Reticular Bodies Research Articles

Treatment of cats with conjunctivities and keratites for chlamidia infection

The article presents the results of research and treatment of cats with chlamydia in Poltava. The most common clinical sign of feline chlamydia is conjunctivitis (57 %) and keratitis (9.5 %). The combination of keratitis with conjunctivitis was found in 6 animals (28 %). Laboratory diagnosis of chlamydial infection was based on detecting chlamydial structures in a light microscope. In mild cases, when studying smears after Romanowski staining, Galbedstedter-Provachek bodies, which are cytoplasmic inclusions characteristic of chlamydia, were found in the material. Inclusions were painted according to Romanovsky-Gimza in blue and contained large reticular bodies, which, and small elementary bodies, were painted in purple-red. They were usually located near the nucleus. In the early stages of development, the parasites reached only 2–4 μm in diameter. At some stages of inclusion, they had a larger diameter and occupied the entire cytoplasm of the cell. The cell nucleus was displaced to the periphery of the membrane and changed shape. In addition to typical inclusions, parasites were often detected at the stage of reticular bodies. As a rule, the cells showed signs of damage in such cases. The treatment of cats with chlamydia was comprehensive. Antichlamydial therapy combines antibacterial therapy and immunocorrection. Cycloferon 12.5 % (aqueous solution for injection) was used as an immunomodulator for treating chlamydia. It was administered intramuscularly at a rate of 10 mg/kg body weight once a day–a total of ten injections. The second course was carried out in three weeks. Application Cycloferon 12.5 % was combined with an antibacterial agent. Azithromycin 10% was used as an antibacterial agent. It was included in the treatment regimen from the second day of application of Cycloferon 12.5 % intramuscularly, 1 ml per 10 kg of body weight; for 14 days (every 72 hours). Vigamox eye drops were instilled one drop into each eye four times a day for 21 days. In persistent conjunctival hyperemia, 1 % Emoxipin was prescribed as a capillary stabilizing agent. It was administered subconjunctivally in two drops (0.2–0.3 ml) twice a day for 15 days. Prodevit was used as a vitamin preparation. It was administered subcutaneously in a dosage of 0.3 ml: once every seven days. In most animals, improvement in clinical condition was noted on days 13–15 after treatment. During this period, there were no signs of purulent conjunctivitis, breathing improved, and wheezing was not heard. In animals diagnosed at the initial stage of the disease, clinical recovery occurred 7–8 days after treatment.

Prevalência de infecção por Chlamydia trachomatis na população feminina brasileira: um artigo de revisão

Chlamydia trachomatis (CT) é uma bactéria Gram negativa que parasita células eucarióticas, com maior frequência à cérvice feminina ocasionando diversas consequências a saúde da mulher. Apresenta duas formas, a primeira denominada de corpos elementares (CE) sendo está a forma infectante e resistente que sobrevive no meio extracelular e a segunda, intitulada de corpos reticulares (CR), forma proliferativa da bactéria. A CT é um patógeno muito virulento, com mais de vinte sorotipos que causam patologias graves nos seres humanos. Foi realizada uma revisão de literatura a partir de estudos de artigos publicados entre os anos de 2009 a 2018, nos idiomas Português, Inglês e língua Espanhola, sendo selecionado um total de 41 artigos. O presente trabalho buscou estimar a prevalência de Chlamydia trachomatis na população brasileira, destacando a epidemiologia, características da bactéria, importância do seu diagnóstico e tratamento. No Brasil, o diagnóstico e acompanhamento das infecções por esse microrganismo ainda não é uma realidade e há poucos estudos vinculados à temática, demonstram alta prevalência da infecção. Diante disso, a falta de acompanhamento e diagnóstico desse patógeno poderá culminar em complicações futuras, visto que o resultado da infecção em longo prazo e a promoção de infertilidade são relatados em vários estudos. As pacientes do sexo feminino são as mais afetadas por esse patógeno, ainda assim, complicações podem ser relatadas no sexo masculino.

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.

Microscopic and immunohistochemical study on the cornification of the developing beak in the turtle Emydura macquarii.

The development and cornification of the ramphoteca (beak) in turtles are not known. The microscopic aspects of beak formation have been analyzed in the pleurodirian turtle Emydura macquarii using histological, immunocytochemical and ultrastructural methods. At embryonic Stage 15 the maxillar beak is originated from discontinuous placodes (one frontal and two oral) formed in the epidermis above and below the mouth that later merge into the epidermis of the beak. The mandibular beak is formed by two lateral placodes. In the placodes, basal keratinocytes in contact with local mesenchymal condensations become columnar, and generate suprabasal cells forming 5-6 layers of embryonic epidermis at Stages 17-20 and a compact shedding alpha-layer at the base of the embryonic epidermis. These keratinocytes contain irregular or aggregated reticular bodies made of 30-40 nm thick strands of coarse filaments, mixed with tonofilaments and sparse lipid droplets. Beneath the shedding layer are present 3-4 layers of keratinocytes accumulating coarse filaments mixed with beta-corneous packets, and underneath spindle-shaped beta-cells differentiate where beta-corneous packets completely replace the reticulate bodies. Differently from scales where corneocytes partially merge, beak corneocytes remain separated but they are joined by numerous interlocking spines. The production of beta-cells in the thick corneous layer of the developing beak, like in claws, occurs before the differentiation of beta-cells in the body scutes. This indicates that a massive mesenchymal condensation triggers beta-differentiation before this process is later activated in most of body scutes of the carapace and plastron. J. Morphol. 277:1309-1319, 2016. © 2016 Wiley Periodicals, Inc.

Amoebal endosymbiont Parachlamydia acanthamoebae Bn9 can grow in immortal human epithelial HEp-2 cells at low temperature; an in vitro model system to study chlamydial evolution.

Ancient chlamydiae diverged into pathogenic and environmental chlamydiae 0.7–1.4 billion years ago. However, how pathogenic chlamydiae adapted to mammalian cells that provide a stable niche at approximately 37°C, remains unknown, although environmental chlamydiae have evolved as endosymbionts of lower eukaryotes in harsh niches of relatively low temperatures. Hence, we assessed whether an environmental chlamydia, Parachlamydia Bn9, could grow in human HEp-2 cells at a low culture temperature of 30°C. The assessment of inclusion formation by quantitative RT-PCR revealed that the numbers of bacterial inclusion bodies and the transcription level of 16SrRNA significantly increased after culture at 30°C compared to at 37°C. Confocal microscopy showed that the bacteria were located close to HEp-2 nuclei and were actively replicative. Transmission electron microscopy also revealed replicating bacteria consisting of reticular bodies, but with a few elementary bodies. Cytochalasin D and rifampicin inhibited inclusion formation. Lactacystin slightly inhibited bacterial inclusion formation. KEGG analysis using a draft genome sequence of the bacteria revealed that it possesses metabolic pathways almost identical to those of pathogenic chlamydia. Interestingly, comparative genomic analysis with pathogenic chlamydia revealed that the Parachlamydia similarly possess the genes encoding Type III secretion system, but lacking genes encoding inclusion membrane proteins (IncA to G) required for inclusion maturation. Taken together, we conclude that ancient chlamydiae had the potential to grow in human cells, but overcoming the thermal gap was a critical event for chlamydial adaptation to human cells.

High‐temperature adapted primitive Protochlamydia found in Acanthamoeba isolated from a hot spring can grow in immortalized human epithelial HEp‐2 cells

To elucidate how ancient pathogenic chlamydiae could overcome temperature barriers to adapt to human cells, we characterized a primitive chlamydia found in HS-T3 amoebae (Acanthamoeba) isolated from a hot spring. Phylogenetic analysis revealed the primitive species to be Protochlamydia. In situ hybridization staining showed broad distribution into the amoebal cytoplasm, which was supported by transmission electron microscopic analysis showing typical chlamydial features, with inclusion bodies including both elementary and reticular bodies. Interestingly, although most amoebae isolated from natural environments show reduced growth at 37°C, the HS-T3 amoebae harbouring the Protochlamydia grew well at body temperature. Although infection with Protochlamydia did not confer temperature tolerance to the C3 amoebae, the number of infectious progenies rapidly increased at 37°C with amoebal lysis. In immortalized human epithelial HEp-2 cells, fluorescence microscopic study revealed atypical inclusion of the Protochlamydia, and quantitative real-time polymerase chain reaction analyses also showed an increase in 16S ribosomal RNA DNA amounts. Together, these results showed that the Protochlamydia found in HS-T3 amoebae isolated from a hot spring successfully adapted to immortalized human HEp-2 cells at 37°C, providing further information on the evolution of ancient Protochlamydia to the present pathogenic chlamydiae.

Lipid chain branching at the iso- and anteiso-positions in complex chlamydia membranes: A molecular dynamics study

Membranes in the intracellular eubacterial parasite Chlamydia trachomatis consist of the elementary body (EB) and reticular body (RB), and contain methyl branches at the iso- and anteiso-positions for some phospholipid chains. Acyl chain branching is the focus of this study. Molecular dynamics simulations were used to study bilayers of 1-13-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (13-MpPPC), 1-14-methylpentadecanoyl-2-palmitoyl-phosphatidylcholine (14-MpPPC), and diphytanoylphosphatidylcholine (DPhPC). These three membranes were simulated at 323K and simulations of DPhPC at 298K were also performed for better comparison to existing experimental data. Two simulations of representative EB and RB membranes of C. trachomatis composed of nine different lipid components were performed at 310.15K, to accurately reflect compositions determined by experiment and physiological conditions. Based on nearly 0.5μs of simulation data, we report that branching increases average lipid surface area, area elastic moduli, and lipid axial relaxation times, while decreasing lipid chain order. Branching also has a distinct effect on electron density profiles. Due to their high cholesterol concentrations, the EB and RB membranes were found to have relatively high area elastic moduli, which may have important biological implications.

Proteome analysis of Chiamydia pneumoniae

This chapter describes proteome analysis of bacteria particularly, Chlamydia pneumoniae. Bacteria are especially well suited for proteome analysis, because of the availability of high quality genomes, the relatively small size of the genomes, and the low level of functional redundancy. All Chlamydia species are obligate intracellular gram-negative bacteria sharing a characteristic biphasic developmental cycle in which they alternate between two morphologically and metabolically distinct forms, the infectious elementary bodies (EB) and the replicative reticular bodies (RB). The described proteome comparison is a novel method to identify candidate secreted proteins for obligate intracellular bacteria. Identification and further investigation of the proteins differing between whole cell lysates and purified bacteria may reveal important Chlamydia pathogenicity factors and vaccine candidates and contribute to the understanding of the intracellular life of Chlamydia.

A dual infection by infectious cuticular epithelial necrosis virus and a Chlamydia -like organism in cultured Litopenaeus vannamei (Boone) in Ecuador

During 1996, microscopic examinations of post larvae and juveniles of moribund Litopenaeus vannamei showed multifocal necrosis in the cuticular epithelial tissues. In addition to these severe degenerative alterations in the epithelial cells typical of infectious cuticular epithelial necrosis virus (ICENV), columnar cells of the epithelium displayed small round intracytoplasmic inclusions in the necrotic tissue. Examination by electron microscopy of affected tissues demonstrated prokaryotic organisms in the cytoplasm of epithelial cells delineated by a distinct cytoplasmic vesicle; the prokaryotic organisms were morphologically similar to the genus Chlamydia. The necrotic tissue also showed the presence of particles of ICENV; the double infection by two different organisms in cuticular epithelial cells has not been reported previously. Two distinct stages in the intracellular development of a Chlamydia-like organism were recognized: (1) pleomorphic elementary bodies (EBs) that were spherical to oval were often observed in the process of division or in forming a common chain of three cells, the cells were surrounded by a rigid cell envelope and the presence of a cap or plaque hexagonally arrayed; (2) the reticular bodies (RBs) were forms often in the process of division. These cells had an electron-dense cytoplasm and contained a loose network of nuclear fibrils and a more fragile cell envelope. Regardless of the development stages of the Chlamydia-like organism within the cytoplasmic vesicles, ICENV particles were observed, either dispersed or in clusters, surrounded or inside the vesicles. The potential adverse impact of this dual infection on shrimp culture should be considered, especially in high-density operations.

Fine structure of Pseudotrypanosoma giganteum of Porotermes, a Trichomonad with a contractile costa

Summary Pseudotrypanosoma giganteum is a large trichomonad which shares many features with other members of the family Trichomonadidae, such as a striated costa with mesh-like structure (B pattern) and a lamella-like undulating membrane similar to that of Trichomonas vaginalis . Microtubules of the pelta have tubular reinforcements like those in the adhesive disc of the trichomonad Cochlosoma anatis . It has a contractile costa and an undulating membrane linked to the costa by microfibrils as well as two “reticular bodies” situated under the basal bodies, as seen in Trichomitopsis termopsidis ; these features characterise the Trichomitopsiinae subfamily . As previously described, the costa is a very thick fibre which is contractile and truly undulates within the cytoplasm.

The LPS localization might explain the lack of protection of LPS-specific antibodies in abortion-causing Chlamydia psittaci infections

Four monoclonal antibodies against chlamydial lipopolysaccharide (LPS) were used to study their localization and distribution in the Chlamydia psittaci AB7 abortion-causing strain by immunoelectron microscopy. A non-embedding technique on whole chlamydiae, together with a post-embedding technique on McCoy cells infected with the strain, were performed. Immunogold labelling was observed on the surface of reticular bodies (RB), but not on elementary bodies (EB). Immunolabelling was observed in ultrathin sections on both sides of the external chlamydial membrane, mainly on the inner side of EB and on the outer side of RB. Immunogold density was higher in EB than in RB; however, the absolute number of gold particles was higher in RB than EB, suggesting a loss of immunolabelling during the transformation of RB into EB. Specific labelling of LPS was also found in electrodense and adielectronic vacuoles near the surface of the cytoplasmic membrane of infected McCoy cells. These results suggest that the lack of protection against some chlamydial strains, despite the presence of anti-LPS specific antibodies, is due to the localization of LPS on the inner side of the external membrane of EB.

Fine Structure of the Gravid Paruterine Organ and Embryonic Envelopes of Mesocestoides lineatus (Cestoda)

Mesocestoides lineatus tetrathyridia were removed from the body cavity of lizards, Anolis caroli- nensis, and fed to hamsters in which they developed to adults. Gravid proglottids were taken from feces for study. Intact proglottids or excised paruterine organs (PO) were processed by standard techniques for TEM. For SEM, PO contents were exposed by subjecting whole proglottids to freon cryofracture or sectioning in Paraplast. Gravid PO's were thick-walled with enclosed eggs distributed throughout a cellular matrix. The wall was of parenchymal origin, containing muscle bundles and various cellular products. Each egg consisted of an oncosphere enclosed by a double-unit oncospheral membrane and a syncytial cytoplasmic envelope containing an embryo- phore. A discrete outer envelope and capsule were absent. Eggs were separated by PO matrix cells with prominent nuclei, mitochondria, intercellular junctions, and much extracellular material. These matrix cells may be related ontogenetically to individual eggs and thus homologous to embryonic envelope cells normally lying outside the embryophore of other cyclophyllids. Some differences in embryonic envelopes presumably were associated with containment of all eggs in the PO which may eliminate the need for individual protective shells. Other components of the PO matrix such as large reticular bodies are of unknown significance.

Toxicity of cadmium to Helisoma anceps and its effect on kidney ultrastructure

When specimens of Helisoma anceps were exposed to water containing 0.1, 1, and 5 ppm cadmium, mortality was proportional to concentration but tissue levels of Cd were not. The shell was the site of highest concentration and total amount of Cd due to adsorption and active accumulation. Concentrations of Cd in the kidney and mantle were equal to or greater than the remaining soft tissue. Ultrastructural changes in the kidney associated with Cd exposure were almost exclusively confined to the nephrocytes. Graded transformation of the residual body to a secondary lysosome was proportional to the Cd concentration of the medium and length of exposure. Atypical nucleoli and reticular bodies appeared in the 1-ppm Cd dose. Only after autolysis of the nephrocytes in 5 ppm did ultrastructural changes occur in the ureter cells. Ultrastructural changes in the kidney epithelia were proportional to Cd dosage but were not necessarily indicative of the tissue burden of Cd.

Electron microscopic study of oospore maturation and germination in an emasculate isolate of Saprolegnia ferax. 3. Changes in organelle status and associations

Changes in the structure of the main cytoplasmic organelles (excluding wall and nuclei) during the formation, maturation, and germination of Saprolegnia ferax oospores have been followed by transmission electron microscopy and their status assessed by stereologic analysis of micrographs. During oosphere differentiation there is a steady accumulation of lipid and dense-body (lipo-phospho glucan complexes) reserves; at the same time the overall status quo of other organelles is maintained. Oospore maturation is associated with the coalescence of dense-body vesicles, partial utilization of neutral lipid reserves, and dramatic decline in most other cell organelles. This is preceded by a marked increase in lipid-associated microbodies (glyoxysomes) which appear to develop from the fragmentation of cisternae of smooth endoplasmic reticulum. At the onset of germination the inner oospore wall is rapidly digested and normal levels of cytoplasmic organelles is restored. The neutral lipid fraction progressively declines with half the reserves being utilized before germ tube emergence. The osmiophilic dense-body vesicle derived ooplast matrix disintegrates and dense-bodylike granules are released into the expanding central vacuole and peripheral cytoplasmic vesicles, which eventually become associated with cup-shaped mitochondria. Unusual reticular bodies (mitochondria), crystalline aggregates (virus particles), and multivesicular bodies are also described in the cytoplasm of germinating spores. The results are discussed in relation to the possible metabolic pathways associated with the utilization of endogenous reserves.