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Cryptosporidium causes severe gastrointestinal disease resulting from the ingestion of oocysts, followed by oocyst excystation in the small intestine and the release of infective sporozoites. An understudied strategy for Cryptosporidium inactivation is purposeful oocyst excystation, as sporozoites do not survive long in the environment. This study showed that C. parvum oocyst excystation was induced by direct contact with various glycosaminoglycans (GAGs), including heparin (Hep), chondroitin sulfate A (CSA), and hyaluronan (HA), assembled on polydopamine (PD)-functionalized surfaces. PD surfaces elicited 97.9 ± 3.6% oocyst attachment, with some of the attached oocysts partially (7.3 ± 1.3%) or fully (4.0 ± 0.6%) excysted after 4 days. The PD-GAG surfaces (GAG concentration = 2 mg/mL) elicited similarly high attachment (>97%) and higher oocyst excystation efficiencies after 4 days. The PD-Hep surfaces elicited the highest number of attached excysted oocysts (11.8 ± 0.63% partially excysted; 11.9 ± 0.49% fully excysted), and the PD-HA surfaces elicited the lowest (8.8 ± 2.1% partially excysted; 7.8 ± 1.2% fully excysted). Surface characterization revealed that the addition of GAGs to the PD surface changed both the surface roughness as well as the surface wettability. Treatment of oocysts with an enzyme that degraded the surface glycocalyx markedly reduced excystation (to <2%) of the oocysts attached to the PD and PD-GAG surfaces. These findings suggest that GAGs provide an important local signal for the excystation of C. parvum oocysts and that certain surface-expressed oocyst receptors are necessary for efficient excystation. These oocyst-receptor relationships may be useful in the design of functionalized surfaces for the purposeful inactivation of oocysts in the environment or in water treatment systems. IMPORTANCE Polydopamine surfaces functionalized with glycosaminoglycans were shown to facilitate the attachment and excystation of Cryptosporidium parvum oocysts. Our findings suggest that a surface-expressed receptor on the oocyst wall plays a key role in excystation, with glycosaminoglycans serving as ligands that trigger the initiation of the process. Future technologies and treatment strategies designed to promote premature excystation of oocysts will minimize the ingestion of sporozoites that initiate infection. Therefore, the results from this study have important implications for the protection of public health from waterborne cryptosporidiosis and may serve as a foundation for engineered surfaces designed to remove oocysts from surface waters or inactivate oocysts in water treatment systems.

Cryptosporidium spp. are protozoan parasites that are transmitted via fecal-oral routes and can exhibit chemical resistance. Chlorine resistance makes it very difficult to eliminate parasites present in contaminated drinking water. While the efficacy of ultraviolet light-emitting diodes (UV-LEDs) against microorganisms has been reported, the efficacy of UV-LEDs against Cryptosporidium spp. has not been fully evaluated. Here, we assessed the efficacy of UV-LEDs with peak wavelengths of 268, 275, 284, and 289 nm against Cryptosporidium parvum at various exposure times, with a fixed exposure distance, using two in vitro methods. Consequently, the time required for 2 log 10 inactivation through the excystation method by UV-LEDs of 268, 275, 284, and 289 nm was estimated as 115.5, 104.1, 37.4, and 30.7 min, respectively. The propidium iodide (PI) and 4',6-diamidino-2-phenylindole (DAPI) staining assays estimated the inactivation time as 311.3, 275.2, 60.6, and 39.1 min, respectively. Our results showed that UV-LED irradiation at longer wavelengths produced higher inactivation activity against C. parvum, which corroborates our previously reported in vivo assay results, although further study is needed to clarify the mechanism., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Cryptosporidiosis is a disease caused by infection with an intestinal coccidian parasite Cryptosporidium. Cryptosporidium species are the second leading cause of diarrheal disease and death in children in developing countries. Until now, no data have been available or published on its prevalence among children with diarrhea in Sudan. Therefore, this paper was designed to determine the prevalence rate of Cryptosporidium among children with diarrhea under 5 years who were admitted to Kosti Teaching Hospital., Methods: A hospital-based cross-sectional study including children under 5 years old admitted to the pediatric section of the hospital between September 2020 and December 2020. A total of one-hundred and fifty stool samples were collected. All stool samples were examined using the modified Ziehl Neelsen (mZN) staining technique and then examined microscopically for Cryptosporidium infection., Results: A total of 150 children were examined out of which 70 presented with diarrhea. A greater prevalence of 19/70 (27.1%) of Cryptosporidium was observed in children with diarrhea than children without diarrhea 7/80 (8.8%). There was a significant relationship between the prevalence of Cryptosporidium and the presence of diarrhea in children under 5 years in the Kosti Teaching Hospital(P < 0.05). It was found that a higher prevalence was registered among children using piped-water sources for drinking., Conclusions: The overall prevalence of parasite detected was 17.3% among children admitted to Kosti Teaching Hospital. The prevalence rate of the infection among Children with diarrhoea was 27.1%. Studying the prevalence rate of cryptosporidiosis among diarrheic children may predict their health status, leading to a better diagnosis, treatment, and, therefore, patients' status improvement.

Infection with protozoa of the genus Cryptosporidium is a leading cause of child morbidity and mortality associated with diarrhea in the developing world. Research on this parasite has been impeded by many technical limitations, including the lack of cryopreservation methods. While cryopreservation of Cryptosporidium oocysts by vitrification was recently achieved, the method is restricted to small sample volumes, thereby limiting widespread implementation of this procedure. Here, a second-generation method is described for cryopreservation of C. parvum oocysts by vitrification using custom high aspect ratio specimen containers, which enable a 100-fold increase in sample volume compared to previous methods. Oocysts cryopreserved using the described protocol exhibit high viability, maintain in vitro infectivity, and are infectious to interferon-gamma (IFN-γ) knockout mice. Importantly, the course of the infection is comparable to that observed in mice infected with unfrozen oocysts. Vitrification of C. parvum oocysts in larger volumes will expedite progress of research by enabling the sharing of isolates among different laboratories and the standardization of clinical trials.

Cryptosporidium is a genus of ubiquitous unicellular parasites belonging to the phylum Apicomplexa. Cryptosporidium species are the second largest cause of childhood diarrhea and are associated with increased morbidity. Accompanying this is the low availability of treatment and lack of vaccines. The major barrier to developing effective treatment is the lack of reliable in vitro culture methods. Recently, our lab has successfully cultivated C. parvum in the esophageal cancer-derived cell line COLO-680N, and has been able to maintain infection for several weeks. The success of this cell line was assessed with a combination of various techniques including fluorescent microscopy and qPCR. In addition, to tackle the issue of long-term oocyst production in vitro, a simple, low-cost bioreactor system using the COLO-680N cell line was established, which produced infectious oocysts for 4 months. This chapter provides details on the methodologies used to culture, maintain, and assess Cryptosporidium infection and propagation in COLO-680N. © 2019 by John Wiley & Sons, Inc., (© 2019 John Wiley & Sons, Inc.)

Members of the genus Cryptosporidium are frequent protozoan pathogens in humans and a wide range of animals. There is no consistently effective treatment against cryptosporidiosis, especially in immunodeficient patients. The present study was carried out to study the therapeutic effects of curcumin against cryptosporidiosis in immunosuppressed BALB/c mice. Mice were divided into five groups and immunosuppressed by dexamethasone. Three groups were inoculated with C. parvum oocysts, administered with curcumin, paromomycin, and without treatment. The reminders were regarded as controls. The oocysts in the fecal smear were counted daily. At days 0, 3, 7, and 11 post-treatment, the mice were sacrificed, and the efficacy of drugs was evaluated by comparing the histopathological alterations in jejunum and ileum, measuring the total antioxidant capacity, and malondialdehyde in the affected tissues. The infection was completely eliminated in the curcumin-treated group, and oocyst shedding stopped with no recurrence after drug withdrawal. On the contrary, paromomycin was unable to eliminate C. parvum infection completely, and oocyst shedding continued even 10 days after the drug withdrawal. Based on these findings, curcumin can be a trustworthy compound for the elimination of infection in immunosuppressed hosts. Further evaluation to find its accurate mechanism of action should be considered., (Copyright © 2018 Elsevier Inc. All rights reserved.)

At present, there are few technologies which enable the detection, identification and viability analysis of protozoan pathogens including Cryptosporidium and/or Giardia at the single (oo)cyst level. We report the use of Microfluidic Impedance Cytometry (MIC) to characterise the AC electrical (impedance) properties of single parasites and demonstrate rapid discrimination based on viability and species. Specifically, MIC was used to identify live and inactive C. parvum oocysts with over 90% certainty, whilst also detecting damaged and/or excysted oocysts. Furthermore, discrimination of Cryptosporidium parvum, Cryptosporidium muris and Giardia lamblia, with over 92% certainty was achieved. Enumeration and identification of (oo)cysts can be achieved in a few minutes, which offers a reduction in identification time and labour demands when compared to existing detection methods.

Worldwide, high incidences of cryptosporidiosis and giardiasis are attributed to livestock waste. Quantitative microbial risk assessment can be used to estimate the risk of livestock related infections from Cryptosporidium parvum and Giardia lamblia. The objective of this paper was to assess the occupational and public health risks associated with management of raw and anaerobically digested livestock waste in two rural communities in Costa Rica based on fomite, soil and crop contamination and livestock waste management exposure pathways. Risks related to cattle waste were greater than swine waste due to cattle shedding more (oo)cysts. Cryptosporidium parvum also posed a greater risk than Giardia lamblia in all exposure pathways due to livestock shedding high loads of Cryptosporidium parvum oocysts and oocysts' lower inactivation rates during anaerobic digestion compared with Giardia lamblia cysts. The risk of infection from exposure to contaminated soil and crops was significantly lower for a community using tubular anaerobic digesters to treat livestock waste compared to a community where the untreated waste was applied to soil. The results indicate that treatment of livestock waste in small-scale tubular anaerobic digesters has the potential to significantly decrease the risk of infection below the World Health Organization's acceptable individual annual risk of infection (10 -4 ).

Faecal samples were obtained from 433 wild birds being treated in wildlife recovery centres in Galicia (Northwest Spain), between February 2007 and September 2009. The birds belonged to 64 species representing 17 different orders. Giardia cysts and Cryptosporidium oocysts were detected by an immunofluorescence antibody test and identified at the molecular level by established PCR-sequencing methods. The overall prevalence of Giardia was 2·1% and that of Cryptosporidium, 8·3%. To our knowledge, this is the first description of Giardia sp. in Tyto alba and Caprimulgus europaeus; and of Cryptosporidium sp. in Apus apus, Athene noctua, C. europaeus, Falco tinnunculus, Morus bassanus, Parabuteo unicinctus and Strix aluco. Furthermore, the first PCR-sequence confirmed detection of Giardia duodenalis assemblage B in, Buteo buteo, Coturnix coturnix and Pica pica; G. duodenalis assemblage D in Garrulus glandarius; and G. duodenalis assemblage F in Anas platyrhynchos; Cryptosporidium parvum in Accipiter nisus, B. buteo, Milvus migrans, Pernis apivorus and P. pica; and Cryptosporidium meleagridis in Streptopelia turtur. The study findings demonstrate the wide spread of Giardia and Cryptosporidium between wild birds.

The prevalence of Cryptosporidium in sheep in the eastern states of Australia has not been well described, therefore a study of the prevalence, oocyst concentration, species and subtypes of Cryptosporidium were assessed from lamb faecal samples at three sampling periods (weaning, post-weaning and pre-slaughter) from eight farms across South Australia, New South Wales, Victoria and Western Australia. A total of 3412 faecal samples were collected from approximately 1182 lambs across the four states and screened for the presence of Cryptosporidium using a quantitative PCR (qPCR) at the actin locus. Positives were typed at the 18S locus and at a second locus using C. parvum and C. hominis specific qPCR primers. The overall prevalence was 16.9% (95% CI: 15.6-18.1%) and of the 576 positives, 500 were successfully genotyped. In general, the prevalence of Cryptosporidium was higher in WA than the eastern states. Cryptosporidium prevalence peaked at 43.9% and 37.1% at Pingelly (WA2) and Arthur River (WA1), respectively during weaning and at Pingelly (WA2) during pre-slaughter (36.4%). The range of oocyst shedding at weaning overall across all states was 63-7.9×10(6) and the median was 3.2 × 10(4) oocysts g(-1). The following species were identified; C. xiaoi (69%-345/500), C. ubiquitum (17.6%-88/500), C. parvum (9.8%-49/500), C. scrofarum (0.8%-4/500), mixed C. parvum and C. xiaoi (2.4%-12/500), C. andersoni (0.2%-1/500) and sheep genotype 1 (0.2%-1/500). Subtyping of C. parvum and C. ubiquitum isolates identified IIa and IId subtype families within C. parvum (with IId as the dominant subtype) and XIIa within C. ubiquitum. This is the first published description of C. parvum subtypes detected in lambs in Australia., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Objective: Cryptosporidium is an important zoonotic parasite in humans and animals worldwide. This study was conducted to investigate the prevalence of Cryptosporidium infection in Iran., Methods: Fecal samples (n=1.749) were collected randomly in asymptomatic sheep from different rural regions of Iran in 2011 to 2012. All samples were examined by using the cold modified Ziehl-Neelsen staining technique., Results: Oocysts of Cryptosporidium was found in 11.3% (198/1749) of samples (9.8 < CI 95% < 12.8). There was a statistical differences among Cryptosporidium infection, age groups (p < 0.0001), and gender (p=0.02)., Conclusion: This study is the first report of Cryptosporidium infection in sheep in different regions of Iran. Therefore, further comprehensive molecular studies in sheep to identify the source of contaminations (animals or humans) and designing control strategies is highly recommended.

The objective of the present study was to evaluate the characteristics of two rapid tests, namely, a faecal smear staining method (Heine staining) and a commercially available immunochromatographic (IC) assay, against a direct immunofluorescent antibody test (IFAT) for the diagnosis of Cryptosporidium infections in 917 faecal samples from calves aged <3 weeks. These rapid tests were performed on non-concentrated faeces using a semi-quantitative approach using a 6-point scale (0-5) for Heine staining according to the number of oocysts per microscopic field, and a 4-point scale (0-3) for the IC assay reflecting the intensity of the positive line compared to the control line. Direct IFAT was performed following a diethyl ether concentration and results were expressed as oocysts per g of faeces (opg). Heine staining showed a sensitivity of 76.7% and a specificity of 90.7%. For faecal samples with ≥ 10,000opg, sensitivity increased to 90.0%. The sensitivity of the IC assay was lower (61.8%) but the specificity was 100%. For faeces with ≥ 100,000opg, the sensitivity of the IC assay reached 81%, indicating some limitation for clinical cryptosporidiosis diagnosis. Additional scoring (1-5) of the Heine staining correlated with the corresponding direct IFAT results, particularly for ranges of 1000 to >1,000,000opg. Additional scoring from 1 to 3 according to the thickness of the sample line for the IC test correlated with increasing levels of Cryptosporidium opg measured by IFAT in the range of 10,000 to >1,000,000opg. In conclusion, both Heine staining and the IC test can be reliably used through a simple semi-quantitative scale for grossly quantifying oocyst output in calf faecal samples., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Cryptosporidium is a zoonotic protozoan parasite with public health importance worldwide. The objectives of this study were to (1) conduct a meta-analysis of published literature for oocyst shedding and diarrhoea outcomes, and (2) develop recommendations for standardization of experimental dose-response studies. Results showed that for the outcome of oocyst shedding in faeces, the covariates 'experimental species', 'immunosuppression', 'oocyst dose' and 'oocyst dose' × 'age' were all significant (P≤0.05). This study suggests that exposing mice, piglets, or ruminants, and using immunosuppressed experimental hosts, is more likely to result in oocyst shedding. For the outcome of diarrhoea in experimentally infected animal species, the key covariates 'experimental species', 'age' and 'immunosuppression' were significant (P≤0.2). Therefore, based on the results of this meta-analysis, these variables should be carefully reported and considered when designing experimental dose-response studies. Additionally, detection of possible publication bias highlights the need to publish additional studies that convey statistically non-significant as well as significant results in the future.

Because of outbreaks of cryptosporidiosis in humans, some Cryptosporidium spp. have become a public health concern. Commercial swine operations can be a source of this protozoan parasite. Although the species distribution of Cryptosporidium is likely dominated by Cryptosporidium suis , a fraction may be comprised of other Cryptosporidium species infectious to humans such as Cryptosporidium parvum . To better understand the survival dynamics of Cryptosporidium spp., oocysts associated with swine operations, 2 experiments were performed to determine die-off rates of C. parvum oocysts in a swine waste lagoon (2009 and 2010) and its spray field (2010 and 2011). Sentinel chambers containing a lagoon effluent suspension of C. parvum oocysts were submerged in the lagoon, and triplicate chambers were removed over time; oocysts were extracted and assayed for viability. For comparative purposes, inactivation rates of Ascaris suum eggs contained in sentinel chambers were also determined. For 2 spray field experiments, air-dried and sieved surface soil was placed in sentinel chambers, hydrated, and inoculated with a lagoon effluent suspension of C. parvum oocysts. Sentinel chambers and control oocysts in PBS contained in microcentrifuge tubes were buried 1.5 cm below the soil surface in 3 blocks. Triplicate chambers and controls were removed over time; oocysts were extracted and assayed for viability. Based on the first order decay equation, days to reach 99% die-off (T(99)) were determined. T(99)-values determined for the 2 lagoon experiments were 13.1 and 20.1 wk, respectively. A T(99)-value for C. parvum in the spray field was significantly longer at 38.0 wk than the control oocysts in PBS at 29.0 wk. The waste lagoon and spray field system of manure management at this large-scale farrowing operation appeared to reduce the load of C. parvum oocysts before they can be hydrologically transported off the operation and reduces their likelihood of contaminating surface waters and threatening public health.

The present study examined the prevalence and genotypes of Cryptosporidium andersoni in cattle in Shaanxi province, China. A total of 2071 fecal samples (847 from Qinchuan cattle and 1224 from dairy cattle) were examined for the presence of Cryptosporidium oocysts, and 70 samples (3.4%) were C. andersoni-positive and those positive samples were identified by PCR amplification of the small subunit ribosomal RNA (SSU rRNA) and the Cryptosporidium oocyst wall protein (COWP) genes. C. andersoni was the only species found in the examined cattle in this province. Fifty-seven C. andersoni isolates were characterized into 5 MLST subtypes using multilocus sequence typing analysis, including a new subtype in the native beef breed Qinchuan cattle. All of these C. andersoni isolates presented a clonal genetic structure. These findings provide new insights into the genetic structure of C. andersoni isolates in Shaanxi province and basic data of Cryptosporidium prevalence status, which in turn have implications for controlling cryptosporidiosis in this province.

Water scarcity leads to an increased use of reclaimed water, which in turn calls for an improvement in water reclamation procedures to ensure adequate quality of the final effluent. The presence of infectious Cryptosporidium oocysts (IOO) in reclaimed water is a health hazard for users of this resource. Here, we gathered information on Cryptosporidium (concentrations, infectivity and genotype) in order to perform quantitative microbial risk assessment (QMRA). Moreover, data concerning the spores of sulphite-reducing clostridia (SRC) were used to undertake QMRA at a screening level. Our results show that the probability of infection (PI) by Cryptosporidium depends on the tertiary treatment type. The mean PI using the exponential dose-response model was 3.69 × 10(-6) in tertiary effluents (TE) treated with UV light, whereas it was 3 log(10) units higher, 1.89 × 10(-3), in TE not treated with this disinfection method. With the β-Poisson model, the mean PI was 1.56 × 10(-4) in UV-treated TE and 2 log(10) units higher, 4.37 × 10(-2), in TE not treated with UV. The use of SRC to perform QMRA of Cryptosporidium showed higher PI than when using directly IOO data. This observation suggests the former technique is a conservative method of QMRA.

A comprehensive quantitative microbial risk assessment (QMRA) of Cryptosporidium infection, considering pathogen removal efficiency, different exposure pathways and different susceptible subpopulations, was performed based on the result of a survey of source water from 66 waterworks in 33 major cities across China. The Cryptosporidium concentrations in source water were 0-6 oocysts/10 L, with a mean value of 0.7 oocysts/10 L. The annual diarrhea morbidity caused by Cryptosporidium in drinking water was estimated to be 2701 (95% confidence interval (CI): 138-9381) cases per 100,000 immunodeficient persons and 148 (95% CI: 1-603) cases per 100,000 immunocompetent persons, giving an overall rate of 149.0 (95% CI: 1.3-606.4) cases per 100,000 population. The cryptosporidiosis burden associated with drinking water treated with the conventional process was calculated to be 8.31 × 10(-6) (95% CI: 0.34-30.93 × 10(-6)) disability-adjusted life years (DALYs) per person per year, which was higher than the reference risk level suggested by the World Health Organization (WHO), but lower than that suggested by the United States Environmental Protection Agency (USEPA). Sixty-six percent of the total health burden due to cryptosporidiosis that occurred in the immunodeficient subpopulation, and 90% of the total DALYs was attributed to adults aged 15-59 years. The sensitivity analysis highlighted the great importance of stability of the treatment process and the importance of watershed protection. The results of this study will be useful in better evaluating and reducing the burden of Cryptosporidium infection., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Dog parks are very popular in urban areas, but there are no current studies attempting to correlate visits to dog parks and risk of colonization by enteric parasites. The purpose of this study was to determine whether dog park visitation is associated with an increased prevalence of enteric parasites or an increase in prevalence of gastrointestinal signs in dogs in northern Colorado. Feces from dogs owned by veterinary students or Veterinary Teaching Hospital staff members were submitted with a completed survey form detailing dog park attendance rates, fecal character scores, and other clinical information. Feces were examined microscopically for parasites after sugar centrifugation, for Giardia spp. cysts and Cryptosporidium spp. oocysts by a commercially available immunofluorescence assay (FA) and the FA positive samples were genotyped after PCR amplification. The Giardia assemblages were determined using the glutamate dehydrogenase (GDH) β-giardin and triose phosphate isomerase (TPI) genes and the Cryptosporidium species were determined using the heat shock protein-70 gene. A total of 129 fecal samples were assayed; 66 were from dog park attending dogs and 63 were from non-dog park-attending dogs. The overall parasite prevalence rate was 7.0% (9 of 129 samples). Dog park attending dogs were more likely to be positive for Giardia or Cryptosporidium than non-dog park-attending dogs (p=0.0279), but there was no association of gastrointestinal signs with dog park attendance or with fecal flotation or FA results. The five Giardia isolates were assemblage C and/or D and the one Cryptosporidium isolate was Ctenocephalides canis., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We describe recent advances in the genomics and population biology of Cryptosporidium parvum and C. hominis, the causative agents of cryptosporidiosis in humans and animals. Many basic aspects of the biology of Cryptosporidium species remain to be investigated and effective drugs to control cryptosporidiosis are not available. Sequencing and annotation of the genome of C. parvum and C. hominis has uncovered unique features of the metabolism of these species. The recently sequenced genome of the gastric species C. muris is providing new insights into the evolution of the genus. Cryptosporidian sequence information has facilitated the identification of polymorphic genetic markers. Genotyping of oocysts excreted by human and animal hosts using such markers has revealed many new species and genotypes, and is leading to a better understanding of the epidemiology of cryptosporidiosis., (© 2011 Blackwell Publishing Ltd.)

Members of the genus Cryptosporidium, which cause the gastrointestinal disease cryptosporidiosis, still represent a significant cause of water-borne disease worldwide. While intensive efforts have been invested in the development of techniques for parasite culture, in vitro growth has been hampered by a number of factors including low levels of infectivity as well as delayed life-cycle development and poor synchronicity. In this study we examined factors affecting the timing of contact between excysted sporozoites and target host cells and the subsequent impact of this upon the establishment of infection. We demonstrate that excystation rate impacts upon establishment of infection and that in our standard assay format the majority of sporozoites are not close enough to the cell monolayer when they are released from the oocyst to successfully establish infection. However, this can be easily overcome by centrifugation of oocysts onto the cell monolayer, resulting in approximately 4-fold increases in sporozoite attachment and subsequent infection. We further demonstrate that excystation procedures can be tailored to control excystation rate to match the assay end purpose and that excystation rate can influence data interpretation. Finally, the addition of both a centrifugation and washing step post-sporozoite attachment may be appropriate when considering the design of in vitro culture experiments for developmental analysis and stage-specific gene expression as this appears to increase the synchronicity of early developmental stages.

The genus Cryptosporidium includes many common parasites infecting animals and humans, and is a major cause of diarrheal illness worldwide. The biology of gastric Cryptosporidium spp., including replication in the stomach, has not been well documented. This study evaluated the viability of Cryptosporidium andersoni sporozoites in gastric environments after excystation and examined the endogenous development and histopathological changes in the stomachs of infected mice, using a novel type of C. andersoni. Sporozoites were affected by low pH (61.6% viability after 3h at pH2.0). Electron microscopy revealed developmental parasites on the gastric foveolae but not on the surface of the gastric mucosa. Histopathological examinations at 1, 2, 4 and 12 weeks p.i. uncovered three different lesions. The gastric mucosa of foveolae filled with parasites was extended and the amount of neutral mucopolysaccharide at the mucosal surface was decreased with the first type of lesion. The gastric mucosa was atrophied, some gastric glands were disrupted and the amount of acid mucopolysaccharide at the mucosal surface was increased with the second type. Finally, the gastric mucosa was slightly extended and goblet cells were present in the gastric mucosa, indicating intestinal metaplasia, in the third type. No parasites were detected in these areas with increased acidic mucin and indications of metaplasia. The results suggest that C. andersoni parasites could not survive in acidic environments for a long period before invading host cells and preferentially develop in neutral sites of the gastric mucosa, resulting in histopathological changes and chronic shedding of oocysts., (Copyright © 2010 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.)

Cryptosporidium parvum is an enteric coccidian parasite that is recognised as a frequent cause of water-borne disease in humans. We report for the first time on use of the in vitro HCT-8 cell culture-quantitative PCR (qPCR) assay and the in vivo SCID-mouse bioassay for evaluating critical factors that reduce or eliminate infectivity of C. parvum after irradiating oocysts in saline solution under varying operational conditions with pulsed UV light. Infections post UV treatments were detected by immunofluorescence (IF) microscopy and by quantitative PCR in cell culture, and by IF staining of faeces and by hematoxylin and eosin staining of intestinal villi in mice. There was a good agreement between using cell culture-qPCR and the mouse assay for determining reduction or elimination of C. parvum infectivity as a consequence of varying UV operating conditions. Reduction in infectivity depended on the intensity of lamp discharge energy applied, amount of pulsing and population size of oocysts (P < or = 0.05). Conventional radiometer was unable to measure fluence or UV dose in saline samples due to the ultra-short non-continuous nature of the high-energy light pulses. Incorporation of humic acid at a concentration above that found in surface water (i.e., < or =10 ppm) did not significantly affect PUV disinfection capability irrespective of parameters tested (P < or = 0.05). These observations show that use of this HCT-8 cell culture assay is equivalent to using the 'gold standard' mouse-based infectivity assay for determining disinfection performances of PUV for treating C. parvum in saline solution., (Copyright 2010 Elsevier B.V. All rights reserved.)

Fecal samples were taken from 132 (103 wild and 29 domestic) aquatic birds on selected areas in Hungary from February 2008 to March 2008. Cryptosporidium oocysts and Giardia cysts were purified from the samples and were viewed via fluorescent antibody staining. Molecular detection tools, such as PCR-sequencing and Loop mediated isothermal amplification (LAMP) were used in order to determine the Cryptosporidium species and Giardia duodenalis assemblages present. All together 6 (5.8%) and 6 (5.8%) samples out of the 103 wild bird samples and 4 (13%) and 7 (24%) samples out of the 29 domestic bird samples have been found to be Cryptosporidium and G. duodenalis positive respectively. The results of this study indicate that aquatic ducks, geese, coot and cormorant can play role in the environmental dissemination of human pathogenic Giardia cysts and Cryptosporidium oocysts in Hungary. To our knowledge, this is the first description of Cryptosporidium sp. in Anser fabalis and Anser anser, furthermore Giardia sp. in Fulica atra, A. fabalis and P. carbo and the first PCR-sequence confirmed detection of C. parvum in A. platyrhynchos and F. atra, G. duodenalis Assemblage A in A. strepera and G. duodenalis Assemblage B in A. anser.

Mussels filter large volumes of water and can concentrate pathogenic organisms, which may act as potential vehicles of transmission to the consumer. A survey study was carried out to investigate the presence of Cryptosporidium protozoan parasites in green mussels (Perna viridis), the smussles pecies most destined for consumption in Thailand. In total, 56 samples were examined from Bangkok (n = 24) and Samut Prakan (n = 32) a wholesale shell-fish markets located at the mouth of the Chao Phraya River. The market for green mussels was closed to the mussel culture placed along the coastal line and this localization may have significant economical impact if the mussels' cultures are found contaminated. Cryptosporidium spp. oocysts were detected by the immunofluorescence antibody method (IFA) in 12.5% of the samples examined. The detection of Cryptosporidium oocysts in green mussels' population of Samut Prakan was higher (15.6%) than in Bangkok market (8.3%). These differences in positive samples from the two locations may be caused by physical, ecological and anthropogenic conditions. This could relay to different contamination levels of marine water by Cryptosporidium oocysts and consequently to contamination of harvested shellfish populations. The results demonstrate that the Cryptosporidium spp. oocysts were found indigenous in mussels from the coastal line of Thailand, indicating that mussels may act as a reservoir of Cryptosporidium foodborne infections for humans.

Cryptosporidium is a zoonotic protozoan that is most often diagnosed in association with diarrhea in 1- to 3-wk-old dairy calves. There are neither consistently effective nor approved antimicrobial drugs for treatment in animals. The objective of this study was to test nitazoxanide (NTZ) as a treatment for cryptosporidiosis in experimentally infected dairy calves. A randomized, controlled, and blinded trial was performed using Holstein bull calves obtained from a large commercial dairy. All births were attended by study personnel and calves were fed 4 L of heat-treated colostrum within 1 h of birth. Calves were randomly assigned to treatment or placebo group and maintained for a 32-feeding (16 d) study period. Twenty-three calves were enrolled with 3 lost to follow up. Thirteen calves were assigned to the treatment group and 7 calves to the placebo group. All calves were inoculated with 1 x 10(6) viable Cryptosporidium parvum oocysts at feeding 3. Treatment was a commercially available NTZ product and the placebo was the carrier of the same product. Nitazoxanide was administered at 1.5 g twice per day for 5 d. Nitazoxanide or placebo treatment began after feeding 10 and when the fecal score was greater than 1 out of 3. Outcome measurements included twice-daily fecal and health scores and a once-daily oocyst count by an immunofluorescent antibody assay. Data were analyzed by nonparametric and time-to-event methods. Measures of passive transfer of antibodies, initial body weight, and onset of oocyst shedding were not different between treatment and control calves. Eighty-five percent of the NTZ-treated calves stopped shedding oocysts by the end of the observation period whereas only 15% of the placebo group stopped shedding. The median number of feedings with a fecal score equal to 3 was 2 in the NTZ group while it was 6 in the placebo group. Calves receiving NTZ were 0.13 times as likely to have severe and sustained diarrhea than control calves (95% confidence interval, 0.02-0.98). Treating calves with NTZ reduced the duration of oocyst shedding and improved fecal consistency.

At different times after inoculation with Cryptosporidium muris, infected CF-1 female mice were immunosuppressed with a single subcutaneous dose of methylprednisolone acetate (MPA; 600 mg/kg). MPA immunosuppression decreases circulating CD3, CD4 and CD8 T-lymphocytes and B-lymphocytes by greater than 90% for approximately 14 days with numbers not returning to pre-suppression levels until after 41 days post-suppression. Immunosuppression was initiated at selected times before, during, and after oocyst production. Immunosuppression initiated prior to oocyst production delayed the start of production by 4-5 days and extended oocyst shedding by 16 days. Initiation of immunosuppression during oocyst production both extended oocyst shedding and greatly increased the number of oocysts shed per day over most of the extended shedding period. Immunosuppression during the decline of oocyst production resulted in only a moderate extension of shedding and a moderate increase in oocyst numbers. Immunosuppression initiated soon after oocyst shedding had ceased resulted in the re-initiation of limited oocyst production for only a few days. Suppression initiated on days 40 and 46 post-infection, 11 and 17 days after oocysts could no longer be detected in the feces, did not result in a resumption of oocyst production. In all cases, where oocyst production was extended or reinitiated, the shedding of oocysts halted between days 45 and 53 post-oocyst inoculation. These studies demonstrate that the effect of MPA immunosuppression depends on the immunologic conditions existing in the host at the time immunosuppression was initiated. Immunosuppression initiated during oocyst production allows an overwhelming parasitism to exist, implying that T- and B-lymphocytes play an important role in moving the host immune process along during this period of the infection. Conversely, severe suppression of T- and B-lymphocytes initiated as oocyst production is decreasing does not result in a complete relapse of the disease suggesting that T- and B-lymphocytes are not critical to the continuation of the immune process after this point. These studies also show that the C. muris infection persists beyond the end of the detection of oocysts in the feces.

A recent review by Brendon King and Paul Monis once again puts Cryptosporidium oocysts under the spotlight. Why is this tough transmission stage so troublesome now? And are future environments likely to assist or hinder its apparent ubiquity? Here, we explore further the fascination and challenge engendered by this parasite transmission stage.

The present study was done to investigate the effects of fucoidan and de-sulfated fucoidan isolated from the sporophyll of Undaria pinnatifida on the C. parvum adhesion to the cultured human intestinal cells and on the C. parvum infection in neonatal mice. The C. parvum adhesion to human Intestinal 407 cells was significantly suppressed by a low dose (1 micro g/ml) of Mekabu fucoidan (1 micro g/ml) (approx. 20.5 oocysts, p<0.0001), but not by de-sulfated fucoidan (approx. 138.2 oocysts), as compared with that (approx. 121.0 oocysts) of phosphate-buffered saline (PBS). The in vivo experiments presented here revealed that C. parvum oocysts in the fucoidan-treated mice was reduced nearly one fifth (approx. 5.4x10(4) oocysts, p<0.02) of the total number of oocysts (approx. 3.0x10(5)) in mice treated with PBS, but no significant effect of de-sulfated fucoidan was observed. These results show that (i) fucoidan effectively inhibits the growth of C. parvum in mice; and (ii) the ester sulfate of fucoidan is an active site to prevent the adhesion of C. parvum to the intestinal epithelial cells. Finally, we concluded that fucoidan might inhibit cryptosporidiosis through the direct binding of fucoidan to the C. parvum-derived functional mediators in the intestinal epithelial cells in neonatal mice.

In the last decade, a major concern for the scientific community has been whether infected animals can serve as reservoirs of Giardia and Cryptosporidium infection for humans. Worldwide, prevalence studies and molecular tools have provided insights into the taxonomy and epidemiology of these protozoa in order to better understand such a relation. This paper presents data on the prevalence and molecular genotyping studies from several sample types from land to sea (humans, companion animals, sheep, cattle, goats, wastewaters, surface water, and shellfish) available in Italy. The contribution of Italian researchers to the international debate on the veterinary significance of these infections and their impact on public health is highlighted and the main objectives to be pursued in the future depicted.

Giardia and Cryptosporidium spp. are parasitic protozoa which are frequent etiologic agents of waterborne diseases. This lecture will summarize the main biological and environmental factors involved in the potential risk for waterborne transmission of giardiosis and cryptosporidiosis, which have caused many outbreaks in different geographical areas. In particular, the current epidemiological situation of these parasitoses in Italy will be analysed, on the basis of research carried out on humans and on the environment. Finally, current methods for evaluating the presence of Giardia cysts and Cryptosporidium oocysts in water and new methods for cyst/oocyst removal from drinking water and wastewater will be examined.

Cryptosporidium muris-like oocysts were isolated from large Japanese field mice, Apodemus speciosus. Morphologically, these oocysts resembled those obtained from a C. andersoni Kawatabi isolate but were smaller in size than those from a C.muris isolate. Following oral inoculation of the oocysts into large Japanese field mice and SCID mice, developing stages were found in the stomach epithelium. The infectivity of the isolate to wild and laboratory mice was slightly different from that of C.muris. DNA sequences of the 18S ribosomal RNA (rRNA) gene of the isolate were not identical to those of any known Cryptosporidium spp.; however, phylogenetic analysis indicated that the isolate was a member of the C.muris cluster. Differences between the isolate and C. muris are not significant at this point; therefore, we propose that this isolate is a novel genotype of C.muris and denote it as C. muris Japanese field mouse genotype.

The occurrence of Cryptosporidium oocysts in feces from a population of wild eastern grey kangaroos inhabiting a protected watershed in Sydney, Australia, was investigated. Over a 2-year period, Cryptosporidium oocysts were detected in 239 of the 3,557 (6.7%) eastern grey kangaroo fecal samples tested by using a combined immunomagnetic separation and flow cytometric technique. The prevalence of Cryptosporidium in this host population was estimated to range from 0.32% to 28.5%, with peaks occurring during the autumn months. Oocyst shedding intensity ranged from below 20 oocysts/g feces to 2.0 x 10(6) oocysts/g feces, and shedding did not appear to be associated with diarrhea. Although morphologically similar to the human-infective Cryptosporidium hominis and the Cryptosporidium parvum "bovine" genotype oocysts, the oocysts isolated from kangaroo feces were identified as the Cryptosporidium "marsupial" genotype I or "marsupial" genotype II. Kangaroos are the predominant large mammal inhabiting Australian watersheds and are potentially a significant source of Cryptosporidium contamination of drinking water reservoirs. However, this host population was predominantly shedding the marsupial-derived genotypes, which to date have been identified only in marsupial host species.

Cryptosporidium parvum excystation and host cell invasion have been characterized in some detail ultrastructurally. However, until recently, the biochemical and molecular basis of host-parasite interactions and parasite- and host-specific molecules involved in excystation, motility and host cell invasion have been poorly understood. This article describes our understanding of Cryptosporidium excystation and the events leading to host cell invasion, and draws from information available about these processes in other apicomplexans. Many questions remain but, once the specific mechanisms are identified, they could prove to be novel targets for drug delivery.

It has been recognized recently that human cryptosporidiosis is usually caused by Cryptosporidium parvum genotype I ("human" C. parvum), which is not found in animals. Compared to C. parvum genotype II, little is known of the biology of invasion of the human-restricted C. parvum genotype I. The aims of the present study were (i) to explore and compare with genotype II the pathogenesis of C. parvum genotype I infection by using an established in vitro model of infection and (ii) to examine the possibility that host-specific cell tropism determines species restriction among C. parvum genotypes by using a novel ex vivo small intestinal primary cell model of infection. Oocysts of C. parvum genotypes I and II were used to infect HCT-8 cells and primary intestinal epithelial cells in vitro. Primary cells were harvested from human endoscopic small-bowel biopsies and from bovine duodenum postmortem. C. parvum genotype I infected HCT-8 cells with lower efficiency than C. parvum genotype II. Actin colocalization at the host parasite interface and reduction in levels of invasion after treatment with microfilament inhibitors (cytochalasin B and cytochalasin D) were observed for both genotypes. C. parvum genotype II invaded primary intestinal epithelial cells, regardless of the species of origin. In contrast, C. parvum genotype I invaded only human small-bowel cells. The pathogenesis of C. parvum genotype I differs from C. parvum genotype II. C parvum genotype I does not enter primary bovine intestinal cells, suggesting that the species restriction of this genotype is due to host tissue tropism of the infecting isolate.

Cryptosporidium parvum has been associated with outbreaks of human illness by consumption of contaminated water, fresh fruits, and vegetables. Free-living nematodes may play a role in pathogen transmission in the environment. Caenorhabditis elegans is a free-living soil nematode that has been extensively studied and serves as a good model to study possible transmission of C. parvum oocysts that may come into contact with produce before harvest. The objective of this study was to determine whether C. elegans could serve as a potential mechanical vector for transport of infectious C. parvum and Cyclospora cayetanensis in agricultural settings and whether C. elegans could ingest, excrete, and protect oocysts from desiccation. Seventy to 85% of worms ingested between 0 and 500 oocysts after 1 and 2 hr incubation with oocysts. Most of the nematodes ingested between 101 and 200 oocysts after 2 hr. Intact oocysts and empty shells were excreted by nematodes. Infectivity was determined by the neonatal assay with different treatments of worms (intact or homogenized) or oocysts or both. Adult C. elegans containing C. parvum kept in water were infective for mice. In conclusion, C. elegans adults can ingest and excrete C. parvum oocysts. Caenorhabditis elegans containing C. parvum oocysts can infect mice but does not seem to protect oocysts from extreme desiccation at 23 C incubation of a day or longer. Cyclospora oocysts were not ingested by C. elegans. The role of free-living nematodes in produce contamination needs to be further examined.

The anticryptosporidial effect of sodium selenite (selenium) was evaluated in a bovine fallopian tube epithelial (BFrE) cell culture system and an immunosuppressed C57BL/6N adult mouse model. Parasite numbers in cell culture were significantly reduced (p<0.01) following treatment with selenium (Se) at concentrations of 6, 9, and 12 microM at 48 h postinoculation (PI) and at 1.5, 3, and 6 microM at 96 h PI. Parasite reduction was greater than 50% at 48 h PI when 9 and 12 microM Se was used, and at 96 h PI when 6 pM Se was used. Such Se-induced reduction of Cryptosporidium parvum infection was significantly (p<0.0001) blocked when using free-radical scavengers such as mannitol (20 mM). A combined solution of mannitol (20 mM) and reduced glutathione (0.5 mM) enhanced the blockage to almost 100%. Adult C57BL/6N mice were immunosuppressed with dexamethasone phosphate administered ad libitum (16 microg/mL) in drinking water and inoculated with 10(5) oocysts/ mouse. Significantly fewer (p<0.001) oocysts were shed in the feces of mice treated with Se administered ad libitum (12 microM) in drinking water than in untreated mice. The survival time of mice was also significantly increased (p<0.001) following Se treatment. Collectively, these results indicate that Se plays an important role in cryptosporidiosis, and oxidative stress caused by Se is probably a major mechanism in inhibition of C. parvum infection.

This paper reports the anti-cryptosporidial effects of, and concomitant amelioration of the histological changes in the gut of neonatal rats with intestinal cryptosporidiosis treated with the dinitroaniline, oryzalin. The ED50 was determined to be 7 mg/kg using twice daily doses administered for 3 consecutive days. A maximum inhibition of 85.5% was achieved at 25 mg/kg and this inhibition remained constant despite increasing the oryzalin dose to 200 mg/kg. Cryptosporidiosis significantly decreased the intestinal villus/crypt (VC) ratio by approximately 50% (duodenum = 2.3, jejunum = 2.5 and ileum = 1.7) when compared to uninfected untreated controls (duodenum = 4.3, jejunum = 5.9 and ileum = 4.5). Treatment with oryzalin doubled the VC ratio in the duodenum, jejunum and ileum following doses of 5 mg, 50 mg and 200 mg/kg respectively. Oryzalin concentrations in the small intestine contents and plasma were determined, using HPLC, at 0.5, 1 and 2 h after dosing. The much greater dose required to return VC ratios to normal in the ileum (200 mg/kg) compared to the duodenum (6.25 mg/kg) appeared to reflect the decreased concentration of the drug in the distal small intestine. Concentrations of oryzalin equivalent to the in vitro IC50 were maintained for 2 h in the first half of the small intestine following a single dose of 100 mg/kg.