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Statistical Evaluation of ELISA Methods for Testing Caprine Paratuberculosis
†Veterinary Research Institute of Thessaloniki, National Agricultural Research Foundation, Thessaloniki, Greece
‡Institutul National de Medicina Veterinara “Pasteur”, Calleo Glulasti br 333, Sector Bucuresti, Romania.
†Medical School University of Thessaly, Papakyriazi 22, Larisa 41222, Greece
§Laboratory of Microbiology of Faculty of Veterinary Medicine, Aristotelian University of Thessaloniki, Greece.
Research was financially supported by the General Secretariat of Research and Technology of the Hellenic Ministry for Development (Joint research program between Greece-Romania, Bilateral-Cooperation).
KEYWORDS: Paratuberculosis, goats, ELISA, statistical evaluation
A survey was conducted to assess the diagnostic value of four enzyme-linked immunosorbent assays (ELISAs): LAM-A, LAM-P, GP, IDEXX-ELISA, and AGIT, for sensitivity and specificity. The assessment was based on two groups of adult goats: group 1 consisted of 44 goats infected with paratuberculosis; group 2 consisted of 73 clinically healthy goats belonging to 12 known paratuberculosis-infected herds in Northern Greece. Sera were taken from 62 known negative goats from two herds that had no clinical history of paratuberculosis and were repeatedly fecal culture test negative. In group 1, the goats were tested using tissue culture or histopathology, and in group 2, they were tested using fecal culture. These tests were used as gold standards. The four ELISA methods were compared using receiver operating characteristics (ROC) analysis. The AGIT method was evaluated using the McNemar’s test. For group 1, all methods had a good overall diagnostic accuracy. Sensitivities (SE) ranged from 0.82 to 0.91 (for LAM-A and GP, SE = 0.91; for LAM-P, SE = 0.87; for IDEXX, SE = 0.82; and for AGIT, SE = 0.84). For group 2, the overall diagnostic accuracy of methods is limited. LAM-A, GP, and IDEXX had identical sensitivities (SE = 0.55) The LAM-P had SE = 0.45. The AGIT had low sensitivity (SE = 0.09). The specificities of ELISA methods ranged from 0.79 to 0.95 (LAM-A = 0.79 and GP = 0,86; for LAM-P = 0.92 and IDEXX = 0.96). The AGIT had high specificity (SP = 1).
Paratuberculosis, or Johne’s disease, is a chronic granulomatous enteritis of ruminants caused by M. a. paratuberculosis (MAP).1 In small ruminants, the only consistent, though nonspecific, clinical sign of the disease is progressive weight loss. Diarrhea is not a constant feature and is often intermittent, although it can be severe in some goats in individual herds.2
The prevalence of the disease in goats in Greece is high, and infection is the cause of significant economic loss. To reduce the infection rate in a flock, detecting and culling infected animals at an early stage of infection and preventing transmission of MAP to young livestock within an infected flock are important. Effective detection of subclinical cases of caprine Johne’s disease is a critical step in the reduction of disease prevalence in infected goat flocks.
The serologic diagnosis of Johne’s disease has long been hampered by a lack of specific and sensitive tests. The practicing veterinarian needs a rapid, economic, reliable test to confirm a case of clinical Johne’s disease and to detect the subclinically infected animal. Sherman et al.3 evaluated an agar gel immunodiffusion (AGIT) test as an aid in differential diagnosis of Johne’s disease. AGIT might be a highly sensitive and specific test in ruminants with clinical signs suggestive of Johne’s disease.
Numerous modifications in enzyme-linked immunosorbent assay (ELISA) methods have been evaluated. Improvements include preabsorbing the test sera with a suspension of M. phlei4 and using an affinity-purified peptide antigen.5 Efforts to identity nonprotein antigens are also being pursued. Sugden et al.6 observed higher sensitivity than the commercial ELISA using the lipoarabinomannan antigen in an experimental ELISA when diagnosing paratuberculosis in sheep.
An ELISA for the detection of antibodies against M. a. paratuberculosis in cattle was evaluated based on a GP (proteinic) antigen. The M. phlei-absorbed ELISA under field condition had a 57% sensitivity and a 99% specificity using fecal culture or histopathology positivity as the gold standard signifying true infection.7
The goal of this study was to assess the diagnostic value of four ELISA tests: LAM-A, LAM-P, GP, IDEXX ELISA, and the AGIT in terms of sensitivity (SE) and specificity (SP).
Materials and methods
A total of 117 adult goats belonging to 12 known paratuberculosis-infected herds in Northern Greece were tested using bacteriology, histopathology, and serologic tests. The 44 clinical cases were selected on the basis of chronic weight loss from herds with established paratuberculosis problems, and the diagnosis was confirmed by microscopical examination, culture, or histopathology. The remaining 73 goats were free of clinical signs.
Serum samples were obtained from all animals described. Sera from 62 known negative goats from two herds that had no clinical history of paratuberculosis and were repeatedly fecal culture test negative were used as negative controls.
Tissues and Feces
Intestinal and lymph node samples were collected from the 44 clinical cases (total 88 samples) and were cultured for M. a. paratuberculosis. Fecal specimens were collected from the 73 goats free of clinical signs and were cultured for MAP.
Tissues and fecal samples were decontaminated with NaOH and inoculated into Herrold’s egg yolk medium with mycobactin j 2mg/L (Merieux).
The IDEXX-ELISA was performed as recommended by the manufacturer (IDDEX, Corporation Portland, ME, U.S.A.). Briefly, serum samples were absorbed with an absorbent for 60 minutes at room temperature. 100 mL of absorbed sera were added to the appropriate wells of the antigen-coated microtiter plates and incubated for 60 minutes at room temperature. Afterwards, 100 mL of horseradish peroxidase (HRPO) protein G conjugate was added to each well. After addition of the substrate reactive with HRPO, the result is indicated by color development. The color is measured using a microplate reader at a single wavelength of 450 nm.
The three ELISA kits, LAM-A, LAM-P, and GP, were performed as recommended by the manufacturer (Institute Pasteur of Bucharest, [AU: Please give location in Romania.]Romania). Serum samples were diluted 1/200 with PBS + 1% BSA. Then, 100 mL of diluted serum were added to the covered microplates and incubated for 15 minutes at 37˚C. Afterward, 100 mL of peroxidase conjugate was added to each well, and 100 mL of ABTS in substrate buffer was also added. The reaction was stopped with NAF in 6 mL deionized water, and the optical densities (OD) were measured at 405 nm.
Sera were examined by AGIT using antigen prepared by ultrasonication of M. a. paratuberculosis strain 316 F.
For each test the optical densities (OD) were recorded. For AGIT the disease status was recorded in binary form (diseased or not diseased).
The four ELISA methods (LAM-P, LAM-A, GP, IDEXX) were compared using ROC curve analysis. The AGIT method was evaluated using the McNemar’s test. In the ROC curve analysis, a curve is constructed by plotting the sensitivity against 1 specificity over a range of cutoff OD values. When the area under the curve (AUC) is equal to its minimum value of 0.5, the diagnostic method has no accuracy at all. The method has the best accuracy when the AUC has its maximum value of 1.8 The best cutoff value of the diagnostic method is defined by the value that best compromises between the highest sensitivity and specificity values, ie, discriminates between diseased and non-diseased animals. The AUC under the ROC curve and the cut-off with the highest sensitivity and specificity for each antigen were calculated.
The significance of the AUC was determined by the method described by Hanley and McNeil,9 in which the standard error of the AUC is calculated parametrically and its 95% confidence interval (CI) is asymptotic. The specificity and sensitivity cut-off differences were evaluated by comparing the confidence intervals.10 The analysis was performed using SPSS and Fortran subroutines.
At necropsy, the 44 clinically affected goats generally had the clear gross lesions of thickened and inflamed ileum, and Ziehl- Neelsen staining revealed clumps or abundant small slender acid-fast bacteria.
In group 1, 25 of 44 tissue specimens were culture positive in Herrold’s medium while the remaining (19) were culture negative but histopathology positive. In group 2, 11 samples were detected as fecal culture positive and 62 as negative. The serologic reactors in four ELISAS and AGIT tests in relation to tissue culture histopathology (group 1) and fecal culture (group 2) are shown in Tables 1 and 2. Table 3 shows the results of control goats in ELISAS and AGIT.
The specificities of ELISA methods ranged from 0.79 to 0.95 (for LAM-A, 0.79; GP, 0.86; LAM-P, 0.92; IDEXX, 0.96). The AGIT had high specificity (SP = 1).
For Group 1
Figure 1 illustrates the ROC curves for the tissue culture and histopathology data. The AUC with the corresponding standard error (SE) of each curve is shown in Table 1. The AUC of each method is significantly different from 0.5 (P < 0.05), and the upper limits of 95% CIs approach the value of 1, indicating that the methods have a good overall diagnostic accuracy. The GP has the highest AUC (0.959 ± 0.027), and the IDEXX has the lowest AUC (0.894 ± 0.041). For selected cutoff values (see Table 5), the methods have and sensitivities ranging from 0.84 to 0.91 (for LAM-A and GP, SE = 0.91; for LAM-P, SE = 0.87; for IDEXX, SE = 0.82). The 95% CIs are overlapping, and the upper limit approaches the value 1. AGIT has SE = 0.84 and SP = 1, and it agrees with the tissue culture and histopathology data (P = 0.023).
Figure 2 illustrates the ROC curves for the fecal culture data. The AUC with the corresponding SE of each curve is shown in Table 6. The AUC of each test is not significantly different from 0.5 (P > 0.05), indicating that the overall diagnostic accuracy of these methods is limited. However, the LAM-A has the highest AUC (0.671 ± 0.089) compared with the other methods, and the LAMP-P has the lowest AUC (0.551 ± 0.121). For selected cutoff values (Table 7), the LAM-A, IDEXX, and GP have identical sensitivities (SE = 0.55). The LAM-P has a slightly lower sensitivity (SE = 0.45). The 95% CIs for the SE and the SP are overlapping; however, only the upper limit for SP of LAM-P is approaching the value 1. The AGIT is not useful at all in terms of sensitivity (SE = 0.09), but it has a perfect specificity (SP = 1). However, the method is associated with the fecal culture results (P < 0.004).
The diagnosis of paratuberculosis in goats and sheep can be difficult, particularly for an individual animal. Serologic tests combined with a clinical examination and a microscopic and bacteriologic examination of feces are commonly used as diagnostic indicators in ruminants. However none of these tests is definitive.
In sheep, culture is often unsuccessful, and no test reliably identifies subclinical forms of the disease.11 For the two groups, the results of four ELISAs did not mark significant differences in test specificity. The exceptions were IDEXX and LAM-P, with estimated specificities of 0.95 and 0.92, respectively. Therefore, IDEXX and LAM-P seem to be more specific than LAM-A and GP ELISA.
In the cases of 44 clinically affected goats, no significant differences were seen among all methods in test sensitivity. LAM-A and GP detected more tissue culture- or histopathology-positive goats (91%) than LAM-P (87%) and IDEXX (82%). These results are comparable to those seen for application of an ELISA technique based on a lipoarabinomannan antigen, with a positive predictive value of 74% and a negative predictive value of 99%, using the ileocecal lymph node culture as the gold standard.12
The usefulness of serologic testing for clinically diseased animals was shown by the high sensitivity of the AGIT and ELISA tests. In the four ELISA tests of the 44 clinically affected goats, the optical densities of 36 sera in LAM-P, 31 in IDEXX, 28 in LAM-A, and 26 in GP were greater than the mean positive control value. Of the 44 culture- or histopathology-positive animals, 37 were AGIT positive and diseased. Of the 11 fecal culture-positive animals, one was AGIT positive but not diseased.
The high antibody levels of these goats appeared to correlate with clumps of or abundant mycobacteria. However, the sensitivity of the ELISA test is highest for animals in the later stages of the disease, usually when the animals develop clinical signs. 13 Animals with clinical symptoms of paratuberculosis in most cases show high titers until breakdown, which facilitates diagnosis.14
Similar studies on goats gave ELISA sensitivities between 54% and 88.2%, with 65.2% to 73% sensitivity for the AGIT test.15-18 In a serologic survey for ovine paratuberculosis, the sensitivity of two commercial ELISA tests was 50.9%.19
For the group of 73 goats free of clinical signs, the results did not mark significant differences in test sensitivity. In cases of clinically healthy goats results of LAM-P ELISA showed a better correlation with those of fecal culture than the findings of LAM-A. This confirms the observations of Jark et al.12 and Gasteiner et al.,14 assigning H-ELISA (Hannover ELISA) high specificity in identifying subclinically infected animals. This is probably due to use of the antigen from M. a. paratuberculosis instead of antigen from M. a. avium applied in case of LAM-A ELISA. LAM-P ELISA clearly detected fecal culture-negative sera in 92%, which LAM-A only did in 79%.
Additionally, the serum samples of fecal culture positive animals were positive in 55% by LAM-A ELISA and only in 45% by LAM-P. These diversities between the results of both ELISA methods are similar with those of Gasteiner et al.14 They estimate that these diversities may be caused by the presence of antibodies that reacted strongly with the antigen of M. a. avium but not or weakly with M. a. paratuberculosis.
LAM-A and GP ELISA had identical sensitivities in clinically infected animals and identical sensitivity in clinically healthy animals but different specificities of 0.79 and 0.86, respectively. Although both methods use different antigens, M. a. avium for LAM-A and M. a. paratuberculosis for GP-ELISA, there is agreement between them.
The sensitivity results are comparable to those seen for application of commercial ELISA in goats for which 54% of fecal shedders were ELISA–positive18 and in cattle for which 57% of fecal culture or histopathology positive samples were ELISA positive.
With all ELISA tests, however, not all fecal culture-positive animals were identified. Milner et al.20 clearly state that animals in the early stages of infection, even if shedding the organism, cannot necessarily be identified by ELISA. This is because months or even years may elapse before seroconversion, limiting the value of the ELISA and any other serologic tests in any scheme for the eradication of paratuberculosis.
Additionally, the clinical signs and the intermittent nature of fecal shedding became a major determinant of sensitivity for ELISA and perhaps for any serologic test. Most goats in the first stage of the disease are lightly infected and have negative fecal cultures and essentially no detectable antibody in serum.
Poor or low sensitivity has also been described for ELISA tests using the LAM antigen21 to detect lightly infected cattle, as well as agar gel immunodiffusion tests.3
AGIT appeared to be specific for both cases of goats and sensitive (0.84) for clinically infected animals. However, is not useful at all in terms of sensitivity (SE = 0.09) in clinically healthy goats. As shown previously by Huchzermeyer and Bastianello,22 the AGIT test did not prove to be an effective method to diagnose paratuberculosis sheep in the first stage of the disease.
Thus, the sensitivity shown in subclinical cases is not sufficient to base a control program on the ELISA alone. However, if ELISA is used in conjunction with one or possibly more methods in a strict testing program, caprine paratuberculosis may be successfully controlled and eventually eradicated from Greece.
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Table 1. Number of Serologic Reactors in Four ELISA and AGIT Tests in Relation to Tissue Culture or Histopathology From Paratuberculosis Clinical Cases
culture or Serologic Tests
44 38+ 40+ 40+ 36+ 37+
6– 4– 4– 8– 7–
Table 2. Number of Serologic Reactors in Four ELISAS and AGIT Tests in Relation to Fecal Culture From Clinically Healthy Goats
11 5+ 6+ 6+ 6+ 1+
6– 5– 5– 5– 10–
Table 3. Results of Control Goats in Four ELISAS and AGIT
62 57– 49– 53– 59– 62–
5+ 13+ 9+ 3+
4. The AUC Values of the ROC
curves for Group 1 With the Corresponding Standard Errors, Significant
Levels, and 95%
Std. Asymptotic Asymptotic
LAM_P .934 .032 .000 .870 .997
LAM_A .947 .028 .000 .892 1.002
GP .959 .027 .000 .906 1.012
IDEXX .894 .041 .000 .813 .974
Table 5. Cutoff Points of the Four ELISA Methods for Group 1 With the Corresponding Sensitivities
Method Cutoff Se
LAM-P 0.45 0.87
LAM-A 0.48 0.91
GP 0.413 0.91
*Standard error is 0.
Table 6. The AUC Values of the ROC Curves for Group 2 With the Corresponding Standard Errors, Significant Levels, and 95% Confidence Intervals
Std. Asymptotic Asymptotic
LAMP_P .551 .121 .589 .315 .788
GP .658 .102 .096 .458 .858
LAMP_A .671 .089 .072 .496 .846
IDEXX .604 .104 .274 .401 .807
7. Cutoff Points of the four ELISA Methods for Group 2 and Control
Method Cutoff Se SP
LAM-P 0.724 0.45 0.92
(0.16, 0.74) (0.85, 0.99)
LAM-A 0.582 0.55 0.79
(0.26, 0.84) (0.69, 0.89)
GP 0.899 0.55 0.86
(0.26, 0.84) (0.77, 0.95)
IDEXX 0.163 0.55 0.95
(0.26, 0.84) (0.91, 0.99)
*Standard error is 0.
Figure 1. ROC curves for comparing the four ELISA methods (LAM-A, LAM-P, GP, IDEXX-ELISA) in group 1). The gold standard was the tissue culture or the histopathology results. The AUC values of the curves with the corresponding standard errors, significant levels, and 95% confidence intervals (CI) are shown.
Figure 2. ROC curves for comparing the four ELISA methods (LAM-A, LAM-P, GP, IDEXX-ELISA) in group 2. The gold standard was the tissue culture or the histopathology results. The AUC values of the curves with the corresponding standard errors, significant levels and 95% confidence intervals (CI) are shown.
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