Detection and species identification of Cryptosporidium from Taiwan feeding animals

In this study, 107 fecal specimens were collected from 40 sampling sites in Taiwan livestock and avian farms to test for Cryptosporidium spp. oocysts. Ten of 107 samples analyzed by enzyme-linked immunosorbent assay showed the presence of Cryptosporidium spp., among which 6 samples were simultaneously confirmed by immunofluorescence assay and polymerase chain reaction. Nucleic acid sequencing of the 18S rRNA gene identified 3 clusters of Cryptosporidium spp. Three Cryptosporidium parvum isolates were from cattle and sheep feces. One Cryptosporidium andersoni isolate was detected from pig feces. The other 2 novel Cryptosporidium genotypes were not similar to any known Cryptosporidium spp. according to the DNA sequences of the 18S rRNA gene.     

Identification of Cryptosporidium oocysts in river water.

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

Identification of Cryptosporidium oocysts in river water

Water samples were collected from four rivers in Washington State and two rivers in California and examined for the presence of Cryptosporidium oocysts. Oocyst-sized particles were concentrated from 20-liter samples of water by membrane filtration, centrifugation, and differential sedimentation. The particle concentrate was then deposited on a 25-mm-diameter membrane filter for oocyst identification by indirect immunofluorescence assay. The identification procedure had a limit of detection of about five oocysts per liter. Cryptosporidium oocysts were found in each of 11 river water samples examined. Concentrations ranged from 2 to 112 oocysts per liter. The finding of Cryptosporidium oocysts in all samples examined from six western rivers is noteworthy in light of recent reports indicating that Cryptosporidium sp. is a significant agent of human and animal disease. This finding suggests that waterborne oocysts of this parasite are more important than was previously recognized. More detailed studies are needed to define geographical and temporal distribution, to assess the viability of waterborne oocysts, and to determine the importance of water as a means of transmission.     

Detection of Cryptosporidium sp. in two new seal species, Phoca vitulina and Cystophora cristata, and a novel Cryptosporidium genotype in a third seal species, Pagophilus groenlandicus, from the Gulf of Maine

Data on the geographic distribution and host specificity of Cryptosporidium spp. are critical for developing an understanding of likely transmission patterns in nature. During a molecular-based survey of fecal samples from 293 terrestrial and aquatic animals in Maine, USA, we detected Cryptosporidium sp. in 11 harbor seals (Phoca vitulina), 1 hooded seal (Cystophora cristata), and 1 harp seal (Pagophilus groenlandicus). None of the terrestrial or freshwater mammal fecal samples or bird samples tested positive for Cryptosporidium sp. However, the sequencing results of the small subunit (ssu) rRNA gene indicate that the seals were infected with an undescribed species of Cryptosporidium , previously isolated only from ringed seals (Phoca hispida) in northern Quebec, Canada. In addition, the Cryptosporidium sp. detected in the harp seal is significantly different from the previously observed Cryptosporidium sp. in other seals. We confirmed the genetic distinctiveness of this Cryptosporidium genotype and the identity of the other Cryptosporidium sp. seal ssu rRNA sequences by using data from the 70-kDa heat shock protein gene. Based on phylogenetic reconstructions of both genes, it seems that either Cryptosporidium canis or C. felis are sister species to the seal associated Cryptosporidium spp. Our findings extend the range of " Cryptosporidium sp. seal" well south of the 55th parallel, add other species to the list of seals affected by Cryptosporidium sp., and highlight the presence of unrecognized population and potentially species level variation in Cryptosporidium.     

Successful hyperimmune bovine colostrum treatment of Savanna monitors (Varanus exanthematicus) infected with Cryptosporidium sp

Therapy based on the protective passive immunity of hyperimmune bovine colostrum (HBC) (raised against Cryptosporidium parvum in cows) was applied to 4 Savanna monitors (Varanus exanthematicus) with gastric Cryptosporidium sp. infections. All lizards were moderately emaciated, and their fecal and gastric lavage samples contained moderate numbers of Cryptosporidium sp. oocysts. The first 3 of 7 gastric HBC treatments at 1-wk interval each decreased the numbers of oocysts in the fecal and gastric samples to undetectable levels. Neither feces nor lavages of the HBC-treated lizards contained Cryptosporidium sp. oocysts after the HBC therapy, whereas such samples of a single control lizard remained positive for oocysts. Two of the HBC-treated lizards died spontaneously due to metastasized carcinoma and septicemia of unknown etiology, respectively, and 2 lizards treated and killed during the experiment were histologically negative for developmental stages of Cryptosporidium sp. The control lizard died spontaneously of septicemia of unknown etiology and contained developmental stages of Cryptosporidium sp. in the gastric region. The HBC therapy was efficacious in V. exanthematicus and is recommended for lizards with gastric cryptosporidiosis.     

Molecular characterization of cryptosporidium oocysts in samples of raw surface water and wastewater

Recent molecular characterizations of Cryptosporidium parasites make it possible to differentiate the human-pathogenic Cryptosporidium parasites from those that do not infect humans and to track the source of Cryptosporidium oocyst contamination in the environment. In this study, we used a small-subunit rRNA-based PCR-restriction fragment length polymorphism (RFLP) technique to detect and characterize Cryptosporidium oocysts in 55 samples of raw surface water collected from several areas in the United States and 49 samples of raw wastewater collected from Milwaukee, Wis. Cryptosporidium parasites were detected in 25 surface water samples and 12 raw wastewater samples. C. parvum human and bovine genotypes were the dominant Cryptosporidium parasites in the surface water samples from sites where there was potential contamination by humans and cattle, whereas C. andersoni was the most common parasite in wastewater. There may be geographic differences in the distribution of Cryptosporidium genotypes in surface water. The PCR-RFLP technique can be a useful alternative method for detection and differentiation of Cryptosporidium parasites in water.     

Prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran

For evaluation of the prevalence of Cryptosporidium sp. infection in diarrheic and non-diarrheic humans in Iran, fecal specimens from diarrheic (n = 129) and non-diarrheic humans (n = 271) were collected and examined for the presence of Cryptosporidium sp. oocysts. The presence of Cryptosporidium sp. oocysts was determined by Ziehl- Neelsen acid-fast staining. Humans were grouped according to their age as follows: younger than 15, 16-25, 26-35, 36-50, and over 51 years. The results showed that the overall prevalence of infection in all 400 samples was 10.8%, but the prevalence (25.6%) in diarrheic humans was higher than that (3.7%) in non-diarrheic humans. Oocysts of Cryptosporidium sp. were detected in the feces of 21.4%, 9.3%, 8.8%, 6.7% and 5.7% of different age groups, respectively. The intensity of oocysts was significantly higher in diarrheic humans than in non-diarrheic ones. There was a significant association between Cryptosporidium sp. infection and occurrence of diarrhea (P < 0.05). The results indicate that Cryptosporidium sp. infection is prevalent in diarrheic humans in Iran.     

Giardia sp. and Cryptosporidium sp. infections in primates in fragmented and undisturbed forest in western Uganda

In June 2005, we collected 115 fecal samples from wild primates in western Uganda and examined them for Cryptosporidium sp. and Giardia sp. with the use of immunofluorescent antibody (IFA) detection. We sampled primates from an undisturbed forest in Kibale National Park and from 3 highly disturbed forest fragments outside the park. Of disturbed forest samples, red colobus (Pilocolobus tephrosceles) and red-tailed guenons (Cercopithecus ascanius) harbored species of Cryptosporidium or Giardia, but black-and-white colobus (Colobus guereza) did not. All primate samples from undisturbed forest were negative for both parasites. Seven of 35 (20%) red colobus and 1 of 20 red-tailed guenons (5%) from forest fragments were infected with either Cryptosporidium sp. or Giardia sp. The presence of Cryptosporidium and Giardia species in primates living in forest fragments, but not in primates in undisturbed forest, suggests that habitat disturbance may play a role in transmission or persistence of these pathogens.     

Genetic diversity of Cryptosporidium spp. in captive reptiles

The genetic diversity of Cryptosporidium in reptiles was analyzed by PCR-restriction fragment length polymorphism and sequence analysis of the small subunit rRNA gene. A total of 123 samples were analyzed, of which 48 snake samples, 24 lizard samples, and 3 tortoise samples were positive for Cryptosporidium: Nine different types of Cryptosporidium were found, including Cryptosporidium serpentis, Cryptosporidium desert monitor genotype, Cryptosporidium muris, Cryptosporidium parvum bovine and mouse genotypes, one C. serpentis-like parasite in a lizard, two new Cryptosporidium spp. in snakes, and one new Cryptosporidium sp. in tortoises. C. serpentis and the desert monitor genotype were the most common parasites and were found in both snakes and lizards, whereas the C. muris and C. parvum parasites detected were probably the result of ingestion of infected rodents. Sequence and biologic characterizations indicated that the desert monitor genotype was Cryptosporidium saurophilum. Two host-adapted C. serpentis genotypes were found in snakes and lizards.     

Detection and characterization of a Cryptosporidium isolate from a southern elephant seal (Mirounga leonina) from the Antarctic peninsula

The presence of Cryptosporidium and Giardia in 221 fecal samples from different species of Antarctic pinnipeds was investigated by immunofluorescence microscopy and PCR. Cryptosporidium, a skunk-like genotype, was detected only in a southern elephant seal. Giardia was not detected. This is the first report of a Cryptosporidium sp. in Antarctic marine mammals.     

Occurrence, source, and human infection potential of cryptosporidium and Giardia spp. in source and tap water in shanghai, china

Genotyping studies on the source and human infection potential of Cryptosporidium oocysts in water have been almost exclusively conducted in industrialized nations. In this study, 50 source water samples and 30 tap water samples were collected in Shanghai, China, and analyzed by the U.S. Environmental Protection Agency (EPA) Method 1623. To find a cost-effective method to replace the filtration procedure, the water samples were also concentrated by calcium carbonate flocculation (CCF). Of the 50 source water samples, 32% were positive for Cryptosporidium and 18% for Giardia by Method 1623, whereas 22% were positive for Cryptosporidium and 10% for Giardia by microscopy of CCF concentrates. When CCF was combined with PCR for detection, the occurrence of Cryptosporidium (28%) was similar to that obtained by Method 1623. Genotyping of Cryptosporidium in 17 water samples identified the presence of C. andersoni in 14 water samples, C. suis in 7 water samples, C. baileyi in 2 water samples, C. meleagridis in 1 water sample, and C. hominis in 1 water sample. Therefore, farm animals, especially cattle and pigs, were the major sources of water contamination in Shanghai source water, and most oocysts found in source water in the area were not infectious to humans. Cryptosporidium oocysts were found in 2 of 30 tap water samples. The combined use of CCF for concentration and PCR for detection and genotyping provides a less expensive alternative to filtration and fluorescence microscopy for accurate assessment of Cryptosporidium contamination in water, although the results from this method are semiquantitative.     

Cryptosporidium and Giardia in marine-foraging river otters (Lontra canadensis) from the Puget Sound Georgia Basin ecosystem

Species of Cryptosporidium and Giardia can infect humans and wildlife and have the potential to be transmitted between these 2 groups; yet, very little is known about these protozoans in marine wildlife. Feces of river otters (Lontra canadensis), a common marine wildlife species in the Puget Sound Georgia Basin, were examined for species of Cryptosporidium and Giardia to determine their role in the epidemiology of these pathogens. Using ZnSO4 flotation and immunomagnetic separation, followed by direct immunofluorescent antibody detection (IMS/DFA), we identified Cryptosporidium sp. oocysts in 9 fecal samples from 6 locations and Giardia sp. cysts in 11 fecal samples from 7 locations. The putative risk factors of proximate human population and degree of anthropogenic shoreline modification were not associated with the detection of Cryptosporidium or Giardia spp. in river otter feces. Amplification of DNA from the IMS/DFA slide scrapings was successful for 1 sample containing > 500 Cryptosporidium sp. oocysts. Sequences from the Cryptosporidium 18S rRNA and the COWP loci were most similar to the ferret Cryptosporidium sp. genotype. River otters could serve as reservoirs for Cryptosporidium and Giardia species in marine ecosystems. More work is needed to better understand the zoonotic potential of the genotypes they carry as well as their implications for river otter health.     

Cryptosporidium pig genotype II diagnosed in pigs from the state of Rio De Janeiro, Brazil

Pigs may represent a source of Cryptosporidium sp. infection to humans. The objective of this study was to identify the Cryptosporidium species present in pigs from the State of Rio de Janeiro, Brazil, and verify what risks pigs represent in the transmission of human cryptosporidiosis, because there is no such information to date in Brazil. Ninety-one samples of pig feces were collected from 10 piggeries in 2 municipalities located in the north and northwest regions of the State of Rio de Janeiro, Brazil. A nested polymerase chain reaction (PCR) protocol to amplify an 830-bp fragment of the small subunit rDNA (SSU rRNA) gene was followed by sequencing of all positive PCR samples. Two samples (2.2%) were Cryptosporidium sp. positive and were identified as pig genotype type II (PGII). This genotype has been observed in an immunocompetent person, in cattle without pigs nearby, and from a potential human source. Its potential for zoonotic transmission is little known and should be rigorously studied.     

Occurrence of Cryptosporidium and Giardia genotypes and subtypes in raw and treated water in Portugal

Aims:  Waterborne outbreaks of diarrhoeal illness reported worldwide are mostly associated with Cryptosporidium spp. and Giardia spp. Their presence in aquatic systems makes it essential to develop preventive strategies for water and food safety. This study was undertaken to monitor the presence of Cryptosporidium and Giardia in a total of 175 water samples, including raw and treated water from both surface and ground sources in Portugal.
Methods and Results:  The samples were processed according to USEPA Method 1623 for immunomagnetic separation (IMS) of Cryptosporidium oocysts and Giardia cysts, followed by detection of oocysts/cysts by immunofluorecence (IFA) microscopy, PCR-based techniques were done on all water samples collected. Out of 175 samples, 81 (46·3%) were positive for Cryptosporidium and 67 (38·3%) for Giardia by IFA. Cryptosporidium spp. and G. duodenalis genotypes were identified by PCR in 37 (21·7%) and 9 (5·1%) water samples, respectively. C. parvum was the most common species (78·9%), followed by C. hominis (13·2%), C. andersoni (5·3%), and C. muris (2·6%). Subtype IdA15 was identified in all C. hominis -positive water samples. S ubtyping revealed the presence of C. parvum subtypes IIaA15G2R1, IIaA16G2R1 and IIdA17G1. Giardia duodenalis subtype A1 was identified.
Conclusions:  The results of the present study suggest that Cryptosporidium spp. and Giardia spp. were widely distributed in source water and treated water in Portugal. Moreover, the results obtained indicate a high occurrence of human-pathogenic Cryptosporidium genotypes and subtypes in raw and treated water samples.
Significance and Impact of the Study:  Thus, water can be a potential vehicle in the transmission of cryptosporidiosis, and giardiasis of humans and animals in Portugal.     

Molecular characterization of Cryptosporidium sp. isolated from northern Alaskan caribou (Rangifer tarandus)

Cryptosporidium sp. was found in 3 out of 49 caribou (Rangifer tarandus) from northern Alaska. Segments of both the 18S ribosomal RNA and the heat shock protein genes were amplified from the caribou isolate and compared with that obtained from an isolate from a wild white-tailed deer (Odocoileus virginianus) in Virginia as well as other species and isolates available from GenBank. Analyses showed the white-tailed deer isolate to be identical with the C. parvum cattle genotype; however, the caribou isolate represents a new genotype closely related to C. serpentis, C. muris, and C. andersoni. Giardia sp. was not detected in any of the caribou samples nor was Cryptosporidium sp. or Giardia sp. detected in any of the 42 moose (Alces alces) samples examined.     

Cryptosporidium sp. and Giardia sp. infections in mountain gorillas (Gorilla gorilla beringei) of the Bwindi Impenetrable National Park, Uganda

For conservation purposes and because of growing ecotourism, some mountain gorilla (Gorilla gorilla beringei) populations have been habituated to humans. Fecal specimens (n = 100) of nonhabituated and human-habituated gorillas (5 populations; 6 age classes) were tested for Cryptosporidium sp. oocysts and Giardia sp. cysts by conventional staining and immunofluorescent antibody (IFA). Cryptosporidium sp. infections (prevalence 11%) were not restricted to very young gorillas but were observed in 3-yr-old to >12-yr-old gorillas; most of the infections (73%) occurred in human-habituated gorillas. The prevalence of Giardia sp. infections was 2%; 1 nonhabituated gorilla was concomitantly infected. Oocysts of Cryptosporidium sp. in the gorilla stools were morphologically, morphometrically, and immunologically undistinguishable from a bovine isolate of Cryptosporidium parvum oocysts. Mean concentration of Cryptosporidium sp. oocysts and Giardia sp. cysts in gorilla stools was 9.39x10(4)/g, and 2.49x10(4)/g, respectively. There was no apparent relationship between oocyst concentration and gorilla age, sex, or habituation status. Most Cryptosporidium sp. infections found in gorillas with closest proximity to people may be a result of the habituation process and ecotourism. This study constitutes the first report of Cryptosporidium sp. infections in the family Pongidae, in the free-ranging great apes, and in the species of gorilla.     

Distribution of cryptosporidium genotypes in storm event water samples from three watersheds in New York

To assess the source and public health significance of Cryptosporidium oocyst contamination in storm runoff, a PCR-restriction fragment length polymorphism technique based on the small-subunit rRNA gene was used in the analysis of 94 storm water samples collected from the Malcolm Brook and N5 stream basins in New York over a 3-year period. The distribution of Cryptosporidium in this study was compared with the data obtained from 27 storm water samples from the Ashokan Brook in a previous study. These three watersheds represented different levels of human activity. Among the total of 121 samples analyzed from the three watersheds, 107 were PCR positive, 101 of which (94.4%) were linked to animal sources. In addition, C. hominis (W14) was detected in six samples collected from the Malcolm Brook over a 2-week period. Altogether, 22 Cryptosporidium species or genotypes were found in storm water samples from these three watersheds, only 11 of which could be attributed to known species/groups of animals. Several Cryptosporidium spp. were commonly found in these three watersheds, including the W1 genotype from an unknown animal source, the W4 genotype from deer, and the W7 genotype from muskrats. Some genotypes were found only in a particular watershed. Aliquots of 113 samples were also analyzed by the Environmental Protection Agency (EPA) Method 1623; 63 samples (55.7%) were positive for Cryptosporidium by microscopy, and 39 (78%) of the 50 microscopy-negative samples were positive by PCR. Results of this study demonstrate that molecular techniques can complement traditional detection methods by providing information on the source of contamination and the human-infective potential of Cryptosporidium oocysts found in water.     

Cryptosporidium,Giardia, and Cyclospora in ancient Peruvians

Twenty-two coprolites of human origin, collected from excavations along the north-central coast of Peru, were examined using fluorescent microscopy for the presence of fecal parasites, with emphasis on Cryptosporidium sp., Giardia sp., and Cyclospora sp. Three samples were positive. One coprolite dated between ca. 2,375 and 1,525 BC contained Giardia sp. cysts. This coprolite corresponded to the Peruvian preceramic period. Another positive coprolite ca. AD 770-830 corresponded to Epoch 3 of the Middle Horizon and contained Cryptosporidium sp. oocysts. The third positive coprolite (corresponding to the Middle Horizon. ca. AD 500-900) contained Giardia sp. cysts. This report demonstrates that Giardia sp. and Cryptosporidium sp. were present in Peruvian coastal populations for at least 4,300 and 1,100 BP.     

Detection of Cryptosporidium--specific coproantigen in human immunodeficiency virus/acquired immunodeficiency syndrome patients by using a commercially available immunoenzymatic assay

The aim of this study was to verify the occurrence of Cryptosporidium infection in 52 human immunodeficiency virus (HIV)/acquired immunodeficiency syndrome (AIDS) patients (group 1) and 38 clinically healthy individuals (group 2) by using enzyme immunoassay (EIA). All fecal samples collected were submitted to the Baermann, Lutz and Ritchie methods, the Safranin/Methylene Blue, and Weber's chromotrope modified Trichrome staining techniques, and EIA. In group 1, parasitological staining techniques and EIA were both positive for Cryptosporidium sp. infection in 3/52 (5.8%) samples and both negative in 45/52 (86.5%) samples, while 4/52 (7.7%) samples were positive in EIA and negative in parasitological staining techniques. Concerning group 2, all samples were negative by EIA and microscopy for Cryptosporidium infection. In conclusion, EIA may be an alternative method for detecting Cryptosporidium-specific coproantigen in HIV/AIDS patients.