A comparison of ovine monocyte-derived macrophage function following infection with Mycobacterium avium ssp. avium and Mycobacterium avium ssp. paratuberculosis

Mycobacterium avium ssp. paratuberculosis causes Johne's disease in ruminants, whereas the antigenically and genetically similar subspecies Mycobacterium avium ssp. avium is less virulent. In this study, we compared one strain of each subspecies for its ability to survive, induce cytokines, suppress MHC class I and II expression and induce apoptosis or necrosis in ovine monocyte-derived macrophages. Both subspecies survived intracellularly and induced the secretion of IL-10. Low levels of TNF-alpha were detected after infection with both subspecies at 4 h. IL-12 was not upregulated after infection. Downregulation of MHC class I and II was evident in response to infection with both M. avium ssp. avium and M. avium ssp. paratuberculosis. No significant cytotoxicity was detectable in ovine macrophages after the addition of bacteria. M. avium ssp. paratuberculosis induced slightly more apoptosis than M. avium ssp. avium. Still the overall rate of apoptosis was very low and both subspecies suppressed LPS-induced macrophage apoptosis.     

Methylation of GPLs in Mycobacterium smegmatis and Mycobacterium avium

Several species of mycobacteria express abundant glycopeptidolipids (GPLs) on the surfaces of their cells. The GPLs are glycolipids that contain modified sugars including acetylated 6-deoxy-talose and methylated rhamnose. Four methyltransferases have been implicated in the synthesis of the GPLs of Mycobacterium smegmatis and Mycobacterium avium. A rhamnosyl 3-O-methytransferase and a fatty acid methyltransferase of M. smegmatis have been previously characterized. In this paper, we characterize the methyltransferases that are responsible for modifying the hydroxyl groups at positions 2 and 4 of rhamnose and propose the biosynthetic sequence of GPL trimethylrhamnose formation. The analysis of M. avium genes through the creation of specific mutants is technically difficult; therefore, an alternative approach to determine the function of putative methyltransferases of M. avium was undertaken. Complementation of M. smegmatis methyltransferase mutants with M. avium genes revealed that MtfC and MtfB of the latter species have 4-O-methyltransferase activity and that MtfD is a 3-O-methyltransferase which can modify rhamnose of GPLs in M. smegmatis.     

Inactivation of Mycobacterium avium ssp. paratuberculosis in milk by UV treatment

Aims:  To determine the effect of UV radiation on the viability of two strains of Mycobacterium avium ssp. paratuberculosis (Map) inoculated into milk.
Methods and Results:  Mycobacterium avium ssp. paratuberculosis in a ultra heat treated milk matrix was subjected to increasing doses of UV-C radiation from 0 to 1836 mJ ml⁻¹ using a pilot-scale UV reactor (20 l capacity). Survival of Map was monitored by culture on Herrold's egg yolk medium, Middlebrook 7H10 medium and the FASTPlaque TB™ phage assay. Differences in sensitivity to UV treatment were observed between strains, however, at 1000 mJ ml⁻¹ a Map kill rate of 0·1–0·6 log₁₀ was achieved regardless of strain used or method employed to enumerate Map. Although the inactivation trend was similar on the culture and phage assay, the former gave a consistently higher viable count.
Conclusions:  The use of UV radiation alone does not represent an alternative to current pasteurization regimes for a large reduction in viable Map in milk.
Significance and Impact of the Study:  To the authors' knowledge the work here represents the first pilot-scale UV treatment process used to assess UV efficacy to inactivate Map in milk. The results are similar to those obtained with a laboratory-scale process indicating the difficulties associated with UV treatment of an opaque liquid and the recalcitrance of Map towards inimical treatments.     

Numerical taxonomy of mycobactin-dependent mycobacteria, emended description of Mycobacterium avium, and description of Mycobacterium avium subsp. avium subsp. nov., Mycobacterium avium subsp. paratuberculosis subsp. nov., and Mycobacterium avium subsp. silvaticum subsp. nov

We performed a numerical taxonomy analysis of 38 Mycobacterium paratuberculosis and related mycobacterial strains, including wood pigeon mycobacteria; this analysis was based on 22 tests, which were selected for their potential discriminative value from a total of 51 tests studied and produced four well-defined clusters. Cluster 1 contained the M. paratuberculosis strains, including two strains isolated from Crohn's disease patients; cluster 2 contained Mycobacterium avium and Mycobacterium intracellulare reference strains; cluster 3 consisted of the wood pigeon mycobacteria; and the only strain in cluster 4 was M. paratuberculosis 316F, which is used for antigen and vaccine production. Strains in cluster 1 were mycobactin dependent even when they were subcultured, whereas strains in cluster 3 were unable to grow on egg medium and their growth was stimulated by pH 5.5. Growth stimulation by pyruvate, resistance to D-cycloserine (50 micrograms/ml), and alkaline phosphatase activity also were characteristics that were useful for discriminating between clusters 1 and 3. The results of previous DNA-DNA hybridization studies have demonstrated that M. avium Chester 1901, M. paratuberculosis Bergey et al. 1923, and the wood pigeon mycobacteria belong to a single genomic species, and we propose that the name of this species should be M. avium. On the basis of the results of previous genomic analyses based on restriction fragment length, the results of polymorphism studies, and DNA patterns determined by field inversion gel electrophoresis as well as the results of our phenotypic study, we propose that the species should be divided into subspecies which correspond to pathogenicity and host range characteristics.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mycobacterium avium subspecies paratuberculosis and Mycobacterium avium subsp. avium infections in a tule elk (Cervus elaphus nannodes) herd

Between 2 August and 22 September 2000, 37 hunter-killed tule elk (Cervus elaphus nannodes) were evaluated at the Grizzly Island Wildlife Area, California, USA, for evidence of paratuberculosis. Elk were examined post-mortem, and tissue and fecal samples were submitted for radiometric mycobacterial culture. Acid-fast isolates were identified by a multiplex polymerase chain reaction (PCR) that discriminates among members of the Mycobacterium avium complex (MAC). Histopathologic evaluations were completed, and animals were tested for antibodies using a Johne's enzyme-linked immunosorbent assay (ELISA) and agar gel immunodiffusion. In addition, 104 fecal samples from tule elk remaining in the herd were collected from the ground and submitted for radiometric mycobacterial culture. No gross lesions were detected in any of the hunter-killed animals. Mycobacterium avium subsp. paratuberculosis (MAP) was cultured once from ileocecal tissue of one adult elk and was determined to be a strain (A18) found commonly in infected cattle. One or more isolates of Mycobacterium avium subsp. avium (MAA) were isolated from tissues of five additional adult elk. Gastrointestinal tract and lymph node tissues from 17 of the 37 elk (46%) examined had histopathologic lesions commonly seen with mycobacterial infection; however, acid-fast bacteria were not observed. All MAC infections were detected from adult elk (P = 0.023). In adult elk, a statistically significant association was found between MAA infection and ELISA sample-to-positive ratio (S/P) > or = 0.25 (P=0.021); four of five MAA culture-positive elk tested positive by ELISA. Sensitivity and specificity of ELISA S/P > or = 0.25 for detection of MAA in adult elk were 50% and 93%, respectively. No significant associations were found between MAC infection and sex or histopathologic lesions. Bacteriologic culture confirmed infection with MAP and MAA in this asymptomatic tule elk herd. The Johne's ELISA was useful in signaling mycobacterial infection on a population basis but could not discriminate between MAA and MAP antibodies. The multiplex PCR was useful in discriminating among the closely related species belonging to MAC.     

Iron-chelating compound from Mycobacterium avium.

A iron-chelating monohydroxamate was isolated from cultures of Mycobacterium avium grown on an iron-limiting medium. The hydroxyamate metabolite was characterized by chemical degradation and spectral measurements as L-alpha-asparaginyl-L-alpha-(N-hydroxy)-asparagine.     

Extensive genomic polymorphism within Mycobacterium avium

We have initiated comparative genomic analysis of Mycobacterium avium subspecies by DNA microarray, uncovering 14 large sequence polymorphisms (LSPs) comprising over 700 kb that distinguish M. avium subsp. avium from M. avium subsp. paratuberculosis. Genes predicted to encode metabolic pathways were overrepresented in the LSPs, and analysis revealed a polymorphism within the mycobactin biosynthesis operon that potentially explains the in vitro mycobactin dependence of M. avium subsp. paratuberculosis.     

Changes in the virulence of Mycobacterium avium after passage through embryonated hens' eggs

Eight-day-old embryonated hen's eggs were used as a model to study Mycobacterium avium virulence. Strains isolated from human patients caused 20-90% mortality when eggs were infected by injection of bacterial suspensions into the amniotic sac. Virulence of examined strains subsequently decreased with passage through eggs to between 0 and 40% mortality in four passages. Virulence of the egg-attenuated strains could be restored by passage through human peripheral blood mononuclear cells. The site of infection in the egg was usually the mesodermal layer of the chorioallantoic membrane. A few small granulomas containing acid-fast bacteria were seen in the liver, but not in other organs. Death of chicken embryos may have resulted from destruction of the mesodermal layer of the chorioallantoic membrane with consequent respiratory failure. PBMCs infected with less virulent egg-passaged strains of M. avium produced higher levels of tumor necrosis factor-alpha than did peripheral blood mononuclear cells infected with more virulent nonpassaged strains.     

Response of Mycobacterium avium to Ultraviolet Irradiation

The survival response of Mycobacterium avium to ultraviolet irradiation demonstrated the presence of a photoreactivating repair system.     

Electrophoretic Mobility of Mycobacterium avium Complex Organisms

The electrophoretic mobilities (EPMs) of 30 Mycobacterium avium complex organisms were measured. The EPMs of 15 clinical isolates ranged from −1.9 to −5.0 μm cm V⁻¹ s⁻¹, and the EPMs of 15 environmental isolates ranged from −1.9 to −4.6 μm cm V⁻¹ s⁻¹ at pH 7.     

Growth and cell division of Mycobacterium avium

The rates of cell division and of protein, DNA and RNA synthesis upon transition of Mycobacterium avium to and from rich medium were examined. The changes in cell morphology (elongation) were also examined by optical and electron microscopy. Upon transfer from poor to rich medium, the rate of synthesis of RNA increased rapidly, followed by an increase in protein synthesis within 3 h and by an increase in DNA synthesis within 7 h; cell division began after a lag of about 10 h. Upon transfer from rich to poor medium, the preshift rates for protein and DNA synthesis changed to postshift rates after 3 h and 7 h, respectively; RNA synthesis stopped immediately, there was a transient fall in total RNA, and synthesis was resumed at a new rate only after 24 h. After the period of adjustment to new medium, the bacteria entered the postshift growth in which cell size, the increase in cell mass (absorbance at 650 nm) and viable counts, and the rates of synthesis of protein, DNA and RNA were constant. Ultrastructural examination of elongated cells during the adjustment period showed that they had septa at different stages of formation, but no evidence of fragmentation was found. It was concluded that cell division in M. avium was by binary fission, and that the notion of a life-cycle was not supported by present findings.     

Electrophoretic Mobility of Mycobacterium avium Complex Organisms

The electrophoretic mobilities (EPMs) of 30 Mycobacterium avium complex organisms were measured. The EPMs of 15 clinical isolates ranged from -1.9 to -5.0 microM cm V(-1) s(-1), and the EPMs of 15 environmental isolates ranged from -1.9 to -4.6 microM cm V(-1) s(-1) at pH 7.     

Differentiation between Mycobacterium avium and Mycobacterium intracellulare by thin-layer chromatography of lipid fraction after incubation with [35S]methionine.

Mycobacterium avium and Mycobacterium intracellulare are differentiated from each other by thin-layer chromatography of lipid fraction extracted after incubation with [35S]methionine. The former contained a petroleum ether-soluble sulfolipid and the latter did not.     

Susceptibility of Mycobacterium avium and Mycobacterium intracellulare to various antibacterial drugs

Mycobacterium avium and M. intracellulare of human and natural sources, identified by the Gen-Probe Rapid Diagnostic System for M. avium Complex (MAC) were studied for susceptibility to eight different drugs. In the case of human isolates of MAC, the following was noted. M. avium showed nearly the same susceptibility to streptomycin, kanamycin, ethambutol, and clofazimine as was seen with M. intracellulare. M. avium was much more resistant to rifampicin and rifabutin than was M. intracellulare, and M. avium was more susceptible to quinolones such as ofloxacin and ciprofloxacin. Conversely, in the case of MAC from natural sources, there was no difference between the susceptibility of M. avium and M. intracellulare to these antibacterial agents.     

Recovery of Mycobacterium avium subspecies paratuberculosis from the natural host for the extraction and analysis in vivo-derived RNA

RNA has been extracted and analysed from in vivo-derived Mycobacterium avium subspecies paratuberculosis recovered from the natural host. The bacteria were selectively extracted from the intestinal tissue of two goats exhibiting clinical signs of Johne's disease. Small intestine was rapidly removed, luminal contents washed away and the mucosa and submucosa harvested. Mycobacteria in this material were released from the macrophages by isotonic lysis and differential centrifugation. RNA was extracted and compared with RNA extracted from bacteria grown in vitro. Real-time polymerase chain reaction was used to analyse the katG gene from the bacterial messenger RNA. The katG mRNA encoding the putative catalase/peroxidase showed differential expression in the in vivo and in vitro-derived samples. We hypothesize that the increase in katG expression for in vivo-derived M. paratuberculosis may represent a response to the oxidative stress encountered within the intra-macrophage environment.     

Mycobacterium avium infection in a silicone-injected breast

A case of atypical mycobacterial infection (M. avium intracellulare) in a silicone-injection augmented breast is described. The silicone injection may have been a contributing factor to the development of this unusual infection. Disseminated M. avium was present in this patient, with breast involvement being suggested by the rapid appearance and disappearance of localized areas of erythema and tenderness. Aggressive treatment of these breast infections while they are still localized may prevent systemic spread. Conventional incision and drainage of the breast abscess combined with multidrug treatment directed against M. avium is the recommended therapy.     

Killing of Mycobacterium avium subspecies paratuberculosis within macrophages

Background  

Mycobacterium avium subspecies paratuberculosis (M. paratuberculosis) is a facultative intracellular pathogen that resides within host macrophages during infection of ruminant animals. We examined survival of M. paratuberculosis infections within cultured macrophages to better understand the interplay between bacterium and host.