An enzyme-linked immunosorbent assay for the detection of antibodies to Mycoplasma gallisepticum in experimentally infected chickens

An indirect enzyme-linked immunosorbent assay (ELISA) was developed and tested for its ability to detect humoral response to Mycoplasma gallisepticum in chickens. Two antigens were used in the solid phase of the assay. Antigen 1 was a membrane-derived sodium dodecyl sulfate (SDS)-solubilized preparation; Antigen 2 was prepared in the same manner as Antigen 1 but was passed through an immunoadsorbent column containing rabbit anti-medium antibodies. Test conditions were optimized for incubation times and temperatures. Antigen, serum, and enzyme conjugate concentrations were standardized, and reproducibility was determined. A baseline value, representing a positive or negative result, was established independently for both antigens. The assay was then used to detect anti-M. gallisepticum antibodies in experimentally infected chickens. Serum samples collected at 0, 2, 5, 7, 10, 14, 21, 28, and 35 days postinfection (PI) were analyzed by serum plate agglutination (SPA), hemagglutination-inhibition (HI), and ELISA with both Antigens 1 and 2. ELISA was found to be less sensitive but more specific than SPA and more sensitive than HI. The ELISA was more sensitive with Antigen 1 than with Antigen 2. The former assay correctly identified 79% of the serum samples positive for M. gallisepticum by 7 days PI and 100% of the positive birds by 35 days PI. When the absorbance values for each group of birds were averaged, the ELISA successfully identified the M. gallisepticum-infected birds as uniformly positive 7 through 35 days PI and correctly identified all other groups negative for M. gallisepticum through 35 days PI.     

An enzyme-linked immunosorbent assay for detecting antibodies to Pasteurella multocida in chickens

An enzyme-linked immunosorbent assay (ELISA) was developed to detect the humoral antibody response in chickens receiving subcutaneous injections of the CU vaccine strain of Pasteurella multocida. Serum samples were collected twice weekly for 3 weeks, and chicken antibody responses were monitored using ELISA. The positive/negative ratio method of analysis was used to determine the antibody titer of vaccinated chickens. After a loge transformation of the ELISA titer, a linear relationship was confirmed between ELISA titer and positive/negative ratio. Regression analysis was used to construct a standard curve and derive an equation from this relationship. Using this equation, only one dilution was needed to determine the antibody titer of any unknown serum sample. The ELISA technique was used to monitor the mean antibody titer of vaccinated chickens over the 3-week period. A classic primary response curve occurred when titer was plotted against time.     

An enzyme-linked immunosorbent assay for detection of antibodies to avian adenovirus and avian adenovirus-associated virus in chickens

An enzyme-linked immunosorbent assay system (ELISA) was adapted for the detection of antibodies to avian adenovirus (AV) and avian adenovirus-associated virus (A-AV). Both before and after exposure, sera from chickens undergoing natural and experimental infections were assayed by ELISA, virus neutralization (VN), and immunodiffusion (ID) for antibody to both CELO virus and A-AV. The ELISA system was found to be comparable to VN for determining antibody concentrations to CELO virus and A-AV. In many cases, ELISA was found to be more sensitive than ID.     

Competitive enzyme-linked immunosorbent assay for the detection of the antibodies specific to akabane virus.

A competitive enzyme-linked immunosorbent assay (C-ELISA) using neutralizing monoclonal antibodies (MAbs) against Akabane virus (AKAV) was developed to detect antibodies to AKAV in cattle sera. The performance of the test using 7 different competitor MAbs was evaluated in sequential serum samples and sera from cattle infected with various bovine arboviruses. The dynamics of the antibody response expressed by percentage of inhibition (PI) in C-ELISA coincided with those of neutralizing antibody titers in sequential serum samples from 2 cattle experimentally infected with AKAV. The value of PI in C-ELISA for convalescent sera from cattle infected with arboviruses correlated with the neutralizing antibody titer to AKAV but was unaffected by the antibodies to other arboviruses. In the validation experiment of C-ELISA using 286 bovine sera previously examined for the AKAV antibody by serum neutralization (SN) test, the relative specificity of C-ELISA was more than 98%, whereas the relative sensitivities of individual MAbs ranged from 49% to 82.2%. Overall agreement between C-ELISA and the SN test varied from 72% to 90% depending on the MAb. These results suggest that the C-ELISA is acceptable as a rapid and specific method for detecting antibodies to AKAV and is a potential alternative to the SN test.     

An enzyme-linked immunosorbent assay for the detection of antibodies to Marek's disease virus

A reproducible enzyme-linked immunosorbent assay (ELISA) using Marek's disease virus (MDV)-infected cells for the detection of antibodies to MDV is described. The optimum number of MDV-infected chicken embryo fibroblasts (CEF) was 5 X 10(4)/well, and test sera were positive at 1:400 dilutions. Compared with a purified virus preparation, MDV-infected CEF produced high specific and low nonspecific reactivities. Wells coated with whole cells could be stored at 4 C or -20 C for at least 3 months without loss of reactivity. With antibody-negative sera, the cutoff absorbency was 0.20 units. The ELISA was 20-to-40-fold more sensitive than indirect immunofluorescence. Homologous combinations of antisera in wells coated with CEF infected with different MDV serotypes were more reactive at higher dilutions than were heterologous combinations. The procedure described is specific and suitable for large-scale screening of both chicken and monoclonal antibodies against MDV.     

A modified enzyme-linked immunosorbent assay for the detection of avian pneumovirus antibodies.

Avian pneumovirus (APV) infection of turkeys in Minnesota was first confirmed in March 1997. Serum samples (n = 5,194) from 539 submissions to Minnesota Veterinary Diagnostic Laboratory were tested by a modified enzyme-linked immunosorbent assay (ELISA). Of these, 2,528 (48.7%) samples from 269 submissions were positive and 2,666 (51.3%) samples from 270 submissions were negative for APV antibodies. Most positive samples were from Kandiyohi, Stearns, Morrison, and Meeker counties in Minnesota. In addition, 10 samples from South Dakota were positive. The sensitivity and specificity of the ELISA test with anti-chicken and anti-turkey conjugates were compared by testing field and experimental sera. The ELISA test with anti-turkey conjugate was more sensitive than that with anti-chicken conjugate. The ELISA tests with antigens prepared with APV strains isolated from Colorado and Minnesota were also compared. No difference was detectable. Currently, the Minnesota Veterinary Diagnostic Laboratory uses an antigen prepared from the Colorado isolate of APV and a goat anti-turkey conjugate in the ELISA test.     

An enzyme-linked immunosorbent assay for detection of antibodies against bovine pestivirus

A competitive enzyme-linked immunosorbent assay was developed and compared with the serum neutralisation test for bovine pestivirus using 508 cattle sera and serial serum samples from a goat hyperimmunized with five bovine pestivirus isolates. There was 96.7% agreement between the two tests. The relative sensitivity of the enzyme-linked immunosorbent assay compared to the serum neutralisation test was 95.2% and the relative specificity was 99.4%. The titres of individual animals in the assay did not show a close correlation with serum neutralisation test titres. This may be because the antibodies measured in the two tests are directed against different viral proteins. The enzyme-linked immunosorbent assay has the advantage of being quicker and cheaper than the serum neutralisation test. The configuration used in the ELISA means sera from all species can be tested for pestivirus antibody using the same set of reagents.     

Blocking enzyme-linked immunosorbent assay for detection of antibodies against bovine enterovirus

A blocking enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibodies against bovine enterovirus (BEV) in bovine sera. In this ELISA, bovine serum samples were allowed to react with captured viral antigens (by specific chicken IgG), before the addition of specific mouse IgG for measuring non-occupied viral epitopes. The ELISA was slightly more sensitive and required a shorter time period than traditional serum neutralisation (SN). Among the 871 bovine serum samples tested so far, the titres produced by this assay had a significant correlation with those recorded by SN. The ELISA could be used as an alternative assay for SN in a large-scale BEV antibody investigation.     

An enzyme-linked immunosorbent assay for the detection of vesicular stomatitis virus antibodies

A liquid-phase enzyme-linked immunosorbent assay (ELISA) was developed for the detection of vesicular stomatitis virus (VSV) types New Jersey (VSV-NJ) and Indiana subtype Indiana I (VSV-IND₁) antibodies in the sera of naturally and experimentally infected cattle, horses and swine. Four different VSV preparations were compared for use as antigen in the ELISA: virus used in neutralization tests, complement-fixation antigen, immunodiffusion ager gel antigen and viral glycoprotein. Comparative antibody titration results of virus neutralization (VN) and ELISA showed no statistically significant difference between serum titers obtained with the four antigens to VSV-NJ (P=0.21) and VSV-IND₁ (P=0.14). The viral glycoprotein antigen was incorporated in the ELISA system because it is non-infectious and induces neutralizing antibodies. The reliability and variability of the ELISA was determined by testing 516 bovine, equine and swine sera which originated from areas free of vesicular stomatitis, and by testing 186 sera from areas where outbreaks occur. ELISA and VN results were correlated (P < 0.001 for both serotypes), and no statiscafically difference was found between replications of the tests. The ELISA allows the testing of a larger number of sera in a shorter time than is possible with the VN test and it can be used in diagnostic laboratories in VSV-free areas for the support of epidemiological surveillance programs.     

An enzyme-linked immunosorbent assay for the detection of bovine antibodies to vesicular stomatitis virus

An indirect enzyme-linked immunosorbent assay (ELISA) was developed to detect bovine antibody to vesicular stomatitis virus (VSV). Serum samples from cows experimentally infected with the New Jersey serotype of VSV (VSV-NJ) were assayed by the ELISA and serum-neutralization (SN) assay. The ELISA was as sensitive as the SN assay in detecting bovine antibody to VSV. The correlation between SN titers and ELISA values at absorbance at 405 nm was statistically significant. The ELISA was not specific for VSV-NJ, however, and could detect serum samples positive to the Indiana serotype of VSV that had SN titers of greater than or equal to 480. Nonspecific reactions were due to cross-reactive group-specific viral proteins that are shared by both serotypes. The cross-reactivity allows the use of a single rapid test in identifying both serotypes of VSV from the other exotic vesicular diseases, especially foot-and-mouth disease. The ELISA titers of serum samples positive for VSV-NJ were comparable with the corresponding SN titers of each sample. The sensitivity, rapidity, and ease of the ELISA system and the use of a single test in identifying both serotypes of VSV from the other exotic vesicular diseases make this ELISA suitable as a rapid diagnostic assay for VS.     

An enzyme-linked immunosorbent assay for detection of antibodies to exogenous avian leukosis virus

A microplate enzyme-linked immunosorbent assay (ELISA) for detecting antibodies to avian leukosis virus (ALV) of subgroups A and B in infected chickens was developed with the use of Rous-associated virus (RAV)-1 (subgroup A) and RAV-2 (subgroup B) antigens purified by sucrose-gradient centrifugation. The antigen was used for ELISA after treatment with Triton X-100. In the ELISA, the subgroup viral antigen reacted strongly with homologous antiserum but also reacted with heterologous antiserum. Tests with serum absorbed with purified homologous and heterologous virus and tests for antigen-blocking by group-specific antibodies to ALV revealed that the reaction was caused mainly by subgroup-specific antibodies. The ELISA was 8 to 32 times more sensitive than the virus-neutralization (VN) test and detected antibodies to ALV earlier than the VN test in chickens infected experimentally with RAV-1 and RAV-2. In field application of the ELISA, 44.2% of 484 chicken sera were positive for RAV-1 and/or RAV-2 antigen, and 80.4% of flocks were positive. These findings indicate that ELISA is superior to the VN test in sensitivity, simplicity, rapidity, and applicability for large-scale field surveys for ALV infection.     

An enzyme-linked immunosorbent assay for antibodies to Hepatozoon canis

Canine hepatozoonosis is a tick-borne protozoal disease caused in the Old World and South America by Hepatozoon canis. An enzyme-linked immunosorbent assay (ELISA) using purified H. canis gamont antigen was applied for the detection of antibodies reactive with H. canis. Evaluation of the ELISA with sera from naturally infected parasitemic dogs indicated that it was sensitive (86%), specific (97%), and comparable to the indirect fluorescent antibody test (IFAT) for the detection of H. canis antibodies. A variable degree of serologic cross-reactivity was found between sera from H. americanum-infected dogs and the H. canis antigen. Dogs experimentally infected with H. canis seroconverted 1-4 weeks post-infection (PI). Antibody levels peaked at 7-9 weeks PI and gradually declined thereafter remaining above the cut-off value until the conclusion of the study 7 months PI. The ELISA will be valuable for serological evaluation of dogs suspected of exposure to H. canis and for epidemiological studies.     

A monoclonal antibody-blocking enzyme-linked immunosorbent assay for the detection of serovar-specific antibodies to Haemophilus paragallinarum

Two monoclonal antibody-blocking enzyme-linked immunosorbent assays (B-ELISAs) were developed to detect serovar-specific antibodies to Haemophilus paragallinarum. One assay detected antibodies against serovar A and the other antibodies against serovar C. The assays were evaluated with sera derived from disease-free chickens as well as chickens experimentally immunized and/or challenged with H. paragallinarum strains 0083 (serovar A), Modesto (serovar C), or HP31 (serovar C). When tested with 440 negative sera (170 from a specific-pathogen-free and 30 from each of nine commercial layer flocks), both tests gave only a single false-positive reaction. The use of the B-ELISAs with the experimentally produced sera showed the assays to be serovar specific. With the exception of one serum, the serovar A B-ELISA detected antibodies only in the chickens vaccinated with 0083. Similarly, with the exception of one serum, the serovar C B-ELISA detected antibodies only in those chickens vaccinated with Modesto or those chickens challenged with HP31. Overall, the serovar A B-ELISA had a specificity of 99.7% and a sensitivity of 78.7%, whereas the serovar C B-ELISA had a specificity of 99.8% and a sensitivity of 64.7%.     

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.     

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.     

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.     

Rapid enzyme-linked immunosorbent assay for detecting antibodies to canine parvovirus

A rapid screening assay for determining antibodies to canine parvovirus in dog serum using monoclonal antibodies and enzyme-linked immunosorbent assay (ELISA) technology was developed. The ELISA could be read visually, and the results correlated well with serum neutralization (SN) and hemagglutination inhibition (HI) titers. Sera with SN less than or equal to 1:4 or HI less than or equal to 1:10 had an 87.9% correlation with ELISA and sera with SN greater than or equal to 1:64 or HI greater than or equal to 1:80 had a 94.4% correlation. The assay took only 10 to 15 minutes to perform and did not require specialized equipment. The ELISA should be useful in monitoring dogs for the presence of maternal antibodies against parvovirus and for determining seroconversion after vaccination.     

Evaluation of an enzyme-linked immunosorbent assay for the detection of antibodies to caprine arthritis-encephalitis virus in goat serum.

An indirect enzyme-linked immunosorbent assay (ELISA), was evaluated for its ability to detect serum antibodies against caprine arthritis-encephalitis virus (CAEV). The ELISA was compared to three other serological immunoassays, agar gel immunodiffusion test (AGIDT), immunoblot assay (IBA), and a fixed-cell immunoperoxidase assay (FCIPA). A total of 511 samples, from 40 farms representing a variety of goat breeds and ages were tested. An estimate of the ELISA sensitivity and specificity was made, relative to combined test results of the three other CAEV serological assays. The degree of agreement of test results among these four assays was evaluated. The number of positives detected by the ELISA, AGIDT, IBA and IPA tests was 193, 154, 204 and 163, respectively. Of the 511 sera tested, 172 were positive to any two or all three of these tests, and were defined as reference positive. A total of 237 samples were negative to all three reference tests, and were defined as reference negative. Relative to these references, the ELISA had a point estimate of 98.3% sensitivity and 97.9% specificity. There was good agreement between the ELISA and the other three assays with a kappa statistic of agreement greater than 0.7 for all three comparisons. The ELISA is therefore considered a suitable assay, with high sensitivity and specificity, for detection of antibodies to CAEV in serum.     

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.