Do white-footed mice, the main reservoir of the Lyme disease pathogen in the United States, clinically respond to the borrelial tenancy?

ABSTRACT

As white-footed mice, Peromyscus leucopus, are considered the primary animal reservoir of Borreliella burgdorferi sensu stricto (Bb), the main agent of Lyme disease (LD) in the United States, these animals represent the most relevant model to study borrelial spirochetes in the context of their natural life cycle. Previous studies have consistently demonstrated that although white-footed mice respond immunologically to the invasion of the Lyme pathogen, P. leucopus adults do not develop a clinically detectable disease. This tolerance, which is common for mammalian reservoirs of different pathogens, contrasts with detrimental anti-borrelial responses of C3H mice, a widely used animal model of LD, which always result in a clinical manifestation (e.g., arthritis). The current investigation is a follow-up of our recent study that already showed a relative quiescence of the spleen transcriptome for Bb-infected white-footed mice compared to the infected C3H mice. In an effort to identify the mechanism behind this tolerance, in this study, we have evaluated an extensive list of hematological and biochemical parameters measured in white-footed mice after their 70-day-long borrelial infection. Despite missing reference intervals for Peromyscus mice, our sex- and age-matched uninfected controls allowed us to assess the blood and serum parameters. In addition, for our assessment, we also utilized behavioral, immunological, and histological analyses. Collectively, by using the metrics reported herein, the present results have demonstrated clinical unresponsiveness of P. leucopus mice to the borrelial infection, presenting no restriction to a long-term host–pathogen co-existence.

INTRODUCTION

The reservoir competence of the white-footed mouse, Peromyscus leucopus, whose role is to amplify and sustain the tick-borne pathogen of Lyme disease (LD) in its enzootic cycle, was repeatedly demonstrated by numerous studies (1–14). Borreliella burgdorferi sensu stricto (Bb), the key LD genospecies in the United States (U.S.), is part of Borreliella burgdorferi sensu lato complex that also comprises over 20 other borrelial genospecies (7). LD, also known as Lyme Borreliosis, is an emerging and the most widespread tick-borne disease in the Northen Hemisphere, with an estimated incidence rate of almost half a million annual cases in the U.S. by itself (15, 16).

Despite numerous advantages of inbred Mus musculus (M.m.) strains (e.g., reproducibility, ease of breeding, cost-effectiveness, availability of genomic resources) (17), the rodents of genus Peromyscus, colloquially termed as deer mice (18), undoubtedly represent the most relevant animal model to study the enzootic cycle of Bb. Given that the families of Cricetidae and Muridae that include the Peromyscus and Mus genera, respectively, are estimated to have diverged from each other approximately 25 million years ago (18), it is well expected to observe distinct phenotypic responses to borrelial infection between M.m. and Peromyscus mice.

Most of studies have consistently demonstrated that Bb-infected Peromyscus adult mice develop no clinical disease but immunologically respond to the presence of Bb spirochetes in their tissues (3, 5, 6, 19–25). The exception includes some evidence of systemic neurological disease that was supported by culture-based detection of Bb spirochetes in the brain of 45% wild-caught P. leucopus adults (26). A more recent investigation also reported some histological evidence of mild carditis in white-footed mice experimentally infected with Bb strain B31 (27). Thus, the tolerance of Peromyscus mice to Bb spirochetal invasion and persistence is not absolute, likely depending on a delicate balance between robust yet non-sterilizing anti-Bb immune responses (23, 28–31).

The mechanisms underlying the tolerance of Peromyscus mice to borrelial infection have not been defined to date. To address this gap, our recent studies have analyzed spleen transcriptomes of P. leucopus and C3H/HeJ (C3H) mice in response to the persistent Bb infection (28, 32). The results demonstrated that, when compared to the respective uninfected controls, P. leucopus and C3H mice that had been infected with Bb strain 297 for 70 days responded differently. Specifically, the transcriptomic response of the white-footed mice was much more quiescent with a total number of differentially expressed genes (DEGs) being several folds lower (81 DEGs) than that of the infected C3H mice (421 DEGs) (28). Approximately 57% and 43% of the 81 DEGs were down- and upregulated, respectively, while the 421 DEGs comprised about 62% downregulated and 38% upregulated genes. As the current investigation is a follow-up of our published study (28), the design of the previous infectivity experiment has been replicated, yet this time we also included both male and female mice and a higher number of animals per sex. C3H mice were similarly selected as a comparative species as this strain consistently exhibits clinical signs of LD (e.g., arthritis) (33–37). To complement our published transcriptome findings, the present work involved behavioral, hematological, biochemical, histological, and immunological analyses.

MATERIALS AND METHODS

Bacterial strain and mouse infection

Wild-type B. burgdorferi strain 297 was used for the infectivity study. Bacterial cells were cultivated in laboratory-made liquid Barbour-Stoenner-Kelly medium with 6% rabbit sera (Gemini Bio-Products, California, USA; referred to here as BSK-II) at 35°C under 2.5% CO₂.

Ten female and 10 male C3H/HeJ (C3H) mice were acquired from the Jackson Laboratories (Maine, USA), whereas 10 females and 10 males of domesticated and restricted free-mating Peromyscus leucopus lenville mice (LL stock) were purchased from The Peromyscus Genetic Stock Center (the University of South Carolina, South Carolina, USA) (38). C3H mice were separated by sex and infection status (two or three mice per cage), and each P. leucopus mouse was housed individually.

To infect mice, a 100-µL inoculum containing ~1 × 10⁵ spirochetes was subcutaneously (s.c.) administered in the scapular region of each animal. C3H and P. leucopus mice were challenged at 7 and 6–10 weeks of age, respectively. The presence or lack of Bb infection in each mouse was verified by culturing a piece of pinna, part of the heart, part of the bladder, and cartilaginous tips of both tibiotarsal joints harvested at days 72 (all C3H mice) and 73 (all P. leucopus mice) postinoculation (pi) in BSK-II with the antibiotic cocktail (0.02 mg mL⁻¹ phosphomycin, 0.05 mg mL⁻¹ rifampicin, and 2.5 mg mL⁻¹ amphotericin B). Days 72 and 73 pi will be referred to here as day 70 pi. Dates of birth and ages of the mice at the time of inoculation and at the time of their sacrifice are provided in Tables S1 and S2. The lack of infection in each control (never challenged) animal was also confirmed by culturing ear pinnae sampled at the time of mouse sacrifice. Heart tissues were cultured in 3 mL of BSK-II utilizing 8-mL polystyrene tubes (Becton Dickinson Labware, New Jersey, USA); whereas tissues of bladder, tibiotarsal joint, or ear were cultivated in 1.0 mL of BSK-II using 1.7-mL polypropylene microcentrifuge tubes (Denville Scientific Inc., Massachusetts, USA) at 35°C under 2.5% CO₂ for up to 4 weeks. Cultures were weekly assessed by dark-field microscopy to confirm the presence or lack of live spirochetes.

Behavioral assessment

To detect whether mice would endure any pain associated with Bb infection, both Bb-infected P. leucopus and C3H mice as well as their uninfected controls were visually assessed by using a previously developed mouse grimace scale (MGS) (39). The mice were also evaluated for signs of hunched posture (0 – no hunch, 1 – mild hunch, and 2 – major hunch) and their activity (0 – normal activity [e.g., eating, drinking, walking around normally], 1 – abnormal behavior [e.g., appearing sick, abnormal movement]) (40). The clinical assessment of each animal was measured independently by three trained observers (Comparative Medicine Program residents and personnel) 5 days prior to and biweekly after the Bb challenge.

Blood and serum analyses

Mouse blood was collected in EDTA tubes (BD Microtainer Tubes, Becton, Dickinson and Company, New Jersey, USA). Blood cells were measured using the mouse setting of VETSCAN HM5 Hematology Analyzer (Zoetis, New Jersey, USA) within 4 hours of blood collection in the Texas A&M Preclinical Phenotyping Core (TPPC) of Texas A&M Institute for Genome Sciences and Society (TIGSS). The analyzed parameters included total white blood cell count (WBC), lymphocyte count (LYM), monocyte count (MON), granulocyte (basophils, eosinophils, and neutrophils) count (GRA), lymphocyte percentage (LYM%), monocyte percentage (MON%), granulocyte percentage (GRA%), red blood cell count (RBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width with standard deviation (RDW-SD) and coefficient of variation (RWD-CV), platelet count (PLT), platelet crit (PCT), mean platelet volume (MPV), and platelet distribution width with standard deviation (PDW-SD) and coefficient of variation (PDW-CV). Quality control was performed with VetScan HM5 Control Normal Level (Zoetis, Kalamazoo, Michigan, USA) on each day of measurement.

The levels of haptoglobin (HAPT), iron (Fe), and thyroxine 4 (T4) in serum samples were determined by using DXC 700 AU Chemistry Analyzer (Beckman Coulter Ireland Inc., Ireland) in TPPC (TIGSS). The parameters and settings of each test were applied according to the manufacturer’s recommendation. When a sample volume was not sufficient, serum was diluted with Med water (Med Water Systems, Utah, USA) to meet the minimum required volume set by the analyzer, and this dilution was considered for the respective calculation.

Cytokine and chemokine measurements

Serum samples were analyzed by using ProcartaPlex Mouse Cytokine & Chemokine Convenience Panel 1A 36-Plex (Thermo Fisher Scientific, Massachusetts, USA) on Bio-Plex 200 system (Bio-Rad, California, USA) following the manufacturer’s instructions. Targeted analytes included ENA-78(LIX), Eotaxin, G-CSF/CSF-3, GM-CSF, Gro-alpha/KC, IFN-α, IFN-γ, IL-1α, IL-1β, IL-10, IL-12p70, IL-13, IL-15/IL-15R, IL-17A, IL-18, IL-2, IL-22, IL-23, IL-27, IL-28, IL-3, IL-31, IL-4, IL-5, IL-6, IL-9, IP-10, LIF, MCP-1, MCP-3, M-CSF, MIP-1α, MIP-1β, MIP-2, RANTES, and TNF-α.

Histopathology

Hearts, tibiotarsal joints, urinary bladder, and both kidneys were collected and fixed in 10% neutral buffered formalin immediately upon tissue harvest. The joints were decalcified for 4 days prior to trimming in fixative decalcifier solution (Formical-4 Decalcifier; StatLab Medical Products, Texas, USA). Joints, bladders, and kidneys were sagittally cut in half. Hearts were horizontally cut in several pieces. The heart, urinary bladder, and left kidney were processed together in one cassette, and the right kidney and joints were processed in separate cassettes each. The tissues were then embedded in paraffin, cut as 4-µm sections, and stained with hematoxylin and eosin (H&E) using the standard procedures. The histological sections were scored in a blind manner by a board-certified pathologist.

The histologic sections of the heart were scored based on the scoring system adapted from our previous publication (41): 0 for no changes (no inflammation in the myocardium), 1 for minimal changes (minimal inflammation of lymphocytes and neutrophils [single inflammatory cells] and edema in the myocardium), 2 for mild changes (mild inflammation of lymphocytes and neutrophils [clusters of inflammatory cells] and edema in the myocardium), 3 for mild to moderate changes (mild clusters of lymphocytes and neutrophils and edema in the myocardium), and 4 for moderate to severe changes (moderate to severe inflammation of lymphocytes and neutrophils [larger foci of inflammation] and edema in the myocardium).

For joints, we also used our previously applied scoring system (41). Specifically, the following criteria were separately evaluated: synovial hyperplasia (0, no change; 1, mild; 2, moderate; 3, severe; 4, severe with papilliform growth), exudate within joint and/or tendon sheath (0, no change; 1, <10 inflammatory cells [neutrophils]; 2, 10–49 inflammatory cells; 3, 50–100 inflammatory cells; 4, >100 inflammatory cells), superficial inflammation/resorption of bone (0, no change; 1, mild; 2, moderate; 3, moderate to severe; 4, severe), and overall inflammation (0, no change; 1, 1%–24% of inflammatory cells [neutrophils, macrophages, lymphocytes, plasma cells, and mast cells]; 2, 25%–40%; 3, 41%–60%; 4, 61%–100%). The overall score was calculated as an average of different scores.

The histologic sections of kidneys were scored from 0 to 4 as defined: 0 for no inflammation; 1 for focal, mild inflammation (<1%); 2 for multifocal, mild inflammation (1%–10%); 3 for multifocal, moderate inflammation (11%–25%); and 4 for multifocal-coalescing, severe inflammation (>25%). An overall score of kidney scores was calculated using the average of both scores.

The histologic sections of the urinary bladder were scored from 0 to 4 as defined: 0 for no inflammation; 1 for minimal inflammation (<1%); 2 for mild lymphoid hyperplasia (1%–10%); 3 for moderate lymphoid hyperplasia (11%–25%); and 4 for severe lymphoid hyperplasia (>25%). In three of the P. leucopus mice (one uninfected and two 297-infected) and four of the C3H mice (one uninfected, three 297-infected), the urinary bladder was lost during processing due to the small size. The score was set to 0 if no urinary bladder was present for evaluation. An overall score that combined the kidney, joint, and urinary bladder scores was also calculated.

Statistics

The statistical analyses were performed by using SAS software (version 9.4, SAS Institute Inc., North Carolina, USA). For the blood and serum parameters as well as multiplex immunoassay values, the t-test was performed to evaluate a difference between sexes (females vs males), infection status (infected vs uninfected mice), and mouse models (C3H vs P. leucopus mice). The SAS PROC MIXED procedure was used to perform Tukey’s multiple comparison test. The model included the group and sex as fixed effects, and the interaction between the group and sex was also considered. Least squared means were computed for each combination of the group and sex. The difference was considered statistically significant at 5% significance level (P < 0.05).

RESULTS

The culture results showed that all the tissues (pinnae, bladders, hearts, and tibiotarsal joints) harvested from 297-challenged mice at day 70 pi had consistently tested positive by culture, demonstrating the 100% infectivity rate for both P. leucopus and C3H mice (Table 1). Expectedly, the respective uninfected control animals were culture-negative upon harvest.

TABLE 1.

Culture results of tissues harvested from B. burgdorferi 297-infected and uninfected C3H and P. leucopus mice

Tissue harvested for culture upon sacrifice	No. of cultures positive/total no. tested
	297-infected C3H mice (Group A)	Uninfected C3H mice (Group B)	297-infected P. leucopus mice (Group C)	Uninfected P. leucopus mice (Group D)
Ear pinna	10/10	0/9a	9/9a	0/9a
Bladder	10/10	ntb	9/9	nt
Heart	10/10	nt	9/9	nt
Tibiotarsal joint	10/10	nt	9/9	nt
Total	10/10	nt	9/9	nt

^a There was a total of three mortalities—one per group B, C, and D.

^b nt denotes non-tested.

To detect any clinical signs of LD in the Bb-infected mice, each animal including uninfected controls was observed once prior to Bb challenge and at biweekly intervals over the 70-day period pi. To minimize any bias, the mice were visually evaluated by three well-trained observers for the presence of facial expressions indicating pain as well as other welfare indicators such as posture and activity status as detailed (39, 40). The results showed that during the live observations, both infected and uninfected P. leucopus and C3H mice had no abnormal activity as evidenced by their normal eating, drinking, and walking patterns. Moreover, all mice developed no hunches and were consistently given a score of zero on the MGS scale, indicating the lack of Bb infection-associated pain (data not shown).

The averaged values of 23 blood and serum parameters measured in samples from all P. leucopus and C3H males and females prior to and post-Bb challenge (day 70 pi) are presented in Table 2. These clinical parameters were statistically compared between the infected and uninfected groups for each sex and the two sexes combined within and between P. leucopus and C3H mice for each time point (day 0 and day 70 pi). Table S3 contains all the performed comparisons and the respective significant P values (<0.05) with the greater or less sign, indicating which group had a statistically higher or lower parameter, respectively. The day-0 data analysis demonstrated that the uninfected males and females of C3H mice had significant sex-specific differences in a total of 10 clinical parameters, eight of which had consistently higher values in the males (WBC, LYM, MON, MCH, MPV, PDW-CV, PDW-CV, HAPT). In contrast, the uninfected males and females of P. leucopus mice differed only in their T4 level, which was significantly higher in the males (P = 0.0173; 3.25 vs 2.60 µg/dL). When the day-0 data comparisons were performed between the uninfected C3H and P. leucopus mice, most of the clinical parameters were significantly different between the two mouse models regardless of their sex. Far fewer significant differences were observed within C3H mouse groups for the day-70 data, where most of sex-specific differences were detected for only either uninfected (RDW-CV, MPV, PDW-SD, and PDW-CV) or infected (GRA and MCV) C3H mice. MCHC was the only parameter that was consistently higher in the males than in the females of C3H mice regardless of their infection status (Table 2; Table S3). When the infected and uninfected C3H mice were compared, there were also only a few significant differences detected for each sex. Of note, the level of Fe was lower in the infected animals for both females and males. Furthermore, the analysis of day-70 data for P. leucopus mice showed almost no differences between the groups regardless of the sex and/or infection status with the exceptions being MCH, PDW-CV, Fe, and T4. Finally, significant differences were identified for most of the parameters when the comparisons were performed between C3H and P. leucopus mice within their infection status (Table 2; Table S3).

TABLE 2.

Hematological and biochemical parameters of the blood sampled from B. burgdorferi 297-infected and uninfected C3H and P. leucopus mice

Blood parametersa	Uninfected mice (day 0)				297-infected mice (day 70 postinoculation)				Uninfected control mice (day 70 postinoculation)
	C3H		P. leucopus		C3H		P. leucopus		C3H		P. leucopus
	Female (n = 10)	Male (n = 10)	Female (n = 10)	Male (n = 10)	Female (n = 5)	Male (n = 5)	Female (n = 4)	Male (n = 4)	Female (n = 4)	Male (n = 4)	Female (n = 4)	Male (n = 4)
WBC (109/L)	5.68b (1.33)	8.02 (1.96)	5.99 (2.20)	5.48 (1.97)	9.25 (2.67)	9.30 (3.36)	5.86 (1.29)	5.20 (1.34)	5.22 (3.17)	6.98 (0.47)	4.03 (1.50)	3.88 (2.17)
LYM (109/L)	4.48 (1.25)	6.48 (1.70)	5.34 (1.99)	4.62 (1.82)	7.38 (2.24)	6.65 (3.33)	4.88 (1.00)	4.57 (1.04)	3.84 (2.51)	5.56 (0.68)	3.63 (1.15)	3.29 (1.96)
MON (109/L)	0.12 (0.09)	0.39 (0.34)	0.16 (0.10)	0.16 (0.26)	0.60 (0.56)	0.42 (0.42)	0.22 (0.26)	0.04 (0.02)	0.18 (0.19)	0.11 (0.09)	0.08 (0.07)	0.03 (0.02)
GRA (109/L)	1.07 (0.28)	1.15 (0.45)	0.49 (0.53)	0.70 (0.34)	1.27 (0.33)	2.22 (0.75)	0.77 (0.35)	0.59 (0.42)	1.21 (0.54)	1.32 (0.28)	0.32 (0.35)	0.57 (0.29)
LYM (%)	78.26 (5.92)	80.58 (4.16)	89.46 (6.00)	83.53 (7.75)	79.60 (4.13)	69.14 (10.27)	83.58 (6.52)	88.65 (5.78)	71.23 (6.05)	79.38 (4.75)	91.40 (4.89)	83.60 (5.95)
MON (%)	2.35 (1.86)	4.69 (3.72)	2.97 (2.50)	2.37 (2.91)	5.82 (4.55)	3.90 (3.10)	3.40 (3.34)	0.75 (0.30)	3.20 (3.16)	1.53 (1.37)	1.85 (1.73)	0.68 (0.05)
GRA (%)	19.39 (5.07)	14.73 (4.73)	7.59 (6.43)	14.11 (8.25)	14.56 (5.36)	26.94 (13.13)	13.00 (5.42)	10.60 (5.98)	25.53 (7.75)	19.10 (4.61)	6.75 (5.27)	15.75 (5.99)
RBC (1012/L)	9.09 (1.27)	9.57 (0.55)	11.24 (1.25)	10.95 (1.35)	9.99 (0.74)	9.44 (0.87)	12.06 (2.52)	11.19 (0.86)	9.25 (0.28)	9.76 (0.41)	11.04 (1.75)	11.72 (1.58)
HGB (g/dL)	13.49 (2.19)	14.72 (1.01)	13.69 (1.90)	13.32 (1.82)	14.50 (1.24)	13.80 (1.50)	14.15 (2.75)	12.13 (1.10)	13.55 (0.64)	14.68 (0.97)	12.75 (2.40)	12.93 (2.01)
HCT (%)	48.80 (6.58)	52.13 (2.74)	42.44 (5.34)	40.30 (5.35)	53.57 (4.13)	47.49 (4.86)	43.15 (7.37)	37.70 (4.96)	52.05 (4.11)	52.03 (1.86)	38.46 (5.64)	39.23 (4.56)
MCV (fL)	53.60 (1.65)	54.60 (1.08)	37.80 (1.40)	36.70 (1.42)	53.80 (2.59)	50.20 (1.30)	36.25 (2.50)	33.50 (1.73)	56.50 (3.51)	53.25 (0.50)	34.75 (1.26)	33.75 (1.50)
MCH (pg)	14.80 (0.48)	15.36 (0.30)	12.17 (0.93)	12.16 (0.52)	14.52 (0.27)	14.62 (0.40)	11.80 (0.41)	10.85 (0.45)	14.65 (0.34)	15.05 (0.37)	11.53 (0.48)	11.03 (0.52)
MCHC (g/dL)	27.58 (1.11)	28.20 (0.71)	32.27 (1.97)	33.06 (1.10)	27.14 (1.34)	29.02 (1.12)	32.70 (1.14)	32.40 (2.60)	26.10 (1.20)	28.18 (0.86)	33.08 (1.60)	32.90 (1.61)
RDW-SD (fL)	37.97 (2.73)	38.13 (0.96)	27.66 (1.33)	27.12 (1.09)	40.18 (2.39)	37.18 (1.78)	27.53 (0.72)	26.38 (2.83)	39.85 (1.79)	38.88 (0.97)	26.38 (0.72)	26.38 (0.97)
RDW-CV (%)	18.80 (0.84)	18.53 (0.22)	19.76 (0.66)	19.92 (0.42)	19.72 (0.54)	19.50 (0.59)	20.65 (1.22)	20.98 (1.16)	18.55 (0.39)	19.20 (0.22)	20.68 (0.90)	21.20 (1.17)
PLT (109/L)	330.10 (99.97)	355.30 (81.20)	128.40 (32.09)	145.80 (61.40)	478.80 (140.71)	320.20 (125.62)	134.25 (48.07)	117.75 (67.73)	422.25 (135.19)	366.25 (149.81)	133.75 (36.11)	135.00 (86.52)
PCT (%)	0.27 (0.09)	0.30 (0.08)	0.07 (0.02)	0.08 (0.03)	0.43 (0.13)	0.27 (0.12)	0.07 (0.03)	0.07 (0.03)	0.35 (0.13)	0.33 (0.13)	0.08 (0.02)	0.08 (0.05)
MPV (fL)	8.03 (0.47)	8.44 (0.37)	5.31 (0.15)	5.29 (0.21)	8.90 (0.37)	8.16 (0.90)	5.45 (0.30)	5.63 (0.30)	8.25 (0.37)	8.85 (0.26)	5.88 (0.90)	6.20 (1.24)
PDW-SD (fL)	13.86 (1.78)	16.24 (1.51)	6.69 (0.51)	6.56 (0.75)	16.06 (2.18)	15.00 (2.86)	6.15 (0.57)	6.90 (0)	13.60 (1.23)	16.90 (1.43)	7.25 (0.70)	7.25 (0.70)
PDW-CV (%)	37.58 (1.45)	39.50 (1.05)	28.06 (1.06)	27.61 (1.65)	39.28 (1.68)	38.40 (2.32)	26.78 (1.27)	28.40 (0)	37.43 (1.09)	39.95 (0.96)	28.78 (0.75)	28.05 (0.70)
Fe (μg/dL)	335.36 (34.59)	303.95 (25.26)	452.42 (69.51)	402.04 (52.93)	261.40 (32.42)	261.40 (11.93)	369.00 (46.09)	272.00 (125.40)	315.75 (32.78)	291.50 (14.25)	452.25 (67.62)	214.75 (80.12)
T4 (μg/dL)	6.76 (0.93)	7.44 (0.65)	2.60 (0.66)	3.25 (0.42)	5.66 (0.75)	6.40 (0.90)	2.90 (0.47)	3.70 (0.75)	5.33 (0.46)	6.13 (0.99)	2.68 (0.34)	2.48 (0.65)
HAPT (mg/dL)	0.24 (0.76)	1.49 (1.15)	7.94 (5.44)	9.42 (3.30)	6.57 (7.06)	3.11 (1.94)	7.68 (4.11)	7.97 (2.60)	0.00 (0.00)	0.91 (0.90)	3.45 (3.05)	5.56 (3.41)

^a WBC, total white blood cell count; LYM, lymphocyte count; MON, monocyte count; GRA, granulocyte (basophils, eosinophils, and neutrophils) count; LYM (%), lymphocyte percentage; MON (%), monocyte percentage; GRA (%), granulocyte percentage; RBC, red blood cell count; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW-SD and RDW-CV, red blood cell distribution width with standard deviation and coefficient of variation, respectively; PLT, platelet count; PCT, platelet crit; MPV, mean platelet volume; PDW-SD and PDW-CV, platelet distribution width with standard deviation and coefficient of variation, respectively; Fe, iron concentration; T4, thyroxine concentration; HAPT, heptoglobin concentration.

^b Provided are mean values, and in parentheses are standard deviations.

To compare the immunological responses of C3H and P. leucopus mice to Bb infection, several cytokines and chemokines were measured in mouse sera sampled at the time of their sacrifice. Unfortunately, out of 36 targeted analytes, we were able to only generate the data for 20 molecules. The mean values of the 20 cytokines and chemokines and P values for the inter-group comparisons are presented in Table 3; Table S4. When the levels were compared between the infected and uninfected mice for each mouse model, only a few differences were detected. Specifically, the Bb-infected C3H mice had significantly higher levels of IL-12p70 (3.91 vs 2.75 pg/mL), MCP3 (430.28 vs 220.06 pg/mL), MIP-1β (3.13 vs 1.38 pg/mL), and TNF-α (5.29 vs 3.42 pg/mL) than their uninfected C3H controls. By contrast, in the Bb-infected P. leucopus mice, the significant differences were detected only for two molecules, IL-22 and RANTES, with their levels being decreased and elevated by 3.4 and 8 times, respectively, compared to the uninfected P. leucopus mice (Table 3; Table S4). The inter-species comparisons demonstrated noticeably higher number of significant differences in the measured cytokine and chemokine levels for both infected and uninfected animals. Specifically, the Bb-infected P. leucopus mice had significantly lower levels of Eotaxin (117.24 vs 653.00 pg/mL), IL-12p70 (0.53 vs 3.91 pg/mL), IP-10 (4.85 vs 81.29 pg/mL), and TNF-α (0.55 vs 5.29 pg/mL); and about 10 times higher levels of IL-2 (12.66 vs 1.32 pg/mL) and IL-27 (32.41 vs 3.18 pg/mL) compared to the infected C3H mice (Fig. 4). Consistently, as opposed to the uninfected C3H mice (620.69 pg/mL), a significantly lower level of Eotaxin was observed in the uninfected P. leucopus mice (192.38 pg/mL). Finally, significantly higher levels of IFN-γ (5.07 vs 0.58 pg/mL) and IL-22 (50.13 vs 12.19 pg/mL) and lower levels of MCP3 (9.05 vs 220.06 pg/mL) and RANTES (9.16 vs 114.54 pg/mL) were detected in the uninfected P. leucopus mice compared to the uninfected C3H mice (Table 3; Table S4).

TABLE 3.

Levels of cytokines and chemokines measured in B. burgdorferi 297-infected and uninfected C3H and P. leucopus mice

Cytokines/chemokines	C3H		P. leucopus
	297-infected mice (Group A)	Uninfected controls (Group B)	297-infected mice (Group C)	Uninfected controls (Group D)
ENA78	690.55a	801.06	n/ab	n/a
Eotaxin	653.00	620.69	117.24	192.38
GCSF-CSF3	2.78	2.66	n/a	3.10
GM-CSF	1.88	0.79	2.02	2.33
Gro-α-KC	10.65	22.10	5.43	16.56
IFN-γ	0.58	0.58	0.16	5.07
IL-2	1.32	0.28	12.66	14.44
IL-12p70	3.91	2.75	0.53	1.13
IL-13	12.13	53.11	22.40	n/a
IL-22	17.88	12.19	14.68	50.13
IL-27	3.18	27.14	32.41	1.88
IP-10	81.29	87.43	4.85	0.45
LIF	28.38	5.43	7.30	8.91
MCP1	19.35	19.82	n/a	n/a
MCP3	430.28	220.06	0.62	9.05
MIP-1α	4.33	20.16	15.46	2.06
MIP-1β	3.13	1.38	n/a	n/a
MIP-2	5.17	4.22	n/a	n/a
RANTES	105.06	114.54	73.57	9.16
TNF-α	5.29	3.42	0.55	0.36

^a Values are expressed in picograms per milliliter.

^b n/a denotes non-available.

The outcome of Bb infection in both mouse models was also evaluated by histopathological analysis of the heart, tibiotarsal joint, urinary bladder, and kidney. The histological scores for all mice are presented in Table S5, and the group comparisons that had significant differences are provided in Table S6. All heart tissues (myocarditis) were scored as zero for all infected and uninfected P. leucopus and C3H mice including one Bb-infected mouse with mild lymphoplasmacytic inflammation near auricle and one uninfected C3H male with focal, severe, leukocytoclastic vasculitis near auricle. Regardless of the sex, the histological scores of all joint parameters were consistently significantly higher (P < 0.0001) in the infected C3H mice compared to their sex-specific uninfected C3H controls, reiterating that this laboratory strain consistently develop Bb-induced arthritis (Table S6; Fig. 1). Of note, the sex-specific difference was detected between the overall inflammation scores of joints, which were significantly higher in the infected C3H females than in the infected male counterparts (P = 0.0129; Fig. 2). In contrast, the infected C3H males developed more severe cystitis (P = 0.0011) and nephritis (P = 0.0359) compared to the infected C3H females (Fig. 3). The results also showed that the infected C3H males developed cystitis and nephritis when compared to the uninfected C3H males (P = 0.0006 and P < 0.0001, respectively) or females (P < 0.0150 and P = 0.0043, respectively). However, the borrelial infection did not induce any significant inflammation in bladders and kidneys of the C3H females. Collectively, the present histopathology data demonstrated that some detected differences in the response of C3H mice to the persistent borrelial infection were sex-dependent. In contrast to the inbred mouse model, the Bb 297 infection did not induce any histologically significant inflammation in the examined tissues of P. leucopus mice (Fig. 1 and 3; Table S5).

Fig 1.

Histologic appearance of the tibiotarsal joint in B. burgdorferi 297-infected P. leucopus (A and C) and C3H mice (B and D). (A) There are only minimal pathologic changes in the tibiotarsal joint of a 297-infected P. leucopus mouse. 20× magnification. H&E stain. (B) There are severe pathologic changes in the tibiotarsal joint of a 297-infected C3H mouse including moderate inflammation, synovial hyperplasia, and surface inflammation with resorption of the bone. 20× magnification. H&E stain. (C) Higher magnification of A. The mild synovial hyperplasia (arrow) and few inflammatory cells (asterisk) are not significant. 200× magnification. H&E stain. (D) Higher magnification of B. Note severe inflammation (asterisk), moderate synovial hyperplasia (arrow), and neutrophils (arrow heads) within the space between tendon and tendon sheath. 200× magnification. H&E stain. T, tendon; TS, tendon sheath.

Fig 2.

Sex-specific differences in inflammation of the tibiotarsal joint (A and B), kidney (C and D), and urinary bladder (E and F) detected in B. burgdorferi 297-infected C3H mice. (A) Tibiotarsal joint from a 297-infected C3H male. There are multifocal, small areas of inflammation within the tendon sheath (arrow). 20× magnification. H&E stain. (B) Tibiotarsal joint from a 297-infected C3H female. There are multifocal, extensive areas of inflammation within the tendon sheath (arrows). 20× magnification. H&E stain. (C) Kidney from a 297-infected C3H male. There is moderate perivascular inflammation (arrow). 40× magnification. H&E stain. (D) Kidney from a 297-infected C3H female. There is minimal inflammation (arrow) in the pelvis. 40× magnification. H&E stain. (E) Urinary bladder from a 297-infected C3H male. There is multifocal, moderate lymphoid hyperplasia (arrow). 40× magnification. H&E stain. (F) Urinary bladder from a 297-infected C3H female. There is mild lymphocytic inflammation (arrow). 40× magnification. H&E stain. T, tendon; TS, tendon sheath.

Fig 3.

Histologic appearance of the kidney and urinary bladder in B. burgdorferi 297-infected P. leucopus (A and C) and C3H mice (B and D). (A) Urinary bladder of a 297-infected P. leucopus mouse. There are no significant pathologic findings. 40× magnification. H&E stain. (B) Urinary bladder of a 297-infected C3H mouse. Note multifocal, moderate lymphoid hyperplasia (arrow) in the submucosa. 40× magnification. H&E stain. (C) Kidney of a 297-infected P. leucopus mouse. There is a minimal lymphoplasmacytic inflammation near the renal pelvis (asterisk), which is not significant. 100× magnification. H&E stain. (D) Kidney of a 297-infected C3H mouse. There is multifocal, moderate interstitial lymphoplasmacytic nephritis (asterisks). 100× magnification. H&E stain.

DISCUSSION

Susceptibility of Peromyscus mice to borrelial infection

The culture results of the present study demonstrated that, similar to C3H mice, 100% P. leucopus mice became infected with Bb 297 when s.c. challenged with ~1 × 10⁵ spirochetes per mouse. Moreover, all four tissues (the pinna, bladder, heart, and tibiotarsal joint) consistently tested positive for every challenged mouse. This 100% infectivity rate for P. leucopus mice is fully consistent with the result of our preceding study (28) as well as an earlier investigation, where another reservoir-competent Peromyscus species (42, 43), P. maniculatus, was used (29). In the latter study, all deer mice s.c. challenged with ~1 × 10⁵ cells per animal were susceptible to wild-type strains, 297 and B31, which was evidenced by all culture-positive pinna, heart, bladder, and joint tissues sampled at day 28 pi. Invariably, when Bb challenge was performed via spirochete-harboring nymphs of Ixodes scapularis, 100% of P. maniculatus mice became also susceptible to the two wild types. Moreover, all P. maniculatus mice developed culture-detectable spirochetemia when challenged with attenuated B31-derived mutants (ΔvlsE and svlsE) via either needle or I. scapularis nymphs (29). Despite the ΔvlsE and svlsE clones lacked the functional antigenic variation system required to establish a persistent infection in immunocompetent mice (29, 44–47), both mutants were able to establish spirochetemia in all deer mice (29). Collectively, these published culture results contrasted the outcome of a recent investigation, which unexpectedly reported a significantly impaired infectivity rate of Bb B31 for P. leucopus mice (27). In that study, after challenging with B31-carrying I. scapularis nymphs, all mice tested negative by pinna culture for week 4 pi, and only about half of the animals turned positive by week 12 pi (27). Inconsistent with their own culture results, the droplet PCR data of the same study demonstrated that pinnae from all challenged mice tested positive for weeks 4 through 12 pi (27). Taken together, the previous and present data have demonstrated that, under the described experimental conditions, the two Peromyscus species, P. leucopus and P. maniculatus, are fully susceptible to Bb 297 and B31, presenting no restriction to the borrelial infection.

Lack of pathology and behavioral abnormality in P. leucopus mice

The current histopathology results have shown that C3H mice developed borrelia-induced arthritis, which has been repeatedly demonstrated by numerous early and more recent investigations (33–37, 41, 48). As the present data were also analyzed by sex, we were able to identify sex-specific differences for C3H mice: Bb infection-induced cystitis and nephritis were only detectable in males. In contrast, no significant pathological lesions in the heart, tibiotarsal joint, urinary bladder, or kidney were identified in the Bb-infected P. leucopus mice, which strongly indicates the lack of disease processes in the respective organs, thus supporting some earlier findings (6). The absence of pathology may well account for the lack of detectable behavioral abnormalities and pain-reflecting facial expressions in the infected P. leucopus mice. However, the behavioral metrics, which also remained normal for the infected C3H mice during the entire 70-day period, did not corroborate with the significant pathological lesions observed in these inbred mice. It is well possible that the behavioral assessment tools applied in this study were insufficiently sensitive to reflect clinical manifestations of arthritis, nephritis, and/or cystitis in the Bb-infected C3H mice. To overcome this potential caveat, more objective quantitative phenotyping methods (e.g., DigiGait, open field, rotarod) could be used in the future. Of note, over the 70-day period, no signs of seizures or paralysis were detected in any Bb-infected P. leucopus or C3H mice, which discouraged us from collecting neural tissues (e.g., brains, spinal cords) for histopathological analysis. Overall, our results, demonstrating the lack of clinical signs in Bb-infected white-footed mice, are consistent with the findings of several prior investigations (3, 22, 49, 50). By analyzing a high number of P. leucopus captures (n = 2,181), an earlier study involving a capture-mark-recapture test provided a strong piece of evidence against a negative impact of Bb infection on the survivability of white-footed mice in nature (49). Another study demonstrated no difference in wheel-running activity between uninfected controls and P. leucopus mice presumably infected with (or at least exposed to) Bb via I. scapularis nymphs (22). The infection status of the latter experimental group was only confirmed serologically by ELISA (22). The results of a more recent investigation, which quantified the effect of varying borrelial loads on the movement as well as foraging and self-grooming behavior of the white-footed mouse, suggested only a potential reduction of self-grooming with no other noticeable differences recorded (50).

Missing reference intervals and clinical unresponsiveness to borrelial infection

In this study, we have measured a total of 23 blood parameters in uninfected P. leucopus (n = 20) and C3H mice (n = 20), which allowed us to contribute to previously published, but still scarce, data sets on hematological parameters of white-footed mice (22, 51, 52). For convenience of the reader, the data of three previous and our present investigations are compiled in Table 4. Upon comparison, some blood parameters (e.g., WBC, LYM, MON, RBC) were found to be less consistent than others (e.g., HGB, HCT, MCV, MCH) with monocyte and platelet values being variable by 2.5- to fourfold (Table 4). When the values of uninfected C3H mice were contrasted with the respective data of another inbred strain, C57BL/6 mice (51), about two- to threefold differences were also observed for some parameters (e.g., RDW-CV, PLT, MPV). Overall, the high variability in the hematological parameters of P. leucopus mice observed between the four studies could potentially be explained by a variety of factors including the outbred nature of the white-footed mouse itself as well as different analytical methods and instrumentations utilized. In the future, a significantly higher number of deer mice are needed to generate sufficient amount of data for establishing currently missing age- and sex-specific reference intervals. Given their commercial availability and that Peromyscus species are directly relevant models to study the Bb enzootic cycle under controlled experimental conditions, the authors hope that this report will prompt the scientific community to collectively invest into generating reliable and accurate reference ranges.

TABLE 4.

Hematological parameters obtained by the current and earlier investigations from uninfected P. leucopus mice

Blood parametersa	This study (the day-0 data)		(52)		(51)		(22)
	Female (n = 10)	Male (n = 10)	Female (n = 19–20)	Male (n = 20)	Female (n = 45)	Male (n = 29)	Male (n = 10)
WBC (109/L)	5.99 (2.20)b	5.48 (1.97)	8.41 (2.72)	8.76 (2.40)	8.8 (0.4)	8.2 (0.5)	7.76 (1.25)
LYM (109/L)	5.34 (1.99)	4.62 (1.82)	8.02 (2.54)	8.00 (2.39)	7.0 (0.3)	6.5 (0.4)	6.32 (0.98)
MON (109/L)	0.16 (0.10)	0.16 (0.26)	0.12 (0.04)c	0.09 (0.08)	0.38 (0.02)	0.38 (0.03)	0.11 (0.05)
NEU (109/L)	n/m	n/m	0.35 (0.27)	0.66 (0.70)	1.3 (0.1)	1.2 (0.1)	0.81 (0.14)
LYM (%)	89.46 (6.00)	83.53 (7.75)	95.0 (4.3)	91.2 (8.8)	n/md	n/m	n/m
MON (%)	2.97 (2.50)	2.37 (2.91)	2.60 (3.05)c	0.95 (0.83)	n/m	n/m	n/m
NEU (%)	n/m	n/m	4.26 (2.83)	7.75 (8.53)	n/m	n/m	n/m
RBC (1012/L)	11.24 (1.25)	10.95 (1.35)	10.9 (1.0)	12.0 (0.8)	11.2 (0.3)	11.4 (0.3)	14.00 (1.44)
HGB (g/dL)	13.69 (1.90)	13.32 (1.82)	14.3 (1.4)	13.0 (1.0)	13.0 (0.3)	13.2 (0.4)	n/m
HCT (%)	42.44 (5.34)	40.30 (5.35)	41.1 (3.9)	44.6 (3.3)	41.1 (1.1)	43.0 (1.4)	n/m
MCV (fL)	37.80 (1.40)	36.70 (1.42)	37.5 (3.0)	37.2 (2.4)	36.7 (0.4)	37.8 (0.5)	n/m
MCH (pg)	12.17 (0.93)	12.16 (0.52)	13.1 (0.8)	10.8 (0.7)	11.6 (0.1)	11.6 (0.2)	n/m
MCHC (g/dL)	32.27 (1.97)	33.06 (1.10)	34.9 (2.1)	29.1 (0.5)	n/m	n/m	n/m
RDW-SD (fL)	27.66 (1.33)	27.12 (1.09)	n/m	n/m	n/m	n/m	n/m
RDW-CV (%)	19.76 (0.66)	19.92 (0.42)	n/m	n/m	18.4 (0.2)	17.6 (0.2)	n/m
PLT (109/L)	128.40 (32.09)	145.80 (61.40)	368 (131)	421 (145)	400 (33)	421 (42)	n/m
PCT (%)	0.07 (0.02)	0.08 (0.03)	n/m	n/m	n/m	n/m	n/m
MPV (fL)	5.31 (0.15)	5.29 (0.21)	4.83 (0.86)	4.61 (0.49)	3.56 (0.04)	3.54 (0.05)	n/m
PDW-SD (fL)	6.69 (0.51)	6.56 (0.75)	n/m	n/m	n/m	n/m	n/m
PDW-CV (%)	28.06 (1.06)	27.61 (1.65)	n/m	n/m	n/m	n/m	n/m

^a WBC, total white blood cell count; LYM, lymphocyte count; MON, monocyte count; NEU, neutrophil count; LYM (%), lymphocyte percentage; MON (%), monocyte percentage; NEU (%), neutrophil percentage; RBC, red blood cell count; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RDW-SD and RDW-CV, red blood cell distribution width with standard deviation and coefficient of variation, respectively; PLT, platelet count; PCT, platelet crit; MPV, mean platelet volume; PDW-SD and PDW-CV, platelet distribution width with standard deviation and coefficient of variation, respectively.

^b Provided are mean values, and in parentheses are standard deviations.

^c Measurements were performed on five mice.

^d n/m denotes not measured.

Despite the lack of established reference intervals, the sex- and age-matched uninfected controls allowed us to statistically evaluate the blood and serum parameters of the infected animals. The sex-combined data revealed no changes in any of the 23 parameters measured in the P. leucopus mice after the 70-day infection period. This is in agreement with an earlier study that also did not find any differences in LYM, MON, RBC, and WBC counts between 9 uninfected and 10 serologically borrelia-positive P. leucopus males (22). Overall, these clinical findings are supported by the complete absence of pathological lesions, altogether reiterating that LD is not developed in the white-footed mouse. However, the analysis of C3H mouse data also demonstrated only a few changes even though these inbred mice did develop significant arthritis, cystitis, and/or nephritis upon Bb infection. These negative findings were unexpected and are worth investigating. Moreover, those few significant differences identified in C3H mice were mainly sex-associated, suggesting a hormonal influence on the mouse anti-borrelial response. The similar trend of unresponsiveness was also observed for cytokines and chemokines in both mouse models. Among the 23 analytes measured in the Bb-infected P. leucopus mice, the levels of only six cytokines and chemokines (Eotaxin, IL-2, IL-12p70, IL-27, IP-10, and TNF-α) were significantly different from those of the infected C3H mice (Fig. 4). A potential involvement of these immunoregulatory molecules in the LD tolerance of the white-footed mouse warrants further investigation. Finally, it is worth mentioning that the persistent borrelial infection in C3H mice was characterized by increased levels of IL-12p70, MCP3, MIP-1β, and TNF-α, which could be suggestive of subclinical inflammatory state. The latter was also supported by elevated levels of haptoglobin, an acute phase protein (53), and decreased iron levels in the infected C3H mice compared to their uninfected C3H controls (Fig. 5). Of note, the increase in proinflammatory cytokines, such as TNF-α, has been documented to reduce iron availability through hepcidin-regulated sequestration of iron in macrophages and enterocytes (54).

Fig 4.

Serum cytokine and chemokine profile differences in B. burgdorferi 297-infected C3H and P. leucopus (PL) mice. Dots represent the levels of TNF-α (A), IL-12p70 (B), IP-10 (C), Eotaxin (D), IL-2 (E), and IL-27 (F) measured for individual infected mice at day 70 postinoculation. Error bars represent SD (standard deviation). Statistical analysis was performed by t-test.

Fig 5.

B. burgdorferi 297-induced infection in C3H mice causes serum cytokine, chemokine, and biochemical changes suggestive of a proinflammatory response. Dots represent the levels of TNF-α (A), IL-12p70 (B), MIP-1β (C), MCP-3 (D), iron (Fe) (E), and HAPT (F) measured for individual mice at day 70 postinoculation. Error bars represent SD (standard deviation). Statistical analysis was performed by t-test.

In summary, the present investigation is the first to have extensively assessed clinical hematology phenotype of white-footed mice upon their persistent borrelial infection. Although the respective reference intervals have not been established for P. leucopus mice, their age- and sex-matched uninfected controls allowed us to statistically assess the blood and serum parameters. Together, the current findings, which also included biochemical, immunological, histological, and behavioral results, demonstrated clinical unresponsiveness of P. leucopus mice to the long-term infection of the LD pathogen. Overall, the present results supported the previously documented quiescent transcriptomic response of P. leucopus mice to the borrelial tenancy (28, 32). An essential thread of our successive studies appears to be the ability of Bb spirochetes to evade and downregulate host defenses. These properties, acted upon by different host influences, may produce few symptoms in P. leucopus and chronic symptomatic infection in C3H mice and humans.

ETHICS APPROVAL

Mice were housed in an animal facility at Texas A&M University, which is accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International. All the experiments described in this study were approved by the Institutional Animal Care and Use Committee of Texas A&M University (IACUC 2017-0390) and were performed following the Public Health Service Policy on Humane Care and Use of Laboratory Animals (2002), Guide for the Care and Use of Agricultural Animals in Research and Teaching (2010), and Guide for the Care and Use of Laboratory Animals (2011).

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/iai.00382-24.

Table S1. iai.00382-24-s0001.docx.

The age of P. leucopus mice at the beginning of the infectivity study and at the time of sacrifice.

Table S2. iai.00382-24-s0002.docx.

The age of C3H mice at the beginning of the infectivity study and at the time of sacrifice.

Table S3. iai.00382-24-s0003.xlsx.

Significant differences in hematological and biochemical parameters identified between B. burgdorferi 297-infected and uninfected C3H and P. leucopus mice.

Table S4. iai.00382-24-s0004.xlsx.

Levels of cytokines and chemokines measured in B. burgdorferi 297-infected and uninfected C3H and P. leucopus mice.

Table S5. iai.00382-24-s0005.xlsx.

The histological scores of hearts, tibiotarsal joints, urinary bladders, and kidneys harvested from B. burgdorferi 297-infected and uninfected C3H and P. leucopus mice.

Table S6. iai.00382-24-s0006.docx.

Significant histological differences of least squares means detected between B. burgdorferi 297-infected and uninfected C3H and P. leucopus groups.

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