Contribution of environmental mycobacteria to false-positive serum ELISA results for paratuberculosis

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.     

Bacteriological investigations about paratuberculosis in dairy herds in Switzerland

In Switzerland clinical bovine paratuberculosis is registered sporadically with on average seven outbreaks per year. Our present studies are aimed to investigate the prevalence of Mycobacterium avium ssp. paratuberculosis (MAP)-infections in the Swiss cattle population and, therefore methods to culture MAP from bovine feces as well as a commercially available ELISA to detect MAP-specific antibodies are evaluated by using fecal samples and blood sera from herds with cases of clinical paratuberculosis. A series of molecular methods i.e. PCR-coupled RFLP analysis of the IS1311-insertion element of M. avium, PCR-coupled RFLP-analysis of the mycobacterial rpoB-gene, and DNA analysis of the mycobacterial 16S rRNA gene are used to identify mycobacterial isolates grown from bovine feces. Up to now, MAP was detected by culture in 12 of 155 (7.7%) animals from herds with paratuberculosis. A rather striking result is the finding of atypical mycobacteria in feces of 75 cattle (48.3%). Among these isolates, M. avium ssp. avium, M. thermoresistibile, and M. hassiacum/M. buckleii have been identified so far.     

Environmental mycobacteria in soil and water on beef ranches: association between presence of cultivable mycobacteria and soil and water physicochemical characteristics

Exposure to environmental mycobacteria has been reported to be a factor contributing to false-positive results on bovine serological tests detecting antibodies to Mycobacterium avium subsp. paratuberculosis (Mptb). This study was conducted to investigate the association between recovery of mycobacteria from the environment of cattle and both (i) historically high or low seroprevalence to Mptb, and (ii) soil and water physicochemical characteristics. Eighty-two samples (soil and water) from nine beef cattle ranches in South-central and South Texas were assessed for the presence of mycobacteria. Twelve mycobacterial species were cultured from soil and water from four herds; no Mptb were detected in environmental samples. A positive culture of environmental mycobacteria from soil was significantly associated with lower pH and calcium as well as higher iron, zinc and manganese contents. Beef cattle are likely to be exposed to environmental mycobacteria that may contribute to false-positive results on ELISAs for Mptb infection. Exposure rates to these mycobacteria likely vary across small geographical areas and may be related to soil and/or water physicochemistry.     

Prevalence of and risk factors for paratuberculosis in purebred beef cattle

OBJECTIVE: To estimate the prevalence of paratuberculosis in purebred beef cattle in Texas and identify risk factors for seropositivity. DESIGN: Epidemiologic survey. ANIMALS: 4,579 purebred cattle from 115 beef ranches in Texas. PROCEDURE: Blood was collected, and serum was analyzed for antibodies with a commercial ELISA. Fecal samples were collected and frozen at -80 degrees C until results of the ELISA were obtained, and feces from seropositive cattle were submitted for mycobacterial culture. Herd owners completed a survey form on management factors. RESULTS: Results of the ELISA were positive for 137 of the 4,579 (3.0%) cattle, and 50 of the 115 (43.8%) herds had at least 1 seropositive animal. Results of mycobacterial culture were positive for 10 of the 137 (7.3%) seropositive cattle, and 9 of the 50 (18%) seropositive herds had at least 1 animal for which results of mycobacterial culture were positive. Risk factors for seropositivity included water source, use of dairy-type nurse cows, previous clinical signs of paratuberculosis, species of cattle (Bos taurus vs Bos indicus), and location. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that seroprevalence of paratuberculosis among purebred beef cattle in Texas may be greater than seroprevalence among beef cattle in the United States as a whole; however, this difference could be attributable to breed or regional differences in infection rates or interference by cross-reacting organisms. Veterinarians should be aware of risk factors for paratuberculosis as well as the possibility that unexpected serologic results may be found in some herds.     

Familial and herd-level associations with paratuberculosis enzyme-linked immunosorbent assay status in beef cattle

A cross-sectional study was performed to determine the odds of having a positive paratuberculosis ELISA result if the dam was ELISA positive in Texas beef cattle, adjusted for individual and herd-level risk factors for seropositivity. Texas beef cattle (n = 2,621) were tested for paratuberculosis by using a commercial ELISA and microbiologic culture of feces for Mycobacterium avium subsp. paratuberculosis (MAP). Pedigree data were collected to identify dam-and sire-offspring pairs. Bayesian mixed-effects logistic regression was used to estimate the odds of seropositivity associated with age, dam ELISA status, sire ELISA status, herd size, herd history of clinical paratuberculosis, within-herd seroprevalence, within-herd fecal MAP prevalence, and within-herd fecal non-MAP Mycobacterium spp. prevalence. Herd of residence was included as a random effect to account for the correlation of observations within the same herd. Statistically probable associations were observed between ELISA status and herd fecal MAP prevalence [OR (odds ratio) 1.28 per 1% increase; P < 0.001] and herd seroprevalence (OR 1.21 per 1% increase; P < 0.001). The association with dam ELISA status was small (OR 1.35) and not highly probable (P = 0.69). Results indicate that use of dam ELISA status to make culling decisions in beef cattle may not improve the success of paratuberculosis control programs. Alternative strategies may be more effective for reducing the odds of seropositivity.     

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.     

Seroprevalence of and agroecological risk factors for Mycobacterium avium subspecies paratuberculosis and neospora caninum infection among adult beef cattle in cow-calf herds in Alberta, Canada

A province-wide cross-sectional seroprevalence and agroecological risk factor study of Mycobacterium avium subspecies paratuberculosis (MAP) and Neospora caninum (NC) infection among cattle in 100 cow-calf herds in Alberta was conducted. The seroprevalence of MAP in adult cattle was 1.5% across all herds. Using a widely accepted herd test cutpoint of 2 or more seropositive cows out of 30 animals tested, 7.9% of herds were estimated to be infected (95% confidence interval (CI): 2.3-23.4%). Seroprevalence of MAP differed by agroecological region; specifically, cattle and herds in areas with high soil pH (> 7.0), southern latitudes, and arid climates had a moderately reduced risk of infection (P < 0.10). Seroprevalence of NC infection was 9.7% among adult beef cattle province-wide--these levels also varied by agroecological region--with 91.0% of herds infected overall.     

Comparison of fecal culture and F57 real-time polymerase chain reaction for the detection of Mycobacterium avium subspecies paratuberculosis in Swiss cattle herds with a history of paratuberculosis

Background

Bovine paratuberculosis is an incurable chronic granulomatous enteritis caused by Mycobacterium avium subspecies paratuberculosis (MAP). The prevalence of MAP in the Swiss cattle population is hard to estimate, since only a few cases of clinical paratuberculosis are reported to the Swiss Federal Food Safety and Veterinary Office each year.Fecal samples from 1,339 cattle (855 animals from 12 dairy herds, 484 animals from 11 suckling cow herds, all herds with a history of sporadic paratuberculosis) were investigated by culture and real-time polymerase chain reaction (PCR) for shedding of MAP.

Results

By culture, MAP was detected in 62 of 445 fecal pools (13.9%), whereas PCR detected MAP in 9 of 445 pools (2.0%). All 186 samples of the 62 culture-positive pools were reanalyzed individually. By culture, MAP was grown from 59 individual samples (31.7%), whereas PCR detected MAP in 12 individual samples (6.5%), all of which came from animals showing symptoms of paratuberculosis during the study. Overall, MAP was detected in 10 out of 12 dairy herds (83.3%) and in 8 out of 11 suckling cow herds (72.7%).

Conclusions

There is a serious clinically inapparent MAP reservoir in the Swiss cattle population. PCR cannot replace culture to identify individual MAP shedders but is suitable to identify MAP-infected herds, given that the amount of MAP shed in feces is increasing in diseased animals or in animals in the phase of transition to clinical disease.     

Familial associations with paratuberculosis ELISA results in Texas Longhorn cattle

The objective of this cross-sectional study was to estimate familial associations with paratuberculosis ELISA status in beef cattle. Texas Longhorn cattle (n=715) greater than 2years of age were sampled for paratuberculosis testing using ELISA and fecal culture. Diagnostic test results were indicative of substantial numbers of false-positive serological reactions consistent with environmental exposure to non-MAP Mycobacterium spp. Associations between ancestors and paratuberculosis ELISA status of offspring were assessed using conditional logistic regression. The association between ELISA status of the dam and her offspring was assessed using linear mixed-effect models. Significant associations were identified between some ancestors and offspring ELISA status. The odds of being classified as "suspect" or greater based on ELISA results were 4.6 times greater for offspring of dams with similarly increased S:P ratios. A significant positive linear association was also observed between dam and offspring log-transformed S:P ratios. Results indicate that there is familial aggregation of paratuberculosis ELISA results in beef cattle and suggest that genetic selection based on paratuberculosis ELISA status may decrease seroprevalence. However, genetic selection may have minimal effect on paratuberculosis control in herds with exposure to non-MAP Mycobacterium spp.     

Comparison of subjective and objective test evaluations for use of Mycobacterium phlei-adsorbed serum in a dot-enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in cattle.

Use of a dot-ELISA with serum adsorbed with Mycobacterium phlei or with nonadsorbed serum was compared. In addition, results attained using visual observation were compared with those obtained using a densitometer. Infection status of cattle was determined by results of culture of feces from a number of cattle with various degrees of exposure (low prevalence and test-negative) and disease manifestation (clinical suspect vs subclinical infection). Two paratuberculosis-negative herds, fecal culture-confirmed clinically suspect cases of paratuberculosis, and cows from 2 paratuberculosis-infected herds with diagnosis confirmed on the farm (low infection rate) were tested. Significant (P less than 0.05) increase in the dot-ELISA response was found in cattle with heavy M paratuberculosis shedding when nonadsorbed and adsorbed sera were used, compared with the response in cattle that were fecal culture-negative or were shedding M paratuberculosis at lower amounts. Paratuberculosis was diagnosed by visual determination in 29 of 44 (65.9%) of fecal culture-positive, clinically suspect cattle when nonadsorbed serum was used. Results of the visual test were negative in 85 of 93 (91.4%) of the fecal culture-negative cattle when nonadsorbed serum was used. However, when using M phlei-adsorbed serum, the sensitivity of the visual determination decreased to 34.1% (15/44), and the specificity increased to 97.8% (91/93).(ABSTRACT TRUNCATED AT 250 WORDS)     

ELISA and fecal culture for paratuberculosis (Johne's disease): sensitivity and specificity of each method

The sensitivity and specificity of the ELISA and fecal culture tests for paratuberculosis in dairy cattle are examined. ELISA and fecal culture data from seven dairy herds where both fecal cultures and ELISA testing was done concurrently are included. A cohort of 954 cattle including 697 parturient adults, cultured every 6 months from 10 herds followed over 4 years served as the basis to determine fecal culture sensitivity. The fecal culture technique utilized a 2g sample with centrifugation and double incubation. Of the 954 cattle cohort of all ages (calf to adult) that were fecal sampled on the first herd visit, 79 were culture positive. An additional 131 animals were detected as culture positive over the next seven tests at 6-month intervals. The sensitivity of fecal culture to detect infected cattle on the first sampling was 38%. Of the 697 parturient cattle cohort, 67 were positive on the first fecal culture, while an additional 91 adult cattle were culture positive over the next seven tests, resulting in a sensitivity of 42% on the first culture of the total animals identified as culture positive. Animals culled from the herds prior to being detected as infected and animals always fecal culture negative with culture positive tissues at slaughter are not included in the calculations. Both groups of infected cattle will lower the apparent sensitivity of fecal culture. Infected dairy herds tested concurrently with both fecal culture and ELISA usually resulted in more than twofold positive animals by culture compared to ELISA.The classification of infected cattle by the extent of shedding of Mycobacterium paratuberculosis in the feces helps define the relative proportion of cattle in each group and therefore the likelihood of detection by the ELISA test. ELISA has a higher sensitivity in animals with a heavier bacterial load, i.e. high shedders (75%) compared to low shedders (15%). Repeated testing of infected herds identifies a higher proportion of low shedders which are more likely to be ELISA negative. Thus, the sensitivity of the ELISA test decreases with repeated herd testing over time, since heavy shedders will be culled first from the herds.     

Age, geographic, and temporal distribution of fecal shedding of Cryptosporidium parvum oocysts in cow-calf herds

OBJECTIVE: To evaluate fecal shedding of Cryptosporidium parvum from California cow-calf herds with respect to age, geographic region, temporal effects, and association with watery feces. ANIMALS: Cows and calves from 38 beef cow-calf operations. PROCEDURE: Fecal specimens were collected and examined for C parvum oocysts, using immunofluorescent microscopy. Associations between age, geographic region, month of collection, watery feces, and likelihood of shedding C parvum were evaluated. RESULTS: 3.9% of cattle were shedding C parvum oocysts. Prevalence of shedding among calves ranged from 0 to 13%, and was 0.6% among cattle > or = 12 months old. The odds of shedding C parvum among 2-month-old calves were 41 times greater than among cattle > 4 months old. The odds of shedding C parvum among cattle tested in May were 8.7 times greater than among cattle tested during June, July, or August. The odds of infected individuals having watery feces were 3 to 4 times greater than for noninfected individuals, but the etiologic fraction was only 8 to 9%. CONCLUSIONS AND CLINICAL RELEVANCE: Substantial fecal shedding of C parvum by cow-calf herds was limited to calves 1 to 4 months old, with low prevalence detected in older animals. Risk of contamination of watersheds with C parvum was limited to those periods when young calves were in the herd. Although the odds of having watery feces were greater for animals infected with C parvum than for noninfected animals, the low etiologic fraction suggests that most calves with watery feces were not infected with C parvum.     

Evaluation of enzyme-linked immunosorbent assays performed on milk and serum samples for detection of paratuberculosis in lactating dairy cows

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.     

Evaluation of bacteriologic culture of individual and pooled fecal samples for detection of Mycobacterium paratuberculosis in dairy cattle herds

OBJECTIVES: To determine the sensitivity of bacteriologic culture of pooled fecal samples in detecting Mycobacterium paratuberculosis, compared with bacteriologic culture of individual fecal samples in dairy cattle herds. STUDY DESIGN: Cross-sectional study. ANIMALS: 24 dairy cattle herds. PROCEDURE: Individual and pooled fecal samples were submitted for bacteriologic culture, and results were compared between these groups. RESULTS: Ninety-four and 88% of pooled fecal samples that contained feces from at least 1 animal with high (mean, > or = 50 colonies/tube) and moderate (mean, 10 to 49 colonies/tube) concentrations of M paratuberculosis, respectively, were identified by use of bacteriologic culture of pooled fecal samples. Prevalences of paratuberculosis determined by bacteriologic culture of pooled and individual fecal samples were highly correlated. CONCLUSIONS AND CLINICAL RELEVANCE: Bacteriologic culture of pooled fecal samples provided a valid and cost-effective method for the detection of M paratuberculosis infection in dairy cattle herds and can be used to estimate prevalence of infection within a herd.     

Assessing familial aggregation of paratuberculosis in beef cattle of unknown pedigree

The objective of this study was to assess genetic similarity of beef cattle using microsatellite markers and to use this information to describe familial aggregation of paratuberculosis test results in Texas beef cattle. Paratuberculosis testing was performed on 2622 adult beef cattle using two commercially available serum ELISAs and radiometric fecal culture. Pedigree records were collected for registered purebred herds and herds with sufficiently detailed production records to identify parent-offspring pairs. Cases were defined as cattle with at least one positive paratuberculosis test result. Three controls were matched by herd of residence for each case. All parent-offspring pairs, cases, and controls were genotyped for 12 microsatellites. Bayesian analysis of allele frequency data was used to describe population substructure and assign individual cattle into groups of genetically similar cattle. The proportion of known parent-offspring pairs assigned to the same cluster was used to assess the validity of the approach to identify familial structure. Conditional logistic regression was used to describe the association between cluster assignment and paratuberculosis test-status matched by herd. Nine clusters of genetically similar individuals were identified and were supported by the proportion of parent-offspring pairs assigned to the same clusters. Increased odds of having at least one positive paratuberculosis test result were identified for two clusters compared to the cluster with the lowest proportion of positive paratuberculosis test results after conditioning on herd. The results of this study demonstrate that population substructure can be used to describe familial aggregation of paratuberculosis test results in beef cattle of unknown pedigree.     

Evaluation of four commercial serum ELISAs for detection of Mycobacterium avium subsp. paratuberculosis infection in dairy cows

During the first 3 months of 2006, a study was performed on four dairy cattle herds with a history of clinical paratuberculosis, to evaluate different enzyme-linked immunosorbent assays (ELISAs). Serum samples obtained from 326 animals were analysed using four ELISAs to detect antibodies to Mycobacterium avium subsp. paratuberculosis (MAP). Kappa (kappa) concordance coefficients in pairwise comparisons of the ELISA outcomes ranged up to 0.22 (linear kappa) and 0.25 (quadratic kappa). When the borderline positives obtained were considered as negatives, kappa values remained low (kappa up to 0.19). Having performed the serological tests, faecal samples were then obtained from 55 animals (including all animals testing positive in two or more ELISAs) from the same herds. Faecal culture and faecal polymerase chain reaction (PCR) for the detection of MAP were negative in all cases. The results indicate that neither the currently available serum ELISAs nor faecal culture and PCR are effective for the early detection of MAP in dairy cattle.     

Association between soil type and paratuberculosis in cattle herds

OBJECTIVE: To determine the association between soil type and paratuberculosis in cattle. SAMPLE POPULATION: Soil samples and test results for paratuberculosis in 92 Indiana cattle herds. PROCEDURE: Testing records from herds in which > or = 20 cattle were tested for paratuberculosis by use of an ELISA between 1998 and 2002 were identified. Soil type was characterized on the basis of herd location. Clusters of herds with seroprevalence greater than the median seroprevalence were identified. Association between clusters and soil types was estimated by logistic regression, adjusted for herd type (dairy or beef). RESULTS: A spatial cluster of greater than the median seroprevalence was identified in northeast Indiana. Soils with low silt content were associated (odds ratio [OR], 7.2; 95% confidence interval [CI], 2.1 to 24.5) with this cluster. Adjusting for herd type did not substantially alter this association (OR, 6.7). Herds located in areas with sandy loam (OR, 6.2; 95% CI, 1.4 to 27.4) and loam (OR, 3.6; 95% CI, 1.0 to 13.2) soils were also more likely to be within the cluster of greater than the median seroprevalence. Herds located in areas of silt loam soils were less likely (OR, 0.2; 95% CI, 0.1 to 0.7) to be included in this cluster. CONCLUSIONS AND CLINICAL RELEVANCE: Spatial distribution of herds with greater than the median seroprevalence of paratuberculosis was associated with soil characteristics. Survival of Mycobacterium avium subsp paratuberculosis may be enhanced by silt or sand content in loamy soils. These results may be used to modify paratuberculosis control programs.     

Environmental contamination with Mycobacterium avium subsp. paratuberculosis in endemically infected dairy herds

Environmental contamination with Mycobacterium avium subsp. paratuberculosis (MAP) is thought to be one of the primary sources of infection for dairy cattle. The exact link between fecal shedding of MAP by individual cows and environmental contamination levels at the herd level was explored with a cross-sectional analysis of longitudinally collected samples on 3 dairy farms. Composite samples from multiple environmental sites in 3 commercial dairy herds in the Northeast US were cultured quarterly for MAP, providing 1131 samples (133 (11.8%) were culture-positive), and all adult animals in the herds were tested biannually by fecal culture (FC), for 6 years. Of the environmental sites sampled, manure storage areas and shared alleyways were most likely to be culture-positive. Environmental sample results were compared to FC results from either the concurrent or previous sampling date at both the herd and the pen level. At the herd level, a 1 log unit increase in average fecal shedding increased the odds of a positive non-pen environmental sample by a factor of 6 and increased the average amount of MAP in non-pen samples by 2.9 cfu/g. At the pen level, a 1 log unit increase in average fecal shedding in the pen increased the odds of a positive environment by a factor of 2.4 and the average amount of MAP was increased by 3.5 cfu/g. We were not able to model the relationship between non-pen environmental sample status and the distance between shedding animals and the sample's location, and neighboring pens did not significantly affect the results of the pen-level analysis. The amount of MAP in pen-level samples and the probability of a pen testing positive for MAP were both positively but non-significantly correlated with the number of animals in the pen shedding >30 cfu/g of MAP. At least 6 environmental samples met the criteria for the U.S. Voluntary Bovine Johne's Disease Control Program on 47 of the 72 sampling dates; of these, 19 of the 47 FC-positive sampling dates were positive by the 6-sample environmental testing method, resulting in a herd sensitivity of 0.40 (95% CI: 0.26-0.54). None of the 3 FC-negative sampling dates produced positive environmental samples. Although environmental sampling can be used as a tool in understanding the level of MAP infection in a herd or pen, it did not appear to be a sensitive diagnostic method for herd positivity in these low prevalence herds, and its use may require caution.     

Evaluation of four commercial enzyme-linked immunosorbent assays for the diagnosis of bovine paratuberculosis in Chilean dairy herds.

The accuracy of 4 commercial enzyme-linked immunosorbent assays (ELISAs) for diagnosis of bovine paratuberculosis was compared using sera from 53 Mycobacterium avium subsp. paratuberculosis (MAP) fecal culture-positive dairy cows (cases) and sera from 345 dairy cattle resident in 11 fecal culture-negative herds on 2 consecutive occasions 1 year apart (controls). The specificity of all 4 ELISA kits was >99%, and their diagnostic sensitivity ranged from 30.2% to 41.5%. Pairwise comparison of ELISAs found no significant differences (McNemar's chi-square test > 0.05), and assay agreement for categorical assay interpretation (positive or negative) was high (>98%) with kappa values ranging from 0.84 to 0.95. Receiver operating characteristic (ROC) curve analysis and the corresponding area under the ROC curves indicate that kit B had the highest overall accuracy. Thus, all 4 ELISA kits for bovine paratuberculosis had comparable accuracy when tested on Chilean dairy cattle, with kit B having a slight statistical advantage based on ROC area under the curve analysis. This suggests that any of the 4 kits could be appropriate for herd certification and for paratuberculosis control programs on Chilean dairy cattle.     

Effects of infectious young stock on results of certification, surveillance and control programmes for paratuberculosis in dairy herds

In many epidemiological models for paratuberculosis, it is assumed that infected young stock (<2 years of age) do not shed Mycobacterium avium subsp. paratuberculosis (MAP) before adulthood. If this assumption were true, the effective separation of young stock from adult cattle (≥ 2 years) would largely prevent postnatal infections, provided that uninfected adult cattle are highly resistant to infection. However, this assumption is in contrast with observed faecal shedding of MAP in young stock. Consequently, this assumption may have resulted in an underestimation of the effects of MAP transmission in herds participating in certification-, surveillance-, and control programmes for paratuberculosis. Therefore, the aim of the present study was to evaluate the long-term effects of transmission of MAP amongst young stock on key output parameters of certification-, surveillance-, and control programmes for paratuberculosis in simulated closed dairy herds. Closed Dutch dairy herds participating in a paratuberculosis programme were simulated with a stochastic model, JohneSSim. Various test schemes, preventive management measures, distributions of age at onset of faecal shedding and rates of effective contacts between young stock were simulated. The results indicate that transmission of MAP amongst young stock has no relevant effects on the animal-level prevalence and milk quality of herds that are certified in a paratuberculosis programme. However, transmission of MAP amongst young stock increased the economic losses due to paratuberculosis and costs of participation in a programme. Moreover, it substantially decreased the beneficial effect of the separation of young stock from adult cattle on the probability of being certified. However, even in the presence of transmission of MAP amongst young stock, preventive management measures to separate young stock from adult cattle remain important.