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.

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.

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.

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.

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.

First Draft Genome for a Burkholderia mallei Isolate Originating from a Glanderous Mule from Brazil.

Burkholderia mallei is the etiological agent of glanders. Here, we present the draft genome sequence of Burkholderia mallei strain 16-2438_BM#8 that was isolated from a mule found dead in Pernambuco, northeast Brazil. It is the first available genomic sequence from a strain isolated on the American continent.

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.

Effects of a chronic stress treatment on vaccinal response in lambs.

Farming systems can expose animals to chronic mild stress which is known to induce negative affective state. Affective state in animals, as in humans, can be assessed through behavioral cues. This study aimed to describe the effect of a chronic mild stress, known to induce a negative affective state, on sheep health through their response to vaccination. The study used 15 lambs subjected to a model of chronic mild stress for 15 weeks and 15 lambs reared under conventional farming as a control group. After 7 weeks of stressful treatment, the lambs were individually exposed to a judgment bias test to assess a putative stress-induced 'pessimism.' After 15 weeks of stressful treatment, antibody immune response was measured after an injection of a live vaccine challenge (Chlamydia abortus attenuated vaccine strain 1B). Stressed lambs displayed a pessimistic-like perception in the judgment bias test, revealing a negative affective state. Stressed and control animals showed different immunological reactions to vaccine challenge: stressed sheep had lower hemoglobin concentrations and higher platelet, granulocyte and acute-phase protein concentrations. Antibody response induced by the vaccine strain was not different between stressed and control sheep. Our results suggest that negative affective state induced by chronic stress treatment may induce a stronger inflammatory response to vaccine challenge in sheep. Improvement of animal health may be achieved through consideration of stressors that may affect the emotional and immunological state of sheep.

Can Chlamydia abortus be transmitted by embryo transfer in goats?

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP.

Interlaboratory ring trial to evaluate CFT proficiency of European laboratories for diagnosis of glanders in equids.

To evaluate the routine complement fixation test (CFT) used to detect Burkholderia mallei antibodies in equine sera, an interlaboratory proficiency test was held with 24 European laboratories, including 22 National Reference Laboratories for glanders. The panels sent to participants were composed of sera with or without B mallei antibodies. This study confirmed the reliability of CFT and highlighted its intralaboratory reproducibility. However, the sensitivity of glanders serodiagnosis and laboratory proficiency may be improved by standardising critical reagents, including antigens, and by developing a standard B mallei serum.

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.

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.

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.

Chlamydiaceae and chlamydial infections in sheep or goats.

Chlamydiae induce a range of pathological syndromes in small ruminants. Abortion is the most common clinical expression of the infection that causes important economic losses and presents a risk to human health, particularly in pregnant women. The present paper gives an overview of chlamydial infections in sheep and goats, focusing specifically on abortion and on recent data brought by cellular and genomic approaches regarding genotyping, virulence of strains, epidemiology, diagnosis, pathogenesis and control of the disease.