Over 13 months, 465 beavers, foxes, muskrats, otters, and raccoons were trapped in four counties in eastern Maryland and examined by molecular methods for microsporidia. A two-step nested PCR protocol was developed to amplify a 392-bp fragment of the internal transcribed spacer region of the rRNA gene of Enterocytozoon spp., with the use of primers complementary to the conserved regions of published nucleotide sequences. Fifty-nine PCR-positive samples were sequenced. Multiple alignments of these sequences identified 17 genotypes of Enterocytozoon spp. (WL1 to WL17); of these, 15 have not been reported before. Most of the genotypes were found in multiple species of wildlife and belonged to a major group consisting of all the previously described Enterocytozoon bieneusi genotypes from human and domestic animals. Some of the isolates from muskrats and raccoons formed two distinct groups. Results of this study indicate that fur-bearing mammals, especially those closely associated with surface water, can be a potential source of human-pathogenic E. bieneusi. However, there are also host-adapted Enterocytozoon genotypes in wildlife, which may represent species different from E. bieneusi and have no apparent public health significance. This is the first report of E. bieneusi in wildlife.

The protozoan parasite Cryptosporidium parvum is known to occur widely in both source and drinking water and has caused waterborne outbreaks of gastroenteritis. To improve monitoring, the U.S. Environmental Protection Agency developed method 1622 for isolation and detection of Cryptosporidium oocysts in water. Method 1622 is performance based and involves filtration, concentration, immunomagnetic separation, fluorescent-antibody staining and 4,6-diamidino-2-phenylindole (DAPI) counterstaining, and microscopic evaluation. The capsule filter system currently recommended for method 1622 was compared to a hollow-fiber ultrafilter system for primary concentration of C. parvum oocysts in seeded reagent water and untreated surface waters. Samples were otherwise processed according to method 1622. Rates of C. parvum oocyst recovery from seeded 10-liter volumes of reagent water in precision and recovery experiments with filter pairs were 42% (standard deviation [SD], 24%) and 46% (SD, 18%) for hollow-fiber ultrafilters and capsule filters, respectively. Mean oocyst recovery rates in experiments testing both filters on seeded surface water samples were 42% (SD, 27%) and 15% (SD, 12%) for hollow-fiber ultrafilters and capsule filters, respectively. Although C. parvum oocysts were recovered from surface waters by using the approved filter of method 1622, the recovery rates were significantly lower and more variable than those from reagent grade water. In contrast, the disposable hollow-fiber ultrafilter system was compatible with subsequent method 1622 processing steps, and it recovered C. parvum oocysts from seeded surface waters with significantly greater efficiency and reliability than the filter suggested for use in the version of method 1622 tested.Cryptosporidium parvum, a coccidian protozoan parasite, remains a risk to drinking water consumers despite extensive efforts put forth by water providers and the U.S. Environmental Protection Agency (EPA) (7,12,14,15,20,21,22). Oocysts are present in many environmental waters because Cryptosporidium is not only a human pathogen but also a zoonotic pathogen infecting livestock, as well as feral animals, in many watersheds used as sources of drinking water. Oocysts persist in the environment and are resistant to the chlorine disinfection routinely used for drinking water (2,11,13,17,23). Therefore, physical removal by chemical pretreatment and filtration is the primary means for reducing oocysts in source waters. When deficiencies in chemical pretreatment and filtration processes occur, oocysts can breach the treatment system and cause disease outbreaks of the magnitude of the 1993 Milwaukee cryptosporidiosis outbreak (14).Detection of Cryptosporidium oocysts in raw water sources is considered an important component in the management, prevention, and control of Cryptosporidium in drinking water supplies. Methods have been developed to detect C. parvum in both raw source waters and finished drinking waters. The EPA developed an Information Collection Rule requiring large munici...

Of 471 specimens examined from foxes, raccoons, muskrats, otters, and beavers living in wetlands adjacent to the Chesapeake Bay, 36 were positive for five types of Cryptosporidium, including the C. canis dog and fox genotypes, Cryptosporidium muskrat genotypes I and II, and Cryptosporidium skunk genotype. Thus, fur-bearing mammals in watersheds excreted host-adapted Cryptosporidium oocysts that are not known to be of significant public health importance.The enteric parasites in the genus Cryptosporidium can be transmitted through ingestion of contaminated water (5). However, the sources of contamination are not clearly identified. Cryptosporidium spp. have been reported to infect a wide range of wild mammals (15). Among them, wild rodents have received particular attention, and it has been suggested that they may serve as reservoirs of Cryptosporidium infection for domestic animals and humans (1-6, 13-15, 18). Since the 1993 cryptosporidiosis outbreak in Milwaukee, Wis., the water industry in the United States has been striving to provide Cryptosporidium-free drinking water through stringent treatment practices and source water protection. Although there has been speculation that wild mammals serve as potential sources of watershed contamination with Cryptosporidium oocysts infectious for humans (1-3, 7), the actual role of wildlife in the contamination of source water with human-pathogenic Cryptosporidium spp. remains unknown.Results of recent studies indicate a strong host adaptation for Cryptosporidium (22). Eight species of Cryptosporidium have been identified as pathogens in humans: C. parvum, C. hominis, C. meleagridis, C. felis, C. canis, C. muris, and Cryptosporidium pig and cervine species (8-12, 20, 21). Of these, C. hominis, C. parvum, and C. meleagridis have been found most frequently, whereas the others have been identified mostly in clinical case reports involving a few persons. Thus, unless wild mammals can be shown to be a source of these three species, they do not represent a significant risk as a source of water contamination affecting humans under normal circumstances. The present study was conducted to determine if Cryptosporidium infections are present in wild mammals (beavers, muskrats, otters, raccoons, and foxes) living in Chesapeake Bay watersheds and, if so, to determine the prevalence and species of Cryptosporidium by molecular methods. Results of the study provide the first genetically based data on the role of wildlife in Cryptosporidium contamination in watersheds.Wildlife fecal specimen collection and genomic DNA extraction. A total of 471 fecal specimens were collected during January 2001 and January 2002 from 87 beavers, 237 muskrats, 20 otters, 51 raccoons, and 76 foxes trapped in the Caroline (Marshy Hope Creek, Federalsburg), Charles (Clifton Creek, Newburg), Dorchester (Hunting Creek, Hurlock), and Talbot (Choptank River, Easton) counties of Maryland. With few exceptions, most of the trapped animals were older than 12 months. Details of the animal sources were described previ...

The steps of two immunofluorescent-antibody-based detection methods were evaluated for their efficiencies in detecting Giardia cysts and Cryptosporidium oocysts. The two methods evaluated were the American Society for Testing and Materials proposed test method for Giardia cysts and Cryptosporidium oocysts in low-turbidity water and a procedure employing sampling by membrane filtration, Percoll-Percoll step gradient, and immunofluorescent staining. The membrane filter sampling method was characterized by higher recovery rates in all three types of waters tested: raw surface water, partially treated water from a flocculation basin, and filtered water. Cyst and oocyst recovery efficiencies decreased with increasing water turbidity regardless of the method used. Recoveries of seeded Giardia cysts exceeded those of Cryptosporidium oocysts in all types of water sampled. The sampling step in both methods resulted in the highest loss of seeded cysts and oocysts. Furthermore, much higher recovery efficiencies were obtained when the flotation step was avoided. The membrane filter method, using smaller tubes for flotation, was less time-consuming and cheaper. A serious disadvantage of this method was the lack of confirmation of presumptive cysts and oocysts, leaving the potential for false-positive Giardia and Cryptosporidium counts when cross-reacting algae are present in water samples.