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.     

Feasibility of pooled-sample testing for the detection of porcine reproductive and respiratory syndrome virus antibodies on serum samples by ELISA

Surveillance of porcine reproductive and respiratory syndrome (PRRS) in negative sow farms is usually performed by testing for the presence of antibodies against PRRS virus in serum with a commercial ELISA test. The objective of this study was to evaluate the feasibility of pooling serum samples for detection of PRRS virus antibodies by ELISA. The effect of pool size on the sensitivity and specificity of the ELISA test was evaluated by testing true positive samples and false positive samples, respectively, diluted in negative sera. The effect of three different cut-off values for the interpretation of the diagnostic test (0.4, 0.3 and 0.2) was evaluated as well. Furthermore, the obtained sensitivity and specificity estimates were used to calculate the herd sensitivity and herd specificity of surveillance protocols in different scenarios. The results showed that pooling serum samples to detect PRRSV antibodies resulted in a decrease in sensitivity and an increase in specificity, compared to testing individual samples, while the reduction of the s/p cut-off value recommended by the manufacturer (0.4) had the opposite effect. We describe an approach that can increase the herd sensitivity of a surveillance protocol for breeding herds, while maintaining high herd specificity and low testing costs. This can be achieved by sampling a larger number of animals and running the samples in pools. Therefore, the conventional monitoring protocols based on ELISA on individual samples can be improved by using pooled-sample testing.     

非洲猪瘟病毒抗体检测间接ELISA方法的建立

以基因工程表达的非洲猪瘟病毒VP73蛋白作为包被抗原,建立了间接ELISA方法,用以检测猪血清中抗非洲猪瘟VP73蛋白的抗体。该方法对非洲猪瘟标准阳性血清的检测灵敏度可以达到1∶2 560,与同类进口ELISA试剂盒相当。此方法只特异性检出非洲猪瘟阳性血清,而对猪传染性胸膜肺炎等5种猪传染病阳性血清的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复性试验结果发现,检测同一份血清的变异系数小于10%,表明其重复性较好。包被好的酶标板37℃放置5d后,对同一份血清的检测敏感性无明显变化,初步表明其稳定性较好。利用建立的间接ELISA方法和进口ELISA试剂盒分别对150份血清样品进行非洲猪瘟血清抗体检测,结果表明本方法的特异性和敏感性分别为99.1%和94.3%,2种方法检测结果的符合率为98%。以上试验表明,本试验建立的间接ELISA方法具有良好的特异性和敏感性、较好的重复性和稳定性,可以满足临床检测的需求。     

Sample handling substantially affects Johne's ELISA

Detection methods for Mycobacterium avium subsp. paratuberculosis (MAP) are imperfect, yet crucial for diagnosis of Johne's disease. Our purpose was to test for significant and biologically relevant changes in Johne's ELISA results associated with how field-collected blood samples were transported to the laboratory, prepared and stored prior to testing, while removing potential confounding by test kit and laboratory variables. Blood samples were collected from 21 cows that previously had MAP ELISA scores ranging from negative to highly positive. Samples for immediate laboratory processing were subjected to different transportation temperatures (on ice, 26 °C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), but were tested using the same ELISA kit in the same laboratory. Samples for laboratory processing after one week of storage were subjected to different storage temperatures (4 °C, −20 °C) and preparation methods (serum separated, hemolyzed and serum separated, clotted whole blood), and again were tested using the same ELISA kit in the same laboratory. Finally, samples were evaluated by time to processing (one day, one week) and storage temperature (4 °C, −20 °C). Data were checked for normality and analyzed with repeated measures ANOVAs. Significantly (P = 0.027) higher MAP ELISA scores were recorded for whole blood and hemolyzed samples transported at 26 °C than serum separated samples. Sample storage for one week at −20 °C resulted in significantly (P < 0.001) lower MAP ELISA scores, regardless of handling method, compared to samples stored at 4 °C for one week. Method of sample preparation, as well as transportation temperature and medium-term storage temperature, affects MAP ELISA results. Such discrepancies will inevitably result in improper classification of MAP-infected cattle, impeding both biosecurity measures on uninfected farms and MAP control programs.     

Use of an enzyme-linked immunosorbent assay for detection of infection with pseudorabies virus on a herd basis.

The use of an ELISA that can differentiate between swine infected with pseudorabies virus (PRV) and swine vaccinated with a specific PRV vaccine was evaluated on an individual and herd basis, and a system for interpreting ELISA results on a herd basis was developed. In 17 herds, recently introduced replacement gilts, seronegative for PRV, were vaccinated with a thymidine kinase- and glycoprotein X (gpX)-deleted vaccine. After vaccination, blood samples were collected from these gilts approximately every 1 to 2 months for up to 19 months. Serum samples were analyzed for antibodies to gpX antigen, using a commercially available ELISA kit according to the manufacturer's protocol. Herd status was determined as positive, suspect, or negative, according to the serum sample:negative control (S:N) values of the samples collected from the herd. From the 17 herds, 130 evaluations were performed. On 49 (38%) of the 130 herd evaluations, 1 or more gilts had suspect test results. Additional testing was required in 19 (39%) of these 49 herd evaluations to determine the PRV infection status of the herd. Status of herds having gilts with suspect results and no positive results was usually negative after retesting. Herds having gilts with positive results were unlikely to have negative status after retesting.     

The suitability of ELISA for the detection of antibodies against Mycobacterium avium ssp. paratuberculosis in bulk milk samples from Rhineland-Palatinate

Paratuberculosis or Johne's Disease, caused by Mycobacterium avium subspecies paratuberculosis (MAP), is a notifiable disease in Germany which produces enormous economical losses in dairy farms. At present,there is no confirmed data about the actual number of infected livestock herds in Germany. A countrywide monitoring program to evaluate the prevalence in dairy herds would only be economically feasible on the basis of bulk milk testing. In this study, we evaluated two ELISA test kits (SVANOVIR Ptb-ELISA, IDEXX-M.pt. Milk test kit) for the detection of antibodies against MAP in bulk milk. First, the Paratuberculosis-status of the herd derived from the history of the farm was used as a gold standard. Paratuberculosis-negative farms were tested negative with each test, but paratuberculosis-positive or Paratuberculosis-serologically-positive farms were detected only in one case (Svanovir) or three cases (IDEXX), respectively. Even if inconclusive results are counted as positive, 82.9 % (Svanovir) or 80 % (IDEXX) of the paratuberculosis-positive or serologically paratuberculosis positive farms were not detected. Nevertheless, a re-validation of both ELISAs by means of ROC and TG-ROC analyses was attempted by searching for ideal cut-offs, optimised for bulk milk. If a high specificity was selected, no acceptable sensitivity could be reached.The best results were obtained using a sensitivity of 32.3 % at a specificity of 100 % (Svanovir). With a small change of the cut-off value, the sensitivity increased to still 57 %, but this reduced the specificity to 67 %. Similar results were obtained with the IDEXX-ELISA. We then evaluated the Svanovir-ELISA for the detection of bulk milk samples on the basis of the current paratuberculosis prevalence within 69 dairy herds from Rhineland-Palatinate using individual milk samples.When the bulk milk samples were tested in two different laboratories using the same ELISA, considerable differences in the results became evident. Nearly all samples were tested with a higher relative test result in one laboratory, which often led to differences in the classification of the prevalence levels.The estimated within-herd seroprevalences ranged between 0 % and 37 %.There was little agreement between the historical paratuberculosis herd status and the within-herd prevalence in milk serum, as reflected in a kappa-index of 0.146.To determine the sensitivity and specificity of the bulk milk ELISA by ROC and TG-ROC analysis, 116 bulk milk samples were used that had been obtained from the 69 dairy herds participating in the study. The optimal ratio of sensitivity (81 %) and specificity (77 %) relative to a "gold standard" was obtained when the cut-off was set at the 10 % level. These values for sensitivity and specificity were better than those obtained in an evaluation of the same ELISA in which the historical Paratuberculosis herd-status was used as a "gold standard." The results of this study question the suitability of the available ELISAs for bulk milk testing.Taking into account that the Svanovir-ELISA for individual milk samples has a sensitivity of 60 96% relative to the blood serum variant of the test, and that the latter has also a limited sensitivity due to the pathogenesis of paratuberculosis, the available test systems examined in this Study do not seem to be suitable for herd diagnosis by using bulk milk samples.     

Survey of Mycobacterium avium subspecies paratuberculosis serological status in beef herds on community pastures in Saskatchewan

Johne's disease is a well recognized problem in dairy herds. Relatively little information is available on either the prevalence or the control of Johne's disease in commercial cow-calf operations. In the fall of 1999, blood samples were collected during pregnancy testing from cows on community pastures in Saskatchewan. Sera from these cows were analyzed using a commercial ELISA for antibodies to Mycoplasma avium subspecies paratuberculosis. All cows from each herd examined at the community pastures were sampled. Of the 1799 samples tested, 15 had sample to positive (S/P) ratios greater than 0.25 and were considered positive (apparent sample prevalence, 0.8%; 95% CI, 0.4% to 1.5%). If we assume test sensitivity of 25% and specificity of 98% as recommended by the National Johne's Working Group, the true sample prevalence is not significantly different from 0.0%. The ELISA S/P results for the antibody test-positive animals ranged from 0.27 to 2.5. If a herd was classified as positive based on one test-positive animal, the average herd apparent prevalence was 15.2% (95% CI, 7.1% to 28.6%). If the potential for false-positive results was considered with 2 or more positive animals being required for positive herd status, the herd prevalence was 3.0% (95% CI, 0.4% to 13.4%). Because of the very low prevalence in cow-calf herds, future research to identify risk factors and control points should target problem herds and utilize a case-control study design.     

An estimate of specificity for a Johne's disease absorbed ELISA in northern Australian cattle

OBJECTIVE: To estimate the specificity of an absorbed enzyme-linked immunosorbent assay kit for Johne's disease (JD) when used in mature cattle populations resident in northern Australia. DESIGN: Blood samples were collected from beef cattle in northern Queensland, the Northern Territory and northern Western Australia, and from dairy cattle in northern Queensland. The specificity of a serological test for JD was estimated by testing the blood samples with an absorbed ELISA kit. Further samples were collected from cattle with positive ELISA results to determine the presence or absence of infection with Mycobacterium avium subsp paratuberculosis. PROCEDURE: During 1995 and 1996, blood, tissue and gut contents were collected from beef cattle at abattoirs in Queensland and the Northern Territory; and blood and faecal samples were collected from dairy cattle in herds assessed to be most at risk for JD in northern Queensland. The blood samples were tested using an absorbed ELISA kit. Tissues and gut contents from beef cattle that had positive ELISA results were cultured for M. avium subsp paratuberculosis, and tissues were examined histologically. Faecal samples from dairy cattle with positive ELISA results were cultured for M. avium subsp paratuberculosis. RESULTS: Estimates of specificity for this absorbed ELISA in mature northern Australian cattle were 98.0% (97.0 to 98.8%, 95% CI) in beef cattle, and 98.3% (96.7 to 99.3%, 95% CI) in dairy cattle. CONCLUSION: Estimates of specificity in this study were lower for beef cattle from the Northern Territory and northern Western Australia and for dairy cattle from northern Queensland than those quoted from studies on cattle in southern Western Australia. This should be considered when serological testing using the JD ELISA is carried out on northern Australian cattle.     

Comparative study of diagnostic testing for feline leukemia virus infection.

Results of commercially available diagnostic test kits and commercial laboratory test results were compared for ability to detect FeLV antigen. Results of the immunofluorescent antibody (IFA) test were compared with test kit ELISA results and with results of a system in which samples were applied to an absorbent material, dried, sent to a laboratory, eluted, and assayed by a plate ELISA. Test kits were generally highly sensitive and specific, compared with the IFA test performed at a commercial laboratory. Feline heterophile antibody, specific for mouse immunoglobulin, was detected in approximately 0.14 to 0.57% of the cat population. Test kits B, E, and D contain reagents that correct for antimouse antibodies. During 1989 and 1990, 2,229 feline serum samples were tested for FeLV antigen (gsa p27); positive ELISA results were obtained for 204 (9%) of the samples. Results for 32 (1.4%) samples were interpreted as equivocal (color development slightly exceeded that of the negative control, but was much less than that of the positive control). Collectively, the data indicate that when testing serum or saliva, a negative test result may be a good predictor that a cat is not infected. In populations of cats in which FeLV prevalence is low, a positive test result may not be reliable and thus, a confirmatory test should be performed.     

Comparison and interpretation of diagnostic tests for feline immunodeficiency virus infection.

Feline sera were submitted to the Cornell Feline Health Center (n = 497) or to the New York State Diagnostic Laboratory (n = 1,565) for feline immunodeficiency virus (FIV) testing. Some sera (n = 166) were submitted for confirmation of previous FIV-positive results; 151 of these sera had been tested at the referring veterinary practice or laboratory, using an in-house ELISA. Excluding the samples submitted for confirmation, a total of 173 samples (9.1%) were FIV-positive; 11.6% of the clinically ill or high-risk cats and 0.49% of the healthy, low risk cats were positive for FIV antibody. A commercially available ELISA for detection of antibody to FIV was evaluated in relation to the immunofluorescent antibody (IFA) test and the immunoblot assay. The ELISA was interpreted according to the manufacturer's instructions, with the ratio of sample optical density to positive control optical density (S/P) determining a positive or negative result. The ELISA results based on the S/P interpretation were compared with a kinetics-based (KELA) interpretation of the ELISA. The KELA values were reported as positive, negative, or equivocal. Using the immunoblot as the standard, ELISA (S/P interpretation) had sensitivity of 0.93 and specificity of 0.98, whereas the IFA test had sensitivity of 0.95 and specificity of 0.98. However, the sensitivity and specificity of the ELISA (S/P interpretation) were markedly reduced for sample results falling in the KELA equivocal range, indicating that equivocal results were valid interpretations for some sera. A high number (22.5%) of the samples submitted for confirmation of a positive result from use of the in-house ELISA were determined to be negative for FIV antibody.(ABSTRACT TRUNCATED AT 250 WORDS)     

An estimate of specificity for a Johne's disease absorbed ELISA in northern Australian cattle

Objective To estimate the specificity of an absorbed enzyme-linked immuno-sorbent assay kit^d for Johne's disease (JD) when used in mature cattle populations resident in northern Australia.
Design Blood samples were collected from beef cattle in northern Queensland, the Northern Territory and northern Western Australia, and from dairy cattle in northern Queensland. The specificity of a serological test for JD was estimated by testing the blood samples with an absorbed ELISA kit. Further samples were collected from cattle with positive ELISA results to determine the presence or absence of infection with Mycobacterium avium subsp paratuberculosis .
Procedure During 1995 and 1996, blood, tissue and gut contents were collected from beef cattle at abattoirs in Queensland and the Northern Territory; and blood and faecal samples were collected from dairy cattle in herds assessed to be most at risk for JD in northern Queensland. The blood samples were tested using an absorbed ELISA kit. Tissues and gut contents from beef cattle that had positive ELISA results were cultured for M avium subsp paratuberculosis , and tissues were examined histo-logically. Faecal samples from dairy cattle with positive ELISA results were cultured for M avium subsp paratuberculosis .
Results Estimates of specificity for this absorbed ELISA in mature northern Australian cattle were 98.0% (97.0 to 98.8%, 95% CI) in beef cattle, and 98.3% (96.7 to 99.3%, 95% CI) in dairy cattle.
Conclusion Estimates of specificity in this study were lower for beef cattle from the Northern Territory and northern Western Australia and for dairy cattle from northern Queensland than those quoted from studies on cattle in southern Western Australia. This should be considered when serological testing using the JD ELISA is carried out on northern Australian cattle.     

Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India

Country lacks indigenous diagnostic kits against Johne's disease in animals. Indigenous ELISA and IS 900 PCR kits, originally developed for goats and sheep, have been adapted for screening of lactating cows. Multiple diagnostic tests were used to screen 26 lactating dairy cows against Johne's disease. Milk ELISA was evaluated with fecal culture, milk culture and fecal PCR. Of the 26 samples from lactating cows, 84.6, 96.1, 88.4 and 23.0% were positive in fecal culture, milk culture, m-ELISA and m-PCR, respectively. Comparatively milk sediment and milk fat culture detected 84.6 and 76.9% cows positive, respectively. Comparatively fecal culture and milk culture detected 84.6 and 96.1% cows positive, respectively. M-ELISA detected 11.5, 0.0, 11.5, 61.0 and 15.3%, cows as negative, suspected, low positive, positive and strong positive, respectively. There was good correlation between milk and fecal culture with m-ELISA. Three negative cows in m-ELISA were also detected in milk and fecal culture. Of the 26 decontaminated fecal samples, 23.0% cows were positive using specific IS 900 f-PCR. Comparative evaluation of m-ELISA with fecal and milk culture showed agreement in 80.7 and 84.6% cows, respectively. Sensitivity of m-ELISA with respect to fecal and culture was 90.9 and 95.6%, respectively. Comparative evaluation of four tests (milk culture, fecal culture, m-ELISA and f-PCR) showed that only 15.3% cows were detected in all the four tests. In three tests (fecal and milk culture and m-ELISA), 57.6% cows were detected positive. None of the cow was exclusively detected in f-PCR. Of the four diagnostic tests used milk culture was most sensitive (96.15%), followed by fecal culture (86.6%), m-ELISA (76.9%) and IS 900 PCR (23.0%) for the diagnosis of bovine Johne's disease (BJD). Milk ELISA detected only one cow extra, which was negative in milk culture. In view of the simplicity, rapidity and efficacy present milk ELISA kit employing soluble protoplasmic antigen from native Map 'Bison type' genotype of goat origin can be reliable for screening of bovine population against Johne's disease in India.     

Comparative evaluation of the agar gel immunodiffusion test and two commercial ELISA kits for the serodiagnosis of equine infectious anemia

Selected sets of serum samples of horses were tested blindly in a comparative investigation for antibodies against Equine Infectious Anemia (EIA) virus. Three commercial kits were used, a well-established agar-gel immuno-diffusion kit which our laboratory has been using routinely for 14 years on one hand, a competitive ELISA kit (CELISA) and a non-competitive ELISA kit on the other hand. The American EIA Reference Laboratory in Ames cotested 56 serum samples with the same 3 products, with highest-level correlation, thereby ascertaining full dependability of our own results. Five EIA experts supplied us critically weak or doubtfully reacting serum samples of experimentally infected horses together with their own test results, by necessity limited to the then available AGID in most instances. A high degree of correlation was found between our and their AGID results. In our own laboratory good correlation was found between the AGID test and one lot of the CELISA product. Time of seroconversion was coincident in some experimentally infected horses, partly AGID, partly CELISA proved more sensitive. Another lot of the CELISA product deteriorated completely long before the warranted validity, an unpleasant finding experienced by many other laboratories alike. The non-competitive ELISA product showed unacceptable inter-lot differences, oscillation between positive and negative results on consecutive samples of one and the same horse, never reacted with the weak positive International Reference Serum, and one lot deteriorated well beyond its expiration date. We discuss our results with the background: high sensitivity versus false-positive horses and advocate to maintain at their present sensitivity levels the AGID and the CELISA tests and not to push them further, as would be technically possible.     

3种ELISA试剂盒检测抗牛支原体抗体的比较

为比较3种抗牛支原体(M.bovis)血清抗体的ELISA试剂盒检测效果,本实验应用3种检测M.bovis血清抗体ELISA诊断试剂盒对38份阳性样品(自然感染19份,人工感染18份)和37份阴性样品进行检测.结果表明:本实验室制备的HVRI试剂盒与商品化试剂盒Kit 1的检测结果和综合检测结果符合率分别达到92%和96%;而商品化试剂盒Kit 2的检测结果与综合检测结果符合率仅为74.67%.一致性检验结果显示:HVRI试剂盒与Kit 1的一致性较高;Kit 2与HVRI试剂盒、Kit 2与Kit 1有中度的一致性.此外,3种试剂盒对牛传染性胸膜肺炎国际标准血清(PS2)的检测结果显示HVRI试剂盒和Kit 1均为为阴性,Kit 2为阳性.因此,HVRI试剂盒与Kit 1更适于M.bovis检测和开展流行病学调查.     

Evaluation of serological tests for detecting tick-borne encephalitis virus (TBEV) antibodies in animals

Tick-borne encephalitis (TBE) in animals is not well understood yet. TBE virus (TBEV) serology in several host species could be valuable for epidemiological analyses in the field as well as for the detection of clinical cases. However, performance and suitability of the available test systems are not well assessed. Therefore, we evaluated two commercial TBEV-ELISA kits in a pilot study and compared them for their suitability in veterinary applications. For this purpose, we tested 163 field collected goat sera and evaluated the results by serum neutralization test (SNT) as "gold standard". Twenty-eight SNT positive sera (17.2%) were detected. The best suited ELISA kit was used for determination of a species-specific cutoff for horses, cattle, sheep, goats, pigs, mice, dogs, rabbits and monkeys with defined sera from animals without known or with improbable contact to TBEV. The level of non-specific ELISA results does not only differ between animal species but may also be influenced by the age of the tested animals. The number of sera which tested false positive by ELISA was higher in older than in young sheep. In order to obtain defined polyclonal sera as references, two dogs, cattle, goats, sheep, rabbits and pigs each, as well as one horse and 90 mice were immunized four times with a commercially available TBEV vaccine. In conclusion, our results demonstrated that commercial TBEV-ELISA kits are suitable for application in veterinary medicine for both, verification of clinical TBE cases and epidemiological screening. However, positive ELISA results should be verified by SNT. Only a very low number of false negative ELISA-results were found.     

重组N蛋白抗原检测PRRSV抗体ELISA的研究Ⅱ.试剂盒阴阳性临界值的测定及其与IDEXX公司试剂盒的比较

对临床上采集的899份猪血清样本。用以纯化的重组N蛋白为包被抗原建立的检测猪繁殖与呼吸综合征病毒（PRRSV）抗体的间接ELISA进行检测．运用统计学方法摸清了检测结果的分布规律。并扣国外IDEXX公司PRRSV抗体检测试剂盒同时对460份血清样本进行检测。结果表明。2种方法的符合率为91．73％。利用TG—ROC软件确定了自制的ELISA酶标板（NP—ELISA）的临界值。并标定试剂盒的特异性扣敏感性均为92．6％。ELISA的结果判定标准是：当以血清样本L为标准参考阳性血清时，样品与阳性血清的比值（S／P）小于或等于0．4为阴性；S／P在0．4与0．5之间为可疑；S／P大于或等于0．5为阳性。与IDEXX公司PRRSV抗体检测试剂盒对临床样品的检测结果进行比较后，初步判定所建立的ELISA之所以出现较多的假阴性。可能是目前临床上出现了PRRSV欧洲型所致。     

Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses.

Recently a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting antibody against H1N1 swine influenza virus (SIV) has been made available to diagnosticians and veterinary practitioners. Because the hemagglutination inhibition (HI) test has been considered the standard test for SIV serology, diagnostic performance of the new ELISA was evaluated using positive (n = 60) and negative (n = 188) serum samples from young pigs with known status of SIV infection and compared with that of the HI test. Both ELISA and HI test identified all negative animals correctly. None of the serum samples (n = 64) from pigs inoculated with H3N2 SIV was positive by ELISA for SIV antibody. The H1N1 SIV antibody detectable by ELISA appears to develop more slowly in comparison with antibody detectable by HI test. Although antibody was detected by HI test in all inoculated animals (n = 20) by day 7 postinoculation (PI), antibody was detected by ELISA in 0%, 75%, and 100% of the inoculated animals on days 7, 14, and 28 PI, respectively. Discrepancy in test results between the 2 serologic tests appeared to be because of differences in antibody isotypes detected by each test. Enzyme-linked immunosorbent assay mainly detected IgG antibody, whereas the HI test detects IgM antibody very efficiently as well as IgG antibody. Collectively, the commercial ELISA is highly specific for antibody to H1N1 SIV but may not identify positive animals at the early stage of infection as effectively as the HI test, particularly when SIV is introduced to a na?ve swine population.     

Evaluation of four commercial enzyme-linked immunosorbent assays for the diagnosis of bovine paratuberculosis in Chilean dairy herds.

The accuracy of 4 commercial enzyme-linked immunosorbent assays (ELISAs) for diagnosis of bovine paratuberculosis was compared using sera from 53 Mycobacterium avium subsp. paratuberculosis (MAP) fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal culture-negative herds on 2 consecutive occasions 1 year apart (controls). The specificity of all 4 ELISA kits was >99%, and their diagnostic sensitivity ranged from 30.2% to 41.5%. Pairwise comparison of ELISAs found no significant differences (McNemar's chi-square test > 0.05), and assay agreement for categorical assay interpretation (positive or negative) was high (>98%) with kappa values ranging from 0.84 to 0.95. Receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curves indicate that kit B had the highest overall accuracy. Thus, all 4 ELISA kits for bovine paratuberculosis had comparable accuracy when tested on Chilean dairy cattle, with kit B having a slight statistical advantage based on ROC area under the curve analysis. This suggests that any of the 4 kits could be appropriate for herd certification and for paratuberculosis control programs on Chilean dairy cattle.     

The sensitivity and specificity of a modified ELISA for the diagnosis of Johne's disease from a field trial in cattle

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycobacterium paratuberculosis in cattle was evaluated in three herds known to have Johne's disease. Prior to testing, the plasma was absorbed with dried Mycobacterium phlei in order to remove cross-reacting antibody specificities. The sensitivity and specificity of the ELISA were calculated after repeatedly testing 327 cattle in the infected herds. Of these, 53 animals had one or more positive faecal cultures or had post-mortem histopathological evidence of infection. The other 274 had three or more negative faecal culture results, and were regarded as non-infected for the purpose of evaluation of the ELISA. Using these criteria for the presence or absence of infection, the M. phlei-absorbed ELISA under field conditions had a 57% sensitivity and a 98.9% specificity. The sensitivity of the absorbed ELISA depended on the stage of disease of the animal under test. In general, it appeared that animals in the more advanced stages of disease were absorbed ELISA positive, whereas those in the early stages of infection were not detected. These results indicate that the M. phlei-absorbed ELISA has an important role as a test for the diagnosis and control of Johne's disease in cattle.     

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.