Grantee Research Project Results
2002 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 , 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, 2002 through March 1, 2003
Project Amount: $525,000
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
Objective:
One objective of this research project is to evaluate and optimize a modified continuous flow centrifugation (CFC) method for recovery of protozoa (Cryptosporidium spp., Giardia lamblia, 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. The second objective of this research project is to develop Enterocytozoon bieneusi and Encephalitozoon intestinalis detection techniques, which include the production of specific antibodies against both protozoa with a view to develop specific and sensitive methods using immunomagnetic separation (IMS). After the concentration of oocysts/cysts/spores from raw or large volumes of drinking water, samples require sensitive and specific detection systems. A combined method for oocysts and cysts using the IMS already exists. We will develop monoclonal antibodies (mAb) and rabbit polyclonal antibodies against E. bieneusi and against E. intestinalis, and develop, optimize, and evaluate infectivity and viability assays for Cryptosporidium spp, G. lamblia, and Microsporidia spp. recovered from turbid and large volumes of water. The infectivity/viability of recovered oocysts/cysts/spores is important, and it raises three questions: (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 third phase of this research project included further optimization of the CFC for recovery of Microsporidia spp. from 10-50 L of water, simultaneous recovery of all three protozoa (C. parvum, G. lamblia, and Microsporidium) from volumes of 10, 50, and 1,000 L of water and viability testing after recovery to ascertain that the CFC method does not cause parasite inactivation. We have completed the characterization of a panel of monoclonal antibodies (mAbs) against E. intestinalis, and currently are in the midst of deriving mAbs against E. bieneusi and further optimization of RNA-based viability assays.
Optimization and Standardization of CFC. We have performed further studies to optimize the CFC method for C. parvum; the C. parvum isolate was obtained from Tufts University Veterinary School and was originally isolated from a symptomatic human. This isolate is referred to as the GCH1 isolate, which is maintained by passage through calves and is purified by saturated sodium chloride flotation followed by Percoll and 30 percent Nycodenz/PBS flotation. The age of this isolate at the time of these recovery trials was approximately 3 months.
The second C. parvum isolate was obtained from the Sterling Parasitology Laboratory, Tucson, AZ, and is known as the Iowa isolate. The oocysts were isolated from the feces by discontinuous sucrose gradients followed by microcentrifuge-scale cesium chloride gradients. The age of this isolate at the time of these recovery trials was approximately 2 months. All purified oocysts were stored at 4°C in 0.01 percent Tween 20 solution containing 100 U of penicillin, 100 µg of streptomycin, and 100 µg of gentamicin per mL of oocyst.
Enumeration of Oocyst Spike Dose. For each stock of oocysts, the spike dose was initially enumerated with a hemacytometer and then diluted accordingly to yield approximately 100-200 oocysts per 100 µL volumes. Each diluted working suspension was enumerated by using the U.S. Environmental Protection Agency Method 1623, utilizing enumeration of 10 replicate wells by immunofluorescence microscopy. The spiking procedure for 10 L samples of various matrices involved introducing the oocysts into a 10-L carboy, and the flow rate of all the presented data was 0.7 L/minute. The logic of the specified flow rate was to enable continuous concentration of 1,000 L in 24 hours.
Elution and Detection of Oocysts. After the desired sample volumes had passed through the PCFC, flow through the inlet port was terminated; however, the bowl was allowed to operate for a short interval to ensure that the residual sample volume within the CFC bowl had been reduced to below 250 mL. Concentrated elution buffer (5 mL) was injected via the inlet port of the PCFC bowl to yield a final elution buffer concentration of 1 percent sodium dodecyl sulphate with 0.01 percent Tween 80 and 0.001 percent antifoam A. The bowl was clamped in an upright position in a wrist shaker (the same type for the Envirochek) and shaken vigorously for 10 minutes. The bowl orientation was changed to 90° and shaking was continued for 10 minutes. Finally, the bowl orientation was changed to 270° and further shaking for 10 minutes was employed. Each bowl was inverted, a hole was drilled into the base (we have two prototypes, the second enables decantation through the inlet port), and the eluant in the bowl was discharged into a 250-mL centrifuge tube (the residual + the buffer total volume always is approximately 250 mL). All 250-mL centrifuge tubes were subjected to centrifugation (1,050 xg; 10 minutes), the supernatant was aspirated to 10 mL, and the entire concentrate was transferred into an individual Leighton tube for immunomagnetic separation. IMS and staining with FITS–mAb was performed as described in Method 1623 (see Table 1).
Table 1. Percentage Oocyst Recoveries From 10-L Spiked Samples of Various Matrices Using Overall PCFC Procedure at a Flow Rate of 0.7-L/minute and 8,000 rpm
Trial 1, N = 2 |
Trial 2, N = 2 |
|
DI Water | 29.4 |
35.3 |
St. Albans Tap Water | 24.5 |
10.5 |
Source Water | 46.9 |
46.4 |
The following data was generated at Tufts University, School of Veterinary Medicine, the Division of Infectious Disease, North Grafton, MA, during 2001-2002. Samples were spiked directly into the carboy contents and processed using the PCFC + Method 1623, from Section 13 onward (using the Dynal IMS and Waterborne staining kit) (see Tables 2-9).
Table 2. Recovery Efficiency of Cryptosporidium Oocysts From 10 L of Filtered Tap Water (Turbidity = 0.5 NTU) at 3,000 xg and Different Flow Rates
Flow (L/min) |
Oocysts Spiked +/- 35% |
Oocysts Recovered |
Recovery (%) |
Average and S.D. (%) |
0.5 |
50 |
69 |
138 |
|
0.5 |
50 |
35 |
70 |
|
0.5 |
50 |
70 |
140 |
|
0.5 |
50 |
38 |
76 |
106 +/- 38.2 |
0.75 |
155 |
192 |
124 |
|
0.75 |
155 |
287 |
185 |
|
0.75 |
155 |
133 |
86 |
|
0.75 |
155 |
100 |
64.5 |
|
0.75 |
57 |
62 |
108.8 |
|
0.75 |
57 |
20 |
35.1 |
|
0.75 |
57 |
15 |
26.3 |
|
0.75 |
57 |
17 |
30 |
82.5 +/- 55.3 |
Table 3. Recovery Efficiency of Cryptosporidium Oocysts From 50 L of Filtered Tap Water (Lab Cartridge Filtration Unit, Pores Size 1 Micron), (Turbidity = 0.5 NTU) at 3,000 xg and Different Flow Rates
Flow (L/min) |
Oocysts Spiked +/- 35% |
Oocysts Recovered |
Recovery (%) |
Average and S.D. (%) |
0.5 |
90 |
21 |
23.3 |
|
0.5 |
90 |
77 |
85.6 |
|
0.5 |
90 |
62 |
68.9 |
|
0.5 |
90 |
102 |
113.3 |
72.8 +/- 37.7 |
0.75 |
90 |
134 |
148.9 |
|
0.75 |
90 |
76 |
84.4 |
|
0.75 |
90 |
137 |
152.2 |
128.5 +/- 38.2 |
1.0 |
90 |
30 |
33.3 |
|
1.0 |
90 |
77 |
85.5 |
|
1.0 |
90 |
50 |
55.5 |
|
1.0 |
90 |
53 |
58.9 |
58.3 +/- 21.4 |
Table 4. Recovery Efficiency of G. lamblia Cysts From 50 L of Filtered Tap Water (Lab Cartridge Filtration Unit, Pores Size 1 Micron), (Turbidity = 0.5 NTU) at 3,000 xg and Different Flow Rates.
Flow (L/min) |
Oocysts Spiked +/- 35% |
Oocysts Recovered |
Recovery (%) |
Average and S.D. (%) |
0.75 |
95 |
122 |
128.4 |
|
0.75 |
95 |
110 |
115.8 |
|
0.75 |
95 |
105 |
110.5 |
|
0.75 |
95 |
127 |
133.7 |
|
0.75 |
1,080 |
870 |
80.5 |
|
0.75 |
1,080 |
915 |
84.7 |
|
0.75 |
1,080 |
1,238 |
114.6 |
|
0.75 |
1,080 |
1,401 |
129.7 |
112.2 +/- 20 |
1.0 |
1,080 |
1,500 |
138.9 |
|
1.0 |
1,080 |
945 |
87.5 |
|
1.0 |
1,080 |
863 |
79.9 |
|
1.0 |
1,080 |
751 |
69.5 |
93.95 +/- 30.9 |
Table 5. Recovery Efficiency of C. parvum Oocysts (250 Oocysts, S.D. +/-15%) Spiked into 10 L of Secondary Effluent at a Centrifugation Force of 3,000 xg and Flow Rate of 0.75 L/minute
Turbidity (NTU) |
Spiking Procedure |
Oocysts Recovered |
Recovery % |
Total Recovery +/- S.D.% |
4.6 |
Standard |
117 |
46.8 |
|
2.8 |
“ |
170 |
68 |
|
“ |
101 |
40.4 |
||
Rinse PBS\T20 |
151 |
60.4 |
||
Rinse PBS\T20 |
167 |
66.8 |
56.48 +/- 12.3 |
Table 6. Recovery Efficiency of C. parvum Oocysts From 1,000 L of Filtered (Lab Cartridge Filtration Unit, Pores Size 1 Micron) Tap Water (Turbidity = 0.5 NTU) at 3,000 xg and Flow Rate of 0.75 L/minute
Recovery % |
Total Oocysts Recovered |
Total Oocysts Spiked |
N= |
Oocysts Spiked/Experiment S.D. = +/- 32% |
54 |
26 |
48 |
2 |
24 |
15.4 |
4 |
26 |
1 |
26 |
65 |
229 |
352 |
8 |
44 |
127.7 |
69 |
54 |
1 |
54 |
19 |
22 |
116 |
2 |
58 |
20 |
12 |
60 |
1 |
60 |
72 |
381 |
528 |
8 |
66 |
63.5 |
559 |
880 |
11 |
80 |
125.5 |
266 |
212 |
2 |
106 |
41.25 |
99 |
240 |
2 |
120 |
13.6 |
35 |
258 |
2 |
129 |
14.5 |
55 |
380 |
2 |
190 |
11.3 |
22 |
194 |
1 |
194 |
Table 7. Recoveries From 20 E. intestinalis Spores S.D. +/- 45 Percent Spiked in 10 L of Tap Water, Centrifuged at 7,800 rpm, at a Flow Rate of 0.75 L/minute. Average recovery = 65 percent +/-34.8.
# Spores Recovered |
% Recovery |
12 |
60 |
5 |
25 |
22 |
110 |
Table 8. Recoveries for the PCFC When Spiking Three Parasites (Giardia, Cryptosporidium, E. intestinalis), in 10 L of Tap Water
Trial # | Crypto Spiked S.D. +/-35% | Crypto Recover % | Giardia Spiked | Giardia Recover % | Micro Spiked | Micro Recover % |
1 | 200 | 40 | 200 | 115 | 225 | 35.5 |
2 | 200 | 64 | 200 | 118 | 225 | 82.8 |
3 | 120 | 45 | 240 | 94 | 220 | 44.5 |
4 | 120 | 31.6 | 240 | 80 | 220 | 3.6 |
5 | 240 | 30.8 | 240 | 71.6 | 500 | 81.6 |
6 | 240 | 93.3 | 240 | 96.6 | 500 | 80.6 |
7 | 240 | 26.6 | 240 | 65 | 500 | 94.4 |
8 | 240 | 110.8 | 240 | 59.2 | 500 | 87 |
9 | 170 | 42.3 | 115 | 111.3 | 112 | 114.3 |
10 | 170 | 47 | 115 | 112.2 | 112 | 57.1 |
11 | 170 | 72.3 | 115 | 68.7 | 112 | 78.6 |
Average % ± S.D. | 61.95 ± 32 | 83.9 ± 34.8 | 71.6 ± 42.5 | |||
For trials 1-4, E. intestinalis were formalin fixed (purchased from waterborne); for trials 5-11, spores were viable (Tufts stock). |
Table 9. Percent Recoveries by PCFC When 50 L of Tap Water are Spiked With Three Parasites (Giardia, C. parvum, E. intestinalis)
Trial # |
Crypto Spiked |
% Recovery |
Giardia Spiked |
% Recovery |
Micro Spiked |
Recovery % |
1 | 1,500 |
75 |
2,500 |
37.6 |
500 |
78.4 |
2 | 1,500 |
71 |
2,500 |
82.6 |
500 |
82.4 |
3 | 500 |
84 |
2,500 |
58.2 |
500 |
54 |
4 | 500 |
37.4 |
2,500 |
10.6 |
500 |
N.A. |
Average % ± S.D. | 57.5 ± 38.7 |
44.6 ± 35 |
53.6 ± 46.5 |
The recoveries for C. parvum-, G. Lamblia-, and E. intestinalis-spiked various volumes of water and turbidity were impressive. The recoveries of all three pathogens simultaneously were particularly impressive after numerous repeated spiking experiments; we are pleased with the reproducibility and the consistency of this system, which is now ready for validation. Additional data compilation and statistical analysis will be needed to compare the results to the current methods.
Development of Monoclonal Antibodies for Detection of Microsporidia
Immunomagnetic Separation. The mAb 17C12, specific for E. intestinalis, and the mAb CG9, crossreactive with other encephalitozoons, were selected for coating magnetic beads. We described the generation of these and other mAbs in last year's progress report. Dynabeads M-450 tosylactivated (DYNAL) superparamagnetic beads were used for coating. The mAb coating was performed according to the manufacturer's instructions with Protein A purified 17C12 and CG9. We attempted three times, however, and were unsuccessful in purifying E. intestinalis with the mAb-coated beads. We also utilized Dynabeads precoated with goat anti-mouse IgG to capture our mAbs first, and then used them to purify E. intestinalis. The final attempts were made to coat protein A purified 17C12 and CG9 with Spherotech beads (Spherotech). Thus far, we have been unsuccessful in consistently purifying E. intestinalis by immunomagnetic separation. Further fine-tuning will be required. We are considering having them purified commercially. This will be performed once we have monoclonal antibodies against E. bieneusi, which are more medically important (see below).
Derivation of Monoclonal Antibodies Against E. Bieneusi. E. Bieneusi remains the most important waterborne pathogen associated with chronic diarrhea and wasting, particularly in people with immunodeficiencies such as HIV/AIDS. We have failed earlier to generate antibodies using HIV-infected macaques challenged with E. bieneusi to generate sufficient spores for immunization. Recently, we have been able to collect sufficient E. bieneusi spores from HIV patients from Uganda, under another award from the National Institutes of Health. These spores were purified, concentrated, and used to immunize mice for hybridoma production. The mice produced an antibody to the spores, and the first fusion was performed recently. We now are confident that we will have fully characterized E. bieneusi monoclonal antibodies within 6 months.
Optimization of the RNA-Based Viability Assay
Rationale. Efforts to develop a marker for C. parvum oocyst infectivity based on the viral double-stranded RNA present in C. parvum type 1 and type 2 [1] were continued. The advantage of this marker over the previously used b-tubulin mRNA [2] is its higher abundance and the absence of homologous sequence in the parasite genome. In the previous report, we described experiments to monitor the postmortem decay of this dsRNA using a reverse transcription-polymerase chain reaction (RT-PCR) assay targeted at a 173 basepair fragment of the short (S) dsRNA genomic segment. These experiments showed that this transcript persists in inactivated oocysts and this assay was therefore unsuitable to determine oocyst infectivity.
Results. Our next approach was to increase the size of the dsRNA amplification fragment, assuming that postmortem decay would be easier to detect when amplifying larger fragments. This assumption is based on the larger probability of nucleolytic degradation of long RNA fragments. Second, we targeted the very 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 all of these experiments, it became obvious that PCR amplification efficiency declined with increasing amplicon size (see Figure 1). We tried to address this problem by moving the RT primer farther downstream and using closely spaced PCR primers, but were not successful in improving the amplification efficiency.
Figure 1. Amplification of Two Fragments of the Viral S dsRNA Transcript. RT-PCR amplification using the benchmark assay with LV1/LV4 primers (173 bp) was better than with a second primer set targeting a 290 bp amplicon located at the 5' end of the viral genome. Lane M, 100 bp markers; lanes 3-9, 3 C. parvum type 2 isolates; lane 10, no RT negative control.
Experiments to exploit the viral dsRNA genome as a marker of oocyst infectivity were not successful. Although small PCR amplicon readily detect the S dsRNA, this approach failed to detect post-mortem decay of the RNA. Changes in the PCR assay that aim to amplify larger fragments were hampered by a low efficiency of amplification. We speculate that this problem is due to the double-stranded nature of this transcript, as this structure may inhibit reverse transcription of long distances. The double-stranded structure also may explain the post-mortem persistence of the RNA.
References:
Khramtsov NV, Woods KM, Nesterenko MV, Dykstra CC, Upton SJ. Virus-like, double-stranded RNAs in the parasitic protozoan Cryptosporidium parvum. Molecular Microbiology 1997;26:289-300.
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.
Future Activities:
We have received a 12-month funded extension for this project. Although the laboratory-based studies for Year 3 of the project have been completed and are included in this report, the Final Report, as instructed, will be after Year 4 of the project at the completion of the extension. The extension was requested, approved, and funded, and it will validate the CFC method under U.S. Environmental Protection Agency guidelines with a view to make it available for general use. We will conduct and complete the validation of the CFC parasite concentration from large volumes of water.
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.