Protocol for the cryopreservation of Cryptosporidium isolates using enteroids.

A major bottleneck in the progress of Cryptosporidium research is the lack of accessible cryopreservation of Cryptosporidium oocysts. Here, we present a protocol for the cryopreservation of Cryptosporidium isolates using enteroids. We describe the steps for the establishment of enteroid cultures and cryopreservation of C. parvum-infected HCT-8 cultures. We then detail procedures for the recovery and propagation of frozen parasites using enteroids. For complete details on the use and execution of this protocol, please refer to Deng et al.1.

Cryptosporidium impacts epithelial turnover and is resistant to induced death of the host cell.

Infection with the apicomplexan parasite Cryptosporidium is a leading cause of diarrheal disease. Cryptosporidiosis is of particular importance in infants and shows a strong association with malnutrition, both as a risk factor and as a consequence. Cryptosporidium invades and replicates within the small intestine epithelial cells. This is a highly dynamic tissue that is developmentally stratified along the villus axis. New cells emerge from a stem cell niche in the crypt and differentiate into mature epithelial cells while moving toward the villus tip, where they are ultimately shed. Here, we studied the impact of Cryptosporidium infection on this dynamic architecture. Tracing DNA synthesis in pulse-chase experiments in vivo, we quantified the genesis and migration of epithelial cells along the villus. We found proliferation and epithelial migration to be elevated in response to Cryptosporidium infection. Infection also resulted in significant cell loss documented by imaging and molecular assays. Consistent with these observations, single-cell RNA sequencing of infected intestines showed a gain of young and a loss of mature cells. Interestingly, enhanced epithelial cell loss was not a function of enhanced apoptosis of infected cells. To the contrary, Cryptosporidium-infected cells were less likely to be apoptotic than bystanders, and experiments in tissue culture demonstrated that infection provided enhanced resistance to chemically induced apoptosis to the host but not bystander cells. Overall, this study suggests that Cryptosporidium may modulate cell apoptosis and documents pronounced changes in tissue homeostasis due to parasite infection, which may contribute to its long-term impact on the developmental and nutritional state of children. IMPORTANCE: The intestine must balance its roles in digestion and nutrient absorption with the maintenance of an effective barrier to colonization and breach by numerous potential pathogens. An important component of this balance is its constant turnover, which is modulated by a gain of cells due to proliferation and loss due to death or extrusion. Here, we report that Cryptosporidium infection changes the dynamics of this process increasing both gain and loss of enterocytes speeding up the villus elevator. This leads to a much more immature epithelium and a reduction of the number of those cells typically found toward the villus apex best equipped to take up key nutrients including carbohydrates and lipids. These changes in the cellular architecture and physiology of the small intestine may be linked to the profound association between cryptosporidiosis and malnutrition.

Intestinal cDC1s provide cues required for CD4+ T cell-mediated resistance to Cryptosporidium.

Cryptosporidium is an enteric pathogen and a prominent cause of diarrheal disease worldwide. Control of Cryptosporidium requires CD4+ T cells, but how protective CD4+ T cell responses are generated is poorly understood. Here, Cryptosporidium parasites that express MHCII-restricted model antigens were generated to understand the basis for CD4+ T cell priming and effector function. These studies revealed that parasite-specific CD4+ T cells are primed in the draining mesenteric lymph node but differentiate into Th1 cells in the gut to provide local parasite control. Although type 1 conventional dendritic cells (cDC1s) were dispensable for CD4+ T cell priming, they were required for CD4+ T cell gut homing and were a source of IL-12 at the site of infection that promoted local production of IFN-γ. Thus, cDC1s have distinct roles in shaping CD4+ T cell responses to an enteric infection: first, to promote gut homing from the mesLN, and second, to drive effector responses in the intestine.

Comparison of Resistance to gamma-Irradiation between Cryptosporidium parvum and Cryptosporidium muris Using In Vivo Infection

In the genus Cryptosporidium, there are more than 14 species with different sizes and habitats, as well as different hosts. Among these, C. parvum and C. hominis are known to be human pathogens. As C. parvum can survive exposure to harsh environmental conditions, including various disinfectants or high doses of radiation, it is considered to be an important environmental pathogen that may be a threat to human health. However, the resistance of other Cryptosporidium species to various environmental conditions is unknown. In this study, resistance against gamma-irradiation was compared between C. parvum and C. muris using in vivo infection in mice. The capability of C. muris to infect mice could be eliminated with 1,000 Gy of gamma-irradiation, while C. parvum remained infective in mice after up to 1,000 Gy of gamma-irradiation, although the peak number of oocysts per gram of feces decreased to 16% that of non-irradiated oocysts. The difference in radioresistance between these 2 Cryptosporidium species should be investigated by further studies.

Relación entre el gradiente discontinuo de sacarosa y la viabilidad de ooquistes de Cryptosporidium sp / Relationship between discontinuous sucrose gradient and viability of Cryptosporidium sp. oocysts

Cryptosporidium sp. causes self-limiting gastroenteritis in immunocompetent individuals and can produce a life-threatening chronic diarrhea in immunodeficient patients. In order to obtain populations of selectively infective oocysts for inoculation in biological experiments, we developed an operational protocol for the enrichment of viable oocysts from crude fecal material. Using either fresh or formaldehyde-fixed feces as sources of viable and nonviable oocysts, respectively, we fractionated the samples on parallel discontinuous sucrose gradients and evaluated oocyst viability at different banding densities by an in vitro excystation assay. The formaldehyde-inactivated fecal samples formed no bands after centrifugation and 91.66 of the oocysts became concentrated in the pellet. Fresh fecal samples formed three bands at densities 1.062, 1.092, and 1.121, in addition to a sediment. Here the viability of the gradient-sedimented oocysts was 92.3 overall, and of those in the second band 100. Modifications in oocyst permeability thus seems to alter their sedimentation characteristics so that consequent distribution on sucrose gradients can be correlated with oocyst viability. Discontinuous sucrose density gradient sedimentation would thus constitute a simple and rapid mean to obtain viable oocysts for use in biological models both in vivo and in vitro.(AU)

Cryptosporidium y agua / Cryptosporidium and water

During the last years, cryptosporidiosis has been recognized as an important cause of diarrheal disease. According to different references, water has been an important vehicle responsible for the transmission in many epidemic outbreaks. The high number of oocysts eliminated by human and animal hosts, as well as low infectious dose and its resistance to desinfectants are some factors which contribute to the infectious risk. It has been also pointed that bacteriologically acceptable water may contain parasites. In order to minimize such risks, it is desirable to protect the water supplies and to use multiple barriers (coagulation, sedimentation and filtration) in drinking water treatment. The aim of this work is to provide information about the different aspects conditioning the presence of Cryptosporidium in drinking water.(AU)