Characterization of acyl carrier protein and LytB in Babesia bovis apicoplast.

The apicoplast is a highly specialized organelle that mediates required functions in the growth and replication of apicomplexan parasites. Despite structural conservation of the apicoplast among different parasite genera and species, there are also critical differences in the metabolic requirements of different parasites and at different stages of the life cycle. To specifically compare apicoplast pathways between parasites that have both common and unique stages, we characterized the apicoplast in Babesia bovis, which has only intraerythrocytic asexual stages in the mammalian host, and compared it to that of Plasmodium falciparum, which has both asexual intraerythrocytic and hepatic stages. Specifically focusing on the type II fatty acid (FASII) and isoprenoid (MEP) biosynthesis pathways, we searched for pathway components and retention of active sites within the genome, localized key components [acyl carrier protein (ACP) and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB)] to the apicoplast, and demonstrated that the N-terminal bipartite signals of both proteins are required and sufficient for trafficking to the apicoplast lumen. Using specific pharmacologic inhibition, we demonstrated that MEP biosynthesis may be disrupted and its presence is required for intraerythrocytic growth of B. bovis asexual stages, consistent with the genomic pathway analysis and with its requirement in the asexual erythrocytic stages of P. falciparum. In contrast, FASII biosynthesis may or may not be present and specific drug targets did not have any inhibitory effect to B. bovis intraerythrocytic growth, which is consistent with the lack of requirement for P. falciparum intraerythrocytic growth. However, genomic analysis revealed the loss of FASII pathway components in B. bovis whereas the pathway is intact for P. falciparum but regulated to be expressed when needed (hepatic stages) and silent when not (intraerythrocytic stages). The results indicate specialized molding of apicoplast biosynthetic pathways to meet the requirements of individual apicomplexan parasites and their unique intracellular niches.

Light microscopy diagnosis of Babesia bovis and Babesia bigemina kinetes in the haemolymph of artificially infected Boophilus microplus engorged female ticks.

The length, width and position of the nucleus of Babesia bovis and Babesia bigemina kinetes from the haemolymph of Boophilus microplus engorged female ticks were recorded. Additionally, the shape of Babesia bovis kinetes were registered as curved, semi-curved or straight. To this aim Boophilus microplus tick larvae from a colony free of Babesia were fed on splenectomised calves artificially infected with either Babesia bovis or Babesia bigemina pathogenic strains. Six engorged female ticks showing an infection of at least ten mature kinetes of Babesia bovis in a sample of haemolymph 5 days after detachment were also monitored 7, 9 and 10 days after collection. The same procedure was followed with six engorged female ticks infected with Babesia bigemina. One hundred and twenty kinetes of each species of Babesia were evaluated. The mean length +/- standard deviation and ranges for Babesia bovis kinetes were 14.30 +/- 0.922 microns and 11.9-16.3 microns, while the corresponding measures for the kinetes of Babesia bigemina were 11.27 +/- 0.900 microns and 9.0-13.1 microns (P < 0.001, t-test). The width was 3.33 +/- 0.315 microns, 2.6-4.0 microns for Babesia bovis and 2.24 +/- 0.287 microns, 1.5-2.8 microns for Babesia bigemina kinetes (P < 0.001). The most common position of the nucleus was central for both species of Babesia. A total of 58% of Babesia bovis kinetes showed the typical curved tail. No effect of time post-collection and individual host ticks in the kinete of Babesia bigemina was found while an unexpected influence of individual host tick in the width of Babesia bovis kinetes was detected (P < 0.01, analysis of variance). The overlap in the sizes of kinetes from both species of Babesia makes it difficult to apply the results to ticks of unknown babesial infection status. This finding is further complicated by the intra-specific size variations of Babesia kinetes from different geographical origins.