The aim of the present study was to develop enzyme linked immunosorbent assays (ELISAs) for
detection of specific antibodies against Babesia bovis and B.bigemina for use in epidemiological
studies of bovine babesiosis in Brazil. These tests were developed using purified proteins of each
parasite, which were identified as species-specific in a comprehensive immunochemical
characterisation of different stocks of Babesia parasites.
The review of the literature on bovine babesiosis covers the geographical distribution, transmission
and life cycle, and in vitro cultivation of both species, as well as immunology, diagnosis, epidemiology
and control ofthe disease and its current situation in Brazil.
The thesis first describes a series of studies on the in vitro culture of B. bovis, which include the
evaluation of the effect of different sera on in vitro growth, the incorporation of feeder cells (bovine
aortic endothelial and mouse peritoneal cells) into cultures, cloning of one isolate by in vitro limiting
dilutions, initiation of cultures from low parasitaemia blood, and several attempts to establish African
isolates ofB.bigemina in vitro.
Methods for concentration of Babesia infected red blood cells and free parasites were then examined.
These included the use of differential hypotonic lysis, density gradient centrifugation, induction of free
merozoites into culture supernatant and techniques for fractionation of exoantigens present in culture
supernatant, namely high performance liquid chromatography.
Somatic components and exoantigens of different stocks of parasites were characterised
immunochemically by Western immuno-blotting, and immunoprecipitation of -^S-methionine labelled
proteins by acrylamide gel electrophoresis, using a wide variety of sera which included calf sera
experimentally raised against different stocks of each parasite, sera collected in the field in Brazil and
a panel of monoclonal antibodies previously produced against B. bovis and characterised by IFAT,
Western immuno-blotting and ELISA in the present study. The aim of the immunochemical
characterisation was to identify antigens common to all stocks yet species-specific with potential use
for the development of ELISAs. Antigenic diversity amongst stocks of B.bovis is reported.
For the development of the ELISAs, calf serum samples of known status and serum samples collected
in endemic areas in Brazil, Malawi and Mozambique were utilised to compare assays using crude
antigen preparations of each Babesia species and purified specific proteins. Four B.bovis stockconserved proteins from somatic components were electro-eluted from acrylamide gels and used to
develop an ELISA. Amongst these, a 56 kDa protein was found to be a potential candidate to replace
the crude parasite extract in immunodiagnostic assays.
Several B.bigemina specific proteins were also identified in the immunochemical analysis and four
candidates were electro-eluted from acrylamide gels. However all purified B.bigemina proteins crossreacted with B.bovis antisera in ELISA. The complementary use of a crude and cross-reactive
B.bigemina antigen together with the 56 kDa B.bovis-specific protein in ELISA to elucidate the
epidemiology of these diseases is recommended.
Future prospects and potential application in Brazil of these ELISA antigens for use in the detection of
anti-B.bovis and anfi-B.bigemina antibodies are discussed with particular reference to their role in
defining the epidemiology of bovine babesiosis in the dairy cattle of Minas Gerais State, Brazil.