Lumpy skin disease (LSD) is a transboundary viral disease of cattle, including Asian water buffalo and yaks, and certain wild ruminants (e.g., African buffalo, giraffe, wildebeest, eland, and Arabian oryx). It is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus (family Poxviridae) together with goatpox virus (GTPV) and sheeppox virus (SPPV). High nucleotide identity and serological cross-reactivity among these viruses hinder differential diagnosis. The aim of this study was to develop a Duplex TaqMan-MGB qPCR Assay for Differential Detection of Chinese Epidemic Lumpy Skin Disease Virus Strains and Goatpox Virus. We developed a duplex TaqMan-MGB real-time PCR (qPCR) assay targeting the LSDV GPCR and GTPV RPO30 loci. Virus-specific primers and MGB probes were designed, and the reaction was optimized for single-tube, two-target detection. The assay showed no cross-amplification, limits of detection of 1 × 101 copies/μL (LSDV) and 1 × 101 copies/μL (GTPV), and coefficients of variation < 1%. The assay was applied to 175 yak-derived field specimens from Qinghai-Tibet Plateau, of which 16 and 36 were positive for LSDV and GTPV, respectively. LSDV- and GTPV-positive samples showed specific amplification in the FAM and VIC channels, respectively. The duplex format enables concurrent detection and unambiguous differentiation of LSDV and GTPV and is compatible with high-throughput screening. This sensitive, specific, and reproducible assay supports surveillance and control of LSD in endemic and at-risk regions.

Congenital babesiosis is rarely reported globally. We report a 74-day-old male infant presented with fever, pallor, and severe, life-threatening haemolytic anaemia (haemoglobin: 45 g/L). The infant had 16% parasitemia with ring forms evident on peripheral blood smear. Babesia microti infection was confirmed in both the mother and infant by PCR and metagenomic next- generation sequencing. Genetic analysis revealed an identical strain in bot. Treatment with intravenous azithromycin and oral atovaquone/proguanil resulted in rapid clearance of parasitemia and resolution of anaemia. This first molecularly confirmed case of congenital B. microti transmission in China demonstrates vertical transmission from an asymptomatic mother. It underscores the need for heightened clinical suspicion in neonates with unexplained haemolytic anaemia in endemic regions and highlights critical gaps in access to essential anti-babesia therapies.

Toxoplasma gondii, a globally prevalent apicomplexan parasite, causes severe morbidity and mortality in immunocompromised individuals and livestock. Current therapies exhibit limited efficacy against chronic bradyzoite stages and face drug resistance. Here, we screened imidocarb and its derivatives for anti-Toxoplasma activity. Among 20 synthesized compounds, squaramide derivative 18 (SA-18) emerged as a promising candidate with nanomolar EC50 against tachyzoites, potent inhibition of bradyzoite differentiation, and > 100-fold selectivity index. Transcriptomic analysis revealed transcriptional dysregulation in parasite metabolic pathways and downregulation of bradyzoite marker BAG1. In vivo studies exhibited significant parasite load reduction in murine models, albeit with suboptimal survival rates. These findings highlight SA-18 as a novel candidate for toxoplasmosis therapy, warranting further optimization for clinical translation.

Ticks and tick-borne pathogens (TBPs) threaten livestock productivity and public health worldwide, and climate-land-use change is expanding vector habitats, elevating tick-borne disease risk. However, TBP diversity and risk in Hubei Province, central China, remain insufficiently defined within a One Health framework. We conducted a province-wide, multiple ecoregion survey sampling questing ticks, ticks parasitizing cattle and sheep, and host blood. Tick species were identified by morphology and 16S rRNA sequencing. DNA from ticks and blood was screened using PCR and sequencing for bacterial and protozoan TBPs, including Rickettsia, Anaplasma, Ehrlichia, Borrelia, Babesia, and Theileria. We collected 1648 ticks representing 10 species in three genera, dominated by Rhipicephalus microplus (56.92%) and Haemaphysalis longicornis (37.93%). Diverse TBPs were detected in vectors and hosts, including zoonotic agents such as Anaplasma phagocytophilum, A. capra, A. ovis, Rickettsia monacensis, R. helvetica, and R. aeschlimannii. Several tick species and pathogens were recorded in Hubei for the first time. Coinfections were common (3.58% of ticks and 36.24% of hosts), most frequently Anaplasma with Theileria. Multivariable logistic regression identified geographic location, season, grazing practices, and host type as independent predictors of pathogen prevalence. Phylogenetic analyses revealed novel genotypes and cross-regional clustering, suggesting pathogen movement and the possible emergence of new variants. These findings indicate that central China is an emerging hotspot for diverse, cocirculating TBPs with zoonotic potential and underscore the need for risk-based, cross-sector surveillance and integrated tick control at the animal-human-environment interface.

Babesia gibsoni is the infectious agent of canine babesiosis, a vector-borne infection that poses a global threat to the canine health. As B. gibsoni is an erythrocytic intracellular parasite, the completion of its genome and transcriptome sequencing and analysis facilitates the elucidation of the mechanism of B. gibsoni residue in the erythrocyte. The main function of red blood cells (RBCs) is oxygen delivery; thus, B. gibsoni may be exposed to high levels of oxidative stress. To date, no report is available on the mechanism by which B. gibsoni survives oxidative stress inside the RBCs. In this study, the thioredoxin peroxidase, an important type of peroxidoxin, was identified from B. gibsoni, with 255 amino acids and a molecular weight of 27.7 kDa. There are two conserved “VCP” domains at the N- and C-termini, respectively, indicating that this gene was a 2-Cys peroxiredoxin belonging to the PTZ00137 superfamily. It was named BgTPx-2 and was detected to be located in the B. gibsoni-infected erythrocytes through an indirect immunofluorescence assay using the polyclonal antibody against the recombinant TPx-2. Additionally, its antioxidant activity was analyzed by mixed-function oxidation assay, and BgTPx-2 could protect the pBluescript SK ( +) plasmid from oxidative damage, suggesting an antioxidant function of BgTPx-2. Moreover, the immunogenicity of BgTPx-2 was tested by Western blotting and ELISA using the serum of beagle dogs infected with B. gibsoni, and the positive serum exhibited a detectable and significant antibody response against BgTPx-2 on day 4 and day 9 post-infection, respectively.

Abstract Background Human babesiosis is caused by several species within the Babesia genus, primarily Babesia microti, Babesia duncani, and Babesia divergens, all of which infect human red blood cells (RBCs). Clinically, the disease manifests with symptoms such as fever, anemia, jaundice, and hemoglobinuria, with B. microti being the most prevalent of these species. Our previous research has shown that B. microti primarily relies on lactate dehydrogenase (LDH)-mediated anaerobic glycolysis, rather than the tricarboxylic acid cycle (TCA cycle), to generate ATP for its intracellular survival. Because LDH is a promising drug target, it can be inhibited by compounds such as gossypol and 3,5-dihydroxy-2-naphthoxylic acid (DHNA). In this study, we conduct a structure-based optimization of DHNA, leading to the development of a novel library of compounds derived from its structure. Methods Two compounds were identified and synthesized through molecular docking, on the basis of the crystal structure of Babesia microti lactate dehydrogenase (BmLDH). The effects of these compounds were evaluated using several methods, including surface plasmon resonance (SPR) assays, enzyme activity inhibition tests, in vitro growth inhibition assays against B. microti, and mammalian cytotoxicity tests. Results Compounds target A (TA) (−36.0) and B (TB) (−43.8), both exhibiting low CDOCKER energy values, achieved final purities of 96.6% and 97.5%, respectively. Surface plasmon resonance (SPR) experiments showed that TA and TB had comparable dissociation constant (K D) values of 11.3 × 10−6 M and 13.2 × 10−6 M, respectively. However, enzyme activity inhibition assays indicated that TB was more potent, with an half-maximal inhibitory concentration (IC50) value of 23.8 μM, compared with TA’s IC50 of 71.6 μM. Additionally, TB demonstrated a strong ability to inhibit the in vitro growth of B. microti, with an IC50 value of 111.7 μM. Conclusions In this study, two compounds capable of inhibiting the growth of B. microti were obtained. Although both compounds showed moderate inhibitory activity against recombinant BmLDH (rBmLDH) and the growth of B. microti, there is potential to enhance their efficacy through further structural modifications, particularly of compound TB. Graphical Abstract

Parasitic diseases caused by Leishmania spp. create considerable health concerns in animals, resulting in a considerable financial impact. They causes a complex infection in equines, affecting weight gain, skin, liver, and spleen. To date, there is a lack of reports on the occurrence of Leishmania in equines in Southern Punjab, Pakistan, highlighting the need for molecular epidemiological surveillance. The current study focused on determining the prevalence of Leishmania in the equine population from District Rahim Yar Khan, Southern Punjab, Pakistan, through amplification of mitochondrial (Cytochrome b) and nuclear (18S rRNA) genes of the parasite. For this purpose, a total of 384 equine - i.e. horses, mules, and donkeys - blood specimens, determined by calculation of the sample size formula, were obtained from District Rahim Yar Khan. The parasite was examined through the Microhematocrit method under the microscope. Leishmania was detected from the buffy coat layer after centrifugation of blood-filled microhematocrit tubes. To detect and characterize Leishmania spp.at the molecular level, DNA extraction from blood samples was carried out using standardized commercial kits, followed by PCR amplification. Information on potential risk factors was gathered through a structured questionnaire. The overall prevalence of Leishmania infection was observed to be 2.1 % via microscopy and 7.3 % and 8.8 % by amplification of the 18S rRNA and Cytochrome b genes using molecular methods. A significantly higher infection percentage was observed in female animals compared to males, and in older and underweight animals compared to younger and healthier ones. Additionally, the infection was non-significantly (P ≥ 0.05) more prevalent in gestating, non-dewormed, symptomatic, and poor body condition animals. Phylogenetic and sequence analyses confirmed that the identified gene sequences clustered within the Leishmania (Leishmania) infantum clade, consistent with strains reported in different animal hosts from various regions. In conclusion, the nuclear gene, i.e., 18S rRNA proved to be a more sensitive molecular marker for detecting Leishmania infection in equines compared to the mitochondrial gene, i.e., Cytochrome b.

This study aimed to investigate the interaction between imidocarb dipropionate (IMDP) and double-stranded DNA, as understanding its mechanism of action is crucial for optimizing its use as a veterinary antiprotozoal agent. Using calf thymus DNA as a model, we systematically explored the binding of IMDP to DNA via UV-vis absorption spectroscopy, Competitive displacement assays, thermal denaturation analysis, circular dichroism spectroscopy, ion interference experiments, viscosity measurement, and molecular docking. Results indicated that IMDP binds to DNA with a decrease in hypochromicity rates of 23.95% and 22.17%. Notably, the Tm value rose from 69 °C to 71 °C upon binding, and the circular dichroism spectrum peaks remained nearly unchanged, suggesting a groove binding mode with minimal impact on DNA conformation and viscosity. Our findings confirm that IMDP binds to double-stranded DNA by interacting within the DNA groove, supporting its potential as a DNA-targeting antiparasitic drug.

Babesiosis is a tick-borne parasitic disease that poses a significant risk to both animal and human health. A comprehensive understanding of Babesia biology necessitates the application of advanced laboratory techniques. This review explores recent advancements in gene editing technologies of Babesia, emphasizing the foundational importance of in vitro culture systems. We highlight the historical challenges encountered in establishing effective in vitro culture and discuss the need for optimizing these methods to enhance gene editing efficiency. Here, we describe recent progress in Babesia transfection, different gene manipulation systems, and the applications of gene editing. This review aims to provide essential insights and technical guidance for future studies in Babesia genetics, highlighting the transformative potential of gene manipulation in combating this important parasitic disease.

AbstractApicomplexan parasites predominantly generate ATP and lactic acid through glycolysis and anaerobic glucose metabolism, incorporating CO2 into glycolysis via a stage-dependent phosphoenolpyruvate carboxylase (PEPC) mechanism. Although the role of PEPC in plant and bacterial carbon fixation is well documented, its function within Babesia remains largely unexplored. This study employs reverse genetics to probe the biological role of PEPC in Babesia gibsoni, noting its conservation across similar protozoa, suggesting a pivotal and conserved biological function. Western blotting and immunofluorescence (IFA) experiments using the BgPEPC-3 × Flag strain revealed that the BgPEPC protein has a molecular weight of 105 kDa and localizes predominantly to the cytoplasm. Attempts to knock out the PEPC gene in BgPEPC-3 × Flag strains failed under standard media conditions, succeeded only with the addition of 5 mM malate, an upstream metabolite of oxaloacetic acid (OAA). In addition to malate, the downstream metabolite of OAA can also partially compensate for the phenotypic defects caused by PEPC deficiency. This intervention alleviated severe growth deficits, underscoring the critical role of aspartate in the parasite lifecycle. Moreover, metabolic inhibitors such as L-cycloserine and triazamidine, which target aspartate aminotransferase and mitochondrial functions, respectively, demonstrated increased efficacy against BgPEPC knockout strains. The lack of a compensatory response to malic acid supplementation underscores the integral role of BgPEPC in intermediary carbon metabolism and its necessity in providing aspartate as a precursor to pyrimidine synthesis. Collectively, these findings suggest that PEPC could be a potential target for future drug development against B. gibsoni infections. Graphical Abstract

Abstract Background Babesia duncani is a pathogen within the phylum Apicomplexa that causes human babesiosis. It poses a significant threat to public health, as it can be transmitted not only through tick bites but also via blood transfusion. Consequently, an understanding of the gene functions of this pathogen is necessary for the development of drugs and vaccines. However, the absence of conditional gene knockdown tools has hindered the research on this pathogen. The auxin-inducible degron (AID) system is a rapid, reversible conditional knockdown system widely used in gene function studies. Thus, there is an urgent need to establish the AID system in B. duncani to study essential gene functions. Methods The endogenous genes of the Skp1-Cullin-F-box (SCF) complex in B. duncani were identified and confirmed through multiple sequence alignment and conserved domain analysis. The expression of the F-box protein TIR1 from Oryza sativa (OsTIR1) was achieved by constructing a transgenic parasite strain using a homologous recombination strategy. Polymerase chain reaction (PCR), western blot, and indirect immunofluorescence assay (IFA) were used to confirm the correct monoclonal parasite strain. The degradation of enhanced green fluorescent protein (eGFP) tagged with an AID degron was detected through western blot and live-cell fluorescence microscopy after treatment of indole-3-acetic acid (IAA). Results In this study, Skp1, Cul1, and Rbx1 of the SCF complex in B. duncani were identified through sequence alignment and domain analysis. A pure BdTIR1 strain with expression of the OsTIR1 gene was constructed through homologous recombination and confirmed. This strain showed no significant differences from the wild type (WT) in terms of growth rate and proportions of different parasite forms. The eGFP tagged with an AID degron was successfully induced for degradation using 500 μM IAA. Grayscale analysis of western blot indicated a 61.3% reduction in eGFP expression levels, while fluorescence intensity analysis showed a 77.5% decrease in fluorescence intensity. Increasing the IAA concentration to 2 mM accelerated eGFP degradation and enhanced the extent of degradation. Conclusions This study demonstrated the functionality of the AID system in regulating protein levels by inducing rapid degradation of eGFP using IAA, providing an important research tool for studying essential gene functions related to invasion, egress, and virulence of B. duncani. Moreover, it also offers a construction strategy for apicomplexan parasites that have not developed an AID system. Graphical Abstract

Babesia orientalis is an intra-erythrocytic protozoan parasite that causes babesiosis in water buffalo. The genome of B. orientalis has been reported and various genes have been accurately annotated, including heat shock proteins (HSP). Three B. orientalis HSPs (HSP90, HSP70 and HSP20) have been previously identified as potential antigenic targets. Here, a new validation strategy for the chaperone activities and cell protection characteristics of the three HSPs was developed in vitro. BoHSP20, BoHSP70 and BoHSP90B were amplified from cDNA, followed by cloning them into the pEGFP-N1 vector and transfecting the vector plasmid separately into 293T and Hela mammalian cells. Their expression and localization were determined by fluorescence microscopy. The biological functions and protein stability were testified through an analysis of the fluorescence intensity duration. Their role in the protection of cell viability from heat-shock treatments was examined by MTT assay (cell proliferation assay based on thiazolyl blue tetrazolium bromide). Fusion proteins pEGFP-N1-BoHSP20, pEGFP-N1-BoHSP70, and pEGFP-N1-BoHSP90B (pBoHSPs: pBoHSP20; pBoHSP70 and pBoHSP90B) were identified as 47 kDa/97 kDa/118 kDa with a 27 kDa GFP tag, respectively. Prolonged fluorescent protein half-time was observed specifically in pBoHSPs under heat shock treatment at 55 °C, and BoHSP20 showed relatively better thermotolerance than BoHSP70 and BoHSP90B. Significant difference was found between pBoHSPs and controls in the cell survival curve after 2 h of 45 °C heat shock. Significant biological properties of heat stress-associated genes of B. orientalis were identified in eukaryote by a new strategy. Fusion proteins pBoHSP20, pBoHSP70 and pBoHSP90B showed good chaperone activity and thermo-stability in this study, implying that BoHSPs played a key role in protecting B. orientalis against heat-stress environment during parasite life cycle. In conclusion, the in vitro model explored in this study provides a new way to investigate the biological functions of B. orientalis proteins during the host–parasite interaction.

Babesia gibsoni, a unicellular eukaryotic parasite causing babesiosis in dog, is primarily transmitted through tick feeding. The intraerythrocytic stage, during which the parasite reproduce within the host's red blood cells, is a vital part of Babesia's life cycle. Continuous in vitro culture B. gibsoni provides an opportunity to study its biological processes. The establishment and development of gene editing systems for Babesia offer a powerful tool to investigate the functions of important genes in specific biological processes. This protocol expands on the existing techniques for in vitro culture and genes editing of B. gibsoni. Specifically, we describe a continuous in vitro culture method employing VP-SFM as a base medium, supplemented with Albumax I and small amount of canine serum (2.5 %), This method, designed for long-term culture, achieving high parasitemia and facilitates subclone culture. By employing homology-dependent repair pathways, the gene editing method utilizing introducing homologous fragments and electroporation can effectively manipulate the genetic of B. gibsoni. This protocol would contribute to the reproducibility of experiments and the overall reliability of research findings.

Babesia orientalis, a protozoan parasite transmitted by the tick Rhipicephalus haemaphysaloides, holds significant economic importance along the Yangtze River. Key factors in the host invasion process include rhoptry neck proteins (RON2, RON4, and RON5) and apical membrane antigen 1 (AMA1). However, the intricacies of the interaction between AMA1 and RONs remain incompletely elucidated in B. orientalis. To better understand these crucial invasion components, the RON4 gene of B. orientalis (BoRON4) was cloned and sequenced. RON4 is 3468 base pairs long, encodes 1155 amino acids, and has a predicted molecular weight of 130 kDa. Bioinformatics analysis revealed a unique region (amino acid residues 109–452) in BoRON4, which demonstrates higher sensitivity to epitope activity. The BoRON4 gene was strategically truncated, amplified, and cloned into the pGEX-6p-1 vector for fusion expression. We successfully used the mouse polyclonal antibody to identify native BoRON4 in B. orientalis lysates. Furthermore, the corresponding BoRON4 protein band was detected in the water buffalo serum infected with B. orientalis, while no such band was observed in the control. Additionally, I-TASSER and Discovery Studio software were used to predict the tertiary structures of BoRON4 and its ligands, CH-PKA and CH-complex. These ligands can serve as lead compounds for the development of anti-babesiosis drugs. In conclusion, BoRON4 emerges as a promising candidate antigen for distinguishing water buffalo infected with B. orientalis from their normal counterparts. This study positions BoRON4 as a potential diagnostic antigen for babesiosis in water buffalo, contributing valuable insights to the field of parasitology.

Babesia duncani (B. duncani), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, B. duncani PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for B. duncani. Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of B. duncani, providing insights for babesiosis prevention and drug development.

The VP-SFM as a base medium supplemented with AlbuMAX I and a low concentration of canine serum (2.5%) allowed the continuous in vitro culture of Babesia gibsoni at both small and large volumes, which was to meet different experimental needs, such as long-term culture and obtaining high parasitemia and subclone culture. The establishment of in vitro culture systems allows researchers to better understand the metabolism and growth patterns of Babesia .

The whole genome of B. gibsoni was first sequenced, annotated, and disclosed. The key part of genome composition, four chromosomes, was comparatively analyzed for the first time.

Human babesiosis is an emerging tick-borne disease, caused by haemoprotozoa genus of Babesia. Cases of transfusion-transmitted and naturally acquired Babesia infection have been reported worldwide in recent years and causing a serious public health problem. Babesia duncani is one of the important pathogens of human babesiosis, which seriously endangers human health. The in vitro culture systems of B. duncani have been previously established, and it requires fetal bovine serum (FBS) to support long-term proliferation. However, there are no studies on serum-free in vitro culture of B. duncani. In this study, we reported that B. duncani achieved long-term serum-free culture in VP-SFM AGTTM (VP-SFM) supplemented with AlbuMaxTM I. The effect of adding different dilutions of AlbuMaxTM I to VP-SFM showed that 2 mg/mL AlbuMaxTM I had the best B. duncani growth curve with a maximum percentage of parasitized erythrocytes (PPE) of over 40%, and it can be used for long-term in vitro culture of B. duncani. However, the commonly used 20% serum-supplemented medium only achieves 20% PPE. Clearly, VP-SFM with 2 mg/mL AlbuMaxTM I (VP-SFMA) is more suitable for the in vitro proliferation of B. duncani. VP-SFM supplemented with CD lipid mixture was also tested, and the results showed it could support the parasite growth at 1:100 dilution with the highest PPE of 40%, which is similar to that of 2 mg/mL AlbuMaxTM I. However, the CD lipid mixture was only able to support the in vitro culture of B. duncani for 8 generations, while VP-SFMA could be used for long-term culture. To test the pathogenicity, the VP-SFMA cultured B. duncani was also subjected to hamster infection. Results showed that the hamster developed dyspnea and chills on day 7 with 30% PPE before treatment, which is similar to the symptoms with un-cultured B. duncani. This study develops a unique and reliable basis for further understanding of the physiological mechanisms, growth characteristics, and pathogenesis of babesiosis, and provides good laboratory material for the development of drugs or vaccines for human babesiosis and possibly other parasitic diseases.

Babesia gibsoni, the causative agent of canine babesiosis, belongs to the phylum Apicomplexa. The development of in vitro culture technology has driven research progress in various kinds of omics studies, including transcriptomic analysis of Plasmodium spp. between in vitro and in vivo environments, which has prompted the observation of diagnostic antigens and vaccine development. Nevertheless, no information on Babesia spp. could be obtained in this respect, which greatly hinders the further understanding of parasite growth and development in the blood stage.In this study, considerable changes in the morphology and infectivity of continuous in vitro cultured B. gibsoni (Wuhan isolate) were observed compared to in vivo parasites. Based on these changes, B. gibsoni (Wuhan isolate) was collected from both in vivo and in vitro cultures, followed by total RNA extraction and Illumina transcriptome sequencing. The acquired differentially expressed genes (DEGs) were validated using qRT-PCR, and then functionally annotated through several databases. The gene with the greatest upregulation after in vitro culture was cloned from the genome of B. gibsoni (Wuhan isolate) and characterized by western blotting and indirect immunofluorescence assay for detecting the native form and cellular localization.Through laboratory cultivation, multiple forms of parasites were observed, and the infectivity of in vitro cultured parasites in dogs was found to be lower. Based on these changes, Illumina transcriptome sequencing was conducted, showing that 377 unigenes were upregulated and 334 unigenes were downregulated. Notably, an AP2 transcription factor family, essential for all developmental stages of parasites, was screened, and the transcriptional changes in these family members were tested. Thus, the novel AP2 transcription factor gene (BgAP2-M) with the highest upregulated expression after in vitro adaptation was selected. This gene comprises an open reading frame (ORF) of 1989 base pairs encoding a full-length protein of 662 amino acids. BgAP2-M contains one AP2 domain and one ACDC conserved domain, which may be involved in the nuclear biology of parasites. The prepared polyclonal antibodies against the BgAP2-M peptides further detected a native size of ~ 73 kDa and were localized to the nuclei of B. gibsoni.This study presents a thorough transcriptome analysis of B. gibsoni in vivo and in vitro for the first time, contributing to a detailed understanding of the effects of environmental changes on the growth and development of parasites in the blood stage. Moreover, it also provides a deeper investigation for the different members of the ApiAP2 transcription factor family as various life stage regulators in Babesia spp.