Vector competence studies with hard ticks and Borrelia burgdorferi sensu lato spirochetes: A review

Abstract

Use of emerging technology allowing for identification of genetic material from pathogens and endosymbionts in ticks collected from humans, domestic animals, wildlife, or the environment has resulted in an avalanche of new data on tick-microorganism associations. This rapidly growing stream of new information is a tremendous resource but also presents challenges, including how detection of pathogen genetic material in ticks should best be interpreted. There is a tendency in the more recent published literature to incorrectly use the term “vector” based on detection of pathogen genetic material from tick species not experimentally confirmed to serve as vectors of the pathogen in question. To serve as a vector of a horizontally maintained pathogen, such as a Borrelia burgdorferi sensu lato (s.l.) Lyme borreliosis spirochete, the tick species in question must be capable of acquiring the pathogen while feeding in the larval or nymphal stage on an infectious host, maintaining it transstadially through the molt, and then transmitting the pathogen to a naïve host while feeding in the subsequent nymphal or adult stage. This review examines the experimental evidence for and against species of hard (ixodid) ticks from different genera to serve as vectors of B. burgdorferi s.l. spirochetes. Of the 18 Ixodes species ticks evaluated to date, 13 were experimentally confirmed as vectors of B. burgdorferi s.l. spirochetes. These studies focused primarily on the three major Lyme borreliosis agents: Borrelia burgdorferi sensu stricto, Borrelia afzelii, and Borrelia garinii. In striking contrast, none of 8 tick species from other genera (1 Amblyomma species, 5 Dermacentor species, and 2 Haemaphysalis species) evaluated to date were unequivocally experimentally confirmed as vectors of B. burgdorferi s.l. spirochetes. The strength of the evidence for or against each tick species to serve as a vector of B. burgdorferi s.l. spirochetes is discussed together with key knowledge gaps and research challenges.

1. Rationale for tick vector competence studies with Borrelia burgdorferi sensu lato spirochetes

Epidemiological studies conducted in the United States in the late 1970s indicated a linkage between Lyme borreliosis (Lyme disease in North America) and bites by Ixodes ticks (Steere and Malawista, 1979). The etiological agent of this illness remained elusive until 1981, when a spirochete was discovered in host-seeking Ixodes scapularis ticks (including the junior synonym, Ixodes dammini) collected in New York state (Burgdorfer et al., 1982). Soon thereafter, this spirochete also was isolated from Lyme borreliosis patients (Benach et al., 1983; Steere et al., 1983). The new disease agent was named Borrelia burgdorferi (Johnson et al., 1984a). Examination of other tick species closely related to I. scapularis (other members of the Ixodes ricinus/Ixodes persulcatus species complex [Filippova et al., 1999; Keirans et al., 1999]) confirmed that B. burgdorferi was present in a suite of four notorious human-biting ticks with a collective distribution spanning a very large portion of the northern hemisphere: I. scapularis in eastern North America (Burgdorfer et al., 1982; Anderson et al., 1983; Bosler et al., 1983); Ixodes pacificus in far western North America (Burgdorfer et al., 1985); and I. ricinus and I. persulcatus in Eurasia (Barbour et al., 1983; Burgdorfer et al., 1983; Ackermann et al., 1984; Korenberg et al., 1987, 1988; Kryuchechnikov et al., 1988; Zhang, 1989; Ai et al., 1990; Miyamoto et al., 1991).

Another major development was the delineation in the early 1990s of B. burgdorferi into three species, associated with different clinical manifestations in afflicted humans, within the Borrelia burgdorferi sensu lato (s.l.) species complex: Borrelia burgdorferi sensu stricto (s.s.), Borrelia afzelii, and Borrelia garinii (Baranton et al., 1992; Canica et al., 1993). The description of new species within the B. burgdorferi s.l. complex is still ongoing with the most recent global count at 21 named species. In addition to the three major causative agents of human illness mentioned above, the B. burgdorferi s.l. complex also includes several species occasionally associated with human illness and other species with unknown pathogenicity to humans (Rudenko et al., 2011; Margos et al., 2016, 2017; Tables 1–2). The global burden of Lyme borreliosis is still poorly defined, but the United States alone now typically documents >30,000 annual reported cases (Rosenberg et al., 2018) and estimates of the true number of annual cases is tenfold higher (Hinckley et al., 2014; Nelson et al., 2015). Moreover, a recent study including 17 countries in western Europe produced an estimate of >200,000 Lyme borreliosis cases diagnosed annually (Sykes and Makiello, 2016).

Table 1.

Overview of outcomes for Ixodes tick species evaluated in vector competence studies with Borrelia burgdorferi sensu lato spirochetes.

		Experimental evidence for vector competence of Ixodes ticksc
		affinis	angustus	arboricola	cookei	dentatus	frontalis	hexagonus	holocyclus	jellisoni	minor	muris	ovatus	pacificus	persulcatus	ricinus	scapularis	sinensis	spinipalpis
Borrelia burgdorferi sensu lato complex species	Geographic range b	NA/ NT	NA/ PA	OR/ PA	NA/ NT	NA	PA	PA	AU	NA	NA/ NT	NA	OR/ PA	NA	OR/ PA	PA	NA/ NT	OR	NA/ NT
Major cause of human illness
Borrelia burgdorferi sensu stricto	NA/PA/OR	Yes	Yes		No			Yes	No		Yes	Yes		Yes		Yes	Yes		Yes
Borrelia afzelii	PA/OR															Yes	Yes
Borrelia garinii	PA/OR			No			No						No		Yes	Yes	Yes	Yes
Minor cause of human illness
Borrelia bavariensis	PA
Borrelia bissettiae	NA/PA										Yes			Yes			Yes		Yes
Borrelia kurtenbachii	NA/PA
Borrelia lusitaniae	PA
Borrelia mayonii	NA																Yes
Borrelia spielmanii	PA						No
Borrelia valaisiana	PA/OR			No
Unknown human pathogenicity
Borrelia americana	NA
Borrelia andersonii	NA
Borrelia californiensis	NA									Yes
Borrelia carolinensis	NA
Borrelia chilensis	NT
Borrelia japonica	PA
Borrelia lanei	NA
Borrelia sinica	PA/OR
Borrelia tanukii	PA
Borrelia turdi	PA
Borrelia yangtzensis	PA/OR
Wild typea					No	Yes				Yes				Yes		Yes	Yes		Yes

^aUncharacterized Borrelia burgdorferi sensu lato spirochetes.

^bNA, Nearctic; NT, Neotropical; PA, Palearctic; AT, Afrotropical; OR, Oriental; AU, Australian. Based on zoogeographic regions of the world and tick distributions as defined in Gugliemone et al. (2014), and distributions of B. burgdorferi s.l. spirochetes from various sources.

^cYes, vector competence was confirmed experimentally; No, vector competence was evaluated experimentally but could not be confirmed; Blank space, tick species not yet evaluated for this B. burgdorferi s.l. species.

Table 2.

Overview of outcomes for Amblyomma (A.), Dermacentor (D.), and Haemaphysalis (H.) tick species evaluated in vector competence studies with Borrelia burgdorferi sensu lato spirochetes.

		Experimental evidence for vector competencec
		A. americanum	D. andersoni	D. nuttalli	D. occidentalis	D. silvarum	D. variabilis	H. concinna	H. longicornis
Borrelia burgdorferi sensu lato complex species	Geographic range b	NA	NA	PA	NA	PA	NA/NT	OR/PA	AU/NA/OR/PA d
Major cause of human illness
Borrelia burgdorferi sensu stricto	NA/PA/OR	No	No		No		No		No
Borrelia afzelii	PA/OR
Borrelia garinii	PA/OR			No		No		No	No
Minor cause of human illness
Borrelia bavariensis	PA
Borrelia bissettiae	NA/PA	No					No
Borrelia kurtenbachii	NA/PA
Borrelia lusitaniae	PA
Borrelia mayonii	NA
Borrelia spielmanii	PA
Borrelia valaisiana	PA/OR
Unknown human pathogenicity
Borrelia americana	NA
Borrelia andersonii	NA
Borrelia californiensis	NA
Borrelia carolinensis	NA
Borrelia chilensis	NT
Borrelia japonica	PA
Borrelia lanei	NA
Borrelia sinica	PA/OR
Borrelia tanukii	PA
Borrelia turdi	PA
Borrelia yangtzensis	PA/OR
Wild typea			No		No		No

^aUncharacterized Borrelia burgdorferi sensu lato spirochetes.

^bNA, Nearctic; NT, Neotropical; PA, Palearctic; AT, Afrotropical; OR, Oriental; AU, Australian. Based on zoogeographic regions of the world and tick distributions as defined in Gugliemone et al. (2014), and distributions of B. burgdorferi s.l. spirochetes from various sources.

^cYes, vector competence was confirmed experimentally; No, vector competence was evaluated experimentally but could not be confirmed; Blank space, tick species not yet evaluated for this B. burgdorferi s.l. species.

^dRecently established in the Nearctic (United States) (Beard et al., 2018).

The early descriptions of natural B. burgdorferi s.l. infection in I. scapularis, I. pacificus, I. ricinus, and I. persulcatus mentioned above resulted in an initial wave of experimental studies to formally demonstrate the vector competence of these four human-biting I. ricinus/I. persulcatus species complex ticks for B. burgdorferi s.l. (see references in Tables 3–6). Additional studies with Ixodes ticks have targeted other notable human-biting species as well as species that more rarely bite humans but may be involved in enzootic maintenance cycles. These include: Ixodes affinis, Ixodes angustus, Ixodes cookei, Ixodes dentatus, Ixodes jellisoni, Ixodes minor, Ixodes muris, and Ixodes spinipalpis [including the junior synonym, Ixodes neotomae] in North America (Table 7); Ixodes arboricola, Ixodes frontalis, Ixodes hexagonus, Ixodes ovatus, and Ixodes sinensis in Eurasia (Table 8); and Ixodes holocyclus in Australia (Table 8).

Table 3.

Detailed results for studies to evaluate the vector competence of the Nearctic/Neotropical tick Ixodes scapularis for Borrelia burgdorferi sensu lato spirochetes.

Spirochete species and source/ isolate	Hosts used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection				Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Geographic origin of ticksf	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hostk	No. hosts known to have been exposed to at least 1 infected tickk	No. hosts for which infection was confirmed after tick feeding
Uncharacterized Borrelia burgdorferi sensu lato
Wild strain	–	–	NY	–	–	–	–	–	–	Femalej	White rabbit	≥2	8	8	Burgdorfer et al., 1982
Wild strain	–	–	NY	–	–	–	–	–	–	Femalej	White rabbit	10	1	1	Burgdorfer, 1984
Wild strain	White rabbit	Needlec	NY	Larva	No data	No data	Nymph	90	8i	–	–	–	–	–	Burgdorfer, 1984
Wild strain	White rabbit	Needlec	OK	Larva	No data	No data	Nymph	316	22i	Nymph	White rabbit	≥6	1	1	Burgdorfer and Gage, 1986
Wild strain	White rabbit	Tick bited	OK	Larva	No data	No data	Nymph	198	31i	–	–	–	–	–	Burgdorfer and Gage, 1986
Wild strain	White rabbit	Tick bited	MA	Larva	No data	No data	Nymph	34	71	–	–	–	–	–	Telford and Spielman, 1989
Wild strain	–	–	NY	–	–	–	–	–	–	Femalej	White rabbit	3–8	4	4	Piesman et al., 1991
Wild strain	White mouse	Needlee	MA	Larva	No data	No data	Nymph	30	40	–	–	–	–	–	Maupin et al, 1994
Wild strain	White mouse	Needlee	MA	Larva	60	65g	Nymph	20	75	Nymph	White mouse	7	1	1	Dolan et al., 1997
Wild strain	–	–	CT/MD/NJ/NY	–	–	–	–	–	–	Femalej	White rabbit	3–28	14	14	Piesman et al, 1999
Wild strain	–	–	MI/MN/WI	–	–	–	–	–	–	Femalej	White rabbit	4–14	3	3	Piesman et al, 1999
Wild strain	–	–	FL/GA/MS/SC	–	–	–	–	–	–	Femalej	White rabbit	0	0	0 (out of 12)l	Piesman et al, 1999
Wild strain	–	–	CT/NJ/NY	–	–	–	–	–	–	Nymphj	White mouse	1	16	15	des Vignes et al., 2001
WI210	Hamster	Tick bited	MA/NY	Larva	No data	No data	Nymph	90	98	–	–	–	–	–	Piesman, 1993
LI-231	Hamster	Needlee	–	Larva	82	28h	Nymph	271	45	–	–	–	–	–	Barker et al., 1993
LI-231	Rat	Needlee	–	Larva	No data	No data	Nymph	70	36	–	–	–	–	–	Barker et al., 1993
LI-231	M. monax a	Needlee	–	Larva	No data	No data	Nymph	57	23	–	–	–	–	–	Barker et al., 1993
006 strain	White mouse	Tick bited	MA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	4	Richter et al, 1996
006 strain	White mouse	Tick bited	MA	Larva	No data	No data	Nymph	No data	No data	Nymph	P. leucopus a	No data	No data	4	Richter et al, 1996
MI-119	White mouse	Needlee	NJ/NY	Larva	40	75g	Nymph	30	90	–	–	–	–	–	Piesman and Happ, 1997
MI-128	White mouse	Needlee	NJ/NY	Larva	20	10g	Nymph	10	90	–	–	–	–	–	Piesman and Happ, 1997
MI-129	White mouse	Needlee	NJ/NY	Larva	40	78g	Nymph	30	80	Nymph	White mouse	No data	4	4	Piesman and Happ, 1997
Valhalla	Hamster	Tick bited	–	–	–	–	–	–	–	Nymph	Hamster	No data	3	3	Levin et al, 1995
Valhalla	O. palustris a	Tick bited	–	Larva	No data	No data	Nymph	694	75	Nymph	O. palustris a	No data	No data	2	Levin et al, 1995
Valhalla	P. leucopus a	Tick bited	–	Larva	No data	No data	Nymph	No data	No data	Nymph	P. leucopus a	1	38	31	Levin and Fish, 2000
Borrelia burgdorferi sensu stricto
JD1	P. leucopus a	Tick bited	MA	Larva	No data	No data	Nymph	No data	>90	Nymph	Hamster	1–3	6	5	Piesman et al., 1987b
JD1	P. leucopus a	Tick bited	MA	Larva	No data	No data	Nymph	No data	>90	Nymph	P. leucopus a	1–3	7	7	Piesman et al, 1987b
JD1	Hamster	Tick bited	MA	Larva	No data	No data	Nymph	No data	No data	Nymph	Hamster	1	No data	6	Piesman et al, 1987a
JD1	P. leucopus a	Tick bited	–	Larva	No data	No data	Nymph	215	92	Nymph	P. leucopus a	1–3	10	10	Mather et al, 1990
JD1	Hamster	Tick bited	MA	Larva	18	61g	Nymph	24	75	–	–	–	–	–	Piesman and Stone, 1991
JD1	White rabbit	Needlee	OK	Larva	200	20h	Nymph	No data	No data	Nymph	White rabbit	No data	No data	6	Mukolwe et al., 1992
JD1	Hamster	Tick bited	MA/NY	Larva	No data	No data	Nymph	180	99	–	–	–	–	–	Piesman, 1993
JD1	Hamster	Tick bited	MA	Larva	132	44h	Nymph	73	88	Nymph	Hamster	1–14	4	4	Piesman and Sinsky, 1988
JD1	P. leucopus a	Tick bited	–	Larva	75	53h	Nymph	161	89	–	–	–	–	–	Mather and Mather, 1990
JD1	White mouse	Needlee	LA	Larva	34	79g	Nymph	10	90	Nymph	White mouse	9	1	1	Jacobs et al., 2003
B31	Hamster	Tick bited	MA/NY	Larva	No data	No data	Nymph	90	84	–	–	–	–	–	Piesman, 1993
B31	White mouse	Needlee	MA	Larva	60	77g	Nymph	20	85	Nymph	White mouse	9	1	1	Dolan et al., 1997
B31	White mouse	Needlee	NY	Larva	20	85g	Nymph	20	90	Nymph	White mouse	No data	No data	2	Dolan et al., 1998
B31	White mouse	Needlee	LA	Larva	51	90g	Nymph	10	90	Nymph	White mouse	3–4	2	2	Jacobs et al., 2003
B31	White mouse	Tick bited	CT	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	1–4	8	7	Goddard et al., 2015
B31	White mouse	Tick bited	LA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	1–4	6	5	Goddard et al., 2015
B31	White mouse	Tick bited	MS	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	1–4	4	4	Goddard et al., 2015
NC-2	White mouse	Needlee	NJ/NY	Larva	30	100g	Nymph	40	100	Nymph	White mouse	No data	4	4	Piesman and Happ, 1997
SH2-82	Hamster	Needlee	GA	Larva	36	89h	Nymph	107	88	Nymph	Hamster	No data	No data	3	Ryder et al., 1992
SH2-82	Hamster	Needlee	GA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	5	Sanders and Oliver, 1995
SH2-82	Hamster	Needlee	GA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	1	Oliver et al., 1993
SI-1	White mouse	Needlee	NJ/NY	Larva	30	73g	Nymph	20	75	–	–	–	–	–	Piesman and Happ, 1997
SI-1	Hamster	Needlee	GA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	2	Oliver et al., 1993
SI-1	Hamster	Needlee	MA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	4	Oliver et al, 1993
CA4	Hamster	Tick bited	MA/NY	Larva	No data	No data	Nymph	90	58	–	–	–	–	–	Piesman, 1993
CA5	Hamster	Tick bited	MA/NY	Larva	No data	No data	Nymph	30	70	–	–	–	–	–	Piesman, 1993
CA5	Hamster	Tick bited	–	Larva	20	25g	Nymph	43	40	Nymph	Hamster	No data	No data	2	Lane et al., 1994
BL206	White mouse	Needlee	CT	Larva	No data	No data	Nymph	No data	82	Nymph	P. leucopus a	No data	No data	6	Derdakova et al., 2004
B348	White mouseb	Needlee	CT	Larva	No data	No data	Nymph	No data	97	Nymph	P. leucopus a	No data	No data	6	Derdakova et al., 2004
B348	P. leucopus a	Tick bited	–	Larva	No data	No data	Nymph	No data	>90	–	–	–	–	–	States et al., 2017
BBC13	P. leucopus a	Tick bited	–	Larva	No data	No data	Nymph	No data	>95	–	–	–	–	–	States et al., 2017
Borrelia bissettiae
MI-6	Hamster	Needlee	GA	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	5	Sanders and Oliver, 1995
MI-6	Hamster	Needlee	GA	Larva	No data	No data	Nymph	52	27	Nymph	S. hispidus a	No data	No data	2	Sanders and Oliver, 1995
Borrelia mayonii
MN14-1420	White mouse	Needlee	CT	Larva	No data	No data	Nymph	241	13	Nymph	White mouse	1–3	13	9	Dolan et al., 2016
MN14-1420	White mouse	Tick bited	CT/MN	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	1–2	20	13	Dolan et al., 2017b
MN14-1420	White mouse	Tick bited	CT	Larva	No data	No data	Nymph	301	13	Nymph	White mouse	1	9	4	Eisen et al., 2017
MN14-1420	White mouse	Tick bited	MN	Larva	No data	No data	Nymph	268	12	Nymph	White mouse	1	6	2	Eisen et al., 2017
MN-17-4755	White mouse	Tick bited	–	Larva	No data	No data	Nymph	20	55	Nymph	P. leucopus a	1–10	23	21	Parise et al., 2020
Borrelia afzelii
Pgau.C3	White mouse	Needlee	NY	Larva	20	90g	Nymph	20	85	Nymph	White mouse	No data	No data	2	Dolan et al., 1998
Borrelia garinii
VS286	White mouse	Needlee	NY	Larva	20	10g	Nymph	20	0	Nymph	White mouse	No data	No data	1	Dolan et al., 1998
VSBP	White mouse	Needlee	NY	Larva	20	5g	Nymph	20	0	Nymph	White mouse	No data	No data	0 (out of 4)l	Dolan et al, 1998

^aLaboratory colony of ground hogs (Marmota monax), marsh rice rats (Oryzomys palustris), white-footed mice (Peromyscus leucopus), or cotton rats (Sigmodon hispidus).

^bImmunodeficient white mouse strain.

^cInoculated with a suspension containing material from field-collected I. scapularis ticks.

^dBite by infected I. scapularis ticks.

^eInoculated with a suspension containing cultured spirochetes.

^fNortheast and Mid-Atlantic region: CT, MA, MD, NJ, NY; Midwestern region: MI, MN, OK, WI; Southeast region: FL, GA, LA, MS, SC.

^gLarvae harvested within the first 2 wk after completing their blood meal.

^hLarvae harvested within the first 2 d after completing their blood meal.

ⁱIncluding only nymphs resulting from larvae fed 14–30 d post-infection.

^jField-collected infected ticks.

^kBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

^lThe study presented no evidence that any of these naïve hosts were exposed to an infected tick.

Table 6.

Detailed results for studies to evaluate the vector competence of the Palearctic/Oriental tick Ixodes persulcatus for Borrelia burgdorferi sensu lato spirochetes.

Spirochete species and source/ isolate	Host used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection			Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hostf	No. hosts known to be exposed to at least 1 infected tickf	No. hosts for which infection was confirmed after tick feeding
Uncharacterized Borrelia burgdorferi sensu lato
Wild strain	Wild hosta	Tick bitea	Nymph	No data	No data	Adult	200	4e	–	–	–	–	–	Korenberg et al., 1988
Borrelia garinii
Wild strain	White mouse	Needleb	Larva	No data	>90d	Nymph	No data	40– 50	Nymph	White mouse	No data	No data	≥1g	Sun et al., 2003a
JEM3	Jird	Needleb	Larva	20	15d	Nymph	68	38	–	–	–	–	–	Nakao and Miyamoto, 1994
JEM4	Jird	Needleb	Larva	20	0d	Nymph	68	21	–	–	–	–	–	Nakao and Miyamoto, 1994
JEM5	Jird	Needleb	Larva	30	30d	Nymph	102	56	–	–	–	–	–	Nakao and Miyamoto, 1994
JEM6	Jird	Needleb	Larva	40	67d	Nymph	136	74	–	–	–	–	–	Nakao and Miyamoto, 1994
JEM7	Jird	Needleb	Larva	30	17d	Nymph	102	39	–	–	–	–	–	Nakao and Miyamoto, 1994
JME8	Jird	Needleb	Larva	30	43d	Nymph	102	37	–	–	–	–	–	Nakao and Miyamoto, 1994
JEM6	Jird	Tick bitec	Larva	No data	No data	Nymph	No data	>90	Nymph	Jird	No data	No data	15	Sato and Nakao, 1997

^aUnknown naturally infected reservoir host.

^bInoculated with a suspension containing cultured spirochetes.

^cBite by infected I. persulcatus ticks.

^dLarvae harvested within the first 2 d after completing their blood meal.

^eDemonstration of transstadial passage from naturally infected nymphs to adults following nymphal feeding on naïve hosts.

^fBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

^gThe study noted that transmission occurred but it was not clear how many animals had evidence of infection.

Table 7.

Detailed results for studies with Borrelia burgdorferi sensu lato spirochetes to evaluate the vector competence of Ixodes ticks (excluding Ixodes scapularis and Ixodes pacificus) occurring in the Nearctic (NA) Zoogeographic region and in some cases with ranges extending also to the Neotropic (NT) or Palearctic (PA) Zoogeographic regions.

Spirochete species and source/isolate	Host used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection			Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hosti	No. hosts known to have been exposed to at least 1 infected ticki	No. hosts for which infection was confirmed after tick feeding
Ixodes affinis (NA/NT)
B. burgdorferi s.s. (SI-1)	–	–	–	–	–	–	–	–	No data	No data	No data	No data	No dataj	Oliver et al., 2003
Ixodes angustus (NA/PA)
B. burgdorferi s.s. (CA4)	White mouse	Needled	Larva	No data	No data	Nymph	50	12	Nymph	P. maniculatus b	No data	No data	1	Peavey et al., 2000
B. burgdorferi s.s. (CA4)	P. maniculatus b	Needled	Larva	No data	No data	Nymph	98	8	Nymph	P. maniculatus b	0	0	0 (out of 9)k	Peavey et al., 2000
Ixodes cookei (NA/NT)
B. burgdorferi s.l. (LI-231)a	Hamster	Needled	Larva	59	5f	Nymph	92	5	–	–	–	–	–	Barker et al., 1993
B. burgdorferi s.l. (LI-231)a	Rat	Needled	Larva	No data	No data	Nymph	50	16	–	–	–	–	–	Barker et al., 1993
B. burgdorferi s.l. (LI-231)a	M. monax b	Needled	Larva	No data	No data	Nymph	100	4	Nymph	M. monax b	0	0	0 (out of 1)k	Barker et al., 1993
B. burgdorferi s.s. (SH2-82)	Hamster	Needled	Larva	36	14f	Nymph	30	0	Nymph	Hamster	No data	No data	0 (out of 3)k	Ryder et al., 1992
Ixodes dentatus (NA)
B. burgdorferi s.l. (Wild)a	White rabbit	Tick bitee	Larva	No data	No data	Nymph	45	47	Nymph	White rabbit	1	1	1	Telford and Spielman, 1989
Ixodes jellisoni (NA)
B. burgdorferi s.l. (CA444)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	21	14	–	–	–	–	–	Lane et al., 1999
B. burgdorferi s.l. (CA445)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	28	25	Nymph	D. californicus b	4	1	1	Lane et al., 1999
B. burgdorferi s.l. (CA447)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	43	2	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA404)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	3	0	Nymph	D. californicus b	1	1	1	Lane et al., 1999
B. californiensis (CA409)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	30	23	Nymph	D. californicus b	7	1	1	Lane et al., 1999
B. californiensis (CA442)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	32	3	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA443)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	7	0	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA446)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	13	15	Nymph	D. californicus b	1	1	0	Lane et al., 1999
Ixodes minor (NA/NT)
B. burgdorferi s.s. (unknown)	–	–	–	–	–	–	–	–	No data	No data	No data	No data	No datal	Oliver et al., 2003
B. bissettiae (unknown)	–	–	–	–	–	–	–	–	No data	No data	No data	No data	No datal	Oliver et al., 2003
Ixodes muris (NA)
B. burgdorferi s.s. (B31)	White mouse	Tick bitee	Larva	12	67g	Nymph	No data	38	Nymph	White mouse	1–3	4	1	Dolan et al., 2000
Ixodes spinipalpis (NA/NT)
B. burgdorferi s.l. (Wild)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	76	33	Nymph	2 hamsters, 1 P. trueib, and 1 N. fuscipesb	1	4	4	Brown and Lane, 1992
B. burgdorferi s.l. (Wild)a	White mouse	Needled	Larva	60	70g	Nymph	20	80	Nymph	White mouse	1–6	3	3	Dolan et al., 1997
B. burgdorferi s.l. (CA444)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	21	10	–	–	–	–	–	Lane et al., 1999
B. burgdorferi s.l. (CA445)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	32	3	–	–	–	–	–	Lane et al., 1999
B. burgdorferi s.l. (CA447)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	28	4	–	–	–	–	–	Lane et al., 1999
B. burgdorferi s.s. (B31)	White mouse	Needled	Larva	60	58g	Nymph	20	70	Nymph	White mouse	1–6	2	2	Dolan et al., 1997
B. burgdorferi s.s. (CA4)	P. maniculatus b	Needled	Larva	No data	No data	Nymph	141	12	–	–	–	–	–	Peavey et al., 2000
B. burgdorferi s.s. (CA4)	White mouse	Needled	Larva	No data	No data	Nymph	12	8	–	–	–	–	–	Eisen et al., 2003
B. bissettiae (Wild)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	No data	No data	Nymphh	M. ochrogaster b	No data	No data	2	Burkot et al., 2000
B. bissettiae (CA389)	White mouse	Needled	Larva	No data	No data	Nymph	19	0	–	–	–	–	–	Eisen et al., 2003
B. bissettiae (N271)	White mouse	Needled	Larva	No data	No data	Nymph	62	2	Nymph	White mouse	1	1	1	Eisen et al., 2003
B. bissettiae (N501)	White mouse	Needled	Larva	No data	No data	Nymph	56	4	Nymph	White mouse	2	1	1	Eisen et al., 2003
B. bissettiae (CA589)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	10	80	Nymph	P. maniculatus b	2	1	1	Eisen et al., 2003
B. bissettiae (CA590)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	4	75	–	–	–	–	–	Eisen et al., 2003
B. bissettiae (CA591)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	20	35	Nymph	P. maniculatus b	2–12	4	4	Eisen et al., 2003
B. bissettiae (CA592)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	8	38	Nymph	P. maniculatus b	4–5	2	2	Eisen et al., 2003
B. californiensis (CA404)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	33	6	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA409)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	43	5	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA442)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	28	0	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA443)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	11	0	–	–	–	–	–	Lane et al., 1999
B. californiensis (CA446)	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	20	25	–	–	–	–	–	Lane et al., 1999

^aUncharacterized Borrelia burgdorferi sensu lato spirochetes.

^bLaboratory colonies of deer mice (Peromyscus maniculatus), ground hogs (Marmota monax), California kangaroo rats (Dipodomys californicus), piñon mice (Peromyscus truei), dusky-footed woodrats (Neotoma fuscipes), or prairie voles (Microtus ochrogaster).

^cField-collected and naturally tick-bite infected dusky-footed woodrats (Neotoma fuscipes) or California kangaroo rats (Dipodomys californicus).

^dInoculated with a suspension containing cultured spirochetes.

^eBite by infected I. scapularis ticks.

^fLarvae harvested within 2 d after completing their blood meal.

^gLarvae harvested within the first 2 wk after completing their blood meal.

^hNaturally infected nymphs recovered as fed larvae from field-collected prairie voles.

ⁱBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

^jEvidence for vector competence limited to the following statement: “The closely related but usually non-human biting I. affinis also experimentally transmitted the B. burgdorferi s.s. isolate SI-1 (J.H.O., A.M.J., and C.W.B., unpublished data).”.

^kThe study presented no evidence that any naïve host was exposed to an infected tick.

^lEvidence for vector competence limited to the following statement: “Although I. minor is not currently considered a member of the Ixodes ricinus species complex, as are I. scapularis and I. affinis, it is an efficient vector of B. bissettii and B. burgdorferi s.s. (J.H.O., J. B. Phillips, C.W.B., L.G., T.L., and A.M.J., unpublished data).”.

Table 8.

Detailed results for studies with Borrelia burgdorferi sensu lato spirochetes to evaluate the vector competence of Ixodes ticks (excluding Ixodes ricinus and Ixodes persulcatus) occurring in the Palearctic (PA), Oriental (OR), or Australian (AU) zoogeographic regions.

Spirochete species and source/isolate	Host used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection			Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hosth	No. hosts known to have been exposed to at least 1 infected tickh	No. hosts for which infection was confirmed after tick feeding
Ixodes arboricola (OR/PA)
B. garinii (Wild)	Birda	Tick biteb	Larva	No data	No data	Nymph	164	3	Nymph	Birda	0	0	0 (out of 4)i	Heylen et al., 2014
B. valaisiana (Wild)	Birda	Tick biteb	Larva	No data	No data	Nymph	164	3	Nymph	Birda	0	0	0 (out of 4)i	Heylen et al., 2014
Ixodes frontalis (PA)
B. garinii (Wild)	Birda	Tick biteb	Larva	No data	No data	Nymph	113	2	Nymph	Birda	0	0	0 (out of 4)i	Heylen et al., 2014
B. spielmanii (Wild)	Birda	Tick biteb	Larva	No data	No data	Nymph	113	<1	Nymph	Birda	0	0	0 (out of 4)i	Heylen et al., 2014
Ixodes hexagonus (PA)
B. burgdorferi s.s. (B31)	–	–	Nymphe	6	67f	Adult	47	70	Female	White mouse	1	4	4	Gern et al., 1991
Ixodes holocyclus (AU)
B. burgdorferi s.s. (JD1)	Hamster	Tick bitec	Larva	36	17f	Nymph	84	0	–	–	–	–	–	Piesman and Stone, 1991
Ixodes ovatus (OR/PA)
B. garinii (JEM3)	Jird	Needled	Larva	20	80g	Nymph	68	0	–	–	–	–	–	Nakao and Miyamoto, 1994
B. garinii (JEM4)	Jird	Needled	Larva	10	20g	Nymph	34	0	–	–	–	–	–	Nakao and Miyamoto, 1994
B. garinii (JEM5	Jird	Needled	Larva	10	10g	Nymph	34	0	–	–	–	–	–	Nakao and Miyamoto, 1994
B. garinii (JEM6)	Jird	Needled	Larva	20	70g	Nymph	68	0	–	–	–	–	–	Nakao and Miyamoto, 1994
B. garinii (JEM7)	Jird	Needled	Larva	20	25g	Nymph	68	0	–	–	–	–	–	Nakao and Miyamoto, 1994
B. garinii (JME8)	Jird	Needled	Larva	10	40g	Nymph	34	0	–	–	–	–	–	Nakao and Miyamoto, 1994
Ixodes sinensis (OR)
B. garinii (Wild)	White mouse	Needled	Larva	50	96g	Nymph	50	56	Nymph	White mouse	No data	No data	9	Sun et al., 2003b
B. garinii (Wild)	White mouse	Needled	Nymph	100	98g	Adult	56	57	Adult	White mouse	No data	No data	6	Sun et al., 2003b

^aBird (Parus major).

^bBite by infected I. ricinus ticks.

^cBite by infected I. scapularis ticks.

^dInoculated with a suspension containing cultured spirochetes.

^eNymphal ticks infected via capillary feeding before taking a blood meal on an uninfected rabbit host.

^fLarvae or nymphs harvested within the first 2 wk after completing their blood meal.

^gLarvae or nymphs harvested within the first 2 d after completing their blood meal.

^hBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

ⁱThe study presented no evidence that any of these naïve hosts were exposed to an infected tick.

In North America, another wave of vector competence studies was spurred by the description of B. burgdorferi s.l. spirochetes from naturally infected non-Ixodes human-biting ticks: Amblyomma americanum, Dermacentor occidentalis, and Dermacentor variabilis (Schulze et al., 1984; Anderson et al., 1985; Lane and Lavoie, 1988; Magnarelli and Anderson, 1988). The resulting vector competence evaluations included four major non-Ixodes human-biting ticks in the United States: A. americanum and D. variabilis in the east, Dermacentor andersoni in the Rocky Mountain region and D. occidentalis in the far west (Table 9). Later, two Eurasian human-biting Dermacentor species, Dermacentor nuttalli and Dermacentor silvarum, also were evaluated experimentally (Table 9). Finally, some of the most recent vector competence studies for B. burgdorferi s.l. spirochetes have focused on Haemaphysalis ticks (Haemaphysalis concinna and Haemaphysalis longicornis), spurred in part by the ongoing emergence in Lyme borreliosis-endemic areas of the United States of the invasive H. longicornis (Table 9).

Table 9.

Detailed results for studies with Borrelia burgdorferi sensu lato spirochetes to evaluate the vector competence of Amblyomma, Dermacentor, or Haemaphysalis ticks occurring in the Nearctic (NA), Palearctic (PA), Oriental (OR), or Australian (AU) zoogeographic regions.

Spirochete species and source/isolate	Host used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection			Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hosth	No. hosts known to have been exposed to at least 1 infected tickh	No. hosts for which infection was confirmed after tick feeding
Amblyomma americanum (NA)
B. burgdorferi s.s. (JD1)	Hamster	Tick bited	Larva	118	4f	Nymph	218	0	–	–	–	–	–	Piesman and Sinsky, 1988
B. burgdorferi s.s. (JD1)	P. leucopus b	Tick bited	Larva	16	19f	Nymph	33	0	–	–	–	–	–	Mather and Mather, 1990
B. burgdorferi s.s. (JD1)	White rabbit	Needlee	Larva	200	0f	Nymph	No data	No data	Nymph	White rabbit	No data	No data	0 (out of 6)i	Mukolwe et al., 1992
B. burgdorferi s.s. (B31)	White mouse	Tick bited	Larva	No data	0g	Nymph	No data	0	–	–	–	–	–	Soares et al., 2006
B. burgdorferi s.s. (NC-2)	White mouse	Needlee	Larva	30	0g	Nymph	40	0	Nymph	White mouse	0	0	0 (out of 2)i	Piesman and Happ, 1997
B. burgdorferi s.s. (SI-1)	Hamster	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	Hamster	No data	No data	0 (out of 15)i	Oliver et al., 1993
B. burgdorferi s.s. (SI-1)	White mouse	Needlee	Larva	20	0g	Nymph	10	0	–	–	–	–	–	Piesman and Happ, 1997
B. burgdorferi s.s. (SH2-82)	Hamster	Needlee	Larva	36	19f	Nymph	60	2	Nymph	Hamster	No data	No data	0 (out of 3)i	Ryder et al., 1992
B. bissettiae (MI-6)	Hamster	Needlee	Larva	No data	No data	Nymph	105	0	Nymph	White mouse	No data	No data	0 (out of 23)i	Sanders and Oliver, 1995
Dermacentor andersoni (NA)
B. burgdorferi s.l. (Wild)a	White mouse	Needlee	Larva	90	17g	Nymph	30	0	Nymph	White mouse	0	0	0 (out of 2)i	Dolan et al., 1997
B. burgdorferi s.s. (B31)	White mouse	Needlee	Larva	90	11g	Nymph	30	0	Nymph	White mouse	0	0	0 (out of 2)i	Dolan et al., 1997
Dermacentor mittalli (PA)
B. garinii (Wild)	White mouse	Needlee	Larva	No data	50f	Nymph	No data	0	Nymph	White mouse	No data	No data	0j	Sun et al., 2003a
B. garinii (Wild)	White mouse	Needlee	Nymph	No data	60f	Adult	No data	0	Adult	White mouse	No data	No data	0j	Sun et al., 2003a
Dermacentor occidentalis (NA)
B. burgdorferi s.l. (Wild)a	Wild rodentsc	Tick bitec	Larva	No data	No data	Nymph	44	0	–	–	–	–	–	Brown and Lane, 1992
B. burgdorferi s.s. (CA5)	Hamster	Tick bited	Larva	20	10g	Nymph	40	0	–	–	–	–	–	Lane et al., 1994
Dermacentor silvarum (PA)
B. garinii (Wild)	White mouse	Needlee	Larva	No data	40f	Nymph	No data	0	Nymph	White mouse	No data	No data	0j	Sun and Xu, 2003
B. garinii (Wild)	White mouse	Needlee	Nymph	No data	50f	Adult	No data	0	Adult	White mouse	No data	No data	0j	Sun and Xu, 2003
Dermacentor variabilis (NA/NT)
B. burgdorferi s.l. (MI-119)a	White mouse	Needlee	Larva	23	61g	Nymph	21	10	–	–	–	–	–	Piesman and Happ, 1997
B. burgdorferi s.l. (MI-128)a	White mouse	Needlee	Larva	20	10g	Nymph	10	0	–	–	–	–	–	Piesman and Happ, 1997
B. burgdorferi s.l. (MI-129)a	White mouse	Needlee	Larva	30	37g	Nymph	30	0	Nymph	White mouse	0	0	0 (out of 18)i	Piesman and Happ, 1997
B. burgdorferi s.s. (JD1)	Hamster	Tick bited	Larva	49	14f	Nymph	77	0	–	–	–	–	–	Piesman and Sinsky, 1988
B. burgdorferi s.s. (JD1)	P. leucopus b	Tick bited	Larva	75	28f	Nymph	150	0	–	–	–	–	–	Mather and Mather, 1990
B. burgdorferi s.s. (JD1)	White rabbit	Needlee	Larva	200	0f	Nymph	No data	No data	Nymph	White rabbit	No data	No data	0 (out of 6)i, k	Mukolwe et al., 1992
B. burgdorferi s.s. (B31)	White mouse	Tick bited	Larva	No data	>0g	Nymph	No data	>0	Nymph	White mouse	No data	No data	0j	Soares et al., 2006
B. burgdorferi s.s. (NC-2)	White mouse	Needlee	Larva	47	28g	Nymph	49	2	Nymph	White mouse	0	0	0 (out of 6)i	Piesman and Happ, 1997
B. burgdorferi s.s. (SI-1)	White mouse	Needlee	Larva	48	58g	Nymph	30	0	–	–	–	–	–	Piesman and Happ, 1997
B. bissettiae (MI-6)	Hamster	Needlee	Larva	No data	No data	Nymph	105	0	Nymph	White mouse	No data	No data	0 (out of 21)i	Sanders and Oliver, 1995
Haemaphysalis concinna (OR/PA)
B. garinii (Wild)	White mouse	Needlee	Larva	No data	70–80f	Nymph	No data	0	Nymph	White mouse	No data	No data	0j	Sun and Xu, 2003
B. garinii (Wild)	White mouse	Needlee	Nymph	No data	70f	Adult	No data	0	Adult	White mouse	No data	No data	0j	Sun and Xu, 2003
Haemaphysalis longicornis (AU/NA/OR/PA)
B. garinii (Wild)	White mouse	Needlee	Larva	No data	60–70f	Nymph	No data	0	Nymph	White mouse	No data	No data	0j	Sun et al., 2003a
B. garinii (Wild)	White mouse	Needlee	Nymph	No data	60–70f	Adult	No data	0	Adult	White mouse	No data	No data	0j	Sun et al., 2003a
B. burgdorferi s.s. (B31)	White mouse	Tick bited	Larva	32	56f	Nymph	520	0	–	–	–	–	–	Breuner et al., 2020

^aUncharacterized Borrelia burgdorferi sensu lato spirochetes.

^bLaboratory colony of white-footed mice (Peromyscus leucopus).

^cField-collected and naturally tick-bite infected dusky-footed woodrats (Neotoma fuscipes) or California kangaroo rats (Dipodomys californicus).

^dBite by infected I. scapularis ticks.

^eInoculated with a suspension containing cultured spirochetes.

^fLarvae or nymphs harvested within 2 d after completing their blood meal.

^gLarvae or nymphs harvested within the first 2 wk after completing their blood meal.

^hBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

ⁱThe study presented no evidence that any naïve host was exposed to an infected tick.

^jThe study presented no evidence that any naïve host was exposed to an infected tick, and the number of hosts used was not given.

^kOne of these rabbits was seroreactive but no spirochetes were isolated from tissues (liver, spleen, heart, kidneys, and urinary bladder) taken at necropsy; the evidence for this rabbit was therefore not considered sufficient to conclude that infection was confirmed. In contrast, rabbits exposed to I. scapularis ticks infected with the same spirochete isolate in this study uniformly were both seroreactive and yielded tissues from which spirochetes were cultured.

The most recent review of tick vector competence studies with B. burgdorferi s.l. spirochetes was published nearly two decades ago (Eisen and Lane, 2002). Although relatively few studies have been published since then, the overall context has changed considerably due to the still ongoing description of new species within the B. burgdorferi s.l. complex. Consequently, as shown in Table 1, not even the most intensively studied tick species, I. scapularis, has been evaluated experimentally with all species within the B. burgdorferi s.l. complex known to occur within the tick’s geographical range. Moreover, use of emerging technology allowing for detection of genetic material from pathogens and endosymbionts in ticks collected from humans, domestic animals, wildlife, or the environment has resulted in an avalanche of new data on tick-microorganism associations, including B. burgdorferi s.l. spirochetes (Kurilshikov et al., 2015; Cross et al., 2018; Greay et al., 2018; Zhuang et al., 2018; Thapa et al., 2019; Tokarz et al., 2019). This rapidly growing stream of new information is a tremendous resource but it also presents major challenges, including how detection of genetic material from B. burgdorferi s.l. in ticks should best be interpreted. There is a tendency in the more recent published literature to incorrectly use the term “vector” based on detection of pathogen genetic material from tick species that have not been experimentally confirmed to serve as vectors of the pathogen in question. This review both serves as a reminder of the concept of vector competence and provides an upto-date single resource for the state of our knowledge regarding tick vector competence for B. burgdorferi s.l. spirochetes.

Tables were compiled to (i) provide an overview of qualitative outcomes for tick species evaluated in vector competence studies with B. burgdorferi s.l. spirochetes (Tables 1–2) and (ii) present important details for individual studies (Tables 3–9). Within these tables and the specific descriptions of transmission studies by tick species (sections 4–8), B. burgdorferi s.l. refers to uncharacterized spirochetes and represent either wild strains from field-collected ticks or animals or named isolates which have yet to be assigned to a species in the published literature. The zoogeographic regions used to describe the broad geographical ranges of tick and B. burgdorferi s.l. species in the Tables follow those used in the tick species descriptions in “The Hard Ticks of the World” by Gugliemone et al. (2014). In the text, I also sometimes use the more familiar terms of North America and Eurasia to broadly describe where different species of ticks and B. burgdorferi s.l. spirochetes occur. Published literature was queried by searching the Scopus database using combinations of the key words “vector”, “tick”, and “Borrelia” together with “transmission”, “transmit”, or “competence”. The snowball technique, which identifies additional publications based on referenced materials, was then employed to identify additional publications of interest.

The issue of duration of tick attachment required for transmission of B. burgdorferi s.l. spirochetes is not addressed here because this topic was examined in a recent separate review (Eisen, 2018). All data presented in this review are based on ticks allowed to feed to repletion on the host; in some cases this includes subsets of data for ticks fed to repletion in studies that also examined spirochete acquisition or transmission during partial blood meals. Data were interpreted and broad conclusions drawn bearing in mind that the studies were conducted over more than 35 years and with substantial methodological variation, including the species and strain of experimental host, the route of initial host infection, and the methods used to detect spirochetes in ticks or hosts.

2. Conceptual underpinnings for tick vector competence studies with Borrelia burgdorferi sensu lato spirochetes

Enzootic maintenance of B. burgdorferi s.l. spirochetes was recognized early on to primarily involve horizontal transmission between vector ticks and reservoir hosts (Anderson et al., 1983; Bosler et al., 1983; Levine et al., 1985; Donahue et al., 1987). Consequently, experimental tick vector competence studies with B. burgdorferi s.l. spirochetes have included demonstration of three distinct processes: (i) acquisition of spirochetes by uninfected ticks feeding on infectious experimental hosts, (ii) maintenance of spirochetes through the molt to the next life stage (transstadial passage), and (iii) transmission of spirochetes to naïve hosts during a subsequent blood meal (Eisen and Lane, 2002; Kahl et al., 2002). A tick species should not be considered a vector of B. burgdorferi s.l. spirochetes unless all three processes have been experimentally demonstrated. In the case of field-derived data, detection of B. burgdorferi s.l. spirochetes in fed ticks (of any life stage) collected from wild animal hosts are only indicative of acquisition of spirochetes from an infected host, whereas detection of B. burgdorferi s.l. in host-seeking nymphal or adult ticks indicate that spirochetes were both acquired during the blood meal in the preceding life stage and passed transstadially through the molt (Eisen and Lane, 2002; Kahl et al., 2002). However, field-derived data alone can never satisfy the final criterion of vector competence: unequivocal demonstration of transmission of B. burgdorferi s.l. spirochetes by feeding ticks.

It also is worth noting another transmission scenario, which in light of new knowledge has become less relevant for B. burgdorferi s.l. spirochetes: demonstration of transmission by transovarially infected larval ticks where infection was passed from the female to her eggs and the resulting larvae. Although the importance of transovarial transmission of B. burgdorferi s.l. spirochetes in the four major Ixodes vectors has been hotly debated (Randolph, 1994), a consensus is building that early records of field-collected infected unfed larvae (based on microscopy or immunofluorescence assays) may represent the more recently described relapsing fever group spirochete, Borrelia miyamotoi (Rollend et al., 2013). Borrelia miyamotoi occurs naturally in Ixodes ticks across the northern hemisphere (Wagemakers et al., 2015), and was demonstrated to be passed transovarially and transmitted by the resulting larvae for both I. ricinus and I. scapularis (Scoles et al., 2001; van Duijvendijk et al., 2016; Breuner et al., 2018). To the best of my knowledge, there is only a single study where B. burgdorferi s.l. spirochetes, characterized using methodology capable of distinguishing them from B. miyamotoi, were demonstrated to be transmitted by Ixodes larvae to an experimental host: van Duijvendijk et al. (2016) found that feeding of field-collected I. ricinus larvae on laboratory mice resulted in infection with B. afzelii. However, the use of field-collected larval ticks in such transmission experiments can be problematic unless it is clearly documented that the larvae had not previously taken a partial blood meal on another (potentially infectious) host that perished in the early stages of their blood meal, thus forcing them to seek another host to acquire the amount of blood required to complete their molt (Piesman, 1991). Previous studies demonstrated that I. scapularis and I. persulcatus ticks allowed to attach to an infectious host for 24−48 h and thereafter removed and placed on a new, naïve experimental host can effectively transmit B. burgdorferi s.s. while completing their blood meal (Shih and Spielman, 1993; Nakao and Sato, 1996).

Experimental animal hosts used in tick vector competence studies with B. burgdorferi s.l. spirochetes have, depending in part on the targeted tick species, typically included rabbits and various species of rodents, including natural tick hosts as well as laboratory animals (see Tables 3–9). Early experimental studies tended to use rabbit hosts (Burgdorfer et al., 1982, 1983; Burgdorfer, 1984; Kornblatt et al., 1984; Burgdorfer and Gage, 1986) but this soon gave way to use of rodents as these animals proved more effective sources to infect feeding immature ticks with B. burgdorferi s.l. spirochetes (Burgdorfer and Gage, 1987; Donahue et al., 1987; Piesman et al., 1987a, b; Piesman and Sinsky, 1988; Mather et al., 1990). Moreover, rodents develop long-lasting spirochete infections (Burgdorfer and Gage, 1987; Donahue et al., 1987; Piesman, 1988; Brown and Lane, 1992; Gern et al., 1994; Lindsay et al., 1997; Lane et al., 1999; States et al., 2017; Dolan et al., 2017a) readily detectable by culture of infected tissue, such as biopsies from ear or internal organs, including bladder, kidney, and spleen (Johnson et al., 1984b; Anderson et al., 1985, 1986; Piesman et al., 1987b; Schwan et al., 1988; Sinsky and Piesman, 1989). For tick species and life stages that feed willingly on mice, or can be compelled to take a blood meal from mice when confined to feeding capsules, outbred strains of the white mouse (Mus musculus) provide practical animal models for vector competence studies with rodent-associated B. burgdorferi s.l. spirochetes (Piesman, 1993). White mice infected with B. burgdorferi s.l. spirochetes via tick bite become highly infectious to feeding uninfected immature ticks within a predictable window of time post-infection (3–5 wk) and they also are excellent naïve hosts in the final transmission step with infected ticks because ear biopsies provide a reliable source of tissue for spirochete culture. Use of a natural tick host, such as the white-footed mouse (Peromyscus leucopus) or deer mouse (Peromyscus maniculatus) in North America, is another option that adds an element of realism to the experimental study.

For the initial step of spirochete acquisition via feeding by uninfected ticks, it is advisable to use experimental hosts that were infected via tick bite rather than needle inoculation in order to ensure that the spirochetes introduced into the host expressed surface proteins associated with tick transmission rather than artificial culturing (Gern et al., 1993; Piesman, 1993; Randolph and Nuttall, 1994). Moreover, for tick species suspected to be poor vectors of B. burgdorferi s.l. spirochetes it is critically important to examine both freshly fed ticks (to demonstrate spirochete acquisition) and molted resulting ticks of the next life stage (to document transstadial passage). There are several examples of studies where a non-Ixodes tick species effectively acquired spirochetes while feeding (infection prevalence >50 % in freshly fed ticks) but the infection was uniformly lost in the molt to the next life stage (Piesman and Happ, 1997; Sun and Xu, 2003; Sun et al., 2003a; Breuner et al., 2020). In cases where there is a high likelihood of efficient spirochete acquisition and transstadial passage, such as for Ixodes ticks, studies often bypass testing of freshly fed larvae to maximize the number of molted nymphs available for pathogen detection and demonstration of spirochete transmission by the nymphs (Tables 3–9). As part of providing evidence for transstadial passage of B. burgdorferi s.l., it is advisable to not rely solely on detection of spirochete DNA in the molted ticks but also demonstrate the presence of viable spirochetes via culture or feeds on naïve experimental hosts.

In the final transmission step, it is important to consider the purpose of the vector competence study. Because mass feeding of infected ticks can result in more effective transmission of B. burgdorferi s.l. spirochetes compared to use of a single or a few ticks (reviewed by Eisen, 2018), it may be useful to restrict the number of feeding ticks to match the typical number of that species and life stage found on a natural reservoir host (in the context of enzootic transmission) or to aim for a number projected to include only a single infected tick (in the context of transmission to humans). This final transmission step can be readily achieved in scenarios where a large proportion of the ticks both acquire and pass spirochetes transstadially but is extremely challenging when the prevalence of infection in the molted ticks is near zero. As an evaluation of transmission by infected ticks requires actual feeding by infected ticks it is critically important to test the fed ticks recovered from each host for presence of spirochetes in order to ascertain which individual experimental hosts were exposed to at least one infected tick versus only fed upon by uninfected ticks.

Seminal studies on experimental transmission of B. burgdorferi s.l. by Ixodes ticks used a model where field-collected adult ticks were fed upon New Zealand white rabbits (Burgdorfer et al., 1982, 1983; Burgdorfer, 1984). Following the recognition that the nymphal stage of I. scapularis is the principal vector of Lyme borreliosis spirochetes to humans in the northeastern United States (Spielman et al., 1985; Piesman et al., 1987c; Lane et al., 1991), there was a shift toward an experimental model with B. burgdorferi s.l. acquisition by larvae and the final transmission step involving infected nymphs. These developments produced a practical experimental system where laboratory-reared rodents infected via tick bite are used to infect feeding larval ticks with the B. burgdorferi s.l. species under study and the resulting molted nymphs then are fed on naïve animals to demonstrate transmission (Piesman, 1993). Variations of this experimental system may be needed for tick species or life stages that are unwilling to feed on rodents or B. burgdorferi s.l. spirochetes adapted to non-mammalian hosts, for example avian-associated ticks and B. garinii variants (Kurtenbach et al., 2002; Heylen et al., 2014). Unequivocal demonstration of transmission of viable spirochetes from infected ticks to hosts should include either culture of spirochetes from host tissues or acquisition of spirochetes from the hosts by uninfected (xenodiagnostic) ticks. Serology can serve as a complement for demonstration of spirochete exposure but is not recommended as a stand-alone method in a transmission experiment.

There have been efforts to employ artificial means to infect ticks with B. burgdorferi s.l. spirochetes, including use of artificial feeders containing infected blood (Burkot et al., 2001), ingestion of culture containing spirochetes via capillary-feeding before or after a blood meal (Monin et al., 1989; Gern et al., 1991, 1993; Li and Lane, 1996; Fingerle et al., 2002), immersion of ticks into culture containing spirochetes (Policastro and Schwan, 2003; Fiserova et al., 2008), and microinjection of spirochetes into the tick hemocoel (Johns et al., 1998, 2000; Urbanova et al., 2017). Although such techniques can be useful complements to the traditional animal model, they also introduce biological deviations from the natural host-tick transmission chain which makes the results of transmission studies more difficult to interpret. One specific problem is the ingestion by ticks of spirochetes not exposed to a host’s immune system and therefore not necessarily expressing the phenotype seen in a natural or experimental host-tick transmission chain.

3. Limitations to tick vector competence studies with Borrelia burgdorferi sensu lato spirochetes

When considering the results of tick vector competence studies, it is important to recognize their limitations. A tick species that experimentally proves incapable or very inefficient to serve as a vector for a given B. burgdorferi s.l. species will not play a major role as a vector in nature. However, it is equally important to recognize that experimental demonstration of vector competence does not necessarily imply that the tick species in question is important either as an enzootic vector (it may not feed commonly on B. burgdorferi s.l. reservoir hosts) or a vector to humans (its questing behavior may not be conducive to making contact with people). Field studies are critical to provide all the data needed for an accurate assessment of the potential (vector potential/vectorial capacity) of an experimentally confirmed vector tick species to contribute to infection of wildlife or human illness (Spielman et al., 1984; Kahl et al., 2002). In some cases there also may be interest in examining vector competence of notorious human-biting tick vectors for human-pathogenic B. burgdorferi s.l. spirochetes that are currently not present within the distribution range of the tick but conceivably could be introduced in the future. The demonstration of vector competence of the Nearctic I. scapularis for two Palearctic Lyme borreliosis spirochetes, B. afzelii and B. garinii, is perhaps the best example of such as study (Dolan et al., 1998).

As noted previously, the still ongoing description of new species within the B. burgdorferi s.l. complex has far outpaced the vector competence studies (Tables 1–2). For example, I. scapularis has been experimentally demonstrated to serve as a vector of B. burgdorferi s.s., Borrelia mayonii, and Borrelia bissettiae (Table 3) but has yet to be evaluated experimentally for several other B. burgdorferi s.l. complex spirochetes occurring naturally in the eastern United States: Borrelia americana, Borrelia andersonii, Borrelia carolinensis, and Borrelia kurtenbachii. The same scenario, with vector competence studies conducted for only a subset of their associated B. burgdorferi s.l. complex spirochetes, holds true also for the other three major global vectors of Lyme borreliosis spirochetes to humans: I. pacificus, I. ricinus and I. persulcatus (Table 1). Because of the potential for further delineation of species within the B. burgdorferi s.l. complex, it is recommended to use well characterized B. burgdorferi s.l. isolates in vector competence studies. Should further delineation occur in the future, the B. burgdorferi s.l. species to which such isolates are re-assigned can most likely be elucidated. In contrast, vector competence studies using “wild type” B. burgdorferi s.l. spirochetes may be difficult to re-interpret and therefore less informative.

The ideal design for a vector competence study involves early generation ticks from a laboratory colony established using field-collected specimens from the same general geographical area as the B. burgdorferi s.l. species and strain/isolate under evaluation. Variation in genotype and phenotype (including infectivity for rodents and ticks) within a single B. burgdorferi s.l. species (Piesman, 1993; Lane et al., 1994; Nakao and Miyamoto, 1994; Peavey and Lane, 1996; Piesman and Happ, 1997; Piesman et al., 1999; Crippa et al., 2002; Eisen et al., 2003; Derdakova et al., 2004; Tonetti et al., 2015; States et al., 2017) can make this challenging, especially when also addressing a tick species with a large distribution range and geographical variation in genotype and phenotype across tick populations. For example, I. scapularis occurs widely across different ecological settings in the eastern United States and displays genetic variation as well as distinct behavioral differences to the north versus south (Van Zee et al., 2015; Eisen et al., 2016; Arsnoe et al., 2019). Within this broad geographical range, there also is considerable genetic and phenotypic variability within B. burgdorferi s.s. (Mathiesen et al., 1997; Qiu et al., 2002; Bunikis et al., 2004; Hoen et al., 2009; Brisson et al., 2010). To address this complex scenario, vector efficiency was assessed in a series of studies using northern and southern I. scapularis populations and B. burgdorferi s.s. isolates originating from different parts of the tick’s geographical range (Oliver et al., 1993; Sanders and Oliver, 1995; Piesman and Happ, 1997; Jacobs et al., 2003; Goddard et al., 2015).

Experimental studies with tick species expected to be poor vectors of B. burgdorferi s.l. spirochetes are especially challenging. One logical end-point for such studies is to demonstrate that spirochetes acquired during a blood meal are not passed to the subsequent life stage (Piesman and Sinsky, 1988; Mather and Mather, 1990; Brown and Lane, 1992; Lane et al., 1994; Breuner et al., 2020), indicating that the tick species under evaluation is not a vector. This, however, requires a large sample size for a statistically robust evaluation. For example, as reported by Breuner et al. (2020), lack of B. burgdorferi s.s. infection in 520 examined H. longicornis nymphs fed as larvae on infectious hosts still resulted in an upper 95 % confidence limit estimate for infection prevalence of 0.7 %. Thus, the effort required to conclude that a given tick species is unlikely to be a vector is considerable. An even more challenging scenario arises when B. burgdorferi s.l. spirochetes are passed transstadially but only for a small percentage of ticks, as recorded in a small subset of experiments with A. americanum and D. variabilis (Ryder et al., 1992; Piesman and Happ, 1997; Soares et al., 2006). Based on the low infection prevalence in the remaining ticks, it can be challenging to subsequently succeed in exposing naïve hosts to infected, rather than only uninfected, feeding ticks. As a case in point, none of the three studies mentioned above succeeded in unequivocally exposing a single animal to an infected tick. Moreover, as shown in Table 9, despite numerous attempts there is still a complete lack of documented exposures of experimental hosts to the feeding by a transstadially infected Amblyomma, Dermacentor, or Haemaphysalis tick.

A final emerging issue is the potential impact of the tick microbiome on vector competence studies (Narasimhan and Fikrig, 2015; Gall et al., 2016; de la Fuente et al., 2017; Kwan et al., 2017; Varela-Stokes et al., 2017; Couper et al., 2019). For example, our understanding of the impact of co-infection with multiple pathogens within a single tick on the transmission of B. burgdorferi s.l. spirochetes is very limited (Piesman et al., 1987a; Levin and Fish, 2000) despite common reports of co-infection in field-collected ticks (Diuk-Wasser et al., 2016; Moutailler et al., 2016; Eisen and Eisen, 2018). In addition, colony ticks considered “pathogen-free” and used in experimental studies may harbor endosymbionts and other microorganisms potentially impacting the outcome of a vector competence or vector efficiency study, positively or negatively. This is simultaneously very intriguing and quite frustrating as it may lead to a better understanding of pathogen transmission dynamics, and perhaps also to new ways of interrupting spirochete transmission, while also presenting new challenges for data interpretation. Future transmission studies may face the added challenge of having to incorporate broad metagenomics data on the microbiome of the individual ticks used in the experiments into the analysis model, potentially resulting in a need to increase the number of animals exposed in order to achieve adequate statistical power of the study design.

4. Vector competence studies with Nearctic/Neotropical Ixodes ticks

To date, 10 species of Nearctic/Neotropical Ixodes ticks have been examined in vector competence studies with B. burgdorferi s.l. spirochetes (Tables 3–4,7). Of these 10 tick species, 9 were experimentally confirmed as vectors of B. burgdorferi s.l. spirochetes. The strength of the evidence for or against vector competence is outlined for each tick species below.

Table 4.

Detailed results for studies to evaluate the vector competence of the Nearctic tick Ixodes pacificus for Borrelia burgdorferi sensu lato spirochetes.

Spirochete species and souree/isolate	Hosts used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection			Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hostj	No. hosts known to have been exposed to at least 1 infected tickj	No. hosts for which infection was confirmed after tick feeding
Uncharacterized Borrelia burgdorferi sensu lato
Wild strain	White rabbit	Tick bitec	Larva	No data	No data	Nymph	120	12h	–	–	–	–	–	Burgdorfer, 1984
Wild strain	Wild rodentsa	Tick bitea	Larva	No data	No data	Nymph	80	24	–	–	–	–	–	Brown and Lane, 1992
Wild strain	Wild rodentsa	Tick bitea	Larva	No data	No data	Nymph	418	40	–	–	–	–	–	Brown and Lane, 1996
Wild strain	–	–	–	–	–	–	–	–	Femalei	White rabbit	1	2	2	Piesman et al., 1999
006 strain	White mouse	Tick bitec	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	0 (out of 14)k	Richter et al., 1996
006 strain	White mouse	Tick bitec	Larva	No data	No data	Nymph	No data	No data	Nymph	P. leucopus b	No data	No data	0 (out of 5)k	Richter et al., 1996
Borrelia burgdorferi sensu stricto
CA4	Hamster	Tick bitec	Larva	No data	No data	Nymph	70	13	–	–	–	–	–	Piesman, 1993
CA4	White rabbit	Needled	Larva	259	3g	Nymph	140	6	Nymph	P. maniculatus b	1–2	6	5l	Lane et al., 1994
CA4	P. maniculatus b	Needled	Larva	No data	No data	Nymph	107	0	–	–	–	–	–	Lane et al., 1994
CA4	White rabbit	Needled	Larva	No data	No data	Nymph	No data	20–60	Nymph	P. maniculatus b	1–2	10	8	Peavey and Lane, 1995
CA4	P. maniculatus b	Tick bitee	Larva	No data	No data	Nymph	103	34	–	–	–	–	–	Peavey and Lane, 1995
CA4	P. maniculatus b	Needled	Larva	No data	No data	Nymph	182	10	–	–	–	–	–	Eisen et al., 2003
CA4	White mouse	Needled	Larva	No data	No data	Nymph	200	19	–	–	–	–	–	Eisen et al., 2003
CA5	Hamster	Tick bitec	Larva	No data	No data	Nymph	30	27	–	–	–	–	–	Piesman, 1993
CA5	Hamster	Tick bitec	Larva	20	25g	Nymph	18	33	–	–	–	–	–	Lane et al., 1994
CA7	Hamster	Needled	Larva	No data	No data	Nymph	60	2	–	–	–	–	–	Lane et al., 1994
CA10	P. maniculatus b	Needled	Larva	No data	No data	Nymph	300	<1	–	–	–	–	–	Eisen et al., 2003
CA11	P. maniculatus b	Needled	Larva	No data	No data	Nymph	32	3	–	–	–	–	–	Lane et al., 1994
JD1	Hamster	Tick bitec	Larva	No data	No data	Nymph	No data	No data	Nymph	Hamster	2	1	1	Lane et al., 1994
JD1	Hamster	Tick bitec	Larva	No data	No data	Nymph	No data	No data	Nymph	P. maniculatus b	1	1	0	Lane et al., 1994
Borrelia bissettiae
CA389	P. maniculatus b	Needled	Larva	No data	No data	Nymph	42	0	–	–	–	–	–	Eisen et al., 2003
CA389	White mouse	Needled	Larva	No data	No data	Nymph	350	<1	–	–	–	–	–	Eisen et al., 2003
CA559	White mouse	Needled	Larva	No data	No data	Nymph	240	0	–	–	–	–	–	Eisen et al., 2003
CA589	P. maniculatus b	Tick bitef	Larva	No data	No data	Nymph	12	42	–	–	–	–	–	Eisen et al., 2003
CA591	P. maniculatus b	Tick bitef	Larva	No data	No data	Nymph	32	43	–	–	–	–	–	Eisen et al., 2003
CA592	P. maniculatus b	Tick bitef	Larva	No data	No data	Nymph	12	75	Nymph	P. maniculatus b	1	3	3	Eisen et al., 2003
CA589/591/592	P. maniculatus b	Tick bitef	Larva	No data	No data	Nymph	No data	No data	Nymph	P. maniculatus b	1–6	4	4	Eisen et al., 2003
CA589/591/592	P. maniculatus b	Tick bitef	Larva	No data	No data	Nymph	No data	No data	Nymph	P. maniculatus b	No data	No data	6	Eisen et al., 2003

^aField-collected and naturally tick-bite infected dusky-footed woodrats (Neotoma fuscipes) or California kangaroo rats (Dipodomys californicus).

^bLaboratory colonies of deer mice (Peromyscus maniculatus) or white-footed mice (Peromyscus leucopus).

^cBite by infected I. scapularis ticks.

^dInoculated with a suspension containing cultured spirochetes.

^eBite by infected I. pacificus ticks.

^fBite by infected I. spinipalpis ticks.

^gLarvae harvested within the first 2 wk after completing their blood meal.

^hIncluding only nymphs resulting from larvae fed 14–30 d post-infection.

ⁱField-collected infected ticks.

^jBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

^kThe study presented no evidence that any of these naïve hosts were exposed to an infected tick.

^lFive mice had ear biopsies positive for spirochetes by culture and were seroreactive. A sixth mouse had ear biopsy negative for spirochetes by culture but was seroreactive; this mouse was not included in the number for which infection was confirmed.

4.1. Ixodes scapularis

Ixodes scapularis (a member of the Ixodes ricinus/Ixodes persulcatus species complex) has been confirmed as a vector of North American uncharacterized B. burgdorferi s.l. spirochetes (wild strains, 006 strain, MI129, and Valhalla), B. burgdorferi s.s. (JD1, B31, NC-2, SH2–82, SI-1, BL206, and B348), B. mayonii (MN14–1420 and MN17–4755) and B. bissettiae (MI-6) (Burgdorfer et al., 1982; Burgdorfer, 1984; Burgdorfer and Gage, 1986; Piesman et al., 1987a, 1987b, 1991, 1999; Piesman and Sinsky, 1988; Mather et al., 1990; Mukolwe et al., 1992; Ryder et al., 1992; Oliver et al., 1993; Lane et al., 1994; Levin et al., 1995; Sanders and Oliver, 1995; Richter et al., 1996; Dolan et al., 1997, 1998, 2016, 2017b; Piesman and Happ, 1997; Levin and Fish, 2000; des Vignes et al., 2001; Jacobs et al., 2003; Derdakova et al., 2004; Goddard et al., 2015; Eisen et al., 2017; Table 3). Moreover, I. scapularis was confirmed as a vector of Palearctic B. afzelii (Pgau.C3) and B. garinii (VS286) (Dolan et al., 1998).

The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was highly variable (<10 to 98 %) across uncharacterized B. burgdorferi s.l. spirochetes (Burgdorfer, 1984; Burgdorfer and Gage, 1986; Telford and Spielman, 1989; Barker et al., 1993; Piesman, 1993; Maupin et al., 1994; Levin et al., 1995; Dolan et al., 1997; Piesman and Happ, 1997). For isolates of B. burgdorferi s.s., the efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was moderate to high (40–100 %), with most studies exceeding 80 % (Piesman et al., 1987b; Piesman and Sinsky, 1988; Mather and Mather, 1990; Mather et al., 1990; Piesman and Stone, 1991; Ryder et al., 1992; Piesman, 1993; Lane et al., 1994; Dolan et al., 1997, 1998; Piesman and Happ, 1997; Jacobs et al., 2003; Derdakova et al., 2004; States et al., 2017). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was lower (27 %) for B. bissettiae (Sanders and Oliver, 1995). For B. mayonii, the efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was low (12–13 %) for a human-derived isolate (MN14–1420) but higher (55 %) for a rodent-derived isolate (MN17–4755) (Dolan et al., 2016; Eisen et al., 2017; Parise et al., 2020; Table 3).

As shown in Table 3, some of the experiments demonstrating spirochete transmission to naïve hosts also include data for the number of animals known to have been exposed to infected ticks whereas in other cases these important data were not reported. Considering only those studies where the number of animals known to have been exposed to at least one infected tick was reported, the transmission rate (defined here as: number of hosts with evidence of spirochete exposure / number of hosts exposed to infected ticks) was 91 % (85/93) for uncharacterized B. burgdorferi s.l. spirochetes, 94 % (50/53) for B. burgdorferi s.s., and 69 % (49/71) for B. mayonii (Table 3). In all cases, this included subsets of naïve hosts exposed to a single infected tick. Including all studies presenting evidence for host infection following exposure to I. scapularis ticks, the total numbers of recorded instances of transmission to an individual host reach 95 for uncharacterized B. burgdorferi s.l. spirochetes (nymphal and female ticks), 93 for B. burgdorferi s.s. (nymphs), 7 for B. bissettiae (nymphs), 49 for B. mayonii (nymphs), 2 for B. afzelii (nymphs), and 1 for B. garinii (nymphs) (Table 3). Finally, as shown in Table 3, the evidence to date indicates that I. scapularis is a highly efficient vector for B. burgdorferi s.s. regardless of the geographical origins of the combination of tick population and spirochete isolate used in the transmission experiment.

4.2. Ixodes pacificus

Ixodes pacificus (a member of the Ixodes ricinus/Ixodes persulcatus species complex) has been confirmed as a vector of uncharacterized B. burgdorferi s.l. spirochetes (wild strain), B. burgdorferi s.s. (CA4 and JD1), and B. bissettiae (CA 592) (Lane et al., 1994; Peavey and Lane, 1995; Piesman et al., 1999; Eisen et al., 2003; Table 4). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was low to moderate (12–40 %) across uncharacterized B. burgdorferi s.l. spirochetes (Burgdorfer, 1984; Brown and Lane, 1992, 1996); highly variable (<5 to 60 %) for isolates of B. burgdorferi s.s. (Piesman, 1993; Lane et al., 1994; Peavey and Lane, 1995; Eisen et al., 2003); and highly variable (<5 to 75 %) for isolates of B. bissettiae (Eisen et al., 2003). Across the evaluated isolates, known exposure of 27 naïve hosts to infected nymphs (n = 1–6 per host) resulted in documented spirochete transmission to 23 of the hosts (85 % transmission rate across exposed hosts): this included 2 recorded instances of transmission for uncharacterized B. burgdorferi s.l. spirochetes, 14 for B. burgdorferi s.s., and 7 for B. bissettiae (Table 4). An additional 6 instances of transmission of B. bissettiae were recorded in a study where it was not clear how many naïve hosts may have been exposed to infected ticks (Eisen et al., 2003).

4.3. Ixodes angustus

Ixodes angustus has been confirmed as a vector of B. burgdorferi s.s. (CA4) (Peavey et al., 2000; Table 7). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was low (8–12 %). A single instance of transmission to a host by feeding nymphs was recorded but it is not clear how many infected nymphs fed on the animal.

4.4. Ixodes dentatus

Ixodes dentatus has been confirmed as a vector of uncharacterized B. burgdorferi s.l. spirochetes (Telford and Spielman, 1989; Table 7). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was moderate (47 %). Known exposure of 1 naïve host to a single infected nymph resulted in transmission to the host.

4.5. Ixodes jellisoni

Ixodes jellisoni (a member of the Ixodes ricinus/Ixodes persulcatus species complex) has been confirmed as a vector of uncharacterized B. burgdorferi s.l. spirochetes (CA445) and B. californiensis (CA404, CA409, and CA446; Margos et al., 2016) (Lane et al., 1999; Table 7). Although the evidence is limited to a single study, the evaluation did include multiple B. californiensis isolates. The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was low (≤25 %). Known exposure of 3 naïve hosts each to 1–7 nymphs infected with B. californiensis resulted in 2 instances of transmission to the host (67 % transmission rate across exposed hosts). The yet uncharacterized B. burgdorferi s.l. spirochete isolate (445) performed similarly in the study and one instance of transmission to a host was recorded (Table 7).

4.6. Ixodes muris

Ixodes muris has been confirmed as a vector of B. burgdorferi s.s. (B31) (Dolan et al., 2000; Table 7). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was moderate (38 %). Known exposure of 4 naïve hosts each to 1–3 infected nymphs resulted in a single instance of transmission to a host (25 % transmission rate across exposed hosts).

4.7. Ixodes spinipalpis

Ixodes spinipalpis has been confirmed as a vector of uncharacterized B. burgdorferi s.l. spirochetes (wild strains), B. burgdorferi s.s. (B31), and B. bissettiae (wild strain, N271, N501, and CA 592) (Brown and Lane, 1992; Dolan et al., 1997; Burkot et al., 2000; Eisen et al., 2003; Table 7). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was highly variable (<5 to 80 %) across uncharacterized B. burgdorferi s.l. spirochetes (Brown and Lane, 1992; Dolan et al., 1997; Lane et al., 1999); highly variable (<10 to 70 %) for isolates of B. burgdorferi s.s. (Dolan et al., 1997; Peavey et al., 2000; Eisen et al., 2003); highly variable (<5 to 80 %) for isolates of B. bissettiae (Eisen et al., 2003); and low (≤25 %) for isolates of B. californiensis (Lane et al., 1999). Across the evaluated isolates, known exposure of 18 naïve hosts to infected nymphs (n = 1–12 per host) uniformly resulted in spirochete transmission to the hosts (100 % transmission rate across exposed hosts): this included 7 recorded instances of transmission for uncharacterized B. burgdorferi s.l. spirochetes, 2 for B. burgdorferi s.s., and 9 for B. bissettiae (Table 7). An additional 2 instances of transmission of B. bissettiae was recorded in a study where it was not clear how many naïve hosts may have been exposed to infected ticks (Burkot et al., 2000). The study (Lane et al., 1999) with spirochetes later characterized as B. californiensis (CA404, CA409, CA442, CA443 and CA446; Margos et al., 2016) did not proceed to evaluate transmission by infected I. spinipalpis ticks.

4.8. Ixodes affinis and Ixodes minor

Ixodes affinis (a member of the Ixodes ricinus/Ixodes persulcatus species complex) has reportedly been confirmed as a vector of B. burgdorferi s.s. (SI-1), and Ixodes minor as a vector of B. burgdorferi s.s. and B. bissettiae (Oliver, 1996; Oliver et al., 2003). However, as indicated in Table 7, the evidence for vector competence remains limited to references to unpublished data, and therefore must be classified as weak.

4.9. Ixodes cookei

The vector competence of I. cookei was evaluated for an uncharacterized B. burgdorferi s.l. spirochete (LI-231) and B. burgdorferi s.s. (SH2–82) in two studies (Ryder et al., 1992; Barker et al., 1993; Table 7). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was low (4–16 %) for the uncharacterized B. burgdorferi s.l. spirochete and zero for B. burgdorferi s.s. Unfortunately, in the last step to demonstrate spirochete transmission by feeding ticks there was no evidence of a single host having been exposed to an infected tick (Table 7). Consequently, the evidence against I. cookei as a vector of Lyme borreliosis spirochetes is weak and further studies are justified to clarify the vector competence status of this tick species.

5. Vector competence studies with Palearctic/Oriental/Australian Ixodes ticks

To date, 8 species of Palearctic/Oriental/Australian Ixodes ticks have been examined in vector competence studies with B. burgdorferi s.l. spirochetes (Tables 5–6, 8). Of these 8 tick species, 4 were experimentally confirmed as vectors of B. burgdorferi s.l. spirochetes. The strength of the evidence for or against vector competence is outlined for each tick species below.

Table 5.

Detailed results for studies to evaluate the vector competence of the Palearctic tick Ixodes ricinus for Borrelia burgdorferi sensu lato spirochetes.

Spirochete species and source/isolate	Host used as source of infection for feeding ticks		Spirochete acquisition by ticks fed on hosts with active infection			Transstadial passage of spirochetes to molted ticks of the next life stage			Spirochete transmission by ticks fed on naïve hosts					Reference
	Species	Route of spirochete infection	Life stage	No. examined	% infected	Life stage	No. examined	% infected	Life stage	Host	No. infected ticks known to have fed per hosti	No. hosts known to be exposed to at least 1 infected ticki	No. hosts for which infection was confirmed after tick feeding
Uncharacterized Borrelia burgdorferi sensu lato
Wild	–	–	–	–	–	–	–	–	Femaleh	White rabbit	1–19	7	7	Burgdorfer et al., 1983
Wild	White rabbit	Needleb	Larva	No data	No data	Nymph	185	16g	–	–	–	–	–	Burgdorfer, 1984
Wild	Wild rodentsa	Tick bitea	Larva	No data	No data	Nymph	1254	50	–	–	–	–	–	Gern et al., 1994
Wild (including B. afzelii)	Gerbil	Tick bitec	Larva	No data	No data	Nymph	162	88	Nymph	Gerbil	No data	No data	6	Kahl et al., 1998; Olaf Kahl (personal communication)
Borrelia burgdorferi sensu stricto
Wild	–	–	–	–	–	–	–	–	Nymphh	White mouse	No data	No data	2	Hu et al., 2001
Wild	–	–	–	–	–	–	–	–	Nymphh	White mouse	No data	No data	3	Hu et al., 2003
ZS7	White mouse	Tick bited	Larva	No data	No data	Nymph	No data	42–88	–	–	–	–	–	Gern et al., 1993
ZS7	White mouse	Tick bited	Larva	No data	No data	Nymph	No data	70	Nymph	White mouse	No data	3	3	Gern and Rais, 1996
B31	White mouse	Needlee	Larva	20	10f	Nymph	20	0	–	–	–	–	–	Dolan et al., 1998
ZS7, NE1849	White mouse	Needlee	Larva	No data	No data	Nymph	No data	60–70	Nymph	White mouse	No data	No data	2	Crippa et al., 2002
Borrelia afzelii
Wild	–	–	–	–	–	–	–	–	Larvah	White mouse	No data	No data	2	van Duijvendijk et al., 2016
Wild	–	–	–	–	–	–	–	–	Nymphh	White mouse	No data	No data	9	Hu et al., 2001
Wild	–	–	–	–	–	–	–	–	Nymphh	White mouse	No data	No data	31	Hu et al., 2003
Pgau.C3	White mouse	Needlee	Larva	20	85f	Nymph	20	90	Nymph	White mouse	No data	No data	3	Dolan et al., 1998
NE496, NE2963	White mouse	Needlee	Larva	No data	No data	Nymph	No data	60–80	Nymph	White mouse	No data	No data	2	Crippa et al., 2002
NE4053	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	2	Tonetti et al., 2015
NE4053	White mouse	Tick bitee	Larva	No data	No data	Nymph	26	4	–	–	–	–	–	Tonetti et al., 2015
NE5046	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	4	Tonetti et al., 2015
NE5046	White mouse	Tick bitec	Larva	No data	No data	Nymph	52	92	–	–	–	–	–	Tonetti et al., 2015
NE36	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	4	Tonetti et al., 2015
NE36	White mouse	Tick bitee	Larva	No data	No data	Nymph	52	67	–	–	–	–	–	Tonetti et al., 2015
E61	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	3	Tonetti et al., 2015
E61	White mouse	Tick bitec	Larva	No data	No data	Nymph	39	54	–	–	–	–	–	Tonetti et al., 2015
P/sto	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	4	Tonetti et al., 2015
P/sto	White mouse	Tick bitec	Larva	No data	No data	Nymph	52	15	–	–	–	–	–	Tonetti et al., 2015
NE4054	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	4	Tonetti et al., 2015
NE4054	White mouse	Tick bitec	Larva	No data	No data	Nymph	39	31	–	–	–	–	–	Tonetti et al., 2015
NE4049	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	8	Tonetti et al., 2015
NE4049	White mouse	Tick bitec	Larva	No data	No data	Nymph	117	85	–	–	–	–	–	Tonetti et al., 2015
NE4051	White mouse	Needlee	Larva	No data	No data	Nymph	No data	No data	Nymph	White mouse	No data	No data	9	Tonetti et al., 2015
NE4051	White mouse	Tick bitec	Larva	No data	No data	Nymph	115	70	–	–	–	–	–	Tonetti et al., 2015
CB43	White mouse	Tick bitec	Larva	No data	No data	Nymph	No data	90	Nymph	White mouse	No data	No data	10	Pospisilova et al., 2019
Borrelia garinii
Wild	–	–	–	–	–	–	–	–	Nymphh	White mouse	No data	No data	7	Hu et al., 2001
Wild	–	–	–	–	–	–	–	–	Nymphh	White mouse	No data	No data	7	Hu et al., 2003
VS286	White mouse	Needlee	Larva	20	5f	Nymph	20	0	Nymph	White mouse	No data	No data	3	Dolan et al., 1998
VSBP	White mouse	Needlee	Larva	20	10f	Nymph	20	0	Nymph	White mouse	No data	No data	0 (out of 3)j	Dolan et al., 1998

^aField-collected and naturally tick-bite infected yellow-necked mice (Apodemus flavicollis) or wood mice (Apodemus sylvaticus).

^bInoculated with a suspension containing material from field-collected I. scapularis ticks.

^cBite by infected I. ricinus ticks.

^dNymphal ticks infected via capillary feeding before taking their blood meal.

^eInoculated with a suspension containing cultured spirochetes.

^fLarvae harvested within the first 2 wk after completing their blood meal.

^gIncluding only nymphs resulting from larvae fed 14–30 d post-infection.

^hField-collected infected ticks.

ⁱBy examination of either fed ticks or the resulting unfed ticks of the next life stage; or elucidated via transmission to uninfected ticks in a co-feeding experiment. Listed as no data when the number was not clearly stated in the publication.

^jThe study presented no evidence that any of these naïve hosts were exposed to an infected tick.

5.1. Ixodes ricinus

Ixodes ricinus (a member of the Ixodes ricinus/Ixodes persulcatus species complex) has been confirmed as a vector of uncharacterized B. burgdorferi s.l. spirochetes (wild strains), B. burgdorferi s.s. (wild strains, ZS7, and NE1849), B. afzelii (wild strains, Pgau.C3, NE496, NE2963, NE4053, NE5046, NE36, E61, P/sto, NE4054, NE4049, NE4051, and CB43), and B. garinii (wild strains and VS286) (Burgdorfer et al., 1983; Gern and Rais, 1996; Dolan et al., 1998; Kahl et al., 1998; Hu et al., 2001, 2003; Crippa et al., 2002; Tonetti et al., 2015; van Duijvendijk et al., 2016; Pospisilova et al., 2019; Table 5). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was low to moderate (16–50 %) across uncharacterized B. burgdorferi s.l. spirochetes (Burgdorfer, 1984; Gern et al., 1994), highly variable (<5 to 88 %) for isolates of B. burgdorferi s.s. (Gern et al., 1993; Gern et al., 1993; Dolan et al., 1998; Crippa et al., 2002), and highly variable (<5 to 90 %) for isolates of B. afzelii (Dolan et al., 1998; Crippa et al., 2002; Tonetti et al., 2015; Pospisilova et al., 2019). No similar robust data were presented for B. garinii. Very few studies on I. ricinus have presented data on the number of hosts known to have been exposed to infected ticks, but in both studies presenting such data (Burgdorfer et al., 1983; Gern and Rais, 1996) all hosts exposed to ticks infected with uncharacterized B. burgdorferi s.l. spirochetes (n = 7 hosts) or B. burgdorferi s.s. (n = 3 hosts) became infected. Including all studies presenting evidence for host infection following exposure to I. ricinus ticks, the total numbers of recorded instances of transmission to individual hosts reach 13 for uncharacterized B. burgdorferi s.l. spirochetes (nymphal and female ticks), 10 for B. burgdorferi s.s. (nymphs), 95 for B. afzelii (larval and nymphal ticks), and 17 for B. garinii (nymphs) (Table 5).

5.2. Ixodes persulcatus

Ixodes persulcatus (a member of the Ixodes ricinus/Ixodes persulcatus species complex) has been confirmed as a vector of B. garinii (wild strain and JEM 6) (Sato and Nakao, 1997; Sun et al., 2003a; Table 6). The efficiency of spirochete acquisition by larval ticks and transstadial passage to the nymphal stage was highly variable (21 to >90 %) across B. garinii isolates but in the 30–60 % range for most examined isolates (Nakao and Miyamoto, 1994; Sato and Nakao, 1997; Sun et al., 2003a). Overall, there were a minimum of 16 recorded instances of transmission to a naïve host by infected nymphal ticks, including 15 for the JEM6 isolate (Table 6). The transmission rate across animals exposed to infected ticks could not be calculated as there is no information in the relevant publications regarding the number of hosts known to have been exposed to infected ticks. I am not aware of any published experimental evaluation of the vector competence of I. persulcatus specifically for B. afzelii, but it seems a near certainty that an experimental study would confirm vector competence. Ixodes persulcatus is commonly naturally infected with B. garinii as well as B. afzelii spirochetes in Russia, East Asia, and Japan (Postic et al., 1997; Korenberg et al., 2002; Miyamoto and Masuzawa, 2002). It also should be noted that - in contrast to I. ricinus, I. scapularis, and I. pacificus for which the nymphal stage is considered the main vector of B. burgdorferi s.l. spirochetes to humans (Eisen and Lane, 2002) - the females of I. persulcatus are the main vectors of B. burgdorferi s.l. spirochetes to humans (Korenberg, 1994; Korenberg et al., 2002).

5.3. Ixodes sinensis

Ixodes sinensis has been confirmed as a vector of B. garinii (wild strains) (Sun et al., 2003b; Table 8). The efficiency of spirochete acquisition by larval or nymphal ticks and transstadial passage to the resulting nymphal or adult stages was moderate (56–57 %). Exposure of naïve hosts to potentially infected ticks (nymphs or adults) resulted in 15 recorded instances of transmission to an individual host. The transmission rate could not be calculated as it is not clear how many hosts, in total, were exposed to infected ticks.

5.4. Ixodes hexagonus

Ixodes hexagonus has been confirmed as a vector of B. burgdorferi s.s. (B31) (Gern et al., 1991; Table 8). Nymphal ticks infected via capillary feeding before taking a blood meal on an uninfected rabbit host were shown to maintain infection after molting to the adult stage, and the resulting female ticks transmitted spirochetes to all 4 hosts known to have been exposed to an infected female tick.

5.5. Ixodes arboricola and Ixodes frontalis

The vector competence of the ornithophilic ticks I. arboricola and I. frontalis was evaluated using wild strains of B. garinii (both tick species), B. spielmanii (I. frontalis only), and B. valaisiana (I. arboricola only) (Heylen et al., 2014; Table 8). The efficiency of spirochete acquisition by larval ticks (from infected birds) and transstadial passage to the resulting nymphal stage was low (0.9–3.0 %) across combinations of tick and spirochete species. Subsequent attempts to feed infected nymphs on naïve avian hosts were unsuccessful as none of the ticks fed on the birds were found to be infected (Table 8). Consequently, as no single host has yet been unequivocally exposed to an infected tick, the evidence against I. arboricola and I. frontalis as vectors of Lyme borreliosis spirochetes is weak and additional studies are justified.

5.6. Ixodes ovatus

The vector competence of I. ovatus was evaluated for B. garinii (JEM3, JEM4, JEM5, JEM6, JEM7, and JEM8) (Nakao and Miyamoto, 1994; Table 8). Larval ticks acquired spirochetes from infected hosts with variable efficiency (10–80 % of freshly fed larvae found to be infected across isolates, with an overall infection prevalence of 47 %) but there was no evidence of transstadial passage to the nymphal stage (none of 306 molted nymphs found to be infected). As there was no evidence of transstadial spirochete passage, there was no attempt to feed resulting nymphs on naïve hosts. Although the evidence against I. ovatus as a vector of the evaluated, human-derived B. garinii strains is robust, it nevertheless would be worthwhile to evaluate the vector competence of this tick species using rodent-associated isolates of B. afzelii and B. garinii.

5.7. Ixodes holocyclus

The vector competence of I. holocyclus was evaluated for B. burgdorferi s.s. (JD1) (Piesman and Stone, 1991; Table 8). Larval ticks acquired spirochetes from infected hosts with low efficiency (17 % of freshly fed larvae found to be infected) but there was no evidence of transstadial passage to the nymphal stage (none of 84 molted nymphs found to be infected). As there was no evidence of transstadial spirochete passage, there was no attempt to feed resulting nymphs on naïve hosts. Based on the limited number of ticks examined to assess transstadial spirochete passage and the use of a single isolate of a single species (B. burgdorferi s.s.), the evidence against I. holocyclus as a vector of Lyme borreliosis spirochetes is weak. Further studies are justified to clarify the vector competence status of this tick species for the species of Lyme borreliosis spirochetes most likely to occur in or be introduced to Australia.

6. Ixodes tick species likely to serve as vectors of Borrelia burgdorferi sensu lato spirochetes but still lacking formal experimental demonstration of vector competence

The seabird tick, Ixodes uriae, is perhaps the best example of a tick species which almost certainly is a vector of Lyme borreliosis spirochetes but where experimental demonstration of vector competence is still lacking (due to logistical challenges of conducting laboratory studies with this tick). There is very strong evidence from several field studies, including in settings lacking other tick species, to indicate that enzootic transmission cycles of B. burgdorferi s.l. spirochetes, particularly B. garinii, are maintained by I. uriae and seabirds (Olsen et al., 1993, 1995; Bunikis et al., 1996; Gylfe et al., 1999; Smith et al., 2006; Munro et al., 2017). Other examples of tick species where field evidence is strong enough to justify experimental transmission studies to formally demonstrate vector competence include the Palearctic ticks, Ixodes trianguliceps and Ixodes pavlovskyi, and the Palearctic/Oriental tick, Ixodes granulatus (Doby et al., 1990; Gorelova et al., 1996, 2001; Hubbard et al., 1998; Chao et al., 2009, 2012; Korenberg et al., 2010, 2015; Kovalevskii et al., 2013; Hou et al., 2015). Ixodes trianguliceps primarily infests small mammals, whereas I. granulatus and I. pavlovskyi have broader host ranges and occasionally bite humans. Based on the uniformly successful vector competence studies with B. burgdorferi s.l. spirochetes with the 6 members of the I. ricinus/I. persulcatus species complex evaluated to date (I. affinis, I. jellisoni, I. pacificus, I. ricinus, I. persulcatus, and I. scapularis), it seems likely that not only I. pavlovskyi but also the 7 additional remaining tick species within the complex (the Palearctic/Oriental Ixodes gibbosus, Ixodes hyatti, Ixodes kashmiricus, Ixodes kazakstani, Ixodes nipponensis, and Ixodes nuttallianus; and the Neotropical Ixodes pararicinus) would prove vectors of B. burgdorferi s.l. spirochetes if evaluated experimentally. A final tick species that deserves mention as a likely vector of B. burgdorferi s.l. spirochetes is the newly described Ixodes inopinatus, which occurs in southern Europe and northern Africa, and is closely related to I. ricinus (Estrada-Peña et al., 2014).

7. Vector competence studies with Nearctic/Neotropical Amblyomma, Dermacentor and Haemaphysalis ticks

To date, 5 species of Nearctic/Neotropical Amblyomma, Dermacentor, and Haemaphysalis ticks have been examined in vector competence studies with B. burgdorferi s.l. spirochetes (Table 9). None of these 5 tick species were experimentally confirmed as vectors of any evaluated B. burgdorferi s.l. spirochete. The strength of the evidence against vector competence is outlined for each tick species below.

7.1. Amblyomma americanum

The vector competence of A. americanum was evaluated for B. burgdorferi s.s. (JD1, B31, NC-2, SI-1, and SH2–82), and B. bissettiae (MI-6) (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; Soares et al., 2006; Table 9). The efficiency of spirochete acquisition by larval ticks was 0–19 % for isolates of B. burgdorferi s.s. (Piesman and Sinsky, 1988; Mather and Mather, 1990; Mukolwe et al., 1992: Ryder et al., 1992; Piesman and Happ, 1997). A single instance of transstadial spirochete transmission to a resulting nymph was recorded (Ryder et al., 1992), but the overall infection prevalence in resulting nymphs was very low: 0.3 % (1/361) for B. burgdorferi s.s., and 0 % (0/105) for B. bissettiae (Table 9). Five studies attempted to feed infected nymphs on naïve hosts but the uniformly negative outcomes (no hosts showing evidence of spirochete exposure) are difficult to interpret because none of the ticks fed on the animals were found to be infected in one study (Piesman and Happ, 1997) and the infection status of the fed nymphs was not examined in the other four studies (Mukolwe et al., 1992; Ryder et al., 1992; Oliver et al., 2003; Sanders and Oliver, 1995). Consequently, no single host has yet been unequivocally exposed to the feeding by an infected A. americanum tick in a vector competence study with Lyme borreliosis spirochetes.

7.2. Dermacentor andersoni

The vector competence of D. andersoni was evaluated for uncharacterized B. burgdorferi s.l. spirochetes (wild strain) and B. burgdorferi s.s. (B31) (Dolan et al., 1997; Table 9). Larval ticks acquired spirochetes from infected hosts with low efficiency (11–17 % of freshly fed larvae were infected) but there was no evidence of transstadial passage to the nymphal stage (none of 60 molted nymphs were infected). Subsequent attempts to feed infected nymphs on naïve hosts were unsuccessful as none of the ticks fed on the animals were found to be infected (Table 9). Consequently, no single host has yet been exposed to the feeding by an infected D. andersoni tick in a vector competence study with Lyme borreliosis spirochetes.

7.3. Dermacentor occidentalis

The vector competence of D. occidentalis was evaluated for uncharacterized B. burgdorferi s.l. spirochetes (wild strains) and B. burgdorferi s.s. (CA5) (Brown and Lane, 1992; Lane et al., 1994; Table 9). Larval ticks acquired spirochetes from infected hosts with low efficiency (10 % of freshly fed larvae were infected) but there was no evidence of transstadial passage to the nymphal stage (none of 84 molted nymphs were infected). As there was no evidence of transstadial spirochete passage, no attempt was made to feed resulting nymphs on naïve hosts.

7.4. Dermacentor variabilis

The vector competence of D. variabilis was evaluated for uncharacterized B. burgdorferi s.l. spirochetes (MI-119, MI-128, and MI-129), B. burgdorferi s.s. (JD1, B31, NC-2, and SI-1), and B. bissettiae (MI-6) (Piesman and Sinsky, 1988; Mather and Mather, 1990; Mukolwe et al., 1992; Sanders and Oliver, 1995; Piesman and Happ, 1997; Soares et al., 2006; Table 9). The efficiency of spirochete acquisition by larval ticks varied from 10 to 61% across uncharacterized B. burgdorferi s.l. spirochetes (Piesman and Happ, 1997) and 0–58 % for isolates of B. burgdorferi s.s. (Piesman and Sinsky, 1988; Mather and Mather, 1990; Mukolwe et al., 1992; Piesman and Happ, 1997). A few instances of transstadial spirochete transmission to the resulting nymphs were recorded (Piesman and Happ, 1997; Soares et al., 2006), but the overall infection prevalence in resulting nymphs was very low: 3.3 % (2/61) for uncharacterized B. burgdorferi s.l. spirochetes, 0.3 % (1/306) for B. burgdorferi s.s., and 0 % (0/105) for B. bissettiae (Table 9). Four studies attempted to feed infected nymphs on naïve hosts but the outcomes are difficult to interpret because none of the ticks fed on the animals were found to be infected in one study (Piesman and Happ, 1997) and the infection status of the fed nymphs was not examined in the other three studies (Mukolwe et al., 1992; Sanders and Oliver, 1995; Soares et al., 2006). Of the 51 hosts included in these transmission experiments, 50 showed no evidence of spirochete exposure following tick feeding. The remaining host, a rabbit, reportedly seroconverted after exposure to D. variabilis nymphs, but there was no evidence presented for infection in the nymphal ticks fed on the rabbit and no spirochetes could be isolated from rabbit tissues (liver, spleen, heart, kidneys, and urinary bladder) taken at necropsy (Mukolwe et al., 1992). In contrast, rabbits exposed to I. scapularis ticks infected with the same spirochete isolate in the study uniformly were both seroreactive and yielded tissues from which spirochetes were cultured (Mukolwe et al., 1992). Consequently, no single host has yet been unequivocally exposed to the feeding by an infected D. variabilis tick in a vector competence study with Lyme borreliosis spirochetes.

7.5. Haemaphysalis longicornis (North American population)

The vector competence of a North American population of H. longicornis was evaluated for B. burgdorferi s.s. (B31) (Breuner et al., 2020; Table 9). Larval ticks efficiently acquired spirochetes from infected hosts (56 % of fed ticks harvested within 1 d after repletion were infected) but there was no evidence of transstadial spirochete passage as all 520 examined resulting nymphs tested negative for B. burgdorferi s.s. As there was no evidence of transstadial spirochete passage, no attempt was made to feed resulting nymphs on naïve hosts. Similar results for an Asian population of H. longicornis are described in section 8.4.

8. Vector competence studies with Palearctic/Oriental Dermacentor and Haemaphysalis ticks

To date, 4 species of Palearctic/Oriental Dermacentor and Haemaphysalis ticks have been examined in vector competence studies with B. burgdorferi s.l. spirochetes (Table 9). None of these 4 tick species were experimentally confirmed as vectors of any evaluated B. burgdorferi s.l. spirochete. The strength of the evidence against vector competence is outlined for each tick species below.

8.1. Dermacentor nuttalli

The vector competence of D. nuttalli was evaluated for B. garinii (wild strains) (Sun et al., 2003a; Table 9). Larval or nymphal ticks efficiently acquired spirochetes from infected hosts (50–60 % of fed ticks harvested 1 d after repletion were infected) but spirochetes could no longer be detected from fed larval ticks harvested 8 d after repletion or nymphal ticks harvested 12 d after repletion (Sun et al., 2003a). Moreover, there was no evidence of transstadial passage to the resulting nymphs or adults examined (no data presented for numbers of ticks tested). The subsequent attempts to feed infected ticks on naïve hosts had uniformly negative outcomes (no hosts showing evidence of spirochete exposure) but are difficult to interpret because the infection status of the fed ticks was not examined (Table 9). Consequently, no single host has yet been unequivocally exposed to the feeding by an infected D. nuttalli tick in a vector competence study with Lyme borreliosis spirochetes.

8.2. Dermacentor silvarum

The vector competence of D. silvarum was evaluated for B. garinii (wild strains) (Sun and Xu, 2003; Table 9). Larval or nymphal ticks efficiently acquired spirochetes from infected hosts (40–50 % of fed ticks harvested 1 d after repletion were infected) but spirochetes could no longer be detected from fed larval ticks harvested 8 d after repletion or nymphal ticks harvested 15 d after repletion (Sun and Xu, 2003). Moreover, there was no evidence of transstadial passage to the resulting nymphs or adults examined (no data presented for numbers of ticks tested). The subsequent attempts to feed infected ticks on naïve hosts had uniformly negative outcomes (no hosts showing evidence of spirochete exposure) but are difficult to interpret because the infection status of the fed ticks was not examined (Table 9). Consequently, no single host has yet been unequivocally exposed to the feeding by an infected D. silvarum tick in a vector competence study with Lyme borreliosis spirochetes.

8.3. Haemaphysalis concinna

The vector competence of H. concinna was evaluated for B. garinii (wild strains) (Sun and Xu, 2003; Table 9). Larval or nymphal ticks efficiently acquired spirochetes from infected hosts (70–80 % of fed ticks harvested 1 d after repletion were infected) but the prevalence of infection fell to <10 % for fed larval ticks harvested 8 d after repletion or fed nymphal ticks harvested 15 d after repletion (Sun and Xu, 2003). Moreover, there was no evidence of transstadial passage to the resulting nymphs or adults examined (no data presented for numbers of ticks tested). The subsequent attempts to feed infected ticks on naïve hosts had uniformly negative outcomes (no hosts showing evidence of spirochete exposure) but are difficult to interpret because the infection status of the fed ticks was not examined (Table 9). Consequently, no single host has yet been unequivocally exposed to the feeding by an infected H. concinna tick in a vector competence study with Lyme borreliosis spirochetes.

8.4. Haemaphysalis longicornis (Asian population)

The vector competence of an Asian population of H. longicornis was evaluated for B. garinii (wild strains) (Sun et al., 2003a; Table 9). Larval or nymphal ticks efficiently acquired spirochetes from infected hosts (60–70 % of fed ticks harvested 1 d after repletion were infected) but spirochetes could no longer be detected from fed larval ticks harvested 8 d after repletion or nymphal ticks harvested 20 d after repletion (Sun et al., 2003a). Moreover, there was no evidence of transstadial passage to the resulting nymphs or adults examined (no data presented for numbers of ticks tested). The subsequent attempts to feed infected ticks on naïve hosts had uniformly negative outcomes (no hosts showing evidence of spirochete exposure) but are difficult to interpret because the infection status of the fed ticks was not examined (Table 9). Consequently, no single host has yet been unequivocally exposed to the feeding by an infected H. longicornis tick in a vector competence study with Lyme borreliosis spirochetes. Similar results for a North American population of H. longicornis are described in section 7.5.

9. Conclusions and future directions

A reasonable goal for each zoogeographic region is to conduct experimental vector competence studies for combinations of the most commonly human-biting Ixodes ticks and geographically relevant potentially human-pathogenic B. burgdorferi s.l. species. In North America, both I. scapularis and I. pacificus were confirmed as vectors of the major Lyme borreliosis spirochete, B. burgdorferi s.s., and I. scapularis also was shown to be a vector of the recently recognized, human-pathogenic B. mayonii (Table 1). Both I. scapularis and I. pacificus also were confirmed as vectors of the potentially human-pathogenic B. bissettiae, but neither species has yet been evaluated for the more recently recognized, potentially human-pathogenic B. kurtenbachii (Table 1). The Eurasian tick I. ricinus has been experimentally confirmed as a vector of the three major Eurasian Lyme borreliosis spirochetes (B. burgdorferi s.s., B. afzelii, and B. garinii) but not for other geographically relevant B. burgdorferi s.l. species occasionally associated with human disease (Borrelia bavariensis, B. bissettiae, B. kurtenbachii, Borrelia lusitaniae, Borrelia spielmanii, and Borrelia valaisiana) (Table 1). The other major human-biting Eurasian tick, I. persulcatus, has to date been experimentally confirmed as a vector for B. garinii but not for either B. afzelii or B. burgdorferi s.s. (Table 1). Although demonstrations of vector competence are worth pursuing to complete the evidence base, it is nevertheless important to keep in mind that the four above-mentioned major human-biting species within the Ixodes ricinus/Ixodes persulcatus complex uniformly have proven to serve as vectors for every B. burgdorferi s.l. species evaluated experimentally to date.

Efforts have been more sporadic for other Ixodes species, with vector competence for B. burgdorferi s.l. spirochetes experimentally confirmed for an additional 9 species (I. affinis, I. angustus, I. dentatus, I. hexagonus, I. jellisoni, I. minor, I. muris, I. sinensis, and I. spinipalpis). The evidence is weakest for I. affinis and I. minor (based on unpublished data), and renewed studies to confirm the vector competence of these ticks for B. burgdorferi s.s. are merited. As outlined in sections 4–5 above, arguments can be made for renewed studies on all 5 species of Ixodes ticks (I. arboricola, I. cookei, I. frontalis, I. holocyclus, and I. ovatus) where previous studies failed to demonstrate vector competence for B. burgdorferi s.l. spirochetes. Although none of these tick species are likely to be highly efficient vectors, which greatly increases the effort required to demonstrate their vector competence, it is overstated to label them incapable of serving as vectors of Lyme borreliosis spirochetes without the benefit of additional studies. As noted in section 6, several additional Ixodes species merit experimental evaluation as vectors of geographically relevant Lyme borreliosis spirochetes: among others these include I. granulatus, I. pavlovskyi, I. trianguliceps and I. uriae.

Despite shortcomings of individual studies on the vector competence of different species of Amblyomma, Dermacentor, and Haemaphysalis ticks for B. burgdorferi s.l. spirochetes, the collective evidence indicates that ticks of these genera are unlikely to contribute more than minimally, if at all, to transmission of Lyme borreliosis spirochetes. Overall, extensive studies involving more than 1600 ticks have produced only a handful (n = 4) of records of spirochete acquisition followed by transstadial spirochete passage in individual ticks, including 3 records for D. variabilis and 1 record for A. americanum (Table 9). Moreover, there is still no single documented instance of an infected Amblyomma, Dermacentor, or Haemaphysalis tick having fed on a naïve host in a transmission experiment. This again underscores the difficulty of experimental evaluations involving tick species expected to at best be inefficient vectors of B. burgdorferi s.l. spirochetes. To produce even a few infected ticks for the final step of demonstrating transmission from infected ticks to naïve hosts, extreme effort may be needed in the initial step of feeding uninfected ticks of the preceding life stage on infected hosts. Moreover, the importance of documenting the infection status of the ticks having fed on naïve hosts in the final step cannot be overemphasized: if all ticks that fed on the hosts were free of spirochetes nothing can be concluded regarding the capacity of infected ticks to transmit spirochetes while feeding.

Several factors probably contribute to Amblyomma, Dermacentor, and Haemaphysalis ticks being poor vectors for B. burgdorferi s.l. spirochetes. Soares et al. (2006) demonstrated that spirochete acquisition by larvae fed on mice infected with B. burgdorferi s.s. (B31) was highly effective for I. scapularis, moderately effective for D. variabilis, and ineffective for A. americanum. Moreover, B. burgdorferi s.l. spirochetes acquired during a larval blood meal are effectively passed to the resulting nymphs for I. scapularis (similar prevalence of infection from fed larvae to molted nymphs) but ineffectively for D. variabilis and A. americanum (decreasing prevalence of infection from fed larvae to molted nymphs) (Piesman and Sinsky, 1988; Mather and Mather, 1990; Ryder et al., 1992; Piesman and Happ, 1997). Another contributing factor to consider is the presence in Amblyomma, Dermacentor, and Haemaphysalis ticks of antimicrobial peptides (defensins) that are lytic to B. burgdorferi s.l. spirochetes (Johns et al., 2000, 2001a, 2001b; Sonenshine et al., 2005; Todd et al., 2007; Chrudimska et al., 2014; Sun et al., 2017). Future research to better understand the mechanisms resulting in some ticks being permissive and others refractory to infection with Lyme borreliosis spirochetes is warranted.