Grantee Research Project Results
Final Report: A Novel Method To Detect and Enumerate Viable Cryptosporidium parvum Oocysts in Water Using Integrated Cell Culture-rRNA In Situ Hybridization
EPA Contract Number: 68D99056Title: A Novel Method To Detect and Enumerate Viable Cryptosporidium parvum Oocysts in Water Using Integrated Cell Culture-rRNA In Situ Hybridization
Investigators: Hsu, Fu-Chih
Small Business: MAS Technology Corporation dba Environmental Health Laboratories
EPA Contact:
Phase: I
Project Period: September 1, 1999 through March 1, 2000
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (1999) RFA Text | Recipients Lists
Research Category: Small Business Innovation Research (SBIR)
Description:
The goal of this project is to develop a sensitive and specific method for detecting viable Cryptosporidium parum oocysts by integrating cell culture-rRNA hybridization. Unique sequences (25-35 bases) to Cryptosporidium parvum will be selected using multiple sequence alignment on 18s rRNA from different Cryptosporidium species. Oligonucleotide probes will be synthesized and labeled with digoxigenin for in situ hybridization. Probes will be evaluated for sensitivity and specificity using different Cryptosporidium species. A special cell line, HCT-8 cell will be used to detect viable C. parvum oocysts. Determination of HTC-8 cell specificity for different species of Cryptosporidium will also be conducted. A chemiluminiscent detection using a very sensitive x-ray film or a colorimetric detection will be used to visualize DNA-RNA hybrids. The developed method using integrated cell culture-rRNA hybridization will be used to detect viable oocysts in different spiking matrix.Summary/Accomplishments (Outputs/Outcomes):
Oligonucleotide Probe Development and Testing
Probe C1 showed high specificity to discriminate C. parvum and C. muris. No C. muris oocysts or reproductive stages of C. muris were found in HCT-8 cell layers after inoculation for 48 h. This demonstrated that the differences determined in the sequence alignment between C. parvum and C. muris were correct. Since no cross-reaction in probe C1 was found by the multiple sequence alignment among Cryptosporidium species, probe C1 should not react with other species of Cryptosporidium. However, more research needs to be done to document the specificity of probe C1. Another probe (C3) did not react with C. parvum and C. muris strains tested in this study. Probe C3 was designed to detect genotype I of C. parvum but not the one we tested (genotype II). By combining probes C1 and C3, all currently known strains of C. parvum should be detected.
We have generated another oligonucleotide probe (C6) targeting 18sRNA for both genotypes I and II of C. Parvum. This 30 base DNA probe is 100 % matched among 6 strains of C. parvum and about 50 % matched to other species of Cryptosporidium. To evaluate the specificity and sensitivity of the probe C6, we performed infectivity assay using HCT-8 cells inoculated with increasing concentrations of C. parvum and C. muris oocysts respectively. After 42 h. incubation, in- situ hybridization using DIG-labeled C6 probe was performed and followed by colorimetric detection. The results indicated that C6 could detect C. parvum genotype II but not C. muris. In our laboratory we are currently using the Iowa strain of C. parvum which belongs to genotype II. C. parvum genotype I is not commercially available yet to perform this test. According to the sequence alignment, we expect that C6 should be able to recognize both genotypes I and II. However, this probe should be tested by the genotype I and other species of Cryptosporidium in the future experiments.
Fluorescent labeled Antibody Evaluation
To determine infectious doses in HCT-8 cells for C. parvum, a commercially available fluorescein-labeled antibody (A600FL; Waterborne, INC) against sporozoites of C. parvum was also evaluated and compared to 18s rRNA probes in this study. Infectious foci with bright green color and high stain intensity were observed. Under DIC observation (1000 X), in some clusters, the oval shaped meroit can be observed revealing the intracellular cryptosporidial reproductive stages. The manufacturer claims this antibody will exhibit only a slight reaction against C. parvum oocysts. When A600FL was used to directly stain C. parvum and C. muris oocysts, no oocysts were detected under epifluorescent microscopy. Moreover, no foci were found in HCT-8 cell layers inoculated with C. muris oocysts. Based on these results, A600FL may be specific enough to detect reproductive stages of C. parvum when combined with HCT-8 cell cultures. However, it may be useful to determine A600FL specificity with other species of Cryptosporidium. It seems that the infection detection limit using HCT-8 cells is somewhere between 100 and 1000 oocysts. However, a vital dye (DAPI) shows only one third of the oocysts contain intact DNA. The DAPI positive oocysts count may reflect the number of oocysts that may have the ability to infect cells. Therefore, the detection limit is estimated to between 30 and 330 viable oocysts. We confirmed that the detection sensitivity was 30-50 oocysts using multiple chamber slides.
Chemiluminescent detection
The background image of chemiluminescent detection was so bright that the infected foci could not be distinguished from the negative control. To improve chemiluminescent detection, an endogenous cellular alkaline phosphatase inhibitor, a cell culture insert (membrane), and a different hybridization formula were tested. Unfortunately, none of them resulted in satisfactory improvement in the background. Due to the difficulties in chemiluminescent detection, an alternative detection method, the colorimetric detection followed by in-situ hybridization (ISH), was used to detect infectious foci. Infectious foci detected by colorimetric detection are very unique and easily observed by the bright-field microscopy.
Detection of infectious C. parvum oocysts from spiked water
To determine if oocysts recovered from spiked samples are still infectious, concentrates recovered by the ICR method and Method 1622 were inoculated into HCT-8 cells. IMS was also used to remove debris from the sample concentrates before inoculation to cell culture. Comparing ICR method and Method 1622, both gave similar number of recovered oocysts (220 and 208, respectively). It appears that the oocysts recovered by the ICR method were not infectious due to harsh processing procedures, which may damage the oocysts. When 500 oocysts were spiked into 10 liters of reagent water and 208 oocysts were recovered using Method 1622, infectious oocysts were detected by HCT-8 cell culture. Based on these results, it is possible to detect infectious oocysts recovered from spiked water samples using Method 1622. However, the detection sensitivity in the matrix spikes was not yet determined.
Conclusions:
We have developed a new method to detect and enumerate viable Cryptosporidium parvum using integrated cell culture-rRNA in-situ hybridization. The DNA Probes C1, C3 and C6 can be used to detect C. parvum specifically and they do not cross react with C. muris. Based on the sequence alignment results, probe C6 should recognize both genotypes I and II of C. parvum. Probes C1 and C3 are designed to detect C. parvum genotype II and I, respectively. When using different combination of these three probes in cell-culture rRNA in-situ hybridization, we should be able to not only detect and enumerate C. parvum but also distinguish genotypes of C. parvum. Moreover, the colorimetric detection of these DIG-labeled probes allows for easier observation under regular light microscope because no epifluorescent microscopy is required. However, different strains of C. parvum genotypes I and II should be studied further in Phase II Research Project.A600FL antibody and probes C-1, 3, and 6 are specifically detecting the reproductive stages of C. parvum. For protozoan challenge studies involving UV, ozone, or other disinfectants, A600FL may be adequate to use for evaluating log reduction when the test microorganisms are C. parvum oocysts. For the field samples, in situ hybridization with rRNA probe has more specificity to detect viable or infectious C. parvum. However, to apply in situ hybridization using rRNA probe to environmental samples, it is important to improve the detection sensitivity to10 to 30 oocysts in the final concentrates.
Detection of infectious oocysts in cell culture depends on several factors. The detection sensitivity in this spiking study required about 200 oocysts which is 2-7 times higher than is needed when infecting cell culture directly with oocysts (30-100 oocysts). One of the possibilities is that processing procedures will not recover all oocysts and may cause a certain degree of damage to oocysts and decrease their infectivity. The results also indicate Method 1622 is better than the ICR Method in recovering oocysts for submitting to the cell culture infectivity assay.
It has been demonstrated that we can employ DNA probes to determine if oocysts are Cryptosporidium parvum and can employ cell culture to determine their infectivity. Additionally, we have shown the feasibility of using two probes to determine the genotype of C. parvum present. These attributes are sufficient to provide data for treatment studies employing large numbers of oocysts for log-removal studies. However, additional research is needed to improve the sensitivity of the assay before it can be routinely employed by water supplies to monitor finished drinking water for infectious Cryptosporidium parvum oocysts.
Supplemental Keywords:
Cryptosporidium, parvum, Oligonucleotide, probe, HCT-8 cell, rRNA, Hybridization, In-situ., RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Wastewater, Environmental Chemistry, Monitoring/Modeling, Analytical Chemistry, Environmental Microbiology, Biochemistry, Environmental Monitoring, Drinking Water, Environmental Engineering, alternative disinfection methods, cryptosporidium parvum oocysts, monitoring, aquatic ecosystem, wastewater treatment, detection, exposure and effects, chemiluminescent detection system, wastewater remediation, exposure, in situ hybridization, cell physiology, chemiluscent detection system, alternative technology, analytical methods, cryptosporidium , treatment, microbial risk management, contaminant removal, drinking water contaminants, drinking water treatmentThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.