Real-Time PCR for Simultaneous Detection and Quantification of Borrelia burgdorferi in Field-Collected Ixodes scapularis Ticks from the Northeastern United States

Abstract

The density of spirochetes in field-collected or experimentally infected ticks is estimated mainly by assays based on microscopy. In this study, A high degree of spirochete aggregation among infected ticks (variance-to-mean ratio of 24,877; moment estimate of k ‫؍‬ 0.279) was observed. From the frequency distribution data and previously published transmission studies, we estimated that a minimum of 300 organisms may be required in a host-seeking nymphal tick to be able to transmit infection to mice while feeding on mice. These data indicate that real-time qPCR is a reliable approach for simultaneous detection and quantification of B. burgdorferi infection in field-collected ticks and can be used for ecological and epidemiological surveillance of Lyme disease spirochetes.Hard ticks (Ixodidae) in the Ixodes persulcatus complex, such as Ixodes scapularis and Ixodes pacificus in North America, Ixodes ricinus in Europe, and Ixodes persulcatus in far eastern Russia and Asia, are the principal vectors of the Lyme disease spirochete, Borrelia burgdorferi, and several other tick-borne pathogens (2, 25). The incidence of human Lyme disease in areas where it is endemic is correlated with the abundance and prevalence of Ixodes ticks infected with B. burgdorferi (34). Moreover, studies of laboratory animals have suggested that the efficiency of host infection is determined in part by the number of spirochetes inoculated using a needle or deposited by the tick at the time of feeding (21, 27). Thus, monitoring B. burgdorferi density in host-seeking ticks will provide more accurate data for assessment of population risk of Lyme disease and the potential variability of disease manifestations after tick bites.Currently, the number of spirochetes in field-collected or experimentally infected Ixodes ticks is estimated mainly by microscopy-based assays in which the spirochetes are counted directly on tick midgut smears stained with fluorescently labeled specific antibodies (8, 9, 27) or on silver-stained histological sections of ticks (11,14). For specific detection of B. burgdorferi in field-collected adult I. scapularis ticks and monitoring of the spirochete kinetics during the tick life cycle, an OspA antigen-capture enzyme-linked immunosorbent assay (ELISA) was also developed (3-5). These methods are laborintensive, which limits the number of ticks that can be analyzed. Moreover, the sensitivities of these methods are relatively low, e.g., a lower detection limit of approximately 150 spirochetes was reported for the antigen-capture ELISA (5).Real-time PCR has features that allow for rapid detection of infectious pathogens in environmental or experimentally infected animal tissues and clinical specimens (38). The technique has recently been employed to detect the presence of B. burgdorferi DNA in I. ricinus ticks from Switzerland (15), to quantify the spirochete loads in experimentally infected animal tissues (23,24,35), and to differentiate the three B. burgdorferi sensu lato species that are pathogenic to humans in Europe (22,2...

Prevalence and Diversity ofBorreliaSpecies in Ticks That Have Bitten Humans in Sweden

“…1), which agrees with the range and medians found in field-collected nymph and adult ticks in the northeastern United States (34). We also observed a significantly higher number of Borrelia cells in adults than in nymphs.…”

Overexpression of CsrA (BB0184) Alters the Morphology and Antigen Profiles ofBorrelia burgdorferi

“…The RNA was treated twice at 37°C for 45 min with DNase I to remove any contaminating DNA, and the total RNA was quantified spectrophotometrically. In order to evaluate the purity of the RNA sample, real-time PCR was done using recA primers (recAFq and recARq) to detect contaminating DNA (55,80). The RNA samples devoid of contaminating DNA were reverse transcribed to cDNA using TaqMan reverse transcription reagents (Applied Biosystems, Foster City, CA).…”

“…Real-time PCRs were set up with SYBR green PCR master mix with various oligonucleotide primers (Table 3) specific to ospC, ospA, dbpA, p66, BBA64, flaB, and BBK32 at a final concentration of 100 nM, and quantitative real-time PCR was done using the ABI Prism 7300 system (Applied Biosystems). The induction of each gene in ES25 relative to that in MSK5 or ML23/pBSV2 was normalized to the levels of recA as previously described (55,80). The threshold cycle (C T ) values of each of the genes from three independent experiments were averaged following normalization, and the levels of induction were determined with the ⌬⌬C T method, where the quantity of each transcript was determined by the equation 2 Ϫ⌬⌬CT , where C T is the cycle number of the detection threshold, as described previously (42,73).…”

Diagnosis of Lyme Borreliosis

“…A real-time PCR assay was first used for quantitation of B. burgdorferi DNA in tissues from experimentally infected laboratory mice (208,242). Subsequently, it has been employed to analyze the number of spirochetes in field mice (33,349), dogs (330), field-collected or laboratory-infected tick vectors (103,260,347), and clinical specimens of patients with LB (177, 296; reviewed in reference 346). Also, real-time PCR assays have been utilized to genotype the pathogenic B. burgdorferi sensu lato species in both ticks and EM patients in Europe (207,261,276).…”