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Journal of Veterinary Diagnostic Investigation Vol. 18 Issue 6, 583-586
Copyright © 2006 by the American Association of Veterinary Laboratory Diagnosticians
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Brief Communication

Comparison of direct fluorescent antibody staining and real-time polymerase chain reaction for the detection of Borrelia burgdorferi in Ixodes scapularis ticks

Gwenn Gaumond, Allison Tyropolis, Sarah Grodzicki and Sandra Bushmich1

Correspondence: 1Corresponding Author: Sandra Bushmich, Department of Pathobiology and Veterinary Science, College of Agriculture and Natural Resources, University of Connecticut, 61 North Eagleville Rd, U-3089, Storrs, CT 06269


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Borrelia burgdorferi, the agent responsible for causing Lyme disease in humans and animals, is transmitted via the bite of infected Ixodes spp. ticks. Ticks removed from humans and animals are routinely tested by diagnostic laboratories to determine if they are infected with these bacteria. The objective of this study was to compare the efficacy of 2 commonly used methods, direct fluorescent antibody staining and real-time polymerase chain reaction (PCR), for the detection of B. burgdorferi in Ixodes scapularis ticks. One hundred and twenty-seven adult I. scapularis ticks collected in Connecticut, a Lyme disease endemic area, were tested, and results were compared. Results showed 24.8% ticks tested positive for Borrelia spp. by fluorescent antibody testing and 32.5% ticks were positive for B. burgdorferi by real-time PCR testing. When ticks were grouped into categories by level of engorgement (unengorged, partially engorged, and fully engorged), 95% of unengorged ticks, 90.5% of partially engorged, and 86.8% of engorged ticks tested were in agreement. Ten of the 127 ticks examined were too dehydrated to be tested by the fluorescent antibody technique; half of these tested positive by PCR. Real-time PCR appears to be the better of these 2 methods for the diagnosis of this bacterial infection in I. scapularis ticks.

Key Words: Borrelia burgdorferi • fluorescent antibody staining • Ixodes scapularis • PCR

Lyme disease was first described in 1975 in 3 Connecticut towns when 39 children and 12 adults developed recurrent asymmetric swelling and pain in a few large joints, most often the knee.21 Over the past 30 years, the incidence of this disease has increased. Today, Lyme disease is the most common arthropod borne disease in the USA, affecting over 170,000 people.17 Lyme disease has also been reported in Europe,12 Asia,1 and Canada.4 This disease affects both humans and animals. Lyme disease causes a wide range of symptoms in humans, including an annular rash (erythema migrans),2 arthritic and flu-like symptoms,6 and neurologic symptoms, including facial nerve paralysis.13 Affected animals, including dogs, horses, and cattle, most often present with a shifting large joint lameness, sometimes associated with fever, and a change in behavior.11 The spirochete Borrelia burgdorferi is the causative agent of this disease.20 This bacterium is transmitted by the tick vector Ixodes scapularis, also known as the black-legged tick or the deer tick.19 Testing of I. scapularis ticks for B. burgdorferi is often requested of diagnostic laboratories in Lyme disease endemic areas. Confirmation of a B. burgdorferi positive tick bite can speed diagnosis of the disease; if symptoms/clinical signs develop, treatment can be initiated quickly, improving the therapeutic response.7 There are several methods for detection of B. burgdorferi in ticks. Some of these methods include dark-field microscopy, direct and indirect fluorescent antibody (FA) testing, traditional polymerase chain reaction (PCR), and real-time PCR. Previous studies compared FA testing and traditional PCR.9,14 These studies used unfed laboratory-raised or field-collected I. scapularis ticks of unknown blood engorgement status. The purpose of this study was to compare the detection rates of B. burgdorferi in adult I. scapularis ticks by real-time PCR and direct FA tests. The ticks used in this experiment were of varying degrees of blood engorgement, most of which were collected from horses, mimicking the engorgement status of ticks received for testing by diagnostic laboratories.

One hundred and twenty-seven adult I. scapularis ticks, 20 unengorged (kindly provided by K. Stafford III, Connecticut Agricultural Experiment Station, New Haven, CT) and 107 engorged or partially engorged (collected from horses by study participants) from Lyme disease endemic regions of Connecticut, were used for this study. The same ticks were used for both tests. Each tick was first tested by FA, then frozen and tested by real-time PCR by using recA primers,15 at a later date.

Fluorescent antibody staining was accomplished by mixing a small amount of tick midgut fluid with 2–3 drops of 1x phosphate buffered saline solution (PBS) on a glass slide, air-drying, fixing in acetone for 10 minutes, then air-drying again. Three to 5 drops of fluorescein-labeled goat anti-Borrelia spp. antibody,a diluted 1:30, were added to the slide, and incubated in a moisturized chamber at 37°C for 30 minutes. Slides were then soaked in 1x PBS for 5 minutes, rinsed with distilled water, and air-dried. Slides were examined by using a microscopeb with a reflected light fluorescence attachmentc powered by a mercury lamp,d at a wavelength of 490 nm, for the presence of fluorescing spirochetes.

Real-time PCR was conducted by using a commercially available thermal cycler.e Published recA primers15 specific for B. burgdorferi sensu stricto were used to generate a product 222 bp in length. The published primers did not react with any other Borrelia spp., nor did they react with Bordetella pertussis; Bordetella parapertussis; 3 different Pneumococcus strains; Beta-hemolytic Streptococcus C, G; and A, Salmonella typhimurium; Salmonella enteritidis; Anaplasma phagocytophilum; or Mycobacterium tuberculosis (Johanna Makinen, personal communication). Tick DNA was extracted by using a commercial kit.f The manufacturer's protocol was followed, except that samples were heated to 95°C for 15 minutes immediately after the overnight digestion at 56°C. The recA amplification was carried out in 20-µl reactions that contained 20 pmol of each primer,g 2x Sybr Green,h and appropriate amounts of distilled water. The extracted tick DNA was diluted 1:10 with distilled water. Samples were amplified and analyzed on channel 1 of the real-time PCR machine by using the following conditions:18 an initial incubation of 15 minutes at 95°C, followed by 55 cycles of 15 seconds at 94°C, 30 seconds at 59°C, 11 seconds at 72°C, and 5 seconds at 77°C. Melting curve profiles were generated for each amplified product by using the real-time PCR machine over a temperature range of 55°C to 94°C at 0.1°C per second. By using samples of PBS spiked with known numbers of B. burgdorferi, the sensitivity of this protocol was determined to be reliably 25 organisms/ml, which is consistent with findings of a previous study.5

One hundred and twenty-seven adult I. scapularis ticks were collected for this study. Ten of these ticks were either crushed or dried out, preventing a reliable FA, leaving 117 ticks (20 unengorged, 21 partially engorged, and 76 engorged) available for both FA and PCR analysis. Data were analyzed according to the method used by Luster et. al 10 by using a 2 x 2 contingency table. Concordance was defined as the probability of correctly determining the presence or the absence of B. burgdorferi in the I. scapularis tick by using the formula (a + d)/ (a + b + c + d) x 100% (Table 1).


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  		Table 1 Concordance value table template.

 
Results showed 29/117 (24.8%) ticks positive for Borrelia spp. by FA testing and 38/117 (32.5%) ticks positive for B. burgdorferi by real-time PCR testing (Table 2). The overall concordance value for the experiment was 88.9%. Of the 13/117 test results that differed between the 2 techniques (Table 2), 11/13 (84.6%) were FA negative and PCR positive. Two of the 13 samples (15.4%) with discordant results were FA positive and PCR negative. One of these samples had only 1 fluorescing organism noted on the FA test. Results were further divided into tick engorgement categories: unengorged, partially engorged, and engorged (Fig. 1). There were 10/20 unengorged, 7/21 partially engorged, and 12/76 engorged ticks positive by FA. Eleven of 20 unengorged, 9/21 partially engorged, and 18/76 engorged ticks tested positive by real-time PCR. This resulted in concordance values of 95%, 90.5%, and 86.8% in unengorged, partially engorged, and engorged ticks, respectively (Table 2). Ticks positive by either test were taken as a separate group, and test agreements were compared. Ten of 11 (90.9%) positive, unengorged ticks, 7/9 (78%) positive, partially engorged ticks, and 10/20 (50%) positive, engorged ticks were in agreement (Fig. 2).


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  		Table 2 Test results.

 

Figure 18061001
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  		Figure 1 Template for concordance value tables.

 

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  		Figure 2 Comparison of FA and PCR techniques for the detection of Borrelia burgdorferi, in Ixodes scapularis ticks at various stages of engorgement.

 
The fluorescein-labeled goat anti-Borrelia spp. antibodya used in this study is specific for B. burgdorferi, Borrelia hermsii, Borrelia anserina, and Borrelia coriaceae, but has not been tested for cross-reactivity to treponemes. The tick vectors of B. hermsii and B. coriaceae are the Ornithodoros spp. ticks, O. hermsi, and O. coriaceus, respectively.8,16 The tick vector for B. anserina is an Argasidae spp. tick.3 Because these species of Borrelia spp. are not usually found in the I. scapularis tick, their presence in the I. scapularis ticks used in this experiment is highly unlikely. In addition, B. hermsii and B. coriacae are generally found in the western USA,8,16 not in Connecticut, where the ticks for this study were collected. The recA primers used in this study were previously shown to be specific to B. burgdorferi.15 Based on these data, the results of both the FA and the PCR tests in this study should be specific for B. burgdorferi, and false positives because of cross reactivity are unlikely.

Culture is used as the "gold standard" in many studies to demonstrate the efficacy of a new diagnostic technique for B. burgdorferi. However, in this study, it was decided that the main objective of the study was to compare the results of the 2 previously mentioned diagnostic tests with each other. The same tick was used for both tests. Splitting each tick into thirds, instead of halves, may have further complicated the results, because less of the tick would be used for each test, which may have posed a problem, especially in ticks infected with low numbers of B. burgdorferi.

Results showed strong agreement between FA and real-time PCR testing. Most (11/13) results that differed between tests were PCR positive and FA negative. Polymerase chain reaction appears to be more sensitive than the FA technique used. The increased sensitivity seemed most obvious when testing engorged ticks. FA may be less reliable in engorged ticks because of the increased content of the midgut; blood in the midgut may dilute the number of B. burgdorferi present and hinder the ability to visualize the organisms microscopically.

Two of the 13 discordant results were FA positive and PCR negative. It is conceivable that these ticks were infected with low numbers of B. burgdorferi, and, in the process of midgut dissection for the FA test, most of the B. burgdorferi were expelled onto the microscope slide, leaving few in the remaining tick body, which was subsequently used for DNA extraction. By operating under the assumption that real-time PCR should be the more sensitive test, all samples (4) that initially tested FA positive, but PCR negative, were retested by using real-time PCR. The justification for reamplifing these samples was based on data published in a previous study, where only 60%–70% of tick samples spiked with 12.5 B. burgdorferi per milliliter tested positive for B. burgdorferi.5 The thought being that these samples may have been just at or below the detection limit of 25 B. burgdorferi per milliliter. Fluorescent antibody testing could not be repeated for any samples, because the ticks were frozen and subsequently desiccated. For these 4 samples, the extracted DNA was reamplified, resulting in 2 positive samples; 1 sample was very weakly positive between 50 and 55 cycles, and one was a weak positive after 43 cycles, reflecting probable low numbers of B. burgdorferi in the partially dissected tick. It is possible that reamplifing all extracted samples that tested negative (not just the FA positive, PCR negative) may have resulted in more PCR-positive samples, samples with very low numbers of B. burgdorferi, very close to the detection limit of this test. It is likely that more accurate real-time PCR results will occur in situations when only real-time PCR is performed on each tick, and the whole tick can be used for DNA extraction, instead of a partially dissected one, as was necessary in this study.

When the ticks that tested positive were examined separately as a group, the trends seen in the overall study became more obvious. Agreement between the 2 tests decreases as the ticks fill with blood. FA testing may be nearly as accurate as real-time PCR for testing unengorged ticks, but it is clearly the inferior test when partially engorged, engorged, or desiccated ticks are tested for B. burgdorferi.

Polymerase chain reaction offers the additional advantage of the ability to test desiccated ticks (not possible with this FA technique). Ten of the original 127 ticks had to be excluded from the study because of desiccation. Five of these ticks were positive for B. burgdorferi by real-time PCR. These positives would have been missed if FA was used solely to test ticks for the presence of B. burgdorferi. Ixodes scapularis ticks submitted to the Connecticut Veterinary Medical Diagnostic Laboratory for B. burgdorferi testing are sometimes received in a desiccated state, preventing testing by fluorescent antibody staining. The results of this study show that real-time PCR is a better test than fluorescent antibody staining for the determination of B. burgdorferi presence in I. scapularis ticks, especially when tick condition and level of engorgement are variable.


   Acknowledgments
 
The authors thank Kirby Stafford III, chief scientist at the Connecticut Agricultural Experiment Station, New Haven, CT, and Lisa Dinsmore, barn manager, at the 1st Company Governor's Horse Guard, Avon, CT, for providing ticks used in this experiment. We would also like to thank Marilyn Jezek at the Connecticut Veterinary Medical Diagnostic Laboratory, University of Connecticut, for assistance with the fluorescent antibody staining technique. In addition, we would like to thank the many students and staff members at the University of Connecticut Department of Animal Science Horse Unit, Storrs, CT, for their help with this study.


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From the Department of Pathobiology and Veterinary Science, University of Connecticut (full address same as corresponding author). Back

a. Kirkegaard and Perry Laboratories, Gathersburg, MD. Back

b. BH-2, Olympus, Tokyo, Japan. Back

c. BH-RFL-W, Olympus, Tokyo, Japan. Back

d. Mercury-100, Chiu Technical Corp., Glen Cove, NY. Back

e. LightCycler 2.0, Roche Diagnostics Corp., Indianapolis, IN. Back

f. NucleoSpin Tissue Kit, BD Biosciences, Palo Alto, CA. Back

g. Fisher Oligos, Pittsburgh, PA. Back

h. Quantitect SYBR Green PCR Kit, Qiagen, Valencia, CA. Back


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