The apicoplast genomes of two taxonomic units of Babesia from sheep

Highlights

• The apicoplast genomes of two Babesia taxa were directly sequenced.

• The relationships of these and other related apicomplexans were assessed.

• Markers for future epidemiological and population genetic studies were provided.

• This sequencing-bioinformatic approach should be broadly applicable.

Abstract

The apicoplast (ap) is a unique, non-photosynthetic organelle found in most apicomplexan parasites. Due to the essential roles that this organelle has, it has been widely considered as target for drugs against diseases caused by apicomplexans. Exploring the ap genomes of such parasites would provide a better understanding of their systematics and their basic molecular biology for therapeutics. However, there is limited information available on the ap genomes of apicomplexan parasites. In the present study, the ap genomes of two operational taxonomic units of Babesia (known as Babesia sp. Lintan [Bl] and Babesia sp. Xinjiang [Bx]) from sheep were sequenced, assembled and annotated using a massive parallel sequencing-based approach. Then, the gene content and gene order in these ap genomes (∼30.7 kb in size) were defined and compared, and the genetic differences were assessed. In addition, a phylogenetic analysis of ap genomic data sets was carried out to assess the relationships of these taxonomic units with other apicomplexan parasites for which complete ap genomic data sets were publicly available. The results showed that the ap genomes of Bl and Bx encode 59 and 57 genes, respectively, including 2 ribosomal RNA genes, 25 transfer RNA genes and 30–32 protein-encoding genes, being similar in content to those of Babesia bovis and B. orientalis. Ap gene regions that might serve as markers for future epidemiological and population genetic studies of Babesia species were identified. Using sequence data for a subset of six protein-encoding genes, a close relationship of Bl and Bx with Babesia bovis from cattle and B. orientalis from water buffalo was inferred. Although the focus of the present study was on Babesia, we propose that the present sequencing-bioinformatic approach should be applicable to organellar genomes of a wide range of apicomplexans of veterinary importance.

Introduction

Babesiosis is a socioeconomically important disease of humans and other animals caused by tick-borne apicomplexans of the genus Babesia. This disease can have a major, adverse economic impact on the health and productivity of livestock animals, particularly ruminants, as a consequence death, reduced meat and milk production, increased sterility and abortion rates and/or costs associated with treatment or prevention (Bock et al., 2004, Uilenberg, 2006, Schnittger et al., 2012), and is an ongoing problem, particularly in tropical and subtropical regions of Australia, Asia, Africa and the Americas. Most economic impact worldwide appears to relate to babesiosis of cattle (Schnittger et al., 2012, Gohil et al., 2013), caused by Babesia bovis, B. bigemina and/or B. divergens, but the socioeconomic importance of babesiosis in small ruminants is also acknowledged to be considerable (Uilenberg, 2006). The main causative agents of sheep and goats are B. ovis, B. motasi and B. crassa, transmitted by ticks of the genera Rhipicephalus and Haemaphysalis (Uilenberg, 2006), each of which can cause relatively severe disease. However, in China, other distinct taxa of Babesia have been reported in small ruminants. For instance, Babesia sp. Lintan (Bl) (Guan et al., 2002) and Babesia sp. Xinjiang (Bx) (Guan et al., 2001) have been recorded; these taxa have marked differences in vector specificity, virulence and pathogenicity (Liu et al., 2007). Interestingly, while the former taxon (Bl) is transmitted by Haemaphysalis spp. and causes mild to severe disease, the latter (Bx) is transmitted by Hyalomma anatolicum and usually relates to subclinical infection (Liu et al., 2007).

Most apicomplexan protists, including Babesia, harbour a plastid-like organelle, termed the apicoplast (ap), which was derived from a secondary endosymbiotic event with green algae (McFadden, 2011). This unique organelle is believed to play a critical role in essential metabolism of the parasite, including the synthesis of fatty acids, haem, iron-sulphur clusters and isoprenoids (Fichera and Roos, 1997, Vaishnava and Striepen, 2006, van Dooren and Striepen, 2013). Therefore, the ap genome represents a target for drugs against apicomplexans (Wiesner et al., 2008, Chakraborty, 2016). These ap genomes also provide data sets to explore the taxonomy and evolutionary relationships of apicomplexans.

However, there is surprisingly little information on ap genomes of apicomplexans and none for Babesia taxa of small ruminants. To date, complete ap genomes have been sequenced and/or characterised for B. bovis, B. orientalis, B. microti, Cyclospora cayetanensis, Eimeria tenella, Leucocytozoon caulleryi, Plasmodium chabaudi chabaudi, Theileria parva, Th. equi and Toxoplasma gondii (Table 1). While most of these studies used PCR and/or cloning-based approaches (Cai et al., 2003, Sato et al., 2013, Garg et al., 2014, Imura et al., 2014, Huang et al., 2015), some have utilized direct, deep sequencing of total genomic DNA (Gardner et al., 2005, Brayton et al., 2007, Kappmeyer et al., 2012, Tang et al., 2015).

In present study, Illumina technology was used to sequence the ap genomes of Bl and Bx directly from genomic DNA and a customised bioinformatics approach to annotated them. A phylogenetic analysis of the ap genomic data sets was conducted to assess the relationships of Bl and Bx with other apicomplexan parasites for which complete ap genomic data were publicly available. The results of the present study suggest that the sequencing-bioinformatic approach should be readily applicable to other protists of veterinary importance.