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PubMed2006

Development of luminescent Mycobacterium avium subsp. paratuberculosis for rapid screening of vaccine candidates in mice.

Mycobacterium avium subsp. paratuberculosis is a slowly growing mycobacterial species, requiring 6 to 8 weeks of culture before colonies can be counted visually. Here, we describe the development of luminescent M. avium subsp. paratuberculosis expressing luxAB genes of Vibrio harveyi and its use for vaccine testing in an experimental mouse model, replacing fastidious CFU counting by rapid luminometry.

Animals↗
PubMed1986

Immunohistochemical distribution of ferritin, lactoferrin, and transferrin in granulomas of bovine paratuberculosis.

Granulomatous lesions of bovine paratuberculosis contained ferritin, lactoferrin, and a small amount of transferrin, as demonstrated by the immunohistochemical method. Macrophages in the normal bovine ileum did not contain lactoferrin and transferrin; however, ferritin was found in individual macrophages of Peyer's patches. These results may help elucidate the relationship between intracellular growth of Mycobacterium paratuberculosis and the presence of iron-binding proteins in the granulomas.

Animals↗
PubMed1997

Study of T-lymphocyte subsets of healthy and Mycobacterium avium subsp. paratuberculosis-infected cattle.

The relative contributions of T-lymphocyte subsets to host defense in cattle infected with Mycobacterium avium subsp. paratuberculosis is reported. The subsets were purified with appropriate monoclonal antibodies and a magnetic bead column separation system, and their purity was verified by flow cytometry. Biological activity of each subset, expressed as lymphoproliferation and gamma interferon (IFN-gamma) production, was measured in response to phytohemagglutinin (PHA) and an M. avium antigen preparation (A-PPD). IFN-gamma was measured by antibody capture enzyme-linked immunosorbent assay. The results showed a correlation between proliferation and IFN-gamma production in response to A-PPD but not to PHA. In response to PHA, CD4+ lymphocytes were the most prolific producers of IFN-gamma. CD8+ lymphocytes produced IFN-gamma to a lesser extent, whereas gammadelta+ T lymphocytes produced little or no IFN-gamma. Differences observed between the amount of IFN-gamma produced by CD4+ versus CD8+ cells and CD4+ versus gammadelta+ cells were significant (P < 0.01), but those between peripheral blood mononuclear cells (PBMC) and CD4+ T cells were not. Similar responses to A-PPD were observed except that PBMC produced higher levels of IFN-gamma than did CD4+ T cells. These data for cattle are similar to observations made for other animal species, where CD4+ cells are the major type of T lymphocytes producing IFN-gamma. They further suggest that whatever the role gammadelta+ T cells may play in paratuberculosis, it is not likely to be mediated by IFN-gamma production.

Animals↗
PubMed1965

UTILIZATION OF EXTERNAL GROWTH FACTORS BY INTRACELLULAR MICROBES: MYCOBACTERIUM PARATUBERCULOSIS AND WOOD PIGEON MYCOBACTERIA.

Wheeler, William C. (Johns Hopkins University-Leonard Wood Memorial Leprosy Research Laboratory, Baltimore, Md.), and John H. Hanks. Utilization of external growth factors by intracellular microbes: Mycobacterium paratuberculosis and wood pigeon mycobacteria. J. Bacteriol. 89:889-896. 1965.-The extent to which the intracellular growth of microbes is dependent upon capacities for growth in vitro has been investigated by use of organisms which require a specific factor, mycobactin, for isolation and growth on conventional mycobacterial media. Similarities between growth on bacteriological media and within tissue cells have been demonstrated by examining the responses of Mycobacterium paratuberculosis and wood pigeon mycobacteria to mycobactin, iron, CO(2), and glycerol, and to the low pH which characterizes phagocytic vacuoles. The results indicate that success of intracellular infections depends upon the independent growth of microbes and that the phagocytic vacuoles of sheep and mouse macrophages seem to be freely accessible to bacterial growth factors, minerals, and substrates. Because these factors did not modify the appearance, overall metabolism, or surface properties of macrophages, it was concluded that the intracellular growth of microbes is not determined solely by the components, metabolism, or immunological properties of host cells, but is influenced to an important degree by compounds and conditions provided by extracellular environments.

Animals↗
PubMed2002

Molecular characterization of pigmented and nonpigmented isolates of Mycobacterium avium subsp. paratuberculosis.

Five pigmented isolates of Mycobacterium avium subsp. paratuberculosis were examined by pulsed-field gel electrophoresis (PFGE), IS900 restriction fragment length polymorphism (IS900-RFLP), and IS1311 polymorphism analysis using PCR. All of the pigmented isolates exhibited one of three distinct PFGE profiles with SnaBI, designated 9, 10, and 11, and with SpeI, designated 7, 8, and 9, which generated three multiplex profiles designated [9-7], [10-8], and [11-9]. All of the pigmented isolates had the same IS900-RFLP BstEII and PvuII profiles. The IS900-RFLP BstEII profile was new, but the IS900-RFLP PvuII profile corresponded to PvuII type 6 of a sheep strain described by Cousins and colleagues (D. V. Cousins, S. N. Williams, A. Hope, and G. J. Eamens, Aust. Vet. J. 78:184-190, 2000). IS1311-PCR analysis typed all of the pigmented isolates as sheep (S) strains. The genetic relationship between pigmented and nonpigmented isolates was investigated by using multiplex PFGE data from the analysis of both the 5 pigmented isolates and 88 nonpigmented isolates of M. avium subsp. paratuberculosis from a variety of host species and geographic locations. It was possible to classify the isolates into two distinct types designated type I, comprising the pigmented isolates, and type II, comprising the nonpigmented isolates, which exhibit a very broad host range.

Animals↗
PubMed1988

Role of M cells and macrophages in the entrance of Mycobacterium paratuberculosis into domes of ileal Peyer's patches in calves.

Ligated ileal loops of calves were inoculated with live and heat-killed Mycobacterium paratuberculosis and were examined by light and electron microscopy. At 5 hours after inoculation, acid-fast bacilli were in subepithelial macrophages, but not in M cells covering domes. At 20 hours, more than 50 acid-fast bacilli per cross section were in subepithelial macrophages in domes. Both living and heat-killed bacilli passed into domes. Addition of anti-M. paratuberculosis bovine serum to the inoculum enhanced entry of bacteria into domes. By electron microscopy, intact bacilli with electron-transparent zones (peribacillary spaces) were in the supranuclear cytoplasm of M cells at 20 hours. M cells also contained vacuoles, including electron-dense material interpreted as degraded bacilli. Subepithelial and intraepithelial macrophages contained bacilli and degraded bacterial material in phagosomes. These results suggest that calf ileal M cells take up bacilli, and that subepithelial and intraepithelial macrophages secondarily accept bacilli or bacterial debris which are expelled from M cells.

Animals↗
PubMed2005

Inflammatory cytokine gene expression in different types of granulomatous lesions during asymptomatic stages of bovine paratuberculosis.

The granulomatous lesions in bovine paratuberculosis have been classified into two types, i.e., the lepromatous type and the tuberculoid type. To clarify the immunopathologic mechanisms at the site of infection, we compared inflammatory cytokine gene expression between the two types of lesions. Samples were obtained from noninfected control cows (n = 5) and naturally infected cows (n = 7) that were diagnosed by enzyme-linked immunosorbent assay (ELISA) and fecal culture test. Although none of the infected cows showed clinical signs, tuberculoid lesions were observed in five cows (tuberculoid group) and lepromatous lesions in two cows (lepromatous group). Among the cytokines examined by reverse transcription-polymerase chain reaction (RT-PCR), Th2-type cytokines interleukin-4 (IL-4) and IL-10, and Th1-type cytokine IL-2 were expressed more significantly in the lepromatous group than in the tuberculoid (P < 0.01) and noninfected groups (P < 0.05). No statistical differences were observed in the expression of interferon-gamma, IL-1 beta, TNF-alpha, and GM-CSF among lepromatous, tuberculoid, and noninfected groups. Expression of proinflammatory cytokine IL-12 mRNA, however, did not differ among the three groups; IL-18 was expressed at lower levels in the lepromatous group than in the tuberculoid group and the noninfected group (P < 0.0001). Moreover, the number of cells in which IL-18 mRNAs were detected by in situ hybridization was markedly decreased in the lepromatous group. These results indicate that the formation of lepromatous-type lesions or tuberculoid-type lesions may be influenced by alterations in Th1/Th2-type cytokine production and that IL-18 may play an important role in a Th1-to-Th2 switch in paratuberculosis.

Animals↗
PubMed2005

Expression of interleukin-10 and suppressor of cytokine signaling-3 associated with susceptibility of cattle to infection with Mycobacterium avium subsp paratuberculosis.

OBJECTIVE: To determine functional characteristics of monocytes obtained from cows with subclinical infection with Mycobacterium avium subsp paratuberculosis (MAP) that may have predisposed those cows to becoming infected with MAP SAMPLE POPULATION: Monocytes obtained from 5 uninfected cows and 5 cows subclinically infected with MAP in a herd with a high prevalence of paratuberculosis (ie, Johne's disease). PROCEDURES: Monocytes from uninfected and subclinically infected cows were incubated with MAP for 2, 6, 24, 72, or 96 hours. Variables measured included expression of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-10, IL-12, transforming growth factor-beta, and suppressor of cytokine signaling-3 (SOCS-3); apoptosis of monocytes; acidification of phagosomes; and killing of MAP. RESULTS: Monocytes from infected cows had greater expression of IL-10 and SOCS-3 at 2 hours of coincubation with MAP and lower expression of TNF-alpha and IL-12 when results for all incubation times were combined. Monocytes from infected cows had a greater capacity to acidify phagosomes. No differences were observed in the rate of apoptosis or capacity of monocytes to kill MAP organisms. CONCLUSIONS AND CLINICAL RELEVANCE: Monocytes obtained from cows with subclinical infection with MAP had upregulated expression of IL-10 and SOCS-3 within the first 2 hours after exposure to MAP organisms. Although this did not inhibit acidification of phagosomes, apoptosis of monocytes, or attenuation of the capacity to kill MAP organisms, it may have attenuated the capacity of mononuclear phagocytes to initiate inflammatory and adaptive immune responses.

Animals↗
PubMed1999

Hepatic encephalopathy associated with paratuberculosis in a goat.

A 3-year-old pregnant Nubian goat that was examined because of weight loss, weakness, and change in attitude was determined to be infected with Mycobacterium paratuberculosis. Signs of depressed attitude, trembling, and ataxia were consistent with hepatic encephalopathy, which was confirmed by detection of hyperammonemia. These signs were consistent with histopathologic lesions in the liver and brain. Changes in energy balance and the hypoproteinemia that often develop in goats with paratuberculosis may lead to fat infiltration of the liver, hepatic insufficiency and, ultimately, hepatoencephalopathy.

Animals↗
PubMed1975

Intravenous johnin and tuberculin tests in cattle vaccinated with Mycobacterium paratuberculosis cells and subsequently inoculated with Mycobacterium bovis.

Calves at 30 days of age were vaccinated with a killed whole-cell Mycobacterium paratuberculosis vaccine. Four months later, these calves were inoculated with Mycobacterium bovis. The intravenous tuberculin and johnin tests were applied both before and after inoculation. The results of the hematologic investigation had extremes at both high and low values and were too unsuitable for statistical analysis. The intravenous tuberculin test is considered unsuitable for diagnosis of bovine tuberculosis in cattle vaccinated against paratuberculosis.

Animals↗
PubMed1992

Prophylactic effect of monensin sodium against experimentally induced paratuberculosis in mice.

Monensin sodium (0, 15, or 30 mg/kg of complete feed) was fed ad libitum for 1 week to female mice (strain C57BL6/J) that were genetically susceptible to infection with Mycobacterium paratuberculosis. Ten mice in each of the 3 groups were inoculated intraperitoneally with M paratuberculosis (10(9) organisms). Sterile saline solution was injected intraperitoneally into 10 other mice in each group. Rations were continued for 50 days, then mice were euthanatized, and body weight, splenic weight, and hepatic weight for each mouse were recorded. Ratios of body weight to splenic weight and of body weight to hepatic weight were calculated for each mouse. Hepatic granulomas in 50 light microscopic fields were counted, and presence of acid-fast organisms in those granulomas was recorded. Infected mice given monensin had higher body weight and fewer hepatic granulomas than did mice not given monensin. Although hepatic granulomas were fewer in these mice, they contained acid-fast organisms. Effects of 15 mg of monensin and those of 30 mg of monensin/kg of complete feed were not different.

Animals↗
PubMed2004

[A study of the epidemiological behaviour of bovine paratuberculosis using time series in Tandil in the province of Buenos Aires, Argentina].

The authors present the results of a study conducted using time series data from the 1993--2001 period in order to characterise the clinical behaviour of bovine paratuberculosis. The case data, confirmed by macroscopic examination, enzyme-linked immunosorbent assay (ELISA) and comparative tuberculin test, came from the herd health register, located in Tandil in the province of Buenos Aires, Argentina. The upper limit of customary variation was observed in April, with an incidence of 2.5%. In 1997, 1999, 2000 and 2001 the disease was epidemic and a peak of 5.6% occurred in March 1999. Over the long term a rise in the total annual incidence was observed, from 0.7% in 1993 to 10.2% in 2001. Knowledge about the epidemiology of paratuberculosis will help to control the disease and minimise its impact on the national economy, and will also provide new information for use in public health.

Animals↗
PubMed2005

[Paratuberculosis in cattle. Reflections on the interactions with Crohn disease in humans. A literature review].

Based on more than 30 literature references, the author tries to explain the phenomena of paratuberculosis in cattle. He gives a short description of the origin of the infection, the disease categories, the diagnostic problems and eradication. Also the human infection with the Mycobacterium known as Mycobacterium avium supspecies paratuberculosis (MAP)--a recognized pathogen in animals causing some or possibly all cases of Crohn's disease will be discussed. The conclusion is that until now no study gives evidence that MAP may be transmitted to humans through foods derived from cattle with MAP infection.

Animals↗
PubMed1990

[Biologic characteristics of mycobacterium strains isolated from cattle from herds with clinical paratuberculosis].

In the period from 1983 to 1986, bacteriological examination for paratuberculosis was performed in 263 samples of lymph nodes, intestinal mucous membrane and excrements of cattle, kept on a farm where clinical paratuberculosis occurred. Seventy-nine strains of mycobacteria were isolated during the culturing. On selective agar medium with mycobactin as the growth stimulator, 71 strains were isolated which had failed to grow on the conventional mycobacterium-culturing media. In the subculture, the dependence of mycobacteria on the mycobactin declined and the number of mycobacterium strains growing in the subculture on conventional mycobacting-free media doubled. Two thirds of the mycobacteria which did not depend on mycobactin during growth exhibited the same antigenic properties as Mycobacterium avium 1, 2, 3, 8 during serotypification. Ability to induce sensibility to PPD avian tuberculin or paratuberculin was demonstrated during the bioassays of mycobactin. Almost a half of the strains inducing animals' sensitivity to the above-mentioned allergens were found to be virulent to pullets that had tuberculosis in their parenchymatous organs. Of the laboratory animals, the highest virulence of the mycobactin-dependent mycobacterium strains was demonstrated in mice subjected to intravenous infection, accompanied by hyperplasia of the spleen, with reisolation of the mycobacterium culture within six eight weeks after infection.

Animals↗
PubMed1990

Use of an enzyme-linked immunosorbent assay to estimate prevalence of paratuberculosis in cattle of Florida.

A survey was conducted from 1986 through 1987, for which an ELISA was used to obtain information on the prevalence of Mycobacterium paratuberculosis infection in cattle of Florida. Results revealed prevalence of 8.6% in beef cattle and 17.1% in dairy cattle. In beef and dairy cattle, prevalence increased with increasing herd size. It was concluded that ELISA-detectable circulating antibodies to M paratuberculosis are widespread in cattle of Florida.

Animals↗
PubMed1989

Genetic relatedness among Mycobacterium paratuberculosis and M. avium complex.

Total DNA was extracted from M. paratuberculosis (ATCC 19698) and from M. avium complex (ATCC 25291) cultivated on RVB-10 enriched liquid media. Restriction endonuclease analysis of total DNA was performed with 34 enzymes and DNA digestion profiles were compared. Fifteen enzymes revealed important differences between the two species. Two pairs of enzymes (EcoRII, BstNI) and (MboI, Sau3AI) provide evidence for the presence of dcmI and dam methylation in DNA of M. avium complex and M. paratuberculosis. The differences in DNA fragments of these two species could be of potential value in differentiating these clinically significant mycobacteria.

DNA Probes↗
PubMed1989

Serodiagnosis of ovine paratuberculosis, using lipoarabinomannan in an enzyme-linked immunosorbent assay.

The use of lipoarabinomannan (LAM; obtained from Mycobacterium paratuberculosis) in and ELISA (LAM-ELISA) to test 75 sheep sera from a paratuberculosis-infected flock resulted in an approximate threefold increase in sensitivity (from 23.5% to 70.6%), compared with the use of Annau's polysaccharide in a complement fixation test (P-CFT). Even after manipulation of the LAM-ELISA cut-off value to produce a specificity of 100% to match that of the P-CFT, the sensitivity still was approximately twofold greater than that of the P-CFT. Anti-bovine monoclonal antiglobulin-enzyme conjugates matched commercially available anti-ovine polyclonal antiglobulin-enzyme conjugates with respect to sensitivity and specificity. False-positive results were found to be less frequent after combining 2 serodiagnostic tests, LAM-ELISA and D antigenagar gel immunodiffusion, resulting in an increase in specificity from 88.1% to 95.2%. The repeatability of true seropositive and seronegative results was found to be 89.5% and 91.1%, respectively, for sera obtained less than or equal to 1 month prior to slaughter and 91.7% and 95.5%, respectively, for reanalysis of sera obtained at the time of slaughter.

Animals↗
PubMed1989

Comparison of two methods for isolation of Mycobacterium paratuberculosis from bovine fecal samples.

Fecal samples from 131 cattle clinically suspect for paratuberculosis were cultured bacteriologically, using the traditional sedimentation processing method and a processing method that included a centrifugation step. Of 16 samples that were contaminated, 6 were culture-positive on at least 1 medium and by 1 processing method. Ten of 131 (7.6%) fecal samples processed by both methods were lost because of contamination. The number of culture-positive samples (using both processing methods) were 65 of 121 (53.7%) on media without miconazole and 60 of 121 (49.6%) on media with miconazole. Seven of the 121 (5.8%) samples were culture-positive, using centrifugation, after 16 weeks' incubation at 37 C. Thirteen of 60 (21.7%) isolates were obtained only with centrifugation, and 10 of these had low colony counts, suggesting that a centrifugation step may have concentrated microorganisms that would have gone undetected without centrifugation. Six of 60 (10%) isolates positive for M paratuberculosis on the sedimentation method were negative on the centrifugation method. Contamination rates were significantly (P less than 0.001) increased when centrifugation was used. The miconazole significantly (P less than 0.001) decreased contamination rates when centrifugation was used.

Animals↗