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 ^1*, Edward Jarroll ¹, Peter Wallis ¹ and Harry van Keulen ¹

¹ 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)

^* To whom correspondence should be addressed. E-mail: E-mail: erlan001{at}umn.edu.

Submitted on February 17, 2005
Accepted on 22 February 2005

	Abstract

In this study we describe the development of fluorescent oligonucleotide probes to variable regions in the SSU of 16S rRNA in three distinct Giardia species. Sense and anti-sense probes (17-22 mer) to variable regions 1, 3, and 8 were labeled with digoxygenin or selected fluorochomes (FluorX^TM, Cy3^TM or Cy5^TM). 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 G. lamblia, G. muris, and G. ardeae were shown to specifically stain rRNA only within cysts or trophozoites of that 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.

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

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