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Development of Species-specific rDNA Probes for Giardia by Multiple Fluorescent In Situ Hybridization Combined with Immunocytochemical Identification of Cyst Wall Antigens

Stanley L. Erlandsen, Edward Jarroll, Peter Wallis and Harry van Keulen

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

In this study, we describe the development of fluorescent oligonucleotide probes to variable regions in the small subunit of 16S rRNA in three distinct Giardia species. Sense and antisense probes (17–22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX, Cy3, or Cy5). Optimal results were obtained with fluorochome-labeled oligonucleotides for detection of rRNA in Giardia cysts. Specificity of fluorescent in situ hybridization (FISH) was shown using RNase digestion and high stringency to diminish the hybridization signal, and oligonucleotide probes for rRNA in Giardia lamblia, Giardia muris, and Giardia ardeae were shown to specifically stain rRNA only within cysts or trophozoites of those species. The fluorescent oligonucleotide specific for rRNA in human isolates of Giardia was positive for ten different strains. A method for simultaneous FISH detection of cysts using fluorescent antibody (genotype marker) and two oligonucleotide probes (species marker) permitted visualization of G. lamblia and G. muris cysts in the same preparation. Testing of an environmental water sample revealed the presence of FISH-positive G. lamblia cysts with a specific rDNA probe for rRNA, while negative cysts were presumed to be of animal or bird origin. (J Histochem Cytochem 53:917–927, 2005)

Key Words: cysts • fluorescent in situ hybridization • Giardia lamblia • Giardia muris • Giardia ardeae • rDNA probes • trophozoites

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