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D Veal

Publications and source records attributed to D Veal.

15 recordsLinked to original sources
PubMed2004

Determination of Cryptosporidium parvum oocyst viability by fluorescence in situ hybridization using a ribosomal RNA-directed probe.

AIMS: Fluorescence in situ hybridization (FISH) has been proposed for species-specific detection, and viability determination of Cryptosporidium parvum oocysts. FISH-based viability determination depends on rRNA decay after loss of viability. We examined the effects of RNase(s) and RNase inhibitors on FISH of C. parvum. METHODS AND RESULTS: FISH was performed using a 5'-Texas red-labelled DNA oligonucleotide probe at 1 pM microl(-1). Intact and heat-permeabilized oocysts were treated with 1-100 microg ml(-1) RNase. FISH of intact oocysts appeared unaffected by exogenous RNase if this was neutralized before permeabilization. FISH fluorescence of heat-killed oocysts stored in phosphate-buffered saline at room temperature decayed by 1/2 after 55 h, but remained detectable after 6 days. Addition of vanadyl ribonucleoside complex (VRC) extended rRNA half-life of heat-permeabilized oocysts to 155 h. CONCLUSIONS: Extended rRNA half-life may result in viability overestimation using FISH. RNase pretreatment before FISH is recommended to destroy residual rRNA in recently killed oocysts. Incorporation of 1-10 mM l(-1) VRC before FISH permeabilization steps should neutralize RNase activity. SIGNIFICANCE AND IMPACT OF THE STUDY: Elimination of FISH fluorescence of nonviable C. parvum is desirable. Use of RNase and VRC is suggested to reduce numbers of false-positive 'viable' oocysts.

Animals↗
PubMed2004

A flow cytometric assay for global estimation of tyrosine phosphorylation associated with capacitation of spermatozoa from two marsupial species, the tammar wallaby (Macropus eugenii) and the brushtail possum (Trichosurus vulpecula).

The phosphorylation of tyrosine residues in cellular proteins is a major signal transduction event during sperm capacitation. In this study protein phosphorylation was monitored using a fluorescein isothiocyanate (FITC)-labeled antiphosphotyrosine monoclonal antibody and a flow cytometric procedure optimized for sperm. Using this technique, the correlation between tyrosine phosphorylation and sperm capacitation was examined in two marsupial species, the brushtail possum and the tammar wallaby and compared with that of ram spermatozoa. The levels of tyrosine phosphorylation in sperm from all three species were increased by the addition of cyclic AMP (cAMP) and vandate, a phosphotyrosine phosphatase inhibitor and were decreased by the addition of the phosphotyrosine kinase inhibitor, staurosporine. Oviductal conditioned media (CM) induced a progressive increase in tyrosine phosphorylation in both marsupial species and also induced morphological transition from a streamlined to a 'T'-shape configuration in brushtail possum spermatozoa but not in tammar wallaby spermatozoa. Transition to the 'T'-shape orientation associated with capacitation in marsupial spermatozoa was observed by 2 h of incubation in both species when tyrosine phosphorylation was increased by higher levels of cAMP i.e. 5 mM dibutyryl cAMP plus 3 mM pentoxyphylline. Thus the tyrosine phosphorylation trigger with CM may differ in these two marsupial species. Ram sperm tyrosine phosphorylation could be increased by addition of lower levels of cAMP (1 mM). These results support the finding that tyrosine phosphorylation is associated with sperm capacitation in marsupials. Similar results were obtained by using SDS PAGE/Western blot analysis of tyrosine phosphorylation in the brushtail possum spermatozoa. The specificity, efficiency and sensitivity of the procedure described here make it applicable for routine assessment of capacitation in large numbers of samples and in other species.

Animals↗
PubMed2002

Comparison of tests for viable and infectious Cryptosporidium parvum oocysts.

The purpose of this study was to compare different assays for viable Cryptosporidium parvum incubated in water at a temperature commonly found in the environment. C. parvum oocysts were stored in sterile water for 9 months at 15 degrees C. A sample was removed monthly and analyzed by five different assays to determine oocyst viability. Mouse infection and cell culture showed that C. parvum oocysts remained viable and infectious when stored for 7 months at this temperature. Fluorescence in situ hybridization (FISH) using probes directed to ribosomal RNA was also applied to these oocysts. The proportion of FISH-positive oocysts was 70-80% for the first 2 months of storage, decreased and remained nearly constant at 40-50% for 3-7 months, then decreased to 20% by 8 months, and to 0% by 9 months. Amylopectin content and mRNA for amyloglucosidase (CPAG), as measured by RT-PCR, decreased much more rapidly. By 3 months and for the remainder of the incubation period, amylopectin content was 20% of the original amount present in the oocysts. The CPAG RT-PCR signal at 3 months was 50% of that observed after 1 month storage, 20% at 4 months, and was not detected thereafter. Thus, results from cell culture and mouse infection assay exhibited the best agreement, the FISH assay showed modest agreement with these assays, and CPAG RT-PCR and the amylopectin assay displayed marginal agreement with the other three assays.

Amylopectin↗
PubMed2001

Detection of oxytetracycline production by Streptomyces rimosus in soil microcosms by combining whole-cell biosensors and flow cytometry.

Combining the high specificity of bacterial biosensors and the resolution power of fluorescence-activated cell sorting (FACS) provided qualitative detection of oxytetracycline production by Streptomyces rimosus in soil microcosms. A plasmid containing a transcriptional fusion between the tetR-regulated P(tet) promoter from Tn10 and a FACS-optimized gfp gene was constructed. When harbored by Escherichia coli, this plasmid produces large amounts of green fluorescent protein (GFP) in the presence of tetracycline. This tetracycline biosensor was used to detect the production of oxytetracycline by S. rimosus introduced into sterile soil. The tetracycline-induced GFP-producing biosensors were detected by FACS analysis, enabling the detection of oxytetracycline encounters by single biosensor cells. This approach can be used to study interactions between antibiotic producers and their target organisms in soil.

Biosensing Techniques↗
PubMed2000

A novel two-color flow cytometric assay for the detection of Cryptosporidium in environmental water samples.

BACKGROUND: Cryptosporidium is an important waterborne pathogen. Detection of Cryptosporidium in concentrated water samples depends on oocyst isolation using immunomagnetic separation (IMS) and/or fluorescence-activated cell sorting (FACS), followed by confirmation using immunofluorescence staining (IFA) and fluorescence microscopy. These methods require highly trained microscopists for oocyst identification and confirmation. Analysis is hampered due to the presence of autofluorescent particles coupled with particles binding nonspecifically with the monoclonal antibodies (mAbs) used for detection. Flow cytometry (FCM) has the potential to be a more specific method for oocyst detection, but such a system would require more than one selection parameter. METHODS: Various mAbs from commercial suppliers were paired with CRY104-PE and evaluated. The mAb combination that best discriminated stained oocyst from detritus was optimized and compared to Cryptosporidium detection utilizing one-color IFA/FACS. RESULTS: A highly specific two-color assay employing the IgG(1) mAb CRY104 was developed. The assay resulted in reductions, up to 20-fold, in the number of non-Cryptosporidium particles detected. The addition of a second selection parameter improved microscopic analysis times and simplified oocyst confirmation by microscopists. CONCLUSIONS: A two-color assay employing competing surface mAbs reduces the number of fluorescent particles sorted, thus improving FCM detection methods for Cryptosporidium.

Animals↗
PubMed2000

New chromogenic identification and detection of Staphylococcus aureus and methicillin-resistant S. aureus.

This paper describes a new chromogenic plate medium, CHROMagar Staph aureus (CHROMagar, Paris, France), for the identification of Staphylococcus aureus on the basis of colony pigmentation. The abilities of CHROMagar Staph aureus, thermostable nuclease (DNase), and mannitol salt agar (MSA) to identify S. aureus isolates (n = 114) and discriminate between S. aureus and coagulase-negative staphylococci (CoNS; n = 22) were compared. CHROMagar Staph aureus proved to be more sensitive and specific than DNase and MSA, allowing a reliable, simple, and rapid method for the identification of S. aureus isolates. All CoNS encountered in this study with the exception of S. chromogenes could be easily differentiated from S. aureus on this medium. The supplementation with 4 microgram of oxacillin or methicillin per ml allowed simple identification of methicillin resistance in hospital-acquired S. aureus strains which show multiple-drug resistance profiles. Community-acquired methicillin-resistant S. aureus strains showing non-multi-drug resistance profiles require further evaluation on this new chromogenic medium. Methicillin or oxacillin resistance of all S. aureus isolates was confirmed by the detection of penicillin-binding protein 2a, encoded by the mecA gene, using the latex slide agglutination MRSA-Screen test (PBP 2' Test, DR900M; Oxoid).

Bacterial Proteins↗
PubMed1998

Flow cytometry and cell sorting for yeast viability assessment and cell selection.

Yeast suspensions were analysed by flow cytometry after dye staining for determination of total and viable cell densities. Results were comparable to traditional colony counting and, in addition, provided further information on the percentage of total cells that were viable. The flow cytometric methods provided results within 20 min whereas colony counts were not available until 36 h. We evaluated a number of fluorescent dyes: ChemChrome Y (CY), oxonol (Ox), propidium iodide (PI), Fungolight and rhodamine 123, for accurate determination of viability of industrial yeast cultures and freshly re-hydrated high activity dried yeast (HADY). PI, Ox and CY gave the most conclusive live/dead discrimination and were the simplest to use. Culturing after dye staining and cell sorting demonstrated that the yeast remained viable after cell sorting and incubation with PI, CY or Ox. The methods, therefore, permit physical selection of individual yeast cells from populations of mixed viability. Sorting demonstrated that PI stained non-culturable cells whilst CY stained culturable cells. Analysis of yeast stained simultaneously with CY and PI or with Ox and PI demonstrated that PI and CY assays were in mutual agreement with respect to viability assessments. The Ox assay was in agreement with CY and PI for live/heat-killed mixtures. However, for re-hydrated HADY, Ox stained a significantly (P < or = 0.05) higher proportion of cells than did PI.

Colony Count, Microbial↗
PubMed1998

Viable Cryptosporidium parvum oocysts exposed to chlorine or other oxidising conditions may lack identifying epitopes.

The intestinal protozoan parasite Cryptosporidium parvum is a known cause of water-borne disease in humans. The detection of Cryptosporidium oocysts in water samples relies upon the use of fluorescently labelled antibodies, preferably using flow cytometry and epifluorescence microscopy. Here we demonstrate that four commercially available antibodies recognise a similar set of immunodominant epitopes on the oocyst wall. These epitopes appear to be carbohydrate in nature and are labile to chlorine treatment and oxidising conditions. Sodium hypochlorite and sodium meta-periodate reduced the ability of the antibodies to detect Cryptosporidium oocysts. Damage to the epitopes did not necessarily reduce the viability of oocysts. This finding may be important for the water industry, where naturally occurring oxidising conditions or sanitizing treatments could produce viable oocysts that are undetectable using standard protocols.

Animals↗
PubMed1998

Water quality in rural Australia.

Grab samples of drinking water collected from reservoirs and from creeks flowing over pristine land, farmland or land having mixed use were analysed for their physicochemical and microbiological characteristics. A significant difference between sites for conductivity and sites for pH was noted using a two-way ANOVA. No significant interactions were detected between any of the other parameters: Giardia, Cryptosporidium, Escherichia coli, coliforms, plate count, turbidity or rainfall.

Animals↗
PubMed1998

Rapid method for fluorescent in situ ribosomal RNA labelling of Cryptosporidium parvum.

A method for fluorescence in situ hybridization (FISH) is described that requires less than 1 h duration. Oocysts were resuspended in 50% ethanol and incubated at 80 degrees C for 10 min for simultaneous fixation and permeabilization. Samples were than incubated with the oligonucleotide probe at 48 degrees C for more than 30 min. The rRNA binding specificity of the optimized protocol was confirmed. FISH was found to be valuable as a second label for oocysts presumptively identified immunofluorescently, but required more than an order of magnitude signal amplification for independent use. The number of oligonucleotide probes bound per oocyst was compared with the copy number of 18S rRNA molecules per oocyst to provide a measure of the labelling efficiency of the FISH method. Hybridization kinetics were also analysed. These data indicate that significant further increases in the brightness of FISH-labelled oocysts cannot be achieved by further optimization of the pre-treatment and hybridization conditions.

Animals↗
PubMed1998

Evaluation of fluorochromes for flow cytometric detection of Cryptosporidium parvum oocysts labelled by fluorescent in situ hybridization.

Oligonucleotide probes specific to Cryptosporidium parvum (CRY1) were conjugated with a range of fluorochromes. The fluorescence after in situ hybridization (FISH) labelling of oocysts and controls was assessed. The objective was to determine the most suitable conjugate for FISH labelling, followed by analysis with a 488 nm laser flow cytometer. The most promising candidate was fluorescein isothiocyanate but only when linked to the CRY1 probe via an 18-carbon spacer arm consisting of six ethylene glycol moieties. The use of the spacer increased fluorescent signals fivefold compared with an equivalent probe in which the FITC was linked directly to the 5'-amino group of the DNA.

Animals↗
PubMed1998

Chromogenic tube test for presumptive identification or confirmation of isolates as Candida albicans.

This report describes a new, modified, simple, and cost-effective method for the use of CHROMagar Candida (CHROMagar Company, Paris, France) for the presumptive identification of isolates as Candida albicans after preliminary growth. Sixty randomly selected clinical isolates were evaluated, including 38 of C. albicans. With incubation at 37 degrees C for 24 h, the sensitivity and specificity appeared to be excellent and the test performed better than the traditional germ tube test. However, at earlier times, C. tropicalis isolates gave false-positive results.

Candida albicans↗
PubMed1996

Observation of single-cell fluorescence spectra in laser flow cytometry.

We report the first demonstration of directly recording fluorescence spectra of single cells in flow cytometry. An intensified, 512-element photodiode array was used in conjunction with a dispersing prism to capture the fluorescence emission spectra of Coulter ImmunoCheck calibration beads and Dictyostelium discoideum spores stained with the indocarbocyanine derivative CY3, fluorescein isothiocyanate, or R-phycoerythrin. The demonstration was made feasible by enhancing the signal-to-noise ratio of the detection process by using a fast gating technique applied to the detector. Results show that the complete fluorescence spectra of individual stained cells contain information that is normally not captured by conventional flow cytometers. By using the spectrographic flow cytometer, all this information is recorded, allowing small features and shifts in the fluorescence spectra of labelled particles to be studied.

Animals↗
PubMed1994

Application of flow cytometric methods for the routine detection of Cryptosporidium and Giardia in water.

Cryptosporidium and Giardia are common causes of waterborne disease. The currently used methods of detecting these organisms in water rely on filtration capture, immunofluorescence labelling, and epifluorescence microscopy. These methods are inefficient, labour intensive, and require a highly skilled microscopist. We describe an alternative technique using flocculation concentration, followed by flow cytometry with fluorescence activated cell sorting. Environmental samples were analysed, and protozoan-like particles were sorted and collected before confirmation with epifluorescence microscopy. The technique was found to be significantly more sensitive and considerably faster than the conventional methods.

Animals↗