FamilyWaddliaceae

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Formally described by Rurangirwa et al., 1999, the type strain is W. chondrophila WSU 86-1044 which was isolated from pooled lung and liver tissues of an aborted bovine foetus [Dilbeck et al., 1990]. At first, the agent appeared to be a new member of the rickettsiae because it showed cross-reactions with antisera directed against Cowdria ruminantium [a tick-borne Rickettsiales agent of ruminants; the cross-reacting antigen was not identified]. The organism replicated within intracytoplasmatic vacuoles and had a development cycle comparable to that of chlamydiae and ehrlichiae [Kocan et al., 1990; Corsaro et al., 2003]. In contrast to chlamydiae, replication was not inhibited by penicillin [Dilbeck et al., 1990]. Phylogenetic relationship to chlamydiae was shown by analysis of the 16S ribosomal (r)DNA [Rurangirwa et al., 1999; Everett, 2000]. Close association of W. chondrophila reticulate bodies with host cell mitochondria [Fig. 1] gave rise to the species name. W. chondrophila has the characteristic Chlamydiales insertions and deletions [Griffiths and Gupta, 2002]. A second species, W. malayensis, was isolated from fruit bat excreta in Malaya [Chua 2003; Chua et al., 2005].

Taxonomic Description of Waddliaceae fam. nov

Waddliaceae (Waddli.a’ce.ae M.L. fem. n. Waddlia the type genus of the family; aceae ending to denote a family; M.L. fem. pl. Waddliaceae the Waddlia family.

Waddliaceae currently includes the type genus, Waddlia. The description of this family is identical at present to that of `micro-organism WSU 86-1044′ (Dilbeck et al., 1990; Kocan et al., 1990) which are obligate intracellular organisms resistant to penicillin. They grow well in BT producing multiple cytoplasmic vacuoles and Gram-negative, periodic acid�Schiff negative and non-acid-fast inclusions. The inclusions contain coccoid organisms ranging from 0�2 to 0�5 um in size. The BT infectivity is abolished by tetracycline and / or chloroform treatment. The organism multiplies by binary fission and has two developmental forms: the dense form which is infective, and the reticulated form, usually associated with mitochondria, which undergoes binary fission. These organisms do not react with antisera used for typing chlamydiae or rickettsiae. The 16S rDNA of the Waddliaceae strains are>90% similar to ribosomal genes in WSU 86-1044T. The family Waddliaceae belongs to the order Chlamydiales and is a sister taxon of the Chlamydiaceae because the ribosomal genes are 80�90% similar to ribosomal genes in the Chlamydiaceae. Phylogenetic analyses of the Waddliaceae 16S rDNA sequence is presented here. At present this family comprises a single genus, the type genus Waddlia [Rurangirwa et al., 1999].

Taxonomic Description of Waddlia gen. nov.

Waddlia [Wadd’li.a. N.L. fem. n. Waddlia arbitrary name derived from the abbreviation WADDL (Washington Animal Disease Diagnostic Laboratory)]. Members of the genus Waddlia have 16S rDNA that is >90% similar to that of the type species, Waddlia chondrophila strain WSU 86-1044T [Rurangirwa et al., 1999].

Waddlia chondrophila

Taxonomic Description of Waddlia chondrophila sp. nov

Waddlia chondrophila (chon.dro’phi.la. Gr. n. chondros clump; Gr. adj. philos, a friendly to; M.L. chondrophila liking clumps, in reference to the association of the organism with cellular mitochondria). The species Waddlia chondrophilacurrently includes only the type strain, WSU 86-1044T ( = ATCC VR 1470T). Waddlia chondrophila was isolated from the tissues of a first-trimester aborted bovine foetus. The description of this species is identical to that of`microorganism WSU 86-1044T [Dilbeck et al., 1990; Kocan et al., 1990]. The full length of Waddlia chondrophila 16S rDNA sequence is 15 � 25 % different from the 16S rDNA of Chlamydiaceae spp., fitting into the 80�90% identity range that makes this organism a member of the order Chlamydiales, but not a member of the family Chlamydiaceae. The Waddlia chondrophila 16S rDNA sequence is 15.8% different from that of Simkania negevensis strain ZT, while it is 12.8% different from that of Parachlamydia acanthamoebae strain Bn9 T, which excludes it from Simkaniaceae and Parachlamydiaceae families of the order Chlamydiales (Everett et al., 1999. For a newly identified strain to be described as a member of the Waddliaceae, a nearly full-length rDNA of the new strain may only differ from Waddlia chondrophila 16S rDNA by <10% [Rurangirwa et al., 1999].


As indicated by the official description above, organism WSU was originally isolated from the tissues of a first trimester aborted bovine foetus [Dilbeck et al., 1990]. A cytopathic effect was observed within 2 – 3 days of inoculation of pooled spleen and liver homogenate into bovine turbinate (BT) cell cultures. The organism was resistant to penicillin or gentamicin but sensitive to tetracycline.

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Fig. 1. Electron micrograph of Waddlia chondrophila strain 2032/99 in McCoy cells. Chlamydia-like particles were located within the cytoplasm in vacuoles. Infected vacuoles were surrounded by host cell mitochondria. (Photo: H. Granzow, BFAV, Insel Riems, Germany). [Double click images to expand].

Subsequently an agent with similar properties and morphology was isolated from the heart of an aborted bovine foetus [Henning et al., 2002]. This latter organism was also resistant to penicillin and failed to react with antisera direct against Chlamydiaceae or Simkania negevensis. 16S and partial 23S ribosomal RNA sequences suggested that the isolate 2032/99 was W. chondrophila or a closely related organism. 16SrRNA sequencing has also suggested the presence of W. chonodrophila in an Australian mammal, the Gilbert’s Potoroo [Bodett et al., 2003].

Animal and human infections with W. chondrophila

W. chondrophila type strain WSU 86–1044 was implicated as an abortigenic agent in 1986 when it was detected in lung, liver, and other tissues of an aborted bovine foetus in the USA [Dilbeck et al., 1990] and was subsequently isolated in Germany from a septic, still born calf [Henning et al., 2002]. However, its overall significance as an agent of bovine abortion is still unclear. In the USA a serological study indicated a highly significant association between antibody to W. chondrophila and bovine abortion (p<0.00001). However, a PCR-based study of the incidence of chlamydia-related abortions in cattle from the Graubunden region of Switzerland, found chlamydia-like organisms in 43 of 235 cases. Sequencing the PCR products revealed no evidence of Wadllia infection although, surprisingly, 9 cases were associated with Parachlamydia acanthamoebae [Borel et al., 2006; 2007].

The question arises whether Waddlia might be associated with human abortion. Waddlia – related phylotypes have been identified by PCR in human samples [Corsaro et al., 2002; 2003]. Baud et al., 2007; 2008 found that anti-Waddlia antibody reactivity by immunofluorescence and immunoblot was higher in UK women who had sporadic and recurrent miscarriages (n=69 & 200 respectively) than in control women (n= 169; p<0.001), with odds ratios ranging from 4.9 – 6.2.

Cultivation and detection

Cultivation of W. chondrophila was performed using bovine turbinate cells, P388D1 mouse macrophage cells [Kocan et al., 1990], Buffalo Green Monkey (BGM) cells, McCoy cell cultures and human diploid fibroblasts cells [Henning et al., 2002]. The cells were grown at 35 or 37� C in Eagle minimal essential medium [Dilbeck et al., 1990], RPMI [Kocan et al., 1990] or in a medium which was composed of medium RPMI 1640 and PFEK-1 [Henning et al., 2002], supplemented with 5 or 10 % foetal calf serum. Additionally, the agent can be propagated in serum free cell cultures using medium Nephros LP (unpublished data). Penicillin, gentamicin sulphate, streptomycin, vancomycin, and amphotericin B may be used as antibiotics [Dilbeck et al, 1990; Henning et al., 2002]. After an incubation period of 28 to 36 h large vacuoles filled with particles can be observed in the infected cells. Inclusions can be stained using Gim�nez [Fig. 2] or Giemsa [Fig. 3] stains [Dilbeck et al., 1990; Henning et al., 2002].

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Fig. 2. Waddlia chondrophila strain 2032/99 in Buffalo Green Monkey cells, Gim�nez stain. Waddlia _ = red coccoid particles (Photo: L. Minke, BFAV, Wusterhausen, Germany). Fig. 3. Waddlia chondrophila strain 2032/99 in Buffalo Green Monkey cells, Giemsa stain. (Photo: L. Minke, BFAV, Wusterhausen, Germany).

Interestingly, W. chondrophila can be propagated in amoebal hosts, i.e. Hartmannella vermiformis, among other free-living amoeba. Transmission electron microscopy of Hartmannella trophozoites infected with Waddlia revealed typical morphological stages of a Chlamydia-like life-cycle, including the presence of elementary and reticulate bodies [Fig. 4]. Thus, free-living amoebae may be an environmental reservoir for W. chondrophila [Michel et al., 2003].

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Fig. 4. Hartmannella vermiformis (strain Os 101) infected with Waddlia chondrophila strain 2032/99. Waddlia are replicating within vacuoles (v) or separately within the cytoplasm. N = Nucleus with large nucleosome; arrows indicate various endoparasitic stages of Waddlia, mainly elementary bodies. (Photo: R. Michel, Zentrales Institut-BW, Koblenz, Germany).

Field specimens can be screened for W. chondrophila and related organisms using a PCR test which is specific for all bacteria in the order Chlamydiales [Everett et al., 1999; See: Speciation PCR. Further details of PCR probes and of cell culture are given in the review of Corsaro and Greub, 2006.

See also: Evolutionary divergence of chlamydiae in the chlamydial evolution section.

[Klaus Henning, September 2003 updated by MEW March 2008]

Waddlia malayensis

During a search for the natural reservoir of Nipah virus encephalitis, urine was collected on plastic sheeting underneath roosting sites of the fruit bat Eonycteris spelaea at Gua Tempurong in northern Malaysia. From the urine 23 isolates of infectious agent capable of causing cytopathogenic effect (CPE) in Vero cells within 5 – 7 days of culture were obtained [Chua, 2003; Chua et al., 2005]. The organism grew rapidly in cell culture in the absence of cycloheximide [PICKMEW comment: the authors say chlorhexidine]. Inclusions visible by phase-contrast microscopy could be detected within 48 to 72 h postinfection of Vero cells. These inclusions could be stained by both Giemsa and the Schiff reaction, but not with Microtrak C. trachomatis MOMP-specific monoclonal antibody. Transmission electron microscopy revealed a typical chlamydial dimorphic developmental cycle. The 16S rRNA gene, the 16S-23S rRNA intergenic spacer and part of the 23SrRNA gene were sequenced bidirectionally [Chua et al., 2005]. The gene signatures had 91% identity with W. chondrophila sequences AF346001 and AF042496. Like W. chondrophila the organism was able to grow in a variety of host cells, was resistant to penicillin and streptomycin but sensitive to tetracycline, and inclusions were surrounded by mitochondria. Unlike W. chondrophila the host was markedly different and its inclusions were intensely Schiff stain positive suggesting a glycogen-like matrix. It was suggested this is a distinct species within the Waddliaceae and the authors proposed the name W. malayensis [Chua et al., 2005].

[PICK MEW comment: No formal taxonomic description of the proposed new species has been published in IJSEM or elsewhere. The requirement for the type isolate of the proposed species to be deposited in two culture collections in two different countries has not been met. W. malayensis is not included in the IJSEM validation lists to date. This is unlikely to be a formally valid species proposal; see TaxonomyRules].

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References

Baud, D., Regan, L. & Greub, G. (2008). Emerging role of Chlamydia and Chlamydia-like organisms in adverse pregnancy outcomes. Current Opinion in Infectious Diseases Feb; 21 (1): 70 – 76.

Baud, D., Thomas, V., Arafa A., Regan, L. & Greub, G. (2007). Waddlia chondrophila, a potential agent of human fetal death. Emerg Infect Dis 13(8), 1239 – 1243. Full paper (html)

Bodett, T. J., Viggers, K., Warren, K., Swan, R., Conaghty, S., Sims, C. et al. (2003). Wide range of Chlamydiale types detected in native Australian mammals. Vet Microbiol. 96, 177 – 187.

Borel N. et al., (2006). Chlamydia-related abortions in cattle from Graubunden, Switzerland. Vet Pathol 43 (5), 702 – 708.

Borel N, Ruhl S, Casson N, Kaiser C, Pospischil A, Greub G. (2007). Parachlamydia spp. and related Chlamydia-like organisms and bovine abortion. Emerg Infect Dis. Full paper (html)

Chua, K. B. (2003). A novel approach for collecting samples from fruit bats for isolation of infectious agents. Microbes and Infection 5, 487 – 490.

Chua, P.K., Corkill, J.E., Hooi, P.S., Cheng, S.C., Winstanley, C. and Hart, C.A. (2005). Isolation of Waddlia malaysiensis, a novel intracellular bacterium, from fruit bat ( Eonycteris spelaea). Emerging Infect. Dis. 11 (2), 271-277. Full paper(html)

Corsaro, D. & Greub, G. (2006). Pathogenic potential of novel chlamydiae and diagnostic approaches to infections due to these obligate intracellular bacteria. Clinical Microbiology Reviews 19 (2), 283 – 297. Full paper (html) Full paper [Acrobat] [Useful review]

Corsaro, D., Venditti, D. & Valassina, M. (2002). New parachlamydial 16S rDNA phylotypes detected in human clinical samples. Research Microbiology 153, 563 – 567.

Corsaro, D., Valassina, M. & Venditti, D. (2003). Increasing diversity within Chlamydiae._Critical Reviews of Microbiology 29, 37 – 78. [Excellent scholarly review and taxonomic study]

Dilbeck, P. M., et al., (1990). Isolation of a previously undescribed rickettsia from an aborted bovine fetus. Journal of Clinical Microbiol_ogy 28, 814 – 816.

Dilbeck-Robertson, P., M. M. McAllister, D. Bradway, and J. F. Evermann. (2003). Results of a new serologic test suggest an association of Waddlia chondrophila with bovine abortion. J. Vet. Diagn. Investig. 15: 568-569

Everett, K. D. E. (2000). Chlamydia and Chlamydiales: more than meets the eye. Veterinary Microbiology 75, 100 – 126. [Review article].

Everett, K. D. E., Hornung, L. J. & Andersen, A. A. (1999). Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests. Journal of Clinical Microbiology 37, 575 – 580. Full article

Griffiths, E. & Gupta, R. S. (2002). Protein signatures distinctive of chlamydial species: horizontal transfers of cell wall biosynthesis genes glmU from archaea to chlamydiae and murA between chlamydiae and Streptomyces. Microbiology 148, 2541 – 2549.

Haider S, Collingro A, Walochnik J, Wagner M, Horn M. (2008). Chlamydia-like bacteria in respiratory samples of community-acquired pneumonia patients. FEMS Microbiol Lett. Feb 27 [Epub ahead of print].

Henning, K., Schares, G., Granzow, H., Polster, U., Hartmann, M., Hotzel, H., Sachse, K., Peters, M. & Rauser M (2002). _Neospora caninum_ and Waddlia chondrophila strain 2032/99 in a septic stillborn calf. Veterinary Microbiology 85, 285 – 292.

Kocan, K. M., Crawford, T. B., Dilbeck, P. M., Evermann, J. F. & McGuire, T. C. (1990). Development of a rickettsia isolated from an aborted bovine fetus. Journal of Bacteriology 172, 5949 – 5955.

Michel, R., Steinert, M., Z�ller, L., Haur�der, B & Henning, K. (2003). Cocultivation of protozoa and the chlamydia-like bacterium Waddlia chondrophila isolated from an aborted bovine foetus in Germany (submitted).

Rurangirwa, F. R., et al., (1999). Analysis of the 16S rRNA gene of microorganism WSU 86-1044 from an aborted calf 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.