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%.     

Comparison of two enzyme-linked immunosorbent assays for diagnosis of Mycobacterium avium subsp. paratuberculosis.

Enzyme-linked immunosorbent assays (ELISAs) are used in Johne's disease (JD) control programs as a first screening for presence of the disease in a herd. A high sensitivity of the ELISA is therefore important, yet the commonly used ELISAs have relatively low sensitivity. The inclusion of an absorption phase, although improving specificity, potentially decreases sensitivity. Sera and feces of 383 adult dairy cows in 8 herds were used to compare the test characteristics of an absorbed and a nonabsorbed indirect ELISA for the detection of JD. The absorbed ELISA is based on a protoplasmic antigen, whereas the nonabsorbed uses a lipoarabinomannan-based antigen. The potential advantage of the nonabsorbed ELISA is that it may be less specific and more sensitive. Two herds certified free of JD were used to compare the specificity of the ELISAs. The other herds used to compare sensitivity were either infected with Mycobacterium avium subsp. paratuberculosis or had unknown status. Using fecal culture as a gold standard, the diagnostic specificity for the absorbed and nonabsorbed ELISAs were 98.4% and 87.9%, respectively. The diagnostic sensitivity was 72.4% and 65.5% for the absorbed and the nonabsorbed ELISA, respectively. Furthermore, a comparison using a fecal DNA probe as the comparison standard resulted in both ELISAs having a sensitivity of 61.9%. Agreement between the 2 ELISAs was moderate, with a kappa statistic of 0.58. The nonabsorbed ELISA did not have a higher sensitivity and had a lower specificity than the absorbed ELISA. Therefore, in this population, there was no advantage gained with using the nonabsorbed ELISA.     

Agreement between three ELISAs for Mycobacterium avium subsp. paratuberculosis in dairy cattle

During a 10-month period in 1999, 994 serum and tissue samples were collected from dairy cows at slaughter in eastern Canada. The sources of these cattle were from all four Atlantic Canadian provinces along with some cows from the state of Maine. The sera were used to assess the agreement of three commercially available ELISAs for Mycobacterium avium subsp. paratuberculosis. Two ELISAs were indirect absorbed ELISAs licensed for use in North America, the third was an indirect non-absorbed ELISA licensed for use in Europe. Overall, there was poor agreement between the three ELISAs. The highest and lowest kappa values were 0.33 and 0.18, which is fair and poor agreement, respectively. However, when only tissue culture-positive cattle were compared, the ELISAs had better agreement (kappa=0.37-0.51). The proportions of positive tests, however, were significantly different among the three ELISAs. The poor agreement among the three ELISAs is as concerning as the fact that these tests have low sensitivity. The implications are greatest when the tests are used at the cow level to make individual animal decisions, which is not the recommended method on the product labels. At the cow level, if the result obtained from one ELISA is positive, using a different ELISA in a pre-clinical animal has a high likelihood of giving a different result due to low predictive values of positive test results.     

An Enzyme-linked Immunosorbent Assay using Immonoaffinity-purified Antigen in the Diagnosis of Caprine Paratuberculosis and Its Comparison with Conventional ELISAs

An enzyme-linked immunosorbent assay (APA-ELISA) using an immunoaffinity-purified antigen was developed and compared with the unabsorbed and absorbed ELISA procedures, using a crude antigenic preparation, for its efficacy in detecting antibodies in goat sera against Mycobacterium avium paratuberculosis. Serum samples from 89 goats belonging to three different flocks, two with a history and evidence of paratuberculosis and one without it, were subjected to each ELISA, which had been standardized on known positive sera from goats experimentally infected with paratuberculosis. Faecal culture, faecal examination and histopathology were used as indicators of infection. The diagnostic sensitivities of the unabsorbed, absorbed and APA-ELISA were 81.8%, 77.3% and 77.3% and the specificities were 90.6%, 93.7% and 96.8%, respectively. The positive predictive values of APA-ELISA (94.4%) was the highest, followed by absorbed ELISA (80.9%) and unabsorbed ELISA (72.0%). The negative predictive values for APA-ELISA, absorbed ELISA and unabsorbed ELISA were 93.0%, 92.7% and 93.8%, respectively. The results indicated the value of APA-ELISA in avoiding the need to absorb individual test sera with Mycobacterium phlei and giving more consistent results than the absorbed ELISA. The APA-ELISA was also better than the other two procedures in terms of specificity and positive predictive values.     

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.     

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.     

Serological and molecular detection of <Emphasis Type="Italic">Mycobacterium avium</Emphasis> subsp. <Emphasis Type="Italic">paratuberculosis</Emphasis> in cattle of dairy herds in Colombia

The objective of this study is the detection of Mycobacterium avium subsp. paratuberculosis (MAP) by serum enzyme-linked immunosorbent assay (ELISA), fecal polymerase chain reaction (PCR), and fecal culture in Colombian dairy herds. Serum and fecal samples from asymptomatic cows (n = 307) of 14 dairy herds were tested for MAP by an unabsorbed ELISA test (ELISA-A). Serum and fecal samples from positive ELISA-A animals (n = 31) were further tested by an absorbed ELISA test (ELISA-B) and PCR. Fecal samples from animals of herds positive by ELISA-A and PCR (n = 105) were inoculated onto three different culture media. ELISA-A produced positive results in 10% of the serum samples and 71% of the herds. ELISA-B and PCR results were positive in two and six serum and fecal samples from positive ELISA-A animals, respectively. Fecal samples were negative for MAP on all culture media. The results of this study confirmed the presence of MAP in local dairy herds and the difficulties of MAP detection in asymptomatic animals by ELISA, PCR, and fecal culture.     

Evaluation of five enzyme immunoassays compared with the cytotoxicity assay for diagnosis of Clostridium difficile-associated diarrhea in dogs.

Clostridium difficile-associated-diarrhea (CDAD) is a nosocomial infection in dogs. Diagnosis of this infection is dependent on clinical signs of disease supported by laboratory detection of C. difficile toxins A or B, or both, in fecal specimens via enzyme-linked immunosorbent assay (ELISA). Unfortunately, to the authors' knowledge, commercially available ELISAs have not been validated in dogs to date. We evaluated 5 ELISAs done on 143 canine fecal specimens (100 diarrheic and 43 nondiarrheic dogs) and on 29 C. difficile isolates. The results of each ELISA were compared with the cytotoxin B tissue culture assay (CTA). Clostridium difficile was isolated from 23% of the fecal specimens. Eighteen of the 143 fecal specimens were toxin positive (15 diarrheic and 3 nondiarrheic dogs). On the basis of multiplex polymerase chain reaction (PCR) analysis for toxin-A and -B genes, 72% of the isolates were toxigenic. The carriage rate of toxigenic isolates in diarrheic dogs was higher than that in the nondiarrheic dogs; however, these differences were not statistically significant. A good correlation was found between CTA, PCR, and culture results. The ELISAs done on fecal specimens collected from diarrheic dogs had low sensitivity (7-33%). In contrast, ELISA for toxin A or B, or both, performed on toxigenic isolates had high sensitivity (93%). These results suggest that commercially available human ELISAs are inadequate for the diagnosis of canine C. difficile-associated diarrhea when tested on fecal specimens. In contrast, the Premier ToxinA/B and Techlab ToxinA/B ELISAs may be useful for the diagnosis of canine CDAD when used on toxigenic isolates.     

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.     

Diagnosis of paratuberculosis by fecal culture and ELISA on milk and serum samples in two types of Chilean dairy goat herds.

Fecal culture has been the primary method used to diagnose paratuberculosis in goats. It is laborious, slow, and expensive. Validation of enzyme-linked immunosorbent assays (ELISAs) on milk samples could make paratuberculosis testing more widely available for goat farmers. The aim of this study was to determine the accuracy of serum and milk ELISAs for paratuberculosis, relative to fecal culture, in Chilean dairy goats. Eight dairy goat herds were selected. Feces, blood, and milk samples were collected from all female goats >2 years old. Fecal samples were cultured using Herrold egg yolk medium with mycobactin J and antibiotics. Serum and milk samples were tested using a commercial ELISA kit for Mycobacterium avium subsp. paratuberculosis antibody detection. A total of 383 goats were tested by ELISA and fecal culture. The sensitivity of ELISA on serum and milk relative to fecal culture was 74.3% (95% CI: 59.8-88.8) and 60% (95% CI: 43.8-76.2), respectively. The corresponding values for ELISA specificity based on the percentage of non- M. avium subsp. paratuberculosis-infected goats testing ELISA-negative were 98.6% (95% CI: 96.6-100) and 99.3% (95% CI: 97.9-100) on serum and milk, respectively. Proportions of positive results for serum and fecal samples were significantly different, whereas the proportions of positive results for milk and fecal samples were not significantly different. The milk ELISA had a moderate level of agreement with fecal culture results (Kappa = 0.57). The paratuberculosis ELISA on goat milk samples may be a cost-effective, accurate alternative to fecal culture.     

Evaluation of highly sensitive indigenous milk ELISA kit with fecal culture, milk culture and fecal-PCR for the diagnosis of bovine Johne's disease (BJD) in India

Country lacks indigenous diagnostic kits against Johne's disease in animals. Indigenous ELISA and IS 900 PCR kits, originally developed for goats and sheep, have been adapted for screening of lactating cows. Multiple diagnostic tests were used to screen 26 lactating dairy cows against Johne's disease. Milk ELISA was evaluated with fecal culture, milk culture and fecal PCR. Of the 26 samples from lactating cows, 84.6, 96.1, 88.4 and 23.0% were positive in fecal culture, milk culture, m-ELISA and m-PCR, respectively. Comparatively milk sediment and milk fat culture detected 84.6 and 76.9% cows positive, respectively. Comparatively fecal culture and milk culture detected 84.6 and 96.1% cows positive, respectively. M-ELISA detected 11.5, 0.0, 11.5, 61.0 and 15.3%, cows as negative, suspected, low positive, positive and strong positive, respectively. There was good correlation between milk and fecal culture with m-ELISA. Three negative cows in m-ELISA were also detected in milk and fecal culture. Of the 26 decontaminated fecal samples, 23.0% cows were positive using specific IS 900 f-PCR. Comparative evaluation of m-ELISA with fecal and milk culture showed agreement in 80.7 and 84.6% cows, respectively. Sensitivity of m-ELISA with respect to fecal and culture was 90.9 and 95.6%, respectively. Comparative evaluation of four tests (milk culture, fecal culture, m-ELISA and f-PCR) showed that only 15.3% cows were detected in all the four tests. In three tests (fecal and milk culture and m-ELISA), 57.6% cows were detected positive. None of the cow was exclusively detected in f-PCR. Of the four diagnostic tests used milk culture was most sensitive (96.15%), followed by fecal culture (86.6%), m-ELISA (76.9%) and IS 900 PCR (23.0%) for the diagnosis of bovine Johne's disease (BJD). Milk ELISA detected only one cow extra, which was negative in milk culture. In view of the simplicity, rapidity and efficacy present milk ELISA kit employing soluble protoplasmic antigen from native Map 'Bison type' genotype of goat origin can be reliable for screening of bovine population against Johne's disease in India.     

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.     

Development of a blocking enzyme-linked immunosorbent assay for detection of turkey antibodies to Mycoplasma meleagridis

A blocking enzyme-linked immunosorbent assay (B-ELISA) was developed to detect antibodies to Mycoplasma meleagridis (MM) in turkey sera. This assay was based on two mouse monoclonal antibodies recognising all MM strains tested but none of seven avian mycoplasmal species tested. Furthermore, their binding to the Tween 20 antigen was inhibited by serum from MM-infected birds. The B-ELISA test format was optimized. The cut-off was determined using a set of sera from MM-free turkeys. This B-ELISA was then compared with a commercial indirect ELISA (I-ELISA). Specificities of the two ELISA tests were not significantly different (100 or 99%, respectively). The sensitivity of B-ELISA was significantly higher than the I-ELISA when I-ELISA suspicious results were considered as negative. Testing sera from experimentally MM-infected animals showed that serum plate agglutination (SPA) test detected positive birds before both ELISA methods. Samples were collected in MM-infected commercial flocks and analyzed by SPA, ELISAs, MM-PCR or culture. Results showed that the sensitivity of the B-ELISA appeared superior to the I-ELISA. Moreover, the ability to detect maternal antibodies makes it a useful tool for eradication or control of MM infections.     

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.     

Temporal patterns of diagnostic results in serial samples from cattle with advanced paratuberculosis infections.

As part of investigating diagnostic strategies for Mycobacterium avium subsp. paratuberculosis (Map), serial results from polymerase chain reaction (PCR) on extraintestinal tissues (blood, milk, and liver) were compared with those from more conventional detection methods including serum enzyme-linked immunosorbent assay (ELISA), fecal culture, and fecal PCR. Three cows previously identified as being subclinically infected with Map were selected for the study. Blood, milk, and feces were collected daily and liver biopsies were obtained weekly for a 30-day period. Unexpectedly, a substantial daily variation in serum ELISA sample to positive (S/P) ratios was observed in all 3 cows. In contrast, fecal culture results were consistently positive. However, whereas fecal culture colony counts were consistently high for 2 cows throughout the study, colony counts from the third cow varied from day to day. Diagnostic sensitivity of PCR for fecal, blood, milk, and liver samples in these advanced subclinically infected cows was 87%, 40%, 96%, and 93%, respectively.     

Factors influencing the isolation of Mycobacterium avium subsp. paratuberculosis from bovine fecal samples.

A modified procedure was used for culture of Mycobacterium paratuberculosis (Mptb) from bovine feces. Bovine fecal samples were decontaminated with NaOH, exposed to a mixture of oxalic acid and malachite green, incubated in a mixture of neomycin and amphotericin B. Decontaminated specimens were inoculated onto modified L?wenstein-Jensen medium. Specimens processed by high-speed centrifugation showed growth earlier than specimens prepared by low-speed centrifugation. However, the overall number of positive cultures at 16 weeks was not different for the 2 methods. When infected dairy herds were sampled 4 times at 6-month intervals and culture-positive cows were culled, the prevalence of infected cattle declined over time. After selective culling, the cattle left in the herds shed low numbers of Mptb, which explains why it took longer for cultures to become positive. No heifers younger than 11 months were culture positive, but heifers 13-14 months of age were more frequently culture positive than were heifers of any other age. The 16-week culture period is needed with this method to detect cattle shedding low numbers of Mptb. High-speed centrifugation of samples does not increase the efficiency of identification of animals shedding Mptb.     

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

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

Comparison of two swine Mycoplasma hyopneumoniae enzyme-linked immunosorbent assays for detection of antibodies from vaccinated pigs and field serum samples.

Mycoplasma hyopneumoniae (Mhyo) causes mycoplasmal pneumonia, an economically important disease of swine. Serodiagnosis of Mhyo is based on the current available commercial enzyme immunoassays for detection of swine antibodies against Mhyo, which are the indirect enzyme-linked immunosorbent assay (ELISA) and the blocking ELISA (B-ELISA). Because of the limited information available for these ELISAs, these 2 assays were compared by testing 347 serum samples collected from vaccinated pigs at 0, 13, 28, 43, and 62 days postimmunization (DPI), 50 samples from nonvaccinated pigs, and 1,013 field serum samples. The results of comparison study showed that the specificity for both ELISAs was 99.2% generated from 139 non-vaccinated negative samples. The sensitivities for indirect ELISA generated from samples collected from animals that received the vaccine at DPI 13, 28, 43, and 62 were 0%, 95.7%, 88.4%, and 92.6%, respectively, whereas the sensitivities for B-ELISA were 0%, 98%, 100%, and 97%, respectively. The overall agreement of 96.7% and 80.3% was generated between 2 ELISAs from negative and vaccinated pigs and from field samples, respectively.     

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.     

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

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