Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization

Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1–2 copies of HPV DNA type 16 and 10–50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1–2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells. Considered altogether our results shows that in situ hybridization is a powerful technique to detect small amounts of nucleic acid sequences but the choice of the technique for cell examination is important. Single genes of HPV were visualized most efficiently by association of FISH with LSCM or quantitative microscopy with an intensified CCD camera.     

Phylogenetic evidence for two new insect-associated Chlamydia of the family Simkaniaceae

On the basis of 16S-23S ribosomal DNA analyses, the whitefly Bemisia tabaci (Sternorrhyncha, Aleyrodidae) and the eriococcid Eriococcus spurius (Sternorrhyncha, Eriococcidae) were each found to harbor novel related chlamydial species within the family Simkaniaceae. The generic designation Fritscheagen. nov. is proposed to accommodate the two species, F. bemisiaesp. nov. and F. eriococci sp. nov. The finding of chlamydial 16S-23S ribosomal DNA in B. tabaci is consistent with a previous electron microscopy study which found that bacteriocytes of this species contain structures that we consider to resemble the elementary and reticulate bodies of chlamydia (Costa HS, Westcot DM, Ullman DE, Rosell R, Brown JK, Johnson MW. Protoplasma 189:194-202, 1995). The cloning and sequencing of a 16.6 kilobase DNA fragment from F. bemisiae indicated that it contains six genes encoding for proteins similar to those found in other species of chlamydia. These results extend the range of organisms that harbor chlamydia.     

Patterns of immunoenhancement and suppression induced by Chlamydia trachomatis in vivo and in vitro

We investigated the cycle of immune enhancement and suppression seen in mice infected with Chlamydia trachomatis by using in vivo and in vitro model systems. BALB/c mice injected intravenously with chlamydia reveal a three- to seven-fold increase in numbers of plaque-forming cells producing antibodies against sheep red blood cells (SRBC), when immunized with SRBC 0 to 5 days after chlamydia infection. When mice are injected with SRBC 10 to 15 days after initial chlamydia infection, the specific anti-SRBC plaque-forming cell response is suppressed two- to three-fold. In vitro, low numbers (2 to 5 X 10(6) bacteria/ml) of chlamydia stimulate potent proliferative responses by B lymphocytes while high numbers (25 X 10(6) bacteria/ml) of bacteria generate strong, general T suppressor activity. This model has important implications for regulation of immune responses that arise at different times during chlamydial infections, as well as for the potential effectiveness of chlamydial vaccines.     

Detection and Differentiation of Chlamydiae by Fluorescence In Situ Hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and Parachlamydia. The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Detection and differentiation of chlamydiae by fluorescence in situ hybridization

Chlamydiae are important pathogens of humans and animals but diagnosis of chlamydial infections is still hampered by inadequate detection methods. Fluorescence in situ hybridization (FISH) using rRNA-targeted oligonucleotide probes is widely used for the investigation of uncultured bacteria in complex microbial communities and has recently also been shown to be a valuable tool for the rapid detection of various bacterial pathogens in clinical specimens. Here we report on the development and evaluation of a hierarchic probe set for the specific detection and differentiation of chlamydiae, particularly C. pneumoniae, C. trachomatis, C. psittaci, and the recently described chlamydia-like bacteria comprising the novel genera Neochlamydia and PARACHLAMYDIA: The specificity of the nine newly developed probes was successfully demonstrated by in situ hybridization of experimentally infected amoebae and HeLa 229 cells, including HeLa 229 cells coinfected with C. pneumoniae and C. trachomatis. FISH reliably stained chlamydial inclusions as early as 12 h postinfection. The sensitivity of FISH was further confirmed by combination with direct fluorescence antibody staining. In contrast to previously established detection methods for chlamydiae, FISH was not susceptible to false-positive results and allows the detection of all recognized chlamydiae in one single step.     

Some aspects of the immune response of koalas (Phascolarctos cinereus) and in vitro neutralization of chlamydia psittaci (koala strains)

Abstract Western-blot analysis was used to study the reaction of koala antisera, two specific polyclonal antibodies and one monoclonal antibody, with chlamydial antigens in koalas infected with Chlamydia psittaci . The koala sera recognized four C. psittaci surface antigens, corresponding to the major outer membrane protein (39.5 kDa), 31 kDa protein, 18 kDa protein and lipopolysaccharide. The S25-23 LPS specific monoclonal antibody inhibited chlamydial infection (55–67%) with both koala strains (type I and type II). Both koala antiserum and rabbit polyclonal antibodies against either type of chlamydia significantly reduced the number of infected cells resulting from type II infections at a dilution of 1 in 20. Rabbit antiserum against type II was effective in neutralizing infection by type II elementary bodies, but was less effective against type I infection. In addition, no koala antiserum was effective in neutralizing type I infection.     

Localization of TLR2 and MyD88 to Chlamydia trachomatis inclusions. Evidence for signaling by intracellular TLR2 during infection with an obligate intracellular pathogen

Chlamydia trachomatis is an obligate intracellular gram-negative pathogen and the etiologic agent of significant ocular and genital tract diseases. Chlamydiae primarily infect epithelial cells, and the inflammatory response of these cells to the infection directs both the innate and adaptive immune response. This study focused on determining the cellular immune receptors involved in the early events following infection with the L2 serovar of C. trachomatis.We found that dominant negative MyD88 inhibited interleukin-8 (IL-8) secretion during a productive infection with chlamydia. Furthermore, expression of Toll-like receptor (TLR)-2 was required for IL-8 secretion from infected cells, whereas the effect of TLR4/MD-2 expression was minimal. Cell activation was dependent on infection with live, replicating bacteria, because infection with UV-irradiated bacteria and treatment of infected cells with chloramphenicol, but not ampicillin, abrogated the induction of IL-8 secretion. Finally, we show that both TLR2 and MyD88 co-localize with the intracellular chlamydial inclusion, suggesting that TLR2 is actively engaged in signaling from this intracellular location. These data support the role of TLR2 in the host response to infection with C. trachomatis. Our data further demonstrate that TLR2 and the adaptor MyD88 are specifically recruited to the bacterial or inclusion membrane during a productive infection with chlamydia and provide the first evidence that intracellular TLR2 is responsible for signal transduction during infection with an intracellular bacterium.     

Gold-FISH: A new approach for the in situ detection of single microbial cells combining fluorescence and scanning electron microscopy

A novel fluorescence in situ hybridisation (FISH) method is presented that allows the combination of epifluorescence and scanning electron microscopy (SEM) to identify single microbial cells. First, the rRNA of whole cells is hybridised with horseradish peroxidase-labelled oligonucleotide probes and this is followed by catalysed reporter deposition (CARD) of biotinylated tyramides. This facilitates an amplification of binding sites for streptavidin conjugates covalently labelled with both fluorophores and nanogold particles. The deposition of Alexa Fluor 488 fluoro-nanogold–streptavidin conjugates was confirmed via epifluorescence microscopy and cells could be quantified in a similar way to standard CARD–FISH approaches. To detect cells by SEM, an autometallographic enhancement of the nanogold particles was essential, and allowed the in situ localisation of the target organisms at resolutions beyond light microscopy. Energy dispersive X-ray spectroscopy (EDS) was used to verify the effects of CARD and autometallography on gold deposition in target cells.     

The effect of cortisol on glycogen and fructose-1,6-bisphosphatase in baby hamster kidney cells infected with Chlamydia trachomatis

This study was designed to assay changes in glycogen synthesis which may occur as a result of cortisol treatment of chlamydial-infected cells. Monolayers of baby hamster kidney (BHK) cells, unlabeled and prelabeled with [6-14C]glucose, were treated with various concentrations of cortisol before (pretreated) or during (post-treated) infection with Chlamydia trachomatis. At designated times after absorption, the cells were harvested and assayed for total glycogen and 14C accumulation in glycogen. The total amount of glycogen accumulated in cells during the period of greatest chlamydial glycogen synthesis (36 h) was not affected by cortisol treatment. Cortisol treatment appeared to have retarded the accumulation of glycogen in treated infected cells until 30 h after infection. Treated infected cells prelabeled with [6-14C]glucose accumulated a greater amount of 14C in glycogen than untreated infected cells. All cortisol-treated, infected cells exhibited elevated levels of fructose-1,6-bisphosphatase activity, whereas untreated infected cells did not. The hypothesis that cortisol affects chlamydia multiplication by altering the intracellular environment of the host cell is compatible with the results obtained.     

Production, reproductive, and metabolic factors associated with chlamydial seropositivity and reproductive tract antigens in dairy herds with fertility disorders

While chlamydial infections cause abortions in cattle, its role in other reproductive disorders is uncertain. This study identified the risk factors for chlamydial infection in herds with history of subfertility. We investigated the possible effects of coinfections, different metabolic parameters, abortion, ovarian cysts, pathological vaginal discharge, length of the open period, milk yield, housing conditions and age. In cows from 34 farms with elevated reproductive disorders, 41.5% had antibodies against chlamydia, while chlamydia antigen was detected in the vagina and uterus of 46.7%. A statistical relationship between seropositivity and antigen positivity was not found. Abortion (OR = 6.6) and loose housing (OR = 2.3) were risk factors for the presence of chlamydia antibodies. Furthermore, there were significant relationships between metabolic disorders and chlamydial infections. Increased levels of beta-hydroxybutaric acid (OR = 6.8) and hypocalcaemia (OR = 6.0) often accompanied chlamydia antigen in the vagina. Increased age (OR = 1.2) and pathological vaginal discharge (OR = 2.4) were identified as risk factors for chlamydia antigen in the vagina. The largest risk factor was for the association of ovarian cysts (OR = 21.5) with uterine antigen. In conclusion, chlamydial infection in dairy herd cows is best understood as a multifactorial disease.     

Behavior of mitochondria during eupyrene and apyrene spermatogenesis in the silkworm,Bombyx mori (Lepidoptera), investigated by fluorescence in situ hybridization and electron microscopy

Summary Changes in the morphology and quantity of mitochondria and mitochondrial DNA during eupyrene and apyrene spermatogenesis in the silkworm were examined by electron microscopy and by fluorescence in situ hybridization with a 2 kb silkworm mitochondrial DNA clone (pBmMtE2). In the eupyrene spermatogenesis, the spermatocytes at early prophase I contained only a small amount of cytoplasm and showed a rather faint signal. As the cells grew larger in the later prophase I, the signal grew stronger. In the eupyrene spermatids, an especially strong signal was evident in the nebenkerns, in which all the cell's mitochondria were aggregated, and the strong fluorescence was maintained in mitochondrial derivatives. On the other hand, the apyrene cells were markedly smaller throughout spermatogenesis, showing much fainter signals for mitochondrial DNA than the eupyrene. Electron microscopy disclosed considerable differences in the behavior of mitochondria between the apýrene and the eupyrene cells. The observed qualitative and/or quantitative differences in the mitochondria may have some physiological bearing on the spermatogenesis of the two types of sperm.Abbreviations FISH fluorescence in situ hybridization - FITC fluorescein isothiocyanate - kb kilo base pair - PI propidium iodide - PBS phosphate-buffered saline     

Proportion of prokaryotes enumerated as viruses by epifluorescence microscopy

It is well known that there are prokaryotes small in size (e.g. ultra-microprokaryotes) that pass through a 0.2-μm filter. As bacterial and viral abundances are determined by epifluorescence microscopy and the differentiation between them is based on particle size, some bacteria can be erroneously enumerated as viruses, namely in marine waters where bacteria are small. However, there is no information on the proportion of prokaryotes that could be misidentified as viruses by epifluorescence microscopy. In this work, we assessed, in water samples collected in the estuarine system Ria de Aveiro (Portugal), the proportion of prokaryotes that could be counted as viruses by the current widespread epifluorescence microscopy and, for the first time, by fluorescence in situ hybridization (FISH). The total number of particles was determined on membranes of 0.2 and 0.02 μm after staining with 4′,6-diamidino-2-phenylindole (DAPI), and the number of prokaryotes (Bacteria and Archaea) was determined by FISH for both pore size membranes. The results show that, in the marine zone of the estuarine system, 28 % of particles enumerated as virus-like particles were prokaryotes, but, in the brackish water zone, only 13 % of the particles counted as viruses were actually prokaryotic cells. Epifluorescence microscopy overestimates viral abundance, and also the ratio viruses:prokaryotes, and this error must be taken into consideration because it can vary significantly within a system. In fact, in the marine zone of an estuarine system, the overestimation of viral abundance can be twice as high as in the brackish water zone.     

Trafficking of chlamydial antigens to the endoplasmic reticulum of infected epithelial cells

Confinement of the obligate intracellular bacterium Chlamydia trachomatis to a membrane-bound vacuole, termed an inclusion, within infected epithelial cells neither prevents secretion of chlamydial antigens into the host cytosol nor protects chlamydiae from innate immune detection. However, the details leading to chlamydial antigen presentation are not clear. By immunoelectron microscopy of infected endometrial epithelial cells and in isolated cell secretory compartments, chlamydial major outer membrane protein (MOMP), lipopolysaccharide (LPS) and the inclusion membrane protein A (IncA) were localized to the endoplasmic reticulum (ER) and co-localized with multiple ER markers, but not with markers of the endosomes, lysosomes, Golgi nor mitochondria. Chlamydial LPS was also co-localized with CD1d in the ER. Since the chlamydial antigens, contained in everted inclusion membrane vesicles, were found within the host cell ER, these data raise additional implications for antigen processing by infected uterine epithelial cells for classical and non-classical T cell antigen presentation.     

Light and electron microscopy of virus inclusions inAmaranthus lividus cells

Summary Amaranthus plants infected with a virus of rod-shaped particles showed under the light microscope intracytoplasmic amorphous and crystalline inclusions.The submicroscopic organization of mesophyll cells from infectedAmaranthus leaves by electron microscopy is described. Besides big crystalline inclusions, long dark inclusions correspondent to needle-like inclusions observed by light microscopy are definable in the cytoplasm. The amorphous inclusion bodies were formed by an overgrown protrusion of vacuolate cytoplasm containing virus particles, long very dark stained inclusions forming dense bands and rings, normal elements of the cytoplasm such as mitochondria, endoplasmic reticulum and ribosomes, and some spherosomes. Inclusions and virus particles were not found in chloroplasts, mitochondria or nuclei of infected cells.     

CCD microscopy and image analysis of cells and chromosomes stained by fluorescence in situ hybridization

Summary This paper reviews methods and applications of CCD microscopy for analysing cells and chromosomes subjected to fluorescence in situ hybridization (FISH). The current status of indirect and direct FISH staining methods with respect to probe labelling, detection sensitivity, multiplicity and DNA resolution is summarized. Microscope hardware, including special multi-band pass filters and CCD cameras required for FISH analysis, is described. Then follows a detailed discussion of current and emerging applications such as the automated enumeration of chromosomal abnormalities (counting of dots in interphase cells), comparative genomic hybridization, automated evaluation of radiation-induced chromosomal translocations, and high-resolution DNA mapping on highly extended chromatin. Finally, the limitations of the present methodology and future prospects are discussed.The Historical Journal lecture for 1993 presented by Dr Tanke to a joint meeting of the Royal Microscopical society of cell Biology, Clinical Cytology, Electron Microscopy and Pathology and the Belgian Socities in Brussels, Belgium on 2 September 1993.This work was supported financially by the Netherlands Organization for Scientific Research (NWO) and in part by Imagenetics, Framingham, MA, USA.     

Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples

A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other microorganisms and debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observing characteristic internal morphological features with phase-contrast microscopy. With this method, Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished surface water supplies during several recent outbreaks.     

Phylogenetic Evidence for Two New Insect-Associated Chlamydia of the Family <Emphasis Type="Italic">Simkaniaceae</Emphasis>

On the basis of 16S–23S ribosomal DNA analyses, the whitefly Bemisia tabaci (Sternorrhyncha, Aleyrodidae) and the eriococcid Eriococcus spurius (Sternorrhyncha, Eriococcidae) were each found to harbor novel related chlamydial species within the family Simkaniaceae. The generic designation Fritschea gen. nov. is proposed to accommodate the two species, F. bemisiae sp. nov. and F. eriococci sp. nov. The finding of chlamydial 16S–23S ribosomal DNA in B. tabaci is consistent with a previous electron microscopy study which found that bacteriocytes of this species contain structures that we consider to resemble the elementary and reticulate bodies of chlamydia (Costa HS, Westcot DM, Ullman DE, Rosell R, Brown JK, Johnson MW. Protoplasma 189:194–202, 1995). The cloning and sequencing of a 16.6 kilobase DNA fragment from F. bemisiae indicated that it contains six genes encoding for proteins similar to those found in other species of chlamydia. These results extend the range of organisms that harbor chlamydia. Received: 27 August 2002 / Accepted: 4 September 2002     

Use of immunofluorescence and phase-contrast microscopy for detection and identification of Giardia cysts in water samples.

A method was developed in which indirect immunofluorescence and phase-contrast microscopy are used for rapid detection and identification of Giardia cysts in raw and finished water supplies. When anti-Giardia cyst antiserum and fluorescein conjugate were applied to known Giardia cysts on membrane filters, the cysts fluoresced bright green when they were illuminated by UV light. This procedure permitted individual cysts to be quickly located even in samples heavily contaminated with other microorganisms and debris. The identity of presumptive Giardia cysts located in this way could then be confirmed by observing characteristic internal morphological features with phase-contrast microscopy. With this method, Giardia cysts were detected and their identities were confirmed in samples taken from raw and finished surface water supplies during several recent outbreaks.