Geographic differences in genetic locus linkages for Borrelia burgdorferi.

Borrelia burdorferi genotype in the northeastern United States is associated with Lyme borreliosis severity. Analysis of DNA sequences of the outer surface protein C gene and rrs-rrlA intergenic spacer from extracts of Ixodes spp. ticks in 3 US regions showed linkage disequilibrium between the 2 loci within a region but not consistently between regions.

Most bacterial pathogens comprise a variety of strains in various proportions. For Borrelia burgdorferi, an agent of Lyme borreliosis, strains differ in their reservoir host preferences (), propensities to disseminate in humans (,), and prevalences in ticks by geographic area (,). Strain identification of B. burgdorferi now is predominantly based on DNA sequences of either of 2 genetic loci: 1) the plasmid-borne, highly polymorphic ospC gene, which encodes outer surface protein C (,), or 2) the intergenic spacer (IGS) between the rrs and rrlA rDNA, here called IGS1. Other loci for genotyping are the plasmid-borne ospA gene () and the rrfA-rrlB rDNA intergenic spacer, here called IGS2 (). The apparent clonality of B. burgdorferi was justification for inferring strain identity from a single locus (,), but the extent of genomewide genetic exchange in this species may have been underestimated ().

Given reports of an association between disease severity and B. burgdorferi genotype (,), prediction of a strain’s virulence potential from its genotype has clinical, diagnostic, and epidemiologic relevance. But is a single locus sufficient for this assessment?

The Study

To investigate this issue, we determined sequences of ospC and IGS1 loci, and in selected cases the ospA and IGS2 loci, in 1,522 DNA extracts from B. burgdorferi–infected Ixodes scapularis nymphs collected from the northeastern, mid-Atlantic, and north-central United States during the summers of 2004, 2005, 2006, and 2007, as described (,). We also included results from 214 infected I. pacificus nymphs collected in Mendocino County, California (); 20 infected I. pacificus adults from Contra Costa County, California (J. Bunikis and A.G. Barbour, unpub. data); and 10 B. burgdorferi genomes (strains B31, ZS7, 156a, 64b, 72a, 118a, WI91-23, 94a, 29805, and CA-11.2a), for which sequences are publicly available (www.ncbi.nlm.nih.gov). Multilocus sequence typing (MLST), based on 8 chromosomal housekeeping genes, had been carried out for several strains represented in the extracts (Table) (,). The corresponding MLST types of the 10 genome sequences were assigned by reference to a B. burgdorferi MLST database (http://borrelia.mlst.net) (). For this study, we also determined the MLST type of strain CA8.

The methods for 1) DNA extraction from ticks (), 2) PCR amplification of ospC, ospA, and IGS1 (), 3) amplification of IGS2 (), and 4) amplification of 8 chromosomal loci for MLST () have been described. Sequences for both strands were determined from either PCR products or cloned fragments with custom primers (). We followed the basic nomenclature of Wang et al. () until, after exhausting the alphabet, we assigned both a letter and, arbitrarily, the number 3 (e.g., C3) when a new nucleotide sequence differed by >8% from known ospC alleles. We distinguished ospC variants with <1% sequence difference by adding a lowercase letter, e.g., Da and Db. Except for ospC D3 and Oa, novel polymorphisms were confirmed in at least 1 other sample. To simplify IGS1 nomenclature, we numbered types sequentially, beginning with the original 9 types (); ospA alleles () and IGS2 loci were likewise sequentially numbered. The Table A1 provides accession numbers for all sequences, as well as original and revised names for IGS1 sequences.

Table A1

GenBank accession numbers of sequences of Borrelia burgdorferi in this study*

Strain name	ospC allele	ospC accession no.	Former IGS1† name	Revised IGS1 name	IGS1 accession no.	ospA allele	ospA accession no.	IGS2‡ name	IGS2 accession no.
B31	A	AE000792	1A	1	AE000783	1	AE000790	1	GQ463603
CA4	A	EU377746	1A-684	10	EU377801	23	GQ443123
CA6	A	EU377748	1A-684	10	EU377803
2206617	A	AE000792	1A-684/672	11	GQ478289	22	GQ443122
64b	Ba	CP001422	3A	3	ABKA02000001	3	CP001421
B373	Ba	EU377779	3B	3	EU377795
51405UT	Ba	EU375825	6A	6	EU375815	14	GQ443114
ZS7	Bb	NC_011724	3D	16	NC_011728	28	CP001199
JD1	Ca	DQ437462	5G	24	DQ437478
BL515	Ca	EU377774	5G	24	EU377790
OC4	Da	AF029863	5A	5	AY275201
516113	Da	AY275217	5A	5	AY275201	5	GQ433636	4	GQ463606
424404	Db	GQ478283	5A	5	AY275201	18	GQ443118	7	GQ463609
CA-11.2a	Db	CP001484	5A-239	19	ABJY02000007	27	CP001473
N40	E	AY275221	9A	9	AY275211	9	M57248
B348	E	AF467875	9C	9	AF467863
990503	Fa	AY275225	4C	17	GQ130198
B156	Fa	EU377776	4C	17	EU377792
MI407	Fb	EF537433	4D	18	EF537367
1469205	Fc	GQ478285	4D	18	EF537367	13	GQ443113	6	GQ463608
MR616	G	EU377771	6B	26	EU377787
72a	G	CP001375	6B	26	ABGJ02000006	9	CP001370
1468503	G	AY275223	5C	22	GQ130201	21	GQ443121
B509	Ha	EU377781	2D	12	EU377797
156a	Hb	CP001271	2D	12	ABCV02000001	2	CP001257
519014UT	Hb	EU375831	2D	12	EU375823
519512	Hb	GQ478286	2D	12	EU375823
CA92-0953	Hb	EU377751	2D-713	13	EU377806
B500	Ia	AF467878	7A	7	AF467866
B331	Ia	AF467874	7A	7	AF467862	7	GQ443107
1472505	Ia	AY275219	7A	7	AY275205	10	GQ443110
WI91-23	Ia	CP001446	7A	7	ABJW02000006	11	CP001447
CA92-1096	Ib	EU377752	7A	7	EU377807
CA337	Ib	EU377752	7A	7	EU377807	30	GU815347
118a	J	CP001535	5B	20	ABGI02000001	8	CP001542
297	K	AY275214	2B	2	AY275192	2	X85442	2	GQ463604
501604	K	AY275214	2A	2	AY275191
149901	K	AY275214	2E	14	GQ120104	31	GU815348
47703UT	L	EU375832	2E	14	GQ120104
29805	M	CP001550	6A	6	ABJX02000028	2	CP001554	3	GQ463605
CA92-1337	M	EU377753	6A	6	EU377808
MR661	N	EU377775	4A	4	EU377791	4	GQ433635
500203	N	AY275216	4A	4	AY275199
MI418	N	EF537430	5E	23	EF537363
51108	N	AY275216	5E	23	GQ130203
501427	Oa	FJ997281	6C	27	AY275204
2207807	Ob	FJ997282	6A	6	ABJX02000028
23509	T	AY275222	8C	28	AY275209
1476702	T	AY275222	8C-808	29	GQ478288	20	GQ443120
94a	Ua	CP001493	8A	8	ABGK02000002	8	CP001500
B485	Ua	EU377769	8A	8	EU377785
48802	Ua	AY275220	8A	8	ABGK02000002	16	GQ443116
2207116	Ua	EU377769	8A	8	EU377785	12	GQ443112	10	GQ463612
426905	Ub	GQ478287	8E	30	GQ130197	8	GQ443108	9	GQ463611
2206613	A3	EF592541	2E	14	GQ120104	19	GQ443119
2250201	B3	EF592542	5E	23	GQ130203	17	GQ443117
50202	C3	EF592543	4C	17	GQ130198	15	GQ443115	5	GQ463607
2150902	D3	EF592544	New	31	GQ478290
2127701	E3	EF592545	5B	20	GQ130200			8	GQ463610
HRT25	E3	EF592545	5A-725	21	EU886975	24	GQ443124
LMR28	E3	EF592545	5A	5	AY275201	25	GQ443125
1456802	F3	EF592547	5A	5	AY275201
CA8	H3	FJ932733	5A8	25	EU886974	26	GQ247743
CA11	I3	FJ932734	4C	17	GQ130198
CA12	I3	FJ932734	4C	17	GQ130198

*Boldface indicates new accession number from this study.
†IGS1, rrs-rrlA intergenic spacer region.
‡IGS2, rrf-rrlB intergenic spacer.

For 741 Ixodes ticks from northeastern and north-central United States or from northern California, 1 ospC allele was identified and sequenced. In the remaining samples, we found a mixture of strains or evidence of >2 ospC and/or >2 IGS sequences (). In 678 (91%) of the 741 samples with a single ospC, the allele could be matched with particular IGS1 (Table). We identified 9 unique ospC sequences: Fc, Ob, Ub, A3, B3, C3, D3, E3, and F3, all from the north-central United States. Alleles H3 and I3 of California were recently reported by Girard et al. (). Of 32 codon-aligned ospC sequences, 6 pairs and 1 trio (Fa, Fb, and Fc) differed in sequence by <1% (Figure, panel A). Nine novel IGS1 sequences, numbered 24–31 and 33, were discovered in samples from which ospC alleles were determined.

Figure

A) Bayesian and maximum-likelihood phylogenetic inference of outer surface protein C (ospC) gene sequences and B) concatenated multilocus sequence typing (MLST) sequences of Borrelia burgdorferi. Sequences were aligned by codon. Labels at the tips refer to ospC alleles (A) or MLST (ST) and linked ospC alleles (B; Table). Consensus phylograms were the output of the MrBayes version 3.1.2 algorithm (http://mrbayes.csit.fsu.edu). There were 500,000 generations with the first 1,000 discarded. Nodes with posterior probabilities of >0.5 are indicated by values above the branches. Below the branches are integer values for nodes with support of >500 of 1,000 bootstrap iterations of the maximum-likelihood method, as carried out with the PhyML 3.0 algorithm (www.atgc-montpellier.fr/phyml). For both data sets and both algorithms, the models were general time reversible with empirical estimations of the proportions of invariant sites and gamma shape parameters. Scale bars indicate genetic distance. GenBank accession numbers for sequences are given in the Table A1.

When we confined analysis to samples from northeastern states, we confirmed linkage disequilibrium between ospC and IGS1 loci (,,). However, when results from north-central states and California were included, a different picture emerged (Table, Figure, panel B). Most of the ospC alleles showed concordance with the chromosomal loci; monophyletic MLST showed either the same ospC allele or a minor variant of it. However, in several instances, the ospC alleles were linked to different IGS1 sequences, different ospA sequences, and/or different MLST with internal nodes in common. We observed this linkage for ospC alleles A, G, Hb, and N. In the case of ospC Hb, the shared internal node was deep.

We applied the Simpson index of diversity, as implemented by Hunter and Gaston (), to the data in the Table to compare the discriminatory power (DP) of genotyping on the basis of a combination of ospC and IGS1 sequences with genotyping by 8-locus MLST (). For double-locus typing, there were 43 types were found for 678 strains; DP value was 0.96. For MLST in this data set, 36 types were shown for 554 strains; DP was 0.95. In the study of Hoen et al. in which selection was made for geographic isolation, 37 types were distributed among 78 strains; DP was 0.97 ().

Conclusions

Dependence on a single locus for typing may falsely identify different lineages as the same, especially when the samples come from different regions. Other loci may be as informative as ospC or IGS1, but the abundance of extant sequences for these loci justifies their continued use. Uncertainties about the linkage of ospC and IGS1 usually can be resolved by sequencing the ospA allele (Table). IGS2 provided little additional information in this study.

One interpretation of these findings is that lateral gene transfer of all or nearly all of an ospC gene has occurred between different genetic lineages. We previously had not detected recombination at the IGS1 locus on the chromosome (), but there may be recombination at other chromosomal loci, as well as plasmid loci (). Besides extending the understanding of the geographic structuring of the B. burgdorferi population, the results indicate that the ospC allele does not fully represent the complexity of B. burgdorferi lineages; thus, inferring phenotypes on the basis of this single locus should be made with caution.

Table

Linkages between ospC alleles and other loci in Borrelia burgdorferi strains*

ospC	IGS1	Geographic region*	Representative cultured isolate or tick sample†	IGS1-ospC associations‡	ospA	IGS2	MLST§
A	1	1, 2	B31	45/52	1	1	1
A	11	2	2206617	4/4	22	1	55
A	10	3	CA4, CA6	14/18	23	1	2
Ba	3	1	64b, B373	39/41	3	1	7,58,59
Ba	6	2	51405UT	7/9	14	1	30
Bb	16	4	ZS7	–	28	–	20
C	24	1	JD1, BL515	10/10	8	5	11
Da	5	1	516113	13/14	5	4	38
Db	5	2	424404	13/15	18	7	51
Db	19	3	CA11.2A	16/16	27	4	70
E	9	1, 2	N40, B348	17/19	9	1	19
Fa	17	1, 2, 3	B156	61/64	3	4	8
Fb	18	2	MI407	14/19	8	6	–
Fc	18	2	1469205	7/8	13	6	56
G	26	1	72a, MR616	10/11	9	4	14
G	22	2, 3	1468503	9/10	21	4	48,49
Ha/Hb	12	1	B509/156a	13/13	2	2	4
Hb	12	2	519014UT	56/65	11	2	32
Hb	13	3	CA92-0953	20/20	23	2	6
Ia	7	1	B500, B331	12/16	7	4	15,16
Ia	7	2	WI91-23	5/5	11	4	71
Ib	7	3	CA92-1096	–	30	4	17
J	20	1, 2	118a	3/5	8	4	34
K	2	1	297	67/68	2	2	3
K	14	2	149901	7/10	31	2	–
L	14	2	47703UT	23/25	8	2	29
M	6	1	29805	4/4	2	3	12
M	6	2, 3	CA92-1337	16/16	17	3	13
N	4	1	MR661, 500203	41/41	4	10	9,36
N	23	2	51108	8/10	2	1	43
Oa	27	1	501427	1/1	–	–	54
Ob	6	2	2207807	6/7	2	–	–
T	28	1	23509	16/16	8	4	37
T	29	2	1476702	10/11	20	4	46
Ua	8	1	94a, B485	19/19	8	4	18
Ua	8	2	48802	4/4	16	4	47
Ua	17	2	2207116	4/4	12	10	–
Ub	30	2	426905	3/3	8	9	–
A3	14	2	2206613	6/6	19	2	–
B3	23	1, 2	2250201	3/3	17	1	57
C3	17	2	50202	6/9	15	5	–
D3	31	2	2150902	1/1	–	–	–
E3	20	2	2127701	4/4	8	8	52
E3	21	3	HRT25	12/12	24	–	–
E3	5	3	LMR28	12/12	25	–	–
F3	5	2	1456802	8/12	8	4	–
H3	25	3	CA8	37/40	26	4	(72)
I3	17	3	CA11, CA12	5/5	27	4	–

*Regions: 1, northeastern United States; 2, north-central United States; 3, northern California; 4, western Europe; osp, outer surface protein; IGS, intergenic spacer; MLST, multilocus sequence typing; –, MLST not determined.
†Tick samples () are indicated by italics; strains with genome sequences are indicated in boldface.
‡Number of tick extracts with the listed IGS1 locus (numerator)/number of extracts with the listed ospC allele (denominator).
§MLST from (,) or this study (in parentheses).