Development of effective living vaccines against bovine babesiosis--the longest field trial?

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.

Babesia bovis: proteins of virulent and avirulent parasites passaged through ticks and splenectomized or intact calves.

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.

Biological comparisons between a laboratory-maintained and a recently isolated field strain of Boophilus microplus.

Biological differences between two strains of Boophilus microplus were examined. The A-strain of ticks had been maintained at the laboratory for many years and the N-strain was recently isolated, being a composite strain derived from ticks from different sources in the field. In three experiments, up to three times as many N-strain ticks grew to maturity than did A-strain ticks, although A-strain ticks matured earlier. N-strain ticks were 17 to 60% heavier, and laid 50 to 100% more eggs than A-strain ticks. N-strain eggs were significantly more fertile than A-strain eggs. Unfed, N-strain larvae survived much longer than A-strain larvae. The reduced vitality of the A-strain is attributed to its long history in the laboratory during which time it has become biologically disadvantaged through inbreeding. These results suggest that some laboratory-maintained strains of ticks may be unsuitable for ecological or acaricide studies.

Preparation and laboratory testing of a frozen vaccine containing Babesia bovis, Babesia bigemina and Anaplasma centrale.

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.

Serological comparison of strains of Babesia bovis occurring in Australia and Mozambique.

An antigen prepared in Australia and antisera collected from cattle having had field infections of babesiosis were taken to Mozambique for a serological comparison of strains of babesia bovis from the 2 countries. The reactivity of antisera collected in Mozambique against the Australian antigen was similar to that of the imported antisera. This suggested a close serological relationship between b. bovis of the 2 countries. The practical implication of this finding is that Australian vaccine should protect cattle being introduced into southern Africa from B. bovis-free environments.

Development of a highly infective Babesia bigemina vaccine of reduced virulence.

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.

Some aspects of the epidemiology and control of bovine babesiosis in Australia.

A short account of the epidemiology and control of babesiosis in Australia is presented. Epidemiological topics discussed include differences in the transmission of Babesia bovis and B. bigemina by the cattle tick, Boophilus microplus and the relative prevalence, disease incidence and pathogenicity of B. bovis and B. bigemina. Circumstances under which babesiosis occurs in Australia are described. In the Section on control, only vaccination is discussed. Changes in the preparation of babesial vaccines, particularly those resulting in a highly infective vaccine containing relatively avirulent B. bovis are described. Fluctuations in demand, such as the increase from about 100,000 to over 1,000,000 doses in 4 years in the mid-1960s are shown. An unexpected increase in the use of A. centrale in 1973 is discussed, and the supply of B. bigemina for cattle exported from Australia reported.

Evaluation of an indirect fluorescent antibody test to diagnose Babesia equi infection in horses.

An indirect fluorescent antibody (IFA) test for the diagnosis of Babesia equi infections was evaluated. Antigen prepared by conventional methods was of high quality in one instance and of lesser quality in a second when possible autofluorescence of the horse blood caused inconvenience in reading tests. Tests on 14 horses shown by parasitological means to be either infected (9) or uninfected (5) produced reactions at dilutions of 1/270 to 1/7290 for infected and at 1/10 to 1/90 for uninfected animals. The accuracy of the test was further demonstrated during investigations of 701 horses in 3 states of Australia. The 30 horses reacting at 1/270 to 1/2430 were from 33 imported to 3 different farms in Australia from a common source. Investigations of crossreactivity between B. equi and B. bovis of cattle suggested that B. bovis would not interfere with the test for B. equi, but that the reverse was possible.

Biological differences between African Theileria mutans (Theiler 1906) and two benign species of Theileria of cattle in Australia and Britain.

Serological and morphological comparisons between the theilerias found in Australian and British cattle and of these with African Theileria mutans (Theiler 1906) showed that the Australian and British parasites were similar to each other but different from T. mutans. Further investigations are required to determine taxonomic affinities between the Australian and British theilerias and T. sergenti, a similar parasite found in eastern Asia.

Infection rates and outbreaks of disease due to Babesia argentina in unvaccinated cattle on 5 beef properties in south-eastern Queensland.

Serums from unvaccinated groups in 5 herds of beef-cattle in South-East Queensland were tested for antibodies to Babesia argentina at intervals while the cattle were increasing in age from about 6 months. An indirect fluorescent antibody test was used. Infection rates, indicating the proportions of the groups that had been exposed to tick-transmitted infection were 49.2, 56.9 and 69.1% for cattle aged approximately 6, 12 and 18 months, respectively. The degree to which cattle were infested with the vector, Boophilus microplus, was estimated. There appeared to be a strong correlation between infection rate and tick incidence. Four serologically negative animals died of either confirmed or suspected babesiosis during the sampling period. Sickness was observed in 5 others. The serological status of 57 changes from negative to positive without symptoms being observed, indicating relatively low mortality and morbidity rates in the enzootic situation studied.

Vaccination with Babesia argentina in 5 beef herds in south-eastern Queensland.

Observations on the use of a vaccine containing Babesia argentina in 5 partly susceptible beef herds in south-eastern Queensland were made on 1,029 female breeding cattle over a period of 4 years. Groups averaging about 20 heifers were given 0, 1, 2 or 3 vaccinations. Incidence derived from groups experiencing clinical attacks were 17.9% for unvaccinated cattle and 1.2% for vaccinates. Increasing the number of vaccinations did not appear to increase protection. The one clinical manifestation of infection with B. bigemina was associated with a concurrent reaction to vaccination with Anaplasma centrale. There were no cases of haemolytic anaemia in new-born calves. No severe reactions followed primary vaccination, but 2 revaccinated animals became sick.

Piroplasmosis in buffaloes and its serological diagnosis based on a homology between buffalo and bovine immunoglobulins.

Babesia argentina, Babesia bigemina and Theileria mutans were transmitted experimentally from water buffaloes (Bubalus bubalis) to splenectomised Bos taurus calves. Buffaloes were positive to an indirect fluorescent antibody test for B. argentina when reagents of bovine origin were used. The formation of similar patterns during immunoelectrophoresis suggested a homology of buffalo and bovine serum proteins, particularly IgG.