Current advances in serological and molecular diagnosis of Schistosoma mekongi infection.

Schistosomiasis, a neglected tropical disease, caused by blood flukes belonging to the genus Schistosoma; it persists as a public health problem in selected regions throughout Africa, South America, and Asia. Schistosoma mekongi, a zoonotic schistosome species endemic to the Mekong River in Laos and Cambodia, is one of the significant causes of human schistosomiasis along with S. japonicum, S. mansoni, S. haematobium and S. intercalatum. Since its discovery, S. mekongi infection has been highly prevalent in communities along the Mekong River. Although surveillance and control measures have shown success in recent years, more robust diagnostic tools are still needed to establish more efficient control and prevention strategies to achieve and sustain an elimination status. Diagnosis of S. mekongi infection still relies on copro-parasitological techniques, commonly made by Kato-Katz stool examination. Serological techniques such as enzyme-linked immunosorbent assay (ELISA) may also be applicable but in a limited setting. Targeted molecular and serological tools specific to the species, on the other hand, have been limited. This is due, in part, to the limited research and studies on the molecular biology of S. mekongi since genome information of this species has not yet been released. In this review, current advances, and gaps and limitations in the molecular and immunological diagnosis of S. mekongi are discussed.

Aurantoside L, a New Tetramic Acid Glycoside with Anti-Leishmanial Activity Isolated from the Marine Sponge Siliquariaspongia japonica.

A new tetramic acid glycoside, aurantoside L (1), was isolated from the sponge Siliquariaspongia japonica collected at Tsushima Is., Nagasaki Prefecture, Japan. The structure of aurantoside L (1) composed of a tetramic acid bearing a chlorinated polyene system and a trisaccharide part was elucidated using spectral analysis. Aurantoside L (1) showed anti-parasitic activity against L. amazonensis with an IC50 value of 0.74 µM.

Erratum: Ascofuranone antibiotic is a promising trypanocidal drug for nagana.

No abstract available.

Tabanus chrysurus is a potential biological vector of Trypanosoma (Megatrypanum) theileri in Japan.

Several species of horse flies (Diptera: Tabanidae) are known as vectors of Trypanosoma (Megatrypanum) theileri and T. theileri-like trypanosomes; these host-parasite relationships were established based on the developmental stages of these parasites discovered in the hindgut of horse flies. T. theileri and T. theileri-like trypanosomes have been detected in cattle and wild deer in Japan; however, the vector horse fly species remains unidentified. Therefore, in this study, we aimed to identify the potential horse fly species serving as vectors of T. theileri in Japan. A total of 176 horse flies were collected between June to September 2020 and 2021 in Tokachi, Hokkaido, Japan. The T. theileri infection in the captured horse flies was determined by PCR and microscopic analyses of their midgut and hindgut. Additionally, the trypanosome, microscopically detected in a horse fly, was molecularly characterized and phylogenetically analyzed using 18S rRNA and partial cathepsin L-like protein gene (CATL) sequence of the trypanosome. The microscopy and PCR analyses revealed 0.57% and 35.8% prevalence of T. theileri in horse flies, respectively. Epimastigote stages of T. theileri, adhered to the hindgut epithelial cells of Tabanus chrysurus via flagella or actively moving in the lumen of the gut, were detected. Phylogenetic analysis revealed the connection of isolated trypanosomes with T. theileri in the TthI clade. These results suggest that Ta. chrysurus is a potential vector of T. theileri.

Ascofuranone antibiotic is a promising trypanocidal drug for nagana.

Trypanosomosis is a disease complex which affects both humans and animals in sub-Saharan Africa, transmitted by the tsetse fly and distributed within the tsetse belt of Africa. But some trypanosome species, for example, Trypanosoma brucei evansi, T. vivax, T. theileri and T. b. equiperdum are endemic outside the tsetse belt of Africa transmitted by biting flies, for example, Tabanus and Stomoxys, or venereal transmission, respectively. Trypanocidal drugs remain the principal method of animal trypanosomosis control in most African countries. However, there is a growing concern that their effectiveness may be severely curtailed by widespread drug resistance. A minimum number of six male cattle calves were recruited for the study. They were randomly grouped into two (T. vivax and T. congolense groups) of three calves each. One calf per group served as a control while two calves were treatment group. They were inoculated with 105 cells/mL parasites in phosphate buffered solution (PBS) in 2 mL. When parasitaemia reached 1 × 107.8 cells/mL trypanosomes per mL in calves, treatment was instituted with 20 mL (25 mg/kg in 100 kg calf) ascofuranone (AF) for treatment calves, while the control ones were administered a placebo (20 mL PBS) intramuscularly. This study revealed that T. vivax was successfully cleared by AF but the T. congolense group was not cleared effectively.Contribution: There is an urgent need to develop new drugs which this study sought to address. It is suggested that the AF compound can be developed further to be a sanative drug for T. vivax in non-tsetse infested areas like South Americas.

Prophylactic activity of orally administered dry-heat-sterilized Acremonium egyptiacum against Trypanosoma congolense-induced animal African trypanosomosis.

Animal African trypanosomosis (AAT) is an important global disease of livestock that causes economic losses of up to 4.5 billion US dollars per year. Thus, eliminating AAT in endemic countries will improve agricultural productivity and economic growth. To prevent AAT, vector control and the development of prophylactic drugs are crucial. Ascofuranone (AF) is a bioactive fungal compound with proven in vitro trypanocidal potency and in vivo treatment efficacy. However, the complex stereoselective synthesis of AF has prevented its cost-effective industrial production. Recently, a genetically modified strain of Acremonium egyptiacum fungus that produces a high yield of AF was developed. Therefore, we hypothesized that the oral administration of the AF-producing fungus itself may be effective against AAT. Hence, this study aimed to evaluate the prophylactic activity of orally administered dry-heat-sterilized A. egyptiacum against Trypanosoma congolense IL3000 infection using a mouse model. The survival rate was significantly prolonged (p = 0.009), and parasitemia was suppressed in all AF-fungus-treated groups (Group 1-9) compared with that in the untreated control group (Group 10). Hence, the trypanocidal activity of AF was retained after dry-heat-sterilization of the AF-producing fungus and that its oral administration effectively prevented AAT. Since AAT is endemic to rural areas with underdeveloped veterinary infrastructure, dry-heat-sterilized A. egyptiacum would be the most cost-effective potential treatment for AAT.

Molecular detection and internal transcribed spacer-1 sequence diversity of Trypanosoma evansi in goats from Cavite, Philippines.

Goat production is an important source of livelihood and food. Goats may serve as reservoir of surra affecting livestock production. Here, forty-two free-roaming goats from Cavite, Philippines were screened using two primer sets, Trypanosoma brucei minisatellite chromosome for initial detection and the internal transcribed spacer 1 (ITS-1) to determine phylogeny. Initial PCR detection showed that 19/42 (45%) goats were positive, much higher than the rate previously reported in goats from Cebu (34%). The infectivity rate was higher in male (56%) than in female (42%) and the rate was higher in young ≤1 year old (100%) than in adult >1 year old (43%). Phylogenetic analysis of the ITS-1 sequences between T. evansi goat samples and other isolates indicate potential interspecies transmission.

Expression profile analysis of the transient receptor potential (TRPM) channel, a possible target of praziquantel in Schistosoma japonicum.

The WHO considers schistosomiasis, which is controlled by the mass administration of the drug praziquantel (PZQ), to be a neglected tropical disease. Despite its clinical use for over four decades, PZQ remains the only choice of chemotherapy against this disease. Regarding the previous studies that demonstrated that PZQ activates the transient receptor potential (TRP) channel in Schistosoma mansoni (Sm.TRPMPZQ), the expression profile of the ortholog of this channel gene (Smp_246790.5) in S. japonicum (EWB00_008853) (Sj.TRPMPZQ) was analyzed. The relative expression of this gene in various stages of the parasite lifecycle was analyzed by quantitative real-time reverse transcription-PCR (qRT-PCR), and the expression of Sj.TRPMPZQ was observed by immunohistochemical staining using anti-serum against the recombinant Sj.TRPMPZQ protein. qRT-PCR revealed the significantly lower mRNA expression in the snail stage in comparison to other stages (p < 0.01). The relative quantity of the Sj.TRPMPZQ expression for paired females, unpaired males, and eggs was 60%, 56%, and 68%, respectively, in comparison to paired males that showed the highest expression (p < 0.05). Interestingly, immunostaining demonstrated that Sj.TRPMPZQ is expressed in the parenchyma which contains muscle cells, neuronal cells and tegument cells in adult worms. This may support the two major effects of PZQ-worm paralysis and tegument disruption-induced by channel activation. Moreover, the channel was expressed in both the eggshell and the miracidia inside, but could not be observed in sporocyst. These results suggest that the expression of Sj.TRPMPQZ corresponds to the known sensitivity of S. japonicum to PZQ.

Efficacy of oral administration of ascofuranone with and without glycerol against Trypanosoma congolense.

In many developing countries, trypanosomosis in animals results in the reduction of livestock productivity. Since trypanosomosis is endemic to rural areas where medical and veterinary infrastructure is underdeveloped, development of affordable and easy-to-maintain drugs for treatment and prophylaxis against trypanosomosis is necessary. To this end, in this study, we evaluated the efficacy of oral administration of ascofuranone (AF), with and without glycerol (GOL), against trypanosomosis, using a mouse model. We used T. congolense IL3000, the most virulent animal-infecting trypanosome, and BALB/c mice in this study. Eight mice were assigned to either of Groups 1-7: non-infected, untreated, AF 10, 20, 30, 50, and 100 mg/kg with or without GOL, respectively. In the experiment with AF administered with GOL, survival rates were 0% in Group 2 (untreated) and Group 3 (AF 10 mg/kg), 37.5% in Group 4 (AF 20 mg/kg) and Group 5 (AF 30 mg/kg), 50% in Group 6 (AF 50 mg/kg), and 100% in Group 7 (AF 100 mg/kg). In groups in which AF was administered without GOL, survival rates were 0% in Group 2 (untreated), Group 3 (AF 10 mg/kg), Group 4 (AF 20 mg/kg), Group 5 (AF 30 mg/kg), and Group 6 (AF 50 mg/kg), and 12.5% in Group 7 (AF 100 mg/kg), with one mouse surviving till the end of the observation period. The results of the analysis showed that survival rates were significantly higher in all groups (Groups 3-7) than in the untreated group (Group 2) (p < 0.05). Furthermore, a comparison of groups with or without GOL at the same AF concentration revealed that the survival rate was significantly higher in the group treated with GOL. These results suggest that the treatment efficacy of AF against animal trypanosomosis caused by T. congolense is greater when co-administered with GOL, and that oral administration of AF could be a new therapeutic strategy for animal African trypanosomosis.

Seasonal Variation and Factors Affecting Trypanosoma theileri Infection in Wild Sika Deer (Ezo Sika Deer Cervus nippon yesoensis) in Eastern Hokkaido.

Trypanosoma (Megatrypanum) spp. are isolated from domestic and wild ruminants, including deer, worldwide. The prevalence of trypanosomes in mammals is influenced by a number of factors such as host age and vector abundance. However, the seasonal variation of and factors affecting trypanosome infection in the wild deer population remain elusive. In this study, we analyzed the seasonal variation in trypanosome prevalence and the factors that affect Trypanosoma theileri Laveran, 1902, infection in wild sika deer (Ezo sika deer) Cervus nippon yesoensis (Heude, 1884) in Eastern Hokkaido through a two-year survey. Seasonal variation in the prevalence of trypanosome infection in the deer population ranged from 0 to 41% as per hematocrit concentration and 17 to 89% as per PCR results. In general, the prevalence of T. theileri by PCR in 2020 was higher than that in 2019. Moreover, the prevalence was significantly higher in the aged population than among the younger population. These findings may explain why individual conditions and sampling season were associated with trypanosome prevalence. This is the first study to investigate the seasonal variation in and risk factors affecting trypanosome infection in wild deer.

Spherical Body Protein 4 from Babesia bigemina: A Novel Gene That Contains Conserved B-Cell Epitopes and Induces Cross-Reactive Neutralizing Antibodies in Babesia ovata.

Bovine babesiosis is a tick-transmitted disease caused by intraerythrocytic protozoan parasites of the genus Babesia. Its main causative agents in the Americas are Babesia bigemina and Babesia bovis, while Babesia ovata affects cattle in Asia. All Babesia species secrete proteins stored in organelles of the apical complex, which are involved in all steps of the invasion process of vertebrate host cells. Unlike other apicomplexans, which have dense granules, babesia parasites instead have large, round intracellular organelles called spherical bodies. Evidence suggests that proteins from these organelles are released during the process of invading red blood cells, where spherical body proteins (SBPs) play an important role in cytoskeleton reorganization. In this study, we characterized the gene that encodes SBP4 in B. bigemina. This gene is transcribed and expressed in the erythrocytic stages of B. bigemina. The sbp4 gene consists of 834 nucleotides without introns that encode a protein of 277 amino acids. In silico analysis predicted a signal peptide that is cleaved at residue 20, producing a 28.88-kDa protein. The presence of a signal peptide and the absence of transmembrane domains suggest that this protein is secreted. Importantly, when cattle were immunized with recombinant B. bigemina SBP4, antibodies identified B. bigemina and B. ovata merozoites according to confocal microscopy observations and were able to neutralize parasite multiplication in vitro for both species. Four peptides with predicted B-cell epitopes were identified to be conserved in 17 different isolates from six countries. Compared with the pre-immunization sera, antibodies against these conserved peptides reduced parasite invasion in vitro by 57%, 44%, 42%, and 38% for peptides 1, 2, 3, and 4, respectively (p < 0.05). Moreover, sera from cattle infected with B. bigemina cattle contained antibodies that recognized the individual peptides. All these results support the concept of spb4 as a new gene in B. bigemina that should be considered a candidate for a vaccine to control bovine babesiosis.

Evaluation of Crude and Recombinant Antigens of Schistosoma japonicum for the Detection of Schistosoma mekongi Human Infection.

Asian schistosomiasis caused by the blood fluke Schistosoma mekongi is endemic in northern Cambodia and Southern Lao People's Democratic Republic. The disease is mainly diagnosed by stool microscopy. However, serodiagnosis such as enzyme-linked immunosorbent assay (ELISA) with soluble egg antigen (SEA), has been shown to have better sensitivity compared to the stool examination, especially in the settings with a low intensity of infection. To date, no recombinant antigen has been assessed using ELISA for the detection of S. mekongi infection, due to the lack of genome information for this schistosome species. Thus, the objective of this study is to evaluate several recombinant S. japonicum antigens that have been developed in our laboratory for the detection of S. mekongi infection. The crude antigen SjSEA and recombinant antigens Sj7TR, SjPCS, SjPRx-4, and SjChi-3 were evaluated in ELISA using serum samples positive for S. mekongi infection. The cross-reaction was checked using sera positive for Ophistorchis viverrini. ELISA results showed that S. japonicum SEA at low concentrations showed better diagnostic performance than the recombinant antigens tested using the archived serum samples from Cambodia. However, further optimization of the recombinant antigens should be conducted in future studies to improve their diagnostic performance for S. mekongi detection.

Cloning, Expression and Evaluation of Thioredoxin Peroxidase-1 Antigen for the Serological Diagnosis of Schistosoma mekongi Human Infection.

Schistosoma mekongi, a blood fluke that causes Asian zoonotic schistosomiasis, is distributed in communities along the Mekong River in Cambodia and Lao People's Democratic Republic. Decades of employing numerous control measures including mass drug administration using praziquantel have resulted in a decline in the prevalence of schistosomiasis mekongi. This, however, led to a decrease in sensitivity of Kato-Katz stool microscopy considered as the gold standard in diagnosis. In order to develop a serological assay with high sensitivity and specificity which can replace Kato-Katz, recombinant S. mekongi thioredoxin peroxidase-1 protein (rSmekTPx-1) was expressed and produced. Diagnostic performance of the rSmekTPx-1 antigen through ELISA for detecting human schistosomiasis was compared with that of recombinant protein of S. japonicum TPx-1 (rSjTPx-1) using serum samples collected from endemic foci in Cambodia. The sensitivity and specificity of rSmekTPx-1 in ELISA were 89.3% and 93.3%, respectively, while those of rSjTPx-1 were 71.4% and 66.7%, respectively. In addition, a higher Kappa value of 0.82 calculated between rSmekTPx-1 antigen ELISA and Kato-Katz confirmed better agreement than between rSjTPx-1 antigen ELISA and Kato-Katz (Kappa value 0.38). These results suggest that ELISA with rSmekTPx-1 antigen can be a potential diagnostic method for detecting active human S. mekongi infection.

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.

Vitellogenin-2 Accumulation in the Fat Body and Hemolymph of Babesia-Infected Haemaphysalis longicornis Ticks.

The protozoan parasite Babesia spp. invades into tick oocytes and remains in the offspring. The transovarial transmission phenomenon of Babesia in ticks has been demonstrated experimentally, but the molecular mechanisms remain unclear. Babesia invasion into oocytes occurs along with the progression of oogenesis. In the present study, to find the key tick factor(s) for Babesia transmission, we focused on molecules involved in yolk protein precursor (vitellogenin, Vg) synthesis and Vg uptake, which are crucial events in tick oogenesis. With a Haemaphysalis longicornis tick-Babesia ovata experimental model, the expression profiles of Akt, target of rapamycin, S6K, GATA, and Vg, Vg synthesis-related genes, and Vg receptor (VgR) and autophagy-related gene 6 (ATG6), Vg uptake-related genes, were analyzed using real-time PCR using tissues collected during the preovipositional period in Babesia-infected ticks. The expression levels of H. longicornis Vg-2 (HlVg-2) and HlVg-3 decreased in the fat body of Babesia-infected ticks 1 day after engorgement. In the ovary, HlVg-2 mRNA expression was significantly higher in Babesia-infected ticks than in uninfected ticks 1 and 2 days after engorgement and decreased 3 days after engorgement. HlVgR expression was significantly lower in Babesia-infected ticks than in uninfected ticks 2 and 4 days after engorgement. HlATG6 had a lower gene expression in Babesia-infected ticks compared to uninfected ticks 2 days after engorgement. Additionally, western blot analysis using protein extracts from each collected tissue revealed that H. longicornis Vg-2 (HlVg-2) accumulate in the fat body and hemolymph of Babesia-infected ticks. These results suggest that Vg uptake from the hemolymph to the ovary was suppressed in the presence of B. ovata. Moreover, HlVg-2 knockdown ticks had a lower detection rate of B. ovata DNA in the ovary and a significant reduction of B. ovata DNA in the hemolymph compared with control ticks. Taken together, our results suggest that accumulated HlVg-2 is associated with Babesia infection or transmission in the tick body. These findings, besides previous reports on VgR, provide important information to elucidate the transovarial transmission mechanisms of pathogens in tick vectors.

Serological evaluation of the schistosome's secretory enzyme phytochelatin synthase and phosphoglycerate mutase for the detection of human Schistosoma japonicum infection.

Secretory enzymes from Schistosoma japonicum are promising candidate antigens in the diagnosis of schistosomiasis. Our previous studies have proven that thioredoxin peroxidase-1 (SjTPx-1) is useful for the detection of this parasitic disease in humans, water buffaloes, and dogs. In this study, we evaluated two more secretory enzymes namely phosphoglycerate mutase (SjPGM) and phytochelatin synthase (SjPCS) with SjTPx-1 as the reference antigen. SjPGM was shown to have good diagnostic potentials in animal samples in previous studies, whereas SjPCS was chosen because of its absence in the mammalian hosts. Serum samples including 96 endemic negative controls, 107 schistosomiasis japonica positive samples, and 31 samples positive for other parasitic trematode infections (Clonorchis sinensis, Opisthorchis viverrini, Paragonimus westermani) were tested with the antigens using enzyme-linked immunosorbent assay. Results showed that SjPCS detected more positive samples and had fewer cross-reactions than SjPGM. With 85.05% sensitivity and 93.55% specificity, SjPCS can therefore be used in the detection of human schistosomiasis.

A simple and efficient miracidium hatching technique for preparing a single-genome DNA sample of Schistosoma japonicum.

In this study, a simple and efficient miracidium hatching technique (MHT) protocol for preparing a single-genome DNA of Schistosoma japonicum was proposed. The protocol was designed with 96-well plates to collect a miracidium for single-genome DNA preparation, and the effects of lighting conditions on hatching rates were evaluated. The highest hatching rate was recorded under sunlight (92.4%), followed by fluorescent light (88.0%), and the lowest rate was recorded under the dark condition (4.7%). The results suggested for the first time, to our knowledge, that sunlight was efficient for this simple MHT protocol. Successful amplification of microsatellite marker genes using DNA isolated from a single miracidium also confirmed the quality of the single-genome DNA for subsequent applications.

The GP-45 Protein, a Highly Variable Antigen from Babesia bigemina, Contains Conserved B-Cell Epitopes in Geographically Distant Isolates.

In B. bigemina, the 45 kilodaltons glycoprotein (GP-45) is the most studied. GP-45 is exposed on the surface of the B. bigemina merozoite, it is believed to play a role in the invasion of erythrocytes, and it is characterized by a high genetic and antigenic polymorphism. The objective of this study was to determine if GP-45 contains conserved B-cell epitopes, and if they would induce neutralizing antibodies. The comparative analysis of nucleotide and amino acids sequences revealed a high percentage of similarity between field isolates. Antibodies against peptides containing conserved B-cell epitopes of GP-45 were generated. Antibodies present in the sera of mice immunized with GP-45 peptides specifically recognize B. bigemina by the IFAT. More than 95% of cattle naturally infected with B. bigemina contained antibodies against conserved GP-45 peptides tested by ELISA. Finally, sera from rabbits immunized with GP-45 peptides were evaluated in vitro neutralization tests and it was shown that they reduced the percentage of parasitemia compared to sera from rabbits immunized with adjuvant. GP-45 from geographically distant isolates of B. bigemina contains conserved B-cell epitopes that induce neutralizing antibodies suggesting that this gene and its product play a critical role in the survival of the parasite under field conditions.

Therapeutic Efficacy of Orally Administered Nitrofurantoin against Animal African Trypanosomosis Caused by Trypanosoma congolense Infection.

Animal African trypanosomosis (AAT) leads to emaciation and low productivity in infected animals. Only six drugs are commercially available against AAT; they have severe side effects and face parasite resistance. Thus, the development of novel trypanocidal drugs is urgently needed. Nitrofurantoin, an antimicrobial, is used for treating bacterial urinary tract infections. Recently, we reported the trypanocidal effects of nitrofurantoin and its analogs in vitro. Furthermore, a nitrofurantoin analog, nifurtimox, is currently used to treat Chagas disease and chronic human African trypanosomiasis. Thus, this study was aimed at evaluating the in vivo efficacy of nitrofurantoin in treating AAT caused by Trypanosoma congolense. Nitrofurantoin was orally administered for 7 consecutive days from 4 days post-infection in T. congolense-infected mice, and the animals were observed for 28 days. Compared to the control group, the treatment group showed significantly suppressed parasitemia at 6 days post-infection. Furthermore, survival was significantly prolonged in the group treated with at least 10 mg/kg nitrofurantoin. Moreover, 100% survival and cure was achieved with a dose of nitrofurantoin higher than 30 mg/kg. Thus, oral nitrofurantoin administration has potential trypanocidal efficacy against T. congolense-induced AAT. This preliminary data will serve as a benchmark when comparing future nitrofurantoin-related compounds, which can overcome the significant shortcomings of nitrofurantoin that preclude its viable use in livestock.

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.