MiR-4521 affects the propagation of Cryptosporidium parvum in HCT-8 cells through targeting foxm1 by regulating cell apoptosis.

Cryptosporidium parvum could regulate the expression of microRNAs of epithelial cells to facilitate its intracellular propagation. MiR-4521 has been reported to play an important role during the development and progression of tumors and infectious diseases by regulating cell proliferation, apoptosis, and autophagy. However, the implication of miR-4521 during C. parvum infection was still unknown. In this study, the expression of miR-4521 was found to be upregulated in HCT-8 cells infected with C. parvum from 8 h post-infection (pi) to 48 hpi, and its upregulation would be related with the TLR/NF-κB signal pathway during C. parvum infection. One potential target of miR-4521, foxm1, was down-regulated in HCT-8 cells from 24 hpi to 48 hpi, and the expression of foxm1 was negatively regulated by miR-4521. The target relationship between miR-4521 and foxm1 was further validated by using dual luciferase reporter assay. Further studies showed that miR-4521 promoted the propagation of C. parvum in HCT-8 cells through targeting foxm1 by regulating BCL2-mediating cell apoptosis. These results contribute to further understanding of the regulatory mechanisms of host miRNAs during Cryptosporidium infection.

Myeloperoxidase as a biomarker for intestinal-brain axis dysfunction induced by malnutrition and Cryptosporidium infection in weanling mice.

Cryptosporidiosis is a waterborne protozoal infection that may cause life-threatening diarrhea in undernourished children living in unsanitary environments. The aim of this study is to identify new biomarkers that may be related to gut-brain axis dysfunction in children suffering from the malnutrition/infection vicious cycle, necessary for better intervention strategies. Myeloperoxidase (MPO) is a well-known neutrophil-related tissue factor released during enteropathy that could drive gut-derived brain inflammation. We utilized a model of environmental enteropathy in C57BL/6 weanling mice challenged by Cryptosporidium and undernutrition. Mice were fed a 2%-Protein Diet (dPD) for eight days and orally infected with 107-C. parvum oocysts. C. parvum oocyst shedding was assessed from fecal and ileal-extracted genomic DNA by qRT-PCR. Ileal histopathology scores were assessed for intestinal inflammation. Prefrontal cortex samples were snap-frozen for MPO ELISA assay and NF-kb immunostaining. Blood samples were drawn by cardiac puncture after anesthesia and sera were obtained for serum amyloid A (SAA) and MPO analysis. Brain samples were also obtained for Iba-1 prefrontal cortex immunostaining. C. parvum-infected mice showed sustained stool oocyst shedding for six days post-infection and increased fecal MPO and inflammation scores. dPD and cryptosporidiosis led to impaired growth and weight gain. C. parvum-infected dPD mice showed increased serum MPO and serum amyloid A (SAA) levels, markers of systemic inflammation. dPD-infected mice showed greater MPO, NF-kB expression, and Iba-1 immunolabeling in the prefrontal cortex, an important brain region involved in executive function. Our findings suggest MPO as a potential biomarker for intestinal-brain axis dysfunction due to environmental enteropathy.

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

ETHNOPHARMACOLOGICAL RELEVANCE: For 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 METHODS: This 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. RESULTS: Our 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. CONCLUSION: Allium sativum L. displayed high efficacy as a potential therapeutic agent against Cryptosporidium, which supports its traditional usage in parasite diseases.

A productive immunocompetent mouse model of cryptosporidiosis with long oocyst shedding duration for immunological studies.

OBJECTIVES: Studies on the pathogenesis and immune responses of Cryptosporidium infection and development of drugs and vaccines use mostly immunocompromised mouse models. In this study, we establish an immunocompetent mouse model of cryptosporidiosis with high intensity and long duration of infection. METHODS: We have obtained a Cryptosporidium tyzzeri isolate from laboratory mice, and infect adult C57BL/6 J mice experimentally with the isolate for determinations of infectivity, infection patterns, pathological changes, and transcriptomic responses. RESULTS: The isolate has an ID50 of 5.2 oocysts, with oocyst shedding lasting at high levels for >2 months. The oocyst shedding is boosted by immunosuppression of animals and suppressed by paromomycin treatment. The isolate induces strong inflammatory and acquired immune responses, but down-regulates the expression of α-defensins in epithelium. Comparative genomics analysis has revealed significant sequence differences from other isolates in subtelomeric genes. The down-regulation of the expression of α-defensins may be responsible for the high-intensity and long-lasting infection in this animal model. CONCLUSIONS: The immunocompetent mouse model of cryptosporidiosis developed has the advantages of high oocyst shedding intensity and long oocyst shedding duration. It provides an effective mechanism for the propagation of Cryptosporidium, evaluations of potential therapeutics, and studies of pathogen biology and immune responses.

Anticryptosporidium Efficacy of Olea europaea and Ficus carica Leaves Extract in Immunocompromised Mice Associated with Biochemical Characters and Antioxidative System.

Cryptosporidiosis is caused by an opportunistic protozoan parasite (Cryptosporidium parvum and C. hominis) known as a parasite of humans, especially children and immunocompromised patients. The current study was designed to evaluate the therapeutic efficacy of a mixture of fig and olive leaf extracts as an alternative medicinal plant. Parasitological examination for oocysts in the stool and histopathological alterations in the small intestines were examined. Additionally, biochemical analyses of liver and kidney functions in addition to antioxidant parameters such as superoxide dismutase (SOD), glutathione peroxidase (GSH) and catalase (CAT) in the plasma were evaluated. Our results showed that marked reduction in oocysts shedding and amelioration in intestinal histopathological changes and hepatic or renal functions were detected in all treated groups compared to the control infected group. Additionally, the treated groups with tested extracts at ratios 1:3 and 1:5 showed a significant decrease in the number of oocysts compared to the other treated groups. Results exhibited a significant increase in the plasma SOD, CAT and GSH levels in treated groups compared to the infected control one. This study suggested that a mixture of fig and olive leaf extracts is a convenient promising therapeutic agent for Cryptosporidiosis.

Cryptosporidium rhoptry effector protein ROP1 injected during invasion targets the host cytoskeletal modulator LMO7.

The parasite Cryptosporidium invades and replicates in intestinal epithelial cells and is a leading cause of diarrheal disease and early childhood mortality. The molecular mechanisms that underlie infection and pathogenesis are largely unknown. Here, we delineate the events of host cell invasion and uncover a mechanism unique to Cryptosporidium. We developed a screen to identify parasite effectors, finding the injection of multiple parasite proteins into the host from the rhoptry organelle. These factors are targeted to diverse locations within the host cell and its interface with the parasite. One identified effector, rhoptry protein 1 (ROP1), accumulates in the terminal web of enterocytes through direct interaction with the host protein LIM domain only 7 (LMO7) an organizer of epithelial cell polarity and cell-cell adhesion. Genetic ablation of LMO7 or ROP1 in mice or parasites, respectively, impacts parasite burden in vivo in opposite ways. Taken together, these data provide molecular insight into how Cryptosporidium manipulates its intestinal host niche.

Respiratory cryptosporidiosis in Malawian children with diarrheal disease.

BACKGROUND: Respiratory cryptosporidiosis has been documented in children with diarrhea. We sought to describe the dynamics of respiratory involvement in children hospitalized with gastrointestinal (GI) diarrheal disease. METHODS: We conducted a prospective, observational longitudinal study of Malawian children 2-24 months hospitalized with diarrhea. Nasopharyngeal (NP) swabs, induced sputum and stool specimens were collected. Participants that were positive by Cryptosporidium PCR in any of the three compartments were followed up with fortnightly visits up to 8 weeks post-enrollment. RESULTS: Of the 162 children recruited, participants had mild-moderate malnutrition (mean HAZ -1.6 (SD 2.1)), 37 (21%) were PCR-positive for Cryptosporidium at enrollment (37 stool, 11 sputum, and 4 NP) and 27 completed the majority of follow-up visits (73%). Cryptosporidium was detected in all compartments over the 4 post-enrollment visits, most commonly in stool (100% at enrollment with mean cycle thresholds (Ct) of 28.8±4.3 to 44% at 8 weeks with Ct 29.9±4.1), followed by sputum (31% at enrollment with mean Ct 31.1±4.4 to 20% at 8 weeks with Ct 35.7±2.6), then NP (11% with mean Ct 33.5±1.0 to 8% with Ct 36.6±0.7). Participants with Cryptosporidium detection in both the respiratory and GI tract over the study period reported respiratory and GI symptoms in 81% and 62% of study visits, respectively, compared to 68% and 27%, respectively, for those with only GI detection, and had longer GI shedding (17.5±6.6 v. 15.9±2.9 days). CONCLUSION: Cryptosporidium was detected in both respiratory and GI tracts throughout the 8 weeks post-enrollment. The development of therapeutics for Cryptosporidium in children should target the respiratory as well as GI tract.

Pathology of cryptosporidiosis in raccoons: case series and retrospective analysis, 1990-2019.

Cryptosporidiosis is an intestinal protozoal disease of public health importance caused by Cryptosporidium spp. Despite the high synanthropism of raccoons, studies describing the pathology of Cryptosporidium spp. infections in this species are lacking. Therefore, we characterized the pathology of cryptosporidiosis in 2 juvenile raccoons. In addition, we conducted a retrospective search of the database of the California Animal Health and Food Safety laboratory for 1990-2019 and found 6 additional cases of cryptosporidiosis in raccoons. Sequencing of cryptosporidia was performed in one autopsied raccoon, and PCR on formalin-fixed, paraffin-embedded tissues in archived cases. The Cryptosporidium skunk genotype (CSkG), a strain of zoonotic relevance, was detected in 6 of 8 cases (75%). Frequently, cryptosporidiosis was associated with enteritis, eosinophilic infiltrates, villus atrophy or blunting and/or fusion, and crypt abscesses or necrosis. In 7 of the 8 cases, there was confirmed concurrent coinfection with canine distemper virus; 1 case was coinfected with canine parvovirus. Although crypt necrosis is considered a classic lesion of canine parvoviral infection in mesocarnivores and not a hallmark of cryptosporidiosis, results suggest that canine distemper virus is capable of mimicking such lesions in combination with cryptosporidia and immunosuppression.

The immunomodulatory activity of secnidazole-nitazoxanide in a murine cryptosporidiosis model.

Introduction. Cryptosporidium parvum causes intestinal parasitic infections affecting both immunosuppressed and immunocompetent individuals.Gap statement. Given the absence of effective treatments for cryptosporidiosis, especially in immunodeficient patients, the present study was designed to assess the therapeutic efficacy of secnidazole (SEC) and its combination with nitazoxanide (NTZ) in comparison to single NTZ treatment in relation to the immune status of a murine model of C. parvum infection.Methodology. The infected groups were administered NTZ, SEC or NTZ-SEC for three or five successive doses. At days 10 and 12 post-infection (p.i.), the mice were sacrificed, and the efficacy of the applied drugs was evaluated by comparing the histopathological alterations in ileum and measuring the T helper Th1 (interferon gamma; IFN-γ), Th2 [interleukin (IL)-4 and IL-10] and Th17 (IL-17) cytokine profiles in serum.Results. The NTZ-SEC combination recorded the maximal reduction of C. parvum oocyst shedding, endogenous stages count and intestinal histopathology, regardless of the immune status of the infected mice. The efficacy of NTZ-SEC was dependent on the period of administration, as the 5 day-based treatment protocol was also more effective than the 3 day-based one in terms of immunocompetence and immunosuppression. The present treatment schedule induced an immunomodulatory effect from SEC that developed a protective immune response against C. parvum infection with reduced production of serum IL-17, IFN-γ, IL-4 and IL-10.Conclusions. Application of NTZ-SEC combined therapy may be useful in treatment of C. parvum, especially in cases involving immunosuppression.

Cryptosporidium abrahamseni n. sp. (Apicomplexa: Cryptosporidiiae) from red-eye tetra (Moenkhausia sanctaefilomenae).

The morphological, biological, and molecular characterisation of Cryptosporidium piscine genotype 7 from red-eye tetras (Moenkhausia sanctaefilomenae) are described, and the species name Cryptosporidium abrahamseni n. sp. is proposed. Histological analysis of intestinal tissue identified large numbers of Cryptosporidium organisms along the epithelial lining of the intestine. Sequence and phylogenetic analysis at 18S rRNA (18S) and actin loci conducted on intestinal scrapings revealed that C. abrahamseni n. sp. was genetically distinct from other Cryptosporidium species. At the 18S locus, it was most closely related to C. huwi (3.2% genetic distance) and exhibited genetic distances ranging from 5.9 to 6.5% (C. molnari) to 14.9% (C. scolpthalmi) from all other Cryptosporidium species. At the actin locus, the genetic distances were larger and C. abrahamseni n. sp. exhibited 10.3% genetic distance from C. huwi, and 17.6% (C. molnari) to 28% (C. canis) genetic distance from other Cryptosporidium spp. Phylogenetic analysis of concatenated 18S and actin sequences confirmed that C. abrahamseni n. sp. shares the closest genetic relationship with C. huwi (6.7% genetic distance), while the genetic distance between C. abrahamseni n. sp. and other Cryptosporidium spp. ranged from 12.1% (C. molnari) to 20.4% (C. canis). Based on genetic and histological data, C. abrahamseni n. sp. is validated as a separate species.

Stem cell-derived enteroid cultures as a tool for dissecting host-parasite interactions in the small intestinal epithelium.

Toxoplasma gondii and Cryptosporidium spp. can cause devastating pathological effects in humans and livestock, and in particular to young or immunocompromised individuals. The current treatment plans for these enteric parasites are limited due to long drug courses, severe side effects or simply a lack of efficacy. The study of the early interactions between the parasites and the site of infection in the small intestinal epithelium has been thwarted by the lack of accessible, physiologically relevant and species-specific models. Increasingly, 3D stem cell-derived enteroid models are being refined and developed into sophisticated models of infectious disease. In this review, we shall illustrate the use of enteroids to spearhead research into enteric parasitic infections, bridging the gap between cell line cultures and in vivo experiments.

Cryptosporidium infections in sheep farms from Italy.

Cryptosporidiosis is recognized as being a significant cause of gastrointestinal illness due to its wide range of vertebrate hosts, including humans. Infection with Cryptosporidium spp. is especially common in young domestic ruminants (calves, lambs and goat kids) and has been associated with economic losses worldwide. In contrast to cattle, to date, detailed studies on Cryptosporidium infections in sheep from Europe are still limited; thus, their importance as reservoirs of Cryptosporidium species with implications on animal and public health still needs to be clarified. This study evaluates the prevalence and zoonotic potential of Cryptosporidium spp. in sheep farms in Italy. A total of 915 individual faecal samples divided into three different animal categories were collected from 61 sheep farms. Each sample was examined by microscopy of faecal smears stained by modified Ziehl-Neelsen and by biomolecular techniques. Cryptosporidium oocysts were detected in 10.1% of the animals examined and in 34.4% of the farms. The prevalence of Cryptosporidium spp. was significantly higher (χ2 = 51.854; P < 0.001) in diarrhoeic samples than in pasty or normal faeces. Genotype analyses showed the presence of two Cryptosporidium species: C. parvum and C. ubiquitum. Subtyping analysis of C. parvum isolates revealed the presence of subtypes IIa15G2R1 and IIdA20G1 and of subtype XIIa for C. ubiquitum. These findings have public health implications since both Cryptosporidium species identified are considered zoonotic, and C. parvum is the second-most common Cryptosporidium species infecting humans. Our data reveal that lambs, especially those excreting diarrhoeic faeces, may be important reservoirs of Cryptosporidium. We also highlight the need to establish adequate control and monitoring programmes for the control of this infection in sheep farms primarily through coprological monitoring.

Plasma amino acid status is useful for understanding intestinal mucosal damage in calves with cryptosporidiosis.

We hypothesize that some amino acid abnormalities in diarrheic calves are useful for understanding intestinal mucosal damage, as in humans. However, few reports have revealed the relationship between intestinal mucosal damage and plasma amino acids in diarrheic calves. Therefore, the aim of present study was to investigate whether there is a relationship between the amino acid status and plasma diamine oxidase (DAO) activity, which is known to be a biomarker for intestinal mucosal damage in diarrheic calves. Twenty Holstein calves aged 12.6 ± 4.2 days old were enrolled in this study. In the diarrhea group (n = 10), there were yellow loose feces within the rectum and Cryptosporidium parvum (C. parvum) was detected in all fecal samples. These calves were clinically normal except for diarrhea. All calves in the control group (n = 10) appeared to be healthy based on clinical findings with normal feces production and the absence of C. parvum. Plasma amino acid concentrations and DAO activity were measured. The relationships between plasma DAO activity and the concentration of each plasma amino acid were investigated using Spearman's rank test. The plasma DAO activity was significantly lower in the diarrhea group (176.1 ± 60.1 IU mL-1) than in the control group (309.3 ± 74.8 IU mL-1) (p < 0.001). Furthermore, positive correlations were observed when comparing plasma DAO activity with histidine, proline, cystine, arginine, and glutamine concentrations. As a result of relationship between plasma DAO activity and amino acid status, it was concluded that plasma amino acid status is useful for understanding intestinal mucosal damage in calves with cryptosporidiosis.

Health sequelae of human cryptosporidiosis in industrialised countries: a systematic review.

BACKGROUND: Cryptosporidium is a protozoan parasite which is a common cause of gastroenteritis worldwide. In developing countries, it is one of the most important causes of moderate to severe diarrhoea in young children; in industrialised countries it is a cause of outbreaks of gastroenteritis associated with drinking water, swimming pools and other environmental sources and a particular concern in certain immunocompromised patient groups, where it can cause severe disease. However, over recent years, longer-term sequelae of infection have been recognised and a number of studies have been published on this topic. The purpose of this systematic review was to examine the literature in order to better understand the medium- to long-term impact of cryptosporidiosis. METHODS: This was a systematic review of studies in PubMed, ProQuest and Web of Science databases, with no limitations on publication year or language. Studies from any country were included in qualitative synthesis, but only those in industrialised countries were included in quantitative analysis. RESULTS: Fifteen studies were identified for qualitative analysis which included 3670 Cryptosporidium cases; eight studies conducted in Europe between 2004-2019 were suitable for quantitative analysis, including five case-control studies. The most common reported long-term sequelae were diarrhoea (25%), abdominal pain (25%), nausea (24%), fatigue (24%) and headache (21%). Overall, long-term sequelae were more prevalent following infection with Cryptosporidium hominis, with only weight loss and blood in stool being more prevalent following infection with Cryptosporidium parvum. Analysis of the case-control studies found that individuals were 6 times more likely to report chronic diarrhoea and weight loss up to 28 months after a Cryptosporidium infection than were controls. Long-term abdominal pain, loss of appetite, fatigue, vomiting, joint pain, headache and eye pain were also between 2-3 times more likely following a Cryptosporidium infection. CONCLUSIONS: This is the first systematic review of the long-term sequelae of cryptosporidiosis. A better understanding of long-term outcomes of cryptosporidiosis is valuable to inform the expectations of clinicians and their patients, and public health policy-makers regarding the control and prevention of this infection. Systematic review registration PROSPERO Registration number CRD42019141311.

Plasma histidine concentrations as a specific biomarker for intestinal mucosal damage in calves with cryptosporidiosis.

Specific alterations in plasma histidine concentrations and diamine oxidase (DAO) activity were recently reported as a potential biomarker for intestinal mucosal damage in diarrheic calves. However, there are no data on the comparison of precision between histidine concentration and DAO activity in bovine plasma. The aim of the present study was to compare precision of histidine concentrations and DAO activities in plasma as a biomarker for the Cryptosporidium parvum (C. parvum)-associated intestinal mucosal damage in diarrheic calves. Thirty-two Holstein calves aged 12.2 ± 4.1 days old were enrolled in the present study; they were divided into C. parvum (n = 9), diarrhea (n = 11), and control (n = 12) groups based on the presence or absence of diarrhea and with or without C. parvum infection. Receiver operating characteristic (ROC) curves were used to characterize the sensitivity and specificity of each parameter for the C. parvum-associated intestinal mucosal damage. The proposed cut-off points for plasma histidine concentrations and plasma DAO activities for cryptosporidiosis in calves based on ROC analyses were < 55.8 nM and < 246.0 IU/ml, respectively. The sensitivities and specificities of the proposed diagnostic cut-offs were 88.9% and 82.6% for plasma histidine concentrations and 100.0% and 34.8% for plasma DAO activities, respectively. It was concluded that plasma histidine concentrations may be superior to plasma DAO activities as a specific biomarker for the C. parvum-associated intestinal mucosal damage in diarrheic calves.

Long-term production effects of clinical cryptosporidiosis in neonatal calves.

Cryptosporidiosis can have a devastating effect in neonatal calves, resulting in diarrhoea, dehydration and, in severe cases, death of the animal. The disease is caused by Cryptosporidium spp. and is one of the most common causes of calf enteritis in the UK. The parasite is very difficult to remove from the farm, as the oocysts have a tough outer wall which enables the parasite to survive for several months in moist temperate environmental conditions and it is difficult to kill oocysts with common disinfectants used on a farm. If appropriate management practises are applied, the disease is usually self-limiting and most calves will recover. It has been shown, in studies with children and in lambs, that severe clinical cryptosporidiosis can result in long-term growth and cognitive impairment compared with individuals with no obvious signs of the disease. This study measured the long-term growth rate of beef calves on farm by comparing groups of animals that had suffered differing degrees of clinical severity of cryptosporidiosis as neonates. A group of 27 beef calves were enrolled in the study and monitored from birth to 6 months of age. The calves were scored for severity of cryptosporidiosis and weighed at regular intervals. The average difference in weight gain, at 6 months, between a group of calves that had severe cryptosporidiosis as neonates and a group of calves with no clinical signs of infection was 34 kg. Those calves that had experienced severe cryptosporidiosis as neonates showed a significantly reduced live weight gain compared with those calves showing no clinical signs of infection (P = 0.034). Therefore, the impact of severe cryptosporidiosis in neonatal calves has longer term effects on weight gain and production efficiency, resulting in the parasite having a greater impact on cattle production than previously thought.

Correlates of pathological lesions associated with respiratory cryptosporidiosis prevalence in shot red grouse Lagopus lagopus scotica from moors in northern England.

Infection of wild red grouse Lagopus lagopus scotica by Cryptosporidium baileyi was first diagnosed in 2010. Within three years, signs of infection were reported from grouse on half of all grouse moors in northern England, bringing severe concerns of economic losses to grouse shooting. A total of 45,914 red grouse shot from 10 moors in northern England between 2013 and 2018 were visually screened for signs of respiratory cryptosporidiosis. Prevalence varied with age, being twice as high in juveniles (4.5%) as in adults (2.4%). It also varied nine-fold between moors and three-fold between years. Prevalence was highest in grouse shot later in the shooting season. Our results are consistent with the concept that disease incidence is highest in naïve juveniles that have previously not been exposed to infection, with prevalence dropping as birds develop immunity. We found no evidence of increased prevalence over time, and fears of escalated disease prevalence, bringing with it increased mortality and lowered productivity, that may have significant impacts on the economic viability of shoots, have not yet been realized. We recommend continued annual screening for clinical signs amongst shot birds, better hygiene associated with potential reservoirs of infection, and practices that both improve the detection and selective culling of diseased individuals and generally reduce overall grouse densities.

Mouse Models for Use in Cryptosporidium Infection Studies and Quantification of Parasite Burden Using Flow Cytometry, qPCR, and Histopathology.

Cryptosporidiosis threatens life of young children in developing countries and newborn calves around the world. No vaccine or therapy can prevent or cure this diarrhea-inducing enteric disease caused by Cryptosporidium spp. protozoan parasites. There is an essential need to discover new therapeutic drugs efficient in reducing parasite burden in infected individuals. Research therefore relies on reliable small animal models of cryptosporidiosis. Here, we present excellent mouse models which can efficiently mimic pathogenesis of human and bovine cryptosporidiosis. We also describe methods to purify C. parvum oocysts from stool and intestine of infected mice to facilitate oocyst quantification. Moreover, we present protocols using flow cytometry, quantitative polymerase chain reaction, and histopathology to accurately quantify parasite burden in stool or intestine samples.

Calf Clinical Model of Cryptosporidiosis for Efficacy Evaluation of Therapeutics.

Cryptosporidiosis, caused by the apicomplexan parasite Cryptosporidium parvum, is a moderate-to-severe diarrheal disease now recognized as one of the leading causes of morbidity and mortality in livestock globally, and in humans living in resource-limited parts of the world, particularly those with AIDS or malnourished individuals. This recognition has fueled efforts for the discovery of effective therapeutics. While recent progress in drug discovery has been encouraging, there are presently no acceptably effective parasite-specific drugs for the disease. The urgent need for new drug discovery or drug repurposing has also increased the need for refined animal models of clinical disease for therapeutic efficacy evaluation. Here, we describe an acute model of cryptosporidiosis using newborn calves to evaluate well-defined clinical and parasitological parameter outcomes, including the effect on diarrhea severity and duration, oocyst numbers produced, and multiple measures of clinical health. The model is highly reproducible and provides unequivocal direct measures of treatment efficacy on diarrhea severity and parasite replication.

Cryptosporidium parvum alters glucose transport mechanisms in infected enterocytes.

The parasite Cryptosporidium parvum Tyzzer 1912 destroys parts of the intestinal brush border membrane which is important for the uptake of nutrients like glucose. In this study, glucose transport mechanisms of the host cells (IPEC-J2 cells) infected by C. parvum were investigated. The mRNA expression levels of glucose transporters (GLUT) 1 and 2 and Na+-coupled glucose transporter (SGLT) 1 were compared in infected and uninfected cells over an infection time of 24-96 h by RT-qPCR. Furthermore, the protein expression of SGLT 1 and GLUT 2 was quantified in western blot studies. While the protein expression of SGLT 1 was not altered in infected cells, mRNA expression of SGLT 1 and GLUT 1 was significantly increased 24 h p. i. and decreased 96 h p. i. The mRNA expression of GLUT 2 was significantly decreased 24 h, 72 h, and 96 h p. i. and also correlated significantly with the infection dose at 72 h p. i. In contrast to that, the protein expression of GLUT 2 was significantly increased 48 h p. i., associated with a significantly higher intracellular glucose level in infected cells compared with control cells at that time point of infection. This points to an adaptation of the host cells' glucose uptake taking place in the acute phase of the infection. A better understanding of these molecular mechanisms following a C. parvum infection may probably lead to an improvement of therapy strategies in the future.