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Serological testing of horses for antibodies to the spirochete Borrelia Burgdorferi using enzyme-linked immunosorbent assay and western immunoblotting.

KRISTEN HAGSTOZ (1)

JAMES MCGOVERN (1)

MARYBETH HAMORSKI (2)

JANE A. ARMSTRONG (1)

ABSTRACT Blood samples were collected from 100 horses in the Northwest area of New Jersey, a region of the United States known to be endemic for borreliosis (Lyme Disease). Horses were asymptomatic for borreliosis at the time of sampling. Sera were examined by the enzyme-linked immunosorbent assay (ELISA) and western immunoblot assay using whole cell preparations as antigen to detect equine IgG antibodies to the spirochete Borrelia burgdorferi. Antibody titers to B. burgdorferi were detected in 90 (90%) serum samples by ELISA, with 15 (17%) of the positive horses having reciprocal antibody titers of 10,240 or greater. On western blot analysis, 11 of the 12 (92%) of the samples with the highest ELISA titers reacted with the B. burgdorferi whole-cell lysate. The seven reactive bands had molecular weights of 85, 60, 46, 41, 39.5, 31 kilodaltons (kDa). The negative control samples were unreactive in both assays. The results of the ELISA and western immunoblot analysis of the horses included in this study confirm exposure to the spirochete, but are not a valid indicator of borreliosis in this endemic area.

KEY WORDS: Borrelia burgdorferi, Lyme Disease, Borreliosis, Enzyme-Linked Immunosorbent Assay (ELISA), Western Immunoblotting

INTRODUCTION

Borreliosis is currently the most frequently diagnosed tick borne bacterial disease in the United States (Lyme Disease--United States 1991-1992, 1993). Lyme disease is endemic in three regions of the country: the Northeast, northern Mid-west, and in areas of California. Borreliosis is caused by the spirochete Borrelia burgdorferi which is transmitted by the deer tick, Ixodes scapularis, to humans and other animals. The white- footed mouse, Peromyscus leucopus, is thought to be the enzootic reservoir of the spirochete. The nymphal to adult stages of the deer tick feed on wild and domestic animal species.

Although Lyme borreliosis was first formally recognized in the late 1970s, B. burdorferi DNA has been identified in museum specimens of deer ticks collected in the 1940s from Long Island, New York (Persing et al., 1990). In humans, progression of the disease occurs in three stages. In the first stage, a rash or a localized inflammation may develop two to thirty days after exposure. Anorexia, lethargy, body aches, and fever may also occur. The second stage is characterized by musculoskeletal pain affecting various areas of the body, with neurological, urinary and cardiovascular complications in some cases. The third stage usually involves the onset of arthritis that primarily affects the larger joints. Severe neurologic complications such as meningitis, paralysis, and dementia can develop at this stage. The clinical signs of borreliosis in horses can be variable and may mimic other equine diseases.

A number of serological tests have been used for the diagnosis of borreliosis; these include the indirect fluorescent antibody test (IFA), enzyme-linked immunosorbent assay (ELISA), and western immunoblot analysis. All of these tests rely on the detection of antibodies produced by the horse in response to the spirochete surface antigens.

Early studies performed at the time the disease was first recognized investigated seroprevalence of the infection in horses and found marked differences between sample groups depending on geographic location (Bernard et al., 1990; Cohen et al., 1988; Marcus et al., 1985; Fikrig et al., 1993; Cohen et al., 1992; Browning et al., 1993).

The purpose of this study was to examine the prevalence of equine infection with B. burgdorferi in an established endemic area. The presence of antibodies in sera was determined using ELISA and western immunoblot procedures.

MATERIAL AND METHODS

Sampling design

Blood samples were collected from horses that resided in Hunterdon, Morris, Sussex and Warren Counties in northern New Jersey. Samples were collected by an equine practitioner and analyzed between July of 1996 and January 1998. The clinical history of each horse included in the study was recorded. Blood samples were collected from horses that were considered to be in a state of normal health at the time of collection. None of the horses had a previous history of borreliosis.

Serologic analyses

Blood was aseptically collected from the jugular vein in a Vacutainer tube, SST gel and clot activator (1) and centrifuged at 2000 rpm for 10 minutes. Serum was aspirated and dispensed into a 1.5 ml microfuge tubes and stored at -40[degrees]C. Serum samples were analyzed within one month of collection.

ELISA

Sera were tested by ELISA, an enzyme-linked immunosorbent assay method (Magnarelli et al., 1989) for the presence of antibodies to B.burgdorferi. Three analyses were performed for each serum sample. Washed, whole cell preparations of B. burgdorferi (strain 2591) were obtained from the laboratory of L. Magnarelli (Magnarelli et al., 1989) and coated to wells at a concentration of 2-3ug of protein/ml. Protein concentration was determined by standard method. (2) Non-specific binding sites on the polystyrene plates were blocked with goat serum (0.5% in phosphate buffered saline solution). Peroxidase-labelled affinity purified polyvalent antibody to horse immunoglobulin G (IgG) heavy and light chain was used as the secondary antibody. (3) The optical density (O.D.) values at 405 nanometers were obtained using a microtiter plate photometer. (2) The highest serial serum dilution in which antibody bound to absorbed antigen was reported as the reciprocal titer. For each serum dilution, negative serum samples were anal yzed in triplicate and the cut-off O.D. value was calculated by adding 3 SD to the mean (Magnarelli et al., 1989). Absorbance values exceeding the cut-off values were considered positive. Positive serum samples of known titer were analyzed for each serum dilution.

Western Immunoblot Analysis

Serum samples were submitted to the Veterinary Diagnostic Laboratory, Cornell University, New York State College of Veterinary Medicine, Ithaca, New York. Western blot analysis, a diagnostic test based on serum antibody recognition of B. burgdorferi whole cell lysate antigens, was performed (Dressler et al., 1993). Two analyses were performed for each sample.

RESULTS

Clinical Records

All horses were considered clinically normal. A review of clinical records revealed no evidence of symptoms relevant to the diagnosis of borreliosis and therefore the horses were not suspected of having current or past borreliosis. None of the horses included in the study had a record of prior immunization to B. burgdorferi.

Serum Antibody Titers

Serum samples from 100 horses were analyzed for antibody titers to B. burgdorferi by ELISA. Antibody titers against B. burgdorferi whole cell preparations were detected in ninety of the 100 (90%) serum samples using anti-horse IgG secondary antibody. Results were reported as reciprocal antibody titers and ranged from 1:640 to 1:40,960 (Fig. 1). Horses with antibody titers of 1:160 or greater are considered seropositive; therefore, 90% of the horses were seropositive. Fifteen (17%) of the reactive serum samples had reciprocal titers of 10,240 or greater. Ten (10%) of the serum samples were considered negative.

Specificity of Antibodies to Spirochete Antigens

Samples with a reciprocal titer of 10,240 or greater were additionally tested by western immunoblot analysis. Analysis confirmed the ELISA results for eleven of the twelve horses with the highest titers. Eleven of the serum samples reacted with the B. burgdorferi whole cell lysates and exhibited distinct reactivity with at least three of the immunodominant proteins (Fig. 2). Seven reactive bands with the molecular masses of 85, 60, 46, 41, 39.5, 35, and 31 (kDa) were visualized. Three bacterial proteins, flagellin (41 kD) OSP B (35 kD), and OSP A (31 kD) used as an indication of antibody specificity for B. burgdorferi were identified. Negative control samples were unreactive.

DISCUSSION

This study showed that 90% of the horses surveyed in four counties in northern New Jersey had antibodies to B. burgdorferi using whole cell lysates. All of the horses with antibody titers were asymptomatic at the time they were sampled. This even applied to seropositive individuals with the highest ELISA titers and positive western immunoblot results. The large percentage of positive samples indicates that substantial numbers of horses in the area encounter ticks and are exposed to the spirochete. At the time of the study, there was no evidence of any correlation between the titer of the antibody response and a history of illness suggestive of borreliosis. Although it would have been of interest to follow the health history of these horses, participation in the study was voluntary and many horses are no longer available to continue the study.

The results of a previous study (Bernard et al. 1990) indicated that 18% of the equine population surveyed in New Jersey tested positive. Cohen et al. (1988) reported an overall seroprevalence of 12% in areas of New Jersey and Pennsylvania but reported 60% in horses residing on one farm in central New Jersey were serologically positive to B. burgdorferi. The findings of this study suggest that there has been an increase in the percentage of seropositive horses in the state. The use of B. burgdorferi whole cell lysates for ELISA can result in false-positive reactions due to antibody reactivity to flagellin and other common antigens (Magnarelli et al., 2000). The previous equine studies also used whole cell lysates, and therefore this cannot explain the increase in seropositive horses. Analysis of the serum samples by ELISA, IFA or western immunoblot assay using recombinant antigens of B. burgdorferi could be used to eliminate any false-positive samples.

It can be concluded from this study that the presence of antibody is not necessarily an indicator of disease in horses in this endemic area. It is likely that the seropositive horses were infected with B. burgdorferi but remained asymptomatic. The appearance of multiple reactive bands in the western blots suggests that the horses were in the later stages of infection (Magnarelli et al., 2000). This may indicate that the spirochete persists within these individuals in the absence of any clinical manifestations of disease. Were this to be true, the equine species could act as a host and chronic carrier of the spirochete. Based on the widespread evidence of infection as suggested in this study it would be worth investigating the possible long-term health effects of repeated exposure to B. burgdorferi or chronic infection with the organism.

AKNOWLEDGEMENTS

The authors wish to thank Dr. Louis Magnerelli and his staff for their contributions and Eric Stewart Roberts for his technical assistance.

(1.)CENTENARY COLLEGE, DEPARTMENT OF MATHEMATICS AND NATURAL SCIENCES HACKETTSTOWN, NEW JERSEY 07840

(2.)CALIFON ANIMAL HOSPITAL CALIFON, NEW JERSEY 07830

(1.)Becton Dickinson, Franklin Lakes, New Jersey

(2) Bio-Rad Laboratories, Richmond, CA

(3) Kirkegaard & Perry Laboratories, Gaithersburg, MD

BUSHMICH, S.L. 2001. Lyme Disease in Horses. www.aaep.org/ownereducation/lyme

LITERATURE CITED

BERNARD W., D. COHEN, E. BOSLER, AND D. ZAMOS. 1990. Serologic survey for Borrelia burgdorferi antibody in horses referred to a mid-Atlantic veterinary teaching hospital. J Am Vet Med Assoc. 196(8): 1255-1268.

BROWNING A., S. CARTER, A. BARNES, C. MAY, AND D. BENNETT. 1993. Lameness associated with Borrelia burgdorferi infection in the horse. Vet Rec. 132: 610-611.

COHEN D., E. BOSLER, W. BERNARD, D. MEIRS II, R. EISNER, AND T. SCHULZE. 1988. Epidemiologic studies of lyme disease in horses and their public health significance. Ann NY Acad of Sciences. 539:244-257.

COHEN N., F. HECK, B. HEIM. D. FLAD, E. BOSLER, AND D.COHEN. 1992. Seroprevalence of antibodies to Borrelia burgdorferi in a population of horses in central Texas. J Am Vet Med Assoc. 201(7): 1030-1034.

DRESSLER F., J. WHALEN, B. REINHARDT, AND A. STEERE. 1993. Western blotting in the serodiagnosis of lyme disease. J Inf Dis. 167:392-400.

FIKRIG E., L MAGNARELLI, M. CHEN, J. ANDERSON, AND R. FLAVELL. 1993. Serologic analysis of dogs, horses, and cottontail rabbits for antibodies to an antigenic flagellar epitope of Borrelia burgdorferi. J Clin Microbiol. 31(9): 2451-2355.

LYME DISEASE--UNITED STATES, 1991--1992. 1993. Mor and Mort Wkly Rep. 42(18):345-348.

MAGNARELLI L, AND J. ANDERSON. 1989. Class-specific and polyvalent enzyme -linked immunosorbent assays for detection of antibodies to Borrelia burgdorferi in equids. J Am Vet Med Assoc. 195(10):1365-1368.

MAGNARELLI L., J. IJDO, A. VAN ANDEL, C. Wu, S. PADULA, AND E. FIKRIG. 2000. Serologic confirmation of Ehrlichia equi and Borrelia burgdorferi infectiors in horses from the northeastern United States. J Am Vet Med Assoc. 217(7):1045-1050.

MARCUS L, M. PATTERSON, R. GILFILLAN, AND P. URBAND. 1985. Antibodies to Borrelia burgdorferi in New England Horses: serologic survey. Am J Vet Res. 46(12):2570-2571.

PERSING D. S. TELFORD III, P. RYS, D. DODGE, T. WHITE, S. MALAWISTA, AND A. SPEIELMAN 1990. Detection of Borrelia burgdorferi DNA in museum specimens of Ixodes dammini ticks. Science. 249:1420-1423.

[Figure 1 omitted]
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Article Details
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Author:Hagstoz, Kristen; McGovern, James; Hamorski, Marybeth; Armstrong, Jane A.
Publication:Bulletin of the New Jersey Academy of Science
Geographic Code:1USA
Date:Sep 22, 2001
Words:2086
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