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.

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.

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.

Improved Monte Carlo clinical electron beam modelling.

PURPOSE: An EGSnrc based electron model was developed and validated for an Elekta Synergy® 160-leaf Agility™ linear accelerator. It was able to reproduce measured central axis (CAX) percentage depth dose (PDD) curves and off-axis profiles (OAPs) within 2%/2 mm, and relative output factors (ROFs) within 3%. METHODS: BEAMnrc component modules were used to model the accelerator accurately according to vendor supplied specifications. The electron beam focal spot size and input energy spectrum were determined through their effects on electron CAX PDDs and OAPs as benchmarked against water tank data. Phase space files were used as source input in DOSXYZnrc water phantom simulations. Dose distributions were calculated for six electron nominal energies, 11 field sizes and two source-to-surface distances. RESULTS: The full width at half maximum of the focal spot (assuming a Gaussian intensity distribution) was determined to be 1.50 mm. An asymmetrical input electron energy spectrum with a low-energy tail produced good agreement with measured data and solved the match in the build-up (BU) region for all electron energies used in this study. CONCLUSIONS: The improved input electron spectra for the electron model could predict dose distributions within 2%/2 mm of measured data. The model's success is embedded in the asymmetrical energy spectrum which provided a valuable free parameter which, by fine adjustment, improved the match in the BU region of dose distributions. Furthermore, focal spot parameters could be determined by means of iterative simulations.

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.

Gap analysis of Mycoplasma bovis disease, diagnosis and control: An aid to identify future development requirements.

There is a worldwide problem of disease caused by Mycoplasma (M.) bovis in cattle; it has a significant detrimental economic and animal welfare impact on cattle rearing. Infection can manifest as a plethora of clinical signs including mastitis, pneumonia, arthritis, keratoconjunctivitis, otitis media and genital disorders that may result in infertility and abortion. Current diagnosis and control information are reviewed and analysed to identify gaps in knowledge of the causative organism in respect of the disease pathology, diagnosis and control methods. The main considerations are as follows: no vaccines are commercially available; antimicrobial resistance is increasing; diagnostic and antimicrobial sensitivity testing needs to be improved; and a pen-side test would facilitate more rapid diagnosis and implementation of treatment with antimicrobials. More data on host susceptibility, stress factors, immune response and infectious dose levels are required. The impact of asymptomatic carriers, M. bovis survival in the environment and the role of wildlife in transmitting the disease also needs investigation. To facilitate development of vaccines, further analysis of more M. bovis genomes, its pathogenic mechanisms, including variable surface proteins, is required, along with reproducible disease models.

Bronchoalveolar lavage for diagnosis of tuberculosis infection in elephants.

Tuberculosis (TB) has been known to affect elephants for thousands of years. It was put into spotlight when few circus elephants were diagnosed carrying Mycobacterium (M.) tuberculosis. Because of the zoonotic risk and high susceptibility to M. tuberculosis, periodic testing was enacted since, in captive breeding programmes. Presently, trunk wash is the recommended diagnostic procedure for TB. Trunk wash, however, puts the operator at risk, has low sensitivity, and is prone to contamination. Here, bronchoalveolar lavage is described for the first time for TB diagnosis in elephants. Bronchial, trunk and mouth fluids were investigated using bacterial culture, M. tuberculosis complex (MTC)-specific real-time quantitative PCR (qPCR) and mycobacterial genus-specific qPCR for overall presence of mycobacteria or mycobacterial DNA including bacteria or DNA of closely related genera, respectively, in 14 elephants. Neither bacteria of the MTC nor their DNA were identified in any of the elephants. Yet, 25% of the cultures grew non-tuberculous mycobacteria (NTM) or closely related bacterial species. Furthermore, 85% of the samples contained DNA of NTM or closely related bacterial genera. This finding might explain continued false-positive results from various serological tests. From a zoonotic point of view, bronchoalveolar lavage is safer for the testing personal, has higher probability of capturing MTC and, through PCR, identifies DNA NTM in elephants. Yet, necessary endoscopic equipment, animal sedation and access to a TB reference laboratory might pose challenging requirements in remote conditions in some elephant range countries.

Fibrinolysis in trauma: a review.

Fibrinolytic dysregulation is an important mechanism in traumatic coagulopathy. It is an incompletely understood process that consists of a spectrum ranging from excessive breakdown (hyperfibrinolysis) and the shutdown of fibrinolysis. Both hyperfibrinolysis and shutdown are associated with excess mortality and post-traumatic organ failure. The pathophysiology appears to relate to endothelial injury and hypoperfusion, with several molecular markers identified in playing a role. Although there are no universally accepted diagnostic tests, viscoelastic studies appear to offer the greatest potential for timely identification of patients presenting with fibrinolytic dysregulation. Treatment is multimodal, involving prompt hemorrhage control and resuscitation, with controversy surrounding the use of antifibrinolytic drug therapy. This review presents the current evidence on the pathophysiology, diagnostic challenges, as well as the management of this hemostatic dysfunction. LEVEL OF EVIDENCE: Level III.

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.

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

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

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

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

Global multilocus sequence typing analysis of Mycoplasma bovis isolates reveals two main population clusters.

Mycoplasma bovis is a major bovine pathogen associated with bovine respiratory disease complex and is responsible for substantial economic losses worldwide. M. bovis is also associated with other clinical presentations in cattle, including mastitis, otitis, arthritis, and reproductive disorders. To gain a better understanding of the genetic diversity of this pathogen, a multilocus sequence typing (MLST) scheme was developed and applied to the characterization of 137 M. bovis isolates from diverse geographical origins, obtained from healthy or clinically infected cattle. After in silico analysis, a final set of 7 housekeeping genes was selected (dnaA, metS, recA, tufA, atpA, rpoD, and tkt). MLST analysis demonstrated the presence of 35 different sequence types (STs) distributed in two main clonal complexes (CCs), defined at the double-locus variant level, namely, CC1, which included most of the British and German isolates, and CC2, which was a more heterogeneous and geographically distant group of isolates, including European, Asian, and Australian samples. Index of association analysis confirmed the clonal nature of the investigated M. bovis population, based on MLST data. This scheme has demonstrated high discriminatory power, with the analysis showing the presence of genetically distant and divergent clusters of isolates predominantly associated with geographical origins.

Chlamydiosis in British Garden Birds (2005-2011): retrospective diagnosis and Chlamydia psittaci genotype determination.

The significance of chlamydiosis as a cause of mortality in wild passerines (Order Passeriformes), and the role of these birds as a potential source of zoonotic Chlamydia psittaci infection, is unknown. We reviewed wild bird mortality incidents (2005-2011). Where species composition or post-mortem findings were indicative of chlamydiosis, we examined archived tissues for C. psittaci infection using PCR and ArrayTube Microarray assays. Twenty-one of 40 birds tested positive: 8 dunnocks (Prunella modularis), 7 great tits (Parus major), 3 blue tits (Cyanistes caeruleus), 2 collared doves (Streptopelia decaocto, Order Columbiformes), and 1 robin (Erithacus rubecula). Chlamydia psittaci genotype A was identified in all positive passerines and in a further three dunnocks and three robins diagnosed with chlamydiosis from a previous study. Two collared doves had genotype E. Ten of the 21 C. psittaci-positive birds identified in the current study had histological lesions consistent with chlamydiosis and co-localizing Chlamydia spp. antigens on immunohistochemistry. Our results indicate that chlamydiosis may be a more common disease of British passerines than was previously recognized. Wild passerines may be a source of C. psittaci zoonotic infection, and people should be advised to take appropriate hygiene precautions when handling bird feeders or wild birds.

[Efficacy of doxycycline for treatment of chlamydiosis in flocks of racing and fancy pigeons]. / Wirksamkeit von Doxycyclin bei Chlamydiosen in Taubenbeständen.

OBJECTIVE: Evaluation of the clinical efficacy of doxycycline application via drinking water in the treatment of chlamydiosis in infected, clinically ill flocks of racing and fancy pigeons (Columba livia f. domestica) under field conditions. MATERIAL AND METHODS: In the scope of a field study, 13 pigeon flocks with clinically manifest ornithosis were treated over a period of 25 days with a novel doxycycline formulation via drinking water. Infection with Chlamydia psittaci had been confirmed beforehand by molecular-biological investigation of organ material from dissected pigeons in each flock. Therapeutic success was evaluated by PCR-analysis of triple swabs from 10 animals of each flock on day 25 of treatment and 7 days after completion of the therapy, as well as by repeated individual and daily herd examination. RESULTS: The causative agent Chlamydia psittaci together with additionally documented atypical chlamydia species in six of the 13 flocks could thereby no longer be detected. Furthermore, a resolution of clinical symptoms was achieved in each of the treated pigeon flocks. CONCLUSION: Application of doxycycline in the described therapy regimen proved successful for the treatment of ornithosis. In addition, excellent acceptance and tolerance of the deployed formulation could be confirmed in praxis.

Serological and immunohistochemical HER-2/neu statuses do not correlate and lack prognostic value for ovarian cancer patients.

The serodiagnostics of extracellular domain (ECD) HER-2/neu has turned into an evidenced-based tumour marker for HER-2/neu-positive breast cancer patients. This study investigated the clinical relevance of immunohistochemical and serum HER-2/neu in 44 patients with advanced ovarian cancer. The Hercept-Test® from DAKO Diagnostics was used to analyse immunohistochemical HER-2/neu expression. The HER-2/neu ECD in serum was determined quantitatively by Bayer Immuno 1™ Immunoanalyser. The HER-2/neu serum values were correlated to the clinical course of disease and to established prognostic factors, i.e. progression-free and overall survival. Some 23% of patients (n = 11) expressed HER-2/neu serum levels higher than 15 ng/mL, whereas only 7.7% (n = 2) of the patients examined by immunohistochemistry showed a HER-2/neu overexpression of the tissue. None of them revealed an overexpression of HER-2/neu ECD by serodiagnostics. HER-2/neu overexpression did not correlate significantly to any of the analysed prognostic factors. According to progression-free and overall survival, there was no significant difference between serologically HER-2/neu-positive or negative patients. For ovarian cancer patients, neither high HER-2/neu serum levels, nor immunohistochemically determined HER-2/neu positivity, appear to predict the course of disease. This study shows a lack of association between the immunohistochemical HER-2/neu status and the serum level of solute extracelluar HER-2/neu domain.

Tick-borne encephalitis (TBE) virus prevalence and virus genome characterization in field-collected ticks (Ixodes ricinus) from risk, non-risk and former risk areas of TBE, and in ticks removed from humans in Germany.

Tick-borne encephalitis (TBE) is recognized as the most important viral tick-borne zoonosis in 27 countries in Europe. In this study, ticks were collected in Germany from two non-risk areas in the states of Saxony-Anhalt and Mecklenburg-Western Pomerania, where several single human TBE cases have occurred in recent years. Ticks were also collected from a region in Thuringia, known to be a former risk area for TBE virus (TBEV), where numerous human cases were reported between 1960 and 1975. Detection of TBEV RNA was conducted by real-time RT-PCR. No TBEV was detected in any field-collected ticks. However, ticks were also collected from volunteers living in Bavaria. Three of 239 ticks from this collection were positive for TBEV genome and two genetically distinct TBEV strains were detected and characterized.

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri.

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Chlamydophila psittaci infections in humans during an outbreak of psittacosis from poultry in Germany.

In 2005, an outbreak of severe respiratory disease in a mixed poultry flock that was infected with Chlamydophila (C.) psittaci led to dissemination of the infection to at least 100 small poultry farms in 11 districts of Central Germany. At the same time, a total of 24 persons in contact with poultry from one of the flocks reported flu-like symptoms to their physician, thus suggesting zoonotic transmission. Within 3 weeks, seven individuals had to be hospitalized, with three of them requiring intensive care. Analysis of ompA sequences from chlamydial isolates and directly from clinical samples revealed the presence of both genotype A and E/B of C. psittaci at the source of the outbreak and in contact flocks. Genotype A was also detected in the three severely ill patients. The findings of the present study demonstrate the high zoonotic potential of avian chlamydiae. To ensure speedy eradication of psittacosis in poultry flocks and effective treatment of infected humans, fast, sensitive and species-specific detection of the causative agent is essential, as well as close collaboration between regional public health services, attending physicians and the diagnostic laboratories involved.

An investigation of the aetiological role of bovine herpesvirus 4 in bovine endometritis.

Uteri from 31 infertile cattle were examined for the presence of bovine herpesvirus 4 (BoHV-4) by nested polymerase chain reaction (PCR). Samples were also tested for bacteria, including chlamydiae and Mycoplasma bovis. BoHV-4 was detected by PCR in 27/31 (87.1%) samples, but the presence and amount of viral DNA was not correlated with histological and bacteriological findings. Arcanobacterium pyogenes, Histophilus somni and Pasteurella multocida were isolated from five cows with endometritis. Chlamydiae were detected in four cases (12.9%), but only two of these had endometritis. The study does not support a role for BoHV-4 as primary agent in bovine endometritis.