The use of a ribosomal RNA targeted oligonucleotide probefor fluorescent labelling of viable Cryptosporidiumparvum oocysts
G. Vesey, N. Ashbolt 1, E.J. Fricker 2, D. Deere, K.L. Williams, D.A. Veal and M. Dorsch 3
Macquarie University Centre for Analytical Biotechnology, School of Biological Sciences, Macquarie University,1School of Civil Engineering, University of New South Wales, Australia, 2Thames Water Utilities Ltd, SpencerHouse Laboratories, Reading, UK and 3School of Immunology and Microbiology, University of New South Wales,Australia
6323/07/97: received 17 July 1997, revised 12 November 1997 and accepted 26 January 1998
G. VESEY, N. ASHBOLT, E.J. FRICKER, D. DEERE, K.L. WILLIAMS, D.A. VEAL AND M. DORSCH. 1998.
A fluorescence in situ hybridization (FISH) technique has been developed for the fluorescentlabelling of Cryptosporidium parvum oocysts in water samples. The FISH technique employsa fluorescently labelled oligonucleotide probe (Cry1 probe) targeting a specific sequence inthe 18S ribosomal RNA (rRNA) of C. parvum. Hybridization with the Cry1 probe resultedin fluorescence of sporozoites within oocysts that were capable of excystation, while oocyststhat were dead prior to fixation did not fluoresce. Correlation of the FISH method withviability as measured by in vitro excystation was statistically highly significant, with acalculated correlation coefficient of 0·998. Examination of sequence data for Cryptosporidiumspp. other than C. parvum suggests that the Cry1 probe is C. parvum-specific. In addition,19 isolates of C. parvum were tested, and all fluoresced after hybridization with the Cry1probe. Conversely, isolates of C. baileyi and C. muris were tested and found not to fluoresceafter hybridization with the Cry1 probe. The fluorescence of FISH-stained oocysts was notbright enough to enable detection of oocysts in environmental water concentrates containingautofluorescent algae and mineral particles. However, in combination withimmunofluorescence staining, FISH enabled species-specific detection and viabilitydetermination of C. parvum oocysts in water samples.
INTRODUCTION
Cryptosporidium parvum is a protozoan parasite that causesgastrointestinal disease in humans. The infectious form of C.parvum, an environmentally resistant oocyst, can be found inmany surface waters (LeChevallier et al. 1991; Rose et al.1991; Smith et al. 1991). Although oocyst densities in surfacewaters are normally low, numerous outbreaks of crypto-sporidiosis have been documented in which contaminateddrinking water has been identified as the source of infection.Often these outbreaks have been traced to water treatmentplants which have met all current microbiological and chemi-cal standards (Richardson et al. 1991; Mackenzie et al. 1994;Lisle and Rose 1995). Many of these outbreaks have been
Correspondence to: Graham Vesey, School of Biological Sciences, MacquarieUniversity, NSW 2109, Australia (e-mail: [email protected]).
attributed to gross contamination of the untreated water sup-ply with oocysts. Since the occurrence of these outbreaks,many water supply companies have considered it necessary tomonitor untreated water sources for the presence of oocysts.
The detection of C. parvum in water is difficult and atpresent it is not possible to culture C. parvum reliably fromenvironmental samples. Current detection methods rely onimmunological techniques employing monoclonal antibodies(mAbs) to label oocysts fluorescently within concentratedwater samples. Samples are then examined using epi-fluorescence microscopy to detect the fluorescing oocysts.The technique has been reported to be labour-intensive andto suffer from low sensitivity, as well as giving false-positiveand false-negative results (Whitmore and Carrington 1993;Clancy et al. 1994; Nieminski et al. 1995).
Some of the problems associated with the detection ofCryptosporidium have been overcome by the application of
430 G. VESEY ET AL.
flow cytometry (Vesey et al. 1993a, 1994a; Hoffman et al.1997). However, the mAbs currently employed to detect C.parvum oocysts can react with both viable and non-viableoocysts and cross-react with Cryptosporidium species that arenot infectious to humans (Rose et al. 1989; Graczyk et al.1996). Therefore, immunofluorescence alone does not dis-criminate C. parvum from other Cryptosporidium species, noris the viability of oocysts determined. The presence of deadoocysts, or Cryptosporidium spp. other than C. parvum, indrinking water is of little significance to public health. Thereis therefore a requirement for an effective method for detect-ing viable C. parvum oocysts in water that is applicable toroutine monitoring of water supplies.
Amann et al. (1990) have pioneered a technique known asfluorescence in situ hybridization (FISH) to label bacteriaspecifically (reviewed by Amann et al. 1995). The FISH tech-nique employs fluorescently labelled oligonucleotide probestargeted to specific sequences of ribosomal RNA (rRNA).Molecules of rRNA are ideal targets for fluorescently labellednucleic acid probes for several reasons: (i) high sensitivity canbe achieved as the target molecules may be present in veryhigh numbers; (ii) a denaturation step is not required duringthe procedure as the target region is often at least partiallysingle-stranded and (iii) rRNA has a short half-life and shouldonly be present in a high copy number in viable or recentlyviable cells (Amann et al. 1995; Vesey et al. 1995). Further-more, the degree of specificity of a probe can be designed.Regions of rRNA sequences have evolved at different ratesand target sequences range from highly conserved (through-out a phylogenetic kingdom) to highly variable (up to strain-specific) (Amann et al. 1995).
The FISH technique has recently been reported for fluor-escent labelling of Cryptosporidium oocysts (Vesey et al. 1995).The technique was useful for determining oocyst viabilitybecause rRNA within non-viable oocysts was degraded. Theprobes that were used in this earlier study were, however,specific to a sequence of rRNA present in all eukarya andwere therefore not specific to Cryptosporidium oocysts in watersamples because of the presence of other eukaryotic cells suchas algae.
In this report the use of an oligodeoxynucleotide probethat is potentially specific for C. parvum was investigated forlabelling oocysts in water samples. The specificity of thisprobe was investigated and a method for labelling C. parvumoocysts in water samples developed.
MATERIALS AND METHODS
Cryptosporidium oocysts
Cryptosporidium parvum oocysts purchased from MoredunAnimal Health Ltd (Midlothian, UK) were used for allexperiments unless stated otherwise. Oocysts were supplied
in 1ml samples containing approximately 109 oocysts. Alter-natively, C. parvum oocysts were purified from pooled faecesof naturally infected neonatal calves in Sydney, Australia.Faecal samples were centrifuged (2000 g, 10min) and resus-pended in water twice and then resuspended in 5 volumes of1% NaHCO3. Fat was then extracted twice with 1 volume ofether followed by centrifugation (2000 g, 10min). Pellets wereresuspended in water and filtered through a layer of pre-wettednon-absorbent cotton wool. Oocysts were then overlaid onto5–10 volumes of 55% (w/v) sucrose and centrifuged (2000 g,20min). Oocysts were collected from the interface and thesucrose flotation step repeated until no visible contaminatingmaterial could be detected. Purified oocysts were surface-sterilized with ice-cold 70% (v/v) ethanol for 30min, washedonce in phosphate-buffered saline (150mmol l−1 NaCl,15mmol l−1 KH2PO4, 20mmol l−1 Na2HPO4, 27mmol l−1
KCl, pH 7·4) (PBS) and stored in PBS at 4 °C.Samples of human faeces containing C. parvum oocysts
and samples of partially purified C. parvum oocysts frombovine or human faeces were kindly supplied by ProfessorHuw Smith (Scottish Parasite Reference Laboratory,Stobhill, Glasgow, UK). Additional samples of human faecescontaining C. parvum oocysts were kindly supplied by DrDavid Casemore (Public Health Laboratory Service, Rhyl,UK). Faecal samples were mixed with sufficient PBS toenable pipetting and stored at 4 °C until used.
Cryptosporidium muris oocysts (strain RN66), cultured inBalb/c mice and purified using density gradient centri-fugation, and C. baileyi-infected Leghorn chick faeces, wereobtained from Waterborne Ltd (New Orleans, USA). Oocystswere stored at 4 °C in PBS until use.
A sample of partially purified C. baileyi oocysts was kindlysupplied by Dr Janet Catchpole (Ministry of Agriculture,Fisheries and Food, Weybridge, UK).
Samples of snake faeces containing Cryptosporidium oocystswere kindly supplied by Peter Mirtschin (Venom Supplies,Tanunda, South Australia).
Monoclonal antibodies
A fluorescein isothiocyanate (FITC) conjugated mAb specificto the surface of Cryptosporidium oocysts was purchased fromCelLabs (Brookvale, NSW, Australia). A second mAb(CRY26), also specific to the surface of Cryptosporidiumoocysts (Vesey 1996), was purified and conjugated to FITCor phycoerythrin (PE) as described previously (Haugland1995).
Eukaryotic and bacterial specific oligodeoxynucleotideprobes
A probe (EUK) complementary to an 18S rRNA regionconserved for Eukarya (5?-ACC AGA CTT GCC CTC C-3?)
DETECTION OF VIABLE CRYPTOSPORIDIUM PARVUM USING FISH 431
(Amann et al. 1990) was used as a positive control forstaining Cryptosporidium oocysts. A second probe (EUB338)complementary to a 16S rRNA region conserved for all bac-teria (5?-GCT GCC TCC CGT AGG AGT-3?) (Amannet al. 1990) was used as a negative control in all experiments.Synthetic probes, labelled with a single molecule of FITCvia an amino linker to the 5? end and purified using highperformance liquid chromatography (HPLC), were suppliedby Biotech International (Perth, Australia).
Design of a specific FISH probe for Cryptosporidiumparvum
An aliquot (50ml) of oocyst stock suspension was pelleted bycentrifugation at 13 000 g for 2min, resuspended in 10mmoll−1 Tris-HCl, 1mmol l−1 ethylenediaminetetraacetic acid,pH 8, and repeatedly (six times) frozen in a mixture of dryice and ethanol for 30 s and thawed by boiling for 2min. Afterincubation with 1% (w/v) sodium dodecylsulphate (SDS)and proteinase K (100mgml−1) for 1 h at 37 °C the lysate wasextracted with phenol:chloroform:isoamyl alcohol (25:24:1v/v/v).Nucleic acidwas precipitatedwith 1 volume of 4 mol l−1
ammonium acetate and 2 volumes of isopropanol, washedwith 70% (v/v) ethanol, dried, and finally dissolved in dis-tilled water.
Complete 18S rRNA sequences comprising the speciesC. parvum, C. muris and C. baileyi were obtained throughthe EMBL and GenBank data bases. The sequences weremanually aligned. One particular region appeared to have thepotential to discriminate C. parvum from the other speciesincluded in the analysis. To examine the validity of the pub-lished 18S rRNA sequence of C. parvum, a short stretch ofC. parvum ribosomal DNA including the putative proberegion was sequenced. Two primers targeting the conserved5?- and 3?-ends of the 18S rDNA were designed (forwardprimer 5?-AAC CTG GTT GAT CCT GCC-3? and reverseprimer 5?-GGT TCA CCT ACG GAA ACC-3?) andemployed to amplify the gene using the polymerase chainreaction as described previously for 16S rDNA (Dorsch andStackebrandt 1992). The probe region was then sequencedusing a reverse primer (5?-CCT TCC ATA AAG TCGAGT-3?) complementary to a sequence approximately 50nucleotides downstream of the 3?-end of the target region(Dorsch and Stackebrandt 1992). The results verified thevalidity of the published C. parvum 18S rRNA sequence(EMBL). A secondary structure model for the 18S rRNA ofangiosperms (Schmidt-Puchta et al. 1989) was used to selectpotential targets within single-stranded regions of the nativeribosome. Six sequences were identified as being specificto C. parvum (Table 1). A fluorescently labelled probe wasdesigned for sequence number 6 (5? CGG TTA TCC ATGTAA GTA AAG 3?)(Cry1 probe). In terms of the secondarystructure model of tomato rRNA as published by Schmidt-
Puchta et al. (1989) the probe targets occupy the positionsbetween 138 and 160 on the 18S rRNA of C. parvum. It shouldbe noted that these nucleotide positions are not derived froman internationally accepted numbering system as such ascheme does not exist for 18S rRNA/DNA.
Synthesizing Cryptosporidium parvum -specificoligonucleotide probes and conjugating withfluorochromes
Probes were synthesized and conjugated with a single mole-cule of the fluorochromes Texas Red, Cascade Blue, BOD-IPY FL or tetramethylrhodamine B isothiocyanate (TRITC)via an amino-linker on the 5?-end by Molecular Probes (Eug-ene, USA), with CY3 via an amino-linker on the 5?-end byOswel Ltd (University of Southampton, Southampton, UK)and with FITC by Boehringer Mannheim (Sydney,Australia). Alternatively, probes were synthesized and lab-elled with one, two or three molecules of Fluorescein-ONphosphoramidite (Clontech Laboratories, Palo Alto, CA,USA) using a Beckman Oligo 1000 DNA synthesizer (Beck-man Instruments, Gladesville, Australia). The Fluorescein-ON phosphoramidite is a nucleotide analogue that carries afluorescent label which does not disrupt normal base pairing.Labelling of the probe was achieved by replacing thymine oradenine bases with Fluorescein-ON phosphoramidite (Table2). The FITC-labelled probe from Boehringer Mannheimwas used for all experiments unless otherwise stated.
Fixation of oocysts
Oocysts were fixed using a method modified from that pre-viously described by Wallner et al. (1993) for the fixation ofyeasts and bacteria. One volume of oocyst suspension, faecalsample or seeded water concentrate was mixed with 3 volumesof fresh cold 4% (w/v) paraformaldehyde in PBS, and heldat 4 °C for 1 h. The oocysts were washed three times bypelleting with centrifugation (13 000 g, 30 s) and resuspendingin PBS. The sample was then centrifuged (13 000 g, 30 s) andresuspended in cold (– 20 °C) absolute ethanol and stored at4 °C. All samples, unless stated otherwise, were hybridizedwithin 1 h of fixation.
Hybridization
Fixed samples of either pure oocysts, water concentratesseeded with oocysts or faecal samples were hybridized withthe probe by mixing 10ml oocyst suspension with 100mlhybridization buffer (0·9mol l−1 NaCl, 20mmol l−1 Tris-HCl, 0·1% SDS, pH7·2) pre-warmed to 48 °C and 10ml ofprobe (approximately 5 ngml−1 in distilled water). The sam-ple was mixed and incubated at 48 °C for 1 h. The samplewas then washed and resuspended in PBS, pre-warmed to
*Numbering system of nucleotides according to that published by Schmidt-Puchta et al.(1989).†The Cry1 target.
Table 2 Position of Fluorescein-ONphosphoramidite molecules in theoligonucleotide probe Cry1,synthesized with one, two orthree molecules of Fluorescein-ONphosphoramidite substitutedfor thymine or adenine bases
*A Fluorescein-ON phosphoramidite molecule is designated by an F.
48 °C. Samples were then allowed to cool to room tem-perature and analysed using flow cytometry or epi-fluorescence microscopy.
The hybridization temperature and the pH and SDS con-centration of the hybridization buffer were varied to deter-mine the conditions that resulted in maximum fluorescenceof oocysts. Hybridization temperatures of 22, 48, 55, 60, 70and 80 °C and SDS concentrations of 0·01, 0·1, 0·5 and 1%(w/v) were tested.
An RNase inhibitor, RNASIN, isolated from human pla-centa, obtained from Promega (Sydney, Australia) was incor-porated during the hybridization and washing procedures todetermine if staining of oocysts was improved. RNASIN wasadded at a concentration of 1ml ml−1 of hybridization orwashing buffer.
Treatments to increase oocyst wall permeability
Three procedures (heating, exposure to detergents andexposure to organic solvents) were evaluated as pre-hybridization treatments to increase the fluorescence of Cry1-stained oocysts. Oocyst stock solution (100ml) was fixed andthen diluted 1 in 50 in PBS (approximately 2× 107 oocystsml−1) before being treated. Samples (50ml) were incubatedat 100, 90, 80, 70, 60, 50, 37, 30, 20 or 4 °C for 30min.
Further samples (50ml) of oocysts were mixed with 3volumes of one of the following solvents (concentrations arev/v in PBS): 70% acetone; 10% dimethyl sulphoxide; 50%
ethanol; 70% formic acid and 70% acetic acid. Alternatively,oocysts were exposed to a series of ethanol concentrations bymixing oocysts with three volumes of 50%, 75%, 90% and95% ethanol at 15min intervals. Treated oocysts were washedtwice by centrifuging at 13 000 g for 2min, removing anddiscarding the supernatant fluid and resuspending in PBS.
Other samples (50ml) of oocysts were mixed with 3 vol-umes of a 5% (w/v for all except Nonidet P40 and Triton X-100 which were v/v) solution in PBS of one of the followingdetergents: SDS; deoxycholic acid; cetyltrimethylammonium bromide (CTAB); dodecyltrimethyl ammoniumbromide (DTAB); 3-[(3-cholamidopropyl) dime-thylammonio] 1-propane-sulphonate (CHAPS); octyl-phenolethylene-oxide (Nonidet P40) and iso-octylphenoxypolyethoxyethanol (Triton X-100). Samples were incubatedat 70 °C for 30min and then washed twice by centrifuging at13 000 g for 2min, removing and discarding the supernatantfluid and resuspending in PBS. All samples were immediatelyhybridized with FITC-labelled Cry1 probe and analysed asdescribed above.
Excystation
In vitro excystation was performed as described by Robertsonet al. (1993) on six different samples of oocysts purified frombovine faeces. Oocyst suspensions to be tested (100ml) weremixed with 1ml of acidified (pH 2·75) Hanks balanced saltsolution (HBSS), and incubated at 37 °C for 30min. Samples
DETECTION OF VIABLE CRYPTOSPORIDIUM PARVUM USING FISH 433
were then washed and resuspended (13 000 g, 2min) in 100mlof PBS, pH 7·4, with 10ml of 1% (w/v) sodium deoxycholatein Hanks minimal essential medium and 10ml of 2·2% (w/v)sodium hydrogen carbonate in HBSS and incubated at 37 °Cfor 4 h. Samples were then fixed by mixing with 10% (v/v)formalin in PBS for 5min at 4 °C, centrifuged (13 000 g,2min), the supernatant fluid removed and discarded and thepellet resuspended in 100ml of PBS plus 2% (w/v) bovineserum albumin (PBS-BSA). An equal volume of FITC-con-jugated CelLabs or CRY26 mAb was added and samplesincubated at 37 °C for 20min. Samples were then examinedusing epifluorescence microscopy and Differential Inter-ference Contrast (DIC) optics, and the proportion of emptyoocysts, partially excysted oocysts and non-excysted oocystsdetermined. At least 100 oocysts were counted in all samples.The percentage excystation was calculated as follows:
no. of empty oocysts + no. of partially excysted oocyststotal no. of oocysts counted
100
It was necessary to account for any oocysts that were emptyprior to the excystation procedure. Therefore, the term ‘no.of empty oocysts’ in the numerator of the above equation wascalculated after subtracting the number of empty oocystspresent before excystation from the number present afterexcystation.
Probing of excysting oocysts
To determine if the amount of rRNA within oocysts increasedprior to or during excystation, oocysts were excysted asdescribed above but incubation was stopped after 5, 15 and30min and the oocysts fixed. Samples were then hybridizedwith FITC-labelled Cry1 probe and analysed using flow cyto-metry.
Staining oocysts with the Cry1 probe and a mAb
Oocysts hybridized with either the Cascade Blue-, TexasRed-, CY3- or TRITC-conjugated Cry1 probe were washedtwice by centrifuging (13 000 g, 2min), removing the super-natant fluid before resuspending in PBS-BSA plus 0·1%(v/v) RNASIN. An equal volume of FITC-conjugated Cel-Labs mAb or CRY26 mAb was then added and incubated at37 °C for 30min. Samples were examined using epi-fluorescence microscopy.
Alternatively, samples were hybridized with FITC-con-jugated Cry1 probe and then labelled as above but with PE-conjugated CRY26 mAb. Samples were then analysed usingflow cytometry.
Anti-FITC antibodies were used in an attempt to amplify thefluorescence signal. A biotinylated anti-FITC rabbit poly-clonal antibody (Molecular Probes) was added to stained andwashed oocysts at the working dilution (1 in 20) in PBS-BSAand 0·1% (v/v) RNASIN and incubated at 37 °C for 20min.The sample was then washed and resuspended in PBS-BSAand 0·1% (v/v) RNASIN. A neutralite-avidin Cascade Blueconjugate (Molecular Probes) was then added at workingdilution (1:20) and incubated at 37 °C for 20min. The samplewas then washed as before and examined using epi-fluorescence microscopy.
Environmental samples
Portions (10–50%) of sample concentrates of river and res-ervoir water samples (10 or 20 l) that had been collected fromvarious sites within the Sydney region and concentrated byflocculation (Vesey et al. 1993b) were pooled to create a com-posite sample. A portion of these samples had previouslybeen analysed using flow cytometry (Vesey et al. 1993a, 1994)and determined not to contain detectable numbers of oocysts(³ 0·1 oocysts l−1). Aliquots (100ml) were seeded with vari-ous numbers of oocysts, hybridized with the Cry1 probe andanalysed using flow cytometry or epifluorescence microscopy.Some samples were stained with FITC conjugated CRY26mAb as described above, after hybridization with CascadeBlue-, Texas Red-, CY3- or TRITC-conjugated Cry1 probe.
Sample analysis
Flow cytometry was performed using a Coulter Elite flowcytometer (Coulter, Brookvale, Australia), Coulter XL flowcytometer, BD FACScan flow cytometer or epifluorescencemicroscopy as described previously (Vesey et al. 1993a, 1994).
Statistical analysis
Data were entered into Microsoft Excel (Microsoft Corpor-ation, Redmond, USA). The hypothesis that the means fromdifferent samples were equal was tested using analysis ofvariance. A significance level of 0·05 was used to evaluatecritical values for the F statistic.
RESULTS
Fluorescent staining of oocysts
Examination of oocysts that had been hybridized with theEuk rRNA probe by epifluorescence microscopy revealedboth fluorescent and non-fluorescent oocysts. Fluorescentstaining was located inside the oocysts. Examination of the
434 G. VESEY ET AL.
fluorescent oocysts using DIC optics revealed intact oocystswith a small gap between the oocyst wall and the internalstructures. In comparison, non-fluorescent oocysts frequentlyhad a ruptured oocyst wall often with a large gap betweenthe oocyst wall and the internal structures. Empty oocyststhat fluoresced after FISH were not observed.
Examination of oocysts stained with the Cry1 probe usingepifluorescence microscopy revealed almost identical resultsto those observed with the EUK probe. However, as judgedby eye, fluorescence was dimmer than that observed with theEUK probe. Results were similar for FITC-, Cascade Blue-,Texas Red-, BODIPY FL-, CY3- and TRITC-conjugatedCry1 probes. Texas Red- and CY3-conjugated probesappeared somewhat brighter, as judged by eye, than thatobtained using any of the other conjugates.
Flow cytometric analysis of oocysts stained with FITC-labelled EUK probe and FITC-labelled Cry1 probe revealeda fluorescent population and a non-fluorescent population.The Cry1-stained oocysts had a mean fluorescence intensityof 102·8 with a standard deviation of 1 (n� 3). The EUK-stained oocysts were almost three times brighter with a meanfluorescence intensity of 285 and a standard deviation of 6·3.Fluorescent labelling of oocysts was not observed after FISHwith EUB338.
Optimizing hybridization conditions
Hybridization temperatures of above 48 °C resulted in weakfluorescence of full oocysts and some fluorescence of emptyoocysts and bacteria when the Cry1 probe was used. A hybrid-ization temperature of 48 °C resulted in fluorescence of onlyfull oocysts and a temperature of 22 °C produced no detect-able fluorescence. A temperature of 48 °C was thereforechosen as the optimal hybridization temperature. A similarincrease in non-specific binding was observed with the nega-tive control probe (EUB338) when hybridization was per-formed at temperatures greater than 48 °C.
An SDS concentration of 0·5% (w/v) in the hybridizationbuffer resulted in oocysts that fluoresced significantly(P³ 0·05) brighter than with lower SDS concentrations. Nosignificant difference (P³ 0·05) was observed between thefluorescence of oocysts hybridized using 0·5 or 1% (w/v)SDS.
Incorporating multiple fluorochromes into the Cry1probe
Flow cytometric analysis of oocysts stained with the Cry1probe containing two or three molecules of Fluorescein-ONphosphoramidite revealed a significantly (P³ 0·05) brighterfluorescence signal than staining with the Cry1 probe con-taining a single molecule of Fluorescein-ON phos-phoramidite. There was no significant difference between the
brightness of oocysts stained with probes incorporating twoor three molecules of FITC. No increase was recorded in thefluorescence of empty oocysts or bacteria in samples stainedwith the probe that contained two molecules of Fluorescein-ON phosphoramidite; however, samples stained with theprobe that contained three molecules of Fluorescein-ONphosphoramidite showed increased fluorescence of emptyoocysts and bacteria.
Increasing the permeability of the oocyst wall
Treatment of oocysts with heat prior to hybridization at48 °C with the Cry1 probe resulted in significantly brighterfluorescence when temperatures of 70, 80, 90 or 100 °C wereused (Fig. 1).
Treatment of oocysts with an ethanol series prior to hybrid-ization with the Cry1 probe resulted in significantly(P³ 0·05) brighter fluorescence compared with all otherorganic solvents and the control (Table 3). Treatment withformic acid resulted in very bright staining but this wasdetermined to be non-specific binding of the probe since theEUB338 probe also bound to oocysts. No staining with theEUB338 probe occurred for oocysts treated with all otherorganic solvents.
Treatment of oocysts at 50 °C with all detergents exceptCHAPS and Nonidet P40, prior to hybridization with the
Fig. 1 Mean fluorescence intensity of oocysts heated for 30 minin (Ž), 5% dodecyltrimethyl ammonium bromide or (ž),phosphate-buffered saline at a range of temperatures prior tohybridization at 48 °C with fluorescein isothiocyanate-labelled Cry1 probe. Results are the means of three separateexperiments and standard deviations are shown with error bars
DETECTION OF VIABLE CRYPTOSPORIDIUM PARVUM USING FISH 435
Table 3 Mean fluorescence of oocysts treated with organicsolvents prior to staining with fluorescein isothiocyanate-labelled Cry1 probe. Results are the means of three separateanalyses of 2000 oocysts using the FACScan flow cytometer—–––––––––––––––––––––––––––––––––––––––––––––––––––––
Organic solvent Mean fluorescence Standard deviation—–––––––––––––––––––––––––––––––––––––––––––––––––––––10% DMSO 36 1·870% Acetone 38·7 2·570% Acetic acid 31·6 2·250% Ethanol 37·4 1·4Ethanol series 42·3* 2·1No organic solvent 37·1 1·7—–––––––––––––––––––––––––––––––––––––––––––––––––––––
*Significant (P ³ 0·05) difference shown between oocyststreated with an ethanol series and all other oocysts. DMSO,dimethyl sulphoxide.
Cry1 probe, produced an increase in the degree of fluor-escence compared with the PBS control (P³ 0·05)(Table 4).Statistical analysis showed that DTAB and CTAB (bothcationic detergents) produced a significantly brighter levelof fluorescence than any of the other detergent treatments(P³ 0·05). There was no significant difference (P³ 0·05)between the level of fluorescence of oocysts treated withCTAB or DTAB.
Combining the DTAB treatment with each of the organicsolvent treatments did not produce significantly brighter fluor-escence than DTAB alone (P³ 0·05). Combining theDTAB treatment with heat at 80 °C produced significantly
Table 4 Mean fluorescence of oocysts treated with detergentsprior to staining with the Cry1 probe. Results are the means of threeseparate analyses of 2000 oocysts—–––––––––––––––––––––––––––––––––––––––––––––––––––––
brighter fluorescence (P³ 0·05) than heat alone or DTAB at50 °C (Fig. 1). Heating at 100 °C with DTAB resulted infaintly stained oocysts.
Staining excysting oocysts with FISH probes
Significantly brighter fluorescence (P³ 0·05) was observedfor oocysts that had been treated with the excystation pro-cedure prior to hybridization with the Cry1 probe. Therewas, however, no significant difference (P³ 0·05) betweenthe fluorescence intensity of oocysts treated with excystationand then with DTAB at 80 °C before staining with the Cry1probe, and oocysts treated with only DTAB at 80 °C beforestaining.
Amplification of the fluorescent signal
The use of anti-FITC antibodies did not successfully amplifyfluorescence after FISH. When oocysts were hybridized withFITC-labelled Cry1 probe then labelled with an anti-FITCbiotinylated mAb followed by an avidin Cascade Blue conju-gate, the blue fluorescence from the Cascade Blue was dim,as judged by eye. Fluorescence did not appear brighter thanoocysts stained with the Cascade Blue-conjugated Cry1probe. Green fluorescence of the FITC was observed eventhough the anti-FITC mAb should quench the fluorescenceof FITC. This suggests that complete binding of the anti-FITC mAb to the Cry1-FITC conjugated probe was notoccurring. A control sample of oocysts stained with FITC-conjugated CelLabs mAb and then reacted with the anti-FITC probe lost all green fluorescence as expected.
Specificity of the Cry1 oligonucleotide probe
The Cry1 probe reacted with all samples of viable C. parvumoocysts against which it was tested. These included six dif-ferent batches of oocysts (a single strain) from MoredunAnimal Health Ltd, 14 samples of C. parvum isolates fromhumans, two bovine isolates from the UK and six bovineisolates from Sydney.
No reaction occurred with a sample of C. muris oocystsand both samples of C. baileyi oocysts, even though positivereactions were recorded for the EUK probe. Oocysts in fourdifferent samples of snake faeces tested negative for both theCry1 and EUK probes, suggesting that the oocysts lackedrRNA. Insufficient oocysts were present to determine theviability using excystation.
Staining purified oocysts with the Cry1 probe and amonoclonal antibody
Examination, using epifluorescence microscopy, of oocystsstained with either the Cascade Blue-, Texas Red-, CY3- or
436 G. VESEY ET AL.
TRITC-conjugated Cry1 probe and counterstained with aFITC-labelled CRY26 mAb revealed bright green fluor-escence of the oocyst wall, typical of mAb staining, whenexamined with the green filter block. Examination of theoocysts, with the appropriate (u.v. for Cascade Blue, redfor Texas Red, CY3 and TRITC) filter blocks, revealedfluorescence within the oocysts typical of FISH staining. TheCascade Blue-stained oocysts showed dim blue fluorescence.The Texas Red-, CY3- and TRITC-stained oocysts fluor-esced bright red, with the Texas Red and CY3 appearingbrighter than the TRITC.
Incorporation of RNASIN in the PBS-BSA was found tobe necessary. When RNASIN was omitted and FISH-stainedoocysts were mixed with antibody, the fluorescence intensityof the FISH-stained oocysts was observed to rapidly decrease.The fluorescence of the FISH stain decreased to an unde-tectable level, as judged by eye, within 5min.
The use of the Cry1 probe with environmental samples
It was found that RNASIN had to be incorporated into thehybridization buffer (33 units ml−1) to enable staining ofoocysts in environmental samples. Staining of oocysts wasnot observed when RNASIN was omitted.
Oocysts seeded into environmental samples, hybridizedwith the Cry1 probe (with RNASIN) and examined usingepifluorescence microscopy showed identical fluorescence tostained samples of pure oocysts. No fluorescence of otherparticles in the sample was observed except for auto-fluorescence. The autofluorescence of the debris particles,however, made detection of oocysts extremely difficult. Flowcytometric analysis of similar samples showed that oocystswere not fluorescing brightly enough to be detected abovethe autofluorescence of some debris particles (Fig. 2).
Dual labelling oocysts with PE-conjugated CRY26 mAband FITC-conjugated Cry1 probe enabled flow cytometricanalysis of large numbers of oocysts seeded into water con-centrates. Figure 3 shows the two dot-plots used to enumerateoocysts. The PE fluorescence measured in fluorescence detec-tor 2 (FL2) was used as the discriminator and an area on adot-plot of FL2 vs 90° light scatter (90LS) was defined thatenclosed the PE-stained oocysts. By using this area as gate fora dot-plot of the FITC fluorescence measured in fluorescencedetector 1 vs 90LS it was possible to measure the fluorescencefrom the FISH probe. Two populations can be seen on thedot-plot that represent oocysts stained with the Cry1 probeand those that are not (Fig. 3).
Comparison of viability determined by FISH and in vitroexcystation
The results of comparing oocyst viability, as measured byexcystation, and viability, determined by staining with the
Cry1-specific probe, for six different batches of oocysts iso-lated from calf faeces are presented in Table 5. Percentageviabilities determined by the two methods significantly cor-related (r2 � 0·998), never differed by more than 10% and,with one exception, were not significantly different(P³ 0·05).
DISCUSSION
It has been demonstrated previously that FISH using ribo-somal RNA targeted probes can be used for assessing theviability of cryptosporidium oocysts. Oocysts that containedfluorescing sporozoites after hybridization with the probeswere considered viable, and oocysts which did not fluoresce,dead (Vesey et al. 1995). It is reasonable to hypothesize thatdead oocysts did not stain because loss of viability is linkedwith a breakdown in membrane integrity followed by rapidRNA degradation.
In vitro excystation is currently considered the standardwith which methods for determining Cryptosporidium oocystviability are compared. However, the technique can only beapplied to samples containing high numbers of oocysts andis, therefore, not suitable for the analysis of water samples.In this study, results from comparing measurements of oocystviability using FISH with a C. parvum-specific probe andmeasuring viability using in vitro excystation produced verysimilar results for all samples of oocysts analysed. Correlationof the FISH assay with excystation was statistically highlysignificant, with a calculated correlation coefficient of 0·998.Similar findings have been reported previously when theEUK probe was used to label oocysts (Vesey et al. 1995). Dueto the large amount of algal and other eukaryotic cells presentin water samples, the application of the EUK (eukarya-spec-ific) probe to the detection of oocysts in water is limited.In this study a C. parvum-specific probe (Cry1 probe) wassuccessfully designed and tested. Many regions within the18S rRNA cannot be used for FISH because the structure ofthe ribosome rRNA includes regions that are not accessiblefor oligonucleotide probes (Amann et al. 1995). This studydemonstrated that the Cry1 probe recognizes an accessibleregion of the ribosome. However, the intensity of fluorescenceof oocysts stained with the Cry1 probe was almost three timesless than oocysts stained with the EUK probe. This suggeststhat the probe was not binding optimally, possibly becausethe target region is less exposed than that to which EUKbinds.
The Cry1 probe appeared to be specific to C. parvum. Theoligonucleotide probe reacted with 19 different isolates of C.parvum and did not react with a strain of C. muris nor withtwo strains of C. baileyi. No other particles present in thewater samples tested reacted with the probe. Further strainsof Cryptosporidium species should be tested, includingadditional strains of C. parvum and C. baileyi together with
DETECTION OF VIABLE CRYPTOSPORIDIUM PARVUM USING FISH 437
Fig. 2 Flow cytometric dot-plots of analysis of (a) water concentrate and (b) Cry1-stained dodecyltrimethyl ammonium bromide-treated (80 °C) purified oocysts. Note the oocysts appear in the same position as some autofluorescent debris particles
strains of C. muris, C. meleagridis, C. serpentis and C. nasorumto determine the specificity of the oligonucleotide probe. Todate, however, we have been unable to acquire additionalstrains of Cryptosporidium species other than C. parvum.
After the Cry1 probe was designed the sequence of the18S rRNA of a Cryptosporidium species that affects guineapigs, C. wrairi, was submitted to the EMBL database. TheCry1 sequence matches perfectly the sequence of C. wrairi.It is doubtful at present if C. wrairi is in fact a separate speciesfrom C. parvum (O’Donoghue 1995) but care should be takenwith the interpretation of results from the use of Cry1 withsamples that may be contaminated with guinea pig faeces.
Inhibition of RNases was necessary when environmentalsamples were stained with the FISH technique or whenFISH-stained oocysts were suspended in a buffer containingBSA. This was probably due to RNAases, present in environ-mental samples and BSA, degrading the rRNA of the oocystsand supports the hypothesis that oocysts that are non-viablewhen fixed do not stain due to rRNA degradation.
Cryptosporidium-specific monoclonal antibodies are anexcellent tool for fluorescent labelling of Cryptosporidiumoocysts in water samples. However, the FISH technique hasseveral advantages over immunofluorescence with mAbs. Theoligonucleotide probes can be labelled with a range of fluoro-chromes, are inexpensive to produce (compared with mAbs)
and are stable for several years (Wallner et al. 1993). Thecurrently employed Cryptosporidium-specific mAbs recognizean antigen on the surface of oocysts that is present on bothlive and dead oocysts. Furthermore, the mAbs cross-reactwith Cryptosporidium species other than C. parvum (Rose et al.1989; Graczyk et al. 1996) and bind to debris particles presentin water samples (Vesey et al. 1991; Campbell et al. 1993)making detection and identification of oocysts difficult. Incomparison, the Cry1 probe recognizes rRNA that is onlypresent in viable oocysts, is potentially C. parvum-specificand does not bind to debris particles present in water samples.However, it should be noted that, at this time, the Cry1 probehas only been tested on a small number of Cryptosporidiumspecies. Furthermore, the use of the probe on chemicallydisinfected oocysts has yet to be evaluated.
The FISH method can be used to determine oocystviability in samples that have been stored. The samples canbe fixed and then stored in ethanol at 4 °C until analysis(Vesey et al. 1995). Alternative methods of determiningoocyst viability such as excystation (Robertson et al. 1993)and exclusion of fluorogenic dyes (Campbell et al. 1992) canonly be applied to fresh samples.
The fluorescence signal from oocysts stained with FISHwas not bright enough to enable it to be used alone fordetecting oocysts in water concentrates. The autofluorescence
438 G. VESEY ET AL.
Fig. 3 Flow cytometric dot-plots of analysis of water concentrate seeded with oocysts, stained with (a) phycoerythrin (PE)-conjugatedCRY26 monoclonal antibody and (b) fluorescein isothiocyanate-conjugated Cry1 probe and analysed using the FACScan. Thepopulation in area R1 represents PE-labelled oocysts; (b) is gated by R1. FISH, Fluorescence in situ hybridization
Table 5 Comparison of oocyst percentage viability determinedby excystation and by fluorescence in-situ hybridization (FISH) withCry1 on six different samples of oocysts isolated from bovinefaeces. Data are means of three replicates—–––––––––––––––––––––––––––––––––––––––––––––––––––––
*100 oocysts were examined for each determination.†Standard deviations are given in brackets.
of many particles of debris was brighter than stained oocysts.Attempts to increase the brightness of the FISH stain weremade using several different approaches. An increase influorescence was observed after optimizing the hybridizationtemperature, increasing the SDS concentration in the hybrid-ization buffer and performing a detergent pre-treatment. Theobserved increase in fluorescence of the oocysts was probably
due to improved permeability of the oocyst wall. It has beenreported previously that the addition of detergent can increasethe permeability of cells, enabling more oligonucleotide probeto enter (Amann et al. 1992).
Treatment of oocysts under excystation conditions priorto staining increased the fluorescence after hybridization withlabelled Cry1. There was, however, no significant difference(P³ 0·05) in the fluorescence intensity between oocysts pre-treated under excystation conditions followed by DTAB at80 °C compared with a treatment with DTAB at 80 °C alone.The absence of an increase in fluorescence was not a resultof excystation of sporozoites from all oocysts. Samples ofoocysts that were fixed just 5 or 15min into the excystationtreatment contained oocysts with motile sporozoites withinthem at the time of fixation. Therefore, the hypothesis thatsporozoites may manufacture additional rRNA prior to orduring excystation is not supported by these experiments. Itis more likely that the excystation procedure increases thepermeability of oocysts to the Cry1 probe. Oocysts musttherefore contain sufficient rRNA while in the environmentto enable excystation and subsequent infection of the hostcells.
Increasing the number of fluorescein molecules incor-porated into the Cry1 probe from one to two resulted in
DETECTION OF VIABLE CRYPTOSPORIDIUM PARVUM USING FISH 439
significantly brighter oocysts. Increasing the amount offluorescein to three molecules, however, resulted in the probebinding non-specifically. Similar findings have been reportedwith the use of FISH probes labelled with multiple fluoro-chromes for staining bacteria (Wallner et al. 1993).
Amplification of the fluorescence of Cry1-stained oocystsusing an anti-FITC mAb was not successful, possibly becausethe mAb did not efficiently penetrate the oocyst wall. ThemAb (an IgG) was more than 10 times larger than the Cry1probe (120 kDa compared with³ 10 kDa, respectively).
The fluorescence of the Cry1 probe within oocysts seededinto water concentrates could be detected when the sameoocysts were labelled with a surface-specific mAb. The Cry1probe and the mAb must be labelled with different colouredfluorochromes. This dual-labelling method enables the detec-tion of oocysts in water samples and simultaneous deter-mination of the oocyst viability and possible confirmationthat the oocyst is C. parvum. The technique can be combinedwith immunofluorescence staining by performing hybrid-ization with the Cry1 probe (labelled with a red fluorochromesuch as Texas Red or a blue fluorochrome such as CascadeBlue) prior to staining with the FITC-conjugated mAb. Thesample would then be examined as normal using epi-fluorescence microscopy with optical filters suitable forFITC. When green fluorescing oocyst-like particles aredetected they can then be examined using optical filters suit-able for the red- or blue-labelled Cry1 probe, to determine ifthe particle has stained with the Cry1 probe.
It was demonstrated that the dual labelling technique canalso be combined with the flow cytometry method (Veseyet al. 1993a, 1994a) that is routinely used for the detection oflow numbers of Cryptosporidium oocysts in water samples.The Cry1 probe was successfully used to label oocysts withCascade Blue or Texas Red that had been seeded into watersamples before staining with FITC-conjugated CRY26 mAband analysing and sorting oocyst-like particles using the EpicsElite flow cytometer. The Cry1 labelling was not used as aparameter to define which particles to sort. Sorting was sim-ply based on the FITC staining of the oocysts and performedas described previously. Sorted particles were examined withepifluorescence microscopy. Oocysts detected from the greenfluorescence of the mAb were then examined for red flu-orescence of Cry1 staining using appropriate optical filters.
This is the first report of a technique that can be usedto label C. parvum oocysts specifically fluorescently. Whencombined with antibody staining and flow cytometry thetechnique is suitable for detection of low numbers of oocystsin water samples. As currently available antibodies react withCryptosporidium species other than C. parvum (Rose et al.1989; Graczyk et al. 1996), when a water utility detects oocystsin drinking water, the oocysts could actually be a speciesthat is not infectious to man. Furthermore, the currentlyemployed methods cannot determine the viability of the
oocysts. Here we have demonstrated that FISH can be usedto discriminate C. parvum from other Cryptosporidium species.Furthermore, we have demonstrated that FISH can be usedto determine the viability of both freshly excreted oocystsand oocysts that have been in water for long periods (Veseyet al. 1995). Using the Cry1 probe in a FISH technique alongwith antibody staining and flow cytometry overcomes thelimitations of the current methods. The detection of lownumbers of C. parvum oocysts in water samples and thedetermination of oocyst viability are now possible.
ACKNOWLEDGEMENTS
This work was supported by Australian Water Technologies.Aspects of this work have been submitted as Australian patentapplication number 54920/96. The authors wish to thankBecton Dickinson for the loan of the FACScan flow cyto-meter, Jose Wins for technical assistance, Gerd Winter forproviding purified oocysts and Marc Wilkinson for designingand synthesizing the Fluorescein ON-labelled oligo-nucleotide probes. Daniel Deere gratefully acknowledgesreceipt of Research Fellowships from the Royal Society (UK)and the Wain Foundation of the British Biotechnology andBiological Sciences Research Council.
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