Indole metabolites generated by microbiota inhibit Cryptosporidium growth.

Funkhouser-Jones et al. recently identified gut metabolites that affected Cryptosporidium growth. A key focus, indole, was shown to inhibit the parasite in vivo and in vitro by decreasing the host mitochondria function and the membrane potential of parasite mitosomes. These findings help clarify the role microflora and metabolites play in host resistance.

Development and evaluation of a molecular based protocol for detection and quantification of Cryptosporidium spp. in wastewater.

Infections caused by protozoan parasites are a major public health concern globally. These infections are commonly diagnosed during water-borne outbreaks, necessitating accurate and highly sensitive detection procedures to assure public health protection. Current molecular techniques are challenged by several factors, such as low parasite concentration, inefficient DNA extraction methods, and inhibitors in environmental samples. This study focused on the development and validation of a molecular protocol for DNA extraction, efficient protozoan (oo)cyst recovery and quantification of protozoan parasites from wastewater using droplet digital polymerase chain reaction (ddPCR). Five DNA extraction methods, including commercial kits, custom phenol-chloroform, and in-house modified methods, were evaluated. The efficiency of each method was assessed via spectrophotometric analysis and ddPCR amplification using specific primers. Lastly, the developed protocol was evaluated for the detection and quantification of Cryptosporidium parvum in wastewater from different regions in South Africa. The conventional phenol-chloroform extraction method yielded the highest DNA concentration of 223 (±0.71) ng/µl and detected the highest number of Cryptosporidium parvum (1807 (±0.30) copies/ddPCR reaction) compared to other methods evaluated in this study. Additionally, the phenol-chloroform method demonstrated high sensitivity in extracting DNA from as few as one cyst/L of Cryptosporidium parvum, corresponding to 5.93 copies/ddPCR reaction. It was also observed that analysis of both the filtered supernatant and pellets after centrifugation improves the recovery efficiency of oocysts from wastewater by 10.5%, resulting in a total recovery of 64.1%. This optimized protocol was successfully applied to measure protozoan concentration in wastewater from different regions in South Africa. The improved DNA extraction and quantification method proposed in this study would be effective in monitoring protozoan concentration in the environment, which will help in instituting mitigation measures to reduce water-borne infections.

Challenges for Cryptosporidium Population Studies.

Cryptosporidiosis is ranked sixth in the list of the most important food-borne parasites globally, and it is an important contributor to mortality in infants and the immunosuppressed. Recently, the number of genome sequences available for this parasite has increased drastically. The majority of the sequences are derived from population studies of Cryptosporidium parvum and Cryptosporidium hominis, the most important species causing disease in humans. Work with this parasite is challenging since it lacks an optimal, prolonged, in vitro culture system, which accurately reproduces the in vivo life cycle. This obstacle makes the cloning of isolates nearly impossible. Thus, patient isolates that are sequenced represent a population or, at times, mixed infections. Oocysts, the lifecycle stage currently used for sequencing, must be considered a population even if the sequence is derived from single-cell sequencing of a single oocyst because each oocyst contains four haploid meiotic progeny (sporozoites). Additionally, the community does not yet have a set of universal markers for strain typing that are distributed across all chromosomes. These variables pose challenges for population studies and require careful analyses to avoid biased interpretation. This review presents an overview of existing population studies, challenges, and potential solutions to facilitate future population analyses.

Prevalence of cryptosporidiosis among children with diarrhoea under five years admitted to Kosti teaching hospital, Kosti City, Sudan.

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.

Supply and demand-heme synthesis, salvage and utilization by Apicomplexa.

The Apicomplexa phylum groups important human and animal pathogens that cause severe diseases, encompassing malaria, toxoplasmosis, and cryptosporidiosis. In common with most organisms, apicomplexans rely on heme as cofactor for several enzymes, including cytochromes of the electron transport chain. This heme derives from de novo synthesis and/or the development of uptake mechanisms to scavenge heme from their host. Recent studies have revealed that heme synthesis is essential for Toxoplasma gondii tachyzoites, as well as for the mosquito and liver stages of Plasmodium spp. In contrast, the erythrocytic stages of the malaria parasites rely on scavenging heme from the host red blood cell. The unusual heme synthesis pathway in Apicomplexa spans three cellular compartments and comprises enzymes of distinct ancestral origin, providing promising drug targets. Remarkably given the requirement for heme, T. gondii can tolerate the loss of several heme synthesis enzymes at a high fitness cost, while the ferrochelatase is essential for survival. These findings indicate that T. gondii is capable of salvaging heme precursors from its host. Furthermore, heme is implicated in the activation of the key antimalarial drug artemisinin. Recent findings established that a reduction in heme availability corresponds to decreased sensitivity to artemisinin in T. gondii and Plasmodium falciparum, providing insights into the possible development of combination therapies to tackle apicomplexan parasites. This review describes the microeconomics of heme in Apicomplexa, from supply, either from de novo synthesis or scavenging, to demand by metabolic pathways, including the electron transport chain.

A chicken embryo model for the maintenance and amplification of Cryptosporidium parvum and Cryptosporidium baileyi oocysts.

Cryptosporidium is a genus of apicomplexan parasites that inhabit the respiratory and gastrointestinal tracts of vertebrates. Research of these parasites is limited by a lack of model hosts. This study aimed to determine the extent to which infection at the embryo stage can enhance the propagation of Cryptosporidium oocysts in chickens. Nine-day-old chicken embryos and one-day-old chickens were experimentally infected with different doses of Cryptosporidium baileyi and Cryptosporidium parvum oocysts. Post hatching, all chickens had demonstrable infections, and the infection dose had no effect on the course of infection. Chickens infected as embryos shed oocysts immediately after hatching and shed significantly more oocysts over the course of the infection than chickens infected as one-day-olds. In chickens infected as embryos, C. baileyi was found in all organs except the brain whereas, C. parvum was only found in the gastrointestinal tract and trachea. In chickens infected as one-day-olds, C. baileyi was only found in the gastrointestinal tract and trachea. Chickens infected as embryos with C. baileyi died within 16 days of hatching. All other chickens cleared the infection. Infection of chickens as embryos could be used as an effective and simple model for the propagation of C. baileyi and C. parvum.

Cryptosporidium Oocyst Purification Using Discontinuous Gradient Centrifugation.

Many laboratory studies in cryptosporidial research require a source of purified oocysts. Sources can include experimentally infected laboratory animals or from samples collected from naturally infected animals and from clinical cases of human cryptosporidiosis. Purification of oocysts can be accomplished with readily available laboratory equipment including tabletop centrifuges and microcentrifuges. Following purification, oocysts can be stored in antibiotic-supplemented buffers or in 2.5% aqueous potassium dichromate for over 6 months. Ultimately, oocyst viability and infectivity decline to less than 10% after 1 year, so if isolates are expected to be maintained, serial passage in a suitable host at ≤6-month intervals is recommended. Oocysts purified as described in this chapter are suitable for animal infection studies, cell culture studies, and a wide range of molecular biological studies, environmental studies, drug testing, and disinfection studies.

Production and Purification of Functional Cryptosporidium Glycoproteins by Heterologous Expression in Toxoplasma gondii.

Development of an effective vaccine against cryptosporidiosis is a medical and veterinary priority. However, many putative Cryptosporidium vaccine candidates such as surface and apical complex antigens are posttranslationally modified with O- and N-linked glycans. This presents a significant challenge to understanding the functions of these antigens and the immune responses to them. Isolation of large amounts of native antigen from Cryptosporidium oocysts is expensive and is only feasible for C. parvum antigens. Here, we describe a method of producing recombinant, functional Cryptosporidium glycoprotein antigens in Toxoplasma gondii. These functional recombinant proteins can be used to investigate the role of glycotopes in Cryptosporidium immune responses and parasite-host cell interactions.

Two- and Three-Dimensional Bioengineered Human Intestinal Tissue Models for Cryptosporidium.

Conventional cell cultures utilizing transformed or immortalized cell lines or primary human epithelial cells have played a fundamental role in furthering our understanding of Cryptosporidium infection. However, they remain inadequate with respect to their inability to emulate in vivo conditions, support long-term growth, and complete the life cycle of the parasite. Previously, we developed a 3D silk scaffold-based model using transformed human intestinal epithelial cells (IECs). This model supported C. parvum infection for up to 2 weeks and resulted in completion of the life cycle of the parasite. However, transformed IECs are not representative of primary human IEC.Human intestinal enteroids (HIEs) are cultures derived from crypts that contain Lgr5+ stem cells isolated from human biopsies or surgical intestinal tissues; these established multicellular cultures can be induced to differentiate into enterocytes, enteroendocrine cells, goblet cells, Paneth cells, and tuft cells. HIEs better represent human intestinal structure and function than immortalized IEC lines. Recently, significant progress has been made in the development of technologies to culture HIEs in vitro. When grown in a 3D matrix, HIEs provide a spatial organization resembling the native human intestinal epithelium. Additionally, they can be dissociated and grown as monolayers in tissue culture plates, permeable supports or silk scaffolds that enable mechanistic studies of pathogen infections. They can also be co-cultured with other human cells such as macrophages and myofibroblasts. The HIEs grown in these novel culture systems recapitulate the physiology, the 3D architecture, and functional diversity of native intestinal epithelium and provide a powerful and promising new tool to study Cryptosporidium-host cell interactions and screen for interventions ex vivo. In this chapter, we describe the 3D silk scaffold-based model using transformed IEC co-cultured with human intestinal myofibroblasts and 2D and 3D HIE-derived models of Cryptosporidium, also co-cultured with human intestinal myofibroblasts.

Investigation of the Physical, Chemical Characteristics and Microbial Contamination of the Indoor Swimming Pools

Objective: The aim of this study was to investigate the physical, chemical and microbiological contamination of indoor swimming pools. Methods: Pool water specimens were collected using a plastic polypropylene sterilized bottle. The physical and chemical qualities of the waters were analyzed in terms of temperature, turbidity, pH, and free residual chlorine, with the standard methods for the examination of water. Bacteriological (routine methods) and parasitological (molecular methods) tests were carried out on pools water. Results: The mean temperature, pH, and residual chlorine of the indoor pools were 31.2 °C, 7.6 and 1.5 mg/L, respectively. Turbidity was not observed in any of the pools. The pH and temperature values were in standard ranges in 92.3% and 15.4% of the waters of swimming pools, respectively. The prevalence rates of bacterial and amoebic contaminations of the water in the swimming pools were 53.8% and 46.2%, respectively. One pool (7.7%) was contaminated with both bacteria and amoeba. Streptococcus viridans, Staphylococcus epidermidis, Pseudomonas stutzeri, Cryptosporidium and Bacillus spp. were isolated from the pool waters. Conclusion: In this study, some microorganisms were identified from the water pools. Effective management of swimming pools and proper control of the physical, chemical and microbiological property of water pools can produce the healthy recreational activity.

A Stem-Cell-Derived Platform Enables Complete Cryptosporidium Development In Vitro and Genetic Tractability.

Despite being a frequent cause of severe diarrheal disease in infants and an opportunistic infection in immunocompromised patients, Cryptosporidium research has lagged due to a lack of facile experimental methods. Here, we describe a platform for complete life cycle development and long-term growth of C. parvum in vitro using "air-liquid interface" (ALI) cultures derived from intestinal epithelial stem cells. Transcriptomic profiling revealed that differentiating epithelial cells grown under ALI conditions undergo profound changes in metabolism and development that enable completion of the parasite life cycle in vitro. ALI cultures support parasite expansion > 100-fold and generate viable oocysts that are transmissible in vitro and to mice, causing infection and animal death. Transgenic parasite lines created using CRISPR/Cas9 were used to complete a genetic cross in vitro, demonstrating Mendelian segregation of chromosomes during meiosis. ALI culture provides an accessible model that will enable innovative studies into Cryptosporidium biology and host interactions.

A Genetically Tractable, Natural Mouse Model of Cryptosporidiosis Offers Insights into Host Protective Immunity.

Cryptosporidium is a leading cause of diarrheal disease and an important contributor to early childhood mortality, malnutrition, and growth faltering. Older children in high endemicity regions appear resistant to infection, while previously unexposed adults remain susceptible. Experimental studies in humans and animals support the development of disease resistance, but we do not understand the mechanisms that underlie protective immunity to Cryptosporidium. Here, we derive an in vivo model of Cryptosporidium infection in immunocompetent C57BL/6 mice by isolating parasites from naturally infected wild mice. Similar to human cryptosporidiosis, this infection causes intestinal pathology, and interferon-γ controls early infection while T cells are critical for clearance. Importantly, mice that controlled a live infection were resistant to secondary challenge and vaccination with attenuated parasites provided protection equal to live infection. Both parasite and host are genetically tractable and this in vivo model will facilitate mechanistic investigation and rational vaccine design.

Identification of a potent benzoxaborole drug candidate for treating cryptosporidiosis.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children and causes chronic diarrhea in AIDS patients, but the only approved treatment is ineffective in malnourished children and immunocompromised people. We here use a drug repositioning strategy and identify a promising anticryptosporidial drug candidate. Screening a library of benzoxaboroles comprised of analogs to four antiprotozoal chemical scaffolds under pre-clinical development for neglected tropical diseases for Cryptosporidium growth inhibitors identifies the 6-carboxamide benzoxaborole AN7973. AN7973 blocks intracellular parasite development, appears to be parasiticidal, and potently inhibits the two Cryptosporidium species most relevant to human health, C. parvum and C. hominis. It is efficacious in murine models of both acute and established infection, and in a neonatal dairy calf model of cryptosporidiosis. AN7973 also possesses favorable safety, stability, and PK parameters, and therefore, is an exciting drug candidate for treating cryptosporidiosis.

Role of filtration in managing the risk from Cryptosporidium in commercial swimming pools - a review.

Most commercial swimming pools use pressurised filters, typically containing sand media, to remove suspended solids as part of the water treatment process designed to keep water attractive, clean and safe. The accidental release of faecal material by bathers presents a poorly quantified risk to the safety of swimmers using the pool. The water treatment process usually includes a combination of maintaining a residual concentration of an appropriate biocide in the pool together with filtration to physically remove particles, including microbial pathogens, from the water. However, there is uncertainty about the effectiveness of treatment processes in removing all pathogens, and there has been growing concern about the number of reported outbreaks of the gastrointestinal disease cryptosporidiosis, caused by the chlorine-resistant protozoan parasite Cryptosporidium. A number of interacting issues influence the effectiveness of filtration for the removal of Cryptosporidium oocysts from swimming pools. This review explains the mechanisms by which filters remove particles of different sizes (including oocyst-sized particles, typically 4-6 µm), factors that affect the efficiency of particle removal (such as filtration velocity), current recommended management practices, and identifies further work to support the development of a risk-based management approach for the management of waterborne disease outbreaks from swimming pools.

Parasitic contamination of vegetables marketed in Arba Minch town, southern Ethiopia.

BACKGROUND: Consumption of unwashed, raw or unhygienically prepared fruits and vegetables act as potential source for the spread of various parasitic diseases. Moreover, the level of contamination and species of contaminant parasites vary from place to place because of variations in environmental and human factors. Therefore local determination of the level of contamination and associated factors is important for efficient intervention of infections acquired via those food items. METHODS: A Cross-sectional study was conducted among purchased vegetables in selected markets of Arba Minch town from January to March, 2018. A structured questionnaire was used to capture data about factors associated with parasitic contamination of vegetables in the marketing phase. Selected vegetables were purchased and processed for examination of parasitic contamination using direct wet mount, iodine wet mount and modified zeihl Neelson staining following standard protocols. All data were analyzed using SPSS version 20.0. RESULTS: Among 347 vegetable samples examined, 87(25.1%) were contaminated with at least one parasite species. Tomato (35.0%) was the most commonly contaminated vegetable while green pepper (10.6%) was the least contaminated one. Entameoba histolytica/dispar (29, 8.4%) was the commonest parasitic contaminant detected followed by Giardia lamblia (24, 6.9%) and oocyst of Cryptosporidium species (5.8%). Vegetable type (X2 = 13.5; p = 0.009) and source of vegetables (X2 = 24.1; p < 0.001) were significantly associated with parasitic contamination of vegetables. CONCLUSION: Parasitic contamination rate among marketed vegetables in the present study is significantly considerable. Entameoba histolytica /dispar was the most frequently detected parasite. We recommend to the local public health sector to establish a system for continuous monitoring of contamination of vegetables sold at local markets.

A suite of phenotypic assays to ensure pipeline diversity when prioritizing drug-like Cryptosporidium growth inhibitors.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

Toxoplasma gondii and Other Zoonotic Protozoans in Mediterranean Mussel ( Mytilus galloprovincialis) and Blue Mussel ( Mytilus edulis): A Food Safety Concern?

Mediterranean mussels ( Mytilus galloprovincialis) and blue mussels ( Mytilus edulis) are among the most consumed fishery products, but they are frequent vehicles of foodborne infection worldwide. In this study, we investigated the occurrence and seasonality of zoonotic protozoans in mussels farmed or sold at retail outlets in Italy. We collected and tested 1,440 M. galloprovincialis and 180 M. edulis. Pooled samples were molecularly tested for Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii and then sequenced. Sixty-two (45.9%; 95% confidence interval, 37.5 to 54.3%) mussel pools tested positive for one or more of the investigated pathogens. Both Mytilus species and samples from all the investigated areas harbored pathogens. Mussels were statistically more contaminated by Cryptosporidium spp., followed by T. gondii and G. duodenalis assemblage A, and M. galloprovincialis was more contaminated than M. edulis ( P < 0.01). Contamination was more likely in mussels at retail outlets ( P < 0.05) than in those from farms and in mussels collected in spring ( P < 0.01) than in other seasons. This is the first report of T. gondii found in M. galloprovincialis in Italy and in M. edulis in Europe. The detection of zoonotic protozoans in a widely consumed food source indicates the need for a more detailed microbiological risk analysis, especially considering that bivalve mollusks are often consumed raw worldwide.

Past and future trends of Cryptosporidium in vitro research.

Cryptosporidium is a genus of single celled parasites capable of infecting a wide range of animals including humans. Cryptosporidium species are members of the phylum apicomplexa, which includes well-known genera such as Plasmodium and Toxoplasma. Cryptosporidium parasites cause a severe gastro-intestinal disease known as cryptosporidiosis. They are one of the most common causes of childhood diarrhoea worldwide, and infection can have prolonged detrimental effects on the development of children, but also can be life threatening to HIV/AIDS patients and transplant recipients. A variety of hosts can act as reservoirs, and Cryptosporidium can persist in the environment for prolonged times as oocysts. While there has been substantial interest in these parasites, there is very little progress in terms of treatment development and understanding the majority of the life cycle of this unusual organism. In this review, we will provide an overview on the existing knowledge of the biology of the parasite and the current progress in developing in vitro cultivation systems. We will then describe a synopsis of current and next generation approaches that could spearhead further research in combating the parasite.

Cryptosporidium infection is associated with reduced growth and diarrhoea in goats beyond weaning.

Cryptosporidium and Giardia are common parasites of ruminant livestock worldwide. These parasites are associated with diarrhoea outbreaks in young goats (pre-weaning), but the impacts on health and productivity for older goats (post-weaning) are not well understood. Here we show Cryptosporidium faecal shedding is associated with reduced growth and diarrhoea in goats aged approximately 9-15 months. Goats were sampled four times at one-month intervals. Faecal shedding for a range of pathogens were determined using quantitative PCR and sequencing (Cryptosporidium, Giardia, Eimeria, Salmonella, Campylobacter), and microscopy (trichostrongylid nematode worm egg count and Entamoeba). Cryptosporidium faecal shedding was associated with 1.5 kg lower growth for the one-month period following sampling. Specifically, C. xiaoi was associated with 1.9 kg lower growth in the following month. This is the first report of production impacts associated with C. xiaoi in ruminants older than 3 months of age. Cryptosporidium shedding was associated with an over 4-fold increase in risk of diarrhoea, with C. parvum associated with 10-fold and C. ubiquitum associated with 16-fold increase in risk of diarrhoea. Notably, C. xiaoi shedding was not associated with increased risk of diarrhoea. Giardia shedding was associated with looser faecal consistency, but not diarrhoea. Higher Eimeria oocyst counts were weakly associated with lower live weight, poorer body condition and looser faecal consistency. Shedding of other enteric pathogens were not associated with impacts on live weight, growth or diarrhoea risk. This study challenges the two notions that Cryptosporidium infections only impact health and productivity of goats during the pre-weaning period, and that Cryptosporidium (and specifically C. xiaoi) infections in the absence of diarrhoea are asymptomatic. Recognising the potential for impacts of Cryptosporidium infection on growth rates in the absence of diarrhoea will support improved design for experiments testing impacts of Cryptosporidium on ruminant health and production. Improved understanding of the role of protozoan infections on animal health has implications for the management of goats in order to reduce adverse impacts on farm profitability, animal welfare and public health risk.

Modelling Cryptosporidium infection in human small intestinal and lung organoids.

Stem-cell-derived organoids recapitulate in vivo physiology of their original tissues, representing valuable systems to model medical disorders such as infectious diseases. Cryptosporidium, a protozoan parasite, is a leading cause of diarrhoea and a major cause of child mortality worldwide. Drug development requires detailed knowledge of the pathophysiology of Cryptosporidium, but experimental approaches have been hindered by the lack of an optimal in vitro culture system. Here, we show that Cryptosporidium can infect epithelial organoids derived from human small intestine and lung. The parasite propagates within the organoids and completes its complex life cycle. Temporal analysis of the Cryptosporidium transcriptome during organoid infection reveals dynamic regulation of transcripts related to its life cycle. Our study presents organoids as a physiologically relevant in vitro model system to study Cryptosporidium infection.