(Borrelia burgdorferi) in - Journal of Clinical Microbiology - American ...

2 downloads 0 Views 2MB Size Report
RUSSELL T. GREENE,'* RICHARD L. WALKER,2 WILLIAM L. NICHOLSON,2 HANS W. HEIDNER,2. JAY F. LEVINE,2 ELIZABETH C. BURGESS, MICHAEL ...
JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 1988, p. 648-653 0095-1137/88/04648-06$02.00/0 Copyright © 1988, American Society for Microbiology

Vol. 26, No. 4

Immunoblot Analysis of Immunoglobulin G Response to the Lyme Disease Agent (Borrelia burgdorferi) in Experimentally and Naturally Exposed Dogs RUSSELL T. GREENE,'* RICHARD L. WALKER,2 WILLIAM L. NICHOLSON,2 HANS W. HEIDNER,2 JAY F. LEVINE,2 ELIZABETH C. BURGESS, MICHAEL WYAND,4 EDWARD B. BREITSCHWERDT,1 AND HERMAN A. BERKHOFF2 Departments of Companion Animal and Special Species Medicine' and Microbiology, Pathology and Parasitology,2 North Carolina State University School of Veterinary Medicine, Raleigh, North Carolina 27606; Research Animal Resources Center and Department of Medicine, School of Veterinary Medicine, University of Wisconsin-Madison, Madison, Wisconsin 537063; and New England Regional Primate Research Center, Southborough, Massachusetts 017724 Received 17 August 1987/Accepted 8 January 1988

Immunoblots were used to study the immunoglobulin G response to Borrelia burgdorferi in experimentally and naturally exposed dogs. Adsorption studies confirmed that the antibodies were specific for B. burgdorferi. Experimentally exposed dogs were asymptomatic. Naturally exposed dogs included both asymptomatic animals and animals showing signs compatible with Lyme disease. Naturally exposed dogs were from four geographic regions of the country. No differences were detected between immunoblot patterns of naturally exposed symptomatic or asymptomatic dogs from different areas of the country. The immunoblot patterns obtained with sera from experimentally exposed dogs were different from those obtained with sera from naturally exposed dogs and were characterized by reactivity toïfewer and different protein bands. Immunoblot analysis using an OspA-protein-producing Escherichia coli recombinant showed that experimentally exposed dogs produced antibodies to OspA, whereas naturally exposed dogs did not. Modifications of the immune response over time, different routes of antigen presentation, and strain variation are factors postulated to account for thé observed differences.

Lyme disease is a tick-borne, spirochetal disease with clinical manifestations in humans including dermatitis, carditis, neuritis, and arthritis (17). Initial reports of Lyme disease in dogs described arthritis as the only clinical manifestation (10, 12, 13), but a more recent report described skin lesions, lameness, vomiting, and abortion (8). Since the causative organism, Borrelia burgdorferi, has been difficult to isolate from affected human and canine patients, serology has been widely used as a diagnostic aid (6, 8, 10, 12, 13, 17). The exact nature of the canine humoral response is poorly defined. An elevated serologic titer in the presence of compatible clinical signs has been used to diagnose Lyme borreliosis (8, 10, 13). However, previous reports have shown that high antibody titers may develop in asymptomatic dogs (10, 13). In addition, B. burgdorferi has been isolated from the blood of some dogs with low antibody titers (6, 12). These findings emphasize the difficulties associated with making a definitive diagnosis of canine borreliosis. In humans, it takes approximately 1 to 1.5 months after exposure for specific immunoglobulin G (IgG) titers to become detectable (7, 16). These titers slowly increase during the progression of the disease and often peak months to years after clinical remission. Immunoblots have been used to demonstrate expansion of the IgG response, involving up to 11 antigens late in the disease (1, 7). In-depth knowledge of the canine serologic response is lacking. In experimental studies, IgG titers increased within 21 days and remained elevated for up to 10 months after dogs were infected with B. burgdorferi (5; R. T. Greene, J. F. Levine, E. B. Breitschwerdt, R. L. Walker, H. A. Berkhoff, J. Cullen, and W. L. Nicholson, Am. J. Vet. Res., in press). *

In natural infections it is difficult to determine the time of exposure, the interval before a specific IgG response becomes detectable, and the total length of time that the IgG response remains elevated. It is not known whether the results obtained after experimental exposure can be extrapolated to natural exposures since experimentally exposed

dogs have failed to demonstrate clinical signs. Accordingly, immunoblots were performed on canine sera positive for antibodies to B. burgdorferi by enzyme-linked immunosorbent assay (ELISA) and indirect immunofluorescence assay (IFA). Naturally exposed dogs were both asymptomatic and symptomatic, whereas experimentally exposed dogs were asymptomatic. Serum samples from naturally exposed dogs were collected in four geographical regions of the country. The goals of this study were (i) to determine whether clinical signs could be related to differences in immunoblot patterns, (ii) to determine whether immunoblot pattern differences existed among dogs from different geographical regions, and (iii) to evaluate whether immunoblot pattern differences existed between naturally and experimentally exposed dogs. (This report represents a portion of a thesis submitted by R. T. Greene as partial fulfillment of the requirements for the Ph.D. degree from North Carolina State University, Raleigh.) MATERIALS AND METHODS

Experimentally infected animals. Eight laboratory-reared, male -Beagles were experimentally exposed to B. burgdorferi. The experimental design and clinical and serological results have been described elsewhere (Greene, Levine, Breitschwerdt, Walker, Berkhoff, Cullen, and Nicholson, in press). Briefly, infected nymphal ticks were allowed to feed

Corresponding author. 648

VOL. 26, 1988

on the skin of one group of four dogs. After 56 days, when none of these dogs seroconverted, they were inoculated intravenously with 3 x 108 spirochetes. The isolate of B. burgdorferi was obtained by dissection of a nymphal Ixodes dammini tick. The second group of four dogs initially received a subcutaneous inoculation of 500 organisms. After 56 days, when only two of these dogs had seroconverted, they were inoculated intraperitoneally with 3 x 108 spirochetes. The sera used in this study were obtained from these dogs 15 to 30 days after the second exposure. The IFA titers were .1:512, and the ELISA values (a ratio that relates the absorbance of the test serum with that of positive and negative control sera, such that the ELISA values for previously unexposed dogs are often less than 10) were .50.0 (Greene, Levine, Breitschwerdt, Walker, Berkhoff, Cullen, and Nicholson, in press). These dogs remained asymptomatic for Lyme disease for the duration of the study. Naturally exposed animals. Positive serum samples were obtained from symptomatic dogs in endemic areas. The animals were presented to their local veterinarians with the primary complaint of lameness. The diagnosis of Lyme disease was made based on clinical signs of fever and lameness or joint pain and a positive serologic response. The endemic areas included Connecticut (12 dogs), Wisconsin (7 dogs), and Maryland (10 dogs) (14). Sera from these dogs had IFA titers .1:512 and ELISA values -50.0. Positive serum samples from dogs without clinical signs were obtained when serologic surveys were performed on dogs from North Carolina (four dogs), Maryland (six dogs), and Wisconsin (three dogs). All sera from these dogs had IFA titers -1:512 and ELISA values -50.0. Bacterial strains. The bacterial strain used as an antigen source for the immunoblots was B. burgdorferi B31 (ATCC 35210; American Type Culture Collection, Rockville, Md.). In the adsorption studies, Borrelia anserina (provided by H. J. Barnes, North Carolina State University, Raleigh), Leptospira interrogans serovar canicola (provided by C. Willis, Rollins Animal Disease Diagnostic Laboratory, Raleigh, N.C.), and Escherichia coli ATCC 35218 were used. To evaluate the reactivity of sera against the major outer surface protein (OspA), E. coli DHSa (Bethesda Research Laboratories, Inc., Gaithersburg, Md.) containing plasmid pTRH44 [DH5at(pTRH44)] which codes for the production of OspA was used (9). E. coli DH5a without pTRH44 served as a negative control (both strains were provided by A. G. Barbour, University of Texas Health Science Center, San

Antonio). Serologic assays. The IFA and ELISA procedures have been described elsewhere (R. T. Greene, J. F. Levine, E. B. Breitschwerdt, and H. A. Berkhoff, Am. J. Vet. Res., in press; Greene, Levine, Breitschwerdt, Walker, Berkhoff, Cullen, and Nicholson, in press). Monoclonal antibodies. Genus (H9724)- and species (H5332)-specific monoclonal antibodies (MAbs) (provided by A. G. Barbour) and a MAb directed against an outer surface protein (HT5S) (provided by T. G. Schwan, Rocky Mountain Laboratories, Hamilton, Mont.) were used to locate major proteins in the immunoblots. H9724 recognizes a 41-kilodalton (kDa) flagellar protein, H5332 is directed against the 31-kDa OspA protein, and HT5S recognizes the 34-kDa major outer surface protein (OspB) (2, 3, 4). Electrophoresis and immunoblotting. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed on whole-cell lysates of B. burgdorferi by a modification of methods described by Laemmli (11). For a whole-cell lysate, 2-week-old cultures of B. burgdorferi were centrifuged at

IgG RESPONSE TO B. BURGDORFERI IN DOGS

649

10,000 x g for 30 min at 4°C and washed three times with phosphate-buffered saline (pH 7.38) (Greene, Levine, Breitschwerdt, and Berkhoff, in press; Greene, Levine, Breitschwerdt, Walker, Berkhoff, Cullen, and Nicholson, in press). The pellet was resuspended in enough sterile phosphate-buffered saline that 100 ,ul of a 1:4 dilution of the suspension had an A410 of 0.165 in a micro-ELISA reader (Dynatech Laboratories, Inc., Alexandria, Va.). Samples (30 ,ul) were diluted in an equal volume of sample buffer containing 60 mM Tris hydrochloride (pH 6.8) (Bio-Rad Laboratories, Richmond, Calif.), 2% sodium dodecyl sulfate (Bio-Rad), 5% 2-p-mercaptoethanol, 10% glycerol, and 0.00125% (wt/vol) bromophenol blue. This mixture was heated to 95°C for 5 min. The lysates were electrophoresed in a discontinuous sodium dodecyl sulfate-polyacrylamide gel, using a 4% acrylamide stacking gel and a 12% acrylamide resolving gel. The molecular masses of the proteins were determined by coelectrophoresis of known standards (Bio-Rad). Electrophoresis was performed at a constant current of 35 mA for 3 to 4 h. Proteins were electrophoretically transferred from the unstained gel to a nitrocellulose membrane (18). Briefly, transfer to nitrocellulose membranes was effected electrophoretically in Tris (25 mM)-glycine (192 mM) buffer (pH 8.3) at 30 V (100 mA) overnight in a Transblot cell (Bio-Rad). After transfer was complete, 4- to 5-mm strips of the nitrocellulose membrane were cut and placed into capped tubes (16 by 150 mm; Fisher Scientific Co., Raleigh, N.C.). The strips were then blocked for 1 h at room temperature (ca. 25°C) by using 3 ml of 20 mM Tris-buffered saline (TBS; pH 7.5) with 3% (wt/vol) gelatin, with constant rocking. The strips were washed three times for 5 min per wash with 0.05% TweenTBS. The strips were incubated for 1 h with constant rocking at 25°C with 2 ml of the test serum diluted 1:50 in 1% gelatin-TBS (antibody buffer). The wash procedure was repeated, and the strips were incubated for 1 h at room temperature with 2 ml of a 1:500 dilution of horseradish peroxidase-conjugated goat anti-dog IgG (heavy chain specific) (Kirkegaard & Perry Laboratories, Gaithersburg, Md.) in antibody buffer. The strips were washed twice with Tween-TBS and then once with TBS. The nitrocellulose strips were developed by the addition of a freshly prepared solution of 0.05% (wt/vol) 4-chloro-1-naphthol (Bio-Rad), 0.015% hydrogen peroxide, and 20% methanol in TBS. The reaction was stopped by washing the strips with cold distilled water. The top and bottom edges of the strips were used for alignment. When immunoblots were performed with MAbs, 2 ml of a 1:50 dilution of the MAb was incubated similarly to the test serum and 2 ml of a 1:3,000 dilution of horseradish peroxidase-conjugated goat anti-mouse IgG (heavy and light chain specific) (Bio-Rad) was used as a second antibody. Adsorption. For the adsorption procedures, whole-cell suspensions of B. burgdorferi, B. anserina, L. interrogans serovar canicola, and E. coli were prepared as described above for electrophoresis. By using the micro-ELISA reader, suspensions were prepared such that 100 pul of 1:4 dilutions of the suspensions had an A410 of 0.222. To demonstrate the specificity of the reactions, serum from one dog from each group was adsorbed with the B. burgdorferi suspension, and one serum sample was adsorbed with all three organisms. A 0.5-ml portion of the bacterial suspension to be used for adsorption was centrifuged at 8,200 x g for 10 min at 25°C. The supernatant was removed, and 10 pul of the test serum diluted with 0.5 ml of antibody buffer was used to resuspend the organisms. This suspension

650

GREENE ET AL.

was incubated for 30 min at 37°C. The suspension was then centrifuged at 8,200 x g for 10 min, and the supernatant was used to suspend the new pellet of the same organism. A total of three adsorptions with each organism were performed. After the adsorption steps, immunoblotting was performed with the final supernatant, as described above. The 0.5 ml of diluted, adsorbed serum was added to 1.5 ml of antibody buffer to make a total of 2 ml of a 1:50 dilution of the serum sample, as described above for the immunoblot procedure. As a control, the test serum was processed by the adsorption procedure with the adsorption antigen excluded. Use of recombinants to evaluate OspA reactivity. Recommendations for bacterial growth medium and procedures for preparation of recombinant cells were made by A. G. Barbour (personal communication). The E. coli recombinant DH5a(pTRH44) was grown overnight at 37°C in 10 ml of broth containing 1% Casamino Acids (Difco Laboratories, Detroit, Mich.), 0.5% yeast extract (Difco), 52 mM NaCl, 26 mM KCl, 20 mM HEPES (N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid) (Sigma Chemical Co., St Louis, Mo.), 10 mM MgCl2, and 50 ,ug of sodium ampicillin per ml. The broth inoculated with the control strain E. coli DHSa did not contain ampicillin. After overnight growth, 1.5-ml aliquots were centrifuged at 8,200 x g for 10 min at 25°C. The pellets were suspended in 1 ml of phosphate-buffered saline. The washed cells were then centrifuged as described above, and the supernatant was discarded. The pellets were stored at -100°C for 15 min and then suspended in 200 pl of 2 x electrophoresis sample buffer. The sample was boiled for 5 min, allowed to cool, and then centrifuged at 8,200 x g for 10 min at 25°C. The supernatant was loaded into 4-cm-wide wells for electrophoresis. Electrophoresis and transfer to nitrocellulose was performed as described above. Before immunoblotting, the positive sera from experimentally and naturally exposed dogs were adsorbed with E. coli DH5a. The concentration of the adsorption antigen and the procedure for the adsorption are described above. RESULTS When sera from naturally exposed dogs were adsorbed with B. burgdorferi, reactivity to most protein bands was eliminated (Fig. 1). At areas corresponding to some bands which reacted strongly with unadsorbed sera, faint reactivity could be seen after adsorption. When B. anserina, L. interrogans serovar canicola, and E. coli were used, there was little to no reduction in the reactivity to any bands (Fig. 1). The immunoblots obtained with sera from naturally exposed dogs from the different areas of the country were similar (Fig. 2 and Table 1). Most sera recognized at least 15 protein bands (Fig. 2). There were approximately 10 polypeptide bands that sera from most dogs recognized strongly (major bands), and an additional 7 to 10 bands showed weak reactivity (minor bands). There were some major variations among dogs, but for a geographical area, all protein bands were adequately represented (Table 1). The major bands recognized by sera from most dogs were approximately 83, 66, 61 to 60, 41 to 39, 31 to 29, 17, and 15 kDa. The minor bands corresponded to 90, 75, 50, 48, 34, 27, 24, 22, and 19 kDa. There were no apparent differences in serum reactivity between naturally exposed dogs that were exhibiting clinical signs and those that were not (Fig. 2 and 3 and Table 1). All of the sera from the experimentally exposed dogs reacted similarly. In contrast to the naturally exposed dogs, the experimentally exposed dogs had different immunoblot pat-

j~ 4-5.

J. CLIN. MICROBIOL.

terns (Fig. 2 and 3). The sera from the experimentally exposed dogs reacted against a limited number of antigens, often less than 6, whereas the sera from the naturally exposed dogs often reacted with at least 15 protein bands. One major antigen recognized by the sera from the experimentally exposed dogs was a broad band in the 31-kDa region (Fig. 2 and 3). The species-specific MAb reacted to this protein in a similar manner (Fig. 3). The sera from naturally exposed dogs reacted against a narrower band located near the bottom of this region (Fig. 2 and 3). In addition, sera from the experimentally exposed dogs consistently reacted against a 34-kDa band which was only occasionally recognized by the sera from naturally exposed dogs. The reactivity of MAb H5332 in the immunoblot obtained with the recombinant DH5a(pTRH44) demonstrated the OspA band at approximately 31 kDa (Fig. 4). No reactivity was seen when H5332 was reacted with DH5a. A thin band in the OspA region was present when sera from experimentally exposed dogs were reacted with DH5a(pTRH44). No corresponding band was seen when the same sera were reacted with DH5a. When serum from a naturally exposed dog was reacted with DH5a(pTRH44), no band was seen in the OspA region (Fig. 4). There was a dark band at approximately 66 kDa in the immunoblot with DH5a. A corresponding band, but much lighter, was seen when the serum was reacted with DH5a(pTRH44). DISCUSSION Diagnosis of canine borreliosis is often based on positive serologic response in conjunction with compatible clinical 1

2

5 Mws

3

4

_

" _--92.5

--66.2

X = x::

_

-1.

~ ~

Ifs -.`A:

4

:. c;[

*: w.

Of

21.5

14.5

FIG. 1. Immunoblot analysis of whole-cell suspensions of B. burgdorferi after adsorption of sera with organisms. Lanes: 1, no adsorption; 2, adsorbed with B. burgdorferi; 3, adsorbed with B. anserina; 4, adsorbed with L. interrogans serovar canicola; 5, adsorbed with E. coli. MWS, Molecular weight standards (molecular weights in thousands).