Development of a deep amplicon sequencing method to determine the proportional species composition of piroplasm haemoprotozoa as an aid in their control

Abstract

Piroplasmosis is caused by tick-borne haemoprotozoa of the genera Theileria and Babesia. These parasitic infections can cause serious impact on the health of livestock and production. Multiple piroplasm species can infect a single host, but reliable molecular diagnostic tools are needed with which to understand the composition of these complex parasite communities. Theileria and Babesia vary in their epidemiology, drug sensitivity, pathogenicity and interaction of co-infecting species, but are similar in the 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 pools with random amounts of the parasites and amplified with four different numbers of PCR cycles to assess sequence representation of 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.

New insight into genetic diversity of zoonotic‐potential Ancylostoma ceylanicum in stray cats living in Bangkok, Thailand, based on deep amplicon sequencing

“…to demonstrate species composition in coinfection of blood protozoa such as piroplasms (Theileria parva, T. annulate, Babesia bigemina, and Bartonella bovis) and Trypanosoma spp. (Trypanosoma brucei, T. theileri, T. congolense, and Tabanus vivax) (Chaudhry et al, 2019;Yasein et al, 2022). Further, because of deep amplicon sequencing ability to detect minor population parasites, this method was used to detect low-density Plasmodium falciparum infection (Early et al, 2019).…”

Contrasting population genetics of cattle- and buffalo-derivedTheileria annulatacausing tropical theileriosis

“…Peripheral blood smears were prepared and stained with 4% Giemsa, then visualised under 1000x oil immersion to detect piroplasm infection. ‘Haemoprotobiome’ sequencing was performed on piroplasm-positive blood samples to confirm the presence of T. annulata (Chaudhry et al, 2019). Genomic DNA was isolated from T. annulata positive samples by lysis with GS buffer and proteinase K as described in the TIANamp Blood DNA Kit (TIANGEN Biotech Co. Ltd, Beijing) and stored at −20°C.…”

“…Genomic DNA was isolated from positive samples by lysis with GS buffer containing proteinase K as described in the TIANamp Blood DNA Kit (TIANGEN Biotech Co. Ltd, Beijing) and stored at −20°C. ‘Haemoprotobiome’ high-throughput sequencing (Chaudhry et al, 2019) was performed on piroplasm-positive blood samples to confirm the presence of T. annulata .…”

Simultaneous genotyping of snails and infecting trematode parasites using high‐throughput amplicon sequencing

“…In this study, we make use of the increasingly accessible and affordable next generation sequencing (NGS) technology of high‐throughput amplicon sequencing (HTAS) to develop an efficient and comprehensive method for simultaneous identification of parasitic trematodes and their snail hosts, thereby preserving the host–parasite relationships. HTAS, which involves multiplexing and pooling, is commonly used in metabarcoding studies (Braukmann et al, 2019; Cruaud et al, 2017; Herbold et al, 2015)and has been successfully applied to identify parasite infections from DNA extracts of their hosts (Chaudhry et al, 2019; Cooper et al, 2018). By multiplexing various markers in one single PCR and pooling numerous samples per sequencing run (Bybee et al, 2011; Cruaud et al, 2017; Herbold et al, 2015) , the study of snail–trematode communities becomes scalable.…”