Lehrstuhl für Mikrobiologie, Technische Universität München, Am Hochanger 4, D-83530 Freising, Germany1
Central Institute of the Federal Armed Forces Medical Service, D-56065 Koblenz, Germany2
Department of Laboratory Medicine, University of Washington, Seattle, WA 98195, USA3
Author for correspondence: Michael Wagner. Tel: +49 8161 715444. Fax: +49 8161 715475. e-mail: wagner{at}mikro.biologie.tu-muenchen.de
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Keywords: Hartmannella, endoparasite, Parachlamydiaceae, Neochlamydia hartmannellae, Chlamydia
Abbreviations: EB, elementary body; FLA, free-living amoebae; RB, reticulate body
The GenBank accession number for the sequence reported in this paper is AF177275.
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
A significant fraction of environmental and clinical FLA isolates harbour, like many other protozoa (Heckmann & Görtz, 1992 ; Preer & Preer, 1984
), bacterial endocytobionts (Fritsche et al., 1993
; Michel et al., 1995
). Recent studies have begun to elucidate the phylogenetic diversity of FLA-associated endocytobionts by applying the rRNA approach. The majority of the endocytobionts identified thus far are related to bacterial genera currently recognized as important human pathogens. For example, Legionella-related, Rickettsia-related and Chlamydia-related organisms are known to occur in FLA (Amann et al., 1997
; Birtles et al., 1996
; Fritsche et al., 1999
). In addition, several endocytobionts which group phylogenetically with the Paramecium caudatum symbiont Caedibacter caryophilus (Springer et al., 1993
) are known to proliferate within Acanthamoeba spp. (Horn et al., 1999
). Whereas the relationship between hosts and endocytobionts remains largely unexplored, there is increasing evidence that some FLA endocytobionts are of medical importance. Endocytobiont-mediated increase of Acanthamoeba cytopathogenicity in tissue culture suggests that these intracellular bacteria enhance FLA virulence (Fritsche et al., 1998
). Furthermore, some of the endocytobionts have been implicated as causative agents for disease, as indicated by the presence of specific antibodies against Chlamydia-related endocytobionts of Acanthamoeba in blood from respiratory-disease patients, and by the detection of Parachlamydia-like 16S rDNA sequences in specimens from bronchitis patients (Birtles et al., 1997
; Ossewaarde & Meijer, 1999
).
This report describes the investigation of coccoid bacterial endocytobionts of Hartmannella vermiformis strain A1Hsp isolated from the water conduit system of a dental unit, by (i) transfection experiments, (ii) electron microscopy, and (iii) the rRNA approach including comparative 16S rRNA sequence analysis and fluorescence in situ hybridization using 16S rRNA-targeted oligonucleotide probes and confocal laser scanning microscopy. Portions of this work were presented as an abstract at the 99th General Meeting of the American Society for Microbiology in Chicago, IL, 1999.
![]() |
METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Extracellular growth of H. vermiformis endoparasites.
Attempts to culture the Hartmannella endocytobionts extracellularly included cultivation on blood agar (Becton Dickinson), Casiton-agar (Biotest-Heipha) and SCGYE (De Jonckheere, 1977 ) at incubation temperatures of 20 and 30 °C. Both whole amoeba cells and filter-purified endocytobionts from lysed amoeba cells were transferred to the respective media. If no growth was observed after 14 d incubation, cultures were considered negative.
Transfection experiments.
Following lysis of endocytobiont-infected H. vermiformis cells from 45-d-old cultures by freezethawing, the coccoid bacterial endocytobionts were filter-purified (1·2 µm membrane filter). An aliquot of 80 µl of the resulting suspension was added to strains of different species of FLA, growing either in SCGYE medium or on NN-agar plates covered with a lawn of Enterobacter cloacae. The host range of the endocytobiont was investigated by transfection experiments with 14 different strains of FLA (Table 1), and one strain of Dictyostelium discoideum isolated from human nasal mucosa. Infection of each host species was monitored by phase-contrast microscopy. After 21 d incubation at 30 or 20 °C the host was considered resistant to infection if no infected cells nor any marked reduction in amoebal numbers (which may have resulted from parasitic activity of the endocytobiont) were observed.
|
DNA isolation, amplification of 16S rDNA, cloning and sequencing.
Simultaneous isolation of DNA from the amoebae and their endocytobionts was performed using a modified UNSET procedure (Hugo et al., 1992 ). Amoebae and their endocytobionts were harvested from axenic cultures by centrifugation (2000 g, 3 min), washed twice with double-distilled water, resuspended in 500 µl UNSET lysis buffer (8 M urea, 0·15 M NaCl, 2% SDS, 0·001 M EDTA and 0·1 M Tris/HCl at pH 7·5) and incubated at 60 °C for 5 min. Lysates were extracted twice with phenol/chloroform (Roth) and DNA was precipitated for 3 h at -20 °C with 2 vols absolute ethanol. After centrifugation (10000 g, 10 min) at 4 °C the ethanol was removed and the pellet was washed twice with 80% ice-cold ethanol to remove residual salts. The pellet was air-dried and resuspended in 30 µl double-distilled water.
Oligonucleotide primers targeting 16S rDNA signature regions which are conserved within the Chlamydiales were used for PCR to obtain near-full-length bacterial 16S rRNA gene fragments. The nucleotide sequences of the forward and reverse primers used for amplification were 5'-CGGATCCTGAGAATTTGATC-3' and 5'-TGTCGACAAAGGAGGTGATCCA-3' (Pudjiatmoko et al., 1997 ). 16S rDNA amplification reactions were performed in a thermal capillary cycler (Idaho Technology) using reaction mixtures including 15 pM of each primer, 0·25 µg BSA ml-1, 2 mM MgCl2 reaction buffer and 2·5 IU Taq DNA polymerase (Promega). Thermal cycling was carried out as follows: an initial denaturation step at 94 °C for 30 s followed by 30 cycles of denaturation at 94 °C for 15 s, annealing at 52 °C for 20 s, and elongation at 72 °C for 30 s. Cycling was completed by a final elongation step at 72 °C for 5 min. A negative control was performed using a reaction mixture without added DNA. Amplified products were directly ligated into the cloning vector pCRII-TOPO and transformed into competent Escherichia coli (TOP10 cells) according to the instructions of the manufacturer (Invitrogen). Nucleotide sequences of the cloned DNA fragments were determined by the dideoxynucleotide method (Sanger et al., 1977
) by cycle sequencing of purified plasmid preparations (Qiagen) with a Thermo Sequenase Cycle Sequencing Kit (Amersham Life Science) and an automated DNA sequencer (Li-Cor) under conditions recommended by the manufacturers. Dye-labelled vector-specific primers M13/pUC V (5'-GTAAAACGACGGCCAGT-3') and M13/pUC R (5'-GAAACAGCTATGACCATG-3') were applied.
Phylogenetic analysis.
The 16S rDNA sequences obtained were added to the rDNA sequence database of the Technische Universität München (encompassing more than 16000 published and unpublished homologous small-subunit rDNA primary structures) by use of the program package ARB (O. Strunk and others, unpublished; program available through the homepage of the Technische Universität München: http://www.mikro.biologie.tu-muenchen.de). Alignment of the new rDNA sequences was performed by using the ARB automated alignment tool (version 2.0). The alignments were refined by visual inspection and by secondary-structure analysis. Phylogenetic analyses were performed by applying the ARB parsimony, distance matrix and maximum-likelihood methods to different data sets. To determine the robustness of the phylogenetic trees, analyses were performed with and without the application of various filtersets to exclude highly variable positions.
Fluorescence in situ hybridization and confocal laser scanning microscopy.
The following oligonucleotide probes were used: (i) EUB338 (5'-GCTGCCTCCCGTAGGAGT-3'), targeting most, but not all, members of the domain Bacteria (Amann et al., 1990 ; Daims et al., 1999
), and (ii) S-S-ParaC-0658-a-A-18 (5'-TCCATTTTCTCCGTCTAC-3'), previously designed as complementary to a signature region of the 16S rRNA of the Parachlamydia-related endosymbionts of Acanthamoeba spp. strains UWC22 and TUME1 (T. R. Fritsche and others, unpublished; probe designation according to the standard proposed by Alm et al., 1996
). Oligonucleotides were synthesized and directly 5'-labelled with 5(6)-carboxyfluorescein-N-hydroxysuccinimide ester (FLUOS), or the hydrophilic sulphoindocyanine fluorescent dye Cy3 (Interactiva).
For in situ hybridization, Hartmannella cells were harvested from 4 ml liquid broth culture by centrifugation (2000 g, 3 min), washed twice with double-distilled water, and resuspended in 0·05% agarose. Twenty microlitres of this suspension was spotted on a glass slide, air-dried and subsequently dehydrated in 80% ethanol for 10 s. Hybridization was performed using the hybridization buffer (including 30% formamide) and the buffer washing (containing 112 mM NaCl, without SDS) described by Manz et al. (1992) . Slides were examined using a confocal laser scanning microscope (LSM 510, Carl Zeiss) in combination with a helium-neon laser (543 nm) and an argon-krypton laser (488 nm). Image analysis processing was performed with the standard software package delivered with the instrument (version 2.01).
![]() |
RESULTS AND DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
At present, we can only speculate on the ecological significance of the suppression of FLA cyst formation by some endocytobionts. A possible clue might be provided by the observation that Escherichia coli (Steinert et al., 1998 ) is eradicated from artificially infected Acanthamoeba cells during cyst formation. Thus, prevention of cyst formation might be a protection mechanism for parasitic endocytobionts, which would be negatively affected by the differentiation of FLA into resting forms. This strategy would contrast with the one used by Legionella pneumophila (Steinert et al., 1998
; Kilvington & Price, 1990
) and several other obligate Acanthamoeba endocytobionts (Fritsche et al., 1999
; Horn et al., 1999
), which survive host-cell cyst formation and thus directly benefit from cyst-mediated host resistance against unfavourable environmental conditions.
Chlamydia-like life cycle and parasitic behaviour of the H. vermiformis endocytobiont
Electron micrographs revealed a Chlamydia-like morphology and developmental cycle of the endocytobionts (Fig. 1). Stages showing binary fission resembling those of reticulate bodies (RBs, 0·40·6 µm in diameter) of Chlamydia could be observed. Additionally, the highly condensed coccoid stages (0·50·6 µm in diameter) are similar to elementary bodies (EBs) of Chlamydia. While RBs of the H. vermiformis endocytobiont clearly possess a Gram-negative type cell wall, results of electron microscopic analysis of its EBs are ambiguous. However, since the EBs of the Hartmannella endocytobiont showed an outer membrane, we consider them as Gram-negative. This is in noticeable contrast to the Gram-positive type of cell wall which has been observed for the Chlamydia-related endoparasite of Acanthamoeba sp. strain BN9 (P. acanthamoebae; Amann et al., 1997
). In contrast to Chlamydia species and Parachlamydia-related endocytobionts of Acanthamoeba (Amann et al., 1997
; T. R. Fritsche and others, unpublished), RBs and EBs of the Hartmannella endocytobiont were not surrounded by vacuoles and were thus located directly within the cytoplasm of the host cell, indicating that the endocytobionts possess an escape mechanism from the phagosomes.
|
Natural stability of this hostparasite association would require an amoebal generation time shorter than the period between parasite infection and host cell lysis. The aggressive parasitic behaviour of the endocytobiont within its Hartmannella host suggests a limited adaptation of host and parasite caused by a relatively short evolutionary relationship. Hartmannella species may have only recently been infected by these parasites, suggesting their origin from another protist species. Limited adaptation of the endoparasite to the H. vermiformis host is also suggested by the suppression of cyst formation, which might protect the parasites from eradication but which may decrease the fitness of the association against environmental stress.
Host range of the obligate endoparasite of H. vermiformis
Standard cultivation techniques failed to support extracellular growth of the Hartmannella endoparasites, suggesting that Hartmannella cells are necessary for its growth. This finding is in accordance with the obligate intracellular growth of other endocytobionts of FLA (Amann et al., 1997 ; Fritsche et al., 1993
).
The host range of the Hartmannella endoparasite was determined by transfection experiments encompassing a recently isolated D. discoideum strain and 14 different strains of FLA belonging to the genera Hartmannella, Acanthamoeba, Naegleria and Willaertia (Table 1). Except for the original host H. vermiformis A1Hsp and two more H. vermiformis strains, only D. discoideum strain Berg25 could be successfully infected. Whereas the extent of parasitic behaviour of the investigated endocytobionts varied slightly between the different H. vermiformis host strains, aggregation and stalk and fruiting body formation of D. discoideum were not disturbed by the endocytobionts and endoparasite-free spores were formed.
There are remarkable differences in host range between the investigated Hartmannella endoparasite and the Acanthamoeba endoparasite P. acanthamoebae. The Hartmannella endoparasite is not able to infect the tested Acanthamoeba strains, including A. castellanii C3, which is a suitable host for P. acanthamoebae (Amann et al., 1997 ). Conversely, P. acanthamoebae is unable to infect H. vermiformis strains, which serve as host for the investigated Hartmannella endoparasite (R. Michel, personal communication). Future studies are required to elucidate the molecular mechanism of a specific recognition system that may mediate specificity of infection.
Phylogeny and in situ identification of the H. vermiformis endoparasite
Near-full-length 16S rDNA amplicons (1529 bp) retrieved from mixed genomic DNA of amoebal hosts and bacterial endoparasites were successfully cloned and sequenced. Comparative sequence analysis revealed that the retrieved 16S rRNA sequence displayed highest similarity values with 16S rRNA sequences of members of the Chlamydiales (Table 2). In particular, the investigated Hartmannella endoparasites are moderately related to the Acanthamoeba parasite P. acanthamoebae strain Bn9 (92%), the only validly described member of the new family Parachlamydiaceae (Everett et al., 1999
). It should be noted that even higher sequence similarities of between 96·5% and 97·1% to the Hartmannella parasite were calculated for 16S rRNA sequences of two recently investigated Parachlamydia-related endosymbionts of Acanthamoeba (Table 2
; T. R. Fritsche et al., unpublished).
|
|
|
Description of Neochlamydia gen. nov.
Neochlamydia (Ne.o.chla.mydi.a L. fem. n.; Neochlamydia referring to the modest phylogenetic relationship to the Chlamydiaceae).
Phylogenetic position: order Chlamydiales, family Parachlamydiaceae. Members of the genus Neochlamydia should have a 16S rDNA that is >95% identical to the 16S rDNA of the type species, Neochlamydia hartmannellae strain A1Hsp.
Description of Neochlamydia hartmannellae sp. nov.
Neochlamydia hartmannellae (hartmann.el.lae. L. gen. sing. n. of Hartmannella, taxonomic name of a genus of Hartmannellidae; pertaining to the name of the host amoeba, Hartmannella vermiformis strain A1Hsp, in which the organism was first discovered).
Gram-negative reticulate bodies and Gram-negative elementary bodies; coccoid morphology; 0·40·6 µm in diameter. Basis of assignment: 16S rDNA sequence accession number AF177275, nucleotide probe S-S-ParaC-0658-a-A-18 (5'-TCCATTTTCTCCGTCTAC-3'). Not cultivated on cell-free media; obligate intracytoplasmatic parasite of H. vermiformis strain A1Hsp and other H. vermiformis strains, therein preventing cyst formation. Host range: able to multiply in D. discoidum, but not in Acanthamoeba spp.; mesophilic (2030 °C). Isolated from the water conduit system of a dental unit (Lahnstein, Germany). Type strain, A1Hsp (=ATCC 50802).
Diversity within the Chlamydiales and clinical aspects of N. hartmannellae
In a more general perspective, our results and the recent identification of four Parachlamydia-related acanthamoebal endocytobionts (T. R. Fritsche and others, unpublished), a Chlamydia-like bovine intracellular organism (Waddlia chondrophila; Rurangirwa et al., 1999 ) and a Chlamydia-related organism observed within tissue culture (Simkania negevensis; Kahane et al., 1999
) demonstrate a previously unrecognized diversity within the Chlamydiales. Interestingly, the order Chlamydiales still exclusively comprises obligate intracellular bacteria, some of which have developed mechanisms to survive and exploit uptake by protozoa. The adaptation to intracellular growth in the ubiquitously distributed FLA could have functioned as a preadaptation of Chlamydia-like ancestors to survival within other host cells of higher eukaryotes, including humans; this raises the question of the clinical significance of members of the family Parachlamydiaceae. Few studies have addressed this important issue. Among these, Birtles et al. (1997)
screened for the presence of specific antibodies against Parachlamydia-like endocytobionts of Acanthamoeba sp. (Halls coccus, displaying more than 99% 16S rDNA similarity to P. acanthamoebae) in blood sera from patients with pneumonia of undetermined cause, and found positively reacting sera that did not react with Chlamydia psittaci, C. trachomatis or C. pneumoniae. These researchers therefore suggested that Halls coccus be considered potentially pathogenic for humans. Another remarkable finding was the recovery of novel Chlamydia-like 16S rDNA sequence fragments from specimens of respiratory-disease patients, recently reported by Ossewaarde & Meijer (1999)
. The sequence similarities of these sequences and N. hartmannellae range between 72% and 84%. Reliable phylogenetic analysis of the Chlamydia-like sequences and the 16S rRNA sequence of N. hartmannellae could not be performed, due to the short length of the Chlamydia-like sequence fragments (approx. 220 bp). A stable tree topology could not be obtained by applying different treeing methods and data sets. Further research is needed to clarify whether the FLA endocytobionts of the family Parachlamydiaceae are indeed able to infect humans.
Concluding remarks
In conclusion, we have identified an obligate endoparasite of H. vermiformis, provisionally classified as Neochlamydia hartmannellae, as a new member of the family Parachlamydiaceae. These findings broaden our knowledge of the phylogenetic diversity within the Chlamydiales. Although it is too early to draw conclusions on the clinical significance of these bacteria, the detection of these organisms in FLA suggests that FLA may act as a general reservoir for Chlamydia-like organisms. More detailed knowledge is needed on the natural habitats, diversity, physiology and virulence of members of the family Parachlamydiaceae.
![]() |
ACKNOWLEDGEMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() |
---|
Amann, R., Binder, B. J., Olson, R. J., Chisholm, S. W., Devereux, R. & Stahl, D. A. (1990). Combination of 16S rRNA-targeted oligonucleotide probes with flow cytometry for analyzing mixed microbial populations. Appl Environ Microbiol 56, 1919-1925.[Medline]
Amann, R., Springer, N., Schönhuber, W., Ludwig, W., Schmid, E., Müller, K. & Michel, R. (1997). Obligate intracellular bacterial parasites of Acanthamoebae related to Chlamydia spp. Appl Environ Microbiol 63, 115-121.[Abstract]
Birtles, R. J., Rowbotham, T. J., Raoult, D. & Harrison, T. G. (1996). Phylogenetic diversity of intra-amoebal legionellae as revealed by 16S rRNA gene sequence comparison. Microbiology 142, 3525-3530.[Abstract]
Birtles, R. J., Rowbotham, T. J., Storey, C., Marrie, T. J. & Raoult, D. (1997). Chlamydia-like obligate parasite of free-living amoebae. Lancet 349, 925-926.[Medline]
Daims, H., Schleifer, K.-H. & Wagner, M. (1999). Probe EUB338 is insufficient for the detection of all Bacteria: development and evaluation of a more comprehensive probe set. Syst Appl Microbiol 22, 438-448.
De Jonckheere, J. F. (1977). Use of an axenic medium for differentiation between pathogenic and non-pathogenic Naegleria fowleri isolates. Appl Environ Microbiol 33, 751-757.[Medline]
Everett, K. D., Bush, R. M. & Andersen, A. A. (1999). Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 49, 415-440.[Abstract]
Fritsche, T. R., Gautom, R. K., Seyedirashti, S., Bergeron, D. L. & Lindquist, T. D. (1993). Occurrence of bacteria endosymbionts in Acanthamoeba spp. isolated from corneal and environmental specimens and contact lenses. J Clin Microbiol 31, 1122-1126.[Abstract]
Fritsche, T. R., Sobek, D. & Gautom, R. K. (1998). Enhancement of in vitro cytopathogenicity by Acanthamoeba spp. following acquisition of bacterial endosymbionts. FEMS Microbiol Lett 166, 231-236.[Medline]
Fritsche, T. R., Horn, M., Seyedirashti, S., Gautom, R. K., Schleifer, K.-H. & Wagner, M. (1999). In situ detection of novel bacterial endosymbionts of Acanthamoeba spp. phylogenetically related to members of the Rickettsiales. Appl Environ Microbiol 65, 206-212.
Gautom, R. & Fritsche, T. R. (1995). Transmissibility of bacterial endosymbionts between isolates of Acanthamoeba spp. J Eukaryot Microbiol 42, 452-456.[Medline]
Heckmann, K. & Görtz, H.-D. (1992). Prokaryotic symbionts of ciliates. In The Prokaryotes, pp. 3865-3890. Edited by A. Balows, H. G. Trüper, M. Dworkin, W. Harder & K.-H. Schleifer. New York & Heidelberg: Springer.
Horn, M., Fritsche, T. R., Gautom, R. K., Schleifer, K.-H. & Wagner, M. (1999). Novel bacterial endosymbionts of Acanthamoeba spp. related to the Paramecium caudatum symbiont Caedibacter caryophilus. Environ Microbiol 1, 357-368.[Medline]
Hugo, E. R., Gast, R. J., Byers, T. J. & Stewart, V. J. (1992). Purification of amoeba mtDNA using the UNSET procedure. In Protocols in Protozoology, pp. D7·17·2. Edited by J. J. Lee & A. T. Soldo. Lawrence, KS: Allen Press.
Kahane, S., Everett, K. D., Kimmel, N. & Friedman, M. G. (1999). Simkania negevensis strain ZT: growth, antigenic and genome characteristics. Int J Syst Bacteriol 49, 815-820.[Abstract]
Kilvington, S. & Price, J. (1990). Survival of Legionella pneumophila within cysts of Acanthamoeba polyphaga following chlorine exposure. J Appl Bacteriol 68, 519-525.[Medline]
Ludwig, W., Strunk, O., Klugbauer, S., Klugbauer, N., Weizenegger, M., Neumaier, J., Bachleitner, M. & Schleifer, K.-H. (1998). Bacterial phylogeny based on comparative sequence analysis. Electrophoresis 19, 554-568.[Medline]
Manz, W., Amann, R., Ludwig, W., Wagner, M. & Schleifer, K.-H. (1992). Phylogenetic oligonucleotide probes for the major subclasses of Proteobacteria: problems and solutions. Syst Appl Microbiol 15, 593-600.
Michel, R., Hauröder-Philippczyk, B., Müller, K. D. & Weishaar, I. (1994). Acanthamoeba from human nasal mucosa infected with an obligate intracellular parasite. Eur J Protistol 30, 104-110.
Michel, R., Müller, K. D. & Schmid, E. N. (1995). Ehrlichia-like organisms (KSL1) observed as obligate intracellular parasites of Saccamoeba species. Endocytobiosis Cell Res 11, 69-80.
Ossewaarde, J. & Meijer, A. (1999). Molecular evidence for the existence of additional members of the order Chlamydiales. Microbiology 145, 411-417.[Abstract]
Page, F. C. (1988). A New Key to Freshwater and Soil Gymnamoebae. Ambleside, UK: Freshwater Biological Association.
Preer, J. R.Jr & Preer, L. B. (1984). Endosymbionts of protozoa. In Bergeys Manual of Systematic Bacteriology, pp. 795-811. Edited by N. R. Krieg & J. G. Holt. Baltimore: Williams & Wilkins.
Pudjiatmoko, Fukushi, H., Ochial, Y., Yamaguchi, T. & Hirai, K. (1997). Phylogenetic analysis of the genus Chlamydia based on 16S rRNA gene sequences. Int J Syst Bacteriol 47, 425431.
Rodriguez-Zaragoza, S. (1994). Ecology of free living amoebae. Crit Rev Microbiol 20, 225-241.[Medline]
Rurangirwa, F. R., Dilbeck, P. M., Crawford, T. B., McGuire, T. C. & McElwain, T. F. (1999). Analysis of the 16S rRNA gene of microorganism WSU 86-1044 from an aborted bovine foetus reveals that it is a member of the order Chlamydiales: proposal of Waddliaceae fam. nov., Waddlia chondrophila gen. nov., sp. nov. Int J Syst Bacteriol 49, 577-581.[Abstract]
Sanger, F. N. S. & Coulson, A. R. (1977). DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 7, 5463-5467.
Springer, N., Ludwig, W., Amann, R., Schmidt, H. J., Görtz, H.-D. & Schleifer, K.-H. (1993). Occurrence of fragmented 16S rRNA in an obligate bacterial endosymbiont of Paramecium caudatum. Proc Natl Acad Sci USA 90, 9892-9895.[Abstract]
Steinert, M., Birkness, K., White, E., Fields, B. & Quinn, F. (1998). Mycobacterium avium bacilli grow saprozoically in coculture with Acanthamoeba polyphaga and survive within cyst walls. Appl Environ Microbiol 64, 2256-2261.
Visvesvara, G. S. (1995). Pathogenic and opportunistic free-living amebae. In Manual of Clinical Microbiology, pp. 1196-1203. Edited by P. R. Murray. Washington, DC: American Society for Microbiology.
Received 24 September 1999;
revised 22 December 1999;
accepted 24 January 2000.