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Vesicle formation at endomembranes requires the selective concentration of cargo by coat proteins. Conserved adapter protein complexes at the Golgi (AP-3), the endosome (AP-1), or the plasma membrane (AP-2) with their conserved core domain and flexible ear domains mediate this function. These complexes also rely on the small GTPase Arf1 and/or specific phosphoinositides for membrane binding. The structural details that influence these processes, however, are still poorly understood. Here we present cryo-EM structures of the full-length stable 300 kDa yeast AP-3 complex. The structures reveal that AP-3 adopts an open conformation in solution, comparable to the membrane-bound conformations of AP-1 or AP-2. This open conformation appears to be far more flexible than AP-1 or AP-2, resulting in compact, intermediate, and stretched subconformations. Mass spectrometrical analysis of the cross-linked AP-3 complex further indicates that the ear domains are flexibly attached to the surface of the complex. Using biochemical reconstitution assays, we also show that efficient AP-3 recruitment to the membrane depends primarily on cargo binding. Once bound to cargo, AP-3 clustered and immobilized cargo molecules, as revealed by single-molecule imaging on polymer-supported membranes. We conclude that its flexible open state may enable AP-3 to bind and collect cargo at the Golgi and could thus allow coordinated vesicle formation at the trans-Golgi upon Arf1 activation.

Serological analysis of Chlamydia (C.) spp. infections is still mainly based on micro-immunofluorescence and ELISA. To overcome the limitations of conventional serology, we have designed a novel microarray carrying 52 synthetic peptides representing B-cell epitopes from immunodominant proteins of all 11 chlamydial species. The new assay has been validated using monospecific mouse hyperimmune sera. Subsequently, serum samples from cattle, sheep and humans with a known history of chlamydial infection were examined. For instance, the specific humoral response of sheep to treatment with a C. abortus vaccine has been visualized against a background of C. pecorum carriership. In samples from humans, dual infection with C. trachomatis and C. pneumoniae could be demonstrated. The experiments revealed that the peptide microarray assay was capable of simultaneously identifying specific antibodies to each Chlamydia spp. The actual assay represents an open platform test that can be complemented through future advances in Chlamydia proteome research. The concept of the highly parallel multi-antigen microarray proven in this study has the potential to enhance our understanding of antibody responses by defining not only a single quantitative response, but also the pattern of this response. The added value of using peptide antigens will consist in unprecedented serodiagnostic specificity.

Tc toxins are bacterial protein complexes that inject cytotoxic enzymes into target cells using a syringe-like mechanism. Tc toxins are composed of a membrane translocator and a cocoon that encapsulates a toxic enzyme. The toxic enzyme varies between Tc toxins from different species and is not conserved. Here, we investigate whether the toxic enzyme can be replaced by other small proteins of different origin and properties, namely Cdc42, herpes simplex virus ICP47, Arabidopsis thaliana iLOV, Escherichia coli DHFR, Ras-binding domain of CRAF kinase, and TEV protease. Using a combination of electron microscopy, X-ray crystallography and in vitro translocation assays, we demonstrate that it is possible to turn Tc toxins into customizable molecular syringes for delivering proteins of interest across membranes. We also infer the guidelines that protein cargos must obey in terms of size, charge, and fold in order to apply Tc toxins as a universal protein translocation system., Tc toxins are a major class of bacterial toxin translocation systems that inject toxic enzymes into target cells. Here the authors present functional and structural data showing that the toxic enzyme can be replaced by other small proteins and identify prerequisites required for successful translocation, which could facilitate the development of functional Tc-based protein injection devices.

Selecting particles from digital micrographs is an essential step in single-particle electron cryomicroscopy (cryo-EM). As manual selection of complete datasets—typically comprising thousands of particles—is a tedious and time-consuming process, numerous automatic particle pickers have been developed. However, non-ideal datasets pose a challenge to particle picking. Here we present the particle picking software crYOLO which is based on the deep-learning object detection system You Only Look Once (YOLO). After training the network with 200–2500 particles per dataset it automatically recognizes particles with high recall and precision while reaching a speed of up to five micrographs per second. Further, we present a general crYOLO network able to pick from previously unseen datasets, allowing for completely automated on-the-fly cryo-EM data preprocessing during data acquisition. crYOLO is available as a standalone program under http://sphire.mpg.de/ and is distributed as part of the image processing workflow in SPHIRE., Thorsten Wagner et al. present SPHIRE-crYOLO, a particle picking software for selecting particles from digital micrographs in cryoEM data. After training, the method automatically recognizes particles with high recall and precision, simplifying data pre-processing.

eLife 7, e38017 (2018). doi:10.7554/eLife.38017, α-Xenorhabdolysins (Xax) are α-pore-forming toxins (α-PFT) that form 1–1.3 MDa large pore complexes to perforate the host cell membrane. PFTs are used by a variety of bacterial pathogens to attack host cells. Due to the lack of structural information, the molecular mechanism of action of Xax toxins is poorly understood. Here, we report the cryo-EM structure of the XaxAB pore complex from Xenorhabdus nematophila and the crystal structures of the soluble monomers of XaxA and XaxB. The structures reveal that XaxA and XaxB are built similarly and appear as heterodimers in the 12–15 subunits containing pore, classifying XaxAB as bi-component α-PFT. Major conformational changes in XaxB, including the swinging out of an amphipathic helix are responsible for membrane insertion. XaxA acts as an activator and stabilizer for XaxB that forms the actual transmembrane pore. Based on our results, we propose a novel structural model for the mechanism of Xax intoxication., Published by eLife Sciences Publications, Cambridge

Tc toxins secrete toxic enzymes into host cells using a unique syringe-like injection mechanism. They are composed of three subunits, TcA, TcB and TcC. TcA forms the translocation channel and the TcB-TcC heterodimer functions as a cocoon that shields the toxic enzyme. Binding of the cocoon to the channel triggers opening of the cocoon and translocation of the toxic enzyme into the channel. Here we show in atomic detail how the assembly of the three components activates the toxin. We find that part of the cocoon completely unfolds and refolds into an alternative conformation upon binding. The presence of the toxic enzyme inside the cocoon is essential for its subnanomolar binding affinity for the TcA subunit. The enzyme passes through a narrow negatively charged constriction site inside the cocoon, probably acting as an extruder that releases the unfolded protein with its C terminus first into the translocation channel.

In the literature, studies of Chlamydia infection in birds have usually been confined to the search for Chlamydia (C., formerly Chlamydophila) psittaci, so that little is known about the presence of other chlamydial agents. In the present study, cloacal swabs and faeces samples of urban pigeons have been examined by real-time PCR, DNA microarray assays and partial ompA sequencing. Whilst C. psittaci was the predominant chlamydial agent in this pigeon population (75.8% of all Chlamydiaceae positives), the combined use of highly specific and sensitive molecular assays facilitated the detection of atypical serovars of C. psittaci, as well as other species of Chlamydia, such as C. abortus. Detection of C. pecorum and C. trachomatis from an avian host is reported here for the first time. Rather unexpectedly, 19.5% of all Chlamydiaceae-positive cases turned out to be infected with non-classified organisms. The considerable prevalence of these novel agents raises the question of their epidemiological importance and possible role as pathogens. Future surveys in domestic and wild birds will have to take the extended variety of chlamydial organisms into account.

Mycoplasma bovis infection of cattle tends to persist in affected herds and can be resistant to treatment. Given that the level of shedding of the organism can reflect the state of ongoing infection, a study was established to quantify the degree of such shedding in milk samples from infected herds using a novel real-time PCR technique. While the M. bovis load in herds with clinical disease was significantly higher than in disease-free herds (P < 0.001), infection persisted in the latter. Similarly, M. bovis was detected more frequently in conjunctival swabs from calves with respiratory disease when compared with samples from animals that did not exhibit such clinical signs. This novel real-time PCR assay is specific for M. bovis and can detect bacterial loads as low as 100 colony forming units/mL of milk.

Reports of canine chlamydiosis are infrequent, possibly because the pathogen is rarely considered to be a cause of disease in dogs. This report presents details of Chlamydophila psittaci infection in four bitches with recurrent keratoconjunctivitis, severe respiratory distress and reduced litter size (up to 50% stillborn or non-viable puppies) in a small dog-breeding facility in Germany. Cell culture and immunofluorescence examination of conjunctival, nasal and pharyngeal swabs revealed chlamydial inclusions. PCR and sequencing of ompA amplification products confirmed the presence of Cp. psittaci genotype C. The zoonotic potential of the pathogen was illustrated by evidence of disease in two children that lived on the premises with the infected dogs. There was circumstantial evidence to suggest infection of dogs and humans may have followed the introduction of two canaries and a parrot to the household. The persistent nature of the chlamydial infection suggests that dogs may be reservoirs of Cp. psittaci, but this putative role and whether or not dogs shed the pathogen require further investigation.

To assess long-term effects of naturally occurring infection with Chlamydophila spp. on animal health, 25 calves were grouped according to their chlamydial carrier status and checked for health parameters from 2 to 7 months of age. Monthly PCR testing revealed persistent or frequently recurring infections with Chlamydophila pecorum and Chlamydophila abortus in Group 2 (Chl, n = 13), but not in Group 1 (Chl-, n = 12). Despite the absence of any clinical illness, calves in Group 2 showed significantly higher body temperatures (subfebrile), lower bodyweights, reduced serum iron concentrations, lower total haemoglobin and haematocrit values. Counting and flow cytometric differentiation of peripheral white blood cells revealed a general decrease in leukocytes in Group 2. At necropsy, follicular bronchiolitis was found in 10/13 calves in Group 2 but in none of Group 1, and the weight of pharyngeal tonsils was significantly higher in Group 2. In conclusion, naturally occurring infections with Chlamydophila species in calves were found to be associated with chronic effects on animal health at a subclinical level. (C) 2007 Elsevier Ltd. All rights reserved

Introduction: The bacterium Mycoplasma capricolum subsp. capricolum is known as a pathogen in goats. There have been no reports on a zoonotic potential so far. Case presentation: A case of septicaemia and meningoencephalitis in a 62-year-old patient has been associated with infection by M. capricolum subsp. capricolum. No other infectious agent could be detected. Conclusion: Although it was impossible to identify the source of infection, coincidental contact with small ruminants or consumption of food products from goats during a tourist trip may have played a role.

The obligatory intracellular bacterium Chlamydophila psittaci is the causative agent of psittacosis in birds and humans. The capability of this zoonotic pathogen to develop a persistent phase is likely to play a role in chronicity of infections, as well as in failure of antibiotic therapy and immunoprophylaxis. To elucidate three different in vitro models for transition of C. psittaci to persistence (iron depletion, penicillin G treatment, and gamma interferon [IFN-γ] exposure), a set of 27 genes was examined by mRNA expression analysis using quantitative real-time PCR. While the phenotypical characteristics were the same as in other chlamydiae, i.e., aberrant morphology of reticulate bodies, loss of cultivability, and rescue of infectivity upon removal of inducers, the transcriptional response of C. psittaci to persistence-inducing factors included several new and distinctive features. Consistent downregulation of membrane proteins, chlamydial sigma factors, cell division protein, and reticulate body-elementary body differentiation proteins from 24 h postinfection onward proved to be a general feature of C. psittaci persistence. However, other genes displayed considerable variations in response patterns from one model to another, which suggests that there is no persistence model per se. In contrast to results for Chlamydia trachomatis , late shutdown of essential genes in C. psittaci was more comprehensive with IFN-γ-induced persistence, which is probably due to the absence of a functional tryptophan synthesis operon.

Viral protein R (Vpr) of human immunodeficiency virus, type 1 (HIV-1) is the major virion-associated accessory protein that affects a number of biological functions in the retroviral life cycle, including promotion of the transport of the preintegration complex into the nucleus and the induction of G2 host cell cycle arrest. Our recent investigation of the conformational heterogeneity of the proline residues in the N terminus of Vpr suggested a functional interaction between Vpr and a host peptidylprolyl cis/trans isomerase (PPIase) that might regulate the cis/trans interconversion of the imidic bond within the conserved proline residues of Vpr in vivo. Using surface plasmon resonance spectroscopy, Far Western blot, and pulldown experiments a physical interaction of Vpr with the major host PPIase cyclophilin A (CypA) is now demonstrated. The interaction domain involves the N-terminal region of Vpr including an essential role for proline in position 35. The CypA inhibitor cyclosporin A and non-immunosuppressive PPIase inhibitors such as NIM811 and sanglifehrin A block expression of Vpr without affecting pre- or post-translational events such as transcription, intracellular transport, or virus incorporation of Vpr. Similarly to CypA inhibition, Vpr expression is also reduced in HIV-1 infected CypA-/- knock-out T cells. This study thus shows that in addition to the interaction between CypA and HIV-1 capsid occurring during early steps in virus replication, CypA is also important for the de novo synthesis of Vpr and that in the absence of CypA activity, the Vpr-mediated cell cycle arrest is completely lost in HIV-1-infected T cells.

Antimicrobial treatment of chlamydial infections is known to be of limited efficacy. In this study, effects of doxycycline (D), usually the drug of choice, were compared with the combined therapy of doxycycline and rifampicin (R) in a bovine model of respiratory Chlamydia psittaci infection. After intrabronchial inoculation of the pathogen, 30 animals were assigned to five groups (n = 6 per group): untreated controls, monotherapy with D (5 mg kg(-1)day(-1) or 10 mg kg(-1)day(-1)), and combination therapy of D and R (600 mg day(-1)). Treatment continued until day 14 post inoculation (d.p.i.). Clinical signs, inflammatory markers, and pathological findings confirmed successful infection in all animals. Reisolation of the pathogen was possible in 4/6 untreated animals and in 4/12 animals treated with D alone until 4 d.p.i., but in none of the calves of the two D + R groups. Pathogen detection was possible in all animals without significant differences among groups. Severity of disease and time course of its resolution, assessed by clinical and pathological findings as well as inflammatory parameters, were not significantly different between untreated controls and calves receiving D alone or in combination with R. Regardless of the treatment regimen, all groups recovered clinically and cleared the infection within 2 weeks.

Einleitung: Chlamydia psittaci (Cp)-Infektionen konnen bei Mensch und Tier respiratorische Erkrankungen unterschiedlichen Schweregrades verursachen. Unter Verwendung eines etablierten Kalber-Modells [1, 2] waren die Ziele der vorliegenden Studie 1.) die pathophysiologische Charakterisierung der pulmonalen Reaktionsmechanismen sowie 2.) der klinische Vergleich mit naturlich infizierten Kalbern. Methoden: Je 21 Kalbern wurden 108 Einschluss-bildende Einheiten Cp oder Zellsuspension intrabronchial inokuliert. 1 – 2 Tage nach Inokulation (dpi) wurden drei gesunde, naive Sentinel-Kalber mit den Cp-infizierten Tieren vergesellschaftet. Ausgangswerte wurden ab einer Woche vor Inokulation gewonnen. Analysiert wurden Ergebnisse des klinischen Scorings, der nicht-invasiven Lungenfunktionsdiagnostik (Spirometrie, Kapnovolumetrie, Impulsoszillo-resistometrie) und der Blutproben (Zellkomposition, Konzentration des Lipopolysaccharid-bindenden Proteins (LBP), Gehalt chlamydialer DNA, Antikorper Bildung). Wahrend der Sektionen (n = 3 an 2, 3, 4, 7, 10, 14 & 35 dpi) wurde broncho-alveolare Lavageflussigkeit (BALF) gewonnen und zytologisch untersucht. Ergebnisse: Nach experimenteller Inokulation erreichte die fieberhafte, von respiratorischen Symptomen gepragte Allgemeininfektion ihren Hohepunkt 2 – 3 dpi und klang im Laufe einer Woche ab, ohne das Ausgangsniveau wieder zu erreichen. Dieser Verlauf spiegelte sich auch in der LBP-Konzentration wider. Trotz kurzen, flachen Atmungsmusters war 2 – 3dpi das Atemminutenvolumen erhoht. Anderungen der Atmungsmechanik resultierten aus Obstruktionen und Restriktionen, die bis 11dpi nachweisbar waren. Der Gehalt an Lymphozyten (L) im Blut sank nach Cp Inokulation signifikant (bis 4 dpi), wahrend stab- und segmentkernige neutrophile Granulozyten (N) anstiegen (2 dpi Leukozytose). Beide Zellfraktionen wurden zum Entzundungsherd Lunge rekrutiert (BALF: ↑N: 2 – 7 dpi, ↑L : 7 – 10 dpi). Nach Erregerkontakt war chlamdiale DNA bei 67% der Cp-exponierten Kalber im Blut nachweisbar, wobei der Gehalt in der experimentell infizierten Gruppe signifikant hoher war als bei den Sentinels, deren Infektion auch deutlich milder verlief. In beiden Gruppen fanden sich geringe DNA-Mengen bis zum Studienende (35 dpi) im Blut. Eine humorale Immunantwort war nur bei 67% der experimentell inokulierten Tiere nachweisbar, begann fruhestens 10 dpi und sank teilweise schon zum Studienende hin wieder ab. Diskussion: Die Freiheit von Symptomen nach Cp-Infektion erlaubt keinen Ruckschluss bezuglich der Elimination des Erregers oder dem Aufbau einer humoralen Immunantwort. Literatur: [1] Ostermann C et al. Vet J 2013; 196:351 – 359. [2] Reinhold P et al. PLoS One. 2012;7:e30125.7.

Summary Campylobacter jejuni strains are able to produce at least two different cytotoxins called “cytolethal distending toxin” (CLDT) and “cytolethal rounding toxin” (CLRT). In this study, we investigated the corresponding changes in CHO-K1 cells using the cell counter and analyzer system CASY 1. Determination of the cell volume after toxin treatment of the cells is a useful criterion for differentiation between the cytotoxic activities produced by Campylobacter strains. Incubation of the cells with crude CLDT resulted in a decrease in the cell count combined with a dramatic increase of the mean cell volume in comparison to the control culture. A decrease in the cell count was also seen as a response to CLRT preparations, while this toxin had no effect on the mean cell volume determined. It was shown that only CLDT caused histone-associated DNA fragments in the cytoplasm of CHO-K1 cells indicating an apoptotic pathway of cell death. In addition, the polymerase chain reaction (PCR) was employed to screen Campylobacter strains for the presence of the cdtB gene sequence, which was detectable in all strains investigated.

Infectious bovine keratoconjunctivitis (IBK) has significant economic consequences and a detrimental impact on animal welfare. Although Moraxella (Mor.) bovis is the primary causative agent, the role of other bacteria, such as Mor. ovis, Mor. bovoculi and Mycoplasma (Myc.) bovoculi, is not well understood. To assess the prevalence of infection with these organisms, and to correlate this with outbreaks of IBK, conjunctival samples from four herds of cattle in Germany of differing IBK status were examined. Herds were selected to represent a hypothetical course of IBK ranging from the pre-outbreak stage (herd 1), to the acute disease stage (herd 2), to a stage where treatment had ceased (herd 3). Unaffected animals were also included (herd 4). To facilitate effective, sensitive sample analysis, a new real-time PCR for Myc. bovoculi was developed and used in concert with established real-time PCR protocols for Myc. bovis and Moraxella spp. Herds 1 and 2 showed similarly high rates of detection for Myc. bovoculi (92.5% and 84.0%, respectively), whereas herds 3 and 4 had a lower prevalence (35.5% and 26.2%, respectively). Mor. bovis and Mor. ovis were more prevalent in herd 1 (32.5% and 87.5%, respectively) and herd 2 (38% and 58%, respectively) than herd 3 (10.4% and 1.3%, respectively) and herd 4 (9.8% and 31.1%, respectively). Mor. bovoculi was the only pathogen that correlated with clinical signs of IBK; at 20% prevalence, it was almost exclusively detected in herd 2. The results indicate that herds with high Myc. bovoculi prevalence are more predisposed to outbreaks of IBK, possibly due to a synergistic interaction with Moraxella spp.

Chlamydia (C.) psittaci is the causative agent of psittacosis, a zoonotic disease in birds and man. In addition, C. psittaci has been repeatedly found in domestic animals and is, at least in calves, also able to induce respiratory disease. Knowledge about transmission routes in cattle herds is still deficient, and nothing is known about differences in host response after either experimental or natural exposure to C. psittaci. Therefore, our recently developed respiratory infection model was exploited to evaluate (i) the presence of the pathogen in blood, excretions and air, (ii) the possibility of transmission and (iii) clinical symptoms, acute phase and immune response until 5 weeks after exposure. In this prospective study, intrabronchial inoculation of 10(8) inclusion-forming units of C. psittaci (n = 21 calves) led to reproducible acute respiratory illness (of approximately one week), accompanied by a systemic inflammatory reaction with an innate immune response dominated by neutrophils. Excretion and/or exhalation of the pathogen was sufficient to transmit the infection to naïve sentinel calves (n = 3) co-housed with the infected animals. Sentinel calves developed mild to subclinical infections only. Notably, excretion of the pathogen, predominantly via feces, occurred more frequently in animals naturally exposed to C. psittaci (i.e. sentinels) as compared to experimentally-inoculated calves. The humoral immune response was generally weak, and did not emerge regularly following experimental infection; however, it was largely absent after naturally acquired infection.

This study aimed to establish and evaluate a bovine respiratory model of experimentally induced acute C. psittaci infection. Calves are natural hosts and pathogenesis may resemble the situation in humans. Intrabronchial inoculation of C. psittaci strain DC15 was performed in calves aged 2-3 months via bronchoscope at four different challenge doses from 10(6) to 10(9) inclusion-forming units (ifu) per animal. Control groups received either UV-inactivated C. psittaci or cell culture medium. While 10(6) ifu/calf resulted in a mild respiratory infection only, the doses of 10(7) and 10(8) induced fever, tachypnea, dry cough, and tachycardia that became apparent 2-3 days post inoculation (dpi) and lasted for about one week. In calves exposed to 10(9) ifu C. psittaci, the respiratory disease was accompanied by severe systemic illness (apathy, tremor, markedly reduced appetite). At the time point of most pronounced clinical signs (3 dpi) the extent of lung lesions was below 10% of pulmonary tissue in calves inoculated with 10(6) and 10(7) ifu, about 15% in calves inoculated with 10(8) and more than 30% in calves inoculated with 10(9) ifu C. psittaci. Beside clinical signs and pathologic lesions, the bacterial load of lung tissue and markers of pulmonary inflammation (i.e., cell counts, concentration of proteins and eicosanoids in broncho-alveolar lavage fluid) were positively associated with ifu of viable C. psittaci. While any effect of endotoxin has been ruled out, all effects could be attributed to infection by the replicating bacteria. In conclusion, the calf represents a suitable model of respiratory chlamydial infection. Dose titration revealed that both clinically latent and clinically manifest infection can be reproduced experimentally by either 10(6) or 10(8) ifu/calf of C. psittaci DC15 while doses above 10(8) ifu C. psittaci cannot be recommended for further studies for ethical reasons. This defined model of different clinical expressions of chlamydial infection allows studying host-pathogen interactions.

The respiratory pathogen Chlamydia psittaci naturally occurs in bovine herds and was recently shown to impair calf health in a dose-dependent manner. The aim of this study was to determine whether the functional consequences and immunological reactions of infection were dose related by quantifying the consequences of acute respiratory chlamydial infection on respiratory signs, disturbances of pulmonary gas exchange, response of the innate immune system, and acute-phase reaction. Fourteen calves were challenged intrabronchially with different C. psittaci doses (from 106 to 109 inclusion-forming units (ifu) per animal). Ten controls received either UV-inactivated chlamydiae or cell culture medium. Compared to the controls, all animals challenged with live C. psittaci developed hypoxaemia linked to reduced haemoglobin oxygen saturation, increased alveolar–arterial oxygen partial pressure difference (A-aO2) and pulmonary shunt, with symptoms following a dose-dependent pattern. Increases in lipopolysaccharide-binding protein (LBP) and leukocytes were also dose-dependent and accompanied by a regenerative left shift in neutrophil granulocytes. With the exception of LBP, which reflected the load of chlamydial cell components in the host, pathophysiological reactions were only detected in calves challenged with viable chlamydiae. These results indicate that the pathophysiological consequences of respiratory C. psittaci infections are strongly dependent on the challenge dose of chlamydiae. For further studies, challenge doses between 106 and 108 ifu/calf are recommended.

Current typing methods of Chlamydia (C) trachomatis are mainly based on the diversity of the ompA gene, which is coding for the major outer membrane protein A. The present study aimed at facilitating genotyping of strains of this obligate intracellular human pathogen by developing a DNA microarray assay using the ArrayTube (TM) format for individual samples and the ArrayStrip (TM) format for higher throughput. The new test is exploiting multiple discriminatory sites by involving a total of 61 oligonucleotide probes representing genotype-specific polymorphisms in variable domains 1, 2 and 4 of the ompA gene. After multiplex amplification of these domains using biotinylated primers, the sample is hybridized in the microarray vessel under highly stringent conditions. The resulting binding pattern is genotype specific, thus allowing direct identification. We were able to show that DNA from each of the currently accepted genotypes (serovars) yielded a unique, theoretically expected and distinct hybridization pattern. The assay was also shown to be highly sensitive as a dilution containing the equivalent of 1 inclusion-forming unit was still correctly genotyped. In addition, when 62 clinical samples were examined and compared to PCR-RFLP typing results, the genotype was correctly identified by the DNA microarray in all cases. The present test is easy to handle and economically affordable, and it allows genotyping of C. trachomatis to be accomplished within a working day, thus lending itself for epidemiological studies and routine diagnosis. (C) 2010 Elsevier Ltd. All rights reserved

Three cases of atypical pneumonia in individuals working at a poultry slaughterhouse prompted an epidemiological survey in 10 poultry farms that had supplied birds. Using a Chlamydiaceae-specific real-time PCR assay, chlamydial agents were detected in 14 of 25 investigated flocks. Rather unexpectedly, Chlamydophila psittaci was identified only in one of the positive flocks, whereas ArrayTube DNA microarray testing indicated the presence of a new, so far unclassified member of the genus Chlamydophila. For further characterization of the agent involved, positive cloacal swabs were used to inoculate embryonated chicken eggs and isolates were obtained from 6 different flocks. Sequencing of 16S rRNA genes revealed nearly identical sequences of all samples. Alignment with representative sequences of Chlamydiaceae showed the separate position of the present strains outside the currently recognized species of Chlamydophila, but clearly within this genus. In contrast, partial ompA gene sequences displayed considerable diversity among the isolates, which had already been observed in restriction enzyme analysis of ompA PCR products. These data suggest that each farm had been infected with a different strain of this new chlamydial agent, the zoonotic potential and the exact taxonomic status of which have yet to be defined.

The avian and human pathogen Chlamydophila (C) psittaci represents a genetically heterogeneous species. To facilitate epidemiological surveys, more rapid yet highly specific molecular tests are needed. Currently used typing methods, i.e. serotyping and PCR-RFLP, have only limited sensitivity and are incapable of covering the wide spectrum of naturally occurring types of C psittaci strains. In the present study, a new DNA microarray assay based on the ArrayTube(R) (AT) technology was used to genotype C. psittaci in 98 isolates and 23 clinical tissue samples. The present array carries 35 oligonucleotide probes derived from variable domains 2 and 4 of the ompA gene. The assay proved highly sensitive, allowing correct genotyping of DNA from 2 inclusion-forming units. The results of DNA microarray genotyping of cultured strains proved highly concordant with the data from PCR-RFLP typing and serotyping. Sequencing of the ompA gene served as the reference test to verify the accuracy of AT genotyping results. In 15 instances (15.3%), strains were successfully typed by the AT assay, while serotyping and/or PCR-RFLP genotyping failed to produce unambiguous results. Eleven of these samples were ompA sequenced to confirm the AT findings. In addition to the currently accepted nine ompA genotypes, the microarray test was shown to recognise new provisional genotypes, such as Mat116 and YP84. In conclusion, the new AT assay proved to be suitable for rapid, sensitive and reproducible genotyping of C psittaci strains and can be recommended for routine diagnosis. (C) 2008 Elsevier B.V. All rights reserved

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.

While DNA microarrays have become a widely accepted tool for mRNA expression monitoring, their use in rapid diagnosis of bacterial and viral pathogens is only emerging. So far, insufficient sensitivity and high costs have been the major limiting factors preventing more widespread use of microarray platforms in direct testing of clinical samples. In the present study, a total of 339 samples, among them 293 clinical specimens from animals and humans, were examined by the ArrayTube™ (AT) DNA microarray assay to detect chlamydial DNA and identify the species of Chlamydia and Chlamydophila involved. Samples included nasal and conjunctival swabs, formalin-fixed, paraffin-embedded and fresh organ tissue, milk, feces and cell culture. Notably, the AT test was shown to detect mixed infections in clinical samples. The calculated median sensitivity of 0.81 over the entire panel of clinical samples was comparable to conventional 16S PCR, but slightly lower than real-time PCR and other PCR assays. However, when a panel of long-time stored swab samples was excluded from the calculation, the sensitivity was clearly higher (0.87) and equivalent to that of real-time PCR. Altogether, the data demonstrate the suitability of this DNA microarray assay for routine diagnosis.

Chlamydia psittaci is a zoonotic bacterium with a wide host range that can cause respiratory disease in humans and cattle. In the present study, effects of treatment with macrolides and quinolones applied alone or in combination with rifampicin were tested in a previously established bovine model of respiratory C. psittaci infection. Fifty animals were inoculated intrabronchially at the age of 6-8 weeks. Seven served as untreated controls, the others were assigned to seven treatment groups: (i) rifampicin, (ii) enrofloxacin, (iii) enrofloxacin + rifampicin, (iv) azithromycin, (v) azithromycin + rifampicin, (vi) erythromycin, and (vii) erythromycin + rifampicin. Treatment started 30 hours after inoculation and continued until 14 days after inoculation (dpi), when all animals were necropsied. The infection was successful in all animals and sufficient antibiotic levels were detected in blood plasma and tissue of the treated animals. Reisolation of the pathogen was achieved more often from untreated animals than from other groups. Nevertheless, pathogen detection by PCR was possible to the same extent in all animals and there were no significant differences between treated and untreated animals in terms of local (i.e., cell count and differentiation of BALF-cells) and systemic inflammation (i.e. white blood cells and concentration of acute phase protein LBP), clinical signs, and pathological findings at necropsy. Regardless of the reduced reisolation rate in treated animals, the treatment of experimentally induced respiratory C. psittaci infection with enrofloxacin, azithromycin or erythromycin alone or in combination with rifampicin was without obvious benefit for the host, since no significant differences in clinical and pathological findings or inflammatory parameters were detected and all animals recovered clinically within two weeks.

The use of conventional battery cages for hens will be prohibited in Germany in 2007. Only few studies, however, have considered the differences between battery cages and alternative systems with regard to infectious diseases. The existing gaps in the current knowledge need to be closed by research and measures must be developed that will prevent the spread of viral, bacterial, and parasitic infections in alternative poultry housing systems. With regard to virus infections, avian influenza requires particular attention. Since wild birds, particularly anseriformes, represent a reservoir for avian influenza viruses, free-ranging poultry is much more at risk of infection than birds in closed hen-houses. Appropriate measures must prevent direct contact with wild birds and transmission via contaminated water, feed, or equipment. Several bacterial infections of poultry represent zoonoses. Salmonella and Campylobacter are considered as particularly important. To avoid a potential increase in the risk of infection for consumers due to poultry keeping systems that might favour infections with bacterial zoonotic agents, there is a special need for research in this area. With regard to parasitic infections, coccidioses may cause problems in alternative poultry housing systems, and lead to considerable economic consequences. The epidemiological situation concerning infections with Histomonas meleagridis needs to be analysed. Since all compounds that had been used for prophylactic or therapeutic purposes in the past have been banned, there is a need to develop new drugs which are safe for animals and humans.

Mycoplasmas comprise a conglomerate of pathogens and commensals occurring in humans and animals. The genus Mycoplasma alone contains more than 120 species at present, and new members are continuously being discovered. Therefore, it seems promising to use a single highly parallel detection assay rather than develop separate tests for each individual species. In this study, we have designed a DNA microarray carrying 70 oligonucleotide probes derived from the 23S rRNA gene and 86 probes from the tuf gene target regions. Following a PCR amplification and biotinylation step, hybridization on the array was shown to specifically identify 31 Mycoplasma spp., as well as 3 Acholeplasma spp. and 3 Ureaplasma spp. Members of the Mycoplasma mycoides cluster can be recognized at subgroup level. This procedure enables parallel detection of Mollicutes spp. occurring in humans, animals or cell culture, from mono- and multiple infections, in a single run. The main advantages of the microarray assay include ease of operation, rapidity, high information content, and affordability. The new test's analytical sensitivity is equivalent to that of real-time PCR and allows examination of field samples without the need for culture. When 60 field samples from ruminants and birds previously analyzed by denaturing-gradient gel electrophoresis (DGGE) were tested by the microarray assay both tests identified the same agent in 98.3% of the cases. Notably, microarray testing revealed an unexpectedly high proportion (35%) of multiple mycoplasma infections, i.e., substantially more than DGGE (15%). Two of the samples were found to contain four different Mycoplasma spp. This phenomenon deserves more attention, particularly its implications for epidemiology and treatment.

Background The currently used genotyping system for the avian zoonotic pathogen Chlamydophila (C.) psittaci has evolved from serology and is based on ompA sequence variations. It includes seven avian and two non-avian genotypes. Restriction enzyme cleavage of the amplified ompA gene and, less frequently, ompA sequencing are being used for examination, but, beside methodological limitations, an increasing number of recently tested strains could not be assigned to any established genotype. Results Comprehensive analysis of all available ompA gene sequences has revealed a remarkable genetic diversity within the species C. psittaci, which is only partially covered by the present genotyping scheme. We suggest adjustments and extensions to the present scheme, which include the introduction of subgroups to the more heterogeneous genotypes A, E/B and D, as well as six provisional genotypes representing so far untypable strains. The findings of sequence analysis have been incorporated in the design of a new DNA microarray. The ArrayTube™ microarray-based ompA genotyping assay has been shown to discriminate among established genotypes and identify so far untyped strains. Its high specificity, which allows detection of single-nucleotide polymorphisms, is due to the parallel approach consisting in the use of 35 hybridization probes derived from variable domains 2 and 4 of the ompA gene. Conclusion The traditional genotyping system does not adequately reflect the extent of intra-species heterogeneity in ompA sequences of C. psittaci. The newly developed DNA microarray-based assay represents a promising diagnostic tool for tracing epidemiological chains, exploring the dissemination of genotypes and identifying non-typical representatives of C. psittaci.

Background To study the specific antibody response to infection with Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), the agent of Contagious Bovine Pleuropneumonia (CBPP), we examined three panels of sera collected during three experimental infection trials in African cattle. The methods used included an in-house complement fixation test (CFT), a commercially available CFT, a competitive antibody ELISA (cELISA) and the immunoblotting test (IBT). In addition, lung tissue samples were examined by culture. Results A total of 89% (51/59) of all experimentally infected animals tested positive on at least one of the serological tests throughout the trial. The specific antibody titres to the MmmSC infection became positive first by CFT (6 to 9 days post infection [dpi]), followed by IBT (9 to 13 dpi) and cELISA (13 to 16 dpi). Individual animals were found to display remarkably distinct seroconversion patterns, which allowed their classification into i) early high responders, ii) late high responders, and iii) low responders. In accordance with other studies, none of the present serological tests was capable of detecting all CBPP infected animals. Conclusion Comparison of the assays' performance in terms of sensitivity and specificity raises serious questions as to their reliability for identification of infected individuals in the field. In view of these limitations, a combination of CFT and cELISA can markedly improve CBPP diagnosis at single-animal level.