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

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

Simkania negevensis may produce long-lasting infections in human pneumocytes and endometrial cells.

Simkania negevensis is a novel Chlamydia-related bacterium and the founding member of the Simkaniaceae family within the Chlamydiales order. Little is known about the biology and pathogenesis of this bacterium. So far, S. negevensis has been considered as an amoebal symbiont, but its natural host remains unknown. Moreover, evidence of human exposition has been reported worldwide and an association with pneumonia and bronchiolitis is suspected. Here, we evaluated the ability of S. negevensis to replicate in potential environmental reservoirs, namely amoebae and arthropods, as well as in mammalian cells (Vero cells, pneumocytes and endometrial cells) and further evaluated the characteristics of its replicative vacuole. We demonstrated that S. negevensis efficiently replicates in all cell lines tested, with the shortest doubling time and an increased adhesion observed in pneumocytes. Our work highlights the specificities of the Simkania-containing vacuole compared to other Chlamydiales; contrarily to Chlamydia trachomatis, S. negevensis does not disrupt the Golgi apparatus. Importantly, our work suggests that S. negevensis infection is associated with few cytopathic effects and might persist for a prolonged time in infected cells. Further evaluation of its implication in human diseases is required; an implication in chronic or subacute respiratory infections might be suspected.

Respective IL-17A production by γδ T and Th17 cells and its implication in host defense against chlamydial lung infection.

The role of IL-17A is important in protection against lung infection with Chlamydiae, an obligate intracellular bacterial pathogen. In this study, we explored the producers of IL-17A in chlamydial lung infection and specifically tested the role of major IL-17A producers in protective immunity. We found that γδT cells and Th17 cells are the major producers of IL-17A at the early and later stages of chlamydial infection, respectively. Depletion of γδT cells in vivo at the early postinfection (p.i.) stage, when most γδT cells produce IL-17A, failed to alter Th1 responses and bacterial clearance. In contrast, the blockade of IL-17A at the time when IL-17A was mainly produced by Th17 (day 7 p.i.) markedly reduced the Th1 response and increased chlamydial growth. The data suggest that the γδ T cell is the highest producer of IL-17A in the very early stages of infection, but the protection conferred by IL-17A is mainly mediated by Th17 cells. In addition, we found that depletion of γδ T cells reduced IL-1α production by dendritic cells, which was associated with a reduced Th17 response. This finding is helpful to understand the variable role of IL-17A in different infections and to develop preventive and therapeutic approaches against infectious diseases by targeting IL-17A.

Evolution of hosts paying manifold costs of defence.

Hosts are expected to incur several physiological costs in defending against parasites. These include constitutive energetic (or other resource) costs of a defence system, facultative resource costs of deploying defences when parasites strike, and immunopathological costs of collateral damage. Here, we investigate the evolution of host recovery rates, varying the source and magnitude of immune costs. In line with previous work, we find that hosts paying facultative resource costs evolve faster recovery rates than hosts paying constitutive costs. However, recovery rate is more sensitive to changes in facultative costs, potentially explaining why constitutive costs are hard to detect empirically. Moreover, we find that immunopathology costs which increase with recovery rate can erode the benefits of defence, promoting chronicity of infection. Immunopathology can also lead to hosts evolving low recovery rate in response to virulent parasites. Furthermore, when immunopathology reduces fecundity as recovery rate increases (e.g. as for T-cell responses to urogenital chlamydiosis), then recovery and reproductive rates do not covary as predicted in eco-immunology. These results suggest that immunopathological and resource costs have qualitatively different effects on host evolution and that embracing the complexity of immune costs may be essential for explaining variability in immune defence in nature.

Chlamydial biology and its associated virulence blockers.

Chlamydiales are obligate intracellular parasites of eukaryotic cells. They can be distinguished from other Gram-negative bacteria through their characteristic developmental cycle, in addition to special biochemical and physical adaptations to subvert the eukaryotic host cell. The host spectrum includes humans and other mammals, fish, birds, reptiles, insects and even amoeba, causing a plethora of diseases. The first part of this review focuses on the specific chlamydial infection biology and metabolism. As resistance to classical antibiotics is emerging among Chlamydiae as well, the second part elaborates on specific compounds and tools to block chlamydial virulence traits, such as adhesion and internalization, Type III secretion and modulation of gene expression.

Identification and characterization of a novel porin family highlights a major difference in the outer membrane of chlamydial symbionts and pathogens.

The Chlamydiae constitute an evolutionary well separated group of intracellular bacteria comprising important pathogens of humans as well as symbionts of protozoa. The amoeba symbiont Protochlamydia amoebophila lacks a homologue of the most abundant outer membrane protein of the Chlamydiaceae, the major outer membrane protein MOMP, highlighting a major difference between environmental chlamydiae and their pathogenic counterparts. We recently identified a novel family of putative porins encoded in the genome of P. amoebophila by in silico analysis. Two of these Protochlamydiaouter membrane proteins, PomS (pc1489) and PomT (pc1077), are highly abundant in outer membrane preparations of this organism. Here we show that all four members of this putative porin family are toxic when expressed in the heterologous host Escherichia coli. Immunofluorescence analysis using antibodies against heterologously expressed PomT and PomS purified directly from elementary bodies, respectively, demonstrated the location of both proteins in the outer membrane of P. amoebophila. The location of the most abundant protein PomS was further confirmed by immuno-transmission electron microscopy. We could show that pomS is transcribed, and the corresponding protein is present in the outer membrane throughout the complete developmental cycle, suggesting an essential role for P. amoebophila. Lipid bilayer measurements demonstrated that PomS functions as a porin with anion-selectivity and a pore size similar to the Chlamydiaceae MOMP. Taken together, our results suggest that PomS, possibly in concert with PomT and other members of this porin family, is the functional equivalent of MOMP in P. amoebophila. This work contributes to our understanding of the adaptations of symbiotic and pathogenic chlamydiae to their different eukaryotic hosts.

A natural freshwater origin for two chlamydial species, Candidatus Piscichlamydia salmonis and Candidatus Clavochlamydia salmonicola, causing mixed infections in wild brown trout (Salmo trutta).

Gill disease in salmonids is characterized by a multifactorial aetiology. Epitheliocystis of the gill lamellae caused by obligate intracellular bacteria of the order Chlamydiales is one known factor; however, their diversity has greatly complicated analyses to establish a causal relationship. In addition, tracing infections to a potential environmental source is currently impossible. In this study, we address these questions by investigating a wild brown trout (Salmo trutta) population from seven different sites within a Swiss river system. One age class of fish was followed over 18 months. Epitheliocystis occurred in a site-specific pattern, associated with peak water temperatures during summer months. No evidence of a persistent infection was found within the brown trout population, implying an as yet unknown environmental source. For the first time, we detected 'Candidatus Piscichlamydia salmonis' and 'Candidatus Clavochlamydia salmonicola' infections in the same salmonid population, including dual infections within the same fish. These organisms are strongly implicated in gill disease of caged Atlantic salmon in Norway and Ireland. The absence of aquaculture production within this river system and the distance from the sea, suggests a freshwater origin for both these bacteria and offers new possibilities to explore their ecology free from aquaculture influences.

Chlamydiaceae infections in pig.

Chlamydiaceae are Gram-negative obligate intracellular bacteria. They are responsible for a broad range of diseases in animals and humans. In pigs, Chlamydia suis, Chlamydia abortus, Chlamydia pecorum and Chlamydia psittaci have been isolated. Chlamydiaceae infections in pigs are associated with different pathologies such as conjunctivitis, pneumonia, pericarditis, polyarthritis, polyserositis, pseudo-membranous or necrotizing enteritis, periparturient dysgalactiae syndrome, vaginal discharge, return to oestrus, abortion, mummification, delivery of weak piglets, increased perinatal and neonatal mortality and inferior semen quality, orchitis, epididymitis and urethritis in boars. However, Chlamydiaceae are still considered as non-important pathogens because reports of porcine chlamydiosis are rare. Furthermore, Chlamydiaceae infections are often unnoticed because tests for Chlamydiaceae are not routinely performed in all veterinary diagnostic laboratories and Chlamydiaceae are often found in association with other pathogens, which are sometimes more easily to detect. However, recent studies have demonstrated that Chlamydiaceae infections in breeding sows, boars and piglets occur more often than thought and are economically important. This paper presents an overview on: the taxonomy of Chlamydiaceae occurring in pigs, diagnostic considerations, epidemiology and pathology of infections with Chlamydiaceae in pigs, public health significance and finally on prevention and treatment of Chlamydiaceae infections in pigs.

Epitheliocystis in Atlantic salmon, Salmo salar L., farmed in fresh water in Ireland is associated with 'Candidatus Clavochlamydia salmonicola' infection.

Intracellular inclusions containing chlamydia-like organisms are frequently observed in the gill epithelial cells of Atlantic salmon, Salmo salar L., cultured in fresh water in Ireland. In this study, the causative agent was identified in four separate freshwater sites, using 16s rRNA sequencing, as 'Candidatus Clavochlamydia salmonicola'. Histopathology and real-time (RT) PCR were used to further assess infections. The prevalence of infection ranged from 75-100% between sites and infection intensity was highly variable. No significant lesions were associated with these infections. As a diagnostic tool, RT-PCR proved marginally more sensitive than histopathology. The fate of 'Candidatus Clavochlamydia salmonicola' in Atlantic salmon post-seawater transfer was investigated in a 12-week marine longitudinal study. Both RT-PCR and histopathological examination indicate that the organism disappears from the gills 4-6 weeks post-transfer.

Inclusion proteins of Chlamydiaceae.

Chlamydiaceae are obligate intracellular pathogens with family members among the etiological agents of several human diseases, such as blinding trachoma, sexually transmitted disease (Chlamydia trachomatis) and pneumonia (Chlamydophila pneumoniae, Chlamydophila psittaci). The bacteria replicate intracellularly in a membrane-bound vacuole termed inclusion. The chlamydial inclusion is effectively separated from eukaryotic endocytic pathways. More than two decades ago it was already speculated that Chlamydiae might modify the inclusion membrane through the insertion of chlamydial-derived components. However, because the classical genetic approaches cannot be applied to manipulate these bacteria, it took more than 10 years before definitive proof was obtained that Chlamydiae indeed actively modify the inclusion membrane by the insertion of proteins of chlamydial origin, first observed by Rockey et al. in 1995. This review will focus on the structural and functional aspects of inclusion proteins of Chlamydiaceae, thereby summarizing data obtained by in vitro studies and comparative genomics.

Mixed infections in vitro with different Chlamydiaceae strains and a cell culture adapted porcine epidemic diarrhea virus.

Assuming a synergistic or additive effect of Chlamydiaceae in coexistence with other enteropathogenic agents, the viral/bacterial interaction between a cell culture adapted porcine epidemic diarrhea virus (ca-PEDV) and different Chlamydiaceae strains was studied in vitro. Vero cells were dually infected with ca-PEDV and one of the three chlamydial strains Chlamydia trachomatis S45, Chlamydophila abortus S26/3 or Chlamydophila pecorum 1710S. Three experimental protocols were designed varying the inoculation sequence. Cell layers were first inoculated with Chlamydiaceae and 20 h later with ca-PEDV in protocol one. In protocol two, both agents were administered concurrently, whereas in protocol three, ca-PEDV was applied 20 h in advance of the Chlamydiaceae. Immunofluorescence techniques, immunohistochemical (IH) staining and electron microscopy were subsequently employed to investigate the cell layers. Using indirect immunofluorescence (IF) labeling, all mixed infections revealed dually infected cells, however, only incidentally and in low numbers. Characteristically, ca-PEDV syncytia with one or more chlamydial inclusions were detected but dually infected single cells were absent. Some syncytial cells contained enlarged C. abortus or C. pecorum inclusions with abnormally large developmental forms. In comparison with simultaneously conducted monoinfections, larger chlamydial inclusions were observed in dually infected cell layers. Experiments with C. trachomatis showed significantly increased numbers of chlamydial inclusions in dually infected cell layers compared to monoinfected ones. These findings indicate an influence of ca-PEDV on the chlamydial developmental cycle and in the case of C. trachomatis, a positive effect on chlamydial colonization in mixed infections.

In silico inference of inclusion membrane protein family in obligate intracellular parasites chlamydiae.

Chlamydiae are obligate intracellular pathogens that proliferate only inside a vacuole, called an inclusion. Chlamydial Inc proteins are known to be a major component of the inclusion membrane, but little is known about the gene number and function. The Inc proteins share very low sequence similarity but a similar hydropathy profile among them. Using the hydropathy profile, we computationally searched the open reading frames (ORFs) having a similar profile and predicted 90 and 36 ORFs (Inc-like ORFs) as candidates for Inc proteins in Chlamydia pneumoniae J138 and Chlamydia trachomatis serovar D, respectively. On the other hand, only a few Inc-like ORFs were found in organisms other than chlamydiae, suggesting that the Inc-like ORFs are specific to chlamydiae. Comparative genome analysis also revealed that the Inc-like ORFs have multiplied and diverged as paralogues and orthologues in the chlamydial genomes, and that some Inc-like ORFs lacked the N-terminal portion or encoded the split form. The data suggest that these gene products constitute a large protein family and may play an important role in chlamydial infection, growth and survival in the host cell.

Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms.

The current taxonomic classification of Chlamydia is based on limited phenotypic, morphologic and genetic criteria. This classification does not take into account recent analysis of the ribosomal operon or recently identified obligately intracellular organisms that have a chlamydia-like developmental cycle of replication. Neither does it provide a systematic rationale for identifying new strains. In this study, phylogenetic analyses of the 16S and 23S rRNA genes are presented with corroborating genetic and phenotypic information to show that the order Chlamydiales contains at least four distinct groups at the family level and that within the Chlamydiaceae are two distinct lineages which branch into nine separate clusters. In this report a reclassification of the order Chlamydiales and its current taxa is proposed. This proposal retains currently known strains with > 90% 16S rRNA identity in the family Chlamydiaceae and separates other chlamydia-like organisms that have 80-90% 16S rRNA relatedness to the Chlamydiaceae into new families. Chlamydiae that were previously described as 'Candidatus Parachlamydia acanthamoebae' Amann, Springer, Schönhuber, Ludwig, Schmid, Müller and Michel 1997, become members of Parachlamydiaceae fam. nov., Parachlamydia acanthamoebae gen. nov., sp. now. 'Simkania' strain Z becomes the founding member of Simkaniaceae fam. nov., Simkania negevensis gen. nov., sp. nov. The fourth group, which includes strain WSU 86-1044, was left unnamed. The Chlamydiaceae, which currently has only the genus Chlamydia, is divided into two genera, Chlamydia and Chlamydophila gen. nov. Two new species, Chlamydia muridarum sp. nov. and Chlamydia suis sp. nov., join Chlamydia trachomatis in the emended genus Chlamydia. Chlamydophila gen. nov. assimilates the current species, Chlamydia pecorum, Chlamydia pneumoniae and Chlamydia psittaci, to form Chlamydophila pecorum comb. nov., Chlamydophila pneumoniae comb. nov. and Chlamydophila psittaci comb. nov. Three new Chlamydophila species are derived from Chlamydia psittaci: Chlamydophila abortus gen. nov., sp. nov., Chlamydophila caviae gen. nov., sp. nov. and Chlamydophila felis gen. nov., sp. nov. Emended descriptions for the order Chlamydiales and for the family Chlamydiaceae are provided. These families, genera and species are readily distinguished by analysis of signature sequences in the 16S and 23S ribosomal genes.