Theileria annulata is an economically important protozoan parasite (Api-complexa) which cycles between bovine and invertebrate tick hosts. Within the bovid, sporozoites invade leucocytes and develop into macroschizonts: macroschizonts divide, initially by binary fission, in synchrony with the newly transformed host cell, but later merogony takes place, producing merozoites which invade erythrocytes. These are subsequently ingested by the tick. The post-macroschizont life cycle stages are poorly defined, and this experimental project was aimed at investigating the basic biology of these stages at the molecular level. The polypeptide complement of the infected erythrocyte membrane has been investigated. The precise location of piroplasm polypeptides in the infected cells was not determined, but the experimental results indicate that several (most notably molecules of 122kDa, 98-100kDa & 77kDa) are associated with the erythrocyte membrane. Monoclonal antibodies have been raised against infected erythrocytes. By immunofluorescence microscopy, several antibodies recognise, mainly, either the outer perimeter of the piroplasm, vesicular structures (dots), or molecules located within the piroplasm. For the purpose of strain differentiation, a panel of these monoclonal antibodies distinguish between un-cloned preparations of Ankara, Hissar and Gharb stocks. The vast majority of the monoclonal antibodies (27 cloned lines) are stage specific and do not recognise slide preparations of macroschizonts or sporozoites. Merogony has been induced in vitro, in recently transformed macroschizont infected cell lines, and four anti-piroplasm monoclonal antibodies recognise preparations of merozoites. Immunoelectron microscopy results have shown that antibody 5E1 recognises the surface of heat induced merozoites. Western blot analysis suggests that the 30kDa and 120kDa polypeptides, recognised by antibody 5E1, also elicit an antibody response in the bovine host. These antigens are preliminary candidates for part of a molecular sub-unit vaccine against the disease (Tropical theileriosis) caused by T.annulata. The T.annulata gene, which encodes the epitope determining antibody 5E1 has been cloned from a lambda gtl 1 genomic expression library. A model system for investigating the molecular mechanisms of stage differentiation has been developed. Macroschizont merogony can be induced in recently infected cell lines by elevating culture temperatures. Similar heat-treatment, of the same cell line after prolonged passage, fails to result in the differentiation into merozoites. The epitope recognised by antibody 5E1 is stage specific to merozoite and piroplasm stages and preliminary Northern slot-blot analysis, with the cloned gene sequence, suggests that expression is regulated at the level of transcription.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:651532 |
| Date | January 1989 |
| Creators | Glascodine, Jane |
| Publisher | University of Edinburgh |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://hdl.handle.net/1842/13916 |
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