Evaluation of a Canine C 6 ELISA Lyme Disease Test for the Determination of the Infection Status of Cats Naturally Exposed to Borrelia burgdorferi*

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1 Veterinary Therapeutics Vol. 4, No. 2, Summer 2003 Evaluation of a Canine C 6 ELISA Lyme Disease Test for the Determination of the Infection Status of Cats Naturally Exposed to Borrelia burgdorferi* Steven A. Levy, VMD a Thomas P. O Connor, PhD b Jancy L. Hanscom, BS b Paulette Shields, BS b a Durham Veterinary Hospital PC 178 Parmelee Hill Road Durham, CT b IDEXX Laboratories Department of Research and Development Companion Animal Group Westbrook, ME ABSTRACT The efficacy of a commercially available inoffice kit (SNAP 3Dx, IDEXX Laboratories) for detection of antibodies directed against an invariable region (IR 6 ) of the B. burgdorferi surface protein VlsE (Vmp-like sequence, Expressed), a surface antigen of the spirochete recognized during active infection has been evaluated in dogs. The present study was conducted to determine whether this in-office test could be useful for detection of antibodies to B. burgdorferi in cats. Cats owned by clients of a veterinary hospital located in an area hyperendemic for Lyme disease were included in the study. When possible, cats with an outdoor lifestyle, bite wounds, or current tick infestation were recruited for the study to help ensure that animals with a likelihood of exposure to natural infection by B. burgdorferi would be included in the test group. Of the 24 cats tested, 17 samples were positive for antibodies to B. *Funding for this study was provided by IDEXX Laboratories, Westbrook, ME. burgdorferi by the C 6 ELISA kit. For all 17 of these samples, a duplicate sample tested by immunofluorescent assay (IFA) was in agreement with the ELISA. Five samples were negative by both assays. Two samples that were negative by the C 6 ELISA test had low IFA titers (1:100). One of these two discrepant samples was negative and one was positive for antibodies to B. burgdorferi by the Western blot test. It was concluded that the C 6 ELISA test performed with good agreement with the IFA and Western blot tests for detection of antibody to B. burgdorferi in the majority of cats tested. The test offers the advantages of producing a result rapidly (approximately 8 minutes), and it requires only two drops of serum, plasma, or whole blood. INTRODUCTION Canine Lyme borreliosis is a multisystemic illness caused by the spirochete Borrelia burgdorferi and vectored by ticks in the Ixodes ricinus complex In endemic areas, 50% or more of unvaccinated dogs have been reported 172

2 S. A. Levy, T. P. O Connor, J. L. Hanscom, and P. Shields to be infected by the organism, 3,4,10,11 and significant clinical syndromes involving limb and joint abnormalities have been reported. 1 6,9 12 Cats living in endemic Lyme disease areas have been reported to exhibit signs of limb and joint disorders and to be positive for antibodies induced by exposure to Borrelia burgdorferi. 13 Cats suspected of having Lyme disease also have demonstrated fever and anorexia. Signs are often intermittent and can resemble those observed from other conditions, such as bite-wound cellulites or trauma of unknown cause. Treatment with appropriate antibiotics (frequently amoxicillin) has resulted in a resolution of these signs. a The efficacy of a commercially available inoffice kit (SNAP 3Dx, IDEXX Laboratories) for detection of antibodies directed against an invariable region (IR 6 ) of the B. burgdorferi surface protein VlsE (Vmp-like sequence, Expressed), a surface antigen of the spirochete recognized during active infection, has been evaluated in dogs. 23,24 The C 6 ELISA test is not cross-reactive with antibodies induced by vaccination with either recombinant B. burgdorferi outer-surface protein A or wholecell bacterin. In addition to antibodies induced by infection with B. burgdorferi, the test detects antibodies induced by infection with Ehrlichia canis and antigen produced by Dirofilaria immitis. In dogs, the test is highly sensitive and specific, 23 but its application for determination of the infection status of cats living in an area highly endemic for Lyme disease had not been previously evaluated in a clinic setting. The present study was conducted to determine whether this test could provide a simple, rapid, and economical means for the practicing veterinarian to determine whether a cat has been infected by B. burgdorferi. Samples were tested immediately follow- a Steven A. Levy: Unpublished data, Durham Veterinary Hospital, Durham, CT, ing collection using the in-clinic C 6 ELISA test kit. An additional aliquot was frozen for subsequent laboratory analysis by immunofluorescent antibody (IFA) assay; discrepant samples were tested by Western blot (WB). MATERIALS AND METHODS Cats and Sample Collection Cats (n = 24) owned by clients at the Durham Veterinary Hospital PC in Durham, CT, an area hyperendemic for Lyme disease, were included in the study. When possible, cats with an outdoor lifestyle, bite wounds, or current tick infestation were recruited for the study to help ensure that animals with a likelihood of exposure to natural infection by B. burgdorferi would be included in the test group. A wholeblood sample was collected from each cat and serum or plasma was extracted from each sample. The C 6 ELISA test can be used with canine whole blood, serum, or plasma. All samples were initially tested in the clinic using the C 6 ELISA diagnostic kit. Serum or plasma from each cat was frozen for later evaluation by IFA, WB, or both. C 6 ELISA Test The ELISA for antibody to the C 6 peptide was performed using the commercial test kit (SNAP 3Dx) licensed for use in dogs. The assay uses a proprietary device, which provides reversible chromatographic flow of sample, and automatic, sequential flow of wash and enzyme substrate. The C 6 synthetic peptide was conjugated to bovine serum albumin (BSA) and to horseradish peroxidase (HRP) using standard methods. 25 The BSA peptide conjugate was deposited onto a porous flow matrix, which was spotted using semiautomatic dispensers. The HRP C 6 peptide conjugate was contained in a conjugate diluent containing HRP-labeled antiheartworm antibody, HRP-labeled E. canis peptide conjugate, nonspecific proteins, and 173

3 Veterinary Therapeutics Vol. 4, No. 2, Summer 2003 detergent. Two drops of test sample were mixed with five drops of conjugate and applied to the flow matrix as described in the kit package insert. Only the designated site for B. burgdorferi antibody testing was used on the device; samples from cats in this study were PC NC Figure 1. Examples of Western blot assay results for detection of Borrelia burgdorferi antibodies in serum samples from selected cats in a clinical evaluation of an in-office canine C 6 ELISA test. Bands 1, 2, and 3 are results from three cats that were positive for B. burgdorferi antibodies by the C 6 ELISA test and by immunofluorescent assay. Bands 4 and 5 were from two cats that were negative for B. burgdorferi antibodies by the C 6 ELISA test and by immunofluorescent assay. The PC band is the B. burgdorferi antibody positive feline control; the NC band is the B. burgdorferi antibody negative feline control. not evaluated for heartworm antigen or E. canis antibodies. The B. burgdorferi antibodies (if present) in the sample would bind to the synthetic peptide HRP conjugate and to the synthetic peptide BSA conjugate. The deposited BSA C 6 peptide conjugate was exposed to wash and substrate reagents in the course of the assay. The C 6 ELISA test was considered positive if a blue color developed in the area of deposition of the BSA C 6 peptide conjugate. Immunofluorescent Assay An indirect IFA was performed for all samples using whole-cell B. burgdorferi antigen coated IFA slides (Bion Enterprises) and fluorescein isothiocyanate (FITC) labeled goat antifeline IgG (Jackson ImmunoResearch). Dilutions of test samples were made in phosphate-buffered saline and incubated with the antigen-coated IFA slides. The slides were washed, incubated with FITC-labeled conjugate, and viewed using ultraviolet light microscopy. Samples with IFA titers 1:100 or greater were considered positive. Any sample tested by IFA that was not in agreement with results obtained by the C 6 ELISA test was subsequently tested by WB. Western Blot Assay WB tests for B. burgdorferi antibodies were performed at the Connecticut Veterinary Diagnostic Laboratory at the University of Connecticut, Storrs, CT (Figure 1). Purified B. burgdorferi was disrupted with sodium dodecyl sulfate, separated on a 12% polyacrylamide gel, and transferred to nitrocellulose. Individual strips were blocked and incubated with a 1:100 dilution of the sample for 90 minutes. The strips were washed and incubated for 60 minutes with affinity purified goat antifeline HRP conjugate. The substrate incubation was stopped by removing the substrate solution 174

4 S. A. Levy, T. P. O Connor, J. L. Hanscom, and P. Shields TABLE 1. C 6 ELISA and Immunofluorescent Assay (IFA) Results for Feline Serum or Plasma Samples Tested for Antibodies to Borrelia burgdorferi in a Veterinary Clinic in an Area Endemic for Lyme Disease CAT ID C 6 ELISA IFA Titer* F1 Positive 1:400 F2 Negative Negative F3 Negative Negative F4 Positive 1:400 F5 Negative 1:100 F6 Negative Negative F7 Positive 1:100 F8 Positive 1:100 F9 Positive 1:100 F10 Positive 1:100 F11 Negative 1:100 F12 Positive 1:100 F13 Positive 1:400 F14 Positive 1:100 F15 Positive 1:400 F16 Positive 1:1600 F17 Positive 1:1600 F18 Negative Negative F19 Positive 1:100 F20 Positive 1:100 F21 Negative Negative F22 Positive 1:400 F23 Positive 1:100 F24 Positive 1:400 *IFA titers 1:100 or higher were considered positive. Results shown in bold indicate tests that did not have agreement between the two assays. and washing the strips four or five times with deionized water. WB bands produced by individual samples were identified by comparison with bands produced by known positive and negative control feline serum samples. RESULTS Results of C 6 ELISA and IFA analyses are shown in Table 1. Of the 24 cats tested, 17 samples were positive for antibodies to B. burgdorferi by the C 6 ELISA kit. For all 17 of these samples, the corresponding sample tested by IFA was in agreement with the ELISA. Five samples were negative by both assays. Two samples that were negative by the C 6 ELISA test (Cats F5 and F11) had low IFA titers (1:100). These two discrepant samples were tested by WB, and by this assay, Cat F5 was negative and Cat F11 was positive for antibodies to B. burgdorferi. DISCUSSION The significance of Lyme disease as a clinical entity in cats is not as clearly understood as it is in dogs. Naturally exposed cats have been reported to have positive titers to B. burgdorferi and exhibit clinic signs, such as fever and lameness. 13 These cats do appear to respond to antibiotic therapy. Yet, the development of a laboratory model for feline Lyme disease has been problematic. One laboratory has reported success in producing infections in cats but has had no success in producing clinical signs or pathology. 26 However, another laboratory was able to produce infection, clinical signs, and pathologic changes in affected tissues. 27 The common occurrence of bite wound trauma in cats further confounds the clinical picture because wounds may often be small and hidden by the fur coat. Many cats with fever and lameness are treated 175

5 Veterinary Therapeutics Vol. 4, No. 2, Summer 2003 for a presumptive diagnosis of bite wound cellulites, even if no wounds are found. Using the canine C 6 ELISA, a majority of the cats sampled in this study (71%) demonstrated evidence of infection by B. burgdorferi. This C 6 ELISA test, which is capable of detecting antibodies to B. burgdorferi in whole blood, serum, or plasma from dogs, was found to be capable of detecting antibodies to B. burgdorferi in feline serum and plasma samples. It may also be appropriate for use with feline whole-blood samples; however, this was not tested in the current study. The correlation of positive results by C 6 ELISA test with positive results by the IFA assay was 100%, and all five samples negative by IFA were also negative using the C 6 in-clinic test kit. However, two samples that were negative according to the C 6 ELISA test were positive by IFA (titer = 1:100). Of these two discrepant samples, one was positive and one was negative by WB testing. These two samples likely represent weak positive samples or cross-reactive negative samples that are very close to the cutoff for these assays. When a veterinarian is presented with a cat exhibiting signs of limb or joint abnormality, fever, and anorexia, but with no clear evidence of bite wound or other trauma, it would be useful to know the status of the cat for B. burgdorferi infection. After ruling out other diagnoses, veterinarians could consider testing the animal by the C 6 ELISA for antibodies to B. burgdorferi, particularly in areas endemic for Lyme disease. A presumptive diagnosis of Lyme disease could be made for cats positive by this test. This diagnostic paradigm has served well for dogs with clinical cases of Lyme disease 1,2 and may similarly serve as a clinical diagnostic standard for cats. A consideration for further study and clinical impact is the effect of coinfection by Anaplasma phagocytophila (formerly known as Ehrlichia equi), the agent of granulocytic ehrlichiosis also transmitted by Ixodes scapularis ticks. An earlier study revealed that 73% of cats positive for infection with B. burgdorferi were also positive for the granulocytic ehrlichiosis organism. a Although the clinical significance of granulocytic ehrlichiosis in cats is not clearly understood, many tick-borne infections have been reported to be more likely to produce clinical disease following infection by multiple organisms. Identifying a cat with signs of Lyme disease and a positive C 6 ELISA test is an indication that the cat has been parasitized by I. scapularis, the vector of both Lyme disease and granulocytic ehrlichiosis. The risk of coinfection may be an indication to choose an antibiotic (such as doxycycline or tetracycline, both of which have activity against both B. burgdorferi and A. phagocytophila) as therapy for these cats. The population of cats chosen for sampling in this pilot study included cats that were likely to have tick exposure based on outside lifestyle, bite wounds, and tick parasitism. The authors recognize the importance of sampling a large number of cats on a random basis to identify the rate of infection in cats with varying risk factors, including lifestyle (inside versus outside), use of various tick control products, and age at time of test (which correlates with the duration of potential tick exposure). In summary, the C 6 ELISA test (SNAP 3Dx) was in good agreement with the IFA and WB tests for detection of antibody to B. burgdorferi in the majority of cats tested. The test offers the advantages of producing a result rapidly (approximately 8 minutes), and it requires only two drops of serum, plasma or whole blood). ACKNOWLEDGMENTS The authors wish to thank Dr. Sandra Bushmich and Marilyn Jezek from the Connecticut Veterinary Diagnostic Laboratory at the University of Connecticut for conducting the Western blot evaluations in this study. 176

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