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PubMed1999

Hormonal modulation of phagocytosis and intracellular growth of Mycobacterium avium ss. paratuberculosis In bovine peripheral blood monocytes.

In this study, we evaluated the effects of several hormones (i.e. growth hormone, prolactin, vitamin D3, luteinizing hormone, oxytocin) on the phagocytosis and intracellular survival of Mycobacterium avium ss. paratuberculosis within bovine peripheral blood monocytes. Phagocytosis of M. avium ss. paratuberculosis declined in a dose-dependent manner when monocytes were exposed to increasing amounts of recombinant bovine growth hormone, with little phagocytosis occurring at a growth hormone concentration of 50 ng/ml. The other hormones tested had little effect on phagocytosis. Continuous exposure of bovine monocytes to bovine growth hormone (10 ng per ml) resulted in enhanced intracellular bacillary growth. This was detected within 3 days of monocyte infection, and resulted in a 1 Log10 greater number of M. avium ss. paratuberculosis in growth hormone treated, than control, monocytes at 12 days of infection. When monocytes were incubated with growth hormone for only the first 5 days of a 12 day incubation period, a further increase in bacillary multiplication was observed. A similar increase in bacillary multiplication was observed when M. avium ss. paratuberculosis monocytes were incubated with prolactin for the first 5 days of a 12 day incubation period. These data indicate that varying levels of growth hormone and prolactin can affect the intracellular multiplication of M. avium ss. paratuberculosis in bovine monocytes.

Animals↗
PubMed1996

No Mycobacterium paratuberculosis found in Crohn's disease using polymerase chain reaction.

M. paratuberculosis has been considered as a putative causative factor of Crohn's disease. However, its detection in diseased tissue samples using the polymerase chain reaction yielded conflicting results. We validated this technique for the detection of mycobacteria (any species) and M. paratuberculosis before applying it to 72 intestinal biopsies from patients with Crohn's disease (N = 36), ulcerative colitis (N = 13), and control subjects (N = 23). Possible polymerase chain reaction inhibitors were detected by spiking template DNA with the equivalent of two M. paratuberculosis genomes. Mycobacteria were found in 17/36 (47%), 6/13 (46%), and 13/23 (57%) tissue samples of Crohn's disease, ulcerative colitis, and controls, respectively. No M. paratuberculosis were detected in any sample. It is concluded that mycobacteria are present with a similar frequency in the intestinal tissues or luminal inclusions of patients with inflammatory bowel disease and of those unaffected by the disease. Our data do not support a role for M. paratuberculosis in Crohn's disease.

Adult↗
PubMed1988

Intracellular iron storage and the pathogenesis of paratuberculosis. Comparative studies with other mycobacterial, parasitic or infectious conditions of veterinary importance.

The distribution of iron and mycobacteria was examined in the intestinal tract of ruminants with naturally-occurring M. paratuberculosis infection and compared with mycobacterial infections in several species. This distribution was compared with that of iron in chronic lesions caused by other microbial or parasitic agents. In the clinical form of paratuberculosis in cattle, sheep and goats there was marked lymphangiectasis and a high proportion of the granulomatous lesions contained siderotic macrophages with a high mycobacterial content. In cattle with preclinical lesions of granulomatous enteropathy, the greatest number of acid-fast organisms was present in siderotic, non-differentiated, ileo-caecal macrophages; concurrent mast cell-associated allergic enteropathy was also apparent in the duodenum, proximal and mid-ileum of most animals. In paratuberculosis-affected herds, a high proportion of non-productive cows were without classical granulomatous change but had cultural or immunological evidence of M. paratuberculosis infection and similar allergic catarrhal enteropathy of the upper intestinal tract. Interstitial haemorrhage of the ileocaecal valve, with the accumulation of haemosiderin and ferritin in undifferentiated macrophages was observed in some of these cattle and also in others with experimentally-induced copper deficiency and acute ostertagiasis. Colonisation of the ileo-caecal or caecal glandular crypts by large, apparently saprophytic acid-fast organisms indicated regional tolerance to such organisms in all cattle. In other mycobacterioses such as bovine or avian tuberculosis, undifferentiated, siderotic macrophages containing mycobacteria were also seen in early granulomas, but epithelioid and giant cell differentiation invariably led to the disappearance of intracellular iron and a reduction in mycobacterial numbers. In possums in which epithelioid and giant cells did not occur in response to M. bovis infection, siderosis persisted in many macrophages and overwhelming mycobacterial multiplication occurred. These studies indicate that, in most infections with mycobacteria, differentiation of macrophages radically reverses their iron acquisitive properties, creating an intracellular environment unsuitable for mycobacterial multiplication. It seems likely that allergically mediated microvascular haemorrhage, local tolerance of commensal mycobacteria and attenuation of the macrophage siderosis reversal mechanism provide unique conditions for early, uninhibited, intracellular multiplication of M. paratuberculosis in the ileo-caecal valve of certain mature ruminants.

Animals↗
PubMed1993

Effects of administration of anti-CD4 and anti-CD8 monoclonal antibodies on Mycobacterium paratuberculosis infection in intragastrically challenged mice.

In this study, we examined the roles of CD4+ and CD8+ cells in resistance to experimental paratuberculosis. Mice received purified anti-CD4 or anti-CD8 monoclonal antibodies before intragastric challenge with Mycobacterium paratuberculosis and on a biweekly basis for six months. This resulted in sustained depletion of CD4+ and CD8+ cells as verified by flow cytometry analysis of spleen cells from M. paratuberculosis infected mice. Depletion of CD4+ or CD8+ cells did not enhance fecal shedding of M. paratuberculosis, bacillary multiplication in the liver and ceca, nor histopathologic damage to the intestinal tract, mesenteric lymph nodes, spleen and liver. These data suggest that cells other than CD4+ or CD8+ cells are important for host defense in experimental paratuberculosis.

Animals↗
PubMed1989

Analysis of restriction endonuclease fragment patterns of DNA from Mycobacterium paratuberculosis.

The DNA from 31 isolates and a reference strain of Mycobacterium paratuberculosis was digested individually with restriction endonucleases BstE II and Pst I. DNA fragments were separated by gel electrophoresis and analyzed. The isolates were from 23 American states, Argentina and Nova Scotia. Twenty-seven were isolated from cattle, two from goats and two from sheep. With the exception of one isolate from cattle, all had restriction endonuclease fragment patterns identical to the fragment patterns for the reference strain, M. paratuberculosis ATCC 19698T. These results confirm other reports and indicate that organisms identified as typical M. paratuberculosis isolates are genetically very similar. It may be possible to use restriction endonuclease analysis to differentiate isolates of M. paratuberculosis from other slowly growing mycobacteria. The genetic similarity also indicates that it may be possible to develop a diagnostic probe that is specific for M. paratuberculosis.

Animals↗
PubMed1991

A DNA probe for the detection of Mycobacterium paratuberculosis.

A genomic library of DNA extracted from Mycobacterium paratuberculosis was constructed in the expression vector lambda gt 11. The library was screened by plaque hybridization with labelled M. paratuberculosis genomic DNA as probe. Strongly hybridizing plaques were isolated and their DNA extracted and characterised for M. paratuberculosis specificity by hybridization to DNA from other Mycobacteriaceae. A clone was obtained which was specific for M. paratuberculosis. DNA from this clone could detect 7 ng M. paratuberculosis DNA.

Animals↗
PubMed1992

The cellular immunology of bovine paratuberculosis: the predominant response is mediated by cytotoxic gamma/delta T lymphocytes which prevent CD4+ activity.

Peripheral blood T-cell subsets were obtained from an experimentally sensitized bovine and from nine bovines naturally infected with Mycobacterium paratuberculosis and tested for their ability to respond to antigen. It was determined that following antigen challenge, proliferative responses followed a biphasic pattern: an initial CD4+ proliferative response, a period of anergy, and a final response governed by gamma/delta T lymphocytes. The anergic phase was characterized by a dramatic drop in peripheral blood CD4+ cells; the nature of the non-responsiveness could not be determined. The anergic phase was followed by increased proliferative responses of non-MHC restricted gamma/delta T lymphocytes. Although CD4+ cells had the ability to proliferate in response to M. paratuberculosis antigens in the absence of gamma/delta T cells, antigen-primed CD4+ lymphocytes failed to incorporate [3H]-thymidine in the presence of gamma/delta T cells and M. paratuberculosis antigen. It was concluded that M. paratuberculosis-specific gamma/delta T lymphocytes have immunoregulatory function and exhibit cytotoxic activity against antigen-primed CD4+ helper cells. The data suggest that the inability of effector cell populations to prevent intracellular proliferation of M. paratuberculosis may be a result of cytotoxic killing of the T helper lymphocyte population required for macrophage activation.

Animals↗
PubMed2004

A molecular beacon-based real-time NASBA assay for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.

A molecular beacon-based real-time NASBA assay for detection and identification of Mycobacterium avium subsp. paratuberculosis has been developed. It targets and amplifies sequences from the dnaA gene which are specific for this bacterium. The assay includes an internal amplification control, to allow identification of inhibited reactions. The assay was tested against 18 isolates of M. avium subsp. paratuberculosis, 17 other mycobacterial strains and 25 non-mycobacterial strains, and was fully selective in that it detected all the targets but none of the non-targets. The lowest number of cells which the assay can detect with 99% probability is 150-200 cells per reaction (as determined using pure culture suspensions). Using centrifugation and nucleic acid extraction as sample treatment, the assay was able to consistently detect 10(3) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(4) M. avium subsp. paratuberculosis cells in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium subsp. paratuberculosis.

Animals↗
PubMed2005

Real-time PCR-based methods for detection of Mycobacterium avium subsp. paratuberculosis in water and milk.

A real-time PCR assay for quantitative detection of Mycobacterium avium paratuberculosis has been developed. It targets and amplifies sequences from the IS900 insertion element which is specific for this bacterium, and includes an internal amplification control. The assay was tested against 18 isolates of M. avium paratuberculosis, 17 other mycobacterial strains, and 25 non-mycobacterial strains, and was fully selective. It is capable of detecting <3 genomic DNA copies with 99% probability or alternatively, using cells directly in the reaction, 12 cells can be detected with 99% probability. Using prior centrifugation, the assay was able to consistently and quantifiably detect 10(2) M. avium paratuberculosis cells in 20 ml artificially contaminated drinking water. With a simple detergent and enzymatic sample pretreatment before centrifugation and nucleic acid extraction, the assay was able to consistently detect 10(2) M. avium paratuberculosis in 20 ml artificially contaminated semi-skimmed milk. The assay will be a useful addition to the range of diagnostic tools available for the study of M. avium paratuberculosis.

Animals↗
PubMed2004

A novel IS element, ISMpa1, in Mycobacterium avium subsp. paratuberculosis.

A novel insertion element belonging to the IS110 family was identified in Mycobacterium avium subsp. paratuberculosis. The IS element, ISMpa1, is 1500 bp and has one ORF encoding a putative transposase. Three copies of ISMpa1 were identified in the M. avium subsp. paratuberculosis genome. The element had inserted into the 3' end of the highly conserved mycobacterial genes prrB and a homologue of M. tuberculosis Rv1593c, and between a putative cytochrome p450 oxygenase and a putative hydrolase. The IS element was present in all (n = 11) M. avium subsp. paratuberculosis strains but not detected in most other mycobacterial species examined, including 10 M. avium subsp. avium isolates of human, avian and porcine origin. However two porcine isolates of M. avium subsp. avium and the reference strain IWGMT49 did harbour ISMpa1. These three strains belong to a previously described subgroup of M. avium subsp. avium based on IS1245 restriction fragment length polymorphism (RFLP) pattern and serovars. All of the M. avium subsp. paratuberculosis strains examined had an identical RFLP pattern when probed with sequences corresponding to the 5' end of ISMpa1, whereas a different pattern was seen in the positive M. avium subsp. avium strains. This novel IS element might be a useful tool in strain classification of M. avium subsp. avium and also for the identification of M. avium subsp. paratuberculosis when used in combination with IS900.

DNA Transposable Elements↗
PubMed2000

Binding of bovine growth hormone to. Mycobacterium avium ss paratuberculosis.

Mycobacterium avium ss paratuberculosis causes a chronic progressive enteritis in cattle and other ruminants referred to as Johne's disease. It also has been suggested by some as possibly being associated with Crohn's disease in humans. In a previous study we observed that incubation of bovine monocytes with recombinant bovine growth hormone (bGH) altered the ingestion and intracellular growth of M. avium ss paratuberculosis in vitro. This led us to investigate whether bGH also has a direct effect on M. avium ss paratuberculosis. We observed that addition of bGH (5 microg/ml) had a direct inhibitory effect on the growth of M. avium ss paratuberculosis in Middlebrook 7H9 broth. In contrast, the growth of Mycobacterium smegmatis was unaffected, even at a bGH concentration of 50 microg/ml. Using 125I-bGH we observed high affinity binding (Kd = 1.32 nM) of bGH to M. avium ss paratuberculosis, with an estimated 204 binding sites per bacillus. To the best of our knowledge, this is the first report of a mammalian hormone binding to this important enteric pathogen.

Animals↗
PubMed2001

An evaluation of a modified interferon-gamma assay for the detection of paratuberculosis in dairy herds.

Whole blood samples were obtained from multiple dairy herds in Pennsylvannia and in Wisconsin which were previously determined to be infected with Mycobacterium paratuberculosis (MpS) (Johne's disease) by fecal culture. Blood samples were shipped overnight to the National Animal Disease Center (NADC) in Ames, IA for processing and interferon-gamma (IFN-gamma) analysis. Blood samples were incubated alone (non-stimulated) or with concanavalin A (ConA), a T-cell mitogen used as a positive control in the assay, for 18h. In addition, samples were incubated with M. avium purified protein derivative (AvPPD), M. bovis purified protein derivative (BoPPD), or a whole cell sonicate of M. paratuberculosis for 18h to elicit antigen-specific IFN-gamma production. After incubation, plasma was harvested and analyzed for IFN-gamma by ELISA. Values for IFN-gamma for non-stimulated blood samples (background) were consistently low for animals in all herds evaluated. In contrast, ConA stimulation of blood samples evoked a significant secretion of IFN-gamma regardless of infection status or fecal culture results for individual cows, indicating that immune cells were still viable after overnight shipment and capable of responding to stimulation. Antigen-specific IFN-gamma results were positively correlated with infection status as determined by previous fecal shedding and/or current fecal shedding of M. paratuberculosis. Accuracy of the IFN-gamma assay for correctly predicting infection status of individual cows in the herds with low levels of infection ranged from 50 to 75% when used as a single test. Combined use of the IFN-gamma test and a commercial ELISA antibody test accurately predicted infection status of 73% of cows from a dairy herd with a high level of M. paratuberculosis infection and 90% from a well-characterized group of dairy cows at the NADC. These results indicate that the antigen-specific IFN-gamma assay is a very sensitive diagnostic tool for detection of subclinical paratuberculosis in cattle and may be useful on an individual animal basis to remove infected animals from the herd.

Animals↗
PubMed2001

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis.

Cytokine gene expression in ileal tissues of cattle infected with Mycobacterium paratuberculosis was evaluated. The effects of infection with M. paratuberculosis on cytokine production may influence immune regulation at the site of colonization, resulting in the chronic inflammatory state associated with the latter stages of this disease. Ileal samples were obtained at necropsy from noninfected control cows (n=8) and clinically infected cows (n=7) and processed for immunohistochemistry and in situ hybridization. Cows infected with M. paratuberculosis were in the latter stages of disease with clinical signs such as weight loss, watery diarrhea, and inappetence. Among cytokines we studied, interleukin-1alpha (IL-1alpha), IL-1beta, IL-6, and interferon-gamma (IFN-gamma) were expressed significantly more in infected animals than in noninfected control animals. The expression of tumor necrosis factor-alpha (TNF-alpha), however, was not different between the two groups of cattle. In addition, immunohistochemical staining demonstrated that the number of resident macrophages in the ileum of infected animals was three times greater than that of noninfected cows. In contrast to this, ileal tissues from noninfected control animals contained 1.5 times more neutrophils than the ileal tissues from cows infected with M. paratuberculosis. These data demonstrate that localized ileal cytokine production is different between cows chronically infected with M. paratuberculosis and noninfected control cows.

Animals↗
PubMed2002

Survival tactics of M. paratuberculosis in bovine macrophage cells.

Mycobacterium paratuberculosis (M. paratuberculosis) is a facultative intracellular bacterium with the ability to survive and proliferate inside the vesicles of macrophage cells. How M. paratuberculosis and other mycobacteria survive in this hostile environment is not well understood. Present research findings can be divided into three possibilities: (1) mycobacteria may interfere with host protein expression and trafficking to stop vesicle maturation, (2) mycobacteria may express proteins that interfere with macrophage activation in a more direct manner, or (3) mycobacteria may enter macrophages in such a way as to avoid the normal process of activation via Toll-like receptors and other, as yet unknown mechanisms. Research thus far has predominately centered on possible macrophage/mycobacteria protein interactions. To more completely define how mycobacteria interfere with the process of phagosome maturation our group has recently taken a functional genomics approach, allowing the macrophage to "tell" us what host genes may be affected by phagocytosis of mycobacteria. We used DDRT-PCR to examine differences in macrophage cell gene expression during phagocytosis of E. coli and M. paratuberculosis. Macrophage cells not exposed to any phagocytosis target and in the process of phagocytosing latex beads were used as negative and positive controls, respectively. To date, we have identified 380 DDRT-PCR amplicons corresponding to transcripts whose expression profiles appear to be altered during the general process of phagocytosis. Dot-blot and Northern blot hybridizations with a subset of these amplicons were performed to confirm results observed with DDRT-PCR. Our preliminary results indicate that macrophage gene expression profiles change dramatically following phagocytosis and that gene expression profiles following phagocytosis of M. paratuberculosis are different than those following phagocytosis of E. coli or latex beads.

Animals↗
PubMed2000

Dutch paratuberculosis programme history, principles and development.

Organised disease control started in the Netherlands in the 18th century with governmental attempts to eradicate cattle plague. At the beginning of the 20th century, the dairy industry and cattle breeding organisations initiated a programme to control infectious diseases by means of a complex system of rewards and penalties. This was also the reason for establishing the Animal Health Service in Friesland in 1919. The history of programmes to control paratuberculosis in sheep, goats, and cattle in various countries is described. The vaccination of young animals seems to be an effective measure in the prevention of clinical paratuberculosis, although changes in management and hygiene practices are also important. A control programme for infectious cattle diseases has a number of phases (a lifecycle) and different components. Two components are essential for success, namely: open and regular communication with farmers, veterinary practitioners, and other people involved and a good registration and identification system for cattle, herds, and veterinary practitioners. The Dutch paratuberculosis programme has 10 herd status levels: 5-10 for non-suspect herds and 1-4 for infected herds or herds of unknown status. The higher the status, the greater the chance that a herd is free of paratuberculosis. An outline is given of the Dutch paratuberculosis programme including its objectives, basic principles for eradication, communication plan, legal action, logistic considerations, and complementary research programme.

Animal Husbandry↗
PubMed2001

Incidence of Mycobacterium paratuberculosis in raw sheep and goats' milk in England, Wales and Northern Ireland.

A blind survey of 104 raw sheep and goats' milk samples (90 goat, 14 sheeps) from bulk tanks on farms throughout England, Wales and Northern Ireland was carried out over a 5-month period (January-May 1998) in order to determine the incidence of Mycobacterium paratuberculosis. Each milk sample (100 ml) was divided into two 50ml portions. One portion was decontaminated with 0.75% hexadecylpyridinium chloride for 5h before culture on slopes of Herrold's egg yolk medium and in BACTEC radiometric medium. The second portion was subjected to immunomagnetic separation followed by IS900 PCR (IMS-PCR). The IMS-PCR assay was employed in order to provide a more rapid indication of the presence of M. paratuberculosis in each milk sample than is possible by culture. Information on the Johne's disease status of the sheep and goat herds that took part in the survey was not sought at the time of milk sampling. However, it subsequently emerged that at least some of the herds whose bulk milk was tested during this study were previously or currently infected with Johne's disease. Overall, during this survey one raw goats' milk sample tested positive for the presence of M. paratuberculosis by IMS-PCR (<1% of milk samples tested) but no viable M. paratuberculosis were isolated by culture. The results of this study suggest that bulk raw sheep and goats' milk from these regions of the UK may not represent significant vehicles of transmission of M. paratuberculosis to humans.

Animals↗
PubMed1999

Effect of higher pasteurization temperatures, and longer holding times at 72 degrees C, on the inactivation of Mycobacterium paratuberculosis in milk.

Raw cow's milk spiked with 10(6) cfu ml-1 of Mycobacterium paratuberculosis was subjected to heat treatments of 72, 75, 78, 80, 85 or 90 degrees C for 15 s, and 72 degrees C for 20 and 25 s, using laboratory pasteurizing units. Three bovine strains of Myco. paratuberculosis were studied (NCTC 8578, B2 and DVL 943). Each strain was subjected to all the heat treatments indicated on three separate occasions. Although each of the heat treatments achieved a substantial (5-6 log10) reduction in numbers of viable Myco. paratuberculosis, small numbers of the organism (4-16 cfu 10 ml-1) survived in a proportion of the milk samples at each of the higher temperatures investigated, right up to 90 degrees C for 15 s. A longer holding time of 25 s at 72 degrees C was found to be more effective at inactivating Myco. paratuberculosis. Only one of the three strains studied, B2, yielded small numbers of survivors after heating at 72 degrees C for 20 s, but it was completely inactivated by extending the holding time at 72 degrees C by a further 5 s to 25 s. It was concluded that a longer holding time is more likely to achieve the complete inactivation of Myco. paratuberculosis in milk than a higher pasteurization temperature.

Animals↗
PubMed1998

Effect of high-temperature, short-time (HTST) pasteurization on milk containing low numbers of Mycobacterium paratuberculosis.

The efficacy of high-temperature, short-time (HTST) pasteurization (72 degrees C/15 s) when low numbers (< or = 10(3) cfu ml-1) of Mycobacterium paratuberculosis are present in milk was investigated. Raw cows' milk spiked with Myco. paratuberculosis (10(3) cfu ml-1, 10(2) cfu ml-1, 10 cfu ml-1, and 10 cfu 50 ml-1) was subjected to HTST pasteurization using laboratory pasteurizing units. Ten bovine strains of Myco. paratuberculosis were tested in triplicate. Culture in BACTEC Middlebrook 12B radiometric medium detected acid-fast survivors in 14.8% and 10% of HTST-pasteurized milk samples at the 10(3) and 10(2) cfu ml-1 inoculum levels, respectively, whereas conventional culture on Herrold's egg yolk medium containing mycobactin J detected acid-fast survivors in only 3.7% and 6.7% of the same milk samples. IS900-based PCR confirmed that these acid-fast survivors were Myco. paratuberculosis. No viable Myco. paratuberculosis were isolated from HTST-pasteurized milk initially containing either 10 cfu ml-1 or 10 cfu 50 ml-1.

Animals↗