SEARCH · Search PubMed
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Mongolian gerbils (Meriones unguiculatus) were exposed to Mycobacterium paratuberculosis by intraperitoneal and oral routes. Fourteen weeks after exposure, the gerbils were necropsied and tissues were cultured and examined microscopically. Results of the study showed that gerbils can be infected with M paratuberculosis but are not suitable for studies of paratuberculosis.
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.
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.
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.
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.
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.
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.
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.
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.
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.
The susceptibility of C57BL/6 mice to oral inoculation with Myobacterium paratuberculosis was evaluated histopathologically. Granulomatous lesions containing acid-fast bacteria developed in the mesenteric lymph nodes in over 50% of the mice by 11 months after inoculation. The results suggest that C57BL/6 mice may be useful for studying infection, pathogenesis, and other aspects of paratuberculosis.
Changes in the number and distribution of lymphocyte subsets were investigated in the intestinal mucosa and mesenteric lymph nodes of three goats with natural paratuberculosis, comparisons being made with a single uninfected control animal. Lesions in the naturally infected goats varied from small granulomata with scarce epithelioid or multinucleated giant cells, containing few or no bacilli, in the intestine (tuberculoid type) to an extensive, diffuse epithelioid cell infiltrate containing numerous bacilli in the gut and mesenteric lymph nodes (lepromatous type). The number and distribution of lymphocyte subsets in the control were consistent with data reported from other non-infected goats. However, in the goats with paratuberculosis, significant changes were observed in the number and distribution of CD4+ and CD8+ T lymphocytes, the changes being related to the severity of the lesions. In the intestinal mucosa of the goat with tuberculoid lesions no significant changes were observed, but in the cortical area of mesenteric lymph nodes the number of CD4+ T lymphocytes decreased and the number of CD8+ T lymphocytes increased. In the two goats with lepromatous lesions, there was a decrease in the CD4+ T subpopulation and an increase of CD8+ T lymphocytes in the lamina propria of the intestine and particularly in the cortical area of the mesenteric lymph nodes, the CD4:CD8 ratio (< 1) being the opposite of that observed in healthy goats. Because of the small numbers of animals, further studies including additional animals are needed to confirm these preliminary results, which suggest that the progression of paratuberculous lesions may be due to an ineffective host immune response attributable to the CD8+ T lymphocyte subset that "downregulates" the activity of the CD4+ T lymphocytes required for macrophage activation.
Characterisation of the T-cell subsets in intestinal lesions in sheep with paratuberculosis may contribute to our understanding of the pathogenesis of this disease. To determine the phenotype and distribution of lymphocytes in the normal sheep intestinal mucosa and in Mycobacterium avium subspecies paratuberculosis infected sheep, immunohistochemistry was performed on 12 normal sheep and 18 naturally infected, clinically diseased sheep of which 12 showed lepromatous and six tuberculoid forms of the disease. Immunoperoxidase staining was carried out on frozen sections of ileum using monoclonal antibodies against ovine CD4, CD8, and gamma delta T-cell receptor (TCR) markers. In all three sample groups, cells appeared to be non-randomly distributed throughout the lamina propria. Higher densities of lymphocytes were present in villus than in crypt areas. CD8+ cells were located principally around the epithelial basement membrane, whereas CD4+ cells were localised towards the central villus area of the lamina propria. Lymphocytes bearing the gamma delta T-cell receptor were more widely distributed, both in epithelial and lamina propria compartments. Ileum with tuberculoid lesions had higher densities of CD4 and gamma delta T-cell subsets while lepromatous lesions had lower densities of CD4 and CD8 cells compared with normal tissues. The median relative percentage of CD4+ cells was increased and that of CD8+ cells decreased in tuberculoid cases, with a corresponding increase in the CD4:CD8 ratio, while the relative percentage of gamma delta + cells was increased in lepromatous cases.
An absorbed ELISA for detection of antibodies against Mycobacterium paratuberculosis was performed on serum samples obtained from the Missouri Animal Health Laboratory. Samples from 1954 Missouri cattle representing 89 herds were randomly selected from samples submitted for brucellosis testing. The apparent seroprevalence of paratuberculosis in dairy cattle (8 +/- 3%) was similar to that in beef cattle (5 +/- 2%). When herds were classified as dairy or beef, 74% (14 of 19) of dairy herds and 40% (27 of 68) of beef herds were positive.
Infection of the calf soon after birth by oral ingestion of M. paratuberculosis organisms from feces of infected cows is the most important method of transmission of paratuberculosis. The severity and rate of progression of the disease are dependent on the quantity of organisms in the exposure and the age of the animal. Only a small dose of organisms may be required to establish infection in a newborn calf, and overwhelming age-related resistance by introduction of a large dose of organisms to an adult cow is probably possible. Other means of transmission such as transplacental and direct excretion in the milk and colostrum should not be ignored and should be a particular concern primarily in advanced stages of infection when heavy fecal shedding is detected.
The currently available and accurate diagnostic tests for paratuberculosis are reviewed. Technical issues concerning test performance are briefly mentioned and the advantages, disadvantages, and comparative accuracy are described. The article concludes with recommendations on which tests to use in eight different situations requiring laboratory diagnostics for paratuberculosis.
To obtain insights into the pathogenic mechanisms involving Mycobacterium paratuberculosis in Crohn's disease (CD) we questioned if the strains of M. paratuberculosis isolated from CD are distinguishable from those involved in Johne's disease (JD), a chronic granulomatous enteritis in cattle. Accordingly we compared human and animal strains at the DNA level, both by the analysis of restriction fragment length polymorphism (RFLP) in and around the insertion sequence IS 900 and by the arbitrarily primed chain reaction (AP-PCR). Results are in favour of a common clonal origin for the 4 strains isolated from CD and for 8 of the 11 strains isolated from cattle and sheep JD.
BALB/c mice were infected intraperitoneally with Mycobacterium paratuberculosis and, after allowing the infection to progress for 30 days, were treated with rifabutin at 0, 12.5, 25, and 50 mg/kg of body weight. Rifabutin was administered in drinking water under conditions of water deprivation, whereby the entire daily dose was delivered within a 1-h period. Animals were killed at biweekly intervals from time zero of treatment to 180 days. Spleens and livers from each animal were examined by quantitative bacteriologic culture and histopathology. Restricted water availability was found to be a viable alternative to daily gavage for single-dose bolus administration. Infection, as assessed by bacterial counts, was reduced only in animals that received 50 mg of rifabutin per kg. In these animals, bacterial counts in the liver and spleen were reduced from 7.2 x 10(5) +/- 4.1 x 10(4) and 6.5 x 10(5) +/- 4.1 x 10(4) to 3.0 x 10(3) +/- 1.8 x 10(2) and 3.1 x 10(3) +/- 2.2 x 10(2), respectively, over the 6-month treatment period. Rifabutin may be an appropriate chemotherapeutic drug for long-term treatment of M. paratuberculosis infection and should be considered in any multidrug regimen.