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PubMed1985

Characterization of Mycobacterium paratuberculosis of bovine, caprine, and ovine origin by gas-liquid chromatographic analysis of fatty acids in whole-cell extracts.

The cellular fatty acid composition of Mycobacterium paratuberculosis from bovine, ovine, and caprine origin (grown on Herrold's egg yolk medium) was determined by gas-liquid chromatography (GLC) and mass spectrometry and was compared with that of 19 other Mycobacterium spp. A species-specific fatty acid of M paratuberculosis was not demonstrable. Gas-liquid chromatographic analysis of cellular fatty acids was used to develop flow charts for the rapid identification of M paratuberculosis and other Mycobacterium spp. Two charts for distinguishing organisms were developed; one chart was based on GLC alone and the other based on GLC, growth rate, and chromogenicity. Seemingly, the GLC was reliable for detecting Mycobacterium spp rapidly.

Animals↗
PubMed1985

Lymphocyte blastogenesis, complement fixation, and fecal culture as diagnostic tests for paratuberculosis in North American wild ruminants and domestic sheep.

The efficacy of the lymphocyte blastogenesis and complement-fixation tests and fecal culture for detection of Mycobacterium paratuberculosis infection was assessed in bighorn sheep (Ovis canadensis), elk (Cervus elaphus nelsoni), mule deer (Odocoileus hemionus), white-tailed deer (O virginianus), bighorn X mouflon (O musimon) hybrid sheep, and domestic sheep. Spontaneously infected bighorns were tested at the time of capture; experimentally infected animals were tested monthly for 12 months or periodically for 36 months. Lymphocyte blastogenesis tests were conducted with peripheral blood mononuclear cells and protein antigens of M avium, M bovis, and M paratuberculosis. Best diagnostic results were obtained when M avium purified-protein derivative was used as antigen and 20% bovine fetal serum was incorporated in the culture medium; a positive test was defined as a stimulation index greater than or equal to 3.5. Test sensitivity and specificity, respectively, were 82% and 94% in hybrid sheep and were 72% and 100% in domestic sheep. Sensitivity and specificity, respectively, were 39% and 94% in elk and 53% and 92% in deer. When infection was determined in spontaneously infected bighorns by culture of M paratuberculosis and/or the presence of acid-fast bacilli in characteristic microscopic lesions, sensitivity was 75% and specificity was 87%. Fecal cultures and the complement-fixation tests seldom correctly identified infected animals.

Animals↗
PubMed1977

Application of the direct leukocyte-migration agarose test in cattle from a Mycobacterium paratuberculosis-infected herd.

Using johnin as the specific antigen, the direct leukocyte-migration agarose test was applied to 10 cows from a Mycobacterium paratuberculosis-infected herd. Nine of the cows did not have signs of paratuberculosis; 1 cow was clinically affected. The cows were slaughtered and, on the basis of histopathologic and cultural examinations of the intestines, were classified as infected or noninfected. Six of the healthy cows from which the bacterium was not isolated and the clinically affected cow reacted with migration indices lower than those of 3 healthy animals that were found to harbor the bacterium. It is suggested that the long incubation period of paratuberculosis could to some extent be due to immunologic tolerance.

Animals↗
PubMed1983

Eastern white-tailed deer as a reservoir of ruminant paratuberculosis.

Ten white-tailed deer were shot and killed at a Connecticut farm that had a 6-year history of bovine paratuberculosis, and organs from these animals were examined for evidence of paratuberculosis. Mycobacterium paratuberculosis was isolated from the cecal lymph node, terminal ileum, and ileocecal valve of 1 deer and from the cecal lymph node of another. Characteristic lesions and acid-fast bacilli were not observed. It was concluded that infected deer could serve as sources of infection for domestic stock.

Animals↗
PubMed1981

Bovine paratuberculosis III. An evaluation of a whole blood lymphocyte transformation test.

A whole blood lymphocyte transformation test was used to examine cattle with varying degrees of infection with Mycobacterium paratuberculosis. Minimally infected animals characteristically responded to johnin purified protein derivative in the lymphocyte transformation test but did not routinely react on serological and/or skin testing. Heavily infected animals showed considerable variation in their lymphocyte transformation responses to antigen and some of them were consistently unresponsive. Antigen induced lymphocyte transformation reactions were recorded in 7.6 to 41.5% of uninfected animals whose infection status was determined by bacteriology and/or histopathology. The number of positive reactions recorded in uninfected animals depended on the population, the larger percentage being found in a herd with a proven history of paratuberculosis. The potential of lymphocyte transformation as a diagnostic test for bovine paratuberculosis is discussed.

Animals↗
PubMed1978

Effect of dexamethasone on Mycobacterium paratuberculosis infection in hamsters.

Sixty hamsters were randomly divided into 4 equal groups: group 1--given only dexamethasone, group 2--given dexamethasone and exposed to Mycobacterium paratuberculosis, group 3--exposed to M paratuberculosis but not treated, and group 4--used as normal controls. Group 2 hamsters (treated with dexamethasone and exposed to the bacterium) had significantly higher bacterial counts in spleen and aggregated lymphatic nodules (Peyser's patches) than did those in group 3. None of the hamsters exposed to M paratuberculosis (groups 2 and 3) developed clinical signs of disease. All hamsters treated with the drug (groups 1 and 2) grew more slowly than did the hamsters not treated.

Animals↗
PubMed1977

Application of the direct leukocyte-migration agarose test in cattle naturally infected with Mycobacterium paratuberculosis.

Four cows with persistent diarrhea and complement-fixing antibodies to johnin were examined, using the leukocyte-migration agarose test (LMAT) with johnin as the specific antigen. The diagnosis of paratuberculosis (Johne's disease) was microscopically verified at necropsy. As control animals, 3 healthy cows and 3 cows with disease other than paratuberculosis were examined, using LMAT. The migration indices in the group of cows with paratuberculosis were significantly lower (less than 0.001) than those in the group of control cows. Furthermore, the migration zones of the unstimulated peripheral blood leukocytes in the former group were significantly smaller (less than 0.001) than those of the control group.

Animals↗
PubMed1985

Comparison of the pathogenicity of Mycobacterium paratuberculosis and Mycobacterium spp isolated from the wood pigeon (Columba palumbus-L).

The pathogenicity of 4 strains of mycobacteria isolated from wood pigeons and 2 strains of Mycobacterium paratuberculosis was compared in calves, mice and chickens. Three of the 4 wood pigeon strains and the 2 M. paratuberculosis strains produced clinical Johne's disease in calves. All 6 strains were pathogenic for mice and the 4 wood pigeon strains were pathogenic for chickens. The strains were not agglutinated by antisera to strains of the M. avium complex and all were mycobactin-dependent. It was concluded that the wood pigeon isolates may constitute a distinct group with the pathogenic capability of M. avium in chickens and M. paratuberculosis in calves.

Animals↗
PubMed1990

Effects of colonial morphology and tween 80 on antimicrobial susceptibility of Mycobacterium paratuberculosis.

Smooth (Sm) and rough (Rg) colonial types of Mycobacterium paratuberculosis ATCC 19698 and two clinical isolates were tested to examine their growth responses in medium containing antimicrobial agents. Susceptibility tests were done in Middlebrook 7H12B medium with and without Tween 80 and one of the following antimicrobial agents: streptomycin, isoniazid, rifampin, ethambutol, ciprofloxacin, and penicillin G. Growth responses in the presence of antimicrobial agents led to the following observations. (i) In the absence of Tween, Rg colony types were more resistant than Sm colony types; (ii) the addition of Tween 80 significantly increased the susceptibility of both Sm and Rg colony types; however, the increase was greater with the Sm colony types. These studies showed that the antimicrobial susceptibility of M. paratuberculosis was significantly affected when Tween 80 was present in either the primary culture medium or the drug susceptibility test medium. In the absence of the perturbing influence of Tween 80, M. paratuberculosis was resistant to the antimicrobial agents tested.

Anti-Bacterial Agents↗
PubMed1983

Iron-binding compounds of Mycobacterium avium, M. intracellulare, M. scrofulaceum, and mycobactin-dependent M. paratuberculosis and M. avium.

Fifty-three strains of M. avium and related species all produced one or more exochelins, the extracellular iron-binding compounds of the mycobacteria, when grown iron deficiently. Only those strains which could grow without the addition of mycobactin (i.e., mycobactin independent) produced mycobactin, the intracellular iron-binding compound of the mycobacteria. Exochelins varied from 20 to 2,000 micrograms per g of cell dry weight; mycobactins were between 1 and 10 mg per g of cell dry weight. M. paratuberculosis (13 strains) and 13 strains of M. avium, both species dependent upon mycobactin for growth, failed to produce spectrophotometrically detectable amounts of mycobactin (less than 0.2 microgram per g of cell dry weight), although mycobactin could be recognized in one strain of M. avium grown with an additional supply of salicylate and examined by a radiolabeling technique. On repeated subculture three of the mycobactin-dependent strains of M. avium, but none of those of M. paratuberculosis, lost their mycobactin dependence and on reexamination were found to produce their own mycobactin at 0.3 mg per g of cell dry weight. It is concluded that mycobactin biosynthesis is probably strongly repressed in the mycobactin-dependent strains rather than being a genetic deletion. The exochelins, when examined by high-pressure thin-layer chromatography were revealed as being multiples of similar compounds, with up to 20 individual iron-binding compounds being recognizable with some strains. It is argued that the exochelins represent the single most important means of iron acquisition in mycobacteria growing in vitro and in vivo, and their elaboration by the fastidious M. paratuberculosis and related species explains how these organisms are able to grow in vivo in the absence of an external supply of mycobactin.

Chromatography, Thin Layer↗
PubMed1990

T-cellular immune reactions (in macrophage inhibition factor assay) against Mycobacterium paratuberculosis, Mycobacterium kansasii, Mycobacterium tuberculosis, Mycobacterium avium in patients with chronic inflammatory bowel disease.

A mycobacterial aetiology has been suggested for Crohn's disease. A slow growing mycobacterium, biochemically and genetically identical to M paratuberculosis, the causative agent of enteritis in ruminants (Johne's disease), has been isolated from gut specimens of patients affected by Crohn's disease. If M paratuberculosis or other mycobacteria play a role in the pathogenesis of Crohn's disease, then patients may have been sensitised to these mycobacteria or show an anergy immune reaction. We therefore investigated the T-cell mediated immune response to sonicates of M paratuberculosis, M kansasii, M avium, and M tuberculosis in 35 patients with Crohn's disease, 28 with ulcerative colitis, and 25 controls using a macrophage inhibition factor assay on peripheral blood lymphocytes. Two types of reaction patterns were identified--that is, 'responders' (subjects with a macrophage inhibition factor assay in which a dose response relation was present and a percentage of inhibition exceeding 20%), and 'non-responders'. There was no significant difference in the prevalence of responders (59%-80%) and non-responders (20%-41%) to these mycobacteria between the group of Crohn's disease, ulcerative colitis, and control group. We found also that a large proportion of controls showed T-cell immunisation to the mycobacteria which supports the contention that the antigens are practically commensal. Our results do not support the proposed involvement of mycobacteria in the pathogenesis of Crohn's disease.

Adult↗
PubMed1986

Biochemical characteristics of various strains of Mycobacterium paratuberculosis.

Biochemical activities of 20 wild-type strains and of 2 laboratory strains of Mycobacterium paratuberculosis were evaluated. Biochemical activities evaluated were growth at 30 C, 37 C, and 42 C; production of urease, niacin, pyrazinamidase, arylsulfatase, and catalase; hydrolyzation of Tween 80; reduction of nitrate and tellurite; and growth in 5% NaCl. Antimicrobial susceptibility to thiophene-2-carboxylic acid hydrazide (10 micrograms/ml), neotetrazolium chloride (1:40,000), streptomycin (2 micrograms/ml), rifampin (0.25 micrograms/ml), and isoniazid (10 micrograms/ml) also was determined. Generally, M paratuberculosis was biochemically inactive, with only a few strains producing pyrazinamidase and maintaining catalase activity after heating. All strains grew optimally at 37 C, grew slightly at 30 C, and did not grow at 42 C. Wild-type strains did not grow in the presence of neotetrazolium chloride, streptomycin, and rifampin, and grew in the presence of thiophene-2-carboxylic acid hydrazide and isoniazid. Although biochemical evaluation can be used as an aid in the identification of M paratuberculosis, growth rate, and mycobactin dependency remain major criteria for positive identification.

Amidohydrolases↗
PubMed1981

Lack of intracellular degradation of Mycobacterium paratuberculosis by bovine macrophages infected in vitro and in vivo: light microscopic and electron microscopic observations.

Blood-derived macrophages from clinically healthy cows and cows infected with Mycobacterium paratuberculosis were cultured in vitro and inoculated with heat-killed M paratuberculosis. Five weeks after infection, acid-fast bacteria continued to be demonstrated in the macrophages. Macrophage cultures also were infected with living M paratuberculosis and were examined by electron microscopy. Four weeks after infection, intact bacteria were found in all cultures. Electron microscopic examinations of intestinal macrophages from infected cows also demonstrated intracellular intact bacteria.

Animals↗
PubMed1993

The cellular immunology of bovine paratuberculosis: immunity may be regulated by CD4+ helper and CD8+ immunoregulatory T lymphocytes which down-regulate gamma/delta+ T-cell cytotoxicity.

In a previous investigation, we obtained evidence that the major histocompatibility complex (MHC)-restricted proliferative response of CD4+ lymphocytes to Mycobacterium paratuberculosis antigens was depressed in naturally infected and immunized animals. Findings suggested that depression of the response was attributable to an abrogation in the ability of CD4+ cells to respond to specific antigens and/or the actual loss of antigen-reactive cells. In vitro cell experiments indicated that the depression was associated with the presence of gamma/delta+ T cells that modulated CD4+ cell function. Examination of additional animals confirmed and extended these observations and showed that the ability of gamma/delta+ T cells to regulate CD4+ responses were blocked by the presence of CD8+ cells. CD4+ T cells from some exposed animals incorporated [3H]-thymidine in the presence of CD8+, gamma/delta+ cells and/or antigen and antigen-presenting cells, but CD4+ cell proliferation was abrogated when CD8+ were excluded from the assays. Likewise, gamma/delta+ T-cell proliferation was abrogated when CD8+ cells were present. The mechanism by which CD8+ cells blocked gamma/delta+ T-cell responses could not be determined, however, the observed effect resembled the veto cell phenomenon. The data suggest that the development of protective immunity against M. paratuberculosis may be dependent on the capacity of CD8+ cells to modulate the regulatory activity of gamma/delta+ T populations.

Animals↗
PubMed2000

Construction and screening of Mycobacterium paratuberculosis insertional mutant libraries.

Mycobacterium paratuberculosis causes Johne's disease, a common wasting disease in ruminants. As a first step in studying virulence mechanisms, libraries of random mutants were produced in two M. paratuberculosis strains by using the conditionally replicating shuttle phasmid phAE94 which contains the transposon Tn5367. Two thousand mutants were screened for auxotrophy, carbon source preference, and altered cell wall. Genes interrupted by insertion were identified for seven mutants isolated from the screening process. Two mutants had insertions in putative genes involved in synthesis of the cell wall.

Animals↗
PubMed1993

Comparison of polymerase chain reaction tests and faecal culture for detecting Mycobacterium paratuberculosis in bovine faeces.

A polymerase chain reaction (PCR) test for M. paratuberculosis was developed based on a 218 bp segment of a DNA insertion sequence, IS900, that is specific for this organism. The method involved two consecutive amplification reactions, with the second set of primers being nested inside the first set. The method reliably detected 50 organisms/g faeces. This PCR test was applied to 32 bovine faecal specimens containing high, moderate or low numbers of M. paratuberculosis organisms as determined by culture. The PCR test detected all specimens containing > or = 1600 colony forming units (cfu)/g faeces, six of ten specimens with 160-480 cfu/g faeces but only two of 13 specimens containing < or = 112 cfu/g faeces. The sensitivity of this test was better than that of a commercial PCR test which was carried out on the same faecal specimens.

Animals↗
PubMed1994

Comparison of a complement fixation test, a gel diffusion test and two absorbed and unabsorbed ELISAs for the diagnosis of paratuberculosis in sheep.

A complement fixation test for paratuberculosis, a gel diffusion test and two enzyme-linked immunosorbent assays (ELISA) were evaluated using sera from Mycobacterium paratuberculosis infected and non-infected sheep. Gross pathology and histopathology were used as parameters of infection. The two ELISAs, one of which is commercially available for testing cattle, were used before and after sera had been absorbed with a soluble sonicate of Mycobacterium phlei. Differences between the various tests and between ELISAs before and after absorption were non-significant (P > 0.05) in non-infected sheep or in animals with gross or histopathological lesions. The specificity of all the tests was at least 97%. Sensitivity in histopathologically positive sheep was at least 98%. Sheep from infected flocks but without histopathological lesions showed serological results which were poorly correlated between the various tests.

Animals↗