Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens.     

Monoclonal antibodies against the M-protein and carbohydrate antigens of histoplasmin characterized by the enzyme-linked immunoelectrotransfer blot method.

Monoclonal antibodies (MAbs) of two different specificities were produced by immunizing mice with the semipurified M antigen of histoplasmin. One type, from clone CB4, was an immunoglobulin M that precipitated a polysaccharide present in histoplasmin and also formed immunoprecipitates with a cross-reactive polysaccharide present in extracts of Blastomyces dermatitidis and Coccidioides immitis. The second type of MAb, from clone EC2, was an immunoglobulin G that reacted in the enzyme-linked immunoelectrotransfer blot (EITB) assay with a doublet of proteins with an apparent molecular size of 70 to 75 kilodaltons. This molecule is proposed as the authentic M protein antigen that is recognized by M antibodies in sera from mice and rabbits immunized with Histoplasma capsulatum and from persons with histoplasmosis. The M factor also occurs in an abundant disulfide-bridged dimer which has a molecular size of 150 kilodaltons and is nonimmunoreactive under the conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Clinical demonstration of isolation of Nocardia asteroides on buffered charcoal-yeast extract media.

Nocardia asteroides was isolated only from sputum samples, obtained from three patients with pulmonary nocardiosis, that had been cultured onto buffered charcoal-yeast extract (BCYE) and selective BCYE media as part of laboratory workups for Legionella species. A decontamination procedure with low-pH pretreatment (KCl-HCl solution) had been performed on the sputa prior to culture onto the BCYE media because direct cultures on the media were overgrown with commensal microflora. Chalky white colonies, 0.5 to 1.0 mm in diameter, that were subsequently identified as N. asteroides grew well on the BCYE media. Thus, the techniques and the selective media used for Legionella species were useful for isolating Nocardia species from sputum.     

Investigation of cross-reactions against Trichinella spiralis antigens by enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot assay in patients with various diseases.

Data regarding cross-reactions against Trichinella spiralis in humans are scarce and controversial. For this reason, we tested serum samples from patients with typhoid fever, brucellosis, toxoplasmosis, amoebiasis, cysticercosis, trichocephaliasis, ascariasis, and onchocerciasis against an antigenic extract of T. spiralis infective larvae in an enzyme-linked immunosorbent assay (ELISA) and an enzyme-linked immunoelectrotransfer blot (EITB) assay. All except one serum sample from the group of patients with onchocerciasis were negative in the ELISA; in the EITB assay, only faint bands were observed with the samples from patients with onchocerciasis and ascariasis and negative results were obtained with the samples from patients with other diseases. In conclusion, cross-reactions were found only in the groups of patients with other nematode infections and were of very low magnitude, most of them virtually negative.     

Comparison of paraffin baiting and conventional culture techniques for isolation of Nocardia asteroides from sputum.

Of 1,510 sputum samples examined from 1,016 patients with bronchopulmonary disorders, Nocardia asteroides was isolated from 67 samples by paraffin baiting, as compared with only 30 isolations by the conventional technique of culturing on Sabouraud dextrose agar. This higher efficacy of the paraffin bait technique was found to be statistically significant (P less than 0.001).     

DNA probes for the identification of Nocardia asteroides.

DNA probes for the rapid identification of Nocardia asteroides were obtained by constructing a genomic library of strain GUH-2 in the lambda cloning vector EMBL3. Of 50 recombinant clones tested, 2 were identified that hybridized with 31% of the N. asteroides strains in a reference collection without cross-hybridization with related members of the Actinomycetales. Additional libraries were then generated from selected strains of N. asteroides that had failed to hybridize with any of the GUH-2 clones. Four additional clones were obtained from these strains which, when pooled, provided DNA probes specific for all of the N. asteroides strains tested.     

Adherence of Nocardia asteroides within the murine brain.

Nonlethal infection of BALB/c mice with Nocardia asteroides GUH-2 (GUH-2) produces a variety of neurological signs, including an L-dopa-responsive movement disorder in 10 to 15% of the infected population. To study nocardial interactions with the brain, we characterized the attachment of GUH-2 within specific regions through the use of microdissection. Following an intravenous injection of a single-cell suspension of log-phase GUH-2, viable cells were recovered from all regions of the brain, and the distribution of the nocardiae was independent of the size of the inoculum. In addition, two mutants of GUH-2 were found to possess significantly altered binding characteristics with regard to both the percentage of the inoculum bound per brain and the relative distribution of adherence to regions of the brain, when compared with the parental strain. These results indicated that GUH-2 bound throughout the murine brain and suggested that GUH-2 utilized specific receptors to facilitate this attachment.     

Immune responses to the protein, carbohydrate and lipid antigens of Nocardia asteroides in experimental nocardiosis in mice

The intravenous injection of Nocardia asteroides into mice produced systemic nocardiosis involving all the vital organs. Infection of the kidneys and adrenals was more persistent and progressive than in other organs as evidenced by increased bacterial counts and histopathological findings. During the course of the experimental infection, no humoral immune response was detected against various protein antigens up to 4 weeks after challenge, but significant cell-mediated immunity (CMI) was found. Phospholipid antigens elicited only a humoral immune response. The increased CMI responses with protein antigens correlated well with the decreasing bacterial load, which suggested that CMI against proteins was important in the pathogenesis of this disease.     

Site-specific growth of Nocardia asteroides in the murine brain.

The growth of Nocardia asteroides GUH-2 and two mutants (NG-49 and I-38-syn) in regions of the brains of BALB/c mice was determined by microdissection and viable counting. GUH-2 grew throughout the murine brain but at different growth rates that depended on the specific location. The rate of increase in total CFU per brain during GUH-2 infection was unaffected by the inoculum size; however, in five of eight brain regions, an alteration in the inoculum size resulted in altered nocardial growth rates. Mutant NG-49 showed a significantly slower rate of increase in total CFU per brain than did the parental strain, GUH-2, and significantly decreased growth rates in seven brain regions. Mutant I-38-syn showed a rate of increase in total CFU per brain similar to that of the parental strain; however, this mutant grew significantly faster in the cerebellum and pons-medulla. Growth appeared to be a necessary precursor to the cellular damage that resulted in the variety of neurological disorders observed in mice infected with N. asteroides GUH-2, because mutant NG-49 exhibited a decreased ability to grow in specific regions of the brain and did not induce signs of neurological damage. In contrast, mutant I-38-syn induced neurological signs in a larger percentage of the infected animals than did parental strain GUH-2 and grew better in certain regions of the brain than did the parental strain. Furthermore, there appeared to be a relationship between the growth of N. asteroides in the substantia nigra and the induction of an L-dopa-responsive head shake that was observed in some of the mice following a sublethal intravenous injection of N. asteroides GUH-2.     

Site-specific invasion of the basal ganglia by Nocardia asteroides GUH-2

Nocardia asteroides GUH-2 (GUH-2) invades the nigrostriatal region of the brain in mice [15]. Selective dopaminergic neuronal dropout in the substantia nigra results in parkinsonian changes characterized by movement disorders responsive to L-dopa [15]. This is the only reported example of an experimental bacterial model for parkinsonism. Following i.v. inoculation of GUH-2 into the non-human primate Macaca fasicularius, the nocardiae preferentially invaded and grew within the basal ganglia (substantia nigra, caudate, putamen, and globus pallidus) often without inducing apparent inflammation. Reduced, limited growth of nocardiae occurred in the white matter of the cerebral cortex, medulla, and hippocampus, whereas neither significant adherence to nor growth within the meninges was observed. Twenty-four hours after injection, nocardial cells were found within capillary endothelial cells, the basal lamina, neurons, astroglia and in axonal extensions. The bacteria, in endothelial cells, were surrounded by a unit membrane, but in the basal lamina they appeared to be free and not membrane bound. After the organisms passed into the brain parenchyma, the nocardiae once again became surrounded by membrane, often being encapsulated by numerous layers with the innermost layer tightly adherence to the bacterial surface. There was a propensity for nocardial growth within and along myelinated axons, either with or without disruption to the surrounding myelin sheath. There was electron microscopic evidence that the nocardiae induced a neurodegenerative response especially in the substantia nigra region. Thus, the early interactions of GUH-2 within the primate brain appeared to be similar to those reported in the mouse. Received: 23 September 1999     

In vitro cellular immune response to homologous and heterologous antigens in rabbits sensitized by five species of Mycobacterium and Nocardia asteroides.

Rabbits were sensitized with killed Mycobacterium tuberculosis, M. kansasii, M. avium, M. scrofulaceum, M. fortuitum, and Nocardia asteroides, and their response to homologous and heterologous antigens was assessed in vitro by direct and indirect macrophage migration inhibition tests. The antigens were obtained by disintegration of bacterial mass and by purification of the supernatants by ultracentrifugation. In the direct test, hypersensitivity to homologous antigen was most marked with M. tuberculosis, M. kansasii, and M. avium (migration indices [MI] = 0.42 to 0.50), but was significantly weaker with organisms possessing a lower degree of pathogenic activity (MI for M. fortuitum and N. asteroides = 0.70 and 0.72, respectively). Reactivity to heterologous antigens was also highest in animals sensitized with strongly pathogenic species, approximating normal values in rabbits sensitized with weak pathogens. In the indirect test, the strongest responses were obtained again to homologous antigens (MI = 0.42 to 0.67), and they differed more markedly from reactions to heterologous antigens than in the direct test. The weakest activity of heterologous antigens was again found with M. fortuitum and N. asteroides, where MIs were 0.82 to 0.93.     

Identification of a 17-kilodalton Fasciola hepatica immunodiagnostic antigen by the enzyme-linked immunoelectrotransfer blot technique.

Sera obtained from human patients, calves, sheep, and rabbits infected with Fasciola hepatica were tested by the Falcon assay screening test enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) techniques with Fasciola hepatica excretory-secretory antigens in order to evaluate their immunodiagnostic potential. The study included sera from 13 patients infected with F. hepatica or a history suggesting fascioliasis, 5 patients infected and treated with bithionol or praziquantel (3 were cured with bithionol), 10 patients infected with Schistosoma mansoni, 6 infected with Trichinella spiralis, and 13 controls and sera from calves, sheep, and rabbits with a primary F. hepatica infection. By FAST-ELISA with F. hepatica excretory-secretory antigens, the serum samples from fascioliasis patients gave the highest absorbance values, and the schistosomiasis patient sera gave intermediate values compared with a normal human serum control. Also by FAST-ELISA, the values for serum from patients with fascioliasis decreased steadily after cure, reaching normal levels 20 to 47 weeks postcure. In contrast, the serum from two patients who had been treated but were not yet cured had high levels of antibodies for up to 3 years of infection. By EITB, the serum samples from humans, rabbits, cattle, and sheep with fascioliasis recognized two antigenic polypeptides of 17 and 63 kilodaltons (kDa) in the form of sharp bands. For humans, this recognition lasted for at least 3 years of infection. Sera from individuals with schistosomiasis mansoni or trichinosis or from normal controls did not recognize the 17-kDa F. hepatica antigenic polypeptide. However, serum from one human with S. mansoni and one with T. spiralis infection has slight bands in the 63-kDa region, suggesting cross-reactivity. Reactivity to the 17-kDa polypeptide was absent in fascioliasis patients at 1 year postcure. Reactivity to the 63-kDa polypeptide was significantly diminished in fascioliasis patients at 1 year postcure. The sera from rabbits with a primary F. hepatica infection also recognized both the 17- and 63-kDa antigenic polypeptides by week 4 of infection. Reactivity to both antigens diminished significantly 6 weeks postcure and disappeared by 8 weeks postcure. The sera from infected cattle and sheep recognized these two antigenic polypeptides by week 8 of infection. These studies suggest that the 17-kDa F. hepatica excretory secretory antigen is an excellent candidate for the immunodiagnosis of acute and chronic fascioliasis. Purification of this antigen and its application to quantitative serologic tests will permit further analysis of its predictive value to evaluate cure.     

L-dopa-responsive movement disorder caused by Nocardia asteroides localized in the brains of mice.

Nocardia asteroides can cause infections in the brain of humans and a variety of animals. In mice, invasion of the central nervous system results in specific neurologic signs. Following intravenous injection of various doses of log-phase N. asteroides GUH-2 into female BALB/c mice, localization and growth of nocardial cells within the brains were determined, histopathological sections were prepared, and Nissl substance and tyrosine hydroxylase immunoreactivity were observed. Mice were monitored for the development of neurologic signs, and their responsiveness to L-dopa was determined. It was shown that nocardial cells became localized within specific regions of the brain and then underwent rapid growth followed by a delayed clearance, and there was no inflammatory response at the site of invasion for 24 h. Mice that received a subclinical dose of nocardiae developed specific neurologic signs that emerged following the elimination of nocardial cells from the brain. On the basis of the specific signs, mice could be divided into distinct groups. One group consisted of animals that had a form of hemiparesis that did not respond to L-dopa. They expressed a deviation of the head and a tendency to roll, and when suspended by the tail they would spin rapidly. The second group of mice developed a rhythmic, uncontrolled vertical shake of the head (four to five times per s) tremulous movement, stooped posture, restlessness, and no signs of hemiparesis. The head shakes were temporarily stopped by treatment with L-dopa. Mice that expressed head shakes had a loss of Nissl substance and tyrosine hydroxylase immunoreactivity in the neurons of the substantia nigra and ventral tegmental areas of the brain. Hyaline inclusion bodies that resembled Lewy bodies were found in the neurons of mice with head shake 1 month after infection. Therefore, mice infected with N. asteroides may serve as a model for studying parkinsonian signs and other degenerative diseases involving extrapyramidal and pyramidal systems.     

Distribution of serotypes of Nocardia asteroides from animal, human, and environmental sources.

The antigenic types of 129 isolates of Nocardia asteroides from diverse clinical, environmental, and geographic origins were determined. The majority of the isolates studied were of bovine (56) or human (44) origin; 11 were derived from six species of animals other than cattle, and 10 were isolated from environmental sources; the source of 8 strains could not be determined. Testing culture filtrate antigens against four standard reference sera in a gel diffusion precipitin test established the antigenic type of 95.3% of the isolates. After excluding strains that weighted the data because of common infection, the distribution of serotypes was examined according to the origin of the isolate. Type I was the most frequently encountered serotype (31.9%); types III (15.0%) and IV (20.4%) were also observed frequently, as was the antigenic mixture III + IV (14.2%). There was an apparent difference in frequency of type III and IV antigens among isolates of bovine and human origin; type III made up 20.0% of the bovine isolates and 13.6% of the human isolates, whereas type IV constituted 10.0% of bovine and 27.3% of human isolates.     

Use of enzyme-linked immunosorbent assay and enzyme-linked immunoelectrotransfer blot for the diagnosis and monitoring of neurocysticercosis

A total of 70 proven cases of neurocysticercosis from la Réunion (Indian Ocean) were studied with enzyme-linked immunoassay (ELISA) and immunoelectrotransfer blot (EITB) to detect specific antibodies in serum and cerebrospinal fluid (CSF). Absorbance levels of antibody to crudeTaenia solium cyst extract as an antigen were compared with EITB banding-pattern and computed tomography-scan results. The EITB analysis of sera and CSF from patients with active neurocysticercosis, confirmed with characteristic brain-scan imaging and highest ELISA absorbance, regularly revealed two bands with molecular weights of 13 and 14 kDa, respectively. These low-molecular-weight fractions are potential markers of active cerebral cysticercosis, a result obtained in the simple epidemiological situation of La Réunion (Indian Ocean). A parallel study is underway in Madagascar, where cross-reactivities with other parasitic diseases, includingSchistosoma infections, may interfere.     

Differences in the interactions of Nocardia asteroides with macrophage, endothelial, and astrocytoma cell lines.

An in vitro model for studying host cell interactions with Nocardia asteroides was developed. Thus, macrophage cell lines J774A.1 and P388D1, pulmonary artery endothelium cell line CPAE, rat glial tumor cell line C6, and human astrocytoma cell lines CCF-STTG1 and U-373 MG were infected with either log- or stationary-phase cells of N. asteroides GUH-2, and the host cell-nocardia interactions were determined by light microscopy and electron microscopy. Polyclonal antinocardial antibody did not enhance uptake of nocardiae by any of these cell lines; however, log-phase cells of GUH-2 infected a higher percentage of J774A.1 and P388D1 than did stationary-phase organisms. When cells infected with stationary-phase GUH-2 were incubated for 6 h, filaments developed, which indicated that nocardial growth had occurred. In J774A.1 and P388D1, only 31 to 57% of the total stationary-phase coccobacillary cells that were phagocytized formed filaments within 6 h. This indicated that there was some inhibition of growth of the phagocytized nocardiae within these macrophage cell lines; however, the nocardiae grew within the endothelial (> 87% filaments) and astrocytoma (100% filaments) cell lines. Microfilament inhibitor cytochalasin B inhibited uptake of GUH-2 by macrophages and other cell lines, except that there was no effect on uptake of nocardial cells by astrocytoma cell line U-373 MG. Scanning and transmission electron microscopy showed phagocytosis of GUH-2 by the different cell lines. In cytochalasin B-treated cells, nocardiae were shown to penetrate through the cell surface and become internalized in a manner distinct from typical phagocytosis, suggesting that filamentous forms of this organism have a phagocytosis-independent invasion factor. The extent of this cytochalasin-resistant cellular penetration by the nocardiae differed in the different cell lines.     

Effect of intravenous silica on the course of Nocardia asteroides pneumonia.

Silica, a known toxin of mononuclear phagocytes, was administered intravenously to mice during Nocardia asteroides pneumonia. Mice that received silica had a sevenfold decrease in the number of peripheral blood monocytes and developed more severe N. asteroides pneumonia than control mice. Lung histology in mice that received silica resembled that of mice with impaired cell-mediated immunity. These results are most consistent with the explanation that silica injures blood monocytes and impairs their contributions to pulmonary host defense.     

Opacification of Middlebrook agar as an aid in distinguishing Nocardia farcinica within the Nocardia asteroides complex.

Among 58 aerobic actinomycetes isolated from different sources and geographical locations, none of 23 Nocardia asteroides isolates, at 18 N. farcinica isolates, 1 of 5 N. otitidiscaviarum isolates, and 1 of 4 Rhodococcus species isolates opacified Middlebrook 7H10 medium. Within the N. asteroides complex, this characteristic, together with growth at 45 degrees C and resistance to each of erythromycin, cefotaxime, and tobramycin, provides a simple means of distinguishing N. farcinica from N. asteroides.     

Molecular study of persistence of Nocardia asteroides and Nocardia otitidiscaviarum strains in patients with long-term nocardiosis.

Three Nocardia asteroides and five Nocardia otitidiscaviarum isolates recovered from three patients with long-term nocardiosis were compared by random amplification of polymorphic DNA fingerprinting, antibiotic susceptibility testing, and enzymatic characterization. Results obtained with primer DKU49 (P. Palittapongarnpim, S. Chomyc, A. Fanning, and D. Kunimoto, J. Infect. Dis. 167:975-978, 1993) provide evidence that patient A was infected by two N. asteroides strains during a single episode of nocardiosis and that patients B and C remained infected by the same strain, respectively. Resistance to minocycline that was present in the first isolate recovered from patient B reverted to intermediate resistance in the second isolate and reverted to susceptibility in the third isolate. Resistance to penicillin G and beta-lactams was acquired by the second isolate obtained from patient C.