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.

Evaluation of three ELISAs for Mycobacterium avium subsp. paratuberculosis using tissue and fecal culture as comparison standards.

Three serum ELISAs for detection of antibodies against Mycobacterium avium subsp. paratuberculosis (Mptb) were evaluated against culture of tissue and feces samples from 994 dairy cows collected at slaughter. Culture of ileum and associated lymph nodes for Mptb were positive for 160 (16.1%) of the 994 cows and 36 (3.6%) were fecal culture-positive for Mptb. Two of the ELISAs evaluated were absorbed indirect assays and the third was a non-absorbed indirect assay. Estimated sensitivities of the absorbed ELISAs when compared to tissue culture were 8.8% and 6.9%, while the unabsorbed ELISA had a sensitivity of 16.9%. Specificities were 97.6%, 96.0% and 90.8%, respectively. When compared to fecal culture, the sensitivities of the absorbed ELISAs were 16.6% and 13.9%, respectively, and the sensitivity of the unabsorbed ELISA was 27.8%. Specificities were 97.1%, 95.9% and 90.1%, respectively. Area under the curve (AUC) of receiver operator characteristic curves for the absorbed ELISAs when tissue culture was the standard were 0.553 and 0.547, while the unabsorbed ELISA had an AUC of 0.540. When fecal culture was the comparison standard, the AUC of the absorbed ELISAs was 0.575 and 0.574, while the unabsorbed ELISA was 0.529. Overall, the sensitivities of the ELISAs when compared to tissue culture were low. The apparent advantage of the unabsorbed ELISA with respect to sensitivity is at the cost of lowered specificity and test accuracy.

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.

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)

Immune-mediated hemolytic anemia and thrombocytopenia in a foal.

A one-month-old Quarter Horse filly had unilateral epistaxis, hyphema, icterus, petechial hemorrhages in the oral, nasal, conjunctival, and vulvar mucous membranes, anemia, thrombocytopenia, negative antinuclear test result, and a positive direct Coombs' test result. Megakaryocytes or cell-associated IgG (fluorescent antibody and immunoperoxidase stains) were not found in bone marrow biopsy specimens. Treatment consisted of glucocorticoids, antibiotics, and a single whole blood transfusion. The foal responded well to treatment, did not develop relapses of the disease, and was clinically normal one year after treatment.

Malignant melanoma in a goat.

Malignant melanoma was identified in a crossbred goat. It involved the coronary band area and the medial claw of the right hindfoot, which was removed surgically. The goat was reexamined 1 month after tumor removal and found to have widespread metastases with concurrent neurologic signs.

Herd prevalence and geographic distribution of, and risk factors for, bovine paratuberculosis in Wisconsin.

A random sample of Wisconsin dairy herds, stratified by herd size, were tested for paratuberculosis by use of an absorbed ELISA procedure. The ELISA was optimized for overall accuracy by means of receiver operating characteristic curve analysis, and had a sensitivity and specificity of 50.9 and 94.9%, respectively. Herd prevalence was analyzed for correlation with responses to a management practices questionnaire completed by the herd owners. One hundred and fifty-eight herds and 4,990 cattle were tested. Of these, 50% of herds and 7.29% of cattle had positive test results. Calculation of true prevalence from the apparent prevalence indicated that 4.79% of cattle and 34% of the Wisconsin dairy herds tested had serologic evidence of paratuberculosis. Among the 54 herds classified as positive on the basis of true prevalence estimation, the mean number of test positive cattle was 20.3%. The geographic distribution of herds with positive results was not uniform. More infected herds were found in the southern and western districts of Wisconsin than in the eastern district. The west-central district had a larger number of infected herds than did other districts. By use of chi 2 analysis, the only management factor found to be significantly associated with herd prevalence was housing of calves after weaning (P = 0.03). Specifically, in herds with higher prevalence, calves were separated after weaning into calf barns and hutches rather than into pens in the cow barn more often than in herds with lower prevalence. This factor was also considered significant by use of logistic regression analysis.(ABSTRACT TRUNCATED AT 250 WORDS)

Epidemiologic study of on-farm management practices associated with prevalence of Mycobacterium paratuberculosis infections in dairy cattle.

OBJECTIVE: To use an on-farm recording form to quantity the effect of specific management practices on apparent prevalence of Mycobacterium paratuberculosis in dairy cattle herds. DESIGN: Epidemiologic survey. ANIMALS: 26 commercial Wisconsin dairy farms. PROCEDURES: An instrument was developed on the basis of literature review and expert interviews to quantify on-farm management practices associated with increased apparent prevalence of M paratuberculosis. On-farm visits were conducted to assess how specific management practices were conducted. Apparent prevalence of M paratuberculosis infection was measured for all animals > 20 months old on all farms, using a commercial ELISA. Regression analysis was used to identify management variables that were significantly associated with apparent prevalence of M paratuberculosis. RESULTS: Regression analysis (R2 = 0.90) identified that high scores for environmental conditions, newborn calf care, grower calf care, bred heifer care, and manure handling were significantly associated with M paratuberculosis apparent prevalence in Wisconsin dairy herds. CLINICAL IMPLICATIONS: Environmental conditions, newborn calf care, grower calf management, bred heifer management, and manure handling factors may serve as a prioritized checklist for instructing owners and managers where to place emphasis in changing management practices to limit M paratuberculosis prevalence. Likewise, the factors identified as having low association with apparent prevalence may be de-emphasized in control programs, allowing dairy managers to focus time and finances on more effective components of an M paratuberculosis control program.

Johne's disease eradication and control: regulatory implications.

The time has come for the livestock industry and the veterinary profession to take Johne's disease seriously in the United States. The continued spread of the disease with accompanying economic hardship to infected herds and the possibility that M. paratuberculosis may be a food-borne pathogen makes inaction a costly proposition. Efforts have started at the national level with NJWG to have guidelines written for a national control program for Johne's disease. A national control program is desirable because it would provide uniformity to control efforts. Veterinarians and livestock owners should be aware of the effort and let their views be known. In addition, national program guidelines already have been written (see the appendix) to certify test-negative herds for paratuberculosis. States that do not offer the certification program should consider it. A list of test-negative herds for Johne's disease that livestock owners could use to buy low risk animals would be a significant step forward in our efforts to control Johne's disease. Federal regulations concerning Johne's disease are outdated and should be changed to facilitate participation by the livestock industry in a Johne's disease control and eradication program.

Comparison of the Johne's absorbed EIA and the complement-fixation test for the diagnosis of Johne's disease in cattle.

A commercially available absorbed ELISA for the diagnosis of Johne's disease (JD) (paratuberculosis) in cattle, the Johne's Absorbed EIA, was compared with the conventional complement-fixation test (CFT) used in Australia. Stored plasma from 3 Victorian dairy herds with a history of JD, sera from specimens submitted from animals showing clinical signs of JD and sera from the US National Repository for Paratuberculosis Specimens were used to determine the sensitivity of each test. The EIA detected 48.8% of 43 Australian animals with subclinical JD, while the CFT detected only 12 (21.4%) of 56 subclinically affected cattle. Of 150 subclinically infected US cattle, the EIA detected 47.3% and the CFT detected 52.0%. The EIA detected 59.7% of animals which at the time of sampling were shedding Mycobacterium paratuberculosis in their faeces, but showed no clinical signs of JD, while the CFT detected 57.3%. The EIA correctly identified 88.2% of 136 histologically confirmed clinical cases, and the CFT detected 83.4%. The specificity of each test was determined by testing sera collected at slaughter from animals residing in a known JD-free area of Australia, and from samples from the US National Repository of Paratuberculosis Specimens collected from certified-free herds in Wisconsin. The EIA was found to have a specificity of 99.8% when 998 Australian animals were used as the test population, and 99.0% when 196 US animals were used. The specificity of the CFT using Australian samples was 96.9% and 95.2% using American samples.

Evaluation of a commercial enzyme-linked immunosorbent assay for Johne's disease.

A new commercial kit for diagnosis of bovine paratuberculosis (Johne's disease), called the Johne's Absorbed EIA (enzyme immunoassay; Commonwealth Serum Laboratories, Parkville, Victoria, Australia), was evaluated by using serum specimens from the National Repository for Paratuberculosis Specimens. The evaluation was specifically designed to measure test sensitivity and specificity for detection of dairy cattle with subclinical paratuberculosis. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of cattle without diarrhea or chronic weight loss. Animals designed as free of the disease originated exclusively from four herds in Wisconsin that were certified to be free of disease. The kit had a sensitivity of 47.3% for serum specimens from 150 infected cattle. The test detected 59.7% of animals that shed M. paratuberculosis in their feces, as defined by conventional fecal culture, at the time of serum collection. Testing of 196 serum specimens from cattle without paratuberculosis yielded two false-positive results; the test specificity was thus 99.0%. Decision analysis procedures on the economics of using the kit in a test-and-cull disease control program indicated it would be cost-effective in any herd with a true paratuberculosis prevalence of greater than or equal to 3%. Comparison of the sensitivity and specificity of the Johne's Absorbed EIA with those of other tests for detection of subclinical paratuberculosis indicated that it may be the most accurate commercially available test at present and better than standard complement fixation test used in the United States.

Evaluation of four serological tests for bovine paratuberculosis.

The standard complement fixation (CF) test, a commercial agarose gel immunodiffusion (AGID) test (ImmuCell Corporation, Portland, Maine), and two commercial enzyme-linked immunosorbent assays (ELISAs; Allied Laboratories, Glenwood Springs, Colo. [Allied ELISA], and the CSL, Limited, [Parkville, Victoria, Australia] enzyme immunoassay [CSL ELISA]) were evaluated by using sera from the Repository for Paratuberculosis Specimens. The case definition of subclinical bovine paratuberculosis was isolation of Mycobacterium paratuberculosis from fecal samples or internal organs of dairy cattle without diarrhea or weight loss. Animals designated as free of the disease originated exclusively from certified paratuberculosis-free herds in Wisconsin. None of the cattle evaluated had been vaccinated for paratuberculosis. Among 177 M. paratuberculosis-infected cattle, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA had test sensitivities of 38.4, 26.6, 58.8, and 43.4%, respectively, and specificities of 99.0, 100.0, 95.4, and 99.0%, respectively. Only 108 of the infected cattle were confirmed by culture or by a commercial DNA probe (IDEXX Corporation, Portland, Maine) to be shedding the organism in fecal samples at the time of serological testing. Among the 108 M. paratuberculosis fecal shedders, the CF test, the AGID test, the Allied ELISA, and the CSL ELISA were positive for 54.6, 40.7, 65.7, and 56.5% of the cows, respectively; and among the 69 cows that were nonshedders, the tests were positive for 14.5, 4.3, 47.8, and 24.6% of the cows, respectively. There was a significant difference (P < 0.05) in the rate of positive test results for all four tests between these two groups of animals. The CF test performed well when it was compared with the other serological tests if a titer of >/- 1.8 was classified as a positive test result.

Metabolic changes due to experimentally induced rupture of the bovine urinary bladder.

Metabolic changes were studied in the serum, saliva and peritoneal fluid of 5 healthy yearling feedlot steers after experimentally induced urinary bladder rupture. There were statistically significant decreases in serum, saliva and peritoneal fluid sodium and chloride values and significant increases in serum, saliva and peritoneal fluid urea nitrogen, creatinine and phosphorus values. Serum calcium, pH, bicarbonate, and base excess decreased significantly. Potassium did not change significantly in serum but did increase significantly in the saliva. The hemogram and peritoneal fluid analysis failed to provide clinicopathologic evidence of peritonitis. The average time of death or euthanasia after bladder rupture was 13.6 days with a range of 8-21 days. No single biochemical parameter could be identified which would allow prediction of the approaching time of death or duration of the disease process. There was no peritonitis at necropsy indicating that urine was not irritating to the bovine peritoneal cavity. Extracellular replacement fluid with or without sodium bicarbonate supplementation appeared to be the fluid of choice for correction of fluid and electrolyte imbalances in steers with ruptured urinary bladders. The ratio between serum and peritoneal fluid creatinine concentrations appears to be valuable for the clinical diagnosis of ruptured urinary bladders in steers.

Enhanced radiometric detection of Mycobacterium paratuberculosis by using filter-concentrated bovine fecal specimens.

A commercial radiometric medium, BACTEC 12B, was modified by addition of mycobactin, egg yolk suspension, and antibiotics (vancomycin, amphotericin B, and nalidixic acid). Decontaminated bovine fecal specimens were filter concentrated by using 3-microns-pore-size, 13-mm-diameter polycarbonate filters, and the entire filter was placed into the radiometric broth. Comparison of the radiometric technique with conventional methods on 603 cattle from 9 Mycobacterium paratuberculosis-infected herds found that of 75 positive specimens, the radiometric technique detected 92% while conventional methods detected 60% (P less than 0.0005). Only 3.9% of radiometric cultures were contaminated. To measure the effect of filter concentration of specimens on the detection rate, 5 cattle with minimal and 5 with moderate ileum histopathology were sampled weekly for 3 weeks. M. paratuberculosis was detected in 33.3% of nonfiltered specimens and 76.7% of filtered specimens (P less than 0.005). Detection rates were directly correlated with the severity of disease, and the advantage of specimen concentration was greatest on fecal specimens from cattle with low-grade infections. Detection times were also correlated with infection severity: 13.4 +/- 5.9 days with smear-positive specimens, 27.9 +/- 8.7 days with feces from cows with typical subclinical infections, and 38.7 +/- 3.8 days with fecal specimens from cows with low-grade infections. Use of a cocktail of vancomycin, amphotericin B, and nalidixic acid for selective suppression of nonmycobacterial contaminants was better than the commercial product PANTA (Becton Dickinson Microbiologic Systems, Towson, Md.) only when specimens contained very low numbers of M. paratuberculosis. Radiometric culture of filter-concentrated specimens generally doubled the number of positive fecal specimens detected over conventional methods, making it a useful tool for diagnosis and control of bovine paratuberculosis.

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.