Skip to main content Accessibility help
×
Home

Quantitative PCR to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in Daphnia

  • D. REFARDT (a1) (a2) and D. EBERT (a1) (a2)

Abstract

Reliable detection, discrimination and quantification of parasites are important for host-parasite studies and diagnostics. Microsporidial infections are problematic in this respect. Their discrimination and quantification using light microscopy is difficult because spores are the only light microscopically visible form of the parasite and they offer few distinct characters. We developed a quantitative PCR (qPCR) assay based on SYBR Green chemistry to quantify the microsporidia Glugoides intestinalis, Octosporea bayeri and Ordospora colligata in their host, the freshwater crustacean Daphnia magna. The assay allows the quantification of infection intensities in whole animals and is more than an order of magnitude more sensitive than light microscopy. Sampling and DNA extraction account for more than 90% of the residual variance in infection intensity data and this variance considerably impairs the resolution of qPCR. Where higher resolution is required, we propose using the ratio of parasite to host DNA as the measure of infection intensity. We show that this measure is robust and greatly improves resolution of qPCR. Additionally, this method can be applied to compare samples of unequal volume.

Copyright

Corresponding author

Zoologisches Institut der Universität Basel, Evolutionsbiologie, Vesalgasse 1, CH-4051 Basel, Switzerland. Tel: +41612760375. Fax: +41612670362. E-mail: dominik.refardt@gmail.com

References

Hide All

REFERENCES

Bedhomme, S., Agnew, P., Sidobre, C. and Michalakis, Y. ( 2004). Virulence reaction norms across a food gradient. Proceedings of the Royal Society of London, B 271, 739744. DOI: 10.1098/rspb.2003.2657.
Bustin, S. A. ( 2000). Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. Journal of Molecular Endocrinology 25, 169193. DOI: 10.1677/jme.0.0250169.
Candotti, D., Temple, J., Owusu-Ofori, S. and Allain, J.-P. ( 2004). Multiplex real-time quantitative RT-PCR assay for hepatitis B virus, hepatitis C virus, and human immunodeficiency virus type 1. Journal of Virological Methods 118, 3947. DOI: 10.1016/j.jviromet.2004.01.017.
Canning, E. U., Refardt, D., Vossbrinck, C. R., Okamura, B. and Curry, A. ( 2002). New diplokaryotic microsporidia (Phylum Microsporidia) from freshwater bryozoans (Bryozoa, Phylactolaemata). European Journal of Protistology 38, 247265. DOI: 10.1078/0932-4739-00867.
Capaul, M. and Ebert, D. ( 2003). Parasite-mediated selection in experimental Daphnia magna populations. Evolution 57, 249260.
Cheesman, S. J., de Roode, J. C., Read, A. F. and Carter, R. ( 2003). Real-time quantitative PCR for analysis of genetically mixed infections of malaria parasites: technique validation and applications. Molecular and Biochemical Parasitology 131, 8391. DOI: 10.1016/S0166-6851(03)00195-6.
Decaestecker, E., Declerck, S., De Meester, L. and Ebert, D. ( 2005). Ecological implications of parasites in natural Daphnia populations. Oecologia 144, 382390. DOI: 10.1007/s00442-005-0083-7.
Ebert, D. ( 1994). Virulence and local adaptation of a horizontally transmitted parasite. Science 265, 10841086.
Ebert, D. ( 2005). Ecology, Epidemiology, and Evolution of Parasitism in Daphnia [Internet]. National Library of Medicine, National Center for Biotechnology Information, Bethesda (MD). http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books
Ebert, D., Hottinger, J. W. and Pajunen, V. I. ( 2001). Temporal and spatial dynamics of parasite richness in a Daphnia metapopulation. Ecology 82, 34173434.
Ebert, D., Lipsitch, M. and Mangin, K. L. ( 2000). The effect of parasites on host population density and extinction: Experimental epidemiology with Daphnia and six microparasites. American Naturalist 156, 459477.
Fischer, O. and Schmid-Hempel, P. ( 2005). Selection by parasites may increase host recombination frequency. Biology Letters 1, 193195. DOI: 10.1098/rsbl.2005.0296.
Green, J. ( 1974). Parasites and epibionts of Cladocera. Transactions of the Zoological Society of London 32, 417515.
Hester, J. D., Varma, M., Bobst, A. M., Ware, M. W., Lindquist, H. D. A. and Schaefer III., F. W. ( 2002). Species-specific detection of three human-pathogenic microsporidial species from the genus Encephalitozoon via fluorogenic 5′ nuclease PCR assays. Molecular and Cellular Probes 16, 435444. DOI: 10.1006/mcpr.2002.0442.
Higuchi, R., Fockler, C., Dollinger, G. and Watson, R. ( 1993). Kinetic PCR analysis: Real-time monitoring of DNA amplification reactions. Bio/Technology 11, 10261030.
Jírovec, O. ( 1936). Über einige in Daphnia magna parasitierende Mikrosporidien. Zoologischer Anzeiger 116, 136142.
Larsson, J. I. R., Ebert, D., Mangin, K. L. and Vávra, J. ( 1998). Ultrastructural study and description of Flabelliforma magnivora sp. n. (Microspora: Duboscqiidae), a microsporidian parasite of Daphnia magna (Crustacea: Cladocera: Daphniidae). Acta Protozoologica 37, 4152.
Larsson, J. I. R., Ebert, D. and Vávra, J. ( 1997). Ultrastructural study and description of Ordospora colligata gen. et sp. nov. (Microspora, Ordosporidae fam. nov.), a new microsporidian parasite of Daphnia magna (Crustacea, Cladocera). European Journal of Protistology 33, 432443.
Larsson, J. I. R., Ebert, D., Vávra, J. and Voronin, V. N. ( 1996). Redescription of Pleistophora intestinalis Chatton, 1907, a microsporidian parasite of Daphnia magna and Daphnia pulex, with establisment of the new genus Glugoides (Microspora, Glugeidae). European Journal of Protistology 32, 251261.
Lass, S. and Ebert, D. ( 2005). Apparent seasonality of parasite dynamics: analysis of cyclic prevalence patterns. Proceedings of the Royal Society of London, B. 273, 199206. DOI: 10.1098/rspb.2005.3310.
Lively, C. M. and Dybdahl, M. F. ( 2000). Parasite adaptation to locally common host genotypes. Nature 405, 6791474. DOI: 10.1038/35015069.
Mangin, K. L., Lipsitch, M. and Ebert, D. ( 1995). Virulence and transmission modes of two microsporidia in Daphnia magna. Parasitology 111, 133142.
Menotti, J., Cassinat, B., Porcher, R., Sarfati, C., Derouin, F. and Molina, J. M. ( 2003 a). Development of a real-time polymerase-chain-reaction assay for quantitative detection of Enterocytozoon bieneusi DNA in stool specimens from immunocompromised patients with intestinal microsporidiosis. Journal of Infectious Diseases 187, 14691474.
Menotti, J., Cassinat, B., Sarfati, C., Liguory, O., Derouin, F. and Molina, J. M. ( 2003 b). Development of a real-time PCR assay for quantitative detection of Encephalitozoon intestinalis DNA. Journal of Clinical Microbiology 41, 14101413. DOI: 10.1128/JCM.41.4.1410–1413.2003.
Mouton, L., Dedeine, F., Henri, H., Boulétreau, M., Profizi, N. and Vavre, F. ( 2004). Virulence, multiple infections and regulation of symbiotic population in the Wolbachia-Asobara tabida symbiosis. Genetics 168, 181189. DOI: 10.1534/genetics.104.026716.
Mucklow, P. T., Vizoso, D. B., Jensen, K. H., Refardt, D. and Ebert, D. ( 2004). Variation in phenoloxidase activity and its relation to parasite resistance within and between populations of Daphnia magna. Proceedings of the Royal Society of London, B 271, 11751183. DOI: 10.1098/rspb.2004.2707.
Pfaffl, M. W. ( 2004). Quantification strategies in real-time PCR. In A–Z of Quantitative PCR ( ed. Bustin, S. A.), pp. 78120. International University Line (IUL), La Jolla, CA.
Pulkkinen, K. and Ebert, D. ( 2004). Host starvation decreases parasite load and mean host size in experimental populations. Ecology 85, 823833.
Rozen, S. and Skaletsky, H. J. ( 2000). Primer3 on the WWW for general users and for biologist programmers. In Bioinformatics Methods and Protocols: Methods in Molecular Biology ( ed. Krawetz, S. and Misener, S.), pp. 365386. Humana Press, Totowa, NJ. http://frodo.wi.mit.edu/
Stirnadel, H. A. and Ebert, D. ( 1997). Prevalence, host specificity and impact on host fecundity of microparasites and epibionts in three sympatric Daphnia species. Journal of Animal Ecology 66, 212222.
Vizoso, D. B. and Ebert, D. ( 2004). Within-host dynamics of a microsporidium with horizontal and vertical transmission: Octosporea bayeri in Daphnia magna. Parasitology 128, 3138. DOI: 10.1017/S0031182003004293.
Vizoso, D. B., Lass, S. and Ebert, D. ( 2005). Different mechanisms of transmission of the microsporidium Octosporea bayeri: a cocktail of solutions for the problem of parasite permanence. Parasitology 130, 501509. DOI: 10.1017/S0031182004006699.
Wasson, K. and Barry, P. A. ( 2003). Molecular characterization of Encephalitozoon intestinalis (Microspora) replication kinetics in a murine intestinal cell line. Journal of Eukaryotic Microbiology 50, 169174. DOI: 10.1111/j.1550-7408.2003.tb00112.x.

Keywords

Quantitative PCR to detect, discriminate and quantify intracellular parasites in their host: an example from three microsporidians in Daphnia

  • D. REFARDT (a1) (a2) and D. EBERT (a1) (a2)

Metrics

Full text views

Total number of HTML views: 0
Total number of PDF views: 0 *
Loading metrics...

Abstract views

Total abstract views: 0 *
Loading metrics...

* Views captured on Cambridge Core between <date>. This data will be updated every 24 hours.

Usage data cannot currently be displayed