An unusual murine behavior following infection with log-phase Nocardia asteroides type 6 strain GUH-2 (Nocardia cyriacigeorgica GUH-2)

Nocardia asteroides (Nocardia cyriacigeorgica) strain GUH-2 infects the brains of mice following intravenous injection. Non-lethal infections resulted in a transitory increase of bacterial numbers in the brain followed by the development of permanent impaired movements at a time when bacteria appeared to be eliminated from the brain. These signs included headshake, rigidity, stooped posture, dyskinesia, retropulsion, and abnormal tail positioning in approximately 20% of infected animals. The attached video presents a typical mouse following infection with this organism, as compared to an age- and gender-matched uninfected control mouse.     

Comparison of PCR and culture for detection of Nocardia asteroides in brain specimens from experimentally infected BALB/c mice

Systemic infection of BALB/c mice with Nocardia asteroides strain GUH-2 results in widespread replication of the organism in the brain, followed by its immune-mediated clearance. The present study compared the sensitivity of polymerase chain reaction (PCR) to bacterial culture for detection of cerebral nocardial infection in this experimental system. Mice (n=4/time point) were administered N. asteroides by intravenous injection, and brain specimens were evaluated for Nocardia by PCR and culture at post-infection days 2, 7, 14 and 21. Nocardia was detected by PCR in all infected animals on post-infection days 2, 7, and 14, and in one of four mice on post-infection day 21; in contrast, the organism was detected by culture only on post-infection days 2 and 7. These findings suggest that PCR may be more sensitive than culture for the detection of low numbers of Nocardia in the brain.     

Characterization of 2,3-dihydroxybenzoic acid from Nocardia asteroides GUH-2.

Culture filtrates of virulent Nocardia asteroides GUH-2 after growth in acetate minimal medium displayed an absorbance maximum at 320 nm. After isolation by polyamide extraction and anion chromatography, a UV-active compound with this absorbance was shown to be 2,3-dihydroxybenzoic acid (DHB) by nuclear magnetic resonance, gas chromatographic, and mass spectrometric techniques. DHB production under several culture conditions was quantified by a standard high-pressure liquid chromatography assay. Under iron deficiency conditions, N. asteroides GUH-2 excreted up to 11 mg of DHB per liter into the culture medium. No DHB was detected when N. asteroides GUH-2 was grown in an iron-rich medium. With the less virulent strain N. asteroides 10905, DHB was not found under any condition tested.     

Pathogenic and virulence characterization of colonial mutants of Nocardia asteroides GUH-2

The pathogenicities in mice (comparing LD50 determinations) of two mutant strains and one wild-type strain of Nocardia asteroides GUH-2, each possessing a colonial morphology distinct from the other, were compared at respective stages of growth. Despite the three strains' colinear growth curves and similar physiological properties, unique patterns of pathogenicity emerged for each strain upon analysis. Ultrastructural and fatty acid profiles of cultures at the various growth stages were monitored. The mutant strain SCII-A1 was consistently less virulent than the other strains of N. asteroides GUH-2 (SCII-P and SCII-C). Further, its fatty acid profiles as well as the shape and consistency of its colonies differed greatly from those of the wild-type strain. The fatty acid composition and the colonial morphology of strain SCII-C more closely resembled those of the parent, although its virulence was both greater than (before 28 h of growth) and less than the parent's depending upon the specific stage of growth. The comparative degrees of cellular fragmentation and complexity, as determined by scanning and transmission electron microscopy, were found to coincide with changes in relative degrees of pathogenicity.     

Cutting edge: protective response to pulmonary injury requires gamma delta T lymphocytes

Gamma delta intraepithelial lymphocytes are thought to coordinate responses to pathogens that penetrate the epithelial barrier. To directly test this, mice were inoculated with Nocardia asteroides. At doses that were nonlethal for control mice, gamma delta-deficient mice became severely ill and died within 14 days. Histologic examination of these lungs demonstrated the presence of severe tissue damage and unimpeded bacterial growth in the gamma delta-deficient mice compared with neutrophilic lesions and clearance of the organism in control mice. Interestingly, ozone exposure that targets a comparable lung region also resulted in diffuse epithelial necrosis associated with a similar lack of neutrophil recruitment in gamma delta-deficient mice. These data demonstrate that gamma delta intraepithelial lymphocytes can protect the host from pathogenic and nonpathogenic insults by targeting the inflammatory response to epithelial necrosis.     

Effect of growth stage on mycolic acid structure in cell walls of Nocardia asteroides GUH-2

Mycolic acids were extracted from the cell walls of Nocardia asteroides GUH-2 during different phases of growth at 37 degrees C. These were subjected to structural analysis by combining thin-layer chromatography and gas-liquid chromatography with UV and infrared spectrophotometry and mass spectroscopy of both methyl esters and trimethyl silyl derivatives. By analyzing the fragmentation patterns of these derivatives by three different methods of mass spectroscopy combined with gas-liquid chromatographic separation, the different structural subclasses of mycolic acids were quantitated. Significant qualitative and quantitative modifications of specific mycolic acid subclasses occurred in the cell walls of N. asteroides GUH-2 that were growth stage dependent. The mycolic acids that were predominant in the log phase were polyunsaturated (greater than 2 double bonds per molecule), with long chain lengths and even carbon atom numbers (i.e., C54, C56). In contrast, those that were prominent in the stationary phase were more saturated (few or no double bonds) and of shorter overall carbon chain length (less than or equal to C52). Furthermore, stationary-phase cells had significantly increased amounts of mycolic acids with odd-numbered carbon chain lengths (i.e., C49, C51, C53).     

Immunodiffusion studies of some Nocardia strains

Forty-three strains of Nocardia, one of Actinomadura and two of Nocardiopsis were studied using the comparative immunodiffusion technique. Three reference precipitation systems were employed: one represented Nocardia asteroides N10, one N. asteroides ATCC 19247, and one N. otitidis-caviarum ATCC 14629. One tight cluster was formed by the N. otitidis-caviarum strains and another tight cluster was formed by some of the N. asteroides strains studied. However, other strains of N. asteroides were distinct from the latter cluster. Furthermore, N. asteroides ATCC 19247, which is the type strain, differed from most ot the N. asteroides strains tested. Strains of the species N. asteroides, N. brasiliensis, N. farcinica and N. otitidis-caviarum were found to be closely related, while N. amarae strains differed slightly from this group. The strains referred to Actinomadura and Nocardiopsis were clearly distinct from the three Nocardia reference strains; nevertheless, three antigens common to these genera were revealed.     

Genome sequence of the human- and animal-pathogenic strain Nocardia cyriacigeorgica GUH-2

The pathogenic strain Nocardia cyriacigeorgica GUH-2 was isolated from a fatal human nocardiosis case, and its genome was sequenced. The complete genomic sequence of this strain contains 6,194,645 bp, an average G+C content of 68.37%, and no plasmids. We also identified several protein-coding genes to which N. cyriacigeorgica's virulence can potentially be attributed.     

Tinctorial Properties of Spherical Bodies in Broth Cultures of Nocardia asteroides GUH-2

In yeast extract-supplemented brain heart infusion (BHI) broth cultures of Nocardia asteroides GUH-2, many spherical bodies (SBs) were frequently seen nearby filamentous cells. They showed no Gram-positivity when Gram stain was applied. When acridine orange stain was applied, many of them showed different green fluorescence from bright orange fluorescence of the filamentous nocardiae under ultraviolet light. Their acid-fastness appeared to depend on the presence of paraffin. Using the polymerase chain reaction (PCR) method, 16S rRNA genes were detected in SB-containing broth cultures inoculated with culture filtrates from broth cultures of the strain and identical to that of N. asteroides. These results suggest that SBs are cell wall-defective (CWD) forms which result from the spontaneous mutation of N. asteroides GUH-2.     

Purification and properties of a unique superoxide dismutase from Nocardia asteroides

A unique form of superoxide dismutase was isolated and characterized from Nocardia asteroides GUH-2. This enzyme contains 1 to 2 g atoms each of Fe, Mn, and Zn per mol and exhibits spectral properties suggestive of Fe- or Mn-containing superoxide dismutases. Its Mr = 100,000, and it is composed of four subunits of equal size which are not covalently joined. The amino acid composition of the enzyme was more closely related to the Mn- or Fe-containing enzymes of Mycobacterium species and was least related to the Cu-Zn enzyme of eukaryotes. Azide at 1 and 20 mM inhibits the activity 10 and 41%, respectively, and 5 mM H2O2 inhibits 40%, but 1 or 5 mM cyanide caused trivial effect. The immunofluorescent staining, which was specific for superoxide dismutase of N. asteroides, indicated the association of this enzyme to the outer cell wall of the organism. Further, the enzyme was shown to be selectively secreted into the medium.     

Cell wall modification resulting from in vitro induction of L-phase variants of Nocardia asteroides.

The chemical composition of the cell walls of several L-form revertants derived from Nocardia asteroides 10905 was determined at different stages of growth. It was observed that each L-form revertant had a cell well that differed from that of the parental strain when grown under identical conditions. In some strains the peptidolipid and mycolic acid components were affected the most, whereas in other strains the fatty acid, sugar, and mycolic acid moieties were altered. Shifts in mycolic acid size were prominent, whereas the basic peptidoglycan structure appeared to be affected the least. Both the method used to induce the L-form of N. asteroides 10905 and the length of time these organisms were maintained in the wall-less state affected the degree of cell wall modification during the reversion process. Thus, removal of the cell wall appeared to potentiate and select for mutational alterations within the cell envelope of N. asteroides, and these changes resulted in altered cellular and colonial morphology.     

Intrauterine infection/inflammation during pregnancy and offspring brain damages: Possible mechanisms involved

Intrauterine infection is considered as one of the major maternal insults during pregnancy. Intrauterine infection during pregnancy could lead to brain damage of the developmental fetus and offspring. Effects on the fetal, newborn, and adult central nervous system (CNS) may include signs of neurological problems, developmental abnormalities and delays, and intellectual deficits. However, the mechanisms or pathophysiology that leads to permanent brain damage during development are complex and not fully understood. This damage may affect morphogenic and behavioral phenotypes of the developed offspring, and that mice brain damage could be mediated through a final common pathway, which includes over-stimulation of excitatory amino acid receptor, over-production of vascularization/angiogenesis, pro-inflammatory cytokines, neurotrophic factors and apoptotic-inducing factors.     

Monoamine Changes in the Brain of BALB/c Mice Following Sub-Lethal Infection with Nocardia asteroides (GUH-2)

BALB/c mice injected intravenously with a single, sub-lethal dose of Nocardia asteroides GUH-2 develop several levodopa responsive movement disorders. These included head-shake, stooped posture, bradykinesia, and hesitation to forward movement (6). The changes in monoamine levels in the brain of these mice were determined. There was a significant loss of dopamine with greatly increased dopamine turnover in the neostriatum 7 to 29 days after infection. These effects were specific for dopaminergic neurons since minimal changes were found in neostriatal norepinephrine and serotonin even though serotonin turnover was increased. Changes in monoamine metabolism were not limited to the neostriatum. There were reduced levels of serotonin and norepinephrine with increased serotonin turnover in the cerebellum. One year after infection, dopamine metabolism had returned to near normal levels, but many of the movement disorders persisted. Specific changes in neurochemistry did not always appear to correspond with these impairments. Nevertheless, these data are similar to those reported in MPTP treated BALB/c mice.     

Nocardia asteroides (Eppinger) Blanchard in the soils of the Ukrainian SSR]

The distribution of Nocardia asteroides (Eppinger) Blanchard, the main causative agent of nocardiosis in humans and animals, in the soils of the Ukrainian SSR was studied. This species was found to inhabit cultivated soils in various soil-climatic zones of the Ukrainian SSR. The isolated strains proved to be heterogeneous in their properties. N. asteroides fell into 2 groups: acetamidase-negative (A) and acetamidase-positive (B). Newly isolated N. asteroides belonging to the acetamidase-positive group were shown to be always pathogenic, whereas the organisms of the acetamidase-negative group could be pathogenic and non-pathogenic in white mice.     

Experimental pathogenicity of isolates of Nocardia asteroides, Nocardia brasiliensis and Nocardia caviae from different sources

The experimental pathogenicity of 14 isolates of Nocardia brasiliensis, 15 of N. asteroides, and 5 of N. caviae was investigated for the white Swiss mice inoculated intraperitoneally and in the foot pad, and for the guinea-pig and the hamster (Mesocricetus auratus) both inoculated intratesticularly. The guinea-pig was remarkably sensitive to N. asteroides, with an apparent relationship between pathogenicity and thermotolerance, confirming previous observations. Mice were in general less susceptible to this species. In both guinea-pigs and hamsters it was possible to observe typical granules with or without clubs. N. caviae was highly pathogenic for the guinea-pig and the hamster but no mycetomas were produced in the mice inoculated in the foot pad. Isolates of N. brasiliensis from natural sources were scarcely virulent for the different animals. Those of human origin produced significant lesions in the mice inoculated intraperitoneally with granules. Foot pad inoculation of mice with N. brasiliensis caused mycetomas in several animals.     

Genetic model of some human hereditary diseases: features of behavior, neurochemistry and morphology of the brain in mice of the 101/HY line

Studies of behavior, neurophysiological reactions, neuromediator synthesis and brain structure of mice of the 101/HY strain (including those of the authors) are reviewed. This mouse strain is characterized by a chromosomal instability because of a recessive mutation mutator-1 (mut-1) and the defective DNA excision repair. Experimental studies revealed a number of behavioral and neurological deviations in the 101/HY as compared to the CBA and the C3H strains. These are abnormalities in spatial orientation, altered fear and anxiety reactions, anomalous locomotion, seizure developing in response to agents of various nature, and disturbances of the central nervous system, both structural and biochemical. Genome instability results in a number of neurological mutations, that may lead to the phenotypical effects observed in the 101/HY mice. Since the 101/HY mice partially display signs of severe human hereditary diseases caused by chromosomal instability and defective DNA repair, they can serve as a promising genetic model for these and other diseases related to impairment of the central nervous system.     

Mouse pathogenicity studies of Nocardia asteroides complex species and clinical correlation with human isolates

Abstract Nocardia asteroides complex organisms derived from human specimens between 1979 and 1992 were identified on the species level. Of 117 N. asteroides complex organisms, 34 (29%) were N. farcinica , 28 (24%) were N. nova , and 55 (47%) were N. asteroides sensu stricto . An analysis of the specimen sites from which the organisms were derived showed that isolates derived from blood, brain, or bone marrow were more likely to be N. farcinica than the other two species. A study of the virulence of ten strains of each species was undertaken, using a mouse model with intravenous inoculation. The 50% lethal doses (LD₅₀) for N. farcinica were significantly lower than those of the other two species. LD₅₀ values for N. nova and N. asteroides were not significantly different. The above data confirming the greater virulence of N. farcinica support the identification of species within the N. asteroides complex.     

Pathogenicity of Nocardia transvalensis for laboratory mice

Nocardia transvalensis was found to be virulent for laboratory mice both by intraperitoneal and intravenous routes of inoculation, the latter route producing a more progressive and disseminating infection. Cortisone administration was found to enhance the susceptibility of mice, the LD₅₀ for the cortisone treated mice being five times less than that for the untreated animals. In the tissues of cortisone treated mice, N. transvalensis grew as dispersed filaments with coccobacillary forms or sometimes their loose aggregates, whereas in normal animals granule formation was a conspicuous feature. The pathogenicity of N. transvalensis is compared with that of N. asteroides, N. brasiliensis and N. caviae from published reports.     

Immunochemical characterization of Nocardia asteroides antigens: support for a single species concept

Nocardia asteroides strains are highly heterogeneous. They show morphological, physiological, and immunological differences. In a previous study, we delineated seven immunotypes of N. asteroides. In the present study, we compared the culture filtrate antigens of these immunotypes by antigen-antibody crossed-immunoelectrophoresis and by rocket electrophoresis. We have also compared the antigen preparations by two-dimensional electrophoresis. While unique components constitute the major portion of the components, the results indicate that similar components are present in the culture filtrates of all strains. This finding supports the view of retaining all the immunotypes in the species Nocardia asteroides rather than designating different species such as N. farcinica and N. sebivorans.