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The Chlamydiaceae are a family of obligate intracellular bacteria known for their unique biphasic developmental cycle. Chlamydial are associated with various host organisms, including humans, and have been proposed as emerging pathogens. Genomic studies have significantly enhanced our understanding of chlamydial biology, host adaptation, and evolutionary processes. In this study, we conducted a complete pangenome association analysis (pan-GWAS) using 101 genomes from the Chlamydiaceae family to identify differentially represented genes in Chlamydia and Chlamydophila , revealing their distinct evolutionary strategies for interacting with eukaryotic hosts. Our analysis identified 289 genes with differential abundance between the two clades: 129 showed a strong association with Chlamydia and 160 with Chlamydophila . Most genes in Chlamydia were related to the type III secretion system, while Chlamydophila genes corresponded to various functional categories, including translation, replication, transport, and metabolism. These findings suggest that Chlamydia has developed a high dependence on mammalian cells for replication, facilitated by a complex T3SS for intracellular manipulation. In contrast, the metabolic and functional diversity in Chlamydophila allows it to colonize a broad range of hosts, such as birds, reptiles, amphibians, and mammals, making it a less specialized clade.

Southern greater gliders (Petauroides volans) are endangered Australian marsupials for which there is little health data currently available. Chlamydia pecorum is the only reported pathogen of greater gliders and infects a broad range of hosts, including other marsupials, ruminants, swine and birds. Conjunctival and cloacal swabs collected from thirty-two southern greater gliders across southeastern New South Wales, Australia were screened for Chlamydia spp. by 23S real-time PCR. None of the ninety-six swabs tested returned a positive result for Chlamydia DNA. We found no evidence of Chlamydia infection among the four geographically disparate populations sampled, suggesting that Chlamydia may not pose a widespread health risk to southern greater gliders in this region, based on the current data. However, various clinical signs of ophthalmic pathology were observed, warranting further investigation to determine their underlying aetiology., Competing Interests: Declarations. Ethics approval: All procedures were approved by the University of Wollongong research ethics committee (approval number AE19/02) and performed in accordance with the ethical standards laid out in the NSW Animal Research Act and the Australian Code for the Care and Use of Animals for Scientific Purposes. This research was conducted under a NSW Scientific Licence (SL101968) granted by the Department of Climate Change, Energy, the Environment and Water. Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature B.V.)

Introduction: This study employs bulk RNA sequencing, PCR, and ELISA assays to analyze the pathological factors affecting the outcomes of C. pecorum ocular infections in koalas. It investigates the immune responses and gene expression profiles associated with various stages of koala ocular chlamydiosis., Methods: A cohort of 114 koalas from Queensland, Australia were assessed, with 47% displaying clinical signs of ocular disease. Animals were classified into three cohorts: acute active disease (G1), chronic active disease (G2), and chronic inactive disease (G3), along with subclinical Chlamydia pecorum positive (H2) and healthy (H1) cohorts., Results: Analysis of clinical, microbiological, humoral immune and cellular immune biomarkers revealed varying chlamydial loads and anti-chlamydial IgG levels across disease grades, with a negative correlation observed between ocular chlamydial load and anti-chlamydial IgG. Koala ocular mucosa gene expression analysis from 27 koalas identified shared expression pathways across disease cohorts, with a significant upregulation of IFNγ expression and tryptophan metabolism in all disease stages., Discussion: These findings help elucidate immune response dynamics and molecular pathways underlying koala ocular chlamydiosis, providing insights crucial for disease management strategies., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Phillips, Madden, Gillett, Quigley, Jelocnik, Bommana, O’Meally, Timms and Polkinghorne.)

Pinkeye is a highly contagious disease of goats with different aetiologies. Surveys in Lao PDR have identified eye lesions typical of pinkeye as a common condition, however, this has not been confirmed diagnostically, and the responsible pathogens have not been identified. A matched case-control study was implemented in 70 goat holdings from Savannakhet province, Lao PDR, to detect agents causing pinkeye and conduct phylogenetic analysis of the identified pathogens. Fifty eye swabs from goats with infected eyes (cases) and 50 paired samples from unaffected cohorts (controls) were collected from 25 holdings. Samples were tested using quantitative PCR assays targeting known pinkeye pathogens at the genus and species levels. The prevalence of pathogens in case and control goats was as follows: Mycoplasma conjunctivae (94% and 74% respectively, P = 0.006, OR = 5.5), Chlamydia pecorum (4%, 10%), Moraxella ovis (30%, 30%), Moraxella bovis (0%, 0%) and Moraxella bovoculi (0%, 0%). M. conjunctivae was present in a high proportion of goats in both groups revealing that Lao goats are carriers of M. conjunctivae. However, the mean log 10 genome copy number/µL of DNA extract was significantly higher in case goats than control goats (P < 0.05). Thus, M. conjunctivae is likely the principal causative agent of pinkeye in Lao goats with carrier status converting to clinical infection following corneal damage or other causative factors. M. conjunctivae detected in samples from different goats and districts showed low genetic diversity. Identifying the causes of pinkeye in Lao goats will assist in designing appropriate treatment and control strategies., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Our study was designed to investigate the original spectrum of feline respiratory tract infection and to provide a scientific basis for the clinical diagnosis and treatment of feline respiratory infections and for precise prevention and control measures. A total of 400 cats with upper respiratory tract infections from animal hospitals in 12 provinces in China were examined from November 2022 to October 2023 to investigate the epidemiology of feline calicivirus (FCV), feline herpes virus type 1 (FHV-1), influenza A virus (IAV), Mycoplasma felis, Chlamydia felis, and Bordetella bronchiseptica through loop-mediated isothermal amplification (LAMP) with microfluidic chip detection. The results showed that 396 of the 400 samples tested were positive for at least one of these pathogens, with an overall detection rate of 99.00%. The detection rates were as follows: FCV, 36.00% (144/400); M. felis, 34.00% (136/400); FHV-1, 21.50% (86/400); C. felis, 15.75% (63/400); B. b, 13.00% (52/400); IAV, 4.50% (18/400). There were no statistically significant differences in the detection rates of respiratory pathogens between different sexes, ages, seasons, breeds, or regions (P > 0.05). There were 88 mixed infections, giving a total mixed infection rate of 22.00% (88/400). It is worth noting that the detection rate of FCV at different ages and of FHV-1 in different sexes showed significant differences (P < 0.05). The highest rate of FCV infection was found in animals that were 1 to 2 years old, and the rate of FHV-1 infection in male cats was higher than that in female cats. The results showed that the spectrum of feline respiratory pathogens is complex, with diverse epidemiological characteristics and mixed infections, and some differences among different respiratory pathogens were found with regard to the sex, age, and breed of the cat. Studies should be continued to provide a scientific basis for precise prevention and control of feline respiratory diseases., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Austria, part of Springer Nature.)

Feline upper respiratory tract disease (URTD) is a common but complicated disease that occurs in domestic cats, worldwide. 396 cats in Guangxi Province, China were screened for URTD-associated pathogens from March 2022 to August 2023. Mycoplasma felis was found to be the most prevalent infectious agent with a positivity rate of 24.75 %, followed by feline calicivirus (FCV), Chlamydia felis, feline herpesvirus 1 (FHV-1) and feline influenza A virus (FeIAV) with rates of 15.91, 11.62, 5.56 and 1.52 %, respectively. In particular, C. felis and M. felis were found in 13 of 55 co-infected cats. Of the 46 C. felis-positive samples, one strain, named as GXNN36, was successfully isolated using chicken embryos and it was characterized both in vivo and in vitro. For the cat studies, both high- and low-dose challenged groups showed severe conjunctivitis, accompanied by transient fever and respiratory symptoms. C. felis replicated well in turbinate, trachea and lung tissues with high copy numbers and the infection subsequently spread to the livers, spleens, pancreas, kidneys, hearts and intestines. These findings will help our understanding of the role of C. felis in feline URTD and provide a valuable model to evaluate the efficacy of vaccines and therapeutic remedies in the future., Competing Interests: Declaration of Competing Interest This manuscript has been reviewed by all authors. There are no potential conflicts of interest., (Copyright © 2024 Elsevier B.V. All rights reserved.)

To overcome shortcomings in discriminating Chlamydia pecorum strains infecting the koala (Phascolarctos cinereus) at the local level, we developed a novel genotyping scheme for this pathogen to inform koala management at a fine-scale subpopulation level. We applied this scheme to two geographically distinct koala populations in New South Wales, Australia: the Liverpool Plains and the Southern Highlands to South-west Sydney (SHSWS). Our method provides greater resolution than traditional multi-locus sequence typing, and can be used to monitor strain emergence, movement, and divergence across a range of fragmented habitats. Within the Liverpool Plains population, suspected recent introduction of a novel strain was confirmed by an absence of genetic diversity at the earliest sampling events and limited diversity at recent sampling events. Across the partially fragmented agricultural landscape of the Liverpool Plains, diversity within a widespread sequence type suggests that this degree of fragmentation may hinder but not prevent spread. In the SHSWS population, our results suggest movement of a strain from the south, where diverse strains exist, into a previously Chlamydia-free area in the north, indicating the risk of expansion towards an adjacent Chlamydia-negative koala population in South-west Sydney. In the south of the SHSWS where koala subpopulations appear segregated, we found evidence of divergent strain evolution. Our tool can be used to infer the risks of strain introduction across fragmented habitats in population management, particularly through practices such as wildlife corridor constructions and translocations., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Avian chlamydiosis is a bacterial infectious disease of birds, considered until recently caused only by Chlamydia psittaci, that now includes the newly described species C. buteonis, C. avium, and C. gallinacea, associated with several avian hosts. Since its recognition as a species in 2014 and having chickens as one of its main hosts, C. gallinacea has already been described in backyard poultry on all continents. The present study aimed to survey by molecular techniques the presence and species of Chlamydia spp. in backyard chickens from three states of the southern region of Brazil (Paraná-PR, Santa Catarina-SC, and Rio Grande do Sul-RS). DNA extracted from cloacal swab samples were tested by polymerase chain reaction (PCR) for different species of Chlamydia, namely Chlamydiaceae (23 S rRNA gene), C. psittaci (ompA gene), C. avium (enoA gene) and C. gallinacea (gidA and enoA genes). The 16 S rRNA gene was used for sequencing and phylogenetic analysis. A total of 582 backyard chicken samples were collected and grouped in 238 pools, from 134 properties in 59 municipalities. Chlamydiaceae was detected in 25.2% (60/238) of the samples, in 38.8% (52/134) of the properties and in 66.1% (39/59) of the municipalities. None of the samples yielded positive PCR results for C. psittaci or C. avium. For C. gallinacea, the overall percentage was 16.3% (39/238) according to the results of gidA and enoA genes. Sequence analysis confirmed that the samples corresponded to C. gallinacea. This is the first report of C. gallinacea in Brazil., (© 2024. The Author(s) under exclusive licence to Sociedade Brasileira de Microbiologia.)

The review presents modern view on the global problem of chlamydial infections. Current nomenclature of chlamydiae was adduced. Epidemiology, etiology, clinical features, pathogenesis, diagnosis and treatment of chlamydiosis received full coverage. The potential involvement of chlamydiae in the progression of various infectious and somatic diseases was revealed. Special attention was paid to pathomorphological alterations in human tissues, which develop during primary infection with chlamydia as well as during chronic infection. Key problems were demonstrated: underestimation of prevalence of chlamydiae among humans in worldwide clinical practice, the difficulty of detection of extragenital chlamydiosis, the lack of effective methods for diagnosis and treatment of persistent forms, the paucity of descriptions of pathomorphological picture of human chlamydiosis, the absence of specific prevention of infection.

Avian chlamydiosis is an acute or chronic disease of birds after infection by Chlamydia . Although Chlamydia psittaci is the primary agent of the disease, two additional species, Chlamydia avium and Chlamydia gallinacea , have also been recognized as potential disease agents. Therefore, the diagnosis of avian chlamydiosis requires differential identification of these avian Chlamydia species. The objective of the present study was to develop a multiplex real-time polymerase chain reaction (PCR) assay to rapidly differentiate between these three species of avian Chlamydia ( C. psittaci , C. avium , and C. gallinacea ) as well as to detect the genus Chlamydia . Specific genetic regions of the three species were identified by comparative analysis of their genome sequences. Also, the genus-specific region was selected based on 23S rRNA sequences. PCR primers and probes specific to the genus and each species were designed and integrated in the multiplex real-time PCR assay. The assay was highly efficient (94.8-100.7%). It could detect fewer than 10 copies of each target sequence of the genus and each species. Twenty-five Chlamydia control and field DNA samples were differentially identified while 20 other bacterial strains comprising 10 bacterial genera were negative in the assay. This assay allows rapid, sensitive, and specific detection of the genus and the three species of avian Chlamydia in a single protocol that is suitable for routine diagnostic purposes in avian diagnostic laboratories.

Chlamydial infections, caused by a group of obligate, intracellular, gram-negative bacteria, have health implications for animals and humans. Due to their highly infectious nature and zoonotic potential, staff at wildlife rehabilitation centers should be educated on the clinical manifestations, prevalence, and risk factors associated with Chlamydia spp. infections in raptors. The objectives of this study were to document the prevalence of chlamydial DNA shedding and anti-chlamydial antibodies in raptors admitted to five wildlife rehabilitation centers in California over a one-year period. Chlamydial prevalence was estimated in raptors for each center and potential risk factors associated with infection were evaluated, including location, species, season, and age class. Plasma samples and conjunctiva/choana/cloaca swabs were collected for serology and qPCR from a subset of 263 birds of prey, representing 18 species. Serologic assays identified both anti-C. buteonis IgM and anti-chlamydial IgY antibodies. Chlamydial DNA and anti-chlamydial antibodies were detected in 4.18% (11/263) and 3.14% (6/191) of patients, respectively. Chamydial DNA was identified in raptors from the families Accipitridae and Strigidae while anti-C.buteonis IgM was identified in birds identified in Accipitridae, Falconidae, Strigidae, and Cathartidae. Two of the chlamydial DNA positive birds (one Swainson's hawk (Buteo swainsoni) and one red-tailed hawk (Buteo jamaicensis)) were necropsied, and tissues were collected for culture. Sequencing of the cultured elementary bodies revealed a chlamydial DNA sequence with 99.97% average nucleotide identity to the recently described Chlamydia buteonis. Spatial clusters of seropositive raptors and raptors positive for chlamydial DNA were detected in northern California. Infections were most prevalent during the winter season. Furthermore, while the proportion of raptors testing positive for chlamydial DNA was similar across age classes, seroprevalence was highest in adults. This study questions the current knowledge on C. buteonis host range and highlights the importance of further studies to evaluate the diversity and epidemiology of Chlamydia spp. infecting raptor populations., Competing Interests: Dr. Branson Ritchie is the Director of the Infectious Disease Laboratory, College of Veterinary Medicine, Univ. of GA, Athens, GA and contributed in-kind support by providing the EBA serology, qPCR testing and whole genome sequencing for this project.

Chlamydiaceae are obligate intracellular bacteria with a broad host range. Several studies have found chlamydial species that are genetically intermediate between Chlamydia psittaci and Chlamydia abortus in various avian species. One of these intermediate Chlamydia species, found in a red-shouldered hawk ( Buteo lineatus ), was recently classified as a new species Chlamydia buteonis . This newly described Chlamydia species has, so far, only been reported in hawks exhibiting clinical signs of conjunctivitis, dyspnea, and diarrhea. In the present study, fecal samples of 5 gyrfalcons ( Falco rusticolus ), 3 gyr/peregrine falcon hybrids ( Falco rusticolus × Falco peregrinus ), and 15 falcons of unknown species presented to falcon clinics on the Arabian Peninsula were shipped to the Vetsuisse Faculty, University of Zurich (Zurich, Switzerland), for examination for the presence of Chlamydiaceae. A step-wise diagnostic approach was performed to identify the chlamydial species involved. Chlamydiaceae were detected in 21/23 falcons by a family-specific real-time quantitative PCR (qPCR). Further identification with a 23S ribosomal RNA-based microarray assay and 16S conventional PCR and sequencing yielded inconclusive results, indicating the presence of an intermediate Chlamydia species. Because none of the falcons tested positive for Chlamydia psittaci by specific qPCR, all 23 samples were subjected to a Chlamydia buteonis -specific qPCR, which was positive in 16/23 samples. Detailed information regarding clinical history was available for 8 falcons admitted to a falcon clinic in Dubai, United Arab Emirates. Six of those birds that were presented to the clinic because of loss of performance and poor general condition, including vomiting and diarrhea, were positive for C buteonis . In 2 birds without clinical disease signs admitted for a routine health examination, 1 was positive for C buteonis , and 1 was negative. It is yet unknown whether Chlamydia buteonis causes disease in birds, but the findings in this study indicate that Chlamydia buteonis may be an infectious pathogen in falcon species.

Chlamydiae are highly successful strictly intracellular bacteria associated with diverse eukaryotic hosts. Here we analyzed metagenome-assembled genomes of the "Genomes from Earth's Microbiomes" initiative from diverse environmental samples, which almost double the known phylogenetic diversity of the phylum and facilitate a highly resolved view at the chlamydial pangenome. Chlamydiae are defined by a relatively large core genome indicative of an intracellular lifestyle, and a highly dynamic accessory genome of environmental lineages. We observe chlamydial lineages that encode enzymes of the reductive tricarboxylic acid cycle and for light-driven ATP synthesis. We show a widespread potential for anaerobic energy generation through pyruvate fermentation or the arginine deiminase pathway, and we add lineages capable of molecular hydrogen production. Genome-informed analysis of environmental distribution revealed lineage-specific niches and a high abundance of chlamydiae in some habitats. Together, our data provide an extended perspective of the variability of chlamydial biology and the ecology of this phylum of intracellular microbes.

Chlamydia is a known pathogen in both saltwater and freshwater crocodiles. However, the exact species/strain has not been clearly identified. In this study, we successfully cultivated Siamese crocodile Chlamydia in McCoy cells at a temperature of 30°C. Electron microscopy; phylogeny based on nine conserved taxonomically informative markers, on ompA, or on seven housekeeping genes; and whole-genome sequencing and analysis of the isolate confirmed the identity of the isolate as a new member of the genus Chlamydia, a new species that we name Chlamydia crocodili., Competing Interests: The authors have declared that no competing interests exist.

Chlamydia (C.) pecorum, an obligate intracellular bacterial species commonly found in ruminants, can also occur in pigs. However, its significance as a potential porcine pathogen, or commensal, is still unclear. In a previous study (Hoffmann et al. 2015), mixed infections of C. suis and C. pecorum were detected in 14 Swiss fattening pig farms. Using these samples, we aimed to investigate the infection dynamics of C. suis and C. pecorum mixed infections in these farms. In addition, we analyzed the genetic diversity of Swiss porcine C. pecorum strains in relation to globally circulating strains. In total, 1284 conjunctival and rectal swabs from 391 pigs, collected at the beginning and end of the fattening period, were tested during the course of this study. We determined the bacterial loads of C. suis and C. pecorum using species-specific real-time PCR (qPCR) and compared these results to already existing DNA-microarray and Chlamydiaceae qPCR data. Overall, C. suis and Chlamydiaceae copy numbers decreased in the course of the fattening period, whereas C. pecorum copy numbers increased. No association was found between clinical signs (conjunctivitis, lameness and diarrhea) and the bacterial loads. Preventive antibiotic treatment at the beginning of the fattening period significantly lowered the chlamydial load and outdoor access was associated with higher loads. Proximity to the nearest ruminants correlated with increased C. pecorum loads, indicating that C. pecorum could be transmitted from ruminants to pigs. Multi-locus sequence typing (MLST) and major outer membrane protein (ompA) genotyping revealed two novel sequence types (STs) (301, 302) and seven unique ompA genotypes (1-7) that appear to form a specific clade separate from other European C. pecorum strains., (Copyright © 2021 The Author(s). Published by Elsevier B.V. All rights reserved.)

Members of the Chlamydia genus are known to cause disease in both humans and animals. A variety of other species in the order Chlamydiales are increasingly being discovered and emerging as potential pathogens, yet there are scarce data on the diversity, prevalence and impacts of these pathogens in wild birds. To address this gap, we investigated which Chlamydiales species are present in a wild population of a common Australian parrot, the Crimson Rosella (Platycercus elegans). We collected cloacal swabs and serum from 136 individuals in south-eastern Australia, over two years, and tested several predictors of prevalence: age, sex, season and breeding status. We used multiple PCR assays to determine bacterial prevalence in cloacal swabs and a solid-phase ELISA to determine seroprevalence. We found Chlamydiales PCR prevalence of 27.7% (95% CI 20.2, 36.2) and identified at least two families (Chlamydiaceae and Parachlamydiaceae). Regarding known chlamydial avian pathogens, we found C. psittaci at 6.2% (95% CI 2.7, 11.8) and C. gallinacea at 4.6% (95% CI 1.7, 9.8) prevalence. We also identified at least two potentially novel Chlamydiales species, of unknown pathogenicity. Sex and breeding status predicted Chlamydiales PCR prevalence, with females more likely to be infected than males, and non-breeding birds more likely to be infected than breeding birds. Seroprevalence was 16% (95% CI 8.8, 25.9). Season and breeding status were strong predictors of seroprevalence, with highest seroprevalence in autumn and in non-breeding birds. Our results reveal a diversity of Chlamydiales species in this abundant wild host, and indicate that host-specific and temporal factors are associated with infection risk. Our findings suggest that wild parrots are a reservoir of both known and novel Chlamydiales lineages, of zoonotic and pathogenic potential., (© 2020 Blackwell Verlag GmbH.)

Chlamydiae have been known for more than a century as major pathogens of humans. Yet they are also found ubiquitously in the environment where they thrive within protists and in an unmatched wide range of animals. This review summarizes recent advances in understanding chlamydial diversity and distribution in nature. Studying these environmental chlamydiae provides a novel perspective on basic chlamydial biology and evolution. A picture is beginning to emerge with chlamydiae representing one of the evolutionarily most ancient and successful groups of obligate intracellular bacteria., (Copyright © 2020 The Author. Published by Elsevier Ltd.. All rights reserved.)

Chlamydial disease control is increasingly utilised as a management tool to stabilise declining koala populations, and yet we have a limited understanding of the factors that contribute to disease progression. To examine the impact of host and pathogen genetics, we selected two geographically separated south east Queensland koala populations, differentially affected by chlamydial disease, and analysed koala major histocompatibility complex (MHC) genes, circulating strains of Chlamydia pecorum and koala retrovirus (KoRV) subtypes in longitudinally sampled, well-defined clinical groups. We found that koala immunogenetics and chlamydial genotypes differed between the populations. Disease progression was associated with specific MHC alleles, and we identified two putative susceptibility (DCb 03, DBb 04) and protective (DAb 10, UC 01:01) variants. Chlamydial genotypes belonging to both Multi-Locus Sequence Typing sequence type (ST) 69 and ompA genotype F were associated with disease progression, whereas ST 281 was associated with the absence of disease. We also detected different ompA genotypes, but not different STs, when long-term infections were monitored over time. By comparison, KoRV profiles were not significantly associated with disease progression. These findings suggest that chlamydial genotypes vary in pathogenicity and that koala immunogenetics and chlamydial strains are more directly involved in disease progression than KoRV subtypes.

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., (Copyright © 2020 Elsevier GmbH. All rights reserved.)

Chlamydia spp. are a group of obligate intracellular pathogens causing a number of diseases in animals and humans. Avian chlamydiosis (AC), caused by Chlamydia psittaci (C. psittaci) as well as new emerging C. avium, C. gallinacea and C. ibidis, have been described in nearly 500 avian species worldwidely. The Crested Ibis (Nipponia nippon) is a world endangered avian species with limited population and vulnerable for various infections. To get a better understanding of the prevalence of Chlamydia spp. in the endangered Crested Ibis, faecal samples were collected and analysed. The results confirmed that 20.20% (20/99) of the faecal samples were positive for Chlamydiaceae and were identified as C. ibidis with co-existence of C. psittaci in one of the 20 positive samples. In addition, ompA sequence of C. psittaci obtained in this study was classified into the provisional genotype Matt116, while that of C. ibidis showed high genetic diversity, sharing only 77% identity with C. ibidis reference strain 10-1398/6. We report for the first time the presence of C. ibidis and C. psittaci in the Crested Ibis, which may indicate a potential threat to the endangered birds and should be aware of the future protection practice.

Following the occurrence of sudden death cases in a zoo reptile collection, histological analyses conducted on tissues from two common adders suggested an infection due to Chlamydia. The survey was extended to 22 individual snakes from the same collection and a PCR analysis targeting a conserved gene in Chlamydiaceae revealed bacterial shedding in six of them. The infection resolved spontaneously in one snake whereas another one succumbed one month later. The antibiotic treatment administered (marbofloxacin) to the remaining four PCR positive animals stopped the mortalities and the shedding. Analysis of the 16S and 23S ribosomal gene sequences identified C. serpentis, a recently described novel chlamydial species in snakes. A PCR tool for a quick and specific identification of this new chlamydial species was developed in this study., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Chlamydia spp. and Chlamydia-like organisms are able to infect vertebrates such as mammals, reptiles and birds, but also arthropods and protozoans. Since they have been detected in bats and bat feces, we expected Chlamydiae bacteria to also be present in the mite Spinturnix myoti, an ectoparasite of mouse-eared bats (Myotis spp.). The prevalence of Chlamydiales in 88 S. myoti was 57.95% and significantly depended on bat host species. In addition, the prevalence was significantly different between bat species living in sympatry or in allopatry. While there was uninterpretable sequencing for 16 samples, eight showed best BLAST hit identities lower than 92.5% and thus corresponded to new family-level lineages according to the established taxonomy cut-off. The four remaining sequences exhibited best BLAST hit identities ranging from 94.2 to 97.4% and were taxonomically assigned to three different family-level lineages, with two of them belonging to the Parachlamydiaceae, one to the Simkaniaceae, and one to the Chlamydiaceae. These results highlighted for the first time the presence of Chlamydia-like organisms and the possible zoonotic origin of Chlamydia sp. in S. myoti ectoparasites of bats, and therefore suggest that these ectoparasites may play a role in maintaining and/or transmitting members of the Chlamydiae phylum within Myotis spp. bat populations. Our results further highlight that the wide diversity of bacteria belonging to the Chlamydiae phylum is largely underestimated., (© K. Thiévent et al., published by EDP Sciences, 2020.)

Chlamydiaceae are obligate intracellular bacterial pathogens for humans and animals. A recent study highlighted that a Chlamydiaceae intermediary between C. psittaci and C. abortus can infect hawks. Here, an isolate was obtained upon passage of cloacal and conjunctival sac material collected from a female hatch-year red-shouldered hawk (Buteo lineatus) in cultured cells. The diseased bird, one of 12 birds housed in a rehabilitation center, developed conjunctivitis and later died. Swabs from both sites tested positive for Chlamydia using the QuickVue Chlamydia test. The isolate, named RSHA, tested negative in qPCR assays specific for C. psittaci and C. abortus, respectively. Analysis of the 16S rRNA, 23S rRNA and whole genome sequences as well as MLST, ANIb and TETRA values reveal that C. psittaci and C. abortus are the closest relatives of RSHA. However, the overall results strongly suggest a phylogenetic intermediate position between these two species. Therefore, we propose the introduction of a new species designated Chlamydia buteonis with RSHA T as the type strain., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Purpose: Koala retrovirus (KoRV-A) is 100  % prevalent in northern Australian (Queensland and New South Wales) koala populations, where KoRV-B has been associated with Chlamydia pecorum disease and the development of lymphosarcoma. In southern populations (Victoria and South Australia), KoRV-A is less prevalent and KoRV-B has not been detected in Victoria, while the current prevalence in South Australian populations is unknown but is thought to be low. This study aimed to determine (i) the prevalence of KoRV in the two largest South Australian koala populations [Kangaroo Island (KI) and Mount Lofty Ranges (MLR)], (ii) KoRV subtype and (iii) if an association between KoRV and C. pecorum exists., Methodology: Wild koalas were sampled in KI ( n =170) between 2014 and 2017 and in MLR ( n =75) in 2016. Clinical examinations were performed, with blood collected for KoRV detection and typing by PCR., Results: KoRV prevalence was 42.4  % [72/170, 95 % confidence interval (CI): 34.9-49.8  %] in KI and 65.3  % (49/75, 95 % CI: 54.6-76.1  %) in MLR. Only KoRV-A, and not KoRV-B, was detected in both populations. In MLR, there was no statistical association between KoRV and C. pecorum infection (P =0.740), or KoRV and C. pecorum disease status ( P=0.274), although KoRV-infected koalas were more likely to present with overt C. pecorum disease than subclinical infection (odds ratio: 3.15, 95 % CI: 0.91-5.39)., Conclusion: KoRV-A is a prevalent pathogen in wild South Australian koala populations. Future studies should continue to investigate KoRV and C. pecorum associations, as the relationship is likely to be complex and to differ between the northern and southern populations.

Introduction and Objectives: Chlamydia (C.) felis can cause infection which may be associated with conjunctivitis and/or respiratory tract disease, particularly in kittens, but could also be the cause of the disease in adult cats. Infection is more common in multi-cat environments. The zoonotic potential of C. felis appears low, but exposure to this microorganism is possible by handling the affected cats, by contact with their aerosol, and also via fomites., Material and Methods: In the study, 140 cats of various breeds from Košice region in Slovakia were studied. Conjunctival samples were obtained from 71 clinically healthy cats (50.7%) and 69 cats with clinical signs of conjunctivitis and upper respiratory tract impairment (49.3%). Cats were divided into 4 groups according to breed and type of environment in which they lived. In the 1 st group were cats kept inside only (n=33), in the 2 nd group, free-roaming cats (n=50), the 3 rd group comprised stray cats, taken from the streets (n=28), and the 4 th group included cats kept in shelters or deposit devices (n=29). Molecular method PCR and DNA sequencing was used as the diagnostic method., Results: Overall positivity was 17.1%. Of the 24 positive cats, the highest positivity was detected in the population of stray cats (35.7%) and shelter cats (31%). In the group of free-roaming cats, 10% had positivity. No positive animals were detected in the group of cats kept inside only. It was also found that the risk of C. felis in cats with clinical signs of disease was more than 7-fold higher than in cats without clinical signs of conjunctivitis and respiratory tract., Conclusions: The obtained results show that cats, especially stray and shelter cats, can be important sources of feline chlamydiosis, and due to their close contact with people they can present a risk for transmission.

Chlamydiae are globally widespread obligate intracellular bacteria, which several species are a well-recognized threat to human and animal health. In Australia, the most successful chlamydial species are the infamous koala pathogen C. pecorum, and C. psittaci, an emerging pathogen associated with zoonotic events. Little is known about infections caused by other chlamydial organisms in Australian livestock or wildlife. Considering that these hosts can be encountered by humans at the animal/human interface, in this study, we investigated genetic diversity of chlamydial organisms infecting Australian domesticated and wild ungulates. A total of 185 samples from 129 domesticated (cattle, horses, sheep, and pigs) and 29 wild (deer) ungulate hosts were screened with C. pecorum and C. psittaci species-specific assays, followed by a screen with pan-Chlamydiales assay. Overall, chlamydial DNA was detected in 120/185 (65%) samples, including all ungulate hosts. Species-specific assays further revealed that C. pecorum and C. psittaci DNA were detected in 27% (50/185) and 6% (11/185) of the samples, respectively, however from domesticated hosts only. A total of 46 "signature" 16S rRNA sequences were successfully resolved by sequencing and were used for phylogenetic analyses. Sequence analyses revealed that genetically diverse novel as well as traditional chlamydial organisms infect an expanded range of ungulate hosts in Australia. Detection of the C. psittaci and C. pecorum in livestock, and novel taxa infecting horses and deer raises questions about the genetic make-up and pathogenic potential of these organisms, but also concerns about risks of spill-over between livestock, humans, and native wildlife., (© 2019 Blackwell Verlag GmbH.)

Chlamydiosis, caused by Chlamydia pecorum, is regarded as an important threat to koala populations. Across the koala's geographical range, disease severity associated with C. pecorum infection varies, with pathogen diversity and strain pathogenicity being likely important factors. To examine C. pecorum diversity on a sub-population level a Multi-Locus Sequence Typing (MLST) scheme, containing the housekeeping genes; gatA, oppA&#95;3, hflX, gidA, enoA, hemN and fumC, was used to type strains from two sub-populations of koalas from the Liverpool Plains, NSW, Australia, with different disease expressions. Typing of samples from 2015 to 2017, revealed a significant association between sequence type ST 69 and clinical disease and a significant difference in sequence type frequencies between sub-populations. Sequence type ST 69 has previously been identified in both subclinical and clinically diseased koalas indicating that these markers alone are not illustrative of pathogenicity. However, recent emergence of this sequence type in a naïve population may explain the differing disease expressions. Sequence types ST 73 and ST 69 have been described in koalas across a broad geographic range, indicating multiple introduction events and/or a limited veracity of the MLST loci to explore fine scale epidemiological investigations, particularly those examining the interface between pathogenic strain and disease outcome., (Copyright © 2019. Published by Elsevier B.V.)

Developed two decades ago as a molecular method to provide definite characterization of a bacterial isolate, Multilocus Sequence Typing (MLST) is today globally adopted as a universal fine-detailed molecular typing tool and has been applied to numerous pathogenic and nonpathogenic bacterial as well eukaryotic organisms. MLST utilizes DNA sequence of several conserved housekeeping (HK) genes which are assigned an allelic number, which then collectively constitute an allelic profile or sequence type (ST), a "molecular barcode" of the interrogated bacterial strain or a eukaryotic organism. Here, we describe the principles and molecular approaches for generating MLST data for an analysis of a bacteria in the order Chlamydiales, using a Chlamydia pecorum-specific MLST scheme as an example.

The accelerating growth of genomic and proteomic information for Chlamydia species, coupled with unique biological aspects of these pathogens, necessitates bioinformatic tools and features that are not provided by major public databases. To meet these growing needs, we developed ChlamBase, a model organism database for Chlamydia that is built upon the WikiGenomes application framework, and Wikidata, a community-curated database. ChlamBase was designed to serve as a central access point for genomic and proteomic information for the Chlamydia research community. ChlamBase integrates information from numerous external databases, as well as important data extracted from the literature that are otherwise not available in structured formats that are easy to use. In addition, a key feature of ChlamBase is that it empowers users in the field to contribute new annotations and data as the field advances with continued discoveries. ChlamBase is freely and publicly available at chlambase.org., (© The Author(s) 2019. Published by Oxford University Press.)

Background: Chlamydia infects multiple sites within hosts, including the gastrointestinal tract (GIT). In certain hosts, gastrointestinal infection is linked to treatment avoidance and self-infection at disease susceptible sites. GIT C. pecorum has been detected in livestock and koalas, however GIT prevalence rates within the koala are yet to be established., Methods: Paired conjunctival, urogenital and rectal samples from 33 koalas were screened for C. pecorum and C. pecorum plasmid using 16S rRNA and CDS5-specific quantitative PCR assays, respectively. Amplicon sequencing of 359 bp ompA fragment was used to identify site-specific genotypes., Results: The overall C. pecorum prevalence collectively (healthy and clinically diseased koalas) was 51.5%, 57.6% and 42.4% in urogenital, conjunctival and gastrointestinal sites, respectively. Concurrent urogenital and rectal Chlamydia was identified in 14 koalas, with no cases of GIT only Chlamydia shedding. The ompA genotype G dominated the GIT positive samples, and genotypes A and E' were dominant in urogenital tract (UGT) positive samples. Increases in C. pecorum plasmid per C. pecorum load (detected by PCR) showed clustering in the clinically diseased koala group (as assessed by scatter plot analysis). There was also a low correlation between plasmid positivity and C. pecorum infected animals at any site, with a prevalence of 47% UGT, 36% rectum and 40% faecal pellet., Conclusions: GIT C. pecorum PCR positivity suggests that koala GIT C. pecorum infections are common and occur regularly in animals with concurrent genital tract infections. GIT dominant genotypes were identified and do not appear to be related to plasmid positivity. Preliminary results indicated a possible association between C. pecorum plasmid load and clinical UGT disease., Competing Interests: Endeavour Veterinary Ecology Pty Ltd is a commercial company. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

We describe a case of community-acquired pneumonia due to Chlamydia caviae in a patient with no direct animal exposure, raising questions about the zoonotic reservoirs and potential transmission routes. Genotyping of Chamydia isolates that cause pneumonia should be performed for a precise diagnosis and to initiate adequate infection control measures.

Chlamydia pecorum in koalas ( Phascolarctos cinereus) is considered a sexually transmitted infection. Analysis of samples from koala joeys (<1 yr) suggested that mother-to-young direct transmission was also occurring. Further, evidence suggested that joeys from vaccinated mothers were less likely to contract infections than joeys with unvaccinated mothers.

Chlamydia abortus is an obligate intracellular Gram-negative bacteria, which can infect animals and human, including goats. However, little information on C. abortus infection is available in goats in Hunan province, subtropical China. To investigate the seroprevalence and risk factors of C. abortus infection in goats in Hunan province, China, a total of 911 goat blood samples were collected randomly from 14 herds having number of goats ranging from 1000 to 3000 from March 2014 to December 2015. Seropositive animals were found in 11 out of 14 (78.57%) goat herds with seroprevalence ranging from 0.00% to 29.94% in individual herds. Overall, the seroprevalence of C. abortus infection was different among regions (southern Hunan: 1.78%; northeast Hunan: 5.47%; and west Hunan: 15.29%), gender (male: 4.58% and female: 9.10%), seasons (spring: 5.97%; summer: 2.61%; autumn: 16.88%; and winter: 10.94%), and ages (year ≤1: 2.39%; 1 < year ≤2: 9.58%; 2 < year ≤3: 9.16%; and year >3: 17.57%). Risk factors for C. abortus infection were associated with region, season, and age in this study. To our knowledge, this is the first document to demonstrate the existence of C. abortus infection in goats, and the seroprevalence was 8.45% out of 911 goats in Hunan province.

The Chlamydiales order is composed of obligate intracellular bacteria and includes the Chlamydiaceae family and several family-level lineages called Chlamydia-related bacteria. In this review we will highlight the conserved and distinct biological features between these two groups. We will show how a better characterization of Chlamydia-related bacteria may increase our understanding on the Chlamydiales order evolution, and may help identifying new therapeutic targets to treat chlamydial infections., (Copyright © 2017 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Background: The live, temperature-attenuated vaccine strain 1B of Chlamydia abortus, the aetiological agent of ovine enzootic abortion (OEA), has been implicated in cases of vaccine breakdown. The aim of this study was to understand the nature of this attenuation through sequencing of the vaccine parent strain (AB7) and the derived mutant strains 1B and 1H, as well as to clarify the role of the vaccine strain in causing disease through comparative whole genome analysis., Methods: Whole genome sequencing was performed on: vaccine parent strain AB7; N-methyl-N'-nitro-N-nitrosoguanidine (NTG)-induced temperature attenuated mutant strain 1B grown from the commercial live vaccines Cevac Chlamydia and Enzovax; strain 1H a reverted NTG mutant; and 5 strains isolated from cases of OEA originating from animals from the original vaccine safety trial (2 strains) or from vaccinated ewes or ewes exposed to vaccinated animals (3 strains)., Results: We confirmed that AB7 is in a different lineage from the reference strain S26/3. The genome of vaccine strain 1B contains ten single nucleotide polymorphisms (SNPs) created by the NTG treatment, which are identical to those found in strain 1H. The strains from OEA cases also cluster phylogenetically very tightly with these vaccine strains., Conclusions: The results show that C. abortus vaccine strain 1B has an identical genome sequence to the non-attenuated "reverted mutant" strain 1H. Thus, the protection of the 1B vaccine is unlikely to be due to the NTG induced SNPs and is more likely caused by the administration of high doses of C. abortus elementary bodies that stimulate protective immunity. Vaccine-identical strains were also isolated from cases of disease, as well as strains which had acquired 1-3 SNPs, including an animal that had not been vaccinated with either of the commercial live OEA vaccines, indicating that the 1B vaccine strain may be circulating and causing disease., (Copyright © 2018. Published by Elsevier Ltd.)

Recent comparative genomics studies have suggested that horizontal gene transfer (HGT) is one of the major processes in bacterial evolution. In this study, HGT events of 64 Chlamydia strains were investigated based on the pipeline employed in HGTree database constructed in our recent study. Tree reconciliation method was applied in order to calculate feasible HGT events. Following initial detection and an evaluation procedure, evidence of the HGT was identified in 548 gene families including 42 gene families transferred from outside of Chlamydiae phylum with high reliability. The donor species of inter-phylum HGT consists of 12 different bacterial and archaeal phyla, suggesting that Chlamydia might have even more various host range than in previous reports. In addition, each species of Chlamydia showed varying preference towards HGT, and genes engaged in HGT within Chlamydia and between other species showed different functional distribution. Also, examination of individual gene flows of niche-specific genes suggested that many of such genes are transferred mainly within Chlamydia genus. Our results uncovered novel features of HGT acting on Chlamydia genome evolution, and it would be also strong evidence that HGT is an ongoing process for intracellular pathogens. We expect that the results provide more insight into lineage- and niche-specific adaptations regarding their infectivity and pathogenicity.

Chlamydia species have recently been recognized as emerging pathogens in snakes. However, isolation of novel snake chlamydiae is critical and their growth characteristics are largely unknown. In this study, two novel chlamydial species are described: Chlamydia serpentis and Chlamydia poikilothermis, isolated after attempts on 23 cloacal and choanal swabs from 18 PCR-positive captive snakes originating from different Swiss snake collections. Isolation success, growth curve and infectivity rates over a 48-hour time period were dependent on temperature (37 °C for C. serpentis, 28 °C for C. poikilothermis). C. serpentis and C. poikilothermis were sensitive to tetracycline and moxifloxacin during evaluation by in vitro antibiotic susceptibility assay but intermediate to resistant (2-4 μg/ml) to azithromycin. Whole genome sequencing of the isolates provided proof of the novel species status, and gives insights into the evolution of these branches of genus Chlamydia.

Until recently, Chlamydia psittaci was considered to be the only etiological agent of avian chlamydiosis, but two new avian species, Chlamydia gallinacea and Chlamydia avium, have recently been described in poultry and pigeons or psittacine birds, respectively. The aim of this study was to explore the occurrence of C. psittaci and C. gallinacea in backyard chickens in Italy. Cloacal swabs were taken from 160 asymptomatic chickens reared in 16 backyard farms. Samples were tested for C. psittaci and C. gallinacea by specific real-time polymerase chain reaction assays, with 24 (15%) of the 160 chickens resulting positive for C. gallinacea. To attempt chlamydial isolation, new samples were obtained from two farms harboring a high prevalence (60% and 70%, respectively) of C. gallinacea-positive chickens. In total, eight C. gallinacea and one C. psittaci isolates were successfully recovered from 13 chickens. C. gallinacea was confirmed to be the endemic chlamydial species in chickens, with a high ompA intraspecies diversity. The presence of viable C. psittaci and C. gallinacea demonstrated by isolation from chickens in backyard farms poses a potential public health problem.

A retrospective study of 109 skin biopsies with granuloma annulare (GA) or morphea histology from patients with suspected tick bite was performed. Biopsies were tested for cutaneous Borrelia burgdorferi DNA using PCR. The same biopsies were analysed for tick-borne novel agents, Chlamydia-related bacteria (members of the Chlamydiales order), using a PCR-based method. Borrelia DNA was detected in 7/73 (9.6%) biopsies with GA and in 1/36 (2.8 %) biopsies with morphea, while Chlamydiales DNA was found in 53/73 (72.6%) biopsies with GA and 25/34 (73.4%) biopsies with morphea. All Borrelia DNA-positive GA samples were also positive for Chlamydiales DNA. The Chlamydiales sequences detected in GA were heterogeneous and contained Waddliaceae and Rhabdochlamydiaceae bacteria, which are also present in Ixodes ricinus ticks, while the Chlamydiales sequences detected in morphea closely resembled those found in healthy skin. In conclusion, tick-mediated infections can trigger GA in some cases, while correlation of either Borrelia or Chlamydiales with morphea is unlikely.

The expanding field of bacterial genomics has revolutionized our understanding of microbial diversity, biology and phylogeny. For most species, DNA extracted from culture material is used as the template for genome sequencing; however, the majority of microbes are actually uncultivable, and others, such as obligate intracellular bacteria, require laborious tissue culture to yield sufficient genomic material for sequencing. Chlamydiae are one such group of obligate intracellular microbes whose characterization has been hampered by this requirement. To circumvent these challenges, researchers have developed culture-independent sample preparation methods that can be applied to the sample directly or to genomic material extracted from the sample. These methods, which encompass both targeted [immunomagnetic separation-multiple displacement amplification (IMS-MDA) and sequence capture] and non-targeted approaches (host methylated DNA depletion-microbial DNA enrichment and cell-sorting-MDA), have been applied to a range of clinical and environmental samples to generate whole genomes of novel chlamydial species and strains. This review aims to provide an overview of the application, advantages and limitations of these targeted and non-targeted approaches in the chlamydial context. The methods discussed also have broad application to other obligate intracellular bacteria or clinical and environmental samples.

In poultry several Chlamydia species have been detected, but Chlamydia psittaci and Chlamydia gallinacea appear to be most prevalent and important. Chlamydia psittaci is a well-known zoonosis and is considered to be a pathogen of poultry. Chlamydia gallinacea has been described more recently. Its avian pathogenicity and zoonotic potential have to be further elucidated. Within the Netherlands no data were available on the presence of Chlamydia on poultry farms. As part of a surveillance programme for zoonotic pathogens in farm animals, we investigated pooled faecal samples from 151 randomly selected layer farms. On a voluntary base, 69 farmers, family members or farm workers from these 151 farms submitted a throat swab. All samples were tested with a generic 23S Chlamydiaceae PCR followed by a species specific PCR for C. avium, C. gallinacea and C. psittaci. C. avium and psittaci DNA was not detected at any of the farms. At 71 farms the positive result could be confirmed as C. gallinacea. Variables significantly associated with the presence of C. gallinacea in a final multivariable model were 'age of hens,' 'use of bedding material' and 'the presence of horses.' The presence of C. gallinacea was associated with neither clinical signs, varying from respiratory symptoms, nasal and ocular discharges to diarrhoea, nor with a higher mortality rate the day before the visit. All throat swabs from farmers, family members or farm workers tested negative for Chlamydia DNA, giving no further indication for possible bird-to-human (or human-to-bird) transmission.

Peptidoglycan is the predominant stress-bearing structure in the cell envelope of most bacteria, and also a potent stimulator of the eukaryotic immune system. Obligate intracellular bacteria replicate exclusively within the interior of living cells, an osmotically protected niche. Under these conditions peptidoglycan is not necessarily needed to maintain the integrity of the bacterial cell. Moreover, the presence of peptidoglycan puts bacteria at risk of detection and destruction by host peptidoglycan recognition factors and downstream effectors. This has resulted in a selective pressure and opportunity to reduce the levels of peptidoglycan. In this review we have analysed the occurrence of genes involved in peptidoglycan metabolism across the major obligate intracellular bacterial species. From this comparative analysis, we have identified a group of predicted 'peptidoglycan-intermediate' organisms that includes the Chlamydiae, Orientia tsutsugamushi, Wolbachia and Anaplasma marginale. This grouping is likely to reflect biological differences in their infection cycle compared with peptidoglycan-negative obligate intracellular bacteria such as Ehrlichia and Anaplasma phagocytophilum, as well as obligate intracellular bacteria with classical peptidoglycan such as Coxiella, Buchnera and members of the Rickettsia genus. The signature gene set of the peptidoglycan-intermediate group reveals insights into minimal enzymatic requirements for building a peptidoglycan-like sacculus and/or division septum., (© 2017 The Authors. Molecular Microbiology Published by John Wiley & Sons Ltd.)

Obligate intracellular bacteria entirely depend on the metabolites of their host cell for survival and generation of progeny. Due to their lifestyle inside a eukaryotic cell and the lack of any extracellular niche, they have to perfectly adapt to compartmentalized intracellular environment of the host cell and counteract the numerous defense strategies intrinsically present in all eukaryotic cells. This so-called cell-autonomous defense is present in all cell types encountering Chlamydia infection and is in addition closely linked to the cellular innate immune defense of the mammalian host. Cell type and chlamydial species-restricted mechanisms point a long-term evolutionary adaptation that builds the basis of the currently observed host and cell-type tropism among different Chlamydia species. This review will summarize the current knowledge on the strategies pathogenic Chlamydia species have developed to subvert and overcome the multiple mechanisms by which eukaryotic cells defend themselves against intracellular pathogens.

Background: Chlamydia suis is an important, globally distributed, highly prevalent and diverse obligate intracellular pathogen infecting pigs. To investigate the prevalence and genetic diversity of C. suis in China, 2,137 nasal, conjunctival, and rectal swabs as well as whole blood and lung samples of pigs were collected in 19 regions from ten provinces of China in this study., Results: We report an overall positivity of 62.4% (1,334/2,137) of C. suis following screening by Chlamydia spp. 23S rRNA-based FRET-PCR and high-resolution melting curve analysis and confirmatory sequencing. For C. suis-positive samples, 33.3 % of whole blood and 62.5% of rectal swabs were found to be positive for the C. suis tetR(C) gene, while 13.3% of whole blood and 87.0% of rectal swabs were positive for the C. suis tet(C) gene. Phylogenetic comparison of partial C. suis ompA gene sequences revealed significant genetic diversity in the C. suis strains. This genetic diversity was confirmed by C. suis-specific multilocus sequence typing (MLST), which identified 26 novel sequence types among 27 examined strains. Tanglegrams based on MLST and ompA sequences provided evidence of C. suis recombination amongst the strains analyzed., Conclusions: Genetically highly diverse C. suis strains are exceedingly prevalent in pigs. As it stands, the potential pathogenic effect of C. suis on pig health and production of C. suis remains unclear and will be the subject of further investigations. Further study is also required to address the transmission of C. suis between pigs and the risk of 'spill-over' and 'spill-back' of infections to wild animals and humans.

Chlamydiae are obligate intracellular bacteria comprising important human pathogens and symbionts of protists. Molecular evidence indicates a tremendous diversity of chlamydiae particularly in marine environments, yet our current knowledge is based mainly on terrestrial representatives. Here we provide first insights into the biology of marine chlamydiae representing three divergent clades. Our analysis of single-cell amplified genomes revealed hallmarks of the chlamydial lifestyle, supporting the ancient origin of their characteristic developmental cycle and major virulence mechanisms. Surprisingly, these chlamydial genomes encode a complete flagellar apparatus, a previously unreported feature. We show that flagella are an ancient trait that was subject to differential gene loss among extant chlamydiae. Together with a chemotaxis system, these marine chlamydiae are likely motile, with flagella potentially playing a role during host cell infection. This study broadens our view on chlamydial biology and indicates a largely underestimated potential to adapt to different hosts and environments.

A variety of Chlamydia species belonging to the Chlamydiaceae family have been reported in reptilian hosts but scarce data about their occurrence in turtles and tortoises are available. In this study, research was conducted to acquire information on invasive alien species (IAS) of turtles and indigenous turtles and tortoises, living both free and in captivity, as possible reservoirs of Chlamydiaceae. Analysis of specimens (pharyngeal and cloacal swabs and tissues) from 204 turtles and tortoises revealed an overall Chlamydiaceae prevalence of 18.3% and 28.6% among free-living and captive animals respectively, with variable levels of shedding. Further testing conducted with a species-specific real-time PCR and microarray test was unsuccessful. Subsequently sequencing was applied to genotype the Chlamydiaceae-positive samples. Almost the full lengths of the 16S rRNA and ompA genes as well as the 16S-23S intergenic spacer (IGS) and 23S rRNA domain I were obtained for 14, 20 and 8 specimens respectively. Phylogenetic analysis of 16S rRNA amplicons revealed two distinct branches. Group 1 (10 specimens), specific to freshwater turtles and reported here for the first time, was most closely related to Chlamydia (C.) pneumoniae strains and the newly described Candidatus C. sanzinia. Group 2 (four specimens), detected in Testudo spp. samples, showed highest homology to C. pecorum strains but formed a separate sub-branch. Finally, molecular analysis conducted on positive samples together with their geographical distribution in places distant from each other strongly suggest that Group 1 specimens correspond to a new species in the Chlamydiaceae family. In-depth studies of Chlamydia spp. from turtles and tortoises are needed to further characterise these atypical strains and address arising questions about their pathogenicity and zoonotic potential.

Advances in culture-independent methods have meant that we can more readily detect and diagnose emerging infectious disease threats in humans and animals. Metagenomics is fast becoming a popular tool for detection and characterisation of novel bacterial pathogens in their environment, and is particularly useful for obligate intracellular bacteria such as Chlamydiae that require labour-intensive culturing. We have used this tool to investigate the microbial metagenomes of Chlamydia-positive cloaca and choana samples from snakes. The microbial complexity within these anatomical sites meant that despite previous detection of chlamydial 16S rRNA sequences by single-gene broad-range PCR, only a chlamydial plasmid could be detected in all samples, and a chlamydial chromosome in one sample. Comparative genomic analysis of the latter revealed it represented a novel taxon, Ca. Chlamydia corallus, with genetic differences in regards to purine and pyrimidine metabolism. Utilising statistical methods to relate plasmid phylogeny to the phylogeny of chromosomal sequences showed that the samples also contain additional novel strains of Ca. C. corallus and two putative novel species in the genus Chlamydia. This study highlights the value of metagenomics methods for rapid novel bacterial discovery and the insights it can provide into the biology of uncultivable intracellular bacteria such as Chlamydiae.

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial-animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial 'Candidatus Syngnamydia', and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: 'Candidatus Syngnamydia medusae', 'Candidatus Medusoplasma mediterranei' and 'Candidatus Tenacibaculum medusae'., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

A 5-yr-old, captive, hatched, female false gharial (Tomistoma schlegelii) presented with a 1-mo history of cervical spinal curvature. Antemortem diagnostics, including blood work, electromyography, muscle biopsies, and advanced imaging tests, were either within reference ranges or did not identify any specific etiology. Necropsy revealed extensive, marked, chronic granulomatous encephalomyelitis along with neuronal necrosis, rarefaction, gliosis, and astrocytosis of the white and gray matter of the cerebrum, cerebellum, brainstem, and spinal cord. Pan-chlamydiae polymerase chain reaction protocols for the 16S ribosomal RNA and ompA genes were performed on samples of spinal cord and brain, and both resulted in amplicons. Sequencing of the products revealed that they were positive for a novel Chlamydia species. Infections by members of the phylum Chlamydiae have been reported in a diverse range of vertebrate hosts, including crocodilians. Chlamydia spp. infections are likely underdiagnosed because of a paucity of diagnostic techniques specific for detection. This is the first case report of a novel Chlamydia species associated with severe granulomatous encephalomyelitis in a false gharial.

Chlamydia suis infections are endemic in domestic pigs in Europe and can lead to conjunctivitis, pneumonia, enteritis and reproductive failure. Currently, the knowledge on the zoonotic potential of C. suis is limited. Moreover, the last decades, porcine tetracycline resistant C. suis strains have been isolated, which interfere with treatment of chlamydial infections. In this study, the presence of C. suis was examined on nine Belgian pig farms, using Chlamydia culture and a C. suis specific real-time PCR in both pigs and farmers. In addition to diagnosis for C. suis, the farmers' samples were examined using a Chlamydia trachomatis PCR. Additionally, the Chlamydia isolates were tested for the presence of the tet(C) resistance gene. C. DNA was demonstrated in pigs on all farms, and eight of nine farmers were positive in at least one anatomical site. None of the farmers tested positive for C. trachomatis. Chlamydia suis isolates were obtained from pigs of eight farms. Nine porcine C. suis isolates possessing a tet(C) gene were retrieved, originating from three farms. Moreover, C. suis isolates were identified in three human samples, including one pharyngeal and two rectal samples. These findings suggest further research on the zoonotic transfer of C. suis from pigs to humans is needed., (© 2015 Blackwell Verlag GmbH.)