Introduction

Bovine babesiosis caused by Babesia bovis and B. bigemina is an acute and persistent tick-borne disease with a high negative economic impact worldwide. The disease is characterized by high mortality and morbidity in susceptible animals that develop fever, anemia, jaundice, weight lost, reduction in milk production and, in severe cases, death. Animals that survive an acute infection become long-term asymptomatic carriers of the parasites. Persistently infected animals are therefore a continuous source for transmission by ticks and the maintenance of herd immunity1.

B. bigemina is usually regarded as having relatively reduced virulence compared to B. bovis however, B. bigemina is also responsible for important economic losses worldwide and improved methods of control are urgently needed. Currently control of babesiosis is achieved by acaricides and live-attenuated vaccines, but these approaches have serious limitations, including the development of acaricide resistance by ticks, and current efforts are focused on the development of novel and more effective recombinant protein sub-unit vaccines or genetically-attenuated parasites. The development of stable transfection systems for B. bovis2,3, have already facilitated several new avenues of research, including functional gene characterization4 and novel vaccine development5,6, but such methods are not available for B. bigemina. The development of genetic manipulation tools for B. bigemina will advance our understanding of parasite biology, gene function and improved control of bovine babesiosis6,7,8,9. A recent study described an effective B. bigemina promoter, interspecies (B. bovis and B. bigemina) activity for the elongation factor (ef)−1α promoters, and a method for incorporating exogenous DNA into B. bigemina, but appropriate selectable markers and a permissible site for stable integration of transfected genes remained undefined. However, initial B. bovis transfection systems were based on the use of the blasticidin and blasticidin deaminase (BSD) as a selectable marker, and the ef-1α locus as a permissible site for targeting exogenous gene integration2. Importantly, the availability of a partial B. bigemina genome (http://www.sanger.ac.uk/resources/downloads/protozoa/babesia-bigemina.html) and the previous characterization of the B. bigemina ef-1α locus10, indicate that the structure of this locus is essentially identical in both B. bigemina and B. bovis. Together, these observations suggests that it would be feasible to use a common strategy for gene integration in both B. bovis and B. bigemina, based on targeting one of the two identical ef-1α open reading frames (orfs) present in the ef-1α locus.

Here, we describe for the first time a stable transfection system for B. bigemina based on integration of the egfp-bsd gene under the control of the ef-1α promoter, into the ef-1α locus of B. bigemina, after drug selection with blasticidin. In addition, the same plasmid used for transfection of B. bigemina was able to insert and express foreign sequences in the ef-1α locus of B. bovis, thus expanding the options available for the genetic manipulation of Babesia sp. parasites more generally.

Materials and Methods

Ethics statement

Animals (Bos taurus Holstein steers, 12–20 months old) were used as blood donors for the maintenance of in vitro cultures of B. bigemina and B. bovis and were approved by the Institutional Animal Care and Use Committee (protocol 2013–66). All experiments performed in this study were conducted in accordance with the Protocol of Animal Usage Number 2013–66 approved by the University of Idaho IACUC Committee.

In vitro parasite culture

B. bigemina (Puerto Rico strain)11 and B. bovis (T3Bo strain)12 were propagated in continuous microaerophilic stationary-phase culture as previously described. Briefly, B. bigemina and B. bovis cultures were grown in 96-well plates in bovine red blood cells (RBC) at 5% or 10% hematocrit, respectively, using HL-1 culture media at pH 7.2, and were incubated at 37 °C in an atmosphere of 5% CO2 and 90% N210.

Evaluation of sensitivity of B. bigemina and B. bovis to blasticidin

B. bigemina and B. bovis parasites were cultured in 180 µl of culture medium containing 5% and 10% bovine RBC, respectively, in a 96-well plate with different concentrations of blasticidin: 1, 1.2, 1.5, 1.8, 2, 4, 6, 8, 10, 12 and 14 μg/μl. Media without blasticidin was used as a control. The initial parasitemia was 0.2%. One hundred and fifty µl of culture media was daily replaced with corresponding amount of blasticidin. Percentage of parasitized erythrocytes (PPE) was monitored daily by Diff-Quik stained smears every 24 hr over a period of 72 hr by light microscopy (1000 × magnification). This experiment was carried out in triplicate.

Parasite DNA extraction

Genomic DNA (gDNA) and plasmid DNA (pDNA) was extracted from cultured B. bigemina and B. bovis using the Qiagen Blood core kit according to the manufacturer’s instructions.

Plasmid constructs

The 3′ and 5′ insertion target regions for stable transfection in the ef-1-alpha-B orf were generated by PCR with B. bigemina gDNA isolated from tissue culture cells utilizing the following sets of primers: Bbig-EF-orf-B-3′-BamHI-F and Bbig-EF-orf-B-3′-BamHI-R primers for the 3′ region of B. bigemina ef (expected size of 678 base pair (bp) amplicon]; Bbig-EF-orf-B-5′-Xho-F and Bbig-EF-orf-B-5′-Xho-R for the 5′ region of B. bigemina ef (expected size of 674 bp amplicon). These PCR products were cloned into the TOPO-TA 2.1 (Life technologies) cloning vector for sequence confirmation. The 3′ and 5′ insertion regions were then digested from the cloning vectors respectively with BamHI and XhoI restriction enzyme and the amplicons were isolated and recovered from a 1% agarose gel. The plasmid containing the ef-1α-A promoter and luciferase gene along with the 3′ rap-1a stop region used in the B. bigemina transient transfection10 was digested with EcoRI restriction enzyme to remove the luciferase gene and the resulting linearized vector now containing just the B. bigemina ef-1a-A promoter and B. bigemina 3′ rap-1a stop region was re-circularized by ligation and transformed into TOP-10 E. coli competent cells (Life Technologies). The purified vector was then digested with BamHI restriction enzyme and the 674 bp amplicon corresponding to 3′ ef-1α-B orf insertion region was ligated into this linear vector, transformed into TOP-10 cells and sequenced to confirm the correction orientation of the insertion. The resulting vector was digested with XhoI restriction enzyme and a similar procedure was used to ligate the 5′ ef-1α-B orf insertion target region into the vector. After confirming that both the 3′ and 5′ insertion sites were present in the correct orientation by sequencing, the vector was prepared for further ligation by digestion with EcoRI restriction enzyme. A synthetic egfp-bsd fusion gene from the p6-Cys-EKO plasmid (GenBank Accession number KX247384)13 containing EcoRI restriction sites was digested with EcoRI to remove the egfp-bsd fragment. This egfp-bsd fragment was isolated on a gel and the recovered fragment ligated into the vector containing the 3′ and 5′ B. bigemina insertion regions and the rap-1a 3′ stop region to generate the final stable transfection vector designated pbig-ef-egfp-bsd. The plasmid pbig-ef-egfp-bsd was used to transform TOP-10 cells, and plasmid was purified with the Qiagen Endotoxin free Plasmid Maxi Kit following the manufacturer’s instructions prior to transfection. The pBluescript (pBS) plasmid was used as a negative control in the transfection experiments.

Transfection of B. bigemina

The transfection of B. bigemina-infected RBC was performed as described by Suarez et al.2. Briefly, B. bigemina iRBC with PPE ~20% was centrifuged at 600 xg for 5 min, and the cells washed once with 1 ml of Cytomix buffer (120 mM KCl, 0.15 mM CaCl2, 10 mM K2HPO4/KH2PO4 pH 7.6). Twenty μg of pbig-ef-egfp-bsd plasmid in 55 μl of cytomix was then gently mixed with 40 μl of washed B. bigemina iRBC then transferred to 0.2 cm electroporation cuvette and transfected by electroporation using a BioRad Gene Pulser II system at 1.2 kV + 25 µF + 200 Ω. After transfection, cells were immediately transferred to a 24-well plate containing 1.2 ml of HL-1 media with 5% of bovine RBC and 8 μg/μl of bsd to select for egfp-bsd expressing transgenic parasites.

Transfection of B. bovis

The process for transfection of B. bovis parasites using plasmid pbig-ef-egfp-bsd was performed as described above for B. bigemina. After transfection, cells were immediately transferred to a 24-well plate containing 1.2 ml of HL-1 media with 10% of bovine RBC and 3 μg/μl of bsd to select for egfp-bsd expressing transgenic parasites.

Confirmation of stable integration

Fluorescence microscopy

Two µl of either B. bigemina- (bigemina-big-ef-egfp-bsd) or B. bovis- (bovis-big-ef-egfp-bsd) transfected or wild-type cultures (non-transfected parasites) were observed by fluorescence microscopy (630 × magnification).

Quantifying in vitro growth of B. bigemina and B. bovis

B. bigemina (bigemina-big-ef-egfp-bsd) and B. bigemina wild-type parasites were cultured in 180 µl of culture medium containing 5% bovine RBC, in the absence of blasticidin and in the presence of 8 μg/μl of blasticidin. B. bovis (bovis-big-ef-egfp-bsd) and B. bovis wild-type parasites were cultured in 180 µl of culture medium containing 10% bovine RBC in the presence of 3 μg/μl of blasticidin or in the absence of blasticidin (positive control). The initial parasitemia was 0.2% and parasites were cultured in triplicate in 96-well plates. Medium (150 µl) was replaced daily. PPE was monitored every 24 hr up to 72 hr by Diff-Quik-stained RBC smears by optical microscope under 1000 × amplification. This experiment was carried out in triplicate.

Southern blot analysis

Genomic DNA was extracted from wild-type B. bigemina and B. bovis parasites, bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd with linearized pBS plasmid. One and half µg of gDNA were digested overnight with 10 units/µl of BgIII restriction enzyme and electrophoresis was carried out on a 1% agarose gel containing SYBR green dye. Three DIG-labeled were used: B. bigemina rap-1 gene, a B. bovis msa-1 gene, an egfp-bsd containing complete orf of egfp gene, a B. bigemina ef-1α gene (fwd 5′- CGCCTTTAGGGGCTTTACAACTCTGC-3′, rev 5′-CATTCCTGCTTAACGATACAGGC-3′) and a B. bovis ef-1α gene (fwd 5′-TGACATCTTTGAGAAATCTTAATGC-3′, rev 5′-ATCAGACAGATGTCTACTGAACTCGA GC-3′). Probes were DIG-labeled using a PCR DIG Probe Synthesis Kit (Roche) and hybridized with Dig Easy Hyb solution (Roche). Hybridizations of the DIG-labeled probes where detected using anti-digoxygenin Fab alkaline phosphatase conjugated (Roche) diluted 1/10.000 in blocking buffer, as described previously2. Roche Dig labeled DNA marker II was used as standard molecular marker, pBS control plasmid without Babesia promoter (pBS promoterless control plasmid) was used as plasmid control (Pr-) and big-ef-egfp-bsd plasmid control was used as a negative control.

PCR amplification and sequencing

Sets of primers were designed to confirm the integration of plasmid bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd by specific amplification of a DNA fragments surrounding the 5′ recombination site, the 3′ recombination site and the locus (Table 1). Amplicons were cloned into pCR™2.1-TOPO vector (Life Technologies) according to the manufacturer’s instructions and nucleotide sequences were confirmed by Sanger sequencing (ABI 3730).

Table 1 List of primers.
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Immunoblot analysis

Proteins were extracted from cultured wild-type B. bigemina and B. bovis, bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd and were used for immunoblot analysis as previously described2. Briefly, the immunoblots were incubated with mouse monoclonal antibody (MAb) against B. bigemina rap-1 protein (64/4.10.3)14 diluted to 2 µg/ml; B. bovis and bovis -big-ef-egfp-bsd were incubated with mouse MAb against B. bovis rap-1 protein (23/53.156.77) diluted to 2 µg/ml; wild-type B. bigemina and B. bovis, bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd were incubated with rabbit anti-GFP diluted to 1:5,000. Membranes were subsequently incubated with HRP-conjugated goat anti-mouse IgG diluted 1:10,000 (on 64/4.10.3 and 23/53.156.77 antibody) or with HRP-conjugated goat anti-rabbit IgG diluted 1:5,000 (on anti-GFP antibody) for 45 minutes at RT. Chemiluminescent detection employed ECL™ western blotting substrate followed by exposure to X-film. Pre-immune mouse serum or non-infected bovine RBC were used as controls.

Statistical analysis

Statistical significance was determined using ANOVA (GraphPad Prism 7 software). P < 0.05 were considered statistically significant.

Results

Growth inhibitory concentrations of blasticidin in B. bigemina and B. bovis

First we compared the inhibitory concentrations of blasticidin on in vitro cultured B. bigemina and B. bovis. Parasites were cultured in the presence of different concentrations of blasticidin ranging from 1 µg/µl to14 µg/µl, and the PPE was calculated daily up to 72 hr (Suppl. Figure 1a and b). The results show that increasing blasticidin concentrations above 4 μg/μl correlated with decreasing PPE for B. bigemina. The calculated IC50 found for B. bigemina and B. bovis was 3 µg/µl and 0.8 µg/µl, respectively. However 8 µg/µl and 3 µg/µl of blasticidin completely inhibited the growth of B. bigemina and B. bovis respectively (Suppl. Figure 1a and b), demonstrating that blasticidin as an appropriate selective inhibitory drug for developing a stable transfection system for B. bigemina.

Generation of B. bigemina and B. bovis transfected lines stably expressing egfp-bsd under the control of a B. bigemina ef-1α promoter

B. bigemina and B. bovis parasites were electroporated with plasmid pbig-ef-egfp-bsd (Fig. 1a–c) followed by culturing in the presence of inhibitory doses of blasticidin. The transfection plasmid pbig-ef-egfp-bsd was designed for targeting the integration of the egfp-bsd selectable marker gene into the ef-1α locus of B. bigemina (Fig. 1b). Blasticidin resistant and green fluorescent B. bigemina and B. bovis parasites emerged 15 and 18 days after electroporation respectively, but no parasites were detectable in culture wells containing either control mock transfected or non-transfected or wild-type B. bovis and B. bigemina parasites growing in the presence of inhibitory doses of blasticidin (data not shown). The blasticidin-resistant parasites were maintained on blasticidin containing cultures for 2 months before analyzed for phenotypic and genotypic characterization. Analysis by fluorescence microscopy revealed intracellular expression of eGFP protein in the blasticidin-resistant B. bigemina and B. bovis transfected parasites, which were termed bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd, respectively (Fig. 2a). As expected, no eGFP fluorescence was observed in B. bigemina and B. bovis wild-type control parasites (Fig. 2b).

Figure 1
figure 1

Experimental strategy used for the stable transfection of B. bigemina and B. bovis cell lines. (a) Schematic representation of the target ef-1α locus of B. bigemina in B. bigemina parasites (bigemina-big-ef-egfp-bsd). (b) Schematic representation of the stable transfection plasmid pbig-ef-egfp-bsd used for electroporation. (c) Schematic representation of the target ef-1α locus of B. bigemina in B. bovis parasites (bovis-big-ef-egfp-bsd). BgIII: restriction enzyme.

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Figure 2
figure 2

Analysis of B. bigemina and B. bovis parasites by fluorescence. (a) Bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd transfected parasites observed under bright field, green laser (eGFP), blue laser (DAPI) and merge images. (b) B. bigemina and B. bovis wild-type parasites observed under bright field, green laser (eGFP), blue laser (DAPI) and merge images. Amplification 630x.

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Phenotypic comparison between non-transfected and transfected Babesia parasites

The in vitro growth rate of bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd parasite lines, and wild-type parasites were compared. Notably, bigemina-big-ef-egfp-bsd parasites grew three times faster than wild-type parasites (P < 0.05) while bigemina-big-ef-egfp-bsd parasites grew at similar rate regardless of the presence or absence of blasticidin (Fig. 3a). However, as expected, B. bigemina wild-type parasites did not grow in the presence of blasticidin (Fig. 3a).

Figure 3
figure 3

In vitro growth curve of B. bigemina and B. bovis parasites up to 72 hr. (a) B. bigemina growth curve. (b) B. bovis growth curve. Tbg + bsd: bigemina-big-ef-egfp-bsd parasites in the presence of blasticidin; Tbg-bsd: bigemina-big-ef-egfp-bsd parasites in the absence of blasticidin; wt Bbg + bsd: B. bigemina wild-type parasites in the presence of blasticidin; wt Bbg-bsd: B. bigemina wild-type parasites in the absence of blasticidin; Tbo + bsd: bovis-big-ef-egfp-bsd parasites in the presence of blasticidin; Tbo-bsd: bovis-big-ef-egfp-bsd parasites in the absence of blasticidin; wt Bbo + bsd: B. bovis wild-type parasites in the presence of blasticidin; wt Bbo-bsd: B. bovis wild-type parasites in the absence of blasticidin. Normalized PPE values (Y axis) obtained from Babesia spp. in the presence or absence of blasticidin (X axis). Media was used as negative controls for no inhibition. Error bars indicate standard deviations for each sample tested from triplicate culture. Data were compared using ANOVA analysis. *Represents P < 0.05 indicating a statistically significant difference between groups.

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The bovis-big-ef-egfp-bsd parasites showed a higher rate of growth (P < 0.05) in blasticidin-free culture media (Fig. 3b) when compared to growth of bovis-big-ef-egfp-bsd parasites in the presence of blasticidin or the growth of wild-type parasites. Growth rates for big-ef-egfp-bsd and wild-type parasites in the presence or absence of blasticidin were indistinguishable (Fig. 3b). B. bovis wild-type parasites did not grow in the presence of blasticidin (Fig. 3b).

Genotypic and proteomic characterization of stable B. bigemina and B. bovis transfected parasites

The genotypic characterization of transfected parasites was performed by Southern blot analysis, PCR and sequencing of PCR products. Genomic DNA from transfected bigemina-big-gfp-bsd and bovis-big-gfp-bsd cell lines, non-transfected B. bovis and B. bigemina parental strains, and plasmid pbig-ef-egfp-bsd, were analyzed in Southern blots using B. bovis msa-1 (Fig. 4a), B. bigemina rap-1 (Fig. 4b), egfp (Fig. 4c) and ef-1α (Fig. 4d) specific dig-labeled probes. The presence of a single band hybridizing with egfp-bsd probe, only in the transfected parasites is consistent with a single site integration of the exogenous transfected egfp-bsd gene in both bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd cell lines (green boxes, Fig. 4c). Probing the blots with an ef-1α specific probe that hybridizes with sequences that are not included in the transfection constructs reveal an increase in the size of the ef-1α locus in the transfected parasites as a result of the insertion of the transfected genes. The calculated size of the BgIII restriction size containing the ef-1α locus of wild type B. bigemina and B. bovis are 16.4 and 18.8 Kb, respectively. However, the size of the locus, as detected by the specific labeled probe, was increased to 18.6 and 21 Kb, respectively, in both transfected parasite lines which matches with the predicted size of the stably inserted DNA (2.2 kb). As expected, the rap-1 and msa-1 probes react with identical patterns in transfected and non-transfected gDNA. Taken together, these results are consistent with stable integration of a single egfp-bsd gene copy in both parasite species.

Figure 4
figure 4

Southern blot analysis using dig-labeled probes against: (a) B. bigemina rap-1a; (b) B. bovis msa-1; (c) egfp and (d) ef-1α. 1: wild-type B. bigemina gDNA digested with BgIII; 2: wild-type B. bigemina gDNA undigested; 3: bigemina-big-ef-egfp-bsd digested with BgIII; 4: bigemina-big-ef-egfp-bsd undigested; DM: Dig labeled DNA marker II; Pr-: pBS promoterless control plasmid; C-: pbig-ef-egfp-bsd plasmid control; 5: wild-type B. bovis gDNA digested with BgIII; 6: wild-type B. bovis gDNA undigested; 7: bovis-big-ef-egfp-bsd digested with BgIII; 8: bovis-big-ef-egfp-bsd undigested. Vertical black stripes on the blot indicate cropping of the blot. A full image of the original blot can be seen in Supplementary Info section.

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We then designed a PCR aimed at demonstrating correct integration of the exogenous gene into the B. bigemina ef-1α locus using primers based on the transfected egfp gene and sequences adjacent to the ef-1α locus of B. bigemina and B. bovis that are not present in the transfection plasmids (Figs 5 and 6, respectively). Genomic DNA derived from stably transfected and non-transfected (wild-type) control B. bigemina parasites were amplified by PCR using the set primers: A) egfp-Fwd and ef1α-Rev; and B) egfp-Fwd and egfp-Rev (Fig. 5 and Table 1). The expected band of 2.2 kb was observed only upon amplification of the transfected parasite line bigemina-big-ef-egfp-bsd but not on the gDNA derived from the wild-type B. bigemina parasites (Fig. 5a). The sequence of the 2.2 kb PCR amplicon was consistent with integration of the transfected egfp-bsd gene and its flanking regions into the B ef-1α gene of B. bigemina by homologous recombination (GenBank accession nr: MG234552). In addition, control PCR reactions using primers representing sequences present only in the transfection plasmid (Fig. 5b) (egfp-Fwd and egfp-Rev) only amplify a similar fragment in the transfected line bigemina-big-ef-egfp-bsd and transfection plasmid pbig-ef-egfp-bsd, but not on the gDNA derived from the wild-type B. bigemina parasites.

Figure 5
figure 5

PCR integration analysis in B. bigemina using two different set of primers. (a) egfp-Fwd and ef1α-Rev. (b) egfp-Fwd and egfp-Rev. Line 1: wild-type B. bigemina gDNA; Line 2: bigemina-big-ef-egfp-bsd; C-: pbig-ef-egfp-bsd plasmid control; MW: molecular size ladder in bp, 1 Kb Plus DNA ladder.

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Figure 6
figure 6

PCR integration analysis in B. bovis culture using two different sets of primers. (a) egfp-Fwd and Bbov-UpS-efB-Rev. (b) rap-1a-Fwd and rap-1a-Rev. Line 1: bovis-big-ef-egfp-bsd gDNA; line 2: wild-type gDNA B. bovis; MW: molecular size ladder in bp, 1 Kb Plus DNA ladder. (c) Alignments among the regions of insertion of the transfected genes into bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd parasites. Vertical black stripes on the blot indicate where the image was cropped. Full image of the original agar electrophoresis gel can be seen in Supplementary Info section.

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PCR amplifications performed using similar sets of primers on transfected bovis-big-ef-egfp-bsd and wild-type B. bovis parasites are shown in Fig. 6. Integration PCR using the set of primers egfp-Fwd and Bbov-UpS-efB-Rev yielded a ~2.2 kb band (Fig. 6a and Table 1) in gDNA of bovis-big-ef-egfp-bsd but not in gDNA from wild-type B. bovis parasites. Sequencing of the 2.2 kb amplicon is consistent with integration of the transfected egfp-bsd gene and its flanking regions into the B ef-1α gene of B. bovis by homologous recombination (GenBank accession nr: MG234553). Interestingly, sequencing of the PCR amplicon demonstrates the generation of a theoretically predictable hybrid partial B. bigemina-B. bovis ef-1α molecule in the transfected parasite. A comparison among the sequences of the hybrid portion of the molecule and the ef-1α DNA sequence of B. bovis and B. bigemina for the region involved in the insertion of the transfected genes into the genomes of the parasites is shown in Fig. 6b. Figure 6c also show alignments among the regions of insertion of the transfected genes into B. bovis and B. bigemina transfected parasites. PCR amplifications using primers rap-1a-Fwd and rap-1a-Rev confirmed the presence of the rap-1a gene in gDNA from transfected and wild-type parasites (Fig. 6b). An amplicon of ~320 bp was obtained (Fig. 6b).

We also performed immunoblotting to confirm expression of eGFP-BSD by the transfected B. bigemina and B. bovis parasites. As can be seen in the immunoblot, no reactivity was observed for any of the antibodies used and uninfected bovine RBC (Fig. 7a and lanes 5). The wild-type B. bigemina and bigemina-big-ef-egfp-bsd reacted against B. bigemina rap-1 protein (~50 kDa) and the B. bovis and bovis-big-ef-egfp-bsd reacted against B. bovis rap-1 protein (~50 kDa) (Fig. 7b,c) using anti-RAP-1 MAbs as positive controls. Also, anti-GFP antibodies reacted with proteins present in both bigemina-big-ef-egfp-bsd and bovis-big-ef-egfp-bsd parasite lines with the expected molecular weight of the GFP-BSD fusion protein (~40 kDa), but not in wild-type B. bigemina and B. bovis (Fig. 7d).

Figure 7
figure 7

Immunoblot analysis. (1) wild-type B. bigemina. (2) bigemina-big-ef-egfp-bsd. (3) wild-type B. bovis. (4) bovis-big-ef-egfp-bsd. (5) Uninfected bovine RBC. Samples were incubated with antibodies: (a) pre-immune mouse serum; (b) anti-B. bigemina rap-1 MAb. (c) anti-B. bovis rap-1 MAb. (d) anti-GFP MAb. (M) molecular size ladder in kDa indicated by arrows. Vertical black stripes on the blot indicate cropping of the blot. A full image of the original blot can be seen in Supplementary Info section.

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Taken together, Southern blot, PCR and immunoblot data confirmed stable integration of the egfp-bsd gene into the genomes of both B. bovis and B. bigemina with demonstrated expression of the transfected exogenous genes.

Discussion

Here, a stable transfection of B. bigemina and B. bovis using identical B. bigemina insertion and gene regulatory sequences that can be used for functional gene characterization or for the delivery of exogenous antigens by Babesia spp. parasites is described.

Importantly, the transfected egfp-bsd gene integrated as a single copy in the expected ef-1α locus in transfected B. bigemina parasites and no episomal forms of exogenous DNA bigemina-big-ef-gfp-bsd were detectable in parasites that were selected with blasticidin for at least two months.

A similar pattern of specific integration by means of homologous recombination for the exogenous transfected egfp-bsd gene into the expected ef-1α locus was also found to occur in B. bovis parasites using a plasmid transfection vector designed for integration into the B. bigemina ef-1a locus, despite the occurrence of sequence divergence in the ef-1α gene among the two species. Sequence comparisons among the ef-1α orf of B. bovis and B. bigemina show a level of identity of 87.45%. (Suppl. Figure 2 and Suppl. Table 1). Remarkably, that level of identity was sufficient to allow specific integration of the pbig-ef-egfp-bsd gene into the ef-1α locus of B. bovis genome, generating a hybrid ef-1α molecule in transfected B. bovis. The DNA sequence comparisons between ef-1α locus of B. bigemina and B. bovis (Suppl. Figure 2) provide hints on the mechanisms of homologous recombination operating in the parasite. Additionally, and consistent with previous findings10, the ef-1α promoter of B. bigemina was able to generate expression levels of the egfp-bsd gene that are sufficient to sustain growth of transfected parasites at high levels of blasticidin. The strategy for exogenous gene insertion used in this study makes expression of the transfected egfp-bsd gene by the “native” B. bigemina promoter theoretically possible, but this possibility is highly unlikely since the homologous or heterologous ef-1α promoter region (~700 bp), located immediately downstream the truncated ef-1α orf contain numerous stop codons. In addition, we previously demonstrated heterologous promoter function using transient transfection, where the transfected gene is expressed under the control of the sequences present in the transfection plasmid, and without the intervention of the original promoters, supporting the contention that the exogenous integrated promoter is indeed responsible for the expression of the egfp-bsd gene in the stably transfected parasites. Transfected B. bigemina parasites grew three times faster than non-transfected (wild-type) parasites. This might be due to changes in the regulation of the expression of ef-1α locus in the transfected parasites. It will be interesting to determine whether these changes affect the ability and efficiency of the parasite to infect bovine and tick hosts, and whether they are associated with parasite virulence.

The ability to transfect B. bigemina and B. bovis using B. bigemina insertion and promoter sequences also has important implications for improving the design of Babesia sp.-based vectored vaccines6. On one hand, a vaccine delivery platform based on B. bigemina transfected parasites might be more advantageous compared with B. bovis since the former parasite is known to cause less severe clinical disease in cattle, and, in contrast to B. bovis, does not result in microvascular sequestration of iRBC in the host. In contrast to B. bovis, B. bigemina parasites can be cleared from persistently infected animals, and dual B. bovis-B. bigemina infections are frequent in cattle in endemic areas15,16,17.

Possible future applications of this transfection platform include the use of attenuated B. bigemina-transfected parasites that express B. bovis and/or vector tick antigens that induce parasite and/or vector controlling immunity during subclinical persistent infection. These vectored vaccines might become ideal to generate protective immunity effective against both bovine parasites and their vector.

It will also be necessary to test the functionality of the B. bigemina ef-1α promoter in distinct life stages using transfected B. bigemina and B. bovis and parasites. These observations could have practical consequences for developing improved methods for the study and control of these parasites at different life-cycle stages.

In summary, we describe an efficient method for the stable transfection of B. bigemina that can be used for the future development of novel vaccines. These will require assuring that the transfected parasites are safe to deploy and that the genetic modifications do not result in undesirable phenotypic characteristics in potential vaccine candidate strains. The observation that the B. bigemina-specific construct can also be effectively and specifically used to transfect B. bovis parasites expands the range of possibilities toward the development of novel vaccines. Future work is aimed at determining whether transfected B. bigemina parasites are able to express exogenous or homologous antigens as vaccine platforms, and work as an effective method for functional genetic analysis in B. bigemina. The use of B. bigemina promoters in stably transfected B. bovis parasites also expands the toolbox available for the genetic manipulation of this parasite towards improved gene function characterization and vaccine development.