Elucidating Transmission Patterns of Endemic Mycobacterium avium subsp. paratuberculosis Using Molecular Epidemiology.

Mycobacterial diseases are persistent and characterized by lengthy latent periods. Thus, epidemiological models require careful delineation of transmission routes. Understanding transmission routes will improve the quality and success of control programs. We aimed to study the infection dynamics of Mycobacterium avium subsp. paratuberculosis (MAP), the causal agent of ruminant Johne's disease, and to distinguish within-host mutation from individual transmission events in a longitudinally MAP-defined dairy herd in upstate New York. To this end, semi-annual fecal samples were obtained from a single dairy herd over the course of seven years, in addition to tissue samples from a selection of culled animals. All samples were cultured for MAP, and multi-locus short-sequence repeat (MLSSR) typing was used to determine MAP SSR types. We concluded from these precise MAP infection data that, when the tissue burden remains low, the majority of MAP infections are not detectable by routine fecal culture but will be identified when tissue culture is performed after slaughter. Additionally, we determined that in this herd vertical infection played only a minor role in MAP transmission. By means of extensive and precise longitudinal data from a single dairy herd, we have come to new insights regarding MAP co-infections and within-host evolution.

Impact of the shedding level on transmission of persistent infections in Mycobacterium avium subspecies paratuberculosis (MAP).

Super-shedders are infectious individuals that contribute a disproportionate amount of infectious pathogen load to the environment. A super-shedder host may produce up to 10,000 times more pathogens than other infectious hosts. Super-shedders have been reported for multiple human and animal diseases. If their contribution to infection dynamics was linear to the pathogen load, they would dominate infection dynamics. We here focus on quantifying the effect of super-shedders on the spread of infection in natural environments to test if such an effect actually occurs in Mycobacterium avium subspecies paratuberculosis (MAP). We study a case where the infection dynamics and the bacterial load shed by each host at every point in time are known. Using a maximum likelihood approach, we estimate the parameters of a model with multiple transmission routes, including direct contact, indirect contact and a background infection risk. We use longitudinal data from persistent infections (MAP), where infectious individuals have a wide distribution of infectious loads, ranging upward of three orders of magnitude. We show based on these parameters that the effect of super-shedders for MAP is limited and that the effect of the individual bacterial load is limited and the relationship between bacterial load and the infectiousness is highly concave. A 1000-fold increase in the bacterial contribution is equivalent to up to a 2-3 fold increase in infectiousness.

Longitudinal data collection of Mycobacterium avium subspecies Paratuberculosis infections in dairy herds: the value of precise field data.

Longitudinal infection data on Mycobacterium avium subspecies paratuberculosis (MAP) was collected on three dairy farms in Northeastern United States during approximately 10 years. Precise data on animal characteristics and animal location within farm were collected on these farms. Cows were followed over time with regard to MAP status during biannual fecal and serum sampling and quarterly serum sampling. Approximately 13 000 serum samples, 6500 fecal samples and 2000 tissue samples were collected during these years. Prevalence of positive samples was 1.4% for serological samples, 2.2% in fecal samples and 16.7% in tissue samples. Infection dynamics of MAP was studied and resulted in a number of potential changes in our understanding of MAP infection dynamics. First, a high prevalence of MAP infection was observed in these herds due to lifetime follow up of cows, including slaughter. Second, two distinctly different infection patterns were observed, so called non-progressors and progressors. Non-progressors were characterized by intermittent and low shedding of MAP bacteria and a virtual absence of a humoral immune response. Progressors were characterized by continuous and progressive shedding and a clearly detectable and progressive humoral immune response. Strain typing of MAP isolates on the three farms identified on two of three farms a dominant strain type, indicating that some strains are more successful in terms of transmission and infection progression. Continuous high quality longitudinal data collection turned out to be an essential tool in our understanding of pathobiology and epidemiology of MAP infections in dairy herds.

Differences in intermittent and continuous fecal shedding patterns between natural and experimental Mycobacterium avium subspecies paratuberculosis infections in cattle.

The objective of this paper is to study shedding patterns of cows infected with Mycobacterium avium subsp. paratuberculosis (MAP). While multiple single farm studies of MAP dynamics were reported, there is not large scale meta-analysis of both natural and experimental infections. Large difference in shedding patterns between experimentally and naturally infected cows were observed. Experimental infections are thus probably driven by different pathological mechanisms. For further evaluations of shedding patterns only natural infections were used. Within such infections, the transition to high shedding was studied as a proxy to the development of a clinical disease. The majority of studied cows never developed high shedding levels. Those that do, typically never reduced their shedding level to low or no shedding. Cows that eventually became high shedders showed a pattern of continuous shedding. In contrast, cows with an intermittent shedding pattern had a low probability to ever become high shedders. In addition, cows that start shedding at a younger age (less than three years of age) have a lower hazard of becoming high shedders compared to cows starting to shed at an older age. These data suggest the presence of three categories of immune control. Cows that are intermittent shedders have the infection process under control (no progressive infection). Cows that start shedding persistently at a young age partially control the infection, but eventually will be high shedders (slow progressive infection), while cows that start shedding persistently at an older age cannot effectively control the infection and become high shedders rapidly.

Back to the real world: connecting models with data.

Mathematical models for infectious disease are often used to improve our understanding of infection biology or to evaluate the potential efficacy of intervention programs. Here, we develop a mathematical model that aims to describe infection dynamics of Mycobacterium avium subspecies paratuberculosis (MAP). The model was developed using current knowledge of infection biology and also includes some components of MAP infection dynamics that are currently still hypothetical. The objective was to show methods for parameter estimation of state transition models and to connect simulation models with detailed real life data. Thereby making model predictions and results of simulations more reflective and predictive of real world situations. Longitudinal field data from a large observational study are used to estimate parameter values. It is shown that precise data, including molecular diagnostics on the obtained MAP strains, results in more precise and realistic parameter estimates. It is argued that modeling of infection disease dynamics is of great value to understand the patho-biology, epidemiology and control of infectious diseases. The quality of conclusions drawn from model studies depend on two key issues; first, the quality of biology that has gone in the process of developing the model structure; second the quality of the data that go into the estimation of the parameters and the quality and quantity of the data that go into model validation. The more real world data that are used in the model building process, the more likely that modeling studies will provide novel, innovative and valid results.

Persistence of Mycobacterium avium subsp. paratuberculosis in soil, crops, and ensiled feed following manure spreading on infected dairy farms.

The goal of this study was to determine the persistence of Mycobacterium avium subsp. paratuberculosis (MAP) in soil, crops, and ensiled feeds following manure spreading. This bacterium was often found in soil samples, but less frequently in harvested feeds and silage. Spreading of manure on fields used for crop harvest is preferred to spreading on grazing pastures.

Persistance deMycobacterium aviumssp.paratuberculosisdans le sol, les récoltes et l'ensilage après l'épandage de fumier dans des fermes laitières infectées. Le but de cette étude était de déterminer la persistance de Mycobacterium avium ssp. paratuberculosis (MAP) dans le sol, les récoltes et l'ensilage après l'épandage de fumier. Cette bactérie se trouvait souvent dans des échantillons de sol, mais moins fréquemment dans les récoltes d'aliments pour le bétail et l'ensilage. L'épandage de fumier dans les champs utilisés pour la récolte des cultures est préféré à l'épandage dans les pâturages.(Traduit par Isabelle Vallières).

Cost-effectiveness of diagnostic strategies to identify Mycobacterium avium subspecies paratuberculosis super-shedder cows in a large dairy herd using antibody enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, and bacterial culture.

Diagnostic strategies to detect Mycobacterium avium subsp. paratuberculosis (MAP) super-shedder cows in dairy herds have been minimally studied. The objective of the current study was to compare the cost-effectiveness of strategies for identification of MAP super-shedders on a California dairy herd of 3,577 cows housed in free-stall pens. Eleven strategies that included serum or milk enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR) or culture of environmental samples, pooled or individual cow fecal samples, or combinations thereof were compared. Nineteen super-shedders (0.5%) were identified by qPCR and confirmed by culture as cows shedding ≥ 10,000 colony forming units (CFU)/g feces (median of 30,000 CFU/g feces). A stratified random sample of the study herd based on qPCR results of fecal pools was the most sensitive (74%) strategy and had the highest cost ($5,398/super-shedder). The reference strategy with the lowest cost ($1,230/super-shedder) and sensitivity (47%) included qPCR testing of fecal samples from ELISA-positive lactating (milk) and nonlactating (serum) cows housed in pens with the highest MAP bioburden. The most cost-effective alternative to the reference was to perform qPCR testing of fecal samples from ELISA-positive cows (milk and serum for milking and dry cows, respectively) for a sensitivity of 68% and cost of $2,226/super-shedder. In conclusion, diagnostic strategies varied in their cost-effectiveness depending on the tests, specimen type, and labor costs. Initial qPCR testing of environmental samples from free-stall pens to target cows in pens with the highest MAP bioburden for further testing can improve the cost-effectiveness of strategies for super-shedder identification.

Quantitative real-time PCR for detection of the neurotoxin gene of Clostridium botulinum type B in equine and bovine samples.

Clostridium botulinum type B is estimated to cause more than 85% of cases of equine botulism in the United States, as well as many outbreaks in cattle. In this study, a quantitative real-time polymerase chain reaction for detection of the neurotoxin gene of C. botulinum type B was compared to the mouse bioassay using 45 positive and 43 negative samples of equine, bovine or associated environmental origin. The sensitivity of the qPCR assay was 96%, whereas the sensitivity of the mouse bioassay was 84%. The specificity of the qPCR assay was 95% and the specificity of the mouse bioassay was 100%.

Meta-analysis of two genome-wide association studies of bovine paratuberculosis.

BACKGROUND: Bovine paratuberculosis (ParaTB) also known as Johne's disease, is a contagious fatal disease resulting from infection by Mycobacterium avium subspecies paratuberculosis (MAP). Previous studies have identified loci associated with ParaTB using different measurements to define cases and controls. The objective of this study was to combine the data from two recent studies to identify genetic loci associated with MAP tissue infection and humoral immune response, defined by MAP ELISA-positive cattle, by comparing cases and control animals for one or both measures of infection. METHODOLOGY/PRINCIPAL FINDINGS: The two populations used for the association analyses were a cohort of MAP tissue infected animals and control Holstein cows from the USA and the second cohort composed of ELISA-positive and ELISA-negative Holstein cows from Italy. Altogether 1190 cattle were genotyped with the Illumina BovineSNP50 BeadChip. SNP markers were removed if the minor allele frequency <0.01 or genotyping failure was >5%. Animals were removed with >5% genotyping failure. Whole genome association analyses were conducted with the GRAMMAR-CG method using two different definitions of control populations. CONCLUSION/SIGNIFICANCE: The analyses identified several loci (P<5 e-05) associated with ParaTB, defined by positive ELISA and presence of bacteria in tissue compared to ELISA and tissue negative animals, on chromosomes 1, 12 and 15 and one unassigned SNP. These results confirmed associations on chromosome 12 and the unassigned SNP with ParaTB which had been found in the Italian population alone. Furthermore, several additional genomic regions were found associated with ParaTB when ELISA and tissue positive animals were compared with tissue negative samples. These loci were on chromosomes 1, 6, 7, 13, 16, 21,23 and 25 (P<5 e-05). The results clearly indicate the importance of the phenotype definition when seeking to identify markers associated with different disease responses.

Low rate of detectable in utero transmission of Mycobacterium avium subspecies paratuberculosis in a dairy herd with a low prevalence of Johne's disease.

In an effort to correlate the likelihood of in utero transmission of Mycobacterium avium subsp. paratuberculosis (MAP), the causal organism of Johne's disease, with the test status of the dam, tissues from neonatal calves borne to known test status cows were cultured for the presence of MAP. Tissues from a single calf borne to a test-positive cow shedding large numbers of organisms in the feces were positive for MAP. The detected overall transmission rate was approximately 2% (1/49), and the detected transmission rate in cows that were fecal culture positive and serum enzyme-linked immunosorbent assay suspect or positive was approximately 4.3% (1/23).

Evolution of the bovine TLR gene family and member associations with Mycobacterium avium subspecies paratuberculosis infection.

Members of the Toll-like receptor (TLR) gene family occupy key roles in the mammalian innate immune system by functioning as sentries for the detection of invading pathogens, thereafter provoking host innate immune responses. We utilized a custom next-generation sequencing approach and allele-specific genotyping assays to detect and validate 280 biallelic variants across all 10 bovine TLR genes, including 71 nonsynonymous single nucleotide polymorphisms (SNPs) and one putative nonsense SNP. Bayesian haplotype reconstructions and median joining networks revealed haplotype sharing between Bos taurus taurus and Bos taurus indicus breeds at every locus, and specialized beef and dairy breeds could not be differentiated despite an average polymorphism density of 1 marker/158 bp. Collectively, 160 tagSNPs and two tag insertion-deletion mutations (indels) were sufficient to predict 100% of the variation at 280 variable sites for both Bos subspecies and their hybrids, whereas 118 tagSNPs and 1 tagIndel predictively captured 100% of the variation at 235 variable sites for B. t. taurus. Polyphen and SIFT analyses of amino acid (AA) replacements encoded by bovine TLR SNPs indicated that up to 32% of the AA substitutions were expected to impact protein function. Classical and newly developed tests of diversity provide strong support for balancing selection operating on TLR3 and TLR8, and purifying selection acting on TLR10. An investigation of the persistence and continuity of linkage disequilibrium (r2≥0.50) between adjacent variable sites also supported the presence of selection acting on TLR3 and TLR8. A case-control study employing validated variants from bovine TLR genes recognizing bacterial ligands revealed six SNPs potentially eliciting small effects on susceptibility to Mycobacterium avium spp paratuberculosis infection in dairy cattle. The results of this study will broadly impact domestic cattle research by providing the necessary foundation to explore several avenues of bovine translational genomics, and the potential for marker-assisted vaccination.

Treatment and chemoprophylaxis for paratuberculosis.

There is no definitive cure for Mycobacterium avium subsp. paratuberculosis (MAP) infections, but several therapeutic agents may be used to alleviate clinical signs of Johne's disease (JD) in ruminants of significant value. Treatment has to be maintained for the life of the animal and treated animals usually continue to shed MAP. No drugs are approved for treatment of JD in the United States; any drug use is "extra-label." Isoniazid, rifampin, and clofazimine are most commonly used for treatment. Monensin, may aid in the prevention of infection in calves and to lower MAP fecal shedding in infected adult cattle.

Evaluation of the in vitro activity of gallium nitrate against Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To evaluate the in vitro susceptibility of various field isolates of Mycobacterium avium subsp paratuberculosis (MAP) to gallium nitrate. SAMPLE: 10 isolates of MAP, including 4 isolated from cattle, 2 isolated from bison, 1 isolated from an alpaca, and 3 isolated from humans. PROCEDURES: The in vitro susceptibility to gallium nitrate was tested by use of broth culture with detection of MAP growth by means of a nonradiometric automated detection method. For each MAP isolate, a series of 7 dilutions of gallium nitrate (concentrations ranging from 200 to 1,000 µM) were tested. Gallium nitrate was considered to have caused 90% and 99% inhibition of the MAP growth when the time to detection for culture of the MAP stock solution and a specific concentration of gallium nitrate was delayed and was similar to that obtained for culture of the MAP stock solution (without the addition of gallium nitrate) diluted 1:10 and 1:100, respectively. RESULTS: Gallium nitrate inhibited MAP growth in all 10 isolates. The susceptibility to gallium nitrate was variable among isolates, and all isolates of MAP were inhibited in a dose-dependent manner. Overall, the concentration that resulted in 90% inhibition ranged from < 200 µM for the most susceptible isolates to 743 µM for the least susceptible isolates. CONCLUSIONS AND CLINICAL RELEVANCE: Gallium nitrate had activity against all 10 isolates of MAP tested in vitro and could potentially be used as a prophylactic agent to aid in the control of MAP infections during the neonatal period.

Application of a peptide-mediated magnetic separation-phage assay for detection of viable Mycobacterium avium subsp. paratuberculosis to bovine bulk tank milk and feces samples.

Naturally contaminated bovine bulk tank milk (n = 44) and feces (n = 39) were tested for the presence of viable Mycobacterium avium subsp. paratuberculosis by a novel peptide-mediated magnetic separation-phage (PMS-phage) assay. Counts of viable M. avium subsp. paratuberculosis cells ranging from 1 to 110 PFU/50 ml of milk and 6 to 41,111 PFU/g of feces were indicated by the PMS-phage assay.

Culture phenotypes of genomically and geographically diverse Mycobacterium avium subsp. paratuberculosis isolates from different hosts.

Mycobacterium avium subsp. paratuberculosis causes paratuberculosis (Johne's disease) in ruminants in most countries. Historical data suggest substantial differences in culturability of M. avium subsp. paratuberculosis isolates from small ruminants and cattle; however, a systematic comparison of culture media and isolates from different countries and hosts has not been undertaken. Here, 35 field isolates from the United States, Spain, Northern Ireland, and Australia were propagated in Bactec 12B medium and Middlebrook 7H10 agar, genomically characterized, and subcultured to Lowenstein-Jensen (LJ), Herrold's egg yolk (HEY), modified Middlebrook 7H10, Middlebrook 7H11, and Watson-Reid (WR) agars, all with and without mycobactin J and some with sodium pyruvate. Fourteen genotypes of M. avium subsp. paratuberculosis were represented as determined by BstEII IS900 and IS1311 restriction fragment length polymorphism analysis. There was no correlation between genotype and overall culturability, although most S strains tended to grow poorly on HEY agar. Pyruvate was inhibitory to some isolates. All strains grew on modified Middlebrook 7H10 agar but more slowly and less prolifically on LJ agar. Mycobactin J was required for growth on all media except 7H11 agar, but growth was improved by the addition of mycobactin J to 7H11 agar. WR agar supported the growth of few isolates. The differences in growth of M. avium subsp. paratuberculosis that have historically been reported in diverse settings have been strongly influenced by the type of culture medium used. When an optimal culture medium, such as modified Middlebrook 7H10 agar, is used, very little difference between the growth phenotypes of diverse strains of M. avium subsp. paratuberculosis was observed. This optimal medium is recommended to remove bias in the isolation and cultivation of M. avium subsp. paratuberculosis.

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis in a longitudinal study of three dairy herds.

The objective of this study was to evaluate whether cows that were low shedders of Mycobacterium avium subsp. paratuberculosis were passively shedding or truly infected with M. avium subsp. paratuberculosis. We also investigated whether it is possible that these M. avium subsp. paratuberculosis-infected animals could have been infected as adults by contemporary high-shedding animals (supershedders). The M. avium subsp. paratuberculosis isolates were obtained from a longitudinal study of three dairy herds in the northeastern United States. Isolates were selected from fecal samples and tissues at slaughter from all animals that were culture positive at the same time that supershedders were present in the herds. Shedding levels (CFU of M. avium subsp. paratuberculosis/g of feces) for the animals at each culture-positive occasion were determined. Using a multilocus short-sequence-repeat technique, we found 15 different strains of M. avium subsp. paratuberculosis from a total of 142 isolates analyzed. Results indicated herd-specific infection patterns; there was a clonal infection in herd C, with 89% of isolates from animals sharing the same strain, whereas herds A and B showed several different strains infecting the animals at the same time. Tissues from 80% of cows with at least one positive fecal culture (other than supershedders) were culture positive, indicating a true M. avium subsp. paratuberculosis infection. The results of M. avium subsp. paratuberculosis strain typing and observed shedding levels showed that at least 50% of low shedders have the same strain as that of a contemporary supershedder. Results of this study suggest that in a dairy herd, more of the low-shedding cows are truly infected with M. avium subsp. paratuberculosis than are passively shedding M. avium subsp. paratuberculosis. The sharing of strains between low shedders and the contemporary supershedders suggests that low shedders may have been infected by environmental exposure of M. avium subsp. paratuberculosis.

Development of a predictive model for detection of Mycobacterium avium subsp. paratuberculosis in faeces by quantitative real time PCR.

This study focused on the development of a reliable and cost-efficient DNA isolation procedure for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in faeces by previously developed IS900 and F57 quantitative real time PCR (qPCR) and their comparison with culture. The recovery of MAP DNA from the spiking experiments ranged from 29.1 to 102.4% of the input amount of MAP with median 37.9%. The limit of detection was determined to be 1.03 × 10(4) for F57 qPCR and 6.87 × 10(2)MAP cells per gram of faeces for IS900 qPCR, respectively. The developed technique for DNA isolation was coupled with IS900 qPCR and compared to traditional MAP culture using a cohort of 1906 faecal samples examined from 12 dairy cattle farms in our laboratory. From those 1906 original faecal samples, 875 were positive by IS900 qPCR and 169 by culture. None of the culture positive samples was negative by IS900 qPCR. This data facilitated development of a predictive model capable of estimating the probability of being culture positive by estimating the absolute number of MAP per gram of faeces as determined IS900 qPCR without performing the culture.

Correlation between Herrold egg yolk medium culture and real-time quantitative polymerase chain reaction results for Mycobacterium avium subspecies paratuberculosis in pooled fecal and environmental samples.

Real-time quantitative polymerase chain reaction (qPCR) testing for Mycobacterium avium subspecies paratuberculosis (MAP) in fecal samples is a rapid alternative to culture on Herrold egg yolk medium (HEYM), the traditional antemortem reference test for MAP. Although the sensitivity and specificity of these 2 tests have been estimated based on dichotomized test results, the correlation between real-time qPCR threshold cycle (Ct) values and colony-forming units (CFU) on HEYM for fresh and thawed samples has not been evaluated. The objectives of the present study were to estimate the correlation and association between Ct and CFU in fresh and thawed pooled fecal and environmental samples. Results of HEYM culture of 1,997 pooled fecal samples from cows in 14 herds, and 802 environmental samples from 109 dairies nationwide were negatively (inversely) correlated with their respective real-time qPCR results. The Spearman's rank correlation between Ct and CFU was good (-0.66) in fresh and thawed pooled fecal samples, and excellent (-0.76) and good (-0.61) in fresh and thawed environmental samples, respectively. The correlation varied from good (-0.53) to excellent (-0.90) depending on the number of samples in a fecal pool. Truncated regression models indicated a significant negative association between Ct and CFU in fecal pools and environmental samples. The use of real-time qPCR instead of HEYM can yield rapid, quantitative estimates of MAP load and allow for incorporation of real-time qPCR results of pooled and environmental samples in testing strategies to identify dairy cow groups with the highest MAP shedding.

GSEA-SNP identifies genes associated with Johne's disease in cattle.

SNP-based gene-set enrichment analysis from single nucleotide polymorphisms, or GSEA-SNP, is a tool to identify candidate genes based on enrichment analysis of sets of genes rather than single SNP associations. The objective of this study was to identify modest-effect genes associated with Mycobacterium avium subsp. paratuberculosis (Map) tissue infection or fecal shedding using GSEA-SNP applied to KEGG pathways or Gene Ontology (GO) gene sets. The Illumina Bovine SNP50 BeadChip was used to genotype 209 Holstein cows for the GSEA-SNP analyses. For each of 13,744 annotated genes genome-wide located within 50 kb of a Bovine SNP50 SNP, the single SNP with the highest Cochran-Armitage Max statistic was used as a proxy statistic for that gene's strength of affiliation with Map. Gene-set enrichment was tested using a weighted Kolmogorov-Smirnov-like running sum statistic with data permutation to adjust for multiple testing. For tissue infection and fecal shedding, no gene sets in KEGG pathways or in GO sets for molecular function or cellular component were enriched for signal. The GO biological process gene set for positive regulation of cell motion (GO:0051272, q = 0.039, 5/11 genes contributing to the core enrichment) was enriched for Map tissue infection, while no GO biological process gene sets were enriched for fecal shedding. GSEA-SNP complements traditional SNP association approaches to identify genes of modest effects as well as genes with larger effects as demonstrated by the identification of one locus that we previously found to be associated with Map tissue infection using a SNP-by-SNP genome-wide association study.

Absorbed EVELISA: a diagnostic test with improved specificity for Johne's disease in cattle.

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.