Grantee Research Project Results
Final Report: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water
EPA Grant Number: R828043Title: Development and Evaluation of Methods for the Concentration, Separation, Detection, and Viability/Infectivity of Three Protozoa from Large Volume of Water
Investigators: Tzipori, Saul , Widmer, Giovanni , Buckholt, Michael , Zuckermann, Udi
Institution: Tufts University
EPA Project Officer: Hahn, Intaek
Project Period: March 1, 2000 through March 1, 2003
Project Amount: $525,000
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The overall objective of this research project was to develop, optimize, and evaluate a portable continuous flow centrifuge (PCFC) for concentration of Cryptosporidium, Giardia, and microsporidia from large volumes of water (up to 1,000 L). Special funding for Year 4 of the project by the Science To Achieve Results (STAR) program allowed the validation of the PCFC method by independent water testing laboratories, as required by the U.S. Environmental Protection Agency (EPA) Office of Water, to qualify as Method 1623.
The specific objectives of this research project were to:
- evaluate and optimize a modified continuous flow centrifugation (CFC) method for recovery of protozoa (Cryptosporidium spp., Giardia , and Microsporidia spp.) from turbid and large volumes of water;
- develop Encephalitozoon bieneusi and E. intestinalis detection techniques;
- and develop, optimize, and evaluate infectivity and viability assays for Cryptosporidium spp., Giardia, and microsporidia spp. recovered from turbid and large volumes of water.
Summary/Accomplishments (Outputs/Outcomes):
Objective 1: Evaluation and Optimization of a Modified CFC method for Recovery of Protozoa From Turbid and Large Volumes of Water
The CFC method allows for the concentration of oocysts, cysts, and spores from large volumes of water, and for continuous monitoring of their presence in water, as opposed to one-time sampling of existing methods. This method is efficient, portable, rapid, and easy to operate.
Data Analysis and Discussion. This work was performed in collaboration with four independent, EPA-approved water testing laboratories. Study results demonstrate that PCFC meets and exceeds the acceptance criteria for Method 1623 and is capable of concentrating Cryptosporidium oocysts from 50 L of source water. Because laboratory 1 failed to get acceptable recoveries for Giardia spiked into turbid source water, the research project could not demonstrate that each laboratory in the project could achieve acceptable recoveries in the matrices tested for each organism. The mean recovery in laboratory #1 for Giardia in source water was less than 1 percent under the minimum acceptable value of 15 percent. One explanation for this result could be the interaction of the cysts with the material suspended within the 50 L of greater than 10 nephelometric turbidity units (NTU) source matrix during the process, which could lead to their degradation. Previous testing of the PCFC with greater waters having greater turbidities demonstrated much higher recoveries.
Laboratory #4 failed to get acceptable results for Initial Precision and Recovery 1 (IPR1), Matrix Spike (MS) 1, and MS 2. During training, this laboratory demonstrated the ability to perform acceptable results when using the PCFC. The cause for the poor results during the validation was not identified . A potentially significant confounding variable, however, was identified. During the training, the laboratory performed the wrist shaking by using its own old model wrist shaker. Because this model did not enable setting the desired rpm and orientation of the clamps, they were supplied with a current Tuft’s wrist shaker model. Because of the expiration date of the spike samples, the laboratory did not have time to practice with the new wrist shaker. These factors suggest that the likely explanation for the low recoveries of the source water samples was insufficient elution of the oocysts/cysts from the bowl.
Objective 2: Development of E. bieneusi and E. intestinalis Detection Techniques
Concentration of oocysts/cysts/spores from raw or large volume drinking water samples requires sensitive and specific detection systems. A combined method for oocysts and cysts using the immunomagnetic separation already exists. We proposed to develop monoclonal antibodies (mAbs) and rabbit polyclonal antibodies against E. bieneusi and E. intestinalis. During the 3-year funding period, we successfully generated immune reagents against two microsporidia, E. intestinalis, and the clinically more significant E. bieneusi.
E. Intestinalis Spores . E. intestinalis, E. cuniculi, and E. hellem were grown at 37°C on monolayers of rabbit kidney cells (RK13). Spores that were extruded periodically into the culture medium were collected from several flasks and purified.
E. Bieneusi Spores . A combination of isopycnic Percoll gradient and continuous sucrose gradient (40% w/w - 60% w/w) centrifugations were performed to purify E. bieneusi spores from feces (obtained from Uganda). Stock of 100 percent isotonic Percoll was made by mixing 90 ml of Percoll (Sigma) with 10 ml of 10 ´ phosphate buffered saline (PBS). One ml fecal suspension containing E. bieneusi as determined by PCR was mixed with 9 ml of 80 percent Percoll (8 parts 100% stock Percoll mixed with 2 parts sterilized distilled water) and centrifuged in Beckman’s L-70 ultracentrifuge using SW 41 Ti rotor at 11,000 rpm for 1 hour at 10 °C. The sample separated into 2 two bands and a pellet. Band one was at the top of the tube,. and Band two (bottom band) was just above the pellet at the bottom of the tube. The bands and the pellet were analyzed for the presence of spores by Calcofluor white staining. The bands, but not the pellet, contained spores. The two bands were further subjected to continuous sucrose gradient centrifugation.
Nine ml of continuous sucrose gradient (40% w/w - 60% w/w) were prepared in ultracentrifuge tubes of SW41 Ti rotor. Each tube was overlaid with 1.5 ml of either the top band or the bottom band of Percoll purified material. Tubes were again centrifuged in Beckman’s ultracentrifuge using SW 41 Ti rotor at 25,000 rpm for 24 hours at 10 °C. The top band of the Percoll did not separate into clear bands. The bottom band of Percoll, however, separated into three bands (top, middle, and bottom) and a pellet. The middle band contained more than 98 percent E. bieneusi spores (see Figure 1) and less than 2 percent of other microorganisms and debris. The yield of purified spores after a combination of Percoll and sucrose gradient was 9.1 percent of the starting material.
Figure 1. Transmission Electron Micrographs of Purified E. bieneusi Spores Obtained After Isopycnic Percoll and Sucrose Gradient Centrifugations. Purified spores (arrowhead), as well as a few contaminants (arrow) are shown (Bar = 2 µ).
Production of Monoclonal and Polyclonal Antibodies. Rabbit polyclonal antibodies and mouse mAbs, which react with spore walls of E. bieneusi and E. intestinalis , have been produced. The polyclonal antibodies against E. bieneusi reacted only with E. bieneusi by immunofluorescence (IF). Conversely, rabbit polyclonal serum against E. intestinalis was cross-reactive with E. hellem and E. cuniculi, but not with E. bieneusi . These findings show the lack of antigenic cross-reaction between spore wall antigens of E. bieneusi and other Encephalitozoon spp.
Ten mAbs (5 IgG2a isotype, 1 IgG2b, 2 IgG1, and 2 IgM) have been produced against E. intestinalis. Six mAbs (CG9, 5D2, C2B5, C2G11, 17C12, and LB5) are against spore wall antigens and detect E. intestinalis spores by IF. Figure 2 shows reactivity of 17C12 with the spore wall antigens. Four (CG9, 5D2, C2B5, and C2G11) of these mAbs also detect E. cuniculi and E. hellem. Two mAbs (17C12 and LB5) identify only E. intestinalis spores. The rest of the mAbs react in ELISA but not in IF. The mAbs C2G9 and 17C12 (both mouse IgG2a isotypes), selected for evaluation as diagnostic reagents, identify all Encephalitozoon spp. and only E. intestinalis, respectively, in feces. Both antibodies did not recognize any bacteria, fungi, or any other protozoa in the feces.
Figure 2. Reactivity of the mAb 17C12 With Spore Wall by Immunogold Electron Microscopy (A, bar = 200 nm) and Confocal IF (B)
All nine mouse mAbs (8 IgM isotype and 1 IgG2a isotype) react with the spore wall antigens. The mAbs 2G4 (IgM isotype, Figure 3) and 1B7 (IgG2a isotype), selected for evaluation as diagnostic reagents, identify only E. bieneusi in feces.
Figure 3. Specific IF Detection of E. bieneusi Spores by Mouse Anti-E. bieneusi mAb 2G4 in Fecal Smear
Immunomagnetic Separation. Several of these antibodies have been evaluated as reagents for immunomagnetic separation. We have not been very successful in purifying spores by immunomagnetic separation. These findings are in agreement with other studies (Accoceberry, et al., 2001), which have reported purification of only about 1 percent of the starting material by immunomagnetic separation.
Objective 3: Development, Optimization, and Evaluation of Infectivity and Viability Assays for Protozoa Recovered From Turbid and Large Volumes of Water
The infectivity/viability of recovered oocysts/cysts/spores is important for answering the following questions:
- Is water treatment effective in inactivating these pathogens?
- Do the concentration and separation processes impact infectivity/viability?
- Can molecular fingerprinting of oocysts/cysts/spores help determine the source/origin of contamination?
Conventional methods for detecting waterborne oocysts of Cryptosporidium and cysts of Giardia do not discriminate between infectious and dead oocysts and cysts. As a consequence of this limitation, the public health risk of oocysts and cysts detected during routine water monitoring cannot be assessed. For Cryptosporidium , the two approaches for assessing the infectivity of oocysts are based on the ability of infectious oocysts to infect cultured cells or laboratory animals. The advantages and drawbacks of the various methods have been previously discussed (Bukhari, et al., 2000; Rochelle, et al., 2002), but these methods require resources that are not available to typical water monitoring facilities and also have long turn-around periods.
In an attempt to develop a molecular viability assay capable of discriminating between infectious and dead oocysts, we studied the possibility of using C. parvum RNA transcripts as markers of infectivity. The rationale for this approach is that most mRNA transcripts decay upon cell death and are rapidly degraded. Initially, the post-mortem decay of C. parvum β-tubulin mRNA, ribosomal RNA, and a third anonymous transcript were investigated using reverse-transcription (RT) PCR (Widmer, et al., 1999). Although the β-tubulin transcript was found to accurately predict oocyst infectivity in mice because of a fast rate of post-mortem decay, it also was found that the low abundance of this transcript made its use as a viability marker difficult. This was particularly a concern when working with oocysts recovered from raw water samples, which are typically present at low concentrations. We therefore explored the feasibility of using RNA transcribed from a double-stranded viral RNA genome present in C. parvum and C. hominis(Khramtsov, et al., 1997). The rationale for this approach was two fold. (1) Because multiple copies of the viral genome are present in each sporozoite, oocysts are expected to contain many more copies of viral transcripts then genomic transcripts such as b-tubulin mRNA. (2) Because the template for the viral mRNA is an RNA genome, no DNA copies of the viral genome are present in the cell. Consequently, no homologous DNA, which can serve as template during PCR, is available, eliminating the possibility of false positive results.
Experimental Approach. We amplified a 173 bp fragment from the short viral genomic segment (S-dsRNA) and examined whether the viral transcript would be a suitable marker of oocyst infectivity. To this aim, purified oocysts were heat-inactivated or inactivated by three cycles of freeze-thawing. Inactivated oocysts then were incubated at room temperature for 0-96 hours to assess the rate of post-mortem degradation of the viral RNA. Experiments with heat-inactivated oocysts, however, demonstrated that this fragment of viral RNA was too stable to be used as a marker of viability. Surprisingly, the lack of apparent decay also was observed in oocysts, which had lost infectivity following storage for 17 and 19 months at 4ºC. We assumed that the double-stranded structure of the viral genomic RNA is resistant to degradation and interfered with the assay.
Our next approach was to increase the size of the dsRNA amplification fragment, assuming post-mortem decay would be easier to detect when amplifying larger fragments. This assumes a larger probability of nucleolytic degradation of long RNA fragments. We also targeted the 5' end of the S-dsRNA to determine whether the location of the amplicon on the dsRNA transcript might affect the rate of decay. In these experiments, it became obvious that PCR amplification efficiency declined with increasing amplicon size. We tried to address this problem by moving the RT primer further downstream and using closely spaced PCR primers, but were not successful in improving amplification efficiency.
Conclusions:
Conclusions. Experiments correlating the presence of intact β-tubulin transcripts with mouse infectivity demonstrated the validity of using specific mRNA as markers of oocyst infectivity. Because of the limitations of the method, there is a need for transcripts that are expressed in oocysts at a level high enough to enable their detection by RT PCR and contain an intron to make the discrimination between amplicons originating from cDNA and genomic DNA unambiguous. In light of the small proportion of genes in C. parvum and C. hominis , which contain introns (Abrahamsen, et al., 2004; Xu, et al., 2004), more work is needed to identify mRNA transcripts meeting these criteria. The fact that oocysts are non-dividing stages of the parasite and presumably have only limited metabolic activity, suggests that many genes may not be transcribed, limiting the choice of potential infectivity markers.
Summary and Conclusions
We have demonstrated that the PCFC method, after extensive optimization under laboratory conditions, was shown to be superior to the currently available concentration techniques, which are based on filtration. As compared with filtration, the PCFC method is easier to use, less expensive, portable, faster, and has the potential to sample large volumes (at least 1,000 L), which makes it ideal for continuous monitoring of a water supply. We also were able to show that with further manipulation, the PCFC method is able to concentrate all three targeted enteric protozoa simultaneously including Cryptosporidium, Giardia, and microsporidia.
The PCFC method was subsequently validated by independent water testing laboratories, under the direction of the EPA Office of Water, and was approved as Method 1623. This made it possible to commercialize the method. In addition, we were able to develop a method to concentrate E. bieneusi, the clinically most significant of the Microsporidia (see Figure 1). This allowed us to generate sufficient purified spores to produce polyclonal and monoclonal antibodies against E. bieneusi and E. intestinalis. This should facilitate the development of immune-based sensitive detection methods similar to those available for Giardia and Cryptosporidium.
References:
Accoceberry I, Thellier M, Datry A, sportes-Livage I, Biligui S, Danis M, Santarelli X. One-step purification of Enterocytozoon bieneusi spores from human stools by immunoaffinity expanded-bed adsorption. Journal of Clinical Microbiology 2001;39(5):1947-1951.
Abrahamsen MS, Templeton TJ, Enomoto S, Abrahante JE, Zhu G, Lancto CA, Deng MQ, Liu C, Widmer G, Tzipori S, Buck GA, Xu P, Bankier AT, Dear PH, Konfortov BA, Spriggs HF, Iyer L, Anantharaman V, Aravind L, Kapur V. Complete genome sequence of the apicomplexan, Cryptosporidium parvum. Science 2004;304(5669):441-445.
Bukhari Z, Marshall MM, Korich DG, Fricker CR, Smith HV, Rosen J, Clancy JL. Comparison of Cryptosporidium parvum viability and infectivity assays following ozone treatment of oocysts. Applied and Environmental Microbiology 2000;66(7):2972-2980.
Khramtsov NV, Woods KM, Nesterenko MV, Dykstra CC, Upton SJ. Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum. Molecular Microbiology 1997;26(2):289-300.
Rochelle PA, Marshall MM, Mead JR, Johnson AM, Korich DG, Rosen JS, De Leon R. Comparison of in vitro cell culture and a mouse assay for measuring infectivity of Cryptosporidium parvum. Applied and Environmental Microbiology 2002;68(8):3809-3817.
Widmer G, Orbacz EA, Tzipori S. Beta-tubulin mRNA as a marker of Cryptosporidium parvum oocyst viability. Applied and Environmental Microbiology 1999;65(4):1584-1588.
Xu P, Widmer G, Wang YP, Ozaki LS, Alves JM, Serrano MG, Puiu D, Manque P, Akiyoshi D, Mackey AJ, Pearson WR, Dear PH, Bankier AT, Peterson DL, Abrahamsen MS, Kapur V, Tzipori S, Buck GA. The genome of Cryptosporidium hominis. Nature 2004;431(7012):1107-1112.
Journal Articles on this Report : 3 Displayed | Download in RIS Format
Other project views: | All 4 publications | 3 publications in selected types | All 3 journal articles |
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Sheoran AS, Feng X, Singh I, Chapman-Bonofiglio S, Kitaka S, Hanawalt J, Nunnari J, Mansfield K, Tumwine JK, Tzipori S. Monoclonal antibodies against Enterocytozoon bieneusi of human origin. Clinical and Diagnostic Laboratory Immunology 2005;12(9):1109-1113. |
R828043 (Final) |
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Zhang Q, Singh I, Sheoran A, Feng X, Nunnari J, Carville A, Tzipori S. Production and characterization of monoclonal antibodies against Enterocytozoon bieneusi purified from Rhesus Macaques. Infection and Immunity 2005;73(8):5166-5172. |
R828043 (Final) |
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Zuckerman U, Tzipori S. Portable continuous flow centrifugation and method 1623 for monitoring of waterborne protozoa from large volumes of various water matrices. Journal of Applied Microbiology 2006;100(6):1220-1227. |
R828043 (Final) |
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Supplemental Keywords:
Cryptosporidium, Microsporidia, Enterocytozoon bieneusi, Encephalitozoon intestinalis, Giardia, protozoa, water, ecosystem protection/environmental exposure and risk, health, physical aspects, drinking water, health risk assessment, monitoring/modeling, physical processes, risk assessments, analytical methods, assays, assessment technology, bacteria, concentration device, detection, exposure, exposure and effects, human health risk, infective dose, infectivity, infectivity assays, measurement, microbial contamination, microbial monitoring, microbial risk management, microbiological organisms, microorganism, monitoring, pathogenic protozoa, public water systems,, RFA, Health, PHYSICAL ASPECTS, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, Health Risk Assessment, Risk Assessments, Monitoring/Modeling, Physical Processes, Drinking Water, public water systems, microbial contamination, enterocytozoon , concentration device, microbial monitoring, monitoring, measurement , detection, waterborne disease, bacteria, microbiological organisms, encephalitozoon, assays, infective dose, exposure and effects, exposure, infectivity assays, cryptosporidium , analytical methods, microbial risk management, measurement, microorganism, pathogenic protozoa, infectivity, Giardia, microsporidia, assessment technology, cryptosporidiumProgress and Final Reports:
Original AbstractThe 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.