INTRODUCTION

The beaver (Castor canadensis) is distributed broadly throughout North America. They reside in ponds and streams that humans sometimes use for recreation or sources of drinking water, and consequently have been implicated as sources of waterborne disease. Waterborne pathogens of public health concern include some species and genotypes of Microsporidia, Giardia, and Cryptosporidium, but the role of the beaver as a source of these organisms is not known, is not firmly established, or has been implicated from circumstantial data. The best evidence that beavers serve as a source of waterborne parasites comes from transmission studies in which Giardia spp. cysts from humans were used experimentally to infect beavers, and those from beavers were used to infect humans experimentally (Table 1). Studies of intestinal protozoa in beavers have relied almost exclusively on microscopy (Table 2), much the same as surveys that have relied heavily on microscopic methods for the detection of spores, oocysts, and cysts of these pathogens in other wildlife. Although microscopy has been helpful in identifying many organisms at the generic or higher taxonomic levels, neither morphologic features nor application of immunofluorescence reagents have proven sufficient for identification or differentiation of the multiple species and strains of Microsporidia, Giardia, and Cryptosporidium, each of which parasitize a specific range of hosts. Highly sensitive and specific molecular methods have proven helpful for parasite detection in two ways: when parasite numbers are so low in some specimens that microscopic methods fail to detect them, and when morphologically identical organisms can be distinguished only by identifying genetic differences. Therefore, in addition to the classical and more routinely used microscopic methods, and because of the potential importance of beavers as sources of waterborne pathogens, the present study was conducted by molecular methods to determine the identity of any species of Microsporidia, Giardia, and Cryptosporidium excreted by beavers.

MATERIALS AND METHODS

Feces from 62 beavers live-trapped and then released in northeastern, central, and western Massachusetts (Fig. 1) were shipped to the USDA laboratory in Beltsville, MD. Feces were collected in spring and fall in 2002, 2003, and 2004. Three age groups were considered (adults, subadults, and kits). The present study reports analyses from 27 adults (15 males and 12 females), 31 subadults (22 males and 9 females), and 4 kits (2 males and 2 females).

Fifteen grams of feces were placed into a 50-ml tube and thoroughly mixed with 35 ml dH₂O. The fecal suspension was passed through a 45-μm screen, collected into a second 50-ml tube, and adjusted to a final volume of 50 ml with dH₂O. The tubes were centrifuged at 1,500 g for 15 min, the supernatant discarded and the fecal pellet resuspended in 25 ml dH₂O. Twenty-five milliters of CsCl (1.4 g/ml) was added to each tube and the samples were thoroughly mixed. The samples were subjected to a second centrifugation at 250 g for 20 min. Following the second centrifugation, the top 4 ml of supernatant was aspirated from each sample and transferred into a 15-ml tube. Deionized H₂O was added to all tubes to bring the final volume to 15 ml. Samples were washed twice with dH₂O and the final pellet was suspended in 500 μl of dH₂O. Portions of this 500-μl suspension were used for immunofluorescence analysis or molecular analysis, as described below.

For the immunofluorescence analysis of species of Cryptosporidium and Giardia, a 100-μl aliquot of the fecal suspension was transferred to a microcentrifuge tube and washed once with dH₂O. The pellet was suspended in 25 μl of premixed MeriFluor™ reagents (Meridian Diagnostics, Cincinnati, Ohio, USA). Premixed MeriFluor™ reagents were prepared as follows. MeriFluor test reagent and counterstain were mixed with 3 ml sterile PBS. Two microliters of the feces–stain suspension was transferred to one well of a three-well slide (Cel-line, HTC, Portsmouth, New Hampshire, USA); a coverslip was placed on the slide and slides were examined at 400× with the use of a Zeiss Axioskop microscope equipped with epifluorescence and an FITC-Texas Red dual-wavelength filter (Chroma Technology Corporation, Rockingham, Vermont, USA).

For the immunofluorescence analysis of Microsporidia 15 μl of suspended pellet was aspirated, pipetted into a poly-l-lysine-coated, 11.5-mm-diameter ring on a glass microscope slide (Erie Scientific, Portsmouth, NH), stained with Calcofluor white (Becton Dickinson and Company, Sparks, Maryland, USA), and examined by fluorescence microscopy (Carl Zeiss Microimaging Inc., Thornwood, New York, USA).

For the molecular analysis, total DNA was extracted from each CsCl-cleaned fecal sample using a DNeasyTissue Kit (Qiagen, Valencia, California, USA) with a slightly modified protocol. The protocol, described below, utilized reagents provided by the manufacturer. A total of 50 μl of processed feces were suspended in 180 μl of ATL buffer and thoroughly mixed by vortexing. To this suspension, 20 μl of Proteinase K (20 mg/ml) was added, and the sample was thoroughly mixed. Following an overnight incubation of the mixture at 55°C, 200 μl of AL buffer was added. The remaining protocol followed manufacturer's instructions with one exception. To increase the quantity of recovered DNA, the nucleic acid was eluted in 100 μl of AE buffer.

For Giardia spp., fragments of the ssrRNA (∼292 bp), β-giardin (∼750 bp), and TPI (∼500 bp) genes were amplified by PCR as previously described.5,17,28 For Cryptosporidium spp. a fragment of the ssrRNA gene (∼800 bp) was amplified by PCR as previously described.32 For species of Microsporidia, generic primers that amplify a fragment of the ssrRNA gene (∼292 bp) as well as primers specific for Enterocytozoon bieneusi that amplify the ITS region as well as a portion of the flanking large and small subunit ribosomal RNA genes were used.4,14 PCR products were analyzed on 1% agarose gel and visualized by ethidium bromide staining.

PCR positives were purified with the use of EXO-SAP enzyme (USB Corporation, Cleveland, Ohio, USA). Purified products were sequenced with the same PCR primers used for the original amplification in 10-μl reactions, Big Dye™ chemistries, and an ABI3100 sequencer analyzer (Applied Biosystems, Foster City, California, USA). Each sample was sequenced in both directions. Sequence chromatograms from each strand were aligned and inspected with the use of Lasergene software (DNASTAR, Inc., Madison, Wisconsin, USA). All PCR-positive samples were sequenced. To determine the Giardia sp. genotype, each sequence for all three gene fragments was independently compared to GenBank sequences of Giardia spp. genotypes.

RESULTS

Spores of Microsporidia were not detected by fluorescence microscopy of Calcofluor-stained fecal smears. The PCR method, using both generic primers for Microsporidia and specific primers for E. bieneusi, also failed to detect species of Microsporidia in fecal specimens.

Cysts of Giardia spp. were detected by immunofluorescence microscopy in feces from four subadult and kit beavers, and their presence was confirmed by PCR (Table 3). Gene sequence data for these PCR products indicated that the four beavers were excreting the G. duodenalis genotype equivalent to the zoonotic Assemblage B. Nucleotide sequences of the TPI, 16s rRNA, and β-giardin genes of G. duodenalis isolates from beavers in the current study, representing Assemblage B were deposited in GenBank under accession numbers DQ789112 through DQ789116.

Based on immunofluorescence microscopy, oocysts of Cryptosporidium spp. were identified in the feces from two (numbers 23 and 25) of the four Giardia spp. positive beavers (Table 3). However, when specimens from these beavers were examined by PCR, using primers for the ssrRNA gene, they were negative (Table 3).

DISCUSSION

Microsporidia are intracellular parasites consisting of more than 1,200 species in 143 genera that infect invertebrate and vertebrate hosts.7 At least six genera including 15 species have been reported to infect humans, primarily the immunocompromised. The most common microsporidian that infects humans is E. bieneusi, but little is known of reservoirs or routes of infection.7 However, recently, E. bieneusi was isolated from domesticated animals and wildlife including cattle (Bos taurus), pigs (Sus scrofa), dogs (Canis familiaris), a cat (Felis domesticus), beavers (Castor canadensis), muskrats (Ondatra zibethica), otters (Lutra canadensis), raccoons (Procyon lotor), and foxes (Vulpes vulpes).20,26,29 Of 100 beavers trapped in Maryland, 14 were found positive for E. bieneusi by nucleotide sequence analysis of the ITS region of the rRNA gene.29 Five of these 14 isolates were identical to genotypes isolated from humans, 5 were identical to genotypes isolated from pigs, and 5 were unique to beavers. In contrast, none of the beavers in the present study were found infected with species of Microsporidia. Because beavers have been examined for species of Microsporidia only in Massachusetts (present study) and Maryland29 too few animals have been examined to provide meaningful data on the prevalence, age and sex distribution, sources, and other factors to characterize these infections.

Beavers have been thought to be a major source of Giardia spp.–contaminated drinking water for reasons both obvious and deductive: Beavers are found associated with water and some are infected with Giardia spp. (Table 2), they have been experimentally infected with Giardia spp. cysts derived from humans (Table 1), and beavers excreting Giardia spp. cysts have been found in the vicinity of contaminated drinking water supplies. However, despite efforts by many investigators of waterborne outbreaks of giardiasis, the source of Giardia spp. cysts in the water has never been irrecusably determined. For many years, morphology and cross-transmission studies were the only methods available for identifying cysts and trophozoites from various hosts.11 Immunofluorescence microscopy facilitated recognition of cysts, but this technique had a major drawback. Antibodies to the cyst wall were not species or subspecies specific, and therefore, because most cysts appeared similar in size and shape, they were indistinguishable.2 Isoenzyme patterns of isolates provided a refined method to distinguish isolates and place them in zymodemes,3,25 but this method was both time consuming and required a great deal of expertise to interpret the intricate patterns. Molecular methods have now provided a universally accepted tool for facilitating the identification of Giardia spp. isolates at the genetic level.28 The present study has identified Giardia spp. cysts based on immunofluorescence microscopy, and based on sequencing three different genes, TPI, ssrRNA, and β-giardin, indicating that four young beavers were excreting cysts of the zoonotic assemblage B of G duodenalis. In another study, beavers in Texas were examined over a period of 2 years and identified as juveniles or adults, and as males or females.9 However, the species of Giardia was not determined and neither sex nor age differences were found to be related to infection.9

Cryptosporidium, a protozoan parasite responsible for numerous drinking water and recreational water outbreaks of diarrhea worldwide, has been reported to infect over 150 species of animals.31 Of 16 species and over 30 genotypes, each with a limited host range, C. parvum, a zoonotic pathogen, is the species reported most often and with the widest range of hosts. In a study involving 481 fur-bearing mammals (beaver, fox, muskrat, otter, and raccoon) trapped in Maryland, 8% were found infected by Cryptosporidium spp. utilizing RFLP analysis of PCR, but none of the 87 beavers were found infected.33 Of 19 European beavers (Castor fiber) examined in Poland, oocysts were detected in two Ziehl-Neelsen stained specimens by bright-field microscopy and immunofluorescence microscopy,1 but the species were not determined. Although the present report is the first to document the presence of Cryptosporidium spp. in C. canadensis in North America by immunofluorescence microscopy, molecular methods were unable to provide the data necessary to determine the species or genotype. It is interesting to note that all four infected beavers were young animals. Similarly, a multiyear prevalence study of cryptosporidiosis in dairy cattle has found a much higher rate of infection in calves than in more mature animals.27