Evaluation of a commercial enzyme-linked immunosorbent assay for detection of Borrelia burgdorferi exposure in dogs

OBJECTIVE: To evaluate the effectiveness of a commercially available ELISA kit for detecting antibodies against Borrelia burgdorferi in dogs. SAMPLE POPULATION: Banked sera from 440 military working dogs were used for serologic analyses. PROCEDURE: Serum samples were analyzed for antibodies against B burgdorferi by use of a commercially available ELISA and subsequently by western blot analysis as a confirmatory test. RESULTS: Results from the ELISA indicated that 89 (20%) samples were positive for exposure to B burgdorferi or canine Lyme disease vaccine, and 351 (80%) were negative. Follow-up testing by western blot analysis indicated that results for 109 (25%) samples were positive and 331 (75%) were negative for exposure. All samples that had positive results on the ELISA also had positive results on western blot analysis (true positives). Of the 351 samples that had negative results on the ELISA, only 331 had negative results on western blot analysis (true negatives). The remaining 20 samples had positive results on western blot analysis. By use of a standard 2 x 2 table, it was determined that the ELISA had a sensitivity of 82%, specificity of 100%, positive predictive value of 100%, and negative predictive value of 94%. CONCLUSIONS AND CLINICAL RELEVANCE: The commercial ELISA kit evaluated in this study appeared to lack adequate sensitivity for detecting all potential cases of borreliosis in dogs. The ELISA was also unable to discriminate natural exposure from exposure attributable to vaccination, which could complicate interpretation of positive results and treatment of dogs with clinical signs.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for the detection of allergen-specific IgE antibodies in dogs

Twenty-eight atopic dogs, 22 pruritic, non-atopic dogs and 10 healthy dogs were ELISA tested. For calculations of diagnostic specificity and sensitivity, positive ELISA test results in non-atopic dogs were considered false positive results. The absence of any positive results in the atopic dogs was considered false negative results. The atopic dogs were tested both with ELISA and an intradermal test, utilising allergen extracts from the same manufacturer, to determine the frequency of positive allergen reactions in the ELISA test compared with the intradermal test. The Prausnitz-Küstner test was performed to evaluate the significance of a positive ELISA test result. Based on cross-tabulations with clinically defined atopic dermatitis, the ELISA test showed a sensitivity of 53.6% and a specificity of 84.4%. The correlation between the ELISA and the intradermal test was poor. Positive Prausnitz-Küstner tests were not obtained using sera from dogs that were intradermal test negative for the tested allergens, even though sera had high levels of IgE as measured by the ELISA. These findings question the significance of a positive ELISA test result and indicate that the test is not measuring functional allergen-specific IgE.     

A preliminary study on the use of enzyme-linked immunosorbent assay (ELISA) for detection of Trichinella spiralis infections in dogs

An enzyme-linked immunosorbent assay (ELISA) was developed to detect Trichinella spiralis infections in dogs using rabbit anti-canine IgG-horseradish peroxidase prepared according to the improved periodate method and an antigen purified from T. spiralis larvae by Sephadex G-200 chromatography. Sixty-six canine sera were tested for trichinosis by the ELISA and it showed a detection rate which was significantly higher than that by trichinoscopy. This antigen of T. spiralis appeared not to cross-react with the sera of dogs infected with Ancylostoma caninum or Taenia spp. A comparison of ortho-phenylenediamine and 5-amino-2-hydroxybenzoic acid as substrates in the ELISA did not reveal a significant difference. Pieces of filter paper saturated with a defined quantity of whole blood can be substituted for serum as a source material for the test. The relationship between worm burden and the absorbance value in ELISA is discussed.     

Impact of Giardia vaccination on asymptomatic Giardia infections in dogs at a research facility

Feces were collected from 107 asymptomatic dogs at a research facility in Guelph, Ontario. The prevalence of Giardia infection was 11% (12/107). To assess the effectiveness of Giardia vaccination for treatment of Giardia carriage, 9 additional asymptomatic Giardia antigen-positive dogs were brought into the facility. The Giardia antigen-positive dogs were then randomly allocated to receive either vaccine (n = 10) or a saline placebo (n = 10). Feces were then monitored monthly for 6 mo for Giardia antigen and Giardia cysts. At weeks 4, 8, 12, and 16 following vaccination, there were more Giardia-positive dogs in the vaccinated group (10/10, 9/10, 9/10, 8/10, respectively) compared with the controls (7/10, 7/10, 8/10, 4/10, respectively). At week 20, an equal number of dogs (5/10) were Giardia positive, and at week 24, fewer dogs were positive in the vaccinated group than in the control group (2/10 versus 5/10, respectively). However, there was no significant difference between the 2 groups. Vaccination was, therefore, not an effective treatment for asymptomatic canine Giardia infections in this setting.     

The detection of canine autoantibodies to thyroid antigens by enzyme-linked immunosorbent assay, hemagglutination and indirect immunofluorescence.

Autoantibodies to thyroid antigens in serums from 34 clinically hypothyroid dogs were detected by various methods. Antibodies to thyroglobulin were detectable by enzyme-linked immunosorbent assay (ELISA) in 59%, by chromic chloride hemagglutination in 53% and by indirect immunofluorescence on formalin-fixed, paraffin-embedded, trypsin-digested thyroid tissue in 73% of samples. Antibody to thyroid microsomal antigen was detectable by ELISA in 29% of serums. Indirect immunofluorescence showed cytoplasmic fluorescence of thyroid follicular cells in several serums, however, this could not be confirmed as specific for microsomal antigen by absorption trials. Hemagglutination tests using commercially available tanned red cells coated with human antigens and indirect immunofluorescence assays on formalin-fixed tissue without trypsin digestion, on Bouin's fixed tissue, or on cryostat, methanol-fixed sections, were insensitive. Cryostat sections without methanol fixation were unsuitable due to tissue fragility. No method was recommended for routine diagnostic use. The ELISA test, because of its convenience, may be useful as a screening aid or adjunct to the diagnosis of thyroid disease.     

Monoclonal-antibody-mediated enzyme-linked immunosorbent assay for detection of reticuloendotheliosis viruses

An enzyme-linked immunosorbent assay (ELISA) is described for the detection of reticuloendotheliosis viruses (REVs). The assay uses a mixture of monoclonal antibodies (MCAs) prepared against a 62-kilodalton REV envelope glycoprotein (gp62) to capture antigen, rabbit anti-REV serum as detection antibody, and peroxidase-conjugated anti-rabbit IgG as indicator antibody. The MCAs were reactive with REV strain T, chick syncytial virus, and duck infectious anemia virus but unreactive against Marek's disease and avian leukosis viruses. The ELISA was compared with complement fixation test and REV immunofluorescent assay of infected fibroblasts, plasmas, and egg albumen from infected chickens. The lower limit of gp62 detection was about 120 ng of REV protein. Limit dilution of infectious REV was detected after 7-8 days of cultivation of infected fibroblasts.     

Development of an indirect enzyme-linked immunosorbent assay for the detection of leptospiral antibodies in dogs.

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources, expertise, or facilities to perform the microscopic agglutination test (MAT). Thus, there is a need for a rapid and simple serological test that could be used in any diagnostic laboratory. In this study, a genus-specific, heat-stable antigenic preparation from Leptospira interrogans serovar pomona was used in an enzyme-linked immunosorbent assay (ELISA) for the detection of leptospiral antibodies in dog sera. This antigenic preparation reacted with rabbit antisera against L. interrogans serovars bratislava, autumnalis, icterohaemorrhagiae and pomona and with rabbit antiserum against L. kirschneri serovar grippotyphosa. The ELISA showed a relative specificity of 95.6% with 158 dog sera which were negative at a dilution of 1:100 in the MAT for serovars pomona, bratislava, icterohaemorrhagiae, autumnalis, hardjo, and grippotyphosa. The relative sensitivity of this assay with 21 dog sera that revealed serovars MAT titres of > or =100 to different serovars was 100%. This assay is easily standardized, technically more advantageous than MAT, and uses an antigenic preparation that can be routinely prepared in large amounts. It was concluded that this ELISA is sufficiently sensitive test to be used as an initial screening test for the detection of leptospiral antibodies in canine sera, with subsequent confirmation of positive test results with the MAT.     

Evaluation of antigen-coating procedures of enzyme-linked immunosorbent assay method for detection of trypanosomal antibodies

Research was undertaken to improve the antigen-coating step of indirect enzyme-linked immunosorbent assay (ELISA) method through the use of polystyrene 96-well plates precoated with antigenically stabile crude trypanosomal antigens. The plates were precoated with antigens, air dried and sealed before being packed in plastic bags with silica gel desiccant packets. Such plates stored at +4 and +37 degrees C provided an assay performance, which was superior to that of plates freshly coated with antigens from a frozen stock. Antigen-precoated plates consistently proved stable after storage up to +50 degrees C for at least 1 year. The accuracy of the assay was not affected, i.e. trypanosomal antibody-positive sera were clearly discriminated from trypanosomal antibody-negative negative sera. In contrast, lyophilized trypanosomal antigens lacked stability on storage at +37 degrees C for longer than 1 month. It was concluded that the routine use of antigen precoated polystyrene plates for the enzyme immunoassay technique will contribute to improved assay robustness at an acceptable diagnostic proficiency. The modified coating procedure will also provide an improved quality assurance and standardization procedure for the assay, which is required to allow the reliable detection of trypanosomal antibodies and comparison of data from different laboratories.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of transmissible gastroenteritis virus antibodies

An enzyme-linked immunosorbent assay (ELISA) using a detergent-solubilized antigen of purified virus was developed for detection of antibody against porcine transmissible gastroenteritis (TGE) virus in swine serum. The ELISA demonstrated antibody responses in pigs immunized intramuscularly with the attenuated TO-163 strain of TGE virus and in pigs orally infected with the virulent Shizuoka strain of the virus. The results of the ELISA were well correlated with those of the neutralization test. These results indicate the usefulness of the ELISA as a serological tool for TGE virus antibody.     

Evaluation of an enzyme-linked immunosorbent assay for detection of Eperythrozoon suis antibodies in swine.

An ELISA was developed and tested to detect antibodies to Eperythrozoon suis in swine. Results were compared with those of the indirect hemagglutination (IHA) test. Antigen isolated from swine heavily infected with E suis was used for both tests. Comparison of the ELISA with the IHA test revealed a significant (P less than 0.001) correlation between results. Of 114 samples obtained from 9 swine infected with E suis, 87.7% were seropositive (titer greater than or equal to 200) via the ELISA, and 80.7% were seropositive (titer greater than or equal to 20) via the IHA test. The sensitivity of the ELISA was greater than that of the IHA test. All blood samples obtained from specific-pathogen-free swine tested negative for E suis antibody. Cross-reactions were not observed between E suis antigen and antisera against various swine and cattle disease agents using ELISA. We concluded that the ELISA may be used for rapid and effective diagnosis of infection with E suis in swine.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of Corynebacterium pseudotuberculosis infection in sheep

Two enzyme-linked immunosorbent assays to measure antibody to the cell wall antigen (C-ELISA) and toxin antigen (T-ELISA) of C. pseudotuberculosis were evaluated on serum from 6 separate groups of sheep. For sheep naturally infected with C. pseudotuberculosis the sensitivity and specificity of the C-ELISA was 76% and 73% respectively and for the T-ELISA 67% and 77% respectively. For sheep slaughtered one year after artificial infection the sensitivity of both tests was greater than or equal to 83% and the specificity greater than or equal to 72%. For sheep slaughtered 4 months after artificial infection the specificity of both tests was less than 30% while the mean sensitivity was 85%. The C-ELISA in conjunction with the T-ELISA detected 92% of sheep with lung lesions.     

Evaluation of an enzyme-linked immunosorbent assay for detection of antibodies to Fasciola hepatica in milk

Antibodies against Fasciola hepatica were detected in serum and individual milk samples of dairy cattle using an ELISA. Percentage positivity (PP) values in milk samples were related to serum PP values and were not influenced by days into lactation. The correlation coefficient between serum and individual milk samples was highly significant (r=0.84, P<0.005). The correlation coefficient between herd seroprevalence and herd milk antibody prevalence was 0.96. The correlation coefficient between prevalence measured by faecal egg count and both seroprevalence and milk antibody prevalence within the herd was 0.87. The diagnostic sensitivity and specificity for milk were 92% (95% CI=89-96) and 88% (95% CI=85-91), respectively, when the serum test was considered as a gold standard. In conclusion, the level of antibody to F. hepatica in milk is significantly correlated with the antibody level in serum and this ELISA is suitable as a means of routine veterinary diagnosis of exposure to F. hepatica in cattle and an alternative to testing sera.     

The use of enzyme-linked immunosorbent assay systems for serology and antigen detection in parvovirus, coronavirus and rotavirus infections in dogs in The Netherlands.

Complex trapping blocking (CTB) enzyme-linked immunosorbent assays (ELISAs) and indirect ELISAs for the detection of antibodies to canine parvovirus (CPV), canine coronavirus (CCV) and rotavirus in sera of dogs were established. Double antibody sandwich ELISAs for the detection of CPV-, CCV- and rotavirus antigens in fecal samples were also developed. Both the serological and antigen-detection ELISAs were used to screen samples from dogs in The Netherlands, with or without a history of acute diarrhea. It was shown that the results of the respective serological ELISAs correlated well and that CPV was the major cause of virus-induced acute diarrhea in dogs in The Netherlands.     

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 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.     

Use of electron microscopy and an enzyme-linked immunosorbent assay for the detection of rotaviruses in neonatal calf diarrhea.

Electron microscopy and an enzyme-linked immunosorbent assay were compared for the diagnosis of rotaviruses associated with neonatal calf diarrhea. One hundred percent correlation was observed when 125 samples were tested by both techniques. Both techniques were equally efficient in detecting rotaviruses. The enzyme-linked immunosorbent assay was more sensitive but being specific could not detect other viruses.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of Mycoplasma hyopneumoniae antibody in porcine serum

An enzyme-linked immunosorbent assay (ELISA) for detecting antibody to Mycoplasma hyopneumoniae in porcine serum is described. The results are presented as an ELISA ratio, calculated by dividing the absorbance of the test sample by the mean absorbance of control negative sera. In known infected pigs, the ELISA ratio was highest when the serum concentration applied to the ELISA plate was diluted 1 in 20 in PBS - Tween. Mean ELISA ratios ranged from 1.2 +/- 0.3 for pigs without porcine enzootic pneumonia (PEP) lesions to 5.5 +/- 1.5 for pigs observed with a PEP lesion reacting positively with immunofluorescent histopathology. Pigs observed with typical PEP lesions at slaughter, but not confirmed by immunofluorescent histopathology had a mean ELISA ratio of 4.9 +/- 1.7. The ELISA was highly sensitive (95.6%) and specific (98.8%) when pig sera from commercial piggeries of known M hyopneumoniae infection status were assessed. No cross-reactivity with serum from a pig hyperimmunised with killed M flocculare was detected, and reactivity with serum from another pig hyperimmunised with killed M hyorhinis showed only weak cross-reactivity, which failed to reach the ELISA positive threshold (ELISA ratio 3) for M hyopneumoniae.     

The enzyme-linked immunosorbent assay for detecting antibodies against Dirofilaria immitis in dogs

Antisera prepared in mice by injection of antigens from Dirofilaria immitis, Toxocara canis, Dipylidium canium and Fasciola hepatica and sera from Dirofilaria-infected and non-infected dogs were tested at different dilutions using an enzyme-linked immunosorbent assay (ELISA). For this ELISA, adult D. immitis antigen was fractionated by gel filtration methods and then absorbed with immunoadsorbent-fixed IgG fractions from mouse sera immunized with various parasites. The results indicated satisfactory discrimination between antisera to D. immitis and those to other parasites. A significant ELISA O.D. value was considered to be greater than or equal to 0.30 with a 1 : 250 dilution of the dog sera. However, the lack of significant differences between the O.D. value of microfilaraemic and amicrofilaraemic infections was observed. These facts suggest that the use of immunoadsorbent chromatography for canine dirofilariasis is especially useful for purifying antigens and eliminating cross-reactions against other parasitic infections, when immunological methods are used for serodiagnosis.     

Diagnosis of Fasciola sp. infections in cattle by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) was evaluated for diagnosis of experimental or naturally occurring Fasciola sp. infections in cattle. The positive rate for the ELISA in calves inoculated with Fasciola metacercariae were 21.1% by 2 weeks postinoculation (PI), 94.6% by 4 weeks PI and 100% by 6-21 weeks PI. The positive rate for the immunodiffusion test (Ouchterlony test) reached 91.7% by 2 weeks PI, however, it dropped to 77.8% by 10 weeks PI. The positive rate for the fecal egg examination was 0% by 10 weeks PI, 77.8% by 12 weeks PI and 100% by 14-21 weeks PI. The practical application of ELISA was tested by using 165 cows raised under field condition. All the 24 cows that were positive both in the fecal egg examination and the Ouchterlony test were ELISA positive. Of the 6 cows that were egg positive and Ouchterlony negative, 5 showed ELISA positive reactions. Of the 27 cows that were egg negative and Ouchterlony positive, 24 were ELISA positive. Of the 108 cows that were egg negative and Ouchterlony negative, 90 were ELISA negative. However, the other 18 cows had ELISA positive reactions. Our results suggested that the ELISA using crude adult antigen was superior to the Ouchterlony test and fecal egg examination for diagnosis of experimental or naturally occurring Fasciola sp. infections in cattle.