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. Author manuscript; available in PMC: 2019 Apr 11.
Published in final edited form as: J Med Entomol. 2018 May 4;55(3):501–514. doi: 10.1093/jme/tjx250

Amblyomma americanum (Acari: Ixodidae) Ticks Are Not Vectors of the Lyme Disease Agent, Borrelia burgdorferi (Spirocheatales: Spirochaetaceae): A Review of the Evidence

Ellen Y Stromdahl 1,8, Robyn M Nadolny 1, Graham J Hickling 2, Sarah A Hamer 3, Nicholas H Ogden 4, Cory Casal 1, Garrett A Heck 1,5, Jennifer A Gibbons 6, Taylor F Cremeans 1, Mark A Pilgard 7
PMCID: PMC6459681  NIHMSID: NIHMS1021381  PMID: 29394366

Abstract

In the early 1980s, Ixodes spp. ticks were implicated as the key North American vectors of Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) (Spirocheatales: Spirochaetaceae), the etiological agent of Lyme disease. Concurrently, other human-biting tick species were investigated as potential B. burgdorferi vectors. Rashes thought to be erythema migrans were observed in patients bitten by Amblyomma americanum (L.) (Acari: Ixodidae) ticks, and spirochetes were visualized in a small percentage of A. americanum using fluorescent antibody staining methods, sparking interest in this species as a candidate vector of B. burgdorferi. Using molecular methods, the spirochetes were subsequently described as Borrelia lonestari sp. nov. (Spirocheatales: Spirochaetaceae), a transovarially transmitted relapsing fever Borrelia of uncertain clinical significance. In total, 54 surveys from more than 35 research groups, involving more than 52,000 ticks, have revealed a low prevalence of B. lonestari, and scarce B. burgdorferi, in A. americanum. In Lyme disease-endemic areas, A. americanum commonly feeds on B. burgdorferi-infected hosts; the extremely low prevalence of B. burgdorferi in this tick results from a saliva barrier to acquiring infection from infected hosts. At least nine transmission experiments involving B. burgdorferi in A. americanum have failed to demonstrate vector competency. Advancements in molecular analysis strongly suggest that initial reports of B. burgdorferi in A. americanum across many states were misidentified B. lonestari, or DNA contamination, yet the early reports continue to be cited without regard to the later clarifying studies. In this article, the surveillance and vector competency studies of B. burgdorferi in A. americanum are reviewed, and we conclude that A. americanum is not a vector of B. burgdorferi.

Keywords: Amblyomma americanum, Borrelia burgdorferi, prevalence, vector competency


Sylvatic transmission cycles of the spirochetal bacterium Borrelia burgdorferi (Johnson, Schmid, Hyde, Steigerwalt and Brenner) (Spiro cheatales: Spirochaetaceae), the etiological agent of Lyme disease, are widespread in the eastern United States. Ixodes scapularis Say (Acari: Ixodidae) ticks are recognized as the key vector in the eastern half of the United States; however, other human-biting ticks – particularly Amblyomma americanum (L.) (Acari: Ixodidae) feed on many of the same wildlife reservoirs, leading to concerns that this tick species also may contribute to the risk of acquiring Lyme disease. Here we review the literature describing 30 yr of surveillance of A. americanum for Borrelia spp. and summarize the results of multiple vector competency studies undertaken on this tick. This body of research overwhelmingly indicates that A. americanum is not a vector of B. burgdorferi.

Discovery of B. burgdorferi and Investigation of Possible Vector Ticks

Lyme disease was first recognized and described in the United States in 1975 (Steere et al. 1977). Willy Burgdorfer and colleagues discovered the spirochetal agent, later described as B. burgdorferi, in I. scapularis (dammini) ticks from Shelter Island, NY, and demonstrated the vector competency of this tick species (Burgdorfer et al. 1982). The agent was later found to also be transmitted by human-biting Ixodes pacificus Cooley and Kohls (Acari: Ixodidae) in the western United States and by other human-biting Ixodes genus ticks in Eurasia (Lane et al. 1991, Rudenko et al. 2011). Additionally, several Ixodes spp. that do not regularly feed on humans have been implicated as responsible in maintaining sylvatic transmission of B. burgdorferi (Clark et al. 2002). During the early years of Lyme disease research, other common species of anthropophilic ticks were also investigated as potential vectors of the agent of the disease. Some of the publications from the 1980s and 1990s (reviewed below and summarized in Table 1) described spirochetes in A. americanum and identified them as B. burgdorferi. However, these early investigations used microscopy and fluorescent antibody staining methods that depend on subjective interpretation and can detect related spirochete species (Barbour et al. 1996), or remnants of dead spirochetes (Bockenstedt et al. 2012). Many of the studies involved ticks removed while feeding on animal hosts, and spirochetes detected were likely in the bloodmeal in the ticks’ midgut rather than representing infections within the ticks’ bodies. Also, contamination is suspected in some investigations of A. americanum, as results were not repeatable and suspect-positives shared identity with the Borrelia strain used for positive control (Nelson 1995, Piesman and Happ 1997). Subsequent studies (vide infra) provided further evidence that the presence of other spirochetes, degraded spirochetes, or contamination most likely account for the reports of B. burgdorferi in these early studies.

Table 1.

Initial investigations of Borrelia spp. in A. americanumusing microscopy, immunofluorescent methods, culturing, and PCR

Date of collection Location Source of ticksa Method of analysis Total ticks tested Ticks with spirochetes References
1983 NJ Vegetation Darkfield, DFA (PAb) 44 4 Schulze et al. (1984)
1983–1984 NC Animals DFA (PAb) 512 7 Magnarelli et al. (1986)
1984 NJ Vegetation Darkfield, DFA (PAb), culture 756, 32 larval poolsb 35, 5 larval pools Schulze et al. (1986)
1984–1987 NC Vegetation Darkfield, DFA, (PAb) 1836 1 Levine et al. (1989)
1987 NC, VA Vegetation, animals Darkfield, IFA (MAb) 218 4 Levine et al. (1991)
1988–1989 AL Mixed DFA, IFA (PAb) 150 6 Luckhart et al. (1991)
1988–1989 TX Mixed Culture, darkfield, IFA (MAb) 354 3 Teltow et al. (1991)
1990 AR Mixed IFA (MAb), DFA (PAb) 471 0 Kardatzke et al. (1992)
1988–1990 AL Animals DFA, IFA (PAb) 125 8 Luckhart et al. (1992)
1989–1991 AR Vegetation, animals Darkfield, IFA (MAb) 200 7 Simpson and Hinck (1993)
1989 MO Vegetation IFA (MAb), PCR 1,752 33 Feir et al. (1994)
1990–1992 TX Vegetation, animals DFA (PAb), culture 5,195c 54 Rawlings and Teltow (1994)
1991–1994 VA Vegetation, animals Darkfield, IFA (MAb) 546 1 Sonenshine et al.(1995)
1990–1993 NC Vegetation, animals IFA (MAb) 5,724 51 Ouellette et al. (1997)
1995–1996 MO Vegetation, animals IFA (PAb, MAb) 436 5 Kollars et al. (2000)
1994–1995 SC Vegetation, animals Culture 210 0 Clark et al. (2002)
1999–2000 MS Mixed DFA (PAb) 68 0 Goddard et al. (2003)
Totals 18,597, 32 larvalpools 224

Darkfield, darkfield microscopy; DFA, direct fluorescent antibody test; PAb, polyclonal antibody; IFA, indirect immunoflourescence assay; MAb, monoclonal antibody; PCR, polymerase chain reaction.

a

Mixed = vegetation, human, and wildlife/domestic animals.

b

Culture of spirochetes from ticks in this study was attempted, but none were isolated.

c

Culture of spirochetes from >20,000 additional ticks in this study was attempted, but none were isolated.

Concurrently with these tick surveillance studies, experiments to evaluate the ability of A. americanum to acquire and transmit B. burgdorferi were conducted, and added no evidence to support the role of A. americanum in Lyme disease. Seven studies in the 1980s and 1990s failed to demonstrate vector competency of A. americanum (Piesman and Sinsky 1988, Mather and Mather 1990, Mukolwe et al. 1992, Ryder et al. 1992, Oliver et al. 1993, Sanders and Oliver 1995, Piesman and Happ 1997). Three subsequent studies suggested and demonstrated a salivary mechanism for the apparent inability of A. americanum to acquire or support colonization by B. burgdorferi (Ledin et al. 2005, Soares et al. 2006, Zeidner et al. 2009).

In 1996, phylogenetic analysis of Borrelia DNA sequences amplified from A. americanum led to the identification of Borrelia lonestari sp. nov. (Spirocheatales: Spirochaetaceae), a relapsing fever species distinct from B. burgdorferi (Armstrong et al. 1996, Barbour et al. 1996) and other Lyme disease group spirochetes. This new spirochete became a focus of research after it was detected in both an A. americanum removed from a patient suffering a skin rash and in the medium containing a biopsy of the rash (James et al. 2001), leading to speculation that it was the etiological agent of Southern Tick Associated Rash Illness (STARI), which occurs following bites of A. americanum (Masters et al. 2008). Many of the subsequent investigations of A. lonestari in A. americanum involved screening of ticks using genus-wide or broadly reactive polymerase chain reaction (PCR) primers capable of also detecting B. burgdorferi. Consequently, publications reporting these investigations provide separate data on the prevalences of both B. lonestari and B. burgdorferi infection in A. americanum, as long as sufficient measures – such as nucleotide sequencing, or confirmation with multiple PCR targets – were taken to discriminate among Borrelia species in Borrelia-positive samples (Table 2).

Table 2.

Discovery of B. lonestari and surveillance for Borrelia spp. in A. americanum using PCR and other methods

Reported PCR-positive
Date of collection Location Source of ticksa Method of analysis Screening primer reference Total tested B. burgdorferi B. lonestari References
1995 MD Vegetation Darkfield, IFA, PCR Armstrong et al. (1996) 297 0 1 Armstrong et al. (1996)
1989–1992 MO, NJ, NY, NC, TX Humans, vegetation DFA (PAb), PCR Barbour et al. (1996) 875 0 Unspecified Barbour et al. (1996)
1999 AL Vegetation PCR Barbour et al. (1996) 202 0 2 Burkot et al. (2001)
1997 US Humans PCR Rosa et al. (1991) 222 7b Stromdahl et al. (2001)
1994 MD Vegetation Darkfield, culture, PCR Rich et al. (2001) 388 0 2 Rich et al. (2001)
2001 MO Vegetation PCR Barbour et al. (1996) 214 0 12 pools Bacon et al. (2003)
1999–2000 TN Vegetation PCR Stegall-Faulk et al. (2003) 453 0 2 Stegall-Faulk et al. (2003)
2000–2002 US Humans, vegetation PCR Barbour et al. (1996) 6,334 0 78 Stromdahl et al. (2003)
1999–2000 FL Vegetation PCR Barbouret al. (1996), Johnson et al. (1992) 396 5c 8 Clark (2004)
2001–2003 GA Vegetation PCR Barbour et al. (1996) 398 0 4 Varela et al. (2004b)
2002 MO Vegetation PCR Bacon et al. (2005),Demaerschalck et al. (1995) 654 0 22 pools Bacon et al. (2005)
Unspecified MO Mixed PCR Cyr et al. (2005) 16 4d Cyr et al. (2005)
2003–2004 NJ Vegetation PCR Barbour et al. (1996) 121 0 11 Schulze et al. (2005)
1997–2000 US Mixed PCR, DBH Taft et al. (2005) 269 4e 6 Taft et al. (2005)
1998–2005 US Vegetation PCR Barbour et al. (1996) 2,038 0 54 Mixson et al. (2006)
2004 NJ PCR Stegall-Faulk et al. (2003) 103 0 6 Schulze et al. (2006)
2004–2005 TN Vegetation PCR Haynes et al. (2005) 339 0 0 Jordan et al. (2009)
2005, 2007–2008 MO Vegetation PCR, RLB Rijpkema et al. (1995),Pichon et al. (2003) 1,383 0 18 Allan et al. (2010)
2008 MS Vegetation PCR Barbour et al. (1996) 191 0 3 Castellaw et al. (2010)
2004–2008 TX Humans PCR Barbour et al. (1996) 367 0 4 Williamson et al. (2010)
2008–2010 MO, TX Vegetation PCR Bunikis et al. (2004),Margos et al. (2008) 228 0 3 Yuan (2010)
2008 KY Mixed PCR Bunikis et al. (2004) 108 0 1 Fritzen et al. (2011)
2008 NJ Vegetation PCR Barbour et al. (1996) 281 0 19 Schulze et al. (2011)
2006–2008 AR Animals PCR Barbour et al. (1996) 657 0 107 Fryxell et al. (2012)
2010 US Humans PCR Barbour et al. (1996) 1621 0 13 Stromdahl and Hickling (2012)
2009–2010 FL, GA Humans PCR Clark et al. (2013) 3 2f 0 Clark et al. (2013)
2010–2011 GA Vegetation PCR Barbour et al. (1996) 3061 0 38 Gleim (2013)
2005–2009 GA Vegetation PCR Barbour et al. (1996) 4,236 0 59 Killmaster et al. (2014)
2012 NE Vegetation PCR Barbour et al. (1996) 251 0 4 Maegli et al. (2016)
2013 US Humans PCR Clark et al. (2013) 1097 0 6 Stromdahl et al. (2015)
2005–2006 GA Humans PCR Barbour et al. (1996) 426 0 2 Gleim et al. (2016)
2014 MO Vegetation PCR Barbour et al. (1996) 1,880 0 20 Hudman and Sargentini (2016)
2008–2014 TX Humans PCR Barbour et al. (1996),Williamson (2010) 591 0 8 Mitchell et al. (2016)
2013 AL, FL, GA, TN, SC Vegetation PCR Clark et al. (2005) 590 13g Rudenko et al. (2016)
2010–2012 FL Vegetation PCR Barbour et al. (1996) 260 0 5 Sayler et al. (2016)
2014 FL Vegetation PCR Barbour et al. (1996) 777 0 0 Sayleretal. (2017)
2006–2017 US Humans, mixed PCR TickReport real-time PCR assay 2,483 0 31 TickReport (https://www.tickreport.com/stats), accessed 26 September 2017
Totals 33,810 35 578

Darkfield, darkfield microscopy; IFA, indirect fluorescent antibody testing; PCR, polymerase chain reaction; DFA, direct fluorescent antibody testing; PAb, polyclonal antibody; DBH, dot blot hybridization; RLB, reverse line blot.

a

Mixed = vegetation, human, and wildlife/domestic animals.

b

Contamination was suspected, and these results were re-evaluated in a later publication (Stromdahl et al. 2015).

c

The identity of positive amplicons was confirmed by sequencing in one direction only.

d

Only one gene target was used for PCR, and sequencing data were not presented.

e

The positive samples were not sequenced.

f

The identity of the PCR-positives relied on the detection and sequencing of one gene only.

g

Multilocus sequence typing of 10 gene targets could only confirm 3 of these positives, and not all 10 loci were amplified in each of those three samples.

Since the mid-1980s, at least 35 different research groups have published 54 studies describing the investigation of more than 52,000 A. americanum for B. burgdorferi. Scarce (0.5%) A. americanum were reported as suspect-positive, and B. burgdorferi was never isolated in culture from these ticks. Ten studies evaluating the vector competency and the mechanisms of B. burgdorferi evasion by A. americanum concluded that this tick species has no relevance to the transmission of B. burgdorferi. These studies are discussed in detail below.

Initial Investigations of B. burgdorferi in A. americanum

Detection by investigators in the 1980s and 1990s of a low prevalence (~1%) of Borrelia in more than 18,000 A. americanum (Table 1) demonstrated that the ticks were being exposed to spirochetes in host bloodmeals and presaged the discovery of B. lonestari, a spirochete that can be maintained in A. americanum. The methods of analysis of ticks for Borrelia spp. were typically darkfield microscopy and direct fluorescent antibody testing (DFA) or indirect fluorescent antibody testing (IFA) using polyclonal antibody (PAb) or monoclonal antibody (MAb). In one study, PCR was also used as a final step on samples positive by IFA. Analysis by darkfield or IFA with PAb will detect related Borrelia species (Barbour et al. 1996), so the identity of the species in these studies was not established (Schulze et al. 1984, 1986; Magnarelli et al. 1986; Levine et al. 1989; Rawlings and Teltow 1994; Luckhart et al. 1991, 1992; Kollars et al. 2000). Analysis using IFA and MAb is designed to be specific for B. burgdorferi (Barbour et al. 1983b), and IFA with MAb H5332 has been shown not to react with B. lonestari (Varela et al. 2004a); however, incorrect technique can produce false positives. False-positive results using a monoclonal antibody to the ospA protein (H5332) may arise due to insufficient blocking or washing, or concentrations of primary or secondary antibodies that are too high, all of which can result in nonspecific binding. Ideally, as a negative control studies should use an irrelevant mouse monoclonal antibody of the same immunoglobulin type as the monoclonal antibody against B. burgdorferi. Buffer or medium by itself may be inadequate as a negative control. Most of the studies reviewed here do not present IFA methods in enough detail to evaluate whether sufficient measures were taken to avoid false positives and none involved use of a negative control slide with B. lonestari antigen because the organism had not been characterized or cultured at the time the studies were conducted (Varela et al. 2004a). Furthermore, IFAs with MAbs rely on subjective interpretation and can potentially detect dead cells from host bloodmeals (Allan et al. 2010; Bockenstedt et al. 2012); it has been suggested by researchers investigating B. burgdorferi xenodiagnosis that ticks might acquire dead organisms during feeding (Marques et al. 2014). In the studies using the B. burgdorferi-specific MAb H5332, spirochetes were more often detected in A. americanum collected from animal hosts (Levine et al. 1991, Ouellette et al. 1997), but in some of the studies, spirochetes were detected by MAb H5332 in ticks collected from vegetation (Simpson & Hinck 1993, Ouellette et al. 1997, Feir et al. 1994). Results of MAb IFA in the study from Feir et al. (1994) were not consistently confirmed by subsequent PCR of the samples, and contamination was suspected (Nelson 1995). In one IFA study using MAb H5332, the origin of collection of the single positive tick was not reported, and this study included ticks from both vegetation and animal hosts (Sonenshine et al. 1995). Five studies reported attempts to culture B. burgdorferi from A. americanum ticks; none were successful (Schulze et al. 1986, Teltow et al. 1991, Rawlings and Teltow 1994; Rich et al. 2001; Clark et al. 2002).

Investigations of A. americanum from New Jersey

The first investigations of possible B. burgdorferi transmission by A. americanum were conducted in the 1980s in New Jersey by entomologists from the New Jersey State Department of Health (Schulze et al. 1984, Schulze et al. 1986). In the first study, A. americanum females were removed from two different patient’s erythema migrans-like lesions, prompting a field survey in which spirochetes were detected in 9.1% (4/44) of A. americanum collected on the property where the second patient lived (Schulze et al. 1984). However, the authors observed no motile spirochetes in the infected americanum and remarked that ‘It is not known if nonmotile spirochetes were the result of examining dead or moribund ticks, loss of pathogen viability during transtadial passage, or from indigenous substances within the tick hostile to spirochete vigor or survival’. The second study by the same lead author reported detection of B. burgdorferi in 4.6% (35/756) of adult and nymphal A. americanum and 15.6% (5/32 pools of 15 larvae) of larvae, when identifying spirochetes by darkfield microscopy and DFA testing using fluorescein isothiocyanate (FITC) labeled rabbit anti-B. burgdorferi antiserum. These methods are liable to detect antibodies to infections with other Borrelia spp. (Barbour et al. 1996), or antigens of non-viable, degraded B. burgdorferi spirochetes (Bockenstedt et al. 2012). The researchers were not able to establish cultures in Barbour-Stoenner-Kelly (BSK) medium – the standard medium used for cultivating burgdorferi (Barbour et al. 1983a). Subsequently, spirochetes found in A. americanum collected by the New Jersey group were used to develop the PCR methods instrumental in the first investigation and description of B. lonestari (Barbour et al. 1996).

In later studies by the same New Jersey research group (Schulze et al. 2005, 2006), the findings from the 1980s were no longer described as B. burgdorferi, but rather as ‘spirochetes’, and the authors proposed that the spirochetes previously detected in A. americanum were B. lonestari, not B. burgdorferi. The finding of larval pools infected with spirochetes in Schulze et al. (1986) further suggests that the organism was the transovarially transmitted relapsing fever spirochete B. lonestari, rather than B. burgdorferi, which has not been found to be vertically transmitted by ticks (Stromdahl et al. 2003, Rollend et al. 2013). Investigation of A. americanum adults in Schulze et al. (2005), using PCR with primers designed to amplify all Borrelia spp. (Barbour et al. 1996), followed by sequencing of representative positive amplicons to confirm the species, reported 9.1% (11/121) infected with B. lonestari and none infected with B. burgdorferi. Another investigation of A. americanum adults by Schulze et al. (2006), using primers for B. burgdorferi/B. lonestari (Stegall-Faulk et al. 2003), followed by sequencing of amplicons, revealed 5.8% (6/103) infected with B. lonestari and none infected with B. burgdorferi.

Investigations of A. americanum from North Carolina and Virginia

Magnarelli et al. (1986), a group of researchers from Connecticut and North Carolina, examined A. americanum collected from whitetailed deer in North Carolina by DFA staining using FITC-labeled rabbit antibody against B. burgdorferi and detected Borrelia spirochetes in 1.4% (7/512) of the ticks. White-tailed deer are reservoir incompetent for B. burgdorferi (Telford et al. 1988) and are also zooprophylactic, likely because deer blood complement lyses B. burgdorferi in feeding ticks (Bouchard et al. 2013, Roome et al. 2017). Although B. burgdorferi is sometimes detected in I. scapularis collected from deer, these are likely fragments of dead or dying spirochetes, or another species more able to remain viable in whitetailed deer, such as Borrelia miyamotoi sp. nov. (Spirocheatales: Sp irochaetaceae) (Han et al. 2016) or B. lonestari (Moyer et al. 2006, Varela-Stokes 2007). DFA and polyclonal rabbit antiserum used in these analyses are liable to cross-react with other Borrelia spp. or identify dead and degraded B. burgdorferi spirochetes.

Levine et al. (1989), from North Carolina State University, used darkfield microscopy and DFA with polyclonal antisera to screen 1,836 North Carolina A. americanum adults and nymphs collected in 1984–1987 from vegetation. Spirochetes were found in nine A. americanum ticks using darkfield microscopy, but only one tick reacted to the polyclonal antisera. This same research group next removed ticks from hosts and vegetation in North Carolina and Virginia in 1987 and examined them for Borrelia with IFA using a B. burgdorferi species-specific MAb H5332 (Levine et al. 1991). Borrelia was not detected in 151 questing A. americanum, but was found in 6.0% (4/67) of A. americanum removed from animals (four larvae removed from two raccoons). The MAb H5332 was designed to be specific for B. burgdorferi (Barbour et al. 1983b) and has been found to be nonreactive with B. lonestari (Varela et al. 2004a), so, as is always the case for detection of pathogens in engorged ticks collected from hosts, this might indicate spirochetes or their remnants in host blood, rather than viable infection of the tick itself. Sonenshine et al. (1995), at Old Dominion University, collected ticks from vegetation and animal hosts at several sites in eastern Virginia in 1991–1994 and used darkfield microscopy and IFA with MAb H5332 to detect B. burgdorferi in 0.2% (1/546) of A. americanum. Ticks examined in this study were either questing or removed from animal hosts; the origin of the positive tick was not specified in the article, so it is possible the spirochetes came from host blood.

The North Carolina State University group again collected A. americanum from raccoons and by flagging vegetation in North Carolina from 1990 to 1993 (Ouellette et al. 1997). Examination by IFA using B. burgdorferi-specific MAb H5332 revealed a very low prevalence of spirochetes, 0.2% (6/2,985 in the questing ticks), and a slightly higher prevalence of 1.7% (45/2,739) in ticks removed from raccoons. These investigators also sampled the raccoons and were able to culture spirochetes from the blood of 26% (23/87) of the animals; however, none of the IFA-positive ticks were removed from culture-positive raccoons. The spirochetes detected in the questing ticks may have been undigested fragments of spirochetes from previous infected bloodmeals (Allan et al. 2010, Bockenstedt et al. 2012).

Investigations of A. americanum from Texas

Investigations in 1988–1989 by the Texas Department of Health reported isolation of spirochetes identified by culture in 3/354 A. americanum pools collected from vegetation, animal hosts, and humans in Texas (Teltow et al. 1991). These cultured spirochetes were inoculated into mice in preparation for testing the vector competence of three human-biting tick species, including A. americanum. However, the isolates proved not to be infectious for mice, so vector competence trials were precluded. These isolates had pulsed field gel electrophoretic patterns and plasmid profiles that were indistinguishable from high-passage B. burgdorferi strain B31, which is suggestive of cross-contamination of cultures during primary isolation (Piesman and Happ 1997). In a second study of A. americanum collected from vegetation and animal hosts by the same research group (Rawlings and Teltow 1994), spirochetes were detected in 1.0% (54/5,195) of ticks using DFA testing that did not discriminate among Borrelia species. Attempts to culture spirochetes from additional ticks collected in this study failed. Spirochetes in A. americanum collected in this study were also used in the first investigation and description of B. lonestari, where authors (including G. J. Teltow, author of the reports mentioned in this paragraph) proposed that ‘the majority if not all of the spirochetes previously noted in A. americanum ticks were B. lonestari sp. nov. and not B. burgdorferi’ (Barbour et al. 1996). In more recent studies of human-biting ticks submitted to the Texas Department of State Health Services, all spirochetes found by PCR in A. americanum were determined by nucleotide sequencing to be B. lonestari, including 0.8% (4/367) in 2004–2008 (Williamson et al. 2010) and 1.4% (8/591) in 2008–2014 (Mitchell et al. 2016). In another investigation of A. americanum from Texas, Yuan (2010), at University of Texas at Houston, reported B. lonestari, and no B. burgdorferi, in 1.5% (3/186) of ticks tested by PCR and multilocus sequence typing (MLST).

Investigations of A. americanum from Alabama and Mississippi

Luckhart et al. (1991), of Auburn University, detected spirochetes in 4.0% (6/150) of A. americanum removed in 1988 and 1989 from white-tailed deer and vegetation in Alabama using DFA and IFA with anti-B. burgdorferi PAb, which are not B. burgdorferi-specific tests (as described above). The six positive ticks were recovered feeding on deer, so detection of other Borrelia species or fragments of dead spirochetes cannot be ruled out. In another Alabama study using DFA and IFA with anti-B. burgdorferi PAb, the same Auburn University group reported spirochetes from 6.4% (8/125) of A. americanum removed from hunter-killed deer (three of the positives were from the same deer), but again, the species identity of these spirochetes was not established (Luckhart et al. 1992). Almost a decade later, Auburn researchers used Borrelia genus-wide primers and sequencing (Barbour et al. 1996) to identify 1.0% (2/202) of questing A. americanum as being infected with B. lonestari, not B. burgdorferi; PCR of these ticks using B. burgdorferi-specific primers was negative (Burkot et al. 2001).

Collaborators from the CDC and the Mississippi Department of Health used DFA to examine 68 A. americanum collected in 1999 and 2000 from vegetation, deer, dogs, and humans in Mississippi; none were positive for Borrelia spp. spirochetes (Goddard et al. 2003).

Investigations of A. americanum from Arkansas

A U.S. Army entomology group examined ticks collected from vegetation, human, and animal hosts in Arkansas in 1990 using IFA with the B. burgdoferi-specific MAb H5332 (Kardatzke et al. 1992). No B. burgdoferi infections were detected in 471 A. americanum. Selected samples of IFA-negative ticks were also tested using DFA and FITC-labeled rabbit anti-B. burgdorferi PAb to determine whether other spirochetes were missed by the more specific test, and none were detected. Simpson and Hinck (1993), Arkansas State University, also investigated A. americanum from Arkansas for B. burgdorferi. Two hundred A. americanum were collected in 1989–1991 from vegetation and animal hosts and were first examined for spirochetes by darkfield microscopy. Those slides on which spirochetes were detected were then examined using IFA with MAb H5332. Spirochetes were observed in five females and two nymphs of 200 (3.5%) A. americanum, and authors reported that the majority of the IFA-positive A. americanum were collected from vegetation. Again, this might indicate spirochetes or their remnants in host blood, rather than viable infection of the tick itself (Allan et al. 2010, Bockenstedt et al. 2012). Simpson and Hinck did not provide details of IFA methods and reference an article that describes DFA (Anderson and Magnarelli 1984); therefore, it is not possible to evaluate their precautions to avoid false positives.

Investigations of A. americanum from Missouri

Using IFA with MAb H5332, Feir et al. (1994) visualized spirochetes in 1.9% (33/1752) of A. americanum collected from vegetation in 1989 in Missouri, and subsequent PCR of the tick smear material from the IFA slides amplified B. burgdorferi in a number of these ticks (both Dermacentor variabilis (Say) (Acari: Ixodidae) and A. americanum were investigated, but the number of PCR-positive of each species is not reported). However, the PCR was not congruent with the IFA results; some IFA-negative samples were PCR-positive, and some IFA-positive samples were PCR negative. Perhaps the concentration of the secondary antibody, at a 1:20 dilution, was high enough to cross-react with other Borrelia species. Although two primer sets, one for a 371 bp chromosomal target, and one for 16S rRNA, were used on a sample of the A. americanum ticks, burgdorferi was only amplified using the chromosomal target PCR. Sequencing was performed on only one A. americanum tick smear PCR amplicon. A contemporaneous critique of this article questioned the author’s methods and conclusions because B. burgdorferi was never isolated from the ticks and their PCR analysis used material removed from IFA slides, not ticks (Nelson 1995). Two subsequent articles, both co-authored by E. M. Masters, also an author of Feir et al. (1994), cite the Feir article as evidence of B. lonestari, not B. burgdorferi stating ‘Approximately 2% of A. americanum ticks are infected with a spirochete different from both B. burgdorferi and the other Borrelia genospecies recognized as causes of Lyme disease in Eurasia. Barbour et al. proposed the name B. lonestari species novum. This borrelial species appears to be closely related to B. theileri, the cause of bovine borreliosis.’ (Wormser et al. 2005b). The second article (Wormser et al. 2005a) contains a similar statement. In the face of the inadequate evidence from the Feir article itself, and the re-evaluation of the results by co-author Masters, it is remarkable that this article continues to be cited as support for the role of A. americanum in the transmission of B. burgdorferi (Rudenko et al. 2016).

Four subsequent investigations of Missouri A. americanum using PCR found only B. lonestari and no B. burgdorferi. Researchers from Georgia Southern University tested A. americanum collected from animals and vegetation in Missouri in 1995 and 1996 with IFA using both PAb and MAb (H5332). Five ticks (5/436 = 1.1%) were positive with the PAb, but negative with the MAb, and authors suggested that this might indicate infection with B. lonestari (Kollars et al. 2000). A study by Centers for Disease Control and Prevention (CDC) and U.S. Army investigators using PCR primers for both the 16S rDNA and the flaB gene of all Borreliae (Barbour et al. 1996), plus sequencing, identified a minimum infection rate of 5.6% (12/214) for B. lonestari and no B. burgdorferi in pools of A. americanum adults and nymphs (Bacon et al. 2003). Additional analysis of another population of Missouri A. americanum by the same research team revealed only B. lonestari; all 654 (114 pools) of A. americanum were tested using primers specific for B. lonestari and also primers specific for the ospA gene of B. burgdorferi (Demaerschalck et al. 1995). Twenty-two pools (yielding a maximum likelihood estimate of 3.8% [22/654]) were positive for B. lonestari with none positive for B. burgdorferi (Bacon et al. 2005). In 2010, a Missouri team from Washington University and St. Louis Children’s Hospital examined 1,383 questing nymphal A. americanum using PCR that amplified the 16SrDNA gene of B. lonestari and B. burgdorferi and the 23S-5S intergenic spacer region of B. burgdorferi; 1.3% (18/1383) contained B. lonestari, and none were positive for B. burgdorferi (Allan et al. 2010). In yet another study of ticks from Missouri, Yuan (2010) investigated A. americanum adults collected from vegetation and detected neither B. burgdorferi nor B. lonestari in 42 ticks using primers that amplified the 16S-23S intergenic spacer region, the recG gene and the uvrA gene of B. burgdorferi and B. lonestari.

Two other studies of Missouri A. americanum report PCR detection of Borrelia spp. and B. burgdorferi (Cyr et al. 2005, Hudman and Sargentini 2016), but problems with methodology undermine the credibility of these results. Cyr et al. (2005) present insufficient evidence to support their report of B. burgdorferi in a small sample of A. americanum collected from vegetation, humans, and dogs. Oddly, sequencing of the positive amplicons is described in the Materials and Methods section, but results of the sequencing are not reported. Detection of 4 of 16 A. americanum positive for B. burgdorferi was remarkable, and some explanation of these findings should have been put forward. Hudman and Sargentini (2016), of A.T. Still University, investigated A. americanum collected from vegetation in Missouri and described detection of B. burgdorferi in 0.3% (5/1,880) of adult and nymphal ticks using the primers from Barbour et al. (1996) and sequencing. However, the authors recognized that the evidence of one gene only was insufficient to confirm the identity of B. burgdorferi and therefore reported these samples as positive for Borrelia spp. only. Furthermore, the primers used were not specific for B. burgdorferi; both internal and external primer sets of the nested PCR used in this study would amplify Borrelia spp. other than B. burgdorferi (Barbour et al. 1996).

Investigations of A. americanum from South Carolina

A group from Georgia Southern University attempted culture of Borrelia from 210 A. americanum adults collected from vegetation in South Carolina in 1994 and 1995, but no isolations were obtained (Clark et al. 2002).

Transmission/Vector Competency Trials

Arthropod species may be incompetent as vectors for a pathogen for three possible reasons: 1) failure to acquire the pathogen while feeding on an infected host; 2) inability of the pathogen to persist in the vector; or 3) inability of the tick to subsequently transmit the pathogen to another vertebrate host even if the arthropod can acquire and maintain infection (Ledin et al. 2005). Since 1988, there have been nine studies to our knowledge using animal experiments to assess vector competence of A. americanum for B. burgdorferi, and in none was vector competence demonstrated. The strains of B. burgdorferi used in these trials have been diverse and from a wide range of geographic areas. Five of these animal transmission experiments used strains of B. burgdorferi from northeastern Lyme disease-endemic areas, JDI and SH2-82 (Piesman and Sinsky 1988, Mather and Mather 1990, Mukolwe et al. 1992, Ryder et al. 1992, Soares et al. 2006). One experiment used the SI-1 strain of B. burgdorferi from a cotton mouse and I. scapularis ticks from Georgia (Oliver et al. 1993). Another experiment used the MI-6 strain of B. burgdorferi from Florida (later identified as Borrelia bissettii sp. nov. (Spirocheatales: Spirochaetaceae) by Lin et al. 2002), the northeastern strain SH2-82 as a positive control, and ticks collected in Georgia (Sanders and Oliver 1995). Another study involved 34 strains isolated from the northeastern, southeastern, midwestern, Rocky Mountain, Pacific, and southwestern regions (Piesman and Happ 1997). In two of these experiments, A. americanum larvae acquired spirochetes during feeding upon infectious hosts, but all of these larvae became spirochete-negative before molting to the nymphal stage (Piesman and Sinsky 1988, Mather and Mather 1990). In another study involving A. americanum larvae fed on hamsters infected with B. burgdorferi, a single nymph (1 per 60 nymphs) retained infection through the molt and was positive by IFA for B. burgdorferi. However, nymphal A. americanum that had fed on infected hosts did not transmit infection when fed on uninfected hamsters (Ryder et al. 1992). In one study, researchers not only fed A. americanum larvae by themselves on infected mice but also fed A. americanum and I. scapularis together on the mice. Co-feeding with I. scapularis significantly increased bacterial uptake by A. americanum during feeding, but no spirochetes were detectable in the A. americanum by 14 d after feeding (Soares et al. 2006). There has been no successful transmission of B. burgdorferi between infected and naïve hosts by A. americanum (Mukolwe et al. 1992, Ryder et al. 1992, Oliver et al. 1993, Sanders and Oliver 1995, Piesman and Happ 1997). In all nine studies, successful experimental transmission of B. burgdorferi by I. scapularis acted as a positive control on the experimental conditions. Subsequently, discovery of a protein in the saliva of A. americanum that destroyed B. burgdorferi has provided a biological explanation for the observed lack of vector competency suggesting that the incompetency of A. americanum is mostly due to their inability to acquire viable B. burgdorferi spirochetes (Ledin et al. 2005, Zeidner et al. 2009).

Discovery of B. lonestari and Surveillance for Borrelia spp. in A. americanum Using PCR

Repeated detection of Borrelia in A. americanum with microscopy and immunofluorescent methods (described above) – juxtaposed with repeated failure of A. americanum to maintain and transmit B. burgdorferi in laboratory studies – strongly suggested infection of A. americanum by a distinct Borrelia species. Evidence of this hypothesized novel Borrelia species was first published in 1996 for A. americanum from Maryland (Armstrong et al. 1996) and from Missouri, New Jersey, New York, North Carolina, and Texas (Barbour et al. 1996). Researchers from Harvard School of Public Health conducted a detailed epidemiological/entomological study of ticks and tick-bite victims in Maryland and investigated A. americanum using IFA with polyclonal rabbit antiserum to B. burgdorferi sensu lato, which identified spirochetes in 1.0% (7/685) of ticks (Armstrong et al. 2001). These IFA-positive samples were analyzed using fla gene PCR, and amplicon sequencing plus phylogenetic analysis revealed a spirochete close to the relapsing fever spirochete Borrelia theileri (Laveran) (Spirocheatales: Spirochaetaceae), which later was identified as B. lonestari. None of the ticks contained B. burgdorferi, and attempts to cultivate the spirochete in BSK II and Kelly’s medium failed (Armstrong et al. 1996, Rich et al. 2001). Another research group also conducted molecular analyses of the Borrelia detected in A. americanum from a variety of locations and described the spirochete as B. lonestari (Barbour et al. 1996); Borrelia sequences detected in A. americanum by both groups were identical (Rich et al. 2001).

The description of B. lonestari (Armstrong et al. 1996, Barbour et al. 1996, Rich et al. 2001) and its detection in both an A. americanum tick removed from a patient suffering a skin rash and the supernatant of the patient’s skin biopsy sample (James et al. 2001) raised the possibility of B. lonestari being the etiologic agent of a condition that became known as STARI (Masters et al. 2008). This prompted numerous PCR surveys of Borrelia in A. americanum throughout its range. Many of the investigators screened ticks using broadly reactive or generic Borrelia primers, often flaB gene nested primers from the article first describing B. lonestari (Barbour et al. 1996). These primers amplify Borrelia strains potentially associated with human illness, including Borrelia americana sp. nov. (Spirocheatales: Spiro chaetaceae), Borrelia andersonii sp. nov. (Spirocheatales: Spirochae taceae), and B. bissettii (Stromdahl et al. 2015), as well as B. burgdorferi, B. lonestari and B. miyamotoi. Other research teams used different primer sets specific for B. lonestari and for B. burgdorferi.

Ultimately, no further human case studies linked B. lonestari with STARI patients, and it is no longer thought to be a human pathogen (Philipp et al. 2006; Wormser et al. 2005a,b). Nevertheless, the surveillance performed in these numerous studies, summarized in Table 2, provides extensive evidence of the absence of B. burgdorferi in A. americanum. Over 33,000 A. americanum, from locations throughout the range of the tick, have been tested for Borrelia spp. in this manner in 37 studies from more than 25 research groups, yielding a prevalence of ~1.7% of B. lonestari. In six of these surveys, from four research groups, PCR detection of 35 samples (~0.1%) positive for B. burgdorferi was reported. Only three of these PCR-positives from one study were characterized in detail (Rudenko et al. 2016), and laboratory contamination was suspected in another (Stromdahl et al. 2001, 2015).

Investigations Identifying Only B. lonestari and Not B. burgdorferi Using Primers From Barbour et al. (1996) and Sequencing of All Amplicons

Nine of the surveys, from seven different research groups from seven states (Alabama, Florida, Georgia, Missouri, Nebraska, New Jersey, and Texas), using flagellin gene primers from Barbour et al. (1996) and sequencing of all amplicons, identified B. lonestari and no other Borrelia spp. in 1.7% (79/5,771) of A. americanum removed from vegetation and humans (Burkot et al. 2001, Bacon et al. 2003, Varela et al. 2004b, Williamson et al. 2010, Gleim 2013, Maegli et al. 2016, Gleim et al. 2016, Mitchell et al. 2016, Sayler et al. 2016). Bacon et al. (2003) took extra steps to verify their results by using an additional PCR for a Borrelia genus-specific 16S rRNA gene and a PCR for B. burgdorferi ospA as a control for false positives. Similarly, Mitchell et al. (2016) tested all A. americanum in their study with primers for the 16S rDNA of genus Borrelia, and, as with the PCR with the primers from Barbour et al. (1996), none were found positive for B. burgdorferi. Sayler and the University of Florida research group cited above conducted a second study using the primers from Barbour et al. (1996) to investigate 777 additional Florida A. americanum, and none of the ticks tested were positive (Sayler et al. 2017). To investigate the possibility of false negatives in this study, genomic DNA from all tested tick specimens was visualized on agarose gels and quantified using a Qubit Fluorometer; intact, high molecular weight DNA was verified in all samples.

Investigations Identifying Only B. lonestari and Not B. burgdorferi Using Primers From Barbour et al. (1996) and Multilocus PCR Electrospray Ionization Mass Spectrometry

In another study, from U.S. Army entomology (Stromdahl and Hickling 2012), 1,621 A. americanum removed from humans were tested using the primers from Barbour et al. (1996). This effort yielded 24 Borrelia-positive samples that were then tested further using specific PCRs for B. lonestari (Bacon et al. 2004) and B. burgdorferi (Straubinger 2000). Nine of 1,621 (0.6%) were positive in the B. lonestari PCR, but none were positive for B. burgdorferi. Ten of the 15 tick samples that were positive in the generic Borrelia PCR, but negative in both the B. lonestari and B. burgdorferi-specific PCRs, were sent to Ibis Biosciences for further analysis using a multilocus PCR electrospray ionization mass spectrometry (PCR/ESI-MS) Borrelia identification and genotyping assay (Crowder et al. 2010). PCR/ESI-MS analysis determined that two samples were Borrelia-negative and four were positive for B. lonestari. The remaining three samples, and one of the B. lonestari-positive samples, were positive for the B. burgdorferi flagellin primer but negative for seven other Borrelia primers. Attempts to clone and sequence the flagellin amplicon from these samples were unsuccessful. The PCR/ESI-MS assay targets the same region of the flagellin gene used in the initial screening (Barbour et al. 1996), so amplicon contamination from the positive control could have been responsible for these flagellin primer detections. In total, 0.8% (13/1,621) of A. americanum adults and nymphs were confirmed positive for B. lonestari by multiple PCRs, three suspects were amplicon contamination or perhaps amplification of remnants of Borrelia, five suspects were not identified to species, and no B. burgdorferi was found.

Investigations Identifying Only B. lonestari and No B. burgdorferi Using Primers From Barbour et al. (1996) and Sequencing of a Selection of Amplicons

Seven of the studies in Table 2 also involved the use of flagellin gene primers from Barbour et al. (1996) and reported detection of B. lonestari only, and no B. burgdorferi, in 2.1% (297/13,858) of A. americanum from human, animals, and vegetation, but in these cases, only representative amplicons were sequenced, so detection of other Borrelia cannot be ruled out. Most of these researchers (Stromdahl et al. 2003; Schulze et al. 2005, 2011; Mixson et al. 2006; Castellaw et al. 2010; Killmaster et al. 2014) reported all PCR-positives as B. lonestari. Fryxell et al. (2012) sequenced 66% (107/161) of the amplicons from PCR-positive ticks and reported those not sequenced as ‘Borrelia spp.’.

U.S. Army entomologists (Stromdahl et al. 2003) investigated ticks from the entire geographic range of A. americanum in the United States and detected 78 Borrelia positives. Of these, 68 were sequenced, and all were B. lonestari. Furthermore, all 78 of these positive samples tested negative in a PCR specific for B. burgdorferi ospA (Rosa et al. 1991). Mixson et al. (2006), from CDC and other academic and public health laboratories, used the Barbour flagellin primers to investigate A. americanum from nine states: Florida, Georgia, Iowa, North Carolina, New Jersey, New York, Oklahoma, Rhode Island, and South Carolina. Ticks from all states except Florida, Iowa, Oklahoma, and Rhode Island were PCR-positive. Only selected samples were sequenced, and all were identified as B. lonestari. Similarly, New Jersey public heath entomologists detected Borrelia in americanum from New Jersey using the Barbour flagellin primers, selected samples were sequenced, and all were identified as B. lonestari (Schulze et al. 2005, 2011). Castellaw et al. (2010) used the same primers to test A. americanum from Mississippi and detected Borrelia in 2.6% (5/191) of the ticks. Three of these were sequenced and were identified as B. lonestari. Killmaster et al. (2014) also used the Barbour primers to test 4,236 A. americanum from Georgia and reported 59 ticks positive for B. lonestari and none for B. burgdorferi, but only 10% were confirmed by sequencing.

Fryxell et al. (2012) used the primers from Barbour et al. (1996) to test 657 A. americanum removed from deer and dogs in Arkansas. Of these, 161 produced amplicons and sequencing of 107 identified all as B. lonestari, and none as B. burgdorferi. A single unidentified nymphal Amblyomma spp. removed from a deer was identified by sequencing as B. burgdorferi; however, over 100 ticks of another Amblyomma species, Amblyomma maculatum Koch (Acari: Ixodidae), were collected from deer and dogs in this survey, and B. burgdorferi was identified and confirmed by sequencing in two of these, so it cannot be assumed that the tick was A. americanum. This was the first reported detection of B. burgdorferi in A. maculatum, and a subsequent PCR study examining unfed ticks was undertaken to investigate the potential of this tick to transmit B. burgdorferi (Lee et al. 2014). In this study, no B. burgdorferi was detected in PCR of 306 adult A. maculatum using primer sets for both the flaB and 16S rRNA genes, and 97 adult A. maculatum using only the 16S rRNA PCR. However, two ticks contained a novel reptile-associated Borrelia. This suggests that the B. burgdorferi found in the Fryxell study had been acquired during the bloodmeal and was not being maintained by the tick.

Investigations Identifying No B. burgdorferi Using Primers Other than Barbour et al. (1996)

Nine reports of A. americanum describing the detection of only B. lonestari (and no other Borrelia spp.), or no Borrelia, used primer sets other than those of Barbour et al. (1996), and together they revealed a B. lonestari prevalence of 1.3% (96/7372; Stegall-Faulk et al. 2003, Bacon et al. 2005, Schulze et al. 2006, Jordan et al. 2009, Allan et al. 2010, Yuan 2010, Fritzen et al. 2011, Stromdahl et al. 2015, https://www.tickreport.com/stats (Accessed 26 September 2017).

Primers designed by researchers at Middle Tennessee State University that amplify the flagellin gene of B. lonestari and B. burgdorferi were used to test Tennessee A. americanum (Stegall-Faulk et al. 2003). Two of 453 (0.4%) were positive at the gel band size indicating B. lonestari, and identity of both as B. lonestari was confirmed with dot blot hybridization of PCR products. One of the samples was sequenced to further confirm identity. Bacon et al. (2005) tested 114 pools (654 total ticks) of A. americanum using primers specific for B. lonestari and also primers specific for the ospA gene of B. burgdorferi (Demaerschalck et al. 1995). Twenty-two pools were positive for B. lonestari with none positive for B. burgdorferi. Schulze et al. (2006) also used the primers from Stegall-Faulk et al. (2003) to test A. americanum from New Jersey. Six of 103 (5.8%) americanum samples produced gel bands at the size indicating B. lonestari, and none were positive for B. burgdorferi. All six samples were sequenced to confirm identity. Different flagellin primers designed by the Middle Tennessee State University research group to amplify both B. lonestari and B. burgdorferi were used to test 399 A. americanum collected in Tennessee, but no tick samples were positive for either target (Jordan et al. 2009). As described earlier in this review, the Missouri team from Washington University and St. Louis Children’s Hospital examined 1,383 A. americanum using PCR for B. lonestari and B. burgdorferi; 1.3% (18/1383) contained B. lonestari, and none were positive for B. burgdorferi (Allan et al. 2010). Universal 16S rDNA primers (Pichon et al. 2003) and primers for the 23S-5S intergenic spacer of Borrelia (Rijpkema et al. 1995) were used for the PCR screen, and a reverse line blot assay for B. burgdorferi and B. lonestari reconfirmed the positives.

Yuan (2010) at University of Texas at Houston used primers designed to amplify the 16S-23S intergenic spacer of both B. burgdorferi and B. lonestari (Bunikis et al. 2004), plus MLST modified to identify the recG and uvrA genes from B. burgdorferi and B. lonestari (Margos et al. 2008) and reported B. lonestari in 1.5% (3/186) of A. americanum from Texas, no Borrelia in 42 A. americanum from Missouri, and no B. burgdorferi in any of these 228 ticks.

A group from the Tennessee Department of Health and the Kentucky Department for Public Health also used primers for the Borrelia 16S-23S intergenic spacer from Bunikis et al. (2004) to detect B. lonestari in 0.9% (1/108), and no B. burgdorferi in A. americanum collected from animals in Kentucky. The identity of the Borrelia spp. in this sample of ticks was confirmed by sequencing. These ticks were also assessed with B. burgdorferi-specific ospA primers from Demaerschalck et al. (1995), and none of the ticks were positive (Fritzen et al. 2011).

Researchers from U.S. Army entomology, CDC, two academic laboratories, and Ibis Biosciences used Borrelia flagellin gene primers from Clark et al. (2013) to screen 1,097 A. americanum removed from humans throughout the range of the tick (Stromdahl et al. 2015). Nine suspect-positives (five pools and four individuals) were all confirmed as B. lonestari by subsequent PCRs targeting the 16S rRNA qPCR for Borrelia (Tsao et al. 2004), the 16S-23S intergenic spacer region of Borrelia (Bunikis et al. 2004), and in the eight Borrelia PCRs of the Ibis Biosciences PCR/ESI-MS system (Crowder et al. 2010).

The Laboratory of Medical Zoology (LMZ) at the University of Massachusetts offers a tick identification and pathogen testing service. The crowd-sourced program provides a public surveillance database of human-biting ticks, their feeding status, and submitted and tested by LMZ (https://www.tickreport.com/stats). From 2006 until 2017, the LMZ tested 2,483 human-biting A. americanum for B. burgdorferi using a TaqMan qPCR assay (Xu et al. 2016). None of these A. americanum were positive for B. burgdorferi, while 31 (1.11%) were positive for B. lonestari.

Investigations Identifying B. lonestari, B. burgdorferi, Or Both in A. americanum

Six of the molecular surveys listed in Table 2 report identification of B. burgdorferi in A. americanum (Stromdahl et al. 2001, Clark 2004, Cyr et al. 2005, Taft et al. 2005, Clark et al. 2013, Rudenko et al. 2016). In the earliest article, the B. burgdorferi-positive PCR of A. americanum was likely due to contamination, as authors explained in subsequent publications (Stromdahl et al. 2001, 2015). Four of the surveys that reported B. burgdorferi presented insufficient evidence (PCR of one gene only, sequencing in one direction only, products not sequenced) to definitively confirm the identity of the PCR products (Clark 2004, Cyr et al. 2005, Taft et al. 2005, Clark et al. 2013). In the sixth study, Rudenko et al. (2016) confirmed species identity by MLST/MLSA of up to 10 genes, but only a very small number of ticks (3) ticks were so characterized.

Stromdahl et al. (2001) used PCR to investigate A. americanum collected in 1997 for B. burgdorferi and reported a minimum infection rate of 11.7% (26/222), though only 3.2% (7/222) produced amplicons in PCRs for two different gene targets. In a later publication, these PCR results were re-evaluated and contamination was suspected because all occurred in the first year of the study when PCR was initially implemented, and no positive results were obtained across large numbers of samples in all subsequent years (Stromdahl et al. 2015). Amplicons from the 1997 tick PCRs were not sequenced.

In a study that involved ticks collected from vegetation in Florida, Clark (2004) reported detection of B. burgdorferi in 1.3% (5/396) of A. americanum. Identity of positive amplicons was confirmed by sequencing in one direction only, and the various PCR methods used on each positive sample are not precisely described. Of 252 A. americanum ticks tested using flaB primers from Johnson et al. (1992), five tested positive for B. burgdorferi. It appears that these were also tested using primers from Barbour et al. (1996, Tables 3 and 5; Clark 2004), and only one tick was positive for B. burgdorferi. Some of the samples positive with the primers from Johnson et al. (1992) were tested using nested ospA primers (Guttman et al. 1996, Guy et al. 1991) and 5S-23S primers (Rijpkema et al. 1995), but no A. americanum ticks were positive for B. burgdorferi. Most of the samples positive with the primers from Johnson et al. (1992) were tested using nested p66 primers (Rosa et al. 1991), and two A. americanum were positive for B. burgdorferi.

Cyr et al. (2005), as described earlier in this review, presented insufficient evidence to support their report of a improbably high incidence of B. burgdorferi in a small sample of A. americanum (4/16 = 25%); the 95% confidence interval for finding 4 of 16 ticks positive is 0.08–52%. The investigators stated that this occurred because the tick collections for their study were purposely made in areas of Missouri suspected of being ‘hot spots’ for Lyme disease, and PCR using the same primers produced positive results testing skin biopsies from Missouri patients suspected of having Lyme disease. This result is inconsistent with the much larger surveys reported here. In the absence of confirmation by another test, this report should be viewed cautiously. Novel findings of B. burgdorferi in tick species should be supported by characterization of multiple gene targets and evaluation of cross-reactivity with other Borrelia, but in this instance, only one gene target, 16S rDNA, was used for the tick samples and for the human samples that corroborated the findings in the tick samples. The sequencing data from the positive ticks and skin samples were not presented therefore could not be analyzed. A search for articles citing Cyr et al. (2005) identified only one other study using these primers and again, authors report ‘anomalous’ infection rates detected in I. scapularis ticks collected in Maryland (Carroll and Cyr 2005). These novel primers should have been assessed in conjunction with other, more frequently used, assays for Borrelia spp.

Taft et al. (2005) tested ticks using primers of their own design targeting the flagellin gene of both B. burgdorferi and B. lonestari and reported 1.5% (4/269) of A. americanum positive for B. burgdorferi and 2.2% (6/269) positive for B. lonestari. The B. burgdorferi-positive A. americanum were two adults and two nymphs collected from vegetation. The positive samples were reconfirmed via dot blot hybridization with probes internal to the amplicon that differentiated B. burgdorferi and B. lonestari; however, the B. burgdorferi-positive samples were not sequenced, so these tick are best considered ‘suspect-positive’.

Clark et al. (2013) described A. americanum as a vector of the agent of Lyme disease and presented as evidence the detection of B. burgdorferi by PCR in two A. americanum ticks removed from two patients. Confirmation of the identity of B. burgdorferi in these two PCR-positive A. americanum relied on the detection and sequencing of one gene only (flaB). The 429 nucleotide flaB sequence amplified from one of the ticks (collected from Patient 4) was 100% identical to the B. burgdorferi B31 PCR control (NC_001318.1 sequence, GenBank), but was 99.8% (429/430-bp) similar to the flaB sequence from the blood of Patient 4 from whom the tick was collected. The 456 nucleotide flaB sequence amplified from the other tick (collected from Patient 7) was 99.6% (453/455-bp) identical to the B31 PCR control and 99.6% (451/453) identical to B. burgdorferi in an EM biopsy from Patient 7. The number of sequencing reads used to determine their sequences is not stated for this study, so it is unclear whether the sequence differences between the patient samples and the B31 control were real or due to sequencing errors. The B. burgdorferi sequences amplified from the Patient 4 blood sample and the Patient 7 skin sample did not match those obtained from the same patient’s attached A. americanum tick, so there is no evidence that the B. burgdorferi DNA found in the patient samples came from B. burgdorferi in the attached tick.

Rudenko et al. (2016) reported 2.2% (13/590) of A. americanum to be PCR-positive using B. burgdorferi flagellin gene primers, but MLST of 10 gene targets could only confirm 3 of these positives, and not all 10 loci were amplified in each of those 3 samples. B. burgdorferi is readily amplified from Ixodes spp. ticks; weak PCR signals for B. burgdorferi in A. americanum tick samples may indicate amplification of remnants of a bloodmeal from a previous life stage (Allen et al. 2010, Bockenstedt et al. 2012, Marques et al. 2014), rather than active infection with viable spirochetes. It should also be emphasized that infrequent detection by molecular methods of B. burgdorferi DNA in A. americanum does not indicate that these ticks are capable of transmission.

Discussion and Conclusions

Early studies suggested the presence of B. burgdorferi in A. americanum ticks in the United States. However, in almost all cases, Borreliae were detected using methods that were not Borrelia species-specific; spirochetes that were detected were likely other species, or transient infections detected in engorged ticks collected from hosts infected with B. burgdorferi transmitted by other sympatric vector-competent tick species. More recently, extensive surveillance, using methods that discriminate among Borrelia species, has only rarely detected B. burgdorferi in A. americanum ticks, and most of these observations could have resulted from the presence of B. burgdorferi DNA that was most likely from an infectious host bloodmeal. Definitive experimental infections using a diverse array of B. burgdorferi strains have repeatedly failed to demonstrate vector competency of A. americanum for B. burgdorferi.

A hypothesis of ‘selective compatibility’—i.e., that certain strains of B. burgdorferi in the southeastern United States may be better adapted to development in ticks other than Ixodes spp.—was proposed by Luckhart et al. (1991). Rudenko et al. (2016) revisited the possible role of A. americanum as a Lyme vector, citing as evidence findings of ‘B. burgdorferi’ in A. americanum from New Jersey (Schulze et al. 1984), Missouri (Feir et al. 1994), and Texas (Teltow et al. 1991). However, authors of these three studies subsequently revised their findings to ‘B. lonestari’ (Barbour et al. 1996; Schulze et al. 2005, 2006; Wormser et al. 2005a,b). Rudenko et al. (2016) reported detecting three questing A. americanum positive for B. burgdorferi by PCR and MLST and suggested that when compatible spirochete strains meet an appropriate tick population, maintenance and transmission could occur. If so, then these strains are likely very rare, as transmission did not occur in two laboratory experiments involving A. americanum and B. burgdorferi strains from the region of origin of these positive ticks (Oliver et al. 1993, Sanders and Oliver 1995). Borrelia burgdorferi has never been successfully cultured from A. americanum; motile spirochetes have never been described from A. americanum, and the studies reviewed in Tables 1 and 2 report no conclusive evidence of B. burgdorferi infection in extensive surveillance of more than 52,000 A. americanum. Throughout the entire range of A. americanum (Springer et al. 2014), the tick is constantly being exposed to Borreliae, although sympatric tick populations and host species compositions change. By some mechanism, most likely borreliacidal salivary components, the tick resists colonization by B. burgdorferi. While the existence of some compatible spirochete strains and A. americanum tick populations cannot be entirely ruled out, how transmission cycles could be maintained in nature at the vanishingly low prevalence levels detected in surveillance of A. americanum ticks remains an important question that needs to be addressed.

PCR and other detection methods have limitations when trying to answer the question of whether or not A. americanum has the ability to transmit/vector B. burgdorferi to humans. Most important to note is that the detection of Borrelia DNA does not mean living Borrelia are present. It has been shown that pieces of dead B. burgdorferi can elicit antigenic responses that are identified by immunofluorescent staining, and contain DNA that can be detected by PCR (Bockenstedt et al. 2012), and DNA from the blood cells of a previous host blood-meal often persists through the tick’s molt and has been detected in between 45 and 63% of questing nymphal and adult A. americanum ticks (Allan et al. 2010, Harmon et al. 2015). Therefore, the rare detection of B. burgdorferi DNA in questing A. americanum should not be over-interpreted, and these rare detections need to be put in context with the comparatively high prevalence of infection in questing ticks of species known to be vector-competent. PCR data alone may reveal pathogen DNA, but the more useful information remains unknown, including whether the DNA came from live or dead bacteria within the tick’s body, or dead bacteria residing in the tick’s midgut that are remnants of the tick’s last meal.

The possibility of false-positive test results is increased when using PCR (particularly nested PCR) to detect bacterial DNA because the products of DNA amplification that give the test result can be due to contamination or false priming if the PCR protocol is not sufficiently stringent (Lo and Chan 2006). There is greater confidence in PCR test results when multiple PCR targets are used and when amplicons are sequenced. Careful review of sequences of PCR amplicons is needed to rule out the possibility of contamination of PCR reactions by DNA from positive controls and strains grown in the laboratory, and to ensure that there is no carry-over contamination from other tested samples. PCR, sequencing, and analysis of highly variable DNA genes or multiple genes can assist in assuring that DNA contamination did not occur with the positive DNA control or other lab strains if they do not match the sequences of these strains. DNA sequencing should be done on both strands of a PCR product, and if Sanger sequencing is employed, a sufficiently large number of reads should be used to determine the consensus sequence to assure sequencing errors did not occur.

Confirmation of a suspect-positive result by reproducing the result with multiple real-time PCR assays can have benefits. The ability to consistently reproduce positive results with other assays is suggestive that PCR amplicon contamination has not been occurring, but it cannot rule out genomic DNA contamination of a sample. Proper DNA extraction controls, PCR controls, and sequencing are needed to do this. Performing real-time PCR assays instead of standard or nested PCR in general reduces the likelihood that amplicon contamination will occur since the PCR products do not need to be pipetted from an open tube in order to be used in downstream PCRs or run on an agarose gel, both of which increase the chance of laboratory contamination with PCR products. In addition, real-time PCR assays that use a standard curve can provide information on the approximate DNA copy number present in a tick so as to provide information on whether the detected pathogen is present in biologically meaningful numbers.

Successful culture isolation of B. burgdorferi from a tick removed from an animal host does not distinguish between exposure to B. burgdorferi in the bloodmeal versus active infection of the tick, nor does it determine whether the spirochetes would survive transtadially until the tick feeds again at the next life stage, or be successfully transmitted to a new host during the next bloodmeal.

Culturing of host-seeking ticks, or of ticks removed from hosts and allowed to molt to the next life stage before culturing, could answer the question of whether some strains of B. burgdorferi can live through a molt. Transmission studies would then be required to verify whether these ticks could transmit the spirochete to their next bloodmeal host.

The role of A. americanum in the transmission of Lyme disease remains controversial in the minds of a very few scientists, yet public sentiment has kept the possibility of ‘Lyme disease from lone star ticks’ in the spotlight. Although advancements in molecular analysis strongly suggest that initial reports of B. burgdorferi in A. americanum across many states were in fact misidentified B. lonestari or DNA contamination, early reports continue to be cited without mention of the later clarifying studies; consequently, the search for a population of A. americanum that can transmit the Lyme disease spirochete has been ongoing. We suggest that the few studies implicating A. americanum as a vector of B. burgdorferi have not yet met the burden of proof for their assumption. This review of extensive surveillance and vector competency studies of B. burgdorferi in A. americanum studies supports the conclusion that A. americanum is not a consequential factor in Lyme disease ecology and epidemiology. Information pertaining to the geographical distribution of infected ticks is quite important so that practicing physicians adopt the appropriate level of concern in a given patient population. There is no epidemiological need whatsoever to invoke another vector to explain Lyme disease prevalence and distribution in the United States.

Acknowledgments

We thank Stephanie Cinkovich for technical review, and Stephen Rich for unpublished data from the University of Massachusetts Laboratory of Medical Zoology and insightful comments on drafts of the manuscript. The views expressed in this article are those of the authors and do not reflect the official policy or position of the Department of the Army, Department of Defense or the U.S. Government. Some of the authors, as employees of the U.S. Government (E.Y.S., R.M.N., G.A.H, C.C., and T.F.C.), conducted the work as part of their official duties.

References Cited

  1. Allan BF, Goessling LS, Storch GA, and Thach RE. 2010. Blood meal analysis to identify reservoir hosts for Amblyomma americanum ticks. Emerg. Infect. Dis 16: 433–440. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Armstrong PM, Rich SM, Smith RD, Hartl DL, Spielman A, and Telford SR 3rd. 1996. A new Borrelia infecting Lone Star ticks. Lancet. 347: 67–68. [DOI] [PubMed] [Google Scholar]
  3. Armstrong PM, Brunet LR, Spielman A, and Telford SR 3rd. 2001. Risk of Lyme disease: perceptions of residents of a Lone Star tick-infested community. Bull. World Health Organ 79: 916–925. [PMC free article] [PubMed] [Google Scholar]
  4. Bacon RM, Gilmore RD Jr, Quintana M, Piesman J, and Johnson BJ. 2003. DNA evidence of Borrelia lonestari in Amblyomma americanum (Acari: Ixodidae) in southeast Missouri. J. Med. Entomol 40: 590–592. [DOI] [PubMed] [Google Scholar]
  5. Bacon RM, Pilgard MA, Johnson BJ, Raffel SJ, and Schwan TG. 2004. Glycerophosphodiester phosphodiesterase gene (glpQ) of Borrelia lonestari identified as a target for differentiating Borrelia species associated with hard ticks (Acari:Ixodidae). J. Clin. Microbiol 42: 2326–2328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bacon RM, Pilgard MA, Johnson BJ, Piesman J, Biggerstaff BJ, and Quintana M. 2005. Rapid detection methods and prevalence estimation for Borrelia lonestari glpQ in Amblyomma americanum (Acari: Ixodidae) pools of unequal size. Vector Borne Zoonotic Dis. 5: 146–156. [DOI] [PubMed] [Google Scholar]
  7. Barbour AG, Burgdorfer W, Hayes SF, Péter O, and Aeschlimann A. 1983a. Isolation of a cultivable spirochete from Ixodes ricinus ticks of Switzerland. Curr. Microbiol 8:123–126. [Google Scholar]
  8. Barbour AG, Tessier SL, and Todd WJ. 1983b. Lyme disease spirochetes and ixodid tick spirochetes share a common surface antigenic determinant defined by a monoclonal antibody. Infect. Immun 41: 795–804. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Barbour AG, Maupin GO, Teltow GJ, Carter CJ, and Piesman J. 1996. Identification of an uncultivable Borrelia species in the hard tick Amblyomma americanum: possible agent of a Lyme disease-like illness. J. Infect. Dis 173: 403–409. [DOI] [PubMed] [Google Scholar]
  10. Bockenstedt LK, Gonzalez DG, Haberman AM, and Belperron AA. 2012. Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy. J. Clin. Invest 122: 2652–2660. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Bouchard C, Leighton PA, Beauchamp G, Nguon S, Trudel L, Milord F, Lindsay LR, Bélanger D, and Ogden NH. 2013. Harvested white-tailed deer as sentinel hosts for early establishing Ixodes scapularis populations and risk from vector-borne zoonoses in southeastern Canada. J. Med. Entomol 50: 384–393. [DOI] [PubMed] [Google Scholar]
  12. Bunikis J, Tsao J, Garpmo U, Berglund J, Fish D, and Barbour AG. 2004. Typing of Borrelia relapsing fever group strains. J. Emerg. Infect. Dis 10: 1161–1164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, and Davis JP. 1982. Lyme disease-a tick-borne spirochetosis? Science. 216: 1317–1319. [DOI] [PubMed] [Google Scholar]
  14. Burkot TR, Mullen GR, Anderson R, Schneider BS, Happ CM, and Zeidner NS. 2001. Borrelia lonestari DNA in adult Amblyomma americanum ticks, Alabama. Emerg. Infect. Dis 7: 471–473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Carroll JF, and Cyr TL. 2005. A note on the densities of Ixodes scapularis (Acari:Ixodidae) and white-tailed deer on the campus of the National Institutes of Standards and Technology, Maryland, USA. Proc. Entomol. Soc. Wash 107: 973–976. [Google Scholar]
  16. Castellaw AH, Showers J, Goddard J, Chenney EF, and Varela-Stokes AS. 2010. Detection of vector-borne agents in lone star ticks, Amblyomma americanum (Acari: Ixodidae), from Mississippi. J. Med. Entomol 47: 473–476. [DOI] [PubMed] [Google Scholar]
  17. Clark K 2004. Borrelia species in host-seeking ticks and small mammals in northern Florida. J. Clin. Microbiol 42: 5076–5086. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Clark KL, Oliver JH Jr, James AM, Durden LA, and Banks CW. 2002. Prevalence of Borrelia burgdorferi sensu lato infection among rodents and host-seeking ticks in South Carolina. J. Med. Entomol 39: 198–206. [DOI] [PubMed] [Google Scholar]
  19. Clark K, Hendricks A, and Burge D. 2005. Molecular identification and analysis of Borrelia burgdorferi sensu lato in lizards in the southeastern United States. Appl. Environ. Microbiol 71: 2616–2625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Clark KL, Leydet B, and Hartman S. 2013. Lyme borreliosis in human patients in Florida and Georgia, USA. Int. J. Med. Sci 10: 915–931. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Crowder CD, Matthews HE, Schutzer S, Rounds MA, Luft BJ, Nolte O, Campbell SR, Phillipson CA, Li F, Sampath R, et al. 2010. Genotypic variation and mixtures of Lyme Borrelia in Ixodes ticks from North America and Europe. PLos One. 5: e10650. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Cyr TL, Jenkins MC, Hall RD, Masters EJ, and McDonald GA. 2005. Improving the specificity of 16S rDNA-based polymerase chain reaction for detecting Borrelia burgdorferi sensu lato-causative agents of human Lyme disease. J. Appl. Microbiol 98: 962–970. [DOI] [PubMed] [Google Scholar]
  23. Demaerschalck I, Ben Messaoud A, De Kesel M, Hoyois B, Lobet Y, Hoet P, Bigaignon G, Bollen A, and Godfroid E. 1995. Simultaneous presence of different Borrelia burgdorferi genospecies in biological fluids of Lyme disease patients. J. Clin. Microbiol 33: 602–608. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Feir D, Santanello CR, Li BW, Xie CS, Masters E, Marconi R, and Weil G. 1994. Evidence supporting the presence of Borrelia burgdorferi in Missouri. Am. J. Trop. Med. Hyg 51: 475–482. [PubMed] [Google Scholar]
  25. Fritzen CM, Huang J, Westby K, Freye JD, Dunlap B, Yabsley MJ, Schardein M, Dunn JR, Jones TF, and Moncayo AC. 2011. Infection prevalences of common tick-borne pathogens in adult lone star ticks (Amblyomma americanum) and American dog ticks (Dermacentor variabilis) in Kentucky. Am. J. Trop. Med. Hyg 85: 718–723. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Fryxell RT, Steelman CD, Szalanski AL, Kvamme KL, Billingsley PM, and Williamson PC. 2012. Survey of Borreliae in ticks, canines, and white-tailed deer from Arkansas, U.S.A. Parasit. Vectors 5: 139. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Gleim ER 2013. The effects of long-term prescribed burning on ticks and tick-borne pathogen prevalence Ph.D. dissertation, The University of Georgia, Athens, GA. [Google Scholar]
  28. Gleim ER, Garrison LE, Vello MS, Savage MY, Lopez G, Berghaus RD, and Yabsley MJ. 2016. Factors associated with tick bites and pathogen prevalence in ticks parasitizing humans in Georgia, USA. Parasit. Vectors 9: 125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Goddard J, Sumner JW, Nicholson WL, Paddock CD, Shen J, and Piesman J. 2003. Survey of ticks collected in Mississippi for Rickettsia, Ehrlichia, and Borrelia species. J. Vector Ecol 28: 184–189. [PubMed] [Google Scholar]
  30. Guttman DS, Wang PW, Wang IN, Bosler EM, Luft BJ, and Dykhuizen DD. 1996. Multiple infections of Ixodes scapularis ticks by Borrelia burgdorferi as revealed by single-strand conformation polymorphism analysis. J. Clin. Microbiol 34: 652–656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Guy EC, and Stanek G. 1991. Detection of Borrelia burgdorferi in patients with Lyme disease by the polymerase chain reaction. J. Clin. Pathol 44: 610–611. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Han S, Hickling GJ, and Tsao JI. 2016. High Prevalence of Borrelia miyamotoi among Adult Blacklegged Ticks from White-Tailed Deer. Emerg. Infect. Dis 22: 316–318. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Harmon JR, Scott MC, Baker EM, Jones CJ, and Hickling GJ. 2015. Molecular identification of Ehrlichia species and host bloodmeal source in Amblyomma americanum L. from two locations in Tennessee, United States. Ticks Tick Borne Dis. 6: 246–252. [DOI] [PubMed] [Google Scholar]
  34. Haynes JM, Prescott LP, Seipelt RL, and Wright SM. 2005. Detection of Borrelia burgdorferi sequences in a biopsy from a Tennessee patient. J. TN Acad. Sci 80: 57–59. [Google Scholar]
  35. Hudman DA, and Sargentini NJ. 2016. Detection of Borrelia, Ehrlichia, and Rickettsia spp. in ticks in northeast Missouri. Ticks Tick Borne Dis. 7: 915–921. [DOI] [PubMed] [Google Scholar]
  36. James AM, Liveris D, Wormser GP, Schwartz I, Montecalvo MA, and Johnson BJ. 2001. Borrelia lonestari infection after a bite by an Amblyomma americanum tick. J. Infect. Dis 183: 1810–1814. [DOI] [PubMed] [Google Scholar]
  37. Johnson BJ, Happ CM, Mayer LW, and Piesman J. 1992. Detection of Borrelia burgdorferi in ticks by species-specific amplification of the flagellin gene. Am. J. Trop. Med. Hyg 47: 730–741. [DOI] [PubMed] [Google Scholar]
  38. Jordan BE, Onks KR, Hamilton SW, Hayslette SE, and Wright SM. 2009. Detection of Borrelia burgdorferi and Borrelia lonestari in birds in Tennessee. J. Med. Entomol 46: 131–138. [DOI] [PubMed] [Google Scholar]
  39. Kardatzke JT, Neidhardt K, Dzuban DP, Sanchez JL, and Azad AF. 1992. Cluster of tick-borne infections at Fort Chaffee, Arkansas: Rickettsiae and Borrelia burgdorferi in ixodid ticks. J. Med. Entomol 29: 669–672. [DOI] [PubMed] [Google Scholar]
  40. Killmaster LF, Loftis AD, Zemtsova GE, and Levin ML. 2014. Detection of bacterial agents in Amblyomma americanum (Acari: Ixodidae) from Georgia, USA, and the use of a multiplex assay to differentiate Ehrlichia chaffeensis and Ehrlichia ewingii. J. Med. Entomol 51: 868–872. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Kollars TM Jr, Oliver JH, Kollars PG Jr, James AM, Richardson A, Durden LA, Miles DO, and Masters EJ. 2000. Phenotypic variation in Borrelia burgdorferi sensu lato in ticks (Acari: Ixodidae) and isolates from Missouri, USA. Int. J. Acarol 26: 167–172. [Google Scholar]
  42. Lane RS, Piesman J, and Burgdorfer W. 1991. Lyme borreliosis: relation of its causative agent to its vectors and hosts in North America and Europe. Annu. Rev. Entomol 36: 587–609. [DOI] [PubMed] [Google Scholar]
  43. Ledin KE, Zeidner NS, Ribeiro JM, Biggerstaff BJ, Dolan MC, Dietrich G, Vredevoe L, and Piesman J. 2005. Borreliacidal activity of saliva of the tick Amblyomma americanum. Med. Vet. Entomol 19: 90–95. [DOI] [PubMed] [Google Scholar]
  44. Lee JK, Smith WC, McIntosh C, Ferrari FG, Moore-Henderson B, and Varela-Stokes A. 2014. Detection of a Borrelia species in questing Gulf Coast ticks, Amblyomma maculatum. Ticks Tick Borne Dis. 5: 449–452. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Levine JF, Apperson CS, Nicholson WL. 1989. The occurrence of spirochetes in ixodid ticks in North Carolina. J. Entomol. Sci 24: 594–602. [Google Scholar]
  46. Levine JF, Sonenshine DE, Nicholson WL, and Turner RT. 1991. Borrelia burgdorferi in ticks (Acari: Ixodidae) from coastal Virginia. J. Med. Entomol 28: 668–674. [DOI] [PubMed] [Google Scholar]
  47. Lin T, Oliver J,H Jr, and Gao L. 2002. Genetic diversity of the outer surface protein C gene of southern Borrelia isolates and its possible epidemiological, clinical, and pathogenetic implications. J. Clin. Microbiol 40: 2572–2583. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Lo YM, and Chan KC. 2006. Setting up a polymerase chain reaction laboratory. Methods Mol. Biol 336: 11–18. [DOI] [PubMed] [Google Scholar]
  49. Luckhart S, Mullen GR, and Wright JC. 1991. Etiologic agent of Lyme disease, Borrelia burgdorferi, detected in ticks (Acari: Ixodidae) collected at a focus in Alabama. J. Med. Entomol 28: 652–657. [DOI] [PubMed] [Google Scholar]
  50. Luckhart S, Mullen GR, Durden LA, and Wright JC. 1992. Borrelia sp. in ticks recovered from white-tailed deer in Alabama. J. Wildl. Dis 28: 449–452. [DOI] [PubMed] [Google Scholar]
  51. Maegli A, Loy JD, and Cortinas R. 2016. Note on Ehrlichia chaffeensis, Ehrlichia ewingii, and “Borrelia lonestari” infection in lone star ticks (Acari: Ixodidae), Nebraska, USA. Ticks Tick Borne Dis. 7: 154–158. [DOI] [PubMed] [Google Scholar]
  52. Magnarelli LA, Anderson JF, Apperson CS, Fish D, Johnson RC, and Chappell WA. 1986. Spirochetes in ticks and antibodies to Borrelia burgdorferi in white-tailed deer from Connecticut, New York State, and North Carolina. J. Wildl. Dis 22: 178–188. [DOI] [PubMed] [Google Scholar]
  53. Margos G, Gatewood AG, Aanensen DM, Hanincová K, Terekhova D, Vollmer SA, Cornet M, Piesman J, Donaghy M, Bormane A, et al. 2008. MLST of housekeeping genes captures geographic population structure and suggests a European origin of Borrelia burgdorferi. Proc. Natl. Acad. Sci. USA 105: 8730–8735. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Marques A, Telford SR 3rd, Turk SP, Chung E, Williams C, Dardick K, Krause PJ, Brandeburg C, Crowder CD, Carolan HE, et al. 2014. Xenodiagnosis to detect Borrelia burgdorferi infection: a first-in-human study. Clin. Infect. Dis 58: 937–945. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Masters EJ, Grigery CN, and Masters RW. 2008. STARI, or Masters disease: Lone Star tick-vectored Lyme-like illness. Infect. Dis. Clin. North Am 22: 361–376, viii. [DOI] [PubMed] [Google Scholar]
  56. Mather TN, and Mather ME. 1990. Intrinsic competence of three ixodid ticks (Acari) as vectors of the Lyme disease spirochete. J. Med. Entomol 27: 646–650. [DOI] [PubMed] [Google Scholar]
  57. Mitchell EA, Williamson PC, Billingsley PM, Seals JP, Ferguson EE, and Allen MS. 2016. Frequency and distribution of Rickettsiae, Borreliae, and Ehrlichiae detected in human-parasitizing ticks, Texas, USA. Emerg. Infect. Dis 22: 312–315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Mixson TR, Campbell SR, Gill JS, Ginsberg HS, Reichard MV, Schulze TL, and Dasch GA. 2006. Prevalence of Ehrlichia, Borrelia, and Rickettsial agents in Amblyomma americanum (Acari: Ixodidae) collected from nine states. J. Med. Entomol 43: 1261–1268. [DOI] [PubMed] [Google Scholar]
  59. Moyer PL, Varela AS, Luttrell MP, Moore VA 4th, Stallknecht DE, and Little SE. 2006. White-tailed deer (Odocoileus virginianus) develop spirochetemia following experimental infection with Borrelia lonestari. Vet. Microbiol 115: 229–236. [DOI] [PubMed] [Google Scholar]
  60. Mukolwe SW, Kocan AA, Barker RW, Kocan KM, and Murphy GL. 1992. Attempted transmission of Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) (JDI strain) by Ixodes scapularis (Acari: Ixodidae), Dermacentor variabilis, and Amblyomma americanum. J. Med. Entomol 29: 673–677. [DOI] [PubMed] [Google Scholar]
  61. Nelson JA 1995. Evidence supporting the presence of Borrelia burgdorferi in Missouri. Infect. Dis. Clin. Prac 4: 110–111 [Google Scholar]
  62. Oliver JH Jr, Chandler FW Jr, Luttrell MP, James AM, Stallknecht DE, McGuire BS, Hutcheson HJ, Cummins GA, and Lane RS. 1993. Isolation and transmission of the Lyme disease spirochete from the southeastern United States. Proc. Natl. Acad. Sci. USA 90: 7371–7375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Ouellette J, Apperson CS, Howard P, Evans TL, and Levine JF. 1997. Tick-raccoon associations and the potential for Lyme disease spirochete transmission in the coastal plain of North Carolina. J. Wildl. Dis 33: 28–39. [DOI] [PubMed] [Google Scholar]
  64. Philipp MT, Masters E, Wormser GP, Hogrefe W, and Martin D. 2006. Serologic evaluation of patients from Missouri with erythema migrans-like skin lesions with the C6 Lyme test. Clin. Vaccine Immunol 13: 1170–1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
  65. Pichon B, Egan D, Rogers M, and Gray J. 2003. Detection and identification of pathogens and host DNA in unfed host-seeking Ixodes ricinus L. (Acari: Ixodidae). J. Med. Entomol 40: 723–731. [DOI] [PubMed] [Google Scholar]
  66. Piesman J, and Sinsky RJ. 1988. Ability to Ixodes scapularis, Dermacentor variabilis, and Amblyomma americanum (Acari: Ixodidae) to acquire, maintain, and transmit Lyme disease spirochetes (Borrelia burgdorferi). J. Med. Entomol 25: 336–339. [DOI] [PubMed] [Google Scholar]
  67. Piesman J, and Happ CM. 1997. Ability of the Lyme disease spirochete Borrelia burgdorferi to infect rodents and three species of human-biting ticks (blacklegged tick, American dog tick, lone star tick) (Acari:Ixodidae). J. Med. Entomol 34: 451–456. [DOI] [PubMed] [Google Scholar]
  68. Rawlings JA, and Teltow GJ. 1994. Prevalence of Borrelia (Spirochaetaceae) spirochetes in Texas ticks. J. Med. Entomol 31: 297–301. [DOI] [PubMed] [Google Scholar]
  69. Rich SM, Armstrong PM, Smith RD, and Telford SR 3rd. 2001. Lone star tick-infecting borreliae are most closely related to the agent of bovine borreliosis. J. Clin. Microbiol 39: 494–497. [DOI] [PMC free article] [PubMed] [Google Scholar]
  70. Rijpkema SG, Molekenboer MJ, Schouls LM, Jongejan F, and Schellekens JG. 1995. Simultaneous detection and genotyping of three genomic groups of Borrelia burgdorferi sensu lato in Dutch Ixodes ricinus ticks by characterization of the amplifi ed intergenic spacer region between 5S and 23S rRNA genes. J. Clin. Microbiol 33: 3091–3095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  71. Rollend L, Fish D, and Childs JE. 2013. Transovarial transmission of Borrelia spirochetes by Ixodes scapularis: a summary of the literature and recent observations. Ticks Tick Borne Dis. 4: 46–51. [DOI] [PubMed] [Google Scholar]
  72. Roome A, Hill L, Al-Feghali V, Murnock CG, Goodsell JA, Spathis R, and Garruto RM. 2017. Impact of white-tailed deer on the spread of Borrelia burgdorferi. Med. Vet. Entomol 31: 1–5. [DOI] [PubMed] [Google Scholar]
  73. Rosa PA, Hogan D, and Schwan TG. 1991. Polymerase chain reaction analyses identify two distinct classes of Borrelia burgdorferi. J. Clin. Microbiol 29: 524–532. [DOI] [PMC free article] [PubMed] [Google Scholar]
  74. Rudenko N, Golovchenko M, Grubhoffer L, and Oliver JH Jr. 2011. Updates on Borrelia burgdorferi sensu lato complex with respect to public health. Ticks Tick Borne Dis. 2: 123–128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  75. Rudenko N, Golovchenko M, Clark K, Oliver JH, and Grubhoffer L. 2016. Detection of Borrelia burgdorferi sensu stricto in Amblyomma americanum ticks in the southeastern United States: the case of selective compatibility. Emerg. Microbes Infect 5: e48. [DOI] [PMC free article] [PubMed] [Google Scholar]
  76. Ryder JW, Pinger RR, and Glancy T. 1992. Inability of Ixodes cookei and Amblyomma americanum nymphs (Acari: Ixodidae) to transmit Borrelia burgdorferi. J. Med. Entomol 29: 525–530. [DOI] [PubMed] [Google Scholar]
  77. Sanders FH Jr, and Oliver JH Jr. 1995. Evaluation of Ixodes scapularis, Amblyomma americanum, and Dermacentor variabilis (Acari: Ixodidae) from Georgia as vectors of a Florida strain of the Lyme disease spirochete, Borrelia burgdorferi. j. Med. Entomol 32: 402–406. [DOI] [PubMed] [Google Scholar]
  78. Sayler KA, Loftis AD, Beatty SK, Boyce CL, Garrison E, Clemons B, Cunningham M, Alleman AR, and Barbet AF. 2016. Prevalence of tick-borne pathogens in host-seeking Amblyomma americanum (Acari: Ixodidae) and Odocoileus virginianus (Artiodactyla: Cervidae) in Florida. J. Med. Entomol 53: 949–956. [DOI] [PubMed] [Google Scholar]
  79. Sayler K, Rowland J, Boyce C, and Weeks E. 2017. Borrelia burgdorferi DNA absent, multiple Rickettsia spp. DNA present in ticks collected from a teaching forest in North Central Florida. Ticks Tick Borne Dis. 8: 53–59. [DOI] [PubMed] [Google Scholar]
  80. Schulze TL, Bowen GS, Bosler EM, Lakat MF, Parkin WE, Altman R, Ormiston BG, and Shisler JK. 1984. Amblyomma americanum: A potential vector of Lyme disease in New Jersey. Science 224: 601–603. [DOI] [PubMed] [Google Scholar]
  81. Schulze TL, Lakat MF, Parkin WE, Shisler JK, Charette DJ, and Bosler EM. 1986. Comparison of rates of infection by the Lyme disease spirochete in selected populations of Ixodes dammini and Amblyomma americanum (Acari: Ixodidae). Zentralbl. Bakteriol. Mikrobiol. Hyg. A 263: 72–78. [DOI] [PubMed] [Google Scholar]
  82. Schulze TL, Jordan RA, Schulze CJ, Mixson T, and Papero M. 2005. Relative encounter frequencies and prevalence of selected Borrelia, Ehrlichia, and Anaplasma infections in Amblyomma americanum and Ixodes scapularis (Acari: Ixodidae) ticks from central New Jersey. J. Med. Entomol 42: 450–456. [DOI] [PubMed] [Google Scholar]
  83. Schulze TL, Jordan RA, Healy SP, Roegner VE, Meddis M, Jahn MB, and Guthrie DL Sr. 2006. Relative abundance and prevalence of selected Borrelia infections in Ixodes scapularis and Amblyomma americanum (Acari: Ixodidae) from publicly owned lands in Monmouth County, New Jersey. J. Med. Entomol 43: 269–275. [DOI] [PubMed] [Google Scholar]
  84. Schulze TL, Jordan RA, White JC, Roegner VE, and Healy SP. 2011. Geographical distribution and prevalence of selected Borrelia, Ehrlichia, and Rickettsia infections in Amblyomma americanum (Acari: Ixodidae) in New Jersey. J. Am. Mosq. Control Assoc 27: 236–244. [DOI] [PubMed] [Google Scholar]
  85. Simpson KK, and Hinck LW. 1993. The prevalence of Borrelia burgdorferi, the Lyme disease spirochete, in ticks and rodents in Northeast Arkansas. Proc. Arkansas Acad. Sci 47:110–114. [Google Scholar]
  86. Soares CA, Zeidner NS, Beard CB, Dolan MC, Dietrich G, and Piesman J. 2006. Kinetics of Borrelia burgdorferi infection in larvae of refractory and competent tick vectors. J. Med. Entomol 43: 61–67. [DOI] [PubMed] [Google Scholar]
  87. Sonenshine DE, Ratzlaff RE, Troyer J, Demmerle S, Demmerle ER, Austin WE, Tan S, Annis BA, and Jenkins S. 1995. Borrelia burgdorferi in eastern Virginia: comparison between a coastal and inland locality. Am. J. Trop. Med. Hyg 53: 123–133. [DOI] [PubMed] [Google Scholar]
  88. Springer YP, Eisen L, Beati L, James AM, and Eisen RJ. 2014. Spatial distribution of counties in the continental United States with records of occurrence of Amblyomma americanum (Ixodida: Ixodidae). J. Med. Entomol 51: 342–351. [DOI] [PMC free article] [PubMed] [Google Scholar]
  89. Steere AC, Malawista SE, Snydman DR, Shope RE, Andiman WA, Ross MR, and Steele FM. 1977. Lyme arthritis: an epidemic of oligoarticular arthritis in children and adults in three connecticut communities. Arthritis Rheum. 20: 7–17. [DOI] [PubMed] [Google Scholar]
  90. Stegall-Faulk T, Clark DC, and Wright SM. 2003. Detection of Borrelia lonestari in Amblyomma americanum (Acari: Ixodidae) from Tennessee. J. Med. Entomol 40: 100–102. [DOI] [PubMed] [Google Scholar]
  91. Straubinger RK 2000. PCR-Based quantification of Borrelia burgdorferi organisms in canine tissues over a 500-Day postinfection period. J. Clin. Microbiol 38: 2191–2199. [DOI] [PMC free article] [PubMed] [Google Scholar]
  92. Stromdahl EY, and Hickling GJ. 2012. Beyond Lyme: aetiology of tick-borne human diseases with emphasis on the south-eastern United States. Zoonoses Public Health. 59 (Suppl 2): 48–64. [DOI] [PubMed] [Google Scholar]
  93. Stromdahl EY, Evans SR, O’Brien JJ, and Gutierrez AG. 2001. Prevalence of infection in ticks submitted to the human tick test kit program of the U.S. Army Center for Health Promotion and Preventive Medicine. J. Med. Entomol 38: 67–74. [DOI] [PubMed] [Google Scholar]
  94. Stromdahl EY, Williamson PC, Kollars TM Jr, Evans SR, Barry RK, Vince MA, and Dobbs NA. 2003. Evidence of Borrelia lonestari DNA in Amblyomma americanum (Acari: Ixodidae) removed from humans. J. Clin. Microbiol 41: 5557–5562. [DOI] [PMC free article] [PubMed] [Google Scholar]
  95. Stromdahl EY, Nadolny RM, Gibbons JA, Auckland LD, Vince MA, Elkins CE, Murphy MP, Hickling GJ, Eshoo MW, Carolan HE, et al. 2015. Borrelia burgdorferi not confirmed in human-biting Amblyomma americanum ticks from the southeastern United States. J. Clin. Microbiol 53: 1697–1704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  96. Taft SC, Miller MK, and Wright SM. 2005. Distribution of borreliae among ticks collected from eastern states. Vector Borne Zoonotic Dis. 5: 383–389. [DOI] [PubMed] [Google Scholar]
  97. Telford SR 3rd, Mather TN, Moore SI, Wilson ML, and Spielman A. 1988. Incompetence of deer as reservoirs of the Lyme disease spirochete. Am. J. Trop. Med. Hyg 39: 105–109. [DOI] [PubMed] [Google Scholar]
  98. Teltow GJ, Fournier PV, and Rawlings JA. 1991. Isolation of Borrelia burgdorferi from arthropods collected in Texas. Am. j. Trop. Med. Hyg 44: 469–474. [DOI] [PubMed] [Google Scholar]
  99. Tsao JI, Wootton JT, Bunikis J, Luna MG, Fish D, and Barbour AG. 2004. An ecological approach to preventing human infection: vaccinating wild mouse reservoirs intervenes in the Lyme disease cycle. Proc. Natl. Acad. Sci. USA 101: 18159–18164. [DOI] [PMC free article] [PubMed] [Google Scholar]
  100. Varela AS, Luttrell MP, Howerth EW, Moore VA, Davidson WR, Stallknecht DE, and Little SE. 2004a. First culture isolation of Borrelia lonestari, putative agent of southern tick-associated rash illness. J. Clin. Microbiol 42: 1163–1169. [DOI] [PMC free article] [PubMed] [Google Scholar]
  101. Varela AS, Moore VA, and Little SE. 2004b. Disease agents in Amblyomma americanum from northeastern Georgia. J. Med. Entomol 41: 753–759. [DOI] [PubMed] [Google Scholar]
  102. Varela-Stokes AS 2007. Transmission of bacterial agents from lone star ticks to white-tailed deer. J. Med. Entomol 44: 478–483. [DOI] [PubMed] [Google Scholar]
  103. Williamson PC, Billingsley PM, Teltow GJ, Seals JP, Turnbough MA, and Atkinson SF. 2010. Borrelia, Ehrlichia, and Rickettsia spp. in ticks removed from persons, Texas, USA. Emerg. Infect. Dis 16: 441–446. [DOI] [PMC free article] [PubMed] [Google Scholar]
  104. Wormser GP, Masters E, Liveris D, Nowakowski J, Nadelman RB, Holmgren D, Bittker S, Cooper D, Wang G, and Schwartz I. 2005a. Microbiologic evaluation of patients from Missouri with erythema migrans. Clin. Infect. Dis 40: 423–428. [DOI] [PMC free article] [PubMed] [Google Scholar]
  105. Wormser GP, Masters E, Nowakowski J, McKenna D, Holmgren D, Ma K, Ihde L, Cavaliere LF, and Nadelman RB. 2005b. Prospective clinical evaluation of patients from Missouri and New York with erythema migrans-like skin lesions. Clin. Infect. Dis 41: 958–965. [DOI] [PubMed] [Google Scholar]
  106. Xu G, Mather TN, Hollingsworth CS, and Rich SM. 2016. Passive surveillance of Ixodes scapularis (Say), their biting activity, and associated pathogens in Massachusetts. Vector Borne Zoonotic Dis. 16: 520–527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  107. Yuan DT, 2010: A metagenomic study of the tick midgut UT GSBS Dissertations and Theses. Paper 85. University of Texas Graduate School of Biomedical Sciences, Houston, TX: http://digitalcommons.library.tmc.edu/utgsbs_dissertations8/85. [Google Scholar]
  108. Zeidner N, Ullmann A, Sackal C, Dolan M, Dietrich G, Piesman J, and Champagne D. 2009. A borreliacidal factor in Amblyomma americanum saliva is associated with phospholipase A2 activity. Exp. Parasitol 121: 370–375. [DOI] [PubMed] [Google Scholar]

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