Theileria annulata sporozoite surface antigen expressed in Escherichia coli elicits neutralizing antibody.

Abstract

Theileria annulata is an economically important protozoan parasite that threatens an estimated 250 million cattle with the disease tropical theileriosis. Development of a defmed subunit vaccine is one means of trying to develop control measures against the disease. To this end we have characterized a surface antigen complex of the infective stage (sporozoite), by using a monoclonal antibody that neutralizes sporozoite infectivity in vitro. We have cloned the gene coding for this complex and have demonstrated that a fusion protein expressed from a fragment of this gene elicits strong neutralizing antibodies. Furthermore we provide data on the structure and expression of this gene. In particular we show that the region of the gene, expressed in one clone, codes for a protein segment relatively rich in proline residues. Also we demonstrate that expression of this gene appears to be stage specific, transcripts being present only in the sporoblast and sporozoite stages. The relevance of these rmdings to the production of a defined subunit vaccine is discussed.Tropical theileriosis threatens an estimated 250 million cattle in the Mediterranean littoral, North Africa, the Middle East, the Indian subcontinent, and Central Asia and acts as a major constraint on livestock production and improvement in many developing countries (1). The disease is caused by a tickborne protozoan, Theileria annulata, which enters the bovine host as the infective sporozoite stage in the saliva of a feeding tick. These sporozoites rapidly invade mononuclear cells in which the macroschizont stage develops; subsequently these parasites are liberated, whereupon they invade erythrocytes producing the piroplasm stage of the life cycle (2). Present methods of vaccination depend upon establishing active infection (3) and thus incur both the risk of causing clinical disease and the problems of handling live parasite material under tropical conditions. For these reasons we are actively engaged in identifying parasite antigens capable of inducing a protective immune response (4-6) using similar approaches to those followed by investigators working on Theileria parva, the cause of East Coast fever (7-11). Here we describe an antisporozoite monoclonal antibody (mAb) that exhibits significant sporozoite neutralizing activity and provide data on the nature of the polypeptides recognized by this antibody. In addition we describe the isolation of two recombinant DNA clones that express the epitope defined by this mAb and we report our findings on the structure and expression of this gene(s). § MATERIALS AND METHODSParasite Material. Three uncloned isolates of Theileria annulata were used in this study: they are called Ankara, from Turkey (12); Hissar, from India (13); and Gharb, from Morocco (14). Sporozoites for mAb production, immunofluorescence tests, inhibition assays, and immunoblotting were obtained from Theileria annulata-infected adult ticks (Hyalomma anatolicum anatolicum). These ticks were fed on rabbits for 3 days to stimulate sporozoite m...

Identification of candidate transmission-blocking antigen genes in Theileria annulata and related vector-borne apicomplexan parasites

“…The gene ( TA03755 ) encoding the major sporozoite surface antigen SPAG-1 [42] and a gene encoding a putative papain cysteine protease were placed in this category. TA10955 was found to display peak expression at Day 15 of the tick time-course, indicating that the encoded protein may not be present until the later part of the life cycle in the tick.…”

Validation of the indirect TaSP enzyme-linked immunosorbent assay for diagnosis of Theileria annulata infection in cattle

“…It is difficult, however, to standardize antigens purified from parasite crude material, and there is also the requirement of experimental animals for parasite production. These problems have been circumvented by the use of several recombinant parasite antigens in ELISA, of which the merozoite rhoptry antigen Tams-1 and a sporozoite antigen SPAG-1 has been developed and used (Gubbels et al 2000;Ilhan et al 1998;Williamson et al 1989). However, the sensitivity of SPAG-1 has not been determined, while the sensitivity and specificity of Tams-1 was determined using the two-graph receiver-operating characteristic (TG-ROC) (Gubbels et al 2000).…”

Preliminary studies on the effect of Anaplasma marginale antibodies ingested by Dermacentor andersoni ticks (Acari:Ixodidae) with their blood meal on infections in salivary glands

“…In addition, stage-specific proteins have been demonstrated on the protozoan haemoparasites of cattle, Theileria and Babesia. Unique antigens were demonstrated on Theileria sporozoites, including those of Theileria parva (Musoke et al, 1982;Dobbelaere et al, 1985a,b;Nene et al, 1992) and Theileria annulata (Miranpuri, 1986;Williamson et al, 1989;Kachani et al, 1992). The sporozoite stage of T. parva is neutralized with an mAb directed against a sporozoite-specific antigen.…”