Development of a Method for the Concentration and Recovery of Microsporidia from Tap Water

Abstract

Microsporidia are obligate intracellular parasites. Microsporidian spores infect a wide variety of hosts, including humans. The spores may be found in infected hosts' urine and feces, thus waterborne transmission is possible. This study details method development for the detection of microsporidia in tap water. In this study, filtration, centrifugation, purification, and detection parameters were optimized for the detection of microsporidia. The Pall-Gelman Envirocheck sampling capsule (Pall Gelman, Ann Arbor, MI) was chosen as the filter element. Optimal centrifugal force for spore recovery was 1500 x g. Additionally, it was determined that eluting microsporidia spores in a detergent elution buffer solution had a detrimental effect on spore recovery. A direct examination of the concentrate resulted in a greater recovery with less variability than subjecting the sample concentrate to a Percoll-sucrose density gradient purification step. The staining method employed for the detection spores was Calcofluor white (Sigma, St. Louis, MO). Percent recoveries for 10 L tap water samples (n = 5) using the Envirocheck sampling capsule without a density gradient purification step were 26.1+/-13.4 compared to 25+/-13.8 for samples subjected to a density gradient purification step.

Environmental Monitoring of Opportunistic Protozoa in Rivers and Lakes in the Neotropics Based on Yearly Monitoring

“…They are the most prevalent microsporidia identified in humans (Didier and Weiss 2006) and other mammals (Keeling and Fast 2002). The spores are the infective agent (Barbosa et al 2009) because they are resistant to environmental conditions (Stine et al 2005) and to the usual water treatment procedures (Hoffman et al 2003). Consequently, transmission involves waterborne routes (Karanis et al 2007).…”

Cytometric Approach for Detection of Encephalitozoon intestinalis , an Emergent Agent

“…However, in immunocompromised patients, it causes chronic diarrhea, which tends to disseminate, with the kidney being the major organ affected (8,9). Spores may spread to the environment from infected patients via feces, urine, and/or other body fluids and tissues (9,24). Consequently, the routes of transmission may involve person-to-person contact as well as waterborne or food-borne contamination, especially in developing countries with poor sanitation (11,25).…”

“…Only a small number of cases of Encephalitozoon intestinalis infection have been documented (25); E. intestinalis (reclassified from Septata intestinalis) has been detected in AIDS patients in developed countries and in Africa, as well as in other immunocompromised individuals, including patients who have undergone transplantation (8,9,24).…”

“…Consequently, the routes of transmission may involve person-to-person contact as well as waterborne or food-borne contamination, especially in developing countries with poor sanitation (11,25). The microsporidial spores are usually very resistant to environmental conditions (24) and to the usual water treatment procedures (10), and they remain infective for long periods of time, especially when they are protected from desiccation (9). As a result of these characteristics, the U.S. Environmental Protection Agency placed microsporidia in first place as a candidate contaminant for drinking water (10).…”

“…Such methods are very time-consuming and are subject to human error (8,16). Although molecular studies are highly sensitive and specific, they are too complex, too difficult for use for routine analysis, and expensive and are unable to assess spore viability (24). Flow cytometry (FC) allows both morphofunctional evaluation and quantification of individual microorganisms; it additionally provides significant advantages and a high degree of specificity, especially when it is combined with specific monoclonal or polyclonal antibodies against the E. intestinalis spore wall (8,9,16,25).…”