LETTERS

Novel - formed by using forward primer 16S sequences, identifi ed by a BLAST FOR2 (5′-CGT GGA TGA GGC ATG (www.ncbi.nlm.nih.gov/BLAST) like Organisms as CAA GTC GA-3′) and reverse primer similarity search of the European Cause of Bovine 16S REV2 (5′-CAA TCT CTC AAT Molecular Biology Laboratory/Gen- Abortions, UK CCG CCT AGA CGT CTT AG-3′) to Bank database, plus alignment of an generate amplicons of ≈260 bp (5). outgroup of 7 non-Chlamydiales se- To the Editor: Despite the world- Negative-control reactions contained quences. A Bayesian phylogenetic tree wide economic impact of, and welfare DNA-free water instead of extracted (Markov Chain Monte Carlo settings: issues associated with, infectious bo- DNA. PCR products were purifi ed 2 runs of 625,000 generations; burn-in vine abortifacients, as well as poten- (QIAquick PCR Purifi cation Kit; of 125,000 generations; trees sampled tial zoonotic threats to human health, QIAGEN, Crawley, UK) before direct every 100 generations) was then esti- accurate diagnosis of the causes of sequencing by using the PCR primers mated with a general time reversible + abortion is uncommon (1). This poor and dideoxy chain termination/cycle Γ nucleotide substitution model by us- diagnosis could be explained in part sequencing on an ABI 3730XL DNA ing the MrBayes program (6) launched by the lack of identifi cation of infec- sequencer (MWG Operon, Ebersberg, from the TOPALi v2 package (7). tious abortifacient agents. Germany). Despite the short sequence length Although abortus After the initial PCR, 22 (26.5%) of the 15 samples, the tree was well is a known etiologic agent of rumi- of the 83 fetal samples tested were resolved with the Chlamydiales se- nant abortion, several novel species Chlamydiales positive. Serologic, quences and formed 3 clusters (Chla- of Chlamydia-like organisms have bacteriologic, and histopathologic mydiaceae, Rhabdochlamydiaceae/ recently emerged as putative ruminant examination of fetal tissues identi- , and Parachlamydiace- abortifacients. chondrophila fi ed no other infectious abortifacient ae/Waddliaceae/Criblamydiaceae) was isolated from the brain and ner- agents in the Chlamydiales-positive (Figure). Two of these sequence vous tissue of an aborted bovine fetus samples. Sequence information was clusters represented 10 and 5 of the in Germany (2), and Parachlamydia successfully obtained for 15 of these samples, whereas no samples were acanthamoebae and other unidenti- 22 samples with forward and reverse represented in the cluster containing fi ed Chlamydia-like species were primers; sequences ranged from 140 the , which includes identifi ed in 18.3% of bovine placenta bp to 194 bp (European Molecular C. abortus. Most (10/15) sequences samples in Switzerland (3,4). Given Biology Laboratory/GenBank acces- were found in the cluster containing the paucity of information about the sion nos. GQ919016–GQ919030). the . This fi nding causes of infectious bovine abortion These 15 short sequences were care- agrees with those of the aborted bo- and the high prevalence of Chlamyd- fully aligned to a representative set of vine placenta studies in Switzerland ia-like organisms in the samples from 22 similar Chlamydiales 16S rDNA (3,4) and provides further evidence Switzerland, we attempted to deter- mine whether such organisms can be detected in bovine fetal tissues in the United Kingdom. Pooled tissue samples compris- ing brain, heart, and/or placenta (de- pending on availability) were obtained from bovine fetuses submitted for di- agnosis to the Scottish Agricultural College Disease Surveillance Centre, Dumfries, Scotland, UK, during 2008. Tissue pools were homogenized by us- ing a Precellys bead mill homogenizer (Bertin Technologies, Ann Arbor, MI, USA), and DNA was extracted by us- ing the Wizard Genomic DNA Puri- fi cation Kit (Promega, Southampton, UK) according to manufacturer’s in- Figure. Bayesian phylogenetic tree demonstrating the relationship of 15 isolated organisms structions. A pan-Chlamydiales PCR from the older chlamydiales samples to known chlamydial species. Cand., Candidatus; R., for the 16S rDNA sequence was per- ; P., Protochlamydia.

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 8, August 2010 1323 LETTERS that Parachlamydia-like species may This work was funded by the Scot- Dis. 2009;15:120–1. DOI: 10.3201/ play a substantial role in bovine abor- tish Government Rural and Environment eid1501.080911 10. Lamoth F, Aeby S, Schneider A, Jaton- tion in mainland Europe and the Unit- Research and Analysis Directorate and the Ogay K, Vaudaux B, Greub G. Parachla- ed Kingdom. Four of the remaining Biological and Biotechnology Sciences mydia and Rhabdochlamydia in premature 5 samples clustered with members of Research Council. neonates. Emerg Infect Dis. 2009;15:2072– the family Rhabdochlamydiaceae; the 75. DOI: 10.3201/eid1512.090267 fi fth sequence (CLBUK3), although Nicholas Wheelhouse, Address for correspondence: Nicholas present in the same Rhabdochlamy- Frank Katzer, Frank Wright, Wheelhouse, Moredun Research Institute, diaceae/Simkaniaceae cluster, ap- and David Longbottom Pentlands Science Park, Bush Loan, Edinburgh, peared to be more distinct from other Author affi liations: Moredun Research Insti- Scotland EH26 0PZ, UK; email: nick. family members. tute, Edinburgh, Scotland, UK (N. Wheel- [email protected] The identifi cation of these organ- house, F. Katzer, D. Longbottom); and isms in such a large percentage of the Scottish Crop Research Institute, Dundee, bovine fetal tissue samples tested may Scotland, UK (F. Wright) indicate a role for these organisms in undiagnosed bovine abortions in the DOI: 10.3201/eid1608.091878 United Kingdom and Europe and may be a zoonotic source of infection for References humans. Indeed, considerable evidence 1. Cabell E. Bovine abortion: aetiology and West Nile Virus supports a role for Parachlamydia spp. investigations. In Pract. 2007;29:455–63. Knowledge among in human pneumonia, whereas Rhab- 2. Henning K, Schares G, Granzow H, Pol- dochlamydia spp. is a suspected cause ster U, Hartmann M, Hotzel H, et al. Neo- Hispanics, spora caninum and Waddlia chondrophila (8). In addition, evidence suggests strain 2032/99 in a septic stillborn calf. San Diego County, that P. acanthamoebae crosses the hu- Vet Microbiol. 2002;85:285–92. DOI: California, USA, man placenta to the unborn fetus (9). 10.1016/S0378-1135(01)00510-7 Also, the presence of both parachla- 3. Borel N, Ruhl S, Casson N, Kaiser C, 2006 mydial and rhabdochlamydial DNA Pospischil A, Greub G. Parachlamydia spp. and related Chlamydia-like organ- To the Editor: West Nile virus in the lung secretions of hospitalized isms and bovine abortion. Emerg Infect (WNV), spread by infected mosqui- premature human neonates recently Dis. 2007;13:1904–7. toes, is a serious public health threat correlated with increased medical in- 4. Ruhl S, Casson N, Kaiser C, Thoma R, throughout the United States and terventions and increased duration of Pospischil A, Greub G, et al. Evidence for Parachlamydia in bovine abortion. can cause life-altering and even fa- hospital stay (10). Vet Microbiol. 2009;135:169–74. DOI: tal disease (1). In San Diego County, We demonstrate the presence of 10.1016/j.vetmic.2008.09.049 California, the human infection rate Parachlamydiaceae and Rhabdochla- 5. Ossewaarde JM, Meijer A. Molecular was 0.18 per 100,000 persons dur- mydiaceae species in bovine abortions evidence for the existence of additional members of the order Chlamydiales. ing 2003–2006 (5 cases, 1 locally in the United Kingdom. Given the Microbiology. 1999;145:411–7. DOI: acquired) and then increased to 0.52 zoonotic potential and the economic 10.1099/13500872-145-2-411 and 1.17 per 100,000 persons in 2007 and welfare impacts of bovine abor- 6. Ronquist F, Huelsenbeck JP. MrBayes and 2008, respectively, despite few tion on the agricultural sector, further 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. changes in surveillance activities (2). studies are required to understand the 2003;19:1572–4. DOI: 10.1093/bioinfor- Community-based mosquito control incidence and pathogenic roles of matics/btg180 programs, adoption of personal pro- these organisms in both humans and 7. Milne I, Lindner D, Bayer M, Husmeier tective behavior (PPB), and education animals. These studies should include D, McGuire G, Marshall DF, et al. TOPA- Li v2: a rich graphical interface for evo- are the most effective ways to prevent broader molecular epidemiologic stud- lutionary analyses of multiple alignments human WNV infection because no ies, as well as detailed histologic/im- on HPC clusters and multi-core desktops. specifi c antiviral drug treatment or munohistochemical investigations and Bioinformatics. 2009;25:126–7. DOI: vaccine exists (1,3). Although WNV- organism recovery through culture of 10.1093/bioinformatics/btn575 8. Greub G. Parachlamydia acanthamoebae, associated illness has occurred in all infected placental and fetal tissues. an emerging agent of pneumonia. Clin racial and ethnic groups, Hispanics are Microbiol Infect. 2009;15:18–28. DOI: potentially at risk because of language Acknowledgment 10.1111/j.1469-0691.2008.02633.x 9. Baud D, Goy G, Gerber S, Vial Y, and cultural barriers to obtaining in- We thank the Scottish Agricultural Hohlfeld P, Greub G. Evidence of ma- formation regarding WNV prevention College Disease Surveillance Centre, ternal-fetal transmission of Parachla- (4). San Diego County Department of Dumfries, for sample collection. mydia acanthamoebae. Emerg Infect

1324 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 16, No. 8, August 2010