Point-of-Use Removal of Cryptosporidium parvum from Water

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Point-of-use removal of Cryptosporidium parvum from water: Independent effects of disinfection by silver nanoparticles and silver ions and by physical filtration in ceramic porous media Lydia S. Abebe, Yi-Hsuan Su, Richard L. Guerrant, Nathan S. Swami, and James A. Smith Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.5b02183 • Publication Date (Web): 23 Sep 2015 Downloaded from http://pubs.acs.org on October 8, 2015

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Environmental Science & Technology

Body weight (% change)

110 Uninfected Heat treated Pro-capped AgNPs treated PVP-capped AgNPs treated AgNO3 treated Infected

100

90

80

0

1

2 3 4 5 6 Time Post-Infection (days)

7

Figure 1 Plots of percent body weight change of mice fed Cryptosporidium parvum oocysts for a 7-d post-infection period. Each data set represents a different oocyst disinfection method as well as positive (infected) and negative (uninfected) controls. Error bars represent standard error for each measurement.

ACS Paragon Plus Environment


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Pro-capped AgNPs treated

AgNO3 treated

PVP-capped AgNPs treated

Infected

No. of oocysts/10 mg stool

105

104

103

102

101

1

2

Time Post-Infection (days)

3

Figure 2 Cryptosporidium parvum oocyst concentration in stool shed from mice as a function of postinfection time for different oocyst disinfection treatments. Each data set represents a different oocyst disinfection method as well as positive (infected) and negative (uninfected) controls. Error bars represent standard error for each measurement. *Heat treated group not represented in graph, shedding was below detectable limit.


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Figure 3 High resolution contrast images illustrating the morphological differences in the oocysts after disinfectant treatments. First column shows phase contrast images and second column shows Nomarski differential interference contrast images under different treatments: (a and b) Untreated; (c and d) Heat-treated; (e and f) PVP-coated ACS Paragon Environment AgNP treated; (g and h) Proteinate-coated AgNP treated; (i and Plus j) AgNO 3 treated oocysts of Cryptosporidium parvum.


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Figure 4 Typical images from excystation studies on the oocysts: (a) Untreated; (b) Heat-treated; (c) PVP AgNP treated; (d) Proteinate AgNP and (e) AgNO3 treated oocysts of Cryptosporidium parvum. Dotted red arrows show normal excystation behavior of the oocysts and solid yellow arrows show the lack of excystation.


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3

Log Removal

2

1

0

10-mesh / 10%

16-mesh / 11%

16-mesh / 10%

16-mesh / 9%

20-mesh / 10%

Disk Types

Figure 5 Mean log removal of C. parvum by ceramic disks. The labeling system for the disk types refers to the mesh size, and the percentage of sawdust in the ceramic disk. The error bars represent the standard error of the mean.



Part Procedure I. In vivo assessment of C. parvum DNA Stool Mini ool Mini Procedure QIAamp DNA Stool Mini Procedure infectivity with and without Ag deactivation

Part II. C. parvum oocyst removal by filtration

1x109 oocysts/ 200 µL 1x107 oocysts/ 0.6 mL

PBS

ibitEX Tablet

PBS

Ag + PBS

4 pairs of ceramic disks with varying porosity

Mouse model

Measure C. parvum removal

Weigh mice and quantify Adsorb Adsorb Adsorb C. parvum in mouse stool inhibitors with inhibitors with oocysts inhibitors with InhibitEX matrix InhibitEX matrix InhibitEX matrix


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Point-of-use removal of Cryptosporidium parvum from water: Independent effects of disinfection by silver nanoparticles and silver ions and by physical filtration in ceramic porous media Lydia S. Abebe*,1, Yi-Hsuan Su2, Richard L. Guerrant3, Nathan S. Swami2, and James A. Smith4 1

Environmental Sciences and Engineering Department, University of North Carolina Chapel Hill, North Carolina 27599-7400 2 Department of Electrical and Computer Engineering, University of Virginia, P.O. Box 400743 Charlottesville, Virginia 22904-4742 3 Department of Medicine, University of Virginia, P.O. Box 801379 Charlottesville, Virginia 22904-4742 4 University of Virginia, Department of Civil and Environmental Engineering Charlottesville, VA, USA 22904-4742 Abstract Ceramic water filters (CWFs) impregnated with silver nanoparticles are a means of household-level water treatment. CWFs remove/deactivate microbial pathogens by employing two mechanisms: metallic disinfection and physical filtration. Herein we report on the Independent effects of silver salt and nanoparticles on Cryptosporidium parvum (C. parvum) and the removal of C. parvum by physical filtration in porous ceramic filter media. Using a murine (mouse) model, we observed that treatment of oocysts with silver nitrate and proteinate-capped silver nanoparticles resulted in decreased infection relative to untreated oocysts. Microscopy and excystation experiments were conducted to support the disinfection investigation. Heat and proteinate-capped silver-nanoparticle treatment of oocysts resulted in morphological modifications and decreased excystation rates of sporozoites. Subsequently, disk-shaped ceramic filters were produced to investigate the transport of C. parvum. Two factors were varied: sawdust size and clay-to-sawdust ratio. Five disks were prepared with combinations of 10, 16, and 20 mesh sawdust and sawdust percentage that ranged from 9 to 11%. C. parvum removal efficiencies ranged from 1.5 log (96.4%) to 2.1 log (99.2%). The 16-mesh/10% sawdust had the greatest mean reduction of 2.1-log (99.2%), though there was no statistically significant difference in removal efficiency. Based on our findings, physical filtration and silver nanoparticle disinfection likely contribute to treatment of C. parvum for silver impregnated ceramic water filters, although the contribution of physical filtration is likely greater than silver disinfection. INTRODUCTION



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One of the most problematic waterborne diseases is Cryptosporidiosis.

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Cryptosporidiosis is caused by Cryptosporidium, a protozoan parasite that infects the

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mammalian gastrointestinal epithelium 1, 2. The parasite consists of oocysts that are 4-6

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µm in size, have a low infectious dose, and cause diarrhea that is self-limiting in

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immunocompetent individuals, but is potentially fatal to immunocompromised

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individuals, especially those with human immunodeficiency virus (HIV) 1, 3. Young

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children are also vulnerable, according to a study in 7 countries in Africa and Asia,

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Cryptosporidium was the second most common pathogen contributing to moderate-to-

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severe diarrhea in children aged under 5 years 4. Cryptosporidiosis cases have been

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reported in six continents in both developed and developing areas 2. Genetic studies have

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identified 20 species of Cryptosporidium, of which Cryptosporidium parvum (C. parvum)

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is attributed to the majority of human infections 2.

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The deactivation and removal of C. parvum from water supplies has proven to be

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a particularly difficult problem, both in the developed world and within communities of

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the developing world 5, 6. Relative to other waterborne pathogens, oocyst-forming C.

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parvum is resistant to disinfection by hypochlorous acid, chlorine dioxide, and

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chloramine 7. Therefore, due to the recalcitrant nature of C. parvum, its treatment is

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typically addressed through filtration and pretreatment with coagulation-flocculation to

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optimize its physical removal 8. In resource-limited settings in developing countries,

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centralized treatment facilities are often scarce and point-of-use (POU) household

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treatment methods are the primary alternative 9.

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Several meta-analyses have emphasized the importance of improved water quality on the reduction of diarrheal disease, and suggest POU technologies as effective


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methods of improving water quality at the household level 10-14. Studies have identified

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ceramic water filters as a promising technology based on performance and social

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acceptability, which results in higher long term use 13, 15.

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Ceramic water filters (CWFs) were developed in the 1980s in Guatemala out of

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growing demand for decentralized, household water treatment systems. Multiple

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organizations have helped developed ceramic filter production facilities in developing-

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world settings. One such organization is Potters for Peace (PfP). PfP has established over

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30 filter factories that produce CWFs in Africa, Asia, and Central and South America.

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CWFs gained popularity because the filters are produced from locally sourced materials,

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are cost-effective, are easy to use, and have demonstrated laboratory and field

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effectiveness 15-20.

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The filters resemble a ceramic pot. They are porous, and are flat or rounded at the

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base. The ceramic media is suspended in a 5-gallon plastic bucket with a lid. Water is

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poured into the filter and percolates through into the lower reservoir. Water in this

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reservoir is then accessed through a spigot attached at the bottom. The ceramic media are

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either impregnated or coated with silver nanoparticles (AgNPs). Silver nanoparticles have

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demonstrated antimicrobial properties, and do not pose adverse health risks below the

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Drinking Water Quality Guidelines

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purified through physical filtration and chemical disinfection (caused by the silver

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impregnated into the ceramic matrix).

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21-24

. As a result of percolation and silver, the water is

Although there is little data on the mechanism of silver nanoparticle disinfection

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for protozoa, there is mechanistic information for bacterial pathogens. For bacteria,

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disinfection is believed to be caused by the release of silver ions in combination with



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reactive oxygen species 25. Direct nanoparticle-pathogen interaction is not responsible for

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disinfection 26, 27. Silver ions are hypothesized to deactivate microbial enzymes by

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binding to thiol groups (–SH) 28. Other theories suggest that silver ions enter the

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microbial cell and intercalate between purine and pyramiding base pairs. This disrupts

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hydrogen bonding between the two anti-parallel strands and denatures the cellular DNA

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28

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stabilize nanoparticle size during synthesis, affects the nanoparticle’s physiochemical

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properties and as a result modifies the release of silver ions and reactive oxygen species

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(ROS) 29, 30. Morones et al. determined AgNPs with sizes ranging from 1 to 10 nm

. Some studies suggest that the presence of a capping agent, which are necessary to

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demonstrated the greatest antibacterial effect 21. Additionally, nanoparticle shape and size

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affects the release of ions and ROS, therefore silver nanoparticles have an advantage over

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ions alone due to their increased surface area, which allows for greater contact area to the

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binding site and a slow release of Ag+ and ROS 23. Furthermore, the effects of silver nanoparticles on the infectivity of C. parvum

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oocysts have, to our knowledge, have never been studied despite the global significance

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of this protozoan pathogen to human health. These information gaps are in part caused by

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the difficulties associated with quantifying the viability and infectivity of C. parvum.

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Unlike other common bacterial pathogens, C. parvum does not reproduce outside the

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mammalian gastrointestinal track. Therefore, to positively determine infectivity, murine

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models must be employed 31. These models combine cryptosporidial infection with

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malnutrition based on trends from longitudinal cohort studies, which have demonstrated

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short- and long-term impacts of cryptosporidiosis on growth and development of children

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32-35

. Studies have shown malnutrition greatly increases the risk for developing severe


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cryptosporidial infections 36, 37. Thus, murine models were developed with malnourished

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weaned mice, which have demonstrated severely impaired growth leading to substantially

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heavier infections 37-39.

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To date, to our knowledge, no studies have systematically studied the transport of

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C. parvum through ceramic water filters. Bielefeldt et al. observed silver increased

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removal with virus-sized microspheres, but no difference was observed for C. parvum-

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sized microspheres, which are larger 40. Oyanedel-Craver and Smith observed the use of

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AgNPs significantly improved E. coli removal, wherein no bacteria were detected in

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effluent samples, resulting in 100% removal 17.

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For the first time, to our knowledge, we present the results of in vivo studies to

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determine the effects of silver nanoparticles and silver ions on the infectivity of C.

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parvum. Additionally, to substantiate the studies on modifications to the infectivity of

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Cryptosporidium parvum oocysts after treatment with disinfectants using the murine

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models, we herein examine the morphological and functional alterations to the oocysts by

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optical micrography and analysis of the excystation behavior, respectively, as induced by

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various disinfectants (including silver nanoparticles and silver ions). Second, using

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porous ceramic media fabricated by methods common to point-of-use ceramic water

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filters, we quantify the removal of C. parvum oocysts by sorption and physical filtration

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and examine the effects of the porous media on this removal process. Finally, we

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conclude with evaluating the removal of C. parvum performance of the porous ceramic

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media using WHO health-based water-technology recommendations for household water

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treatment 41.



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MATERIALS AND METHODS

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In Vivo Studies

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Animals and malnutrition

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The protocol described herein is in accordance with the Institutional Animal Care

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and Use Committee (IACUC) policies of the University of Virginia. Weaned 21-day-old

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female C57BL/6 mice were purchased from Charles River Laboratories, Inc. Upon

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arrival, mice were acclimated, weighed, and distributed in groups. On day 28 of life, mice

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received chow containing 2% protein (Harlan Laboratories, Madison, WI). The animals

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remained on this diet for 7 days to establish malnutrition before infection and for the rest

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of the experiment post-infection. Diet and water were given ad libitum. Each mouse was

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weighed on a daily basis throughout the length of the experiment. Stool samples were

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obtained through the gentle stroking on the abdomen and collected on a daily basis from

148

the start of the infection until the end of the experiment. The first day of infection is

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recorded as day 0. The mice were observed for 7 days post-infection.

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Preparation of oocyst.

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C. parvum oocysts were purchased from Waterborne, Inc. Oocysts arrived in a

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stock solution of 1 X 109 per 50 mL phosphate-buffered saline (PBS) solution. A

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hemocytometer was used to quantify the number of oocysts for each infection.

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Preparation of oocysts required washing and suspension in deionized water. Each

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infected mouse received freshly prepared 1 X 107 oocysts in 100 µL of PBS via oral

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gavage. Mice receiving treated C. parvum were administered inoculums from the same

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freshly prepared batch. Inoculums with no treatment were refrigerated along with


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inoculums with treatment for the duration of the treatment contact time. All groups were

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gavaged on the same day.

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Preparation of treatments

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C. parvum oocysts were subject to the following treatment groups that include

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heat as a positive control (90° C for 5 minutes), silver nitrate (100 mg/L of Ag+ for 30

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minutes) and silver nanoparticles (100 mg/L of metallic silver Ag0 for 30 minutes). An

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aqueous silver suspension for treatment of C. parvum was prepared in deionized (DI)

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water. Proteinate-capped and polyvinylpyrrolidone-capped silver nanoparticles were

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prepared. Proteinate-capped silver was purchased from Laboratorios Argenol. Argenol

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silver nanoparticles have a mean diameter of 15 nm in size

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powder with 7.5 % silver content. 1000 mg/L of 10 nm polyvinylpyrrolidone (PVP)-

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capped AgNPs were purchased from nanoComposix based on the silver content reported

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by nanoComposix. Finally, silver nitrate (AgNO3) solution was prepared with silver

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nitrate (crystalline) from Fisher Scientific.

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Preparation of treatment groups

173

42

and arrive as dry solid

Mice were separated into the following groups: (I) Phosphate-buffered saline

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(PBS); (II) Heat-treated C. parvum; (III) Proteinate-capped-AgNP-treated C. parvum;

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(IV) PVP-capped-AgNP-treated C. parvum; (V) AgNO3-treated C. parvum; and (VI) C.

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parvum. Treatment groups are summarized in the Supporting Information Section, Table

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S1 (Supporting Information).

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DNA extraction for parasite detection

179 180

Stools collected from the mice were stored at -20°C until extraction. DNA was extracted from stool samples using reagents from Qiagen QIAamp DNA Stool Kit



181

(Qiagen Inc., Germantown, Maryland) and QIAcube, which automates the extraction

182

process. Minor modifications were made to the traditional extraction process for the

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extraction of DNA from mouse stool as described in Costa et al. 38, 43.

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Quantitative PCR for C. parvum

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Detection of C. parvum oocysts was performed using an iCycler iQ Multicolor

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quantitative PCR detection system (BioRad) with the use of known primers. A master

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mix consisting of 12.5 µL of Bio-Rad iQ SYBR Green Supermix (Bio-Rad Laboratories,

188

Hercules, California), 5.5 µL DEPC-treated nuclease free sterile water (Fisher Scientific,

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Pittsburgh, Pennsylvania) and 1.0 µL of forward and reverse primers (Invitrogen,

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Carlsbad, California) was prepared for qPCR. The primers target the 18s rRNA gene of

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the parasite (forward: 59-CTGCGAATGGCTCATTATAACA-39; reverse: 59-

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AGGCCAATACCCTACCG-TCT-39; GenBank no. AF164102). Bio-Rad iCycler

193

multicolor PCR Detection System using iCycler softwater (version 3.0) were used to

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perform detection of oocysts. The amplification progression used as described by Costa

195

et al. 38, 43. Threshold cycle (Ct) values were obtained from each run and were transformed

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into the number of organisms per sample of stool. Finally, results from C. parvum

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detection were expressed in log counts per 10 mg of stool (average weight of stool is 10

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mg).

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Statistical analyses

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Mouse weight was expressed in percent change based on body weight on day 0.

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Weight and shedding analysis were performed using GraphPad Prism Version 5.0c using

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Bonferroni post-tests (Two-way ANOVA).

203 204

Excystation Studies


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Disinfection treatments C. parvum oocysts were subject to disinfectants that include heat (90° C for 5

206 207

minutes), silver nitrate (100 mg/L of Ag+) and silver nanoparticles (100 mg/L of metallic

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silver Ag0 for 30 minutes) treatments.

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Excystation assay on oocysts. After each disinfection treatment, oocyst excystation was studied. Each oocyst

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suspension (20 L) was treated with 10% bleach (Bleach-Rite) for 30 minutes in a

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micro-centrifuge tube at room temperature, followed by vortexing every 10 minutes for a

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total of 3 times. The suspension was incubated in a micro-vial chamber for an hour using

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25 mm x 25 mm cover glass for real-time recording of the excystation; i.e. release of

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sporozoites from the oocyst. The excystation was examined under an inverted microscope

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using a 63X oil immersion phase and DIC objective lens. At least ten different fields of

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views were taken (Hammatsu Orca Flash4) and results from at least 300 oocysts were

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used to calculate the net excystation rate for each treatment. The percentage of the

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excystation was calculated using the following equation:

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=

221 222 223 224 225

Ceramic disk transport studies

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filters

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used is from the Mukondeni Pottery Cooperative in Mashamba in Limpopo Province,

228

South Africa. According to XRD analysis, the predominant clay mineral is smectite

229

The particle size distribution of the clay was analyzed using a Saturn DigiSizer II, results

# #

× 100

44

.

Disk synthesis Ceramic disks were manufactured to mimic transport through ceramic water 17

. The disks were manufactured using clay, sawdust, and water. The clay that was


45

.


230

yielded a mean of 110 µm (90 % of the clay particles were finer than 240 µm, 50% were

231

finer than 90 µm, and 10% were finer than 11 µm). We chose to test clay from

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Mukondeni because clay from this region is being used at a recently established ceramic

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filter factory that has been created by the non-profit organization, PureMadi, the

234

University of Virginia, the University of Venda, and Rotary International. As

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aforementioned, C. parvum is problematic in developing countries, particularly where

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there is a confluence of poor quality water and HIV prevalence. This unfortunate

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confluence exists in Limpopo and further motivated testing the clay from this region.

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The sawdust was acquired from a lumber mill in Ruckersville, Virginia.

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Table S2 (Supporting Information) summarizes the five clay and sawdust

240

combinations investigated in this study. The clay and sawdust were mixed and combined

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with water. The mixture was divided into four equal portions. Each portion was then

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placed in a 6.5-cm-diameter polyvinylchloride cylindrical mold, and compressed for 1

243

min at 1000 psi. The compacted mixture yielded a disk that was approximately 1.5 cm

244

thick. The disks were air dried at room temperature for 3 d. The disks were then placed

245

into an electric kiln and were subjected to the following temperature program: from room

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temperature to 600 ˚C, the temperature increased at a rate of 150 ˚C/h; from 600 to 900

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C, the rate increased at 300 ˚C/h. The temperature was held at 900 ˚C for 3 hr.

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C. parvum transport

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The experiments were performed using a flexible-wall permeameter, a high

250

performance liquid chromatography (HPLC) pump, and a three-way stopcock connected

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to the inflow and a 1 mL syringe. A 0.005 N CaSO4 solution was prepared and used as

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the inflow solution. The HPLC pump maintained a 0.6 mL/min inflow rate of the PBS


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17

253

solution. The chosen inflow rate was calculated by Oyanedel-Craver and Smith

to

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approximately correspond to a whole-filter flow rate of 1.5 L/h. Pore volume for each

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disk was determined based on the difference in weight of the saturated disk and dry disk

256

and the density of water (1 g/mL). Porosity was determined by dividing pore volume by

257

the total volume of the disk.

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C. parvum transport experiments were performed in duplicate on each ceramic

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disk. Each disk was re-fired in the kiln in between the first and second run to combust

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any previously deposited C. parvum. A 0.6 mL pulse of 1 x 107 C. parvum oocysts was

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injected into the filter and 1 mL of effluent was collected every 5 min for 90 min.

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Quantification of C. parvum

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Minor modifications were made to the DNA extraction protocol for mouse stool.

264

Effluent water samples were stored at 4 ˚C until extraction. Extraction from effluent

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samples was conducted by centrifuging at 5000 rpm for 5 minutes in 2 mL Qiagen

266

sample collection tubes to spin down oocysts. The supernatant was discarded and the

267

remaining sample was resuspended in 400 µL of ASL buffer and heated at 95 ˚C for 5

268

min before loading into the QiaCube as described by Costa et al 38, 43. DNA was

269

extracted using reagents from Qiagen QIAamp DNA Stool Kit (Qiagen Inc.,

270

Germantown, Maryland) in the QIAcube, which automates the extraction process. Minor

271

modifications were made to the traditional extraction process for the extraction of DNA.

272

The modifications made to the protocol are described in Costa et al. 38, 43. Detection of C.

273

parvum oocysts was conducted as stated in previous section titled Quantitative PCR for

274

C. parvum.

275

RESULTS



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In Vivo Studies Animal studies were used to investigate the effect of silver salts and nanoparticles

278

on C. parvum to determine whether oocysts treated with different disinfection

279

technologies will result in reduced weight decrement and number of parasites shed in

280

stool relative to experiments with untreated oocysts. The results shown in Figure 1 give

281

mean ± standard error of the mean (SEM). Weight at day 0 is considered 100% and

282

changes after day 0 were recorded as increases or decreases relative to day 0. The

283

“infected” group demonstrated a mean loss of 12% body weight by day 3 post-infection,

284

where C. parvum infection was at its peak. Mice that consumed heat-treated oocysts

285

maintained the growth pattern of the “uninfected” group and 102.8% (~3% gain) of body

286

weight at day 3 (P

Point-of-Use Removal of Cryptosporidium parvum from Water

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