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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.

Between 1959 and 1996, research was performed to change a vaccine against babesiosis in Australia and to improve it as actual or threatened untoward field responses became apparent. The most significant change occurred in 1964 with the traditionally used carriers of Babesia being replaced as vaccine donors by acutely infected splenectomised calves. This ensured the infectivity of the vaccine and was fortuitously associated with a reduction in the virulence of Babesia bovis in vaccine. Since then, more than 27 million doses of highly infective vaccine have been supplied from the laboratory at Wacol near Brisbane. This vaccine reduced serious losses from babesiosis in vaccinated cattle in Australia to very low levels and has now gained acceptance worldwide. Research to ensure the continuing effectiveness of the vaccine has proved to be essential.

The virulence of a strain of Babesia bigemina was reduced by syringe passaging at 3 to 16-week intervals in a series of 7 calves. The calves were splenectomised 1 to 14 weeks after inoculation to induce the relapse parasitaemias used for passaging. Parasites taken at relapse from the last 3 calves in the series were inoculated into splenectomised calves from which highly parasitised blood for vaccine was obtained. The vaccine produced mild infections in 32 recipient cattle. When challenged either 5 weeks or 7 months after vaccination, the cattle had substantial immunity to a heterologous strain of B. bigemina.

Babesia argentina was repeatedly exposed to imidocarb by transmitting parasites from infected ticks in a series of 4 groups of nonsplenectomised calves that had been treated prophylactically at 2 mg/kg. As the number of exposures to imidocarb increased, the parasites more readily infected the treated calves, indicating increased tolerance to the drug. Tests comparing parasites not previously exposed to imidocarb with those exposed 4 times showed that a dose of 3 mg/kg imidocarb completely cured subclinical infections with non-exposed, but not exposed, parasites; a dose of 1 mg/kg controlled acute infections with either nonexposed or exposed parasites; and the virulence of the parasite was apparently unaffected by the exposures.

Two strains of Babesia bovis that had been serially blood passaged in splenectomized calves 27 to 33 times, a procedure known to have reduced their virulence for normal cattle, were shown to have low pathogenicity for replete, female Boophilus microplus. In comparison with a strain of B. bovis unmodified by repeated blood passage, the two modified strains infected higher proportions of ticks and produced comparable numbers of morphologically similar parasites in their haemolymph, but killed significantly fewer of them. Red discolouration of haemolymph was observed in many ticks infected with the unmodified strain, but in none of those infected with the modified strains. It is suggested that the modified strains have lost a quality causing pathological effects on the gut cells of infected ticks.

Observations were made on the effects of five different methods of laboratory maintenance on the infectivity and virulence of Babesia bigemina for the tick Boophilus microplus. The original isolate was highly infective and virulent, causing premature death of engorged female ticks and reduced egg production. Maintenance of the strain by syringe passage in unsplenectomised calves at six to 10 week intervals reduced both its infectivity and virulence for ticks. When slow passages were preceded by a series of rapid passages in splenectomised calves, the changes to the strain were less pronounced. The other three procedures, rapid syringe passage in splenectomised calves and tick passage in either splenectomised or intact calves, had no statistically significant effect on the characteristics measured.

Two strains of Babesia bovis that were known to have lost infectivity for the normal tick vector, Boophilus microplus, due to repeated blood passaging in cattle, were studied to determine whether the strains would regain infectivity for ticks during longstanding infections. Parasitaemias were monitored in 4 chronically infected calves that were regularly infested with ticks. Two strains of ticks known to be susceptible to infection with unmodified strains of B. bovis were used. Adult female ticks that dropped from the calves on days that a parasitaemia was evident were tested for B. bovis infection. Sixty-six batches of ticks collected up to 279 days after infection of the calves produced 14 pools of larvae, none of which transmitted infection. Primary infections established from the chronic infections by subinoculation at 200, 259 and 333 days after infection of the calves were also not transmitted by ticks.

Procedures used to prepare and test frozen vaccine against bovine tick fever are described. Blood from splenectomised calves infected separately with Babesia bovis, Babesia bigemina and Anaplasma centrale was diluted in the ratio of 3:1 with 8 M dimethyl sulphoxide in phosphate buffered saline and cooled at rates between 5 degrees C and 110 degrees C per minute to -196 degrees C. After varying periods of storage up to 369 days, blood was thawed by immersing containers in a 40 degrees C water bath and its infectivity tested in a total of 150 nonsplenectomised cattle. Subcutaneous inoculation of the cryopreserved blood, and 10-fold and 50-fold dilutions prepared from it, showed that high infectivity of all three parasites was retained during storage. The use of frozen vaccine exported from Australia to Trinidad and Tobago is briefly discussed.

Pure strains of Babesia bovis, Babesia bigemina and Anaplasma marginale were isolated from cattle infected with all 3 species as well as Theileria sp. and Eperythrozoon teganodes, using only transmission by the tick, Boophilus microplus. Unengorged adult ticks transferred to susceptible cattle transmitted A. marginale, but not Babesia. Engorged adults gave rise to progeny that transmitted Babesia, B. bovis by larvae and B. bigemina by male ticks. The Theileria and E. teganodes were not transmitted by the ticks and thus did not appear in calves used for isolating the pure strains of Babesia and A. marginale.

Passage of the avirulent vaccine (K) strain of Babesia bovis (KA) through either Boophilus microplus ticks, intact calves, or intact calves and then ticks, resulted in two distinct protein and protein antigen profiles as analyzed by two-dimensional gel electrophoresis of biosynthetically labeled proteins and immunoprecipitates. Different degrees of expression of two major acidic antigens of KA designated Ka1 (Mr 47,500) and Ka2 (Mr 43,000) were observed. Ka1 was apparently lost following passage of KA B. bovis through intact calves but was strongly represented in the parasite population following a single tick passage. In contrast, passage through ticks of the virulent KV B. bovis (from which KA was derived by passage in splenectomized calves) did not lead to strong representation of the Ka1 protein although there was increased representation of another major acidic protein antigen, designated KV (Mr 35,000). These data suggest that the previously recognized reversion to a strain-dependent basal antigenic type in the tick vector depends also on intrastrain characteristics such as virulence and strain heterogeneity. The data suggest that KA is a more heterogeneous population than KV although cloned isolates are required to establish this point. Comparable syringe passage of another strain of B. bovis, designated C strain, through splenectomized calves resulted in less marked differences between the putative CA and CV B. bovis. This may explain the less stable avirulence of CA compared to KA B. bovis. Various selection pressures must act, in either the tick or the vertebrate host, on subpopulations in heterogeneous isolates to produce the changes described in protein antigen profiles of B. bovis. The possible relevance of changes in representation of proteins to biological characteristics of B. bovis (such as virulence and tick transmissibility) is discussed.

Blood containing either Babesia bovis, Babesia bigemina or Anaplasma centrale was mixed with an equal volume of 3 M glycerol in phosphate-buffered saline with or without glucose and then stored in liquid nitrogen for 2-30 days. After being thawed, the parasitized blood was subjected to various procedures, including dilution up to 1000-fold followed by incubation at 30 or 4 degrees C for 8 h, before infectivity of the parasites was tested in a total of 70 cattle. The results showed that the blood cryopreserved with glycerol remained highly infective after thawing, despite dilution and incubation for 8 h at 30 degrees C. The results have practical application in the use of frozen, live vaccines against bovine babesiosis and anaplasmosis.

Twenty Bos taurus cattle were vaccinated with either live commercial Babesia bovis vaccine, live parasites from in vitro culture or non-living supernatant antigen (NLSA) derived from in vitro culture and combined with the adjuvant saponin. Heterologous strain challenge 10 weeks later indicated that cattle vaccinated with live parasites from either source were strongly protected, those given 2 doses of NLSA 2 weeks apart were partially protected, and those given one dose of NLSA were poorly protected. Enzyme immunoassay detected comparable, increasing levels of specific babesial antibody in all vaccinated cattle during the 2 to 3 weeks following vaccination, after which levels in cattle given NLSA decreased. Antibody to bovine blood group factors was detected in 4 of the 10 animals given NLSA. Titres peaked after 3 to 4 weeks and then declined rapidly.

Observations were made on the morphology and infectivity of Babesia bigemina parasites in the gut of Boophilus microplus ticks. The babesia parasites were derived from stabilates of a laboratory strain, prepared at intervals during several years of blood-passaging in cattle. Increased numbers of a particular developmental stage of the parasite, considered to be a type of strahlenkörper, were observed in ticks ingesting parasites derived from the latter stages of passaging. The presence of large numbers of these parasites was associated with reduced infectivity of the strain for ticks.

The development and morphology of Babesia bigemina in the gut of Boophilus microplus ticks were studied during laboratory maintenance of the babesia by four methods: tick transmission in unsplenectomised (intact) calves; tick transmission in splenectomised calves; syringe passage at intervals of five days or less in splenectomised calves; and syringe passage at intervals of six weeks or more in intact calves. The first method had no apparent effect on the development of B bigemina in ticks compared to that of the original isolate. The other three methods had obvious effects, the most pronounced being increased numbers of babesial forms with processes, particularly during early stages of development. The findings emphasise the importance of maintaining laboratory strains of parasites by natural means in life cycle studies.