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PubMed2006

Consensus recommendations on diagnostic testing for the detection of paratuberculosis in cattle in the United States.

The report provided here contains a simplified set of diagnostic testing recommendations. These recommendations were developed on the basis of research funded by the USDA-Animal and Plant Health Inspection Service-Veterinary Services through a cooperative agreement. The report is intended to provide simple, practical, cost-effective consensus testing recommendations for cattle herds that are not enrolled in the US Test-Negative Program. The information has been reviewed by paratuberculosis (Johne's disease) experts at the USDA and academic centers as well as stakeholders in various segments of the cattle industry. The recommendations were accepted by the National Johne's Working Group and Johne's Disease Committee of the US Animal Health Association during their annual meetings in October 2006. The report is intended to aid veterinarians who work with cattle producers in the United States. The recommendations are based on information available up to October 2006. There is a paucity of large-scale, high-quality studies of multiple tests conducted on samples obtained from the same cattle. It is understood that there may be special circumstances that require deviation from these recommendations. Furthermore, as new information becomes available and assays are improved and their accuracy is critically evaluated, changes to these recommendations may be necessary.

Animals↗
PubMed2002

Effect of pasteurization on survival of Mycobacterium paratuberculosis in milk.

Mycobacterium paratuberculosis (Mptb) is the causative agent of Johne's disease of ruminant animals including cattle, goats, and sheep. It has been suggested that this organism is associated with Crohn's disease in humans, and milk is a potential source of human exposure to this organism. A total of 18, including 7 regular batch and 11 high temperature short time (HTST) pasteurization experiments, were conducted in this study. Raw milk or ultra-high temperature pasteurized milk samples were spiked at levels of 10(3), 10(5), and 10(7) cfu of Mptb/ml. Escherichia coli and Mycobacterium bovis BCG strains at 10(7) cfu/ml were used as controls. Pasteurization experiments were conducted using time and temperature standards specified in the Canadian National Dairy Code: regular batch pasteurization method: 63 degrees C for 30 min, and HTST method: 72 degrees C for 15 s. The death curve of this organism was assessed at 63 degrees C. No survivors were detected after 15 min. Each spiked sample was cultured in Middlebrook 7H9 culture broth and Middlebrook 7H11 agar slants. Samples selected from 15 experiments were also subjected to BACTEC culture procedure. Survival of Mptb was confirmed by IS900-based PCR of colonies recovered on slants. No survivors were detected from any of the slants or broths corresponding to the seven regular batch pasteurization trials. Mptb survivors were detected in two of the 11 HTST experiments. One was by both slant and broth culture for the sample spiked to 10(7) cfu/ml of Mptb, while the other was detected by BACTEC for the sample spiked to 10(5) cfu/ml. These results indicate that Mptb may survive HTST pasteurization when present at > or = 10(5) cfu/ml in milk. A total of 710 retail milk samples collected from retail store and dairy plants in southwest Ontario were tested by nested IS900 PCR for the presence of Mptb. Fifteen percent of these samples (n = 110) were positive. However, no survivors were isolated from the broth and agar cultures of 44 PCR positive and 200 PCR negative retail milk samples. The lack of recovery of live Mptb from the retail milk samples tested may be due to either the absence of live Mptb in the retail milk samples tested or the presence of low number of viable Mptb which were undetected by the culture method used in this study.

Animals↗
PubMed2005

Effects of seasonal climatic conditions on the diagnosis of Mycobacterium avium subspecies paratuberculosis in dairy cattle.

Validity of Johne's disease programs and control protocols that rely on established cut points [e.g., specified sample-to-positive (S/P) ratios] for ELISA serological tests depends on interpreted results that are not susceptible to variable test accuracy. It was hypothesized that seasonal variability exists in serological response to Mycobacterium avium subsp. paratuberculosis (MAP) infection. Further, a reciprocal response may occur, resulting in greater risk of fecal shedding in subclinically infected animals. A testing regimen was invoked that included multiple testing of individual adult cows during the 4 seasons. Serum was collected on a cyclic, monthly basis from 3 randomly selected cohorts of dairy cows, and fecal samples were collected from the 20% of cows with the greatest ELISA test S/P ratios. Staggered, quarterly sampling was continued for 1 yr, and at the conclusion, serum was analyzed en masse. The ELISA outcome values (i.e., S/P ratio) were treated both as categorical and continuous variables. The potential lagged effects of temperature-related seasonality on S/P ratio, as well as the potential for a change in test result caused by temperature were assessed. Results for fecal culture were analyzed on a categorical scale and compared with the ELISA results to explore the possibility of reciprocal fecal shedding. No significant seasonal effects on either S/P ratios or the proportion of cows seropositive to MAP were observed. Furthermore, no evidence was found linking temperature-related seasonality to a reciprocal increase in the risk of fecal culture positivity for MAP.

Animals↗
PubMed2002

Mycobacterium avium subsp. paratuberculosis infection in a patient with HIV, Germany.

Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne disease in ruminants, has been incriminated as the cause of Crohn disease in humans. We report the first case of human infection with MAP in a patient with HIV; infection was confirmed by obtaining isolates from several different specimen types.

AIDS-Related Opportunistic Infections↗
PubMed2004

Comparison of PCR prescreening to two cultivation procedures with PCR confirmation for detection of Mycobacterium avium subsp. paratuberculosis in U.S. Department of Agriculture fecal check test samples.

Mycobacterium avium subsp. paratuberculosis (MAP) is the etiologic agent in Johne's disease in cattle and causes diarrhea, decreased milk production, emaciation, and frequently death. The ability to detect MAP rapidly and accurately is an integral part of herd management. However, detection of this bacterium is complicated due to its slow division time and its ability to enter dormancy. Culture methods are considered the "gold standard," but they have their limitations. Many enzyme-linked immunosorbent assay methods and conventional PCR methods have been used as diagnostic tools. The present study compares the results of a PCR prescreen to two culture methods of detection paired with confirmatory PCR to determine the most accurate, rapid, and sensitive method using U.S. Department of Agriculture (USDA) fecal check samples. This study involving two laboratories (Marshfield Clinic Laboratories, using solid culture medium [Herrold's egg yolk agar], and TREK Diagnostic Systems Research and Development, using liquid culture medium [ESP Culture System II]) showed that the PCR prescreening method used in this study lacked specificity and sensitivity as a stand-alone test in fecal samples. However, the combination of liquid enrichment culture using the ESP II system, and PCR confirmation with the hspX primer set, was not only 100% sensitive and specific but also correlated with viable MAP and USDA culture results.

Animals↗
PubMed2005

Detection of viable Mycobacterium avium subsp. paratuberculosis in retail pasteurized whole milk by two culture methods and PCR.

Cattle with Johne's disease can shed live Mycobacterium avium subsp. paratuberculosis (MAP) in their milk, and MAP can survive under simulated commercial pasteurization conditions. In several studies conducted in the United Kingdom and Canada, MAP DNA has been detected in retail pasteurized milk samples; however, in one study in the United Kingdom viable MAP was identified in commercially pasteurized milk. A double-blind study involving two laboratories was undertaken to evaluate retail pasteurized whole milk in the United States. Marshfield Clinic Laboratories used solid culture medium (Herrold's egg yolk agar slants with mycobactin J and amphotericin B, nalidixic acid, and vancomycin), and TREK Diagnostic Systems, Research and Development used liquid culture medium (ESP culture system). Cultures at both laboratories were confirmed by PCR. A total of 702 pints of retail whole milk were purchased in three of the top five milk-producing states (233 from California, 234 from Minnesota, and 235 from Wisconsin) over a 12-month period and were tested for the presence of viable MAP. The criteria used for identifying samples as positive for viable MAP were similar to those followed by most laboratories (positive culture with PCR confirmation). The combined data from the two laboratories revealed the presence of viable MAP in 2.8% of the retail whole milk pints tested. Although the number of samples containing viable MAP was similar among states (P > 0.05), there was a seasonal effect on the presence of viable MAP in retail milk (P = 0.05). More MAP-positive samples were identified during the third quarter of the year (July through September). Of the 22 brands of retail milk tested, 12 (55%) yielded at least one sample positive for viable MAP.

Animals↗
PubMed2006

Growth of Mycobacterium avium subsp. paratuberculosis in the presence of hexadecylpyridinium chloride, natamycin, and vancomycin.

A design-of-experiments approach was used to examine the effect of hexadecylpyridinium chloride (HPC), alone or in combination with the antibiotics vancomycin and natamycin, on the growth of Mycobacterium avium subsp. paratuberculosis (MAP). At concentrations above 74.4 microg/ml, HPC had a highly significant detrimental effect on the growth of MAP, whereas natamycin at 10.8 and 21.6 microg/ml and vancomycin at 5.2 and 10.4 microg/ml did not have such an effect. Titration of the amount of HPC tolerated by MAP indicated that growth can occur in the presence of 24.8 microg/ml or lower. Processing of bovine fecal specimens indicated that reducing the concentration of HPC from 32.22 to 1.07 mg/ml during decontamination may improve detection when cultures are grown on solid medium but not when cultures are grown in liquid medium. Further investigation into optimizing HPC concentration during processing of fecal samples is warranted. Natamycin, in conjunction with vancomycin, may be useful for controlling fungal contamination during isolation of MAP from fecal samples.

Animals↗
PubMed2006

Application of an F57 sequence-based real-time PCR assay for Mycobacterium paratuberculosis detection in bulk tank raw milk and slaughtered healthy dairy cows.

A light cycler-based real-time PCR assay that targets the F57 sequence was used to collect data on the prevalence of Mycobacterium avium subsp. paratuberculosis (MAP) in 100 bulk tank raw milk samples and in a population of 101 slaughtered dairy cattle. The assay's reproducible detection limit in total genomic DNA templates isolated from 10-ml samples of MAP-spiked raw milk was 100 cells per ml. Similarly, the evaluation of MAP-spiked bovine feces also demonstrated that the assay had a reproducible detection limit of 100 cells if they were contained within 200 mg of fecal sample material. Among the 100 bulk tank milk samples that were tested, we found 3 samples (3%) to be positive for MAP In the slaughterhouse part of the study, 8.9% (9 of 101) of the cows were positive for MAP DNA in fecal samples, 4.9% (5 of 101) in mesenteric lymph nodes, 0.9% (1 of 101) in ileum tissue, and 3.6% (3 of 84) in milk. Meanwhile, for 2.9% (3 of 101) of the culled cows, MAP DNA was detected in samples of diaphragmatic muscles.

Abattoirs↗
PubMed1975

Age of cattle as related to resistance to infection with Mycobacterium paratuberculosis.

Two 1-month-old calves, four 9-month old calves, and 4 adult (5 to 11 years old) cattle were exposed to Mycobacterium paratuberculosis. At 150 days after exposure, all cattle were killed and necropsied, and at that time, tissues of the 1-month-old calves had more bacilli and lesions than did tissues of the 9-month-old calves or the adult cattle. All cattle responded to immunologic tests during the experiment.

Administration, Oral↗
PubMed2002

Paucibacillary paratuberculosis in a goat.

A 5-year-old Saanen doe was presented with emaciation, good appetite, normal feces, and a dry flaky hair coat. Johne's disease was tentatively diagnosed. Gross postmortem findings were a thickened, corrugated ileum and enlarged edematous mesenteric lymph nodes. Histologically, there was extensive lymphocytic infiltration of the ileal lamina propria. Mycobacterium paratuberculosis could not be identified or cultured.

Animals↗
PubMed2003

Estimation of paratuberculosis prevalence in dairy cattle in a province of Korea using an enzyme-linked immunosorbent assay: application of Bayesian approach.

To draw inferences about the sensitivity and specificity of the newly developed ELISA test for bovine paratuberculosis (PTB) diagnosis and posterior distribution on the prevalence of PTB in a province of Korea, we applied Bayesian approach with Gibbs sampler to the data extracted from the prevalence study in 1999. The data were from a single test results without a designated gold test. The prevalence estimates for PTB in study population ranged 3.2-5.3% for conservative and 6.7-7.1% for liberal, depending on the priors used. The simulated specificities of the ELISA close to one another, ranging 84.7-90.6%, whereas the sensitivity was somewhat spread out depending largely on the priors with a range of 46.4-88.2%. Our findings indicate that the ELISA method appeared useful as a screening tool at a minimum level in comparison to other diagnostic tests available for this disease in terms of sensitivity. However, this advantage comes at a cost of having low specificity of the test.

Animals↗
PubMed2004

Immunological response to Mycobacterium avium subsp. paratuberculosis in chickens.

Enzyme-linked immunosorbent assay (ELISA) and culture are 2 common diagnostic tests for detecting Mycobacterium avium subsp. paratuberculosis (Map) in Johne's disease, but they are not as sensitive as polymerase chain reaction (PCR). However, inhibitors can coextract with the target DNA and cause interference in PCR. Development of an immune capture assay followed by PCR amplification can alleviate this problem. In this study, we were able to induce an immune response in chickens using heat or formalin inactivated Map. The purified immunoglobulin (Ig)Y has a molecular weight of 160 kDa. The titers were at 1:6400 and 1:12 800 at weeks 5 to 6 and 8 to 9, respectively, as determined by the IDEXX modified ELISA kit for Johne's disease. The IgY produced from inactivated bacterial cells had no effect on its ability to recognize live Map cells as illustrated by immunofluorescence assay and immune capture PCR results.

Animals↗
PubMed2006

Inhibition of phagosome maturation and survival of Mycobacterium avium subspecies paratuberculosis in polymorphonuclear leukocytes from Crohn's disease patients.

BACKGROUND: Mycobacterium avium subspecies paratuberculosis (MAP) is an intracellular pathogen that is known to parasitize macrophages. MAP is the known etiological agent of Johne's disease and implicated in the etiology of Crohn's disease. MATERIAL/METHODS: In this study, the survival of human-derived MAP isolate following phagocytosis was evaluated using murine macrophage cell line J774A.1 and polymorphonuclear cells (PMNC's) from six Crohn's disease patients. PMNC's from five healthy individuals and four ulcerative colitis patients, as well as Escherichia coli and Mycobacterium tuberculosis, were included as controls (MOI 10:1). Maturation of the phagosome was determined by evaluating the presence of stage specific markers on the surface of the phagosomal membrane. The endosomal protein, transferrin receptor, and the lysosomal protein, Lamp-1, were then immunostained with Cy-5 conjugated secondary antibodies, and colocalization of bacteria with each marker was evaluated separately using confocal scanning laser microscopy (CSLM). RESULTS: In both models, colocalization of viable MAP and M. tuberculosis with the early endosomal marker occurred with a higher frequency than did association with the late lysosomal marker, as compared to live E. coli, and all dead bacterial species. Using differential live/dead staining and fluorescent microscopy, survival of M. tuberculosis and MAP was calculated to be 85% and 79%, respectively compared to only 14% for E. coli. CONCLUSIONS: Overall, MAP survival in murine macrophages and human PMNCs appears to mimic M. tuberculosis, suggesting the ability of this microorganism to resist phagolysosome fusion, by maintaining association with the early endosomes. The data supports MAP virulence in humans.

Animals↗
PubMed1991

Experimental disease in young chickens induced by a Mycobacterium paratuberculosis isolate from a patient with Crohn's disease.

The susceptibility of young chickens to infection with an isolate of Mycobacterium paratuberculosis which had been recovered from diseased tissues of a patient with Crohn's disease was determined. Two-week-old Leghorn-Cochin chicks were inoculated with strain Linda. Six birds received 10(7) organisms orally, six intraperitoneally, and five intracardially. Six uninoculated birds served as contact controls. Birds from each group were necropsied at two-week intervals. Focal granulomatous lesions occurred in two of six chickens which were inoculated orally, in six of six inoculated intraperitoneally, in three of five inoculated intracardially, but in none of six controls. Of the 11 birds with lesions, acid-fast bacilli were demonstrated in five. Immunoperoxidase reactivity paralleled the presence of acid-fast bacilli.

Animals↗
PubMed1990

Diagnostic value of intravenously administered johnin purified protein derivative (PPD) in bovine paratuberculosis.

The diagnostic value of intravenously administered johnin purified protein derivative (PPD) was studied in 45 cattle of different age, coming from herds infected by, or free from, Mycobacterium paratuberculosis. In addition to observing the clinical symptoms, the animals' sera were assayed for specific antibodies by the complement fixation (CFT) and immunodiffusion (AGID) tests. The blastogenic transformation of peripheral lymphocytes was determined on the basis of 3HTdR incorporation. Changes in the neutrophilic leucocyte/lymphocyte ratio of the blood were also monitored. Detection of the pathogen in the faeces was attempted by microscopic examination and by culturing. Combined evaluation of responses elicited by intravenously administered johnin PPD can be a valuable aid in recognizing infected animals, particularly those among the heifer progeny of infected cows.

Animals↗
PubMed1990

Comparison of the complement-fixation and agar gel immunodiffusion tests for diagnosis of subclinical bovine paratuberculosis.

The performance of the serum complement fixation (CF) test was compared with that of a serum agar gel immunodiffusion (AGID) test on 74 subclinically infected and 154 uninfected cattle in 6 commercial midwestern dairy herds with Mycobacterium paratuberculosis infection and on 30 cattle in a herd that was free of infection. Infection status of cattle within herds was established by performance of a series of 3 or more fecal cultures and of ileocecal lymph node cultures of culled cattle. In cattle with subclinical infection detected by culturing, the sensitivity estimates of the CF and AGID tests were 10.8% (3.6% SE) and 18.9% (4.5% SE), respectively. In the cattle classified as disease free, the specificity estimates of the CF and AGID tests were 97.4% (1.3% SE) and 99.4% (0.6% SE), respectively. Neither set of estimates was significantly different. Negative test results obtained with the use of either test in apparently normal cattle from suspect herds should be interpreted with caution because both tests suffer from low sensitivities in subclinically infected animals. However, the AGID test may be more useful in regulatory situations in which the CF test is currently used because the AGID test is easier to perform and to interpret.

Animals↗