Comparative studies of antigen 21 in Mycobacterium and Nocardia species: possible taxonomic relationships with Mycobacterium leprae.

Studies of Mycobacterium leprae, Mycobacterium tuberculosis and Nocardia caviae in comparison with each other and with other Mycobacterium and Nocardia species were performed on the basis of antigen 21 intramolecular heterogeneity. Three different antisera were used: rabbit anti-Mycobacterium smegmatis antiserum, rabbit anti-Nocardia asteroides antiserum, and a lepromatous serum pool. With reference to each of the three antiserum sources used the strains were ranked in an order of relatedness or sharing of determinants. The three antisera showed distinctly different antigen 21 antibody specificities reflecting the species origin of the immunogen. The present investigations confirmed that antigen 21 of N. caviae shares determinants with antigens from Mycobacterium strains which were not present in corresponding antigens of all other Nocardia strains tested. M. tuberculosis, as judged by antigen 21 analysis, occupies a position separate from both the slow-growing and the fast-growing mycobacterial clusters in accordance with accepted taxonomic relationships. An interesting possibility of establishing a position for M. leprae in relation to other mycobacterial species was apparent. The order of relatedness among the strains studied went from M. leprae to M. tuberculosis to N. caviae to Mycobacterium avium to Mycobacterium fortuitum, the last two being representatives of the slow-growing and fast-growing mycobacteria. It can therefore be concluded that evidence from antigen 21 analysis indicates that M. leprae is more closely related to M. tuberculosis than to the other strains investigated.     

First helminthological data on Iberian vipers: Helminth communities and host-parasite relationships

European vipers are ambush predators with sporadic feeding events, thereby maintaining the digestive tract empty for long periods. According to previous studies relating lizards’ dietary habits and their helminth faunas, we predict poor gastrointestinal helminth communities in vipers. To test this hypothesis, we have examined the digestive tract of 86 specimens of Vipera aspis (L., 1758) and V. latastei Boscá, 1878, from several localities of the Iberian Peninsula. We found adults of only two nematode species Kalicephalus viperae (Rud., 1819) and Ophidascaris sp. and cysts adhering to the external wall on the stomach in case of two other nematode species Ascarops strongylina (Rud., 1819) and Spirurida gen. sp. All these nematodes are common parasite species in snakes, although Ophidascaris sp. has never before been recorded in Vipera sp. The low prevalence and small number of parasite species in Iberian vipers matched their low feeding rates. However, our results contrast with studies in Poland and Belarus of V. berus species, in which nematodes, as well as trematodes, are common and abundant. Rainfall rates are lower in the Iberian Peninsula than in eastern Central Europe, where amphibians are more available and consumed by V. berus. Amphibians, intermediate hosts for these helminths, have been recorded only sporadically as prey for V. aspis and V. latastei, thus supporting the absence of trematodes in both Iberian viper species. Among populations of Iberian vipers, the prevalence of parasites correlates with the feeding rate (i.e. percentage of vipers with prey), suggesting a linkage between the two parameters. In conclusion, our results suggest that several factors, including climatic characteristics of localities, feeding rates of predators, and type of prey consumed, influence the number and type of parasites in Iberian vipers.     

Modified Thayer-Martin medium for recovery of Nocardia species from contaminated specimens.

Specimens submitted for isolation of Nocardia species are frequently contaminated with other bacteria. Although decontamination with sodium hydroxide or benzalkonium chloride can remove these bacteria, this treatment is also toxic for Nocardia species. We demonstrate that modified Thayer-Martin medium can be used for selective isolation of Nocardia species.     

Isolation and characterization of bacteriophage NTR1 infectious for Nocardia transvalensis and other Nocardia species

Virus Genes - We describe here the isolation and characterization of the bacteriophage, NTR1 from activated sludge. This phage is lytic for Nocardia transvalensis, Nocardia brasiliensis and...     

Cell culture of snails. Their use in the study of immunologic host-parasite relationships in schistosomiasis]

The host-parasite relationship between Schistosoma mansoni and the snail Biomphalaria glabrata is so complex that its experimental study needs some particularly working methods: for instance molluscan organs and tissue's cultures. In the experiment described we report that digestive gland's cells cultures were grown on synthetic cultural medium. This one contained a physiological salt solution and a basal nutrient medium consisting of foetal calf serum, chicken embryo extract and amino-acids. In this case we observed good growth and multiplication of the digestive gland cells. This preliminary experiment has to be used in immunological studies regarding the origin and the mode of acquisition of the snail antigen by the cercariae membrane.     

First clinical isolates of Nocardia carnea, Nocardia elegans, Nocardia paucivorans, Nocardia puris and Nocardia takedensis in Japan.

Five aerobic actinomycete strains isolated from patients in Japan were assigned provisionally to the genus Nocardia based on morphological and physiological characteristics. The five strains, IFM 10481, IFM 0668, IFM 0901, IFM 0583 and IFM 0342, were not classified into any Nocardia species reported as infectious agents in Japan. Therefore, they were studied further to determine their specific taxonomic positions. Detailed chemotaxonomic and physiologic characterization and 16S rDNA sequence data of the five strains showed that they belonged to respective species of Nocardia carnea, N. elegans, N. paucivorans, N. puris and N. takedensis. This is the first isolation report of these five Nocardia species from patients in Japan.     

Nocardia asteroides and Nocardia brasiliensis infections in mice.

A model for Nocardia asteroides and Nocardia brasiliensis infections in Swiss white mice has been established without the addition to the inocula of any form of adjuvant. Serial histopathological studies revealed that these two actinomycetes cause lesions that are quite different in their features. An acute suppurative abscess characterizes the lesions of N. asteroides. In the case of N. brasiliensis infections a granuloma is produced in which a striking feature is the presence of large numbers of foam-laden macrophages, although occasional exceptions to this pattern were noted. Electron microscopic studies demonstrated that these macrophages contain within their cytoplasm organisms in varying stages of degeneration. Repeated mortality studies in mice failed to demonstrate differences in mortality rates produced by N. asteroides and N. brasiliensis. Thus, despite relatively trivial biochemical and antigenic differences between these two species of Nocardia, the local pathogenic response is quite different. The presence in the "brasiliensis lesion" of foamy macrophages with intracellular organisms is reminiscent of the histopathological features of lepromatous leprosy and of disseminated Myocobacterium bovis infection when this occurs in the immune suppressed situation. It is possible that N. brasiliensis infection produces a depression of cellular immunity that modifies the local host response to the organism.     

Organisms designated as Nocardia asteroides drug pattern type VI are members of the species Nocardia cyriacigeorgica

Nocardia cyriacigeorgica has recently been described as an "emerging" pathogen. However, DNA-DNA hybridization results confirm that Nocardia asteroides drug pattern type VI, which has long been recognized as a common and significant pathogen in the United States, belongs to the species N. cyriacigeorgica.     

Nocardia species as an etiologic agent in Parkinson's disease: serological testing in a case-control study.

To test the hypothesis that Nocardia spp. may be an etiologic factor in Parkinson's disease (PD), we used a serodiagnostic panel to determine if PD patients had antibodies specific for Nocardia spp. To validate the serological test panel, sera from healthy volunteers and from patients with culture-proven nocardiosis (n = 307) were compared in part 1 of the study. The sensitivity of the panel was 88% for detection of culture-proven nocardial infections, and specificity was 85% (excluding cross-reactive leprosy cases). In part 2, no difference in seropositivity was found when PD patients were compared with their age- and gender-matched controls (n = 140). We found a high exposure rate of humans to nocardial antigens, especially among men and older individuals. Our results offer no support to the hypothesis that Nocardia spp. are causative in PD; however, it is possible that serological testing may not be optimal for detection of nocardial central nervous system infection.     

Novel method for rapid identification of Nocardia species by detection of preformed enzymes.

The purpose of the present study was to devise a method for the identification of Nocardia species that is more technically simple, accurate, and rapid than current standard methods of identification. We focused on a commercial bacteria identification system that contained chromogenic test substrates. Two MicroScan products were selected for use in the study on the basis of their content of chromogenic and conventional substrates. They were the Rapid Anaerobe Identification and the HNID panels. A total of 85 strains of Nocardia representing five species were used in the study. All isolates were identified as Nocardia species by the use of standard methods. The beta-naphthylamide-labeled substrate L-pyrrolidonyl-beta-naphthylamide (PYR), the nitrophenyl-labeled substrate p-nitrophenyl-alpha-D-mannopyranoside (MNP), and indoxyl phosphate were found to be useful for identification purposes. N. farcinica and N. nova were the only species positive for PYR, whereas N. brasiliensis was the only species that hydrolyzed MNP. All strains of N. brasiliensis, N. otitidiscavarium, and N. farcinica were positive for indoxyl phosphate, whereas strains of N. nova and N. asteroides sensu stricto were always negative. Agreement between the standard and enzymatic identification methods was 100%. In summary, detection of preformed enzymes appears to be a simple and reproducible method for the identification of Nocardia spp.     

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.     

Antigenic relationships and rapid identification of Peptostreptococcus species.

Antisera against whole cells of each Peptostreptococcus species (P. anaerobius, P. micros, P. parvulus, and P. productus) were produced in rabbits. When these antisera were reacted against sonically disrupted cells and culture supernatant fluids in Ouchterlony tests, lines of identity were obtained among the antigens from all the species and uninoculated culture medium. When the antisera were subsequently absorbed with the dehydrated culture medium used to grow the peptostreptococci, all cross-reactions in heterologous antigen-antibody combinations were eliminated, leaving only species-specific precipitin arcs. These absorbed antisera, specific for each Peptostreptococcus species by Ouchterlony tests, were used for rapid identification studies. Staphylococcus aureus-bearing protein A was sensitized with each absorbed antiserum. These reagents produced specific coagglutination reactions with suspensions of each Peptostreptococcus reference strain and with 16 clinical isolates. No cross-reactions occurred with the Streptococcus intermedius, Peptococcus magnus, or Peptococcus asaccharolyticus strains tested.     

Nocardia wallacei sp. nov. and Nocardia blacklockiae sp. nov., human pathogens and members of the "Nocardia transvalensis Complex"

Nocardia isolates that share the property of in vitro amikacin resistance are grouped together by some authors in the Nocardia transvalensis complex. Our examination of 13 isolates that are amikacin resistant has revealed the existence of three distinct species. Sequence analysis of the 16S rRNA, 65-kDa heat shock protein, and secA1 genes, coupled with DNA-DNA hybridization, indicated that "N. asteroides drug pattern IV," "N. transvalensis new taxon 1," and N. transvalensis sensu stricto should each be considered a distinct species. The phenotypic and molecular characteristics of the proposed new species Nocardia wallacei (N. asteroides drug pattern IV) and N. blacklockiae (N. transvalensis new taxon 1) are presented and compared with those of N. transvalensis sensu stricto. The relative genetic diversity of isolates best placed with the species N. blacklockiae is also discussed. Case studies demonstrating the pathogenicity of N. wallacei and N. blacklockiae are presented. The type strain of N. wallacei is ATCC 49873 (DSM 45136), and that of N. blacklockiae is ATCC 700035 (DSM 45135).     

Identification of Nocardia species from clinical isolates using MALDI-TOF mass spectrometry

The identification of Nocardia isolates still represents a challenge for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) despite its acceptance for most bacterial and fungal isolates. In this study we evaluate the identification of Nocardia isolates using direct spotting and an updated database.Overall, 82 Nocardia isolates belonging to 13 species were identified by DNA sequence analysis of the 16S rRNA and secA1 genes. Nine of these well-characterized isolates from 6 Nocardia species were used to create an in-house library. The remaining 73 isolates were directly spotted on the target plate and on-plate protein extraction was performed. The protein spectra obtained were analyzed by MALDI-TOF MS using the BDAL database (Bruker Daltonics) updated with 6,903 MSPs or the combination of this commercial database and our in-house library.As a result, the use of the commercial database alone and in combination with the in-house library yielded 94.5% and 95.9% of correct species-level identifications, respectively, No isolate was misidentified at the genus level with either database. Besides, the use of the in-house library allowed the species-level identification of a N. otitidiscaviarum isolate that could only be identified at the genus-level with a score value <1.6 using the commercial database.In conclusion, the implementation of the direct spotting method and the in-house database provided a high rate of correct species assignment of Nocardia isolates despite the low number of isolates added. Further addition of well-characterized Nocardia isolates may ensure the rapid, accurate and inexpensive identification of most isolates encountered in the routine of the microbiology laboratory.     

Analysis of secA1 gene sequences for identification of Nocardia species

Molecular methodologies, especially 16S rRNA gene sequence analysis, have allowed the recognition of many new species of Nocardia and to date have been the most precise methods for identifying isolates reliably to the species level. We describe here a novel method for identifying Nocardia isolates by using sequence analysis of a portion of the secA1 gene. A region of the secA1 gene of 30 type or reference strains of Nocardia species was amplified using secA1-specific primers. Sequence analysis of 468 bp allowed clear differentiation of all species, with a range of interspecies similarity of 85.0% to 98.7%. Corresponding 16S rRNA gene sequences of a 1,285-bp region for the same isolates showed a range of interspecies similarity of 94.4 to 99.8%. In addition to the type and reference strains, a 468-bp fragment of the secA1 gene was sequenced from 40 clinical isolates of 12 Nocardia species previously identified by 16S rRNA gene sequence analysis. The secA1 gene sequences of most isolates showed >99.0% similarity to the secA1 sequences of the type or reference strain to which their identification corresponded, with a range of 95.3 to 100%. Comparison of the deduced 156 amino acid sequences of the SecA1 proteins of the clinical isolates showed between zero and two amino acid residue differences compared to that of the corresponding type or reference strain. Sequencing of the secA1 gene, and using deduced amino acid sequences of the SecA1 protein, may provide a more discriminative and precise method for the identification of Nocardia isolates than 16S rRNA gene sequencing.