Host-Adapted Cryptosporidium spp. in Canada Geese (Branta canadensis)

The prevalence and distribution of Cryptosporidium spp. in the fecal droppings of the free-living waterfowl Canada geese were examined at 13 sites in Ohio and Illinois. On the basis of the analysis of the small-subunit rRNA gene by PCR, followed by restriction fragment length polymorphism analysis and DNA sequencing, 49 (23.4%) of 209 fecal specimens collected from 10 sites (76.9%) were positive for Cryptosporidium spp. The following five Cryptosporidium species and genotypes were identified: Cryptosporidium goose genotype I (in 36 specimens), Cryptosporidium goose genotype II (in 9 specimens), Cryptosporidium duck genotype (in 1 specimen), Cryptosporidium parvum (in 4 specimens), and C. hominis (in 2 specimens). Cryptosporidium goose genotype I was the most prevalent parasite and was found at all five Cryptosporidium-positive sites in Ohio and at four of five positive sites in Illinois, followed by Cryptosporidium goose genotype II, which was found at two of five positive sites in Ohio and at four of five positive sites in Illinois. Cryptosporidium goose genotype II was detected for the first time, and it is phylogenetically related to goose genotype I and the duck genotype. All three genotypes have not so far been reported in humans, and their pathogenicity in geese has not been determined. Only 10.2% of the Cryptosporidium-positive specimens had C. parvum and C. hominis. The results of this study indicate that Canada geese might only serve as accidental carriers of cryptosporidia infectious to humans and probably play a minor role in the animal-to-human transmission cycle of the pathogen.     

Phylogenetic Analysis of the Hypervariable Region of the 18S rRNA Gene of Cryptosporidium Oocysts in Feces of Canada Geese (Branta canadensis): Evidence for Five Novel Genotypes

To assess genetic diversity in Cryptosporidium oocysts from Canada geese, 161 fecal samples from Canada geese in the United States were analyzed. Eleven (6.8%) were positive for Cryptosporidium spp. following nested PCR amplification of the hypervariable region of the 18S rRNA gene. Nine PCR products from geese were cloned and sequenced, and all nine diverged from previously reported Cryptosporidium 18S rRNA gene sequences. Five sequences were very similar or identical to each other but genetically distinct from that of Cryptosporidium baileyi; two were most closely related to, but genetically distinct from, the first five; and two were distinct from any other sequence analyzed. One additional sequence in the hypervariable region of the 18S rRNA gene isolated from a cormorant was identical to that of C. baileyi. Phylogenetic analysis provided evidence for new genotypes of Cryptosporidium species in Canada geese. Results of this study suggest that the taxonomy of Cryptosporidium species in geese is complex and that a more complete understanding of genetic diversity among these parasites will facilitate our understanding of oocyst sources and species in the environment.     

Assessment of Methods for Detection of Infectious Cryptosporidium Oocysts and Giardia Cysts in Reclaimed Effluents

This study evaluates the occurrence of Cryptosporidium oocysts and Giardia cysts in reclaimed effluents if method 1623 with the Envirochek capsule filters (standard and high-volume [HV] filters) and a modified version of the Information Collection Rule method (ICR) with the polypropylene yarn-wound cartridge filter are used. The recovery efficiency of the analytical methods was evaluated with samples of reagent, tap, and reclaimed water by using flow cytometer-sorted spike suspensions. (Oo)cyst recovery efficiency determined filter performance and method reproducibility in the water matrix tested. Method 1623 with the Envirochek HV capsule filter generated significantly higher recovery rates than did the standard Envirochek filter and the modified ICR method. Notwithstanding, large variations in recovery rates (>80%) occurred with samples of reclaimed water, and none of the water quality parameters analyzed in the reclaimed effluents could explain such variability. The highest concentrations of indigenous oocysts were detected by method 1623 with the HV filter, which provided a sufficient number of oocysts for further confirmation of infectious potential. Confirmation of species and potential infectivity for all positive protozoan samples was made by using a nested PCR restriction fragment polymorphism assay and the focus detection method most-probable-number assay, respectively. The methodology and results described in the present investigation provide useful information for the establishment of pathogen numeric standards for reclaimed effluents used for unrestricted irrigation.     

Seasonal Enumeration of Fecal Coliform Bacteria from the Feces of Ring-Billed Gulls (Larus delawarensis) and Canada Geese (Branta canadensis)

Water suppliers have often implicated roosting birds for fecal contamination of their surface waters. Geese and gulls have been the primary targets of this blame although literature documenting the fecal coliform content of these birds is quite limited. To determine the actual fecal coliform concentrations of these birds, fecal samples from 249 ring-billed gulls and 236 Canada geese in Westchester County, N.Y., were analyzed over a 2-year period. Results indicate that gull feces contain a greater average concentration of fecal coliform bacteria per gram (3.68 × 10⁸) than do goose feces (1.53 × 10⁴); however, average fecal sample weights of the geese were more than 15 times higher than those of the gulls.     

Occurrence of Cryptosporidium Oocysts and Giardia Cysts in Sewage in Norway

Samples of sewage influent from 40 sewage treatment works (STW) throughout Norway were examined for Cryptosporidium oocysts and Giardia duodenalis cysts. Both parasites were detected frequently (80% of STW were Cryptosporidium positive; 93% of STW were Giardia positive) and at maximum concentrations of >20,000 parasites/liter. The data suggest giardiasis is more widespread, and/or occurs with greater infection intensity, than cryptosporidiosis in Norway. STW serving higher person equivalents were more likely to be positive and had higher parasite concentrations. Parasite concentrations were used to estimate the proportion of contributing populations that could be clinically infected. For Cryptosporidium, the highest estimates were up to 5 per 100,000 individuals for two populations in eastern Norway. For Giardia, the highest estimate was 40 infected per 100,000 persons (approximately five times the usual national annual average) contributing to an STW in western Norway. As this population experienced a large waterborne giardiasis outbreak 6 months after sampling, it can be speculated that regular challenge with Giardia may occur here. Most Giardia isolates in sewage influent were assemblage A, although some assemblage B isolates were detected. There was substantial heterogeneity, but most samples contained isolates similar to genotype A3. Removal efficiencies at two STW with secondary treatment processes were estimated to be approximately 50% for Cryptosporidium and >80% for Giardia. An STW with minimal treatment had negligible removal of both parasites. Many STW in Norway have minimal treatment and discharge effluent into rivers and lakes, thus, risk of contamination of water courses by Cryptosporidium and Giardia is considerable.     

Giardia Cysts and Cryptosporidium Oocysts in Membrane-Filtered Municipal Wastewater Used for Irrigation

A wastewater tertiary treatment system based on membrane ultrafiltration and fed with secondary-treated municipal wastewater was evaluated for its Giardia cyst and Cryptosporidium oocyst removal efficiency. Giardia duodenalis (assemblages A and B) and Cryptosporidium parvum were identified in feed water but were found in filtered water only during occasional failure of the filtration system.     

Detection of Infectious Cryptosporidium parvum Oocysts in Mussels (Mytilus galloprovincialis) and Cockles (Cerastoderma edule)

Infective Cryptosporidium parvum oocysts were detected in mussels (Mytilus galloprovincialis) and cockles (Cerastoderma edule) from a shellfish-producing region (Gallaecia, northwest Spain, bounded by the Atlantic Ocean) that accounts for the majority of European shellfish production. Shellfish were collected from bay sites with different degrees of organic pollution. Shellfish harboring C. parvum oocysts were recovered only from areas located near the mouths of rivers with a high density of grazing ruminants on their banks. An approximation of the parasite load of shellfish collected in positive sites indicated that each shellfish transported more than 10³ oocysts. Recovered oocysts were infectious for neonatal mice, and PCR-restriction fragment length polymorphism analysis demonstrated a profile similar to that described for genotype C or 2 of the parasite. These results demonstrate that mussels and cockles could act as a reservoir of C. parvum infection for humans. Moreover, estuarine shellfish could be used as an indicator of river water contamination.     

Fate of Cryptosporidium Oocysts and Giardia Cysts in the Norwegian Aquatic Environment over Winter

We investigated the survival of Cryptosporidium oocysts and Giardia cysts during winter in an aquatic environment (approximate temperature measurements between 1 and 7°C) in Norway, using morphology and uptake of dyes as indicators of viability. Previous research has shown that in the terrestrial environment, shear forces caused by freeze and thaw cycles probably cause the parasites to be inactivated. Such forces occurred infrequently in the aquatic environment, as freezing of the water around the parasites was not observed during the study period (although freezing of the water surface did occur). The rate of decline in viability (log₁₀ N _t/N ₀) was similar in control and experimental environments for both parasites; no Cryptosporidium oocysts with viable morphology were detected after approximately 20 weeks and no Giardia cysts with apparently viable morphology could be detected after 1 month. These results suggest that infection with these parasites in Norway is not usually from transmission stages that have overwintered in the Norwegian environment.     

Survival of Infectious Cryptosporidium parvum Oocysts in Seawater and Eastern Oysters (Crassostrea virginica) in the Chesapeake Bay

Oocysts of Cryptosporidium parvum placed in artificial seawater at salinities of 10, 20, and 30 ppt at 10°C and at 10 ppt at 20°C were infectious after 12 weeks. Those placed in seawater at 20 ppt and 30 ppt at 20°C were infectious for 8 and 4 weeks, respectively. These findings suggested that oocysts could survive in estuarine waters long enough to be removed by filter feeders such as oysters. Thereafter, 30 Eastern oysters, Crassostrea virginica, were collected with a dredge or with hand tongs at each of six sites within Maryland tributaries of the Chesapeake Bay in May and June and in August and September of 1997. Hemocytes and gill washings from all oysters were examined for the presence of Cryptosporidium oocysts and Giardia cysts by immunofluorescence microscopy utilizing a commercially available kit containing fluorescein isothiocyanate-conjugated monoclonal antibodies. Giardia was not detected by this method from any of the 360 oysters examined. Presumptive identification of Cryptosporidium oocysts was made in either hemocytes or gill washings of oysters from all six sites both times that surveys were conducted. In addition, during August and September, for each of the six sites, hemocytes from the 30 oysters were pooled and gill washings from the oysters were pooled. Each pool was delivered by gastric intubation to a litter of neonatal mice to produce a bioassay for oocyst infectivity. Intestinal tissue from two of three mice that received gill washings from oysters collected at a site near a large cattle farm and shoreline homes with septic tanks was positive for developmental stages of C. parvum. These findings demonstrate for the first time that oysters in natural waters harbor infectious C. parvum oocysts and can serve as mechanical vectors of this pathogen.     

Method for Detection and Enumeration of Cryptosporidium parvum Oocysts in Feces, Manures, and Soils

Eight concentration and purification methods were evaluated to determine percentages of recovery of Cryptosporidium parvum oocysts from calf feces. The NaCl flotation method generally resulted in the highest percentages of recovery. Based on the percentages of recovery, the amounts of fecal debris in the final oocyst preparations, the relatively short processing time (<3 h), and the low expense, the NaCl flotation method was chosen for further evaluation. Extraction efficiency was evaluated by using oocyst concentrations of 25, 50, 10², 10³, 10⁴, and 10⁵ oocysts g of bovine feces⁻¹. The percentages of recovery ranged from 10.8% (25 oocysts g⁻¹) to 17.0% (10⁴ oocysts g⁻¹) (r² = 0.996). A conservative estimate of the detection limit for bovine feces is ca. 30 oocysts g of feces⁻¹. Percentages of recovery were determined for six different types of animal feces (cow, horse, pig, sheep, deer, and chicken feces) at a single oocyst concentration (10⁴ oocysts g⁻¹). The percentages of recovery were highest for bovine feces (17.0%) and lowest for chicken feces (3.2%). Percentages of recovery were determined for bovine manure after 3 to 7 days of storage. The percentages of recovery ranged from 1.9 to 3.5% depending on the oocyst concentration, the time of storage, and the dispersing solution. The percentages of oocyst recovery from soils were evaluated by using different flotation solutions (NaCl, cold sucrose, ZnSO₄), different dispersing solutions (Triton X-100, Tween 80, Tris plus Tween 80), different dispersion techniques (magnetic stirring, sonication, blending), and different dispersion times (5, 15, and 30 min). Twenty-five-gram soil samples were used to reduce the spatial variability. The highest percentages of recovery were obtained when we used 50 mM Tris–0.5% Tween 80 as the dispersing solution, dispersion for 15 min by stirring, and saturated NaCl as the flotation solution. The percentages of oocyst recovery from freshly spiked sandy loam, silty clay loam, and clay loam soils were ca. 12 to 18, 8, and 6%, respectively. The theoretical detection limits were ca. 1 to 2 oocysts g of soil⁻¹ depending on the soil type. The percentages of recovery without dispersant (distilled H₂O or phosphate-buffered saline) were less than 0.1%, which indicated that oocysts adhere to soil particles. The percentages of recovery decreased with storage time, although the addition of dispersant (Tris-Tween 80) before storage appeared to partially prevent adhesion. These data indicate that the NaCl flotation method is suitable for routine detection and enumeration of oocysts from feces, manures, soils, or soil-manure mixtures.     

Sensitive and Rapid Detection of Viable Giardia Cysts and Cryptosporidium parvum Oocysts in Large-Volume Water Samples with Wound Fiberglass Cartridge Filters and Reverse Transcription-PCR

[This corrects the article on p. 1743 in vol. 64, PMID: 9572946.].     

Avian pox infection in a Canada goose (Branta canadensis)

Lesions suggestive of Avian Pox were found on a debilitated Canada goose (Branta canadensis). These were demonstrated histologically to be characteristic poxvirus lesions and poxvirus particles were seen using electron microscopy. The virus was successfully transmitted to domestic geese but not to leghorn chickens or to domestic ducks.     

An approach to the assessment of change in the numbers of Canada Geese Branta canadensis and Greylag Geese Anser anser in southern Britain

Capsule Population change in geese was assessed using an approach that requires a relatively small sampling effort.

Aims During the 1999 breeding season a survey was carried out to determine if the numbers of introduced Canada and re-established Greylag Geese in southern Britain had changed since 1988–91 and whether any change had occurred in areas with previously high or low Canada Goose densities.

Methods A randomized stratified sample of 246 tetrads from the 24 156 tetrads covered between 1988–91 in this area, as part of the New Atlas of Breeding Birds, were resurveyed. Eight habitat categories were used in the stratification and were based on 1-km-square summary data obtained from the CEH Land Cover Map of Great Britain (water cover and urbanization) and LANDCLASS stratification (upland/lowland). The five habitat categories with the highest densities of Canada Geese and the greatest variance in numbers were sampled.

Results Between 1989 and 1999, the number of Canada Geese on land with over 5% water cover and on lowland with some water cover increased by on average 156%, an average rate of increase of 9.9% per annum. Southern Britain probably now holds a minimum of 82 000 Canada Geese. Between 1989 and 1999, the number of Greylag Geese on land with over 5% water cover and on lowland with some water cover increased by on average 214%, an average rate of increase of 12% per annum. Southern Britain probably now holds a minimum of 30 000 Greylag Geese.

Conclusion Maximum densities of Canada Geese may have been reached in high-density habitats but their numbers are still increasing very rapidly. Greylag Geese are increasing even more rapidly.     

Effect of Sample Holding Time on Recovery of Cryptosporidium Oocysts and Giardia Cysts from Water Samples

U.S. Environmental Protection Agency methods for analysis of water for Cryptosporidium and Giardia stipulate maximum sample holding times which are not always practical to comply with. A spiking experiment indicated that holding times of up to 2 weeks had no significant effect on recovery of these parasites from 10-liter samples of raw water in plastic carboys.     

Isolation of Actinobacillus suis from a Canada goose (Branta canadensis)

Blindness from conjunctivitis caused by Actinobacillus suis was found in a Canada goose (Branta canadensis) from a wildlife refuge in Kentucky. Lesions were not observed elsewhere and other body organs were grossly normal. Actinobacillus spp. in birds is rare and this is apparently the first report of conjunctivitis resulting from this infection in waterfowl.     

Computer-Assisted Laser Scanning and Video Microscopy for Analysis of Cryptosporidium parvum Oocysts in Soil, Sediment, and Feces

A computer-assisted laser scanning microscope equipped for confocal laser scanning and color video microscopy was used to examine Cryptosporidium parvum oocysts in two agricultural soils, a barnyard sediment, and calf fecal samples. An agar smear technique was developed for enumerating oocysts in soil and barnyard sediment samples. Enhanced counting efficiency and sensitivity (detection limit, 5.2 x 10(sup2) oocysts(middot)g [dry weight](sup-1)) were achieved by using a semiautomatic counting procedure and confocal laser scanning microscopy to enumerate immunostained oocysts and fragments of oocysts in the barnyard sediment. An agarose-acridine orange mounting procedure was developed for high-resolution confocal optical sectioning of oocysts in soil. Stereo images of serial optical sections revealed the three-dimensional spatial relationships between immunostained oocysts and the acridine orange-stained soil matrix material. In these hydrated, pyrophosphate-dispersed soil preparations, oocysts were not found to be attached to soil particles. A fluorogenic dye permeability assay for oocyst viability (A. T. Campbell, L. J. Robertson, and H. V. Smith, Appl. Environ. Microbiol. 58:3488-3493, 1992) was modified by adding an immunostaining step after application of the fluorogenic dyes propidium iodide and 4(prm1),6-diamidino-2-phenylindole. Comparison of conventional color epifluorescence and differential interference contrast images on one video monitor with comparable black-and-white laser-scanned confocal images on a second monitor allowed for efficient location and interpretation of fluorescently stained oocysts in the soil matrix. This multi-imaging procedure facilitated the interpretation of the viability assay results by overcoming the uncertainties caused by matrix interference and background fluorescence.