Efficacy of South African Babesia bovis vaccine against field isolates.

A high-passage Babesia bovis vaccine containing only one genotype population was, although protective, inferior compared to the immunity afforded by a lower passage of the same strain containing two populations. The 24 times serially passaged South African B. bovis S vaccine strain contain only a single parasite population (Bv80 allele A 558bp). Forty-four field isolates sampled were all found different with regard to the number and composition of the parasite populations present in each isolate. The extensive genotypic diversity in South Africa and the limited genotypic diversity observed in the S24 vaccine, raised the question on its ability to protect against such diverse populations. The 6 isolates selected for challenge in the current study originated from geographically distinct populations that also possessed thirteen unique genotypes based on the Bv80 gene and included strains that resulted in clinical disease. The strain coverage was therefore much greater than in previous studies on the protective ability of the S24 vaccine. Challenge of vaccinated cattle indicated that the vaccine gave adequate protection against 5/6 isolates. Protection against the remaining isolate proved inadequate. However, field observations in the region where this isolate originated from, showed only minor mortalities in vaccinated animals compared to losses experienced in unvaccinated herds. This study demonstrated the ability of the South African B. bovis S24 vaccine to protect cattle against challenge from local field isolates containing single or multiple parasite populations.

Co-transmission of the non-transmissible South African Babesia bovis S24 vaccine strain during mixed infection with a field isolate.

The South African Babesia bovis live blood vaccine, originating from a field isolate attenuated by 23 serial syringe passages in splenectomized calves, has lost the ability to infect the natural vector Rhipicephalus (Boophilus) microplus. In this study, infection with mixed parasites from the vaccine strain and a field isolate, resulted in transmission of both genotype populations. Comparing the field isolate and transmitted combination indicated no significant difference in their virulence, while challenge of vaccinated cattle with these isolates showed the ability of the vaccine to protect against both. Limiting dilution of the transmitted combination, followed by infection of splenectomized cattle (n=34) yielded no single infections for the vaccine strain genotype, seven clonal lines of the field isolate and one mixture of vaccine strain and field isolate. Only one of two field isolate clonal lines selected for vector transmission study was transmitted. Showing that B. bovis isolates can contain both tick transmissible and non-transmissible subpopulations. The findings of this study also indicate the probability of vaccine co-infection transmission occurring in the field, which may result in new genotype populations of B. bovis. However, the impact of this recombination with field isolates is considered negligible since a genotypically diverse population of B. bovis is already present in South Africa.

Genotypic diversity in Babesia bovis field isolates and vaccine strains from South Africa.

Genotypic diversity in Babesia bovis (cause of Asiatic redwater in cattle) vaccine strains and field isolates from South Africa were investigated using the Bv80 gene as well as microsatellites. The S11 vaccine strain possessed both A and B alleles of the Bv80 gene, as well as genotypic diversity within each allele type as defined by repeat variation resulting in different amplicon sizes. Rapid serial passage of vaccine strain from passage S10 to S24 resulted in loss of genotypic diversity that yielded a single allele A genotype with an amplicon size of 558 bp. This suggested that clonal selection occurred during rapid passaging. Extensive genotypic diversity exists in 44 field isolates characterized with both Bv80 A and B alleles, but can be readily distinguished from the S24 vaccine strain using either the Bv80 allele specific PCR assays or using multi-locus micro-satellite typing. This indicated that no recent documented clinical cases of Asiatic redwater were caused by the reversion to virulence of the current vaccine strain.

Serological responses to Babesia bovis vaccination in cattle previously infected with Babesia bigemina.

Serological responses of field cattle (260) on a farm in KwaZulu-Natal, South Africa were determined before and after vaccination with the commercial Babesia bovis live-blood vaccine, using the indirect fluorescent antibody test (IFAT). All the cattle tested negative for B. bovis antibodies before vaccination while 83% of them had significant antibody titres (>or=1/80) to Babesia bigemina, indicating a high degree of natural exposure to the latter parasite. By Day 60 post-vaccination only 53% of the cattle had seroconverted to B. bovis. This raised the question as to why only half of the vaccinated cattle had seroconverted. The possibility of previous exposure to B. bigemina infection interfering with the development of detectable antibodies to B. bovis was therefore investigated under controlled conditions. It was found that simultaneous vaccination with B. bigemina and B. bovis (n=6), and B. bigemina vaccination followed by B. bovis vaccination (n=12), had no effect on the animals' immune responses to B. bovis vaccination. All of these cattle developed a significant antibody response. However, only 58% of cattle (n=12) which had previously been inoculated with the B. bigemina field isolate, obtained from the trial farm, seroconverted (>or=1/80) after B. bovis vaccination, yet parasites for B. bovis could be demonstrated microscopically in all of the animals in this group. These findings confirmed the serology results from the field trial. When challenged with a B. bovis field isolate, cattle in this group did not show clinical reactions compared with an unvaccinated control group. The judicious use of IFAT to establish vaccination success obtained with the current South African B. bovis vaccine is indicated.

In vitro cultivation of a south African isolate of an Anaplasma sp. in tick cell cultures.

This paper describes the first successful in vitro cultivation of a South African isolate of an Anaplasma sp., initially thought to be Anaplasma marginale, in the continuous tick cell line IDE8. Blood from a bovine naturally infected with A. marginale kept on the farm Kaalplaas (28 degrees 08' E, 25 degrees 38' S) was collected, frozen, thawed and used as inoculum on confluent IDE8 cell cultures. Twenty days after culture initiation small intracellular colonies were detected in a Cytospin smear prepared from culture supernatant. Cultures were passaged on Day 34. Attempts to infect IRE/CTVM18 cell cultures with the Kaalplaas isolate derived from IDE8 cultures failed, whereas a reference stock of A. marginale from Israel infected IRE/CTVM18 tick cell cultures. Attempts to infect various mammalian cell lines (BA 886, SBE 189, Vero, L 929, MDBK) and bovine erythrocytes, kept under various atmospheric conditions, with tick cell-derived Anaplasma sp. or the Israeli strain of A. marginale failed. Molecular characterization revealed that the blood inoculum used to initiate the culture contained both A. marginale and Anaplasma sp. (Omatienne) whereas the organisms from established cultures were only Anaplasma sp. (Omatjenne).

Effect of diminazene block treatment on live redwater vaccine reactions.

One third of the manufacturer's prescribed dose of diminazene has long been used to block treat the South African unfrozen Babesia bigemina and Babesia bovis (redwater) vaccine reactions, with no known adverse effects. It is known that the inhibitory effect of antibabesial drugs is more pronounced in animals inoculated with the frozen vaccine than those with the unfrozen vaccine. Reports of vaccine failures in some animals in which diminazene was used for block treatment of the reactions following inoculation with frozen South African redwater vaccine led us to reinvestigate the required waiting period before treatment and the reduced dose necessary for successful treatment and development of immunity. Results from febrile reactions in cattle following vaccination indicated day 7 as the optimal day for administering block treatment. Treatment of B. bigemina vaccine reactions in cattle on day 7 at a level of 0.35 mg/kg (1/10, fraction of the normal dose) diminazene killed all the parasites while B. bovis vaccine parasites survived treatment using diminazene at levels between 0.35 mg/kg and 1.16 mg/kg. However, various other factors, such as the degree of natural resistance of different cattle breeds and individual animals, the accuracy of diminazene content according to the manufacturer's label claim and the accuracy of the drug dose administered, all influence the successful immunization of animals. Consequently block treating of Babesia vaccines with diminazene on day 7 after vaccination is not recommended.

Residual effect of antibabesial drugs on the live redwater blood vaccines.

It has been demonstrated that the attenuated organisms used in the unfrozen South African Babesia bovis and B. bigemina (redwater) vaccines are susceptible for longer periods to the residual effect of the anti-babesial drugs diminazene and imidocarb dipropionate than the virulent field strains. Reports of vaccine failures in some animals vaccinated with the frozen South African redwater vaccines after prophylactic treatment with imidocarb dipropionate have led us to reinvestigate the validity of the recommended prescribed waiting periods. Results indicated that waiting periods before administration of the frozen B. bovis and B. bigemina vaccines in animals that have been treated with diminazene at 3.5 mg/kg live weight, compare favorably with results initially obtained for the unfrozen vaccines at 4 and 8 weeks, respectively. However, the inhibitory effect of imidocarb dipropionate at 3.0 mg/kg live weight on the infectivity of both frozen B. bovis and B. bigemina vaccines is longer than previously anticipated and necessitated changing the minimum waiting periods before administration of these vaccines from 8 to 12 weeks and 16 to 24 weeks, respectively.

Evaluation of a 3 ml heartwater (cowdriosis) infective blood vaccine dose.

Three milliliters of blood from the present commercially produced heartwater infective blood vaccine (Ball3 stock) was experimentally tested in sheep and cattle for infectivity and efficacy. Results obtained for this vaccine dose were statistically not different from results for the prescribed 5 ml vaccine dose.