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Paratuberculosis is one of the complex livestock infections whose control has largely been hampered due to the lack of efficacious diagnostics. Present study optimized plate ELISA assay for the diagnosis and screening of paratuberculosis using recombinant secretory proteins. Five secretory antigens (2677c, 3547c, 4308c, 1693c, and 2168c) were produced in the recombinant system using the E. coli host and used for the optimization of the assay. These proteins were selected because of their prior proven specificity and antigenicity as humoral immunity markers. The assay was first optimized using traditional ELISA reader and then the performance was evaluated using a handheld ELISA reader. Findings were identical in both traditional ELISA reader as well as handheld ELISA reader. Optimized ELISA was found reproducible using different batches of the recombinant antigens as well as in terms of the inter and intra assay %CV values. The present ELISA has a sensitivity and specificity of 91.6% and 100%, respectively. Also, rELISA revealed AUC ROC and Youden index J of 0.95 and 0.91, respectively. In conclusion, assay conditions of MAP-recombinant protein-based ELISA were optimized and the optimized ELISA ODs can be read using portable handheld ELISA reader. Thereby, opening a future window to develop assay for onsite testing., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiological agent of Johne's disease, a chronic granulomatous enteritis with a high global prevalence in dairy cattle. This disease causes significant economic loss in the dairy industry and has been challenging to control, as current diagnostic assays are low in sensitivity and specificity, and previously developed vaccines do not prevent infection and face regulatory concerns due to interference with bovine tuberculosis diagnostics. To remediate this issue, positive and negative immune markers were created in a MAP strain as a step towards a vaccine capable of differentiating infected from vaccinated animals (DIVA). A gene coding for an immunogenic protein (MAP1693c) in the MAP genome was replaced with a library of epitope-tagged immunogenic genes (pepA) via a stable allelic exchange method. These markers were evaluated in a calf infection trial, where Holstein-Friesian dairy calves were inoculated at two weeks of age with either the marked strain or the parent strain, or remained uninfected controls. Cellular immune responses to the markers were measured using an interferon gamma release assay (IGRA). There were no MAP1693c marker-specific differences in cellular immune responses between infection groups. A scrambled version of the HA (human influenza hemagglutinin) epitope, but not the actual HA epitope, induced a significant IFN-γ response in marker-infected calves compared to WT-infected and uninfected groups at 4.5 months post-inoculation. This scrambled HA epitope thus holds potential as a diagnostic tool as part of a DIVA vaccine for Johne's disease., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The aim of our study was to evaluate the flock sensitivity and specificity of fecal qPCR and serum ELISA using pooled samples for screening paratuberculosis in French sheep. Using individual feces with low or high qPCR Ct values from ewes sampled in 14 infected flocks, a total of 555 pools of size 5, 10 and 20 were created by diluting individual materials in negative feces and analysed using a commercial IS900 qPCR kit. The relative performances of pooled serum ELISA analysis were evaluated based on the analysis of 181 different pools of size 5 and 10, composed of individual serum samples of various individual S/P values. Results showed that for pools of size 5, 10 or 20, individual fecal samples with low Ct values were invariably detected. Conversely fecal samples with high Ct values were associated with a lower detection rate in both pools of size 5 (87.0% to 90.0%), 10 (63.0% to 70.7%) and 20 (46.7% to 60.0%). After lowering the decision threshold to 25% and 15% for serum pools of size 5 and 10 respectively, the pooled serum ELISA relative sensitivity ranged between 62.2% and 100.0% depending on the composition of the pools. Finally, a simulation study was carried out to evaluate the performances of 16 screening strategies at flock level, with varying pool size (5 to 20) and number (5 to 60). The use of pooled serum ELISA led to very false positive detection rate ranging between 37.6% and 91.8% in paratuberculosis free flocks and prevents its further use in that context. For infection prevalence ≤ 5%, the flock sensitivity based on pooled fecal qPCR ranged between 39.0% (5 pools of size 10) and 99.9% (300 sampled individuals, with pools of size 5,10 or20), and was always above 93% when the infection prevalence was greater or equal to 15%. We conclude that pooled-fecal qPCR but not pooled-serum ELISA could be a useful tool to detect sheep flocks infected with paratuberculosis., Competing Interests: The authors have declared that no competing interests exist.

Testing large quantities of samples in order to detect one or more test-positive sample(s) is expensive and time-consuming. It is possible to optimize this process by pooling samples. Two frameworks to produce different hierarchical and non-hierarchical pooling schemes were tested and compared to standard pooling. Their efficiency and the potential savings were determined as a function of prevalence and the number of pooled samples. The potential benefit of pooling samples is dependent upon the changes in the analytical sensitivity and specificity of the test used when diluting test-positive samples by pooling. To illustrate this, the sensitivity of antibody ELISA on pooled samples of bovine milk for Salmonella Dublin, Mycobacterium avium spp. paratuberculosis, and bovine virus diarrhea was tested. For these milk assays, the analytical sensitivity decreased rapidly with increasing pool sizes. The efficiency of pooling is usually only measured by the number of tests performed, yet real savings depend on all the costs involved in the pooling process. These may differ between laboratories depending on the available equipment and the salaries of the technicians, among other factors. Therefore, several cost parameters were introduced to describe the total cost and thereby calculate the total savings. In terms of overall savings, both tested schemes were potentially optimal depending on the prevalence, possible pool size, and the cost of retesting. For the pool sizes of interest in this study, the three-stage hierarchical pooling scheme was often marginally more efficient in terms of the total number of tests. However, if the price of re-pooling was high, the two-stage scheme performed better in terms of total savings. In addition, for low prevalences and the possibility of pooling a large number of samples, the two-stage non-hierarchical test may be more efficient, both in terms of number of tests and overall cost. In order to apply these results in different laboratory settings, a free Shiny WebApp was developed, to compare several pooling schemes with different cost parameters., (Copyright © 2016 Elsevier B.V. All rights reserved.)

An enzyme-linked immunosorbent assay (ELISA) was developed and optimised using a Mycobacterium avium subspecies paratuberculosis (MAP) antigen prepared from a C strain (316v) passed through a French press. The optimised assay was evaluated with a panel of sera from MAP infected (n = 66) and uninfected (n = 1,092) sheep. Animals in the MAP infected category were positive on either tissue culture or histopathology but were of unknown serum antibody status. The diagnostic performance and cost of the assay were compared with those of a commercial ELISA (IDEXX). At 99.8% diagnostic specificity the assay showed a diagnostic sensitivity of 23% (95% CI: 15.1-35.8) compared with 36.4% (95% CI: 25.8-48.4) for the commercial ELISA (McNemar's test: chi-square 5.82, p < 0.05). The sensitivities were 5.9% (95% CI: 1-26.9), 27.9% (95% CI: 14.7-45.7) and 35% (95% CI: 18.1-56.7), for low grade, paucibacillary and multibacillary lesion grades, respectively. The cost of the commercial assay kit was 2.7 to 5.2 times greater than that of the 316v ELISA for an equivalent number of tests, the multiple depending on the number of plates processed per run. For flock-level surveillance, to account for the lower sensitivity of the 316v ELISA compared with the commercial ELISA, sample sizes would be increased but the test cost would still be lower. The 316v assay will be useful for diagnosis of Johne's disease in sheep flocks, particularly in developing countries where labour costs are low relative to the cost of consumables.

Johne's disease (JD), caused by Mycobacterium avium subspecies paratuberculosis (MAP), is an important gastrointestinal disease of cattle worldwide because of the economic losses encountered in JD-affected herds. These losses include reduction in milk yield in cows, premature culling and reduced carcass weight of culled diseased animals. In the U.S. dairy industry, economic losses from reduced productivity associated with JD are estimated to cost between $200 and $250 million annually. The development of non-laboratory-based assays would support more frequent testing of animals for JD and could improve its control. Conductometric biosensors combine immunomigration technology with electronic signal detection and have been adapted for the detection of IgG antibody against MAP. In the present study, a capture membrane with limited variability in the immunomigration channel and an optimal concentration of the secondary anti-bovine antibody used in a previously developed conductometric biosensor were compared with a commercially available antibody detection ELISA in their evaluation of JD, using samples of serum from cattle whose JD status where unknown. There was a moderate strength of agreement (kappa = 0.41) between the two assays. Findings from this preliminary study support the continued development of conductometric biosensors for use in the diagnosis of JD.

Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis (MAP), can cause considerable economic losses in affected herds. Early diagnosis of JD is hampered by the chronic nature of the disease with a slow subclincal progression. The aim of the present study was to challenge the hypothesis that lymphatic fluid is of diagnostic value in the early stages of the disease. Lymphatic fluid from 122 animals was collected and tested for MAP by nested PCR for IS900 and compared to the results of testing for MAP in feces (culture), blood and milk (ELISA) in 110 of these samples. MAP was detected by PCR in 27.1% of the lymph samples. Agreement between the tests was poor: 6.9% of the lymph positive cows were also positive in all other tests applied, and 69.0% had negative results in fecal culture, blood and milk ELISA. Resampling of 25 cows after 8 to 12 and 16 to 20 months revealed 20.0% lymph positive animals at the first, 5.5% at the second and 27.8% at the third sampling, respectively. Only one cow showed positive lymph-PCR results at more than one sampling date. Lymph-positive cows had a 7.2 times greater likelihood of being culled within 8 to 12 months after sampling, compared to negative cows, mainly due to other health issues than JD. It can be concluded, that lymphatic fluid might be promising for the detection of early MAP-infection in cows, but further studies to elucidate the potential of this diagnostic approach are needed., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The enzyme-linked immunosorbent assay (ELISA) is the diagnostic test most commonly used in efforts to control paratuberculosis in domestic ruminants. However, commercial ELISAs have not been validated for detecting antibodies against Mycobacterium avium subsp. paratuberculosis in wild animals. In this study, we compared the sensitivities and specificities of five ELISAs using individual serum samples collected from 41 fallow deer with or without histopathological lesions consistent with paratuberculosis. Two target antigenic preparations were selected, an ethanol-treated protoplasmic preparation obtained from a fallow deer M. avium subsp. paratuberculosis isolate (ELISAs A and B) and a paratuberculosis protoplasmic antigen (PPA3) (ELISAs C and D). Fallow deer antibodies bound to the immobilized antigens were detected by using a horseradish peroxidase (HRP)-conjugated anti-fallow deer IgG antibody (ELISAs A and C) or HRP-conjugated protein G (ELISAs B and D). A commercially available assay, ELISA-E, which was designed to detect M. avium subsp. paratuberculosis antibodies in cattle, sheep, and goats, was also tested. Although ELISAs A, C, and E had the same sensitivity (72%), ELISAs A and C were more specific (100%) for detecting fallow deer with lesions consistent with paratuberculosis at necropsy than was the ELISA-E (87.5%). In addition, the ELISA-A was particularly sensitive for detecting fallow deer in the latent stages of infection (62.5%). The antibody responses detected with the ELISA-A correlated with both the severity of enteric lesions and the presence of acid-fast bacteria in gut tissue samples. In summary, our study shows that the ELISA-A can be a cost-effective diagnostic tool for preventing the spread of paratuberculosis among fallow deer populations., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

A desirable test to diagnose infections with Mycobacterium avium subsp. paratuberculosis facilitates identification of infected cattle prior to the state of M. avium subsp. paratuberculosis shedding. This study aimed at adjusting a flow cytometry (FC)-based assay, using intact M. avium subsp. paratuberculosis bacteria as the antigen, for diagnosis of M. avium subsp. paratuberculosis infections in calves. Serum samples were collected from experimentally infected (n = 12) and naturally exposed (n = 32) calves. Samples from five calves from positive dams were analyzed to determine the dynamics of maternal antibodies. Samples from adult cattle with defined infection status served as the standard (18 M. avium subsp. paratuberculosis shedders, 22 M. avium subsp. paratuberculosis free). After preadsorption with Mycobacterium phlei, sera were incubated with M. avium subsp. paratuberculosis and M. avium subsp. avium bacterial suspensions, respectively, followed by the separate detection of bovine IgG, IgG1, IgG2, and IgM attached to the bacterial surface. M. avium subsp. paratuberculosis-specific sample/positive (S/P) ratios were compared to enzyme-linked immunosorbent assay (ELISA) S/P ratios. In adult cattle, the FC assay for IgG1 had a sensitivity of 78% at a specificity of 100%. Maternally acquired antibodies could be detected in calves up to 121 days of life. While all but two sera taken at day 100 ± 10 postnatum from naturally exposed calves tested negative, elevated S/P ratios (IgG and IgG1) became detectable from 44 and 46 weeks postinoculation onwards in two calves infected experimentally. Even with the optimized FC assay, M. avium subsp. paratuberculosis-specific antibodies can only occasionally be detected in infected calves less than 12 months of age. The failure to detect such antibodies apparently reflects the distinct immunobiology of M. avium subsp. paratuberculosis infections rather than methodological constraints.

The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne's disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies., The objectives of this study were to identify risk factors associated with i) a Mycobacterium avium subsp. paratuberculosis (MAP)-antibody milk enzyme-linked immunosorbent assay (MAP milk ELISA)-positive herd status, and ii) the within-herd MAP milk ELISA-positive prevalence in Canadian dairy herds. This prospective cohort study was conducted between 2005 and 2009 on 226 herds in Ontario and western Canada, which participated in a voluntary risk assessment (RA)-based Johne’s disease control program. Two MAP milk ELISA and risk assessments and a previsit survey were available per herd. The overall farm RA scores alone could not be used to predict whether a herd would test positive for MAP antibodies. However, the results of this study indicated that increasing the likelihood of exposing calves to MAP through certain management practices, as assessed with the RA, increased the likelihood of a herd being test-positive for MAP antibodies.

Johne's disease (JD) or paratuberculosis, caused by Mycobacterium avium ssp. paratuberculosis (MAP), is one of the most economically important diseases of dairy cattle. Control of JD could be achieved by good herd management practices, and diagnosis; however, this approach has been hampered by the low sensitivity of currently available enzyme-linked immunosorbent assay (ELISA) tests. In our previous study, we developed a sensitive serum ELISA test, ethanol-vortex enzyme-linked immunosorbent assay (EVELISA), using ethanol extract of MAP. The objective of this study is to demonstrate that the EVELISA can be used for detection of anti-MAP antibodies in milk samples. In this study, we tested and optimized concentrations of antigen, milk, and secondary antibody for better differentiation of milk samples of cattle with MAP infections from those of cattle in JD-free herds. We evaluated five environmental mycobacteria as absorbents of cross-reactive antibodies in milk and found that the mycobacteria had no significant effect on EVELISA results. Using the optimized conditions, a total of 57 milk samples from Holstein dairy cattle (37 animals found positive on the fecal polymerase chain reaction test and 20 animals from JD-free herds) were tested for anti-MAP antibody in milk by using the EVELISA method. The average of ELISA values in the JD-positive milk samples (mean±SD=0.355±0.455) was significantly higher than that in the JD-negative milk samples (mean±SD=0.071±0.011). These results warrant further studies for evaluation and validation of the EVELISA for milk testing of cattle for JD.

Although the interferon-gamma (IFN-γ) assay for measurements of cell-mediated immune (CMI) responses to paratuberculosis PPD (johnin) has been available for close to 20 years, the assay has not yet emerged as the long desired test to identify infected animals at an early time point. Among other issues, this relates to problematic interpretation of the test results and maybe an over-expectation of what can be deducted from this kind of test given the chronic nature and slow development of infection of paratuberculosis. Over a number of years a modified IFN-γ assay with addition of recombinant bovine IL-12 to the PPDj stimulation of blood samples from the heifer group in more than 20 Danish dairy herds which also perform surveillance of MAP antibodies in milk have been performed. The results indicate that IFN-γ assay results are specific for paratuberculosis, but the IFN-γ assay result of an individual animal cannot establish whether the animal is infected or predict the future progression of disease in this animal. The IFN-γ assay should thus be used on a group of animals to test the level of exposure to paratuberculosis bacteria the animals have experienced, and thereby assist in maintaining rational in-herd management procedures and in the establishment of paratuberculosis status of a given herd. Indeed, for any diagnostic test applied in paratuberculosis, both the diagnostic target condition and the purpose of the diagnostic testing must be considered before any meaningful estimates of sensitivity or specificity can be given., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis heat shock protein 70 (MAPHsp70) is an immunodominant antigen, which can be used as a subunit vaccine against bovine paratuberculosis. In the present study, we evaluated the immunogenic activities of MAPHsp70 expressed by DNA vaccine in chicken and the use of prepared specific avian IgY antibodies for western blotting and ELISA methods. The gene encoding MAP Hsp70 was subcloned into the eukaryotic expression vector, pcDNA3.1, and the recombinant plasmid (pcDNA3.1-MAP Hsp70) transfected into COS-7 cells. Chickens were also immunized with pcDNA3.1-MAP Hsp70, and egg yolk antibodies extracted from eggs were collected after immunization. DNA-designed IgY antibody was used in Western blotting analysis to detect the expression of MAPHsp70, and in a sandwich ELISA to assess the prevalence of anti-MAPHsp70 antibodies in cattle serum. Western blotting results indicate the expression of rMAP hsp70 in COS-7 cells and sandwich ELISA could detect anti-MAPHsp70 antibodies in 7.5% of cows. Chicken immunization with pcDNA3.1-MAPHsp70 could demonstrate the effective production of anti-MAPHsp70 IgY antibodies. Monospecific anti-MAPHsp70 antibody generated in chickens is useful for detection of MAPHsp70 peptide in cell culture and MAP lysate.

Of the non-ruminant wildlife species known to harbor Mycobacterium avium paratuberculosis (MAP), the rabbit (Oryctolagus cuniculus) is thought to pose the greatest risk of transmission to cattle. We analyzed 80 hunter-harvested wild rabbits from a core study area in southern Spain, and sera from 157 wild rabbits sampled opportunistically on seven additional sites. Gross lesions compatible with paratuberculosis were observed in two of 80 necropsied rabbits. Histopathology revealed focal to diffuse multibacillary MAP-compatible lesions in 8 of 10 rabbits examined. Presence of MAP was confirmed in one rabbit with gross lesions by positive amplification curves for both IS900 and ISMAP02. However, no isolate was obtained from 47 samples by culture. We adapted an indirect ELISA for the detection of MAP antibodies. At the established cut-off of 0.5, 6 of 237 wild rabbit sera (2.5%) yielded a positive ELISA result. Antibodies were detected in rabbits from 3 of 8 sampling sites. Considering the increasing relevance of MAP infection for animal health, these results open a challenging field for future research., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p<0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases., (Copyright © 2009 Elsevier Ltd. All rights reserved.)

The use of enzyme-linked immunosorbent assays (ELISAs) is recommended for Johne's disease (JD) control in dairy herds. In 2006, we developed a novel ELISA test for JD, named EVELISA (ELISA using ethanol extract of Mycobacterium avium subsp. paratuberculosis), which showed higher sensitivity than commercial ELISA tests. To further investigate the performance of EVELISA, we obtained 38 serum samples from cattle in a JD-free herd with suspected cases of serological false-positive reactions. When these samples were tested using the EVELISA and a commercial ELISA test, more than 70% of the samples were falsely identified as JD positive. Antibodies in the serum samples reacted strongly with antigens of various environmental mycobacteria, suggesting the presence of cross-reactive antibodies in the samples. The possible cross reactions in the EVELISA were inhibited markedly by the use of Mycobacterium phlei antigens for antibody absorption. When these samples were tested, 8 samples were classified as positive for JD by the EVELISA with the antibody absorption, whereas 27 samples were classified as positive for JD by the commercial ELISA. For an estimation of tentative sensitivity and specificity, the ELISA tests were performed on 38 serum samples from JD-negative herds with no suspected cases of serological false-positive reaction and 68 samples from cattle diagnosed as positive for M. avium subsp. paratuberculosis infection by fecal culture test. Sensitivity and specificity of the EVELISA with preabsorption of serum with M. phlei ("ethanol vortex absorbed-ELISA" or EVA-ELISA) were estimated to be 97.1% and 100%, respectively, whereas those of the commercial ELISA were 48.5% and 97.4%, respectively. Further, in 85 fecal culture-negative cattle in JD-positive herds, higher sensitivity of the EVA-ELISA than the commercial ELISA was demonstrated by a Bayesian analysis. This study indicates that the EVA-ELISA may form a basis for a sensitive diagnostic test with a higher level of specificity than that of the current commercial ELISA test.

Johne's disease, caused by Mycobacterium avium, subspecies paratuberculosis (MAP) is becoming increasingly widespread on dairy farms worldwide, due in part, to the absence of vaccine/drug or curative modalities.  This spread is of concern since MAP is at the center of a controversy as to its role in Crohn's disease.  None of the methods presently available to define paratuberculosis in cattle have been examined for their ability to assess progression/regression of any treatment or intervention of this disease   The research presented herein, therefore was designed to assess the reliability and accuracy of available ante-mortem assays to predict disease change of individual animals undergoing a probiotic, potentially therapeutic, treatment.  Paratuberculosis positive (n = 75) and negative (n = 10) animals were longitudinally monitored over their natural lifetimes with specific serum antibody and fecal shedding assays, and for development of end-stage clinical disease.  Longitudinal, increasing/decreasing serum ELISA values were associated with, and predictive of, progression/regression of disease.  Changes in fecal shedding and serum AGID were of value at only specific stages.  Documentation that ELISA-positive animals were positive for paratuberculosis was done by a compilation of ELISA-independent assays--succumbing with end-stage clinical disease, autopsy, AGID, and MAP fecal shedding.

The etiology of Crohn's disease (CD) is unresolved, but it is likely that an interplay of host genetic factors and environmental triggers is relevant. Mycobacterium paratuberculosis (MAP) has been focused upon as one of these triggers because it causes a similar chronic inflammatory bowel disease in animals. However, the differences among MAP antigens isolated from humans (H-MAP) and cattle (B-MAP) have not been well characterized. In this study, culture filtrate (CF) proteins from MAP isolates were tested with sera from CD patients and healthy controls in enzyme-linked immunosorbent assay (ELISA). Antibody produced by seven CD patients reacted differently according to the antigen source: strong reactivity was seen to H-MAP CF, but not to B-MAP CF. Six proteins, ModD, PepA, transaldolase, EchA9, MAP2120c, and MAP2950c, in H-MAP CF reacting specifically with CD patient sera were identified by liquid chromatography-electrospray ionization-MS. Bioinformatic analysis revealed that ModD and PepA were the same proteins reacting with sera from cattle infected with MAP. The elevated antibody responses of CD patients to rModD and rPepA were confirmed by ELISA (P<0.001). These results support previous studies showing ModD and PepA as key antigens for the diagnosis of MAP infections. The study also identified additional proteins potentially useful in the design of assays for human MAP infections.

Disseminated infection (DI) of Mycobacterium avium subspecies paratuberculosis (MAP) in cattle may impair cow health, potentiate spread of disease, and is a potential food-safety risk. The objectives of this study were to determine the association between severity of histologic enteric lesions and the occurrence of DI, clinical signs, and positive fecal culture and serum ELISA results. Bacteriologic fecal culture and serum ELISA were performed on 40 dairy cows from MAP-infected herds. Cows were classified as having DI if MAP was isolated from any of 11 extra-intestinal tissues collected postmortem. A grade of 0-3, corresponding to the severity of histologically evident granulomatous inflammation was determined for sections of ileum, jejunum, mesenteric lymph node, and ileocolic lymph node. An overall intestinal inflammation (OII) grade of 0-3 was assigned to each cow. The proportion of cows with DI increased with tissue-specific lesion grade and OII grade. All cows with grade 3 inflammation in any single tissue had DI, however, some cows with DI had grade 1 inflammation or no lesions. In general, there was a positive association between OII grade and clinical signs, gross enteric lesions, and positive ELISA and fecal culture results. However, 12% of OII grade 0 cows had clinical signs (explained by other conditions recognized with necropsy), and the proportion of positive ELISA results was lower for OII grade 3 cows relative to grade 2 cows. Although MAP dissemination may occur early in the disease process, histopathology of intestinal tissues may be used to detect a substantial proportion of DI cows.

Surveillance and monitoring are important for measuring the level of disease in a population, documenting changes in prevalence over time, determining high-risk areas for implementation of control measures, eradicating disease, and documenting freedom from disease. The documentation of freedom from disease has importance for international trade and the resumption of production after an outbreak. Johne's disease (JD) is an example of an endemic disease of cattle that has variable prevalence related to environmental and animal-level factors. Three methods of sample collection were used to describe the prevalence and distribution of JD seropositivity in Texas. Sampled cattle were: (1) extensively managed herds, (2) market cattle, and (3) clinically ill cattle examined by practicing veterinarians throughout Texas. Samples were evaluated for JD using a commercially available serum ELISA. Proportion of seropositive samples was compared and spatial distributions were evaluated for clustering. Difference of JD seropositivity was observed among the three sample populations suggesting that estimation of disease prevalence is dependant upon the source of samples.

The objective of this study was to estimate heritability of antibody response to Mycobacterium avium ssp. paratuberculosis (MAP) in 4,524 German Holstein cows by applying linear and threshold models. Data were collected within a paratuberculosis voluntary control program in Thuringia, Germany, in 2005. The MAP-positive prevalence of the 12 farms in the data set varied between 5 and 36.9%. A nearly linear increase in prevalence was observed from 2- to 3-yr-old cows, whereas prevalence declined in cows older than 5 yr. This could be explained by greater culling rates associated with increasing age. Classification as MAP positive, questionable, and negative was available for all cows, and the optical density values of the Svanovir ELISA test existed for 2,084 of the animals originating from 6 farms. The heritability estimates of linear and threshold animal and sire models were compared. For the available data sets with an average of 8 progeny per sire, animal models were more robust and yielded more reliable results than did sire models. Heritability estimates from sire models led to overestimation of genetic variances because of a low number of progeny per sire and average relationship within sire progeny of greater than one-fourth, as expected between half-sibs. For all animal models, a heritability of about 0.1 was estimated for antibody response to MAP. Furthermore, it can be concluded that for the estimation of breeding values for antibody response to MAP optical density values of the ELISA test as a normally distributed trait (log-transformed) should be used rather than MAP status (positive or negative) as a binary trait because of the greater heritability and more robust parameter estimates when sire or animal models are used.

We previously reported that protein antigens of serodiagnostic potential were more abundant in culture filtrates than cellular extracts from liquid cultures of Mycobacterium avium subsp. paratuberculosis (D. Cho and M. T. Collins, Clin. Vaccine Immunol. 13:1155-1161, 2006). Based on this observation, a novel enzyme-linked immunosorbent assay (ELISA) using antigens secreted by young (early- to mid-log-phase) cultures of M. avium subsp. paratuberculosis JTC303 (a low-passage isolate originating from the ileum of a Holstein bull) in mycobactin-supplemented Watson-Reid medium (pH 6.0) was developed and evaluated using a previously described panel of bovine sera (M. T. Collins et al., Clin. Diagn. Lab. Immunol. 12:685-692, 2005) that included 444 paratuberculosis cases and 412 controls. The new assay, called JTC-ELISA, had a significantly higher diagnostic sensitivity and an equivalent specificity compared to those of five commercial paratuberculosis ELISA kits. By receiver-operating characteristic analysis, the JTC-ELISA had the highest area under the curve of the six assays evaluated. The JTC-ELISA was particularly sensitive at detecting low-level fecal shedders of Mavium subsp. paratuberculosis (40%; the sensitivity of the commercial kits was 20%). The JTC-ELISA works effectively on both serum and milk samples for the detection of cattle with subclinical M. avium subsp. paratuberculosis infections, providing a cost-effective diagnostic tool to support paratuberculosis control programs in cattle herds.

Five commercially available ELISA tests for the detection of antibodies against Mycobacterium avium subsp. paratuberculosis in bovine serum were evaluated at the individual animal level using sera from 286 paratuberculosis-free and 110 paratuberculosis-infected dairy cattle. Sensitivity (Se) and specificity (Sp) of the tests were estimated after determination of the cut-off dtheta by TG-ROC analysis or using the cut-off values recommended by the manufacturers, respectively. When the dtheta cut-off values were applied, the five ELISA tests showed sub-optimal Se and Sp. Adopting the cut-offs recommended by the manufacturers, the Sp of four of the five ELISA increased, two tests reaching Sp > or = 99.0%. Test sensitivity clearly depended on the disease state of the animals examined. Se was significantly higher in clinically diseased than in latently infected dairy cattle. Calculation of the positive and negative predictive values indicated that, depending on the test, a considerable proportion of false positive and false negative results have to be expected. Therefore, the suitability of antibody detection for the diagnosis of paratuberculosis in individual animals is questioned.

Mycobacterium avium subspecies paratuberculosis (MAP) disseminated infection in dairy cattle affects animal health and productivity and is also a potential public health concern. The study objectives were to characterize MAP disseminated infection in dairy cattle and to determine the role of antemortem tests in detecting cattle with disseminated infection. Forty culled dairy cows representing a variety of serum enzyme-linked immunosorbent assay (ELISA) results and body conditions were selected for the study. The physical condition of the cows was assessed via clinical examination prior to euthanasia and blood and feces were collected and tested by serum ELISA and fecal culture, respectively. Fifteen tissues were aseptically collected from each cow during necropsy and cultured for isolation of MAP. Disseminated infection was diagnosed when MAP was isolated in tissues other than the intestines or their associated lymph nodes (LNs) and was distinguished from infection found only in the gastrointestinal tissues and from absence of infection. Of the 40 cows in the study, 21 had MAP disseminated infection. Results showed that 57% (12/21) of cows with disseminated infection had average to heavy body condition and no diarrhea. Cows with disseminated infection had no to minimal gross pathologic evidence of infection in 37% (8/21) of cases. Only 76% (16/21) of cows with disseminated infection had positive historical ELISA results and only 62% (13/21) had a positive ELISA at slaughter. Thus, antemortem evidence of MAP infection was lacking in a high proportion of cows where MAP disseminated infection was confirmed.

Methods to improve the ELISA test to detect Mycobacterium avium subsp. paratuberculosis have been explored over several years. Previously, selected recombinant proteins of M. avium subspecies paratuberculosis were found to be immunogenic in cattle with Johne's disease. In the present study, antibody responses of infected and healthy sheep were evaluated using 18 purified recombinant proteins in an ELISA-based format for the serodiagnosis of ovine paratuberculosis. These selected recombinant proteins represent heat shock proteins, hypothetical proteins and cell surface proteins of M. avium subsp. paratuberculosis. Whereas, Map0862 (a gene uniquely present in M. avium subspecies paratuberculosis) and Map3786 encoded protein solicited the strongest antibody response in infected sheep. The protein encoded by Map2116c showed the weakest antibody response among the animals tested. Although none of the recombinant proteins detected all 11 infected sheep singly, antibodies to Map0862 were detected in 9 of 11 (81%) infected sheep. Furthermore, ovine responses to these selected antigens were assessed temporally over the course of 1 year during which we found a spiking effect rather than an incremental increase of antibody reactivity. This study evaluated multiple M. avium subsp. paratuberculosis recombinant proteins in an ELISA-based format for sheep.

Background: Many non-tuberculous mycobacteria synthesize abundant glycopeptidolipids (GPLs). These surface-located GPLs are involved in pathogenicity by interfering with the host immune system. In Mycobacterium avium subsp. avium (Mav), GPLs consist of a lipopeptide core composed of a tetrapeptide O-linked to mono- and oligo-saccharides. The biosynthesis pathway of the simplest GPLs is now relatively well understood and involves probably more than fifteen genes. Whereas it is very obvious that most, if not all, of the Mav isolates produce GPLs, the picture is not as clear for M. avium subsp. paratuberculosis (Map), the etiologic agent of Johne's disease in cattle, and several conflicting data have been produced., Methods: Biochemical analysis of a large set of characterized Map isolates showed that all Map strains tested produce a lipopentapeptide (L5P) instead of GPLs. To provide a genomic basis for the synthesis of this compound, the recently published genome sequence of Map was explored using in silico methods. Even though Map produces a lipopeptide rather than GPL, its genome contains nevertheless a locus highly similar to the GPL biosynthetic pathway of Mav. We showed that the module composition of the non-ribosomal protein synthase (Nrp) of Map, the enzyme involved in the synthesis of the peptidyl moiety, is dramatically different from that of other GPL producers such as M. smegmatis (Ms) and Mav and is in agreement with the amino acid content of the L5P. We also showed that the peptidyl moiety of the L5P is a target for a strong specific humoral response in Map infected animals., Conclusions: These genomic and biochemical differences may help to unambiguously distinguish Map from Mav and also from M. bovis, to reclassify related strains of the Map species and to allow the convenient and specific diagnosis of paratuberculosis.

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.

Prior to establishing a control and prevention program for Johne's disease in cattle in Galicia (northwest Spain), a survey was conducted to estimate the prevalence of the disease. For this survey, 61,069 animals of at least 1-year of age from 2735 randomly selected herds were bled and samples analyzed with a commercial ELISA. The estimated true individual-level prevalences--assuming the manufacturer's reported test sensitivity of 48.5% and specificity of 98.9%--were 3.02% in dairy cattle, 1.03% in beef cattle and 2.83% in animals from farms with both dairy and beef cattle. True herd prevalences (with herds declared positive if one or more animals tested positive) were 10.69% for dairy herds, 0% for beef herds and 2.71% for mixed herds. When herds were declared positive if at least two animals tested positive, true herd prevalences were 14.75% for dairy herds, 1.47% for beef herds and 12.01% for mixed herds. Assuming a higher specificity of 99.4%, true individual-level prevalences increased to 4.03% in dairy herds, 2.07% in beef herds and 3.84% in mixed herds. Herd prevalences were 27.77%/18.79%, 2.78%/2.40% and 5.70%/12.24% (using the one/two-animal cut-offs) in dairy, beef and mixed herds, respectively. In conclusion, these results seem to indicate that a small percentage of cows and a rather high percentage of dairy herds in this region are MAP-seropositive.

Paratuberculosis (Johne's disease), which is widely distributed throughout the world, is caused by Mycobacterium avium subspecies paratuberculosis (MAP). Diagnosis of subclinically infected cattle is challenging and is especially problematic in herds with low prevalence of MAP. The aim of this long-term study was the comparison of different diagnostic tests for MAP and specific antibodies in a herd with low prevalence of MAP. Three different commercially available serum-ELISA (Svanovir-ELISA, Svanova, Uppsala, Sweden; IDEXX-ELISA, IDEXX Laboratories, Maine, USA; Pourquier-ELISA, Institut Pourquier, Montpellier, France) and two milk ELISA (Svanovirm-ELISA Svanova, Uppsala, Sweden; Pourquier-ELISA, Institut Pourquier, Montpellier, France) were compared. Apart from these indirect diagnostic tests, two methods for the detection of the etiologic agent (bacteriologic culture and real-time PCR of faecal samples) were performed. In January 2005 the first and in April 2005 the second herd investigation of all animals older than 2 years (n=335) were carried out. Blood, milk and faecal samples were taken. From November 2005 until April 2006 follow up investigations were performed. For this purpose, blood-, milk- and faecal samples were monthly taken from 63 selected animals. The highest number of blood- and milk samples with a detectable antibody-level was found by the Svanovir-ELISA. There was a significant correlation between serum- and milk- Svanovir-ELISA results, whereas the agreement between ELISA and faecal culture/PCR was low. Significant correlations between Svanovir-serum-ELISA results and milk somatic cell counts could be registered. Moreover, there was significant agreement between IDEXX-serum-ELISA results with the age and number of lactations of the cows, as well as the mother's MAP-status.

In Chile, Mycobacterium avium subsp. paratuberculosis (Map) has been isolated on several occasions and clinical cases have been reported. Nevertheless, diagnostic tests have not yet been validated for this agent in the Chilean setting. The objective of the study was to validate a commercial ELISA to detect Map shedding dairy cows in management conditions, prevalence and stages of infection existing in Southern Chile, utilising different statistical approaches. Blood and faeces were collected from 1333 lactating cows in 27 dairy herds (both large commercial and smallholder dairy farms) between September 2003 and August 2004. Within the herds up to a maximum of 100 dairy cows were selected based on age (>or=3 years old) and, if present, clinical signs of a Map infection. In herds with less than 100 cows, all cows >or=3 years old were sampled. Blood samples were tested using a commercial ELISA kit (IDEXX Laboratories, Inc.). Faecal samples were cultured on Herrold's Egg Yolk Medium (HEYM). Latent class models (i.e. maximum likelihood (ML) methods and Bayesian inference) were used to determine the validity of the ELISA. Map was cultured from 54 (4.1%) cows and 10 (37.0%) herds, which were all large, commercial dairy herds. As a result of empty cells in the cross-tabulations, the ML model provided the same results as the validation with faecal culture as the gold-standard. In the Bayesian model, the Se and Sp of the ELISA were estimated to be 26% (95% CI: 18-35%) and 98.5% (95% CI: 97.4-99.4%), respectively. For faecal culture, the Se was 54% (95% CI: 46-62%) and the Sp was 100% (95% CI: 99.9-100%). Interestingly, the prevalence in the smallholder dairy farms was estimated to be 8% even though there were no faecal culture positive cows detected in those herds. There was no significant correlation between the two tests. The advantage of Bayesian inference is that the Se and Sp of both tests are obtained in one model relative to the (latent) true disease status, the model can handle small datasets and empty cells and the estimates can be corrected for the correlation between tests when the tests are not conditionally independent. Therefore, Bayesian analysis was the preferred method for Map that lacks a gold-standard and usually has low cow-level prevalence.

Objective: To evaluate the seroprevalence of paratuberculosis by use of 2 commercial ELISAs in association with prevalence of fecal shedding of mycobacteria within beef cattle herds., Design: Cross-sectional field study., Animals: Six beef herds (affected herds; 522 cattle) with and 3 geographically matched herds (181 cattle) without high seroprevalence of paratuberculosis., Procedures: Blood and fecal samples were collected from adult cattle and assessed for serum anti-Mycobacterium avium subsp paratuberculosis (MAP) antibodies with 2 commercial ELISA kits and submitted for bacterial culture for MAP and environmental bacteria (termed environmental mycobacteria) via a radiometric method, respectively. Species of mycobacterial isolates were identified, and sensitivities and specificities of the 2 ELISAs were compared., Results: Compared with comparison cattle, cattle from affected herds were 9.4 times as likely to have environmental mycobacteria isolated from feces. Among the 6 affected and 3 comparison herds, the proportions of cattle shedding environmental mycobacteria were 0.225 (range, 0.1 to 0.72) and 0.04 (range, 0 to 0.06), respectively. Although relative MAP- detection specificities (compared with bacterial culture of feces) were different between the 2 ELISAs, sensitivities were not. Nine environmental mycobacterial species were identified from participating herds. All affected herds apparently had > or = 1 bovid infected with MAP, although MAP was not isolated from any cattle in comparison herds., Conclusions and Clinical Relevance: In beef herds with persistently high rates of false- positive ELISA results, which may be associated with recovery of environmental myco- bacteria from feces, organism detection via bacterial culture of feces or PCR assay should direct paratuberculosis control measures.

Objective: To evaluate the effect of exposure to environmental mycobacteria on results of 2 commercial ELISAs for paratuberculosis in cattle., Design: Experimental trial., Animals: 19 weaned crossbred beef calves., Procedures: Calves were inoculated SC with 1 of 5 mycobacterial isolates (3 calves/isolate) derived from herds with high proportions of false-positive serologic reactions for paratuberculosis, Mycobacterium avium subsp paratuberculosis (MAP; positive control inoculum; 2 calves), or mineral oil (negative control inoculum; 2 calves). Sera were assessed at intervals by use of 2 ELISAs (A and B) for paratuberculosis in cattle, and all calves underwent tuberculosis testing at the end of the study., Results: Neither mineral oil-inoculated calf had positive results with either ELISA during the study. Both MAP-inoculated calves were identified as seropositive via ELISA-A, and 1 calf was identified as seropositive via ELISA-B. By use of ELISA-A, > or = 1 false-positive reaction over time was detected in 2, 3, 3, and 1 of the 3 calves injected with Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, or Mycobacterium terrae, respectively. By use of ELISA-B, only M scrofulaceum induced false-positive reactions (2/3 calves). Calves that had at least 1 positive ELISA-A result were more likely to be classified as suspect reactors via the caudal fold tuberculosis test., Conclusions and Clinical Relevance: False-positive serologic reactions may occur during use of commercially available ELISAs for paratuberculosis in calves experimentally exposed to environmental mycobacteria; naturally occurring exposures with these mycobacteria may represent a cause for high proportions of false-positive serologic reactions for paratuberculosis in some cattle herds.

Low sensitivity (Se) of diagnostic tools is often mentioned as a major problem in the control of paratuberculosis (PTB) and much effort is put into the improvement of these tests. The hypothetical perspectives of improving the Se of a milk-antibody ELISA (hereafter: milk-ELISA) used in test-&-cull strategies against PTB in dairy cattle were investigated by simulations. The current Se varies between 10 and 80%, increasing with increasing lactation stage, parity and infection stage. We simulated the effects on a dairy herd's production of improving this Se to 80% (independent of these factors) and assumed no concomitant decrease in specificity. By using a PTB model called PTB-Simherd, 12 scenarios were simulated to study three test-&-cull strategies in each of four herds with 200 dairy cows. To show the maximal effect of using test-&-cull with such an improved test we simulated three strategies: (1) no testing, (2) testing with milk-ELISA test with the current Se and culling of positive cows immediately and (3) testing with milk-ELISA test with a Se improved to 80% and culling positive cows immediately. The four herds were defined by a moderate (25%) or high (80%) initial true within-herd prevalence (including young stock), and a poor or good heat-detection success of 40 or 60%. We assumed that these factors influenced the effects of improving the Se of the milk-ELISA. Management both concerning calf management and in general was specified to represent a typical Danish herd. Using an improved milk-ELISA was predicted to reduce the prevalence of PTB more effectively than the current ELISA, and over 10 years bring the production of a herd with moderate initial prevalence up to a production level comparable to a non-infected herd (unlike if the current ELISA had been used). In a herd with high initial prevalence (80%) milk production was increased more by using the improved milk-ELISA, but after 10 years the replacement rate was still very high causing problems with having enough recruitment animals-especially in high-prevalence herds with poor reproductive performance. Economically important measurements in all four herds benefited from the improvement of the test over a 10-year period. However, in the first 3-5 years the improved test would be more expensive to use than the current test, due to increased replacement (reduced net annual revenue per cow euro15 on average) but after that, net annual revenue increased continuously; after 10 years it was euro70-90 higher, than if the current milk-ELISA was used. Also, the milk-ELISA test with its current Se seemed to be profitable already after 2 years in high-prevalence herds using a test-&-cull strategy based on the milk-ELISA alone.

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.

Paratuberculosis (Johne's disease) is a chronic infectious disease of ruminants, caused by Mycobacterium avium subspecies paratuberculosis (MAP). Because of its long incubation period, high economic losses, difficulties in diagnosis and possible links to Morbus Crohn in humans, paratuberculosis is one of the most important diseases of ruminants today. An abattoir-based nationwide survey on the occurrence of MAP in the Austrian cattle population was performed using serology (SVANOVIR-ELISA) as well as culture, ZN-stain and IS900-PCR on faeces and lymph node samples. A total of 756 Austrian slaughter cattle were serologically, bacteriologically and molecularbiologically tested for the occurrence of MAP and specific antibodies respectively. Samples were collected following a statistical plan to obtain balanced specimens from the whole country. Nineteen per cent of the animals tested were serological positive, 10.1% gave an inconclusive result and 70.9% showed no specific antibodies against MAP. Only in four individuals MAP could be detected by stain, bacteriology or Polymerase Chain Reaction. The calculated prevalence of 19.0% positive cattle, each representing one farm, showing specific antibodies against MAP is rather high in terms of animal-level but low in herd level prevalence compared with other countries. When this study is compared with a similar study performed in Austria 1999, a significant increase of positive cattle and farms could be seen in Austria.

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.

Our objective was to define the role of monensin sodium in protecting cows from being milk-ELISA positive for paratuberculosis in Ontario, Canada dairy herds. In total, 4933 dairy cows from 94 herds were enrolled in this cross-sectional study. Forty-four of the enrolled herds were selected purposively by their herd veterinarian and another 50 herds were randomly selected from a local milk production-recording agency. A herd-management survey was completed on each farm during the months of May through August 2003. During this same time-period, composite milk samples were collected from all lactating cows and tested with a milk-ELISA for antibodies to Mycobacterium avium subspecies paratuberculosis. Analyses were stratified according to the paratuberculosis history of the herds. In the 48 herds in which paratuberculosis had not been diagnosed before, the use of calf hutches and monensin in milking cows were both associated with reduced odds of a cow testing positive (OR=0.19 and 0.21, respectively). In the 46 herds with a prior history of paratuberculosis, feeding monensin to the breeding-age heifers was associated with decreased odds of a cow testing positive (OR=0.54). Monensin use might be associated with milk-ELISA positivity, but its impact on the transmission of paratuberculosis remains unknown.

The present study was designed to evaluate a commercial ELISA kit (Institut Pourquier) for the diagnosis of ovine and caprine paratuberculosis under Australian conditions and to compare its accuracy with the existing AGID test. The sensitivity of the ELISA in sheep and goats was 34.9% and 56.4%, with a specificity of 98.8% and 100.0%, respectively. Sensitivity of AGID was 13.8% for sheep and 39.5% for goats, with specificity of 100.0% for both species. The sensitivity of the ELISA in sheep depended on the category of histological lesions. AGID and ELISA were conditionally independent, and appeared to detect overlapping but distinct subgroups of infected animals. The ELISA was significantly more sensitive than the AGID. The ELISA was simple to perform, robust and repeatable. Coefficients of variation of <12.0% were observed for positive and negative controls included on 193 plates over a 10-month period and there was a high level of intraassay repeatability with 12.0% of the duplicate samples having CV of >15.0%.

Our objective was to evaluate the effects of time and temperature on whole blood used in the gamma interferon enzyme-linked immunosorbent assay (IFN-gamma ELISA) for paratuberculosis along with evaluating four potential positive controls, and four different mycobacterial antigens for the ELISA. Nine adult Holstein cattle naturally infected with Mycobacterium avium ssp. paratuberculosis were used in a randomized complete block design. Forty-nine blood tubes were collected from each animal and held at 48.9, 37.8, 26.7, 21.1, 15.6 and 4.4 degrees C for 0, 4, 8, 12, 18, 24, 32, 48 and 72 h. Each blood tube was tested with four mycobacterial antigens (two johnin PPDs, an avain PPD and a whole cell sonicate) and four potential positive controls [concanavalin A (conA), phytohaemagglutinin A (PHA), pokeweed mitogen (PWM) and Staphylococcus enterotoxin A (SEA)]. After incubation for 24 h, the plasma was assayed with a commercial IFN-gamma ELISA. Blood stored at 21.1 and 15.6 degrees C maintained the highest ELISA optical densities (OD) over time with severe reduction in OD values at or above 37.8 degrees C. None of the potential positive controls exactly mimicked the antigen response. SEA and PWM were able to elicit a response after the whole blood quit responding to the antigen and conA underestimated the responsiveness. Phytohemagglutinin A was similar to the antigens on an average, but there was significant disagreement among samples. The PPDs were more potent at stimulating IFN-gamma production than the whole cell sonicate. In conclusion, whole blood should be stored/transported at ambient room temperature and stimulated within 12 h of collection.

During a 10-month period in 1999, 994 serum and tissue samples were collected from dairy cows at slaughter in eastern Canada. The sources of these cattle were from all four Atlantic Canadian provinces along with some cows from the state of Maine. The sera were used to assess the agreement of three commercially available ELISAs for Mycobacterium avium subsp. paratuberculosis. Two ELISAs were indirect absorbed ELISAs licensed for use in North America, the third was an indirect non-absorbed ELISA licensed for use in Europe. Overall, there was poor agreement between the three ELISAs. The highest and lowest kappa values were 0.33 and 0.18, which is fair and poor agreement, respectively. However, when only tissue culture-positive cattle were compared, the ELISAs had better agreement (kappa=0.37-0.51). The proportions of positive tests, however, were significantly different among the three ELISAs. The poor agreement among the three ELISAs is as concerning as the fact that these tests have low sensitivity. The implications are greatest when the tests are used at the cow level to make individual animal decisions, which is not the recommended method on the product labels. At the cow level, if the result obtained from one ELISA is positive, using a different ELISA in a pre-clinical animal has a high likelihood of giving a different result due to low predictive values of positive test results.

Cows from 2 California dairies were tested for paratuberculosis at the end of lactation by using fecal culture and a commercially available serum enzyme-linked immunosorbent assay (ELISA) test kit. Individual cow characteristics and production variables were evaluated along with ELISA testing results as predictors of fecal culture status. In multivariable logistic regression analysis, age and a herd-standardized version of 305-day mature equivalent (305 ME) milk production were significant predictors of fecal culture status after adjusting for herd, quarter of the study year, and ELISA sample-to-positive (S/P) ratio. The area under a nonparametric receiver operating characteristic curve was significantly greater for a multivariable model that included age and the level of milk production when compared with a model without these covariates. In conclusion, consideration of cow-level covariates was useful as an aid in predicting Mycobacterium avium ssp. paratuberculosis (MAP) fecal culture status. For a given ELISA S/P ratio, older cows and those with lower 305 ME milk production relative to other cows in the herd were significantly more likely to be shedding MAP in their feces at the end of lactation.

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7x) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD.

Two thousand nine hundred fifty-two serum samples, collected once or twice annually from 545 cows of known fecal culture status were tested for antibodies to Mycobacterium avium subsp. paratuberculosis using a commercially available enzyme-linked immunosorbent assay (ELISA) test. Overall, 13.5% of the samples from 282 infected cows had positive ELISA results, but when tested multiple times, 38.3% of the cows had at least 1 serum sample with positive results. Among 263 fecal culture-negative cows, 98.1% of the serum samples had negative ELISA results, but when tested multiple times, 7.8% of the cows had at least 1 positive ELISA sample. Fecal culture was positive on a test before the first positive ELISA in 50 cows, ELISA was positive before fecal culture in 12 cows, and in 38 cows, both tests became positive at the same testing time. An additional 174 cows were positive on fecal culture and always negative on ELISA until culled. For cows that had ELISA sample:positive (S/P) ratios below the cutoff point, the change in S/P between sequential tests was evaluated to determine whether a rise in S/P could predict infection status. In this study, change in S/P was not a useful predictor of infection status in seronegative cows.

Three serum ELISAs for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Mptb) were evaluated against culture of tissue and feces samples from 994 dairy cows collected at slaughter. Culture of ileum and associated lymph nodes for Mptb were positive for 160 (16.1%) of the 994 cows and 36 (3.6%) were fecal culture-positive for Mptb. Two of the ELISAs evaluated were absorbed indirect assays and the third was a non-absorbed indirect assay. Estimated sensitivities of the absorbed ELISAs when compared to tissue culture were 8.8% and 6.9%, while the unabsorbed ELISA had a sensitivity of 16.9%. Specificities were 97.6%, 96.0% and 90.8%, respectively. When compared to fecal culture, the sensitivities of the absorbed ELISAs were 16.6% and 13.9%, respectively, and the sensitivity of the unabsorbed ELISA was 27.8%. Specificities were 97.1%, 95.9% and 90.1%, respectively. Area under the curve (AUC) of receiver operator characteristic curves for the absorbed ELISAs when tissue culture was the standard were 0.553 and 0.547, while the unabsorbed ELISA had an AUC of 0.540. When fecal culture was the comparison standard, the AUC of the absorbed ELISAs was 0.575 and 0.574, while the unabsorbed ELISA was 0.529. Overall, the sensitivities of the ELISAs when compared to tissue culture were low. The apparent advantage of the unabsorbed ELISA with respect to sensitivity is at the cost of lowered specificity and test accuracy.

Current assays used to detect Mycobacterium bovis infection lack accuracy, especially for recently infected animals, or are impractical for rapid field diagnostic applications. To overcome these limitations with serological assays, a synthetic peptide derived from early secretory antigenic target 6 (ESAT6-p) and a recombinant major secreted immunogenic protein (rMPB70) of M. bovis were used in an enzyme-linked immunosorbent assay (EIA), an immunochromatographic assay (ICGA), and a latex bead agglutination assay (LBAA). Sera from noninfected, M. bovis-infected, or M. avium subsp. paratuberculosis-infected (by natural and experimental routes) animals were evaluated. Receiver operating characteristic analysis comparing optical density values from the EIA with results of bacterial culture or skin test, the reference test, established suitable cutoff values for assessing sensitivity and specificity. The EIA and LBAA, respectively, had sensitivities of 98.6 and 94.8%, specificities of 98.5 and 92.6%, and kappa values of 0.97 and 0.88 with ESAT6-p. The EIA, ICGA, and LBAA, respectively, had sensitivities of 96.8, 83.0, and 86.7%, specificities of 90.1, 99.4, and 97.8%, and kappa values of 0.87, 0.85, and 0.83 with rMPB70. Examination of serial samples of sera collected from experimentally M. bovis-infected cattle and deer revealed that ESAT6-p-specific responses developed early after infection whereas responses to rMPB70 developed later in the course of disease. The advantage of the LBAA and ICGA as initial tests for multiple species is a rapid reaction obtained in 2 to 3 h by LBAA or 20 min by ICGA without species-specific secondary antibodies under field conditions, thus allowing immediate segregation of suspect animals for further testing before culling.

Antibody detection-based tests for paratuberculosis offer speed and economy, 2 diagnostic test attributes important to animal industries with narrow profit margins. Application of such tests to individual milk samples instead of serum samples can further improve testing efficiency and decrease testing cost. Accuracy of a commercial bovine paratuberculosis enzyme-linked immunosorbent assay (ELISA) adapted for use on goat serum and milk samples was determined. Fecal, blood, and milk samples were collected from 159 goats belonging to 2 Wisconsin goat herds with a prior history of paratuberculosis and 1 herd of 50 goats from a paratuberculosis-free Wisconsin herd. Fecal samples were cultured using the modified BACTEC 12B media. Sera were tested according to the manufacturer's instructions for bovine samples. Milk samples were centrifuged and mixed with the ELISA kit's Mycobacterium phlei-containing diluent at a ratio of 1:2. Using fecal culture as the "gold standard," the sensitivity of the ELISA on goat serum was 64% and the sensitivity of the ELISA on goat milk was 48%. The milk ELISA had higher agreement with fecal culture results (kappa = 0.525) than the serum ELISA (kappa = 0.425). ELISA specificity was 100% on both serum and milk. Regression analysis also showed good correlation between serum and milk S/P values (r2 = 0.67). Although less sensitive, the ELISA on goat milk samples appears to offer a useful, low-cost alternative for detection of goats with paratuberculosis that have progressed to the stage of shedding M. paratuberculosis in their feces.

Five diagnostic tests based on enzyme-linked immunosorbent assay (ELISA) technology for bovine paratuberculosis were evaluated by using individual serum or milk samples from 359 dairy cattle in seven paratuberculosis-free herds and 2,094 dairy cattle in seven Mycobacterium paratuberculosis-infected dairy herds. Three independent laboratories using three different culture procedures completed fecal cultures for M. paratuberculosis on these cattle and found 417 cows to be shedding M. paratuberculosis in their feces. An animal that was fecal culture positive for M. paratuberculosis by any of the three laboratories was considered a confirmed case of infection. The specificity of three ELISAs (two on serum and one on milk) was > or =99.8%. The specificity of the remaining two ELISAs, both done on serum, was 94.9 and 84.7%. Four of the five ELISAs evaluated produced similar sensitivity in detecting fecal culture-positive cattle (27.8 to 28.9%). Serum ELISA "D" had the lowest specificity (84.7%) and the highest sensitivity (44.5%), but if the cutoff value defining a positive test was changed from 125 to 250% (of the positive control) the sensitivity and specificity, 31.8 and 97.5%, respectively, were comparable to those of the other four assays. If the case definition for M. paratuberculosis infection was based on the culture results of a single laboratory instead of the combined results of three laboratories, ELISA sensitivity estimates were 45.7 to 50.0%. With the exception of ELISA D, assay agreement was high (kappa 0.66 to 0.85) for categorical assay interpretations (positive or negative), but linear regression of quantitative results showed low correlation coefficients (r(2) = 0.40 to 0.68) due to the fact that ELISA results for some cows were high in one assay but low in another assay. Likelihood ratio analysis showed a direct relationship between the magnitude of ELISA result and the odds of a cow shedding M. paratuberculosis in its feces. If used judiciously and interpreted quantitatively, these ELISAs are useful tools in support of paratuberculosis control programs in dairy herds.

Objective: To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis., Animals: 1043 cattle from 10 herds in Michigan., Procedure: Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium., Results: Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant., Conclusions and Clinical Relevance: No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.

Objective: To determine whether results obtained for milk and serum samples with ELISAs intended for diagnosis of paratuberculosis in dairy cows were comparable to results obtained by means of mycobacterial culture of fecal samples., Design: Cross-sectional study., Animals: 689 lactating dairy cows in 9 Ontario herds., Procedure: Milk, serum, and fecal samples were obtained from all cows. Fecal samples were submitted for mycobacterial culture. Serum samples were tested with a commercially available ELISA for antibodies against Mycobacterium paratuberculosis, and preserved milk samples were tested with an indirect ELISA for antibodies against M paratuberculosis., Results: Results were positive for 130 of the 689 (18.9%) serum samples, 77 of the 689 (11.1%) milk samples, and 72 of the 689 (10.4%) fecal samples. The level of agreement between results for milk and serum samples was only moderate. Proportions of positive results for serum and fecal samples were significantly different, but proportions of positive results for milk and fecal samples were not significantly different. In addition, results for milk samples had a higher level of agreement with results of mycobacterial culture than did results for serum samples., Conclusions and Clinical Relevance: Results suggest that the indirect ELISA used on milk samples may be a convenient method of detecting paratuberculosis in dairy herds.

Several serological tests for detection of antibodies to Mycobacterium avium ssp. paratuberculosis, which is the causative agent of Johne's disease are validated, some of which are available commercially. These tests differ in sensitivity and specificity. Test results reported to farmers or veterinarians are therefore dependent upon the test in use. In the present study, three commercially available tests are used to test 2748 bovine sera from 119 Bavarian herds serologically. Serological results are compared with individual animals and on herd level. A scheme for serological testing and classification of herds as well as of individual animals based on a combination of serological test results is proposed.