587548 research-article2015
VDIXXX10.1177/1040638715587548Comparison between serologic tests for EGA and LBSchvartz et al.
Brief Communication
Comparison between available serologic tests for detecting antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi in horses in Canada
Journal of Veterinary Diagnostic Investigation 1–7 © 2015 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638715587548 jvdi.sagepub.com
Gili Schvartz,1 Tasha Epp, Hilary J. Burgess, Neil B. Chilton, Katharina L. Lohmann
Abstract. To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalenceadjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted. Key words: Anaplasma phagocytophilum; Borrelia burgdorferi; Canada; comparison; horses; serology. Equine granulocytic anaplasmosis (EGA) caused by Anaplasma phagocytophilum and Lyme borreliosis (LB) caused by Borrelia burgdorferi are important tick-borne diseases in horses, and coexposure to both organisms has been reported in the United States.16 In North America, both bacteria are transmitted by the black-legged tick (Ixodes scapularis), which occurs in the northeastern, midwestern, and southeastern United States, and the western black-legged tick (Ixodes pacificus), which occurs along the western coast of the United States.10,25 There are also an increasing number of established populations of both tick species in southern Canada,13,17 and an expansion of the geographic range of I. scapularis to the north and west as a consequence of climatic changes has been predicted.13,17 Cases of EGA1,3,21 and LB2 have been reported in Canadian horses. Diagnosis of clinical cases of EGA or LB is typically based on the manifestation of clinical signs, geographic location and likelihood of exposure to tick vectors, and detection of the infectious organism or host antibody response.4,16,24 The available serologic tests employ different test methodologies and target antigens (Table 1), which should be taken into account when attempting to achieve a diagnosis in a clinically suspect case. The use of serologic testing merely to determine exposure status is complicated by the inability to
rely on clinical data; we previously observed that a point-ofcare enzyme-linked immunosorbent assay (pocELISA) lacked agreement with a laboratory-based A. phagocytophilum indirect fluorescent antibody test (IFAT) and B. burgdorferi ELISA–Western blot (ELISA-WB) combination, respectively, when used for this purpose in a low-prevalence population.19 Given that a perceived increase in disease risk in Canada may result in more frequent “screening” of horses for exposure, we expanded our investigation to all currently available serologic tests. Fifty equine serum samples that were originally submitted for equine infectious anemia testing in April 2013 were supplied by Prairie Diagnostic Services Inc. (Saskatoon, Saskatchewan, Canada) in accordance with the laboratory’s confidentiality rules, and the study was approved by the Animal Research Ethics Board at the University of Saskatchewan (protocol no. 20120015). The samples were From the Departments of Large Animal Clinical Sciences (Schvartz, Epp, Lohmann) and Veterinary Pathology (Burgess), Western College of Veterinary Medicine, and the Department of Biology (Chilton), University of Saskatchewan, Saskatoon, Saskatchewan, Canada. 1 Corresponding Author: Gili Schvartz, Doodaim 23, Neve Noy, BeerSheva, 84854 Israel. [email protected]
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
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Schvartz et al.
Table 1. Comparison between methodology, time between infection and antibody detection, sensitivity, and specificity of serological tests for equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB).* Test method
Time to antibody detection
Antigen
EGA (Anaplasma phagocytophilum) pocELISA P44
NR
IFAT
2 weeks11†
Whole organism
LB (Borrelia burgdorferi) C6 (synthetic, pocELISA resembles IR6 of VlsE) ELISA-WB Whole organism (ELISA) IFAT Whole organism
Sensitivity 100% relative to IFAT7; 87% relative to IFAT24 NR
Specificity 100% relative to IFAT7; 99.8% relative to IFAT24 NR
NR
100% relative to WB7; 95% relative to WB7; 63% in experimental 100% in experimental infection12 infection12 May be low in first weeks NR after infection4 NR NR
Multiplex assay Osp A
3 weeks23‡
49%22
85%22
2–3 weeks23 8 weeks23
80%22 86%22
79%22 69%22
Osp C Osp F
3–4 weeks in dogs14
Other Marketed for use in dogs IFAT is considered the “gold standard”24 Marketed for use in dogs, detects only active infection7 Two-tier approach4 May be less sensitive than ELISA4 Good agreement with C6 ELISA23
* pocELISA = point-of-care enzyme-linked immunosorbent assay; IFAT = indirect fluorescent antibody test; WB = Western blot; Osp = outer surface protein; NR = not reported. Superscript numbers correspond to references. † In experimentally infected horses. ‡ In animals vaccinated with an Osp A vaccine.
Table 2. Reference guidelines for interpretation of serologic tests for equine granulocytic anaplasmosis as provided by the veterinary diagnostic laboratories.* IFAT1 1,250–26,000)‡
Negative Weak positive Moderate positive Strong positive No Borrelia burgdorferi–specific antibody reactivity. Some antibody reactivity, insufficient in specificity or quantity to be graded positive. A follow-up sample in 3–4 weeks should show development of B. burgdorferi–specific antibodies if infection is present. Reactivity to a small number of B. burgdorferi–specific antigens. Usually indicates an early response to B. burgdorferi infection but may also be seen as infection resolves. A follow-up sample in 3–4 weeks should distinguish between a recent or resolving B. burgdorferi infection. Multiple B. burgdorferi–specific antibody bands, as well as nonspecific bands. The quantity and intensity of bands are less than that of a strong positive blot. If treating, consider retesting in 8 weeks to follow the serological response to treatment. Indicates B. burgdorferi infection of many months duration as indicated by multiple B. burgdorferi– specific (as well as nonspecific) antibody bands, generally accompanied by a high (strong positive) ELISA titer. Negative Low–moderate. The animal may be recently infected or infected with cross-reacting spirochetes. Retesting of a convalescent sample in 3–4 weeks is recommended. Fairly high, may indicate a recent infection. Cross-reaction with other spirochetes is less likely. Negative values for antibodies to all 3 Osp antigens are predictive that the horse is not infected. Equivocal values can indicate very early infection or can be induced by nonspecific serum reaction. If there are no positive values for any of the 3 Osp antigens, the horse should be retested in 2–3 weeks to confirm or exclude early infection. Typically observed in vaccinated animals; in horses, antibodies to Osp A also seem to rise during infection. If antibodies to Osp C and/or Osp F are positive along with Osp A, the horse should be considered as infected with B. burgdorferi. Positive values for antibodies to Osp C only indicate early infection. Antibodies to Osp A can also be elevated during early infection. Positive values for antibodies to Osp F only are predictive for chronic infection stages. Positive values for antibodies to Osp C and Osp F in the same sample are indicators for an infection that occurred several weeks ago and is moving toward the chronic infection stage.
* Test results that were considered seropositive for the purpose of the study are in boldface. IFAT = indirect fluorescent antibody test; ELISA = enzymelinked immunosorbent assay; Osp = outer surface protein. † Assessment as seropositive or seronegative was based on the Western blot results; samples with an ELISA titer 1,000) or positive anti–Osp F titer (>1,250) were considered seropositive. || For the ELISA-WB combination, samples with positive ELISA titers (≥1:160) that were confirmed by WB were considered seropositive; all positive WB results were either weak positive (+) or moderate positive (++). Samples with negative ELISA results were not retested by WB.
agreement between WB and the Lyme multiplex assay when testing sera from horses with unknown history of infection with B. burgdorferi. The criteria and cutoff values we chose
to categorize samples as seropositive or seronegative may have influenced the agreement between tests. Although follow-up testing may have been helpful in some instances, it
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
6
Schvartz et al.
was not possible in the context of our study. It is worth mentioning that an increase in the cutoff values for the B. burgdorferi IFAT to >1:1,280 and for the ELISA-WB to a moderate positive WB would have resulted in worse agreement of each test with the pocELISA and worse agreement between the 2 tests, effectively reducing agreement based on kappa to less than that expected by chance alone (data not shown). In addition to methodological considerations, we consider it likely that some of our results represent false positives. False-positive results may explain why individual samples showed rather strongly positive results in 1 test but were negative in several or all other tests, and are supported by an expected low-positive predictive value of serologic tests in this to date low-prevalence population of horses. In summary, our results show that available serologic tests lacked agreement when used to assess the exposure to A. phagocytophilum or B. burgdorferi of horses from a lowprevalence population. We, therefore, caution against the use of serologic “screening” in the absence of clinical suspicion and recommend that methodological details of the available tests as well as the potential for cross-reactivity and false positive results are taken into account. As stated earlier, our study is not suitable to draw any conclusions with regard to sensitivity and specificity of the employed tests in a clinical context. While there may be insufficient information to unequivocally support individual serologic tests as the “gold standard” in horses, previous studies reported high sensitivity and specificity of available serologic tests7,23,24 when known positive and negative samples were tested. Previous reports in other populations of horses also stated much better agreement between diagnostic tests.7,12,23,24 Acknowledgments We thank Prairie Diagnostic Services Inc. (Saskatoon, Saskatchewan, Canada) for providing the samples for this study.
Sources and manufacturers a. SNAP 4Dx Plus ELISA, IDEXX Laboratories Inc., Westbrook, ME. b. Anaplasma phagocytophilum 12-well IFA substrate slides, Fuller Laboratories, Fullerton, CA. Tested at the Michigan State University Diagnostic Center for Population & Animal Health, Lansing, MI. c. Anaplasma phagocytophilum IFA substrate slide, VMRD Inc., Pullman, WA. Tested at the Connecticut Veterinary Medical Diagnostic Laboratory, Storrs, CT. d. Lyme ELISA and Lyme Western blot, Connecticut Veterinary Medical Diagnostic Laboratory, Storrs, CT. e. Borrelia burgdorferi 12-well FA substrate slides, VMRD Inc., Pullman, WA. Tested at the Michigan State University Diagnostic Center for Population & Animal Health, Lansing, MI. f. Equine Lyme multiplex assay, Animal Health Diagnostic Center, Cornell University, Ithaca, NY.
Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Equine Health Research Fund at the Western College of Veterinary Medicine, University of Saskatchewan (grant 414214) with in-kind support from IDEXX Laboratories (Westbrook, Maine).
References 1. Berrington A, et al. A case of Ehrlichia equi in an adult horse in British Columbia. Can Vet J 1996;37:174–175. 2. Burgess B. British Columbia. Lyme disease in horses. Can Vet J 1988;29:393–394. 3. Burgess H, et al. Granulocytic anaplasmosis in a horse from Saskatchewan. Can Vet J 2012;53:886–888. 4. Butler CM, et al. Borrelia burgdorferi infections with special reference to horses. A review. Vet Q 2005;27:146–156. 5. Byrt T, et al. Bias, prevalence and kappa. J Clin Epidemiol 1993;46:423–429. 6. Centers for Diseases Control and Prevention. Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep 1995;44:590–591. 7. Chandrashekar R, et al. Serologic diagnosis of equine borreliosis: evaluation of an in-clinic enzyme-linked immunosorbent assay (SNAP® 4Dx®). Int J Appl Res Vet Med 2008;6: 145–150. 8. Chang YF, et al. Antibiotic treatment of experimentally Borrelia burgdorferi-infected ponies. Vet Microbiol 2005;107: 285–294. 9. Dohoo I, et al. Attributes of the test per se. In: Veterinary Epidemiologic Research. 2nd ed. Charlottetown, Prince Edward Island, Canada: VER Inc., 2009:97–100. 10. Foley JE, et al. Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. J Vector Ecol 2004;29:41–50. 11. Franzén P, et al. Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum. J Vet Intern Med 2005;19:232–239. 12. Johnson AL, et al. Validation of an in-clinic enzyme-linked immunosorbent assay kit for diagnosis of Borrelia burgdorferi infection in horses. J Vet Diagn Invest 2008;20:321–324. 13 Leighton PA, et al. Predicting the speed of tick invasion: an empirical model of range expansion for the Lyme disease vector Ixodes scapularis in Canada. J Appl Ecol 2012;49: 457–464. 14. Liang FT, et al. Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay. J Clin Microbiol 2000;38:4160–4166. 15. Madigan JE, Gribble D. Equine ehrlichiosis in northern California: 49 cases (1968–1981). J Am Vet Med Assoc 1987;190:445–448.
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
Comparison between serologic tests for EGA and LB 16. Magnarelli LA, et al. Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States. J Am Vet Med Assoc 2000;217:1045–1050. 17. Ogden NH, et al. Active and passive surveillance and phylogenetic analysis of Borrelia burgdorferi elucidate the process of Lyme disease risk emergence in Canada. Environ Health Perspect 2010;118:909–914. 18. Ogden NH, et al. Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada. Appl Environ Microbiol 2011;77:3244–3254. 19. Schvartz G, et al. Seroprevalence of equine granulocytic anaplasmosis and Lyme borreliosis in Canada as determined by a point-of-care ELISA. Can Vet J. In press. 20. Sim J, Wright CC. The kappa statistic in reliability studies: use, interpretation, and sample size requirements. Phys Ther 2005;85:257–268. 21. Uehlinger FD, et al. Granulocytic anaplasmosis in a horse from Nova Scotia caused by infection with Anaplasma phagocytophilum. Can Vet J 2011;52:537–540. 22. Wagner B, et al. Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods. Vet Immunol Immunopathol 2011;144:374–381.
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23. Wagner B, et al. Antibodies to OspC, OspF and C6 antigens as indicators for infection with Borrelia burgdorferi in horses. Equine Vet J 2013;45:533–537. 24. Veronesi F, et al. Evaluation of the performance of a rapid enzyme-linked immunosorbent assay in the detection of Anaplasma phagocytophilum antibodies in horses. Vector Borne Zoonotic Dis 2014;14:317–323. 25. Woldehiwet Z. The natural history of Anaplasma phagocytophilum. Vet Parasitol 2010;167:108–122.
Authors’ contributions G Schvartz drafted the manuscript. G Schvartz and KL Lohmann contributed to acquisition, analysis, and interpretation of data. T Epp contributed to analysis and interpretation of data. HJ Burgess contributed to acquisition and interpretation of data. NB Chilton contributed to interpretation of data. G Schvartz, T Epp, HJ Burgess, NB Chilton, and KL Lohmann substantially contributed to conception and design; critically revised manuscript; gave final approval; and agree to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
VDIXXX10.1177/1040638715587548Comparison between serologic tests for EGA and LBSchvartz et al.
Brief Communication
Comparison between available serologic tests for detecting antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi in horses in Canada
Journal of Veterinary Diagnostic Investigation 1–7 © 2015 The Author(s) Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/1040638715587548 jvdi.sagepub.com
Gili Schvartz,1 Tasha Epp, Hilary J. Burgess, Neil B. Chilton, Katharina L. Lohmann
Abstract. To investigate the agreement between available serologic tests for the detection of antibodies against Anaplasma phagocytophilum and Borrelia burgdorferi, 50 serum samples from horses of unknown clinical status and at low risk for infection were tested. In addition to a point-of-care enzyme-linked immunosorbent assay (pocELISA), the evaluated tests included 2 indirect fluorescent antibody tests (IFATs) for antibodies against A. phagocytophilum and an IFAT, an ELISA confirmed with Western blot, and the Lyme multiplex assay for antibodies against B. burgdorferi. For each pair-wise comparison between serologic tests, the difference in the proportion of seropositive results as well as kappa and the prevalenceadjusted, bias-adjusted kappa were calculated. The proportion of seropositive results differed significantly in each pairwise comparison of tests for detection of antibodies against A. phagocytophilum, and between the pocELISA and IFAT as well as between the pocELISA and Lyme multiplex assay for detection of antibodies against B. burgdorferi. Agreement based on kappa varied from poor to fair while agreement was improved when evaluating prevalence-adjusted, bias-adjusted kappa. Lack of agreement may be explained by differences in methodology between the evaluated tests, cross-reactivity or false-positive and false-negative tests. In addition to the limitations of serologic test interpretation in the absence of clinical disease, this data suggest that screening of horses for exposure to tick-borne diseases in nonendemic areas may not be warranted. Key words: Anaplasma phagocytophilum; Borrelia burgdorferi; Canada; comparison; horses; serology. Equine granulocytic anaplasmosis (EGA) caused by Anaplasma phagocytophilum and Lyme borreliosis (LB) caused by Borrelia burgdorferi are important tick-borne diseases in horses, and coexposure to both organisms has been reported in the United States.16 In North America, both bacteria are transmitted by the black-legged tick (Ixodes scapularis), which occurs in the northeastern, midwestern, and southeastern United States, and the western black-legged tick (Ixodes pacificus), which occurs along the western coast of the United States.10,25 There are also an increasing number of established populations of both tick species in southern Canada,13,17 and an expansion of the geographic range of I. scapularis to the north and west as a consequence of climatic changes has been predicted.13,17 Cases of EGA1,3,21 and LB2 have been reported in Canadian horses. Diagnosis of clinical cases of EGA or LB is typically based on the manifestation of clinical signs, geographic location and likelihood of exposure to tick vectors, and detection of the infectious organism or host antibody response.4,16,24 The available serologic tests employ different test methodologies and target antigens (Table 1), which should be taken into account when attempting to achieve a diagnosis in a clinically suspect case. The use of serologic testing merely to determine exposure status is complicated by the inability to
rely on clinical data; we previously observed that a point-ofcare enzyme-linked immunosorbent assay (pocELISA) lacked agreement with a laboratory-based A. phagocytophilum indirect fluorescent antibody test (IFAT) and B. burgdorferi ELISA–Western blot (ELISA-WB) combination, respectively, when used for this purpose in a low-prevalence population.19 Given that a perceived increase in disease risk in Canada may result in more frequent “screening” of horses for exposure, we expanded our investigation to all currently available serologic tests. Fifty equine serum samples that were originally submitted for equine infectious anemia testing in April 2013 were supplied by Prairie Diagnostic Services Inc. (Saskatoon, Saskatchewan, Canada) in accordance with the laboratory’s confidentiality rules, and the study was approved by the Animal Research Ethics Board at the University of Saskatchewan (protocol no. 20120015). The samples were From the Departments of Large Animal Clinical Sciences (Schvartz, Epp, Lohmann) and Veterinary Pathology (Burgess), Western College of Veterinary Medicine, and the Department of Biology (Chilton), University of Saskatchewan, Saskatoon, Saskatchewan, Canada. 1 Corresponding Author: Gili Schvartz, Doodaim 23, Neve Noy, BeerSheva, 84854 Israel. [email protected]
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
2
Schvartz et al.
Table 1. Comparison between methodology, time between infection and antibody detection, sensitivity, and specificity of serological tests for equine granulocytic anaplasmosis (EGA) and Lyme borreliosis (LB).* Test method
Time to antibody detection
Antigen
EGA (Anaplasma phagocytophilum) pocELISA P44
NR
IFAT
2 weeks11†
Whole organism
LB (Borrelia burgdorferi) C6 (synthetic, pocELISA resembles IR6 of VlsE) ELISA-WB Whole organism (ELISA) IFAT Whole organism
Sensitivity 100% relative to IFAT7; 87% relative to IFAT24 NR
Specificity 100% relative to IFAT7; 99.8% relative to IFAT24 NR
NR
100% relative to WB7; 95% relative to WB7; 63% in experimental 100% in experimental infection12 infection12 May be low in first weeks NR after infection4 NR NR
Multiplex assay Osp A
3 weeks23‡
49%22
85%22
2–3 weeks23 8 weeks23
80%22 86%22
79%22 69%22
Osp C Osp F
3–4 weeks in dogs14
Other Marketed for use in dogs IFAT is considered the “gold standard”24 Marketed for use in dogs, detects only active infection7 Two-tier approach4 May be less sensitive than ELISA4 Good agreement with C6 ELISA23
* pocELISA = point-of-care enzyme-linked immunosorbent assay; IFAT = indirect fluorescent antibody test; WB = Western blot; Osp = outer surface protein; NR = not reported. Superscript numbers correspond to references. † In experimentally infected horses. ‡ In animals vaccinated with an Osp A vaccine.
Table 2. Reference guidelines for interpretation of serologic tests for equine granulocytic anaplasmosis as provided by the veterinary diagnostic laboratories.* IFAT1 1,250–26,000)‡
Negative Weak positive Moderate positive Strong positive No Borrelia burgdorferi–specific antibody reactivity. Some antibody reactivity, insufficient in specificity or quantity to be graded positive. A follow-up sample in 3–4 weeks should show development of B. burgdorferi–specific antibodies if infection is present. Reactivity to a small number of B. burgdorferi–specific antigens. Usually indicates an early response to B. burgdorferi infection but may also be seen as infection resolves. A follow-up sample in 3–4 weeks should distinguish between a recent or resolving B. burgdorferi infection. Multiple B. burgdorferi–specific antibody bands, as well as nonspecific bands. The quantity and intensity of bands are less than that of a strong positive blot. If treating, consider retesting in 8 weeks to follow the serological response to treatment. Indicates B. burgdorferi infection of many months duration as indicated by multiple B. burgdorferi– specific (as well as nonspecific) antibody bands, generally accompanied by a high (strong positive) ELISA titer. Negative Low–moderate. The animal may be recently infected or infected with cross-reacting spirochetes. Retesting of a convalescent sample in 3–4 weeks is recommended. Fairly high, may indicate a recent infection. Cross-reaction with other spirochetes is less likely. Negative values for antibodies to all 3 Osp antigens are predictive that the horse is not infected. Equivocal values can indicate very early infection or can be induced by nonspecific serum reaction. If there are no positive values for any of the 3 Osp antigens, the horse should be retested in 2–3 weeks to confirm or exclude early infection. Typically observed in vaccinated animals; in horses, antibodies to Osp A also seem to rise during infection. If antibodies to Osp C and/or Osp F are positive along with Osp A, the horse should be considered as infected with B. burgdorferi. Positive values for antibodies to Osp C only indicate early infection. Antibodies to Osp A can also be elevated during early infection. Positive values for antibodies to Osp F only are predictive for chronic infection stages. Positive values for antibodies to Osp C and Osp F in the same sample are indicators for an infection that occurred several weeks ago and is moving toward the chronic infection stage.
* Test results that were considered seropositive for the purpose of the study are in boldface. IFAT = indirect fluorescent antibody test; ELISA = enzymelinked immunosorbent assay; Osp = outer surface protein. † Assessment as seropositive or seronegative was based on the Western blot results; samples with an ELISA titer 1,000) or positive anti–Osp F titer (>1,250) were considered seropositive. || For the ELISA-WB combination, samples with positive ELISA titers (≥1:160) that were confirmed by WB were considered seropositive; all positive WB results were either weak positive (+) or moderate positive (++). Samples with negative ELISA results were not retested by WB.
agreement between WB and the Lyme multiplex assay when testing sera from horses with unknown history of infection with B. burgdorferi. The criteria and cutoff values we chose
to categorize samples as seropositive or seronegative may have influenced the agreement between tests. Although follow-up testing may have been helpful in some instances, it
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
6
Schvartz et al.
was not possible in the context of our study. It is worth mentioning that an increase in the cutoff values for the B. burgdorferi IFAT to >1:1,280 and for the ELISA-WB to a moderate positive WB would have resulted in worse agreement of each test with the pocELISA and worse agreement between the 2 tests, effectively reducing agreement based on kappa to less than that expected by chance alone (data not shown). In addition to methodological considerations, we consider it likely that some of our results represent false positives. False-positive results may explain why individual samples showed rather strongly positive results in 1 test but were negative in several or all other tests, and are supported by an expected low-positive predictive value of serologic tests in this to date low-prevalence population of horses. In summary, our results show that available serologic tests lacked agreement when used to assess the exposure to A. phagocytophilum or B. burgdorferi of horses from a lowprevalence population. We, therefore, caution against the use of serologic “screening” in the absence of clinical suspicion and recommend that methodological details of the available tests as well as the potential for cross-reactivity and false positive results are taken into account. As stated earlier, our study is not suitable to draw any conclusions with regard to sensitivity and specificity of the employed tests in a clinical context. While there may be insufficient information to unequivocally support individual serologic tests as the “gold standard” in horses, previous studies reported high sensitivity and specificity of available serologic tests7,23,24 when known positive and negative samples were tested. Previous reports in other populations of horses also stated much better agreement between diagnostic tests.7,12,23,24 Acknowledgments We thank Prairie Diagnostic Services Inc. (Saskatoon, Saskatchewan, Canada) for providing the samples for this study.
Sources and manufacturers a. SNAP 4Dx Plus ELISA, IDEXX Laboratories Inc., Westbrook, ME. b. Anaplasma phagocytophilum 12-well IFA substrate slides, Fuller Laboratories, Fullerton, CA. Tested at the Michigan State University Diagnostic Center for Population & Animal Health, Lansing, MI. c. Anaplasma phagocytophilum IFA substrate slide, VMRD Inc., Pullman, WA. Tested at the Connecticut Veterinary Medical Diagnostic Laboratory, Storrs, CT. d. Lyme ELISA and Lyme Western blot, Connecticut Veterinary Medical Diagnostic Laboratory, Storrs, CT. e. Borrelia burgdorferi 12-well FA substrate slides, VMRD Inc., Pullman, WA. Tested at the Michigan State University Diagnostic Center for Population & Animal Health, Lansing, MI. f. Equine Lyme multiplex assay, Animal Health Diagnostic Center, Cornell University, Ithaca, NY.
Declaration of conflicting interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was supported by the Equine Health Research Fund at the Western College of Veterinary Medicine, University of Saskatchewan (grant 414214) with in-kind support from IDEXX Laboratories (Westbrook, Maine).
References 1. Berrington A, et al. A case of Ehrlichia equi in an adult horse in British Columbia. Can Vet J 1996;37:174–175. 2. Burgess B. British Columbia. Lyme disease in horses. Can Vet J 1988;29:393–394. 3. Burgess H, et al. Granulocytic anaplasmosis in a horse from Saskatchewan. Can Vet J 2012;53:886–888. 4. Butler CM, et al. Borrelia burgdorferi infections with special reference to horses. A review. Vet Q 2005;27:146–156. 5. Byrt T, et al. Bias, prevalence and kappa. J Clin Epidemiol 1993;46:423–429. 6. Centers for Diseases Control and Prevention. Recommendations for test performance and interpretation from the Second National Conference on Serologic Diagnosis of Lyme Disease. MMWR Morb Mortal Wkly Rep 1995;44:590–591. 7. Chandrashekar R, et al. Serologic diagnosis of equine borreliosis: evaluation of an in-clinic enzyme-linked immunosorbent assay (SNAP® 4Dx®). Int J Appl Res Vet Med 2008;6: 145–150. 8. Chang YF, et al. Antibiotic treatment of experimentally Borrelia burgdorferi-infected ponies. Vet Microbiol 2005;107: 285–294. 9. Dohoo I, et al. Attributes of the test per se. In: Veterinary Epidemiologic Research. 2nd ed. Charlottetown, Prince Edward Island, Canada: VER Inc., 2009:97–100. 10. Foley JE, et al. Ecology of Anaplasma phagocytophilum and Borrelia burgdorferi in the western United States. J Vector Ecol 2004;29:41–50. 11. Franzén P, et al. Acute clinical, hematologic, serologic, and polymerase chain reaction findings in horses experimentally infected with a European strain of Anaplasma phagocytophilum. J Vet Intern Med 2005;19:232–239. 12. Johnson AL, et al. Validation of an in-clinic enzyme-linked immunosorbent assay kit for diagnosis of Borrelia burgdorferi infection in horses. J Vet Diagn Invest 2008;20:321–324. 13 Leighton PA, et al. Predicting the speed of tick invasion: an empirical model of range expansion for the Lyme disease vector Ixodes scapularis in Canada. J Appl Ecol 2012;49: 457–464. 14. Liang FT, et al. Characterization of a Borrelia burgdorferi VlsE invariable region useful in canine Lyme disease serodiagnosis by enzyme-linked immunosorbent assay. J Clin Microbiol 2000;38:4160–4166. 15. Madigan JE, Gribble D. Equine ehrlichiosis in northern California: 49 cases (1968–1981). J Am Vet Med Assoc 1987;190:445–448.
Downloaded from vdi.sagepub.com at Bobst Library, New York University on July 10, 2015
Comparison between serologic tests for EGA and LB 16. Magnarelli LA, et al. Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infections in horses from the northeastern United States. J Am Vet Med Assoc 2000;217:1045–1050. 17. Ogden NH, et al. Active and passive surveillance and phylogenetic analysis of Borrelia burgdorferi elucidate the process of Lyme disease risk emergence in Canada. Environ Health Perspect 2010;118:909–914. 18. Ogden NH, et al. Investigation of genotypes of Borrelia burgdorferi in Ixodes scapularis ticks collected during surveillance in Canada. Appl Environ Microbiol 2011;77:3244–3254. 19. Schvartz G, et al. Seroprevalence of equine granulocytic anaplasmosis and Lyme borreliosis in Canada as determined by a point-of-care ELISA. Can Vet J. In press. 20. Sim J, Wright CC. The kappa statistic in reliability studies: use, interpretation, and sample size requirements. Phys Ther 2005;85:257–268. 21. Uehlinger FD, et al. Granulocytic anaplasmosis in a horse from Nova Scotia caused by infection with Anaplasma phagocytophilum. Can Vet J 2011;52:537–540. 22. Wagner B, et al. Development of a multiplex assay for the detection of antibodies to Borrelia burgdorferi in horses and its validation using Bayesian and conventional statistical methods. Vet Immunol Immunopathol 2011;144:374–381.
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23. Wagner B, et al. Antibodies to OspC, OspF and C6 antigens as indicators for infection with Borrelia burgdorferi in horses. Equine Vet J 2013;45:533–537. 24. Veronesi F, et al. Evaluation of the performance of a rapid enzyme-linked immunosorbent assay in the detection of Anaplasma phagocytophilum antibodies in horses. Vector Borne Zoonotic Dis 2014;14:317–323. 25. Woldehiwet Z. The natural history of Anaplasma phagocytophilum. Vet Parasitol 2010;167:108–122.
Authors’ contributions G Schvartz drafted the manuscript. G Schvartz and KL Lohmann contributed to acquisition, analysis, and interpretation of data. T Epp contributed to analysis and interpretation of data. HJ Burgess contributed to acquisition and interpretation of data. NB Chilton contributed to interpretation of data. G Schvartz, T Epp, HJ Burgess, NB Chilton, and KL Lohmann substantially contributed to conception and design; critically revised manuscript; gave final approval; and agree to be accountable for all aspects of the work in ensuring that questions relating to the accuracy or integrity of any part of the work are appropriately investigated and resolved.
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