Previous Article | Next Article 
Journal of Clinical Microbiology, March 1999, p. 575-580, Vol. 37, No. 3
0095-1137/99/$00.00+0
Rapid Detection of the Chlamydiaceae and
Other Families in the Order Chlamydiales: Three PCR
Tests
Karin D. E.
Everett,*
Linda J.
Hornung, and
Arthur
A.
Andersen
Avian and Swine Respiratory Diseases Research
Unit, USDA Agricultural Research Service, National Animal Disease
Center, Ames, Iowa 50010
Received 6 October 1998/Accepted 8 December 1998
 |
ABSTRACT |
Few identification methods will rapidly or specifically detect all
bacteria in the order Chlamydiales, family
Chlamydiaceae. In this study, three PCR tests based on
sequence data from over 48 chlamydial strains were developed for
identification of these bacteria. Two tests exclusively recognized the
Chlamydiaceae: a multiplex test targeting the
ompA gene and the rRNA intergenic spacer and a TaqMan test
targeting the 23S ribosomal DNA. The multiplex test was able to detect
as few as 200 inclusion-forming units (IFU), while the TaqMan test
could detect 2 IFU. The amplicons produced in these tests ranged from
132 to 320 bp in length. The third test, targeting the 23S rRNA gene,
produced a 600-bp amplicon from strains belonging to several families
in the order Chlamydiales. Direct sequence analysis of this
amplicon has facilitated the identification of new chlamydial strains.
These three tests permit ready identification of chlamydiae for
diagnostic and epidemiologic study. The specificity of these tests
indicates that they might also be used to identify chlamydiae without
culture or isolation.
| |
INTRODUCTION |
The order Chlamydiales
has been recently shown to include four families of obligately
intracellular bacteria that infect vertebrates or amoebae, the
Chlamydiaceae, Parachlamydiaceae,
Simkaniaceae, and Waddliaceae (8, 23).
Members of these families are explicitly identified by DNA sequence
analysis; a new study indicates that the Chlamydiales may be
comprised of even more lineages (19). The oldest family in
this order is the Chlamydiaceae, which was proposed in 1957 (21). The Chlamydiaceae are antigenically and genetically diverse, belonging to two genera and nine species (8). Inclusions formed in host cells by these bacteria are recognized, but not necessarily distinguished from one another, by
microscopy and staining methods. At one time, all chlamydiae were
thought to be recognized by immunohistochemical staining or serological
techniques that were believed to be Chlamydiaceae specific.
Occasionally, however, specimens that could not be confirmed by
techniques positively specific for the Chlamydiaceae
were cultured or isolated (4, 6, 9, 26). The
Chlamydiaceae are all identified by monoclonal antibodies
(MAbs) that recognize the lipopolysaccharide epitope
Kdo-(2
8)-Kdo-(24)-Kdo (Kdo is 3-deoxy-D-manno-octulosonic acid) (3, 5, 13,
18). These MAbs have been used to detect new groups in the
Chlamydiaceae (22) and to identify
Chlamydiaceae in novel hosts (10, 12, 26). MAb
staining may be done directly on smears or may require days or even
weeks for laboratory culture of chlamydiae in host cell monolayers.
PCR and other DNA-based tests for chlamydiae have tended to be specific
for groups within the Chlamydiaceae. Tests targeting the
ompA gene have shown some promise as tools for
identification of all Chlamydiaceae (16, 25, 27).
However, the DNA sequence of ompA is highly variable, and it
has been difficult to find segments conserved in all species that could
be targeted by a single set of primers to amplify a short,
characteristic PCR product. The rRNA operon contains many segments that
are conserved among all the Chlamydiaceae, but this locus
has been used only for identifying specific species, strains, or groups
of strains. Efforts to detect and identify chlamydiae are important
because chlamydiae not only cause disease but also interact
synergistically with viruses or with other bacteria, increasing the
virulence of these organisms (20, 28). In humans, livestock,
and birds, chlamydiae cause reproductive, respiratory, cardiovascular,
gastrointestinal, central nervous system, and systemic disease, as well
as conjunctivitis and arthritis (2, 3, 7, 11, 15, 17).
In this report, ribosomal DNA (rDNA) sequences from over 60 Chlamydiaceae strains and ompA sequences from 48 Chlamydiaceae strains, many of the sequences extending 300 or more bases past the ompA stop codon, were used to design
two PCR tests for specific and rapid detection of all species belonging
to the Chlamydiaceae. A third PCR test that recognized the
Chlamydiaceae as well as members of newer families in the
Chlamydiales was developed. These tests facilitate the
identification of strains belonging to these families.
| |
MATERIALS AND METHODS |
Template DNA sources and preparation.
Sources of the
chlamydial strains used for these tests have been described previously
(5, 7, 14). Chlamydial template DNA was prepared by reducing
alkaline lysis. The first step in reducing alkaline lysis was to pellet
chlamydiae and/or chlamydia-infected cells by centrifugation
(10,000 × g). The pellet was resuspended in 30 mM Tris
(pH 9.0)-10 mM EDTA (pH 9.0)-50 mM dithiothreitol and incubated for
1 h at 37°C. An equal volume of 1% Nonidet P-40 was then added
to each sample, as was DNase-free RNase (Boehringer Mannheim
Biochemicals, Indianapolis, Ind.; 2.5 µg for a 200-µl mixture).
Some samples (of strains R27 and GPICT) were divided into
two aliquots after the addition of Nonidet P-40, and one aliquot of
each was not treated with RNase so that control studies in the presence
of RNA could be done. All aliquots were incubated for 1 h at
37°C and then extracted with phenol-chloroform and chloroform
(24). DNA from Chlamydophila psittaci
6BCT was also prepared by CsCl gradient centrifugation for
a series of dilution controls (24). DNA from lysates of
Waddlia chondrophila WSU 86-1044T and
Simkania negevensis ZT were provided by Fred
Rurangirwa and Maureen Friedman, respectively.
Nonchlamydial template DNA was prepared by several means. Lysis under
alkaline reducing conditions as described above was used to prepare DNA
from mycoplasmas provided by Janet Saupe (National Veterinary Services
Laboratory, USDA (Animal and Plant Health Inspection Service, Ames,
Iowa) and from Vero cells, which were the host cells in which
chlamydiae were grown. CsCl-prepared DNA from Campylobacter,
Arcobacter, Listeria, Erysipelothrix,
and Helicobacter species was provided by Irene V. Wesley and
Sharon Franklin (National Animal Disease Center, USDA (Agricultural
Research Service, Ames, Iowa). Salmonella DNA from isolated
colonies was prepared by boiling in Tris-EDTA and provided by Alan
Baetz (National Animal Disease Center). DNAs from lysates of
Verrucomicrobium spinosum and Legionella
pneumophila were provided by Peter Janssen (University of
Melbourne, Melbourne, Australia) and Paul S. Hoffman (Dalhousie
University, Halifax, Nova Scotia, Canada), respectively. Pasturella, Bordetella, Salmonella,
Staphylococcus, Streptococcus, and
Escherichia coli field isolates from swine were provided by Douglas G. Rogers (University of Nebraska, Lincoln).
Test setup and controls.
All DNA templates were tested by
using three different sets of PCR primers. Identical template arrays
were set up on multiwell PCR plates, and each 50-µl PCR mixture for
chlamydiae included 0.25 µg of template (determined
spectrophotometrically). The concentrations of other templates ranged
from 0.25 to 2.0 µg/reaction mixture. Chlamydial templates included
RNase-treated preparations, several preparations that contained RNA and
DNA, and the RNA-DNA preparations to which RNase was added along with
the PCR reagents just before amplification. Six controls without
template were included on every plate.
Sensitivity.
Each plate containing an array of templates
also included a series of 10-fold dilutions of Chlamydophila
psittaci 6BCT template DNA to assess the sensitivity
of each assay. CsCl-purified 6BCT DNA was quantitated
spectrophotometrically and had an
A260/A280 ratio of 1.90. Electrophoresis of this DNA on a 1% agarose gel showed that most of
the DNA was >12 kbp in length. PCR of all templates on each plate was
performed at one time.
Test sensitivity was also determined for specific quantities of
inclusion-forming units (IFU) by using Renografin-purified
infectious
elementary bodies (EBs) of
C. psittaci NJ1. A dilution
series of EBs was prepared on ice, and approximately 2,000 Vero
cells
were added to each aliquot to provide a carrier for the
small numbers
of EBs. The aliquots were then immediately centrifuged
to provide an
EB-cell pellet from which DNA was prepared for PCR.
Microtiter plates
containing monolayers of Vero cells were also
infected with the
serially diluted NJ1 EBs. IFU were scored in
duplicate at 20 and
42 h by microimmunofluorescence using MAb
NJ1/D3 (
1).
Primers and PCR conditions.
The PCR primers used in these
tests are summarized in Table 1 and
illustrated in Fig. 1. Because the
control template DNAs were obtained from many laboratories, RNase was
included in the amplification reaction mixtures to ensure that RNA did
not interfere with control template amplification. RNase, per se, did
not interfere with PCR amplification. To test whether chlamydial RNA
affected amplification, some aliquots of R27 and GPICT
template DNA were also prepared without RNase. To test whether template
integrity affected amplification, specific aliquots of GPICT template that had been damaged with DNase so that no
template of >12 kbp could be detected were prepared.
View larger version (6K):
[in this window]
[in a new window]
|
FIG. 1.
Map of primer loci. 1, test 1 multiplex primers; 2, test
2 TaqMan primers and probe; 3, test 3 primers.
|
|
For test 1, primer IGF exactly matched the 16S/23S intergenic spacer of
all known
Chlamydiaceae; primer IGR spanned the start
site
of the 23S rRNA gene of all known
Chlamydiaceae, with a
deliberate
2-base mismatch in the center. Primer 1260 recognized all
known
ompA genes starting 48 bases before the stop codon;
primer TGLY
complemented a tRNA
Gly located approximately
270 bp downstream of the
ompA stop codon
in all known
Chlamydiaceae. These primers were used for PCR amplification
of template DNA in a GeneAmp PCR System 9600 thermocycler with
Taq DNA polymerase (Boehringer Mannheim). Reactions were
prepared
on ice in a 1× PCR mix containing Mg
2+ (1.5 mM;
Boehringer Mannheim), with or without added MgCl
2
(Mg
2+ final concentration, 4.0 mM) and with or without 1 µl of RNase
(0.5 µg) in each 50-µl reaction mix. Cycling
conditions for PCR
were 40 cycles of 30 s at 94°C, 15 s at
55°C, and 30 s at 72°C,
followed by incubation for 5 min at
72°C. Electrophoresis of 5
µl of each 50-µl reaction mix
separated the multiplex PCR products
in a VisiGel separation matrix
(Stratagene, La Jolla, Calif.).
The DNA was visualized with ethidium
bromide, which was included
in the
gel.
The primer set in test 2 (Table
1) was designed for use with the TaqMan
sequence detection system (Perkin-Elmer, Foster City,
Calif.) to target
the 23S rRNA gene. This primer set included
primers TQF and TQR, which
were specific for all known
Chlamydiaceae,
and a
fluorescent-labeled probe which annealed between primers
TQF and TQR.
Reaction mixtures were prepared at room temperature
with 2.5 mM
MgCl
2, 400 µM dUTP, 200 µM each dATP, dCTP, and dGTP,
1× TaqMan buffer A, 0.25 µl (1.25 U) of AmpliTaq Gold DNA polymerase
(Perkin-Elmer; AmpErase UNG was not included), 0.15 µM each primer,
0.1 µM probe, and 1 µl of RNase in each 50-µl reaction mixture.
The reaction mixtures were incubated for 10 min at 94°C and then
immediately subjected to 40 cycles of 15 s at 95°C and 1 min at
59°C. After cycling, the reaction mixtures were held at 4°C for
less than 1 h prior to fluorometric TaqMan reading. The mixtures
were frozen for
storage.
The primers in test 3 were U23F, which matched the sequence just after
the start of the 23S rRNA gene, and 23SIGR, which complemented
the
sequence approximately 600 bases downstream (Table
1) (
8).
These primers have been shown to PCR amplify the 23S rRNA signature
sequence, which has been designated for use in distinguishing
species
belonging to the
Chlamydiaceae and to other families in
Chlamydiales (
8). A BLAST search of the GenBank
database with
these primers suggested that they might also amplify
other bacterial
templates. These primers had high melting temperatures
to ensure
that they would anneal without regard for a few mismatches.
Reaction
mixtures were prepared on ice with
Taq DNA
polymerase, as in test
1, and with 1 µl of RNase in each 50-µl
reaction volume. Cycling
conditions for PCR were 35 cycles of 30 s
at 94°C, 15 s at 61°C,
and 30 s at 72°C, followed by
incubation for 5 min at 72°C. Electrophoresis
of 5 µl of each
positive 50-µl reaction mixture provided a single
600-bp PCR product
that could be visualized with ethidium bromide
in the VisiGel
separation
matrix.
Sequence analysis and primer synthesis.
Oligonucleotide
primers for tests 1 and 3 were prepared by the Iowa State University
DNA Sequencing and Synthesis Facility, Ames. Test 2 primers and probe
were prepared by Perkin-Elmer. The sequences upon which these tests
were based are available from GenBank.
| |
RESULTS |
Test 1: specific detection of the Chlamydiaceae.
Multiplex PCR amplified a 320-bp ompA/tRNAGly
PCR product and a 240-bp rRNA intergenic spacer product from each of
the nine species in Chlamydiaceae (Fig.
2). The sizes were consistent with sizes expected from sequence data, with the exact sizes varying in accordance with known sequence differences. Using test 1, PCR products were not
amplified from any of a wide variety of other bacterial DNAs (Table
2). DNA templates from the species most
closely related to the Chlamydiaceae, i.e., S. negevensis and W. chondrophila, did not amplify.
Assayed dilutions of C. psittaci NJ1 IFU showed that when 4 mM Mg2+ was included in the PCR buffer, as few as 170 IFU
could be detected (Table 3).
Amplification of a dilution series of CsCl-purified C. psittaci 6BCT template DNA indicated that the test
could detect as little as 0.7 pg of 6BCT template DNA (500 target chromosomes) (Fig. 2; Table 4).
When the concentration of Mg2+ was reduced from 4 to 1.5 mM, the sensitivity was reduced by 4 logs. When heavily fragmented DNA
template was used or when RNA was present, the sensitivity was reduced
by 2 to 4 logs.
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 2.
Test 1 multiplex detection of the
Chlamydiaceae. (A) PCR amplification of the
Chlamydiaceae and several other bacterial strains, in 1.5 mM
Mg2+; (B) multiplex PCR amplification of 10-fold dilutions
of the 6BCT template, in 4.0 mM Mg2+.
|
|
Test 2: specific detection of the Chlamydiaceae.
The
primers and fluorescent probe designed for specific PCR detection of
the Chlamydiaceae in the TaqMan 7200 sequence detection system were strongly positive for all nine species in the
Chlamydiaceae (Fig. 3; Table 2). The primers generated only
negative scores with DNA from S. negevensis and W. chondrophila, which are closely related to the
Chlamydiaceae; negative scores were generated with all other
bacterial templates examined (Table 2). The sensitivity of plus/minus
computer scoring of the test was set by the level of background
fluorescence produced in six no-template controls (Fig.
3). A detailed examination of the raw
fluorescence spectra showed that chlamydial fluorescence intensity
appeared as a broad plateau at 10,000 U (Fig. 3C). When chlamydial
template was serially diluted to less than 50 target molecules (Table
4), the intensity of fluorescence at 515 nm was reduced to background
levels (Fig. 3B and C). After PCR, the raw spectra from some
nonchlamydial templates were of intermediate fluorescence intensity
(not reaching the 10,000-nm plateau and not background). Qualitatively,
these intermediate spectra could not be distinguished from the raw
spectra produced by using extremely dilute chlamydial template. The DNA concentrations of these nonchlamydial DNA templates were high (0.25 to
2.0 µg/reaction mixture), relative to the concentration of chlamydial
template that was sufficiently dilute to generate a comparable signal
(<70 fg/reaction mixture). To characterize the intermediate
nonchlamydial signals, template DNAs from the nonchlamydial bacteria
that had produced the highest signals were diluted 10-fold and retested
(data not shown). The levels of fluorescence from these diluted
templates were at background levels, indicating that nonchlamydial
template could not produce a strongly positive fluorescent signal
plateau.
View larger version (104K):
[in this window]
[in a new window]
|
FIG. 3.
Test 2, TaqMan results and output, showing automated
+/ scoring and fluorescence intensity at 515 nm for each sample. Each
experiment had its own set of no-template controls for automated
calculation of background fluorescence. (A) Column 1, rows 1 to 6, no-template controls; column 1, rows 7 and 8, and column 2, rows 1 to
7, nine species of the Chlamydiaceae (Table 2). All other
columns and rows used template DNA from the strains with negative PCR
results shown in Table 2, including those listed in footnote
a. (B) Column 1, rows 1 to 6, no-template controls; column
1, rows 7 and 8, and column 2, rows 1 to 8, 10-fold dilutions of
6BCT template DNA. (C) Raw spectra corresponding to the
6BCT dilutions shown in panel B, column 2. Left panel,
template only; right panel, the six no-template controls and one of the
template samples.
|
|
By using the TaqMan test, as few as 1.7 IFU of
C. psittaci
NJ1 EBs could be detected (Table
3). In the 6BC
T template
dilution series, the sequence detection system scored
a positive
fluorescent signal when as little as 70 fg of
C. psittaci 6BC
T template was used for PCR (50 targets) (Table
4).
Fluorescence
intensity decreased as the template concentration was
reduced
until the raw spectral fluorescence was equivalent to the
background
fluorescence (Fig.
3). The sensitivity of the TaqMan assay
decreased
by at least 4 logs when samples were prepared by boiling
rather
than by the reducing alkaline lysis method. Sensitivity was not
diminished, however, when damaged template was used or when RNA
was
present in the
sample.
Test 3: detection of the Chlamydiales.
PCR of a wide
variety of template DNAs using the 23S rRNA signature sequence primers
resulted in PCR amplicons from all Chlamydiaceae, from
S. negevensis and W. chondrophila, and from
several nonchlamydial species (Fig. 4;
Table 2). All of these PCR products were approximately the same size,
based on gel electrophoresis. A number of the PCR products were
subjected to direct sequence analysis using the amplification primers.
These gave 23S rRNA sequences as expected from both chlamydial and
nonchlamydial templates. Assayed dilutions of C. psittaci
NJ1 EBs showed that as few as 170 IFU could be detected when 4 mM
Mg2+ was used in the PCR mixture (Table 3). Amplification
of a dilution series of 6BCT template DNA indicated that
the test could detect as little as 0.7 pg of C. psittaci
6BCT template DNA (500 targets) (Table 4). When damaged
template DNA was used, the sensitivity was reduced by 2 to 4 logs. When the concentration of Mg2+ was reduced from 4 to 1.5 mM, the
sensitivity was reduced by another 1 log. When RNA was present, the
amount of PCR product generated was further reduced.
| |
DISCUSSION |
This study has characterized three different PCR tests that can be
used for the identification of chlamydiae. The TaqMan and multiplex
tests were specific for members of the family Chlamydiaceae, and they targeted the rRNA operon and/or the ompA gene.
These tests are so specific for the Chlamydiaceae that they
may be useful for the screening of field specimens. The third test
targeted 23S rDNA segments that were conserved among all families
belonging to the order Chlamydiales, including the
Chlamydiaceae. This test also recognized some nonchlamydial
bacterial templates and therefore is recommended primarily for
characterizing isolates. Test 3 is extremely important because a
specific way to identify new chlamydial families has not previously
existed. All three tests were designed to generate short PCR products
so that amplification and electrophoresis time would be minimized,
enzyme and amplification conditions would not be limiting, and poor
template integrity would not prevent detection.
Test 2, the TaqMan test, was more sensitive than test 1 and test 3 by
approximately 2 logs. This test required no pipetting or handling of
PCR products following the initial setup of the PCR mix. The
TaqMan test amplified a 132-bp PCR product, in comparison to the 320- and 240-bp multiplex products and the 600-bp test 3 product. The TaqMan
test could detect as few as two target molecules in a 50-µl reaction
mixture, as determined by assay of a dilution series of C. psittaci NJ1 IFU. This plus/minus assay required 100 min of PCR
and 10 min of automated reading for 90 samples and six controls.
Test 1, the multiplex gel detection test, was also specific for the
Chlamydiaceae. The limit of detection for this test was 200 to 500 target molecules. Primer set 1260-TGLY targeted the 3' end of
the ompA gene, and primer set IGF-IGR targeted the
intergenic spacer of the rRNA operon. Having two PCR products provides
confirmation of the identity of positive specimens yet also helps to
ensure that mutations in as-yet-undiscovered chlamydiae will not
entirely prevent detection by this test. The TGLY primer, complementing sequence just downstream of ompA, was an exact match to all
known Chlamydiaceae strains. Primer IGF, located within the
rRNA intergenic spacer, was also an exact match to all known strains.
Primers 1260 and IGR each had two or more bases that did not match all chlamydial template sequences. Primer 1260 mismatches were overcome by
making this primer extra long, whereas the 2-base mismatch in primer
IGR was designed to ensure a mismatch with every chlamydial template.
IGR mismatches enhanced the specificity of the primer, because close to
its natural annealing termperature, this primer would be readily
dissociated from template if there were additional mismatches.
Annealing by this primer could even be made more tenuous with a low
Mg2+ concentration. Figure 2 illustrates this effect: the
6BCT ribosomal PCR product was poorly amplified in 1.5 mM
Mg2+ (Fig. 2A) but well amplified in 4 mM Mg2+
(Fig. 2B). This discrimination helps to ensure the specificity of the
ribosomal primers for the Chlamydiaceae.
Test 3 used a primer set that amplified domain I of the 23S rRNA. This
segment is a signature sequence for chlamydial species, genera, and
families (8). The test identified chlamydia-like isolates
that were not recognized by the tests that were specific for the
Chlamydiaceae. Identification was done by directly
sequencing the 600-bp PCR product with the U23F and 23SIGR
amplification primers (8) and then using this sequence in a
BLAST search of the GenBank database. The primer set amplified as few
as 500 targets. It was used to PCR amplify and sequence the 23S rRNA gene of S. negevensis and W. chrondrophila,
species which belong to two new families in the
Chlamydiales.
Currently, because of the difficulties involved in chlamydial detection
and identification, our understanding of the Chlamydiales has been primarily limited to strains found in hosts of economic importance. The PCR tests described in this report make it possible to
recognize all of these chlamydiae and new strains, as well, with good
sensitivity. Furthermore, the PCR products generated in these analyses
(including the TaqMan products) can be directly sequenced to identify
the species or strain, if desired. The availability of these assays
facilitates the study of the epidemiology of chlamydiae and may also
improve diagnostic capability. By using these tests, biodiversity
studies can be reasonably and affordably undertaken, with the assurance
that the outcome will be consistent with our current understanding of
chlamydial phylogeny.
| |
ACKNOWLEDGMENTS |
We thank Alan Baetz, Harlan D. Caldwell, Lee Ann Campbell, Pam M. Dilbeck, Sharon Franklin, Maureen Friedman, Peter Janssen, Paul S. Hoffman, Douglas G. Rogers, Fred Rurangirwa, and Irene V. Wesley for
providing DNA or strains used in this study.
| |
ADDENDUM IN PROOF |
The new family, genus, and species names for bacteria in the order
Chlamydiales that are used in this work will become valid upon the publication of references 8 and 23.
| |
FOOTNOTES |
*
Corresponding author. Present address: Department of
Medical Microbiology/Parasitology, College of Veterinary Medicine,
University of Georgia, Athens, GA 30602-7371. Phone: (706) 542-5823. Fax: (706) 542-5771. E-mail: keverett{at}calc.vet.uga.edu or
kdeeverett{at}hotmail.com.
| |
REFERENCES |
| 1.
|
Andersen, A. A., and R. A. Van Deusen.
1988.
Production and partial characterization of monoclonal antibodies to four Chlamydia psittaci isolates.
Infect. Immun.
56:2075-2079[Abstract/Free Full Text].
|
| 2.
|
Balin, B. J.,
H. C. Gérard,
E. J. Arking,
D. M. Appelt,
P. J. Branigan,
J. T. Abrams,
J. A. Whittum-Hudson, and A. P. Hudson.
1998.
Identification and localization of chlamydia pneumoniae in the Alzheimer's brain.
Med. Microbiol. Immunol.
187:23-42[Medline].
|
| 3.
|
Birtles, R. J.,
T. J. Rowbotham,
C. Storey,
T. J. Marrie, and D. Raoult.
1997.
Chlamydia-like obligate parasite of free-living amoebae.
Lancet
349:925-926[Medline].
|
| 4.
|
Bocklisch, V. H.,
C. Ludwig, and S. Lange.
1990.
Chlamydien als Abortursache beim Pferd.
Berl. Muench. Tieraerztl. Wochenschr.
104:119-124.
|
| 5.
|
Dilbeck, P. M.,
J. F. Evermann,
T. B. Crawford,
A. C. S. Ward,
C. W. Leathers,
C. J. Holland,
C. A. Mebus,
L. L. Logan,
F. R. Rurangirwa, and T. C. McGuire.
1990.
Isolation of a previously undescribed rickettsia from an aborted bovine fetus.
J. Clin. Microbiol.
28:814-816[Abstract/Free Full Text].
|
| 6.
|
Dilbeck, P. M.,
J. F. Evermann,
S. Kraft, and S. Tyler.
1985.
Equine chlamydial infections: comparative diagnostic aspects with bovine and ovine chlamydiosis, p. 285.
In
M. W. Vorheis (ed.), Proceedings of the 28th Annual Meeting of the American Association of Veterinary Laboratory Diagnosticians. American Association of Veterinary Laboratory Diagnosticians, Milwaukee, Wis.
|
| 7.
|
Everett, K. D. E., and A. A. Andersen.
1997.
The ribosomal intergenic spacer and domain I of the 23S rRNA gene are phylogenetic markers for Chlamydia spp.
Int. J. Syst. Bacteriol.
47:461-473[Abstract/Free Full Text].
|
| 8.
| Everett, K. D. E., R. M. Bush, and
A. A. Andersen. 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., in press.
|
| 9.
|
Forster, J.-L.,
M. M. Wittenbrink,
H.-J. Häni,
L. Corboz, and A. Pospischil.
1997.
Absence of Chlamydia as an aetiological factor in aborting mares.
Vet. Rec.
141:424[Free Full Text].
|
| 10.
|
Groff, J. M.,
S. E. LaPatra,
R. J. Munn,
M. L. Anderson, and B. I. Osburn.
1996.
Epitheliocystis infection in cultured white sturgeon (Acipenser transmontanus): antigenic and ultrastructural similarities of the causative agent to the chlamydiae.
J. Vet. Diagn. Invest.
8:172-180[Abstract/Free Full Text].
|
| 11.
|
Herring, A. J.
1993.
Typing Chlamydia psittaci a review of methods and recent findings.
Br. Vet. J.
149:455-475[Medline].
|
| 12.
|
Huchzermeyer, F. W.
1997.
Public health risks of ostrich and crocodile meat.
Rev. Sci. Technol.
16:599-604[Medline].
|
| 13.
| Kahane, S., K. D. E. Everett, N. Kimmel, and
M. G. Friedman. Simkania negevensis, strain
ZT: growth, antigenic and genome characteristics. Int. J. Syst. Bacteriol., in press.
|
| 14.
|
Kahane, S.,
R. Gonen,
C. Sayada,
J. Elion, and M. G. Friedman.
1993.
Description and partial characterization of a new chlamydia-like microorganism.
FEMS Microbiol. Lett.
109:329-334[Medline].
|
| 15.
|
Kahane, S.,
D. Greenberg,
M. G. Friedman,
H. Haikin, and R. Dagan.
1998.
High prevalence of S. negevensis, a novel chlamydia-like bacterium, in infants with acute bronchiolitis.
J. Infect. Dis.
177:1425-1427[Medline].
|
| 16.
|
Kaltenböck, B.,
N. Schmeer, and R. Schneider.
1997.
Evidence for numerous omp1 alleles of porcine Chlamydia trachomatis and novel chlamydial species obtained by PCR.
J. Clin. Microbiol.
35:1835-1841[Abstract].
|
| 17.
|
Lieberman, D.,
S. Kahane,
D. Lieberman, and M. G. Friedman.
1997.
Pneumonia with serological evidence of acute infection with the chlamydia-like microorganism "Z."
Am. J. Respir. Crit. Care Med.
156:578-582[Abstract/Free Full Text].
|
| 18.
|
Löbau, S.,
U. Mamat,
W. Brabetz, and H. Brade.
1995.
Molecular cloning, sequence analysis, and functional characterization of the lipopolysaccharide biosynthetic gene kdtA encoding 3-deoxy-alpha-D-manno-octulosonic acid transferase of Chlamydia pneumoniae strain TW-183.
Mol. Microbiol.
18:391-399[Medline].
|
| 19.
|
Meijer, A., and J. M. Ossewaarde.
1998.
Broad range Chlamydia PCR detects previously unrecognized Chlamydia sequences: a new genus in the family Chlamydiaceae?, p. 523-526.
In
R. S. Stephens, G. I. Byrne, G. Christiansen, I. N. Clarke, J. T. Grayston, R. G. Rank, G. L. Ridgway, P. Saikku, J. Schachter, and W. E. Stamm (ed.), Chlamydial infections: proceedings of the Ninth International Symposium on Human Chlamydial Infection. International Chlamydia Symposium San Francisco, Calif.
|
| 20.
|
Pospischil, A., and R. D. Wood.
1987.
Intestinal Chlamydia in pigs.
Vet. Pathol.
24:568-570[Medline].
|
| 21.
|
Rake, G. W.
1957.
Family II. Chlamydiaceae Rake, Fam. Nov., p. 957-968.
In
R. S. Breed, E. G. D. Murray, and N. R. Smith (ed.), Bergey's manual of systematic bacteriology, 7th ed. The Williams & Wilkins Co., Baltimore, Md.
|
| 22.
|
Rogers, D. G.,
A. A. Andersen,
A. Hogg,
D. L. Nielsen, and M. A. Huebert.
1993.
Conjunctivitis and keratoconjunctivitis associated with chlamydiae in swine.
J. Am. Vet. Med. Assoc.
203:1321-1323[Medline].
|
| 23.
| Rurangirwa, F. R., P. M. Dilbeck, T. B. Crawford, T. C. McGuire, and T. F. McElwain. 16S DNA
sequence of WSU 86-1044 microorganism from an aborted bovine fetus
reveals it is a member of the order Chlamydiales: proposal
of Waddliaceae fam. nov., Waddlia chondrophila,
gen. nov., sp. nov. Int. J. Syst. Bacteriol., in press.
|
| 24.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Press, Cold Spring Harbor, N.Y.
|
| 25.
|
Sayada, C.,
E. Denamur,
J. Orfila,
F. Catalan, and J. Elion.
1991.
Rapid genotyping of the Chlamydia trachomatis major outer membrane protein by the polymerase chain reaction.
FEMS Microbiol. Lett.
83:73-78.
|
| 26.
|
Vanrompay, D.,
R. Ducatelle, and F. Haesebrouck.
1992.
Diagnosis of avian chlamydiosis: specificity of the modified Giménez staining on smears and comparison of the sensitivity of isolation in eggs and three different cell cultures.
J. Vet. Med. B
39:105-112.
|
| 27.
|
Yoshida, H.,
Y. Kishi,
S. Shiga, and T. Hagiwara.
1998.
Differentiation of Chlamydia species by combined use of polymerase chain reaction and restriction endonuclease analysis.
Microbiol. Immunol.
42:411-414[Medline].
|
| 28.
|
Zahn, I.,
L. Szeredi,
I. Schiller,
U. Straumann-Kunz,
E. Bürgi,
F. Guscetti,
E. Heinen,
L. Corboz,
T. Sydler, and A. Pospischil.
1995.
Immunohistochemical determination of Chlamydia psittaci/pecorum and C. trachomatis in the piglet gut.
J. Vet. Med. B
42:226-276.
|
Journal of Clinical Microbiology, March 1999, p. 575-580, Vol. 37, No. 3
0095-1137/99/$00.00+0
This article has been cited by other articles:
-
Kutlin, A., Roblin, P. M., Kumar, S., Kohlhoff, S., Bodetti, T., Timms, P., Hammerschlag, M. R.
(2007). Molecular characterization of Chlamydophila pneumoniae isolates from Western barred bandicoots. J Med Microbiol
56: 407-417
[Abstract]
[Full Text]
-
Jansen, B. D., Heffelfinger, J. R., Noon, T. H., Krausman, P. R., deVos, J. C. Jr.
(2006). Infectious keratoconjunctivitis in bighorn sheep, silver bell mountains, Arizona, USA.. J Wildl Dis
42: 407-411
[Abstract]
[Full Text]
-
Griffiths, E., Petrich, A. K., Gupta, R. S.
(2005). Conserved indels in essential proteins that are distinctive characteristics of Chlamydiales and provide novel means for their identification. Microbiology
151: 2647-2657
[Abstract]
[Full Text]
-
Everson, J. S., Garner, S. A., Fane, B., Liu, B.-L., Lambden, P. R., Clarke, I. N.
(2002). Biological Properties and Cell Tropism of Chp2, a Bacteriophage of the Obligate Intracellular Bacterium Chlamydophila abortus. J. Bacteriol.
184: 2748-2754
[Abstract]
[Full Text]
-
Hartley, J. C., Kaye, S., Stevenson, S., Bennett, J., Ridgway, G.
(2001). PCR Detection and Molecular Identification of Chlamydiaceae Species. J. Clin. Microbiol.
39: 3072-3079
[Abstract]
[Full Text]
-
Smieja, M., Chong, S., Natarajan, M., Petrich, A., Rainen, L., Mahony, J. B.
(2001). Circulating Nucleic Acids of Chlamydia pneumoniae and Cytomegalovirus in Patients Undergoing Coronary Angiography. J. Clin. Microbiol.
39: 596-600
[Abstract]
[Full Text]
-
Angen, O., Jensen, J., Lavritsen, D. T.
(2001). Evaluation of 5' Nuclease Assay for Detection of Actinobacillus pleuropneumoniae. J. Clin. Microbiol.
39: 260-265
[Abstract]
[Full Text]
-
Killgore, G. E., Holloway, B., Tenover, F. C.
(2000). A 5' Nuclease PCR (TaqMan) High-Throughput Assay for Detection of the mecA Gene in Staphylococci. J. Clin. Microbiol.
38: 2516-2519
[Abstract]
[Full Text]
Top
Abstract
Introduction
