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Detection of antibodies to Borrelia burgdorferi in naturally infected horses in the USA by enzyme-linked immunosorbent assay using whole-cell and recombinant antigens
Research in Veterinary Science 79 (2005) 99–103 www.elsevier.com/locate/rvsc
Detection of antibodies to Borrelia burgdorferi in naturally infected horses in the USA by enzyme-linked immunosorbent assay using whole-cell and recombinant antigens L. Magnarelli b
a,*
, E. Fikrig
b
a The Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, CT 06504, USA Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
Accepted 24 November 2004
Abstract Blood samples were collected from 98 horses suspected of having borreliosis or granulocytic ehrlichiosis in Connecticut and New York State, USA during 1985, 1995, and 1996. Serum antibodies to Borrelia burgdorferi were detected by an enzyme-linked immunosorbent assay (ELISA), based on whole-cell and recombinant antigens, in 82 (84%) horses. Of the 181 sera tested, 59% were positive, using whole-cell antigens, compared to 48% with protein (p)37 and 35% with VlsE antigens. An ELISA containing either of these fusion proteins can be used as an adjunct to general screening by an ELISA or immunoblotting in animals not vaccinated for this disease. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Borrelia burgdorferi; Antibodies; Enzyme-linked immunosorbent assays; Horses
1. Introduction Lyme borreliosis is a multisystemic disease caused by Borrelia burgdorferi sensu lato in North America and Eurasia where Ixodes pacificus, Ixodes persulcatus, Ixodes ricinus, or Ixodes scapularis ticks occur. In the United States and Europe, B. burgdorferi sensu stricto causes disease in humans and domesticated animals. Borrelia afzelii, Borrelia garinii, and Borrelia lusitaniae, closely related bacteria, are also recognized or suspected pathogens in Europe (van Dam et al., 1993; Busch et al., 1996; Collares-Pereira et al., 2004). Laboratory diagnosis of Lyme borreliosis in humans, horses, and dogs has relied mainly on the detection of serum antibodies. Whole-cell lysates of B. burgdorferi have been used extensively in enzyme-linked immunosorbent assays *
Corresponding author. Tel.: +1 203 974 8466; fax: +1 203 974 8502. E-mail address: [email protected] (L. Magnarelli).
0034-5288/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.11.009
(ELISA), but there is potential for false positive reactions (Magnarelli et al., 2000a) because heat shock proteins and other antigens are shared among different species of bacteria. In the United States, a two-step laboratory process is followed, where sera positive by ELISA are re-tested by Western blot analysis. Immunoblotting has been helpful in verifying ELISA results in horses (Magnarelli et al., 2000b). However, in other instances, co-migrations of proteins having similar molecular masses can appear as clusters of narrowly separated bands, which make interpretations difficult. To improve the specificity of ELISA, various recombinant antigens of B. burgdorferi have been used to screen human, equine, and canine sera for antibodies (Lawrenz et al., 1999; Liang et al., 1999, 2001; Magnarelli et al., 2000a, 2001). Of the recombinant antigens evaluated, preparations of the surface- exposed lipoprotein VlsE were more sensitive and specific for B. burgdorferi in tests of human sera (Magnarelli et al.,
100
L. Magnarelli, E. Fikrig / Research in Veterinary Science 79 (2005) 99–103
2002; Bacon et al., 2003; Schulte-Spechtel et al., 2003; Philipp et al., 2003). The objectives of the present study were to: (1) evaluate a full-length recombinant VlsE1HIS antigen in polyvalent ELISA with a panel of previously tested North American horse sera; (2) compare results with those of ELISAs containing other select recombinant antigens or whole cells of B. burgdorferi.
2. Materials and methods 2.1. Sources of serum samples Veterinarians collected 181 blood samples from 98 privately owned, ill horses in Connecticut and New York State, USA during 1985, 1995, and 1996, as previously described (Magnarelli et al., 1997, 2000b). Briefly, sera were obtained from animals before antibiotic treatment in passive surveillance programs for suspected B. burgdorferi or Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) infections. The animals lived in areas infested by I. scapularis ticks, were of mixed breeds and ages, and were not immunized with wholecell B. burgdorferi bacterin or with vaccines directed to outer surface protein (Osp)A of this pathogen. Clinically affected horses showed the following signs of possible tick-borne infections: lethargy, low-grade fever (38.6– 39.1 °C), and single or multiple swollen joints resulting in lameness, or stiffness and reluctance to move. Forty positive serum samples, analyzed earlier (Magnarelli et al., 2000b) and determined to have antibodies to B. burgdorferi (strain 2591) by polyvalent ELISA or immunoblotting methods (Magnarelli et al., 2000b) were re-tested in the present study. These samples were stored at 60 °C at The Connecticut Agricultural Experiment Station. Eight positive and 15–34 negative horse serum samples selected from archived collections were used to standardize tests and to assess the reproducibility of the ELISA. 2.2. Serologic testing A polyvalent ELISA, developed and evaluated earlier (Magnarelli et al., 2000b), was used to establish antibody titers to whole-cell or purified recombinant antigens of B. burgdorferi. The following antigens were tested separately: outer surface protein (Osp)A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE. All antigens except VlsE were cloned, expressed, and purified as glutathione S-transferase (GST) fusion proteins in Escherichia coli. VlsE was expressed and purified as a polyhistidine-tagged fusion protein. Details of the methods of production for these recombinant reagents have been reported earlier (Fikrig et al., 1997; Lawrenz et al., 1999; Magnarelli et al., 2002).
The materials and methods used to determine cut-off values for positive sera have been described previously (Magnarelli et al., 1997, 2000b), with the exception of VlsE and p35 (47 kDa fibronectin-binding protein). Based on checkerboard titrations, the optimal concentrations of VlsE and p35 antigens for coating to flatbottomed, polystyrene plates (Nunc A/S, Roskilde, Denmark) were 1 and 5 lg/ml, respectively. Fifteen to twenty-seven negative control sera used earlier (Magnarelli et al., 1997, 2000b) were diluted in phosphate buffer saline solution (PBSS) to 1:160, 1:320, and 1:640. Net absorbance values, differences in optical density (OD) readings with or without antigen for each serum dilution, were calculated. Statistical analyses (mean plus 3 SD) of net absorbance values were used to determine critical regions for each serum dilution. In analyses of 27 negative horse sera with VlsE antigen, net OD values of 0.04 and 0.03 were considered positive for serum dilutions of 1:160 and P1:320, respectively. In tests of 15 negative horse sera with the p35 antigen, cut-off values of 0.10 and 0.05 were established for sera diluted to 1:160 and P1:320. The commercially produced affinity-purified horseradish peroxidase-labeled goat antihorse (H & L chains specific) immunoglobulins (Ig) (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) were tested at a working dilution of 1:4000 in PBSS. There were controls for positive and negative sera, the conjugate, PBSS, and GST reagents. Positive human sera (Magnarelli et al., 2002) were used to verify reactivities of all antigens. Tests on reproducibility for 24 positive and 3 negative horse sera were conducted to assess variability of results. A positive result for a given horse was based on the detection of serum antibodies to whole cells or to two or more recombinant antigens. In previous works (Magnarelli et al., 1997, 2000b, 2001, 2002), there was little or no cross-reactivity when homologous antibodies to Leptospira interrogans serovars, Ehrlichia canis, Rickettsia rickettsii, Treponema pallidum, and Borrelia recurrentis were screened with recombinant antigens of B. burgdorferi by an ELISA. Additional tests were conducted with 13 horse sera positive for antibodies to A. phagocytophilum and 12 horse sera containing antibodies to Neorickettsia risticii to further assess specificity with the p37 and VlsE antigens. The homologous antibody titers ranged from 1:80 to 1:40,960; 7 samples had titration endpoints greater than 1:1280.
2.3. Statistical analyses Significant differences in seropositivity rates were determined by performing a z-test (SigmaStat, SPSS, Chicago, IL, USA). The Yates correction was applied in all calculations.
L. Magnarelli, E. Fikrig / Research in Veterinary Science 79 (2005) 99–103
101
Positive reactions were recorded for all antigens tested. The majority (73%) of the sera had titration endpoints ranging from 1:160 to 1:1280. Of the 477 total positive reactions recorded, 208 (44%) had antibody titers of 1:640–1:1280, followed by results ranging from 1:160 to 1:320 (n = 139 positives), 1:2560 to 1:5,120 (n = 95), and 1:10,240 to 1:40,960 (n = 35). Horse sera reacted more frequently with 2 or more recombinant antigens than to a single fusion protein (Table 2). Of the 371 total positive reactions, sera reacted most often with 3 (n = 93 positives) or 4 recombinant antigens (n = 85). Twenty-one sera had antibodies to 7 recombinant antigens. When results for selected pairs of antigens were compared, serum reactivities to both p37 and VlsE antigens (n = 46) exceeded those recorded for p37 and OspA (n = 15) or VlsE and OspA (n = 20). Serum antibodies also were detected when p37 and OspF (n = 26), p37 and OspB (n = 24), VlsE and OspF (n = 24), and p35 and p37 (n = 22) antigens were tested separately. Serum reactivities to OspA and OspB were infrequent (n = 8).
3. Results There was serologic evidence of exposure to B. burgdorferi in 82 (84%) of the 98 ill horses. Serum reactivities to whole-cell B. burgdorferi antigen (46% and 65%) exceeded seropositivity values for separate recombinant antigens. Of the 9 recombinant antigens evaluated (Table 1), seropositivities for the p37 (40% and 51%) and VlsE (28% and 39%) reagents were elevated. Differences in percent positive values for horse sera obtained in 1995–1996 and tested with whole-cell or p37 antigens were statistically significant (z = 2.105, P = 0.035). However, differences in seropositivity values for p37 (51%) and VlsE test results (39%) for this study group or for these antigens tested with sera obtained in 1985 (40% and 28%) were not statistically significant (z = 1.772, P = 0.076) and (z = 1.155, P = 0.248), respectively. Geometric mean antibody titers were highest when whole cells were tested, but similar results also were recorded when OspF and p41-G antigens were incorporated into an ELISA.
Table 1 Results of antibody analyses for Connecticut horse sera tested with whole-cell or recombinant antigens of B. burgdorferi by polyvalent enzyme-linked immunosorbent assays Antigens
Numbera (%) of positive sera
Reciprocal antibody titers Meansb
Maximum
Whole cell OspA OspB OspC OspE OspF p35 p37 p41-G VlsE a b
1985
1995–1996
1985
1995–1996
1985
1995–1996
26 (46) 15 (26) 4 (7) 4 (7) 12 (21) 14 (25) 7 (12) 23 (40) 16 (28) 16 (28)
80 (65) 20 (16) 31 (25) 13 (11) 1 (2) 20 (16) 34 (27) 63 (51) 30 (24) 48 (39)
10,240 640 640 320 640 2560 640 5120 2560 10,240
20,480 5120 5120 640 320 20,480 2560 5120 20,480 10,240
1463 640 453 190 427 820 580 361 761 515
3263 874 669 288 320 2079 962 593 1650 668
Numbers of horse sera tested in 1985 (n = 57) and 1995–1996 (n = 124). Geometric means for positive results only.
Table 2 Frequency distributions of serologic reactivities of horse sera with one or more recombinant antigens of B. burgdorferi by polyvalent enzyme-linked immunosorbent assays Number of antigens
Numbera (%) seropositive OspA
1 2 3 4 5 6 7 Totals a
OspF
p35
p37
p41-G
VlsE
8 10 4 6 4 1 2
OspB 7 7 7 7 3 2 2
OspC 1 3 5 4 2 1 1
OspE 1 1 7 2 0 0 2
1 2 5 12 9 3 2
3 11 8 8 6 2 3
15 14 23 17 11 3 3
0 3 14 14 9 3 3
5 8 20 15 10 3 3
35
35
17
13
34
41
86
46
64
Refers to the total number of positive reactions to recombinant antigens listed in each respective column. For example, there were 8 sera that reacted only to OspA, 10 sera that reacted to OspA and 1 other recombinant antigen, 4 sera that reacted to OspA and 2 other antigens, etc.
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Serologic test results for an ELISA with whole-cell, VlsE, or p37 antigens were compared to determine concordance. Of the 181 sera analyzed, 90 (50%) were positive or negative to all 3 antigen preparations. When results for VlsE and p37 antigens were compared, 68% were either positive or negative. Agreement also was noted when results were compared for whole-cell and VlsE antigens (68%) or whole-cell and p37 antigens (64%). In analyses of 25 horse sera, containing specific antibodies to A. phagocytophilum or N. risticii, there was minor cross-reactivity. One serum sample, with antibodies to A. phagocytophilum (titer of 1:1280), had reacted with the p35 antigen of B. burgdorferi at a serum dilution of 1:320. The remaining sera were non-reactive to p35 and VlsE antigens. When positive sera from 24 horses were re-analyzed with whole-cell or one or more recombinant antigens on different days (n = 70 replicates), detection of antibodies was confirmed in 53 (76%) tests. Results for the remaining 17 sera changed from positive to negative; titration endpoints for 16 sera were 1:640 or less when first tested. The antibody titer for 1 serum sample dropped from 1:1280 to negative. Reproducibility of antibody titers for 7 positive sera varied with the antigens tested. For example, results for OspC and p41G varied by 2-fold or less in replicated trials performed on different days, whereas changes in antibody titers were 4-fold or less when sera were tested with the VlsE antigen. Similar variations in titers were noted when sera were re-tested with the remaining antigens, although titer declines of 8-fold were recorded in 13 instances.
4. Discussion Sera from horses with suspected B. burgdorferi infections frequently reacted to whole-cell or multiple recombinant antigens of B. burgdorferi in an ELISA. These findings support results of Western blot analysis (Magnarelli et al., 2000b), which show multiple bands, and indicate that horses mount broad humoral responses when they are infected by this bacterium. Of the recombinant antigens evaluated, p37 and VlsE were most suitable. Because of the high specificities obtained, an ELISA containing either of these antigens could be used in North America to help confirm B. burgdorferi infections in horses not vaccinated for this organism. A wide range of antibody titers was noted. Humoral responses to B. burgdorferi vary among mammalian hosts (Engstrom et al., 1995; Magnarelli et al., 2000b; Hovius et al., 2000). It is unknown when horses in the present study were initially infected by B. burgdorferi, how many times a given animal was bitten by infected
ticks, or how many horses actually had active B. burgdorferi infections. Also, antibody titers will depend, in part, on host immune responses to an infectious agent that differentially expresses immunodominant surface proteins. For example, multiple different Erp proteins are produced on the outer membrane of B. burgdorferi during mammalian infections (Stevenson et al., 2002); considerable variation in Erp sequences has been documented for different B. burgdorferi isolates. Moreover, severity of disease and duration of infection are additional factors to consider. Dogs with signs of canine borreliosis have stronger antibody responses to B. burgdorferi than infected dogs without signs of disease (Hovius et al., 2000). It is likely that the seropositive horses in our study were in different stages of infection and that concentrations of antibodies changed over time. Positive horse sera reacted to multiple recombinant antigens. In human beings, there is marked expansion in the number of reactive antigens as IgG antibodies are produced several weeks after the onset of illness. During the early phase of infection, IgM antibodies specific to relatively few antigens, such as flagellin, VlsE, and OspC, are detected (Dressler et al., 1993; Engstrom et al., 1995; Magnarelli et al., 2002). Based on results of the present study, we conclude that most seropositive animals were either repeatedly exposed to B. burgdorferi over extended periods or were persistently infected. The strong reactions of sera to VlsE antigen indicates that this variable outer surface lipoprotein is expressed by B. burgdorferi in infected horses. For human sera, the full-length recombinant antigen or a peptide corresponding to the invariant IR6 region of the VlsE antigen was judged to be most sensitive and specific in an ELISA (Lawrenz et al., 1999; Liang et al., 1999, 2000; Magnarelli et al., 2002; Bacon et al., 2003; Schulte-Spechtel et al., 2003). In humans, who had localized or disseminated B. burgdorferi infections, a reduction in antibodies specific to the C6 peptide is believed to be an indicator of clearance of infection (Philipp et al., 2003). However, other authors (Peltomaa et al., 2003) reported that C6 reactions persisted in persons following treatment. Nonetheless, an ELISA with VlsE antigen by itself or possibly fused to another key antigen, such as p37, to form a composite (i.e., chimeric) reagent may facilitate laboratory diagnosis of equine borreliosis. Incorporation of a VlsE antigen into an ELISA may be useful for testing horse sera in Europe, where B. burgdorferi and closely related Borrelia species occur. In analyses of sera from persons infected by B. burgdorferi in the USA and Europe, there was demonstrable presence of antibodies reacting to a synthetic peptide based on the VlsE IR6 of B. garinii (strain Ip90) (Liang et al., 1999, 2001). The invariable domains of the VlsE molecule may serve as a universal probe for the serodiagnosis of B. burgdorferi in different continents (Heikkila et al., 2003). However, the heterogeneity of the C-terminal se-
L. Magnarelli, E. Fikrig / Research in Veterinary Science 79 (2005) 99–103
quence of VlsE (Liang et al., 2000) may affect assay sensitivity. Further testing of horse sera from Europe is needed to determine the utility of the VlsE antigen as a diagnostic method there.
Acknowledgments We thank Tia Mastrone for technical assistance. This work was supported, in part, by federal Hatch funds administered by the United States Department of Agriculture and grants from the United States Centers for Disease Control and Prevention (CCU-106581), the National Institutes of Health (PO-1-AI-30548 and AI-49988), the Mathers Foundation, the Arthritis Foundation, the State of Connecticut (Charles Goodyear Award), and the American Horse Show Association. Erol Fikrig is a recipient of a clinical scientist award in translational research from the Burroughs Wellcome Fund. The authors are grateful to Steven J. Norris of the University of Texas Medical School (Houston, Texas, USA) for providing the VlsE antigen.
References Bacon, R.M., Biggerstaff, B.J., Schriefer, M.E., Gilmore, R.D., Philipp, M.T., Steere, A.C., Wormser, G.P., Marques, A.R., Johnson, B.J., 2003. Serodiagnosis of Lyme disease by kinetic Elisas using recombinant VlsE1 or peptide antigens of Borrelia burgdorferi compared with two-tiered testing using whole-cell lysates. Journal of Infectious Diseases 87, 1187–1199. Busch, U., Hizo-Teufel, R., Boehmer, R., Fingerle, V., Nitschko, H., Wilske, B., Preac-Mursic, V., 1996. Three species of Borrelia burgdorferi sensu lato (B. burgdorferi sensu stricto, B. afzelii, and B. garinii) identified from cerebrospinal fluid isolates by pulsed field gel electrophoresis and Pcr. Journal of Clinical Microbiology 34, 1072–1078. Collares-Pereira, M., Couceiro, S., Franca, I., Kurtenbach, K., Schafer, S.M., Vitorino, L., Goncalves, L., Baptista, S., Vieira, M.L., Cunha, C., 2004. First isolation of Borrelia lusitaniae from a human patient. Journal of Clinical Microbiology 42, 1316–1318. Dressler, F., Whalen, J.A., Reinhardt, B.N., Steere, A.C., 1993. Western blotting in the serodiagnosis of Lyme disease. Journal of Infectious Diseases 167, 392–400. Engstrom, S.M., Shoop, E., Johnson, R.C., 1995. Immunoblot interpretation criteria for serodiagnosis of early Lyme disease. Journal of Clinical Microbiology 33, 419–427. Fikrig, E., Barthold, S.W., Sun, W., Feng, W., Telford III, S.R., Flavell, R.A., 1997. Borrelia burgdorferi P35 and P37 proteins expressed in vivo, elicit protective immunity. Immunity 6, 531–539. Heikkila, T., Huppertz, H.-I., Seppala, I., Sillapaa, H., Saxen, H., Lahdenne, P., 2003. Recombinant or peptide antigens in the serology of Lyme arthritis in children. Journal of Infectious Diseases 187, 1888–1894. Hovius, J.W.R., Hovius, K.E., Oei, A., Houwers, D.J., Van Dam, A.P., 2000. Antibodies against specific proteins of and immobilizing activity against three strains of Borrelia burgdorferi sensu lato can be found in symptomatic but not in infected asymptomatic dogs. Journal of Clinical Microbiology 38, 2611–2621.
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Lawrenz, M.B., Hardham, J.M., Owens, R.T., Nowakowski, J., Steere, A.C., Wormser, G.P., Norris, S.J., 1999. Human antibody responses to VlsE antigenic variation protein of Borrelia burgdorferi. Journal of Clinical Microbiology 37, 3997–4004. Liang, F.T., Steere, A.C., Marques, A.R., Johnson, B.J.B., Miller, J.N., Philipp, M.T., 1999. Sensitive and specific serodiagnosis of Lyme disease by enzyme-linked immunosorbent assay with a peptide based on an immunodominant conserved region of Borrelia burgdorferi VlsE. Journal of Clinicial Microbiology 37, 3990–3996. Liang, F.T., Aberer, E., Cinco, M., Gern, L., Hu, C.M., Lobet, Y.N., Ruscio, M., Voet Jr., P.E., Weynants, V.E., Philipp, M.T., 2000. Antigenic conservation of an immunodominant invariable region of the VlsE lipoprotein among European pathogenic genospecies of Borrelia burgdorferi SL. Journal of Infectious Diseases 182, 1455–1462. Liang, F.T., Bowers, L., Philipp, M.T., 2001. C-terminal invariable domain of VlsE is immunodominant but its antigenicity is scarcely conserved among strains of Lyme disease spirochetes. Infection and Immunity 69, 3224–3231. Magnarelli, L.A., Flavell, R.A., Padula, S.J., Anderson, J.F., Fikrig, E., 1997. Serologic diagnosis of canine and equine borreliosis: use of recombinant antigens in enzyme-linked immunosorbent assays. Journal of Clinical Microbiology 35, 169–173. Magnarelli, L.A., IJdo, J.W., Padula, S.J., Flavell, R.A., Fikrig, E., 2000a. Serologic diagnosis of Lyme borreliosis by using enzymelinked immunosorbent assays with recombinant antigens. Journal of Clinical Microbiology 38, 1735–1739. Magnarelli, L.A., IJdo, J.W., Van Andel, A.E., Wu, C., Padula, S.J., Fikrig, E., 2000b. Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States. Journal of the American Veterinary Medical Association 217, 1045–1050. Magnarelli, L.A., Levy, S.A., IJdo, J.W., Wu, C., Padula, S.J., Fikrig, E., 2001. Reactivity of dog sera to whole-cell or recombinant antigens of Borrelia burgdorferi by Elisa and immunoblot analysis. Journal of Medical Microbiology 50, 889–895. Magnarelli, L.A., Lawrenz, M., Norris, S.J., Fikrig, E., 2002. Comparative reactivity of human sera to recombinant VlsE and other Borrelia burgdorferi antigens in class-specific enzyme-linked immunosorbent assays for Lyme borreliosis. Journal of Medical Microbiology 51, 649–655. Peltomaa, M., Mchugh, G., Steere, A.C., 2003. Persistence of the antibody response to the VlsE sixth invariant region (IR6) peptide of Borrelia burgdorferi after successful antibiotic treatment of Lyme disease. Journal of Infectious Diseases 187, 1178–1186. Philipp, M.T., Marques, A.R., Fawcett, P.T., Dally, L.G., Martin, D.S., 2003. C6 test as an indicator of therapy outcome for patients with localized or disseminated Lyme borreliosis. Journal of Clinical Microbiology 41, 4955–4960. Schulte-Spechtel, U., Lehnert, G., Liegl, G., Fingerle, V., Heimerl, C., Johnson, B.J.B., Wilske, B., 2003. Significant improvement of the recombinant Borrelia-specific immunoglobulin G immunoblot test by addition of VlsE and DbpA homologue derived from Borrelia garinii for diagnosis of early neuroborreliosis. Journal of Clinical Microbiology 41, 1299–1303. Stevenson, B., El-Hagen, N., Hines, M.A., Miller, J.C., Babb, K., 2002. Differential binding of host complement inhibitor factor H by Borrelia burgdorferi Erp surface proteins: a possible mechanism underlying the expansive host range of Lyme disease spirochetes. Infection and Immunity 70, 491–497. van Dam, A.P., Kuiper, H., Vos, K., Widjojokusumo, A., De Jongh, B.M., Spanjaard, L., Ramselaar, C.P., Kramer, M.D., Dankert, J., 1993. Different genospecies of Borrelia burgdorferi are associated with distinct clinical manifestations of Lyme borreliosis. Clinical Infectious Diseases 17, 708–717.
Detection of antibodies to Borrelia burgdorferi in naturally infected horses in the USA by enzyme-linked immunosorbent assay using whole-cell and recombinant antigens L. Magnarelli b
a,*
, E. Fikrig
b
a The Connecticut Agricultural Experiment Station, P.O. Box 1106, New Haven, CT 06504, USA Section of Rheumatology, Department of Internal Medicine, Yale University School of Medicine, New Haven, CT 06520, USA
Accepted 24 November 2004
Abstract Blood samples were collected from 98 horses suspected of having borreliosis or granulocytic ehrlichiosis in Connecticut and New York State, USA during 1985, 1995, and 1996. Serum antibodies to Borrelia burgdorferi were detected by an enzyme-linked immunosorbent assay (ELISA), based on whole-cell and recombinant antigens, in 82 (84%) horses. Of the 181 sera tested, 59% were positive, using whole-cell antigens, compared to 48% with protein (p)37 and 35% with VlsE antigens. An ELISA containing either of these fusion proteins can be used as an adjunct to general screening by an ELISA or immunoblotting in animals not vaccinated for this disease. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Borrelia burgdorferi; Antibodies; Enzyme-linked immunosorbent assays; Horses
1. Introduction Lyme borreliosis is a multisystemic disease caused by Borrelia burgdorferi sensu lato in North America and Eurasia where Ixodes pacificus, Ixodes persulcatus, Ixodes ricinus, or Ixodes scapularis ticks occur. In the United States and Europe, B. burgdorferi sensu stricto causes disease in humans and domesticated animals. Borrelia afzelii, Borrelia garinii, and Borrelia lusitaniae, closely related bacteria, are also recognized or suspected pathogens in Europe (van Dam et al., 1993; Busch et al., 1996; Collares-Pereira et al., 2004). Laboratory diagnosis of Lyme borreliosis in humans, horses, and dogs has relied mainly on the detection of serum antibodies. Whole-cell lysates of B. burgdorferi have been used extensively in enzyme-linked immunosorbent assays *
Corresponding author. Tel.: +1 203 974 8466; fax: +1 203 974 8502. E-mail address: [email protected] (L. Magnarelli).
0034-5288/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2004.11.009
(ELISA), but there is potential for false positive reactions (Magnarelli et al., 2000a) because heat shock proteins and other antigens are shared among different species of bacteria. In the United States, a two-step laboratory process is followed, where sera positive by ELISA are re-tested by Western blot analysis. Immunoblotting has been helpful in verifying ELISA results in horses (Magnarelli et al., 2000b). However, in other instances, co-migrations of proteins having similar molecular masses can appear as clusters of narrowly separated bands, which make interpretations difficult. To improve the specificity of ELISA, various recombinant antigens of B. burgdorferi have been used to screen human, equine, and canine sera for antibodies (Lawrenz et al., 1999; Liang et al., 1999, 2001; Magnarelli et al., 2000a, 2001). Of the recombinant antigens evaluated, preparations of the surface- exposed lipoprotein VlsE were more sensitive and specific for B. burgdorferi in tests of human sera (Magnarelli et al.,
100
L. Magnarelli, E. Fikrig / Research in Veterinary Science 79 (2005) 99–103
2002; Bacon et al., 2003; Schulte-Spechtel et al., 2003; Philipp et al., 2003). The objectives of the present study were to: (1) evaluate a full-length recombinant VlsE1HIS antigen in polyvalent ELISA with a panel of previously tested North American horse sera; (2) compare results with those of ELISAs containing other select recombinant antigens or whole cells of B. burgdorferi.
2. Materials and methods 2.1. Sources of serum samples Veterinarians collected 181 blood samples from 98 privately owned, ill horses in Connecticut and New York State, USA during 1985, 1995, and 1996, as previously described (Magnarelli et al., 1997, 2000b). Briefly, sera were obtained from animals before antibiotic treatment in passive surveillance programs for suspected B. burgdorferi or Anaplasma phagocytophilum (formerly Ehrlichia phagocytophila) infections. The animals lived in areas infested by I. scapularis ticks, were of mixed breeds and ages, and were not immunized with wholecell B. burgdorferi bacterin or with vaccines directed to outer surface protein (Osp)A of this pathogen. Clinically affected horses showed the following signs of possible tick-borne infections: lethargy, low-grade fever (38.6– 39.1 °C), and single or multiple swollen joints resulting in lameness, or stiffness and reluctance to move. Forty positive serum samples, analyzed earlier (Magnarelli et al., 2000b) and determined to have antibodies to B. burgdorferi (strain 2591) by polyvalent ELISA or immunoblotting methods (Magnarelli et al., 2000b) were re-tested in the present study. These samples were stored at 60 °C at The Connecticut Agricultural Experiment Station. Eight positive and 15–34 negative horse serum samples selected from archived collections were used to standardize tests and to assess the reproducibility of the ELISA. 2.2. Serologic testing A polyvalent ELISA, developed and evaluated earlier (Magnarelli et al., 2000b), was used to establish antibody titers to whole-cell or purified recombinant antigens of B. burgdorferi. The following antigens were tested separately: outer surface protein (Osp)A, OspB, OspC, OspE, OspF, protein (p) 41-G, p35, p37, and VlsE. All antigens except VlsE were cloned, expressed, and purified as glutathione S-transferase (GST) fusion proteins in Escherichia coli. VlsE was expressed and purified as a polyhistidine-tagged fusion protein. Details of the methods of production for these recombinant reagents have been reported earlier (Fikrig et al., 1997; Lawrenz et al., 1999; Magnarelli et al., 2002).
The materials and methods used to determine cut-off values for positive sera have been described previously (Magnarelli et al., 1997, 2000b), with the exception of VlsE and p35 (47 kDa fibronectin-binding protein). Based on checkerboard titrations, the optimal concentrations of VlsE and p35 antigens for coating to flatbottomed, polystyrene plates (Nunc A/S, Roskilde, Denmark) were 1 and 5 lg/ml, respectively. Fifteen to twenty-seven negative control sera used earlier (Magnarelli et al., 1997, 2000b) were diluted in phosphate buffer saline solution (PBSS) to 1:160, 1:320, and 1:640. Net absorbance values, differences in optical density (OD) readings with or without antigen for each serum dilution, were calculated. Statistical analyses (mean plus 3 SD) of net absorbance values were used to determine critical regions for each serum dilution. In analyses of 27 negative horse sera with VlsE antigen, net OD values of 0.04 and 0.03 were considered positive for serum dilutions of 1:160 and P1:320, respectively. In tests of 15 negative horse sera with the p35 antigen, cut-off values of 0.10 and 0.05 were established for sera diluted to 1:160 and P1:320. The commercially produced affinity-purified horseradish peroxidase-labeled goat antihorse (H & L chains specific) immunoglobulins (Ig) (Kirkegaard and Perry Laboratories, Gaithersburg, MD, USA) were tested at a working dilution of 1:4000 in PBSS. There were controls for positive and negative sera, the conjugate, PBSS, and GST reagents. Positive human sera (Magnarelli et al., 2002) were used to verify reactivities of all antigens. Tests on reproducibility for 24 positive and 3 negative horse sera were conducted to assess variability of results. A positive result for a given horse was based on the detection of serum antibodies to whole cells or to two or more recombinant antigens. In previous works (Magnarelli et al., 1997, 2000b, 2001, 2002), there was little or no cross-reactivity when homologous antibodies to Leptospira interrogans serovars, Ehrlichia canis, Rickettsia rickettsii, Treponema pallidum, and Borrelia recurrentis were screened with recombinant antigens of B. burgdorferi by an ELISA. Additional tests were conducted with 13 horse sera positive for antibodies to A. phagocytophilum and 12 horse sera containing antibodies to Neorickettsia risticii to further assess specificity with the p37 and VlsE antigens. The homologous antibody titers ranged from 1:80 to 1:40,960; 7 samples had titration endpoints greater than 1:1280.
2.3. Statistical analyses Significant differences in seropositivity rates were determined by performing a z-test (SigmaStat, SPSS, Chicago, IL, USA). The Yates correction was applied in all calculations.
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Positive reactions were recorded for all antigens tested. The majority (73%) of the sera had titration endpoints ranging from 1:160 to 1:1280. Of the 477 total positive reactions recorded, 208 (44%) had antibody titers of 1:640–1:1280, followed by results ranging from 1:160 to 1:320 (n = 139 positives), 1:2560 to 1:5,120 (n = 95), and 1:10,240 to 1:40,960 (n = 35). Horse sera reacted more frequently with 2 or more recombinant antigens than to a single fusion protein (Table 2). Of the 371 total positive reactions, sera reacted most often with 3 (n = 93 positives) or 4 recombinant antigens (n = 85). Twenty-one sera had antibodies to 7 recombinant antigens. When results for selected pairs of antigens were compared, serum reactivities to both p37 and VlsE antigens (n = 46) exceeded those recorded for p37 and OspA (n = 15) or VlsE and OspA (n = 20). Serum antibodies also were detected when p37 and OspF (n = 26), p37 and OspB (n = 24), VlsE and OspF (n = 24), and p35 and p37 (n = 22) antigens were tested separately. Serum reactivities to OspA and OspB were infrequent (n = 8).
3. Results There was serologic evidence of exposure to B. burgdorferi in 82 (84%) of the 98 ill horses. Serum reactivities to whole-cell B. burgdorferi antigen (46% and 65%) exceeded seropositivity values for separate recombinant antigens. Of the 9 recombinant antigens evaluated (Table 1), seropositivities for the p37 (40% and 51%) and VlsE (28% and 39%) reagents were elevated. Differences in percent positive values for horse sera obtained in 1995–1996 and tested with whole-cell or p37 antigens were statistically significant (z = 2.105, P = 0.035). However, differences in seropositivity values for p37 (51%) and VlsE test results (39%) for this study group or for these antigens tested with sera obtained in 1985 (40% and 28%) were not statistically significant (z = 1.772, P = 0.076) and (z = 1.155, P = 0.248), respectively. Geometric mean antibody titers were highest when whole cells were tested, but similar results also were recorded when OspF and p41-G antigens were incorporated into an ELISA.
Table 1 Results of antibody analyses for Connecticut horse sera tested with whole-cell or recombinant antigens of B. burgdorferi by polyvalent enzyme-linked immunosorbent assays Antigens
Numbera (%) of positive sera
Reciprocal antibody titers Meansb
Maximum
Whole cell OspA OspB OspC OspE OspF p35 p37 p41-G VlsE a b
1985
1995–1996
1985
1995–1996
1985
1995–1996
26 (46) 15 (26) 4 (7) 4 (7) 12 (21) 14 (25) 7 (12) 23 (40) 16 (28) 16 (28)
80 (65) 20 (16) 31 (25) 13 (11) 1 (2) 20 (16) 34 (27) 63 (51) 30 (24) 48 (39)
10,240 640 640 320 640 2560 640 5120 2560 10,240
20,480 5120 5120 640 320 20,480 2560 5120 20,480 10,240
1463 640 453 190 427 820 580 361 761 515
3263 874 669 288 320 2079 962 593 1650 668
Numbers of horse sera tested in 1985 (n = 57) and 1995–1996 (n = 124). Geometric means for positive results only.
Table 2 Frequency distributions of serologic reactivities of horse sera with one or more recombinant antigens of B. burgdorferi by polyvalent enzyme-linked immunosorbent assays Number of antigens
Numbera (%) seropositive OspA
1 2 3 4 5 6 7 Totals a
OspF
p35
p37
p41-G
VlsE
8 10 4 6 4 1 2
OspB 7 7 7 7 3 2 2
OspC 1 3 5 4 2 1 1
OspE 1 1 7 2 0 0 2
1 2 5 12 9 3 2
3 11 8 8 6 2 3
15 14 23 17 11 3 3
0 3 14 14 9 3 3
5 8 20 15 10 3 3
35
35
17
13
34
41
86
46
64
Refers to the total number of positive reactions to recombinant antigens listed in each respective column. For example, there were 8 sera that reacted only to OspA, 10 sera that reacted to OspA and 1 other recombinant antigen, 4 sera that reacted to OspA and 2 other antigens, etc.
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Serologic test results for an ELISA with whole-cell, VlsE, or p37 antigens were compared to determine concordance. Of the 181 sera analyzed, 90 (50%) were positive or negative to all 3 antigen preparations. When results for VlsE and p37 antigens were compared, 68% were either positive or negative. Agreement also was noted when results were compared for whole-cell and VlsE antigens (68%) or whole-cell and p37 antigens (64%). In analyses of 25 horse sera, containing specific antibodies to A. phagocytophilum or N. risticii, there was minor cross-reactivity. One serum sample, with antibodies to A. phagocytophilum (titer of 1:1280), had reacted with the p35 antigen of B. burgdorferi at a serum dilution of 1:320. The remaining sera were non-reactive to p35 and VlsE antigens. When positive sera from 24 horses were re-analyzed with whole-cell or one or more recombinant antigens on different days (n = 70 replicates), detection of antibodies was confirmed in 53 (76%) tests. Results for the remaining 17 sera changed from positive to negative; titration endpoints for 16 sera were 1:640 or less when first tested. The antibody titer for 1 serum sample dropped from 1:1280 to negative. Reproducibility of antibody titers for 7 positive sera varied with the antigens tested. For example, results for OspC and p41G varied by 2-fold or less in replicated trials performed on different days, whereas changes in antibody titers were 4-fold or less when sera were tested with the VlsE antigen. Similar variations in titers were noted when sera were re-tested with the remaining antigens, although titer declines of 8-fold were recorded in 13 instances.
4. Discussion Sera from horses with suspected B. burgdorferi infections frequently reacted to whole-cell or multiple recombinant antigens of B. burgdorferi in an ELISA. These findings support results of Western blot analysis (Magnarelli et al., 2000b), which show multiple bands, and indicate that horses mount broad humoral responses when they are infected by this bacterium. Of the recombinant antigens evaluated, p37 and VlsE were most suitable. Because of the high specificities obtained, an ELISA containing either of these antigens could be used in North America to help confirm B. burgdorferi infections in horses not vaccinated for this organism. A wide range of antibody titers was noted. Humoral responses to B. burgdorferi vary among mammalian hosts (Engstrom et al., 1995; Magnarelli et al., 2000b; Hovius et al., 2000). It is unknown when horses in the present study were initially infected by B. burgdorferi, how many times a given animal was bitten by infected
ticks, or how many horses actually had active B. burgdorferi infections. Also, antibody titers will depend, in part, on host immune responses to an infectious agent that differentially expresses immunodominant surface proteins. For example, multiple different Erp proteins are produced on the outer membrane of B. burgdorferi during mammalian infections (Stevenson et al., 2002); considerable variation in Erp sequences has been documented for different B. burgdorferi isolates. Moreover, severity of disease and duration of infection are additional factors to consider. Dogs with signs of canine borreliosis have stronger antibody responses to B. burgdorferi than infected dogs without signs of disease (Hovius et al., 2000). It is likely that the seropositive horses in our study were in different stages of infection and that concentrations of antibodies changed over time. Positive horse sera reacted to multiple recombinant antigens. In human beings, there is marked expansion in the number of reactive antigens as IgG antibodies are produced several weeks after the onset of illness. During the early phase of infection, IgM antibodies specific to relatively few antigens, such as flagellin, VlsE, and OspC, are detected (Dressler et al., 1993; Engstrom et al., 1995; Magnarelli et al., 2002). Based on results of the present study, we conclude that most seropositive animals were either repeatedly exposed to B. burgdorferi over extended periods or were persistently infected. The strong reactions of sera to VlsE antigen indicates that this variable outer surface lipoprotein is expressed by B. burgdorferi in infected horses. For human sera, the full-length recombinant antigen or a peptide corresponding to the invariant IR6 region of the VlsE antigen was judged to be most sensitive and specific in an ELISA (Lawrenz et al., 1999; Liang et al., 1999, 2000; Magnarelli et al., 2002; Bacon et al., 2003; Schulte-Spechtel et al., 2003). In humans, who had localized or disseminated B. burgdorferi infections, a reduction in antibodies specific to the C6 peptide is believed to be an indicator of clearance of infection (Philipp et al., 2003). However, other authors (Peltomaa et al., 2003) reported that C6 reactions persisted in persons following treatment. Nonetheless, an ELISA with VlsE antigen by itself or possibly fused to another key antigen, such as p37, to form a composite (i.e., chimeric) reagent may facilitate laboratory diagnosis of equine borreliosis. Incorporation of a VlsE antigen into an ELISA may be useful for testing horse sera in Europe, where B. burgdorferi and closely related Borrelia species occur. In analyses of sera from persons infected by B. burgdorferi in the USA and Europe, there was demonstrable presence of antibodies reacting to a synthetic peptide based on the VlsE IR6 of B. garinii (strain Ip90) (Liang et al., 1999, 2001). The invariable domains of the VlsE molecule may serve as a universal probe for the serodiagnosis of B. burgdorferi in different continents (Heikkila et al., 2003). However, the heterogeneity of the C-terminal se-
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quence of VlsE (Liang et al., 2000) may affect assay sensitivity. Further testing of horse sera from Europe is needed to determine the utility of the VlsE antigen as a diagnostic method there.
Acknowledgments We thank Tia Mastrone for technical assistance. This work was supported, in part, by federal Hatch funds administered by the United States Department of Agriculture and grants from the United States Centers for Disease Control and Prevention (CCU-106581), the National Institutes of Health (PO-1-AI-30548 and AI-49988), the Mathers Foundation, the Arthritis Foundation, the State of Connecticut (Charles Goodyear Award), and the American Horse Show Association. Erol Fikrig is a recipient of a clinical scientist award in translational research from the Burroughs Wellcome Fund. The authors are grateful to Steven J. Norris of the University of Texas Medical School (Houston, Texas, USA) for providing the VlsE antigen.
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