2001 Progress Report: Development and Evaluation of Procedures for Detection of Infectious Microsporidia in Source WatersEPA Grant Number: R828042
Title: Development and Evaluation of Procedures for Detection of Infectious Microsporidia in Source Waters
Investigators: Rochelle, Paul A. , Leitch, Gordon , Visvesvara, Govinda
Current Investigators: Rochelle, Paul A. , Johnson, Anne M. , Leitch, Gordon , Visvesvara, Govinda
Institution: Metropolitan Water District of Southern California , Morehouse School of Medicine
Current Institution: Metropolitan Water District of Southern California , Centers for Disease Control and Prevention , Morehouse School of Medicine
EPA Project Officer: Nolt-Helms, Cynthia
Project Period: May 1, 2000 through May 1, 2002
Project Period Covered by this Report: May 1, 2000 through May 1, 2001
Project Amount: $294,635
RFA: Drinking Water (1999) RFA Text | Recipients Lists
Research Category: Drinking Water , Water
The overall objectives of this research project are to: (1) develop methods to recover microsporidia from water; (2) determine the viability and infectivity of detected spores; and (3) use the methods to assess the occurrence of microsporidia in untreated source waters. The specific objectives of this research project are to: (1) evaluate concentration and purification approaches for recovery of microsporidial spores from environmental water samples; (2) evaluate molecular and microscopic methods for detection of Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Nosema algare; (3) optimize and compare in vitro viability and infectivity assays for microsporidia recovered from water; and (4) determine the prevalence of infectious microsporidia in natural water sources by combining the most suitable elements of specific objectives 1, 2, and 3 into a validated analytical procedure.
The microsporidia group of protozoa, particularly E . bieneusi and Encephalitozoon spp., are considered as emerging pathogens that represent serious threats to public health. Some microsporidia can lead to disseminated infection in almost every organ of the human body in immunocompromised patients; they also can infect immunocompetent individuals. Because many animals can carry microsporidia, it is possible that surface waters can be contaminated and consequently may serve as a route of transmission to humans. Very little is known, however, about the occurrence of microsporidia in environmental water sources, and there is a critical need to determine the role drinking water plays in the epidemiology of this group of parasites. Moreover, there are no routine methods for detection of microsporidia in water.
The recovery and purification methods that will be evaluated include filtration of water samples using a variety of different filter formats and porosities, immunomagnetic separation, and density gradient centrifugation. Optimized PCR assays and microscopic methods will be used for detection and identification. Viability will be assessed using a spore germination assay coupled with a nucleic acid stain, a fluorescent dye exclusion assay, and phase contrast microscopy. Infectivity will be determined by inoculating spores into cell cultures and detecting infections using molecular and microscopic methods.
This research project is expected to produce optimized and validated methods for the detection of infectious microsporidia in environmental water samples. We also will obtain information on the prevalence of microsporidia in environmental waters. This information will allow the water industry and public health officials to determine the extent of microsporidia contamination in water and hence determine whether water represents a significant route of transmission for these parasites.
Commercially available capsule filters with an absolute porosity of 1 μm were evaluated for recovery of E. intestinalis spores along with custom-made capsule filters with porosities of 0.55 μm and 0.88 μm. A suspension of microsporidia spores was seeded into 10 L of treatment plant effluent water (< 0.1 NTU) and filtered through the capsules using the protocol described in U.S. Environmental Protection Agency (EPA) Method 1623 for Cryptosporidium and Giardia. The filtrate was passed through a 0.2 μm track-etch membrane. The membrane was placed in a 50 mL centrifuge tube with 5 mL of eluting buffer and vortexed for 5 minutes to release the spores from the membrane. A 100 μL aliquot was placed on a well slide, stained with a chitin-specific stain, and enumerated by microscopy. The 0.8 μm filter captured 3-20 percent of the spores, approximately the same as the 1.0 μm standard Envirocheck capsule used for EPA Method 1623, whereas the recovery efficiencies obtained with the 0.55 μm capsule filter generally were higher (38 ± 18 %) compared to 66 percent recovery of Cryptosporidium oocysts with the 1 μm capsule filter. Initial trials with centrifugal filters consisting of a modified nylon membrane in a centrifuge tube resulted in recovery efficiencies of 52 percent, 22 percent, and 36 percent, with porosities of 0.2 μm, 0.3 μm, and 0.45 μm, respectively. Experiments with flat membrane filters demonstrated a threefold increase in spore retention by 0.1 μm porosity filters compared to 0.6 μm filters. These filtration studies indicated that the small porosity of filters necessary to recover microsporidia spores will limit the volume of untreated source water that can be analyzed for the presence of microsporidia. Alternative filtration systems that were evaluated included compressed foam disc filters and a tangential flow device. Neither of these approaches matched the performance of the capsule filters, and they were far more complicated and time consuming to operate.
A variety of procedures were evaluated for detection and enumeration of recovered microsporidia spores. Two different polyclonal antibodies directed against Encephalitozoon spp. were tested. Both antibodies demonstrated nonspecific staining with considerable background staining, particularly with environmental samples. In addition, a significant number of spores did not stain at all. Nonantibody-based stains such as a modified trichrome method and Calcofluor were found to be effective only in nonenvironmental samples. The environmental samples had unacceptable amounts of background staining. Ultimately, a staining solution containing the fluorescent dye Cellufluor was selected as the most consistent method for enumerating spike preparations. An indirect immunomagnetic purification method yielded recovery efficiencies of up to 44 percent, with acid disassociation reducing the efficiency to 34 percent.
For molecular detection methods, three commercially available extraction kits were tested to determine which generated the highest yield of PCR-compatible DNA from treatment plant effluent water and environmental water concentrates seeded with a range of spore densities. The quality of the extraction was determined by PCR using primers specific to all Encephalitozoon spp. Published amplification primers were evaluated for their sensitivity and specificity in detecting E. intestinalis, E. hellem, and E. cuniculi. E. hellem-specific primers amplified the expected fragment from E. hellem spores but not from E. intestinalis or E. cuniculi spores. The E. cuniculi primers also were found to be specific for E. cuniculi DNA only. The primers reported to be specific for E. intestinalis, however, amplified DNA from all three species. Although it appears that these primers were not specific for E. intestinalis, the possibility that the initial samples of E. hellem and E. cuniculi spores were contaminated with E. intestinalis spores could not be precluded. To determine if the primers were amplifying E. intestinalis DNA in the E. hellem and E. cuniculi samples, PCR amplicons from all three species were sequenced and the sequences compared to database sequences. The sequences showed the highest homology with the E. intestinalis sequence, implying that the E. hellem and E. cuniculi samples were contaminated with E. intestinalis spores. This has been a consistent problem with spore preparations and indicates the need for rigorous quality control in spore propagation procedures.
A cell culture-based infectivity assay also was developed for microsporidia spores. A variety of cell lines (MDCK, MA104, HCT-8, Caco-2, RK13) were evaluated for their ability to support infection with E. intestinalis spores. Although infection was observed in some of these cell lines, particularly RK13 and MDCK cells, the level of infection was low, with little spore propagation in all cells except RK13. Infection in RK13 cells was rapid and led to effective spore propagation. A confluent monolayer of RK13 cells in a 75 cm2 flask produced more than 1 x 108 spores within 1 week of inoculation with a low dose of E. intestinalis spores. The final infectivity assay involved inoculating spores onto monolayers of RK13 cells in 48-well culture plates, incubating for 72 hours at 35°C, and detecting infection by RT-PCR following RNA extraction. The 50 percent infectious dose for this assay was 45 spores for E. intestinalis.
We will actively seek alternative antibodies that will be used for further evaluations of immunomagnetic purification and spore staining. In addition, a wider range of PCR primers will be screened for improved specificity. The most appropriate primers will be used to develop a quantitative PCR detection assay. The cell culture-based infectivity assay will be used to assess the efficacy of disinfectants such as ultraviolet light, ozone, and chlorine dioxide for inactivation of microsporidia spores.
The progress of this research project has demonstrated the limitations of developing recovery and detection methods for novel microorganisms based on adapting existing techniques. It is apparent that the biological understanding of microsporidia is still evolving, and more information is needed on the morphology of different spores types, nucleic acid sequences, and antigenicity of spores. An efficient recovery procedure will need effective antibodies with high avidity and specificity that can be used for both immunomagnetic separation purification and immunofluorescent detection of spores.