Chlamydiales : Proposal of Waddliaceae Fam. Nov., Waddlia Chondrophila Gen
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International Journal of Systematic Bacteriology (1999), 49, 577-581 Printed in Great Britain Analysis of the 16s rRNA gene of micro- organism 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. nova Fred R. Rurangirwa,'a2 Pamela M. Dilbeckf2Timothy B. Crawford,l Travis C. McGuirel and Terry F. McElwain1g2 Author for correspondence: Fred R. Rurangirwa. Fax: + 1 509 335 8529. e-mail : ruvuna@ vetmed.wsu.edu Department of Veterinary The structural gene encoding the 165 rRNA of the new obligate intracellular Microbiology and organism presently designated WSU 86-10UTwas sequenced and analysed to Pathology' and Washington Animal Diseases Diagnostic establish its phylogenetic relationships. The 16s rDNA sequence was most La boratoryz, Washington closely related to those of chlamydia1 species, having 84.7-85-3O/O sequence State University, Pullman, similarity, while it had 724-73-2 YO similarity with rickettsia-like organisms. WA 99164-7040, USA When the sequences of the four species of chlamydiae (Chlamydophila psittaci, Chlamydia trachomatis, Chlamydophilapneumoniae and Chlamydophila pecorum) were compared, they had > 93 O/o sequence similarity indicating that WSU 86-10UTwas not close enough to be in the same family as current Chlamydiaceae members. However, based on the 84*7-85*3%165 rDNA sequence similarity of WSU 86-10UTand other previously described characteristics, WSU 86-1WTbelongs to a novel family within the order Chlamydiales; hence, the proposal of Waddliaceae fam. nov., Waddlia chondmphila gen. nov., sp. nov. Keywords: chlamydia, phylogeny, 16s rRNA, 16s rDNA sequence, abortion INTRODUCTION cytoplasmic inclusions that ranged in size from 0.2 to 0.4 pm (Dilbeck et al., 1990). Electron microscopy The organism WSU 86- 1044T,was originally isolated confirmed that the organisms multiplied within a from tissues of a first-trimester aborted bovine foetus cytoplasmic vacuole with a developmental life cycle at the Washington Animal Disease Diagnostic Lab- resembling that of erhlichiae and chlamydiae (Dilbeck oratory (Dilbeck et al., 1990). A cytopathic effect was et al., 1990; Kocan et al., 1990). The organisms observed within 2-3 d after the initial inoculation of occurred in two forms ; a reticulated form found within bovine turbinate (BT) cell cultures with pooled spleen a cytoplasmic vacuole, and a dense infective form that and liver homogenates. The organism was serially was released from the cells. passaged numerous times and consistently induced cytopathic effect. It replicated rapidly to high levels, Presently, WSU 86-1044T is characterized as an ob- reaching peak titres exceeding 1Os 50 % tissue-culture- ligate intracellular organism, which replicates within infective doses (TCID,,) per ml within 3 d (Dilbeck et cytoplasmic vacuoles, exhibiting structural charac- al., 1990). Replication was inhibited by tetracycline, teristics compatible with those of rickettsiae and et al., chlamydiae (Dilbeck et al., 1990; Kocan et al., 1990). but not by penicillin or gentamicin (Dilbeck 86- 1990). Light microscopy revealed organisms within Serological identification of WSU 1044T was not successful as the organism did not react with mono- Rickettsia. ., ., .. , . , ., . , . , . ., .. , , .. , . ., .. .. .. , . , ,. , . , . , , , .. , . , .. ,. .. .. .. , . .. .. .. clonal or Dolvclonal antisera to a varietv of Abbreviations: BT, bovine turbinate; TCID,,, 50 % tissue-culture-infective Coxiella? whlbachia, Anaplasma Or dhlamYdia sPP: dose. (Dilbeck et al., 1990). However, it did react weakly The GenBank accession number for the 165 rRNA gene sequence of WSU with antisera to Cowdria rW?'linantium.Thus, wsu 86- 86-1OUT is AF042496. 1044T has not been taxonomically classified. 00838 0 1999 IUMS 577 F. R. Rurangirwa and others Progress in molecular biology using PCR and PCR amplification was done in 100 pl reaction mixture and sequencing of DNA encoding 16s rRNA has enabled Taq DNA polymerase from Gibco-BRL. Typically, a tube determination of the precise phylogenetic position of contained 10 pl10 x PCR buffer made of 200 mM Tris/HCl bacterial species, particularly intracellu- (pH 8.4) and 500 mM KCl, 5 p150 mM MgCl,, 2 pl 10 mM each obligate dinucleotide mix (Invitrogen), 0-5 pl 50 mM of the reverse lar bacteria which express few phenotypic characters and forward primers, 79.5 pl double-distilled water, 2 pl (Hills et al., 1996; Roux & Raoult, 1995). The primers containing 0.25 pg target DNA and 0.5 pl(2.5 U) Taq DNA are chosen from sequences that are highly conserved Polymerase. Before addition of the target DNA and Taq among the phylogenetic group referred to as the polymerase a wax gem (Perker-Elmer) was added to the tube eubacteria (Wilson et al., 1990; Woese, 1987), but and heated at 75 "C for 5 min and then allowed to cool to which are not found in eukaryotes, archaea or mito- room temperature. Amplification was performed in a chondria. It is therefore possible to amplify only GeneAmp PCR System 9600 Thermal Cycler (Perkin-Elmer) bacterial 16s rDNA sequences even in the presence of in which the target DNA was denatured by incubation at nucleic acids from other types of organisms. Because 95 "C for 5 rnin followed by 35 cycles of denaturation (94 "C the nucleotide sequences found in 16s rDNAs vary in for 1 min), primer annealing (55 "C for 3 min) and primer extension (72 "C for 3 min). At the end of the cycling, the fashion phylogenetic an orderly throughout the tree, reaction mixture was held at 72 "C for 7 rnin and cooled to they have been useful for the study of molecular 4 "C. The size of the PCR product was verified by agarose gel evolution (Woese, 1987). Thus 16s rRNA or 16s electrophoresis of 10 p1 reaction mixture (Sambrook et al., rDNA sequencing is one of the most powerful and 1989). precise methods for determining the distant as well as Cloning, sequencing and sequence analysis. The amplified close (intrageneric) genealogical relationships of bac- fragment was ligated into the EcoRI site of vector pCR 2.1 teria (Hills et al., 1996; Swofford et al., 1996; Woese, (Invitrogen) and used to transform Escherichia coli (one- 1987). shot cell INVaF' Competent Cells ; Invitrogen). Individual, In an effort to classify the agent WSU 86-1044T, the transformed colonies were grown in Luria Broth, DNA was DNA encoding the 16s rRNA was PCR-amplified and isolated and then digested with EcoRI. DNA fragments were sequenced. Comparison of the 16s rDNA sequence separated in 1.5% agarose gel and stained with ethidium with other 16s rDNA sequences in GenBank indicated bromide to verify the size of the fragment ligated into the plasmid. Nucleotide sequencing of the recombinant inserts that WSU 86-1044T belonged to the order from selected colonies was performed by the Laboratory for Chlarnydiales. However, the similarity was not Biotechnology and Bioanalysis, Washington State Univer- sufficient to allow its classification in any of the families sity, using the Perkin Elmer Applied Biosystems Prism Dye within the order. Terminator Kit and analysed on an ABI 373 DNA In a recent paper by Everett et al. (1999) on a revised Sequencer. Cloning the amplified fragment into pCR 2.1 classification scheme for the order Chlarnydiales, enabled sequencing of the whole amplicon, initially using Chlamydia pneumoniae, Chlamydia pecorum M 13 forward and reverse primers (Invitrogen) approxi- and mately 100 bases up- and downstream from the EcoRI Chlamydia psittaci were reclassified to Chlamydophila cloning site, and subsequently with commercially gen. nov. as Chlamydophila pneurnoniae comb. nov., synthesized specific primers (Life Technologies) selected as Chlamydophila pecorum comb, nov. and Chlamydo- sequence information was obtained. Table 1 shows the phila psittaci comb. nov., respectively. Their new primers used to sequence through the entire 16s rDNA names will be used throughout this paper. amplicon of the WSU 86-1044T.Sequence analysis programs REFORMAT, GAP, REVERSE, PILEUP, DISTANCES, NEIGHBOR and METHODS GROWTREE in the University of Wisconsin Genetics Com- puter Group (GCG), version 8 (1994), were used for DNA WSU 86-1044T. The isolation of the organism from pooled analyses (Devereux et al., 1984). GenBank search for foetal liver/lung homogenate has been described previously similarities was accomplished using BLAST and FASTA (Dilbeck et al., 1990). The organism was cloned at the 8th programs on-line (Pearson & Lipman, 1988). Comparison of passage by three serial limiting dilutions in 96-well plates of the WSU 86-1044T 16s rDNA sequence with published bovine turbinate (BT) cells (ATCC CRL- 1390) (Dilbeck et sequences of other organisms, most of which were obligate al., 1990). Cloned organisms at the 13th passage in BT cells intracellular bacteria, was performed both by PILEUP and by with a titre of 1 x lo6', TCID,, were used in these studies. DISTANCES analysis to calculate the Kimura two-parameter DNA extraction. Genomic DNA was prepared from WSU distances. Sequences of the following micro-organisms 86-1044' passaged in bovine turbinate cells. The super- (GenBank accession no. and strain designation in par- natants were harvested when the cytopathic effect was entheses) were compared : Chlamydophila (formerly advanced and stored at -70 "C. For use, the fluid was Chlamydia) psittaci (M 13769, strain 6BCT), Chlamydia thawed and clarified at 500 g for 10 min, and the organisms trachomatis (M59 178, strain 434T),Chlamydophila (formerly