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Background: Enzyme-linked immunosorbent assays (ELISAs) are the most widely used diagnostic tools in bovine paratuberculosis (bPTB) control. However, their diagnostic accuracy may be compromised by bovine tuberculosis (bTB) infection, as both diseases share diagnostic targets., Methods: The bPTB and bTB infection status of 228 animals was determined using microbiological tissue culture as a reference test. The diagnostic performance (sensitivity, specificity, likelihood ratios and predictive values) of the bPTB-ELISA on blood serum samples, taking into account the bPTB animal-level prevalence of the area and the bTB status of the animals, was evaluated., Results: A sensitivity of 40.7% (95% confidence interval [CI]: 27.5%-53.9%) and a specificity of 94.7% (95% CI: 91.4%-98.0%) were obtained for bPTB-ELISA in all animals. A bPTB-ELISA-positive animal would have a post-test probability of 70% or more of being infected in areas with a bPTB prevalence of 23% or more. A negative bPTB-ELISA result, in areas with a bPTB prevalence of 41% or less, would rule out the disease with more than 70% certainty. In bTB-positive animals, sensitivity increased (94.4% [95% CI: 81.4%-100%] vs. 25.1% [95% CI: 11.8%-38.4%]) and specificity decreased (82.6% [95% CI: 71.8%-93.4%] vs. 99.4% [95% CI: 98.0%-99.9%]). The bPTB-ELISA is a good tool to rule out bPTB co-infection in bTB-positive animals, while in bTB-negative animals, it allows confirmation of disease with more than 70% probability if disease prevalence is 6% or more., Limitations: The observed differences could be enhanced by the effect of frequent application of the intradermal tuberculin test, which was unknown in the animals studied., Conclusions: These results provide useful guidance for the application and interpretation of ELISA as a tool for bPTB disease control., (© 2023 British Veterinary Association.)

Background: Bovine tuberculosis (bTB) diagnosis is impaired by numerous factors including cross-reactivity with Mycobacterium avium subspecies paratuberculosis , which causes Johne's disease (JD). In addition, the effect of repeated bTB-intradermal testing on the performance of JD diagnostic tests is not fully understood. This study aimed to evaluate the impact of repeated bTB-intradermal tests under field conditions in Spain on the JD serological status of cattle., Methods: bTB-positive herds (n=264) from Castilla-y-Leon region were selected and matched with officially tuberculosis-free control herds. The association between JD and bTB status at the herd level was assessed using conditional logistic regression and, in herds with both JD-positive and bTB-positive animals, a Bayesian hierarchical mixed-effect model was used for individual-level analysis., Results: A significantly higher risk of being JD positive (OR: 1.48; 95 per cent CI: 1.01 to 2.15) was found for bTB-positive herds compared with controls. Individual results indicated that cattle tested more than three times per year, within the last 90 days and more than 12 months were more likely to be JD positive. A skin test-related boost in antibody response could be the cause of an apparent increase of the sensitivity of the JD-absorbed ELISA., Conclusion: The results demonstrate the interaction between bTB repeated testing and JD individual and herd-level results and this improved knowledge will facilitate the design of more effective control programmes in herds coinfected with two of the most important endemic diseases affecting cattle in Spain., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

This retrospective case-control study investigates the relationship between seroconversion to Mycobacterium avium subspecies paratuberculosis (MAP) and raised somatic cell count (SCC). The study consists of 112 case cows from three dairy farms in the UK; for each case cow with a positive antibody titre, there was a seronegative control cow for comparison. Seroconversion was monitored using milk ELISA antibody titres for MAP taken at quarterly intervals. SCCs were recorded at the time a positive antibody titre was first recorded as well as at the previous and subsequent milk recording in order to explore a temporal relationship between the two events. The previous and subsequent milk recordings were a month before and after seroconversion was identified. The results showed that cows that were infected with MAP had an increased SCC around the time that they first became seropositive, providing evidence for a temporal relationship between the two events; high SCCs were particularly prevalent before and at the time of first detecting seroconversion. The explanation is being discussed that potentially an underlying, currently not studied, factor may be predisposing both events, the progression of paratuberculosis is predisposing the host to mastitis, or indeed intramammary infections help initiate paratuberculosis progression., Competing Interests: Competing interests: None declared., (© British Veterinary Association 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

The area under the receiver operating characteristic (ROC) curve (AUC) is used as a performance metric for quantitative tests. Although multiple biomarkers may be available for diagnostic or screening purposes, diagnostic accuracy is often assessed individually rather than in combination. In this paper, we consider the interesting problem of combining multiple biomarkers for use in a single diagnostic criterion with the goal of improving the diagnostic accuracy above that of an individual biomarker. The diagnostic criterion created from multiple biomarkers is based on the predictive probability of disease, conditional on given multiple biomarker outcomes. If the computed predictive probability exceeds a specified cutoff, the corresponding subject is allocated as 'diseased'. This defines a standard diagnostic criterion that has its own ROC curve, namely, the combined ROC (cROC). The AUC metric for cROC, namely, the combined AUC (cAUC), is used to compare the predictive criterion based on multiple biomarkers to one based on fewer biomarkers. A multivariate random-effects model is proposed for modeling multiple normally distributed dependent scores. Bayesian methods for estimating ROC curves and corresponding (marginal) AUCs are developed when a perfect reference standard is not available. In addition, cAUCs are computed to compare the accuracy of different combinations of biomarkers for diagnosis. The methods are evaluated using simulations and are applied to data for Johne's disease (paratuberculosis) in cattle., (Copyright © 2015 John Wiley & Sons, Ltd.)

Commercial ELISA kits are widely used in the diagnosis of paratuberculosis of dairy cattle. It is critically important to understand the influences on these test results and their relation to faecal culture (FC) results in order to interpret the findings and to make decisions concerning serial testing and control measures. A total of 1021 cattle (423 FC positive, 598 FC negative) from 14 Mycobacterium avium subspecies paratuberculosis (MAP) positive herds were tested with four ELISA systems and FC simultaneously to calculate the kappa coefficients for the agreement of the different ELISA systems as well as find influencing factors. For the agreement of FC and ELISA, the kappa coefficients were low and ranged from 0.19 to 0.24, whereas, results of the different ELISA were consistently high (0.74-0.90). Agreement with FC was enhanced with the duration of control (P≤0.001) and the lactation number (P≤0.01), and reduced with within-herd prevalence (P≤0.001). There were substantial differences in the detection rate of low (15-24 per cent) and high (85-100 per cent) MAP shedders. In conclusion, the factors shown to influence test sensitivity, should be taken into account for validation and interpretation of ELISA tests. The benefit of serial ELISA testing is low.

The receiver operating characteristic (ROC) curve is commonly used for evaluating the discriminatory ability of a biomarker. Measurements for a diagnostic test may be subject to an analytic limit of detection leading to immeasurable or unreportable test results. Ignoring the scores that are beyond the limit of detection of a test leads to a biased assessment of its discriminatory ability, as reflected by indices such as the associated area under the curve (AUC). We propose a Bayesian approach for the estimation of the ROC curve and its AUC for a test with a limit of detection in the absence of gold standard based on assumptions of normally and gamma-distributed data. The methods are evaluated in simulation studies, and a truncated gamma model with a point mass is used to evaluate quantitative real-time polymerase chain reaction data for bovine Johne's disease (paratuberculosis). Simulations indicated that estimates of diagnostic accuracy and AUC were good even for relatively small sample sizes (n=200). Exceptions were when there was a high per cent of unquantifiable results (60 per cent) or when AUC was < or =0.6, which indicated a marked overlap between the outcomes in infected and non-infected populations.

A stratified matched-pair study is often designed for adjusting a confounding effect or effect of different trails/centers/ groups in modern medical studies. The relative risk is one of the most frequently used indices in comparing efficiency of two treatments in clinical trials. In this paper, we propose seven confidence interval estimators for the common relative risk and three simultaneous confidence interval estimators for the relative risks in stratified matched-pair designs. The performance of the proposed methods is evaluated with respect to their type I error rates, powers, coverage probabilities, and expected widths. Our empirical results show that the percentile bootstrap confidence interval and bootstrap-resampling-based Bonferroni simultaneous confidence interval behave satisfactorily for small to large sample sizes in the sense that (i) their empirical coverage probabilities can be well controlled around the pre-specified nominal confidence level with reasonably shorter confidence widths; and (ii) the empirical type I error rates of their associated test statistics are generally closer to the pre-specified nominal level with larger powers. They are hence recommended. Two real examples from clinical laboratory studies are used to illustrate the proposed methodologies.

We develop a novel semiparametric modeling framework involving mixtures of Polya trees for screening data with the dual purpose of diagnosing infection or disease status and of assessing the accuracy of continuous diagnostic measures. In this framework, we obtain (i) predictive probabilities of 'disease' based on continuous diagnostic test outcomes in conjunction with other information, including relevant covariates and results from one or more independent binary diagnostic tests. An example would be the modeling of a serum enzyme-linked immunosorbent assay (ELISA) procedure for detecting antibodies to an infectious agent when used in conjunction with culture for antigen detection. Our second goal is to (ii) characterize measures of diagnostic performance of continuous tests by estimating receiver-operating characteristic curves and area under the curve, primarily when such extra information is available. When true disease status is unknown, parametric and nonparametric analyses require sufficient separation between the distributions of outcome values for the diseased and nondiseased populations. However, this overlap becomes less problematic when additional information in the form of either an informative 'prior' that is based on real (preferably data-based) scientific input, or when additional information, or both, are available. The additional information can be used to distinguish 'diseased' from 'nondiseased' individuals. We present an example using simulated data that illustrates this point. We also present an example involving data from an animal-health survey for Johne's disease, where the performance of a serum ELISA is evaluated using additional information obtained from fecal culture. Issues related to identifiability and partial identifiability are also discussed., ((c) 2008 John Wiley & Sons, Ltd.)

Monoclonal antibodies (mAbs) against a recombinant carboxyl terminus of the 34 kDa protein of Mycobacterium paratuberculosis were produced in mice. Two of the mAbs cross-reacted with Mycobacterium avium and Mycobacterium intracellulare in both an elisa and immunoblot. The recombinant protein also reacted with polyclonal sera produced in rabbits against all three mycobacteria, indicating the presence of cross-reactive epitopes in the protein. To determine the reactivity of cattle sera against epitopes recognised by the mAbs, competition assays between bovine sera and the mAbs were carried out. Two mAbs were significantly inhibited by sera from cattle that were naturally infected with M paratuberculosis. The results indicate that epitopes on the carboxyl terminus of the 34 kDa protein common to M paratuberculosis, M avium and M intracellulare readily induce antibody production in naturally infected cattle. These epitopes reduce the diagnostic specificity of the carboxyl terminus of the 34 kDa protein, which was originally thought to contain only M paratuberculosis-specific epitopes.

Thirty-two sheep with clinical paratuberculosis and 43 normal, healthy control animals were tested for serum antibodies to Mycobacterium avium subspecies paratuberculosis with the absorbed enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion (AGID) test. All the sheep were necropsied and the diseased cases were categorised as having either multibacillary ('lepromatous') or paucibacillary ('tuberculoid') intestinal lesions. The ELISA and AGID test were highly sensitive when testing the multibacillary group (86.4 per cent and 100 per cent, respectively) but the sensitivity of the tests in the paucibacillary group was significantly lower (10 to 50 per cent and 30 per cent, respectively). These findings were related to the ELISA optical density readings, with the multibacillary samples having values significantly greater than those of the paucibacillary and control groups, and the optical density values appearing to correlate with the numbers of mycobacteria present in the intestinal lesions. These results indicate both the usefulness of serological testing in the diagnosis of the multibacillary form of paratuberculosis and the difficulty in identifying animals with the paucibacillary form of the disease.

An outbreak of Johne's disease in a herd of farmed red deer was studied for four years. Serological, histopathological and cultural techniques were used to monitor the progress of the disease, and delayed type hypersensitivity skin tests were also applied. The results of the serological tests showed that they were poor predictors of future clinical cases and did not consistently identify animals harbouring mycobacteria. The histopathological methods provided a sensitive and specific means of confirming the infection. The skin tests had a low sensitivity and the results were poorly correlated with the serological results in seropositive animals. A vaccination policy was instituted which was accompanied by a change in the pattern of disease. Although the histopathological evidence suggested that the infection was still occurring, there was a marked reduction in the incidence of clinical disease. Vaccinated animals showed a good response to the skin test.

Paratuberculosis was diagnosed in one 18-month-old and two 30-month-old hinds in a herd of 70 red deer (Cervus elaphus) in Ireland. Loss of condition and intermittent diarrhoea were the main clinical findings. Clumps of acid-fast organisms were found in the faeces of the three deer. Post mortem examination of one deer showed a slight swelling and pallor of the intestinal tract and associated lymph nodes. Histopathology showed a severe, granulomatous enteritis and lymphadenitis, with extensive cellular infiltration, notably with epithelioid macrophages containing numerous acid-fast organisms. Mycobacterium paratuberculosis was isolated from intestinal and lymph node samples. Paratuberculosis was also confirmed in one of nine clinically normal, yearling stags, sampled at slaughter. Complement fixation tests and enzyme-linked immunosorbent assays gave higher readings for clinically affected deer than healthy ones. Acid soil on the farm was believed to be a contributory cause.

Gastrointestinal tract tissues from cattle with suspected clinical Johne's disease (JD), in which acid-fast bacilli were not identified in mucosal smears, were examined histopathologically. Twenty-two per cent were positive and 4 per cent were suggestive of JD. Failure to identify mycobacteria in mucosal smears appeared to result from the presence of mild, often focal mucosal lesions which contained relatively few bacilli. Ten of the 22 histopathologically positive cases had complement fixation titres for JD but several false-positive titres also occurred.

A tuberculin testing trial was carried out in eight counties of south-eastern England to compare the specificity for bovine tuberculosis of Weybridge human PPD with that of Rotterdam bovine PPD. The matching of these two tuberculins for potency in naturally infected cattle had already been established, the bovine PPD being approximately one-and-a-half times more potent than the human PPD per unit of weight. In 1110 cattle in 25 herds with histories of long-standing freedom from tuberculosis and in which non-specific tuberculin sensitivity was present, cross reactions were less to the bovine PPD than to the human PPD, showing that in the environment of this trial the bovine PPD was more specific than the human PPD. Induration diameter was a satisfactory alternative to skin thickening as a measure of tuberculin reactions in cattle under field conditions. Due to the steep slope of the dose-response curves of the avian PPD in the different groups of non-tuberculous cattle, the discriminating power of the comparative test, using avian and mammalian tuberculins, was less at lower doses of tuberculin. Concentrations of 1-0 mg per ml of bovine PPD and 0-5 mg per ml of avian PPD are recommended for use in a comparative tuberculin test.

Paratuberculosis was studied for three years in a large goat herd. Post mortem lesions were seen mainly in the ileum and mesenteric lymph nodes where caseation was often present. In three successive years the annual faecal excretor rate was 15, 9 and 13 per cent despite removal of positives. Faecal culture and an agar gel immunodiffusion test proved useful for detecting the disease in live goats, but complement fixation and tuberculin tests were of less value. The prospects of controlling the disease are discussed.

Sera from 775 adult milking cattle on 13 farms in Friesland and from 359 of the same cattle taken during the following year, were examined for evidence of infection with M johnei by the complement fixation, fluorescent antibody, haemagglutination, haemagglutination-lysis and immunodiffusion tests. Eighteen animals, not clinically affected but which had serologically positive tests were slaughtered and M johnei was isolated in cultures from 16. The fluorescent antibody test was as sensitive as the complement fixation test. The other tests were relatively insensitive and would only be of value as confirmatory tests in the presence of reactions to the first two tests. It is concluded that the use of the fluorescent antibody and the complement fixation test together was of value in detecting sub-clinical Mycobacterium johnei infection.

Since 1962, when the control of Johne's disease was last reviewed by the BVA technical development committee there have been no dramatic advances in diagnostic methods for Johne's disease or in methods of control. However, there has been a slow, but steady increase in knowledge of the various aspects of the disease. This article attempts to set out what is known and, more important, not known about the subject.

A field trial on a country-wide basis was undertaken to compare the specificity for bovine tuberculosis of single and comparative tuberculin tests in cattle using either Weybridge human or Weybridge bovine PPD. The tests were made on 10,305 cattle in 179 herds distributed throughout all regions of England, Scotland and Wales. Results showed that a comparative tuberculin test using avian PPD with either human or bovine PPD had a much higher efficiency than a single injection of mammalian tuberculin in the neck of cattle, and confirmed that a comparative test is still essential in the British environment. Weybridge bovine PPD gave significantly better discrimination between tuberculous and non-tuberculous cattle than Weybridge human PPD when used together with avian PPD in a comparative tuberculin test. The diameter of induration gave an absolute measure of the extent of oedema, if present, and induration diameter used in conjunction with skin thickening increased the sensitivity and specificity of the test. Rules of interpretation were developed and are presented for an intradermal comparative tuberculin test in cattle using Weybridge avian and bovine PPDs.