Prevalence of Giardia spp. in beaver and muskrat populations in northeastern states and Minnesota: detection of intestinal trophozoites at necropsy provides greater sensitivity than detection of cysts in fecal samples

Surveys of the prevalence of the intestinal protozoan Giardia spp. in animal populations have relied almost exclusively on the detection of cysts in fecal samples. We have determined the prevalence of Giardia spp. in beaver and muskrat populations in four northeastern states and Minnesota by using both the detection of trophozoites in mucosal scrapings from live-trapped animals at necropsy and the detection of cysts in fecal samples collected from kill-trapped animals. In muskrats the prevalence of Giardia infection was 36.6% by cyst detection in fecal samples (n = 790) from kill-trapped animals and 95.9% in live-trapped muskrats when the intestinal contents were analyzed for the presence of trophozoites (n = 219). Similarly, in beavers, Giardia infection was 9.2% by cyst detection in fecal samples (n = 662) from kill-trapped beavers and 13.7% in live-trapped animals examined for the presence of intestinal trophozoites (n = 302). The detection of trophozoites in mucosal scrapings from live-trapped animals consistently yielded a significantly higher prevalence for both muskrats and beavers than did the method based on detection of cysts in the fecal samples. The prevalence of Giardia infection in juvenile and adult live-trapped muskrats was similar (92.5 and 94.4%, respectively), but the prevalence in juvenile live-trapped beavers (23.2%) was significantly greater than that seen in the adult animals (12.6%). No difference in Giardia prevalence on the basis of sex was seen in either animal species. Regional variation, often statistically significant, was seen in the prevalence of Giardia in beavers in the northeastern states and Minnesota, but was not detected for muskrats.     

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin.

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cross-species transmission of Giardia spp.: inoculation of beavers and muskrats with cysts of human, beaver, mouse, and muskrat origin

Giardia cysts isolated from humans, beavers, mice, and muskrats were tested in cross-species transmission experiments for their ability to infect either beavers or muskrats. Giardia cysts, derived from multiple symptomatic human donors and used for inoculation of beavers or muskrats, were shown to be viable by incorporation of fluorogenic dyes, excystation, and their ability to produce infections in the Mongolian gerbil model. Inoculation of beavers with 5 x 10(5) Giardia lamblia cysts resulted in the infection of 75% of the animals (n = 8), as judged by the presence of fecal cysts or intestinal trophozoites at necropsy. The mean prepatent period was 13.1 days. An infective dose experiment, using 5 x 10(1) to 5 x 10(5) viable G. lamblia cysts collected by fluorescence-activated cell sorting, demonstrated that doses of between, less than 50, and less than 500 viable cysts were required to produce infection in beavers. Scanning electron microscopy of beaver small intestine revealed that attachment of G. lamblia trophozoites produced lesions in the microvillous border. Inoculation of muskrats with G. lamblia cysts produced infections when the dose of cysts was equal to or greater than 1.25 x 10(5). The inoculation of beavers with Giardia ondatrae or Giardia muris cysts did not produce any infection; however, the administration to muskrats of Giardia cysts of beaver origin resulted in the infection of 62% of the animals (n = 8), with a prepatent period of 5 days. Our results demonstrated that beavers and muskrats could be infected with Giardia cysts derived from humans, but only by using large numbers of cysts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reservoirs of Giardia spp. in southwestern Alberta

A survey of potential hosts of Giardia spp. was carried out during 1982 and 1983 in the Kananaskis Valley and Banff National Park, Alberta, Canada. Diagnosis was based mainly on fecal analysis but a few animals were examined at necropsy and scrapings from the small intestine analyzed. A total of 304 specimens was examined from humans (Homo sapiens L.) and a variety of animal species. Cysts and/or trophozoites of Giardia were found in 10.5% of the specimens examined. Positive samples were found from 20 of 21 red-backed voles (Clethrionomys gapperi Vigors), two of six meadow voles (Microtus pennsylvanicus Ord), one of three long-tailed voles (Microtus longicaudus Allen), five of 50 deer mice (Peromyscus maniculatus Mearns), and two of 58 beavers (Castor canadensis Kuhl). Cysts obtained from a beaver were successfully introduced to gerbils (Meriones unguiculatus Milne-Edwards) and the trophozoites obtained were cultured in vitro.     

Prevalence of and associated risk factors for shedding Cryptosporidium parvum oocysts and Giardia cysts within feral pig populations in California.

Populations of feral pigs (Sus scrofa) may serve as an environmental reservoir of Cryptosporidium parvum oocysts and Giardia sp. cysts for source water. We conducted a cross-sectional study to determine the prevalence of and associated demographic and environmental risk factors for the shedding of C. parvum oocysts and Giardia sp. cysts. Feral pigs were either live-trapped or dispatched from 10 populations located along the coastal mountains of western California, and fecal samples were obtained for immunofluorescence detection of C. parvum oocysts and Giardia sp. cysts. We found that 12 (5.4%) and 17 (7.6%) of 221 feral pigs were shedding C. parvum oocysts and Giardia sp. cysts, respectively. The pig's sex and body condition and the presence of cattle were not associated with the probability of the shedding of C. parvum oocysts. However, younger pigs (< or = 8 months) and pigs from high-density populations (> 2.0 feral pigs/km2) were significantly more likely to shed oocysts compared to older pigs (> 8 months) and pigs from low-density populations (< or = 1.9 feral pigs/km2). In contrast, none of these demographic and environmental variables were associated with the probability of the shedding of Giardia sp. cysts among feral pigs. These results suggest that given the propensity for feral pigs to focus their activity in riparian areas, feral pigs may serve as a source of protozoal contamination for surface water.     

Observations on Giardia of Budgerigars

ABSTRACT. Sick budgerigars from a local aviary were found to be infected with Giardia. The trophozoites were of the Giardia duodenalis type as defined by Filice with elongate median bodies pointed on one or both ends and more or less perpendicular to the long axis of the body. Using three fixation-staining methods, and material from three birds, the length ranged from 10 to 18 μm and the width from 4.5 to 11 μm with a mean length to width ratio of 2. Attempts to culture the trophozoites in vitro from intestinal scrapings were unsuccessful. Also attempts to transmit the infection by fecal cysts to canaries and mice failed. It is proposed that the budgerigar form be called Giardia duodenalis , race psittaci .     

Excystation and culturing of human and animal Giardia spp. by using gerbils and TYI-S-33 medium.

Mongolian gerbils were used as an animal model to excyst and host Giardia spp. isolated from meadow voles, dogs, beavers, and humans. Both cysts and trophozoites were used to establish infections. Gerbils were infected with Giardia duodenalis from beaver, dog, and human sources, and the trophozoites were extracted and cultured in Diamond TYI-S-33 medium. The use of gentamicin and ampicillin in the medium, coupled with treatment of gerbils with gentamicin before they were sacrificed, permitted the elimination of trophozoite purification techniques before culturing. An extract of whole bovine calf blood, CLEX, was substituted for fetal bovine serum in TYI-S-33 medium and was found to be both adequate and less expensive.     

Excystation and culturing of human and animal Giardia spp. by using gerbils and TYI-S-33 medium

Mongolian gerbils were used as an animal model to excyst and host Giardia spp. isolated from meadow voles, dogs, beavers, and humans. Both cysts and trophozoites were used to establish infections. Gerbils were infected with Giardia duodenalis from beaver, dog, and human sources, and the trophozoites were extracted and cultured in Diamond TYI-S-33 medium. The use of gentamicin and ampicillin in the medium, coupled with treatment of gerbils with gentamicin before they were sacrificed, permitted the elimination of trophozoite purification techniques before culturing. An extract of whole bovine calf blood, CLEX, was substituted for fetal bovine serum in TYI-S-33 medium and was found to be both adequate and less expensive.     

Small rodents and other mammals associated with mountain meadows as reservoirs of Giardia spp. and Campylobacter spp.

Sixty-five percent (469 of 722) of the fecal samples collected from small rodents in the central Washington Cascade mountains were positive for Giardia spp. Trapping studies showed that microtines of the genus Microtus were heavily infected with the parasite. Morphologically the cysts and trophozoites were of the Giardia duodenalis type. Small-rodent populations appear to maintain their infection throughout the year. Our data suggest that there is no difference in the percentage of positive animals in areas receiving a lot of human use as opposed to animals in those areas receiving very little or no human use. Giardia spp. were also found in elk and beaver fecal samples. Campylobacter spp. were recovered infrequently from the small rodents inhabiting alpine meadows. Of 551 specimens cultured, less than 1% were positive for the bacterium, and the isolates were identified as Campylobacter coli. Water voles were susceptible to a human isolate of Campylobacter jejuni and shed the bacterium for several weeks. C. jejuni was also isolated from a bear fecal sample collected from a protected watershed. Our studies indicate that microtines and possibly other small rodents inhabiting mountain meadows have a potential to act as a reservoir for both Giardia spp. and Campylobacter spp. Because these animals may carry human pathogens, they should be included in animal surveys designed to assess the health risks associated with mountain watersheds.     

Small rodents and other mammals associated with mountain meadows as reservoirs of Giardia spp. and Campylobacter spp

Sixty-five percent (469 of 722) of the fecal samples collected from small rodents in the central Washington Cascade mountains were positive for Giardia spp. Trapping studies showed that microtines of the genus Microtus were heavily infected with the parasite. Morphologically the cysts and trophozoites were of the Giardia duodenalis type. Small-rodent populations appear to maintain their infection throughout the year. Our data suggest that there is no difference in the percentage of positive animals in areas receiving a lot of human use as opposed to animals in those areas receiving very little or no human use. Giardia spp. were also found in elk and beaver fecal samples. Campylobacter spp. were recovered infrequently from the small rodents inhabiting alpine meadows. Of 551 specimens cultured, less than 1% were positive for the bacterium, and the isolates were identified as Campylobacter coli. Water voles were susceptible to a human isolate of Campylobacter jejuni and shed the bacterium for several weeks. C. jejuni was also isolated from a bear fecal sample collected from a protected watershed. Our studies indicate that microtines and possibly other small rodents inhabiting mountain meadows have a potential to act as a reservoir for both Giardia spp. and Campylobacter spp. Because these animals may carry human pathogens, they should be included in animal surveys designed to assess the health risks associated with mountain watersheds.     

The detection of Giardia muris and Giardia lamblia cysts by immunofluorescence in animal tissues and fecal samples subjected to cycles of freezing and thawing

The effects of freezing and thawing on the detection of selected Giardia spp. cysts were investigated using immunofluorescence, bright field microscopy, and low voltage scanning electron microscopy (SEM). Giardia muris cysts were obtained from either animal carcasses, fecal pellets, or isolated cyst preparations, whereas Giardia lamblia cysts were isolated from fecal samples. These samples were stained using an immunofluorescence technique after 1-3 freezing (-16 C) and thawing (20 C) cycles. Cysts were detected successfully by immunofluorescence in all samples. However, in those samples subjected to freeze-thawing, the cyst walls often became distorted and then were not detectable by bright field microscopy. Low voltage SEM demonstrated that the filaments in the distorted cyst wall underwent rearrangements of interfilament spacing. Quantitation of cyst recovery after freezing and thawing demonstrated that a substantial loss occurred after 1 cycle of alternating temperature when low concentrations of cysts were used, but not with high concentrations of cysts. Cyst recovery, after 3 freezing and thawing cycles, was dramatically lowered irrespective of the initial cyst concentration. These results demonstrated that immunofluorescence was an effective technique for the detection of Giardia spp. cysts in frozen samples and would suggest that freezing and thawing of fecal samples could prevent the detection of cysts when only bright field microscopy was employed.     

Novel and canine genotypes of Giardia duodenalis in harbor seals ( Phoca vitulina richardsi)

Feces of harbor seals (Phoca vitulina richardsi) and hybrid glaucous-winged/western gulls (Larus glaucescens / occidentalis) from Washington State's inland marine waters were examined for Giardia and Cryptosporidium spp. to determine if genotypes carried by these wildlife species were the same genotypes that commonly infect humans and domestic animals. Using immunomagnetic separation followed by direct fluorescent antibody detection, Giardia spp. cysts were detected in 42% of seal fecal samples (41/97). Giardia-positive samples came from 90% of the sites (9/10) and the prevalence of positive seal fecal samples differed significantly among study sites. Fecal samples collected from seal haulout sites with over 400 animals were 4.7 times more likely to have Giardia spp. cysts than samples collected at smaller haulout sites. In gulls, a single Giardia sp. cyst was detected in 4% of fecal samples (3/78). Cryptosporidium spp. oocysts were not detected in any of the seals or gulls tested. Sequence analysis of a 398 bp segment of G. duodenalis DNA at the glutamate dehydrogenase locus suggested that 11 isolates originating from seals throughout the region were a novel genotype and 3 isolates obtained from a single site in south Puget Sound were the G. duodenalis canine genotype D. Real-time TaqMan PCR amplification and subsequent sequencing of a 52 bp small subunit ribosomal DNA region from novel harbor seal genotype isolates showed sequence homology to canine genotypes C and D. Sequence analysis of the 52 bp small subunit ribosomal DNA products from the 3 canine genotype isolates from seals produced mixed sequences at could not be evaluated.     

Presence of Giardia spp. and absence of Salmonella spp. in New Jersey muskrats (Ondatra zibethicus)

Of 220 muskrat fecal specimens collected from 12 sites in southwestern New Jersey, 154 (70%) were found to contain cysts of the protozoan parasite Giardia spp. Cysts from selected muskrat fecal specimens infected Mongolian gerbils, but attempts to cultivate trophozoites removed from these gerbils were unsuccessful. Salmonella spp. were not detected in any of the muskrat fecal specimens.     

Presence of Giardia spp. and absence of Salmonella spp. in New Jersey muskrats (Ondatra zibethicus).

Of 220 muskrat fecal specimens collected from 12 sites in southwestern New Jersey, 154 (70%) were found to contain cysts of the protozoan parasite Giardia spp. Cysts from selected muskrat fecal specimens infected Mongolian gerbils, but attempts to cultivate trophozoites removed from these gerbils were unsuccessful. Salmonella spp. were not detected in any of the muskrat fecal specimens.     

Reduction in fecal excretion of Giardia cysts: effect of cholestasis and diet

Bile is a major growth factor for the proliferation of Giardia spp. trophozoites in the small intestine and, at high concentrations, stimulates encystment of trophozoites. This report demonstrates that surgical cholestasis to interrupt the flow of bile from liver to intestine or the use of bile-binding resins in the diet can both dramatically decrease the fecal excretion of Giardia muris cysts. Cholestasis produced a 3 log reduction in excretion of G. muris cysts within 24 hr of surgery and a 4 log reduction after 3 days. Sham controls showed no difference in cyst excretion from presurgical control values. Two isocaloric diets were studied: a control diet (N) of Purina mouse chow containing 5% celufil and an experimental diet (CR) containing 5% cholestyramine, a resin that binds bile. Compared with the N diet, the CR diet was associated with reductions in cyst excretion of 3 logs within 1 day. Despite lowered excretion of G. muris cysts in mice fed the cholestyramine diet, the trophozoite recovery from the duodenum was similar with both diets. Cyclic feeding of the CR diet and the N diet at 3-day intervals produced significant oscillations (changes of 3-4 logs) in fecal cyst shedding. The significant reductions in fecal excretion of cysts observed with agents that bind bile suggests that diets capable of binding bile might be a therapeutic means to minimize the fecal excretion of cysts and thereby may help to reduce the risk of spreading giardiasis through fecal-oral contamination.     

Prevalence of Giardia sp. in a beaver colony and the resulting environmental contamination

The prevalence of Giardia sp. in a beaver (Castor canadensis) colony in Colorado was determined by the collection and analysis of fecal samples over a period of 14 mo. Environmental contamination was monitored through the use and analysis of water filter samples. Beaver shed cysts of Giardia sp. in their feces throughout the year with temporal variations in the prevalence, and became infected as kits and remained infected as juveniles and adults. Beaver served as amplification hosts for Giardia sp. and contaminated surface waters downstream from their dams in late spring and early fall. In slow moving waters the cysts of Giardia sp. settled rapidly. Muskrats (Ondatra zibethicus) were the only other species of wildlife shedding cysts of Giardia sp. on the study area.     

High-resolution electron microscopic evidence for the filamentous structure of the cyst wall in Giardia muris and Giardia duodenalis

High-resolution morphological studies of the cyst wall of Giardia spp. were performed using low-voltage scanning electron microscopy (LVSEM) and transmission electron microscopy (TEM). The cyst wall was composed of membranous and filamentous layers. The membranous layer consisted of an inner and an outer cyst membrane separated by a thin layer of cytoplasm. The filamentous layer contained individual filaments that ranged from 7 to 20 nm in diameter when measured by LVSEM, formed a dense meshwork with branches or interconnections, and were occasionally arranged on the surface in whorled patterns. Cysts of Giardia muris from mice, Giardia duodenalis from dogs, pigs, voles, beavers, muskrats, and humans, and Giardia psittaci from a bird (parakeet), possessed an essentially identical wall composed of filaments. Inducement of excystation in viable Giardia cysts produced a dramatic increase in the interfilament spacing over an entire cyst, but none was observed in heat-killed or chemically fixed control cysts. These results demonstrated that the cyst wall of Giardia spp. was composed of a complex arrangement of filaments, presumably formed during the process of encystment.     

Seasonal and biogeographical patterns of gastrointestinal parasites in large carnivores: wolves in a coastal archipelago

Parasites are increasingly recognized for their profound influences on individual, population and ecosystem health. We provide the first report of gastrointestinal parasites in gray wolves from the central and north coasts of British Columbia, Canada. Across 60 000 km(2), wolf feces were collected from 34 packs in 2005-2008. At a smaller spatial scale (3300 km(2)), 8 packs were sampled in spring and autumn. Parasite eggs, larvae, and cysts were identified using standard flotation techniques and morphology. A subset of samples was analysed by PCR and sequencing to identify tapeworm eggs (n=9) and Giardia cysts (n=14). We detected ≥14 parasite taxa in 1558 fecal samples. Sarcocystis sporocysts occurred most frequently in feces (43·7%), followed by taeniid eggs (23·9%), Diphyllobothrium eggs (9·1%), Giardia cysts (6·8%), Toxocara canis eggs (2·1%), and Cryptosporidium oocysts (1·7%). Other parasites occurred in ≤1% of feces. Genetic analyses revealed Echinococcus canadensis strains G8 and G10, Taenia ovis krabbei, Diphyllobothrium nehonkaiense, and Giardia duodenalis assemblages A and B. Parasite prevalence differed between seasons and island/mainland sites. Patterns in parasite prevalence reflect seasonal and spatial resource use by wolves and wolf-salmon associations. These data provide a unique, extensive and solid baseline for monitoring parasite community structure in relation to environmental change.     

Detection of Giardia duodenalis antigen in human fecal eluates by enzyme-linked immunosorbent assay using polyclonal antibodies

The present study developed and standardized an enzime-linked immunosorbent assay (ELISA) to detect Giardia antigen in feces using rabbit polyclonal antibodies. Giardia cysts were purified from human fecal samples by sucrose and percoll gradients. Gerbils (Meriones unguiculatus) were infected to obtain trophozoites. Rabbits were inoculated with either cyst or trophozoite antigens of 14 Colombian Giardia isolates to develop antibodies against the respective stages. The IgG anti-Giardia were purified by sequential caprylic acid and ammonium sulfate precipitation. A portion of these polyclonal antibodies was linked to alkaline phosphatase (conjugate). One hundred and ninety six samples of human feces, from different patients, were tested by parasitologic diagnosis: 69 were positive for Giardia cysts, 56 had no Giardia parasites, and 71 revealed parasites other than Giardia. The optimal concentration of polyclonal antibodies for antigen capture was 40 g/ml and the optimal conjugate dilution was 1:100. The absorbance cut-off value was 0.24. The parameters of the ELISA test for Giardia antigen detection were: sensitivity, 100% (95% CI: 93.4-100%); specificity, 95% (95% CI: 88.6-97.6%); positive predictive value, 91% (95% CI: 81.4-95.9%); and negative predictive value, 100% (95% CI: 96.1-100%). This ELISA will improve the diagnosis of Giardia infections in Colombia and will be useful in following patients after treatment.     

A novel Giardia duodenalis assemblage A subtype in fallow deer

The molecular identification of species and genotypes of Giardia spp. infecting wild mammals represents the most reliable tool to understand the role played by these animals as reservoirs of cysts infectious for human and other animals. Of 139 fecal samples collected from fallow deer (Dama dama L.) hunted in a Natural Reserve of northern Italy, the prevalence of Giardia sp. was 11.5% (16 of 139 animals), and it was higher in fawns than in older animals. Fragments of the betagiardin and triose phosphate isomerase (tpi) genes were successfully polymerase chain reaction amplified and sequenced from 8 isolates. No sequence variation was observed between isolates at the 2 genetic loci. Sequence and phylogenetic analyses identified a Giardia duodenalis subtype that clusters with assemblage A isolates and that shows homologies of 98 and 97% at the beta-giardin and tpi loci, respectively, compared with the A1 subtype. Because the G. duodenalis subtype found in fecal samples of fallow deer has never been detected previously, its role as a pathogen for humans and domestic animals is unknown, but, considering its genetic distinctiveness, it is likely to be low.