Complete genome sequence of a Salmonella enterica strain of serovar Agona associated with an outbreak in France.

We report here the complete genome of one Salmonella Agona strain isolated in 2017 from a dried milk powder in France.

Isolation of Burkholderia pseudomallei from a goat in New Caledonia: implications for animal and human health monitoring and serological tool comparison.

BACKGROUND: Melioidosis is a serious bacterial infection caused by Burkholderia pseudomallei, a gram-negative bacterium commonly found in soil and water. It can affect both humans and animals, and is endemic in regions such as Southeast Asia and Northern Australia. In recent years, there have been reports of an emergence of human melioidosis in other areas, including New Caledonia. RESULTS: During standard laboratory analysis in New Caledonia in 2021, a strain of B. pseudomallei was isolated from a goat. The strain was characterized using both MLST and WGS techniques and was found to cluster with previously described local human strains from the area. In parallel, several serological tests (CFT, ELISA, Luminex (Hcp1, GroEL, BPSS1840), arrays assay and a latex agglutination test) were performed on animals from the farm where the goat originated, and/or from three other neighboring farms. Using two commercial ELISA kits, seropositive animals were found only on the farm where the infected goat originated and tests based on recombinant proteins confirmed the usefulness of the Hcp1 protein for the diagnosis of melioidosis in animals. CONCLUSIONS: Despite the regular reports of human cases, this is the first confirmed case of melioidosis in an animal in New Caledonia. These results confirm the presence of the bacterium in the region and highlight the importance of vigilance for both animal and human health. It is critical that all health partners, including breeders, veterinarians, and biologists, work together to monitor and prevent the spread of the disease.

Detection, Characterization and Sequencing of BTV Serotypes Circulating in Cuba in 2022.

In Cuba, despite a high sero-prevalence of bluetongue virus (BTV), circulating serotypes remain unknown. The aim of this study was to identify circulating BTV serotypes in farms throughout the western region of Cuba. Blood samples were collected from 200 young cattle and sheep between May and July 2022 for virological analyses (PCR, viral isolation and virus neutralization) and genome sequencing. The results confirmed viral circulation, with viro-prevalence of 25% for BTV. The virus was isolated from 18 blood samples and twelve BTV serotypes were identified by sequencing RT-PCR products targeting the segment 2 of the BTV genome (BTV-1, 2, 3, 6, 10, 12, 13, 17, 18, 19, 22 and 24). Finally, the full genome sequences of 17 Cuban BTV isolates were recovered using a Sequence Independent Single Primer Amplification (SISPA) approach combined to MinION Oxford Nanopore sequencing technology. All together, these results highlight the co-circulation of a wide diversity of BTV serotypes in a quite restricted area and emphasize the need for entomological and livestock surveillance, particularly in light of recent changes in the global distribution and nature of BTV infections.

Exploring Long-Read Metagenomics for Full Characterization of Shiga Toxin-Producing Escherichia coli in Presence of Commensal E. coli.

The characterization of Shiga toxin-producing Escherichia coli (STEC) is necessary to assess their pathogenic potential, but isolation of the strain from complex matrices such as milk remains challenging. In previous work, we have shown the potential of long-read metagenomics to characterize eae-positive STEC from artificially contaminated raw milk without isolating the strain. The presence of multiple E. coli strains in the sample was shown to potentially hinder the correct characterization of the STEC strain. Here, we aimed at determining the STEC:commensal ratio that would prevent the characterization of the STEC. We artificially contaminated pasteurized milk with different ratios of an eae-positive STEC and a commensal E. coli and applied the method previously developed. Results showed that the STEC strain growth was better than the commensal E. coli after enrichment in acriflavine-supplemented BPW. The STEC was successfully characterized in all samples with at least 10 times more STEC post-enrichment compared to the commensal E. coli. However, the presence of equivalent proportions of STEC and commensal E. coli prevented the full characterization of the STEC strain. This study confirms the potential of long-read metagenomics for STEC characterization in an isolation-free manner while refining its limit regarding the presence of background E. coli strains.

Genomic analysis of 61 Chlamydia psittaci strains reveals extensive divergence associated with host preference.

BACKGROUND: Chlamydia (C.) psittaci, the causative agent of avian chlamydiosis and human psittacosis, is a genetically heterogeneous species. Its broad host range includes parrots and many other birds, but occasionally also humans (via zoonotic transmission), ruminants, horses, swine and rodents. To assess whether there are genetic markers associated with host tropism we comparatively analyzed whole-genome sequences of 61 C. psittaci strains, 47 of which carrying a 7.6-kbp plasmid. RESULTS: Following clean-up, reassembly and polishing of poorly assembled genomes from public databases, phylogenetic analyses using C. psittaci whole-genome sequence alignment revealed four major clades within this species. Clade 1 represents the most recent lineage comprising 40/61 strains and contains 9/10 of the psittacine strains, including type strain 6BC, and 10/13 of human isolates. Strains from different non-psittacine hosts clustered in Clades 2- 4. We found that clade membership correlates with typing schemes based on SNP types, ompA genotypes, multilocus sequence types as well as plasticity zone (PZ) structure and host preference. Genome analysis also revealed that i) sequence variation in the major outer membrane porin MOMP can result in 3D structural changes of immunogenic domains, ii) past host change of Clade 3 and 4 strains could be associated with loss of MAC/perforin in the PZ, rather than the large cytotoxin, iii) the distinct phylogeny of atypical strains (Clades 3 and 4) is also reflected in their repertoire of inclusion proteins (Inc family) and polymorphic membrane proteins (Pmps). CONCLUSIONS: Our study identified a number of genomic features that can be correlated with the phylogeny and host preference of C. psittaci strains. Our data show that intra-species genomic divergence is associated with past host change and includes deletions in the plasticity zone, structural variations in immunogenic domains and distinct repertoires of virulence factors.

Combination of whole genome sequencing and supervised machine learning provides unambiguous identification of eae-positive Shiga toxin-producing Escherichia coli.

Introduction: The objective of this study was to develop, using a genome wide machine learning approach, an unambiguous model to predict the presence of highly pathogenic STEC in E. coli reads assemblies derived from complex samples containing potentially multiple E. coli strains. Our approach has taken into account the high genomic plasticity of E. coli and utilized the stratification of STEC and E. coli pathogroups classification based on the serotype and virulence factors to identify specific combinations of biomarkers for improved characterization of eae-positive STEC (also named EHEC for enterohemorrhagic E.coli) which are associated with bloody diarrhea and hemolytic uremic syndrome (HUS) in human. Methods: The Machine Learning (ML) approach was used in this study on a large curated dataset composed of 1,493 E. coli genome sequences and 1,178 Coding Sequences (CDS). Feature selection has been performed using eight classification algorithms, resulting in a reduction of the number of CDS to six. From this reduced dataset, the eight ML models were trained with hyper-parameter tuning and cross-validation steps. Results and discussion: It is remarkable that only using these six genes, EHEC can be clearly identified from E. coli read assemblies obtained from in silico mixtures and complex samples such as milk metagenomes. These various combinations of discriminative biomarkers can be implemented as novel marker genes for the unambiguous EHEC characterization from different E. coli strains mixtures as well as from raw milk metagenomes.

Avian Chlamydia abortus Strains Detected in Galápagos Waved Albatross (Phoebastria irrorata).

Galápagos Penguin (Spheniscus mendiculus), Flightless Cormorant (Phalacrocorax harrisi), and Waved Albatross (Phoebastria irrorata) are among the most vulnerable species to natural and anthropogenic factors in the Galápagos Islands. In 2017, a dedicated study was conducted to detect Chlamydiaceae on cloacal swabs collected from 59 albatrosses, 68 penguins, and 10 cormorants in different islands and sites in the Galápagos Archipelago. A real-time PCR method targeting the conserved 23S ribosomal RNA gene of the Chlamydiaceae family detected the presence of the bacterium only in albatrosses from Punta Suárez, Española Island, with 21 positive samples (35.6%), whereas negative results were obtained with available real-time PCR systems specific to Chlamydia psittaci and Chlamydia abortus. Multilocus sequence typing (MLST) of the most strongly positive samples revealed a new sequence type closely related to the recently described avian strains of C. abortus. For a quick identification, a new real-time PCR system that allows the detection of all strains (avian and ruminant) belonging to the C. abortus species has been developed. Applied to a second set of samples from 31 albatrosses collected at Punta Suárez, Española Island, in 2018, the new real-time PCR system confirmed the presence of this bacteria in this group of birds, with the same new MLST sequence type.

Hybrid Assembly from 75 E. coli Genomes Isolated from French Bovine Food Products between 1995 and 2016.

Here, we report the complete (or near-complete) genome sequences of 75 Escherichia coli isolates, including 71 stx-positive E. coli isolates, isolated in France between 1995 and 2016 from food of bovine origin. Genomes were assembled using a combination of long- and short-read sequencing.

Features of Mycobacterium bovis Complete Genomes Belonging to 5 Different Lineages.

Mammalian tuberculosis (TB) is a zoonotic disease mainly due to Mycobacterium bovis (M. bovis). A current challenge for its eradication is understanding its transmission within multi-host systems. Improvements in long-read sequencing technologies have made it possible to obtain complete bacterial genomes that provide a comprehensive view of species-specific genomic features. In the context of TB, new genomic references based on complete genomes genetically close to field strains are also essential to perform precise field molecular epidemiological studies. A total of 10 M. bovis strains representing each genetic lineage identified in France and in other countries were selected for performing complete assembly of their genomes. Pangenome analysis revealed a "closed" pangenome composed of 3900 core genes and only 96 accessory genes. Whole genomes-based alignment using progressive Mauve showed remarkable conservation of the genomic synteny except that the genomes have a variable number of copies of IS6110. Characteristic genomic traits of each lineage were identified through the discovery of specific indels. Altogether, these results provide new genetic features that improve the description of M. bovis lineages. The availability of new complete representative genomes of M. bovis will be useful to epidemiological studies and better understand the transmission of this clonal-evolving pathogen.

Evaluation of high molecular weight DNA extraction methods for long-read sequencing of Shiga toxin-producing Escherichia coli.

Next generation sequencing has become essential for pathogen characterization and typing. The most popular second generation sequencing technique produces data of high quality with very low error rates and high depths. One major drawback of this technique is the short reads. Indeed, short-read sequencing data of Shiga toxin-producing Escherichia coli (STEC) are difficult to assemble because of the presence of numerous mobile genetic elements (MGEs), which contain repeated elements. The resulting draft assemblies are often highly fragmented, which results in a loss of information, especially concerning MGEs or large structural variations. The use of long-read sequencing can circumvent these problems and produce complete or nearly complete genomes. The ONT MinION, for its small size and minimal investment requirements, is particularly popular. The ultra-long reads generated with the MinION can easily span prophages and repeat regions. In order to take full advantage of this technology it requires High Molecular Weight (HMW) DNA of high quality in high quantity. In this study, we have tested three different extraction methods: bead-based, solid-phase and salting-out, and evaluated their impact on STEC DNA yield, quality and integrity as well as performance in MinION long-read sequencing. Both the bead-based and salting-out methods allowed the recovery of large quantities of HMW STEC DNA suitable for MinION library preparation. The DNA extracted using the salting-out method consistently produced longer reads in the subsequent MinION runs, compared with the bead-based methods. While both methods performed similarly in subsequent STEC genome assembly, DNA extraction based on salting-out appeared to be the overall best method to produce high quantity of pure HMW STEC DNA for MinION sequencing.

A One-Health Approach to Investigating an Outbreak of Alimentary Tick-Borne Encephalitis in a Non-endemic Area in France (Ain, Eastern France): A Longitudinal Serological Study in Livestock, Detection in Ticks, and the First Tick-Borne Encephalitis Virus Isolation and Molecular Characterisation.

Tick-borne encephalitis virus' (TBEV) geographic range and the human incidence are increasing throughout Europe, putting a number of non-endemic regions and countries at risk of outbreaks. In spring 2020, there was an outbreak of tick-born encephalitis (TBE) in Ain, Eastern France, where the virus had never been detected before. All patients but one had consumed traditional unpasteurised raw goat cheese from a local producer. We conducted an investigation in the suspected farm using an integrative One Health approach. Our methodology included (i) the detection of virus in cheese and milk products, (ii) serological testing of all animals in the suspected farm and surrounding farms, (iii) an analysis of the landscape and localisation of wooded area, (iv) the capture of questing ticks and small mammals for virus detection and estimating enzootic hazard, and (v) virus isolation and genome sequencing. This approach allowed us to confirm the alimentary origin of the TBE outbreak and witness in real-time the seroconversion of recently exposed individuals and excretion of virus in goat milk. In addition, we identified a wooded focus area where and around which there is a risk of TBEV exposure. We provide the first TBEV isolate responsible for the first alimentary-transmitted TBE in France, obtained its full-length genome sequence, and found that it belongs to the European subtype of TBEV. TBEV is now a notifiable human disease in France, which should facilitate surveillance of its incidence and distribution throughout France.

Chlamydia Species and Related Risk Factors in Poultry in North-Western Italy: Possible Bird-to-Human Transmission for C. gallinacea.

Chlamydiaceae are obligatory intracellular bacteria causing acute and chronic diseases in animals and humans worldwide, with recently discovered species with a still unclear pathogenic potential (i.e., C. gallinacea). In Italy, Chlamydiaceae infections are underestimated both in animals and humans. To estimate the prevalence of Chlamydiaceae species in poultry and occupationally exposed workers on farm, a cross-sectional study was carried out in north-western Italy. A total of 2063 samples from 83 commercial and 31 backyard poultry farms were analysed using real-time PCRs for Chlamydiaceae screening and species typing. Chlamydiaceae were detected in 23 farms, with a herd prevalence of 20.2% (95%CI: 13.2-28.7), higher in backyard farms (38.7%; 95%CI: 21.8-57.8) compared to commercial ones (13.3%; 95%CI: 6.8-22.5). C. gallinacea was found in 18 chicken farms, both commercial and backyard, and C. psittaci only in 3 backyard farms. Exposure to wild birds and factors related to biosecurity resulted the main risk factors associated with Chlamydia positivity. Out of the 113 sputum samples collected from farmers, 16 tested positive to Chlamydiaceae, with a prevalence of 14.2% (95%CI: 8, 3-22). To the best of our knowledge, for the first time at international level, C. gallinacea was detected in humans with farmer positivity associated with farm infectious status, suggesting a bird-to-human transmission.

Molecular characterization of Burkholderia mallei strains isolated from horses in Brazil (2014-2017).

Glanders is an infectious zoonosis caused by Burkholderia (B.) mallei that mainly affects equids. The objective of this work was to provide additional knowledge on the diversity of the strains circulating in Brazil. Six Burkholderia mallei isolates obtained during necropsies of glanderous horses between 2014 and 2017 in two different states (Pernambuco and Alagoas) were analyzed by polymerase chain reaction-high-resolution melting (PCR-HRM). While four strains (9902 RSC, BM_campo 1, BM_campo 3 and UFAL2) clustered in the L3B2 branch, which already includes the Brazilian 16-2438_BM#8 strain, two strains (BM_campo 2.1 and BM_campo 2.2) clustered within the L3B3sB3 branch, which mostly includes older isolates, from Europe and the Middle East. Whole genome sequencing of two of these strains (UFAL2 and BM_campo 2.1), belonging to different branches, confirmed the HRM typing results and refined the links between the strains, including the description of the L3B3Sb3Gp1SbGp1 genotype, never reported so far for contemporary strains. These results suggest different glanders introduction events in Brazil, including a potential link with strains of European origin, related to colonization or trade.

A step forward for Shiga toxin-producing Escherichia coli identification and characterization in raw milk using long-read metagenomics.

Shiga toxin-producing Escherichia coli (STEC) are a cause of severe human illness and are frequently associated with haemolytic uraemic syndrome (HUS) in children. It remains difficult to identify virulence factors for STEC that absolutely predict the potential to cause human disease. In addition to the Shiga-toxin (stx genes), many additional factors have been reported, such as intimin (eae gene), which is clearly an aggravating factor for developing HUS. Current STEC detection methods classically rely on real-time PCR (qPCR) to detect the presence of the key virulence markers (stx and eae). Although qPCR gives an insight into the presence of these virulence markers, it is not appropriate for confirming their presence in the same strain. Therefore, isolation steps are necessary to confirm STEC viability and characterize STEC genomes. While STEC isolation is laborious and time-consuming, metagenomics has the potential to accelerate the STEC characterization process in an isolation-free manner. Recently, short-read sequencing metagenomics have been applied for this purpose, but assembly quality and contiguity suffer from the high proportion of mobile genetic elements occurring in STEC strains. To circumvent this problem, we used long-read sequencing metagenomics for identifying eae-positive STEC strains using raw cow's milk as a causative matrix for STEC food-borne outbreaks. By comparing enrichment conditions, optimizing library preparation for MinION sequencing and generating an easy-to-use STEC characterization pipeline, the direct identification of an eae-positive STEC strain was successful after enrichment of artificially contaminated raw cow's milk samples at a contamination level as low as 5 c.f.u. ml-1. Our newly developed method combines optimized enrichment conditions of STEC in raw milk in combination with a complete STEC analysis pipeline from long-read sequencing metagenomics data. This study shows the potential of the innovative methodology for characterizing STEC strains from complex matrices. Further developments will nonetheless be necessary for this method to be applied in STEC surveillance.

A New SNP-Based Genotyping Method for C. psittaci: Application to Field Samples for Quick Identification.

Chlamydia (C.) psittaci is the causative agent of avian chlamydiosis and human psittacosis. In this study, we extracted single-nucleotide polymorphisms (SNPs) from the whole genome sequences of 55 C. psittaci strains and identified eight major lineages, most of which are host-related. A combined PCR/high-resolution melting (HRM) assay was developed to screen for eight phylogenetically informative SNPs related to the identified C. psittaci lineages. The PCR-HRM method was validated on 11 available reference strains and with a set of 118 field isolates. Overall, PCR-HRM clustering was consistent with previous genotyping data obtained by ompA and/or MLST analysis. The method was then applied to 28 C. psittaci-positive samples from animal or human cases. As expected, PCR-HRM typing results from human samples identified genotypes linked to ducks and pigeons, a common source of human exposure, but also to the poorly described Mat116-like genotype. The new genotyping method does not require time-consuming sequencing and allows a quick identification of the source of infection.

A genetic variant of Burkholderia mallei detected in Kuwait: Consequences for the PCR diagnosis of glanders.

Glanders is a contagious zoonotic disease caused by Burkholderia mallei. Following the detection of glanders positive horses using the OIE complement fixation test, the tissues of two horses were analysed by PCR. While PCR systems targeting the Burkholderia pseudomallei complex gave positive signals, the species-specific PCR systems targeting B. mallei (fliP-IS407A) and B. pseudomallei (orf11)-the OIE recommended targets-resulted in negative signals. However, the presence of B. mallei in these tissues was confirmed with a recently described B. mallei-specific real-time PCR system and genotyping with MLST- and SNP-based methods, performed on the most positive tissue, identified a genotype closely related to B. mallei strains recently isolated in the Middle East. This study leads to recommendations regarding the use of PCR systems for the molecular diagnosis of glanders, especially in regions where the circulating B. mallei strains have not yet been fully genetically characterized.

Comparative Genome Analysis of 33 Chlamydia Strains Reveals Characteristic Features of Chlamydia Psittaci and Closely Related Species.

To identify genome-based features characteristic of the avian and human pathogen Chlamydia(C.) psittaci and related chlamydiae, we analyzed whole-genome sequences of 33 strains belonging to 12 species. Using a novel genome analysis tool termed Roary ILP Bacterial Annotation Pipeline (RIBAP), this panel of strains was shown to share a large core genome comprising 784 genes and representing approximately 80% of individual genomes. Analyzing the most variable genomic sites, we identified a set of features of C. psittaci that in its entirety is characteristic of this species: (i) a relatively short plasticity zone of less than 30,000 nt without a tryptophan operon (also in C. abortus, C. avium, C. gallinacea, C. pneumoniae), (ii) a characteristic set of of Inc proteins comprising IncA, B, C, V, X, Y (with homologs in C. abortus, C. caviae and C. felis as closest relatives), (iii) a 502-aa SinC protein, the largest among Chlamydia spp., and (iv) an elevated number of Pmp proteins of subtype G (14 in C. psittaci, 14 in Cand. C. ibidis). In combination with future functional studies, the common and distinctive criteria revealed in this study provide important clues for understanding the complexity of host-specific behavior of individual Chlamydia spp.

Chlamydiosis in a Gouldian Finch (Erythrura gouldiae).

Avian chlamydiosis is an infection caused by obligate intracellular and Gram-negative bacteria belonging to the family Chlamydiaceae and has been reported in more than 450 avian species distributed in 30 orders. In particular, a high prevalence of infection has been demonstrated in wild passerine populations, including both asymptomatic and clinically ill individuals, suggesting a role of these avian species as important carriers. In May 2018, avian chlamydiosis was diagnosed in a 1-year-old male Gouldian finch (Erythrura gouldiae) at the Turlock Branch of the California Animal Health and Food Safety Laboratory System. The bird belonged to an outdoor aviary with mixed avian species, including Gouldian finches, doves (Geopelia cuneata and Spilopelia chinensis), and psittacines (Aratinga, Psittacula, Pyrrhura, and Trichoglossus sp.). Severe respiratory distress and mortality were noted among the finches. Gross and histopathologic lesions were concentrated in the liver and spleen, with a mild involvement of the upper respiratory tract. Chlamydia spp. were detected in the spleen and kidney by real-time PCR and were further confirmed by immunohistochemistry. Subsequently, Chlamydia psittaci was isolated from the liver and spleen and characterized as a CP3-like strain (genotype B). In addition, viral particles compatible with circovirus were identified in the liver by direct electron microscopy. To the authors' knowledge, this is the first report of avian chlamydiosis with hepatic viral particles consistent with circovirus infection in a Gouldian finch.

Reporte de caso- Clamidiosis en un pinzón diamante de Gould (Chloebia gouldiae). La clamidiosis aviar es una infección causada por bacterias intracelulares y Gramnegativas obligadas que pertenecen a la familia Chlamydiaceae y se ha reportado en más de 450 especies de aves distribuidas en 30 órdenes. En particular, se ha demostrado una alta prevalencia de infección en poblaciones de paseriformes silvestres, incluyendo individuos asintomáticos y clínicamente enfermos, lo que sugiere un papel de estas especies aviares como portadores importantes. En mayo del año 2018, se diagnosticó clamidiosis aviar en un pinzón diamante de Gould (Chloebia gouldiae) de un año de edad remitido a la sede en Turlock del Sistema de Laboratorios de Salud Animal y Seguridad Alimentaria del Estado de California. El ave pertenecía a un aviario al aire libre con especies mixtas de aves, incluyendo los diamantes de Gould, palomas (Geopelia cuneata y Spilopelia chinensis) y psitacinas (Aratinga, Psittacula, Pyrrhura y Trichoglossus sp.). Se observaron problemas respiratorios severos y mortalidad entre los pinzones. Las lesiones macroscópicas e histopatológicas se concentraron en el hígado y el bazo, con problemas leves en el tracto respiratorio superior. Se detectó Chlamydia spp. en el bazo por PCR en tiempo real y fueron confirmados por inmunohistoquímica. Posteriormente, se aisló Chlamydia psittaci del hígado y el bazo y se caracterizó como una cepa de tipo CP3 (genotipo B). Además, se identificaron partículas virales compatibles con circovirus en el hígado mediante microscopía electrónica directa. Según el conocimiento de los autores, este es el primer informe de clamidiosis aviar con partículas virales hepáticas compatibles con infección por circovirus en un pinzón diamante de Gould.

Chlamydia psittaci in fulmars on the Faroe Islands: a causative link to South American psittacines eight decades after a severe epidemic.

A psittacosis epidemic linked to fulmar hunting occurred on the Faroe Islands in the 1930s. This study investigates a plausible explanation to the 20% human mortality in this outbreak. Phylogenetic analysis showed that Chlamydia psittaci isolated from fulmars were closely related to the highly virulent 6BC strains from psittacines and are compatible with an acquisition by fulmars of an ancestor of the 6BC clade in the 1930s. This supports the hypothesis that the outbreak on the Faroe Islands started after naïve fulmars acquired C. psittaci from infected dead parrots thrown overboard when shipped to Europe in the 1930s.

Cross-sectional study on Chlamydiaceae prevalence and associated risk factors on commercial and backyard poultry farms in Mexico.

Chlamydiaceae infections in poultry are mainly due to Chlamydia psittaci and Chlamydia gallinacea. While C. psittaci has long been known to affect birds and to have zoonotic potential, C. gallinacea is a newly described species that has been found to be widespread in chickens. As no data were available regarding the presence of Chlamydiaceae in Mexican poultry, a cross-sectional survey to detect the presence of Chlamydiaceae on commercial and backyard farms was carried out in eight federal states of Mexico with a high poultry density. Individual cloacal swabs were collected on 14 large-scale commercial poultry farms with controlled environment houses, 23 large-scale commercial poultry farms with open-sided houses, and 16 backyard farms. Samples were tested using a specific Chlamydiaceae real-time PCR technique. Chlamydial species were subsequently identified by a species-specific real-time PCR method. Information on potential risk factors was collected through a questionnaire. Logistic regression was performed to identify risk factors associated with Chlamydiaceae-positive results at the farm level on commercial farms. For backyard farms, a mixed-effect logistic regression model was used to consider information collected either at the animal or at the farm level. Overall, 7.1 % (n = 1/14) of controlled environment commercial farms, 26.1 % (n = 6/23) of open-sided commercial farms, and 75.0 % (n = 12/16) of backyard farms were Chlamydiaceae-positive. Apparent prevalence increased inversely to the level of confinement (controlled environment vs open-sided poultry houses vs backyards). Chlamydia gallinacea was the only chlamydial species detected. On commercial farms, egg-laying hen flocks had 6.7 times higher odds of being Chlamydiaceae-infected than broilers flocks (OR = 6.7, 95 % CI: 1.1-44.3, p = 0.04). On backyard farms, two variables were significantly associated with Chlamydiaceae infection: the lack of antibiotic use (OR = 8.4, 95 % CI: 1.84-38.49, p = 0.006), and an impaired health status (OR=8.8, 95 % CI: 1.9-38.9, p = 0.004). Further studies should be carried out to investigate the impact of C. gallinacea infection on egg quality and production performance in egg-laying hen flocks.