Cryptosporidium serpentis oocysts and microsporidian spores in feces of captive snakes

Fecal smears of 90 snakes, 29 lizards, and 8 turtles and tortoises were tested for Cryptosporidium spp. oocysts and microsporidian spores. Microsporidian spores measured mean = 3.7 microm in length and mean = 2.3 microm in width and were present in feces of 19 snakes and 1 lizard (16%); 13 of these snakes also shed Cryptosporidium serpentis oocysts. The oocysts were numerous in all positive samples, whereas microsporidian spores were always sparse, irrespective if whether fecal samples contained the oocysts. Retrospective examination of reptile clinical records revealed that all animals shedding microsporidian spores died naturally due to diseases, pathologic conditions, and clinical problems or were killed due to severe cryptosporidiosis. The present study indicates that microsporidian infections in reptiles have the features of an opportunistic infection.     

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.     

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.     

Cryptosporidium sp. Infections in Green Turtles, Chelonia mydas, as a Potential Source of Marine Waterborne Oocysts in the Hawaiian Islands

For the first time, Cryptosporidium sp. oocysts were identified in fecal and intestinal samples from free-ranging marine turtles, Chelonia mydas, from the Hawaiian Islands. The oocysts produced positive reactions with commercial test kits recommended for the detection of human-infectious waterborne oocysts of Cryptosporidium parvum.     

Host specificity of Cryptosporidium sp. isolated from chickens

The host specificity of Cryptosporidium sp. infecting chickens was evaluated by oral inoculation of oocysts into 6 different species of neonatal rodents, adult nude mice (athymic), neonatal conventional and gnotobiotic pigs, turkeys, muscovy ducks and bobwhite quail. Examinations of tissue sections, ileal mucosal smears, fecal flotations and stained feces failed to reveal any infections in the mammalian species examined. Oocysts were observed in the feces, and developmental stages were observed in tissue sections, of turkeys and muscovy ducks but not bobwhite quail. This study indicates that Cryptosporidium sp. infections in avian species are probably not a zoonotic threat to humans.     

Isolation of Cryptosporidium sp. oocysts from human fecal samples]

Experiments using two sequential discontinuous sucrose gradients, performed with 12%, 18%, 21% and 24% solutions, for the isolation of Cryptosporidium sp. oocysts from human fecal samples were undertaken. The sucrose gradients were centrifuged at 4000 rpm during 20 min. at 10 degrees C or room temperature. After that, 3 bands were observed. Oocysts were recovered mainly from the second band (75.5% after the first gradient, and 44.4% in the next). The two sequential discontinuous sucrose gradients would permit an efficient isolation of Cryptosporidium sp. oocysts from human fecal samples.     

Isolation and identification of Cryptosporidium from various animals in Korea. III. Identification of Cryptosporidium baileyi from Korean chicken]

Each of SPF chicken (Hi-Line strain, 2-day-old males) was inoculated with 2.5 or 5 x 10(4) oocysts by stomach tube. The oocyst was the medium type of Cryptosporidium previously isolated from Korean chicken origin, and passed in 2-day-old SPF chicken. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of the developmental stages, the bursa of Fabricius of the chicken was examined by transmission electron microscopy (TEM) on the 12th day postinoculation. The prepatent period for 8 chicken was 5.9 days postinoculation on the average, and the patent period was 12.9 days. The number of oocysts discharged per day for the chicken was reached peak on day 12 postinoculation on the average. A large number of oocysts was found in fecal samples obtained from inoculated chicken on days 8-14 postinoculation. The ultrastructural feature of almost every developmental stage of the medium type from chicken was very similar to that of Cryptosporidium previously isolated from mammalia including human and birds except for the attachment site of C. muris to the mucus cell from mammalia, but dimension of the oocysts from fecal samples of the medium type was different from those of C. meleagridis and mammalia origin. The above results reveal that the medium type of Cryptosporidium of Korean chicken origin is identified as Cryptosporidium baileyi.     

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.     

Waterborne Giardia cysts and Cryptosporidium oocysts in the Yukon, Canada.

Several outbreaks of waterborne giardiasis have occurred in southern Canada, but nothing has been reported from the Canadian North. The objective of this study was to collect information relevant to waterborne giardiasis and cryptosporidiosis in the Yukon including epidemiological data and analyses of water, sewage, and animal fecal samples. Remote, pristine water samples were found to be contaminated with Giardia cysts (7 of 22 or 32%) but not with Cryptosporidium oocysts. Giardia cysts were found in 21% (13 of 61) of animal scats, but no Cryptosporidium oocysts were observed (small sample size). Whitehorse's drinking water was episodically contaminated with Giardia cysts (7 of 42 or 17%) and Cryptosporidium oocysts (2 of 42 or 5%). Neither were found in Dawson City's water supply. The only water treatment in the Yukon is chlorination, but contact times and free chlorine residuals are often too low to provide adequate protection by disinfection. Raw sewage samples from the five largest population centers in the Yukon contained 26 to 3,022 Giardia cysts and 0 to 74 Cryptosporidium oocysts per liter. Treated sewage from Whitehorse contained fewer Giardia cysts but more Cryptosporidium oocysts on average. Both were detected in Lake Laberge, downstream of Whitehorse, which has a history of fecal coliform contamination. Daily monitoring of raw sewage from the suburbs of Whitehorse showed a summertime peak of Giardia cysts and occasional Cryptosporidium oocysts after springtime contamination of drinking water. Despite this evidence, epidemiological data for the Yukon showed an endemic infection rate of only 0.1% for giardiasis (cryptosporidiosis is not notifiable).(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Diseases and parasites in wolves of the Riding Mountain National Park region, Manitoba, Canada

We examined wolf (Canis lupus) blood and fecal samples from the Riding Mountain National Park (RMNP) region of Manitoba, Canada. In 601 fecal samples collected during two study periods in RMNP and the Duck Mountain Provincial Park and Forest (DMPPF) we found gastrointestinal helminth eggs from Alaria sp. (15.5%), Capillaria sp. (1.0%), taeniid tapeworms (30.8%), Toxascaris sp. (1.7%), Toxocara sp. (0.2%), Trichuris sp. (2.2%), and Moniezia sp. (0.5%). In addition, we found Demodex sp. (0.2%) and the protozoal cysts/oocysts of Sarcocystis sp. (37.3%), Cryptosporidium sp. (1.2%), coccidia (Isospora sp. or Eimeria sp.) (1.7%), and Giardia sp. (29.5%). No fecal shedding of canine parvovirus (CPV, n=387) was detected. All 18 blood samples collected in RMNP showed CPV exposure and eight of 18 blood samples indicated canine distemper virus (CDV) exposure. One wolf died from CDV. Our results are consistent with previous findings on pathogens affecting wolves and with high Giardia sp. prevalence in wolves inhabiting agricultural regions.     

Isolation and identification of Cryptosporidium from various animals in Korea. II. Identification of Cryptosporidium muris from mice

Each of SPF mice(Scl: ICR strain, 3-week-old males) was inoculated with 5 x 10(4) oocysts of Cryptosporidium by stomach tube. The oocysts were large type one which was previously isolated from Korean mice, and passaged in 3-week-old SPF mice. The patterns of oocyst discharge were monitored daily, and in order to observe the ultrastructure of developmental stages the stomach of the mice was examined by transmission electron microscopy (TEM) at 4 weeks post-inoculation. The prepatent period for 6 mice was 5.6 days post-inoculation on the average, and the patent period was 63.2 days. The number of oocysts discharged per day from the mice reached peak on day 36.6 post-inoculation on the average. A large number of oocysts were found in fecal samples obtained from inoculated mice on days 30-50 post-inoculation. C. muris was larger than C. parvum at almost every developmental stages, the size difference being 1.4 times in oocysts, 2.4 times in sporozoites, 1.6 times in merozoites, and 1.5 times in microgametes. The ultrastructural features of the attachment site of C. muris to the mucus cells were remarkably different from those of C. parvum and its closely related species. The anterior projection of the protozoa (C. muris), the outer aspect of which was surrounded by a thick filamentous process of the host cell, has not been reported at any developmental stages of C. parvum or its closely related species. The size of the oocysts of strain RN 66 was larger than that of Korean mice origin. The above results reveal that the large type Cryptosporidium of Korean mice origin is identified as Cryptosporidium muris and this type was named as C. muris (strain MCR).     

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.     

PCR-Mediated Recombination between Cryptosporidium spp. of Lizards and Snakes

ABSTRACT: The presence or absence of genetic recombination has often been used as one of the criteria for Cryptosporidium species designation and population structure delineation. During a recent study of cryptosporidiosis in reptiles that were housed in the same room, 4 lizards were found to have concurrent infections of C. serpentis (a gastric parasite) and C. saurophilum (an intestinal parasite), and 6 snakes were concurrently infected with C. serpentis, C. sattrophitmm and a new Cryptosporidium as indicated by PCR-RFLP analysis of the SSU rRNA gene. DNA sequence analysis of cloned PCR products confirmed the diagnosis of mixed infections. Surprisingly, it appeared that 11 of the 22 clones (8 and 14 clones from a lizard and a snake, respectively) had chimeric sequences of two Cryptosporidium spp. BootScan analysis indicated the existence of recombinants among the cloned sequences and detection of the informative sites confirmed the BootScan results. Because the probability for genetic recombination between gastric and intestinal parasites is small, these hybrid sequences were likely results of PCR artifacts due to the presence of multiple templates. This was confirmed by PCR-sequencing analysis of single-copy templates using diluted DNA samples. Direct sequencing of 69 PCR products from 100-to 1.000-fold diluted DNAs from the same snake and lizard produced only sequences of C. serpentis, C. saurophilum and the unnamed Cryntosnoridium. , sp. Thus, care should be taken to eliminate PCR artifacts when determining the presence of genetic recombination or interpreting results of population genetic studies.     

Indirect immunofluorescent detection of oocysts of Cryptosporidium parvum in the feces of naturally infected raccoons (Procyon lotor)

Fecal samples from 100 wild raccoons were examined for the presence of oocysts of Cryptosporidium parvum using a commercially available indirect immunofluorescent detection procedure. Thirteen (13%) of the samples were positive for oocysts. All positive samples were from juvenile raccoons. Over 61% of the infected samples contained moderate to large numbers of oocysts. Raccoons may serve as potential reservoirs for transmission of C. parvum.     

Hepatozoon kisrae n. sp. infecting the lizard Agama stellio is transmitted by the tick Hyalomma cf. aegyptium

Hepatozoon kisrae n. sp. was found infecting a starred lizard at a site in southeastern Samaria, Palestine. These lizards were also hosts to the ixodid tick Hyalomma cf. aegyptium, which was demonstrated to be the vector of this hemogregarine. Hepatozoon and tick infections occurred in lizards within a very restricted locality; at a second site, nearby, ticks occurred without Hepatozoon infection. Micro- and macromeronts occurred mainly in the lungs, while cyst-like merogonic stages, mainly dizoic, occurred in the liver. Mature intraerythrocytic gametocytes were stout and encapsulated. Development from oocysts to sporocysts took place in the tick hemocoel, and was examined by transmission electron microscopy. Lizards were successfully infected when fed on sporocyst-infected ticks or viscera of infected lizards. Ticks become infected when fed on infected lizards; sporogony was complete when the ticks reached adult stage, over 40 days after initial attachment.     

The identification of Cryptosporidium species (protozoa) in Ifsahan, Iran by PCR-RFLP analysis of the 18S rRNA gene

Cryptosporidium is an important protozoan that cause diarrheal illness in humans and animals. Different species of Cryptosporidium have been reported and it is believed that species characteristics are an important factor to be considered in strategic planning for control. We therefore analyzed oocysts from human and animal isolates of Cryptosporidium by PCR-RFLP to determine strain variation in Isfahan. In total, 642 human fecal samples from children under five years of age, immunocompromised patients, and high risk persons and 480 randomly selected rectal specimens of cows and calves in Isfahan were examined. Microscopic examination showed that 4.7% (30/642) of human samples and 6.2% (30/480) of animal samples were infected with Cryptosporidium. After identification of the samples infected with the parasite, oocysts were purified and their DNA was extracted. We used PCR-RFLP analysis of a 1750-bp region of 18S rRNA gene to identify Cryptosporidium species. The human samples were infected with Cryptosporidium parvum II, C. muris, C. wrairi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520951). The cattle samples were identified as C. parvum II, C. muris, C. wrairi, C. serpentis, C. baileyi, and a new genotype of Cryptosporidium (GenBank accession numbers: DQ520952). Also we found a new genotype infecting both human and cattle samples (GenBank accession numbers: DQ520950). In addition to demonstrating the widespread occurrence of most species of Cryptosporidium, C. parvum, we also observed extensive polymorphism within species. Furthermore, the occurrence of the same species of parasite in both animal and human samples shows the importance of the animal-human cycle.     

Prevalence and molecular identification of Cryptosporidium isolates from pet lizards and snakes in Italy

In order to acquire prevalence and genetic data on Cryptosporidium infections in captive lizards and snakes kept as pets, a survey was conducted on 150 individual reptiles from southern Italy. Fecal samples were preserved in 5% formalin and analyzed using a commercial immunofluorescence assay (IFA) for the detection of Cryptosporidium oocysts and Giardia cysts. IFA revealed the presence of Cryptosporidium oocysts in nine of the 150 samples examined (6.0%), precisely in 6/125 snakes (4.8%) and in 3/25 lizards (12.0%); all fecal samples tested negative for the presence of Giardia cysts. Molecular characterization based on nested PCR amplification and sequencing of the SSU-rRNA gene, revealed the presence of Cryptosporidium serpentis in three samples from snakes (Boa constrictor constrictor, Elapheguttata guttata guttata and Python molurus).     

A case of human cryptosporidiosis in Czechoslovakia

A case of human cryptosporidiosis, the first one reported in Czechoslovakia, is described. The disease was diagnosed by the presence of Cryptosporidium oocysts in the feces. The methods used independently to identify oocysts were the fecal flotation technique employing a saturated solution of sucrose and the microscopic examination of stained fecal smears. The patient was a 4-year-old boy with watery diarrhea of 5 days' duration who was kept on a diet and treated with a suspension of Endiaron N Spofa. Excretion positivity for Cryptosporidium oocysts in the feces was detectable at 3 and 5 days after appearance of first clinical manifestations. Bacteriological examination was repeatedly negative. This finding leaves little doubt as to existence of human cryptosporidiosis in Czechoslovakia, but what remains obscure is its overall contribution to the etiology of diarrheal diseases encountered in the population.