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The objectives of this study were to improve the practicality of an in-vitro cell culture-based infectivity assay and assess procedures for simplification. In addition, potential uses of such a method were demonstrated. An in-vitro infectivity assay was developed using monolayers of human cells grown in chambers on glass microscope slides. Infections were detected by reverse transcriptase-polymerase chain reaction (RT-PCR) targeting heat shock protein (hsp70) mRNA. Only infectious oocysts invaded human cells and produced various stages of the C. parvum life cycle which actively transcribed hsp70 mRNA. Extensive control infections demonstrated that inoculum oocysts were not detected by this method. The sensitivity of this assay under ideal conditions, combined with immunomagnetic purification of oocysts, was 10 oocysts or less. To improve the robustness of the assay, a variety of procedures for RNA extraction and RT-PCR were evaluated; a two-step mRNA extraction protocol followed by a two step RT-PCR amplification were most efficient. The level of infection was determined semi-quantitatively by measuring the amount of amplicon produced based on signal intensities. This infectivity assay is now used routinely to assess infectivity and has been used to investigate the inactivation of C. parvum oocysts by UV irradiation. However, because tube-based RT-PCR amplifications are difficult to quantitate, an in-situ hybridization (SH) procedure was developed which allowed for simple enumeration of infections directly on the cell monolayers. The original ISH protocols were simplified using commercially available ISH kits and were used to follow the progression of infection over time. Includes 8 references, table, figures.

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Edition: Vol. - No. Published: 01/01/1999File Size: 1 file , 330 KB