Presence of DNA from Chlamydia-like organisms in the nasal cavities of grey seal pups (Halichoerus grypus) and three different substrates present in a breeding colony.

BACKGROUND: Chlamydia-like organisms (CLO) have been found to be present in many environmental niches, including human sewage and agricultural run-off, as well as in a number of aquatic species worldwide. Therefore, monitoring their presence in sentinel wildlife species may be useful in assessing the wider health of marine food webs in response to habitat loss, pollution and disease. We used nasal swabs from live (n = 42) and dead (n = 50) pre-weaned grey seal pups and samples of differing natal substrates (n = 8) from an off-shore island devoid of livestock and permanent human habitation to determine if CLO DNA is present in these mammals and to identify possible sources. RESULTS: We recovered CLO DNA from 32/92 (34.7%) nasal swabs from both live (n = 17) and dead (n = 15) seal pups that clustered most closely with currently recognised species belonging to three chlamydial families: Parachlamydiaceae (n = 22), Rhabdochlamydiaceae (n = 6), and Simkaniaceae (n = 3). All DNA positive sediment samples (n = 7) clustered with the Rhabdochlamydiaceae. No difference was found in rates of recovery of CLO DNA in live versus dead pups suggesting the organisms are commensal but their potential as opportunistic secondary pathogens could not be determined. CONCLUSION: This is the first report of CLO DNA being found in marine mammals. This identification warrants further investigation in other seal populations around the coast of the UK and in other areas of the world to determine if this finding is unique or more common than shown by this data. Further investigation would also be warranted to determine if they are present as purely commensal organisms or whether they could also be opportunistic pathogens in seals, as well as to investigate possible sources of origin, including whether they originated as a result of anthropogenic impacts, including human waste and agricultural run-off.

Prevalence and phylogeny of Chlamydiae and hemotropic mycoplasma species in captive and free-living bats.

BACKGROUND: Bats are hosts for a variety of microorganisms, however, little is known about the presence of Chlamydiales and hemotropic mycoplasmas. This study investigated 475 captive and free-living bats from Switzerland, Germany, and Costa Rica for Chlamydiales and hemotropic mycoplasmas by PCR to determine the prevalence and phylogeny of these organisms. RESULTS: Screening for Chlamydiales resulted in a total prevalence of 31.4%. Positive samples originated from captive and free-living bats from all three countries. Sequencing of 15 samples allowed the detection of two phylogenetically distinct groups. These groups share sequence identities to Chlamydiaceae, and to Chlamydia-like organisms including Rhabdochlamydiaceae and unclassified Chlamydiales from environmental samples, respectively. PCR analysis for the presence of hemotropic mycoplasmas resulted in a total prevalence of 0.7%, comprising free-living bats from Germany and Costa Rica. Phylogenetic analysis revealed three sequences related to other unidentified mycoplasmas found in vampire bats and Chilean bats. CONCLUSIONS: Bats can harbor Chlamydiales and hemotropic mycoplasmas and the newly described sequences in this study indicate that the diversity of these bacteria in bats is much larger than previously thought. Both, Chlamydiales and hemotropic mycoplasmas are not restricted to certain bat species or countries and captive and free-living bats can be colonized. In conclusion, bats represent another potential host or vector for novel, previously unidentified, Chlamydiales and hemotropic mycoplasmas.

ATYPICAL CHLAMYDIACEAE IN WILD POPULATIONS OF HAWKS ( BUTEO SPP.) IN CALIFORNIA.

Chlamydiaceae bacteria infect many vertebrate hosts, and previous reports based on polymerase chain reaction (PCR) assays and serologic assays that are prone to cross-reaction among chlamydial organisms have been used to describe the prevalence of either DNA fragments or antibodies to Chlamydia spp. in wild raptorial populations. This study reports the PCR-based prevalence of Chlamydiaceae DNA that does not 100% match any avian or mammalian Chlamydiaceae in wild populations of hawks in California Buteo species. In this study, multimucosal swab samples ( n = 291) for quantitative PCR (qPCR) and plasma ( n = 78) for serology were collected from wild hawks. All available plasma samples were negative for antibodies using a C. psittaci-specific elementary body agglutination test (EBA; n = 78). For IgY antibodies all 51 available samples were negative using the indirect immunofluorescent assay. The overall prevalence of Chlamydiaceae DNA detection in wild Buteo species sampled was 1.37% (4/291) via qPCR-based analysis. Two fledgling Swainson's hawks ( Buteo swainsoni) and two juvenile red-tailed hawks ( Buteo jamaicensis) were positive by qPCR-based assay for an atypical chlamydial sequence that did not 100% match any known C. psittaci genotype. Positive swab samples from these four birds were sequenced based on the ompA gene and compared by high-resolution melt analysis with all known avian and mammalian Chlamydiaceae. The amplicon sequence did not 100% match any known avian chlamydial sequence; however, it was most similar (98.6%) to C. psittaci M56, a genotype that is typically found in muskrats and hares. Culture and full genome sequence analysis of Chlamydia spp. isolated from diseased hawks will be necessary to classify this organism and to better understand its epizootiology and potential health impact on wild Buteo populations in California.

Diverse Chlamydia-like agents associated with epitheliocystis infection in two cyprinid fish species, the common carp (Cyprinus carpio L.) and the gibel carp (Carassius auratus gibelio L.).

During a general annual fish health survey in natural waters and ponds, epitheliocystis infections were recorded in fingerlings of two cyprinid fish species, the cultured common carp and the wild gibel carp. Benign and heavy infections were equally observed without mortality. In addition to the general health inspection of fish, histopathological examinations of infected gills and molecular biological investigations of separated epitheliocysts were performed. Epitheliocysts were formed both in the interlamellar epithelial cells and in the lamella-free multilayered epithelium of the gill filaments. At the early stage of infection darkstaining inclusion bodies densely stuffed with some pathogenic agents were located at the centre of the cell, while in a progressive stage of the process inclusion bodies within the host cells were disseminated in the cytoplasm and stained pale. Molecular studies demonstrated three different agents related to Neochlamydia, Protochlamydia and Piscichlamydia based on sequence analysis of short regions of the 16S rRNA gene. Among them, Piscichlamydia is a primary fish pathogen, while Neochlamydia and Protochlamydia mostly infect free-living amoebae but have adapted thoroughly to fish.

Flow cytometry as an improved method for the titration of Chlamydiaceae and other intracellular bacteria.

Chlamydiaceae is a family of intracellular bacteria causing a range of diverse pathological outcomes. The most devastating human diseases are ocular infections with C. trachomatis leading to blindness and genital infections causing pelvic inflammatory disease with long-term sequelae including infertility and chronic pelvic pain. In order to enable the comparison of experiments between laboratories investigating host-chlamydia interactions, the infectious titer has to be determined. Titer determination of chlamydia is most commonly performed via microscopy of host cells infected with a serial dilution of chlamydia. However, other methods including fluorescent ELISpot (Fluorospot) and DNA Chip Scanning Technology have also been proposed to enumerate chlamydia-infected cells. For viruses, flow cytometry has been suggested as a superior alternative to standard titration methods. In this study we compared the use of flow cytometry with microscopy and Fluorospot for the titration of C. suis as a representative of other intracellular bacteria. Titer determination via Fluorospot was unreliable, while titration via microscopy led to a linear read-out range of 16 - 64 dilutions and moderate reproducibility with acceptable standard deviations within and between investigators. In contrast, flow cytometry had a vast linear read-out range of 1,024 dilutions and the lowest standard deviations given a basic training in these methods. In addition, flow cytometry was faster and material costs were lower compared to microscopy. Flow cytometry offers a fast, cheap, precise, and reproducible alternative for the titration of intracellular bacteria like C. suis. © 2016 International Society for Advancement of Cytometry.

Hymenobacter rubidus sp. nov., bacterium isolated from a soil.

Strain DG7B(T) was isolated from a soil sample collected in Seoul, Republic of Korea and was observed to be a gram-negative, short-rod shaped and non-motile bacterium. Its 16S rRNA gene sequence is closely related to those of Hymenobacter terrae DG7A(T) (97.8 % similarity), H. soli PB17(T) (97.5 %), H. glaciei VUG-A130(T) (96.4 %), H. saemangeumensis GSR0100(T) (95.7 %), H. ruber PB156(T) (95.3 %), and H. antarcticus VUG-A42aa(T) (95.3 %). The low levels of DNA-DNA relatedness (<50.3 %) with the above species identified strain DG7B(T) as a novel species in the genus Hymenobacter. The genomic DNA G+C content was determined to be 54.9 %. Growth of strain DG7B(T) was observed at 12-30 °C (optimum at 25 °C) and pH 6.0-11.0 (optimum at pH 7). The cells tolerate <0.5 % NaCl. A UV-visible scan of an ethanol extract of the whole cell pigment showed absorbance peaks at 264.5, 320.0, and 481.5 nm, so the pigment type was determined to be 2'-hydroxyflexixanthin. Chemotaxonomic data showed that strain DG7B(T) possesses menaquinone-7 as the predominant isoprenoid quinone, sym-homospermidine as the major polyamine, phosphatidylethanolamine as the predominant polar lipid and iso-C15:0, anteiso-C15:0 and summed feature 3 (C16:1 ω7c/C16:1 ω7c) as the major fatty acids. Strain DG7B(T) showed low-level resistance to ultraviolet C. Based on the polyphasic analysis, it is concluded that strain DG7B(T) (=KCTC 32553(T) = KEMB 9004-166(T) = JCM 30008(T)) should be classified as the type strain of a novel Hymenobacter species, for which the name Hymenobacter rubidus sp. nov. is proposed.

A new intracellular bacterium, Candidatus Similichlamydia labri sp. nov. (Chlamydiaceae) producing epitheliocysts in ballan wrasse, Labrus bergylta (Pisces, Labridae).

Certain wrasse species (Labridae) are used as cleaner fish in salmon farms on the Norwegian coast, reducing salmon louse intensities. The pathogen repertoire of wrasse in Norway is poorly known, and the objective of the present study is to describe a novel intracellular bacterium detected in Norwegian Labrus bergylta. Histological examination of gill tissues from ballan wrasse, L. bergylta, revealed epitheliocysts occurring basally to the secondary lamellae in the interlamellar epithelium. Ultrastructurally, these had bacteria-filled inclusions with thickened membranes and radiating ray-like structures (actinae). 16S rRNA gene sequences from the gill bacteria showed the highest (97.1 %) similarity to Candidatus Similichlamydia latridicola from the gills of the latrid marine fish Latris lineata in Australia and 94.9 % similarity to Candidatus Actinochlamydia clariae, causing epitheliocystis in the freshwater catfish Clarias gariepinus in Uganda. A total of 47 gill samples from L. bergylta from Western Norway were screened by real time RT-PCR with an assay targeting Candidatus Actinochlamydiaceae 16S rRNA. Prevalence was 100 %. We propose the name Candidatus Similichlamydia labri sp. nov. for this new agent producing gill epitheliocysts in L. bergylta.

Quantification of chlamydiae in the endangered Houston toad (Anaxyrus houstonensis).

Two primer set/probe combinations targeting variable regions on the 23S rRNA gene were designed to detect and quantify chlamydiae in DNA extracted from brain swabs of the endangered Houston toad (Anaxyrus houstonensis) using SYBRGreen- and Taqman-based quantitative polymerase chain reaction (qPCR). Prevalence and abundance values for samples were generally different between SYBRGreen- and Taqman-based detection methods, with higher specificity observed for Taqman-based detection. Of the 314 samples analyzed, initial screening with SYBRGreen-based qPCR retrieved 138 positive samples, of which 52 were confirmed by Taqman-based analyses as chlamydiae. All of these samples were subsequently identified as Chlamydia pneumoniae by specific qPCR and confirmed by comparative sequence analyses of 23S rRNA gene amplicons. These results demonstrate the usefulness of our developed qPCR methods to screen for and verify prevalence of chlamydiae in DNA of brain swabs, and ultimately specifically identify and quantify chlamydiae, specifically C. pneumoniae in these samples.

[Specific motifs in the genomes of the family Chlamydiaceae].

Specific motifs in the genomes of the family Chlamydiaceae were discussed. The search for genetic markers ofbacteria identification and typing is an urgent problem. The progress in sequencing technology resulted in compilation of the database of genomic nucleotide sequences of bacteria. This raised the problem of the search and selection of genetic targets for identification and typing in bacterial genes based on comparative analysis of complete genomic sequences. The goal of this work was to implement comparative genetic analysis of different species of the family Chlamydiaceae. This analysis was focused to detection of specific motifs capable of serving as genetic marker of this family. The consensus domains were detected using the Visual Basic for Application software for MS Excel. Complete coincidence of segments 25 nucleotide long was used as the test for consensus domain selection. One complete genomic sequence for each of 8 bacterial species was taken for the experiment. The experimental sample did not contain complete sequence of C. suis, because at the moment of this research this species was absence in the database GenBank. Comparative assay of the sequences of the C. trachomatis and other representatives of the family Chlamydiaceae revealed 41 common motifs for 8 Chlamydiaceae species tested in this work. The maximal number of consensus motifs was observed in genes of ribosomal RNA and t-RNA. In addition to genes of r-RNA and t-RNA consensus motifs were observed in 5 genes and 6 intergene segments. The gene CTL0299, CTLO800, dagA, and hctA consensus motifs detected in this work can be regarded as identification domains of the family Chlamydiaceae.

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.

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.

Microbial and pathological findings in farmed Atlantic salmon Salmo salar with proliferative gill inflammation.

Proliferative gill inflammation (PGI) is an important cause of loss in seawater-farmed Atlantic salmon in Norway. Several microbes have been associated with PGI, including the commonly but not exclusively observed inclusions (epitheliocysts) within the gill lamellae related to infection with 'Candidatus Piscichlamydia salmonis'. Atlantic salmon transferred in the spring of 2004 to 12 seawater farms situated in mid- and southwest Norway were sampled throughout that year. Outbreaks of PGI, as evaluated by clinical examination, histology, and mortality data, were diagnosed in 6 of 7 farms in southwest Norway but not in the 5 farms studied in mid-Norway. Generally, mortality started 3 to 5 mo after seawater transfer and outbreaks lasted at least 1 to 3 mo. 'Ca. P. salmonis' was detected by real-time PCR only in fish from PGI-affected farms and our results indicate an association between 'Ca. P. salmonis' load and PGI severity. Likewise, although widely distributed in all 12 farms studied, epitheliocyst prevalence and number per fish as observed by histology appears associated with PGI prevalence and severity. However, the occurrence of epitheliocysts showed no association with molecular detection of 'Ca. P. salmonis', suggesting that at least 1 other organism is responsible for many of the observed inclusions. A microsporidian, Desmozoon lepeophtherii, was identified at high prevalence regardless of fish and farm PGI status, but at higher loads in fish with PGI. Our results support a multifactorial etiology for PGI in which 'Ca. P. salmonis', an unidentified epitheliocyst agent, and the microsporidian are contributing causes. No evidence for the involvement of Atlantic salmon paramyxovirus in PGI development was identified in the present study. High water temperatures and ectoparasites probably exacerbated mortality.

Identification of phylogenetic position in the Chlamydiaceae family for Chlamydia strains released from monkeys and humans with chlamydial pathology.

Based on the results of the comparative analysis concerning relatedness and evolutional difference of the 16S-23S nucleotide sequences of the middle ribosomal cluster and 23S rRNA I domain, and based on identification of phylogenetic position for Chlamydophila pneumoniae and Chlamydia trichomatis strains released from monkeys, relatedness of the above stated isolates with similar strains released from humans and with strains having nucleotide sequences presented in the GenBank electronic database has been detected for the first time ever. Position of these isolates in the Chlamydiaceae family phylogenetic tree has been identified. The evolutional position of the investigated original Chlamydia and Chlamydophila strains close to analogous strains from the Gen-Bank electronic database has been demonstrated. Differences in the 16S-23S nucleotide sequence of the middle ribosomal cluster and 23S rRNA I domain of plasmid and nonplasmid Chlamydia trachomatis strains released from humans and monkeys relative to different genotype groups (group B-B, Ba, D, Da, E, L1, L2, L2a; intermediate group-F, G, Ga) have been revealed for the first time ever. Abnormality in incA chromosomal gene expression resulting in Chlamydia life development cycle disorder, and decrease of Chlamydia virulence can be related to probable changes in the nucleotide sequence of the gene under consideration.

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.

Characterization of Chlamydiaceae species using PCR and high resolution melt curve analysis of the 16S rRNA gene.

AIM: To design a rapid diagnostic test to differentiate species belonging to the family Chlamydiaceae. METHODS AND RESULTS: Five oligonucleotide sets each targeting various conserved regions of the genome of six species (Chlamydia muridarum, C. suis, C. trachomatis, Chlamydophila felis, Cp. pneumoniae and Cp. psittaci) belonging to the family Chlamydiaceae were tested for their suitability for polymerase chain reaction (PCR) and high resolution melt (HRM) curve analysis to differentiate Chlamydiaceae species. Three of the oligonucleotide sets were able to detect all six reference species used in this study, but only one set (16SG) could clearly differentiate between them by HRM curve analysis. The PCR-HRM curve analysis confidence percentages correlated strongly with the nucleotide sequence identities. Clinical specimens from a number of animal species suspected of chlamydiosis were tested with the newly developed 16SG PCR-HRM curve analysis and sequenced to confirm the infecting species. It was demonstrated that PCR-HRM using the 16SG oligonucleotide set could relate the infecting Chlamydiaceae species to the most similar (based on 16S rRNA gene nucleotide sequence) reference species tested. Although Cp. pecorum was not included initially as a reference species in this assay, inclusion of a field isolate of Cp. pecorum as a reference allowed two koala specimens to be correctly identified. CONCLUSION: PCR-HRM analysis using the oligonucleotide set 16SG is a robust, simple and rapid technique for differentiation of at least the Chlamydiaceae species used in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: This technique allowed for the rapid detection and identification of the six Chlamydiaceae reference species and may be useful for identification of uncharacterized Chlamydiaceae species or for use in animal species where occurrence of the disease has not been fully investigated.

Detection of Chlamydiaceae in ocular swabs from Australian pre-export feedlot sheep.

Infectious Ovine Keratoconjunctivitis (IOK) is a contagious ocular disease of sheep. A range of organisms have been observed as the aetiological agents of IOK. In this study, the presence of chlamydial pathogens (C. pecorum, C. abortus, C. psittaci) in conjunctival swabs was tested for. The swabs were collected from sheep with varying grades of IOK in an Australian pre-export feedlot. The sheep had been rejected from a shipment because of the eye disease. The relative contribution of chlamydial pathogens to IOK and the rejection of animals was evaluated. In total, 149 conjunctival swabs were taken from rejected sheep (IOK Grades 1 to 6; n = 126) as well as those with healthy eyes (Grade 0; n = 23). Screening for chlamydial pathogens was done using species-specific qPCR assays. Chlamydial DNA was detected in 35.6% (53/149) of conjunctival samples. C. pecorum was the most predominant species with an overall prevalence of 28.9% (43/149). C. psittaci prevalence was 6.7% (10/149). Both organisms were detected in healthy as well as IOK-affected eyes. All swabs tested negative for C. abortus. The results from this study demonstrate that Chlamydia spp can be readily detected in sheep presenting with IOK. The zoonotic C. abortus was not detected in any of the samples in this study, providing further evidence to the suggestion that this pathogen remains absent from Australia. Although the exact contribution of Chlamydia spp in the IOK pathogenesis is unclear, such studies are anticipated to be of benefit to Australian domestic and live export production systems.

Survey on Chlamydiaceae in cloacal swabs from Swiss turkeys demonstrates absence of Chlamydia psittaci and low occurrence of Chlamydia gallinacean.

In Switzerland, domestic turkey meat is a niche product. Turkeys are fattened on mixed family-based farms scattered across the country, with most providing access to an uncovered outdoor pasture for the birds. Swiss fattening turkeys may therefore get infected with Chlamydiaceae via wild birds or their faeces, potentially shedding these bacteria at a later stage. The aim of the present study was to acquire baseline data about the shedding of Chlamydiaceae in clinically unremarkable Swiss fattening turkeys at slaughter, potentially exposing slaughterhouse workers to infection. In this large-scale study, 1008 cloacal swabs of Swiss turkeys out of 53 flocks from 28 different grow-out farms with uncovered outdoor pasture were collected over the course of 14 months and examined for the occurrence of Chlamydiaceae by a family-specific 23S-rRNA real-time PCR. Positive samples were further analyzed by Chlamydia psittaci (C. psittaci)-specific real-time PCR and the Arraymate DNA Microarray for species identification. All samples were negative for C. psittaci, but seven swabs out of one flock were tested positive for Chlamydia gallinacea (0.7%). Although turkeys with access to pasture may have contact with Chlamydiaceae-harbouring wild birds or their faeces, the infection rate in Swiss turkeys was shown to be low.

Chlamydiaceae in wild, feral and domestic pigeons in Switzerland and insight into population dynamics by Chlamydia psittaci multilocus sequence typing.

Feral pigeons, common wood pigeons and Eurasian collared doves are the most common representatives of the Columbidae family in Switzerland and are mostly present in highly populated, urban areas. Pigeons may carry various members of the obligate intracellular Chlamydiaceae family, particularly Chlamydia (C.) psittaci, a known zoonotic agent, and C. avium. The objective of the study was to identify the infection rates of common free-roaming pigeons for different Chlamydia species with the overall aim to assess the risk pigeons pose to public health. In this study, 431 pigeons (323 feral pigeons, 34 domestic pigeons, 39 Eurasian collared doves, 35 common wood pigeons) from several geographic locations in Switzerland were investigated for the presence of Chlamydiaceae. Samples consisted of pooled choanal-cloacal swabs (n = 174), liver samples (n = 52), and paired swab and liver samples from 205 pigeons (n = 410). All 636 samples were screened using a Chlamydiaceae family-specific 23S rRNA real-time PCR (qPCR). Subsequent species identification was performed by DNA-microarray assay, sequencing of a 16S rRNA gene fragment and a C. psittaci specific qPCR. In total, 73 of the 431 pigeons tested positive for Chlamydiaceae, of which 68 were positive for C. psittaci, four were C. avium-positive and one pigeon was co-infected with C. avium and C. psittaci. The highest infection rates were detected in feral (64/323) and domestic pigeons (5/34). Common wood pigeons (2/35) and Eurasian collared doves (2/39) revealed lower infection rates. Additionally, multilocus sequence typing of twelve selected C. psittaci-positive samples revealed closely related sequence types (ST) between and within different Swiss cities. Furthermore, liver and corresponding swab samples from the same bird were colonized by the same ST. Considering the high infection rates of C. psittaci in domestic and feral pigeons, close or frequent contact to these birds poses a human health risk.

Prevalence of Chlamydiaceae and Mollicutes on the genital mucosa and serological findings in dairy cattle.

It was the aim of this project to obtain information on the prevalence of Chlamydiaceae and Mollicutes and their potential importance for reproductive problems in cattle. Cervical or vaginal swabs were taken from 644 animals in 196 farms and blood samples were collected from 375 cattle. Out of the animals, 6.8% had aborted within the last 12 months, 2.6% showed clinical vaginitis and 11.6% clinical endometritis. Chlamydiaceae were detected and identified by PCR followed by restriction fragment length polymorphism (RFLP) analysis. For the detection and identification of Mollicutes cultivation procedures, biochemical differentiation and serological identification were used. Sera were tested for antibodies against Chlamydiaceae and Mycoplasma (M.) bovis by ELISA and against M. bovigenitalium by Western blot analysis. Chlamydophila (Cp.) abortus was found in three cervical swabs. Cp. pecorum was detected in 9% of cervical or vaginal swabs. The majority of Cp. species found was Cp. pecorum and thus fertility problems caused by Cp. abortus are limited. M. bovis was found in only one genital swab. M. bovigenitalium was rarely diagnosed (3% of cervical and 2% of vaginal swabs). M. bovigenitalium was found more often in cattle having aborted (4/32 animals) than in cattle without history of abortion (5/220, p<0.05). Ureaplasma (U.) diversum existed in 12% of cervical and 36% of vaginal swabs and was found in 8 out of 17 animals with vaginitis. Out of the animals tested, 44.9% were seropositive for Chlamydiaceae, 14.8% for M. bovis and 27.3% for M. bovigenitalium.

Detection of Chlamydiaceae and Chlamydia-like organisms on the ocular surface of children and adults from a trachoma-endemic region.

Trachoma, the leading infectious cause of blindness, is caused by Chlamydia trachomatis (Ct), a bacterium of the phylum Chlamydiae. Recent investigations revealed the existence of additional families within the phylum Chlamydiae, also termed Chlamydia-like organisms (CLOs). In this study, the frequency of Ct and CLOs was examined in the eyes of healthy Sudanese (control) participants and those with trachoma (case). We tested 96 children (54 cases and 42 controls) and 93 adults (51 cases and 42 controls) using broad-range Chlamydiae and Ct-specific (omcB) real-time PCR. Samples positive by broad-range Chlamydiae testing were subjected to DNA sequencing. Overall Chlamydiae prevalence was 36%. Sequences corresponded to unclassified and classified Chlamydiae. Ct infection rate was significantly higher in children (31.5%) compared to adults (0%) with trachoma (p < 0.0001). In general, 21.5% of adults and 4.2% of children tested positive for CLOs (p = 0.0003). Our findings are consistent with previous investigations describing the central role of Ct in trachoma among children. This is the first study examining human eyes for the presence of CLOs. We found an age-dependent distribution of CLO DNA in human eyes with significantly higher positivity in adults. Further studies are needed to understand the impact of CLOs in trachoma pathogenicity and/or protection.