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MassTag polymerase chain reaction (PCR) is a platform that enables microbe detection using primers labeled through a photocleavable link with tags that vary in molecular weight. After multiplex PCR, tags are released by ultraviolet irradiation and analyzed by mass spectroscopy. The identification of a microbe in a sample is determined by its cognate tags. Here we describe establishment and implementation of a MassTag PCR panel for surveillance of microbes implicated in tick-vectored infectious diseases.
Ticks can harbor many pathogens; thus, a single tick bite may result in polymicrobial infections (Benach et al. 1985, Magnarelli et al. 1995, Krause et al. 1996, Mitchell et al. 1996). Common human-biting ticks associated with pathogen transmission in the United States include Ixodes scapularis, Amblyomma americanum, Dermacentor variabilis, and Dermacentor andersoni (Bratton and Corey 2005). Lyme disease, the most common vector-borne disease in the United States, is caused by the spirochete Borrelia burgdorferi and transmitted by I. scapularis ticks (Burgdorfer et al. 1982). In addition, I. scapularis can transmit Anaplasma phagocytophilum bacteria, the eti-ologic agent of human granulocytic anaplasmosis, and the protozoan Babesia microti, the agent of babesiosis (Spielman et al. 1979, Pancholi et al. 1995). Other microorganisms detected in I. scapularis, notably Bartonella species (spp.), may have a role in tick-borne infections; however, whether they are transmitted by ticks remains to be determined (Chang et al. 2001, Adelson et al. 2004, Holden et al. 2006). Dermacentor ticks (both D. variabilis and D. andersoni) are vectors of Rickettsia rickettsii, the etiologic agent of Rocky Mountain spotted fever, and Fran-cisella tularensis, the etiologic agent of tularemia (McDade and Newhouse 1986, Goethert et al. 2004). Amblyomma americanum can transmit Ehrlichia chaffeensis, the etiologic agent of human monocytic ehrlichiosis, as well as F. tularensis (Childs and Paddock 2003). A. americanum ticks can also harbor Borrelia lones-tari, a Borrelia species of unclear pathogenicity related to relapsing fever Borreliae (Fukunaga et al. 1996, James et al. 2001). Coxiella burnetii has been detected in a wide variety of ticks, including Dermacentor and Amblyomma, and natural tick transmission to humans documented, although this is considered to contribute only a minor component of human acute Q fever (Maurin and Raoult 1999).
We recently described the application of a multiplex polymerase chain reaction (PCR) method for microbial surveillance wherein primers are attached to tags of varying mass that serve as digital signatures for their genetic targets (Briese et al. 2005). Tags are cleaved from primers and recorded by mass spectroscopy enabling sensitive, multiplex microbial detection. The method, MassTag PCR, has been implemented for differential diagnosis of respiratory infection and hemorrhagic fevers (Briese et al. 2005, Lamson et al. 2006, Palacios et al. 2006, Renwick et al. 2007, Briese et al. 2008). In this report, we describe a MassTag PCR assay adapted for rapid screening of field-collected ticks for multiple pathogens. The ease and efficiency of the assay allows rapid analysis of large sample numbers.
Species- and genus-specific PCR primer sets were designed from multiple nucleotide sequence alignments of target pathogens using Greene SCPrimer, a program based on set cover theory (Jabado et al. 2006; Tables 1 and and2).2). Prior to committing to synthesis and conjugation of mass tagged primers, primer set performance was assessed using unmodified primers in singleplex and multiplex PCR assays wherein products were detected by electrophoresis in ethidium bromide-stained agarose gels. Primer pairs were first tested for specificity in singleplex PCR reactions containing their cognate and irrelevant targets. Thereafter, all primer sets were combined in multiplex reactions to assess for interference in performance. Primer sets that passed quality control tests were conjugated to mass tags (operon) and incorporated into the panel. DNA standards used for assay development were cloned by PCR from pathogen DNA and ligated into pGEM-T Easy vector (Promega). In our laboratory singleplex PCR assays are typically pursued using 25 ng of tick DNA. Thus, sensitivity assays were performed with 10-fold dilutions of linearized plasmid in a background of 25 ng/μL of tick DNA. For tick assays, live adult ticks were collected in 2006 and 2007 from Suffolk County, New York. Individual ticks were homogenized in sterile H2O, and total DNA was isolated using Qiaprep DNA kit (Qiagen, Valencia, CA). DNA was resuspended in 20 μL of water, of which 2 μL was used in the MassTag PCR reaction. The specificity of pathogen detection in ticks by MassTag PCR was confirmed by singleplex PCR followed by sequencing of amplification products.
A MassTag PCR panel was designed to detect known or suspected pathogens transmitted by ticks endemic on the east coast of the united states, including A. phagocytophilum, B. microti, Bartonella spp., B. burgdorferi sensu lato, B. lonestari, Coxiella burnetti, Ehrlichia spp., F. tularensis, and R. rickettsi. specificity was tested using relevant and irrelevant targets. All primer sets amplified only their cognate targets. To model conditions encountered in field samples, sensitivity was tested using serially diluted linearized DNA standards in a background of I. scapularis, D. variabilis, and A. americanum DNA (Fig. 1). Sensitivity for detection of Ehrlichia and R. rickettsii was <200 copies/reaction; sensitivity for other targets was <20 copies/reaction. To test assay performance in environmental samples, we isolated DNA from 88 individual adult I. scapularis ticks collected in Suffolk County, New York in 2006 (Fig. 2). MassTag PCR detected B. burgdorferi in 51 (58%) of ticks analyzed (Table 3). For confirmation of assay fidelity, we elected 25 MassTag positive samples for singleplex PCR amplification and sequencing of a 676-bp fragment of the B. burgdorferi ospA gene (Fig. 2A). All 25 MassTag B. burgdorferi positive samples were positive by singleplex PCR assays. We also selected 15 MassTag negative samples, all of which were negative in singleplex PCR assays. Other agents detected by MassTag PCR included A. phagocytophilum (14 I. scapularis ticks, four of which also contained B. burgdorferi) and B. microti (five I. scapularis ticks, one of which also contained B. burgdorferi; Tables 3 and and4).4). To test fidelity of these assays, a 112-bp portion of Anaplasma-specific mspA gene sequence was cloned from 10 I. scapularis ticks positive for A. phagocytophilum in MassTag PCR. Sequence analysis confirmed the presence of A. phagocytophilum. Ten samples negative for A. phagocytophilum in MassTag PCR were also negative by singleplex PCR (Fig. 2C). Similar fidelity assays were performed for B. microti positive (five samples) and negative samples (10 samples). Singleplex PCR confirmed MassTag results (Fig. 2B). In one I. scapularis tick, we detected a triple infection with B. microti, B. burgdorferi, and A. phagocytophilum (Fig. 3).
MassTag PCR assays employ both species- and genus-specific primers (Table 1). Two I. scapularis ticks were positive in MassTag PCR assays with genus-specific Bartonella primers. A 460-bp informative region of the 16S rRNA was amplified by PCR (Table 2)and sequenced to enable speciation. Both contained Bartonella henselae. B. lonestari was not detected in any specimen from I. scapularis; however, four ticks contained a closely related species, Borrelia miyamotoi. For confirmation, a 965-bp region of the flaB gene was amplified by PCR (Table 2)and sequenced to enable speciation. All four sequences were B. miyamotoi. Like B. lonestari, B. miyamotoi is a species grouped to the relapsing fever Borreliae. it has been previously reported in I. scapularis (scoles et al. 2001). one of the I. scapularis ticks infected with B. miyamotoi was also coinfected with B. burgdorferi. In another sample, we detected a mixed infection with B. burgdorferi, A. phagocytophilum, and B. miyamotoi (Table 4).
In additional experiments, we screened Dermacentor and Amblyomma ticks collected in Suffolk County, in the spring of 2007. DNA from a total of 40 D. variabilis and 55 A. americanum ticks was isolated and screened by MassTag PCR. B. lonestari was detected in three A. americanum ticks and Ehrlichia chaffensis in two other Amblyomma ticks. The presence of these pathogens was confirmed by sequencing.
Our results indicate that MassTag PCR is an efficient tool for surveillance of tick microflora. Each assay, comprising up to 20 primer pairs (20 different microbial genetic targets), costs $15 and allows detection of coinfections as well as single infection with sensitivity similar to that obtained with singleplex assays. We typically use a 96-well plate format to simultaneously run 1920 tests. Tagged primers are available from commercial vendors; primers and protocols are freely available; costs for mass spectrometry instruments are approximately $75,000. Although we have not tested for the presence of tick-borne pathogens in human materials, based on previous work in differential diagnosis of respiratory diseases (Lamson et al. 2006, Renwick et al. 2007, Briese et al. 2008) and hemorrhagic fevers (Palacios et al. 2006), the assays reported here may also be of utility in clinical microbiology.
We thank Courtney Bolger for Francisella tularensis fopA DNA and Brian Fallon and Gustavo Palacios for helpful comments.
Work reported here was supported by NIH awards AI070411, HL83850, NS047537, and U54AI5758 (Northeast Biodefense Center-Lipkin).