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

Abstract

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 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 testudinis.

Introduction

The spur-thighed tortoise (Testudo graeca) is one of the five species of Mediterranean tortoises 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 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 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 ownership of these animals has led to the return of many turtles to wildlife recovery centers.

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

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 organisms of the family Chlamydiaceae.