Evaluation of antibody-ELISA and real-time RT-PCR for the diagnosis and profiling of bluetongue virus serotype 8 during the epidemic in Belgium in 2006

In 2006 bluetongue (BT) emerged for the first time in North-Western Europe. Reliable diagnostic tools are essential in controlling BT but data on the diagnostic sensitivity (Se) and specificity (Sp) are often missing. This paper aims to describe and analyse the results obtained with the diagnostics used in Belgium during the 2006 BT crisis. The diagnosis was based on a combination of antibody detection (competitive ELISA, cELISA) and viral RNA detection by real-time RT-PCR (RT-qPCR). The performance of the cELISA as a diagnostic tool was assessed on field results obtained during the epidemic and previous surveillance campaigns. As the infectious status of the animals is unknown during an epidemic, a Bayesian analysis was performed. Both assays were found to be equally specific (RT-qPCR: 98.5%; cELISA: 98.2%) while the diagnostic sensitivity of the RT-qPCR (99.5%) was superior to that of the cELISA (87.8%). The assumption of RT-qPCR as standard of comparison during the bluetongue virus (BTV) epidemic proved valid based on the results of the Bayesian analysis. A ROC analysis of the cELISA, using RT-qPCR as standard of comparison, showed that the cut-off point with the highest accuracy occurred at a percentage negativity of 66, which is markedly higher than the cut-off proposed by the manufacturer. The analysis of the results was further extended to serological and molecular profiling and the possible use of profiling as a rapid epidemiological marker of the BTV in-field situation was assessed. A comparison of the serological profiles obtained before, during and at the end of the Belgian epidemic clearly showed the existence of an intermediate zone which appears soon after BTV (re)enters the population. The appearance or disappearance of this intermediate zone is correlated with virus circulation and provides valuable information, which would be entirely overlooked if only positive and negative results were considered.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.     

Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.     

Development of a capture ELISA for the detection of antibodies to enteropathogenic Escherichia coli (EPEC) in rabbit flocks using intimin-specific monoclonal antibodies

A capture enzyme-linked immunosorbent assay (cELISA) was developed using intimin-specific monoclonal antibodies to detect specific antibody in rabbits that have been in contact with enteropathogenic Escherichia coli (EPEC). Sera from 121 EPEC-negative, minimum-disease-level (MDL) rabbits were used for negative controls, and sera from 25 MDL rabbits, experimentally infected with EPEC of bio-/serotype 3-/O15, for positive controls. These were used to determine a cut-off value for a positive cELISA result. The value selected gave the test a sensitivity of 80.0% and a specificity of 98.4% on an individual level. At this value, a flock level sensitivity and specificity of 79.2 and 85.2%, respectively were calculated for a flock with a prevalence of seven per cent, if 40 animals were tested, and a minimum of two reactors were obtained. The test characteristics improve with increasing prevalence. To evaluate the diagnostic potential of the cELISA, sera from 40 to 50 slaughter rabbits per flock from 25 rabbit flocks with bacteriologically determined EPEC status were tested. The results demonstrated that this test can be a useful tool to determine the EPEC status of a rabbitry, provided that it is used at regular intervals.     

Principles and practical application of the receiver-operating characteristic analysis for diagnostic tests

We review the principles and practical application of receiver-operating characteristic (ROC) analysis for diagnostic tests. ROC analysis can be used for diagnostic tests with outcomes measured on ordinal, interval or ratio scales. The dependence of the diagnostic sensitivity and specificity on the selected cut-off value must be considered for a full test evaluation and for test comparison. All possible combinations of sensitivity and specificity that can be achieved by changing the test's cut-off value can be summarised using a single parameter; the area under the ROC curve. The ROC technique can also be used to optimise cut-off values with regard to a given prevalence in the target population and cost ratio of false-positive and false-negative results. However, plots of optimisation parameters against the selected cut-off value provide a more-direct method for cut-off selection. Candidates for such optimisation parameters are linear combinations of sensitivity and specificity (with weights selected to reflect the decision-making situation), odds ratio, chance-corrected measures of association (e. g. kappa) and likelihood ratios. We discuss some recent developments in ROC analysis, including meta-analysis of diagnostic tests, correlated ROC curves (paired-sample design) and chance- and prevalence-corrected ROC curves.     

Evaluation of the sensitivity and specificity of bovine tuberculosis diagnostic tests in naturally infected cattle herds using a Bayesian approach

Test-and-slaughter strategies have been the basis of bovine tuberculosis (BT) eradication programs worldwide; however, eradication efforts have not succeeded in certain regions, and imperfect sensitivity and specificity of applied diagnostic techniques have been deemed as one of the possible causes for such failure. Evaluation of tuberculosis diagnostic tools has been impaired by the lack of an adequate gold standard to define positive and negative individuals. Here, a Bayesian approach was formulated to estimate for the first time sensitivity (Se) and specificity (Sp) of the tests [single intradermal tuberculin (SIT) test, and interferon-gamma (IFN-γ) assay] currently used in Spain. Field data from the first implementation of IFN-γ assay (used in parallel with SIT test 2-6months after a first disclosure SIT test) in infected beef, dairy and bullfighting cattle herds from the region of Castilla and Leon were used for the analysis. Model results suggested that in the described situation: (i) Se of SIT test was highly variable (40.1-92.2% for severe interpretation, median=66-69%), and its Sp was high (>99%) regardless interpretation criteria; (ii) IFN-γ assay showed a high Se (median=89-90% and 83.5% for 0.05 and 0.1 cut-off points respectively) and an acceptable Sp (85.7% and 90.3% for 0.05 and 0.1 thresholds) and (iii) parallel application of both tests maximized the combined Se (95.6% using severe SIT and 0.05 cut-off point in the IFN-γ assay). These results support the potential use of the IFN-γ assay as an ancillary technique for routine BT diagnosis.     

Cut-off points for aggreate herd testing in the presence of disease clustering and correlation of test errors

In order to test if disease is present in a large herd, an investigator will often subject only a small sample of animals to a fallible diagnostic test. The herd is declared positive for disease if the number of test-positive animals is greater than or equal to a previously chosen cut-off value. Such a test, called an aggregate test, has a sensitivity and specificity that depends on the sample size, the cut-off point and the sensitivity and specificity of the individual test. It also depends on the distribution of the disease among the herds being tested and on the fact that factors such as herd-level seropositivity may cause some herds to be more prone to testing errors than others. In this paper, we use the beta-binomial distribution to model all these factors and thereby calculate and tabulate aggregate test sensitivities and specificities under a variety of conditions. Receiver operating characteristic (ROC) curve methodology permits the choice of optimum sample sizes and cut-off values. We also investigate the situation in which an investigator may be willing to miss detecting the disease if the prevalence in the herd is low. A compiled FORTRAN program for the calculation of aggregate test cut-off point properties, including positive and negative predictive values, is available from the authors.     

Validation of a commercially available cELISA test for canine neosporosis against an indirect fluorescent antibody test (IFAT)

A commercially available competitive enzyme-linked immunosorbent assay (cELISA, VMRD^®) was validated for the detection of Neospora caninum antibodies in the serum of dogs, using as a reference test an indirect fluorescent antibody test (IFAT, Fuller^®). A partial verification approach was used. A total of 618 dogs were screened with cELISA and a subset of positive and negative sera (n = 237) were then tested with IFAT. Naïve relative sensitivity (SE_nv) and naïve relative specificity (SP_nv) of cELISA were calculated and then corrected (SE_corr; SP_corr) for studies with partial validation. Results showed a SE_nv of 72% and a SP_nv of 89.3%; corrected estimates showed a SE_corr of 47% and a SP_corr of 96%. ROC analysis showed that the cutoff recommended by the manufacturer (30%) corresponded to the highest naïve sensitivity (72%) combined with a good naïve specificity (90%) of cELISA. Corrected estimates of SE and SP for partial verification method revealed that SE of the cELISA is lower and SP is higher than naïve estimates. The results suggest to use this test for confirmation of a clinical suspicion of neosporosis, and to use some techniques for adjustment of misclassification in prevalence and risk-factor studies.     

Analysis of the diagnostic accuracy of the gamma interferon assay for detection of bovine tuberculosis in U.S. herds

The goal of this study was to evaluate the test sensitivity (SE) and specificity (SP) of the gamma interferon (G-IFN) assay used for the detection of bovine tuberculosis (bTB) in U.S. cattle herds. In addition, the study assessed the association between G-IFN test results and bTB status of cattle, and explored different cut off values for classification of test results in adult cattle using receiver operating characteristics (ROC) curve analysis. Test SE was estimated using a population of 87 confirmed infected cattle from 14 herds distributed in 6 states. Test SP was estimated using a population of 4123 cattle representing 3000 premises in 3 states. These animals were from bTB free areas, accredited bTB free herds, or herds that were historically bTB free based on the absence of lesions found at slaughter and historical records of negative tests performed for bTB surveillance. The distribution of G-IFN results and its association with bTB infection status was also explored in a group of 914 exposed cattle in which infection was not confirmed. The results showed that the SE of the G-IFN for a cut-off value ≥0.1 was 83.9% (76.1, 91.6). The SP of the G-IFN was 90.7% (95% CI: 89.8, 91.6), 97% (95% CI: 96.5, 97.5), and 98.6%(95% CI: 98.2, 98.9), for cut off values of 0.1, 0.3, and 0.5, respectively. For a cut off value ≥0.1, the likelihood ratio of a positive G-IFN test was 9.03 (95% CI: 7.90, 10.31), and the likelihood ratio of a negative G-IFN test was 0.18 (95% CI: 0.11, 0.29). The area under the ROC curve was 0.976 (95% CI: 0.97, 0.98), characteristic of a highly accurate test. ROC analysis also showed that lower cut-off values, such as 0.1, have high SE with suitable SP for use in parallel testing, while cut-off values ranging between 0.3 and 0.6 provide the high SP desired in series-testing protocols with lower SE values. Findings from this study indicated that the G-IFN performs with high accuracy in the field, yielding SE and SP estimates comparable to those reported in previous evaluations (Ryan et al., 2000; Ameni et al., 2000; de la Rua-Domenech et al., 2006; Gormley et al., 2006).     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

Evaluation of a Portable Meter to Measure Ketonemia and Comparison with Ketonuria for the Diagnosis of Canine Diabetic Ketoacidosis

Background: The diagnosis of canine diabetic ketoacidosis (DKA) usually is based on measurement of urinary acetoacetate (ketonuria). In humans, this test is less sensitive and specific than blood 3-β-hydroxybutyrate (ketonemia) evaluation.
Hypothesis: Ketonemia measurement using a portable meter is more accurate than ketonuria determination with a dipstick to diagnose canine DKA.
Animals: Seventy-two client-owned diabetic dogs with ketonemia, ketonuria, or both.
Methods: Prospective observational study. Based on blood bicarbonate concentration and anion gap, dogs were divided into 2 groups: patients with DKA ( n = 25); patients with diabetic ketosis ( n = 47). Sensitivity, specificity, and positive and negative likelihood ratio (LR) at different cut-off points were determined for both ketonemia and ketonuria. Receiver operating characteristic (ROC) analysis was used to assess the accuracy of each diagnostic test to diagnose DKA.
Results: With regard to ketonemia, cut-off values of 2.3 and 4.3 mmol/L revealed 100% sensitivity and 100% specificity, respectively, whereas cut-off values of 2.8 and 3.5 mmol/L showed a −LR of 0.05 and a + LR of 13.16, respectively. With regard to ketonuria, a cut-off value of 1+ revealed 92% sensitivity, 40% specificity, and −LR of 0.20, whereas a cut-off value of 3+ revealed 44% sensitivity, 94% specificity, and +LR of 6.89. The areas under the ROC curves for the ketonemia and ketonuria tests were significantly different (0.97 and 0.81, respectively, P = .003).
Conclusions and Clinical Importance: Measurement of ketonemia is accurate and more effective than measurement of ketonuria to diagnose canine DKA.     

Quantitative assessment of clinical signs for the detection of classical swine fever outbreaks during an epidemic

The performance of clinical signs as a diagnostic test for the detection of classical swine fever (CSF) outbreaks during the 1997-1998 CSF epidemic in The Netherlands was evaluated by constructing and analysing a receiver operating characteristic (ROC) curve. This curve assesses the discriminating ability of a diagnostic test over a range of test signals. The cut-off values for a defined diagnostic test to detect CSF outbreaks were set by different combinations of clinical signs observed. The area under the ROC curve, which is a quantitative measure of test performance, was significantly (P<0.001) larger than the area under the random ROC curve. This indicates that clinical signs have a significantly higher performance as a diagnostic test for the detection of CSF than for flipping a coin. However, the gain in diagnostic performance compared to a random process is not as much as we would wish it to be. The optimal efficient diagnostic test combined a sensitivity of 72.7% with a specificity of 52.7%, with a combination of the following clinical signs: unsteady gait/ataxia, not eating, not reacting to antibiotic treatment, conjunctivitis, hard faecal pellets.     

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.     

Development of a competitive enzyme-linked immunosorbent assay for detection of turkey coronavirus antibodies

A competitive enzyme-linked immunosorbent assay (cELISA) was developed for detection of turkey coronavirus (TCV) antibodies. The cELISA utilized a recombinant baculovirus (Autographa californica nuclear polyhedrosis virus)-expressed TCV nucleocapsid (N) protein and biotin-labeled TCV N protein-specific monoclonal antibody. Sensitivity and specificity of the cELISA for detection of TCV antibodies were determined by comparison with the indirect fluorescent antibody test (IFAT) with 1269 reference, experimentally derived, and field-origin sera. Sera with discordant cELISA and IFAT results were further evaluated by western immunoblot analyses. The cELISA detected antibodies specific for TCV and infectious bronchitis virus, a closely related coronavirus, but did not detect antibodies specific for other avian viruses. A high degree of concordance was observed between the cELISA and IFAT; sensitivity and specificity of the cELISA relative to IFAT were 92.9% and 96.2%, respectively. Western immunoblot analyses provided additional evidence of cELISA specificity. The findings indicate that the cELISA is a rapid, sensitive, and specific serologic test for detection of TCV antibodies in turkeys.     

Seroprevalences for ovine enzootic abortion in Switzerland

Our aim was to assess the seroprevalence of Chlamydophila (Cd) abortus (Chlamydia psittaci serotype 1), denoted ovine enzootic abortion (OEA), in the Swiss sheep population. A competitive enzyme-linked immunosorbent assay (cELISA) was adapted for the investigation of pooled serum samples (pool approach) and receiver-operator characteristic (ROC) analysis was applied to define the cut-off of the pool approach. At a cut-off value of 30% inhibition, the flock-level pooled sensitivity and specificity were 92.9% and 97.6% when compared to classifying the flock based on individual-animal samples.

Subsequently, sera from 775 randomly selected flocks out of 11 cantons of Switzerland were investigated using the pool approach. The cantons included in the study represented 72% of the Swiss sheep flocks and 76% of Swiss sheep population. Antibodies against Cd. abortus were found in almost 19% (144) of the 775 examined sheep flocks. Test prevalences were adjusted for the imperfect test characteristics using the Rogan–Gladen estimator and Bayesian inference. Seroprevalence was highest (43%) in the canton Graubünden. In the remaining 10 cantons the seroprevalence ranged from 2 to 29%. The cELISA in combination with testing pooled sera and statistical methods for true prevalence estimation provided a good survey tool at lower costs and time when compared to other approaches.     

Validation of a Neospora caninum iscom ELISA without a gold standard

Neospora caninum is an intracellular parasite which causes abortion in cattle worldwide. One problem in the validation of the different methods for demonstration of this parasite is the lack of an appropriate gold standard. To validate an immunostimulating complex (iscom) enzyme-linked immunoassay (ELISA) used to detect antibodies to N. caninum, sera from 244 cattle in five Swedish dairy herds infected with N. caninum were analysed. The sera also were analysed by a standard indirect-fluorescent antibody test (IFAT). The results obtained by the two tests were compared using the Gibbs sampler. Gibbs sampling is a latent-class approach based on Bayesian statistics; neither test is assumed to be more correct in stating the true status of infection. The Gibbs sampler was run using both informative and non-informative prior probabilities. We also simulated different cut-offs in the iscom ELISA (providing data to inform selection of optimal cut-off values for different applications).The ELISA produced fewest incorrect test results over all at a cut-off value of 0.200. The sensitivity and specificity at this cut-off were 99 and 96%, respectively. The IFAT had a high specificity (99%) but a lower sensitivity (78%) than expected-confirming that the IFAT cannot be treated as a true gold standard. Sensitivity and specificity of the ELISA were presented in a two-graph receiver operating characteristic (TG-ROC) plot. Any cut-off between 0.150 and 0.300 will have both sensitivity and specificity > or =95%. Optical densities of < or =0.150 and > or =0.550 (or > or =0.350) were suggested as limits to rule out and rule in infection, respectively.     

Performance characteristics of an enzyme-linked immunosorbent assay performed in milk for the detection of liver fluke (Fasciola hepatica) infection in cattle

AIM: To determine the performance characteristics of an enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against liver fluke (Fasciola hepatica) in bovine milk. METHODS: Serum and milk from liver fluke infected and non-infected cattle was assayed in a commercially available enzyme-linked immunosorbent assay. Serum test results were used to determine the "gold standard" infection status of cattle and milk ELISA results assessed by ROC analysis. RESULTS: ROC analysis suggested changes to the ELISA protocol, arriving at milk dilutions assayed considerably higher than those suggested by the manufacturer. With those changes, the ELISA performed with high sensitivity and specificity, 95 and 98.2%, respectively, for individual bovine milks (relative to sera). For bovine tank milks, sensitivity was lower, with bulk milks only testing positive if 60% or more of cattle milking in the herd were infected. CONCLUSIONS: The analysis of the ELISA's performance when used on individual bovine milks demonstrated high sensitivity and specificity. ROC analyses optimised the assay conditions and cut-off point suggested by the manufacturer for this commercial diagnostic assay. This would help with the identification and control of fasciolosis, enabling simpler sample collection.     

Performance evaluation of two serological tests for contagious bovine pleuropneumonia (CBPP) detection in an enzootic area using a Bayesian framework

A Bayesian approach, allowing for conditional dependence between two tests was used to estimate without gold standard the sensitivities of complement fixation test (CFT) and competitive enzyme-linked immunosorbent assay test (cELISA) and the serological prevalence of CBPP in a cattle population of the Central Delta of the Niger River in Mali, where CBPP is enzootic and the true prevalence and animals serological state were unknown. A significant difference (P = 0.99) was observed between the sensitivities of the two tests, estimated at 73.7% (95% probability interval [PI], 63.4-82.7) for cELISA and 42.3% (95% PI, 33.3-53.7) for CFT. Individual-level serological prevalence in the study population was estimated at 14.1% (95% PI, 10.8-16.9). Our results indicate that in enzootic areas, cELISA performs better in terms of sensitivity than CFT. However, negative conditional sensitivity dependence between the two tests was detected, implying that to achieve maximum sensitivity, the two tests should be applied in parallel.     

Estimation of receiver-operating characteristic curves to determine accuracy of a competitive enzyme-linked immunosorbent assay for the serodiagnosis of Brucella infection in domestic water buffalo (Bubalus bubalis) and cattle

OBJECTIVE: To estimate receiver-operating characteristic (ROC) curves for a competitive ELISA (c-ELISA) that is used in serodiagnosis of brucellosis in water buffalo and cattle, to determine the most appropriate positive cutoff value for the c-ELISA in confirmation of infection, and to evaluate species differences in c-ELISA function. SAMPLE POPULATION: Sera from 4 herds of cattle (n = 391) and 4 herds of water buffalo (381). PROCEDURE: Serum samples were evaluated for Brucella-specific antibodies by use of a c-ELISA. On the basis of previous serologic test results, iterative simulation modeling was used to classify animals as positive or negative for Brucella infection without the use of a gold standard. Accuracy of c-ELISA for diagnosis of infection was compared between cattle and water buffalo by comparison of areas under ROC curves. RESULTS: A positive cutoff value of 30% inhibition for c-ELISA yielded sensitivity and specificity estimates, respectively, of 83.9 and 92.6% for cattle and 91.4 and 95.4% for water buffalo. A positive cutoff value of 35% inhibition yielded sensitivity and specificity estimates, respectively, of 83.9 and 96.2% for cattle and 88.0 and 974% for water buffalo. Areas under ROC curves were 0.94 and 0.98 for cattle and water buffalo, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: ROC curves can be estimated by use of iterative simulation methods to determine optimal cutoff values for diagnostic tests with quantitative outcomes. A cutoff value of 35% inhibition for the c-ELISA was found to be most appropriate for confirmation of Brucella infection in cattle and water buffalo.     

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.