abien Vorimoreb, Nadia Vicari f, Simone Magninof, Elisabeth Liebler-Tenorioa,nke Ruettgera, Patrik M. Bavoil g, Frank T. Hufertd, Ramon Rosselló-Mórah, Manja Marzc
Institute of Molecular Pathogenesis, Friedrich-Loeffler-Institut (Federal Research Institute for Animal Health), Jena, GermanyUniversity Paris-Est, Anses, Animal Health Laboratory, Bacterial Zoonoses Unit, Maisons-Alfort, FranceFaculty of Mathematics and Computer Science, Friedrich-Schiller-Universität, Jena, GermanyDepartment of Virology, Universitätsmedizin Göttingen, GermanyInstitute for Genome Sciences & Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, MD, USAIstituto Zooprofilattico Sperimentale della Lombardia e dell’Emilia Romagna “Bruno Ubertini”, Sezione Diagnostica di Pavia, Pavia, ItalyDepartment of Microbial Pathogenesis, University of Maryland, Baltimore, MD, USAInstitut Mediterrani d’Estudis Avanc ats, Esporles, Illes Balears, Spain
r t i c l e i n f o
rticle history:eceived 3 June 2013eceived in revised form 9 December 2013ccepted 12 December 2013
The family Chlamydiaceae with the recombined single genus Chlamydia currently comprises nine species,all of which are obligate intracellular organisms distinguished by a unique biphasic developmental cycle.Anecdotal evidence from epidemiological surveys in flocks of poultry, pigeons and psittacine birds haveindicated the presence of non-classified chlamydial strains, some of which may act as pathogens. In thepresent study, phylogenetic analysis of ribosomal RNA and ompA genes, as well as multi-locus sequenceanalysis of 11 field isolates were conducted. All independent analyses assigned the strains into two dif-ferent clades of monophyletic origin corresponding to pigeon and psittacine strains or poultry isolates,respectively. Comparative genome analysis involving the type strains of currently accepted Chlamydi-aceae species and the designated type strains representing the two new clades confirmed that the latter
6S rRNA gene sequenceulti-locus sequence analysis
could be classified into two different species as their average nucleotide identity (ANI) values were alwaysbelow 94%, both with the closest relative species and between themselves.
In view of the evidence obtained from the analyses, we propose the addition of two new species tothe current classification: Chlamydia avium sp. nov. comprising strains from pigeons and psittacine birds(type strain 10DC88T; DSMZ: DSM27005T, CSUR: P3508T) and Chlamydia gallinacea sp. nov. comprising
e stra T T T
strains from poultry (typ
ntroduction
Microorganisms commonly known under the trivial name ofhlamydiae comprise a group of obligate intracellular bacteria with
Please cite this article in press as: K. Sachse, et al., Evidence for the existenChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Micr
unique biphasic developmental cycle. The phylum Chlamydiaeurrently encompasses a single class Chlamydia and a single orderhlamydiales [15].
In 1966, Page introduced the genus Chlamydia within the familyChlamydiaceae and the order Chlamydiales [27]. Chlamydia (C.) tra-chomatis and C. psittaci were the only two species known until the1980s, and their distinction was based on biochemical characteris-tics, morphology and developmental replication [28]. Accordingly,strains accumulating glycogen in inclusions and being suscepti-ble to sulfadiazine were assigned to C. trachomatis, while thosenot accumulating glycogen and resistant to sulfadiazine becameC. psittaci. After the definition of two further species, C. pneumo-niae [10] and C. pecorum [5], it became evident that the former two
ce of two new members of the family Chlamydiaceae and proposal ofobiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
species were very heterogeneous in terms of host species, tissuetropism and genetic markers. Based on extensive analysis of ribo-somal RNA gene sequences, Everett et al. [4] addressed this problemby dividing the family Chlamydiaceae into the genera Chlamydia
nd Chlamydophila (Cp.) comprising three and six species, respec-ively, i.e. C. trachomatis, C. muridarum and C. suis, as well as Cp.bortus, Cp. caviae, Cp. felis, Cp. pecorum, Cp. pneumoniae, and Cp.sittaci. While the extended list of species has been universallyccepted, the rationale for the introduction of two different generaas been disputed [1,38]. The Subcommittee on the Taxonomy ofhe Chlamydiae proposed the return to the combined genus Chlamy-ia comprising all known species of the family [12], and the latestdition of Bergey’s Manual already features the single genus [18].o conduct the reunification of the genus formally, a request forn opinion is to be submitted to the Judicial Commission of theCSP. Therefore, in the face of the forthcoming adjustment and tovoid confusion, we are using the single genus Chlamydia in thisaper.
As the membership of the order Chlamydiales has been repeat-dly extended by the addition of new families and species in theast decade, the likelihood of the family Chlamydiaceae includ-
ng more members than currently accepted also appears to beigh [3,6,22]. The use of highly specific DNA-based diagnosticssays enabled the detection of atypical strains from poultry [6,22],igeons [8,34] and ibis [39]. However, reports in the literaturerovided only anecdotal evidence of the identity of non-classifiedaxa and their possible epidemiological dissemination. It is neces-ary to characterise all available isolates in order to identify theiraxonomic position. In the present paper, we describe the phe-otypic and genetic properties of 11 so far non-classified isolatesnd conclude that they should be assigned to two new species,. avium sp. nov. comprising strains from pigeons and psittacineirds and C. gallinacea sp. nov. comprising strains from poul-ry.
aterials and methods
acterial strains
Strain 10DC88 was isolated from spleen tissue of a perishedsittacine bird (genus Polytelis) from a breeder flock in the fed-ral state of North Rhine Westphalia, Germany, in 2010. The birdad shown signs of catarrhal enteritis and post-mortem examina-ion revealed hepatosplenomegaly. Several other birds of the sameock had also died with similar signs. Other Chlamydiaceae speciesere not detected.
Strain 11DC96 was isolated from spleen tissue of a young pigeonColumba livia) from a breeder flock in Saxony, Germany, in 2011.ymptoms of respiratory disease and diarrhoea had been present inhe flock of approximately 100 birds for about a year. Other micro-ial agents identified in this bird included C. psittaci, Mycoplasmapp., Trichomonas gallinae, and Spironucleus columbae.
Strain 12DC97 was isolated from faeces of a young breederigeon showing respiratory symptoms and general decrease in per-ormance in 2012. The clinical signs had been observed in the flock62 birds, state of North Rhine Westphalia, Germany) for about aear. The bird also carried C. psittaci, Mycoplasma spp. and Candidalbicans.
Strains 08-1274/03, 08-1274/13, 08-1274/19, 08-1274/21, and8-1274/23 originated from cloacal swabs of chickens. The birdsere from different poultry farms in the region of Charente (France)
nd showed no clinical signs. Details were published previously22]. Strain 10-743 SC13 was isolated from a cloacal swab of anrban pigeon in Paris, France.
Strains PV3515/3 and PV7344/2 were isolated from intestinal
Please cite this article in press as: K. Sachse, et al., Evidence for the existenChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Micr
ontents and intestinal tissue of urban pigeons sampled in Milannd Ferrara in the regions of Lombardy and Emilia-Romagna, Italy,n 1996 and 1997, respectively. Basic metadata of the cultured iso-ates included in the present study are listed in Table 1.
PRESS Microbiology xxx (2014) xxx– xxx
Isolation and propagation in cell culture
Buffalo green monkey (BGM) cells in minimal essential medium(MEM, Lonza, Cologne, Germany) with 5% serum were seededinto Trac bottles containing glass coverslips (Bibby Sterilin Ltd.,Staffordshire, UK) and incubated at 37 ◦C with 5% CO2 in a fullyhumidified cabinet for 4 days. All cell monolayers were examinedfor confluent growth on the day of inoculation. In each case, dif-ferent amounts of crushed and ultrasonicated (ten 8-s pulses at anamplitude of 80%; Branson 450D Sonifyer) samples were inoculatedinto six Trac bottles. After inoculation, the bottles were centrifugedat 3000 × g and 37 ◦C for 60 min and subsequently incubated for2 h. The MEM medium was then replaced with serum-free mediumNephros LP (Lonza), and the latter was renewed after 18 h.
Three days after inoculation, a single coverslip was fixed withmethanol, and the monolayer was stained with IMAGEN Chlamy-dia (Oxoid Ltd., Cambridgeshire, UK), an immunofluorescence kitbased on a genus-specific monoclonal antibody. A sample was con-sidered positive when inclusions of typical chlamydial morphologyappeared as bright apple-green smooth-edged spots after two pas-sages.
Transmission electron microscopy
Cell culture aliquots of strains 10DC88 and 08-1274/3 werefixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, postfixedin 2% osmic acid and embedded in araldite. Ultrathin sectionswere prepared as described previously [9] and examined with atransmission electron microscope (TECNAI 12, FEI, Eindhoven, TheNetherlands).
Real-time PCR for detection of the new species
The protocol for detection of atypical chlamydial strains frompoultry to be designated C. gallinacea was published recently [41].The assay targets the 16S rRNA gene locus and includes primersACC Fw (5′-CGAACGAAATAACACTTCGGTGTTG-3′) and ACC Rv (5′-ACATACCACATTCGGTATTAGCGGT-3′), as well as probe ACC Pr (5′-FAM-GTGGCGGAAGGGTTAGTAAT-TAMRA-3′).
C. avium, which includes pigeon and psittacine strains, can bespecifically detected using the methodology of Zocevic et al. [42].Briefly, primers APC Fw (5′-CATGCAAGCTATTGAGAAAAGTGGT-3′)and APC Rv (5′-CCTTGATATGTACGTGTTTTCTCG-3′), and probeAPC Pr (5′-FAM-CACCCCTGGTGAAGATATTTCCTTAGCAT-TAMRA3′) targeting the enoA gene locus were used in a TaqMan assay.
Sequencing of the ompA and ribosomal RNA genes from fieldisolates
Sequences of an approximately 1500-nt segment comprisingthe 16S ribosomal (r) RNA gene, were determined from amplifi-cation products of chromosomal DNA using primer pairs 16S1/rp2[31], 16SF2/23SIGR, as well as 16SIGF [4] and the newly designed16SF2-rev (CCATGATGTGACGGGCGG).
Sequencing of the ompA gene involved primers CTU/CTL[36] or CTU/ompA-rev [35]. Purified amplification products weresequenced by Eurofins MWG (Ebersberg, Germany).
Multi-locus sequence analysis (MLSA)
The MLSA system used was comprised of four genomic loci:gatA (aspartyl/glutamyl-tRNA amidotransferase subunit A), hflX
ce of two new members of the family Chlamydiaceae and proposal ofobiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
(GTP-binding protein), enoA (enolase) and gidA (tRNA uracil-5-methyltransferase), as described previously for C. psittaci [30]. Therelevant segments of the first three genes were amplified usingprimers for the homologs in C. psittaci. Primers for the gidA locus
K. Sachse et al. / Systematic and Applied Microbiology xxx (2014) xxx– xxx 3
Table 1Cultured isolates of new chlamydial species described in this study.
Designation Source Year Country Species MLSAa sequence type GenBank acc. no. (16S rRNA/ompA)
PV3515/3 Pigeon, intestinal contents 1996 Italy C. avium a KF366256/KF366262PV7344/2 Pigeon, intestine 1997 Italy C. avium a KF366257/KF36626410-743 SC13 Pigeon, cloacal swab 2010 France C. avium a KF366258/KF36626310DC88 Parrot, spleen 2010 Germany C. avium b KF366255/KF36626111DC96 Pigeon, spleen 2011 Germany C. avium a KF366259/KF36626512DC97 Pigeon, faeces 2012 Germany C. avium a KF366260/KF36626608-1274/3 Chicken, cloacal swab 2008 France C. gallinacea c GQ398026/GQ39803308-1274/13 Chicken, cloacal swab 2008 France C. gallinacea c GQ398027/GQ39803508-1274/19 Chicken, cloacal swab 2008 France C. gallinacea d GQ398028/GQ39803608-1274/21 Chicken, cloacal swab 2008 France C. gallinacea e GQ398029/GQ39803708-1274/23 Chicken, cloacal swab 2008 France C. gallinacea c GQ398031/GQ398032
a MLSA type based on sequence analysis of four housekeeping genes (i.e. enoA, gatA, gidA and hflX).
Table 2Primers used for multi-locus sequence analysis of C. avium and C. gallinacea.
Target gene Forward primer (name,sequence)
Reverse primer Reference
gatA YPgatA5GCYTTAGAGTTAAGARATGCT
YPgatA6GAKCCWCCWGTATCAGAWCC
[30]
hflX YPhflX5GCTTCTARRGTACTTTTAAATG
YPhflX6ATWTTAGAGATCTTTGCTAGYCG
[30]
gidA Biovar AgidAGGAGTCWCTACWAAAGAAGG
gidA2TCGTAYTGYACATCRAAAGG
[31]
Biovar BgidA biovB forwTGGTCGTATCCAAGGAGTAACAAC
gidA biovB revTTCTAGCCCATGGACAGAACG
This study
enoA YPenoA5 YPenoA6 [30]
R
wP
P
3Ta3chp5sp1veDr
G
DDaDgNuKR
CCWATGATGAAYCTYATTAACGG
= A or G; S = G or C; W = A or T; Y = C or T; M = A or C; K = G or T.
ere newly designed based on the 10DC88 genome sequence.rimer sequences are given in Table 2.
reparation of DNA for genome sequencing
Genomic DNA of designated type strains was prepared from0 mL of BGM cell cultures harvested from a 75-cm2 culture flask.he suspension was centrifuged at 20,000 × g and 8 ◦C for 40 min,nd the supernatant was discarded. The pellet was resuspended in0 mL PBS buffer and centrifuged at 500 × g for 10 min to removeell debris. The supernatant was transferred to a fresh tube andigh-speed centrifuged as detailed above. The pellet was resus-ended in PBS containing 1% (w/v) SDS and subjected to digestion at6 ◦C for 30 min by adding 50 �L of 0.2% (w/v) proteinase K. Furtherteps of DNA enrichment and purification included sodium acetaterecipitation (400 �L 5 M KAc, 30 min on ice), centrifugation at3,200 × g for 5 min, extraction of the supernatant with an equalolume of phenol-chloroform-isoamyl alcohol (25:24:1, v/v/v) andthanol precipitation of the aqueous phase at −20 ◦C overnight. TheNA pellet was dissolved in 200 �L TE buffer. Real-time PCR testing
evealed that the content of chlamydial DNA was higher than 99%.
enome sequencing and assembly
For 454 shotgun pyrosequencing, 50 �L (5 �g) of the genomicNA from strain 10DC88 was further purified using the GenomicNA Clean & Concentrator Kit (Zymo Research, Freiburg, Germany)nd eluted with 20 �L TE buffer. The concentration of genomicNA was determined using a NanoDrop ND-1000 (Peqlab, Erlan-en, Germany). Two different libraries were constructed. First, a
Please cite this article in press as: K. Sachse, et al., Evidence for the existenChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Micr
extera library was generated from 50 ng of purified genomic DNAsing the Roche Titanium-compatible Nextera DNA Sample Prepit (Epicentre Biotechnologies, Madison, WI). In parallel, a Rocheapid Library was generated from 500 ng of purified genomic DNA
TCTTCYTCAGCRAGGCCATCT
using the GS Titanium Rapid Library Prep Kit (Roche Diagnostics,Mannheim, Germany). Each library was sequenced in one lane ofa 4-lane picotitre plate on a Roche/454 FLX pyrosequencer withTitanium chemistry. The Nextera library yielded 221,735 quality-filtered reads and the Roche Rapid library contained 221,163quality-filtered reads.
The complete genome and plasmid sequences were assembledusing a combination of GS De Novo Assembler v. 2.6, SeqManNGen v. 4.0.1 and SeqMan Pro v. 9.1.0. The median coverage of the1,041,169-bp genome of strain 10DC88 was 33-fold and the mediancoverage of the 7099 bp plasmid was 267-fold.
The genome sequences of strains 10DC88 and 08-1274/3 havebeen deposited in the NCBI database under GenBank accessionnumbers CP006571 and AWUS00000000, respectively.
Annotation of non-coding (nc) RNA genes
tRNA search was conducted using the local version of tRNAscan-SE v.1.23 with parameters -omlfrF [23]. rRNAs were identified withRNammer v. 2.1 and options S bac -m lsu,ssu,tsu -gff [19]. For otherncRNAs, known homologous sequences were downloaded fromRFAM v.10.1, [7] and homology searches were performed usingBLAST v.2.2.25 with an E-value below 10−4 and Infernal v.1.1rc1[26] using the co-variance models of the RFAM database. To enablecomparison with other Chlamydiaceae species, ncRNA analysis wasperformed on strains 10DC88, 08-1274/3 and all other chlamydialgenomes available at the NCBI website on December 2012.
Phylogenetic analysis based on rRNA genes
ce of two new members of the family Chlamydiaceae and proposal ofobiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
In comparative analysis of chlamydial genomes, phylogenetictrees were based on multiple sequence alignments of the annotated16S rRNA (I), 23S rRNA (II), and all those ncRNAs (III) identified inthe genomes using the highest-scoring (Infernal bitscore) copy per
cRNA family. For I, II, and III, the multiple alignments were cre-ted using the program L-INS-I of the MAFFT package [16], with000 iterations as a module in the EPoS framework for phyloge-etic analysis [13]. Based on these alignments, a neighbour-joiningree (Kimura correction model, 1000 bootstrap replicates) was con-tructed using Waddlia chondrophila as an outgroup. Additionally,he sequence identity of the best scored 16S (I) and 23S rRNA (II)opies among all analysed Chlamydiaceae species were pairwiseomputed. Insertions, deletions and mutations were also counted,nd they were normalised based on lengths.
For 16S rRNA analysis of field isolates, the newequences were added to the latest update of the LTP [40]www.arb-silva.de/projects/living-tree). Sequences were alignedsing the SINA alignment software implemented in the ARB soft-are package [24]. Tree reconstructions using the 16S rRNA gene
equences were performed using either the maximum likelihoodlgorithm RAxML version 7.0 with the GTRGAMMA model [37] orhe neighbour-joining algorithm using the Jukes–Cantor corrections implemented in the ARB package [24] and all homologous pos-tions common to all members of the domain Bacteria. Bootstrapnalyses were performed by analysing 100 replicates.
ree reconstructions using the MLSA concatenate and the ompAenes
For the MLSA concatenate and the ompA genes, alignments wereenerated using ClustalX [20]. In order to remove hypervariableositions and indels, the alignments were sieved using the programblocks [2]. The resulting filtered alignments were then used for
ree reconstructions with different algorithms (neighbour-joiningnd maximum likelihood), as implemented in the ARB programackage [24], or RaxMl [37].
airwise comparison of genome sequences
The parameters of the tetranucleotide signature frequencyorrelation coefficient (tetranucleotide regression) and aver-ge nucleotide identity (ANIb) were determined as describedreviously [32] using the program JSpecies (www.imedea.uib.s/jspecies) with the default parameters.
esults
istory of cases involving non-classified strains of Chlamydiaceaepecies
In the last few years, diagnostic investigations of chlamydialnfection in birds in Germany, France and Italy have produced
number of unclear findings, whereby the chlamydial agentppeared to be different from the established species. In 2005,xamination of clinical samples from an outbreak in a mixed poul-ry farm in Germany led to the detection of Chlamydiaceae-positiveet C. psittaci-negative avian strains [6]. The chickens carrying thosetrains showed no signs of disease. Three years later, cases of atyp-cal pneumonia in workers of a poultry slaughterhouse in Francerompted an epidemiological investigation in the breeder flocks.urprisingly, C. psittaci was present only in one of the 25 flocksxamined, whereas non-classified strains of Chlamydiaceae genet-cally related to those previously mentioned were isolated fromeven different flocks, including strain 08-1274/3 [22]. Meanwhile,urther studies have revealed the occurrence of the agent in Europe,
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hina [41] and Australia [33].In addition, a retrospective survey of urban pigeons in Italy in
006 revealed the presence of genetically similar strains. In 2009, closely related yet uncultured strain was identified in northern
PRESS Microbiology xxx (2014) xxx– xxx
Germany in a flock of psittacine birds suffering from clinical psitta-cosis with fatal cases. A similarly severe outbreak in a psittacinebreeder flock occurred in western Germany a year later, whichled to the isolation of strain 10DC88. Notably, no other potentialpathogen was found in either of the psittacine flocks. Elsewhere,pigeons proved to be a major host of this agent when geneti-cally related strains were detected in urban pigeons in France [8]and Germany [34]. In addition, the OIE Reference Laboratory forChlamydiosis identified strains that can now be assigned to thenew Chlamydia spp. from cases of disease with unclear aetiology inpoultry (n = 5) and psittacine birds (n = 5) in the last five years.
Phylogenetic analysis of 16S ribosomal RNA gene sequences offield isolates
To determine the genealogical position of 11 hitherto non-classified chlamydial strains, their almost complete 16S rRNA geneswere amplified and sequenced. Phylogenetic reconstruction of thenew sequences relative to all classified chlamydial species revealedthat all new isolates were affiliated to an independent branchwithin the genus (Fig. 1). The sequences branched into two dif-ferent clades: one corresponding to the poultry isolates, includingstrain 08-1274/3T, and another comprising the pigeon isolates andpsittacine strain 10DC88T. All 16S rRNA sequences within a cladewere more than 99% identical, whereas sequences in one clade weremaximally 97.3% identical to their counterparts in the other clade(Table S1). Sequence identity of the new isolates to the closest chla-mydial clade, which included C. abortus, C. caviae, C. felis and C.psittaci, ranged from 96.4% to 98.1%, whereas sequence identitywithin members of this clade of established species ranged from97.8% to 99.6%.
Sequence identity between the new isolates and the remainingspecies of the genus was well below 96%. The tree topology shownin Fig. 1 corresponds to a maximum likelihood reconstruction, butreconstructions based on neighbour-joining algorithms showedidentical topology (data not shown).
The same clustering pattern has been reproduced by aligningsequences of the ompA gene, which encodes the major outer mem-brane protein, but sequence variation was expectedly greater andthe resulting dendrogram was more branched (Fig. S1).
Based on these findings and the data shown below, we proposethe introduction of two new species, C. avium sp. nov. representingthe pigeon/psittacine clade and C. gallinacea sp. nov. comprising thepoultry clade.
Genome analysis of the designated type strains and comparisonwith other Chlamydiaceae species
The genome size of C. avium type strain 10DC88T was deter-mined to be 1,041,169 bp, while that of the C. gallinacea type strain08-1274/3T was 1,045,134 bp. For comparison with other chlamyd-ial genomes, a number of selected parameters have been calculatedand they are shown in Table 3. Thus, the genomes of both strainswere smaller than those of C. pneumoniae, C. psittaci, C. felis, C. caviaeand C. abortus, but were comparable in size to C. trachomatis andC. muridarum. The G + C contents of 36.9 and 37.9 mol% were thelowest among Chlamydiaceae species, compared to the minimumamong the other species of 41.3 mol%. Regarding non-coding RNAs,their G + C content was above the genome average, even though thedifference was relatively small in the case of strain 08-1274/3T.
In both genomes, a single rRNA operon was identified encod-ing 16S, 23S and 5S rRNAs, which seems to be characteristic for
ce of two new members of the family Chlamydiaceae and proposal ofobiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
members of the former genus Chlamydophila. Analysis involvingfull-length 16S and 23S rRNA genes from genomes available atGenBank using Bayesian and neighbour-joining algorithms con-sistently showed C. avium strain 10DC88T being placed within
Please cite this article in press as: K. Sachse, et al., Evidence for the existence of two new members of the family Chlamydiaceae and proposal ofChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Microbiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
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K. Sachse et al. / Systematic and Applied Microbiology xxx (2014) xxx– xxx 5
Fig. 1. Phylogenetic reconstruction based on almost complete 16S rRNA genes from type strains of established Chlamydiceae species and field isolates of the new speciesC. avium and C. gallinacea. Construction of the tree was based on the RAxML algorithm without any filter removing hypervariable positions. Bootstrap values indicate thestability of the branches based on 100 replicates. The bar indicates 1% sequence divergence. For numerical sequence identity values see Table S1.
Table 3Comparison of basic genomic features of C. avium and C. gallinacea with those of established species of Chlamydiaceae.
a Using the novel BacProt software. BacProt predicts protein-coding genes using a lineage-specific model generated on-the-fly. This allows more accurate predictions thanthrough generic approaches. The tool is suitable for newly sequenced bacterial genomes, even if the phylogenetic classification is not known. Putative gene functions aredetermined according to homologs from related species. Reference: Lechner M, Findeiss S, Stadler PF, Marz M. 2013. BacProt: Genome-wide annotation of proteins in bacteria(in preparation).
b According to NCBI (incl. pseudogenes).c Probably includes a temporary local copy.
Table 4Pairwise sequence identity values for 16S (upper right) and 23S (lower left) ribosomal RNA gene loci from genome sequences of Chlamydiaceae species type strains, includingC. avium and C. gallinacea.
C. avium C. gallinacea C. psittaci C. abortus C. caviae C. felis C. pecorum C. pneumoniae C. trachomatis C. suis C. muridarum
he family Chlamydiaceae, but forming a separate branch in thelade comprised of the species C. psittaci, C. abortus, C. felis and C.aviae (Fig. S2). The results of this analysis have been condensedn Table 4, which gives the numerical values of pairwise sequencedentity among Chlamydiaceae species for the 16S and 23S rRNAene loci. These figures confirmed the close relatedness betweentrains 10DC88T and 08-1274/3T (98.24/98.67% identity), as well aso C. psittaci, C. abortus, C. caviae, and C. felis, with sequence identityalues above 95.8% for both loci. The other five chlamydial specieshowed lower identity to the new taxa, but were still clearly abovehe 90% level recommended for genus assignment.
Additional comparative analysis of RNomes (Fig. S3) androteomes (data not shown) derived from currently available chla-ydial genome sequences led to phylogenetic reconstructions in
ull agreement with the present rRNA analysis.With 39 tRNAs identified, the proposed C. avium and C. gal-
inacea type strains possessed one more methionine tRNA thanther Chlamydiaceae species (Table 3). These tRNAs are processedy ribonuclease P (RNaseP), of which one copy was found in eachhlamydial genome. The other known house-keeping ncRNAs werelso found in one copy per genome (i.e. transfer-messenger RNAtmRNA)), which has properties of tRNA as well as mRNA and recy-les stalled ribosomes, and the signal recognition particle (SRP)NA, which belongs to the SRP ribonucleotide complex and con-ributes to signal peptide binding and release.
Notably, a 6S RNA candidate in C. avium was identified, whicheems to be closely related to the 6S RNA of flavobacteria. Theutative sequence was extremely short (118 nt) and conservedmong several Chlamydiaceae species, although literature reportsf 6S RNA in other chlamydiae are absent. To date, we have notet been able to identify the respective transcript, but it will bemportant to conduct further investigations because 6S RNA inter-cts with the RNA polymerase holoenzyme during the stationaryhase to repress transcription of genomic DNA. The 7099-bp plas-id of strain 10DC88T has been designated pDC88. It contains seven
redicted open reading frames, but no non-coding RNA. Strain8-1274/3T was found to harbour a plasmid of 7096 bp with fiverovisionally identified genes, and it was designated p1274.
NI correspondence among the genomes of strains 10DC88T,8-1274/3T and other Chlamydiaceae species
Pairwise comparisons of the newly sequenced genomes withhose of the type strains of the closest relatives have beenonducted using the JSpecies package by determining ANInd tetranucleotide regressions. ANI parameters closely mirrorNA–DNA hybridisation values, and have, therefore, been sug-ested as a substitute for the latter [32].
The current recommendation for the genomic circumscriptionf species by means of pairwise comparisons consists of tetranu-leotide regression values above 0.999 and ANI above 94–96% [32].n the present case, although the fully assembled and annotatedenome sequence of 08-1274/3T is not available yet, the draftenome sequence data could be used adequately for the cal-ulations. Direct comparison of 08-1274/3T and 10DC88T led toalues of 0.9866 (tetranucleotide regression) and 80.96% (ANI),hus confirming that both monophyletic clades may constituteifferent genomospecies. The results in Table 5 show maximalalues of 0.9277 (tetranucleotide regression) and 74.04% (ANI)etween 10DC88T and the type strains of C. felis (Fe/C-56T) and. caviae (GPICT), respectively. Similarly, the highest values of the8-1274/3T genome against the closest relative type strain were
Please cite this article in press as: K. Sachse, et al., Evidence for the existenChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Micr
t 0.9466 tetranucleotide regression vs. C. felis (Fe/C-56T), and at3.63% ANI vs. C. caviae GPICT. The results confirmed that 10DC88T
nd 08-1274/3T would represent two new genomospecies withinhe genus. It is worth noting here that all members of the same
PRESS Microbiology xxx (2014) xxx– xxx
clade (i.e. C. psittaci, C. felis, C. caviae and C. abortus, as well as C. tra-chomatis and C. muridarum) shared values above 80% for ANI and0.898 for tetranucleotide regression.
Multi-locus sequence analysis (MLSA) of field isolates
To test the clonal diversity of the isolates, we adapted theMLST approach that has already been used for established speciesof the Chlamydiaceae [29,30] (http://pubmlst.org/chlamydiales/).This system based on seven essential genes (enoA, fumC, gatA,gidA, hemN, hflX and oppA) has so far identified 46 differentsequence types (or allelic profiles) among the accepted Chlamy-diaceae species.
However, we have not yet been able to identify all homologs ofthe seven genes in the field strains of this study. In the strains ofC. avium, the oppA gene has not been localised, and in C. gallinaceastrains only the enoA, gatA, gidA and hflX genes could be amplified,so the present MLSA was restricted to these four loci.
Phylogenetic analysis of concatenated sequences resulted in atree reconstruction (Fig. 2) with a topology almost identical to thatof the 16S rRNA tree in Fig. 1, with the isolates examined formingtwo distinct monophyletic clusters of pigeon and poultry strains,respectively. The results in Table 1 indicated that the diversityof sequence types (STs) was limited, particularly among C. aviumstrains. The highest degree of variation was seen at the gidA (5 ST)and hflX (5 ST) loci. For enoA, two sequence types were identified inaccordance with the animal host. Based on the analysis conductedon the four housekeeping genes, five different allelic profiles des-ignated “a” to “e” were identified among the 11 isolates of bothchlamydial species. In contrast to rRNA data, the pigeon isolatesof C. avium appeared to be more homogeneous than the poultrystrains of C. gallinacea because they could all be grouped in thesame ST a, and only the psittacine strain 10DC88T formed a distincttype b.
Phenotypic characterisation of the field isolates
All isolates of the new taxa could be propagated under condi-tions routinely used for culture of chlamydiae (i.e. in cell culture),such as in BGM cells or embryonated chicken eggs. Confocal laserscanning microscopic images of cultured C. gallinacea type strain08-1274/3T had been shown to display intracellular inclusionsreminiscent of other chlamydial agents [22]. The electron micro-graphs of both designated type strains in Fig. 3A and B shows singleand multiple inclusions of variable size (2–10 �m in diameter) ininfected cells. Small inclusions contained predominantly reticulatebodies (RBs), while a mix of RBs and elementary bodies (EBs) wasseen in the larger inclusions. EBs were small (240–330 nm in diam-eter), round and electron dense. RBs appeared larger (450–1100 nmin diameter), round to oval and more electron lucent. Differentstages of division by binary fission were seen in RBs. In addi-tion, budding of up to eight daughter cells from a single RB wasobserved. Occasionally, intermediate developmental forms withelectron-dense nuclei were found. Mitochondria and stacks of Golgimembranes were located close to the inclusion membrane in amultifocal distribution. These observations indicated a biphasicdevelopmental cycle as seen in all chlamydiae. The observed lengthof such a cycle was at approximately 60–72 h (vs. 36–48 h for typical
ce of two new members of the family Chlamydiaceae and proposal ofobiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
strains of other Chlamydia spp.). In immunofluorescence testing, allstrains showed a positive reaction with a monoclonal antibody toChlamydiaceae LPS that is part of the IMAGEN Chlamydia Kit (Oxoid,Wesel, Germany).
Please cite this article in press as: K. Sachse, et al., Evidence for the existence of two new members of the family Chlamydiaceae and proposal ofChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Microbiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
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Fig. 2. Phylogenetic reconstruction based on the alignment of concatenated sequences included in MLST analysis of C. avium and C. gallinacea isolates. The results are inferredfrom concatenated enoA, gidA, gatA and hflX genes. The tree has been reconstructed using the RAxML algorithm, and the numbers on nodes indicate the bootstrap supportof each branch after 100 replicates. The scale bar shows the percentage sequence similarities.
Fig. 3. Electron microscopic images of BGM cell culture infected with C. avium strain 10DC88T (A) and C. gallinacea strain 08-1274/3T (B). Bar = 1.5 �m. (A) Two inclusionsare depicted (1 and 2, contoured with hatched lines). Inclusion 1 contains a mixture of RBs (R), EBs (thin arrows) and a few intermediate bodies (I). Inclusion 2 consistspredominantly of RBs. Binary fission and budding events are marked with a thick arrow and arrowheads, respectively. Mitochondria (M) and stacks of Golgi membranes (G)are closely associated with the inclusion membrane. (B) The large inclusion contains predominantly RBs (R), many intermediate forms (I) and a few EBs (thin arrows).
8 K. Sachse et al. / Systematic and Applied Microbiology xxx (2014) xxx– xxx
Table 5Pairwise comparison of the available genome sequences of the type strains of Chlamydiaceae species based on average nucleotide identity (ANI; lower triangle) andtetranucleotide signature correlation index (upper triangle).
Rapid detection methods are available for both species. Thessay specific for C. gallinacea strains is based on the 16S rRNA geneocus [41], while strains of C. avium can be specifically detectedsing a target in the enolase A (enoA) gene [42]. All field strains
ncluded in this study have been examined using these two real-ime PCR protocols. The results confirmed the assignment to theespective species in accordance with the source of isolation (seeable 1). Examination of dilution series of cell culture aliquots con-aining defined quantities of inclusion-forming units (ifu) revealedetection limits in the order of 10 ifu for C. avium and 1 ifu for C.allinacea. It is possible to conduct both assays in a single real-timeCR run using a duplex mode.
To investigate the new agents’ capability to elicit a humoralmmune response, rabbit hyperimmune sera to C. gallinacea strain8-1274/3 and C. avium strain 10-743 SC13 were tested by West-rn immunoblotting. The results indicated that specific antibodiesere present in the sera, which preferably reacted with antigens
f the homologous species, but also showed cross-reactions withntigens of the other species (data not shown).
iscussion
vidence for the existence of two additional members of the genushlamydia
Analysis of 11 field isolates has shown that the criteria forssignment to two new species within the genus Chlamydia areulfilled (Table 4 and Fig. 1). First of all, 16S rRNA gene sequencedentity to members of the family Chlamydiaceae was higher than0% for all new strains, thus justifying assignment to this family.he criterion that 16S or 23S rRNA sequences of new isolates shoulde ≥95% identical to the type strain [4] in order to be included inhe same genus has been repeatedly challenged, and more flexibil-ty in this relatively heterogeneous family has been recommended11,38]. Analysis of ribosomal sequences extracted from whole-enome sequences confirmed that the two new species were eachther’s closest relatives, followed by C. psittaci, with ribosomalequence identities higher than 98% (Table 4). In contrast, the per-entages varied only from 99.68% (99.49% in 23S) to 98.31% (98.09%n 23S) among C. psittaci, C. abortus, C. felis and C. caviae, all of whichad been grouped under C. psittaci sensu lato before the revision in999, whereas C. pneumoniae and C. suis were more distant fromach other with 93% identity for 16S and 23S rDNA.
The field isolates clustered with their respective type strainsnd separately from the established Chlamydia spp. in all analyses
Please cite this article in press as: K. Sachse, et al., Evidence for the existenChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Micr
i.e. 16S rRNA (Fig. 1 and Table S1), ompA sequences (Fig. S1) andLSA (Fig. 2)). Calculated genomic parameters of the designated
ype strains also supported their classification into separate speciesTable 3).
Although the number of strains characterised to date is limited,an observed trend is that genetic diversity among C. gallinaceastrains may be higher than among C. avium strains, as three dif-ferent MLSA types were identified in the former and only two inthe latter. The fact that ompA sequences were distinct among allstrains (except 08-1274/3T and 08-1274/13, which were isolatedfrom different flocks of the same farm) indicates clonal diversity.
In conclusion, since genomic comparisons between 10DC88T
and 08-1274/3T on the one hand and the type strains of theestablished Chlamydia spp. on the other hand led to ANI andtetranucleotide regression values below the thresholds circum-scribing species [32], and since the minimal standards for definitionof chlamydial species [12] were fulfilled, we propose that thenew isolates are members of two new species to be classified asC. avium sp. nov. with type strain 10DC88T (DSMZ: DSM27005T;CSUR: P3508T) and C. gallinacea sp. nov. with type strain 08-1274/3T
(DSMZ: DSM27451T; CSUR: P3509T), respectively.
Description of C. avium sp. nov.
C. avium (a’vi.um. L. fem. gen. pl. avium of the birds, becausebirds are the only known hosts of the strains identified to date).
C. avium strains occur in pigeons and psittacine birds. The pres-ence of the agent in other avian species seems possible, but hasyet to be investigated. C. avium can be recovered from cloacalswabs, faeces or, in clinical cases, internal organs of their hosts.The natural route of transmission seems to involve dust particlesor aerosolised dry faeces. While the majority of infected carri-ers remain asymptomatic, there are indications of a facultativepathogenic role, possibly also in concert with C. psittaci. The poten-tial for zoonotic infection of humans is unknown.
C. avium strains can be grown in cell culture, where they dis-play the typical chlamydial morphology, as well as in embryonatedchicken eggs. Shape, size and distribution of inclusions resemblethose seen in C. psittaci-infected cells. EBs and RBs are comparablein size and morphology to those of C. psittaci and C. trachomatis.While the presence of EBs and RBs indicates a biphasic develop-mental cycle, the different composition of inclusions with onlyRBs or a mixture of RBs and EBs within the same cell may reflectrepeated or asynchronous infections of the same cell. As reportedfor other chlamydiae, mitochondria and stacks of Golgi membraneswere closely associated with the inclusions [14,25].
C. avium 16S rRNA genes that have been sequenced to daterevealed sequence diversity of less than 1% (Table S1). Identifica-
ce of two new members of the family Chlamydiaceae and proposal ofobiol. (2014), http://dx.doi.org/10.1016/j.syapm.2013.12.004
tion of members of the species can be obtained using real-time PCR,as described in Materials and methods, and can also be based on16S rDNA and ompA sequences. The type strain is 10DC88T (DSMZ:DSM27005T; CSUR: P3508T).
C. gallinacea (gal.li.na’ce.a. L. fem. adj. gallinacea, pertaining to aomestic fowl, because all the strains identified to date have beenrom domestic poultry).
C. gallinacea strains occur in chicken, guinea fowl, turkey andossibly other domestic poultry. The agent can be recovered fromloacal swabs, faeces and internal organs of the hosts. Transmissionccurs through dust and faeces. So far, no evidence of a pathogenicotential has emerged, but it cannot be ruled out that C. gallinaceaontributes to clinical symptoms in birds, for instance in mixednfection with C. psittaci. Furthermore, the potential for zoonoticnfection has been suggested [21].
Like all other Chlamydiaceae species, C. gallinacea strains can berown in cell culture and embryonated chicken eggs. The cellu-ar morphology possesses the typical features of other chlamydialpecies.
Sequence diversity of the 16S rRNA genes currently charac-erised is less than 1% (Table S1). Identification of members of thepecies can be conducted using real-time PCR, as described in Mate-ials and methods section, and can also be based on 16S rRNA andmpA sequences.
The type strain is 08-1274/3T (DSMZ: DSM27451T; CSUR:3509T).
ossible epidemiological importance of the new agents
From the data available to date, it seems likely that C. avium and. gallinacea are widely disseminated, particularly among pigeonsnd poultry, respectively. Systematic studies focusing on these newgents have not been conducted yet, and the accuracy of futurefforts will largely depend on the use of the right diagnostic tools.reliminary data from breeder pigeon flocks in Germany indicatehat C. avium was detected in 4 of 27 (14.8%) flocks [17]. In a recenttudy in urban pigeons in France, 10 of 125 (8%) Chlamydiaceae-ositive samples proved positive for C. avium [8]. Another survey inrban pigeons in Germany revealed that 19.5% of all Chlamydiaceae-ositive cases involved non-classified chlamydial organisms, buthe proportion of C. avium in that panel was not determined [34].he prevalence in psittacine birds cannot be assessed as only a fewndividual cases have been examined.
In the case of C. gallinacea, a recent study showed that it occurredn poultry flocks of four European countries and China, where itsrevalence even surpassed that of C. psittaci [41].
While both agents have been predominantly encountered insymptomatic chickens and pigeons, we recently found indicationsf a pathogenic role of C. avium, as it was identified in several out-reaks of respiratory disease in psittacine birds in Germany, whereeither other chlamydiae nor relevant bacterial or viral pathogensere detected. It is also possible that C. avium strains, such as
1DC97, contributed to respiratory disease and/or diarrhoea inreeder flocks of pigeons. Moreover, we also reported unexplainedases of atypical pneumonia in slaughterhouse workers that werexposed to chickens carrying C. gallinacea [22], which raises theossibility of a zoonotic potential. Further investigations will helpo clarify the importance of C. avium and C. gallinacea as emergingathogens.
cknowledgements
The authors thank Jochen Kilwinski (Arnsberg), Maria-Elisabeth
Please cite this article in press as: K. Sachse, et al., Evidence for the existenChlamydia avium sp. nov. and Chlamydia gallinacea sp. nov. Syst. Appl. Micr
rautwald-Junghanns (Leipzig), Tiziana Rampin (Milan), Mariaenzi (Bologna) for providing samples that led to the isolation oftrains included in the present study. Excellent technical assistancey Sabine Scharf, Christine Grajetzki, Simone Bettermann, Sergio
[
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PRESS Microbiology xxx (2014) xxx– xxx 9
Vigo, Iris Labalestra, and Andrea Manfredini is gratefully acknowl-edged. This work is dedicated to the memory of the late SergioVigo, who contributed with exceptional skill to isolating the Italianstrains.
Part of this study was financially supported by the Federal Min-istry of Higher Education and Research (BMBF) of Germany underGrant no. 01 KI 0720 “Zoonotic chlamydiae – Models of chronic andpersistent infections in humans and animals” and grant number01KI0710 “Emerging arthropod-borne viral infections in Germany:Pathogenesis, diagnostics and surveillance”.
Sequence data acquisition and analysis were funded in partby federal funds from the National Institute of Allergy andInfectious Diseases, National Institutes of Health, US Depart-ment of Health and Human Services under contract numberHHSN272200900007C.
RRM acknowledges the funding by the Spanish Plan NacionalCGL 2012-39627-C03-03 and Consolider Ingenio CE-CSD2007-0005 projects, both co-financed with FEDER support from theEuropean Union. The funders had no role in study design, datacollection and analysis, decision to publish, or preparation of themanuscript.
Appendix A. Supplementary data
Supplementary data associated with this article can befound, in the online version, at http://dx.doi.org/10.1016/j.syapm.2013.12.004.
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