Differential diagnosis of tick-borne diseases and population genetic analysis of Babesia bovis and Babesia bigemina.

Abstract: Tick-borne diseases are a constraint to livestock production in many developing countries. They are responsible for high morbidity and mortality resulting in decreased production of meat, milk and other livestock by-products. The most important tick-borne diseases of livestock in sub-Saharan Africa are East coast fever (caused by Theileria parva), babesiosis (caused by Babesia bigemina and Ba. bovis), anaplasmosis (caused by Anaplasma marginale) and heartwater (caused by Ehrlichia ruminantium). Despite their economic importance, information on the epidemiology of these diseases in many countries is often lacking or inadequate, resulting in inappropriate disease control strategies being implemented. The availability of specific, sensitive and cost-effective diagnostic methods is important in the design and implementation of effective disease control strategies. In this study PCR assays based on the 18S and 16S rRNA gene sequences, that could identify Theileria / Babesia and Anaplasma / Ehrlichia pathogens of cattle respectively, were developed. In addition, PCR assays based on the β-tubulin gene that could detect T. parva, Ba. bigemina, Ba. bovis and T. taurotragi, and PCR assays based on the cytochrome b gene that could diagnose infection by Ba. bigemina and Ba. bovis were also developed. When the 18S and 16S rRNA gene PCR assays were combined into a multiplex PCR assay, Ba. bigemina and E. ruminantium DNA did not amplify and some non-specific bands were observed following agarose gel electrophoresis. The β-tubulin gene multiplex PCR assay for the diagnosis of T. parva, Ba. bovis and Ba. bigemina worked relatively well when used on laboratory-derived parasite DNA preparations. However, when it was used on field samples collected on FTA cards, multiple non-specific bands were observed after agarose gel electrophoresis of the PCR products. The 18S and 16S rRNA gene PCR assays were used for an epidemiological study of tick-borne diseases of cattle in Central and Eastern Zambia in the wet and dry seasons. All the disease pathogens under study (T. parva, T. mutans, T. taurotragi, Ba. bovis, Ba. bigemina, Anaplasma spp and E. ruminantium) were prevalent in all the regions of the country in both seasons. However, variation was observed in the prevalence of these pathogens between the regions and the seasons. A number of risk factors, associated with the occurrence of tick-borne pathogens in cattle and the tick burdens observed on cattle in the wet season were determined. A negative association was observed between the number of co-infecting pathogens and the erythrocyte packed cell volume (PCV) of carrier cattle. Using recently available genome sequences, mini- and microsatellite markers were developed for population genetic analysis of Ba. bovis and Ba. bigemina parasite populations. Ba. bovis isolates from Zambia and Turkey and Ba. bigemina isolates from Zambia were used in the population genetic analysis. High levels of genetic diversity were observed for both parasites. Population genetic analysis of the Zambian and Turkish Ba. bovis populations, using eight genetic markers showed that the two populations were sub-structured. The Zambian population comprised a single randomly mating population, while the Turkish population comprised two genetically distinct subpopulations. Population genetic analysis of the Ba. bigemina parasites from Zambia showed that this parasite population was in linkage disequilibrium. Further, analysis of the Ba. bigemina population using STRUCTURE showed that it was genetically sub-structured into five distinct subgroups. However, the resulting sample size of each subgroup was too small to definitely determine whether they were panmictic. These results provide an improved understanding of the epidemiology of bovine Babesia parasites in Turkey and Zambia.