Antiparasitic Evaluation of Aquiluscidin, a Cathelicidin Obtained from Crotalus aquilus, and the Vcn-23 Derivative Peptide against Babesia bovis, B. bigemina and B. ovata.

Babesiosis is a growing concern due to the increased prevalence of this infectious disease caused by Babesia protozoan parasites, affecting various animals and humans. With rising worries over medication side effects and emerging drug resistance, there is a notable shift towards researching babesiacidal agents. Antimicrobial peptides, specifically cathelicidins known for their broad-spectrum activity and immunomodulatory functions, have emerged as potential candidates. Aquiluscidin, a cathelicidin from Crotalus aquilus, and its derivative Vcn-23, have been of interest due to their previously observed antibacterial effects and non-hemolytic activity. This work aimed to characterize the effect of these peptides against three Babesia species. Results showed Aquiluscidin's significant antimicrobial effects on Babesia species, reducing the B. bigemina growth rate and exhibiting IC50 values of 14.48 and 20.70 µM against B. ovata and B. bovis, respectively. However, its efficacy was impacted by serum presence in culture, and it showed no inhibition against a B. bovis strain grown in serum-supplemented medium. Conversely, Vcn-23 did not demonstrate babesiacidal activity. In conclusion, Aquiluscidin shows antibabesia activity in vitro and its efficacy is affected by the presence of serum in the culture medium. Nevertheless, this peptide represents a candidate for further investigation of its antiparasitic properties and provides insights into potential alternatives for the treatment of babesiosis.

Stable expression of red fluorescent protein-blasticidin deaminase fusion gene (rfp-bsd) as a selectable marker for DNA transfection in Babesia ovata.

The red fluorescent protein(rfp)-blasticidin deaminase(bsd) fusion gene was transfected into Babesia ovata by electroporation with the plasmid DNA and selected with 15 µg/mL of blasticidin S under the in vitro culture condition. The transfected parasite with episomal DNA was selected and cultured for further analysis based on the presence of the rfp-bsd fusion gene by PCR and expression of the fusion protein by immunofluorescence antibody test under fluorescence microscopy for 2 months after the transfection. The results are the first, to our knowledge, to demonstrate the expression and stability of the episomal rfp-bsd fusion gene under the control of actin promoter as a selectable marker for the transfection system in B. ovata.

Establishment of a stable transfection and gene targeting system in Babesia divergens.

Babesia divergens is an emerging tick-borne pathogen considered as the principal causative agent of bovine babesiosis in Europe with a notable zoonotic risk to human health. Despite its increasing impact, considerable gaps persist in our understanding of the molecular interactions between this parasite and its hosts. In this study, we address the current limitation of functional genomic tools in B. divergens and introduce a stable transfection system specific to this parasite. We define the parameters for a drug selection system hdhfr-WR99210 and evaluate different transfection protocols for highly efficient generation of transgenic parasites expressing GFP. We proved that plasmid delivery into bovine erythrocytes prior to their infection is the most optimal transfection approach for B. divergens, providing novel evidence of Babesia parasites' ability to spontaneously uptake external DNA from erythrocytes cytoplasm. Furthermore, we validated the bidirectional and symmetrical activity of ef-tgtp promoter, enabling simultaneous expression of external genes. Lastly, we generated a B. divergens knockout line by targeting a 6-cys-e gene locus. The observed dispensability of this gene in intraerythrocytic parasite development makes it a suitable recipient locus for further transgenic application. The platform for genetic manipulations presented herein serves as the initial step towards developing advanced functional genomic tools enabling the discovery of B. divergens molecules involved in host-vector-pathogen interactions.

Complete mitochondrial genome analyses confirm that bat Polychromophilus and ungulate Plasmodium constitute a distinct clade independent of other Plasmodium species.

In recent phylogenetic studies, bat Polychromophilus and ungulate Plasmodium, two relatively understudied haemosporidian parasites within the Apicomplexa phylum, have often been overlooked. Instead, the focus has been primarily on haemosporidian parasites in primates, rodents, and birds. Several phylogenetic analyses of bat Polychromophilus have relied on limited datasets and short informative DNA sequences. As a result of these inherent limitations, the substantiation of their evolutionary stance has encountered a diminished degree of robust validation. This study successfully obtained complete mitochondrial genome sequences from 11 Polychromophilus parasites originating from Hipposideros gentilis and Myotis siligoensis bats for the first time. Additionally, the authors have sequenced the apicoplast caseinolytic protease C genes from Polychromophilus murinus and a potentially new Polychromophilus species. These mitochondrial genomes range in length from 5994 to 6001 bp and consist of three protein-coding genes (PCGs), seven small subunit ribosomal RNA genes (SSU rRNA), 12 large subunit ribosomal RNA genes (LSU rRNA), and seven miscellaneous RNA genes. Phylogenetic analyses using Bayesian Inference and Maximum Likelihood methods indicated robust support for the grouping of ungulate Plasmodium and bat Polychromophilus in a single clade separate from other Plasmodium spp., confirming previous reports, albeit with stronger evidence in this study. The divergence between Polychromophilus in bats and Plasmodium in ungulates occurred approximately 29.61 to 55.77 million years ago (Mya), with a node age estimated at 40.63 Mya. These findings highlight that the genus Plasmodium, which includes species found in ungulates, birds, reptiles, and other mammals, does not form a monophyletic group. By incorporating Polychromophilus in bats and Plasmodium in ungulates, this study contributes significantly to understanding the phylogenetic relationships within the Haemosporida order. It provides valuable insights into the evolutionary history and interconnections among these diverse parasites, thereby expanding knowledge in this field.

Chromosome-level genome assembly of Babesia caballi reveals diversity of multigene families among Babesia species.

BACKGROUND: Babesia caballi is an intraerythrocytic parasite from the phylum Apicomplexa, capable of infecting equids and causing equine piroplasmosis. However, since there is limited genome information available on B. caballi, molecular mechanisms involved in host specificity and pathogenicity of this species have not been fully elucidated yet. RESULTS: Genomic DNA from a B. caballi subclone was purified and sequenced using both Illumina and Nanopore technologies. The resulting assembled sequence consisted of nine contigs with a size of 12.9 Mbp, rendering a total of 5,910 protein-coding genes. The phylogenetic tree of Apicomplexan species was reconstructed using 263 orthologous genes. We identified 481 ves1-like genes and named "ves1c". In contrast, expansion of the major facilitator superfamily (mfs) observed in closely related B. bigemina and B. ovata species was not found in B. caballi. A set of repetitive units containing an open reading frame with a size of 297 bp was also identified. CONCLUSIONS: We present a chromosome-level genome assembly of B. caballi. Our genomic data may contribute to estimating gene expansion events involving multigene families and exploring the evolution of species from this genus.

Molecular Detection and Identification of Piroplasm in Cattle from Kathmandu Valley, Nepal.

BACKGROUND: Tick-borne protozoan parasites (TBPPs) cause significant problems for domestic animals' health in Nepal. TBPPs are routinely diagnosed by labor-intensive blood smear microscopy. In Nepal, there are some reports of Babesia and Theileria in cattle, although species identification is rarely performed. Therefore, we performed conventional nested PCR (nPCR) followed by sequence analysis to identify TBPP species infecting cattle in Nepal. METHODS: One hundred and six blood samples were collected from cattle in the Kathmandu Valley. Thin blood smears were prepared for microscopic examination. Parasite DNA was extracted from the blood, and nPCR and sequencing were performed to identify the TBPPs present. RESULTS: Among the 106 samples, 45 (42.5%) were positive for piroplasm (Babesia spp. and Theileria spp.) via microscope observation and 56 (52.8%) samples were positive via nPCR. The obtained PCR products were used for direct sequencing, and we identified the species as B. bigemina, B. bovis, T. annulate and T. orientalis. Phylogenetic analyses showed that the B. bovis, B. bigemina and T. orientalis sequences from this study belonged to each species clade. On the other hand, T. annulate was divided into two clades in the analysis, and our T. annulate sequences were also divided in these two clades. The piroplasm-positive cattle showed lower hemoglobin and red blood cells than healthy cattle. CONCLUSIONS: To the best of our knowledge, this study is the first to apply molecular detection and species determination of TBPPs in cattle in Nepal. The results of this study may be used as a starting point for the development of successful TBPP surveillance and prevention programs in Nepal.

Target Protein Expression on Tetrahymena thermophila Cell Surface Using the Signal Peptide and GPI Anchor Sequences of the Immobilization Antigen of Cryptocaryon irritans.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites".

Detection of Babesia bovis using loop-mediated isothermal amplification (LAMP) with improved thermostability, sensitivity and alternative visualization methods.

Bovine babesiosis is one of the most economically important tick-borne diseases in tropical and subtropical countries. A conventional microscopic diagnosis is typically used because it is inexpensive and expeditious. However, it is highly dependent on well-trained microscopists and tends to be incapable of detecting subpatent and chronic infections. Here, we developed a novel nucleic acid-based amplification method using loop-mediated isothermal amplification (LAMP) in conjunction with a colori-fluorometric dual indicator for the rapid and accurate detection of Babesia bovis based on the mitochondrial cytochrome b gene. We aimed to improve the thermostability, sensitivity, specificity, and alternative visualization of LAMP-based methods. We assessed its diagnostic performance compared to two conventional PCR agarose gel electrophoresis (PCR-AGE) methods. The thermostability of LAMP reaction mixtures and DNA templates in variable conditions was also assessed. In addition, we evaluated alternative visualization methods using different light sources including neon, LED, and UV lights. We found that the LAMP-neon was ten times more sensitive than the PCR-AGE, while the LAMP-LED and LAMP-UV were 1,000 times more sensitive. The current LAMP method showed no cross-amplification with uninfected cattle DNA or other common blood parasites in cattle, including Babesia bigemina, Theileria orientalis, Anaplasma marginale, and Trypanosoma evansi. In addition, the developed LAMP method has good thermostability and the potential for on-site utility as B. bovis DNA could still be detected up to 72 h after initial preparation. Our findings suggested that the developed LAMP method provides an alternative approach for B. bovis detection with sensitivity higher than PCR-AGE diagnostics, high specificity, and the flexibility to use neon, LED, and UV light sources for positive signal observations.

Myzomyia and Pyretophorus series of Anopheles mosquitoes acting as probable vectors of the goat malaria parasite Plasmodium caprae in Thailand.

Unlike malaria parasites in humans, non-human primates, rodents, and birds, ungulate malaria parasites and their vectors have received little attention. As a result, understanding of the hosts, vectors, and biology of ungulate malaria parasites has remained limited. In this study, we aimed to identify the vectors of the goat malaria parasite Plasmodium caprae. A total of 1019 anopheline and 133 non-anopheline mosquitoes were collected from goat farms in Thailand, where P. caprae-infected goats were discovered. Anopheline mosquitoes were identified using molecular biological methods that target the cytochrome c oxidase subunit 1 (cox1), the cytochrome c oxidase subunit 2 (cox2) genes, and the internal transcribed spacer 2 (ITS2) region. Pool and individual mosquitoes were tested for P. caprae using the head-thorax parts that contain the salivary glands, with primers targeting three genetic markers including cytochrome b, cytochrome c oxidase subunit 1, and 18S small subunit ribosomal RNA genes. Additionally, goat blood samples were collected concurrently with mosquito surveys and screened to determine the status of malaria infection. This study revealed nine mosquito species belonging to six groups on goat farms, including Hyrcanus, Barbirostris, Subpictus, Funestus, Tessellatus, and Annularis. The DNA of P. caprae was detected in Anopheles subpictus and Anopheles aconitus. This is the first time An. subpictus and An. aconitus have been implicated as probable vectors of P. caprae.

Molecular characterization of anopheline mosquitoes from the goat malaria-endemic areas of Thailand.

Despite the fact that over a 100 anopheline mosquito species have been identified as human malaria vectors, little is known about ungulate malaria vectors. Consequently, we focused on investigating the bionomics and genetic characterizations of anopheline mosquitoes in goat malaria-endemic regions. We also attempted to screen for ungulate malaria potential vectors. A total of 1019 female anopheline mosquitoes were collected from six goat farms in four provinces of Thailand from 2020 to 2021. Mosquitoes were morphologically identified and subsequently confirmed using the mitochondrial DNA barcoding region-cytochrome oxidase c subunit I (MtDNA-COI), mitochondrial DNA-cytochrome c oxidase subunit II (MtDNA-COII), and ribosomal DNA internal transcribed spacer 2 (rDNA-ITS2) sequences. The current study reveals the genetic characteristics and distribution of nine mosquito species within the Anopheles and Cellia subgenera. Four dominant species, including Anopheles peditaeniatus, Anopheles subpictus, Anopheles vagus, and Anopheles aconitus exhibited significant intraspecific gene flow within their corresponding species. Although malaria parasites were not found in 126 mosquito pools, meaning more investigation is necessary, the current study adds to the existing DNA barcoding data collection and improves the current understanding of the genetic structure and distribution of anopheline mosquito species, which could be useful for effective control of mosquito-borne diseases.

Phosphatidylinositol 4-kinase is a viable target for the radical cure of Babesia microti infection in immunocompromised hosts.

Human babesiosis is a global emerging tick-borne disease caused by infection with intra-erythrocytic parasites of the genus Babesia. With the rise in human babesiosis cases, the discovery and development of new anti-Babesia drugs are essential. Phosphatidylinositol 4-kinase (PI4K) is a widely present eukaryotic enzyme that phosphorylates lipids to regulate intracellular signaling and trafficking. Previously, we have shown that MMV390048, an inhibitor of PI4K, showed potent inhibition against Babesia species, revealing PI4K as a druggable target for babesiosis. However, twice-administered, 7-day regimens failed to clear Babesia microti parasites from the immunocompromised host. Hence, in this study, we wanted to clarify whether targeting PI4K has the potential for the radical cure of babesiosis. In a B. microti-infected SCID mouse model, a 64-day-consecutive treatment with MMV390048 resulted in the clearance of parasites. Meanwhile, an atovaquone (ATO) resistant parasite line was isolated from the group treated with ATO plus azithromycin. A nonsynonymous variant in the Y272C of the cytochrome b gene was confirmed by sequencing. Likewise, MMV390048 showed potent inhibition against ATO-resistant parasites. These results provide evidence of PI4K as a viable drug target for the radical cure of babesiosis, which will contribute to designing new compounds that can eradicate parasites.

Advances in understanding red blood cell modifications by Babesia.

Babesia are tick-borne protozoan parasites that can infect livestock, pets, wildlife animals, and humans. In the mammalian host, they invade and multiply within red blood cells (RBCs). To support their development as obligate intracellular parasites, Babesia export numerous proteins to modify the RBC during invasion and development. Such exported proteins are likely important for parasite survival and pathogenicity and thus represent candidate drug or vaccine targets. The availability of complete genome sequences and the establishment of transfection systems for several Babesia species have aided the identification and functional characterization of exported proteins. Here, we review exported Babesia proteins; discuss their functions in the context of immune evasion, cytoadhesion, and nutrient uptake; and highlight possible future topics for research and application in this field.

Efficacy of the Antimalarial MMV390048 against Babesia Infection Reveals Phosphatidylinositol 4-Kinase as a Druggable Target for Babesiosis.

The present study aimed to evaluate the anti-Babesia effect of MMV390048, a drug that inhibits Plasmodium by targeting the phosphatidylinositol 4-kinase (PI4K). The half inhibitory concentration (IC50) of MMV390048 against the in vitro growth of Babesia gibsoni was 6.9 ± 0.9 µM. In immunocompetent mice, oral treatment with MMV390048 at a concentration of 20 mg/kg effectively inhibited the growth of B. microti (Peabody mjr strain). The peak parasitemia in the control group was 30.5%, whereas the peak parasitemia in the MMV390048-treated group was 3.4%. Meanwhile, MMV390048 also showed inhibition on the growth of B. rodhaini (Australia strain), a highly pathogenic rodent Babesia species. All MMV390048-treated mice survived, whereas the mice in control group died within 10 days postinfection (DPI). The first 7-day administration of MMV390048 in B. microti-infected, severe combined immunodeficiency (SCID) mice delayed the rise of parasitemia by 26 days. Subsequently, a second 7-day administration was given upon recurrence. At 52 DPI, a parasite relapse (in 1 out of 5 mice) and a mutation in the B. microti PI4K L746S, a MMV390048 resistance-related gene, were detected. Although the radical cure of B. microti infection in immunocompromised host SCID mice was not achieved, results from this study showed that MMV390048 has excellent inhibitory effects on Babesia parasites, revealing a new treatment strategy for babesiosis: targeting the B. microti PI4K.

Protozoan and Rickettsial Pathogens in Ticks Collected from Infested Cattle from Turkey.

Diseases caused by tick-transmitted pathogens including bacteria, viruses, and protozoa are of veterinary and medical importance, especially in tropical and subtropical regions including Turkey. Hence, molecular surveillance of tick-borne diseases will improve the understanding of their distribution towards effective control. This study aimed to investigate the presence and perform molecular characterization of Babesia sp., Theileria sp., Anaplasma sp., Ehrlichia sp., and Rickettsia sp. in tick species collected from cattle in five provinces of Turkey. A total of 277 adult ticks (males and females) were collected. After microscopic identification, tick pools were generated according to tick species, host animal, and sampling sites prior to DNA extraction. Molecular identification of the tick species was conducted through PCR assays. Out of 90 DNA pools, 57.8% (52/90) were detected to harbor at least 1 pathogen. The most frequently-detected pathogens were Babesia bovis, with a minimum detection rate of 7.9%, followed by Ehrlichia sp. (7.2%), Theileria annulata (5.8%), Coxiella sp. (3.3%), Anaplasma marginale (2.5%), Rickettsia sp. (2.5%), and B. occultans (0.7%). Rickettsia sp. identified in this study include Candidatus Rickettsia barbariae, R. aeschlimannii, and Rickettsia sp. Chad. All sequences obtained from this study showed 99.05−100% nucleotide identity with those deposited in GenBank (query cover range: 89−100%). This is the first molecular detection of Rickettsia sp. Chad, a variant of Astrakhan fever rickettsia, in Turkey. Results from this survey provide a reference for the distribution of ticks and tick-borne pathogens in cattle and expand the knowledge of tick-borne diseases in Turkey.

Myzorhynchus series of Anopheles mosquitoes as potential vectors of Plasmodium bubalis in Thailand.

Ungulate malaria parasites and their vectors are among the least studied when compared to other medically important species. As a result, a thorough understanding of ungulate malaria parasites, hosts, and mosquito vectors has been lacking, necessitating additional research efforts. This study aimed to identify the vector(s) of Plasmodium bubalis. A total of 187 female mosquitoes (133 Anopheles spp., 24 Culex spp., 24 Aedes spp., and 6 Mansonia spp. collected from a buffalo farm in Thailand where concurrently collected water buffalo samples were examined and we found only Anopheles spp. samples were P. bubalis positive. Molecular identification of anopheline mosquito species was conducted by sequencing of the PCR products targeting cytochrome c oxidase subunit 1 (cox1), cytochrome c oxidase subunit 2 (cox2), and internal transcribed spacer 2 (ITS2) markers. We observed 5 distinct groups of anopheline mosquitoes: Barbirostris, Hyrcanus, Ludlowae, Funestus, and Jamesii groups. The Barbirostris group (Anopheles wejchoochotei or Anopheles campestris) and the Hyrcanus group (Anopheles peditaeniatus) were positive for P. bubalis. Thus, for the first time, our study implicated these anopheline mosquito species as probable vectors of P. bubalis in Thailand.

Brachylaima phaedusae n. sp. (Trematoda: Brachylaimidae) from door snails in Japan.

The metacercarial infections of door snails (Gastropoda: Clausiliidae) with unknown species of the genus Brachylaima (Trematoda: Brachylaimidae) have recently been reported in eastern Honshu and Kyushu, Japan. A large scale snail survey was carried out to clarify their taxonomic status. From the period of 2015 to 2020, a total of 1239 land snails (768 door snails and 471 others) were collected from 32 localities in Honshu, Shikoku, and Kyushu. The resulting trematode isolates were identified as Brachylaima sp. by mitochondrial DNA barcoding. The sporocysts were found only a few from Megalophaedusa sublunellata (Clausiliidae), Tauphaedusa subaculus (Clausiliidae), and Aegista trochula (Camaenidae), while the metacercariae were frequently detected from 14 species of Clausiliidae and 2 species of other families. Although Brachylaima sp. showed a broad range of intermediate hosts, door snails seem to be very important to drive the life cycle. The gravid adults of Brachylaima sp. was experimentally raised from metacercariae using immunosuppressed mice. Morphological, phylogenetical, and ecological considerations prompted us to propose Brachylaima phaedusae n. sp. for this unknown species. The definitive hosts of the new species are completely unknown. The wide geographic distribution and high genetic diversity of the new species suggest a possibility that the definitive host is ground-foraging birds, which prefer door snails.

Distinct effects on the secretion of MTRAP and AMA1 in Plasmodium yoelii following deletion of acylated pleckstrin homology domain-containing protein.

Plasmodium, the causative agents of malaria, are obligate intracellular organisms. In humans, pathogenesis is caused by the blood stage parasite, which multiplies within erythrocytes, thus erythrocyte invasion is an essential developmental step. Merozoite form parasites released into the blood stream coordinately secrets a panel of proteins from the microneme secretory organelles for gliding motility, establishment of a tight junction with a target naive erythrocyte, and subsequent internalization. A protein identified in Toxoplasma gondii facilitates microneme fusion with the plasma membrane for exocytosis; namely, acylated pleckstrin homology domain-containing protein (APH). To obtain insight into the differential microneme discharge by malaria parasites, in this study we analyzed the consequences of APH deletion in the rodent malaria model, Plasmodium yoelii, using a DiCre-based inducible knockout method. We found that APH deletion resulted in a reduction in parasite asexual growth and erythrocyte invasion, with some parasites retaining the ability to invade and grow without APH. APH deletion impaired the secretion of microneme proteins, MTRAP and AMA1, and upon contact with erythrocytes the secretion of MTRAP, but not AMA1, was observed. APH-deleted merozoites were able to attach to and deform erythrocytes, consistent with the observed MTRAP secretion. Tight junctions were formed, but echinocytosis after merozoite internalization into erythrocytes was significantly reduced, consistent with the observed absence of AMA1 secretion. Together with our observation that APH largely colocalized with MTRAP, but less with AMA1, we propose that APH is directly involved in MTRAP secretion; whereas any role of APH in AMA1 secretion is indirect in Plasmodium.

Gliding motility of Plasmodium merozoites.

Plasmodium malaria parasites are obligate intracellular protozoans that use a unique form of locomotion, termed gliding motility, to move through host tissues and invade cells. The process is substrate dependent and powered by an actomyosin motor that drives the posterior translocation of extracellular adhesins which, in turn, propel the parasite forward. Gliding motility is essential for tissue translocation in the sporozoite and ookinete stages; however, the short-lived erythrocyte-invading merozoite stage has never been observed to undergo gliding movement. Here we show Plasmodium merozoites possess the ability to undergo gliding motility in vitro and that this mechanism is likely an important precursor step for successful parasite invasion. We demonstrate that two human infective species, Plasmodium falciparum and Plasmodium knowlesi, have distinct merozoite motility profiles which may reflect distinct invasion strategies. Additionally, we develop and validate a higher throughput assay to evaluate the effects of genetic and pharmacological perturbations on both the molecular motor and the complex signaling cascade that regulates motility in merozoites. The discovery of merozoite motility provides a model to study the glideosome and adds a dimension for work aiming to develop treatments targeting the blood stage invasion pathways.

Recent Advances in Molecular Genetic Tools for Babesia.

Development of in vitro culture and completion of genome sequencing of several Babesia parasites promoted the efforts to establish transfection systems for these parasites to dissect the gene functions. It has been more than a decade since the establishment of first transfection for Babesia bovis, the causative agent of bovine babesiosis. However, the number of genes that were targeted by genetic tools in Babesia parasites is limited. This is partially due to the low efficiencies of these methods. The recent adaptation of CRISPR/Cas9 for genome editing of Babesia bovis can accelerate the efforts for dissecting this parasite's genome and extend the knowledge on biological aspects of erythrocytic and tick stages of Babesia. Additionally, glmS ribozyme as a conditional knockdown system is available that could be used for the characterization of essential genes. The development of high throughput genetic tools is needed to dissect the function of multigene families, targeting several genes in a specific pathway, and finally genome-wide identification of essential genes to find novel drug targets. In this review, we summarized the current tools that are available for Babesia and the genes that are being targeted by these tools. This may draw a perspective for the future development of genetic tools and pave the way for the identification of novel drugs or vaccine targets.