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.

An enzyme-linked immunosorbent assay for detection of Theileria parva antibodies in cattle using a recombinant polymorphic immunodominant molecule.

Field and experimental bovine infection sera were used in immunoblots of sporozoite and schizont lysates of Theileria parva to identify candidate diagnostic antigens. Four parasite antigens of Mr 67,000 (p67), 85,000 (the polymorphic immunodominant molecule, PIM), 104,000 (p104), and 150,000 (p150) were selected for a more detailed analysis. The p67 and p104 antigens were present only in the sporozoite lysates, whereas PIM and p150 were found in both sporozoite and schizont lysates. The four antigens were expressed as recombinant fusion proteins and were compared with each other in an enzyme-linked immunosorbent assay (ELISA) and in the whole-schizont-based indirect fluorescent antibody test (IFAT) in terms of their ability to detect antibodies in sera of experimentally infected cattle. The PIM-based ELISA provided a higher degree of sensitivity and specificity than did the ELISA using the other three recombinant antigens or the IFAT. Further evaluation of the PIM-ELISA using experimental sera derived from cattle infected with different hemoparasites and field sera from endemic and nonendemic T. parva areas showed that the assay had a sensitivity of > 99% and a specificity of between 94% and 98%.

Humoral immune responses to Theileria parva in cattle as measured by two-dimensional western blotting.

Humoral immune responses to schizont antigens from six stocks of Theileria parva were compared by two-dimensional Western blotting using sera from cattle that had been infected with a T. parva stock or a clone. Isoelectric points of a polymorphic immunodominant molecule (PIM) of schizonts that induces strong antibody responses in cattle ranged from acidic to basic. Molecular masses (Mr) of the PIM of the respective T. parva stocks were as follows: T. parva Muguga, 86 kDa; Mariakani, 83 kDa; Marikebuni, 83 kDa; Uganda, 83 kDa; T. parva Boleni, 83 kDa; and T. parva 7014, 100 kDa. Among nine cattle infected with T. parva Muguga, four produced antibodies to a basic antigen having an Mr of 32 kDa. The PIM of T. parva Muguga, T. parva Boleni, and T. parva 7014 reacted strongly with serum obtained from an animal that had been infected with T. parva Muguga. Two-dimensional Western blotting using antischizont monoclonal antibodies enabled us to differentiate between stocks of T. parva.

A recombinant sporozoite surface antigen of Theileria parva induces protection in cattle.

At present immunization against Theileria parva is by infection with live sporozoites and simultaneous treatment with a long-acting oxytetracycline. This method has major limitations in that live organisms are used and the immunity engendered is parasite stock specific. In an attempt to develop an alternative immunization procedure, the gene encoding p67, a major surface antigen of sporozoites, has been expressed by using the plasmid expression vector pMG1. The gene, which has been characterized previously, encodes 709 amino acid residues, contains a single intron of 29 base pairs, and is only transcribed during sporogony. The recombinant p67 sequences were fused to the first 85 amino acid residues derived from a nonstructural gene (NS1) of influenza virus A. Immunization with a partially purified recombinant antigen emulsified in 3% saponin induced sporozoite neutralizing antibodies in cattle and provided protection in six of nine animals on homologous challenge with T. parva sporozoites. This recombinant antigen is therefore a candidate for development of a vaccine against T. parva.

Identification of a Theileria mutans-specific antigen for use in an antibody and antigen detection ELISA.

Purified piroplasms of Theileria mutans were used to immunize BALB/c mice to generate monoclonal antibodies (MoAbs). The MoAbs recognized an antigen of a relative molecular mass of 32 kDa in Western blots. This antigen was also recognized by sera from cattle which had recovered naturally from experimental tick-transmission or infections induced by the blood stages of T. mutans. The MoAbs did not react, in indirect immunofluorescence or enzyme-linked immunosorbent assays (ELISA), with the common haemoparasites of cattle, namely, T. parva, T. annulata, Babesia bigemina, B. bovis, Anaplasma marginale, Trypanosoma congolense, T. vivax or T. brucei. An antigen capture ELISA was established with two of the MoAbs which recognized different epitopes on the 32 kDa molecule. Using this test it was possible to detect circulating antigens or immune complexes in sera collected from cattle during the acute or chronic phases of infection. When the purified 32 kDa protein was used as antigen in a micro-ELISA to detect circulating antibodies in both experimental and field cattle sera, it was found that the titres of antibodies ranged between 1:20 and 1:10,240. Results of this study indicate that the antigen and immune complex capture assays and the antibody detection ELISA can be complementary in the immunodiagnosis of acute and chronic T. mutans infections. Moreover, the tests are useful in the differential diagnosis of the disease and for epidemiological studies.

Immune effector mechanisms involved in the control of parasitaemia in Trypanosoma brucei-infected wildebeest (Connochaetes taurinus).

The course of Trypanosoma brucei infection in the wildebeest (Connochaetes taurinus) was studied. A low but persistent parasitaemia developed in all five wildebeest following intravenous inoculation with 1 X 10(8) organisms of clone ILTat 2.1. Unlike cattle controls, however, the wildebeest did not develop anaemia. In both cattle and wildebeest, radioimmunoassay studies revealed a classical sequence of production of IgM, IgG1 and IgG2 antibodies which had the capacity to bind to the corresponding purified variable surface glycoprotein and to neutralize the infectivity of ILTat 2.1. Investigations into the interaction between post-infection sera, trypanosomes and freshly isolated peripheral blood leucocytes (PBL) of wildebeest and cattle showed that sera from the wildebeest had a higher capacity to induce adherence of trypanosomes to homologous PBL. The adherence and phagocytosis-inducing activity resided in the IgM fraction. Cross-testing of the antibodies and PBL revealed that wildebeest IgM antibodies induced high adherence indices when tested on cattle PBL. High adherence indices were also observed when cattle IgM antibodies were tested on PBL of wildebeest. It was concluded that the phagocytic system of the wildebeest was superior to that of cattle, that freshly prepared wildebeest PBL bear receptors for wildebeest as well as cattle IgM, and that cattle PBL bear a receptor for wildebeest IgM that would appear to be different from that for cattle IgM.