Potentially Infectivity Assay for Giardia Iamblia Cysts
Year Released: 2012
Type: Scientific Investigation
Funding Partner: Bureau of Reclamation
Total Investment: $244,714.93 (Cash: $175,000, In-Kind: $69,714.93)
Principal Investigator: Giovanni Widmer, Tufts Cummings School of Veterinary Medicine
Cysts of the protozoan parasite Giardia lamblia are found worldwide in surface water, in wastewater, and in treatment plant effluents. If ingested, infectious cysts release a trophozoite that can initiate an infection by colonizing the small intestine and dividing to high numbers. Immunofluorescence assays that are routinely used to microscopically detect cysts in water concentrates or fecal samples do not differentiate between infectious cysts and cysts that are unable to cause an infection. In experimental settings, laboratory rodents are needed to assess infectivity, but this approach is not practical for water monitoring.
Goals and Objectives
The project developed a molecular assay that can rapidly discriminate between infectious cysts and cysts unable to cause an infection. Messenger RNA (mRNA) was evaluated as a molecular marker of infectivity. In living cells mRNA levels are regulated by the rate of synthesis (transcription) and decay. Upon cell death, most mRNA transcripts decay and become undetectable. Such mRNA transcripts can rapidly and specifically be monitored using established polymerase chain reaction (PCR) technology.
The project was divided into three specific aims: 1) Identify mRNA transcripts which are up-regulated in Giardia lamblia cysts incubated at 37°C. Purified cysts were incubated at 37°C and pH 2 to mimic the conditions in the host’s gastro-intestinal tract, and induce excystation. RNA was extracted from induced and control cysts, and genes expressing different levels of mRNA were identified on Giardia DNA microarrays. 2) Identify mRNA transcripts which rapidly decay in dead Giardia lamblia cysts. 3) Assay implementation: Reverse transcriptase (RT) PCR protocols which specifically amplify G. lamblia mRNA transcripts identified in Specific Aim 1 and 2 were developed. Selected transcripts which are differentially regulated when cysts are exposed to 37°C, and which rapidly decay in dead cyst were amplified by RT PCR to assess the validity of the approach.
Findings and Conclusions
The mRNA population (transcriptome) of live cysts and of cysts inactivated by aging, heat, and ultraviolet (UV) irradiation was surveyed using oligonucleotide microarrays. For this analysis, mRNA is copied in bulk to complementary DNA (cDNA) and fluorescently labeled to generate a probe. Microarrays were hybridized with such probes to obtain a quantitative representation of the complete cyst transcriptome. Statistical analysis of replicate microarrays revealed that, as compared to trophozoites (the dividing stage in the parasite’s life cycle), only about 1/20 of the mRNA transcripts was detected. The analysis of replicate microarray data set identified about 200 mRNA transcripts in live cysts. As an alternative approach, cysts were induced to excyst and the transcriptome of excysting cysts compared to that of control cysts. Selected transcripts expressed at a high level in infectious cysts and absent from inactivated (dead) cysts, or overexpressed in excysting cysts, were evaluated using reverse transcriptase PCR (RT-PCR). These analyses confirmed differential mRNA levels in live and inactivated cysts. These findings did not apply to cysts irradiated with a lethal dose of 254-nm UV light, a treatment that had little impact on the cyst transcriptome. This observation is consistent with the mode of action of UV irradiation, which does not act by killing microorganisms but by inducing mutations in the DNA.
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