Molecular characterization and recombinant expression of Babesia ovis SBP4 protein as a potential serodiagnostic antigen

Abstract

Ovine babesiosis caused by Babesia ovis is a tick-borne disease that affects sheep in enzootic regions of Southern Europe, Middle East, and Asia, resulting in significant economic loss. Despite its clinical relevance, only a few useful serological markers are available for accurate diagnosis. In the present study, we characterized the B. ovis spherical body protein 4 (SBP4) using in silico, molecular, and immunological approaches, and evaluated its preliminary activity with sera naturally infected sheep, suggesting its potential for future serodiagnostic application. The full-length sbp4 gene was identified via genome analysis, revealing a 984 bp intronless open reading frame encoding a 327-amino acid protein with a predicted signal peptide. Codon-optimized for expression in Escherichia coli, the sbp4 gene was cloned and inserted into the pET-29b(+) vector. Expression in Escherichia coli C43(DE3) produced soluble recombinant rBoSBP4 protein, which was purified via affinity chromatography. The ~ 37 kDa protein was confirmed by SDS‒PAGE and recognized by anti-His antibodies. Western blot and indirect ELISA using sera from naturally B. ovis-infected sheep demonstrated that rBoSBP4 is specifically recognized during infection, whereas negative control sera showed no reactivity. The immunization of rabbits with rBoSBP4 also induced a strong antibody response, as determined by ELISA. These results indicate that rBoSBP4 is both immunogenic and antigenically specific, supporting its potential utility in the development of serological diagnostic assays for ovine babesiosis.

Introduction

Babesia ovis, one of the causative agents of ovine babesiosis in southern Europe, North Africa, the Middle East and East Asia, is a tick-borne pathogen of the phylum Apicomplexa^1,2,3,4,5. Other causative agents of ovine babesiosis include B. motasi, B. crassa, Babesia sp. Xinjiang and recently reported species that vary in prevalence across different regions^4,6. Babesia ovis affects mainly sheep and leads to an acute clinical disease with signs including fever, anemia, icterus, and hemoglobinuria^7,8,9. The parasite is transmitted by the Rhipicephalus bursa during the blood feeding^7,8,10,11. The transmission of the parasite by the tick vector occurs through both transstadial and transovarial routes^4,7,11. This vector‒parasite relationship plays a key role in the epidemiology and spread of the parasite in endemic regions¹².

In Babesia species, spherical body proteins (SBPs) are the predominantly proteins expressed in the intraerythrocytic stage of the parasite. These proteins are known for their strong antigenic properties, making them promising targets for both diagnostic and immunological applications¹³. Within the SBP protein family, particular emphasis has been placed on SBP4. It has been recently reported that the gene encoding SBP4 in certain Babesia species is conserved among different geographical isolates of the parasites and demonstrates excellent performance when used as an antigen for a serological assay^14,15,16,17. More importantly, SBP4 has been found to be secreted throughout the parasite’s life cycle in vertebrate hosts, thereby causing continuous and intense immunological stimulation during infection^14,15,16.

In contrast to bovine Babesia spp., there are few reports on the secretory proteins of B. ovis, the most important causative agent of ovine babesiosis. The first study on the immunogenic proteins of B. ovis was performed by Sevinc et al.¹⁸, where 5 immunoreactive proteins were recognized by SDS‒PAGE and Western blot (WB) procedures in two experimentally infected lambs. Two antigens of B. ovis recombinant antigens, designated B. ovis secreted antigen 1 (BoSA1) and B. ovis secreted antigen 2 (BoSA2), were also found in further investigations by the same group of researchers¹⁹. There is currently no information on SBPs in ovine Babesia infections. Identifying potential antigens for the development of more effective diagnostic methods and vaccines is still essential for the control of ovine babesiosis.

On the basis of the potential value of SBP4 as a diagnostic and immunogenic antigen of bovine Babesia species^15,17, we aimed to characterize its ortholog in B. ovis and evaluate its molecular and immunoreactive features. Nevertheless, there has been no molecular or immunological characterization of SBP4 in B. ovis thus far. In this study, we aimed to design specific primers targeting the full-length B. ovis sbp4 gene, perform its molecular amplification and cloning, and produce the corresponding recombinant protein in Escherichia coli. Furthermore, the immunogenicity of the purified protein was assayed by indirect ELISA (iELISA) and WB with sera from both naturally infected sheep and immunized rabbits via affinity-purified proteins to obtain specific polyclonal antibodies. This study is the first attempt to describe SBP4 in B. ovis and to assess its molecular, and immunoreactive features using in silico, molecular, and serological approaches.

Results

In silico characterization of the SBP4 protein

The sbp4 gene was retrieved from the genome of the B. ovis Selcuk isolate (GenBank Accession No: BLIY00000000), previously annotated as a putative spherical body protein (GenBank Accession No: GFE53746), and publicly available through the study by Yamagishi et al.²⁰. The gene is 984 bp long and encodes a 327 amino acid protein. The gene did not have any introns according to the sequence analysis, suggesting an uninterrupted open reading frame. Signal peptide predictions via SignalP 6.0 identified a cleavable N-terminal signal sequence, suggesting that the protein may be directed to a secretory pathway (Supplementary Fig. 1a). The prediction of the transmembrane region was performed via TMHMM 2.0, and the results revealed no transmembrane domain, which suggests that the protein is non-membranous and soluble. The structural topology of the SBP4 protein was predicted using PROTTER, which revealed several extracellular surface-exposed loops (Supplementary Fig. 1b). These regions are potentially accessible to host antibodies and therefore relevant for immunological detection and antigen design.

Synteny analysis via the PiroplasmaDB platform revealed that the sbp4 locus in B. ovis shares a conserved genomic context with orthologs in B. bovis, B. bigemina, B. ovata and B. caballi, suggesting evolutionary conservation at the chromosomal level (Fig. 1). To further assess the molecular relationship between B. ovis SBP4 and its orthologs, pairwise amino acid identity analysis was conducted. The SBP4 protein of B. ovis showed the highest similarity with B. orientalis (44.6%), B. bovis (43.3%), and Babesia sp. Xinjiang (38.2%), while comparatively lower identities were observed with B. caballi (33.6%), B. ovata (32.2%), and B. bigemina (31.9%).

Fig. 1

Synteny map of the sbp4 gene in different Babesia species. Ef G2: Elongation Factor G, VW: Von Willebrand factor type A, SBP4: Spherical Body Protein 4, HP: Hypothetical Protein, Fib: Fibronectin Type III Domain Containing 3C1-Like, FAD Ox: FAD-Dependent Oxidoreductase, RPS 16: Ribosomal Protein RPS16, Rib Prot L 13: Ribosomal Protein RPL13.

A three-dimensional structural model of the codon-optimized SBP4 protein was generated via the Phyre2 server. The predicted model had an overall compact structure with dimensions of approximately X: 35.569 Å, Y: 29.121 Å, and Z: 23.562 Å (Fig. 2a). The use of two different visualization styles in Fig. 2b and c provides complementary structural insights, highlighting both the global protein fold and the potential surface-exposed regions inferred from docking simulations. However, these images are not intended to define precise immunogenic epitopes.

Fig. 2

Predicted 3D structure of the recombinant SBP4 protein. (a) Ribbon model generated via Phyre2. (b) Cartoon view. (c) Liquorice view from docking-based modeling (HDOCK) to illustrate overall folding and side-chain exposure. These visualizations do not represent experimentally confirmed immunogenic regions.

PCR amplification of the sbp4 gene

The full-length sbp4 gene of B. ovis, spanning 984 bp, was successfully amplified via several primer combinations specifically designed in this study. The primer binding sites and their relative genomic positions are illustrated in Fig. 3a. All primer sets produced amplification products of the expected sizes; however, the most distinct and reliable band was consistently obtained via the Bosbp4outF1-Bosbp4outR3 primer pair (Fig. 3b). On the basis of these results, this primer set was selected for subsequent amplification.

Using the optimized primer pair, the sbp4 gene was successfully amplified from B. ovis Alacakaya stabilate, as well as from DNA samples derived from field isolates collected across eight geographically distinct provinces of Türkiye: Adana, Bingöl, Diyarbakır, Elazığ, Gaziantep, Kahramanmaraş, Malatya, and Siirt. These findings confirm that the selected primers efficiently target a conserved region of sbp4, allowing its amplification across diverse B. ovis strains. Multiple sequence alignment of the translated SBP4 amino acid sequences revealed a high degree of conservation among all the isolates. Only a single amino acid substitution was detected in the extreme C-terminal region (position 325), suggesting minimal variability across geographically distinct field strains (Supplementary Fig. 2). This high level of conservation supports the potential of SBP4 as a stable and broadly applicable antigen for the serological detection of B. ovis. The sequences have been submitted to GenBank under accession numbers PP471910, PV737600-PV737607. To evaluate the evolutionary relationship of the SBP4 protein from B. ovis with its orthologs in other Babesia species, a phylogenetic tree was constructed based on the amino acid sequences. The analysis revealed a high degree of sequence conservation among Turkish B. ovis isolates and clustered them distinctly from Babesia species (Supplementary Fig. 3).

Fig. 3

Reactions with combinations of primers for the target B. ovis sbp4 gene. (a) Schematic representation of the sbp4 gene locus in the B. ovis genome relative to flanking genes (Ef G2 and a hypothetical protein). The arrows show the locations and orientations of the designed primers for PCR amplification. (b) Agarose gel electrophoresis of PCR products produced by different primer pairs for the sbp4 gene.

Recombinant expression and purification of rBoSBP4

The codon-optimized gene sbp4 (957 bp) was inserted into the pET-29b(+) vector without the native signal peptide and with an additional N-terminal ATG and a C-terminal 6×His tag. Transformants of the recombinant plasmids were identified in E. coli BL21(DE3) and C43(DE3) strains. Among the tested E. coli expression strains, C43(DE3) repeatedly yielded the rBoSBP4 protein with greater purity and less degradation than BL21(DE3). The optimal expression was achieved with the culture being induced at an OD₆₀₀ of 0.7 using 0.5–1 mM IPTG, followed by overnight shaking at 18 °C. Protein yield was optimized to improve recombinant protein solubility and adequate structure folding. After cell lysis by high-pressure homogenization, the lysates were separated into soluble and insoluble fractions via centrifugation. SDS‒PAGE and Western blot analyses revealed that the supernatant fraction included the largest population of expressed rBoSBP4 protein, suggesting that it was soluble and in its native conformation. The protein concentrations of all bacterial lysates were determined using the Bradford assay, and 30 µg of total protein was loaded per lane for SDS-PAGE and Western blot analyses. Western blot analysis using an HRP-conjugated goat polyclonal anti-6×His antibody detected a distinct band compatible with the expected molecular mass (~ 37 kDa), consistent with the predicted size of the recombinant His-tagged SBP4 protein. The apparent molecular weight of rBoSBP4 was further verified by SDS-PAGE under reducing conditions (data not shown). These results indicate that rBoSBP4 is efficiently expressed in a soluble form when our optimized conditions are used. The supernatants containing active protein were purified using a HisTrap Ni-NTA affinity column on an ÄKTA purification system via a single-step elution scheme. Western blot analysis of the supernatant fractions revealed that rBoSBP4 was expressed in E. coli C43(DE3) under the optimum conditions. A prominent ~ 37 kDa band was present in the IPTG-induced samples, and no signal was detected in the non-induced and non-transformed controls. At 0.5–1 mM IPTG, the signal was more intense (Fig. 4).

Fig. 4

Western blot analysis of recombinant rBoSBP4 expression in E. coli C43(DE3) under different IPTG induction conditions. Lanes 1–4: transformed colonies induced with 0.5 mM (lanes 1–2) and 1.0 mM IPTG (lanes 3–4), supernatant fractions harvested at OD₆₀₀ = 0.7. Lane 5: non-transformed control E. coli C43(DE3) induced with 1.0 mM IPTG. Lane 6: transformed colonies without IPTG induction. A distinct band with a mass compatible with approximately 37 kDa was detected in IPTG-induced samples, corresponding to the expected size of His-tagged rBoSBP4. No signal was detected in the non-induced or non-transformed controls. M: molecular-weight marker.

Western blot and iELISA analysis of naturally infected Sera

The immunoreactivity of the purified rBoSBP4 protein was evaluated via western blotting with sera from naturally B. ovis-infected sheep. Reducing SDS‒PAGE and Western blotting analysis were carried out on the supernatant fraction containing soluble rBoSBP4, which was confirmed by anti-His detection via Western blotting. The membranes were blocked in 5% skim milk and incubated with infected serum (1:500 in blocking buffer) overnight at 4 °C. Bound antibodies were detected with a secondary antibody (Protein A/G-HRP) after washing.

A single, specific band of ~ 37 kDa was detected when sera from sheep naturally infected with B. ovis were blotted, indicating that rBoSBP4 can be recognized by antibodies elicited during natural infection. There was no reactivity with sera from uninfected controls, further confirming the antigenic specificity of the recombinant protein (Fig. 5a). The immunoreactivity of rBoSBP4 was assessed via an iELISA using sera from sheep naturally infected with B. ovis, as confirmed by microscopy and PCR (results not shown). The purified recombinant protein was used to coat microtiter plates, and the serum samples were tested at a 1:100 dilution. Sera from five animals naturally infected with B. ovis exhibited a very strong ELISA signal, whereas the negative control serum showed no detectable reactivity (Fig. 5b). These results are in agreement with the Western blot analysis performed following SDS-PAGE under reducing conditions, showing that rBoSBP4 is specifically recognized by antibodies produced during natural B. ovis infection.

Fig. 5

Evaluation of the immunoreactivity of recombinant rBoSBP4 in sera from naturally B. ovis-infected sheep. (a) Western blot analysis of five positive field serum samples (lanes 1–5) and one negative control serum sample (lane 6). A distinct ~ 37 kDa band was detected in all positive samples. M: molecular weight marker. (b) Indirect ELISA results showing strong reactivity of rBoSBP4-coated wells with sera from naturally infected sheep, while no significant signal was observed with negative control serum. The data are presented as the means, and the statistical analysis revealed a significant difference (**p < 0.01).

Rabbit immunization and polyclonal antibody response

To determine the immunogenicity of rBoSBP4, New Zealand white rabbits were immunized with purified recombinant protein according to a standard immunization procedure with Freund’s adjuvant. Sera obtained two weeks after the last booster were analyzed by iELISA for antibodies specific to rBoSBP4. Thus, the iELISA values for optical density obtained for the post-immune sera were considerably greater than those measured with the pre-immune sera, which is consistent with a primary humoral response (Fig. 6). These results indicated that rBoSBP4 is immunogenic and can induce the immune system to produce specific antibodies in rabbits in the experiments outlined in the present study.

Fig. 6

Indirect ELISA analysis of sera collected from rabbits immunized with recombinant rBoSBP4. Sera were tested at a 1:100 dilution. The X-axis represents the serum collection point: 0 = pre-immune serum; 1–4 = post-immunization bleeds collected after each successive round of immunization. The data are presented as the means, and the statistical analysis revealed a significant difference (**: p < 0.01).

Discussion

The availability of the B. ovis Selçuk isolate genome has opened the door to identifying new molecular targets for understanding and managing ovine babesiosis²⁰, a disease that remains significant in regions such as southern Europe, the Middle East, and Asia⁴. Although B. ovis is known to cause serious clinical outcomes in sheep, current diagnostic and preventive tools remain insufficient. In our study, we focused on the SBP4 protein, describing its molecular features and evaluating its reactivity with host antibodies using recombinant expression. The data presented here may help advance the search for more accurate and species-specific serological markers for this pathogen.

Through in silico analyses of the recently published B. ovis Selçuk isolate genome, we identified a 984 bp intronless sbp4 gene encoding a 327-amino acid protein with a predicted N-terminal signal peptide. The gene presented conserved synteny and domain architecture with SBP4 orthologs from other Babesia species¹⁶. Sequencing of field isolates from eight different geographical areas of Türkiye indicated that B. ovis SBP4 has a highly conserved amino acid sequence among strains circulating within the country. There was only one specific amino acid substitution in the C-terminus (Supplementary Fig. 2). This minor polymorphism demonstrates that SBP4 is indeed a genetically stable antigen; however, the functional or diagnostic significance of this slight heterogeneity is not known.

SBP4 is a highly immunogenic and conserved protein and has been used as a serodiagnostic antigen in bovine babesiosis caused by B. bovis and B. bigemina^14,15,16,17. Our data for B. ovis align with previous reports in other Babesia species and support the consideration of SBP4 as a promising antigen for serological detection within the genus. Furthermore, the strong antibody response observed in rabbits immunized with rBoSBP4 confirms its immunogenicity and warrants its further evaluation in future vaccine development efforts.

While the lack of WB validation using rabbit sera is a limitation, stable iELISA responses strongly indicate the induction of antigen-specific antibodies. Furthermore, the fact that sera from naturally infected sheep were used for western blotting and iELISA contributes to the practical significance of the results and highlights the antigenic potential of rBoSBP4. All the naturally infected sera used in this study were post-acute samples, but it remains unknown whether SBP4-specific antibody responses occur in the early or chronic phase of infection. Additionally, although the negative control sera used in this work validated epitope specificity, wider investigations into different populations and disease contexts would further validate conclusions about diagnostic specificity. Finally, the immune response was determined only at the humoral level; thus, an additional study is needed to evaluate the cell response and even the protective prophylaxis of rBoSBP4 in challenged animals. These efforts are necessary to determine the value of rBoSBP4 in serodiagnosis and vaccine research on ovine babesiosis caused by B. ovis. Further experiments will be required to demonstrate native SBP4 expression in merozoites by WB and to evaluate cross-reactivity using sera from animals infected with other ovine Babesia species. These data will be essential to conclusively validate the diagnostic specificity of rBoSBP4.

Conclusion

This is the first report to fully describe the molecular and immunological characteristics of SBP4 in B. ovis and to suggest that this protein is a promising novel recombinant antigen for serodiagnosis and vaccine development against B. ovis infection. The rBoSBP4 protein is expressed as a soluble protein and has been shown to be highly immunogenic, inducing strong antibody responses in both naturally infected sheep and immunized rabbits. Sequence analysis of field isolates obtained from eight provinces throughout Türkiye revealed high amino acid conservation, indicating its diagnostic potential for regional isolates. Nevertheless, the limited polymorphism of the C-terminal region means that it is necessary to carry out comparative studies with other B. ovis SBP4 strains from other endemic countries to guarantee global diagnostic value. Future studies should evaluate the duration and kinetics of the antibody response to SBP4 after experimental infection in sheep. Due to its stable immunoreactivity and sequence conservation, SBP4 warrants further investigation as a potential serology marker and candidate vaccine antigen for ovine babesiosis caused by B. ovis.

Materials and methods

In silico analysis of the B. ovis sbp4 gene and bioinformatics characterization

The presence of the sbp4 gene in B. ovis was confirmed by analyzing the genome of the B. ovis Selcuk isolate from Türkiye (GenBank Accession No: BLIY00000000)²⁰. The putative gene was annotated as the spherical body protein B. ovis (GenBank Accession No: GFE53746).

The signal peptide and transmembrane domain regions were identified via SMART (http://smart.embl-heidelberg.de), PROSITE (https://prosite.expasy.org), SignalP 6.0 (https://services.healthtech.dtu.dk), and TMHMM 2.0 (http://www.cbs.dtu.dk). Membrane topology and subcellular localization predictions were further supported by visualization via PROTTER (http://wlab.ethz.ch/protter).

Multiple sequence alignments were conducted between SBP4 from B. ovis and its orthologs from other Babesia species (e.g., B. bovis, B. bigemina, and B. caballi) via Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/) to assess its conservation and homology. Furthermore, to determine the genomic location and structural conservation of sbp4 in other Babesia species (B. bovis, B. bigemina, B. ovata, and B. caballi), synteny map analyses were performed via the PiroplasmaDB (https://piroplasmadb.org/piro/app).

Structural modeling and docking studies were carried out to predict the 3D structure and potential binding faces of the recombinant SBP4 protein. The codon-optimized amino acid chain of SBP4 was uploaded to the Phyre2 server (http://www.sbg.bio.ic.ac.uk/phyre2/) and modeled in intensive mode. The homology prediction model is represented in rainbow colors (from the N- to the C-terminus). Protein‒protein docking was subsequently performed with the HDOCK server (http://hdock.phys.hust.edu.cn/). The SBP4 model was analyzed in both cartoon and liquorice visualization styles to assess surface accessibility and potential ligand-binding regions.

Primer design, PCR amplification, and phylogenetic analysis

The primer pairs were designed from the B. ovis sequence of the sbp4 gene and the genomic sequences of the gene flanking regions (loci BaOVIS_011490, BaOVIS_011510 encoding translation elongation factor G and hypothetical protein, respectively) available from the PiroplasmaDB database (https://piroplasmadb.org).

The primers used were generated with the Primer Quest™ Tool of Integrated DNA Technologies (https://www.idtdna.com/pages/tools/primerquest) (Supplemental Table 1). PCRs were carried out with Phusion^® High Fidelity PCR Master Mix with GC buffer (NEB, M0532S). The 20-µL reaction mixture contained 10 µL of 2× Phusion Master Mix, 1 µL each of forward and reverse primers, 1 µL of genomic DNA and 7 µL of nuclease-free water. The template DNA was extracted from the blood of a sheep experimentally infected with B. ovis (Alacakaya strain)⁷ via the GeneJET Genomic DNA Purification Kit (Thermo Scientific) according to the manufacturer’s instructions. The PCR-amplified products were resolved on a 1.5% agarose gel, stained with ethidium bromide, and viewed under UV light. The size of the expected amplicon was 1122–1302 bp.

Apart from the B. ovis reference Alacakaya stabilate, DNA samples of field isolates previously established to be B. ovis positive by PCR were included in the evaluation of the primers formulated. These samples were collected from 8 provinces in Türkiye (Adana, Bingöl, Diyarbakır, Elazig, Gaziantep, Kahramanmaraş, Malatya and Siirt). The correctness of the amplified products was confirmed by next-generation sequencing of the PCR products and de novo assembly of the corresponding reads. The consensus sequences validated the full-length structure of the sbp4 gene, which was further analyzed to define the codons for gene optimization and synthetic gene design.

The evolutionary relationship of the B. ovis SBP4 protein with orthologous sequences from other Babesia species was evaluated using MEGA11 software. Amino acid sequences were aligned, and a phylogenetic tree was constructed using the Maximum Likelihood method based on the Le_Gascuel_2008 model²¹. Initial trees were generated using Neighbor-Join and BioNJ algorithms with the JTT matrix.

All procedures involving animals were approved by the Local Ethics Committee for Animal Experiments of Firat University, Animal Experiment Ethic Committee, under protocol number 2023/03 − 02. All methods were performed in accordance with the relevant guidelines and regulations. Rabbits were obtained from the Elazig Veterinary Control Institute, Elazig, Türkiye. Sheep were privately owned and naturally infected animals, submitted by local veterinarians for diagnostic purposes, with owner consent obtained prior to sample collection.

Cloning and expression of the Recombinant SBP4 protein

Following successful PCR amplification, the sbp4 coding sequence of B. ovis was subjected to next-generation sequencing (NGS). De novo assembly of the sequencing data was performed by a commercial sequencing service provider (Ficus Biotechnology, Ankara, Türkiye) to verify the integrity and accuracy of the amplified gene.

Codon-optimized sequences were ordered from a commercial provider (GenScript, Piscataway, NJ, USA), after which the verified sequence was codon optimized for improved translation in Escherichia coli. The natural signal peptide was deleted from the 5′ end of the gene, a start codon (ATG) was inserted, and a 6×His tag encoding sequence was appended at the 3′ end of the gene to improve the later process of purification. The final optimized gene construct was synthesized and cloned and inserted into the pET-29b(+) expression vector.

Chemically competent E. coli cell lines were transformed with recombinant plasmids via calcium chloride heat shock. The strains DH5α and TOP10 were used for cloning and amplification of plasmids, whereas the strains BL21(DE3) and C43(DE3) were used for protein expression. The transformants were screened on LB plates containing 50 µg/mL kanamycin, and the positive colonies were reconfirmed via plasmid extraction and restriction enzyme digestion^22,23.

For recombinant protein expression analysis, starter cultures were inoculated and diluted 1:250 in fresh LB broth. Induction was performed at various optical densities (OD₆₀₀ = 0.6, 0.7, 0.8, and stationary phase), IPTG concentrations (0.05 mM and 1 mM), and incubation temperatures (18 °C, 25 °C, and 37 °C) to identify optimal expression conditions. The cell pellets were harvested postinduction and processed for protein purification and analysis^22,23.

Protein purification and SDS‒PAGE analysis

The expression cultures of the E. coli BL21(DE3) and C43(DE3) strains were harvested via centrifugation at 5.000 × g for 10 min at 4 °C after induction, and the cell pellets were washed once with phosphate-buffered saline (PBS), resuspended in a small volume of cold PBS, and loaded into the feed tank of a high-pressure homogenizer (M-110P Microfluidizer, Microfluidics, USA)^22,23.

Cell lysis was performed by mechanical rupture at 18.000 psi. Homogenization was achieved via the microchannel system of an instrument comprising diamond-coated Z-shaped chambers with channel widths of 84 μm. This setup allows high-pressure flow and shear forces to finely disintegrate bacterial cells without any chemical lysis, which will maintain the native structure of the recombinant proteins. Protein concentrations in all bacterial lysates were determined using the Bradford protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer’s instructions. The lysate was centrifuged at 15,000 × g for 30 min at 4 °C to separate the soluble protein fraction from the cell debris. The supernatant was collected for affinity purification of the recombinant His-tagged SBP4 protein. The soluble fraction of the recombinant SBP4 protein was affinity purified with a nickel‒nitrilotriacetic acid (Ni‒NTA) chromatographic system. The purification process was carried out under native conditions via HisTrap™ FF columns on an FPLC system (ÄKTA, pure) according to the manufacturer’s instructions. Binding, washing, and elution buffers were supplemented with appropriate concentrations of imidazole to ensure specific binding and efficient elution of the His-tagged recombinant protein. The imidazole concentrations used were 20 mM for binding, 40 mM for washing, and 500 mM for elution. The eluted fractions were analyzed by SDS‒PAGE via a 12% polyacrylamide gel to evaluate the purity and approximate molecular weight of the recombinant protein. The protein bands were visualized via Coomassie Brilliant Blue staining^22,23.

Western blot analysis

Western blot analysis was carried out to ascertain the synthesis and immunogenicity of the recombinant SBP4 protein by employing anti-His tag antibodies and the sera of naturally infected sheep. Sera used for WB and iELISA analyses were obtained from five sheep naturally infected with B. ovis in Elazig Province, Türkiye. Infection was previously confirmed by clinical signs, microscopy, and species-specific PCR. Blood samples were collected during the postacute phase of infection after clinical recovery. Sera were separated by centrifugation and stored at − 80 °C until use. Negative control serum was obtained from a clinically healthy, PCR-negative sheep from a non-endemic area.

After SDS‒PAGE, the proteins were electrophoretically transferred to nitrocellulose membranes via a wet transfer system at 100 V, and the membranes were blocked with 5% (w/v) skim milk in TBST (Tris-buffered saline with 0.1% Tween-20) for 1 h at room temperature to block non-specific binding. For immunoreactivity testing, the membranes were incubated overnight at 4 °C with a 1:500 dilution of naturally infected sheep serum in blocking buffer. The membranes were washed with TBST, followed by incubation with peroxidase-conjugated Pierce™ recombinant protein A/G (#catalog number: 32490, Thermo Scientific) for 1 h at room temperature at a final concentration of 1000 ng/mL. After further washing, an enhanced chemiluminescence (ECL) detection solution was added, and the signal was photographed in a dark room via a chemiluminescence imaging system. Clear immunoreactive bands were observed in concentrated elution fractions, which indicated the expression and immunogenicity of the recombinant SBP4 protein with the antibodies present in naturally infected animal sera. Uncropped original blot images, including membrane edges and replicate results, are provided in Supplementary Fig. 4 to ensure full transparency of the WB data.

Rabbit immunization and antibody production

To prepare polyclonal antibodies against rBoSBP4, healthy New Zealand White rabbits (2–3 months old) were used for immunization. In the first immunization, 500 µg of purified rBoSBP4 protein in 1 mL of solution (500 µL of 1× PBS and 500 µL of Freund’s Complete Adjuvant; Sigma‒Aldrich, USA) was injected subcutaneously into each rabbit as the primary immunization. The antigen‒adjuvant mixture was injected subcutaneously at four locations in the cervical area, with 250 µL in each location. Three booster immunizations were administered at two-week intervals. For each booster, 150 µg of rBoSBP4 was emulsified in 250 µL of PBS and 250 µL of Freund’s incomplete adjuvant. The entire 500 µL injection volume at the two sites was administered subcutaneously. Two weeks after the last booster, blood was taken from the central ear vein of each rabbit. The blood was allowed to clot and was subsequently centrifuged to obtain the serum, which was stored at -80 °C until use^13,24,25,26. The rabbit sera were checked for specific anti-rBoSBP4 antibodies via iELISA.

Indirect ELISA

An iELISA was carried out to determine antigenicity of the rBoSBP4 protein and the specific antibodies in the sera of rabbits and naturally infected sheep. In brief, 96-well plates (Immulon^® 2HB 96-well Microtiter EIA Plate, Thermo Scientific) were coated overnight at 4 °C with 100 µL of purified rBoSBP4 (2 µg/mL) in carbonate-bicarbonate coating buffer (pH 9.6) per well. After coating, the wells were washed three times with PBS-T (0.05% Tween-20 in PBS) and then blocked with 5% skim milk solution in PBST for 1 h at 37 °C (200 µL/well). The plates were then supplemented with 1:100 dilutions of rabbit serum or sheep serum in blocking buffer. A volume of 100 µL of each serum sample was added to the well, incubated at 37 °C for 1 h and then washed; 100 µL of HRP-conjugated secondary antibody was added to each well. Rabbit or sheep sera were treated with goat anti-rabbit IgG-HRP or protein A/G-HRP (100 ng/mL) (Pierce™ recombinant protein A/G, Thermo Scientific), respectively, diluted in blocking buffer according to the manufacturer´s protocol. The plates were washed, incubated at 37 °C for 1 h, and washed again, and 100 µL of TMB substrate solution was then added to each well. Color development was stopped at 10 min with 50 µL of 1 M H₂SO₄. The absorbance was recorded at 450 nm with a personal microplate reader (BioTek ELx800). The negative control wells contained serum from uninfected animals. All the samples were tested in duplicate, and a cutoff value was determined on the basis of the mean OD of the negative controls plus three standard deviations.

Statistical analysis

All ELISA data were analyzed via GraphPad Prism version 8 (GraphPad Software, San Diego, CA, USA). Indirect ELISA was performed using sera collected from both experimentally immunized rabbits and naturally Babesia ovis-infected sheep to evaluate the immunoreactivity of the recombinant rBoSBP4 antigen. In rabbit studies, antibody responses at different immunization time points were compared with those of pre-immune sera. Sera from naturally infected sheep were compared with those from non-infected sheep. The non-parametric Kruskal‒Wallis test was used to assess significant differences between groups, followed by Dunn’s multiple comparison test. The data are presented as the means, and p values less than 0.05 were considered statistically significant.