RAPID COMMUNICATION |
Development of Species-specific rDNA Probes for Giardia by Multiple Fluorescent In Situ Hybridization Combined with Immunocytochemical Identification of Cyst Wall Antigens
Department of Genetics, Cell Biology, and Developmental Biology, University of Minnesota Medical School, Minneapolis, Minnesota (SLE); Department of Biology, Northeastern University, Boston, Massachusetts (EJ); Hyperion Research Ltd, Medicine Hat, Alberta, Canada (PW); and Department of Biological, Geological, and Environmental Sciences, Cleveland State University, Cleveland, Ohio (HvK)
Correspondence to: Dr. Stanley L. Erlandsen, Department of Genetics, Cell Biology, and Development, 6-160 Jackson Hall, University of Minnesota Medical School, Minneapolis, MN 55455. E-mail: erlan001{at}umn.edu
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Summary |
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Key Words: cysts fluorescent in situ hybridization Giardia lamblia Giardia muris Giardia ardeae rDNA probes trophozoites
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Introduction |
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Classical approaches to parasitology, based on host specificity and trophozoite morphometrics, recognized over forty different species of Giardia (Kulda and Nohnková 1978
), but the development of molecular techniques and nucleotide sequencing of nucleic acids have firmly identified six species based on sequence of rRNA or unique morphological characteristics: Giardia lamblia, Giardia ardeae, Giardia muris, Giardia psittaci, Giardia agilis, and Giardia microti (van Keulen et al. 1993
,1998
; Adam 2000
; Thompson et al. 2000
). Initial morphological studies of Giardia led Filice (1952)
to suggest that three morphological groups exist based on several criteria, including the shape of a microtubule-rich organelle called the median body. These three groups were: Giardia duodenalis, Giardia muris, and Giardia agilis. However, G. dudoenalis (syn. G. lamblia, Giardia intestinalis) should not be used as a species name because the described morphological criteria also fit the description for G. microti, G. ardeae, G. psittaci, characterized as distinct species on the basis of molecular sequencing of rRNA (van Keulen et al. 1993
, 1998
). Therefore, in this manuscript, the term "Giardia lamblia" will be used to describe isolates of Giardia that have been obtained from humans.
Phylogenetically, rRNA is conserved in function, organization, and sequence, and most cells contain thousands of copies within ribosomes, making this molecule an ideal target for development of genus- and species-specific probes. rRNA sequencing has been used in over 2000 microorganisms and cells, including five different species of Giardia, to establish phylogenetic relationships (van Keulen et al. 1993,1998
). rRNA constitutes 90% to 95% of cellular RNA and contains three forms, namely a 16S small subunit (SSU), a 5S SSU, and a 23S large subunit (Waters and McCutchan 1990
). Although rRNAs are highly conserved in structure, most of the diversity in nucleotide substitutions and deletions occurs in Giardia spp. within the 16S SSU, and these nucleotide alterations tend to be concentrated in nine variable regions.
Molecular approaches to characterizing or detecting Giardia spp. have included chromosomal band analysis (Campbell et al. 1990), PCR analysis, detection of heat shock protein genes (Abbaszadegan et al. 1992
; Mahbubani et al. 1992
), genetic analysis of glutamate dehydrogenase locus (Monis et al. 1996
), sequence variation in rRNA and rDNA (Weiss et al. 1992
, van Keulen et al. 1993
,1998
), sequence of giardin genes (Caccio et al. 2002
), and the use of oligonucleotide microarrays (Wang et al. 2004
). In the last two decades, non-radioactive hybridization methods using enzymatic or fluorescent markers have proved to be useful tools for multiple labeling of nucleic acid targets (Höfler, 1987
; Nederlof et al. 1990
; Speel et al. 1994
). Detection of multiple mRNAs and antigens within the same cell or tissue has been described (Dirks et al. 1991
; Trembleau et al. 1993
), and our laboratory has also used fluorescent in situ hybridization (FISH) for the detection of 16S SSU rRNA in fixed Giardia trophozoites in archival pathology specimens of human small intestine (Macechko et al. 1998
).
The aim of this study was to develop FISH methods for detecting rRNA in Giardia spp. cysts. Oligonucleotides specific for rRNA-variable regions in different species of Giardia were labeled with haptens or fluorochromes. A method was developed for identifying Giardia spp. cysts in environmental samples using immunochemical localization of cyst wall antigens, and speciation of these cysts was accomplished by simultaneous FISH with multiple fluorochrome-labeled probes emitting at different wavelengths.
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Materials and Methods |
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DNA Probe Synthesis and Labeling
Sense and antisense oligonucleotide probes (1722 mer) corresponding to rDNA in variable regions 1, 3, and 8 of the 16S SSU rDNA molecule (Figure 1)
were synthesized on an Applied Biosystems 394 DNA synthesizer in the Microchemical Facility at the University of Minnesota. Probes were tailed at the 3' end with digoxygenin-11-UTP and dATP with terminal transferase (Boehringer Mannheim, Indianapolis, IN) following the manufacturer's instructions to keep tail length below 100 base pairs, or oligonucleotides were labeled via a 5' aminolink with one of the carboxymethylindocyanine dyes, including, FluorX-CTP (absorption 494 nm; emission 520 nm), Cy3-CTP (absorption 552 nm; emission 567 nm), or Cy5-CTP (absorption 648 nm; emission 665 nm) (Biological Detection Systems, Inc., Pittsburgh, PA; Amersham, Chicago, IL). All probes were purified by high-pressure liquid chromatography before use. The melting temperature (Tm) of each probe was calculated based on hybridization in 2x SSC (1x SSC = 0.15 M sodium chloride, 0.015 M sodium citrate) in 50% formamide.
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Prehybridization and Hybridization
Air-dried slides were rehydrated in 0.1 M PBS containing 7.5% sucrose, and charge was blocked by immersing for 10 min in 0.25% acetic anhydride in 0.1 M triethanolamine at pH 8.0. Slides were then rinsed for 2 min in 2x SSC, followed by dehydration in an ascending ethanol series (50%, 70%, 95%, 100%), and air dried. Cells were rehydrated in prehybridization mix [2x SSC, 50% formamide, 5x Denhardt's solution (0.02% polyvinylpyrrolidone, 0.02% Ficoll, and 0.02% bovine serum albumin), and 1 mg salmon sperm DNA] for 60 min at room temperature. Oligonucleotide probes (1 ng/µl) in 50% formamide in 4x SSC were denatured by boiling for 10 min, cooled immediately to 4C, and then added to slides that were heated for an additional 10 min at 85C, followed by hybridization overnight at 37C in moist chambers. Following hybridization, slides were rinsed sequentially in 4x SSC, 2x SSC, and 1x SSC.
Alkaline Phosphatase Incubation for Digoxygenin-labeled Probes
After the last stringency wash, slides were immersed for 2 hr in a blocking solution (0.1 M Tris, 1% normal sheep serum, 0.3% dry milk, 0.1% Triton X-100, and 0.05% sodium azide) on a slow rotary shaker. The slides were drained and then incubated overnight with a 1:100 dilution of sheep anti-digoxygenin Fab fragments conjugated with alkaline phosphatase (Boehringer Mannheim). Slides were then rinsed at room temperature for 30 min with TBS, pH 7.0, followed by 30 min in PBS, pH 8.0. Alkaline phosphatase activity was revealed by incubation in 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium. The enzyme staining reaction was stopped by incubating the slides in 10 mM Tris containing 0.1 mM EDTA at pH 8.0, and slides were mounted in an aqueous medium.
Immunocytochemistry and Multiple rDNA Hybridization
Multiple labeling of rRNA in cysts was accomplished by mixing 1 ng/µl each of oligonucleotide probes specific for G. lamblia (Cy5 conjugate) and G. muris (FluorX conjugate) and denaturing the probes by boiling for 10 min. These probes were cooled to 4C, and the mixture was applied to slides, incubated for 10 min at 85C, and hybridized overnight at 37C. Following stringency washes, single rDNA hybridization with Cy3-conjugated probes were directly immunostained for cyst wall antigens using a fluorescein-conjugated mouse monoclonal antibody (RD348; Meridian Diagnostics, Cincinnati, OH), whereas double rDNA hybridization with Cy5 and FluorX oligonucleotide conjugates were counterstained for cyst wall antigens using rabbit anti-Giardia lamblia cyst antibody (GLMB; provided by Judy Sauch, Environmental Protection Agency, Cincinnati, OH) and sheep-anti-rabbit IgG conjugated with Cy3.
Controls
Hybridization controls consisted of: (a) omitting the specific probe or use of unlabeled probe; (b) using sense and antisense probes for the same variable rDNA region; (c) RNase (100 µg/ml) digestions of cells in 2x SSC for 30 min at room temperature, oxidized RNase (no enzyme activity) at 100 µg/ml in 2x SSC for 30 min at room temperature; (d) use of 100:1 ratio of unlabeled probe to fluorochrome-labeled probe for hybridization; (e) exposure only to concentrations of free Cy3 dye equivalent to that in labeled probe to determine whether nonspecific staining occurred via absorption of free dye; (f) use of high-stringency (0.1x SSC at either 50C or 65C, depending on probe) exceeding probe Tm by 5C to10C; and (g) comparing staining of specific oligonucleotide probes with other known Giardia spp.
Microscopy
A Zeiss photomicroscope (Carl Zeiss; Thornwood, NY) equipped with brightfield, phase contrast, and Nomarski optics was used with 20x, 40x oil, and 63x oil planapochromatic objective lenses for evaluating digoxygenin-labeled probes. Fluorescent imaging of conjugated probes was accomplished with a BioRad MRC-600 laser scanning confocal microscope (BioRad; Hercules, CA) equipped with a dual krypton-argon laser. Enhancement of images was achieved by processing in Confocal Assistant, a program developed by Dr. Todd Brelje at the University of Minnesota.
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Results |
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Discussion |
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In the last decade, genetic analysis has revealed considerable levels of diversity in G. lamblia (Thompson et al. 2000). Isolates of G. lamblia fall into two major assemblages (A, B) based on a unique trinucleotide in the 16S SSU rRNA molecule (Homan et al. 1992
; van Keulen et al. 1995
; Thompson et al. 2000
); and more recently, primers specific for PCR amplification of the triose phosphate isomerase gene distinguished between assemblages A and B (Wang et al. 2004
). Slight variations within each assemblage have been detected, and attempts to characterize which assemblage contains Giardia strains that are pathogenic in humans have led to contradictory results (Homan and Mank 2001
; Read et al. 2002
). Design of fluorescent oligonucleotide probes to distinguish multiple species of Giardia, and also to detect subtypes in assemblages within the G. lamblia species, would be valuable for epidemiologic evaluations of outbreaks of giardiasis and to address the question of zoonotic potential for this disease.
Detection of Giardia in environmental water and fecal samples has been investigated using PCR amplification with gene probes for giardin genes (Mahbubani et al. 1992; Caccio et al. 2002
), SSU rRNA (Abbaszadegan et al. 1991
; Rochelle et al. 1997
; Ghosh et al. 2000
), 18S rRNA (Weiss et al. 1992
), mRNA (Kauener and Stinear 1998
) triose phosphate isomerase (McIntyre et al. 2000
; Amar et al. 2003
), variant-specific surface protein genes (Ey et al. 1997
), and genes for heat shock protein (Rochelle et al. 1997
) have been used to detect Giardia cysts with a sensitivity of less than one cyst per milliliter. However, detection in environmental samples was highly variable, occasionally giving negative results even when 105 cysts were present, or differentiation between known Giardia species was not achieved. Major drawbacks in using gene probes on environmental samples have been nonspecific absorption of probes on debris within the samples, generation of nonspecific DNA fragments, and inhibition of amplification due to contaminants within the sample.
In this study, we have shown that FISH with rDNA probes can positively identify G. lamblia in water samples under outbreak conditions (Figure 5E). The application of this technique to routine monitoring could be an important improvement in the analysis of water samples, especially using the enhanced recovery offered by US Environmental Protection Agency Method 1623, because results could be obtained more quickly than by PCR methods. The identification of cysts that are not G. lamblia and do not correspond to isolates known to infect humans would help refine risk evaluation and would prevent unnecessary boil water advisories. It would also require that "zero tolerance" regulatory policies such as that adopted in Canada during the late 1990s (since rescinded) need to be re-evaluated to allow for the occurrence of species that are not infective to humans.
In summary, we believe that further refinement of our microscopic FISH approach using immunochemical identification of genotypic markers (cyst wall antigens) in combination with species-specific fluorescent oligonucleotides may provide a convenient and specific method for the simultaneous detection of different G. lamblia strains or other species (animal or bird origin). Use of multiple fluorescent probes for genus- and species-specific genes, combined with either microarray technology (Wang et al. 2004) or the use of radio-labeled fluorescent DNA probes (Dauwerse et al. 1992
) for FISH, together with spectral imaging, should permit simultaneous evaluation of multiple genes within the same cyst. This approach should prove to be valuable for epidemiologic investigation of environmental water samples or clinical human fecal samples.
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Acknowledgments |
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Footnotes |
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Literature Cited |
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Abbaszadegan M, Gerba CP, Rose JB (1991) Detection of Giardia cysts with a cDNA probe and applications to water samples. Appl Environ Microbiol 57:927931[Medline]
Abbaszadegan M, Huber MS, Pepper IL, Gerba CP (1992) Detection of viable Giardia cysts in water samples using polymerase chain reaction. Proceedings of the Water Technology Conference, American Waterworks Association, Miami, FL. 529548
Adam RD (2000) The Giardia lamblia genome. Int J Parasitol 30:475484[CrossRef][Medline]
Amar CFL, Dear PH, McLauchlin J (2003) Detection and genotyping by real-time PCR/RFLP analysis of Giardia duodenalis from human faeces. J Med Microbiol 52:681683
Caccio SM, De Giacomo M, Pozio E (2002) Sequence analysis of the ß-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol 32:10231030[CrossRef][Medline]
Campbell SR, van Keulen H, Erlandsen SL, Senturia JB, Jarroll EL (1990) Giardia spp.: Comparison of electrophoretic karyotypes. Exp Parasitol 71:470482[CrossRef][Medline]
Dauwerse JB, Wiegant J, Raap AK, Breuning MH, van Ommen GJB (1992) Multiple colors by fluorescent in situ hybridization using radio-labeled DNA probes create a molecular karyotype. Hum Mol Genet 1:593598[Abstract]
Dirks RW, Van Giljlswijk RPM, Voolis MA, Smit AB, Bogerd J, van Minnen J, Raap AK, Van der Ploeg M (1991) 3'-end fluorochromized and haptenized oligonucleotides as in situ hybridization probes for multiple simultaneous RNA detection. Exp Cell Res 194:310315[CrossRef][Medline]
Erlandsen SL, Bemrick WJ (1987) SEM evidence for a new species, Giardia psittaci. J Parasitol 73:623629[Medline]
Erlandsen SL, Rasch E (1994) The DNA content of trophozoites and cysts of Giardia lamblia by microdensitometric quantitation of Feulgen staining and examination by laser scanning confocal microscopy. J Histochem Cytochem 42:14131416
Ey PL, Mansouri M, Kulda J, Nohynkova E, Monis PT, Andrews RH, Mayrhofer G (1997) Genetic analysis of Giardia from hoofed farm animals reveals artiodactyl-specific and potentially zoonotic genotypes. J Euk Microbiol 44:626635[Medline]
Filice FP (1952) Studies on the cytology and life history of Giardia from the laboratory rat. California Publ Zool 57:53146
Ghosh S, Debnath A, Sil A, De S, Chattopadhyay DJ, Das P (2000) PCR detection of Giardia lamblia in stool: targeting intergenic spacer regions of multicopy rRNA gene. Mol Cell Probes 14:181189[CrossRef][Medline]
Höfler H (1987) What's new in "in situ hybridization." Pathol Res Pract 182:421430[Medline]
Homan WL, Mank TG (2001) Human giardiasis: genotype linked differences in clinical symptomatology. Int J Parasitol 31:823826
Homan WL, van Enckevort FHJ, Limper EHJ, van Eys GJJM, Schoone GJ, Kasprzak W, Majewska AC, et al. (1992) Comparison of Giardia isolates from different laboratories by isoenzyme analysis and DNA probes. Parasitol Res 78:316323[CrossRef][Medline]
Kauener C, Stinear T (1998) Sensitive and rapid detection of viable Giardia cysts and Cryptosporidium parvum oocysts in large-volume water samples with wound fiberglass cartridge filters and reverse transcriptase PCR. Appl Environ Microbiol 64:17431749
Kulda J, Nohnková E (1978) Flagellates of the human intestine and of intestines of other species. In Krier, JP, ed. Parasitic Protozoa, vol 2, Intestinal flagellates, histomonads, trichomonads, amoeba, opalinids, and ciliates. New York, Academic Press, 1138
Macechko PT, van Keulen H, Jarroll EL, Mulgrew T, Gurien A, Erlandsen SL (1998) FISH detection of Giardia trophozoites in archival pathology specimens of human small intestine. Microsc Microanal 4:397403[Medline]
Mahbubani MH, Bej AK, Perlin HH, Schaeffer FW III, Jakubowski W, Atlas RM (1992) Differentiation of Giardia duodenalis from other Giardia spp. by using polymerase chain reaction and gene probes. J Clin Microbiol 30:7478[Abstract]
McIntyre L, Hoang L, Ong CSL, Lee P, Isaac-Renton JL (2000) Evaluation of molecular techniques to biotype Giardia duodenalis collected during an outbreak. J Parasitol 86:172177[Medline]
Monis PT, Mayerhoefer G, Andrew RH, Homan WL, Limper L, Ey PL (1996) Molecular genetic analysis of Giardia intestinalis isolates at the glutamate dehydrogenase locus. Parasitol 112:112
Nederlof PM, van der Flier S, Wiegant J, Raap AK, Tanke HJ, Ploem JS, van der Ploeg M (1990) Multiple fluorescence in situ hybridization. Cytometry 11:126131[Medline]
Read C, Walters J, Robertson ID, Thompson RCA (2002) Correlation between genotype of Giardia duodenalis and diarrhea. Int J Parasitol 32:229231[CrossRef][Medline]
Rochelle PA, De Leon R, Stewart MH, Wolfe RL (1997) Comparison of primers and optimization of PCR conditions for detection of Cryptosporidium parvum and Giardia lamblia in water. Appl Environ Microbiol 63:106114[Abstract]
Schupp DG, Erlandsen SL (1987) Determination of Giardia muris cyst viability by differential interference contrast or bright field microscopy. J Parasitol 73:723729[Medline]
Speel EJM, Jansen MPHM, Ramaekers FCS, Hopman AHN (1994) A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry. J Histochem Cytochem 42:12991307
Thompson RCA, Hopkins RM, Homan WL (2000) Nomenclature and genetic groupings of Giardia infecting mammals. Parasitol Today 16:210213[CrossRef][Medline]
Trembleau A, Roche D, Calas A (1993) Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section. J Histochem Cytochem 41:489498
van Keulen H, Feely DE, Macechko PT, Jarroll EL, Erlandsen SL (1998) The sequence of Giardia small subunit rRNA shows that voles and muskrats are parasitized by a unique species Giardia microti. J Parasitol. 84:294300[Medline]
van Keulen H, Gutell RR, Gates MA, Campbell SR, Erlandsen SL, Jarroll EL, Kulda J, et al. (1993) Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Girdia ardeae, G. muris, G. duodenalis and Hexamita sp. FASEB J 7:223231
van Keulen H, Homan WL, Erlandsen SL, Jarroll EL (1995) A three nucleotide signature sequence in small subunit rRNA divides human Giardia into two different genotypes. J Eukaryot Microbiol 42:392394[Medline]
van Keulen H, Macechko PT, Wade S, Schaaf S, Wallis PM, Erlandsen SL (2002) Presence of human Giardia in domestic, farm and wild animals, and environmental samples suggests a zoonotic potential for giardiasis. Vet Parasitol 108:97107[CrossRef][Medline]
Wang Z, Vora GJ, Stenger DA (2004) Detection and genotyping of Entoamoeba histolytica, Entoamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray. J Clin Microbiol 42:32623271
Wallis PM, Primrose B, Robertson WJ (1998) Outbreak of waterborne giardiasis caused by sewage contamination of drinking water. Env Hlth Rev 42:4451
Waters AP, McCutchan TF (1990) Ribosomal RNA: nature's own polymerase-amplified target for diagnosis. Parasitol Today 6:5659[CrossRef][Medline]
Weiss JB, van Keulen H, Nash TE (1992) Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction. Mol Biochem Parasitol 54:7386[CrossRef][Medline]
World Health Organization (1996) The World Health Report 1996. Brussels, World Health Organization
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