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.

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.

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.

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.

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.

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.

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.

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.

Chlamydiae in human intestinal biopsy samples.

Chlamydia trachomatis is frequently detected in anorectal specimens from men and women. A recent hypothesis suggests that C. trachomatis is a natural commensal organism asymptomatically colonizing the gastrointestinal tract. In this study, we investigated the presence of chlamydial DNA and antigen in intestinal biopsy samples taken during colonoscopy. Cases (n = 32) were patients whose histopathology reports included the term 'chlamydia', suggesting a possible history of infection. Control patients (n = 234) did not have chlamydia mentioned in their histopathology report and all tested negative for Chlamydiaceae DNA by 23S ribosomal RNA-based real-time PCR. Amongst the cases, C. trachomatis DNA was detected in the appendix and colon of two female and one male patients. Chlamydia abortus DNA was present in the colon of a fourth female patient. Thus, chlamydial DNA could be demonstrated in intestinal biopsy samples proximal to the anorectal site and inclusions were identified in rectum or appendix of two of these patients by immunohistochemistry. However, the findings in two cases were compatible with sexually acquired C. trachomatis. The identification of C. trachomatis DNA/antigen does not prove the presence of active infection with replicating bacteria. Larger prospective studies on fresh tissue samples are required to confirm the data obtained in this study.

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.

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.

Infectious keratoconjunctivitis and occurrence of Mycoplasma conjunctivae and Chlamydiaceae in small domestic ruminants from Central Karakoram, Pakistan.

Infectious keratoconjunctivitis (IKC) is a contagious eye disease primarily caused by Mycoplasma conjunctivae in domestic and wild Caprinae. Chlamydophila species have also been detected in ruminants with IKC. The objectives of this study are to investigate the ocular infection of M. conjunctivae and Chlamydiaceae and assess its interaction in relation to IKC in sheep and goats from remote communities around the Central Karakoram National Park in Pakistan, performing a combination of cross-sectional and case-control study design. Mostly asymptomatic and endemic infections of M. conjunctivae and Chlamydiaceae were found in sheep (19.3 per cent and 4.5 per cent, respectively) and goats (9.5 per cent and 1.9 per cent, respectively) from all communities, assessed by qPCR. Prevalence significantly differed between species only for M. conjunctivae (P=0.0184), which was also more prevalent in younger sheep (P<0.01). Chlamydophila pecorum was identified by sequencing and was related with IKC only when coinfection with M. conjunctivae occurred, which suggest a synergic interaction. Cluster analysis of M. conjunctivae strains revealed higher diversity of strains than expected, evidenced interspecific transmission and suggested a higher local livestock trade than previously assumed. These results highlight the widespread occurrence of M conjunctivae in sheep worldwide and its implications for wildlife should be assessed from a conservation perspective.

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.

Chlamydial infections in wildlife-conservation threats and/or reservoirs of 'spill-over' infections?

Members of the order Chlamydiales are biphasic intracellular pathogens known to cause disease in both humans and animals. As we learn more about the genetic diversity of this group of pathogens, evidence is growing that these bacteria infect a broader range of animal hosts than previously thought. Over 400 host species are now documented globally with the majority of these being wild animals. Given the impact of chlamydial infections on humans and domesticated animals, the identification of members of the order Chlamydiales in wildlife raises significant questions over a) their impact on animal health and b) the relationships to those strains also found in humans and domestic animals. In some species such as the iconic marsupial, the koala, the conservation impact is known with chlamydial infections associated with debilitating disease, however, in general, little is known about the pathogenic potential of Chlamydiae infecting most wildlife hosts. Accumulating evidence suggests contact with wild animals is a risk factor for infections in domestic animals and/or humans. Beyond the well-recognised zoonotic pathogen, Chlamydia psittaci, a range of studies have now reported traditional pathogens in the family Chlamydiaceae such as Chlamydia pecorum, Chlamydia suis, Chlamydia pneumoniae and Chlamydia abortus in wild animals. The spectre of cross-host transmission 'spill-over' and 'spill-back' in the epidemiology of infections is of potential concern, however, comprehensive epidemiological studies are lacking for most of these. Accurate evaluation of the significance of chlamydial infections in wildlife is otherwise hampered by i) the cross-sectional nature of most impact studies, ii) a lack of standardised diagnostic approaches, iii) limited study sizes, and iv) biases associated with opportunistic sampling.

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.

Detection of atypical Chlamydiaceae in roe deer (Capreolus capreolus).

Investigations on fecal samples, vaginal swabs and sera from roe deer (Capreolus capreolus) in south-western France led to the detection of a non-classified Chlamydiaceae strain. A total of 85 vaginal swabs were sampled from roe deer that had been captured in 2012 (n=42) and 2013 (n=43). Using a Chlamydiaceae family-specific real-time PCR, only one vaginal swab out of the 42 samples done in 2012 tested positive and was subsequently identified as Chlamydia (C.) psittaci. In contrast, 6/43 vaginal swab samples were positive in 2013. Four of these positive samples came from a single group of roe deer, captured in the Fabas plain. Fecal samples from this group of 9 females were subsequently analyzed, with 6 of them testing positive with the Chlamydiaceae-specific PCR. All positive samples collected in 2013 were negative when re-tested with C. abortus-, C. pecorum- and C. suis-specific real-time PCR assays. Sera from this group of 9 females were analyzed with two immunoassays (recomLine and ELISA). Whereas intense positive reactions with C. pneumoniae antigens were observed for all sera when tested with the recomLine test, none was positive with the C. abortus specific ELISA test. Comparative sequence analysis of the 16S, 23S rRNA and ompA gene sequences from 3 animals, as well as the MLST analysis from 2 animals, showed that this roe deer group likely harbored the same bacterium related to members of the family Chlamydiaceae. Notably, the roe deer strain formed a separate entity different from the currently recognized chlamydial species, with C. trachomatis, C. suis and C. muridarum appearing as its closest relatives.

Prevalence of Chlamydial Infections in Fattening Pigs and Their Influencing Factors.

Chlamydial infections in pigs are associated with respiratory disease, diarrhea, conjunctivitis and other pathologies. The aim of this study was to define the prevalence of Chlamydiaceae in Swiss fattening pigs by applying sensitive and specific detection methods and to correlate prior antibiotic treatment and farm related factors with differences in prevalence. Conjunctival and fecal swabs were collected from 636 pigs in 29 Swiss fattening pig farms with and without antibiotic treatment, at the beginning and the end of the fattening period. The swabs were screened by real-time PCR for Chlamydiaceae. For the chlamydial detection and species-identification, a DNA-microarray analysis was performed. All farms were positive for Chlamydiaceae with 94.3 and 92.0% prevalence in fecal swabs as well as 45.9 and 32.6% in conjunctival swabs at the first and second time points, respectively. Antibiotic treatment could not clear the infection on herd level. Potential contact with wild boars was a significant risk factor, while hygiene criteria did not influence chlamydial prevalence. A correlation of chlamydial positivity to diarrhea, but not to conjunctivitis was evident. Chlamydia suis was the predominant species. Mixed infections with C. suis and C. pecorum were common, with a substantial increase in C. pecorum positivity at the end of the fattening period, and this finding was associated with ruminant contact. C. abortus was detected in one conjunctival swab. In this study, C. suis inhabited the intestinal tract of nearly all examined pigs, implying a long-term infection. C. pecorum was also common and might be transmitted to pigs by ruminants.

The contribution of temperature, exposure intensity and visible light to the inhibitory effect of irradiation on acute chlamydial infection.

Water-filtered infrared A (wIRA) is radiation with a spectrum ranging from 780 to 1400 nm. Chlamydiaceae are obligate intracellular bacteria associated with various diseases in both animals and humans. A recent in vitro study demonstrated that wIRA combined with visible light (wIRA/VIS) has potential as a non-chemical method for the treatment of chlamydial infections without adversely affecting the cell viability. The aim of this study was to investigate the influence of various factors on the effect of wIRA/VIS on acute chlamydial infection, namely the impact of temperature, exposure intensity and infectious dose (multiplicity of infection) as well as the efficacy of the visible light component.We demonstrate that non-thermal effects contribute to the inhibition of acute chlamydial infection. Visible light enhances the inhibitory effect of wIRA on extracellular bacteria (elementary bodies or EBs).Moreover, the inhibitory effect of wIRA/VIS following treatment of EBs prior to infection correlated with increased irradiation intensity. The infectivity of mature chlamydial inclusions was significantly reduced upon wIRA/VIS exposure at all irradiation intensities investigated, suggesting the contribution of host cell factors to the anti-chlamydial effect of wIRA/VIS in the late stage of the developmental cycle. The effect of irradiation was not influenced by the infectious dose.

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.