Genetic and phenotypic analysis of the pathogenic potential of two novel Chlamydia gallinacea strains compared to Chlamydia psittaci.

Chlamydia gallinacea is an obligate intracellular bacterium that has recently been added to the family of Chlamydiaceae. C. gallinacea is genetically diverse, widespread in poultry and a suspected cause of pneumonia in slaughterhouse workers. In poultry, C. gallinacea infections appear asymptomatic, but studies about the pathogenic potential are limited. In this study two novel sequence types of C. gallinacea were isolated from apparently healthy chickens. Both isolates (NL_G47 and NL_F725) were closely related to each other and have at least 99.5% DNA sequence identity to C. gallinacea Type strain 08-1274/3. To gain further insight into the pathogenic potential, infection experiments in embryonated chicken eggs and comparative genomics with Chlamydia psittaci were performed. C. psittaci is a ubiquitous zoonotic pathogen of birds and mammals, and infection in poultry can result in severe systemic illness. In experiments with embryonated chicken eggs, C. gallinacea induced mortality was observed, potentially strain dependent, but lower compared to C. psittaci induced mortality. Comparative analyses confirmed all currently available C. gallinacea genomes possess the hallmark genes coding for known and potential virulence factors as found in C. psittaci albeit to a reduced number of orthologues or paralogs. The presence of potential virulence factors and the observed mortality in embryonated eggs indicates C. gallinacea should rather be considered as an opportunistic pathogen than an innocuous commensal.

Intranasal immunization with inactivated chlamydial elementary bodies formulated in VCG-chitosan nanoparticles induces robust immunity against intranasal Chlamydia psittaci challenge.

Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

Complement and Chlamydia psittaci: Non-Myeloid-Derived C3 Predominantly Induces Protective Adaptive Immune Responses in Mouse Lung Infection.

Recent advances in complement research have revolutionized our understanding of its role in immune responses. The immunomodulatory features of complement in infections by intracellular pathogens, e.g., viruses, are attracting increasing attention. Thereby, local production and activation of complement by myeloid-derived cells seem to be crucial. We could recently show that C3, a key player of the complement cascade, is required for effective defense against the intracellular bacterium Chlamydia psittaci. Avian zoonotic strains of this pathogen cause life-threatening pneumonia with systemic spread in humans; closely related non-avian strains are responsible for less severe diseases of domestic animals with economic loss. To clarify how far myeloid- and non-myeloid cell-derived complement contributes to immune response and resulting protection against C. psittaci, adoptive bone marrow transfer experiments focusing on C3 were combined with challenge experiments using a non-avian (BSL 2) strain of this intracellular bacterium. Surprisingly, our data prove that for C. psittaci-induced pneumonia in mice, non-myeloid-derived, circulating/systemic C3 has a leading role in protection, in particular on the development of pathogen-specific T- and B- cell responses. In contrast, myeloid-derived and most likely locally produced C3 plays only a minor, mainly fine-tuning role. The work we present here describes authentic, although less pronounced, antigen directed immune responses.

Regeneration of Pulmonary Tissue in a Calf Model of Fibrinonecrotic Bronchopneumonia Induced by Experimental Infection with Chlamydia Psittaci.

Pneumonia is a cause of high morbidity and mortality in humans. Animal models are indispensable to investigate the complex cellular interactions during lung injury and repair in vivo. The time sequence of lesion development and regeneration is described after endobronchial inoculation of calves with Chlamydia psittaci. Calves were necropsied 2-37 days after inoculation (dpi). Lesions and presence of Chlamydia psittaci were investigated using histology and immunohistochemistry. Calves developed bronchopneumonia at the sites of inoculation. Initially, Chlamydia psittaci replicated in type 1 alveolar epithelial cells followed by an influx of neutrophils, vascular leakage, fibrinous exudation, thrombosis and lobular pulmonary necrosis. Lesions were most extensive at 4 dpi. Beginning at 7 dpi, the number of chlamydial inclusions declined and proliferation of cuboidal alveolar epithelial cells and sprouting of capillaries were seen at the periphery of necrotic tissue. At 14 dpi, most of the necrosis had been replaced with alveoli lined with cuboidal epithelial cells resembling type 2 alveolar epithelial cells and mild fibrosis, and hyperplasia of organized lymphoid tissue were observed. At 37 dpi, regeneration of pulmonary tissue was nearly complete and only small foci of remodeling remained. The well-defined time course of development and regeneration of necrotizing pneumonia allows correlation of morphological findings with clinical data or treatment regimen.

Virulence-related comparative transcriptomics of infectious and non-infectious chlamydial particles.

BACKGROUND: Members of the phylum Chlamydiae are obligate intracellular pathogens of humans and animals and have a serious impact on host health. They comprise several zoonotic species with varying disease outcomes and prevalence. To investigate differences in virulence, we focused on Chlamydia psittaci, C. abortus and Waddlia chondrophila. Most threatening is C. psittaci, which frequently infects humans and causes psittacosis associated with severe pneumonia. The closest relative of C. psittaci is C. abortus, which shares the vast majority of genes but less frequently infects humans, and causes stillbirth and sepsis. W. chondrophila is more distantly related, and occasional human infections are associated with respiratory diseases or miscarriage. One possible explanation for differences in virulence originate from species-specific genes as well as differentially expressed homologous virulence factors. RESULTS: RNA-sequencing (RNA-Seq) was applied to purified infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs) in order to elucidate the transcriptome of the infectious and replicative chlamydial states. The results showed that approximately half of all genes were differentially expressed. For a descriptive comparison, genes were categorised according to their function in the RAST database. This list was extended by the inclusion of inclusion membrane proteins, outer membrane proteins, polymorphic membrane proteins and type III secretion system effectors. In addition, the expression of fifty-six known and a variety of predicted virulence and immunogenic factors with homologs in C. psittaci, C. abortus and W. chondrophila was analysed. To confirm the RNA-Seq results, the expression of nine factors was validated using real-time quantitative polymerase chain reaction (RT-qPCR). Comparison of RNA-Seq and RT-qPCR results showed a high mean Pearson correlation coefficient of 0.95. CONCLUSIONS: It was shown that both the replicative and infectious chlamydial state contained distinctive transcriptomes and the cellular processes emphasised in EBs and RBs differed substantially based on the chlamydial species. In addition, the very first interspecies transcriptome comparison is presented here, and the considerable differences in expression of homologous virulence factors might contribute to the differing infection rates and disease outcomes of the pathogens. The RNA-Seq results were confirmed by RT-qPCR and demonstrate the feasibility of interspecies transcriptome comparisons in chlamydia.

Molecular evidence to suggest pigeon-type Chlamydia psittaci in association with an equine foal loss.

Chlamydia psittaci is an important avian pathogen with spillover from infected wild and domesticated birds also posing a risk to human health. We recently reported a case of C. psittaci equine placentitis associated with further spillover to humans. Molecular typing of this case revealed it belonged to the 6BC clade of C. psittaci, a globally distributed highly virulent set of strains, typically linked to infection spillover from parrots. Equine chlamydiosis associated with C. psittaci infection has previously been reported elsewhere in countries where parrots are not endemic, however, raising questions over the identity of infecting C. psittaci strains and the potential infection reservoirs. In this study, we describe the detection and molecular characterization of C. psittaci in a case of equine abortion in southern Queensland. Equine placenta and fresh liver and lung tissue from the necropsied foetus were positive by C. psittaci-specific qPCR. Chlamydia psittaci-specific multilocus sequence typing and ompA genotyping were used to further characterize the detected equine strains and an additional strain obtained from a dove from a different geographic region presenting with psittacosis. Molecular typing of this case revealed that the infecting equine strains were closely related to the C0sittaci detected in dove, all belonging to an evolutionary lineage of C. psittaci strains typically associated with infections of pigeons globally. This finding suggests a broader diversity of C. psittaci strains may be detected in horses and in association with reproductive loss, highlighting the need for an expansion of surveillance studies globally to understand the epidemiology of equine chlamydiosis and the associated zoonotic risk.

Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform.

The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms.

The cellular ceramide transport protein CERT promotes Chlamydia psittaci infection and controls bacterial sphingolipid uptake.

Chlamydiaceae are bacterial pathogens that cause diverse diseases in humans and animals. Despite their broad host and tissue tropism, all Chlamydia species share an obligate intracellular cycle of development and have evolved sophisticated mechanisms to interact with their eukaryotic host cells. Here, we have analysed interactions of the zoonotic pathogen Chlamydia psittaci with a human epithelial cell line. We found that C. psittaci recruits the ceramide transport protein (CERT) to its inclusion. Chemical inhibition and CRISPR/Cas9-mediated knockout of CERT showed that CERT is a crucial factor for C. psittaci infections thereby affecting different stages of the infection including inclusion growth and infectious progeny formation. Interestingly, the uptake of fluorescently labelled sphingolipids in bacteria inside the inclusion was accelerated in CERT-knockout cells indicating that C. psittaci can exploit CERT-independent sphingolipid uptake pathways. Moreover, the CERT-specific inhibitor HPA-12 strongly diminished sphingolipid transport to inclusions of infected CERT-knockout cells, suggesting that other HPA-12-sensitive factors are involved in sphingolipid trafficking to C. psittaci. Further analysis is required to decipher these interactions and to understand their contributions to bacterial development, host range, tissue tropism, and disease outcome.

Deficiency of LIGHT signaling pathway exacerbates Chlamydia psittaci respiratory tract infection in mice.

LIGHT, a costimulatory member of the immunoglobulin superfamily (Ig SF), can greatly impact T cell activation. The role of the LIGHT signaling pathway in chlamydial infection was evaluated in mice following respiratory tract infection with Chlamydia psittaci. Compared with wild type (WT) mice, LIGHT knockout (KO) mice showed significant reduction of body weight, much lower survival rate, higher bacterial burden, prolonged infection time courses and more severe pathological changes in lung tissue. The mRNA levels of IFN-γ, TNF-α, IL-17 and IL-12 in the lung tissue of LIGHT KO mice were significantly lower than those in WT mice. While there was no obvious difference in the percentages of CD4+ and CD8+ T cells in the spleens of the two groups of mice, there was a markedly elevated percentage of CD4+ CD25+ FoxP3+ Treg cells in LIGHT KO mice. Together, these results demonstrate that the LIGHT signaling pathway is not only required for inflammatory cytokine production as part of the host response to chlamydial infection, but also influences the differentiation of CD4+ CD25+ FoxP3+ Treg cells, both of which may be essential for control of C. psittaci respiratory tract infection.

Analysis of Polymorphic Membrane Protein Expression in Cultured Cells Identifies PmpA and PmpH of Chlamydia psittaci as Candidate Factors in Pathogenesis and Immunity to Infection.

The polymorphic membrane protein (Pmp) paralogous families of Chlamydia trachomatis, Chlamydia pneumoniae and Chlamydia abortus are putative targets for Chlamydia vaccine development. To determine whether this is also the case for Pmp family members of C. psittaci, we analyzed transcription levels, protein production and localization of several Pmps of C. psittaci. Pmp expression profiles were characterized using quantitative real-time PCR (RT-qPCR), immunofluorescence (IF) and immuno-electron microscopy (IEM) under normal and stress conditions. We found that PmpA was highly produced in all inclusions as early as 12 hpi in all biological replicates. In addition, PmpA and PmpH appeared to be unusually accessible to antibody as determined by both immunofluorescence and immuno-electron microscopy. Our results suggest an important role for these Pmps in the pathogenesis of C. psittaci, and make them promising candidates in vaccine development.

Chlamydia-host cell interaction not only from a bird's eye view: some lessons from Chlamydia psittaci.

Chlamydia psittaci causes psittacosis/ornithosis in birds and is an economically important pathogen for poultry farming. It also infects nonavian domestic animals as well as rodents, and is a zoonotic human pathogen responsible for atypical pneumonia. The bacterium efficiently disseminates in host organisms causing pulmonary and systemic disease. Its rapid entry, fast replication cycle, and tight control of intracellular transport routes contribute to the host-to-host transmission and efficient growth observed with C. psittaci. Recent studies have revealed that the pathogen copes better than other chlamydial strains with proinflammatory effectors produced during the early immune reaction of infected hosts. These features likely contribute to successful infections and might explain the potent adaptation and evasion characteristics of the agent. Current findings on cell-autonomous, innate, and adaptive defenses against C. psittaci provide novel insights into the concerted immune mechanisms involved in the clearance of the pathogen. Further in-depth studies on C. psittaci and other related agents in cellular as well as animal models are needed to develop more efficient antichlamydial therapies and vaccination strategies.

Chlamydia psittaci infection increases mortality of avian influenza virus H9N2 by suppressing host immune response.

Avian influenza virus subtype H9N2 (H9N2) and Chlamydia psittaci (C. psittaci) are frequently isolated in chickens with respiratory disease. However, their roles in co-infection remain unclear. We tested the hypothesis that C. psittaci enhances H9N2 infection through suppression of host immunity. Thus, 10-day-old SPF chickens were inoculated intra-tracheally with a high or low virulence C. psittaci strain, and were simultaneously vaccinated against Newcastle disease virus (NDV). Significant decreases in body weight, NDV antibodies and immune organ indices occurred in birds with the virulent C. psittaci infection, while the ratio of CD4+/CD8+ T cells increased significantly compared to that of the lower virulence strain. A second group of birds were inoculated with C. psittaci and H9N2 simultaneously (C. psittaci+H9N2), C. psittaci 3 days prior to H9N2 (C. psittaci/H9N2), or 3 days after H9N2 (H9N2/C. psittaci), C. psittaci or H9N2 alone. Survival rates were 65%, 80% and 90% in the C. psittaci/H9N2, C. psittaci+H9N2 and H9N2/C. psittaci groups, respectively and respiratory clinical signs, lower expression of pro-inflammatory cytokines and higher pathogen loads were found in both C. psittaci/H9N2 and C. psittaci+H9N2 groups. Hence, virulent C. psittaci infection suppresses immune response by inhibiting humoral responses and altering Th1/Th2 balance, increasing mortality in H9N2 infected birds.

Ocorrência de Chlamydophila psittaci em pombos (Columba livia) na cidade de Salvador, Bahia / Occurrence of the pigeon Chlamydophila psittaci in pigeons (Columba livia) within the city of Salvador, Bahia

A existência de populações numerosas de pombos (Columba livia) em centros urbanos, em quase todo o mundo, tem se tornado um risco à saúde pública em vista das zoonoses que podem transmitir. A infecção por Chlamydophila psittaci foi avaliada em pombos que frequentam áreas públicas, como praças, ruas e pontos turísticos na cidade de Salvador, Bahia, por meio da PCR em amostras de fezes frescas, suabes cloacais e orofaríngeos. O estudo revelou uma frequência de infecção por C. psittaci de 11,7% (16/137) dos pombos, e oito dos 10 locais pesquisados apresentavam aves infectadas. A detecção de C. psittaci em amostras de cloaca e orofaringe foi maior (15,8% - 3/19) que em amostras de fezes frescas (11% - 13/118). Os resultados demonstram a ocorrência de infecção por C. psittaci em pombos que habitam as áreas públicas da cidade de Salvador e apontam para a necessária elaboração de medidas de controle e monitoramento das populações de pombos urbanos, bem como de ações voltadas à conscientização da sociedade sobre os riscos à saúde pública.

The existence of numerous pigeon (Columba livia) populations in urban centers, in most of the world, has become a public-health risk given the zoonoses they can transmit. Infection with Chlamydophila psittaci was evaluated in pigeons that frequent public areas, such as squares, streets and tourist sites in the city of Salvador, through PCR from fresh feces samples, cloacal swabs and oropharyngeals. The study revealed a frequency of infection with C. psittaci of 11.7% (16/137) in pigeons, and infected birds were found in eight of the ten locales researched. The detection of C. psittaci in samples of cloaca and oropharyngs was greater (15.8% - 3/19) than in the samples of fresh feces (11% - 13/118). The results show the occurrence of infection with C. psittaciem in pigeons that live in public areas in the city of Salvador and points to the necessity of elaborating control and monitoring measures for the populations of urban pigeons as well as developing actions that will raise society´s awareness of these risks to public health.

SINC, a type III secreted protein of Chlamydia psittaci, targets the inner nuclear membrane of infected cells and uninfected neighbors.

SINC, a new type III secreted protein of the avian and human pathogen Chlamydia psittaci, uniquely targets the nuclear envelope of C. psittaci-infected cells and uninfected neighboring cells. Digitonin-permeabilization studies of SINC-GFP-transfected HeLa cells indicate that SINC targets the inner nuclear membrane. SINC localization at the nuclear envelope was blocked by importazole, confirming SINC import into the nucleus. Candidate partners were identified by proximity to biotin ligase-fused SINC in HEK293 cells and mass spectrometry (BioID). This strategy identified 22 candidates with high confidence, including the nucleoporin ELYS, lamin B1, and four proteins (emerin, MAN1, LAP1, and LBR) of the inner nuclear membrane, suggesting that SINC interacts with host proteins that control nuclear structure, signaling, chromatin organization, and gene silencing. GFP-SINC association with the native LEM-domain protein emerin, a conserved component of nuclear "lamina" structure, or with a complex containing emerin was confirmed by GFP pull down. Our findings identify SINC as a novel bacterial protein that targets the nuclear envelope with the capability of globally altering nuclear envelope functions in the infected host cell and neighboring uninfected cells. These properties may contribute to the aggressive virulence of C. psittaci.

Cross-species transmission and emergence of novel viruses from birds.

Birds, the only living member of the Dinosauria clade, are flying warm-blooded vertebrates displaying high species biodiversity, roosting and migratory behavior, and a unique adaptive immune system. Birds provide the natural reservoir for numerous viral species and therefore gene source for evolution, emergence and dissemination of novel viruses. The intrusions of human into natural habitats of wild birds, the domestication of wild birds as pets or racing birds, and the increasing poultry consumption by human have facilitated avian viruses to cross species barriers to cause zoonosis. Recently, a novel adenovirus was exclusively found in birds causing an outbreak of Chlamydophila psittaci infection among birds and humans. Instead of being the primary cause of an outbreak by jumping directly from bird to human, a novel avian virus can be an augmenter of another zoonotic agent causing the outbreak. A comprehensive avian virome will improve our understanding of birds' evolutionary dynamics.

Host-pathogen interactions in specific pathogen-free chickens following aerogenous infection with Chlamydia psittaci and Chlamydia abortus.

Although Chlamydia (C.) psittaci infections are recognized as an important factor causing economic losses and impairing animal welfare in poultry production, the specific mechanisms leading to severe clinical outcomes are poorly understood. In the present study, we comparatively investigated pathology and host immune response, as well as systemic dissemination and expression of essential chlamydial genes in the course of experimental aerogeneous infection with C. psittaci and the closely related C. abortus, respectively, in specific pathogen-free chicks. Clinical signs appeared sooner and were more severe in the C. psittaci-infected group. Compared to C. abortus infection, more intense systemic dissemination of C. psittaci correlated with higher and faster infiltration of immune cells, as well as more macroscopic lesions and epithelial pathology, such as hyperplasia and erosion. In thoracic air sac tissue, mRNA expression of immunologically relevant factors, such as IFN-γ, IL-1ß, IL-6, IL-17, IL-22, LITAF and iNOS was significantly stronger up-regulated in C. psittaci- than in C. abortus-infected birds between 3 and 14 days post-infection. Likewise, transcription rates of the chlamydial genes groEL, cpaf and ftsW were consistently higher in C. psittaci during the acute phase. These findings illustrate that the stronger replication of C. psittaci in its natural host also evoked a more intense immune response than in the case of C. abortus infection.