Diagnostic evaluation of nested PCR and microscopy for Cryptosporidiosis in goats: can COWP gene based qRT-PCR be useful in assessment of active Cryptosporidial infections?

The present research delved into the transmission patterns, diagnostic methods, molecular traits, and phylogenetic analysis of Cryptosporidium species. The research was undertaken to enhance comprehension of the epidemiology and the potential for zoonotic transmission. A total of 80 goat-kid samples were tested, 7 were confirmed positive by mZN microscopy and 12 by nested-PCR. By PCR, 18SSUrRNA, HSP70, and GP60 amplicons were tested for Cryptosporidium. The restriction enzymes viz., SspI, VspI and MboII were used to genotype 12 Cryptosporidium positive samples by which C. parvum and C. bovis mixed infections were detected. Quantitative reverse transcription real-time PCR was used to transcriptionally screen the COWP-subunit genes to assess the severity of the infection in goat-kids, which showed upregulation of COWP6 and COWP4, while COWP9 and COWP3 genes were downregulated. A silent mutation was found at the codon CCA→CCC, which is being reported for the first time in goat field isolates. Phylogenetic and sequencing analyses confirmed the presence of the anthropozoonotic IIe subtype.

Cost-effective In Vivo and In Vitro Mouse Models for Evaluating Anticryptosporidial Drug Efficacy: Assessing Vorinostat, Docetaxel, and Baicalein.

BACKGROUND: Cryptosporidiosis is a significant diarrheal disease in humans and animals. Immunodeficient mice are the primary small animal models, but their high costs and specialized breeding/housing requirements limit in vivo drug testing. Numerous anticryptosporidial lead compounds identified in vitro remain untested in vivo. METHODS: Cryptosporidium tyzzeri, a natural mouse parasite closely related to Cryptosporidium parvum and Cryptosporidium hominis, was isolated to establish an infection model in immunocompetent mice. The model was validated using classic anticryptosporidial drugs (paromomycin and nitazoxanide) and then employed to assess the efficacy of 3 new leads (vorinostat, docetaxel, and baicalein). An in vitro culture of C. tyzzeri was also developed to complement the animal model. RESULTS: Chronic C. tyzzeri infection was established in chemically immunosuppressed wild-type mice. Paromomycin (1000 mg/kg/d) and nitazoxanide (100 mg/kg/d) demonstrated efficacy against C. tyzzeri. Vorinostat (30 mg/kg/d), docetaxel (25 mg/kg/d), and baicalein (50 mg/kg/d) were highly effective against C. tyzzeri infection. In vitro, nitazoxanide, vorinostat, docetaxel, and baicalein exhibited low to submicromolar efficacy against C. tyzzeri. CONCLUSIONS: Novel in vivo and in vitro models have been developed for cost-effective anticryptosporidial drug testing. Vorinostat, docetaxel, and baicalein show potential for repurposing and/or optimization for developing new anticryptosporidial drugs.

Assessment microbial risks for the presence of Cryptosporidium spp. and Giardia spp. based on the surveillance of the water supply systems in Colombia, 2014-2018.

Cryptosporidium spp. and Giardia spp. cause gastrointestinal diseases of zoonotic origin as well transmitted from person to person, being various reported outbreaks associated with water. The infecting (oo)cyst forms of these parasites are highly resistant to water treatments such as chlorine disinfection and fast filtration. The objective of this study was to assess the microbial risk of infection and symptomatic illness by the ingestion of Giardia spp. and Cryptosporidium spp. in water for human consumption in Colombia, based on the results of water quality surveillance. The detection method was according to the USEPA method 1623. Concentration data of the different points of distribution were grouped according to the pathogen and type of treatment (no treatment; chlorine treatment; chlorine treatment + coagulant). Annual microbial risks of infection and symptomatic diseases were estimated using the quantitative microbial risk assessment approach that included parasite concentrations, the dose-response model, the ingestion rates of water by children and adults, and the morbidity rate of the diseases. The mean annual microbial risk of infection for Giardia spp. was 29.8% for treated water and 50.4% for untreated water, while being 6.0% and 17.7%, respectively, for Cryptosporidium spp. Microbial risk of symptomatic illness for Giardia spp, was 8.2% for treated water and 13.9% for untreated water, while being 3.6% and 10.6%, respectively, for Cryptosporidium spp. The estimated annual microbial risks of infection exceeded the acceptable value of 10-4 (0.01%) recommended by USEPA. Results obtained in this study suggest the need to reduce the microbial risk of infection to protozoan parasites by improving the water treatment, by adopting better handling practices for livestock manure and treatment processes of human feces. PRACTITIONER POINTS: The presence of Cryptosporidium spp was identified in 28 (6.2%) samples and Giardia spp in 29 (6.4%) in water for human consumption in Colombia. The mean annual risk of symptomatic illness due to infection by Giardia spp or Cryptosporidium spp ranges from 33.6%, for treated water, to 58.1%, for untreated water. Annual risks ingestion of protozoa studying in water for human exceed of 10-4 (0.01%) recommended by USEPA.

Assessment of a Multiplex PCR for the Simultaneous Diagnosis of Intestinal Cryptosporidiosis and Microsporidiosis: Epidemiologic Report from a French Prospective Study.

Microsporidiosis and cryptosporidiosis are associated with chronic diarrhea in immunocompromised patients. The objectives of this study were to: i) assess a multiplex quantitative PCR assay targeting Cryptosporidium spp and the microsporidian Enterocytozoon bieneusi and Encephalitozoon spp, and ii) provide an update on the epidemiology of these pathogens. A prospective study was conducted from January 2017 to January 2019. Performance of the assay was assessed, and all cryptosporidia and microsporidia isolates were genotyped. The sensitivity of the multiplex PCR method reached 1 copy/µL for each targeted pathogen. The sensitivity of co-proantigen testing in the diagnosis of cryptosporidiosis was 73%. The sensitivity of microscopy in the diagnosis of cryptosporidiosis was 64%, and microsporidiosis, 50%. Among the 456 patients included, 14 were positive for Cryptosporidium spp (4 different species); 5, for E. bieneusi; and 2, for Encephalitozoon intestinalis. The overall prevalence of cryptosporidia was 3.1%, and of microsporidia, 1.5%; in kidney transplant recipients (n = 82), corresponding values were 7.3% and 2.4% (6 and 2 patients), respectively. Two cases of E. intestinalis infection were diagnosed in children who had traveled to the tropics. This study is the first to assess a multiplex quantitative PCR method for the simultaneous diagnosis of intestinal microsporidiosis and cryptosporidiosis. The highest prevalences of both pathogens were observed in kidney transplant recipients.

Importance of Distributional Forms for the Assessment of Protozoan Pathogens Concentrations in Drinking-Water Sources.

The identification of appropriately conservative statistical distributions is needed to predict microbial peak events in drinking water sources explicitly. In this study, Poisson and mixed Poisson distributions with different upper tail behaviors were used for modeling source water Cryptosporidium and Giardia data from 30 drinking water treatment plants. Small differences (<0.5-log) were found between the "best" estimates of the mean Cryptosporidium and Giardia concentrations with the Poisson-gamma and Poisson-log-normal models. However, the upper bound of the 95% credibility interval on the mean Cryptosporidium concentrations of the Poisson-log-normal model was considerably higher (>0.5-log) than that of the Poisson-gamma model at four sites. The improper choice of a model may, therefore, mislead the assessment of treatment requirements and health risks associated with the water supply. Discrimination between models using the marginal deviance information criterion (mDIC) was unachievable because differences in upper tail behaviors were not well characterized with available data sets ( n<30 ). Therefore, the gamma and the log-normal distributions fit the data equally well but may predict different risk estimates when they are used as an input distribution in an exposure assessment. The collection of event-based monitoring data and the modeling of larger routine monitoring data sets are recommended to identify appropriately conservative distributions to predict microbial peak events.

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR.

BACKGROUND: Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA. METHODS: We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106-1); (ii) diluted template DNA from pure oocysts (×10-×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105-1 and 5 × 104-50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10-×1000). RESULTS: Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations. CONCLUSIONS: For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

Foodborne Parasitic Diseases in Europe: Social Cost-Benefit Analyses of Interventions.

Social cost-benefit analysis (SCBA) can be used to evaluate the benefit to society as a whole of a particular intervention. Describing preliminary steps of an SCBA for two foodborne parasitic diseases, echinococcosis and cryptosporidiosis, indicates where data are needed in order to identify those interventions of greatest benefit.

Cryptosporidium genotyping in Europe: The current status and processes for a harmonised multi-locus genotyping scheme.

Due to the occurrence of genetic recombination, a reliable and discriminatory method to genotype Cryptosporidium isolates at the intra-species level requires the analysis of multiple loci, but a standardised scheme is not currently available. A workshop was held at the Robert Koch Institute, Berlin in 2016 that gathered 23 scientists with appropriate expertise (in either Cryptosporidium genotyping and/or surveillance, epidemiology or outbreaks) to discuss the processes for the development of a robust, standardised, multi-locus genotyping (MLG) scheme and propose an approach. The background evidence and main conclusions were outlined in a previously published report; the objectives of this further report are to describe 1) the current use of Cryptosporidium genotyping, 2) the elicitation and synthesis of the participants' opinions, and 3) the agreed processes and criteria for the development, evaluation and validation of a standardised MLG scheme for Cryptosporidium surveillance and outbreak investigations. Cryptosporidium was characterised to the species level in 7/12 (58%) participating European countries, mostly for human outbreak investigations. Further genotyping was mostly by sequencing the gp60 gene. A ranking exercise of performance and convenience criteria found that portability, biological robustness, typeability, and discriminatory power were considered by participants as the most important attributes in developing a multilocus scheme. The major barrier to implementation was lack of funding. A structured process for marker identification, evaluation, validation, implementation, and maintenance was proposed and outlined for application to Cryptosporidium, with prioritisation of Cryptosporidium parvum to support investigation of transmission in Europe.

Development of a Cryptosporidium-arsenic multi-risk assessment model for infant formula prepared with tap water in France.

Tap water is used in France to reconstitute powder infant formula, although it is not sterile and possibly contaminated by microbiological and chemical hazards. The present study aims to quantify risks of using tap water in France for the preparation of infant formula, during the first six months of life. Cryptosporidium and arsenic were selected as hazards of greatest concern in microbiology and chemistry, respectively. A probabilistic model was developed using French (when available) and European (alternatively) data. Second order Monte Carlo simulation was used to separate uncertainty and variability of inputs. Outputs were expressed at the individual level as probability of illness and at the population level, using a common metric, the DALY (Disability Adjusted Life Year). Two scenarios of milk preparation were considered: with un-boiled or boiled tap water. Consuming infant formula rehydrated with un-boiled tap water during the first six months of life led to a total of 2250 DALYs per 100,000 infants (90% uncertainty interval [960; 7650]) for Cryptosporidium due to diarrhea, and 1 DALY [0.4; 2] for arsenic due to expected lifetime risk of lung and bladder cancer as a result of early exposure in life. For the entire population, boiling water would suppress the risk from Cryptosporidium. In contrast, the incremental cancer risk was low at the population level but elevated for 5% of the population exposed to high levels of arsenic. A stringent monitoring of tap water supply points should be continued. This multi-risk assessment model could help public health authorities and managers in evaluating both microbiological and chemical safety issues associated with using infant formula prepared with tap water.

First detection of Cryptosporidium parvum in falcons (Falconiformes): Diagnosis, molecular sequencing, therapeutic trial and epidemiological assessment of a possible emerging disease in captive falcons.

Cryptosporidiosis in raptors and falcons is well-known to be caused by Cryptosporidium baileyi and associated mainly with respiratory pathology. This report presents the diagnosis of an atypical cryptosporidiosis event caused by Cryptosporidium parvum, that to the authors' knowledge, is a case observed for the first time in falcons. Two falcons (Gyrfalcon x Peregrine hybrids) were presented for annual check without any clinical signs. Hematology, biochemistry, fecal and crop parasitology, radiographic and endoscopic examinations were performed. Endoscopy revealed microcystic formation of the caudal lung field in the two falcons, adhesions and air sac alterations. Sampling and subsequent cytology revealed fungal spores and acid fast stain organisms (identified as Cryptosporidium spp.). Feces and affected lung tissue was further send for Cryptosporidium spp.-DNA detection. Fecal samples and lung tissue tested positive for Cryptosporidium spp. gp60 gene by PCR. By sequence analysis of the gp60 gene locus, diagnosis of C. parvum was confirmed with 100% homology. Despite the fact that falcons didn't recover after 1 month of therapy, eight months after the initial examination they were clinically healthy and had satisfactory flying performance. No other falcons were observed with C. parvum infections in the facility so far. The possible source, infection route and implications are discussed.

Viability Assessment of Cryptosporidium parvum Oocysts by Vital Dyes: Dry Mounts Overestimate the Number of "Ghost" Oocysts.

Viability assessment of Cryptosporidium parvum oocysts is crucial for evaluation of the public health significance of this important zoonotic protozoon. Viability is commonly assessed in wet mounts after acid pretreatment and staining with fluorogenic vital dyes. However, in some studies, oocyst viability is evaluated in dry mounts after staining in suspension. Here, we evaluate the effect of acid pretreatment in nine replicate samples and compare the assessment of oocyst viability after evaluation in wet and dry mounts, respectively. Although acid pretreatment had no significant effect on the viability scores, data obtained by scoring oocysts in dry mounts resulted in ∼25% underestimation of the proportion of viable oocyst (82.5% ± 0.9% [wet mount +acid], 57.7% ± 2.3% [dry mount, ÷ acid], 76.0% ± 1.7% [wet mount, ÷ acid]), while the proportions of nonviable oocysts (DAPI+/PI+) were comparable for wet and dry mounts (9.7% ± 0.4% [wet mount +acid], 12.1 ± 1.5% [dry mount, ÷ acid], 15.5% ± 1.1% [wet mount, ÷ acid]).

Checking the detail in retail: Occurrence of Cryptosporidium and Giardia on vegetables sold across different counters in Chandigarh, India.

Fresh produce has been recognized as a vehicle of infection for protozoan parasites, particularly Cryptosporidium, and, to a lesser extent, Giardia. For both parasites, outbreaks associated with fresh produce have been documented. Although documented outbreaks tend to be from industrialized countries, contamination of fresh produce with these parasites is a global issue. In developing countries, infections with these parasites are often endemic in the community, and basic infrastructure and hygiene measures may be inadequate, thus the likelihood of contamination of fresh produce with these parasites may be higher. Realization of the importance of this transmission route comes against a backdrop of raw salads and more Western culinary habits gaining a foothold, and fresh produce being encouraged as part of the diet due to their associated health benefits. However, if consumption of uncooked fresh produce is going to increase its market sector in India, it is important that it is safe. In this study, various types of fresh produce obtained from three types of vendors in Chandigarh, a major city in Northern India, were analyzed for contamination with Cryptosporidium oocysts and Giardia cysts using a method that has been previously validated in inter-laboratory spiking experiments. A total of 284 samples of different fresh produce items were analyzed, obtained from the different retailers situated in different societal layers of the city. The overall prevalence of contamination of fresh produce with these parasites was just under 11%, with 6% of the vegetables contaminated with Cryptosporidium oocysts, and 5% with Giardia cysts. Contaminated vegetables included turnip, cabbage, carrot, chili, coriander, cucumber, radishes, and tomatoes. Molecular analyses identified contamination with Cryptosporidium parvum and Giardia duodenalis of Assemblage A and Assemblage D, indicating that contamination from animals may be of relevance. Although the prevalence of contamination is similar to those reported in previous studies, the levels of contamination on some items of fresh produce were relatively high. Although the different socioeconomic areas of Chandigarh from which the samples were obtained was not associated with likelihood of contamination, fresh produce from supermarkets had heavier contamination with Cryptosporidium oocysts than fresh produce purchased through other sales outlets. The results are discussed in relation to the fresh produce chain and sales models in Chandigarh, both in terms of where contamination may occur and the potential importance of fresh produce as a transmission vehicle.

Economic Assessment of Waterborne Outbreak of Cryptosporidiosis.

In 2007, a waterborne outbreak of Cryptosporidium hominis infection occurred in western Ireland, resulting in 242 laboratory-confirmed cases and an uncertain number of unconfirmed cases. A boil water notice was in place for 158 days that affected 120,432 persons residing in the area, businesses, visitors, and commuters. This outbreak represented the largest outbreak of cryptosporidiosis in Ireland. The purpose of this study was to evaluate the cost of this outbreak. We adopted a societal perspective in estimating costs associated with the outbreak. Economic cost estimated was based on totaling direct and indirect costs incurred by public and private agencies. The cost of the outbreak was estimated based on 2007 figures. We estimate that the cost of the outbreak was >€19 million (≈€120,000/day of the outbreak). The US dollar equivalent based on today's exchange rates would be $22.44 million (≈$142,000/day of the outbreak). This study highlights the economic need for a safe drinking water supply.

Cryptosporidium and Giardia in Africa: current and future challenges.

Cryptosporidium and Giardia are important causes of diarrhoeal illness. Adequate knowledge of the molecular diversity and geographical distribution of these parasites and the environmental and climatic variables that influence their prevalence is important for effective control of infection in at-risk populations, yet relatively little is known about the epidemiology of these parasites in Africa. Cryptosporidium is associated with moderate to severe diarrhoea and increased mortality in African countries and both parasites negatively affect child growth and development. Malnutrition and HIV status are also important contributors to the prevalence of Cryptosporidium and Giardia in African countries. Molecular typing of both parasites in humans, domestic animals and wildlife to date indicates a complex picture of both anthroponotic, zoonotic and spill-back transmission cycles that requires further investigation. For Cryptosporidium, the only available drug (nitazoxanide) is ineffective in HIV and malnourished individuals and therefore more effective drugs are a high priority. Several classes of drugs with good efficacy exist for Giardia, but dosing regimens are suboptimal and emerging resistance threatens clinical utility. Climate change and population growth are also predicted to increase both malnutrition and the prevalence of these parasites in water sources. Dedicated and co-ordinated commitments from African governments involving "One Health" initiatives with multidisciplinary teams of veterinarians, medical workers, relevant government authorities, and public health specialists working together are essential to control and prevent the burden of disease caused by these parasites.

Prevalence and multilocus genotyping of Cryptosporidium andersoni in dairy cattle and He cattle in Xinjiang, China.

Cryptosporidium andersoni is the predominant species in post-weaned and adult cattle in China. The aim of this study was to determine the prevalence and understand the transmission of cattle cryptosporidiosis in the Xinjiang Uyghur Autonomous Region, China, a total of 1827 fecal samples (436 from He cattle and 1391 from dairy cattle) were examined for the presence of C. andersoni-like oocysts by microscopy after Sheather's sugar flotation technique. The overall prevalence of C. andersoni-like was 3.8% (70/1827) and all the C. andersoni-like isolates were identified as C. andersoni at the SSU rRNA locus. Among the C. andersoni isolates, a total of 60 isolates were successfully characterized into eight multilocus sequence typing (MLST) subtypes using MLST analysis at the four microsatellite/minisatellite loci (MS1, MS2, MS3 and MS16), and three new subtypes were identified. The MLST subtype A4,A4,A4,A1 showed a predominance and a wide distribution among the eight MLST subtypes obtained in the investigated areas. The MLST subtypes A2,A4,A2,A1 and A4,A5,A2,A1 showed a unique distribution in the investigated areas. A linkage disequilibrium analysis showed the presence of an epidemic population genetic structure of C. andersoni isolated from dairy and He cattle in Xinjiang. These findings provide new insights into the genetic structure of C. andersoni isolates and are also helpful to explore the infection source of C. andersoni in cattle in Xinjiang, China.

The Burden of Cryptosporidium Diarrheal Disease among Children < 24 Months of Age in Moderate/High Mortality Regions of Sub-Saharan Africa and South Asia, Utilizing Data from the Global Enteric Multicenter Study (GEMS).

BACKGROUND: The importance of Cryptosporidium as a pediatric enteropathogen in developing countries is recognized. METHODS: Data from the Global Enteric Multicenter Study (GEMS), a 3-year, 7-site, case-control study of moderate-to-severe diarrhea (MSD) and GEMS-1A (1-year study of MSD and less-severe diarrhea [LSD]) were analyzed. Stools from 12,110 MSD and 3,174 LSD cases among children aged <60 months and from 21,527 randomly-selected controls matched by age, sex and community were immunoassay-tested for Cryptosporidium. Species of a subset of Cryptosporidium-positive specimens were identified by PCR; GP60 sequencing identified anthroponotic C. parvum. Combined annual Cryptosporidium-attributable diarrhea incidences among children aged <24 months for African and Asian GEMS sites were extrapolated to sub-Saharan Africa and South Asian regions to estimate region-wide MSD and LSD burdens. Attributable and excess mortality due to Cryptosporidium diarrhea were estimated. FINDINGS: Cryptosporidium was significantly associated with MSD and LSD below age 24 months. Among Cryptosporidium-positive MSD cases, C. hominis was detected in 77.8% (95% CI, 73.0%-81.9%) and C. parvum in 9.9% (95% CI, 7.1%-13.6%); 92% of C. parvum tested were anthroponotic genotypes. Annual Cryptosporidium-attributable MSD incidence was 3.48 (95% CI, 2.27-4.67) and 3.18 (95% CI, 1.85-4.52) per 100 child-years in African and Asian infants, respectively, and 1.41 (95% CI, 0.73-2.08) and 1.36 (95% CI, 0.66-2.05) per 100 child-years in toddlers. Corresponding Cryptosporidium-attributable LSD incidences per 100 child-years were 2.52 (95% CI, 0.33-5.01) and 4.88 (95% CI, 0.82-8.92) in infants and 4.04 (95% CI, 0.56-7.51) and 4.71 (95% CI, 0.24-9.18) in toddlers. We estimate 2.9 and 4.7 million Cryptosporidium-attributable cases annually in children aged <24 months in the sub-Saharan Africa and India/Pakistan/Bangladesh/Nepal/Afghanistan regions, respectively, and ~202,000 Cryptosporidium-attributable deaths (regions combined). ~59,000 excess deaths occurred among Cryptosporidium-attributable diarrhea cases over expected if cases had been Cryptosporidium-negative. CONCLUSIONS: The enormous African/Asian Cryptosporidium disease burden warrants investments to develop vaccines, diagnostics and therapies.

Occurrence and quantitative microbial risk assessment of Cryptosporidium and Giardia in soil and air samples.

BACKGROUND: Cryptosporidium oocysts and Giardia cysts can be transmitted by the fecal-oral route and may cause gastrointestinal parasitic zoonoses. These zoonoses are common in rural zones due to the parasites being harbored in fecally contaminated soil. This study assessed the risk of illness (giardiasis and cryptosporidiosis) from inhaling and/or ingesting soil and/or airborne dust in Potam, Mexico. METHODS: To assess the risk of infection, Quantitative Microbial Risk Assessment (QMRA) was employed, with the following steps: (1) hazard identification, (2) hazard exposure, (3) dose-response, and (4) risk characterization. RESULTS: Cryptosporidium oocysts and Giardia cysts were observed in 52% and 57%, respectively, of total soil samples (n=21), and in 60% and 80%, respectively, of air samples (n=12). The calculated annual risks were higher than 9.9 × 10(-1) for both parasites in both types of sample. CONCLUSIONS: Soil and air inhalation and/or ingestion are important vehicles for these parasites. To our knowledge, the results obtained in the present study represent the first QMRAs for cryptosporidiosis and giardiasis due to soil and air inhalation/ingestion in Mexico. In addition, this is the first evidence of the microbial air quality around these parasites in rural zones.

Assessment of the viability of Cryptosporidium parvum oocysts with the induction ratio of hsp70 mRNA production in manure.

Determining the presence of viable Cryptosporidium parvum oocysts in complex environmental matrices in hygiene control can prevent the contamination of water resources and food with this pathogen. This study assessed the induction ratio of hsp70 mRNA production by heat shock in different oocysts as a marker of viability. Using different procedures for (m)RNA extraction directly from manure and reverse transcription real-time qPCR, this study found slightly increased hsp70 mRNA contents in viable oocysts that were heat shock induced at 45°C for 20 min compared to not induced oocysts (1.6 fold induction in average). Prolonging the heat shock treatment to 2h did not further increase the copy numbers. Heat shock by consecutive stimuli, such as freezing and then heating, did not yield significantly higher copy numbers than the 45°C treatment. There was a certain background level of hsp70 mRNA in viable oocysts that were not exposed to heat shock, indicating a constitutive production of the transcripts in the oocysts. The production of hsp70 mRNA induced by heat shock in oocysts aged for 9 months that exhibited reduced viability was lower than in fresher oocysts (induction ratio<1.2). No production of hsp70 mRNA by heat shock was detected in 12 months old oocysts that were not viable in the excystation test. Oocysts inactivated at 75°C for 30 min were not able to respond to heat shock, and low amount of copies were occasionally measured only in total RNA extracts, but not in mRNA extracts that were purified directly with an oligo (dT)25 based system. The induction ratio of hsp70 mRNA varied according to the viability of the organisms in a sample. Copy numbers of ß-tubulin mRNA in viable oocysts were lower than hsp70 mRNA, therefore the latter is more suitable to detect low numbers of oocysts by RT-qPCR.

Assessment of Cryptosporidium parvum infection in immunocompetent and immunocompromised mice and its role in triggering intestinal dysplasia.

OBJECTIVES: There is an association between chronic inflammation and cancer, including colon cancer. Cryptosporidium parvum is a protozoan parasite that infects the gastrointestinal epithelial cells causing several parasitological and pathological changes. It is incriminated in the development of colorectal cancer in immunosuppressed individuals. Cyclin D1 expression is essential for cell cycle progression and its overexpression has been reported in colorectal cancer. This work aimed to study the gastrointestinal changes, including parasitological and pathological changes, induced by C. parvum infection in both immunocompetent and in chemically immunosuppressed mice, together with immunohistochemical assessment of cyclin D1 expression in infected tissues. In addition, the effectiveness of nitazoxanide (NTZ) in the treatment of cryptosporidiosis was evaluated. METHODS: This study included six groups of mice: group I, infected; group II, infected and immunosuppressed; group III, infected and treated with NTZ; group IV, infected, immunosuppressed, and treated with NTZ; and groups V and VI representing non-infected controls. Mice were subjected to stool examination for oocyst counts and were later sacrificed for intestinal dissection and routine histopathological examination of pathological changes; the endogenous developmental stages of the parasite were counted and immunohistochemical staining was carried out for the determination of cyclin D1. RESULTS: Group II showed the highest numbers of oocysts shed and endogenous developmental stages compared to the other groups. Intestinal dysplastic changes were seen only in groups I and II, where these changes were in favor of group II compared to group I. High-grade dysplasia was seen in four out of 20 mice in group II and was significantly associated with the number of endogenous developmental stages of C. parvum. NTZ was effective in the treatment of Cryptosporidium infection, with a greater effect in group III than in group IV. CONCLUSIONS: C. parvum is one of the infectious agents that may induce intestinal dysplasia, including the high-grade category, which occurs particularly in the presence of immune suppression states and elevated endogenous parasite loads. Cyclin D1 is a good and useful marker for the detection of intestinal dysplasia. The effectiveness of NTZ is dependent on the immune status of the infected host.

Cryptosporidium parvum infection in SCID mice infected with only one oocyst: qPCR assessment of parasite replication in tissues and development of digestive cancer.

Dexamethasone (Dex) treated Severe Combined Immunodeficiency (SCID) mice were previously described as developing digestive adenocarcinoma after massive infection with Cryptosporidium parvum as soon as 45 days post-infection (P.I.). We aimed to determine the minimum number of oocysts capable of inducing infection and thereby gastrointestinal tumors in this model. Mice were challenged with calibrated oocyst suspensions containing intended doses of: 1, 10, 100 or 10(5) oocysts of C. parvum Iowa strain. All administered doses were infective for animals but increasing the oocyst challenge lead to an increase in mice infectivity (P = 0.01). Oocyst shedding was detected at 7 days P.I. after inoculation with more than 10 oocysts, and after 15 days in mice challenged with one oocyst. In groups challenged with lower inocula, parasite growth phase was significantly higher (P = 0.005) compared to mice inoculated with higher doses. After 45 days P.I. all groups of mice had a mean of oocyst shedding superior to 10,000 oocyst/g of feces. The most impressive observation of this study was the demonstration that C. parvum-induced digestive adenocarcinoma could be caused by infection with low doses of Cryptosporidium, even with only one oocyst: in mice inoculated with low doses, neoplastic lesions were detected as early as 45 days P.I. both in the stomach and ileo-caecal region, and these lesions could evolve in an invasive adenocarcinoma. These findings show a great amplification effect of parasites in mouse tissues after challenge with low doses as confirmed by quantitative PCR. The ability of C. parvum to infect mice with one oocyst and to develop digestive adenocarcinoma suggests that other mammalian species including humans could be also susceptible to this process, especially when they are severely immunocompromised.