Evaluation of conventional and radiometric fecal culture and a commercial DNA probe for diagnosis of Mycobacterium paratuberculosis infections in cattle.

Radiometric (RCM) and conventional fecal culture (HEY) and a commercial polymerase chain reaction/DNA probe were evaluated as diagnostic tests for subclinical paratuberculosis in dairy cattle using fecal specimens from a repository of paratuberculosis specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis, by conventional or radiometric culture, from fecal samples or internal organs of dairy cattle without diarrhea or chronic weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. Among 182 infected cattle, RCM and HEY fecal culture and the DNA probe had test sensitivities of 54.4%, 45.1% and 33.5%, respectively. Fecal samples from only 111 of the M. paratuberculosis-infected cows tested positive by at least one of the three tests and these cows were designated as fecal shedders; the remaining 71 were considered to have prepatent infections. Among the 111 M. paratuberculosis fecal shedders, RCM, HEY and the probe detected the organism in 89.2%, 73.8% and 55.0% of the fecal specimens, respectively. Herd prevalence significantly affected the sensitivity of all three diagnostic tests (p less than 0.05) but only affected the fecal shedder detection efficiency of the DNA probe (p less than 0.01). No positive DNA probe results were found on 100 randomly selected fecal samples from cows in four certified paratuberculosis-free herds, thus the DNA probe was 100% specific. Probe analyses could be performed in 24 h or less. Time to complete the culture-based tests was 12 wk for HEY and 7 wk for RCM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of Mycobacterium paratuberculosis infection on production, reproduction, and health traits in US Holsteins

Our objective was to estimate the effect of Mycobacterium paratuberculosis infection on milk, fat, and protein yield deviations, pregnancy rate, lactation somatic cell score, and projected total months in milk (productive life). A serum ELISA and fecal culture for M. paratuberculosis were performed on 4375 Holsteins in 232 DHIA herds throughout the US. Primarily first through third lactation cows (99% of total) were assayed for infection. Trait information (except productive life) was obtained for the lactation concurrent with disease tests. Productive life was total months in milk through a cow's life, which was projected if a cow was still milking. For most analyses, case definition for M. paratuberculosis infection was defined as either an ELISA S/P ratio>or=0.25 or a positive fecal culture for M. paratuberculosis or both. To determine if diagnostic test affected estimates, case definition was redefined to include only cows with ELISA S/P ratios>or=0.25 or only fecal culture-positive cows. Linear models were used to estimate effect of M. paratuberculosis infection on traits. M. paratuberculosis-infected cows (7.89% of cows) produced 303.9 kg less milk/lactation, 11.46 kg less fat/lactation, and 9.49 kg less protein/lactation (P相似文献   

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.     

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)     

Evaluation of two absorbed enzyme-linked immunosorbent assays and a complement fixation test as replacements for fecal culture in the detection of cows shedding Mycobacterium avium subspecies paratuberculosis.

Control of paratuberculosis in dairy herds is based on preventing the transmission of Mycobacterium avium subsp. paratuberculosis (Mptb) from cows to calves by management measures, supported by removal of cows excreting these bacteria by the fecal route (Mptb shedders). Fecal culture is the most accurate test for identifying Mptb shedders, but this technique is expensive and takes up to 16 weeks for results to be available. Serologic tests are inexpensive, rapid, and easy to perform. Of serologic tests, the complement fixation test (CFT) and absorbed enzyme-linked immunosorbent assay (ELISA) are the serologic tests used most frequently; the CFT is considered less accurate than the ELISA with respect to sensitivity and specificity. The commonly accepted absorbed ELISA is from the Australian Central Serum Laboratory. However, a European supplier has marketed a second ELISA that is supposed to be more sensitive in detecting Mptb shedders. These 2 absorbed ELISAs, designated ELISA-A and ELISA-B, and an in-house CFT were compared with data from 2 serum panels. The Mptb shedding panel consisted of sera from 198 culture-positive cows from 53 infected herds. The method used for culture of fecal samples was a modified J?rgensen method on individual samples. The Mptb shedder detection rate by the 3 serologic tests ranged from 29.8% to 39.4%. Detection rate for ELISA-A was lower than that for ELISA-B and CFT. For all 3 tests, detection rate was dependent on the level of Mptb shedding and the age of the animals. Detection rates increased as cattle age increased to 4 years. The specificity panel was initially composed of sera from 811 cows randomly selected from 41 herds without clinical paratuberculosis that were negative for Mptb based on whole-herd fecal culture. The modified J?rgensen method for culture was used on pooled fecal samples. Serologic test specificity ranged from 93.4% to 99.8%. The specificity of ELISA-A was higher than that of ELISA-B and CFT. Specificity of ELISA-B between herds was 75-100%. Specificity of CFT between herds was 62-100%. The low specificity of ELISA-B and CFT could not be explained by a higher sensitivity for Mptb-infected cows before onset of shedding, because in the 19 herds with 8 more subsequent negative whole-herd fecal cultures in the 4 years after sampling, specificity was not improved. The insufficient specificity of ELISA-B was not corrected sufficiently by heightening the cutoff value because Mptb shedder detection rate was lowered to 28.9%, equal to that of ELISA-A, and specificity only rose to 97%, much lower than that of ELISA-A. Taking into account the different test characteristics, serologic tests are a cost-effective alternative to fecal culture in high-prevalence herds. For certification programs, only ELISA-A is recommended because in a large number of nonsuspect herds specificity remained almost 100%.     

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.     

Bovine fetal infection with Mycobacterium paratuberculosis

In utero transmission of Mycobacterium paratuberculosis, the causative agent of paratuberculosis in cattle, has been suggested. Tissue specimens were obtained at a packing plant from pregnant dairy cows and their fetuses and from cows with clinical signs of paratuberculosis and from their fetuses. Specimens were processed according to methods described for isolating M paratuberculosis from bovine tissues and were incubated on Herrold egg yolk medium for 16 weeks. Presumed positive specimens were confirmed to be M paratuberculosis, using acid-fast staining and subculturing. Of 407 lymph nodes from cows, 34 (8.4%) were culture positive for M paratuberculosis; 9 of 34 (26.4%) of these culture-positive cows had fetuses from which specimens were also culture positive. The results estimated the risk of fetal infection with M paratuberculosis to be 26.4% (95% confidence interval between 11.3 and 40.7%).     

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.     

Decrease of Johne's disease prevalence and incidence in six Minnesota, USA, dairy cattle herds on a long-term management program

The objective of this prospective longitudinal field study was to describe changes in prevalence of seroconversion and fecal shedding and changes in incidence rate of seroconversion, fecal shedding and culling of milk cows for clinical signs of Johne's disease (JD) in six Minnesota (USA) herds participating to the JD Demonstration Herd Project (JDDHP) from 2000 to December 2005. Changes in prevalence and incidence rate were evaluated in light of the owner's compliance to the JDDHP using a risk assessment (RA) score. Adult cows were tested regularly using serum ELISA and bacterial fecal culture to evaluate progress made throughout the control program. Logistic regression was used to evaluate the association between the risk for a cow to test positive and the year on the program. After 5 years of follow-up, the proportion of cows that tested positive to serum ELISA and fecal culture (all positive cultures as well as moderate to heavy shedders only) decreased significantly from the first to the last year (8-3.1%, 10.4-5.6% and 3.1-1.5%, respectively). Cox proportional hazards regression was used to evaluate change of incidence rate across birth cohorts. Birth cohorts were defined by birth date of the animals with the reference cohort or oldest cohort being already 12-24 months of age at the onset of the long-term management program. All cohorts were censored at 45 months of age. Compared to cows from the reference cohort, cows from cohorts that could have benefitted from the JDDHP in their young age (less than 12 months of age at the start of the program or born later) were significantly less at risk of seroconversion and fecal shedding (hazard ratios for seroconversion, any fecal shedding and heavy shedding less than 0.63, 0.67 and 0.62, respectively). For the three herds achieving good management changes with a risk assessment score under 30 at their last year of the study, the cohorts that were born after the program was instituted did better than those born before the start of the program, implying that the program could have helped around birth as well for those herds. This study suggests that reduction of environmental contamination of heifers up to a year of age may have had some impact on the success of the program. The JDDHP appears more beneficial for herds achieving a better reduction of their RA score with a decrease risk for infection in very young calves.     

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.     

The association between Mycobacterium avium subsp. paratuberculosis fecal shedding or clinical Johne's disease and lactation performance on two Minnesota, USA dairy farms

Lactation performance of cows infected with Mycobacterium avium subsp. paratuberculosis (Map) was previously studied using only serum ELISA as a diagnostic method. This study evaluated on two dairy farms in Minnesota, USA the lactation performance (measures of health, production, reproduction, and survival) of cows shedding Map in feces before calving and of cows culled with clinical signs consistent with Johne's disease (JD) during the subsequent lactation. Fecal samples were collected from 1052 cows within 21 day before calving and tested for Map with bacterial culture. Producers’ observed signs of clinical disease (milk fever, retained placenta, metritis, ketosis, displaced abomasum, lameness, mastitis, pneumonia, and JD) and production and reproduction data were recorded for each cow. The association between fecal shedding or clinical JD and lactation performance was evaluated. Logistic regression was used to evaluate the association with any clinical and subclinical diseases as the outcome. General linear model was used to evaluate the association with milk production, and survival analysis techniques were used to evaluate the association with days in the study before culling and days from calving to conception. In 84 cows (8% of 1052 cows) fecal samples were positive for Map (46% light, 26% moderate, and 28% heavy shedders). In multivariable analysis, light, moderate, and heavy fecal shedding cows produced on average 537, 1403, and 1534 kg, respectively, less milk per lactation and 1.4, 5.2, and 7.5 kg, respectively, less milk per day than fecal negative cows. Fecal culture positive cows were less likely to be bred and conceive. In the multivariable analysis the 56 cows culled with presumed JD produced approximately 1500 kg/lactation or 5 kg/day less than all other cows. The negative economic impact implied by decreased lactation performance in cows shedding Map or with clinical JD may motivate producers to implement programs to control Map infection and subsequent JD.     

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).     

Development and application of quantitative polymerase chain reaction assay based on the ABI 7700 system (TaqMan) for detection and quantification of Mycobacterium avium subsp. paratuberculosis.

Numerous reports have described diagnostic methods based on the polymerase chain reaction (PCR) used to detect Mycobacterium avium subsp. paratuberculosis, the causative agent of Johne's disease. The result of conventional PCR tests has been only qualitative, either positive or negative; it does not present any quantitative information about the number of the agents in the specimen. A quantitative PCR method (IS900 TaqMan) was developed to measure the number of M. a. paratuberculosis organisms present in field and clinical samples. The sensitivity of IS900 TaqMan was 1 colony-forming unit (CFU) for M. a. paratuberculosis ATCC 19698. The specificity of the method was determined by testing 14 mycobacterial species (M. abscessus, M. asiaticum, M. avium subsp. avium, M. bovis, M. fortuitum subsp. fortuitum, M. intracellulare, M. kansasii, M. marinum, M. phlei, M. scrofulaceum, M. simiae, M. smegmatis, M. terrae, and M. ulcerans) and 9 nonmycobacterial species (Borrelia burgdorferi, Chlamydia psittaci, Ehrlichia canis, E. equi, E. risticii, Escherichia coli, E. coli O157:H7, Streptococcus equi, and S. zooepidemicus). Even at high cell numbers (10(5) CFU/reaction), most of the organisms tested negative for the IS900 insertion element except M. marinum and M. scrofulaceum. This finding for M. scrofulaceum was consistent with previous reports that several M. scrofulaceum-like isolates were positive for IS900. Those isolates had 71-79% homology with M. a. paratuberculosis in the region of IS900. When used in conjunction with the new liquid medium-based ESP culture system II for bovine clinical fecal samples, IS900 TaqMan confirmed that the ESP II-positive samples contained 10(5)-10(6) CFU/ml of M. a. paratuberculosis. All of the 222 ESP II-positive and acid-fast bacilli-positive samples tested in this study were positive by IS900 TaqMan. IS900 TaqMan was also useful in the study of growth characteristics of 3 groups of M. a. paratuberculosis strains in bovine fecal samples from 3 shedding levels (heavy, medium, and low) based on cell numbers measured by Herrold egg yolk (HEY) agar culture. When cultured in ESP medium, M. a. paratuberculosis reached 10(5)-10(6) CFU/ml within 2 weeks for heavy shedders, 3-4 weeks for medium, and 6-8 weeks for low shedders. No significant growth was observed after up to 5 weeks of incubation for some of low shedders. No or extremely slow growth characteristic of low shedders might be a possible explanation for frequent false-negative results by HEY. The detection time was dependent on the inoculum size and the growth rate of M. a. paratuberculosis. Generation times were inversely proportional to the shedding level: 1-2 days for medium and heavy shedders and >4 days for low shedders. IS900 TaqMan could be a useful tool for determining viable cell counts by measuring changes in cell numbers over the incubation period.     

The effect of subclinical Mycobacterium paratuberculosis infection on days open in Michigan, USA, dairy cows

A prospective cohort study design was used to evaluate the impact of subclinical Mycobacterium paratuberculosis infection on days open in a sample of Michigan dairy herds with a history of cows positive for M. paratuberculosis diagnosed by fecal culture. Participating herds were tested and productivity and reproduction records were monitored for 18 months. All cows > or = 24 months old were tested for M. paratuberculosis infection using the ELISA and radiometric fecal-culture (RFC) techniques. Test-negative cows were re-tested at the conclusion of the monitoring period. Multivariable regression models were used. Using both tests in parallel, the overall sample apparent prevalence for M. paratuberculosis infection was 41.8% (223/533 animals tested). Adjusting for diagnostic sensitivity and specificity, this resulted in a calculated sample true prevalence of 59.9%. ELISA-positive cows (on average) had a 28-day increase in days open when compared to ELISA-negative cows (p=0.02). The diagnostic method used to define a case altered the apparent association between paratuberculosis test status and days open. Fecal culture was a less-effective diagnostic tool for use in herds with a high prevalence of infected animals. The increase in days open in the ELISA-positive cows was an indication that perhaps reduced estrus expression or an increased post-partum anestrous period occurred in the subclinically infected ELISA-positive animals. This might have been due to a negative energy balance associated with M. paratuberculosis infection.     

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.     

Accuracy assessment and screening of a dairy herd with paratuberculosis by three different ELISAs

Although the culture of Mycobacterium avium subsp. paratuberculosisis is the gold standard for the diagnosis of paratuberculosis, this bacterium is difficult to grow. In contrast, serological tests like ELISAs are inexpensive, rapid, and easy to perform. The aims of this study were to evaluate the accuracy of three different ELISAs: one with the commercial antigen PPA-3, another one with L5P (a recently described lipopentapeptide), and a third one with an in-house antigen whole cell lysates (WCL) of M. avium (MAA) strain D4ER (Study 1), and to compare them with other tests for paratuberculosis (PTB) diagnosis (Study 2). In Study 1, the sensitivities of the three ELISAs tested were 74.1%, 37% and 74.1%, respectively, whereas their specificities were 98.9%, 100% and 100%, respectively. In Study 2, we compared the three above-mentioned ELISAs with the intradermal reaction test using Avian PPD (PPDa) and fecal culture associated with Ziehl-Neelsen stain and PCR tests, in a dairy herd with 4.6% of cows with clinical signs of PTB. The results showed that fecal samples from 14 cows (16%) were culture-positive and that fecal samples from nine cows (10%) were PPDa-positive. Most of these animals (culture-positive and PPDa-positive) were detected as positive with any of the three ELISAs tested. Serological results showed that 31% of the animals were positive to ELISA-PPA-3, 17% to ELISA-L5P and 42.5% to ELISA-WCL. The combination of these three ELISAs identified 50.6% of the animals as positive in the infected herd. In particular, the results show that the locally developed ELISA seems to be useful for identifying many infected animals in a herd.     

The results of using faecal culture as confirmation test of paratuberculosis-seropositive dairy cattle

A total of 15,822 cattle aged 3 years and older, belonging to 378 randomly selected herds, were tested for paratuberculosis using an absorbed enzyme-linked immunosorbent assay (ELISA); 3.3% tested positive. This percentage was lowest for the group of cattle aged 3-4 years (2.3%) and highest for cattle with the age of 5-6 years (4.5%). The mean Sample to Positive (S/P) ratio of seropositive cattle vaccinated against paratuberculosis was higher (0.75 +/- 0.33) than that of seropositive, non-vaccinated cattle (0.58 +/- 0.26). Faecal samples of 422 ELISA-positive cattle were cultured for the presence of Mycobacterium avium subsp. paratuberculosis, 12% of these were contaminated. The percentage of non-contaminated samples with positive culture results was 17.3%, with a substantial difference between vaccinated (1.7%) and non-vaccinated cattle (20.2%). Of the positive cultures, the number of colonies varied from 1-10 (22% of cultures), 11-100 (22%), to more than 100 (55%). The percentage of ELISA-positive, non-vaccinated cattle tested culture-positive was positively correlated with the magnitude of the S/P ratio. This percentage varied from 12% (S/P ratio 0.3-0.5) to 58% (S/P ratio > 1.1), a result that might have implications for interpretation of the test. In this study, the percentage of ELISA-positive cattle with positive faecal culture results was limited and these individuals were mostly moderate to heavy shedders.     

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.     

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.