Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov. prevail in diverse enzootic transmission cycles.

Two species of the genus Borrelia, Borrelia bissettiae sp. nov. and Borrelia californiensis sp. nov., were first described by Postic and co-workers on the basis of genetic analyses of several loci. Multilocus sequence analysis of eight housekeeping loci confirmed that these two Borrelia genomospecies are distinct members of the Borrelia burgdorferi sensu lato complex. B. bissettiae sp. nov. was initially described in transmission cycles involving Neotoma fuscipes wood rats and Ixodes pacificus ticks in California, and Neotoma mexicana and Ixodes spinipalpis in Colorado. The preferred host of B. californiensis sp. nov. appears to be the California kangaroo rat, Dipodomys californicus; Ixodes jellisoni, I. spinipalipis and I. pacificus ticks are naturally infected with it. Thus, the ecological associations of the two genomospecies and their genetic distance from all other known Borrelia genomospecies species justify their description as separate genomospecies: B. bissettiae sp. nov. (type strain DN127T = DSM 17990T =  CIP 109136T) and B. californiensis (type strain CA446T = DSM 17989T = ATCC BAA-2689T).

Lyme borreliosis (LB) is caused by several species of bacteria belonging to the LB group of spirochaetes, also referred to as Borrelia burgdorferi sensu lato. B. burgdorferi sensu lato is a heterogeneous species complex that currently consists of at least 20 recognized or proposed genomospecies. These bacteria are maintained in natural transmission cycles among vertebrate reservoir hosts and ticks of the Ixodes persulcatus species complex or other species of the genus Ixodes, such as Ixodes spinipalpis (Brown & Lane, 1992, 1996; Kurtenbach ).

The strain designated the Borrelia bissettiae sp. nov. type strain, DN127T, was isolated from a questing Ixodes pacificus tick collected in Del Norte County, California, during the 1980s (Bissett & Hill, 1987). Additional strains of this genomospecies have been isolated from I. pacificus or Ixodes neotomae (now I. spinipalpis) in California and Colorado (Bissett & Hill, 1987; Brown & Lane, 1992; Maupin ; Schneider ) and from rodents captured in the Chicago area of Illinois (Picken & Picken, 2000). Postic proposed that these strains constitute a distinct genomospecies within the B. burgdorferi sensu lato complex and named it B. bissettii sp. nov. In the USA, B. bissettiae enzootic transmission cycles were also found in some southern states involving Ixodes affinis and various rodent-host species (Oliver ). In the far west of the USA, B. bissettiae was associated with the dusky-footed woodrat, Neotoma fuscipes Baird (Brown ). In the same geographical region, B. bissettiae was detected in a host-seeking avian tick, Ixodes auritulus, co-infected with B. burgdorferi (Padgett & Bonilla, 2011) and, more recently, in the blood of several bird species and I. pacificus immatures infesting birds (Newman ). In the latter study, the infection prevalence in I. pacificus larvae was much lower for B. bissettiae than it was for B. burgdorferi; thus, the role of birds as either primary or secondary reservoir hosts for B. bissettiae remains to be established.

Of the tick species known to transmit B. bissettiae sp. nov. in the USA, i.e. I. pacificus and I. spinipalpis in the far west and south-west, and I. affinis in the south-east (Bissett & Hill, 1987; Lin , 2003; Maupin ), only I. pacificus attaches to humans with any frequency. This may partly explain why B. bissettiae is not considered to be a human pathogen in the USA (Maupin ). On the other hand, B. bissettiae occasionally infects humans in northern California as demonstrated by the presence of its DNA in a few serum specimens, but signs or symptoms suggestive of clinical Lyme disease are lacking in this region (Girard ).

In Europe, B. bissettiae sp. nov. DNA has been detected in human patients (Picken , b; Rudenko , 2009; Strle ), and in questing Ixodes ricinus ticks (Hulínská ; Tappe ). One human isolate of B. bissettiae (PGeb) was obtained from a German patient without a history of travel, providing direct evidence that B. bissettiae occurs in Europe (Fingerle ) (Fig. 1).

Fig. 1.

Molecular phylogenetic analysis of Borrelia bissettiae sp. nov. (dark grey dots) and Borrelia californiensis sp. nov. (light grey dot) strains. The evolutionary history was inferred by using the maximum-likelihood method based on the Tamura–Nei model (Tamura & Nei, 1993). The tree with the highest log-likelihood ( − 21015.8975) is shown. Bootstrap values (500 replications) are shown next to nodes. Initial tree(s) for the heuristic search were obtained by applying the neighbour-joining method to a matrix of pairwise distances estimated using the maximum composite likelihood (MCL) approach. A discrete Gamma distribution was used to model evolutionary rate differences among sites [5 categories (+G, parameter = 0.2602)]. All positions containing gaps and missing data were eliminated. There were a total of 4779 positions in the final dataset. Evolutionary analyses were conducted in mega6 (Tamura ). Bar, 0.001 substitutions per site.

In phylogenetic analyses of the rrf-rrl intergenic spacer (IGS) region of B. burgdorferi sensu lato, strains CA443 and CA446 from northern California fell into a clade well separated from all other known genomospecies, a finding consistent with them representing a distinct genomospecies for which the name Borrelia californiensis sp. nov. was proposed (Postic ). Twenty-three Borrelia strains mainly isolated from the California kangaroo rat (Dipodomys californicus) clustered together with strains CA404, CA443 and CA446 (Postic ). Those data suggest that all such strains belong to the genomospecies B. californiensis sp. nov., and that D. californicus is a primary reservoir host of this genomospecies. Strains CA443 and CA446 investigated by multilocus sequence analysis (MLSA) with eight housekeeping genes formed a distinct clade that differed from all other species of the genus Borrelia (Margos ) (Fig. 1). Genetic-distance analysis confirmed the distinctness of these strains from other described species of the genus Borrelia (Margos ).

The samples used for studies of the two genomospecies are listed in Table 1.

Table 1.

Borrelia bissettiae sp. nov. and B. californiensis sp. nov. isolates from California and Colorado, USA, evaluated in previous studies

ST*	Strain ID	Country of origin	Region	Genomospecies	Biological source of isolate	Year of collection	Typed with:	pubMLST ID/GenBank accession no.
156	CA128	USA	Mendocino County, CA	B. bissettiae	I. neotomae (now I. spinipalpis) ex N. fuscipes	1991	MLSA	1002
282	CA370	USA	Alameda County, CA	B. bissettiae	N. fuscipes ear biopsy	1992	MLSA	1003
283	CA371	USA	Alameda County, CA	B. bissettiae	N. fuscipes ear biopsy	1992	MLSA	1004
270	CA389	USA	Alameda County, CA	B. bissettiae	I. pacificus	1993	MLSA	1005
272	DN127-Cl9-2/p7	USA	Del Norte County, CA	B. bissettiae	I. pacificus	1985	MLSA	1006
273	gom93-268	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex Neotoma mexicana	1993	MLSA	1007
273	gom93-274	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1008
273	gom93-275	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1009
273	gom93-278	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1010
160	gom93-283	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1011
273	gom93-284	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1012
274	gom93-286	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1013
273	gom93-287	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1014
271	gom93-296	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1015
273	gom93-297	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1016
158	gom93-299	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1017
275	gom93-305	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex Peromyscus difficilis	1993	MLSA	1018
276	gom93-310	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1019
277	gom93-501	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1020
277	gom93-543	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1021
277	gom93-544	USA	Larimer County, CO	B. bissettiae	I. spinipalpis ex N. mexicana	1993	MLSA	1022
667	PGeb	Germany	Baden-Württemberg	B. bissettiae	Human	1996	MLSA	1874
447	CA443	USA	Mendocino County, CA	B. californiensis	D. californicus ear biopsy	1995	MLSA	1450
447	CA446	USA	Mendocino County, CA	B. californiensis	D. californicus ear biopsy	1995	MLSA	1277
na	CA552	USA	Mendocino County, CA	B. californiensis	Ixodes jellisoni ex D. californicus	1998	rrf-rrl IGS	AY182059
na	CA507	USA	Mendocino County, CA	B. californiensis	D. californicus	1997	rrf-rrl IGS	AY182056
na	CA504	USA	Mendocino County, CA	B. californiensis	D. californicus	1997	rrf-rrl IGS	AY182055
na	CA502	USA	Mendocino County, CA	B. californiensis	D. californicus	1997	rrf-rrl IGS	AY182054
na	CA462	USA	Mendocino County, CA	B. californiensis	D. californicus	1996	rrf-rrl IGS	AY182053
na	CA448	USA	Mendocino County, CA	B. californiensis	D. californicus	1995	rrf-rrl IGS	AY182052
na	CA442	USA	Mendocino County, CA	B. californiensis	D. californicus	1995	rrf-rrl IGS	AF073254
na	CA411	USA	Mendocino County, CA	B. californiensis	D. californicus	1994	rrf-rrl IGS	AY182048
na	CA31	USA	Mendocino County, CA	B. californiensis	D. californicus	1990	rrf-rrl IGS	AJ006372
na	CA22	USA	Mendocino County, CA	B. californiensis	D. californicus	1990	rrf-rrl IGS	AY177631
na	CA134	USA	Mendocino County, CA	B. californiensis	I. pacificus ex D. californicus	1991	rrf-rrl IGS	AY182042
na	CA468	USA	Mendocino County, CA	B. californiensis	D. californicus	1996	rrf-rrl IGS	AY177641
na	CA404	USA	Mendocino County, CA	B. californiensis	D. californicus	1993	rrf-rrl IGS	AJ006371
na	CA33	USA	Mendocino County, CA	B. californiensis	D. californicus	1990	rrf-rrl IGS	AY177632
na	CA20	USA	Mendocino County, CA	B. californiensis	D. californicus	1990	rrf-rrl IGS	AY180239
na	CA142	USA	Mendocino County, CA	B. californiensis	D. californicus	1991	rrf-rrl IGS	AY182043
na	CA409	USA	Mendocino County, CA	B. californiensis	D. californicus	1993	rrf-rrl IGS	AF073255
na	CA547	USA	Mendocino County, CA	B. californiensis	D. californicus	1998	rrf-rrl IGS	AY177642
na	CA445	USA	Mendocino County, CA	B. californiensis	D. californicus	1995	rrf-rrl IGS	AF073256

na, Not applicable.

st, sequence type.

Description of Borrelia bissettiae sp. nov.

Borrelia bissettiae [bis.set′ti.ae. N.L. gen. n. bissettiae of Bissett, proposed in honour of Dr Marjorie L. Bissett, who isolated and described this spirochaete along with her co-worker Warren Hill (Bissett & Hill, 1987)].

Cells are helical, approximately 0.2 μm by 20 μm, and stain well with Giemsa stain. Unstained cells can be visualized by dark-field microscopy. Flexible and motile with rotational and forward/backwards movement. Cells can be cultured in vitro under microaerophilic conditions (Johnson ) using liquid media such as Barbour–Stoenner–Kelly (BSK) medium. Optimal growth occurs at 33–34 °C.

The type strain, DN127T, was isolated from a questing I. pacificus tick in the late 1980s. It has been deposited in the German Microbial Strain Collection ( = DSM 17990T) and at the Institut Pasteur, Paris, France ( = CIP 109136T). B. bissettiae can be distinguished from other genomospecies of the genus Borrelia via sequences of the 5S–23S IGS, the rrs locus and by MLSA (Margos ; Postic ). The B. bissettiae group is heterogeneous as shown by 5S–23S rRNA IGS (Postic ), MLSA analyses and by the size of the 16S–23S rRNA IGS fragment (Bunikis ) that approximates 1000 or 1100 bp (unpublished data). This bacterium is maintained in nature in diverse transmission cycles involving various rodent reservoir hosts and certain tick species of the genus Ixodes. Strains of this species have been found in the USA and Europe. The mean DNA G+C content of the type strain is 27 mol%.

Description of sp. nov.

Borrelia californiensis (ca.li.for.ni.en′sis N.L. fem. adj. californiensis belonging to California, from where the type strain was isolated) was proposed by Postic .

Cells are helical, approximately 0.2 μm by 20 μm, and stain well with Giemsa stain. Unstained cells can be visualized by dark-field microscopy. Flexible and motile with rotational and forward/backwards movement. Cells can be cultured in vitro under microaerophilic conditions (Johnson ) using liquid media such as BSK medium. Optimal growth occurs at 33–34 °C.

The type strain, CA446T, was isolated from an ear-punch biopsy excised from a male D. californicus captured in November 1995 by Kerry A. Padgett at the University of California Hopland Research and Extension Center in Mendocino County, California. It has been deposited in the American Type Culture Collection ( = ATCC BAA-2689T) and the German Microbial strain collection ( = DSM 17989T). Sequence analysis of the rrf-rrl intergenic spacer and the rrs and flagellin genes differentiates B. californiensis from B. bissettiae (Postic ). B. californiensis strains are also distinguishable from all other LB species by using two different MLSA schemes (Margos ; Postic ). B. californiensis seems a rather homogeneous species. So far, it is restricted in distribution to northern California where its primary vertebrate host is the California kangaroo rat, Dipodomys californicus (Brown & Lane, 1992, 1996; Lane & Brown, 1991). Known vectors include Ixodes jellisoni, I. pacificus and I. spinipalpis. The mean DNA G+C content of the type strain is 27 mol%.