International Journal for Parasitology 47 (2017) 701–710

Contents lists available at ScienceDirect

International Journal for Parasitology

journal homepage: www.elsevier.com/locate/ijpara

Invited Review Advances in the application of genetic manipulation methods to apicomplexan parasites ⇑ ⇑ C.E. Suarez a,b, , R.P. Bishop b,c, H.F. Alzan b,d, W.A. Poole e, B.M. Cooke e, a Animal Disease Research Unit, USDA-ARS, Washington State University, 3003 ADBF, P.O. Box 646630, Pullman, WA 99164, USA b Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, WA 99164, USA c The Paul G. Allen School for Global Animal Health, Washington State University, Pullman, WA, USA d Parasitology and Animal Diseases Department, National Research Center, Dokki, Giza, Egypt e Biomedicine Discovery Institute and Department of Microbiology, Monash University, Victoria 3800, Australia article info abstract

Article history: Apicomplexan parasites such as Babesia, Theileria, Eimeria, Cryptosporidium and Toxoplasma greatly Received 23 July 2017 impact animal health globally, and improved, cost-effective measures to control them are urgently Received in revised form 24 August 2017 required. These parasites have complex multi-stage life cycles including obligate intracellular stages. Accepted 24 August 2017 Major gaps in our understanding of the biology of these relatively poorly characterised parasites and Available online 8 September 2017 the diseases they cause severely limit options for designing novel control methods. Here we review potentially important shared aspects of the biology of these parasites, such as cell invasion, host cell Keywords: modification, and asexual and sexual reproduction, and explore the potential of the application of rel- Apicomplexan atively well-established or newly emerging genetic manipulation methods, such as classical transfec- Genetic manipulation Transfection tion or gene editing, respectively, for closing important gaps in our knowledge of the function of Gene editing specific genes and proteins, and the biology of these parasites. In addition, genetic manipulation meth- CRISPR/Cas9 ods impact the development of novel methods of control of the diseases caused by these economically important parasites. Transient and stable transfection methods, in conjunction with whole and deep genome sequencing, were initially instrumental in improving our understanding of the molecular biol- ogy of apicomplexan parasites and paved the way for the application of the more recently developed gene editing methods. The increasingly efficient and more recently developed gene editing methods, in particular those based on the CRISPR/Cas9 system and previous conceptually similar techniques, are already contributing to additional gene function discovery using reverse genetics and related approaches. However, gene editing methods are only possible due to the increasing availability of in vitro culture, transfection, and genome sequencing and analysis techniques. We envisage that rapid progress in the development of novel gene editing techniques applied to apicomplexan parasites of vet- erinary interest will ultimately lead to the development of novel and more efficient methods for disease control. Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction parasitic diseases of veterinary importance, such as tick and mos- quito borne diseases caused by Babesia, Theileria and, albeit to a Globalisation and climate change are driving forces that are lesser extent, Plasmodium (Giles et al., 2014; Karbowiak, 2014; rapidly changing our world. These unforeseen factors cause rapid Novikov and Vaulin, 2014; Dantas-Torres, 2015; Tokarevich et al., expansion of previously controlled or geographically contained 2017). In addition, these rapid global changes favour the expansion and importance of other diseases such as toxoplasmosis, neosporo- sis, eimeriosis, and cryptosporidiosis. The common feature among these diseases is that they are all caused by apicomplexan para- ⇑ Corresponding authors at: Animal Disease Research Unit, Agricultural Research sites. If uncontrolled, some of these parasites can severely impact Service, United States Department of Agriculture, Pullman, WA 99164-6630, USA. Fax: +1 509 335 8328 (C.E. Suarez). Fax: +61 3 9902 9222 (B.M. Cooke). the production of food, but in addition, they can also compromise E-mail addresses: [email protected] (C.E. Suarez), [email protected] human health. Other negative consequences include increased aca- (B.M. Cooke). ricide and drug resistance, and the rapid geographical expansion of http://dx.doi.org/10.1016/j.ijpara.2017.08.002 0020-7519/Published by Elsevier Ltd on behalf of Australian Society for Parasitology. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). 702 C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710 vectors, parasites and other pathogens by extensive human migra- in high cattle losses in eastern sub-Saharan Africa with severe tion and global transportation of goods and merchandise. These economic consequences. Eimeria includes various species capable new realities are among the most important and unanticipated of causing coccidiosis in animals, including cattle, sheep, goats, public health challenges of the current century and require urgent and poultry (Chartier and Paraud, 2012). The more divergent attention. Cryptosporidium parasites cause important intestinal disorders The Apicomplexa are a large and diverse ancient phylum of in livestock, including diarrhoea, and have high zoonotic obligate intracellular protozoan parasites that are defined by the potential. presence of an apical complex structure, and include more than Collectively, this group of apicomplexan parasites is responsible 6000 species (Seeber and Steinfelder, 2007). They are arguably for poorly controlled acute and persistent infections of veterinary the most successful group of obligate parasitic protozoa responsi- importance. Increased strategic and basic research in this field will ble for life-threatening diseases in domestic, food, and wild ani- ultimately lead to enhanced control methods, improved animal mals. In particular, certain apicomplexan parasites, such as health, productivity in crop-livestock and pastoralist systems Babesia, Theileria, Cryptosporidium, Eimeria, and Toxoplasma have and, ultimately, human health globally. a high global negative impact on animal health. The global cost of tick transmitted pathogens in the livestock sector alone, of 2. Basic biological features of apicomplexan parasites which a significant percentage derives from apicomplexan para- sites, was estimated at US $17 billion, approximately 20 years This phylum of parasitic alveolate protozoans comprises obli- ago (de Castro, 1997). More recently, the global impact of the gate endoparasites of animals that share apical complex structures. incidence of neosporosis alone in the cattle industries of 10 Apicomplexan parasites are mostly motile, using gliding mecha- selected major countries was estimated to be more than US nisms based on myosin motors. The alveolar structure of these pro- $1218 million per annum (Reichel et al., 2013). Therefore tozoans consists of flattened vesicles contained in three membrane improved strategies to control these important parasites are layers which are penetrated by micropores. The apical complex clearly overdue. provides orientation for specific interactions with host cells, and Apicomplexan parasites typically have complex life cycles and contains the secretory organelles needed for host cell invasion, the ability to invade multiple vertebrate and invertebrate hosts including the micronemes (or microspheres in the case of Theile- and cells thus imposes severe challenges to most currently avail- ria), typically a pair of rhoptries, and dense granules or spherical able control strategies. Their long-term co-evolution with their bodies (Bonnin et al., 1995; Kats et al., 2006; Gubbels and hosts has produced sophisticated adaptation mechanisms, result- Duraisingh, 2012; Kemp et al., 2013; Swapna and Parkinson, ing in parasites that are able to effectively manipulate innate and 2017), and other structures such as the conoid (except for the Aco- adaptive host immune responses and, in some cases, transform noida Babesia and Theileria parasites) and polar rings. The apical their host’s cells. complex is a secretory structure that is required for invasion of Until recently, numerous gaps in our knowledge and under- host cells. However, in Theileria, the apical complex plays a greater standing of the biology of these parasites and the diseases they role in establishment after internalisation, rather than invasion. cause have severely limited options for designing new methods Also ‘micronemes’ in the strict sense are not discernable by elec- for control. Research aimed at filling basic key knowledge gaps, tron microscopy, although ‘microspheres’ can be observed in these including the role of certain genes in the regulation of life stage parasites. transitions, the molecular mechanisms involved in host cell inva- Apicoplasts are a chloroplastic remnant derived from a symbi- sion, avoidance of host immunity, and parasite transmission, otic organism that invaded a precursor of dinoflagellates and api- through the application of recently developed gene manipulation complexans, and are also shared among most apicomplexans methods, will contribute toward the identification of critical (Lim and McFadden, 2010; McFadden and Yeh, 2017). However, parasite-encoded pathways. These research outcomes can be plastid DNA has never been identified in Cryptosporidium, and it exploited to enhance rational design of novel methods for control, is likely that this parasite lost this organelle in evolutionary history particularly vaccines and drugs. (Sato, 2011). Given the importance of apicomplexan parasites as pathogens, The genome of apicomplexans is haploid, and the parasites can new research strategies are urgently required to accelerate dis- reproduce asexually by mitosis (merogony and sporogony), and coveries leading to improved control. The relatively recent appli- sexually by the fusion of gametes (generated by gametogony, cation of a myriad of emerging ‘‘omics” disciplines, together with derived from merozoites), concomitant with meiosis occurring in other state-of-the-art molecular biology techniques including zygotes (Smith et al., 2002). In the case of Theileria, and Babesia, biochemical, immunological, and in vitro culture techniques in gamete fusion and meiosis occurs in the arthropod tick vector. apicomplexan research, are already enhancing our understanding These reproductive events may occur in a single host in the case of the biology of these parasites and creating exciting new of monoxenous species, such as Crytosporidium, or in different hypotheses. In this review, we focus on recent research and pos- hosts, for heteroxenous species such as Toxoplasma, Neospora, Thei- sible future directions involving genetic manipulation approaches leria and Babesia. The latter develop in definitive hosts, where they for Toxoplasma, Neospora, Babesia, Theileria, Eimeria, and Cryp- undergo sexual reproduction, and in non-definitive hosts by non- tosporidium. Toxoplasma gondii is a highly adaptable parasite with sexual mechanisms (e.g. endodyogeny in Toxoplasma, merogony the ability to infect a wide range of hosts, and has become a in Theileria and Babesia, and schizogony in Theileria)(Smith et al., ‘model organism’ for the Apicomplexa. It can cause abortions in 2002). infected animals and has the potential to produce a large zoono- An aspect shared among protozoans, also essential to their tic impact, especially when it infects immuno-compromised pathogenicity, is their ability to disseminate and colonise new hosts. Neospora caninum is highly related to Toxoplasma and also hosts. Transmission strategies are also variable among the Apicom- responsible for abortions in livestock. Babesia spp. are tick-borne plexa. These parasites are diverse and can use distinct modes of intraerythrocytic parasites that may infect livestock and most transmission including direct (ie: through intimate body contact domestic animals, causing anaemia and high levels of mortality for example); faecal-oral (e.g., cyst stages of Cryptosporidium and in naïve adult animals. The tick-borne Theileria parasites are Eimeria), vector-borne (Babesia, Theileria, Plasmodium) or preda- unique due to their ability to transform host leucocytes, resulting tor–prey transmission (Toxoplasma gondii). Thus, while Babesia C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710 703 and Theileria parasites are transmissible by ticks and Plasmodium the long term selection of more resistant individuals. Infectious via mosquitoes, Cryptosporidium, Eimeria, Toxoplasma and Neospora organisms co-evolve with their hosts, exerting a powerful and con- are transmitted via oocysts that are highly resistant to environ- tinuous evolutionary pressure on host populations. Other parasite mental factors. factors mediate cytoadhesion facilitating the persisting infection of the host (as is the case for Babesia bovis) while simultaneously evading the immune system of the host by antigenic variation 3. Parasite life cycles (Allred and Al-Khedery, 2004; Dzikowski and Deitsch, 2006; Wahlgren et al., 2017). For these reasons some virulence factors, Parasites can have definitive and intermediate hosts. Definitive especially those involved in interaction with effectors generated hosts provide the environment for the sexual reproduction of the by the immune system of the hosts, might represent appropriate parasites and are obligatory to the life cycle. Intermediate hosts targets for vaccine development. The use of gene manipulation can also be obligatory for completion of the life cycle, but do not approaches for the phenotypic characterisation of mutants that provide the appropriate environment for sexual reproduction. Api- are deficient or have partially functional virulence factors can be complexan parasites can use asexual and sexual reproductive a powerful method to characterise these factors and thereby help mechanisms with the different forms of reproduction occurring to define vaccine candidates or novel drug targets in apicomplexan in distinct hosts, typically a combination of invertebrate and verte- parasites. brate hosts, and consequently many apicomplexans, including Babesia and Theileria, circulate between their definitive inverte- brate and intermediate vertebrate hosts. 5. Genomes and gene conservation Infections caused by Babesia and Theileria parasites normally initiate mammalian host infection following inoculation of sporo- Complete and annotated genomes for apicomplexan parasites zoite stage parasites by the arthropod vectors, specifically ticks addressed in this review are currently available (http://eupathdb. (Babesia and Theileria), whereas Toxoplasma, Eimeria, and Cryp- org/eupathdb/). Detailed comparisons of these genomes permits tosporidium infections can be caused by exposure of the hosts to identification of conserved molecules involved in common and cysts. likely essential pathways, including parasite survival and persis- The life cycle of Theileria is broadly similar to that of Babesia tence, that can be the target of gene manipulation techniques, spp. However, uniquely among protozoa, the schizont stage of cer- and for the rational design of therapeutic interventions. However, tain Theileria (including Theileria parva, Theileria anulata, and it is often challenging, based on sequence conservation alone, to Theileria lestoquardi) induce transformation of the bovine/ovine exploit these data to assign a specific function and to identify leukocytes after invasion by the sporozoites. These multinucleate those genes that represent optimal targets or candidates for ther- schizonts immortalise ruminant leukocytes by a process similar apeutic interventions. Because such conserved proteins might be to virally-induced cancers of mammalian cells (reviewed by essential to the parasite, it can be challenging to use gene knock- Dobdelaere and Huessler, 1999) a process that is reversible out (KO) approaches for their functional characterisation. How- in vitro by buparavaquone (Marsolier et al., 2015). The transformed ever, these comparisons also allow the identification of species- cells are easily maintained in culture, but the intracellular nature specific genes and gene families supporting species-specific adap- of the schizont makes Theileria difficult to manipulate genetically. tations resulting in distinct life-cycles and infection strategies. For The subsequent red cell-infective stages exhibit more limited repli- instance, several molecules associated with the apical complex, cation than Babesia and are therefore more difficult to culture for the defining morphological feature of the Apicomplexa, responsi- genetic manipulation. ble for mediating attachment, invasion and establishment within While most of the life cycles of apicomplexan parasites are host cells, are mostly conserved among this major group of para- understood or at least partially characterised at the cellular level, site genera of veterinary importance. These include proteins very little is known about the events underpinning these pro- expressed in conserved secretory organelles such as rhoptries, cesses at the molecular and genetic levels, one important example micronemes and spherical bodies/dense granules that release their being the regulation of genes involved in life cycle transitions. contents during invasion in the merozoite asexual stages, includ- Our ability to disrupt such events may lead to novel methods of ing MIC-1, AMA-1, and TRAP1 (Gaffar et al., 2004a, 2004b; control, but more basic research is needed before this can be Cérède et al., 2005; Silva et al., 2010; Salama et al., 2013; attempted in a targeted fashion. These shortcomings can be Templeton and Pain, 2016). In addition, the microneme protein addressed using transformation methods which can provide SPATR conserved in Plasmodium, Toxoplasma, Eimeria and Neospora important insights on the role of genes in the development of was found to contribute to both invasion and virulence (Huynh the life cycle of the parasites (Pieszko et al., 2015; Alzan et al., et al., 2014). However no obvious homologues have been identified 2016a,b). in Babesia, Theileria or Cryptosporidium. Several genes encoding for Infection involves multiple levels of host-parasite interactions, proteins responsible for functions associated with the develop- and thus development of disease is multifactorial. On one hand, ment of sexual stages and essential to sexual reproductive mech- the susceptibility of the host depends on age, gender, nutritional anisms are also consistently conserved, including HAP 2, CCp, and physiological state, and it immunological competence. How- and 6-cys proteins (Bastos et al., 2013; Alzan et al., 2016a, b). Fur- ever parasite properties such as pathogenicity, or expression of thermore, these parasites also share key gene regulatory mecha- intrinsic virulence factors, are also important. nisms, such as those mediated by the members of the Ap2, HMG and Myb gene families encoding for transcription factors (Alzan 4. Parasite virulence factors et al., 2017). The value of such comparative genomics is enhanced by using model apicomplexan organisms for functional studies to Parasite molecules responsible for inducing pathology, known identify the role of key molecules involved in essential mecha- as virulence factors or virulence determinants, often correlate nisms, such as invasion and sexual stage development (Kim and directly with the capacity of the parasite to survive and reproduce. Weiss, 2004). These studies are facilitated by the use of genetic However, these factors may also adversely affect host survival and manipulation techniques, as described further in the subsequent fecundity. This applies pressure on host populations resulting in sections. 704 C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710

6. Methods for genetic manipulation A simplified chronology of the events leading to the develop- ment of GMM in apicomplexan parasites of interest is depicted Important constraints to research on apicomplexan parasites in Fig. 1. have included the difficulties associated with the maintenance of in vitro parasite cultures, the availability of complete, well anno- 7. Genetic modification using transfection tated genomes, and practical tools for their genetic manipulation. However, in recent years, several of the apicomplexan species have Transfection consists of the introduction of nucleic acid mole- been successfully cultured and in some case multiple genomes cules (DNA or RNA) into eukaryotic cells. The transferred nucleic sequenced. The application of robust GMMs is indispensable for acid, usually DNA, might reside as extra-chromosomal replicating expanding our understanding of gene function and for allowing episomes in the nucleus of the transfected cells, or it may also be targeted modifications of parasite genotypes. Current availability stably inserted into chromosomes, typically by homologous of genome sequences provides new opportunities for developing recombination mechanisms. Transfection methods can be used basic research leading to a better understanding of the life-style, for multiple applications including the study of promoter function gene regulation, metabolism, and invasion mechanisms, poten- and gene regulation, gene function using reverse genetic tially leading to the rational design of improved methods of con- approaches, and for vaccine development purposes. Using stable trol. The predominant source of this genomic information is transfections, genes can be targeted and deleted by including EuPathDB (http://eupathdb.org/eupathdb/)(Aurrecoechea et al., appropriate homologous 50 and 30 flanking regions that are incor- 2017), which currently provides specialised services for porated into the transfecting plasmid DNA. Stable transfected par- Plasmodium spp. (PlasmoDB, (http://plasmodb.org/plasmo/)), asite lines can be also used as vaccine delivery systems (Oldiges coccidians (ToxoDB, http://toxodb.org/toxo/), piroplasms et al., 2016). The phenotypic characterisation of genetically- (PiroplasmaDB, http://piroplasmadb.org/piro/), and Cryptosporid- transformed parasite lines can provide important clues about gene ium spp. (CryptoDB, http://cryptodb.org/cryptodb/), and the function during different stages of the parasite life cycle (Alzan T. parva DB (available at http://erepository.uonbi.ac.ke/handle/ et al., 2017). 11295/14283)(Visendi et al., 2011). The initial report of the successful application of transfection to Currently vaccines using attenuated parasites are available for the parasites of interest in this review was the description of a preventing Babesia and Theileria annulata acute infections, but the transient transfection method for Toxoplasma gondii (Soldati and mechanisms of attenuation remain unknown and these vaccines Boothroyd, 1993). This transient transfection method facilitated have several important limitations. The limitations of the Babesia the development of a method for the stable transfection of this vaccines are discussed in detail elsewhere (Florin-Christensen organism (Kim et al., 1993). Importantly, transient transfection is et al., 2014). The molecular basis of attenuation of the T. annulata a useful approach for defining appropriate electroporation param- attenuated cell culture vaccine generated by passaging infected eters (Suarez et al., 2007) and to identify and test the function and monocytes is also so far unknown. Furthermore, although vacci- efficacy of regulatory elements (promoters and termination sig- nation is efficacious, application to date has been relatively lim- nals) mediating gene expression and regulation. Shortly thereafter, ited and confined to certain countries in North Africa and the transient and stable transfection methods were also developed for Middle East, particularly Tunisia (Darghouth et al., 1999). In the some species of Plasmodium parasites (Goonewardene et al., 1993; case of T. parva, there is currently no live attenuated vaccine. Van Dijk et al., 1995), and for Neospora caninum, where they were An ‘infection and treatment’ (ITM) procedure is available involv- easily derived from the work previously performed on the related ing the concomitant delivery of a potentially lethal dose of sporo- Toxoplasma (Howe and Sibley, 1997). An Eimeria tenella transient zoites together with a long acting formulation of oxytetracycline. transfection system was first reported in 1998 (Kelleher and Although ITM is effective and increasingly adopted by pastoralists Tomley, 1998), followed by the description of the transient expres- in certain countries it is expensive, and difficult to produce and sion of the YFP and RFP reporter genes (Hao et al., 2007) and stable standardise (di Giulio et al., 2009). Consequently, new improved transfection, described by Yan et al. (2009) (Fig. 1). Briefly, tran- vaccines based on subunit antigens or in parasites that do not sient transfection techniques are designed to introduce and have the risk of reverting to virulence and are not transmissible express foreign DNA, usually in the form of a plasmid, into a nucle- by their vectors are needed. GMMs are research tools that enable ated cell in a non-stable manner. Thus, in transient transfection, investigation of multiple aspects of parasite biology and identifi- the introduced plasmid nucleic acid does not integrate into the cation of novel approaches for control, including identification of genome of the target cells, and the transfected genes will not be virulence factors, subunit vaccine components, and parasite trans- replicated. Transient transfection methods were also developed mission factors. These techniques are continuously evolving and for B. bovis (Suarez et al., 2004, 2006; Suarez and McElwain, are being applied to increasing numbers of organisms. Other 2008, 2010, 2007) T. parva (De Goeyse et al., 2015) C. parvum (Li possible applications include the use of GMMs to generate et al, 2009), and T. annulata (Adamson et al., 2001). Transient attenuated parasites expressing foreign genes coding for exoge- transfection methods proved to be useful for the definition of pro- nous or stage-specific endogenous protective antigens for the moters in B. bovis and later in Babesia bigemina (Silva et al., 2016), development of novel vaccine delivery platforms. The most and defined a baseline protocol for stable transfection systems at widely used genetic manipulation methods include classic trans- least for T. gondii (Kim et al., 1993) and B. bovis (Suarez and fections based on the insertion of exogenous DNA using McElwain, 2009, 2010). Subsequently, similar parasite introduc- homologous recombination mechanisms, and more recently tion systems will likely be an essential first step for implementa- CRISPR/CAS9, which has recently been successfully applied to tion of CRISPR/Cas9 editing methods, described below. Transient Crytposporidium and Toxoplasma, as described below, and earlier transfection plasmids typically include a reporter gene or a gene incarnations of gene editing such as TALENs and Zinc-finger that needs to be expressed transiently (such as required for cur- nucleases, based on programmable nucleases. By analogy rent CRISPR/Cas9 methods), placed under the transcriptional con- with multicellular eukaryotes the latter, particularly CRISPR/ trol of a promoter with transcription and translation regulatory CAS9, are potentially more flexible and powerful (Ran et al., regions located at the 50 and 30 ends, respectively. An appropriate 2013; Hsu et al., 2014). amount of the transient transfection plasmid is then introduced C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710 705

Fig. 1. Timeline of selected landmark publications on the development of genetic manipulation methods in Toxoplasma, Eimeria, Babesia, Cryptosporidium, Theileria, and Neospora. Moon, sun, and star shapes indicate transient, stable transfection, and CRISPR/Cas9 editing, respectively.

into the target cells using the most effective methods. These (Huynh and Carruthers, 2009). However, the efficiency of exoge- include those relying on physical treatments such as electropora- nous gene integration can also be affected by the specific DNA base tion, nucleofection, biolistic delivery (gene gun), or microinjection, composition of the target cells, as in the case of the A + T rich gen- and those relying on chemical entities, such as liposomes ome of P. falciparum (Gardner et al., 2002). Interestingly, Toxo- (Kepczynski and Róg, 2016). plasma parasites are also difficult to engineer using classic Stable transfection techniques are based on the ability of the transfection technologies because they have the ability to ran- parasites to insert genetic material into their genomes using domly insert the foreign DNA in sites different from those specifi- homologous recombination mechanisms, in a fashion allowing cally targeted. This occurs, at least in part, due to the action of the expression of the transfected genes. As for transient transfection, non-homologous end joining (NHEJ) repair mechanisms based on these techniques rely either on the use of liposomes such as lipo- the activity of a gene encoding the KU80 protein. This limitation fectamine or on the application of a controlled electrical pulse, has been recently addressed by preparing a genetically trans- such as in electroporation, or later in nucleofection. These proce- formed T. gondii line lacking the KU80 gene, which makes it amen- dures allow the incorporation of exogenous DNA, usually in the able to gene targeting using homologous recombination form of a circular or linearised plasmid, into the nuclear compart- mechanisms (Huynh and Carruthers, 2009). In contrast, in ment of a eukaryotic cell. Therefore, basic steps involved include: B. bovis-specific transfection seems to be an efficient technique, (i) identification of a suitable genetic marker to select for at least in terms of targeting, when applied to this organism transgenic parasites (‘selectable marker’); (ii) preparation of a (Suarez et al., 2015). Differences in the gene repertoires and transfection plasmid vector containing, at a minimum, a select- genetic structure of other proteins involved in gene repair mecha- able marker gene under the control of a suitable promoter and nisms between B. bovis and T. gondii, together with differential reg- 50 and 30 regions to target integration of the construct into the ulation of their expression, might also help to explain the genome; (iii) a liposome, or electroporation/nucleofection proto- differences observed among the mechanisms of gene repair operat- col which minimises impact on the viability of the target cells; ing in these two organisms. Thus, stable transfection techniques for (iv) a method for selection of transfected parasites (Suarez and B. bovis allowed highly specific KO and KO reversion experiments McElwain, 2010). that are needed to study gene function (Asada et al., 2012a,b; Specific integration of the transfected gene(s) at the intended Suarez et al., 2015, 2015). Recent progress in Babesia transfection site into the genome of the target parasite depends upon the oper- technology includes the demonstration of a method for functional ation of homologous recombination mechanisms. The efficiency of gene analysis by generating gene KO followed by gene function the integration process in protozoan parasites is highly variable recovery (Asada et al., 2015), and the demonstration of cross- among species or cells as it heavily depends on the available species promoter function (Silva et al., 2016). This later study DNA repair mechanisms, as has been demonstrated for Toxoplasma describes the ability of a B. bovis ef-1a promoter to function effi- 706 C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710 ciently in B. bigemina. This observation suggests that common reg- possibility of using different gene manipulation strategies. Thus, ulatory signals should exist, allowing the control of promoter func- introduction of simple mutations resulting in gene inactivation tions in both closely related parasite species. or disruption can be generated by creating a double break fol- Progress with transfection and genetic manipulation of Theileria lowed by NHEJ. This repair mechanism can generate either inser- has lagged behind other members of the phylum Apicomplexa. As tions or deletions (indels) in the target gene resulting in already mentioned transient transfection has been described for frameshifts that disrupt the continuity of the open reading frame T. annulata (Adamson et al., 2001) and T. parva (De Goeyse et al., of the gene, usually leading to the functional knock-out of the 2015) sporozoites using both lipofectin or nucleofection tech- gene. If the objective is the insertion of foreign genes, such as niques to introduce foreign DNA with parasite 50 and 30 sequences reporter genes, it will be necessary to add donor plasmid DNA driving expression. However, the process is inefficient, in part containing the gene to be inserted, with the addition of homol- because a large number of sporozoites are required, that have to ogous flanking regions, to facilitate accurate targeting. In this be generated by feeding ticks on infected cattle, and there has been case, the insertion of the foreign gene will likely be mediated limited experimentation with optimising conditions for introduc- by HDR mechanisms. Importantly, new discoveries on the mech- tion of foreign DNA. These studies represent proof of concept, but anisms of DNA repair in apicomplexan parasites have revealed have not yet been used extensively for applications such as identi- the participation of certain proteins such as rad51 and KU80. fication of features such as key nucleotides defining promoter As discussed earlier, targeted mutation of the KU80 gene sequences. resulted in a T.a gondii mutant line that is more efficient for gene To develop stable transfection systems for Theileria, it will be targeting, since it favours the KU80-independent HDR mecha- necessary to transfect sporozoites with a selectable marker and nism and prevents random incorporation of transfected genes, then use these sporozoites to infect bovine leukocytes. This will commonly observed in this parasite. This cell line is thus ideally enable dissection of parasite-host interactions of the intracellular suited for gene function analysis in Toxoplasma using homolo- schizont parasite with its bovine/ovine host cell. A key constraint gous recombination KO approaches (Huynh and Carruthers, for T. parva and T. annulata stable transfection to date has been 2009; Smolarz et al., 2014). the identification of a schizont-specific selectable marker that The specific design of gene editing experiments depends on can be used on in vitro cultured Theileria-transformed host cells. the programming nuclease of choice. The currently available pro- Recently a novel secreted parasite-specific peptidyl prolyl iso- grammable gene editing methods include the use of engineered merase (PPI) that is convincingly implicated in bovine host cell proteins such as zinc-finger nucleases (ZFNs) and transcription immortalisation has been identified in T. annulata, with a corre- activator-like effector nucleases (TALENs). More recently RNA- sponding homologue in T. parva (Marsolier et al., 2015). It has guided engineered nucleases (RGENs), have become almost rou- further been shown that the protein represents one of the targets tine in multicellular eukaryotes. Despite the improved target of the anti-Theileria drug buparvaquone. A mutation in the para- specificity of TALENs, the RGEN methods have the advantages site PPI that confers resistance to buparvaquone has been identi- of a simpler design, higher versatility, and lower cost compared fied and incorporation of this mutation confers resistance to the with the earlier described techniques requiring protein engineer- drug (Marsolier et al., 2015). This may provide a solution to the ing. Briefly, the ZFNs attach DNA cutting domains derived from current lack of a selectable marker for stable transfection of the prokaryote Flavobacterium okeanokoites to proteins called Theileria. zinc fingers that can be customised to recognise certain 3 bp In addition to stable integration of the targeted sequences into DNA codes. On the other hand, TALENs fuse the same cutting the genome, stable transfected cell lines can be obtained with domains to different proteins called TAL effectors. Both ZFNs non-integrated episomes. In these cases, the episomal recombi- and TALENs require two cutting domains in order to cleave nant DNA is replicated and segregated into the daughter target double-stranded DNA. Excellent reviews on the use of ZFN and cells, sometimes in the form of concatemers (Asada et al., TALEN approaches for gene editing are available elsewhere (Ma 2012a,b). and Liu, 2015). The most widely used RGEN method is based on the CRISPR/ Cas9 system. Greater detail on the discovery and function of the 8. Gene editing using programmable nucleases CRISPR/Cas9 system has been described in recent reviews (Lander, 2016; Wright et al., 2016). Briefly, this bacterial host Targeted genetic editing methods that allow precise modifica- defense system is divided into three types based on the specific tions in a genome have been developed recently. These methods Cas proteins involved. Only the more simple Type II system is used offer great potential for the manipulation of the genomes of api- for gene editing, and is essentially based on a single effector Cas9 complexan parasites, especially where transfection methods protein, although other putative effectors can now also be used. based solely on homologous recombination typically exhibit very Briefly, the acronym CRISPR is derived from ‘‘clustered regularly- low efficiency, as is the case for Plasmodium. A key factor dra- interspaced short palindromic repeats”, which together with the matically increasing efficiency of programmable nucleases is Cas (‘‘CRISPR Associated” proteins) endonucleases, such as Cas9, their ability to generate blunt-ended double strand breaks (DSBs) are part of an adaptive immune system that controls bacterio- in the target DNA of interest. The DSB results in the activation of phage pathogens of bacteria and archaebacteria (Wright et al., repair systems in the cells, such as error-prone NHEJ mecha- 2016). This bacterial immune system provides RNA-mediated nisms, which can repair the break without the presence of donor immunity against viruses and plasmids based on copying and homologous DNA. Alternatively, the breaks can be repaired by specifically cleaving exogenous genetic materials. It has been rea- homology directed repair (HDR) mechanisms in the presence of lised that this system could also be used to edit DNA in any homologous donor double or single strand DNA, leading to the prokaryotic or eukaryotic cell if the necessary components are pro- insertion of exogenous genetic material. However, some apicom- vided. Together, CRISPR and Cas9 are able to target and cut almost plexan parasites, such as Plasmodium, and Cryptosporidium lack any DNA in vivo and, together with transfection techniques, they the NHEJ pathway (Vinayak et al., 2015; Calhoun et al., 2017). have quickly become important as efficient and specific tools for The existence of these alternative pathways also suggests the gene editing. C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710 707

The most commonly used CRISPR/Cas9 systems are adapted potential for improving the control of parasites of veterinary from Streptococcus pyogenes. A CRISPR-Cas9 system specifically interest. cleaves a DNA sequence through a two-stage recognition pro- The importance of T. gondii as a model apicomplexan was fur- cess. Initially, as depicted in detail below, a Cas9-sgRNA complex ther emphasised through development of a CRISPR/Cas9 based will be able to attach stably to a DNA sequence only if an appro- genome-wide genetic screen for the identification of T. gondii priate, short (often only a few bp) protospacer-adjacent motif essential genes (Sidik et al., 2016). The experimental approach (PAM) is located in close proximity. Therefore, an important involved the application of a genome-wide genetic screen adapted advantage of the CRISPR/Cas9 type II system is its simplicity, for T. gondii based on CRISPR/Cas9, designed to assess the contribu- since only three components are required to achieve site- tion of each gene from the parasite Toxoplasma gondii during infec- specific DNA recognition and cleavage. These include a CRISPR tion of human fibroblasts (Sidik et al., 2016). This approach RNA (crRNA) and a trans-activating crRNA (tracrRNA) which allowed the identification of an apicomplexan-conserved invasion are required in order to guide the Cas9 enzyme to its target factor termed claudin-like apicomplexan microneme protein sequence. These two elements (crRNA and tracrRNA) are usually (CLAMP). Furthermore, approximately 200 essential Toxoplasma combined into a single synthetic guide RNA (sgRNA). According genes identified in this CRISPR/Cas9 screen database had homo- to experimental design and specific gene targeting the sgRNAs logues in all sequenced apicomplexan genomes. Therefore both can be designed to include the specific base sequence that the data generated and the novel approach has potential for appli- matches the target gene of interest. In that way, the complex cation to other apicomplexan parasites. can redirect the Cas9 enzyme to almost any target sequence. A The use of genetic transformation methods greatly accelerates novel on-line tool, the ‘‘Eukaryotic Pathogen gRNA Design Tool” our knowledge of the genetics of apicomplexan parasites and the (EuPaGDT; available at http://grna.ctegd.uga.edu), which identi- development of new vaccines. A website to guide the design of fies guide RNA (gRNA) in input gene(s) can be used for appropri- CRISPR tools in protozoan pathogens is a useful resource that is ate gRNA design for many eukaryotic pathogens (Peng and currently freely available (http://grna.ctegd.uga.edu/batch.html). Tarleton, 2015). The S. pyogenes Cas9 endonuclease, which also Finally, different strategies for the selection of edited parasites, incorporates nuclear localisation signals (NLS), preferably with or without the use of selectable markers, have also been pub- requires an NGG PAM (with ‘‘N” representing any nucleotide lished (Mogollon et al., 2016). base followed by two guanine, or ‘‘G” bases). However, NAG and NGA PAM motifs can also sometimes be recognised. The 9. Recent examples on the use of gene editing methods on 20 bp long sequence in the guide RNA then recognises the apicomplexan parasites of veterinary importance homologous DNA target sequence by Watson–Crick base pairing. If the 20 bp target sequence is confirmed by specific binding to Toxoplasma gondii and N. caninum are closely related apicom- the target sequence, allosteric activation of two nuclease plexans. A heterologous platform for expression of foreign genes domains of Cas9, RuvC and HNH will result in dual cleavage based on transfected N. caninum has been recently developed. This and, accordingly, a complete double-strand break in the target system might be useful for analysing specific gene functions, the sequence. Clearly, the specificity of any CRISPR-Cas9 system identification of new vaccine components against the related depends heavily on the proper design of the gRNA. This can T. gondii and to investigate the biology of the parasites and the sometimes be effected using algorithms that minimise the likeli- molecules responsible for the phenotypic differences among the hood of off-target effects. In other words, designing CRISPR/Cas9 highly related N. caninum and T. gondii (Zhang et al., 2010; Mota gene editing experiments requires the design of a 20 nucleotide et al., 2017). guiding RNA (sgRNA) that can hybridise specifically with Cryptosporidia are unusual among apicomplexan parasites. sequences in the target gene. All the sequences encoding the They are able to complete their full life cycle inside a single host, gRNA, the gene encoding Cas9 (including the NLS) and donor lack apicoplasts, parasitize target cells using alternative invasion DNA need to be provided to the target cells for expression in mechanisms, feed from the parasitised cell using a unique orga- the form of plasmid DNA. Co-expression of these DNAs can be nelle, and contain a DNA-less atypical mitochondria (mitosome). achieved using a single vector or multiple vector strategies. Thus, Their 9.1 Mb genome (Abrahamsen et al., 2004), encoding for example, a single vector can include the genetic information 3950 genes, contains numerous genes acquired by lateral transfer necessary for the co-expression of Cas9, sgRNA and donor DNA. from other microorganisms (Beverley, 2015). In addition, Cryp- This can be achieved using transient transfection of an appropri- tosporidium spp. lack NHEJ DNA repair mechanisms. Water- ately designed and engineered plasmid with each gene under the borne cryptosporidia are an important cause of gastroenteritis transcriptional control of distinct promoters known to be func- and diarrhoea in animals and in humans, and improved control tional in the target parasite. This is now facilitated in B. bovis is needed. However, the identification of Cryptosporidium viru- by the discovery that at least one heterologous B. bigemina pro- lence factors have been delayed by difficulties associated with moter is also active in this parasite (Silva et al., 2016). Gene the ability to maintain the parasite in long-term in vitro cultures editing based on CRISPR/Cas9 has been used successfully for and the lack of genetic manipulation tools. As was the case for genetic analysis of several apicomplexan parasites of veterinary Toxoplasma, Plasmodium and Babesia, transient transfection meth- importance (Cui and Yu, 2016), including C. parvum (Vinayak ods were initially established using the nanoluciferase gene under et al., 2015) and T. gondii (Shen et al., 2014a, b). In contrast to the control of regulatory parasite sequences. Initially, optimal T. gondii, the low level of non-homologous or random integration transfection conditions were established, allowing the identifica- of exogenous transfected genes in B. bovis suggests that this par- tion of C. parvum 50 and 30 regulatory regions and the selection asite uses mainly HDR rather than NHEJ repair mechanisms. of a selectable marker. Then, researchers were able to develop a Despite possible off-target cleavage and other potential limi- CRISPR/Cas9 gene editing system used for the repair of a co- tations, gene editing procedures can be used for understanding transfected mutated nLuc gene in C. parvum (Vinayak et al., gene function, generation of mutated attenuated parasites, or 2015), and to knock out the thymidine kinase gene using a classic as a tool for the development of novel vaccines with the targeted deletion approach. 708 C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710

In contrast to Cryptosporidium, T. gondii is relatively easy to cul- References ture in vitro and can be efficiently transfected. However, reverse genetic approaches using conventional transfection techniques Abrahamsen, M.S., Templeton, T.J., Enomoto, S., Abrahante, J.E., Zhu, G., Lancto, C.A., Deng, M., Liu, C., Widmer, G., Tzipori, S., Buck, G.A., Xu, P., Bankier, A.T., Dear, P. were difficult to implement in this parasite due to the relatively H., Konfortov, B.A., Spriggs, H.F., Iyer, L., Anantharaman, V., Aravind, L., Kapur, V., high rate of non-targeted insertions and low targeting frequencies. 2004. Complete genome sequence of the apicomplexan, Cryptosporidium As mentioned previously, this is due to unspecific integration by parvum. Science 304 (5669), 441–445. KU-80-mediated activity of the NHEJ DNA repair pathway, which Adamson, R., Lyons, K., Sharrard, M., Kinnaird, J., Swan, D., Graham, S., Shiels, B., Hall, R., 2001. Transient transfection of Theileria annulata. Mol. Biochem. Parasitol. does not rely on DNA sequence homology. These limitations can 114, 53–61. now be overcome either by using KU80 mutant strains of T. gondii Alzan, H.F., Knowles, D.P., Suarez, C.E., 2016a. Comparative bioinformatics analysis for reverse genetic analysis, or by using more specific gene target- of transcription factor genes indicates conservation of key regulatory domains among Babesia bovis, Babesia microti, and Theileria equi. PLoS Negl. Trop. Dis. 10 ing methods such as the CRISPR/Cas9 method. Recent advances (11), e0004983. using efficient gene editing methods have been recently reported Alzan, H.F., Lau, A.O., Knowles, D.P., Herndon, D.R., Ueti, M.W., Scoles, G.A., (Shen et al., 2014a,b; Sidik et al., 2014). Subsequently, Sidik et al. Kappmeyer, L.S., Suarez, C.E., 2016b. Expression of 6-Cys gene superfamily defines Babesia bovis sexual stage Development within Rhipicephalus microplus. (2016) developed a genome –wide screen in order to define essen- PLoS One 11 (9), e0163791. tial apicomplexan genes using a gene editing method based on Alzan, H.F., Silva, M.G., Davis, W.C., Herndon, D.R., Schneider, D.A., Suarez, C.E., 2017. CRISPR/Cas9. In addition, combination of an auxin-inducible Geno- and phenotypic characteristics of a transfected Babesia bovis 6-Cys-E knockout clonal line. Parasit. Vectors 10, 214. degron (AID) and genome editing techniques resulted in a novel Allred, D.R., Al-Khedery, B., 2004. Antigenic variation and cytoadhesion in Babesia system for examining essential proteins in T. gondii (Brown et al., bovis and Plasmodium falciparum: different logics achieve the same goal. Mol. 2017). Biochem. Parasitol. 134, 27–35. Asada, M., Goto, Y., Yahata, K., Yokoyama, N., Kawai, S., Inoue, N., Kaneko, O., Kawazu, S., 2012a. Gliding motility of Babesia bovis merozoites visualized by 10. Conclusions time-lapse video microscopy. PLoS One 7, e35227. Asada, M., Tanaka, M., Goto, Y., Yokoyama, N., Inoue, N., Kawazu, S., 2012b. Stable expression of green fluorescent protein and targeted disruption of thioredoxin We have briefly reviewed general features of apicomplexan par- peroxidase-1 gene in Babesia bovis with the WR99210/dhfr selection system. asites of veterinary importance and current gene manipulation Mol. Biochem. Parasitol. 181, 162–170. methods that can be used to improve our understanding of their Asada, M., Yahata, K., Hakimi, H., Yokoyama, N., Igarashi, I., Kaneko, O., Suarez, C.E., Kawazu, S., 2015. Transfection of Babesia bovis by double selection with genetics and biology. These parasites have a very complex biology WR99210 and blasticidin-S and its application for functional analysis of and diverse reproductive choices, giving them enormous survival thioredoxin peroxidase-1. PLoS One 10, e0125993. advantages, while providing significant challenges for efficient Aurrecoechea, C., Barreto, A., Basenko, E.Y., Brestelli, J., Brunk, B.P., Cade, S., Crouch, K., Doherty, R., Falke, D., Fischer, S., Gajria, B., Harb, O.S., Heiges, M., Hertz- and improved methods of control. Genetic modification methods Fowler, C., Hu, S., Iodice, J., Kissinger, J.C., Lawrence, C., Li, W., Pinney, D.F., are being continuously improved and expanded in their applica- Pulman, J.A., Roos, D.S., Shanmugasundram, A., Silva-Franco, F., Steinbiss, S., tions, and it is likely that gene editing methods will be soon applied Stoeckert Jr., C.J., Spruill, D., Wang, H., Warrenfeltz, S., Zheng, J., 2017. EuPathDB: to the study of all the organisms discussed in this review. These the eukaryotic pathogen genomics database resource. Nucleic Acids Res. 45, D581–D591. approaches may lead to more efficient identification of target Bastos, R.G., Suarez, C.E., Laughery, J.M., Johnson, W.C., Ueti, M.W., Knowles, D.P., molecules in these parasites that can be used for developing novel 2013. Differential expression of three members of the multidomain adhesion strategies of control. For instance, interrogation of the role and CCp family in Babesia bigemina, Babesia bovis and Theileria equi. PLoS One 8, e67765. function of the secreted proteins that mediate the transformation Beverley, S.M., 2015. Parasitology: CRISPR for Cryptosporidium. Nature 523, 413– of Theileria-infected lymphocytes (many of which have already 414. been predicted through in silico identification of predicted Bonnin, A., Gut, J., Dubremetz, J.F., Nelson, R.G., Camerlynck, P., 1995. Monoclonal antibodies identify a subset of dense granules in Cryptosporidium parvum zoites secreted schizont proteins), and apical complex molecules secreted and gamonts. J. Eukaryot. Microbiol. 42, 395–401. by Toxoplasma, Babesia, and Crytosporidium. The rapid pace of pro- Brown, K.M., Long, S., Sibley, L.D., 2017. Plasma membrane association by N- gress of GMMs, together with other tools, will result in the emer- acylation governs PKG function in Toxoplasma gondii. mBio 8 (3). Calhoun, S.F., Reed, J., Alexander, N., Mason, C.E., Deitsch, K.W., Kirkman, L.A., 2017. gence of novel integrated methods for the functional analysis and Chromosome end repair and genome stability in Plasmodium falciparum. mBio 8 modification of genomes, leading to improved understanding of (4). apicomplexan and other parasite lifestyles, and ultimately, to the Cérède, O., Dubremetz, J.F., Soête, M., Deslée, D., Vial, H., Bout, D., Lebrun, M., 2005. Synergistic role of micronemal proteins in Toxoplasma gondii virulence. J. Exp. rational design of improved methods for the control of currently Med. 201, 453–463. important animal infectious parasitic diseases that threaten the Chartier, C., Paraud, C., 2012. Coccidiosis due to Eimeria in sheep and goats, a review. chain of food production, and /or a source of infections in humans. Small Ruminant Res. 103 (1), 84–92. While control of these diseases will be facilitated by the applica- Cui, Y., Yu, L., 2016. Application of the CRISPR/Cas9 gene editing technique to research on functional genomes of parasites. Parasitol. Int. 65, 641–644. tion of GMMs, ultimately, the solutions to multifaceted sanitary Dantas-Torres, F., 2015. Climate change, biodiversity, ticks and tick-borne diseases: problems affecting food production and animal and human health the butterfly effect. Int. J. Parasitol. Parasites Wildl. 4 (3), 452–461. will require better and more coordinated management of diverse Darghouth, M.A., Bouttour, A., Kilan, M., 1999. Tropical Theileriosis in Tunisia epidemiology and control. Parasitologia 41 (Suppl. 1), 33–36. scientific, cultural, economic, and political resources available de Castro, J.J., 1997. Sustainable tick and tickborne disease control in livestock worldwide. improvement in developing countries. Vet. Parasitol. 71, 77–97. De Goeyse, I., Jansen, F., Madder, M., Hayashida, K., Berkvens, D., Dobbelaere, D., Geysen, D., 2015. Transfection of live, tick derived sporozoites of the protozoan Apicomplexan parasite Theileria parva. Vet. Parasitol. 208, 238– Acknowledgements 241. Di Giulio, D., Lynen, G., Morzaria, S., Oura, C., Bishop, R., 2009. Live immunization HFA was supported by a scholarship provided by the Egyptian against east coast fever-an update. Trends Parasitol. 25, 85–92. Dobdelaere, D., Huessler, V., 1999. Transfomation of leukocytes by Theileria parva government, Ministry of High Education and Scientific Research. and Theileria annulata. Ann. Rev. Microbiol. 53, 1–4. Thanks to Jacob Laughery and Paul Lacy for their assistance in Dzikowski, R., Deitsch, K., 2006. Antigenic variation by protozoan parasites: insights the preparation of the manuscript. We acknowledge support by from Babesia bovis. Mol. Microbiol. 59, 364–366. Florin-Christensen, M., Suarez, C.E., Rodriguez, A.E., Flores, D.A., Schnittger, L., 2014. the United States Department of Agriculture-Agriculture Research Vaccines against bovine babesiosis: where we are now and possible roads Service Current Research Information System Project No. 5348- ahead. Parasitology 28, 1–30. 32000-028-00D. Gaffar, F.R., Yatsuda, A.P., Franssen, F.F., de Vries, E., 2004a. (a). Erythrocyte invasion by Babesia bovis merozoites is inhibited by polyclonal antisera directed against C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710 709

peptides derived from a homologue of Plasmodium falciparum apical membrane Ran, F.A., Hsu, P.D., Wright, J., Agarwala, V., Scott, D.A., Zhang, F., 2013. antigen 1. Infect. Immun. 72, 2947–2955. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 11, 2281– Gaffar, F.R., Yatsuda, A.P., Franssen, F.F., de Vries, E., 2004b. (b). A Babesia bovis 2308. merozoite protein with a domain architecture highly similar to the Reichel, M.P., Alejandra, Ayanegui-Alcérreca M., Gondim, L.F., Ellis, J.T., 2013. What thrombospondin-related anonymous protein (TRAP) present in Plasmodium is the global economic impact of Neospora caninum in cattle – the billion dollar sporozoites. Mol. Biochem. Parasitol. 136, 25–34. question. Int. J. Parasitol. 43, 133–142. Gardner, M.J., Hall, N., Fung, E., White, O., Berriman, M., Hyman, R.W., et al., 2002. Salama, A.A., Terkawi, M.A., Kawai, S., Aboulaila, M., Nayel, M., Mousa, A., Zaghawa, Genome sequence of the human malaria parasite Plasmodium falciparum. A., Yokoyama, N., Igarashi, I., 2013. Specific antibody to a conserved region of Nature 419, 498–511. Babesia apical membrane antigen-1 inhibited the invasion of B. bovis into the Giles, J.R., Peterson, A.T., Busch, J.D., Olafson, P.U., Scoles, G.A., Davey, R.B., erythrocyte. Exp. Parasitol. 135, 623–628. Pound, J.M., Kammlah, D.M., Lohmeyer, K.H., Wagner, D.M., 2014. Invasive Sato, S., 2011. The apicomplexan plastid and its evolution. Cell. Mol. Life Sci. 68, potential of cattle fever ticks in the southern United States. Parasit. Vectors 17 1285–1296. (7), 189. Seeber, F., Steinfelder, S., 2007. Diversity, nomenclature, and taxonomy of protists. Goonewardene, R., Daily, J., Kaslow, D., Sullivan, T.J., Duffy, P., Carter, R., Mendis, K., Syst. Biol. 56 (4), 684–689. Wirth, D., 1993. Transfection of the malaria parasite and expression of firefly Shen, B., Brown, K.M., Lee, T.D., Sibley, L.D., 2014a. Efficient gene disruption luciferase. Proc. Natl. Acad. Sci. 90, 5234–5236. in diverse strains of Toxoplasma gondii using CRISPR/CAS9. mBio 5, e01114– Gubbels, M.J., Duraisingh, M.T., 2012. Evolution of apicomplexan secretory e1214. organelles. Int. J. Parasitol. 42, 1071–1081. Shen, B., Buguliskis, J.S., Lee, T.D., Sibley, L.D., 2014b. Functional analysis of Hao, L., Liu, X., Zhou, X., Li, J., Suo, X., 2007. Transient transfection of Eimeria tenella rhomboid proteases during Toxoplasma invasion. mBio 5, e01795– using yellow or red fluorescent protein as a marker. Mol. Biochem. Parasitol. e1814. 153 (2), 213–215. Sidik, S.M., Hackett, C.G., Tran, F., Westwood, N.J., Lourido, S., 2014. Efficient Howe, D.K., Sibley, L.D., 1997. Development of molecular genetics for genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS One 9, Neospora caninum: a complementary system to Toxoplasma gondii. Methods 2, e100450. 123–133. Sidik, S.M., Huet, D., Ganesan, S.M., Huynh, M.H., Wang, T., Nasamu, A.S., Thiru, P., Hsu, P.D., Lander, E.S., Zhang, F., 2014. Development and applications of CRISPR- Saeij, J.P., Carruthers, V.B., Niles, J.C., Lourido, S., 2016. A genome-wide CRISPR Cas9 for genome engineering. Cell 157, 1262–1278. screen in Toxoplasma identifies essential apicomplexan genes. Cell 166 (6), Huynh, M.H., Boulanger, M.J., Carruthers, V.B., 2014. A conserved apicomplexan 1423–1435.e12. microneme protein contributes to Toxoplasma gondii invasion and virulence. Silva, M.G., Knowles, D.P., Suarez, C.E., 2016. Identification of interchangeable cross- Infect. Immun. 82, 4358–4368. species function of elongation factor-1 alpha promoters in Babesia bigemina and Huynh, M.H., Carruthers, V.B., 2009. Tagging of endogenous genes in a Toxoplasma Babesia bovis. Parasit. Vectors 9, 576. gondii strain lacking Ku80. Eukaryot. Cell 8, 530–539. Silva, M.G., Ueti, M.W., Norimine, J., Florin-Christensen, M., Bastos, R.G., Goff, W.L., Karbowiak, G., 2014. The occurrence of the Dermacentor reticulatus tick–its Brown, W.C., Oliva, A., Suarez, C.E., 2010. Babesia bovis expresses a expansion to new areas and possible causes. Ann. Parasitol. 60 (1), neutralization-sensitive antigen that contains a microneme adhesive repeat 37–47. (MAR) domain. Parasitol. Int. 59, 294–297. Kats, L.M., Black, C.G., Proellocks, N.I., Coppel, R.L., 2006. Plasmodium rhoptries: how Smith, T.G., Walliker, D., Ranford-Cartwright, L.C., 2002. Sexual differentiation and things went pear-shaped. Trends Parasitol. 22, 269–276. sex determination in the Apicomplexa. Trends Parasitol. 18, 315–323. Kelleher, M., Tomley, F.M., 1998. Transient expression of beta-galactosidase in Smolarz, B., Wilczyn´ ski, J., Nowakowska, D., 2014. DNA repair mechanisms and differentiating sporozoites of Eimeria tenella. Mol. Biochem. Parasitol. 97 (1–2), Toxoplasma gondii infection. Arch. Microbiol. 196, 1–8. 21–31. Soldati, D., Boothroyd, J.C., 1993. Transient transfection and expression Kemp, L.E., Yamamoto, M., Soldati-Favre, D., 2013. Subversion of host cellular in the obligate intracellular parasite Toxoplasma gondii. Science 260, functions by the apicomplexan parasites. FEMS Microbiol. Rev. 37, 607–631. 349–352. Kepczynski, M., Róg, T., 2016. Functionalized lipids and surfactants for specific Suarez, C.E., Johnson, W.C., Herndon, D.R., Laughery, J.M., Davis, W.C., 2015. applications. Biochim. Biophys. Acta 1858, 2362–2379. Integration of a transfected gene into the genome of Babesia bovis occurs by Kim, K., Soldati, D., Boothroyd, J.C., 1993. Gene replacement in Toxoplasma gondii legitimate homologous recombination mechanisms. Mol. Biochem. Parasitol. with chloramphenicol acetyltransferase as selectable marker. Science 262, 911– 202, 23–28. 914. Suarez, C.E., Lacy, P., Laughery, J., Gonzalez Ma.G., McElwain T., 2007. Optimization Kim, K., Weiss, L.M., 2004. Toxoplasma gondii: the model apicomplexan. Int. J. of Babesia bovis transfection methods. Parassitologia 49, 67–70. Parasitol. 34, 423–432. Suarez, C.E., McElwain, T.F., 2008. Transient transfection of purified Babesia bovis Lander, E.S., 2016. The heroes of CRISPR. Cell 164, 18–28. merozoites. Exp. Parasitol. 118, 498–504. Li, W., Zhang, N., Liang, X., Li, J., Gong, P., Yu, X., Ma, G., Ryan, U.M., Zhang, X., 2009. Suarez, C.E., McElwain, T.F., 2009. Stable expression of a GFP-BSD fusion protein in Transient transfection of Cryptosporidium parvum using green fluorescent Babesia bovis merozoites. Int. J. Parasitol. 39 (3), 289–297. protein (GFP) as a marker. Mol. Biochem. Parasitol. 168 (2), 143–148. Suarez, C.E., McElwain, T.F., 2010. Transfection systems for Babesia bovis: a review of Lim, L., McFadden, G.I., 2010. The evolution, metabolism and functions of the methods for the transient and stable expression of exogenous genes. Vet. apicoplast. Philos. Trans. R. Soc. Lond. B Biol. Sci. 365, 749–763. Parasitol. 167, 205–215. Ma, D., Liu, F., 2015. Genome editing and its applications in model organisms. Suarez, C.E., Norimine, J., Lacy, P., McElwain, T.F., 2006. Characterization and Genomics Proteomics Bioinformatics 13, 336–344. gene expression of Babesia bovis elongation factor-1a. Int. J. Parasitol. 36, 965– Marsolier, J., Perichon, M., DeBarry, J.D., Villoutreix, B.O., Chluba, J., Lopez, T., 973. Garrido, C., Zhou, X.Z., Lu, K.P., Fritsch, L., Ait-Si-Ali, S., Mhadhbi, M., Medjkane, Suarez, C.E., Palmer, G.H., LeRoith, T., Florin-Christensen, M., Crabb, B., McElwain, T. S., Weitzman, J.B., 2015. Theileria parasites secrete a prolyl isomerase to F., 2004. Intergenic regions in the rhoptry associated protein-1 (rap-1) locus maintain host leukocyte transformation. Nature 520, 378–382. promote exogenous gene expression in Babesia bovis. Int. J. Parasitol. 34, 1177– McFadden, G.I., Yeh, E., 2017. The apicoplast: now you see it, now you don’t. Int. J. 1184. Parasitol. 47, 137–144. Swapna, L.S., Parkinson, J., 2017. Genomics of apicomplexan parasites. Crit. Rev. Mogollon, C.M., van Pul, F.J., Imai, T., Ramesar, J., Chevalley-Maurel, S., de Roo, G.M., Biochem. Mol. Biol. 52, 254–273. Veld, S.A., Kroeze, H., Franke-Fayard, B.M., Janse, C.J., Khan, S.M., 2016. Rapid Templeton, T.J., Pain, A., 2016. Diversity of extracellular proteins during the generation of marker-free P. falciparum fluorescent reporter lines using transition from the ’proto-apicomplexan’ alveolates to the apicomplexan modified CRISPR/Cas9 constructs and selection protocol. PLoS One 11, obligate parasites. Parasitology 143, 1–17. e0168362. Tokarevich, N., Tronin, A., Gnativ, B., Revich, B., Blinova, O., Evengard, B., 2017. Mota, C.M., Chen, A.L., Wang, K., Nadipuram, S., Vashisht, A.A., Wohlschlegel, J.A., Impact of air temperature variation on the ixodid ticks habitat and tick-borne Mineo, T.W.P., Bradley, P.J., 2017. New molecular tools in Neospora caninum for encephalitis incidence in the Russian Arctic: the case of the Komi Republic. Int. studying apicomplexan parasite proteins. Sci. Rep. 7, 3768. J. Circumpolar Health 76 (1), 1298882. Novikov, Y.M., Vaulin, O.V., 2014. Expansion of Anopheles maculipennis s.s. Van Dijk, M., Waters, A., Janse, C., 1995. Stable transfection of malaria parasite blood (Diptera: Culicidae) to northeastern Europe and northwestern Asia: causes stages. Science 268, 1358–1362. and consequences. Parasit. Vectors 22 (7), 389. Vinayak, S., Pawlowic, M.C., Sateriale, A., Brooks, C.F., Studstill, C.J., Bar-Peled, Y., Oldiges, D.P., Laughery, J.M., Tagliari, N.J., Leite Filho, R.V., Davis, W.C., da Silva, Vaz Cipriano, M.J., Striepen, B., 2015. Genetic modification of the diarrhoeal I., Jr., Termignoni C., Knowles D.P., Suarez C.E., 2016. Transfected Babesia bovis pathogen Cryptosporidium parvum. Nature 523, 477–480. expressing a tick GST as a live vector vaccine. PLoS Negl. Trop. Dis. 10, Visendi, P., Ng’ang’a, W., Bulimo, W., Bishop, R., Ochanda, J., de Villiers, E.P., 2011. e0005152. TparvaDB: a database to support Theileria parva vaccine development. Database Peng, D., Tarleton, R., 2015. EuPaGDT: a web tool tailored to design CRISPR guide (Oxford) 2011 (bar015), 2011. http://dx.doi.org/10.1093/database/bar015. RNAs for eukaryotic pathogens. Microb. Genom. 1 (4), e000033. Print. Pieszko, M., Weir, W., Goodhead, I., Kinnaird, J., Shiels, B., 2015. ApiAP2 factors as Wahlgren, M., Goel, S., Akhouri, R.R., 2017. Variant surface antigens of candidate regulators of stochastic commitment to merozoite production in Plasmodium falciparum and their roles in severe malaria. Nat. Rev. Microbiol. Theileria annulata. PLoS Negl. Trop. Dis. 9, e0003933. 15, 479–491. 710 C.E. Suarez et al. / International Journal for Parasitology 47 (2017) 701–710

Wright, A.V., Nuñez, J.K., Doudna, J.A., 2016. Biology and applications of Zhang, G., Huang, X., Boldbaatar, D., Battur, B., Battsetseg, B., Zhang, H., Yu, L., Li, Y., CRISPR systems: harnessing nature’s toolbox for genome engineering. Cell Luo, Y., Cao, S., Goo, Y.K., Yamagishi, J., Zhou, J., Zhang, S., Suzuki, H., Igarashi, I., 164, 29–44. Mikami, T., Nishikawa, Y., Xuan, X., 2010. Construction of Neospora caninum Yan, W., Liu, X., Shi, T., Hao, L., Tomley, F.M., Suo, X., 2009. Stable transfection of stably expressing TgSAG1 and evaluation of its protective effects against Eimeria tenella: constitutive expression of the YFP-YFP molecule throughout the Toxoplasma gondii infection in mice. Vaccine 28 (45), 7243–7247. life cycle. Int. J. Parasitol. 39 (1), 109–117.