte and not present in uninfected erythrocytes. Furthermore, cross-reactions with Babesia bigemina antisera suggested that serological cross-reactivity in bovine Babesia species is at least due in part to lipid or lipid-associated antigens.     

Isolation of Fasciola hepatica genus-specific antigens

Santiago de Weil N., Hillyer G. V. and Pacheco E. 1984. Isolation of Fasciola hepatica genus-specific antigens. International Journal for Parasitology14: 197–206. The Fasciola hepatica antigens which induce antibody formation in acute fascioliasis were isolated by acid elution after reacting an F. hepatica tegument antigen extract with a CNBr-Sepharose 4B column coupled with IgG obtained from the serum of rabbits infected with fascioliasis for 6–10 weeks. These isolated antigens were further separated by gel filtration using a column packed with Sephacryl S-200. In this manner three major peaks were obtained. The best serologic antigens were found in peak 2 which had a mol. wt range of 14,000–43,000. This peak contains genus-specific F. hepatica antigens which are highly reactive with fascioliasis serum. These antigens do not cross-react with either Schistosoma mansoni or with bovine serum albumin by gel diffusion. Monitoring by ELISA and gel diffusion with heterologous and homologous antisera showed that as purification by antibody affinity chromatography proceeded, cross reactivity with S. mansoni was eliminated. The rabbit antiserum obtained against peak 2, when tested by immunoelectrophoresis with a crude F. hepatica extract shows one main band identical to the main band observed with serum from acutely infected rabbits. Up to two other minor bands can be detected using concentrated homologous antisera. Fractions obtained from preparative iso-electric focusing of the F. hepatica tegument extract were reacted with sera from rabbits with acute fascioliasis. Two main bands were observed in immunodiffusion with antigens eluting in a pH range of 7.4–8.7. When these fractions were monitored with anti peak 2 antisera, two precipitin bands appeared with antigens eluting in a pH range of 7.4–7.9. The F. hepatica genus-specific antigen pool was applied to ELISA to evaluate its ability to detect antibody in a primary F. hepatica infection in rabbits. A rise in absorbance values could be detected by 2 weeks of infection, reached high levels by 6 weeks and remained high through 28 weeks of infection.     

The specificity and significance of the inhibition of Fc receptor binding by anti H-2 sera

The possibility that Ia antigens are unique among H-2 antigens in their relationship to the Fc receptor was investigated in an EA rosette assay. Antibody specific for antigens in various regions of theH-2 complex was incubated with mouse cells, and the ability of the cells to form rosettes with antibody-coated chicken erythrocytes was tested. Antibody raised against the H-2 antigens of Ia-negative tumor cells was highly effective in inhibiting rosette formation. A variety of antisera againstK-, I-, andD-region antigens tested in recombinant mice inhibited EA rosette formation, suggesting that antigens in each of these regions could be detected in rosette inhibition. The F(ab′)₂ fragments of all antisera tested also produced specific EA rosette inhibition. Finally, antibody against Ia antigens failed to inhibit bone marrow RFCs, although antibody against H-2K and H-2D antigens did inhibit. Although H-2 serology is in a state of rapid change at present, it must be concluded that in this assay, antibody against antigens in theK andD regions as well as theI region can inhibit EA rosette formation. Inhibition of these rosettes by anti H-2 sera is therefore not due to a special association of Ia antigens with Fc receptors.     

Serological and vaccination studies with bloodstream and culture forms of Trypanosoma musculi

Khazindar S. H. and Dusanic D. G. 1982. Serological and vaccination studies with blood-stream and culture forms for Trypanosoma musculi. International Journal for Parasitology12: 257–264. Trypanosoma musculi bloodstream forms (BSF) were collected from immunosuppressed infected mice and extracted with phosphate buffered saline (PBS). ML-15, O'Daly's and LMC media, each containing 5% fetal calf serum (FCS) and a dialysate medium were investigated to identify the medium providing the optimal growth of T. musculi culture forms (CF). Because of the ease of preparation, ML-15 containing 5% FCS was selected and the culture forms were harvested when the parasites attained concentrations of at least 1 × 10⁷ trypanosomes/ml. Cellular antigens present in PBS extracts of the BSF and CF parasites were analyzed with rabbit antisera by crossed immunoelectrophoresis and tandem crossed immuno-electrophoresis. Absorptions of rabbit antisera with CF, BSF, media and normal mouse blood extracts were performed on immunoabsorbent affinity columns prior to crossed immunoelectrophoresis to further study the unique and shared antigens of the parasites. A minimum of 13 antigens were shared by these trypanosomes. Four antigens appeared to be unique to BSF and a single antigen to CF. In immunization studies, two groups of C3H/Anf mice were immunized with the equivalent of 1 × 10⁸ frozen-thawed BSF or CF/injection. Two groups of five animals injected with PBS or uninoculated medium and one untreated group served as controls. Animals in each group received 6 injections administered at 3-day intervals. Three days following the last injection, all animals were challenged with 1 × 10⁴ BSF. Hemacytometer counts were performed every 4 days until no parasites were seen in wet blood preparations of the untreated group. None of the animals inoculated with BSF homogenate displayed parasitemias, while animals inoculated with CF homogenate were found to be infected. Parasitemias in mice immunized with CF were lower than those of the control mice.     

Polymorphism of erythrocyte antigens of spotted reindeer in Primorski Krai and prospects for its use in selection

First attempt to study polymorphism of erythrocyte antigens of spotted reindeer Cervus (Cervus) nippon Temminck, 1838 blood was made. With the aid of a set of monospecific antisera against blood groups of cattle and pigs, natural antisera of spotted reindeer, iso- and heteroimmune antisera against factors of erythrocytes of spotted reindeer, antigenic picture of blood in cattle of five Primorsk region farms was revealed. Indeces of genetic similarity between populations studied have been calculated and the scheme of their relationships presented.     

Production and properties of reaginic antibodies in rabbits infected with Clonorchis sinensis or Schistosoma japonicum

The production of reaginic antibodies detected by homologous passive cutaneous anaphylaxis (PCA) was demonstrated in all rabbits experimentally infected with either Clonorchis sinensis or Schistosoma japonicum. The antibodies appeared in the sera as early as 3 weeks after exposure and persisted with relatively high titers for at least 7 weeks in some animals. The antisera of rabbits infected with C. sinensis were found to be cross reactive against heterologous trematode antigens, although PCA titers were less than 3% of the titer by the homologous antigen; no cross reaction was observed between S. japonicum antiserum and the heterologous antigens. PCA activity of the antisera was completely destroyed in some samples by heat treatment at 56 C for 2 hr, but partially in the others even after heating for 6 hr. However, the physicochemical properties of these antibodies were analogous to human IgE; the PCA activity was eluted with 0.035 M phosphate buffer from a DEAE-cellulose column and recovered in the ascending portion of the IgG peak by Sephadex G-200 gel filtration. PCA activity was found in a β region in preparative agar electrophoresis.