Epitheliocystis in Greater Amberjack: Evidence of a Novel Causative Agent, Pathology, Immune Response and Epidemiological Findings.

Epitheliocystis is a fish gill disease caused by a broad range of intracellular bacteria infecting freshwater and marine fish worldwide. Here we report the occurrence and progression of epitheliocystis in greater amberjack reared in Crete (Greece). The disease appears to be caused mainly by a novel Betaproteobacteria belonging to the Candidatus Ichthyocystis genus with a second agent genetically similar to Ca. Parilichlamydia carangidicola coinfecting the gills in some cases. After a first detection of the disease in 2017, we investigated epitheliocystis in the following year's cohort of greater amberjack juveniles (cohort 2018) transferred from inland tanks to the same cage farm in the open sea where the first outbreak was detected. This cohort was monitored for over a year together with stocks of gilthead seabream and meagre co-farmed in the same area. Our observations showed that epitheliocystis could be detected in greater amberjack gills as early as a month following the transfer to sea cages, with ionocytes at the base of the gill lamellae being initially infected. Cyst formation appears to trigger a proliferative response, leading to the fusion of lamellae, impairment of gill functions and subsequently to mortality. Lesions are characterized by infiltration of immune cells, indicating activation of the innate immune response. At later stages of the outbreak, cysts were no longer found in ionocytes but were observed in mucocytes at the trailing edge of the filament. Whole cysts appeared finally to be expelled from infected mucocytes directly into the water, which might constitute a novel means of dispersion of the infectious agents. Molecular screening indicates that meagre is not affected by this disease and confirms the presence of previously described epitheliocystis agents, Ca. Ichthyocystis sparus, Ca. Ichthyocystis hellenicum and Ca. Similichlamydia spp., in gilthead seabream. Prevalence data show that the bacteria persist in both gilthead seabream and greater amberjack cohorts after first infection.

Iron Regulation in Elderly Asian Elephants (Elephas maximus) Chronically Infected With Mycobacterium tuberculosis.

Restriction of nutrients to pathogens (nutritional immunity) is a critical innate immune response mechanism that operates when pathogens such as Mycobacterium tuberculosis have the potential to evade humoral immunity. Tuberculosis is of growing concern for zoological collections worldwide and is well-illustrated by infections of Asian and African elephants, where tuberculosis is difficult to diagnose. Here, we investigated hematological parameters and iron deposition in liver, lung, and spleen of three Asian elephants (Elephas maximus) infected with Mycobacterium tuberculosis. For reference purposes, we analyzed tissue samples from control M. tuberculosis-negative elephants with and without evidence of inflammation and/or chronic disease. Molecular analyses of bacterial lesions of post mortally collected tissues confirmed M. tuberculosis infection in three elephants. DNA sequencing of the bacterial cultures demonstrated a single source of infection, most likely of human origin. In these elephants, we observed moderate microcytic anemia as well as liver (mild), lung (moderate) and spleen (severe) iron accumulation, the latter mainly occurring in macrophages. Macrophage iron sequestration in response to infection and inflammation is caused by inhibition of iron export via hepcidin-dependent and independent mechanisms. The hepatic mRNA levels of the iron-regulating hormone hepcidin were increased in only one control elephant suffering from chronic inflammation without mycobacterial infection. By contrast, all three tuberculosis-infected elephants showed low hepcidin mRNA levels in the liver and low serum hepcidin concentrations. In addition, hepatic ferroportin mRNA expression was high. This suggests that the hepcidin/ferroportin regulatory system aims to counteract iron restriction in splenic macrophages in M. tuberculosis infected elephants to provide iron for erythropoiesis and to limit iron availability for a pathogen that predominantly proliferates in macrophages. Tuberculosis infections appear to have lingered for more than 30 years in the three infected elephants, and decreased iron availability for mycobacterial proliferation may have forced the bacteria into a persistent, non-proliferative state. As a result, therapeutic iron substitution may not have been beneficial in these elephants, as this therapy may have enhanced progression of the infection.

Metagenomic Analysis of Fish-Associated Ca. Parilichlamydiaceae Reveals Striking Metabolic Similarities to the Terrestrial Chlamydiaceae.

Chlamydiae are an example of obligate intracellular bacteria that possess highly reduced, compact genomes (1.0-3.5 Mbp), reflective of their abilities to sequester many essential nutrients from the host that they no longer need to synthesize themselves. The Chlamydiae is a phylum with a very wide host range spanning mammals, birds, fish, invertebrates, and unicellular protists. This ecological and phylogenetic diversity offers ongoing opportunities to study intracellular survival and metabolic pathways and adaptations. Of particular evolutionary significance are Chlamydiae from the recently proposed Ca. Parilichlamydiaceae, the earliest diverging clade in this phylum, species of which are found only in aquatic vertebrates. Gill extracts from three Chlamydiales-positive Australian aquaculture species (Yellowtail kingfish, Striped trumpeter, and Barramundi) were subject to DNA preparation to deplete host DNA and enrich microbial DNA, prior to metagenome sequencing. We assembled chlamydial genomes corresponding to three Ca. Parilichlamydiaceae species from gill metagenomes, and conducted functional genomics comparisons with diverse members of the phylum. This revealed highly reduced genomes more similar in size to the terrestrial Chlamydiaceae, standing in contrast to members of the Chlamydiae with a demonstrated cosmopolitan host range. We describe a reduction in genes encoding synthesis of nucleotides and amino acids, among other nutrients, and an enrichment of predicted transport proteins. Ca. Parilichlamydiaceae share 342 orthologs with other chlamydial families. We hypothesize that the genome reduction exhibited by Ca. Parilichlamydiaceae and Chlamydiaceae is an example of within-phylum convergent evolution. The factors driving these events remain to be elucidated.

Ca. Endozoicomonas cretensis: A Novel Fish Pathogen Characterized by Genome Plasticity.

Endozoicomonas bacteria are generally beneficial symbionts of diverse marine invertebrates including reef-building corals, sponges, sea squirts, sea slugs, molluscs, and Bryozoans. In contrast, the recently reported Ca. Endozoicomonas cretensis was identified as a vertebrate pathogen, causing epitheliocystis in fish larvae resulting in massive mortality. Here, we described the Ca. E. cretensis draft genome, currently undergoing genome decay as evidenced by massive insertion sequence (IS element) expansion and pseudogene formation. Many of the insertion sequences are also predicted to carry outward-directed promoters, implying that they may be able to modulate the expression of neighbouring coding sequences (CDSs). Comparative genomic analysis has revealed many Ca. E. cretensis-specific CDSs, phage integration and novel gene families. Potential virulence related CDSs and machineries were identified in the genome, including secretion systems and related effector proteins, and systems related to biofilm formation and directed cell movement. Mucin degradation would be of importance to a fish pathogen, and many candidate CDSs associated with this pathway have been identified. The genome may reflect a bacterium in the process of changing niche from symbiont to pathogen, through expansion of virulence genes and some loss of metabolic capacity.

Ca. Similichlamydia in Epitheliocystis Co-infection of Gilthead Seabream Gills: Unique Morphological Features of a Deep Branching Chlamydial Family.

The Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) bacterial superphylum constitutes a broad range of organisms with an intriguing array of ultrastructural morphologies, including intracellular membranes and compartments and their corresponding complex genomes encoding these forms. The phylum Chlamydiae are all obligate intracellular bacteria and, although much is already known of their genomes from various families and how these regulate the various morphological forms, we know remarkably little about what is likely the deepest rooting clade of this phylum, which has only been found to contain pathogens of marine and fresh water vertebrates. The disease they are associated with is called epitheliocystis; however, analyses of the causative agents is hindered by an inability to cultivate them for refined in vitro experimentation. For this reason, we have developed tools to analyse both the genomes and the ultrastructures of bacteria causing this disease, directly from infected tissues. Here we present structural data for a member of the family Ca. Similichlamydiaceae from this deep-rooted clade, which we have identified using molecular tools, in epitheliocystis lesions of gilthead seabream (Sparus aurata) in Greece. We present evidence that the chlamydial inclusions appear to develop in a perinuclear location, similar to other members of the phylum and that a chlamydial developmental cycle is present, with chlamydial forms similar to reticular bodies (RBs) and elementary bodies (EBs) detected. Division of the RBs appeared to follow a budding process, and larger RBs with multiple condensed nucleoids were detected using both transmission electron microscopy (TEM) and by focused-ion beam, scanning electron microscopy (FIB-SEM). As model hosts, fish offer many advantages for investigation, and we hope by these efforts to encourage others to explore the biology of fish pathogens from the PVC.

Culture-independent genomics of a novel chlamydial pathogen of fish provides new insight into host-specific adaptations utilized by these intracellular bacteria.

Several Chlamydiales families are associated with epitheliocystis, a common condition of the fish gill epithelium. These families share common ancestors with the Chlamydiaceae and environmental Chlamydiae. Due to the lack of culture systems, little is known about the biology of these chlamydial fish pathogens. We investigated epitheliocystis in cultured Orange-spotted grouper (Epinephelus coioides) from North Queensland, Australia. Basophilic inclusions were present in the gills of 22/31 fish and the presence of the chlamydial pathogen in the cysts was confirmed by in situ hybridization. Giant grouper (Epinephelus lanceolatus) cultured in the same systems were epitheliocystis free. 16S rRNA gene sequencing revealed a novel member of the Candidatus Parilichlamydiaceae: Ca. Similichlamydia epinephelii. Using metagenomic approaches, we obtained an estimated 68% of the chlamydial genome, revealing that this novel chlamydial pathogen shares a number of key pathogenic hallmarks with the Chlamydiaceae, including an intact Type III Secretion system and several chlamydial virulence factors. This provides additional evidence that these pathogenic mechanisms were acquired early in the evolution of this unique bacterial phylum. The identification and genomic characterization of Ca. S. epinephelii provides new opportunities to study the biology of distantly-related chlamydial pathogens while shining a new light on the evolution of pathogenicity of the Chlamydiaceae.

A Zebrafish Model for Chlamydia Infection with the Obligate Intracellular Pathogen Waddlia chondrophila.

Obligate intracellular chlamydial bacteria of the Planctomycetes-Verrucomicrobia-Chlamydiae (PVC) superphylum are important pathogens of terrestrial and marine vertebrates, yet many features of their pathogenesis and host specificity are still unknown. This is particularly true for families such as the Waddliacea which, in addition to epithelia, cellular targets for nearly all Chlamydia, can infect and replicate in macrophages, an important arm of the innate immune system or in their free-living amoebal counterparts. An ideal pathogen model system should include both host and pathogen, which led us to develop the first larval zebrafish model for chlamydial infections with Waddlia chondrophila. By varying the means and sites of application, epithelial cells of the swim bladder, endothelial cells of the vasculature and phagocytosing cells of the innate immune system became preferred targets for infection in zebrafish larvae. Through the use of transgenic zebrafish, we could observe recruitment of neutrophils to the infection site and demonstrate for the first time that W. chondrophila is taken up and replicates in these phagocytic cells and not only in macrophages. Furthermore, we present evidence that myeloid differentiation factor 88 (MyD88) mediated signaling plays a role in the innate immune reaction to W. chondrophila, eventually by Toll-like receptor (TLRs) recognition. Infected larvae with depleted levels of MyD88 showed a higher infection load and a lower survival rate compared to control fish. This work presents a new and potentially powerful non-mammalian experimental model to study the pathology of chlamydial virulence in vivo and opens up new possibilities for investigation of other members of the PVC superphylum.

Epitheliocystis Distribution and Characterization in Brown Trout (Salmo trutta) from the Headwaters of Two Major European Rivers, the Rhine and Rhone.

We present a first description of the distribution and characterization of epitheliocystis infections in brown trout (Salmo trutta) from the upper catchments of two major European rivers, the Rhine and the Rhone. Overall, epitheliocystis was widely distributed, with 70% of the Rhine and 67% of the Rhone sites harboring epitheliocystis positive brown trout. The epitheliocystis agents Candidatus Piscichlamydia salmonis and Candidatus Clavichlamydia salmonicola could be identified in both catchments, although their relative proportions differed from site to site. Additionally, in two rivers in the Rhine catchment, a new species of Candidatus Similichlamydia was identified. Based on the histology, infection intensity, and severity of pathological changes were significantly more pronounced in mixed chlamydial infections, whereas single infections showed only low numbers of cysts and mild pathology. Infections could be found over a wide range of temperatures, which showed no correlation to infection prevalence or intensity.

Host-Associated Genomic Features of the Novel Uncultured Intracellular Pathogen Ca. Ichthyocystis Revealed by Direct Sequencing of Epitheliocysts.

Advances in single-cell and mini-metagenome sequencing have enabled important investigations into uncultured bacteria. In this study, we applied the mini-metagenome sequencing method to assemble genome drafts of the uncultured causative agents of epitheliocystis, an emerging infectious disease in the Mediterranean aquaculture species gilthead seabream. We sequenced multiple cyst samples and constructed 11 genome drafts from a novel beta-proteobacterial lineage, Candidatus Ichthyocystis. The draft genomes demonstrate features typical of pathogenic bacteria with an obligate intracellular lifestyle: a reduced genome of up to 2.6 Mb, reduced G + C content, and reduced metabolic capacity. Reconstruction of metabolic pathways reveals that Ca Ichthyocystis genomes lack all amino acid synthesis pathways, compelling them to scavenge from the fish host. All genomes encode type II, III, and IV secretion systems, a large repertoire of predicted effectors, and a type IV pilus. These are all considered to be virulence factors, required for adherence, invasion, and host manipulation. However, no evidence of lipopolysaccharide synthesis could be found. Beyond the core functions shared within the genus, alignments showed distinction into different species, characterized by alternative large gene families. These comprise up to a third of each genome, appear to have arisen through duplication and diversification, encode many effector proteins, and are seemingly critical for virulence. Thus, Ca Ichthyocystis represents a novel obligatory intracellular pathogenic beta-proteobacterial lineage. The methods used: mini-metagenome analysis and manual annotation, have generated important insights into the lifestyle and evolution of the novel, uncultured pathogens, elucidating many putative virulence factors including an unprecedented array of novel gene families.

Emerging pathogens of gilthead seabream: characterisation and genomic analysis of novel intracellular ß-proteobacteria.

New and emerging environmental pathogens pose some of the greatest threats to modern aquaculture, a critical source of food protein globally. As with other intensive farming practices, increasing our understanding of the biology of infections is important to improve animal welfare and husbandry. The gill infection epitheliocystis is increasingly problematic in gilthead seabream (Sparus aurata), a major Mediterranean aquaculture species. Epitheliocystis is generally associated with chlamydial bacteria, yet we were not able to localise chlamydial targets within the major gilthead seabream lesions. Two previously unidentified species within a novel ß-proteobacterial genus were instead identified. These co-infecting intracellular bacteria have been characterised using high-resolution imaging and genomics, presenting the most comprehensive study on epitheliocystis agents to date. Draft genomes of the two uncultured species, Ca. Ichthyocystis hellenicum and Ca. Ichthyocystis sparus, have been de novo sequenced and annotated from preserved material. Analysis of the genomes shows a compact core indicating a metabolic dependency on the host, and an accessory genome with an unprecedented number of tandemly arrayed gene families. This study represents a critical insight into novel, emerging fish pathogens and will be used to underpin future investigations into the bacterial origins, and to develop diagnostic and treatment strategies.

The emergence of epitheliocystis in the upper Rhone region: evidence for Chlamydiae in wild and farmed salmonid populations.

We present the first study comparing epitheliocystis in a wild and farmed salmonid in Europe. Sampling three tributaries to the Lake Geneva, including one from headwaters to river mouth, revealed an unequal distribution of epitheliocystis in brown trout (Salmo trutta). When evaluated histologically and comparing sites grouped as wild versus farm, the probability of finding infected trout is higher on farms. In contrast, the infection intensities, as estimated by the number of cysts per gill arch, were higher on average and showed maximum values in the wild trout. Sequence analysis showed the most common epitheliocystis agents were Candidatus Piscichlamydia salmonis, all clustering into a single clade, whereas Candidatus Clavichlamydia salmonicola sequences cluster in two closely related subspecies, of which one was mostly found in farmed fish and the other exclusively in wild brown trout, indicating that farms are unlikely to be the source of infections in wild trout. A detailed morphological analysis of cysts using transmission electron microscopy revealed unique features illustrating the wide divergence existing between Ca. P. salmonis and Ca. C. salmonicola within the phylum Chlamydiae.

Environmental marine pathogen isolation using mesocosm culture of sharpsnout seabream: striking genomic and morphological features of novel Endozoicomonas sp.

Aquaculture is a burgeoning industry, requiring diversification into new farmed species, which are often at risk from infectious disease. We used a mesocosm technique to investigate the susceptibility of sharpsnout seabream (Diplodus puntazzo) larvae to potential environmental pathogens in seawater compared to control borehole water. Fish exposed to seawater succumbed to epitheliocystis from 21 days post hatching, causing mortality in a quarter of the hosts. The pathogen responsible was not chlamydial, as is often found in epitheliocystis, but a novel species of the γ-proteobacterial genus Endozoicomonas. Detailed characterisation of this pathogen within the infectious lesions using high resolution fluorescent and electron microscopy showed densely packed rod shaped bacteria. A draft genome sequence of this uncultured bacterium was obtained from preserved material. Comparison with the genome of the Endozoicomonas elysicola type strain shows that the genome of Ca. Endozoicomonas cretensis is undergoing decay through loss of functional genes and insertion sequence expansion, often indicative of adaptation to a new niche or restriction to an alternative lifestyle. These results demonstrate the advantage of mesocosm studies for investigating the effect of environmental bacteria on susceptible hosts and provide an important insight into the genome dynamics of a novel fish pathogen.

Evaluation of zebrafish as a model to study the pathogenesis of the opportunistic pathogen Cronobacter turicensis.

Bacteria belonging to the genus Cronobacter spp. have been recognized as causative agents of life-threatening systemic infections, primarily in premature, low-birth weight and/or immune-compromised neonates. Knowledge remains scarce regarding the underlying molecular mechanisms of disease development. In this study, we evaluated the use of a zebrafish model to study the pathogenesis of Cronobacter turicensis LMG 23827(T), a clinical isolate responsible for two fatal sepsis cases in neonates. Here, the microinjection of approximately 50 colony forming units (CFUs) into the yolk sac resulted in the rapid multiplication of bacteria and dissemination into the blood stream at 24 h post infection (hpi), followed by the development of a severe bacteremia and larval death within 3 days. In contrast, the innate immune response of the embryos was sufficiently developed to control infection after the intravenous injection of up to 10(4) CFUs of bacteria. Infection studies using an isogenic mutant devoid of surviving and replicating in human macrophages (ΔfkpA) showed that this strain was highly attenuated in its ability to kill the larvae. In addition, the suitability of the zebrafish model system to study the effectiveness of antibiotics to treat Cronobacter infections in zebrafish embryos was examined. Our data indicate that the zebrafish model represents an excellent vertebrate model to study virulence-related aspects of this opportunistic pathogen in vivo.

Twenty years of research into Chlamydia-like organisms: a revolution in our understanding of the biology and pathogenicity of members of the phylum Chlamydiae.

Chlamydiae are obligate intracellular bacteria that share a unique but remarkably conserved biphasic developmental cycle that relies on a eukaryotic host cell for survival. Although the phylum was originally thought to only contain one family, the Chlamydiaceae, a total of nine families are now recognized. These so-called Chlamydia-like organisms (CLOs) are also referred to as 'environmental chlamydiae', as many were initially isolated from environmental sources. However, these organisms are also emerging pathogens, as many, such as Parachlamydia sp., Simkania sp. and Waddlia sp., have been associated with human disease, and others, such as Piscichlamydia sp. and Parilichlamydia sp., have been documented in association with diseases in animals. Their strict intracellular nature and the requirement for cell culture have been a confounding factor in characterizing the biology and pathogenicity of CLOs. Nevertheless, the genomes of seven CLO species have now been sequenced, providing new information on their potential ability to adapt to a wide range of hosts. As new isolation and diagnostic methods advance, we are able to further explore the richness of this phylum with further research likely to help define the true pathogenic potential of the CLOs while also providing insight into the origins of the 'traditional' chlamydiae.

Candidatus Syngnamydia venezia, a novel member of the phylum Chlamydiae from the broad nosed pipefish, Syngnathus typhle.

Chlamydia are obligate intracellular bacteria and important pathogens of humans and animals. Chlamydia-related bacteria are also major fish pathogens, infecting epithelial cells of the gills and skin to cause the disease epitheliocystis. Given the wide distribution, ancient origins and spectacular diversity of bony fishes, this group offers a rich resource for the identification and isolation of novel Chlamydia. The broad-nosed pipefish (Syngnathus typhle) is a widely distributed and genetically diverse temperate fish species, susceptible to epitheliocystis across much of its range. We describe here a new bacterial species, Candidatus Syngnamydia venezia; epitheliocystis agent of S. typhle and close relative to other chlamydial pathogens which are known to infect diverse hosts ranging from invertebrates to humans.

Immunohistochemical expression of Bax and Bak in canine non-neoplastic tissues.

Apoptosis is critical for embryonic development, maintenance of tissue homeostasis and protection against malignant transformation. The Bcl-2 family of proteins plays a key role in intrinsic apoptosis by controlling the integrity of the outer mitochondrial membrane, and the multidomain pro-apoptotic Bcl-2 family members Bax and Bak are essential components of this pathway. The aim of this study was to provide data on the expression of these proteins in normal canine tissues. Two antibodies against Bax recognising different conformations of the protein and one antibody against Bak were validated by immunohistochemistry and immunoblotting using canine recombinant proteins and keratinocytes treated with ultraviolet light. The antibodies were used immunohistochemically to label a wide panel of histologically normal tissues assembled on tissue microarrays. In addition, a subset of the tissues was evaluated by Western blot analysis. Immunohistochemical and Western blot analyses revealed that both Bax and Bak are widely expressed in non-neoplastic tissues from adult dogs. Immunohistochemistry showed almost exclusively cytoplasmic labelling and prominent labelling of epithelial cells. In lymph nodes, immunohistochemical labelling was diffuse for both proteins and showed enhanced intensities in the mantle zones for Bax and the germinal centres for Bak. Strong reactivity for the active conformation of Bax was detected only in enterocytes and Leydig cells and in scattered lymphocytes. These data indicate widespread expression of Bax and Bak in normal canine tissues. Knowledge of the expression of Bax and Bak in normal tissues is a prerequisite in assessing the role of these proteins in canine neoplastic disease.

Evidence for Chlamydia in wild mammals of the Serengeti.

Only limited information is available on the presence of Chlamydiaceae in wildlife, a deficit that is particularly acute concerning mammalian wildlife in Africa. In a retrospective analysis of organ material from an earlier study on wild mammals from the Seregenti National Park, 521 samples from 54 animals of 14 mammalian species were investigated. The presence of Chlamydiaceae was analyzed using molecular methods and immunohistochemistry. Chlamydial DNA was detected by real-time polymerase chain reaction in formalin-fixed and paraffin-embedded tissues from large ruminants (African buffaloes, Syncerus caffer, n=4) and a large predator (spotted hyena, Crocuta crocuta, n=1). Microarray results revealed Chlamydia abortus in all cases, confirmed by sequencing of selected samples, and a mixed infection with Chlamydia abortus and Chlamydia pneumoniae in an African buffalo. This is the first report of Chlamydiaceae in African wildlife of the Serengeti area.

Health of farmed fish: its relation to fish welfare and its utility as welfare indicator.

This brief review focuses on health and biological function as cornerstones of fish welfare. From the function-based point of view, good welfare is reflected in the ability of the animal to cope with infectious and non-infectious stressors, thereby maintaining homeostasis and good health, whereas stressful husbandry conditions and protracted suffering will lead to the loss of the coping ability and, thus, to impaired health. In the first part of the review, the physiological processes through which stressful husbandry conditions modulate health of farmed fish are examined. If fish are subjected to unfavourable husbandry conditions, the resulting disruption of internal homeostasis necessitates energy-demanding physiological adjustments (allostasis/acclimation). The ensuing energy drain leads to trade-offs with other energy-demanding processes such as the functioning of the primary epithelial barriers (gut, skin, gills) and the immune system. Understanding of the relation between husbandry conditions, allostatic responses and fish health provides the basis for the second theme developed in this review, the potential use of biological function and health parameters as operational welfare indicators (OWIs). Advantages of function- and health-related parameters are that they are relatively straightforward to recognize and to measure and are routinely monitored in most aquaculture units, thereby providing feasible tools to assess fish welfare under practical farming conditions. As the efforts to improve fish welfare and environmental sustainability lead to increasingly diverse solutions, in particular integrated production, it is imperative that we have objective OWIs to compare with other production forms, such as high-density aquaculture. However, to receive the necessary acceptance for legislation, more robust scientific backing of the health- and function-related OWIs is urgently needed.

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.

Chlamydia abortus YhbZ, a truncated Obg family GTPase, associates with the Escherichia coli large ribosomal subunit.

The stringent stress response is vital for bacterial survival under adverse environmental conditions. Obligate intracellular Chlamydia lack key stringent response proteins, but nevertheless can interrupt the cell cycle and enter stasis or persistence upon amino acid starvation. A possible key protein retained is YhbZ, a homologue of the ObgE guanosine triphosphatase (GTPase) superfamily connecting the stringent stress response to ribosome maturation. Curiously, chlamydial YhbZ lacks the ObgE C-terminal domain thought to be essential for binding the large ribosomal subunit. We expressed recombinant Chlamydia abortus YhbZ and showed it to be a functional GTPase, with similar activity to other Obg GTPase family members. As Chlamydia are resistant to genetic manipulation, we performed heterologous expression and gradient centrifugation experiments in Escherichia coli and found that, despite the missing C-terminal domain, C. abortus YhbZ co-fractionates with the E. coli 50S large ribosomal subunit. In addition, overexpression of chlamydial YhbZ in E. coli leads to growth defects and elongation, as reported for other Obg members. YhbZ did not complement an E. coli obgE temperature-sensitive mutant, indicating the C-terminal acidic domain may have an additional role. This data supports a role for YhbZ linking the chlamydial stress response to ribosome function and cellular growth.