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PubMed1999

Contribution to the diagnosis of Johne's disease in cattle. Comparative studies on the validity of Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test in detecting Mycobacterium paratuberculosis in faeces from cattle.

In the present study, 132 selected faecal samples from clinically affected and subclinically infected cattle from dairy herds known to be affected by Johne's disease were investigated for the presence of Mycobacterium paratuberculosis using Ziehl-Neelsen staining, faecal culture and a commercially available DNA-Probe test. The sensitivity was 36.4% for Ziehl-Neelsen staining, 85.6% for faecal culture and 47.7% for the DNA-Probe test. Proving the presence of acid-fast bacteria in 49.3% of the samples from clinically affected cattle and 19.3% of those from subclinically infected cattle, Ziehl-Neelsen staining had the lowest detection rate of the three tests under investigation. Faecal culture showed the highest detection rate of M. paratuberculosis in samples from both clinically affected (84.0%) and subclinically infected (87.7%) animals. The DNA-Probe test showed a positive result in 68.0% of the samples from clinically affected cattle and 21.1% of those from subclinically infected cattle. Ziehl-Neelsen staining proved unreliable in diagnosing Johne's disease. Faecal culture was the most sensitive method for detecting M. paratuberculosis both in clinically affected and subclinically infected cattle. The sensitivity of a commercially available DNA-Probe test has to be enhanced to enable a quick and reliable diagnosis of Johne's disease.

Animals↗
PubMed1992

Induction of cellular immune reactions by A36, an antigen complex of Mycobacterium paratuberculosis: comparison of A36 and johnin components.

Paratuberculosis (Johne's disease) is a chronic enteritis syndrome of ruminants, which is due to infection by Mycobacterium paratuberculosis. Cutaneous testing with proteins extracted from a mycobacterial culture fluid (johnin-PPD) is currently used to evaluate the cellular immune status. We have compared the components of johnin-PPD with those of the A36 complex, a thermostable macromolecular antigen (TMA) present in the cytoplasm and associated with the cell wall of M. paratuberculosis. The presence in the johnin-PPD of fifteen A36 components has been shown by Western blotting. Moreover, monoclonal antibodies, which bind respectively to the 65-kDa M. leprae heat shock protein, the 28-kDa M. leprae superoxide dismutase, and M. tuberculosis lipoarabinomannan, recognized components of the johnin-PPD. The ability of A36 to trigger delayed hypersensitivity reactions in sensitized rabbits, and to induce the proliferation of T lymphocytes from the lymph nodes of A36-sensitized mice, matched that of johnin-PPD. The homology levels of T epitopes between A36 and the TMA complexes of M. phlei, M. bovis, M. tuberculosis and M. avium were estimated, in a lymphoproliferation assay, to be 51, 52, 59 and 94% respectively. A strong cross-reactivity of A36 with an M. leprae sonicate was also observed by cutaneous testing. The A36 components within the 45.2-26.8-kDa and the 21.6-19.8-kDa ranges were proved to induce the proliferation of T lymphocytes from sensitized mice. This work supports the possible use of the A36 complex, and of some of its components, for cutaneous tests and lymphocyte proliferation assays, in order to monitor cellular immunity in Johne's disease.

Animals↗
PubMed2001

Isolation and diagnostic potential of ISMav2, a novel insertion sequence-like element from Mycobacterium avium subspecies paratuberculosis.

A novel Mycobacterium avium ssp. paratuberculosis (M. paratuberculosis) specific insertion sequence has been identified by representational difference analysis and designated as ISMav2. ISMav2 has no similarity to known mycobacterial IS elements but shows more than 50% identity to a non-composite transposon of Streptomyces coelicolor at the DNA and protein level. ISMav2 is present in at least three copies on the genome as assessed by Southern blot analysis and its potential value as a diagnostic tool was confirmed by PCR analyses on 79 M. paratuberculosis field isolates, nine M. avium ssp. avium isolates, and the reference strains of nine other mycobacterial species.

Base Sequence↗
PubMed2002

Putative in vitro expressed gene fragments unique to Mycobacterium avium subspecies paratuberculosis.

By a suppression subtractive hybridization based method, nine novel Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) fragments of between 318 and 596 bp have been identified and characterized. Database search revealed little or no similarity with other mycobacteria. The uniqueness and diagnostic potential of seven of these fragments in relation to M. paratuberculosis closest relative Mycobacterium avium subsp. avium (M. avium) was confirmed by species-specific PCR and Southern blot. Furthermore, RT-PCR indicated that eight of the nine fragments originate from areas of the genome that are expressed in vitro.

Blotting, Southern↗
PubMed1993

Search for Mycobacterium paratuberculosis DNA in tissue from patients with sarcoidosis by enzymatic gene amplification.

The etiology of sarcoidosis has not yet been established, but several mycobacterial species have been implicated in the pathogenesis of this disease. We have used a nested primer polymerase chain reaction (PCR) specific for the IS900 Mycobacterium paratuberculosis insertion element to analyse paraffin-embedded mediastinal lymphoid tissue from 18 patients with pulmonary sarcoidosis verified by mediastinoscopy. Only the positive control contained M. paratuberculosis-specific DNA. The present study does not support a role for M. paratuberculosis in the pathogenesis of sarcoidosis. However, further studies are needed in order to clarify the possible role of other mycobacteria in this disease.

Adolescent↗
PubMed1986

A reappraisal of the complement fixation test using soluble Mycobacterium avium antigen for the detection of M. paratuberculosis infection in cattle.

Serums from 263 cattle suspected of having paratuberculosis on the basis of clinical signs, were tested for antibodies to Mycobacterium paratuberculosis with a complement fixation test (CFT) employing a heat extracted, soluble M. avium antigen. Microscopic examination confirmed that 172 (65.4%) clinically affected animals had paratuberculosis, the remainder being disease-free. The specificity and sensitivity of the CFT was 92.3% and 74.4% respectively. Phenol treatment of serums before testing was compared with no treatment and was found to have no significant effect on the CFT titres. Results obtained are discussed in relation to the cause of false negative and false positive reactions.

Animals↗
PubMed2001

Effect of turbulent-flow pasteurization on survival of Mycobacterium avium subsp. paratuberculosis added to raw milk.

A pilot-scale pasteurizer operating under validated turbulent flow (Reynolds number, 11,050) was used to study the heat sensitivity of Mycobacterium avium subsp. paratuberculosis added to raw milk. The ATCC 19698 type strain, ATCC 43015 (Linda, human isolate), and three bovine isolates were heated in raw whole milk for 15 s at 63, 66, 69, and 72 degrees C in duplicate trials. No strains survived at 72 degrees C for 15 s; and only one strain survived at 69 degrees C. Means of pooled D values (decimal reduction times) at 63 and 66 degrees C were 15.0 +/- 2.8 s (95% confidence interval) and 5.9 +/- 0.7 s (95% confidence interval), respectively. The mean extrapolated D72 degrees C was <2.03 s. This was equivalent to a >7 log10 kill at 72 degrees C for 15 s (95% confidence interval). The mean Z value (degrees required for the decimal reduction time to traverse one log cycle) was 8.6 degrees C. These five strains showed similar survival whether recovery was on Herrold's egg yolk medium containing mycobactin or by a radiometric culture method (BACTEC). Milk was inoculated with fresh fecal material from a high-level fecal shedder with clinical Johne's disease. After heating at 72 degrees C for 15 s, the minimum M. avium subsp. paratuberculosis kill was >4 log10. Properly maintained and operated equipment should ensure the absence of viable M. avium subsp. paratuberculosis in retail milk and other pasteurized dairy products. An additional safeguard is the widespread commercial practice of pasteurizing 1.5 to 2 degrees above 72 degrees C.

Animals↗
PubMed1998

Identification and characterization of a novel extracellular ferric reductase from Mycobacterium paratuberculosis.

A novel extracellular mycobacterial enzyme was identified in the ruminant pathogen Mycobacterium paratuberculosis. The enzyme was capable of mobilizing iron from different sources such as ferric ammonium citrate, ferritin, and transferrin by reduction of the metal. The purified reductase had a calculated Mr of 17,000, was sensitive to proteinase K treatment, and had an isoelectric point of pH 9. Analysis of the amino acid composition revealed glycine, serine, asparagine (or aspartic acid), and glutamine (or glutamic acid) as the most frequently occurring residues. Enzymatic activity was highest at 37 degrees C and between pH 5 and 10. The calculated Km and Vmax for ferric ammonium citrate were 0.213 mM and 0.345 mM min(-1) mg(-1), respectively. Using a specific antireductase antibody in immunoelectron microscopy, we were able to detect the enzyme associated with intracellular mycobacteria in naturally M. paratuberculosis-infected bovine tissue. We prepose that the reductase of M. paratuberculosis represents an alternative strategy of mycobacteria to mobilize ferric iron and discuss its potential role in bacterial evasion of intracellular defense mechanisms.

Animals↗
PubMed2003

Enhanced expression of interleukin-1alpha and tumor necrosis factor receptor-associated protein 1 in ileal tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis.

Infection with Mycobacterium avium subsp. paratuberculosis is associated with high levels of morbidity, decreased production, and early culling in dairy cattle. Clinical symptoms of Johne's disease include persistent diarrhea, inappetence, and resultant weight loss due to chronic inflammation of the small intestine. Although the presence or absence of intestinal lesions cannot be used as a definitive indicator of M. avium subsp. paratuberculosis infection, most infected cattle exhibit significant changes to intestinal mucosa, with the focus of pathology surrounding the ileal cecal junction. Typical pathology of M. avium subsp. paratuberculosis infection includes inflammation, thickening of the lumenal wall, and hyperplasia in draining lymph nodes. To further understand the pathology of Johne's disease, we compared the gene expression profiles of ileal tissues from Johne's disease-positive (n = 6), and Johne's disease-negative (n = 5) Holstein cattle. Gene expression profiles were compared with a bovine total leukocyte (BOTL-3) cDNA microarray. Genes that were expressed at significantly higher levels (>1.5-fold; P < 0.05) in tissues from Johne's disease-infected animals relative to noninfected animals included those encoding tumor necrosis factor receptor-associated protein 1 (TRAF1), interleukin-1alpha (IL-1alpha), MCP-2, N-cadherin, and beta1 integrin (CD29). Dramatic upregulation of IL-1alpha (21.5-fold) and TRAF1 (27.5-fold) gene expression in tissues of Johne's disease-positive cows relative to tissues from control cows was confirmed by quantitative real-time PCR. Western blot analysis confirmed that IL-1alpha and TRAF1 mRNA levels resulted in increased protein expression in tissues of Johne's disease-positive cattle relative to tissues from control cattle. High levels of IL-1alpha can produce symptoms similar to those found in clinical Johne's disease. Taken together, the data presented in this report suggest that many outward symptoms of Johne's disease may be due to IL-1alpha toxicity. In addition, enhanced levels of TRAF1 could result in cells within the lesions of Johne's disease-positive cattle that are highly resistant to TNF-alpha-induced signaling.

Animals↗
PubMed1985

Isolation of Mycobacterium paratuberculosis from intestinal mucosa and mesenteric lymph nodes of goats by use of selective Dubos medium.

To isolate Mycobacterium paratuberculosis from contaminated material, a selective medium, selective Dubos medium (SDubos), was developed by supplementing conventional Dubos medium (CDubos) with carbenicillin, polymyxin, trimethoprim, and amphotericin B. The intestine and mesenteric lymph nodes of 1,501 goats were cultured in parallel on SDubos and CDubos after decontamination with oxalic acid. The contamination rate was reduced more than 150 times by the use of SDubos. The number of positive specimens (18, or 1.2%), i.e., those which revealed growth of M. paratuberculosis, was too small to evaluate the number of specimens which could have been missed due to contamination on CDubos. However, the number of specimens positive on SDubos (16, or 89%) showed that the antibiotics used were not harmful to M. paratuberculosis.

Animals↗
PubMed1987

Crohn's disease-isolated mycobacteria are identical to Mycobacterium paratuberculosis, as determined by DNA probes that distinguish between mycobacterial species.

DNA extracted from an unclassified Crohn's disease-isolated Mycobacterium strain was cloned. The recombinant clones were radiolabeled and hybridized to restriction digests of mycobacterial DNA transferred to nylon membranes. Restriction fragment length polymorphisms (RFLPs) were identified that distinguished between mycobacterial DNA samples. Quantitative estimates of frequencies of DNA base substitution were also obtained. No RFLPs were detected between the DNA of three unclassified Crohn's disease-isolated mycobacteria and Mycobacterium paratuberculosis, although several RFLPs were detected that distinguished between M. paratuberculosis and both M. avium complex serovars 2 and 5. The frequency of DNA base substitution between M. paratuberculosis and M. avium complex serovar 2 was measured as 0.87 (+/- 1.2)%.

Cloning, Molecular↗
PubMed1991

Gen-Probe Rapid Diagnostic System for the Mycobacterium avium complex does not distinguish between Mycobacterium avium and Mycobacterium paratuberculosis.

Three reference and 16 field strains of Mycobacterium paratuberculosis were tested with the Gen-Probe Mycobacterium avium complex DNA probe (Gen-Probe Inc., San Diego, Calif.). All reference strains and 12 of 16 field strains gave positive hybridization results with the probe. This study shows that the M. avium complex probe does not distinguish between M. avium and M. paratuberculosis and indicates heterogeneity in the 16S rRNA gene of M. paratuberculosis.

Animals↗
PubMed1977

Effect of freezing on the viability of Mycobacterium paratuberculosis in bovine feces.

Three bovine fecal specimens were cultured for Mycobacterium paratuberculosis before freezing and after frozen storage at -70 degrees C for 3 and 15 weeks. The losses in viability from 0 to 3 weeks of storage were significant (P = 0.01) for all three samples. The losses in viability between 3 and 15 weeks of storage were not significant (P greater than 0.05) for two specimens in which the M. paratuberculosis occurred naturally, but the loss was significant (P = 0.01) in a simulated specimen (M. paratuberculosis culture added to feces from a healthy cow).

Animals↗
PubMed1987

Economic losses due to paratuberculosis in dairy cattle.

The results of a study of the economic losses caused by paratuberculosis in dairy cattle are reported. The losses in production and the determination of lost future income due to premature disposal are emphasised. A decrease in milk production of 19.5 per cent compared with the lactation two years before culling was recorded in animals showing clinical signs of paratuberculosis. The decrease in production in the last lactation but one compared with the previous lactation was 5 per cent. In animals with non-clinical forms of paratuberculosis these decreases in production were 16 per cent and 6 per cent, respectively.

Age Factors↗
PubMed2001

Presence of Mycobacterium avium subspecies paratuberculosis in suspensions of ovine trichostrongylid larvae produced in faecal cultures artificially contaminated with the bacterium.

A reference strain of Mycobacterium avium subspecies paratuberculosis was added to faecal larval cultures of Haemonchus contortus, Ostertagia circumcincta and Trichostrongylus colubriformis. Samples of the larvae produced were cultured for the presence of the bacterium in modified BACTEC 12B medium, both before and after exposure to gamma irradiation. The water used to wash the larvae off the faecal cultures was also tested for the presence of the bacterium. Positive growth was confirmed as M. avium subspecies paratuberculosis by IS900 polymerase chain reaction and restriction endonuclease analysis of the product. M. avium subspecies paratuberculosis was detected in the unirradiated larval suspensions and wash waters of all three nematode species, and in the irradiated H. contortus larval suspension.

Animals↗
PubMed1978

Pathological evaluation of paratuberculosis in naturally infected cattle.

Thirty-two of 51 cattle infected with Mycobacterium paratuberculosis had chronic enteritis, chronic lymphangitis or mesenteric lymphadenopathy, or all three, at slaughter. Granulomatous inflammatory lesions were mild to advanced and predominantly involved the distal small intestine. Rectal involvement was seen only in five cattle. Fourteen had microgranulomas in the liver. There were three cytological forms of macrophages: histiocytic, polygonal and epithelioid. The latter two types had engulfed moderate numbers of acid-fast bacilli. The histiocytic macrophages usually were packed with acid-fast bacilli. Except in the liver and occasionally its nodes, remote lesions of paratuberculosis were not found in other organs. One animal had endocardial and aortic calcifications. Most cattle with signs of diarrhea had globule leukocytes in or around myenteric ganglion cells. The thymus of 3- to 8-year-old cattle with clinical signs frequently had mild to advanced involution. The thymus of similarly aged infected animals without clinical signs, and of paratuberculosis-negative animals, had not involuted.

Animals↗
PubMed1993

Use of a dot enzyme-linked immunosorbent assay on absorbed sera for the diagnosis of bovine paratuberculosis.

This study describes the response of cattle to a dot enzyme-linked immunosorbent assay (ELISA) using sera absorbed with Mycobacterium phlei. Results obtained by visual observation are compared with those obtained using a densitometer. Infection status of cattle was determined by faecal culture. Cattle of different levels of exposure and disease manifestation were examined. A significantly higher dot ELISA response was observed (using both absorbed and non-absorbed sera) in animals with heavy shedding of M. paratuberculosis than in animals which tested negative by faecal culture or shed M. paratuberculosis at lower levels (P < 0.05). Paratuberculosis was diagnosed by visual determination of dot ELISA results using non-absorbed sera in 29 of 44 (65.9%) clinically-suspect animals giving positive results by faecal culture, and 85 of 93 (91.4%) cattle testing negative by faecal culture. With absorbed sera, the sensitivity of visual determination decreased to 15 of 44 (34.1%), while specificity increased to 91 of 93 (97.8%). Approximately 75% of cattle yielding positive results by dot ELISA were heavy bacterial shedders (> 1,500 colonies/g of faeces) at the time of serological testing. Comparison of the dot ELISA results determined visually with results obtained by objective densitometric measurement showed compatible specificity. Sensitivity of the dot ELISA was 65.9% for non-absorbed sera using visual evaluation and 87.5% using densitometric evaluation at a cut-off optical density value of 0.2. For absorbed sera, the values were 34.1% and 82.5%, respectively.

Absorption↗
PubMed2002

Cell sorting of formalin-treated pathogenic Mycobacterium paratuberculosis expressing GFP.

GFP is widely used as a molecular tool for the study of microbial pathogens. However, the manipulation of these pathogenic microorganisms poses a health threat to the laboratory worker, requiring biosafety level II or III containment. Although the GFPfluorophore is tolerant toformalin, a thorough analysis of this treatment on fluorescent output in prokaryotic systems has not been described. In addition, the analysis of microorganisms expressing GFP often depends on specialized equipment, which may not be housed in biosafety level II or III laboratories. Therefore, we sought to develop a safe and effective method for manipulating the GFP-expressing pathogenic bacterium Mycobacterium avium subsp, paratuberculosis (M. paratuberculosis) utilizing a formalin treatment that would permit the analysis of GFP fluorescence without requiring stringent biosafety containment. We demonstrate that formalin-treated M. paratuberculosis expresses 50% less fluorescence than viable cells, but this reduction is still compatible with spectrofluorometry and cell sorting. Furthermore, plasmid DNA that expresses GFP can be recovered efficiently from nonviable, sorted fluorescent cells. This approach is flexible, provides an additional margin of safety for laboratory personnel, and can be easily applied to other pathogenic microorganisms expressing GFP.

Blotting, Southern↗