Confirmation of Tick Bite by Detection of Antibody to Ixodes Calreticulin Salivary Protein

I. scapularis infestation of C3H/HeN and BALB/c mice.Pathogen-free I. scapularis ticks were obtained from a colony maintained at the University of Connecticut Health Center. Ticks were maintained at 22°C in 97% relative humidity and oversaturated potassium sulfate and with a 16-h:8-h light-dark cycle. Ticks were placed on female C3H/HeN or BALB/c mice. Larvae (200 to 300 per mouse) or nymphs (10 to 40 per mouse) were applied to the entire body of a mouse. Ticks were then left to feed to completion over 3 to 7 days, and the engorged ticks were collected (4). In some instances, a second infestation was performed after mice were housed for 14 days. Two to three months after the last infestation, mouse blood was collected by retro-orbital bleeding, allowed to clot, and centrifuged at 150 × g for 10 min at 4°C to collect sera prior to storage at −30°C for further use. All animal experiments were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee of the University of Connecticut Health Center.

Human study population.The first study group consisted of residents of Block Island, Rhode Island, who developed Lyme disease, babesiosis, or human granulocytic anaplasmosis (HGA) and enrolled in our tick-borne illness study between 1995 and 2000 as previously described (8). These subjects agreed to a history and physical examination and submission of an acute-phase and a convalescent-phase blood sample. For the purposes of this study, we included only the 10 subjects who reported no tick bite prior to illness and who had enrolled in a biannual serosurvey on Block Island for determination of antibodies to the agents of Lyme disease, babesiosis, and HGA prior to development of tick-borne illness. Thus, we were able to test serum samples for antibody against tick salivary protein before, during, and after development of tick-borne illness in these subjects.

The second study group consisted of 234 Block Island residents who enrolled in our 2004 serosurvey but did not experience symptomatic Lyme disease, babesiosis, or HGA. They were asked to complete a questionnaire that included information on tick bite within the previous year and tick-associated itch, an indication of the intensity of tick exposure, as well as provide a blood sample for antibody to tick salivary protein.

Finally, we enrolled seven subjects from the Mansfield Family Practice in Mansfield, Connecticut, who had experienced an I. scapularis tick bite within the previous 2 days of enrollment during the summer of 2005 and who submitted the tick that bit them for identification and estimate of engorgement level. They were asked to provide a history, undergo a physical examination, provide a blood sample to determine their antibody against tick salivary protein, and to return 4 to 6 weeks later for a clinical examination and for blood testing for antibody against tick salivary protein.

Positive control sera consisted of pooled acute-phase sera from Connecticut residents who had experienced Lyme disease within the previous 3 months and whose sera contained anti-B. burgdorferi antibody. Negative control sera were obtained from two residents of Iceland, where no vector ticks are found, and from three children between 1 to 2 years of age living in Connecticut whose sera were obtained for routine serum electrolyte testing. Sera were extracted immediately after blood drawing and maintained frozen at −80°C until testing. Negative control sera were individually tested in all experiments. Written informed consent was obtained from study participants in accordance with human experimentation guidelines approved by the institutional review boards at Connecticut Children's Medical Center and the Harvard School of Public Health.

Insect cell culture and media for preparation of recombinant I. scapularis calreticulin antigen. Trichoplusia ni cells (BTI-TN-5B1-4, High 5; Invitrogen, Carlsbad, CA) used in protein expression were grown at 27°C in Express 5 serum-free medium (Invitrogen), supplemented with 17 mM l-glutamine, 10 μg/ml gentamicin (GIBCO, Carlsbad, CA), and 10 μg/ml blasticidin (Invitrogen). Cells were started as adherent cultures and then used to inoculate 65-ml to 125-ml spinner flask suspension cultures at an initial density of 1.0 × 10⁶ cells/ml. Once a density of 6 × 10⁶ to 7 × 10⁶ cells/ml was reached, cells were harvested for processing.

RACE synthesis for preparation of recombinant Ixodes calreticulin antigen.Prior to dissection, partially fed female I. scapularis ticks were washed with sterile 0.1 M phosphate-buffered saline (PBS; pH 7.4). Salivary glands were then removed, rinsed into 500 μl of sterile PBS containing Complete EDTA-free protease inhibitor cocktail (Roche, Indianapolis, IN), and transferred immediately into a 2.0-ml cryovial containing 1.0 ml RNALater RNA storage solution (Ambion, Austin, TX). The suspensions of glands were then stored at −80°C until use.

Poly(A)⁺ mRNA was isolated from tick salivary glands with the Oligotex Direct mRNA Micro kit (QIAGEN, Valencia, CA) following the manufacturer's protocol. First-strand cDNA was isolated from tick salivary gland poly(A)⁺ mRNA and used directly in 5′- and 3′-rapid amplification of cDNA ends (RACE) PCR, using the SMART RACE cDNA amplification kit (Clontech, Mountain View, CA) according to the manufacturer's instructions. Calreticulin-specific oligonucleotide primers were designed from the consensus sequence resulting after alignment of published calreticulin sequences from the ticks Amblyomma americanum (U07708) and Boophilus microplus (AF420211). Sequences were as follows: sense primer Calret3, 5′-ACTCGGGCTTGTCCCAGTCCTCGGG-3′; and antisense primer Calret5, 5′-AAGCACGAGCAGAACATCGACTGCG-3′. These primers created overlapping 5′- and 3′-RACE products, respectively, that were joined by restriction digestion and ligation using restriction site XhoI in the region of the overlap to create the full-length cDNA. The resulting 1,548-bp cDNA was then used as template to generate a PCR product containing the entire 5′ end of the gene and the 3′ end up to the stop codon. Oligonucleotide primers used for this purpose were CalretFFL (5′-GGCTTCTAATACGACTCACTATAGGG-3′) and CalretRFL (5′-CACAAGTTCCTCGTGGTCGTGCTTG-3′). The stop codon was eliminated in order to allow fusion of the C-terminal region of the expressed protein to a His₆ tag to facilitate protein purification. The 1,349-bp PCR product was then cloned into the pIB/V5-His-TOPO expression vector (Invitrogen), which contains the blasticidin resistance gene for selection of cells that are stably transfected. Transcription of the calreticulin insert was driven by the baculovirus Orgyia pseudotsugata immediate-early 2 promoter (OpIE2) (28). The resulting construct, pIB/Calreticulin, was then sequenced in both directions using standard dideoxynucleotide sequencing procedures (19). After alignment of published calreticulin protein sequences from various Ixodes tick species (Ixodes woodi, Ixodes ricinus, Ixodes persulcatus, Ixodes pararicinus, Ixodes pacificus, Ixodes pavlovskyi, Ixodes ovatus, Ixodes nipponensis, Ixodes muris, Ixodes minor, Ixodes jellisoni, and Ixodes affinis) with the I. scapularis used in this study, we found that the difference in amino acid sequences between species was negligible, with an average percent identity of approximately 98% among all sequences. These data indicate that antibody response to recombinant Ixodes scapularis calreticulin antigen would be the same as that against any other recombinant Ixodes calreticulin antigen.

Endotoxin-free pIB/Calreticulin recombinant plasmid was purified using the EndoFree plasmid maxi kit (QIAGEN) and transfected into High 5 cells according to protocols supplied by the manufacturer. Stably transfected cells were maintained routinely in medium containing blasticidin at a final concentration of 10 μg/ml. For medium collection, cells were grown to a density of greater than 6 7 × 10⁶ to 7 × 10⁶ cells/ml, at which point media were collected and centrifuged at 6,000 × g to remove cells and particulate matter. Cell culture supernatants were then buffer exchanged with PBS (pH 7.2) and concentrated at least 10× with a 250-ml stirred cell (Millipore, Billerica, MA) fitted with low-protein-binding regenerated cellulose membranes (10-kDa molecular mass cutoff).

For purification by immobilized-metal affinity chromatography, concentrates were loaded onto a Ni⁺²-nitriloacetic acid (QIAGEN) column that was preequilibrated and washed with 50 mM NaH₂PO₄, 500 mM NaCl, and 10 mM imidazole (pH 8.0). His-tagged protein was eluted with 50 mM NaH₂PO₄, 500 mM NaCl, and 250 mM imidazole (pH 8.0). Eluted fractions were dialyzed against PBS and concentrated using Amicon Ultra 15 concentrators (10-kDa molecular mass cutoff; Millipore). Proteins were quantified by means of the bicinchoninic acid assay (27), and purity was assessed by discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (10) and immunoblot analysis (3).

ELISA for detecting antibody against Ixodes recombinant calreticulin antigen.The ELISA is a quantitative microtiter method for detecting immunoglobulin G (IgG) antibody to recombinant Ixodes calreticulin purified from High 5 cells. This ELISA method is a modification of that described by Magnarelli et al. (11). Recombinant calreticulin was added to alternate wells of flat-bottom microdilution plates at a final concentration of 5 μg per well. In the intervening wells, 50 μl of PBS (1.54 mM KH₂PO₄, 2.7 mM Na₂HPO₄ · 7H₂O, 154 mM NaCl, pH 7.2) was added to test for nonspecific binding. The plates were blocked with PBS (200 μl) containing 5% horse serum (JR Scientific, Woodland, CA) and 0.01% dextran sulfate (Sigma) and washed five times with PBS-Tween 20. Sera from selected patients with a previous history of tick exposure (diluted from 1:40 to 1:320) were added to matching wells and incubated for 1 h at 37°C and washed as previously described. After incubation for 1 h with peroxidase-labeled anti-human IgG, plates were washed again as described above, and the reactions were visualized with 2,2′-azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt/tetramethyl benzidine (ABTS/TMB) as a substrate. Optical density readings at 414 nm were taken on a μQuant plate reader (BioTek, Winooski, VT). The optical density of the positive control minus that for the nonspecific binding well was standardized to 1.0 for IgG. A sample was considered as reactive if the net absorbance (antigen well minus the nonspecific binding well) was 3 standard deviations or more than the mean absorbance of the PBS-containing comparison wells. A reactive serum was defined as one reacting at a dilution equal to or greater than 1:80 for IgG.

Western blot assays for detection of antibody against Ixodes recombinant calreticulin antigen.For immunoblot analysis, tick salivary gland extract protein or purified recombinant calreticulin protein was separated, along with molecular mass standards (Bio-Rad, Hercules, CA), by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions using 12% gels (28) and transferred to nitrocellulose membranes using a Mini Trans-Blot electrophoretic transfer cell (Bio-Rad). Human sera were diluted 1:2,000 in blocking buffer (10 mm Tris-HCl, 300 mM NaCl, pH 7.4, 5% nonfat dry milk) and incubated for 1 h at room temperature (11). Antigen binding was detected with horseradish peroxidase-conjugated goat anti-human IgG antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) diluted 1:10,000 in blocking buffer and reacted for 1 h at room temperature. Antigen-antibody complexes were visualized by chemiluminescence (Supersignal; Pierce, Rockford, IL) on radiography film (Kodak, Rochester, NY).

Statistical analysis.Fisher's exact test was used to compare two proportions (2-by-2 contingency tables). SAS 9 for Windows (SAS Institute Inc., Cary, NC) was used for statistical analysis.

Antibody against Ixodes recombinant calreticulin protein using a mouse model.C3H/HeN and BALB/c mice were exposed to deer ticks and tested for antibody against Ixodes recombinant calreticulin. Larval or nymphal I. scapularis ticks were allowed to feed to repletion on mice, and in some instances, the infestation was repeated 2 weeks later. Blood was obtained 2 to 3 months after the last infestation for determination of serum calreticulin antibody by ELISA. Sera from mice exposed to deer ticks contained anticalreticulin antibody at concentrations of 1:80 to 1:160, while sera from tick-naïve mice were nonreactive (Table 1). Mice exposed to deer ticks develop antibody against I. scapularis recombinant calreticulin.

Development of antibody against Ixodes calreticulin protein in people exposed to deer ticks who develop tick-borne infection.We tested the sera of 10 Block Island subjects who had experienced Lyme disease, babesiosis, and/or HGA; reported no tick bite prior to infection; and submitted a serum sample before (as part of the biannual serosurvey), during, and within 3 months after the onset of illness. Seven of the 10 subjects had no detectable I. scapularis calreticulin ELISA antibody prior to development of infection (Table 2). All of these subjects seroconverted during or within 3 months of acute infection. Among the three subjects from whom long-term follow-up sera were available, calreticulin-specific antibody was detectable at 17 months but not at 29 months after infection in one person and present at 3 to 4 months but absent at 15 and 27 months after infection in the other two. Three of the 10 subjects had detectable calreticulin antibody prior to developing infection despite no recollection of a previous tick bite. As residents of Block Island, these subjects were very likely to have had previous tick bites. One of the three experienced a twofold rise in antibody during the interval before and after infection, while the titer did not change in the other two subjects. Positive control sera derived from pooled acute-phase sera of several patients experiencing Lyme disease were reactive against Ixodes recombinant calreticulin antigen, while no such antibodies were detected in the sera of two subjects who lived in Iceland, where no deer ticks are found, and in three children 1 to 2 years of age whose sera were obtained for serum electrolyte testing (Table 2). Antibody against Ixodes calreticulin is detectable by ELISA in people following a deer tick bite(s) but may fall to undetectable levels as early as 15 months following tick exposure.

Confirmation that antibody detected by Ixodes recombinant calreticulin ELISA is directed at calreticulin.We used a Western blot assay with Ixodes recombinant calreticulin as the antigen to test the sera of five of the same Lyme disease subjects who had developed antibody against Ixodes calreticulin as measured by ELISA. In each of five Lyme disease serum pairs, a reactive calreticulin band was absent in sera collected before the development of Lyme disease and present in sera obtained after the development of Lyme disease (Fig. 1). A sample of positive control sera from pooled acute-phase sera of several patients experiencing Lyme disease also was reactive against the Ixodes calreticulin protein band. In contrast, sera obtained from a control subject with no history of tick bites who lived in Iceland failed to react to the Ixodes calreticulin protein band.

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FIG. 1.

Western blots of sera against Ixodes recombinant calreticulin (10 μg/strip) from people before (lanes B) and within 3 months after (lanes A) development of Lyme disease. The ELISA antibody results of these subjects are shown in Table 2. The positive control consists of pooled acute-phase sera from patients experiencing Lyme disease. The negative control consists of serum from a resident of Iceland.

Development of antibody against Ixodes calreticulin protein in people exposed to deer ticks who do not develop tick-borne infection.We determined whether anticalreticulin antibody forms in people who were bitten by I. scapularis ticks but who removed these ticks before transmission of tick-borne infection could develop. Seven subjects who experienced a tick bite within the previous 2 days submitted the I. scapularis tick that bit them and a blood sample. The sera from each of these subjects reacted against Ixodes calreticulin antigen within 1 to 3 days after exposure. Serum samples were obtained from four of these people 4 weeks after exposure, and all were reactive, although only one increased above the previous titer (Table 3). The same positive and negative control sera used in previous experiments (as depicted in Table 2) were reactive and nonreactive, respectively. People who experience a deer tick bite but no tick-borne infection develop antibody against Ixodes calreticulin antigen.

Association between self-reports of tick bite and the presence of anti-Ixodes calreticulin antibody.We tested the sera of 234 residents of Block Island who participated in the 2004 Block Island serosurvey for antibody against Ixodes recombinant calreticulin. We compared their antibody response with a history of tick bite within the previous year, tick bite-associated itch, and the presence of antibody against B. burgdorferi and Babesia microti (Table 4). None had experienced symptomatic tick-borne illness within the previous year, and most provided a complete history and sufficient sera for testing, although some residents did not respond to questions about tick bite or tick-associated itch. Only 6% (n = 14) had detectable antibody against Ixodes calreticulin. A significant association was found between seroreactivity to Ixodes recombinant calreticulin and the presence of antibody against B. burgdorferi and Babesia microti, but not with a history of tick bite or tick-associated itch. The sera from about half of the subjects whose sera contained antibody against B. burgdorferi and Babesia microti were nonreactive against calreticulin antigen, presumably because these subjects had experienced Lyme disease or babesiosis a year or more before their sera were tested for anticalreticulin antibody. The persistence of anticalreticulin antibody generally appears to be less than that for antibody directed against either the Lyme disease or babesial pathogens. The presence of detectable anti-Ixodes calreticulin antibody is associated with previous B. burgdorferi and B. microti infection but not with self-reports of previous tick bite or tick-associated itch.