Passive Tick Surveillance: Exploring Spatiotemporal Associations of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae),Babesia microti (Piroplasmida: Babesiidae),and Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae) Infection in Ixodes scapularis (Acari: Ixodidae)

Abstract

Ixodes scapularis transmits a group of pathogens, including Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum, the causative agents for Lyme disease, babesiosis, and anaplasmosis, respectively. I. scapularis ticks submitted by state residents to the Connecticut Agricultural Experiment Station-Tick Testing Laboratory between 2015 and 2018 were screened using standard PCR and pathogen-specific primers. Infection and coinfection prevalence in I. scapularis was estimated to assess differences in infection status by life stage (nymph or adult female), county, and year, as well as whether infection with B. burgdorferi changes the likelihood of infection with either B. microti or A. phagocytophilum. Of the 11,254 I. scapularis acquired in Connecticut, 40.7% tested positive for at least one pathogen and the remaining 59.3% were negative. Most I. scapularis ticks tested positive for a single pathogen (33.6%), and only 7.2% were infected with more than one pathogen, of which 93.2% were identified with dual infection and 6.8% tested positive for all three pathogens. Adults were more likely than nymphs to be infected or coinfected with these pathogens. Furthermore, we found that ticks were 74% more likely to be infected with B. microti and 98% more likely to be infected with A. phagocytophilum if infected with B. burgdorferi compared with those not infected. We did not find spatial differences in infection or coinfection prevalence, but between 2015 and 2018, the likelihood that a tick was coinfected increased with time. These results from Connecticut, an endemic state for Lyme disease with long-established populations of I. scapularis, suggest that the increased likelihood of coinfection prevalence over time may have significant implications for clinical diagnosis, course, severity, and treatment of human disease cases.

Introduction

I xodes (Ixodes) scapularis (Acari: Ixodidae) is currently responsible for transmitting seven pathogens to humans (Eisen et al. 2017), of which the three most common are Borrelia burgdorferi (Spirochaetales: Spirochaetaceae), Babesia microti (Piroplasmida: Babesiidae), and Anaplasma phagocytophilum (Rickettsiales: Anaplasmataceae), causing Lyme disease, babesiosis, and anaplasmosis, respectively (Piesman and Spielman 1979, Burgdorfer et al. 1982, Pancholi et al. 1995, Telford et al. 1996, Eisen and Eisen 2018). The incidence and geographic range of these three diseases continue to increase in the United States (Joseph et al. 2011, Stafford et al. 2014, Dahlgren et al. 2015, Centers for Disease Control and Prevention 2016, 2017, 2018, Eisen and Eisen 2018).

The preferred hosts for immature I. scapularis are the white-footed mouse (Peromyscus leucopus) and other small mammals, which serve as the primary reservoir for the three aforementioned pathogens (Spielman 1976, Spielman et al. 1981, Mather et al. 1989). As adults, I. scapularis prefer larger mammalian hosts, primarily the white-tailed deer, Odocoileus virginianus (Piesman and Spielman 1979). Human infection is incidental, and occurs when humans encroach upon habitat that harbors ticks and their hosts.

Given the shared rodent/mammal/tick transmission cycle, these pathogens will occur where I. scapularis is or will become established (Nelder et al. 2016), and with its continued geographic spread, coinfections may also increase. Coinfection in ticks could lead to concurrent human infection with B. burgdorferi and B. microti or A. phagocytophilum, which may complicate diagnosis, lead to insufficient treatment, and increase the severity of disease (Thomas et al. 2001, Belongia 2002, Swanson et al. 2006, Horowitz et al. 2012, Caulfield and Pritt 2015, Diuk-Wasser et al. 2016).

Connecticut, with endemic populations of I. scapularis throughout its eight counties (Dennis et al. 1998, Eisen et al. 2016) and one of the highest incidence rates of Lyme disease, babesiosis, and anaplasmosis in the United States (Centers for Disease Control and Prevention 2016, 2017, 2018), provides an ideal location to investigate infection and coinfection prevalence (Hamer et al. 2014). In this study, we estimate the prevalence of infection and coinfection in I. scapularis with B. burgdorferi, B. microti, and A. phagocytophilum in ticks passively collected in Connecticut between 2015 and 2018. We aim to assess differences in infection status by life stage (nymph and adult female), county, and year as well as whether infection with B. burgdorferi changes the likelihood of infection with either B. microti or A. phagocytophilum.

Materials and Methods

Tick collections and passive surveillance

As part of the passive surveillance program, ticks recovered from humans are submitted by residents, health departments, and physicians' offices to the Connecticut Agricultural Experiment Station-Tick Testing Laboratory (CAES-TTL) for pathogen testing. On the tick submission form, the person submitting the tick must enter their, or their patient's town of residence and provide information on the likely town the tick was acquired, if it is known to be different from the town of residence. Only ticks submitted and acquired from Connecticut are retained for analysis. Ticks are examined under a dissecting microscope and identified to species with standard keys and taxonomic references (Keirans and Litwak 1989, Durden and Keirans 1996), and only I. scapularis ticks are screened for evidence of infection with B. burgdorferi, B. microti, and A. phagocytophilum.

I. scapularis pathogen screening

Screening of ticks was conducted by extracting genomic DNA using DNAzol BD (Molecular Research Center, Cincinnati, OH) according to the manufacturer's recommendations with modifications. Briefly, ticks were washed twice with autoclaved diH₂O and macerated in microtubes containing 400 μL DNAzol BD using a sterile copper BB and a vibration mill (Model MM301; Retsch, Haan, Germany). The homogenates were centrifuged at 14,000 g for 10 min, and supernatant was transferred into a new microtube. After adding 3 μL of Polyacryl Carrier (Molecular Research Center) to the supernatant, DNA was then precipitated by using absolute ethyl alcohol. The DNA pellet was washed twice with 75% ethyl alcohol, air dried briefly, reconstituted in 30 μL of 1 × TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA), and stored at −20°C for further analysis.

Isolated DNA served as template in subsequent PCR to screen for infection with B. burgdorferi using specific primer sets for flagellin (Barbour et al. 1996), the 16S ribosomal RNA (rRNA) (Gazumyan et al. 1994), and Osp A (Persing et al. 1990) genes. A Taq PCR Core Kit (Qiagen, Germantown, MD) was used for PCRs according to the manufacturer's recommendation. A detailed description of these methods is provided elsewhere (Williams et al. 2018). Ticks were initially screened for B. burgdorferi using flagellin gene (Barbour et al. 1996) followed by screening with 16S rRNA (Gazumyan et al. 1994). If the screening results using the two genes were in accordance, the specimen was considered positive, otherwise, the Osp A gene (Persing et al. 1990) was also used for confirmation purpose.

Screening for evidence of infection with B. microti was performed by using a primer set for recombinant DNA (rDNA) (Persing et al. 1992). The cycling conditions were as follows: an initial reaction activation step of 95°C for 10 min, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 60°C for 1 min, and extension at 72°C for 1 min. The final cycle was completed with 10 min of extension at 72°C.

Screening for A. phagocytophilum was carried out by using a specific primer set for 16S rRNA (Steiner et al. 2006, Lee et al. 2014). Cycling conditions included an initial reaction activation step of 95°C for 2 min, followed by 40 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 30 s, and extension at 72°C for 1 min. The final cycle was completed with 5 min of extension at 72°C. All PCRs were performed with Veriti or the GeneAmp PCR System 9700 (Applied Biosystems, Foster City, CA). PCR-amplified products were run on 1–2% agarose gel, stained with ethidium bromide, visualized under ultraviolet light, and documented using GelDoc system (UVP, Upland, CA).

Statistical analysis

I. scapularis infection prevalence, calculated as the total positive divided by the total tested, was determined for each pathogen as well as for coinfections. We assessed differences in I. scapularis infection and coinfection prevalence with logistic regression to determine differences by life stage, spatial differences at the county level, and temporal differences. We also evaluated if infection with B. burgdorferi increased the likelihood of infection with either B. microti or A. phagocytophilum using multivariable logistic regression, where B. burgdorferi infection prevalence was included as an explanatory variable in each model. All models were adjusted for tick life stage (nymph versus adult female) and year by including them as covariates in the models, and significance was assessed at p < 0.05. All data processing and analysis were conducted in R (R Core Team 2017).

Results

There were 11,254 I. scapularis ticks tested at the CAES-TTL between 2015 and 2018. Overall (both adult females and nymphs), 40.7% (n = 4,583) tested positive for one or more pathogens and the remainder 59.3% (n = 6,671) were uninfected (Table 1). The prevalences of infection with B. burgdorferi, B. microti, and A. phagocytophilum were 32.9% (n = 3,708), 8.1% (n = 907), and 7.4% (n = 828), respectively. The majority of infections were single (82.4%; n = 3,778) and only 7.2% (n = 805) of I. scapularis ticks tested positive for multiple pathogens, of which 93.2% (n = 750) were infected with two and 6.8% (n = 55) with all three pathogens. Overall, just 0.5% of all ticks tested positive for three pathogens.

Table 1.

Prevalence of Infection and Coinfection in Ixodes scapularis by Tick Life Stage

	Adult (%)	Nymph (%)	Total (%)
Tested	8,084	3,170	11,254
Positive	3,742 (46.3)	841 (26.5)	4,583 (40.7)
Single	3,033 (37.5)	745 (3.5)	3,778 (33.6)
Coinfected	709 (8.8)	96 (3.0)	805 (7.2)
Total Bb	3,047 (37.7)	661 (20.9)	3,708 (32.9)
Total Bm	739 (9.1)	168 (5.3)	907 (8.1)
Total Ap	716 (8.9)	112 (3.5)	828 (7.4)
Single Bb	2,354 (29.1)	567 (17.9)	2,921 (26)
Single Bm	370 (4.6)	101 (3.2)	471 (4.2)
Single Ap	309 (3.8)	77 (2.4)	386 (3.4)
BbBm	302 (3.7)	61 (1.9)	363 (3.2)
BbAp	340 (4.2)	29 (0.9)	369 (3.3)
BmAp	16 (0.2)	2 (0.1)	18 (0.2)
All three	51 (0.6)	4 (0.1)	55 (0.5)
Uninfected	4,342 (53.7)	2,329 (73.5)	6,671 (59.3)

Tested are all tick submissions acquired in Connecticut between 2015 and 2018. Positive refers to all ticks that tested positive for any of the three pathogens. Single infections are a summation of the three single infections (Single Bb, Single Bm, and Single Ap). Coinfected ticks are a summation of all double and triple infections (BbBm, BbAp, BmAp, and All 3). Total Bb, Total Bm, and Total Ap refer to any tick that tested positive for each pathogen regardless of coinfection status with the other pathogens. Single Bb, Single Bm, and Single Ap refer to ticks with single infections. BbBm, BbAp, BmAp, and all three refer to coinfections. Uninfected ticks are those that did not test positive for any of the three pathogens.

Ap, Anaplasma phagocytophilum; Bb, Borrelia burgdorferi; Bm, Babesia microti.

Of the 3,708 ticks infected with B. burgdorferi, 78.8% (n = 2,921) were single infections, 9.8% (n = 363) with B. microti, 10.0% (n = 369) with A. phagocytophilum, and 1.5% (n = 55) with all three pathogens. Of the 907 ticks infected with B. microti, 52.0% (n = 471) were single infections, 40.0% (n = 363) with B. burgdorferi, 2.0% (n = 18) with A. phagocytophilum, and 6.1% (n = 55) with all 3 pathogens. Of the 828 ticks infected with A. phagocytophilum, 46.6% (n = 386) were single infections, 44.6% (n = 369) with B. burgdorferi, 2.2% (n = 18) with B. microti, and 6.6% (n = 55) with all 3 pathogens.

Of the 11,254 I. scapularis, 28.2% (n = 3,170) were nymphs and 71.8% (n = 8,084) were adult females (Table 1). Compared with adults, nymphs were significantly less likely to be infected with B. burgdorferi (56% less likely; odds ratio [OR] = 0.44, 95% confidence interval [CI] = 0.40–0.48, p < 0.001), B. microti (44% less likely; OR = 0.56, 95% CI = 0.47–0.66, p < 0.001), A. phagocytophilum (62% less likely; OR = 0.38, 95% CI = 0.31–0.46, p < 0.001), or be coinfected (68% less likely; OR = 0.32, 95% CI = 0.26–0.40, p < 0.001).

There were no significant differences in I. scapularis infection or coinfection across counties (Tables 2 and 3 and Fig. 1). The overall effect of county in the logistic regression models was not significant (B. burgdorferi: χ² = 7.2, df = 7, p = 0.41; B. microti: χ² = 4.8, df = 7, p = 0.69; A. phagocytophilum: χ² = 14.2, df = 7, p = 0.05; coinfections: χ² = 10.8, df = 7, p = 0.15).

FIG. 1.

Prevalence of infection and coinfection of Ixodes scapularis with Borrelia burgdorferi, Babesia microti, and Anaplasma phagocytophilum in the eight counties in Connecticut and across the 4 years, 2015–2018. Note the change in scale across graphs.

Table 2.

Prevalence of Infection and Coinfection in Ixodes scapularis Nymphs by County

	Fairfield	Hartford	Litchfield	Middlesex	New Haven	New London	Tolland	Windham	Total (%)
Tested	1,917	149	196	119	564	123	71	31	3,170
Positive	506 (26)	35 (23)	57 (29)	37 (30)	147 (26)	37 (30)	18 (25)	4 (13)	841 (27)
Single	454 (24)	31 (21)	43 (22)	35 (29)	129 (23)	31 (25)	18 (25)	4 (13)	745 (24)
Coinfected	52 (3)	4 (3)	14 (7)	2 (2)	18 (3)	6 (5)	0	0	96 (3)
Total Bb	390 (20)	28 (19)	49 (25)	32 (27)	112 (20)	30 (24)	17 (24)	3 (10)	661 (21)
Total Bm	103 (5)	9 (6)	11 (6)	7 (6)	28 (5)	8 (7)	1 (1)	1 (3)	168 (5)
Total Ap	68 (4)	2 (1)	11 (6)	0	26 (5)	5 (4)	0	0	112 (4)
Single Bb	339 (18)	24 (16)	35 (18)	30 (25)	95 (17)	24 (20)	17 (24)	3 (10)	567 (18)
Single Bm	64 (3)	5 (3)	2 (1)	5 (4)	19 (3)	4 (3)	1 (1)	1 (3)	101 (3)
Single Ap	51 (3)	2 (1)	6 (3)	0	15 (3)	3 (3)	0	0	77 (2)
BbBm	35 (2)	4 (3)	9 (5)	2 (2)	7 (1)	4 (3)	0	0	61 (2)
BbAp	13 (1)	0	5 (3)	0	9 (2)	2 (2)	0	0	29 (1)
BmAp	1 (<1)	0	0	0	1 (<1)	0	0	0	2 (<1)
All three	3 (<1)	0	0	0	1 (<1)	0	0	0	4 (<1)
Uninfected	1,411 (74)	114 (77)	139 (71)	82 (70)	417 (74)	86 (70)	53 (75)	27 (87)	2,329 (73)

See Table 1 legend for explanations.

Table 3.

Prevalence of Infection and Coinfection in Ixodes scapularis Adults by County

	Fairfield	Hartford	Litchfield	Middlesex	New Haven	New London	Tolland	Windham	Total (%)
Tested	4,250	688	786	245	1,675	216	155	69	8,084
Positive	1,961 (46)	301 (44)	375 (48)	110 (45)	775 (46)	107 (50)	76 (49)	37 (54)	3,742 (46)
Single	1,576 (37)	256 (37)	303 (39)	90 (37)	637 (38)	80 (37)	59 (38)	32 (46)	3,033 (38)
Coinfected	385 (9)	45 (7)	72 (9)	20 (8)	138 (8)	27 (13)	17 (11)	5 (7)	709 (9)
Total Bb	1,603 (38)	242 (35)	305 (39)	94 (38)	622 (37)	90 (42)	59 (38)	32 (46)	3,047 (38)
Total Bm	394 (9)	67 (10)	72 (9)	16 (7)	154 (9)	18 (8)	14 (9)	4 (6)	739 (9)
Total Ap	377 (9)	40 (6)	76 (10)	22 (9)	146 (9)	27 (13)	20 (13)	8 (12)	716 (9)
Single Bb	1,227 (29)	198 (29)	235 (30)	74 (30)	487 (29)	63 (29)	43 (28)	27 (39)	2,354 (29)
Single Bm	200 (5)	38 (6)	37 (5)	5 (2)	74 (4)	8 (4)	7 (5)	1 (1)	370 (5)
Single Ap	149 (4)	20 (3)	31 (4)	11 (4)	76 (5)	9 (4)	9 (6)	4 (6)	309 (4)
BbBm	157 (4)	25 (4)	27 (3)	9 (4)	68 (4)	9 (4)	6 (4)	1 (1)	302 (4)
BbAp	191 (4)	16 (2)	37 (5)	9 (4)	58 (3)	17 (8)	10 (6)	2 (3)	340 (4)
BmAp	9 (<1)	1 (<1)	2 (<1)	0	3 (<1)	0	1 (<1)	0	16 (<1)
All three	28 (<1)	3 (<1)	6 (<1)	2 (<1)	9 (<1)	1 (<1)	0	2 (<1)	51 (<1)
Uninfected	2,289 (54)	387 (56)	411 (52)	135 (55)	900 (54)	109 (50)	79 (51)	32 (46)	4,342 (54)

See Table 1 legend for explanations.

There were significant temporal trends in I. scapularis infection and coinfection prevalence (Tables 4 and 5 and Fig. 1). After adjusting for tick life stage, with each year the odds of infection with B. burgdorferi, A. phagocytophilum, and coinfection (compared to none) increased by 9% (OR = 1.09, 95% CI = 1.05–1.13, p < 0.001); A. phagocytophilum increased by 43% (OR = 1.43, 95% CI = 1.33–1.54, p < 0.001); coinfection increased by 22% (OR = 1.22, 95% CI = 1.13–1.31, p < 0.001); and B. microti (compared with none) decreased by 9% (OR = 0.91, 95% CI = 0.86–0.97, p = 0.005). Driving the increase in coinfections were those between B. burgdorferi and A. phagocytophilum (OR = 1.46, 95% CI = 1.31–1.63, p < 0.001) because we note no change in coinfections between B. burgdorferi and B. microti over time (OR = 0.93, 95% CI = 0.85–1.03, p = 0.175).

Table 4.

Prevalence of Infection and Coinfection in Ixodes scapularis Nymphs by Year

	2015	2016	2017	2018	Total
Tested	853	665	812	840	3,170
Positive	302 (35)	164 (25)	174 (21)	201 (24)	841 (27)
Single	260 (30)	147 (22)	156 (19)	182 (22)	745 (24)
Coinfected	42 (5)	17 (3)	18 (2)	19 (2)	96 (3)
Total Bb	235 (28)	124 (19)	142 (17)	160 (19)	661 (21)
Total Bm	72 (8)	37 (6)	30 (4)	29 (3)	168 (5)
Total Ap	38 (4)	20 (3)	21 (3)	33 (4)	112 (4)
Single Bb	194 (23)	107 (16)	125 (15)	141 (17)	567 (18)
Single Bm	43 (5)	27 (4)	15 (2)	16 (2)	101 (3)
Single Ap	23 (3)	13 (2)	16 (2)	25 (3)	77 (2)
BbBm	27 (3)	10 (2)	13 (2)	11 (1)	61 (2)
BbAp	13 (2)	7 (1)	3 (<1)	6 (<1)	29 (<1)
BmAp	1 (<1)	0	1 (<1)	0	2 (<1)
All three	1 (<1)	0	1 (<1)	2 (<1)	4 (<1)
Uninfected	551 (65)	501 (75)	638 (79)	639 (76)	2,329 (73)

See Table 1 legend for explanations.

Table 5.

Prevalence of Infection and Coinfection in Ixodes scapularis Adults by Year

	2015	2016	2017	2018	Total
Tested	1,533	1,457	3,047	2,047	8,084
Positive	672 (44)	592 (41)	1,292 (42)	1,186 (58)	3,742 (46)
Single	559 (26)	511 (35)	1,060 (35)	903 (44)	3,033 (38)
Coinfected	113 (7)	81 (6)	232 (8)	283 (14)	709 (9)
Total Bb	524 (34)	488 (33)	1,091 (36)	944 (46)	3,047 (38)
Total Bm	187 (12)	114 (8)	193 (6)	245 (12)	739 (9)
Total Ap	79 (5)	75 (5)	253 (8)	309 (15)	716 (9)
Single Bb	412 (27)	407 (28)	862 (28)	673 (33)	2,354 (29)
Single Bm	109 (7)	64 (4)	87 (3)	110 (5)	370 (5)
Single Ap	38 (2)	40 (3)	111 (4)	120 (6)	309 (4)
BbBm	72 (5)	46 (3)	90 (3)	94 (5)	302 (4)
BbAp	35 (2)	31 (2)	126 (4)	148 (7)	340 (4)
BmAp	1 (<1)	0	3 (<1)	12 (<1)	16 (<1)
All three	5 (<1)	4 (<1)	13 (<1)	29 (1)	51 (<1)
Uninfected	861 (56)	865 (59)	1,755 (58)	861 (42)	4,342 (54)

See Table 1 legend for explanations.

Finally, we found that infection with B. burgdorferi significantly increased the likelihood of coinfection. After adjusting for life stage and year, we found that if a tick was infected with B. burgdorferi it was significantly more likely to be infected with B. microti (OR = 1.74, 95% CI = 1.51–1.99, p < 0.001) or A. phagocytophilum (OR = 1.98, 95% CI = 1.71–2.29, p < 0.001).

Discussion

B. burgdorferi infection prevalence in I. scapularis adults and nymphs tested at the CAES-TTL between 2015 and 2018 was 37.7% and 20.9% (Tables 4 and 5), respectively, which fall within the ranges previously reported from the northeastern United States 24–64% for adults (Courtney et al. 2003, Schulze et al. 2005, Walk et al. 2009, Tokarz et al. 2010, Aliota et al. 2014, Prusinski et al. 2014, Hutchinson et al. 2015) and 10–23% for nymphs (Daniels et al. 1998, Schulze et al. 2013, Diuk-Wasser et al. 2014, Hersh et al. 2014, Prusinski et al. 2014). We calculated B. microti infection prevalence in I. scapularis adults and nymphs at 9.1% and 5.3%, respectively. These estimates are similar to other studies in the northeastern United States, which report B. microti infection prevalence in adults between 2.5% and 20% (Walk et al. 2009, Tokarz et al. 2010, Aliota et al. 2014, Prusinski et al. 2014, Hutchinson et al. 2015) and in nymphs between 4% and 8% (Schulze et al. 2013, Diuk-Wasser et al. 2014, Hersh et al. 2014, Prusinski et al. 2014).

A. phagocytophilum infection prevalence in I. scapularis adults and nymphs in the present study was 8.9% and 3.5%, respectively. The documented range for A. phagocytophilum infection prevalence in the northeastern United States in adults is 0.1–20% (Courtney et al. 2003, Schulze et al. 2005, Walk et al. 2009, Tokarz et al. 2010, Aliota et al. 2014, Hutchinson et al. 2015, Prusinski et al. 2014), and in nymphs is 2.7–13% (Daniels et al. 1998, Hersh et al. 2014, Prusinski et al. 2014).

Coinfection prevalence was also different by life stage (Table 1). Around 3.7% of adults and 1.9% of nymphs were coinfected with B. burgdorferi and B. microti, whereas 4.2% of adults and 0.9% of nymphs were coinfected with B. burgdorferi and A. phagocytophilum. 0.6% of adults and 0.1% of nymphs tested positive for all three pathogens. These estimates are as within those reported previously in the northeastern United States: B. burgdorferi and B. microti coinfection in adults (1–17%) (Walk et al. 2009, Tokarz et al. 2010, Aliota et al. 2014, Hutchinson et al. 2015, Prusinski et al. 2014) and nymphs (1–6.7%) (Schulze et al. 2013, Hersh et al. 2014, Prusinski et al. 2014); and B. burgdorferi and A. phagocytophilum coinfection in adults (1.5–16%) (Courtney et al. 2003, Schulze et al. 2005, Walk et al. 2009, Tokarz et al. 2010, Aliota et al. 2014, Hutchinson et al. 2015, Prusinski et al. 2014) and nymphs (0.5–2.5%) (Daniels et al. 1998, Hersh et al. 2014, Prusinski et al. 2014).

Triple infection prevalence is also within the range, although on the lower end of what others have found (0.3–5%) (Tokarz et al. 2010, Hersh et al. 2014, Xu et al. 2016). Our estimated infection and coinfection prevalence derived from passive surveillance data are consistent with those derived from active surveillance previously reported in the northeastern United States, supporting the utility of passive surveillance data to determine infection and coinfection prevalence in ticks and evaluate the risk of human infection with tick-borne pathogens.

Nymphs may pose the greatest risk for human transmission due to their size (Eisen et al. 2017), but greater adult submissions (72%) justify their inclusion in our analysis. Nymphs were 56%, 44%, 62%, and 68% less likely than adults to be infected with B. burgdorferi, B. microti, A. phagocytophilum, or be coinfected, respectively. Only 3% of nymphs were coinfected in comparison to 9% of the adults.

It is understandable that adults would have higher infection and coinfection prevalence than nymphs. Because there is no evidence of transovarial transmission (i.e., from one generation to the next) of these pathogens, larvae hatch uninfected. Nymphs, having fed once, can only acquire pathogens from one host but adults, having fed twice (once as larvae and once as nymphs), can obtain pathogens from two hosts (Ginsberg 2008, Eisen and Eisen 2018). Additionally, if one pathogen increases the risk of acquisition of a second pathogen, there may be even greater infection prevalence in adults compared with nymphs (Nieto and Foley 2009).

Although we note variability in tick infection and coinfection prevalence across counties (Fig. 1), we could not identify a significant difference in the overall effect of county on the odds of infection or coinfection. Studies have found spatial differences in infection prevalence (Steiner et al. 2008, Aliota et al. 2014, Dibernardo et al. 2014, Diuk-Wasser et al. 2014, Lee et al. 2014, Nelder et al. 2014, Prusinski et al. 2014, Feldman et al. 2015, Johnson et al. 2017, Egizi et al. 2018). Our analysis was based on public submissions weighted toward more populated areas in Connecticut (75% of submissions came from Fairfield and New Haven Counties), which may undermine detection of spatial differences in contrast to studies based on drag sampling with more even surveillance.

Spatial variability may depend on larger underlying study areas, for example Canadian provinces (Dibernardo et al. 2014), U.S. states (Johnson et al. 2017) or zones created by aggregating counties (Prusinski et al. 2014, Egizi et al. 2018). That we did not find spatial variability could be attributable to the scale of the analysis. If we looked at smaller or larger scales, we may be able to find significant spatial differences (Daniels et al. 1998). On the other hand, some studies do not find spatial differences (Daniels et al. 1998, Swanson et al. 2006, Schulze et al. 2013). And in other studies, spatial differences were found in B. microti or A. phagocytophilum but not in B. burgdorferi infection prevalence (Phelps 2014, Hutchinson et al. 2015, Xu et al. 2016), which may be explained by how long the pathogens have been established.

In Connecticut, B. microti infection in white-footed mice and human cases of babesiosis and anaplasmosis have been shown to be higher in the southeastern part of the state (Gacek et al. 2014, Stafford et al. 2014, Esponda and Nelson 2015). Spatial differences in human risk could result from differences in tick density, human behavior, diagnosis, or other factors and not due to tick infection prevalence (Little et al. 2019). Or because I. scapularis and these pathogens are long established in Connecticut (Anderson et al. 1991), spatial differences that were once distinct are no longer the case.

We identified temporal trends in infection and coinfection prevalence between 2015 and 2018. Specifically, the likelihood of single infection with B. burgdorferi, A. phagocytophilum, and coinfection increased, whereas infection with B. microti decreased over time. Driving the increase in coinfection over time were coinfections of B. burgdorferi and A. phagocytophilum. Previous studies investigating more than 2 years of data have reported temporal variability in infection or coinfection prevalence (Holman et al. 2004, Schulze et al. 2013, Prusinski et al. 2014), but we found only one other study that established a temporal trend (Nelder et al. 2014).

It is important to note that the duration and period over which studies are conducted might influence the conclusions. Also using data from the CAES-TTL, we reported a decline in B. burgdorferi infection prevalence in Connecticut between 2007 and 2017 (Little et al. 2019). But if we look at B. burgdorferi infection prevalence between 1996 and 2017, we find a slight increase with time (OR = 1.019, 95% CI = 1.016–1.022, p < 0.001); likewise, between 2015 and 2018 we find an increase in B. burgdorferi infection prevalence over time. These varying results, dependent upon the years investigated, mandate longitudinal data collection and analysis to determine if infection and coinfection prevalence are generally stable but fluctuate or if they are actually increasing (or decreasing).

Differences in infection and coinfection prevalence by life stage and years in the present study were accounted for in the logistic regression models. After adjusting for life stage and year, we found that infection with B. burgdorferi increased the likelihood of infection with B. microti and A. phagocytophilum by 74% and 98%, respectively. Other studies have also reported that B. burgdorferi increases the likelihood of infection with B. microti (Piesman et al. 1986, Diuk-Wasser et al. 2014, Hersh et al. 2014, Phelps 2014, Prusinski et al. 2014) and A. phagocytophilum (Swanson et al. 2006, Hamer et al. 2014). However, in a few other investigations no associations between B. burgdorferi and B. microti (Steiner, et al. 2008, Walk et al. 2009, Hutchinson et al. 2015), or between B. burgdorferi and A. phagocytophilum (Courtney et al. 2003, Steiner et al. 2008, Walk et al. 2009, Dibernardo et al. 2014, Hersh et al. 2014, Hutchinson et al. 2015) have been reported.

In addition to B. burgdorferi increasing the likelihood of infection with either B. microti or A. phagocytophilum, we found equivalent rates of coinfection between B. burgdorferi and either B. microti or A. phagocytophilum (3%). Given these findings, it seems that over time coinfections would also increase. However, between 2015 and 2018, we only note an increase in B. burgdorferi and A. phagocytophilum coinfection prevalence and no change in B. burgdorferi and B. microti coinfection prevalence.

Our estimated nymphal coinfection prevalence in 2015 (B. burgdorferi and B. microti = 3%; B. burgdorferi and A. phagocytophilum = 2%) were lower than those from Minnesota in 2015 (B. burgdorferi and B. microti = 4%; B. burgdorferi and A. phagocytophilum = 4%) (Johnson et al. 2018). Given that the tick and pathogens have been established longer and coinfection prevalence estimates are lower in Connecticut compared with Minnesota, coinfection prevalence may level off or even decrease.

B. burgdorferi and B. microti were discovered within a few years of each other in the same geographical area near southeastern Connecticut (Spielman 1994), yet B. burgdorferi incidence is still higher than B. microti incidence (Diuk-Wasser et al. 2014, Walter et al. 2016, Eisen and Eisen 2018). This discrepancy may be attributed to barriers to B. microti transmission: (1) transmission of B. microti from infectious hosts to I. scapularis is not as efficient (Mather et al. 1996, Diuk-Wasser et al. 2014, Goethert et al. 2018), and (2) the survival rate of B. microti relative to B. burgdorferi is lower in overwintering I. scapularis (Piesman et al. 1986). Higher tick densities may be necessary to support B. microti transmission (Mather et al. 1996) and infection with B. burgdorferi may promote infection with B. microti (Dunn et al. 2014, Diuk-Wasser et al. 2016). In areas where both these conditions are met, B. microti infection prevalence may continue to increase (Diuk-Wasser et al. 2014).

Connecticut has long established populations of I. scapularis and high incidence of Lyme disease and babesiosis (Dahlgren et al. 2015, Menis et al. 2015, Centers for Disease Control and Prevention 2016, 2017, 2018). In this study, we find that a tick infected with B. burgdorferi is more likely to be infected with B. microti than an uninfected tick. Yet we find a decrease in B. microti infection prevalence and no change in B. burgdorferi and B. microti coinfection over time. Analysis of coinfection prevalence over a longer period of time in future studies will help to better understand the dynamics of infection and coinfection in I. scapularis.

Whether there is an association between B. burgdorferi and A. phagocytophilum infection in ticks remains debated. Two variants of A. phagocytophilum, the human active strain (A. phagocytophilum-ha) and a nonpathogenic variant (A. phagocytophilum-v1) may be responsible for the divergent findings. O. virginianus is the primary reservoir for A. phagocytophilum-v1 and P. leucopus is the primary reservoir for A. phagocytophilum-ha (Massung et al. 2005, Steiner et al. 2008). A. phagocytophilum-ha was the only variant found in mice in Pennsylvania (Courtney et al. 2003) and nymphs were 2/3 more likely to be infected with A. phagocytophilum-ha than A. phagocytophilum-v1 in New York (Keesing et al. 2014). It reasons that coinfection with A. phagocytophilum-v1and B. burgdorferi may be independent while A. phagocytophilum-ha and B. burgdorferi may be associated.

A study in New York State that did not distinguish between variants, found an association in adults but not in nymphs (Prusinski et al. 2014), which may be due to underlying host differences. Our study did not differentiate between variants either; however, we found an association between B. burgdorferi and A. phagocytophilum infection for both life stages. Stratifying by life stage, we find B. burgdorferi increased the likelihood of infection with A. phagocytophilum in nymphs (OR = 1.62, 95% CI = 1.05–2.43, p = 0.024) and in adults (OR = 2.13, 95% CI = 1.83–2.49, p < 0.001). Because the association between B. burgdorferi and A. phagocytophilum for nymphs held in our study, and the fact that nymphs are unlikely to be infected with A. phagocytophilum-v1 (Courtney et al. 2003, Keesing et al. 2014), we infer that A. phagocytophilum-ha infections could be prevalent and even increasing over time.

In the present study, we only discussed three of the seven I. scapularis-borne human pathogens (Eisen et al. 2017). Other pathogens may also be expanding geographically and increasing in incidence, which warrant further investigations on their prevalence and associations.

Animal models suggest synergistic effects of pathogens and increased severity of disease (Zeidner et al. 2000, Thomas et al. 2001, Belongia 2002, Moro et al. 2002, Bhanot and Parveen 2019) with some exceptions (Levin and Fish 2000, 2001, Coleman et al. 2005). While retrospective studies have not shown an increase in the severity of disease (Wang et al. 2000), prospective studies that are able to distinguish simultaneous from sequential infections have shown that coinfections increase the severity and duration of illness (Krause et al. 1996, 2002). Coinfections in humans may not only manifest with more severe symptoms but also lead to challenges in diagnosis and improper treatment (Swanson et al. 2006).

In this study, we show that adult and nymph I. scapularis ticks can be coinfected and the increased likelihood of coinfection prevalence over time may have serious implications for the clinical diagnosis, course, severity, and treatment of human tick-borne disease cases.

Footnotes

Acknowledgments

The authors are grateful to Dr. Durland Fish for his advice on approaching the analysis. They thank Alex Diaz, a research technician at the CAES-TTL, as well as Mallery Breban and numerous other seasonal research assistants for technical help. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention.

Author Disclosure Statement

No competing financial interests exist.

Funding Information

The CAES-TTL is funded by the State of Connecticut. This publication was supported in part by the cooperative agreement number, U01 CK000509, funded by the Centers for Disease Control and Prevention.

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