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PubMed2002

Paratuberculosis in a mandrill (Papio sphinx).

A 2.5-year-old captive female mandrill (Papio sphinx) died following a protracted course of intermittent abdominal bloat, diarrhea, and severe weight loss. Necropsy revealed emaciation and marked gastrointestinal distention with gas and ingesta. Histologic evaluation revealed severe diffuse granulomatous enterocolitis and mesenteric lymphadenitis with massive numbers of 1-2-microm acid-fast bacilli within macrophages. Additionally, there was moderate to severe multifocal myocardial and vascular amyloidosis, moderate multifocal pyogranulomatous interstitial pneumonia with no acid-fast bacteria, and moderate multifocal glossal candidiasis. Samples of feces, ileum, and colon were positive for Mycobacterium avium subsp. paratuberculosis by radiometric culture and a polymerase chain reaction-amplified DNA probe specific for the insertion sequence IS900 of this organism.

Animals↗
PubMed2003

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.

Animals↗
PubMed2003

Pooled fecal culture sampling for Mycobacterium avium subsp. paratuberculosis at different herd sizes and prevalence.

A stochastic spreadsheet model was developed to obtain estimates of the costs of whole herd testing on dairy farms for Mycobacterium avium subsp. paratuberculosis (Map) with pooled fecal samples. The optimal pool size was investigated for 2 scenarios, prevalence (a low-prevalence herd [< or = 5%] and a high-prevalence herd [> 5%]) and for different herd sizes (100-, 250-, 500- and 1,000-cow herds). All adult animals in the herd were sampled, and the samples of the individuals were divided into equal sized pools. When a pool tested positive, the manure samples of the animals in the pool were tested individually. The individual samples from a negative pool were assumed negative and not tested individually. Distributions were used to model the uncertainty about the sensitivity of the fecal culture at farm level and Map prevalence. The model randomly allocated a disease status to the cows (not shedding, low Map shedder, moderate Map shedder, and heavy Map shedder) on the basis of the expected prevalence in the herd. Pooling was not efficient in 100-cow and 250-cow herds with low prevalence because the probability to detect a map infection in these herds became poor (53% and 88%) when samples were pooled. When samples were pooled in larger herds, the probability to detect at least 1 (moderate to heavy) shedder was > 90%. The cost reduction as a result of pooling varied from 43% in a 100-cow herd with a high prevalence to 71% in a 1,000-cow herd with a low prevalence. The optimal pool size increased with increasing herd size and varied from 3 for a 500-cow herd with a low prevalence to 5 for a 1,000-cow herd with a high prevalence.

Animals↗
PubMed2004

Detection of Mycobacterium avium subsp. paratuberculosis in bovine fecal samples: comparison of three polymerase chain reaction-based diagnostic tests with a conventional culture method.

Three commercially available assays, designed to specifically detect the presence of Mycobacterium avium subsp. paratuberculosis (MAP) in fecal samples by IS900-PCR, were compared with a conventional culture method. Fecal samples from 100 dairy cows were tested. Fifty-four (67.5%) of 80 culture-positive samples were positive for an assay that detects MAP DNA by dot spot hybridization of polymerase chain reaction products (kit A), 48 (60%) were positive by an assay using ethidium bromide staining for agar gel visualization of amplification products (kit B), and 49 (61.3%) were positive by an assay in which amplified products are detected by a colorimetric detection system (kit C). Relative sensitivity of all tests increased in proportion to the presence of MAP in fecal samples. Specificity was 100% based on results from 20 culture-negative samples from an MAP-free herd.

Animals↗
PubMed2005

Amplified fragment length polymorphism to detect clonal diversity and distribution of Mycobacterium avium subspecies paratuberculosis in selected Minnesota dairy cattle.

The molecular ecology of Mycobacterium avium subspecies paratuberculosis (MAP), the causative agent of Johne's disease, is not well understood in the United States. In this study, a DNA fingerprinting method, amplified fragment length polymorphism (AFLP), was used to subtype the pathogen and assess the clonal diversity of MAP in Minnesota dairy herds. Fifty-six fecal culture test-positive isolates from various Minnesota counties and culture dates were analyzed in this study. The AFLP identified 11 profiles with 50% of isolates representing 1 major profile. The major profile was distributed across the state. The genetic diversity of bovine MAP clones in Minnesota based on AFLP analysis of this data appears to be relatively low.

Animals↗
PubMed2005

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.

Animals↗
PubMed2006

Longitudinal study of ELISA seroreactivity to Mycobacterium avium subsp. paratuberculosis in infected cattle and culture-negative herd mates.

Two thousand nine hundred fifty-two serum samples, collected once or twice annually from 545 cows of known fecal culture status were tested for antibodies to Mycobacterium avium subsp. paratuberculosis using a commercially available enzyme-linked immunosorbent assay (ELISA) test. Overall, 13.5% of the samples from 282 infected cows had positive ELISA results, but when tested multiple times, 38.3% of the cows had at least 1 serum sample with positive results. Among 263 fecal culture-negative cows, 98.1% of the serum samples had negative ELISA results, but when tested multiple times, 7.8% of the cows had at least 1 positive ELISA sample. Fecal culture was positive on a test before the first positive ELISA in 50 cows, ELISA was positive before fecal culture in 12 cows, and in 38 cows, both tests became positive at the same testing time. An additional 174 cows were positive on fecal culture and always negative on ELISA until culled. For cows that had ELISA sample:positive (S/P) ratios below the cutoff point, the change in S/P between sequential tests was evaluated to determine whether a rise in S/P could predict infection status. In this study, change in S/P was not a useful predictor of infection status in seronegative cows.

Animals↗
PubMed2006

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.

Animals↗
PubMed2006

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis in bovine feces utilizing the ESP para-JEM liquid culture system.

A testing scheme for the detection of Mycobacterium avium subsp. paratuberculosis (MAP) in broth cultures of bovine fecal samples carried out in ESP para-JEM System was evaluated. The scheme included acid-fast staining (on signal-positive and signal-negative samples), and confirmation by PCR for 2 MAP-specific targets and subculture of all acid-fast positive PCR-negative samples. Two hundred and fifty bovine fecal samples were evaluated for the presence of MAP using this scheme. Thirty-seven (15%) of 250 fecal samples had a positive culture result when the proposed testing scheme was used, compared to 14 (6%) positive results when using the standard ESP para-JEM protocol (requiring samples to have a positive signal from the system, a positive acid-fast stain, and a positive IS900 PCR result), and 20 (8%) positives when conventional culture was performed on Herrold egg yolk (HEY) media. A preliminary comparison of real-time and conventional PCR on DNA extracted from 15 MAP-positive broth cultures by 3 different protocols suggested that conventional PCR may be a better choice for the confirmation of the presence of MAP in the liquid cultures than real-time PCR.

Animals↗
PubMed2006

Comparison of three DNA preparation methods for real-time polymerase chain reaction confirmation of Mycobacterium avium subsp. paratuberculosis growth in an automated broth culture system.

Three methods of harvesting DNA from broth culture tubes for quantitative real-time polymerase chain reaction (qrtPCR) confirmation of Mycobacterium avium subsp. paratuberculosis (MAP) were evaluated. A commercial DNA extraction kit, the boil method (boiling for 5 minutes), or direct addition of broth culture media to the PCR reaction mix were tested. Samples were evaluated at 8 or 11 days of incubation and at the time of instrument-signal culture-positive. In total, when tested at time to instrument signal positive, 10/10 (100%) of samples extracted by the commercial method were positive on qrtPCR, whereas 9/10 (90%) were positive after the boil method, and 6/10 (60%) were positive after the direct method. Increased volumes of egg-yolk emulsion added to the culture tubes prolonged the number of cycles to threshold positive for the samples that were not subjected to commercial extraction or boiling. Samples were not reliably positive when tested at 8 or 11 days of incubation. The boil method appears to represent a reasonable time- and money-saving method to harvest DNA for qrtPCR confirmation of MAP in broth culture at time to instrument signal positive.

Analysis of Variance↗
PubMed1993

Reproducibility of a commercial enzyme-linked immunosorbent assay for bovine paratuberculosis among eight laboratories.

Interlaboratory reproducibility of an absorbed enzyme-linked immunosorbent assay (ELISA) kit for detection of bovine serum antibodies to Mycobacterium paratuberculosis was evaluated. A panel of 30 bovine sera (15 positives and 15 negatives) was tested in triplicate microtiter wells on each of 2 days at 8 different laboratories. One laboratory had invalid results because of positive or negative serum control optical density (OD) readings beyond the acceptable range specified by the kit. The coefficient of variation (CV) for mean OD values was influenced by low ODs on test negative sera at 2 laboratories, thus the CVs on positive sera were considered a more representative measure of kit reproducibility. Between-well CVs averaged 6.7% +/- 2.8% (mean +/- standard deviation), and between-day CVs averaged 14.5% +/- 9.8% among the 7 laboratories with valid assays on the 15 positive sera. The OD values were converted to positive or negative classifications for each assay well, and the results were compared. Among 1,392 assays in 7 laboratories, 98.6% were in agreement. Eleven of 18 discrepant results were due to a sample that consistently gave OD values near the cutoff for a positive test. Exclusion of that serum from the analysis resulted in a 99.8% rate of agreement among laboratories. Results indicated that the absorbed ELISA kit provided reproducible results within and between laboratories.

Analysis of Variance↗
PubMed1999

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 Löwenstein-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.

Age Factors↗
PubMed1999

Mycobacterium paratuberculosis detection in bovine feces is improved by coupling agar culture enrichment to an IS900-specific polymerase chain reaction assay.

The feasibility of coupling an agar culture enrichment step with gene amplification (ACE-PCR) as a means to improve turnaround time and detect Mycobacterium paratuberculosis (Mpt) in the presence of contaminants was investigated. Fecal samples from 463 Pennsylvania dairy cows were cultured in duplicate sets. One replicate from each set was processed and interpreted according to standard culture (SC) protocol, whereas cultures from the second replicate were harvested at 6 weeks postinoculation; DNA extracts from the harvested material were evaluated by a polymerase chain reaction (PCR) test for the Mpt-specific IS900 gene. One hundred seventy-six of 463 culture sets were positive by either method. One hundred sixty-five of these (94%) were ACE-PCR positive, and 151 (86%) were positive by SC. Eleven SC-positive samples were ACE-PCR negative, and 9 ACE-PCR-positive samples were negative by SC; these discrepancies could be a consequence of a low organism burden (< or = 5 organisms/g) or slow growth rate of Mpt in cultures of these samples. One hundred thirty-nine of 463 culture sets (30%) were reported as inconclusive because of culture contamination according to SC protocol; 16 of these (11.5%) were ACE-PCR positive. Seventy-four ACE-PCR-positive sets (42% of all positives) were negative or inconclusive by SC at 6 weeks postinoculation. Agar culture enrichment prior to IS900 PCR testing significantly improves Mpt culture turnaround time and sensitivity.

Agar↗
PubMed2002

Detection of Mycobacterium avium subspecies paratuberculosis specific IS900 insertion sequences in bulk-tank milk samples obtained from different regions throughout Switzerland.

BACKGROUND: Since Mycobacterium avium subspecies paratuberculosis (MAP) was isolated from intestinal tissue of a human patient suffering Crohn's disease, a controversial discussion exists whether MAP have a role in the etiology of Crohn's disease or not. Raw milk may be a potential vehicle for the transmission of MAP to human population. In a previous paper, we have demonstrated that MAP are found in raw milk samples obtained from a defined region in Switzerland. The aim of this work is to collect data about the prevalence of MAP specific IS900 insertion sequence in bulk-tank milk samples in different regions of Switzerland. Furthermore, we examined eventual correlation between the presence of MAP and the somatic cell counts, the total colony counts and the presence of Enterobacteriaceae. RESULTS: 273 (19.7%) of the 1384 examined bulk-tank milk samples tested IS900 PCR-positive. The prevalence, however, in the different regions of Switzerland shows significant differences and ranged from 1.7% to 49.2%. Furthermore, there were no statistically significant (p >> 0.05) differences between the somatic cell counts and the total colony counts of PCR-positive and PCR-negative milk samples. Enterobacteriaceae occur as often in IS900 PCR-positive as in PCR-negative milk samples. CONCLUSION: This is the first study, which investigates the prevalence of MAP in bulk-tank milk samples all over Switzerland and infers the herd-level prevalence of MAP infection in dairy herds. The prevalence of 19.7% IS900 PCR-positive bulk-milk samples shows a wide distribution of subclinical MAP-infections in dairy stock in Switzerland. MAP can therefore often be transmitted to humans by raw milk consumption.

Animals↗
PubMed2006

New PCR systems to confirm real-time PCR detection of Mycobacterium avium subsp. paratuberculosis.

BACKGROUND: Johne's disease, a serious chronic form of enteritis in ruminants, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). As the organism is very slow-growing and fastidious, several PCR-based methods for detection have been developed, based mainly on the MAP-specific gene IS900. However, because this gene is similar to genes in other mycobacteria, there is a need for sensitive and reliable methods to confirm the presence of MAP. As described here, two new real-time PCR systems on the IS900 gene and one on the F57 gene were developed and carefully validated on 267 strains and 56 positive clinical faecal samples. RESULTS: Our confirmatory PCR systems on IS900 were found sensitive and specific, only yielding weak false positive reactions in one strain for each system. The PCR system on F57 did not elicit any false positives and was only slightly less sensitive than our primary IS900-system. DNA from both naturally infected and spiked faeces that tested positive with our primary system could be confirmed with all new systems, except one low-level infected sample that tested negative with the F57 system. CONCLUSION: We recommend using the newly constructed DH3 PCR system on the F57 gene as the primary confirmatory test for PCR positives, but should it fail due to its lower sensitivity, the DH1 and DH2 PCR systems should be used.

Animals↗
PubMed2001

Economic aspects of disease monitoring with special reference to bovine paratuberculosis.

Monte Carlo simulation models were used to evaluate the feasibility and potential results of a proposed national survey of the prevalence of bovine paratuberculosis (PTB) in dairy herds in Norway. The expected herd prevalence was assumed to be 0.2% in the simulations. Infected herds were classified as detected if 1 animal was sero-positive. With a sample size of 6000 herds at least 1 truly infected herd was detected in 99% of the iterations. The low sensitivity of the ELISA test, the assumed low herd prevalence, the typical low within-herd prevalence of PTB and the small herd sizes in Norway all present problems in detection of the disease. The results showed that the ratio between false-positive herds and true positive herds detected had a median of 70:1. At the assumed herd prevalence of 0.2% and a cost/test of 70 NOK the median cost of detecting 1 infected herd was approximately 900,000 NOK. If 2 positive reactors were needed to classify a herd "infected" the median cost of detecting 1 infected herd was 5,055,000 NOK. Our results suggest that a randomized national prevalence survey would not be feasible, due to the low probability of detecting infected herds and because of the high number of false-positive reactions that would be expected.

Animals↗
PubMed2006

Seroprevalence of Mycobacterium avium subspecies paratuberculosis in Korean black goats (Capra hircus aegagrus).

In total, 582 sera from 116 black goat herds were analyzed by a commercially available ELISA kit to monitor the seroprevalence of Mycobacterium avium subspecies paratuberculosis (Mpt) in Korean black goats (Capra hircus aegagrus). The mean number of goats sampled per herd was 5.11, 4.66, and 5.38 for the northern, central, and southern regions of Korea, respectively. The apparent regional prevalence of Mpt was estimated at 18.2-38.2% and 4.6-15.3% for herds and goats, respectively. The Mpt-positive goats were predominantly detected in the south (n=28), compared to either the northern (n=9) or central (n=11) regions (chi=14.459, P<0.05). Our findings indicate that Mpt is prevalent among the goat population, but regional variation exists.

Animals↗
PubMed2006

Pathology of naturally occurring paratuberculosis in water buffaloes (Bubalus bubalis).

Gross and histologic lesions of paratuberculosis were studied in water buffaloes. Small intestines and associated mesenteric lymph nodes of 405 water buffaloes were examined. Of these, 20 animals having visible changes of intestinal thickening, mucosal corrugations, and enlargement of mesenteric lymph nodes exhibited histologic alteration characteristics of mild to moderate granulomatous inflammation. The histologic lesions observed in these animals were classified into 3 grades on the basis of type of cellular infiltration, granuloma formation, and presence of acid-fast bacilli. Grade-1 lesions observed in 8 animals were marked by the presence of scattered epithelioid macrophages amid large number of lymphocytes in the intestinal villi and in the paracortical regions of the associated mesenteric lymph nodes. Another 8 animals classified under grade-2 revealed microgranulomas, infiltration with a larger number of epithelioid macrophages besides lymphocytes in the intestinal villi, and granulomas in the mesenteric lymph nodes. Grade-3 lesions observed in 4 animals were characterized by the presence of epithelioid granulomas and giant cells in the intestines and the mesenteric lymph nodes. The Ziehl-Neelsen's stained tissue sections revealed acid-fast bacilli in grade-3 and -2 animals and acid-fast granular debris in grade-1 animals. Among these 20 buffaloes, 14 (70%) were positive in the IS900 specific polymerase chain reaction and 6 (30%) were positive in the bacterial culture.

Animals↗