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A NOVEL DETECTION METHOD FOR ENCEPHALITOZOON HELLEM IN WATER
Citation:
Hester, J. D., H.D A. Lindquist, A. M. Bobst, AND F W. Schaefer III. A NOVEL DETECTION METHOD FOR ENCEPHALITOZOON HELLEM IN WATER. Presented at International Symposium on Waterborne Pathogens, Milwaukee, WI, August 29-September 1, 1999.
Impact/Purpose:
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa.
2) Perform field tests of devices or methods that have been developed under this task.
3) Evaluate these methods or devices in a variety of water matrices and parasite concentrations.
This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
Description:
Microsporidia are a ubiquitous group of protozoan pathogens, containing over 100 genera and hundreds of species. This group has been found in all animal groups. Recently, members of this group have been found even in man. The transmission state of all members of this group is a spore that is unique and diagnostic. Encephalitozoon hellem, a microsporidian that infects man, has been recovered from the central nervous system and eye. Encephalitozoon hellem is even known to disseminate throughout the human body to other sites like the urinary tract from where spores may be released to the environment. It can logically be concluded that there is potential for waterborne transmission of this pathogenic agent. Of all cases of waterborne gastroenteritis, only about fifty percent are attributable to a specific etiologic agent. Lack of specific, sensitive, and practical methods to identify pathogens in a matrix as complex as water contributes to this problem. Until adequate detection methods for these pathogens in water are developed, there is no way to know whether water is a significant route of transmission. The purpose of this research was to develop a method useful for the detection of E. hellem in water.
Antibodies currently employed to detect human microsporidia spores have been shown to cross-react with other species not associated with pathogenicity in humans or not associated with human hosts at all. In order to overcome this problem with antibody specificity, a fluorescence in situ hybridization (FISH) technique for E. hellem spores was developed. The FISH technique employed a 6-carboxyfluorescein (6-FAM) labeled oligonucleotide probe targeting a specific sequence in the 18S ribosomal RNA (rRNA) of E. hellem. Samples of E. hellem spores, which have undergone FISH, are examined using a fluorescent microscope. Encephalitozoon hellem spores used in this study were cultured in Rabbit Kidney (RK-13:ATCC CCL-37) monolayers. Spores were purified from cell culture supernatants using Percoll buoyant density gradient centrifugation. Both transmission and scanning electron microscopy were used to insure quality and homogeneity of the spores produced in culture.
The signal from purified FISH stained spores was bright enough to enable detection in reagent water and in Ohio River water concentrates as well. Preliminary specificity testing of the FISH probe in bacteria, algae, and protozoans found in environmental samples has shown no cross-reactivity. This is the first report of a FISH probe being used to label E. hellem spores specifically. There is some evidence that FISH technique maybe useful as a conservative indicator of viability, since rRNA within non-viable E. hellem spores should be degraded. Initial dual labeling employing an antibody against the spore surface in conjunction with the FISH probe has been shown possible. These data demonstrate the potential for using an antibody against the E. hellem spore coat for detection and using a FISH probe to confirm the identity of this clinically important microsporidian in environmental water.