Failure of a recombinant Babesia bovis antigen to protect cattle against heterologous strain challenge.

Groups of cattle were inoculated subcutaneously with (i) a recombinant DNA-derived Babesia bovis protein (KaBbl-GZ) fused to beta-galactosidase and combined with adjuvants, or (ii) native beta-galactosidase (GZ) plus adjuvant, or (iii) adjuvant only or (iv) a live, attenuated B bovis vaccine. KaBbl-GZ was produced in the lambda gt11-amp3 system as a 5-10 kD babesial polypeptide linked to GZ. KaBbl has previously been shown to be an immunodominant antigen of B bovis, localised at the apex of the parasite, and present in a range of B bovis strains. High levels of GZ antibodies were observed in KaBbl-GZ and GZ inoculated cattle, but specific KaBbl antibodies could not be detected by ELISA. Five months after primary inoculation, all cattle were blood challenged with a virulent heterologous B bovis strain. Despite four inoculations with KaBbl-GZ, significant protection against the challenge was not observed.

A microplate enzyme-linked immunosorbent assay for measuring antibody to Anaplasma marginale in cattle serum.

An enzyme-linked immunosorbent assay (ELISA) method is described for measuring antibody against Anaplasma marginale in cattle serum. This method was more sensitive and objective than a previously described ELISA method for A. marginale and possible reasons for this are discussed. All 83 cattle experimentally infected with A. marginale (81) or A. centrale (2) developed demonstrable specific antibody but the serums of 98.8% of 839 cattle from cattle tick-free areas did not react by ELISA; 378 serums containing antibody to Babesia bovis were tested for cross reactions in the A. marginale ELISA. There were no significant cross-reactions except when cattle had been inoculated at least twice with B. bovis-infected erythrocytes, presumably due to antibodies reacting with erythrocyte material in the ELISA antigen. The ELISA detected antibodies for more than 3 years after infection, at least 2 years longer than did a complement fixation test. When A. marginale infections in cattle were eliminated by long acting oxytetracycline, their serums ceased to react by ELISA. An ELISA score for serum antibody level was shown to have a statistically significant correlation with ELISA titre.

Immune responses of cattle following vaccination with living and non-living Babesia bovis antigens.

Twenty-four yearling Hereford (Bos taurus) cattle were vaccinated against Babesia bovis using either live parasites or non-living antigens obtained from the supernatant of in vitro cultures. A single dose of live parasites was given subcutaneously, while the non-living supernatant antigen (NLSA) was combined with saponin and 2 doses given, 2 weeks apart. Following vaccination with live parasites, serum antibodies remained at high levels for 6 months, but the lymphocyte transformation response was low and lasted only 10-18 days. In contrast, NLSA vaccination was followed, after 21-28 days, by a peak of serum antibodies which then slowly declined. The lymphocyte transformation response in these animals was much higher and persisted for 6 months. Following heterologous challenge all unvaccinated cattle had severe reactions and required treatment to prevent death. Cattle vaccinated with live parasites had mild reactions with only 1 of the 12 requiring treatment. Cattle vaccinated with NLSA were only partially protected and 6 of the 12 required treatment.

Metabolism of Babesia parasites in vitro. Glucose and energy metabolism of B. bovis.

The glucose and oxygen consumption, glycolytic ratio, lactate and pyruvate production and change in adenylate energy charge (AEC) and ATP concentration of B strain B. bovis-infected erythrocytes were measured. Although less than 5% of erythrocytes were infected, glucose and oxygen consumption in basal salts solution (BSS) were more than 2.5 times, and pyruvate production 7 times, those of uninfected erythrocytes. The glucose consumption of B. bovis alone was similar to previously reported values for B. rodhaini alone but the glycolytic ratio of B. bovis was less than half that previously reported for B. rodhaini. Pre-incubation concentrations of ATP in B strain B. bovis-infected erythrocytes were significantly lower than in uninfected erythrocytes but increased considerably following incubation in BSS, in contrast to B. rodhaini in which ATP concentrations were previously shown to decrease significantly following incubation in basal salts medium. When monitored every 4 h during 24 h incubation in BSS, the glucose consumption and lactate production of two strains of B. bovis were very similar. The glycolytic ratio of K strain B. bovis in RPMI 1640 medium plus 15% bovine plasma was double that in BSS. Both strains of B. bovis were infective after 24 h incubation in basal medium, in marked contrast to B. rodhaini which had been shown to lose infectivity.

Metabolism of Babesia parasites in vitro. Change in adenylate energy charge and infectivity of Babesia rodhaini-infected erythrocytes.

Studies were undertaken to investigate a possible correlation between the change, during experimental in vitro treatments, in adenylate energy charge (AEC) of B. rodhaini-infected erythrocytes and the infectivity of the parasites for mice. When the AEC and infectivity of B. rodhaini-infected erythrocytes were modified by incubation in basal salts media containing various substrates, parasite infectivity correlated significantly (r2 = 0.83, p less than 0.01) with change in AEC. However, B. rodhaini frozen with and without cryoprotectant demonstrated large changes in infectivity but relatively small changes in AEC. The final AEC values were similar to those of unfrozen organisms, although infectivity was greatly depressed. The results suggest that, when in vitro treatment changed the adenine nucleotide pool (ATP + ADP + AMP) of infected erythrocytes, change in AEC correlated with infectivity. When the adenylate pool was unchanged, no such correlation was evident. Thus, the AEC can be a useful index of parasite infectivity in some, but not necessarily in all, in vitro systems. Data on the relative utilization by B. rodhaini of various substrates, obtained when AEC and infectivity of infected erythrocytes were modified, are also reported and discussed.

Development of a Babesia rodhaini mouse assay for quality control of the serum component of a bovine babesiosis vaccine.

The rodent babesia, Babesia rodhaini, survived equally well in basal medium containing either 10 per cent rat serum or 10 per cent bovine serum. As a result, a B rodhaini mouse assay is now performed routinely to determine the suitability of batches of bovine serum for use in a commercial Babesia bovis vaccine issued in Australia. Heat inactivation of bovine serum at 56 degrees C for two hours did not affect the survival of either B bovis or B rodhaini.

Babesia bovis: comparison of culture-derived parasites, non-living antigen and conventional vaccine in the protection of cattle against heterologous challenge.

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.

A microplate enzyme immunoassay for detecting and measuring antibodies to Babesia bovis in cattle serum.

A microplate enzyme immunoassay (EIA) is described for measuring IgG antibody to Babesia bovis in cattle serum. B. Bovis antibody status (whether positive or negative) and the amount of B. Bovis antibody (EIA score), were measured by comparison with reference serums. The EIA was shown to be specific for B. Bovis, and EIA score correlated well with EIA titre. Comparison of EIA with the Indirect Fluorescent Antibody Test (IFAT) showed more than 95% agreement between the methods and disagreement in only 1.6% of serum samples tested. The remaining 3.2% were positive by EIA and suspected positive by IFAT. The EIA was shown, by titrating positive serums, to be more sensitive than IFAT, which explained its tendency to detect more positive serums than IFAT. EIA detected B. bovis antibody in experimentally infected cattle by day 14 post infection (pi) and for at least 268 days pi. EIA score for B. bovis antibody in immune cattle increased significantly (p less than 0.05) following heterologous strain challenge.

Metabolism of Babesia parasites in vitro. Glucose and energy-metabolism and survival of Babesia rodhaini in a basal medium with and without adenosine.

During in vitro incubation in Krebs Ringer phosphate (KRP) medium, rat erythrocytes about 20% parasitized with B. rodhaini used two to four times as much glucose as unparasitized erythrocytes. However, the AtP concentration and the ATP production of parasitized erythrocytes decreased. For example, before incubation the ATP concentration in parasitized erythrocytes was significantly higher thant unparasitized erythrocytes, but following 0.5 h incubation it was reduced to about the same as in unparasitized erythrocytes. Addition of 5 X 10(-3) M adenosine to the basal medium increased the ATP production and the adenylate energy charge of parasitized erythrocytes. Infectivity tests in susceptible mice showed that, despite these seemingly favourable changes in energy metabolism, adenosine had no detectable effect on the survival of B. rodhaini. Absence of infectivity showed the inability of KRP medium to support B. rodhaini for 22 h in vitro with or without adenosine. Adenosine also greatly decreased the glucose uptake by parasitized erythrocytes, apparently by competitive inhibition, since lactate production was unchanged.

Metabolism of Babesia parasites in vitro. amino acid production by Babesia rodhaini compared to Plasmodium berghei.

The in vitro amino acid production by Babesia rodhaini, Plasmodium berghei and uninfected rat erythrocytes was determined following 4 and 18 h of incubation in Krebs Ringer medium. Both parasites produced excess free amino acid. B. rodhaini produced up to 150 times and P. berghei up to 1,100 times as much free amino acid as parasite-free rat erythrocytes. The composition of excess amino acids produced by both parasites had a statistically significant concordance with the amino acid composition of rat haemoglobin, suggesting that haemoglobin was probably the main source of the amino acids produced by the parasites.

Metabolism of Babesia parasites in vitro. Comparison of several zwitterion buffers for the in vitro incubation of Babesia rodhaini.

The effect on the haemoprotozoan parasite Babesia rodhaini of adding a zwitterion buffer, HEPES, to a basal medium was studied by comparing the glucose uptake, ATP concentration, medium pH and infectivity of parasitized rat erythrocytes every 4 h during 24 h incubation in either Krebs Ringer phosphate (KRP) or KRP plus 28 mM HEPES. KRP plus HEPES was found to be superior to KRP for the maintenance of glucose uptake, erythrocyte ATP concentration, medium pH and infectivity of the parasite. Having established that HEPES improved the basal KRP medium, HEPES was then compared to three other buffers TES, BES and TRIS, to determine their relative effects on B. rodhaini during 18 h of incubation. At 28 and 56 mM concentration there was no significant difference in glucose uptake or lactate production among the 4 buffers. However, TES maintained the medium pH best, and BES preserved the infectivity of the parasite best. Further comparison of TES and BES at 10, 20, 30 and 40 mM established 50 mM TES to be best for buffering the pH of the medium and maintaining infectivity of B. rodhaini.

Identification and turnover of glycosaminoglycans in rat kidneys.

The turnover of sulfate label in crude glycosaminoglycan fractions from rat kidney cortex, medulla, and papilla has been determined. Heparan sulfate, chondroitin sulfate, dermatan sulfate, and hyaluronate have been separated electrophoretically and their specific activities determined after injection of labeled sulfate or glucose. The half-lives of the sulfated glycosaminoglycans are within the ranges found for other organs and tissues, but hyaluornate has a somewhat faster turnover in the kidney than elsewhere.