of G418 Sulfate at 4 × 105 cells/ml to be seeded in 384-well plates. For this, the cell suspension (25 µl; ∼10,000 cells/well) was dispensed into all 384 wells of the plate. To distribute cells evenly, mixing was by aspirating and immediately re-dispensing 10 µl of the applied cell suspension three times. Plates were incubated overnight at 37◦C under 5% CO2. On the next day, cells were prepared for the assay following the kit's instructions: the old media in the assay plate was fully removed by inverting the plate and gently blotting it on paper towels, and media was replaced with 45 µl of Fluo-8 AM loading dye (1×). The plate was incubated at 37◦C under 5% CO2 for 30 min, then equilibrated at room temperature in the dark for 30 min. The screening of the small molecule library was achieved by a dual-addition assay. The "first addition" consisted of 5 µl of either the blank solvent or a 10 × solution of the potential antagonist transferred from the 384-well library plate, to reach 1 × concentration in the wells. After 5 min incubation with cells, a second addition of 5.6 µl of 10 µM kinin receptor-specific agonist peptide (FFFSWGamide) resulted in a final concentration of 1 µM. The calcium fluorescence signal was read by a Varioskan LUXTM (Thermos Scientific, Waltham, MA, United States) plate reader set for fluorescence plate-mode with Ex/Em = 490/525 nm and kept at 29◦C. Endpoint responses were read immediately after the first addition and 5 min after the second addition of agonist. The plate was read from both forward and reverse orientations by rotating the plate (180◦ ) on the instrument to compensate the variation in signal kinetics during the lapse in plate reading, because there was a 2 min lag time between the readings of the first and the last well. The response to each addition was represented as the average value of both forward and reverse plate readings (**Supplementary Tables S3**, **S4**, Panels B,C). The antagonist activity was calculated as the percentage of the response to 1 µM FFFSWGa of cells that had been incubated with the putative antagonist compound in comparison to the response of cells that had been incubated with blank solvent only. A compound was considered to have antagonistic activity, if it minimally inhibited 50% of the response to the agonist FFFSWGa (1 µM) applied in the second addition. These candidates were selected for further validation in a dose-response kinetic calcium mobilization bioluminescence assay.

## Kinetic, Dose-Response Calcium Mobilization Bioluminescence Assay

The calcium bioluminescence assay was used for the kinetic analysis of dose-responses to the compounds identified in the primary screen. Aequorin is the calcium reporter, transiently expressed in the BMLK3 cells (Lu et al., 2011b). This "dualaddition" kinetic assay was conceived to characterize the diverse activity patterns the putative ligands may display. The "first addition" consists of the compound being tested, either a small molecule or a peptidomimetic; the bioluminescence response elicited is measured for 30 s (if the compound is an agonist there will be bioluminescence response during this first 30 s period). Immediately after, at 32 s, a "second addition" with a single concentration of agonist follows (FFFSWGa, 1 µM), and the response continues to be measured for another 30 s. It is important to emphasize that the response measured after this addition of agonist is influenced by the presence of the unknown compound applied in the first addition. If the compound is an antagonist, the response to the agonist applied in the second addition will be reduced with respect to that of the positive control (buffer + agonist). The pharmacological activity of the unknown molecule can be determined based on the integrated area under the bioluminescence response curve registered during both these 30 s periods (total bioluminescence expressed as average bioluminescence units per second). That is, it can be inferred if the unknown molecule is a full agonist, full antagonist, partial agonist, partial antagonist, or an allosteric modulator. This approach is widely applied in GPCR drug discovery (Ma et al., 2017).

Selected small molecules were solubilized in 1 × DMEM medium (Gibco R , Invitrogen) with 10% DMSO and prepared from 500 nM to 500 µM as 5× of the final concentration. In addition, three peptidomimetic ligands of neurokinin receptors were purchased from Tocris R Bioscience: one agonist, hemokinin 1 (human), and two antagonists, antagonist G and spantide I. These peptidomimetics were solubilized in 80% acetonitrile: 0.01% trifluoroacetic acid and then aliquoted (100 nm per tube) and freeze-dried; the dry peptidomimetics were stored at −20◦C before use. For the assay, the peptidomimetics were solubilized and diluted in assay buffer (1 × DMEM) containing 1% DMSO from 10 nM to 100 µM as 10× of the final concentrations in the assay. All compounds, either small molecules or peptidomimetics were tested in three independent replicates, each with 2-3 wells as pseudo-replicates. Responses from each assay were calculated as the average of individual responses from the pseudoreplicate wells.

The cells preparation was described in detail elsewhere (Lu et al., 2011b). In brief, the BMLK3 and vector-only cells were cultured to ∼ 90% confluency in T-25 flasks. The cells were trypsinized and suspended in maintenance medium, counted, and diluted to 1 × 105 cells/ml; 2 ml of this cell suspension was placed into each well of 6-well-plates (CELLSTAR R , Greiner Bio-one). After overnight incubation, when the cells reached a confluency of 40–60%, old medium was replaced with 1 ml of serum-reduced Opti-MEMTM medium (Gibco R , Invitrogen) in each well. Following the instructions of the transfection reagent manufacturer, cells in each well of the 6-well plate were transiently transfected with 1 µg mtAequorin/pcDNA1 plasmid mixed in 4 µl of FuGENE6 (Promega, Madison, WI, United States) and 96 µl of Opti-MEMTM medium. After 6 h of incubation, the old medium was replaced with 2 ml of F-12K medium with FBS (10%) (antibiotic-free medium). Following 24 h incubation, cells were trypsinized, seeded into white/clear 96-well-plates (CELLSTAR R , Greiner Bio-one) (20,000 cells/well in 100 µl of antibiotic-free medium), and incubated overnight until they reached optimal confluence of 80%. BMLK3 cells and vector only cells were prepared in the same plate to test different concentrations of each compound. To reconstitute the aequorin-complex, cells were incubated with 90 µl per well of calcium-free DMEM (1×) containing coelenterazine (5 µM) (RegisTM Technology, Inc., Morton Grove, IL, United States). After 3 h of incubation in the dark, cells were ready for the assay.

The "dual-addition" assays were performed with a ClariostarTM (BMG LABTECH, Chicago, IL, United States) plate reader set at 29◦C and for bioluminescence and "well" mode at 469 nm emission wavelength. The two additions were performed by the built-in injectors of the ClariostarTMplate reader. The first addition was performed after 2 s of initiating the recording of the bioluminescence response, which continued for 30 s. This addition applied either 22.5 µl (5×) of certain small molecule antagonist at a specific concentration or blank solvent, or 10 µl of peptidomimetic (10×). At 32 s, a second addition of 12.5 µl of agonist peptide FFFSWGa was executed with the second injector into the same well containing the test compound or blank solvent, to reach a final concentration of 1 µM. For peptidomimetics, 11 µl were applied. Recording continued for another 30 s; in sum, the kinetic response of each well was recorded for a total of 65 s at 1 s intervals. The same procedure was performed for each concentration of the ligand tested. The kinetic response to both additions were recorded immediately after addition for 30 s, with 1 s interval, and is expressed as bioluminescence units (BU) per second. The total response of the cells to each addition was represented as the percentage of the averaged BU per second obtained in each of the two 30 s-ranges, relative to the average BU to the PC (blank solvent + 1 µM FFFSWGa) in the 2nd range (average BU of the PC curve from 35 to 65 s).

### Effect of a Prolonged Pre-incubation With Various Concentrations of TKSM14 on the Agonist-Induced Response

Because TKSM14 displayed antagonistic activity in the fluorescence assay but not in the calcium bioluminescence assay, a modified calcium bioluminescence assay was performed in which the incubation time (previously 30 s) was increased to 5 min, as in the fluorescence assay. The first addition was performed manually to add 22.5 µl of the blank buffer or different dosages of the TKSM14 (5 × solutions), to achieve final concentrations of 1, 10, 30, 50, and 100 µM. The second addition was performed with the built-in injector of the ClariostarTM plate reader (12.5 µl of the 10× agonist FFFSWGa solution for a final concentration of 1 µM), as described in the previous section. The response to the agonist in the second addition was recoded for 30 s, at 1 s intervals. Similarly, as before, three independent assays were performed, with each assay containing 3–4 pseudo-replicates. Responses from each assay were calculated as the average of individual responses from the pseudo-replicate wells. The antagonistic activity was represented as a percentage of the average bioluminescence units obtained during the 30 s after the second addition (wells containing both the small molecule being analyzed and FFFSWGa), divided by the average bioluminescence units obtained from wells containing FFFSWGa only.

## Statistical Analyses

All statistical analyses were performed with Prism 6.0 (GraphPad Software, La Jolla, CA, United States). In the end-point fluorescence assay, dose-response curves and IC50 values were calculated with "log [inhibitor] vs. response – Variable slope (four parameters)" function in Prism 6.0. IC50s of the antagonists were defined as the concentration of antagonist that inhibits agonist response in the mid-range of the respective fitted doseresponse curve (this is not the concentration that inhibits 50% of the agonist response). In the kinetic calcium bioluminescence assay, to determine the statistical significance of the inhibitory effect from various concentrations of the same compound, the response after the second addition for each concentration within each molecule was compared to the corresponding blank control by one-way ANOVA (n = 3) followed by Tukey's multiple comparisons test.

## RESULTS

Our long-term approach for discovering novel ligands for arthropod GPCRs is summarized in **Figure 1**. In this study, we focused on processes on the left of the figure, by using transcriptomics and genomic data to clone the kinin cDNA, followed by forward pharmacological approaches toward the identification of novel ligands for the kinin receptor of the southern cattle tick, R. microplus.

### R. microplus cDNA Sequence and Gene Structure

We first identified the predicted 332 amino acid residue protein sequence of the putative kinin precursor of Ix. scapularis from a genome fragment (DS680282: 583-1410) (Gulia-Nuss et al., 2016). Although it was reported that the gene encoded 19 kinins (Gulia-Nuss et al., 2016), the exact sequences of kinins were not provided. Herein, we listed the sequence of the 19 paracopies of putative bioactive kinins from Ix. scapularis (**Table 1**). Using this curated precursor protein as query, the tBLASTn results against WGS assembly and TSA of R. microplus (taxid: 6941) identified a genomic DNA fragment of accession number LYUQ01126194.1 and four transcripts with high similarities. Our cloning of the putative kinin precursor cDNA from R. microplus verified the full-length cDNA sequence is 1,398 bp long encoding a 1,227 bp open reading frame (ORF). The predicted precursor protein of 409 amino acid residues has a predicted molecular mass of 44.72 kDa (GenBank QDO79406) (**Supplementary Figure S2**). The 50 - and 30 -untranslated regions (UTRs) are 85 bp and 86 bp, respectively.

Alignment of the cloned cDNA to the genome sequence revealed that the 50 -end was missing in the genome, as determined by BLAST (**Figure 2A**). We were able to obtain this missing sequence by genomic PCR amplification using a primer designed based on the sequence of the cDNA 50 -end (**Supplementary Figure S1** and **Supplementary Table S1**). By further amplification of the genomic DNA with gene-specific primers we sequenced the putative kinin gene of R. microplus, that is encoded in approximately 4.1 kb (GenBank MK875970) (**Figure 2B**), composed of two exons and one intron. The first exon is 140 bp long and the second exon is 1,253 bp long; in between there is an intron of ∼ 2,700 bp. Although the genomic DNA and cDNA sequences obtained were from different strains of R. microplus, the alignment of their ORF region showed 99% identity.

## Analysis of the R. microplus Kinin Precursor

The first 38 amino acid residues of the R. microplus kinin precursor were predicted with SignalP v 5.0 as belonging to a signal peptide with only 50% probability, denying such conclusive identification. Seventeen paracopies of putative bioactive kinins were encoded by this single cDNA (**Table 1**). Three of them

TABLE 1 | Predicted bioactive kinins from Rhipicephalus microplus and Ixodes scapularis.

Under name, ordinal numbers reflect the order in which each sequence appears from the 50 to 30 direction either in the cDNA (R.m.) or in the predicted gene (Ix.). The bolded predicted molecular weights were similar to previously predicted kinin masses (approximately 1,009 Da) (Neupert et al., 2005).

(Rmkinin 10, 13, and 15) have a predicted molecular weight of about 1,009 Da, which was the mass of putative R. microplus kinin detected through mass spectrometry in a previous study (Neupert et al., 2005). The R. microplus kinins showed the canonical critical residues and necessary characteristics for activity of the insect kinins (FX1X2WGa): Phe and Trp at the first and fourth position of the pentapeptide minimally active core, respectively, as well as the amidated Gly at the fifth position. Noticeably, the second variable position (X2) was dominated by Pro; only two out of 17 kinin paracopies from R. microplus have a different amino acid other than Pro on the X2 position (**Table 1**). This consensus pattern of the kinin C-terminal pentapeptide (FX1PWGa) was prevalent in other tick species as well (**Table 1** and **Figure 3A**).

#### Kinin Precursors From Other Tick Species

1398 bp cDNA (2) obtained in this study.

Using the R. microplus kinin precursor as query for TBLASTN against the transcriptomes of all Acari (taxid: 6933) on NCBI, we predicted amino acid sequences of the putative kinin precursors from another seven tick species. Four full amino acid sequences were obtained, those from the relapsing fever tick, Ornithodoros turicata, the castor bean tick, Ix. ricinus, the brown dog tick, R. sanguineus, and the curated kinin precursor of Ix. scapularis.

The precursor from Ix. scapularis was predicted by aligning the kinin precursors deduced from three transcripts and the genomic scaffold. Missing sequences were identified from each of these: the transcripts provided a sequence of 23 amino acid residues at the N-terminus containing part of the signal peptide, and an additional C-terminal sequence of 23 amino acid residues followed by the stop codon. These sequences are absent in the genomic scaffold. However, the protein deduced from the transcriptome lack 40 amino acid residues, which are present in the genomic scaffold, distributed in two gaps within positions 250–293 in the alignment (see **Supplementary Figure S3**). These

FIGURE 3 | Amino acid sequences of kinin precursors from eight tick species. (A) Amino acid sequences were manually curated after mining the available transcriptomes on NCBI. The number of kinin paracopies in each precursor are denoted in the parentheses. The gaps within the protein are denoted with ". . .." The signal peptides predicted by SignalP 5.0 were underlined. The predicted cleavage sites are in bold, and potential bioactive kinins containing the kinin C-terminal motif, FX1X2WG-amide, are highlighted with gray color. The sequence of kinin precursor from Ixodes scapularis was curated based on the putative kinin gene in the genome scaffold (DS680282), while the additional N- and C- terminal sequences were deduced from sequences of three transcripts (Supplementary Figure S3). The number 1 to the left of the Amblyomma sculptum sequence points to an unusual cleavage site (K) or a potential sequencing error at this site (K followed by G) (in black square frame). The version numbers are listed on the top of each sequence (accession numbers are identical except do not contain the 0.1 at the end). (B) Sequence logos of the kinins from eight tick species. The logos were created by WebLogo (Crooks et al., 2004) using the amino acid sequences from each tick kinin precursor between predicted cleavage sites (highlighted in panel A). The overall height of each letter stack indicates the sequence conservation at that position (measured in bits), whereas the height of amino acid symbols within the stack reflects the relative frequency of the corresponding residue at that position. On the X axis, the numbers refer to the amino acid position within the alignment, which was anchored to the conserved C-terminal amidated-glycine residue, similarly as shown in the DINeR database (Yeoh et al., 2017). Toward the N-terminus, logos begin at the position within the alignment where bits are above 0 value.  The dashed-line indicates the bit value of the amino acid residue at the first variable position (X1) of the kinin C-terminal pentapeptide motif (FX1X2WGamide).

gaps may have arisen from errors in the transcriptome assembly because this genomic region encodes four repeats of the sequence "DTFGPWGGKR." Therefore, the hypothetical kinin precursor from Ix. scapularis, is predicted as a protein of 378 amino acid residues (**Figure 3A**).

Three partial kinin precursors were predicted from the American dog tick, Dermacentor variabilis, and from Amblyomma sculptum and Hyalomma dromedarii. The former two were predicted with a gap retained in both amino acid sequences (**Figure 3A**). Whereas the H. dromedarii precursor, while missing approximately 70 amino acid residues at the N-terminus, should contain all the kinin paracopies as inferred from the alignment of tick kinin precursors (**Supplementary Figure S3**). Similarly, the deduced amino acid sequence of D. variabilis kinin precursor appeared to miss the same 70 residues sequence at the N-terminus (**Supplementary Figure S3**). Among the seven deduced protein sequences, only the A. sculptum kinin precursor lacked the stop codon. Among the eight tick kinin precursors analyzed, SignalP 5.0 predicted the signal peptide from those of O. turicata, Ix. scapularis, Ix. ricinus, and A. sculptum (**Figure 3A**). For O. turicata, the signal peptide includes the first 28 amino acid residues, with a cleavage site between residues A and S (**Figure 3A**). For the latter three species, the signal peptide includes the first 30 amino acid residues, with the cleavage site between C and Q for Ixodes spp., and between A and Q for A. sculptum.

Putative enzyme cutting sites, characterized by contiguous basic residues (Veenstra, 2000), and those for kinin sequences were predicted in the translated precursor sequences (**Figure 3A**). The number of kinin paracopies was high, especially in hard ticks. In the curated full-length kinin precursor of four hard tick species, 17 paracopies of kinins were predicted, in average. The transcript of the only soft tick kinin gene analyzed (O. turicata) is predicted to encode 10 kinin paracopies.

Within the functional C-terminal pentapeptide (FX1X2WGamide) core of insect kinins, the first (F), fourth (W), and fifth (G) amino acid residues are highly conserved, and similar conservation was observed in tick kinins. However, the second variable position predominantly featured proline in all eight tick species (**Figure 3B**), and the first variable position showed lower conservation in all analyzed tick species. Asparagine (N) predominately occupied the X1 position in the soft tick O. turicata kinins, while in the Ixodes ticks (Prostriata), the X1 position was equally occupied by either N, G, or S. The amino acid residue in the X1 position was even less conserved (lower bits value) in ticks of the Metastriata group (**Figures 3B**, **4**–**8**).

#### Phylogenetic Analysis of Kinin Precursors

To establish how conserved the kinin precursors are between ticks and other insect blood feeders, a phylogenetic Bayesian analysis was performed with the putative kinin precursors from eight tick species and those known from five blood-feeding insect species (**Figure 4**). Bhatt et al. (2014) validated the in vivo activity of the R. prolixus kinin sequences used to construct this tree, and the three aedeskinins are active on the cognate receptor (Pietrantonio et al., 2005). The kinins from C. lectularius were detected in the CNS by mass spectrometry by Predel et al. (2017).

The number of kinin paracopies in different arthropod species varied dramatically. However, the selection pressure behind these putative insertions or deletions is unclear. The number of kinin copies was high in ticks and some hematophagous insects, but not in all (**Figure 4**). With the support of the phylogenetic unrooted tree, we found that the kinin precursors from ticks and insects were separated into two different groups. Similarly, within each branch, arthropods that are phylogenetically closer to each other clustered in the same group, indicating substantial conservation of kinin precursors across broad taxonomic groups. The only exception was that a soft tick, O. turicata, clustered with Ixodes ticks (hard ticks) but with a posterior node probability of 0.76. Insect kinin precursors, even those from species with several paracopies as in the case of ticks (i.e., up to 15 in R. prolixus), were more similar among themselves than to tick kinin precursors (**Supplementary Figure S4**). Thus, the number of kinin paracopies, does not appear to differentiate sequences of ticks from those of insects.

## Small Molecule Screening on Recombinant Tick Kinin Receptor

Toward the discovery of novel "small-molecule" ligands (Lipinski, 2016) for the target GPCR, we initiated a small-scale screening with the dual-addition assay we developed. Because

kinin receptors are invertebrate-specific GPCRs, there is no available commercial library of ligands from any ortholog mammalian GPCR (**Figure 1**). To explore the opportunity for discovery of antagonists, we selected a small-molecule library of known antagonists of those mammalian GPCRs that have the highest amino acid sequence identity to the tick kinin receptor. These are the neurokinin receptors (NK1-3 receptors), with approximately 36% amino acid sequence identity to the tick kinin receptor, as per Blast analyses in NCBI. The preliminary screening of fourteen small molecules was performed with an end-point calcium fluorescence assay. The screening of these small molecules was with 22 concentrations of antagonists prepared in 2% DMSO, performed on the BMLK3 cell line only. Four compounds, TKSM02, TKSM10, TKSM13 and TKSM14 showed some antagonistic activities, as they showed increasing values of fluorescence with decreasing concentrations (**Supplementary Table S3**, panel D, rows C, K, N, and O). It was noticed during the plate preparation that compounds TKSM04, TKSM05, TKSM08, TKSM09 and TKSM14 may have precipitated out when prepared in 2% DMSO at 1 mM. Indeed, fluorescence endpoint readings of some of them confirmed this suspicion due to randomness of their values. Therefore, a second small-molecule plate was made in 10% DMSO with ten selected compounds prepared in 10 serial concentrations (**Supplementary Table S4**, panel A). These small molecules included the four active small molecules from the first screen; those four more hydrophobic molecules that had precipitated; and the small molecules TKSM06 and TKSM07 that had showed erratic activity in the first screen (**Supplementary Table S3**, panel D, rows G and H). When TKSM02 and TKSM14 were

tested after being prepared in 10% DMSO, they showed dosedependent antagonistic activities that allowed the estimation of IC50s and confidence intervals (**Figures 5A,D**). TKSM02 acted as a full antagonist with IC50 = 45 µM (95% confidence interval of 31–61 µM) (**Figure 5A**). Three small molecules showed partial antagonism: TKSM14, with IC50 = 23 µM (95% confidence interval of 17–30 µM), only inhibited 60% of the agonist response at the highest concentrations tested of 35 and 50 µM (**Figure 5D**), and TKSM10 and TKSM13 did not have meaningful IC50 values because their 95% confidence intervals were too wide (**Figures 5B,C**). Nevertheless, TKSM10 exhibited antagonist activity at concentrations above 70 µM and inhibited 80% of agonist response when applied at 100 µM (**Figure 5B**). Although TKSM13 showed an overall weak antagonistic activity across concentrations, it acted as a potential antagonist (**Figure 5C**) as it inhibited approximately 50% of the agonist response at concentrations above 25 µM. The rest of the compounds re-tested (TKSM04-TKSM09) did not show any activity (**Supplementary Table S4**, panel D).

The small molecule library prepared for the second screening of potentially insoluble molecules (10% DMSO), was screened on both BMLK3 cells and "vector only" cells to test for the tick receptor specificity of the agonist responses observed after the first addition of small molecules, because the endogenous expression of NK receptors in CHO-K1 cells is assumed. Counter screening on vector only cells (**Supplementary Table S4**). confirmed that none of the agonistic responses observed after the first addition were specific for the tick kinin receptor because the vector only cells showed equivalent agonistic responses to these molecules as BMLK3 cells did (**Supplementary Table S4**,

FIGURE 5 | Four small molecules antagonized the tick kinin receptor activity. Only four molecules out of the 10 tested exhibited antagonistic activities. (A–D) Dose-responses of the tick kinin receptor (in the BMLK3 cell line) to small-molecule ligands of mammalian neurokinin receptors were measured in a calcium fluorescence assay. Ten dosages were applied for each compound, starting from 100 µM (except TKSM14, starting from 50 µM) and following a 1:1.4 dilution series. The antagonist activity of 10 selected small molecules was tested by first dispensing the blank buffer or small molecules at various concentration [Log (Molar) on the X-axis] from the library plate into the assay plate. This was followed by incubation with the cells for 5 min, after which the kinin agonist (1 µM FFFSWGa) of the tick kinin receptor was dispensed into all wells and incubated for another 5 min. The end-point relative fluorescence units (RFU) from each well was represented as the average RFU (left Y-axis) of two end-point reads obtained by inverting the orientation of the plate in a Varioskan LUXTM (Thermos Scientific, Waltham, MA, United States) plate reader. Additionally, the right Y-axis represents the percentage of RFU relative to the averaged responses from cells to the PC (blank buffer + agonist). Non-linear dose-response curves IC50s were defined as the concentration of antagonist that inhibits agonist response in the mid-range of its respective fitted dose-response curve and they are marked with a cross (×) on the curve. The IC50s and corresponding 95% confidence intervals of IC50S were calculated with "log(inhibitor) vs. response- Variable slope (four parameters)" in GraphPad 6.0. The IC50s shown here are not the concentrations that inhibit 50% of the agonist response.

panel B, compare row F, wells 2-12 to row F, wells 22 to 13). Therefore, only antagonistic activities remain to be demonstrated in tick bioassays.

#### Kinetic Responses to Selected Small Molecules and Peptidomimetics in a Dual-Addition Calcium Bioluminescence Assay

The primary screen using fluorescence provided only one endpoint reading after 5 min of incubation with cells. A kinetic calcium bioluminescence assay was subsequently performed with molecules identified in the first screen. This was to determine the kinetic responses during the first 30 s after the addition of compound of unknown activity (small molecule or peptidomimetic) and the response after the subsequent addition of known agonist (1 µM FFFSWGa) (**Figure 6**). In contrast to the results of the fluorescence assay, nearly no agonist responses were detected in the kinetic assay in the first 30 s after the addition of small molecules (**Figures 6A–E**, right panel, black bars). The statistical analyses of antagonist activities based on the responses after the agonist addition were calculated by one-way ANOVA followed by Tukey's multiple comparison test among all dosages. Consistent with the preliminary screen, TKSM02 was the most potent antagonist on the tick kinin receptor and inhibited 82.7 ± 1.8% of the FFFSWGa agonist response at 100 µM (**Figure 6A**; black curve in the left panel and in the histogram, last gray bar). The bioluminescence response of TKSM02 at 100 µM was significantly lower than the responses to the four lower concentrations tested from 100 nM–30 µM (histogram, four bars to the left), and significantly lower than the response to agonist only (Blank bar), as expected (p < 0.0001) (**Figure 6A**). A dose-dependent trend of antagonistic responses was observed for TKSM10 from 1 to 100 µM (**Figure 6B**, decreasing bar heights in histogram), with the lowest cell response being 84 ± 6% of the response to agonist only (16% inhibition). However, the

FIGURE 6 | Kinetic dose-responses of the BMLK3 cell line to five selective small molecules. Kinetic bioluminescence responses of tick kinin receptor recombinant cells to (A–D) four antagonist candidates (n = 3), and (E) TKSM03 [negative control (n = 1)] in the "dual-addition" calcium bioluminescence assay. All small molecules were tested at five different concentrations from 100 nM to 100 µM. Two additions were performed by the two injectors built-in the ClariostarTM plate reader (BMG LABTECH) as follows: the first addition of various concentrations of small molecules (5×) or corresponding blank buffer, was performed at 2 s after initiation of the recording. The response to the first addition was recorded for 30 s with 1 s intervals (first range). At 32 s, the second addition of the agonist of tick kinin receptor used in this study (1 µM FFFSWGa) was performed in the same well, and the cell response was recorded for another 30 s with 1 s intervals (second range). Curves on the left of each panel depict in different colors the kinetic response in bioluminescence units (BU) per second to each concentration (4–9 wells) from three independent assays (except TKSM03 n = 2 wells from one assay). The Y-axis represents the percentage BU of each concentration normalized to the maximal BU of the positive control (blank buffer + agonist, blue curve) (mean ± SEM). The histogram on the right of each panel shows sequentially the total BU responses of cells to the two additions, the first (black) and second (gray) range recordings, respectively. The total response in each range was expressed as the percentages of the average BU (per second) obtained in each of the two 30 s-ranges, relative to the average units to the PC in the second range (average BU of the blue curve at 35–65 s). In each independent assay, the total response to each concentration of each small molecule in the first addition or to the agonist in the second addition was calculated as the average of 2–3 pseudo-replicate wells (Y axis: mean ± SEM; n = 3 independent assays). None of the tested compounds showed agonist activity. Statistical differences in inhibitory activities between different concentrations of each compound were analyzed by one-way ANOVA (n = 3) followed by Tukey's multiple comparison test (p ≤ 0.05) with GraphPad 6.0 (GraphPad Software, La Jolla, CA, United States). Bars with different letter superscripts indicate significant differences.

increasing trend of inhibition with higher concentrations was not detected as statistically significant by ANOVA (p = 0.08). The ANOVA for TKSM13 detected significant variation in the response among concentrations (p = 0.02) and Tukey's test detected a significant decrease in the response to the agonist of 72 ± 19% at 100 µM with respect to the 133 ± 17% response to 10 µM concentration (p < 0.05) (**Figure 6C**). Unexpectedly, TKSM14 did not show any antagonistic activity in the calcium bioluminescence assay, which was inconsistent with the result of the primary screen in the fluorescence assay. We then asked whether increasing the preincubation time to 5 min in the bioluminescence assay (previously, 30 s), similar to the fluorescence assay, could reveal TKSM14 antagonistic activity. Indeed, when the preincubation with TKSM14 was 5 min, a dose-dependent antagonistic response was detected after agonist addition (**Figures 7A,B**). Tukey's test showed significant antagonistic activities of TKSM14 at both 50 µM (63 ± 8%) and 100 µM (55 ± 4%) which inhibited ∼ 40 and 45% of the agonist response (**Figure 7A**, orange and black curves on the left panel and last two bars on the histogram to the right). This activity matched the result observed in the first screen. This suggested that TKSM14 might have a lower binding affinity, reflected in the longer incubation time with the cells required to block the receptor.

Compound TKSM03 was chosen as a negative control for the bioluminescence assay with small molecules due to its lack of activity in the fluorescence screen. Its lack of activity was confirmed in the bioluminescence assay, as this compound did not decrease the agonist response even at the highest concentration tested of 100 µM (**Figure 6E**, histogram on right). The corresponding black curve on the left panel (**Figure 6E**) could be misleading at first sight, giving the impression that TKSM03 could be an antagonist, as the black curve appeared to be lower than other curves. However, it must be emphasized that although the maximal bioluminescence units per second at about 40 s is diminished with respect to the other concentrations, the area under the curve did not change with respect to that of the blank control (**Figure 6E**, right panel). This is why it is important to consider the total, integrated bioluminescence response and not only the peak response.

In addition, three peptidomimetic ligands of mammalian neurokinin receptors were tested using this approach. These ligands of NK receptors showed neither agonistic nor antagonistic activities on the tick kinin receptor within the tested concentration range (1 nM–10 µM) (**Figures 8A–C**) (ANOVA p > 0.1 for all three peptidomimetics). In **Figure 8B**, a slight agonistic activity of antagonist G at 1-, 3- and 10-µM can be detected, peaking at about 14 s; the same activity is reflected in the histogram to the right. However, this activity was only observed in the first independent replication and could not be seen in the two other independent replications. This agonist response was also not detected in the vector only cells (not shown). We do not know what could have caused this effect.

#### DISCUSSION

The insect kinins were named leucokinins after first being isolated from the roach Leucophaea maderae for their myotropic activity (Holman et al., 1986). The kinin neuropeptide signaling system is pleiotropic in insects, regulating physiological functions both at the central and peripheral levels. They regulate myotropic and diuretic activities, orchestrate innate behaviors during preecdysis and influence feeding behavior (Veenstra et al., 1997; Kim et al., 2006; Kersch and Pietrantonio, 2011; Schooley et al., 2012; Kwon et al., 2016; Pietrantonio et al., 2018). However, the kinin functions in ticks have remained elusive. In the tick R. microplus LKR was immunolocalized on the midgut outer surface, and LKR-silenced females displayed variations in gut discoloration, had a reduction in body weight of ∼30%, reduced weight of their egg masses, and experienced decreased egg hatching. Thus, LKR silencing in female ticks caused a reproductive fitness cost, perhaps related to defects in heme metabolism because some guts from silenced females were completely white (Brock et al., 2019). In agreement, the LKR transcript of the tick R. sanguineous was apparently expressed at higher levels in the gut and salivary glands than in other tissues, as inferred from RT-PCR analyses (Lees et al., 2010).

Most information on tick GPCRs was obtained from recombinant receptor systems (Yang et al., 2013, 2015; Gross et al., 2015; Gondalia et al., 2016; Kim et al., 2018). Transcripts for

(A) The kinetic responses of BMLK3 cells to the agonist (1 µM FFFSWGa) after being incubated with various concentrations of TKSM14 (1–100 µM) or blank solvent for 5 min. The response was recorded for 30 s at 1 s intervals by the ClariostarTM plate reader (BMG LABTECH). The kinetic response is represented as the percentage of the maximal bioluminescence units (BU) of the positive control (PC = blank buffer + agonist, blue curve) in each assay (Mean ± SEM, n = 9–10 wells from three independent assays). (B) The total cellular response in each well is expressed as the percentage of the average BU (per second) obtained in the 30 s relative to the average response units to the PC in 30 s. Three independent assays were performed. Responses from each assay were calculated as the average of individual responses from 3-4 pseudo-replicate wells (Y axis: mean ± SEM). Statistical differences in inhibitory activities between different treatment were analyzed by one-way ANOVA (n = 3) followed by Tukey's multiple comparison test (p ≤ 0.05) with GraphPad 6.0 (GraphPad Software, La Jolla, CA, United States). Bars with different letter superscripts indicate significant differences.

tick GPCRs are often expressed in vivo at very low levels, which complicates their physiological characterization. For example, results of qRT-PCR after gene silencing is often highly variable in diverse tick tissues due to their normal low expression levels (Brock et al., 2019). To address this challenge, additional future approaches to elucidate the physiological function of the tick kinin system through loss-of-function experiments include the knockdown of the endogenous kinin neuropeptide transcript and a pharmacological approach to block receptor function. With these approaches in mind, we cloned the kinin cDNA from R. microplus, and developed a dual addition assay to discover antagonists of the receptor to block the response to endogenous ligands and impair the normal physiology of ticks (**Figure 1**). To expand our current knowledge on the kinin system of the cattle fever tick, R. microplus, we used a forward pharmacological approach (**Figure 1**). Herein, we integrated the cloning of the cDNA encoding the tick endogenous kinins from the synganglion of female R. microplus and predicted kinin sequences from other Acari.

Kinins arose before the shared common ancestor of Mollusca, Insecta and Acari, and the lymnokinin receptor (leucokininlike) was functionally characterized in the pond snail, Lymnaea stagnalis (Cox et al., 1997; Grimmelikhuijzen and Hauser, 2012).

In the cattle fever tick, we identified a single cDNA that encodes 17 potential bioactive kinins, all different (**Table 1**). This is the first report for the cloning of a kinin-encoding cDNA from any tick species. Similar to R. microplus, the additional tick species analyzed also showed an expansion in the number of predicted kinins (**Figure 3**), underscoring the potential importance of the kinin system in ticks. Most of the R. microplus kinins featured the conserved C-terminal pentapeptide motif found in insects (FX1X2WGa). We predict the kinins reported in this study are active because they satisfy the minimal requirement for activity and we have experimental evidence of activity of similar kinin analogs and insect kinins on the R. microplus receptor expressed in CHO-K1 cells (Holmes et al., 2003; Taneja-Bageshwar et al., 2006, 2009; Xiong et al., 2019). However, the variable position X1 that in insect kinins is occupied by His, Asn, Ser or Tyr (Torfs et al., 1999), is different in some of the R. microplus kinins, featuring instead Gly, Ala, Thr, or Arg (Bold amino acids in **Table 1**). R. microplus retained the same variable residues as in the insect kinins in position X2 (Ser/Pro/Ala) (Torfs et al., 1999), with Pro being more frequent in tick kinins (**Table 1**). These seventeen predicted kinins await to be synthesized and tested in the receptor functional assay to verify their biological activities.

The genome analysis of Ix. scapularis identified a putative kinin-encoding gene and four genes encoding kinin receptors (Gulia-Nuss et al., 2016). In contrast, BLAST of R. microplus LKR against the updated genome assembly (Rm2.0; NCBI) of this species identified only one receptor and did not reveal kinin receptor paralogs. The same sequence of 1,481 bp was identified in two contigs (sequences ID LYUQ01138740.1 and LYUQ01085891.1), and it matched the 30 -half of the R. microplus LKR cDNA with 99.9% identity. Therefore, it appears that in R. microplus there is only one kinin receptor gene, or perhaps an identical duplicate. In the genomes of six non-tick chelicerate species (spiders, the house dust mite and a scorpion), five species have one gene for both the kinin peptide and receptor, except that the African social velvet spider (Stegodyphus mimosarum) has 3 LKR paralogs (Veenstra, 2016). The transcript expression level of kinin and its receptor(s) seems to be generally low in ticks because transcripts were not reported in transcriptomes from synganglia of females of R. microplus from Texas, Ix. scapularis

FIGURE 8 | Kinetic dose-responses of the BMLK3 cell to three peptidomimetic ligands of mammalian neurokinin receptors in the "dual-addition" calcium bioluminescence assay. Kinetic bioluminescence responses of tick kinin receptor recombinant cells to (A–C) three peptidomimetics (1 nM–10 µM, n = 3). Two additions were performed by the two injectors built-in the ClariostarTM plate reader (BMG LABTECH) as follows: the first addition of various concentrations of peptidomimetics (10×), or corresponding blank buffer, was performed at 2 s after initiation of the recording. The response to the first addition was recorded for 30 s with 1 s intervals (first range). At 32 s, the second addition of the agonist of tick kinin receptor (1 µM FFFSWGa) was performed in the same well, and the cellular response was recorded for another 30 s with 1 s intervals (second range). Curves on the left of each panel depict in different colors the kinetic response in bioluminescence units (BU) per second to each concentration (8–9 wells from three independent assays). The Y-axis represents the percentage BU of each concentration normalized to the maximal BU of the positive control (blank buffer + agonist, blue curve) (mean ± SEM). The histogram on the right of each panel shows sequentially the total BU responses of cells to the two additions, the first and second range recordings, respectively. The total responses in each range was expressed as the percentages of the average BU (per second) obtained in each of the two 30 s-ranges, relative to the average units to the PC in the second range (average BU of the blue curve at 35–65 s). In each independent assay, the total response to each concentration of each peptidomimetic in the first addition or to the agonist in the second addition was calculated as the average of 2–3 pseudo-replicate wells (Y axis: mean ± SEM, n = 3 independent assays). None of the tested compounds showed agonist activity (for B panel, first range, see explanation in text). Statistical differences in inhibitory activities between different concentrations of each compound were analyzed by one-way ANOVA (n = 3) but no statistical differences were detected.

and O. turicata (Egekwu et al., 2014, 2016; Guerrero et al., 2016). As the kinin receptor transcript level is low in ticks (as well as the level of receptor protein (Brock et al., 2019), it is tempting to speculate that kinin signaling may be regulated by changes in the expression and release of their expanded kinin peptide repertoire. A detailed temporal quantitative analysis of receptor and kinin precursor mRNA transcripts is still lacking. It is also yet unknown whether the high copy number of the tick kinins will result in increased peptide expression (i.e., concentration) or in sustained, constitutive expression. It is worth noting, however, that kinins are active at nanomolar levels (low concentrations) (Holmes et al., 2003; Xiong et al., 2019).

Kinins arose before the shared common ancestor of Mollusca, Insecta and Acari, and the lymnokinin receptor (leucokininlike) was functionally characterized in the pond snail, Lymnaea stagnalis (Cox et al., 1997; Grimmelikhuijzen and Hauser, 2012). Among ticks, the Argasidae are considered a basal group to the Ixodidae (Geraci et al., 2007). Arthropod molecular phylogenetic studies have suggested Prostriata are more ancient than Metastriata (Jeyaprakash and Hoy, 2009). Although the pentapeptide core of tick kinins is largely conserved, it is noteworthy that more ancient species show higher conservation on the X1 position of this core, with asparagine (N), glycine (G) or serine (S) being more frequent. Further, although the Prostriata and Metastriata have a similar number of kinin paracopies (∼17), the tick kinins of the Metastriata showed more frequent amino acid substitutions than those of the Prostriata. Specifically, Ix. scapularis and Ix. ricinus (Prostriata) have multiple iterations of the same kinin sequence (DTFGPWGa) in the kinin precursor, whereas ticks from the Metastriata showed no repetition in the sequence of kinin paracopies (**Table 1** and **Figure 3A**).

The unrooted phylogenetic tree revealed kinin sequence differences between ticks and insect blood feeders (**Figure 4**). Although the number of paracopies does not appear to differentiate kinin sequences of ticks from those of insects, we have experimental evidence that kinin receptors from insects and ticks have different ligand structure-activity relationships, as manifested in the differential potency of the same set of kinin analogs (Taneja-Bageshwar et al., 2006; Xiong et al., 2019). Therefore, differences in the sequences of the mature kinin peptides between ticks and insects further support this differential activation. Additionally, we reiterate that kinins have pleiotropic functions in insects and therefore, it is possible that they are similarly pleiotropic in ticks. We speculate this pleiotropism could be achieved in ticks either by diversification of receptors or ligands. In Ixodes (Prostriata), despite low variation in the kinin sequences, up to four kinin receptors may be present (Gulia-Nuss et al., 2016). In contrast, Rhipicephalus microplus appears to have only one receptor but exhibits high variation in the sequences of the kinin ligands (**Table 1**). As a mechanism for pleiotropism, there is evidence that in the mosquito renal organ different kinin analogs promote differential downstream tissue responses, either fluid secretion or changes membrane voltage (Schepel et al., 2010), apparently through the same receptor because only one kinin receptor has been identified in this tissue (Lu et al., 2011a).

To advance the discovery of novel ligands toward application, we tested potential synthetic ligands (small molecules and peptidomimetics) of mammalian neurokinin receptors on the recombinant tick receptor (**Figure 1**). Our previous success with a kinin mimetic, named 1728, that elicits aversive behavior in the mosquito and inhibits the sugar neuron provided the proof of principle to pursue this approach (Kwon et al., 2016). The NK receptors are considered "pseudo-orthologs" of the arthropod kinin receptor because while tachykinins exist in both mammals and arthropods, the kinin system is arthropod-specific (Nachman et al., 1999). To accelerate discovery and reduce the cost of screening a commercial GPCR-specific small molecule library and selected peptidomimetics, we developed a dual-addition functional assay using two reporters for calcium, in fluorescence and bioluminescence modes, respectively. A comparable pipeline was developed by Ejendal et al. (2012) for validating the dopamine receptor of Ix. scapularis as acaricide target, except their functional assay detects cAMP as the secondary messenger. Using a reverse pharmacological approach, Duvall et al. (2019) recently discovered a novel small molecule ligand of the mosquito neuropeptide receptor, NPY receptor, which discourages the biting behavior of mosquito (Duvall et al., 2019).

In this study, through the primary screening of a small molecule library of NK receptor antagonists, and their secondary validation by a bioluminescence assay, a small molecule, named TKSM02, was identified as a "full antagonist" of the tick kinin receptor with an IC50 of 45 µM (**Figure 5A**), as it fully blocked receptor activity at 100 µM. Although of weak potency, we believe this is the first antagonist reported for a kinin receptor of any arthropod species. However, it should be noted that we used a generic kinin receptor agonist, FFFSWGa, in the antagonist screen. Therefore, the antagonists identified with this screen may not similarly antagonize endogenous tick kinins on the cognate receptor. Overall, ligands of NK receptors displayed more than a 10,000-fold reduced potency on the tick kinin receptor. The quantitative functional data reported here will provide valuable information to constrain the ligand-receptor interaction surface in computational modeling.

Agonists and antagonist peptidomimetics of NK receptors were tested: hemokinin 1 (human) is an agonist of the NK1 receptor with active concentration at the nanomolar level, antagonist G is a broad antagonist on NK receptors, and spantide I is a selective potent antagonist of the NK1 receptor (**Supplementary Table S2**). Our results provided additional functional evidence that the tick kinin receptor is pharmacologically different from the insect tachykinin receptor as none of the peptidomimetic ligands of NK receptors showed any activity on the tick kinin receptor. However, spantide I antagonizes insect tachykinins in recombinant tachykinin receptors of the stable fly and fruit fly at 1 and 50 µM, respectively (Torfs et al., 2002; Poels et al., 2009).

In summary, we report for the first time putative tick kinin sequences. Further, our small molecule screening results confirmed in one direction the target selectivity of the kinin receptor for arthropod vector control, as only one of the small molecule NK antagonists was active. The dual addition assay we developed is amenable and ready for the high throughput screening of random small molecule libraries on the tick kinin receptor or other tick neuropeptide receptors signaling through the calcium cascade. While receptor "hits" of random molecules are expected, the next frontier is to develop suitable tick bioassays to elucidate the physiological functions of kinins that could be impaired by these synthetic ligands.

#### DATA AVAILABILITY

fphys-10-01008 August 5, 2019 Time: 14:47 # 18

The DNA sequence of the PCR product verifying the R. microplus kinin gene sequence with mRNA sequence of the kinin precursor were uploaded in NCBI with GenBank accession number (MK875970). The raw data of the fluorescence dualaddition assay are under **Supplementary Tables S3**, **S4**. The raw data of the calcium bioluminescence assay can be made available upon request.

#### AUTHOR CONTRIBUTIONS

PP and CX designed the study, developed the fluorescence assay, performed the data analyses and wrote the manuscript. CX was responsible for performing the molecular cloning, cell culture and calcium bioluminescence assay. DB advised on composition of small molecule library. CX and DB performed the 384-wellplate calcium fluorescence assay. All authors edited and approved the final manuscript.

#### REFERENCES

#### FUNDING

This work was supported by NIFA-AFRI Animal Health and Well-Being Award 2016-67015-24918 to Patricia V. Pietrantonio and Kevin Temeyer and from competitive funds from the Texas A&M AgriLife Research Insect Vector Diseases Grant Program (FY19-21) to PP. NIFA also supports PP program through Hatch project TX (TEX0-2-9206), accession 1002279 (Y 2018-2023).

#### ACKNOWLEDGMENTS

We thank Distinguished Professor James C. Sacchettini (Deptartment of Biochemistry and Biophysics, Texas A&M University) for the support of his staff and for the use of facilities for high throughput screening. Dr. Wen Dong is acknowledged for training CX in the use of the Cybio R system. We thank the A.W.E.S.O.M.E. faculty group in the College of Agriculture and Life Sciences at Texas A&M University for assistance with editing the manuscript. We acknowledge Professor James Woolley for his advice on the construction of the unrooted phylogenetic tree. We also thank both reviewers for their input to improving this manuscript.

#### SUPPLEMENTARY MATERIAL

The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fphys. 2019.01008/full#supplementary-material

library screening and comparative pharmacological analyses. Insect Biochem. Molec. 42, 846–853. doi: 10.1016/j.ibmb.2012.07.011

choice across a large model space. Syst. Biol. 61, 539–542. doi: 10.1093/sysbio/ sys029

**Conflict of Interest Statement:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2019 Xiong, Baker and Pietrantonio. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Experimental *Ixodes ricinus*-Sheep Cycle of *Anaplasma phagocytophilum* NV2Os Propagated in Tick Cell Cultures

Consuelo Almazán1 \*, Lisa Fourniol 1 , Clotilde Rouxel 1 , Pilar Alberdi 2 , Christelle Gandoin1 , Anne-Claire Lagrée1 , Henri-Jean Boulouis 1 , José de la Fuente2,3 and Sarah I. Bonnet 1 \*

1 UMR BIPAR, INRAE, Ecole Nationale Vétérinaire d'Alfort, ANSES, Université Paris-Est, Maisons-Alfort, France, 2 SaBio, Instituto de Investigación en Recursos Cinegéticos IREC (CSIC-UCLM-JCCM), Ciudad Real, Spain, 3 Department of Veterinary Pathobiology, Center for Veterinary Health Sciences, Oklahoma State University, Stillwater, OK, United States

#### *Edited by:*

Armanda Bastos, University of Pretoria, South Africa

#### *Reviewed by:*

Eliane Esteves, University of São Paulo, Brazil Itabajara Silva Vaz Jr., Federal University of Rio Grande do Sul, Brazil

#### *\*Correspondence:*

Consuelo Almazán<EMAIL_ADDRESS>Sarah I. Bonnet<EMAIL_ADDRESS>
#### *Specialty section:*

This article was submitted to Veterinary Infectious Diseases, a section of the journal Frontiers in Veterinary Science

*Received:* 05 September 2019 *Accepted:* 15 January 2020 *Published:* 06 February 2020

#### *Citation:*

Almazán C, Fourniol L, Rouxel C, Alberdi P, Gandoin C, Lagrée A-C, Boulouis H-J, de la Fuente J and Bonnet SI (2020) Experimental Ixodes ricinus-Sheep Cycle of Anaplasma phagocytophilum NV2Os Propagated in Tick Cell Cultures. Front. Vet. Sci. 7:40. doi: 10.3389/fvets.2020.00040 The causative agent of tick-borne fever and human granulocytic anaplasmosis, Anaplasma phagocytophilum, is transmitted by Ixodes ricinus, and is currently considered an emerging disease throughout Europe. In this study, we established a model of A. phagocytophilum sheep infection and I. ricinus transmission using the European Norway variant 2 ovine strain (NV2Os) propagated in both IDE8 and ISE6 tick cells. Two sheep were inoculated with IDE8 tick cells infected with NV2Os. Both sheep developed A. phagocytophilum infection as determined by qPCR and PCR, the presence of fever 4 days post inoculation (dpi), the observation of morulae in granulocytes at 6 dpi, and the detection of A. phagocytophilum antibodies at 14 dpi. A. phagocytophilum was detected by PCR in skin, lung, small intestine, liver, spleen, uterus, bone marrow, and mesenteric lymph node from necropsies performed at 14 and 15 dpi. One sheep was infested during the acute phase of infection with I. ricinus nymphs from a pathogen-free colony. After molting, A. phagocytophilum transstadial transmission in ticks was validated with qPCR positive bacterial detection in 80% of salivary glands and 90% of midguts from female adults. Infected sheep blood collected at 14 dpi was demonstrated to be able to infect ISE6 tick cells, thus enabling the infection of two additional naive sheep, which then went on to develop similar clinical signs to the sheep infected previously. One of the sheep remained persistently infected until 115 dpi when it was euthanized, and transmitted bacteria to 70 and 2.7% of nymphs engorged as larvae during the acute and persistent infection stages, respectively. We then demonstrated that these infected nymphs were able to transmit the bacteria to one of two other naive infested sheep. As expected, when I. ricinus females were engorged during the acute phase of infection, no A. phagocytophilum transovarial transmission was detected. The development of this new experimental model will facilitate future research on this tick-borne bacterium of increasing importance, and enable the evaluation of any new tick/transmission control strategies.

Keywords: *Anaplasma phagocytophilum*, *Ixodes ricinus*, sheep, NV2Os, tick cell cultures

## INTRODUCTION

Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), is an obligate intracellular Gram negative bacterium mainly transmitted by I. scapularis and I. pacificus in the United States, I. persulcatus in Asia, and I. ricinus in Europe (1). A. phagocytophilum is the causative agent of human granulocytic anaplasmosis (HGA) and tick-borne fever (TBF), affecting both humans and a variety of domestic and wild animal species (2–4). Tick-borne fever was first identified in sheep from Scotland in 1932 (5), and the discovery of the etiological disease agent followed in 1940 (6). Throughout Europe, sheep are exposed to A. phagocytophilum (7)—with seroprevalence as high as 80% in sheep grazing in tick-infested Norwegian pastures—resulting in considerable economic and animal welfare consequences (8, 9).

The wide range of potential hosts, as well as incidence and severity of the disease in a particular host appear to vary according to geographical region (1). A. phagocytophilum infects granulocytes, mostly neutrophils, and exists as macrocolonies or morulae within intracytoplasmic vacuole (10, 11). Approximatively within a week of exposure to an infectious tick bite, TBF disease becomes clinically evident and is characterized by fever, leukopenia, marked neutropenia, and thrombocytopenia (12). In sheep, A. phagocytophilum infection causes a fever lasting from 1 to 2 weeks, and which may vary according to animal age, the A. phagocytophilum variant, the host breed and its immunological status (13). Anorexia, depression (14), reduced weight gain (15), as well as abortions (16) have also been reported. In addition, because of the severe hematological and immune disorders associated with A. phagocytophilum infection, animals are more susceptible to secondary infections, including tick pyemia, caused by Staphylococcus spp. (14).

A. phagocytophilum has a large number of genetic variants which vary in virulence and clinical manifestation, and which can be differentiated by sequencing 16S rRNA (9) or mps4 genes (17, 18). Four variants that differ in pathogenicity and immunogenicity were identified as circulating in sheep flocks from Norway (9). Among them, A. phagocytophilum variant 1 (var1) is believed to be associated with the majority of fatal TBF cases in sheep (19). In contrast, A. phagocytophilum variant 2 (var2), which is also frequently found circulating in naturallyinfected sheep, produces a less severe clinical manifestation with shorter periods of fever and bacteremia, and a less severe neutropenia (20–22). Indeed, as it produces mild TBF in sheep, A. phagocytophilum var2 could be a useful model for studying bacterial infection processes in sheep, tick transmission modalities, and could also be used in vaccine trials.

Tick cell lines are well-established systems in which tick-borne pathogens—including Anaplasma species—can be propagated (23). Both HGA and equine granulocytic anaplasmosis variants were initially cultivated in the human promyelocytic cell line HL-60 (24), and then in IDE8 and ISE6 tick cell lines derived from I. scapularis embryos (25, 26). TBF variants were successfully cultivated in these tick cell lines, including variants isolated from sheep, such as the Old Sourhope strain, which, after three culture passages, was shown to be infectious in susceptible sheep (27). The A. phagocytophilum Norway var2 ovine strain (NV2Os), isolated from Norwegian sheep (9), was also successfully propagated in IDE8 embryonic tick cells (28), but, to the best of our knowledge, sheep infection from this cell culture has never been experimentally achieved to date.

The present study aims to investigate whether the A. phagocytophilum NV2Os propagated in both IDE8 and ISE6 tick cells can be used in the laboratory to experimentally recreate the entire bacterial transmission cycle from sheep to ticks and from ticks to sheep, thus creating an experimental model in which both tick-host-pathogen interactions, as well as novel tick/transmission control strategies, such as anti-tick or anti-transmission vaccines, can be studied. We report that the development of this model enabled successful sheep infection, the production of infected I. ricinus from both acute and persistently A. phagocytophilum-infected sheep, as well as the re-transmission of the bacteria to naïve sheep by these ticks.

## MATERIALS AND METHODS

#### Experimental Design

The experimental design of the study is presented in **Figure 1**. Two 9-month-old Romane breed female sheep (identification numbers 128 and 320) were inoculated with IDE8 tick cells infected with A. phagocytophilum NV2Os into the jugular vein, and euthanized at 14 and 15 days post inoculation (dpi), due to clinical signs of distress. During the acute phase of A. phagocytophilum infection at 6 dpi, sheep 320 was infested with I. ricinus nymphs. In addition, infected blood obtained from sheep 320 was used to assess whether NV2Os could be propagated in ISE6 tick cells. These A. phagocytophilum-infected ISE6 tick cells were then intrajugularly inoculated into two 11-month-old Romane breed female sheep (identification numbers 381 and 648). Unexpectedly, sheep 648 died at 12 dpi, and no necropsy could be performed. Sheep 381 however, recovered after the acute phase and remained without signs of infection until 115 dpi, when it was euthanized. A. phagocytophilum tick transmission was evaluated by infesting both sheep 381 and 648 during the acute phase of infection at 7 and 6 dpi, respectively, as well as sheep 381 during the persistent phase of infection at 108 dpi with I. ricinus larvae, and the infection assessed after molting into nymphal stages. The infectious status of nymphs molted from larvae fed on infected sheep 381 and 648 was assessed by feeding them on two naive PreAlps breed sheep (identification numbers 572 and 615). For sheep 128 and 320, blood samples were obtained every day from day 0 to 14 and 15 dpi, respectively. Sheep 648 was bled every day from day 0 until 12 dpi, when it died. Sheep 381 was bled every day from day 0 to 15 dpi, and then every 15 days until 115 dpi. Sheep infected with I. ricinus nymphs (572 and 615) were bled every 5 days from day 0 to day 25 post tick infestation.

#### Culture of *A. phagocytophilum* NV2Os in IDE8 and ISE6 Tick Cells

IDE8 and ISE6 embryonic tick cell cultures were maintained according to Munderloh and Kurtti (29) and Munderloh et

al. (30), respectively. Healthy tick cells from both cell lines were propagated in L-15B medium, whereas infected cells were cultured in L-15B supplemented with 0.1% NaHCO3 and 10 mM HEPES and the pH was adjusted to 7.5. Both uninfected and infected IDE8 and ISE6 cells were maintained at 31 and 34◦C, respectively. The A. phagocytophilum NV2Os was propagated in IDE8 tick cells (approximate passage of 110) as described by Alberdi et al. (28). A. phagocytophilum infection was propagated by transferring 1/10th of an infected IDE8 cell culture to a new flask of healthy cells, every 4 days. To determine the level of infection, 60 µl of RPMI medium with suspended cells were centrifuged on slides with a cytocentrifuge Shandon Cytospin (Thermo Fisher Scientific, Sweden) for 5 min at 1,000 rpm, stained using the Hemacolor R staining kit (Merck, Darmstadt, Germany) and observed under a light microscope.

Blood from A. phagocytophilum-infected sheep 320 collected at 14 dpi was used to infect ISE6 tick cells. A 500 µl drop of blood was added to two 25 cm² culture flasks of ISE6 cells (approximate passage of 90). Cytocentrifuged slides were stained twice a week using Hemacolor R kit (Merck Millipore, Darmstadt, Germany) in order to determine the level of infection. Once infection reached 70%, cells (∼1 × 107 infected cells) were collected and centrifuged for 5 min at 200 × g, then resuspended in 2 ml of sterile RPMI to prepare the inoculum (31).

#### Sheep Inoculation

Four 9-month-old Romane breed female sheep, originally from the INRA Experimental Animal Center at Bressonvilliers, France, and reared in a secure sheepfold at the Biomedical Research Center (CRBM) facilities, National School of Veterinary Medicine of Alfort (ENVA), were used. First, two sheep identified with numbers 128 and 320 (**Figure 1**) were inoculated with 1 ml of medium containing 1 × 107 A. phagocytophilum-infected IDE8 cells with a 70% infection level into the jugular vein with a 6 cc syringe and a 20-gauge 1" needle (Terumo) after skin disinfection. Two months later, the two other sheep, with identification numbers 381 and 648 (**Figure 1**) were inoculated with 1 ml of medium containing 5 × 106 ISE6 cells infected with A. phagocytophilum obtained from sheep 320 and with a 70% infection level.

#### Detection of *A. phagocytophilum* in Sheep's Blood by PCR and Determination of Infection Levels by Quantitative PCR (qPCR)

PCR detection of A. phagocytophilum msp4 was performed using DNA obtained from blood collected daily, from day 0 to 14 or 15 dpi in sheep inoculated with IDE8 infected cells (sheep 320 and 128, respectively), and from day 0 to 12 or 15 dpi in sheep inoculated with ISE6 infected cells (sheep 648 and 381, respectively). For sheep infected by I. ricinus nymphs (572, 615), PCR detection was performed every 5 days from 0 to 25 days post tick infestation and until euthanasia. Blood was collected from the jugular vein, and drawn into 10 ml EDTA vacutainer tubes. The PCR protocol described by Kocan et al. was followed (32). Briefly, DNA was extracted using a NucleoSpin Blood kit (Macherey-Nagel, Germany) from 200 µl of blood. PCR reactions were then performed using the oligonucleotide primers msp4- F (5′ -CCTTGGCTGCAGCACCACCTG-3′ ), and msp4-R (5′ - TGCTGTGGGTCGTGACGCG-3′ ), in 20 µl of final volume using the Takara Ex Taq system (Bio Europe, France). For positive and negative controls, DNA from A. phagocytophiluminfected ISE6 tick cells and nuclease free water were used, respectively. PCR products were visualized by 2% agarose gel electrophoresis. PCR products were purified using the PCR Clean-Up kit (Macherey Nagel, Germany), and were sequenced by the Eurofins sequencing service (France). The obtained sequences were submitted to the BLAST (basic local alignment search tool) platform to search for sequences with homology to A. phagocytophilum NV2Os.

A. phagocytophilum infection levels in sheep were determined by qPCR targeting the msp4 gene using DNA obtained from blood collected every 3 days from sheep 320 and 128, starting at day 0 until euthanasia (14 and 15 dpi, respectively), and from sheep 648 and 381 until 12 and 15 dpi respectively. For sheep 381, qPCR was performed every 15 days until 115 dpi. Samples were processed according to the protocol described by Reppert et al. (31). DNA concentration was evaluated with a NanoDropTM 2000 (Thermo Scientific, USA) and 20 ng of DNA was then mixed in a 20 µl reaction containing the primers qmsp4F (5-ATGAATTACAGAGAATTGCTTGTAGG-3), and qmsp4R (5-TTAATTGAAAGCAAATCTTGCTCCTATG-3) using the SsoAdvancedTM SYBR R Green Supermix (Bio-Rad, Hercules, CA, USA). Reactions were performed in a LightCycler R 480 (Roche Life Science, Indianapolis, IN, USA). The Ovis aries aldolase B gene (ALDOB) was used for normalization, with the primers Oa-AldBF (5′ -CCCATCTTGCTATCCAGGAA-3 ′ ) and Oa-AldBR (5′ -TACAGCAGCCAGGACCTTCT-3′ ). The same mix without DNA, or DNA from infected ISE6 cell culture were used as negative and positive controls, respectively. Triplicate values from each sample were normalized by calculating the ratio of A. phagocytophilum msp4 DNA to the averaged ALDOB gene. The standard errors of the averaged normalized values of the mean were determined and values analyzed by the Student's t-test (p = 0.05).

#### Detection of *A. phagocytophilum* Intracellular Inclusions in Blood Smears

Blood smears were performed on blood samples collected daily in EDTA tubes, starting at day 0 until 14 dpi for sheep 320, 15 dpi for sheep 128 and 381, and 12 dpi for sheep 648. Blood smears were stained using the Hemacolor R staining kit (Merck, Darmstadt, Germany). Slides were observed under a light microscope and 100 white cells per slide were examined to determine the percentage of infected neutrophils. When basophilic inclusions consistent with A. phagocytophilum organisms were found, slides were then examined under an Imager 21 Zeiss microscope adapted to an AxioCam HRC Zeiss camera to record images.

#### Gross Lesions and Detection of *A. phagocytophilum* in Sheep Tissues

In order to detect gross lesions, necropsies were performed immediately after euthanasia, which was carried out via intravenous injection of 18% sodium pentobarbital. Euthanasia occurred at 14 and 15 dpi for sheep 320 and 128 during the acute phase of the infection, and at day 115 for sheep 381 during the persistent phase of the infection. For PCR detection of the A. phagocytophilum msp4 gene, ∼1 cm2 of skin from the tick infestation area, and samples from sheep lung, myocardium, liver, spleen, stomach, small intestine, lymph nodes, brain, uterus, ovary, cerebellum, bone marrow, kidney, and gallbladder were obtained, deposited into sterile tubes and stored at −80◦C until later use. DNA was extracted using a NucleoSpin R tissue kit (Macherey-Nagel, Germany) from 25 mg of each tissue. DNA concentration was evaluated with a NanoDropTM 2000 (Thermo Scientific, USA) and PCR reactions were performed using 20 ng of DNA in a final volume of 20 µl as described for blood in section Detection of A. phagocytophilum in Sheep's Blood by PCR and Determination of Infection Levels by Quantitative PCR (qPCR).

#### Indirect Immunofluorescence Antibody Assay (IFA)

The detection of anti-A. phagocytophilum antibodies was performed using the indirect immunofluorescence antibody assay (IFA) on serum samples obtained at day 0 (for negative controls), and at 14 dpi and 15 dpi from sheep 320 and 128, respectively; and every 15 days until 115 dpi for sheep 381. Blood samples were collected from the jugular vein, and serum obtained by centrifugation. Serial dilutions of serum starting from 1:50 were analyzed using the Mega-Screen Fluo A. phagocytophilum-coated slides (MegaCor, Hoerbranz, Austria), following manufacturer's instructions. Briefly, the A. phagocytophilum-coated slides were incubated with serial dilutions of serum for 30 min at 37◦C. Slides were rinsed with PBS and incubated with 25 µl of 1:50 fluorescein isothiocyanateconjugated rabbit anti-sheep IgG (H + L) (Jackson Immuno Research, Cambridge, UK) in Evans blue solution (Biomerieux, Hampshire, UK) for counterstaining. After incubation for 30 min at 37◦C, slides were rinsed and mounting medium (Bio-Rad, Hercules, CA, USA) was added. Finally, slides were analyzed with a fluorescent microscope. A titer of 1.69 (Log10 reciprocal of 1:50) or higher was considered positive (8).

#### Tick Infestation

I. ricinus ticks originally collected from the Sénart Forest, France (coordinates 48◦ 40′ 00′′N 2◦ 29′ 00′′E), and maintained as a pathogen-free colony reared at 22◦C with 95% relative humidity and a 12-h light/dark cycle as previously described (33), were used for sheep infestation. For feeding, ticks were enclosed into stockinet cells attached to the back of each sheep, following a protocol adapted from cattle and rabbits (34, 35). Nine hundred nymphs of I. ricinus were engorged on sheep 320 when A. phagocytophilum was detected by PCR, during the highest fever spike (6 dpi) (**Figure 1**). Engorged nymphs were collected after 6 days of feeding. Collected engorged ticks were cleaned and reared until they molted into adults. The transmission of A. phagocytophilum to I. ricinus ticks during acute infection of sheep was tested by infesting 950 naive nymphs on sheep 381 and 2,000 naive larvae on sheep 381 and 648 at 7 and 6 dpi, respectively (**Figure 1**). Engorged larvae were collected after 3–5 days of feeding and then incubated in order to test for A. phagocytophilum presence in both engorged ticks and recently molted nymphs. Transovarial transmission of A. phagocytophilum was tested by infesting 15 adult couples on sheep 381 at 7 dpi. Engorged females were collected after 9 days of feeding and reared until oviposition. The transmission of A. phagocytophilum to I. ricinus ticks during persistent infection of sheep was tested by infesting 4,000 naive larvae on sheep 381 at 108 dpi (**Figure 1**). Finally, in order to test if nymphs molted from larvae fed on A. phagocytophilum-infected sheep 381 and 648 were able to transmit the bacteria to naive sheep, two 8-monthold male PreAlps breed sheep, identified as 572 and 615, were infested with 45 nymphs each (**Figure 1**).

## Detection of *A. phagocytophilum* in Ticks

A. phagocytophilum detection by qPCR was performed using DNA obtained from salivary glands and midguts from adult ticks engorged as nymphs on infected sheep 320, whole nymphs engorged as larvae on sheep 381 and 648, as well as egg masses obtained from females engorged on sheep 381 (**Figure 1**). DNA was extracted from each individual tick with the NucleoSpin tissue kit (Macherey Nagel, Germany), and 20 ng of DNA was mixed in a 20 µl reaction containing the primers msp2-F (5- ATGGAAGGTAGTGTTGGTTATGGTATT-3), and msp2-R (5- TTGGTCTTGAAGCGCTCGTA), adapted from the protocol described by Courtney et al. (36), using the SsoAdvancedTM SYBR R Green Supermix (Bio-Rad, Hercules, CA, USA). Reactions were performed in triplicate in a LightCycler R 480 (Roche Life Science, Indianapolis, IN, USA). Normalization was performed using the tick ribosomal protein S4 gene (rps4) with primers rps4F 5- GGTGAAGAAGATTGTCAAGCAGAG-3 and rps4R 5- TGAAGCCAGCAGGGTAGTTTG-3 (37).

## RESULTS

### *A. phagocytophilum* NV2Os Propagated in Both IDE8 and ISE6 Tick Cells Can Infect Sheep

The two sheep (128, 320) inoculated with the A. phagocytophilum NV2Os propagated in IDE8 tick cells developed an infection as demonstrated by clinical signs, observation of morulae inclusions in neutrophils, positive PCR and qPCR results, anti-A. phagocytophilum antibody detection, as well as the presence of lesions detected during necropsies after euthanasia. The first sign of infection was increased temperature by the 3rd dpi with peaks of 41.5◦C at 5 and 6 dpi in sheep 128 and 320, respectively (**Figure 2A**). After 7 dpi, a decrease in appetite, and the presence of nasal discharge and conjunctivitis were observed in both animals. Sheep 320 maintained a temperature over 40◦C until 13 dpi, at which point nasal discharge and lacrimal secretions increased, it then became lethargic with tachypnea, prostration, and stopped drinking water. Due to animal welfare regulations and ethics, euthanasia was performed at 14 and 15 dpi for sheep 320 and 128, respectively. A. phagocytophilum msp4 PCR was positive from 5 dpi until 12 dpi in both sheep (**Figure 2B**). For both sheep, qPCR-determined A. phagocytophilum infection levels increased after 3 dpi, showed the highest fold change at 6 dpi, and then decreased drastically at 9 dpi. However, A. phagocytophilum DNA remained at detectable levels until 14 dpi in sheep 128 (**Figure 3A**). The statistical analysis of the averaged normalized values of the mean indicated that the fold change was

FIGURE 2 | A. phagocytophilum infection in sheep experimentally infected with NV2O-infected IDE8 tick cells. (A) Temperature (◦C) recorded daily from day 0 post-infection to the day of euthanasia for sheep 128 and 320; (B) PCR detection of A. phagocytophilum msp4 gene in sheep 320 blood samples from day 0 to day 12 post-infection, MM, molecular marker; +C, positive control; –C, negative control; (C) Lung necropsy of sheep 320 14 days post infection. Arrows indicate patches of red coloration contrasting with the pink normal color.

significant (p = 0.05) from 3 dpi until 14 dpi. A. phagocytophilum morulae were found in neutrophils from both infected sheep at 6 and 7 dpi, whereas no other blood infection was detected (**Figure 4**). The highest cellular infection level was observed at 8 dpi with 47 and 60% of neutrophils infected in sheep 128 and 320, respectively (**Figure 4**). Few morulae were seen at 10 dpi and no further intracellular inclusions were found after 12 dpi.

The main gross lesions observed during necropsies of the two sheep 128 and 320 at 14 and 15 dpi corresponded to a moderate hepatomegaly and splenomegaly. The spleens from both sheep showed rounded borders and mild enlargement. While the use of barbiturates for euthanasia may have contributed to the observed mild splenomegaly, the absence of bleeding during necropsy suggests that it was mainly due to infection with A. phagocytophilum. In addition, in contrast to a normal pink lung color, patches of red coloration in sheep 320 lungs were observed (**Figure 2C**), as well as abundant bronchial and tracheal liquid. Mesenteric, axillar, and mandibular lymph nodes from both sheep were also mildly increased in size. Lesions were not detected in the remainder of examined tissues. A. phagocytophilum msp4 PCR on necropsies yielded positive results in lung, liver, spleen, uterus, and the small intestine from sheep

128, while in sheep 320, skin, spleen, lung, small intestine, bone marrow, and mesenteric lymph nodes were positive (**Table 1**).

The two sheep (381, 648) inoculated with A. phagocytophilum propagated in ISE6 tick cells showed clinical signs similar to sheep 128 and 320. Increased temperature was observed at 5 dpi with a 42◦C peak at 6 dpi. Sheep 648 presented with other clinical signs, such as trembling, total loss of appetite, and excessive salivation, and died at 12 dpi. Sheep 381, on the other hand, recovered after the febrile process, without any clinical signs until 115 dpi, when it was euthanized. A. phagocytophilum msp4 PCR was positive for both sheep at 5 dpi, and sheep 381 became negative at 15 dpi (data not shown). qPCR-Determined infection levels showed the highest fold change at 6 dpi in sheep 648, and at 9 dpi in sheep 381 followed by a drastic decrease at 12 dpi for both sheep (**Figure 3B**). For sheep 381, significant values (p = 0.05) were obtained until the end of the experiment, except at 60 dpi. Granular inclusions consistent with A. phagocytophilum morulae were detected in both sheep, with the highest percentages of infected neutrophils at 8 dpi with 47 and 60% in sheep 381 and 648, respectively. No other blood pathogens were identified via the blood smears from the two sheep, including sheep 648 that died at 12 dpi. For sheep 381, except for a mild splenomegaly, no gross lesion was observed at 115 dpi and all tested tissues were negative for A. phagocytophilum by PCR (**Table 1**).

Antibody detection by IFA indicated that the four sheep, 128, 320, 381, and 648 were negative prior to experimental inoculations (titer ≤ 1.6), and sheep 128, 320, and 381 became positive at 14 dpi (titer = 2.0). Sheep 648 died at 12 dpi before seroconversion, and sheep 381 remained positive with antibody titers between 2.0 and 2.77 from 14 to 115 dpi, until the animal was euthanized.

#### Transmission of *A. phagocytophilum* NV2Os by *I. ricinus*

The percentages of engorged ticks collected after feeding on infected sheep, as well as the rate of A. phagocytophilum detection in molted ticks are shown in **Table 2**. On all sheep, 67.5 and 11.3% of larvae and nymphs underwent successful engorgement, respectively. On sheep 320 inoculated with A. phagocytophilum-infected IDE8 tick cells, 17.7% of tick nymphs became engorged, and 50% underwent molting. A. phagocytophilum transstadial transmission was demonstrated by qPCR in 8 salivary glands (80%) and 9 midgut samples (90%) obtained from 10 dissected females infected at the nymphal stage. For sheep 648 inoculated with A. phagocytophilum-infected ISE6 tick cells, 46.2% of tick larvae became engorged, and where no transstadial transmission was detected in 10 recentlymolted nymphs engorged at the larval stage. However, when testing sheep 381—also infected with ISE6 cells—during both the acute (7 dpi) and persistent phases of infection (108 dpi), A. phagocytophilum acquisition and transstadial transmission from larvae to nymphs was demonstrated. In fact, at 7 dpi, 7 of the 10 recently-molted nymphs infected at the larval stage were positive for A. phagocytophilum infection, whereas at 108 dpi, only 2.7% (2/72) were positive despite an 85.3% larval engorgement success rate. At 7 dpi, only 4.5% of nymphs became engorged, with 34.9% of engorged nymphs then molting into adults. Following the engorgement of 15 pairs of adult ticks on infected sheep 381 at 7 dpi, A. phagocytophilum transovarial transmission was tested using DNA extracted from five egg masses obtained from engorged females, and all samples were negative (data not shown).

Finally, the transmission of A. phagocytophilum by I. ricinus nymphs fed as larvae on infected sheep 381 and 648, was tested by infesting two PreAlps sheep (572 and 615) with 45 nymphs each. To achieve this, a mixed pool of nymphs from both sheep was used, leading to an estimated 42% infection level in each tick batch. Nymph engorgement was as low as 3/45 (7%) in sheep 572, and 10/45 (22%) in sheep 615. Although sheep 572 remained healthy and A. phagocytophilum was not detected, sheep 615 developed hyperthermia above 40◦C with inappetence, lethargy, and reluctance to drink water, which led us to perform euthanasia at 15 days post nymph infestation. These clinical signs were then similar to what was previously observed during an NV2Os infection, and this infection was confirmed by positive qPCR bacterial detection in blood samples as early as the 5th day post tick infestation. Sequencing the PCR-amplified msp4 gene demonstrated that the isolate corresponded to A. phagocytophilum NV2Os, Gene Bank accession number CP015376.1.

#### DISCUSSION

As the etiological agent causing both HGA in humans and TBF in domestic animals, A. phagocytophilum receives growing scientific interest due to its importance in public health, to livestock welfare, and national economies. In the United Kingdom, there are an estimated 300,000 cases of tick pyaemia each year caused by immunosuppression in A. phagocytophilum-infected animals (14). A similarly high number of A. phagocytophilum-infected lambs has been reported in Norway ( 8). For livestock exposed to tick-infested pastures, current control methods are based on the use of long-acting antibiotics—given before animals are moved from tick-free environments into tick-infested pasture—and by reducing tick infestation with acaricides. Due to the multiple disadvantages of such chemical control measures (development of resistance, environmental hazard, contamination of milk, and meat products with drug residues, high cost), new approaches are urgently needed. The development of such approaches requires the establishment of relevant laboratory models. Therefore, the purpose of the present study was to develop an I. ricinus-sheep model that enabled us to mimic the natural A. phagocytophilum infection cycles of both sheep and ticks. This would facilitate future studies on tick-host-pathogen interactions, as well as enabling the evaluation of new control methods, such as anti-tick vaccines or transmission-blocking vaccines. It is well-known that for both vertebrates and ticks, tick-borne pathogen acquisition differs between natural infection via tick bites and experimental infection through injection due to, among other reasons, salivaassisted transmission mechanisms (38).

In thisstudy, only a limited number of sheep were included for this first experiment which aimed to validate our experimental model. Therefore, infection follow-up results and clinical observations should be taken with caution, and should ideally be reproduced to take into account any individual sheep variation and be generalized to the NV2Os/Romane breed sheep pair.

It has been reported that the delay in detecting A. phagocytophilum in sheep may vary according to the infectious dose, bacterial genotypes, infection source (infected blood or cell culture), as well as to the immune status and susceptibility of animal breeds [see review in ( 1)]. Here we report for the first time that the Norway variant 2 ovine strain (NV2Os) propagated in both IDE8 and ISE6 tick cell cultures was able to successfully infect Romane breed female sheep. Following infection with NV2Os-infected IDE8 cells, the bacteria were detected in sheep blood at 4 dpi by qPCR, 5 dpi by conventional PCR and 6 dpi by microscopic examination of blood smears, which is consistent with the assumed sensitivity of these detection techniques. Such a prepatent period in sheep is shorter than what was reported with the human strain NY-18 (10–21 dpi) (31 , 32), longer than with the var1 (13 , 22), but in accordance with what was observed with var2 in Norwegian Dala breed lambs (22). According to qPCR results, bacteremia peaked at 6 dpi, except for sheep 381 that recovered after the acute phase with a peak at 9 dpi following infection with A. phagocytophilum NV2Os propagated in ISE6 cells. Nevertheless, blood smears demonstrated a detection lag, with the maximum number of infected neutrophils (47 and 60%) at 8 dpi for sheep infected with IDE8 cells, suggesting also that

TABLE 2 | Ixodes ricinus ticks collected after feeding on A. phagocytophilum-infected sheep during both the acute or the persistent phases of infection, and detection of infection in engorged and molted ticks.

qPCR detection is more reliable. The observed initial bacteremia period with a mean duration of 10.75 (±1.7) days, as determined by qPCR, was also similar to that reported by Granquist et al. for the Var2 where the mean was 11.4 (±1.8) (22). Lastly, infection monitoring in sheep 381 and positive qPCR bacterial detection until 115 dpi confirmed that A. phagocytophilum can establish long-term infections in immune competent sheep, and has previously been reported for as long as 6–25 months (22, 39– 41). Such persistent cyclic activity was suggested to be linked to variant-specific antigen immune responses to the bacteria (42, 43), and is epidemiologically very important in the maintenance of A. phagocytophilum infection in the field, especially during periods of no tick activity.

The indirect immunofluorescence assay, adapted to detect antibodies against A. phagocytophilum, and which has been extensively used in experimental and natural infections (8, 9, 21, 22) was used to test seroconversion in infected sheep. Our findings showed that antibodies were detected in all infected sheep at 14 dpi. Although the titers varied during the persistent infection of sheep 381, the results remained positive until 115 dpi, when the animal was euthanized. These results are consistent with studies in naturally- and experimentally-infected sheep (9, 13, 22).

In accordance with previous studies using the same strain but in Norwegian Dala sheep by Stuen et al. and Granquist et al. (21, 22), the first clinical sign was an increase in temperature up to 41.5◦C between 3 and 6 dpi dependent on the sheep. However, a discrepancy was observed that may be explained by the genetic susceptibility of the French breeds used in this study, where the febrile process lasted between 8 and 9 days here and 2.2–3 days in their studies. All infected sheep also showed signs of loss of appetite and lethargy until euthanasia, except for sheep 381 that recovered after the acute phase of infection, and showed no clinical signs until euthanasia at 115 dpi.

Few studies have been performed in order to evaluate the existence of tissue that may act as A. phagocytophilum reservoirs in persistently-infected sheep and which could explain the maintenance of the bacteria in the field, especially during periods of vector tick non-activity. When the Suffolk breed was infected with the NY-18 human strain, no bacteria could be detected in any internal organ 1 month after infection, even with quantitative PCR (31). However, when the Norwegian Dala breed was infected with a Norway ovine field isolate, several organs (bone marrow, intestinal and bladder walls, kidney, lymph node, and thymus) were PCR positive 3 months after infection (44). Here we observed positive PCR results in several organs from sheep euthanized 2 weeks after the infection, but no infection was detected by PCR during necropsies performed at 115 days post infection. These varying results may reflect differences due to the bacterial strain or sheep breed, but the main conclusion is that more experiments with more animals are needed to answer this question. However, preferential involvement of lymphoid tissue may suggest that these tissues could represent a source of infected cells that, when released into the blood, would enable tick infection (44). As for the lesions, A. phagocytophilum infection has been reported to be associated with moderate tissue damage, which targets lymphoid tissues and the spleen in particular (31, 32, 45), as confirmed by our study.

Experimentally-infected sheep here proved to be excellent hosts for the production of I. ricinus ticks infected with ovine NV2Os, with feeding success rates of 67, 11, and 60% for larvae, nymphs, and female adults, respectively. Unlike laboratory animals, such as mice or rabbits, the large size of sheep makes it possible to feed many ticks at once without animal suffering in the absence of infection. This result is very encouraging considering the fact that in an experimental study with I. scapularis, Kocan et al. reported larvae feeding failure but, like us, poor performance of nymphs feeding on sheep (32). After engorgement during the acute phase, ticks acquired A. phagocytophilum, with an estimation of 80% acquisition in both engorged larvae and nymphs (data not shown). Similar A. phagocytophilum acquisition percentages in field populations of I. ricinus have been reported in a study performed in the United Kingdom. There, sheep naturally exposed to tick-infested pasture for a minimum of 4 weeks, resulted in 59 and 72% of PCR-positive engorged larvae and nymphs, respectively, after feeding on sheep. It must be taken into consideration however, that nymphs may have been previously infected (46).

In this last study, the authors reported that after molting, engorged larvae led to 18.5% of infected nymphs, where engorged nymphs led to 48% of infected adults (46). Here, higher transstadial infection rates were recovered after feeding larvae and nymphs on A. phagocytophilum-experimentally-infected sheep during the acute phase, with 70% of nymphs infected, and 80 and 90% of female tick salivary glands and midguts infected, respectively. The differences could be due to bacterial strain and sheep breed, and/or to the fact that in our study, ticks were fed during the acute phase of the infection, at the point where bacteria reached their highest peak in blood, as demonstrated by qPCR. Indeed, in the study performed by Ogden and coworkers, ticks were collected from sheep undergoing both acute and postacute phases of infection at different time points over 9 weeks (46). In addition, it has been reported that tick acquisition efficiency gradually declines from high levels in the acute and immediate post-acute infection phases, to low levels in sheep that have been infected for much longer periods (47).

This last observation was confirmed by our results on transstadial transmission efficiency of A. phagocytophilum in I. ricinus ticks fed during acute or persistent infection of the sheep 381 with 70% and only 2.7% infection levels in molted nymphs, respectively.  Indeed, in nymphs or adults, which ingest larger volumes of blood and consequently have access to higher infectious bacterial doses, we can expect higher infection percentages. In addition, it has been suggested that a greater number of feeding adult ticks stimulates bacterial multiplication at the tick bite site and thus increases the A. phagocytophilum infectious dose for ticks (48, 49). Here, only larvae were used to infest sheep during the persistent infection, when both larvae and nymphs were used during the acute infection, which may also explain the observed difference in tick infection levels. However, bacterial transmission to ticks from persistently-infected sheep with no clinical signs, no positive PCR bacterial detection in blood, and for which all necropsies were PCR negative, has a real epidemiological importance for bacteria and disease maintenance and propagation in the field.

Our results also showed that larvae which had acquired A. phagocytophilum NV2Os from an infected sheep were able to retransmit it, as nymphs, to a naive sheep, thus validating that the transstadial transmitted bacteria remained infectious. This is the first demonstration of a complete NV2Os transmission cycle, from an in vitro bacterial culture to I. ricinus ticks. Finally, the failure to detect bacteria in eggs laid by adult ticks engorged on infected sheep during the acute phase, confirms that transovarial transmission of A. phagocytophilum is not likely to occur, in correlation with previous studies (32, 46).

## CONCLUSION

Here we established a sheep model of IDE8 and ISE6 tick cell culture-propagated A. phagocytophilum NV2Os infection and tick transmission successfully mimicking the entire transmission cycle in the laboratory. Previously, sheep were experimentally infected with A. phagocytophilum NV2Os by inoculating blood obtained from infected sheep, but to our knowledge, this is the first time that this strain propagated in IDE8 or ISE6 tick cells has been used to infect sheep, and which produced clinical signs due to A. phagocytophilum infection. Our results also showed that not only were infected sheep able to transmit the bacteria to ticks during the acute phase of the infection, but also during the chronic persistent infection phase, a finding which has important epidemiological significance. Moreover, we demonstrated that nymphs infected on sheep during the preceding infection stage were also able to transmit the bacteria to naive sheep. The establishment of such a model opens an entire spectrum of possibilities in which to study the molecular events of A. phagocytophilum infection in both vertebrate and invertebrate hosts, tick-host-pathogen interactions, as well as testing the efficacy of vaccine candidates against I. ricinus and A. phagocytophilum.

## DATA AVAILABILITY STATEMENT

The datasets generated for this study are available on request to the corresponding author.

## ETHICS STATEMENT

The animal study was reviewed and approved by ComEth Anses/ENVA/UPEC; Permit Number<PHONE_NUMBER>395004, ComEth Anses/ENVA/UPEC; Permit Number<PHONE_NUMBER>257726.

## AUTHOR CONTRIBUTIONS

CA and SB conceived and designed the study, analyzed the results, and wrote the paper. JF advised on experimental design. CA and LF performed animal experiments and performed laboratory analyses. H-JB, CA, and LF performed necropsies. PA and CR performed the cell cultures. CG performed IFA. PA, CR, and A-CL edited the manuscript. All authors read and approved the final paper.

## FUNDING

This study received funding from the French Government's Investissement d'Avenir program, Laboratoire d'Excellence Integrative Biology of Emerging Infectious Diseases (grant n ◦ANR-10-LABX-62-IBEID) and from the SATTIdF Innov.

## ACKNOWLEDGMENTS

We thank Snorre Stuen (Norwegian University of Life Sciences) for providing the A. phagocytophilum NV2Os strain. Benoit Lecuelle, Francis Moreau, and Michael Jacob (Biomedical Research Center, Ecole Nationale Vétérinaire d'Alfort) are acknowledged for their technical assistance during the sheep infections. We are in debt to Jean-Luc Servely (Bio-Pole, Ecole Nationale Vétérinaire d'Alfort) for his valuable imaging assistance. Gilberto Reyes de Luna, MSc student from the University of Queretaro, Mexico, is also acknowledged for his assistance on sheep infection and tick infestation, as well as DNA isolation from sheep tissues.

## REFERENCES

phagocytophilum in sheep. Clin Diagn Lab Immunol. (2003) 10:692– 5. doi: 10.1128/cdli.10.4.692-695.2003

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2020 Almazán, Fourniol, Rouxel, Alberdi, Gandoin, Lagrée, Boulouis, de la Fuente and Bonnet. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Tick Cell Lines in Research on Tick Control

Ahmed Al-Rofaai and Lesley Bell-Sakyi\*

Department of Infection Biology, Institute of Infection and Global Health, University of Liverpool, Liverpool, United Kingdom

Ticks and the diseases they transmit are of huge veterinary, medical and economic importance worldwide. Control of ticks attacking livestock and companion animals is achieved primarily by application of chemical or plant-based acaricides. However, ticks can rapidly develop resistance to any new product brought onto the market, necessitating an ongoing search for novel active compounds and alternative approaches to tick control. Many aspects of tick and tick-borne pathogen research have been facilitated by the application of continuous cell lines derived from some of the most economically important tick species. These include cell lines derived from acaricide-susceptible and resistant ticks, cell sub-lines with in vitro-generated acaricide resistance, and genetically modified tick cells. Although not a replacement for the whole organism, tick cell lines enable studies at the cellular and molecular level and provide a more accessible, more ethical and less expensive in vitro alternative to in vivo tick feeding experiments. Here we review the role played by tick cell lines in studies on acaricide resistance, mode-of-action of acaricides, identification of potential novel control targets through better understanding of tick metabolism, and anti-tick vaccine development, that may lead to new approaches to control ticks and tick-borne diseases.

Keywords: Rhipicephalus microplus, Ixodes spp., control, tick cell line, acaricide, resistance, metabolism, antitick vaccine

#### INTRODUCTION

Ticks are obligate, haematophagous ectoparasites. Like mosquitoes, they cause problems to their hosts both from skin irritation and damage during blood feeding and by transmitting numerous pathogens including viruses, bacteria and protozoa. Consequently, ticks and tick-borne pathogens (TBPs) are of huge veterinary, medical and economic importance due to their negative impact on the health of humans and domestic animals (de la Fuente et al., 2008). Therefore, many control strategies have been devised to eliminate or avoid ticks and reduce the spread of TBPs, such as personal protection, landscape management, chemicals, vaccination, and biological control (Stafford et al., 2017). Amongst these, chemical control using a variety of acaricidal compounds is still the main strategy for controlling ticks affecting livestock and companion animals. However, many tick species develop resistance to these chemicals, which has led to attempts to find natural alternatives by studying the effects of some natural products on ticks and TBPs (Rodriguez-Vivas et al., 2018). Globally, the most economically important species, the invasive Asian cattle tick

#### Edited by:

Itabajara Silva Vaz Jr., Federal University of Rio Grande do Sul, Brazil

#### Reviewed by:

Eliane Esteves, University of São Paulo, Brazil Ulrike G. Munderloh, University of Minnesota Twin Cities, United States

#### \*Correspondence:

Lesley Bell-Sakyi<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Invertebrate Physiology, a section of the journal Frontiers in Physiology

Received: 16 December 2019 Accepted: 12 February 2020 Published: 25 February 2020

#### Citation:

Al-Rofaai A and Bell-Sakyi L (2020) Tick Cell Lines in Research on Tick Control. Front. Physiol. 11:152. doi: 10.3389/fphys.2020.00152

**304**

Rhipicephalus (Boophilus) microplus, is also the most notorious for rapid development of resistance to every new acaricidal product brought onto the market (Jonsson and Hope, 2007; Abbas et al., 2014). Acaricide-resistant R. microplus are widespread throughout tropical and sub-tropical Asia, Latin America and Australia (Abbas et al., 2014), and are rapidly spreading in Southern, Central and West Africa (Nyangiwe et al., 2018; Silatsa et al., 2019).

Tick cell lines have an invaluable role to play in many aspects of tick and TBP research including tick biology, hostvector-pathogen relationships, genetic manipulation, genomics and proteomics (Bell-Sakyi et al., 2007). In addition, tick cell lines play a major role as essential tools in in vitro studies to examine and assess the impact of chemical molecules and vaccines against ticks and TBPs, as well as to investigate the mechanisms of resistance and tick metabolism which can lead to the development of novel approaches to control ticks and TBPs (Bell-Sakyi et al., 2018). While >60 continuous cell lines have now been established from 16 ixodid and three argasid tick species (Bell-Sakyi et al., 2018), studies on tick and pathogen control have predominantly centered around cell lines derived from, not surprisingly, R. microplus. A smaller number of studies have used cell lines derived from other tropical Rhipicephalus species of veterinary importance, from Ixodes ricinus, a European tick that feeds on a wide range of domestic and wild animal hosts as well as attacking humans, and from the medically important North American species Ixodes scapularis (**Table 1**).

This Mini Review focuses on the role of tick cell lines in studies on control of ticks and the few studies dealing with approaches to combined control of ticks and TBPs. While tick cell lines have been used in many studies contributing to aspects of pathogen control per se, these are beyond the scope of our review. Here we review selected studies to highlight how tick cell lines can contribute to tick control research, bearing in mind that results gained in vitro should be validated in vivo.

## TICK CELL LINES IN STUDIES ON ACARICIDE RESISTANCE

Tick cell lines have been investigated for their potential as cheap and ethical tools to study acaricide resistance. Some studies focused on establishing drug-resistant cell lines which provide a useful tool to study and understand the mechanisms of resistance, an important step toward improving detection and prevention of tick resistance against acaricides. Tick cell lines are also a useful resource for identifying genes responsible for acaricide resistance and some published studies have focused on this aspect.

## Generation and Application of Acaricide-Resistant Tick Cell Lines

Cossio-Bayugar et al. (2002b) generated three organophosphateresistant R. microplus cell sub-lines by exposing the R. microplus BmVIII-SCC cell line (Holman, 1981), originally derived from embryos of acaricide-susceptible ticks, to gradually increasing concentrations of the organophosphate compound coumaphos (**Table 2**). They then assessed the levels of esterase, an enzyme that, when present at high levels, is responsible for causing resistance by modifying the organophosphate target site to be less sensitive. After exposure to coumaphos, there were higher levels of esterase in the resistant sub-lines compared to the original susceptible cell line. These findings, which were in line with previous in vivo studies (Miranda et al., 1995; Rosario-Cruz et al., 1997), validated the use of tick cell lines to study development of resistance in ticks, which could thereafter help to determine a suitable approach to formulating acaricides that would be less likely to induce resistance.

This group then used the coumaphos-resistant R. microplus cell sub-lines to evaluate the mechanisms that may contribute to acaricide resistance by further characterizing them compared to susceptible control cells (Cossio-Bayugar et al., 2002a). In addition to higher esterase levels, they found that the resistant cells exhibited higher levels of intracellular calcium and glutathione, decreased glutathione S-transferase activity, and reduced plasma and mitochondrial membrane potentials. Their results were consistent with the in vivo observations of increased esterase activity in organophosphate-resistant ticks, as well as resistance mechanisms found in other cell systems. One of the coumaphos-resistant R. microplus cell sublines played an important role in a further study (Cossio-Bayugar et al., 2005) that identified amino acid substitutions in the protein encoded by the phospholipid hydroperoxide glutathione peroxidase (PHGPx) gene in susceptible and resistant R. microplus, highlighting the possibility of its relationship with acaricide resistance in these tick populations.

A study conducted by Pohl et al. (2014) also focused on understanding the mechanisms of acaricide resistance in R. microplus. In this study, the authors established an ivermectin-resistant sub-line (BME26-IVM) of the R. microplus embryo-derived cell line BME26 (Kurtti et al., 1988; Esteves et al., 2008) by exposing susceptible cells to increasing concentrations of ivermectin (**Table 2**) over 46 weeks. Using the BME26-IVM sub-line, the contribution of the ATP-binding cassette (ABC) transporter mechanism was investigated by evaluating mRNA expression using quantitative RT-PCR. This study demonstrated increased levels of ABC transporter gene transcripts in the ivermectin-resistant cells compared to the original susceptible BME26 cells.

#### Effect of Acaricide Treatment of "Susceptible" Tick Cell Lines on Expression of Genes Associated With Resistance

Taking a different approach and using a cell line expected to be susceptible to ivermectin, Mangia et al. (2016) evaluated the expression of selected members of the ABC transporter subfamily B genes encoding P-glycoproteins (PgPs) in the I. ricinus embryo-derived cell line IRE/CTVM19 following treatment with ivermectin. The authors showed that ABC pump expression was not significantly modulated by ivermectin treatment, and expression of one of the subfamily genes was not detected. Interestingly, this study revealed that the IRE/CTVM19 cell line was able to tolerate a much higher concentration of ivermectin

TABLE 1 | Tick cell lines used in studies on tick control reviewed in this article.

TABLE 2 | Acaricides and pesticides used with tick cell lines in studies on tick control reviewed in this article.

(30 µg/ml) than the R. microplus-derived line BME26 that did not survive after exposure to 12.5 µg/ml ivermectin (Pohl et al., 2014). This could have been due to biological differences between the two tick species from which the cell lines were derived, differences in the phenotypic composition of the cell lines and/or in the culture conditions used. This should be considered when conducting such in vitro experiments using tick cell lines.

In a further study, Mangia et al. (2018) treated IRE/CTVM19 cultures with different concentrations of the acaricides permethrin, amitraz and fipronil (**Table 2**). They assessed cell viability, morphology, metabolic activity and expression of ABC genes at day 10 post-treatment. In general, the results showed that fipronil had the greatest significant negative effects on cell viability and metabolic activity, followed by permethrin, while amitraz only caused a significant decrease in these parameters at the highest concentration tested. The negative effects of fipronil and permethrin were confirmed by their effects on ABC gene expression with upregulation of ABCB6, ABCB8, and ABCB10, while amitraz had little or no effect on expression of any of the ABC genes tested.

#### Exploiting Tick Cell Lines Derived From Susceptible and Resistant Ticks to Identify Genes Involved in Acaricide Resistance

For some of the currently available R. microplus cell lines, the acaricide resistance status of the parent ticks from which the

cultures were derived is known. As already mentioned, the parent BmVIII-SCC and BME26 cells used by, respectively, Cossio-Bayugar et al. (2002b) and Pohl et al. (2014) to derive acaricide-resistant sub-lines in vitro were originally susceptible to the compounds used, implying that the parent ticks of both lines, originally from Mexico, were also acaricide-susceptible. Two additional R. microplus cell lines, BME/CTVM2 (Bell-Sakyi, 2004) and BME/CTVM4 (Bell-Sakyi et al., 2007), were derived from known acaricide-susceptible ticks from Costa Rica, while two other cell lines, BME/CTVM5 (Bell-Sakyi et al., 2007) and BME/CTVM6 (Bell-Sakyi, 2004) were derived from Colombian ticks known to be resistant to organophosphates, organochlorines and amitraz (Koh-Tan et al., 2016). While these cells whose natural resistance status derives from their parent ticks offer a potentially very interesting complement to the in vitro-derived resistant R. microplus cell lines of Cossio-Bayugar et al. (2002b) and Pohl et al. (2014), to date they have only been exploited in a single study. Koh-Tan et al. (2016) investigated the expression of the β-adrenergic octopamine receptor (βAOR) gene, associated with amitraz resistance, and the ATP-binding cassette B10 (ABCB10) gene, associated with macrocyclic lactone resistance, in tick cell lines derived from R. microplus, including BmVIII-SCC, BME/CTVM2, BME/CTVM5 and BME/CTVM6, Rhipicephalus appendiculatus, Rhipicephalus sanguineus, and Rhipicephalus evertsi (**Table 1**). Expression of the βAOR gene was detected in all the Rhipicephalus spp. cell lines, but in BME/CTVM6, derived from amitraz-resistant ticks, a novel larger amplicon was detected. This was found to encode a 12-amino acid insertion identified as a duplication of the immediately upstream sequence; the insertion was also present in genomic DNA from BME/CTVM5 cells, similarly derived from amitraz-resistant ticks, but transcription of the novel amplicon was not detectable. Expression of the ABC10 gene was approximately seven-fold higher in BME/CTVM6 cells compared to all other cell lines examined including BME/CTVM5. These findings suggest that BME/CTVM6 cells in particular could be a useful model system for studying resistance to multiple acaricide groups in R. microplus; further experiments are needed to determine whether additional genes are mutated in this cell line.

### TICK CELL LINES ELUCIDATE NOVEL ASPECTS OF TICK CELL METABOLISM TO IDENTIFY POTENTIAL CONTROL TARGETS

Tick cell lines have also played a valuable role in the study of tick cell metabolism which can help in developing novel control methods. De Abreu et al. (2013) investigated the role of protein kinase B (AKT) and glycogen synthase kinase 3 (GSK3) in glycogen metabolism and cell viability using the R. microplus cell line BME26, and the results revealed an antagonistic role for AKT and GSK3. A reduction in AKT caused a decrease in glycogen content, cell viability and altered cell membrane permeability, whereas the inhibition of GSK3 promoted an increase in glycogen content.

To better understand energy metabolism during embryonic development in ticks, Da Silva et al. (2015) used the BME26 cell line to evaluate several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism. Their results showed that several genes displayed mutual regulation in response to glucose treatment, and the transcription of gluconeogenic genes in tick cells is controlled by GSK3.

The BME26 cell line also played an essential and effective role in the first report on characterization of a cell cycle protein in arachnids, and the reversal of its functions with an inhibitor (Gomes et al., 2013). The BME26 cell line was used in an in vitro inhibition assay to determine the inhibitory effects of roscovitine, a purine derivative, on tick cyclin-dependent kinases (CDKs), which are essential for cell cycle progression, and the results suggested that CDKs may present an alternative strategy for designing a novel acaricide that targets both oogenesis and embryogenesis processes.

The I. scapularis embryo-derived tick cell line ISE6 (Kurtti et al., 1996) is the one of the most widely used tick cell lines due to its susceptibility to a wide range of TBPs (Oliver et al., 2015). ISE6 cells were used by Cabezas-Cruz et al. (2017) to study possible control of infection with the obligate intracellular bacterium Anaplasma phagocytophilum, causative agent of disease in humans, ruminants, horses and dogs, by increasing the synthesis of phosphoenolpyruvate from tyrosine. Ferritins (FER) are iron-storage proteins that act to store excess iron available in the cellular iron pool by the binding and oxidation of the ferrous ion (Fe2+) and the formation of ferric ion (Fe3+) in the FER core cavity (Galay et al., 2015). Hernandez et al. (2018) were able to induce intracellular ferritin (FER1) protein expression by exposing ISE6 cells to various concentrations of ferrous sulfate. They showed that silencing of FER1 expression by RNA interference led to a decrease in ISE6 cell proliferation and an increase in mortality, concluding that ISE6 cells could be a good tool to further understanding of the mechanism of FER1 action. Thereafter, Hernandez et al. (2019) characterized the activity of an iron-inducible tick promoter derived from the tick Haemaphysalis longicornis, previously identified by Kusakisako et al. (2018b), in ISE6 cells, which could be a valuable tool in the development of a gene-manipulation system to control ticks and TBPs. Kusakisako et al. (2018a) established methods to visualize H2O2 in ISE6 cells using an intracellular H2O2 probe and observed that paraquat, known to induce oxidative stress in mammalian cells (**Table 2**), was also effective in this respect in ISE6 tick cells. Iron derived from host blood may react with oxygen in the tick's body and lead to high levels of H2O2, causing oxidative stress such as DNA strand breaks, lipid peroxidation and degradation of other molecules (Kusakisako et al., 2018a; Hernandez et al., 2019).

#### GENETIC MANIPULATION OF TICK CELL LINES

The development of new strategies for control of ticks could be greatly enhanced by exploring genetic manipulation of ticks

and tick cell lines. Demonstrating proof of concept, Kurtti et al. (2006) first reported stable transfection of the I. scapularis cell line ISE6 with a gene encoding a fluorescent protein using the Sleeping Beauty transposon system. In a different approach, Cossio-Bayugar and Miranda-Miranda (2007) reported retroviral transfection of primary R. microplus cell cultures with green fluorescent protein; fluorescence was detectable for up to 2 weeks following retroviral transfection. Using yet another approach, Machado-Ferreira et al. (2015) achieved the expression of transgenes in R. microplus ticks and the cell line BME/CTVM2 using Agrobacterium tumefaciens T-DNA constructs; evidence of transcription and protein synthesis were detectable for 2– 3 weeks by fluorescence microscopy, western blot analyses and RT-PCR. These studies demonstrate that genetic manipulation approaches used with other animal cell systems can equally be applied to tick cell lines, and raise the possibility of modifying ticks to render them incapable of developing acaricide resistance.

#### TICK CELL LINES CONTRIBUTE TO ANTI-TICK VACCINE DEVELOPMENT

Vaccination with recombinant tick antigens represents a promising tick control strategy. Vaccines containing recombinant tick antigens demonstrated advantages including reduction in acaricide application, cost-effectiveness and reducing the prevalence of tick-borne diseases through reducing exposure of cattle to infected ticks. However, the efficacies of these vaccines vary considerably between tick species and geographic location (de la Fuente et al., 2007). Tick cell lines have played a central role in identifying tick protective antigens to produce a variety of vaccines that contribute to the prevention and control of tick infestations and TBP infections. For example, three embryo-derived tick cell lines, R. microplus BME/CTVM2 and I. scapularis IDE8 (Munderloh et al., 1994) and ISE6, were used in a study conducted by Antunes et al. (2014) to identify tick proteins involved in tick-pathogen interactions. The aim was to identify candidate protective antigens that could be used as vaccines for the control of cattle infestations with R. microplus ticks and infection with the TBPs Anaplasma marginale and Babesia bigemina transmitted by R. microplus. As an important part of the experiment, antibodies produced against the R. microplus protein subolesin were tested using immunofluorescence and showed positive reactions in the three cell lines, but no effect was observed on pathogen DNA levels. In contrast, using the I. scapularis cell line ISE6 and the I. ricinus cell line IRE/CTVM20 (Bell-Sakyi et al., 2007), Contreras et al. (2017) determined that two candidate tick-protective antigens – I. scapularis lipocalin (ISCW005600) and lectin pathway inhibitor (AAY66632), and their I. ricinus homologs – constitute candidate protective antigens for the control of tick infestations as well as A. phagocytophilum infection. They showed that anti ISCW005600 and AAY66632 IgG significantly increased the percentage of apoptotic tick cells and decreased pathogen infection, which could help in the development of effective antitick and anti-pathogen vaccines.

#### CONCLUSION

Ticks and TBPs have huge negative impacts on global livestock and public health and national economies. Scientific research has focused on creating and developing a variety of strategies to prevent and control ticks and TBPs. Tick cell lines represent essential tools in this area of research: in vitro-generated acaricide-resistant cell lines, cell lines derived from acaricideresistant and susceptible ticks, genetically modified cell lines and cell lines supporting growth of intracellular TBPs all have a role to play. Experimental parameters can be easily controlled, effects at the cellular and molecular level can be determined, and cell lines reduce, while not ultimately replacing, the need for expensive in vivo studies involving maintenance of tick colonies and feeding on laboratory animals. As the numbers of cell lines derived from economically important tick species increase, so do the opportunities for exploiting them in a wide range of studies. For example, the potential for developing high-throughput preliminary screens of novel, potentially acaricidal compounds using cell lines from multiple tick species remains unexplored; such screens could greatly increase the number of active compounds identified for further testing at relatively low cost, and could be applied both to compound libraries held by pharmaceutical companies and to natural plant products. It is anticipated that gene editing of tick cell lines by technologies such as CRISPR will add another facet to the control portfolio. The Tick Cell Biobank (Bell-Sakyi et al., 2018), which houses all the cell lines mentioned in this review as well as cell lines derived from many other species of veterinary and medical concern, will continue to underpin research on tick and TBP control.

#### AUTHOR CONTRIBUTIONS

AA-R drafted the manuscript, LB-S edited the draft and both authors produced and agreed the final version.

## FUNDING

AA-R is supported by the Council for At-Risk Academics (CARA) and the University of Liverpool. LB-S is supported by the United Kingdom Biotechnology and Biological Sciences Research Council grant number BB/P024270/1.

## ACKNOWLEDGMENTS

We would like to thank staff of the Council for At-Risk Academics (CARA) and Dr. Benjamin Makepeace for their instrumental roles in bringing AA-R to the University of Liverpool.

## REFERENCES

**Conflict of Interest:** The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer UM declared a past co-authorship with one of the authors LB-S to the handling Editor.

Copyright © 2020 Al-Rofaai and Bell-Sakyi. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Global Transcription Profiles of Anaplasma phagocytophilum at Key Stages of Infection in Tick and Human Cell Lines and Granulocytes

Curtis M. Nelson1 , Michael J. Herron1 , Xin-Ru Wang1 , Gerald D. Baldridge1 , Jonathan D. Oliver 2 and Ulrike G. Munderloh1 \*

1 Department of Entomology, College of Food, Agriculture, and Natural Resource Sciences, University of Minnesota, Minneapolis, MN, United States, 2 Division of Environmental Health Sciences, School of Public Health, University of Minnesota, Minneapolis, MN, United States

#### Edited by:

Itabajara Silva Vaz Jr, Federal University of Rio Grande Do Sul, Brazil

#### Reviewed by:

Pilar Alberdi, Institute of Game and Wildlife Research (CSIC), Spain Andréa Cristina Fogaça, University of São Paulo, Brazil

> \*Correspondence: Ulrike G. Munderloh<EMAIL_ADDRESS>
#### Specialty section:

This article was submitted to Veterinary Infectious Diseases, a section of the journal Frontiers in Veterinary Science

Received: 29 October 2019 Accepted: 13 February 2020 Published: 06 March 2020

#### Citation: