Parvum Oocysts Research Articles

A Simple Alcohol-based Method of Oocyst Inactivation for Use in the Development of Detection Assays for Cryptosporidium

Cryptosporidium spp. are obligate, intracellular parasites that cause life-threatening diarrhea among children and immunocompromised adults. Transmission occurs by the fecal-oral route following ingestion of thick-walled oocysts that can contaminate, persist, and resist disinfection in water and food. Sodium hypochlorite, peroxides, ozone, formaldehyde, and ammonia are suitable disinfectants against Cryptosporidium oocysts. Effective concentrations of these chemicals can be toxic and not practical for downstream research use of non-viable oocysts. Oocyst inactivation approaches such as UV light, heat, and treatments with ethanol or methanol are generally more accessible for routine lab use, yet their applicability in Cryptosporidium assay development is limited. The aims of this study were to evaluate methods of inactivation of Cryptosporidium oocysts that can be readily applied in the laboratory and test the utility of whole inactive oocysts in quantitative PCR (qPCR). Experiments were performed on C. parvum oocysts subjected to heat (75 °C/10 min) or treated with increasing concentrations of ethanol and methanol over time. Viability assays based on propidium iodide (PI) staining, in vitro excystation, and infection of the Hct-8 cell line were used to evaluate the efficacies of the treatments. Excystation of sporozoites was not impaired with 24 h exposures of oocysts to 50% ethanol or methanol, even though significant PI incorporation was observed. Concentrations of ≥70% of these chemicals were required to completely inhibit excystation and infection of Hct-8 cells in vitro. Inactivated oocysts stored for up to 30 days at 4 °C retained cyst wall integrity and antigenicity as observed by light microscopy and immunofluorescence. Moreover, non-viable oocysts applied directly in qPCR assays of the COWP gene were useful reference reagents for the identification and quantification of Cryptosporidium in spiked water samples. In summary, we have established a practical approach to inactivate C. parvum oocysts in the laboratory that is suitable for the development of detection or diagnostic assays targeting the parasite.

On-chip-based electrochemical biosensor for the sensitive and label-free detection of Cryptosporidium

Cryptosporidium, an intestinal protozoan pathogen, is one of the leading causes of death in children and diarrhea in healthy adults. Detection of Cryptosporidium has become a high priority to prevent potential outbreaks. In this paper, a simple, easy to fabricate, and cost-effective on-chip-based electrochemical biosensor has been developed for the sensitive and label-free detection of Cryptosporidium oocysts in water samples. The sensor was fabricated using standard lithography using a mask with a 3-electrode design and modified by self-assembling a hybrid of a thiolated protein/G and the specific anti-Cryptosporidium monoclonal antibodies (IgG3). The electrochemical impedance spectroscopy (EIS) was employed to quantitate C. parvum in the range of 0 to 300 oocysts, with a detection limit of approximately 20 oocysts/5 µL. The high sensitivity and specificity of the developed label-free electrochemical biosensor suggest that this novel platform is a significant step towards the development of fast, real-time, inexpensive and label-free sensing tool for early warning and immediate on-site detection of C. parvum oocysts in water samples, as compared to the traditional methods (such as PCR and microscopy). Furthermore, under optimized conditions, this label-free biosensor can be extended to detect other analytes and biomarkers for environmental and biomedical analyses.

Sponges (Porifera: Spongillida) as ecological indicators for parasitic protozoans Cryptosporidium and Giardia infective stages in freshwater ecosystems

Sponges filter large volumes of water by actively pumping it through their bodies to acquire nutrients, while Cryptosporidium and Giardia are aquatic ecosystems' primary faecal parasitic pollutants. Five aquatic matrices each of surface water (SW), water sediments (WS), substrate-associated biofilms (SAB), as well as five sponges collected from Lake Buhi were investigated for the presence of parasitic protozoan (oo) cysts. Modified Kinyoun's acid-fast stain (MK) was used to detect Cryptosporidium, while immunofluorescence (IFT) staining was employed to detect both Cryptosporidium and Giardia. Scanning Electron Microscopy (SEM) was used to visualize the skeletal sponge structures and simulated contamination with C. parvum oocysts. Aquatic matrices of WS and SAB demonstrated a 20% (3/15) positivity for Cryptosporidium spp. oocysts with MK and IFT. Overall, the study site prevalence of Cryptosporidium spp. oocysts in aquatic matrices were 60% (3/5). Freshwater sponges demonstrated 100% (5/5) Cryptosporidium and 20% (1/5) Giardia (oo) cysts positivity through IFT. Sponges were ascribed to Radiospongilla cfr. philippinensis (Annandale, 1909) based on diagnostic morphotraits of the siliceous skeleton, i.e., megascleres and gemmuloscleres identified under light microscopy (LM) and SEM. Sponges spiked with Cryptosporidium parvum oocysts simulated parasitic protozoan contamination and demonstrated that oocysts could fit through and be trapped within the sponge aquiferous canals. The presence of Cryptosporidium spp. oocysts in aquatic matrices and Cryptosporidium and Giardia spp. (oo) cysts in Radiospongilla cfr. philippinensis demonstrated the contamination of Porifera with infective stages of both parasites. This study submits a promising perspective on the role of freshwater sponges as ecological indicators, i.e. reservoirs and potential biomonitors for the contamination of infective stages of waterborne parasitic protozoans in aquatic ecosystems.

Is Cryptosporidium a hijacker able to drive cancer cell proliferation?

The pathophysiological mechanisms of Cryptosporidium infection are multifactorial and not completely understood. Some advances achieved recently revealed that the infection by Cryptosporidium parvum induces cytoskeleton remodeling and actin reorganization through the implication of several intracellular signals involving, for example, PI3K, Src, Cdc42 and GTPases. It has also been reported that the infection by C. parvum leads to the activation of NF-κβ, known to induce anti-apoptotic mechanisms and to transmit oncogenic signals to epithelial cells. Despite the growing evidence about the hijacking of cellular pathways, potentially being involved in cancer onset, this information has rarely been linked to the tumorigenic potential of the parasite. However, several evidences support an association between Cryptosporidium infection and the development of digestive neoplasia. To explore the dynamics of Cryptosporidium infection, an animal model of cryptosporidiosis using corticoid dexamethasone-treated adult SCID (severe combined immunodeficiency) mice, orally infected with C. parvum or Cryptosporidium muris oocysts was implemented. C. parvum-infected animals developed digestive adenocarcinoma. When mechanisms involved in this neoplastic process were explored, the pivotal role of the Wnt pathway together with the alteration of the cytoskeleton was confirmed. Recently, a microarray assay allowed the detection of cancer-promoting genes and pathways highly up regulated in the group of C. parvum infected animals when compared to non-infected controls. Moreover, different human cases/control studies reported significant higher prevalence of Cryptosporidium infection among patients with recently diagnosed colon cancer before any treatment when compared to the control group (patients without colon neoplasia but with persistent digestive symptoms). These results suggest that Cryptosporidium is a potential oncogenic agent involved in cancer development beyond the usual suspects. If Cryptosporidium is able to hijack signal transduction, then is very likely that this contributes to transformation of its host cell. More research in the field is required in order to identify mechanisms and molecular factors involved in this process and to develop effective treatment interventions.

Potential effect of methanol leaf extracts from three Podocarpus species on experimental cryptosporidiosis.

Cryptosporidiosis causes diarrhea in both immunocompetent and immunocompromised individuals, with acute manifestations occurring particularly in children and the elderly. Up till now, there is no curative therapy for cryptosporidiosis, so discovery of new classes of drugs are of great importance. This study aimed to examine the effect of methanol leaves extracts of the three Podocarpus species; P. macrophyllus (Thunb.), P. gracilior (Pilg.) and P. elongatus (Aiton) L' Hér. ex Pers and their combination on Cryptosporidium parvum (C. parvum) in experimentally infected mice in comparison with the commercially used drug, Nitazoxanide. As well as spectrophotometric estimation of the total phenolic and flavonoid content of these extracts was done. Results revealed that treatment with these three Podocarpus extracts and their combination showed a significant reduction of the number of C. parvum oocyst shed in the stool of infected mice compared to infected control group and Nitazoxanide- infected treated group at P < 0.001. The combination of the three Podocarpus extracts was the most effective treatment showing the lowest number of oocysts shedding in comparison with other used extracts and Nitazoxanide. Histopathological inspection of sections from ilium and colon displayed signs of improvement after treatment with P. macrophyllus and P. gracilior extracts and more remarkable improvement when the three extracts were combined. It was concluded that the three Podocarpus species extracts used in this study had a promising anti-Cryptosporidium activity especially when they were combined.

Age and episode-associated occurrence of Cryptosporidium species and subtypes in a birth-cohort of dairy calves.

The role of species-specific immunity in infection patterns of Cryptosporidium spp. in humans and farm animals is not well understood. In the present study, the dynamics of Cryptosporidium infections in a natural cryptosporidiosis model was examined using genotyping, subtyping and whole genome sequencing tools. In a cross-sectional survey of Cryptosporidium spp. in 934 dairy cattle on one farm, marked age-associated differences in the distribution of Cryptosporidium species and C. bovis subtypes were observed. In a closely followed longitudinal birth cohort study of 81 calves over a 9-month period, shedding of C. parvum oocysts by the IIdA19G1 subtype started at 4 days, peaked at 2weeks and ended mostly by 4weeks. In contrast, the shedding of C. bovis oocysts started at 2weeks, peaked initially at 6weeks and had a second wave during 15th to 23rd weeks. For C. ryanae, calves had mostly only one episode of infection by one subtype, with accumulative infection increasing much slower than C. parvum and C. bovis. Overall, the accumulative infection rates and mean duration of oocyst shedding for calves in the cohort were 97.4% (76/78) and 2.3weeks, 100.0% (80/80) and 3.9weeks, and 78.7% (63/80) and 3.2weeks for C. parvum, C. bovis and C. ryanae, respectively. The oocyst shedding intensity was much lower in C. bovis and C. ryanae infections compared with C. parvum infection, and in the second episode of C. bovis infection compared with the first episode. The two episodes of C. bovis infections were caused by different genome types that differed mostly in nine genes. Cryptosporidium parvum infection was associated with the occurrence of watery diarrhoea. Data from the natural history study of cryptosporidiosis indicate that despite the existence of acquired immunity against homologous pathogens, neonatal animals experience waves of Cryptosporidium infections by different species and genome types.

ATP Purinergic Receptor P2X1-Dependent Suicidal NETosis Induced by Cryptosporidium parvum under Physioxia Conditions.

Simple SummaryCryptosporidium parvum, a zoonotic apicomplexan, is one of the leading causes of severe diarrheal disease worldwide and a major contributor to early infant and neonatal mortality. In vivo, sporozoites of C. parvum must invade enterocytes where they develop in a unique intracellular extracytoplasmic niche. Polymorphonuclear neutrophils (PMN) are the most common leukocytes in the immune defense system of the host, the first to be transferred to C. parvum-infected gut mucosa, and are increasingly recognized as important cells in the fight against other intestinal parasites. We investigated the role of ATP purinergic receptor P2X1, glycolysis, plasma membrane monocarboxylate transporters (MCTs) of lactate, extracellular acidification rates (ECAR), and mitochondrial oxygen consumption rates (OCR), as well as Notch signaling in C. parvum-triggered NETs formation in exposed bovine PMN under intestinal physioxic (5% O2) as well as hyperoxic (21% O2) conditions. Both C. parvum stages, i.e., sporozoites and oocysts, strongly induce suicidal NETosis in a P2X1-dependent manner, suggesting anti-cryptosporidial effects not only through firm sporozoite entrapment and hampered sporozoite excystation, but also probably through direct exposure to NETs-derived toxic molecules.Cryptosporidiosis is a zoonotic intestinal disease that affects humans, wildlife, and neonatal cattle, caused by Cryptosporidium parvum. Neutrophil extracellular traps (NETs), also known as suicidal NETosis, are a powerful and ancient innate effector mechanism by which polymorphonuclear neutrophils (PMN) battle parasitic organisms like protozoa and helminths. Here, C. parvum oocysts and live sporozoites were utilized to examine suicidal NETosis in exposed bovine PMN under both 5% O2 (physiological conditions within small intestinal tract) and 21% O2 (normal hyperoxic conditions in research facilities). Both sporozoites and oocysts induced suicidal NETosis in exposed PMN under physioxia (5% O2) and hyperoxia (21% O2). Besides, C. parvum-induced suicidal NETosis was affirmed by total break of PMN, co-localization of extracellular DNA decorated with pan-histones (H1A, H2A/H2B, H3, H4) and neutrophil elastase (NE) by means of confocal- and immunofluorescence microscopy investigations. C. parvum-triggered NETs entrapped sporozoites and impeded sporozoite egress from oocysts covered by released NETs, according to scanning electron microscopy (SEM) examination. Live cell 3D-holotomographic microscopy analysis visualized early parasite-induced PMN morphological changes, such as the formation of membrane protrusions towards C. parvum while undergoing NETosis. Significant reduction of C. parvum-induced suicidal NETosis was measured after PMN treatments with purinergic receptor P2X1 inhibitor NF449, under both oxygen circumstances, this receptor was found to play a critical role in the induction of NETs, indicating its importance. Similarly, inhibition of PMN glycolysis via 2-deoxy glucose treatments resulted in a reduction of C. parvum-triggered suicidal NETosis but not significantly. Extracellular acidification rates (ECAR) and oxygen consumption rates (OCR) were not increased in C. parvum-exposed cells, according to measurements of PMN energetic state. Treatments with inhibitors of plasma membrane monocarboxylate transporters (MCTs) of lactate failed to significantly reduce C. parvum-mediated NET extrusion. Concerning Notch signaling, no significant reduction was detected after PMN treatments with two specific Notch inhibitors, i.e., DAPT and compound E. Overall, we here describe for the first time the pivotal role of ATP purinergic receptor P2X1 in C. parvum-mediated suicidal NETosis under physioxia (5% O2) and its anti-cryptosporidial properties.

Garlic (Allium sativum Linnaeus) improved inflammation and reduced cryptosporidiosis burden in immunocompromised mice

Ethnopharmacological relevanceFor thousands of years, garlic (Allium sativum Linnaeus) has been consumed in food and health by numerous civilizations. Cryptosporidium (C.) parvum is an apicomplexan parasite that causes a gastrointestinal disease, with the most common symptoms being watery diarrhea. Although several substances have been tried for its anti-cryptosporidial action, there is no effective treatment for Cryptosporidium disease, especially in immunocompromised individuals. The present study aimed firstly to characterize the bio-active compounds in Allium sativum L. and secondly to evaluate its efficacy as a therapy for cryptosporidiosis especially in immunocompromised mice. Materials and methodsThis was accomplished by evaluating the parasitological and histopathological parameters in the experimentally infected immunocompetent and immunocompromised mice. Also, the cytokine profile during the experimental time was recorded through the measuring of T helper (h)1, Th2 and Th17 cells cytokines. Immunosuppressed mice were given 0.25 μg/g per day of dexamethasone orally, before infection with Cryptosporidium parvum oocysts, for fourteen consecutive days. Starting 10 days post infection (PI), nitazoxanide (100 mg/kg per day) or Allium sativum (50 mg/kg per day) was given orally for fourteen consecutive days. ResultsOur results showed that oocyst shedding, on the 32nd day PI, in immunocompromised infected group treated with Allium sativum (354.11, 99.35% PR) showed a significant decrease when compared to its corresponding group treated with nitazoxanide (4369.14, 92.05% PR). On the 32nd day PI, all cytokines levels have been decreased to levels that were similar to those of their uninfected corresponding control groups; also, the histopathological changes and the loss in animals’ body weight had been improved. Treatment with nitazoxanide did not result in infection clearance or a reduction in the increased cytokines' levels. ConclusionAllium sativum L. displayed high efficacy as a potential therapeutic agent against Cryptosporidium, which supports its traditional usage in parasite diseases.

Curcumin mitigates Cryptosporidium parvum infection through modulation of gut microbiota and innate immune-related genes in immunosuppressed neonatal mice

Cryptosporidium parvum is a major cause of diarrheal disease in immature or weakened immune systems, mainly in infants and young children in resource-poor settings. Despite its high prevalence, fully effective and safe drugs for the treatment of C. parvum infections remain scarce, and there is no vaccine. Meanwhile, curcumin has shown protective effects against C. parvum infections. However, the mechanisms of action and relationship to the gut microbiota and innate immune responses are unclear. Immunosuppressed neonatal mice were infected with oocysts of C. parvum and either untreated or treated with a normal diet, curcumin or paromomycin. We found that curcumin stopped C. parvum oocysts shedding in the feces of infected immunosuppressed neonatal mice, prevented epithelial damage, and villi degeneration, as well as prevented recurrence of infection. Curcumin supplementation increased the relative abundance of Bacteroidetes and decreased the relative abundance of Firmicutes and Proteobacteria in mice infected with C. parvum as shown by 16S rRNA gene sequencing analysis. The relative abundance of Lactobacillus, Bacteroides, Akkermansia, Desulfovibrio, Prevotella, and Helicobacter was significantly associated with C. parvum infection inhibited by curcumin. Curcumin significantly (P < 0.01) suppressed IFN-γ and IL -18 gene expression levels in immunosuppressed neonatal C. parvum-infected mice. We demonstrate that the therapeutic effects curcumin are associated with alterations in the gut microbiota and innate immune-related genes, which may be linked to the anti-Cryptosporidium mechanisms of curcumin.

Chitosan Protects Immunosuppressed Mice Against Cryptosporidium parvum Infection Through TLR4/STAT1 Signaling Pathways and Gut Microbiota Modulation.

Cryptosporidium parvum infection is very common in infants, immunocompromised patients, or in young ruminants, and chitosan supplementation exhibits beneficial effects against the infection caused by C. parvum. This study investigated whether chitosan supplementation modulates the gut microbiota and mediates the TLR4/STAT1 signaling pathways and related cytokines to attenuate C. parvum infection in immunosuppressed mice. Immunosuppressed C57BL/6 mice were divided into five treatment groups. The unchallenged mice received a basal diet (control), and three groups of mice challenged with 1 × 106 C. parvum received a basal diet, a diet supplemented with 50 mg/kg/day paromomycin, and 1 mg/kg/day chitosan, and unchallenged mice treated with 1 mg/kg/day chitosan. Chitosan supplementation regulated serum biochemical indices and significantly (p < 0.01) reduced C. parvum oocyst excretion in infected mice treated with chitosan compared with the infected mice that received no treatment. Chitosan-fed infected mice showed significantly (p < 0.01) decreased mRNA expression levels of interferon-gamma (IFN-γ) and tumor necrosis factor-α (TNF-α) compared to infected mice that received no treatment. Chitosan significantly inhibited TLR4 and upregulated STAT1 protein expression (p < 0.01) in C. parvum-infected mice. 16S rRNA sequencing analysis revealed that chitosan supplementation increased the relative abundance of Bacteroidetes/Bacteroides, while that of Proteobacteria, Tenericutes, Defferribacteres, and Firmicutes decreased (p < 0.05). Overall, the findings revealed that chitosan supplementation can ameliorate C. parvum infection by remodeling the composition of the gut microbiota of mice, leading to mediated STAT1/TLR4 up- and downregulation and decreased production of IFN-γ and TNF-α, and these changes resulted in better resolution and control of C. parvum infection.

The Effects of Commercially Available Syzygium aromaticum, Anethum graveolens, Lactobacillus acidophilus LB, and Zinc as Alternatives Therapy in Experimental Mice Challenged with Cryptosporidium parvum.

BackgroundWaterborne cryptosporidiosis is the second cause of diarrhea in young children and immunocompromised hosts after rotavirus. Except for nitazoxanide (NTZ), there is no accredited cryptosporidiosis treatment to date. Therefore, there is an urgent need to find an effective and safe treatment for cryptosporidiosis. This study aimed to investigate the possible anti-protozoal effects of Syzygium aromaticum (clove) oil, Anethum graveolens (dill) seeds oil, Lactobacillus acidophilus LB, and zinc against Cryptosporidium parvum in comparison to NTZ.MethodsBesides the negative control, mice from six experimental groups (T1–T6) were infected with Cryptosporidium parvum oocysts. On the seventh day post-infection (PID), mice from five groups were treated for 8 consecutive days with NTZ, clove oil, dill seed oil, Lactobacillus acidophilus LB, and zinc commercial forms (T2–T5). Oocysts shedding rate, differences of mice body weight, serum IL10, and TNF-α, cryptosporidial antigen, and cd3 at the intestinal mucosa were evaluated at the end of the experiment.ResultsThe mean of the C. parvum oocysts’ shedding rate was significantly lower in all treated groups than in the non-treated group. The oocysts reduction rate was the highest in zinc-treated mice (98.3%), Lactobacillus acidophilus LB and dill-treated groups (95.77%), and the NTZ-treated group (91.55%). Clove oil was the least effective, with a 74.65% reduction rate. Excluding the clove oil-treated group, immunohistochemical analysis revealed the clearance of the Cryptosporidium antigen in the intestinal tissue in all treated groups.ConclusionThe study has provided a rational basis for using these safe, cheap, and commercially available alternatives in treating cryptosporidiosis combined with NTZ.

Use of a natural herbal-based feed additive containing isoquinoline alkaloids in newborn calves with cryptosporidiosis

Cryptosporidium infections are one of the most prevalent causes of diarrhea in calves and considered to be one of the major sources of economic loss in livestock production. A global trend is currently underway, in identifying natural and sustainable alternatives to support animal husbandry and production. Isoquinoline alkaloids are known for their anti-inflammatory, antimicrobial, and antioxidant properties in the promotion of gut health. Thus, an experiment was designed to evaluate the effects of natural, herbal-based feed isoquinoline alkaloids to support calves experimentally inoculated with Cryptosporidium parvum. Twenty-six calves were randomly divided into control (CN) (n = 13) and treatment (SG) (n = 13) groups. The SG group received 5 g of feed additive in every milk feeding from 1 to 21 days of age. The CN group received milk without any additives. All calves were orally inoculated on the third day of life with 1 × 106Cryptosporidium parvum oocysts. The animals were evaluated daily, from 3 to 30 days of age, for the occurrence, duration, and intensity of diarrhea. Calves with a base deficit of ≥ 9 mEq/L were hydrated to aid recovery. The SG calves showed a higher average weight gain between 14 and 21 days of age, without mortality and with reduced intensity and duration of diarrhea. In contrast, calves in the CN group showed more serious acid-base disorders, required more hydration support, and had a mortality rate of 15.4 %. These results showed that calves supplemented with isoquinoline alkaloids had decreased intensity and duration of symptoms, reduced requirement for supportive therapy, and prevented mortality among animals.

Discrimination of waterborne pathogens, Cryptosporidium parvum oocysts and bacteria using surface-enhanced Raman spectroscopy coupled with principal component analysis and hierarchical clustering

Waterborne pathogens (parasites, bacteria) are serious threats to human health. Cryptosporidium parvum is one of the protozoan parasites that can contaminate drinking water and lead to diarrhea in animals and humans. Rapid and reliable detection of these kinds of waterborne pathogens is highly essential. Yet, current detection techniques are limited for waterborne pathogens and time-consuming and have some major drawbacks. Therefore, rapid screening methods would play an important role in controlling the outbreaks of these pathogens. Here, we used label-free surface-enhanced Raman Spectroscopy (SERS) combined with multivariate analysis for the detection of C. parvum oocysts along with bacterial contaminants including, Escherichia coli, and Staphylococcus aureus. Silver nanoparticles (AgNPs) are used as SERS substrate and samples were prepared with simply mixed of concentrated AgNPs with microorganisms. Each species presented distinct SERS spectra. Principal component analysis (PCA) and hierarchical clustering were performed to discriminate C. parvum oocysts, E. coli, and S. aureus. PCA was used to visualize the dataset and extract significant spectral features. According to score plots in 3 dimensional PCA space, species formed distinct group. Furthermore, each species formed different clusters in hierarchical clustering. Our study indicates that SERS combined with multivariate analysis techniques can be utilized for the detection of C. parvum oocysts quickly.

Development of cell-imprinted polymer surfaces for Cryptosporidium capture and detection

Cryptosporidium parvum is waterborne parasite that can cause potentially life-threatening gastrointestinal disease and is resistant to conventional water treatment processes, including chlorine disinfection. The current Environmental Protection Agency-approved method for oocyst detection and quantification is expensive, limiting the ability of water utilities to monitor complex watersheds thoroughly to understand the fate and transport of C. parvum oocysts. In this work, whole cell imprinting was used to create selective and sensitive surfaces for the capture of C. parvum oocysts in water. Cell-imprinted Polydimethylsiloxane (PDMS) was manufactured using a modified stamping approach, and sensitivity and selectivity were analyzed using different water chemistries and different surrogate biological and non-biological particles. The overall binding affinity was determined to be less than that of highly specific antibodies, but on par with standard antibodies and immune-enabled technologies. These initial results demonstrate the potential for developing devices using cell-imprinting for use in waterborne pathogen analysis.

CRISPR/Cas12a-powered immunosensor suitable for ultra-sensitive whole Cryptosporidium oocyst detection from water samples using a plate reader

Waterborne pathogens, such as Cryptosporidium parvum, pose a major threat to public health globally, and this requires screening of drinking and environmental water for low number of contaminating microbes. However, current detection approaches generally require trained experts with sophisticated instruments, and are not suitable for large-scale screening and rapid outbreak response. Recent advances in ultrasensitive CRISPR/Cas-based biosensing continue to expand the range of detectable molecular targets, however single microbes could not be directly detected so far, especially in environmental samples. Here, we report an ultrasensitive CRISPR/Cas12a-powered immunosensing method suitable for microbial detection which links antibody-based recognition with CRISPR/Cas12a-based fluorescent signal amplification through an antibody-DNA conjugate. This approach is shown here to detect whole 4 µm size Cryptosporidium parvum oocysts with a linear range from 6.25 – 1600 oocysts/mL, at a maximum sensitivity of single oocyst per sample. Its potential to apply to various complex sample matrices has also been demonstrated. After sample dilution by factor of 10, we were able to detect 10 oocysts from a back-wash mud samples from water treatment plate. This method uses the same experimental setup (plate reader) as a conventional ELISA assay thus reducing the need for microscopy-based identification of Cryptosporidium, which represents the gold-standard but requires high level expertise and time-consuming manual counting. This work highlights the potential of CRISPR/Cas-based biosensing for water quality assessment and ultrasensitive whole pathogen detection.

Detection of Cryptosporidium Parvum Oocysts in Artificially Contaminated Filter Backwash Water and Ozone Treatment at Pilot Scale

ABSTRACT Waterborne diseases are a relevant concern for public health systems since commonly applied treatment techniques may not remove all water contaminants. Cryptosporidium spp. oocysts are an issue in water treatment plants due to their reduced size and resistance to the disinfection process (e.g., chlorination). Moreover, oocysts retained on the filter media might recirculate amongst the filter backwash water (FBW). This study aimed to detect Cryptosporidium parvum oocysts artificially inoculated on the FBW and evaluate the ozone treatment performance. A synthetic FBW underwent three concentration methods followed by immunomagnetic separation: calcium carbonate flocculation, direct centrifugation, and direct centrifugation with the 7X ICN dispersion solution. The latter method was selected as it presented less interference on oocyst viability (37.2% reduction) and higher recovery (22.1%) on preliminary assays. The recovery for the commercial suspensions was 15.4 ± 3.3%, although the analytical quality performed with EasySeed® suspension obtained a recovery of 2.8 ± 0.8%. These limitations and methodologies for protozoan detection are challenging due to low recoveries, especially in complex matrices. Finally, FBW ozonation was performed on a pilot scale, and the propidium iodide dye indicated oocyst viability decreased after treatment. Oocyst inactivation was 2.83 log and 3.44 log for dosages of 7.5 mg O3 L−1 for 10 min (i.e., 75 mg min L−1) and 10 mg O3 L−1 for 5 min (i.e., 50 mg min L−1), respectively. Disinfection is a crucial pathway for addressing outbreak scenarios, and ozone treatment should be further studied.

A COMPARISON OF RAPID DIAGNOSTIC TEST AND MODIFIED ZIEHL-NEELSEN METHOD IN THE DIAGNOSIS OF CRYPTOSPORIDIOSIS.

Background: Cryptosporidium parvum as a leading cause of persistent diarrhoea in developing countries causes a more severe illness in patients infected with human immunodeficiency virus (HIV). The intermittent shedding of Cryptosporidium parvum oocysts in stool, even in patients with massive diarrhoea, makes diagnosis difficult. This study evaluated the validity of Crypto-Strip test in comparison to the modified Ziehl-Neelsen for the diagnosis of Cryptosporidium infection using polymerase chain reaction (PCR) as gold standard. Methods: This was a hospital based cross-sectional study of 100 HIV positive patients with diarrhoea at a tertiary health facility in Jos, Nigeria. We collected 15mls of stool sample and relevant information from patients who gave informed consent. The oocytes of Cryptosporidium parvum were identified in the stool samples using modified Ziehl-Neelsen stain and Rapid diagnostic test (Crypto-Strip). We also ran all samples using polymerase chain reaction (PCR). Statistical analysis was with statistical package for Social Sciences (SPSS) version 21. Result: Out of the 100 stool samples analysed, 13 (13%) were positive for cryptosporidiosis by PCR. Crypto- Strips recorded 12 (12%) positive with one (1) being false negative as compared to PCR. Conversely, eight (8%) of the 100 samples collected were positive for Cryptosporidium oocysts using modified Ziehl Neelsen (mZN) stain. Out of the eight positives by mZN stain, two were false positive as both tested negative by the PCR. The Crypto-Strip test kit had a sensitivity of 92.3% and a specificity of 100.0% and also positive and negative predictive values of 100.0% and 98.9%, respectively. Whereas, the modified Ziehl Neelsen stain had a sensitivity of 46.2% and a specificity of 97.7% with a positive and negative predictive value of 75.0% and 92.4%, respectively. Conclusion: In this study, we have shown that Crypto-Strip test was a better diagnostic method for the diagnosis of crypto

Successful experimental infant baboon model for childhood cryptosporidiosis studies

BackgroundCryptosporidiosis causes high morbidity and mortality in children under 2 years of age globally. The lack of an appropriate animal model that mimics the pathogenesis of disease in humans has hampered the development and testing of potential therapeutic options. This study aimed to develop and validate an infant baboon infection model of cryptosporidiosis.MethodsEighteen immunocompetent weaned infant baboons aged 12 to 16 months were used. The animals were n = 3 controls and three experimental groups of n = 5 animals each inoculated with Cryptosporidium parvum oocysts as follows: group 1: 2 × 104, group 2: 2 × 105, group 3: 2 × 106 followed by daily fecal sampling for oocyst evaluation. Blood sampling for immunological assay was done on the day of infection and weekly thereafter until the end of the experiment, followed by necropsy and histopathology. Statistical analysis was performed using R, SPSS, and GraphPad Prism software. Analysis of variance (ANOVA) and Bonferroni post hoc tests were used for comparison of the means, with p < 0.05 considered as a significant difference. Correlation coefficient and probit analysis were also performed.ResultsIn all experimental animals but not controls, the onset of oocyst shedding occurred between days 2 and 4, with the highest oocyst shedding occurring between days 6 and 28. Histological analysis revealed parasite establishment only in infected animals. Levels of cytokines (TNF-α, IFN-γ, and IL-10) increased significantly in experimental groups compared to controls.ConclusionFor developing a reproducible infant baboon model, 2 × 104 oocysts were an effective minimum quantifiable experimental infection dose.Graphic abstract

Diagnostic Methods for the Detection of Cryptosporidium parvum Oocysts in Faeces

Diagnostic Methods for the Detection of Cryptosporidium parvum Oocysts in Faeces

Analysis of Cryptosporidium spp. from clinical samples by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

To purify Cryptosporidium spp. oocysts from clinical stool samples and evaluate using an up-to-date mass spectrometry protocol producing high-quality reference spectra. A refined purification protocol was developed for oocysts from stools, involving salt flotation and potassium bromide density centrifugation. Purified oocysts were prepared for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) by formic acid extraction, and the extracts analysed using the Bruker MALDI Biotyper system. Individual spectral markers were identified by their specific mass peaks. Cryptosporidium parvum oocysts (Iowa strain) propagated in vivo, and C. parvum (n=2) and Cryptosporidium hominis (n=1) oocysts from clinical stool samples produced distinct spectra that were considered specific to Cryptosporidium spp. with no evidence of contamination. The production of distinct spectra demonstrated the utility of the purification method for oocysts from clinical stool samples and provided reference spectra. The use of MALDI-TOF MS and other mass spectrometry techniques has been limited previously to C. parvum oocysts propagated in vivo. Appropriate purification of oocysts can achieve sufficient biomass, enabling analysis by MALDI-TOF MS and potentially other mass spectrometry platforms, facilitating peptide and protein discovery and identification of biomarkers from a much wider range of Cryptosporidium spp. from natural infections.