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 : proposal of Waddliaceae fam. nov., 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 -like organisms. WA 99164-7040, USA When the sequences of the four species of ( psittaci, 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 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 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 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 pelleted at 20000 g for 30 min. DNA was prepared from the Chlamydia) pneumoniae (249873, strain TW-1 83T), sedimented WSU 86- 1044Torganisms using Puregene DNA Chlamydophila (formerly Chlamydia) pecorum (U7 3 78 2), Isolation Kit (Gentra Systems). Simkania negevensis (L27666, strain Z'), Parachlamydia acanthamoebae (Y07556, strain Bn9T), Rickettsia rickettsii PCR amplification. The DNA encoding the 16s rRNA was Cowdria ruminantium in vitro (M21293, strain R), [X61659, strain amplified via PCR (White, 1993) by using a primer Anaplasma marginale set (forward 5' AGA GTT TGA TCC TGG 3' and reverse 5' Crystal Springs (Zimbabwe) isolate], (M603 13), Ehrlichia phagocytophila (M73220, strain 0s') TAC CTT GTT ACG ACT T 3') selected from the conserved Escherichia coli sequences at the beginning and the end of the eubacterial and (M24996). kingdom 16s rDNA (Wilson et al., 1990; Woese, 1987). The neighbour-joining method and tree construction were

578 International Journal of Systematic Bacteriology 49 Waddliaceae, a new family in the order Chlamydiales performed by using the NEIGHBOR and GROWTREE programs, genic cross-reactivity between WSU 86- 1044T and respectively. All sequences were aligned using the PILEUP Chlamydia ruminantium (Dilbeck et al., 1990) was not multiple-sequence alignment program. reflected in significant rDNA sequence identity.

RESULTS DISCUSSION PCR product Analysis of 16s rDNA of WSU 86-1044T indicated Conserved eubacterial rDNA primers amplified a that the abortion agent was closely related to the major WSU 86-1044T fragment slightly above 1.5 kb. members of the order Chlamydiales, having When the plasmid with the insert was isolated and 84.5-85.3 YO sequence similarity. The relatively high digested with EcoRI endonuclease, three fragments degree of similarity of WSU 86-1044T 16s rDNA to resulted comprising the vector pCR 2.1 (3.9 kb), one 16s rDNA from the members of the order Chla- insert fragment slightly longer than 0.8 kb, and a mydiales provided justification for its inclusion in this second insert fragment of approximately 0.7 kb, indi- group/order. However, when the rDNA sequences of cating an internal EcoRI site in the amplicon. An four members from the family Chlamydiaceae were EcoRI site at nucleotide positions 679-685 from the 5’ aligned they had sequence similarities of 93.8-96.7 %. of WSU 86- 1044T was confirmed by sequence analysis. Therefore the lower level of sequence similarity betw- An EcoRI site also was reported approximately 680 een WSU 86-1044T and the members of the Chla- nucleotides from the 5’ end of rDNA for Chlamydo- mydiaceae family did not justify inclusion of WSU 86- phila (formerly Chlamydia) psittaci (Weisburg 1044Tinto this family (Kahane et al., 1995; Everett & et al., 1986). The sequence of Chlamydophila psittaci Andersen, 1997; Everett et al., 1999; Takahashi et al., rDNA obtained from GenBank has an EcoRI site 1997). between nucleotides 681 and 687. It has been shown that WSU 86-1044T is morpho- logically similar to rickettsiae and chlamydiae but is Sequences biochemically and antigenically distinct (Dilbeck et al., et al., The sequence of the WSU 86-1044T 16s rDNA 1990; Kocan 1990). Two developmental forms consisted of 1526 nucleotides (400 A, 300 C, 453 G, 333 (dense bodies and reticulate bodies) of WSU 86-1044T organisms were observed in intracytoplasmic vacuoles T). On-line BLAST and FASTA searches of GenBank for by electron microscopy. WSU 86- 1044T was com- similar sequences revealed that WSU 86- 1044T rDNA in vitro, was > 80 YOsimilar to chlamydiae. The first 30 items of pletely resistant to penicillin whereas all known the search indicated similarity to the four known chlamydiae (Chlamydophila psit taci, Chlamydia trac- homatis, Chlamydophila pneumoniae and Chlamyd- Chlamydia trachomatis (434T) ophila pecorum), small subunit of chlamydia rRNA, and to Simkania negevensis (Kahane et al., 1995; Chlamydophila psittaci (6BCT) Everett et al., 1999). Similarity ranking by the Ribo- somal Database Project (RDP) at the University of Chlamydophila pecorum - Illinois Urbana-Champaign Department of Micro- biology indicated that the WSU 86-1044T rDNA Chlamydophila pneumoniae (TW-183T) sequence was most similar to Chlamydophila psit taci. Parachlamydia acanthamoebae (BngT) Phylogenetic analyses Simkania negevensis (zT) A new classification of the order Chlamydiales has been proposed in which the currently known Waddlia chondrophila (WSU 86-1044T) strains with > 90% 16s rRNA similarity form the Chlamydiaceae family. The chlamydia-like organisms Anaplasma marginale which have 80-90% 16s rRNA similarity form two new families : and Eh rlichia p hag ocytoph ila (0ST) (Everett et al., 1999). We compared 16s rDNA iL sequences of some members of the Chlamydiaceae, Cowdria ruminantium Simkaniaceae and Parachlamydiaceae (Everett et al., (Crystal Spring [Zimbabwe] Isolate) 1999), WSU 86- 1044T,and some intracellular parasites Rickettsia rickettsii (strain ATCC VR 891) from the Rickettsiaceae to assess their percentage sequence similarity. WSU 86- 1044T exhibited the Escherichia coli greatest similarity to members of the order Chlamydi- ...... , ...... , . . , ...... , , .. , ...... , . , ...... , . , . , ...... , ...... ales (> 84.5 Yo). Three rickettsia species, which Fig. I. Dendogram (based on the distances calculated by PILEUP included Cowdria ruminantium, had values ranging analysis) showing relationship between WSU 86-1 OMT, from 72 to 72.9% while one Ehrlichia species had chlamydiae and rickettsiae. Bar indicates 10 nucleotide sequence similarities of 72-1 YO.The reported anti- substitutions.

International Journal of Systematic Bacteriology 49 579 F. R. Rurangirwa and others

Table 1. Primers for sequencing the rDNA amplicon of WSU 86-1044l

Primer Sequence (postion/source)

Forward F1 (M13) 5’ GTTTTCCCAGTCACGACGTTGTA (Invitrogen) F2 5‘ GCTCACCAAGGCTAAGACGTC (277-298) F3 5’ CTAGCTTTGACCTGACGCTGAT (752-774) F4 5’ GAATCTGCAACTCGGCTCCATG (1 323-1345) Reverse Rl (M13) 5’ TTGTGAGCGGATAACAATTTC (Invitrogen) R1 5’ CATCCTAAATGCTGGCAAC (392-373) R2 5’ CACCGCTACATGTGGAATTCC (843-822) R4 5’ GATCCTCTCTAGCACCATATCC (1358-1 336) chlamydia are sensitive (Moulder, 199 1). Furthermore, multiplies by binary fission and has two developmental WSU 86-1044Tdid not react with either polyclonal or forms: the dense form which is infective, and the monoclonal antibodies used to type chlamydia, reticulated form, usually associated with mitochon- although there was some reactivity with antibodies to dria, which undergoes binary fission. These organisms Chlamydia rurninantium (Dilbeck et al., 1990). do not react with antisera used for typing chlamydiae Waddliaceae Evolutionary distance values calculated using or rickettsiae. The 16s rDNA of the strains are > 90 YOsimilar to ribosomal genes in WSU DISTANCES were used to construct the phylogenetic tree 86- 1044T. The family Waddliaceae belongs to the order shown in Fig. 1. The tree shows the distinctions Chlamydiales Chlarnydiaceae between chlamydiae and rickettsiae. Although WSU and is a sister taxon of the 86- 1044T, Simkania negevensis and Parachlamydia because the ribosomal genes are 80-90% similar to acanthamoebae formed a cluster, the three are as far ribosomal genes in the Chlamydiaceae. Phylogenetic apart from one another (84-6-87-2 YO sequence simi- analyses of the Waddliaceae 16s rDNA sequence is larity) as they are from the chlamydiae. presented here. At present this family comprises a single genus, the type genus Waddlia. The genetic data described herein, and the morpho- logical similarity (Kocan et al., 1990), are consistent with a close relationship between WSU 86-1044Tand Description of Waddlia gen. nov. the chlamydiae. However, since rDNA sequence simi- Waddlia [Wadd’li.a. N.L. fem. n. Waddlia arbitrary larity of > 90% has been suggested as a criterion for name derived from the abbreviation WADDL classifying chlamydia-like organisms within the same (Washington Animal Disease Diagnostic-Labora- family of the order Chlamydiales (Everett et al., 1999), tory)l. WSU 86-1044T having < 90% similarity does not fit into any of the present families in the order Members of the genus Waddlia have 16s rDNA that is Chlamydiales. Therefore, we propose that WSU 86- > 90% similar to that of the type species, Waddlia 1044T be classified as Waddliaceae fam. nov., Waddlia chondrophila strain WSU 86-1044T. chondrophila gen. nov., sp. nov. Description of Waddlia chondrophila sp. nov. Description of Waddliaceae fam. nov. Waddlia chon drop h ila (chon .dro ’phi. la. Gr. n . Waddliaceae (Wadd’li.a’ce .ae M .L. fem. n. Waddlia chondros clump; Gr. adj. philos, -a friendly to; M.L. the type genus of the family; -aceae ending to denote a chondrophila liking clumps, in reference to the as- family; M.L. fem. pl. Waddliaceae the Waddlia sociation of the organism with cellular mitochondria). family). The species Waddlia chondrophila currently includes Waddliaceae currently includes the type genus, only the type strain, WSU 86-1044T (= ATCC VR Waddlia. The description of this family is identical at 1470T). Waddlia chondrophila was isolated from the present to that of ‘micro-organism WSU 86- 1044’ tissues of a first-trimester aborted bovine foetus. The (Dilbeck et al., 1990; Kocan et al., 1990) which are description of this species is identical to that of ‘micro- obligate intracellular organisms resistant to penicillin. organism WSU 86-1044’ (Dilbeck et al., 1990; Kocan They grow well in BT producing multiple cytoplasmic et al., 1990). The full length of Waddlia chondrophila vacuoles and Gram-negative, periodic acid-Schiff 16s rDNA sequence is 15.0 0.25 YOdifferent from the negative and non-acid-fast inclusions. The inclusions 16s rDNA of Chlamydiaceae spp., fitting into the contain coccoid organisms ranging from 0.2 to 0.5 ym 80-90% identity range that makes this organism a in size. The BT infectivity is abolished by tetracycline member of the order Chlamydiales, but not a member and/or chloroform treatment. The organism of the family Chlamydiaceae. The Waddlia

580 International Journal of Systematic Bacteriology 49 chondrophila 16s rDNA sequence is 15-8% different species, and standards for the identification of organisms. Int J from that of Simkania negevensis strain ZT, while it is Syst Bacteriol49, 41 5440. 12.8 YO different from that of Parachlamydia Hills, D. M., Mable, B. K., Larson, A., Davis, 5. K. & Zimmer, E. A. acanthamoebae strain BnST, which excludes it from (1996). Nucleic acids IV : sequencing and cloning. In Molecular Simkaniaceae and Parachlamydiaceae families of the Systematics, 2nd edn, pp. 321-381. Edited by D. M. Hills, C. order Chlamydiales (Everett et al., 1999). For a newly Moritz & B. K. Mable. Sunderland, MA: Sinauer. identified strain to be described as a member of Kahane, S., Metzer, E. & Friedman, M. G. (1995). Evidence that Waddliaceae, a nearly full-length rDNA of the new the novel microorganism ‘Z’ may belong to a new genus in the strain may only differ from Waddliu chondrophilu 16s family Chlamydiaceae. FEMS Microbiol Lett 126, 203-208. rDNA by < 10%. Kocan, K. M., Crawford, T. B., Dilbeck, P. M., Evermann, 1. F. & McGuire, T. C. (1990). Development of rickettsia isolated from an aborted bovine fetus. J Bacterioll72, 5949-5955. ACKNOWLEDGEMENTS Moulder, W. 1. (1991). Interaction of chlamydia and host cells in We appreciate the help with the GCG Program from Ms vitro. Mierobiol Rev 55, 143-190. Teresa Harkins and Dr Dorothy French, and invaluable Pearson, W. R. & Lipman, D. 1. (1988). Improved tools for assistance from many staff members of WADDL in the biological sequence comparison. Proc Natl Acad Sci USA 85, process of doing this work. We are particularly indebted to 2444-2448. Dr Karin Everett for giving us access to the proposed new Roux, V. & Raoult, D. (1995). Phylogenetic analysis of the genus classification of the order Chlamydiales before it was Rickettsia by 16s rDNA sequencing. Res Microbiol 146, published. Our thanks also go to Professor Dr Hans G. 38 5-3 96. Triiper for checking the appropriateness of the name, and to Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989). Molecular Yvonne McGehee and Jing Cui for preparation of the Cloning: a Laboratory Manual, sequence material. 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory. Swofford, D. L., Olsen, G. J., Waddell, P. J. & Hills, D. M. (1996). REFERENCES Phylogenetic inference. In Molecular Systematics, 2nd edn, pp. 407-514. Edited by D. M. Hills, C. Moritz & B. K. Mable. Devereux, J., Haeberli, P. & Smithies, 0. (1984). A comprehensive Sunderland, MA : Sinauer. set of sequence analysis programs for the VAX. Nucleic Acids Res 12, 387-395. Takahashi, T., Masuda, M., Tsuruno, T., Mori, Y., Takashima, I., Hiramune, T. & Kikuchi, N. (1997). Phylogenetic analyses of Dilbeck, P. M., Evermann, 1. F., Crawford, T. B. & 7 other authors Chlamydia psittaci strains from birds based on 16s rRNA gene (1990). Isolation of previously undescribed rickettsia from an sequence. J Clin Microbiol35, 2908-29 14. aborted bovine fetus. J Clin Microbiol28, 8 14-8 16. Weisburg, W. G., Hatch, T. P. & Woese, C. R. (1986). Eubacterial Everett, K. D. E. & Andersen, A. A. (1997). The ribosomal origin of chlamydiae. J Bacteriol 167, 570-574. intergenic spacer and domain 1 of the 23s rRNA gene are phylogenetic markers for Chlamydia spp. Int J Syst Bacteriol White, B. A. (1993). PCR Protocols: Current Methods and 47,461473. Applications. Totowa, NJ : Humana Press. Everett, K. D. E., Bush, R. M. & Andersen, A. A. (1999). Emended Wilson, K. H., Blitchington, R. B. & Greene, R. C. (1990). Ampli- description of the order Chlamydiales, proposal of Para- fication of bacterial 16s ribosomal DNA with polymerase chain chlamydiaceae fam. nov. and Simkaniaceae fam. nov., each reaction. J Clin Microbiol28, 942-1946. containing one monotypic genus, revised of the Woese, C. R. (1987). Bacterial evolution. Microbiol Rev 51, family Chlamydiaceae including a new genus and five new 22 1-27 1.

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