A novel multi-variate immunological approach, reveals immune variation associated with environmental conditions, and co-infection in the koala (Phascolarctos cinereus).

External signs of disease are frequently used as indicators of disease susceptibility. However, immune profiling can be a more effective indicator to understand how host responses to infection may be shaped by host, pathogen and environmental factors. To better inform wildlife health assessment and research directions, we investigated the utility of a novel multivariate immunophenotyping approach examining innate and adaptive immune responses in differing climatic, pathogen co-infection and demographic contexts across two koala (Phascolarctos cinereus) populations in New South Wales: the Liverpool Plains (LP), and Southern Highlands to South-west Sydney (SHSWS). Relative to the comparatively healthy SHSWS, the LP had greater and more variable innate immune gene expression (IL-1ß, IL-6), and KoRV transcription. During extreme heat and drought, koalas from the LP displayed upregulation of a stress pathway gene and reduced adaptive immune genes expression, haematocrit and plasma protein, suggesting the possibility of environmental impacts through multiple pathways. In those koalas, KoRV transcription status, Chlamydia pecorum infection loads, and visible urogenital inflammation were not associated with immune variation, suggesting that immune markers were more sensitive indicators of real-time impacts than observed disease outcomes.

Investigation of Chlamydia pecorum in livestock from Switzerland reveals a high degree of diversity in bovine strains.

Chlamydia pecorum is a widespread veterinary chlamydial species causing endemic infections in livestock, such as ruminants and pigs, globally. However, there is limited contemporary knowledge on infecting strain diversity in various hosts. This study aimed to evaluate the genetic diversity of C. pecorum strains infecting Swiss livestock through C. pecorum genotyping and phylogenetic analyses in comparison to the global population, while also assessing chlamydial strains for plasmid carriage. A total of 263 C. pecorum positive samples from clinically healthy ruminant and pig herds (Bovines = 216, sheep = 25, pigs = 14) as well as placentae from eight C. pecorum positive ruminant abortion cases from other Swiss herds were investigated. The ompA and Multi-Locus sequence typing revealed novel C. pecorum genotypes, and bovine strains exhibited considerable genetic diversity, contrasting with lower diversity in sheep and pig strains. C. pecorum plasmid was detected in 100.0% of sheep (41/41) and pig (255/255) samples, and in 69.4% of bovine samples (150/216). In contrast, no plasmid was detected in the eight C. pecorum-positive ruminant abortion cases either representing plasmid-less strains or possibly escaping PCR detection due to autolysis of the placenta. This study supports the genetic diversity of C. pecorum strains, particularly in bovines, and identifies novel sequence types in Swiss livestock.

Diversity and genetic characterization of Chlamydia isolated from Siamese crocodiles (Crocodylus siamensis).

Chlamydiosis, an infection caused by several Chlamydia species, has been reported in Nile, saltwater, and Siamese crocodiles. Despite its widespread reports in various countries, including Thailand, genetic information on Chlamydia species remains limited. This study presents a whole-genome-based characterization of Siamese crocodile-isolated Chlamydia. The results showed that Siamese crocodile Chlamydia contained a single circular chromosome with a size of 1.22-1.23 Mbp and a plasmid with a size of 7.7-8.0 kbp. A plasmid containing eight coding sequences (CDSs) was grouped in a ß lineage. A chromosome sequence had approximately 1,018-1,031 CDSs. Chlamydial factors involving virulence were documented in terms of the presence of cytotoxins and several virulence factors in the chromosomes of Siamese crocodile Chlamydia. The analysis of antimicrobial resistance genes in the Chlamydia genome revealed that the most common resistance genes were associated with aminoglycosides, fluoroquinolones, macrolides, tetracyclines, and cephalosporins, with loose matching (identities between 21.12 % and 74.65 %). Phylogenetic analyses, encompassing the assessments of both whole proteome and nine taxonomic markers, revealed that Siamese crocodile Chlamydia was separated into three lineages (lineages I-III) with high bootstrapping statistic support. Interestingly, isolate 12-01 differed genetically from the others, suggesting that it is a new member of Chlamydia. The study findings indicate that Siamese crocodiles are susceptible hosts to Chlamydia, involving more than one species. This study is the first employing the highest number of whole-genome data on Siamese crocodile Chlamydia and provides better insights into pathogen genetics.

Detection of a novel genotype of Chlamydia buteonis in falcons from the Emirates.

Chlamydiaceae are a family of obligate intracellular bacterial pathogens that affect both humans and animals. Recently, a new species named Chlamydia (C.) buteonis was isolated from hawks. In this study, we aimed to investigate the prevalence of Chlamydiaceae in 60 falcons that underwent a routine health check at a specialized clinic in Dubai, United Arab Emirates. Using real-time PCR, we analyzed cloacal and tracheal swabs from these birds and found that 39 of them tested positive for Chlamydiaceae. Subsequent real-time PCR assays specific for C. psittaci, C. abortus, C. avium, and C. gallinacea yielded negative results, while testing positive for C. buteonis. Analysis of ompA and MLST sequences indicated a highly conserved group of strains within this set of samples, but with sequences distinct from the C. buteonis RSHA reference strains and other C. buteonis strains isolated from hawks in the United States. Two strains were further isolated by cell culture and sequenced using whole-genome sequencing, confirming the clustering of these falcon strains within the C. buteonis species, but in a separate clade from the previously identified hawk strains. We also developed a SNP-based PCR-HRM assay to distinguish between these different genotypes. Overall, our findings suggest a high prevalence of C. buteonis in falcons in Dubai and highlight the importance of monitoring this pathogen in birds of prey.

The Chlamydia-related Waddlia chondrophila encodes functional type II toxin-antitoxin systems.

Bacterial toxin-antitoxin (TA) systems are widespread in chromosomes and plasmids of free-living microorganisms, but only a few have been identified in obligate intracellular species. We found seven putative type II TA modules in Waddlia chondrophila, a Chlamydia-related species that is able to infect a very broad series of eukaryotic hosts, ranging from protists to mammalian cells. The RNA levels of Waddlia TA systems are significantly upregulated by iron starvation and novobiocin, but they are not affected by antibiotics such as ß-lactams and glycopeptides, which suggests different mechanisms underlying stress responses. Five of the identified TA modules, including HigBA1 and MazEF1, encoded on the Waddlia cryptic plasmid, proved to be functional when expressed in a heterologous host. TA systems have been associated with the maintenance of mobile genetic elements, bacterial defense against bacteriophages, and persistence upon exposure to adverse conditions. As their RNA levels are upregulated upon exposure to adverse conditions, Waddlia TA modules may be involved in survival to stress. Moreover, as Waddlia can infect a wide range of hosts including free-living amoebae, TA modules could also represent an innate immunity system to fight against bacteriophages and other microorganisms with which Waddlia has to share its replicative niche.IMPORTANCEThe response to adverse conditions, such as exposure to antibiotics, nutrient starvation and competition with other microorganisms, is essential for the survival of a bacterial population. TA systems are modules composed of two elements, a toxic protein and an antitoxin (protein or RNA) that counteracts the toxin. Although many aspects of TA biological functions still await to be elucidated, TAs have often been implicated in bacterial response to stress, including the response to nutrient starvation, antibiotic treatment and bacteriophage infection. TAs are ubiquitous in free-living bacteria but rare in obligate intracellular species such as chlamydiae. We identified functional TA systems in Waddlia chondrophila, a chlamydial species with a strikingly broad host range compared to other chlamydiae. Our work contributes to understand how obligate intracellular bacteria react to adverse conditions that might arise from competition with other viruses/bacteria for the same replicative niche and would threaten their ability to replicate.

Longitudinal study of Chlamydia pecorum in a healthy Swiss cattle population.

Chlamydia pecorum is a globally endemic livestock pathogen but prevalence data from Switzerland has so far been limited. The present longitudinal study aimed to get an insight into the C. pecorum prevalence in Swiss cattle and investigated infection dynamics. The study population consisted of a bovine herd (n = 308) located on a farm in the north-eastern part of Switzerland. The herd comprised dairy cows, beef cattle and calves all sampled up to five times over a one-year period. At each sampling timepoint, rectal and conjunctival swabs were collected resulting in 782 samples per sampled area (total n = 1564). Chlamydiaceae screening was performed initially, followed by C. pecorum-specific real-time qPCR on all samples. For C. pecorum-positive samples, bacterial loads were determined. In this study, C. pecorum was the only chlamydial species found. Animal prevalences were determined to be 5.2-11.4%, 38.1-61.5% and 55-100% in dairy cows, beef cattle and calves, respectively. In all categories, the number of C. pecorum-positive samples was higher in conjunctival (n = 151) compared to rectal samples (n = 65), however, the average rectal load was higher. At a younger age, the chlamydial prevalence and the mean bacterial loads were significantly higher. Of all sampled bovines, only 9.4% (29/308) were high shedders (number of copies per µl >1,000). Calves, which tested positive multiple times, either failed to eliminate the pathogen between sampling timepoints or were reinfected, whereas dairy cows were mostly only positive at one timepoint. In conclusion, C. pecorum was found in healthy Swiss cattle. Our observations suggested that infection takes place at an early age and immunity might develop over time. Although the gastrointestinal tract is supposed to be the main infection site, C. pecorum was not present in rectal samples from dairy cows.

Genetic markers of Chlamydia pecorum virulence in ruminants support short term host-pathogen evolutionary relationships in the koala, Phascolarctos cinereus.

In ruminants infected with Chlamydia pecorum, shorter lengths of coding tandem repeats (CTR) within two genes, the inclusion membrane protein (incA) and Type III secretor protein (ORF663), have been previously associated with pathogenic outcomes. In other chlamydial species, the presence of a chlamydial plasmid has been linked to heightened virulence, and the plasmid is not ubiquitous in C. pecorum across the koala's range. We therefore investigated these three markers: incA, ORF663 and C. pecorum plasmid, as potential indicators of virulence in two koala populations in New South Wales with differing expression of urogenital chlamydiosis; the Liverpool Plains and one across the Southern Highlands and South-west Sydney (SHSWS). We also investigated the diversity of these loci within strains characterised by the national multi-locus sequence typing (MLST) scheme. Although CTR lengths of incA and ORF663 varied across the populations, they occurred only within previously described pathogenic ranges for ruminants. This suggests a relatively short-term host-pathogen co-evolution within koalas and limits the utility of CTR lengths for incA and ORF663 as virulence markers in the species. However, in contrast to reports of evolution of C. pecorum towards lower virulence, as indicated by longer CTR lengths in ruminants and swine, CTR lengths for ORF663 appeared to be diverging towards less common shorter CTR lengths within strains recently introduced to koalas in the Liverpool Plains. We detected the plasmid across 90% and 92% of samples in the Liverpool Plains and SHSWS respectively, limiting its utility as an indicator of virulence. It would be valuable to examine the CTR lengths of these loci across koala populations nationally. Investigation of other hypervariable loci may elucidate the evolutionary trajectory of virulence in C. pecorum induced disease in koalas. Profiling of virulent strains will be important in risk assessments for strain movement to naïve or susceptible populations through translocations and wildlife corridor construction.

Draft genomes of novel avian Chlamydia abortus strains from Australian Torresian crows (Corvus orru) shed light on possible reservoir hosts and evolutionary pathways.

Chlamydia abortus, an obligate intracellular bacterium, is a major causative agent of reproductive loss in ruminants, with zoonotic potential. Though this pathogen is primarily known to infect livestock, recent studies have detected and isolated genetically distinct avian strains of C. abortus from wild birds globally. Before this study, only five avian C. abortus genomes were publicly available. Therefore, we performed culture-independent probe-based whole-genome sequencing on clinical swabs positive for avian C. abortus obtained from Australian Torresian crows (Corvus orru) in 2019 and 2020. We successfully obtained draft genomes for three avian C. abortus strains (C1, C2 and C3), each comprising draft chromosomes with lengths of 1 115 667, 1 120 231 and 1 082 115 bp, and associated 7 553 bp plasmids, with a genome completeness exceeding 92 %. Molecular characterization revealed that these three strains comprise a novel sequence type (ST333), whilst phylogenetic analyses placed all three strains in a cluster with other avian C. abortus genomes. Interestingly, these three strains share a distant genomic relation (2693 single nucleotide variants) with the reference strain 15-58d/44 (ST152), isolated from a Eurasian magpie (Pica pica) in Poland, highlighting the need for more publicly available genomes. Broad comparative analyses with other avian C. abortus genomes revealed that the three draft genomes contain conserved Chlamydia genomic features, including genes coding for type III secretion system and polymorphic membrane proteins, and potential virulence factors such as the large chlamydial cytotoxin, warranting further studies. This research provides the first avian C. abortus draft genomes from Australian birds, highlighting Torresian crows as novel reservoir hosts for these potential pathogens, and demonstrates a practical methodology for sequencing novel Chlamydia genomes without relying on traditional cell culture.

Epidemiology of Chlamydia sp. infection in farmed Siamese crocodiles (Crocodylus siamensis) in Thailand.

BACKGROUND: Although Chlamydia sp. causes widespread disease outbreaks in juvenile crocodiles in Thailand, data regarding the epidemiology, and risk factors of such infections are limited. The aim of this study was to investigate the prevalence and possible risk factors associated with Chlamydia sp. infections on Siamese crocodile (Crocodylus siamensis) farms in Thailand. A cross-sectional study was conducted from July to December 2019. Samples were collected from 40 farms across six regions in Thailand. Conjunctival, pharyngeal, and cloacal swab samples were analyzed for Chlamydiaceae nucleic acids using semi-nested PCR followed by phylogenetic analysis based on the ompA gene fragment. Risk factors of infection were analyzed using chi-square and univariate regression to calculate odds ratios. RESULTS: The prevalence of Chlamydia sp. infection across all regions was 65%. The ompA phylogenetic analysis showed that Chlamydia sp. detected in this study was genetically closely related to Chlamydia crocodili and Chlamydia caviae. The risk factors for infection were water source, reusing treated wastewater from the treatment pond, not disposing of leftover food, low frequency of water replacement in the enclosure of juvenile crocodiles, and lack of water replacement after the death of a crocodile. CONCLUSION: The prevalence of Chlamydia sp. infection in farmed crocodiles in Thailand was 65% during the study period. Cloacal swabs were superior to conjunctival and pharyngeal swabs due to their higher sensitivity in detecting Chlamydia sp., as well as their lower invasiveness. Good management and biosecurity in crocodile farming can reduce the risk of Chlamydia sp.

Whole genome sequencing characteristics of Chlamydia psittaci caprine AMK-16 strain, a promising killed whole cell veterinary vaccine candidate against chlamydia infection.

Chlamydia psittaci is a primary zoonotic pathogen with a broad host range causing severe respiratory and reproductive system infection in animals and humans. To reduce the global burden of C. psittaci-associated diseases on animal welfare and health and to control the pathogen spread in husbandry, effective vaccines based on promising vaccine candidate(s) are required. Recently, the caprine C. psittaci AMK-16 strain (AMK-16) demonstrated a high level of protection (up to 80-100%) in outbred mice and pregnant rabbits immunized with these formaldehyde-inactivated bacteria against experimental chlamydial wild-type infection. This study investigated the molecular characteristics of AMK-16 by whole-genome sequencing followed by molecular typing, phylogenetic analysis and detection of main immunodominant protein(s) eliciting the immune response in mouse model. Similarly to other C. psittaci, AMK-16 harbored an extrachromosomal plasmid. The whole-genome phylogenetic analysis proved that AMK-16 strain belonging to ST28 clustered with only C. psittaci but not with Chlamydia abortus strains. However, AMK-16 possessed the insert which resulted from the recombination event as the additional single chromosome region of a 23,100 bp size with higher homology to C. abortus (98.38-99.94%) rather than to C. psittaci (92.06-92.55%). At least six of 16 CDSs were absent in AMK-16 plasticity zone and 41 CDSs in other loci compared with the reference C. psittaci 6BC strain. Two SNPs identified in the AMK-16 ompA sequence resulted in MOMP polymorphism followed by the formation of a novel genotype/subtype including three other C. psittaci strains else. AMK-16 MOMP provided marked specific cellular and humoral immune response in 100% of mice immunized with the inactivated AMK-16 bacteria. Both DnaK and GrpE encoded by the recombination region genes were less immunoreactive, inducing only a negligible T-cell murine immune response, while homologous antibodies could be detected in 50% and 30% of immunized mice, respectively. Thus, AMK-16 could be a promising vaccine candidate for the development of a killed whole cell vaccine against chlamydiosis in livestock.

Chlamydia suis displays high transformation capacity with complete cloning vector integration into the chromosomal rrn-nqrF plasticity zone.

IMPORTANCE: The obligate intracellular Chlamydia genus contains many pathogens with a negative impact on global health and economy. Despite recent progress, there is still a lack of genetic tools limiting our understanding of these complex bacteria. This study provides new insights into genetic manipulation of Chlamydia with the opportunistic porcine pathogen Chlamydia suis, the only chlamydial species naturally harboring an antibiotic resistance gene, originally obtained by horizontal gene transfer. C. suis is transmissible to humans, posing a potential public health concern. We report that C. suis can take up vectors that lack the native plasmid, a requirement for most chlamydial transformation systems described to date. Additionally, we show that C. trachomatis, the most common cause for bacterial sexually transmitted infections and infectious blindness worldwide, can be transformed with C. suis vectors. Finally, the chromosomal region that harbors the resistance gene of C. suis is highly susceptible to complete vector integration.

A Recombinase-Aided Amplification Assay for the Detection of Chlamydia felis.

Chlamydia felis is an important zoonotic agent for humans and various animals. A recombinase-aided amplification (RAA) assay was developed for detecting C. felis. RAA can be performed in a closed tube at 39°C within 30 min. The detection limit was 10.6 copies of the C. felis plasmid DNA per reaction. No positive signals for other pathogens were detected. The coincidence rate of RAA and conventional PCR was 95.24% (20/21) and 100% (96/96) for positive and negative samples, respectively. The established RAA assay is a simple, rapid, highly sensitive, and specific method for detecting C. felis.

Evaluation of a biosecurity survey approach for contamination by Chlamydia pecorum in koala rehabilitation, field capture, and captive settings.

Transmission of Chlamydia pecorum between koalas is a potential risk in field capture or rehabilitation settings, where koalas are held in proximity to each other, or equipment is shared between animals. Given the impact of C. pecorum on koala welfare and population viability it is surprising that quarantine and disinfection protocols in a koala rehabilitation facility or capture settings have not previously been evaluated. This study aimed to evaluate an approach, based on the detection of chlamydial DNA and cell viability, to determine the degree of environmental contamination within a koala care facility. Various fomite sites associated with koala care at a koala rehabilitation facility in New South Wales, Australia were identified as potential sources of chlamydial contamination, following exposure to koalas known to be infected with C. pecorum. Fomite sites were swabbed following exposure, and again after decontamination procedures were carried out. Samples were tested for the presence of chlamydial DNA using qPCR and viability using both RT-qPCR and cell culture. From a total of 239 sampling events, 30 tested qPCR positive for chlamydial DNA, with 19 and 11 samples corresponding to pre-decontamination and post-decontamination events respectively. Detection of chlamydial DNA appeared to be most common in the examination room, especially on fomite sites in direct contact with koalas. Physical removal of chlamydial DNA, or its degradation by the elements, appeared to be more common on outdoor enclosures, clothing, and hands. Based on the cell culture assay, of the pre-decontamination samples with chlamydial DNA, eight had viable chlamydial cells, two of these at low levels. Of the post-decontamination samples with chlamydial DNA, one had a moderate number, and one had a very low number of viable chlamydial cells. RT-qPCR was unsuccessful in determining cell viability due to low yields of RNA and high levels of contaminants from the environmental samples. The outcomes of this study provide a knowledge base for the design of future biosecurity evaluation guidelines in captive and koala rehabilitation facilities. The higher incidence of chlamydial DNA detection by qPCR than viable organism highlights the need to use viability assays in similar studies. However, further investment is still needed to optimise these methods and improve sensitivity for complex environmental samples.

The Fish Pathogen "Candidatus Clavichlamydia salmonicola"-A Missing Link in the Evolution of Chlamydial Pathogens of Humans.

Chlamydiae like Chlamydia trachomatis and Chlamydia psittaci are well-known human and animal pathogens. Yet, the chlamydiae are a much larger group of evolutionary ancient obligate intracellular bacteria that includes predominantly symbionts of protists and diverse animals. This makes them ideal model organisms to study evolutionary transitions from symbionts in microbial eukaryotes to pathogens of humans. To this end, comparative genome analysis has served as an important tool. Genome sequence data for many chlamydial lineages are, however, still lacking, hampering our understanding of their evolutionary history. Here, we determined the first high-quality draft genome sequence of the fish pathogen "Candidatus Clavichlamydia salmonicola", representing a separate genus within the human and animal pathogenic Chlamydiaceae. The "Ca. Clavichlamydia salmonicola" genome harbors genes that so far have been exclusively found in Chlamydia species suggesting that basic mechanisms important for the interaction with chordate hosts have evolved stepwise in the history of chlamydiae. Thus, the genome sequence of "Ca. Clavichlamydia salmonicola" allows to constrain candidate genes to further understand the evolution of chlamydial virulence mechanisms required to infect mammals.

Recent advances in genetic systems in obligate intracellular human-pathogenic bacteria.

The ability to genetically manipulate a pathogen is fundamental to discovering factors governing host-pathogen interactions at the molecular level and is critical for devising treatment and prevention strategies. While the genetic "toolbox" for many important bacterial pathogens is extensive, approaches for modifying obligate intracellular bacterial pathogens were classically limited due in part to the uniqueness of their obligatory lifestyles. Many researchers have confronted these challenges over the past two and a half decades leading to the development of multiple approaches to construct plasmid-bearing recombinant strains and chromosomal gene inactivation and deletion mutants, along with gene-silencing methods enabling the study of essential genes. This review will highlight seminal genetic achievements and recent developments (past 5 years) for Anaplasma spp., Rickettsia spp., Chlamydia spp., and Coxiella burnetii including progress being made for the still intractable Orientia tsutsugamushi. Alongside commentary of the strengths and weaknesses of the various approaches, future research directions will be discussed to include methods for C. burnetii that should have utility in the other obligate intracellular bacteria. Collectively, the future appears bright for unraveling the molecular pathogenic mechanisms of these significant pathogens.

Clinical identification and microbiota analysis of Chlamydia psittaci- and Chlamydia abortus- pneumonia by metagenomic next-generation sequencing.

Introduction: Recently, the incidence of chlamydial pneumonia caused by rare pathogens such as C. psittaci or C. abortus has shown a significant upward trend. The non-specific clinical manifestations and the limitations of traditional pathogen identification methods determine that chlamydial pneumonia is likely to be poorly diagnosed or even misdiagnosed, and may further result in delayed treatment or unnecessary antibiotic use. mNGS's non-preference and high sensitivity give us the opportunity to obtain more sensitive detection results than traditional methods for rare pathogens such as C. psittaci or C. abortus. Methods: In the present study, we investigated both the pathogenic profile characteristics and the lower respiratory tract microbiota of pneumonia patients with different chlamydial infection patterns using mNGS. Results: More co-infecting pathogens were found to be detectable in clinical samples from patients infected with C. psittaci compared to C. abortus, suggesting that patients infected with C. psittaci may have a higher risk of mixed infection, which in turn leads to more severe clinical symptoms and a longer disease course cycle. Further, we also used mNGS data to analyze for the first time the characteristic differences in the lower respiratory tract microbiota of patients with and without chlamydial pneumonia, the impact of the pattern of Chlamydia infection on the lower respiratory tract microbiota, and the clinical relevance of these characteristics. Significantly different profiles of lower respiratory tract microbiota and microecological diversity were found among different clinical subgroups, and in particular, mixed infections with C. psittaci and C. abortus resulted in lower lung microbiota diversity, suggesting that chlamydial infections shape the unique lung microbiota pathology, while mixed infections with different Chlamydia may have important effects on the composition and diversity of the lung microbiota. Discussion: The present study provides possible evidences supporting the close correlation between chlamydial infection, altered microbial diversity in patients' lungs and clinical parameters associated with infection or inflammation in patients, which also provides a new research direction to better understand the pathogenic mechanisms of pulmonary infections caused by Chlamydia.

Genome Dynamics and Temperature Adaptation During Experimental Evolution of Obligate Intracellular Bacteria.

Evolution experiments with free-living microbes have radically improved our understanding of genome evolution and how microorganisms adapt. Yet there is a paucity of such research focusing on strictly host-associated bacteria, even though they are widespread in nature. Here, we used the Acanthamoeba symbiont Protochlamydia amoebophila, a distant relative of the human pathogen Chlamydia trachomatis and representative of a large group of protist-associated environmental chlamydiae, as a model to study how obligate intracellular symbionts evolve and adapt to elevated temperature, a prerequisite for the pivotal evolutionary leap from protist to endothermic animal hosts. We established 12 replicate populations under two temperatures (20 °C, 30 °C) for 510 bacterial generations (38 months). We then used infectivity assays and pooled whole-genome resequencing to identify any evolved phenotypes and the molecular basis of adaptation in these bacteria. We observed an overall reduction in infectivity of the symbionts evolved at 30 °C, and we identified numerous nonsynonymous mutations and small indels in these symbiont populations, with several variants persisting throughout multiple time points and reaching high frequencies. This suggests that many mutations may have been beneficial and played an adaptive role. Mutated genes within the same temperature regime were more similar than those between temperature regimes. Our results provide insights into the molecular evolution of intracellular bacteria under the constraints of strict host dependance and highly structured populations and suggest that for chlamydial symbionts of protists, temperature adaptation was facilitated through attenuation of symbiont infectivity as a tradeoff to reduce host cell burden.

Genome organization and genomics in Chlamydia: whole genome sequencing increases understanding of chlamydial virulence, evolution, and phylogeny.

The genus Chlamydia contains important obligate intracellular bacterial pathogens to humans and animals, including C. trachomatis and C. pneumoniae. Since 1998, when the first Chlamydia genome was published, our understanding of how these microbes interact, evolved and adapted to different intracellular host environments has been transformed due to the expansion of chlamydial genomes. This review explores the current state of knowledge in Chlamydia genomics and how whole genome sequencing has revolutionised our understanding of Chlamydia virulence, evolution, and phylogeny over the past two and a half decades. This review will also highlight developments in multi-omics and other approaches that have complemented whole genome sequencing to advance knowledge of Chlamydia pathogenesis and future directions for chlamydial genomics.

Detection of Chlamydia abortus in aborted chorioallantoises of horses from Western Canada.

Chlamydiae are reported to cause abortion in several species, however the association between Chlamydia sp. and equine abortions is poorly understood. A zoonotic transfer event of C. psittaci from aborted equine tissues in Australia has emphasized the need to better understand the prevalence of this pathogen in equine populations. The prevalence of chlamydia in equine abortions in North America has not been investigated thoroughly. We examined 99 formalin-fixed, paraffin-embedded placental samples submitted between 2009 and 2020 from equine abortions in Western Canada using chlamydia-specific 16S rRNA conventional PCR testing; 26 of 99 submissions tested positive for chlamydial DNA. Most of these submissions (n = 17) had no final diagnosis noted on their original pathology reports. DNA sequencing identified 22 of the 26 cases as C. abortus; 21 of the 22 C. abortus-positive samples were positive on chlamydial immunohistochemistry. These findings contrast with studies in Europe that found a low prevalence of chlamydiae using similar methodology. The high prevalence of the potentially zoonotic C. abortus identified in our study suggests that more substantial biosecurity protocols may be warranted for equine foaling, abortion, and stillbirth in Western Canada to prevent zoonotic transfer of the pathogen.