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PubMed2002

The immunogenicity of Mycobacterium paratuberculosis 85B antigen.

Mycobacterium paratuberculosis (MPT) is the etiological agent of paratuberculosis. The disease is prevalent throughout the world, and exacts a heavy financial toll. At present, the only means of controlling this disease are culling or vaccination. The existing vaccines are not very efficient and produce a long-lasting local reaction at the point of injection and induce anti-bodies/delayed-type hypersensitivity (DTH) reaction that cannot be differentiated from those of naturally infected animals. New potent acellular vaccines that allow discrimination between infected and vaccinated animals are necessary to improve the control of this disease. We have isolated, overexpressed and purified the 85B antigen of MPT, and characterized the immune response induced by this antigen in mice. Our results showed that the recombinant MPT 85B (rMPT 85B) antigen induced a high production of interferon (IFN)gamma, interleukin (IL)-6, IL-10 and nitric oxide (NO). Spleen cells from mice immunized with rMPT 85B in Ribi adjuvant produced a higher level of IL-10 and NO than spleen cells of mice immunized with rMPT 85B only. In contrast, the addition of Ribi to the immunization protocol resulted in a lower amount of IFNgamma released by spleen cells. The levels of spleen cells proliferation in mice vaccinated with the rMPT 85B protein alone or with rMPT 85B with Ribi adjuvant were, respectively, four times or five times greater than in the control mice. The Ribi adjuvant induced significantly higher anti-85B antibody production of all classes tested and increased the IgG1/IgG2a ratio. DTH responses in mice footpads were observed only in mice immunized with rMPT 85B emulsified in Ribi. rMPT 85B induced both a Th1 and Th2 type of immune response with the later slightly more pronounced when the vaccination protocol comprised Ribi as an adjuvant. The rMPT 85B antigen elicited a strong immune response and can be considered as a potential candidate for a future acellular vaccine.

Acyltransferases↗
PubMed1991

Lysis and fractionation of Mycobacterium paratuberculosis and Escherichia coli by matrix solid-phase dispersion.

A novel method for the lysis and subsequent fractionation of bacterial constituents from Mycobacterium paratuberculosis strain 19698 (M. paratuberculosis) and Escherichia coli strain DH5 alpha utilizing the technique of matrix solid-phase dispersion (MSPD) is described. Bacteria were blended with octadecylsilyl (C18) derivatized silica to obtain cellular lysis. The blended material was used to prepare a column which was sequentially eluted with solvents of increasing polarity. Fractionation of cellular components was confirmed by analysis of the solvent extracts. The possible applicability of the MSPD technique as a general method for the lysis and fractionation of bacterial components is proposed.

Bacteriological Techniques↗
PubMed1988

IgG, IgM and IgA in the serum of cattle naturally infected with Mycobacterium paratuberculosis.

Serum IgG, IgM and IgA antibody response in 20 cattle naturally infected with Mycobacterium paratuberculosis and in 15 non-infected cattle were measured by enzyme-linked immunosorbent assay. A strong IgG response was detected in 16 (80%) of the infected animals. Diagnostic levels of IgM were detectable in all of the infected animals as well as in 8 (53%) of the non-infected animals. Animals with paratuberculosis had a very weak specific serum IgA response and this appears to be of little value in detection of infection in these animals.

Animals↗
PubMed1988

The effect of different levels of iron intake on the multiplication of Mycobacterium paratuberculosis in C57 and C3H mice.

Two groups of C57 and C3H mice of 5 weeks of age were infected via the intraperitoneal route with 2.0 mg (wet weight) of Mycobacterium paratuberculosis. These were maintained with a similar number of segregated and non-inoculated mice of the same strains under specially controlled conditions of low, medium and high iron intake. Mice were killed and bled at 7 months post-infection and assessments of haematological parameters and the degree of mycobacterial granulomatous involvement of abdominal and other tissues were made. In addition, the total mycobacterial numbers visible in macrophages in standardized histological sections of liver, spleen and bone marrow in the presence or absence of stainable iron storage compounds were assessed using a double staining technique for iron and mycobacteria. Moderate to marked anaemia in both C57 and C3H mice on low iron intake, irrespective of infection, indicated that an effective low iron status was achieved in the animals by dietary manipulation. Medium and high iron intake groups exhibited normal haematological parameters. Iron storage compounds were readily visible in liver microgranulomas of mice on medium and high, but not on low iron intake. In liver, spleen and bone marrow samples, mycobacterial counts in iron-containing microgranulomas were significantly higher than in those without stainable iron. Increased frequencies of residual and progressive infection were associated with increased iron intake. The greater susceptibility of the C57 strain was evident from the significantly higher liver microgranuloma counts, higher mycobacterial numbers and greater progressive infection when compared with the C3H strain. These findings in mice strongly suggest that slow multiplication of M. paratuberculosis is enhanced in iron-replete compared with iron-deficient macrophages. This enhancement occurs despite the capacity of the less susceptible strain of mouse to limit the spread of the organism within the body.

Animals↗
PubMed1989

The specificity of antibody response in experimental and natural bovine paratuberculosis studied by crossed immunoelectrophoresis with intermediate gel.

The sonicate antigen (MPS) of a local strain (IVRI) of Mycobacterium paratuberculosis and a commercial lysate of Strain 18 were analysed using hyperimmune rabbit and calf antisera to MPS in crossed immunoelectrophoresis with intermediate gel (CIE-ig) and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The rabbit antiserum was more potent than the calf antiserum and it precipitated 35 and 15 antigens, respectively, among MPS and lysate antigens. SDS-PAGE resolved 50 and 32 peptides among these antigens respectively, of which, 35 and 15 were precipitated by rabbit antiserum. A CIE-ig reference system, with 30 MPS antigens, was standardized and used to analyse antibody specificities among sera derived from animals experimentally and naturally infected with bovine paratuberculosis. Fourteen antigens of MPS were found to be reactive with these sera and among these, Antigens 2 and 5 were found to be serodominant; sonicate antigens of M. bovis BCG and M. avium did not contain these antigens. Both were high molecular weight (greater than 60 kDa) antigens which may be of serodiagnostic value.

Animals↗
PubMed1995

Characterization by restriction endonuclease analysis and DNA hybridization using IS900 of bovine, ovine, caprine and human dependent strains of Mycobacterium paratuberculosis isolated in various localities.

DNA of 90 mycobactin-dependent strains of Mycobacterium paratuberculosis, isolated in 9 countries, was digested with restriction endonuclease PstI and hybridized with a DNA fragment containing insertion sequence IS900. Bovine strains (n = 73) were isolated from 61 animals in 17 herds, ovine strains (n = 15) from 13 animals in 3 herds and the set was completed by 1 caprine and 1 human (Linda) strain. Three types, tentatively designated A (n = 37), B (n = 51) and C (n = 2) were differentiated by restriction fragment length polymorphism (RFLP). Of the bovine strains 27, 45 and 1 were classified as belonging to types A, B and C, respectively; of the 15 ovine strains 10 and 5 belonged to types A and B, respectively; the caprine strain belonged to type C. The human strain Linda, isolated in the U.S.A., from a man with Crohn's disease, belonged to B type. A certain degree of type uniformity was observed among strains isolated within one herd in the course of several years, and the prevalence of a single type was also recorded within individual regions of the Czech Republic and Slovakia. Type A was identified in the course of 2 years in a sheep farm with frequent sales and purchases of animals, and type C was demonstrated in a goat kept in the same farm. Differences between RFLP types of the strains isolated from mesenteric lymph nodes and intestinal mucosa were found in one cow and one sheep. Selected strains of M. paratuberculosis RFLP type A (14 strains) and B (18 strains) were digested with restriction endonuclease BstEII. All the strains of A type were classified into C1 group and all the strains of B type into Cx group.

Animals↗
PubMedDate not supplied

Characterization of macrophages and occurrence of T cells in intestinal lesions of subclinical paratuberculosis in goats.

The granulomatous lesions of subclinical paratuberculosis of goats were examined with emphasis on phenotypic characteristics of macrophages and the presence of different subpopulations of T cells. The macrophages in the granulomatous lesions were morphologically homogeneous in histological sections but showed varying expression of the macrophage marker CD68 (a glycoprotein found mainly in late endosomal and lysosomal membranes) and varying acid phosphatase activity. The lesional macrophages showed decreased expression of complement receptor 3 and major histocompatibility complex proteins, which are markers associated with phagocytosis and antigen-presentation, respectively. The granulomas showed low proliferation activity as measured by the proliferation-associated protein Ki-67, indicating that most cells were recruited to the lesions. Few apoptotic cells were demonstrated by the TUNEL technique, suggesting a low cell turnover in the lesions. CD4(+) T cells constituted the main T-cell population among the CD68(+) macrophages in the granulomatous lesions, and few CD8(+) T cells and gamma delta T cells were observed within the lesions, suggesting the limited ability of these cells to influence the granulomatous lesions in caprine subclinical paratuberculosis. Both WC1(+) and WC1(-) gamma delta T cells were present in the small intestinal wall, but the latter were the more numerous. No difference in the numbers of these cells was observed between the subclinically infected animals and control animals.

Animals↗
PubMed2003

Monte Carlo simulation of flock-level sensitivity of abattoir surveillance for ovine paratuberculosis.

We used Monte Carlo simulation to estimate distributions for flock-level sensitivity of abattoir-based surveillance for ovine paratuberculosis as currently practised in New South Wales, Australia. Probability distributions were used as input variables for within-flock prevalence, years-infected and individual animal-level sensitivity and specificity of gross pathology as a screening test for the presence of paratuberculosis. Distributions used as inputs for the size of abattoir-slaughter groups were based on existing abattoir-surveillance data from NSW. Predicted flock-level sensitivity depended on within-flock prevalence and the number of animals examined and was sensitive to estimates of animal-level sensitivity and specificity. The median probability of detection of an infected flock based on the examination of one abattoir line was predicted not to exceed 0.95 unless the within-flock prevalence was > or =7%. If the within-flock prevalence was 2%, the probability distribution of flock-level sensitivity had a median of 0.73, with 80% of values lying between 0.55 and 0.84. Improvement in the flock-level sensitivity could be achieved by submitting more than three gross pathology-positive specimens per line, if available-but the degree of improvement depended on the number of sheep slaughtered (line size) and the within-flock prevalence. At 2% prevalence, a median flock-level sensitivity of 0.95 could be obtained in lines of >390 sheep if six gross pathology-positive specimens were submitted. We concluded that abattoir surveillance based on identification of gross pathology as a screening test is not a sensitive tool for detecting recently infected flocks or flocks which have a moderate or lower prevalence of infected animals. But-with relatively minor modifications of the protocol currently in use-it could become a key component of a surveillance programme which included additional testing strategies for small flocks.

Abattoirs↗
PubMed2006

A stochastic model simulating paratuberculosis in a dairy herd.

Paratuberculosis (PTB) causes severe economic losses to farmers and the infection has very complex effects (many indirect) on the production of a dairy herd. These indirect effects have not or only briefly been described by earlier PTB-simulation models, and therefore they were included in a new model called PTB-Simherd. Our aim was to develop the basis for a decision-support tool which can predict herd-specific production-related effects from introduction of different control strategies against PTB. The PTB-Simherd is a dynamic, stochastic, and mechanistic Monte-Carlo model simulating a dairy herd including young stock. Paratuberculosis and relevant control strategies against this infection were built into an existing herd simulation model. The model simulates epidemiological and production related consequences of PTB and control strategies against it in the herd. It also reflects indirect effects of PTB and control strategies through effects on replacements and herd demographics. Every animal in the herd is specified with biological parameters (including PTB state and test results) and it is updated in weekly time-steps. Management is specified at herd level with 353 parameters of which 78 are related to PTB. To demonstrate the basic characteristics of the model, scenarios with varying infection risks (sensitivity analyses) plus scenarios with seven different control strategies in two herds with good and poor reproduction were simulated for 10 years. Breaking of infection routes turned out to be the only strategy predicted to reduce the true prevalence of PTB in a herd. Supplementing this strategy with test-&-cull strategies had limited effect on prevalence and using test-&-cull alone just delayed the increase in prevalence. The effects of different PTB-control strategies on the production (especially sale/purchase of heifers, feed consumption and prevalences of other diseases) were predicted to be affected by other conditions like heat-detection success, replacement% and herd demographics--which were again affected by PTB infection of the herd. These links and indirect effects of control strategies thus seem important to include when modeling and predicting effects of PTB control in dairy herds.

Animals↗
PubMed1990

Experimental infection of calves with an apparently specific goat-pathogenic strain of Mycobacterium paratuberculosis.

Four calves and four goat kids were inoculated perorally with a Norwegian goat-pathogenic strain of Mycobacterium paratuberculosis. None of the calves developed clinical disease, pathological lesions or humoral antibody response, but the organism was reisolated from the small intestine and mesenteric lymph nodes when the calves were slaughtered after 7, 12, 14 and 18 months, respectively. As one of the goats died of non-specific causes and one did not become infected, two remained as positive controls. One of these became subclinically and one clinically infected, but both showed distinct histopathological lesions at necropsy. Both were shown to be positive in the complement fixation test (CFT) and the enzyme-linked immunosorbent assay (ELISA) but only the clinically affected goat proved positive in the agar gel immunodiffusion (AGID) test. The AGID test was found to be of low diagnostic value, and the ELISA was as sensitive as the CFT in detecting infection at an early stage. However, when infection was finally established, the ELISA titres became far higher than the CFT titres. The results confirm previous experience in Norway, that the current Norwegian strain of M. paratuberculosis has little or no pathogenicity for cattle.

Animals↗
PubMed1990

Intestinal lesions resembling paratuberculosis in a wild rabbit (Oryctolagus cuniculus).

One of three feral rabbits from a colony on a hill grazing used to sustain farmed red deer in north-east Scotland, had chronic intestinal lesions resembling paratuberculosis (Johne's disease), but similar lesions were not found in 29 juvenile and adult rabbits killed subsequently on the same ground. Acid- and alcohol-fast bacteria were observed in fixed tissues, but material for culture was not obtained from the affected rabbit, thus the species of Mycobacterium involved was not identified. The farmed deer had experienced recently a severe outbreak of paratuberculosis.

Animals↗
PubMed1994

Experimental infection of vaccinated and non-vaccinated lambs with Mycobacterium paratuberculosis.

A study of the pathogenesis of paratuberculosis and the effects of vaccination was carried out on 17 Rasa lambs, allocated to four groups. Vaccination seemed to accelerate the progress of the infection, and led quickly to healing. The only site of early lesions was the interfollicular areas in the Peyer's patches. This would suggest an explanation both for the location of paratuberculous lesions in the ileum in clinical cases, and for the lower susceptibility to infection of adult animals, in which the intestinal organized lymphoid tissue is greatly diminished. Thus, a critical role of the Peyer's patch in the establishment of M. paratuberculosis infection is suspected.

Animals↗
PubMed1993

Susceptibility of Balb/c, C57/B6 and C57/B10 mice to infection with Mycobacterium paratuberculosis.

Balb/C, C57/B10 and C57/B6 mice were examined for their susceptibility to disseminated Mycobacterium paratuberculosis infection after intraperitoneal inoculation with a suspension of organisms containing mineral oil. Animals were examined monthly by histopathology and bacterial tissue counts of liver and spleen over a 6-month period. Only Balb/c mice maintained a steady infection with an average of 4.1 x 10(5) +/- 7.8 x 10(3) and 8.1 x 10(5) +/- 2.6 x 10(4) colony forming units (cfu) per gram of liver and spleen, respectively, during the course of the study. In contrast, C57/B10 mice reduced the bacterial counts in the liver and spleen from 6.8 x 10(4) and 1.3 x 10(5) to 7.1 x 10(2) and 4.3 x 10(3), respectively during the first 120-150 days after infection. The reduction in cfu was associated with the development of caseous necrotic lesions. C57/B10 mice were of intermediate resistance, slowly reducing cfu in the liver, but not the spleen, during the 6-month period. Balb/c was found to be a suitable mouse strain for the study of chronic M. paratuberculosis infection.

Animals↗
PubMed1996

The pathology of ovine paratuberculosis: gross and histological changes in the intestine and other tissues.

In sheep clinically affected with paratuberculosis, two distinct forms of microscopical pathology were recognized, related to a high or a low degree of mycobacterial colonization ("multibacillary" or "paucibacillary" presence). These forms were characterized by different types of cellular infiltrate in the ileal mucosa and submucosa. Statistical analysis demonstrated strong correlations between the presence of large numbers of acid-fast organisms and macrophage infiltration, and between small numbers and lymphocyte infiltration. Correlations also existed between high numbers of acid-fast bacteria and a positive serum antibody test results; and between the presence of giant cells and lymphocytes in the gut. This study suggests that in ovine paratuberculosis the same clinical and gross pathological changes can result from different pathogenetic mechanisms.

Animals↗
PubMed1999

Interferon-gamma and interleukin-2 release by lymphocytes derived from the blood, mesenteric lymph nodes and intestines of normal sheep and those affected with paratuberculosis (Johne's disease).

This study sought to determine if T-cell cytokine responses to mycobacterial infections in sheep were similar to those in other species and if such responses correlated with prevailing gut pathology. Lymphocytes were isolated from the blood (PBL), mesenteric lymph nodes (MLN) and ileal lamina propria (LPL) of control sheep and of sheep with clinical Johne's disease due to infection with Mycobacterium avium ssp. paratuberculosis (M.a. paratuberculosis). These animals had previously been categorised into two groups exhibiting either the 'tuberculoid' (paucibacillary) form of lesion or the 'lepromatous' (multibacillary) form. Lymphocytes were examined for their capacity, following stimulation with johnin-PPD, to release interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) characteristic of the Th1 subset of MHC Class II-restricted CD4+ (helper) T-cells in other species. The expression of the two cytokines appeared related to the type of histological lesion observed. Antigen-stimulated lymphocytes from the tuberculoid group exhibited greater release of IFN-gamma and IL-2 than lymphocytes from the lepromatous group suggesting a Th1-type of response in the former in which expression of IFN-gamma by PBL showed a significant positive correlation with that expressed by MLN and LPL. Lymphocytes from animals with lepromatous lesions released lesser mycobacterium-induced IFN-gamma and IL-2 indicating a diminished role for a Th1 subset in this group of sheep. Differences in cytokine expression were much more apparent with lymphocytes which were derived from MLN.

Animals↗
PubMed1996

Economic impact of paratuberculosis.

The economic impact of paratuberculosis has been estimated in dairy cattle for several areas of the United States and in some other dairy-intensive areas of the world. Losses are primarily due to decreased milk production and unrealized income related to premature culling. Milk production losses in the range of 15% have been reported in paratuberculosis-infected cows. Additional economic costs include diagnostic testing, control measures, and accelerated cull and replacement rates.

Animal Husbandry↗
PubMed2003

The ranging behaviour and habitat use of rabbits on farmland and their potential role in the epidemiology of paratuberculosis.

Grazing herbivores avoid grass swards contaminated with faeces as the ingestion of faeces is a common route of micro- and macro-parasite transmission. The recent novel finding that herbivores do not avoid grass swards contaminated with rabbit faeces suggests that disease risk posed to herbivores by rabbits is determined by rabbit ranging and excretory behaviour. Using as a case study rabbits and the risk Mycobacterium avium sub-species paratuberculosis (M. a. paratuberculosis) poses to cattle, the interaction between rabbits and grazing pasture was studied on an infected farm in the east of Scotland in spring and autumn 2000. Radio telemetry, burrow surveys and faecal pellet count data were collected on two areas (Areas 1 and 2) of the farm with different habitat mosaics, to study the potential effects of season and habitat on the spatial distribution of rabbits faeces and thus disease in the environment. Twenty one rabbits were radio tracked and a total of 902 fixes collected. Mean home range sizes (100% minimum convex polygons) were between 2.0 and 7.1 ha per rabbit per season. Home ranges were significantly larger in spring, and in Area 1 which had more moor and woodland and less rough pasture. Rabbits used rough pasture most in Area 1 and gorse scrub in Area 2. In both areas, significantly more burrows were located in gorse scrub than in any other habitat. Most faecal pellets were deposited on the moorland habitat of Area 2 in autumn. In habitats to which grazing livestock had access, the mean rate of faecal deposition was 8571 pellets per ha per day. The greatest risk of disease transmission occurred in habitats of poor grazing quality (e.g., gorse scrub) which were used by rabbits for burrowing and thus contained high concentrations of faeces. The findings of the study are discussed in relation to management practices aimed at reducing disease risk to livestock, including the fencing of scrub and the reduction of rabbit population size to prevent expansion of rabbit burrows from scrub into grazing pastures.

Animal Feed↗
PubMed2002

Seroreactivities against Saccharomyces cerevisiae and Mycobacterium avium subsp. paratuberculosis p35 and p36 antigens in Crohn's disease patients.

Crohn's disease (CD) is an idiopathic chronic inflammatory bowel disease (IBD). Accurate diagnosis of the disease is of great clinical importance to assess its prognosis and success of therapy. Recent studies have validated and confirmed the potential utility of anti-Saccharomyces cerevisiae (baker's yeast; ASCA) IgG/IgA antibodies and anti-M. avium ss. paratuberculosis p35/p36 antibodies, separately, as serological markers to identify patients with CD. The efficacy of these markers was evaluated in the same patients with Crohn's disease. The anti-ASCA IgA/IgG and the anti-M. avium ss. paratuberculosis p35/p36 antibodies were positive in 60% (36/60) and 86.7% (52/60) of CD patients, respectively. When all the serologic markers were considered, the sensitivity in detecting CD was increased to 95.0% (57/60); 21 of 24 ASCA-negative patients were p35/p36-positive and five of eight of p35/p36-negative patients were ASCA-positive. This investigation further establishes the utility of p35 and p36 recombinant clones for the diagnosis of CD, and reveals the complimentary role of ASCA and p35 and p36 for effective detection of CD. Larger studies are needed to investigate the combined use of these serologic markers for the diagnosis of CD.

Antibodies, Bacterial↗