Parvum Oocysts Research Articles

Transforming Growth Factor-β, Reverse IFN-γ Activation of Intestinal Epithelial Cells during Cryptosporidium parvum Infection

Background: The intracellular parasite Cryptosporidium parvum (C. parvum) is a causative agent of diarrhea in humans and potentially fatal in AIDS patients. The parasites develop in enterocytes and are transmitted mainly by the fecal-oral route. In developing countries, infections are more common in children and may be associated with malabsorption and malnutrition. Enterocytes are an active component of intestinal mucosal immunity and their resistance to infection can be mediated by more than one mechanism including responding to cytokine signals. IFN-γ activates various mechanisms in infected enterocytes to kill invading pathogens. In contrast, transforming growth factor β (TGF-β) is crucial in downregulating inflammation caused by the Th1 response. Aim of Study: This study aims to investigate the role of IFN-γ and TGF-β in host immunity against C. parvum infection. Methods: To determine the role of IFN- on the development of C. parvum in vitro, HT-29 monolayers were incubated for 24h with varying concentrations of IFN-. Following infection with C. parvum oocyst, the cells were re-incubated with cytokines for a further 24h before being fixed and stained with Giemsa. The parasites were then counted using a Zeiss Axioplan microscope at x1000 magnification with oil-immersion in 20 random fields across the diameter of the coverslip. Results: IFN- was found to have a marked inhibitory effect on C. parvum infection. The effect of IFN-γ was partially reversed by TGF-β, which produces a significant dose-dependent antagonist of IFN-γ activity. IFN-γ mediated its action by modification of intracellular Fe2+ concentration.

Analytical challenges and perspectives of assessing viability of Giardia muris cysts and Cryptosporidium parvum oocysts by live/dead simultaneous staining

ABSTRACT Giardia and Cryptosporidium are pathogenic protozoa often present in the environment in their infective form(cysts and oocysts). These parasites are very resistant to disinfection, which makes them important target organisms in environmental quality monitoring and sanitation. Viability assessment provides an interpretation of cell inactivation, and it can be evaluated by membrane integrity as well as enzyme activity, using different staining methods. These are straightforward and adequate to laboratories that lack infrastructure for molecular-based technologies or animal infectivity tests. This study investigated simultaneous staining by a commercial live/dead kit, in order to assess viability of Cryptosporidium parvum oocysts and Giardia muris cysts, comparing it to propidium iodide (PI) incorporation, a common stain applied in viability estimation. Results suggested that, although the central hypothesis of one-panel visualization (α = 0.05) was met, simultaneous staining impaired (oo)cyst detection by immunofluorescence assay (IFA), which was found to be essential to enumeration, as the live/dead test led to poor (oo)cyst labelling or a 10-fold lower recovery when carried out concomitantly to IFA. As for the viability assessment itself, although red dye uptake occurred as expected by dead or weakened organisms, neither live G. muris cysts or C. parvum oocysts present any green fluorescence by esterase metabolism. This may have been caused by low enzyme activity in the infective form and/or wall thickness of these parasites. The results do not exclude the possibility of simultaneous fluorescence staining for protozoa, but it is a starting point for a broader analysis, that may consider, for instance, different incubation conditions.

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

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

Therapeutic Efficacy of Ginger (Zingiber officinale), Ginseng (Panax ginseng) and Sage (Salvia officinalis) Against Cryptosporidium parvum in Experimentally Infected Mice

Cryptosporidium parvum (C. parvum) is a worldwide zoonotic protozoan parasite infects most mammalian hosts causing a major health problem. The present study investigated the efficacy of ginger (Zingiber officinale), ginseng (Panax ginseng) and sage (Salvia officinalis) methanolic extracts on the progression of cryptosporidiosis in the experimental mice.Forty five mice experimentally infected with C. parvum were treated with medicinal plants extracts (ginger, ginseng and sage) as compared to the reference drug, Nitazoxanide (NTZ). Mice fecal smears were examined daily for 4 weeks post infection (PI). The results interpreted by oocysts count and histological examination of mice intestinal mucosa.The obtained results recorded that there was a statistically significant reduction in oocyst shedding in high dose ginger, ginseng and NTZ treated groups till no oocysts were found at days 21 and 23 PI, respectively. The infected non-treated, NTZ and low dose of both, ginseng and sage treated mice groups returned to excrete oocysts at low levels at day 27 PI while the other animal groups’ feces were still negative for C. parvum oocysts. The histopathological examination showed that NTZ, high dose ginger and ginseng treated mice had more protective and curative effect on infected mice intestinal epithelium in comparison with other treatments used.In conclusion, these results proved the therapeutic efficacy of ginger, ginseng and sage medicinal plants against the C. parvum in experimentally infected mice, and that theginger extract had an obvious effect on infected mice than other treatments and such results could be adapted in similar infections in susceptible animals and man.

EFFECT OF CRYPTOSPORIDIUM PARVUM INFECTION ON DNA DAMAGE AND CYTOKINES GENE EXPRESSION IN BLOOD OF IMMUNOCOMPROMISED MICE

There are few data regarding the genotoxic and immunomodulatory effects of Cryptosporidium parvum (Family: Cryptosporidiidae) infection in vivo. Therefore, the present study evaluated the effect of Cryptosporidium parvum infection on DNA and gene expressions of interleukin-6 (Il-6) and tumor necrosis factor-α (Tnf-α) in blood of the immunocompromised mice. Fifty adult male Swiss albino mice were used in the present study; these were randomly divided into five groups (10 mice/each group) as follows: group I served as a healthy, uninfected control group; groups II-IV were immunosuppressed by dexamethasone, and uninfected, or infected (orally) with 3.0×103 (low dose) or 1.0×104 (high dose) Cryptosporidium parvum oocysts, respectively; group V did not receive dexamethasone and infected with 1.0×104 Cryptosporidium parvum oocysts. DNA damage of leucocytes was assessed by flow cytometry and comet assay; the gene expression of the cytokines was performed using real-time polymerase chain reaction. The results revealed that infection with the low or the high dose of Cryptosporidium parvum induced DNA damage in the leucocytes of the immunosuppressed mice, as revealed by the elevations in tail length and tail moment of the comet assay compared with the healthy and immunosuppressed, uninfected, mice. In addition, significant decreases and increases in the expression of Il-6 and Tnf-α genes, respectively, were recorded in leucocytes of the immunosuppressed, Cryptosporidium parvum-infected mice in comparison with the healthy and immunosuppressed, uninfected, mice. In conclusion, Cryptosporidium infection induced DNA damage and augmented the changes induced in the Il-6and Tnf-α gene expressions in the leucocytes of the immunosuppressed mice.

Experimental study of heat-killed oocysts of Cryptosporidium Parvum in Balb/ c Mice

The present study investigated the possibility of infecting Mus musculus mice, experimentally, with cryptosporidiosis by inoculation with Cryptosporidium parvum oocysts, after treatment with temperatures (70 °C) and non-treated oocysts, single or double doses, aiming at enrichment of information related to existence of oocysts infected, outside the body of the host when subjected to temperatures. Criteria taken into consideration are the period spent by the parasite inside the body of the host, number of oocytes shed by the parasite with the host’s stool, total and differential leukocyte count. The Results of the present study shows: 1- Shedding of Oocysts started at the 4th day post infection by non-treated Oocysts, at single dose, and continued until the day 35 post infection, whereas with the challenge dose it continued until the 17th day post infection. 2- Temperature at (70Co) killed the Oocysts as infection of mice didn’t occur. 3- Generally, a gradual increase in total and differential leukocyte count on infection with non-treated Oocysts at day 3 post infection.

Cold Atmospheric Plasma Jet Inactivates Cryptosporidium parvum Oocysts on Cilantro

Cilantro was recently identified as a vehicle for protozoan illness. Current postharvest practices are not sufficient to inactivate protozoa on cilantro. Cold plasma is an emerging nonthermal waterless technology with potential applications in food processing that are currently being investigated to enhance the safety of herbs. The purpose of this study was to determine the impact of cold atmospheric plasma (CP) on the viability of Cryptosporidium parvum oocysts on cilantro. C. parvum oocysts were inoculated onto cilantro and treated with a CP jet for 0, 30, 90, and 180 s at a working distance of 10 cm with a flow of 1.42 × 10-3 m3/s. Oocyst viability was determined using HCT-8 cell culture infectivity assays. Overall, each treatment significantly reduced oocyst infectivity compared with the 0-s treatment control (P ≤ 0.02). Log inactivations of oocysts observed on cilantro were 0.84, 1.23, and 2.03 for the 30-, 90-, and 180-s treatment times, respectively. Drying and darkening of cilantro leaves was observed with treatments longer than 30 s. CP can reduce C. parvum infectivity on cilantro. With further research and optimization, this treatment technology has potential applications in postharvest processing of cilantro.

CHENOPODIUM AMBROSIOIDES OIL EXTRACT REDUCED CRYPTOSPORIDIUM PARVUM DEVELOPMENT IN VIVO

Cryptosporidium is a widespread parasite transmitted through contaminated food and water; it could lead to severe gastrointestinal disease in immunocompromised hosts. To date, no specific therapy proved to be effective against Cryptosporidium parvum (C. parvum). The present work studied the efficacy of Chenopodium ambrosioides (CA) oil extract on C. parvum infected dexamethasone (DEX) immunosuppressed mice. Infected im unosuppressed mice (n=70) were divided into 14 groups of five mice each according to different concentrations of received treatments either CA oil extract at doses of 75, 100, 125, 150 & 175mg/kg/day alone or in combination with nanoparticles or nitazoxanideat doses of 50 & 100mg/kg/day alone or in combination with nanoparticles. Five infected non-treated mice were included as an infected control group; five non- infected & non-treated mice were enrolled as a normal control one. Anti-Cryptosporidium efficacies of different treatment regimens were evaluated in the 16th mice groups microscopically by detection of C. parvum oocysts in fecal pellets and histopathological examination of their small intestines. The results showed that different concentrations of CA oil extract induced a significant reduction in the mean number of C. parvum oocysts. The rate of oocysts reduction was parallel to the increasing concentrations of the drug. The inhibitory effects of CA oil extract on the parasite were enforced by adding nanoparticles. Histopathological examination of mice intestine revealed that CA did not show toxic effect on intestinal mucosa even with high doses.

CHENOPODIUM AMBROSIOIDES OIL EXTRACT REDUCED CRYPTOSPORIDIUM PARVUM DEVELOPMENT IN VIVO

Cryptosporidium is a widespread parasite transmitted through contaminated food and water; it could lead to severe gastrointestinal disease in immunocompromised hosts. To date, no specific therapy proved to be effective against Cryptosporidium parvum (C. parvum). The present work studied the efficacy of Chenopodium ambrosioides (CA) oil extract on C. parvum infected dexamethasone (DEX) immunosuppressed mice. Infected im unosuppressed mice (n=70) were divided into 14 groups of five mice each according to different concentrations of received treatments either CA oil extract at doses of 75, 100, 125, 150 & 175mg/kg/day alone or in combination with nanoparticles or nitazoxanideat doses of 50 & 100mg/kg/day alone or in combination with nanoparticles. Five infected non-treated mice were included as an infected control group; five non- infected & non-treated mice were enrolled as a normal control one. Anti-Cryptosporidium efficacies of different treatment regimens were evaluated in the 16th mice groups microscopically by detection of C. parvum oocysts in fecal pellets and histopathological examination of their small intestines. The results showed that different concentrations of CA oil extract induced a significant reduction in the mean number of C. parvum oocysts. The rate of oocysts reduction was parallel to the increasing concentrations of the drug. The inhibitory effects of CA oil extract on the parasite were enforced by adding nanoparticles. Histopathological examination of mice intestine revealed that CA did not show toxic effect on intestinal mucosa even with high doses.

Identification of Cryptosporidium parvum Oocysts among Hospitalized Children under-5years in Northeastern Nigeria

Cryptosporidium parvum is among the major pathogens causing diarrheal diseases in children. It is of major public health significance due to its low infectious dose and its oocysts are highly resistant to chlorination, common household disinfectants and survive long periods in the environment. This study was designed to evaluate the occurrence of Cryptosporidium parvum oocysts in stool of hospitalized children under-5 years. One hundred and fifty (150) stool samples were collected from one hundred and fifty children (Male:Female = 1:1.08, Mean Age±S.D=22.08 months ± 21.02) and were processed using the modified Ziehl-Nelson method for identification of protozoan oocysts. Out of the one hundred and fifty (150) stool samples analyzed, 16 tested positive to oocysts of C. parvum, which gives a parasite prevalence rate of 10.7%. This was observed to be higher among male patients (52.0%) and children between the age 32-41 months (31.3%). Parasite prevalence in relation to age of patients was statistically not significant (X2=0.105, DF=1, P-value = 0.74591, p<0.05). Other intestinal protozoan parasites identified include Entamoeba histolytica (1.33%) and Giardia lamblia (2.60%).

Efficacy of ultraviolet light-emitting diodes (UV-LED) at four different peak wavelengths against Cryptosporidium parvum oocysts by inactivation assay using immunodeficient mice.

As an alternative to using ultraviolet (UV) lamps, which are made with mercury that is toxic to the environment and human health, UV light-emitting diodes (UV-LEDs) are expected to be effective for inactivating microorganisms in water. Although UV-LEDs have been reported to be effective against bacteria and viruses, the effectiveness of UV-LEDs against Cryptosporidium parasites has not been fully evaluated. As we report here, we have developed an in vivo quantitative inactivation assay for C. parvum oocysts using immunodeficient mice. Using the assay, we evaluated the effectiveness of treatment by UV lamp (254nm) at approximately 1000μJ/cm2 (for 3s at a distance of 95mm) compared to inactivation by commercially available UV-LEDs (with peak wavelengths of 268, 275, 284, and 289nm). The shed patterns of oocysts after treatment with 284- and 289-nm wavelength UV-LEDs were significantly delayed compared to that after treatment with a UV lamp. These findings provide the first suggestion that UV-LEDs are effective against these parasites, as assessed using commercially available 350-mA UV-LEDs under conditions of fixed exposure distance and time.

Effect of Ginsenoside-Rh2 and Curcurbitacin-B on Cryptosporidium parvum in vitro

Ginsenoside-Rh2 and cucurbitacin-B (CuB) are secondary metabolites of Ginseng (Panax ginseng) and Cucurbitaceae plants respectively. We assessed the anticryptosporidial activity of these two functional compounds in a cell culture model of cryptosporidiosis. The highest concentration of each compound that was not toxic to the host cells was used to assess the activity against C. parvum during infection/invasion and growth in HCT-8 cell monolayers. Monolayers were infected with pre-excysted C. parvum oocysts. Infected monolayers were incubated at 37 °C for 24 h and 48 h in the presence of different concentrations of each test compound. A growth resumption assay was performed by incubating infected monolayers in the presence of compounds for 24 h followed by a second 24-h incubation in the absence of compound. To screen for invasion inhibiting activity, freshly excysted C. parvum sporozoites were pre-treated with different concentrations of compounds prior to adding them to the cell monolayers. Paromomycin, a known inhibitor of C. parvum, and DMSO were used as positive and negative control, respectively. The level of infection was initially assessed using an immunofluorescent assay and quantified by real-time PCR. Both compounds were found to strongly inhibit C. parvum intracellular development in a dose-dependent manner. IC50 values of 25 μM for a 24 h development period and 5.52 μM after 48 h development were measured for Rh2, whereas for CuB an IC50 value of 0.169 μg/ml and 0.118 μg/ml were obtained for the same incubation periods. CuB also effectively inhibited resumption of growth, an activity that was not observed with Rh2. CuB was more effective at inhibiting excystation and/or host cell invasion, indicating that this compound also targets extracellular stages of the parasite.

Filter Backwash Water and Floated Residue Containing Pathogenic Protozoa: Detection Method and Treatment Alternatives

Treatments using ozone for filter backwash water (FBW) and calcium oxide for floated residue (FR) were evaluated adopting bench-scale testing for the inactivation of Giardia spp. cysts and Cryptosporidium parvum oocysts. The protocol chosen for protozoa detection involved following the concentration step by direct centrifugation (adding ICN 7X cleaning solution at 1.0%) and purification by immunomagnetic separation (IMS). The FR treatment with calcium oxide (dosage of 23 mg CaO 100 mL−1 and 3-day contact time at 25 °C) proved to be efficient, as no parasites were detected after the treatment. The reduction of calcium oxide dosage (16 mg CaO 100 mL−1 and 3-day contact time at 25 °C) was insufficient to inactivate the protozoa, since potentially viable organisms were identified using propidium iodide (PI). Concerning the disinfection conditions with ozone (5-min and 10-min contact time and dosage of 10 mg O3 L−1 and 7.5 mg O3 L−1, respectively), there was complete removal of the target organisms, as no protozoa were detected after the FBW treatment. From the results obtained, the tested treatments can be considered promising alternatives for water treatment plants (WTPs). However, the costs incurred from these treatments have to be considered.

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

ABSTRACT 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.

A Structural Bioinformatics Study of Calcium Dependent Protein Kinase 6 (CDPK6) of Cryptosporidium Parvum for Identification of Novel Therapeutics

Cryptosporidium parvum is an obligate intracellular protozoan parasite that infects the intestinal tract of mammals and causes cryptosporidiosis. C. parvum oocysts are ingested by a host that reproduces sexually and asexually in the intestinal lining cells, resulting in diarrhea. Calcium Dependent Protein Kinases (CDPKs) are found in the apicomplexan, algae, and plants; however, they are reported in vertebrates and are regarded as excellent drug targets for pharmaceutical interventions. CDPKs of Cryptosporidium are probably involved in the regulation of invasion and egress process during the infection of the host cells. Calcium-dependent protein kinase6 (CDPK6) (cgd4_3330) is a hypothetical protein of the CDPKs family which was originated from the plants and also a few species of protozoa. These proteins regulate several biological processes of apicomplexan for example Cryptosporidium, Plasmodium, and Toxoplasma. In this study, an attempt is made to address the sequences similarity and phylogenetic study of CDPK6 among apicomplexans CDPKs family. Furthermore, the three-dimensional structure determination of the target protein was performed through a molecular modeling approach (MODELLER v9.21 software) followed by virtual screening (GOLD software suite & Glide software) of small molecule inhibitors from five different datasets. The best inhibitor Tres Cantos Antimalarial Set (TCAMS) _11730 reported a binding affinity of -8.2 kcal/mol towards the CDPK6 of C. parvum through molecular docking studies (AutoDock v4.2.6 software). Furthermore, the reliability and validation of the binding mode of the inhibitor were performed through a complex molecular dynamics simulation in an aqueous environment with GROMACS 2018.3 software for a time interval of 100 ns. The findings of the study advocate that the best inhibitor may be considered for industrial pharmaceutical research in future.

Protozoa and Virus Disinfection by Silver- and Copper-Embedded Ceramic Tablets for Water Purification

Point-of-use water treatment (PoUWT) technologies have been shown to reduce incidences of waterborne diseases. However, their effectiveness is limited against the most harmful pathogens, such as protozoa and viruses, due to the inability to deliver effective and safe levels of disinfectant. This paper studies disinfection mechanisms and efficiency of silver and copper against Cryptosporidium parvum, Giardia lamblia, and MS2 bacteriophage through a novel PoUWT method, a ceramic tablet embedded with metallic ions. Results demonstrate that the PoUWT method can deliver safe and effective levels of silver and copper to inhibit sporozoite metabolic ability, and thus disinfect protozoa. Excystation reduced from 74% to 14% for Cryptosporidium parvum oocysts treated with silver-embedded ceramic tablets. Similar results were observed among Giardia lamblia oocysts and copper-treated oocysts. Fluorescence microscopy demonstrated two mechanisms for protozoa disinfection by metallic ions: (1) structural damage of oocyst walls, and (2) alteration of sporozoite structure. Also, MS2 bacteriophage viability reduced by 89% when treated with ceramic tablet treatment. Using the PoUWT method in this study, silver and copper show promise as disinfecting agents for protozoa.

In vitro Model for Cryptosporidium parvum Infection

ABSTRACT Background: The in vitro culture system of C. parvum has been investigated using numerous cell lines that support the growth of different stages of the parasite which is less expensive and more convenient for the study of host-parasite interactions than in vivo models. Objective of the study: to evaluate in vitro models of C. parvum and to establish an in vitro culture system for further research studies. Material and Methods: Confluent HT-29 Human Colorectal Adenocarcinoma cell monolayers were infected with 4x105 viable C. parvum oocysts, suspended in 200 l cell culture medium (DMEM). Cells were then incubated for 2 hours at 37°C. Cell monolayers that grow on 13 mm glass coverslips were washed twice with PBS and fixed in methanol for 1 min, stained with 10% Giemsa at different periods (6, 24, 48 and 72 h) and then mounted on glass slides. The parasites were counted, and the obtained data were statistically analyzed. Results: It was found that the HT-29 cell line supports the in vitro cultivation of C. parvum. The maximum numbers of parasites were obtained by 6 h and 24 h post-infection. Only immature stages were observed in the cell line after 6 h of infection. However, there were many immature and mature asexual stages in the cell monolayer 24 and 48 post infection. The maximum numbers of parasites were counted in cell monolayer infected by 4x105 oocysts. Conclusion: It was concluded that the HT-29 cell line supports the in vitro cultivation of C. parvum protozoan parasite.

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).

Comparison of viability and phagocytic responses of hemocytes withdrawn from the bivalves Mytilus edulis and Dreissena polymorpha, and exposed to human parasitic protozoa

Bivalve molluscs are now considered indicator species of aquatic contamination by human parasitic protozoa. Nonetheless, the possible effects of these protozoa on the immune system of their paratenic hosts are poorly documented. The aim of this study was to evaluate the effects of two protozoa on hemocyte viability and phagocytosis from two mussels, the zebra mussel (freshwater habitat) and the blue mussel (seawater habitat). For these purposes, viability and phagocytic markers have been analysed on hemocytes from mussels without biological stress (control hemocytes), and on hemocytes exposed to a biological stress (Toxoplasma gondii and Cryptosporidium parvum oocysts). We report, for the first known time, the interactions between protozoa and hemocytes of mussels from different aquatic environments. Zebra mussel hemocytes showed a decrease in phagocytosis of fluorescent microbeads after exposure to both protozoa, while blue mussel hemocytes reacted only to T. gondii oocysts. These decreases in the ingestion of microbeads can be caused by competition between beads and oocysts and can be influenced by the size of the oocysts. New characterisations of their immune capacities, including aggregation, remain to be developed to understand the specificities of both mussels.

Microfluidic diffraction phase microscopy and its application in parasites measurement

This paper proposes a method of using diffraction phase microscopy combined with microfluidic chip to quantitatively measure waterborne parasites. A diffraction phase microscopy system is built up by combining interferometry with optical microscope to achieve high sensitivity real-time measurement of parasites. Based on soft lithographic techniques, a double-layered microfluidic chip with U-shaped trapping structures is designed and fabricated for high throughput single parasites trapping. Ficoll solution with the same refractive index as polydimethylsiloxane (PDMS) is introduced into the microfluidic chamber to eliminate significant artifacts in phase imaging originating from diffraction at the edges of trapping structures. The accuracy of the system is verified using standard polystyrene microspheres of different diameters, and the error of maximum phase shift does not exceed 3%. 100 Giardia Lamblia (G. Lamblia) cysts and 100 Cryptosporidium Parvum (C. Parvum) oocysts are measured using this system. The phase maps of the parasites are obtained from the interferograms. The morphological parameters and quantitative optical volume difference distribution of the two kind of waterborne parasites are obtained by analyzing the quantitative phase maps. Quantitative data provides the basis for understanding their physiological characteristics. The microfluidic diffraction phase microscopy system has simple structure, good stability and high measurement accuracy, and has great potential for real-time monitoring and label-free quantitative studies of single microorganism.