We conclude that the competitive inhibition ELISA is a useful alternative to the card agglutination test for detection of A marginale or A centrale infection in cattle. The assay should be particularly useful for epidemiological applications such as prevalence studies and control programs.

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Monoclonal antibodies, directed against a 58-kDa Babesia bigemina merozoite antigen that reacted strongly with immune sera from experimentally and naturally infected cattle in Western blots, were used to develop a competitive-inhibition enzyme-linked immunosorbent assay (ELISA). As based on the testing of 70 antibody-positive sera from experimentally infected cattle and 166 antibody-negative sera collected in non-endemic areas of Australia, the sensitivity and specificity of the ELISA were 95.7% and 97.0%, respectively. In sequential sera collected from six calves during the course of experimental B. bigemina infections the ELISA detected seroconversion at about 10 days post-inoculation. The specificity of the ELISA was not affected by the presence of antibodies to B. bovis, Anaplasma marginale or Theileria buffeli. In 42 sera from cattle experimentally infected with B. bovis but negative for B. bigemina the specificity of the ELISA was 95.2%. The use of a competitive-inhibition ELISA format detecting only antibody directed against a single epitope on the 58-kDa antigen appears to have overcome many of the specificity problems that have plagued serological tests for B. bigemina in the past. The test should be useful for epidemiology studies, particularly in areas where B. bovis and B. bigemina have overlapping distributions.

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57containing mixes of parasite strains collected from multiple donor animals are not economic to produce due to the cost of quality assurance and quarantine of donor animals. It would be more cost effective to produce multi-strain vaccines in single donor animals co-infected with more than one vaccine strain. The question arises as to whether heterologous strains of B bovis can coexist in the same host.As part of our continuing research to better understand and prevent vaccine failures, we have developed and applied molecular markers for B bovis for studying relationships among field and vaccine strains. 7-9 These markers include a single copy gene, designated BV80 10 and a 40kDa merozoite antigen (T40). Amplification of the BV80 gene using PCR allows differentiation between strains on the basis of the size of the amplicon. The T40 antigen is present in the Dixie strain of B bovis 6 but absent from many others. 8 We describe an experiment in which these markers were used to examine the relationship between pairs of B bovis strains during co-infection of splenectomised calves. The main objective was to determine whether two antigenically distinct strains of B bovis could be passaged simultaneously in splenectomised calves, an approach that might allow the production of mixed-strain vaccines with a broader antigenic spectrum than can be achieved with single-strain vaccines.Two attenuated vaccine strains (K and Dixie) and two virulent field isolates (G100 and F11) were chosen so that only one attenuated strain and one virulent isolate possessed the T40 antigen and all were readily separated in the BV80 PCR. Pairs of strains and/or isolates for co-infections (Table 1) were chosen so that virulent/virulent, virulent/attenuated and attenuated/attenuated combinations of parasites were tested and only one strain or isolate from each pairing contained the T40 antigen. Experimental Bos taurus calves were obtained from dairy herds in a Boophilus-free area of south eastern Queensland and confirmed free of antibodies to Babesia spp using an IFAT. A total of seven calves were splenectomised and maintained under tick-free conditions. Four were infected with one of the four strains/isolates of B bovis and used to supply parasitised blood for coinfection experiments. The three remaining calves were infected with mixed strains (Table 1).PCR was used to amplify the BV80 B bovis genomic marker. Sample preparation for, and the resolution, sensitivity and specificity of the BV80 PCR are described by Lew et al. 6 Amplified fragments were separated and sized against a 100 bp ladder (Promega Corp, WI, USA) by electrophoresis in Visigel (Stratagene, CA, USA) according to the manufacturers instructions. The presence of T40 B bovis antigen on merozoites was detected using the T40 IFAT conducted as described by Molloy et al, 8 except that the monospecific rabbit antiserum to T40 was preabsorbed with 5% uninfected bovine RBC to reduce background fluorescence.B bovis infected blood for co-infection experiments was collected from four splenec...