Attenuation of virulence in an apicomplexan hemoparasite results in reduced genome diversity at the population level.

BACKGROUND: Virulence acquisition and loss is a dynamic adaptation of pathogens to thrive in changing milieus. We investigated the mechanisms of virulence loss at the whole genome level using Babesia bovis as a model apicomplexan in which genetically related attenuated parasites can be reliably derived from virulent parental strains in the natural host. We expected virulence loss to be accompanied by consistent changes at the gene level, and that such changes would be shared among attenuated parasites of diverse geographic and genetic background. RESULTS: Surprisingly, while single nucleotide polymorphisms in 14 genes distinguished all attenuated parasites from their virulent parental strains, all non-synonymous changes resulted in no deleterious amino acid modification that could consistently be associated with attenuation (or virulence) in this hemoparasite. Interestingly, however, attenuation significantly reduced the overall population's genome diversity with 81% of base pairs shared among attenuated strains, compared to only 60% of base pairs common among virulent parental parasites. There were significantly fewer genes that were unique to their geographical origins among the attenuated parasites, resulting in a simplified population structure among the attenuated strains. CONCLUSIONS: This simplified structure includes reduced diversity of the variant erythrocyte surface 1 (ves) multigene family repertoire among attenuated parasites when compared to virulent parental strains, possibly suggesting that overall variance in large protein families such as Variant Erythrocyte Surface Antigens has a critical role in expression of the virulence phenotype. In addition, the results suggest that virulence (or attenuation) mechanisms may not be shared among all populations of parasites at the gene level, but instead may reflect expansion or contraction of the population structure in response to shifting milieus.

Viability assays of intra-erythrocytic organisms using fluorescent dyes.

Three intra-erythrocytic tick fever organisms of cattle (Babesia bovis, Babesia bigemina and Anaplasma centrale) were subjected to a range of stressors, including heat, storage over time, specific chemotherapy and cryopreservation. Various stains, both alone and in combination, were used in an attempt to assess viability of these organisms before and after the stressors were applied. Carboxyfluorescein diacetate succinimidyl ester (CFSE) stained live Babesia spp. very well while fluorescein diacetate (FDA) stained A. centrale successfully. Propidium iodide (PI) and ethidium-homodimer-1 (Eth-D) were used as counter stains to identify dead organisms. Stain combinations allowed differentiation between living and dead Babesia organisms after exposure to heat and after chemotherapy. PI and Eth-D as counter stains were of little value after deglycerolisation of cryopreserved organisms. Possible reasons for this limited success in determining death or viability of tick fever organisms after some treatments include the impermeability of red blood cells to PI and Eth-D counter stains or the loss of live and/or dead organisms during sample processing.

Observation of a novel Babesia spp. in Eastern Grey Kangaroos (Macropus giganteus) in Australia.

The roles and epidemiological features of tick-borne protozoans are not well elicited in wildlife. Babesia spp. are documented in many domestic animals, including cattle, horses, pigs, dogs and cats. Three cases affecting eastern grey kangaroos are described. The kangaroos exhibited neurological signs, depression and marked anaemia, and microscopic examination of blood smears revealed intraerythrocytic piroplasms. One to seven intraerythrocytic spherical, oval, pyriform and irregularly-shaped parasites consistent with Babesia spp. were seen in the blood smears and the percentage of infected erythrocytes was estimated to be approximately 7% in each case. Data suggest that the tick vector for this kangaroo Babesia sp. is a Haemaphysalis species. For Case 2, ultrastructural examination of the erythrocytes of the renal capillaries showed parasites resembling Babesia spp. and 18 of 33 erythrocytes were infected. DNA sequencing of the amplified 18S rDNA confirmed that the observed intraerythrocytic piroplasms belong to the genus Babesia. The phylogenetic position of this new kangaroo Babesia sp. (de novo Babesia macropus), as a sister species to the new Australian woylie Babesia sp., suggests a close affinity to the described Afro-Eurasian species Babesia orientalis and Babesia occultans suggesting perhaps a common ancestor for the Babesia in kangaroos.