Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites

Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites Taisei Takahashi, Jae Seung Lee, Apinya Arnuphapprasert, Atefeh Fathi, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9469245/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Babesia bovis merozoite surface antigen 2 (MSA-2) are proteins encoded by msa multigene family and implicated in erythrocyte recognition and invasion, yet their roles remain only partially understood. We investigated spatiotemporal differences of three MSA-2 paralogs (MSA-2a1, MSA-2a2, and MSA-2b) by using epitope-specific rabbit antisera. Alignment of amino acid sequences of B. bovis T2Bo strain and Texas strain C1 clone showed that MSA-2a1 is highly conserved (99.6% identity) among the parasites, whereas MSA-2a2 (84%) and MSA-2b (94%) are more variable; notably, the least conserved paralog, MSA-2a2, harbored a contiguous 24 amino acid deletion. Indirect immunofluorescence assays using the antisera revealed apical localization of all three proteins in intraerythrocytic parasite. Following merozoite purification and brief incubation, localization of MSA-2a1 was observed on the merozoite surface before the attachment, whereas MSA-2a2 and MSA-2b remained at the apical end. Moreover, growth-inhibition assays, antiserum Ab-1 recognizing MSA-2a2 and MSA-2b inhibited 90.4% at 48 h incubation, whereas Ab-2 recognizing MSA-2a1 and MSA-2a2 inhibited only 13.1%. These results suggest functional difference among MSA-2 paralogs: MSA-2a1 was secreted to the surface before attachment, while MSA-2a2 and MSA-2b remain inside the merozoite at the onset of attachment. Taken together, MSA-2a1, MSA-2a2, and MSA-2b appear structurally similar, yet functionally distinct proteins that may contribute to the coordinated control of erythrocyte invasion. Babesia bovis MSA-2 family erythrocyte invasion Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Babesia bovis is a tick-transmitted intraerythrocytic protozoan parasite of the phylum Apicomplexa that causes bovine babesiosis. The sporozoites are transmitted from the salivary glands of infected ticks to cattle during blood feeding and undergoes asexual replication inside of host red blood cells (RBCs) (Santos et al., 2023 ; Suarez et al., 2019 ). An important step during the intraerythrocytic stage of B. bovis is the invasion to host RBCs. This process is mediated by multiple proteins derived from the apical organelles (Suarez et al., 2019 ; Yokoyama et al., 2006 ). Particularly, merozoite surface antigen (MSA) family proteins are expressed on the surface of both B. bovis merozoites and sporozoites, and are presumed to play essential roles in the early stages of recognition and attachment to host RBC membranes during invasion (Florin-Christensen et al., 2002 ; Mosqueda et al., 2002 ; Wilkowsky et al., 2003 ). In a study using B. bovis Mexico Mo7 strain, one MSA-1 antigen and four members of the MSA-2 antigens (MSA-2a1, MSA-2a2, MSA-2b, and MSA-2c) were identified (Florin-Christensen et al., 2002 ). MSA-2 family genes were reported to be arranged in tandem within the genome and confirmed to contain open reading frames (ORFs) without introns (Florin-Christensen et al., 2002 ). Comparative analysis between the Mexico Mo7 strain and the Argentina R1A strain revealed that MSA-2 proteins exhibit relatively high levels of amino acid sequence conservation (Florin-Christensen et al., 2002 ). These antigens have glycosylphosphatidylinositol (GPI) signal sequence, and most of them have been reported to contain B-cell epitopes with immunogenic potential (Florin-Christensen et al., 2002 ; Mosqueda et al., 2002 ). Notably, the presence of distinct B-cell epitopes has been reported for MSA-2a1, MSA-2b, and MSA-2c (Florin-Christensen et al., 2002 ). These findings may indicate that the genetic diversity detected among the MSA-2 genes reflects functional divergence among their encoded antigens. However, previous studies have not fully characterized the localization and function of each MSA-2 paralog during parasite egress to invasion. In this study, we generated two epitope-specific rabbit antisera, Ab-1 and Ab-2, which recognize MSA-2a2 and MSA-2b, and MSA-2a1 and MSA-2a2, respectively. These antibodies were used to examine the localization patterns of the proteins during invasion, and their neutralization activity was assessed. MATERIALS AND METHODS In vitro culture B. bovis Texas strain C1 clone was maintained in purified bovine RBCs (Japan BioSerum, Japan) using GIT medium (Fujifilm Wako Pure Chemical, Japan) under a microaerophilic stationary-phase culture system (Hakimi et al., 2025 ). Parasites were cultured at 10% hematocrit in 1 mL of medium per well in 24-well culture plates (AS ONE, USA). Parasitemia levels were monitored daily by staining thin blood smears with Giemsa solution. The whole genome sequence analysis and transcriptome analysis of C1 clone have completed in the previous study (Hakimi et al., 2025 ). Production of anti-MSA antibodies Two antisera targeting MSA-2 proteins were generated. One of the antisera (Ab-1) was prepared against the amino acid sequence corresponding to residues 132–149 (FLNDNPQRLLADKNGEVT) of MSA-2a2, which is conserved on MSA-2a2 and MSA-2b (Fig. 1 , Fig. S1 ). The another anti-MSA-2 antiserum (Ab-2) was prepared against residues 234–251 (SPQGPTAESPSQADHPTK) of MSA-2a2, a region that is shared by both MSA-2a1 and MSA-2a2 (Fig. 1 , Fig. S1 ). Due to the high sequence similarity among paralogs, two conserved regions shared between MSA-2a2 and either MSA-2a1 or MSA-2b were selected. Both antisera were produced by immunizing rabbits with their respective synthetic peptides (Cosmo Bio, Japan). In brief, the peptides were synthesized and conjugated to keyhole limpet hemocyanin. Each rabbit was immunized with its corresponding peptide four times (400 µg for first immunization, 200 µg from second immunization) and sacrificed to obtain antisera. Protein extraction and Western blotting Protein extraction and Western blotting Infected red blood cells (iRBCs) were mixed with 0.15% saponin in PBS for cell lysis and washed twice with a protease inhibitor solution (PI, cOmplete Mini EDTA-free, Roche Diagnostics GmbH, Germany). Proteins were extracted using a solution consisting of 1% Triton X, 2% SDS with PBS-PI solution, followed by three times of freeze-thaw cycles. The extracted proteins were separated by polyacrylamide gel electrophoresis using a 5–20% gradient gel (ATTO, Japan). Following electrophoresis, the proteins were transferred onto a polyvinylidene difluoride membrane (Clear Blot P+ membrane, ATTO, Japan) using a transfer apparatus (ATTO, Japan). The transferred membrane was incubated in Blocking One (Nacalai Tesque Inc., Japan) for 60 min for the blocking step, and then reacted overnight with Ab-1 or Ab-2 serum diluted 1:500. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega, USA) was used as the secondary antibody, diluted 1:25,000, and reacted 2 hours. After reaction with the secondary antibody, the membrane was washed three times with PBS containing 0.05% of Tween 20. Protein bands on the membrane were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) and detected with a CCD camera (LuminoGraph II EM, ATTO, Japan). Merozoite purification Two mL of iRBCs with over 5% parasitemia were centrifuged at 3,000 g for 1 min. The pellet was resuspended in cooled PBS to a final volume of 20 mL and filtered twice using 2.0 µm pore size polycarbonate membrane filters (Isopore filter, Merck, USA). The filtrate containing free merozoite was centrifuged at 3,000 g for 10 min at 10 ℃ and washed twice with PBS. After the final wash, the merozoites were resuspended in prewarmed GIT medium at 37 ℃. During merozoite purification, the merozoites were kept cool, and Ca 2+ /Mg 2+ -free PBS was used to prevent merozoite activation. Indirect immunofluorescence assay (IFA) The blood smears of the B. bovis were fixed using a 1:1 (v/v) mixture of acetone-methanol mixture at -30 ℃ for 10 min. After fixation, the slides were washed with PBS and incubated for 30 min at room temperature with PBS-0.05% Tween 20 (v/v) mixed with 10% of normal goat serum. The smears were then incubated with Ab-1 and Ab-2 as the primary antibody at 37 ℃ for 60 min. Primary antibodies were diluted 1:100 in 1% normal goat serum-PBS. As a negative control, 1% of normal goat serum-PBS was applied. Subsequently, Alexa Fluor 488-conjugated goat anti-rabbit IgG and Hoechst 33342 were used as secondary antibody and nuclear stain, respectively, and incubated at 37 ℃ for 60 min. The secondary antibody and Hoechst were diluted 1:200 and 1:200, respectively, with PBS-0.05% Tween 20 (v/v). Fluorescence images were acquired using a confocal microscope (Leica TCS SP5 Confocal Microscope, Leica microsystems, Germany). Neutralization assay The antisera Ab-1 and Ab-2 were applied for in vitro neutralization assay. The antisera were filtered using 0.22 µm pore-size filters before usage. For each well in a 96-well plate, 90 µL of GIT medium, 10 µL of iRBCs, and 10 µL of either immune serum or pre-immune serum were mixed. Parasitemia of iRBCs were adjusted to 0.5% and added to the wells. All experimental conditions were prepared in triplicate wells and three independent experiments were performed. Parasitemia levels were assessed daily for 2 consecutive days by counting around 2,000 RBCs per sample. RESULTS Comparative analysis of MSA-2a1, 2a2, and 2b between B. bovis T2Bo and C1 clones The MSA-2 sequence of the C1 clone was obtained from the whole genome sequencing data performed on the former experiment (Hakimi et al., 2025 ), and aligned using Clustal W. A comparison of the MSA-2a1 amino acid sequences between the T2Bo and C1 clone revealed 99.6% identity (Fig. S1 ). MSA-2a2, sequence comparison exhibited 84% amino acid identity between the two strains (Fig. S1 ). A total of 27 amino acid differences were identified, along with a deletion of a continuous 24 amino acid region. This deletion was located between amino acid positions 212 and 235 in the T2Bo strain. The MSA-2b sequence showed 94% amino acid identity between the T2Bo and C1 strains (Fig. S1 ). In the C1 clone, MSA-2a1 and MSA-2a2 showed 86.3% amino acid identity, while lower identity values were observed between MSA-2a1 and MSA-2b (54.8%) and between MSA-2a2 and MSA-2b (65.8%) (Fig. 1 ). Generation and evaluation of two antisera, Ab-1 and Ab-2 Two antisera against MSA-2 (Ab-1 and Ab-2) were generated by immunizing rabbits with synthetic peptides corresponding to regions of MSA-2. The site highlighted in orange corresponds to the epitope used to generate Ab-1, which is identical in both MSA-2a2 and MSA-2b (Fig. 1 , Fig. S1 ). The site highlighted in blue corresponds to the epitope used to generate Ab-2, which is identical between MSA-2a1 and MSA-2a2 (Fig. 1 , Fig. S1 ). Due to the high sequence similarity among MSA-2a1, MSA-2a2, and MSA-2b, no appropriate region was found to elicit paralogs-specific amino acids sequences for peptide antibody production. Therefore, two conserved regions shared between MSA-2a2 and either MSA-2a1 or MSA-2b were selected. The specificity of Ab-1 and Ab-2 was evaluated by Western blotting using protein extracted from the parasite (Fig. 2 , Fig. S2 ). The Ab-1 detected two bands at approximately 29 kDa and 33 kDa. The Ab-2 also detected two bands at 33 kDa and 41 kDa. The 29 kDa band detected by Ab-1 was expected to represent MSA-2b, while the 41 kDa band observed using Ab-2 corresponds to MSA-2a1. Notably, both antisera detected a 33 kDa band, which was consistent with MSA-2a2. Localization of MSA-2a1, MSA-2a2, and MSA-2b by indirect immunofluorescence assay To investigate the localization of MSA-2a1, MSA-2a2, and MSA-2b, IFA was conducted using Ab-1 and Ab-2. Thin blood smears of iRBC were reacted with each antisera. Reaction with Ab-1 showed significant green fluorescence within the parasites, with intense accumulation of MSA-2a2 and MSA-2b at the apical end in both single and binary forms (Fig. 3 , Single and Binary). Similarly, Ab-2 also exhibited comparable fluorescence patterns, indicating that MSA-2a1 and MSA-2a2 are localized to the apical end. To further assess the secretion timing of MSA-2 proteins during merozoite egress to invasion, IFA was conducted on purified merozoites. After merozoite filtration, three types of smears were prepared including (1) free merozoites collected immediately after filtration, (2) merozoites incubated in GIT medium for 5 minutes, and (3) merozoites incubated with RBCs in GIT medium for 5 minutes. The first sample was used to evaluate MSA-2 localization on free merozoites, while the second and third samples were prepared to examine temporal changes in MSA-2 localization after egress. On the free merozoites immediately post-filtration, both antisera detected fluorescence with strong accumulation at the apical end (Fig. 3 , Post-filtration). After five minutes of incubation in GIT medium, Ab-1 staining remained concentrated at the apical end, whereas Ab-2 showed circular staining, which indicated localization of MSA-2a1 to the merozoite surface (Fig. 3 , GIT). Similarly, in merozoites incubated with GIT medium and RBCs, Ab-1 still showed apical localization, while Ab-2 showed circular localization. The Ab-2-stained merozoite was observed in close contact with an RBC, indicating surface expression of MSA-2a1 during erythrocyte attachment. However, these results suggested MSA-2a2 and MSA-2b were still not secreted at this moment. Inhibitory effect of anti–MSA-2 antisera on the growth of B. bovis A neutralization assay was performed to assess whether two antisera (Ab-1 and Ab-2) inhibit the growth of B. bovis . The Ab-1 antiserum, which reacts with MSA-2a2 and MSA-2b, significantly reduced parasite growth compared to the corresponding pre-immune serum. After 2 days of culture, the pre-immune serum exposed group reached 5.54 ± 0.38% parasitemia, whereas those treated with Ab-1 serum exhibited only 0.98 ± 0.18% parasitemia (Fig. 4 A). The calculated inhibition rate for Ab-1 was 90.4 ± 3.6% (Fig. 4 B). In contrast, the Ab-2 serum, which reacts with MSA-2a1 and MSA-2a2, did not significantly inhibit parasite growth. The parasitemia levels were 4.30 ± 0.02% for the Ab-2 exposed group and 5.15 ± 0.45% for the pre-immune serum group, resulting in an inhibition rate of 13.1 ± 8.4% for Ab-2. DISCUSSION In this study, we investigated the MSA-2 proteins of B. bovis (MSA-2a1, MSA-2a2, and MSA-2b), the GPI-anchored antigens secreted on the merozoite surface (Mosqueda et al., 2002 ; Rodriguez et al., 2014 ). We generated two epitope-specific antisera (Ab-1 and Ab-2) to analyze their secretion timing and localization of the proteins during the egress to invasion and assessed antibody-mediated growth inhibition. Together, our data indicate that while all three paralogs were apically localized at the timing of egress, MSA-2a1 transiently secreted to the surface at pre-attachment, whereas MSA-2a2 and MSA-2b remained apical organelle. In addition, two antisera had different ability to inhibit parasite growth supporting paralog specific roles during invasion. Between the T2Bo and C1 clones (Fig. S1 ), MSA-2a1 was highly conserved, whereas MSA-2a2 and MSA-2b contained multiple substitutions and deletions; notably, MSA-2a2 of C1 clone harbored a contiguous 24 amino acid deletion, representing the greatest divergence. Similar inter-strain differences have been documented across other B. bovis populations. In a study comparing the Mexico Mo7 and Argentina R1A strains, MSA-2c was the most conserved (88.7% amino-acid identity); among the three paralogs examined in this study, MSA-2a1 showed 83.6% identity (second highest overall), followed by MSA-2b (79.1%) and MSA-2a2 (69.4%) (Florin-Christensen et al., 2002 ). In addition, Florin-Christensen and colleagues reported an apparent recombination region between MSA-2a2 and MSA-2b (Florin-Christensen et al., 2002 ). In our study, this region contained the Ab-1-targeted epitope (FLNDNPQRLLADKNGEVT), and antiserum Ab-1 significantly inhibited the growth of live B. bovis (Fig. 1 and Fig. 4 ). Together, these results may indicate that this region is an antibody-accessible epitope shared by both MSA-2a2 and MSA-2b in live parasites and can be functionally targeted. Localization analysis indicated that MSA-2a1, MSA-2a2, and MSA-2b were localized to the apical region of the merozoites, and MSA-2a1 secreted to the parasite surface prior to the attachment (Fig. 3 ). Former studies have reported that each paralogue have expressed on the surface of merozoites by live IFA using the free or purified merozoites (Mosqueda et al., 2002 ; Wilkowsky et al., 2003 ). However, precise localization and secretion timing of MSA-2 paralogues were unclear. Therefore, this study analyzed the merozoite just after the purification and substantial incubation by medium, and IFA images were observed by confocal microscopy. The observed images suggested that MSA-2a1 functions during the initial stage of erythrocyte attachment, whereas MSA-2a2 and MSA-2b remain within the apical organelles and may be released later during the internalization process. In the comparative neutralization assay, Ab-1 (MSA-2a2/MSA-2b) strongly inhibited parasite growth by 90.4% at 48 h, whereas Ab-2 (MSA-2a1/MSA-2a2) caused only a 13.1%inhibition. The Ab-2 recognizes both MSA-2a1 and MSA-2a2, and both antisera were raised against overlapping peptide epitopes; therefore, paralog-specific effects may be confounded and epitope placement may influence neutralization efficiency. However, these results may indicate that the contribution of the MSA-2a subfamily, particularly MSA-2a1, to invasion inhibition is limited, while MSA-2b likely plays a pivotal role in the invasion process. This interpretation aligns with previous reports showing that antibodies against MSA-2 proteins significantly inhibit merozoite invasion (Mosqueda et al., 2002 ; Wilkowsky et al., 2003 ), although the present findings further differentiate the inhibitory effects among individual MSA-2 paralogs. Taken together, these data suggest that, although the three molecules belong to the same protein family, they perform distinct roles at different stages of erythrocyte invasion. Further work defining the organelle harboring MSAs, regulatory signals of secretion, and identification of RBC ligands will delineate coordinated invasion mechanism of this parasite. Declarations Acknowledgements This work was conducted at the Joint Usage/Research Center for the control of protozoan diseases, National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, and the Joint Usage/Research Center for overcoming zoonotic diseases, International Institute for Zoonosis Control, Hokkaido University. Author contributions Conceptualization: Masahito Asada, Junya Yamagishi, Methodology: Taisei Takahashi, Apinya Arnuphapprasert, Atefeh Fathi, Formal analysis and investigation: Taisei Takahashi, Jae Seung Lee, Apinya Arnuphapprasert, Atefeh Fathi, Kota Komatsu, Miruka Silviane, Morakot Kaewthamasorn, Junya Yamagishi, Masahito Asada, Writing - original draft preparation: Jae Seung Lee, Taisei Takahashi, Writing - review and editing: Masahito Asada, Jae Seung Lee, Taisei Takahashi, Apinya Arnuphapprasert, Atefeh Fathi, Kota Komatsu, Miruka Silviane, Morakot Kaewthamasorn, Junya Yamagishi, Funding acquisition: Junya Yamagishi, Masahito Asada, Resources: Masahito Asada, Junya Yamagishi, Supervision: Masahito Asada. Funding This work was supported by JSPS KAKENHI Grant Number 24K02270 to J.Y. and 26K01907 to M.A. Data availability The genome sequence of B. bovis was obtained from our previous study (Hakimi et al., 2025). The whole genome sequence is available from the corresponding author. Ethics approval and consent to participate All experiments were carried out according to the approval from biosafety committee of Obihiro University of Agriculture and Veterinary Medicine (No. 202407). Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Florin-Christensen, M., Suarez, C.E., Hines, S.A., Palmer, G.H., Brown, W.C., McElwain, T.F., 2002. The Babesia bovis merozoite surface antigen 2 locus contains four tandemly arranged and expressed genes encoding immunologically distinct proteins. Infect. Immun. 70, 3566–3575. https://doi.org/10.1128/IAI.70.7.3566-3575.2002 Hakimi, H., Yamagishi, J., Sakaguchi, M., Fathi, A., Lee, J.S., Verocai, G.G., Kawazu, S.I., Asada, M., 2025. ves1α genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells. PLoS Pathog. 21, 1–16. https://doi.org/10.1371/journal.ppat.1012583 Mosqueda, J., McElwain, T.F., Palmer, G.H., 2002. Babesia bovis Merozoite Surface Antigen 2 Proteins Are Expressed on the Merozoite and Sporozoite Surface, and Specific Antibodies Inhibit Attachment and Invasion of Erythrocytes. Infect. Immun. 70, 6448–6455. https://doi.org/10.1128/IAI.70.11.6448-6455.2002 Rodriguez, A.E., Florin-Christensen, M., Flores, D.A., Echaide, I., Suarez, C.E., Schnittger, L., 2014. The glycosylphosphatidylinositol-anchored protein repertoire of Babesia bovis and its significance for erythrocyte invasion. Ticks Tick. Borne. Dis. 5, 343–348. https://doi.org/10.1016/j.ttbdis.2013.12.011 Santos, J.H.M., Siddle, H. V., Raza, A., Stanisic, D.I., Good, M.F., Tabor, A.E., 2023. Exploring the landscape of Babesia bovis vaccines: progress, challenges, and opportunities. Parasites and Vectors 16, 1–12. https://doi.org/10.1186/s13071-023-05885-z Suarez, C.E., Alzan, H.F., Silva, M.G., Rathinasamy, V., Poole, W.A., Cooke, B.M., 2019. Unravelling the cellular and molecular pathogenesis of bovine babesiosis: is the sky the limit? Int. J. Parasitol. 49, 183–197. https://doi.org/10.1016/j.ijpara.2018.11.002 Wilkowsky, S.E., Farber, M., Echaide, I., Torioni De Echaide, S., Zamorano, P.I., Dominguez, M., Suarez, C.E., Florin-Christensen, M., 2003. Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle. Mol. Biochem. Parasitol. 127, 133–141. https://doi.org/10.1016/S0166-6851(02)00329-8 Yokoyama, N., Okamura, M., Igarashi, I., 2006. Erythrocyte invasion by Babesia parasites: Current advances in the elucidation of the molecular interactions between the protozoan ligands and host receptors in the invasion stage. Vet. Parasitol. 138, 22–32. https://doi.org/10.1016/j.vetpar.2006.01.037 Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9469245","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":636969996,"identity":"30d82f32-4251-49a4-a6e2-a8c3ed0fa055","order_by":0,"name":"Taisei Takahashi","email":"","orcid":"","institution":"Obihiro University of Agriculture and Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Taisei","middleName":"","lastName":"Takahashi","suffix":""},{"id":636969997,"identity":"0e49baee-35d6-45f8-9545-22710849aa93","order_by":1,"name":"Jae Seung Lee","email":"","orcid":"","institution":"Obihiro University of Agriculture and Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jae","middleName":"Seung","lastName":"Lee","suffix":""},{"id":636969998,"identity":"548d2029-3a27-4202-a1f8-619e44a33bf3","order_by":2,"name":"Apinya Arnuphapprasert","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Apinya","middleName":"","lastName":"Arnuphapprasert","suffix":""},{"id":636969999,"identity":"15369e40-61fa-4947-b402-7a116cfb7ed6","order_by":3,"name":"Atefeh Fathi","email":"","orcid":"","institution":"Obihiro University of Agriculture and Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Atefeh","middleName":"","lastName":"Fathi","suffix":""},{"id":636970000,"identity":"791e8ca8-430b-4bfd-9ae9-c1afcc78c2b6","order_by":4,"name":"Kota Komatsu","email":"","orcid":"","institution":"Obihiro University of Agriculture and Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Kota","middleName":"","lastName":"Komatsu","suffix":""},{"id":636970001,"identity":"b483d36b-3487-4eeb-9bab-f1a7b0e0dc67","order_by":5,"name":"Miruka Silviane","email":"","orcid":"","institution":"Obihiro University of Agriculture and Veterinary Medicine","correspondingAuthor":false,"prefix":"","firstName":"Miruka","middleName":"","lastName":"Silviane","suffix":""},{"id":636970002,"identity":"9bb37fb2-e4bc-4d0c-91e8-2bda6bc3e2d1","order_by":6,"name":"Morakot Kaewthamasorn","email":"","orcid":"","institution":"Chulalongkorn University","correspondingAuthor":false,"prefix":"","firstName":"Morakot","middleName":"","lastName":"Kaewthamasorn","suffix":""},{"id":636970004,"identity":"4b145cef-d310-428b-ac2a-56e6c3a335df","order_by":7,"name":"Junya Yamagishi","email":"","orcid":"","institution":"Hokkaido University","correspondingAuthor":false,"prefix":"","firstName":"Junya","middleName":"","lastName":"Yamagishi","suffix":""},{"id":636970007,"identity":"eb3c281b-5010-4925-9ecb-67db45df4273","order_by":8,"name":"Masahito Asada","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYLACHgYbBgNmHoYDUH4CMVrSSNdymMEASBIHdNuPP/zwpua8nDk778HDFRUM8vwNDM8e4NNidibHWHLOsdvGls18CQfPnGEwnHGAId0Ar5YDOQzSPGy3Ezcc5jE42NjGwLiBgSFNAq+W888f/+b5dw6uxZ6wlhsJZtK8bQfgWhKJ0PLGzHJuXzLQL0AtDWckkmccJuSX8+mPb7z5Zidnzn/G+GNDhY1tf3tP2gN8WtAB0EnMPGmk6AAD9mMkaxkFo2AUjIJhDQCCWErgvxaGSQAAAABJRU5ErkJggg==","orcid":"","institution":"Obihiro University of Agriculture and Veterinary Medicine","correspondingAuthor":true,"prefix":"","firstName":"Masahito","middleName":"","lastName":"Asada","suffix":""}],"badges":[],"createdAt":"2026-04-20 08:39:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9469245/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9469245/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":109320836,"identity":"cd03aa08-6482-4062-bc92-f023c2ad3971","added_by":"auto","created_at":"2026-05-15 13:32:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7389706,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAmino acid sequence alignment of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. bovis \u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003eC1 clone MSA-2a1, MSA-2a2 and MSA2b\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThree amino acid sequences including MSA-2a2, MSA-2a1 and MSA-2b were aligned using ClustalW. Identical residues among the sequences are indicated by dots (·) and gaps are indicated in dashed lines (-). Amino acid sequences highlighted in orange (MSA-2a2 and MSA-2b) and in blue (MSA-2a2 and MSA-2a1) indicate the peptides used to obtain rabbit antisera Ab-1 and Ab-2, respectively.\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/535f0c8ae68900432f902d4f.png"},{"id":109320838,"identity":"c672a1cb-e0cc-4597-bbd6-de336601d2f8","added_by":"auto","created_at":"2026-05-15 13:32:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1378032,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eWestern blot analysis of MSA detection by two rabbit anti-MSA antibodies (Ab-1 and Ab-2)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBands at 33 and 29 kDa in the Ab-1 blot indicate the reaction with MSA-2a2 and MSA-2b, respectively. Bands at 41 and 33 kDa in the Ab-2 blot indicate the reaction with MSA-2a1 and MSA-2a2, respectively.\u003c/p\u003e","description":"","filename":"Fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/9eb4c0a17af27125ad319fad.png"},{"id":109320840,"identity":"982c4055-7c92-40d8-99d5-5e75a8595c25","added_by":"auto","created_at":"2026-05-15 13:32:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":8594625,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLocalization analysis of MSA-2a1, MSA-2a2, and MSA-2b by immunofluorescence assay (IFA) using Ab-1 and Ab-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIFA images of MSA-2 protein localization in \u003cem\u003eB. bovis\u003c/em\u003e. The blood smears were prepared on glass slides and free merozoites were obtained by filtering the iRBC. The terms post-filtration, GIT, and GIT+RBC indicate free merozoites obtained immediately after filtration, after 5 minutes of incubation in GIT following filtration, and after 5 minutes of incubation in GIT with RBC following filtration, respectively. The parasites were stained with anti-MSA antibodies (Ab-1 or Ab-2, green). The nuclei of the parasites were stained with Hoechst 33342 (blue). Overlay images indicate bright field, anti-MSA-2 antibodies, and nuclei (blue). Scale bar = 7.5 μm.\u003c/p\u003e","description":"","filename":"Fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/48039454991f50c12afa180e.png"},{"id":109320841,"identity":"8bb0fa3d-c606-4974-894a-c1afb787c9e0","added_by":"auto","created_at":"2026-05-15 13:32:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1431546,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eInhibition of \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003eB. bovis\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e growth by rabbit antisera against MSA-2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) Growth of \u003cem\u003eB. bovis\u003c/em\u003e in cultures supplemented with Ab-1 or Ab-2 antisera, compared to corresponding preimmune serum. Initial parasitemia was 0.5% and parasitemia levels were monitored for 2 days with daily culture medium replacement. The red and blue lines represent Ab-1 and Ab-2 serum-treated groups, respectively. The pink and sky blue lines indicate their corresponding pre-immune serum controls. The graph is shown as mean ± standard error (SE) for three independent experiments. Statistically significant differences were observed between the Ab-1 serum and its corresponding pre-immune serum groups on both day 1 and day 2, as determined by one-way ANOVA followed by Tukey’s \u003cem\u003epost-hoc\u003c/em\u003e test (** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01; *** \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001). In contrast, no significant differences were detected between the Ab-2 serum and its pre-immune control. (B) The inhibition rates were calculated by normalized parasitemia at day 1 or 2, and comparing the results to those of the corresponding pre-immune serum. The sky blue and orange bars indicate the Ab-1 and Ab-2 serum-treated groups, respectively.\u003c/p\u003e","description":"","filename":"Fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/e7f459b387561081ac1ee1d8.png"},{"id":109406286,"identity":"b984e599-3d36-42aa-9eca-5b00c5afa8c9","added_by":"auto","created_at":"2026-05-17 13:27:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":17355303,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/9685a566-0f32-4b02-ac7e-34720e169807.pdf"},{"id":109405545,"identity":"44d28536-fc4d-4120-b6f6-d748c04d9412","added_by":"auto","created_at":"2026-05-17 13:18:52","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":7025708,"visible":true,"origin":"","legend":"","description":"","filename":"FigS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/bd89bcbc1f12483806112282.docx"},{"id":109405623,"identity":"1b9b7a72-8cf3-4d38-a1c2-c6bdadcc4a45","added_by":"auto","created_at":"2026-05-17 13:19:25","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1506329,"visible":true,"origin":"","legend":"","description":"","filename":"FigS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9469245/v1/76cb8a80037e73f9066527e7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003e \u003cem\u003eBabesia bovis\u003c/em\u003e is a tick-transmitted intraerythrocytic protozoan parasite of the phylum Apicomplexa that causes bovine babesiosis. The sporozoites are transmitted from the salivary glands of infected ticks to cattle during blood feeding and undergoes asexual replication inside of host red blood cells (RBCs) (Santos et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Suarez et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). An important step during the intraerythrocytic stage of \u003cem\u003eB. bovis\u003c/em\u003e is the invasion to host RBCs. This process is mediated by multiple proteins derived from the apical organelles (Suarez et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Yokoyama et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). Particularly, merozoite surface antigen (MSA) family proteins are expressed on the surface of both \u003cem\u003eB. bovis\u003c/em\u003e merozoites and sporozoites, and are presumed to play essential roles in the early stages of recognition and attachment to host RBC membranes during invasion (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Mosqueda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wilkowsky et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn a study using \u003cem\u003eB. bovis\u003c/em\u003e Mexico Mo7 strain, one MSA-1 antigen and four members of the MSA-2 antigens (MSA-2a1, MSA-2a2, MSA-2b, and MSA-2c) were identified (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). MSA-2 family genes were reported to be arranged in tandem within the genome and confirmed to contain open reading frames (ORFs) without introns (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Comparative analysis between the Mexico Mo7 strain and the Argentina R1A strain revealed that MSA-2 proteins exhibit relatively high levels of amino acid sequence conservation (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These antigens have glycosylphosphatidylinositol (GPI) signal sequence, and most of them have been reported to contain B-cell epitopes with immunogenic potential (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Mosqueda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Notably, the presence of distinct B-cell epitopes has been reported for MSA-2a1, MSA-2b, and MSA-2c (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThese findings may indicate that the genetic diversity detected among the MSA-2 genes reflects functional divergence among their encoded antigens. However, previous studies have not fully characterized the localization and function of each MSA-2 paralog during parasite egress to invasion. In this study, we generated two epitope-specific rabbit antisera, Ab-1 and Ab-2, which recognize MSA-2a2 and MSA-2b, and MSA-2a1 and MSA-2a2, respectively. These antibodies were used to examine the localization patterns of the proteins during invasion, and their neutralization activity was assessed.\u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro culture\u003c/h2\u003e \u003cp\u003e \u003cem\u003eB. bovis\u003c/em\u003e Texas strain C1 clone was maintained in purified bovine RBCs (Japan BioSerum, Japan) using GIT medium (Fujifilm Wako Pure Chemical, Japan) under a microaerophilic stationary-phase culture system (Hakimi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Parasites were cultured at 10% hematocrit in 1 mL of medium per well in 24-well culture plates (AS ONE, USA). Parasitemia levels were monitored daily by staining thin blood smears with Giemsa solution. The whole genome sequence analysis and transcriptome analysis of C1 clone have completed in the previous study (Hakimi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eProduction of anti-MSA antibodies\u003c/h3\u003e\n\u003cp\u003eTwo antisera targeting MSA-2 proteins were generated. One of the antisera (Ab-1) was prepared against the amino acid sequence corresponding to residues 132\u0026ndash;149 (FLNDNPQRLLADKNGEVT) of MSA-2a2, which is conserved on MSA-2a2 and MSA-2b (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The another anti-MSA-2 antiserum (Ab-2) was prepared against residues 234\u0026ndash;251 (SPQGPTAESPSQADHPTK) of MSA-2a2, a region that is shared by both MSA-2a1 and MSA-2a2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Due to the high sequence similarity among paralogs, two conserved regions shared between MSA-2a2 and either MSA-2a1 or MSA-2b were selected. Both antisera were produced by immunizing rabbits with their respective synthetic peptides (Cosmo Bio, Japan). In brief, the peptides were synthesized and conjugated to keyhole limpet hemocyanin. Each rabbit was immunized with its corresponding peptide four times (400 \u0026micro;g for first immunization, 200 \u0026micro;g from second immunization) and sacrificed to obtain antisera.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eProtein extraction and Western blotting\u003c/h3\u003e\n\u003cdiv class=\"Heading\"\u003eProtein extraction and Western blotting\u003c/div\u003e \u003cp\u003eInfected red blood cells (iRBCs) were mixed with 0.15% saponin in PBS for cell lysis and washed twice with a protease inhibitor solution (PI, cOmplete Mini EDTA-free, Roche Diagnostics GmbH, Germany). Proteins were extracted using a solution consisting of 1% Triton X, 2% SDS with PBS-PI solution, followed by three times of freeze-thaw cycles. The extracted proteins were separated by polyacrylamide gel electrophoresis using a 5\u0026ndash;20% gradient gel (ATTO, Japan). Following electrophoresis, the proteins were transferred onto a polyvinylidene difluoride membrane (Clear Blot P+ membrane, ATTO, Japan) using a transfer apparatus (ATTO, Japan). The transferred membrane was incubated in Blocking One (Nacalai Tesque Inc., Japan) for 60 min for the blocking step, and then reacted overnight with Ab-1 or Ab-2 serum diluted 1:500. Horseradish peroxidase (HRP)-conjugated anti-rabbit IgG (Promega, USA) was used as the secondary antibody, diluted 1:25,000, and reacted 2 hours. After reaction with the secondary antibody, the membrane was washed three times with PBS containing 0.05% of Tween 20. Protein bands on the membrane were visualized using the Immobilon Western Chemiluminescent HRP Substrate (Millipore, USA) and detected with a CCD camera (LuminoGraph II EM, ATTO, Japan).\u003c/p\u003e\n\u003ch3\u003eMerozoite purification\u003c/h3\u003e\n\u003cp\u003eTwo mL of iRBCs with over 5% parasitemia were centrifuged at 3,000 g for 1 min. The pellet was resuspended in cooled PBS to a final volume of 20 mL and filtered twice using 2.0 \u0026micro;m pore size polycarbonate membrane filters (Isopore filter, Merck, USA). The filtrate containing free merozoite was centrifuged at 3,000 g for 10 min at 10 ℃ and washed twice with PBS. After the final wash, the merozoites were resuspended in prewarmed GIT medium at 37 ℃. During merozoite purification, the merozoites were kept cool, and Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-free PBS was used to prevent merozoite activation.\u003c/p\u003e\n\u003ch3\u003eIndirect immunofluorescence assay (IFA)\u003c/h3\u003e\n\u003cp\u003eThe blood smears of the \u003cem\u003eB. bovis\u003c/em\u003e were fixed using a 1:1 (v/v) mixture of acetone-methanol mixture at -30 ℃ for 10 min. After fixation, the slides were washed with PBS and incubated for 30 min at room temperature with PBS-0.05% Tween 20 (v/v) mixed with 10% of normal goat serum. The smears were then incubated with Ab-1 and Ab-2 as the primary antibody at 37 ℃ for 60 min. Primary antibodies were diluted 1:100 in 1% normal goat serum-PBS. As a negative control, 1% of normal goat serum-PBS was applied. Subsequently, Alexa Fluor 488-conjugated goat anti-rabbit IgG and Hoechst 33342 were used as secondary antibody and nuclear stain, respectively, and incubated at 37 ℃ for 60 min. The secondary antibody and Hoechst were diluted 1:200 and 1:200, respectively, with PBS-0.05% Tween 20 (v/v). Fluorescence images were acquired using a confocal microscope (Leica TCS SP5 Confocal Microscope, Leica microsystems, Germany).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eNeutralization assay\u003c/h2\u003e \u003cp\u003eThe antisera Ab-1 and Ab-2 were applied for \u003cem\u003ein vitro\u003c/em\u003e neutralization assay. The antisera were filtered using 0.22 \u0026micro;m pore-size filters before usage. For each well in a 96-well plate, 90 \u0026micro;L of GIT medium, 10 \u0026micro;L of iRBCs, and 10 \u0026micro;L of either immune serum or pre-immune serum were mixed. Parasitemia of iRBCs were adjusted to 0.5% and added to the wells. All experimental conditions were prepared in triplicate wells and three independent experiments were performed. Parasitemia levels were assessed daily for 2 consecutive days by counting around 2,000 RBCs per sample.\u003c/p\u003e \u003c/div\u003e"},{"header":"RESULTS","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eComparative analysis of MSA-2a1, 2a2, and 2b between B. bovis T2Bo and C1 clones\u003c/h2\u003e \u003cp\u003eThe MSA-2 sequence of the C1 clone was obtained from the whole genome sequencing data performed on the former experiment (Hakimi et al., \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2025\u003c/span\u003e), and aligned using Clustal W. A comparison of the MSA-2a1 amino acid sequences between the T2Bo and C1 clone revealed 99.6% identity (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). MSA-2a2, sequence comparison exhibited 84% amino acid identity between the two strains (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). A total of 27 amino acid differences were identified, along with a deletion of a continuous 24 amino acid region. This deletion was located between amino acid positions 212 and 235 in the T2Bo strain. The MSA-2b sequence showed 94% amino acid identity between the T2Bo and C1 strains (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). In the C1 clone, MSA-2a1 and MSA-2a2 showed 86.3% amino acid identity, while lower identity values were observed between MSA-2a1 and MSA-2b (54.8%) and between MSA-2a2 and MSA-2b (65.8%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eGeneration and evaluation of two antisera, Ab-1 and Ab-2\u003c/h2\u003e \u003cp\u003eTwo antisera against MSA-2 (Ab-1 and Ab-2) were generated by immunizing rabbits with synthetic peptides corresponding to regions of MSA-2. The site highlighted in orange corresponds to the epitope used to generate Ab-1, which is identical in both MSA-2a2 and MSA-2b (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). The site highlighted in blue corresponds to the epitope used to generate Ab-2, which is identical between MSA-2a1 and MSA-2a2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Due to the high sequence similarity among MSA-2a1, MSA-2a2, and MSA-2b, no appropriate region was found to elicit paralogs-specific amino acids sequences for peptide antibody production. Therefore, two conserved regions shared between MSA-2a2 and either MSA-2a1 or MSA-2b were selected. The specificity of Ab-1 and Ab-2 was evaluated by Western blotting using protein extracted from the parasite (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, Fig. \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). The Ab-1 detected two bands at approximately 29 kDa and 33 kDa. The Ab-2 also detected two bands at 33 kDa and 41 kDa. The 29 kDa band detected by Ab-1 was expected to represent MSA-2b, while the 41 kDa band observed using Ab-2 corresponds to MSA-2a1. Notably, both antisera detected a 33 kDa band, which was consistent with MSA-2a2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLocalization of MSA-2a1, MSA-2a2, and MSA-2b by indirect immunofluorescence assay\u003c/h2\u003e \u003cp\u003eTo investigate the localization of MSA-2a1, MSA-2a2, and MSA-2b, IFA was conducted using Ab-1 and Ab-2. Thin blood smears of iRBC were reacted with each antisera. Reaction with Ab-1 showed significant green fluorescence within the parasites, with intense accumulation of MSA-2a2 and MSA-2b at the apical end in both single and binary forms (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Single and Binary). Similarly, Ab-2 also exhibited comparable fluorescence patterns, indicating that MSA-2a1 and MSA-2a2 are localized to the apical end.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo further assess the secretion timing of MSA-2 proteins during merozoite egress to invasion, IFA was conducted on purified merozoites. After merozoite filtration, three types of smears were prepared including (1) free merozoites collected immediately after filtration, (2) merozoites incubated in GIT medium for 5 minutes, and (3) merozoites incubated with RBCs in GIT medium for 5 minutes. The first sample was used to evaluate MSA-2 localization on free merozoites, while the second and third samples were prepared to examine temporal changes in MSA-2 localization after egress.\u003c/p\u003e \u003cp\u003eOn the free merozoites immediately post-filtration, both antisera detected fluorescence with strong accumulation at the apical end (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Post-filtration). After five minutes of incubation in GIT medium, Ab-1 staining remained concentrated at the apical end, whereas Ab-2 showed circular staining, which indicated localization of MSA-2a1 to the merozoite surface (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e, GIT). Similarly, in merozoites incubated with GIT medium and RBCs, Ab-1 still showed apical localization, while Ab-2 showed circular localization. The Ab-2-stained merozoite was observed in close contact with an RBC, indicating surface expression of MSA-2a1 during erythrocyte attachment. However, these results suggested MSA-2a2 and MSA-2b were still not secreted at this moment.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eInhibitory effect of anti\u0026ndash;MSA-2 antisera on the growth of B. bovis\u003c/h2\u003e \u003cp\u003eA neutralization assay was performed to assess whether two antisera (Ab-1 and Ab-2) inhibit the growth of \u003cem\u003eB. bovis\u003c/em\u003e. The Ab-1 antiserum, which reacts with MSA-2a2 and MSA-2b, significantly reduced parasite growth compared to the corresponding pre-immune serum. After 2 days of culture, the pre-immune serum exposed group reached 5.54\u0026thinsp;\u0026plusmn;\u0026thinsp;0.38% parasitemia, whereas those treated with Ab-1 serum exhibited only 0.98\u0026thinsp;\u0026plusmn;\u0026thinsp;0.18% parasitemia (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The calculated inhibition rate for Ab-1 was 90.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). In contrast, the Ab-2 serum, which reacts with MSA-2a1 and MSA-2a2, did not significantly inhibit parasite growth. The parasitemia levels were 4.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% for the Ab-2 exposed group and 5.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45% for the pre-immune serum group, resulting in an inhibition rate of 13.1\u0026thinsp;\u0026plusmn;\u0026thinsp;8.4% for Ab-2.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we investigated the MSA-2 proteins of \u003cem\u003eB. bovis\u003c/em\u003e (MSA-2a1, MSA-2a2, and MSA-2b), the GPI-anchored antigens secreted on the merozoite surface (Mosqueda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Rodriguez et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). We generated two epitope-specific antisera (Ab-1 and Ab-2) to analyze their secretion timing and localization of the proteins during the egress to invasion and assessed antibody-mediated growth inhibition. Together, our data indicate that while all three paralogs were apically localized at the timing of egress, MSA-2a1 transiently secreted to the surface at pre-attachment, whereas MSA-2a2 and MSA-2b remained apical organelle. In addition, two antisera had different ability to inhibit parasite growth supporting paralog specific roles during invasion.\u003c/p\u003e \u003cp\u003eBetween the T2Bo and C1 clones (Fig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e), MSA-2a1 was highly conserved, whereas MSA-2a2 and MSA-2b contained multiple substitutions and deletions; notably, MSA-2a2 of C1 clone harbored a contiguous 24 amino acid deletion, representing the greatest divergence. Similar inter-strain differences have been documented across other \u003cem\u003eB. bovis\u003c/em\u003e populations. In a study comparing the Mexico Mo7 and Argentina R1A strains, MSA-2c was the most conserved (88.7% amino-acid identity); among the three paralogs examined in this study, MSA-2a1 showed 83.6% identity (second highest overall), followed by MSA-2b (79.1%) and MSA-2a2 (69.4%) (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In addition, Florin-Christensen and colleagues reported an apparent recombination region between MSA-2a2 and MSA-2b (Florin-Christensen et al., \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). In our study, this region contained the Ab-1-targeted epitope (FLNDNPQRLLADKNGEVT), and antiserum Ab-1 significantly inhibited the growth of live \u003cem\u003eB. bovis\u003c/em\u003e (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e and Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Together, these results may indicate that this region is an antibody-accessible epitope shared by both MSA-2a2 and MSA-2b in live parasites and can be functionally targeted.\u003c/p\u003e \u003cp\u003eLocalization analysis indicated that MSA-2a1, MSA-2a2, and MSA-2b were localized to the apical region of the merozoites, and MSA-2a1 secreted to the parasite surface prior to the attachment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Former studies have reported that each paralogue have expressed on the surface of merozoites by live IFA using the free or purified merozoites (Mosqueda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wilkowsky et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). However, precise localization and secretion timing of MSA-2 paralogues were unclear. Therefore, this study analyzed the merozoite just after the purification and substantial incubation by medium, and IFA images were observed by confocal microscopy. The observed images suggested that MSA-2a1 functions during the initial stage of erythrocyte attachment, whereas MSA-2a2 and MSA-2b remain within the apical organelles and may be released later during the internalization process.\u003c/p\u003e \u003cp\u003eIn the comparative neutralization assay, Ab-1 (MSA-2a2/MSA-2b) strongly inhibited parasite growth by 90.4% at 48 h, whereas Ab-2 (MSA-2a1/MSA-2a2) caused only a 13.1%inhibition. The Ab-2 recognizes both MSA-2a1 and MSA-2a2, and both antisera were raised against overlapping peptide epitopes; therefore, paralog-specific effects may be confounded and epitope placement may influence neutralization efficiency. However, these results may indicate that the contribution of the MSA-2a subfamily, particularly MSA-2a1, to invasion inhibition is limited, while MSA-2b likely plays a pivotal role in the invasion process. This interpretation aligns with previous reports showing that antibodies against MSA-2 proteins significantly inhibit merozoite invasion (Mosqueda et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Wilkowsky et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2003\u003c/span\u003e), although the present findings further differentiate the inhibitory effects among individual MSA-2 paralogs.\u003c/p\u003e \u003cp\u003eTaken together, these data suggest that, although the three molecules belong to the same protein family, they perform distinct roles at different stages of erythrocyte invasion. Further work defining the organelle harboring MSAs, regulatory signals of secretion, and identification of RBC ligands will delineate coordinated invasion mechanism of this parasite.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003eThis work was conducted at the Joint Usage/Research Center for the control of protozoan diseases, National Research Center for Protozoan Diseases,\u0026nbsp;Obihiro University of Agriculture and Veterinary Medicine, and the Joint Usage/Research Center for overcoming zoonotic diseases, International Institute for Zoonosis Control, Hokkaido University.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003eConceptualization: Masahito Asada, Junya Yamagishi, Methodology: Taisei Takahashi, Apinya Arnuphapprasert, Atefeh Fathi, Formal analysis and investigation: Taisei Takahashi, Jae Seung Lee, Apinya Arnuphapprasert, Atefeh Fathi, Kota Komatsu, Miruka Silviane, Morakot Kaewthamasorn, Junya Yamagishi, Masahito Asada, Writing - original draft preparation: Jae Seung Lee, Taisei Takahashi, Writing - review and editing: Masahito Asada, Jae Seung Lee, Taisei Takahashi, Apinya Arnuphapprasert, Atefeh Fathi, Kota Komatsu, Miruka Silviane, Morakot Kaewthamasorn, Junya Yamagishi, Funding acquisition: Junya Yamagishi, Masahito Asada, Resources: Masahito Asada, Junya Yamagishi, Supervision: Masahito Asada.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e This work was supported by JSPS KAKENHI Grant Number 24K02270 to J.Y. and 26K01907 to M.A.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003eThe genome sequence of \u003cem\u003eB. bovis\u003c/em\u003e was obtained from our previous study (Hakimi et al., 2025). The whole genome sequence is available from the corresponding author.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003eAll experiments were carried out according to the approval from biosafety committee of Obihiro University of Agriculture and Veterinary Medicine (No. 202407).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003eThe authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eFlorin-Christensen, M., Suarez, C.E., Hines, S.A., Palmer, G.H., Brown, W.C., McElwain, T.F., 2002. The Babesia bovis merozoite surface antigen 2 locus contains four tandemly arranged and expressed genes encoding immunologically distinct proteins. Infect. Immun. 70, 3566\u0026ndash;3575. https://doi.org/10.1128/IAI.70.7.3566-3575.2002\u003c/li\u003e\n\u003cli\u003eHakimi, H., Yamagishi, J., Sakaguchi, M., Fathi, A., Lee, J.S., Verocai, G.G., Kawazu, S.I., Asada, M., 2025. ves1\u0026alpha; genes expression is the major determinant of Babesia bovis-infected erythrocytes cytoadhesion to endothelial cells. PLoS Pathog. 21, 1\u0026ndash;16. https://doi.org/10.1371/journal.ppat.1012583\u003c/li\u003e\n\u003cli\u003eMosqueda, J., McElwain, T.F., Palmer, G.H., 2002. Babesia bovis Merozoite Surface Antigen 2 Proteins Are Expressed on the Merozoite and Sporozoite Surface, and Specific Antibodies Inhibit Attachment and Invasion of Erythrocytes. Infect. Immun. 70, 6448\u0026ndash;6455. https://doi.org/10.1128/IAI.70.11.6448-6455.2002\u003c/li\u003e\n\u003cli\u003eRodriguez, A.E., Florin-Christensen, M., Flores, D.A., Echaide, I., Suarez, C.E., Schnittger, L., 2014. The glycosylphosphatidylinositol-anchored protein repertoire of Babesia bovis and its significance for erythrocyte invasion. Ticks Tick. Borne. Dis. 5, 343\u0026ndash;348. https://doi.org/10.1016/j.ttbdis.2013.12.011\u003c/li\u003e\n\u003cli\u003eSantos, J.H.M., Siddle, H. V., Raza, A., Stanisic, D.I., Good, M.F., Tabor, A.E., 2023. Exploring the landscape of Babesia bovis vaccines: progress, challenges, and opportunities. Parasites and Vectors 16, 1\u0026ndash;12. https://doi.org/10.1186/s13071-023-05885-z\u003c/li\u003e\n\u003cli\u003eSuarez, C.E., Alzan, H.F., Silva, M.G., Rathinasamy, V., Poole, W.A., Cooke, B.M., 2019. Unravelling the cellular and molecular pathogenesis of bovine babesiosis: is the sky the limit? Int. J. Parasitol. 49, 183\u0026ndash;197. https://doi.org/10.1016/j.ijpara.2018.11.002\u003c/li\u003e\n\u003cli\u003eWilkowsky, S.E., Farber, M., Echaide, I., Torioni De Echaide, S., Zamorano, P.I., Dominguez, M., Suarez, C.E., Florin-Christensen, M., 2003. Babesia bovis merozoite surface protein-2c (MSA-2c) contains highly immunogenic, conserved B-cell epitopes that elicit neutralization-sensitive antibodies in cattle. Mol. Biochem. Parasitol. 127, 133\u0026ndash;141. https://doi.org/10.1016/S0166-6851(02)00329-8\u003c/li\u003e\n\u003cli\u003eYokoyama, N., Okamura, M., Igarashi, I., 2006. Erythrocyte invasion by Babesia parasites: Current advances in the elucidation of the molecular interactions between the protozoan ligands and host receptors in the invasion stage. Vet. Parasitol. 138, 22\u0026ndash;32. https://doi.org/10.1016/j.vetpar.2006.01.037\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Babesia bovis, MSA-2 family, erythrocyte invasion","lastPublishedDoi":"10.21203/rs.3.rs-9469245/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9469245/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e \u003cem\u003eBabesia bovis\u003c/em\u003e merozoite surface antigen 2 (MSA-2) are proteins encoded by \u003cem\u003emsa\u003c/em\u003e multigene family and implicated in erythrocyte recognition and invasion, yet their roles remain only partially understood. We investigated spatiotemporal differences of three MSA-2 paralogs (MSA-2a1, MSA-2a2, and MSA-2b) by using epitope-specific rabbit antisera. Alignment of amino acid sequences of \u003cem\u003eB. bovis\u003c/em\u003e T2Bo strain and Texas strain C1 clone showed that MSA-2a1 is highly conserved (99.6% identity) among the parasites, whereas MSA-2a2 (84%) and MSA-2b (94%) are more variable; notably, the least conserved paralog, MSA-2a2, harbored a contiguous 24 amino acid deletion. Indirect immunofluorescence assays using the antisera revealed apical localization of all three proteins in intraerythrocytic parasite. Following merozoite purification and brief incubation, localization of MSA-2a1 was observed on the merozoite surface before the attachment, whereas MSA-2a2 and MSA-2b remained at the apical end. Moreover, growth-inhibition assays, antiserum Ab-1 recognizing MSA-2a2 and MSA-2b inhibited 90.4% at 48 h incubation, whereas Ab-2 recognizing MSA-2a1 and MSA-2a2 inhibited only 13.1%. These results suggest functional difference among MSA-2 paralogs: MSA-2a1 was secreted to the surface before attachment, while MSA-2a2 and MSA-2b remain inside the merozoite at the onset of attachment. Taken together, MSA-2a1, MSA-2a2, and MSA-2b appear structurally similar, yet functionally distinct proteins that may contribute to the coordinated control of erythrocyte invasion.\u003c/p\u003e","manuscriptTitle":"Localization and functional difference of MSA-2 paralogs in Babesia bovis merozoites","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-15 13:32:29","doi":"10.21203/rs.3.rs-9469245/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-05-06T13:55:09+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T10:31:58+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T01:47:06+00:00","index":"","fulltext":""},{"type":"submitted","content":"Parasitology Research","date":"2026-04-20T08:35:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"parasitology-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pare","sideBox":"Learn more about [Parasitology Research](http://link.springer.com/journal/436)","snPcode":"436","submissionUrl":"https://submission.nature.com/new-submission/436/3","title":"Parasitology Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"12e69b66-a3ef-453b-ac6a-51a7db3818c4","owner":[],"postedDate":"May 15th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"4","date":"2026-05-06T13:55:09+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-15T13:32:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-15 13:32:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9469245","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9469245","identity":"rs-9469245","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}