Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis

Journal Pre-proof Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis K. Laroucau, N. Ortega, F. Vorimore, R. Aaziz, A. Mitura, M. Szymanska-Czerwinska, M. Cicerol, J. Salinas, K. Sachse, M.R. Caro

PII:

S0723-2020(20)30019-9

DOI:

https://doi.org/10.1016/j.syapm.2020.126071

Reference:

SYAPM 126071

To appear in:

Systematic and Applied Microbiology

Received Date:

16 September 2019

Revised Date:

30 January 2020

Accepted Date:

4 February 2020

Please cite this article as: Laroucau K, Ortega N, Vorimore F, Aaziz R, Mitura A, Szymanska-Czerwinska M, Cicerol M, Salinas J, Sachse K, Caro MR, Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis, Systematic and Applied Microbiology (2020), doi: https://doi.org/10.1016/j.syapm.2020.126071

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Systematic and Applied Microbiology Detection of a novel Chlamydia species in captive spur-thighed tortoises (Testudo graeca) in southeastern Spain and proposal of Candidatus Chlamydia testudinis. Laroucau K1*#, Ortega N2#, Vorimore F1, Aaziz R1, Mitura A3, Szymanska-Czerwinska M3, Cicerol M2, Salinas J2, Sachse K4, Caro MR2.

1 University

Paris-Est, ANSES, Animal Health Laboratory, Bacterial Zoonoses Unit. Maisons-Alfort,

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France

Departamento de Sanidad Animal, Facultad de Veterinaria, Campus Regional de Excelencia

internacional, Campus Mare Nostrum, Universidad de Murcia, Spain

Department of Cattle and Sheep Diseases/Laboratory of Serological Diagnosis, National

Veterinary Research Institute, Pulawy, Poland

Friedrich-Schiller-Universität Jena, Institut f. Bioinformatik, 07743 Jena, Germany

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* Corresponding author. ANSES, Animal Health Laboratory, Bacterial Zoonoses Unit. 14 rue Pierre et Marie Curie, Maisons-Alfort, 94700, France. Phone: (33) 1 49 77 13 00, Fax: (33) 1 49 77 13 44, E-mail address: [email protected]

equal contribution

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Abstract

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#

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The spur-thighed tortoise (Testudo graeca) is an endangered Mediterranean tortoise that lives in North Africa, Southern Europe and Southwest Asia. In the wake of recent legislation making their keeping as domestic animals illegal, many of these animals have been returned to wildlife recovery centers in Spain. In the present study, a population of such tortoises showing signs of ocular disease and nasal discharge was examined for the presence of Chlamydia spp. Cloacal, conjunctival and/or choanal swabs were collected from 58 animals. Using a real-time PCR specific

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for the family Chlamydiaceae, 57/58 animals tested positive in at least one sample. While only a few samples proved positive for C. pecorum, sequencing of the 16S rRNA gene revealed a sequence identical to previously published sequences from specimens of German and Polish tortoises. Whole-genome sequences obtained from two conjunctival swab samples, as well as ANIb, TETRA values and a scheme based on 9 taxonomic marker genes revealed that the strain present in the Spanish tortoises represented a new yet non-classified species, with C. pecorum being its closest relative. We propose to designate the new species Candidatus Chlamydia

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testudinis. Keywords: Testudo graeca, Chlamydiaceae, WGS, real-time PCR.

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Introduction

The spur-thighed tortoise (Testudo graeca) is one of the five species of Mediterranean tortoises

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belonging to the family Testudinidae and the genus Testudo. Native from North Africa [13], this species is perfectly adapted to the Iberian ecosystems of the Southeast Mediterranean [11]. In the

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last decades, wild animals were taken away from their natural habitat and kept as pets in captivity [28]. Listed in the Appendix II of Washington Convention (Convention on International Trade in

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Endangered Species of Wild Fauna and Flora [7]), this species is now categorized as an endangered and vulnerable species, and a recently adopted law in Spain prohibiting private

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ownership of these animals has led to the return of many turtles to wildlife recovery centers.

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Like many other animals, turtles can be carriers of pathogens and, therefore, pose a potential risk to human health [3, 8]. Based on earlier reports from the literature, the obligate intracellular bacteria of the family Chlamydiaceae have to be considered as possible infecting agents. This family currently consists of the single genus Chlamydia with 14 characterized species: C. abortus, C. avium, C. buteonis, C. caviae, C. felis, C. gallinacea, C. muridarum, C. pecorum, C. pneumoniae, C. poikilothermis, C. psittaci, C. serpentis, C. suis, and C. trachomatis [5, 22, 31]. In recent years,

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new candidate species were proposed based on molecular detection and identification methods, i.e. Candidatus C. ibidis isolated from ibis [39], as well as new taxa from snakes [37, 38]. Chlamydial infections show a variety of manifestations, ranging from inapparent to clinical disease, such as respiratory disorders, gastroenteritis, encephalomyelitis, conjunctivitis, arthritis, and abortion. C. pneumoniae and C. trachomatis are mainly associated with infections in humans, while other chlamydiae infect animals, with potential zoonotic implication [24]. Strains of Chlamydia spp. have been found in many reptiles with and without clinical signs, including

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turtles, iguanas, crocodiles, and pythons [1, 15, 16, 17, 26, 35, 36]. The identification of chlamydial organisms related to C. pneumoniae or C. caviae in snakes led to the recent proposal of new taxa:

C. poikilothermis, C. serpentis as well as Candidati C. corallus and C. sanzinia [35, 37, 38]. In

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tortoises, non-classified chlamydial strains closely related to C. pecorum were reported in the literature [16, 26].

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The aim of this study was to screen for Chlamydia spp. in captive tortoises with ocular pathologies and nasal discharge and to characterize the detected strain(s) in order to extend our knowledge on

 Samples

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Material and methods

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organisms of the family Chlamydiaceae.

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Samples were collected from 58 spur-thighed tortoises, mostly with ocular pathology and nasal

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discharge. All were adults and kept in captivity for years. Their owners had voluntarily handed them in to the wildlife recovery center located in Murcia (South-East of Spain). A total of 58 cloacal swabs, 52 conjunctival swabs (all from animals with ocular pathology such as blepharitis, conjunctival congestion or keratitis), and six choanal swabs (from animals with no apparent clinical signs) were collected and stored in 250 µL of 6M guanidine thiocyanate in order to improve the performance and efficacy of the PCR methods [20].

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 Direct detection of Chlamydiaceae in samples For DNA extraction, the NucleoSpin Tissue kit (Macherel-Nagel) was used. The DNA was eluted with 100 µL of elution buffer (5 mM Tris/HCl, pH 8.5), checked for purity and concentration by UV spectrophotometry (NanoDrop one. Thermo Scientific), and stored at -20 °C before analysis. Each DNA extract was examined using Chlamydiaceae-specific real-time PCR targeting the conserved 23S rRNA gene [12]. Cq values were automatically determined by the software (Via7, Applied).  DNA-based characterization

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Species-specific real-time PCR Chlamydiaceae-positive samples were all re-examined using C. psittaci, C. abortus, and C. pecorum-specific real-time PCR assays [27].

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Amplification for 16S rRNA sequencing and sequence analysis

Partial amplification of the 16S rRNA gene sequence (1400 bp) was performed as previously

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reported using primers 16S1/rp2 [21] and the sequencing was performed at Eurofins Genomics (Germany). Sequences determined in this study were aligned with previously published sequences

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that were relevant for phylogenetic and epidemiological considerations. Sequence data were analyzed using the Bionumerics software package version 7.6 (Applied-Maths, Saint-Martens-

coefficient.

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Latem, Belgium). Cluster analysis was conducted using the categorical parameter and the UPGMA

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 Whole-genome sequencing

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Whole-genome sequencing was performed using an Illumina MiSeq platform (Illumina) according to the manufacturer’s instructions and using the Nextera XT kit (Illumina) for library preparation. The MiSeq run was carried out on the DNA preparation, with paired-end reads of 150 bp using MiSeq V2 reagents. The raw reads were trimmed using trimmomatic [2] and assembled de novo using SPAdes 3.11.1 [4]. Scaffolds from SPAdes were blasted on the nucleotide database of NCBI to remove non-Chlamydial sequences. Reads were mapped by BWA [23] on remaining Scaffolds

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to calculate the coverage, the percentage of chlamydial reads and to correct assembly errors. Corrected scaffolds were ordered by CAR [25] against C. pecorum E58 genome (NC_015408.1). Draft genomes were evaluated using Quast v4.6.3 [14] and annotated using prokka v1.13 [33]. The genome sequences of the chlamydia strains 17-3921_L77 and 17-3921_L98 amplified from two conjunctival swab samples have been submitted to ENA under project PRJEB34668.  Phylogenetic and genome analysis The parameters of the tetranucleotide signature frequency correlation coefficient (tetranucleotide

using the program JSpecies v1.2.1 with default parameters.

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regression) and average nucleotide identity (ANIb) were determined as described previously [30]

16S rRNA, 23S rRNA and nine phylogenetical markers previously described [29] were extracted

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from the assembled genomic sequences of the two tortoise specimens and compared with reference sequences deposited in public databases. Phylogenetic tree for the 16S rRNA and 23S

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rRNA were reconstructed with Geneious v7 (www.geneious.com) using the Neighbor-Joining method from a mafft alignment. The similarity matrix was calculated with Jukes-Cantor model.

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For the taxonomic markers scheme, amino acid sequences were aligned with mafft v7 [19] and concatenated to reconstruct a reference phylogeny using RAxML v8 [34], with the LG+G+I model

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and 100 bootstrap replicates. Pairwise amino acid sequence identities were calculated based on the mafft alignment by Geneious v7.

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 Real-time PCR for detection of the new species

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Specific primers F (5‘-TTT CGG CTT CGT CCA GAT CTC-3’) and R (5’- AGG TTG CTC CAG ATC CTG AC-3’), as well as a specific probe P (5’-FAM- TGG AAT CCC TTT CAA GGT AAA GTC T TAMRA 3’) for the 4-diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE) gene locus were designed using the primer3 software. Real-time PCR amplifications were performed as previously described [22] with modified annealing temperature of 60ºC. The specificity of the new real-time PCR system was evaluated in silico and on 30 Chlamydiaceae strains (Table S1).

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Results Examination of swabs collected from 58 spur-thighed tortoises using a Chlamydiaceae-specific real-time PCR assay demonstrated that all except one harbored chlamydiae. Indeed, chlamydial DNA was detected in 97% (56/58, Cq values ranging from 20.9 to 39.9, median 30.2) of the cloacal samples, in all choanal (6/6, Cq values ranging from 22.3 to 32.2, median 28.4) and 71% (37/52, Cq values ranging from 17.8 to 35.6, median 25.6) of the conjunctival samples. High chlamydial

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loads (Cq values around 17-20) were observed in six specimens. When re-tested with C. psittaci, C. abortus and C. pecorum species-specific real-time PCR systems, all samples proved negative, with a few of them giving signal intensities around the Cq threshold for C. pecorum (data not

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shown).

In order to clarify the species identity of the chlamydial strain(s) present in these tortoise specimens,

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the 16S rRNA gene was partially sequenced (1400 bp) from four of the strongest positives among conjunctival samples (i.e. specimens 17-3921_L77, 17-3921_L98, 17-3921_M03 and 17-

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3921_M06 with Cq values of 18.3, 17.8, 18.9 and 19.3, respectively). All four sequences proved identical and BLASTN search revealed 100% sequence identity with chlamydiae reported from a

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turtle sample in Germany (V1242-01) [16] and two Polish tortoises (clones 16-10083 and 16-10076) [26], all of which closely related to C. pecorum (data not shown).

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As the sample material was not sufficient to attempt cell culture isolation of the chlamydial strain,

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DNA from two of the positive conjunctival samples (i.e. specimens 17-3921_L77 and 17-3921_L98) were submitted to whole-genome sequencing using Illumina technology. The raw reads were analyzed using bioinformatics pipelines that included Blast searches of the NCBI and ENA bacterial genome databases. As a result, a high rate (more than 65%) of Chlamydia-related sequences was identified from these swabs samples (Figure S1 and data not shown).

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Following extraction of Chlamydiaceae-related sequences, 13 and 15 contigs with a total length of 929’685 and 1’002’085 bp, were obtained from samples 17-3921_L77 and 17-3921_L98, respectively (Table S2). A complete plasmid sequence of 7’652 bp was identified in both samples, which shared 76% similarity with the plasmid of C. pecorum (data not shown). The complete 16S and 23S rRNA gene sequences were extracted from the whole-genome sequences. The 16S rRNA sequences and the partial sequences mentioned above shared 100% sequence identity. Sequences were then aligned with corresponding sequences of representative

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strains of all established Chlamydia species (Figure 1). Sequence homology to established Chlamydia spp. was greater than 94% for 16S rRNA and greater than 92% for 23S rRNA genes,

thus confirming the taxonomic position of the chlamydial organism within the genus Chlamydia and

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its close relatedness to C. pecorum (Table S3).

Pairwise comparison of the two genomes from tortoise samples with sequences of the Chlamydia

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spp. strains was conducted to determine the average nucleotide identity (ANIb) and tetranucleotide regression parameters. The results in Table 1 show maximal values of 0.8809/0.8808

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(tetranucleotide regression) and 73.49% (ANIb) between the tortoise strains and type strains of C. psittaci (6BC) and C. pecorum (E58), respectively. Using a recently proposed classification scheme

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for the order Chlamydiales that is based on a set of nine taxonomically relevant proteins [29], the sequence similarity between gene products extracted from the tortoise genome sequences and

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homologs from validly published chlamydial species was calculated. While results presented in

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Figure 2 reveal C. pecorum to be the most closely related species, all values obtained for the tortoise chlamydiae proved below the accepted cut-off for species assignment, i.e. RpoN < 96%, FtsK < 98%, PepF < 96%, Adk < 95%, HemL < 95%, DnA ≥ 70%, SucA ≥ 64%, Hyp325 ≥ 57% and Fabl ≥ 78%. A specific qPCR assay for detection of this new species was designed based on the 4diphosphocytidyl-2-C-methyl-D-erythritol kinase (ispE) gene locus. This gene has the advantage of 7

being present in the genomes of all Chlamydia spp., with specific point mutations identified in the two tortoise chlamydia sequences. All Chlamydiaceae-positive DNA samples from the 58 tortoises tested positive with this new PCR assay, whereas no cross-reactivity against the other Chlamydia species was detected (data not shown). Discussion The tortoises examined in this study showed severe clinical signs of rhinitis and conjunctivitis. Although Chlamydia spp. are generally associated with pathologies in humans and domestic

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animals, they have also been frequently found in wildlife, including reptiles and amphibians. Indeed,

the first report was from an African clawed frog (Xenopus laevis) [6], followed by detection of nonclassified chlamydiae related to C. psittaci and C. pneumoniae in various reptiles, such as snakes,

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iguanas and chameleons [1, 9, 18]. Recent advances in molecular epidemiology allowed strains to

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be genotyped more precisely, so that three new taxa closely related to C. pneumoniae, i.e. Candidati C. corallus and C. sanzinia, as well as C. serpentis, and a new species related to C.

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caviae, i.e. C. poikilothermis, were recently described in snakes [35, 37]. The present analysis of whole-genome sequencing data from two clinical specimens allowed the

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identification of a new chlamydial species based on 16SrRNA, 23SrRNA, ANI and Tetra values and was complemented with a taxonomically relevant protein-based classification scheme [29]. The

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current recommendation for circumscription of species by means of pairwise genomic comparison includes tetranucleotide regression values above 0.999 and ANIb above 94–96% [30]. According

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to the protein-based classification scheme proposed by Pillonel et al. [29], sequence similarity values of ≥ 80% for 16S rRNA and 23S rRNA, or ≥ 92.5% for 16S rRNA, ≥ 91% for 23S rRNA justify assignment of the tortoise strains to the order Chlamydiales and family Chlamydiaceae, respectively. Comparative analysis of conserved proteins among representative strains of each member of the genus Chlamydia yields sequence identities that are consistent with assignment to a new species. Altogether, the results show that the criteria for assignment of the tortoise strains 8

to a new chlamydial species within the family Chlamydiaceae were all fulfilled (Tables 1 and S3, Figure 2). As a consequence, we propose to designate this new taxon Candidatus Chlamydia testudinis. The Candidatus rank should be converted into regular species in the future, once a live cell-cultured strain becomes available. The tortoises examined in this study came from private sources and shared the same enclosure since their arrival at the recovery center. A high rate of chlamydia shedding was detected in this population. It is possible that only a few tortoises were initially infected and that the infection spread

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rapidly to other animals, mainly because of the particular living conditions in such centers (overcrowding, stress etc.). Some of these animals also harbored Escherichia coli, which was detected in 36.2% of cloacal samples from these animals (21/58) (manuscript in preparation). Such

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co-infections in reptiles and amphibians could weaken their immune system [32] and modulate the

expression of virulence factors. The presence of E. coli in these tortoises could favor the

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multiplication and transmission of chlamydiae and explain their high prevalence observed in this study compared to similar studies [10, 16]. The rapid spread of chlamydial infection in favorable

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conditions could also be confirmed by data from a Polish study, where 65.7% of Testudo spp. animals originating from private owners and dwelling together in recovery centers for around 10

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years were found positive, but with lower level of chlamydia shedding (Cq values ranging from 24.4 to 37.9, median 31.6) and no obvious clinical symptoms. Moreover, in a group of 48 separately kept

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young Horsfield’s tortoises in the same recovery center, none of the animals tested positive [26].

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Unfortunately, it was not possible to culture the bacteria, so that only DNA was available for further analysis. The question about the significance of the presence of this DNA in these specimens arises, since there is no additional evidence that these animals were infected by this chlamydial bacterium, nor that the bacteria were alive and able to replicate. Real-time PCR results showed that some animals had very high chlamydial loads, which suggests an active infection at the time of sample collection. Using the new PCR assay designed in this study, Candidatus C. testudinis

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has been confirmed in the tortoise samples belonging to the group 2 of the Polish study (Supplementary data) [26], from which the chlamydial organism was previously reported. In addition, the partial 16S rRNA sequence of this new chlamydia species is identical to one of the samples found positive in a German study [16]. These results suggest that the new chlamydial taxon could be widespread in tortoises showing clinical signs or not and further investigations are needed. Although the gold standard is to isolate strains for further characterization, practical constraints can

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be limiting factors, such as sample availability, tissue sample preservation, bacterial load or ad hoc cell line availability for propagation. These constraints can be partially circumvented by direct

genomic sequencing from tissue samples, as recently published [26, 37, 38]. This approach made

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it possible to process and analyze the best biological sample to identify novel infectious agents and

new host species susceptible to chlamydial infection and, eventually, describe new chlamydial

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organisms.

In summary, reptiles and tortoises are an ecological niche for Chlamydia spp. Phylogenetic analysis

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suggests the existence of a new chlamydial species in this population of Spanish turtles, but also in the specimens from a previous study in Poland [26] and possibly in German samples [16]. This

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new Candidatus species forms a separate clade closely related to C. pecorum, within the genus Chlamydia. This finding is in line with previously published data [26]. It extends our knowledge on

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the genetic diversity within the genus and highlights how much remains to be learned about the

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dissemination, epidemiology and host range of chlamydiae. The description of Candidatus Chlamydia testudinis is given in Table 2.

Acknowledgments We thank the Environment Department of the Autonomous Government of Murcia, especially María José Gens, for making this study possible. We would also like to thank the El Valle Wildlife

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Recovery Center, especially veterinarians Ana Cristina Miñano and Luisa Lara Rosales for their help in collecting samples.

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large phylogenies. Bioinformatics. 30(9):1312-3.

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[35] Staub E, Marti H, Biondi R, Levi A, Donati M, Leonard CA, Ley SD, Pillonel T, Greub G, SethSmith HMB, Borel N. 2018. Novel Chlamydia species isolated from snakes are temperaturesensitive and exhibit decreased susceptibility to azithromycin. Sci Rep. 8(1):5660.

[36] Taylor-Brown A, Rüegg S, Polkinghorne A, Borel N. 2015. Characterisation of Chlamydia pneumonia and other novel chlamydial infections in captive snakes. Vet Microbiol. 178(1-2):8893.

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[37] Taylor-Brown A, Bachmann NL, Borel N, Polkinghorne A. 2016. Culture-independent genomic characterisation of Candidatus Chlamydia sanzinia, a novel uncultivated bacterium infecting snakes. BMC Genomics. 17:710. [38] Taylor-Brown A, Spang L, Borel N, Polkinghorne A. 2017. Culture-independent metagenomics supports discovery of uncultivable bacteria within the genus Chlamydia. Sci Rep. 7(1):10661. [39] Vorimore F, Hsia RC, Huot-Creasy H, Bastian S, Deruyter L, Passet A, Sachse K, Bavoil P, Myers G, Laroucau K. 2013. Isolation of a New Chlamydia species from the Feral Sacred Ibis

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ur

na

lP

re

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ro of

(Threskiornis aethiopicus): Chlamydia ibidis. PLoS One. 8(9):e74823.

15

Tables & Figures Figure 1. Phylogenetic reconstruction based on the complete 16S (A) and 23S (B) rRNA sequences from the samples 17-3921_L077 and 17-3921_L078, and from the type strains of Chlamydiaceae species, including Candidatus C. corallus and C. ibidis representatives. The phylogeny was reconstructed by Neighbor-Joining method from a similarity matrix calculated by pairwise alignment. Bootstrap values are shown as percentages. The scale bar indicates the number of substitution per site. Simkania nevegensis

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lP

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ro of

was used as an outgroup.

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Figure 2. Phylogeny of the genus Chlamydia including the new candidatus species. The phylogeny was reconstructed based on the concatenated alignment of 9 phylogenetically informative protein markers, and includes one representative of each species whenever genomic data is available including Candidatus C. corallus. The phylogeny was reconstructed by Neighbor-Joining method. Bootstrap values are shown as percentages. The scale bar indicates the number of amino acid substitutions per site. Simkania nevegensis

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was used as an outgroup.

17

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Table 1. Pairwise comparison of the available genome sequences of the type strains of Chlamydiaceae species based on average nucleotide identity (ANIb, lower triangle)

73.54

--73.55

0.85 05 0.85 04

0.8268 0.8264 0.9022

--70.42

70.43

70.54

70.90

70.55

70.90

69.3 2

---

69.3 9

69.3 6

C. bute onis RSH A

C. psit taci 6BC

C. poikilot hermis S15834K

C. abo rtus S26 /3

C. feli s Fe/ Pn1

C. aviu m 10D C88

C. C. gallin cav acea iae 08Gp/ 1274/ Ic 3

0.85 81 0.85 79

0.8 766 0.8 765

0.86 29 0.86 25

0.872 0 0.871 6

0.8 633 0.8 631

C. murida rum MoPn/ WiessNigg

C. suis SW A-2

0.7 972 0.7 970

0.89 08 0.89 08

0.8 809 0.8 808

0.829 8

0.83 03

0.7 293

0.85 68

0.8 831

0.8464

0.88 32

0.8 739

0.81 14

0.833 2

0.8 921

0.7761

0.7 761

0.7899

0.945 6

0.91 46

0.7 106

0.86 96

0.8 961

0.8681

0.90 39

0.8 769

0.80 94

0.832 6

0.9 114

0.7969

0.7 855

0.7883

0.93 88

0.7 715

0.86 82

0.8 765

0.8786

0.87 05

0.8 763

0.84 02

0.853 8

0.8 953

0.7902

0.7 792

0.7644

0.8 413

0.92 55

0.9 133

0.9326

0.88 80

0.9 082

0.91 53

0.913 8

0.9 131

0.8322

0.8 120

0.7914

0.8898 0.8898

0.8386 0.8381

0.8 441 0.8 437

C. tracho matis D/UW -3/CX

0.90 07 0.90 03

85.64

79.86

C. ibid is 10139 8/6

e-

C. serp entis H151957 -10C

Pr

--100.0 0

1.000 0

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ANI/Tetra 173921_L77 173921_L98 C. pecorum E58 C. pneumoni ae TW183 Candidat us C. corallus G3-2742324 C. serpentis H151957-10C

173921 _L98

C. pneum oniae TW183

na l

173921 _L77

C. peco rum E58

Candi datus C. corall us G32742324 0.817 2 0.816 7

pr

and tetranucleotide signature correlation index (TETRA, upper triangle).

0.7978 0.7974

--80.60 ---

18

68.8 7

69.76

68.56

69.83

68.5 9 69.9 2

70. 96 70. 72

70.22

68.8 4

69.74

69.64

69.8 7

70.49

70.51

69.1 6

69.97

69.82

70.0 8

69.82

69.83

69.55

69.57

69.41

69.43

68.8 7 68.7 7 68.3 8

69.66 69.56

69.53 69.62

68.2 9

69.04

69.19

68.91

69.04

0.8 865

0.8960

0.85 65

0.8 836

0.93 29

0.925 8

0.8 488

0.8077

0.7 728

0.7698

0.9 873

0.9791

0.96 63

0.9 703

0.93 96

0.947 7

0.9 658

0.8551

0.8 295

0.8315

0.98 60

0.9 665

0.92 35

0.941 3

0.9 698

0.8546

0.8 332

0.8428

0.94 04

0.9 569

0.93 84

0.939 6

0.9 685

0.8186

0.7 912

0.7895

0.9 567

0.89 83

0.923 3

0.9 565

0.8358

0.8 213

0.8347

0.92 76

0.947 5 0.986 4

0.9 510 0.9 007

---

71. 02

81.9 4

0.9641

81. 57

69.6 7

70. 45

92.5 7

92. 58

69.5 8 69.2 9

70. 43 69. 94

81.1 3 73.8 6

80. 84 73. 77

69.1 1

69. 65

73.4 7

73. 42

na l

69.72

--93.5 2

Pr

70.20

69.72

---

0.90 73

f

68.40

oo

70.20

67.8 1

pr

70.20

69.06

e-

69.06

Jo ur

C. ibidis 101398/6 C. buteonis RSHA C. psittaci 6BC C. poikilothe rmis S15834K C. abortus S26/3 C. felis Fe/Pn-1 C. avium 10DC88 C. gallinace a 081274/3 C. caviae Gp/Ic C. muridaru m MoPn/Wi ess-Nigg

--80.94 82.10 74.29

74.05

--79.8 2 73.3 0 73.0 1

--73. 70 73. 32

--81.1 2

0.9 114

0.8433 0.8060

0.8 365 0.7 785

0.8303 0.7658

0.8217

0.8 001

0.7885

0.8441

0.8 198

0.8341

0.9 770

0.9722

---

70.19

70.20

69.0 8

69.88

69.70

69.8 9

70. 69

81.5 5

81. 26

86.18

80.2 6

81. 94

74.0 4

73.33

68.02

68.02

67.1 3

67.57

67.66

67.7 3

68. 24

68.4 3

68. 60

68.81

68.5 8

68. 46

68.3 0

68.50

--68. 72

---

19

67.35

67.46

67.3 3

67. 97

68.2 1

68. 36

67.72

67.72

67.2 6

67.54

67.70

67.6 2

68. 13

68.2 3

68. 37

68.38

f

67.0 6

68.3 8

68. 21

67.8 0

68.21

68. 22

81.53

68.2 9

68. 23

68.0 2

68.40

68. 47

80.24

oo

67.72

68.50

pr

67.72

---

0.9718

79. 15 ---

Jo ur

na l

Pr

e-

C. suis SWA-2 C. trachoma tis D/UW3/CX

20

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Table 2. Description of Candidatus Chlamydia testudinis.

Chlamydia

Candidatus Chlamydia testudinis

Genus etymology

Chlamydia trachomatis

testudinis sp. nov. [Candidatus] tes.tu'di.nis. L. gen. n. testudinis, of the genus Testudo, of the tortoise

-

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Specific epithet Species status Species etymology

Chla.my'di.a. Gr. fem. n. chlamys, chlamydis a cloak; N.L. fem. Chlamydia a cloak

na l

Type species of the genus

e-

single genus

Pr

Genus name Species name Genus status

pr

Table x. Description of Candidatus Chlamydia testudinis

21

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Description of the new taxon and diagnostic traits

Jo ur

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Pr

e-

pr

The agent has been detected from conjunctival and cloacal swabs. From the reported cases, it seems likely that Candidatus C. testudinis can cause conjunctivitis and/or nasal discharge, or at least be a contributing factor to these clinical manifestations. Natural routes of transmission have yet to be investigated, but, analogous to most other chlamydiae, direct contact with exudates or fresh feces as well as airborne transmission through aerosolized dry feces and dust particles seems possible. The potential for zoonotic infection of humans is unknown. Members of the species can be specifically identified using real-time PCR targeting the ispE gene locus as described in Materials and Methods. In addition, 16S and 23S rRNA gene sequence alignments can be used for identification. Sequence homology of Candidatus C. testudinis 16S and 23S rRNA genes to those of other Chlamydia spp. is greater than 94 and 92%, respectively, with C. pecorum being its closest relative. No isolate is available at present. Its genome size is approximately 1 Mbp and a plasmid of 7’652 bp was found in the strains investigated so far.

Country of origin Place of origin Date of detection Source of detection Sampling date Latitude

Spain El Valle Wildlife Recovery Center, Murcia 2018 conjunctival swabs month of April 2018 37°55'44.03'' N

22

f

Longitude Genome accession number Genome status Genome size Designation of the Type Strain

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na l

Pr

e-

pr

oo

1°8'35.18'' W PRJEB34668 complete approximately 1 Mbp and a plasmid of 7’652 bp no type strain has been designated yet

23