Conservation, abundance, glycosylation profile, and localization of the TSP protein family in Cryptosporidium parvum.

Cryptosporidium parvum is a zoonotic apicomplexan parasite and a common cause of diarrheal disease worldwide. The development of vaccines to prevent or limit infection remains an important goal for tackling cryptosporidiosis. At present, the only approved vaccine against any apicomplexan parasite targets a conserved adhesin possessing a thrombospondin repeat domain. C. parvum possesses 12 orthologous thrombospondin repeat domain-containing proteins known as CpTSP1-12, though little is known about these potentially important antigens. Here, we explore the architecture and conservation of the CpTSP protein family, as well as their abundance at the protein level within the sporozoite stage of the life cycle. We examine the glycosylation states of these proteins using a combination of glycopeptide enrichment techniques to demonstrate that these proteins are modified with C-, O-, and N-linked glycans. Using expansion microscopy, and an antibody against the C-linked mannose that is unique to the CpTSP protein family within C. parvum, we show that these proteins are found both on the cell surface and in structures that resemble the secretory pathway of C. parvum sporozoites. Finally, we generated a polyclonal antibody against CpTSP1 to show that it is found at the cell surface and within micronemes, in a pattern reminiscent of other apicomplexan motility-associated adhesins, and is present both in sporozoites and meronts. This work sheds new light on an understudied family of C. parvum proteins that are likely to be important to both parasite biology and the development of vaccines against cryptosporidiosis.

Identification of the glycopeptide epitope recognized by a protective Cryptosporidium monoclonal antibody.

Cryptosporidium species are a leading cause of pediatric diarrheal disease and death in low- and middle-income countries and pose a particular threat to immunocompromised individuals. As a zoonotic pathogen, Cryptosporidium can have devastating effects on the health of neonatal calves. Despite its impact on human and animal health, consistently effective drug treatments for cryptosporidiosis are lacking and no vaccine is available. We previously showed that C. parvum mucin-like glycoproteins, gp40, and gp900 express an epitope identified by a monoclonal antibody 4E9. 4E9 neutralized C. parvum infection in vitro as did glycan-binding proteins specific for the Tn antigen (GalNAc-α1-S/T). Here, we show that 4E9 ameliorates disease in vivo in a calf challenge model. The 4E9 epitope is present on C. hominis in addition to C. parvum gp40 and gp900 and localizes to the plasma membrane and dense granules of invasive and intracellular stages. To characterize the epitope recognized by 4E9, we probed a glycan array containing over 500 defined glycans together with a custom-made glycopeptide microarray containing glycopeptides from native mucins or C. parvum gp40 and gp15. 4E9 exhibited no binding to the glycan array but bound strongly to glycopeptides from native mucins or gp40 on the glycopeptide array, suggesting that the antibody epitope contains both peptide and glycan moieties. 4E9 only recognized glycopeptides with adjacent S or T residues in the motif S*/T*-X-S*/T* where X = 0 or 1. These data define the 4E9 epitope and have implications for the inclusion of the epitope in the development of vaccines or other immune-based therapies.

Giardia Detection and Codetection With Other Enteric Pathogens in Young Children in the Vaccine Impact on Diarrhea in Africa (VIDA) Case-Control Study: 2015-2018.

BACKGROUND: Giardia has been associated with reduced risk of diarrhea in children in low-resource settings, but the mechanism underlying this association is unknown. To assess whether Giardia may shape colonization or infection with other enteric pathogens and impact associations with diarrhea, we examined Giardia and enteric pathogen codetection among children <5 years old in Kenya, The Gambia, and Mali as part of the Vaccine Impact on Diarrhea in Africa study. METHODS: We tested for Giardia and other enteric pathogens using enzyme-linked immunosorbent assays and real-time polymerase chain reaction (PCR) on stool, respectively. We evaluated associations between Giardia and enteric pathogen detection using multivariable logistic regression models separately for children with moderate-to-severe diarrhea (MSD, cases) and free of diarrhea (controls). RESULTS: Among 11 039 enrolled children, Giardia detection was more common among controls (35%) than cases (28%, P < .001). Campylobacter coli/jejuni detection was associated with Giardia in controls in The Gambia (adjusted odds ratio [aOR] [95% confidence interval {CI}]: 1.51 [1.22‒1.86]) and cases across all sites (1.16 [1.00‒1.33]). Among controls, the odds of astrovirus (1.43 [1.05‒1.93]) and Cryptosporidium spp. (1.24 [1.06‒1.46]) detection were higher among children with Giardia. Among cases, the odds of rotavirus detection were lower in children with Giardia in Mali (.45 [.30‒.66]) and Kenya (.31 [.17‒.56]). CONCLUSIONS: Giardia was prevalent in children <5 years old and was associated with detection of other enteric pathogens, with differing associations in cases versus controls and by site. Giardia may affect colonization or infection by certain enteric pathogens associated with MSD, suggesting an indirect mechanism of clinical impact.

A Cryptosporidium parvum vaccine candidate effect on immunohistochemical profiling of CD4+, CD8+, Caspase-3 and NF-κB in mice.

BACKGROUND: Cryptosporidium parvum is a protozoan parasite of medical and veterinary importance that causes neonatal diarrhea in many vertebrate hosts. In this study, we evaluated the efficacy of an affinity-purified antigen as a C. parvum vaccine candidate using ileal and liver tissues of experimentally infected neonatal mice by immunohistochemical profiling and immune scoring of CD4+, CD8+, Caspase-3, and nuclear factor kappa B (NF-κB). This vaccine was prepared from the C. parvum oocysts antigen using immune affinity chromatography with cyanogen bromide-activated Sepharose-4B beads. METHODS: Thirty neonatal mice were divided into three groups (10 mice/group): (1) non-immunized non-infected, (2) non-immunized infected (using gastric tubes with a single dose of 1 × 105 of C. parvum oocysts in 250 µl PBS solution 1 h before a meal) and (3) immunized (twice with 40 µg/kg of purified C. parvum antigen at 2-week intervals and then infected with 1 × 105 C. parvum oocysts simultaneously with the second group). After euthanizing the animals on the 10th day, post-infection, their ileal and liver tissues were collected and prepared for immunohistochemistry (IHC) staining to detect CD4+, CD8+, Caspase-3, and NF-κB levels, which are indicators for T helper cells, cytotoxic T cells, apoptosis, and inflammation, respectively. RESULTS: The IHC results showed that CD4+, CD8+, Caspase-3, and NF-κB expression varied significantly (P < 0.001) in both organs in all the groups. We also recorded high CD4+ levels and low CD8+ expression in the non-immunized non-infected mice tissues, while the opposite was observed in the non-immunized infected mice tissues. In the immunized infected mice, the CD4+ level was higher than CD8 + in both organs. While the Caspase-3 levels were higher in the ileal tissue of non-immunized infected than immunized infected mice ileal tissues, the reverse was seen in the liver tissues of both groups. Furthermore, NF-κB expression was higher in the liver tissues of non-immunized infected mice than in immunized infected mice tissues. Therefore, the IHC results and immune-scoring program revealed a significant difference (P < 0.001) in the CD4+, CD8+, Caspase-3, and NF-κB expression levels in both ileal and liver tissues of all mice groups, which might be necessary for immunomodulation in these tissues. CONCLUSIONS: The improvement observed in the immunized infected mice suggests that this vaccine candidate might protect against cryptosporidiosis.

Fate of Cryptosporidium and Giardia through conventional and compact drinking water treatment plants.

Over the past three decades, a notable rise in the occurrence of enteric protozoan pathogens, especially Giardia and Cryptosporidium spp., in drinking water sources has been observed. This rise could be attributed not only to an actual increase in water contamination but also to improvements in detection methods. These waterborne pathogens have played a pivotal role in disease outbreaks and the overall escalation of disease rates in both developed and developing nations worldwide. Consequently, the control of waterborne diseases has become a vital component of public health policies and a primary objective of drinking water treatment plants (DWTPs). Limited studies applied real-time PCR (qPCR) and/or immunofluorescence assay (IFA) for monitoring Giardia and Cryptosporidium spp., particularly in developing countries like Egypt. Water samples from two conventional drinking water treatment plants and two compact units (CUs) were analyzed using both IFA and qPCR methods to detect Giardia and Cryptosporidium. Using qPCR and IFA, the conventional DWTPs showed complete removal of Giardia and Cryptosporidium, whereas Mansheyat Alqanater and Niklah CUs achieved only partial removal. Specifically, Cryptosporidium gene copies removal rates were 33.33% and 60% for Mansheyat Alqanater and Niklah CUs, respectively. Niklah CU also removed 50% of Giardia gene copies, but no Giardia gene copies were removed by Mansheyat Alqanater CU. Using IFA, both Mansheyat Alqanater and Niklah CUs showed a similar removal rate of 50% for Giardia cysts. Additionally, Niklah CU achieved a 50% removal of Cryptosporidium oocysts, whereas Mansheyat Alqanater CU did not show any removal of Cryptosporidium oocysts. Conventional DWTPs were more effective than CUs in removing enteric protozoa. The contamination of drinking water by enteric pathogenic protozoa remains a significant issue globally, leading to increased disease rates. Infectious disease surveillance in drinking water is an important epidemiological tool to monitor the health of a population.

Development of Two Mouse Models for Vaccine Evaluation against Cryptosporidiosis.

Cryptosporidiosis was shown a decade ago to be a major contributor to morbidity and mortality of diarrheal disease in children in low-income countries. A serious obstacle to develop and evaluate immunogens and vaccines to control this disease is the lack of well-characterized immunocompetent rodent models. Here, we optimized and compared two mouse models for the evaluation of vaccines: the Cryptosporidium tyzzeri model, which is convenient for screening large numbers of potential mixtures of immunogens, and the Cryptosporidium parvum-infected mouse pretreated with interferon gamma-neutralizing monoclonal antibody.

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.

Isolation and identification of sporozoite membrane protein of Cryptosporidium parvum and evaluation of calmodulin-like protein immune protection.

Until now, no completely effective parasite-specific drugs or vaccines have been approved for the treatment of cryptosporidiosis. Through the separation and identification of the sporozoite membrane protein of Cryptosporidium parvum (C. parvum), 20 related proteins were obtained. Among them, a calmodulin-like protein (CML) has a similar functional domain-exchange factor hand (EF-hand) motif as calmodulin proteins (CaMs), so it may play a similarly important role in the invasion process. A 663 bp full gene encoding the C. parvum calmodulin-like protein (CpCML) was inserted in pET28a vector and expressed in Escherichia coli. An immunofluorescence assay showed that CpCML was mainly located on the surface of the sporozoites. Three-week-old female BALB/c mice were used for modelling the immunoreactions and immunoprotection of recombinant CpCML (rCpCML) against artificial Cryptosporidium tyzzeri infections. The results indicated a significantly increased in anti-CpCML antibody response, which was induced by the immunized recombinant protein. Compared to rP23 (recombinant P23), GST6P-1 (expressed by pGEX-6P-1 transfected E. coli), GST4T-1 (expressed by pGEX-4T-1 transfected E. coli), glutathione (GSH), adjuvant and blank control groups, rCpCML-immunized mice produced specific spleen cell proliferation in addition to different production levels of IL-2, IFN-γ, TNF-α, IL-4 and IL-5. Additionally, immunization with rCpCML led to 34.08% reduction of oocyst shedding in C. tyzzeri infected mice faeces which was similar to rP23. These results suggest that CpCML may be developed as a potential vaccine candidate antigen against cryptosporidiosis.

Utilization of health belief model in comprehending diarrheal disease dynamics: a case of cryptosporidiosis in Uganda.

BACKGROUND: Diarrheal diseases contribute greatly to the reported global childhood mortality and morbidity with related social, economic consequences. This study was conducted to analyze the utilization of the Health Belief Model (HBM) theory to comprehend diarrheal disease dynamics in Uganda. METHODS: Our study utilized a qualitative cross-sectional design among adult livestock farmers in selected farming communities. A total of 80 individuals were recruited and interviewed through Focus Discussion Groups (FDGs) (n = 6) and Key Informant Interviews (KIIs) (n = 8) to evaluate diarrheal disease dynamics. The scope of dynamics included but not limited to exposure risks, knowledge, and attitudes. Our results were presented using the five (5) constructs of the HBM. RESULTS: Perceived susceptibility; communities believed that both humans and their animals are at high risk of different kinds of diarrheal infections. The farmers believed that majority of these diarrhea infections are hard to treat especially among animals. Perceived severity; farmers believed that diarrheal diseases are characterized by loss of weight, fever, emaciation, dry eyes, severe prolonged diarrhea and sudden death. Perceived barriers; limited knowledge and misconceptions about the diarrheal infections were great inhibitors to successful disease prevention and control. Self-efficacy; farmers had fear of laxity that interventions being suggested and put in place to curb diarrheal diseases such as cryptosporidiosis would wither away with time thus endemicity of the problem in the community. Modifying factors and cues to action; most of the farmers treat animals by themselves based on; probability, traditional knowledge and previous experience. CONCLUSION: Sustained public health interventional activities should therefore be undertaken by both human and animal health sectors with maximum community involvement. Communities suggested the need to increase preventive measures and promote household hygiene efforts to always wash hands with soap and running water in order to reduce the burden of diarrhea diseases such as cryptosporidiosis.

Development of an immunocompetent mouse model susceptible to Cryptosporidium tyzzeri infection.

AIMS: Immunocompromised mice are extensively used in the screening of vaccines and drugs for Cryptosporidium, but this study model does not reflect the real status of infection in immunocompetent animals. This study aimed to provide an optimized animal model for future studies of Cryptosporidium vaccine. METHODS AND RESULTS: Three mouse strains (ICR, BALB/c and KM) with or without immunosuppression were compared after challenge with Cryptosporidium tyzzeri (C tyzzeri). The results indicated that ICR mice shed a greater number of faecal oocysts (20 346 ± 203 oocysts/g) compared with BALB/c (2077 ± 142 oocysts/g) and KM mice (3207 ± 431 oocysts/g) after experimental infection with C tyzzeri (P < .001). However, ICR mouse model is uniquely effective for C tyzzeri, not for other Cryptosporidium spp. such as C parvum. ICR mice were then used to determine the immunoreactions and immunoprotection of P23-DNA vaccine (pVAX1-P23) to C tyzzeri experimental infection. The results showed that a significant increase in anti-P23 antibody levels was induced by the pVAX1-P23 vaccine. Compared to pVAX1, TB and blank control mice, pVAX1-P23 immunized mice produced specific spleen cell proliferation as well as enhanced IL-5, IL-12p70 and IFN-γ production in sera. After challenge with 5 × 106 C tyzzeri oocysts, the oocyst shedding of the pVAX1-P23 immunized group was reduced by 69.94% comparing to the infection control. CONCLUSION: These results provide an optimized animal model for the study of prophylactic vaccines and this model might be applied to other candidates against Cryptosporidium, not only for pVAX1-P23.

Efficacy of halofuginone products to prevent or treat cryptosporidiosis in bovine calves: a systematic review and meta-analyses.

A prior systematic review on the efficacy of halofuginone (HFG) treatment to prevent or treat cryptosporidiosis in bovine calves was inconclusive. We undertook an updated synthesis and meta-analyses on key outcomes for the treatment of calves with HFG. Evaluated outcomes were oocyst shedding, diarrhoea, mortality and weight gain. Experiments had to describe results for same age animals in contemporary arms. Most doses were 100-150 mcg kg-1 day-1. Results were subgrouped by study design, experiments with the lowest risk of bias and lack of industry funding. Eighteen articles were found that described 25 experiments. Most evidence came from randomized controlled trials in Europe. Significantly lower incidence of oocyst shedding, diarrhoea burden and mortality was reported when treatment started before calves were 5 days old. Most studies reported on outcomes for animals up to at least 28 days old. Publication bias was possible in all outcomes and seemed especially likely for diarrhoea outcomes. Beneficial results when HFG treatment was initiated in calves older than 5 days were also found. Prophylactic treatment to prevent cryptosporidiosis is effective in preventing multiple negative outcomes and is beneficial to calf health and will result in a reduction of environmental contamination by Cryptosporidium oocysts.

Impact of Hydrometeorological Events for the Selection of Parametric Models for Protozoan Pathogens in Drinking-Water Sources.

Temporal variations in concentrations of pathogenic microorganisms in surface waters are well known to be influenced by hydrometeorological events. Reasonable methods for accounting for microbial peaks in the quantification of drinking water treatment requirements need to be addressed. Here, we applied a novel method for data collection and model validation to explicitly account for weather events (rainfall, snowmelt) when concentrations of pathogens are estimated in source water. Online in situ ß-d-glucuronidase activity measurements were used to trigger sequential grab sampling of source water to quantify Cryptosporidium and Giardia concentrations during rainfall and snowmelt events at an urban and an agricultural drinking water treatment plant in Quebec, Canada. We then evaluate if mixed Poisson distributions fitted to monthly sampling data ( n = 30 samples) could accurately predict daily mean concentrations during these events. We found that using the gamma distribution underestimated high Cryptosporidium and Giardia concentrations measured with routine or event-based monitoring. However, the log-normal distribution accurately predicted these high concentrations. The selection of a log-normal distribution in preference to a gamma distribution increased the annual mean concentration by less than 0.1-log but increased the upper bound of the 95% credibility interval on the annual mean by about 0.5-log. Therefore, considering parametric uncertainty in an exposure assessment is essential to account for microbial peaks in risk assessment.

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

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

A new passive immune strategy based on IgY antibodies as a key element to control neonatal calf diarrhea in dairy farms.

BACKGROUND: Neonatal diarrhea remains one of the main causes of morbi-mortality in dairy calves under artificial rearing. It is often caused by infectious agents of viral, bacterial, or parasitic origin. Cows vaccination and colostrum intake by calves during the first 6 h of life are critical strategies to prevent severe diarrhea but these are still insufficient. Here we report the field evaluation of a product based on IgY antibodies against group A rotavirus (RVA), coronavirus (CoV), enterotoxigenic Escherichia coli, and Salmonella sp. This product, named IgY DNT, has been designed as a complementary passive immunization strategy to prevent neonatal calf diarrhea. The quality of the product depends on the titers of specific IgY antibodies to each antigen evaluated by ELISA. In the case of the viral antigens, ELISA antibody (Ab) titers are correlated with protection against infection in calves experimentally challenged with RVA and CoV (Bok M, et al., Passive immunity to control bovine coronavirus diarrhea in a dairy herd in Argentina, 2017), (Vega C, et al., Vet Immunol Immunopathol, 142:156-69, 2011), (Vega C, et al., Res Vet Sci, 103:1-10, 2015). To evaluate the efficiency in dairy farms, thirty newborn Holstein calves were randomly assigned to IgY DNT or control groups and treatment initiated after colostrum intake and gut closure. Calves in the IgY DNT group received 20 g of the oral passive treatment in 2 L of milk twice a day during the first 2 weeks of life. Animals were followed until 3 weeks of age and diarrhea due to natural exposure to infectious agents was recorded during all the experimental time. RESULTS: Results demonstrate that the oral administration of IgY DNT during the first 2 weeks of life to newborn calves caused a delay in diarrhea onset and significantly reduced its severity and duration compared with untreated calves. Animals treated with IgY DNT showed a trend towards a delay in RVA infection with significantly shorter duration and virus shedding compared to control calves. CONCLUSIONS: This indicates that IgY DNT is an effective product to complement current preventive strategies against neonatal calf diarrhea in dairy farms. Furthermore, to our knowledge, this is the only biological product available for the prevention of virus-associated neonatal calf diarrhea.

Systematic review of modifiable risk factors shows little evidential support for most current practices in Cryptosporidium management in bovine calves.

Cryptosporidiosis is common in young calves, causing diarrhoea, delayed growth, poor condition and excess mortality. No vaccine or cure exists, although symptomatic onset may be delayed with some chemoprophylactics. Other response and management strategies have focused on nutritional status, cleanliness and biosecurity. We undertook a systematic review of observational studies to identify risk or protective factors that could prevent Cryptosporidium parvum infection in calves. Included studies used multivariate analysis within cohort, cross-sectional or case-control designs, of risk factors among young calves, assessing C. parvum specifically. We tabulated data on characteristics and study quality and present narrative synthesis. Fourteen eligible studies were found; three of which were higher quality. The most consistent evidence suggested that risk of C. parvum infection increased when calves had more contact with other calves, were in larger herds or in organic production. Hard flooring reduced risk of infection and calves tended to have more cryptosporidiosis during warm and wet weather. While many other factors were not found to be associated with C. parvum infection, analyses were usually badly underpowered, due to clustering of management factors. Trials are needed to assess effects of manipulating calf contact, herd size, organic methods, hard flooring and temperature. Other factors need to be assessed in larger observational studies with improved disaggregation of potential risk factors.

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.

The truth about in vitro culture of Cryptosporidium species.

Cryptosporidium research has focused on the development of infection control, and effective therapy that has thus far been hampered by the inability to culture Cryptosporidium in vitro. Other limitations include inadequate animal models, cumbersome screening procedures for chemotherapeutic approaches and a lack of tools for genetic manipulation. These limitations can, however, be eased by the improvement and focused development of in vitro cultivation. The ability to culture relevant Cryptosporidium isolates in vitro and to propagate the life cycle stages that are responsible for causing disease in an infected host is still a critical link. This ability will facilitate other relevant approaches, e.g., the ability to knockout genes and the application of broader screening for drug discoveries and vaccine developments, in combination with new discoveries on the parasite's basic biology, genetic manipulation and new life cycle stages. Success in this effort represents an essential step towards significant progress in the control of cryptosporidiosis.

An evaluation of health protection practices for the investigation and management of Cryptosporidium in England and Wales.

Background: Cryptosporidium is a major cause of gastroenteritis (cryptosporidiosis). Case and outbreak report rates vary geographically, which may in part reflect public health practice. Methods: To examine the public health management of cryptosporidiosis, an online questionnaire was administered to the 28 Health Protection Teams (HPTs) in England and Wales in 2014. Practices for investigation and management of cases and outbreaks were compared. Results: Practice varied among the 24 (86%) respondents in terms of who undertook actions (HPT or Local Authority) to investigate and manage cryptosporidiosis. HPTs without exceedance monitoring detected fewer outbreaks (1/5, 20%) than those with it (13/19, 68%) (P = 0.12), and those that always administered a risk-factor questionnaire detected more outbreaks (12/19, 63%) than those who did this only sometimes (2/5, 40%) (P = 0.62). Significantly more HPTs with a system to detect common exposures reported at least one outbreak (14/19, 74%) compared to HPTs with no system (0/5) (P = 0.01). Conclusions: Applying exceedance monitoring, using a standardized questionnaire taking into account the incubation period for Cryptosporidium, and having a structured system to detect common exposures increased outbreak detection. Information about all cases should be shared between local public health authorities, and current guidance used for the prevention of spread.

Limitations in the screening of potentially anti-cryptosporidial agents using laboratory rodents with gastric cryptosporidiosis.

The emergence of cryptosporidiosis, a zoonotic disease of the gastrointestinal and respiratory tract caused by Cryptosporidium Tyzzer, 1907, triggered numerous screening studies of various compounds for potential anti-cryptosporidial activity, the majority of which proved ineffective. Extracts of Indonesian plants, Piper betle and Diospyros sumatrana, were tested for potential anti-cryptosporidial activity using Mastomys coucha (Smith), experimentally inoculated with Cryptosporidium proliferans Kvác, Havrdová, Hlásková, Danková, Kandera, Jezková, Vítovec, Sak, Ortega, Xiao, Modrý, Chelladurai, Prantlová et McEvoy, 2016. None of the plant extracts tested showed significant activity against cryptosporidia; however, the results indicate that the following issues should be addressed in similar experimental studies. The monitoring of oocyst shedding during the entire experimental trial, supplemented with histological examination of affected gastric tissue at the time of treatment termination, revealed that similar studies are generally unreliable if evaluations of drug efficacy are based exclusively on oocyst shedding. Moreover, the reduction of oocyst shedding did not guarantee the eradication of cryptosporidia in treated individuals. For treatment trials performed on experimentally inoculated laboratory rodents, only animals in the advanced phase of cryptosporidiosis should be used for the correct interpretation of pathological alterations observed in affected tissue. All the solvents used (methanol, methanol-tetrahydrofuran and dimethylsulfoxid) were shown to be suitable for these studies, i.e. they did not exhibit negative effects on the subjects. The halofuginone lactate, routinely administered in intestinal cryptosporidiosis in calves, was shown to be ineffective against gastric cryptosporidiosis in mice caused by C. proliferans. In contrast, the control application of extract Arabidopsis thaliana, from which we had expected a neutral effect, turned out to have some positive impact on affected gastric tissue.

Natural History of Cryptosporidiosis in a Birth Cohort in Southern India.

BACKGROUND: Cryptosporidium is a leading cause of moderate to severe childhood diarrhea in resource-poor settings. Understanding the natural history of cryptosporidiosis and the correlates of protection are essential to develop effective and sustainable approaches to disease control and prevention. METHODS: Children (N = 497) were recruited at birth in semiurban slums in Vellore, India, and followed for 3 years with twice-weekly home visits. Stool samples were collected every 2 weeks and during diarrheal episodes were tested for Cryptosporidium species by polymerase chain reaction (PCR). Serum samples obtained every 6 months were evaluated for seroconversion, defined as a 4-fold increase in immunoglobulin G directed against Cryptosporidium gp15 and/or Cp23 antigens between consecutive sera. RESULTS: Of 410 children completing follow-up, 397 (97%) acquired cryptosporidiosis by 3 years of age. PCR identified 1053 episodes of cryptosporidiosis, with an overall incidence of 0.86 infections per child-year by stool and serology. The median age for the first infection was 9 (interquartile range, 4-17) months, indicating early exposure. Although infections were mainly asymptomatic (693 [66%]), Cryptosporidium was identified in 9.4% of diarrheal episodes. The proportion of reinfected children was high (81%) and there was clustering of asymptomatic and symptomatic infections (P < .0001 for both). Protection against infection increased with the order of infection but was only 69% after 4 infections. Cryptosporidium hominis (73.3%) was the predominant Cryptosporidium species, and there was no species-specific protection. CONCLUSIONS: There is a high burden of endemic cryptosporidiosis in southern India. Clustering of infection is suggestive of host susceptibility. Multiple reinfections conferred some protection against subsequent infection.