Veterinary Microbiology, 26 ( 1991 ) 179-190 Elsevier Science Publishers B.V., A m s t e r d a m

179

Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog R.T. Greene*, R.L. Walker**, W.L. Nicholson and J.F. Levine Department of Microbiology, Pathology, and Parasitology, College of Veterinary Medicine, North Carolina State University, Raleigh, NC 27606, USA (Accepted 4 July 1990)

ABSTRACT Greene, R.T., Walker, R.L., Nicholson, W.L. and Levine, J.F., 1991. Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog. Vet. Microbiol., 26:179-190. An enzyme-linked immunosorbent assay (ELISA) was compared to an indirect immunofluorescence assay (IFA) for detection of IgG antibodies to Borrelia burgdorferi in dog sera. The concordance of the two tests was 93.5% for sera from dogs from Maryland ( n = 93 ), 98.0% for sera from dogs from North Carolina (n = 446 ), and 97.2% for the combined sample groups (n = 539). Twenty-five of the 27 samples with discordant or low positive results were tested, and showed immunoblot reactions to 1 to 10 different bands. Reaction patterns and intensity of the bands were quite variable, and did not explain a reason for the discordance.

INTRODUCTION

Lyme disease (Lyme borreliosis) is a tick-borne spirochetal disease caused by Borrelia burgdorferi. The clinical manifestations of Lyme disease in humans have recently been reviewed in detail (Steere, 1989 ). Infections with B. burgdorferi have also been reported in a number of other mammals, including dogs (Steere, 1989). In dogs, the more common clinical manifestations are lameness, lethargy, and lymphadenopathy (Lissman et al., 1984; Kornblatt *Present address: Justus Liebig Universit~it, Bakteriologie u n d Immunologic, FB 18, Frankfurter Str. 107, D-6300 Giessen, F R G . R e p r i n t request to: Russell T. Greene, c / o Dr. Leroy Coggins, M P P D e p a r t m e n t , N o r t h Carolina State University, 4700 Hillsborougb St, Raleigh, N C 27606, USA. **Present address: California Veterinary Diagnostic Laboratory System, University of California, Davis, CA, 95616, USA.

0378-1135/91/$03.50

© 1991-

Elsevier Science Publishers B.V.

1 ~0

R.T. GREENE ET .sd~.

et al., 1985; Magnarelli et al., 1985b). A few cases of renal disorders (Bosler et al., 1988; Grauer et al., 1988; Magnarelli et al., 1987) heart block (Levy and Duray, 1988), and development of rheumatoid arthritis (Roush et al., 1989) have been reported, but are not yet considered definitive manifestations of the disease. Attempts to isolate B. burgdorferi from infected patients are often unsuccessful. Therefore, the diagnosis of Lyme disease is often based on history of a tick bite, compatible clinical signs, exclusion of other diseases, positive serology, and response to therapy (Steere, 1989). The indirect immunofluorescence assay (IFA) and enzyme-linked immunosorbent assay (ELISA) are both used for detection of antibodies to B. burgdorferi in human sera (Craft et al., 1984; Magnarelli et al., 1984; Russell et al., 1984; Cutler and Wright, 1989). Botla testing methods have been shown to be most sensitive in latestage disease (Magnarelli et al., 1985a; Steere, 1989). It has been shown that dogs are at greater risk of exposure tlaan humans (Eng et al., 1988) and that monitoring the prevalence of antibodies to B. burgdorJi'ri in dogs may be a useful method for identifying geographical areas of potential exposure (Schulze et al., 1986; Eng et al., 1988). Serosurveys for anti-B, burgdorferi antibodies in dogs have commonly used the IFA methodology (Kornblatt et al., 1985; Magnarelli et al., 1985b; Burgess, 1986; Eugster and Angulo, 1986; Rawlings, 1986; Schulze et al., 1986; Magnarelli et al., 1987; Doby et al., 1988; Euzeby and Raffi, 1988; Gilfillan et al., 1988; Greene et al., 1988b; Teitler et al., 1988; Euzeby, 1989; Hansen and Dietz, 1989; Rodgers et al., 1989 ) while ELISAs have been used less often (Magnarelli et al., 1987; Bosler et al., 1988; Bryant et al., 1988; Liu et al., 1988 ). The ELISA is preferable to the IFA for epidemiological surveillance of dogs because of its ease of performance, its potential for automation, and its objective colored end-product. Additionally, the EL1SA methodology is more suited for application as a clinical diagnostic test for use by veterinary practitioners. Therefore, tl~e purpose of this report was to compare an ELISA to an 1FA for detection of IgG antibodies to B. burgdorjkri in dog sera.

MATERIALS

AND METHODS

Bacterial strains Borrelia burgdorferi B31 (American Type Culture Collection 35210), Escherichia coli (ATCC 25922 ), Borrelia anserina Es-m (kindly provided by H. John Barnes, North Carolina State University, Raleigh, NC), and Leptospira interrogans serovar canicola (kindly provided by Carol Willis, Rollins Animal Disease Diagnostic Laboratory, Raleigh, NC) were used in this study.

IFA AND EL1SA T E S T I N G FOR BORRELL4 BURGDORFERI IN DOGS

18 1

Serum samples Canine samples ( n -- 93 ) were collected from counties in eastern Maryland, a known endemic focus for Lyme disease (Thambidurai et al., 1988). Samples (n---446) considered to be from a non-endemic area were collected at the Veterinary Teaching Hospital in Raleigh, NC.

Indirect immunofluorescence assay The IFA for detection of antibodies in clog sera was performed as previously described, using goat-anti-dog IgG (gamma) (Kirkegaard & Perry Laboratories) diluted 1:400 (Greene et al., 1988b). Positive, negative, and saline controls were included on each slide. Titers were expressed as the reciprocal of the highest serum dilution showing distinct fluorescence of at least 50% of the spirochetes in each field. As has been used in other studies, a reciprocal titer less than 64 was considered negative.

Enzyme-linked immunosorbent assay The ELISA was conducted as a modification of that previously described (Greene et al., 1988c). Briefly, spirochetes were washed three times using phosphate buffered saline (pH 7.4) (PBS). After the final wash, the pellet was resuspended in enough PBS that a 1 : 4 dilution of the suspension had an absorbance of 0.222 when measured spectrophotometrically (MR 700 Dynatech EEISA reader) at 410 nm. The suspension was sonicated as described (Greene et al., 1988c) and the protein concentration (Bradford method, BioRed Laboratories, Richmond, CA) adjusted to 0.3 mg/ml. This stock antigen was stored in 50 #1 aliquots at - 7 0 ° C until used, A 1:200 dilution of stock antigen in carbonate-bicarbonate buffer was applied (50/A per well) to the wells of a flat-bottomed, polystyrene plate (Flow Laboratories, McLean, VA). The subsequent assay steps were the same as previously reported (Greene et al., 1988c), using conjugated goat anti-dog IgG (gamma) diluted 1 : 500. Positive control sera were obtained from an outdoor dog residing in an area (Talbot county, MD) where Lyme disease is endemic (Thambidurai et al., 1988). The dog had a history of lameness with swelling in the left carpus. Unidentified ticks were found on the dog. The serum was tested for antinuclear antibodies, rheumatoid factor, and IgG against B. burgdorferi by IFA and immunoblot analysis. To further characterize the positive control, the sera was first adsorbed with B. burgdorferi, L. interrogans serovar canicola, B. anserina, and E. coli and then tested by immunoblot analysis. Pooled sera from three, clinically normal, laboratory-raised Beagle dogs constituted negative control sera. Absorbance values at 410 nm were converted into arbitrary units, termed ELISA values, which were calculated as: ((A~es~-Ancg)/ (Apos-Aneg)) X 100. This value relates the mean (of duplicate wells) absorbance of the test sample (Atest) to that of the positive (Avo~) and negative (An~g) controls (tripli-

182

R.T. GREENE ET AL.

cares). By calculating ELISA values, day-to-day variation in room temperature, and substrate reaction times were minimized. The mean ELISA value, plus three standard deviations (SD) of 100 randomly selected, clinically normal dog sera from North Carolina were used to determine the cut-offpoint for a negative classification. All were non-reactive ( < 16) in the IFA.

Immunoblot analysis Sera samples that had discordant or low positive results were further evaluated by immunoblot analysis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cell B. burgdorferi B31 preparations, protein transfer to nitrocellulose paper, and immunochemical detection of antigens were performed using previously described methods (Greene et al., 1998a). RESULTS

Characterization of ELISA controls The positive control serum used in the ELISA was antinuclear antibody and rheumatoid factor negative, and had a reciprocal IFA titer of 1024. Absorbances of the positive control lay generally in the range 0.8-0.9, with the negative control less than 0.1, and the blanks less than 0.005. Multiple intense bands were evident by immunoblot analysis when the positive control sera were reacted with strain B31. The sera reacted strongly with proteins of apparent molecular weights of 17, 22, 31, 39-41, 60-61, 66, and 83 kilodaltons (kDa) (Fig. 1 ). There appeared to be co-migrating bands in some of the regions where strong reactions occurred. A number of lighter bands were also evident. The reactivity of all bands was almost completely eliminated by adsorption with B. burgdorferi. Faint bands could still be visualized in areas corresponding to the most strongly reacting bands prior to adsorption. There was no appreaciable reduction in reactivity when the sera was adsorbed with B. anserina, L. interrogans serovar canicola, or E. coli (Fig. 1 ). Each individual negative control serum tested negative by IFA and immunoblot analysis for IgG antibodies to B. burgdorferi.

Comparison of ELISA and IFA The mean ELISA value for the 100 randomly selected sera samples was 4.43 with a SD of 11.79. Using the mean plus 3 SD of the negative control samples, a cut-off value of 40 was selected, and samples below 40 were considered negative. Fifty (54%) of the 93 samples from Maryland, and 431 (97%) of the 446 samples from North Carolina were negative by the ELISA (Table 1 ). Inthe IFA, 50 (54%) of the samples from Maryland and 438 (98%)

IFA AND ELISA TESTING FOR BORRELIA BURGDORFERI IN DOGS

1 2

3

4

18 3

5

Fig. I. Reaction patterns by immunoblot analysis obtained with ELISA positive control sera prior to and after adsorption with various bacterial species. Lanes: ( 1 ) unadsorbed serum; (2) adsorbed with Borrelia burgdorferi; (3) adsorbed with B. anserina; (4) adsorbed with Leptospira interrogans serovar canicola; (5) adsorbed with Escherichia coll. Numbers on the left indicate the apparent molecular mass of the protein standards (in kDa).

o f the samples f r o m N o r t h C a r o l i n a h a d r e c i p r o c a l titers less t h a n 64, a n d were c o n s i d e r e d negative. D i s c o r d a n t ( p o s i t i v e in one assay, negative in the o t h e r ) results were rec o r d e d in six o f the samples f r o m M a r y l a n d a n d n i n e o f the samples f r o m N o r t h Carolina. T h i s gives a c o n c o r d a n c e o f 93.5% for the M a r y l a n d samples

] 84

R.T. GREENE ET AL ~

~

~

~

~

~

~

~

~ ~

,~

0

~

O ~

~

0

0 0

t-,I

~

~

e~

eq

oo

0

0 0

~

~

~

e~

~

~-,

,~-

t-~-~ .~-

~,~ 0

~~ -

0 0

0

~

O 0 0

0

e"h e~h~ ~

0

t'q

~,!

~

~

~

0 0

~

t",l

0

0

0

~

~

O

0

0

0

0

0

0

0

0

0

0

0

0

0

r---

0

~"~

O 0 0

r~

0

O0

¢~ ©

~

~ 0

0

0

0

0

O 0 0

0

0

0

0 ~1

~

:..;

~

~-~ ~ I

~

0

0

0

~

0

0

0

~

0

0

0

0

0

0

~

0

0

0

0

0

0

0

~

~ 2 ~ ~

~1

~

0 0 0 ~ ~1

~

Z

IFA ,ANDELISATESTING FOR BORRELLt BURGDORFERI IN DOGS

185

TABLE 2

Median and range of EL1SA values (EV)1 corresponding to reciprocal I FA titers for 5 39 dog sera Reciprocal l FA titer

Comparison of an enzyme-linked immunosorbent assay to an indirect immunofluorescence assay for the detection of antibodies to Borrelia burgdorferi in the dog.

An enzyme-linked immunosorbent assay (ELISA) was compared to an indirect immunofluorescence assay (IFA) for detection of IgG antibodies to Borrelia bu...
839KB Sizes 0 Downloads 0 Views