Anaplasma marginale
Two strains of A. marginale were used in the experiments: Jaboticabal and Palmeira. The Jaboticabal strain was
isolated from bovines in the municipality of Jaboticabal, São Paulo state, southeast
Brazil (21° 15' 18'' S; 48° 19' 20'' W) (
25
</a>). The strain Palmeira was isolated in the beginning of 2,000’s from Holstein dairy
bovines showing clinical anaplasmosis in the municipality of Palmeira das Missões,
Rio Grande do Sul state, southern Brazil (27° 54' 09" S; 53° 18' 26" W). After isolation,
both strains were cryopreserved in liquid nitrogen. </p>
Calves and tick cell line ISE6
Eight male calves (Bos taurus taurus) of the Aberdeen-Angus breed were acquired from the tick-free area in Santa Vitória
do Palmar municipality, Rio Grande do Sul State, Brazil. All of them were seronegative
to A. marginale in ELISA assays (data not shown). All calves, aging from 10 to 14 months and weighting
between 140 and 220 kg, were kept in individual screened stalls. During the experiments,
animals were fed thrice a day with alfafa hay and pelleted food for cattle, and had
free access to water. At the end of the experiment the animals were discarded according
to the procedure approved by the protocol stated in the section “Ethics approval and
consent to participate”. The embryonic cell line ISE6, derived from I. scapularis (
21), was cultured as previously described (
26
</a>). Cell growth and viability were assessed by cell counting in a Neubauer chamber
using optical microscopy after trypan blue staining. </p>
Calf experimental infection and antimicrobial treatment
Calves were experimentally infected by intravenous (jugular vein) inoculation of approximately
106 bovine erythrocytes parasitized by A. marginale strain Jaboticabal (animals J1, J2, J3, and J4) or strain Palmeira (animals P1, P2,
P3, and P4). The parasitemia was monitored daily. To that end, peripheral blood smear
was stained with the Instant-Prov kit for hematological analyses (NewProv Produtos
para Laboratório, Pinhais, PR, Brazil). Then, the number of infected erythrocytes
in 10 different microscopic fields was determined and used to calculate the parasitemia
(% of infected erythrocytes). In addition, the rectal temperature (RT) and the packed
cell volume (PCV) were also daily evaluated. The treatment protocol was based on guidelines
of Brazilian Ministry of Agriculture for antimicrobial efficacy test for new drugs.
After the calves reached a parasitemia > 2%, treatment with injectable antimicrobials
was started, using either OTC (Terramicina, Zoetis, Brazil - 20 mg / kg) (J1, J2,
P1, and P2) or IMD (Imizol, MSD Animal Health, Brazil - 30 mg / kg) (J3, J4, P3, and
P4). Another treatment with the same dose of OTC was repeated if the animal presented
parasitemia 1% three days after the treatment onset. In the case of IMD, the same
treatment was repeated if parasitemia was 1% seven days after the administration
of the first dose. The animal was considered cured when the parasitemia was zero for
at least three consecutive days.
ISE6 cells infection and antimicrobial treatment
The antimicrobials OTC (Sigma-Aldrich, Israel, final concentrations of 1 μg/mL, 4
μg/mL or 16 μg/mL), ENRO (Floxiclin, Biofarm Química e Farmacêutica, Brazil; final
concentrations of 0.25 μg/mL, 1 μg/mL or 4 μg/mL) or IMD (Imicarb Eurofarma Laboratories
S.A., Brazil; final concentrations of 0.25 μg/mL, 1 μg/mL or 4 μg/mL) were added to
ISE6 cells (1.5 106) in L15B infection medium (
27
</a>). After 24 h at 34°C, cells were challenged with <em>A. marginale</em> strain Jaboticabal. To that end, infected bovine blood was defrosted in a water-bath
at 37° C, centrifuged at 5000 g for 10 min at 4° C and the resulting pellet was washed
twice in sterile phosphate-buffered saline (PBS). The final pellet, containing bacteria
and cellular debris, was suspended in L15B infection medium (<a href="#_ENREF_27">
27
</a>) and used as inoculum at a multiplicity of infection (MOI) of 100. After incubation
at 34° C from 1 up to 7 days, the supernatant was discarded, the cell monolayer was
washed with PBS and detached with a Trypsin-EDTA solution (Vitrocell, Brazil). The
cell suspension was centrifuged at 3000 x <em>g</em> for 10 min at 4°C. The genomic DNA (gDNA) was extracted from resulting pellet and
used for bacterial quantification. Five biological replicates were processed for each
group. </p>
Quantification of A. marginale by real-time quantitative PCR
Total gDNA was extracted from ISE6 cells using Kit Invisorb Spin Tissue Mini Kit (STRATEC Molecular, Germany) according to the manufacturer's instructions. The number
of A. marginale in ISE6 cells was determined by quantitative real-time PCR (qPCR) using specific
primers and a hydrolysis probe for msp5, the encoding gene of the major surface protein 5 of A. marginale (
28
</a>). Briefly, the amplification reactions were performed in a final volume of 15 μl
containing 2 μl of gDNA (approximately 100 ng), 2 μl of a mixture of forward and reverse
primers (0.6 μM each), 0.02 μl of TaqMan probe, 7.5 μl of Maxima Probe/ROX qPCR Master
Mix (Thermo Fisher Scientific), and 3.5 μl of ultrapure water. The reactions were
performed on a StepOnePlusTM Real-Time PCR System (Thermo Fisher Scientific) using
the following thermocycler program: 10 min at 95 °C, followed by 40 cycles of 15 s
at 95 °C, 30 s at 60 °C, and 45 s at 72 °C. The cycle of quantification (Cq) values
of reactions using a dilution series of 10<sup>2</sup> to 10<sup>7</sup> copies of <em>msp5</em> cloned into a plasmid (pGEM-®T Easy, Promega, USA) were used to establish a standard
curve for determining the absolute number of <em>A. marginale</em> in each sample. All samples were analyzed in three technical replicates.</p>
Light microscopy
Seven days post-infection, ISE6 cells were collected by scraping and centrifuged onto
microscope slides through five pulses of 10 seconds at 100 rpm each using a Citospin
Brand (Fanem, Brazil). Cells were air-dried and stained with Panoptico (Newprov).
The images were acquired in an inverted microscope (Zeiss Primovert, Gottingen, Germany)
under Plan-NeoFluar 100x/1.30 oil objective, coupled to a digital camera system (Axiocam
105 color, Zeiss) and processed by the Zeiss Software (ZENcore).
Statistical analysis
The statistical significance of the differences in the bacteria number between the
experimental and control groups was evaluated using GraphPad Prism version 7.0 for
Windows (GraphPad Software, San Diego, CA, USA). The Kruskal-Wallis test was used
to assess similarities between the groups, and the Mann-Whitney test was used to compare
the medians. A p-value of less than 0.05 (P < 0.05) was considered statistically significant.