In vitro cultivation of B. bovis
The Texas (T2Bo) strain of B. bovis was maintained with purified bovine RBCs in serum-free GIT culture medium (WAKO Pure Chemical Industrial, Ltd., Osaka, Japan) supplemented with antibiotic-antimycotic solution (Sigma-Aldrich, Tokyo, Japan). Parasites were continuously cultured in an atmosphere of 5% O2 and 5% CO2 at 37°C. The culture medium was changed daily [4].
Prediction, analysis and synthesis of a B-cell epitope
A BbAMA-1 amino acid sequence retrieved from UniProtKB database (entry no. A7ASF6) was subjected to bioinformatics analysis to predict both linear and conformational B-cell epitope in the PAN motif of domain I as described by Rittipornlertrak et al. (2017) [26]. The genetic diversity of the predicted epitope was visualized by multiply aligning the corresponding amino acid sequences from various B. bovis isolates, using the MUSCLE algorithm (http://www.ebi.ac.uk/Tools/msa/muscle/) [44]. In addition, a multiple alignment of complete AMA-1 sequences from Babesia spp. and other apicomplexan parasites was examined by I-TASSER server (https://zhanglab.ccmb.med.umich.edu/I-TASSER/) [45] for the comparative analysis of predicted secondary structures. Moreover, a homology model of the complete BbAMA-1 was constructed with Phyre2 (http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index) [46] and I-TASSER server based on the 3D structure of Babesiadivergens (B. divergens) AMA-1 (BdAMA-1) [47]. The selected epitope was synthesized as a peptide (sBbAMA-1).
Production of rabbit polyclonal anti-sBbAMA-1 antibody
Two New Zealand White female rabbits (Mlac:NZW, National Laboratory Animal Center, Mahidol University, Nakhon Pathom, Thailand) with 3-6 months old were kept individually in cages without bedding at a temperature of 20°C to 24°C, humidity of 55±10 % and a 12/12-hour light/dark cycle at laboratory building, Faculty of Veterinary Medicine, Chiang Mai University, Thailand. Rabbits were maintained with ad libitum access to food and water. To obtain rabbit polyclonal anti-sBbAMA-1 antibody, a rabbit was intramuscularly immunized with 100 µg/ml sBbAMA-1 formulated with Montanide ISA 206 VG (Seppic, Paris, France; 1:1 v/v) 4 times at 2-week interval after housing for 1 week. To obtain a negative control serum, the other rabbit was intramuscularly immunized with 0.5 ml of Montanide adjuvant only. The adverse events were monitored throughout the experiment. Every week, serum samples were collected from the immunized rabbits to determine antibody titer using an indirect enzyme-linked immunosorbent assay (ELISA). Briefly, each well in 96-well immuno plates (Nunc-Immuno Plate MaxiSorp, Intermed, Roskildes, Denmark) was coated with 10 ng of sBbAMA-1 in a coating buffer (0.05 M carbonate bicarbonate buffer, pH 9.6), and then incubated overnight at 4°C. The plates were washed thrice with wash buffer (Phosphate-buffered saline (PBS) containing 0.05% Tween-20) and then blocked with a blocking buffer (5% skim milk in PBS). Rabbit serum samples at dilution of 1:100 in blocking buffer was added and then incubated at 37°C for 1 h. After washing thrice with washing buffer, horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin (Ig) G (KPL, Gaithersburg, MD, USA) at dilution of 1:2,000 in blocking buffer was added and then incubated at 37°C for 1 h. After washing thrice with washing buffer, 3,3’,5,5’-tetramethybenzidine (TMB) substrate (SeraCare Life Sciences, Gaithersburg, MD, USA) was added, and incubated at room temperature in dark. The reaction was stopped with 2 M H2SO4 after 15 min of incubation. The absorbance at 450 nm was measured using an Accu Reader Microplate reader M965 (Metertech, Taipei, Taiwan R.O.C.). Due to the final blood collection of rabbits, the generalized anesthesia was done by intravenous injection of pentobarbitone sodium (Nembutal ®, 20 mg/kg). The blood collection was done by using a 1-in. long, 18-gauge needle for penetrating to jugular vein. Blood was taken until the volume reached 100 ml, then the rabbits were euthanized by intravenous injection of overdosage pentobarbitone sodium (Nembutal®, 90 mg/kg).
Recognition of native BbAMA-1 by Western blot analysis
The B. bovis lysate was prepared as described by Yokoyama et al. (2002) [48] with minor modifications. Briefly, the B. bovis infected RBCs from the in vitro cultures were treated with 0.83% NH4Cl solution for 10 min at 37°C, and then washed thrice with cold PBS. The pellets containing the parasites were suspended in 1 ml of a lysis buffer (50 mM Tris-HCl (pH 7.6), 0.1% Triton X-100, 150 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM dithiothreitol (DTT), 10 mg/ml pepstatin A and 10 mg/ml leupeptin), incubated on ice for 20 min, and then centrifuged at 18,000 × g for 30 min at 4°C. The supernatant of clarified lysate was dialyzed overnight using a dialysis buffer (50 mM Tris-HCl (pH 7.6), 150 mM NaCl, 20% glycerol, 1 mM EDTA, 1 mM PMSF, and 1 mM DTT). The dialysate was centrifuged at 18,000 × g for 30 min at 4°C. Subsequently, the supernatant was analyzed to identify native BbAMA-1 by Western blot analysis. The lysate were separated by 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel. The proteins from SDS-PAGE gel were electrically transferred onto a nitrocellulose membrane (Merck Millipore™, Merck KGaA©, Darmstadt, DEU). The blotting time was 60 min at a constant voltage of 20 V. The membrane was then incubated with a blocking buffer (5% skim milk in PBS) for 1 h at room temperature with gentle shaking. After being washed thrice with wash buffer (PBS containing 0.05% Tween® 20), the membrane was incubated with rabbit polyclonal anti-sBbAMA-1 serum (1:50 dilution) at 4°C overnight. The membrane was probed with HRP-conjugated goat anti-rabbit IgG antibody (1:4,000 dilution; KPL, Gaithersburg, MD, USA). The membrane was incubated with gentle shaking at room temperature for 1 h and then washed three times with wash buffer. Finally, the reactions were visualized using a solution containing 3,3´-diaminobenzidine (DAB; Invitrogen, Carlsbad, CA, USA) and hydrogen peroxide (H2O2; Merck, Germany).
The immunofluorescence antibody test (IFAT)
The immunofluorescence antibody test was carried out as described in a previous study [49] with minor modifications. The B. bovis-infected RBCs were coated on indirect IFAT slides (Matsunami Glass Ind., Ltd, Osaka, Japan), air-dried, and then fixed in cold absolute acetone at -20°C for 5 min. Ten microliters of the rabbit anti-sBbAMA-1 serum diluted at 1:40 in a blocking buffer (5% fetal bovine serum (FBS) in PBS) was added as the first antibody on the fixed smears, and then incubated for 1 h at 37°C in a moist chamber. After washing once with PBST, Alexa-FluorÒ 488 conjugated goat anti-rabbit IgG (Molecular Probes, Invitrogen, Carlsbad, CA, USA) was subsequently applied (1:200 dilution in the blocking buffer) as a secondary antibody, and then incubated for 30 min at 37°C. The slides were washed thrice with PBST, mounted in 50% glycerol-PBS, and then examined using a fluorescent microscope (E400 Eclipse, Nikon, Kawasaki, Japan).
Preparation of free merozoites by cold treatment
The method described by Ishizaki et al. (2016) [50] was used to isolate free merozoite liberated from the infected RBCs. A 5 ml of an in vitro culture of B. bovis with 30% parasitemia was incubated on ice for 2 h, and then the culture was resuspended in 5 ml of GIT medium. The suspension was slowly overlaid onto 2 ml of 30% (1.043 g/ml density) Percoll/ PBS solution (GE Healthcare, Buckinghamshire, UK) at the bottom of a 15 ml centrifuge tube (Corning, Corning, NY, USA). The tube was centrifuged at 280 x g for 5 min, and then at 330 x g for 20 min at 4°C. The medium and free merozoite layers were transferred carefully to a new tube, and then centrifuged at 1500 x g for 5 min at 4°C. The pellet containing free merozoites was washed twice with 20 ml of GIT medium, and then suspended in 1 ml of GIT medium. The concentration of purified merozoites was calculated with a disposable hemo-cytometer (AR Brown, Tokyo, Japan). The viability of the merozoites was determined after staining with 6-carboxyfluorescein diacetate (6-CFDA; Invitrogen Corp., Carlsbad, CA, USA) and propidium iodide (PI; Dojindo, Kumamoto, Japan).
In vitro growth-inhibition assay
The growth-inhibition assay was performed in 96-well culture plates (Nunc, Roskilde, Denmark) as described by Salama et al. (2013) [28]. One hundred and eighty microliters of GIT medium, GIT medium containing anti-sBbAMA-1 serum at 1:5, 1:10, and 1:20 or rabbit anti-Montanide adjuvant serum (negative control) at 1:5 dilution was added into each well in triplicates. Then, 20 µl of bovine RBCs with 1% parasitemia was added into each well. The plate was incubated as described in section 2.1 for 4 days, and the culture medium was replaced everyday with fresh medium containing the indicated dilution of antiserum. Giemsa-stained thin blood smears were prepared every 24 h and parasitemia was monitored by counting the infected RBCs among approximately 1,000 total RBCs, using a light microscope. The percentage of growth inhibition was calculated as the rate of parasitemia reduction in antibody-treated cultures compared to GIT-control. The assay was repeated twice.
In vitro invasion-inhibition assay
The RBC invasion-inhibition assay was performed in triplicate in a 96-well plates (Nunc, Roskilde, Denmark) according to Ishizaki et al. (2016) [50] with some modification. Briefly, purified free merozoites (obtained from B. bovis in vitro culture) together with uninfected bovine RBCs with a multiplicity of infection (MOI) of 4.2 were added to GIT medium containing rabbit anti-sBbAMA-1 serum at 1:5 and 1:10 dilutions. Rabbit anti-Montanide adjuvant serum at dilution of 1:5 and GIT medium were used as control. The culture plate was incubated, and Giemsa-stained thin blood smears were prepared at 1, 2 and 4 h post-incubation. The percentage of parasitemia was evaluated under a light microscope, based on approximately 3,000 observed RBCs. The percentage of invasion-inhibition was calculated based on the parasitemia reduction in antibody-treated cultures compared to GIT-control. The experiments were carried out in two separate trials.
Statistical analyses
The growth- and invasion-inhibition rates were analyzed by independent-samples Student's t test. P values < 0.05 were considered statistically significant.