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PubMed2002

[Inflammatory bowel disease--do microorganisms play a role?].

This review focuses on the potential pathogenic role of microorganisms in relation to inflammatory bowel diseases, i.e. Crohn's disease and ulcerative colitis. Pathogenic microorganism such as Mycobacterium paratuberculosis, measles and mumps viruses, Epstein-Barr virus, and Listeria monocytogenes are discussed, as well as involvement of the normal intestinal flora. Furthermore, the influence of microorganisms in experimental animal colitis models is discussed. The available results are inconclusive, but there seems to be basis for proposing the hypothesis that the inflammation in inflammatory bowel disease reflects an immune imbalance with loss of tolerance for normally harmless antigens in the mucosal microflora.

Animals↗
PubMed2006

Johne's disease in Canada part II: disease impacts, risk factors, and control programs for dairy producers.

Part I of this 2-part review examined the clinical stages, pathophysiology, diagnosis, and epidemiology of Johne's disease, providing information relevant to Canada, where available. In Part II, a critical review of the economic impacts of the disease, risk factors, and important control measures are presented to enable Canadian bovine practitioners to successfully implement control strategies and participate in control programs. In cattle positive by enzyme-linked immunosorbant assay, there is a 2.4 times increase in the risk of their being culled, and their lactational 305-day milk production is decreased by at least 370 kg. Reduced slaughter value and premature culling account for losses of CDN dollars 1330 per year per infected 50-cow herd. Research has failed to show a consistent association between Mycobacterium avium subsp. paratuberculosis test status and reduced fertility or risk of clinical or subclinical mastitis. Host level factors include age and level of exposure, along with source of exposure, such as manure, colostrum, or milk. Agent factors involve the dose of infectious agent and strains of bacteria. Environmental management factors influence the persistence of the bacteria and the level of contamination in the environment. Emphasizing a risk factor approach, various control strategies are reviewed, including a number of national control programs currently in place throughout the world, specifically Australia, The Netherlands, and the United States. By reviewing the scientific literature about Johne's disease, control of the disease could be pursued through informed implementation of rational biosecurity efforts and the strategic use of testing and culling.

Animals↗
PubMed1991

Correction of antigen-specific T cell defects in aged murine gut-associated lymphoid tissues an immune intervention by combined adoptive transfer of an antigen-specific immunoregulatory CD4 T cell subset and interleukin 2 administration.

Gut-associated lymphoid tissues (GALT) from aged mice enterically immunized with Mycobacterium paratuberculosis protoplasmic antigen show hyperreactive humoral immune responses; this hyperresponsiveness can be corrected to a considerable extent, but not entirely, by systemic administration of interleukin 2 (IL 2) alone. The aim of the present study was to determine further whether the hyperreactivity in the antigen-specific humoral immune responses in aged GALT could be fully restored by adoptive transfer of in vitro expanded antigen-specific IL 2-dependent helper (CD4+VV-) T cells from GALT in conjunction with recombinant IL 2 administration. The results show that the age-associated hyperresponsiveness in gut mucosal antigen-specific humoral immune responses can be entirely corrected by adoptive transfer with the antigen-specific GALT T helper cells together with in vivo IL 2 administration. The mechanism of this restoration involves reversal of the decline in antigen-specific CD8+ suppressor T cell functions in aged GALT.

Aging↗
PubMed1999

C1q autoantibodies in HIV infection: correlation to elevated levels of autoantibodies against 60-kDa heat-shock proteins.

Antibodies to solid phase C1q (C1qAb) were determined in 295 serum samples from 132 HIV-infected subjects and in sera from 140 HIV-seronegative healthy individuals as control. An ELISA method applied for the determination of C1qAb in other diseases was used. In part of these sera, other autoantibodies (antibodies reacting with 60-kDa human heat shock protein (hsp60) or mycobacterial hsp65; IgA and IgG class antibodies against the Fab and F(ab')2 moieties of IgG) as well as complement-mediated antibody-dependent enhancement/neutralization (C'-ADE) were also determined. Increased amount of C1qAb was found in HIV-infected subjects as compared with HIV-seronegative controls (P = 0.0138). In 17 of 132 (13.0%) seropositive individuals but only in 7/140 (5.0%) samples from the controls, the amount of C1qAb exceeded the upper limit (95th percentile) of the normal values (P = 0.031). The amount of C1qAb significantly decreased during a follow-up period of 65 months. C1qAb levels were found to strongly correlate to hsp60/65 autoantibodies but did not correlate or only weakly correlated to the amount of anti-Fab or anti-F(ab')2 autoantibodies measured in the same serum samples. Anti-C1q antibodies recognized the solid phase hsp60/65. Three predicted epitope regions of M. paratuberculosis hsp65 were able to bind efficiently C1q antibodies. An inverse correlation was found between C1qAb and C'-ADE, neutralization was more frequent in the sera with detectable C1qAb, whereas sera without C1qAb more likely enhanced HIV infection in vitro.

Antigens, Bacterial↗
PubMed2002

In situ identification of mycobacteria in Crohn's disease patient tissue using confocal scanning laser microscopy.

The diversity in the methodology employed to investigate Crohn's disease (CD) etiology has added significantly to the controversy of the mycobacterial role in this chronic inflammatory bowel disease. Mycobacterium avium subsp paratuberculosis (MAP), a proposed and suspected agent in many CD patients, is a fastidious and very slow grower bacillus, which causes Johne's disease (JD) in cattle. The methodology that has been widely and successfully used for isolation and identification of MAP from and in JD animals is not reliable and has proven to be unsuccessful in achieving the same objectives for CD diagnosis. In this study, a Confocal Scanning Laser Microscopy (CSLM) system has been employed in an attempt to detect MAP in CD patient. In situ hybridization was performed on full thickness tissue using rabbit anti-MAP polyclonal antibody that was adsorbed with E. coli protein extracts. Consequently, MAP was detected in the microvilli region in tissue specimens from CD patient and not in the controls. In the same CD tissue specimen, MAP was not detected when isotype normal rabbit sera was employed. The polyclonal antibody marker may be replaced with monoclonal antibodies, if available, or with MAP-specific-DNA or RNA probes. This technique adds an additional approach to investigate MAP role in CD etiology especially when the culture approach is long and inconsistent.

Animals↗
PubMed1992

Composition and immunogenicity of the polysaccharide components of the thermostable macromolecular antigen group of mycobacterial antigens.

The thermostable macromolecular antigen (TMA) group includes major components of the mycobacterial cell envelope and cytoplasm, which elicit humoral and cellular immune reactions, and seems to play important roles in infectious diseases. The best known member of this group, antigen A60 of Mycobacterium bovis BCG, was previously shown to contain three moieties of polysaccharides, free lipids, and polypeptides. In this work, the TMA polysaccharides of three pathogenic mycobacteria (M. avium, M. bovis and M. paratuberculosis) have been analyzed by coupled gas chromatography-mass spectrometry. In all cases the cores of the TMA complexes were represented by branched glucans of high molecular mass (about 10(6) daltons), for which structural models have been proposed. The immunogenicity of the polysaccharide components from the three TMA was verified with several immunological procedures (immunodiffusion and immunoelectrophoresis of the antigen, and immunoblotting of the corresponding electrofocused immunoglobulins). All tests tallied in showing a negligible immunogenicity of the glucans examined (inability to produce, upon injection, the synthesis of specific immunoglobulins), thus pointing to the protein moiety of TMA as the one responsible for the high immunoreactivity of the complexes.

Animals↗
PubMed1995

Glycosylation of IgG during potentially arthritogenic lentiviral infections.

Agalactosyl IgG [Gal(0)] was first discovered in patients with rheumatoid arthritis (RA). However, the proportion of this glycoform is also raised in tuberculosis and leprosy. This has helped reinforce the suggestion that RA may be triggered by a mycobacterium-like slow bacterial infection. On the other hand, arthritis can occur in mycobacterial diseases, so raised Gal(0) could be associated with a tendency to arthritis, rather than with a particular type of infection. Therefore, we wished to find out whether the percentage of Gal(0) [%Gal(0)] is increased in sheep and goats following infection with maedi visna virus or caprine arthritis encephalitis virus (CAEV), both of which can lead to inflammatory synovitis. We found that the normal level of Gal(0) in these species is much lower than in humans. Goats infected with CAEV or Mycobacterium paratuberculosis (used as a control mycobacterial infection) had a significant increase in %Gal(0), though it was still below the level seen in normal humans. Studies by Western blot confirmed the presence of terminal N-acetylglucosamine on heavy chains, and percentages of Gal(0) comparable to those seen in human RA could be generated by exposing goat IgG to streptococcal beta-galactosidase. The rise in %Gal(0) was greatest in members of infected herds that were just starting to manifest arthritis, and tended to be lower in those in which severe carpitis had developed at the time of bleeding, implying the possibility that raise %Gal(0) may be an early or predisposing event for the development of arthritis.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosamine↗
PubMed1990

Impaired humoral immune responses to mycobacterial antigen in aged murine gut-associated lymphoid tissues.

Senescence-related alterations of local gut mucosal immune responses to enteric mycobacterial antigen (Ag) were examined. Both aged (greater than 24 months old) and young adult (4-5 months old) BALB/c mice were enterically immunized with crude Mycobacterium paratuberculosis (M. paratbc) protoplasmic Ag, and in vitro Ag- and class-specific immunoglobulin (g) production by lymphocytes from gut-associated lymphoid tissues (GALT) (Peyer's patches, PP; mesenteric lymph nodes, MLN) and non-GALT (spleen, SPN) were determined against semipurified M. paratbc Ag. Ag-specific spontaneous immunoglobulin production by aged B cells from both GALT and non-GALT was enhanced only to a minor extent. Similarly, the functional activity of the Ag-specific T (Th) (CD3+, CD4+) cell in both GALT and non-GALT was not profoundly affected by senescence (qualitative preservation). However, that of the suppressor T (Ts) (CD3+, CD8+) cell was considerably diminished (qualtative defect). Thus, oral tolerance (systemic immunologic hyporesponsiveness) to M. paratbc Ag in aged mice is impaired. These age-related changes, manifested as hyperreactive humoral responses to the enteric microbial Ag, are due, at least in part, to hyporeactivity of the Ts cell, resulting in relative hyperfunction of the Ag-specific Th cell, despite the quantitative defect of the latter cell.

Aging↗
PubMed1990

In vivo immunologic intervention in age-related T cell defects in murine gut-associated lymphoid tissues by IL2.

Our recent studies indicate that the aging process impairs gut mucosal humoral immune responses to mycobacterial antigen (Ag), largely owing to defects in T cell function--in particular, that of suppressor T cells. To correct the age-associated Ag-specific T cell-mediated immune alteration recombinant IL2 (50,000 units/s.c./mouse/day) was administered for 3 weeks to the aged (greater than 24 months) mice (BALB/c), which were divided into 4 groups (Gr) [Gr. 1, fed intragastrically (i.g.) with saline; Gr. 2, immunized i.g. with Mycobacterium paratuberculosis (M. paratbc) protoplasmic Ag; Gr. 3, administered IL2 alone; Gr. 4, immunized i.g. with the Ag and given IL2]. In addition, young adult mice were also grouped and treated as the aged. First, we examined the effect of exogenous IL2 on Ag-specific immunoglobulin (Ig) production by gut-associated lymphoid tissues (GALT) (Peyer's patches, PP; mesenteric lymph nodes, MLN) and non-GALT (spleen, SPN) cells. Aged Gr. 4 (treated with both Ag and IL2) GALT and SPN unfractionated cells showed significantly reduced production of Ag-specific IgM, IgG, and IgA, as compared to aged Gr. 2 (treated with Ag alone) cells. Second, in co-culture experiments with aged T and B cells, aged GALT-derived CD8+ suppressor T (Ts)-depleted T cell subsets of Gr. 4 helped Ag-specific IgM and IgA production by GALT B cells, but to a slightly lesser extent, than those of the Gr. 2. GALT CD4+ T cells of aged Gr. 4 augmented IgM and IgA production by GALT B cells nearly to the levels of the corresponding cocultures of the Gr. 2. In contrast to aged Gr. 2 cocultures, GALT CD4+ plus CD8+ cells of aged Gr. 4 decreased IgM and IgA production to a considerable extent, and in those of SPN, IgG production was also diminished. The humoral immune responses of aged unprimed Gr. 1 (treated with saline) and Gr. 3 (treated with IL2 alone) GALT and SPN cells remained almost unchanged. Similarly, in all Gr. from young adult mice, oral tolerance was maintained regardless of IL2 administration. Third, together with the deletion experiments of the Ts cells, the results of the cross experiments, in which the young adult B and CD4+ Th cells and aged CD8+ Ts cells were cocultured, clearly support the view that the corrective mechanism of the humoral immune responses in aged GALT by exogenous IL2 is attributed to the partial recovery of the Ts cell functions.(ABSTRACT TRUNCATED AT 400 WORDS)

Aging↗
PubMed1990

The sensitivity and specificity of a modified ELISA for the diagnosis of Johne's disease from a field trial in cattle.

An enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Mycobacterium paratuberculosis in cattle was evaluated in three herds known to have Johne's disease. Prior to testing, the plasma was absorbed with dried Mycobacterium phlei in order to remove cross-reacting antibody specificities. The sensitivity and specificity of the ELISA were calculated after repeatedly testing 327 cattle in the infected herds. Of these, 53 animals had one or more positive faecal cultures or had post-mortem histopathological evidence of infection. The other 274 had three or more negative faecal culture results, and were regarded as non-infected for the purpose of evaluation of the ELISA. Using these criteria for the presence or absence of infection, the M. phlei-absorbed ELISA under field conditions had a 57% sensitivity and a 98.9% specificity. The sensitivity of the absorbed ELISA depended on the stage of disease of the animal under test. In general, it appeared that animals in the more advanced stages of disease were absorbed ELISA positive, whereas those in the early stages of infection were not detected. These results indicate that the M. phlei-absorbed ELISA has an important role as a test for the diagnosis and control of Johne's disease in cattle.

Animals↗
PubMed1989

Crohn's disease and mycobacteria: two cases of Crohn's disease with high anti-mycobacterial antibody levels cured by dapsone therapy.

Five patients with Crohn's colitis or ileocolitis (CD) refractory to conventional therapy were enrolled in an open trial with dapsone (100 mg/day). This therapy was apparently effective in two out of five patients. In these patients we observed a clinical improvement after one month of therapy and, in the first patient, a complete healing of all the cutaneous and rectal ulcers. In the two responders antibody levels to a soluble extract of M. paratuberculosis (MPSE) were significantly greater than in the three non-responders (P = 0.03); in the first patient, moreover, there was a rise of 39% in antibody titres following the treatment. This rise in antibody levels, that might be expected following death of the pathogen and release of antigen, is similar to that observed after treatment of tuberculosis. Our data suggest that a mycobacterial species or another pathogen that cross-react with those of MPSE, sensitive to dapsone, may in a subset of cases be responsible for the development of CD. This is the first report of clinical cure with an agent active against specific bacterial species, associated with immunologic confirmation of a response.

Adult↗
PubMed1990

Molecular biology of Crohn's disease mycobacteria.

A Glasgow surgeon, T.K. Dalziel, published a detailed description of chronic enteritis in humans in 1913. He proposed that the disease was caused by the same organisms as those responsible for chronic enteritis, Johne's disease, in animals described a few years earlier (1895). Dalziel's dilemma was that he could see acid-fast bacilli in the diseased animal tissues but not in the diseased human tissues. Little real progress in the medical understanding of the causes of chronic enteritis in humans occurred over the next half a century or more. From 1978, a decade of research in many laboratories using improved methods for the culture of environmental mycobacteria showed that these could be grown in bacillary form from about one in five cases of Crohn's disease, from the same proportion of cases of ulcerative colitis, and from about one in ten control tissues. Spheroplasts were grown from two in five cases of Crohn's disease, one in five cases of ulcerative colitis, and rarely from control tissues. The nature of these agents was often uncertain. We describe work which began in 1985 and led rapidly to the identification of IS900, a DNA repetitive element in an uncharacterized Crohn's disease mycobacterial isolate. With other isolates, these were then shown by DNA fingerprinting to be indistinguishable from Mycobacterium paratuberculosis, Johne's bacillus. Similar techniques also demonstrated the wood-pigeon strain of M. avium in some Crohn's disease cultures. This bacillus can also cause chronic enteritis in calves. IS900 is the first of a family of unusual DNA insertion sequences which extend widely throughout environmental mycobacteria. Use of assays based on PCR amplification of highly specific DNA sequences from these insertional elements, and recombinant and synthetic peptides from their predicted proteins, will revolutionize the detection and characterization of these agents. These methods, applied to animal, human and environmental samples, will indicate new ways for the prevention and treatment of chronic enteritis, as well as other disorders associated with infections by environmental mycobacteria.

Actinomycetales↗
PubMed2004

A practical approach to calculate sample size for herd prevalence surveys.

When designing a herd-level prevalence study that will use an imperfect diagnostic test, it is necessary to consider the test sensitivity and specificity. A new approach was developed to take into account the imperfections of the test. We present an adapted formula that, when combined with an existing piece of software, allows improved planning. Bovine paratuberculosis is included as an example infection because it originally stimulated the work. Examples are provided of the trade-off between the benefit (low number of herds) and the disadvantage (large number of animals per herd and exclusion of small herds) that are associated with achieving high herd-level sensitivity and specificity. We demonstrate the bias in the estimate of prevalence and the underestimate of the confidence range that would arise if we did not account for test sensitivity and specificity.

Animals↗
PubMed2005

Sample size determination for estimation of the accuracy of two conditionally independent tests in the absence of a gold standard.

We developed an Excel spreadsheet template (available at http://www.epi.ucdavis.edu/diagnostictests/) to calculate sample sizes to estimate sensitivity and specificity with desired precision in the absence of a gold standard. Calculations are predicated on the use of two conditionally independent tests for screening animals from two populations and are based on the methods of Hui and Walter(1980). Sample size calculations rely on asymptotic normality of maximum likelihood (ML) estimates of parameters. Spreadsheets for calculating standard errors for the parameter estimates and for providing ML estimates using cross-tabulated data also are included. An example of application of the methods to bovine paratuberculosis is presented.

Animals↗
PubMed2004

Expression of adhesion molecules on milk and blood lymphocytes from periparturient dairy cattle with Johne's disease.

Twelve dairy cows infected with Mycobacterium avium subsp. paratuberculosis were monitored for lymphocyte subsets and expression of adhesion molecules on cells in blood and milk at parturition and at intervals up to 21 days post-partum. Using fluorescent antibody labeling of cells and analysis by flow cytometry, we determined percentages of T cell subsets (CD4+, CD8+, gammadelta+) and expression of adhesion molecules (CD62L, LFA-1, LPAM-1, and CD44) on cells from blood and milk of these cows. Significantly higher percentages of CD8+ cells were found in milk than in blood at all time points; there were no significant differences in percentages of CD4+ or gammadelta+ cells. CD62L, LFA-1, and LPAM-1 were expressed on a significantly higher percentage of all T cell subsets in milk than in blood at various times after parturition. No differences were seen in expression of CD44. Increased percentages of T lymphocytes expressing adhesion molecules in milk compared to blood suggest that a migratory population of cells is being selectively recruited to the mammary gland from the circulation.

Animals↗
PubMed2006

Contribution of respiratory burst activity to innate immune function and the effects of disease status and agent on chemiluminescence responses by ruminant phagocytes in vitro.

The mechanisms of interaction between phagocytes and different bacteria that help resolve lung infections or contribute to lung pathology are poorly defined. Alveolar phagocytes (resident macrophages and recruited neutrophils) make a major contribution to innate immunity by mounting a respiratory burst that helps kill internalised bacteria. However, this ability may be altered during or after exposure to infection. This review considers the application and limitations of a variety of analytical methods for oxygen-dependent mechanisms of respiratory burst in phagocytes initiated by soluble and particulate activators. Particular reference is given to the study in vitro of phagocytes from healthy and diseased ruminants during either natural infection with Mycobacterium avium paratuberculosis or experimental infection with Pasteurella multocida or Mannheimia haemolytica.

Animals↗
PubMed2002

A simulation of Johne's disease control.

A dynamic and stochastic simulation model (the "JohneSSim model") was developed to evaluate the economic and epidemiological effects of different strategies for control of paratuberculosis in dairy herds. Animals occupy one of the six defined infection states; the spread of Johne's disease is modeled with five infection routes. Many different dairy farm situations can be simulated. Control strategies that can be simulated are: (1) test-and-cull; (2) calf hygiene management; (3) vaccination and (4) grouping of animals. Losses are caused by: (1) reduced milk production; (2) diagnosis and treatment costs; (3) lower slaughter value of cows and (4) sub-optimal culling. The benefits were calculated as reduction in the losses caused by Johne's disease; the costs of each strategy were calculated on the basis of actual costs of each item; and net present value (NPV) was calculated as benefits minus costs. Herd and prevalence data from The Netherlands and Pennsylvania, USA were used. In both situations, a low true mean prevalence within 20 years could be reached only when all calf management tools were applied. The Dutch control program (PPN) was on average economically attractive (with or without labor costs, the average NPV was Euro 1183 and 12,397, respectively). In Pennsylvania, contract heifer rearing and improved calf hygiene reduced the prevalence effectively and had large economic benefits (US$ 43,917 for 20-year period) if the calves were sent to the heifer facility while very young. Validation with data from 21 infected Dutch dairy farms (as well as face-validation: comparison of the results of the JohneSSim model with experiences of Johne's experts) supported the basic assumptions in the model.

Age Factors↗
PubMed2000

Use of macroporous polypropylene filter to allow identification of bacteria by PCR in human fecal samples.

The detection of pathogenic bacteria directly in human fecal specimens by PCR, requires removal of PCR-inhibitory substances. To investigate whether five different macroporous filters (polypropylene, nylon, polyester, polyethylene, fluorocarbon) could retain polysaccharides, major PCR inhibitors, an in vitro model and human fecal samples were used. The in vitro model consisted of Xanthum gum solutions (3 mg/ml PBS), a bacterial polysaccharide, to which Helicobacter pylori cells were added. Fecal samples from healthy volunteers were spiked with H. pylori and Mycobacterium paratuberculosis cells. Polysaccharide concentrations were significantly reduced only by the polypropylene but not by the other filters. Accordingly, both Xanthum gum solutions and spiked fecal specimens became PCR positive only after filtration with the polypropylene filter. We conclude that this filter can be used to prepare a bacterial DNA template suitable for PCR analysis from human feces.

Bacteria↗