Prevalence of Borrelia burgdorferi, Bartonella spp., Babesia microti, and Anaplasma phagocytophila in Ixodes scapularis Ticks Collected in Northern New Jersey

Abstract

PCR analysis of Ixodes scapularis ticks collected in New Jersey identified infections with Borrelia burgdorferi (33.6%), Babesia microti (8.4%), Anaplasma phagocytophila (1.9%), and Bartonella spp. (34.5%). The I. scapularis tick is a potential pathogen vector that can cause coinfection and contribute to the variety of clinical responses noted in some tick-borne disease patients.

Lyme disease (LD) has been characterized as a multisystem disease caused by the spirochete Borrelia burgdorferi (3). The Centers for Disease Control and Prevention reported 17,730 domestic cases of LD in 2000, making it the most common vector-borne disease in the United States (5). In most patients, antibiotic treatment with doxycycline or amoxicillin has been proven to be a highly effective mode of treatment for acute and late stages of LD (28). However, a subset of patients exhibits persistent symptoms regardless of antibiotic therapeutic intervention.

The primary vertebrate reservoir for B. burgdorferi in the northeastern United States has been identified as the white-footed mouse, Peromyscus leucopus. Both larval and nymphal stages of Ixodes scapularis ticks mainly feed on P. leucopus and can infect human hosts with B. burgdorferi (1). Other pathogens, including Babesia microti and Anaplasma phagocytophila, have also been identified in I. scapularis ticks, and cotransmission with B. burgdorferi has been documented (17, 18, 19).

The present study examined the prevalence of four pathogens in I. scapularis that could potentially be transmitted to humans. Coinfection with two or more of these organisms may complicate LD prognosis. For example, simultaneous LD and babesiosis correlated with a more severe clinical progression than either condition alone (15). I. scapularis ticks were collected by the tick sweep method (4) from February through July 2001, primarily in Union County, N.J., and were identified by standard taxonomic keys (9, 12, 14). Individual ticks were placed in a microcentrifuge tube containing 470 μl of TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM EDTA), 25 μl of 10% sodium dodecyl sulfate, and 12 μl of DNase-free proteinase K (10 mg/ml). A handheld motorized pestle (Kontes, Vineland, N.J.) was used to homogenize each tick lysate. DNA was extracted with phenol-chloroform and recovered by ethanol precipitation. Primers for amplification of genomic DNA were synthesized by Research Genetics (Huntsville, Ala.) and are listed in Table 1. The conditions for PCR amplification of B. burgdorferi, B. microti, Bartonella spp., and A. phagocytophila were as previously described (7, 8, 13, 20). PCR products were analyzed by electrophoresis through a 1% agarose gel containing 0.5-μg/ml ethidium bromide and UV analysis utilizing a Photodocumentation System (Fisher Biotech, Pittsburgh, Pa.). The identity of selected amplicons was confirmed by independent DNA sequencing (SeqWright, Houston, Tex.). Positive controls were included in each PCR experiment and consisted of genomic DNA extracted from pathogens purchased from the American Type Culture Collection (Table 1). Negative controls consisted of the substitution of pyrogen-free water for DNA. Precautions against contamination were maintained as previously described (13).

TABLE 1.

Oligonucleotide primers utilized for species-specific pathogen and l. scapularis amplification

Primer	Oligonucleotide primer sequence	Target gene and reference	Product size (bp)
LY1 (F)	5′-GAAATGGCTAAAGTAAGCGGAATTGTAC-3′	B. burgdorferi ly1 (ATCC 35210) (16)	231
LY2 (R)	5′-CAGAAATTCTGTAAACTAATCCCACC-3′
MRL-7 (F)	5′-GTTTCAGTAGATTTGCCTGG-3′	B. burgdorferi ospA (7)	569
MRL-8 (R)	5′-GCCTGAATTCCAAGCTGCAG-3′
HGE1F	5′-GGATTATTCTTTATAGCTTGCT-3′	A. phagocytophila 16S rDNA (ATCC CRL-10679) (2)	920
HGE3R	5′-TTCCGTTAAGAAGGATCTAATCTC-3′
P24E	5′-GGAATTCCCTCCTTCAGTTAGGCTGG-3′	B. henselae 16S rDNA (ATCC 49882) (20)	279
P12B	5′-CGGGATCCCGAGATGGCTTTTGGAGATTA-3′
Bab1	5′-CTTAGTATAAGCTTTTATACAGC-3′	B. microti (ATCC 30222) (25)	238
Bab4	5′-ATAGGTCAGAAACTTGAATGATACA-3′
IXO-16S-F	5′-TAAACAATTAAAAGCTTTCTT-3′	I. scapularis 16S rDNA (this study)a	215
IXO-16S-R	5′-AATCGCTAAAAACGGAACTTA-3′

^aPrimers derived from data reported previously (3a).

As another species of tick, Dermacentor variabilis, is also very common in the geographic region studied, molecular techniques were adopted to verify all ticks as I. scapularis (Fig. 1). The primers for amplification were IXO-16S-F and IXO-16S-R (Table 1). The PCR conditions were denaturation at 94°C for 3 min followed by 35 cycles of 94°C for 3 min, 58°C for 1 min, and 72°C for 1 min. Each reaction was concluded with a 10-min final extension step at 72°C in a T3 Thermocycler (Biometra, Göttingen, Germany). Amplification products were resolved through a 2% agarose gel containing 0.5-μg/ml ethidium bromide and visualized on a MultiGenius gel documentation and analysis system (Syngene, Frederick, Md.). The specificity of the primers was verified by amplifying known DNA control extracts from I. scapularis and D. variabilis. Through this technique, 16 ticks were excluded from the sampling pool due to nonamplification. This control also confirms the validity of the results in those samples in which no pathogens were detected. From the PCR analyses, it was evident that at least one of the four pathogens was present in 45.8% (49 of 107) of the ticks (Table 2) Also, 15 of 107 ticks (14.0%) contained more than one pathogen. The most common combination was B. burgdorferi and Bartonella spp.

FIG. 1.

Molecular detection of I. scapularis by amplification of 16S rDNA. Lanes: 1, 2, and 5, DNA extracted from I. scapularis; 3 and 4, DNA extracted from D. variabilis; 6, negative water control; M, molecular weight ladder developed in house with bands in decreasing order of size of 2,743, 980, 752, 650 (double intensity), 552, 395, 300, 198, and 104 bp.

TABLE 2.

Identification of I. scapularis ticks infected with B. burgdorferi, B. microti, A. phagocytophila, or B. henselae

Pathogen(s)	No. (%) of ticks PCR positivea
Single infections
B. burgdorferi	36 (33.6)
B. microti	9 (8.4)
A. phagocytophila	2 (1.9)
Bartonella spp.	37 (34.5)
None	58 (54.2)
B. burgdorferi and Bartonella spp. coinfections (includes those listed above)
B. burgdorferi and Bartonella spp.b	9 (8.4)
B. burgdorferi and B. microti	2 (1.9)
Bartonella spp. and B. microti	1 (0.9)
Bartonella spp. and A. phagocytophila	1 (0.9)
B. burgdorferi, Bartonella spp., and A. phagocytophila	1 (0.9)
Bartonella spp., B. microti, and A. phagocytophila	1 (0.9)

^aNumber of ticks positive for each pathogen out of 107 total ticks (confirmed as I. scapularis by the procedure depicted in Fig. 1).

^bCoinfection data overlap with the single-pathogen prevalence percentages.

Transmission of B. burgdorferi to humans causing LD is just one of several possible outcomes from a tick bite. The environment in the tick is suitable for bacterial diversity, and up to 10 clones of B. burgdorferi as well as B. microti and A. phagocytophilum can be simultaneously isolated from a single I. scapularis host (17, 19, 23). In Table 3, the prevalence of the pathogens studied was found to be in agreement with previous reports detailing pathogen detection by PCR methodologies in the northeastern United States, with the exception of the human granulocyltic ehrlichiosis agent, which was at a lower prevalence than all except in northwestern Pennsylvania (11).

TABLE 3.

Reported infection rates of B. burgdorferi, A. phagocytophila; and B. microti in the northeastern United States

Location (reference)	% of infection with:
	B. burgdorferi	A. phagocytophila	B. microti
Westchester County, N.Y. (26)	52	53	NTa
Nantucket Island, Mass. (27)	36	11	9
Hunterdon County, N.J. (11)	43	17	5
Northwest Pennsylvania (11)	61.6	1.9	NT
Southeast Pennsylvania	13	39.8	NT
Union County, N.J. (this study)	31	1.6	8

^aNT, not tested.

This is the first report to assay for Bartonella spp. in field-collected I. scapularis ticks. Previously, we published a clinical case study presenting molecular and serological diagnostic evidence of patients coinfected with B. burgdorferi and Bartonella henselae (13). For two patients, ticks found to contain B. burgdorferi and B. henselae by PCR and identified as I. scapularis were found in their households, one of which was removed from a household cat. Our report was further reinforced by a more recent publication in which both pathogens were detected by PCR in the cerebrospinal fluid of two patients with symptoms suggestive of neuroborreliosis (22). Although the primers in this study were originally selected for the species-specific amplification of B. henselae, this region of the Bartonella 16S ribosomal DNA (rDNA) gene is highly conserved among many species within the genus (2, 21). For example, Multalin alignment analysis revealed that only 5 of 279 nucleotides differ between the B. henselae and B. quintana amplicons, only 2 of which are situated between the oligonucleotide amplification primers (10, 21). Further investigations should seek to amplify more divergent regions of the Bartonella genus that can be utilized for species-specific identification. Members of the Bartonella genus have also been found in ticks and other insects. For example, B. henselae was detected by PCR in 4 of 271 Ixodes ricinus ticks removed from humans in Belluno Province, Italy (24), and Bartonella species were also detected by PCR in 29 of 151 Ixodes pacificus ticks collected in Santa Clara County, Calif. (6).

As treatment for Bartonella infections varies from that prescribed for LD patients, physicians should add Bartonella infections to the list of possible coinfection agents when evaluating patients in regions of tick endemicity, as single- and multiple-pathogen transmission can complicate clinical presentations. Future studies need to clarify that the Bartonella spp. can be passed in culturable form from vector to host and to identify which specific species of Bartonella are present.