Fig 2: High seropositivity for B. burgdorferi and A. phagocytophilum in eastern coyotes. B. burgdorferi B31 and human promyelocytic HL-60 cells (CCL-240; ATCC) and HL-60 cells infected with A. phagocytophilum NCH-1 were cultivated as previously described (14, 15). Cells were harvested by centrifugation, washed, and solubilized in SDS-PAGE buffer. The B. burgdorferi cell lysates (A) and A. phagocytophilum-infected HL-60 cells (B) were separated by SDS-PAGE (AnykD Criterion precast gels; Bio-Rad), immunoblotted, and screened with a 1:1,000 dilution of each plasma sample, as previously described (15). The sector of Pennsylvania from which each animal was harvested is indicated above the immunoblots, with a plus or minus indicating the Ab scoring for each sample (see Table 1, footnote b, for sector abbreviations). The migration positions of native A. phagocytophilum P130 and P44 proteins were determined by screening cell lysate immunoblots with rat anti-P130 and rat anti-P44 antisera (C), generated as previously described (15). Molecular weight markers are indicated.

Fig 3: Composition of chemical elements in tick salivary glands and cement. Chemical elements were characterized by SEM combined with EDS analysis in samples from tick salivary glands (SG) and cement. (A) Representation (%) of the chemical elements at different feeding stages (T1–T3). (B) Changes in the representation (%) of the chemical elements at different feeding stages (T1–T3). The composition of chemical elements was compared at different time points by One-way ANOVA test (https://www.socscistatistics.com/tests/anova/default2.aspx; p < 0.05, n = 2–4 biological replicates). (C) Characterization of α-Gal content in tick SG and cement protein extracts and in comparison with human promyelocytic leukemia HL60 cells (ATCC CCL-240) and pork (Sus scrofa) kidney as negative and positive controls, respectively. A calibration curve with 0.0–1.0 ng α-Gal and O.D. values at 450 nm was constructed using Microsoft Excel for Mac (v. 16.26) to convert ELISA reader values to α-Gal content per sample (R2 = 0.913). The results (average + S.D. of α-Gal/1 μg protein) were compared between pork kidney positive control, salivary gland or cement samples and HL60 cells negative control (black * p < 0.05, n = 3 biological replicates) and between salivary gland and cement samples at different time points (T1–T3) (red * p < 0.05, n = 3 biological replicates) by Student’s t-test with unequal variance. Variations in α-Gal levels at different time points (T1–T3) in both sialome and cementome were compared by one-way ANOVA test (https://www.socscistatistics.com/tests/anova/default2.aspx) (f and p values are shown, n = 3 biological replicates).

Fig 4: Biocompatibility and cytotoxicity studies: (a-i) bright field image of HL-60 [ATCC, CCL-240] cell line with no exposure to printed polymer; (a-ii) bright field image of HL-60 [ATCC, CCL-240] cell line exposed to printed polymer (L indicates 10× magnification, and H indicates 20% increase in digital magnification); (b-i) cell population used for flow cytometry; (b-ii) histogram of PI assay with no PI stain; (b-iii) histogram of PI assay with stain; (c) MTT assay absorbance data comparison for HL-60 [ATCC, CCL-240] cell line exposed to the printed polymer for 1, 3, and 7 days in culture (*p < 0.05, **p < 0.01, and ***p < 0.001).