Assessment of the Immunoprotective Efficacy of Recombinant 14-3-3 Protein and Dense Granule Protein 10 (GRA10) as Candidate Antigens for Rabbit Vaccines against Eimeria intestinalis.

Eimeria intestinalis infects rabbits, causing severe intestinal coccidiosis. Prolonged anticoccidial drug use might lead to coccidia resistance and drug residues in food. Thus, vaccines are required to control rabbit coccidiosis. In this study, recombinant E. intestinalis 14-3-3 and GRA10 proteins (rEi-14-3-3 and rEi-GRA10) were obtained via prokaryotic expression and used as recombinant subunit vaccines. Fifty 30-day-old rabbits were randomly grouped as follows: PBS-uninfected group, PBS-infected group, Trx-His-S control group, and rEi-14-3-3 and rEi-GRA10 immunized groups. The rabbits were subcutaneously immunized twice at 2-week intervals, challenged with 7 × 104 sporulated oocysts, and sacrificed 14 days later. The protective effects were assessed via clinical signs, relative weight gain, oocyst reduction, mean intestinal lesion score, ACI (anticoccidial index), cytokine, and specific antibody levels in sera. The rEi-14-3-3 and rEi-GRA10 groups had higher relative weight gain rates of 81.94% and 73.61% (p < 0.05), and higher oocyst reduction rates of 86.13% and 84.87% (p < 0.05), respectively. The two immunized groups had fewer intestinal lesions (p < 0.05) and higher IgG levels (p < 0.05). Higher levels of IL-2, IL-4, and IFN-γ cytokines in the rEi-14-3-3 group (p < 0.05) and a higher level of IFN-γ in the rEi-GRA10 group (p < 0.05) were observed. The ACI values of the rEi-14-3-3 and rEi-GRA10 groups were 168.24 and 159.91, with good and moderate protective effects, respectively. Both rEi-14-3-3 and rEi-GRA10 induced humoral immunity in the rabbits. In addition, rEi-14-3-3 induced Th1- and Th2-type immune responses. Both recombinant proteins were protective against E. intestinalis infection in rabbits, with rEi-14-3-3 showing a better protective effect.

Isolation of saprophytic filamentous fungi from avian fecal samples and assessment of its predatory activity on coccidian oocysts.

Fungal strains used in the biocontrol of animal gastrointestinal parasites have been mainly isolated from pasture soil, decaying organic matter, and feces from herbivores and carnivores. However, their isolation from birds and assessment of predatory activity against avian GI parasites has been scarce thus far. This research aimed to isolate filamentous fungi from avian fecal samples and evaluate their predatory activity against coccidia. A pool of 58 fecal samples from chickens, laying hens, and peacocks, previously collected between July 2020-April 2021, were used for isolation of filamentous fungi and assessment of their in vitro predatory activity against coccidian oocysts, using Water-Agar medium and coprocultures. The Willis-flotation technique was also performed to obtain concentrated suspensions of oocysts. A total of seven Mucor isolates was obtained, being the only fungal taxa identified, and all presented lytic activity against coccidia. Isolates FR3, QP2 and SJ1 had significant coccidiostatic efficacies (inhibition of sporulation) higher than 70%, while isolates FR1, QP2 and QP1 had coccidicidal efficacies (destruction of the oocysts) of 22%, 14% and 8%, respectively, after 14 days of incubation, being a gradual and time-dependent process. To our knowledge, this is the first report regarding the isolation of native predatory fungi from avian feces and demonstration of their lytic activity against coccidia.

Production performance and economic analysis of broiler chickens after vaccination with a live attenuated vaccine against avian coccidiosis.

The objective of this study was to evaluate the effectiveness of live attenuated commercial vaccine LIVACOX® T against avian coccidiosis upon parasite exposure through its correlation with productive and economic performance, clinical observation, and oocyst excretion of broiler chickens. For this purpose, 420 1-day-old Cobb chicks were divided into five groups of 84 birds: (G1) unvaccinated and unchallenged control; (G2) vaccinated on day 0; (G3) challenged on day 1; (G4) vaccinated on day 0 and challenged on day 14; and (G5) challenged on day 14. For 28 days, the clinical signs of infection, weight and feed conversion of the birds, and excretion of oocysts in the feces were evaluated. Macroscopic analysis of intestinal lesions in birds was also performed. After vaccination in G2, G3, and G4 as well as after challenge in G3, G4, and G5, there was an increase in oocyst excretion. In the analysis of weight gain, the difference in final weight between groups G3 and G4 is - 105.74 g per bird. Therefore, if we multiply this value by the average number of birds slaughtered per day in a medium/large slaughterhouse (250,000), we have 26,435 kg of chicken meat per day of slaughter, representing 581,570 kg of monthly losses (22 days of slaughter/month), or approximately R$3,489,420.00 (US$872,355.00), considering the commercial value at R$6.00/kg (US$ 1.5/kg). Thus, the productive and economic impact of coccidiosis in broiler chickens is evident, and the importance of vaccination to prevent the occurrence of the disease and reduce subsequent loss is highlighted.

Spatially explicit model of the Cryptosporidium and Giardia disease burden from surface and ground waters in urban and rural areas of the Three Gorges Reservoir watershed in Chongqing, China.

Cryptosporidium and Giardia (major causes of diarrhea) are widely distributed in Chinese source waters and threaten human health. A new spatially explicit GloWPa-TGR-Crypt-Giar C1 model is presented to simultaneously estimate mean monthly (oo)cyst concentrations in surface and ground waters in the Three Gorges Reservoir (TGR) watershed. A quantitative risk assessment of protozoal infections considered different source waters, transmission pathways, regions, susceptible subpopulations, and drinking water treatments. Monthly mean Cryptosporidium oocyst and Giardia cyst concentrations ranged between 0.5-19.3 oocysts/10 L and 0.2-5.0 cysts/10 L in surface water, respectively, and 0.007-0.3 oocysts/10 L and 0.002-0. 2 cysts/10 L in groundwater. The cumulative disease burdens attributable to cryptosporidiosis and giardiasis were, respectively, 5.77×10-5 DALYs (disability-adjusted life years/person/year) and 4.63×10-6 DALYs in urban areas, and 6.35×10-4 DALYs and 8.84×10-5 DALYs in rural areas, which were much higher than the reference risk level recommended by the World Health Organization ([Formula: see text] DALYs). The annual burden associated with consuming surface water was calculated to be 3.84×10-4 DALYs for Cryptosporidium and [Formula: see text] DALYs for Giardia, whereas consuming groundwater entailed the lower burdens (1.26×10-5 and 3.50×10-6 DALYs, respectively). Most DALYs were a consequence of consumption of directly supplied surface water. Fifty percent of the health burden was carried by immunodeficiency with HIV. Children (0-4 years) were more likely to have an individual disease burden than adults (15-64 years). Males were more susceptible than females. Improving sanitation through adequate ozone and microfiltration treatment should be considered when attempting to reduce disease burden. Sensitivity analysis highlighted the importance of reducing (oo)cyst loads to protect the watershed. The methodology and results described will help in evaluating and reducing the burden of protozoal infection associated with surface and ground waters in the TGR and similar watersheds.

Comparison of the caecal microbial community structure and physiological indicators of healthy and infection Eimeria tenella chickens during peak of oocyst shedding.

Eimeria tenella (E. tenella), an important intestinal parasite of chicken caeca, causes coccidiosis and brings large economic losses to the poultry industry annually. Gut microorganismal alterations directly affect the health of the body. To understand how E. tenella affects its host, we analysed the changes in caecal microbial diversity and the physiological and morphological changes during the peak of oocyst shedding. Infected and healthy chickens differed significantly in caecal pathology and blood indicators. At the genus level, the abundances of Faecalibacterium, Clostridium, Lachnoclostridium, Gemmiger, Flavonifractor, Pseudoflavonifractor and Oscillibacter were significantly decreased in the infected samples, whereas Escherichia, Nocardia and Chlamydia were significantly increased. Functional gene pathways related to replication, recombination and repair, and transcription were significantly decreased, and functional genes related to metabolism were highly significantly reduced in the infected samples. Furthermore, in the infected samples, E. tenella reduced the haemoglobin levels and red blood cell counts, greatly reduced the beneficial bacteria and increased the potentially pathogenic bacteria. This study provides a research basis for further understanding the pathogenic mechanisms of E. tenella and provides insight for potential new drug development.RESEARCH HIGHLIGHTS First simultaneous description of caecal microbiota and physiological indicators during E. tenella infection.Metagenomics used to explore functional properties of chicken caecal microbiota during E. tenella infection.Caecal microbial compositions and functional genes altered significantly after infection.Blood indicators and caecal morphology were significantly altered in the infected group.

Human health risk assessment for (re)emerging protozoan parasites in surface water used for public supply and recreational activities.

Gastrointestinal diseases caused by protozoan parasites remain a major challenge in developing countries and ingestion of contaminated surface water represents one of the main sources by which these diseases are contracted. This study assessed the risk of infection and diseases caused by Cryptosporidium spp. and Giardia sp. due to ingestion of surface water used for public supply and recreational activities, focusing on the southeastern Brazilian Pardo River and applying the USEPA 1623 method to quantify (oo)cyst concentrations. Infection and disease probabilities due to ingestion of drinking water or during recreational activities were estimated using the Quantitative Microbial Risk Assessment (QMRA) approach. Mean concentrations of Cryptosporidium spp. and Giardia sp. in surface water ranged from 0.2 to 0.4 oocysts L-1 and 0.2 to 4.4 cysts L-1, respectively. Considering public water supply, annual infection probabilities were higher for adults than children and exceeded the USEPA limit; also, disease probabilities were higher for adults than children. For recreational activities, annual infection and disease probabilities were higher for children, followed by men and women. The occurrence of both parasites likely reflects raw sewage discharge, effluent from sewage treatment plants, and diffuse sources of pollution, such as runoff from pasture lands and deforested riparian forest corridors. Our results highlight substantial infection risks by both parasite types after conventional treatment of water used for public supply and also call for careful monitoring of water bodies used for recreational purposes.

Assessment of the Activity of Decoquinate and Its Quinoline-O-Carbamate Derivatives against Toxoplasma gondii In Vitro and in Pregnant Mice Infected with T. gondii Oocysts.

The quinolone decoquinate (DCQ) is widely used in veterinary practice for the treatment of bacterial and parasitic infections, most notably, coccidiosis in poultry and in ruminants. We have investigated the effects of treatment of Toxoplasma gondii in infected human foreskin fibroblasts (HFF) with DCQ. This induced distinct alterations in the parasite mitochondrion within 24 h, which persisted even after long-term (500 nM, 52 days) treatment, although there was no parasiticidal effect. Based on the low half-maximal effective concentration (IC50) of 1.1 nM and the high selectivity index of >5000, the efficacy of oral treatment of pregnant mice experimentally infected with T. gondii oocysts with DCQ at 10 mg/kg/day for 5 days was assessed. However, the treatment had detrimental effects, induced higher neonatal mortality than T. gondii infection alone, and did not prevent vertical transmission. Thus, three quinoline-O-carbamate derivatives of DCQ, anticipated to have better physicochemical properties than DCQ, were assessed in vitro. One such compound, RMB060, displayed an exceedingly low IC50 of 0.07 nM, when applied concomitantly with the infection of host cells and had no impact on HFF viability at 10 µM. As was the case for DCQ, RMB060 treatment resulted in the alteration of the mitochondrial matrix and loss of cristae, but the changes became apparent at just 6 h after the commencement of treatment. After 48 h, RMB060 induced the expression of the bradyzoite antigen BAG1, but TEM did not reveal any other features reminiscent of bradyzoites. The exposure of infected cultures to 300 nM RMB060 for 52 days did not result in the complete killing of all tachyzoites, although mitochondria remained ultrastructurally damaged and there was a slower proliferation rate. The treatment of mice infected with T. gondii oocysts with RMB060 did reduce parasite burden in non-pregnant mice and dams, but vertical transmission to pups could not be prevented.

Does each bead count? A reduced-cost approach for recovering waterborne protozoa from challenge water using immunomagnetic separation.

Giardia duodenalis and Cryptosporidium spp. are two of the most prominent aetiological agents of waterborne diseases. Therefore, efficient and affordable methodologies for identifying and quantifying these parasites in water are increasingly necessary. USEPA Method 1623.1 is a widely used and validated protocol for detecting these parasites in water samples. It consists of a concentration step, followed by parasite purification and visualization by immunofluorescence microscopy. Although efficient, this method has a high cost particularly due to the immunomagnetic separation (IMS) step, which is most needed with complex and highly contaminated samples. Based on this, the present study aimed to determine whether it is possible to maintain the efficiency of Method 1623.1 while reducing the amount of beads per reaction, using as a matrix the challenge water recommended by the World Health Organization. As for Giardia cysts, a satisfactory recovery efficiency (RE) was obtained using 50% less IMS beads. This was evaluated both with a commercial cyst suspension (56.1% recovery) and an analytical quality assessment (47.5% recovery). Although RE rates obtained for Cryptosporidium parvum did not meet Method 1623.1 criteria in any of the experimental conditions tested, results presented in this paper indicated the relevance of the described adaptations, even in challenge water.

Detection of Cyclospora cayetanensis on bagged pre-cut salad mixes within their shelf-life and after sell by date by the U.S. food and drug administration validated method.

Recently, outbreaks of Cyclospora cayetanensis in the U.S. were linked to the consumption of a variety of salads containing romaine and/or iceberg lettuce, carrots and/or red cabbage. The Bacteriological Analytical Manual (BAM) Chapter 19b method was validated for the detection of C. cayetanensis in carrots, cabbage and romaine lettuce, but has not been previously evaluated in ready-to-eat (RTE) salad mixes. In addition, the only samples available for traceback investigations are sometimes leftovers in bad conditions. This study evaluated the validated BAM method for detection of C. cayetanensis in two different RTE mixed salads (mix 1: romaine and iceberg lettuces, carrots, and red cabbage and mix 2: romaine and iceberg lettuces, carrots, red cabbage, radish, and pea pods) in good condition and after their sell by date. Individual samples (25 g) were seeded with five and 200 C. cayetanensis oocysts. Unseeded produce was used as negative control. The method included washing of the produce, concentration and extraction of C. cayetanensis DNA and molecular detection of C. cayetanensis 18 S rRNA gene. As few as five oocysts were detected in both fresh and after sell by date mix salads. All unseeded samples were negative, and all samples of both salad types seeded with 200 oocysts were positive. In samples seeded with 200 oocysts, average 18 S rRNA C. cayetanensis CT values were significantly higher in fresh salad mix 1 compared to fresh salad mix 2; CT values were significantly higher in the after sell by date salads compared to their respective fresh mixes (p < 0.05). In conclusion, the BAM method was able to detect as few as five oocysts even in after sell by date RTE mix salads. However, the differences in detection observed, highlight the importance of evaluating the performance of the validated C. cayetanensis detection method in different food matrices and conditions, in advance for future outbreak investigations.

Giardia spp. cysts and Cryptosporidium spp. oocysts in drinking water treatment residues: comparison of recovery methods for quantity assessment.

Water treatment plant (WTP) residues, e.g. sludge and filter backwash water (FBW), may contain pathogenic microorganisms, as Giardia spp. and Cryptosporidium spp. However, recovering protozoa from such matrices lacks a formal and precise protocol, which is imperative to improve research in their detection, removal and inactivation. The latter includes a deeper challenge as some recovery methods may compromise viability. This study applied different recovery methods for G. muris cysts and C. parvum oocysts spiked into settled sludge and FBW obtained from a bench treatment. Procedures in sludge involved direct centrifugation, alkaline and acid flocculation, including purification by immunomagnetic separation (IMS). FBW samples were tested for membrane filtration (MF) and heated Tween® scrapings followed or not by IMS. Propidium iodide (PI) inclusion was used for oocyst viability evaluation prior and after recovery. Results with purified suspensions lead to higher recovery efficiencies (RE) for C. parvum, which was assumed to relate to poor G. muris fluorescence. Analytical quality assessments were carried out with ColorSeed® for the methods that stood out for each matrix and the results indicated lower RE than when organisms from purified suspensions were recovered. Ferric sulphate flocculation and MF, both followed by IMS reached 32.25% and 11.00% RE for Giardia spp. and 19.61% and 2.00% for Cryptosporidium spp., respectively. All of the tested methods affected oocyst viability. These results encourage further research to overcome the matrices complexity explained in this paper and increase RE, taking effects in protozoa viability into consideration.

Digging into Toxoplasma gondii infections via soil: A quantitative microbial risk assessment approach.

Soil has been identified as an important source of exposure to a variety of chemical and biological contaminants. Toxoplasma gondii is one of those potential biological contaminants associated with serious health effects in pregnant women and immunocompromised patients. Gardening or consumption of homegrown vegetables may present an important route of T. gondii infection via accidental ingestion of soil. In the Netherlands, there is quantitative information on the risk of T. gondii infection via meat products, but not on the risk of infection through soil. The objective of this study was to develop a quantitative microbial risk assessment (QMRA) model for estimating the risk associated with T. gondii exposure via accidental soil ingestion in the Netherlands. In order to obtain the needed information, a magnetic capture method for detection of T. gondii oocysts in soil samples was developed, and T. gondii DNA was detected using qPCR targeting the 529 bp repeat element. The method was shown to provide 95% probability of detection (95% CI: 88-100%) when at least 34 oocysts are present in 25 g of soil. T. gondii DNA was detected in 5 of 148 soil samples with interpretable results (3%, 95% CI: 1.5-7.7%). Results for 18 samples were not interpretable due to PCR inhibition. The estimated amount of oocysts presented in qPCR positive samples was quantified by a linear model, and the amount varied from 8 to 478 in 25 g of soil. The estimated incidence rate of T. gondii infection from the QMRA model via soil varied from 0.3 to 1.8 per 1000 individuals per day. Several data gaps (e.g., soil contamination/ingestion and oocysts viability) have been identified in this study, the structure of the model can be applied to obtain more accurate estimates of the risk of T. gondii infection via soil when data become available.

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.

Assessment of Commercial DNA Cleanup Kits for Elimination of Real-Time PCR Inhibitors in the Detection of Cyclospora cayetanensis in Cilantro.

ABSTRACT: Inhibited reactions have occasionally been observed when cilantro samples were processed for the detection of Cyclospora cayetanensis using quantitative real-time PCR (qPCR). Partial or total inhibition of PCR reactions, including qPCR, can occur, leading to decreased sensitivity or false-negative results. If inhibition occurs, this implies the need for additional purification or cleanup treatments of the extracted DNA to remove inhibitors prior to molecular detection. Our objective was to evaluate the performance of five commercial DNA cleanup kits (QIAquick purification kit from Qiagen [kit 1], OneStep PCR inhibitor removal by Zymo Research [kit 2], NucleoSpin genomic DNA cleanup XS from Macherey-Nagel [kit 3], DNA IQ system by Promega [kit 4], and DNeasy PowerPlant pro kit from Qiagen [5]) to minimize qPCR inhibition using the U.S. Food and Drug Administration-validated Bacteriological Analytical Manual (BAM) Chapter 19b method for detection of C. cayetanensis in cilantro samples containing soil. Each of the five commercial DNA cleanup kits evaluated was able to reduce the qPCR internal amplification control cycle threshold values to those considered to be normal for noninhibited samples, allowing unambiguous interpretation of results in cilantro samples seeded at both a high oocyst level (200 oocysts) and a low oocyst level (10 oocysts). Of the five kits compared, kits 1, 2, and 3 did not show significant differences in the detection of C. cayetanensis, while significantly higher cycle threshold values, indicating lower recovery of the target DNA, were observed from kits 4 and/or 5 in samples seeded with 200 and 10 oocysts (P < 0.05). This comparative study provides recommendations on the use of commercial cleanup kits which could be implemented when inhibition is observed in the detection of C. cayetanensis in cilantro samples using the BAM Chapter 19b method.

Assessment of health risks from recreational exposure to Giardia and Cryptosporidium in coastal bathing waters.

Some Brazilian beaches are impacted by raw or poorly treated sewage. Thus, users (beachgoers, sports people, and children) are exposed to pathogens, which pose health concerns. This study aimed to estimate the probability of infection and disease by Giardia and Cryptosporidium, using the quantitative microbial risk assessment (QMRA), for three groups of bathers: children, adults, and open water swimmers. The concentrations of (oo)cysts were taken from a study run by CETESB (Environmental Company of Sao Paulo State) throughout 2011 and 2012, in which 203 samples were collected monthly and analyzed for (oo)cysts of Giardia and Cryptosporidium. Giardia was present in 43% of the samples, while Cryptosporidium in 13%. Infection probability was higher in beaches with more positive samples for Giardia cysts for the group of open water swimmers. In some cases, the highest annual risk obtained for giardiasis and cryptosporidiosis was 2.4 × 10-1 and 8.9 × 10-3 for open water swimmers, respectively, exceeding the incidence results found in the epidemiological study run in summer of 1999 in São Paulo state coast. The results bring insights to improve environmental quality in order to protect tourists' and residents' wellbeing.

Waterborne protozoan pathogens in environmental aquatic biofilms: Implications for water quality assessment strategies.

Biofilms containing pathogenic organisms from the water supply are a potential source of protozoan parasite outbreaks and a significant public health concern. The aim of the present study was to demonstrate the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens (WBPP) in substrate-associated biofilms (SAB) and compare it to surface water (SW) and sediments with bottom water (BW) counterparts using manual filtration and elution from low-volume samples. For scenario purposes, simulated environmental biofilm contamination was created from in-situ grown one-month-old SAB (OM-SAB) that were spiked with Cryptosporidium parvum oocysts. Samples were collected from the largest freshwater reservoirs in Luzon, Philippines and a University Lake in Thailand. A total of 69 samples (23 SAB, 23 SW, and 23 BW) were evaluated using traditional staining techniques for Cryptosporidium, and Immunofluorescence staining for the simultaneous detection of Cryptosporidium and Giardia. WBPP were found in 43% SAB, 39% SW, and 39% BW of the samples tested in the present study with SAB results reflecting SW and BW results. Further highlights were demonstrated in the potential of using low-volume samples for the detection of parasites in source water. Scanning electron microscopy of OM-SAB samples revealed a naturally-associated testate amoeba shell, while Cryptosporidium oocysts spiked samples provided a visual profile of what can be expected from naturally contaminated biofilms. This study provides the first evidence for the simultaneous and multi-spatial occurrence of waterborne protozoan pathogens in low-volume aquatic matrices and further warrants SAB testing along with SW and BW matrices for improved water quality assessment strategies (iWQAS).

Comparative evaluation of Cryptosporidium infection in malnourished and well-nourished children: Parasitic infections are affected by the interaction of nutritional status and socio-demographic characteristics.

Cryptosporidium, as a small protozoan parasite, is a leading cause of persistent diarrhea in children in developing countries and has both a short and long-term impact on the growth of children. In the present study, Cryptosporidium infection was compared in malnourished and well-nourished children by modified acid-fast staining, nested-polymerase chain reaction (nested-PCR) and loop-mediated isothermal amplification (LAMP) methods. As a case-control study, Cryptosporidium infection in 94 malnourished children was evaluated and compared with those of 188 age and gender-matched well-nourished children. Oocysts of Cryptosporidium were detected by modified acid-fast staining method. The extracted DNA was amplified by nested-PCR and LAMP techniques. In addition, positive amplicons were directly sequenced for phylogenetic analysis. Cryptosporidium oocysts were found in the stools of two (2.12 %) children who were hospitalized and had diarrhea by nested-PCR while three isolates (3.2 %) were found by LAMP. Cryptosporidium-positive children were more malnourished compared to those who were negative for Cryptosporidium infection but this important finding was not statistically significant. C. parvum was the main species of Cryptosporidium detected in malnourished children in northwest Iran. LAMP can be considered as a sensitive field monitoring assay in patients with low parasite burden. Nutritional status and socio-demographic factors may have interactive effects on the incidence and severity of parasitic diseases.

Assessment of differences between DNA content of cell-cultured and freely suspended oocysts of Cryptosporidium parvum and their suitability as DNA standards in qPCR.

BACKGROUND: Although more modern methods are available, quantitative PCR (qPCR) is reproducible, sensitive and specific with instruments and expertise readily available in many laboratories. As such, the use of qPCR in Cryptosporidium research is well established and still widely used by researchers globally. This method depends upon the generation of standards at different concentrations to generate standard curves subsequently used for the quantification of DNA. METHODS: We assessed four types of DNA template used to generate standard curves in drug screening studies involving Cryptosporidium spp.: (i) serially diluted Cryptosporidium parvum oocysts (106-1); (ii) diluted template DNA from pure oocysts (×10-×106 dilution of 106 oocyst DNA template); (iii) oocysts incubated in human ileocecal adenocarcinoma (HCT-8) cells (105-1 and 5 × 104-50); and (iv) diluted DNA template (5 × 104) from cell culture incubated parasites (×10-×1000). RESULTS: Serial dilutions of both cell culture and pure oocyst suspension DNA template yielded better linearity than cell culture derived standards, with dilutions of 106 oocysts exhibiting similar quantification cycle (Cq) values to those obtained from DNA template dilutions of 106 oocysts. In contrast, cell culture incubated oocysts demonstrated significantly higher DNA content than equivalent freely suspended oocysts and diluted DNA template from both cell culture derived and freely suspended oocysts across numerous concentrations. CONCLUSIONS: For many studies involving Cryptosporidium, only relative DNA content is required and as such, the superior linearity afforded by freely suspended oocysts and diluted DNA template (from either cell culture derived standards or freely suspended oocysts) will allow for more accurate relative quantification in each assay. Parasite division in the cell culture standards likely explains the higher DNA content found. These standards, therefore, have the potential to more accurately reflect DNA content in cell culture assays, and despite more modern methods available for absolute quantification, i.e. droplet digital PCR (ddPCR), the ubiquity of qPCR for the foreseeable future encourages further investigation into the reduced linearity observed in these standards such as varying oocyst seeding density, non-linear growth rates and assay efficiency.

Low-cost internal controls for detection of Giardia cysts in water samples.

Giardia cysts stained with hot carbolfuchsin were used as internal controls in a concentration method for surface water samples. The morphological integrity of stained cysts and the stain's stability and intensity were tested with each of the chemical reagents used in the aluminum sulfate flocculation method. No alterations in morphology or color were noted. The stained cyst preparation has a low cost, high stability, and suitability for both light and immunofluorescent microscopy, making it affordable to researchers in low- and middle-income countries.

Quantitative microbial risk assessment of Cryptosporidium in bivalve samples from Manila Bay, Philippines

In the Philippines, consumption of bivalves is very common due to its year-round availability and cheap price. However, many consume bivalves as lightly-cooked or raw. This might pose health hazards because bivalves are filter-feeders which act as vehicles for transmission of several pathogens such as the protozoan parasite Cryptosporidium, the causative agent of cryptosporidiosis. Cryptosporidiosis in humans is manifested by profuse diarrhea and abdominal pain. To determine the risk of acquiring cryptosporidiosis from consumption of bivalves, quantitative microbial risk assessment (QMRA) should be done. This study aimed to determine the risk associated with the consumption of bivalves which are contaminated with Cryptosporidium oocysts. The results indicate that consumption of at least 21 grams of cooked bivalves contaminated with at least 0.1% viable oocysts might pose a risk to consumers, especially to immunocompromised individuals. This estimated risk of infection exceeded the United States Environmental Protection Agency (US EPA) standards (1.0×10-4). Results call for drive of decision-makers to establish an educational or treatment program to reduce the incidence of gastrointestinal infections of the consumers. Improvement of sanitation techniques and hygienic practices will contribute to the decrease of occurrence of the disease.

Cryptosporidium oocyst persistence in agricultural streams -a mobile-immobile model framework assessment.

Rivers are a means of rapid and long-distance transmission of pathogenic microorganisms from upstream terrestrial sources. Pathogens enter streams and rivers via overland flow, shallow groundwater discharge, and direct inputs. Of concern is the protozoal parasite, Cryptosporidium, which can remain infective for weeks to months under cool and moist conditions, with the infectious stage (oocysts) largely resistant to chlorination. We applied a mobile-immobile model framework to assess Cryptosporidium transport and retention in streams, that also accounts for inactivation. The model is applied to California's Central Valley where Cryptosporidium exposure can be at higher risk due to agricultural and wildlife nonpoint sources. The results demonstrate that hyporheic exchange is an important process to include in models characterizing pathogen dynamics in streams, delaying downstream transmission and allowing for immobilization processes, such as reversible filtration in the sediments, to occur. Although in-stream concentrations decrease relatively quickly (within hours), pathogen accumulation of up to 66% of the inputs due to immobilization processes in the sediments and slower moving surface water could result in long retention times (months to years). The model appropriately estimates baseflow pathogen accumulation and can help predict the potential loads of resuspended pathogens in response to a storm event.