Pathology of Tnf-deficient mice infected with Plasmodium chabaudi adami 408XZ.

Tumor necrosis factor alpha (Tnf) plays a pleiotropic role in murine malaria. Some investigations have correlated Tnf with hypothermia, hyperlactatemia, hypoglycemia, and a suppression of the erythropoietic response, although others have not. In this study, we have evaluated parasitemia, survival rate and several pathological features in C57BL/6JTnf(-/-) and C57BL/6JTnf(+/+) mice after infection with Plasmodium chabaudi adami 408XZ. Compared to the C57BL/6JTnf(+/+) mice, C57BL/6JTnf(-/-) mice showed increased parasitemia and decreased survival rate, whereas blood glucose, blood lactate and body weight were not significantly different. However, C57BL/6JTnf(-/-) mice suffered significantly more from severe anemia and hypothermia than C57BL/6JTnf(+/+) mice. These results suggest that Tnf is an important mediator of parasite control, but not of anemia development. We hypothesize that the high mortality observed in the Tnf knock-out mice is due to increased anemia and pathology as a direct result of increased levels of parasitemia.

Immunisation of cattle with cysteine proteinases of Trypanosoma congolense: targetting the disease rather than the parasite.

In order to test the hypothesis that trypanosome cysteine proteinases (CPs) contribute to pathology of trypanosomosis, cattle were immunised with CP1 and/or CP2, the major CPs of Trypanosoma congolense, and subsequently challenged with T. congolense. Immunisation had no effect on the establishment of infection and the development of acute anaemia. However, immunised cattle, unlike control cattle, maintained or gained weight during infection. Their haematocrit and leukocyte counts showed a tendency to recovery after 2-3 months of infection. Cattle immunised with CP2 mounted early and prominent IgG responses to CPs and to the variable surface glycoprotein following challenge. Thus trypanosome CPs may play a role in anaemia and immunosuppression; conversely, anti-CP antibody may modulate the trypanosome-induced pathology.

Protective immune mechanisms against Theileria parva: evolution of vaccine development strategies.

Theileria parva is an intracellular sporozoan parasite that infects and transforms bovine lymphocytes, causing a severe lymphoproliferative disease known as East Coast fever in eastern, central and southern Africa. In this article, Declan McKeever and colleagues summarize the current understanding of immune mechanisms provoked by the parasite with regard to their role in both pathogenesis and protection. In particular, the influence of genomic polymorphism in parasite and host on the development of immunity is discussed, along with the evolution of current vaccine development strategies as a result of immunological research on the disease.

Cloning and characterization of a 150 kDa microsphere antigen of Theileria parva that is immunologically cross-reactive with the polymorphic immunodominant molecule (PIM).

To identify the genes encoding novel immunodominant antigens of Theileria parva a lambda gt11 library of piroplasm genomic DNA was immunoscreened with bovine recovery serum and a gene encoding a 150 kDa antigen (p150) was identified. The predicted polypeptide contains an N-terminal secretory signal sequence and a proline-rich region of repeated amino acid motifs. The repeat region is polymorphic between stocks of T. parva in both copy number and sequence, and analysis of the repeat region from 10 stocks of T. parva revealed 5 p150 variants. A monoclonal antibody (mAb) against the T. parva polymorphic immunodominant molecule (PIM) cross-reacted with the recombinant p150. The p150 has sequence homology with a PIM peptide sequence containing the anti-PIM mAb epitope. Immunoelectron microscopy demonstrated that the p150 antigen, like PIM, is located in the microspheres of the sporozoites and is exocytosed following sporozoite invasion of the host lymphocyte. By immunoelectron microscopy p150 was subsequently transiently detectable on the sporozoite surface and in the lymphocyte cytosol. Immunoblotting showed that p150 is also expressed by the schizont stage, but at much lower levels compared to the sporozoite. These results suggest a major role for p150 in the early events of host-sporozoite interaction.

Characterization of the gene encoding the polymorphic immunodominant molecule, a neutralizing antigen of Theileria parva.

Theileria parva, a tick-transmitted protozoan parasite related to Plasmodium spp., causes the disease East Coast fever, an acute and usually fatal lymphoproliferative disorder of cattle in Africa. Previous studies using sera from cattle that have survived infection identified a polymorphic immunodominant molecule (PIM) that is expressed by both the infective sporozoite stage of the parasite and the intracellular schizont. Here we show that mAb specific for the PIM Ag can inhibit sporozoite invasion of lymphocytes in vitro. A cDNA clone encoding the PIM Ag of the T. parva (Muguga) stock was obtained by using these mAb in a novel eukaryotic expression cloning system that allows isolation of cDNA encoding cytoplasmic or surface Ags. To establish the molecular basis of the polymorphism of PIM, the cDNA of the PIM Ag from a buffalo-derived T. parva stock was isolated and its sequence was compared with that of the cattle-derived Muguga PIM. The two cDNAs showed considerable identity in both the 5' and 3' regions, but there was substantial sequence divergence in the central regions. Several types of repeated sequences were identified in the variant regions. In the Muguga form of the molecule, there were five tandem repeats of the tetrapeptide, QPEP, that were shown, by transfection of a deleted version of the PIM gene, not to react with several anti-PIM mAbs. By isolating and sequencing the genomic version of the gene, we identified two small introns in the 3' region of the gene. Finally, we showed that polyclonal rat Abs against recombinant PIM neutralize sporozoite infectivity in vitro, suggesting that the PIM Ag should be evaluated for its capacity to immunize cattle against East Coast Fever.

Molecular characterization and immunogenicity of neutralization-sensitive Babesia bigemina merozoite surface proteins.

Monoclonal antibodies binding to the surface of live Mexico isolate Babesia bigemina merozoites have defined 4 parasite-encoded surface antigens (36, 45, 55, and 58 kDa) that are potential targets for immune-mediated neutralization of merozoites. In this study, we have characterized the post-translational modification, antigenic polymorphism, and immunogenicity of these 4 proteins. Monoclonal antibody immunoaffinity-purified 36- and 55-kDa polypeptides were identical in gel electrophoresis to immunoprecipitated radiolabeled proteins, while the purified 45-kDa protein consisted of 2 closely spaced polypeptides with relative molecular weights of 45 and 43 kDa. The 36-, 45-, and 55-kDa proteins were post-translationally modified by incorporation of [3H]glucosamine and [3H]myristic acid, suggesting they are integral membrane proteins secured by a phosphatidylinositol anchor. Cross-reactivity studies with monoclonal and monospecific polyclonal antibodies revealed marked antigenic polymorphism of these 3 glycoproteins among diverse geographic isolates. In contrast, none of the polypeptides bound by anti-p58 monoclonal antibody were glycosylated or myristilated. Both monoclonal and monospecific polyclonal antibodies recognizing p58 bound to similar molecular weight proteins in 4 additional isolates of B. bigemina from Mexico, Puerto Rico, St. Croix, and Kenya, suggesting widespread conservation of p58 immunogenic epitopes among geographic isolates. Calves immunized with immunoaffinity purified gp45, gp55, or p58 antigens were able to neutralize the infectivity of merozoites as indicated by significant reductions in the peak parasitemia after experimental challenge. Precise definition and appropriate presentation of neutralization sensitive epitopes on gp45, gp55, or p58 may enhance the merozoite neutralizing immune response in immunized cattle.

Identification of antigens for use in immunodiagnosis of Taenia saginata cysticercosis.

Fifteen metacestode antigens from Taenia saginata were defined by Laurell crossed immunoelectrophoresis and investigated for their potential use in immunodiagnosis of bovine cysticercosis. Several antigens cross reacted with those of some common cattle parasites. Three of the antigens, designated as numbers 4, 8 and 11, were selected on the basis of their restricted cross reactions and were isolated by affinity chromatography. These antigens showed high sensitivity and specificity values in the enzyme-linked immunosorbent assay (ELISA) for serodiagnosis of bovine cysticercosis.

A single exon codes for the enzyme domain of a protozoan cysteine protease.

Theileria parva is an intracellular protozoan parasite of cattle. We have determined the nucleotide sequence of a gene and cDNA coding for a cysteine protease of T. parva. The gene is divided into two exons. The first exon codes for a signal sequence and for part of the "pro" region of the zymogen. The second exon codes for the remainder of the pro region, including residues thought to be involved in zymogen processing, and for the entire enzyme domain. Part of this exon cross-hybridizes, at high stringency, with DNA isolated from Plasmodium falciparum. It is likely that the T. parva gene is functionally active. Parasite extracts contain hydrolytic activity against benzyloxycarbonyl-Phe-Arg-7-amido-4-methylcoumarin. This activity is optimal at pH 6.0 and is inhibited by the cysteine protease inhibitor L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane (E-64). This is, to our knowledge, the first description of a gene coding for a eukaryotic cysteine protease which lacks an intron in DNA coding for the enzyme domain.

Biologic activities of bovine IgG subclasses.

With regard to our studies on the functional properties of bovine IgG1 and IgG2 antibodies, the differences were minimal when homologous systems were evaluated. Both IgG1 and IgG2 fixed bovine complement by the classical pathway, caused homologous passive cutaneous anaphylaxis, caused phagocytosis of coated erythrocytes by cultured monocytes and precipitated with an antigen having multiple unique determinants (ovalbumin). In contrast to IgG2, IgG1 caused neither adherence nor phagocytosis by freshly isolated neutrophils and monocytes. Additionally, IgG2 antibodies to DNP precipitated with DNP19ovalbumin while IgG1 antibodies formed soluble complexes with this antigen.

The bovine lymphoid system. III. A monoclonal antibody specific for bovine cell surface and serum IgM.

Mouse spleen cells from animals immunized with bovine peripheral blood lymphocytes were fused to X63 . Ag8 myeloma cells and the activity of one of the resulting myeloma hybrids was characterized. The product of this clone (B5/4.1.4) binds to pentameric bovine IgM isolated from serum but not to serum IgG1 or IgG2. This reagent also binds to cell surface (monomeric) IgM and can be used in immunofluorescence assays to enumerate IgM-bearing cells in lymphoid cell suspensions and to examine B lymphocytes or B lymphocyte derived cells in tissue sections.

Activation of complement by hydatid cyst fluid of Echinococcus granulosus.

Factors present in hydatid cyst fluid of Echinococcus granulosus were found to interact with complement from several species. This nonimmunologic fixation resulted in the depletion of hemolytically active complement from fresh guinea pig serum, the conversion of human C3 to an electrophoretically faster species and the genration of smooth muscle contracting substances, analogous to anaphylatoxins, in normal rat serum in vitro. Vascular permeability changes were produced in vivo in rats and humans after intradermal inoculation of complement interacting fractions of hydatid fluid. It is suggested that these factors may contribute to the pathogenesis of the shock syndrome which follows intravenous administration of hydatid fluid in normal animals, and to the nonspecificity of immunodiagnostic skin tests for hydatid infection in man and animals.

Antibody-mediated secondary eosinophilic response to Taenia taeniaeformis in the rat.

Normal rats were given intravenous doses of either immune serum or immunoglobulin fractions 24 hr before oral challenge with 1,000 eggs of Taenia taeniaeformis. Total eosinophil counts per mm3 were performed for 3 days prior to and 6 days after challenge. Sensitized rats usually showed sharp peaks of eosinophilia 2 to 6 days after this dose. The pattern of the eosinophilic response was similar to that which occurs after challenge of immune infected rats. The differences in peripheral eosinophil levels in passively immunized and normal rats were statistically significant. Immunoglobulin fractions containing protective IgG2alpha were most effective, but a fraction containing reaginic antibody activity also sensitized rats to give secondary eosinophilic responses. The findings are discussed in relation to the probable contribution of antigen-antibody reactions to the production of secondary eosinophilic responses in experimental cysticercosis.