Frequency and Distribution of Rickettsiae, Borreliae, and Ehrlichiae Detected in Human-Parasitizing Ticks, Texas, USA

Abstract

To describe the presence and distribution of tickborne bacteria and their vectors in Texas, USA, we screened ticks collected from humans during 2008–2014 for Rickettsia, Borrelia, and Ehrlichia spp. Thirteen tick species were identified, and 23% of ticks carried bacterial DNA from at least 1 of the 3 genera tested.

Ticks are vectors for a variety of microorganisms, many of which are known agents of zoonotic disease. Although much current research is focused on areas where these diseases are common, it is crucial to collect data from areas with fewer diagnoses of tickborne illness. In Texas, USA, tickborne diseases caused by Rickettsia, Borrelia, and Ehrlichia bacteria are diagnosed less frequently than in some areas of the United States (1); however, those agents have been documented to occur (2), and many medically relevant tick species, capable of carrying and transmitting these pathogens, are established in various geographic areas of Texas (1). Long-term surveillance data encompassing consecutive seasons and a wide geographic range are necessary to ascertain disease transmission risks associated temporally or geographically with established or emerging tickborne pathogens and their vectors. The University of North Texas Health Science Center Tick-Borne Disease Research Laboratory (UNTHSC-TBDL), the primary tick-testing facility for Texas Department of State Health Services Zoonosis Control (TX DSHS), receives ticks continually throughout the year. The data collected from this testing provide an assessment of the prevalence of tick species and associated tickborne bacterial agents collected in Texas.

The Study

From October 1, 2008, through September 30, 2014, ticks removed from humans were sent by TX DSHS to UNTHSC-TBDL, where they were tested by using PCR-based methods, then underwent by DNA sequence analysis to determine the presence of Rickettsia, Borrelia, and Ehrlichia spp. Morphologic identification of tick species was implemented by entomologists at TX DSHS. Ticks that could not be classified morphologically were identified at UNTHSC-TBDL by sequencing mitochondrial 16S rDNA (data not shown).

Each tick was sent to UNTHSC-TBDL in an individual collection tube. Upon arrival, ticks were processed according to the laboratory’s standard protocol, as described by Williamson et al. (2). After bead pulverization, we extracted DNA using the E.Z.N.A. Mollusc DNA Isolation Kit (Omega Bio-Tek, Norcross, GA, USA) following the manufacturer’s protocol.

DNA from each specimen was screened in duplicate by PCR for Rickettsia, Borrelia, and Ehrlichia spp. as previously described (2) by using primers listed in Table 1. PCR products were evaluated, and presumptive-positive amplicons were purified for sequencing (2). Cycle sequencing reactions were performed in both directions by using BigDye Terminator version 3.1 chemistry (Life Technologies, Carlsbad, CA, USA). Dideoxy chain termination products were detected electrophoretically on an ABI 310 or 3130xL Genetic Analyzer (Life Technologies). Sequence analysis was performed by using Sequencher version 4.8/5.0 (GeneCodes, Ann Arbor, MI, USA). Analyzed sequences were compared with reference data in GenBank (http://blast.ncbi.nlm.nih.gov/). Sequences were submitted to GenBank under accession nos. KP861333–KP861347.

Table 1. Primers used for screening of human-parasitizing tick specimens, Texas, USA, October 1, 2008–September 30, 2014.

Primer name	Gene	Primer sequence, 5′ → 3′	Specificity	Amplicon, bp	Reference
Borrelia spp.
FlaLL	flaB	ACATATTCAGATGCAGACAGAGGT	Genus	664	(3)
FlaRL	flaB	GCAATCATAGCCATTGCAGATTGT	Genus		(3)
FlaLS	flaB	AACAGCTGAAGAGCTTGGAATG	Genus	330	(3)
FlaRS	flaB	CTTTGATCACTTATCATTCTAATAGC	Genus		(3)
BL-Fla 522F	flaB	GGTACATATTCAGATGCAGACAGAGGG	B. lonestari	660	(2)
BL-Fla 1182R	flaB	GCACTTGATTTGCTTGTGCAATCATAGCC	B. lonestari		(2)
BL-Fla 662F	flaB	CTGAAGAGCTTGGAATGCAACCTGC	B. lonestari	198	(2)
BL-Fla 860R	flaB	GAGCTAATCCCACCTTGAGCTGG	B. lonestari		(2)
BL-16S 227F	16S	TCACACTGGAACTGAGATACGGTCC	Genus	693	(2)
BL-16S 920R	16S	GAATTAAACCACATGCTCCACCGC	Genus		(2)
Rickettsia spp.
Rr.190 70P	rompA	ATGGCGAATATTTCTCCAAAA	Genus	532	(4)
Rr.190 602N	rompA	AGTGCAGCATTCGCTCCCCCT	Genus		(4)
BG1–21	rompB	GGCAATTAATATCGCTGACGG	Genus	650	(5)
BG2–20	rompB	GCATCTGCACTAGCACTTTC	Genus		(5)
Ehrlichia spp.
Ehr DSB 330F	dsb	GATGATGTCTGAAGATATGAAACAAAT	Genus	398	(6)
Ehr DSB 728R	dsb	CTGCTCGTCTATTTTACTTCTTAAAGT	Genus		(6)
Ehr map1F	map1	ATTTTTACCTGGTGTGTCCTTTTCTGA	Genus	873	(7)
Ehr map1R	map1	CCTTCCTCCAATTTCTATACC	Genus		(7)
Ehr Pmap2F	map1	GACACCAAGGCAGTATACGG	Genus		(7)
Ehr Pmap2R	map1	CTAAGTCAGTACCAATACCTGCAC	Genus		(7)
Tick DNA
16S-1	mt16S	CCGGTCTGAACTCAGATCAAG	Unknown	300	(8)
16S+2	mt16S	TTGGGCAAGAAGACCCTATGAA	Unknown		(8)

The TX DSHS submitted 1,112 ticks to UNTHSC-TBDL during October 1, 2008–September 30, 2014, of which 1,062 originated in Texas. Thirteen tick species were identified; most were Amblyomma americanum (55.7%), followed by Dermacentor variabilis (15.0%), Rhipicephalus sanguineus (13.0%), Ixodes scapularis (5.6%), A. maculatum (5.4%), and A. cajennense (2.9%). Approximately 23.3% of ticks originating in Texas tested positive for DNA from Rickettsia, Borrelia, or Ehrlichia bacteria (Table 2; Technical Appendix Table). Of these bacteria, most belonged to spotted fever group rickettsiae (SFGR); A. americanum was the most common tick species found to carry an SFGR agent. The most frequent SFGR sequences detected demonstrated 100% identity to Candidatus Rickettsia amblyommii rompA (GenBank accession no. EF194096). Candidatus R. amblyommii was detected in both A. americanum and A. cajennense ticks and showed prevalence rates of 30.3% and 32.3%, respectively. The second most common SFGR rompA sequences were 100% homologous to the previously termed rickettsial I. scapularis endosymbiont, which has been officially named R. buchneri (accession no. KP172259) (9). Five A. maculatum specimens contained DNA sequences identical to R. parkeri rompA (accession no. KC003476). Sequences that shared 100% similarity to 1 specific R. rhipicephali isolate (accession no. U43803) and 99% similarity to other R. rhipicephali rompA isolates (accession nos. EU109175–EU109178) were obtained from 4 D. variabilis ticks. Sequences isolated from 2 D. andersoni ticks were identical to R. peacockii rompA and rompB (accession nos. FM883671 and CP001227, respectively). Tick species was confirmed by sequencing mitochondrial 16S rDNA. Sequences from both specimens aligned 99% with D. andersoni (accession no. EU711343) and 94% with D. variabilis (accession no. L34300). D. andersoni is not known to inhabit Texas (1,10), so this finding could suggest a novel geographic association.

Table 2. Number of positive bacterial DNA sequences identified for each human-parasitizing tick species, Texas, USA, October 1, 2008–September 30, 2014*.

Tick	No. positive
	Borrelia			Ehrlichia chaffeensis	Rickettsia
	UNID	burgdorferi	lonestari		amblyommii†	parkeri	peacockii	rhipicephali	buchneri
Amblyomma americanum	0	0	8	2	179	0	0	0	0
A. cajennense	0	0	0	0	10	0	0	0	0
A. maculatum	2	0	0	0	0	5	0	0	0
Dermacentor variabilis	1	0	0	0	0	0	0	4	0
D. andersoni	0	0	0	0	0	0	2	0	0
Ixodes scapularis	0	1	0	0	0	0	0	0	44
Rhipicephalus sanguineus	0	0	0	0	0	0	0	0	0
Total	3	1	8	2	189	5	2	4	44
*Only tick species originating in Texas that tested positive for Borrelia, Ehrlichia, or Rickettsia spp. by DNA sequence analysis are shown. Additionally, 2 A. maculatum ticks from Texas were positive for Panola Mountain Ehrlichia. UNID, unidentified species. †Candidatus species.

The total prevalence of borreliae detected was 1.1%. DNA sequences sharing 100% identity to B. lonestari were found in 8 A. americanum ticks (1.4%). As seen by Stromdahl et al., the B. lonestari isolates matching sequences in this study depended on the insertion or deletion of a nucleotide triplet, AAG (11). Sequences from 7 tick samples matched 100% with B. lonestari flaB isolates containing the additional triplet (accession no. AY850063), and 1 sequence was identical to B. lonestari flaB isolates lacking the triplet (accession no. AY850064). Of the 8 A. americanum ticks from which the B. lonestari sequences were obtained, 6 were co-infected with Candidatus R. amblyommii. DNA extracts from 1 I. scapularis tick contained a sequence consistent with B. burgdorferi sensu stricto (s.s.) and was co-infected with R. buchneri. The flaB sequence matched 100% to (accession no. CP002228), and 99% to (accession no. CP009656) B. burgdorferi s.s. reference sequences. The Borrelia 16S rDNA sequence showed 100% identity to (accession no. CP009656) and differed by 1 single nucleotide polymorphism from (accession no. CP002228) B. burgdorferi s.s. reference sequences. A flaB gene sequence from 1 D. variabilis tick shared 100% identity with Candidatus B. texasensis (accession no. AF264901). Samples from 2 A. maculatum ticks showed flaB sequences matching 90% identity values to B. turcica (accession no. AB109243), a reptilian Borrelia sp. Those flaB sequences were identical to a novel Borrelia sp. (accession no. KF395230) previously found in A. maculatum ticks in Mississippi and known to share a phylogenetic clade with B. turcica (12). Borrelia 16S rDNA primers produced nonspecific amplification with these 2 samples.

Phylogenetic analysis was performed by using MEGA version 5.1 (http://www.megasoftware.net) using GenBank reference sequences to examine relationships between the Borrelia sp. from this study, B. turcica, and both Lyme disease–associated and relapsing fever borreliae (Figure). The results supported findings by Lee et al. that the novel Borrelia sp. flaB sequences were more closely related to the reptilian Borrelia than the other 2 Borrelia groups (12).

Figure.

Maximum-likelihood tree showing that the novel Borrelia sp. identified in Amblyomma maculatum ticks from Texas in this study (box) and from Mississippi (12) shares a closer phylogenetic relationship to B. turcica than to to other Borreliae groups. Analysis is based on flaB sequences (267 bp). GenBank accession numbers are shown in parentheses. Tree was constructed using the Tamura 3-parameter model with a bootstrap value of 1,000 replicates. Scale bar indicates substitutions per nucleotide position.

Two A. americanum ticks contained DNA sharing 100% identity with Ehrlichia chaffeensis dsb (accession no. CP000236). One of these ticks was co-infected with Candidatus R. amblyommii. Prevalence of E. chaffeensis in the A. americanum specimens tested was 0.34%. In addition, 2 of 42 A. maculatum ticks tested for the emerging pathogen Panola Mountain Ehrlichia sp. (PME) (7) each produced a map1 sequence that was 100% homologous to 2 separate PME reference sequences (accession nos. EU272356, EU272358). These sequences differed from each other by 1 single nucleotide polymorphism. This finding represents a novel association, as A. americanum is the known vector for PME (7). A subset of 141 A. americanum ticks was also tested for PME, with negative results.

Conclusions

Frequency of tickborne zoonoses in Texas remains low compared with some regions of the United States. We report the detection of known pathogens along with bacteria of unknown pathogenicity in human-parasitizing ticks commonly found in Texas. Our findings underscore the importance of better characterization and continued surveillance of the frequency and distribution of tick species and the bacterial agents they carry. Continued monitoring in low-risk areas provides data regarding the presence of potential emerging pathogens and vectors not yet commonly identified, which could pose unidentified threats to public health.

Technical Appendix

Summary of number, identity, and bacterial screening results for ticks collected in Texas, USA, October 2008–September 2014.

Biography

Ms. Mitchell is a tick-borne disease analyst at the University of North Texas Health Science Center Tick-Borne Disease Research Laboratory in the Center for Biosafety and Biosecurity. Her current research focuses on detection, identification, and characterization of pathogens and potential emerging agents of human disease.