Seasonal fluctuations of Babesia bigemina and Rhipicephalus microplus in Brangus and Nellore cattle reared in the Cerrado biome, Brazil | Parasites and vectors
The study was conducted in the municipality of Água Clara, State of Mato Grosso do Sul, Brazil (20°46′24″S 52°32′24″W; altitude: 309 m asl). The climate of this area is characterized as humid tropical, with a dry season of 1 to 3 months and an average temperature > 18°C all months of the year . According to Flumignan et al. , the precipitation distribution pattern in the municipality of Água Clara follows a very consistent pattern in which most precipitation occurs from December to February, gradually decreasing from March to May until the dry season from June to August. A gradual increase in precipitation occurs from September to November until the rainiest months of the year. This study was carried out from June to December 2016 and meteorological data was obtained from the database of the State Government of Mato Grosso do Sul. .
Animals used in the experiment
Nineteen animals (9 Brangus and 10 Nellore) raised on pasture in the growth phase, aged 8 to 10 months and with an average weight of 219.5 kg, were studied. These animals were naturally infested and kept together at a density of 0.6 animals per hectare. The ground consisted of sandy soil with a pasture consisting of Urochloa (brachial) decubens. Acaricidal treatment and prophylactic control of tick-borne diseases (TBD) were not carried out.
Tick count and blood test
Data was collected from June 2016 to June 2017, with 18-day intervals  between tick counts according to the method of Wharton and Utech . Briefly, ticks > 4.5–8 mm in length on both sides of each animal were counted. The taxonomic classification of ticks was carried out according to the system of Pereira et al. . In addition, every 36 days the animals were weighed on a digital scale (Coimma®; Dracena—SP, Brazil) to assess animal welfare. , and blood was drawn from the tail vein using sterile vacuum tubes containing ethylenediaminetetraacetic acid anticoagulant. A total of 228 blood samples (12 from each animal) were collected; these samples were stored at 4°C and transported to the laboratory for serum and genomic DNA (gDNA) extraction.
Genomic DNA was extracted in duplicate from whole blood as previously described. . Briefly, each extraction was performed in a 2 ml microtube containing 300 μl of bovine blood, 2 μl of proteinase K (20 mg/ml) (Sigma-Aldrich, St. Louis, MO, USA) and 500 μl of sodium dodecyl sulphate (20%). Each sample was incubated for 1 h in a 65°C water bath, after which 800 μl of chloroform was added to the microtube and the samples were vigorously vortexed to achieve homogenization. Then 350 μl of protein precipitation solution (6 ml potassium acetate, 1.1 ml glacial acetic acid, 2.9 ml ddH2O) was added and the mixture was centrifuged at 13,000 rpm for 10 min. The aqueous phase was transferred to a new tube, 1 ml of ice-cold 100% ethanol was added to the tube, and the samples were stored at -20°C overnight for DNA precipitation. The samples were then centrifuged at 13,000 rpm for 5 min, the supernatant was discarded and 1 ml of 70% ethanol was added to the tube. The mixture was centrifuged at 13,000 rpm for 2 min and the supernatant discarded. The resulting pellet was oven-dried at 37°C and the DNA was resuspended in 50 μl of ultrapure water and eluted in a water bath for 30 min at 65°C. The amount and purity of each sample were estimated spectrophotometrically with a NanoDrop™ spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA), at absorbance readings of 260 nm and absorbance ratio of 260/280 nm, respectively. The 228 samples corresponding to each animal and collection time point were then stored at -80°C until further use.
These gDNA samples were analyzed by PCR in duplicate following the methodology described previously.  using primers KB-18 (5′-GATGTACAACCTCACCAGAGTACC-3′ forward) and KB-19 (5′-CAACAAAATAGAACCAAGGTCCTAC-3′ reverse), which produce a 262 bp PCR product. The PCR reaction was carried out according to  using the following reagents: 2.5 µl 10× buffer, 0.75 µl MgCl2 (50 mM), 0.5 µl dNTPs (2.5 mM; Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), 0.5 µl forward and reverse primers (10 pmol), 0.3 µl Taq DNA polymerase (Ludwig Biotec, Rio Grande do Sul, Brazil), 1 µl of DNA (100 ng) and ultrapure water to a final volume of 25 µl. Two negative controls (a blood sample obtained from a healthy bovine donor and ultrapure water) and a positive control (a sample obtained from a bovine blood smear positive for B. bigemina; Supplementary File 1: Image S1) were tested. The reactions were performed in a BioRad T100™ thermal cycler (Bio-Rad Laboratories, Hercules, CA, USA), and the cycling parameters were: 95°C for 2 min, followed by 40 cycles of 95°C for 1 min , 60°C for 30 s and 72°C for 1h30 min, with a final extension of 72°C for 7 min.
The final product was visualized in a 1.5% agarose gel stained with ethidium bromide. Twelve samples giving the expected PCR product size for B. bigemina were purified using a PureLink Rapid Gel Extraction Kit (Invitrogen, Thermo Fisher Scientific). These DNA samples were sequenced at the Human Genome and Stem Cell Research Center (Universidade de São Paulo [USP], São Paulo, Brazil) in an automatic sequencer (ABI 3730 DNA Analyzer; Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) with a 48-capillary DNA analysis system. The sequenced PCR products were analyzed with the MEGA X software and the consensus sequences of the analyzed samples have been deposited in GenBank under the accession number MZ542450.1.
Real-time quantitative PCR analysis
Genomic DNA samples were diluted to a concentration of 100 ng/μl and subjected to quantitative real-time PCR (qPCR) analysis to quantify circulating copies of the B. bigemina cytochrome b embarrassed (cbisg) as described in . Absolute quantification was performed using dual quenched hydrolysis primers and probes (PrimeTime® Std qPCR Assay; Integrated DNA Technologies, Coralville, IA, USA) based on the B. bigemina LK054939.1 in GenBank and designed using the PrimerQuest tool (Integrated DNA Technologies), which generated an 88 bp product of the cbisg gene (sense primer: 5′-TGTTCCAGGAGATGTTGATTTCT-3′; probe: 5′-/56-FAmQCGAGTGTGT/Zen/TATCAGAGTATTAACTGAGGT/3IABkFQ/-3′; antisense primer: 5′-GCACTTCGTTATTTCCATGCT -3′). Primer-dimer formation was tested with the OligoAnalyzer tool (https://www.idtdna.com/pages/tools/oligoanalyzer).
In silico specificity was tested using the NCBI BLAST platform (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch). The organisms searched were limited to ‘cattle’, ‘humans’ and ‘babesia’.
The efficiency and reproducibility (Supplementary File 2: Table S1, Supplementary File 3: Figure S1, Supplementary File 4: Figure S2) of the reaction were calculated according to . Serial dilutions (1:10) from 101 at 10ten were used to construct a standard curve with different concentrations of synthetic DNA gBlocks® Gene Fragments (Integrated DNA Technologies) containing the sequence of B. bigemina (5′-TGACCTTTTATTATGTTCCAGGAGATGTTGATTCTTTCGAGTGTGTTATCAGAGTATTAACTGAGGTTAATATGGGTTGGGCACTTCGTTATTTCCATGCTCAATGTGTTTCTTTTGCTTTTTCTTTATGATGTTACATATGTTAAAAGGTTTATG-3′ – also constructed based on the sequence deposited under accession number LK054939.1). Duplicate Positive Controls and Negative Matrix and Negative Control samples were added to each qPCR run.
The reaction volume was 10 μl per well and consisted of 5 μl of Taqman™ Universal PCR Master Mix (Thermo Fisher Scientific), 0.5 μl of each primer (10 μM), 3 μl of Milli-Q H2O and 1 μl of 100 ng/μl of gDNA. The reactions were carried out in duplicate. Ultrapure water was used instead of gDNA as a negative control.
A standard 5-point curve (concentrations of 105 at 10ten gBlocks®) was used in triplicate as an internal control in each 98-well plate. Samples were analyzed using a StepOnePlus™ Real-time PCR system (Thermo Fisher Scientific) using one cycle of hydrolase probe activation of 95°C for 10 min followed by 45 cycles of denaturation at 95°C for 45 s and annealing/extension at 60°C for 1 min.
The reaction signal was recorded during the extension step and the data was analyzed using StepOne v2.3. Guidelines regarding minimum information for publishing quantitative real-time PCR (MIQE) experiments were followed .
Using qPCR results, the number of target DNA molecules in each reaction (copy number [CN]) was calculated according to as follows: CN (L) = (6.022 × 1023 [copies/mol]×concentration [g/mol])/molecular mass (g/l), where 6.022 × 1023 is Avogadro’s number and the molecular mass is the average molecular weight of the double-stranded DNA (330 × 2) multiplied by the size of the cloned fragment.
For the detection of antigens of anti-B. bigemina immunoglobulin G (IgG), the indirect ELISA technique (iELISA) was used according to a protocol based on .
Total antigen of B. bigemina (carried out by the Immunoparasitology Laboratory of the Faculty of Agronomic and Veterinary Sciences [FCAV]/Sao Paulo State University [UNESP], Jaboticabal, SP, Brazil) was diluted to an optimal concentration of 10 μg/ml in a 0.5 M carbonate/bicarbonate buffer (pH 9.6). After 12 h incubation at 4°C, blocking was performed with phosphate buffered saline—Tween 20 (PBS—Tween 20) (pH 7.2, 0.05% PBST) and 6% skim milk powder (Molico®; Nestlé, São Paulo, Brazil). 96-well plates (Nunc Maxisorp™; Thermo Fisher Scientific) were incubated for 90 min at 37°C in a humid chamber.
After three washes with PBST buffer, positive, negative and reference sera were added (all diluted 1:400 in PBST + 5% normal rabbit serum). The plates are then incubated at 37° C. for 90 min in a humid chamber. After three washes with PBST, alkaline phosphatase-conjugated anti-bovine IgG (Sigma-Aldrich, St. Louis, MO, USA) diluted 1:30,000 in PBST + 5% normal rabbit serum was added , and the plates were washed again.
The p-nitrophenyl phosphate alkaline phosphatase substrate (Sigma-Aldrich) was then diluted in 1 mg/ml of diethanolamine buffer (pH 9.8). The plates were sealed in aluminum foil and incubated for 30 min at room temperature, then read at a wavelength of 405 nm on a micro-ELISA reader (BT-100; Embrabio, São Paulo, Brazil).
R version 3.6.3  and R Studio (8.15 build 180 091)  were used for statistical analysis.
The Kolmogorov-Smirnov test was performed to check the normality of the data, then the Mann-Whitney U test was used to compare weight, tick count and B. bigemina cbisg CN gene between breeds because the data did not show a normal distribution.
CNs and tick counts were logarithmicten(n+1)-transformed and then analyzed by Spearman’s rho statistic to estimate a rank-based measure of association between weight, logten(CN) and newspaperten(ticking). A P-value