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PubMed2003

Longitudinal study to investigate variation in results of repeated ELISA and culture of fecal samples for Mycobacterium avium subsp paratuberculosis in commercial dairy herds.

OBJECTIVE: To determine sources and amounts of variation in a kinetics ELISA (KELA) and results of culture of fecal samples for Mycobacterium avium subsp paratuberculosis (MAP) in repeated tests of individual cows. ANIMALS: 112 cows on 6 commercial dairy farms in New York. PROCEDURE: A nonrandom longitudinal study was conducted from January 2001 to March 2002. A KELA was performed monthly, and MAP culture was performed bimonthly. Cow- and herd-level data were collected. The KELA and culture results were analyzed by use of models that corrected for clustering within herds and repeated measures on cows. RESULTS: Cows of second or higher lactation had increased KELA values, compared with values for first-lactation cows. Cows had lowest KELA values during the first 15 days in milk; KELA values increased until 60 days in milk and then stabilized. Moderate and heavy shedders had significantly higher KELA values than culture-negative cows, and KELA values of shedders progressively increased over time. On average, the KELA value was significantly increased 132 days after a cow was first detected to be a moderate shedder and 236 days after a cow was first detected to be a low shedder. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis suggests that KELA results vary on a cow-level on the basis of lactation number and stage of lactation. High KELA values indicate heavy fecal shedding, but the KELA is not useful in identifying low and moderate shedders that can require up to 236 days to have a significant increase in KELA value.

Animals↗
PubMed2004

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.

Animals↗
PubMed2006

Monitoring responses by use of five-color flow cytometry in subsets of peripheral T cells obtained from cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis vaccine.

OBJECTIVE: To monitor by use of 5-color flow cytometry the antigen-specific responses of subsets of peripheral T cells in cattle inoculated with a killed Mycobacterium avium subsp paratuberculosis (MAP) vaccine and to compare results with those for 2 established cell-mediated immunity assays. ANIMALS: 45 female Holstein cattle with negative results for MAP in skin tests conducted at time of inoculation with MAP. PROCEDURES: Cattle were allocated to 4 groups. Cattle of group 1 (n = 12) were 0 to 3 months old and inoculated with a killed MAP vaccine. The 10 cattle of group 2 were the same age as those in group 1 but were not inoculated with MAP vaccine. The 11 cattle of group 3 were 9 to 12 months old and inoculated with killed MAP vaccine. The 12 cattle of group 4 were the same age as those in group 3 but were not inoculated with MAP vaccine. RESULTS: Flow cytometry identified T-cell subsets that responded specifically to the recall antigen. Results of assays for CD25 expression and wholeblood interferon-gamma had the strongest correlation with results for skin tests as well as results with each other. Intracellular expression of interferon-gamma was not correlated as well with results for the other tests. CONCLUSIONS AND CLINICAL RELEVANCE: Flow cytometry can be useful for characterizing the immune response after administration of MAP vaccine and should be evaluated with regard to its sensitivity and specificity when used in detecting cattle naturally infected with MAP.

Animals↗
PubMed2002

Assessment of test results when using a commercial enzyme-linked immunosorbent assay for diagnosis of paratuberculosis in repeated samples collected from adult dairy cattle.

OBJECTIVE: To determine the proportion of adult cattle that change test status when an ELISA for antibodies against Mycobacterium avium subsp paratuberculosis (MAP) is used to assay samples collected twice at variable intervals and to determine whether cows with an initial strong positive result were more likely to maintain positive status, compared with all cows with an initial positive result. DESIGN: Cross-sectional observational study. ANIMALS: 3,757 adult dairy cattle. PROCEDURE: Serum samples were obtained twice from cattle at intervals ranging from 77 to 600 days between collections. Samples were tested with an ELISA for detection of antibodies to MAP. RESULTS: Of 157 cattle with initial positive results (value for the sample divided by the value for positive-control serum [S/P] > or = 0.25), 62 (39.5%) had negative results for the second sample. Of 71 cattle with an initial S/P value > or = 0.40, 13 (18.3%) had a negative result (S/P < 0.25) for the second sample. Of 33 cattle with an initial S/P > or = 0.70, 3 (9.1%) had a negative result (S/P value < 0.25) for the second sample. Interval between collection of samples did not affect results. CONCLUSIONS AND CLINICAL RELEVANCE: Many cows changed ELISA status between samples collected at variable intervals. Cows with an initial high S/P value (> or = 0.70) were more likely to maintain positive status than cows classified as positive on the basis of cutoff values of > or = 0.25 or > or = 0.40. Veterinarians should expect variability in ELISA results when repeated testing of cattle is used as part of an MAP control program.

Animals↗
PubMed2004

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.

Animals↗
PubMed2005

Evaluation of Mycobacterium avium subsp paratuberculosis infection of dairy cows attributable to infection status of the dam.

OBJECTIVE: To estimate the extent to which infection with Mycobacterium avium subsp paratuberculosis (MAP) of cows in a large dairy was attributable to the infection status of their dams. DESIGN: Retrospective longitudinal study. ANIMALS: 625 dam-daughter pairs of Holstein cows. PROCEDURE: Serologic test results were compared between cows and their dams. Logistic regression was used to assess whether a cow's serologic status was associated with its dam's serologic status. Infection with MAP attributable to being born to a seropositive dam was estimated for individual cows and for the herd. RESULTS: Cows with seropositive dams were 6.6 times as likely to be seropositive, compared with cows of seronegative dams. For seropositive cows born to seropositive dams, 84.6% of seropositivity was attributable to being born to a seropositive dam and 15.4% to other exposures, including exposure as calves to flush water that contained feces of adult cattle. For the herd as a whole, the seropositive status in 34% of seropositive cows was attributable to being born to a seropositive dam. CONCLUSIONS AND CLINICAL RELEVANCE: For dairy herds that breed seropositive cows, subsequent transmission of MAP to their daughters, either congenitally or via exposure to feces and colostrum of the dam shortly after birth, can contribute substantially to maintaining prevalence of MAP in a herd. Removal of seropositive, clinically unaffected cows and their daughters would be necessary to reduce infection with MAP attributable to congenital or peri-parturient transmission from dam to daughter.

Animals↗
PubMed2004

Growth, Congo Red agar colony morphotypes and antibiotic susceptibility testing of Mycobacterium avium subspecies paratuberculosis.

OBJECTIVE: Mycobacterium avium subspecies (subsp.) paratuberculosis (MAP) is the causative agent of Johne's disease in ruminants and has been associated with Crohn's disease in humans. We sought to test growth rates and susceptibilities of various strains of MAP in two available growth media. DESIGN: Paired comparison design. METHODS: Using the BACTEC macrobroth radiometric growth system and Congo Red-staining agar media, we determined inherent differences in growth characteristics of three bovine and two human strains of MAP and compared susceptibility results obtained in each growth system. RESULTS: Significant differences were observed in growth rate as well as mycobactin J dependence between strains and between a laboratory-adapted isolate of the same strain in the macrobroth system. Similarly, colonial morphology and Congo Red staining on agar media were observed. Two strains, one human and one bovine, demonstrated a 100% rough transparent colony with white coloration on Congo Red agar, while one bovine isolate exclusively grew as a smooth opaque colony with red coloration on Congo Red agar. The remaining strains exhibited mixtures of these two colonial morphotypes on agar media. Comparative susceptibility results between the BACTEC radiometric macrobroth method and the agar proportionality method showed good correlation for most antibiotics/inhibitors tested. However, erratic or poor growth in the macrobroth system prevented minimal inhibitory concentration determinations for two bovine strains by this method. CONCLUSION: This study demonstrates the variability in the colonial morphology of MAP on Congo Red agar as well as the correlation of antibiotic susceptibility results between the BACTEC macro broth method and the agar proportionality method. This study also emphasizes the need for the development of improved, standardized culture and susceptibility test methods for MAP.

Agar↗
PubMed2002

Variation of the milk antibody response to paratuberculosis in naturally infected dairy cows.

A longitudinal study was performed to determine the course of the milk antibody response in cows presumably infected with Mycobacterium avium subsp. paratuberculosis. Milk samples were collected repeatedly (1 to 10 times) from all lactating cows in seven Danish dairy herds. A total of 4,289 observations from 812 cows was analyzed after exclusion of samples collected after 280 days in milk (DIM). The level of antibodies in the milk samples was assessed using an indirect ELISA. A piece-wise linear random coefficient regression model was specified. The model controlled for the effect of herd, breed, laboratory effects, and age at first calving to estimate parity-specific antibody responses in relation to DIM. Separate antibody profiles were estimated for fecal culture-positive and fecal culture-negative cows. The resulting population average models showed higher antibody levels for fecal culture-positive cows and higher antibody levels with increasing parity. On average, the antibody response was high at the beginning and end of lactation. However, evaluating the cows individually indicated that most cows actually had quite stable ELISA levels throughout lactation, with some cows having higher levels than others. Thus, two criteria seem applicable to assess whether a cow is infected: stability and ELISA level. The random coefficients for each cow were highly significant. Thus, the study suggests that all cows can be classified into one of the four categories by combining the cow-level ELISA characteristics "stability" and "level" as an aid in the diagnosis ofparatuberculosis and thereby substantially increasing the sensitivity of the ELISA.

Animals↗
PubMed2004

Cow-level prevalence of paratuberculosis in culled dairy cows in Atlantic Canada and Maine.

The prevalence of Mycobacterium avium ssp. paratuberculosis (Mptb) in culled dairy cattle in Eastern Canada and Maine was determined to be 16.1% (95% confidence interval 13.8 to 18.3%) based on a systematic random sample of abattoir cattle. Mesenteric lymph nodes and ileum from 984 cows were examined by histologic and bacteriologic methods. Histological testing was far less sensitive than bacteriologic methods for detecting infected cattle. A seasonal pattern of positive cows was also detected, with the highest proportion of cows being Mptb-positive in June (42.5%). Overall, body condition score was not associated with prevalence of Mptb isolation.

Abattoirs↗
PubMed2005

Continuous-data diagnostic tests for paratuberculosis as a multistage disease.

We devised a general method for interpretation of multistage diseases using continuous-data diagnostic tests. As an example, we used paratuberculosis as a multistage infection with 2 stages of infection as well as a noninfected state. Using data from a Danish research project, a fecal culture testing scheme was linked to an indirect ELISA and adjusted for covariates (parity, age at first calving, and days in milk). We used the log-transformed optical densities in a Bayesian network to obtain the probabilities for each of the 3 infection stages for a given optical density (adjusted for covariates). The strength of this approach was that the uncertainty associated with a test was imposed directly on the individual test result rather than aggregated into the population-based measures of test properties (i.e., sensitivity and specificity).

Aging↗
PubMed2006

Evaluation of environmental sampling and culture to determine Mycobacterium avium subspecies paratuberculosis distribution and herd infection status on US dairy operations.

The objectives of this study were to determine the distribution of Mycobacterium avium subspecies paratuberculosis (MAP) in the environment and assess the relationship between the culture status of MAP in the farm environment and herd infection status. The National Animal Health Monitoring System's Dairy 2002 study surveyed dairy operations in 21 states. One component of the study involved collection and culturing of environmental samples for MAP from areas on farms where manure accumulated from a majority of a herd's cows. Operations were selected for inclusion based on perceived risk factors for MAP infection identified in a previously administered questionnaire. Individual animal and environmental samples were collected and used to determine the efficiency of environmental sampling for determination of herd infection status. Individual animal fecal, serum, and milk samples were used to classify herds as infected or not infected based on the presence of at least one test-positive animal in the herd. A total of 483 environmental samples (approximately 5 per farm) were collected, and 218 (45.1%) were culture-positive for MAP. A similar percentage of environmental cultures collected from all designated areas were positive [parlor exits (52.3%), floors of holding pens (49.1%), common alleyways (48.8%), lagoons (47.4%), manure spreaders (42.3%), and manure pits (41.5%)]. Of the 98 operations tested with the environmental sample culture, 97 had individual serum ELISA results, 60 had individual fecal culture results, and 34 had individual milk ELISA results. Sixty-nine of the 98 operations (70.4%) had at least one environmental sample that was culture-positive. Of the 50 herds classified as infected by fecal culture, 38 (76.0%) were identified by environmental culture. Two of the 10 operations classified as not infected based on individual animal fecal culture were environmental culture-positive. Of the 80 operations classified as infected based on serum ELISA-positive results, 61 (76.3%) were identified as environmental-positive, whereas 20 of the 28 (71.4%) operations identified as infected based on milk ELISA were detected by environmental sampling. Environmental sample culturing is less costly than individual animal sampling, does not require animal restraint, and identified more than 70% of infected operations. Environmental sampling is another diagnostic tool that veterinarians and dairy producers can use to determine herd infection status for MAP.

Animals↗
PubMedDate not supplied

[Detection of Mycobacterium avium subspecies paratuberculosis (M. p.) by the polymerase chain reaction (PCR) and culture experiments in animals in Austria].

The polymerase chain reaction and cultural method was applied to detect Mycobacterium avium subspecies paratuberculosis (M. p.) in fecal samples of 165 suspected and of 35 diseased cattle, 18 intestinal tissues and 14 lymph nodes of diseased ruminants as well as organ material (n = 10) were also tested. An agreement (+/+, -/-) in the results between both methods was found in 89.7% of all cases examined. 12.7% of samples of suspected and 9.1% of diseased animals were only positive by PCR. In cases of intestinal lymph node and tissue as well as udder tissue of diseased cattle a total agreement between PCR and culture was observed. Lymph node of lung of one diseased cow was positive only in PCR.

Animals↗
PubMed2002

Survey of Mycobacterium avium subspecies paratuberculosis serological status in beef herds on community pastures in Saskatchewan.

Johne's disease is a well recognized problem in dairy herds. Relatively little information is available on either the prevalence or the control of Johne's disease in commercial cow-calf operations. In the fall of 1999, blood samples were collected during pregnancy testing from cows on community pastures in Saskatchewan. Sera from these cows were analyzed using a commercial ELISA for antibodies to Mycoplasma avium subspecies paratuberculosis. All cows from each herd examined at the community pastures were sampled. Of the 1799 samples tested, 15 had sample to positive (S/P) ratios greater than 0.25 and were considered positive (apparent sample prevalence, 0.8%; 95% CI, 0.4% to 1.5%). If we assume test sensitivity of 25% and specificity of 98% as recommended by the National Johne's Working Group, the true sample prevalence is not significantly different from 0.0%. The ELISA S/P results for the antibody test-positive animals ranged from 0.27 to 2.5. If a herd was classified as positive based on one test-positive animal, the average herd apparent prevalence was 15.2% (95% CI, 7.1% to 28.6%). If the potential for false-positive results was considered with 2 or more positive animals being required for positive herd status, the herd prevalence was 3.0% (95% CI, 0.4% to 13.4%). Because of the very low prevalence in cow-calf herds, future research to identify risk factors and control points should target problem herds and utilize a case-control study design.

Animals↗
PubMed2002

[Diagnosis of paratuberculosis].

Diagnostics of paratuberculosis infection is a difficult and complex field, that causes confusion, lack of understanding and frustration in practitioners, veterinary officers and last but not least farmers. In this review the various diagnostic approaches with their potentials, advantages and disadvantages and their limits are discussed.

Animals↗
PubMed2003

Diagnostic testing patterns of natural Mycobacterium paratuberculosis infection in pygmy goats.

Thirteen pygmy goats (Capra hircus) from a herd naturally infected with Mycobacterium avium ss. paratuberculosis (MPTB) were monitored with 4 diagnostic assays for 2 to 15 mo. Cellular and humoral immune responses to the infection were assessed with assays of gamma interferon (IFNgamma), serum antibody [enzyme-linked immunosorbent assay (ELISA) and agar gel diffusion (AGID)], and radiometric fecal culture. Microscopic examination and radiometric culture of tissue from 12 sites were performed at necropsy. Goats were considered infected if MPTB was isolated from any tissue sample collected at necropsy. Mycobacterial isolates were confirmed as MPTB with an IS900 polymerase chain reaction assay. Ten goats whose antemortem tests indicated infection carried heavy organism burdens at necropsy, both within and beyond the gastrointestinal system. False-negative ELISA, AGID, and/or culture results were obtained in 5 of the 10 confirmed cases during the study period. In 3 goats with sporadic fecal shedding of MPTB or detectable IFNgamma response, or both, no abnormalities were detected at necropsy and no MPTB was isolated from the tissue samples; the antemortem fecal-culture and IFNgamma results were thus considered false-positive. Diagnosticians should be alert to the possibility of both false-positive and false-negative test results for Johne's disease in goats. False-positive fecal-culture results may occur when a high prevalence of infection exists in the herd and the premises are likely to be heavily contaminated. The diverse antemortem testing patterns seen in these goats underscore the importance of using varied diagnostic assays serially or in parallel to increase the likelihood of identifying all infected goats.

Animals↗
PubMed1992

Paratuberculosis vaccine in a large dairy herd.

On a 500-cow dairy farm a total of 866 young calves less than one month old were vaccinated with a heat-killed oil-adjuvated bacterin against Mycobacterium paratuberculosis over a period of five years. The vaccinated calves were tested by faecal microscopy, bacteriology and serology on the day of vaccination, at the age of 3, 6, 9 and 12 months, at breeding age, and on the day of calving. A total of 721 bull calves and 379 female calves served as unvaccinated controls in two groups. The results were evaluated by trend analyses. Vaccination greatly reduced the faecal shedding of mycobacteria as demonstrated by the annual faecal microscopic examinations. During the last 6 months of the experiment only 9 of 612 samples were found positive by microscopy and by bacterial culture. The number of seropositive animals and the antibody titres demonstrated by the complement fixation test (CFT) and agar gel immunodiffusion (AGID) increased during the first three years. Later on, both the number of seropositive animals and CFT titres decreased.

Animals↗
PubMedDate not supplied

[Investigation of serological prevalence and risk factors of paratuberculosis in dairy farms in the state of Mecklenburg-Westpommerania, Germany].

A total of 2,997 serum samples were randomly taken at 59 pre-selected dairy farms covering all districts with an average of 334 animals kept per farm. Samples were analysed for antibodies against Mycobacterium avium subspecies paratuberculosis (M.a.p.) using Svanovir-ELISA. Risk factors on farm characteristics and animal health were recorded at all participating farms by use of a questionnaire. Fifty out of 59 farms (84.7%) showed at least one reactor. The average within herd prevalence was 12.2%. In the univariate analysis of risk factors no significant differences towards farm prevalences were found. Results are discussed with special emphasis on the need for a common national strategy to control the disease in Germany.

Animals↗
PubMed2004

Comparative antibody response of five recombinant antigens in related to bacterial shedding levels and development of serological diagnosis based on 35 kDa antigen for Mycobacterium avium subsp. paratuberculosis.

Eighty-five complex (85A, 85B and 85C), 35-kDa and superoxide dismutase (SOD) were cloned, expressed and purified as antigens in an enzyme-linked immunosorbent assay (ELISA) to compare the serological reactivity of cows with different shedding levels of Mycobacterium avium subsp. paratuberculosis (MPT). Antibody responses to all recombinant antigens positively increased depending on shedding levels. In particular, antibody responses to the 35 kDa were higher than those to the others in all shedder groups. Also, the mean of O. D. values among Ag 85 complex, 85B showed slightly higher response than others with high sensitivity and specificity in all shedder groups. In receiver operating characteristic (ROC) curve analysis, the result of 35 kDa ELISA yielded an area under the curve value of 0.945 (95% confidence interval = 0.895. 0.996), which indicated that this 35 kDa is more accurate indicator of MPT infection than other antigens. At the cut-off point recommended by the ROC curve analysis, the sensitivity and specificity of 35 kDa ELISA were higher than those of other antigens with 93.3% and 86.4%, respectively. Finally, a commercially available ELISA kit was used to clarify 200 positive and 200 negative sera. We then re-tested these serum samples with our ELISA test using the 35-kDa antigens. 35 kDa ELISA and commercial kit showed almost similar results in ROC curve analysis even though two of positive sera in commercial kit were negative in 35 kDa ELISA. The sera, which showed difference in the comparison with commercial ELISA kit, they also did not react with 35 kDa in Western blot. These results suggest that a 35-kDa based ELISA can be useful for detecting MPT infection.

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