Reactivity of serum samples of dogs and horses tested by use of class-specific recombinant-based enzyme-linked immunosorbent assays for detection of granulocytic ehrlichiosis

OBJECTIVE: To test serum samples of dogs and horses by use of class-specific recombinant-based ELISA for establishing a diagnosis of granulocytic ehrlichiosis attributable to infection with organisms from the Ehrlichia phagocytophila genogroup. SAMPLE POPULATION: Serum samples from 43 client-owned dogs and 131 horses (81 with signs of acute illness and 50 without signs of disease). PROCEDURE: Serum samples were analyzed, using ELISA with a recombinant 44-kd protein antigen for IgM and IgG antibodies to the human granulocytic ehrlichiosis (HGE) agent (NCH-1 strain). Western blot analyses, using infected human promyelocytic leukemia cells, were conducted on 38 serum samples of horses and 11 serum samples of dogs to verify reactivity to the 44-kd peptide. RESULTS: IgM or IgG antibodies to the HGE agent were detected in 5 to 28% of dog serum samples and 5 to 37% of horse serum samples. Thirty-five of 38 (92%) horse serum samples had corresponding results on both tests (2 positive results for 26 samples and 2 negative results for 9 samples), using an ELISA for IgG antibodies or immunoblotting for total immunoglobulins. All 11 serum samples of dogs had positive results for both methods. CONCLUSION AND CLINICAL RELEVANCE: These ELISA with recombinant 44-kd antigen are suitable for detecting IgM or IgG antibodies to the HGE agent in serum samples of dogs and horses. Positive results for serum samples of horses from Connecticut, New York, Virginia, and Georgia indicate that the HGE agent is widely distributed in tick-infested areas of the eastern United States.     

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 recombinant enzyme-linked immunosorbent assay for detecting Chlamydophila psittaci antibodies in turkey sera

Chlamydophila psittaci (formerly Chlamydia psittaci) is one of the major pathogens associated with turkey respiratory disease. Devastating outbreaks with high mortality rates, similar to those of 1950 to 1970 in the USA occasionally occur, but respiratory signs without or with low mortality mostly characterize outbreaks now a day. Accurate diagnostic methods should be made available. The present study examined the sensitivity and specificity of a recombinant ELISA (rMOMP ELISA) for detecting Cp. psittaci major outer membrane specific antibodies in turkey sera. Test results were compared to those of immunoblotting and of a competitive ELISA (Chlamydia-psittaci-AK-EIA, R?hm Pharma, Germany) and an indirect ELISA (LPS/LGP) detecting antibodies to the lipopolysaccharide/lipoglycoprotein complex. The rMOMP ELISA was most sensitive as determined on serial dilutions of positive control sera originating from experimentally infected SPF turkeys. The competitive ELISA gave false positives since three negative controls reacted positive. For conventional sera, the sensitivities of the competitive ELISA, immunoblotting and the indirect ELISA were found to be 99.4, 93.1 and 82.2%, respectively, as compared to the rMOMP ELISA (100%). The specificities of the rMOMP ELISA, immunoblotting and the indirect ELISA were found to be 100% while the specificity of the competitive ELISA was only 2.7%. The rMOMP ELISA was chosen to compare the prevalence of chlamydiosis in 2002 with the one from 1992. In 2002, 188 on 200 (94%) turkey sera reacted positive compared to 175 on 200 (87.5%) in 1992 and like 10 years ago all examined farms were seropositive at slaughter. Interestingly, Belgian as well as French farms were seropositive.     

Evaluation of an enzyme-linked immunosorbent assay (ELISA) for the serological diagnosis of sarcoptic mange in dogs

Canine scabies is a challenging disease to diagnose because sarcoptic mites are hard to find on skin scrapings. The purpose of this study was to evaluate a serologic enzyme-linked immunosorbent assay (ELISA) as an aid in the diagnosis of canine scabies. In addition, serum samples were obtained post treatment to determine the duration and persistence of circulating scabies antibodies after resolution of natural infection. Nineteen dogs diagnosed with sarcoptic mange and 38 control dogs were tested. Sixteen scabies-infested dogs showed positive pretreatment ELISA results (84.2% sensitivity). Thirty-four control dogs showed negative ELISA results (89.5% specificity). In the 11 scabies dogs from which multiple post treatment serum samples were obtained, detectable antibodies were not present 1 month after treatment in four cases, but were present for 1-4.5 months post treatment in seven dogs. Our results suggest that this scabies ELISA test is useful in the diagnosis of canine scabies.     

Evaluation of serum and secretory antibody responses to an immunodominant recombinant merozoite surface antigen, p150, using a sensitive enzyme-linked immunosorbent assay

An equation obtained from linear-regression analysis of positive/negative ratios and log of enzyme-linked immunosorbent assay (ELISA) titers of coccidia-immune serum samples was used to accurately predict the antibody titers of chickens immunized with a recombinant merozoite surface protein (p150). Chickens immunized twice intramuscularly with the recombinant p150 antigen emulsified in complete Freund's adjuvant developed a dose-dependent anti-p150 antibody response 14 days after primary immunization. Serum IgG and IgM and secretory biliary IgA antibodies were detected 2 months after primary immunization. Oral challenge with live Eimeria parasites significantly enhanced both the serum and secretory anti-p150 antibody titers. These results indicate that vaccination of chickens with the p150 recombinant merozoite antigen can induce a parasite-specific host immune response.     

Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias.     

Evaluation of a novel enzyme-linked immunosorbent assay for serological diagnosis of porcine proliferative enteropathy

A specific enzyme-linked immunosorbent assay (ELISA) for detection of antibodies to the porcine pathogen Lawsonia intracellularis was developed and evaluated using sera from na?ve, naturally infected as well as experimentally infected pigs. On the basis of 37 serum samples collected from experimentally infected pigs and 62 serum samples from naturally infected pigs the sensitivity of the ELISA was calculated to 98.0%. The specificity of the test was 99.3%, calculated on the basis of 273 serum samples collected in six herds free of L. intracellularis after medicated eradication. The novel ELISA was a specific and sensitive method for detecting specific antibodies, and may be a good alternative to the existing serological tests for L. intracellularis. It may be usable for diagnosis of proliferative enteropathy and for determination of a herd's epidemiologic status.     

Evaluation of an enzyme-linked immunosorbent assay for diagnosis of trichinellosis in swine.

Experimental and field trials were conducted to evaluate an ELISA for its ability to detect Trichinella-infected domestic swine and to compare ELISA results with muscle-digestion test results. The ELISA used was a commercial double-antibody kit, containing an excretory-secretory antigen, and was evaluated principally for epidemiologic use. Experimentally induced infection in swine (4 groups of 3 pigs each; inoculated with 0, 50, 500 or 5,000 larvae) was detected as early as postinoculation week 4, with seroconversion of all inoculated swine by postinoculation week 8. The rate of seroconversion appeared to be affected by initial larval dose, time after inoculation, and immunocompetence of the individual host. Determination of antibody kinetics generally revealed rapidly increasing antibody titer, followed by its steady decrease in most pigs. Once seropositive, however, all pigs remained seropositive for the duration of the 10-week study. Presence of muscle larvae was confirmed in all infected pigs at termination of the study. We recognize that the experimental conditions may not be truly representative of those under which natural infection develops in pigs; however, the ELISA detected an infected pig with muscle larval density of 0.87 larvae/g of tissue. Results of a field trial (n = 310) indicated no muscle digestion test-positive pigs (35 g of diaphragm muscle digested/pig), but 3 samples tested positive by ELISA for specificity of 99.0%.     

Evaluation of the Leishmania recombinant K39 antigen as a diagnostic marker for canine leishmaniasis and validation of a standardized enzyme-linked immunosorbent assay

Canine infections with Leishmania infantum are important as a cause of serious disease in the dog and as a reservoir for human visceral leishmaniasis (VL). Accurate diagnosis of canine infections is essential to the veterinary community and for VL surveillance programs. A standardized ELISA using a purified recombinant antigen (rK39) specific to VL was compared to the immunofluorescent antibody test (IFAT) as the standard. The ELISA was developed, optimized and evaluated using sera from 6368 dogs. The standardized ELISA and IFAT results were highly concordant. The timing and pattern of ELISA and IFAT seroconversion in dogs followed prospectively after natural infections were very similar. Antibodies reacting with rK39 were more common in asymptomatic canine infections than reported for subclinical human VL. The rK39 ELISA is a relatively simple and rapid assay for assessing the infection status of dogs, and is an alternative to IFAT, especially when screening large numbers of samples.     

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.     

Evaluation of an enzyme-linked immunosorbent assay in the diagnosis of bovine tuberculosis

The behavior of the immunoenzymatic system was tested in the serologic diagnosis of bovine tuberculosis in different populations from areas free of the disease, and in other areas with different prevalence. The specificity registered in the 4009 samples showed 94.4%, while the range of sensitivity increased proportionally to the number and distribution of lesions found in the anatomopathological test. Overall the sensitivity of the ELISA was 47%. When animals from groups with different prevalence of the disease were studied, a significant correlation (78%) between the ELISA system and the Intradermal Tuberculin Test (IT) was observed in the groups with low prevalence of the disease, animals reactive to both tests were found in all the units. The results of the work and the inherent advantages of the ELISA technique allow recommending this ELISA in programs for control and/or eradication of bovine tuberculosis.     

Evaluation of a commercially available enzyme-linked immunosorbent assay for the diagnosis of canine sarcoptic mange

This study was designed to assess the accuracy of a commercial enzyme-linked immunosorbent assay for the diagnosis of canine scabies. Serum samples from 37 dogs were examined blind; 12 had sarcoptic mange confirmed by the identification of mites in skin scrapings, 12 were atopic (with positive intradermal reactions to one or more aeroallergens, including Dermatophagoides farinae), and 13 were healthy dogs with no history of skin disease. Optical density values of more than 0.16 were considered positive, 0.145 to 0.16 were considered questionable and less than 0.145 were considered negative. Ten of the 12 dogs with scabies were positive, all 12 atopic dogs were negative, and 11 of the 13 healthy dogs were negative and two were questionable.     

Validation of an in-clinic enzyme-linked immunosorbent assay kit for diagnosis of Borrelia burgdorferi infection in horses.

Confirmation of Borrelia burgdorferi infection in horses has required enzyme-linked immunosorbent assay (ELISA) or Western blot tests performed by reference laboratories. An in-clinic C6 ELISA SNAP kit has been marketed for dogs. This canine kit was evaluated for horses using serum from experimentally infected ponies. Serum samples originated from 2 previous studies. In the first study, 7 ponies were exposed to B. burgdorferi-infected ticks; 4 ponies served as uninfected controls. Serum samples were obtained bimonthly for 9 months. In the second study, 16 ponies were exposed to B. burgdorferi-infected ticks. After confirmation of infection by skin culture, polymerase chain reaction (PCR), and serology, the ponies were allocated to 4 groups that received tetracycline, doxycycline, ceftiofur, or no treatment. Serum samples were obtained monthly, both before and after antibiotic treatments, for 11 months. For the current study, selected samples (n = 220) from both studies were tested with IDEXX SNAP Heartworm Ab/Borrelia burgdorferi Ab/Ehrlichia canis Ab Test Kits. Tested samples included samples taken before infection, from various times postinfection, and after antibiotic treatments. Results from confirmed positive or negative samples were used to determine sensitivity and specificity of the assay. Results indicate that the test kits have fair sensitivity (63%) and very high specificity (100%) for horses recently infected with B. burgdorferi. Validation of this test provides equine practitioners with an inexpensive, in-clinic method to confirm infection, although its moderate sensitivity may result in a moderate chance of a false negative test.     

HPLC purification of recombinant NcGRA6 antigen improves enzyme-linked immunosorbent assay for serodiagnosis of bovine neosporosis

The gene for a dense granule protein (NcGRA6) of Neospora caninum was expressed in Escherichia coli as a His-tag fusion protein and purified by NiNTA affinity chromatography. In a preliminary study, high binding of antibodies from N. caninum-negative cows was observed in enzyme-linked immunosorbent assay (ELISA) using NiNTA-purified NcGRA6. Analysis of NiNTA eluates revealed a significant number of E. coli proteins that co-purified with recombinant NcGRA6. In an attempt to improve the relative sensitivity and specificity of the NcGRA6-based ELISA, the rNcGRA6 eluates were subjected to a secondary purification using reverse phase-high performance liquid chromatography (RP-HPLC). Analysis of RP-HPLC eluates by SDS-PAGE/silver staining revealed the purification of recombinant NcGRA6 from contaminating E. coli proteins. ELISAs using the RP-HPLC purified NcGRA6 (dELISA) or singly purified NcGRA6 (sELISA) for identifying seropositive and seronegative cows in a beef herd experiencing an epidemic outbreak of neosporosis were compared to standard assays based on native tachyzoite protein-immunofluorescence antibody test, immunoblot assay, and ISCOM-ELISA. The relative sensitivity, specificity, and kappa value of the NcGRA6d-ELISA were greatly improved over the NcGRA6s-ELISA when compared to the three native antigen immunoassays. These results indicate that removal of contaminating E. coli proteins improves the performance of recombinant NcGRA6 ELISA in diagnosing bovine neosporosis, and may have applicability to the use of recombinant proteins in diagnosing other infectious agents.     

Detection of Corynebacterium equi-specific antibody in horses by enzyme-linked immunosorbent assay

An enzyme-linked immunosorbant assay was developed to measure naturally occurring Corynebacterium equi specific antibody in horse serum. Antibody against C equi was demonstrated in normal adults and was passively transferred to foals. Adult levels of specific antibody were reached by 5 to 6 months of age in healthy foals. Decreased early antibody levels were demonstrated in a limited number of foals with confirmed C equi infection.     

Preparation of Mycoplasma hyopneumoniae antigen for the enzyme-linked immunosorbent assay.

Methods of preparation of Mycoplasma hyopneumoniae antigens for the enzyme-linked immunosorbent assay to detect specific antibody, and properties of the antigens, are described. The reactivity and specificity of antigen prepared by Sephacryl S-300 column chromatography after treatment of M. hyopneumoniae cells with Tween 20 (S-300 antigen) were superior to those of antigen prepared by Sephadex G-25 column chromatography after treatment with Tween 20, or to lipid antigen. There were no differences among strains MI-3, J and VPP11 of M. hyopneumoniae. The S-300 antigen did not show cross-reactivity against porcine hyperimmune sera produced by M. hyorhinis, M. hyosynoviae, M. hyopharyngis, M. flocculare and Acholeplasma granularum. Antibody was first detected in sera of pigs inoculated intranasally with M. hyopneumoniae at two to four weeks after inoculation and seven to eight weeks after pigs were contact-exposed to the same mycoplasma.     

Development of an enzyme-linked immunosorbent assay for the detection of phenothiazine tranquillisers in horses

An acepromazine (ACP) hapten was synthesised, coupled to bovine serum albumin and injected into a horse to produce antibodies to the drug. A competitive ELISA was developed whereby ACP attached to the solid phase via lysozyme competed with free ACP present in phosphate buffered saline, horse serum or horse urine for limiting amounts of antibody. The assay could detect the presence of ACP and, or, some of its metabolites in horse urine for at least 25 hours after intravenous injection of 0.1 mg kg-1 ACP maleate, but because of non-specific interference, horse serum could not be used. As little as 0.24 micrograms ml-1 ACP or its metabolites could be detected. The level of detection and the ease of performance of the assay make it an attractive alternative to the more complex methods currently available for the screening of horse urine samples at horse races, shows and sales.     

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.     

Validation of a von Willebrand factor antigen enzyme-linked immunosorbent assay and newly developed collagen-binding assay

No single test is comprehensive enough to detect all of the variants of von Willebrand Disease (VWD), making determination of both concentration and function of von Willebrand Factor (VWF) important for an accurate diagnosis. The objective of the study was to validate a newly developed VWF collagen binding assay (VWF:CB) and VWF antigen enzyme-linked immunosorbent assay (ELISA) developed at the Ontario Veterinary College (OVC VWF:Ag). Linearity, sensitivity, and coefficients of variation were determined. The Asserachrom VWF:Ag ELISA was used as the reference assay for this study. Concordance correlation and Bland-Altman plots were used to evaluate agreement between both VWF:Ag assays. The VWF:CB accuracy was assessed by degree of association with the VWF:Ag assays, and the VWF:Ag to VWF:CB ratio. All assays were assessed for their ability to distinguish between VWD negative and VWD positive patients. Linearity, intra-assay coefficients of variation, and inter-assay coefficients of variation were acceptable for both the newly developed VWF:CB (R² = 0.97, average CV = 4.4, and 15, respectively) and OVC VWF:Ag assays (R² = 0.96, average CV = 7.9, and 5.9, respectively). Agreement between the OVC VWF:Ag assay and reference assay was excellent (ρ_c = 0.89), and although differences between assay results precluded interchangeable use of the assays, both successfully distinguished VWD positive and VWD negative dogs (P < 0.0001). The VWF:CB showed a strong association with both VWF:Ag assays (R² = 0.86, 0.82) and VWF:Ag to VWF:CB ratios (≤ 1) were as expected. The excellent performance of both assays in this validation study confirm their reliability and potential for clinical application.