European Chlamydia abortus livestock isolate genomes reveal unusual stability and limited diversity, reflected in geographical signatures.

BACKGROUND: Chlamydia abortus (formerly Chlamydophila abortus) is an economically important livestock pathogen, causing ovine enzootic abortion (OEA), and can also cause zoonotic infections in humans affecting pregnancy outcome. Large-scale genomic studies on other chlamydial species are giving insights into the biology of these organisms but have not yet been performed on C. abortus. Our aim was to investigate a broad collection of European isolates of C. abortus, using next generation sequencing methods, looking at diversity, geographic distribution and genome dynamics. RESULTS: Whole genome sequencing was performed on our collection of 57 C. abortus isolates originating primarily from the UK, Germany, France and Greece, but also from Tunisia, Namibia and the USA. Phylogenetic analysis of a total of 64 genomes shows a deep structural division within the C. abortus species with a major clade displaying limited diversity, in addition to a branch carrying two more distantly related Greek isolates, LLG and POS. Within the major clade, seven further phylogenetic groups can be identified, demonstrating geographical associations. The number of variable nucleotide positions across the sampled isolates is significantly lower than those published for C. trachomatis and C. psittaci. No recombination was identified within C. abortus, and no plasmid was found. Analysis of pseudogenes showed lineage specific loss of some functions, notably with several Pmp and TMH/Inc proteins predicted to be inactivated in many of the isolates studied. CONCLUSIONS: The diversity within C. abortus appears to be much lower compared to other species within the genus. There are strong geographical signatures within the phylogeny, indicating clonal expansion within areas of limited livestock transport. No recombination has been identified within this species, showing that different species of Chlamydia may demonstrate different evolutionary dynamics, and that the genome of C. abortus is highly stable.

Chlamydiaceae in North Atlantic Seabirds Admitted to a Wildlife Rescue Center in Western France.

Birds are the primary hosts of Chlamydia psittaci, a bacterium that can cause avian chlamydiosis in birds and psittacosis in humans. Wild seabirds are frequently admitted to wildlife rescue centers (WRC) at European Atlantic coasts, for example, in connection with oil spills. To investigate the extent of chlamydial shedding by these birds and the resulting risk for animals in care and the medical staff, seabirds from a French WRC were sampled from May 2011 to January 2014. By use of a quantitative PCR (qPCR), 195 seabirds belonging to 4 orders, 5 families and 13 species were examined, of which 18.5% proved to be Chlamydiaceae positive. The highest prevalence of shedders was found in northern gannets (Morus bassanus) (41%), followed by European herring gulls (Larus argentatus) (14%) and common murres (Uria aalge) (7%). Molecular characterization and phylogenetic analysis of qPCR-positive northern gannet samples revealed two variants of a strain closely related to C. psittaci. In European herring gulls and in one common murre, strains showing high sequence similarity to the atypical Chlamydiaceae-like C122 previously found in gulls were detected. Our study shows that seabirds from the northeastern Atlantic Ocean carry several chlamydial organisms, including C. psittaci-related strains. The staff in WRCs should take protective measures, particularly in the case of mass admissions of seabirds.

Assessment of Chlamydia psittaci Shedding and Environmental Contamination as Potential Sources of Worker Exposure throughout the Mule Duck Breeding Process.

Chlamydia psittaci is an obligate intracellular bacterium responsible for avian chlamydiosis, otherwise known as psittacosis, a zoonotic disease that may lead to severe atypical pneumonia. This study was conducted on seven mule duck flocks harboring asymptomatic birds to explore the circulation and persistence of C. psittaci during the entire breeding process and assess the potential sources of worker exposure. Cloacal swabs and air samples were taken on each occasion requiring humans to handle the birds. In parallel, environmental samples, including dust, water, and soil, were collected. Specific real-time PCR analyses revealed the presence of C. psittaci in all flocks but with three different shedding patterns involving ducks about the age of 4, 8, and 12 weeks with heavy, moderate, and low excretion levels, respectively. Air samples were only positive in flocks harboring heavy shedders. Dust in flocks with heavy or moderate shedders carried chlamydial loads strongly associated with the loads detected in avian and soil samples. Environmental contamination, significantly correlated with shedding dynamics, was considered to be the most probable source of exposure. The high prevalence of bacteriophage Chp1 in all flocks, mostly jointly present with chlamydia, suggests an important factor in C. psittaci persistence, thus creating a greater risk for humans. A survey conducted in these flocks regarding farming practices and activities showed that disinfection seems to be the most promising practice for reducing C. psittaci prevalence in ducks and that the place and the duration of action during operations seem to be potential risk factors. Strict adherence to good practices is strongly recommended.

Circulation of Coxiella burnetii in a Naturally Infected Flock of Dairy Sheep: Shedding Dynamics, Environmental Contamination, and Genotype Diversity.

Q fever is a worldwide zoonosis caused by Coxiella burnetii. Domestic ruminants are considered to be the main reservoir. Sheep, in particular, may frequently cause outbreaks in humans. Because within-flock circulation data are essential to implementing optimal management strategies, we performed a follow-up study of a naturally infected flock of dairy sheep. We aimed to (i) describe C. burnetii shedding dynamics by sampling vaginal mucus, feces, and milk, (ii) assess circulating strain diversity, and (iii) quantify barn environmental contamination. For 8 months, we sampled vaginal mucus and feces every 3 weeks from aborting and nonaborting ewes (n=11 and n=26, respectively); for lactating females, milk was obtained as well. We also sampled vaginal mucus from nine ewe lambs. Dust and air samples were collected every 3 and 6 weeks, respectively. All samples were screened using real-time PCR, and strongly positive samples were further analyzed using quantitative PCR. Vaginal and fecal samples with sufficient bacterial burdens were then genotyped by multiple-locus variable-number tandem-repeat analysis (MLVA) using 17 markers. C. burnetii burdens were higher in vaginal mucus and feces than in milk, and they peaked in the first 3 weeks postabortion or postpartum. Primiparous females and aborting females tended to shed C. burnetii longer and have higher bacterial burdens than nonaborting and multiparous females. Six genotype clusters were identified; they were independent of abortion status, and within-individual genotype diversity was observed. C. burnetii was also detected in air and dust samples. Further studies should determine whether the within-flock circulation dynamics observed here are generalizable.

An Unusual Outbreak of Chlamydiosis in Commercial Turkeys Involving the Nasal Glands.

An unusual outbreak of chlamydiosis was diagnosed in 15,000, 13-wk-old organically grown turkeys housed in a semiconfinement housing system. The disease was characterized by unilateral or bilateral swelling above the eye due to mild-to-severe inflammation of the nasal glands in 3%-5% of the birds. Except for a slight drop in feed and water consumption, the birds did not exhibit any respiratory signs, morbidity, and mortality. Chlamydiosis in the turkeys was confirmed by immunofluorescence, immunohistochemistry, and PCR assay of the nasal glands. Other samples such as conjunctiva, lungs, air sacs, heart, liver, spleen, and feces were negative for chlamydia by florescence antibody test in birds submitted over several weeks. Chlamydia psittaci strain B was isolated in chicken egg embryos and typed by multilocus sequence variable number of tandem repeats analysis, multilocus sequence typing, and ompA gene sequencing as a CP3-like strain. This is the first report of a naturally occurring chlamydiosis affecting the nasal glands in turkeys.

Outbreak of psittacosis in a group of women exposed to Chlamydia psittaci-infected chickens.

Eight cases of psittacosis due to Chlamydia psittaci were identified in May 2013 among 15 individuals involved in chicken gutting activities on a mixed poultry farm in France. All cases were women between 42 and 67 years-old. Cases were diagnosed by serology and PCR of respiratory samples. Appropriate treatment was immediately administered to the eight hospitalised individuals after exposure to birds had been discovered. In the chicken flocks, mainly C. gallinacea was detected, a new member of the family Chlamydiaceae, whereas the ducks were found to harbour predominantly C. psittaci, the classical agent of psittacosis. In addition, C. psittaci was found in the same flock as the chickens that the patients had slaughtered. Both human and C. psittaci-positive avian samples carried the same ompA genotype E/B of C. psittaci, which is widespread among French duck flocks. Repeated grassland rotations between duck and chicken flocks on the farm may explain the presence of C. psittaci in the chickens. Inspection by the veterinary service led to temporary closure of the farm. All birds had to be euthanised on site as no slaughterhouses accepted processing them. Farm buildings and grasslands were cleaned and/or disinfected before the introduction of new poultry birds.

Relationships between metals exposure and epidemiological parameters of two pathogens in urban pigeons.

Human activities often generate or increase concentration of chemical compounds including pesticides, hydrocarbons and metals that can potentially affect ecological interactions. We found that elevated levels of zinc in pigeon feathers were associated with both low prevalence of Chlamydiaceae (ornithosis disease) and low intensity of blood pathogens (Haemosporidian parasites). In contrast, high levels of lead in pigeon feathers were associated with high blood pathogens intensities. Our results suggest that metals linked to human activities in cities such as zinc and lead may play a significant role in the ecology of host-parasite interactions and could potentially affect the epidemiology of diseases in the urban environment.

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.

Impact of urban environment and host phenotype on the epidemiology of Chlamydiaceae in feral pigeons (Columba livia).

Chlamydiaceae are obligate intracellular Gram-negative bacteria found all over the world and known to cause various forms of disease in animals and humans. Urban pigeons are known to be an important reservoir of Chlamydia psittaci, the agent of human psittacosis. In this study, we examined the influence of pigeon houses used to regulate pigeon populations and of melanin-based coloration on several epidemiological parameters of Chlamydiaceae in 708 urban pigeons in Paris. We also identified species and genotypes of Chlamydiaceae present in Parisian populations. First, our results revealed that pigeons roosting and breeding in pigeon houses were equally infected by Chlamydiaceae as those that did not. Second, we found that dark melanic pigeons excreted more Chlamydiaceae than pale melanic ones. Finally, species and strain diversities were very low: all samples were of C. psittaci genotype B. Nevertheless, two atypical Chlamydiaceae were identified based on 16S rRNA and ompA sequences. Our study thus highlights the importance of considering environmental and host phenotype when investigating the epidemiology of infectious diseases.

Psittacosis outbreak after participation in a bird fair, Western France, December 2008.

In December 2008, three hospitalized cases of suspected psittacosis infection were notified by respiratory disease clinicians from a local hospital to the Regional Epidemiology Unit of Pays de la Loire, France. They all had attended a bird fair. A retrospective cohort study was conducted among exhibitors and organizers to identify potential risk factors in relation to this fair. Environmental and veterinary investigations were implemented to trace potential sources of infection. We identified two confirmed, two probable and 44 possible cases among participants. The attack rate in exhibitors and organizers was 38% (33/86). The median incubation period was 11 days (range 6-22 days). Individuals located in two particular sectors of the showroom were found to be at double the risk of developing psittacosis (relative rate 2·1, 95% confidence interval 1·03-4·18) than those in other sectors. Pooled faecal samples of birds belonging to a possible case exhibitor tested positive for Chlamydiaceae by PCR. Ventilation conditions in the showroom were inadequate. This investigation allowed the formulation of recommendations to prevent psittacosis in bird exhibitions which are held weekly in France.

Molecular epidemiology of Burkholderia mallei isolates from India (2015-2016): New SNP markers for strain tracing.

Glanders, caused by a bacterium called B. mallei, is primarily an infectious horse and human disease. Although its incidence is rare in developed countries, it is nonetheless prevalent in several geographical areas of the world. There is a lack of cost-effective, rapid and specific molecular typing tools for epidemiological tracing of glanders cases. We previously reported an SNP-based typing method that categorizes global B. mallei strains into three lineages (L1 to L3), as well as additional branches, sub-branches and groups. However, further discrimination of the Indian and Pakistani isolates within the L2B2sB2 sub-branch was not possible due to the lack of sufficient epidemiological markers. In this study, 10 B. mallei strains isolated from four states in India during 2015-2016 were whole genome sequenced; SNP analysis further confirmed their position in the L2B2sB2 branch. To better track the strains, four new markers targeting Indian or Pakistani strains, and specifically targeting sub-groups within the Indian strains, were identified. The new SNP markers were tested and validated on the 10 Indian isolates included in this study as well as on 6 contemporary B. mallei Pakistani strains. These rapid and discriminating typing tools will contribute to the epidemiological monitoring of B. mallei infections, particularly in South Asia and the Middle East, endemic regions of the disease.

Evidence for the existence of a new genus Chlamydiifrater gen. nov. inside the family Chlamydiaceae with two new species isolated from flamingo (Phoenicopterus roseus): Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov.

The family Chlamydiaceae currently comprises a single genus Chlamydia, with 11 validly published species and seven more taxa. It includes the human pathogens Chlamydia (C.) trachomatis, C. pneumoniae and C. psittaci, a zoonotic agent causing avian chlamydiosis and human psittacosis, as well as other proven or potential pathogens in ruminants, birds, snakes, reptiles and turtles. During routine testing of 15 apparently healthy captive flamingos in a zoo in 2011, an atypical strain of Chlamydiaceae was detected by real-time PCR of cloacal swab samples. Sequence analysis of the 16S rRNA gene revealed high similarity to the uncultured Chlamydiales bacterium clone 122, which previously had been found in gulls. As more samples were collected during annual campaigns of the flamingo ringing program in southern France from 2012 to 2015, Chlamydiaceae-specific DNA was detected by PCR in 30.9% of wild birds. From these samples, three strains were successfully grown in cell culture. Ultrastructural analysis, comparison of 16S and 23S rRNA gene sequences, whole-genome analysis based on de novo hybrid-assembled sequences of the new strains as well as subsequent calculation of taxonomic parameters revealed that the relatedness of the flamingo isolates to established members of the family Chlamydiaceae was sufficiently distant to indicate that the three strains belong to two distinct species within a new genus. Based on these data, we propose the introduction of Chlamydiifrater gen. nov., as a new genus, and Chlamydiifrater phoenicopteri sp. nov. and Chlamydiifrater volucris sp. nov., as two new species of the genus.

Development of a microsphere-based immunoassay for the serological detection of glanders in equids.

Burkholderia mallei is the etiologic agent of glanders, an infectious disease of solipeds, with renewed scientific interest due to its increasing incidence in different parts of the world. More rapid, sensitive and specific assays are required by laboratories for confirmatory testing of this disease. A microsphere-based immunoassay consisting of beads coated with B. mallei recombinant proteins (BimA, GroEL, Hcp1, and TssB) has been developed for the serological diagnosis of glanders. The proteins' performance was compared with the OIE reference complement fixation test (CFT) and an indirect enzyme-linked immunosorbent assay (iELISA) on a large panel of sera comprised of uninfected horses (n=198) and clinically confirmed cases of glanders from India and Pakistan (n=99). Using Receiver Operating Characteristics (ROC) analysis and adjusting the cutoff levels, Hcp1 (Se=100%, Sp=99.5%) and GroEL (Se= 97%, Sp=99.5%) antigens exhibited the best specificity and sensitivity. Neither Hcp1 and GroEL proteins, nor iELISA reacted with doubtful and positive CFT samples from glanders free countries which further confirmed the false positive reactions seen in CFT.

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

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.

A cluster of Chlamydia serpentis cases in captive snakes.

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.

Chlamydial infections in feral pigeons in Europe: Review of data and focus on public health implications.

Feral pigeons (Columba livia domestica), which thrive in most European towns and cities, are commonly infected with the zoonotic bacterium Chlamydophila psittaci, the agent of psittacosis (also known as ornithosis) in humans. A number of surveys carried out over the last thirty years across Europe have detected high seropositivity values and high percentages of infection in feral pigeon populations. Overall, when considering data from 11 European countries, seropositivity values to C. psittaci in the sampled populations ranged from 19.4% to 95.6%. In most surveys, the complement fixation test was used, and antibodies were detected in 19.4-66.3% of the samples, with a median of 46.1%. Indirect immunofluorescence and ELISA tests were employed less frequently, but led to the detection of higher percentages of seropositivity (23.7-67.7% and 35.9-95.6%, respectively). Attempts to grow C. psittaci in cell culture or embryonated chicken eggs were successful in 2-42.3% and 0-57.1% of samples, respectively, antigen detection methods were positive in 2.3-40% of samples, while conventional PCR and real-time PCR using different genomic targets detected the organism in 3.4-50% of samples. Twenty-five C. psittaci isolates from pigeons were typed as ompA genotype B (n=14), E (n=10) and E/B (n=1). The huge increase of feral pigeon populations in Europe is a major cause of concern for the detrimental effect of pigeon droppings on environmental hygiene, in addition to the extensive damage due to the fouling of buildings and monuments. The most important pathogenic organism transmissible from feral pigeons to humans is C. psittaci, with 101 cases of disease reported in the literature. Exposure to C. psittaci-contaminated dust, direct contact with pigeons through handling and, to a lesser extent, through pigeon feeding have been identified as hazardous exposures in more than half of the human cases, while loose or transient contacts with feral pigeons have been mentioned in about 40% of the cases. Education initiatives as to the communication of a health risk resulting from contact with pigeons and pigeon excreta should primarily be targeted at individuals who may be exposed to C. psittaci-contaminated dust, such as demolition/construction workers. Recommendations to this category of workers include wearing protective clothes with hoods, boots, gloves and air filter face masks when removing pigeon faeces from roofs, garrets and buildings, especially if working indoors. Monitoring for C. psittaci infections in these workers over time should also be considered. Children should be warned not to handle sick or dead pigeons, and immunocompromised individuals should be advised to carefully limit their contact to feral pigeons. Culling of pigeons by shooting or poisoning is both unethical and ineffective as the place of the killed birds in the population is quickly filled by new juveniles or immigrating birds from neighbouring areas. Pigeon-deterring systems, such as nets and plastic or metal spikes applied to buildings and monuments will prevent their fouling, and the administration of contraceptive drugs may allow size regulation of the pigeon populations. Nevertheless, the measure that will ultimately lead to permanent reduction and will establish healthy sustainable populations is the restriction of indiscriminate feeding by pigeon lovers. The erection of dovecotes and artificial breeding facilities should be considered for providing shelter and a balanced diet to the birds, as well as a chance of interaction for pigeon lovers in a hygienically controlled environment.

Chlamydia buteonis, a new Chlamydia species isolated from a red-shouldered hawk.

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 RSHAT as the type strain.

Abortion storm induced by the live C. abortus vaccine 1B strain in a vaccinated sheep flock, mimicking a natural wild-type infection.

Chlamydia abortus is responsible for enzootic abortion (known as ovine enzootic abortion (OEA) and enzootic abortion of ewes (EAE)) in both sheep and goats and has major economic implications for the farming industry worldwide. A virulence-attenuated mutant strain of C. abortus (strain 1B) is currently commercially available as a live attenuated vaccine for immunization of sheep and goats in several European countries. Following an abortion storm in a French flock of 200 ewes that occurred two years after vaccination of 36 replacement ewes with the commercial 1B vaccine strain, the vaginal swabs of 3 vaccinated and 7 unvaccinated aborted ewes and 12 of the 13 dead fetuses were found to be positive for C. abortus by real-time PCR. Genotyping of the samples, using vaccine-specific SNP markers, identified all as positive for the vaccine-type strain. The recent vaccination of this flock with the attenuated commercial vaccine strain, the large number of abortion cases observed in ewes irrespective of vaccination status, the high C. abortus load detected in vaginal swabs or abortion tissues and the identification of specific vaccine-type markers in these samples strongly suggest that the 1B strain has been transmitted from vaccinated to naïve animals, thus mimicking a natural wild-type infection.

First molecular characterisation of a Brazilian Burkholderia mallei strain isolated from a mule in 2016.

We present the first molecular characterisation based on MLVA and SNP analysis of a strain of Burkholderia mallei isolated from a mule found dead in Brazil in 2016.

High-resolution melting PCR analysis for rapid genotyping of Burkholderia mallei.

Burkholderia (B.) mallei is the causative agent of glanders. A previous work conducted on single-nucleotide polymorphisms (SNP) extracted from the whole genome sequences of 45 B. mallei isolates identified 3 lineages for this species. In this study, we designed a high-resolution melting (HRM) method for the screening of 15 phylogenetically informative SNPs within the genome of B. mallei that subtype the species into 3 lineages and 12 branches/sub-branches/groups. The present results demonstrate that SNP-based genotyping represent an interesting approach for the molecular epidemiology analysis of B. mallei.