Receiver operating characteristic-based assessment of a serological test used to detect Johne’s disease in Israeli dairy herds

The overall accuracy of an enzyme-linked immunosorbent assay (ELISA) used to detect Johne's disease at herd level was explored in relation to an imperfect test (fecal culture) in 57 Israeli dairy herds. Receiver-operating characteristic (ROC) analysis indicated an area under the curve (AUC) that corresponded to a test accuracy of 82.0% (69.5% to 90.9%; 95% confidence), with optimized herd sensitivity and herd specificity of 70.4% and 83.3%, respectively; and predictive values of 79.2 (+) and 75.8% (-). The optimal ELISA cutoff was 3.16% (> 3.16% seropositive cows in a herd), which was associated with likelihood ratios (LR) of 4.22 (+LR) and 0.36 (-LR), and post-test probabilities of 0.79 (+) and 0.17 (-). For herds with < or = 200 cows (n = 19 herds), the 95% confidence interval (CI) for the AUC was 0.62-0.97 and the optimal cutoff was 3.33% (HSe = 87.5, HSp = 81.8); for herds with > 200 but < or = 270 cows (n = 19 herds), the 95% AUC CI was 0.62-0.97 and the optimal cutoff was 1.13% (HSe = 90.0, HSp = 77.78); and for herds with > 270 cows (n = 19 herds), the 95% AUC CI was 0.69-0.99 and the optimal cutoff was 0.7% (HSe = 100.0, HSp = 70.0). The AUC was not influenced by across-herd prevalence [R2 (adjusted) = 0.0, P > 0.05]. Findings may be applied to facilitate targeted sampling of herds similar to those evaluated. For instance, a test cutoff of 0.76% could be considered for "ruling disease in," while a cutoff of 3.7% could be used for "ruling disease out." Caveats that may influence this analysis are discussed.     

Estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis in a sample of Minnesota dairy herds using bacterial culture of pooled fecal samples

The objective of this study was to describe the estimated within-herd prevalence (WHP) of Mycobacterium avium subsp. paratuberculosis (Map) in a sample of infected dairy herds in Minnesota (N = 66) using test results from bacterial culture of pooled fecal samples. Fecal samples were collected from up to 100 cows in each herd and were tested using bacterial culture in pools of 5 cows based on age order. The mean herd size was 222 (44 to 1500) milking cows; the cows were predominantly Holstein. Using a frequentist approach, the within-herd mean individual fecal prevalence was 10% [95% confidence interval (CI) = 4% to 16%] assuming 70% test sensitivity and 99.5% test specificity. Using Bayesian methods, the estimated true within-herd individual cow prevalence was 14% (95% CI = 7% to 27%). Within-herd prevalence was higher in larger dairy herds than in herds with fewer cows. As Map is the causative agent of Johne's disease (JD), the results of this study could contribute to the success of a nationwide control program for this disease.     

Cow-level evaluation of a kinetics ELISA with multiple cutoff values to detect fecal shedding of Mycobacterium avium subspecies paratuberculosis in New York State dairy cows

In control programs for Mycobacterium avium subsp. paratuberculosis (Map), the infection status of the cows in a herd is often obtained by testing (a sample of) the herd with an ELISA that may lack some sensitivity and specificity but that is fast and inexpensive. In New York State (NYS), an unabsorbed kinetics ELISA (KELA) has been used extensively for Map control. The objective of this study was to determine the relative sensitivity and specificity of the KELA for detection of fecal shedding of Map for the NYS dairy cow population, taking into account possible confounders such as different antigen batches and Map prevalence in a herd.

The data for the study consisted of all serum samples from NYS dairy cows with concurrent fecal culture results submitted to the NY Animal Health Diagnostic Laboratory (NYAHDL) between 1991 and 1996 (n = 10,562). The data represented cows with different levels of fecal shedding from herds with different within-herd Map prevalence, including herds that were whole herd fecal culture negative on repeated testing.

The cutoff values were based on the predictive value for fecal shedding obtained with a multiple logistic regression model that included variables for the three antigen batches and the Map prevalence in the herd. The KELA could not distinguish between non-shedders and low shedders (≤30 total colony forming units (TCFU)) and thus the predictive value of the KELA to detect moderate to heavy fecal shedders (>30 TCFU) was modeled. The three cutoff values of 65, 135 and 170 were based on low (<0.2), moderate (<0.80) and high (>0.95) probabilities for moderate to heavy fecal shedding. The sensitivity and specificity values relative to culture were 67% and 95.2%, 31% and 99.7%, and 11% and 99.9% for the three cutoff values, respectively. Cutoff values for the KELA decreased for herds with increasing within-herd Map prevalence. For the best positive predictive value of a KELA for moderate to heavy fecal shedding, the cutoff values should be determined based on the apparent within-herd prevalence in a herd.     

Fecal shedding of Mycobacterium avium subsp. paratuberculosis by dairy cows

Between 1982 and 2000, fecal samples were obtained from 786 cows that were shedding Mycobacterium avium subsp. paratuberculosis (Map). These cows were resident on 93 Pennsylvania dairies (mean herd size, 64 milk cows) that had no or minimal previous testing for Map. Feces were cultured on four tubes of Herrold's egg yolk medium and the distribution of mean Map colony forming units (CFU) was evaluated. Most cows were light (< 10 CFU/tube, 51.4%) or high (> 50 CFU/tube, 30.8%) fecal shedders with fewer cows in the moderate category (10-50 CFU/tube). Of the 786 cows, 192 (24.4%) had colonies in only one of four tubes. In the multivariable negative binomial model, there were significant associations between mean CFU/tube and prevalence, herd size, and season and an interaction between herd size and season. The linear mixed model of continuous tube counts with a random herd effect yielded similar findings with associations with herd size as a continuous variable, season, and an interaction between categorized prevalence and continuous herd size. Variability in CFU/tube was greatest among cows in the same herd, intermediate for replicate tubes from the same cow, and smallest among cows in different herds. Reduction in the number of replicate tubes from four would have reduced the sensitivity of fecal culture for Map by approximately 6% (for three tubes) to 12% (for two tubes).     

Herd-level prevalence of Mycobacterium avium subsp. paratuberculosis infection in United States dairy herds in 2007

Testing of composite fecal (environmental) samples from high traffic areas in dairy herds has been shown to be a cost-effective and sensitive method for classification of herd status for Mycobacterium avium subsp. paratuberculosis (MAP). In the National Animal Health Monitoring System's (NAHMS) Dairy 2007 study, the apparent herd-level prevalence of MAP was 70.4% (369/524 had ≥1 culture-positive composite fecal samples out of 6 tested). Based on these data, the true herd-level prevalence (HP) of MAP infection was estimated using Bayesian methods adjusting for the herd sensitivity (HSe) and herd specificity (HSp) of the test method. The Bayesian prior for HSe of composite fecal cultures was based on data from the NAHMS Dairy 2002 study and the prior for HSp was based on expert opinion. The posterior median HP (base model) was 91.1% (95% probability interval, 81.6 to 99.3%) and estimates were most sensitive to the prior for HSe. The HP was higher than estimated from the NAHMS Dairy 1996 and 2002 studies but estimates are not directly comparable with those of prior NAHMS studies because of the different testing methods and criteria used for herd classification.     

Effects of prevalence and testing by enzyme-linked immunosorbent assay and fecal culture on the risk of introduction of Mycobacterium avium subsp. paratuberculosis-infected cows into dairy herds.

A stochastic simulation model was developed to assess the risk of introduction of Mycobacterium avium subsp. paratuberculosis infection into a dairy herd through purchase of female replacement cattle. The effects of infection prevalence in the source herd(s), number of females purchased, and testing by enzyme-linked immunosorbent assay (ELISA) alone or ELISA and fecal culture as risk mitigation strategies were evaluated. Decisions about negative test results were made on a lot and individual basis. A hypothetical dairy herd, free from M. a. paratuberculosis, which replaced 1 lot (10, 30, or 100) of cows per year, was considered. Probability distributions were specified for the sensitivities and specificities of ELISA and fecal culture, the proportion of infected herds and within-herd prevalence for randomly selected replacement source herds (high prevalence) and herds in level 2 (medium prevalence) and level 3 (low prevalence) of the Voluntary Johne's Disease Herd Status Program (VJDHSP). Simulation results predicted that 1-56% of the lots had at least 1 M. a. paratuberculosis-infected cow. Assuming that ELISA sensitivity was 25%, simulation results showed on a lot basis that between 0.4% and 18% and between 0.1% and 9% were predicted to have at least 1 infected cow not detected by ELISA and by a combination of ELISA and fecal culture, respectively. On an individual cow basis, between 0.1% and 8.3% of ELISA-negative cattle in ELISA-positive lots were estimated to be infected. In both the lot and individual analyses, the probability of nondetection increased with larger lot sizes and greater prevalence. Sensitivity analysis indicated that the effect of a lower ELISA sensitivity (10%) was a variable decrease in mean detection probabilities for all combinations of prevalence and lot size. The benefit of testing introduced cattle with ELISA alone or in combination with fecal culture was found to be minimal if cows were purchased from known, low-prevalence (level 3) herds. The value of testing by ELISA alone or in combination with fecal culture was greatest in high-prevalence herds for all lot sizes. Testing of random-source cattle, bought as herd replacements, can partially mitigate the risk of introduction of M. a. paratuberculosis but not as well as by using low-prevalence source herds (level-3 VJDHSP), with or without testing.     

Simulation of alternatives for the Dutch Johne's disease certification-and-monitoring program

To identify optimal method(s) for certification and subsequent monitoring of Mycobacterium avium subsp. paratuberculosis (Map)-unsuspected herds, certification-and-monitoring schemes were studied using a stochastic simulation model ("JohneSSim"). JohneSSim simulated the within-herd transmission and economic aspects of Map in closed Dutch dairy herds. The model was validated with field observations on Map-unsuspected herds. The current Dutch certification-and-monitoring schemes were compared with 11 alternative schemes in which individual and pooled fecal culture, ELISA, Johnin-intradermal test and gamma-IFN ELISA were used, varying the test frequency, tested age group and number of tested animals. On reaching the 'Map-free' status with the standard certification scheme, 11% of the simulated herds were not truly Map-free. Therefore, the designation 'Map-free' should be changed into, for instance, 'low-risk Map'. In the most-attractive alternative certification scheme, the 'Map-free' status was reached after four herd examinations (at 2-year intervals) consisting of serial testing of all cattle > or = 2 years of age with a pooled fecal culture and individual fecal culture of positive pools. This scheme resulted in lower total and annual discounted costs and a lower animal-level prevalence at reaching the 'Map-free' status compared to the standard scheme, assuming that there was no new introduction of the infection. Schemes to monitor the 'Map-free' status were compared, assuming that this status was reached with the standard certification scheme. In comparison to the standard monitoring scheme, none of the alternative monitoring schemes resulted in both a lower animal-level prevalence of undetected pre-existing Map infections in closed herds, and lower median annual discounted costs. Results of the model were very sensitive to the assumed sensitivity of the fecal culture test and to management measures that prevent within-herd transmission of Map. If these preventive measures were taken, the probability of undetected Map infections in closed 'Map-free' herds was decreased substantially.     

Diagnostic validity and costs of pooled fecal samples and individual blood or fecal samples to determine the cow- and herd-status for Mycobacterium avium subsp. paratuberculosis

Two tests are used on a regular basis to detect Mycobacterium avium subsp. paratuberculosis (Map): ELISA and fecal culture. Fecal culture is considered more sensitive and specific but is costly and requires 3-4 months for results. Pooling of fecal samples of individual animals may reduce the high costs of fecal culture. The objective of the study was to investigate the diagnostic validity and costs for pooling of fecal samples in dairy farms relative to culture or an ELISA on individual samples to determine the cow- or herd-status for Map. Fifty fecal and blood samples per herd were collected in 12 Chilean dairy herds. The sensitivity of pooling was estimated given the pool-size, amount of shedding in the pool and the prevalence in the herd. The sensitivity of the pools relative to individual fecal culture was 46% (95% CI 29-63%) and 48% (28-68%) for pools of 5 and 10 cows, respectively. The sensitivity of the pools was lower in pools with low shedders (26 and 24% for pools of 5 and 10, respectively) than in pools with moderate or heavy shedders (>75% sensitivity). Pools of 10 cows are the better option to determine or monitor the herd status. A whole-herd ELISA is the least expensive way to determine the status of individual cows but has a lower Se and Sp than individual culture.     

Pooled fecal culture sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and prevalence.

A stochastic spreadsheet model was developed to obtain estimates of the costs of whole herd testing on dairy farms for Mycobacterium avium subsp. paratuberculosis (Map) with pooled fecal samples. The optimal pool size was investigated for 2 scenarios, prevalence (a low-prevalence herd [< or = 5%] and a high-prevalence herd [> 5%]) and for different herd sizes (100-, 250-, 500- and 1,000-cow herds). All adult animals in the herd were sampled, and the samples of the individuals were divided into equal sized pools. When a pool tested positive, the manure samples of the animals in the pool were tested individually. The individual samples from a negative pool were assumed negative and not tested individually. Distributions were used to model the uncertainty about the sensitivity of the fecal culture at farm level and Map prevalence. The model randomly allocated a disease status to the cows (not shedding, low Map shedder, moderate Map shedder, and heavy Map shedder) on the basis of the expected prevalence in the herd. Pooling was not efficient in 100-cow and 250-cow herds with low prevalence because the probability to detect a map infection in these herds became poor (53% and 88%) when samples were pooled. When samples were pooled in larger herds, the probability to detect at least 1 (moderate to heavy) shedder was > 90%. The cost reduction as a result of pooling varied from 43% in a 100-cow herd with a high prevalence to 71% in a 1,000-cow herd with a low prevalence. The optimal pool size increased with increasing herd size and varied from 3 for a 500-cow herd with a low prevalence to 5 for a 1,000-cow herd with a high prevalence.     

Sensitivity of test strategies used in the Voluntary Johne's Disease Herd Status Program for detection of Mycobacterium paratuberculosis infection in dairy cattle herds

OBJECTIVE: To evaluate sensitivities at the herd level of test strategies used in the Voluntary Johne's Disease Herd Status Program (VJDHSP) and alternative test strategies for detecting dairy cattle herds infected with Mycobacterium paratuberculosis. DESIGN: Nonrandom cross-sectional study. SAMPLE POPULATION: 64 dairy herds from Pennsylvania, Minnesota, Colorado, Ohio, and Wisconsin. Fifty-six herds had at least 1 cow shedding M. paratuberculosis in feces; the other 8 herds were free from paratuberculosis. PROCEDURE: For all adult cows in each herd, serum samples were tested for antibodies to M. paratuberculosis with an ELISA, and fecal samples were submitted for bacterial culture for M. paratuberculosis. Sensitivities at the herd level (probability of detecting infected herd) of various testing strategies were then evaluated. RESULTS: Sensitivity at the herd level of the testing strategy used in level 1 of the VJDHSP (use of the ELISA to test samples from 30 cows followed by confirmatory bacterial culture of feces from cows with positive ELISA result) ranged from 33 to 84% for infected herds, depending on percentage of cows in the herd with positive bacterial culture results. If follow-up bacterial culture was not used to confirm positive ELISA results, sensitivity ranged from 70 to 93%, but probability of identifying uninfected herds as infected was 89%. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that the testing strategy used in the VJDHSP will fail to identify as infected most dairy herds with a low prevalence of paratuberculosis. A higher percentage of infected herds was detected if follow-up bacterial culture was not used, but this test strategy was associated with a high probability of misclassifying uninfected herds.     

Evaluation of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis in large dairy herds

OBJECTIVE: To evaluate sensitivity of microbial culture of pooled fecal samples for detection of Mycobacterium avium subsp paratuberculosis (MAP) in large dairy herds and assess the use of the method for estimation of MAP prevalence. ANIMALS: 1,740 lactating cows from 29 dairy herds in California. PROCEDURE: Serum from each cow was tested by use of a commercial ELISA kit. Individual fecal samples were cultured and used to create pooled fecal samples (10 randomly selected fecal samples/pool; 6 pooled samples/herd). Sensitivity of MAP detection was compared between Herrold's egg yolk (HEY) agar and a new liquid culture method. Bayesian methods were used to estimate true prevalence of MAP-infected cows and herd sensitivity. RESULTS: Estimated sensitivity for pooled fecal samples among all herds was 0.69 (25 culture-positive pools/36 pools that were MAP positive). Sensitivity increased as the number of culture-positive samples in a pool increased. The HEY agar method detected more infected cows than the liquid culture method but had lower sensitivity for pooled fecal samples. Prevalence of MAP-infected cows was estimated to be 4% (95% probability interval, 2% to 6%) on the basis of culture of pooled fecal samples. Herd-level sensitivity estimate ranged from 90% to 100% and was dependent on prevalence in the population and the sensitivity for culture of pooled fecal samples. CONCLUSIONS AND CLINICAL RELEVANCE: Use of pooled fecal samples from 10 cows was a cost-effective tool for herd screening and may provide a good estimate of the percentage of MAP-infected cows in dairy herds with a low prevalence of MAP.     

Use of simulation modeling to estimate herd-level sensitivity, specificity, and predictive values of diagnostic tests for detection of tuberculosis in cattle

OBJECTIVE: To estimate herd-level sensitivity (HSe), specificity (HSp), and predictive values for a positive (HPVP) and negative (HPVN) test result for several testing scenarios for detection of tuberculosis in cattle by use of simulation modeling. SAMPLE POPULATION: Empirical distributions of all herds (15,468) and herds in a 10-county area (1,016) in Michigan. PROCEDURE: 5 test scenarios were simulated: scenario 1, serial interpretation of the caudal fold tuberculin (CFT) test and comparative cervical test (CCT); scenario 2, serial interpretation of the CFT test and CCT, microbial culture for mycobacteria, and polymerase chain reaction assay; scenario 3, same as scenario 2 but specificity was fixed at 1.0; and scenario 4, sensitivity was 0.9 (scenario 4a) or 0.95 (scenario 4b), and specificity was fixed at 1.0. RESULTS: Estimates for HSe were reasonably high, ranging between 0.712 and 0.840. Estimates for HSp were low when specificity was not fixed at 1.0. Estimates of HPVP were low for scenarios 1 and 2 (0.042 and 0.143, respectively) but increased to 1.0 when specificity was fixed at 1.0. The HPVN remained high for all 5 scenarios, ranging between 0.995 and 0.997. As herd size increased, HSe increased and HSp and HPVP decreased. However, fixing specificity at 1.0 had only minor effects on HSp and HPVN, but HSe was low when the herd size was small. CONCLUSIONS AND CLINICAL RELEVANCE: Tests used for detecting cattle herds infected with tuberculosis work well on a herd basis. Herds with < approximately 100 cattle should be tested more frequently or for a longer duration than larger herds to ensure that these small herds are free of tuberculosis.     

Seroprevalence of Mycobacterium avium subsp paratuberculosis infection among dairy cows in Colorado and herd-level risk factors for seropositivity

OBJECTIVE: To estimate seroprevalence of Mycobacterium avium subsp paratuberculosis (MAP) infection among adult dairy cows in Colorado and determine herd-level factors associated with the risk that individual cows would be seropositive. DESIGN: Cross-sectional observational study. ANIMALS: 10,280 adult (> or = 2 years old) dairy cows in 15 herds in Colorado. PROCEDURE: Serum samples were tested with a commercial ELISA. A herd was considered to be infected with MAP if results of mycobacterial culture of > or = 1 individual cow fecal sample were positive or if > or = 1 culled cow had histologic evidence of MAP infection. RESULTS: 424 of the 10,280 (4.12%) cows were seropositive. Within-herd prevalence of seropositive cows ranged from 0% to 7.82% (mean, 2.6%). Infection was confirmed in 11 dairies. Cows in herds that had imported > or = 8% of their current herd size annually during the preceding 5 years were 3.28 times as likely to be seropositive as were cows in herds that imported < 8%. Cows in herds with > or = 600 lactating cows were 3.12 times as likely to be seropositive as were cows in herds with < 600 lactating cows. Cows in herds with a history of clinical signs of MAP infection were 2.27 times as likely to be seropositive as were cows in herds without clinical signs. CONCLUSIONS AND CLINICAL RELEVANCE: Annual importation rate, herd size, and whether cows in the herd had clinical signs typical of MAP infection were associated with the risk that individual cows would be seropositive for MAP infection.     

When can a veterinarian be expected to detect classical swine fever virus among breeding sows in a herd during an outbreak?

The herd sensitivity (HSe) and herd specificity (Hsp) of clinical diagnosis of an infection with classical swine fever (CSF) virus during veterinary inspection of breeding sows in a herd was evaluated. Data gathered from visits to herds during the CSF outbreak in 1997-1998 in The Netherlands were used for the analysis. Herds were visited one or more times by the same or by different veterinarians. On the basis of the veterinarians' reports, each visit was coded as 0 (negative clinical diagnosis) or 1 (positive clinical diagnosis). The HSe for clinical diagnosis of CSF was modelled as a function of days elapsed since introduction of the virus. The moment of introduction of the CSF virus in the CSF-positive herds was unknown, so for each herd, a probability distribution for the unknown number of days since introduction was derived from serum samples collected at depopulation. The information from the reports of the veterinarians and from the test results of the serum samples at depopulation was combined in a Bayesian analysis. Data from CSF-negative herds were analysed to estimate HSp of clinical diagnosis of CSF. The HSe of clinical diagnosis was 0.5 at 37 days after virus introduction (95% CI: 31, 45) and reached 0.9 at 47 days after virus introduction (95% CI: 41, 54). The estimated herd specificity was 0.72 (95% CI: 0.64, 0.79). Dependence of HSe and HSp on characteristics of the veterinarians and the herds also was studied. Specialisation of the veterinarian significantly, although not markedly, affected the HSe.     

Culture of strategically pooled bovine fecal samples as a method to screen herds for paratuberculosis.

Fecal samples from 733 cows in 11 dairy herds with a low prevalence of paratuberculosis were cultured for the presence of Mycobacterium avium subsp. paratuberculosis both individually and after combining (pooling) in groups of 5. The culture procedure was the modified Jorgensen method, which uses NaOH and oxalic acid for decontamination and modified Lowenstein-Jensen agar slants for cultivation. Pooling was performed by mixing fecal samples from 5 animals ordered by age, herein referred to as strategic pooling. Culture of individual fecal samples detected M. a. paratuberculosis infections in 43 of the 733 cows and 7 of 11 infected herds (herd sensitivity = 64%). Culture of pooled fecal samples detected M. a. paratuberculosis in 28 of 151 pooled samples representing 8 of the infected 11 herds (herd sensitivity = 73%). Feces of the 43 culture-positive cows was included in 32 pools: of these 32 pools, 26 were culture positive and 6 were culture negative. In addition to the 26 positive pools containing feces from cows that were found culture positive on individual fecal samples, another 2 pools were culture positive, although comprised of feces from cows with negative results after culture of individual fecal samples. From the total of 45 infected cows that were found (43 by individual fecal culture and an additional 2 by pooled fecal culture), individual fecal culture detected 43 of these 45 (96%), while pooled fecal culture detected 39 (87%). Culture of strategically pooled fecal samples using the modified Jorgensen method was equivalent in herd sensitivity to the culture of individual fecal samples and is significantly less expensive.     

Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of bovine Johne's disease (BJD) in lactating Indian dairy cattle

Present study is the first attempt to evaluate an indigenous milk ELISA with milk culture, standardize milk PCR, estimate lacto-prevalence of Map and genotype Map DNA from milk samples in few Indian dairy herds. In all 115 cows were sampled from 669 lactating cows in six dairy herds from three districts of North India. Fifty milk samples (four herds) were screened by three tests (milk culture, m-ELISA and m-PCR). Lacto-prevalence of Map in four dairy herds was 84.0% (50.0% in fat and 62.0% in sediment). Screening of both fat and sediment increased the sensitivity of culture. Colonies appeared between 45 and 120 DPI. In indigenous m-ELISA, protoplasmic antigen derived from native Map 'Bison type' strain of goat origin was used. Screening of 115 lactating cows by m-ELISA ('herd screening test') detected 32.1% positive lactating cows (lacto-prevalence). Sensitivity of ELISA was 28.5% and 42.8% in single point cutoff and S/P ratio, respectively. Lacto-prevalence of JD was high in dairy herds (66.6-100.0% by culture and 20.0-50.0% by m-ELISA). DDD farm, Mathura had very high (95.8%) and moderate prevalence of Map and lacto-antibodies, respectively. All cows were clinically suffering from JD. Specific IS 900 PCR was standardized in decontaminated fat and sediment of milk samples. DNA isolated from decontaminated pellets was amplified and characteristic 229 bp band was confirmatory for Map. Of the 50 milk samples, 6.0% were positive in m-PCR. The test needs further standardization. Map DNA were genotyped as Map 'Bison type' by IS 1311 PCR-REA. Of the three tests, milk culture was most sensitive followed by m-ELISA and m-PCR. Map DNA isolated from milk samples of dairy cattle were first time genotyped as Map, 'Bison type' in India. High prevalence of Map in milk of dairy herds, posed major health hazard for calves and human beings.     

Comparison of milk and serum enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subspecies paratuberculosis infection in dairy cattle.

Milk and serum samples from 35 dairy herds in 17 states were evaluated for cow- and herd-level Mycobacterium avium subspecies paratuberculosis (MAP) antibody test agreement. Evaluation of 6,349 samples suggested moderate agreement between milk and serum enzyme-linked immunosorbent assay (ELISA) results, with a kappa value of 0.50. Cow-level sensitivity (Se) for 18 dairy operations with 1,921 animals was evaluated relative to fecal culture results. At the cow level, the milk ELISA relative Se was not significantly different from that of the serum ELISA (21.2 and 23.5%, respectively). Logistic regression models revealed a positive association between lactation number and milk ELISA status. Non-Holstein cows were more likely to test milk ELISA positive than Holstein cows. Cows in the first 2 weeks of lactation and after week 45 of lactation were more likely to test milk ELISA positive than cows between 3 and 12 weeks of lactation. Milk production > 80% of herd average was negatively associated with testing milk ELISA positive. Animals in the West and Midwest regions were less likely than animals in the Southeast region to test ELISA positive by either test. Estimates for herd-level sensitivity for the milk and serum ELISA, relative to fecal culture results, ranged from 56 to 83%. At the cow and herd levels, milk ELISA performed equivalent to serum ELISA using fecal culture as a reference for MAP infection and has the advantage of decreased labor costs on farms that use Dairy Herd Improvement Association testing.     

Herd-level seroprevalence of fasciolosis in cattle in north central Portugal

An epidemiological study of Fasciola hepatica in cattle was implemented in the north central region of Portugal. Both an enzyme-linked immunosorbent assay and an egg shedding quantification technique were used in the follow-up of seven herds. Two of these herds were negative and the other five were positive for F. hepatica. A herd cut-off of value of 0.425 optical density was calculated and herd sensitivity (HSe) and herd specificity (HSp) were defined. Three seroprevalence studies were also implemented in the region with stratification by county sub-regions for a period of 18 months. Overall mean herd prevalence in Vagos of 11, 23 and 48% was progressively found for the three studies, respectively.     

Evaluation of cost-effectiveness of targeted sampling methods for detection of Mycobacterium avium subsp paratuberculosis infection in dairy herds

OBJECTIVE: To investigate the epidemiologic and financial impacts of targeted sampling of subpopulations of cows, compared with random sampling of all cows, for classification of dairy herd infection status for paratuberculosis. ANIMALS: All cows from 4 infected herds with a low-to-moderate prevalence of paratuberculosis and from 1 noninfected herd in California. PROCEDURE: The infection status of each cow was classified on the basis of results of an ELISA or combined ELISA and fecal culture results. Thirteen sampling schemes designed to randomly sample cows on the basis of lactation number, stage of lactation, and milk production were evaluated. Sampling without replacement was used to obtain a probability of herd detection of paratuberculosis for each evaluated sampling method and for simulated sample sizes between 30 and 150 cows. Marginal cost-effectiveness analysis was used to determine the cost increase relative to the increase in detection probability. RESULTS: Sampling cows in the third or higher lactation and > or = 200 days into lactation yielded the highest detection probability in most instances, resulting in a detection probability that was 1.4 to 2.5 times that obtained by sampling 30 cows in the second or higher lactation. Costs of testing via the alternative method with a 95% detection probability were approximately dollar 300 lower in a high-prevalence herd (31%) and dollar 800 lower in a low-prevalence herd (9%), compared with use of the reference method. CONCLUSIONS AND CLINICAL RELEVANCE: Detection of herds with paratuberculosis could be improved, and costs of testing substantially reduced by sampling targeted groups of cows.     

Evaluation of environmental fecal culture for Mycobacterium avium subspecies paratuberculosis detection in dairy herds and association with apparent within-herd prevalence

This study evaluated test characteristics of environmental culture (EC) for the detection of Mycobacterium avium subspecies paratuberculosis (MAP) in 32 herds over a 2-year period. Individual fecal samples were collected every 6 mo and environmental samples every 3 mo. Individual fecal culture was performed on samples from positive pools. Samples were cultured in broth, with confirmatory polymerase chain reaction performed on positive fecal samples. Repeated measures were accounted for using GEE logistic models. Relative to a MAP herd-status based on all pooled fecal culture results collected during the study, sensitivity of a set of 6 EC-samples collected from prescribed locations within the herd environment (EC-6) was 71% [95% confidence interval (CI): 49% to 86%] and specificity was 99% (95% CI: 95% to 100%). Sensitivity of EC increased as apparent within-herd fecal culture prevalence (aWHP) increased. The estimated aWHP increased as the proportion of positive EC-samples within an EC-6 set increased. Environmental culture is an acceptable tool for herd diagnosis of MAP in low-prevalence herds.