Grantee Research Project Results
2000 Progress 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 , Sheoran, Abhineet , Widmer, Giovanni , Buckholt, Michael , Zuckermann, Udi
Current 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 Period Covered by this Report: March 1, 2000 through March 1, 2001
Project Amount: $525,000
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
The objectives of this project are:
- Evaluation and optimization of a modified continuous flow centrifugation (CFC) method for recovery of protozoa (Cryptosporidium spp., Giardia, and Microsporidia spp.) from turbid and large volumes of water. The CFC method allows for 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.
- Development of Enterocytozoon (E.) bieneusi and Encephalitozoon (E.) intestinalis detection techniques that include production of a specific antibody against both protozoa, with a view to develop specific and sensitive methods using immunomagnetic separation (IMS). Concentration of oocysts/cysts/pores from raw or large volumes of drinking water samples requires sensitive and specific detection systems. A combined method for oocysts and cysts using the IMS already exists. We propose to develop monoclonal antibodies (MAb) and rabbit polyclonal antibodies against E. bieneusi and E. intestinalis.
- Development, optimization, and evaluation of infectivity and viability assays for Cryptosporidium spp., Giardia, and Microsporidia spp. recovered from turbid and large volumes of water. The infectivity/viability of recovered oocysts/cysts/spores is important: (1) Is water treatment effective in inactivating these pathogens? (2) Do the concentration and separation processes impact infectivity/viability? and (3) Can molecular fingerprinting of oocysts/cysts/spores help determine the source/origin of contamination?
Progress Summary:
The first phase of this project includes the optimization and standardization of a portable CFC device; and the production of antibodies for detection of parasites from concentrated water.Optimization and Standardization of CFC
The CFC was modified and built from a blood separator into a compact portable unit for concentrating Cryptosporidium (C.) parvum oocysts, Giardia cysts, and Microsporidium spores from various volumes of water (10-1,000 L) and different matrices. Flow rate ranged from 0.5-1.4 L/minute, with a maximum speed of 8,000 rpm (3,000 ? g). After centrifugation, the residual volume was 250 mL, from which oocysts and cysts were dislodged by injecting a concentrated 10 mL elution buffer. The centrifuged residue was transferred for IMS, followed by reaction with specific MAbs, and enumeration by fluorescence microscopy.
The entire procedure, including parasite enumeration, takes approximately 2 hours. For C. parvum oocysts (90 oocysts) spiked into 10 L of filtered tap water, the average recovery (n=12, flow rate = 0.5-0.75\LPM) was 90.3 percent ? 49.8 percent. For the same dose spiked into 50 L (n=11, flow rate = 0.5-1.0\LPM), recovery was 82.7 percent ? 42 percent. For Giardia cysts (95 and 1,080 cysts) spiked into 50 L (same matrix, n=12, flow rate = 0.75 and 1\LPM), the average recovery was 106.1 percent ? 24.4 percent. For a spike dose of 100 oocysts (80 ? 40 oocysts) in 1,000 L of tap water (turbidity = 0.5 NTU, n=43, flow rate = 0.75\LPM), the average recovery was 57.33 percent ? 42.85 percent. For oocysts (250) spiked in 10 L of secondary effluent (turbidity = 2.8-4.6 NTU, n=5, flow rate = 0.75\LPM), the average recovery was 56.5 percent. C. parvum presence was confirmed by immunofluorescence microscopy and by polymerase chain reaction (see Tables 1-5).
Encephalitozoon intestinalis-Specific Antibodies
Parasites. E. intestinalis and E. cuniculi were grown at 37?C on monolayers of rabbit kidney cells (RK13). Spores that were periodically extruded into the culture medium were collected from several flasks and pooled. Most of the debris and unattached RK13 cells were sedimented by low-speed centrifugation at 120 ? g for 10 minutes at 4?C and discarded. The spores in the supernatant were sedimented by relatively high-speed centrifugation at 1,200 ? g for 20 minutes at 4?C. The spore suspension in cold phosphate-buffered saline (PBS) was layered over 45 percent sterile Percoll (Sigma) in PBS and centrifuged at 1,900 ? g for 30 minutes at room temperature. Additional debris and dead RK13 cells were trapped at the PBS-Percoll interface, while spores pelleted. The spores were washed in cold PBS, quantitated with a hemacytometer, and stored at -20?C until use.
Monoclonal Antibodies. Mouse anti-E. intestinalis MAbs were obtained by immunization of BALB/c mice with E. intestinalis spores and subsequent fusion of spleen cells with Ag 8.653 myeloma cells. Hybridoma supernatants were tested by ELISA on E. intestinalis and E. cuniculi lysate-coated plates. Positive supernatants also were tested on methanol-fixed spores by indirect immunofluorescence and in parallel by immunoblotting. Positive hybridomas were cloned twice and iso-typed.
MAbs CE5 and CG9 have been obtained following screening of 1,000 wells from two fusion experiments. Both MAbs react with E. intestinalis as well as E. cuniculi by ELISA and immunoblot. CG9 identifies proteins of about 48,000 and 44,000 daltons, whereas CE5 reacts with proteins of about 70,000; 41,000; and 36,000 daltons. CG9 exhibits strong surface staining of both E. intestinalis and E. cuniculi by indirect immunofluorescence, suggesting that a spore wall antigen located on the surface is detected, whereas CE5 shows no reactivity with either spores.
Polyclonal Antibodies. Polyclonal antibodies have been raised in a rabbit by immunization with E. intestinalis. Antibodies cross-react with E. cuniculi.
Present Status. CG9 presently is being evaluated for its ability to inhibit invasion by mature spores. Both antibodies are being characterized for their cross-reactivity with other bacteria, fungi, and protozoa. Also, two more fusion experiments have been performed to obtain E. intestinalis-specific MAbs.
Purification of Enterocytozoon bieneusi and Production of Antibodies
Purification. Human feces as a source of E. bieneusi spores were obtained from HIV-infected patients. Microsporidia spores were detected by chromotrope-based staining procedure. Positive feces were vortexed in PBS (1:3) and suspension was filtered sequentially through four sieves of pore diameters 425, 280, 106, and 45 µm. Density gradient centrifugation was performed with various concentrations of Percoll (Sigma) and Nycodenz (Sigma). The step Percoll gradient consisted of 2.5 mL of stock isotonic Percoll solution prepared by mixing 10 mL of 10-fold-concentrated PBS and 90 mL of Percoll, 2.5 mL of 67.5 percent stock Percoll, 2.5 mL of 45 percent stock Percoll, and 2.5 mL of 22.5 percent stock Percoll. Three milliliters of the spore suspension were layered over the gradient into a 15-mL centrifuge tube. After centrifugation at 2,500 ? g for 30 minutes at 15?C, four distinct bands were formed. Similarly, three bands were obtained utilizing Nycodenz gradients of 25, 30, and 37 percent. These bands were collected, washed three times in PBS, pelleted at 2,500 ? g for 20 minutes, resuspended in PBS, and tested by PCR and chromotrope staining for the presence of E. bieneusi. All bands were positive by PCR. However, chromotrope staining demonstrated that 30/37 percent band of Nycodenz contained most spores with relatively less bacterial contamination. The average spore size was 0.6 m in breadth and 1.1 m in length, which agrees with the earlier reported size of E. bieneusi. To eliminate or reduce bacterial and fungal load, the 30/37 percent band of Nycodenz was mixed with an antibiotic solution of carbenicillin (10 µg/mL), vancomycin (10 µg/mL), gentamicin (10 µg/mL), cefoperazone (10 µg/mL), and amphotericin B (0.25 µg/mL), and placed at 4?C.
Present Status. Antibiotics are being added daily at the same concentration until sterilization of the preparation as determined by cultivation on blood agar and Sabouraud dextrose agar plates. The sterile concentrate following centrifugation and washing with PBS will be used to immunize rabbits and mice to produce polyclonal and MAbs, respectively. The material we have so far should be sufficient to immunize one rabbit.
Future Activities:
We will continue to optimize the recovery of Microsporidia with the CFC. Once the method of detection with flow cytometer is completed, we will invite Dr. Udi Zuckermann to spend 2 weeks optimizing and combining the method of recovery with that of C. parvum and Giardia. Dr. Sheoran will continue with his effort to produce antibodies against E. bieneusi, and Dr. Widmer has started with the development of Giardia.Journal Articles:
No journal articles submitted with this report: View all 4 publications for this projectSupplemental Keywords:
Cryptosporidium, Microsporidia, Enterocytozoon bieneusi, Encephalitozoon intestinalis, Giardia, protozoa, water., 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.