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PubMed1993

Paratuberculosis in farmed deer: case reports and DNA characterization of isolates of Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis was isolated from farmed deer in New Zealand on 21 occasions over a 7-year period. The number of cases increased during the last 3 years of the study. Clinical paratuberculosis was observed in 5 deer, 3 other cases came from grossly normal animals that reacted to a tuberculin skin test, and the remaining 13 animals had tuberculous lesions that were identified at meat inspection. Pathologic features of the lesions in these 13 cases included necrosis and mineralization in lymph nodes draining the gastrointestinal tract. The histologic changes in these lesions were very similar to those caused by Mycobacterium bovis and members of the M. avium complex. Characterization of 20 of the isolates of M. paratuberculosis by restriction endonuclease analysis and DNA hybridization revealed that 3 of these isolates were identical to New Zealand sheep isolates and 17 were the same as cattle isolates. The source of the cervine infections was not determined.

Animals↗
PubMed1991

Chromatographic purification and characterization of antigens A and D from Mycobacterium paratuberculosis and their use in enzyme-linked immunosorbent assays for diagnosis of paratuberculosis in sheep.

The protein antigens A and D were purified from culture filtrates and sonic extracts of laboratory strains of Mycobacterium paratuberculosis by salt precipitation and chromatography. The characterization of antigen A is shown here, and both antigens were evaluated along with lipoarabinomannan antigen in indirect enzyme-linked immunosorbent assays (ELISA) for the serodiagnosis of ovine paratuberculosis. After anion-exchange (DEAE-5PW) and hydrophobic (phenyl-5PW) chromatography using high-performance liquid chromatography, antigen A showed a prominant band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) at 31 kDa with small amounts of low-molecular-mass proteins but with no evidence of antigen D. A single precipitin arc was evident with purified antigen A in crossed immunoelectrophoresis. The determination of the N-terminal amino acid sequence showed a high degree of homology between the 31-kDa component of antigen A and antigens of the BCG85 complex of Mycobacterium bovis BCG, a total of 24 of 26 residues being identical to those of BCG85C. A prominant SDS-PAGE band at 400 kDa and a single crossed-immunoelectrophoresis arc was also evident for antigen D after gel filtration (Sephacryl S-200), anion-exchange (DEAE-Sephacel), and concanavalin A-Sepharose affinity chromatography. By ELISA, purified antigen A detected antibody in the sera of 18 of 22 paratuberculosis-infected sheep (82% sensitivity), whereas the purified antigen D detected antibody in all 22 infected animals (100% sensitivity). Combined ELISA results showed increased specificity with some loss in sensitivity.

Amino Acid Sequence↗
PubMed2004

Neutralization of interleukin-10 significantly enhances gamma interferon expression in peripheral blood by stimulation with Johnin purified protein derivative and by infection with Mycobacterium avium subsp. paratuberculosis in experimentally infected cattle with paratuberculosis.

Monoclonal antibody neutralization of interleukin-10 (IL-10) increased Johnin purified protein derivative-induced whole-blood gamma interferon (IFN-gamma) secretion 23-fold and also increased IFN-gamma secretion ninefold following in vitro Mycobacterium avium subsp. paratuberculosis infection of peripheral blood mononuclear cells. These results demonstrate the suppressive effect of IL-10 on immune responses to M. avium subsp. paratuberculosis infection in cattle.

Animals↗
PubMed1994

Diagnosis of paratuberculosis in dairy cattle, using enzyme-linked immunosorbent assay for detection of antibodies against Mycobacterium paratuberculosis in milk.

An ELISA containing lipoarabinomannan (LAM) antigen was used to detect antibodies in milk and serum for diagnosis of Mycobacterium paratuberculosis infection in dairy cattle. In experiment 1, milk and serum samples were obtained from 25 cows, and subjected to LAM ELISA testing immediately, and after 1 year of storage at -70 C. Milk samples, with and without a commonly used chemical preservative, were tested. There was no significant difference in LAM ELISA results between fresh and frozen samples or between preserved and unpreserved milk samples. In experiment 2, milk samples were collected daily from 30 cows over a 14-day period. The day-to-day coefficient of variation was 0.19 for milk LAM ELISA and was 0.15 for serum LAM ELISA, with no statistically significant time effect detected. In experiment 3, single milk, serum, and fecal samples were obtained from 764 cows. The fecal samples were cultured for M paratuberculosis to identify infected cows, and the serum and milk samples were subjected to LAM ELISA testing. Results were compared, using the area under the receiver operating characteristic curves. The milk LAM ELISA had specificity (+/- 95% confidence limits) of 87 +/- 8.1% when the cutoff was set at 50% sensitivity, and specificity of 83 +/- 9.1% when sensitivity was set at 60%. The area under the receiver operating characteristic curve was 0.85 +/- 0.03 for the milk ELISA and 0.75 +/- 0.02 for the serum ELISA. In this population of cattle, the milk LAM ELISA had comparable accuracy to serum LAM ELISA, although the milk LAM ELISA was slightly less reproducible (higher coefficient of variation).

Animals↗
PubMed2002

Mycobacterium paratuberculosis heat shock protein 70 as a tool in control of paratuberculosis.

Paratuberculosis in cattle is a chronic intestinal disease in which a distinctive cellular reactivity of a Th1-type preceeds the phase in which antibody titers are easily detectable and the animal becomes clinically ill. During infection with Mycobacterium avium ssp. paratuberculosis (M.a.p), a decrease in CD4 T-helper cells has been observed in the clinical phase. Our ultimate aim is to elicit a cytotoxic reaction against infected macrophages, using recombinant Hsp70 (rHsp70) of M.a.p. as a tool to shuttle antigen into the MHC class I antigen presentation pathway. To investigate the mechanism of rHsp70 as a carrier for antigen into the cell, we studied the interaction between APC and Fitc-labelled rHsp70, using FACS analysis and confocal microscopy. Interaction of rHsp70 with the cell surface of bovine APC, presumably via a receptor, was shown on monocytes, monocyte derived macrophages and dendritic cell (DC). The interaction is detectable on the complete population of freshly derived monocytes, although peak intensity of fluorescence is lower on these cells than on macrophages and DCs. DCs show interaction on a high percentage of the cells, with high intensity, while in the case of macrophages only a subpopulation interacts with rHsp70. Efficient uptake of rHsp70 as compared to OVA is shown. Preincubation of DC with unlabelled rHsp70 leads to a decreased interaction with rHsp70-FITC. DC interacting with rHsp70 in addition showed high expression of MHC I, MHC II, Myd-1 (CD172a) and CD40. Further research will focus on loading of the rHsp70 with M.a.p. antigen for presentation in MHC class I.

Animals↗
PubMed2001

Typing of IS 1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock.

Isolates of Mycobacterium avium subsp. paratuberculosis from nine bison (Bison bison) from Montana, United States of America, were compared with those from other species from the United States of America. All 16 isolates had a novel IS 1311 genotype in which all copies of the element possessed a thymidine-cytosine nucleotide variation at base position 223. Isolates from bison were termed B strain. Thirteen isolates from cattle and goats were polymorphic at this locus, a status previously recognised in isolates from cattle which are termed C strains. Differences in cultural phenotype between B and C strains which were noted upon primary isolation did not manifest upon subculture to Herrold's egg yolk medium or modified Middlebrook 7H10 agar. Rapid differentiation of these strains from each other and from S strains from sheep is possible using polymerase chain reaction and restriction endonuclease analysis. The epidemiology of paratuberculosis in bison in Montana appears to be distinct from that in cattle and other farmed livestock investigated thus far.

Animals↗
PubMed2001

Immune reactions in cattle after immunization with a Mycobacterium paratuberculosis vaccine and implications for the diagnosis of M. paratuberculosis and M. bovis infections.

After immunization of four calves with a live modified Mycobacterium paratuberculosis vaccine the course of the humoral and cell mediated immune reactions was studied during a 2-year clinical investigation. Furthermore, the possibility of shedding of the vaccine strain and the influence of the vaccination on the tuberculin skin test was determined. In addition to standard procedures recently developed diagnostic methods (antibody enzyme-linked immunosorbent assay, interferon-gamma test, polymerase chain reaction) were used. A cell-mediated immune reaction, reflected in an increased, specifically induced, interferon-gamma production developed much earlier (1-2 weeks post-immunization) than humoral immunity (8-16 weeks post-gamma immunization). While the increase in antibody titres was transient, declining to extremely low levels 48-60 weeks post-immunization, cell-mediated immunity remained detectable until the end of the investigation. Spread of the vaccine strain into the body and shedding were never detected during the whole course of the study except for one colon site in one calf. As late as 2 years after vaccine application positive or doubtful skin reactions against M. bovis purified protein derivative were measured, reflecting possible interference of the immunization with the diagnosis of bovine tuberculosis. At the end of the investigation, a positive cell-mediated immune reaction was detected the control animal although clinical, pathological and bacteriological examinations gave no indication for a mycobacterial infection.

Animals↗
PubMed2005

Evaluation of the gamma interferon test for diagnosis of paratuberculosis in goats.

The gamma interferon assay was evaluated for diagnosis of paratuberculosis in goats with special emphasis on false positive reactions. Four categories of herds were tested: (A) herds that had a history of paratuberculosis, had given positive Mycobacterium avium subsp. paratuberculosis fecal samples and were vaccinated against paratuberculosis; (B) herds that had been vaccinated but had never shown clinical signs of paratuberculosis nor given positive M. a. paratuberculosis fecal samples; and (C) non-vaccinated herds without paratuberculosis. To extend the analysis of samples from young goats free of paratuberculosis, animals less than 18 months of age from non-vaccinated herds without paratuberculosis, category D, were included. Heparinized blood was stimulated with purified protein derivate (PPD) from M. a. paratuberculosis for 24 h and plasma was assayed for the presence of gamma interferon. Results were recorded as the difference between OD values of PPD stimulated and control samples. Vaccinated animals from herds with paratuberculosis, category A, showed significant higher gamma interferon responses than animals from vaccinated herds without paratuberculosis, category B. In both these groups the responses were correlated to age with higher responses in younger animals. Some of the vaccinated animals in herds without paratuberculosis had a gamma interferon response lasting for several years, which demonstrate a long lasting interference with diagnostic testing in vaccinated goats. Only three of the 121 non-vaccinated animals free of paratuberculosis in category C had responses against PPD (corrected OD values at 0.2, 0.24 and 0.5), and none of the 255 young animals in category D had corrected OD values exceeding 0.2. This indicates that false positive reactions do not appear to the same extent in young goats as in young cattle. We conclude that the low responses of non-infected goats could make the gamma interferon assay useful in monitoring the paratuberculosis status of non-vaccinated herds. However, more information about the early gamma interferon responses of naturally infected goats and the presence of false negative samples are needed.

Animals↗
PubMed2006

The ability of Mycobacterium avium subsp. paratuberculosis to enter bovine epithelial cells is influenced by preexposure to a hyperosmolar environment and intracellular passage in bovine mammary epithelial cells.

Mycobacterium avium subsp. paratuberculosis is the cause of Johne's disease in cattle and other ruminants. M. avium subsp. paratuberculosis infection of the bovine host is not well understood; however, it is assumed that crossing the bovine intestinal mucosa is important in order for M. avium subsp. paratuberculosis to establish infection. To examine the ability of M. avium subsp. paratuberculosis to infect bovine epithelial cells in vitro, Madin-Darby bovine kidney (MDBK) epithelial cells were exposed to M. avium subsp. paratuberculosis. It was observed that bacteria can establish infection and replicate within MDBK cells. M. avium subsp. paratuberculosis also has been reported to infect mammary tissue and milk, and we showed that M. avium subsp. paratuberculosis infects bovine mammary epithelial cells (MAC-T cell line). Using polarized MAC-T cell monolayers, it was also determined that M. avium subsp. paratuberculosis crosses apical and basolateral surfaces with approximately the same degree of efficiency. Because M. avium subsp. paratuberculosis can be delivered to the naïve host by milk, it was investigated whether incubation of M. avium subsp. paratuberculosis with milk has an effect on invasion of MDBK cells. M. avium subsp. paratuberculosis exposed to milk entered epithelial cells with greater efficiency than M. avium subsp. paratuberculosis exposed to broth medium or water (P < 0.01). Growth of M. avium subsp. paratuberculosis within MAC-T cells also resulted in augmented ability to subsequently infect bovine MDBK cells (P < 0.001). Microarray analysis of intracellular M. avium subsp. paratuberculosis RNA indicates the increased transcription of genes which might be associated with an invasive phenotype.

Animals↗
PubMed2004

Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein facilitates M-cell targeting and invasion through a fibronectin bridge with host integrins.

Efficient attachment and ingestion of Mycobacterium avium subsp. paratuberculosis by cultured epithelial cells requires the expression of a fibronectin (FN) attachment protein homologue (FAP-P) which mediates FN binding by M. avium subsp. paratuberculosis. Invasion of Peyer's patches by M. avium subsp. paratuberculosis occurs through M cells, which, unlike other intestinal epithelial cells, express integrins on their luminal faces. We sought to determine if the interaction between FAP-P of M. avium subsp. paratuberculosis and soluble FN enabled targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo via these surface integrins. Wild-type and antisense FAP-P mutant M. avium subsp. paratuberculosis strains were injected alone or coinjected with blocking peptides or antibodies into murine gut loops, and immunofluorescence microscopy was performed to assess targeting and invasion of M cells by M. avium subsp. paratuberculosis. Nonopsonized M. avium subsp. paratuberculosis preferentially invaded M cells in murine gut loops. M-cell invasion was enhanced 2.6-fold when M. avium subsp. paratuberculosis was pretreated with FN. Invasion of M cells by the antisense FAP-P mutant of M. avium subsp. paratuberculosis was reduced by 77 to 90% relative to that observed for the control strains. Peptides corresponding to the RGD and synergy site integrin recognition regions of FN blocked M. avium subsp. paratuberculosis invasion of M cells by 75 and 45%, respectively, whereas the connecting segment 1 peptide was noninhibitory. Antibodies against the alpha5, alphaV, beta1, and beta3 integrin subunits inhibited M-cell invasion by 52 to 73%. The results indicate that targeting and invasion of M cells by M. avium subsp. paratuberculosis in vivo is mediated primarily by the formation of an FN bridge formed between FAP-P of M. avium subsp. paratuberculosis and integrins on M cells.

Adhesins, Bacterial↗
PubMed2004

Rapid and transient activation of gene expression in peripheral blood mononuclear cells from Johne's disease positive cows exposed to Mycobacterium paratuberculosis in vitro.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is the causative agent of Johne's disease in ruminants. M. paratuberculosis is a slow-growing intracellular bacterium and infections with M. paratuberculosis can persist in a subclinical state for several years. An early and appropriate T cell-mediated cytotoxic response (Th1-like) to M. paratuberculosis infection is often replaced with an antibody or Th 2-like response as infected animals move toward a progressively more clinical state. The reasons for this shift in immune response are unknown. Recent studies suggest that in vitro exposure of peripheral blood mononuclear cells (PBMCs) from Johne's disease positive cows to M. paratuberculosis for 18-24 h results in suppressed expression of numerous immune cell genes. This effect appears at odds with the notion that immune cells from infected cows would respond to M. paratuberculosis-specific antigens in a vigorous and positive manner. In this report, we detail experiments designed to test the hypothesis that many positive changes in PBMC gene expression induced by M. paratuberculosis in vitro are transient, being rapidly suppressed by as yet unknown mechanisms. Our results demonstrate that, indeed, in vitro stimulation with M. paratuberculosis induces rapid changes in infected cow PBMC gene expression (within 2-4 h of exposure) and that many of these changes are no longer evident by 8-16 h of exposure to M. paratuberculosis. Although precise mechanisms responsible for rapid M. paratuberculosis-mediated activation of PBMC gene expression and the loss thereof remain to be determined, our novel results suggest that PBMCs from Johne's disease positive cows are indeed capable of vigorously responding to M. paratuberculosis and that, for many genes, this response is tempered within 8 h of exposure.

Animals↗
PubMed2004

Phagocytosis of M. paratuberculosis fails to activate expression of NADH dehydrogenase and nucleolin-related protein in bovine macrophages.

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular bacterium and causal agent of Johne's disease in cattle. Following phagocytosis, M. paratuberculosis resides and replicates in macrophage phagosomes that fail to mature. Differential display reverse transcription polymerase chain reaction (DDRT-PCR) was used as a high throughput initial screen to begin to test the hypothesis that macrophage gene expression patterns would be differentially affected by M. paratuberculosis when compared to readily degraded bacteria or non-degradable latex beads. Gene expression profiles from immortalized bovine macrophage cells (BOMAC) exposed to M. paratuberculosis were compared to gene expression profiles for BOMAC cells exposed to Escherichia coli, latex beads or PBS. Amplicons representing genes specifically activated or repressed during M. paratuberculosis phagocytosis were cloned for further investigation. Northern blot hybridizations preformed using DDRT-PCR-derived amplicons 3-1-4, 5-2-10, 5-4-2 and 4-1-6 confirmed stimuli dependent differential gene expression. Expression pattern observed for amplicon 3-1-4 represents genes that are up-regulated following phagocytosis of E. coli or latex beads, but not M. paratuberculosis. Amplicon 5-2-10 exhibited a pattern of expression representative of genes that are up-regulated strongly following phagocytosis of E. coli or latex beads but only moderately following M. paratuberculosis phagocytosis. Expression pattern of the gene for amplicon 5-4-2 was representative of genes that are specifically suppressed following M. paratuberculosis phagocytosis, while the amplicon 4-1-6 gene expression pattern represented genes that are generally suppressed following phagocytosis of any of the three stimuli. DNA sequencing and Genbank database analysis of these amplicons revealed that amplicon 3-1-4, whose expression failed to activate following M. paratuberculosis phagocytosis, had high levels of similarity to a Rattus norvegicus nucleolin-related protein (NRP). Amplicon 5-2-10, which increased expression moderately following M. paratuberculosis phagocytosis, was a near perfect match to bovine nicotinamide adenine dinucleotide dehydrogenase (FNADH dehydrogenase) subunit 1 (ND1). Failure to activate these two genes at levels observed following phagocytosis of either E. coli or latex beads may uncover new mechanisms for the survival of M. paratuberculosis within bovine macrophage cells.

Animals↗
PubMed2003

Variation in resistance of Mycobacterium paratuberculosis to acid environments as a function of culture medium.

Acid resistance of Mycobacterium paratuberculosis was examined as a function of growth conditions (i.e., in vitro growth medium and pH). M. paratuberculosis was cultured in either fatty acid-containing medium (7H9-OADC) or glycerol-containing medium (WR-GD or 7H9-GD) at two culture pHs (pHs 6.0 and 6.8). Organisms produced in these six medium and pH conditions were then tested for resistance to acetate buffer at pHs 3, 4, 5, and 6 at 20 degrees C. A radiometric culture method (BACTEC) was used to quantify viable M. paratuberculosis cell data at various acid exposure times, and D values (decimal reduction times, or the times required to kill a 1-log(10) concentration of bacteria) were determined. Soluble proteins of M. paratuberculosis grown under all six conditions were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify proteins that may be associated with acid resistance or susceptibility. The culture medium affected growth rate and morphology: thin floating sheets of cells were observed in 7H9-OADC versus confluent, thick, waxy, and wrinkled pellicles in WR-GD. Culture medium pH affected growth rate (which was highest at pH 6.0), but it had little or no effect on D values for M. paratuberculosis at any test pH. When grown in 7H9-OADC, M. paratuberculosis was more acid resistant at all test pHs (higher D values) than when grown in WR-GD. Glycerol appeared to be the culture medium component most responsible for lower levels of M. paratuberculosis acid resistance. When glycerol was substituted for OADC in the 7H9 medium, D values were significantly lower than those of 7H9-OADC-grown M. paratuberculosis and were approximately the same as those for M. paratuberculosis grown in WR-GD medium. Comparison of the SDS-PAGE protein profiles for M. paratuberculosis cultures grown in 7H9-OADC, WR-GD, or 7H9-GD medium revealed that increased expression of 34.2- and 14.0-kDa proteins was associated with higher levels of acid resistance of M. paratuberculosis grown in 7H9-OADC medium and that 56.6- and 41.3-kDa proteins were associated with lower levels of acid resistance. This is the first report showing that in vitro culture conditions significantly affect growth characteristics, acid resistance, and protein expression of M. paratuberculosis, and the results emphasize the importance of culture conditions for in vitro susceptibility studies.

Acetates↗
PubMed1993

Association of Mycobacterium paratuberculosis infection with reduced mastitis, but with decreased milk production and increased cull rate in clinically normal dairy cows.

Approximately 45 Holstein cows that were Mycobacterium paratuberculosis-positive on the basis of fecal culture results were maintained at any one time in a 210-cow dairy herd. Farm management participated in the New York State Paratuberculosis Eradication Program. Paratuberculosis-positive cows were grouped separately from paratuberculosis-negative cows, but they were otherwise managed identically. During a 1-year study, 180 paratuberculosis-negative cows and 113 clinically normal paratuberculosis-positive cows were identified. Quarter milk samples (n = 6,100) were aseptically collected for microbiologic culture of mastitis pathogens from paratuberculosis-negative cows, and 3,129 quarter samples were obtained from paratuberculosis-positive cows. Dairy Herd Improvement Association (DHIA) records were used to monitor milk somatic cell count linear scores, mature equivalent milk production, new mastitis infections, and chronic mastitis infections. For second-lactation cows greater than 100 days in milk production, and increasing with age beyond that point, paratuberculosis-positive cows had lower mature equivalent milk production than did negative herdmates. Rates of new and chronic mastitis infections, as measured by DHIA linear scores were significantly (P < 0.05, P = 0.05, respectively) lower in cows with nonclinical paratuberculosis. Infected cows were culled from the herd at a faster rate than were paratuberculosis-negative herdmates. Therefore, paratuberculosis was associated with financial loss attributable to reduced milk production and increased culling of infected cows.

Animals↗