Development of a deep amplicon sequencing method to determine the species composition of piroplasm haemoprotozoa

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can seriously impact on the health of livestock and production. Piroplasms of multiple species can be present in a single host, but reliable molecular diagnostic tools are needed in order to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction with co-infecting species, but are similar in that infected animals become persistent carriers after recovery from primary infection, acting as reservoir hosts. Here, we describe for the first time the use of a deep amplicon sequencing platform to identify proportions of piroplasm species in co-infecting communities and develop the concept of a "haemoprotobiome". First, four phenotypically-verified species of Theileria and Babesia were used to prepare mock DNA pools with random numbers of the parasites amplified by four different numbers of PCR cycles to assess sequence representation for each species. Second, we evaluated the detection threshold of the deep amplicon sequencing assay for each of the four species and to assess the accuracy of proportional quantification of all four species. Finally, we applied the assay to the field samples to afford insight of the species composition of piroplasm communities in small and large ruminants in the Punjab province of Pakistan. The "haemoprotobiome" concept has several potential applications in veterinary and human research, including understanding of responses to drug treatment; parasite epidemiology and ecology; species interactions during mixed infections; and parasite control strategies.