Experimental Infection of Calves with Transfected Attenuated Babesia bovis Expressing the Rhipicephalus microplus Bm86 Antigen and eGFP Marker: Preliminary Studies towards a Dual Anti-Tick/Babesia Vaccine

Abstract

Bovine babesiosis, caused by Babesia bovis and B. bigemina, is a major tick-borne disease of cattle with global economic impact. The disease can be prevented using integrated control measures including attenuated Babesia vaccines, babesicidal drugs, and tick control approaches. Vaccination of cattle with the Rhipicephalus microplus Bm86-based recombinant vaccine reduces the fitness of R. microplus and R. annulatus, but several booster inoculations are required to maintain protection. Herein, we generated a stable transfected strain of B. bovis expressing an enhanced GFP (eGFP) and a chimeric version of Bm86 (B. bovis/Bm86/eGFP). The eGFP was expressed in the parasite cytoplasm, whereas Bm86 was displayed on the surface of merozoites. Three splenectomized calves experimentally infected with B. bovis/Bm86/eGFP showed mild signs of acute disease and developed long-lasting antibody responses to B. bovis and native Bm86. No evidence of sequestration of parasites in the cerebral capillaries was found upon postmortem analysis, confirming attenuation of the strain. This is the first report of transfected B. bovis expressing the tick antigen Bm86 on the merozoite surface that elicits an antibody response to native Bm86. These results represent a proof of concept for a novel live, attenuated, tagged dual-vaccine approach to attempt simultaneous control of babesiosis and tick infestation.

Keywords: Babesia bovis; anti-Babesia vaccine; anti-tick vaccine; transfected B. bovis.

Figure 1

Schematic representation of the R. microplus Bm86 and plasmid pEf/Bm86/eGFP: (a) repreScheme 86. (orange box) with the regions encoding for the native N-terminal and C-terminal hydrophobic regions in Bm86. These two regions were removed (dotted lines) to produce the chimeric version of the tick gene (Bm86Ch) encoding for the SigPep of the B. bovis MSA-1 (dark box) and the 6 histidine tag (6xHis) (blue box) minus 100 hydrophobic amino acids at the C terminal end. (b) Representation of pEf/Bm86/eGFP plasmid containing Bm86Ch downstream of the elongation factor 1 (ef-1α) intergenic region (IG) and upstream of the B. bovis RAP-1 3′ regulatory sequence: this plasmid also contains the eGFP-BSD ORF cloned downstream of the B. bovis actin IG and upstream of the B. bovis MSA-1 3′. Additionally, pEf/Bm86/eGFP has 680 nucleotides of the ef-1α ORF B. Also shown is a representation of the B. bovis ef-1α locus where stable integration of pEf/Bm86/eGFP is targeted. The locations of the following primers are also shown in the figure: MSA-1sig F, Bm86 R, eGFP F, BSD R, Ef-A-probe F, and USP-Ef-probe R.

Figure 2

Microscopy analysis of B. bovis/Bm86/eGFP: panels show bright field, 4′,6-diamidino-2-phenylindole (DAPI) staining, eGFP fluorescence, and a merged image. The images were taken 14 days after transfection of the parasites growing in blasticidin selection medium. Bar indicates 5 μM.

Figure 3

PCR demonstrating the integration of Bm86Ch and eGFP-BSD into the B. bovis genome at the expected the ef-1α locus in the parasite line B. bovis/Bm86/eGFP: the PCRs were performed using 1) B. bovis S74-T3Bo gDNA (wild type strain), 2) B. bovis/Bm86/eGFP gDNA, 3) pBluescript plasmid DNA, and 4) pEf/Bm86/eGFP plasmid DNA. The individual panels show the PCR amplicons obtained with primers MSA-1 F/R (a), eGFP F/BSD R (b), MSA-1sig F/Bm86 R (c), eGFP F/UPS-Ef-probe R (d), and Ef-A-probe F/Bm86 (e).

Figure 4

Expression of Bm86Ch and eGFP by B. bovis/Bm86/eGFP demonstrated by Western blot analysis: numbers 1 to 5 indicate the lysate of B. bovis S74-T3Bo, the lysate of transfected B. bovis/Bm86/eGFP, the recombinant eGFP-BSD, the recombinant Bm86, and the R. microplus midgut, respectively. (A) Monoclonal antibody (Ab) anti-MSA-1 Babb35. (B) Rabbit polyclonal Ab anti-GFP. (C) Rabbit polyclonal Ab anti-Bm86. (D) Pre-immune rabbit serum. The protein molecular weight marker (M) is shown on the right side of each immunoblot.

Figure 5

(A) Immunofluorescence assay in permeabilized (upper panels) and non-permeabilized (lower panels) B. bovis/Bm86/eGFP-transfected parasite merozoites: the panels show bright field and staining with DAPI, anti-eGFP antibodies, and anti-Bm86 antibodies. The scale bar is shown in the bright field panels. (B) Immunofluorescence assay in intact, non-permeablilized, extracellular merozoites of the parental S74-T3Bo B. bovis (upper panels) and transfected B. bovis/Bm86/eGFP parasite line (lower panels): the panels show bright field and staining with DAPI, anti-MSA-1 antibodies, and anti-Bm86 antibodies. The scale bar is shown in the bright field panels. Bar indicates 5 μM.

Figure 6

Levels of anti-B. bovis (a) and anti-Bm86 (b) antibodies in B. bovis/Bm86/eGFP-infected calves detected by immunofluorescence assay (IFA) and ELISA, respectively: calves #96 and #89 received one intravenous inoculation with 2 × 10⁸ B. bovis/Bm86/eGFP-infected red blood cells. Calf #97 was inoculated with 50 mL of blood from calf #96 14 days after prime infection. The results of anti-B. bovis serology are presented as a titer where the titration endpoint is reported as the antilog of the serum dilution. The results of anti-Bm86 serology are presented as the optical density (OD) index calculated by the average of the OD of a specific animal sample minus a blank OD divided by the OD of the same animal at time zero minus a blank OD.